WO2015068155A1 - Lipid-polysaccharide conjugates, their preparation and uses thereof - Google Patents

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

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Publication number
WO2015068155A1
WO2015068155A1 PCT/IL2014/050953 IL2014050953W WO2015068155A1 WO 2015068155 A1 WO2015068155 A1 WO 2015068155A1 IL 2014050953 W IL2014050953 W IL 2014050953W WO 2015068155 A1 WO2015068155 A1 WO 2015068155A1
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Prior art keywords
polysaccharide
lipid
another embodiment
conjugate
unsaturated
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PCT/IL2014/050953
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French (fr)
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YEDGAR Saul YEDGAR
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd
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Publication of WO2015068155A1 publication Critical patent/WO2015068155A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • 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
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • 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
    • 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
    • A61K31/734—Alginic acid
    • 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
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels

Definitions

  • This invention provides low molecular weight lipid-polysaccharide 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 (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.
  • the present invention provides a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a mass PL to ratio from about 0.25:15 to about 5:15, respectively.
  • the present invention provides a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) via an amide or ester linkage wherein the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD
  • the polysaccharide is a glycosaminoglycan (GAG). In another embodiment, the polysaccharide is alginate or chitosan.
  • GAG glycosaminoglycan
  • the present invention provides a lipid-polymer conjugate represented b the structure of the general formula (A):
  • 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between L, Z, Y and X is either an amide or an esteric bond; wherein the average molecular weight of said glycosaminoglycan is between 5kD and 20 kD.
  • the present invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain in length from 2 to 30 carbon atoms
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • a phospholipid (PL) with a polysaccharide and a coupling agent, wherein the masspL to masspoiysaccharide ratio from about 0.25:15 to about 5:15, respectively;
  • 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 polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a mass PL to masS p0 i ys;iCCh;iride 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 re resented by the structure of the general formula (A):
  • 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 polysaccharide
  • 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.
  • 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between L, Z, Y and X is either an amide or an esteric bond.
  • the present invention provides a lipid-polymer conjugate
  • PL phospholipid
  • the polysaccharide is a glycosaminoglycan (GAG). In another embodiment, the polysaccharide is alginate or chitosan.
  • GAG glycosaminoglycan
  • the present invention provides a lipid-polysaccharide conjugate comprising a polysaccharide conjugated to a phospholipid wherein the
  • polysaccharide has an average molecular weight between 5 to 90kD.
  • the polysaccharide may have an average molecular weight between 5 to 20kD according to one embodiment.
  • the polysaccharide may be glycosaminoglycan according to one embodiment.
  • the glycosaminoglycan may be hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan or keratan sulfate according to one embodiment.
  • the phospholipid may be a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a
  • the polysaccharide may be alginate according to one embodiment.
  • the polysaccharide may be chitosan according to one embodiment.
  • the phospholipid may comprise palmitic acid or myristic acid according to one embodiment.
  • the phospholipid may be dimyristoyl phosphatidylethanolamine or dipalmitoyl phosphatidylethanolamine according to one embodiment.
  • the polysaccharide may be conjugated to the phospholipid via an amide or ester linkage according to one embodiment.
  • the lipid-polysaccharide conjugate may be prepared by reacting a polysaccharide having an average molecular weight between 5 to 90kD with a phospholipid in a massPL to massPolysaccharide ratio from about 0.25: 15 to about 5:15, respectively according to one embodiment.
  • the massPL to massPolysaccharide ratio may be about 1: 10 according to one embodiment.
  • the present invention also provides a pharmaceutical composition comprising the lipid-polysaccharide conjugate as described according to one embodiment.
  • the present invention further provides a method for treating, inhibiting or
  • lipid-polysaccharide conjugate or pharmaceutical composition as described according to one embodiment.
  • the pathological condition may be selected from the group consisting of eye
  • disease infection, intestinal disease, obstructive respiratory disease, dermatological condition, cystic fibrosis, eye disorder, cardiovascular disease, proliferative disorder, and nervous system disorder according to one embodiment.
  • the pathological condition may be selected from the group consisting of
  • obstructive respiratory disease asthma, allergic rhinitis , Inflammatory Bowel Disease, colitis, Crohn's disease, central nervous system insult, multiple sclerosis, contact dermatitis, atopic dermatitis, psoriasis, cardiovascular disease, including prophylaxis for invasive procedures, invasive cellular proliferative disorders, antioxidant therapy, hemolytic syndromes, sepsis, acute respiratory distress syndrome, tissue transplant rejection syndromes, autoimmune disease, cystic fibrosis, cancer , viral infection, chlamydia infection, dry eye, and hypersensitivity conjunctivitis according to one embodiment.
  • the lipid-polysaccharide conjugate or pharmaceutical composition may be any suitable lipid-polysaccharide conjugate or pharmaceutical composition.
  • 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-phosphatidylethanolarnine conjugate (HyPE) prepared according to Example 5.
  • HyPE hyaluronic acid-phosphatidylethanolarnine 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 45 o) by RAW 264.7 cells in the absence of
  • Fig. 6 depicts the mean XTT reduction (OD 450 ) by LPS-stimulated RAW 264.7 cells.
  • Fig. 7 depicts the mean TNF-a release from RAW 264.7 cells in the absence of LPS.
  • Fig. 8 depicts the mean TNF-a 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 fromRAW 264.7 cells in the absence of LPS.
  • 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.
  • 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
  • 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-a release from RAW 264.7 cells in the absence of LPS.
  • Fig. 25 depicts the mean TNF-a release from LPS-stimulated RAW 264.7 cells.
  • Fig. 26 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS.
  • 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 fromRAW 264.7 cells in the absence of LPS.
  • 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.
  • Fig. 31 depicts the mean PGE 2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
  • Fig. 32 depicts dose-response curves for TNF-a production (+LPS). Data fit using
  • Fig. 33 depicts dose-response curves for IL-6 production (+LPS). Data fit using
  • 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.
  • Fig. 41 depicts inhibition of PLA2-induced RBC haemolysis (IC-50 mg/ml with corresponding polysaccharides) by low molecular weight conjugates according to some embodiments.
  • Fig. 42 depicts inhibition of IL-8 production by low molecular weight conjugates in both normal (corrected cell lines/C38) and Cystic Fibrosis (IB3) according to some embodiments.
  • Fig. 43 depicts inhibition of IL-8 production by low molecular weight conjugates in 16HBE airway epithelial cells transfected with cflr sense (Normal) and anti-sense (Cystic Fibrosis) construct according to some embodiments.
  • DCC refers to dicyclohexylcarbodiimide
  • ED AC refers to l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • BOP refers to Benzotriazole- 1 -yl-oxy-tris-(dimemylamino)-phosphonium hexafiuorophosphate
  • PyBOP refers to benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafiuorophosphate
  • HATU refers to 0-(7-Azabenzotriazole-l-yl)-N, ⁇ , ⁇ ' ⁇ '-tetramethyluronium hexafiuorophosphate
  • TSTU refers to 0-(N-Succ
  • lipid refers to all types of lipids including phospholipids
  • glycerolipids 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 polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a mass PL to mass Po i ysaccharide ratio from about 0.25:15 to about 5:15, respectively. In one embodiment, the ratio is from about 1:5 to 1:50, e.g., 1:10, 1 :20, 1:30 or 1:40.
  • said mass PL to masS p0 i ys;icdi;iride ratio is about 0.25:15.
  • said mass PL to mass Po i ysacC haride ratio is about 0.5:15.
  • said mass PL to mass Po i ysaccharide ratio is about 1 :15.
  • said mass PL to mass Po i ysaccharide ratio is about 2:15.
  • said mass PL to masspoiysaccharide ratio is about 5:15. In one embodiment, the ratio is from about 1:5 to 1:50, e.g., 1:10, 1 :20, 1:30 or 1:40.
  • the present invention provides a lipid-polymer conjugate
  • PL phospholipid
  • the polysaccharide is a glycosaminoglycan (GAG).
  • GAG of the lipid-conjugate compound of this invention may be hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan 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.
  • the polysaccharide is alginate or chitosan.
  • said PL of the lipid-conjugate compound of this invention is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a
  • 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 myristoyl phosphatidylethanolamine.
  • said PL is palmitoyl phosphatidylethanolamine.
  • 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 low molecular weight polysaccharide wherein the average molecular weight of said polysaccharide is between 5kd to 90 kd. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 60 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 40 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD.
