US20110130555A1 - Lipid-polymer conjugates, their preparation and uses thereof - Google Patents

Lipid-polymer conjugates, their preparation and uses thereof Download PDF

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US20110130555A1
US20110130555A1 US12/997,014 US99701410A US2011130555A1 US 20110130555 A1 US20110130555 A1 US 20110130555A1 US 99701410 A US99701410 A US 99701410A US 2011130555 A1 US2011130555 A1 US 2011130555A1
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another embodiment
lipid
gag
unsaturated
polymer conjugate
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Saul Yedgar
Yuval Cohen
Joseph V. Bondi
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Morria Biopharmaceuticals Ltd
Yissum Research Development Co of Hebrew University of Jerusalem
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Morria Biopharmaceuticals Ltd
Yissum Research Development Co of Hebrew University of Jerusalem
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Priority to US12/997,014 priority Critical patent/US20110130555A1/en
Assigned to MORRIA BIOPHARMACEUTICALS, YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM, LTD. reassignment MORRIA BIOPHARMACEUTICALS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YEDGAR, SAUL, BONDI, JOSEPH V., COHEN, YUVAL
Publication of US20110130555A1 publication Critical patent/US20110130555A1/en
Priority to US13/283,020 priority patent/US8859524B2/en
Priority to US13/316,592 priority patent/US8906882B2/en
Priority to US13/788,325 priority patent/US20140005115A1/en
Priority to US14/329,191 priority patent/US20140323434A1/en
Priority to US14/525,111 priority patent/US11013811B2/en
Assigned to MORRIA BIOPHARMACEUTICALS reassignment MORRIA BIOPHARMACEUTICALS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BONDI, JOSEPH V., COHEN, YUVAL
Assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM, LTD. reassignment YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YEDGAR, SAUL
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
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    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention provides low molecular weight lipid-GAG conjugates and methods of use thereof in suppressing, inhibiting, preventing, or treating a pathogenic effect on a cell, including, inter alia, infection with intracellular pathogens.
  • Lipid-conjugates having a pharmacological activity of inhibiting the enzyme phospholipase A2 are known in the prior art.
  • Phospholipase A2 catalyzes the breakdown of phospholipids at the sn-2 position to produce a fatty acid and a lysophospholipid.
  • the activity of this enzyme has been correlated with various cell functions, particularly with the production of lipid mediators such as eicosanoid production (prostaglandins, thromboxanes and leukotrienes), platelet activating factor and lysophospholipids.
  • Lipid-conjugates may offer a wider scope of protection of cells and organisms from injurious agents and pathogenic processes, including the prevention and treatment of microbial infections. Lipid-conjugates may offer a wider scope of protection of cells and organisms from injurious agents and pathogenic processes, including the prevention and treatment of microbial infections.
  • Lipid-conjugates have been subjected to intensive laboratory investigation in order to obtain a wider scope of protection of cells and organisms from injurious agents, pathogenic and inflammatory processes.
  • the present invention provides a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a mass PL to mass GAG ratio from about 0.25:15 to about 5:15, respectively.
  • GAG glycosaminoglycan
  • PL phospholipid
  • the present invention provides a lipid-polymer conjugate comprising a glycosaminglycan (GAG) conjugated to a phospholipid (PL) via an amide or ester linkage wherein the molecular weight of said GAG is between 5 to 20 kD.
  • GAG glycosaminglycan
  • PL phospholipid
  • the present invention provides a lipid-polymer conjugate represented by the structure of the general formula (A):
  • the present invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • the present invention provides a method of treating inflammatory disorders in a subject, said method comprising administering to a subject suffering from an inflammatory disorder a composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a mass PL to mass GAG ratio from about 0.25:15 to about 5:15, respectively.
  • the present invention provides a method for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof comprising the step of administering a compound represented by the structure of the general formula (A):
  • the present invention provides a method of activating NF- ⁇ B, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines comprising the step of administering to a subject a compound represented by the structure of the general formula (A):
  • FIG. 1 depicts a conceptual diagram of the reaction vessel features required to practice the methods of this invention.
  • FIG. 2 depicts an NMR spectrum of a hyaluronic acid-phosphatidylethanolamine conjugate (HyPE) prepared according to Example 5.
  • HyPE hyaluronic acid-phosphatidylethanolamine conjugate
  • FIG. 3 is an HPLC chromatogram of HyPE prepared according to Example 5.
  • FIG. 4 depicts a schematic representation of the in vitro stimulation of RAW 264.7 cells.
  • FIG. 5 depicts the mean XTT reduction (OD 450 ) by RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 6 depicts the mean XTT reduction (OD 450 ) by LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 7 depicts the mean TNF- ⁇ release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 8 depicts the mean TNF- ⁇ release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 9 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 10 depicts the mean IL-6 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 11 depicts the mean IP-10 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 12 depicts the mean IP-10 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 13 depicts the mean PGE 2 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 14 depicts the mean PGE 2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 19 is the chromatogram from the SEC-MALS molecular weight analysis of low molecular weight sodium hyaluronate.
  • the red line pertains to the light scattering signal.
  • the blue line refers to the refractive index signal.
  • FIG. 20 is the SEC-MALS determined distribution of molecular weight of low molecular weight sodium hyaluronate.
  • FIG. 21 is the UV spectrum of sample 208-088 (low molecular weight sodium hyaluronate).
  • FIG. 22 depicts the mean XTT reduction (OD 450 ) by RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 23 depicts the mean XTT reduction (OD 450 ) by LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 24 depicts the mean TNF- ⁇ release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 25 depicts the mean TNF- ⁇ release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 26 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 27 depicts the mean IL-6 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 28 depicts the mean IP-10 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 29 depicts the mean IP-10 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 30 depicts the mean PGE 2 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 31 depicts the mean PGE 2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 36 depicts a photograph of the actual reaction vessel used for the preparation of HyPE.
  • the chiller is behind the reaction vessel and the door on the sound-proof container is open to reveal the ultrasound flow-cell.
  • FIG. 37 depicts a chromatogram of the HyPE reaction from Example 11 after 2 hours.
  • FIG. 38 depicts a chromatogram of the HyPE reaction from Example 11 after 6 hours.
  • FIG. 39 depicts the GPC analysis of final HyPE isolated from Example 11.
  • FIG. 40 depicts the NMR spectrum of final HyPE isolated from Example 11 and treated with 1 drop of 4% NaOD.
  • DCC refers to dicyclohexylcarbodiimide
  • EDAC refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • BOP refers to Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate
  • PyBOP refers to benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
  • HATU refers to O-(7-Azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluronium hexafluorophosphate
  • TSTU refers to O—(N-Succinimidyl)-N,N,N′,N′-t
  • lipid refers to all types of lipids including phospholipids, glycerolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and the like.
  • This invention provides, in one embodiment, a lipid-polymer conjugate which is useful in some embodiments for the treatment of inflammatory disorders.
  • this invention provides a method for the preparation of the lipid-polymer conjugates of this invention. In some embodiments, this invention provides a method for the use of the lipid-polymer conjugates of this invention.
  • this invention provides a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a mass PL to mass GAG ratio from about 0.25:15 to about 5:15, respectively.
  • GAG glycosaminoglycan
  • PL phospholipid
  • said mass PL to mass GAG ratio is about 0.25:15. In another embodiment, said mass PL to mass GAG ratio is about 0.5:15. In another embodiment, said mass PL to mass GAG ratio is about 1:15. In another embodiment, said mass PL to mass GAG ratio is about 2:15. In another embodiment, said mass PL to mass GAG ratio is about 5:15.
  • the present invention provides a lipid-polymer conjugate comprising a glycosaminglycan (GAG) conjugated to a phospholipid (PL) via an amide or ester linkage wherein the molecular weight of said GAG is between 5 to 20 kD.
  • GAG glycosaminglycan
  • PL phospholipid
  • said GAG of the lipid-conjugate compound of this invention is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate.
  • said GAG is hyaluronic acid.
  • said GAG is heparin.
  • said GAG is chondroitin.
  • said GAG is chondroitin sulfate.
  • said GAG is dermatan sulfate, in another embodiment, said GAG is keratan sulfate.
  • said chondroitin sulfate is chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof.
  • said dermatan sulfate is dermatan-6-sulfate, dermatan-4-sulfate or a derivative thereof.
  • said PL of the lipid-conjugate compound of this invention is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol.
  • said PL comprises the residue of palmitic acid, myristic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid or docosahexaenoic acid.
  • said PL is dimyristoyl phosphatidylethanolamine.
  • said PL is dipalmitoyl phosphatidylethanolamine.
  • the polydispersity of said GAG is from about 1 to 1.75. In another embodiment, the polydispersity of said GAG is from about 1.25 to 1.5.
  • the lipid-polymer conjugate of this invention comprises a GAG wherein the average molecular weight of said GAG is between 5 kd to 90 kd. In another embodiment, the average molecular weight of said GAG is between 5 kD to 60 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 40 kD.
  • the average molecular weight of said GAG is between 5 kD to 15 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 20 kD.
  • low molecular weight GAG such as sodium hyaluronate is prepared by acid hydrolysis of sodium hyaluronate as described in Example 9.
  • said acid hydrolysis comprises hydrochloric acid.
  • said acid hydrolysis comprises sulfuric acid.
  • said acid hydrolysis comprises trifluoroacetic acid.
  • said acid hydrolysis comprises hydrobromic acid.
  • said acid hydrolysis comprises acetic acid.
  • the concentration of the acid in said acid hydrolysis is from about 0.1 to 12 molar.
  • the concentration of the acid in said acid hydrolysis is from about 1 to 6 molar.
  • the concentration of the acid in said acid hydrolysis is from about 6 to 12 molar.
  • said acid hydrolysis is carried out at a temperature between 25 degrees Celsius to 100 degrees Celsius. In another embodiment, said acid hydrolysis is carried out at a temperature between 25 degrees Celsius to 50 degrees Celsius. In another embodiment, said acid hydrolysis is carried out at a temperature between 50 degrees Celsius to 100 degrees Celsius.
  • the molecular weight of hyaluronic acid and derivatives is determined by size exclusion chromatography and multiangle light scattering (SEC-MALS) as described in Example 10.
  • SEC-MALS size exclusion chromatography and multiangle light scattering
  • the chromatogram and distribution diagram are stated in FIG. 19 and FIG. 20 whereas the red line pertains to light scattering signal and the blue line to refractive index signal.
  • FIG. 21 illustrates the UV spectrum.
  • Light scattering measurements can provide an absolute measurement of molar mass when used in series with a concentration sensitive detector such as a refractive index detector and if the value of dn/dc (differential refractive index increment) is known. In essence, light scattering measurements automatically provide a column calibration curve for every sample, obviating time-consuming, conformation dependent calibration procedure.
  • the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in a phosphate buffer. In another embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in an acetate buffer. In another embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in a tris buffer. In another embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in a MES buffer.
  • this invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a mass PL to mass GAG ratio from about 0.25:15 to about 5:15, respectively.
  • the average molecular weight of said GAG is between 5 kD to 90 kD.
  • the average molecular weight of said GAG is between 5 kD to 20 kD.
  • the average molecular weight of said GAG is greater than 10 kD.
  • this invention provides a lipid-polymer conjugate represented by the structure of the general formula (A):
  • L is a lipid. In another embodiment L is a phospholipid. In another embodiment, L is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol. In another embodiment, L comprises the residue of palmitic acid, myristic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid or docosahexaenoic acid. In another embodiment, L is dimyristoyl phosphatidylethanolamine. In another embodiment, said L is dipalmitoyl phosphatidylethanolamine.
  • X is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate.
  • X is hyaluronic acid.
  • X is heparin.
  • X is chondroitin.
  • X is chondroitin sulfate.
  • X is dermatan sulfate, in another embodiment, X is keratan sulfate.
  • said chondroitin sulfate is chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof.
  • said dermatan sulfate is dermatan-6-sulfate, dermatan-4-sulfate or a derivative thereof.
  • said lipid-polymer conjugate is represented by the structure of the general formula (I):
  • the molecular weight of said GAG is between 5 to 20 kD.
  • phosphatidylethanolamine (PE) moieties are analogues of the phospholipid in which the chain length of the two fatty acid groups attached to the glycerol backbone of the phospholipid varies from 2-30 carbon atoms length, and in which these fatty acids chains contain saturated and/or unsaturated carbon atoms.
  • alkyl chains attached directly or via an ether linkage to the glycerol backbone of the phospholipid are included as analogues of PE.
  • the PE moiety is dipalmitoyl-phosphatidyl-ethanolamine.
  • the PE moiety is dimyristoyl-phosphatidyl-ethanolamine.
  • Phosphatidyl-ethanolamine and its analogues may be from various sources, including natural, synthetic, and semisynthetic derivatives and their isomers.
  • Phospholipids which can be employed in lieu of the PE moiety are N-methyl-PE derivatives and their analogues, linked through the amino group of the N-methyl-PE by a covalent bond; N,N-dimethyl-PE derivatives and their analogues linked through the amino group of the N,N-dimethyl-PE by a covalent bond, phosphatidylserine (PS) and its analogues, such as palmitoyl-stearoyl-PS, natural PS from various sources, semisynthetic PSs, synthetic, natural and artifactual PSs and their isomers.
  • PS phosphatidylserine
  • phospholipids useful as conjugated moieties in this invention are phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and phosphoatidylglycerol (PG), as well as derivatives thereof comprising either phospholipids, lysophospholipids, phosphatidic acid, sphingomyelins, lysosphingomyelins, ceramide, and sphingosine.
  • PC phosphatidylcholine
  • PI phosphatidylinositol
  • PG phosphoatidylglycerol
  • the phospholipid is linked to the conjugated monomer or polymer moiety through the nitrogen atom of the phospholipid polar head group, either directly or via a spacer group.
  • the phospholipid is linked to the conjugated monomer or polymer moiety through either the nitrogen or one of the oxygen atoms of the polar head group, either directly or via a spacer group.
  • said lipid-polymer conjugate is represented by the structure of the general formula (II):
  • the phosphatidylserine may be bonded to Y, or to X if Y is nothing, via the COO′′ moiety of the phosphatidylserine.
  • said lipid-polymer conjugate is represented by the structure of the general formula (III):
  • said lipid-polymer conjugate is represented by the structure of the general formula (IV):
  • said lipid-polymer conjugate is represented by the structure of the general formula (V):
  • said lipid-polymer conjugate is represented by the structure of the general formula (VI):
  • said lipid-polymer conjugate is represented by the structure of the general formula (VII):
  • said lipid-polymer conjugate is represented by the structure of the general formula (VIII):
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (IX):
  • said lipid-polymer conjugate is represented by the structure of the general formula (IXa):
  • said lipid-polymer conjugate is represented by the structure of the general formula (IXb):
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (X):
  • the compound for use in the present invention is represented by the structure of the general formula (Xa):
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (XI):
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (XII):
  • the compound for use in the present invention is represented by the structure of the general formula (XIIa):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XIII):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XIV):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XV):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XVI):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XVII):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XVIII):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XIX):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XX):
  • said lipid-polymer conjugate is represented by the structure of the general formula (XXI):
  • R 2 of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
  • the compounds (A), (B) (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) as presented hereinabove comprises a Z group.
