CA2179379A1 - Metal chelators - Google Patents
Metal chelatorsInfo
- Publication number
- CA2179379A1 CA2179379A1 CA002179379A CA2179379A CA2179379A1 CA 2179379 A1 CA2179379 A1 CA 2179379A1 CA 002179379 A CA002179379 A CA 002179379A CA 2179379 A CA2179379 A CA 2179379A CA 2179379 A1 CA2179379 A1 CA 2179379A1
- Authority
- CA
- Canada
- Prior art keywords
- gly
- lys
- pro
- compound according
- nleu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
Abstract
Radionuclide chelating compounds are provided for coupling to targetting molecules such as proteins, peptides or antibodies. The resulting labelled targeting molecules may be used in diagnosis and therapy.
Description
-wo95tl7~19 2 1 7q37q PCT/CA91/0(1718 ME TAL ~T'T ~TORS
Field of the InYention This invention is in the f ield of diagnostic imaging, and 5 relates to chemical chelators useful in the radiol~hPll;n~ of agents that target tissues of diagnostic and therapeutic interest.
Backaround to the Invention The art of diagnostic imaging exploits contrasting agents that in binding or localizing site selectively within the body, help to resolve the image of diagnostic interest.
~7Gallium-citrate, for example, has an affinity for 15 tumours and inflamed tissue and, with the aid of sc~nn;n~
tomography, can reveal afflicted body regions to the physician. Other contrasting agents include the metal radionuclides such as 99C~technetium and ~ rhenium, and these have been used to label targetting molecules, such 20 as proteins, peptides and ant;ho~ C that localize at desired regions of the human body.
As targetting agents, proteins and other macromolecules can offer the tissue specificity required for diagnostic 25 accuracy; yet labelling of these agents with metal radionuclides is made difficult by their physical structure. Particularly, protein and peptide targetting agents present "~ uus sites at which r~ nucl ide binding can occur, resulting in a product that is 30 labelled heteroa~nPo~cly. Also, despite their large size, proteins rarely present the structural conf iguration most appropriate f or high af f inity radionuclide binding, i.e. a region incorporating four or more donor atoms that f orm f ivc - h~red rings . As a 35 result, radionuclides are bound typically at the more abundant low-affinity sites, forming unstable complexes.
wo 9S/17~19 PCT/CA9J/007l8 21 7~379 To deal with the problem of background binding, Paik et al (Nucl Med Biol 1985, 12:3) proposed a method whereby labelling of antibody is performed in the presence of excess DPTA (diaminetrimethylenepentaacetic acid), to 5 mask the low affinity binding sites. While the problem of low affinity binding is alleviated by this method, actual binding of the radionuclide, in this case technetium, was consequently also very low. The direct 1 Ahc~l 1 i n~ of proteins having a high proportion of lO cysteine residues also has been demonstrated (Dean et al;
Wo 92/13,572). This approach exploits thiol groups of cysteine residues as high-affinity sites for radionuclide binding, and is n~cecc~rily limited in application to those targetting agents having the required thiol 15 structure.
A promising alternative to the direct l;~h~l 1 in~ Of targetting agents is an indirect approach, in which targetting agent and radionuclide are coupled using a 20 chelating agent. Candidates for use as chelators are those - _ u.-ds that bind tightly to the chosen metal radionuclide and also have a reactive functional group for conjugation with the targetting molecule. For use in labelling peptide and protein-based targetting agents, 25 the chelator is ideally itself peptide-based, to allow the chelator/targetting agent to be synthesized in any desired structural combination using peptide synthesis techniques. For utility in diagnostic imaging, the chelator desirably has characteristics appropriate for 30 its in vivo use, such as blood and renal clearance and extravascular dif fusibility .
S rv Qf the Invention 35 The present invention provides chelators that bind diagnostically useful metals, and can be coupled to targetting agents capable of localizing at body sites of ~ WO 95117419 2 1 7 9 3 7 9 PCTlCA91/00718 diagnostic and therapeutic interest. The chelators of the present invention are peptide analogues designed structurally to present an N3S configuration capable of binding oxo, dioxo and nitrido ions of radi~nllrl; tlPfi such as 99mtechnetium and ~86~88rhenium.
More particularly, and according to one aspect of the invention, there are provided metal chelators of the f ormula:
R
R
~T
Field of the InYention This invention is in the f ield of diagnostic imaging, and 5 relates to chemical chelators useful in the radiol~hPll;n~ of agents that target tissues of diagnostic and therapeutic interest.
Backaround to the Invention The art of diagnostic imaging exploits contrasting agents that in binding or localizing site selectively within the body, help to resolve the image of diagnostic interest.
~7Gallium-citrate, for example, has an affinity for 15 tumours and inflamed tissue and, with the aid of sc~nn;n~
tomography, can reveal afflicted body regions to the physician. Other contrasting agents include the metal radionuclides such as 99C~technetium and ~ rhenium, and these have been used to label targetting molecules, such 20 as proteins, peptides and ant;ho~ C that localize at desired regions of the human body.
As targetting agents, proteins and other macromolecules can offer the tissue specificity required for diagnostic 25 accuracy; yet labelling of these agents with metal radionuclides is made difficult by their physical structure. Particularly, protein and peptide targetting agents present "~ uus sites at which r~ nucl ide binding can occur, resulting in a product that is 30 labelled heteroa~nPo~cly. Also, despite their large size, proteins rarely present the structural conf iguration most appropriate f or high af f inity radionuclide binding, i.e. a region incorporating four or more donor atoms that f orm f ivc - h~red rings . As a 35 result, radionuclides are bound typically at the more abundant low-affinity sites, forming unstable complexes.
wo 9S/17~19 PCT/CA9J/007l8 21 7~379 To deal with the problem of background binding, Paik et al (Nucl Med Biol 1985, 12:3) proposed a method whereby labelling of antibody is performed in the presence of excess DPTA (diaminetrimethylenepentaacetic acid), to 5 mask the low affinity binding sites. While the problem of low affinity binding is alleviated by this method, actual binding of the radionuclide, in this case technetium, was consequently also very low. The direct 1 Ahc~l 1 i n~ of proteins having a high proportion of lO cysteine residues also has been demonstrated (Dean et al;
Wo 92/13,572). This approach exploits thiol groups of cysteine residues as high-affinity sites for radionuclide binding, and is n~cecc~rily limited in application to those targetting agents having the required thiol 15 structure.
A promising alternative to the direct l;~h~l 1 in~ Of targetting agents is an indirect approach, in which targetting agent and radionuclide are coupled using a 20 chelating agent. Candidates for use as chelators are those - _ u.-ds that bind tightly to the chosen metal radionuclide and also have a reactive functional group for conjugation with the targetting molecule. For use in labelling peptide and protein-based targetting agents, 25 the chelator is ideally itself peptide-based, to allow the chelator/targetting agent to be synthesized in any desired structural combination using peptide synthesis techniques. For utility in diagnostic imaging, the chelator desirably has characteristics appropriate for 30 its in vivo use, such as blood and renal clearance and extravascular dif fusibility .
S rv Qf the Invention 35 The present invention provides chelators that bind diagnostically useful metals, and can be coupled to targetting agents capable of localizing at body sites of ~ WO 95117419 2 1 7 9 3 7 9 PCTlCA91/00718 diagnostic and therapeutic interest. The chelators of the present invention are peptide analogues designed structurally to present an N3S configuration capable of binding oxo, dioxo and nitrido ions of radi~nllrl; tlPfi such as 99mtechnetium and ~86~88rhenium.
More particularly, and according to one aspect of the invention, there are provided metal chelators of the f ormula:
R
R
~T
2 0 ~/ ~ 3 ~
wherein R4 R~ and R2 together f orm a 5- or 6 - hered heterocyclic ring which is optionally fused to a 5- or 6-membered ring, wherein either ring is optionally substituted with a conjugating group or with a conjugating group having a targetting molecule coupled thereto;
R3 is selected from H; alkyl; and alkyl substituted by a group sPlectP~l from amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl and imidazolyl;
R~ is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is H or a sulfur protecting group;
wo 9~/17419 PCT/CA94/00718 ~
2~ 7~37~ 4 In an aspect of the invention, chelators of the above formula are provided in a form having a diagnostically or therapeutically useful metal complexed therewith.
5 According to another aspect of the invention, the chelator is provided in a form coupled to a diagnostically or therapeutically useful targetting molecule. An additional aspect of the invention provides the chelator6 coupled to a targetting molecule and in a lO form having a metal complexed therewith.
In another aspect of the invention, targetting molecules are provided having the general sequence: f ormyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-Lys-Asp-X-O~I wherein X is a bond or 15 an amino acid residue; the targetting molecule which may be coupled to chelators of the present invention.
3rie~ Descri~tion of the Fit7~]res 20 Figure l is a graph representing binding affinity of targetting molecules in accordance with an ~mho~ I of the invention.
Figure 2 is a graph representing neu~lu~llic effect of 25 targetting molecules in accordance with an embodiment of the invention.
Detailed Descril~tion of the Invention The invention provides chelators of diagnostically useful metals that when complexed with the metal and in a form coupled to a targetting molecule are useful for delivering the detectable metal to a body site of 35 diagnostic interest. As illustrated in the above f ormula, the chelators are peptidic derivatives that ~ Wo 95/17419 2 1 9 3 7 ~ PCT/CA91/00718 present an N35 conf iguration in which the metal is complexed .
Terms def ining the variables R~ - R4 and T as used 5 hereinabove have the following -~-n;n~
"alkyl" refers to a straight or branched Cl-C8 chain and includes lower C~-C4alkyl;
"alkoxy" refers to straight or branched Cl-C8 alkoxy and includes lower C1-C4alkoxy;
"thiol" refers to a sulfhydryl group that may be substituted with an alkyl group to form a thioether;
"sulfur protecting group" refers to a chemical group that inhibits oxidation of sulfur and includes groups that are cleaved upon chelation of the metal.
Suitable sulfur protecting groups include known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyl and organothio groups.
20 In preferred Pmho~lir Ls of the invention, the chelators conform to the above formula in which: Rl and R2 together form a five or six membered heterocyclic ring such as pyrrole and pyridine, or a f ive or six r ` ed ring fused to a six membered ring such as indole, quinoline 25 and isoquinoline; R3 is selected from H and a hydroxy substituted alkyl group selected from methyl and ethyl and most preferably IIYdLU~Y -thyl; R~ is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is a hydrogen atom or the sulfur 30 protecting group acetamidomethyl (Acm);
In specific ~mho~l;r~ -ts of the invention, the chelators conform to the above general for~ula wherein T is the sulfur protecting group acet~m;~ -thyl (Acm); R3 is H or 35 ~lydLuxy Lhyl; R~ and R2 together form a ring selected from pyridine, pyrrole, indole, quinoline and wo 95/17419 PCT~CA94/00718 isoquinoline; and R~ is a glycine amino acid residue or a glycine residue attached to a targetting peptide.
