U.S. patent application number 12/482986 was filed with the patent office on 2009-12-24 for compositions and methods for modulation of vascular structure and/or function.
This patent application is currently assigned to Marine Polymer Technologies.. Invention is credited to Sergio Finkielsztein, John N. Vournakis.
Application Number | 20090318383 12/482986 |
Document ID | / |
Family ID | 25121948 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090318383 |
Kind Code |
A1 |
Vournakis; John N. ; et
al. |
December 24, 2009 |
COMPOSITIONS AND METHODS FOR MODULATION OF VASCULAR STRUCTURE
AND/OR FUNCTION
Abstract
The present invention relates to compositions comprising
semi-crystalline .beta.-1-4-N-acetylglucosamine polymers (p-GlcNac)
and methods utilizing such polymers modulation of vascular
structure and/or function. The compositions and methods disclosed
are useful for stimulating, in a p-GlcNac concentration-dependent
manner, endothelin-1 release, vasoconstriction, and/or reduction in
blood flow out of a breached vessel, as well as for contributing to
or effecting cessation of bleeding. The methods of the present
invention comprise topical administration of materials comprising
semi-crystalline p-GlcNac polymers that are free of proteins, and
substantially free of single amino acids as well as other organic
and inorganic contaminants, and whose constituent monosaccharide
sugars are attached in a .beta.-1-4 conformation.
Inventors: |
Vournakis; John N.;
(Charleston, SC) ; Finkielsztein; Sergio;
(Chestnut Hill, MA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Marine Polymer
Technologies.
|
Family ID: |
25121948 |
Appl. No.: |
12/482986 |
Filed: |
June 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11542983 |
Oct 3, 2006 |
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12482986 |
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10194740 |
Jul 12, 2002 |
7115588 |
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11542983 |
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09781182 |
Feb 12, 2001 |
7041657 |
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10194740 |
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Current U.S.
Class: |
514/54 ;
536/55.1 |
Current CPC
Class: |
C08B 37/0027 20130101;
A61K 9/0014 20130101; A61K 31/726 20130101; A61K 31/722 20130101;
A61K 9/7007 20130101; C08B 37/003 20130101; A61P 15/00 20180101;
A61K 31/715 20130101; A61K 31/737 20130101; Y10S 514/93
20130101 |
Class at
Publication: |
514/54 ;
536/55.1 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61P 9/00 20060101 A61P009/00; C08B 37/00 20060101
C08B037/00 |
Claims
1. A method for achieving transient, localized, modulation of
vascular structure and/or function, comprising: topically
administering to a patient in need of said modulation, a sufficient
amount of material comprising semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymers, wherein the
polymers are free of protein, substantially free of other organic
contaminants, and substantially free of inorganic contaminants, and
wherein said administering induces at least one transient,
localized physiological response selected from the group consisting
of stimulation of endothelin-1 release, vasoconstriction, and
reduction in blood flow out of a breached vessel, whereby the
patient experiences transient, localized modulation of vascular
structure and/or function.
2. The method of claim 1, wherein the physiological response
comprises stimulation of endothelin-1 release.
3. The method of claim 2, wherein the endothelin-1 is released from
vascular endothelial cells.
4. The method of claim 1, wherein the physiological response
comprises vasoconstriction.
5. The method of claim 1, wherein the physiological response
comprises reduction in blood flow out of a breached vessel.
6. The method of claim 1, wherein the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer comprises about 50 to about 150,000
N-acetylglucosamine monosaccharides covalently attached in a
.beta.-1.fwdarw.4 conformation, and said polymer has a molecular
weight of about 10,000 daltons to about 30 million daltons.
7. The method of claim 6, wherein the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer comprises about 50 to about 50,000
N-acetylglucosamine monosaccharides covalently attached in a
.beta.-1.fwdarw.4 conformation, and said polymer has a molecular
weight of about 10,000 daltons to about 10 million daltons.
8. The method of claim 7, wherein the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer comprises about 50 to about 10,000
N-acetylglucosamine monosaccharides covalently attached in a
.beta.-1.fwdarw.4 conformation, and said polymer has a molecular
weight of about 10,000 daltons to about 2 million daltons.
9. The method of claim 8, wherein the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer comprises about 50 to about 4,000
N-acetylglucosamine monosaccharides covalently attached in a
.beta.-1.fwdarw.4 conformation, and said polymer has a molecular
weight of about 10,000 daltons to about 800,000 daltons.
10. The method of claim 6, wherein the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises at
least one N-acetylglucosamine monosaccharide that is deacetylated,
and wherein at least 40% of said N-acetylglucosamine
monosaccharides are acetylated.
11. The method of claim 1, wherein the patient is a human.
12. The method of claim 1, wherein the material is in the form of a
gel, sponge, film, membrane, foam, spray, emulsion, suspension, or
solution.
13. The method of claim 1, wherein the material is applied directly
to a blood vessel.
14. The method of claim 1, wherein the vascular structure is a
blood vessel selected from the group consisting of capillary, vein,
and artery.
15. The method of claim 14, wherein the blood vessel is a breached
blood vessel.
16. The method of claim 15, whereby the patient experiences
cessation of bleeding.
17. The method of claim 1, wherein the extent of the transient,
localized modulation of vascular structure and/or function is
substantially proportional to the amount of semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine administered.
18. A biodegradable, non-barrier-forming material comprising
semi-crystalline poly-.beta.-1.fwdarw.4 N-acetylglucosamine
polymers comprising about 50 to about 150,000 N-acetylglucosamine
monosaccharides covalently attached in a .beta.-1.fwdarw.4
conformation, free of protein, substantially free of other organic
contaminants, substantially free of inorganic contaminants, and
having a molecular weight of about 10,000 daltons to about 30
million daltons.
19. The material of claim 18, wherein the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises about
50 to about 50,000 N-acetylglucosamine monosaccharides covalently
attached in a .beta.-1.fwdarw.4 conformation and has a molecular
weight of about 10,000 daltons to about 10 million daltons.
20. The material of claim 18, wherein the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises about
50 to about 10,000 N-acetylglucosamine monosaccharides covalently
attached in a .beta.-1.fwdarw.4 conformation and has a molecular
weight of about 10,000 daltons to about 2 million daltons.
21. The material of claim 18, wherein the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises about
50 to about 4,000 N-acetylglucosamine monosaccharides covalently
attached in a .beta.-1.fwdarw.4 conformation and has a molecular
weight of about 10,000 daltons to about 800,000 daltons.
22. The material of claim 18, wherein the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises at
least one N-acetylglucosamine monosaccharide that is deacetylated,
and wherein at least 40% of said N-acetylglucosamine
monosaccharides are acetylated.
23. The material of claim 18, wherein the material is a gel,
sponge, film, membrane, foam, spray, emulsion, suspension, or
solution.
24. A method for treating a patient having a vascular disorder,
comprising: topically administering to a patient in need of such
treatment, a sufficient amount of material comprising
semi-crystalline poly-.beta.-1.fwdarw.4 N-acetylglucosamine
polymers, wherein the polymers are free of protein, substantially
free of other organic contaminants, and substantially free of
inorganic contaminants, and wherein said administering induces at
least one transient, localized physiological response selected from
the group consisting of stimulation of endothelin-1 release,
vasoconstriction, and reduction in blood flow out of a breached
vessel, whereby the patient experiences transient, localized
modulation of vascular structure and/or function, whereby said
administering ameliorates said vascular condition.
25. The method of claim 24, wherein the vascular disorder is
selected from the group consisting of menorrhagia, cerebral
aneurysm, abdominal aneurysm, uterine fibroid lesion, and blood
vessel puncture.
Description
1 INTRODUCTION
[0001] The present invention relates to compositions comprising
semi-crystalline poly-.beta.-1.fwdarw.4-N-acetylglucosamine
(p-GlcNac) polysaccharide polymers and methods utilizing such
polymers for stimulating, in a p-GlcNac concentration-dependent
manner, transient, localized stimulation of endothelin-1 release,
vasoconstriction, and/or reduction in blood flow out of a breached
vessel. These effects, individually and/or collectively, contribute
or lead to cessation of bleeding. More specifically, the methods of
the present invention comprise topical administration of
compositions and materials comprising semi-crystalline polymers of
N-acetylglucosamine that are free of proteins and substantially
free of single amino acids and other organic and inorganic
contaminants, and whose constituent monosaccharide sugars are
attached in a .beta.-1.fwdarw.4 conformation.
2 BACKGROUND
[0002] Vascular homeostasis depends, in part, upon the regulated
secretion of biochemical modulators by endothelial cells. Under
normal physiological conditions, endothelial cells synthesize and
secrete nitric oxide, prostacyclin, PG12, adenosine,
hyperpolarizing factor, tissue factor pathway inhibitor, and
scuplasminogen activator. Endothelial cells also activate
antithrombin III and protein C, which, collectively, mediate
vascular dilation, inhibit platelet adhesion, platelet activation,
thrombin formation and fibrin deposition. Nitric oxide, in
particular, plays a critical role in vascular homeostasis (Pearson,
J. D. (2000) Lupus 9 (3): 183-88; Becker et al., (2000) Z Kardiol
89 (3): 160-7; Schinin-Kerth, V. B. (1999) Transfus Clin Biol 4
(6): 355-63).
[0003] Production of nitric oxide and prostacyclin, which are
powerful vasodilators and inhibitors of platelet aggregation and
activation, underlies the antithrombotic activity of the
endothelium (Yang et al. (1994) Circulation 89 (5): 2666-72).
Nitric oxide is synthesized at a constitutive, basal level from
arginine by nitric oxide synthase, and this synthesis is stimulated
by the vaso-active agents acetylcholine and bradykinin. It has been
shown that inhibition of nitric oxide synthase by the arginine
analogues monomethyl-L-arginine (L-NMMA) and nitro-L-arginine
methyl ester (L-NAME) reduces nitric oxide levels and leads not
only to vasoconstriction, as measured by intravascular ultrasound
imaging, but also to an increase in platelet aggregation (Yao et
al. (1992) Circulation 86 (4): 1302-9; Emerson et al. (1999) Thromb
Haemost 81 (6): 961-66).
[0004] Perturbation of the endothelium as the result of
atherosclerosis, diabetes, postischemic reperfusion, inflammation
or hypertension for example, leads to a prothrombotic state in
which the endothelium elaborates a further set of biochemical
modulators including TNF-.alpha., IL-8, von Willebrand factor,
platelet activating factor, tissue plasminogen activator, and type
1 plasminogen activator inhibitor. (Pearson, J. D. (2000) Lupus 2
(3): 183-88; Becker et al. (2000) Z Kardiol 12 (3): 160-7;
Schinin-Kerth, V. B. (1999) Transfus Clin Biol 6 (6): 355-63). In
addition, the vascular endothelium synthesizes and elaborates the
endothelins, which are the most potent vasoconstrictor peptides
known.
[0005] The endothelins are a family of 21-amino acid peptides,
i.e., endothelin-1, endothelin-2, and endothelin-3, originally
characterized by their potent vasoconstricting and angiogenic
properties (see, e.g., Luscher et al. (1995), Agents Actions Suppl.
(Switzerland) 45: 237-253; Yanagisawa et al. (1988) Nature 332:
411-415). The three isopeptides of the endothelin family,
endothelin-1, endothelin-2, and endothelin-3, possess highly
conserved amino acid sequences that are encoded by three separate
genes (see, e.g., Inoue et al. (1989) Proc Natl Acad Sci USA 86:
2863-67; Saida et al. (1989) J Biol Chem 264: 14613-16). Although
the endothelins are synthesized in several tissues including smooth
muscle cells, endothelin-1 is exclusively synthesized by the
vascular endothelium (Rosendorff, C. (1997) Cardiovasc Drugs 10
(6): 795-802). The endothelins are synthesized as preproendothelins
of two hundred and three amino acids. The endothelin signal
sequence is cleaved and the protein is then further proteolytically
processed to yield the mature, biologically active 21 amino acid
form (see, e.g., Kashiwabara et al. (1989) FEBS Lett 24: 337-40).
Endothelin synthesis is regulated via autocrine mechanisms
including endothelin and non-endothelin converting enzymes as well
as by chymases (Baton et al. (1999) Curr Opin Nephrol Hypertens 8
(5): 549-56). Elaboration of endothelin-1 from the endothelium is
stimulated by angiotensin II, vasopressin, endotoxin, and
cyclosporin inter alia (see e.g. Brooks et al. (1991) Eur J Pharm
194: 115-17) and is inhibited by nitric oxide.
[0006] Endothelin activity is mediated via binding with
preferential affinities to two distinct G protein-coupled
receptors, ET.sub.A and ET.sub.B, in an autocrine/paracrine manner
(see, e.g., Hocher et al. (1997) Eur. J. Clin. Chem. Clin. Biochem.
35 (3): 175-189; Shichiri et al. (1991) J. Cardiovascular
Pharmacol. 17: S76-S78). ET.sub.A receptors are found on vascular
smooth muscle linked to vasoconstriction and have been associated
with cardiovascular, renal, and central nervous system diseases.
ET.sub.B receptors are more complex and display antagonistic
actions. ET.sub.B receptors in the endothelium have the dual roles
of clearance and vasodilation, while ET.sub.B receptors on smooth
muscle cells also mediate vasoconstriction (Dupuis, J. (2000) Can J
Cardiol 16 (1): 903-10). The ET.sub.B receptors on the endothelium
are linked to the release of nitric oxide and prostacycline
(Rosendorff, C. (1997) Cardiovasc Drugs 14 (6): 795-802). There are
a variety of agonists and antagonists of endothelin receptors (Webb
et al. (1997) Medicinal Research Reviews 17 (1): 17-67), which have
been used to study the mechanism of action of the endothelins.
Because endothelin is known to have powerful vasoconstrictive
activity, endothelin antagonists in particular (also termed
"endothelin receptor antagonists" in the art) have been studied
with regard to their possible role in treating human disease, most
notably, cardiovascular diseases such as hypertension, congestive
heart failure, atherosclerosis, restenosis, and myocardial
infarction (Mateo et al. (1997) Pharmacological Res. 36 (5):
339-351).
[0007] Moreover, endothelin-1 has been shown to be involved in the
normal functioning of the menstrual cycle. Menstruation represents
a remarkable example of tissue repair and replacement, involving
the regulated remodeling and regeneration of a new layer of
endometrial tissue lining the uterus. This repair and remodeling
process is remarkable in that it is accomplished without scarring,
a phenomenon generally not seen in other organs of the body.
Defects in that repair process are believed to be the basis of
excessive or abnormal endometrial bleeding in women with documented
menorrhagia as well as in women carrying subcutaneous
levonorgestrel implants (NORPLANT) for contraceptive purposes. In
both of these groups of patients, only very low levels of
endometrial endothelin-1 have been detected as compared with
control populations. Moreover, it has been indicated that
endothelin-1 not only may play a role in effecting cessation of
menstrual bleeding but endothelin-1 may also have a mitogenic
activity required for regenerating and remodeling of endometrial
tissue after menstruation (see Salamonsen et al. 1999, Balliere's
Clinical Obstetrics and Gynaecology (2): 161-79; Goldie 1999,
Clinical and Experimental Pharmacology and Physiology 26: 145-48;
Salamonsen et al. 1999, Clin. Exp. Phamaol. Physiol. 26 (2):
154-57).
[0008] In summary, vascular homeostasis reflects a dynamic balance
between two physiological states mediated by the vascular
endothelium. The first, which has been termed antithrombotic, is
characterized inter alia by the production of nitric oxide,
vasodilation, inhibition of platelet attachment and activation, and
by repression of endothelin-1 synthesis. The second or
prothrombotic physiological state is characterized inter alia by
the production of endothelin-1, vasoconstriction, platelet
activation, and hemostasis (Warner (1999), Clinical and
Experimental Physiology 26: 347-52; Pearson, (2000), Lupus 2(3):
183-88).
[0009] In light of the physiological importance of vascular
homeostasis, there is a need for methods and compositions that are
capable of modulating one or more aspects of the above processes.
More specifically, there is a need for compositions and methods for
the modulation of endothelin release, vasoconstriction, and blood
flow out of a breached vessel and which would therefore be useful
for effecting cessation of bleeding. That is, although such
compositions and methods would act in a manner that is not
dependent upon physical barrier formation, coagulation, or blood
clot formation, such compositions and methods would nevertheless
contribute, inter alia, to the achievement of hemostasis.
Accordingly, such methods and compositions would be expected to
have therapeutic applications for the treatment of diseases or
conditions arising as a consequence of the perturbation of vascular
homeostasis. Moreover, in view of the systemic effects resulting,
e.g., from administration to patients of endothelin-1 antagonists
as described supra, there is an even greater need for compositions
and methods that produce localized and transient physiological
responses, including, but not limited to, stimulation of
endothelin-1 release, in such patients.
3 SUMMARY OF THE INVENTION
[0010] The present invention relates to methods and compositions
for the treatment or amelioration of vascular disorders including
bleeding disorders. More specifically, the invention relates to
compositions comprising semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine (p-GlcNac)
polysaccharide polymers, and use of such polymers in methods to
effect transient localized, modulation of vascular structure and/or
function by, e.g., stimulation of endothelin-1 release,
vasoconstriction, and/or reduction in blood flow out of a breached
vessel, thereby contributing to or effecting cessation of
bleeding.
[0011] The present invention is based in part on the Applicants'
discovery that topical application of semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine (p-GlcNac)
polysaccharide polymers to a vascular surface induces not only
contraction of that vessel, thereby decreasing the lumen of that
vessel, but also induction of a transient, localized stimulation of
endothelin-1 release in those tissues contiguous with the applied
compositions and materials disclosed herein.
[0012] The present invention relates, in one aspect, to a method
for achieving transient, localized, modulation of vascular
structure and/or function in a patient, comprising topical
administration of a material comprising semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymers, which are free
of protein, substantially free of other organic contaminants, and
substantially free of inorganic contaminants. Administration of
these materials induces transient, localized physiological
responses including, but not limited to, stimulation of
endothelin-1 release, vasoconstriction, and reduction in blood flow
out of a breached vessel.
[0013] In one embodiment of the present invention, endothelin-1 is
released from vascular endothelial cells. In other aspects of this
embodiment, endothelin-1 release is stimulated from other
endothelial tissues or from platelets.
[0014] In one embodiment, the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer comprises about 50 to about 4,000
N-acetylglucosamine monosaccharides covalently attached in a
.beta.-1.fwdarw.4 conformation, and has a molecular weight of about
10,000 daltons to about 800,000 daltons.
