U.S. patent application number 12/997014 was filed with the patent office on 2011-06-02 for lipid-polymer conjugates, their preparation and uses thereof.
Invention is credited to Joseph V. Bondi, Yuval Cohen, Saul Yedgar.
Application Number | 20110130555 12/997014 |
Document ID | / |
Family ID | 43085294 |
Filed Date | 2011-06-02 |
United States Patent
Application |
20110130555 |
Kind Code |
A1 |
Yedgar; Saul ; et
al. |
June 2, 2011 |
LIPID-POLYMER CONJUGATES, THEIR PREPARATION AND USES THEREOF
Abstract
This invention provides low molecular weight lipid-GAG and
phospholipids-GAG conjugates and methods of use thereof in
suppressing, inhibiting, preventing, or treating a pathogenic
effect on a cell, including, inter alia, infection with
intracellular pathogens.
Inventors: |
Yedgar; Saul; (Jerusalem,
IL) ; Cohen; Yuval; (New York, NY) ; Bondi;
Joseph V.; (Collegeville, PA) |
Family ID: |
43085294 |
Appl. No.: |
12/997014 |
Filed: |
May 11, 2010 |
PCT Filed: |
May 11, 2010 |
PCT NO: |
PCT/US10/34317 |
371 Date: |
December 9, 2010 |
Current U.S.
Class: |
536/21 ;
536/29.1; 536/29.13 |
Current CPC
Class: |
A61P 9/08 20180101; A61P
31/00 20180101; A61P 11/00 20180101; A61P 17/06 20180101; A61P
25/00 20180101; A61K 47/61 20170801; A61P 19/02 20180101; A61K
47/544 20170801; A61P 17/02 20180101; A61P 31/04 20180101; A61K
31/728 20130101; A61K 31/727 20130101; A61P 17/00 20180101; A61P
37/00 20180101; A61P 29/00 20180101; A61P 1/04 20180101; A61P 9/10
20180101 |
Class at
Publication: |
536/21 ;
536/29.13; 536/29.1 |
International
Class: |
C08B 37/10 20060101
C08B037/10; C08B 37/08 20060101 C08B037/08; C08B 37/00 20060101
C08B037/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2009 |
US |
61/177083 |
Claims
1. A lipid-polymer conjugate comprising a glycosaminoglycan (GAG)
conjugated to a phospholipid (PL) wherein said conjugate is
prepared by reacting said GAG with said PL in a mass.sub.PL to
mass.sub.GAG ratio from about 0.25:15 to about 5:15,
respectively.
2. The lipid-polymer conjugate of claim 0, wherein said mass.sub.PL
to mass.sub.GAG ratio is about 0.25:15.
3. The lipid-polymer conjugate of claim 0, wherein said mass.sub.PL
to mass.sub.GAG ratio is about 0.5:15.
4. The lipid-polymer conjugate of claim 0, wherein said mass.sub.PL
to mass.sub.GAG ratio is about 1:15.
5. The lipid-polymer conjugate of claim 0, wherein said mass.sub.PL
to mass.sub.GAG ratio is about 2:15.
6. The lipid-polymer conjugate of claim 0, wherein said mass.sub.PL
to mass.sub.GAG ratio is about 5:15.
7. The lipid-polymer conjugate of claim 0, wherein said GAG is
hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin
sulfate, dermatan sulfate or keratan sulfate.
8. The lipid-polymer conjugate of claim 0, wherein said PL is a
phosphatidylethanolamine, a phosphatidylserine, a
phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or
a phosphatidylglycerol.
9. The lipid-polymer conjugate of claim 0, wherein said PL
comprises the residue of palmitic acid or myristic acid.
10. The lipid-polymer conjugate of claim 0, wherein said PL is
dimyristoyl phosphatidylethanolamine or dipalmitoyl
phosphatidylethanolamine.
11. The lipid-polymer conjugate of claim 0, wherein the
polydispersity of said GAG is from about 1 to 1.75.
12. The lipid-polymer conjugate of claim 11, wherein the
polydispersity of said GAG is from about 1.25 to 1.5.
13. The lipid-polymer conjugate of claim 0, wherein the average
molecular weight of said GAG is between 5 KD to 90 KD.
14. The lipid-polymerconjugate of claim 13, wherein the average
molecular weight of said GAG is between 5 KD to 20 KD.
15-23. (canceled)
24. A lipid-polymer conjugate comprising a glycosaminglycan (GAG)
conjugated to a phospholipid (PL) via an amide or ester linkage
wherein the molecular weight of said GAG is between 5 to 20 KD and
wherein the polydispersity of said GAG is from about 1 to 1.75.
25. The lipid-polymer conjugate of claim 24, wherein the
polydispersity of said GAG is from about 1.25 to 1.5.
26. The lipid-polymer conjugate of claim 24, wherein said GAG is
hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitin
sulfate, dermatan sulfate or keratan sulfate.
27. The lipid-polymer conjugate of claim 24, wherein said PL is a
phosphatidylethanolamine, a phosphatidylserine, a
phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or
a phosphatidylglycerol.
28. The lipid-polymer conjugate of claim 24, wherein said PL
comprises the residue of palmitic acid or myristic acid.
29. The lipid-polymer conjugate of claim 24, wherein said PL is
dimyristoyl phosphatidylethanolamine or dipalmitoyl
phosphatidylethanolamine.
30. A pharmaceutical composition comprising the lipid-polymer
conjugate of claim 1.
31. A pharmaceutical composition comprising the lipid-polymer
conjugate of claim 24.
Description
FIELD OF THE INVENTION
[0001] This invention provides low molecular weight lipid-GAG
conjugates and methods of use thereof in suppressing, inhibiting,
preventing, or treating a pathogenic effect on a cell, including,
inter alia, infection with intracellular pathogens.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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
[0004] In one embodiment, the present invention provides a
lipid-polymer conjugate comprising a glycosaminoglycan (GAG)
conjugated to a phospholipid (PL) wherein said conjugate is
prepared by reacting said GAG with said PL in a mass.sub.PL to
mass.sub.GAG ratio from about 0.25:15 to about 5:15,
respectively.
[0005] In one embodiment, the present invention provides a
lipid-polymer conjugate comprising a glycosaminglycan (GAG)
conjugated to a phospholipid (PL) via an amide or ester linkage
wherein the molecular weight of said GAG is between 5 to 20 kD.
[0006] In one embodiment, the present invention provides a
lipid-polymer conjugate represented by the structure of the general
formula (A):
##STR00001## [0007] wherein [0008] L is a lipid or a phospholipid;
[0009] Z is either nothing, ethanolamine, serine, inositol,
choline, phosphate, or glycerol; [0010] Y is either nothing or a
spacer group ranging in length from 2 to 30 atoms; [0011] X is a
glycosaminoglycan; and [0012] n is a number from 1 to 70; [0013]
wherein any bond between L, Z, Y and X is either an amide or an
esteric bond; [0014] wherein the molecular weight of said
glycosaminoglycan is between 5 kD and 20 kD.
[0015] In one embodiment, the present invention provides a process
for preparing a compound represented by the structure of the
general formula (I):
##STR00002## [0016] wherein [0017] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0018] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0019] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0020] X is a glycosaminoglycan; and [0021] n is a number from 1 to
70; [0022] 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; [0023]
comprising the steps of: [0024] b. reacting a phospholipid (PL)
with a glycosaminoglycan (GAG) and a coupling agent, wherein the
mass.sub.PL to mass.sub.GAG ratio from about 0.25:15 to about 5:15,
respectively; [0025] c. filtering the reaction mixture from (a) to
generate a filtrate; and [0026] d. extracting a product from a
filtrate.