  • low molecular weight polysaccharide 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 trifiuoroacetic 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. In another embodiment, 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. 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.
  • the molecular weight of hyaluronic acid and derivatives is
  • 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.
  • 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
  • lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a mass PL to mass Po i ys;iCCharide ratio from about 0.25: 15 to about 5:15, respectively.
  • the average molecular weight of said polysaccharide is between 5 kD to 90 kD.
  • the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD .
  • this invention provides a lipid-polymer conjugate represented by the structure of the general formula (A):
  • 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 polysaccharide
  • n is a number from 1 to 70;
  • L is a lipid.
  • L is a phospholipid.
  • L is a phosphatidylemanolamine, a phosphatidylserine, a
  • phosphatidylcholine a phosphatidylinositol, a phosphatidic acid or a
  • 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.
  • L is dimyristoyl phosphatidylemanolamine.
  • said L is dipalmitoyl phosphatidylemanolamine.
  • X is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan 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):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide; and n is a number from 1 to 70;
  • the average molecular weight of said polysaccharide is
  • between 5 to 25 kD e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15- 20kD .
  • Examples of phosphatidylemanolamine (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
  • N,N-dimethyl-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.
  • 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):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • the phosphatidylserine may be bonded to Y, or to X if Y is
  • said lipid-polymer conjugate is represented by the structure of the general formula (III):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phosphatidyl, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (IV):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (V):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide;
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula VII):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (VIII):
  • Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (IX):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (IXa):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (IXb):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide;
  • n is a number from 1 to 70;
  • any bond between the phospholipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (X):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the ceramide phosphoryl, Z, Y and X is either an amide or an esteric bond.
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the ceramide phosphoryl, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (XI):
  • Ri 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 polysaccharide; and n is a number from 1 to 70;
  • said lipid-polymer conjugate is represented by the structure of the of the general formula (XII):
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the ceramide, Z, Y and X is either an amide or an esteric bond.
  • the compound for use in the present invention is
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the ceramide, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XIII):
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the diglyceryl, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XIV):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the glycerolipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XV):
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the glycerolipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XVI):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide;
  • n is a number from 1 to 70;
  • any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XVII):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XVIII):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XIX):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • 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.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XX):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • said lipid-polymer conjugate is represented by the structure of the general formula (XXI):
  • Ri is either hydrogen or a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between the lipid, Z, Y and X is either an amide or an esteric bond.
  • Ri of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
  • R 2 of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
  • Z is a nothing.
  • Z is inositol.
  • Z is choline.
  • Z is glycerol.
  • Z is ethanoleamine.
  • Z is serine.
  • X is a polysaccharide.
  • the polysaccharide is a glycosaminoglycan (GAG).
  • the glycosaminoglycan may be, inter aim, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan, dermatan sulfate, keratin, keratan 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.
  • X is a alginate or chitosan.
  • 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.
  • n is a number from 1 to 70, e.g., from 1 to 50, 1 to 25, 1 to 15;
  • this invention provides lipid-polysaccharide conjugate or phospholipid-polysaccharide 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), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII).
  • said conjugate represented by the structures of the general formulae (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (XIX), (XX), (XXI), and (XXII).
  • the average molecular weight of said polysaccharide is between 5kD to 90 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 60 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 40 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD. In another embodiment, the lipid-polysaccharide conjugate is a phospholipid-polysaccharide 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, -(-0-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:
  • the compounds for use are as listed in Table 1 below.
  • this invention provides a lipid-polymer conjugate represented b the structure of the general formula (B):
  • 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 polysaccharide
  • n is a number from 1 to 10;
  • any bond between L, Z, Y and X is either an amide or an esteric bond.
  • this invention provides a lipid-polymer conjugate represented by the structure of the general formula (XXII):
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 10;
  • n of formula (B) and formula (XXII) is 1-10, in another
  • 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):
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • PL phospholipid
  • this invention provides a process for preparing a compound represented by the structure of the general formula (I):
  • Ri is a linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon atoms;
  • R 2 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 polysaccharide
  • n is a number from 1 to 70;
  • said coupling agent is DCC, ED AC, BOP, PyBOP, HATU, TSTU or any other amide coupling agent.
  • said coupling agent is EDAC.
  • said coupling agent further comprises HOBT or
  • said filtering step comprises a 10 kD centrasette membrane.
  • Ri 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 polysaccharide is between 5 kD to 90 kD. In another embodiment, the average molecular weight of the polysaccharide is between 5 kD to 20 kD. In another embodiment, the average molecular weight of the polysaccharide is between 5 kD to 10 kD. In another embodiment, the average molecular weight of the polysaccharide is between 10 kD to 20 kD. In another embodiment, the average molecular weight of the polysaccharide is between 20 kD to 50 kD. In another embodiment, the average molecular weight of the polysaccharide is between 30 kD to 60 kD.
  • the average molecular weight of the polysaccharide is between 40 kD to 70 kD. In another embodiment, the average molecular weight of the polysaccharide is between 50 kD to 80 kD. In another embodiment, the average molecular weight of the polysaccharide is between 60 kD to 90 kD.
  • hyaluronic acid is used in solution form
  • HA solution is prepared according to Excample 1
  • the process for the preparation of fractionated hyaluronic acid includes ultrafiltration.
  • the ultrafiltration fractionation of hyaluronic acid is as described in Example 2.
  • phosphatidylethanolamine-hyaluronic acid conjugate is prepared by reacting hyaluronic acid with a phosphatidylethanolamine using a coupling agent.
  • HyPE is prepared according to Example 3 using the apparatus depicted in Fig. 1.
  • the GAG-phospholipid conjugate may have the following structure: o
  • PLA2 may function at the site of ester linkage on the phospholipid side chain.
  • the Hyaluronic Acid-Dipalmitoylphosphatidylethanolamine HyPE may have the following structure:
  • Chontroitin Sulfate:Dipalmitoylphosphatidylethanolamine may have the following structure:
  • CSADMPE Sulfate:Dimyristoylphosphatidylethanolamine
  • Alginic acid:Dipalmitoylphosphatidylemanolamine may ha the following structure (site of amide bond indicated by ->):
  • 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
  • ED AC 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. In another embodiment,
  • 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 polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a mass PL to massp 0 i ysaccharide ratio from about 0.25:15 to about 5:15, respectively.
  • a composition comprising a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a mass PL to massp 0 i ysaccharide ratio from about 0.25:15 to about 5:15, respectively.
  • said masspL to masspoiysacchande ratio is about 0.25:15. In another embodiment, said mass PL to massp 0 i ysaccharide ratio is about 0.5:15. In another embodiment, said mass PL to massp 0 i ysaccharide ratio is about 1 :15. In another embodiment, said masspL to massp 0 i ysaccharide ratio is about 2:15. In another embodiment, said massp L to massp 0 i ysacchiiride ratio is about 5:15.
  • said inflammatory disorder is rheumatoid arthritis
  • 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-a 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
  • 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 polysaccharide
  • n is a number from 1 to 70;
  • 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.
  • the present invention provides a method of modulating NF-KB, 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 eneral formula (A): 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 polysaccharide
  • n is a number from 1 to 70;
  • any bond between L, Z, Y and X is either an amide or an esteric bond.
  • 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, antiinflammatory 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 compounds
  • 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, hydrabarnine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandlate, mesylate, methylbromide, methylnitrate, methyl
  • 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 antiinflammatory 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
  • 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.
  • modulating refers to the regulation of biological activities, which may be activating or inhibiting a specific activity or production of a specific gene or protein.
  • 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- polysaccharide conjugate of this invention.
  • 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 palmitoyl phosphatidylethanolamine and heparin. In one embodiment, the compound for use in the present invention comprises palmitoyl
  • the compound for use in the present invention comprises palmitoyl
  • the compound for use in the present invention comprises palmitoyl phosphatidylethanolamine and carboxymethylcellulose.
  • the compound for use in the present invention comprises myristoyl phosphatidylethanolamine and hyaluronic acid.
  • the palmitoyl phosphatidylethanolamine may be dipalmitoyl phosphatidylethanolamine, and the myristoyl phosphatidylethanolamine ma be dimyristoyl
  • the compound for use in the present invention is a
  • 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
  • the compounds may be useful for their antiinflammatory 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 bronchoalveolar 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 modulating NF- ⁇ , 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 impaire wound healing.
  • the compounds as described herein counteract exogenous factors which delay or impaire wound healing.