  • Z is a nothing.
  • Z is inositol.
  • Z is choline.
  • Z is glycerol.
  • Z is ethanoleamine.
  • Z is serine.
  • X is a glycosaminoglycan.
  • the glycosaminoglycan may be, inter alia, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate or a derivative thereof.
  • the chondroitin sulfate may be, inter alia, chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof.
  • X is not a glycosaminoglycan.
  • X is a polysaccharide, which in one embodiment is a hetero-polysaccharide, and in another embodiment, is a homo-polysaccharide.
  • X is a polypyranose.
  • the glycosaminoglycan is a polymer of disaccharide units.
  • the number of the disaccharide units in the polymer is m.
  • m is a number from 2-10,000.
  • m is a number from 2-500.
  • m is a number from 2-1000.
  • m is a number from 50-500.
  • m is a number from 2-2000.
  • m is a number from 500-2000.
  • m is a number from 1000-2000.
  • m is a number from 2000-5000.
  • m is a number from 3000-7000.
  • m is a number from 5000-10,000.
  • a disaccharide unit of a glycosaminoglycan may be bound to one lipid or phospholipid moiety.
  • each disaccharide unit of the glycosaminoglycan may be bound to zero or one lipid or phospholipid moieties.
  • the lipid or phospholipid moieties are bound to the —COOH group of the disaccharide unit.
  • the bond between the lipid or phospholipid moiety and the disaccharide unit is an amide bond.
  • this invention provides lipid-GAG conjugate or phospholipid-GAG conjugate, and methods of use thereof, wherein said conjugate represented by the structures of the general formulae (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XX), and (XXII).
  • the average molecular weight of said GAG is between 5 kD to 90 kD.
  • the average molecular weight of said GAG is between 5 kD to 60 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 40 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 15 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 20 kD. In another embodiment, the lipid-GAG conjugate is a phospholipid-GAG conjugate.
  • Y is nothing.
  • suitable divalent groups forming the optional bridging group (which in one embodiment, is referred to as a spacer) Y are straight or branched chain alkylene, e.g., of 2 or more, preferably 4 to 30 carbon atoms, —CO-alkylene-CO, —NH-alkylene-NH—, —CO-alkylene-NH—, —NH-alkylene-NH, CO-alkylene-NH—, an amino acid, cycloalkylene, wherein alkylene in each instance, is straight or branched chain and contains 2 or more, preferably 2 to 30 atoms in the chain, —(—O—CH(CH 3 )CH 2 —) x — wherein x is an integer of 1 or more.
  • the sugar rings of the glycosaminoglycan are intact.
  • intact refers to closed.
  • intact refers to natural.
  • intact refers to unbroken.
  • the structure of the lipid or phospholipid in any compound according to the invention is intact. In another embodiment, the natural structure of the lipid or phospholipids in any compound according to the invention is maintained.
  • the compounds for use in the present invention are biodegradable.
  • the compounds for use are as listed in Table 1 below,
  • this invention provides a lipid-polymer conjugate represented by the structure of the general formula (B):
  • this invention provides a lipid-polymer conjugate represented by the structure of the general formula (XXII):
  • n of formula (B) and formula (X) is 1-10, in another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 7. In another embodiment, n is 8. In another embodiment, n is 9. In another embodiment, n is 10.
  • this invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • this invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • said coupling agent is DCC, EDAC, BOP, PyBOP, HATU, TSTU or any other amide coupling agent.
  • said coupling agent is EDAC.
  • said coupling agent further comprises HOBT or HOAT.
  • said filtering step comprises a 10 kD centrasette membrane.
  • R 1 is the residue of palmitic acid or the residue of myristic acid.
  • R 2 is the residue of palmitic acid or the residue of myristic acid.
  • the average molecular weight of the glycosaminoglycan is between 5 kD to 90 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 5 kD to 20 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 5 kD to 10 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 10 kD to 20 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 20 kD to 50 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 30 kD to 60 kD.
  • the average molecular weight of the glycosaminoglycan is between 40 kD to 70 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 50 kD to 80 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 60 kD to 90 kD
  • hyaluronic acid is used in solution form.
  • HA solution is prepared according to Example 1,
  • the process for the preparation of fractionated hyaluronic acid includes ultrafiltration.
  • the ultrafiltration fractionation of hyaluronic acid is as described in Example 2.
  • HyPE phosphatidylethanolamine-hyaluronic acid conjugate
  • fractionated HA is used in the preparation of HyPE.
  • fractionated HA is prepared according to Example 3.
  • HyPE is prepared according to Example 11.
  • a coupling reagent is used in the preparation of HyPE according to Example 3.
  • EDAC is used as the coupling reagent.
  • DCC is used as the coupling agent.
  • BOP is used as the coupling agent.
  • PyBOP is used as the coupling agent.
  • HATU is used as the coupling agent.
  • TSTU is used as the coupling agent.
  • the coupling agent used in the preparation of HyPE according to Example 3 comprises HOBT. In another embodiment, the coupling agent comprises HOAT.
  • crude HyPE is processed by an ultrafiltration step.
  • HyPE is subjected to the alkaline ultrafiltration described in Example 4.
  • filtered HyPE is isolated by extraction.
  • HyPE is extracted according to the process described in Example 5.
  • said extraction comprises dichloromethane, ethanol and methanol.
  • this invention provides a method of treating an inflammatory disorder in a subject, said method comprising administering to a subject suffering from an inflammatory disorder a composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a mass PL to mass GAG ratio from about 0.25:15 to about 5:15, respectively.
  • a composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a mass PL to mass GAG ratio from about 0.25:15 to about 5:15, respectively.
  • GAG glycosaminoglycan
  • PL phospholipid
  • said mass PL to mass GAG ratio is about 0.25:15. In another embodiment, said mass PL to mass GAG ratio is about 0.5:15. In another embodiment, said mass PL to mass GAG ratio is about 1:15. In another embodiment, said mass PL to mass GAG ratio is about 2:15. In another embodiment, said mass PL to mass GAG ratio is about 5:15.
  • said inflammatory disorder is rheumatoid arthritis, osteoarthritis, wound healing, dermatitis, restenosis, cystic fibrosis, multiple sclerosis or sepsis.
  • in vitro assays are used to measure the ability of HyPE and HyPE analogs to reduce the expression of pro-inflammatory cytokines.
  • cell-based assays are used according to Example 6, Example 7 and Example 8.
  • expression of IL-6 is measured.
  • expression of TNF- ⁇ is measured.
  • expression of IP-10 is measured.
  • expression of PGE 2 is measured.
  • said composition is administered intravenously. In another embodiment, said composition is administered topically.
  • the present invention provides a method for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof comprising the step of administering a compound represented by the structure of the general formula (A):
  • the present invention provides a method of activating NF- ⁇ B, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines comprising the step of administering to a subject a compound represented by the structure of the general formula (A):
  • compositions comprising Lipid-conjugates in admixture with conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds.
  • excipients i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., vitamins, bronchodilators, steroids, anti-inflammatory compounds, gene therapy, i.e. sequences which code for the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) receptor, surfactant proteins, etc., as will be understood by one skilled in the art.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active
  • the invention provides for the administration of a salt of a compound as described herein as well.
  • the salt is a pharmaceutically acceptable salt, which, in turn may refer to non-toxic salts of compounds (which are generally prepared by reacting the free acid with a suitable organic or inorganic base) and include, but are not limited to, the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate,
  • the route of administration may be parenteral, enteral, or a combination thereof.
  • the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation, nasal aspiration (spray), sublingual, oral, aerosol or suppository or a combination thereof.
  • the dosage regimen will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, etc.
  • the doses utilized for the above described purposes will vary, but will be in an effective amount to exert the desired anti-disease effect.
  • the term “pharmaceutically effective amount” refers to an amount of a compound of formula (I) which will produce the desired alleviation in symptoms or signs of disease in a patient.
  • the doses utilized for any of the above-described purposes will generally be from 1 to about 1000 milligrams per kilogram of body weight (mg/kg), administered one to four times per day, or by continuous IV infusion. When the compositions are dosed topically, they will generally be in a concentration range of from 0.1 to about 10% w/v, administered 1-4 times per day.
  • the use of a single chemical entity with potent anti-oxidant, membrane-stabilizing, anti-proliferative, anti-chemokine, anti-migratory, and anti-inflammatory activity provides the desired protection for a subject with an inflammatory disorder
  • the methods of this invention provide for use of a combination of the compounds described.
  • the compounds for use in the present invention may be provided in a single formulation/composition, or in another embodiment, multiple formulations may be used.
  • the formulations for use in the present invention may be administered simultaneously, or in another embodiment, at different time intervals, which may vary between minutes, hours, days, weeks or months.
  • compositions comprising the compounds for use in the present invention may be administered via different routes, which in one embodiment, may be tailored to provide different compounds at different sites, for example some compounds may be given parenterally to provide for superior perfusion throughout the lung and lymphatic system, and in another embodiment, some formulations/compounds/compositions may be provided via aerosol, or in another embodiment, intranasally, to provide for higher lung mucosal concentration. is there something wrong with this sentence? Seems like you need the word “higher” before mucosal?
  • the compounds for use in the invention may be used for acute treatment of temporary conditions, or may be administered chronically, as needed.
  • concentrations of the compounds will depend on various factors, including the nature of the condition to be treated, the condition of the patient, the route of administration and the individual tolerability of the compositions.
  • the methods of this invention provide for the administration of the compounds in early life of the subject, or in another embodiment, throughout the life of the subject, or in another embodiment, episodically, in response to severity or constancy of symptomatic stages, or in another embodiment.
  • the patients to whom the lipid or PL conjugates should be administered are those that are experiencing symptoms of disease or who are at risk of contracting the disease or experiencing a recurrent episode or exacerbation of the disease, or pathological conditions associated with the same.
  • the term “pharmaceutically acceptable carrier” refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound of the present invention. As such, all of the above-described formulations of the present invention are hereby referred to as “pharmaceutically acceptable carriers.” This term refers to as well the use of buffered formulations wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the compounds and route of administration.
  • sterile solutions preferably oily or aqueous solutions, as well as suspensions or emulsions. It is also possible to freeze-dry the new compounds and use the lyophilates obtained, for example, for the preparation of products for injection.
  • implants or suppositories can be used to administer a lipid-GAG conjugate of this invention.
  • solutions or suspensions of the compounds mixed and aerosolized or nebulized in the presence of the appropriate carrier are particularly for treatment of airway obstruction or congestion.
  • suppositories for enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules.
  • a syrup, elixir, or the like can be used when a sweetened vehicle is employed.
  • suppositories or enema formulations may be the recommended route of administration.
  • Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
  • the methods of the present invention make use of a conjugate as described herein to treat a subject suffering from an inflammatory disorder, reduce or delay the mortality of a subject suffering from an inflammatory disorder or ameliorate symptoms associated with an inflammatory disorder.
  • the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and heparin. In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and chondroitin sulfate. In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and hyaluronic acid. In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and carboxymethylcellulose. In one embodiment, the compound for use in the present invention comprises dimyristoyl phosphatidylethanolamine and hyaluronic acid.
  • the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a glycosaminoglycan.
  • the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a chondroitin sulfate, which is chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof.
  • the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a heparin.
  • the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a hyaluronic acid.
  • the compound for use in the present invention is a dimyristoyl phosphatidylethanolamine conjugated via an amide or ester bond to a hyaluronic acid.
  • the conjugates of this invention display a wide-range combination of cytoprotective pharmacological activities, which are useful in the present invention.
  • the compounds may be useful for their anti-inflammatory effects.
  • Cellular elaboration of cytokines and chemokines serve an important regulatory function in health; however, when a hyperactive response to stress or disease is triggered, these compounds may present in excess and damage tissue, thereby pushing the disease state toward further deterioration.
  • the lipid compounds for use in the methods of this invention possess a combination of multiple and potent pharmacological effects, including inter-alia the ability to inhibit the extracellular form of the enzyme phospholipase A2.
  • the conjugates of this invention are useful in affecting inflammation in a subject with an inflammatory disorder, where the subject is administered lipid-conjugates at pre-symptomatic stages of the disease.
  • a characteristic feature of inflammation in the CF lung is the persistent infiltration of massive numbers of neutrophils into the airways. Although neutrophils help to control infection, when present in great excess, they can be harmful.
  • Major advances in the understanding of the inflammatory process in the CF lung have come from the use of bronchoscopy and bronchioalveolar lavage (BAL) to analyze the inflammatory process in patients who are relatively symptom free and/or do not regularly produce sputum. Recent BAL studies suggest that neutrophil-rich inflammation begins very early, even in infants without clinically apparent lung disease.
  • the lipid/phospholipid conjugates of the present invention may be useful in treating CF, even in presymptomatic stages of disease.
  • the invention provides methods for treating a subject suffering from cystic fibrosis, reducing or delaying the mortality of a subject suffering from cystic fibrosis or ameliorating symptoms associated with cystic fibrosis, and the compounds/compositions/formulations, in one embodiment, diminish or abrogate a deleterious inflammatory response in said subject, or in another embodiment, prevent, treat, reduce the incidence of, reduce the severity of, delay the onset of, or diminish the pathogenesis of an infection is the CF subject.
  • the invention provides methods for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof, while in another embodiment, the invention provides methods of activating NF- ⁇ B, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines.
  • a method for promoting wound healing comprising applying or administering to a wound site to be treated in a subject an effective amount of a composition comprising any conjugate as described herein.
  • a method for promoting wound healing comprising applying or administering to a wound site to be treated in a subject an effective amount of a composition comprising any compound represented by the structure of the general formula (A).
  • promoting wound healing comprises inducing wound healing. In another embodiment, promoting wound healing comprises speeding up wound healing. In another embodiment, promoting wound healing comprises reducing the risk of viral and/or bacterial infection. In another embodiment, promoting wound healing comprises reducing inflammation in or near the wound site.
  • the conjugates as described herein increase the rate of chronic and acute wound healing.
  • the conjugates as described herein counteract mechanisms which delay or impair wound healing.
  • the compounds as described herein counteract exogenous factors which delay or impair wound healing.
  • the conjugates as described herein counteract endogenous factors which delay or impair wound healing.
  • factors include: infection, ulceration particularly through diabetes, circulation problems associated with vascular disease, malnutrition, stress, cancer radiotherapy and/or chemotherapy, compromise of the immune system or simply due to the normal aging process.
  • a method a described herein provides both a therapeutic and a cosmetic approach that promote wound healing processes.
  • wounds include, but are not limited to the following: surgical wounds; bites; burns; acid and alkali burns; cold burn (frostbite), sun burn, minor cuts, major cuts, abrasions, lacerations, wounds caused by gunshot or knife injury; wounds caused by congenital disorders; wounds following dental surgery; periodontal disease; wounds following trauma; tumour associated wounds, which can be classified as malignant cutaneous ulcers related to the primary tumour or metastases; ulcers, leg ulcers; foot ulcers; pressure sores and corneal wounds.
  • the methods of the present invention make use of a conjugate as described herein for treating a subject suffering from arthritis, reducing or delaying the damage to the joints of a subject suffering from arthritis, or ameliorating symptoms associated with arthritis.