[0015] In another embodiment, the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer comprises about 50 to about 10,000
N-acetylglucosamine monosaccharides covalently attached in a
(.beta.-1.fwdarw.4 conformation, and has a molecular weight of
about 10,000 daltons to about 2 million daltons. In yet another
embodiment, the poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer
comprises about 50 to about 50,000 N-acetylglucosamine
monosaccharides covalently attached in a .beta.-1.fwdarw.4
conformation, and has a molecular weight of about 10,000 daltons to
about 10 million daltons. In another embodiment, the
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises about
50 to about 150,000 N-acetylglucosamine monosaccharides covalently
attached in a .beta.1.fwdarw.4 conformation, and has a molecular
weight of about 10,000 daltons to about 30 million daltons.
[0016] In preferred embodiments of the invention, the disclosed
method is used for the treatment of a mammalian patient, and in
more preferred embodiments, for the treatment of a human in need of
such treatment. More specifically, modulation of vascular structure
and/or function is used to effect cessation of bleeding,
particularly in a patient afflicted with a coagulopathy. Such a
disorder can be the result of a genetic defect, such as hemophilia,
or a medical treatment, including for example, administration of
systemic anticoagulants, e.g. coumadin, to a dialysis patient,
cardiac patient, or other patient with an increased risk of vessel
blockage. Similarly, the present method is used to effect a
temporary, localized, reduction in blood flow out of a breached
vessel during surgical repair of an aneurysm or excision of a tumor
or polyp, particularly in a patient having a coagulopahtic
condition, thereby minimizing blood loss during such a procedure.
In other embodiments, the method of the present invention is used
for the treatment of bleeding ulcers or varices, particularly
esophageal varices. While not wishing to be bound by a particular
theory or mechanism, it is believed that such cessation of bleeding
by the methods disclosed herein occurs in a coagulation-independent
manner.
[0017] In other embodiments of the method of the invention, the
p-GlcNac-containing material is topically administered to the skin
of the patient or to the surface of another organ, or the material
may be applied directly to a vascular structure to be modulated,
which may be a capillary, vein, or artery.
[0018] In yet another embodiment of the method of the invention,
where the vascular structure is a breached blood vessel, topical
application of the p-GlcNac-containing materials of the invention
is used to achieve cessation of bleeding.
[0019] In a further embodiment of the invention, the extent of the
transient, localized modulation of vascular structure and/or
function is substantially proportional to the amount of
semi-crystalline poly-.beta.-1.fwdarw.4 N-acetylglucosamine
applied.
[0020] The invention is also directed toward a biodegradable
material comprising semi-crystalline poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymers which are free of protein,
substantially free of other organic contaminants, and are
substantially free of inorganic contaminants. In one embodiment,
the semi-crystalline poly-.beta.-1.fwdarw.4 N-acetylglucosamine
polymers comprise about 50 to about 4,000 N-acetylglucosamine
monosaccharides covalently attached in a .beta.-1.fwdarw.4
conformation and have a molecular weight of about 10,000 daltons to
about 800,000 daltons. In another embodiment, the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises about
50 to about 10,000 N-acetylglucosamine monosaccharides covalently
attached in a .beta.-1.fwdarw.4 conformation, and has a molecular
weight of about 10,000 daltons to about 2 million daltons. In yet
another embodiment, the poly-.beta.-1.fwdarw.4 N-acetylglucosamine
polymer comprises about 50 to about 50,000 N-acetylglucosamine
monosaccharides covalently attached in a .beta.-1.fwdarw.4
conformation, and has a molecular weight of about 10,000 daltons to
about 10 million daltons. In another embodiment, the
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises about
50 to about 150,000 N-acetylglucosamine monosaccharides covalently
attached in a .beta.-1.fwdarw.4 conformation, and has a molecular
weight of about 10,000 daltons to about 30 million daltons.
[0021] In another embodiment, the biodegradable material comprising
semi-crystalline poly-.beta.-1.fwdarw.4 N-acetylglucosamine
polymers is a non-barrier-forming material.
[0022] In still another embodiment, the semi-crystalline
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer comprises at
least one N-acetylglucosamine monosaccharide that is deacetylated.
In other aspects of this embodiment the poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer may comprise about 10%, 20%, 30%, 40%,
50% or 60% deacetylated residues, provided the
partially-deacetylated poly-.beta.-1.fwdarw.4 N-acetylglucosamine
polymer retains its semi-crystalline structure as demonstrated by
sharp, discrete peaks when the polymer is analyzed by IR absorption
spectroscopy, as described in Example 6, below.
4 BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1. Chemical structure of 100% p-GlcNAc. "n" refers to
an integer ranging up to about 150,000.
[0024] FIG. 2. Carbohydrate analysis of p-GlcNAc, Gas
Chromatography-Mass Spectroscopy data. Solid squares represent
p-GlcNAc purified using the acid treatment/neutralization variation
of the Chemical/Biological method, as described in Section 5.3.2,
below.
[0025] FIG. 3A. Circular dichroism spectra of solid membranes of
pure p-GlcNAc.
[0026] FIG. 3B. Circular dichroism spectra of solid membranes of
Deacetylated p-GlcNAc. The disappearance of the 211 nm minimum and
195 nm maximum observed in pure p-GlcNAc (FIG. 3A) indicates
complete deacetylation under the conditions used, as described in
Section 5.4 below.
[0027] FIG. 4A. Infra-red spectra analyses of thin membranes of
pure diatom p-GlcNAc prepared by the mechanical force purification
method, top, and the chemical/biological purification method,
bottom.
[0028] FIG. 4B. Infra-red spectra analyses of two preparations of
commercial "chitin" cast into membranes according to the methods
detailed in Section 5.5, below.
[0029] FIG. 4C. Infra-red spectra analyses of pure p-GlcNAc which
was modified by heat denaturation (top) and by chemical
deacetylation (bottom), according to the methods detailed in
Section 5.4, below.
[0030] FIG. 4D. Infra-red spectrum analysis of a p-GlcNAc membrane
derived from the diatom Thalassiosira fluviatilis, using the
chemical/biological purification method, as detailed in Section
5.3.2, below.
[0031] FIG. 4E. Infra-red spectrum analysis of a p-GlcNAc membrane
prepared by the mechanical force purification method, as described
in Section 53.1, below, following autoclaving.
[0032] FIG. 5A. NMR analysis of p-GlcNAc purified using the
chemical/biological purification method as described in Section
5.3.2, below. Chart depicting peak amplitudes, areas, and ratios
relative to reference controls. Ratio of total areas of peaks.
[0033] FIG. 5B. NMR analysis of p-GlcNAc purified using the
chemical/biological purification method as described in Section
5.3.2. The graph depicts the ratios of total areas of peaks.
[0034] FIGS. 6A-B. Transmission electron micrographs (TEM) of a
p-GlcNAc membrane prepared by the mechanical force purification
method as described in Section 5.3.1, below. Magnification: (FIG.
6A), 4190.times.; (FIG. 6B), 16,250.times..
[0035] FIGS. 7A-B. Transmission electron micro graphs (TEM) of a
p-GlcNAc membrane by HF treatment as described in the discussion of
the chemical/biological purification method in Section 5.3.2,
below. Magnification: (FIG. 7A), 5270.times.; (FIG. 78)
8150.times..
[0036] FIGS. 8A-B. Transmission electron micrographs (TEM) of a
p-GlcNAc membrane prepared by the acid treatment/neutralization
variation of the chemical/biological purification method, as
described in Section 5.3.2, below. Magnification: (FIG. 8A),
5270.times.; (FIG. 8B), 16,700.times..
[0037] FIG. 9A. Scanning electron micrograph depicting a p-GlcNAc
membrane prepared by the acid treatment/neutralization variation of
the chemical/biological purification method as described in Section
5.3.2, below. Magnification: 200.times..
[0038] FIG. 9B. Scanning electron micrograph depicting a p-GlcNAc
membrane prepared by the acid treatment/neutralization variation of
the chemical/biological purification method as described in Section
5.3.2, below. Magnification: 1000.times..
[0039] FIG. 9C. Scanning electron micrograph depicting a p-GlcNAc
membrane prepared by the acid treatment/neutralization variation of
the chemical/biological purification method as described in Section
5.3.2, below. Magnification: 5000.times..
[0040] FIG. 9D. Scanning electron micrograph depicting a p-GlcNAc
membrane prepared by the acid treatment/neutralization variation of
the chemical/biological purification method as described in Section
5.3.2, below. Magnification: 10,000.times..
[0041] FIG. 9E. Scanning electron micrograph depicting a p-GlcNAc
membrane prepared by the acid treatment/neutralization variation of
the chemical/biological purification method as described in Section
5.3.2, below. Magnification: 20,000.times..
[0042] FIGS. 10A-B. Scanning electron micrographs of a pure
p-GlcNAc membrane made from material which was initially produced
using the cell dissolution/neutralization purification method
described in Section 5.3, below, dissolved in
dimethylacetamide/lithium chloride, and reprecipitated in H.sub.2O
into a mat, as described below in Section 5.5. Magnification: (FIG.
10A), 1000.times., (FIG. 10B), 10,000.times..
[0043] FIGS. 11A-B. Scanning electron micrographs of a deacetylated
p-GlcNAc mat. Magnification: (FIG. 11A), 1000.times., (FIG. 11B),
10,000.times..
[0044] FIGS. 12A-B. Photographs of diatoms. Note the p-GlcNAc
fibers extending from the diatom cell bodies.
[0045] FIG. 13. Diagram depicting some of the possible p-GlcNAc and
deacetylated derivatives of the p-GlcNAc starting material.
(Adapted from S. Hirano, "Production and Application of Chitin and
Chitosan in Japan", in "Chitin and Chitosan," 1989, Skjak-Braek,
Anthonsen, and Sanford, eds. Elsevier Science Publishing Co., pp.
37-43.)
[0046] FIG. 14. Transformed NMR data curves, used to obtain areas
for each carbon atom and to then calculate the CH.sub.3 (area) to
C-atom (area) ratios.
[0047] FIG. 15. Typical p-GlcNAc C.sup.13--NMR spectrum. The
individual peaks represent the contribution to the spectrum of each
unique carbon atom in the molecule.
[0048] FIG. 16. Transformed NMR spectrum data representing values
calculated for CH.sub.3 (area) to C-atom (area) ratios. Top:
Graphic depiction of data; bottom: numerical depiction of data.
[0049] FIGS. 17A-G. Three-dimensional p-GlcNAc matrices produced in
various solvents.
[0050] Specifically, the p-GlcNAc matrices were produced in
distilled water (FIG. 17A, FIG. 17D), 10% methanol in distilled
water (FIG. 17B), 25% methanol in distilled water (FIG. 17C), 10%
ethanol in distilled water (FIG. 17E), 25% ethanol in distilled
water (FIG. 17F) and 40% ethanol in distilled water (FIG. 17G).
Magnification: 200.times.. A scale marking of 200 microns is
indicated on each of these figures.
[0051] FIG. 18. A typical standard curve obtained using the
procedure described, below, in Section 18.1. A standard curve such
as this one was used in the lysozyme-chitinase assay also
described, below, in Section 18.1.
[0052] FIG. 19. p-GlcNAc lysozyme digestion data. The graph
presented here depicts the accumulation of N-acetylglucosamine over
time, as p-GlcNAc membranes are digested with lysozyme. The graph
compares the degradation rate of fully acetylated p-GlcNAc to
partially (50%) deacetylated p-GlcNAc, and demonstrates that the
degradation rate for the partially deacetylated p-GlcNAc was
substantially higher than that of the fully acetylated p-GlcNAc
material.
[0053] FIG. 20. p-GlcNAc lysozyme digestion data. The graph
presented here depicts the accumulation of N-acetylglucosamine over
time, as p-GlcNAc membranes are digested with lysozyme. The graph
compares the degradation rate of two partially deacetylated
p-GlcNAc membranes (specifically a 25% and a 50% deacetylated
p-GlcNAc membrane). The data demonstrate that the degradation rate
increases as the percent of deacetylation increases, with the
degradation rate for the 50% deacetylated p-GlcNAc membrane being
substantially higher than that of the 25% deacetylated p-GlcNAc
membrane.
[0054] FIGS. 21A-21E. p-GlcNAc in vivo biodegradability data. FIGS.
21A-21C depict rats which have had prototype 1 (fully acetylated
p-GlcNAc) membrane abdominally implanted, as described, below, in
Section 18.1. FIG. 21A shows a rat at day 0 of the implantation;
FIG. 21B shows a rat at day 14 post-implantation; FIG. 21C shows a
rat at day 21 post-implantation. FIGS. 21D-21E depict rats which
have had prototype 3A (lyophilized and partially deacetylated
p-GlcNAc membrane) abdominally implanted, as described, below, in
Section 18.1. FIG. 21D shows a rat at day 0 of the implantation;
FIG. 21E shows a rat at day 14 post-implantation.
[0055] FIG. 22. Dose-dependent vasoconstriction of isolated aortic
rings by p-GlcNac, either with an intact endothelial layer, panel
A, or after removal of the endothelial layer, panel B. The number
of contraction measurements that were averaged to provide the
values reported at each concentration of p-GlcNac tested, either
with or without an intact endothelial layer, is indicated within
the figure, above each p-GlcNAc concentration tested.
[0056] FIG. 23 A-E. Arterial vasoconstriction by p-GlcNac. FIG. 23
(A) depicts a cross-section of a porcine artery obtained 60 minutes
after application of a gauze dressing to one side of the artery.
FIG. 23 (B) depicts a cross-section of a porcine artery obtained 15
minutes after application of a p-GlcNac membrane to one side of the
artery. FIG. 23 (C) depicts a cross-section of a porcine artery
obtained 60 minutes after application of a p-GlcNac membrane to one
side of the artery. FIG. 23 (D) depicts a cross-section of a
porcine artery obtained 15 minutes after application of a
fibrin-coated collagen dressing to one side of the artery. FIG. 23
(E) depicts a cross-section of a porcine artery obtained 60 minutes
after application of a fibrin-coated collagen dressing to one side
of the artery.
[0057] FIG. 24 Arterial vasoconstriction by p-GlcNac. FIG. 24
depicts the thickness of a porcine arterial wall that either was
(1), or was not (2), in direct contact with the material tested,
for 15 or 60 minutes, as indicated. The materials applied to one
side of the artery were: (A) gauze dressing; (B) and (C) p-GlcNac
membrane; (D) and (E) fibrin-coated collagen dressing.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention relates to compositions and methods
useful for effecting transient, localized modulation of vascular
structure and/or function, by, e.g. (1) stimulation of endothelin-1
release, (2) vasoconstriction, and (3) reduction in blood flow out
of a breached vessel, comprising topical administration of
compositions and materials that comprise semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine (p-GlcNac)
polysaccharide polymers. Stimulation of endothelin-1 release,
vasoconstriction, and reduction in blood flow out of a breached
vessel in a target tissue may be achieved either by direct
application of the materials of the present invention to the target
tissue, or by application of those materials to the skin or other
organ or tissue surface that is adjacent to or contiguous with the
target tissue.
[0059] The present invention is therefore, also directed to
compositions and methods that contribute to or directly effect
cessation of bleeding. Administration of the materials of the
invention, which comprise semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine polymers, results in
stimulation of endothelin-1 release, vasoconstriction, and decrease
in blood flow out of a breached vessel. These physiological
responses, individually and/or collectively, contribute to or
directly effect cessation of bleeding, which may be a capillary,
vein, or artery. While not wishing to be bound by a particular
theory or mechanism, it is believed that such cessation occurs in a
coagulation-independent manner. Moreover, achievement of cessation
of bleeding using the compositions and methods of the present
invention is also not dependent upon formation of a physical
barrier or mechanical matrix that promotes clotting. That is,
according to the present invention, the material need not be a
barrier-forming material that provides a mechanical matrix that
adheres to the site of application and seals the boundaries of the
wound. In contrast, the compositions and methods of the present
invention induce a transient, localized alteration of vascular
structure and/or function, and it is that alteration, which is
independent of clot formation, that, per se, contributes to or
directly effects cessation of bleeding.
[0060] Furthermore, the preferred materials of the compositions and
methods of the present invention comprise fully acetylated
semi-crystalline poly-.beta.-1.fwdarw.4-N-acetylglucosamine
polymers, since, as demonstrated the Examples provided in Sections
16 and 17, as well as FIG. 22, infra, materials comprising
70%-deacetylated poly-.beta.-1.fwdarw.4-N-acetylglucosamine
polymers do not induce vasoconstriction and, therefore will not
decrease the lumen of the vessel and, consequently, will not reduce
blood flow out of a breached vessel.
[0061] This invention is based in part on Applicants' discovery
that topically-applied materials, which need not be barrier-forming
materials, that comprise semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine (p-GlcNac) polymers,
induce vasoconstriction in isolated Sprague-Dawley rat aortic
rings. In this blood-free system, fully acetylated
poly-1-1.fwdarw.4-N-acetylglucosamine induced contraction of the
isolated aortic rings in a concentration-dependent manner. As
demonstrated infra, in the Example presented in Section 17, the
degree of vasoconstriction obtained was substantially proportional
to the concentration of p-GlcNac applied to the isolated aortic
ring. In contrast, 70% deacetylated
poly-.beta.-1.fwdarw.4-N-acetylglucosamine, did not induce
vasoconstriction of the isolated aortic rings, at any concentration
tested.
[0062] This invention is also based in part on Applicants'
discovery that in vivo application of membrane membranes, which are
formed from semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine polymers, to
experimental wounds in arteries, stimulated immediate
vasoconstriction at the site of contact between the arterial tissue
and the applied membrane. Histological analysis of treated tissue
revealed that arterial constriction was greater on the side where
the membrane was applied than on the opposite side of the artery.
Furthermore, immunochemical analyses of these tissue samples also
revealed the presence of a concentration gradient of endothelin-1
release, i.e., stimulation of endothelin-1 release was a localized
physiological response. The extent of the stimulation of
endothelin-1 release was greatest at the surface contacted by the
semi-crystalline poly-.beta.-1.fwdarw.4-N-acetylglucosamine polymer
containing-membrane, and extended into adjacent tissue, although to
an extent that decreased as the distance from the contact surface
increased. A similar, localized stimulation of endothelin-1 release
was observed in spleen tissue contacted with material comprising
semi-crystalline poly-.beta.-1.fwdarw.4-N-acetylglucosamine.
