U.S. patent application number 09/845915 was filed with the patent office on 2001-11-01 for water soluble lipidated arabinogalactan.
This patent application is currently assigned to The University of Montana. Invention is credited to Richards, Geoffrey N..
Application Number | 20010036933 09/845915 |
Document ID | / |
Family ID | 25033934 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036933 |
Kind Code |
A1 |
Richards, Geoffrey N. |
November 1, 2001 |
Water soluble lipidated arabinogalactan
Abstract
Arabinogalactan compositions are provided which are useful in a
wide variety of different biomedical applications. In one
embodiment, water soluble lipidated arabinogalactans are provided
which include arabinogalactan with a limited proportion of
lipophilic groups, such as long-chain hydrocarbon groups,
covalently attached to free hydroxyl groups on the arabinogalactan.
The lipidated arabinogalactans are water soluble and biocompatible
and are useful for a wide variety of different biomedical
applications. The lipidated arabinogalactans can be used, for
example, to inhibit cell adhesion, and to inhibit infection or
inflammation. The lipidated arabinogalactans further may be used as
adjuvants, to inhibit metastasis, and in other therapeutic
applications.
Inventors: |
Richards, Geoffrey N.;
(Missoula, MT) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55401
US
|
Assignee: |
The University of Montana
|
Family ID: |
25033934 |
Appl. No.: |
09/845915 |
Filed: |
April 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09845915 |
Apr 30, 2001 |
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09174232 |
Oct 16, 1998 |
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6258796 |
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09174232 |
Oct 16, 1998 |
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08754225 |
Nov 20, 1996 |
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Current U.S.
Class: |
514/54 ;
536/123 |
Current CPC
Class: |
A61K 47/61 20170801;
C08B 37/006 20130101 |
Class at
Publication: |
514/54 ;
536/123 |
International
Class: |
C08B 037/00; A61K
031/715 |
Claims
What is claimed is:
1. A compound comprising arabinogalactan covalently attached to a
lipophilic group, wherein the compound is water soluble.
2. The compound of claim 1 wherein the solubility of the compound
in water is greater than about 0.1%.
3. The compound of claim 1 wherein the arabinogalactan is
ultrarefined.
4. The compound of claim 3 wherein the arabinogalactan is isolated
from a tree of the genus Larix.
5. The compound of claim 1 wherein the arabinogalactan is selected
from the group consisting of naturally occurring arabinogalactan or
portions thereof, and chemically or biochemically modified
arabinogalactan or portions thereof.
6. The compound of claim 1 wherein the lipophilic group is a long
chain hydrocarbon.
7. The compound of claim 6 wherein the compound comprises
arabinogalactan covalently attached to an aliphatic acid having the
formula HOOC[CH.sub.2].sub.nCH.sub.3, wherein n is between 1 and
90.
8. The compound of claim 6 wherein the compound comprises
arabinogalactan covalently attached to an aliphatic acid having the
formula HOOC[CH.sub.2].sub.nCH.sub.3, wherein n is between 4 and
30.
9. The compound of claim 8 wherein the degree of substitution of
the lipophilic groups in a sample of the compound is between 0.01
to 15%.
10. The compound of claim 1 wherein the lipophilic group is
selected from the group consisting of fat soluble vitamins,
phytosterols, terpenoids, phospholipids, glycerols, and natural or
synthetic fats.
11. An aqueous system further comprising a second compound and an
effective amount of the compound of claim 1 to enhance the water
solubility of the second compound.
12. An aqueous solution comprising the compound of claim 1 in an
amount effective to form a stable emulsion with a non-aqueous
second phase.
13. A composition comprising the compound of claim 1 in a
pharmaceutically acceptable carrier.
14. The composition of claim 13 wherein the lipophilic group
comprises an antineoplastic agent.
15. A composition comprising an antigen and an effective amount of
the compound of claim 1 to serve as an adjuvant in a
pharmaceutically acceptable vehicle.
16. The composition of claim 15 wherein the antigen is a
protein.
17. A method for treating diseases in animals and humans caused by
infectious organisms comprising administering an effective amount
of a water soluble compound, comprising arabinogalactan covalently
attached to a lipophilic group, to alleviate the disease.
18. The method of claim 17 wherein the inflammation is associated
with neutrophil-endothelial cell adhesion.