  • the methods of the present invention make use of a formulation comprising a conjugate as described herein for treating a subject suffering from arthritis, reducing or delaying the damage to the joints of a subject suffering from arthritis, or ameliorating symptoms associated with arthritis.
  • a method of treating a subject suffering from joint pain, swelling within the joint, inflammation within the joint, or a combination thereof comprising the step of administering a composition comprising a conjugate of the invention to the subject.
  • a method of treating a subject suffering from joint pain, swelling within the joint, inflammation within the joint, or a combination thereof comprising the step of injecting into a swelled/inflamed joint a composition comprising a conjugate of the invention.
  • arthritis refers to both rheumatoid arthritis (RA) and osteoarthritis (OA).
  • a compound as described herein inhibits the production of IL-6, IL-8, TNF-alpha, NF- ⁇ B, or their combination, thereby reducing or delaying the damage to the joints of a subject suffering from arthritis.
  • a compound as described herein inhibits the production of IL-6, IL-8, TNF-alpha, NF- ⁇ B, or their combination, thereby ameliorating symptoms associated with arthritis.
  • methods comprising the administration of a conjugate as described herein treat a subject suffering from joint pain, swelling within the joint, inflammation within the joint, or a combination thereof by inhibiting the production of IL-6, IL-8, TNF-alpha, NF- ⁇ B, or their combination.
  • locally administering a composition comprising a conjugate as described herein by intra-joint injection inhibits the production of IL-6, IL-8, TNF-alpha, NF- ⁇ B, or their combination within the joint's cells. In another embodiment, locally administering a composition comprising a conjugate as described herein by intra-joint injection inhibits inflammation within the joint.
  • inflammatory disorders include, but are not limited to, disorders resulting from activation of the immune system.
  • autoimmune disorders are understood to be inflammatory disorders.
  • Such disorders include, but are not limited to, rheumatoid arthritis, osteoarthritis, wound healing, dermatitis, restenosis, cystic fibrosis, central nervous system tissue insult, multiple sclerosis, obstructive respiratory disease, Crohn's disease, cardiovascular disease, atherosclerosis, contact dermatitis, psoriasis, ARDS, or sepsis
  • the invention provides a method of treating a subject suffering from obstructive respiratory disease, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from obstructive respiratory disease.
  • the invention provides a method of treating a subject suffering from colitis, Crohn's disease, or another form of intestinal mucosal injury, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from intestinal mucosal injury, including colitis or Crohn's disease.
  • the invention provides a method of treating a subject suffering from cardiovascular disease, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from a cardiovascular disease.
  • the present invention provides a method of treating a subject suffering from atherosclerosis, including, inter alia, the step of administering to a conjugate of this invention, thereby treating the subject suffering from atherosclerosis.
  • the invention provides a method of treating a subject suffering from central nervous system tissue insult, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from a central nervous system insult.
  • the invention provides a method of treating a subject suffering from multiple sclerosis, including, inter alia, the step of administering to a subject an effective amount of conjugate of this invention, thereby treating the subject suffering from multiple sclerosis.
  • the invention provides a method of treating a subject suffering from contact dermatitis, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from contact dermatitis.
  • the invention provides a of treating a subject suffering from psoriasis, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from psoriasis.
  • the invention provides a method of treating a subject suffering from sepsis, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from sepsis.
  • the invention provides a method of treating a subject suffering from ARDS, comprising the steps of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from ARDS.
  • pharmacological activity of the Lipid-conjugates described herein may be due in part to the nature of the lipid moiety
  • the multiple and diverse combination of pharmacological properties observed for the Lipid-conjugates may represent, in other embodiments, the ability of the conjugate to act essentially as several different drugs in one chemical entity.
  • lung mucosal or lung parenchymal injury as may occur in CF, may be attenuated by any one or all of the pharmaceutical activities of immune suppression, anti-inflammation, anti-oxidation, suppression of nitric oxide production, or membrane stabilization.
  • the invention provides a method of “treating” inflammatory disorders or related diseases or disorders, which in one embodiment, refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove.
  • treating refers to delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing relapse to a disease, decrease the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternate therapeutics.
  • the methods are useful in treating an infection in a subject, wherein the pathogen is a virus or in another embodiment, the pathogen is a bacterium.
  • the infection is with a pathogen which infects the respiratory system, such as mycobacteria, pseudomonas, cryptococcus, streptococcus, reovirus, influenza, or other infections known to those of skill in the art.
  • HA UL 15 was dissolved in 4 L of 0.1% chlorocresol solution with mechanical stirring. To prevent clogging of the ultrafiltration membranes, the HA solution was filtered through a 100 ⁇ m filter followed by a 50 ⁇ m filter followed by a 10 ⁇ m filter, all previously disinfected with 10% hydrogen peroxide and washed with copious amounts of DI water to ensure hydrogen peroxide has been removed (verified with peroxide-detecting strips).
  • DI deionized
  • Example 1 HA solution of Example 1 was loaded into the Centramate system, previously disinfected with 10% hydrogen peroxide and washed with copious amounts of DI water to ensure hydrogen peroxide has been removed (verified with peroxide-detecting strips).
  • the 70 kDa membranes were replaced with 30 kDa membranes and the Centramate system was disinfected with 10% hydrogen peroxide.
  • DPPE dipalmitoylphosphatidylethanolamine
  • HOBT hydroxybenzotriazole
  • a 1 mg/ml solution of LPS (made in 1 ⁇ PBS) was diluted in CM to 10 ⁇ g/ml.
  • RAW 264.7 cells were grown for XX passages (subculture every 3-4 days) in CM at 37° C. with 5% CO 2 .
  • 0.5 ml of cells at 1 ⁇ 106 cells/ml was plated in 24-well tissue culture plates. Cells were allowed to adhere for 30 minutes at 37° C. with 5% CO 2 prior to treatment.
  • the appropriate Test Article, dexamethasone or vehicle control working solutions were added to the cells. Cells were incubated for 1 hour at 37° C. with 5% CO 2 prior to LPS treatment.
  • 110 ⁇ l of CM was added to the -LPS plates.
  • 110 ⁇ l of 10 ⁇ g/ml LPS was added to the +1 ⁇ g/ml LPS plates. The plates were incubated for 24 hours at 37° C. with 5% CO 2 .
  • TNF- ⁇ data relating to high molecular weight HyPE compositions are shown in FIG. 7 , FIG. 8 and FIG. 15 .
  • TNF- ⁇ data relating to low molecular weight HyPE compositions are shown in FIG.
  • IL-6 data relating to high molecular weight HyPE compositions are shown in FIG. 9 , FIG. 10 and FIG. 16 .
  • IL-6 data relating to low molecular weight HyPE compositions are shown in FIG. 26 , FIG. 27 and FIG. 33 .
  • IP-10 data relating to high molecular weight HyPE compositions are shown in FIG. 11 , FIG. 12 and FIG. 17 .
  • IP-10 data relating to low molecular weight HyPE compositions are shown in FIG. 28 , FIG. 29 and FIG. 34 .
  • Raw material of sodium hyaluronate (1.32 MDa) was degraded by acidic hydrolysis.
  • the sample solution was ultrafiltered immediately after degradation.
  • the final product was prepared using spray dryer as in the case of previous samples. In addition it was filtered with 0.2 ⁇ m filter (PALL) before drying to achieve microbial purity.
  • PALL 0.2 ⁇ m filter
  • the chromatography system (Agilent, 1100 Series) consisted of a HPLC pump (G1310A), an automatic injector (G1313A) and the following column system: PL aquagel-OH Mix and PL aquagel-OH 30 (300 ⁇ 7.5 mm, 8 ⁇ m; Agilent Technologies) columns connected in series and thermostated at ambient temperature. Injection volume was 100 ⁇ l.
  • Eluent 0.1 M sodium phosphate buffer pH 7.5
  • Data acquisition and molecular weight calculations were performed using the ASTRA V software, Version 5.3.2.15.
  • the flow rate of mobile phase was maintained at 0.8 ml/min.
  • the specific refractive index increment (dn/dc) of 0,155 mg/ml was used for sodium hyaluronate.
  • the hyaluronan samples were prepared by dissolving of a weighted amount of sample in the phosphate buffer (concentration 20.0 mg/ml). All samples were stirred several hours. The solutions were filtered through syringe filter (0.2 ⁇ m, 25 mm diameter, Whatman) and analysed by HPLC system.
  • Light scattering measurements can provide an absolute measurement of molar mass when used in series with a concentration sensitive detector such as a refractive index detector and if the value of dn/dc (differential refractive index increment) is known.
  • the weight-average molecular weight of hyaluronan was verified by measurements of dextran standard.
  • the determined molecular weight and polydispersity value for low molecular weight hyaluronic acid were 7.86 ⁇ 103 g/mol and 1.32 Mw/Mn, respectively.
  • the chromatogram and distribution diagram are stated in FIG. 19 and FIG. 20 whereas red line pertains to light scattering signal and blue line to refractive index signal.
  • FIG. 21 illustrates the UV spectrum.
  • MES buffer was prepared by dissolving 14.5 g of MES in 75 mL of DI-H 2 O and adjusting the pH to 6.4 with 4N NaOH.
  • 10.0 g of HOBT was dissolved in 225 mL of DI-H 2 O, 60 mL MES buffer, 12 mL of tert-butanol.
  • the pH was adjusted to 6.4 with 4N NaOH.
  • 15.1 g of HA was dissolved in 350 mL of DI-H 2 O.
  • 1.25 g or DPPE was dissolved in 440 mL of tert-butanol and 90 mL DI-H 2 O with heating to 55 deg C.
  • Residual DCM was removed by rotovaporation at 35 deg C. and 200 Torr.
  • the solution was then transferred to a previously cleaned centrasette ultrafiltration system with a 10 kDa membrane and by constant volume diafiltration was washed with 5 L of 1.5% NaHCO 3 to remove residual organic solvents.
  • the pH was then increased by slow addition of 2% Na 2 CO 3 to pH 9.2.
  • the solution was stirred for 1 hour at room temperature. After further washing with 30 L of 1.5% NaHCO 3 the peat at ⁇ 12.5 min had disappeared and the solution was washed with 30 L of DI-H 2 O until pH 7.
  • the solution was then extracted again with 1 L DCM, 1 L MeOH and 0.75 L EtOH.
  • the aqueous layer was extracted again with 400 mL DCM and 50 mL EtOH and finally a third time with 400 mL DCM and 50 mL EtOH.
  • Residual DCM was removed by rotovaporation at 30 deg C. and 200 Torr.
  • diafiltration residual MeOH and EtOH were removed by washing with 15 L DI-H 2 O.
  • the solution was concentrated to 1 L and filtered through a 0.2 ⁇ m filter into a lyoguard container and placed in the lypholizer. It was frozen by lowering the shelf temperature to ⁇ 70 deg C.

Abstract

This invention provides low molecular weight lipid-GAG and phospholipids-GAG conjugates and methods of use thereof in suppressing, inhibiting, preventing, or treating a pathogenic effect on a cell, including, inter alia, infection with intracellular pathogens.

Description

    FIELD OF THE INVENTION
  • This invention provides low molecular weight lipid-GAG conjugates and methods of use thereof in suppressing, inhibiting, preventing, or treating a pathogenic effect on a cell, including, inter alia, infection with intracellular pathogens.
    • BACKGROUND OF THE INVENTION
  • Lipid-conjugates having a pharmacological activity of inhibiting the enzyme phospholipase A2 (PLA2, EC 3.1.1.4) are known in the prior art. Phospholipase A2 catalyzes the breakdown of phospholipids at the sn-2 position to produce a fatty acid and a lysophospholipid. The activity of this enzyme has been correlated with various cell functions, particularly with the production of lipid mediators such as eicosanoid production (prostaglandins, thromboxanes and leukotrienes), platelet activating factor and lysophospholipids. Lipid-conjugates may offer a wider scope of protection of cells and organisms from injurious agents and pathogenic processes, including the prevention and treatment of microbial infections. Lipid-conjugates may offer a wider scope of protection of cells and organisms from injurious agents and pathogenic processes, including the prevention and treatment of microbial infections.
  • Lipid-conjugates have been subjected to intensive laboratory investigation in order to obtain a wider scope of protection of cells and organisms from injurious agents, pathogenic and inflammatory processes.
    • SUMMARY OF THE INVENTION
  • In one embodiment, the present invention provides a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively.
  • In one embodiment, the present invention provides a lipid-polymer conjugate comprising a glycosaminglycan (GAG) conjugated to a phospholipid (PL) via an amide or ester linkage wherein the molecular weight of said GAG is between 5 to 20 kD.
  • In one embodiment, the present invention provides a lipid-polymer conjugate represented by the structure of the general formula (A):
  • Figure US20110130555A1-20110602-C00001
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, phosphate, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between L, Z, Y and X is either an amide or an esteric bond;
      • wherein the molecular weight of said glycosaminoglycan is between 5 kD and 20 kD.
  • In one embodiment, the present invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • Figure US20110130555A1-20110602-C00002
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein if Y is nothing the phosphatidylethanolamine is directly linked to X via an amide bond and if Y is a spacer, said spacer is directly linked to X via an amide or an esteric bond and to said phosphatidylethanolamine via an amide bond;
      • comprising the steps of:
      • b. reacting a phospholipid (PL) with a glycosaminoglycan (GAG) and a coupling agent, wherein the massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively;
      • c. filtering the reaction mixture from (a) to generate a filtrate; and
      • d. extracting a product from a filtrate.
  • In one embodiment, the present invention provides a method of treating inflammatory disorders in a subject, said method comprising administering to a subject suffering from an inflammatory disorder a composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively. In one embodiment, the present invention provides a method for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof comprising the step of administering a compound represented by the structure of the general formula (A):
  • Figure US20110130555A1-20110602-C00003
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between L, Z, Y and X is either an amide or an esteric bond to a subject with high levels of proinflammatory chemokines, cytokines, or a combination thereof.
  • In one embodiment, the present invention provides a method of activating NF-κB, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines comprising the step of administering to a subject a compound represented by the structure of the general formula (A):
  • Figure US20110130555A1-20110602-C00004
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between L, Z, Y and X is either an amide or an esteric bond.
    BRIEF DESCRIPTION OF THE DRAWINGS
  • The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:
  • FIG. 1 depicts a conceptual diagram of the reaction vessel features required to practice the methods of this invention.
  • FIG. 2 depicts an NMR spectrum of a hyaluronic acid-phosphatidylethanolamine conjugate (HyPE) prepared according to Example 5.
  • FIG. 3 is an HPLC chromatogram of HyPE prepared according to Example 5.
  • FIG. 4 depicts a schematic representation of the in vitro stimulation of RAW 264.7 cells.
  • FIG. 5 depicts the mean XTT reduction (OD450) by RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 6 depicts the mean XTT reduction (OD450) by LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 7 depicts the mean TNF-α release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 8 depicts the mean TNF-α release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 9 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 10 depicts the mean IL-6 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 11 depicts the mean IP-10 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 12 depicts the mean IP-10 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 13 depicts the mean PGE2 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 14 depicts the mean PGE2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 15 depicts dose-response curves for TNF-α production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 16 depicts dose-response curves for IL-6 production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 17 depicts dose-response curves for IP-10 production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 18 depicts dose-response curves for PGE2 production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 19 is the chromatogram from the SEC-MALS molecular weight analysis of low molecular weight sodium hyaluronate. The red line pertains to the light scattering signal. The blue line refers to the refractive index signal.