The substituents represented by R~ and R2 together with 5 the ad; acent nitrogen atom f orm a 5- or 6 -membered heterocyclic ring which may be fused to another five or 6iX membered ring. Five and six membered heterocyclic rings include but are not limited to pyrrole, pyrazole, imidazole, pyridine, pyrazine, pyridazine, pyrimidine and l0 triazine. Fused rings include but are not limited to N-containing bicyclics such as guinoline, isoguinoline, indole and purine. Rings containing sulfur atoms e . g.
thiazole and oxygen atoms e. g. oxazole are also Pn, - csed by the present invention .
The heterocyclic ring formed by R~ and R2 may be substituted with a conjugating group that is chemically reactive allowing for coupling a targetting molecule to the chelator. In the preferred case where the targetting 20 molecule is peptidic, the conjugating group is reactive under conditions that do not denature or otherwise adversely affect the peptide. In one Qn~horlir ~ of the invention, the conjugating group is reactive with a functional group of the peptidic targetting molecule such 25 as the carboxy terminus or amino t~rm; n-lc Alternatively, the conjugating group can be reactive with an ~-amino group of a lysine residue. Conjugating groups reactive with amino groups of targetting molecules include carboxyl and activated esters. Conjugating 3 0 groups reactive with carboxyl groups of targetting molecules include amines and hydrazines.
For diagnostic and therapeutic purposes, the chelator per se may be used in combination with a detectable metal 35 capable of forming a complex. Suitable metals include radinn~lrl i~ec such as technetium and rhenium in their various forms such as 99~Tco3+, 99~Tco2+, ReO3+ and ReO2+.
~ Wo 95/17419 PcT/cAs~/on718 More desirably, the chelator is coupled to a targetting molecule that serves to localize the chelated metal to a desired location for diagnostic imaging or for therapy ie. radiation therapy of tumours. Examples of targetting 5 molecules include, but are not limited to, steroids, proteins, peptides, antibodies, nucleotides and saccharides. Preferred targetting molecules include proteins and peptides, particularly those capable of binding with specificity to cell surface L~ctl!l .,L~
l0 characteristic of a particular pathology. For instance, disease states associated with over-expression of particular protein receptors can be imaged by lAhPll;n~
that protein or a receptor binding fragment thereof in accordance with the present invention. Peptide-based 15 targetting molecules can be made by various known methods or in some instances can be commercially obtained. Solid phase synthesis employing alternating t-Boc protection and deprotection is the preferred method of making short peptides which can be an automated process. RPr hin lnt 20 DNA technology is preferred for producing proteins and long fragments thereof.
Chelators of the present invention are peptide derivatives and are most ef f iciently prepared by solid-25 phase peptide synthesis. In general solid-phase synthesis involves the stepwise addition of amino acid residues to a growing peptide chain that is linked to an insoluble support or matrix, such as polystyrene. The C-t~rmin~lC residue of the chelator is first anchored to a 30 commercially available support with its amino group protected with an N-protecting agent such as a t-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl (E~IOC) group. The amino protecting group is removed with suitable deprotecting 35 agents such as TFA in the case of tBOC or piperadine for FMOC and the next amino acid residue ( in N-protected form) is added with a coupling agent such as Wo 95/17419 2 17 ~ 3 7 9 PCT/CAs~/00718 dicyclocarbodiimide (DCC). Upon formation of a peptide bond the reagents are washed from the support. After addition of the final residue, the chelator is cleaved from the support with a suitable reagent such as 5 trifluoroacetic acid (TFA) or hydrogen fluoride (HF) and isolated .
The present invention Pnrc-r~ses chelators incorporating various heterocyclic groups containing a nitrogen atom 10 providcd that it is analogous in ~LLu~:LuLe to an amino acid in that there is a carboxyl carbon, alpha carbon and an alpha nitrogen wherein the alpha carbon and alpha nitrogen are incorporated in a common ring. For example, picolinic acid (pic), dipicolinic acid (dipic), 15 ch~ m;r acid (chel), 2-calboxy~yrazine~ 2-carboxypyrimidine, 2-caLLu-~y~yLLole, 2-quinolinic acid, l-isoquinolinic acid, 3-isoquinolinic acid and the like will behave as a natural amino acid residue in solid phase synthesis by forming a peptidic bond upon reaction 20 of the carboxyl group and a deprotected amino group of a previously added residue. Variation at R3 may be introduced to chelators of the invention simply by incorporation of a desired amino acid residue at the appropriate stage of chain elongation. For example, R3 25 may be a ;1YdL~J~SY ~ ~hyl group by using a serine residue or may be a hydrogen atom by using glycine. Any D or L, naturally occurring or derivati2ed amino acid may be used .
3 0 In accordance with an ~ nt of the present invention, R4 is a targetting molecule that is proteinaceous. Human Immunoglobulin G (HIG), a multisubunit protein, has been directly labelled with technetium-99m and used extensively for imaging sites of 35 inflammation, however smaller peptides are bPr in~ the targetting molecules of choice for their site specificity a~ a result of receptor binding properties and for their = _ _ _ _ _ . _ . . . . . , . .. .. , .. _ _ _ _ _ _ ~
~1 79~79 WO 9S/17419 PCT/CA9-i/00718 ease of preparation. An example of peptidic targetting molecules are Tuftsin antagonists such as Thr-Lys-Pro-Pro-Arg and Lys-Pro-Pro-Arg. Another peptidic targetting molecule useful for imaging inflammation is f~LP (formyl-5 Net-Lys-Phe) and derivatives thereof such as formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys described by Fischman et al in pending Canadian application CA 2,016,235. It is believed that frILP and various derivatives thereof bind to neutrophils and are therefore useful in imaging sites 10 of inf lammation .
In accordance with another aspect of the invention, the present invention provides a peptide useful as a targetting molecule which has the sequence formyl-Nleu-15 Leu-Phe-Nleu-Tyr-Lys-Lys-ASp-X-OH wherein X is a bond or an aminoacid residue. For convenient synthesis of this peptide, X is preferrably a glycine (Gly) residue. In vivo studies have shown this peptide coupled to a chelator of the present invention strongly binds to 20 neutrophils while having a more favourable neuL~ e.-ic profile than native fMLP or the derivative formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys .
Synthesis of a chelator/targetting molecule conjugate 2~ hereinafter referred to as a "conjugate" can be achieved in various ways. When R4 is a peptidic targetting molecule, it is convenient to synthesize the conjugate in toto by starting solid-phase synthesis from the C-tPrm; nllC residue of the targetting molecule and ending 30 with the heterocyclic residue (R~, R2) of the chelator.
Alternatively, a targetting molecule which incoLy.,rc.tes a lysine residue may be coupled to the chelator at R4 by way the ~-amino group of that lysine residue. In this case, the targetting peptide is synthesized as a separate chain 35 from the chelator and is differentially protected at the ~-amino group and N-t~mi ml~ amino group. For example the ~-amino group may be protected with 1- ( 4, 4-dimethyl-WO 95117419 2 1 7 ~ 37 9 PCT/CA9~1On718 ~
2, 6-dioxocyclohexylidine) -ethyl (Dde) while the N-t~rm;nllq amino is FMOC protected. When the targetting molecule 6ynthesis is complete the -amino group is deprotected with hydrazine and is available for reaction 5 with a C-terminus carboxyl group of a chelator while the N-t~rm; nllq amino group is protected.
Targetting molecules may also be coupled to chelators of the invention by way of a conjugating group substituent 10 on the heterocyclic ring of the chelator. For example, a chelator with an amino substituent on the heterocyclic ring upon deprotection will be reactive with the C-terminllq carboxyl group of a peptide targetting molecule.
Such a conjugate may be synthesized as a 6ingle chain 15 starting at the C-t~rmi nllq residue of the chelator and ending with the N-t~rminllq of the targetting molecule.
Alternatively, a peptide targetting molecule may be coupled to the heterocyclic residue by way of its N-t~rmi nl~q when the heterocyclic group has a suitable 20 conjugating group substituent such as a carboxyl group or an activated ester. In this case, the chelator and targetting molecule are synthesized as separate chains and then coupled to form the desired conjugate.
25 In accordance with one aspect of the invention, chelators incorporate a diagnostically or therapeutically useful metal. Inco~ tion of the metal within the chelator can be achieved by various methods common to the art of coordination chemistry. When the metal is the 30 radionuclide technetium-99m, the following general procedure may be used to form a technetium complex. A
chelator solution is formed initially by dissolving the chelator in aqueous alcohol eg. ethanol-water 1:1. The solution is degassed with nitrogen to remove oxygen then 35 the thiol protecting group is removed, for example with sodium hydroxide and heat. The solution is then neutralized with an organic acid such as acetic acid (pH
~ Wo 95/17419 2 ~ 7 9 3 7 9 PcTlcA94Jon7~8 6.0-6.5). In the labelling 6tep, sodium pertechnetate, obtained from a Molybdemun generator, is added to the chelator solution with an amount of stannous chloride - sufficient to reduce the technetium. The solution is mixed and left to react at room temperature and then heated on a water bath. In an alternative method, 1 Ah~l 1 i n~ can be accomplished with the chelator solution adjusted to pH 8. Pertechnetate may be replaced with a solution of technetium complexed with labile ligands suitable for ligand exchange reactions with the desired chelator. Suitable ligands include tartarate, citrate, gluconate and glucoheptonate. Stannous chloride may be replaced with sodium dithionite as the reducing agent if the chelating solution is alternatively adjusted to pH
12-13 for the labelling step. The labelled chelator may be separated from contaminants 99~TCoi and colloidal 99~TCo2 chromatographically, e.g. with a C-18 Sep Pak cartridge activated with ethanol followed by dilute HCl. Eluting with dilute HCl separates the 99~Tcoi, and eluting with EtOH-saline 1:1 brings off the chelator while colloidal 99~Tco2 remains on the column. The chelators of the invention can be coupled to a targetting molecule prior to labelling with the radionuclide, a process referred to as the "bifunctional chelate" method. An alternative approach known as the "prelabelled ligand" method, the chelator is first l Ah~ d with the desired metal and is subsequently coupled to the targetting molecule. This method is advantageous in that the targetting molecule itself is not inadvertently labelled at low affinity binding sites which may render the targetting molecule inactive or may release the metal in vivo.
