WO2010127381A1 - Diagnosis, monitoring, prognosis, prevention and treatment of aneurysms - Google Patents

Diagnosis, monitoring, prognosis, prevention and treatment of aneurysms Download PDF

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Publication number
WO2010127381A1
WO2010127381A1 PCT/AU2009/000579 AU2009000579W WO2010127381A1 WO 2010127381 A1 WO2010127381 A1 WO 2010127381A1 AU 2009000579 W AU2009000579 W AU 2009000579W WO 2010127381 A1 WO2010127381 A1 WO 2010127381A1
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kks
substituted
gene
aneurysm
antagonist
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PCT/AU2009/000579
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French (fr)
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Jonathan Golledge
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James Cook University
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Publication of WO2010127381A1 publication Critical patent/WO2010127381A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/043—Kallidins; Bradykinins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/329—Diseases of the aorta or its branches, e.g. aneurysms, aortic dissection

Definitions

  • This invention relates generally to methods for the diagnosis, monitoring, prognosis, prevention and treatment of aneurysms in individuals.
  • An aneurysm is a localized, blood-filled dilatation of a blood vessel being an artery, vein or heart.
  • Aortic aneurysms are classified by which site within the aorta they occur.
  • Abdominal aortic aneurysms (AAAs), the most common form of aortic aneurysm are found in the abdominal aorta, and thoracoabdominal aortic aneurysms involve both the thoracic and abdominal aorta.
  • An aortic root aneurysm occurs at the commencement of the aorta.
  • Thoracic aortic aneurysms are found within the thoracic aorta and are further classified as ascending, aortic arch, or descending aneurysms depending on the location within the thoracic aortic involved.
  • aneurysms Initially most intact aneurysms are asymptomatic. If left untreated, aneurysms tend to become progressively larger, although the rate of enlargement is unpredictable for any individual. As the size of an aneurysm increases, pain and numbness may be experienced, normally caused by nerve compression. Further, the risk of rupture increases as the size of an aneurysm enlarges. When rupture occurs, symptoms including a drop in blood pressure, increased heart rate, and loss of consciousness are often observed and can result in severe haemorrhage or other complications including sudden death. There is a low survival rate after rupture has occurred, even if surgery is performed. [0006] The result is that aneurysms place a large burden on the health system.
  • AAA for example, is the 10 most common cause of death in men aged over 60 years (Tilson, 2005). In 2004, 470 deaths (5/100,000/yr) and 2,169 hospitalisations (50/100,000/yr) as a result of AAA were recorded in New South Wales, Australia alone (Report by Population Health Division, 2007). This burden is growing as the numbers of identified early AAAs increases, caused by both the growth in the elderly population and an increase in the use of abdominal imaging (Lederle, et al. , 1997).
  • aneurysms are deficient in a number of areas. At this time, for example, there are no blood-based diagnostic tests available. Diagnosis (and monitoring) of intact aneurysms is usually achieved by medical imaging, normally ultrasound. Current imaging protocols simply provide anatomical information on the aorta, such as maximum aortic diameter. AAA 5 for example, is thus diagnosed most commonly using a definition of maximum infrarenal aortic diameter greater than or equal to 30mm. Whilst aortic diameter provides some predictive power with respect to the subsequent behaviour of the aorta, the information provided is incomplete. Some aortas measuring >30mm remain stable for many years with no progression in diameter or risk of rupture.
  • AAAs aneurysms
  • the available treatments for aneurysms are also limited.
  • the only treatments which are currently employed are open or endovascular surgery.
  • Open surgery involves a large laparotomy, clamping of the aneurysm above and below, and replacing the weakened artery with a plastic graft. This operation is associated with a significant mortality of around 5%.
  • Endovascular AAA repair is a more minimally invasive procedure whereby stent grafts are secured within the aneurysm from inside via a groin approach.
  • the technique is associated with a lower mortality of approximately 1% and a smaller rate of perioperative complications and faster recovery than for open surgery.
  • There is some concern over the long term outcome for this procedure since reintervention is not uncommon during long term follow up in up to 20% of patients.
  • AAAs with a maximum diameter ⁇ 55mm Patients with an AAA measuring ⁇ 50mm are thus usually treated conservatively, and simply monitored by regular imaging. Only large aneurysms or those that have ruptured are considered eligible for the surgical or endovascular treatment options. There is therefore an "untreated group" of patients with aneurysms, including those with small aneurysms and those for which these treatments are not an option for other reasons, for example if surgery is considered too high risk.
  • the kallikrein-kinin system represents a metabolic cascade from kininogens to kinins which exert their pharmacological activities by binding specific receptors.
  • the kallikrein-kinin system therefore consists of a large number of proteins and smaller polypeptides, and includes enzymes that activate and deactivate various other components in the system.
  • HMWK high molecular weight kininogen
  • LMWK low molecular weight kininogen
  • HMWK and LMWK have no activity in themselves but are precursors to kinins, including bradykinin and kallidin.
  • HMWK is produced by the liver, while
  • LMWK is protected locally by numerous tissues, and secreted together with tissue kallikrein.
  • the kallikreins of the kallikrein-kinin system are serine proteases that liberate the kinins from the kininogens. Tissue kallikrein is initially secreted as the inactive prokallikrein, and plasma kallikrein is initially secreted as the inactive prekallikrein. Both prokallikrein and prekallikrein require activation by factors XII or other stimuli.
  • tissue kallikrein 1 (KLKl) encoded by the gene KLKl, is the only tissue kallikrein associated with the kallikrein-kinin system.
  • the kinins are small peptides. Bradykinin is a nonapeptide with the amino acid sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and is produced when plasma kallikrein releases it from HMWK.
  • Kallidin (Lys-bradykinin) is a decapeptide with the same amino acid sequence as bradykinin but with the addition of a lysine at the N-terminus. Kallidin is released from HMWK or LMWK by tissue kallikrein.
  • Bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin.
  • Alternative pathways of kinin formation involving enzymes other than kallikreins operate, for example in some disease states.
  • a proportion of kininogens are hydroxylated which leads to the formation of hydroxylated kinins which have similar biological activity to non-hydroxylated kinins.
  • Bradykinin and kallikrein are metabolised by carboxypeptidases.
  • the carboxypeptidases are present in two forms: N circulates and M is membrane-bound.
  • the carboxypeptidases remove arginine residues at the carboxy-terminus of bradykinin and kallidin.
  • Carboxypeptidase metabolites of the kinins include: des-Arg9-BK and Ly s- des-Arg9-BK.
  • kinin when used in reference to humans and most mammals refers to bradykinin and kallidin, the hydroxylated forms of each, and also to carboxy-terminal des-Arg metabolites of bradykinin, kallidin or the hydroxylated forms of each.
  • the kinins exert their pharmacological activities by binding specific kinin receptors: being type 1 (Bl) and type 2 (B2) receptors.
  • Kinin production in vivo is controlled, in part, by endogenous inhibitors of the kallikrein enzymes.
  • the main inhibitors of plasma kallikrein are the Cl inhibitor (Cl-INH - encoded in humans by the gene SERPINGl), ⁇ 2 -macroglobulin (encoded in humans by the gene A2M) and antithrombin III (encoded in humans by the gene SERPINCl).
  • Tissue kallikrein is inhibited by kallistatin which in humans is encoded by the gene SERPINA4.
  • ACE angiotensin converting enzyme
  • neutral endopeptidase deactivate certain peptide mediators, including kinins.
  • altering the levels of components in the kallikrein-kinin system can assist in preventing or treating aneurysms in an individual, and that detecting the presence, determining the prognosis or monitoring the progression of an aneurysm may be achieved by detecting aberrant expression of one or more genes in the kallikrein-kinin system (KKS), as described hereinafter.
  • the present invention provides methods for preventing or treating an aneurysm in an individual. These methods generally comprise administering to the individual an effective amount of a KKS antagonist.
  • KKS antagonists include small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, including those as further described herein.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene is selected from a gene encoding a component of the KKS (e.g.
  • the agent reduces the expression of a gene (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2) or the level or functional activity of an expression product of that gene (e.g. HMWK, LMWK 5 prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des- Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor).
  • the agent increases the expression of a gene (e.g.
  • SERPINGl A2M, SERPINCl, SERPINA4
  • level or functional activity of an expression product of that gene e.g. Cl -INH, ⁇ 2 -macroglobulin, antithrombin III, kallistatin
  • the agent reduces or increases the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Another aspect of the present invention contemplates the use of an effective amount of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist as broadly described herein.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • the present invention resides in the use of an effective amount of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist as broadly described herein.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • the present invention provides methods for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual.
  • One suitable method comprises detecting in the individual the presence of an aberrant gene encoding a component of the KKS or detecting in the individual aberrant expression of a gene encoding a component of the KKS.
  • the aberrant gene is selected from an aberrant KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, or SERPINA4 gene.
  • Another suitable method comprises determining in the individual a level or functional activity of a component in the KKS, which is different than a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component.
  • the method comprises determining an increase or elevation in the level of functional activity of the component (e.g.
  • the method comprises determining a decrease in the level or functional activity of the component (e.g. Cl-INH, ⁇ 2 -macroglobulin, antithrombin III, kallistatin) relative to a normal (e.g. no aneurysm, level measured in the same individual at an earlier time) reference or control level or functional activity of the component.
  • the method is performed on a biological sample obtained from the individual.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • the invention provides methods for identifying agents that antagonise the KKS.
  • One suitable method comprises contacting a preparation with a test agent, wherein the preparation comprises (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence (e.g.
  • a transcriptional control element such as a promoter or a czs-acting sequence
  • a transcriptional control element that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the KKS, or a variant or derivative thereof, , which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i).
  • a detected change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference and/or control level or functional activity in the absence of the test agent indicates that the agent modulates the KKS .
  • Another suitable method comprises contacting a sample of cells expressing a kinin receptor with a kinin and a test agent.
  • the agents identified using the methods broadly described above are useful for preventing or treating an aneurysm in an individual.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Still another aspect of the present invention provides methods of producing an agent comprising a KKS antagonist for preventing or treating an aneurysm in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for antagonising the KKS.
  • the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the aneurysm.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Figure 1 shows some of the results of Example 2.
  • bands of the B2 results are shown in the top line for samples taken from the neck (left hand side) and the body (right hand side) of the aneurysm.
  • the bottom line of Figure 1 shows the bands of the control, ⁇ -actin.
  • Figure 2 also shows some of the results of Example 2.
  • the concentration of both the Bl kinin receptor and B2 kinin receptor are shown.
  • the left result for each of Bl and B2 shows the results from the sample from the aneurysm neck, and the right result for each of Bl and B2 shows the results from the sample from the aneurysm body.
  • Figure 3 shows photographs of an aorta taken from a normal mouse (A — left hand side) and one from a mouse with an aneurysm (B — right hand side) from the experiment described in Example 3.
  • Figure 4 shows the survival rates of the four mice groups from the experiment described in Example 3.
  • an element means one element or more than one element.
  • allelic expression refers to the overexpression or underexpression of a gene encoding a component of the KKS relative to the level of expression of a gene encoding a component of the KKS or a variant thereof in cells obtained from a healthy individual or from an individual with no aneurysm, and/or to a higher or lower level of an expression product of that gene (e.g., transcript or polypeptide) in a biological sample obtained from a healthy individual or from an individual with no aneurysm.
  • transcript or polypeptide e.g., transcript or polypeptide
  • a gene encoding a component of the KKS is aberrantly expressed if the level of expression of the gene is higher by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%,
  • the reference to a level of expression of a gene of expression product of that gene in a healthy individual or an individual with no aneurysm may refer to an average level of expression in a group of healthy individuals or individuals with no aneurysm.
  • alkoxy alkenoxy
  • alkynoxy alkynoxy
  • aryloxy heteroaryloxy
  • heterocyclyloxy acyloxy
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon group and may have a specified number of carbon atoms.
  • Ci-Cio as in “Ci-Cioalkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in linear or branched arrangement.
  • Ci-C 10 alkyl specifically includes, but is not limited to, methyl, ethyl, n- propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl.
  • Alkoxy represents either a cyclic or non-cyclic alkyl group attached through an oxygen bridge. "Alkoxy” therefore encompasses the definitions of alkyl and cycloalkyl above.
  • alkoxy groups include but are not limited to methoxy, oxy ethoxy, n-propyloxy, i-propyloxy, cyclopentyloxy and cyclohexyloxy.
  • alkenyl refers to a non-aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present.
  • C2-C 6 alkenyl means an alkenyl radical having from 2 to 6 carbon atoms.
  • Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl.
  • the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.
  • alkynyl refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present.
  • C 2 -C 6 alkynyl means an alkynyl radical having from 2 to 6 carbon atoms.
  • Alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, 3-methylbutynyl and so on.
  • the straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.
  • substituents may be defined with a range of carbons that includes zero, such as (Co-C 6 )alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as, for example, -CH 2 Ph, -CH 2 CH 2 Ph, CH(CH 3 )CH 2 CH(CH 3 )Ph.
  • alkylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms, more preferably 1 to 12 carbons, even more preferably lower alkylene.
  • the alkylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • exemplary alkylene groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-(CH 2 ) 3 -), cyclohexylene (-C 6 H 10 -), methylenedioxy (-0-CH 2 -O-) and ethylenedioxy (-O-(CH 2 ) 2 -O-).
  • the term "lower alkylene” refers to alkylene groups having 1 to 6 carbons. Preferred alkylene groups are lower alkylene, with alkylene of 1 to 3 carbon atoms being particularly preferred.
  • alkenylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one double bond, more preferably 2 to 12 carbons, even more preferably lower alkenylene.
  • the alkenylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkenylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • the term "lower alkenylene” refers to alkenylene groups having 2 to 6 carbons. Preferred alkenylene groups are lower alkenylene, with alkenylene of 3 to 4 carbon atoms being particularly preferred.
  • alkynylene refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one triple bond, more preferably 2 to 12 carbons, even more preferably lower alkynylene.
  • the alkynylene group is optionally substituted with one or more "alkyl group substituents.” There may be optionally inserted along the alkynylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
  • the term "lower alkynylene” refers to alkynylene groups having 2 to 6 carbons. Preferred alkynylene groups are lower alkynylene, with alkynylene of 3 to 4 carbon atoms being particularly preferred.
  • “Amplification product” refers to a nucleic acid product generated by a nucleic acid amplification technique.
  • antigen-binding molecule a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • Antigenic or immunogenic activity refers to the ability of a polypeptide, fragment, variant or derivative according to the invention to produce an antigenic or immunogenic response in an animal, suitably a mammal, to which it is administered, wherein the response includes the production of elements which specifically bind the polypeptide or fragment thereof.
  • Alkyl means alkyl as defined above which is substituted with an aryl group as defined above, e.g., -CH2 ⁇ henyl, -(CH2)2phenyl, -(CH2)3phenyl, - H2CH(CH3)CH2phenyl, and the like and derivatives thereof.
  • aromatic or aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic.
  • aryl elements include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
  • biologically active fragment is meant a fragment of a full-length parent polypeptide which fragment retains an activity of the parent polypeptide.
  • biologically active fragment includes deletion variants and small peptides, for example of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 contiguous amino acid residues, which comprise an activity of the parent polypeptide.
  • Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Shephard which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson and published by Blackwell Scientific Publications.
  • peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu- C and staphylococcus V8-protease.
  • the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.
  • biological sample refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a patient.
  • the biological sample is a urine, whole blood, serum or plasma sample.
  • cycloalkenyl means a monocyclic unsaturated hydrocarbon group and may have a specified number of carbon atoms.
  • cycloalkenyl includes but is not limited to, cyclobutenyl, cyclopentenyl, 1- methylcyclopentenyl, cyclohexenyl and cyclohexadienyl.
  • cycloalkyl or "aliphatic ring” means a monocyclic saturated aliphatic hydrocarbon group and may have a specified number of carbon atoms.
  • cycloalkyl includes, but is not limited to, cyclopropyl, methyl- cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl.
  • derivative is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art.
  • derivative also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functional equivalent molecules.
  • an aneurysm in the context of treating or preventing an aneurysm is meant the administration of an amount of active ingredient to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for treatment or prophylaxis or improvement of that condition.
  • improvements in an individual include reducing the size of an aneurysm, preventing an increase in the size of an aneurysm in an individual, reducing the number of small aneurysms in an individual, and/or preventing or inhibiting the occurrence of aneurysms in an individual.
  • the effective amount will vary depending upon the health and physical condition of the individual to be treated, the formulation of the composition being administered, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • expression product refers to production of mRNA, translation of RNA message into proteins or polypeptides, or processed forms of those proteins or polypeptides.
  • function refers to a biological, enzymatic, or therapeutic function.
  • the term "gene” as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions.
  • the gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the DNA sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • halo or halogen as used herein is intended to include chloro, fluoro, bromo and iodo.
  • Heteroaralkyl means alkyl as defined above which is substituted with a heteroaryl group, e.g., -CHbpyridinyl, -(CH 2 ) 2 pyrimidinyl, - (CH 2 ) 3 imidazolyl, and the like, and derivatives thereof.
  • heteroaryl or “heteroaromatic,” as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, bezofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
  • heteroaryl is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl.
  • heteroaryiene refers to a bivalent monocyclic or multicyclic ring system, preferably of about 3 to about 15 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms.
  • the heteroaryiene group may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents.
  • Exemplary heteroaryiene groups include, for example, 1,4-imidazolylene.
  • heterocycle means a 5- to 10-membered nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups.
  • heterocyclylalkyl means alkyl as defined above which is substituted with a heterocycle group, e.g., -CHbpyrrolidin-l-yl, -(CH 2 ) 2 piperidin-l-yl, and the like, and derivatives thereof.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • match and mismatch refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • hydrocarbyl as used herein includes any radical containing carbon and hydrogen including saturated, unsaturated, aromatic, straight or branched chain or cyclic including polycyclic groups. Hydrocarbyl includes but is not limited to Ci-Cealkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 10 cycloalkyl, aryl such as phenyl and naphthyl, Ar (C 1 -C 8 )alkyl such as benzyl, any of which may be optionally substituted.
  • Reference herein to "immuno-interactive” includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
  • Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes, avians, reptiles).
  • livestock animals e.g., sheep, cows, horses, donkeys, pigs
  • laboratory test animals e.g., rabbits, mice, rats, guinea pigs, hamsters
  • companion animals e.g., cats, dogs
  • captive wild animals e.g., foxes, deer, dingoes, avians, reptiles.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • modulating is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule.
  • an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule.
  • indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide.
  • a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source of the host.
  • the extract can be obtained from a tissue or a biological fluid isolated directly from the host.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application.
  • PNAs peptide nucleic acids
  • phosphoramidates phosphoramidates
  • phosphorothioates phosphorothioates
  • methyl phosphonates 2-O-methyl ribonucleic acids
  • oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term “polynucleotide” or “nucleic acid” is typically used for large oligonucleotides.
  • operably linked is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.
  • pharmaceutically acceptable carrier or diluent is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to a mammal.
  • Phenylalkyl means alkyl as defined above which is substituted with phenyl, e.g., -CH 2 phenyl, (CH 2 ) 2 phenyl, -(CH 2 ) 3 phenyl, CH 3 CH(CH 3 )CH 2 phenyl, and the like and derivatives thereof. Phenylalkyl is a subset of the aralkyl group.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions as known in the art (see for example Sambrook et al. , Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989).
  • polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide.
  • polynucleotide variant and “variant” also include naturally-occurring allelic variants.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide variant refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter. These terms also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acids.
  • Probe refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly.
  • Pseudohalides are groups that behave substantially similar to halides. Such groups can be used in the same manner and treated in the same manner as halides (X, in which X is a halogen, such as Cl or Br). Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethyl and azide.
  • recombinant polynucleotide refers to a polynucleotide formed in vitro by the manipulation of a polynucleotide into a form not normally found in nature.
  • the recombinant polynucleotide can be in the form of an expression vector.
  • expression vectors include transcriptional and translational regulatory polynucleotide operably linked to the polynucleotide.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
  • reference or control level refers to any suitable reference or control level, including, but not limited to, a normal healthy individual, an individual without an aneurysm, a level measured in the same individual at a different time, and a level measured in a biological sample taken from a different tissue in the same individual or control.
  • reporter molecule as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of a complex comprising an antigen-binding molecule and its target antigen.
  • reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • stereoisomers As used herein, it will also be recognized that the compounds described herein may possess asymmetric centers and are therefore capable of existing in more than one stereoisomeric form.
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centers e.g., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
  • Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector is preferably a viral or viral-derived vector, which is operably functional in animal and preferably mammalian cells.
  • Such vector may be derived from a poxvirus, an adenovirus or yeast.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
  • resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
  • wild-type and "normal” are used interchangeably to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant.
  • underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • KLKl shall mean the KLKl (tissue kallikrein, human kallikrein 1, hKl) gene
  • KLKl shall indicate the protein product or products generated from transcription and translation and alternative splicing of the "KLKl” gene.
  • the present invention is predicated in part on the discovery that the modulation of levels of components in the KKS, can increase or decrease the size of an aneurysm and/or the number of aneurysms and/or the likelihood of aneurysm rupture. It is proposed, therefore, that KKS antagonists will be useful inter alia for the prevention or treatment of an aneurysm, including intact or ruptured aneurysms.
  • the aneurysm may be located in an artery, a vein or the heart. Suitable arterial aneurysms include, but are not limited to, arterial aneurysms that occur at the base of the brain (the circle of Willis) and aortic aneurysms.
  • Suitable aortic aneurysms include, but are not limited to, abdominal aortic aneurysms (AAAs), thoracoabdominal aortic aneurysms, aortic root aneurysms, and thoracic aortic aneurysms.
  • AAAs abdominal aortic aneurysms
  • thoracoabdominal aortic aneurysms thoracoabdominal aortic aneurysms
  • aortic root aneurysms aortic root aneurysms
  • thoracic aortic aneurysms thoracic aortic aneurysms.
  • the present invention provides methods for preventing or treating an aneurysm in an individual, the methods comprising administering to the individual an effective amount of a KKS antagonist.
  • Another aspect of the present invention contemplates the use of an effective amount of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • the present invention resides in the use of an effective amount of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • KKS antagonist refers to any agent which directly or indirectly agonises or antagonises a component in the KKS so as to inhibit or otherwise reduce the metabolic cascade in the KKS.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes a component of the KKS.
  • Representative genes encoding a component of the KKS include KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, and SERPINA4.
  • Representative expression products of genes encoding components of the KKS include kininogen (e.g. HMWK and LMWK), kallikrein precursors (e.g. prekallikrein and prokallikrein), kallikrein (e.g. plasma kallikrein and tissue kallikrein), kinin (e.g. bradykinin, kallidin, des-Arg9-bradykinin, and Lys-des-
  • kininogen e.g. HMWK and LMWK
  • kallikrein precursors e.g. prekallikrein and
  • Arg9-bradykinin kinin receptor (e.g. the kinin Bl receptor and the kinin B2 receptor), Cl-INH, ⁇ 2 -macroglobulin, antithrombin III and kallistatin.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression of a gene encoding a component of the KKS.
  • the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
  • the agent suitably modulates the expression of the gene directly, or modulates an upstream regulator of the expression of the gene, or directly or indirectly modulates the level or functional activity of an expression product of such genes.
  • the agent reduces the expression of a gene (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2) or the level or functional activity of an expression product of that gene (e.g. HMWK, LMWK 5 prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des- Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor).
  • the agent increases the expression of a gene (e.g.
  • the agent reduces or increases the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent.
  • Suitable agents for reducing or abrogating gene expression include, but are not restricted to, oligoribonucleotide sequences, including anti-sense RNA and
  • Anti- sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target sequences.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • the suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • RNA molecules and DNA molecules and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • RNA molecules that mediate RNA interference (RNAi) of a target gene or gene transcript can be used to reduce or abrogate gene expression.
  • RNAi refers to interference with or destruction of the product of a target gene by introducing a single stranded, and typically a double stranded RNA (dsRNA) that is homologous to the transcript of a target gene.
  • dsRNA double stranded RNA
  • dsRNA ⁇ er se and especially dsRNA-producing constructs corresponding to at least a portion of a target gene may be used to reduce or abrogate its expression.
  • RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a target gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts which then anneal to form a dsRNA having homology to a target gene.
  • Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk, R. H., et al, 2000). Therefore, depending on the length of the dsRNA, the RNAi- encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e. , with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene.
  • RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters are preferably at least about 100 nucleotides in length.
  • RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are preferably at least about 200 nucleotides in length.
  • the promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction. Alternatively, the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage.
  • the promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.
  • RNA molecules of about 21 to about 23 nucleotides which direct cleavage of specific mRNA to which they correspond, as for example described by Tuschl et al. in U.S. Patent Application No. 20020086356, can be utilized for mediating RNAi.
  • Such 21-23 nt RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double stranded (as two 21-23 nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').
  • Suitable agents for modulating the level or functional activity of an expression product of a gene include, but are not restricted to small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, as further described herein.
  • antigen-binding molecules can be used to modulate the level or functional activity of an expression product of a gene.
  • binding or activation of the kinin Bl receptor and/or the kinin B2 receptor is the subject of the targeting.
  • inhibition or abrogation of receptor signalling is achieved through reduction in receptor expression, receptor mutation (in particular, but not exclusively, of phosphorylation sites), prevention of receptor aggregation or through approaches that interfere with ligand-receptor interaction including those via blockade of the active binding sites or relevant associated motifs.
  • Such strategies include blocking antibodies to the receptors and small molecule inhibitors of binding. Pharmacological strategies to impair receptor phosphorylation can also be effective.
  • the kinin receptor antagonist may inhibit both the kinin Bl receptor and kinin B2 receptor.
  • the kinin receptor antagonist is a selective kinin Bl receptor antagonist.
  • the kinin receptor antagonist is a selective kinin B2 receptor antagonist.
  • the kinin receptor antagonist is a mutant kinin that binds to and recognises a kinin Bl receptor and/or a kinin B2 receptor but is unable to induce the pharmacological activities induced when a wild-type kinin binds to a kinin receptor.
  • the mutant kinin varies from the corresponding wild-type kinin by the deletion or modification of all or part of the nuclear translocation sequence, which renders it inoperative.
  • the present invention also contemplates the use of receptor antagonistic antigen-binding molecules with varying blocking capacities.
  • Exemplary receptor antagonists include those disclosed herein, including B9430 (Gera et al, 1996), and those disclosed in Blakeney et al 2007, see pages 2967-2971.
  • the subject of the targeting is a component of post-receptor signal transduction.
  • the present invention also contemplates the use in the above method of gene or expression product inhibitors identified according to methods described herein.
  • the present invention provides methods for preventing or treating an aneurysm in an individual, the methods comprising administering to the individual a KKS antagonist.
  • Another aspect of the present invention contemplates the use of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • the present invention resides in the use of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
  • KKS antagonists include small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, including those as further described herein.