  • FIG. 20 is the SEC-MALS determined distribution of molecular weight of low molecular weight sodium hyaluronate.
  • FIG. 21 is the UV spectrum of sample 208-088 (low molecular weight sodium hyaluronate).
  • FIG. 22 depicts the mean XTT reduction (OD450) by RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 23 depicts the mean XTT reduction (OD450) by LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 24 depicts the mean TNF-α release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 25 depicts the mean TNF-α release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 26 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 27 depicts the mean IL-6 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 28 depicts the mean IP-10 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 29 depicts the mean IP-10 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 30 depicts the mean PGE2 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
  • FIG. 31 depicts the mean PGE2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • FIG. 32 depicts dose-response curves for TNF-α production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 33 depicts dose-response curves for IL-6 production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 34 depicts dose-response curves for IP-10 production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 35 depicts dose-response curves for PGE2 production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom=0, Top=100.
  • FIG. 36 depicts a photograph of the actual reaction vessel used for the preparation of HyPE. The chiller is behind the reaction vessel and the door on the sound-proof container is open to reveal the ultrasound flow-cell.
  • FIG. 37 depicts a chromatogram of the HyPE reaction from Example 11 after 2 hours.
  • FIG. 38 depicts a chromatogram of the HyPE reaction from Example 11 after 6 hours.
  • FIG. 39 depicts the GPC analysis of final HyPE isolated from Example 11.
  • FIG. 40 depicts the NMR spectrum of final HyPE isolated from Example 11 and treated with 1 drop of 4% NaOD.
    •
  • It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.
    • DETAILED DESCRIPTION OF THE PRESENT INVENTION
  • In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.
  • Abbreviations used to specify chemicals and reagents used in the processes described herein are readily recognized by one skilled in the art. For the purposes of this invention, it will be understood that DCC refers to dicyclohexylcarbodiimide, EDAC refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), BOP refers to Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate, PyBOP refers to benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, HATU refers to O-(7-Azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluronium hexafluorophosphate, TSTU refers to O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HOBT refers to hydroxybenzotriazole and HOAT refers to 1-hydroxy-7-aza-benzotriazole.
  • Herein, the term “lipid” refers to all types of lipids including phospholipids, glycerolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and the like.
  • This invention provides, in one embodiment, a lipid-polymer conjugate which is useful in some embodiments for the treatment of inflammatory disorders.
  • In some embodiments, this invention provides a method for the preparation of the lipid-polymer conjugates of this invention. In some embodiments, this invention provides a method for the use of the lipid-polymer conjugates of this invention.
  • In one embodiment, this invention provides a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively.
  • In another embodiment, said massPL to massGAG ratio is about 0.25:15. In another embodiment, said massPL to massGAG ratio is about 0.5:15. In another embodiment, said massPL to massGAG ratio is about 1:15. In another embodiment, said massPL to massGAG ratio is about 2:15. In another embodiment, said massPL to massGAG ratio is about 5:15.
  • In one embodiment, the present invention provides a lipid-polymer conjugate comprising a glycosaminglycan (GAG) conjugated to a phospholipid (PL) via an amide or ester linkage wherein the molecular weight of said GAG is between 5 to 20 kD.
  • In another embodiment, said GAG of the lipid-conjugate compound of this invention is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate. In another embodiment, said GAG is hyaluronic acid. In another embodiment, said GAG is heparin. In another embodiment, said GAG is chondroitin. In another embodiment, said GAG is chondroitin sulfate. In another embodiment, said GAG is dermatan sulfate, in another embodiment, said GAG is keratan sulfate.
  • In another embodiment, said chondroitin sulfate is chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. In another embodiment, said dermatan sulfate is dermatan-6-sulfate, dermatan-4-sulfate or a derivative thereof.
  • In another embodiment, said PL of the lipid-conjugate compound of this invention is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol. In another embodiment, said PL comprises the residue of palmitic acid, myristic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid or docosahexaenoic acid. In another embodiment, said PL is dimyristoyl phosphatidylethanolamine. In another embodiment, said PL is dipalmitoyl phosphatidylethanolamine.
  • In another embodiment, the polydispersity of said GAG is from about 1 to 1.75. In another embodiment, the polydispersity of said GAG is from about 1.25 to 1.5.
  • In one embodiment, the lipid-polymer conjugate of this invention comprises a GAG wherein the average molecular weight of said GAG is between 5 kd to 90 kd. In another embodiment, the average molecular weight of said GAG is between 5 kD to 60 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 40 kD.
  • In another embodiment, the average molecular weight of said GAG is between 5 kD to 15 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 20 kD.
  • In one embodiment, low molecular weight GAG, such as sodium hyaluronate is prepared by acid hydrolysis of sodium hyaluronate as described in Example 9. In another embodiment, said acid hydrolysis comprises hydrochloric acid. In another embodiment, said acid hydrolysis comprises sulfuric acid. In another embodiment, said acid hydrolysis comprises trifluoroacetic acid. In another embodiment, said acid hydrolysis comprises hydrobromic acid. In another embodiment, said acid hydrolysis comprises acetic acid. In another embodiment, the concentration of the acid in said acid hydrolysis is from about 0.1 to 12 molar. In another embodiment, the concentration of the acid in said acid hydrolysis is from about 1 to 6 molar. In another embodiment, the concentration of the acid in said acid hydrolysis is from about 6 to 12 molar. In another embodiment, said acid hydrolysis is carried out at a temperature between 25 degrees Celsius to 100 degrees Celsius. In another embodiment, said acid hydrolysis is carried out at a temperature between 25 degrees Celsius to 50 degrees Celsius. In another embodiment, said acid hydrolysis is carried out at a temperature between 50 degrees Celsius to 100 degrees Celsius.
  • In one embodiment the molecular weight of hyaluronic acid and derivatives is determined by size exclusion chromatography and multiangle light scattering (SEC-MALS) as described in Example 10. The chromatogram and distribution diagram are stated in FIG. 19 and FIG. 20 whereas the red line pertains to light scattering signal and the blue line to refractive index signal. FIG. 21 illustrates the UV spectrum.
  • Light scattering measurements can provide an absolute measurement of molar mass when used in series with a concentration sensitive detector such as a refractive index detector and if the value of dn/dc (differential refractive index increment) is known. In essence, light scattering measurements automatically provide a column calibration curve for every sample, obviating time-consuming, conformation dependent calibration procedure.
  • In one embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in a phosphate buffer. In another embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in an acetate buffer. In another embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in a tris buffer. In another embodiment, the hyaluronan samples for SEC-MALS molecular weight determination are prepared by dissolving of a weighted amount of sample in a MES buffer.
  • In another embodiment, this invention provides a pharmaceutical composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively. In another embodiment, the average molecular weight of said GAG is between 5 kD to 90 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 20 kD. In another embodiment, the average molecular weight of said GAG is greater than 10 kD.
  • In one embodiment, this invention provides a lipid-polymer conjugate represented by the structure of the general formula (A):
  • Figure US20110130555A1-20110602-C00005
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, phosphate, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between L, Z, Y and X is either an amide or an enteric bond;
      • wherein the molecular weight of said glycosaminoglycan is between 5 kD and 20 kD.
  • In one embodiment L is a lipid. In another embodiment L is a phospholipid. In another embodiment, L is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol. In another embodiment, L comprises the residue of palmitic acid, myristic acid, myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid or docosahexaenoic acid. In another embodiment, L is dimyristoyl phosphatidylethanolamine. In another embodiment, said L is dipalmitoyl phosphatidylethanolamine.
  • In another embodiment, X is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate. In another embodiment, X is hyaluronic acid. In another embodiment, X is heparin. In another embodiment, X is chondroitin. In another embodiment, X is chondroitin sulfate. In another embodiment, X is dermatan sulfate, in another embodiment, X is keratan sulfate.
  • In another embodiment, said chondroitin sulfate is chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. In another embodiment, said dermatan sulfate is dermatan-6-sulfate, dermatan-4-sulfate or a derivative thereof.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (I):
  • Figure US20110130555A1-20110602-C00006
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan (GAG); and
      • n is a number from 1 to 70;
      • wherein if Y is nothing the phosphatidylserine is directly linked to X via an amide bond and if Y is a spacer, said spacer is directly linked to X via an amide or an esteric bond and to said phosphatidylethanolamine via an amide bond.
  • In another embodiment, the molecular weight of said GAG is between 5 to 20 kD.
  • Examples of phosphatidylethanolamine (PE) moieties are analogues of the phospholipid in which the chain length of the two fatty acid groups attached to the glycerol backbone of the phospholipid varies from 2-30 carbon atoms length, and in which these fatty acids chains contain saturated and/or unsaturated carbon atoms. In lieu of fatty acid chains, alkyl chains attached directly or via an ether linkage to the glycerol backbone of the phospholipid are included as analogues of PE. In one embodiment, the PE moiety is dipalmitoyl-phosphatidyl-ethanolamine. In another embodiment, the PE moiety is dimyristoyl-phosphatidyl-ethanolamine.
  • Phosphatidyl-ethanolamine and its analogues may be from various sources, including natural, synthetic, and semisynthetic derivatives and their isomers.
  • Phospholipids which can be employed in lieu of the PE moiety are N-methyl-PE derivatives and their analogues, linked through the amino group of the N-methyl-PE by a covalent bond; N,N-dimethyl-PE derivatives and their analogues linked through the amino group of the N,N-dimethyl-PE by a covalent bond, phosphatidylserine (PS) and its analogues, such as palmitoyl-stearoyl-PS, natural PS from various sources, semisynthetic PSs, synthetic, natural and artifactual PSs and their isomers. Other phospholipids useful as conjugated moieties in this invention are phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and phosphoatidylglycerol (PG), as well as derivatives thereof comprising either phospholipids, lysophospholipids, phosphatidic acid, sphingomyelins, lysosphingomyelins, ceramide, and sphingosine.
  • For PE-conjugates and PS-conjugates, the phospholipid is linked to the conjugated monomer or polymer moiety through the nitrogen atom of the phospholipid polar head group, either directly or via a spacer group. For PC, PI, and PG conjugates, the phospholipid is linked to the conjugated monomer or polymer moiety through either the nitrogen or one of the oxygen atoms of the polar head group, either directly or via a spacer group.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (II):
  • Figure US20110130555A1-20110602-C00007
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
        • wherein if Y is nothing the phosphatidylserine is directly linked to X via an amide bond and if Y is a spacer, said spacer is directly linked to X via an amide or an esteric bond and to said phosphatidylethanolamine via an amide bond.
  • In one embodiment, the phosphatidylserine may be bonded to Y, or to X if Y is nothing, via the COO″ moiety of the phosphatidylserine.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (III):
  • Figure US20110130555A1-20110602-C00008
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the phosphatidyl, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (IV):
  • Figure US20110130555A1-20110602-C00009
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
        • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (V):
  • Figure US20110130555A1-20110602-C00010
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (VI):
  • Figure US20110130555A1-20110602-C00011
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
        • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (VII):
  • Figure US20110130555A1-20110602-C00012
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
        • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (VIII):
  • Figure US20110130555A1-20110602-C00013
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (IX):
  • Figure US20110130555A1-20110602-C00014
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (IXa):
  • Figure US20110130555A1-20110602-C00015
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (IXb):
  • Figure US20110130555A1-20110602-C00016
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (X):
  • Figure US20110130555A1-20110602-C00017
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the ceramide phosphoryl, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, the compound for use in the present invention is represented by the structure of the general formula (Xa):
  • Figure US20110130555A1-20110602-C00018
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the ceramide phosphoryl, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (XI):
  • Figure US20110130555A1-20110602-C00019
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein if Y is nothing the sphingosyl is directly linked to X via an amide bond and if Y is a spacer, the spacer is directly linked to X and to the sphingosyl via an amide bond and to X via an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (XII):
  • Figure US20110130555A1-20110602-C00020
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the ceramide, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, the compound for use in the present invention is represented by the structure of the general formula (XIIa):
  • Figure US20110130555A1-20110602-C00021
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the ceramide, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XIII):
  • Figure US20110130555A1-20110602-C00022
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, ethanolamine, serine, choline, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the diglyceryl, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XIV):
  • Figure US20110130555A1-20110602-C00023
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the glycerolipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XV):
  • Figure US20110130555A1-20110602-C00024
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the glycerolipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XVI):
  • Figure US20110130555A1-20110602-C00025
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XVII):
  • Figure US20110130555A1-20110602-C00026
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XVIII):
  • Figure US20110130555A1-20110602-C00027
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XIX):
  • Figure US20110130555A1-20110602-C00028
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XX):
  • Figure US20110130555A1-20110602-C00029
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XXI):
  • Figure US20110130555A1-20110602-C00030
      • wherein
      • R1 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Z is either nothing, choline, ethanolamine, serine, inositol, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a physiologically acceptable monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a number from 1 to 70;
      • wherein any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
      • In another embodiment, R1 of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
  • In another embodiment, R2 of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
  • In some embodiments, the compounds (A), (B) (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) as presented hereinabove comprises a Z group. In one embodiment, Z is a nothing. In another embodiment Z is inositol. In another embodiment, Z is choline. In another embodiment, Z is glycerol. In another embodiment, Z is ethanoleamine. In another embodiment, Z is serine.
  • For any or all of the compounds represented by the structures of the general formulae (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII) hereinabove: In one embodiment, X is a glycosaminoglycan. According to this aspect and in one embodiment, the glycosaminoglycan may be, inter alia, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate or a derivative thereof. In one embodiment, the chondroitin sulfate may be, inter alia, chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. In another embodiment, X is not a glycosaminoglycan. In another embodiment, X is a polysaccharide, which in one embodiment is a hetero-polysaccharide, and in another embodiment, is a homo-polysaccharide. In another embodiment, X is a polypyranose.
  • In another embodiment, the glycosaminoglycan is a polymer of disaccharide units. In another embodiment, the number of the disaccharide units in the polymer is m. In another embodiment, m is a number from 2-10,000. In another embodiment, m is a number from 2-500. In another embodiment, m is a number from 2-1000. In another embodiment, m is a number from 50-500. In another embodiment, m is a number from 2-2000. In another embodiment, m is a number from 500-2000. In another embodiment, m is a number from 1000-2000. In another embodiment, m is a number from 2000-5000. In another embodiment, m is a number from 3000-7000. In another embodiment, m is a number from 5000-10,000. In another embodiment, a disaccharide unit of a glycosaminoglycan may be bound to one lipid or phospholipid moiety. In another embodiment, each disaccharide unit of the glycosaminoglycan may be bound to zero or one lipid or phospholipid moieties. In another embodiment, the lipid or phospholipid moieties are bound to the —COOH group of the disaccharide unit. In another embodiment, the bond between the lipid or phospholipid moiety and the disaccharide unit is an amide bond.