19. A method for treating diseases in animals and humans caused by
inflammation, the method comprising administering an effective
amount of a water soluble compound, comprising arabinogalactan
covalently attached to a lipophilic group, to alleviate the
disease.
20. A method for inhibiting metastasis in animals or humans
comprising administering an effective amount of a water soluble
compound, comprising arabinogalactan covalently attached to a
lipophilic group, to alleviate the disease.
21. An improvement in a method for producing an immune response in
an animal or human to an antigen, the method comprising
administering the antigen to the animal or human together with a
compound comprising arabinogalactan covalently attached to a
lipophilic group.
22. The improvement of claim 21 wherein the antigen is a protein.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is generally in the area of methods
for making polysaccharides which include attached lipophilic
groups, but which remain water-soluble.
[0002] Modified polysaccharides have been isolated from natural
sources or synthesized chemically and their biological activities
studied. Starches containing bound protein and lipid groups have
been isolated from plant components such as seeds. See, e.g.,
Tharanathan et al., Starch, 42:247-251 (1990).
[0003] Fatty acid esters of polysaccharides have been produced by
treatment of the polysaccharides with fatty acid chlorides or
anhydrides or by ester exchange, and the alkyl ethers of
polysaccharides have been produced by several methods (Methods in
Carbohydrate Chemistry, vol. 11 (1963) Ed., R. L. Whistler and M.
L. Wolfrom, Academic Press, New York; Section VI and V). However,
most previous studies of this type have involved high degrees of
derivatization with relatively short hydrocarbon chains, such as
acetate esters or methyl ethers. Highly derivatized products of
this type are generally water-insoluble. JP 60233560 to Fujirebio
K.K. discloses a method of measuring lipase activity using as the
enzyme substrate a water soluble fatty acid ester of a low
molecular weight oligosaccharide such as dextran. JP 87209721 to
Sugiyama Industrial Chemical Institute discloses the preparation of
water soluble fatty acid esters of hydrolyzed starch for use as
emulsifiers and detergents.
[0004] The synthesis of cyclodextrin derivatives, modified by the
attachment of fatty acids or alcohols, which may be used as bile
acid absorption agents, is described in DE 4 136 325 to Ahlers et
al. PCT WO 95/12620 to Alpha-Beta Technology, Inc. discloses
derivatized polysaccharide bile acid sequestrants for reducing
cholesterol which include a hydrophobic, cationic ligand coupled to
a polysaccharide substrate.
[0005] Acetylated mannans "acemannans" are long-chain polydispersed
beta-1,4-linked mannan polymers interspersed with O-acetyl groups
which are isolated from the Aloe vera leaf. Acemannans have been
reported to have antitumor activity and to be useful as adjuvants.
Harris et al., Mol. Biother. 3:207-213 (1991). Acetylated mannans
also have been reported to be useful for regulating blood
cholesterol levels; for reducing inflammation and infection; as an
immunostimulant; and as an antiviral. U.S. Pat. Nos. 5,441,943 and
5,308,838 to Carpenter et al.; PCT WO 93/08810 to Carrington Lab,
Inc.; and U.S. Pat. Nos. 5,118,673 and 5,106,616 to Carpenter et
al. Acemannans also have been shown to induce human cytokines.
Marshall et al., Abstract presented at the American Academy of
Allergy and Immunology, Chicago, Ill., March, 1993.
[0006] It is an object of the invention to provide lipid-modified
polysaccharides which are water-soluble, biocompatible and can be
used in a variety of different biomedical applications. It is a
further object of the invention to provide water soluble,
lipid-modified forms of arabinogalactan which can be used in
different applications, for example, to promote the formation of
stable emulsions, to increase drug solubility, and to serve as
adjuvants. It is another object of the invention to provide methods
for making and using a range of such modified forms of
arabinogalactan in different biomedical applications.
SUMMARY OF THE INVENTION
[0007] Arabinogalactan compositions are provided which are useful
in a wide variety of different biomedical applications. In one
embodiment, water soluble lipidated arabinogalactans are provided
which include arabinogalactan with a limited proportion of
hydrophobic groups, such as long-chain hydrocarbon groups,
covalently attached to free hydroxyl groups on the arabinogalactan.
The lipidated arabinogalactans are water soluble and biocompatible
and are useful in a wide variety of different biomedical
applications. The lipidated arabinogalactans can be used, for
example, to inhibit cell adhesion, and to inhibit infection or
inflammation. The lipidated arabinogalactans further may be used as
adjuvants, to inhibit metastasis, and in other therapeutic
applications.