  • Suitable small organic molecules include:
  • Z' is selected from O, S and NH;
  • Q is selected from the group consisting of -NR 4 R 5 , -OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl;
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 4 and R 5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic and where R 4 and R 5 , together with the nitrogen atom pendent thereto are joined to form a heterocyclic, a substituted heterocyclic, a heteroaryl or a substituted heteroaryl group, provide that when R 4 or R 5 is a substituted alkyl group this group is not ⁇ CHR a ⁇ C(O)-NR
  • X is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, nitro, cyano, hydroxyl, alkoxy, substituted alkoxy, carboxy, carboxyl esters, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, acylamino, aminoacyl, and -C(O)NR 7 R 8 wherein R 7 and R 8 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, or R and R together with the nitrogen atom to which they are joined form a heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
  • provisos apply: [0146] A) when Z' is O; R 2 is H; R 3 is 5-(2, 4-dichlorophenyl)-imidazol-4- ylidene-[2-methyl-4-(N-(l-methylsufonyleth-2- -yl)-N-ethylamino)phenyl]-amine; X is H; and R 1 is l-(3-t-butyl-4-hydroxyphenyloxy)tridec-l-yl; then Q is not ethoxy;
  • R 1 is substituted aryl or substituted heteroaryl, the aryl or heteroaryl group is not substituted with -C(0)NH ⁇ W'-C(0)0R b or -C(O)NH ⁇ W' ⁇ C(O)NR b R c , where W is aryl, substituted aryl, heteroaryl or substituted heteroaryl;
  • Exemplary compounds include:
  • A is an acylating group, and preferably a hydrophobic acylating group, or an anti-inflammatory substituent selected from the group consisting of: Aaa,
  • B is an amino acid or substituted amino acid selected from the group consisting of: Bip, Ddip, F5F, F3MF, hPhe, MC2Y, NaI, NMF, OBPY 5 OBrZY, OC2Y,
  • R is a substituted amide, preferably having additional polar character, selected from the group consisting of: Abzp, Aem, Alp, Ambi, Apia, Apyr, AquR, Atmp,
  • Aic 2-Aminoindane-2-carboxylic acid
  • Aq2c Anthraquinone-2-carboxyl
  • AquR (R)-(+)-3-Aminoquinuclidine
  • Atfb 3-Amino-2,5,6-trifluorobenzoyl
  • 4Atfb 4-Aniino-2,3,5,6-tetrafluorobenzoyl
  • Atmp 4-Amino-2,2,6,6-tetramethylpiperidine
  • AtmpO 4-Amino-2,2,6,6-tetramethylpiperidinyloxy
  • B6 3-Hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridylmethyl (Vitamin B6, Pyridoxamine)
  • B6P 3-Hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridylmethyI- 5- phosphate
  • BapR (R)-(-)-l-Benzyl-3-aminopyrrolidine
  • BapS (S)-(+)-l-Benzyl-3-aminopyrrolidine
  • Bcpa bis(4-Chlorophenyl)acetyl
  • Bcpoa bis(4-Chlorophenoxy)acetyl
  • Bdbh (1 S,4S)-(+)-2-Benzyl-2,5-diazabicyclo[2.2. l]heptane
  • Bhp 1-Benzylhomopiperazine
  • Bipa 4-Biphenylacetyl
  • 4Bpc 4-Biphenylcarboxyl
  • Bzac 3-BenzoylacryIoyl
  • Cbp l-(4-Chlorobenzhydrylpiperazine)
  • Chbu 2-Cyclohexylbutyryl
  • Che ⁇ -Cyano-4-hydroxycinnamoyl
  • CHFB 4-Carboxy-hexafluorobutyryl
  • ChI Chlorambucil: 4-[p-(bis[2-Chloroethyl]amino)- phenyljbutyryl
  • Cmioc 3-(2-Chlorophenyl)-5-methylisoxazole-4-carbonyl
  • Daep l-(2-(Diallylamino)ethyl)piperazme
  • tDecl trans-4-(Diethylamino)cinnamyl
  • Dfc Diclofenac: 2-[(2,6-Dichlorophenyl)amino]phenylacetyl
  • cDmap cis-2,6-Dimethyl- 1 -allyl-piperazine
  • CDmm cis-2,6-Dimethylmorpholine
  • Dmmp cis-2,6-Dimethyl- 1 -(methoxycarbonylmethyl)piperazine
  • Esul Exisulindacyl: (Z)-5-Fluoro-2-methyl-[[4- (methylsulfonyl)phenyl]methylene]-lH-mdene-3-acetyl; (cis)
  • Fbhp l-(4-Fluorobenzyl)homopiperazine
  • F5bs Pentafluorobenzenesulfonyl
  • F5bz Pentafluorobenzoyl
  • F3c 2,3,5-Trifluorocinnamoyl
  • ⁇ Fcn ⁇ Fluorocinnamoyl
  • F5F Pentafluorophenylalanine
  • F3MF 4-Trifluoromethylphenylalanine
  • F5pa 2,3,4,5,6-Pentafluorophenylacetyl
  • Fpmp l-bis(4-Fluorophenyl)methylpiperazine
  • Fmpi (Z)-5-Fluoro-2-methyl-(4- ⁇ yridylidene)-3-indenylacetyl
  • F5po 2,3,4,5,6-Pentafluorophenoxyacetyl
  • F5Pt Pentafluorophenylthiocarbamyl
  • Fpdh (1 S,4S)-(-)-2-(4-Fluorophenyl)-2,5- diazabicyclo[2.2. ljheptane
  • HPhe Homo-phenylalanine
  • Indo Indomethacin: l-[p-Chlorobenzoyl]-5-methoxy-2- methylindole-3 acetyl
  • Inp Isonipecotic acid: hexahydroisonicotinic Isoquinolineacetyl
  • KtIc Ketorolac: (.+-.)5-benzoyl-2,3dihydro-lH- ⁇ yrrolizine-l- carboxyl: Toradol
  • Ktpf Ketoprofen: 2-(3-benzoylphenyl)propionyl
  • Matp 4-(Methylamino)-2,2,6,6-tetramethyl ⁇ iperidine
  • Mca 2-Methylcinnamoyl
  • ⁇ Mcn ⁇ -Methylcinnamoyl
  • Mcoa 7-Methoxycoumarin-4-acetyl
  • MC2Y N-Methyl-O-2,6-dichlorobenzyl-tyrosine
  • Mpz 1 -Methylpiperazine
  • MTPA ⁇ -Methoxy- ⁇ -trifluoromethylphenylacetyl
  • Napr Naproxen: ⁇ -Methoxy- ⁇ -methyl ⁇ -Naphthaleneacetyl
  • Nba Norbornane-2-acetyl
  • Nif Niflumic acid, 2-(3-[Trifluoromethyl]aniline)nicotinic acid
  • NMF N-Methylphenylalanine
  • OBPY O-Benzyl-phosphotyrosine
  • OCIY O-2,6-Dichlorobenzyl-3 5 5-diiodo-tyrosme
  • OC2Y 0-2,6-dichlorobenzyl tyrosine
  • Ocp 1-Octylpiperazine
  • Octe 2-Octenoyl
  • Pas p-Aminosalicyloyl
  • PBF p-Bromophenylalanine
  • PCNF p-Cyano-L-phenylalanine
  • Pep l-(2-Phenylethyl)piperazine
  • PFF p-Fluorophenylalanine
  • ⁇ Phc ⁇ -Phenylcinnamoyl
  • ⁇ Ptpa ⁇ -(Phenylthio)phenylacetyl
  • Pxa Pyridoxamine [4-(ammomethyl)-5-hydroxy-6-methyl- 3- pyridinemethanol]
  • CSsa cis-Styrenesulphonylacetyl
  • t ⁇ Sts trans- ⁇ -Styrenesulfonyl
  • Taa l,2,4-Triazole-acetyl
  • Tchc (1R,3R,4S,5R)-1 ,3,4,5-Tetrahydrocyclohexane-l -carboxyl
  • Tf2c trans-3,5-bis(Trifluoromethyl)cinnamoyl
  • 4Tfmb 4-(Trifluoromethoxy)benzoyl
  • Th2n 1 ,2,3 ,4-Tetrahydro-2-naphthoyl
  • Tic Tetrahydroisoquinoline-3-carboxylic acid
  • Tmb Trimethoxybenzoyl
  • Tmbp l-(2,4,6-Trimethylbenzyl)piperazine
  • Tmcc 2,2,3,3-Tetramethylcyclopropanecarboxyl
  • TmpC Carboxy-TEMPO: 4-carboxy-2,2,6,6- tetramethylpiperidinyloxy
  • Representative compounds include:
  • n is an integer from 0 to 4; [0399] p is zero or one; [0400] q is zero or one;
  • W is selected from the group consisting of O, S, and N, wherein:
  • R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
  • each R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted
  • R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0412] or R 7 together with at least one of R 3 and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
  • R 8 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy;
  • alkylene-X a wherein alkylene is optionally substituted and X a is selected from the group consisting of -OH, cyano, and -NR b R b wherein each R b is independently as defined above;
  • Representative compounds include:
  • R 1 is phenyl, 1,2-dichlorophenyl, 3-methylphenyl, 3-bromophenyl, naphth-2-yl, indan-5-yl benzo[l,3]dioxol-5-yl or l,2,3,4-tetrahydronaphth-6-yl;
  • R 2 is hydrogen, halogen, unsubstituted Q-Qalkyl or substituted C 1 -
  • R 3 is hydrogen, halogen or d-C 4 alkyl
  • R 4 is hydrogen or C 1 -C 4 alkyl
  • R 5 is hydrogen or Cj-Qalkyl
  • R 6 is CH 2 OH; tetrazol-5-yl; 1 5 2,4-triazol-5-yl; 1 ,2,3-triazol-5-yl; C(O)OH, C(O)NH 2 ; or ZNH(CH 2 ) n CHR 7 R 8 , wherein Z is -C(O)- or -CH 2 -, n is zero, 1, 2,
  • R 7 is unsubstiruted or substituted Ci-C 4 alkyl, C(O)OH, C(O)OC r
  • R 8 is hydrogen, unsubstituted or substituted C]-C 4 alkyl, unsubstituted or substituted Cs-C 10 aryl or heteroC 5 -C 10 aryl or C 1 -C 4 alkylC 5 -C 10 aryl or Ci-C 4 alkyl- heteroCs- Cioaryl, heteroCs-CiQaryl comprising one or more heteroatoms selected from N, O, and S; and m is 2, 3 or 4,
  • Ci-C 4 alkyl, C 5 -C 10 aryl or heteroCs-Cioaryl, when substituted may be one or more substituents selected from OH, C(O)OH, halogen, Ci-C 4 alkyl, Ci-
  • FRl 73657 involves crystallographic polymorphism, of which crystal hydrates having high purity and good solid stability and easy to handle for formulation into medicines. Three types of crystals referred to as crystal type A, crystal type B and crystal type C, have been found (Japanese Patent Laid-Open No. 316677/1998). However, though having good solid stability and releasability, crystal type A is problematic in that it is often contaminated with crystal type C, making impossible production with stable quality.
  • Crystal type B is the most stable and there is no problem in producing it, but is problematic in that its releasability is much inferior to that of crystal type A.
  • Crystal type C is also problematic in that its solid stability is inferior to that of crystal type B and its releasability is inferior to that of crystal type A.
  • US 7,094,899 describes a vitreous form of FRl 73657 that overcomes these problems. The vitreous form has high purity, good solid stability, good solubility and good releasability and capable of production with stable quality.
  • FR167344 N-[N-[3-[3-bromo-2-methylimdazo[l,2-a]pyridin-8- yl)oxymethyl]2,4-dichloro phenyl]N-methylaminocarbonylmethyl]-4- (dimethylaminocarbonyl)cinnarnylamide hydrochlroide) (also discussed below);
  • WIN-64338 [4- ⁇ (2[ ⁇ bis(cyclohexylamino)methylene ⁇ amino]-3-[2- naphthyl]-l-oxopropyl)amino ⁇ phenyl]methyl)tributylphosphonium chloride monohydrochloride) (also discussed below);
  • Q is selected from the group consisting of -SO 2 R and -CH 2 C(O)R;
  • W is selected from the group consisting of O, S, and N, wherein when W is O or S, then q is zero and when W is N, then q is one;
  • R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
  • each R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, acylamino, aminoacyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, acyl, acyloxy, halogen, nitro, cyano, hydroxy, carboxy, and carboxyl esters;
  • R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy;
  • R together with at least one of R and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
  • p is an integer of from 0 to 3;
  • Exemplary compounds include the following compounds and pharmaceutically acceptable salts thereof:
  • one of bonds characterized by is a double bond and the other two are single bonds;
  • n is an integer from 0 to 4.
  • W is selected from the group consisting of O, S, and N, wherein: [0510] when W is O or S, then q is zero; and when W is N, then q is one;
  • R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
  • each R 3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted
  • R 3 or two or more of R 3 together with the carbon atoms to which they are joined form a fused ring cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
  • R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0516] or R 7 together with at least one of R 3 and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic; [0517] R 8 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0518] and pharmaceutically acceptable salts
  • R 1 nor R 2 is selected from the following:
  • alkylene-X a wherein alkylene is optionally substituted and X a is selected from the group consisting of -OH, cyano, and -NR b R b wherein each R b is independently as defined above;
  • Representative compounds include: [0547] 2-[2-(R,S)-l-(2,4,6-trimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydro-6,-7-dichloroquinoxalin-2-yl]-N-benzylacetamide;
  • R 5A is -X A -R 6A or -N(R 7A )R 8A , wherein X A is piperidinylere or piperazinylene, R 6A is H, C 3 -C 4 alkenyl, C 3 -C 4 alkinyl, Ci-C 4 (alkoxyalkyl), C 1 -C 4 (carboxyalkyl), a C 5 -C 7 heterocyclic group or phenylQ-Qalkyl; [0558] R 7A is amino-C 2 -C 4 alkyl or mono- or di-(Ci-C 5 alkyl)amino-C 2 -
  • R 8A is H, C 1 -C 4 alkyl or has the meanings as given for R 7A ;
  • X 1 is a divalent group of formula IA':
  • n is zero or 1
  • X 3 is CH or N
  • X 4 is a direct bond
  • R 3A is H, C r C 4 alkyl, C 3 -C 6 cycloalkyl, C 3 - C 6 alkenyl, C 3 -C 6 alkinyl, C 7 -C 10 aralkyl or C 6 -C 9 heteroaralkyl
  • R 4A is H and m is 1 or 2 or
  • X 4 is -CH(R 12 )-, R 3A is H and R 4A and R 12 together are propylene and m is 1 , or ethylene and m is 2;
  • X 2 is a divalent group of formula IA" :
  • R 1 ' is d-C 4 alkyl, C 3 -C 6 cloalkyl or -NR 1A R 2A , wherein R 1A and R 2A independently are Q-Qalkyl or, together with the N-atom to which they are attached, represent a 5 to 7 membered heterocyclic ring; and
  • R 9 and R 10 independently are a phenyl or pyddine ring; and salts thereof.
  • Representative compounds include:
  • X 1 and X 2 are independently halo or C ⁇ alkyl
  • R 1 and R 2 are independently hydrogen or C 1-4 alkyl
  • R 3 and R 4 are independently hydrogen or halo; and [0580] R 5 is
  • A is independently halo
  • Y is -(CH 2 )" 1 -, -C(O)- or -S(O)-;
  • R and R are independently C 1-4 alkyl
  • R 3 is selected from
  • Ci -6 alkyl-C 3-7 cycloalkyl the cycloalkyl moiety being optionally substituted with one, two or three substituents independently selected from cyano, amino-
  • R 4 is phenyl substituted at the 2-position with substituent selected from
  • Ci-4alkoxy or C 1-4 alkylthio the Ci ⁇ alkoxy or Ci -4 alkylthio being substituted with one, two or three substituents independently selected from amino, amino-
  • n 0, 1, 2, 3, 4 or 5;
  • Representative compounds include:
  • R 1 is a hydrogen atom or a C 1 -C 4 alkyl group
  • R 2 and R 3 which can be the same or different, are a C 1 -C 4 alkyl group, or R 2 and R 3 , together with the carbon atom which they are linked to, form a cyclic aliphatic group having 3 to 7 carbon atoms or a heterocyclic aliphatic group having 3 to 7 atoms, one or two of which are selected from the group N, O, S and the others being C atoms;
  • R 4 and R 5 which can be the same or different, are a hydrogen atom or a C 1 -C 4 alkyl group
  • X is selected from the group consisting of halogen, OR 1 , SRi, CN, Ci- C 4 alkyl;
  • B has at least one amino group with basic characteristics or a tetraalkylammonium group and can be selected from the group consisting of:
  • T is selected from the group of -NR 7 R 8 , -NR 14 Ri s Ri 9 , -OR 6 ;
  • R 7 and Rg which can be the same or different, are a hydrogen atom, a C 1 -C 4 alkyl group, a cyclohexyl group, or NR 7 R 8 together are a group selected from: i) guanidine optionally substituted with 1 or 2 C 1 -C 4 alkyl or cyclohexyl groups, ii) a 5-7 membered nitrogen heterocycle optionally containing another heteroatom selected from O 3 N 5 S;
  • Y 1 is selected from the group consisting OfNR 7 R 8 , NR 14 R 18 R 19 or from the following residues:
  • Z is selected from the group consisting of H, C 1 -C 6 alkyl, OR 6 , SR 6 , CF 3 , OCOR 65 COR 10 , NHCOR 6 , SO 2 R 6 , SOR 6 , CO 2 R 6 , N(Re) 2 , CI, Br, NO 2 , NH 2 , CN, F, imidazole, phenyl, amidine, guanidine, guanidyl-methyl; [0637] R 9 is selected from the group consisting of hydrogen, ⁇ (CH 2 ) q -L, wherein L is selected from the group of —OH, -NR 5 R 6 , -NR 14 R 1S Ri 9 , amidine optionally substituted with 1 or 2 C 1 -C 4 alkyl groups, guanidine optionally substituted with 1 or 2 Cj-C 4 alkyl groups;
  • R 10 is selected from the group consisting of OR 6 , NR 6 Ri 2 ;
  • Rn is selected from the group consisting of hydrogen, ⁇ (CH 2 ) q -L, — (CH 2 ) p ⁇ NR 4 ⁇ (CH 2 ) q -L;
  • R 12 is a hydrogen atom, Ci-C 6 alkyl, COR 6 ,
  • R 13 is selected from the group consisting of H, C 1 -C 6 alkyl, ⁇ (CH 2 ) p W(CH 2 ) q Y,, Y, -COY, -CH 2 -Y;
  • Rj 5 is selected from the group consisting of hydrogen or straight or branched C ] -C 4 alkyl groups
  • the -NR 16 Ri ? group is a 5-7 membered nitrogen aliphatic heterocycle optionally containing another heteroatom selected from O, S, N;
  • Representative compounds include:
  • fragment L represents a hydrophobic group such as a 5-membered unsaturated ring, 6-membered unsaturated ring, alkene, saturated 3-7 membered ring;
  • fragment E represents carbonyl derivative that in the presence of L, R and Q becomes an activated serine trap
  • fragment R represents a hydrophobic, aromatic ring system such as a 6-membered ring, fused 5-6 membered ring, or fused 6—6 membered ring;
  • fragment Q represents a basic group.
  • Suitable peptides, polypeptides, proteins, and peptidomimetics include: [0665] (A) Peptides having the following sequence:
  • r is any amino acid, preferably Arg;
  • p is any amino acid, preferably Pro;
  • g is amino acid, preferably GIy;
  • X is any amino acid
  • f is any amino acid, preferably Phe;
  • N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in
  • X 1 is a modified amino acid, preferably modified proline or modified serine, more preferably proline, and may be N-terminal;
  • X 2 is any amino acid, preferably proline or phenylalanine
  • X 3 is isoleucine, leucine, or valine, preferably isoleucine or valine;
  • X 4 is any amino acid, preferably valine, isoleucine, or leucine;
  • X 5 is any amino acid, preferably serine, threonine, or proline; and [0681] X 6 is any amino acid, preferably phenylalanine, leucine or arginine;
  • N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613;
  • X 1 is N-terminal and is a modified amino acid, preferably modified proline or modified serine, more preferably modified proline;
  • n is a contiguous sequence of any amino acids of length n, where n can be up to about 50, up to about 30, up to about 10 amino acids or 0 amino acids;
  • X 3 is any amino acid, preferably proline or phenylalanine;
  • X 4 is isoleucine, leucine, or valine, preferably isoleucine or valine;
  • X 5 is any amino acid, preferably valine, isoleucine, or leucine;
  • X 6 is any amino acid, preferably serine, threonine, or proline;
  • X 7 is any amino acid, preferably phenylalanine, leucine, or arginine;
  • N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613;
  • X 2 is a hydrophobic aliphatic residue, preferably alanine, valine, isoleucine, or leucine, but not glycine;
  • X 3 is a hydrophobic aliphatic residue
  • Representative sequences include:
  • modified means any modification at the N-terminal amino acid residue that blocks sequencing by the standard Edman degradation method. Such modifications include, but are not limited to acetylation, amidation, ⁇ -methylthiolation, biotin, carbamylation, citrullination, c-mannosylation, deamidation, n-acyl diglyceride cysteine (tripalmitate), dimethylation, fad, farnesylation, formylation, geranyl-geranyl, gamma-carboxyglutamic acid, o-glcnac, glucosylation (glycation), hydroxylation lipoyl, methylation, myristoylation, palmitoylation, phosphorylation, pyridoxal phosphate, phosphopantetheine, pyrrolidone carboxylic acid, sulfation and trimethylation. Modifications may be on the following modifications, amidation, ⁇ -methylthiolation, biotin, carbamylation, citrullination
  • Xaai is selected from D-arginyl, acetyl lysyl, D-lysyl, sarcosyl, acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon-aminohexanoyl lysyl;
  • X a a2 is selected from prolyl and hydroxyprolyl; ⁇
  • X a a3 is selected from D-l,2,3,4,-tetrahydroisoquinoline-3-carboxyl and "D- ⁇ -(2-naphthyl) alanyl;
  • X a a4 is selected from leucine and isoleucine
  • X aa5 is selected from acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon- aminohexanoyl lysyl;
  • X aa6 is selected from prolyl and hydroxyprolyl; [0717] X aa7 is propyl;
  • X aa g is selected from leucine and isoleucine
  • HOE 140 (or Icatibant) having the following sequence:
  • This peptide, methods for its preparation and its biological activity are discussed in Boa et al. 1991 and many other publications.
  • the peptide is described as being a highly potent, specific and long-acting bradykinin B2-receptor antagonist.
  • the peptide is described as a bradykinin antagonist.
  • NPC 17761 having the following sequence:
  • the peptide is described as being a potent bradykinin antagonist.
  • M is an amide bond
  • x and z are each independently an integer of 1 to 10
  • K is H or an acyl group.
  • Representative peptide analogs include: [0733] Ada-(D)Arg-Arg-cyclo(N ⁇ (l-(6-aminohexylene)Gly-Hyp-Phe-D-
  • Ada is the abbreviated designation for the protecting groups adamantane acetyl.
  • a 0 , B 1 , C 2 , D 3 , and E 4 are basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acids or A 0 is absent;
  • G 6 is an aromatic, aliphatic, heterocyclic, or alicyclic amino acid
  • F 5 , H 7 and J 8 are aromatic, aliphatic, aliphatic heterocyclic, or alicyclic amino acids, provided that at least one of F 5 , H 7 and J 8 is selected from cyclopentane-, cyclohexane- or indane-substituted glycine; and
  • Z is COOH.
  • Representative analogues include: [0747] DArg-Cys-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg;
  • R is selected from the group consisting of Ci-C 6 alkyl, substituted C 1 - C 6 alkyl, C 2 -Cgalkenyl, Ca-Cscycloalkyl, C 3 -Cscycloalkyl substituted Ci-C 6 alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula
  • Xj-Arg-Pro-Xa-Gly-Phe-Ser-Xa- ⁇ [0775] wherein [0776] X 1 is selected from D-arginyl, acetyl lysyl, D-lysyl, sarcosyl, acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon-aminohexanoyl lysyl;
  • X 2 is selected from prolyl and hydroxyprolyl
  • X 3 is selected from D-l,2,3,4-tetrahydroisoquinolme-3-carboxyl and "D-beta-(2-naphthyl) alanyl;
  • X 4 is selected from leucine and isoleucine
  • X 1 is selected from acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcodyl tyrosyl epsilon-aminohexanoyl lysyl and sarcosyl tyrosyl (3,5-iodine) epsilon- aminohexanoyl lysyl;
  • X 2 is selected from prolyl and hydroxyprolyl;
  • X 3 is prolyl;
  • X 4 is selected from leucine and isoleucine.
  • NPC 17751 (described in Mak et al. 1991);
  • NPC 349 (described in Wirth et al. 1995 and Mak et al. 1991);
  • NPC- 1776 (described in Cheronis et al. 1993); [0792] WIN 64338 (described in Sawutz et al. 1995 and elsewhere herein);
  • NPC 18565 D-Arg(Hyp 3 ,D-Hyp 7 (transpropyl),Oic 8 )desArg 9 BK developed by Scios Inc;
  • NPC 18828 D-Arg(Hyp 3 ,D-Hyp 7 (trans thiophenyl),Oic 8 )desArg 9 BK also developed by Scios Inc;
  • KKS antagonists are also disclosed in these references.
  • Phe(4NH 2 ) 4-amino phenylalanine
  • the sequence comprises naturally occurring, non-naturally occurring, or chemically synthesized amino acids, amino acid derivatives, modified amino acids, or combinations thereof.
  • Extracts from at least one plant of the Rosaceae family as disclosed in US 6,586,020 may be prepared from any plant material (including whole plant, or from a plant portion such as the leaves, stems, flowers, petals, roots or from undifferentiated cells) derived from at least one plant of the Rosaceae family using the extraction techniques described in US 6,586,020, including aqueous or alcoholic extracts, or extracts obtained from an organic solvent; and
  • KKS antagonists are known in the art in addition to those specifically described above, including those described in Hirayama, Y. et al. (2002); Hall, J. M. (1997); Campbell et al. (2001); Regoli, et al. (1998); Altamura et al. (1999); Moreau et al. (2005); and Blakeney et al. (2007).
  • B 9430 D-Arg-[Hyp 3 ,Igl 5 ,D-Igl 7 ,Oic 8 ]-bradykinin (discussed herein);
  • HOE 140 (also discussed above) and desArg 9 derivatives of HOE 140;
  • R 493 D-Arg-[Hyp 3 , D-Phe 7 , Leu 8 ]bradykinin
  • NPC 567 D-Arg-[Hyp 3 ,D-Phe 7 ]-bradykinin
  • Martinelline a pyrroloquinoline alkaloid isolated from the roots of the tropical plant Martinella iquitosensis.
  • kinin B 1 -receptor ligands including many described in Regoli et al. (1998) and also including:
  • R-954 (Ac-Orn-[Oic 2 , ⁇ -MePhe 5 , D- ⁇ Nal 7 , Ile 8 ]des-Arg 9 bradykinin);
  • compound 12 (benzodiazepine-based structure); [0845] benzo-sulfonyalmide compounds including compound 12, compound l l and SSR240612;
  • kinin B 2 -receptor ligands including many described in Regoli et al. (1998) and Altamura et al. (1999) and also including: [0847] [Thi 5 ' 8 , D-Phe 7 ]-bradykinin;
  • KKS antagonists including aprotinin and aprotinin-like drugs including ulinastatina nd nafamostat;
  • agents that comprise KKS antagonists are useful for the prevention or treatment of aneurysms.
  • agents can be administered to an individual either by themselves, or in pharmaceutical compositions where they are mixed with a suitable pharmaceutically acceptable carrier or diluent.
  • the agents of the present invention may be conjugated with biological targeting agents which enable their activity to be restricted to particular cell types.
  • biological-targeting agents include substances which are immuno-interactive with cell- specific surface antigens.
  • the agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.
  • Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the drugs of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.
  • the agents can be formulated readily using pharmaceutically acceptable carriers or diluents well known in the art into dosages suitable for oral administration.
  • Such carriers or diluents enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • These carriers or diluents may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.
  • the dose of agent administered to an individual should be sufficient to effect a beneficial response in the individual over time such as reducing the size of an aneurysm, preventing an increase in the size of an aneurysm, reducing the number of small aneurysms, and/or preventing or inhibiting the occurrence of aneurysms.
  • the quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgement of the practitioner.
  • the physician may evaluate the characteristics of the patient, their response to the drug and the safety profile of the drug. In any event, those of skill in the art may readily determine suitable dosages of the agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or other components which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier or diluent which constitutes one or more necessary ingredients.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses.
  • Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Dosage forms of the drugs of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion.
  • Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.
  • controlled release may be effected by using ⁇ other polymer matrices, liposomes or microspheres.
  • the drugs of the invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a test agent, which achieves a half-maximal inhibition or enhancement in activity of a component in the KKS). Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of such drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
  • LD50/ED50 Compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilised.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain KKS antagonistic effects.
  • Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02- 3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5-1200 mg/m2/day, commonly from 0.5-150 mg/m2/day, typically from 5-100 mg/m2/day.
  • the liposomes will be targeted to and taken up selectively by the tissue.
  • the effective local concentration of the agent may not be related to plasma concentration.
  • the present invention also contemplates a method of gene therapy of a mammal.
  • a method of gene therapy of a mammal utilises a gene therapy construct which includes an isolated polynucleotide comprising a nucleotide sequence encoding a component of the kallikrein- kinin system, or a biologically active fragment thereof, wherein the polynucleotide is ligated into a gene therapy vector which provides one or more regulatory sequences that direct expression of the polynucleotide in the mammal.
  • gene therapy vectors are derived from viral DNA sequences such as adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses. Suitable gene therapy vectors currently available to the skilled person may be found, for example, in Robbins et al, 1998.
  • Administration of the gene therapy construct to the mammal may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor.
  • An example of the latter approach would be stem-cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the gene therapy vector comprising a component of the KKS. The stem-cells are cultured for a period and then transferred to the mammal being treated.
  • a polynucleotide encoding a KKS antagonist may be used as a therapeutic or prophylactic composition in the form of a "naked DNA" composition as is known in the art.
  • an expression vector comprising the polynucleotide operably linked to a regulatory polynucleotide (e.g. a promoter, transcriptional terminator, enhancer etc) may be introduced into an animal, preferably a mammal, where it causes production of a KKS antagonist in vivo.
  • a regulatory polynucleotide e.g. a promoter, transcriptional terminator, enhancer etc
  • the step of introducing the expression vector into a target cell or tissue will differ depending on the intended use and species, and can involve one or more of non- viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R.C., (1993).
  • the present invention also provides methods for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual.
  • One suitable method comprises detecting in the individual the presence of an aberrant gene encoding a component of the KKS or detecting in the individual aberrant expression of a gene encoding a component of the KKS.
  • the aberrant gene is selected from an aberrant KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, or SERPINA4 gene.
  • Another suitable method comprises determining in the individual a level or functional activity of a component in the KKS, which is different than a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component.
  • a reference or control level e.g. no aneurysm, level measured in the same individual at an earlier time
  • the level or functional activity of a component in the KKS, which is different than a reference or control level indicates the presence or risk of development of an aneurysm or a negative prognosis.
  • the method is performed in vivo. In other embodiments, the method is performed ex vivo, including in a biological sample obtained from the individual.
  • the method comprises determining an increase or elevation in the level of functional activity of the component (e.g. HMWK, LMWK, prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des- Arg9-bradykinin, Lys-des-Arg9-bradykinin, the kinin B 1 receptor, the kinin B2 receptor) relative to a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component, .
  • the method comprises determining a decrease in the level or functional activity of the component (e.g.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • individual is one that is suspected of having an aneurysm or being at risk of having an aneurysm.
  • determining the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual is achieved by using the methods described above in combination with medical imaging, including ultrasound.
  • the invention also features methods of screening for an agent that antagonises the KKS, including agents that modulate the expression of a gene or the level and/or functional activity of an expression product of that gene, wherein the gene is selected from a gene encoding a component of the KKS, a gene whose expression product modulates directly or indirectly the expression of a gene encoding a component of the KKS, and a gene whose expression product modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
  • the methods comprise: (1) contacting a preparation with a test agent, wherein the preparation contains (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the KKS, or a variant or derivative thereof, which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i) and (2) detecting a change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference or control level and/or functional activity in the absence of the test agent, which indicates that the agent modulates the KKS
  • the methods comprise contacting a sample of cells expressing a kinin receptor with a kinin and a test agent.
  • Candidate test agents encompass numerous chemical classes.
  • Candidate organic molecules comprise functional groups necessary for structural interaction with KKS components including proteins, particularly by way of hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate test agent often comprises cyclical carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate test agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrrolidines, derivatives, structural analogues or combinations thereof.
  • small molecules are particularly preferred as candidate test agents because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc to produce structural analogues.
  • Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.
  • Screening candidate test agents according to the invention can be achieved by any suitable method.
  • the method may include contacting a cell expressing a polynucleotide corresponding to gene encoding a component of the KKS, with an agent suspected of having the modulatory activity and screening for the modulation of the level or functional activity of a protein encoded by the polynucleotide, or the modulation of the level of a transcript encoded by the polynucleotide, or the modulation of the activity or expression of a downstream cellular target of the protein or of the transcript (hereafter referred to as target molecules).
  • target molecules a downstream cellular target of the protein or of the transcript
  • Detecting such modulation can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a streptavidin-biotin detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR).
  • a polynucleotide from which a target molecule of interest is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing. Further, the naturally-occurring or introduced polynucleotide may be constitutively expressed — thereby providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level - or may require activation - thereby providing a model useful in screening for agents that up-regulate expression of an encoded product of the sequence.
  • a polynucleotide may comprise the entire coding sequence which codes for a target protein or it may comprise a portion of that coding sequence (e.g., a binding domain) or a portion that regulates expression of a product encoded by the polynucleotide (e.g., a promoter).
  • a promoter e.g., the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing.
  • detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), luciferase, ⁇ -galactosidase and catecholamine acetyl transferase (CAT). Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.
  • GFP green fluorescent protein
  • CAT catecholamine acetyl transferase
  • the subject of detection could be a downstream regulatory target of the target molecule, rather than the target molecule itself or the reporter molecule operably linked to a promoter of a gene encoding a product the expression of which is regulated by the target protein.
  • These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the polynucleotide encoding the target molecule.
  • the present invention provides assays for identifying small molecules or other compounds (i.e. modulatory agents) which are capable of inducing or inhibiting the level and/or functional activity of target molecules according to the invention.
  • the assays may be performed in vitro using non-transformed cells, immortalised cell lines, or recombinant cell lines.
  • the assays may detect the presence of increased or decreased expression of genes or production of proteins on the basis of increased or decreased mRNA expression (using, for example, the nucleic acid probes disclosed herein), increased or decreased levels of protein products (using, for example, the antigen binding molecules disclosed herein), or increased or decreased levels of expression of a reporter gene (e.g., GFP 5 ⁇ -galactosidase or luciferase) operably linked to a target molecule-related gene regulatory region in a recombinant construct.
  • a reporter gene e.g., GFP 5 ⁇ -galactosidase or luciferase
  • recombinant assays are employed in which a reporter gene encoding, for example, GFP, ⁇ -galactosidase or luciferase is operably linked to the 5' regulatory regions of a target molecule related gene.
  • a reporter gene encoding, for example, GFP, ⁇ -galactosidase or luciferase
  • Such regulatory regions may be easily isolated and cloned by one of ordinary skill in the art.
  • the reporter gene and regulatory regions are joined in-frame (or in each of the three possible reading frames) so that transcription and translation of the reporter gene may proceed under the control of the regulatory elements of the target molecule related gene.
  • the recombinant construct may then be introduced into any appropriate cell type although mammalian cells are preferred, and human cells are most preferred.
  • the transformed cells may be grown in culture and, after establishing the baseline level of expression of the reporter gene, test compounds may be added to the medium.
  • the ease of detection of the expression of the reporter gene provides for a rapid, high throughput assay for the identification of agonists or antagonists of the target molecules of the invention.
  • Compounds identified by this method will have potential utility in modifying the expression of target molecule related genes in vivo. These compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects.
  • these molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modelling, and other routine procedures employed in rational drug design.
  • methods of identifying agents that antagonise the KKS are provided in which a purified preparation of a component of the KKS is incubated in the presence and absence of a candidate agent under conditions in which the component is active, and the level of activity is measured by a suitable assay.
  • a KKS antagonist can be identified by measuring the ability of a candidate agent to decrease kinin - kinin receptor binding. An agent tests positive if it inhibits this activity.
  • methods of identifying agents that antagonise the KKS are provided in which a purified preparation of a component of the KKS is incubated in the presence and absence of a candidate agent under conditions in which kinin is able to bind a kinin receptor, and the level of binding is measured by a suitable assay. An agent tests positive if it inhibits this activity.
  • random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to a target molecule or to a functional domain thereof. Identification of molecules that are able to bind to a target molecule may be accomplished by screening a peptide library with a recombinant soluble target molecule.
  • the target molecule may be purified, recombinantly expressed or synthesised by any suitable technique.
  • Such molecules may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et ah, (1989, supra) in particular Sections 16 and 17; Ausubel et ah, ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998), in particular Chapters 10 and 16; and
  • a target polypeptide according to the invention may be synthesized using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard ⁇ supra) and in Roberge et al. (1995).
  • target polypeptide may be conjugated to any suitable reporter molecule, including enzymes such as alkaline phosphatase and horseradish peroxidase and fluorescent reporter molecules such as fluorescein isothiocynate (FITC), phycoerythrin (PE) and rhodamine. Conjugation of any given reporter molecule, with target polypeptide, may be performed using techniques that are routine in the art.
  • suitable reporter molecule including enzymes such as alkaline phosphatase and horseradish peroxidase and fluorescent reporter molecules such as fluorescein isothiocynate (FITC), phycoerythrin (PE) and rhodamine.
  • target polypeptide expression vectors may be engineered to express a chimeric target polypeptide containing an epitope for which a commercially available antigen-binding molecule exists.
  • the epitope specific antigen- binding molecule may be tagged using methods well known in the art including labelling with enzymes, fluorescent dyes or coloured or magnetic beads.
  • the "tagged" target polypeptide conjugate is incubated with the random peptide library for 30 minutes to one hour at 22° C to allow complex formation between target polypeptide and peptide species within the library. The library is then washed to remove any unbound target polypeptide.
  • the whole library is poured into a petri dish containing a substrate for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3 ',4,4"- diamnobenzidine (DAB), respectively.
  • a substrate for either alkaline phosphatase or peroxidase for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3 ',4,4"- diamnobenzidine (DAB), respectively.
  • BCIP 5-bromo-4-chloro-3-indoyl phosphate
  • DAB 3,3 ',4,4"- diamnobenzidine
  • a chimeric target polypeptide having a heterologous epitope may be used, detection of the peptide/target polypeptide complex may be accomplished by using a labeled epitope specific antigen-binding molecule. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing. 7. Methods of producing agents comprising a KKS antagonist
  • the present invention also provides methods of producing an agent comprising a KKS antagonist for preventing or treating an aneurysm in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for antagonising the KKS.
  • the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the aneurysm.
  • the aneurysm is an aortic aneurysm.
  • An illustrative example of this type of aneurysm includes AAA.
  • Serum was obtained from archived serum from The Health In Men Study (HIMS) previously described (Norman et ⁇ l, 2009) and came from subjects from Western Australia, Australia. Ethical approval from the centre providing the samples and informed consent from all subjects was obtained.
  • HIMS Health In Men Study
  • Serum samples were assayed by ELISA for kallistatin (Duoset, R&D Systems) according to the manufacturer's instructions. Inter-assay coefficient of variation for the assay was ⁇ 10%.
  • Arg 9 -BK produce important physiological effects by stimulating Bl or B2 receptors (Leeb-Lundberg et al, 2005).
  • Protein extraction and Western blotting [0925] Proteins (30 ⁇ g) were extracted and quantitated from human aortic biopsies as previously described in Moran et al, 2005.
  • Proteins were separated using SDS gel electrophoresis (12% SDS; Gradipore) and Western analysis carried out using primary antibodies to Bl receptor (BDKRBl 3 Abeam) and to B2 receptor (BDKRB2, Abeam) and secondary antibody: goat anti-rabbit IgG (Abeam) that was HRP-labelled.
  • SDS gel electrophoresis 12% SDS; Gradipore
  • Western analysis carried out using primary antibodies to Bl receptor (BDKRBl 3 Abeam) and to B2 receptor (BDKRB2, Abeam) and secondary antibody: goat anti-rabbit IgG (Abeam) that was HRP-labelled.
  • mice Male 13 week old ApoE " ' " mice were anaesthetised by intraperitoneal injection of ketamine (150mg/kg) and xylazine (10mg/kg) and a dorsal incision made at the upper back. Alzet® Osmotic miniature pumps (Model 2004) were inserted and the incision sutured.
  • mice were divided into four groups. In three of the four groups the pumps were pre-loaded with human recombinant Angll (Sigma). In the last group, the pumps were pre-loaded with saline. The pumps were loaded so as to deliver 1.44mg/kg/day of Angll or saline over the course of the 28 day experiment. [0941] In addition, the three groups of mice receiving AngII received intraperitoneal injections (2mg/kg/dose) the day prior to pump insertion and every other day throughout the experimental period with a vehicle control, the bradykinin receptor B2 agonist B9972 or the B1/B2 antagonist B9430. [0942] At the conclusion of the experimental period, mice with euthanased by
  • Aortas were dissected from the aortic arch to the iliac bifurcation, taking care to remove adherent adipose tissue and then photographed intact for aortic morphometry. Photographs of an aorta taken from a normal mouse (A - left hand side) and one from a mouse with an aneurysm (B - right hand side) are shown in Figure 3.
  • mice The mean survival time of AngII treated mice was 23 ⁇ 1.5 days with aortic rupture and death occurring in 33% (9/27) mice.
  • mice receiving the B2 agonist B9972 and AngII showed a very high incidence of aortic rupture (68%, 17/25 mice) and mean survival time decreased to 14.5 ⁇ 2 days (p ⁇ 0.01 compared to AngII alone).
  • mice receiving AngII plus the Bl /B2 antagonist B9430 there was significantly less aortic ruptures (8%, 2/24 mice) and the mean survival of this group was 27 days which was significantly different to AngII group (p ⁇ 0.05).
  • Table 8 Maximum aortic diameter in relation to AngII and kinin administration.
  • AngII 1.5Id O. ,32** 1, ,24 ⁇ 0. ,18** 1.54 ⁇ 0.59* 0.79 ⁇ 0, .08 1.27 ⁇ 0.24** + B1/B2 antagonist

Abstract

The present invention relates generally to altering levels of components in the kallikrein-kinin system (KKS) to assist in preventing or treating aneurysms in an individual, and also to detecting the presence, determining the prognosis or monitoring the progression of an aneurysm by detecting aberrant expression of one or more genes in the kallikrein-kinin system (KKS). The invention also relates to methods for identifying agents that antagonise the KKS, and methods of producing agents comprising KKS antagonists for preventing or treating aneurysms.

Description

TITLE OF THE INVENTION
"DIAGNOSIS, MONITORING, PROGNOSIS, PREVENTION AND TREATMENT OF
ANEURYSMS"
FIELD OF THE INVENTION [0001] This invention relates generally to methods for the diagnosis, monitoring, prognosis, prevention and treatment of aneurysms in individuals.
BACKGROUND OF THE INVENTION
/. Aneurysms
[0002] An aneurysm is a localized, blood-filled dilatation of a blood vessel being an artery, vein or heart.
[0003] The exact cause of aneurysms is unclear but it is believed that both genetic factors and environmental factors lead to aneurysms. Risk factors include male gender, smoking, obesity, hypertension, coronary heart disease and dyslipidemia.
[0004] The most common aneurysms are arterial aneurysms that affect the aorta. Aortic aneurysms are classified by which site within the aorta they occur.
Abdominal aortic aneurysms (AAAs), the most common form of aortic aneurysm are found in the abdominal aorta, and thoracoabdominal aortic aneurysms involve both the thoracic and abdominal aorta. An aortic root aneurysm occurs at the commencement of the aorta. Thoracic aortic aneurysms are found within the thoracic aorta and are further classified as ascending, aortic arch, or descending aneurysms depending on the location within the thoracic aortic involved.
[0005] Initially most intact aneurysms are asymptomatic. If left untreated, aneurysms tend to become progressively larger, although the rate of enlargement is unpredictable for any individual. As the size of an aneurysm increases, pain and numbness may be experienced, normally caused by nerve compression. Further, the risk of rupture increases as the size of an aneurysm enlarges. When rupture occurs, symptoms including a drop in blood pressure, increased heart rate, and loss of consciousness are often observed and can result in severe haemorrhage or other complications including sudden death. There is a low survival rate after rupture has occurred, even if surgery is performed. [0006] The result is that aneurysms place a large burden on the health system. AAA, for example, is the 10 most common cause of death in men aged over 60 years (Tilson, 2005). In 2004, 470 deaths (5/100,000/yr) and 2,169 hospitalisations (50/100,000/yr) as a result of AAA were recorded in New South Wales, Australia alone (Report by Population Health Division, 2007). This burden is growing as the numbers of identified early AAAs increases, caused by both the growth in the elderly population and an increase in the use of abdominal imaging (Lederle, et al. , 1997).
[0007] Despite this burden, the management of aneurysms is deficient in a number of areas. At this time, for example, there are no blood-based diagnostic tests available. Diagnosis (and monitoring) of intact aneurysms is usually achieved by medical imaging, normally ultrasound. Current imaging protocols simply provide anatomical information on the aorta, such as maximum aortic diameter. AAA5 for example, is thus diagnosed most commonly using a definition of maximum infrarenal aortic diameter greater than or equal to 30mm. Whilst aortic diameter provides some predictive power with respect to the subsequent behaviour of the aorta, the information provided is incomplete. Some aortas measuring >30mm remain stable for many years with no progression in diameter or risk of rupture. There are even cases of regression of maximum aortic size. To develop a true understanding of the pathological problem other diagnostic aids are urgently required, such as blood based biomarkers or functional imaging modalities. These would enable better prediction of clinical outcomes for aneurysms including progressive enlargement and rupture, and therefore better patient selection for treatment.
[0008] The available treatments for aneurysms are also limited. Currently there are no non surgical treatments which have been shown to slow the progression of aneurysms such as AAAs. The only treatments which are currently employed are open or endovascular surgery. Open surgery involves a large laparotomy, clamping of the aneurysm above and below, and replacing the weakened artery with a plastic graft. This operation is associated with a significant mortality of around 5%. Endovascular AAA repair is a more minimally invasive procedure whereby stent grafts are secured within the aneurysm from inside via a groin approach. The technique is associated with a lower mortality of approximately 1% and a smaller rate of perioperative complications and faster recovery than for open surgery. There is some concern over the long term outcome for this procedure since reintervention is not uncommon during long term follow up in up to 20% of patients.
[0009] Survival of individuals with a ruptured aneurysm is limited, with many patients dying prior to reaching hospital. Of those individuals with a ruptured AAA who are operated on for example, survival is only around 50%. There is currently interest in applying endovascular techniques to patients with ruptured AAA although at present the advantage of endovascular over open surgery as a method for treating this condition is unclear.
[0010] Randomised controlled trials have provided evidence that there is no survival advantage to carrying out elective repair for small aneurysms, for example
AAAs with a maximum diameter <55mm. Patients with an AAA measuring <50mm are thus usually treated conservatively, and simply monitored by regular imaging. Only large aneurysms or those that have ruptured are considered eligible for the surgical or endovascular treatment options. There is therefore an "untreated group" of patients with aneurysms, including those with small aneurysms and those for which these treatments are not an option for other reasons, for example if surgery is considered too high risk.
[0011] In work leading up to the present invention, an hypothesis was pursued that aneurysms are in some way associated with a mechanism in the kallikrein- kinin system. In investigating this hypothesis, the inventors discovered that serum concentrations of kallistatin (a naturally occurring kallikrein inhibitor) were strongly negatively associated with the presence of aneurysms in patients. They also discovered that modulation of the kallikrein-kinin system using kinin receptor agonist and kinin receptor antagonist differentially influenced aneurysm dilatation and rupture. These investigations indicate an important role of components in the kallikrein-kinin system in the pathogenesis of aneurysms .
2. Kallikrein-kinin system
[0012] The kallikrein-kinin system represents a metabolic cascade from kininogens to kinins which exert their pharmacological activities by binding specific receptors. The kallikrein-kinin system therefore consists of a large number of proteins and smaller polypeptides, and includes enzymes that activate and deactivate various other components in the system. [0013] Since discovery of the system in 1909, a considerable amount of research has been investigating the system with one review paper in 2005 noting over 30,000 papers referencing the kallikrein-kinin system in Medline (Moreau et at, 2005). The research means there are now a number of review papers on the kallikrein-kinin system including Moreau et at, 2005; Erdos et at, 2002; and Campbell, D. J., 2001.
2.1 Some components of the kallikrein-kinin system
[0014] In humans the KNGl gene generates both high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) through alternative splicing. HMWK and LMWK have no activity in themselves but are precursors to kinins, including bradykinin and kallidin. HMWK is produced by the liver, while
LMWK is protected locally by numerous tissues, and secreted together with tissue kallikrein.
[0015] The kallikreins of the kallikrein-kinin system are serine proteases that liberate the kinins from the kininogens. Tissue kallikrein is initially secreted as the inactive prokallikrein, and plasma kallikrein is initially secreted as the inactive prekallikrein. Both prokallikrein and prekallikrein require activation by factors XII or other stimuli. Whereas a single gene codes for plasma prekallikrein (KLKBl), there is a large family of tissue prokallikrein genes, although tissue kallikrein 1 (KLKl) encoded by the gene KLKl, is the only tissue kallikrein associated with the kallikrein-kinin system.
[0016] The kinins are small peptides. Bradykinin is a nonapeptide with the amino acid sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and is produced when plasma kallikrein releases it from HMWK. Kallidin (Lys-bradykinin) is a decapeptide with the same amino acid sequence as bradykinin but with the addition of a lysine at the N-terminus. Kallidin is released from HMWK or LMWK by tissue kallikrein.
Bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin. Alternative pathways of kinin formation involving enzymes other than kallikreins operate, for example in some disease states. A proportion of kininogens are hydroxylated which leads to the formation of hydroxylated kinins which have similar biological activity to non-hydroxylated kinins.
[0017] Bradykinin and kallikrein are metabolised by carboxypeptidases. The carboxypeptidases are present in two forms: N circulates and M is membrane-bound. The carboxypeptidases remove arginine residues at the carboxy-terminus of bradykinin and kallidin. Carboxypeptidase metabolites of the kinins include: des-Arg9-BK and Ly s- des-Arg9-BK.
[0018] Generally the term "kinin" when used in reference to humans and most mammals refers to bradykinin and kallidin, the hydroxylated forms of each, and also to carboxy-terminal des-Arg metabolites of bradykinin, kallidin or the hydroxylated forms of each.
[0019] The kinins exert their pharmacological activities by binding specific kinin receptors: being type 1 (Bl) and type 2 (B2) receptors. [0020] Kinin production in vivo is controlled, in part, by endogenous inhibitors of the kallikrein enzymes. The main inhibitors of plasma kallikrein are the Cl inhibitor (Cl-INH - encoded in humans by the gene SERPINGl), α2-macroglobulin (encoded in humans by the gene A2M) and antithrombin III (encoded in humans by the gene SERPINCl). Tissue kallikrein is inhibited by kallistatin which in humans is encoded by the gene SERPINA4.
[0021] Other components of the kallikrein-kinin system include angiotensin converting enzyme (ACE) and neutral endopeptidase. ACE and neutral endopeptidase deactivate certain peptide mediators, including kinins.
[0022] From the foregoing, it is proposed, in accordance with the present invention, that altering the levels of components in the kallikrein-kinin system (KKS) can assist in preventing or treating aneurysms in an individual, and that detecting the presence, determining the prognosis or monitoring the progression of an aneurysm may be achieved by detecting aberrant expression of one or more genes in the kallikrein-kinin system (KKS), as described hereinafter.
SUMMARY OF THE INVENTION
[0023] Accordingly, in one aspect, the present invention provides methods for preventing or treating an aneurysm in an individual. These methods generally comprise administering to the individual an effective amount of a KKS antagonist. Non- limiting examples of suitable KKS antagonists include small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, including those as further described herein. [0024] In some embodiments, the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene is selected from a gene encoding a component of the KKS (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, SERPINA4), a gene whose expression product modulates directly or indirectly the expression of a gene encoding a component of the KKS, and a gene whose expression product modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
[0025] In some embodiments, the agent reduces the expression of a gene (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2) or the level or functional activity of an expression product of that gene (e.g. HMWK, LMWK5 prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des- Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor). In other embodiments, the agent increases the expression of a gene (e.g. SERPINGl, A2M, SERPINCl, SERPINA4) or the level or functional activity of an expression product of that gene (e.g. Cl -INH, α2-macroglobulin, antithrombin III, kallistatin) .
[0026] Suitably, the agent reduces or increases the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent. [0027] In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
[0028] Another aspect of the present invention contemplates the use of an effective amount of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist as broadly described herein.
[0029] In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
[0030] In yet another aspect, the present invention resides in the use of an effective amount of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist as broadly described herein. [0031] In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
[0032] According to another aspect, the present invention provides methods for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual.
[0033] One suitable method comprises detecting in the individual the presence of an aberrant gene encoding a component of the KKS or detecting in the individual aberrant expression of a gene encoding a component of the KKS. In some embodiments, the aberrant gene is selected from an aberrant KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, or SERPINA4 gene.
[0034] Another suitable method comprises determining in the individual a level or functional activity of a component in the KKS, which is different than a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component. In some embodiments, the method comprises determining an increase or elevation in the level of functional activity of the component (e.g. HMWK, LMWK, prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des-Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor) relative to a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component. In other embodiments, the method comprises determining a decrease in the level or functional activity of the component (e.g. Cl-INH, α2-macroglobulin, antithrombin III, kallistatin) relative to a normal (e.g. no aneurysm, level measured in the same individual at an earlier time) reference or control level or functional activity of the component. In some embodiments, the method is performed on a biological sample obtained from the individual.
[0035] In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
[0036] In yet another aspect, the invention provides methods for identifying agents that antagonise the KKS. [0037] One suitable method comprises contacting a preparation with a test agent, wherein the preparation comprises (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence (e.g. a transcriptional control element such as a promoter or a czs-acting sequence) that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the KKS, or a variant or derivative thereof, , which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i). A detected change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference and/or control level or functional activity in the absence of the test agent, indicates that the agent modulates the KKS .
[0038] Another suitable method comprises contacting a sample of cells expressing a kinin receptor with a kinin and a test agent. A detected decrease in level of binding between the kinin receptor and the kinin relative to a reference or control level in the absence of the test agent, indicates that the agent is a KKS antagonist. [0039] In accordance with the present invention, the agents identified using the methods broadly described above are useful for preventing or treating an aneurysm in an individual. In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
[0040] Still another aspect of the present invention provides methods of producing an agent comprising a KKS antagonist for preventing or treating an aneurysm in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for antagonising the KKS. Suitably, the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the aneurysm. In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1 shows some of the results of Example 2. In Figure 1, bands of the B2 results are shown in the top line for samples taken from the neck (left hand side) and the body (right hand side) of the aneurysm. The bottom line of Figure 1 shows the bands of the control, α-actin. [0042] Figure 2 also shows some of the results of Example 2. In Figure 2, the concentration of both the Bl kinin receptor and B2 kinin receptor are shown. The left result for each of Bl and B2 shows the results from the sample from the aneurysm neck, and the right result for each of Bl and B2 shows the results from the sample from the aneurysm body.
[0043] Figure 3 shows photographs of an aorta taken from a normal mouse (A — left hand side) and one from a mouse with an aneurysm (B — right hand side) from the experiment described in Example 3.
[0044] Figure 4 shows the survival rates of the four mice groups from the experiment described in Example 3.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0046] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example,
"an element" means one element or more than one element.
[0047] The term "aberrant expression," as used herein, refers to the overexpression or underexpression of a gene encoding a component of the KKS relative to the level of expression of a gene encoding a component of the KKS or a variant thereof in cells obtained from a healthy individual or from an individual with no aneurysm, and/or to a higher or lower level of an expression product of that gene (e.g., transcript or polypeptide) in a biological sample obtained from a healthy individual or from an individual with no aneurysm. In particular, a gene encoding a component of the KKS is aberrantly expressed if the level of expression of the gene is higher by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%,
300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%, or lower by at least about 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999% than the level of expression of the gene by cells obtained from a healthy individual or from an individual without an aneurysm, and/or relative to the level of expression of the gene in a biological sample obtained from a healthy individual or from an individual without an aneurysm.