An alternative approach for lAh~l 1 ing chelators of the present invention involves techniques described in a co-pending U.S. application 08/152,680 by Pollak et al, filed on 16 November 1993 incorporated herein by reference. Briefly, chelators are immobilized on a solid wo 95117419 2 1 7 9 3 7 9 PCT1CA9~100718 phase support in such a manner that they are released from the support only upon formation of a complex with the 1 AhPl l; n~ metal atom. Thi5 is achieved when the chelator is coupled to a functional group of the support 5 by one of the complexing atoms. Preferably, a complexing sulfur atom is coupled to the support which is functinn~l; 7P~ with sulfur a protecting group such as maleimide .
10 When coupled to a targetting molecule and 1 ~hPl 1 Pcl with a diagnostically useful metal, chelators of the present invention can be used to detect pathological conditions by techniques common in the art. A conjugate labelled with a radionuclide metal such as technetium may be 15 administered to a mammal by intravenous injection in a pharmaceutically acceptable solution such as saline or DMSO. The amount of 1 ~hPl 1 Pd conjugate administered is depPn~Prlt upon the toxicity profile of the chosen targetting molecule as well as the metal . T nt~~ 1; 7~tion 20 of the metal in vivo is tracked by standard scintigraphic techniques at appropriate time intervals subsequent to administration .
The following examples are presented to illustrate 25 certain embodiments of the present invention.
~Y~mnle l PreparatiQn qf Chelators and Conjugates Chelators were synthesized using 9-30 fluorenylmethyloxycarbonyl tFMOC) chemistry on an 2-methoxy-4-alkoxybenzyl alcohol resin preloaded with the protected C-tPrm;nllc residue (Sasrin resin, Bachem Biosciences Inc., ph;l l~plrhi~ PA) using an Applied Biosystems 433A peptide synthesizer (Foster City, CA).
Preparation of Chelators a . Chel-Gly-Cys (Acm) -Gly-OH
_ _ _ wo 9S/17419 2 1 7 ~ ~ 9 PCT/CA94100718 b. DiPic-Gly-Cys (Acm) -Gly-OH
c. Pic-Gly-Cys (Acm) -Gly-OH
- Synthesis began from the Gly residue preloaded on the 5 resin and continued to the final Pic, DiPic or Pic - residue by addition of one of picolinic, dipicolinic or t~hPl ~ m; C acid . The chelator-resin was dried in vacuo for 12 hours. Cleavage of the chelator from the resin involved mixing a cooled 601ution of 9596 trif luoroacetic 10 acid (TFA) and 5% water (lml per 100 mg of peptide-resin) with the peptide-resin for 1. 5 to 2 hours at room temperature. The resin was removed by filtration and washed 3 times with 30 ml t-butyl methyl ether in a 50 ml conical polypropylene centrifuge tube forming a white 15 precipitate. The precipitate was dissolved in water with added acetonitrile. The precipitate was frozen in acetone-dry ice and lyophilized over 12 hours. The resulting white powder was dissolved in water, filtered through a 0.45 ~m syringe filter (Gelman Acrodisc LC
20 PVDF) and purified by ~vt:l,,ed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCN 25 x 10) using 0.1%
TFA in water as buffer A and 0.1~6 TFA in acetonitrile as buffer B. The column was equilibrated with 100:0 buffer A:buffer B and eluted with a linear gradient in 25 min at 25 1 ml/min to 50% buffer B. Fractions were reanalysed on the HPLC and pooled according to matching prof iles . If nP~PC:s~ry the pooled fractions were repurified using the same conditions. The pure fractions were frozen in acetone-dry ice and lyorh i 1 i 7Pd over 10 hours to give a 3 0 white powder .
Preparation of Conjugates d . (Pic-Ser-Cys (Acm) -Gly) -Thr-Lys-Pro-Pro-Arg-OH;
e. (Pic-Ser-Cys (Acm) -Gly) -Lys-Pro-Pro-Arg-OH;
f . 2-Quinolinic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro-Arg-OH;
~o 95117~19 2 1 7 ~ 3 7 9 PCT/CA94100718 ~
g. l-Isoquinolinic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro-Arg -OH;
h. 3-Isoquinolinic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro -Arg-OH;
i. Indole-2-carboxylic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro-Arg-OH; and j . Pyrrole-2-carboxylic acid-Ser-Cys (Acm) -Gly-Thr-Lys -Pro-Pro-Arg-OH .
Synthesi6 began from the Arg residue preloaded on the resin and continued to the Ser residue of the chelator ending with the addition of one of picolinic, 2-quinolinic, l-isoquinolinic~ 3-isoquinolinic, pyrrole-2-carboxylic and indole-2-carboxylic acid. The chelator-peptide-resin was dried in vacuo 12 hours. Cleavage from the resin involved mixing with a solution of 10 ml trifluoroacetic acid (~FA), O.5ml water, O.5ml thioanisole, 0.25ml 1,2-ethanedithiol (EDT) and 0.75g phenol f or 1. 5 to 2 hours at room temperature . The resin was removed by filtration and the peptide washed 3 times with 3 0 ml t-butyl methyl ether in a 50 ml conical polypropylene centrifuge tube forming a white precipitate. The precipitate was dissolved in water with added acetonitrile when solubility problems arose. The precipitate was frozen in acetone-dry ice and lyophilized over 12 hours. The resulting white powder wa6 dissolved in water, filtered through a 0.45 ~m syringe filter (Gelman Acrodisc LC PVDF) and purified by reversed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCM
25 x 10) using 0.1% TFA in water as buffer A and 0.1% TFA
in acetonitrile as buffer B. The column was equilibrated with 100:0 buffer A:buffer B and eluted with a linear gradient in 25 min at 1 ml/min to 50% buffer B.
Fractions were reanalysed on the HPLC and pooled according to matching profiles. If n~C~csAry the pooled fractions were repurified using the same conditions. The ~ wo 95/17419 2 1 7 ~ 3 7 q PCT/CAs~/007l8 pure fractions were frozen in acetone-dry ice and lyophilized over 10 hours to give a white powder.
Preparation of fMLP Conjugates k. (Pic-Gly-Cys-Gly) -~NH-Lys (-Gly-OH) -Tyr-Nleu-Phe-Leu-Nleu-f or 1. (Pic-Gly-Cys (Acm) -Gly) -lNH-Lys (-Gly-OH) -Tyr-Nleu-Phe-Leu-Nleu-f or m. (Pic-Gly-Cys (Acm) -Gly) -~-NH-Lys (-Asp-Gly-OH) -Lys-Tyr-Nleu-Phe-Leu-Nleu-f or Targetting peptides that comprise lysine residues can be coupled to the chelator via the Lys ~-amino group by the following procedure. For ~ u-lds example l(k), 1(1), and 1 (m) . the targetting peptide was initially synthesized from glycine to norleucine by 9-fluorenylmethyloxycarbonyl (FMOC) chemistry using an FMOC-glycine preloaded 2-methoxyl-4-alkoxyl-benzyl alcohol resin and a 1-(4,4-dimethyl-2,6-dioxocyclohexylidine)-ethyl (Dde) orthogonal protected lysine with an Applied Biosystems 433A peptide synthesizer. The fMLP peptide-resin was removed from the synthesizer and dried 12 hours in vacuo to prepare for formylation.
Formic anhydride was prepared by heating acetic anhydride (2 eguivalents) with formic acid (1 equivalent) to 50-C
for 15 minutes followed by cooling to O-C. Formylation of the fMLP peptide involved swelling the peptide-resin in dichloromethane (DCM) (5 ml) followed by swirling with formic anhydride (5ml) for 15 minutes. The formylated fMLP peptide-resin was filtered, washed with DCM and dried in vacuo 12 hours.
Formylated peptide-resin (50 mg/2ml) was swirled with a 296 hydrazine hydrate in N-methylpyrrolidone (NMP) WO 95/17419 PCT/C~94/00718 ~
21 79379 ~16 solution for 3 minutes two times then filtered and washed with DCN and dried in vacuo 12 hours to remove the -amino lysine protecting group (Dde) while leaving the N-t~rmin~ amino group formylated.
The chelator was added to the ~-amino lysine of the fNLP
peptide on the 433A peptide synth~si 7~r. The chelator-peptide-resin was dried in vacuo 12 hours. Cleavage from the resin involved mixing a cooled solution of 95%
trifluoroacetic acid (TFA) and 5% water (1 ml per 100 mg of chelator-peptide-resin) with the chelator-pep~ide-resin for 1.5 to 2 hours at room temperature. The resin wa6 removed by f iltration and washed with 1-3 ml of TFA
to obtain 6-8 ml of a clear yellow liquid. This liguid was 610wly dropped into 30-35 ml of tert-butyl methyl ether in a 50 ml conical polypropylene centrifuge tube f orming a white precipitate . The precipitate was centrifuged at 7000 rpm, O'C for 5 minutes (Sorvall RT
6000, Dupont~, decanted and washed two more times with t-2 0 butyl methyl ether . Following drying under vacuum the precipitate was dissolved in water with added acetonitrile when solubility problems arose. The precipitate was frozen in acetone-dry ice and lyo~hi 1 i 7 d over 10 hours. The resulting white powder was dissoved in dimethylsulfoxide (20 ,uL) and 50:50 acetonitrile:water solution (980 ,uL), filtered through a 0.45 ,um syringe filter (Gelman Acrodisc I.C PVDF), and purified by reversed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCM 25 X 10) using 0.1% TFA in water as buffer A and 0.1% TFA in acetonitrile as buffer B. The column was equilibrated wlth 50:50 buffer A:buffer B and eluted with a linear gradient in 25 min at 1 ml/min to 100% buffer B. Fractions were reanalysed on the HPLC and pooled according to matching prof iles . If n~ Ei~ry, the pooled fractions were repurified using the same conditions. The pure fraction6 were frozen in acetone-WO 95117419 C~ 1 7 9 3 7 9 PCT/C~94100718 dry ice and lyophilized over 12 hours to give a white powder .
le 2 T-AhPl 1 in~J Chelators with 59"Tc - The chelators and conjugates of example 1 (lmg) were dissolved in 2001~L EtOH-water (1:1) in a tube. 100-200~L
sodium pertechnetate (200-600 M;3a, 5-15 mCi), 100 ,uL
phosphate buffer (0.25 M, pH 7.4), and 200 ,uL of a solution containing 50~g stannous chloride dihydrate and 40mg sodium tartrate were added to the tube and capped tightly and placed in a boiling water bath for 10 minutes. In order to achieve adequate separation of the chelators, the solution was then loaded on a C-18 Sep-Pak column activated by washing sequentially with 5ml methanol, 10ml water and 5ml dilute (lmM) HCl to remove Tc04-. Subsequent elution with 2ml EtOH-saline (1:1) removed the chelator while TcOz remained on the column.