[0063] The methods of the present invention comprise topical
administration of materials comprising a therapeutically effective
form and a therapeutically effective amount of semi-crystalline
poly-.beta.-1.fwdarw.4-N-acetylglucosamine polymers, to a patient
in order to achieve transient, localized: (1) enhancement of
endothelin-1 release, (2) vasoconstriction, and/or (3) reduction of
blood flow out of a breached vessel.
[0064] Presented below, is, first, a description of physical
characteristics of the purified p-GlcNac starting material, and of
its reformulations. Next, methods are described for the
purification of the p-GlcNac starting material from microalgae,
preferably diatom, starting sources. Third, reformulations of the
p-GlcNac, and methods for the production of such reformulations are
presented. Finally, uses are presented for the p-GlcNAc, p-GlcNAc
derivatives and/or p-GlcNac reformulations of the starting
material.
[0065] 5.1. p-GlcNac
[0066] The p-GlcNac starting material can be made using techniques
described herein, coupled with the teaching provided in U.S. Pat.
Nos. 5,686,115, 5,624,679, 5,623,064, and 5,622,834, each of which
is hereby incorporated by reference in its entirety. The p-GlcNac
polymers used herein comprise about 50 to about 150,000
N-acetylglucosamine monosaccharides (FIG. 1). The purity of the
p-GlcNac starting material is very high, as evidenced by chemical
and physical criteria. Among these are chemical composition and
non-polysaccharide contaminants. First, chemical composition data
for the p-GlcNac produced using two different purification methods,
both of which are described in Section 5.3, below, is shown in
Table I below. As can be seen, the chemical composition of the
p-GlcNac produced by both methods is, within the bounds of
experimental error, the same as the formula compositions of
p-GlcNac. Second, as is also shown in Table I, the p-GlcNac
produced is free of detectable protein contaminants, is
substantially free of other organic contaminants such as free amino
acids, and is substantially free of inorganic contaminants such as
ash and metal ions (the p-GlcNac starting material may deviate up
to about 2% from the theoretical values of carbon, hydrogen,
nitrogen and oxygen for pure p-GlcNac). Therefore, as used herein,
the terms "substantially free of organic contaminants" and
"substantially free of inorganic contaminants" refer to
compositions of p-GlcNac having the profiles for carbon, hydrogen,
nitrogen and oxygen which deviate no more than about 2% from the
theoretical values, and preferably, the p-GlcNac starting material
contain a profile as exemplified in the Experimental Data on
p-GlcNac mats in Table I (allowing for the percent deviation).
Further, the p-GlcNac starting material exhibits a low percentage
of bound water.
TABLE-US-00001 TABLE I CHEMICAL ANALYSIS DATA (% by weight)
Theoretical Values for Pure p-GlcNac: Carbon - 47.29 Hydrogen -
6.40 Nitrogen - 6.89 Oxygen - 39.41 Protein - 0.00 Experimental
Data on p-GlcNac Mats: (Number of experimental batches for each
membrane type being greater than 30 for each membrane type)
MECHANICAL CHEMICAL/BIO- FORCE METHOD LOGICAL METHOD
Normalized.sup.1 % Dev. Normalized.sup.1 % Dev. Carbon 47.21 .+-.
0.08 -0.17 47.31 .+-. 0.01 +0.04 Hydrogen 6.45 .+-. 0.08 +0.78 6.34
.+-. 0.08 -0.94 Nitrogen 6.97 .+-. 0.18 +0.87 6.94 .+-. 0.16 +0.73
Oxygen 39.55 .+-. 0.36 +0.36 39.41 .+-. 0.10 0.00 Average Values
Average Values Protein 0.00 0.00 Ash 1.30 0.98 Moisture 2.0 1.2
.sup.1Raw analytical data have been normalized to account for ash
and moisture content of the samples.
[0067] The pure p-GlcNac starting material exhibits a carbohydrate
analysis profile substantially similar to that shown in FIG. 2. The
primary monosaccharide of the pure p-GlcNac starting material is
N-acetylglucosamine. Further, the pure p-GlcNac starting material
does not contain the monosaccharide glucosamine.
[0068] The circular dichroism (CD) and sharp infra-red spectra (IR)
of the p-GlcNac starting material are shown in FIG. 3A, and FIGS.
4A, 4D, and 4E, respectively, which present analyses of material
produced using the methods described in Section 5.3, below. Such
physical data corroborates that the p-GlcNac starting material is
of high purity and semi-crystalline. The phrase "semi-crystalline"
refers to the highly ordered nature of the material. One of skill
in the art would readily appreciate that the sharp, well resolved
peaks observed in the infra-red spectra of the p-GlcNAc polymers of
the present invention reflect the highly ordered, crystalline
nature of the material (i.e. "semi-crystalline") examined. That
artisan would also appreciate that broadened, poorly resolved peaks
in such a IR spectra, as for example depicted in FIGS. 4B and 4C,
would indicate loss or lack of a semi-crystalline nature. The
methods used to obtain the CD and IR data are described, below, in
the Working Example presented in Section 6.
[0069] NMR analysis of the pure p-GlcNac starting material exhibits
a pattern substantially similar to that seen in FIGS. 5A, 14, 15
and 16. Such an NMR pattern indicates not only data which is
consistent with the p-GlcNac starting material being a fully
acetylated polymer, but also demonstrates the lack of contaminating
organic matter within the p-GlcNac species. The electron
micrographic structure of the p-GlcNac starting material, as
produced using the methods described in Section 5.3, below and
demonstrated in the Working Examples presented below in Section 8
and 9, is depicted in FIG. 6 through FIG. 9E.
[0070] The p-GlcNac starting material exhibits a high degree of
biocompatability. Biocompatability may be determined by a variety
of techniques, including, but not limited to such procedures as the
elution test, intramuscular implantation, or intracutaneous or
systemic injection into animal subjects. Briefly, an elution test
(U.S. Pharmacopeia XXII, 1990, pp. 1415-1497; U.S. Pharmacopeia
XXII, 1991, Supplement 5, pp. 2702-2703) is designed to evaluate
the biocompatability of test article extracts, and assays the
biological reactivity of a mammalian cell culture line which is
sensitive to extractable cytotoxic articles (such as, for example,
the L929 cell line) in response to the test article. The Working
Example presented in Section 10, below, demonstrates the high
biocompatability of the p-GlcNac starting material.
[0071] 5.2. Methods of Producing Microalgal Sources of p-GlcNac
[0072] 5.2.1. Microalgal Sources of p-GlcNac
[0073] The p-GlcNac starting material is produced by, and may be
purified from, microalgae, preferably diatoms. The diatoms of
several genuses and numerous species within such genuses may be
utilized as p-GlcNac starting sources. Each of these diatoms
produce p-GlcNac. See FIGS. 12A-B for photographs of such diatoms.
The diatoms which may be used as starting sources for the
production of the p-GlcNac starting material include, but are not
limited to members of the Coscinodiscus genus, the Cyclotella
genus, and the Thalassiostra genus, with the Thalassiostra genus
being preferred.
[0074] Among the Coscinodiscus genus, the species of diatom that
may be used to produce the p-GlcNac starting material include, but
are not limited to the concinnus and radiatus species. The diatoms
among the Cyclotella genus which may be used include, but are not
limited to the caspia, cryptica, and meneghiniana species. The
Thalassiosira diatoms that may be utilized to produce the starting
material for the p-GlcNac starting material include, but are not
limited to the nitzschoides, aestivalis, antarctica, deciphens,
eccentrica, floridana, fluviatilis, gravida, guillardii, hyalina,
minima, nordenskioldii, oceanica, polychorda, pseudonana; rotula,
tubifera, rumida, and weissflogii species, with the fluviatilis and
weissflogii species being preferred.
[0075] Diatoms such as those described above may be obtained, for
example, from the culture collection of the Bigelow Laboratory for
Ocean Sciences, Center for Collection of Marine Phytoplankton
(McKown Point, West Boothbay Harbor, Me., 04575).
[0076] 5.2.2. Methods for Growing Diatoms
[0077] Any of the diatoms described in Section 5.2.1, above, may be
grown by utilizing, for example, the methods described in this
section. New diatom cultures are initiated by inoculating, under
aseptic conditions, Nutrient Medium with an aliquot of a mature
diatom culture. The Nutrient Medium must be free of all other
microorganisms, therefore all materials, including water, organic
components, and inorganic components used in the preparation of the
Nutrient Medium must be sterile. In addition, it is mandatory that
all procedures involved in this operation be conducted under
strictly aseptic conditions, i.e., all containers, all transfers of
substances from one vessel to another, etc. must be performed in a
sterile environment. The quantity of Nutrient Medium to be prepared
at one time should not exceed what is necessary to start a new
culture. For example, Fernbach flasks which occupy approximately
one square foot of surface may be used as vessels for the diatom
cultures, and such vessels require one liter of Nutrient Medium for
optimum growth of the diatom organism.
[0078] Preparation of the nutrient medium involves the following
operations:
[0079] a) Acquisition and processing of seawater
[0080] b) Preparation of distilled and deionized water
[0081] c) Preparation of primary nutrient stocks
[0082] d) Preparation of nutrient working stocks
[0083] e) Preparation of the final nutrient medium
[0084] Filtered seawater may be obtained, for example, from the
Marine Biology Laboratory (Woods Hole, Mass.). Seawater containers
should be stored at 5.degree. C. (.+-.2.degree. C.). When required,
the necessary volume of water may be filtered through a Buchner
filtration unit, using a Supor-800 polyether sulfone filter
membrane with 0.8 micron pore size (Gelman, Inc.). The seawater is
then sterilized by autoclaving at, for example, 121.degree. C. for
at least about 15 minutes per liter. On completion of the
sterilization process, the capped flasks are immediately cooled,
preferably by transfer to a cold room capable of allowing the
solutions to reach a temperature of approximately 5.degree. C.
(.+-.2.degree.). When it is to be used, solutions are allowed to
reach room temperature.
[0085] Tap water is distilled and deionized using standard
equipment and procedures, and collected and stored in clean,
securely capped, preferably glass, containers.
[0086] Listed below are formulas which may be followed in preparing
the stock solutions necessary for the preparation of the Nutrient
Medium. It is to be understood that while such formulas are to be
used as guides, it is intended that routine variations of such
formulas which contribute to the preparation of a Nutrient Medium
capable of sustaining microalgal diatom growth sufficient for the
p-GlcNac preparative processes described here also be within the
scope of the present invention.
[0087] I. Trace Metal Primary Stocks (TMPS) [0088] a. 39 mM
CuSO.sub.4.5H.sub.2O (copper [II] sulfate pentahydrate) (9.8 g
copper [II] sulfate/L) [0089] b. 7.5 mM ZnSO.sub.4.7H.sub.2O (Zinc
sulfate heptahydrate) (22 g zinc sulfate/L) [0090] c. 42 mM
CoCl.sub.2.6H.sub.2O (Cobalt [II] chloride hexahydrate) (10 g
cobalt [II] chloride/L) [0091] d. 91 mM MnCl.sub.2.4H.sub.2O
(Manganese [II] chloride tetrahydrate) 18 g manganese [II]
chloride/L) [0092] e. 26 mM NaMoO.sub.4.2H.sub.2O (Sodium molybdate
dihydrate) 6.3 g sodium molybdate/L) [0093] f. 1 mM
H.sub.2SeO.sub.3 (Selenious acid) (0.129 g selenious acid/L).
[0094] Sterile filter each nutrient with a filter of no greater
than 0.2 micron pore size.
[0095] II. Vitamin Primary Stocks (VPS) [0096] a. 1 mg/ml Vitamin
B12b. 0.1 mg/ml Biotin
[0097] Sterile filter both stocks with a filter of no greater than
0.2 micron pore size.
[0098] III. Sodium Salts Working Stocks (SSWS) [0099] a. Sodium
nitrate working stock: 0.88M (75 g NaNO.sub.3/L) [0100] b. Sodium
phosphate monobasic monohydrate working stock: 36.2 mM NaH.sub.2
PO.sub.4.H.sub.2O (5 g NaH.sub.2PO.sub.4.H.sub.2O/L). Sodium
metasilicate monohydrate working stock: 0.11M Na.sub.2
SiO.sub.3.9H.sub.2O (30 g Na.sub.2SiO.sub.3.9H.sub.2O/L) Sterile
filter each of the SSWS with a filter of no greater than 0.2 micron
pore size.
[0101] IV. Trace Metal Working Stocks (TMWS) [0102] 11.7 mM
Na.sub.2 EDTA (Ethylenediamine Tetraacetic acid, disodium salt
dihydrate) (4.36 g/L) [0103] 11.7 mM FeCl.sub.3.6H.sub.2O (Iron
[III] chloride hexahydrate) (3.15 g/L) [0104] 1 ml/L of each of the
six primary trace metal stocks listed above.
[0105] Sterile filter with a filter of no greater than 0.2 micron
pore size. Note that the trace metal working stock must be prepared
fresh weekly.
[0106] V. Vitamin Working Stock (VWS) [0107] 1.0 .mu.g/ml Biotin
(1.0 ml primary Biotin Stock/100 ml) [0108] 1.0 .mu.g/ml Vitamin
B12 (0.1 ml Vitamin B12 primary stock/100 ml) [0109] 0.20 mg/ml of
Thiamine HCl (20 mg Thiamine hydrochloride/100 ml).
[0110] Sterile filter with a filter of no greater than 0.2 micron
pore size. Note that a new Vitamin Working Stock should be prepared
fresh weekly.
[0111] Described below are techniques which may be followed for the
preparation of Nutrient Medium and for diatom culturing. It is to
be understood that, in addition to these techniques, any routine
variation in the formulas and/or procedures described herein which
result in a Nutrient Medium and in procedures capable of sustaining
diatom growth sufficient for the preparative processes described
herein is intended to be within the scope of the present
invention.
[0112] Nutrient Medium may be prepared, for example, as follows: To
each liter of filtered and sterilized seawater may be added 1 ml of
the NaNO.sub.3 working stock. 1 ml of the
NaH.sub.2PO.sub.4.H.sub.2O working stock, 1 ml of the Trace Metal
working stock, and 1 ml of the Na.sub.2SiO.sub.3.9H.sub.2O working
stock. Simultaneously with the addition of Na.sub.2
SiO.sub.3.9H.sub.2O, 2 mls of 1N HCl may be added and the solution
may be shaken to mix. Next, 1.5 m is 1N NaOH may be added and the
solution may again be shaken to mix. Finally, 0.5 ml of the Vitamin
working stock may be added.
[0113] In order to grow a new diatom culture, 7 ml of a mature
culture, (having a cell density within a range of about
1.times.10.sup.5 to about 1.times.10.sup.6 cells/ml.), may be
transferred to a sterile container containing 100 ml of sterile
Nutrient Medium, which may be prepared according to the methods
described above. The inoculated culture may then be incubated for 8
days under the following conditions:
[0114] Temperature: 20 Centigrade Constant illumination.
[0115] Agitation: Gentle swirling of flasks once per day.
[0116] After 8 days of incubation, 80 ml of this incubated culture
may be transferred, under sterile conditions, to 1000 ml of
Nutrient Medium, which may, for example, be contained in a 2.8 L
Fernbach flask, protected by a cotton wool plug covered by
cheesecloth. Such a culture may be allowed to incubate and grow to
the desired cell density, or alternatively, may be used to
inoculate new diatom cultures. Once a culture reaches a desired
cell density, the culture's p-GlcNac fibers may be harvested, and
the p-GlcNac starting material may be purified, using methods such
as those described below in Section 5.3, below.
[0117] CO.sub.2 may be dissolved in the culture solution in order
to maintain a culture pH of approximately 7 to 8, with
approximately 7.4 being preferred. The maintenance of such a
neutral pH environment greatly increases the p-GlcNac yield that
may be obtained from each diatom culture.
[0118] 5.3. Methods for Isolation, Purification, and Concentration
of p-GlcNac Fibers
[0119] Presented in this Section are methods which may be utilized
for the preparation of p-GlcNac fibers from diatom cultures such as
those described, above, in Section 5.2.
[0120] While each of the methods described below for the
purification of p-GlcNac from microalgae, preferably diatom,
starting sources produces very pure, unadulterated,
semi-crystalline p-GlcNac. For example, the p-GlcNac starting
material can be purified via the Mechanical Force method presented
in Section 5.3.1, below. The second method, which is referred to as
the Chemical/Biological method and is described below in Section
5.3.2, produces a much higher average yield than the average
p-GlcNac yield produced by the Mechanical Force method.
Additionally, the acid treatment/neutralization variation described
as part of the Chemical/Biological method of Section 5.3.2, below,
produces extremely long p-GlcNac fibers, with some fibers being in
excess of 100 .mu.m, and containing molecules of the p-GlcNac
polymer of very high molecular weight, as high as 20-30 million
daltons. Molecular weight determination of the p-GlcNac polymeric
starting material is determined using chromatographic and
physiochemical methods well known to those of ordinary skill in the
art including, but not limited to measurement of intrinsic
viscosity.
[0121] 5.3.1. Mechanical Force Method for Preparation of Pure
p-GlcNac
[0122] The p-GlcNac fibers may be separated from diatom cell bodies
by subjecting the contents of the culture to an appropriate
mechanical force. Such a mechanical force may include, but is not
limited to, a shear force generated by, for example, a colloid
mill, an ultrasound device, or a bubble generator, or a cutting
force generated by, for example, a Waring blender.
[0123] The resulting suspension of diatom cell bodies and p-GlcNac
fibers are then segregated. For example, the suspension may be
subjected to a series of centrifugation steps which segregate the
p-GlcNac fibers from the cell bodies, yielding a clear supernatant
exhibiting little, if any, visible flocculent material. A fixed
angle rotor, and a temperature of about 10.degree. C. are preferred
for the centrifugation steps. The speed, duration, and total number
of centrifugation steps required may vary depending on, for
example, the specific centrifugation rotor being used, but the
determination of the values for such parameters will be apparent to
one of ordinary skill in the art.
[0124] The p-GlcNac fibers in the supernatant may then be
concentrated using techniques well known to those of skill in the
art. Such techniques may include, but are not limited to suction
and filtration devices.
[0125] Finally, the concentrated p-GlcNac fibers are washed with,
for example, distilled-deionized water, HCl and ethanol, or other
appropriate solvents, preferably solvents, such as alcohols, in
which both organic and inorganic materials dissolve.