[0027] In one embodiment, the present invention provides a method
of treating inflammatory disorders in a subject, said method
comprising administering to a subject suffering from an
inflammatory disorder a composition comprising a lipid-polymer
conjugate comprising a glycosaminoglycan (GAG) conjugated to a
phospholipid (PL) wherein said conjugate is prepared by reacting
said GAG with said PL in a mass.sub.PL to mass.sub.GAG ratio from
about 0.25:15 to about 5:15, respectively. In one embodiment, the
present invention provides a method for decreasing expression of
proinflammatory chemokines, cytokines, or a combination thereof
comprising the step of administering a compound represented by the
structure of the general formula (A):
##STR00003## [0028] wherein [0029] L is a lipid or a phospholipid;
[0030] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0031] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0032] X is a
glycosaminoglycan; and [0033] n is a number from 1 to 70; [0034]
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.
[0035] In one embodiment, the present invention provides a method
of activating NF-.kappa.B, IL-6, IL-8, or a combination thereof in
human airway epithelial cell lines comprising the step of
administering to a subject a compound represented by the structure
of the general formula (A):
##STR00004## [0036] wherein [0037] L is a lipid or a phospholipid;
[0038] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0039] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0040] X is a
glycosaminoglycan; and [0041] n is a number from 1 to 70; [0042]
wherein any bond between L, Z, Y and X is either an amide or an
esteric bond.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] 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:
[0044] FIG. 1 depicts a conceptual diagram of the reaction vessel
features required to practice the methods of this invention.
[0045] FIG. 2 depicts an NMR spectrum of a hyaluronic
acid-phosphatidylethanolamine conjugate (HyPE) prepared according
to Example 5.
[0046] FIG. 3 is an HPLC chromatogram of HyPE prepared according to
Example 5.
[0047] FIG. 4 depicts a schematic representation of the in vitro
stimulation of RAW 264.7 cells.
[0048] FIG. 5 depicts the mean XTT reduction (OD.sub.450) by RAW
264.7 cells in the absence of LPS. Error bars represent standard
deviations.
[0049] FIG. 6 depicts the mean XTT reduction (OD.sub.450) by
LPS-stimulated RAW 264.7 cells. Error bars represent standard
deviations.
[0050] FIG. 7 depicts the mean TNF-.alpha. release from RAW 264.7
cells in the absence of LPS. Error bars represent standard
deviations.
[0051] FIG. 8 depicts the mean TNF-.alpha. release from
LPS-stimulated RAW 264.7 cells. Error bars represent standard
deviations.
[0052] FIG. 9 depicts the mean IL-6 release from RAW 264.7 cells in
the absence of LPS. Error bars represent standard deviations.
[0053] FIG. 10 depicts the mean IL-6 release from LPS-stimulated
RAW 264.7 cells. Error bars represent standard deviations.
[0054] FIG. 11 depicts the mean IP-10 release from RAW 264.7 cells
in the absence of LPS. Error bars represent standard
deviations.
[0055] FIG. 12 depicts the mean IP-10 release from LPS-stimulated
RAW 264.7 cells. Error bars represent standard deviations.
[0056] FIG. 13 depicts the mean PGE.sub.2 release from RAW 264.7
cells in the absence of LPS. Error bars represent standard
deviations.
[0057] FIG. 14 depicts the mean PGE.sub.2 release from
LPS-stimulated RAW 264.7 cells. Error bars represent standard
deviations.
[0058] FIG. 15 depicts dose-response curves for TNF-.alpha.
production (+LPS). Data fit using Prism 4, Sigmoidal dose-response
curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0059] FIG. 16 depicts dose-response curves for IL-6 production
(+LPS). Data fit using Prism 4, Sigmoidal dose-response curve
(variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0060] FIG. 17 depicts dose-response curves for IP-10 production
(+LPS). Data fit using Prism 4, Sigmoidal dose-response curve
(variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0061] FIG. 18 depicts dose-response curves for PGE.sub.2
production (+LPS). Data fit using Prism 4, Sigmoidal dose-response
curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0062] 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.
[0063] FIG. 20 is the SEC-MALS determined distribution of molecular
weight of low molecular weight sodium hyaluronate.
[0064] FIG. 21 is the UV spectrum of sample 208-088 (low molecular
weight sodium hyaluronate).
[0065] FIG. 22 depicts the mean XTT reduction (OD.sub.450) by RAW
264.7 cells in the absence of LPS. Error bars represent standard
deviations.
[0066] FIG. 23 depicts the mean XTT reduction (OD.sub.450) by
LPS-stimulated RAW 264.7 cells. Error bars represent standard
deviations.
[0067] FIG. 24 depicts the mean TNF-.alpha. release from RAW 264.7
cells in the absence of LPS. Error bars represent standard
deviations.
[0068] FIG. 25 depicts the mean TNF-.alpha. release from
LPS-stimulated RAW 264.7 cells. Error bars represent standard
deviations.
[0069] FIG. 26 depicts the mean IL-6 release from RAW 264.7 cells
in the absence of LPS. Error bars represent standard
deviations.
[0070] FIG. 27 depicts the mean IL-6 release from LPS-stimulated
RAW 264.7 cells. Error bars represent standard deviations.
[0071] FIG. 28 depicts the mean IP-10 release from RAW 264.7 cells
in the absence of LPS. Error bars represent standard
deviations.
[0072] FIG. 29 depicts the mean IP-10 release from LPS-stimulated
RAW 264.7 cells. Error bars represent standard deviations.
[0073] FIG. 30 depicts the mean PGE.sub.2 release from RAW 264.7
cells in the absence of LPS. Error bars represent standard
deviations.
[0074] FIG. 31 depicts the mean PGE.sub.2 release from
LPS-stimulated RAW 264.7 cells. Error bars represent standard
deviations.
[0075] FIG. 32 depicts dose-response curves for TNF-.alpha.
production (+LPS). Data fit using Prism 4, Sigmoidal dose-response
curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0076] FIG. 33 depicts dose-response curves for IL-6 production
(+LPS). Data fit using Prism 4, Sigmoidal dose-response curve
(variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0077] FIG. 34 depicts dose-response curves for IP-10 production
(+LPS). Data fit using Prism 4, Sigmoidal dose-response curve
(variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0078] FIG. 35 depicts dose-response curves for PGE.sub.2
production (+LPS). Data fit using Prism 4, Sigmoidal dose-response
curve (variable slope): Y=Bottom+(Top+Bottom)/(1+10
((LOGIC50-X)*HillSlope)). X is the log of Test Article
concentration, Y is the response. Constraints Bottom=0,
Top=100.
[0079] 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.
[0080] FIG. 37 depicts a chromatogram of the HyPE reaction from
Example 11 after 2 hours.
[0081] FIG. 38 depicts a chromatogram of the HyPE reaction from
Example 11 after 6 hours.
[0082] FIG. 39 depicts the GPC analysis of final HyPE isolated from
Example 11.
[0083] FIG. 40 depicts the NMR spectrum of final HyPE isolated from
Example 11 and treated with 1 drop of 4% NaOD.
[0084] 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
[0085] 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.
[0086] Abbreviations used to specify chemicals and reagents used in
the processes described herein are readily recognized by one
skilled in the art. For the purposes of this invention, it will be
understood that DCC refers to dicyclohexylcarbodiimide, EDAC refers
to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride),
BOP refers to
Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate, PyBOP refers to
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate,
HATU refers to
O-(7-Azabenzotriazole-1-yl)-N,N,N'N'-tetramethyluronium
hexafluorophosphate, TSTU refers to
O--(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate,
HOBT refers to hydroxybenzotriazole and HOAT refers to
1-hydroxy-7-aza-benzotriazole.
[0087] Herein, the term "lipid" refers to all types of lipids
including phospholipids, glycerolipids, sphingolipids, sterol
lipids, prenol lipids, saccharolipids and the like.
[0088] This invention provides, in one embodiment, a lipid-polymer
conjugate which is useful in some embodiments for the treatment of
inflammatory disorders.
[0089] 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.
[0090] In one embodiment, this invention provides a lipid-polymer
conjugate comprising a glycosaminoglycan (GAG) conjugated to a
phospholipid (PL) wherein said conjugate is prepared by reacting
said GAG with said PL in a mass.sub.PL to mass.sub.GAG ratio from
about 0.25:15 to about 5:15, respectively.