The extent of complexation of 99~Tc with chelators was measured by radioactivity of the eluted fractions.
Example J.Ahel 1 i n~ Yield ( % total radioactivity) 1 (a) 7 1 (c) 92 1 (d) 93 1 (e) 92 1 (k) 29 1(1) 63 1 (m) 78 wo 95/17~19 pcTlcA94lon7l8 ~1 7q379 18 Con~ugates l(f) - l(j), were reconstituted (200,u~., lmg/mL
saline) and then injected into 3mL vacutainers with lOO,~IL
pertechnetate (lOmCi) and lOO,uL stannous gluconate (50 ~Lg ~tannous chloride and 1 mg sodium gluconate). The tubes 5 were placed in boiling water bath f or 12 minutes and then f iltered through a Whatman PVDF 5yringe f ilter to collect the ~ AhPl 1 efl conjugate solutions which were further diluted with saline to prepare injectable solutions (2Mbq/mL). The conjugate5 were isolated by HPLC
10 (Beckman) from a (20,uL) sample (before dilution) to determine the lAh~l1 in7 yield by measuring radioactivity.
example T.AhP11 in~ Yield (96) l(f) 94.4 l(g) 96.8 1 (h) 94 l(i) 96.3 l(j) 98.4 T~Y;~ le 3 In vivo imaging and bio~ ribution of chelators and cullju~-ll es For chelators and conjugates of example 1, rat 25 inflammation studies were performed as follows. 2 male Sprague-Dawley rats (Charles River, 250-300g) were injected intrArllcc~ll Arly with 5mg zymosan, a yeast cell wall preparation (20mg for conjugates l(f~ - l(i)) or a virulent E. coli (ATCC 25922, O . lml ûf 1. OXl09 30 organisms/ml) suspension into their right hindlegs 24 hours before imaging. Focal inflammation in the leg was visually detectable after 1 day. lmg (ca. O . 7 ~LMol) of the chelator was dissolved in 50 ,uL of dimethylsulfoxide and added to an ethanol-water mixture (1:1, 200 ,uL). An 35 aliquot of Tc-99m tartarate (ca. 400 r~Bq) was added and ~ wo 95117419 2 1 7 q 3 7 9 PCT/CA9~/007~8 transchelation allowed to proceed for 20 min. at lOO C.
The Tc-99m chelate was purified by elution through a Sep Pak cartridge . The purif ied tracer solution was further diluted with saline to prepare an injectable (200 ~L) 5 containing about 100 uCi (3.7 MBq) of activity.
The rats were anaesthetized with sodium pentobarbital (40 to 50 mg/kg), and the 1 ~h.ol 1 Pd chelator/conjugate solution (200uL) was injected intravenously via the tail 10 vein. Serial whole-body scintigrams were acquired for the first 5 minutes. Subsequently, further images were obtained at 30, 60, and 120 minutes. The rats were then killed with anaesthesia and samples of organs, urine, blood, inflamed muscle (right leg) and non-inflamed 15 muscle ( left leg) were weighed and counted in either a well-type gamma counter or in a gamma dose calibrator.
The dose calculations were made based on assumption that the blood volume constituted 6 . 5% of body weight .
Results are averages for two rats and are corrected for 20 the residual dose in the tail.
WO 95/17419 PCT~CA9.1/00718 21 7937~ 20 E E ~q G ~ ~ NO
- Cc ~! N _ cO ~ ~ N IN
C C e~ 't ~ ~ ~ O
E C E E E E E ", UJ ~
N N N N N
C 0 0 ~ U~ O O U~
'~~ O O O O O O O O O
C
E ~ N
~ E
0 0 ~ U~ 0 ~
.E ~ æ N
~o G ~ ~ 0 C~
N
o G 8 N ~ G r~ g G
o G 1~ ~ N ~ O
I ~q ~ C" Lo ~ Co ~0 0 ~
~ ~, 8 o ,` G r- O N
, ~" ~ cq In ID ~ G G
~_~ .~ O, N _ ~
1::1 G 1~ 0 0 0 ~ IN ~ G G
O . ~ ~ ~ , , G O
D _ O G ~q G G G ~
-E _ '~ ~ o _ C _ _ `' E ~
o WO 95/17~19 PCT/CA94/007 ~ A le 4 ll LLu~hil Binding Assay of fMLP and fMLP
Derivative Conjugates of Example l(l) and l(m) Rat peripheral neutrophils were prepared for binding 5 assay as follows: blood was obtained by cardiac ~ul--;Lu~e - and anticoagulated with acid- citrate dextrose (ACD) (10%). Red blood cells were removed by 5Prl; ation on hydroxyethyl cellulose (1.1%) for 30 min at room temperature and leukocyte-rich supernatant layered onto 65% percol. Centrifugation at 400g for 30 min resulted in a distinct band of mononuclear cells (ly ~-_y~es and monocytes~ which was discarded, the neutrophil rich pellet was rPc~cppn~lpd and ro~~;n;nrJ red blood cells lysed by hypotonic shock using cold water. The ~ ;n;nq 5 neuL~ u~hils were rPcucppnr~ed in Hanks Buffered Salt Solution (HBSS) to the desired concentration. Final neutrophil preparation consisted of cells pooled from up to 10 animals, >90% neutrophils and >9596 viable by Trypan Blue exclusion.
Binding affinity of the fMLP peptide and conjugates 1(1) and l(m) was AcsPcsed by ,- ~;n~ off a constant concentration of tritiated fMlP of known affinity for neutrophil receptors. 106 neutrophils were added to 25 polypropylene plates containing 15n~ tritiated f~LP and varying concentrations of -nl Ah'~l 1 ed test peptide and conjugates in a final volume of 150,uL HBSS. The plate was incubated for 1 hour at room temperature after which cells were harvested by filtration onto glass fibre 30 filter mats (Skatron receptor binding filtermat) using a Skatron cell harvester with 12 well head. Harvested cells were washed with ice-cold saline and air dried.
Filters were then placed in 6ml scintillation vials, 5ml of scintillation fluid added (Ecolume) and vials counted 35 using a liquid scintillation counter. Binding affinity of the f~qLP peptide and conjugates 1(1) and l(m) is illustrated in figure 1 and expressed as ~6 maximal Wo gs/17419 PCT/CA94/00718 ~
tritiated fMLP binding V5. peptide/conjugate concentration. % maximal tritiated fMLP
binding=(specific binding . maximum binding) X 100%.
Specif ic binding was the total binding less non-specif ic binding which was the amount of residual radioactivity bound in the presence of lOuM l~nl ~hPl 1 e~ fMLP. Both 1(1) and 1 (m) had greater binding affinity for neutrophil receptors than native fMLP.
ExamPle 5 Neul~v~ ia Assay of fMLP, fMLP Derivative formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys and Conjugate 1 (m) The effect of fMLP, fMLP derivative formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys and conjugate of example 1 (m) on circulating neutrophil number was assessed using the rat transient neutropenia model. Rats were anaesthetized with 250,uL
somnitol (16mg/rat) and injected via the tail vein at T=0 with the test peptides. At a range of time points after inj ection ( 0, 2, 5, 10, 3 0 min) a 2ml blood sample was taken by cardiac puncture (anticoagulated with 10% ACD).
3 animal were used per time point. For each sample the total white blood cells/ml and % neutrophil6 was det~rm; nPd, the number of neutrophils/ml in each sample being calculated. Within each experiment the number neutrophils/ml after saline injection at all time points was meaned to give a saline control against which the peptides could be compared. The number neutrophils/ml after peptide injection was expressed as a % of the saline control within each experiment.
Referring to figure 2, injections of 5 and 10 nmoles of fMLP produced a dose-~lPrPn(lpnt transient n~:uLLv~ ia~
with a maximal effect occurring 2 min after peptide injection (15 and 9% of control respectively) returning to 93 and 7596 of control values by 30 min after in; ection . 5nmoles of f ormyl-Nleu-Leu-Phe-Nleu-Tyr-Lys produced a smaller maximal reduction in circulating .. . . . . . .. _, . , 2 1 7 ~ 3 7 9 9410071~
wo gS/17419 2 3 PCT/CA
neutrophils (45% o~ control) while l(m) produced only a small transient drop in circulating neutrophils (8096 of control) at 5nmoles.
wherein R4 R~ and R2 together f orm a 5- or 6 - hered heterocyclic ring which is optionally fused to a 5- or 6-membered ring, wherein either ring is optionally substituted with a conjugating group or with a conjugating group having a targetting molecule coupled thereto;
R3 is selected from H; alkyl; and alkyl substituted by a group sPlectP~l from amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl and imidazolyl;
R~ is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is H or a sulfur protecting group;
wo 9~/17419 PCT/CA94/00718 ~
2~ 7~37~ 4 In an aspect of the invention, chelators of the above formula are provided in a form having a diagnostically or therapeutically useful metal complexed therewith.
5 According to another aspect of the invention, the chelator is provided in a form coupled to a diagnostically or therapeutically useful targetting molecule. An additional aspect of the invention provides the chelator6 coupled to a targetting molecule and in a lO form having a metal complexed therewith.
In another aspect of the invention, targetting molecules are provided having the general sequence: f ormyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-Lys-Asp-X-O~I wherein X is a bond or 15 an amino acid residue; the targetting molecule which may be coupled to chelators of the present invention.
3rie~ Descri~tion of the Fit7~]res 20 Figure l is a graph representing binding affinity of targetting molecules in accordance with an ~mho~ I of the invention.
Figure 2 is a graph representing neu~lu~llic effect of 25 targetting molecules in accordance with an embodiment of the invention.
Detailed Descril~tion of the Invention The invention provides chelators of diagnostically useful metals that when complexed with the metal and in a form coupled to a targetting molecule are useful for delivering the detectable metal to a body site of 35 diagnostic interest. As illustrated in the above f ormula, the chelators are peptidic derivatives that ~ Wo 95/17419 2 1 9 3 7 ~ PCT/CA91/00718 present an N35 conf iguration in which the metal is complexed .
Terms def ining the variables R~ - R4 and T as used 5 hereinabove have the following -~-n;n~
"alkyl" refers to a straight or branched Cl-C8 chain and includes lower C~-C4alkyl;
"alkoxy" refers to straight or branched Cl-C8 alkoxy and includes lower C1-C4alkoxy;
"thiol" refers to a sulfhydryl group that may be substituted with an alkyl group to form a thioether;
"sulfur protecting group" refers to a chemical group that inhibits oxidation of sulfur and includes groups that are cleaved upon chelation of the metal.
Suitable sulfur protecting groups include known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyl and organothio groups.