  • the conjugates as described herein counteract endogenous factors which delay or impaire 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- ⁇ , 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- ⁇ , 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- ⁇ , 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- ⁇ , or their combination within the joint's cells.
  • 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, atopic dermatitis, psoriasis, ARDS, or sepsis.
  • the invention provides a method of treating a subject
  • 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 method of treating a subject suffering from atopic 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 atopic dermatitis.
  • the atopic dermatitis is pediatric atopic dermatitis.
  • the atopic dermatitis is adult atopic 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
  • the invention provides a method of treating a subject
  • ARDS 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
  • 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.
  • DPPE dipalmitoylphosphatidylethanolamine
  • hydroxybenzotriazole (HOBT) were dissolved in 940 mL of teri-butanol and 80 mL of water with stirring and heating at 45 °C in a 12L round bottom flask (forming a closed system with the pump and the sonciator, all of which will have been previously autoclaved and/or disinfected with 70% isopropanol).
  • a 12L round bottom flask forming a closed system with the pump and the sonciator, all of which will have been previously autoclaved and/or disinfected with 70% isopropanol.
  • 850 mL of water 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.
  • a 1 mg/ml solution of LPS (made in 1 x 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% C0 2 .
  • 0.5 ml of cells at 1 x 106 cells/ml was plated in 24-well tissue culture plates. Cells were allowed to adhere for 30 minutes at 37°C with 5% C0 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 ⁇ of CM was added to the -LPS plates.
  • 110 ⁇ of 10 ⁇ LPS was added to the +1 g/ml LPS plates. The plates were incubated for 24 hours at 37°C with 5% C0 2 .
  • Luminexbased assay according to the manufacturer's instructions. Data were collected using a Luminex 100 (Luminex Corporation, Austin, TX). Standard curves were generated using a 5-parameter logistic curve-fitting equation weighted by 1/y
  • TNF-a data relating to high molecular weight HyPE compositions are shown in Fig. 7, Fig. 8 and Fig. 15.
  • TNF-a 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.
  • 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 ⁇ filter (PALL) before drying to achieve microbial purity.
  • PALL 0.2 ⁇ filter
  • G1310A an automatic injector (G1313A) and the following column system: PL aquagel-OH Mix and PL aquagel-OH 30 (300 x 7,5 mm, 8 ⁇ ; Agilent Technologies) columns connected in series and thermostated at ambient temperature. Injection volume was 100 ⁇ . 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
  • 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 ⁇ , 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 determined molecular weight and polydispersity value for low molecular weight hyaluronic acid were 7.86 x 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 0 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-H 2 0, 60 mL MES buffer, 12 mL of teri-butanoL The pH was adjusted to 6.4 with 4N NaOH.
  • the molecular weight of HA was 9.54kD.
  • the molecular weight of HA was 10-14 kDa or 10-15kDa.
  • the molecular weight of dipalmitoylphosphatidylethanolamine (DPPE) is 692.
  • DMPE dimyristoylphosphatidylethanolamine
  • HA 15.1 g was dissolved in 350 mL of DI-H 2 0. 1.25 g DPPE or DMPE was dissolved in 440 mL of teri-butanol and 90 mL DI-H 2 0 with heating to 55 deg C.
  • dimyristoylphosphatidylethanolamine may be mixed at a 10: 1 ratio by weight for synthesis.
  • the solutions of HA and HOBT were warmed to 35 deg C and mixed.
  • the DPPE or DMPE 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 ED AC 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 ⁇ 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.
  • 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% NaHC0 3 to remove residual organic solvents. The pH was then increased by slow addition of 2% Na 2 C0 3 to pH 9.2. The solution was stirred for 1 hour at room temperature. After further washing with 30 L of 1.5% NaHC0 3 the peat at -12.5 min had disappeared and the solution was washed with 30 L of DI-H 2 0 until pH 7. To remove any
  • HyPE 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).
  • 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.
  • HyPE is an amphoteric molecule and exhibits some surface active properties in aqueous solution. Maximum solubility is obtained in water and can reach 3% although the viscosity of solutions greater than 2% tends to increase rapidly.
  • Chondroitin Sulfate and Alhinic acid may be obtained from marine and other sources.
  • DMPE has a MW of 677. Chondroitin sulfate-A (15-20kDa) and dipalmitoyl
  • phosphatidylethanolamine or dimyristoylphosphatidylethanolamine may be
  • CSAPE chondroitin sulfate:dipalmitoylphosphatidylemanolamine
  • CSADMPE chondroitin sulfate:dimyristoylphosphatidylemanolamine
  • Alginic Acid of marine origin was isolated.
  • the starting material has a MW of
  • the DPPE has a MW of 692.
  • dipalmitoyl phosphatidylemanolarnine or dimyristoylphosphatidylemanolamine may be mixed at a 10: 1 ratio by weight for synthesis.
  • the solubility of AlgPE is about 20 mg/mL in water. In some experiments, oncentrations greater than
  • 1% nasal spray (100 ⁇ dose) containing HyPE B.I.D. in isotonic buffer containing benzyl alcohol was employed in a six day dosing regimen to 105 subjects.
  • Data shows A.E.'s similar to control (placebo) and decreased cough and headache compared to steroid.
  • the cream is used to treat adult or pediatric atopic dermatitis.
  • HYDMPE (2183+/- 2000 ⁇ g/mL (i.e.
  • Fig. 41 depicts inhibition of PLA2-induced RBC haemolysis (IC-50 mg/ml with corresponding polysaccharides) by low molecular weight conjugates.
  • Fig. 42 depicts inhibition of IL-8 production by low molecular weight conjugates in both normal (corrected cell lines/C38) and Cystic Fibrosis (IB3).
  • Fig. 43 depicts inhibition of IL-8 production by low molecular weight conjugates in

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Abstract

This invention provides low molecular weight lipid-polysaccharide conjugates and methods of use thereof in suppressing, inhibiting preventing, or treating a pathological condition. 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 polysaccharide conjugated to a phospholipid (PL). The present invention provides a method for decreasing expression of proinflammatory chemokines, cytokines, or a combination thereof.

Description

LIPID-POLYSACCHARIDE CONJUGATES, THEIR PREPARATION AND
USES THEREOF
FIELD OF THE INVENTION
[001] This invention provides low molecular weight lipid-polysaccharide 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
[002] 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.
[003] 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
[004] In one embodiment, the present invention provides a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a massPL to
Figure imgf000003_0001
ratio from about 0.25:15 to about 5:15, respectively. [005] In one embodiment, the present invention provides a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) via an amide or ester linkage wherein the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD
[006] In one embodiment, the polysaccharide is a glycosaminoglycan (GAG). In another embodiment, the polysaccharide is alginate or chitosan.
[007] In one embodiment, the present invention provides a lipid-polymer conjugate represented b the structure of the general formula (A):
Figure imgf000004_0001
(A)
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 polysaccharide; 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 average molecular weight of said glycosaminoglycan is between 5kD and 20 kD.
[008] In one embodiment, the present invention provides a process for preparing a compound represented by the structure of the general formula (I):
Figure imgf000004_0002
(I)
wherein
Ri is a linear, saturated, mono -unsaturated, or poly-unsaturated, alkyl chain 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 polysaccharide; 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:
reacting a phospholipid (PL) with a polysaccharide and a coupling agent, wherein the masspL to masspoiysaccharide ratio from about 0.25:15 to about 5:15, respectively;
filtering the reaction mixture from (a) to generate a filtrate; and
extracting a product from a filtrate.
[009] 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 polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a massPL to masSp0iys;iCCh;iride 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 re resented by the structure of the general formula (A):
Figure imgf000005_0001
(A)
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 polysaccharide; 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.
[0010] In one embodiment, the present invention
provides a method of modulating NF-κΒ, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines comprising the step of administering to a subject a compound re resented by the structure of the general formula (A):
Figure imgf000006_0001
(A)
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 polysaccharide; 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.
[0011] In one embodiment, the present invention provides a lipid-polymer conjugate
comprising a polysaccharide conjugated to a phospholipid (PL) via an amide or ester linkage wherein the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD.
[0012] In one embodiment, the polysaccharide is a glycosaminoglycan (GAG). In another embodiment, the polysaccharide is alginate or chitosan.
[0013] In one embodiment, the present invention provides a lipid-polysaccharide conjugate comprising a polysaccharide conjugated to a phospholipid wherein the
polysaccharide has an average molecular weight between 5 to 90kD.