  • In one embodiment, this invention provides lipid-GAG conjugate or phospholipid-GAG conjugate, and methods of use thereof, wherein said conjugate represented by the structures of the general formulae (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XX), and (XXII). In another embodiment, the average molecular weight of said GAG is between 5 kD to 90 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 60 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 40 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 15 kD. In another embodiment, the average molecular weight of said GAG is between 5 kD to 20 kD. In another embodiment, the lipid-GAG conjugate is a phospholipid-GAG conjugate.
  • In one embodiment of the invention, Y is nothing. Non-limiting examples of suitable divalent groups forming the optional bridging group (which in one embodiment, is referred to as a spacer) Y, according to embodiments of the invention, are straight or branched chain alkylene, e.g., of 2 or more, preferably 4 to 30 carbon atoms, —CO-alkylene-CO, —NH-alkylene-NH—, —CO-alkylene-NH—, —NH-alkylene-NH, CO-alkylene-NH—, an amino acid, cycloalkylene, wherein alkylene in each instance, is straight or branched chain and contains 2 or more, preferably 2 to 30 atoms in the chain, —(—O—CH(CH3)CH2—)x— wherein x is an integer of 1 or more.
  • In one embodiment of the invention, the sugar rings of the glycosaminoglycan are intact. In another embodiment, intact refers to closed. In another embodiment, intact refers to natural. In another embodiment, intact refers to unbroken.
  • In one embodiment of the invention, the structure of the lipid or phospholipid in any compound according to the invention is intact. In another embodiment, the natural structure of the lipid or phospholipids in any compound according to the invention is maintained.
  • In one embodiment, the compounds for use in the present invention are biodegradable.
  • In some embodiments, the compounds for use are as listed in Table 1 below,
  • TABLE 1
    Phospholipid Spacer Polymer (m.w.)
    PE None Hyaluronic acid
    (2-2000 kDa)
    Dimyristoyl-PE None Hyaluronic acid
    PE None Heparin
    (0.5-110 kDa)
    PE None Chondroitin sulfate A
    PE None Carboxymethylcellulose
    (20-500 kDa)
    PE Dicarboxylic acid + Polygeline (haemaccel)
    Diamine (4-40 kDa)
    PE None Hydroxyethylstarch
    PE Dicarboxylic acid + Dextran
    Diamine (1-2,000 kDa)
    PE Carboxyl amino group Hyaluronic acid
    (5-20 kDa)
    PE Dicarboxyl group Hyaluronic acid
    (5-20 kDa)
    PE Dipalmitoic acid Hyaluronic acid
    (5-20 kDa)
    PE Carboxyl amino group Heparin
    (5-20 kDa)
    PE Dicarboxyl group Heparin
    (5-20 kDa)
    PE Carboxyl amino group Chondroitin sulfate A
    PE Dicarboxyl group Chondroitin sulfate A
    PE Carboxyl amino group Carboxymethylcellulose
    (5-20 kDa)
    PE Dicarboxyl group Carboxymethylcellulose
    (5-20 kDa)
    PE None Polygeline (haemaccel)
    (5-20 kDa)
    PE Carboxyl amino group Polygeline (haemaccel)
    (5-20 kDa)
    PE Dicarboxyl group Polygeline (haemaccel)
    (5-20 kDa)
    PE Carboxyl amino group Hydroxyethylstarch
    PE Dicarboxyl group Hydroxyethylstarch
    PE None Dextran
    (5-20 kDa)
    PE Carboxyl amino group Dextran
    (5-20 kDa)
    PE Dicarboxyl group Dextran
    (5-20 kDa)
    PE None Chondroitin sulfates
    Dipalmitoyl-PE None Hyaluronic acid
    Dipalmitoyl-PE None Heparin
    Dipalmitoyl-PE None Chondroitin sulfate A
    Dipalmitoyl-PE None Carboxymethylcellulose
    Dipalmitoyl-PE None Polygeline (haemaccel)
    Dipalmitoyl-PE None Hydroxyethylstarch
    Dipalmitoyl-PE None Dextran
    Dimyristoyl-PE None Heparin
    Dimyristoyl-PE None Chondroitin sulfate A
    Dimyristoyl-PE None Carboxymethylcellulose
    Dimyristoyl-PE None Polygeline (haemaccel)
    Dimyristoyl-PE None Hydroxyethylstarch
    Dimyristoyl-PE None Dextran
    PS None Hyaluronic acid
    PS None Heparin
    PS None Polygeline (haemaccel)
    PC None Hyaluronic acid
    PC None Heparin
    PC None Polygeline (haemaccel)
    PI None Hyaluronic acid
    PI None Heparin
    PI None Polygeline (haemaccel)
    PG None Hyaluronic acid
    PG None Heparin
    PG None Polygeline (haemaccel)
  • In one embodiment, this invention provides a lipid-polymer conjugate represented by the structure of the general formula (B):
  • Figure US20110130555A1-20110602-C00031
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, phosphate, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 10;
      • wherein any bond between L, Z, Y and X is either an amide or an esteric bond.
  • In one embodiment, this invention provides a lipid-polymer conjugate represented by the structure of the general formula (XXII):
  • Figure US20110130555A1-20110602-C00032
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 10;
      • wherein if Y is nothing the phosphatidylethanolamine is directly linked to X via an amide bond and if Y is a spacer, said spacer is directly linked to X via an amide or an esteric bond and to said phosphatidylethanolamine via an amide bond.
  • In one embodiment, n of formula (B) and formula (X) is 1-10, in another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 7. In another embodiment, n is 8. In another embodiment, n is 9. In another embodiment, n is 10.
  • In one embodiment, this invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • Figure US20110130555A1-20110602-C00033
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
        • wherein if Y is nothing the phosphatidylethanolamine is directly linked to X via an amide bond and if Y is a spacer, said spacer is directly linked to X via an amide or an esteric bond and to said phosphatidylethanolamine via an amide bond;
      • comprising reacting a phospholipid (PL) with a glycosaminoglycan (GAG) and a coupling agent, wherein the massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively;
  • In one embodiment, this invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • Figure US20110130555A1-20110602-C00034
      • wherein
      • R1 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • R2 is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
        • wherein if Y is nothing the phosphatidylethanolamine is directly linked to X via an amide bond and if Y is a spacer, said spacer is directly linked to X via an amide or an esteric bond and to said phosphatidylethanolamine via an amide bond;
      • comprising the steps of:
      • a. reacting a phospholipid (PL) with a glycosaminoglycan (GAG) and a coupling agent, wherein the massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively;
      • b. filtering the reaction mixture from (a) to generate a filtrate; and
      • c. extracting a product from a filtrate.
  • In another embodiment, said coupling agent is DCC, EDAC, BOP, PyBOP, HATU, TSTU or any other amide coupling agent. In another embodiment, said coupling agent is EDAC. In another embodiment, said coupling agent further comprises HOBT or HOAT.
  • In another embodiment, said filtering step comprises a 10 kD centrasette membrane.
  • In another embodiment, R1 is the residue of palmitic acid or the residue of myristic acid.
  • In another embodiment, R2 is the residue of palmitic acid or the residue of myristic acid.
  • In another embodiment, the average molecular weight of the glycosaminoglycan is between 5 kD to 90 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 5 kD to 20 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 5 kD to 10 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 10 kD to 20 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 20 kD to 50 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 30 kD to 60 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 40 kD to 70 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 50 kD to 80 kD. In another embodiment, the average molecular weight of the glycosaminoglycan is between 60 kD to 90 kD
  • In one embodiment, hyaluronic acid (HA) is used in solution form. In another embodiment, HA solution is prepared according to Example 1,
  • In one embodiment, the process for the preparation of fractionated hyaluronic acid includes ultrafiltration. In another embodiment, the ultrafiltration fractionation of hyaluronic acid is as described in Example 2.
  • In one embodiment, phosphatidylethanolamine-hyaluronic acid conjugate (HyPE) is prepared by reacting a GAG with a PL using a coupling agent. In another embodiment, HyPE is prepared according to Example 3 using the apparatus depicted in FIG. 1.
  • In one embodiment, fractionated HA is used in the preparation of HyPE. In another embodiment, fractionated HA is prepared according to Example 3. In another embodiment, HyPE is prepared according to Example 11.
  • In one embodiment, a coupling reagent is used in the preparation of HyPE according to Example 3. In another embodiment, EDAC is used as the coupling reagent. In another embodiment, DCC is used as the coupling agent. In another embodiment, BOP is used as the coupling agent. In another embodiment, PyBOP is used as the coupling agent. In another embodiment, HATU is used as the coupling agent. In another embodiment, TSTU is used as the coupling agent.
  • In one embodiment, the coupling agent used in the preparation of HyPE according to Example 3 comprises HOBT. In another embodiment, the coupling agent comprises HOAT.
  • In one embodiment, crude HyPE is processed by an ultrafiltration step. In another embodiment, HyPE is subjected to the alkaline ultrafiltration described in Example 4.
  • In one embodiment, filtered HyPE is isolated by extraction. In another embodiment, HyPE is extracted according to the process described in Example 5. In another embodiment, said extraction comprises dichloromethane, ethanol and methanol.
  • In one embodiment, this invention provides a method of treating an inflammatory disorder in a subject, said method comprising administering to a subject suffering from an inflammatory disorder a composition comprising a lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively.
  • In another embodiment, said massPL to massGAG ratio is about 0.25:15. In another embodiment, said massPL to massGAG ratio is about 0.5:15. In another embodiment, said massPL to massGAG ratio is about 1:15. In another embodiment, said massPL to massGAG ratio is about 2:15. In another embodiment, said massPL to massGAG ratio is about 5:15.
  • In another embodiment, said inflammatory disorder is rheumatoid arthritis, osteoarthritis, wound healing, dermatitis, restenosis, cystic fibrosis, multiple sclerosis or sepsis.
  • In one embodiment, in vitro assays are used to measure the ability of HyPE and HyPE analogs to reduce the expression of pro-inflammatory cytokines. In another embodiment, cell-based assays are used according to Example 6, Example 7 and Example 8. In another embodiment, expression of IL-6 is measured. In another embodiment, expression of TNF-α is measured. In another embodiment, expression of IP-10 is measured. In another embodiment, expression of PGE2 is measured.
  • In another embodiment, said composition is administered intravenously. In another embodiment, said composition is administered topically.
  • In one embodiment, the present invention provides a method for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof comprising the step of administering a compound represented by the structure of the general formula (A):
  • Figure US20110130555A1-20110602-C00035
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between L, Z, Y and X is either an amide or an esteric bond to a subject with high levels of proinflammatory chemokines, cytokines, or a combination thereof.
  • In one embodiment, the present invention provides a method of activating NF-κB, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines comprising the step of administering to a subject a compound represented by the structure of the general formula (A):
  • Figure US20110130555A1-20110602-C00036
      • wherein
      • L is a lipid or a phospholipid;
      • Z is either nothing, ethanolamine, serine, inositol, choline, or glycerol;
      • Y is either nothing or a spacer group ranging in length from 2 to 30 atoms;
      • X is a glycosaminoglycan; and
      • n is a number from 1 to 70;
      • wherein any bond between L, Z, Y and X is either an amide or an esteric bond.
    Dosages and Routes of Administration
  • The methods of this invention can be adapted to the use of the therapeutic compositions comprising Lipid-conjugates in admixture with conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., vitamins, bronchodilators, steroids, anti-inflammatory compounds, gene therapy, i.e. sequences which code for the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) receptor, surfactant proteins, etc., as will be understood by one skilled in the art.
  • In one embodiment, the invention provides for the administration of a salt of a compound as described herein as well. In one embodiment, the salt is a pharmaceutically acceptable salt, which, in turn may refer to non-toxic salts of compounds (which are generally prepared by reacting the free acid with a suitable organic or inorganic base) and include, but are not limited to, the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandlate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate, diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts, as well as mixtures of these salts.
  • In one embodiment, the route of administration may be parenteral, enteral, or a combination thereof. In another embodiment, the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation, nasal aspiration (spray), sublingual, oral, aerosol or suppository or a combination thereof. In one embodiment, the dosage regimen will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, etc.
  • In general, the doses utilized for the above described purposes will vary, but will be in an effective amount to exert the desired anti-disease effect. As used herein, the term “pharmaceutically effective amount” refers to an amount of a compound of formula (I) which will produce the desired alleviation in symptoms or signs of disease in a patient. The doses utilized for any of the above-described purposes will generally be from 1 to about 1000 milligrams per kilogram of body weight (mg/kg), administered one to four times per day, or by continuous IV infusion. When the compositions are dosed topically, they will generally be in a concentration range of from 0.1 to about 10% w/v, administered 1-4 times per day.
  • In one embodiment, the use of a single chemical entity with potent anti-oxidant, membrane-stabilizing, anti-proliferative, anti-chemokine, anti-migratory, and anti-inflammatory activity provides the desired protection for a subject with an inflammatory disorder, or in another embodiment, the methods of this invention provide for use of a combination of the compounds described. In another embodiment, the compounds for use in the present invention may be provided in a single formulation/composition, or in another embodiment, multiple formulations may be used. In one embodiment, the formulations for use in the present invention may be administered simultaneously, or in another embodiment, at different time intervals, which may vary between minutes, hours, days, weeks or months.
  • In one embodiment the compositions comprising the compounds for use in the present invention may be administered via different routes, which in one embodiment, may be tailored to provide different compounds at different sites, for example some compounds may be given parenterally to provide for superior perfusion throughout the lung and lymphatic system, and in another embodiment, some formulations/compounds/compositions may be provided via aerosol, or in another embodiment, intranasally, to provide for higher lung mucosal concentration. is there something wrong with this sentence? Seems like you need the word “higher” before mucosal?
  • In one embodiment, the compounds for use in the invention may be used for acute treatment of temporary conditions, or may be administered chronically, as needed. In one embodiment of the invention, the concentrations of the compounds will depend on various factors, including the nature of the condition to be treated, the condition of the patient, the route of administration and the individual tolerability of the compositions.
  • In one embodiment, the methods of this invention provide for the administration of the compounds in early life of the subject, or in another embodiment, throughout the life of the subject, or in another embodiment, episodically, in response to severity or constancy of symptomatic stages, or in another embodiment. In another embodiment, the patients to whom the lipid or PL conjugates should be administered are those that are experiencing symptoms of disease or who are at risk of contracting the disease or experiencing a recurrent episode or exacerbation of the disease, or pathological conditions associated with the same.
  • As used herein, the term “pharmaceutically acceptable carrier” refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound of the present invention. As such, all of the above-described formulations of the present invention are hereby referred to as “pharmaceutically acceptable carriers.” This term refers to as well the use of buffered formulations wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the compounds and route of administration.
  • For parenteral administration, particularly suitable are sterile solutions, preferably oily or aqueous solutions, as well as suspensions or emulsions. It is also possible to freeze-dry the new compounds and use the lyophilates obtained, for example, for the preparation of products for injection.
  • In one embodiment, implants or suppositories, can be used to administer a lipid-GAG conjugate of this invention.
  • For application by inhalation, particularly for treatment of airway obstruction or congestion, solutions or suspensions of the compounds mixed and aerosolized or nebulized in the presence of the appropriate carrier
  • For topical application, particularly for the treatment of skin diseases such as contact dermatitis or psoriasis, admixture of the compounds with conventional creams or delayed release patches is acceptable.