DETAILED DESCRIPTION OF THE INVENTION
[0008] Water soluble lipidated forms of arabinogalactan are
provided which are useful in a wide variety of different
applications, particularly biomedical applications. Water soluble
lipidated arabinogalactan may be used for example, to form stable
emulsions, to increase the solubility of sparingly water soluble
drugs, to inhibit cell adhesion, for metastasis control, as a bile
acid sequestrant, or as an adjuvant. The lipidated arabinogalactan
includes a limited proportion of lipophilic groups, such as long
chain hydrocarbons bonded to the polysaccharide, e.g., by ether or
by ester linkages, to modify its biological activity, while
advantageously retaining water solubility properties. Due to the
water solubility and low toxicity, the lipidated arabinogalactans
are useful in a wide range of therapeutic applications.
[0009] Arabinogalactan
[0010] Arabinogalactan ("AG") is a water-soluble polysaccharide
which can be isolated from trees of the genus Larix, particularly
Larix occidentalis (western larch). Arabinogalactan may constitute
up to 35% of the total heartwood of some species. Stout, "Larch
Arabinogalactan " in Industrial Gums, R. L. Whistler Ed., Academic
Press, New York, pp. 307-310, 1959. It is highly water soluble and
can be purified from larch chips.
[0011] As used herein, the term "arabinogalactan," unless otherwise
specified, includes naturally occurring or synthetic
arabinogalactan, portions of arabinogalactan, such as degradation
products, and chemically or biochemically modified arabinogalactan
or portions thereof which have been modified using methods
available in the art.
[0012] In one preferred embodiment, ultrarefined arabinogalactan is
used to form the lipidated water soluble arabinogalactan
compositions. Methods for the preparation of ultrarefined
arabinogalactan are disclosed in U.S. Pat. No. No. 5,116,969, the
disclosure of which is incorporated herein by reference.
Ultrarefined arabinogalactan of greater than 95%, or optionally,
greater than 99.9% purity (Larex UFT.TM. ) is available from Larex,
International, St. Paul, Minnesota. As defined herein "ultrarefined
arabinogalactan " refers to arabinogalactan, isolated from a plant
source such as trees of the genus Larix, with a purity greater than
95%. In a preferred embodiment, the molecular weight of the
ultrarefined arabinogalactan is between about 10,000 and 30,000
daltons (by size exclusion chromatography with pullulan
reference).
[0013] Arabinogalactan from Larix trees is useful since it is
extremely water-soluble, occurs naturally with a very narrow
molecular weight distribution, and is highly branched and thus not
subject to viscosity problems. Arabinogalactan also is highly
biocompatible and is non-toxic.
[0014] Water Soluble Lipidated Arabinogalactan
[0015] Structure
[0016] Lipidated arabinogalactan molecules which include attached
lipophilic groups but which are water soluble are provided.
Preferably the solubility of the lipidated arabinogalactan compound
is at least about 0.1%. As used herein, the phrase "lipidated
arabinogalactan " refers to arabinogalactan covalently attached to
a lipophilic group. Preferred lipophilic groups include long chain
hydrocarbon groups. Other lipophilic groups include steroids,
terpenes, fat soluble vitamins, phytosterols, terpenoids,
phospholipids, glycerols, and natural or synthetic fats. The
lipophilic group may be attached to the arabinogalactan either
directly or via a linking group. For example, the free hydroxy
groups on the arabinogalactan may be linked to hydrocarbon chains
via an ether or ester linkage.
[0017] The water soluble lipidated arabinogalactan is defined in
one embodiment by the formula:
AG--R--L
[0018] where:
[0019] AG is arabinogalactan;
[0020] L is a lipophilic group, preferably a branched or straight
chain saturated or unsaturated hydrocarbon including between about
one to 90 carbons, for example between about 4 and 30 carbons, or
alternatively between about 10 and 20 carbons, and is most
preferably a C.sub.4-C.sub.30 straight chain saturated hydrocarbon,
for example C.sub.10-C.sub.18 straight chain hydrocarbon; and
[0021] R is a group linking the AG to the lipophilic group,
preferably an ester or ether bond between a hydroxyl on the AG and
a carboxyl or hydroxyl on the lipophilic group. The R group linking
AG and L also may be or contain functional groups such as esters,
thioesters, ethers, sulfonic acid esters, carbonates,
thiocarbomates, carbamates and thiocarbamates.