[0091] In another embodiment, said mass.sub.PL to mass.sub.GAG
ratio is about 0.25:15. In another embodiment, said mass.sub.PL to
mass.sub.GAG ratio is about 0.5:15. In another embodiment, said
mass.sub.PL to mass.sub.GAG ratio is about 1:15. In another
embodiment, said mass.sub.PL to mass.sub.GAG ratio is about 2:15.
In another embodiment, said mass.sub.PL to mass.sub.GAG ratio is
about 5:15.
[0092] In one embodiment, the present invention provides a
lipid-polymer conjugate comprising a glycosaminglycan (GAG)
conjugated to a phospholipid (PL) via an amide or ester linkage
wherein the molecular weight of said GAG is between 5 to 20 kD.
[0093] In another embodiment, said GAG of the lipid-conjugate
compound of this invention is hyaluronic acid, heparin, heparan
sulfate, chondroitin, chondroitin sulfate, dermatan sulfate or
keratan sulfate. In another embodiment, said GAG is hyaluronic
acid. In another embodiment, said GAG is heparin. In another
embodiment, said GAG is chondroitin. In another embodiment, said
GAG is chondroitin sulfate. In another embodiment, said GAG is
dermatan sulfate, in another embodiment, said GAG is keratan
sulfate.
[0094] 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.
[0095] In another embodiment, said PL of the lipid-conjugate
compound of this invention is a phosphatidylethanolamine, a
phosphatidylserine, a phosphatidylcholine, a phosphatidylinositol,
a phosphatidic acid or a phosphatidylglycerol. In another
embodiment, said PL comprises the residue of palmitic acid,
myristic acid, myristoleic acid, palmitoleic acid, oleic acid,
linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic
acid, erucic acid or docosahexaenoic acid. In another embodiment,
said PL is dimyristoyl phosphatidylethanolamine. In another
embodiment, said PL is dipalmitoyl phosphatidylethanolamine.
[0096] 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.
[0097] In one embodiment, the lipid-polymer conjugate of this
invention comprises a GAG wherein the average molecular weight of
said GAG is between 5 kd to 90 kd. In another embodiment, the
average molecular weight of said GAG is between 5 kD to 60 kD. In
another embodiment, the average molecular weight of said GAG is
between 5 kD to 40 kD.
[0098] In another embodiment, the average molecular weight of said
GAG is between 5 kD to 15 kD. In another embodiment, the average
molecular weight of said GAG is between 5 kD to 20 kD.
[0099] In one embodiment, low molecular weight GAG, such as sodium
hyaluronate is prepared by acid hydrolysis of sodium hyaluronate as
described in Example 9. In another embodiment, said acid hydrolysis
comprises hydrochloric acid. In another embodiment, said acid
hydrolysis comprises sulfuric acid. In another embodiment, said
acid hydrolysis comprises trifluoroacetic acid. In another
embodiment, said acid hydrolysis comprises hydrobromic acid. In
another embodiment, said acid hydrolysis comprises acetic acid. In
another embodiment, the concentration of the acid in said acid
hydrolysis is from about 0.1 to 12 molar. In another embodiment,
the concentration of the acid in said acid hydrolysis is from about
1 to 6 molar. In another embodiment, the concentration of the acid
in said acid hydrolysis is from about 6 to 12 molar. In another
embodiment, said acid hydrolysis is carried out at a temperature
between 25 degrees Celsius to 100 degrees Celsius. In another
embodiment, said acid hydrolysis is carried out at a temperature
between 25 degrees Celsius to 50 degrees Celsius. In another
embodiment, said acid hydrolysis is carried out at a temperature
between 50 degrees Celsius to 100 degrees Celsius.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] In another embodiment, this invention provides a
pharmaceutical composition comprising a lipid-polymer conjugate
comprising a glycosaminoglycan (GAG) conjugated to a phospholipid
(PL) wherein said conjugate is prepared by reacting said GAG with
said PL in a mass.sub.PL to mass.sub.GAG ratio from about 0.25:15
to about 5:15, respectively. In another embodiment, the average
molecular weight of said GAG is between 5 kD to 90 kD. In another
embodiment, the average molecular weight of said GAG is between 5
kD to 20 kD. In another embodiment, the average molecular weight of
said GAG is greater than 10 kD.
[0104] In one embodiment, this invention provides a lipid-polymer
conjugate represented by the structure of the general formula
(A):
##STR00005## [0105] wherein [0106] L is a lipid or a phospholipid;
[0107] Z is either nothing, ethanolamine, serine, inositol,
choline, phosphate, or glycerol; [0108] Y is either nothing or a
spacer group ranging in length from 2 to 30 atoms; [0109] X is a
glycosaminoglycan; and [0110] n is a number from 1 to 70; [0111]
wherein any bond between L, Z, Y and X is either an amide or an
enteric bond; [0112] wherein the molecular weight of said
glycosaminoglycan is between 5 kD and 20 kD.
[0113] In one embodiment L is a lipid. In another embodiment L is a
phospholipid. In another embodiment, L is a
phosphatidylethanolamine, a phosphatidylserine, a
phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or
a phosphatidylglycerol. In another embodiment, L comprises the
residue of palmitic acid, myristic acid, myristoleic acid,
palmitoleic acid, oleic acid, linoleic acid, linolenic acid,
arachidonic acid, eicosapentaenoic acid, erucic acid or
docosahexaenoic acid. In another embodiment, L is dimyristoyl
phosphatidylethanolamine. In another embodiment, said L is
dipalmitoyl phosphatidylethanolamine.
[0114] In another embodiment, X is hyaluronic acid, heparin,
heparan sulfate, chondroitin, chondroitin sulfate, dermatan sulfate
or keratan sulfate. In another embodiment, X is hyaluronic acid. In
another embodiment, X is heparin. In another embodiment, X is
chondroitin. In another embodiment, X is chondroitin sulfate. In
another embodiment, X is dermatan sulfate, in another embodiment, X
is keratan sulfate.
[0115] 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.
[0116] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (I):
##STR00006## [0117] wherein [0118] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0119] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0120] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0121] X is a physiologically acceptable monomer, dimer, oligomer
or polymer wherein X is a glycosaminoglycan (GAG); and [0122] n is
a number from 1 to 70; [0123] 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.
[0124] In another embodiment, the molecular weight of said GAG is
between 5 to 20 kD.
[0125] Examples of phosphatidylethanolamine (PE) moieties are
analogues of the phospholipid in which the chain length of the two
fatty acid groups attached to the glycerol backbone of the
phospholipid varies from 2-30 carbon atoms length, and in which
these fatty acids chains contain saturated and/or unsaturated
carbon atoms. In lieu of fatty acid chains, alkyl chains attached
directly or via an ether linkage to the glycerol backbone of the
phospholipid are included as analogues of PE. In one embodiment,
the PE moiety is dipalmitoyl-phosphatidyl-ethanolamine. In another
embodiment, the PE moiety is
dimyristoyl-phosphatidyl-ethanolamine.
[0126] Phosphatidyl-ethanolamine and its analogues may be from
various sources, including natural, synthetic, and semisynthetic
derivatives and their isomers.
[0127] 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.
[0128] 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.
[0129] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (II):
##STR00007## [0130] wherein [0131] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0132] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0133] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0134] X is a physiologically acceptable monomer, dimer, oligomer
or polymer wherein X is a glycosaminoglycan; and [0135] n is a
number from 1 to 70; [0136] 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.
[0137] In one embodiment, the phosphatidylserine may be bonded to
Y, or to X if Y is nothing, via the COO'' moiety of the
phosphatidylserine.
[0138] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (III):
##STR00008## [0139] wherein [0140] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0141] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0142] Z is either
nothing, inositol, choline, or glycerol; [0143] Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; [0144] X is
a physiologically acceptable monomer, dimer, oligomer or polymer
wherein X is a glycosaminoglycan; and [0145] n is a number from 1
to 70; [0146] wherein any bond between the phosphatidyl, Z, Y and X
is either an amide or an esteric bond.
[0147] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (IV):
##STR00009## [0148] wherein [0149] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0150] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0151] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0152] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0153] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0154] n is a number from 1 to 70; [0155] wherein any bond between
the phospholipid, Z, Y and X is either an amide or an esteric
bond.