20 In preferred Pmho~lir Ls of the invention, the chelators conform to the above formula in which: Rl and R2 together form a five or six membered heterocyclic ring such as pyrrole and pyridine, or a f ive or six r ` ed ring fused to a six membered ring such as indole, quinoline 25 and isoquinoline; R3 is selected from H and a hydroxy substituted alkyl group selected from methyl and ethyl and most preferably IIYdLU~Y -thyl; R~ is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is a hydrogen atom or the sulfur 30 protecting group acetamidomethyl (Acm);
In specific ~mho~l;r~ -ts of the invention, the chelators conform to the above general for~ula wherein T is the sulfur protecting group acet~m;~ -thyl (Acm); R3 is H or 35 ~lydLuxy Lhyl; R~ and R2 together form a ring selected from pyridine, pyrrole, indole, quinoline and wo 95/17419 PCT~CA94/00718 isoquinoline; and R~ is a glycine amino acid residue or a glycine residue attached to a targetting peptide.
The substituents represented by R~ and R2 together with 5 the ad; acent nitrogen atom f orm a 5- or 6 -membered heterocyclic ring which may be fused to another five or 6iX membered ring. Five and six membered heterocyclic rings include but are not limited to pyrrole, pyrazole, imidazole, pyridine, pyrazine, pyridazine, pyrimidine and l0 triazine. Fused rings include but are not limited to N-containing bicyclics such as guinoline, isoguinoline, indole and purine. Rings containing sulfur atoms e . g.
thiazole and oxygen atoms e. g. oxazole are also Pn, - csed by the present invention .
The heterocyclic ring formed by R~ and R2 may be substituted with a conjugating group that is chemically reactive allowing for coupling a targetting molecule to the chelator. In the preferred case where the targetting 20 molecule is peptidic, the conjugating group is reactive under conditions that do not denature or otherwise adversely affect the peptide. In one Qn~horlir ~ of the invention, the conjugating group is reactive with a functional group of the peptidic targetting molecule such 25 as the carboxy terminus or amino t~rm; n-lc Alternatively, the conjugating group can be reactive with an ~-amino group of a lysine residue. Conjugating groups reactive with amino groups of targetting molecules include carboxyl and activated esters. Conjugating 3 0 groups reactive with carboxyl groups of targetting molecules include amines and hydrazines.
For diagnostic and therapeutic purposes, the chelator per se may be used in combination with a detectable metal 35 capable of forming a complex. Suitable metals include radinn~lrl i~ec such as technetium and rhenium in their various forms such as 99~Tco3+, 99~Tco2+, ReO3+ and ReO2+.
~ Wo 95/17419 PcT/cAs~/on718 More desirably, the chelator is coupled to a targetting molecule that serves to localize the chelated metal to a desired location for diagnostic imaging or for therapy ie. radiation therapy of tumours. Examples of targetting 5 molecules include, but are not limited to, steroids, proteins, peptides, antibodies, nucleotides and saccharides. Preferred targetting molecules include proteins and peptides, particularly those capable of binding with specificity to cell surface L~ctl!l .,L~
l0 characteristic of a particular pathology. For instance, disease states associated with over-expression of particular protein receptors can be imaged by lAhPll;n~
that protein or a receptor binding fragment thereof in accordance with the present invention. Peptide-based 15 targetting molecules can be made by various known methods or in some instances can be commercially obtained. Solid phase synthesis employing alternating t-Boc protection and deprotection is the preferred method of making short peptides which can be an automated process. RPr hin lnt 20 DNA technology is preferred for producing proteins and long fragments thereof.
Chelators of the present invention are peptide derivatives and are most ef f iciently prepared by solid-25 phase peptide synthesis. In general solid-phase synthesis involves the stepwise addition of amino acid residues to a growing peptide chain that is linked to an insoluble support or matrix, such as polystyrene. The C-t~rmin~lC residue of the chelator is first anchored to a 30 commercially available support with its amino group protected with an N-protecting agent such as a t-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl (E~IOC) group. The amino protecting group is removed with suitable deprotecting 35 agents such as TFA in the case of tBOC or piperadine for FMOC and the next amino acid residue ( in N-protected form) is added with a coupling agent such as Wo 95/17419 2 17 ~ 3 7 9 PCT/CAs~/00718 dicyclocarbodiimide (DCC). Upon formation of a peptide bond the reagents are washed from the support. After addition of the final residue, the chelator is cleaved from the support with a suitable reagent such as 5 trifluoroacetic acid (TFA) or hydrogen fluoride (HF) and isolated .
The present invention Pnrc-r~ses chelators incorporating various heterocyclic groups containing a nitrogen atom 10 providcd that it is analogous in ~LLu~:LuLe to an amino acid in that there is a carboxyl carbon, alpha carbon and an alpha nitrogen wherein the alpha carbon and alpha nitrogen are incorporated in a common ring. For example, picolinic acid (pic), dipicolinic acid (dipic), 15 ch~ m;r acid (chel), 2-calboxy~yrazine~ 2-carboxypyrimidine, 2-caLLu-~y~yLLole, 2-quinolinic acid, l-isoquinolinic acid, 3-isoquinolinic acid and the like will behave as a natural amino acid residue in solid phase synthesis by forming a peptidic bond upon reaction 20 of the carboxyl group and a deprotected amino group of a previously added residue. Variation at R3 may be introduced to chelators of the invention simply by incorporation of a desired amino acid residue at the appropriate stage of chain elongation. For example, R3 25 may be a ;1YdL~J~SY ~ ~hyl group by using a serine residue or may be a hydrogen atom by using glycine. Any D or L, naturally occurring or derivati2ed amino acid may be used .
3 0 In accordance with an ~ nt of the present invention, R4 is a targetting molecule that is proteinaceous. Human Immunoglobulin G (HIG), a multisubunit protein, has been directly labelled with technetium-99m and used extensively for imaging sites of 35 inflammation, however smaller peptides are bPr in~ the targetting molecules of choice for their site specificity a~ a result of receptor binding properties and for their = _ _ _ _ _ . _ . . . . . , . .. .. , .. _ _ _ _ _ _ ~
~1 79~79 WO 9S/17419 PCT/CA9-i/00718 ease of preparation. An example of peptidic targetting molecules are Tuftsin antagonists such as Thr-Lys-Pro-Pro-Arg and Lys-Pro-Pro-Arg. Another peptidic targetting molecule useful for imaging inflammation is f~LP (formyl-5 Net-Lys-Phe) and derivatives thereof such as formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys described by Fischman et al in pending Canadian application CA 2,016,235. It is believed that frILP and various derivatives thereof bind to neutrophils and are therefore useful in imaging sites 10 of inf lammation .
In accordance with another aspect of the invention, the present invention provides a peptide useful as a targetting molecule which has the sequence formyl-Nleu-15 Leu-Phe-Nleu-Tyr-Lys-Lys-ASp-X-OH wherein X is a bond or an aminoacid residue. For convenient synthesis of this peptide, X is preferrably a glycine (Gly) residue. In vivo studies have shown this peptide coupled to a chelator of the present invention strongly binds to 20 neutrophils while having a more favourable neuL~ e.-ic profile than native fMLP or the derivative formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys .
Synthesis of a chelator/targetting molecule conjugate 2~ hereinafter referred to as a "conjugate" can be achieved in various ways. When R4 is a peptidic targetting molecule, it is convenient to synthesize the conjugate in toto by starting solid-phase synthesis from the C-tPrm; nllC residue of the targetting molecule and ending 30 with the heterocyclic residue (R~, R2) of the chelator.
Alternatively, a targetting molecule which incoLy.,rc.tes a lysine residue may be coupled to the chelator at R4 by way the ~-amino group of that lysine residue. In this case, the targetting peptide is synthesized as a separate chain 35 from the chelator and is differentially protected at the ~-amino group and N-t~mi ml~ amino group. For example the ~-amino group may be protected with 1- ( 4, 4-dimethyl-WO 95117419 2 1 7 ~ 37 9 PCT/CA9~1On718 ~
2, 6-dioxocyclohexylidine) -ethyl (Dde) while the N-t~rm;nllq amino is FMOC protected. When the targetting molecule 6ynthesis is complete the -amino group is deprotected with hydrazine and is available for reaction 5 with a C-terminus carboxyl group of a chelator while the N-t~rm; nllq amino group is protected.
Targetting molecules may also be coupled to chelators of the invention by way of a conjugating group substituent 10 on the heterocyclic ring of the chelator. For example, a chelator with an amino substituent on the heterocyclic ring upon deprotection will be reactive with the C-terminllq carboxyl group of a peptide targetting molecule.
Such a conjugate may be synthesized as a 6ingle chain 15 starting at the C-t~rmi nllq residue of the chelator and ending with the N-t~rminllq of the targetting molecule.
Alternatively, a peptide targetting molecule may be coupled to the heterocyclic residue by way of its N-t~rmi nl~q when the heterocyclic group has a suitable 20 conjugating group substituent such as a carboxyl group or an activated ester. In this case, the chelator and targetting molecule are synthesized as separate chains and then coupled to form the desired conjugate.
25 In accordance with one aspect of the invention, chelators incorporate a diagnostically or therapeutically useful metal. Inco~ tion of the metal within the chelator can be achieved by various methods common to the art of coordination chemistry. When the metal is the 30 radionuclide technetium-99m, the following general procedure may be used to form a technetium complex. A
chelator solution is formed initially by dissolving the chelator in aqueous alcohol eg. ethanol-water 1:1. The solution is degassed with nitrogen to remove oxygen then 35 the thiol protecting group is removed, for example with sodium hydroxide and heat. The solution is then neutralized with an organic acid such as acetic acid (pH
~ Wo 95/17419 2 ~ 7 9 3 7 9 PcTlcA94Jon7~8 6.0-6.5). In the labelling 6tep, sodium pertechnetate, obtained from a Molybdemun generator, is added to the chelator solution with an amount of stannous chloride - sufficient to reduce the technetium. The solution is mixed and left to react at room temperature and then heated on a water bath. In an alternative method, 1 Ah~l 1 i n~ can be accomplished with the chelator solution adjusted to pH 8. Pertechnetate may be replaced with a solution of technetium complexed with labile ligands suitable for ligand exchange reactions with the desired chelator. Suitable ligands include tartarate, citrate, gluconate and glucoheptonate. Stannous chloride may be replaced with sodium dithionite as the reducing agent if the chelating solution is alternatively adjusted to pH
12-13 for the labelling step. The labelled chelator may be separated from contaminants 99~TCoi and colloidal 99~TCo2 chromatographically, e.g. with a C-18 Sep Pak cartridge activated with ethanol followed by dilute HCl. Eluting with dilute HCl separates the 99~Tcoi, and eluting with EtOH-saline 1:1 brings off the chelator while colloidal 99~Tco2 remains on the column. The chelators of the invention can be coupled to a targetting molecule prior to labelling with the radionuclide, a process referred to as the "bifunctional chelate" method. An alternative approach known as the "prelabelled ligand" method, the chelator is first l Ah~ d with the desired metal and is subsequently coupled to the targetting molecule. This method is advantageous in that the targetting molecule itself is not inadvertently labelled at low affinity binding sites which may render the targetting molecule inactive or may release the metal in vivo.