[0048] The reference to a level of expression of a gene of expression product of that gene in a healthy individual or an individual with no aneurysm may refer to an average level of expression in a group of healthy individuals or individuals with no aneurysm.
[0049] The term "acyl" either alone or in compound words such denotes a group containing the moiety C=O (and not being a carboxylic acid, ester or amide).
[0050] The terms "alkoxy," "alkenoxy," "alkynoxy," "aryloxy," "heteroaryloxy," "heterocyclyloxy" and "acyloxy" respectively denote alkyl, alkenyl, alkynyl aryl, heteroaryl, heterocyclyl and acyl groups as herein defined when linked by oxygen.
[0051] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon group and may have a specified number of carbon atoms. For example, Ci-Cio, as in "Ci-Cioalkyl" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in linear or branched arrangement. For example, "Ci-C10alkyl" specifically includes, but is not limited to, methyl, ethyl, n- propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl.
[0052] "Alkoxy" represents either a cyclic or non-cyclic alkyl group attached through an oxygen bridge. "Alkoxy" therefore encompasses the definitions of alkyl and cycloalkyl above. For example, alkoxy groups include but are not limited to methoxy, oxy ethoxy, n-propyloxy, i-propyloxy, cyclopentyloxy and cyclohexyloxy.
[0053] If no number of carbon atoms is specified, the term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C6alkenyl" means an alkenyl radical having from 2 to 6 carbon atoms. Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.
[0054] The term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present. Thus, "C2-C6alkynyl" means an alkynyl radical having from 2 to 6 carbon atoms. Alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, 3-methylbutynyl and so on. The straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.
[0055] In certain instances, substituents may be defined with a range of carbons that includes zero, such as (Co-C6)alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as, for example, -CH2Ph, -CH2CH2Ph, CH(CH3)CH2CH(CH3)Ph. [0056] As used herein, "alkylene" refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 1 to about 20 carbon atoms, more preferably 1 to 12 carbons, even more preferably lower alkylene. The alkylene group is optionally substituted with one or more "alkyl group substituents." There may be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-), ethylene (-CH2CH2-), propylene (-(CH2)3-), cyclohexylene (-C6H10-), methylenedioxy (-0-CH2-O-) and ethylenedioxy (-O-(CH2)2-O-). The term "lower alkylene" refers to alkylene groups having 1 to 6 carbons. Preferred alkylene groups are lower alkylene, with alkylene of 1 to 3 carbon atoms being particularly preferred.
[0057] As used herein, "alkenylene" refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one double bond, more preferably 2 to 12 carbons, even more preferably lower alkenylene. The alkenylene group is optionally substituted with one or more "alkyl group substituents." There may be optionally inserted along the alkenylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described. Exemplary alkenylene groups include -CH=CH-CH=CH- and -CH=CH-CH2 . The term "lower alkenylene" refers to alkenylene groups having 2 to 6 carbons. Preferred alkenylene groups are lower alkenylene, with alkenylene of 3 to 4 carbon atoms being particularly preferred.
[0058] As used herein, "alkynylene" refers to a straight, branched or cyclic, preferably straight or branched, bivalent aliphatic hydrocarbon group, preferably having from 2 to about 20 carbon atoms and at least one triple bond, more preferably 2 to 12 carbons, even more preferably lower alkynylene. The alkynylene group is optionally substituted with one or more "alkyl group substituents." There may be optionally inserted along the alkynylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl as previously described.
Exemplary alkynylene groups include -C=C-C=C-, -C=C- and -C=C-CH2-. The term "lower alkynylene" refers to alkynylene groups having 2 to 6 carbons. Preferred alkynylene groups are lower alkynylene, with alkynylene of 3 to 4 carbon atoms being particularly preferred. [0059] "Amplification product" refers to a nucleic acid product generated by a nucleic acid amplification technique.
[0060] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
[0061] "Antigenic or immunogenic activity" refers to the ability of a polypeptide, fragment, variant or derivative according to the invention to produce an antigenic or immunogenic response in an animal, suitably a mammal, to which it is administered, wherein the response includes the production of elements which specifically bind the polypeptide or fragment thereof.
[0062] "Aralkyl" means alkyl as defined above which is substituted with an aryl group as defined above, e.g., -CH2ρhenyl, -(CH2)2phenyl, -(CH2)3phenyl, - H2CH(CH3)CH2phenyl, and the like and derivatives thereof.
[0063] As used herein, "aromatic" or "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. [0064] By "biologically active fragment" is meant a fragment of a full-length parent polypeptide which fragment retains an activity of the parent polypeptide. As used herein, the term "biologically active fragment" includes deletion variants and small peptides, for example of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 contiguous amino acid residues, which comprise an activity of the parent polypeptide. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu- C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.
[0065] The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a patient. Suitably, the biological sample is a urine, whole blood, serum or plasma sample.
[0066] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
[0067] By "corresponds to" or "corresponding to" is meant (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.
[0068] The term "cycloalkenyl" means a monocyclic unsaturated hydrocarbon group and may have a specified number of carbon atoms. For example,
"cycloalkenyl" includes but is not limited to, cyclobutenyl, cyclopentenyl, 1- methylcyclopentenyl, cyclohexenyl and cyclohexadienyl. [0069] The term "cycloalkyl" or "aliphatic ring" means a monocyclic saturated aliphatic hydrocarbon group and may have a specified number of carbon atoms. For example, "cycloalkyl" includes, but is not limited to, cyclopropyl, methyl- cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl. [0070] By "derivative" is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functional equivalent molecules.
[0071] By "effective amount", in the context of treating or preventing an aneurysm is meant the administration of an amount of active ingredient to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for treatment or prophylaxis or improvement of that condition. Non-limiting examples of such improvements in an individual include reducing the size of an aneurysm, preventing an increase in the size of an aneurysm in an individual, reducing the number of small aneurysms in an individual, and/or preventing or inhibiting the occurrence of aneurysms in an individual. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the formulation of the composition being administered, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
[0072] The term "expression product" refers to production of mRNA, translation of RNA message into proteins or polypeptides, or processed forms of those proteins or polypeptides.
[0073] As used herein, the term "function" refers to a biological, enzymatic, or therapeutic function.
[0074] The term "gene" as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions. The gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The DNA sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
[0075] As appreciated by those of skill in the art, "halo" or "halogen" as used herein is intended to include chloro, fluoro, bromo and iodo.
[0076] "Heteroaralkyl" group means alkyl as defined above which is substituted with a heteroaryl group, e.g., -CHbpyridinyl, -(CH2)2pyrimidinyl, - (CH2)3imidazolyl, and the like, and derivatives thereof.
[0077] The term "heteroaryl" or "heteroaromatic," as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, bezofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. [0078] Further examples of "heteroaryl" and "heterocyclyl" include, but are not limited to, the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazoyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofui-anyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom. [0079] As used herein, "heteroaryiene" refers to a bivalent monocyclic or multicyclic ring system, preferably of about 3 to about 15 members where one or more, more preferably 1 to 3 of the atoms in the ring system is a heteroatom, that is, an element other than carbon, for example, nitrogen, oxygen and sulfur atoms. The heteroaryiene group may be optionally substituted with one or more, preferably 1 to 3, aryl group substituents. Exemplary heteroaryiene groups include, for example, 1,4-imidazolylene.
[0080] The term "heterocycle", "heteroaliphatic" or "heterocyclyl" as used herein is intended to mean a 5- to 10-membered nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. [0081] "Heterocyclylalkyl" group means alkyl as defined above which is substituted with a heterocycle group, e.g., -CHbpyrrolidin-l-yl, -(CH2)2piperidin-l-yl, and the like, and derivatives thereof.
[0082] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
[0083] The term "hydrocarbyl" as used herein includes any radical containing carbon and hydrogen including saturated, unsaturated, aromatic, straight or branched chain or cyclic including polycyclic groups. Hydrocarbyl includes but is not limited to Ci-Cealkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C10cycloalkyl, aryl such as phenyl and naphthyl, Ar (C1-C8)alkyl such as benzyl, any of which may be optionally substituted. [0084] Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
[0085] The terms "individual", "patient" and "subject" are used interchangeably herein to refer to individuals of human or other animal origin and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that these terms do not imply that symptoms are present. Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes, avians, reptiles).
[0086] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. [0087] By "modulating" is meant increasing or decreasing, either directly or indirectly, the level or functional activity of a target molecule. For example, an agent may indirectly modulate the level/activity by interacting with a molecule other than the target molecule. In this regard, indirect modulation of a gene encoding a target polypeptide includes within its scope modulation of the expression of a first nucleic acid molecule, wherein an expression product of the first nucleic acid molecule modulates the expression of a nucleic acid molecule encoding the target polypeptide.
[0088] By "obtained from" is meant that a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source of the host. For example, the extract can be obtained from a tissue or a biological fluid isolated directly from the host.
[0089] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotide residues, but the term can refer to molecules of any length, although the term "polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.
[0090] By "operably linked" is meant that transcriptional and translational regulatory polynucleotides are positioned relative to a polypeptide-encoding polynucleotide in such a manner that the polynucleotide is transcribed and the polypeptide is translated.
[0091] By "pharmaceutically acceptable carrier or diluent" is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to a mammal.
[0092] "Phenylalkyl" means alkyl as defined above which is substituted with phenyl, e.g., -CH2phenyl, (CH2)2phenyl, -(CH2)3phenyl, CH3CH(CH3)CH2phenyl, and the like and derivatives thereof. Phenylalkyl is a subset of the aralkyl group.
[0093] The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length. [0094] The terms "polynucleotide variant" and "variant" refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions as known in the art (see for example Sambrook et al. , Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989). These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants.
[0095] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. [0096] The term "polypeptide variant" refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter. These terms also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acids.
[0097] "Probe" refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly.
[0098] As used herein, "pseudohalides" are groups that behave substantially similar to halides. Such groups can be used in the same manner and treated in the same manner as halides (X, in which X is a halogen, such as Cl or Br). Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethyl and azide.
[0099] The term "recombinant polynucleotide" as used herein refers to a polynucleotide formed in vitro by the manipulation of a polynucleotide into a form not normally found in nature. For example, the recombinant polynucleotide can be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory polynucleotide operably linked to the polynucleotide.
[0100] By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
[0101] The term "reference or control level" as used herein refers to any suitable reference or control level, including, but not limited to, a normal healthy individual, an individual without an aneurysm, a level measured in the same individual at a different time, and a level measured in a biological sample taken from a different tissue in the same individual or control.
[0102] By "reporter molecule" as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that allows the detection of a complex comprising an antigen-binding molecule and its target antigen. The term "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
[0103] With reference to the term "stereoisomers" as used herein, it will also be recognized that the compounds described herein may possess asymmetric centers and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centers e.g., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
[0104] By "vector" is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is preferably a viral or viral-derived vector, which is operably functional in animal and preferably mammalian cells. Such vector may be derived from a poxvirus, an adenovirus or yeast. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptll gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.
[0105] The terms "wild-type" and "normal" are used interchangeably to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant.
[0106] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, "KLKl" shall mean the KLKl (tissue kallikrein, human kallikrein 1, hKl) gene, whereas "KLKl" shall indicate the protein product or products generated from transcription and translation and alternative splicing of the "KLKl" gene.
2. Methods of preventing or treating aneurysms
[0107] The present invention is predicated in part on the discovery that the modulation of levels of components in the KKS, can increase or decrease the size of an aneurysm and/or the number of aneurysms and/or the likelihood of aneurysm rupture. It is proposed, therefore, that KKS antagonists will be useful inter alia for the prevention or treatment of an aneurysm, including intact or ruptured aneurysms. The aneurysm may be located in an artery, a vein or the heart. Suitable arterial aneurysms include, but are not limited to, arterial aneurysms that occur at the base of the brain (the circle of Willis) and aortic aneurysms. Suitable aortic aneurysms include, but are not limited to, abdominal aortic aneurysms (AAAs), thoracoabdominal aortic aneurysms, aortic root aneurysms, and thoracic aortic aneurysms.
[0108] Accordingly, the present invention provides methods for preventing or treating an aneurysm in an individual, the methods comprising administering to the individual an effective amount of a KKS antagonist. Another aspect of the present invention contemplates the use of an effective amount of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist. In yet another aspect, the present invention resides in the use of an effective amount of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist. [0109] The term "KKS antagonist" as used herein refers to any agent which directly or indirectly agonises or antagonises a component in the KKS so as to inhibit or otherwise reduce the metabolic cascade in the KKS.
[0110] In some embodiments, the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes a component of the KKS. Representative genes encoding a component of the KKS include KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, and SERPINA4. Representative expression products of genes encoding components of the KKS include kininogen (e.g. HMWK and LMWK), kallikrein precursors (e.g. prekallikrein and prokallikrein), kallikrein (e.g. plasma kallikrein and tissue kallikrein), kinin (e.g. bradykinin, kallidin, des-Arg9-bradykinin, and Lys-des-
Arg9-bradykinin), kinin receptor (e.g. the kinin Bl receptor and the kinin B2 receptor), Cl-INH, α2-macroglobulin, antithrombin III and kallistatin.
[0111] In other embodiments, the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression of a gene encoding a component of the KKS.
[0112] In other embodiments, the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene, wherein the gene encodes an expression product which modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
[0113] The agent suitably modulates the expression of the gene directly, or modulates an upstream regulator of the expression of the gene, or directly or indirectly modulates the level or functional activity of an expression product of such genes.
[0114] In some embodiments, the agent reduces the expression of a gene (e.g. KNGl, KLKl, KLKBl, BDKRBl, BDKRB2) or the level or functional activity of an expression product of that gene (e.g. HMWK, LMWK5 prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des-Arg9-bradykinin, Lys-des- Arg9-bradykinin, the kinin Bl receptor, the kinin B2 receptor). In other embodiments, the agent increases the expression of a gene (e.g. SERPINGl, A2M, SERPINCl, SERPINA4) or the level or functional activity of an expression product of that gene (e.g. Cl-INH, α2-macroglobulin, antithrombin III, kallistatin). [0115] Suitably, the agent reduces or increases the expression of the gene or the level of functional activity of an expression product of that gene by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% relative to the expression, level or functional activity in the absence of the agent.
[0116] Suitable agents for reducing or abrogating gene expression include, but are not restricted to, oligoribonucleotide sequences, including anti-sense RNA and
DNA molecules and ribozymes, that function to inhibit the translation of mRNA. Anti- sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions are preferred.
[0117] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
[0118] Both anti-sense RNA and DNA molecules and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
[0119] Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
[0120] Alternatively, RNA molecules that mediate RNA interference (RNAi) of a target gene or gene transcript can be used to reduce or abrogate gene expression. RNAi refers to interference with or destruction of the product of a target gene by introducing a single stranded, and typically a double stranded RNA (dsRNA) that is homologous to the transcript of a target gene. Thus, in some embodiments, dsRNA^er se and especially dsRNA-producing constructs corresponding to at least a portion of a target gene may be used to reduce or abrogate its expression. RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a target gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts which then anneal to form a dsRNA having homology to a target gene.
[0121] Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk, R. H., et al, 2000). Therefore, depending on the length of the dsRNA, the RNAi- encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e. , with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene. RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters, are preferably at least about 100 nucleotides in length. RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are preferably at least about 200 nucleotides in length. [0122] The promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction. Alternatively, the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage. The promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.
[0123] In other embodiments, RNA molecules of about 21 to about 23 nucleotides, which direct cleavage of specific mRNA to which they correspond, as for example described by Tuschl et al. in U.S. Patent Application No. 20020086356, can be utilized for mediating RNAi. Such 21-23 nt RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double stranded (as two 21-23 nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').
[0124] Illustrative examples of suitable sequences from which the above methods may be based are those disclosed in GenBank, including accession nos NC_000003.10, NT_005612.15, AC_000046.1, NW_921807.1, AC_000135.1,
NWJ)Ol 838884.2, NCJ)OOO 19.8, NTJ) 11109.15, ACJ)00062.1, NW_927284.1, AC_000151.1, NW_001838498.2, NC_000014.7, NT_026437.11, AC_000057.1, NW_925561.1, AC_000146.1, NW_001838115.2, NG_009625.1, NC J)OOOl 1.8, NT_033903.7, AC_000054.1, NW_925106.1, ACJ)OO 143.15 NWJ)01838023.1, NGJ)11717.1, NC_000012.10. NT_009714.16, AC_000055.1, NW_925295.1,
AC_000144.1, NW_001838051.1, NC_000001.9, NT_004487.18, AC_000044.1, NW_926128.1, AC_000133.1, NW_001838533.2, or NW J)Ol 838113.2. Based on these sequences a person of skill in the art would readily know how to prepare suitable agents for reducing or abrogating gene expression as described herein. [0125] Suitable agents for modulating the level or functional activity of an expression product of a gene include, but are not restricted to small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, as further described herein.
[0126] In some embodiments, antigen-binding molecules (including neutralising antibodies and blocking antibodies) can be used to modulate the level or functional activity of an expression product of a gene.
[0127] Suitably, binding or activation of the kinin Bl receptor and/or the kinin B2 receptor is the subject of the targeting. For example, inhibition or abrogation of receptor signalling is achieved through reduction in receptor expression, receptor mutation (in particular, but not exclusively, of phosphorylation sites), prevention of receptor aggregation or through approaches that interfere with ligand-receptor interaction including those via blockade of the active binding sites or relevant associated motifs. Such strategies include blocking antibodies to the receptors and small molecule inhibitors of binding. Pharmacological strategies to impair receptor phosphorylation can also be effective. [0128] In some embodiments, the kinin receptor antagonist may inhibit both the kinin Bl receptor and kinin B2 receptor. In other embodiments, the kinin receptor antagonist is a selective kinin Bl receptor antagonist. In other embodiments, the kinin receptor antagonist is a selective kinin B2 receptor antagonist.
[0129] In some embodiments, the kinin receptor antagonist is a mutant kinin that binds to and recognises a kinin Bl receptor and/or a kinin B2 receptor but is unable to induce the pharmacological activities induced when a wild-type kinin binds to a kinin receptor. In illustrative examples of this type, the mutant kinin varies from the corresponding wild-type kinin by the deletion or modification of all or part of the nuclear translocation sequence, which renders it inoperative. [0130] The present invention also contemplates the use of receptor antagonistic antigen-binding molecules with varying blocking capacities.
[0131] Exemplary receptor antagonists include those disclosed herein, including B9430 (Gera et al, 1996), and those disclosed in Blakeney et al 2007, see pages 2967-2971. [0132] In still other embodiments, the subject of the targeting is a component of post-receptor signal transduction. [0133] The present invention also contemplates the use in the above method of gene or expression product inhibitors identified according to methods described herein.
3. Illustrative agents for preventing or treating aneurysms
[0134] In one aspect, the present invention provides methods for preventing or treating an aneurysm in an individual, the methods comprising administering to the individual a KKS antagonist. Another aspect of the present invention contemplates the use of an agent, which is optionally formulated with a pharmaceutically acceptable carrier or diluent, for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist. In yet another aspect, the present invention resides in the use of an agent in the manufacture of a medicament for preventing or treating an aneurysm in an individual, wherein the agent comprises a KKS antagonist.
[0135] Non-limiting examples of suitable KKS antagonists include small organic molecules, nucleic acids, peptides, polypeptides, proteins, proteoglycans, peptidomimetics, carbohydrates, sugars, lipids or other organic (carbon containing) or inorganic molecules, including those as further described herein.
3.1 Suitable small organic molecules include:
[0136] (A) 3 -amido-5 -substituted pyrazole derivatives and related compounds of formula (I) and (II)
Figure imgf000028_0001
[0137] wherein
[0138] Z' is selected from O, S and NH;
[0139] Q is selected from the group consisting of -NR4R5, -OH, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl;
[0140] R1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0141] R2 and R3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic; [0142] R4 and R5 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic and where R4 and R5, together with the nitrogen atom pendent thereto are joined to form a heterocyclic, a substituted heterocyclic, a heteroaryl or a substituted heteroaryl group, provide that when R4 or R5 is a substituted alkyl group this group is not ~CHRa~C(O)-NRbRc or ~CHRa--C(O)--ORb, wherein Ra is the side chain of a natural or unnatural amino acid, and Rb and Rc are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic;
[0143] X is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, nitro, cyano, hydroxyl, alkoxy, substituted alkoxy, carboxy, carboxyl esters, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, acylamino, aminoacyl, and -C(O)NR7R8 wherein R7 and R8 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, or R and R together with the nitrogen atom to which they are joined form a heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic;
[0144] or pharmaceutically acceptable salts, prodrugs or isomers thereof. [0145] In some embodiments, the following provisos apply: [0146] A) when Z' is O; R2 is H; R3 is 5-(2, 4-dichlorophenyl)-imidazol-4- ylidene-[2-methyl-4-(N-(l-methylsufonyleth-2- -yl)-N-ethylamino)phenyl]-amine; X is H; and R1 is l-(3-t-butyl-4-hydroxyphenyloxy)tridec-l-yl; then Q is not ethoxy;
[0147] B) when Z1 is O; R2 is H; R3 is 4-chlorophenyl; X is aminoacyl; and R1 is methyl or phenyl; then Q is not methyl; [0148] C) when Z' is O; R2 is H; R3 is 4-chlorophenyl or 4-methylphenyl; X is cyano; and R1 is methyl; then Q is not methyl;
[0149] D) when Z' is O; R2 is H; R3 is H; X is H; and R1 is l-[(l-cyclohexyl- 2-(furan-3-yl)-lH-benzoimidazol-5-yl)acylamino]cyclopent-l-yl; then Q is not hydroxy;
[0150] E) when Z' is O; R2 is H; R3 is H; X is H; and R1 is 3-isobutoxy-5- isopropylphenyl or isopropyl; then Q is not methoxy;
[0151] F) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is 2-methyl-3- acetamidopyrazol-5-yl; then Q is not hydroxy or methoxy;
[0152] G) when Z' is O; R2 is H; R3 is H; X is isobutoxycarbonyl or n- propoxycarbonyl; and R1 is methyl; then Q is not methoxy; [0153] H) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is 2-(3- trifluoromethyl-ρhenoxy)pyridine-3-yl; then Q is not ethoxy;
[0154] I) when Z' is O; R2 is 3-cyanobenzyl; R3 is methyl; X is H; and R1 is methyl; then Q is not isopropyl;
[0155] J) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is 1 -methyl-3- (benzyloxycarbonylamino)pyrazol-5-yl; then Q is not N,N-dimethylamino-ethylamino, methoxy or hydroxy;
[0156] K) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is l-methyl-3- (N,N-dimethylaminoethylcarbonylamino)pyrazol-5-yl; then Q is not N,N-dimethylamino- ethylamino; [0157] L) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is 1 -methyl-5- aminopyrazol-3-yl; then Q is notN,N-dimethylaminoethylamino or methoxy;
[0158] M) when Z1 is O; R2 is H; R3 is methyl; X is H; and R1 is 1 -methyl-5- acetamidopyrazol-3-yl-; then Q is not hydroxy or methoxy; [0159] N) when Z1 is O; R2 is H; R3 is methyl; X is H; and R1 is 1 -methyl-5-
(benzyloxycarbonylamino)pyrazol-3-yl; then Q is not N,N-dimethylaminoethylamino, methoxy or hydroxy;
[0160] O) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is l-methyl-5- (N,N-dimethylaminoethylcarbonylamino)pyrazol-3-yl; then Q is not N,N-dimethylamino- ethylamino;
[0161] P) when Z' is O; R2 is H; R3 is methyl; X is H; and R1 is 1 -methyl-5- methylcarbonylaminopyrazol-3-yl; then Q is not l-chloromethyl-5-nitro-2,3-dihydro- l(H)-benzo[e]indol-3-yl or l-chloromethyl-5-amino-2,3-dihydro-l(H)-benzo [e]indol-3- yi; [0162] Q) when Z1 is O; R2 is H; R3 is phenyl; X is benzothiazol-2-yl; and R1 is methyl; then Q is not ethoxy; or
[0163] R) when Q is -NR4R5, -OH, alkoxy or substituted alkoxy; R1 is substituted aryl or substituted heteroaryl, the aryl or heteroaryl group is not substituted with -C(0)NH~W'-C(0)0Rb or -C(O)NH~W'~C(O)NRbRc, where W is aryl, substituted aryl, heteroaryl or substituted heteroaryl;
[0164] and further with the proviso that the compound in Formula (I) is not
[0165] A') 4-bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid; or
[0166] B') 5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid (2- dimethylamino- 1 -methyl-ethyl)-amide.
[0167] Exemplary compounds include:
[0168] 4-Bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid [2-(l-methyl-piperidin-4-yl)-ethyl]-amide;
[0169] (R)-4-Bromo-5-(2-chloro-benzoylamino)-l-phenyl-pyrazole~3- carboxylic acid (2-oxo-azepan-3-yl)-amide; [0170] (S)-4-Bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid (1 -aza-bicyclo[2.2.2]oct-3-yl)-amide;
[0171] (R)-4-Bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid methyl-(l -phenyl-ethyl)-amide; [0172] 4-Bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid
(3-dimethylamino-propyl)-amide;
[0173] (R)-4-Bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid ( 1 -phenyl-2-piperidin- 1 -yl-ethyl)-amide;
[0174] 4-Bromo-5-(2-chloro-benzoylamino)- 1 H-pyrazole-3-carboxylic acid [2-(l -methyl-pyrrolidin-2-yl)-ethyl]-aniide;
[0175] 4-Bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid (5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-amide;
[0176] (R)-4-Bromo-5 -(2-chlorobenzoylamino)- 1 H-pyrazole-3 -carboxylic acid (1 -benzyl-2-oxo-azepin-3-yl)amide; [0177] 4-Bromo-5-(2-chlorό-benzoylamino)-lH-pyrazole-3-carboxylic acid
[2-(4-benzyl-piperazin- 1 -yl)ethyl]amide;
[0178] 4-Chloro-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid (2-oxo-2,3 ,4 , 5 -tetrahydro- 1 H-benzo [b] azepin-3 -yl)amide;
[0179] 4-Bromo-5-(2-chlorobenzoylamino)-l H-pyrazole-3-carboxylic acid [2-(l -pyrimidin-2-yl-piperidin-4-yl)-ethyl]amide;
[0180] 4-Bromo-5-(2-chloro-benzoylamino)- 1 H-pyrazole-3-carboxylic acid (5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrido[3,2-b]azepin-7-yl)amide;
[0181] 5-(3-Chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid [2- (3 ,4,5 ,6-tetrahydro-2H-[ 1 ,4']bipyridin-4-yl)-ethyl] amide; [0182] 4-Chloro-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid
[5-(3-aza-bicyclo[3.2.2]non-3-yl)-l-methyl-2-oxo-2,3-dihydro-lH-benzo[e][- 1 ,4]diazepin-3-yl]amide;
[0183] 5-(2-Chlorobenzoylamino)- 1 -methyl- 1 H-pyrazole-3-carboxylic acid (2-oxo-5-phenyl-2,3-dihydro-lH-benzo[e][l,4]diazepin-3-yl)amide; [0184] 3 -(2-Chlorobenzoylamino)-l -methyl- lH-pyrazole-5-carboxylic acid (2-oxo-5-phenyl-2,3-dihydro-lH-benzo[e][l34]diazepin-3-yl)amide;
[0185] l-(ρyridin-2-yl)-4-methyl-5-(2-chloro-benzoylamino)-lH-pyrazole-3- carboxy- lie acid [2-(3,4,5,6-tetrahydro-2H-[l,4']bipyridin-4-yl)-ethyl]aniide; [0186] 4-bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid
[1 ,4-dimethyl-5-oxo-[l ,4]diazepan-6-yi]amide,
[0187] 5-(2-chloro-benzoylaniino)-lH-pyrazole-3-carboxylic acid [2- (pyridine-4-yl)ethyl] amide ;
[0188] 4-bromo-5-(2-chloro-benzoylamino)-lH-pyrazole-3-carboxylic acid [3-(pyridin-4-yl)-[l,4]diazepin-5-yl]amide; and
[0189] 5-(2-chloro-benzoylamino)- 1 -p-fluorophenyl)-pyrazole-3-carboxylic acid [2-(3,4,5,6-tetrahydro-2H-[l,4]bipyridin-4-yl)-ethyl]amide.