[0014] The polysaccharide may have an average molecular weight between 5 to 20kD according to one embodiment.
[0015] The polysaccharide may be glycosaminoglycan according to one embodiment. [0016] The glycosaminoglycan may be hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan or keratan sulfate according to one embodiment.
[0017] The phospholipid may be a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a
phoshpatidylglycerol according to one embodiment.
[0018] The polysaccharide may be alginate according to one embodiment.
[0019] The polysaccharide may be chitosan according to one embodiment.
[0020] The phospholipid may comprise palmitic acid or myristic acid according to one embodiment.
[0021] The phospholipid may be dimyristoyl phosphatidylethanolamine or dipalmitoyl phosphatidylethanolamine according to one embodiment.
[0022] The polysaccharide may be conjugated to the phospholipid via an amide or ester linkage according to one embodiment.
[0023] The lipid-polysaccharide conjugate may be prepared by reacting a polysaccharide having an average molecular weight between 5 to 90kD with a phospholipid in a massPL to massPolysaccharide ratio from about 0.25: 15 to about 5:15, respectively according to one embodiment.
[0024] The massPL to massPolysaccharide ratio may be about 1: 10 according to one embodiment.
[0025] The present invention also provides a pharmaceutical composition comprising the lipid-polysaccharide conjugate as described according to one embodiment.
[0026] The present invention further provides a method for treating, inhibiting or
suppressing a pathological condition in a subject, comprising administering to the subject a lipid-polysaccharide conjugate or pharmaceutical composition as described according to one embodiment.
[0027] The pathological condition may be selected from the group consisting of eye
disease, infection, intestinal disease, obstructive respiratory disease, dermatological condition, cystic fibrosis, eye disorder, cardiovascular disease, proliferative disorder, and nervous system disorder according to one embodiment.
[0028] The pathological condition may be selected from the group consisting of
obstructive respiratory disease, asthma, allergic rhinitis , Inflammatory Bowel Disease, colitis, Crohn's disease, central nervous system insult, multiple sclerosis, contact dermatitis, atopic dermatitis, psoriasis, cardiovascular disease, including prophylaxis for invasive procedures, invasive cellular proliferative disorders, antioxidant therapy, hemolytic syndromes, sepsis, acute respiratory distress syndrome, tissue transplant rejection syndromes, autoimmune disease, cystic fibrosis, cancer , viral infection, chlamydia infection, dry eye, and hypersensitivity conjunctivitis according to one embodiment.
[0029] The lipid-polysaccharide conjugate or pharmaceutical composition may be
administered orally, intravenously, intranasally intraocularly, intramuscularly, subcutaneously or topically according to one embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] 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:
[0031] Fig. 1 depicts a conceptual diagram of the reaction vessel features required to practice the methods of this invention.
[0032] Fig. 2 depicts an NMR spectrum of a hyaluronic acid-phosphatidylethanolarnine conjugate (HyPE) prepared according to Example 5.
[0033] Fig. 3 is an HPLC chromatogram of HyPE prepared according to Example 5.
[0034] Fig. 4 depicts a schematic representation of the in vitro stimulation of RAW 264.7 cells.
[0035] Fig. 5 depicts the mean XTT reduction (OD45o) by RAW 264.7 cells in the absence of
LPS. Error bars represent standard deviations.
[0036] Fig. 6 depicts the mean XTT reduction (OD450) by LPS-stimulated RAW 264.7 cells.
Error bars represent standard deviations.
[0037] Fig. 7 depicts the mean TNF-a release from RAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0038] Fig. 8 depicts the mean TNF-a release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations. [0039] Fig. 9 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
[0040] Fig. 10 depicts the mean IL-6 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0041] Fig. 11 depicts the mean IP-10 release fromRAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0042] Fig. 12 depicts the mean IP-10 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0043] Fig. 13 depicts the mean PGE2 release from RAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0044] Fig. 14 depicts the mean PGE2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0045] Fig. 15 depicts dose-response curves for TNF-ot production (+LPS). Data fit using Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+ (Top + Bottom)/(l +
Figure imgf000009_0001
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0046] 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)/(l +
Figure imgf000009_0002
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0047] 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)/(l +
Figure imgf000009_0003
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0048] 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)/(l +
Figure imgf000009_0004
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0049] 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. [0050] Fig. 20 is the SEC-MALS determined distribution of molecular weight of low molecular weight sodium hyaluronate.
[0051] Fig. 21 is the UV spectrum of sample 208-088 (low molecular weight sodium
hyaluronate).
[0052] Fig. 22 depicts the mean XTT reduction (OD450) by RAW 264.7 cells in the absence of LPS. Error bars represent standard deviations.
[0053] Fig. 23 depicts the mean XTT reduction (OD450) by LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0054] Fig. 24 depicts the mean TNF-a release from RAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0055] Fig. 25 depicts the mean TNF-a release from LPS-stimulated RAW 264.7 cells.
Error bars represent standard deviations.
[0056] Fig. 26 depicts the mean IL-6 release from RAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0057] Fig. 27 depicts the mean IL-6 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0058] Fig. 28 depicts the mean IP-10 release fromRAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0059] Fig. 29 depicts the mean IP-10 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0060] Fig. 30 depicts the mean PGE2 release from RAW 264.7 cells in the absence of LPS.
Error bars represent standard deviations.
[0061] Fig. 31 depicts the mean PGE2 release from LPS-stimulated RAW 264.7 cells. Error bars represent standard deviations.
[0062] Fig. 32 depicts dose-response curves for TNF-a production (+LPS). Data fit using
Prism 4, Sigmoidal dose-response curve (variable slope): Y=Bottom+ (Top +
Bottom)/(l +
Figure imgf000010_0001
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0063] 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)/(l +
Figure imgf000010_0002
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100. [0064] 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)/(l +
Figure imgf000011_0001
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0065] 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)/(l +
Figure imgf000011_0002
- X)*HiUSlope)). X is the log of Test Article concentration, Y is the response. Constraints Bottom = 0, Top = 100.
[0066] 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.
[0067] Fig. 37 depicts a chromatogram of the HyPE reaction from Example 11 after 2 hours.
[0068] Fig. 38 depicts a chromatogram of the HyPE reaction from Example 11 after 6 hours.
[0069] Fig. 39 depicts the GPC analysis of final HyPE isolated from Example 11.
[0070] Fig. 40 depicts the NMR spectrum of final HyPE isolated from Example 11 and treated with 1 drop of 4% NaOD.
[0071] Fig. 41 depicts inhibition of PLA2-induced RBC haemolysis (IC-50 mg/ml with corresponding polysaccharides) by low molecular weight conjugates according to some embodiments.
[0072] Fig. 42 depicts inhibition of IL-8 production by low molecular weight conjugates in both normal (corrected cell lines/C38) and Cystic Fibrosis (IB3) according to some embodiments.
[0073] Fig. 43 depicts inhibition of IL-8 production by low molecular weight conjugates in 16HBE airway epithelial cells transfected with cflr sense (Normal) and anti-sense (Cystic Fibrosis) construct according to some embodiments.
[0074] 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
[0075] 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.
[0076] 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, ED AC refers to l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), BOP refers to Benzotriazole- 1 -yl-oxy-tris-(dimemylamino)-phosphonium hexafiuorophosphate, PyBOP refers to benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafiuorophosphate, HATU refers to 0-(7-Azabenzotriazole-l-yl)-N, Ν,Ν'Ν'-tetramethyluronium hexafiuorophosphate, TSTU refers to 0-(N-Succinimidyl)-N,N,N,N- tetramethyluronium tetrafiuoroborate, HOBT refers to hydroxybenzotriazole and HOAT refers to l-hydroxy-7-aza-benzotriazole.
[0077] Herein, the term "lipid" refers to all types of lipids including phospholipids,
glycerolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and the like.
[0078] This invention provides, in one embodiment, a lipid-polymer conjugate which is useful in some embodiments for the treatment of inflammatory disorders.
[0079] 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.
[0080] In one embodiment, this invention provides a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a massPL to massPoiysaccharide ratio from about 0.25:15 to about 5:15, respectively. In one embodiment, the ratio is from about 1:5 to 1:50, e.g., 1:10, 1 :20, 1:30 or 1:40.