[0126] The Working Example presented in Section 7, below,
demonstrates the use of this method for the purification of
p-GlcNac.
[0127] 5.3.2. Chemical/Biological Method for Purification of
p-GlcNac
[0128] In this method, p-GlcNac fibers are separated from diatom
cell bodies by subjecting them to chemical and/or biological agents
as described in more detail below.
[0129] Diatom cultures may be treated with a chemical capable of
weakening diatom cell walls, which leads to a release of the
p-GlcNac fibers without altering their length and structure. Such a
chemical may include, but is not limited to, hydrofluoric acid
(HF). Alternatively, a mature diatom culture may be treated with a
biological agent capable of altering a biological process may be
used to inhibit p-GlcNac fiber synthesis, thus releasing the fibers
already present. For example, such an agent may include, but is not
limited to, polyoxin-D, an inhibitor of the enzyme
N-acetylglucosaminyl-P-transferase.
[0130] The cell bodies and p-GlcNac-containing fibers of diatom
cultures treated with a member of the above described chemical or
biological agents are then segregated. For example, the contents of
treated diatom cultures may be allowed to settle such that the
contents of the cultures are allowed to form two distinct layers.
The upper layer will contain primarily the p-GlcNac fibers, while
the bottom layer will contain the cell bodies. The upper p-GlcNac
fiber-containing layer may be siphoned off leaving behind the
settled cellular material of the bottom layer.
[0131] The siphoned off p-GlcNac fiber-containing layer may then be
further purified to remove protein and other unwanted matter by
treatment with a detergent that will not damage the p-GlcNac
fibers. Such a detergent may include, but is not limited to, sodium
dodecyl sulfate (SDS).
[0132] When acid treatment, such as HF treatment, is used to
separate p-GlcNac fibers from diatom cell bodies, a step may be
included for the dispersal of the fibers. Such a step may include,
but is not limited to, the use of mechanical force for fiber
dispersal, such as a step in which the fibers are subjected to the
movements of an orbital shaker.
[0133] Alternatively, the acid-treated suspension may, in an
optional step, be neutralized prior to further purification by
detergent treatment. Such neutralization will, in general, change
the pH of the suspension from approximately 1.8 to approximately
7.0, and may be accomplished by, for example, the addition of an
appropriate volume of 1M Tris (pH 8.0) or the addition of an
appropriate volume of sodium hydroxide (NaOH). Neutralization, in
general, yields pure p-GlcNac fibers of a substantially greater
length than the other purification methods discussed herein.
[0134] The purified p-GlcNac fibers may then be concentrated using
techniques well known to those of skill in the art, such as by
utilizing a suction and filtration device. Finally, the p-GlcNac
fibers are washed, in a series of steps with distilled deionized
water, HCl and ethanol, or other appropriate solvents, preferably
solvents, such as alcohols, in which both organic and inorganic
materials dissolve.
[0135] The Working Example presented, below, in Section 8
demonstrates the successful utilization of such a purification
method.
[0136] The p-GlcNac starting material, or its partially
deacetylated derivative, may be subjected to controlled hydrolysis
conditions, which yield groups of molecules having uniform,
discrete molecular weight and other physical characteristics. Such
hydrolysis conditions may include, for example, treatment with the
enzyme, lysozyme. p-GlcNac may be exposed to lysozyme for varying
periods of time, in order to control the extent of hydrolysis. Such
enzymatic, partial-digestion reactions may also be controlled by
varying the concentration of the substrate, or of the enzyme, or
both the substrate and enzyme, as well as the pH and temperature.
In addition, the rate of hydrolysis may be controlled as a function
of the extent to which the p-GlcNac that is being lysozyme-treated
has been deacetylated. Deacetylation conditions may be as described
earlier in this Section. The more fully a p-GlcNac molecule has
been deacetylated, between about 20 and about 90 percent
deacetylated, the more fully the molecule will be hydrolyzed in a
given time. Changes in physical characteristics, in addition to the
lowering of molecular weight, may be elicited by hydrolysis and/or
deacetylation treatments. The results of a hydrolysis/deacetylation
procedure are presented below in the Working Example of Section 9,
below.
[0137] 5.4. Derivatization of p-GlcNac
[0138] The pure, fully acetylated p-GlcNac starting material may be
derivatized, by utilizing a variety of controlled conditions and
procedures, into a large range of different compounds. See FIG. 13
for a diagram depicting some of these compounds. Such derivatized
compounds may include, but are not limited to, partially
deacetylated p-GlcNac, which has been modified via chemical and/or
enzymatic means, as described in further detail, below.
Additionally, p-GlcNac, or its partially deacetylated derivative,
may be derivatized by being sulfated, phosphorylated, and/or
nitrated. Further, as detailed below, O-sulfonyl, N-acyl, O-alkyl,
N-alkyl, and N-alkylidene and N-arylidene and other derivatives may
be prepared from the p-GlcNac or partially deacetylated p-GlcNac
starting material. The partially deacetylated p-GlcNac starting
material may also be used to prepare a variety of organic salts
and/or metal chelates. Further, the p-GlcNac starting material, or
a derivative thereof, may have attached to it, either covalently or
non-covalently, any of a variety of molecules. Still further, the
p-GlcNac starting material, or a derivative thereof may be
subjected to controlled hydrolysis conditions which yield groups of
molecules having uniform and discrete molecular weight
characteristics. Such materials are useful in the present invention
provided the p-GlcNac polymer retains its semi-crystalline
structure as demonstrated by sharp, discrete peaks when the polymer
is analyzed by IR absorption spectroscopy.
[0139] One or more of the monosaccharide units of the p-GlcNac
starting material may be deacetylated to form a
partially-deacetylated poly-.beta.-1.fwdarw.4-N-acetylglucosamine
species. The deacetylated monomers can be, generally, essentially
randomly distributed throughout the polymer, or may be relative
clustered in discrete subregions within the
poly-(.beta.-1.fwdarw.4-N-acetylglucosamine polymer. A
poly-.beta.-1.fwdarw.4-N-glucosamine species starting material in
which a portion of the monosaccharide units of the
poly-(.beta.-1.fwdarw.4-N-acetylglucosamine species starting
material has been deacetylated will have a molecular weight of up
to about 30 million daltons, comprising about 150,000 glucosamine
monosaccharides covalently attached in a (.beta.-1.fwdarw.4-N
configuration. In one embodiment, at least about 90% of the
glucosamine monosaccharide units of the
poly-.beta.-1.fwdarw.4-N-glucosamine species remain acetylated,
while in other embodiments, at least about 80%, 70%, 60%, 50%, or
40% of the monosaccharide units of the
poly-(.beta.-1.fwdarw.4-N-glucosamine species remain acetylated,
provided the partially-deacetylated poly-.beta.-1.fwdarw.4
N-acetylglucosamine polymer retains its semi-crystalline structure
as demonstrated by sharp, discrete peaks when the polymer is
analyzed by IR absorption spectroscopy, as described in Example 6,
below, and as depicted in FIGS. 4A, 4D, and 4E, in contrast to IR
absorption spectra displayed by non-crystalline p-GlcNac polymers,
as depicted in FIGS. 4B and 4C.
[0140] The p-GlcNac starting material may be deacetylated by
treatment with a base to yield glucosamines with free amino groups.
This hydrolysis process may be carried out with solutions of
concentrated sodium hydroxide or potassium hydroxide at elevated
temperatures. However, to control the extent of deacetylation
precisely and to avoid degradation of the main carbohydrate chain
of the polysaccharide molecule, it is preferable that an enzymatic
procedure utilizing a chitin deacetylase enzyme be used for
p-GlcNac deacylation. Such a deacetylase enzymatic procedure is
well known to those of skill in the art and may be performed as in
(U.S. Pat. No. 5,219,749), which is incorporated herein, by
reference, in its entirety.
[0141] One or more of the monosaccharide units of the p-GlcNac
starting material may be derivatized to contain at least one
sulfate group, or, alternatively, may be phosphorylated or
nitrated, as depicted below.
##STR00001##
where, R and/or R.sub.1, in place of a hydrogen, and/or R.sub.2, in
place of --COCH.sub.3, may be a sulfate (--SHO.sub.3), a phosphate
(--P(OH).sub.2), or a nitrate (--NO.sub.2) group.
[0142] Described below are methods by which such p-GlcNac
derivatives may be prepared. Before performing methods such as
those described in this Section, it may be advantageous to first
lyophilize, freeze in liquid nitrogen, and pulverize the p-GlcNac
starting material.
[0143] Sulphated p-GlcNac derivatives may be generated, by, for
example, a two step process. In the first step, O-carboxymethyl
p-GlcNac may be prepared from the p-GlcNac and/or p-GlcNac
derivatives of the starting material by, for example, utilizing
techniques such as those described by Tokura et al. (Tokura, S. et
al., 1983, Polym. J. 15:485). Second, the sulfation step may be
carried out with, for example, N,N-dimethyl-formamide-sulfur
trioxide, according to techniques well known to those of skill in
the art, such as are described by Schweiger (Schweiger, R. G.,
1972, Carbohydrate Res. 21:219). The resulting product may be
isolated as a sodium salt. Phosphorylated p-GlcNac derivatives of
the starting material may be prepared, for example, by utilizing
techniques well known to those of skill in the art, such as those
described by Nishi et al. (Nishi, N. et al., 1986, in "Chitin in
Nature and Technology," Muzzarelli et al., eds. Plenum Press, New
York, pp. 297-299). Briefly, p-GlcNac/methanesulfonic acid mixture
may be treated with phosphorus pentoxide (in an approximately 0.5
to 4.0 molar equivalent) with stirring, at a temperature of about
0.degree. C. to about 5.degree. C. Treatment may be for about 2
hours. The resulting product may then be precipitated and washed
using standard techniques well known to those of skill in the art.
For example, the sample may be precipitated with a solvent such as
ether, centrifuged, washed with a solvent such as ether, acetone,
or methanol, and dried.
[0144] Nitrated p-GlcNac derivatives may be prepared by utilizing
techniques well known to those of skill in the art, such as those
described by Schorigin and Halt (Schorigin, R. and Halt, E., 1934,
Chem. Ber. 62:1712). Briefly, p-GlcNac and/or a p-GlcNac derivative
may be treated with concentrated nitric acid to form a stable
nitrated product.
[0145] One or more of the monosaccharide units of the p-GlcNac
starting material may contain a sulfonyl group, as depicted
below:
##STR00002##
where R.sub.3 may be an alkyl, an aryl, an alkenyl, or an alkynyl
moiety. Such a derivative may be generated by well known methods
such as the method described in Kurita et al. (Kurita, K. et al.,
1990, Polym. Prep (Am. Chem. Soc., Div. Polym. Chem.) 31:624-625).
Briefly, an aqueous alkali p-GlcNac solution may be reacted with a
chloroform solution of tosyl chloride, and the reaction may then be
allowed to proceed smoothly at low temperatures.
[0146] One or more of the monosaccharides of the p-GlcNac starting
material or its deacetylated derivative may contain one or more
O-acyl groups, as depicted below:
##STR00003##
where R.sub.4 and/or R.sub.5, in place of hydrogen, may be an
alkyl, an alkenyl, or an alkynyl moiety, and R.sub.6 may be an
alkyl, an alkenyl, or an alkynyl moiety. An example of such a
derivative may be generated by well known methods such as those
described by Komai (Komai, T. et al., 1986, in "Chitin in Nature
and Technology," Muzzarelli et al., eds., Plenum Press, New York,
pp. 497-506). Briefly, p-GlcNac may be reacted with any of a number
of suitable acyl chlorides in methanesulfonic acid to yield
p-GlcNac derivatives which include, but are not limited to,
caproyl, capryl, lanoyl, or benzoyl derivatives.
[0147] One or more of the monosaccharides of the deacetylated
p-GlcNac starting material may contain an N-acyl group, as depicted
below:
##STR00004##
where R.sub.7 may be an alkyl, an alkenyl, or an alkynyl moiety.
Such a derivatization may be obtained by utilizing techniques well
known to those of skill in the art, such as the technique described
in Hirano et al. (Hirano, S. et al., 1976, Carbohydrate Research
47: 315-320).
[0148] Deacetylated p-GlcNac is soluble in a number of aqueous
solutions of organic acids. The addition of selected carboxylic
anhydrides to such p-GlcNac-containing solutions, in aqueous
methanolic acetic acid, results in the formation of N-acyl p-GlcNac
derivatives.
[0149] One or more of the monosaccharides of the deacetylated
p-GlcNac starting material or of its deacetylated derivative, may
contain an O-alkyl group, as depicted below:
##STR00005##
where R.sub.8 may be an alkyl, and alkenyl, or a alkynyl moiety.
Such a derivatization may be obtained by using techniques well
known to those of skill in the art. For example, the procedure
described by Maresh et al. (Maresh, G. et al., in "Chitin and
Chitosan," Skjak-Braek, G. et al., eds., 1989, Elsevier Publishing
Co., pp. 389-395). Briefly, deacetylated p-GlcNac may be dispersed
in dimethoxyethane (DME) and reacted with an excess of propylene
oxide. The period of the reaction may be 24 hours, and the reaction
takes place in an autoclave at 40.degree. to 90.degree. C. The
mixture may then be diluted with water and filtered. The DME may be
removed by distillation. Finally, the end-product may be isolated
via lyophilization.
[0150] One or more of the monosaccharide units of the p-GlcNac
starting material may be an alkali derivative, as depicted
below:
##STR00006##
Such a derivative may be obtained by using techniques well known to
those of skill in the art. For example, a method such as that
described by Noguchi at (Noguchi, J. et al., 1969, Kogyo Kagaku
Zasshi 72:796-799) may be utilized Briefly, p-GlcNac may be
steeped, under vacuo, in NaOH (43%, preferably) for a period of
approximately two hours at about 0.degree. C. Excess NaOH may then
be removed by, for example, centrifugation in a basket centrifuge
and by mechanical pressing.
[0151] One or more of the monosaccharide units of the deacetylated
derivative of the p-GlcNac starting material may contain an N-alkyl
group, as depicted below:
##STR00007##
where R.sub.9 may be an alkyl, an alkenyl, or an alkynyl moiety.
Such a derivatization may be obtained by utilizing, for example, a
procedure such as that of Maresh et al. (Maresh, O. et al., in
"Chitin and Chitosan," Skjak-Brack, G. et al., ed. 1989, Elsevier
Publishing Co., pp. 389-395), as described, above, for the
production of O-alkyl p-GlcNac derivatives.
[0152] One or more of the monosaccharide units of the deacetylated
derivative of the p-GlcNac starting material may form a salt as
depicted below:
##STR00008##
where R.sub.11 may be an alkyl, an alkynyl, or an alkynyl moiety.
Such a derivatization may be obtained by using techniques well
known to those of skill in the art. For example, a procedure such
as that described by Austin and Sennett (Austin, P. R. and Sennett,
S., in "Chitin in Nature and Technology," 1986, Muzzarelli, R. A.
A. et al., eds. Plenum Press, pp. 279-286) may be utilized.
Briefly, deacetylated p-GlcNac may be suspended in an organic
medium such as, for example, ethyl acetate or isopropanol, to which
may be added an appropriate organic acid such as, for example,
formic, acetic, glycolic, or lactic acid. The mixture may be
allowed to stand for a period of time (1 to 3 hours, for example).
The temperature of reaction and drying may vary from about
12.degree. C. to about 35.degree. C., with 20.degree. to 25.degree.
C. being preferred. The salts may then be separated by filtration,
washed with fresh medium, and the residual medium evaporated.
[0153] One or more of the monosaccharide units of the deacetylated
derivative of the p-GlcNac starting material may form a metal
chelate, as depicted below:
##STR00009##
where R.sub.12 may be a metal ion, particularly one of the
transition metals, and X is the dative bond established by the
nitrogen electrons present in the amino and substituted amino
groups present in the deacetylated p-GlcNac.
[0154] One or more of the monosaccharide units of the deacetylated
derivative of the p-GlcNac starting material may contain an
N-alkylidene or an N-arylidene group, as depicted below:
##STR00010##
where R.sub.13 may be an alkyl, an alkenyl, an alkynyl, or an aryl
moiety. Such a derivatization may be obtained by using techniques
well known to those of skill in the art. For example, a procedure
such as that described by Hirano et al. (Hirano, S. et al., 1981,
J. Biomed. Mat. Res. 15:903-911) may be utilized. Briefly, an
N-substitution reaction of deacetylated p-GlcNac may be performed
with carboxylic anhydrides and/or arylaldehydes to yield acyl-
and/or arylidene derivatives.
[0155] Further, the p-GlcNac starting material, or its
partially-deacetylated derivative, may be subjected to controlled
hydrolysis conditions, which yield groups of molecules having
uniform, discrete molecular weight and other physical
characteristics. Such hydrolysis conditions may include, for
example, treatment with the enzyme, lysozyme. p-GlcNac may be
exposed to lysozyme for varying periods of time, in order to
control the extent of hydrolysis. In addition, the rate of
hydrolysis may be controlled as a function of the extent to which
the p-GlcNac that is being lysozyme treated has been deacetylated
(see, for example the Examples provided in Section 15, and depicted
in FIGS. 18-20). Such enzymatic, partial digestion reactions may
also be controlled by varying the concentration of the substrate,
the enzyme, or both the substrate and enzyme, as well as the pH and
temperature. In another embodiment, p-GlcNac polymers are reduced
in size by sonication which may be varied not only by the power of
the instrument used but also by the pH, salt concentration, and
temperature of the sample. Solubilization of p-GlcNac or
derivatives thereof are described below in Section 5.5.
Accordingly, by using one or more of these methods, either alone or
in combination with one another, higher molecular weight p-GlcNac
polymers can be hydrolyzed to smaller fragments, which can be
chromatographically separated according to size using, for example,
column chromatography.
[0156] For example, one skilled in the art will vary the extent of
partial digestion of p-GlcNac to provide reaction product having a
desired range of molecular weight. In other embodiments, the
substrate used for partial digestion with lysozyme, is p-GlcNac
that has been sonicated and/or partially de-acetylated. By
combining partial enzymatic digestion with separation techniques,
such as column chromatography, HPLC separations or other techniques
and methods well-known in the art, a skilled artisan can isolate
digestion products with a narrow range of molecular weight
distribution. Moreover, by combining the products of a series of
partial-digestion reactions, one skilled in the art can assemble a
composition comprising p-GlcNac polymers having a wider range of
molecular weight species of semi-crystalline p-GlcNac products,
including, e.g., the populations disclosed herein, that is polymers
comprising from about 50 to about 150,000 monomeric units in one
embodiment, as well as about 50 to about 50,000, about 50 to about
10,000, and about 50 to about 4,000 monomeric units.