[0156] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (V):
##STR00010## [0157] wherein [0158] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0159] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0160] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0161] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0162] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0163] n is a number from 1 to 70; [0164] wherein any bond between
the phospholipid, Z, Y and X is either an amide or an esteric
bond.
[0165] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (VI):
##STR00011## [0166] wherein [0167] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0168] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0169] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0170] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0171] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0172] n is a number from 1 to 70; [0173] wherein any bond between
the phospholipid, Z, Y and X is either an amide or an esteric
bond.
[0174] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (VII):
##STR00012## [0175] wherein [0176] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0177] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0178] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0179] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0180] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0181] n is a number from 1 to 70; [0182] wherein any bond between
the phospholipid, Z, Y and X is either an amide or an esteric
bond.
[0183] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (VIII):
##STR00013## [0184] wherein [0185] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0186] R.sub.2 is either hydrogen
or a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0187] Z
is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; [0188] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0189] X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0190] n is a number from 1 to 70; [0191]
wherein any bond between the phospholipid, Z, Y and X is either an
amide or an esteric bond.
[0192] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the of the general formula
(IX):
##STR00014## [0193] wherein [0194] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0195] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0196] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0197] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0198] X is a physiologically
acceptable monomer, dimer, oligomer, or polymer wherein X is a
glycosaminoglycan; and [0199] n is a number from 1 to 70; [0200]
wherein any bond between the phospholipid, Z, Y and X is either an
amide or an esteric bond.
[0201] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (IXa):
##STR00015## [0202] wherein [0203] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0204] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0205] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0206] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0207] X is a physiologically
acceptable monomer, dimer, oligomer, or polymer wherein X is a
glycosaminoglycan; and [0208] n is a number from 1 to 70; [0209]
wherein any bond between the phospholipid, Z, Y and X is either an
amide or an esteric bond.
[0210] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (IXb):
##STR00016## [0211] wherein [0212] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0213] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0214] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0215] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0216] X is a physiologically
acceptable monomer, dimer, oligomer, or polymer wherein X is a
glycosaminoglycan; and [0217] n is a number from 1 to 70; [0218]
wherein any bond between the phospholipid, Z, Y and X is either an
amide or an esteric bond.
[0219] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the of the general formula (X):
##STR00017## [0220] wherein [0221] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0222] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0223] Z
is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; [0224] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0225] X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a
glycosaminoglycan; and [0226] n is a number from 1 to 70; [0227]
wherein any bond between the ceramide phosphoryl, Z, Y and X is
either an amide or an esteric bond.
[0228] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula
(Xa):
##STR00018## [0229] wherein [0230] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0231] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0232] Z
is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; [0233] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0234] X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a
glycosaminoglycan; and [0235] n is a number from 1 to 70; [0236]
wherein any bond between the ceramide phosphoryl, Z, Y and X is
either an amide or an esteric bond.
[0237] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the of the general formula
(XI):
##STR00019## [0238] wherein [0239] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0240] Y is either nothing or a
spacer group ranging in length from 2 to 30 atoms; [0241] X is a
physiologically acceptable monomer, dimer, oligomer or polymer
wherein X is a glycosaminoglycan; and [0242] n is a number from 1
to 70; [0243] 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.
[0244] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the of the general formula
(XII):
##STR00020## [0245] wherein [0246] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0247] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0248] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0249] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0250] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0251] n is a number from 1 to 70; [0252] wherein any bond between
the ceramide, Z, Y and X is either an amide or an esteric bond.
[0253] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula
(XIIa):
##STR00021## [0254] wherein [0255] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0256] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0257] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0258] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0259] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0260] n is a number from 1 to 70; [0261] wherein any bond between
the ceramide, Z, Y and X is either an amide or an esteric bond.
[0262] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XIII):
##STR00022## [0263] wherein [0264] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0265] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0266] Z is either
nothing, ethanolamine, serine, choline, inositol, or glycerol;
[0267] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0268] X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
[0269] n is a number from 1 to 70; [0270] wherein any bond between
the diglyceryl, Z, Y and X is either an amide or an esteric
bond.
[0271] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XIV):
##STR00023## [0272] wherein [0273] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0274] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0275] Z
is either nothing, choline, ethanolamine, serine, inositol, or
glycerol; [0276] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0277] X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0278] n is a number from 1 to 70; [0279]
wherein any bond between the glycerolipid, Z, Y and X is either an
amide or an esteric bond.
[0280] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XV):
##STR00024## [0281] wherein [0282] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0283] R.sub.2 is either hydrogen
or a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0284] Z
is either nothing, choline, ethanolamine, serine, inositol, or
glycerol; [0285] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0286] X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0287] n is a number from 1 to 70; [0288]
wherein any bond between the glycerolipid, Z, Y and X is either an
amide or an esteric bond.
[0289] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XVI):
##STR00025## [0290] wherein [0291] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0292] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0293] Z
is either nothing, choline, ethanolamine, serine, inositol, or
glycerol; [0294] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0295] X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0296] n is a number from 1 to 70; [0297]
wherein any bond between the lipid, Z, Y and X is either an amide
or an esteric bond.
[0298] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XVII):
##STR00026## [0299] wherein [0300] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0301] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0302] Z
is either nothing, choline, ethanolamine, serine, inositol, or
glycerol; [0303] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0304] X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0305] n is a number from 1 to 70; [0306]
wherein any bond between the lipid, Z, Y and X is either an amide
or an esteric bond.
[0307] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XVIII):
##STR00027## [0308] wherein [0309] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0310] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0311] Z is either nothing, choline, ethanolamine, serine,
inositol, or glycerol; [0312] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0313] X is a physiologically
acceptable monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0314] n is a number from 1 to 70; [0315]
wherein any bond between the lipid, Z, Y and X is either an amide
or an esteric bond.
[0316] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XIX):
##STR00028## [0317] wherein [0318] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0319] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0320] Z is either nothing, choline, ethanolamine, serine,
inositol, or glycerol; [0321] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0322] X is a physiologically
acceptable monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0323] n is a number from 1 to 70; [0324]
wherein any bond between the lipid, Z, Y and X is either an amide
or an esteric bond.
[0325] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XX):
##STR00029## [0326] wherein [0327] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0328] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0329] Z is either nothing, choline, ethanolamine, serine,
inositol, or glycerol; [0330] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0331] X is a physiologically
acceptable monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and [0332] n is a number from 1 to 70; [0333]
wherein any bond between the lipid, Z, Y and X is either an amide
or an esteric bond.
[0334] In another embodiment, said lipid-polymer conjugate is
represented by the structure of the general formula (XXI):
##STR00030## [0335] wherein [0336] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0337] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0338] Z is either nothing, choline, ethanolamine, serine,
inositol, or glycerol; [0339] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0340] X is a physiologically
acceptable monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and n is a number from 1 to 70; [0341] wherein
any bond between the lipid, Z, Y and X is either an amide or an
esteric bond. [0342] In another embodiment, R.sub.1 of formulae
(I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa),
(IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI),
(XVII), (XVIII), (XIX), (XX), (XXI) and (XXII) is a residue of
palmitic acid or a residue of myristic acid.
[0343] In another embodiment, R.sub.2 of formulae (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa),
(XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII),
(XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a
residue of myristic acid.
[0344] In some embodiments, the compounds (A), (B) (III), (IV),
(V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI),
(XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX),
(XX), (XXI) and (XXII) as presented hereinabove comprises a Z
group. In one embodiment, Z is a nothing. In another embodiment Z
is inositol. In another embodiment, Z is choline. In another
embodiment, Z is glycerol. In another embodiment, Z is
ethanoleamine. In another embodiment, Z is serine.