An alternative approach for lAh~l 1 ing chelators of the present invention involves techniques described in a co-pending U.S. application 08/152,680 by Pollak et al, filed on 16 November 1993 incorporated herein by reference. Briefly, chelators are immobilized on a solid wo 95117419 2 1 7 9 3 7 9 PCT1CA9~100718 phase support in such a manner that they are released from the support only upon formation of a complex with the 1 AhPl l; n~ metal atom. Thi5 is achieved when the chelator is coupled to a functional group of the support 5 by one of the complexing atoms. Preferably, a complexing sulfur atom is coupled to the support which is functinn~l; 7P~ with sulfur a protecting group such as maleimide .
10 When coupled to a targetting molecule and 1 ~hPl 1 Pcl with a diagnostically useful metal, chelators of the present invention can be used to detect pathological conditions by techniques common in the art. A conjugate labelled with a radionuclide metal such as technetium may be 15 administered to a mammal by intravenous injection in a pharmaceutically acceptable solution such as saline or DMSO. The amount of 1 ~hPl 1 Pd conjugate administered is depPn~Prlt upon the toxicity profile of the chosen targetting molecule as well as the metal . T nt~~ 1; 7~tion 20 of the metal in vivo is tracked by standard scintigraphic techniques at appropriate time intervals subsequent to administration .
The following examples are presented to illustrate 25 certain embodiments of the present invention.
~Y~mnle l PreparatiQn qf Chelators and Conjugates Chelators were synthesized using 9-30 fluorenylmethyloxycarbonyl tFMOC) chemistry on an 2-methoxy-4-alkoxybenzyl alcohol resin preloaded with the protected C-tPrm;nllc residue (Sasrin resin, Bachem Biosciences Inc., ph;l l~plrhi~ PA) using an Applied Biosystems 433A peptide synthesizer (Foster City, CA).
Preparation of Chelators a . Chel-Gly-Cys (Acm) -Gly-OH
_ _ _ wo 9S/17419 2 1 7 ~ ~ 9 PCT/CA94100718 b. DiPic-Gly-Cys (Acm) -Gly-OH
c. Pic-Gly-Cys (Acm) -Gly-OH
- Synthesis began from the Gly residue preloaded on the 5 resin and continued to the final Pic, DiPic or Pic - residue by addition of one of picolinic, dipicolinic or t~hPl ~ m; C acid . The chelator-resin was dried in vacuo for 12 hours. Cleavage of the chelator from the resin involved mixing a cooled 601ution of 9596 trif luoroacetic 10 acid (TFA) and 5% water (lml per 100 mg of peptide-resin) with the peptide-resin for 1. 5 to 2 hours at room temperature. The resin was removed by filtration and washed 3 times with 30 ml t-butyl methyl ether in a 50 ml conical polypropylene centrifuge tube forming a white 15 precipitate. The precipitate was dissolved in water with added acetonitrile. The precipitate was frozen in acetone-dry ice and lyophilized over 12 hours. The resulting white powder was dissolved in water, filtered through a 0.45 ~m syringe filter (Gelman Acrodisc LC
20 PVDF) and purified by ~vt:l,,ed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCN 25 x 10) using 0.1%
TFA in water as buffer A and 0.1~6 TFA in acetonitrile as buffer B. The column was equilibrated with 100:0 buffer A:buffer B and eluted with a linear gradient in 25 min at 25 1 ml/min to 50% buffer B. Fractions were reanalysed on the HPLC and pooled according to matching prof iles . If nP~PC:s~ry the pooled fractions were repurified using the same conditions. The pure fractions were frozen in acetone-dry ice and lyorh i 1 i 7Pd over 10 hours to give a 3 0 white powder .
Preparation of Conjugates d . (Pic-Ser-Cys (Acm) -Gly) -Thr-Lys-Pro-Pro-Arg-OH;
e. (Pic-Ser-Cys (Acm) -Gly) -Lys-Pro-Pro-Arg-OH;
f . 2-Quinolinic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro-Arg-OH;
~o 95117~19 2 1 7 ~ 3 7 9 PCT/CA94100718 ~
g. l-Isoquinolinic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro-Arg -OH;
h. 3-Isoquinolinic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro -Arg-OH;
i. Indole-2-carboxylic acid-Ser-Cys (Acm) -Gly-Thr-Lys-Pro-Pro-Arg-OH; and j . Pyrrole-2-carboxylic acid-Ser-Cys (Acm) -Gly-Thr-Lys -Pro-Pro-Arg-OH .
Synthesi6 began from the Arg residue preloaded on the resin and continued to the Ser residue of the chelator ending with the addition of one of picolinic, 2-quinolinic, l-isoquinolinic~ 3-isoquinolinic, pyrrole-2-carboxylic and indole-2-carboxylic acid. The chelator-peptide-resin was dried in vacuo 12 hours. Cleavage from the resin involved mixing with a solution of 10 ml trifluoroacetic acid (~FA), O.5ml water, O.5ml thioanisole, 0.25ml 1,2-ethanedithiol (EDT) and 0.75g phenol f or 1. 5 to 2 hours at room temperature . The resin was removed by filtration and the peptide washed 3 times with 3 0 ml t-butyl methyl ether in a 50 ml conical polypropylene centrifuge tube forming a white precipitate. The precipitate was dissolved in water with added acetonitrile when solubility problems arose. The precipitate was frozen in acetone-dry ice and lyophilized over 12 hours. The resulting white powder wa6 dissolved in water, filtered through a 0.45 ~m syringe filter (Gelman Acrodisc LC PVDF) and purified by reversed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCM
25 x 10) using 0.1% TFA in water as buffer A and 0.1% TFA
in acetonitrile as buffer B. The column was equilibrated with 100:0 buffer A:buffer B and eluted with a linear gradient in 25 min at 1 ml/min to 50% buffer B.
Fractions were reanalysed on the HPLC and pooled according to matching profiles. If n~C~csAry the pooled fractions were repurified using the same conditions. The ~ wo 95/17419 2 1 7 ~ 3 7 q PCT/CAs~/007l8 pure fractions were frozen in acetone-dry ice and lyophilized over 10 hours to give a white powder.
Preparation of fMLP Conjugates k. (Pic-Gly-Cys-Gly) -~NH-Lys (-Gly-OH) -Tyr-Nleu-Phe-Leu-Nleu-f or 1. (Pic-Gly-Cys (Acm) -Gly) -lNH-Lys (-Gly-OH) -Tyr-Nleu-Phe-Leu-Nleu-f or m. (Pic-Gly-Cys (Acm) -Gly) -~-NH-Lys (-Asp-Gly-OH) -Lys-Tyr-Nleu-Phe-Leu-Nleu-f or Targetting peptides that comprise lysine residues can be coupled to the chelator via the Lys ~-amino group by the following procedure. For ~ u-lds example l(k), 1(1), and 1 (m) . the targetting peptide was initially synthesized from glycine to norleucine by 9-fluorenylmethyloxycarbonyl (FMOC) chemistry using an FMOC-glycine preloaded 2-methoxyl-4-alkoxyl-benzyl alcohol resin and a 1-(4,4-dimethyl-2,6-dioxocyclohexylidine)-ethyl (Dde) orthogonal protected lysine with an Applied Biosystems 433A peptide synthesizer. The fMLP peptide-resin was removed from the synthesizer and dried 12 hours in vacuo to prepare for formylation.
Formic anhydride was prepared by heating acetic anhydride (2 eguivalents) with formic acid (1 equivalent) to 50-C
for 15 minutes followed by cooling to O-C. Formylation of the fMLP peptide involved swelling the peptide-resin in dichloromethane (DCM) (5 ml) followed by swirling with formic anhydride (5ml) for 15 minutes. The formylated fMLP peptide-resin was filtered, washed with DCM and dried in vacuo 12 hours.
Formylated peptide-resin (50 mg/2ml) was swirled with a 296 hydrazine hydrate in N-methylpyrrolidone (NMP) WO 95/17419 PCT/C~94/00718 ~
21 79379 ~16 solution for 3 minutes two times then filtered and washed with DCN and dried in vacuo 12 hours to remove the -amino lysine protecting group (Dde) while leaving the N-t~rmin~ amino group formylated.
The chelator was added to the ~-amino lysine of the fNLP
peptide on the 433A peptide synth~si 7~r. The chelator-peptide-resin was dried in vacuo 12 hours. Cleavage from the resin involved mixing a cooled solution of 95%
trifluoroacetic acid (TFA) and 5% water (1 ml per 100 mg of chelator-peptide-resin) with the chelator-pep~ide-resin for 1.5 to 2 hours at room temperature. The resin wa6 removed by f iltration and washed with 1-3 ml of TFA
to obtain 6-8 ml of a clear yellow liquid. This liguid was 610wly dropped into 30-35 ml of tert-butyl methyl ether in a 50 ml conical polypropylene centrifuge tube f orming a white precipitate . The precipitate was centrifuged at 7000 rpm, O'C for 5 minutes (Sorvall RT
6000, Dupont~, decanted and washed two more times with t-2 0 butyl methyl ether . Following drying under vacuum the precipitate was dissolved in water with added acetonitrile when solubility problems arose. The precipitate was frozen in acetone-dry ice and lyo~hi 1 i 7 d over 10 hours. The resulting white powder was dissoved in dimethylsulfoxide (20 ,uL) and 50:50 acetonitrile:water solution (980 ,uL), filtered through a 0.45 ,um syringe filter (Gelman Acrodisc I.C PVDF), and purified by reversed-phase HPLC (Beckman System Gold) with a C18 column (Waters RCM 25 X 10) using 0.1% TFA in water as buffer A and 0.1% TFA in acetonitrile as buffer B. The column was equilibrated wlth 50:50 buffer A:buffer B and eluted with a linear gradient in 25 min at 1 ml/min to 100% buffer B. Fractions were reanalysed on the HPLC and pooled according to matching prof iles . If n~ Ei~ry, the pooled fractions were repurified using the same conditions. The pure fraction6 were frozen in acetone-WO 95117419 C~ 1 7 9 3 7 9 PCT/C~94100718 dry ice and lyophilized over 12 hours to give a white powder .
le 2 T-AhPl 1 in~J Chelators with 59"Tc - The chelators and conjugates of example 1 (lmg) were dissolved in 2001~L EtOH-water (1:1) in a tube. 100-200~L
sodium pertechnetate (200-600 M;3a, 5-15 mCi), 100 ,uL
phosphate buffer (0.25 M, pH 7.4), and 200 ,uL of a solution containing 50~g stannous chloride dihydrate and 40mg sodium tartrate were added to the tube and capped tightly and placed in a boiling water bath for 10 minutes. In order to achieve adequate separation of the chelators, the solution was then loaded on a C-18 Sep-Pak column activated by washing sequentially with 5ml methanol, 10ml water and 5ml dilute (lmM) HCl to remove Tc04-. Subsequent elution with 2ml EtOH-saline (1:1) removed the chelator while TcOz remained on the column.