[0081] In another embodiment, said massPL to masSp0iys;icdi;iride ratio is about 0.25:15. In another embodiment, said massPL to massPoiysacCharide ratio is about 0.5:15. In another embodiment, said massPL to massPoiysaccharide ratio is about 1 :15. In another embodiment, said massPL to massPoiysaccharide ratio is about 2:15. In another embodiment, said massPL to masspoiysaccharide ratio is about 5:15. In one embodiment, the ratio is from about 1:5 to 1:50, e.g., 1:10, 1 :20, 1:30 or 1:40.
[0082] In one embodiment, the present invention provides a lipid-polymer conjugate
comprising a polysaccharide conjugated to a phospholipid (PL) via an amide or ester linkage wherein the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD .
[0083] In another embodiment, the polysaccharide is a glycosaminoglycan (GAG). GAG of the lipid-conjugate compound of this invention may be hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan 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.
[0084] 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.
[0085] In another embodiment, the polysaccharide is alginate or chitosan.
[0086] 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 myristoyl phosphatidylethanolamine. In another embodiment, said PL is palmitoyl phosphatidylethanolamine. In another embodiment, said PL is dimyristoyl phosphatidylethanolamine. In another embodiment, said PL is dipalmitoyl phosphatidylethanolamine.
[0087] 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.
[0088] In one embodiment, the lipid-polymer conjugate of this invention comprises a low molecular weight polysaccharide wherein the average molecular weight of said polysaccharide is between 5kd to 90 kd. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 60 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 40 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD.
[0089] In one embodiment, low molecular weight polysaccharide, 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 trifiuoroacetic 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.
[0090] 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
[0091] 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. [0092] 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.
[0093] In another embodiment, this invention provides a pharmaceutical composition
comprising a lipid-polymer conjugate comprising a polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a massPL to massPoiys;iCCharide ratio from about 0.25: 15 to about 5:15, respectively. In another embodiment, the average molecular weight of said polysaccharide is between 5 kD to 90 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD .
[0094] In one embodiment, this invention provides a lipid-polymer conjugate represented by the structure of the general formula (A):
Figure imgf000015_0001
(A)
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 polysaccharide; 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 average molecular weight of said polysaccharide is between 5kD and 20 kD. [0095] In one embodiment L is a lipid. In another embodiment L is a phospholipid. In another embodiment, L is a phosphatidylemanolamine, 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 phosphatidylemanolamine. In another embodiment, said L is dipalmitoyl phosphatidylemanolamine.
[0096] In another embodiment, X is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan 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.
[0097] 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.
[0098] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (I):
Figure imgf000016_0001
(I)
wherein
Ri 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 polysaccharide; 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.
[0099] In another embodiment, the average molecular weight of said polysaccharide is
between 5 to 25 kD, e.g., between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15- 20kD .
[00100] Examples of phosphatidylemanolamine (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.
[00101] Phosphatidyl-ethanolamine and its analogues may be from various sources, including natural, synthetic, and semisynthetic derivatives and their isomers.
[00102] 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.
[00103] 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.
[00104] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (II):
Figure imgf000018_0001
(Π)
wherein
Ri 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 polysaccharide; 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.
[00105] In one embodiment, the phosphatidylserine may be bonded to Y, or to X if Y is
nothing, via the COO" moiety of the phosphatidylserine.
[00106] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (III):
Figure imgf000018_0002
(III)
wherein Ri 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 polysaccharide; 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.
[00107] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (IV):
Figure imgf000019_0001
(IV)
wherein
Ri 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 polysaccharide; 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.
[00108] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (V):
Figure imgf000020_0001
(V)
wherein
Ri 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 polysaccharide; 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.
[00109] In another embodiment, said lipid-polymer conjugate is represented by the structure of
Figure imgf000020_0002
(VI)
wherein
Ri 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 polysaccharide; 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.
[00110] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula VII):
Figure imgf000021_0001
(VII)
wherein
Ri 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 polysaccharide; 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.
[00111 ] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (VIII):
Figure imgf000021_0002
(VIII)
wherein
Ri 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 polysaccharide; 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.
[00112] In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (IX):
Figure imgf000022_0001
(IX)
wherein
Ri 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 polysaccharide; 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.
[00113] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (IXa):
Figure imgf000023_0001
(IXa)
wherein
Ri 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 polysaccharide; 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.
[00114] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (IXb):
Figure imgf000023_0002
(IXb)
wherein
Ri 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 polysaccharide; 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.
[00115] In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (X):
Figure imgf000024_0001
(X)
wherein
Ri 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 polysaccharide; 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.
[00116] In another embodiment, the compound for use in the present invention is represented by the structure of the general formula (Xa):
Figure imgf000025_0001
(Xa)
wherein
Ri 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 polysaccharide; 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.
[00117] In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (XI):
Figure imgf000025_0002
(XI)
wherein
Ri 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 polysaccharide; 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.
[00118] In another embodiment, said lipid-polymer conjugate is represented by the structure of the of the general formula (XII):
Figure imgf000026_0001
(XII)
wherein
Ri 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 polysaccharide; 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.
[00119] In another embodiment, the compound for use in the present invention is
represented by the structure of the general formula (Xlla):
Figure imgf000027_0001
(Xlla)
wherein
Ri 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 polysaccharide; 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.
[00120] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XIII):
Figure imgf000027_0002
(XIII)
wherein
Ri 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 polysaccharide; 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.
[00121] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XIV):
Figure imgf000028_0001
(XIV)
wherein
Ri 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 polysaccharide; 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.
[00122] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XV):
Figure imgf000029_0001
(XV)
wherein
Ri 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 polysaccharide; 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.
[00123] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XVI):
Figure imgf000029_0002
(XVI)
wherein
Ri 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 polysaccharide; 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.
[00124] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XVII):
Figure imgf000030_0001
(XVII)
wherein
Ri 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 polysaccharide; 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.
[00125] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XVIII):
Figure imgf000030_0002
(XVIII)
wherein
Ri 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 polysaccharide; 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.
[00126] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XIX):
Figure imgf000031_0001
(XIX)
wherein
Ri 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 polysaccharide; 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.
[00127] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XX):
Figure imgf000032_0001
(XX)
wherein
Ri 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 polysaccharide; 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.
[00128] In another embodiment, said lipid-polymer conjugate is represented by the structure of the general formula (XXI):
Figure imgf000032_0002
(XXI)
wherein Ri 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 polysaccharide; 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.
[00129] In another embodiment, Ri of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
[00130] In another embodiment, R2 of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a residue of myristic acid.
[00131] In some embodiments, the compounds (A), (B) (III), (IV), (V), (VI), (VII),
(VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (Xlla), (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 a nother embodiment, Z is glycerol. In another embodiment, Z is ethanoleamine. In another embodiment, Z is serine.
[00132] 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), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII) hereinabove: In one embodiment, X is a polysaccharide. In another embodiment, the polysaccharide is a glycosaminoglycan (GAG). According to this aspect and in one embodiment, the glycosaminoglycan may be, inter aim, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, dermatan, dermatan sulfate, keratin, keratan 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 one embodiment, X is a alginate or chitosan.
[00133] 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.
[00134] In one embodiment, n is a number from 1 to 70, e.g., from 1 to 50, 1 to 25, 1 to 15;
2 to 50, 2 to 25, or 2 to 15.
[00135] In one embodiment, this invention provides lipid-polysaccharide conjugate or phospholipid-polysaccharide 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), (Xlla), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII). In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 90 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 60 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5kD to 40 kD. In another embodiment, the average molecular weight of said polysaccharide is between 5 to 20kD, 5 to 15kD, 10 to 20kD, 10-15kD, or 15-20kD. In another embodiment, the lipid-polysaccharide conjugate is a phospholipid-polysaccharide conjugate.
[00136] 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, -(-0-CH(CH3)CH2-)x- wherein x is an integer of 1 or more.
[00137] 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.
[00138] 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.
[00139] In one embodiment, the compounds for use in the present invention are
biodegradable.
[00140] In some embodiments, the compounds for use are as listed in Table 1 below.
Table 1. i. Phospholipid Spacer
Polymer (m.w.)