[0190] These compounds, methods for their preparation and their biological activity are disclosed in US 7,432,379. The disclosed compounds are described as being bradykinin Bl receptor antagonists.
[0191] (B) Acyl amino acid amide derivatives of the formula (III):
A-B-R (III)
[0192] wherein
[0193] A is an acylating group, and preferably a hydrophobic acylating group, or an anti-inflammatory substituent selected from the group consisting of: Aaa,
Aba, Aca, Acre, Aic, Amts, 6Ani, Aq2c, Arac, Aspr, Atfb, 4Atfb, B6, B6P, Baaa, Bbz, Bcin, Bcpa, Bcpoa, Biot, Bipa, 4Bpc, Bphs, tBua, Bzac, Chbu, Che, CHFB, ChI, Chpa, 2CIn, Cmioc, Cpcpc, Dbhc, Dca, DcIa, tDecl, Dfc, Dhq, Dmc, Dmo, 22Dp, Esul, 2-Fa5 3- Fa, F5b, F5bs, F5bz, F3c, F5c, Fein, aFcn, Fmoc, F5pa, Fmpi, F5po, F5ρt, Gbz, Hen, Hmqc, Hor, 2Hyb, 3Iac, 3Ibu, 2Ina, Indo, Inp, KtIc, Ktpf, Mca, aMcn, Mcoa, 34Mdc,
MTPA, INac, INaIa, Nap, Napr, Nba, Octe, Otac, Pac, Pas, Pcin, Pen, Pcnl, βPhc, 3Php, 5Phv, Pic, Piva, Ppr, Ptmb, αPtpa, Pya, Pyre, Pyz, 13cR, Ret, Rio, Saa, Sab, cSdc, Sibu, cSsa, tβSts, Taa, Tchc, Tcpa, Tf2c, 4Tfmb, Thia, Th2n, Tic, Tmb, 4Tmbs, Tmbz, Tmcc, and ZPcn; [0194] B is an amino acid or substituted amino acid selected from the group consisting of: Bip, Ddip, F5F, F3MF, hPhe, MC2Y, NaI, NMF, OBPY5 OBrZY, OC2Y, OCIY5 Pal, PBF, PCNF, PFF, PIF5 PNF, Tic, and Tyr(Bzl); and
[0195] R is a substituted amide, preferably having additional polar character, selected from the group consisting of: Abzp, Aem, Alp, Ambi, Apia, Apyr, AquR, Atmp,
BapR, BapS, Bdbh, Bhp, Btmb, Cbp, Chmp, tCip, 4Clbp, Cpp, Cypp, Daep, Dasd, Depp, cDmap, cDmbp, cDmm, Dmmp, Dmpz, Dpic, Fbhp, 4Fbp, Fpmp, Fpdh, Matp, 4Mbp, Mpz, Ocp, Pep, Pipe, Pipp, Pipz, Pmpz, Ppp, Pypz, 3Qum, or Tmbp;
[0196] and wherein: [0197] Aaa = 1-Adamantaneacetyl
[0198] Aba = 2-cis-4-trans-Abscisic acid
[0199] Abzp = 4- Amino- 1 -benzylpiperidine
[0200] Aca = 1 -Adamantanecarboxyl
[0201] Acre = Acridine-9-carboxyl [0202] Aem = 4-(2-Aminoethyl)-morpholine
[0203] Aic = 2-Aminoindane-2-carboxylic acid
[0204] Alp = l-Allylpiperazine
[0205] Ambi = 2-(Aminomethyl)benzimidazole
[0206] Amp = l-(3-Aminopropyl)-4-methylpiperazine [0207] Amts = 2-Acetamido-4-methyl-5-thiazolesulfonyl
[0208] 6Ani = 6-Aminonicotinoyl
[0209] Apia = l-(3-Aminopropyl)imidazole
[0210] Apyr = 3 -Amino-pyrrolidine
[0211] Aq2c = Anthraquinone-2-carboxyl [0212] AquR = (R)-(+)-3-Aminoquinuclidine
[0213] Arac = Arachidonyl
[0214] Aspr = O-Acetylsalicyl: 2-acetoxybenzoyl
[0215] Atfb = 3-Amino-2,5,6-trifluorobenzoyl [0216] 4Atfb - 4-Aniino-2,3,5,6-tetrafluorobenzoyl
[0217] Atmp = 4-Amino-2,2,6,6-tetramethylpiperidine
[0218] AtmpO = 4-Amino-2,2,6,6-tetramethylpiperidinyloxy
[0219] B6 = 3-Hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridylmethyl (Vitamin B6, Pyridoxamine)
[0220] B6P = 3-Hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridylmethyI- 5- phosphate
[0221] Baaa = 2,2-Bis(acrylamido)acetyl
[0222] BapR = (R)-(-)-l-Benzyl-3-aminopyrrolidine [0223] BapS = (S)-(+)-l-Benzyl-3-aminopyrrolidine
[0224] Bbz = 4-Boronobenzoyl
[0225] Bcin = 4-Boronocinnamoyl
[0226] Bcpa = bis(4-Chlorophenyl)acetyl
[0227] Bcpoa = bis(4-Chlorophenoxy)acetyl [0228] Bdbh = (1 S,4S)-(+)-2-Benzyl-2,5-diazabicyclo[2.2. l]heptane
[0229] Bhp = 1-Benzylhomopiperazine
[0230] Biot - Biotinyl
[0231] Bip = β-(4-Biρhenylyl)alanine
[0232] Bipa = 4-Biphenylacetyl [0233] 4Bpc = 4-Biphenylcarboxyl
[0234] Bphs = 4-Biphenylsulphonyl
[0235] Btmb = 3,5-Bis(trifluoromethyl)benzylamine
[0236] tBua = tert-Butylacetyl
[0237] Bzac = 3-BenzoylacryIoyl [0238] Cbp = l-(4-Chlorobenzhydrylpiperazine)
[0239] 2Ccn = 2-Chlorocinnamoyl
[0240] Chbu = 2-Cyclohexylbutyryl [0241] Che = α-Cyano-4-hydroxycinnamoyl
[0242] CHFB = 4-Carboxy-hexafluorobutyryl
[0243] ChI = Chlorambucil: 4-[p-(bis[2-Chloroethyl]amino)- phenyljbutyryl
[0244] Chmp = 1-Cyclohexylmethylpiperazine [0245] Chpa = α-Cyclohexylphenylacetyl
[0246] tCip = trans- 1 -Cinnamylpiperazine
[0247] 4-Clbp = 1 -(4-Chlorobenzyl)piperazine
[0248] 2CIn = 2-Chloronicotinoyl
[0249] Cmioc = 3-(2-Chlorophenyl)-5-methylisoxazole-4-carbonyl [0250] Cpcpc ^ l-(4-Chlorophenyl)-l-cyclopropanecarboxyl
[0251] Cpp = l-(4-Chlorophenyl)piperazine
[0252] Cypp = l-(4-Cyanophenyl)piperazine
[0253] Daep = l-(2-(Diallylamino)ethyl)piperazme
[0254] Dasd = l,4-Dioxa-8-azaspiro[4.5]decane [0255] Dbhc = 3,6-Di-tert-butyl-4-hydroxycinnamoyl
[0256] Dca = Dicyclohexylacetyl
[0257] DcIa - Dichloroacetyl
[0258] Dcpp = l-(2,3-dichlorophenyl)piperazine
[0259] tDecl = trans-4-(Diethylamino)cinnamyl [0260] Dfc = Diclofenac: 2-[(2,6-Dichlorophenyl)amino]phenylacetyl
[0261] Dhq = 2,3-Dehydroquinuclidine-3-carboxyl
[0262] Dip = 3,3-Diphenylalanine
[0263] cDmap = cis-2,6-Dimethyl- 1 -allyl-piperazine
[0264] cDmbp = cis-2,6-Dimethyl- 1 -benzylpiperazine [0265] Dmc = Dimethoxycinnamoyl
[0266] CDmm = cis-2,6-Dimethylmorpholine [0267] Dmmp = cis-2,6-Dimethyl- 1 -(methoxycarbonylmethyl)piperazine)
[0268] Dmo = 3,7-Dimethyl-6-octenoyl: R-+-Citronellyl
[0269] Dmpz = 2,6-Dimethylpiperazine
[0270] 22Dp = 2,2-Diphenylpropionyl [0271] Dpic = Di-(2-picoyl)amine
[0272] Esul = Exisulindacyl: (Z)-5-Fluoro-2-methyl-[[4- (methylsulfonyl)phenyl]methylene]-lH-mdene-3-acetyl; (cis)
[0273] 2-Fa = 2-Furanacryloyl
[0274] 3-Fa = trans-3 -Furanacryloyl [0275] F5b = 2,3,4,5,6-Pentafluorobenzyl
[0276] Fbhp = l-(4-Fluorobenzyl)homopiperazine
[0277] 4-Fbp = l-(4-Fluorobenzyl)piperazine
[0278] F5bs = Pentafluorobenzenesulfonyl
[0279] F5bz = Pentafluorobenzoyl [0280] F3c = 2,3,5-Trifluorocinnamoyl
[0281] F5c = 2,3,4,5,6-Pentafluorocinnanioyl
[0282] Fein = 4-Formylcinnamoyl
[0283] αFcn = αFluorocinnamoyl
[0284] F5F = Pentafluorophenylalanine [0285] F3MF = 4-Trifluoromethylphenylalanine
[0286] Fmoc = 9-Fluorenylmethoxycarbonyl
[0287] F5pa = 2,3,4,5,6-Pentafluorophenylacetyl
[0288] Fpmp = l-bis(4-Fluorophenyl)methylpiperazine
[0289] Fmpi = (Z)-5-Fluoro-2-methyl-(4-ρyridylidene)-3-indenylacetyl [0290] F5po = 2,3,4,5,6-Pentafluorophenoxyacetyl
[0291] F5Pt = Pentafluorophenylthiocarbamyl [0292] Fpdh = (1 S,4S)-(-)-2-(4-Fluorophenyl)-2,5- diazabicyclo[2.2. ljheptane
[0293] Gbz = 4-Guanidinobenzoyl
[0294] Gun = Guanidyl [0295] Hen = 9-(N-Hydroxycarbamoyl)-nonanoyl
[0296] Hmqc = 3-Hydroxy-2-methyl-4-quinolinecarboxyl
[0297] Hor = (S)-(+)-Hydroorotic acid
[0298] HPhe= Homo-phenylalanine
[0299] 2Hyb = 2-Hydroxybenzoyl [0300] 3Iac = 3-β-Indoleacryloyl
[0301] 3Ibu = Indole-3-butyryl
[0302] IgI = α-2-Indanylglycine
[0303] 2Ina = 2-Indanylacetyl
[0304] Indo = Indomethacin: l-[p-Chlorobenzoyl]-5-methoxy-2- methylindole-3 acetyl
[0305] Inp = Isonipecotic acid: hexahydroisonicotinic Isoquinolineacetyl
[0306] KtIc = Ketorolac: (.+-.)5-benzoyl-2,3dihydro-lH-ρyrrolizine-l- carboxyl: Toradol
[0307] Ktpf= Ketoprofen: 2-(3-benzoylphenyl)propionyl [0308] Matp = 4-(Methylamino)-2,2,6,6-tetramethylρiperidine
[0309] 4-Mbp = l-(4-Methylbenzyl)piperazine
[0310] Mca = 2-Methylcinnamoyl
[0311] αMcn = α-Methylcinnamoyl
[0312] Mcoa = 7-Methoxycoumarin-4-acetyl [0313] MC2Y = N-Methyl-O-2,6-dichlorobenzyl-tyrosine
[0314] 34Mdc = 3 ,4-(methylenedioxy)cinnamoyl
[0315] Mpz = 1 -Methylpiperazine [0316] MTPA = α-Methoxy-α-trifluoromethylphenylacetyl
[0317] lNac = 3-(l-Naphthyl)acryloyl
[0318] NaI = β-Naphthylalanine
[0319] INaIa = Naphthylacetyl [0320] Nap = Naphthoyl
[0321] Napr = Naproxen: ό-Methoxy-α-methyl^-Naphthaleneacetyl
[0322] Nba = Norbornane-2-acetyl
[0323] Nif = Niflumic acid, 2-(3-[Trifluoromethyl]aniline)nicotinic acid
[0324] NMF = N-Methylphenylalanine [0325] OBPY = O-Benzyl-phosphotyrosine
[0326] OBrZY = (O-2-Bromo-Cbz)-tyrosine
[0327] OCIY = O-2,6-Dichlorobenzyl-355-diiodo-tyrosme
[0328] OC2Y = 0-2,6-dichlorobenzyl tyrosine
[0329] Ocp = 1-Octylpiperazine [0330] Octe = 2-Octenoyl
[0331] Otac = (-)-2-Oxo-4-thiazolidinecarboxyl
[0332] Pac = 4-Aminocinnamic acid
[0333] Pal = β-(3-Pyridyl)alanine
[0334] Pas = p-Aminosalicyloyl [0335] PBF = p-Bromophenylalanine
[0336] Pcin = 4-Phenylcinnamoyl
[0337] Pen = α-Phenylcinnamoyl
[0338] PCNF = p-Cyano-L-phenylalanine
[0339] Pcnl = β-Phenylcinnamyl [0340] Pen(Mbzl) = S-(4-methylbenzyl)Penicillamine
[0341] Pep = l-(2-Phenylethyl)piperazine [0342] PFF = p-Fluorophenylalanine
[0343] βPhc = β-Phenylcinnamoyl
[0344] 3Php = 3-Phosphonopropionyl
[0345] 5Phv = 5-Phenylvaleroyl
[0346] Pic = Picolinoyl
[0347] PIF = p-Iodophenylalanine
[0348] Pipe = Piperidine
[0349] Pipp = 4-Piperidinopiperidine
[0350] Pipz = Piperazine
[0351] Piva = Pivaloyl (Trimethylacetyl)
[0352] Pmpz = 1-2-Pyrimidylpiperazine
[0353] PNF = p-Nitro-phenylalanine
[0354] Ppp = l-(3-Phenylpropyl)piperazine
[0355] Ppr = Phenylpropiolyl
[0356] Ptmb = 4-(Trifluoromethyl)benzoyl
[0357] αPtpa = α-(Phenylthio)phenylacetyl
[0358] Pxa = Pyridoxamine [4-(ammomethyl)-5-hydroxy-6-methyl- 3- pyridinemethanol]
[0359] Pya = trans-3-(3-Pyridyl)acryloyl
[0360] Pypz = 1-2-Pyridylpiperazine
[0361] Pyre = Pyridine-3-carboxyl
[0362] Pyz = Pyrazinoyl
[0363] 3Qum = Quinoline-3 -methyl
[0364] 13cR = 13-cis-Retinoyl
[0365] Ret = trans-Retinoyl
[0366] Rio = Ricinoleyl
[0367] Saa = trans-Styrylacetyl [0368] Sab = 4-Sulphamidobenzoyl
[0369] cSdc = cis-Stilbene-4,4'-dicarboxylic
[0370] Sibu = S-(+)-Ibuprofen
[0371] CSsa = cis-Styrenesulphonylacetyl [0372] tβSts = trans-β-Styrenesulfonyl
[0373] Taa = l,2,4-Triazole-acetyl
[0374] Tchc = (1R,3R,4S,5R)-1 ,3,4,5-Tetrahydrocyclohexane-l -carboxyl
[0375] Tcpa = 2,4,5-Trichlorophenoxyacetyl
[0376] Tf2c = trans-3,5-bis(Trifluoromethyl)cinnamoyl [0377] 4Tfmb = 4-(Trifluoromethoxy)benzoyl
[0378] Thia = 3-(2-Thienyl)acryloyl
[0379] Th2n = 1 ,2,3 ,4-Tetrahydro-2-naphthoyl
[0380] Tic = Tetrahydroisoquinoline-3-carboxylic acid
[0381] Tmb = Trimethoxybenzoyl [0382] Tmbp = l-(2,4,6-Trimethylbenzyl)piperazine
[0383] 4Tmbs = 4-(Trifluoromethoxy)benzenesulfonyl
[0384] Tmbz = Trimethoxybenzyl
[0385] Tmcc = 2,2,3,3-Tetramethylcyclopropanecarboxyl
[0386] TmpC = Carboxy-TEMPO: 4-carboxy-2,2,6,6- tetramethylpiperidinyloxy
[0387] Tyr(Bzl) = O-Benzyl-tyrosine
[0388] ZPcn = (Z)-α-Phenylcinnamoyl; (cis)
[0389] Representative compounds include:
[0390] (-)-2-Oxo-4-tmazolidinecarboxyl-O~2,6-dichlorobenzyl tyrosine-4- amino-2,2,6,6-tetramethylpiperidine (Otac-OC2Y-Atmp);
[0391] 9-Fluorenylmethoxycarbonyl-O-2,6-dichlorobenzyl tyrosine-4-amino- 2,2,6,6-tetramethylρiperidine (Fmoc-OC2 Y- Atmp) ; [0392] 2,3,4,5,6-Pentafluorocinnamoyl-O-2,6-dichlorobenzyl tyrosine-2- (Aminomethyl)benzimidazole (F5c-OC2Y-Ambi); and
[0393] 5-benzoyl-2,3dihydro- 1 H-pyrrolizine- 1 -carboxyl-O-2,6- dichlorobenzyl tyrosine-4-amino-2,2,6,6-tetramethylpiperidine (Ktlc-OC2Y-Atmp).
[0394] These compounds, methods for their preparation and their biological activity are disclosed in US 7,427,496. The disclosed compounds are described as being bradykinin antagonists.
[0395] (C) Sulfonylquinoxalone derivatives of the formula (IV):
Figure imgf000042_0001
[0396] wherein
[0397] one of bonds characterized by " is a double bond and the other two are single bonds;
[0398] n is an integer from 0 to 4; [0399] p is zero or one; [0400] q is zero or one;
[0401] Y is selected from the group consisting of =0, =S, -OR8, -NHR8, =NR8, -SR8, and, when Y is -NHR8 or =NR8, R7 and R8, together with the nitrogen atoms to which they are attached, can form a heteroaryl, a substituted heteroaryl, an unsaturated heterocyclic, or a substituted unsaturated heterocyclic; provided that:
[0402] when Y is =0, =S, or =NR8, then the bonds characterized by ~~™*" between the 2-3 and 3-4 position are single covalent bonds and p is one; [0403] when Y is -OR8, -SR8, or -NHR8 and p is zero, then the bond
characterized by "*" between the 3-4 position is a double bond; or
[0404] when Y is -OR8, -SR8, or -NHR8 and p=l and R7 is other than
hydrogen, then the bond characterized by ~~*"~ between the 2-3 position is a double bond; [0405] W is selected from the group consisting of O, S, and N, wherein:
[0406] when W is O or S, then q is zero; and when W is N, then q is one;
[0407] R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0408] R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R1 and R2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; [0409] each R3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, acyl, acyloxy, halogen, nitro, cyano, hydroxy, carboxy, -C(O)OR10 wherein R10 is alkyl, substituted alkyl, aryl, or substituted aryl, and -C(O)NR11R12 wherein R11 and R12 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R11 and R12 together with the nitrogen atoms to which they are joined form a heteroaryl, substituted heteroaryl, heterocyclic a substituted heterocyclic group;
[0410] or two or more of R3 together with the carbon atoms to which they are joined form a fused ring cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic; [0411] R7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0412] or R7 together with at least one of R3 and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
[0413] R8 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy;
[0414] and pharmaceutically acceptable salts thereof.
[0415] In some embodiments, when W=N and Y=O, at least R1 and/or R2 is selected from the group consisting of: [0416] I) alkylene-C(=O)Ra, wherein alkylene is optionally substituted and Ra is selected from the group consisting of hydroxyl, -NRbRb, -ORb, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heterocyclyl, substituted heterocyclyl wherein each Rb is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0417] II) alkylene-Xa, wherein alkylene is optionally substituted and Xa is selected from the group consisting of -OH, cyano, and -NRbRb wherein each Rb is independently as defined above;
[0418] III) -NHRb, wherein Rb is as defined above; [0419] IV) -ORb, wherein Rb is as defined above;
[0420] V) alkylene-heta-C(=O)-CH(Rb)NRbRb, wherein alkylene is optionally substituted and heta is a nitrogen containing heterocycyl attached to the -C(O)- group through a ring nitrogen atom of the heta group and each Rb is as defined above; [0421] VI) alkylene-heta~C(=O)-hetb, wherein alkylene is optionally substituted and heta is as defined above and hetb is a heterocyclyl;
[0422] VII) alkylene-Rc-NRbC(=NRb)NRbRb, wherein alkylene is optionally substituted, each Rb is as defined above and R0 is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0423] VIII) alkylene-R°-NRb C(=O)-NRbRb, wherein alkylene is optionally substituted, each Rb is as defined above and Rc is as defined above;
[0424] IX) alkylene-Rc-alkylene-C(=O)Rb, wherein alkylene is optionally substituted and Rb and Rc are as defined above;
[0425] X) alkylene-R°-C(=O)-alkylene-(Xb)n', wherein alkylene is optionally substituted, Xb is selected from the group consisting of -OH, halo, cyano, and -NRbRb, n' is one except when Xb is halo then n' can be 1-3; and further wherein each Rb is independently as defined above and Rc is as defined above; [0426] XI) alkylene-Rc-C(=O)-Rd, wherein alkylene is optionally substituted and Rc is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic and Rd is selected from the group consisting of substituted alkyl, aryl, heteroaryl, heterocyclic and cycloalkyl; [0427] XII) alkylene-Rc-NRbC(=O)Re wherein alkylene is optionally substituted, Rb and Rc are as defined above, and where Re is substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted heteroaryl;
[0428] XIII) alkynylene-Rd where Rd is as defined above;
[0429] XIV) or where R1 and R2 are joined, together with the nitrogen atom bond thereto, to form a nitrogen containing substituted heterocyclyl with 1 to 2 substituents selected from substituted alkyl, heteroaryl, heterocyclyl;
[0430] XV) alkenylene-Rd where Rd is as defined above; and
[0431] XVI) alkylene-Rc-NRb-C(=NRb)Rb, wherein alkylene is optionally substituted, and each of Rb and R° are as defined above; and further that: [0432] A. when W is N, R1 is hydrogen, R2 is benzyl, R7 is methyl, n is zero, p is one, and Y is =0, then R is not 2,4,6-trimethylphenyl; [0433] B. when W is N5 R1 and R7 are hydrogen, R2 is 2-(ρyrid-4-yl)ethy-l- yl, n is zero, p is one, and Y is =0, then R is not l-methylpyrazol-4-yl;
[0434] C. when W is N, R1 and R7 are hydrogen, R2 is benzyl, n is zero, p is one, and Y is =0, then R is not 2,4-difluorophenyl; [0435] D. when W iS N5 R1, R2 and R7 are hydrogen, n is zero, p is one, and Y is =0, then R is not 2,4-difluorophenyl;
[0436] E. when W is N5 R1 is hydrogen, R2 and R7 are 3-chlorobenzyl, n is zero, p is one, and Y is =0, then R is not 4-chloro-2,5-dimethylphenyl;
[0437] F. when W is N, R1 and R7 are hydrogen, R2 is benzyl, n is zero, p is one, and Y is =0, then R is not phenyl;
[0438] G. when W is N, R1 and R7 are hydrogen, R2 is phenyl, n is zero, p is one, and Y is =0, then R is not quinolin-8-yl; and
[0439] H. when W is N, R1 and R7 are hydrogen, R2 is benzyl, n is zero, p is one, and Y is =0, then R is not thien-2-yl; and with the further proviso excluding the following known compounds :
[0440] I. when R1 and R7 are hydrogen, R2 is 2-methoxyphenyl, n is zero, p is one, and Y' is =0, then R is not 4-methylphenyl; and
[0441] J. when R and R are hydrogen, R is 2-ethoxyphenyl, n is zero, p is one, and Y' is =0, then R is not 4-methylphenyl. [0442] Representative compounds include:
[0443] 2-[2-(R,S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahy- droquinoxalin-2-yl]-N-[l -(R)-I -pyrrolidin-N-ylcarbonyl-2-phenyleth- lyl]acetamide;
[0444] 2-[2-(R,S)- 1 -(4-chloro-2,5-dimethylbenzenesulfonyl)-3 -oxo- 1 ,2,3 ,4- tetra- hydroquinoxalin-2-yl]-N-[l-(S)-carboxamide-2-(indol-3-yl)eth-l-yl]acetamide;
[0445] 2-[2-(R,S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3-,4- tetrahydroquinoxalin-2-yl]-N-[2-(4-hydroxyphenyl)- 1 -(S)-(t-butoxycarbonyl)eth- 1 - yl]acetamide;
[0446] 2-[2-(R,S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahy- droquinoxalm-2-yl]-N-[3-(dimethylamino)prop-l-yl]acetamide; [0447] 2-[2-(S or R)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo- l^jS^-tetrahydroquino- xalin^-ylJ-N-fl-C^-Cpyrrolidin-N-ylcarbony^-S- (guanadino)but- 1 -yl] acetamide;
[0448] 2-[2-(S,R)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetr- ahydroquinoxalin-2-yl]-methylenecarbonyl-[4-(2-aminoethyl)]piperidin- 1 -yl]- acetamide;
[0449] 2-[2-(R or S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo- l,2,3,4-tetrahydroquino- xalin-2-yl]-N-[l-(R)-(pyrrolidm-N-ylcarbonyl)-5-amino-n-pent- 1-yl] acetamide; [0450] 2-[2-(R or S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-
1 ,2,3,4-tetrahydroquino- xalin-2-yl]-N- { 1 -(R)-[pyrrolidin-N-ylcarbonyl]-2-[N-(phenyl)~ piperidin-4-y- 1)] eth- 1-yl} acetamide; and
[0451] 2-[2-(S or R)- 1 -(4-chloro-2,5-dimethylbenzenesulfonyl)-3~oxo- 1 ,2,3 ,4-tetrahydroquino- xalin-2-yl]-N- { 1 -(R)-[pyrrolidin-N-ylcarbonyl]-2-[N-(pyridin-4- yl)-piperid- in-4-yl]eth-l-yl}acetamide.
[0452] These compounds, methods for their preparation and their biological activity are disclosed in US 7,183,281. The disclosed compounds are described as being bradykinin antagonists.
[0453] (D) Sulfonamide derivatives of the formula (V):
Figure imgf000047_0001
[0454] wherein
[0455] R1 is phenyl, 1,2-dichlorophenyl, 3-methylphenyl, 3-bromophenyl, naphth-2-yl, indan-5-yl benzo[l,3]dioxol-5-yl or l,2,3,4-tetrahydronaphth-6-yl; [0456] R2 is hydrogen, halogen, unsubstituted Q-Qalkyl or substituted C1-
Qalkyl; [0457] R3 is hydrogen, halogen or d-C4alkyl; [0458] R4 is hydrogen or C1-C4alkyl; [0459] R5 is hydrogen or Cj-Qalkyl;
[0460] R6 is CH2OH; tetrazol-5-yl; 152,4-triazol-5-yl; 1 ,2,3-triazol-5-yl; C(O)OH, C(O)NH2; or ZNH(CH2)nCHR7R8, wherein Z is -C(O)- or -CH2-, n is zero, 1, 2,
3 or 4;
[0461] R7 is unsubstiruted or substituted Ci-C4alkyl, C(O)OH, C(O)OCr
C4alkyl;
[0462] R8 is hydrogen, unsubstituted or substituted C]-C4alkyl, unsubstituted or substituted Cs-C10aryl or heteroC5-C10aryl or C1-C4alkylC5-C10aryl or Ci-C4alkyl- heteroCs- Cioaryl, heteroCs-CiQaryl comprising one or more heteroatoms selected from N, O, and S; and m is 2, 3 or 4,
[0463] in free form or in form of a salt.