[0022] The water soluble lipidated atabinogalactan is formed by
attaching lipophilic groups to a portion of the free hydroxyl
groups on the arabinogalactan. For example, long chain hydrocarbon
carboxylic acid molecules, having the formula
HOOC[CH.sub.2].sub.nCH.sub.3 where n is between 4 and 30, may be
attached to the free hydroxyl groups on the arabinogalactan.
Alternatively, n may be less than 4, for example, between 1 and 3,
or greater than 30, for example between 30 and 90. Additionally,
the arabinogalactan sample may include different attached
lipophilic groups.
[0023] The degree of substitution of arabinogalactan preferably is
between about 0.01% to 15%, for example, 0.1% to 5%, or optionally
between about 1-3%. As used herein, the phrase "degree of
substitution" or "DS" refers to the percentage of glycose units in
an arabinogalactan sample which carry a lipophilic group, assuming
that all of the glycose units which are substituted are
monosubstituted. In a preferred embodiment, the esterification is
carried out in solution which promotes uniform distribution of
ester groups.
[0024] The degree of substitution of lipophilic hydrocarbon
molecules on the arabinogalactan can be designed and modified for
different applications. It is preferred that the degree of
substitution of hydrocarbon groups on the lipidated arabinogalactan
not be so great as to render the arabinogalactan non-water soluble.
In one preferred embodiment, the degree of substitution is as great
as possible without resulting in making the complex of the
arabinogalactan non-water soluble or poorly soluble in water. The
use of arabinogalactan is advantageous, because a relatively large
degree of substitution with lipophilic hydrocarbons is possible,
e.g., 0.1 to 5%, without loss of water solubility properties. The
water solubility is an important property of the lipidated
arabinogalactan. It is preferred that the water solubility of the
lipidated arabinogalactan be at least about 0.1%, for example, in
one preferred embodiment, between 2 and 40 g/mL. Long-chain esters
of polysaccharides previously prepared have been primarily
water-insoluble. More heavily lipidated AG palmitate is water
insoluble and inactive in certain applications for example as an
adjuvant.
[0025] Synthesis
[0026] The lipidated arabinogalactan may be formed using organic
chemistry reactions available in the art, such as esterification
reactions or etherication reactions, to couple lipophilic groups
such as hydrocarbons to the polysaccharide.
[0027] Esterification
[0028] The water-soluble lipidated arabinogalactan is formed in one
embodiment by the covalent attachment of long-chain hydrocarbons to
the arabinogalactan via an ester linkage. In the esterification
reaction, the arabinogalactan or derivative thereof is reacted with
an anhydride or acid chloride of a long chain carboxylic acid. In
the reaction, partial esterification of the free hydroxy groups in
the arabinogalactan with the long chain carboxylic acid occurs.
[0029] Fatty acids which can be reacted, for example, in anhydride
or acid chloride form, with arabinogalactan ("AG"), in the
esterification reaction include palmitate, stearate, and decanoate.
Exemplary compounds which can be formed include AG palmitate, AG
stearate and AG decanoate. The preferred degree of substitution for
the AG with the hydrocarbon fatty acids is between about 0.01 and
15%, and in one preferred embodiment is between 0.1 and 5%.
[0030] Etherification
[0031] The water soluble lipidated arabinogalactan also may be
formed in an etherification reaction. Partial ethers may be formed
by the reaction of a arabinogalactan, or a derivative thereof, with
long chain alkyl halides or epoxides under alkaline catalysis. For
example, the water-soluble dodecyl ether of arabinogalactan, with a
degree of subsitution ("DS") of 2.6% may be formed by reaction with
dodecyl iodide.
[0032] Adjuvants
[0033] In one embodiment, the lipidated arabinogalactan may be used
as an adjuvant to enhance the imnimunogenicity of an antigen, such
as a virus. The immunogenic composition including the antigen and
lipidated arabinogalactan can be administered by any method known
to those skilled in the art, that does not denature or inactivate
the antigen contained in the composition including oral,
transmembrane and transmucosal administration. Preferably, the
composition is administered parenterally (such as intravenously,
intramuscularly, intraperitoneally), most preferably
subcutaneously. The composition including a mixture of the antigen
and the lipidated arabinogalactan may be administered in
combination with suitable physiologically acceptable carriers known
to those skilled in the art, such as water or saline. The antigen
and lipidated arabinogalactan also may be administered separately,
or, in another embodiment may be covalently conjugated prior to
administration.