[0345] For any or all of the compounds represented by the
structures of the general formulae (A), (I), (II), (III), (IV),
(V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI),
(XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX),
(XX), (XXI), and (XXII) hereinabove: In one embodiment, X is a
glycosaminoglycan. According to this aspect and in one embodiment,
the glycosaminoglycan may be, inter alia, hyaluronic acid, heparin,
heparan sulfate, chondroitin sulfate, keratin, keratan sulfate,
dermatan sulfate or a derivative thereof. In one embodiment, the
chondroitin sulfate may be, inter alia, chondroitin-6-sulfate,
chondroitin-4-sulfate or a derivative thereof. In another
embodiment, X is not a glycosaminoglycan. In another embodiment, X
is a polysaccharide, which in one embodiment is a
hetero-polysaccharide, and in another embodiment, is a
homo-polysaccharide. In another embodiment, X is a
polypyranose.
[0346] 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.
[0347] In one embodiment, this invention provides lipid-GAG
conjugate or phospholipid-GAG conjugate, and methods of use
thereof, wherein said conjugate represented by the structures of
the general formulae (A), (I), (II), (III), (IV), (V), (VI), (VII),
(VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII),
(XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XX), and (XXII).
In another embodiment, the average molecular weight of said GAG is
between 5 kD to 90 kD. In another embodiment, the average molecular
weight of said GAG is between 5 kD to 60 kD. In another embodiment,
the average molecular weight of said GAG is between 5 kD to 40 kD.
In another embodiment, the average molecular weight of said GAG is
between 5 kD to 15 kD. In another embodiment, the average molecular
weight of said GAG is between 5 kD to 20 kD. In another embodiment,
the lipid-GAG conjugate is a phospholipid-GAG conjugate.
[0348] In one embodiment of the invention, Y is nothing.
Non-limiting examples of suitable divalent groups forming the
optional bridging group (which in one embodiment, is referred to as
a spacer) Y, according to embodiments of the invention, are
straight or branched chain alkylene, e.g., of 2 or more, preferably
4 to 30 carbon atoms, --CO-alkylene-CO, --NH-alkylene-NH--,
--CO-alkylene-NH--, --NH-alkylene-NH, CO-alkylene-NH--, an amino
acid, cycloalkylene, wherein alkylene in each instance, is straight
or branched chain and contains 2 or more, preferably 2 to 30 atoms
in the chain, --(--O--CH(CH.sub.3)CH.sub.2--).sub.x-- wherein x is
an integer of 1 or more.
[0349] 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.
[0350] 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.
[0351] In one embodiment, the compounds for use in the present
invention are biodegradable.
[0352] In some embodiments, the compounds for use are as listed in
Table 1 below,
TABLE-US-00001 TABLE 1 Phospholipid Spacer Polymer (m.w.) PE None
Hyaluronic acid (2-2000 kDa) Dimyristoyl-PE None Hyaluronic acid PE
None Heparin (0.5-110 kDa) PE None Chondroitin sulfate A PE None
Carboxymethylcellulose (20-500 kDa) PE Dicarboxylic acid +
Polygeline (haemaccel) Diamine (4-40 kDa) PE None
Hydroxyethylstarch PE Dicarboxylic acid + Dextran Diamine (1-2,000
kDa) PE Carboxyl amino group Hyaluronic acid (5-20 kDa) PE
Dicarboxyl group Hyaluronic acid (5-20 kDa) PE Dipalmitoic acid
Hyaluronic acid (5-20 kDa) PE Carboxyl amino group Heparin (5-20
kDa) PE Dicarboxyl group Heparin (5-20 kDa) PE Carboxyl amino group
Chondroitin sulfate A PE Dicarboxyl group Chondroitin sulfate A PE
Carboxyl amino group Carboxymethylcellulose (5-20 kDa) PE
Dicarboxyl group Carboxymethylcellulose (5-20 kDa) PE None
Polygeline (haemaccel) (5-20 kDa) PE Carboxyl amino group
Polygeline (haemaccel) (5-20 kDa) PE Dicarboxyl group Polygeline
(haemaccel) (5-20 kDa) PE Carboxyl amino group Hydroxyethylstarch
PE Dicarboxyl group Hydroxyethylstarch PE None Dextran (5-20 kDa)
PE Carboxyl amino group Dextran (5-20 kDa) PE Dicarboxyl group
Dextran (5-20 kDa) PE None Chondroitin sulfates Dipalmitoyl-PE None
Hyaluronic acid Dipalmitoyl-PE None Heparin Dipalmitoyl-PE None
Chondroitin sulfate A Dipalmitoyl-PE None Carboxymethylcellulose
Dipalmitoyl-PE None Polygeline (haemaccel) Dipalmitoyl-PE None
Hydroxyethylstarch Dipalmitoyl-PE None Dextran Dimyristoyl-PE None
Heparin Dimyristoyl-PE None Chondroitin sulfate A Dimyristoyl-PE
None Carboxymethylcellulose Dimyristoyl-PE None Polygeline
(haemaccel) Dimyristoyl-PE None Hydroxyethylstarch Dimyristoyl-PE
None Dextran PS None Hyaluronic acid PS None Heparin PS None
Polygeline (haemaccel) PC None Hyaluronic acid PC None Heparin PC
None Polygeline (haemaccel) PI None Hyaluronic acid PI None Heparin
PI None Polygeline (haemaccel) PG None Hyaluronic acid PG None
Heparin PG None Polygeline (haemaccel)
[0353] In one embodiment, this invention provides a lipid-polymer
conjugate represented by the structure of the general formula
(B):
##STR00031## [0354] wherein [0355] L is a lipid or a phospholipid;
[0356] Z is either nothing, ethanolamine, serine, inositol,
choline, phosphate, or glycerol; [0357] Y is either nothing or a
spacer group ranging in length from 2 to 30 atoms; [0358] X is a
glycosaminoglycan; and [0359] n is a number from 1 to 10; [0360]
wherein any bond between L, Z, Y and X is either an amide or an
esteric bond.
[0361] In one embodiment, this invention provides a lipid-polymer
conjugate represented by the structure of the general formula
(XXII):
##STR00032## [0362] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0363] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0364] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0365] X is a glycosaminoglycan; and [0366] n is a number from 1 to
10; [0367] 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.
[0368] In one embodiment, n of formula (B) and formula (X) is 1-10,
in another embodiment, n is 1. In another embodiment, n is 2. In
another embodiment, n is 3. In another embodiment, n is 4. In
another embodiment, n is 5. In another embodiment, n is 6. In
another embodiment, n is 7. In another embodiment, n is 8. In
another embodiment, n is 9. In another embodiment, n is 10.
[0369] In one embodiment, this invention provides a process for
preparing a compound represented by the structure of the general
formula (I):
##STR00033## [0370] wherein [0371] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0372] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0373] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0374] X is a glycosaminoglycan; and [0375] n is a number from 1 to
70; [0376] 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; [0377]
comprising reacting a phospholipid (PL) with a glycosaminoglycan
(GAG) and a coupling agent, wherein the mass.sub.PL to mass.sub.GAG
ratio from about 0.25:15 to about 5:15, respectively;
[0378] In one embodiment, this invention provides a process for
preparing a compound represented by the structure of the general
formula (I):
##STR00034## [0379] wherein [0380] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0381] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0382] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0383] X is a glycosaminoglycan; and [0384] n is a number from 1 to
70; [0385] 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; [0386]
comprising the steps of: [0387] a. reacting a phospholipid (PL)
with a glycosaminoglycan (GAG) and a coupling agent, wherein the
mass.sub.PL to mass.sub.GAG ratio from about 0.25:15 to about 5:15,
respectively; [0388] b. filtering the reaction mixture from (a) to
generate a filtrate; and [0389] c. extracting a product from a
filtrate.
[0390] In another embodiment, said coupling agent is DCC, EDAC,
BOP, PyBOP, HATU, TSTU or any other amide coupling agent. In
another embodiment, said coupling agent is EDAC. In another
embodiment, said coupling agent further comprises HOBT or HOAT.
[0391] In another embodiment, said filtering step comprises a 10 kD
centrasette membrane.
[0392] In another embodiment, R.sub.1 is the residue of palmitic
acid or the residue of myristic acid.
[0393] In another embodiment, R.sub.2 is the residue of palmitic
acid or the residue of myristic acid.