[0464] wherein Ci-C4alkyl, C5-C10aryl or heteroCs-Cioaryl, when substituted may be one or more substituents selected from OH, C(O)OH, halogen, Ci-C4alkyl, Ci-
C6alkoxy, benzyl, pyridinyl or pyrimidinyl
[0465] wherein these compounds may exist in free form or in salt form. [0466] Exemplary compounds include:
[0467] (S)-3-(2-{(4-bromo-2-chloro-benzenesulfonyl)-[2-(indan-5-yloxy)- ethyl]-am- ino}-acetylamino)-4-phenyl-butyric acid; and
[0468] 2,4-dichloro-N-[2-(indan-yloxy)-ethyl]-N-(lH-tetrazol-5- ylmethyl)benzenesulfonamide .
[0469] These compounds, methods for their preparation and their biological activity are disclosed in US 7,109,203. The disclosed compounds are described as exhibiting bradykinin antagonist activity.
[0470] (E) The compound: 8-[3-[N-[(E)-3-(6-acetamidoρyridin-3- yl)acryloylglycyl]-N-methylamino]-2,- 6-dichlorobenzyloxy]-2-methylquinoline having the formula (VI):
Figure imgf000049_0001
[0471] This compound (termed FRl 73657), methods for its preparation and its biological activity are disclosed in Japanese Patent Laid-Open No. 2780/1995 or in Abe et al. 1998. The compound is described as exhibiting bradykinin antagonist activity. [0472] FRl 73657 involves crystallographic polymorphism, of which crystal hydrates having high purity and good solid stability and easy to handle for formulation into medicines. Three types of crystals referred to as crystal type A, crystal type B and crystal type C, have been found (Japanese Patent Laid-Open No. 316677/1998). However, though having good solid stability and releasability, crystal type A is problematic in that it is often contaminated with crystal type C, making impossible production with stable quality. Crystal type B is the most stable and there is no problem in producing it, but is problematic in that its releasability is much inferior to that of crystal type A. Crystal type C is also problematic in that its solid stability is inferior to that of crystal type B and its releasability is inferior to that of crystal type A. [0473] US 7,094,899 describes a vitreous form of FRl 73657 that overcomes these problems. The vitreous form has high purity, good solid stability, good solubility and good releasability and capable of production with stable quality.
[0474] (F) Other non-peptide bradykinin antagonists in addition to FRl 73657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8- quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide) are disclosed in Abe et al. 1998 and also in Salvino et al. 1993., including:
[0475] FR167344 (N-[N-[3-[3-bromo-2-methylimdazo[l,2-a]pyridin-8- yl)oxymethyl]2,4-dichloro phenyl]N-methylaminocarbonylmethyl]-4- (dimethylaminocarbonyl)cinnarnylamide hydrochlroide) (also discussed below); [0476] WIN-64338 ([4-{(2[{bis(cyclohexylamino)methylene}amino]-3-[2- naphthyl]-l-oxopropyl)amino}phenyl]methyl)tributylphosphonium chloride monohydrochloride) (also discussed below);
[0477] WIN-62318 (also discussed below); [0478] CP-2458; [0479] and others.
[0480] (G) Sulfonylbenzodiazpinone acetamide derivatives and related compounds of formula (VII):
Figure imgf000050_0001
[0481] wherein
[0482] Q is selected from the group consisting of -SO2R and -CH2C(O)R;
[0483] W is selected from the group consisting of O, S, and N, wherein when W is O or S, then q is zero and when W is N, then q is one;
[0484] R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0485] R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R1 and R2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic;
[0486] each R3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, acylamino, aminoacyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, acyl, acyloxy, halogen, nitro, cyano, hydroxy, carboxy, and carboxyl esters;
[0487] or two or more of R3 together with the carbon atoms to which they are joined form a fused ring cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic; [0488] R7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy;
[0489] or R together with at least one of R and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
[0490] p is an integer of from 0 to 3;
[0491] or pharmaceutically acceptable salts, prodrugs, tautomers or isomers thereof. [0492] Exemplary compounds include the following compounds and pharmaceutically acceptable salts thereof:
[0493] 3-[3-(R,S)-(4-chloro-2,5-dimethylbenzenesulfonyl)-4-oxo-2,5- benzodiazepin-3-yl]-N-[2-(pyridin-4-yl)ethyl]acetamide;
[0494] 3-[3-(R,S)-(4-chloro-2,5-dimethylbenzenesulfonyl)-2-oxo-2,5- benzodiazepin-3-yl]-N-[2-(N-{pyrid-4-yl}piperidin-4-yl)eth-l-yl]acetamide;
[0495] 3-[3-(R)-(4-chloro-2,5-dimethylbenzenesulfonyl)-4-oxo-2,5- benzodiazepin-3-yl]-N-[2-(pyridin-4-yl)ethyl]acetamide;
[0496] 3-[3-(R,S)-(2,3-dichlorobenzenesulfonyl)-2-oxo-2,5-benzodiazepin-3- yl]-N-[2-(N-{pyridin-2-yl}piperidm-4-yl)eth-l-yl]acetamide; and [0497] 3-[3-(R,S)-(4-chloro-2,5-dimethylphenylcarbonylmethyl)-2-oxo-2,5- benzodia- zepin-3-yl]-N-[2-pyridin-4-yleth-l -yl]acetamide. [0498] These compounds, methods for their preparation and their biological activity are disclosed in US 7,074,783. The disclosed compounds are described as being bradykinin antagonists.
[0499] (H) 1,2,3,4-tetrahydrosulfonylquinoxalone acetamide derivatives and related compounds, represented by formula VIII:
Figure imgf000052_0001
[0500] wherein
[0501] one of bonds characterized by is a double bond and the other two are single bonds; [0502] n is an integer from 0 to 4;
[0503] p is zero or one; [0504] q is zero or one;
[0505] Y is selected from the group consisting of =O, =S, -OR8, -NHR8, =NR8, -SR8, and, when Y is -NHR8 or =NR8, R7 and R8, together with the nitrogen atoms to which they are attached, can form a heteroaryl, a substituted heteroaryl, an unsaturated heterocyclic, or a substituted unsaturated heterocyclic; provided that:
[0506] when Y is =O, =S, or =NR8, then the bonds characterized by between the 2-3 and 3-4 position are single covalent bonds and p is one;
[0507] when Y is -OR8, -SR8, or -NHR8 and p is zero, then the bond
characterized by '""""" " between the 3-4 position is a double bond; or
[0508] when Y is -OR8, -SR8, or -NHR8 and p=l and R7 is other than
hydrogen, then the bond characterized by ^ ~ " between the 2-3 position is a double bond;
[0509] W is selected from the group consisting of O, S, and N, wherein: [0510] when W is O or S, then q is zero; and when W is N, then q is one;
[0511] R is selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0512] R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R1 and R2 together with the nitrogen atom to which they are attached form a heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; [0513] each R3 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, acyl, acyloxy, halogen, nitro, cyano, hydroxy, carboxy, -C(O)OR10 wherein R10 is alkyl, substituted alkyl, aryl, or substituted aryl, and -C(O)NR11R12 wherein R11 and R12 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or R and R together with the nitrogen atoms to which they are joined form a heteroaryl, substituted heteroaryl, heterocyclic a substituted heterocyclic group;
[0514] or two or more of R3 together with the carbon atoms to which they are joined form a fused ring cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic;
[0515] R7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0516] or R7 together with at least one of R3 and the nitrogen and carbon atoms to which they are joined forms a fused ring heteroaryl, substituted heteroaryl, unsaturated heterocyclic or substituted unsaturated heterocyclic; [0517] R8 is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, acyl and acyloxy; [0518] and pharmaceutically acceptable salts thereof.
[0519] In some embodiments, when W=N and Y=O, neither R1 nor R2 is selected from the following:
[0520] I) alkylene-C(=O)Ra, wherein alkylene is optionally substituted and Ra is selected from the group consisting of hydroxyl, -NRbRb, -ORb, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heterocyclyl, substituted heterocyclyl wherein each Rb is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
[0521] II) alkylene-Xa, wherein alkylene is optionally substituted and Xa is selected from the group consisting of -OH, cyano, and -NRbRb wherein each Rb is independently as defined above;
[0522] III) -NHRb, wherein Rb is as defined above; [0523] IV) -ORb, wherein Rb is as defined above;
[0524] V) alkylene-heta-C(=O)-CH(Rb)NRbRb, wherein alkylene is optionally substituted and heta is a nitrogen containing heterocycyl attached to the -C(O)- group through a ring nitrogen atom of the heta group and each Rb is as defined above;
[0525] VI) alkylene-heta-C(=:O)-hetb, wherein alkylene is optionally substituted and heta is as defined above and hetb is a heterocyclyl;
[0526] VII) alkylene-Rc-NRbC(=NRb)NRbRb, wherein alkylene is optionally substituted, each Rb is as defined above and Rc is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic; [0527] VIII) alkylene-R°-NRb C(=O)-NRbRb, wherein alkylene is optionally substituted, each Rb is as defined above and Rc is as defined above; [0528] IX) alkylene-Rc-alkylene-C(=O)Rb, wherein alkylene is optionally substituted and Rb and Rc are as defined above;
[0529] X) alkylene-Rc-C(=O)-alkylene-(Xb)n', wherein alkylene is optionally substituted, Xb is selected from the group consisting of -OH, halo, cyano, and -NRbRb, n' is one except when Xb is halo then n1 can be 1-3; and further wherein each Rb is independently as defined above and Rc is as defined above;
[0530] XI) alkylene-Rc-C(=O)-Rd, wherein alkylene is optionally substituted and Rc is selected from the group consisting of aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic and Rd is selected from the group consisting of substituted alkyl, aryl, heteroaryl, heterocyclic and cycloalkyl;
[0531] XII) alkylene-R°-NRbC(=O)Re wherein alkylene is optionally substituted, Rb and Rc are as defined above, and where Re is substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, cycloalkyl, substituted heteroaryl; [0532] XIII) alkynylene-Rd where Rd is as defined above;
[0533] XIV) or where R1 and R2 are joined, together with the nitrogen atom bond thereto, to form a nitrogen containing substituted heterocyclyl with 1 to 2 substituents selected from substituted alkyl, heteroaryl, heterocyclyl;
[0534] XV) alkenylene-Rd where Rd is as defined above; and [0535] XVI) alkylene-Rc-NRb-C(=NRb)Rb, wherein alkylene is optionally substituted, and each of Rb and Rc are as defined above; and further that:
[0536] A. when W is N, R1 is hydrogen, R2 is benzyl, R7 is methyl, n is zero, p is one, and Y is =0, then R is not 2,4,6-trimethylphenyl;
[0537] B . when W is N, R1 and R7 are hydrogen, R2 is 2-(pyrid-4-yl)ethy- 1 - yl, n is zero, p is one, and Y is =0, then R is not l-methylpyrazol-4-yl;
[0538] C. when W is N, R1 and R7 are hydrogen, R2 is benzyl, n is zero, p is one, and Y is =0, then R is not 2,4-difluorophenyl;
[0539] D. when W is N, R1, R2 and R7 are hydrogen, n is zero, p is one, and Y is =0, then R is not 2,4-difluorophenyl; [0540] E. when W is N, R1 is hydrogen, R2 and R7 are 3-chlorobenzyl, n is zero, p is one, and Y is =0, then R is not 4-chloro-2,5-dimethylphenyl;
[0541] F. when W is N, R1 and R7 are hydrogen, R2 is benzyl, n is zero, p is one, and Y is =0, then R is not phenyl; [0542] G. when W is N, R1 and R7 are hydrogen, R2 is phenyl, n is zero, p is one, and Y is =0, then R is not quinolin-8-yl; and
[0543] H. when W is N, R1 and R7 are hydrogen, R2 is benzyl, n is zero, p is one, and Y is =0, then R is not thien-2-yl; and with the further proviso excluding the following known compounds: [0544] I. when R1 and R7 are hydrogen, R2 is 2-methoxyphenyl, n is zero, p is one, and Y' is =0, then R is not 4-methylphenyl; and
[0545] J. when R1 and R7 are hydrogen, R2 is 2-ethoxyphenyl, n is zero, p is one, and Y' is =0, then R is not 4-methylphenyl.
[0546] Representative compounds include: [0547] 2-[2-(R,S)-l-(2,4,6-trimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydro-6,-7-dichloroquinoxalin-2-yl]-N-benzylacetamide;
[0548] 2-[2-(R,S)-l-(2,3-dichlorobenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydroquinoxalin-2-yl]-N-[2-(pyrid-2-yl)eth- 1 -yl]acetamide;
[0549] 2-[2-(R,S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydroquinoxalin-2-yl] -N-[2-(N-methylpiperidin-4-yl)eth- 1 -yl]acetamide;
[0550] 2~[2-(R,S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydroquinoxalin-2-yl]-N-cyclohexyl-N-ethylacetamide;
[0551] 2-[2-(R,S)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,- 4- tetrahydroquinoxalin-2-yl]-methylenecarbonyl-[3-(R)-t-butoxycarboxamide-pyrrolidin- N-yl]acetamide;
[0552] 2-[2-(R)-l-(4-chloro-2,5-dimethylbenzenesulfonyl)-3-oxo-l,2,3,4- tetrahydroquinoxalin-2-y 1] -N- [2-(N-(3 -methylpyridin-2 -yl)piperidin-4-yl)eth- 1 - yl]acetamide; and
[0553] and pharmaceutically acceptable salts thereof. [0554] These compounds, methods for their preparation and their biological activity are disclosed in US 7,056,937. The disclosed compounds are described as being bradykinin antagonists.
[0555] (I) Sulfonyl amine derivatives of formula (IX):
Figure imgf000057_0001
[0556] wherein
[0557] R5A is -XA-R6A or -N(R7A)R8A, wherein XA is piperidinylere or piperazinylene, R6A is H,
Figure imgf000057_0002
C3-C4alkenyl, C3-C4alkinyl, Ci-C4(alkoxyalkyl), C1-C4(carboxyalkyl), a C5-C7heterocyclic group or phenylQ-Qalkyl; [0558] R7A is amino-C2-C4alkyl or mono- or di-(Ci-C5alkyl)amino-C2-
Csalkyl, and
[0559] R8A is H, C1-C4alkyl or has the meanings as given for R7A; [0560] X1 is a divalent group of formula IA':
— (CHj)11-" — X '(CHa)1n X — N
[0561] wherein n is zero or 1;
[0562] X3 is CH or N;
[0563] (a) X4 is a direct bond, R3A and R4A together are ethylene and m is 2; or
[0564] (b) X4 is a direct bond, R3A is H, CrC4alkyl, C3-C6cycloalkyl, C3- C6alkenyl, C3-C6alkinyl, C7-C10aralkyl or C6-C9heteroaralkyl, R4A is H and m is 1 or 2 or
3; or
[0565] (c) X4 is -CH(R12)-, R3A is H and R4A and R12 together are propylene and m is 1 , or ethylene and m is 2;
[0566] X2 is a divalent group of formula IA" :
Figure imgf000058_0001
[0567] wherein X3 is CH or N; and
[0568] R1 ' is d-C4alkyl, C3-C6cloalkyl or -NR1AR2A, wherein R1A and R2A independently are Q-Qalkyl or, together with the N-atom to which they are attached, represent a 5 to 7 membered heterocyclic ring; and
[0569] R9 and R10 independently are a phenyl or pyddine ring; and salts thereof.
[0570] Representative compounds include:
[0571] {2-(2,2-diphenyl-ethylamino)-5-[4-(4-isopropyl-piperazine-l- carbonyl)-pipe ridine-l-sulfonyl]-phenyl}-morpholin-4-yl-methanone; and
[0572] {2-(2,2-diphenyl-ethylamino)-5-[4-(4-methyl-piperazine-l-carbonyl)- piperid ine- 1 -sulfony 1] -phenyl } -morpholin-4-yl-methanone .
[0573] These compounds, methods for their preparation and their biological activity are disclosed in US 6,958,331. The disclosed compounds are described as exhibiting bradykinin antagonist activity.
[0574] (J) N-benzenesulfonyl L-proline compounds of formula (X):
Figure imgf000058_0002
[0575] or the pharmaceutically acceptable salts thereof [0576] wherein
[0577] X1 and X2 are independently halo or C^alkyl;
[0578] R1 and R2 are independently hydrogen or C1-4alkyl;
[0579] R3 and R4 are independently hydrogen or halo; and [0580] R5 is
[0581] (a) -Cs.pdiazacycloalkyl optionally substituted with C5- \ \ azabicycloalkyl;
[0582] (b) -C3-9azacycloalkyl-NH-(C5-π, azabicycloalkyl optionally substituted with Ci-4alkyl); [0583] (c) -NH-C1-3alkyl-C(O)-C5-πdiazabicycloalkyl;
[0584] (d) -NH-C1-3alkyl-C(O)-NH-C5-1 iazabicycloalkyl, the C5- πazabicycloalkyl being optionally substituted with C1-4alkyl;
[0585] (e) -C^azacycloalkyl optionally substituted with C^azacycloalkyl; or [0586] (f) -NH-Ci-5alkyl-NH-C(O)-C4-9cycloalkyl-NH2. [0587] Exemplary compounds include:
[0588] 8-[[3-[[(2S)-2-[[4-[(3S)-l-azabicyclo[2.2. 2]oct-3-yl]-l- piperazinyl]carbonyl]pyrrolidinyl]sulfonyl]-2. 6-dichlorobenzyl]oxy]-2.4- dimethylquinoline; and
[0589] (2S)-N-[2-(3.8-Diazabicyclo[3.2. l]oct-3-yl)-2-oxoethyl]-l-[[2. 4- dichloro-3-[[(2. 4-dimethyl-8quinolunyl)oxy]methyl]phenyl]sulfonyl]-
2pyrrolidinexcarboxamide.
[0590] These compounds, methods for their preparation and their biological activity are disclosed in US 6,734,306. The disclosed compounds are described as exhibiting bradykinin antagonist activity. [0591] (K) 1,4-dihydropyridine compounds of formula (XI):
Figure imgf000060_0001
[0592] wherein
[0593] A is independently halo;
[0594] Y is -(CH2)"1-, -C(O)- or -S(O)-;
[0595] R and R are independently C1-4alkyl;
[0596] R3 is selected from
[0597] (a) C7-14azacyclo-, azabicylo- or azatricyclo-alkyl, in which the nitrogen atom optionally has a substituent selected from C1-4alkyl, benzyl optionally substituted with one or two substituents independently selected from halo and halosubstituted- C1-4alkyl, C1-4alkoxycarbonyl optionally substituted with one or two halogen atoms and C1-6acyl;
[0598] ' (b) hydrogen, d-7alkyl optionally substituted with one or two substituents independently selected from hydroxy, amino, C1-4alkylamino, di-Q. 4alkylamino, pyridyl, carbamoyl, pyrrolidinylcarbonyl, C1-4alkylaminocarbonyl, piperidinylcarbonyl, morpholinocarbonyl, 2-oxopyrrolidinyl,
Figure imgf000060_0002
cyano, d-βacylamino, 1,1-dioxoisothiazolinyl, 2-oxo-l,3-oxazolidinyl, morpholino, C1- 4alkyl-2-oxopyrrolidinyl, piperidinyl and oxo-piperidinyl;
[0599] (c) piperidinyl optionally substituted on the nitrogen atom with Cj. 4alkyl or C1-4alkoxycarbonyl; [0600] (d) C5-14cycloalkyl, bicycloalkyl or tricycloalkyl, the C5.14cycloalkyl, bicycloalkyl or tricycloalkyl being optionally substituted with one or two substituents independently selected from oxo, hydroxy, amino, C1-4alkylamino, di- Cj- 4alkoxybenzamido, morpholino and oxopyrrolidinyl; [0601] (e) C-Mobicycloalkenyl, benzo-C5-7cycloalkyl or heterocyclic optionally substituted with one or two subtituents independently selected from C1-4alkyl and halo; and
[0602] (f) Ci-6alkyl-C3-7cycloalkyl, the cycloalkyl moiety being optionally substituted with one, two or three substituents independently selected from cyano, amino-
Ci-4alkyl-, Ci-4alkylamino-C1-4alkyl-, Cj-6 acylamino-C1-4alkyl-, CMalkyl-sulfonylamino- C1-4alkyl, amino, oxopyrrolidinyl, C4-7cycloalkylamino-C1-4alkyl,
Figure imgf000061_0001
4alkyl-, hydroxyl, carbamoyl,
Figure imgf000061_0002
(C1-4alkyl)amino-Ci. 4alkyl, di- Ci_4alkylamino, pyrrolidinyl-C1-4alkyl, oxopyrrolidinyl-C1-4alkyl and di-d. 4alkylamino-C1-4alkyl;
[0603] R4 is phenyl substituted at the 2-position with substituent selected from
[0604] (a) C1-4alkyl substituted with one, two or three substituents independently selected from amino, amino-C2-4alkoxy, phenylthio, C1-4alkyl-phenylthio, di-Ci.4alkylamino-C2-4alkoxy, Ci-4alkylamino-C2-4alkoxy,
Figure imgf000061_0003
di-Q.
4alkylamino, hydroxy, C^alkoxy, piperazinyl, oxopyrrolidinyl, pyrrolidinyl, C2- 4alkylenedioxy, Ci-6acyloxy, oxo, morpholino, Ci-4alkylaminocarbonyl-Ci-4acylamino, C ] -4alkoxycarbonyl-C \ _6acylamino, C \ -4alkoxycarbonylpiperazinyl, C \ -6acylpiperazinyl, C1-4alkylthio, heterocyclic-C1-4alkoxy, (di- C1-4alkylamino)(C3-7cycloalkyl)C2-4alkoxy, (C1-4alkylamino)(C3-7cycloalkyl)C2-4alkoxy and (amino)(C3-7cycloalkyl)C2-4alkoxy;
[0605] (b) C5-7alkyl optionally substituted with one, two or three substituents independently selected from amino, amino-C2-4alkoxy, phenylthio, Ci-4alkyl-phenylthio, di-C1-4alkylamino-C2.4alkoxy, C1-4alkylamino-C2-4alkoxy, C^alkylamino, di- C1- 4alkylamino, hydroxy, C1-4alkoxy, piperazinyl, oxopyrrolidinyl, pyrrolidinyl, C2- 4alkylenedioxy, C1-6acyloxy, oxo, morpholino, C1-4alkylaminocarbonyl-Ci-6acylamino,
C1-4alkoxycarbonyl-C1-6acylamino, C1-4alkoxycarbonylpiperazinyl, C1-6acylpiperazinyl, C1-4alkylthio, heterocyclic- C1-4alkoxy, (di-C1-4alkylamino)(C3-7cycloalkyl)C2-4alkoxy, (C] -4alkylamino)(C3-7cycloalkyl)C2-4alkoxy and (amino)(C3-7cycloalkyl)C2-4alkoxy;
[0606] (c) Ci-4alkoxy or C1-4alkylthio, the Ci^alkoxy or Ci-4alkylthio being substituted with one, two or three substituents independently selected from amino, amino-
C2-4alkoxy, phenylthio, Ci-4alkyl-phenylthio, di-C1-4alkylamino-C2-4alkoxy, C1- 4alkylamino-C2-4 alkoxy, Cmalkylamino,
Figure imgf000061_0004
hydroxy, Ci-4alkoxy, piperazinyl, oxopyrrolidinyl, pyrrolidinyl, C2-4alkylenedioxy, C1-6acyloxy, oxo, morpholino, CMalkylaminocarbonyl- Cj-6-acylamino, CMalkoxycarbonyl-Ci-sacylamino, C1-4alkoxycarbonylpiperazinyl, Ci-6acylpiρerazinyl, C1-4alkylthio, heterocyclic-Ci- 4alkoxy, (di-C1-4alkylamino)( C3-7cycloalkyl) C2-4alkoxy, (C1-4alkylamino)(C3. 7cycloalkyl)C2-4alkoxy and (amino)(C3-7cycloalkyl)C2-4alkoxy;
[0607] (d) C5_7alkoxy or C5-7alkylthio, the C5.7a.koxy or C5-7alkylthio being optionally substituted with one, two or three substitiuents independently selected from amino, amino-C2-4alkoxy, phenylthio, C1-4alkyl-phenylthio, di-C1-4alkylamino-C2- 4alkoxy, C1-4alkylamino-C2-4alkoxy, C1-4alkylamino,
Figure imgf000062_0001
hydroxy, Ci- 4alkoxy, piperazinyl, oxopyrrolidinyl, pyrrolidinyl, C2-4alkylenedioxy, C1-6acyloxy, oxo, morpholino , C i ^alkylaminocarbonyl-C \ acylamino , C \ -4alkoxycarbonyl-C \ -6acylamino, CMalkoxycarbonylpiperazinyl, Ci-6acylpiperazinyl, C1-4alkylthio, heterocyclic-Cμ 4alkoxy, (di- C1-4alkylamino)(C3-7cycloalkyl)C2-4alkoxy, (C1-4alkylamino)(C3- 7cycloalkyl)C2-4alkoxy and (amino)(C3-7cycloalkyl)C2-4alkoxy; [0608] (e) amino, C1-4alkylamino, C1-6acylamino, aminoacetylamino, C1-
4alkylsulfonylamino, halosubstituted-CMalkylsulfonylamino, halosubstituted-Ci. 4alkylamino or Ci^alkoxycarbonylaminoacetylamino;
[0609] (f) piperazinylcarbonyl, morpholinocarbonyl, nitro, cyano, hydroxy, C1.4alkylsulfonyl, C1-4alkylsulfonyl or di-C1-4alkylaminosulphenyl; [0610] (g) Ci-4alkylthio, C1-6alkylthio, amino-C1-6acylthio, C1.
4alkylsulfonylthio, halosubstituted-C1-4alkylthio or C1-4alkoxyaminoacetylthio;
[0611] (h) C2-7alkenyl or C2-7alkynyl, the C2-7alkenyl or C2-7alkynyl being optionally substituted with one, two or three substituents independently selected from amino, Q-salkylamino, di-C1-4alkylamino, hydroxy, C1-4alkoxy, piperazinyl, oxopyrrolidinyl, pyrrolidinyl, C2-4alkylenedioxy, halo, C1-6acyloxy, oxo, morpholino, C1.
4alkylaminocarbonyl- Ci-6acylamino, C1-4alkoxycarbonyl-Ci-6acylamino, Ci- 4alkoxycarbonylpiperazinyl, C)-4acylpiperazinyl and C1-4alkylthio; and
[0612] (i) C7-i4azacycloalkyl optionally substituted with one or two substituents independently selected from oxo and C1-4alkyl; [0613] R5 is hydrogen or C1-4alkyl;
[0614] m is 0, 1 or 2; and [0615] n is 0, 1, 2, 3, 4 or 5;
[0616] and pharmaceutically acceptable salts and prodrugs thereof.
[0617] Representative compounds include:
[0618] dimethyl-2-(2-{2-[(2-aminoethoxy)methyl]phenyl}ethyl)-4-(2,6- dichlorophenyl )-6-{2-[4-(8-methyl-8-azabicyclo[3.2. l]oct-3-yl)-l-piperazinyl]-2- oxoethyl}-l,4-dihydro-3,5-pyridinedicarboxylate;
[0619] dimethyl-4-(2,6-dichlorophenyl)-2-[2-(2~hydroxyphenyl)ethyl]-6-{2- [4-(8-met hyl-8-azabicyclo[3.2. l]oct-3-yl)-l-piperazinyl]-2-oxoethyl}-l,4-dihydro-3,5- pyridinedicarboxylate; and [0620] dimethyl-4-(2,6-dichlorophenyl)-2-12-(2-{[2-
(ethylamino)ethoxy]methyl } ρhenyl)ethyl] -6- { 2- [4-(8-methyl- 8 -azabicyclo [3.2. 1 ] oct-3 - yl)-l-piperazinyl]-2-oxoethyl}-l,4-dihydro-3,5-pyridinedicarboxylate.
[0621] These compounds, methods for their preparation and their biological activity are disclosed in US 6,653,313. Processes of preparing these compounds is also disclosed in US 6,649,767. The disclosed compounds are described as exhibiting bradykinin receptor-binding activity and therefore are proposed for use as bradykinin antagonists.