[0034] The antigen can be a cell, bacteria, or virus particle, or
portion thereof, a hormone, a growth factor or an immunogenic
synthetic, recombinant or naturally occurring protein or peptide.
Other examples include an influenza protein, tetanus toxoid, an HIV
protein, a hepatitis B protein and a neisseria gonorrhea
protein.
[0035] Lipidated arabinogalactan is useful as an adjuvant due to
its low toxicity. in comparison to many other available
immunoadjuvants. Many adjuvants, such as Freund's Complete
Adjuvant, are toxic. Freund's adjuvant for example, causes
granulomatous lesions in animals at the site of immunization and
may also cause the recipient of a vaccine to test positive for
tuberculosis, and therefore is only useful for animal research
purposes, not human vaccinations.
[0036] A preferred form of a water soluble lipidated
arabinogalactan for use as an adjuvant is arabinogalactan
substituted with a hydrocarbon group including between about 10 to
18 carbons, wherein the degree of substitution is about 2%. For
example, AG palmitate, with a degree of substitution of about 2%,
may be used as an adjuvant.
[0037] Increasing the Water Solubility of Drugs
[0038] The water soluble lipidated arabinogalactan may be used to
increase the water solubility of drugs which have low solubility in
water, by providing the drug in an aqueous solution of lipidated
arabinogalactan. For example, the concentration of the lipidated
arabinogalactan in the aqueous solution of the drug may be about
0.1 to 20%. The enhancement of the solubility of a sparingly water
soluble drug is described in Example 8.
[0039] The water solubility of any of a wide range of therapeutic
agents can be increased including small organic molecules proteins,
peptides, and nucleic acids. Exemplary drugs include drugs
effecting the nervous system, hormones, analgesics,
antiinflammatory agents, diuretics, antidiuretics, antianginal
agents, antihypertensive agents, antibiotics, antineoplastic
agents, immunomodulators, hematopoietic agents, steroids including
estrogens and progestins, and vitamins.
[0040] The water solubility of sparingly soluble antineoplastic
agents including alkylating agents, steroids, antimetabolites,
antimitotics, DNA intercalators, enzyme inhibitors, DNA synthesis
inhibitors, and lytic agents can be improved. Illustrative agents
include paclitaxel, mitomycin, cisplatin, flutamide, and other
lipophilic agents, as described, for example in Carter and
Livingston, Drugs Available to Treat Cancer, in Principles of
Cancer Treatment, Carter et al., Eds., Chapter 10, pp 111-145,
1982, McGraw-Hill, New York.
[0041] Cell Adhesion
[0042] The water soluble lipidated arabinogalactan can be used in
one embodiment to inhibit cell-adhesion, thereby to prevent or
reduce inflammation or infection.
[0043] Inhibition of Infection
[0044] The arabinogalactan compounds may be used to inhibit
infection of cells by invading microorganisms by interfering with
the binding of the microorganism. Microorganism adherence to and
subsequent invasion of cells during infection is mediated by the
binding of proteins on the surface of the pathogen to animal cell
surface oligosaccharides. Antiadhesive drugs operate to prevent
cell infection by binding to the proteins on the pathogen and
preventing the organism from binding to and infecting the cell,
with the result that the pathogen, such as a bacteria, is washed
away by natural processes which occur on mucosal surfaces for the
clearing of bacteria. Borman, Chemical and Engineering News, Jun.
28, 1993, pp. 27-34, the disclosure of which is incorporated herein
by reference.
[0045] Inhibition of Inflammation
[0046] In another embodiment, the arabinogalactan compounds may be
used to inhibit abnormal inflammation underlying many pathological
states such as rheumatoid arthritis, psoriasis, septic shock,
atherosclerosis, thrombosis, ischemia and reperfusion injury.
[0047] Leukocyte-endothelial cell interactions in the
anti-inflammatory response are mediated by complex signalling and
adhesion molecules as described, for example, in McEver, Current
Opinion in Cell Biology, 4:840-849 (1992), the disclosure of which
is incorporated herein by reference. In the anti-inflammatory
response, white blood cells are recruited to sites of inflammation
on endothelial cells lining the blood vessel wall. The
arabinogalactan compounds may operate by interfering with the
binding of the white blood cells to the endothelial cell surface of
the blood vessel wall. The water-soluble lipidated arabinogalactans
are especially useful in inhibiting the adhesion of neutrophils to
human endothelial cells. This can occur without interference with
the binding of lymphocytes with beneficial effects in treatment of
reperfusion injury for example in heart attacks, angioplasty or
graft rejection. The suppression of inflammatory response may also
be useful in treatment of back injuries.