[0157] Deacetylation conditions may be as described earlier in this
Section. The more fully a p-GlcNac molecule has been deacetylated,
between about 20 and about 90 percent deacetylated, the more fully
the molecule will be hydrolyzed in a given time. Changes in
physical characteristics, in addition to the lowering of molecular
weight, may be elicited by hydrolysis and/or deacetylation
treatments.
[0158] Further, a variety of molecules may be covalently or
non-covalently functionally attached to the deacetylated
derivatives of the p-GlcNac starting material. Such molecules may
include, but are not limited to such polypeptides as growth
factors, such as nerve growth factor, proteases, such as pepsin,
hormones, or peptide recognition sequences such as RGD sequences,
fibronectin recognition sequences, laminin, integrins, cell
adhesion molecules, and the like. See, e.g., the compounds
discussed, below, in Section 5.6.1.1. Covalent attachment of
molecules to the exposed primary amines of deacetylated p-GlcNac
may be accomplished by, for example, chemical attachment utilizing
bi-functional cross-linking reagents that act as specific length
chemical spacers. Such techniques are well known to those of skill
in the art, and may resemble, for example, the methods of Davis and
Preston (Davis, M. and Preston, J. F. 1981, Anal. Biochem.
11:404-407) and Staros et al. (Staros, J. V. et al., 1986, Anal.
Biochem. 156:220-222). Briefly, carboxylic residues on the peptide
to be attached to the deacetylated or partially deacetylated
p-GlcNac starting material may be activated and then crosslinked to
the p-GlcNac. Activation may be accomplished, for example, by the
addition of a solution such as carbodiimide EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to a peptide
solution in a phosphate buffer. Preferably, this solution would
additionally contain a reagent such as sulpho-NHS
(N-hydroxysulphosuccinimide) to enhance coupling. The activated
peptide may be crosslinked to the deacetylated p-GlcNac by mixing
in a high pH buffer, such as carbonate buffer (pH 9.0-9.2).
[0159] The biological activity of the attached peptide (or any
covalently attached molecule) can be maintained by varying the
length of the linker molecule (e.g., the bi-functional crosslinking
compound) utilized to attach the molecule to the p-GlcNac starting
material. An appropriate linker length for a given molecule to be
attached which will not alter the biological activity of the
attached molecule can routinely be ascertained. For example, the
biological, activity (e.g., a therapeutically effective level of
biological activity) of a molecule which has been attached via a
linker of a given length can be tested by utilizing well-known
assays specific for the given molecule being attached.
[0160] Additionally, in order to maintain the biological activity
of the molecule being attached, it may be necessary to utilize a
linker which can be cleaved by an appropriate naturally occurring
enzyme to release the peptide (or any covalently attached
molecule).
[0161] As above, assays commonly employed by those of skill in the
art may be used to test for the retention of the biological
activity of the particular molecule being attached to ensure that
an acceptable level of activity (e.g., a therapeutically effective
level activity) is retained.
[0162] Alternatively, molecules such as those described above may
be non-covalently attached to p-GlcNac and its derivatives using
techniques well known to those of skill in the art. For example, a
molecule or molecules of choice may be mixed with suspensions of
p-GlcNac, or partially deacetylated p-GlcNac solution, with a
p-GlcNac-lactate solution, with a deacetylated or partially
deacetylated p-GlcNac salt solution, or with any p-GlcNac
derivative solution. The mixtures can then be lyophilized.
Molecules become bound to the p-GlcNac matrices following
lyophilization, presumably via hydrophobic, electrostatic and other
non-covalent interactions. Such p-GlcNac formulations are,
therefore, very easy to produce. Further, such formulations can
effectively be achieved with a wide variety of molecules having a
broad spectrum of physical characteristics and water solubility
properties, ranging from the most hydrophobic to the most
hydrophilic. Upon attachment of the molecule or molecules, assays
commonly employed by those of skill in the art to test the activity
of the particular non-covalently attached molecule or molecules can
be used to ensure that an acceptable level of activity (e.g., a
therapeutically effective activity) is achieved with the attached
molecule.
[0163] Alternatively, hybrids comprising p-GlcNac and/or p-GlcNac
derivatives may be formed. Such hybrids may contain any of a number
of natural and/or synthetic materials, in addition to p-GlcNac
and/or p-GlcNac derivatives. For example, hybrids may be formed of
p-GlcNac and/or p-GlcNac derivatives plus one or more extracellular
matrix (ECM) components. Such ECM components may include, but are
not limited to, collagen, fibronectin, glycosaminoglycans, and/or
peptidoglycans. Hybrids may also be formed of p-GlcNac and/or
p-GlcNac derivatives plus one or more synthetic materials such as,
for example, polyethylene. Such a p-GlcNac/polyethylene or p-GlcNac
derivative/polyethylene hybrid may be made by thermally linking the
hybrid components via, for example, autoclaving. Such hybrid
polymers are useful in the present methods, provided the hybrid
polymer retains the p-GlcNac semi-crystalline structure as
demonstrated by sharp, discrete peaks when the polymer is analyzed
by IR absorption spectroscopy, as described in Example 6,
below.
[0164] In the case of a collagen/p-GlcNac hybrid, briefly, a
p-GlcNac suspension and a collagen suspension may be mixed and
lyophilized, and crosslinked, preferably dehydrothermally
crosslinked. The collagen species of such hybrids may be native or
synthetic, and may be of human or non-human, such as bovine, for
example, origin. p-GlcNac/collagen and/or p-GlcNac
derivative/collagen hybrid materials exhibit uniform properties,
and form a porous matrix. The Working Example presented in Section
13 below, demonstrates the formation, properties and usefulness of
such a p-GlcNac/collagen hybrid.
[0165] Additionally, an iodo-p-GlcNac derivative may be
copolymerized with, for example, styrene, for the manufacture of
novel plastic materials. Iodo-p-GlcNac can be prepared by a process
similar to that described by Kurita and Inoue (Kurita, K. and
Inoue, S., 1989, in "Chitin and Chitosan," Skjak-Braek et al.,
eds., Elsevier Science Publishing Co., Inc., p. 365), via
tosylation and iodination of p-GlcNac. The iodo derivative of
p-GlcNac can then be dispersed in nitrobenzene and reacted with
styrene, with tin (IV) chloride being used as a catalyst.
[0166] Hybrids comprising combinations of deacetylated p-GlcNac and
such compounds as, for example sodium alginate, and carboxymethyl
p-GlcNac may be formulated using techniques such as those described
herein. Such combinations may be formed or reformed into, for
example, membranes and fibers.
[0167] Complexes of partially deacetylated p-GlcNac with polyanions
such as, for example, polyacrylic acid or pectin, possessing both
positive and negative charges, may be formulated. The formation of
such complexes may be accomplished according to a method similar to
that described by Mireles et al. (Mireles, C. et al., 1992, in
"Advances in Chitin and Chitosan," Brine, C. J. et al., eds.,
Elsevier Publishers, Ltd.). Partially deacetylated p-GlcNac and
polyacrylic acid, carrageenan or pectin, for example, are dissolved
in HCl and NaCl, respectively, and the reactant solutions, with
equal pH, are mixed. This operation produces effective molecules
possessing both positive and negative characteristics, useful, for
example, in the immobilization of enzymes and therapeutic
compounds.
[0168] 5.5. Reformulations
[0169] The p-GlcNac starting material, as well as its partially
deacetylated derivatives and/or their derivatives, such as those
described above in Section 5.4, may be dissolved and subsequently
reformulated into a variety of shapes and configurations.
[0170] Solution of the p-GlcNac starting material can be achieved
by treatment with dimethyl acetamide (DMA)/lithium chloride.
p-GlcNac may be readily dissolved by stirring in a DMA solution
containing 5% LiCl (by weight of the DMA). Water soluble p-GlcNac
derivatives, such as p-GlcNac salts, may be dissolved in water.
p-GlcNac which has been partially deacetylated may be put into
solution in, for example, a mild acidic solution, such as 1% acetic
acid. p-GlcNac derivatives that are water-insoluble may be put into
solution in organic solvents.
[0171] Derivatization of p-GlcNac in DMA:LiCl with phenyl
isocyanates may be used to produce carbanilates. Further,
derivatization of p-GlcNac in DMA:LiCl with
toluene-p-sulphonylchloride may be used to produce
toluene-p-sulfonate.
[0172] The p-GlcNac starting material, its partially deacetylated
derivatives, and/or their derivatives in solution may then be
precipitated and reformulated into shapes which include, but are
not limited to, mats, strings, microspheres, microbeads, membranes,
fibers, microfibers, powders, and sponges. Further, ultrathin
(i.e., less than about 1 micron thick) uniform membranes may be
formulated.
[0173] Such reformulations may be achieved, by, for example, taking
advantage of the fact that pure p-GlcNac is insoluble in solutions
such as water and alcohol, preferably ethanol. Introduction, by
conventional means, such as by injection, for example, of the
p-GlcNac-containing DMA/LiCl mixture into such a water or alcohol,
preferably ethanol, solution will bring about the reprecipitation,
and therefore reformulation, of the dissolved p-GlcNac. Such a pure
p-GlcNac reformulation is demonstrated in the Working Example
presented, below, in Section 11. In the case of water soluble
p-GlcNac derivatives, reformulations may be achieved by
reprecipitating in such organic solvents as, for example, ethyl
acetate or isopropanol. Reformulations of p-GlcNac which has been
partially deacetylated may be achieved by reprecipitating in an
alkaline solution. Water-insoluble p-GlcNac derivatives may be
reformulated by reprecipitation in aqueous solutions, such as, for
example, water.
[0174] p-GlcNac membranes and three-dimensional p-GlcNac matrices
may be produced via methods which provide for the formation of
controlled average pore sizes within either the membranes or the
matrices. Pore size can be controlled in membranes and matrices by
varying the amount of p-GlcNac material used, and by the addition
of certain solvents such as methanol or ethanol, with ethanol being
preferred, in specific amounts, ranging from about 5% to about 40%,
prior to the formation of membranes and/or matrices. In general,
the greater the percentage of solvent, the smaller the average pore
size formed will be. The Example presented, below, in Section 15,
demonstrates the synthesis and characterization of such porous
p-GlcNac structures.
[0175] In other embodiments, the semi-crystalline p-GlcNac is
formulated as a gel, foam, spray, or as a solution or suspension
comprising microspheres, microbeads, or microfibrils. Such
formulations, therefore may further comprise a suitable amount of a
pharmaceutically acceptable vehicle or carrier so as to provide the
form for proper administration of the semi-crystalline p-GlcNac to
the patient.
[0176] In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in mammals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which a therapeutic agent is
administered. Such pharmaceutical carriers can be liquids, such as
water and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. The pharmaceutical carriers can be saline,
gum acacia, gelatin, starch paste, talc, keratin, colloidal silica,
urea, and the like. In addition, auxiliary, stabilizing,
thickening, lubricating and coloring agents may be used. When
administered to a patient, the p-GlcNac and the pharmaceutically
acceptable carriers are preferably sterile. Saline solutions and
aqueous dextrose and glycerol solutions can be employed as liquid
carriers. Suitable pharmaceutical carriers also include excipients
such as starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene,
glycol, water, ethanol and the like. p-GlcNac compositions, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents.
[0177] Compositions containing p-GlcNac can take the form of
solutions, suspensions, suppositories, emulsions, aerosols, sprays,
or any other form suitable for use. Other examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0178] Although the p-GlcNac formulations and compositions will be
supplied as a pre-mixed dosage form, in other embodiments, the
semi-crystalline p-GlcNac disclosed herein can be supplied
separately, for example as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent, which can be
suspended or dissolved at a desired concentration in a
pharmaceutically acceptable vehicle or solvent prior to use.
[0179] The amount of the semi-crystalline p-GlcNac effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. In addition, in vitro or in vivo
assays can optionally be employed to help identify optimal dosage
ranges. The precise dose of semi-crystalline p-GlcNac to be
employed in the compositions will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. However, semi-crystalline p-GlcNac is
generally topically applied within a range of about 1 mg/cm.sup.2
to about 500 mg/cm.sup.2. In other embodiments, semi-crystalline
p-GlcNac is generally topically applied within a range of about 2
mg/cm.sup.2 to about 100 mg/cm.sup.2, 5 mg/cm.sup.2 to about 50
mg/cm.sup.2, and 10 mg/cm.sup.2 to about 20 mg/cm.sup.2. Effective
doses may be extrapolated from dose-response curves derived from in
vitro or animal model test systems. In addition to those presented
infra in the Examples provided in Sections 16 and 17, other animal
model test systems that are well known in the art, include, without
limitation the following: (a) a porcine model of partial
hepatectomy for evaluating hemostasis treatment as described by
Davidson et al. (Davidson et al. 2000, Br. J. Surg. 82(6): 790-95);
(b) a canine, bleeding ulcer mode, for evaluating treatments
intended to achieve hemostasis is described by Pasricha et al.
(Pasricha et al. 1999 Gastointest Endosc 42(5): 627-31); (c) a
surgical bleeding model in the rat, based upon treatment of liver
incisions, has been described by Sirieix et al (Sirieix et al 1998
Ann Vasc Surg 12(4): 311-16); (d) a method for evaluating
vasoconstriction in isolated rat thoracic aortic rings with intact
endothelium has been described by Kim et al (Kim et al 2000 J Lab
Clin Med 135(2): 180-87; also see Guo et al 1994 Methods Find Exp
Clin Pharmacol 15(5): 347-54); (e) an experimental model intended
to measure both vessel diameter and blood flow through that vessel
in the rabbit has been described by Caron et al (Caron et al 1998
Artif Cells Blood Substit Immobil Biotechnol 26(3): 293-308); (f) a
method permitting the direct observation of uterine microvessels in
the rat, permitting evaluation of the circumferential diameters of
arterioles as a function of the amount of vasoactive agent applied
has been described by Alsip et al (Alsip et al 1996 Am J Obstet
Bynecol 17(2):388-95); and (g) a model system using spontaneously
hypertensive rats has been described by Schiffrin et al, which
inter alia, evaluates the level of immunoreactive endothelin in
blood vessels using radioimmunoassay procedures (Schiffrin et al
1995 Br J Pharmacol 111(8): 1377-81).
[0180] The particular formulation of semi-crystalline p-GlcNac used
will vary depending upon the intended application. For example,
semi-crystalline p-GlcNac may be formulated and manufactured as a
membrane, or bandage etc. for direct application to an accessible
surface. In such formulations, the semi-crystalline p-GlcNac can be
combined with one or other materials, including but not limited to
natural or man-made fibers, and/or reformulated as a copolymer as
described herein. The amount of semi-crystalline p-GlcNac/cm.sup.2
formulated into such a material is determined by the intended use,
e.g., the lower ranges for treating, inter alia, minor cuts and
scrapes, and higher p-GlcNac levels for treatment of more serious
injuries. The size, shape, thickness, and overall composition,
including the total amount of semi-crystalline p-GlcNac formulated
therein, of such materials is similarly determined by the intended
use.
[0181] Where the semi-crystalline p-GlcNac is to be topically
administered to a surface not readily accessible, e.g., oral or
nasal cavities, or deep wounds to the body, the semi-crystalline
p-GlcNac is formulated, inter alia, as a gel, foam, spray,
emulsion, suspension or solution, employing the pharmaceutically
acceptable carriers and vehicles disclosed above. Such
formulations, which usually will be non-barrier forming materials,
generally comprise microspheres, microbeads, or microfibrils formed
from semi-crystalline p-GlcNac, and may further comprise materials,
including but not limited to, natural or man-made fibers, and/or
semi-crystalline p-GlcNac reformulated as a copolymer as described
herein. Again, the amount and/or concentration of semi-crystalline
p-GlcNac included in such formulations is dependent upon the
intended use, and would be apparent to those of skill in the art
and readily determined through routine in vitro and in vivo
testing, especially with animal model systems well known in the
art.
[0182] Since the modulating effects of semi-crystalline p-GlcNac on
vascular structure and/or function are both localized and
transient, administration of formulations comprising
semi-crystalline p-GlcNac may be repeated, at intervals, until the
condition to be corrected is resolved. Generally, such intervals
are about one hour, but they may be shorter or longer, depending on
the nature of the condition treated and the amount of
semi-crystalline p-GlcNac applied. In those instances in which a
composition comprising a semi-crystalline p-GlcNac formulation has
been applied to a relatively non-accessible surface, bio-degradable
compositions and formulations are preferred.
[0183] 5.6. Uses
[0184] The p-GlcNac starting material has a variety of uses,
including modulation of vascular structure and/or function via, for
example, stimulation of endothelin-1 release, vasoconstriction, and
reduction in blood flow out of a breached vessel, as well as
contributing to or effecting cessation of bleeding. The
topically-applied p-GlcNac of the present invention is
biocompatible, biodegradable, nontoxic, and non-pyrogenic. Because
the p-GlcNac materials of the present invention are also
immunoneutral, they do not elicit an immune response in humans, and
therefore are particularly advantageously used in the formulation
of the devices disclosed herein, which include but are not limited
to films, membranes, gels, sponges, microspheres, microbeads,
microfibrils, foams, and sprays. Certain additional materials, such
as natural alginates and, in some cases, synthetic polymers, can
also be used in the construction of such materials and devices, in
combination with the p-GlcNac described herein, provided the
poly-.beta.-1.fwdarw.4 N-acetylglucosamine polymer retains its
semi-crystalline structure as demonstrated by sharp, discrete peaks
when the polymer is analyzed by IR absorption spectroscopy, as
described in Example 6, below. In one embodiment, the p-GlcNac
consists essentially of fully acetylated, semi-crystalline polymers
of .beta.-1.fwdarw.4 N-acetylglucosamine wherein the polymer
comprises about 50 to about 150,000 N-acetylglucosamine
monosaccharides covalently attached in a .beta.-1.fwdarw.4
conformation, free of protein, substantially free of other organic
contaminants, substantially free of inorganic contaminants, and
having a molecular weight of about 10,000 daltons to about 30
million daltons. In other embodiments, the p-GlcNac consists
essentially of fully acetylated, semi-crystalline polymers of
.beta.-1.fwdarw.4 N-acetylglucosamine wherein the polymer comprises
about 50 to about 50,000, about 50 to about 10,000, or about 50 to
about 4,000 N-acetylglucosamine monosaccharides covalently attached
in a .beta.-1.fwdarw.4 conformation, free of protein, substantially
free of other organic contaminants, substantially free of inorganic
contaminants, and having a molecular weight of about 10,000 daltons
to about 10 million daltons, of about 10,000 daltons to about 2
million daltons, and about 10,000 daltons to about 800,000 daltons,
respectively.