  • For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules. A syrup, elixir, or the like can be used when a sweetened vehicle is employed. When indicated, suppositories or enema formulations may be the recommended route of administration.
  • Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
  • It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular situs and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol.
    • Methods of Use
  • In one embodiment of the invention, the methods of the present invention make use of a conjugate as described herein to treat a subject suffering from an inflammatory disorder, reduce or delay the mortality of a subject suffering from an inflammatory disorder or ameliorate symptoms associated with an inflammatory disorder.
  • In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and heparin. In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and chondroitin sulfate. In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and hyaluronic acid. In one embodiment, the compound for use in the present invention comprises dipalmitoyl phosphatidylethanolamine and carboxymethylcellulose. In one embodiment, the compound for use in the present invention comprises dimyristoyl phosphatidylethanolamine and hyaluronic acid.
  • In one embodiment, the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a glycosaminoglycan. In one embodiment, the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a chondroitin sulfate, which is chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. In another embodiment, the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a heparin. In another embodiment, the compound for use in the present invention is a dipalmitoyl phosphatidylethanolamine conjugated via an amide or ester bond to a hyaluronic acid. In another embodiment, the compound for use in the present invention is a dimyristoyl phosphatidylethanolamine conjugated via an amide or ester bond to a hyaluronic acid.
  • In one embodiment, the conjugates of this invention display a wide-range combination of cytoprotective pharmacological activities, which are useful in the present invention. In one embodiment, the compounds may be useful for their anti-inflammatory effects. Cellular elaboration of cytokines and chemokines serve an important regulatory function in health; however, when a hyperactive response to stress or disease is triggered, these compounds may present in excess and damage tissue, thereby pushing the disease state toward further deterioration. In one embodiment, the lipid compounds for use in the methods of this invention, possess a combination of multiple and potent pharmacological effects, including inter-alia the ability to inhibit the extracellular form of the enzyme phospholipase A2.
    • Method of Treating CF
  • In one embodiment, the conjugates of this invention are useful in affecting inflammation in a subject with an inflammatory disorder, where the subject is administered lipid-conjugates at pre-symptomatic stages of the disease. A characteristic feature of inflammation in the CF lung is the persistent infiltration of massive numbers of neutrophils into the airways. Although neutrophils help to control infection, when present in great excess, they can be harmful. Major advances in the understanding of the inflammatory process in the CF lung have come from the use of bronchoscopy and bronchioalveolar lavage (BAL) to analyze the inflammatory process in patients who are relatively symptom free and/or do not regularly produce sputum. Recent BAL studies suggest that neutrophil-rich inflammation begins very early, even in infants without clinically apparent lung disease. Thus, in one embodiment, the lipid/phospholipid conjugates of the present invention may be useful in treating CF, even in presymptomatic stages of disease.
  • Thus, in one embodiment, the invention provides methods for treating a subject suffering from cystic fibrosis, reducing or delaying the mortality of a subject suffering from cystic fibrosis or ameliorating symptoms associated with cystic fibrosis, and the compounds/compositions/formulations, in one embodiment, diminish or abrogate a deleterious inflammatory response in said subject, or in another embodiment, prevent, treat, reduce the incidence of, reduce the severity of, delay the onset of, or diminish the pathogenesis of an infection is the CF subject. In another embodiment, the invention provides methods for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof, while in another embodiment, the invention provides methods of activating NF-κB, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines.
    • Method of Treating Wounds
  • In another embodiment, provided herein a method for promoting wound healing comprising applying or administering to a wound site to be treated in a subject an effective amount of a composition comprising any conjugate as described herein. In another embodiment, provided herein a method for promoting wound healing comprising applying or administering to a wound site to be treated in a subject an effective amount of a composition comprising any compound represented by the structure of the general formula (A).
  • In another embodiment, promoting wound healing comprises inducing wound healing. In another embodiment, promoting wound healing comprises speeding up wound healing. In another embodiment, promoting wound healing comprises reducing the risk of viral and/or bacterial infection. In another embodiment, promoting wound healing comprises reducing inflammation in or near the wound site.
  • In another embodiment, the conjugates as described herein increase the rate of chronic and acute wound healing. In another embodiment, the conjugates as described herein counteract mechanisms which delay or impair wound healing. In another embodiment, the compounds as described herein counteract exogenous factors which delay or impair wound healing. In another embodiment, the conjugates as described herein counteract endogenous factors which delay or impair wound healing. In another embodiment, factors include: infection, ulceration particularly through diabetes, circulation problems associated with vascular disease, malnutrition, stress, cancer radiotherapy and/or chemotherapy, compromise of the immune system or simply due to the normal aging process. In another embodiment, a method a described herein provides both a therapeutic and a cosmetic approach that promote wound healing processes.
  • In another embodiment, wounds include, but are not limited to the following: surgical wounds; bites; burns; acid and alkali burns; cold burn (frostbite), sun burn, minor cuts, major cuts, abrasions, lacerations, wounds caused by gunshot or knife injury; wounds caused by congenital disorders; wounds following dental surgery; periodontal disease; wounds following trauma; tumour associated wounds, which can be classified as malignant cutaneous ulcers related to the primary tumour or metastases; ulcers, leg ulcers; foot ulcers; pressure sores and corneal wounds.
    • Method of Treating Arthritis
  • In another embodiment of the invention, the methods of the present invention make use of a conjugate as described herein for treating a subject suffering from arthritis, reducing or delaying the damage to the joints of a subject suffering from arthritis, or ameliorating symptoms associated with arthritis. In another embodiment of the invention, the methods of the present invention make use of a formulation comprising a conjugate as described herein for treating a subject suffering from arthritis, reducing or delaying the damage to the joints of a subject suffering from arthritis, or ameliorating symptoms associated with arthritis.
  • In another embodiment, provided herein a method of treating a subject suffering from joint pain, swelling within the joint, inflammation within the joint, or a combination thereof comprising the step of administering a composition comprising a conjugate of the invention to the subject. In another embodiment, provided herein a method of treating a subject suffering from joint pain, swelling within the joint, inflammation within the joint, or a combination thereof comprising the step of injecting into a swelled/inflamed joint a composition comprising a conjugate of the invention.
  • It is understood that one skilled in the art recognizes that the term “arthritis” refers to both rheumatoid arthritis (RA) and osteoarthritis (OA).
  • In another embodiment, a compound as described herein inhibits the production of IL-6, IL-8, TNF-alpha, NF-κB, or their combination, thereby reducing or delaying the damage to the joints of a subject suffering from arthritis. In another embodiment, a compound as described herein inhibits the production of IL-6, IL-8, TNF-alpha, NF-κB, or their combination, thereby ameliorating symptoms associated with arthritis. In another embodiment, methods comprising the administration of a conjugate as described herein treat a subject suffering from joint pain, swelling within the joint, inflammation within the joint, or a combination thereof by inhibiting the production of IL-6, IL-8, TNF-alpha, NF-κB, or their combination. In another embodiment, locally administering a composition comprising a conjugate as described herein by intra-joint injection inhibits the production of IL-6, IL-8, TNF-alpha, NF-κB, or their combination within the joint's cells. In another embodiment, locally administering a composition comprising a conjugate as described herein by intra-joint injection inhibits inflammation within the joint.
    • Method of Treating Other Inflammatory Disorders
  • It is understood by one skilled in the art that inflammatory disorders include, but are not limited to, disorders resulting from activation of the immune system. Thus, autoimmune disorders are understood to be inflammatory disorders. Such disorders include, but are not limited to, rheumatoid arthritis, osteoarthritis, wound healing, dermatitis, restenosis, cystic fibrosis, central nervous system tissue insult, multiple sclerosis, obstructive respiratory disease, Crohn's disease, cardiovascular disease, atherosclerosis, contact dermatitis, psoriasis, ARDS, or sepsis
  • In one embodiment, the invention provides a method of treating a subject suffering from obstructive respiratory disease, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from obstructive respiratory disease.
  • In one embodiment, the invention provides a method of treating a subject suffering from colitis, Crohn's disease, or another form of intestinal mucosal injury, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from intestinal mucosal injury, including colitis or Crohn's disease.
  • In one embodiment, the invention provides a method of treating a subject suffering from cardiovascular disease, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from a cardiovascular disease.
  • The present invention provides a method of treating a subject suffering from atherosclerosis, including, inter alia, the step of administering to a conjugate of this invention, thereby treating the subject suffering from atherosclerosis.
  • In one embodiment, the invention provides a method of treating a subject suffering from central nervous system tissue insult, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from a central nervous system insult.
  • In one embodiment, the invention provides a method of treating a subject suffering from multiple sclerosis, including, inter alia, the step of administering to a subject an effective amount of conjugate of this invention, thereby treating the subject suffering from multiple sclerosis.
  • In one embodiment, the invention provides a method of treating a subject suffering from contact dermatitis, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from contact dermatitis.
  • In one embodiment, the invention provides a of treating a subject suffering from psoriasis, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from psoriasis.
  • In one embodiment, the invention provides a method of treating a subject suffering from sepsis, including, inter alia, the step of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from sepsis.
  • In one embodiment, the invention provides a method of treating a subject suffering from ARDS, comprising the steps of administering to a subject an effective amount of a conjugate of this invention, thereby treating the subject suffering from ARDS.
  • While pharmacological activity of the Lipid-conjugates described herein may be due in part to the nature of the lipid moiety, the multiple and diverse combination of pharmacological properties observed for the Lipid-conjugates may represent, in other embodiments, the ability of the conjugate to act essentially as several different drugs in one chemical entity. Thus, for example, lung mucosal or lung parenchymal injury, as may occur in CF, may be attenuated by any one or all of the pharmaceutical activities of immune suppression, anti-inflammation, anti-oxidation, suppression of nitric oxide production, or membrane stabilization.
  • In one embodiment, the invention provides a method of “treating” inflammatory disorders or related diseases or disorders, which in one embodiment, refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove. In one embodiment, treating refers to delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing relapse to a disease, decrease the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternate therapeutics.
  • In one embodiment, the methods are useful in treating an infection in a subject, wherein the pathogen is a virus or in another embodiment, the pathogen is a bacterium. In one embodiment, the infection is with a pathogen which infects the respiratory system, such as mycobacteria, pseudomonas, cryptococcus, streptococcus, reovirus, influenza, or other infections known to those of skill in the art.
  • Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples and preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
    • Example 1 Preparation of Hyaluronic Acid (HA) Solution
  • 4 g of chlorocresol was dissolved in 4 L of deionized (DI) water (0.1% solution). HA UL 15 was dissolved in 4 L of 0.1% chlorocresol solution with mechanical stirring. To prevent clogging of the ultrafiltration membranes, the HA solution was filtered through a 100 μm filter followed by a 50 μm filter followed by a 10 μm filter, all previously disinfected with 10% hydrogen peroxide and washed with copious amounts of DI water to ensure hydrogen peroxide has been removed (verified with peroxide-detecting strips).
    • Example 2 Ultrafiltration Fractionation of Hyaluronic Acid (HA)
  • HA solution of Example 1 was loaded into the Centramate system, previously disinfected with 10% hydrogen peroxide and washed with copious amounts of DI water to ensure hydrogen peroxide has been removed (verified with peroxide-detecting strips).
  • By means of constant volume diafiltration with 70 kDa Omega TFF membranes, 20 L of 0.1% chlorocresol solution, prepared as described in Example 1, was ultrafiltered, collecting the filtrate, the fraction less than 70 kDa, in a carboy, previously disinfected with 10% hydrogen peroxide. The pump speed and valves shall be set such that the retentate flow is ten times the filtrate flow and the feed pressure is less than 40 PSI.
  • The 70 kDa membranes were replaced with 30 kDa membranes and the Centramate system was disinfected with 10% hydrogen peroxide.
  • 5 L of the filtrate, the fraction less than 70 kDa, were loaded into the reservoir and by means of constant volume diafiltration, the remaining 35 L in the carboys of the fraction less than 70 kDa were ultrafiltered. The reservoir volume was reduced to 2 L and an additional 10 L of DI water was ultrafiltered to remove the chlorocresol (confirmed by appropriate GC assay). The reservoir volume was further reduced to 1 L, reducing the pump speed, if necessary, to keep the feed pressure below 40 PSI. The reservoir was then emptied directly into an autoclaved lyoguard container, closed, frozen and lyophilized to yield HA UF 70/30. GPC analysis was performed to ensure that this lot of HA UF 70/30 was consistent with earlier batches. A bioburden assay and an appropriate GC assay for chlorocresol was performed. Karl Fischer analysis was performed to determine the water content of HA UF 70/30.
    • Example 3 HyPE Synthesis Reaction
  • Using the apparatus depicted in FIG. 36, 24 g of 2-(N-morpholino)ethanesulfonic acid (MES) were dissolved in 125 mL of DI water and the pH was adjusted to pH 6.4 by addition of 4 N NaOH.
  • 2.5 g of dipalmitoylphosphatidylethanolamine (DPPE) and 25 g of hydroxybenzotriazole (HOBT) were dissolved in 940 mL of tert-butanol and 80 mL of water with stirring and heating at 45° C. in a 12 L round bottom flask (forming a closed system with the pump and the sonicator, all of which will have been previously autoclaved and/or disinfected with 70% isopropanol). To this was added 850 mL of water and 115 mL of the MES solution. The pH of this solution was adjusted to pH 6.4 by addition of 2.5 N NaOH. 25 g of HA UF 70/30 of Example 2 were then dissolved with stirring and heating at 45° C. 25 g of 1-ethyl-3-(3-dimethylaminoethyl)carbodiimide (EDAC) were then added, the pump and the sonicator were turned on and the system was kept between 40 and 50° C. for 3 hours. GPC analysis was performed to monitor the progress of the reaction. After 3 hours the sonicator and the pump were turned off and the solution was stirred at room temperature overnight. The following day 750 mL of acetonitrile were added to precipitate HyPE. This was allowed to stand for 30 minutes after which the supernatant was removed. To this was added 7.5 L of 2% Na2CO3, previously prepared by dissolving 150 g of Na2CO3 in 7.5 L in DI water. Vigorous mechanical stirring for at least 2 hours hydrolyzed urea related byproducts. The solution was neutralized with 6 N HCl while the temperature was kept at 20-25° C. by passing the solution through a cooled, jacketed flow cell.
    • Example 4 Alkaline Ultrafiltration of HyPE
  • 2.25 kg of NaHCO3 was dissolved in 150 L of 0.1% chlorocresol solution, prepared by dissolving 150 g of chlorocresol in 150 L of DI wate. By means of valves, the closed reaction system was diverted so that the digested, neutralized HyPE solution of Example 3 was pumped from the round bottom flask to the centrasette system. By means of constant volume diafiltration with a 10 kDa Omega TFF membrane, 150 L of 1.5% NaHCO3 in 0.1% chlorocresol solution was ultrafiltered, discarding the filtrate, the fraction less than 10 kDa. The pump speed and valves were set such that the retentate flow was ten times the filtrate flow and the feed pressure was less than 40 PSI. GPC analysis was performed to ensure the disappearance of urea-related peaks at ˜13.2 min and the HOBT peak at ˜17.2 min. The solution was neutralized with 6 N HCl while the temperature was kept at ˜20-25° C. by passing the solution through a cooled, jacketed flow cell.