[0048] Emulsifiers and Surfactants
[0049] The water soluble lipidated arabinogalactan may be used to
form stable emulsions, or may be used as surfactants.
[0050] Bile Acid Sequestrants for Reducing Cholesterol
[0051] The water soluble lipidated arabinogalactan may be used as
an agent for sequestering bile acids to reduce cholesterol. Bile
acid sequestrants can be used to treat hypercholesterolemia by
binding bile acids in the intestine after oral administration and
then carrying them through the small intestine and causing them to
be excreted. Water-soluble lipidated arabinogalactan may be
designed and synthesized with attached lipophilic groups, such as
hydrocarbon groups and then tested in vitro for bile acid binding
ability using assays available in the art. Water-insoluble
lipidated polysaccharides of high substitution (DS>20) have
previously been used for this purpose, as described in PCT WO
95/12620 to Alpha-Beta Technology, the disclosure of which is
incorporated herein by reference, however the use of water-soluble,
lipidated polysaccharides of a low degree of substitution was not
disclosed.
[0052] Metastasis Control
[0053] Arabinogalactan has been shown to reduce tumor metastases in
mice. as described in Hagmar et al., Cellular Pharmacology,
1:87-90, 1994, the disclosure of which is incorporated herein by
reference. Water-soluble lipidated arabinogalactan can be used to
enhance the effect of arabinogalactan in this application. The
lipidated arabinogalactan may be used to reduce metastasis of tumor
cells from different malignancies, including carcinomas, lymphomas,
and sarcomas, which can metastasize to distant sites through the
vasculature.
[0054] Advantages of Arabinogalactan
[0055] Arabinogalactan has unique advantages over other
polysaccharides for many applications including those mentioned
above. It is extremely water-soluble and its concentrated solutions
(e.g. 40%) have low viscosity. Thus when lipid groups are attached
to the molecule it can carry a higher proportion of such groups,
while maintaining water-solubility, compared, e.g., with starch or
cellulose derivatives. Also, the aqueous solutions of the lipidated
derivatives are not viscous and are therefore more biocompatible.
The unusually highly branched structure of the arabinogalactan also
offers exceptional steric accessibility for derivatization and
favorable geometry for the lipid derivatives in solution.
[0056] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLE 1
Assay of Adjuvant Activity of Lipidated Arabinogalactan
[0057] The adjuvant activities of the three arabinogalactan
compounds, arabinogalactan ("AG"), low substitution (DS 1.7%) AG
palmitate (water soluble), and high substitution (DS 7.3%) AG
palmitate (water insoluble) were compared. In the experiment, 25
.mu.g of ovalbumin ("O.A.") was injected into mice in a saline
solution together with 50, 10, 2 or 0 .mu.g (the control) of the
arabinogalactan compounds. The animals were prebled prior to
immunization; injected; bled on days 7 and 14; given a booster
injection on day 21; and bled on days 28 and 35 for antibody
determination. All three arabinogalactan compounds showed some
adjuvant activity in antibody formation in comparison to the
control. The low substitution AG palmitate had the highest adjuvant
activity, however it was 10 to 100 fold weaker than that of
comparable doses of a potent adjuvant, such as monophosphoryl lipid
A. However, the latter is potentially toxic, extremely expensive,
and being water-insoluble, requires complex formulation.
EXAMPLE 2
Inhibition of Cell Adhesion by Arabinogalactans
[0058] The effect of Compounds A-F, listed below in Table 1,
including arabinogalactan, and arabinogalactan with varying degrees
of substitution (DS) of long hydrocarbon chain fatty acids, on
leukocyte-endothelial cell adhesive interactions was evaluated:
1TABLE 1 A. AG (ultrarefined) B. AG stearate (DS 1.7%) C. AG
palmitate (DS 1.7%) D. AG decanoate (DS 2.6%) E. galactan from
fractional hydrolysis of AG (arabinose 1.5%) F. AG dodecyl ether
(DS 1.3%)
[0059] In Vitro Assay
[0060] The in vitro effects of Compound A-F listed in Table I on
leucocyte and endothelial cell adhesion under physiological shear
forces was examined as follows.