[0185] 5.6.1. Stimulation of Endothelin-1 Release
[0186] p-GlcNac materials of the present invention are used, for
example, to stimulate the release of endothelin-1, as demonstrated
successfully in the Example presented in section 16, below.
Stimulation of endothelin-1 release is used, inter alia, for the
treatment of menorrhagia associated with markedly lower levels of
endothelin-1 production by uterine endometrial tissue.
[0187] Stimulation of endothelin-1 release is achieved by topical
application of compositions and materials comprising p-GlcNac to
target tissue of a human or a non-human mammal, including but not
limited to veterinary and companion animals. Such materials and
compositions may comprise certain additional materials, such as
natural alginates and, in some cases, synthetic polymers, in
combination with the p-GlcNac described herein. The p-GlcNac of
such compositions and materials, in preferred embodiments, consists
essentially of fully acetylated, semi-crystalline polymers of
.beta.-1.fwdarw.4 N-acetylglucosamine polymers which are free of
protein, substantially free of other organic contaminants, and are
substantially free of inorganic contaminants, and have a molecular
weight of up to about 30 million daltons.
[0188] Materials of the present invention, which comprise p-GlcNac,
are formulated and applied as, for example, gels, films, membranes,
and sponges. Such materials may also be formulated and applied as a
solution or suspension of microspheres, microbeads, microfibrils,
or as a foam or spray. Accordingly, the materials of the present
invention that comprise p-GlcNac need not be barrier-forming
materials.
[0189] Compositions and materials of the present invention,
comprising p-GlcNac, are applied directly to target tissue, i.e.
tissue in which it is desired to stimulate endothelin-1 release,
which could be, e.g., uterine endometrial tissue of patients
affected by menorrhagia. The target tissue is, generally,
endothelial tissue, and more particularly, will include blood
vessels, which can be arteries, veins, or capillaries. The
materials comprising semi-crystalline p-GlcNac are topically
applied, for example, as a gel, film, membrane, sponge, spray or
foam, as well as a suspension, emulsion, or solution of
microspheres, microbeads, or microfibrils.
[0190] Topical application of the compositions and materials of the
present invention, which comprise p-GlcNac, stimulate, relative to
target tissue untreated with p-GlcNac, release of endothelin-1 in
the target tissue, that is localized, transient, and dependent upon
the dose of p-GlcNac administered. Stimulation of endothelin-1
release is localized in that it is most pronounced in that tissue
in direct contact with the material comprising p-GlcNac, and,
furthermore, the degree of stimulation of endothelin-1 release in
the adjacent tissue diminishes as the distance from the point of
contact between the target tissue and the material comprising
p-GlcNac increases (see e.g. the Example presented in section 16,
infra).
[0191] Stimulation of endothelin-1 release is transient in that the
level of endothelin-1 in tissue contacted with material comprising
semi-crystalline p-GlcNac is greatest shortly after administration
of such materials and declines thereafter to the levels observed
prior to stimulation. That is, the concentration of endothelin-1 in
the contacted tissue is greatest generally not later than 15
minutes after administration of semi-crystalline p-GlcNac, and the
concentration of endothelin-1 returns substantially to the level
observed immediately prior to that contact, within about 60 minutes
after administration of semi-crystalline p-GlcNac (see e.g. the
Example presented in section 16, infra.). Accordingly, in those
instances requiring prolonged stimulation of endothelin-1 release,
additional aliquots or doses of compositions and/or materials
formulated with semi-crystalline p-GlcNac, are applied to the
target tissue, in a sequential manner.
[0192] Stimulation of endothelin-1 release is dose-dependent in
that the level of endothelin-1 released by endothelial tissue
contacted with material comprising p-GlcNac is substantially
proportional to the amount p-GlcNac in that material (for a
representative demonstration of such a "substantially
proportionate" effect, see e.g. the Example presented in section
16, infra.). Accordingly, compositions and materials are formulated
and constructed to comprise that level of p-GlcNac required for the
level of stimulation of endothelin-1 release needed. Determination
of such levels is readily ascertained from routine in vitro
experimentation, and animal model testing. Accordingly, in those
instances in which a greater degree of stimulation of endothelin-1
release is required, compositions and materials are formulated with
an increased concentration of p-GlcNac.
[0193] 5.62. Induction of Vasoconstriction
[0194] p-GlcNac materials of the present invention are used, for
example, to induce vasoconstriction, as demonstrated successfully
in the Examples presented in Sections 16 and 17, below, as well as
depicted in FIG. 22. Vasoconstriction is achieved by topical
application of compositions and materials comprising
semi-crystalline p-GlcNac to target tissue of a human or a
non-human mammal, including but not limited to veterinary and
companion animals.
[0195] Clinical applications for which the topical application of
compositions comprising semi-crystalline p-GlcNac are useful
include, inter alia, use in diagnostic procedures which result in
biopsy wounds in, for example, liver and kidney, or result in
puncture wounds in blood vessels, e.g. cardiac catheterization and
balloon angioplasty procedures. The methods of the present
invention are therefore particularly useful in patients afflicted
with any form of coagulopathy, which may arise from a genetic
defect or from administration of an anticoagulant such as coumadin
or heparin. While not wishing to be bound to any particular theory
or mechanism, it is believed that vasoconstriction elicited by
topical application of semi-crystalline p-GlcNac physically reduces
the size of the puncture wound and thereby facilitates or effects
cessation of bleeding in a manner and by a mechanism that is not
dependent upon clot formation.
[0196] The materials and compositions used in the present invention
may comprise certain additional materials, such as natural
alginates and, in some cases, synthetic polymers, in combination
with the p-GlcNac described herein. The p-GlcNac of such
compositions and materials, in preferred embodiments, consists
essentially of fully acetylated, semi-crystalline polymers of
.beta.-1.fwdarw.4 N-acetylglucosamine, which are free of protein,
substantially free of other organic contaminants, and substantially
free of inorganic contaminants, and having a molecular weight of up
to about 30 million daltons.
[0197] Materials of the present invention, which comprise p-GlcNac,
are formulated as, for example, gels, films, membranes, and
sponges. Such materials may also be formulated an applied as a
solution or suspension of microspheres, microbeads, microfibrils,
or as a spray or foam. Accordingly, the materials of the present
invention that comprise p-GlcNac need not be barrier-forming
materials.
[0198] Compositions and materials of the present invention,
comprising p-GlcNac, are applied to the skin or other tissue
adjacent to or contiguous with the target tissue, or are applied
directly to the target tissue, i.e. tissue or vessel in which it is
desired to induce vasoconstriction. The target tissue or vessel
includes, generally, arteries, veins, or capillaries. The materials
of the present invention which comprise p-GlcNac are topically
applied, for example, as a gel, film, membrane, or sponge, spray or
foam, or as suspension or solution of microspheres, microbeads, or
microfibrils.
[0199] Topical application of the compositions and materials of the
present invention, which comprise p-GlcNac, stimulate
vasoconstriction that is localized, and transient, and dependent
upon the dose of p-GlcNac administered. Induction of
vasoconstriction is localized in that it is most pronounced in
those vessels in direct contact with the material comprising
p-GlcNac, and, furthermore, the degree of stimulation of
vasoconstriction diminishes as the distance from the point of
contact of the material comprising p-GlcNac and the target vessel
increases.
[0200] Stimulation of vasoconstriction is transient in that the
degree of vasoconstriction in the vessel is greatest shortly after
administration of the p-GlcNac materials of the present invention
and declines thereafter to the levels observed prior to
stimulation. That is, the degree of vasoconstriction is greatest
generally not later than 15 minutes after administration of
p-GlcNac, and then declines to substantially control levels, within
about 60 minutes after administration of p-GlcNac. Accordingly, in
those instances requiring prolonged vasoconstriction, additional
aliquots or doses of compositions and/or materials formulated with
p-GlcNac, are applied to the target tissue, in a sequential
manner.
[0201] Induction of vasoconstriction is dose-dependent in that the
degree of vasoconstriction in those vessels contacted with material
comprising p-GlcNac is substantially proportional to the amount
p-GlcNac in that material. Accordingly, compositions and materials
are formulated and constructed to comprise that level of p-GlcNac
required for the degree of vasoconstriction desired. Determination
of such levels of p-GlcNac is readily ascertained from routine in
vitro experimentation, and animal model testing. Accordingly, in
those instances in which a greater induction of vasoconstriction is
required, compositions and materials are formulated with an
increased concentration of p-GlcNac.
[0202] 5.6.3. Reduction in Blood Flow Out of a Breached Vessel
[0203] The methods of the present invention, which comprise topical
administration of material comprising p-GlcNac, are also used, for
example, to reduce blood flow out of a breached vessel in a target
tissue. Clinical uses for the topical application of p-GlcNac to
effect a reduction in blood flow out of a breached vessel, include,
but are not limited to, treatment of abdominal aortic aneurysms,
embolization treatment of tumors, uterine fibroid lesions and
cerebral aneurysms, wounds including, for example, spleen, liver
and blood vessel injuries, and in standard and minimally invasive
surgical procedures, for example, endometriosis surgery and
operations on the gallbladder. In each of these examples, reduction
in blood flow out of a breached vessel as a result of topical
application of p-GlcNac-containing materials, results in a
reduction in blood loss during the procedure. Accordingly, use of
the compositions and methods disclosed herein to bring about
vasoconstriction would be particularly useful for the treatment of
such condition is patients afflicted with any form of coagulopathy,
which may arise from a genetic defect or from administration of an
anticoagulant such as coumadin or heparin.
[0204] Materials and compositions used in the present methods may
comprise certain additional materials, such as natural alginates
and, in some cases, synthetic polymers, in combination with the
p-GlcNac described herein. The p-GlcNac of such compositions and
materials, in preferred embodiments, consists essentially of fully
acetylated, semi-crystalline polymers of .beta.1.fwdarw.4
N-acetylglucosamine wherein the polymer is free of protein,
substantially free of other organic contaminants, substantially
free of inorganic contaminants, and having a molecular weight of up
to about 30 million daltons.
[0205] Materials of the present invention, which comprise p-GlcNac,
are formulated as, for example, as gels, films, membranes, and
sponges. Such materials may also be formulated and applied as a
solution or suspension of microspheres, microbeads, or
microfibrils, and/or applied as foam or spray. Accordingly, the
materials of the present invention that comprise p-GlcNac need not
be barrier-forming materials.
[0206] Compositions and materials of the present invention,
comprising p-GlcNac, are applied either to the skin or other tissue
adjacent to or contiguous with the target tissue, or are applied
directly to the target tissue, i.e. tissue or blood vessel in which
it is desired to reduce blood flow out of a breached vessel. The
target vessel may be an artery, vein, or capillary. The materials
of the present invention, which comprise p-GlcNac, are topically
applied, for example, as a gel, film, membrane, sponge, spray or
foam, or as a suspension or solution of microspheres, microbeads,
and/or microfibrils.
[0207] Topical application of the compositions and materials of the
present invention, which comprise p-GlcNac, induce a reduction in
blood flow out of a breached vessel that is localized, transient,
and dependent upon the dose of p-GlcNac administered. Reduction in
blood flow out of a breached vessel is localized in that it is most
pronounced in vessels in direct contact with the material
comprising p-GlcNac, and, furthermore, the degree of reduction in
blood flow out of a breached vessel diminishes as the distance from
the point of contact between the material comprising p-GlcNac and
the target vessel increases.
[0208] Reduction in blood flow out of a breached vessel is
transient in that the reduction in blood flow contacted with
material comprising p-GlcNac is greatest shortly after
administration of such materials and blood flow out of a breached
vessel thereafter returns to control levels. That is, the degree of
reduction of blood flow out of a breached vessel is greatest
generally not later than 15 minutes after administration of
p-GlcNac, and then blood flow out of a breached vessel returns to
control levels within about 60 minutes after administration of
p-GlcNac. Accordingly, in those instances requiring prolonged
reduction of blood flow out of a breached vessel, additional
aliquots or doses of compositions and/or materials formulated with
p-GlcNac, are applied to the target tissue or vessel, in a
sequential manner.
[0209] Reduction of blood flow out of a breached vessel is
dose-dependent in that the reduction in blood flow out of vessels
contacted with material comprising p-GlcNac is substantially
proportional to the amount p-GlcNac in that material. Accordingly,
compositions and materials are formulated and constructed to
comprise that level of p-GlcNac required for the reduction in blood
flow out of a breached vessel desired. Determination of such levels
is readily ascertained from routine in vitro experimentation, and
animal model testing. Accordingly, in those instances in which a
greater degree reduction in blood flow out of a breached vessel is
required, compositions and materials are formulated with an
increased concentration of p-GlcNac.
[0210] 5.6.4 Specific Indications for Use of the Disclosed
Methods
[0211] Specific instances in which stimulation of endothelin-1
release, vasoconstriction, and/or reduction in blood flow out of a
breached vessel, as well as cessation of bleeding are desired
include, but are not limited to, use in diagnostic procedures which
result in biopsy wounds in, for example, liver and kidney; in
embolization procedures including, but not limited to the
prevention of bleeding following endovascular treatment of
abdominal aortic aneurysms, as well as embolization treatment of
tumors, uterine fibroid lesions and cerebral aneurysms; for
treatment of menorrhagia; in wounds including, for example, spleen,
liver and blood vessel injuries; in standard and minimally invasive
surgical procedures, for example, endometriosis surgery and
operations on the gallbladder; in soft and hard tissue wound
repair, for example, skin wounds and burn healing; in surgical
procedures, in particular, for splenic wounds; and for blood vessel
puncture diagnostic and treatment procedures such as
catheterization and balloon angioplasty procedures.
[0212] The p-GlcNac based starting material, which can be
formulated as a solid material or as a gel, foam, spray, emulsion,
suspension, or solution comprising p-GlcNac microbeads,
microspheres, or microfibrils, can be applied using standard
surgical procedures, and can be used with both standard and
minimally invasive surgical interventions. The gels of the
invention can be delivered, for example, by extrusion from a
syringe type device or in combination with a membrane or film. The
membrane or film can be manufactured from a fully acetylated
p-GlcNac based material or other natural or synthetic
materials.
[0213] In connection with the blood vessel puncture procedures
mentioned above, the compositions and materials of the invention,
which are used to stimulate endothelin-1 secretion,
vasoconstriction, and reduction of blood flow out of a breached
vessel, may be applied at the time when a catheter sheath is being
removed from a blood vessel by applying the p-GlcNac-based material
directly to the skin in conjunction with manual compression, or
introduced into the catheter track. Alternatively, a device that
detects the removal of the catheter sheath from the blood vessel
can be developed using electronic or mechanical systems that
monitor chemical, physical or other differences between the tissue
inside and outside of the vessel. For example, the differential in
fluid dynamics or heat dissipation can be detected when a probe is
removed from the vessel; at that point a signal is sent to initiate
the application of the composition or material comprising p-GlcNac,
which will stimulate release of endothelin-1, induce
vasoconstriction, and/or reduce blood flow out of a breached
vessel.
[0214] The methods of the present invention, which comprise topical
administration of p-GlcNac, preferably fully acetylated, highly
ordered, semi-crystalline polymers of p-GlcNac, to induce
endothelin-1 release, vasoconstriction, and reduction of blood flow
out of a breached vessel may be used in conjunction with those
methods and compositions useful for achieving hemostasis. Such
other methods and compositions include, but are not limited to (1)
application of barrier-forming materials that provide a matrix
impermeable to erythrocytes, and platelets and which may
concentrate circulating factors required for the clotting cascade,
and (2) application to a wound of materials comprising components
of the clotting cascade including, for example, thrombin,
fibrinogen, and Factor 13.
[0215] The methods of the present invention may also be used
prophylactically to minimize the need for, or increase the
efficiency of; methods and compositions for achieving hemostasis
where a need therefor can be anticipated. Examples of such a need
include, but are not limited to removal of polyps during
gastroenterological procedures, excision of tumor tissue, and tooth
extraction. In such instances, the methods of the present invention
are used to induce transient, localized endothelin-1 release,
vasoconstriction, and a reduction in blood flow out of a breached
vessel in those tissues and vessels adjacent to or contiguous with
a target tissue, thereby minimizing subsequent bleeding resulting
from the procedure carried out on the patient.
6. EXAMPLE
Physical Characterization of Preparations of Pure p-GlcNac
[0216] Presented in this Example, are circular dichroism (CD) and
infra-red spectra (IR) analyses of p-GlcNac and deacetylated
p-GlcNac membranes.
[0217] 6.1. Materials and Methods
[0218] p-GlcNac and commercial "chitin" preparations:
[0219] The p-GlcNac used in the CD studies' was prepared using the
Mechanical Force purification method described, above, in Section
53.1.
[0220] Commercial "chitin" was purchased from NovaChem, Ltd., PO
Box 1030 Armdale, Halifax, Nova Scotia, Canada, B3L 4K9.
[0221] The p-GlcNac membranes used in the IR studies were prepared
by either the Mechanical Force purification method as described,
above, in Section 5.3.1, or by the Chemical/Biological purification
method, as described, above, in Section 5.3.2, as indicated
[0222] The commercial "p-GlcNac" preparations were cast into
membranes by dissolving in a dimethylacetamide solution containing
5% lithium chloride, and layering onto distilled, deionized water
until membranes precipitated.
[0223] p-GlcNac derivatives and treatments: The Deacetylated
p-GlcNac used in both the CD and IR studies was prepared by
treatment of the p-GlcNac with 50% NaOH at 60.degree. C. for 2
hours. The heat-denatured p-GlcNac membranes used in the IR studies
were modified by boiling in 0.2 mM EDTA for 3 minutes. p-GlcNac was
autoclaved for 30 minutes at 122.degree. C.
[0224] CD techniques: Solid state CD techniques were carried out
essentially according to Domard (Domard, A., 1986, Int. J.
Macromol. 1:243-246).
[0225] 6.2. Results
[0226] 6.2.1. CD Analysis
[0227] In the CD spectra obtained from untreated p-GlcNac (FIG.