[0394] In another embodiment, the average molecular weight of the
glycosaminoglycan is between 5 kD to 90 kD. In another embodiment,
the average molecular weight of the glycosaminoglycan is between 5
kD to 20 kD. In another embodiment, the average molecular weight of
the glycosaminoglycan is between 5 kD to 10 kD. In another
embodiment, the average molecular weight of the glycosaminoglycan
is between 10 kD to 20 kD. In another embodiment, the average
molecular weight of the glycosaminoglycan is between 20 kD to 50
kD. In another embodiment, the average molecular weight of the
glycosaminoglycan is between 30 kD to 60 kD. In another embodiment,
the average molecular weight of the glycosaminoglycan is between 40
kD to 70 kD. In another embodiment, the average molecular weight of
the glycosaminoglycan is between 50 kD to 80 kD. In another
embodiment, the average molecular weight of the glycosaminoglycan
is between 60 kD to 90 kD
[0395] In one embodiment, hyaluronic acid (HA) is used in solution
form. In another embodiment, HA solution is prepared according to
Example 1,
[0396] 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.
[0397] In one embodiment, phosphatidylethanolamine-hyaluronic acid
conjugate (HyPE) is prepared by reacting a GAG with a PL using a
coupling agent. In another embodiment, HyPE is prepared according
to Example 3 using the apparatus depicted in FIG. 1.
[0398] 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.
[0399] In one embodiment, a coupling reagent is used in the
preparation of HyPE according to Example 3. In another embodiment,
EDAC is used as the coupling reagent. In another embodiment, DCC is
used as the coupling agent. In another embodiment, BOP is used as
the coupling agent. In another embodiment, PyBOP is used as the
coupling agent. In another embodiment, HATU is used as the coupling
agent. In another embodiment, TSTU is used as the coupling
agent.
[0400] 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.
[0401] 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.
[0402] 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.
[0403] In one embodiment, this invention provides a method of
treating an inflammatory disorder in a subject, said method
comprising administering to a subject suffering from an
inflammatory disorder a composition comprising a lipid-polymer
conjugate comprising a glycosaminoglycan (GAG) conjugated to a
phospholipid (PL) wherein said conjugate is prepared by reacting
said GAG with said PL in a mass.sub.PL to mass.sub.GAG ratio from
about 0.25:15 to about 5:15, respectively.
[0404] In another embodiment, said mass.sub.PL to mass.sub.GAG
ratio is about 0.25:15. In another embodiment, said mass.sub.PL to
mass.sub.GAG ratio is about 0.5:15. In another embodiment, said
mass.sub.PL to mass.sub.GAG ratio is about 1:15. In another
embodiment, said mass.sub.PL to mass.sub.GAG ratio is about 2:15.
In another embodiment, said mass.sub.PL to mass.sub.GAG ratio is
about 5:15.
[0405] In another embodiment, said inflammatory disorder is
rheumatoid arthritis, osteoarthritis, wound healing, dermatitis,
restenosis, cystic fibrosis, multiple sclerosis or sepsis.
[0406] 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-.alpha. is measured. In another
embodiment, expression of IP-10 is measured. In another embodiment,
expression of PGE.sub.2 is measured.
[0407] In another embodiment, said composition is administered
intravenously. In another embodiment, said composition is
administered topically.
[0408] In one embodiment, the present invention provides a method
for decreasing expression of proinflammatory chemokines, cytokines,
or a combination thereof comprising the step of administering a
compound represented by the structure of the general formula
(A):
##STR00035## [0409] wherein [0410] L is a lipid or a phospholipid;
[0411] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0412] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0413] X is a
glycosaminoglycan; and [0414] n is a number from 1 to 70; [0415]
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.
[0416] In one embodiment, the present invention provides a method
of activating NF-.kappa.B, IL-6, IL-8, or a combination thereof in
human airway epithelial cell lines comprising the step of
administering to a subject a compound represented by the structure
of the general formula (A):
##STR00036## [0417] wherein [0418] L is a lipid or a phospholipid;
[0419] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0420] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0421] X is a
glycosaminoglycan; and [0422] n is a number from 1 to 70; [0423]
wherein any bond between L, Z, Y and X is either an amide or an
esteric bond.
Dosages and Routes of Administration
[0424] The methods of this invention can be adapted to the use of
the therapeutic compositions comprising Lipid-conjugates in
admixture with conventional excipients, i.e. pharmaceutically
acceptable organic or inorganic carrier substances suitable for
parenteral, enteral (e.g., oral) or topical application which do
not deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include but are not limited to
water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl
alcohols, polyethylene glycols, gelatine, carbohydrates such as
lactose, amylose or starch, magnesium stearate, talc, silicic acid,
viscous paraffin, white paraffin, glycerol, alginates, hyaluronic
acid, collagen, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, hydroxy
methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical
preparations can be sterilized and if desired mixed with auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the
like which do not deleteriously react with the active compounds.
They can also be combined where desired with other active agents,
e.g., vitamins, bronchodilators, steroids, anti-inflammatory
compounds, gene therapy, i.e. sequences which code for the
wild-type cystic fibrosis transmembrane conductance regulator
(CFTR) receptor, surfactant proteins, etc., as will be understood
by one skilled in the art.
[0425] In one embodiment, the invention provides for the
administration of a salt of a compound as described herein as well.
In one embodiment, the salt is a pharmaceutically acceptable salt,
which, in turn may refer to non-toxic salts of compounds (which are
generally prepared by reacting the free acid with a suitable
organic or inorganic base) and include, but are not limited to, the
acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,
bitartrate, borate, bromide, calcium, camsylate, carbonate,
chloride, clavulanate, citrate, dihydrochloride, edetate,
edisylate, estolate, esylate, fumarate, gluceptate, gluconate,
glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,
hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate,
lactate, lactobionate, laurate, malate, maleate, mandlate,
mesylate, methylbromide, methylnitrate, methylsulfate, mucate,
napsylate, nitrate, oleate, oxalate, pamaote, palmitate,
panthothenate, phosphate, diphospate, polygalacturonate,
salicylate, stearate, subacetate, succinate, tannate, tartrate,
teoclate, tosylate, triethiodide, and valerate salts, as well as
mixtures of these salts.
[0426] 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.
[0427] 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.
[0428] In one embodiment, the use of a single chemical entity with
potent anti-oxidant, membrane-stabilizing, anti-proliferative,
anti-chemokine, anti-migratory, and anti-inflammatory activity
provides the desired protection for a subject with an inflammatory
disorder, or in another embodiment, the methods of this invention
provide for use of a combination of the compounds described. In
another embodiment, the compounds for use in the present invention
may be provided in a single formulation/composition, or in another
embodiment, multiple formulations may be used. In one embodiment,
the formulations for use in the present invention may be
administered simultaneously, or in another embodiment, at different
time intervals, which may vary between minutes, hours, days, weeks
or months.
[0429] 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?
[0430] 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.
[0431] 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.
[0432] 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.
[0433] 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.
[0434] In one embodiment, implants or suppositories, can be used to
administer a lipid-GAG conjugate of this invention.
[0435] 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
[0436] For topical application, particularly for the treatment of
skin diseases such as contact dermatitis or psoriasis, admixture of
the compounds with conventional creams or delayed release patches
is acceptable.
[0437] 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.
[0438] 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.
[0439] 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
[0440] 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.
[0441] In one embodiment, the compound for use in the present
invention comprises dipalmitoyl phosphatidylethanolamine and
heparin. In one embodiment, the compound for use in the present
invention comprises dipalmitoyl phosphatidylethanolamine and
chondroitin sulfate. In one embodiment, the compound for use in the
present invention comprises dipalmitoyl phosphatidylethanolamine
and hyaluronic acid. In one embodiment, the compound for use in the
present invention comprises dipalmitoyl phosphatidylethanolamine
and carboxymethylcellulose. In one embodiment, the compound for use
in the present invention comprises dimyristoyl
phosphatidylethanolamine and hyaluronic acid.
[0442] 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.