[0061] Human umbilical vein endothelial cells (HUVEC) were grown to
confluence on the internal surface of collagen type I coated
capillary tubes. Endothelial cells were stimulated for 4 hours with
rIL-.beta. (10 ng, Genzyme), which upregulates adhesion molecules
such as E-selectin within 4-6 hours. Neutrophils and lymphocytes
were collected from normal human donors by venipuncture and
separated from the blood on ficoll-histoplaque density gradients.
The endothelial cell-coated tubes were then integrated into the
closed-loop system. Leukocytes were infused into the assay system
at a concentration of 1.times.10.sup.6-5.times.10.s- up.6 cell/ml
in DMEM plus 20 mM HEPES. Shear forces of 1-3 dynes/cm.sup.2
(standard representation of blood flow) were applied via a variable
speed peristaltic pump, and interactions between the leukocytes and
endothelial cells were monitored by video-microscopy. Comparisons
were made of the binding of leukocytes and endothelial cells
treated with the 6 compounds and untreated controls.
[0062] For lymphocytes, rolling interactions were established for
ten minutes at which point the compounds were infused at increasing
concentrations: 1 .mu.g/ml, 10 .mu.g/ml, and 100 .mu.g/ml added at
5 minute time intervals. Interactions, from time 0 were observed
and recorded to video tape for off-line analysis.
[0063] To observe the effects of the compounds on neutrophil
adhesive interactions, the neutrophils were infused and allowed to
establish rolling for two minutes before the addition of the
compounds. The compounds were added in increasing 1 .mu.g/ml, 10
.mu.g/ml, and 100 .mu.g/ml final concentrations at 2 minute
intervals. The interactions were recorded to video tape.
[0064] Results
[0065] Lymphocytes
[0066] Binding curves illustrated that Compounds A-F had no
significant effect on lymphocyte accumulation on the activated
HUVEC monolayer. Averaged values of three experiments were used to
generate a mean value binding curve and a smoothed fitted binding
curve that showed no significant alteration of lymphocyte adhesion
or rolling by these compounds.
[0067] Thus, compounds A-F did not significantly effect
lymphocyte-HUVEC adhesive rolling interactions. Rolling
interactions of lymphocytes involve multiple adhesion receptor
ligand interactions including L- and E-selectin as well as
.alpha.4/.beta.1 and VCAM-1. Adhesive interactions by these
pathways were not significantly altered in these assays.
[0068] Neutrophils
[0069] Binding curves for compounds A-F in the neutrophil-HUVEC
adhesion study were obtained in addition to control binding curves.
Each experiment was repeated three times. Compound A (repeated
three times) caused an approximate 75% reduction in neutrophil
adhesion during the time course but did not reduce binding to
baseline even at concentrations of 10 .mu.g/ml or 100 .mu.g/ml.
Compounds B and C, in both experimental repeats showed a consistent
90% or greater reduction in neutrophil adhesion beginning
immediately upon addition of the compounds at their lowest
concentration (1 .mu.g/ml).
[0070] Thus, Compounds A, B, and C significantly blocked neutrophil
rolling on activated HUVEC, while Compounds D, E, and F had no
measurable effect. Rolling interactions of neutrophils involve
multiple adhesion receptor ligand interactions including L- and
E-selectin. These adhesive interactions were those most likely to
have been disrupted by compounds A, B, and C. The concentration (1
.mu.g/ml) at which B and C were effective in blocking
neutrophil-HUVEC adhesion are equivalent to the minimal
concentration at which mAb blockers of E-selectin are fully
effective in preventing adhesion.
[0071] The arabinogalactan-containing compounds thus appear to
selectively block neutrophil-endothelial cell adhesion at extremely
low levels without blocking lymphocyte adhesion. Thus, the
compounds are potentially useful in blocking acute inflammatory
associated pathology generated by neutrophil recruitment to sites
of potential ischemic reperfusion injury, or for the treatment of
other disease processes that result in neutrophil recruitment such
as burn associated pathology, heart attack, angioplasty, graft
rejection and acute respiratory diseases.