3A), the expected .pi.-.pi.* and .pi.-.pi.* optically active
electronic transitions (220-185 nM) were observed due to the
presence of the carbonyl group in the acetyl moiety of p-GlcNac.
Such peaks are completely absent in the CD spectrum obtained from
the deacetylated p-GlcNac product, as shown in FIG. 38.
[0228] 6.2.2. IR Spectra Analysis
[0229] The IR spectra obtained in this study are consistent with
the chemical structure of p-GlcNac. Additionally, the sharp
definition of each IR peak is indicative of the presence of an
ordered and regular (i.e., semi-crystalline) structure in the
p-GlcNac fibers. See FIG. 4A for the IR spectrum of p-GlcNac
purified via the Mechanical Force purification method, and FIG. 4D
for the IR spectrum of p-GlcNac purified via the
Chemical/Biological method. For comparison, see FIG. 4B, which
demonstrates the IR spectrum of a commercial "chitin"
preparation.
[0230] The IR spectrum obtained from the autoclaved p-GlcNac
material (FIG. 4E) does not differ visibly from the IR spectrum
observed in FIG. 4A. This data indicates that the p-GlcNac material
may be sterilized by autoclaving with no loss of polymer
structure.
7. EXAMPLE
Purification of p-GlcNac using the Mechanical Force Purification
Method
[0231] In this section, p-GlcNac was purified using the Mechanical
Force technique described above, in Section 5.3.1.
[0232] 7.1. Materials and Method/Results
[0233] Diatom culture conditions: The diatom species Thalassiosira
fluviatilis was grown in culture according the procedures
described, above, in Sections 5.1 and 5.2.
[0234] SEM procedures: The SEM techniques used here are as those
described, below, in Section 12.1.
[0235] p-GlcNac Purification procedure: p-GlcNac was purified from
the diatom culture by utilizing the Mechanical Force technique
described above, in Section 5.3.1. Specifically, the p-GlcNac
fibers were separated from the diatom cell bodies by subjecting the
contents of the culture to three short bursts of top speed mixing
motion in a Waring blender Total time of the three bursts was about
one second. The resulting suspension was centrifuged at 3500 rpm in
a Sorvall GS-4 fixed angle rotor, for 20 minutes at about
10.degree. C. The supernatant was decanted, and centrifuged again,
this time at 4000 rpm, in a Sorvall GS-4 fixed angle rotor for 20
minutes at about 10.degree. C. Once again, the supernatant was
decanted and centrifuged at 4000 rpm at 10.degree. C. The final
supernatant of the third centrifugation was clear, with little, if
any, visible flocs floating in the liquid. The clear supernatant
was decanted into a Buchner filtration unit equipped with a
Supor-800 polyether sulfone filter membrane with 0.8 .mu.m pore
size (Gelman, Inc.), suction was then applied and the liquid was
filtered from the fiber suspension, allowing the fibers to be
collected on the membrane. The collected fibers were washed with 1
liter of distilled, deionized H.sub.2O at 70.degree. C. When almost
all of the water had been drained, fibers were washed, with
suction, with 1 liter of 1N HCl at 70.degree. C. When most of the
acid solution had been drained, the fibers were washed with 1 liter
of distilled, deionized H.sub.2O at 70.degree. C., using suction.
When most of the wash water had been drained, the fibers were
washed with 1 liter of 95% ethanol at room temperature, and vacuum
was applied. The filter membrane on which the white fiber membrane
had been collected was then removed from the filtration unit and
the membrane and its membrane support was dried in a drying oven at
58.degree. C. for 20 minutes, after which the membrane and its
support were placed in a desiccator for 16 hours.
[0236] Following this purification procedure, the yield of p-GlcNac
from a 1000 ml culture was 6.85 milligrams per liter of diatom
culture. SEM photographs of the membrane formed by the collection
of the p-GlcNac fibers via this technique is shown in FIG. 6.
8. EXAMPLE
Purification of p-GlcNac using the Biological/Chemical Purification
Method
[0237] In this section, p-GlcNac was purified using two of the
Chemical/Biological techniques described above, in Section 5.3.2.
Briefly, p-GlcNac was purified via HF treatment, in one case, and
via acid treatment/neutralization in the second case.
[0238] 8.1. Materials and Methods/Results
[0239] Diatom culture conditions: The diatom species Thalassiostra
fluviatilis was grown in a culture according to the procedures
described, above, in Sections 5.1 and 5.2.
[0240] SEM procedures: The techniques utilized in this study were
as described, below, in Section 12.1.
[0241] Purification procedure: First, p-GlcNac was purified by HF
treatment, the results of which are shown in FIG. 7. Specifically,
under a fume hood, 2.42 ml of a 49% (29N) HF solution was added to
the diatom contents of the culture, at room temperature, for each
1000 ml of the volume of the original cell culture, resulting in a
0.07M HF solution. The mixture was then shaken vigorously for about
30 seconds, causing persistent foam to appear over the liquid. The
container was allowed to stand undisturbed for 5-6 hours to allow
heavy particulates to settle. At the end of this time, a layer of
foam had formed, while the liquid itself was divided into two
strata: first, a narrow, very dark green layer resting on the
bottom of the container below a second, much lighter colored
grayish-green and murky phase which represented perhaps 85-90% of
the total volume of liquid. The foam layer was carefully siphoned
off, using a capillary glass tube and vacuum suction. The grayish
cloudy supernatant was then siphoned off, with care being taken not
to disturb the dark bottom layer, which consisted mainly of settled
cell bodies, and was transferred to a separate plastic container.
The grayish cloudy supernatant was allowed to stand undisturbed for
an additional 16 hours. The liquid was initially almost colorless,
light grey, but not transparent. After 16 hours settling time, a
small amount of foam remained on top of the main body of liquid and
a small amount of green matter had settled on the bottom of the
container. The liquid was lighter in color, but still not
transparent. The foam on top of the liquid was siphoned off as
before. The main body of liquid was then carefully siphoned off,
leaving behind the small amount of settled green material at the
bottom of the container. The liquid which had thus been isolated,
contained the majority of the p-GlcNac fibers and some
impurities.
[0242] To remove proteins and other unwanted matter liberated by
the diatoms during the preceding steps in the procedure from the
fiber-containing liquid, the suspension of fibers and cell remnants
was washed with sodium dodecyl sulfate (SDS). Specifically, the
necessary volume of a 20% SDS solution was added to make the final
concentration of the liquid 0.5% SDS by volume. The container
holding the liquid was sealed, secured in a horizontal position on
a shaking machine, and agitated for 24 hours at about 100 shakes a
minute. Soon after shaking began, large flocs of white p-GlcNac
fibers appeared in the suspension, and a considerable amount of
foam accumulated in the head space of the containers. At the end of
the SDS washing, the contents of the containers were transferred to
a Buchner filtration equipment provided with a Supor-800 polyether
sulfone filter membrane, with 0.8 micron pore size (Gelman, Inc.).
The liquid was filtered with suction, and the p-GlcNac fibers in
the liquid were collected on the filter membrane.
[0243] The p-GlcNac fibers collected on the filter membrane were
then washed further. First, the fibers were washed with hot
(70.degree. C.) distilled, deionized H.sub.2O, using three times
the volume of the original suspension. With a water jet using
distilled, deionized H.sub.2O, the white fiber clumps collected on
the filter membrane of the Buchner filter were transferred to a
Waring blender, and the fiber clumps were disintegrated with about
10 short mixing bursts. The suspension of disintegrated fibers was
transferred to a Buchner filter funnel equipped with a polyether
sulfone filter membrane as described above, and the liquid was
removed under suction. The collected fibers were washed with 1000
ml of hot (70.degree. C.) 1N HCl solution, and subsequently further
washed with 1000 ml hot (70.degree. C.) distilled, deionized
H.sub.2O. Finally, the fibers were washed with 1000 ml 95% ethanol
at room temperature, and filtered to dryness. The fiber membrane
and the filter membrane supporting the fiber membrane were then
dried in a drying oven at 58.degree. C. for 20 minutes. The
membrane and membrane support was then placed in a desiccator for
16 hours. The membrane was then carefully detached from the filter
membrane.
[0244] Second, p-GlcNac was purified by using the acid
treatment/neutralization method described, above, in Section 5.3.2.
Specifically, the p-GlcNac was processed as described earlier in
this Section, until prior to the SDS wash step, at which point the
solution was neutralized to a pH of approximately 7.0 by the
addition of a 2.9M Tris solution. The p-GlcNac yield from this
particular purification procedure was 20.20 milligrams per liter of
diatom culture, although, on average, approximately 60 milligrams
per liter diatom culture are obtained. SEM micrographs of membranes
formed during the purification procedure are shown in FIGS. 8A-B
and 9A-9E.
9. EXAMPLE
p-GlcNac Deacetylation
[0245] A p-GlcNac membrane was suspended in an aqueous 50% NaOH
solution. The suspension was heated at 80.degree. C. for 2 hours.
The resulting deacetylated membrane was dried and studied by
scanning electron microscopy, as shown in FIGS. 11A-B.
10. EXAMPLE
p-GlcNac Biocompatibility
[0246] In this Example, it is demonstrated that the p-GlcNac
starting material exhibits no detectable biological reactivity, as
assayed by elution tests, intramuscular implantation in rabbits,
intracutaneous injection in rabbits, and systemic injections in
mice.
[0247] 10.1. Materials and Methods
[0248] 10.1.1. Elution Test
[0249] Conditions for the elution test conformed to the
specifications set forth in the U.S. Pharmacopeia XXII, 1990, pp.
1415-1497 and to U.S. Pharmacopeia XXII, Supplement 5, 1991, pp.
2702-2703.
[0250] Cell culture: Mouse fibroblast L929 cell line (American Type
Culture Collection Rockville, Md.; ATCC No. CCL1; NCTC clone 929)
was utilized. A 24 hour confluent monolayer of L929 cells was
propagated in complete Minimum Essential Medium (MEM).
[0251] p-GlcNac: a solid membrane of p-GlcNac which had been
prepared according to the Mechanical Force method of purification
described, above, in Section 5.3.1, was extracted in 20 ml
serum-supplemented MEM as per U.S. Pharmacopeia XXII (1990)
requirements.
[0252] Controls: Natural rubber was used as a positive control, and
silicone was used as a negative control. Controls were tested in
the same manner as the test article, p-GlcNac.
[0253] Extracts: Extracts were prepared at 37.degree. C., in a
humidified atmosphere containing 5% carbon dioxide, for 24 hours.
Extracts were evaluated for a change in pH, and adjustments were
made to bring the pH to within .+-.0.2 pH units of the original
medium. Adjustments were made with HCl to lower the extract pH or
with NaHCO.sub.3 to raise the extract pH. Extracts were sterile
filtered by passage through a 0.22 micron filter, prior to being
applied to the cell monolayer.
[0254] Dosing: 3 mls of p-GlcNac or control extracts were used to
replace the maintenance medium of cell cultures. All extracts were
tested in duplicate.
[0255] Evaluation Criteria: Response of the cell monolayer was
evaluated either visually or under a microscope. The biological
reactivity, i.e., cellular degeneration and/or malformation, was
rated on a scale of 0 to 4, as shown below. The test system is
suitable if no signs of cellular reactivity (Grade 0) are noted for
the negative control article, and the positive control article
shows a greater than mild reactivity (Grade 2). The test article
(i.e., p-GlcNac) meets the biocompatibility test if none of the
cultures treated with the test article show a greater than mild
reactivity.
TABLE-US-00002 Grade Reactivity Description of Reactivity Zone 0
None Discrete intracytoplasmic granules; No cell lysis 1 Slightly
Not more than 20% of the cells are round, loosely attached, and
without intra- cytoplasmic granules; occasional lysed cells are
present 2 Mild Not more than 50% of the cells are round and devoid
of intracytoplasmic granules; extensive cell lysis and empty areas
between cells 3 Moderate Not more than 70% of the cell layers
contain rounded cells and/or are lysed 4 Severe Nearly complete
destruction of the cell layers
[0256] 10.1.2. Intramuscular Implantations
[0257] Animals: Healthy, New Zealand White Rabbits, male and
female, (Eastern Rabbit Breeding Laboratory, Taunton, Mass.) were
used. Rabbits were individually housed using suspended stainless
steel cages. Upon receipt, animals were placed in quarantine for 8
days, under the same conditions, as for the actual test. Hardwood
chips (Sani-Chips.TM., J. P. Murphy Forest Products, Montvale,
N.J.) were used as non-contact bedding under cages. The animal
facility was maintained at a temperature of 68.degree..+-.3.degree.
F., with a relative humidity at 30-70%, a minimum of 10-13 complete
air exchanges per hour, and a 12-hour light/dark cycle using full
spectrum fluorescent lights. Animals were supplied with commercial
feed (Agway ProLab, Waverly, N.Y.) under controlled conditions and
municipal tap water ad libitum. No known contaminants were present
in the feed, bedding, or water which would be expected to interfere
with the test results. Animals selected for the study were chosen
from a larger pool of animals. Rabbits were weighted to nearest 10
g and individually identified by ear tattoo.
[0258] p-GlcNac: The p-GlcNac used was as described, above, in
Section 10.1.1.
[0259] Implantation Test: Two rabbits were used for each
implantation test. On the day of the test, the animal skin on both
sides of the spinal column was clipped free of fur. Each animal was
anesthetized to prevent muscular movement. Using sterile hypodermic
needles and stylets, four strips of the test p-GlcNac (1 mm.times.1
mm.times.10 mm) were implanted into the paravertebral muscle on one
side of the spine of each of two rabbits (2.5 to 5 cm from the
midline, parallel to the spinal column, and about 2.5 cm from each
other). In a similar fashion, two strips of the USP negative
control plastic RS (1 mm.times.1 mm.times.10 mm) were implanted in
the opposite muscle of each animal. Animals were maintained for a
period of 7 days. At the end of the observation period, the animals
were weighed and euthanized by an injectable barbiturate,
Euthanasia-5 (Veterinary Laboratories, Inc., Lenexa, Kans.).
Sufficient time was allowed to elapse for the tissue to be out
without bleeding. The area of the tissue surrounding the center
portion of each implant strip was examined macroscopically using a
magnifying lens. Hemorrhaging, necrosis, discolorations and
infections were scored using the following scale: 0=Normal, 1=Mild,
2=Moderate, and 3=Severe. Encapsulation, if present, was scored by
first measuring the width of the capsule (i.e., the distance from
the periphery of the implant to the periphery of the capsule)
rounded to the nearest 0.1 mm. The encapsulation was scored as
follows:
TABLE-US-00003 Capsule Width Score None 0 up to 0.5 mm 1 0.6-1.0 mm
2 1.1-2.0 mm 3 Greater than 2.0 mm 4
[0260] The differences between the average scores for the p-GlcNac
and the positive control article were calculated. The test is
considered negative if in each rabbit, the difference between the
average scores for each category of biological reaction for the
p-GlcNac and the positive control plastic implant sites does not
exceed 1.0; or, if the difference between the mean scores for all
categories of biological reaction for each p-GlcNac article and the
average score for all categories for all the positive control
plastic implant sites does not exceed 1.0, for not more than one of
four p-GlcNac strips.
[0261] 10.1.3. Intracutaneous Injections
[0262] Animals: New Zealand white rabbits were used and maintained
as described, above, in Section 10.1.2.
[0263] p-GlcNac: A solid membrane of p-GlcNac which had been
prepared according to the mechanical force method of purification
described, above, in Section 5.3.1, was placed in an extraction
flask, to which 20 ml of the appropriate medium were added.
Extractions were performed by heating to 70.degree. C. for 24
hours. Following this procedure, extracts were cooled to room
temperature. Each extraction bottle was shaken vigorously prior to
administration.
[0264] Intracutaneous Test: On the day of the test, animals were
clipped free of fur on the dorsal side. A volume of 0.2 ml of each
p-GlcNac extract was injected intracutaneously at five sites on one
side of each of two rabbits. More than one p-GlcNac extract was
used per rabbit. At five sites on the other side of each rabbit,
0.2 ml of the corresponding control was injected. Injection sites
were observed for signs of erythema, edema, and necrosis at 24, 48,
and 72 hours after injection. Observations were scored according to
the Draize Scale for the Scoring Skin Reaction (USP Pharmacopeia
XXII, 1990, 1497-1500; USP Pharmacopeia XXII, Supplement 5, 1991,
2703-2705) as shown in Table II, below:
TABLE-US-00004 TABLE II Draize Scale for Scoring Skin Reactions
Value Erythema and Eschar Formation No erythema 0 Very slight
erythema (barely perceptible) 1 Well defined erythema 2 Moderate to
severe erythema 3 Severe erythema (beet redness) to slight 4 eschar
formation (inuries in depth) Total possible erythema score = 4
Edema Formation No edema 0 Very slight erythema (barely
perceptible) 1 Slight edema (edges are well defined by definite
raising) 2 Moderate edema (raised approximately 1 mm and extending
3 beyond area of exposure) Severe edema (raised more than 1 mm and
extending 4 beyond area of exposure) Total possible edema score =
4
[0265] All erythema and edema scores at 24, 48, and 72 hours were
totaled separately and divided by 12 (i.e., 2 animals.times.3
scoring periods.times.2 scoring categories) to determine the
overall mean score for the p-GlcNac versus the corresponding
control Animals were weighed at the end of the observation period
and euthanized by injection of a barbiturate, Euthanasia-5
(Veterinary Laboratories, Inc., Lenexa, Kans.). The results of the
test are met if the difference between the p-GlcNac and the control
means reaction scores (erythema/edema) is 1.0 or less.
[0266] 10.1.4. Systemic Injections
[0267] Animals: Albino Swiss mice (Mus musculus), female, (Charles
River Breeding Laboratories, Wilmington, Mass.) were used. Groups
of 5 mice were housed in polypropylene cages fitted with stainless
steel lids. Hardwood chips (Sanichips.TM., J. P. Murphy Forest
Products, Montvale, N.J.) were used as contact bedding in the
cages. The animal facility was maintained as a limited access area.
The animal rooms were kept at a temperature of
68.degree..+-.3.degree. F., with a relative humidity of 30-70%, a
minimum of 10-13 complete air exchanges per hour, and a 12 hour
light/dark cycle using full spectrum fluorescent lights. Mice were
supplied with commercial feed and municipal tap water ad libitum.
There were no known contaminants present in the feed, bedding, or
water which would be expected to interfere with the test results.