The extent of complexation of 99~Tc with chelators was measured by radioactivity of the eluted fractions.
Example J.Ahel 1 i n~ Yield ( % total radioactivity) 1 (a) 7 1 (c) 92 1 (d) 93 1 (e) 92 1 (k) 29 1(1) 63 1 (m) 78 wo 95/17~19 pcTlcA94lon7l8 ~1 7q379 18 Con~ugates l(f) - l(j), were reconstituted (200,u~., lmg/mL
saline) and then injected into 3mL vacutainers with lOO,~IL
pertechnetate (lOmCi) and lOO,uL stannous gluconate (50 ~Lg ~tannous chloride and 1 mg sodium gluconate). The tubes 5 were placed in boiling water bath f or 12 minutes and then f iltered through a Whatman PVDF 5yringe f ilter to collect the ~ AhPl 1 efl conjugate solutions which were further diluted with saline to prepare injectable solutions (2Mbq/mL). The conjugate5 were isolated by HPLC
10 (Beckman) from a (20,uL) sample (before dilution) to determine the lAh~l1 in7 yield by measuring radioactivity.
example T.AhP11 in~ Yield (96) l(f) 94.4 l(g) 96.8 1 (h) 94 l(i) 96.3 l(j) 98.4 T~Y;~ le 3 In vivo imaging and bio~ ribution of chelators and cullju~-ll es For chelators and conjugates of example 1, rat 25 inflammation studies were performed as follows. 2 male Sprague-Dawley rats (Charles River, 250-300g) were injected intrArllcc~ll Arly with 5mg zymosan, a yeast cell wall preparation (20mg for conjugates l(f~ - l(i)) or a virulent E. coli (ATCC 25922, O . lml ûf 1. OXl09 30 organisms/ml) suspension into their right hindlegs 24 hours before imaging. Focal inflammation in the leg was visually detectable after 1 day. lmg (ca. O . 7 ~LMol) of the chelator was dissolved in 50 ,uL of dimethylsulfoxide and added to an ethanol-water mixture (1:1, 200 ,uL). An 35 aliquot of Tc-99m tartarate (ca. 400 r~Bq) was added and ~ wo 95117419 2 1 7 q 3 7 9 PCT/CA9~/007~8 transchelation allowed to proceed for 20 min. at lOO C.
The Tc-99m chelate was purified by elution through a Sep Pak cartridge . The purif ied tracer solution was further diluted with saline to prepare an injectable (200 ~L) 5 containing about 100 uCi (3.7 MBq) of activity.
The rats were anaesthetized with sodium pentobarbital (40 to 50 mg/kg), and the 1 ~h.ol 1 Pd chelator/conjugate solution (200uL) was injected intravenously via the tail 10 vein. Serial whole-body scintigrams were acquired for the first 5 minutes. Subsequently, further images were obtained at 30, 60, and 120 minutes. The rats were then killed with anaesthesia and samples of organs, urine, blood, inflamed muscle (right leg) and non-inflamed 15 muscle ( left leg) were weighed and counted in either a well-type gamma counter or in a gamma dose calibrator.
The dose calculations were made based on assumption that the blood volume constituted 6 . 5% of body weight .
Results are averages for two rats and are corrected for 20 the residual dose in the tail.
WO 95/17419 PCT~CA9.1/00718 21 7937~ 20 E E ~q G ~ ~ NO
- Cc ~! N _ cO ~ ~ N IN
C C e~ 't ~ ~ ~ O
E C E E E E E ", UJ ~
N N N N N
C 0 0 ~ U~ O O U~
'~~ O O O O O O O O O
C
E ~ N
~ E
0 0 ~ U~ 0 ~
.E ~ æ N
~o G ~ ~ 0 C~
N
o G 8 N ~ G r~ g G
o G 1~ ~ N ~ O
I ~q ~ C" Lo ~ Co ~0 0 ~
~ ~, 8 o ,` G r- O N
, ~" ~ cq In ID ~ G G
~_~ .~ O, N _ ~
1::1 G 1~ 0 0 0 ~ IN ~ G G
O . ~ ~ ~ , , G O
D _ O G ~q G G G ~
-E _ '~ ~ o _ C _ _ `' E ~
o WO 95/17~19 PCT/CA94/007 ~ A le 4 ll LLu~hil Binding Assay of fMLP and fMLP
Derivative Conjugates of Example l(l) and l(m) Rat peripheral neutrophils were prepared for binding 5 assay as follows: blood was obtained by cardiac ~ul--;Lu~e - and anticoagulated with acid- citrate dextrose (ACD) (10%). Red blood cells were removed by 5Prl; ation on hydroxyethyl cellulose (1.1%) for 30 min at room temperature and leukocyte-rich supernatant layered onto 65% percol. Centrifugation at 400g for 30 min resulted in a distinct band of mononuclear cells (ly ~-_y~es and monocytes~ which was discarded, the neutrophil rich pellet was rPc~cppn~lpd and ro~~;n;nrJ red blood cells lysed by hypotonic shock using cold water. The ~ ;n;nq 5 neuL~ u~hils were rPcucppnr~ed in Hanks Buffered Salt Solution (HBSS) to the desired concentration. Final neutrophil preparation consisted of cells pooled from up to 10 animals, >90% neutrophils and >9596 viable by Trypan Blue exclusion.
Binding affinity of the fMLP peptide and conjugates 1(1) and l(m) was AcsPcsed by ,- ~;n~ off a constant concentration of tritiated fMlP of known affinity for neutrophil receptors. 106 neutrophils were added to 25 polypropylene plates containing 15n~ tritiated f~LP and varying concentrations of -nl Ah'~l 1 ed test peptide and conjugates in a final volume of 150,uL HBSS. The plate was incubated for 1 hour at room temperature after which cells were harvested by filtration onto glass fibre 30 filter mats (Skatron receptor binding filtermat) using a Skatron cell harvester with 12 well head. Harvested cells were washed with ice-cold saline and air dried.
Filters were then placed in 6ml scintillation vials, 5ml of scintillation fluid added (Ecolume) and vials counted 35 using a liquid scintillation counter. Binding affinity of the f~qLP peptide and conjugates 1(1) and l(m) is illustrated in figure 1 and expressed as ~6 maximal Wo gs/17419 PCT/CA94/00718 ~
tritiated fMLP binding V5. peptide/conjugate concentration. % maximal tritiated fMLP
binding=(specific binding . maximum binding) X 100%.
Specif ic binding was the total binding less non-specif ic binding which was the amount of residual radioactivity bound in the presence of lOuM l~nl ~hPl 1 e~ fMLP. Both 1(1) and 1 (m) had greater binding affinity for neutrophil receptors than native fMLP.
ExamPle 5 Neul~v~ ia Assay of fMLP, fMLP Derivative formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys and Conjugate 1 (m) The effect of fMLP, fMLP derivative formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys and conjugate of example 1 (m) on circulating neutrophil number was assessed using the rat transient neutropenia model. Rats were anaesthetized with 250,uL
somnitol (16mg/rat) and injected via the tail vein at T=0 with the test peptides. At a range of time points after inj ection ( 0, 2, 5, 10, 3 0 min) a 2ml blood sample was taken by cardiac puncture (anticoagulated with 10% ACD).
3 animal were used per time point. For each sample the total white blood cells/ml and % neutrophil6 was det~rm; nPd, the number of neutrophils/ml in each sample being calculated. Within each experiment the number neutrophils/ml after saline injection at all time points was meaned to give a saline control against which the peptides could be compared. The number neutrophils/ml after peptide injection was expressed as a % of the saline control within each experiment.
Referring to figure 2, injections of 5 and 10 nmoles of fMLP produced a dose-~lPrPn(lpnt transient n~:uLLv~ ia~
with a maximal effect occurring 2 min after peptide injection (15 and 9% of control respectively) returning to 93 and 7596 of control values by 30 min after in; ection . 5nmoles of f ormyl-Nleu-Leu-Phe-Nleu-Tyr-Lys produced a smaller maximal reduction in circulating .. . . . . . .. _, . , 2 1 7 ~ 3 7 9 9410071~
wo gS/17419 2 3 PCT/CA
neutrophils (45% o~ control) while l(m) produced only a small transient drop in circulating neutrophils (8096 of control) at 5nmoles.
Claims (24)
1. A compound of the general formula:
wherein R1 and R together form a 5- or 6-membered heterocyclic ring which is optionally fused to a 5- or 6-membered ring, wherein either ring is optionally substituted with a conjugating group or with a conjugating group having a targetting molecule coupled thereto;
R3 is selected from H; alkyl; and alkyl substituted by a group selected from amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl and imidazolyl;
R4 is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is H or a sulfur protecting group;
wherein R1 and R together form a 5- or 6-membered heterocyclic ring which is optionally fused to a 5- or 6-membered ring, wherein either ring is optionally substituted with a conjugating group or with a conjugating group having a targetting molecule coupled thereto;
R3 is selected from H; alkyl; and alkyl substituted by a group selected from amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl and imidazolyl;
R4 is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is H or a sulfur protecting group;
2. A compound according to claim 1, wherein the ring formed by R1 and R is a five or six membered heterocyclic ring optionally fused to a benzene ring.
3. A compound according to claim 1, wherein R1 and R
together form a ring selected from pyridine, quinoline, isoquinoline, pyrrole and indole.
together form a ring selected from pyridine, quinoline, isoquinoline, pyrrole and indole.
4. A compound according to claim 1, wherein R1 and R2 together form a ring selected from 6-carboxypyridine and 4-hydroxy-6-carboxypyridine.