[0443] In one embodiment, the conjugates of this invention display
a wide-range combination of cytoprotective pharmacological
activities, which are useful in the present invention. In one
embodiment, the compounds may be useful for their anti-inflammatory
effects. Cellular elaboration of cytokines and chemokines serve an
important regulatory function in health; however, when a
hyperactive response to stress or disease is triggered, these
compounds may present in excess and damage tissue, thereby pushing
the disease state toward further deterioration. In one embodiment,
the lipid compounds for use in the methods of this invention,
possess a combination of multiple and potent pharmacological
effects, including inter-alia the ability to inhibit the
extracellular form of the enzyme phospholipase A2.
Method of Treating CF
[0444] In one embodiment, the conjugates of this invention are
useful in affecting inflammation in a subject with an inflammatory
disorder, where the subject is administered lipid-conjugates at
pre-symptomatic stages of the disease. A characteristic feature of
inflammation in the CF lung is the persistent infiltration of
massive numbers of neutrophils into the airways. Although
neutrophils help to control infection, when present in great
excess, they can be harmful. Major advances in the understanding of
the inflammatory process in the CF lung have come from the use of
bronchoscopy and bronchioalveolar lavage (BAL) to analyze the
inflammatory process in patients who are relatively symptom free
and/or do not regularly produce sputum. Recent BAL studies suggest
that neutrophil-rich inflammation begins very early, even in
infants without clinically apparent lung disease. Thus, in one
embodiment, the lipid/phospholipid conjugates of the present
invention may be useful in treating CF, even in presymptomatic
stages of disease.
[0445] Thus, in one embodiment, the invention provides methods for
treating a subject suffering from cystic fibrosis, reducing or
delaying the mortality of a subject suffering from cystic fibrosis
or ameliorating symptoms associated with cystic fibrosis, and the
compounds/compositions/formulations, in one embodiment, diminish or
abrogate a deleterious inflammatory response in said subject, or in
another embodiment, prevent, treat, reduce the incidence of, reduce
the severity of, delay the onset of, or diminish the pathogenesis
of an infection is the CF subject. In another embodiment, the
invention provides methods for decreasing expression of
proinflammatory chemokines, cytokines, or a combination thereof,
while in another embodiment, the invention provides methods of
activating NF-.kappa.B, IL-6, IL-8, or a combination thereof in
human airway epithelial cell lines.
Method of Treating Wounds
[0446] 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).
[0447] 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.
[0448] In another embodiment, the conjugates as described herein
increase the rate of chronic and acute wound healing. In another
embodiment, the conjugates as described herein counteract
mechanisms which delay or impair wound healing. In another
embodiment, the compounds as described herein counteract exogenous
factors which delay or impair wound healing. In another embodiment,
the conjugates as described herein counteract endogenous factors
which delay or impair wound healing. In another embodiment, factors
include: infection, ulceration particularly through diabetes,
circulation problems associated with vascular disease,
malnutrition, stress, cancer radiotherapy and/or chemotherapy,
compromise of the immune system or simply due to the normal aging
process. In another embodiment, a method a described herein
provides both a therapeutic and a cosmetic approach that promote
wound healing processes.
[0449] 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
[0450] 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.
[0451] 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.
[0452] It is understood that one skilled in the art recognizes that
the term "arthritis" refers to both rheumatoid arthritis (RA) and
osteoarthritis (OA).
[0453] In another embodiment, a compound as described herein
inhibits the production of IL-6, IL-8, TNF-alpha, NF-.kappa.B, 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-.kappa.B, 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-.kappa.B, 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-.kappa.B, or their
combination within the joint's cells. In another embodiment,
locally administering a composition comprising a conjugate as
described herein by intra-joint injection inhibits inflammation
within the joint.
Method of Treating Other Inflammatory Disorders
[0454] It is understood by one skilled in the art that inflammatory
disorders include, but are not limited to, disorders resulting from
activation of the immune system. Thus, autoimmune disorders are
understood to be inflammatory disorders. Such disorders include,
but are not limited to, rheumatoid arthritis, osteoarthritis, wound
healing, dermatitis, restenosis, cystic fibrosis, central nervous
system tissue insult, multiple sclerosis, obstructive respiratory
disease, Crohn's disease, cardiovascular disease, atherosclerosis,
contact dermatitis, psoriasis, ARDS, or sepsis
[0455] 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.
[0456] 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.
[0457] 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.
[0458] 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.
[0459] 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.
[0460] 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.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] 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.
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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
[0469] 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 .mu.m filter followed by a 50 .mu.m filter followed
by a 10 .mu.m filter, all previously disinfected with 10% hydrogen
peroxide and washed with copious amounts of DI water to ensure
hydrogen peroxide has been removed (verified with
peroxide-detecting strips).
Example 2
Ultrafiltration Fractionation of Hyaluronic Acid (HA)
[0470] 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).
[0471] By means of constant volume diafiltration with 70 kDa Omega
TFF membranes, 20 L of 0.1% chlorocresol solution, prepared as
described in Example 1, was ultrafiltered, collecting the filtrate,
the fraction less than 70 kDa, in a carboy, previously disinfected
with 10% hydrogen peroxide. The pump speed and valves shall be set
such that the retentate flow is ten times the filtrate flow and the
feed pressure is less than 40 PSI.
[0472] The 70 kDa membranes were replaced with 30 kDa membranes and
the Centramate system was disinfected with 10% hydrogen
peroxide.
[0473] 5 L of the filtrate, the fraction less than 70 kDa, were
loaded into the reservoir and by means of constant volume
diafiltration, the remaining 35 L in the carboys of the fraction
less than 70 kDa were ultrafiltered. The reservoir volume was
reduced to 2 L and an additional 10 L of DI water was ultrafiltered
to remove the chlorocresol (confirmed by appropriate GC assay). The
reservoir volume was further reduced to 1 L, reducing the pump
speed, if necessary, to keep the feed pressure below 40 PSI. The
reservoir was then emptied directly into an autoclaved lyoguard
container, closed, frozen and lyophilized to yield HA UF 70/30. GPC
analysis was performed to ensure that this lot of HA UF 70/30 was
consistent with earlier batches. A bioburden assay and an
appropriate GC assay for chlorocresol was performed. Karl Fischer
analysis was performed to determine the water content of HA UF
70/30.
Example 3
HyPE Synthesis Reaction
[0474] Using the apparatus depicted in FIG. 36, 24 g of
2-(N-morpholino)ethanesulfonic acid (MES) were dissolved in 125 mL
of DI water and the pH was adjusted to pH 6.4 by addition of 4 N
NaOH.
[0475] 2.5 g of dipalmitoylphosphatidylethanolamine (DPPE) and 25 g
of hydroxybenzotriazole (HOBT) were dissolved in 940 mL of
tert-butanol and 80 mL of water with stirring and heating at
45.degree. C. in a 12 L round bottom flask (forming a closed system
with the pump and the sonicator, all of which will have been
previously autoclaved and/or disinfected with 70% isopropanol). To
this was added 850 mL of water and 115 mL of the MES solution. The
pH of this solution was adjusted to pH 6.4 by addition of 2.5 N
NaOH. 25 g of HA UF 70/30 of Example 2 were then dissolved with
stirring and heating at 45.degree. C. 25 g of
1-ethyl-3-(3-dimethylaminoethyl)carbodiimide (EDAC) were then
added, the pump and the sonicator were turned on and the system was
kept between 40 and 50.degree. 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% Na.sub.2CO.sub.3, previously prepared by
dissolving 150 g of Na.sub.2CO.sub.3 in 7.5 L in DI water. Vigorous
mechanical stirring for at least 2 hours hydrolyzed urea related
byproducts. The solution was neutralized with 6 N HCl while the
temperature was kept at 20-25.degree. C. by passing the solution
through a cooled, jacketed flow cell.