[0622] (L) Compounds of the formula (XII) :
Figure imgf000063_0001
[0623] wherein
[0624] R1 is a hydrogen atom or a C1-C4 alkyl group; [0625] R2 and R3, which can be the same or different, are a C1-C4 alkyl group, or R2 and R3, together with the carbon atom which they are linked to, form a cyclic aliphatic group having 3 to 7 carbon atoms or a heterocyclic aliphatic group having 3 to 7 atoms, one or two of which are selected from the group N, O, S and the others being C atoms;
[0626] R4 and R5, which can be the same or different, are a hydrogen atom or a C1-C4 alkyl group;
[0627] X is selected from the group consisting of halogen, OR1, SRi, CN, Ci- C4 alkyl; [0628] B has at least one amino group with basic characteristics or a tetraalkylammonium group and can be selected from the group consisting of:
[0629] NR6(CH2)nNHCOY, NR6(CH2)nN(R6)-Y, NR6(CH2)nN(Y)25 NR6Y, N(Y)2, N(Y)(CH2)PY, and from the residues:
NR15R13
Figure imgf000064_0001
Figure imgf000064_0002
[0630] R6 is a hydrogen atom, Ci-C6 alkyl; [0631] n=l-12; [0632] Y is selected from: hydrogen, (CH2)pYl5 (CH2)PNR6Y1, (CH2)pN(Yi)2, NR5R6, --NReCCH^qY! or from the following residues:
Figure imgf000065_0001
[0633] T is selected from the group of -NR7R8, -NR14Ri sRi9, -OR6;
[0634] R7 and Rg, which can be the same or different, are a hydrogen atom, a C1-C4 alkyl group, a cyclohexyl group, or NR7R8 together are a group selected from: i) guanidine optionally substituted with 1 or 2 C1-C4 alkyl or cyclohexyl groups, ii) a 5-7 membered nitrogen heterocycle optionally containing another heteroatom selected from O3 N5 S;
[0635] Y1 is selected from the group consisting OfNR7R8, NR14R18R19 or from the following residues:
Figure imgf000065_0002
Figure imgf000066_0001
[0636] Z is selected from the group consisting of H, C1-C6 alkyl, OR6, SR6, CF3, OCOR65 COR10, NHCOR6, SO2R6, SOR6, CO2R6, N(Re)2, CI, Br, NO2, NH2, CN, F, imidazole, phenyl, amidine, guanidine, guanidyl-methyl; [0637] R9 is selected from the group consisting of hydrogen, ~(CH2)q-L, wherein L is selected from the group of —OH, -NR5R6, -NR14R1SRi9, amidine optionally substituted with 1 or 2 C1-C4 alkyl groups, guanidine optionally substituted with 1 or 2 Cj-C4 alkyl groups;
[0638] R10 is selected from the group consisting of OR6, NR6Ri2; Rn is selected from the group consisting of hydrogen, ~(CH2)q-L, — (CH2)p~NR4~(CH2)q-L;
[0639] R12 is a hydrogen atom, Ci-C6 alkyl, COR6,
[0640] R13 is selected from the group consisting of H, C1-C6 alkyl, ~ (CH2)pW(CH2)qY,, Y, -COY, -CH2-Y;
[0641] Rj5 is selected from the group consisting of hydrogen or straight or branched C ] -C4 alkyl groups;
[0642] the -NR16Ri? group is a 5-7 membered nitrogen aliphatic heterocycle optionally containing another heteroatom selected from O, S, N;
[0643] the -NRi4R18Ri9 group is a quaternary ammonium group in which: Ri4 is selected from the group consisting of straight or branched Ci-C4 alkyl groups, Ri8 and Ri9, which can be the same or different, are a straight or branched Ci-C4 alkyl group, or -NR]8R19 is a 5-7 membered nitrogen heterocycle optionally containing another heteroatom selected from O, N, S; W=CH2, O, S, NR4, N(R4^; [0644] p=l -6, q=l-6.
[0645] Representative compounds include:
[0646] N-[2-[4-(2-(S)-amino-6-dimethylamino-hexanoyl)-piperazin-l-yl]- 1 , 1 -dimeth- yl-2-oxo-ethyl] -2,4-dichloro-3 -(2-methyl -quinolin-8-yloxy-methyl)- benzensulfonamide trifluoroacetate;
[0647] N- {2-[4-[N-(4-piperidyl)glycyl]-ρiρerazin- 1 ~yl]- 1 , 1 -dimethyl -2-oxo- ethyl} -2,4-dichloro-3-(2-methyl-8-quinolinoxymethyl) -benzene-sulfonamide tris trifluoroacetate;
[0648] N-[I- [4-(2-(R)- Amino-6~guanidino-hexanoyl)-piperazine- 1 -carbonyl]- cyclope- ntyl]-2,4-dichloro-3-(2,4-dimethyl -quinolin-8-yloxymethyl)- benzenesulfonamide tris trifluoroacetate;
[0649] (R)-N- [4-(3 -(S)- Amino-6-guanidino-hexanoyl)-piperazine- 1 - carbonyl]- 1 -met- hyl-propyl]-2,4-dichloro-3 -(2,4-dimethyl -quinolin-8-yloxy-methyl)- benzenesulfonamide tris trifluoroacetate; [0650] 2,4-Dichloro-3-(2,4-dimethyl-quinolin-8-yloxymethyl)-N-{ 1 -[4-(2- piperidin- -4-yl-ethyl)-piperazine-l-carbonyl]-cyclopentyl}-benzene-sulfonamide;
[0651] {3-[(4-{ 1 -[2,4-Dichloro-3-(2,4-dimethyl-quinolin-8-yloxymethyl)- benzenesulfonylamino]-cyclopentanecarbonyl}-piperazine-l-carboximi-doyl)- amino]propyl}-trimethyl-ammonium iris trifluoroacetate; and [0652] N-( 1 - { 4- [(5 - Amino-pentylamino)-methy 1] -piperidine- 1 -carbonyl } - cyclopentyl- )-2,4-dichloro-3-(2,4-dimethyl -quinolin-8-yloxymethyl)- benzenesulfonamide.
[0653] These compounds, methods for their preparation and their biological activity are disclosed in US 7,491,825. The disclosed compounds are described as being specific antagonists of bradykinin B2 receptor.
[0654] (M) A polymorphic form of the compounds 2-(R)-(l-(R)-(3,5- bis(trifluoromethyl)-phenyl)ethoxy)-3 -(S)-(4-fluoro)ρhenyl-4-(3 -(5 -oxo- 1 H,4H- 1,2,4- triazolo)methylmorpholine having the structure:
Figure imgf000068_0001
[0655] These compounds, and the process for its preparation is disclosed in US 6,583,142. The compound is described as a tachykinin receptor antagonist.
[0656] (N) Compounds containing four fragments designated L, E, R and Q, which are joined in linear fashion as follows:
[0657] L - E - R- Q [0658] wherein
[0659] fragment L represents a hydrophobic group such as a 5-membered unsaturated ring, 6-membered unsaturated ring, alkene, saturated 3-7 membered ring;
[0660] fragment E represents carbonyl derivative that in the presence of L, R and Q becomes an activated serine trap;
[0661] fragment R represents a hydrophobic, aromatic ring system such as a 6-membered ring, fused 5-6 membered ring, or fused 6—6 membered ring; and
[0662] fragment Q represents a basic group.
[0663] Also contemplated are pharmaceutically acceptable acid addition salts of this amidine compound. [0664] These compounds and their method of preparation are disclosed in US 6,653,340. The compounds are described as being potent inhibitors of kallikrein and high molecular weight kininogen.
3.2 Suitable peptides, polypeptides, proteins, and peptidomimetics include: [0665] (A) Peptides having the following sequence:
[0666] (i) r-p-p-g-X-X-Pro-f-Arg
[0667] wherein
[0668] r is any amino acid, preferably Arg;
[0669] p is any amino acid, preferably Pro; [0670] g is amino acid, preferably GIy;
[0671] X is any amino acid; and
[0672] f is any amino acid, preferably Phe;
[0673] and wherein the N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in
US 4,801,613;
[0674] or (ii) Xj -X2-X3-X4-X5-PrO-X6-ATg [0675] wherein
[0676] X1 is a modified amino acid, preferably modified proline or modified serine, more preferably proline, and may be N-terminal;
[0677] X2 is any amino acid, preferably proline or phenylalanine;
[0678] X3 is isoleucine, leucine, or valine, preferably isoleucine or valine;
[0679] X4 is any amino acid, preferably valine, isoleucine, or leucine;
[0680] X5 is any amino acid, preferably serine, threonine, or proline; and [0681] X6 is any amino acid, preferably phenylalanine, leucine or arginine;
[0682] and wherein the N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613;
[0683] or (iii) X1-(X2)H-X3-X4-X5-X6-PrO-X7-ATg [0684] wherein [0685] X1 is N-terminal and is a modified amino acid, preferably modified proline or modified serine, more preferably modified proline;
[0686] (X2)n is a contiguous sequence of any amino acids of length n, where n can be up to about 50, up to about 30, up to about 10 amino acids or 0 amino acids;
[0687] X3 is any amino acid, preferably proline or phenylalanine; [0688] X4 is isoleucine, leucine, or valine, preferably isoleucine or valine;
[0689] X5 is any amino acid, preferably valine, isoleucine, or leucine;
[0690] X6 is any amino acid, preferably serine, threonine, or proline; and
[0691] X7 is any amino acid, preferably phenylalanine, leucine, or arginine;
[0692] and wherein the N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613;
[0693] or (iv) Pro-Xi-X2-X3-Pro-Pro-Phe-Arg [0694] wherein [0695] Xi is any amino acid;
[0696] X2 is a hydrophobic aliphatic residue, preferably alanine, valine, isoleucine, or leucine, but not glycine; and
[0697] X3 is a hydrophobic aliphatic residue;
[0698] and wherein the N-terminal amino acid residue is modified and the proline at the position third from the C-terminal end of this sequence is substituted with an aromatic amino acid of the D-configuration or another amino acid as described in US 4,801,613.
[0699] Representative sequences include:
[0700] Pro-Phe-Ile-Val-Pro-Pro-Phe-Arg; and [0701] Pro-Phe-Val-Val-Pro-Pro-Phe-Arg.
[0702] Further exemplary modifications that can be used to create kinin antagonists based on the above peptides (i)-(iv) are described in Kotovych et a (1988).
[0703] The term "modified" as used with reference to the above peptides (i)- (iv), means any modification at the N-terminal amino acid residue that blocks sequencing by the standard Edman degradation method. Such modifications include, but are not limited to acetylation, amidation, β-methylthiolation, biotin, carbamylation, citrullination, c-mannosylation, deamidation, n-acyl diglyceride cysteine (tripalmitate), dimethylation, fad, farnesylation, formylation, geranyl-geranyl, gamma-carboxyglutamic acid, o-glcnac, glucosylation (glycation), hydroxylation lipoyl, methylation, myristoylation, palmitoylation, phosphorylation, pyridoxal phosphate, phosphopantetheine, pyrrolidone carboxylic acid, sulfation and trimethylation. Modifications may be on the -NH2 group or any other group of the terminal amino acid residue.
[0704] These peptides, methods for their preparation and their biological activity are disclosed in US 7,176,280. The disclosed peptides are described as being kinin antagonists.
[0705] (B) Peptide compounds having the sequence:
[0706] (i) Xaa1-Arg-Pro-Xaa2-Gly-Phe-Ser-Xaa3-Xaa4 [0707] wherein [0708] Xaai is selected from D-arginyl, acetyl lysyl, D-lysyl, sarcosyl, acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon-aminohexanoyl lysyl;
[0709] Xaa2 is selected from prolyl and hydroxyprolyl;\
[0710] Xaa3 is selected from D-l,2,3,4,-tetrahydroisoquinoline-3-carboxyl and "D-β-(2-naphthyl) alanyl;
[0711] Xaa4 is selected from leucine and isoleucine
[0712] or
[0713] (ii) Xaa5-Arg-Pro-Xaa6-Gly-Phe-Ser-Xaa7-Xaa8
[0714] wherein [0715] Xaa5 is selected from acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon- aminohexanoyl lysyl;
[0716] Xaa6 is selected from prolyl and hydroxyprolyl; [0717] Xaa7 is propyl;
[0718] Xaag is selected from leucine and isoleucine
[0719] These peptides analogs, methods for their preparation and their biological activity are disclosed in US 7,084,244. The disclosed peptides are described as having bradykinin antagonist activity. [0720] (C) The peptide termed HOE 140 (or Icatibant) having the following sequence:
[0721] D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg
[0722] This peptide, methods for its preparation and its biological activity are discussed in Boa et al. 1991 and many other publications. The review paper Moreau et al. 2005, for example, notes that more than 700 research papers and several clinical studies have used this peptide. The peptide is described as being a highly potent, specific and long-acting bradykinin B2-receptor antagonist.
[0723] (D) The peptide termed B4146 having the following sequence: [0724] D-Arg-Hyp-Pro-Gly-Thi-Ser-D-Pro-Thi-Arg [0725] The biological activity of this peptide is disclosed in Boa et al. 1991.
The peptide is described as a bradykinin antagonist.
[0726] (E) The peptide termed NPC 17761 having the following sequence:
[0727] D-Arg-Arg-Pro-Hyp-Gly-Phe-Ser-D-Hype (7>α«5-thiophenyl)-Oic- Arg [0728] The biological activity of this peptide are disclosed in Otterbein et al.
1993. The peptide is described as being a potent bradykinin antagonist.
[0729] (F) Backbone cyclized peptide analogs of the formula (XIII):
Figure imgf000072_0001
[0730] wherein
[0731] M is an amide bond, x and z are each independently an integer of 1 to 10, and K is H or an acyl group.
[0732] Representative peptide analogs include: [0733] Ada-(D)Arg-Arg-cyclo(Nα(l-(6-aminohexylene)Gly-Hyp-Phe-D-
Asp)-D-Phe-Phe-Arg-OH;
[0734] H-D-Arg-Arg-cyclo(Nα(l-(4-propanoyl))Gly-Hyp-Phe-Nα(- 3-amido- propylene)Gly)-Ser-D-Phe-Phe-Arq-OH; and
[0735] H-D-Arg-Arg-cyclo(Nα(4-propanoyl)Gly-Hyp-Phe-Nα(3-amido- propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH,
[0736] wherein Ada is the abbreviated designation for the protecting groups adamantane acetyl.
[0737] These peptides analogs, methods for their preparation and their biological activity are disclosed in US 7,084,244. The disclosed peptides are described as having bradykinin antagonist activity. Also described in US 7,084,244 are two assay methods for determining if a compound displays bradykinin antagonist activity.
[0738] (G) des-Arg9 bradykinin analogues of the formula: [0739] X - A^ B1 - C2- D3- E4- F5- G6- H7- J8- Z [0740] wherein [0741] X is an aromatic, aliphatic, aromatic-aliphatic, alicyclic, heterocyclic or urethane-type acylating group, or at least one amino acid;
[0742] A0, B1, C2, D3, and E4 are basic or neutral aromatic, aliphatic, heterocyclic, or alicyclic amino acids or A0 is absent;
[0743] G6 is an aromatic, aliphatic, heterocyclic, or alicyclic amino acid; [0744] F5, H7 and J8 are aromatic, aliphatic, aliphatic heterocyclic, or alicyclic amino acids, provided that at least one of F5, H7 and J8 is selected from cyclopentane-, cyclohexane- or indane-substituted glycine; and
[0745] Z is COOH. [0746] Representative analogues include: [0747] DArg-Cys-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg;
[0748] DArg-Lys-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg;
[0749] DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-Tic-Cpg;
[0750] DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Tic-Cpg; [0751] DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DTic-Cpg;
[0752] DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Cpg;
[0753] DArg-Arg-Pro-Hyp-Gly-Cpg-Ser-DCpg-Cpg;
[0754] DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic;
[0755] Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Leu; [0756] DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Leu;
[0757] Gun- DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic;
[0758] DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DIgl-Oic;
[0759] Gun-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DIgl-Oic;
[0760] DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DTic-Cpg; [0761] Lys-Arg-Pro-Hyp-Gly-Igl-Ser-DTic-Cpg;
[0762] Lys-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic; and
[0763] Lys- Lys-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic.
[0764] These compounds, methods for their synthesis and their biological activity are disclosed in US 5,834,431. The disclosed compounds are described as exhibiting selective Bl receptor antagonism.
[0765] (H) Bradykinin sequence-related analogues having the sequence of mammalian bradykinin:
[0766] Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
[0767] but wherein [0768] the L-proline residue at position 7 is substituted with a material having the D-configuration (*) and the formula:
Figure imgf000075_0001
[0769] wherein
[0770] R is selected from the group consisting of Ci-C6alkyl, substituted C1- C6alkyl, C2-Cgalkenyl, Ca-Cscycloalkyl, C3-Cscycloalkyl substituted Ci-C6alkyl, an aryl group, a substituted aryl group, an arylalkyl group, and a group of the formula
R1NHC(O)- where R1 is d-C6alkyl or aryl, and where X is either SOn or oxygen, and n=0, 1, or 2;
[0771] and pharmaceutically acceptable salts thereof.
[0772] These peptides analogs, methods for their preparation and their biological activity are disclosed in US 6,770,741. The disclosed peptides are described as being bradykinin antagonists.
[0773] (I) Compounds having the sequence: [0774] Xj-Arg-Pro-Xa-Gly-Phe-Ser-Xa-^ [0775] wherein [0776] X1 is selected from D-arginyl, acetyl lysyl, D-lysyl, sarcosyl, acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcosyl tyrosyl epsilon-aminohexanoyl lysyl, and sarcosyl tyrosyl (3,5-iodine) epsilon-aminohexanoyl lysyl;
[0777] X2 is selected from prolyl and hydroxyprolyl;
[0778] X3 is selected from D-l,2,3,4-tetrahydroisoquinolme-3-carboxyl and "D-beta-(2-naphthyl) alanyl; and
[0779] X4 is selected from leucine and isoleucine
[0780] or having the sequence:
[0781] Xi-Arg-Pro-X2-Gly-Phe-Ser-X3-X4
[0782] wherein [0783] X1 is selected from acetyl tyrosyl epsilon-aminohexanoyl lysyl, sarcodyl tyrosyl epsilon-aminohexanoyl lysyl and sarcosyl tyrosyl (3,5-iodine) epsilon- aminohexanoyl lysyl;
[0784] X2 is selected from prolyl and hydroxyprolyl; [0785] X3 is prolyl; and
[0786] X4 is selected from leucine and isoleucine.
[0787] These compounds, methods for their preparation and their biological activity are disclosed in US 7,041,785. The disclosed peptides are described as being B1- bradykinin receptor antagonists. [0788] (J) Other peptides described as having a KKS antagonist effect including the peptides termed:
[0789] NPC 17751 (described in Mak et al. 1991);
[0790] NPC 349 (described in Wirth et al. 1995 and Mak et al. 1991);
[0791] NPC- 1776 (described in Cheronis et al. 1993); [0792] WIN 64338 (described in Sawutz et al. 1995 and elsewhere herein);
[0793] des-Arg9-[Leu81 bradykinin (described in Pruneau et al. 1996); and
[0794] Sar4-[D-Phe8]-des-Arg9-bradykinin (described in Gouin et al 1996).
[0795] S 1629 ((Hyp3,Thi5,D-TiC7,OiC8)desArg9BK) developed by Hoechst;
[0796] NPC 18565 (D-Arg(Hyp3,D-Hyp7(transpropyl),Oic8)desArg9BK) developed by Scios Inc;
[0797] NPC 18828 (D-Arg(Hyp3,D-Hyp7(trans thiophenyl),Oic8)desArg9BK) also developed by Scios Inc;
[0798] CP- 1027 (or Bradycor or deltibant) as described in Marmarou, et al. 1999; [0799] NPC-17731 (D-Arg0-[Hyp3,D-HypE(trans-propyl)7, Oic8]BK) as described in Ikemura et al. 1998;
[0800] Other suitable KKS antagonists are also disclosed in these references. [0801] (K) Bisuccinimide-alkane peptide dimer bradykinin antagonists disclosed in Cheronis et al. 1994, Cheronis et al. 1992 and Srivastava et al. 1996, including:
[0802] CP 0127 (also described in Whalley et al. 1992);
[0803] CP 0364;
[0804] and others.
[0805] ((LL)) PPssuueeddooppeptide bradykinin antagonists disclosed in Mavunkel et al.
1996, including:
[0806] NPC 18521;
[0807] NPC 18688;
[0808] and others.
[0809] ((MM)) SSuubbssttrraalte analogues of tissue kallikrein with a length of less than 20 amino acids containing a sequence which corresponds to positions 388 to 390 of human kininogen, and which has a 4-amino phenylalanine (Phe(4NH2)), or a similar modified amino acid, corresponding to position 389.
[0810] In some embodiments the sequence comprises naturally occurring, non-naturally occurring, or chemically synthesized amino acids, amino acid derivatives, modified amino acids, or combinations thereof.
[0811] Such compounds and their method of synthesis are disclosed in US 5,464,820. The compounds are described as inhibitors of tissue kallikrein.
[0812] (N) Monoclonal antibodies produced by cell lines ATCC #HB-8963 and ATCC #HB-8964 as disclosed in US 4,908,431 and US 7,332,161. One of these antibodies specifically recognises the heavy chain of HMWK and LMWK, while the other antibody recognises the light chain of HMWK. [0813] (O) Antibodies that bind to a component of the KKS including the anti-bradykinin antibody α-BK termed AS348. A method for the production of this antibody and its biological activity is disclosed in both US 6,221,845 and US 6,242,210.
[0814] (P) Monospecific antibodies immunologically reactive with a bradykinin B1 receptor as disclosed in US 6,307,027. 3.3 Suitable other KKS antagonists include those isolated from natural sources including:
[0815] Extracts from at least one plant of the Rosaceae family as disclosed in US 6,586,020. These extracts may be prepared from any plant material (including whole plant, or from a plant portion such as the leaves, stems, flowers, petals, roots or from undifferentiated cells) derived from at least one plant of the Rosaceae family using the extraction techniques described in US 6,586,020, including aqueous or alcoholic extracts, or extracts obtained from an organic solvent; and
[0816] Martinelline and L-755807 as described in Altamura et al. (1999) and discussed below.
3.4 Suitable KKS antagonists:
[0817] Many suitable KKS antagonists are known in the art in addition to those specifically described above, including those described in Hirayama, Y. et al. (2002); Hall, J. M. (1997); Campbell et al. (2001); Regoli, et al. (1998); Altamura et al. (1999); Moreau et al. (2005); and Blakeney et al. (2007). [0818] The review paper Regoli et al. (1998), for example, discusses kinin B1 and B2 receptor antagonists including:
[0819] [Leu8]des-Arg9-bradykinin (obtained by replacing the C-terminal Phe residue by Leu;
[0820] Lys-[Leu8]des-Arg9-bradykinin; [0821] R 715 (AcLys-[D-β NaI7, Ile8]desArg9-bradykinin);
[0822] B 9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin) (discussed herein);
[0823] B 9958 (Lys-Lys-[Hyp3,Cpg5,D-Tic7,Cpg8]-desArg9bradykinin);
[0824] HOE 140 (also discussed above) and desArg9 derivatives of HOE 140;
[0825] S 0765 (desArg10-HOE 140 or D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]- desArg9bradykinin);
[0826] [D-Phe7]-bradykinin;
[0827] D-Arg-[Hyρ3, D-Phe7]desArg9bradykinin;
[0828] R 493 (D-Arg-[Hyp3, D-Phe7, Leu8]bradykinin); [0829] NPC 567 (D-Arg-[Hyp3,D-Phe7]-bradykinin) and its homologue (D- Arg-[Hyp3,Thi5l8,D-Phe7]-bradykinin);
[0830] D-Arg-[Hyp3,D-Phe7,Leu8]-bradykinin);
[0831] WIN 64338 (also discussed in Altamura et al (1999) and elsewhere herein), the first nonpeptide competitive kinin B2 antagonist; and
[0832] FR 173657 (also discussed above with reference to Japanese Patent Laid-OpenNo. 2780/1995 or in Abe et al. 1998).
[0833] A later review paper, Altamura et al. (1999) discusses nonpeptide antagonists for kinin receptors including: [0834] WIN 64338 (discussed elsewhere herein) and quinoline and imidazo[l,2,α]pyridine derivatives thereof including FR 167344 (discussed above), FR 165649, FR 173657 (discussed above) and FR 184280, and further derivatives thereof including LF 16.0335, LF 16.0687 and GR 213548X;
[0835] CP-0597 (D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-NChg-Arg) and CP-2055 and CP-2522 developed therefrom;
[0836] Martinelline, a pyrroloquinoline alkaloid isolated from the roots of the tropical plant Martinella iquitosensis; and
[0837] L-755807, a metabolite with a complex chemical structure isolated from a culture of a Microsphaeropsis sp.. [0838] A subsequent review paper, Moreau et al. (2005), discusses KKS antagonists, including:
[0839] kinin B1 -receptor ligands including many described in Regoli et al. (1998) and also including:
[0840] Ac-LyS-[MeAIa6, Leu8]des-Arg9-bradykinin; [0841] B 9858 (Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]-desArg9bradykinin);
[0842] R-954 (Ac-Orn-[Oic2, α-MePhe5, D-βNal7, Ile8]des-Arg9bradykinin);
[0843] PS020990;
[0844] compound 12 (benzodiazepine-based structure); [0845] benzo-sulfonyalmide compounds including compound 12, compound l l and SSR240612;
[0846] kinin B2-receptor ligands including many described in Regoli et al. (1998) and Altamura et al. (1999) and also including: [0847] [Thi5'8, D-Phe7]-bradykinin;
[0848] WIN 62318 (discussed above);
[0849] FR 167344;
[0850] compound 38;
[0851] substituted 1,4-dihydropyridines; and [0852] bradyzide;
[0853] and commercially available KKS antagonists including aprotinin and aprotinin-like drugs including ulinastatina nd nafamostat;
[0854] pasteurised C 1 -INH purified from human blood plasma (pdC 1 INH; Berinert® P); and [0855] DX88, Danazol, and Stanozolol.
[0856] A later review paper, Blakeney et al. (2007), discusses many KKS antagonists, including some discussed herein, including those discussed in the earlier review papers discussed herein, and NPC-18884 (as described in Heitsch, 2002).
4. Prophylactic and therapeutic uses [0857] In accordance with the present invention, it is proposed that agents that comprise KKS antagonists are useful for the prevention or treatment of aneurysms.
[0858] Such agents can be administered to an individual either by themselves, or in pharmaceutical compositions where they are mixed with a suitable pharmaceutically acceptable carrier or diluent. [0859] The agents of the present invention may be conjugated with biological targeting agents which enable their activity to be restricted to particular cell types. Such biological-targeting agents include substances which are immuno-interactive with cell- specific surface antigens. [0860] The agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the drugs of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.
[0861] The agents can be formulated readily using pharmaceutically acceptable carriers or diluents well known in the art into dosages suitable for oral administration. Such carriers or diluents enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers or diluents may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
[0862] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. The dose of agent administered to an individual should be sufficient to effect a beneficial response in the individual over time such as reducing the size of an aneurysm, preventing an increase in the size of an aneurysm, reducing the number of small aneurysms, and/or preventing or inhibiting the occurrence of aneurysms. The quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the drug to be administered, the physician may evaluate the characteristics of the patient, their response to the drug and the safety profile of the drug. In any event, those of skill in the art may readily determine suitable dosages of the agents.
[0863] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or other components which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
[0864] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more drugs as described above with the carrier or diluent which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
[0865] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses. [0866] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
[0867] Dosage forms of the drugs of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release may be effected by using ■ other polymer matrices, liposomes or microspheres.
[0868] The drugs of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
[0869] For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a test agent, which achieves a half-maximal inhibition or enhancement in activity of a component in the KKS). Such information can be used to more accurately determine useful doses in humans.