    • Example 5 Extraction of HyPE
  • An extraction solution was made by mixing 3 L of dichloromethane, 3 L of ethanol and 2.25 L of methanol. 7.5 L of the extraction solution was added to a round bottom flask containing 3 L of crude HyPE solution of Example 4. This was stirred vigorously for 15 minutes after which time it was allowed to stand for 45 min. The lower dichloromethane layer was removed. By means of constant volume diafiltration the solution was washed with 100 L of DI water to remove the methanol and ethanol. GPC analysis was performed to ensure the disappearance of peaks at ˜14 min. The volume was reduced to 3 L and emptied directly into 2 autoclaved lyoguard containers, closed, frozen and lyophilized to yield HyPE. NMR and HPLC data for isolated HyPE are shown in FIG. 2 and FIG. 3.
    • Example 6 IN Vitro Stimulation of RAW 264.7 Cells
  • In vitro stimulation of RAW 364.7 cells was carried out according to the schematic depicted in FIG. 4. Each Test Article was prepared in DMEM (no FBS) at 10 mg/ml (all Test Article concentrations were corrected for moisture content), vortexed, heated at 50° C. for 5 minutes, sonicated for 5 minutes and filtered through a 0.2 micron syringe filter. 2× Test Article working solutions of 0.6 mg/ml, 0.2 mg/ml and 0.06 mg/ml were prepared by diluting the 10 mg/ml stock solutions in CM. A 2.55 mM solution of dexamethasone prepared in ethanol was diluted to 2 μM (2× working solution) in CM. The vehicle control solution was CM. A 1 mg/ml solution of LPS (made in 1×PBS) was diluted in CM to 10 μg/ml. RAW 264.7 cells were grown for XX passages (subculture every 3-4 days) in CM at 37° C. with 5% CO2. 0.5 ml of cells at 1×106 cells/ml was plated in 24-well tissue culture plates. Cells were allowed to adhere for 30 minutes at 37° C. with 5% CO2 prior to treatment. The appropriate Test Article, dexamethasone or vehicle control working solutions were added to the cells. Cells were incubated for 1 hour at 37° C. with 5% CO2 prior to LPS treatment. 110 μl of CM was added to the -LPS plates. 110 μl of 10 μg/ml LPS was added to the +1 μg/ml LPS plates. The plates were incubated for 24 hours at 37° C. with 5% CO2.
    • Example 7 Supernatant Harvesting/XTT Assay
  • Cell culture supernatants were collected after 24 hours of LPS treatment and stored at −30° C. until assayed. 400 μl of media was left in each cell culture well for the XTT assay. 400 μl of media was added to a cell-free culture well for use as a blank in the XTT assay. 200 μl of activated XTT reagent was added to each well. Plates were incubated for 1 hour at 37° C. with 5% CO2. 100 μl was removed from each well and read at 450 nm (630 nm correction) using a ThermoMax microplate reader (Molecular Devices, Sunnyvale, Calif.). XTT data relating to high molecular weight HyPE compositions are shown in FIG. 5 and FIG. 6. XTT data relating to low molecular weight HyPE compositions are shown in FIG. 22 and FIG. 23.
    • Example 8 Cytokine/Chemokine Assays
  • Cell culture supernatants were assayed for IL-6, TNF-α and IP-10 using a Luminexbased assay according to the manufacturer's instructions. Data were collected using a Luminex 100 (Luminex Corporation, Austin, Tex.). Standard curves were generated using a 5-parameter logistic curve-fitting equation weighted by 1/y (StarStation V 2.0; Applied Cytometry Systems, Sacramento, Calif.). Each sample reading was interpolated from the appropriate standard curve. Calculated concentrations were multiplied by the appropriate dilution factor when necessary. TNF-α data relating to high molecular weight HyPE compositions are shown in FIG. 7, FIG. 8 and FIG. 15. TNF-α data relating to low molecular weight HyPE compositions are shown in FIG. 24, FIG. 25 and FIG. 32. IL-6 data relating to high molecular weight HyPE compositions are shown in FIG. 9, FIG. 10 and FIG. 16. IL-6 data relating to low molecular weight HyPE compositions are shown in FIG. 26, FIG. 27 and FIG. 33. IP-10 data relating to high molecular weight HyPE compositions are shown in FIG. 11, FIG. 12 and FIG. 17. IP-10 data relating to low molecular weight HyPE compositions are shown in FIG. 28, FIG. 29 and FIG. 34.
  • Cell culture supernatants were assayed for PGE2 by ELISA following the manufacturer's instructions. Absorbance readings were detected using a ThermoMax microplate reader (Molecular Devices). Standard curves were generated using a 4-parameter logistic curve fitting equation (SoftMax Pro 4.7.1; Molecular Devices). Each sample reading was interpolated from the appropriate standard curve. Duplicate interpolated sample values were averaged and standard deviations were calculated. Calculated concentrations were multiplied by the appropriate dilution factor. PGE2 data relating to high molecular weight HyPE compositions are shown in FIG. 13, FIG. 14 and FIG. 18. PGE2 data relating to low molecular weight HyPE compositions are shown in FIG. 30, FIG. 31 and FIG. 35.
    • Example 9 Preparation of Low Molecular Weight Sodium Hyaluronate
  • Raw material of sodium hyaluronate (1.32 MDa) was degraded by acidic hydrolysis. The sample solution was ultrafiltered immediately after degradation. The final product was prepared using spray dryer as in the case of previous samples. In addition it was filtered with 0.2 μm filter (PALL) before drying to achieve microbial purity.
    • Example 10 SEC-MALS Determination of Molecular Weight
  • The chromatography system (Agilent, 1100 Series) consisted of a HPLC pump (G1310A), an automatic injector (G1313A) and the following column system: PL aquagel-OH Mix and PL aquagel-OH 30 (300×7.5 mm, 8 μm; Agilent Technologies) columns connected in series and thermostated at ambient temperature. Injection volume was 100 μl. Eluent (0.1 M sodium phosphate buffer pH 7.5) was monitored using a DAWN-EOS multi-angle laser light scattering photometer (18-angle, Wyatt Technologies Corporation) and a refractive index detector rEX Optilab (Wyatt Technologies Corporation). Data acquisition and molecular weight calculations were performed using the ASTRA V software, Version 5.3.2.15. The flow rate of mobile phase was maintained at 0.8 ml/min. The specific refractive index increment (dn/dc) of 0,155 mg/ml was used for sodium hyaluronate.
  • The hyaluronan samples were prepared by dissolving of a weighted amount of sample in the phosphate buffer (concentration 20.0 mg/ml). All samples were stirred several hours. The solutions were filtered through syringe filter (0.2 μm, 25 mm diameter, Whatman) and analysed by HPLC system.
  • Light scattering measurements can provide an absolute measurement of molar mass when used in series with a concentration sensitive detector such as a refractive index detector and if the value of dn/dc (differential refractive index increment) is known.
  • In essence, light scattering measurements automatically provide a column calibration curve for every sample, obviating time-consuming, conformation dependent calibration procedure.
  • Known dn/dc and known calibration constant of refractive index detector as calculated method were used. Differential refractive index increment (in mL/g) was determined by using the Wyatt Optilab refractometer.
  • The weight-average molecular weight of hyaluronan was verified by measurements of dextran standard.
  • The determined molecular weight and polydispersity value for low molecular weight hyaluronic acid were 7.86×103 g/mol and 1.32 Mw/Mn, respectively. The chromatogram and distribution diagram are stated in FIG. 19 and FIG. 20 whereas red line pertains to light scattering signal and blue line to refractive index signal. FIG. 21 illustrates the UV spectrum.
    • Example 11 Preparation of HyPE from 9.54 kD Hyaluronic Acid
  • MES buffer was prepared by dissolving 14.5 g of MES in 75 mL of DI-H2O and adjusting the pH to 6.4 with 4N NaOH. Using an apparatus similar to that depicted in FIG. 1, 10.0 g of HOBT was dissolved in 225 mL of DI-H2O, 60 mL MES buffer, 12 mL of tert-butanol. The pH was adjusted to 6.4 with 4N NaOH. 15.1 g of HA was dissolved in 350 mL of DI-H2O. 1.25 g or DPPE was dissolved in 440 mL of tert-butanol and 90 mL DI-H2O with heating to 55 deg C. The solutions of HA and HOBT were warmed to 35 deg C. and mixed. The DPPE solution, at 50 deg C. was then added to afford a clear solution. This was allowed to cool to 43 deg C., when it was added to the flask and circulated through the sonoreactor system (FIG. 36). Some component of the reaction mixture came out of solution and it was necessary to heat the reaction mixture to 49 deg C. with sonication to form a clear solution. 12.5 g of EDAC was added as a powder to the reaction mixture at a temperature of 45 deg C. Sonication began with a power of 180 watts. The reaction was monitored by GPC as shown in FIGS. 37-38 and because the extent of agglomeration, as observed by the ratio of the area of the first peak to that of the second continued to increase, the reaction was allowed to continue beyond the normal 3 h and was continued the next day. The sonication was turned off and the reaction mixture was filtered through a 0.45 μm filter to remove a small amount of rubber debris apparently from the stator. The solution (1200 mL) was extracted with 600 mL DCM and 600 mL MeOH. The resulting emulsion quickly resolved and the aqueous layer was extracted again with 500 mL DCM and 500 mL EtOH. Finally, the aqueous layer was extracted with 250 mL DCM and 250 mL EtOH and left over the weekend. Residual DCM was removed by rotovaporation at 35 deg C. and 200 Torr. The solution was then transferred to a previously cleaned centrasette ultrafiltration system with a 10 kDa membrane and by constant volume diafiltration was washed with 5 L of 1.5% NaHCO3 to remove residual organic solvents. The pH was then increased by slow addition of 2% Na2CO3 to pH 9.2. The solution was stirred for 1 hour at room temperature. After further washing with 30 L of 1.5% NaHCO3 the peat at ˜12.5 min had disappeared and the solution was washed with 30 L of DI-H2O until pH 7. To remove any digestion/ultrafiltration byproducts, such as free palmitic acid, the solution was then extracted again with 1 L DCM, 1 L MeOH and 0.75 L EtOH. The aqueous layer was extracted again with 400 mL DCM and 50 mL EtOH and finally a third time with 400 mL DCM and 50 mL EtOH. Residual DCM was removed by rotovaporation at 30 deg C. and 200 Torr. By constant volume diafiltration residual MeOH and EtOH were removed by washing with 15 L DI-H2O. The solution was concentrated to 1 L and filtered through a 0.2 μm filter into a lyoguard container and placed in the lypholizer. It was frozen by lowering the shelf temperature to −70 deg C. When frozen, vacuum was applied (14 mT) and the shelf temperature was raised to 30 deg C. Five days later 6.134 g of HyPE was recovered with a water-corrected weight of 5.2 g which corresponds to a 42% yield based on 12.5 g (water corrected) of HA. Total phosphorus was found to be 0.28% (dry basis). By LC/MS assay, 1,456 ppm of free EDU were found and after exposure to NaOH 12,557 ppm total EDU was found. No HOBT was detected and MES was less than 80 ppm. GPC of the final product is shown in FIG. 39 and NMR data are shown in FIG. 40.
  • While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (23)

1. A lipid-polymer conjugate comprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said GAG with said PL in a massPL to massGAG ratio from about 0.25:15 to about 5:15, respectively.
2. The lipid-polymer conjugate of claim 0, wherein said massPL to massGAG ratio is about 0.25:15.
3. The lipid-polymer conjugate of claim 0, wherein said massPL to massGAG ratio is about 0.5:15.
4. The lipid-polymer conjugate of claim 0, wherein said massPL to massGAG ratio is about 1:15.
5. The lipid-polymer conjugate of claim 0, wherein said massPL to massGAG ratio is about 2:15.
6. The lipid-polymer conjugate of claim 0, wherein said massPL to massGAG ratio is about 5:15.
7. The lipid-polymer conjugate of claim 0, wherein said GAG is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate.
8. The lipid-polymer conjugate of claim 0, wherein said PL is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol.
9. The lipid-polymer conjugate of claim 0, wherein said PL comprises the residue of palmitic acid or myristic acid.
10. The lipid-polymer conjugate of claim 0, wherein said PL is dimyristoyl phosphatidylethanolamine or dipalmitoyl phosphatidylethanolamine.
11. The lipid-polymer conjugate of claim 0, wherein the polydispersity of said GAG is from about 1 to 1.75.
12. The lipid-polymer conjugate of claim 11, wherein the polydispersity of said GAG is from about 1.25 to 1.5.
13. The lipid-polymer conjugate of claim 0, wherein the average molecular weight of said GAG is between 5 KD to 90 KD.
14. The lipid-polymerconjugate of claim 13, wherein the average molecular weight of said GAG is between 5 KD to 20 KD.
15-23. (canceled)
24. A lipid-polymer conjugate comprising a glycosaminglycan (GAG) conjugated to a phospholipid (PL) via an amide or ester linkage wherein the molecular weight of said GAG is between 5 to 20 KD and wherein the polydispersity of said GAG is from about 1 to 1.75.
25. The lipid-polymer conjugate of claim 24, wherein the polydispersity of said GAG is from about 1.25 to 1.5.
26. The lipid-polymer conjugate of claim 24, wherein said GAG is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate.
27. The lipid-polymer conjugate of claim 24, wherein said PL is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol.
28. The lipid-polymer conjugate of claim 24, wherein said PL comprises the residue of palmitic acid or myristic acid.
29. The lipid-polymer conjugate of claim 24, wherein said PL is dimyristoyl phosphatidylethanolamine or dipalmitoyl phosphatidylethanolamine.