Example 4
Alkaline Ultrafiltration of HyPE
[0476] 2.25 kg of NaHCO.sub.3 was dissolved in 150 L of 0.1%
chlorocresol solution, prepared by dissolving 150 g of chlorocresol
in 150 L of DI wate. By means of valves, the closed reaction system
was diverted so that the digested, neutralized HyPE solution of
Example 3 was pumped from the round bottom flask to the centrasette
system. By means of constant volume diafiltration with a 10 kDa
Omega TFF membrane, 150 L of 1.5% NaHCO.sub.3 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 .about.13.2 min and the
HOBT peak at .about.17.2 min. The solution was neutralized with 6 N
HCl while the temperature was kept at .about.20-25.degree. C. by
passing the solution through a cooled, jacketed flow cell.
Example 5
Extraction of HyPE
[0477] An extraction solution was made by mixing 3 L of
dichloromethane, 3 L of ethanol and 2.25 L of methanol. 7.5 L of
the extraction solution was added to a round bottom flask
containing 3 L of crude HyPE solution of Example 4. This was
stirred vigorously for 15 minutes after which time it was allowed
to stand for 45 min. The lower dichloromethane layer was removed.
By means of constant volume diafiltration the solution was washed
with 100 L of DI water to remove the methanol and ethanol. GPC
analysis was performed to ensure the disappearance of peaks at
.about.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
[0478] 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.degree. C. for 5 minutes, sonicated for 5 minutes and
filtered through a 0.2 micron syringe filter. 2.times. 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
.mu.M (2.times. working solution) in CM. The vehicle control
solution was CM. A 1 mg/ml solution of LPS (made in 1.times.PBS)
was diluted in CM to 10 .mu.g/ml. RAW 264.7 cells were grown for XX
passages (subculture every 3-4 days) in CM at 37.degree. C. with 5%
CO.sub.2. 0.5 ml of cells at 1.times.106 cells/ml was plated in
24-well tissue culture plates. Cells were allowed to adhere for 30
minutes at 37.degree. C. with 5% CO.sub.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.degree. C. with 5% CO.sub.2 prior to LPS treatment. 110 .mu.l
of CM was added to the -LPS plates. 110 .mu.l of 10 .mu.g/ml LPS
was added to the +1 .mu.g/ml LPS plates. The plates were incubated
for 24 hours at 37.degree. C. with 5% CO.sub.2.
Example 7
Supernatant Harvesting/XTT Assay
[0479] Cell culture supernatants were collected after 24 hours of
LPS treatment and stored at -30.degree. C. until assayed. 400 .mu.l
of media was left in each cell culture well for the XTT assay. 400
.mu.l of media was added to a cell-free culture well for use as a
blank in the XTT assay. 200 .mu.l of activated XTT reagent was
added to each well. Plates were incubated for 1 hour at 37.degree.
C. with 5% CO.sub.2. 100 .mu.l was removed from each well and read
at 450 nm (630 nm correction) using a ThermoMax microplate reader
(Molecular Devices, Sunnyvale, Calif.). XTT data relating to high
molecular weight HyPE compositions are shown in FIG. 5 and FIG. 6.
XTT data relating to low molecular weight HyPE compositions are
shown in FIG. 22 and FIG. 23.
Example 8
Cytokine/Chemokine Assays
[0480] Cell culture supernatants were assayed for IL-6, TNF-.alpha.
and IP-10 using a Luminexbased assay according to the
manufacturer's instructions. Data were collected using a Luminex
100 (Luminex Corporation, Austin, Tex.). Standard curves were
generated using a 5-parameter logistic curve-fitting equation
weighted by 1/y (StarStation V 2.0; Applied Cytometry Systems,
Sacramento, Calif.). Each sample reading was interpolated from the
appropriate standard curve. Calculated concentrations were
multiplied by the appropriate dilution factor when necessary.
TNF-.alpha. data relating to high molecular weight HyPE
compositions are shown in FIG. 7, FIG. 8 and FIG. 15. TNF-.alpha.
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.
[0481] Cell culture supernatants were assayed for PGE.sub.2 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.
PGE.sub.2 data relating to high molecular weight HyPE compositions
are shown in FIG. 13, FIG. 14 and FIG. 18. PGE.sub.2 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
[0482] 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 .mu.m filter (PALL) before drying to achieve
microbial purity.
Example 10
SEC-MALS Determination of Molecular Weight
[0483] 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.times.7.5 mm, 8 .mu.m; Agilent Technologies) columns connected
in series and thermostated at ambient temperature. Injection volume
was 100 .mu.l. Eluent (0.1 M sodium phosphate buffer pH 7.5) was
monitored using a DAWN-EOS multi-angle laser light scattering
photometer (18-angle, Wyatt Technologies Corporation) and a
refractive index detector rEX Optilab (Wyatt Technologies
Corporation). Data acquisition and molecular weight calculations
were performed using the ASTRA V software, Version 5.3.2.15. The
flow rate of mobile phase was maintained at 0.8 ml/min. The
specific refractive index increment (dn/dc) of 0,155 mg/ml was used
for sodium hyaluronate.
[0484] 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 .mu.m, 25 mm diameter,
Whatman) and analysed by HPLC system.
[0485] 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.
[0486] In essence, light scattering measurements automatically
provide a column calibration curve for every sample, obviating
time-consuming, conformation dependent calibration procedure.
[0487] 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.
[0488] The weight-average molecular weight of hyaluronan was
verified by measurements of dextran standard.
[0489] The determined molecular weight and polydispersity value for
low molecular weight hyaluronic acid were 7.86.times.103 g/mol and
1.32 Mw/Mn, respectively. The chromatogram and distribution diagram
are stated in FIG. 19 and FIG. 20 whereas red line pertains to
light scattering signal and blue line to refractive index signal.
FIG. 21 illustrates the UV spectrum.
Example 11
Preparation of HyPE from 9.54 kD Hyaluronic Acid
[0490] MES buffer was prepared by dissolving 14.5 g of MES in 75 mL
of DI-H.sub.2O 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.sub.2O, 60 mL MES buffer, 12 mL of
tert-butanol. The pH was adjusted to 6.4 with 4N NaOH. 15.1 g of HA
was dissolved in 350 mL of DI-H.sub.2O. 1.25 g or DPPE was
dissolved in 440 mL of tert-butanol and 90 mL DI-H.sub.2O with
heating to 55 deg C. The solutions of HA and HOBT were warmed to 35
deg C. and mixed. The DPPE solution, at 50 deg C. was then added to
afford a clear solution. This was allowed to cool to 43 deg C.,
when it was added to the flask and circulated through the
sonoreactor system (FIG. 36). Some component of the reaction
mixture came out of solution and it was necessary to heat the
reaction mixture to 49 deg C. with sonication to form a clear
solution. 12.5 g of EDAC was added as a powder to the reaction
mixture at a temperature of 45 deg C. Sonication began with a power
of 180 watts. The reaction was monitored by GPC as shown in FIGS.
37-38 and because the extent of agglomeration, as observed by the
ratio of the area of the first peak to that of the second continued
to increase, the reaction was allowed to continue beyond the normal
3 h and was continued the next day. The sonication was turned off
and the reaction mixture was filtered through a 0.45 .mu.m 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% NaHCO.sub.3 to remove residual organic
solvents. The pH was then increased by slow addition of 2%
Na.sub.2CO.sub.3 to pH 9.2. The solution was stirred for 1 hour at
room temperature. After further washing with 30 L of 1.5%
NaHCO.sub.3 the peat at .about.12.5 min had disappeared and the
solution was washed with 30 L of DI-H.sub.2O 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-H.sub.2O. The solution
was concentrated to 1 L and filtered through a 0.2 .mu.m filter
into a lyoguard container and placed in the lypholizer. It was
frozen by lowering the shelf temperature to -70 deg C. When frozen,
vacuum was applied (14 mT) and the shelf temperature was raised to
30 deg C. Five days later 6.134 g of HyPE was recovered with a
water-corrected weight of 5.2 g which corresponds to a 42% yield
based on 12.5 g (water corrected) of HA. Total phosphorus was found
to be 0.28% (dry basis). By LC/MS assay, 1,456 ppm of free EDU were
found and after exposure to NaOH 12,557 ppm total EDU was found. No
HOBT was detected and MES was less than 80 ppm. GPC of the final
product is shown in FIG. 39 and NMR data are shown in FIG. 40.
[0491] 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.
* * * * *