Figure imgf000035_0001
PE None Heparin
(0.5-110 kDa)
PE None Chondroitin sulfate A
PE None Carboxymethylcellulos e
(20-500 kDa)
PE Dicarboxylic Polygeline (haemaccel) acid + (4-40 kDa)
Diamine
PE None Hydroxyethylstarch
PE Dicarboxylic Dextran
acid + (1-2,000 kDa) Diamine
PE Carboxyl amino Hyaluronic acid
group (5-20 kDa)
PE Dicarboxyl Hyaluronic acid
group (5-20 kDa)
PE Dipalmitoic Hyaluronic acid
acid (5-20 kDa)
PE Carboxyl amino Heparin
group (5-20 kDa)
PE Dicarboxyl Heparin
group (5-20 kDa)
PE Carboxyl amino Chondroitin sulfate A group
PE Dicarboxyl Chondroitin sulfate A group
PE Carboxyl amino Carboxymethylcellulos group e
(5-20 kDa)
PE Dicarboxyl Carboxymethylcellulos group e (5-20 kDa)
PE None Polygeline (haemaccel)
(5-20 kDa)
PE Carboxyl amino Polygeline (haemaccel) group (5-20 kDa)
PE Dicarboxyl Polygeline (haemaccel) group (5-20 kDa)
PE Carboxyl amino Hydroxyethylstarch group
PE Dicarboxyl Hydroxyethylstarch group
PE None Dextran
(5-20 kDa)
PE Carboxyl amino Dextran
group (5-20 kDa)
PE Dicarboxyl Dextran
group (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 Carboxymethylcellulos e
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 Carboxymethylcellulos e
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)
[00141 ] In one embodiment, this invention provides a lipid-polymer conjugate represented b the structure of the general formula (B):
Figure imgf000038_0001
(B)
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 polysaccharide; 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.
[00142] In one embodiment, this invention provides a lipid-polymer conjugate represented by the structure of the general formula (XXII):
Figure imgf000039_0001
(XXII)
Ri 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 polysaccharide; 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.
[00143] In one embodiment, n of formula (B) and formula (XXII) 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.
[00144] In one embodiment, this invention provides a process for preparing a compound represented by the structure of the general formula (I):
Figure imgf000039_0002
(I)
wherein
Ri 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 polysaccharide; 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 polysaccharide and a coupling agent, wherein the massPL to massp ratio from about 0.25:15 to about 5:15, respectively.
[00145] In one embodiment, this invention provides a process for preparing a compound represented by the structure of the general formula (I):
Figure imgf000040_0001
(I)
wherein
Ri 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 polysaccharide; 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:
reacting a phospholipid (PL) with a polysaccharideand a coupling agent, wherein the masspL to massp ratio from about 0.25:15 to about 5:15, respectively; filtering the reaction mixture from (a) to generate a filtrate; and
extracting a product from a filtrate.
[00146] In another embodiment, said coupling agent is DCC, ED AC, 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.
[00147] In another embodiment, said filtering step comprises a 10 kD centrasette membrane.
[00148] In another embodiment, Ri is the residue of palmitic acid or the residue of myristic acid.
[00149] In another embodiment, R2 is the residue of palmitic acid or the residue of myristic acid.
[00150] In another embodiment, the average molecular weight of the polysaccharide is between 5 kD to 90 kD. In another embodiment, the average molecular weight of the polysaccharide is between 5 kD to 20 kD. In another embodiment, the average molecular weight of the polysaccharide is between 5 kD to 10 kD. In another embodiment, the average molecular weight of the polysaccharide is between 10 kD to 20 kD. In another embodiment, the average molecular weight of the polysaccharide is between 20 kD to 50 kD. In another embodiment, the average molecular weight of the polysaccharide is between 30 kD to 60 kD. In another embodiment, the average molecular weight of the polysaccharide is between 40 kD to 70 kD. In another embodiment, the average molecular weight of the polysaccharide is between 50 kD to 80 kD. In another embodiment, the average molecular weight of the polysaccharide is between 60 kD to 90 kD.
[00151] In one embodiment, hyaluronic acid (HA) is used in solution form In another embodiment, HA solution is prepared according to Excample 1,
[00152] 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.
[00153] In one embodiment, phosphatidylethanolamine-hyaluronic acid conjugate (HyPE) is prepared by reacting hyaluronic acid with a phosphatidylethanolamine using a coupling agent. In another embodiment, HyPE is prepared according to Example 3 using the apparatus depicted in Fig. 1. [00154] In one embodiment, the GAG-phospholipid conjugate may have the following structure: o
u
R5 - C- 0— C
O
t!
Q O
I !i ti
H - C - O - P - O - CHZ - CH2 - NH - C
PLA>
H O
[00155] PLA2 may function at the site of ester linkage on the phospholipid side chain.
[00156] In one embodiment, the Hyaluronic Acid-Dipalmitoylphosphatidylethanolamine HyPE) may have the following structure:
Figure imgf000042_0001
[00157] In another embodiment, the Hyaluronic Acid:Dimyristoylphosphatidylethanolamine
Figure imgf000042_0002
I n another embodiment, the Chontroitin Sulfate:Dipalmitoylphosphatidylethanolamine (CSAPE) may have the following structure:
Figure imgf000043_0001
[00158]
[00159] In another embodiment, the Chondroitin
Sulfate:Dimyristoylphosphatidylethanolamine (CSADMPE) may have the following structure:
Figure imgf000043_0002
[00160] In another embodiment, the Alginic acid:Dipalmitoylphosphatidylemanolamine (AlgPE) may ha the following structure (site of amide bond indicated by ->):
Figure imgf000043_0003
[00161] 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.
[00162] In one embodiment, a coupling reagent is used in the preparation of HyPE
according to Example 3. In another embodiment, ED AC 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.
[00163] 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.
[00164] 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.
[00165] 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.
[00166] 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 polysaccharide conjugated to a phospholipid (PL) wherein said conjugate is prepared by reacting said polysaccharide with said PL in a massPL to massp0iysaccharide ratio from about 0.25:15 to about 5:15, respectively.
[00167] In another embodiment, said masspL to masspoiysacchande ratio is about 0.25:15. In another embodiment, said massPL to massp0iysaccharide ratio is about 0.5:15. In another embodiment, said massPL to massp0iysaccharide ratio is about 1 :15. In another embodiment, said masspL to massp0iysaccharide ratio is about 2:15. In another embodiment, said masspL to massp0iysacchiiride ratio is about 5:15.
[00168] In another embodiment, said inflammatory disorder is rheumatoid arthritis,
osteoarthritis, wound healing, dermatitis, restenosis, cystic fibrosis, multiple sclerosis or sepsis. [00169] 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-a is measured. In another embodiment, expression of IP- 10 is measured. In another embodiment, expression of PGE2 is measured.
[00170] In another embodiment, said composition is administered intravenously. In another embodiment, said composition is administered topically.
[00171] 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 eneral formula (A):
Figure imgf000045_0001
(A)
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 polysaccharide; 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.
[00172] In one embodiment, the present invention provides a method of modulating NF-KB, 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 eneral formula (A):
Figure imgf000045_0002
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 polysaccharide; 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.
[00173 ] Dosages and Routes of Administration
[00174] 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, antiinflammatory 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.
[00175] 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, hydrabarnine, 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.
[00176] 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.
[00177] 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. [00178] In one embodiment, the use of a single chemical entity with potent anti-oxidant, membrane-stabilizing, anti-proliferative, anti-chemokine, anti-migratory, and antiinflammatory 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.
[00179] 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?
[00180] 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.
[00181] 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.
[00182] 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.
[00183] The term "modulating" refers to the regulation of biological activities, which may be activating or inhibiting a specific activity or production of a specific gene or protein. 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.
[00184] In one embodiment, implants or suppositories, can be used to administer a lipid- polysaccharide conjugate of this invention.
[00185] 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
[00186] For topical application, particularly for the treatment of skin diseases such as contact dermatitis, atopic dermatitis, or psoriasis, admixture of the compounds with conventional creams or delayed release patches is acceptable.
[00187] 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. [00188] 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.
[00189] 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
[00190] 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.
[00191] In one embodiment, the compound for use in the present invention comprises palmitoyl phosphatidylethanolamine and heparin. In one embodiment, the compound for use in the present invention comprises palmitoyl
phosphatidylethanolamine and chondroitin sulfate. In one embodiment, the compound for use in the present invention comprises palmitoyl
phosphatidylethanolamine and hyaluronic acid. In one embodiment, the compound for use in the present invention comprises palmitoyl phosphatidylethanolamine and carboxymethylcellulose. In one embodiment, the compound for use in the present invention comprises myristoyl phosphatidylethanolamine and hyaluronic acid. The palmitoyl phosphatidylethanolamine may be dipalmitoyl phosphatidylethanolamine, and the myristoyl phosphatidylethanolamine ma be dimyristoyl
phosphatidylethanolamine.