30. A pharmaceutical composition comprising the lipid-polymer conjugate of claim 1.
31. A pharmaceutical composition comprising the lipid-polymer conjugate of claim 24.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130323178A1 (en) * 2012-02-23 2013-12-05 Canon Kabushiki Kaisha Indocyanine green-containing particle and contrast agent for photoacoustic imaging having the particle
WO2015068155A1 (en) * 2013-11-06 2015-05-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Lipid-polysaccharide conjugates, their preparation and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2762224T3 (en) 2011-05-12 2020-05-22 Yissum Res Dev Co Of Hebrew Univ Jerusalem Ltd Liposomes Comprising Polymer Conjugated Lipids and Related Uses
KR101762894B1 (en) 2015-05-19 2017-07-28 광주과학기술원 Cell coated with lipid-conjugated heparin and preparation method thereof

Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2976576A (en) * 1956-04-24 1961-03-28 Wichterle Otto Process for producing shaped articles from three-dimensional hydrophilic high polymers
US3220960A (en) * 1960-12-21 1965-11-30 Wichterle Otto Cross-linked hydrophilic polymers and articles made therefrom
US3431046A (en) * 1964-02-14 1969-03-04 Studies Inc Flexible polyethylene corneal contact lens
US3503942A (en) * 1965-10-23 1970-03-31 Maurice Seiderman Hydrophilic plastic contact lens
US3532679A (en) * 1969-04-07 1970-10-06 Robert Steckler Hydrogels from cross-linked polymers of n-vinyl lactams and alkyl acrylates
US3542461A (en) * 1967-11-20 1970-11-24 Du Pont Contact lens having an index of refraction approximating that of human tears
US3621079A (en) * 1968-10-21 1971-11-16 Patent Structures Inc Graft of hydroxyalkyl methacrylate onto polyvinylpyrrolidone
US3639524A (en) * 1969-07-28 1972-02-01 Maurice Seiderman Hydrophilic gel polymer insoluble in water from polyvinylpyrrolidone with n-vinyl-2-pyrrolidone and methacrylic modifier
US3700761A (en) * 1969-11-28 1972-10-24 Griffin Lab Inc Fabrication of soft plastic contact lens blank
US3758448A (en) * 1971-10-05 1973-09-11 Union Optics Corp Copolymers and hydrogels of unsaturated esters
US3772235A (en) * 1971-10-05 1973-11-13 Union Optics Corp Copolymers and hydrogels of unsaturated heterocyclic compounds
US3786034A (en) * 1972-06-23 1974-01-15 Frigitronics Of Conn Inc Polyurethane polymers for use in soft lenses and method of producing soft lenses
US5464942A (en) * 1990-07-24 1995-11-07 Seikagaku Kogyo Kabushiki Kaisha Phospholipid- or lipid-linked glycosaminoglycan and process for producing the same
US20020049183A1 (en) * 2000-01-10 2002-04-25 Saul Yedgar Use of lipid conjugates in the treatment of disease
US20040087492A1 (en) * 2000-01-10 2004-05-06 Saul Yedgar Use of lipid conjugates in the treatment of diseases
US20040229842A1 (en) * 2000-01-10 2004-11-18 Saul Yedgar Use of lipid conjugates in the treatment of diseases
US20080109236A1 (en) * 1998-04-02 2008-05-08 Deangelis Paul L Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
US20080113935A1 (en) * 2004-03-02 2008-05-15 Saul Yedgar Use of lipid conjugates in the treatment of diseases or disorders of the eye
US20080193538A1 (en) * 2003-10-29 2008-08-14 Teijin Limited Hyaluronic Acid Compound, Hydrogel Thereof and Joint Treating Material

Family Cites Families (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3816571A (en) 1969-11-28 1974-06-11 Warner Lambert Co Fabrication of soft plastic lens
US3803093A (en) 1971-02-25 1974-04-09 C Neefe Methyl methacrylate copolymer which may be hydrated
US3940207A (en) 1971-06-11 1976-02-24 E. I. Du Pont De Nemours And Company Soft, tough low refractive index contact lenses
US4055378A (en) 1971-12-31 1977-10-25 Agfa-Gevaert Aktiengesellschaft Silicone contact lens with hydrophilic surface treatment
US3948871A (en) 1972-04-21 1976-04-06 George H. Butterfield And Son Composition for hard type contact lens with wettable surface
US3875211A (en) 1973-01-22 1975-04-01 Alcolac Inc Process for preparing 2-hydroxyalkylacrylates and 2-hydroxyalkylmethacrylates
US3937680A (en) 1973-05-29 1976-02-10 Global Vision, Inc. Hydrophilic gel terpolymers from hydrophilic n-vinyl monomers, hydroxyalkyl acrylates or methacrylates and polymerizable unsaturated carboxylic acids
JPS5510050B2 (en) 1973-08-13 1980-03-13
JPS5337028B2 (en) 1973-12-11 1978-10-06
JPS543738B2 (en) 1974-05-27 1979-02-26
US4018853A (en) 1974-11-21 1977-04-19 Warner-Lambert Company Crosslinked, hydrophilic rods of pyrrolidone-methacrylate graft copolymers
FR2292989A1 (en) 1974-11-29 1976-06-25 Thomson Csf CONNECTOR FOR OPTICAL FIBER
US4099859A (en) 1974-12-02 1978-07-11 High Voltage Engineering Corporation Contact lens having a smooth surface layer of a hydrophilic polymer
US4064086A (en) 1976-07-13 1977-12-20 National Patent Development Corporation Thermoplastic hydrogels
US4604376A (en) 1982-04-21 1986-08-05 Research Corporation Enteric compounds and complexes
US4654327A (en) 1982-04-21 1987-03-31 Research Corp. Quaternary ammonium complexes of heparin
IT1229075B (en) * 1985-04-05 1991-07-17 Fidia Farmaceutici Topical compsn. contg. hyaluronic acid deriv. as vehicle
JPS59187792A (en) 1983-04-11 1984-10-24 Meito Sangyo Kk Production of phospholipid saccharide derivative using enzymic method
AU6593386A (en) 1985-10-24 1987-05-19 Research Corp. Ltd. Biochemical reagent
DE3607381A1 (en) 1986-03-06 1987-09-10 Pascal Zimmer USE OF DIPHENYLHYDANTOIN AND ITS DERIVATIVES FOR TREATING IMMUNE DISEASES
IL84252A (en) * 1987-10-23 1994-02-27 Yissum Res Dev Co Phospholipase inhibiting compositions
US5169636A (en) 1988-03-17 1992-12-08 Nippon Fine Chemical Co., Ltd. Liposomes
US5034166A (en) 1988-07-21 1991-07-23 Allergan, Inc. Method of molding a colored contact lens
CA2058940C (en) 1989-06-23 2000-05-09 Helen Loughrey Targeted liposomes and methods for liposome-protein coupling
JPH03160498A (en) 1989-11-20 1991-07-10 Oki Electric Ind Co Ltd Speech recognizing device
JPH0418841A (en) 1990-05-14 1992-01-23 Nec Corp Call signal generation device
US5733892A (en) 1990-07-24 1998-03-31 Seikagaku Corporation Metastasis inhibitor composition comprising a phospholipid-linked glycosaminoglycan and method for inhibiting metastasis employing the same
JP2997018B2 (en) * 1990-07-24 2000-01-11 生化学工業株式会社 Phospholipid or lipid-bound glycosaminoglycan
JP2986519B2 (en) * 1990-07-24 1999-12-06 生化学工業株式会社 Phospholipid-bound glycosaminoglycan
JP2986518B2 (en) * 1990-07-24 1999-12-06 生化学工業株式会社 Cancer metastasis inhibitor
JP2536275B2 (en) 1990-11-01 1996-09-18 三菱電機株式会社 Motion detection circuit
ES2084335T3 (en) 1991-02-14 1996-05-01 Baxter Int UNION OF LIPOSOMAS RECOGNITION SUBSTANCES.
US6022866A (en) 1991-07-03 2000-02-08 Hyal Pharmaceutical Corporation Use of hyaluronic acid and forms to prevent arterial restenosis
DE4137540A1 (en) 1991-11-14 1993-05-19 Steigerwald Arzneimittelwerk USE OF PREPARATIONS OF CURCUMA PLANTS
JP3714683B2 (en) 1992-07-30 2005-11-09 生化学工業株式会社 Anti-rheumatic agent
US5512671A (en) 1993-02-16 1996-04-30 Wake Forest University Ether lipid-nucleoside covalent conjugates
US6043231A (en) 1993-03-02 2000-03-28 The Research Foundation Of State Univ. Of New York Inhibition of excessive phospholipase A2 activity and/or production by non-antimicrobial tetracyclines
US5354853A (en) 1993-03-12 1994-10-11 Genzyme Corporation Phospholipid-saccharide conjugates
US5364845C1 (en) 1993-03-31 2002-09-10 Nutramax Lab Inc Glusosamine chondroitin and manganese composition for the protection and repair of connective tissue
WO1996004001A1 (en) 1994-08-05 1996-02-15 Molecular/Structural Biotechnologies, Inc. Site-specific biomolecular complexes
WO1996028544A1 (en) 1995-03-09 1996-09-19 Novo Nordisk A/S A protein-lipid conjugate, consisting of heparin binding protein (hbp) and a ceramide analogue, and its pharmaceutical use in treatment of conditions involving stress injury to cells
US6180596B1 (en) 1995-05-18 2001-01-30 Wisconsin Alumni Research Foundation Methods of inhibiting phospholipase A2 and phospholipase A2 stimulator activities
US5719656A (en) 1995-05-19 1998-02-17 Bowling; Patricia J. Contact lens utilizing stiles-crawford effect
US6033645A (en) 1996-06-19 2000-03-07 Unger; Evan C. Methods for diagnostic imaging by regulating the administration rate of a contrast agent
US5707821A (en) 1995-06-07 1998-01-13 Athena Neurosciences, Inc. Identification of phospholipase A2 inhibitors in Aβ peptide-mediated neurodegenerative disease
JP2001523216A (en) 1995-06-26 2001-11-20 リサーチ．トライアングル．ファーマシューティカルズ Novel adjuvant composition and vaccine formulation comprising the same
US5785975A (en) 1995-06-26 1998-07-28 Research Triangle Pharmaceuticals Adjuvant compositions and vaccine formulations comprising same
US6071532A (en) 1996-10-15 2000-06-06 Emory University Synthesis of glycophospholipid and peptide-phospholipid conjugates and uses thereof
US6090800A (en) 1997-05-06 2000-07-18 Imarx Pharmaceutical Corp. Lipid soluble steroid prodrugs
AU7490098A (en) 1997-05-15 1998-12-08 Genzyme Corporation Cationic amphiphile formulations
US6300308B1 (en) 1997-12-31 2001-10-09 Board Of Regents, The University Of Texas System Methods and compositions for inducing autoimmunity in the treatment of cancers
JP2000120870A (en) 1998-10-15 2000-04-28 Teikoku Piston Ring Co Ltd Piston ring
US6211162B1 (en) 1998-12-30 2001-04-03 Oligos Etc. Inc. Pulmonary delivery of protonated/acidified nucleic acids
US6162787A (en) 1999-04-02 2000-12-19 Immudyne, Inc. Methods for treating arthritis using collagen type II, glucosamine chondroitin sulfate, and compositions
EP1046394A3 (en) 1999-04-19 2001-10-10 ImaRx Pharmaceutical Corp. Novel compositions useful for delivering compounds into a cell
US7893226B2 (en) * 2004-09-29 2011-02-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Use of lipid conjugates in the treatment of diseases
US7504384B2 (en) 2000-01-10 2009-03-17 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of infection
WO2006054304A2 (en) * 2004-11-17 2006-05-26 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of disease
US7393938B2 (en) 2000-01-10 2008-07-01 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
US7772196B2 (en) * 2000-01-10 2010-08-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of lipid conjugates in the treatment of diseases
CA2399039C (en) * 2000-02-03 2009-09-22 Denki Kagaku Kogyo Kabushiki Kaisha Hyaluronic acid gel, method of its production and medical material containing it
US6749813B1 (en) 2000-03-05 2004-06-15 3M Innovative Properties Company Fluid handling devices with diamond-like films
CA2410887C (en) 2000-06-02 2012-07-24 Bracco Research Usa Compounds for targeting endothelial cells, compositions containing the same and methods for their use
US6654460B1 (en) 2000-10-13 2003-11-25 E. Walter Rodgers High-density telecommunication drawer assembly
JP4796708B2 (en) 2001-05-24 2011-10-19 生化学工業株式会社 Cartilage culture substrate and cartilage repair material
JP4266571B2 (en) 2002-05-17 2009-05-20 生化学工業株式会社 Method for producing lipid-bound glycosaminoglycan
US20060023098A1 (en) * 2004-08-02 2006-02-02 Thomas Graen Driving circuit for charge coupled device
CA2617484A1 (en) * 2005-08-03 2007-02-15 Morria Biopharmaceuticals Use of lipid conjugates in cystic fibrosis and applications thereof
WO2007029258A2 (en) * 2005-09-08 2007-03-15 Yissum Research Development Company Use of lipid conjugates in the treatment of infection
US20080183282A1 (en) * 2006-03-09 2008-07-31 Saul Yedgar Use of lipid conjugates for the coating of stents and catheters

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2976576A (en) * 1956-04-24 1961-03-28 Wichterle Otto Process for producing shaped articles from three-dimensional hydrophilic high polymers
US3220960A (en) * 1960-12-21 1965-11-30 Wichterle Otto Cross-linked hydrophilic polymers and articles made therefrom
US3431046A (en) * 1964-02-14 1969-03-04 Studies Inc Flexible polyethylene corneal contact lens
US3503942A (en) * 1965-10-23 1970-03-31 Maurice Seiderman Hydrophilic plastic contact lens
US3542461A (en) * 1967-11-20 1970-11-24 Du Pont Contact lens having an index of refraction approximating that of human tears
US3621079A (en) * 1968-10-21 1971-11-16 Patent Structures Inc Graft of hydroxyalkyl methacrylate onto polyvinylpyrrolidone
US3532679A (en) * 1969-04-07 1970-10-06 Robert Steckler Hydrogels from cross-linked polymers of n-vinyl lactams and alkyl acrylates
US3639524A (en) * 1969-07-28 1972-02-01 Maurice Seiderman Hydrophilic gel polymer insoluble in water from polyvinylpyrrolidone with n-vinyl-2-pyrrolidone and methacrylic modifier
US3721657A (en) * 1969-07-28 1973-03-20 M Seiderman Hydrophilic gel polymers of vinylpyrrolidine and hydroxyalkyl methacrylate
US3700761A (en) * 1969-11-28 1972-10-24 Griffin Lab Inc Fabrication of soft plastic contact lens blank
US3758448A (en) * 1971-10-05 1973-09-11 Union Optics Corp Copolymers and hydrogels of unsaturated esters
US3772235A (en) * 1971-10-05 1973-11-13 Union Optics Corp Copolymers and hydrogels of unsaturated heterocyclic compounds
US3786034A (en) * 1972-06-23 1974-01-15 Frigitronics Of Conn Inc Polyurethane polymers for use in soft lenses and method of producing soft lenses
US5464942A (en) * 1990-07-24 1995-11-07 Seikagaku Kogyo Kabushiki Kaisha Phospholipid- or lipid-linked glycosaminoglycan and process for producing the same
US20080109236A1 (en) * 1998-04-02 2008-05-08 Deangelis Paul L Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
US20020049183A1 (en) * 2000-01-10 2002-04-25 Saul Yedgar Use of lipid conjugates in the treatment of disease
US20040087492A1 (en) * 2000-01-10 2004-05-06 Saul Yedgar Use of lipid conjugates in the treatment of diseases
US20040229842A1 (en) * 2000-01-10 2004-11-18 Saul Yedgar Use of lipid conjugates in the treatment of diseases
US20080193538A1 (en) * 2003-10-29 2008-08-14 Teijin Limited Hyaluronic Acid Compound, Hydrogel Thereof and Joint Treating Material
US20080113935A1 (en) * 2004-03-02 2008-05-15 Saul Yedgar Use of lipid conjugates in the treatment of diseases or disorders of the eye

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130323178A1 (en) * 2012-02-23 2013-12-05 Canon Kabushiki Kaisha Indocyanine green-containing particle and contrast agent for photoacoustic imaging having the particle
WO2015068155A1 (en) * 2013-11-06 2015-05-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Lipid-polysaccharide conjugates, their preparation and uses thereof

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