EXAMPLE 3
Synthesis of Water-Soluble and Water-Insoluble AG Palmitates
[0072] Dry, ultrarefined AG (10.28 g) was dissolved with heating in
dry dimethyl sulfoxide (50 mL) at 100.degree. C. Dry pyridine (25
mL) was added, followed by palmitic anhydride (3.0 g) and stirred
until solution was complete. After 90 min. at 100.degree. C., the
brown solution was cooled to room temperature, ice (5 g) was added
with stirring and the resultant solution then added dropwise with
stirring to redistilled isopropanol (500 mL). The resultant white
powder precipitate was left overnight and then centrifuged, and the
solid washed twice with isopropanol. The solid was dissolved in
water (120 mL), concentrated to about 100 mL and then shaken with
chloroform (100 mL). Three layers formed. The lower layer was
separated and discarded and the upper two layers (the middle layer
being a viscous emulsion) further washed twice with chloroform with
intermdediate centrifugation. The upper layer was then dialyzed
against running water for three days and freeze dried to yield 7.70
g of an off-white solid. The palmitate content was calculated from
the PMR spectrum in D.sub.20 solution by relating the palmitate
CH.sub.2 signal at 1.26 ppm (s, 26 H) to the total glycose CH
integration from 4.3 to 3.1 ppm, assuming that this represented 6 H
per glycose unit. This integration indicated that 1.70% of the
glycose units carried a palmitate ester group.
[0073] The semi-solid middle layer from the above solvent
separation was further washed with water, centrifuged and freeze
dried to a white, water-insoluble product (3.3g), PMR analysis in
d6 dimethyl sulfoxide at 70.degree. C. with integration as above,
indicated that 7.3% of the glycose units carried a palmitate ester
group. This product was partly, but not completely soluble in
boiling water.
EXAMPLE 4
Synthesis of Water-soluble AG Palmitate
[0074] The preparation described in Example 3 was repeated, using
less palmitic anhydride (1 g) and heating for 60 min. only. The
product, after addition of ice, was added dropwise with stirring to
isopropanol (500 mL). The resultant precipitate was filtered,
washed with isopropanol, then boiled with several portions of
chloroform, finally filtered and dried at 50.degree./1 mm. The
product (8.12 g from 8.99 g of AG) was completely soluble in water
at room temperature and PMR analysis indicated that 0.9% of the
glycose units carried a palmitate ester group.
EXAMPLE 5
Synthesis of Water-soluble AG Stearate
[0075] The procedure of Example 4 was repeated with stearic
anhydride (2.0g). Analysis of the final water-soluble product as
above, indicated that 1.7% of the glycose units carried a stearate
ester.
EXAMPLE 6
Synthesis of the Dodecyl Ether of AG
[0076] Dry AG (2.0g) was dissolved with heating in dry dimethyl
sulfoxide (5 mL), cooled to room temperature and stirred for 15
min. with powdered sodium hydroxide (1 g). Dodecyl iodide (0.2 mL)
was added, and the mixture stirred for 30 min. at room temperature.
After filtration, the solution was poured into isopropanol (100 mL)
with stirring, and the resultant white precipitate washed with
isopropanol containing a little acetic acid. After further washing
with isopropanol, the white solid was dissolved in water and
dialyzed for several days before freeze drying. The product was
analyzed by PMR in D.sub.2O solution as in Example 3 to show that
1.3% of the glycose units carried a dodecyl ether group.
EXAMPLE 7
Formation of Oil-water Emulsions with Water-Soluble Lipidated
Arabinogalactan
[0077] An aqueous solution of AG palmitate (2 mL, 1.0% solution, DS
1.7%) was mixed with safflower oil (4 g) and vigorously shaken. A
uniform viscous emulsion formed and no separation of phases was
evident after several days at room temperature.
[0078] When a 1% solution (2 mL) of the same AG palmitate was
shaken with hexane (1 mL) the upper phase became extremely viscous
and opaque and remained in this state for several days.
EXAMPLE 8
Increased Solubility of Progesterone in Aqueous AG Decanoate
[0079] Progesterone was heated with water at 60.degree. C. for 10
min, then held at 25.degree. C. for 20 h. The filtered solution was
analyzed by UV absorption at .lambda.max 250 nm to show that the
saturated solution contained 8.8.times.10.sup.-4% dissolved
progesterone.
[0080] When the above experiment was repeated with a 1% aqueous
solution of AG decanoate (DS 2.59%) the saturated solution
contained 4.7.times.10.sup.-3% progesterone. In the UV analysis of
the AG decanoate solution of the progesterone, the appropriate AG
decanoate solution without progesterone was used as a spectrometer
blank.
* * * * *