[00192] 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.
[00193] 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 antiinflammatory 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
[00194] 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 bronchoalveolar 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.
[00195] 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 modulating NF-κΒ, IL-6, IL-8, or a combination thereof in human airway epithelial cell lines.
Method of Treating Wounds
[00196] 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).
[00197] 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.
[00198] 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 impaire wound healing. In another embodiment, the compounds as described herein counteract exogenous factors which delay or impaire wound healing. In another embodiment, the conjugates as described herein counteract endogenous factors which delay or impaire 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.
[00199] 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
[00200] 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.
[00201] 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.
[00202] It is understood that one skilled in the art recognizes that the term "arthritis" refers to both rheumatoid arthritis (RA) and osteoarthritis (OA).
[00203] In another embodiment, a compound as described herein inhibits the production of IL-6, IL-8, TNF-alpha, NF-κΒ, 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-κΒ, 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-κΒ, 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-κΒ, 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
[00204] 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, atopic dermatitis, psoriasis, ARDS, or sepsis.
[00205] 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.
[00206] 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.
[00207] 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.
[00208] 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.
[00209] 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.
[00210] 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.
[00211] 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.
[00212] In one embodiment, the invention provides a method of treating a subject suffering from atopic 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 atopic dermatitis.
[00213] In one embodiment, the atopic dermatitis is pediatric atopic dermatitis.
[00214] In one embodiment, the atopic dermatitis is adult atopic dermatitis.
[00215] 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.
[00216] 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.
[00217] 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.
[00218] 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.
[00219] 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.
[00220] 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.
[00221] 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
[00222] 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 μπι filter followed by a 50 μπι filter followed by a 10 μπι 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). Hyaluronic acid may be isolated from the cell walls of the Streptococcus zooepidemicus in a fermentation process. EXAMPLE 2
ULTRAFILTRATION FRACTIONATION OF HYALURONIC ACID (HA)
[00223] 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).
[00224] By means of constant volume diafiltration with 70kDa 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.
[00225] The 70kDa membranes were replaced with 30 kDa membranes and the Centramate system was disinfected with 10% hydrogen peroxide.
[00226] 5L of the filtrate, the fraction less than 70 kDa, were loaded into the reservoir and by means of constant volume diafiltration, the remaining 35L in the carboys of the fraction less than 70 kDa were ultrafiltered. The reservoir volume was reduced to 2L and an additional 10L 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
[00227] Using the apparatus depicted in Fig. 36, 24 g of 2-(N-mo holino)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.
[00228] 2.5 g of dipalmitoylphosphatidylethanolamine (DPPE) and 25 g of
hydroxybenzotriazole (HOBT) were dissolved in 940 mL of teri-butanol and 80 mL of water with stirring and heating at 45 °C in a 12L round bottom flask (forming a closed system with the pump and the sonciator, 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 l-ethyl-3-(3- dimethylaminoemyl)carbodiirnide (ED AC) 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% Na2CC>3, previously prepared by dissolving 150 g of Na2C03 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 HC1 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
[00229] 2.25 kg of NaHC03 was dissolved in 150 L of 0.1% chlorocresol solution, prepared by dissolving 150 g of chlorocresol in 150 L of DI water. 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 lOkDa Omega TFF membrane, 150 L of 1.5% NaHC03 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 HC1 while the temperature was kept at - 20-25 °C by passing the solution through a cooled, jacketed flow cell.
EXAMPLE 5
EXTRACTION OF HyPE [00230] 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 3L 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
[00231] 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 X 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 μΜ (2 X working solution) in CM. The vehicle control solution was CM. A 1 mg/ml solution of LPS (made in 1 x 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% C02. 0.5 ml of cells at 1 x 106 cells/ml was plated in 24-well tissue culture plates. Cells were allowed to adhere for 30 minutes at 37°C with 5% C02 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 μΐ of CM was added to the -LPS plates. 110 μΐ of 10 μ^ητΐ LPS was added to the +1 g/ml LPS plates. The plates were incubated for 24 hours at 37°C with 5% C02.
EXAMPLE 7
SUPERNATANT HARVESTING/XTT ASSAY
[00232] Cell culture supematants were collected after 24 hours of LPS treatment and stored at - 30°C until assayed. 400 μΐ of media was left in each cell culture well for the XTT assay. 400 μΐ of media was added to a cell-free culture well for use as a blank in the XTT assay. 200 μΐ of activated XTT reagent was added to each well. Plates were incubated for 1 hour at 37°C with 5% C02. 100 μΐ was removed from each well and read at 450 nm (630 nm correction) using a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA). 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
[00233] Cell culture supernatants were assayed for IL-6, TNF-a and IP- 10 using a
Luminexbased assay according to the manufacturer's instructions. Data were collected using a Luminex 100 (Luminex Corporation, Austin, TX). Standard curves were generated using a 5-parameter logistic curve-fitting equation weighted by 1/y
(StarStation V 2.0; Applied Cytometry Systems, Sacramento, CA). Each sample reading was interpolated from the appropriate standard curve. Calculated concentrations were multiplied by the appropriate dilution factor when necessary. TNF-a data relating to high molecular weight HyPE compositions are shown in Fig. 7, Fig. 8 and Fig. 15. TNF-a 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.
[00234] 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
[00235] 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 μπι filter (PALL) before drying to achieve microbial purity.
EXAMPLE 10
SEC-MALS DETERMINATION OF MOLECULAR WEIGHT
[00236] 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 x 7,5 mm, 8 μπι; Agilent Technologies) columns connected in series and thermostated at ambient temperature. Injection volume was 100 μΐ. 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 (d«/dc) of 0,155 mg/ml was used for sodium hyaluronate.
[00237] 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 μιη, 25 mm diameter, Whatman) and analysed by HPLC system
[00238] 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.
[00239] In essence, light scattering measurements automatically provide a column calibration curve for every sample, obviating time-consuming, conformation dependent calibration procedure. [00240] 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.
[00241] The weight-average molecular weight of hyaluronan was verified by measurements of dextran standard.
[00242] The determined molecular weight and polydispersity value for low molecular weight hyaluronic acid were 7.86 x 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 LOW MOLECULAR WEIGHT HYALURONIC ACID
[00243] MES buffer was prepared by dissolving 14.5 g of MES in 75 mL of DI-H20 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-H20, 60 mL MES buffer, 12 mL of teri-butanoL The pH was adjusted to 6.4 with 4N NaOH. In one experiment, the molecular weight of HA was 9.54kD. In another experiment, the molecular weight of HA was 10-14 kDa or 10-15kDa. In one experiment, the molecular weight of dipalmitoylphosphatidylethanolamine (DPPE) is 692. In another experiment, the molecular weight of dimyristoylphosphatidylethanolamine (DMPE) is 667. 15.1 g of HA was dissolved in 350 mL of DI-H20. 1.25 g DPPE or DMPE was dissolved in 440 mL of teri-butanol and 90 mL DI-H20 with heating to 55 deg C. Hyaluronic acid (10- 15KDa) and dipalmitoyl phosphatidylethanolamine or
dimyristoylphosphatidylethanolamine may be mixed at a 10: 1 ratio by weight for synthesis. The solutions of HA and HOBT were warmed to 35 deg C and mixed. The DPPE or DMPE 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 ED AC 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 μπι 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% NaHC03 to remove residual organic solvents. The pH was then increased by slow addition of 2% Na2C03 to pH 9.2. The solution was stirred for 1 hour at room temperature. After further washing with 30 L of 1.5% NaHC03 the peat at -12.5 min had disappeared and the solution was washed with 30 L of DI-H20 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-H20. The solution was concentrated to 1 L and filtered through a 0.2 μπι filter into a lyoguard container and placed in the lyopholizer. 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. [00244] HyPE is an amphoteric molecule and exhibits some surface active properties in aqueous solution. Maximum solubility is obtained in water and can reach 3% although the viscosity of solutions greater than 2% tends to increase rapidly.
EXAMPLE 12
PREPARATION OF OTHER POLYSACCHARIDE-LIPID CONJUGATES
[00245] The manufacturing processes for CSAPE, HyDMPE, CSADMPE and AlgPE
resemble those used for HyPE described. Chondroitin Sulfate and Alhinic acid may be obtained from marine and other sources.
[00246] Chondroitin sulfate-A of marine origin was isolated by ultrafiltration.