5. A compound according to claim 1 wherein R3 is selected from H and hydroxymethyl.
6. A compound according to claim 1, wherein R4 is selected from -Gly-OH and -Gly-targetting molecule.
7. A compound according to claim 6, wherein the targetting molecule is a peptide.
8. A compound according to claim 7, wherein the peptide has a sequence selected from -NH-Lys-Pro-Pro-Arg-OH; and -NH-Thr-Lys-Pro-Pro-Arg-OH.
9. A compound according to claim 7, wherein the Gly forms an amide linkage with an .epsilon.-amino Lys residue of the peptide selected from:
-.epsilon.-amino Lys (-Gly-OH)-Tyr-Nleu-Phe-Leu-Nleu-formyl;
and -.epsilon.-amino Lys (-Asp-Gly-OH) -Lys-Tyr-Nleu-Phe-Leu-Nleu-formyl.
-.epsilon.-amino Lys (-Gly-OH)-Tyr-Nleu-Phe-Leu-Nleu-formyl;
and -.epsilon.-amino Lys (-Asp-Gly-OH) -Lys-Tyr-Nleu-Phe-Leu-Nleu-formyl.
10. A compound according to claim 1, wherein R1 and R2 together form a pyridine ring; R3 is H; T is Acm; and R4 is selected from:
-Gly-OH; and -Gly-.epsilon.-amino Lys (-Gly-OH) -Tyr-Nleu-Phe-Leu-Nleu-formyl.
-Gly-OH; and -Gly-.epsilon.-amino Lys (-Gly-OH) -Tyr-Nleu-Phe-Leu-Nleu-formyl.
11. A compound according to claim 1, wherein R1 and R2 togethPr form a pyridine ring; R3 is hydroxymethyl; T is Acm; and R4 is selected from:
--Gly--OH;
-Gly-Lys-Pro-Pro-Arg-OH;
-Gly-Thr-Lys-Pro-Pro-Arg-OH;
-Gly-.epsilon.-amino Lys (-Gly-OH)-Tyr-Nleu-Phe-Leu-Nleu-formyl; and -Gly-.epsilon.-amino Lya(-Asp-Gly-OH)-Lys-Tyr-Nleu-Phe-Leu-Nleu-formyl.
--Gly--OH;
-Gly-Lys-Pro-Pro-Arg-OH;
-Gly-Thr-Lys-Pro-Pro-Arg-OH;
-Gly-.epsilon.-amino Lys (-Gly-OH)-Tyr-Nleu-Phe-Leu-Nleu-formyl; and -Gly-.epsilon.-amino Lya(-Asp-Gly-OH)-Lys-Tyr-Nleu-Phe-Leu-Nleu-formyl.
12. A compound according to claim 1, selected from:
2-Quinolinic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH;
1-Isoquinolinic acid-Ser-Cys(Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH;
3-Isoquinolinic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH;
Pyrrole-2-carboxylic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH; and Indole-2-carboxylic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH.
2-Quinolinic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH;
1-Isoquinolinic acid-Ser-Cys(Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH;
3-Isoquinolinic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH;
Pyrrole-2-carboxylic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH; and Indole-2-carboxylic acid-Ser-Cys (Acm)-Gly-Thr-Lys-Pro-Pro-Arg-OH.
13. A compound according to claim 1, wherein R1 and R2 together form a ring selected from a 6-carboxypyridine ring and a 6-carboxy-4-hydroxypyridine ring; R3 is hydroxymethyl; T is Acm; and R4 is -Gly-OH.
14. A compound according to claim 1, in a form complexed with a metal or an oxide or nitride thereof.
15. A compound according to claim 10, wherein the metal is 99mTc.
16. A compound according to claim 11, in a form complexed with 99mTc.
17. A compound according to claim 12, in a form complexed with 99mTc.
18. A method of imaging for sites of in vivo localization of a targetting molecule comprising the steps:
1) administering a diagnostically effective amount of a compound according to claim 15, comprising said targetting molecule; and 2) detecting localization of the compound.
1) administering a diagnostically effective amount of a compound according to claim 15, comprising said targetting molecule; and 2) detecting localization of the compound.
19. A compound of the general formula:
wherein:
R1 and R2 together form a 5- or 6-membered heterocyclic ring which is optionally fused to a 5- or 6-membered ring, wherein either ring is optionally substituted with a conjugating group or with a conjugating group having a targetting molecule coupled thereto;
R3 is selected from H; alkyl; and alkyl substituted by a group selected from amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl and imidazolyl;
R4 is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is H or a sulfur protecting group;
wherein the targetting molecule is a peptide having a sequence:
formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-Lys-Asp-X-OH
and X is a bond or an amino acid residue.
wherein:
R1 and R2 together form a 5- or 6-membered heterocyclic ring which is optionally fused to a 5- or 6-membered ring, wherein either ring is optionally substituted with a conjugating group or with a conjugating group having a targetting molecule coupled thereto;
R3 is selected from H; alkyl; and alkyl substituted by a group selected from amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl and imidazolyl;
R4 is selected from hydroxyl; alkoxy; an amino acid residue; and a targetting molecule; and T is H or a sulfur protecting group;
wherein the targetting molecule is a peptide having a sequence:
formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-Lys-Asp-X-OH
and X is a bond or an amino acid residue.
20. A compound according to claim 19, wherein X is -Gly-.
21. A compound according to claim 19, having the formula:
wherein X is a bond or an amino acid residue.
wherein X is a bond or an amino acid residue.
22. A compound according to claim 21, wherein X is -Gly-.
23. A peptide having the general sequence:
formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-Lys-Asp-X-OH
wherein X is a bond or an amino acid residue.
formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-Lys-Asp-X-OH
wherein X is a bond or an amino acid residue.
24. A peptide according to claim 23, wherein X is -Gly-.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US171,737 | 1993-12-22 | ||
| US08/171,737 US5480970A (en) | 1993-12-22 | 1993-12-22 | Metal chelators |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2179379A1 true CA2179379A1 (en) | 1995-06-29 |
Family
ID=22624941
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002179379A Abandoned CA2179379A1 (en) | 1993-12-22 | 1994-12-22 | Metal chelators |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5480970A (en) |
| EP (1) | EP0733060B1 (en) |
| JP (1) | JPH09506870A (en) |
| AT (1) | ATE296312T1 (en) |
| AU (1) | AU1378395A (en) |
| CA (1) | CA2179379A1 (en) |
| DE (1) | DE69434384T2 (en) |
| WO (1) | WO1995017419A1 (en) |
Families Citing this family (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5443815A (en) * | 1991-11-27 | 1995-08-22 | Diatech, Inc. | Technetium-99m labeled peptides for imaging |
| US5965107A (en) * | 1992-03-13 | 1999-10-12 | Diatide, Inc. | Technetium-99m labeled peptides for imaging |
| EP0730472B1 (en) * | 1993-11-16 | 2006-08-02 | Bracco International B.V. | Immobilized labelling method |
| US5569745A (en) * | 1994-02-25 | 1996-10-29 | Resolution Pharmaceuticals Inc. | Peptide-Chelator conjugates |
| US5662885A (en) * | 1994-07-22 | 1997-09-02 | Resolution Pharmaceuticals Inc. | Peptide derived radionuclide chelators |
| US5665385A (en) * | 1994-12-09 | 1997-09-09 | Sound Nutrition, Inc. | Dietary metal supplements |
| US5804158A (en) * | 1995-05-31 | 1998-09-08 | Resolution Pharmaceuticals Inc. | Sequestered imaging agents |
| US5891418A (en) * | 1995-06-07 | 1999-04-06 | Rhomed Incorporated | Peptide-metal ion pharmaceutical constructs and applications |
| US6027711A (en) * | 1995-06-07 | 2000-02-22 | Rhomed Incorporated | Structurally determined metallo-constructs and applications |
| US5688489A (en) * | 1995-09-15 | 1997-11-18 | Resolution Pharmaceuticals, Inc. | Non-receptor mediated imaging agents |
| GB9708265D0 (en) | 1997-04-24 | 1997-06-18 | Nycomed Imaging As | Contrast agents |
| US6334996B1 (en) * | 1997-12-24 | 2002-01-01 | Resolution Pharmaceuticals Inc. | Chelators that predominantely form a single stereoisomeric species upon coordination to a metal center |
| WO1999040947A2 (en) * | 1998-02-11 | 1999-08-19 | Resolution Pharmaceuticals Inc. | Angiogenesis targeting molecules |
| WO1999051628A1 (en) | 1998-04-03 | 1999-10-14 | Du Pont Pharmaceuticals Company | Radiopharmaceuticals for imaging infection and inflammation and for imaging and treatment of cancer |
| US6872536B1 (en) * | 1998-11-10 | 2005-03-29 | Bracco Imaging S.P.A. | Chemotactic peptide antagonists for imaging sites of inflammation |
| US6409987B1 (en) | 1999-04-07 | 2002-06-25 | Intimax Corporation | Targeted agents useful for diagnostic and therapeutic applications |
| US8263739B2 (en) * | 2000-06-02 | 2012-09-11 | Bracco Suisse Sa | Compounds for targeting endothelial cells, compositions containing the same and methods for their use |
| EP2286843A3 (en) | 2000-06-02 | 2011-08-03 | Bracco Suisse SA | Compounds for targeting endothelial cells |
| US7311893B2 (en) * | 2000-07-25 | 2007-12-25 | Neurochem (International) Limited | Amyloid targeting imaging agents and uses thereof |
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-
1993
- 1993-12-22 US US08/171,737 patent/US5480970A/en not_active Expired - Lifetime
-
1994
- 1994-12-22 JP JP7517075A patent/JPH09506870A/en not_active Ceased
- 1994-12-22 EP EP95904992A patent/EP0733060B1/en not_active Expired - Lifetime
- 1994-12-22 AU AU13783/95A patent/AU1378395A/en not_active Abandoned
- 1994-12-22 CA CA002179379A patent/CA2179379A1/en not_active Abandoned
- 1994-12-22 DE DE69434384T patent/DE69434384T2/en not_active Expired - Fee Related
- 1994-12-22 WO PCT/CA1994/000718 patent/WO1995017419A1/en active IP Right Grant
- 1994-12-22 AT AT95904992T patent/ATE296312T1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DE69434384D1 (en) | 2005-06-30 |
| US5480970A (en) | 1996-01-02 |
| ATE296312T1 (en) | 2005-06-15 |
| JPH09506870A (en) | 1997-07-08 |
| AU1378395A (en) | 1995-07-10 |
| WO1995017419A1 (en) | 1995-06-29 |
| EP0733060A1 (en) | 1996-09-25 |
| DE69434384T2 (en) | 2006-03-23 |
| EP0733060B1 (en) | 2005-05-25 |
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| Date | Code | Title | Description |
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| EEER | Examination request | ||
| FZDE | Discontinued |