[0870] Toxicity and therapeutic efficacy of such drugs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).
[0871] Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain KKS antagonistic effects. Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02- 3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5-1200 mg/m2/day, commonly from 0.5-150 mg/m2/day, typically from 5-100 mg/m2/day.
[0872] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tissue, which is preferably subcutaneous or omental tissue, often in a depot or sustained release formulation.
[0873] Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the tissue.
[0874] In cases of local administration or selective uptake, the effective local concentration of the agent may not be related to plasma concentration.
[0875] The present invention also contemplates a method of gene therapy of a mammal. Such a method utilises a gene therapy construct which includes an isolated polynucleotide comprising a nucleotide sequence encoding a component of the kallikrein- kinin system, or a biologically active fragment thereof, wherein the polynucleotide is ligated into a gene therapy vector which provides one or more regulatory sequences that direct expression of the polynucleotide in the mammal. Typically, gene therapy vectors are derived from viral DNA sequences such as adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses. Suitable gene therapy vectors currently available to the skilled person may be found, for example, in Robbins et al, 1998.
[0876] Administration of the gene therapy construct to the mammal, suitably a human, may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor. An example of the latter approach would be stem-cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the gene therapy vector comprising a component of the KKS. The stem-cells are cultured for a period and then transferred to the mammal being treated. [0877] Delivery of the gene therapy construct to cells or tissues of the mammal or the compatible donor may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g., lipofectin or lipofectamine), electroporation, calcium phosphate or DEAE-dextran-mediated transfection, for example. A discussion of suitable delivery methods may be found in Chapter 9 of Ausubel et al, (1994-1998, supra).
[0878] In an alternate embodiment, a polynucleotide encoding a KKS antagonist may be used as a therapeutic or prophylactic composition in the form of a "naked DNA" composition as is known in the art. For example, an expression vector comprising the polynucleotide operably linked to a regulatory polynucleotide (e.g. a promoter, transcriptional terminator, enhancer etc) may be introduced into an animal, preferably a mammal, where it causes production of a KKS antagonist in vivo.
[0879] The step of introducing the expression vector into a target cell or tissue will differ depending on the intended use and species, and can involve one or more of non- viral and viral vectors, cationic liposomes, retroviruses, and adenoviruses such as, for example, described in Mulligan, R.C., (1993).
5. Methods of detecting, determining prognosis of monitoring aneurysms
[0880] The present invention also provides methods for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual. [0881] One suitable method comprises detecting in the individual the presence of an aberrant gene encoding a component of the KKS or detecting in the individual aberrant expression of a gene encoding a component of the KKS. In some embodiments, the aberrant gene is selected from an aberrant KNGl, KLKl, KLKBl, BDKRBl, BDKRB2, SERPINGl, A2M, SERPINCl, or SERPINA4 gene.
[0882] Another suitable method comprises determining in the individual a level or functional activity of a component in the KKS, which is different than a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component. In specific embodiments, the level or functional activity of a component in the KKS, which is different than a reference or control level indicates the presence or risk of development of an aneurysm or a negative prognosis. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed ex vivo, including in a biological sample obtained from the individual.
[0883] In some embodiments, the method comprises determining an increase or elevation in the level of functional activity of the component (e.g. HMWK, LMWK, prekallikrein, prokallikrein, plasma kallikrein, tissue kallikrein, bradykinin, kallidin, des- Arg9-bradykinin, Lys-des-Arg9-bradykinin, the kinin B 1 receptor, the kinin B2 receptor) relative to a reference or control level (e.g. no aneurysm, level measured in the same individual at an earlier time) or functional activity of the component, . In other embodiments, the method comprises determining a decrease in the level or functional activity of the component (e.g. Cl-INH, α2-macroglobulin, antithrombin III, kallistatin) relative to a normal (e.g. no aneurysm, level measured in the same individual at an earlier time) reference or control level or functional activity of the component.
[0884] In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA. In some embodiments, individual is one that is suspected of having an aneurysm or being at risk of having an aneurysm.
[0885] In some embodiments, determining the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual is achieved by using the methods described above in combination with medical imaging, including ultrasound.
Identification of agents that antagonise the KKS
[0886] The invention also features methods of screening for an agent that antagonises the KKS, including agents that modulate the expression of a gene or the level and/or functional activity of an expression product of that gene, wherein the gene is selected from a gene encoding a component of the KKS, a gene whose expression product modulates directly or indirectly the expression of a gene encoding a component of the KKS, and a gene whose expression product modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
[0887] In some embodiments, the methods comprise: (1) contacting a preparation with a test agent, wherein the preparation contains (i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or (ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates a nucleotide sequence that encodes at least a biologically active fragment of a polypeptide component of the KKS, or a variant or derivative thereof, which is operably linked to a reporter gene; or (iii) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide according to (i) and (2) detecting a change in the level and/or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference or control level and/or functional activity in the absence of the test agent, which indicates that the agent modulates the KKS.
[0888] In some other embodiments, the methods comprise contacting a sample of cells expressing a kinin receptor with a kinin and a test agent. A detected decrease in level of binding between the kinin receptor and the kinin relative to a reference or control level in the absence of the test agent, indicates that the agent is a KKS antagonist.
[0889] Candidate test agents encompass numerous chemical classes. Candidate organic molecules comprise functional groups necessary for structural interaction with KKS components including proteins, particularly by way of hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate test agent often comprises cyclical carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate test agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrrolidines, derivatives, structural analogues or combinations thereof. [0890] In this regard, small molecules are particularly preferred as candidate test agents because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene
(e.g. by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules.
[0891] Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc to produce structural analogues.
[0892] Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.
[0893] Screening candidate test agents according to the invention can be achieved by any suitable method. For example, the method may include contacting a cell expressing a polynucleotide corresponding to gene encoding a component of the KKS, with an agent suspected of having the modulatory activity and screening for the modulation of the level or functional activity of a protein encoded by the polynucleotide, or the modulation of the level of a transcript encoded by the polynucleotide, or the modulation of the activity or expression of a downstream cellular target of the protein or of the transcript (hereafter referred to as target molecules). Detecting such modulation can be achieved utilizing techniques including, but not restricted to, ELISA, cell-based ELISA, inhibition ELISA, Western blots, immunoprecipitation, slot or dot blot assays, immunostaining, RIA, scintillation proximity assays, fluorescent immunoassays using antigen-binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, Ouchterlony double diffusion analysis, immunoassays employing an avidin-biotin or a streptavidin-biotin detection system, and nucleic acid detection assays including reverse transcriptase polymerase chain reaction (RT-PCR). [0894] It will be understood that a polynucleotide from which a target molecule of interest is regulated or expressed may be naturally occurring in the cell which is the subject of testing or it may have been introduced into the host cell for the purpose of testing. Further, the naturally-occurring or introduced polynucleotide may be constitutively expressed — thereby providing a model useful in screening for agents which down-regulate expression of an encoded product of the sequence wherein the down regulation can be at the nucleic acid or expression product level - or may require activation - thereby providing a model useful in screening for agents that up-regulate expression of an encoded product of the sequence. Further, to the extent that a polynucleotide is introduced into a cell, that polynucleotide may comprise the entire coding sequence which codes for a target protein or it may comprise a portion of that coding sequence (e.g., a binding domain) or a portion that regulates expression of a product encoded by the polynucleotide (e.g., a promoter). For example, the promoter that is naturally associated with the polynucleotide may be introduced into the cell that is the subject of testing. In this regard, where only the promoter is utilized, detecting modulation of the promoter activity can be achieved, for example, by operably linking the promoter to a suitable reporter polynucleotide including, but not restricted to, green fluorescent protein (GFP), luciferase, β-galactosidase and catecholamine acetyl transferase (CAT). Modulation of expression may be determined by measuring the activity associated with the reporter polynucleotide.
[0895] In another example, the subject of detection could be a downstream regulatory target of the target molecule, rather than the target molecule itself or the reporter molecule operably linked to a promoter of a gene encoding a product the expression of which is regulated by the target protein. [0896] These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. These methods will also facilitate the detection of agents which bind either the polynucleotide encoding the target molecule or which modulate the expression of an upstream molecule, which subsequently modulates the expression of the polynucleotide encoding the target molecule. Accordingly, these methods provide a mechanism of detecting agents that either directly or indirectly modulate the expression or activity of a target molecule according to the invention. [0897] In a series of embodiments, the present invention provides assays for identifying small molecules or other compounds (i.e. modulatory agents) which are capable of inducing or inhibiting the level and/or functional activity of target molecules according to the invention. The assays may be performed in vitro using non-transformed cells, immortalised cell lines, or recombinant cell lines. In addition, the assays may detect the presence of increased or decreased expression of genes or production of proteins on the basis of increased or decreased mRNA expression (using, for example, the nucleic acid probes disclosed herein), increased or decreased levels of protein products (using, for example, the antigen binding molecules disclosed herein), or increased or decreased levels of expression of a reporter gene (e.g., GFP5 β-galactosidase or luciferase) operably linked to a target molecule-related gene regulatory region in a recombinant construct.
[0898] Thus, for example, one may culture cells which produce a particular target molecule and add to the culture medium one or more test compounds. After allowing a sufficient period of time (e.g., 6-72 hours) for the compound to induce or inhibit the level or functional activity of the target molecule, any change in the level from an established baseline may be detected using any of the techniques described above and well known in the art. Using suitable nucleic acid probes or antigen-binding molecules, detection of changes in the level and or functional activity of a target molecule, and thus identification of the compound as agonist or antagonist of the target molecule, requires only routine experimentation.
[0899] In some embodiments, recombinant assays are employed in which a reporter gene encoding, for example, GFP, β-galactosidase or luciferase is operably linked to the 5' regulatory regions of a target molecule related gene. Such regulatory regions may be easily isolated and cloned by one of ordinary skill in the art. The reporter gene and regulatory regions are joined in-frame (or in each of the three possible reading frames) so that transcription and translation of the reporter gene may proceed under the control of the regulatory elements of the target molecule related gene. The recombinant construct may then be introduced into any appropriate cell type although mammalian cells are preferred, and human cells are most preferred. The transformed cells may be grown in culture and, after establishing the baseline level of expression of the reporter gene, test compounds may be added to the medium. The ease of detection of the expression of the reporter gene provides for a rapid, high throughput assay for the identification of agonists or antagonists of the target molecules of the invention. [0900] Compounds identified by this method will have potential utility in modifying the expression of target molecule related genes in vivo. These compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects. In addition, as described above with respect to small molecules having target polypeptide binding activity, these molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modelling, and other routine procedures employed in rational drug design.
[0901] In other embodiments, methods of identifying agents that antagonise the KKS are provided in which a purified preparation of a component of the KKS is incubated in the presence and absence of a candidate agent under conditions in which the component is active, and the level of activity is measured by a suitable assay. For example, a KKS antagonist can be identified by measuring the ability of a candidate agent to decrease kinin - kinin receptor binding. An agent tests positive if it inhibits this activity.
[0902] In still other embodiments, methods of identifying agents that antagonise the KKS are provided in which a purified preparation of a component of the KKS is incubated in the presence and absence of a candidate agent under conditions in which kinin is able to bind a kinin receptor, and the level of binding is measured by a suitable assay. An agent tests positive if it inhibits this activity.
[0903] In still other embodiments, random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to a target molecule or to a functional domain thereof. Identification of molecules that are able to bind to a target molecule may be accomplished by screening a peptide library with a recombinant soluble target molecule.
The target molecule may be purified, recombinantly expressed or synthesised by any suitable technique. Such molecules may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et ah, (1989, supra) in particular Sections 16 and 17; Ausubel et ah, ("Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998), in particular Chapters 10 and 16; and
Coligan et at, ("Current Protocols in Immunology", (John Wiley & Sons, Inc, 1995- 1997), in particular Chapters 1, 5 and 6. Alternatively, a target polypeptide according to the invention may be synthesized using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard {supra) and in Roberge et al. (1995).
[0904] To identify and isolate the peptide/solid phase support that interacts and forms a complex with a target molecule, suitably a target polypeptide, it may be necessary to label or "tag" the target polypeptide. The target polypeptide may be conjugated to any suitable reporter molecule, including enzymes such as alkaline phosphatase and horseradish peroxidase and fluorescent reporter molecules such as fluorescein isothiocynate (FITC), phycoerythrin (PE) and rhodamine. Conjugation of any given reporter molecule, with target polypeptide, may be performed using techniques that are routine in the art. Alternatively, target polypeptide expression vectors may be engineered to express a chimeric target polypeptide containing an epitope for which a commercially available antigen-binding molecule exists. The epitope specific antigen- binding molecule may be tagged using methods well known in the art including labelling with enzymes, fluorescent dyes or coloured or magnetic beads. [0905] For example, the "tagged" target polypeptide conjugate is incubated with the random peptide library for 30 minutes to one hour at 22° C to allow complex formation between target polypeptide and peptide species within the library. The library is then washed to remove any unbound target polypeptide. If the target polypeptide has been conjugated to alkaline phosphatase or horseradish peroxidase the whole library is poured into a petri dish containing a substrate for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3 ',4,4"- diamnobenzidine (DAB), respectively. After incubating for several minutes, the peptide/solid phase-target polypeptide complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescently tagged target polypeptide has been used, complexes may be isolated by fluorescent activated sorting. If a chimeric target polypeptide having a heterologous epitope has been used, detection of the peptide/target polypeptide complex may be accomplished by using a labeled epitope specific antigen-binding molecule. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing. 7. Methods of producing agents comprising a KKS antagonist
[0906] The present invention also provides methods of producing an agent comprising a KKS antagonist for preventing or treating an aneurysm in an individual. These methods generally comprise: testing a test agent as broadly described above; and synthesising the agent on the basis that it tests positive for antagonising the KKS.
[0907] Suitably, the method further comprises derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the aneurysm. In specific embodiments, the aneurysm is an aortic aneurysm. An illustrative example of this type of aneurysm includes AAA.
[0908] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
EXAMPLES
EXAMPLE 1
[0909] Decreased circulating kallikrein inhibitor in human AAA
[0910] The activity of human kallikrein 1 (hKl) is inhibited by kallistatin (protease inhibitor 4, PI4) both in vitro and in vivo (Diamandis et al, 2000). In this example the circulating concentrations of PI4 in serum from a group of subjects was compared.
Materials and Methods
[0911] Serum was obtained from archived serum from The Health In Men Study (HIMS) previously described (Norman et αl, 2009) and came from subjects from Western Australia, Australia. Ethical approval from the centre providing the samples and informed consent from all subjects was obtained.
[0912] Some characteristics of these subjects are shown in Table 6. [0913] Table 6: Characteristics of subjects in serum kallistatin study (n = 1006)
Characteristic AAA (n = 309) No AAA (n = 697)
Age (years) 72 (69-75) 71 (68-74)
Aortic diameter (mm) 33 (31-33) 22 (20-23)
Males (%) 100 100
Ever smokers (%) 85 63
Diabetes (%) 10 7
Hypertension (%) 51 38
Dyslipidaemia (%) 49 36
Coronary heart disease (%) 44 20
Shown are number (%) for nominal variables and median (inter-quartile range) for continuous variables.
[0914] Serum samples were assayed by ELISA for kallistatin (Duoset, R&D Systems) according to the manufacturer's instructions. Inter-assay coefficient of variation for the assay was <10%.
[0915] Logistic regression analysis was used to examine the relationship between serum kallistatin and AAA using the method disclosed in Golledge et al, 2007.
Results and Discussion [0916] Median (inter-quartile range) PI4 concentrations were 14.86 (11.85-
18.41) μg/mL in the AAA subjects and 17.2 (13.7-21.1) μg/mL in the control (no-AAA) subjects, pO.OOl . Serum PI4 was negatively correlated with aortic diameter (r=-0.21, p<0.001) and negatively associated with AAA after adjusting for other risk factors as shown in Table 7 (below). [0917] Table 7: Kallistatin is negatively associated with AAA when adjusted for other risk factors.
Independent variable Odds ratio (95% CI) P value
Hypertension 1.353 (0.983-1.864) 0.064
Diabetes 1.178 (0.674-2.059) 0.566
Dyslipidaemia 1.162 (0.829-1.63) 0.384
Coronary heart disease 2.569 (1.809-3.649) O.001
Age* 1.305 (1.081-1.575) 0.006
Kallistatin* 0.747 (0.648-0.862) O.001
Smoking 3.557 (2.390-5.292) O.001
* Continuous variables stated per standard deviation increase in units
[0918] This example shows that serum concentrations of the kallikrein inhibitor kallistatin are reduced in patients with AAA.
EXAMPLE 2 [0919] Increased bradykinin receptor expression in human AAA
[0920] Human kallikrein 1 (hKl) generates the kinins bradykinin (BK) and lysyl-bradykinin (Lys-BK) from high and low molecular weight kininogen respectively. These kinins, along with their carboxypeptidase metabolites (des-Arg9-BK and Lys-des-
Arg9-BK) produce important physiological effects by stimulating Bl or B2 receptors (Leeb-Lundberg et al, 2005).
Materials and Methods
[0921] To further investigate the relevance of the kallikrein-kinin system in human AAA, biopsies taken from the aortas of seven patients undergoing AAA repair were examined.
[0922] Sample biopsies from both the diseased aneurysm body (site of maximum aortic dilation) and the relatively normal tissue at the neck of the aneurysm were obtained. [0923] Proteins from these sample biopsies were extracted for semi- quantitative Western blot analysis and immunohistochemistry (IHC) performed for the bradykinin receptors Bl and B2.
[0924] Protein extraction and Western blotting [0925] Proteins (30 μg) were extracted and quantitated from human aortic biopsies as previously described in Moran et al, 2005.
[0926] Proteins were separated using SDS gel electrophoresis (12% SDS; Gradipore) and Western analysis carried out using primary antibodies to Bl receptor (BDKRBl3 Abeam) and to B2 receptor (BDKRB2, Abeam) and secondary antibody: goat anti-rabbit IgG (Abeam) that was HRP-labelled.
[0927] Bands corresponding to Bl and B2 receptors were visualised using enhanced chemiluminescence (ECL Advance™; Amersham Biosciences) and identified on a ChemiDoc™ imaging system (Bio-Rad Laboratories).
[0928] Quantification of protein was performed by comparison of band densities and expressed as mean and standard error of relative band density units
(RDU)/μg protein.
[0929] Proteins were visualised and quantified using the Biorad Chemidoc XRS system.
Results and Discussion [0930] It was found that expression of Bl and B2 were increased in the aneurysm body compared to the aneurysm neck.
[0931] Regarding Bl , the concentration was elevated in the aneurysm body compared to the neck but the difference was not statistically significant.
[0932] Regarding B2, the concentration was elevated in the aneurysm body compared to the neck and this difference was statistically significant (the B2 receptor concentration was 2.7-fold (1.2-3.4, p=0.025) higher in the aneurysm body than in the aneurysm neck). Figure 1 shows the bands of the B2 results (top line) in both the neck (left hand side) and the body (right hand side) of the aneurysm. The bottom line shows the bands of the control, α-actin. [0933] It was also found that the concentration of both Bl and B2 did not differ significantly in the aneurysm neck. However, the concentration of Bl in the aneurysm body was found to be significantly less than the concentration of B2 in the aneurysm body. Figure 2 demonstrates these results. In Figure 2, the left result for each of Bl and B2 shows the results from the sample from the aneurysm neck, and the right result for each of Bl and B2 shows the results from the sample from the aneurysm body.
[0934] Thus this example shows that expression of the kinin B2 receptor is upregulated within biopsies of human AAA.
EXAMPLE 3 [0935] Kinin receptor modulation influences aortic aneurysm formation and rupture in a mouse model
[0936] The human studies discussed in Examples 1 and 2 showed that components of the kallikrein-kinin system are associated with AAA. In order to study if kinins modulate aortic dilatation and inflammation, the effect of the bradykinin receptor B2 agonist B9972 and the B 1 /B2 antagonist B9430 (Gera et al., 1996) were investigated in a mouse model of AAA in which angiotensin II (Angll) is infused subcutaneously in the hyperlipidaemic apolipoprotein deficient mouse.
Materials and Methods
[0937] Experimental aneurysm model I [0938] Mice were purchased from the Animal Resources Centre
(Canningvale, Western Australia, Australia) and all procedures were approved by the James Cook University Animal Ethics Committee.
[0939] Male 13 week old ApoE"'" mice were anaesthetised by intraperitoneal injection of ketamine (150mg/kg) and xylazine (10mg/kg) and a dorsal incision made at the upper back. Alzet® Osmotic miniature pumps (Model 2004) were inserted and the incision sutured.
[0940] The mice were divided into four groups. In three of the four groups the pumps were pre-loaded with human recombinant Angll (Sigma). In the last group, the pumps were pre-loaded with saline. The pumps were loaded so as to deliver 1.44mg/kg/day of Angll or saline over the course of the 28 day experiment. [0941] In addition, the three groups of mice receiving AngII received intraperitoneal injections (2mg/kg/dose) the day prior to pump insertion and every other day throughout the experimental period with a vehicle control, the bradykinin receptor B2 agonist B9972 or the B1/B2 antagonist B9430. [0942] At the conclusion of the experimental period, mice with euthanased by
CO2 asphyxiation, and the aorta perfused using sterile, RNase-free PBS. Aortas were dissected from the aortic arch to the iliac bifurcation, taking care to remove adherent adipose tissue and then photographed intact for aortic morphometry. Photographs of an aorta taken from a normal mouse (A - left hand side) and one from a mouse with an aneurysm (B - right hand side) are shown in Figure 3.
[0943] The outcome of this experiment was assessed by recording aortic rupture (resulting in premature death prior to day 28) and measuring maximum diameter throughout the aorta at death (at rupture or experiment termination at day 28).
Results and Discussion [0944] The mean survival time of AngII treated mice was 23 ± 1.5 days with aortic rupture and death occurring in 33% (9/27) mice.
[0945] Mice receiving the B2 agonist B9972 and AngII showed a very high incidence of aortic rupture (68%, 17/25 mice) and mean survival time decreased to 14.5 ± 2 days (p<0.01 compared to AngII alone). [0946] In mice receiving AngII plus the Bl /B2 antagonist B9430, there was significantly less aortic ruptures (8%, 2/24 mice) and the mean survival of this group was 27 days which was significantly different to AngII group (p<0.05).
[0947] All saline treated mice survived the 28 day experimental period as expected. [0948] The aortic ruptures were either acute (day 4- 11 ) or late (day 18-22) as shown in Figure 4. Aortic rupture following AngII/B2 agonist administration was predominantly acute (day 4-7). The rupture site, observed at necroscopy, was restricted to either the aortic arch or suprarenal region. There were more ruptures in the aortic arch in the AngII/B2 agonist group of mice compared to other groups, where the rupture site was predominantly in the suprarenal aorta. [0949] In addition to increased aortic rupture frequency in the AngII/B2 agonist group, significantly increased arch and infrarenal aortic diameters in this group compared to AngII alone were observed, as was an overall increase in the average maximum aortic diameter when taking into account all four aorta regions (arch, thoracic, suprarenal and infrarenal). In contrast, AngII/Bl/B2 antagonist-treated mice had significantly decreased arch, thoracic, suprarenal and average maximum aortic diameters, compared to AngII alone infused mice. These results are shown in Table 8.
[0950] Table 8: Maximum aortic diameter in relation to AngII and kinin administration.
Diameter (mm)
Intervention Arch Thoracic Suprarenal Infrarenal Average maximum Λ
AngII 1.85±0.45 1.49±0. ,42 2.10±0.74 0.79±0.19 1.56±0.30
AngII 1.5Id=O. ,32** 1, ,24±0. ,18** 1.54±0.59* 0.79±0, .08 1.27±0.24** + B1/B2 antagonist
AngII 2.15±0, .42** 1 .54±0, .35 2.02±0.70 l.Oό±O .28** 1.69±0.31 + B2 agonist
Saline 1.36±0, 1 .H±O.06** 0.99±0.08** 0.69±0 .06** 1.04±0.05**
** PO.01 using Mann Whitney U test compared to AngII alone group * P<0.05 using Mann Whitney U test compared to AngII alone group Λ Calculated by averaging maximum diameters for all four aorta regions in each mouse
[0951] This example shows that aortic rupture was exacerbated by B2 agonism, whereas B1/B2 antagonism protected against Angll-induced rupture. This finding that the agonism and antagonism of bradykinin receptors have opposite effects on aortic rupture and aneurysm formation suggests that kinin pathways are critical in the pathology of aortic aneurysms.
[0952] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. [0953] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
[0954] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.
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Claims

WHAT IS CLAIMED IS:
1. A method for preventing or treating an aneurysm in an individual comprising administering to the individual a KKS antagonist.
2. The method of claim 1, wherein the KKS antagonist modulates the expression of a gene or the level or functional activity of an expression product of the gene.
3. The method of claim 2, wherein the gene is a gene encoding a component of the KKS.
4. The method of claim 2, wherein the gene is a gene whose expression product modulates directly or indirectly the expression of a gene encoding a component of the
KKS.
5. The method of claim 2, wherein the gene is a gene whose expression product modulates directly or indirectly the expression product of a gene encoding a component of the KKS.
6. The method of any one of claims 2 to 5 wherein the KKS antagonist reduces the expression of the gene.
7. The method of any one of claims 2 to 5 wherein the KKS antagonist reduces the level or functional activity of an expression product of the gene.
8. The method of any one of claims 2 to 5 wherein the KKS antagonist increases the expression of the gene.
9. The method of any one of claims 2 to 5 wherein the KKS antagonist increases the level or functional activity of an expression product of the gene.
10. Use of a KKS antagonist for preventing or treating an aneurysm in an individual.
11. The use of claim 12 wherein the KKS antagonist is formulated with a pharmaceutically acceptable carrier or diluent.
12. Use of a KKS antagonist in the manufacture of a medicament for preventing or treating an aneurysm in an individual.
13. A method for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual, the method comprising detecting the presence of an aberrant gene encoding a component of the KKS or of an aberrant expression product of a gene encoding a component of the KKS in the individual, wherein the aberrant gene or the aberrant expression product correlates with the presence or risk of the condition.
14. A method for detecting the presence of an aneurysm, determining the prognosis of an aneurysm, or monitoring the progression of an aneurysm in an individual, the method comprising detecting in the individual a level or functional activity of a component in the KKS5 which is different than a reference or control level or functional activity of the component.
15. A method for identifying an agent that antagonises the KKS for preventing or treating an aneurysm, the method comprising contacting a preparation with a test agent, wherein the preparation comprises:
(i) a polypeptide comprising an amino acid sequence corresponding to at least a biologically active fragment of a polypeptide component of the KKS, or to a variant or derivative thereof; or
(ii) a polynucleotide comprising at least a portion of a genetic sequence that regulates the component, which is operably linked to a reporter gene, wherein a detected change in the level or functional activity of the polypeptide component, or an expression product of the reporter gene, relative to a reference or control level or functional activity in the absence of the test agent, indicates that the agent modulates the KKS and is useful for the treatment or prevention of an aneurysm.
16. A method for identifying an agent that antagonises the KKS for preventing or treating an aneurysm, the method comprising contacting a sample of cells expressing a kinin receptor with a kinin and a test agent, wherein a detected decrease in level of binding between the kinin receptor and the kinin relative to a reference or control level in the absence of the test agent indicates that the agent is a KKS antagonist and is useful for the treatment or prevention of an aneurysm.
17. A method of producing an agent that antagonises the KKS for preventing or treating an aneurysm, the method comprising:
(i) identifying an agent as claimed in claim 15 or 16; and (ii) synthesising the agent on the basis that it tests positive for antagonising the KKS.
18. The method of claim 15, further comprising derivatising the agent.
PCT/AU2009/000579 2009-05-08 2009-05-08 Diagnosis, monitoring, prognosis, prevention and treatment of aneurysms WO2010127381A1 (en)

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WO2022187751A1 (en) * 2021-03-05 2022-09-09 Moore Ella Bradykinin 1 receptor antagonists and uses thereof for prevention and treatment of respiratory complications

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