Dynamic light scattering studies showed the MW to be about 15 kDa. DMPE has a MW of 677. Chondroitin sulfate-A (15-20kDa) and dipalmitoyl
phosphatidylethanolamine or dimyristoylphosphatidylethanolamine may be
mixed at a 10:1 ratio by weight for synthesis. The minimum solubility of
chondroitin sulfate:dipalmitoylphosphatidylemanolamine (CSAPE) or
chondroitin sulfate:dimyristoylphosphatidylemanolamine (CSADMPE) is
about 20 mg/mL in water. In some experiments, oncentrations greater than 30 mg/mL are not used to avoid higher viscosity solutions.
[00247] Alginic Acid of marine origin was isolated. The starting material has a MW of
10-15 kDa. The DPPE has a MW of 692. Alginic Acid (10-20kDa) and
dipalmitoyl phosphatidylemanolarnine or dimyristoylphosphatidylemanolamine may be mixed at a 10: 1 ratio by weight for synthesis. The solubility of AlgPE is about 20 mg/mL in water. In some experiments, oncentrations greater than
30 mg/mL are not used to avoid higher viscosity solutions
EXAMPLE 13
TREATMENT OF RESPIRATORY DISEASE WITH POLYSACCHARIDE-LIPID CONJUGATES
[00248] 1% nasal spray (100 μΐ dose) containing HyPE B.I.D. in isotonic buffer containing benzyl alcohol was employed in a six day dosing regimen to 105 subjects. Data shows A.E.'s similar to control (placebo) and decreased cough and headache compared to steroid.
EXAMPLE 14 TREATMENT OF RESPIRATORY DISEASE WITH POLYSACCHARIDE-LIPID CONJUGATES
[00249] 1% Topical Cream containing HyPE applied B.I.D. for 28 days to 11 subjects. This double blind placebo controlled study showed no adverse effects and significant efficacy versus placebo in treatment of bilateral atopic dermatitis.
[00250] The cream is used to treat adult or pediatric atopic dermatitis.
EXAMPLE 15
TREATMENT OF EYE DISEASE WITH POLYSACCHARIDE-LIPID
CONJUGATES
[00251] Data for the inhibitory activity of HyDMPE in suppressing IL-8 was presented in the summary section of this report. Data obtained from Alcon for its activity in dry eye can be found in the Alcon report already provided.
EXAMPLE 16
TREATMENT OF OTHER DISEASES BY LOW MOLECULAR WEIGHT CONJUGATES
[00252] A summary of data obtain for several of the compounds in a cell proliferation, cell toxicity assay and an assay for inflammation are summarized in the following table 2.
Table 2
Data Summary: Proliferation, Toxicity and Inflammation
Potency (IC50 μ§/ητιΙ. and % Inhibition
Assay Proliferation Toxicity
Cell Origin SMC SMC SMC
IL-
1+PDGF+FG
Stimulation FBS F
233 155 +/-150 Toxicity Observed at
(3097+/- 2000 μg/mL (i.e.
HyPE
(4466 nM 3005nM) 4000nM)
(87%)1 (100%) (2 out of 2)
Not
Significant 109 +/-39 Toxicity Observed at
HYDMPE (2183+/- 2000 μg/mL (i.e.
(2 out of 2) 785nM) 4000nM)
(100%) (2 out of 2) Not
Significant 37+/-52 No Toxicity Observed
CASPE (744+/-
(2 out of 2) 1037nM ) (2 out of )
(100%)
189+/-166 56+/-22 No Toxicity Observed
(3783+/- (1124+/-
AlgPE
3311nM ) 444nM) (2 out of )
(37%) (99%)
1 I n 1 of 2 repeats, EC50 was 223 (87%) with the other repeat not significant
Assay Inflammation
Cell Origin SMC U937 U937-SMC
Stimulation -MCP-1 -TNF Adhesion
8+/-7 69+/-37 361+/-129
(155+/- (1374+/-
HyPE
134nM) 75nM ) (7218+/- 2571nM)
-100% (98%) (97%)
108+/-75 133 +/-5 419+/-45
(2150+/- (2663+/-
HYDMPE
1513nM ) 95nM ) (8390+/-891nM0
(91%) (88%) (100%)
14+/-17 22+/-9 Not Significant
(290+/- (443+/-
CASPE
325nM) 171nM) (2 out of 2)
(98%) (71%)
Not
8 Significant 517
AlgPE
(160n M) (2 out of 2) (10340nM)
(60%)2 (41%)3
2 I n 1 of 2 repeats, EC50 was 8 (60%) with the other repeat not significant
3 I n 1 of 2 repeats, EC50 was 517 (41%) with the other repeat not significant
[00253] Fig. 41 depicts inhibition of PLA2-induced RBC haemolysis (IC-50 mg/ml with corresponding polysaccharides) by low molecular weight conjugates.
[00254] Fig. 42 depicts inhibition of IL-8 production by low molecular weight conjugates in both normal (corrected cell lines/C38) and Cystic Fibrosis (IB3).
[00255] Fig. 43 depicts inhibition of IL-8 production by low molecular weight conjugates in
16HBE airway epithelial cells transfected with cflr sense (Normal) and anti-sense
(Cystic Fibrosis) construct.
[00256] 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

CLAIMS What is claimed is:
1. A lipid-polysaccharide conjugate comprising a polysaccharide conjugated to a phospholipid wherein said polysaccharide has an average molecular weight between 5 to 90kD.
2. The lipid-polysaccharide conjugate of claim 1, wherein said polysaccharide has an average molecular weight between 5 to 20kD.
3. The lipid-polysaccharide conjugate of claim 1, wherein said polysaccharide is glycosaminoglycan.
4. The lipid-polysaccharide conjugate of claim 3, wherein said
glycosaminoglycan is hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, keratan or keratan sulfate.
5. The lipid-polysaccharide conjugate of claim 1, wherein said phospholipid is a phosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a phoshpatidylglycerol.
6. The lipid-polysaccharide conjugate of claim 1, wherein said polysaccharide is alginate.
7. The lipid-polysaccharide conjugate of claim 1, wherein said polysaccharide is chitosan.
8. The lipid-polysaccharide conjugate of claim 1, wherein said phospholipid comprises palmitic acid or myristic acid.
9. The lipid-polysaccharide conjugate of claim 1, wherein said phospholipid is dimyristoyl phosphatidylethanolamine or dipalmitoyl
phosphatidylethanolamine.
10. A lipid-polysaccharide conjugate of claim 1, wherein said polysaccharide is conjugated to said phospholipid via an amide or ester linkage.
11. A lipid-polysaccharide conjugate comprising the lipid-polysaccharide
conjugate of claim 1 , wherein said conjugate is prepared by reacting a polysaccharide having an average molecular weight between 5 to 90kD with said phospholipid in a massPL to massp0iysaccharide ratio from about 0.25:15 to about 5:15, respectively.
12. The lipid-polysaccharide conjugate of claim 11, wherein said massPL to massp0iysaccharide ratio is about 1 :10.
13. A pharmaceutical composition comprising the lipid-polysaccharide conjugate of claim 1.
14. A method for treating, inhibiting or suppressing a pathological condition in a subject, comprising administering to said subject a lipid-polysaccharide conjugate or pharmaceutical composition according to anyone of claims 1-13.
15. The method of claim 14, wherein said pathological condition is selected from the group consisting of eye disease, infection, intestinal disease, obstructive respiratory disease, dermatological condition, cystic fibrosis, eye disorder, cardiovascular disease, proliferative disorder, and nervous system disorder.
16. The method of claim 14, wherein said pathological condition is selected from the group consisting of obstructive respiratory disease, asthma, allergic rhinitis , Inflammatory Bowel Disease, colitis, Crohn's disease, central nervous system insult, multiple sclerosis, contact dermatitis, atopic dermatitis, psoriasis, cardiovascular disease, including prophylaxis for invasive procedures, invasive cellular proliferative disorders, anti-oxidant therapy, hemolytic syndromes, sepsis, acute respiratory distress syndrome, tissue transplant rejection syndromes, autoimmune disease, cystic fibrosis, cancer , viral infection, chlamydia infection, dry eye, and hypersensitivity
conjunctivitis.
17. The method of claim 15 or 16, wherein said drug is administered orally, intravenously, intranasally intraocularly, intramuscularly, subcutaneously or topically.
18. The method of claim 16, wherein said atopic dermatitis is pediatric atopic dermatitis.
19. The method of claim 16, wherein said atopic dermatitis is adult atopic
dermatitis.
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