Animals selected for the study were chosen from a larger pool of
animals. Mice were weighed to the nearest 0.1 g and individually
identified by ear punch.
[0268] p-GlcNac: The samples used were as described, above, in
Section 10.1.1. Extracts were prepared according to the procedures
described in Section 10.1.3, above.
[0269] Systemic Injection Test: Groups of 5 mice were injected with
either p-GlcNac extract or a corresponding control article, in the
same amounts and by the same routes as set forth below:
TABLE-US-00005 Test Article Control or Article Extracts Dosing
Route Dose/Kg Injection Rate 0.9% Sodium Chloride Injection, USP
Intravenous 50 ml 0.1 ml/sec (0.9% NaCl) 1 in 20 Alcohol in 0.9%
Sodium Intravenous 50 ml 0.1 ml/sec Chloride Injection USP
(EtOH:NaCl) Polyethylene Glycol 400 (PEG 400) Intraperitoneal 10 g
-- Cottonseed Oil (CSO) Intraperitoneal 50 ml -- Extracts of the
p-GlcNac prepared with PEG 400, and the corresponding control, were
diluted with 0.9% NaCl, to obtain 200 mg of PEG 400 per ml. For the
Intracutaneous Test, PEG 400 was diluted with 0.9% NaCl to obtain
120 mg of PEG 400 per ml.
[0270] The animals were observed immediately after injection, at 24
hours, 48 hours, and 72 hours after injection. Animals were weighed
at the end of the observation period and euthanized by exposure to
carbon dioxide gas. The requirements of the test are met if none of
the animals treated with the p-GlcNac shows a significantly greater
biological reactivity than the animals treated with the control
article.
[0271] 10.2. Results
[0272] 10.2.1. Elution Test
[0273] The response of the cell monolayer to the p-GlcNac test
article was evaluated visually and under a microscope. No
cytochemical stains were used in the evaluation. No signs of
cellular biological reactivity (Grade 0) were observed by 48 hours
post-exposure to the negative control article or to the p-GlcNac.
Severe reactivity (Grade 4) was noted for the positive control
article, as shown below in Table III:
TABLE-US-00006 TABLE III REACTIVITY GRADES Control Articles
p-GlcNac Negative Positive Time A B A B A B 0 Hours 0 0 0 0 0 0 24
Hours 0 0 0 0 4 4 48 Hours 0 0 0 0 4 4 The p-GlcNac starting
material, therefore, passes requirements of the elution test for
biocompatibility, and, thus, is noncytotoxic.
[0274] 10.2.2. Intramuscular Implantations
[0275] Both rabbits (A and B) tested increased in body weight and
exhibited no signs of toxicity. See Table IV for data. In addition,
there were no overt signs of toxicity noted in either animal.
Macroscopic evaluation of the test and control article implant
sites showed no inflammation, encapsulation, hemorrhage, necrosis,
or discoloration. See Table IV for results. The test, therefore,
demonstrates that the p-GlcNac assayed exhibits no biological
reactivities, in that, in each rabbit, the difference between the
average scores for all of the categories of biological reaction for
all of the p-GlcNac implant sites and the average score for all
categories for all the control implant sites did not exceed
1.0.
TABLE-US-00007 TABLE IV IMPLANTATION TEST (Macroscopic
Observations) Test Article: p-GlcNac Animal Species: Rabbit Test
Control Tissue Site: T1 T2 T3 T4 Average C1 C2 Average Animal #: A
Inflammation 0 0 0 0 0 0 0 0 Encapsulation 0 0 0 0 0 0 0 0
Hemorrhage 0 0 0 0 0 0 0 0 Necrosis 0 0 0 0 0 0 0 0 Discoloration 0
0 0 0 0 0 0 0 Total 0 0 0 0 0 0 MEAN SCORE: 0 0 0 0 0 0 (total/5)
AVERAGE CONTROL VALUE: 0 Animal #: B Inflammation 0 0 0 0 0 0 0 0
Encapsulation 0 0 0 0 0 0 0 0 Hemorrhage 0 0 0 0 0 0 0 0 Necrosis 0
0 0 0 0 0 0 0 Discoloration 0 0 0 0 0 0 0 0 Total 0 0 0 0 0 0 0
MEAN SCORE: 0 0 0 0 0 0 (total/5) AVERAGE CONTROL VALUE: 0
[0276] 10.2.3. Intracutaneous Test
[0277] All of the animals increased in weight. See Table V for
data. There were no signs of erythema or edema observed at any of
the p-GlcNac or control article sites. Overt signs of toxicity were
not observed in any animal. Because the difference between the
p-GlcNac and control article mean reaction scores (erythema/edema)
was less than 1.0, the p-GlcNac meets the requirements of the
intracutaneous test. See Table VI for results. Therefore, as
assayed by this test, the p-GlcNac demonstrates no biological
reactivity.
TABLE-US-00008 TABLE V Intracutaneous and Implant Tests Body
Weights and Clinical Observations Test Article: p-GlcNac Animal
Species: Rabbit Body Weight (Kg) Weight Signs of Group Animal # Sex
Day 0 Day 3 Change Toxicity* 0.9% 23113 Male 2.51 2.55 0.04 None
NaCl & CSO 0.9% 23114 Male 2.43 2.46 0.03 None NaCl & CSO
EtOH: 23115 Male 2.47 2.50 0.03 None NaCl & PEG 400 EtOH: 23116
Male Male 2.63 0.04 None NaCL & PEG 400 Body Weight (Kg) Weight
Signs of Group Animal # Sex Day 0 Day 7 Change Toxicity* Implant A
Male 2.74 2.80 0.06 None B Female 2.66 2.74 0.08 None *Summary of
Observations Day 0 Through Day 7 (Implant) and Day 0 through Day 3
(Intracutaneous).
TABLE-US-00009 TABLE VI INTRACUTANEOUS TEST DRAIZE SCORES Test
Article: p-GlcNac (T = test, C = control) Animal Species: Rabbit
Animal Site Numbers Scoring (ER/ED) Averages ID # Vehicle T-1 C-1
T-2 C-2 T-3 C-3 T-4 C-4 T-5 C-5 Time: T C NaCl Extract 23113 NaCl
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 23114 NaCl 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr.
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0
Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 CSO Extract
23113 CSO 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23114 CSO 0/0 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr.
0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
NaCl/EtOH Extract 23115 NaCl 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 24 hr. 0/0 0/0 EtOH 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48
hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0
Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23116 NaCl
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 EtOH 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 PEG Extract 23115 PEG 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72
hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
23115 PEG 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0
0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0
[0278] 10.2.4. Systemic Test
[0279] All of the mice treated with the p-GlcNac extract or the
control article increased in weight. See Table VII for data. In
addition, there were no overt signs of toxicity observed in any
p-GlcNac or control animal. See Table VI for results. It is
concluded, therefore, that none of the p-GlcNac test animals showed
a significantly greater biological reactivity than the animals
treated with the control article.
TABLE-US-00010 TABLE VII ANIMAL WEIGHTS AND CLINICAL OBSERVATIONS
Body Weight (g) Weight Signs of Group Sex Dose (ml) Animal # Day O
Day 3 Change Toxicity* NaCl Female 1.03 I. 20.6 22.8 2.2 None EtOH
Female 1.06 II. 21.1 23.4 2.3 None Test Female 1.02 III. 20.4 22.6
2.2 None 50 ml/kg Female 1.11 IV. 22.2 24.5 2.3 None Female 1.05 V.
21.0 23.2 2.2 None Mean 21.1 23.3 SD.+-. 0.7 0.7 NaCl: Female 1.04
VI. 20.7 23.2 2.5 None EtOH Female 1.04 VII. 20.8 23.5 2.7 None
Control Female 1.02 VIII. 20.3 22.3 2.0 None 50 ml/kg Female 0.91
IX. 18.2 20.6 2.4 None Female 0.94 X. 1.9 20.9 2.2 None Mean 19.7
22.1 SD.+-. 1.2 1.3 PEG Female 1.02 XI. 20.3 22.7 2.4 None Test
Female 0.96 XII. 19.2 21.4 2.2 None 10 ml/kg Female 0.95 XIII. 18.9
21.6 2.7 None Female 1.05 XIV. 20.9 22.7 1.8 None Female 0.94 XV.
18.7 21.2 2.5 None Mean 19.6 21.9 SD.+-. 1.0 0.7 PEG Female 1.01
XVI. 20.1 22.3 2.2 None Control Female 0.99 XVII. 19.8 22.0 2.3
None 10 g/kg Female 1.10 XVIII. 22.0 24.3 2.3 None Female 1.07 XIX.
21.4 23.6 2.2 None Female 1.03 XX. 20.6 22.4 1.8 None Mean 20.8
22.9 SD.+-. 0.9 1.0 *Summary of observations 0, 4, 24, 48, and 72 h
after injection
11. EXAMPLE
p-GlcNac Reformulation
[0280] In the Working Example presented in this Section, a p-GlcNac
membrane (16.2 mg) was dissolved in 1 ml of a dimethylacetamide
solution containing 5% LiCl. The p-GlcNac-containing solution was
placed in a syringe and extruded into 50 ml of pure water to
precipitate a fiber. The resulting fiber was studied with scanning
electron microscopy, as shown in FIGS. 10A-B.
12. EXAMPLE
p-GlcNac/Collagen Hybrids
[0281] Presented in this Working Example is the formation and
characterization of a p-GlcNac/collagen hybrid material.
[0282] 12.1. Materials and Methods
[0283] Materials: Bovine Type I collagen was used in preparation of
the hybrids described in this study. p-GlcNac was prepared
according to the mechanical force method described, above, in
Section 5.3.2.
[0284] Hybrid preparation: Collagen (10 milligrams/ml) and p-GlcNac
(0.25 milligrams/ml) aqueous suspensions were mixed, in different
ratios, frozen in liquid N.sub.2 (-80.degree. C.), held at
-9.degree. C. for 4 hours, and lyophilized. Material was
dehydrothermally cross-linked under vacuum (approximately 0.030
Torr) at 60.degree. C. for 3 days.
[0285] Cell Culture: Mouse 3T3 fibroblast cells were grown on the
collagen/p-GlcNac hybrids produced. Standard culturing procedures
were followed, and SEM micrographs were taken after 8 days in
culture.
[0286] 12.2. Results
[0287] Collagen and p-GlcNac aqueous suspensions were mixed in
differing ratios (namely, 3:1, 1:1, 2:2, and 1:3 collagen/p-GlcNac
suspension ratios), frozen, lyophilized, and crosslinked. Such a
procedure yielded collagen/p-GlcNac slabs. SEM micrographs of the
resulting materials revealed the porous structure of the hybrid
material, which provides an efficient three-dimensional structure
for the attachment and growth of cells.
13. EXAMPLE
NMR Characterization of Pure Preparations of n-GlcNac
[0288] Presented in this Example is an NMR (nuclear magnetic
resonance) analysis of a pure p-GlcNac preparation.
[0289] 13.1. Materials and Methods
[0290] p-GlcNac preparations: The p-GlcNac used in the NMR studies
described here was prepared using the chemical purification method
described, above, in Section 5.3.2, with hydrofluoric acid utilized
as the chemical reagent.
[0291] NMR techniques: Solid state NMR data was obtained using a
Bruker 500 MHz NMR spectrometer. Computer image analysis was used
to transform the raw NMR spectrum data so as to eliminate
background and to normalize baselines. An example of such
transformed data are shown in FIG. 14. Transformed NMR curves such
as that in FIG. 14 were used to obtain areas for every carbon atom
type, and then to calculate the ratios of CH.sub.3 (area) to C-atom
(area). Such values, obtained as described, are provided in FIG.
16.
[0292] 13.2. Results
[0293] Solid state NMR data was obtained by measuring the
.sup.13C-NMR spectrum of a 500 mg sample of p-GlcNac. A typical NMR
spectrum is shown in FIG. 15. The individual peaks represent the
contribution to the spectrum of each unique carbon atom in the
molecule. The relative percentage of each type of carbon atom in
the molecule was determined dividing the area of the peak generated
by that carbon species by the total sum of the areas under all of
the NMR peaks obtained in the spectrum. Thus, it was possible to
calculate the ratio of each of the atoms of the molecule measured
by a reference atom. All p-GlcNac molecules consist of N-acetylated
glucosamine residues having C1, C2, C3, C4, C5 and C6 atoms, by
definition. The ratio, then, of the area of the N-acetyl CH.sub.3
carbon atom peak to the areas of any of the glucosamine residue
carbon atom peaks, above, should be 1.0 if all of the glucosamine
residues in the polymer are N-acetylated. Data such as those in
FIG. 14 were used to obtain values for the CH.sub.3 (area)
ratios.
[0294] The calculated ratios in FIG. 16 are in many cases equal to
or nearly equal to 1.0, within experimental error, e.g.
CH.sub.3/C2=1.097, CH.sub.3/C6=0.984, CH.sub.3/C5S1.007,
CH.sub.3/C1=0.886. These results are consistent with the conclusion
that the p-GlcNac starting material is free of contaminants and is
fully acetylated (i.e. that essentially 100% of the glucosamine
residues are N-acetylated).
14. EXAMPLE
Synthesis and Biological Characterization of Controlled Pore Size
Three-Dimensional p-GlcNac Matrices
[0295] Described below, are methods for the production of
three-dimensional p-GlcNac based porous matrices having controlled
average pore sizes. Such matrices have a variety of important
applications, including for example, as means for the encapsulation
of cells. Such cell encapsulation compositions are useful as
transplantable cell-based therapeutics, and in other cell and
tissue engineering applications such as in cartilage regeneration.
The capability to manipulate the morphology and dimensionality of
p-GlcNac materials, as demonstrated here, provides a powerful tool
for reformulating p-GlcNac polymers into a variety of shapes,
including without limitation, microbeads and microspheres, which
may be formulated as emulsions, suspensions and/or solutions in a
pharmaceutically acceptable carrier, vehicle, and/or solvent.
[0296] 14.1. Materials and Methods
[0297] p-GlcNac starting material: p-GlcNac was prepared using the
chemical purification method described, above, in Section 5.32,
with hydrofluoric acid utilized as the chemical reagent Matrix
formation: Suspensions (5 mls) containing 20 mg p-GlcNac samples
were made in the solvents listed below in Section 14.2, prior to
lyophilization. Samples were then poured into wells of tissue
culture dishes and frozen at -20.degree. C. The frozen samples were
then lyophilized to dryness, and the resulting three-dimensional
matrices were removed.
[0298] Scanning electron microscopy techniques: The procedures
utilized here were performed as described, above, in Section 12.1.
The images shown in FIGS. 17A-G. are 200.times. magnifications of
the matrix material, and a scale marking of 200 microns is
indicated on each of these figures.
[0299] 14.2. Results
[0300] p-GlcNac suspensions were obtained with each of the
following solvents, as described, above, in Section 14.1:
[0301] A. Distilled water
[0302] B. 10% methanol in distilled water
[0303] C. 25% methanol in distilled water
[0304] D. Distilled water only
[0305] E. 10% ethanol in distilled water
[0306] F. 25% ethanol in distilled water
[0307] G. 40% ethanol in distilled water
[0308] Samples of matrix formed using each of the solvents were
subjected to scanning electron microscopic (SEM) analysis, as shown
in FIGS. 17A-G. These figures reveal that the average matrix pore
size decreases as the percentage of either methanol or ethanol
increases in each suspension.
[0309] Specifically, pore diameter in the two water suspensions
(FIGS. 17A and 17D) approach 200 microns on average. Pore size in
the samples depicted in FIGS. 17C and 17F (25% methanol and
ethanol, respectively) are between 30 and 50 microns on
average.
[0310] The results shown here suggest that while both ethanol and
methanol may be successfully used to control p-GlcNac pore size,
ethanol may be more efficient than methanol.
15. EXAMPLE
Biodegradability of p-GlcNac Materials
[0311] The Example presented in this Section demonstrates that
p-GlcNac starting materials may be prepared which exhibit
controllable in vitro and in vivo biodegradability and rates of
resorption.
[0312] 15.1. Materials and methods
[0313] p-GlcNac materials: Prototype I was made by the method
described, above, in Section 5.3.2, via the chemical method, with
hydrofluoric acid being utilized as the chemical reagent. Prototype
I represented 100% acetylated p-GlcNac.
[0314] The p-GlcNac starting material of prototype 3A was made by
the method described, above, in Section 5.3.2, via the chemical
method, with hydrofluoric acid being utilized as the chemical
reagent. The p-GlcNac material was then deacetylated by the method
described, above, in Section 5.4. Specifically, the p-GlcNac
material was treated with a 40% NaOH solution at 60.degree. C. for
30 minutes. The resulting prototype 3A was determined to be 30%
deacetylated.
[0315] The p-GlcNac starting material of prototype 4 was made by
the method described, above, in Section 5.3.2, via the chemical
method, with hydrofluoric acid being utilized as the chemical
reagent. The p-GlcNac material was then deacetylated by treatment
with a 40% NaOH solution at 60.degree. C. for 30 minutes. Next, the
fibers were suspended in distilled water frozen at -20.degree. C.
and lyophilized to dryness Prototype 4 was also determined to be
30% deacetylated.
[0316] Abdominal implantation model: Sprague Dawley albino rats
were utilized for the abdominal implantation model studies. Animals
were anesthetized and prepared for surgery, and an incision was
made in the skin and abdominal muscles. The cecum was located and
lifted out. A 1 cm.times.1 cm membrane of p-GlcNac material was
placed onto the cecum, and the incision was closed with nylon.
Control animals were those in which no material was placed onto the
cecum.
[0317] Animals were opened at 14 and 21 days post implantation.
Photographs were taken during the implant and explant procedures
(FIGS. 23A-E). Samples of cecum were prepared for histopathology
after the explant procedure.
[0318] p-GlcNac in vitro degradation lysozyme-chitinase assay: The
assay is a colorimetric assay for N-acetyl glucosamine, and was
performed as follows: 150 .mu.l of a reaction sample was pipetted
into 13.times.100 mm glass disposable test tubes, in duplicate 25
.mu.l of 0.25M potassium phosphate buffer (pH 7.1) was added to
each test tube, followed by the addition of 35 .mu.l of 0.8M
potassium borate solution (pH 9.8). Tubes were immediately immersed
into an ice-bath for a minimum of 2 minutes. Samples were then
removed from the ice-bath, 1 ml of freshly prepared DMAB reagent
was added, and the samples were vortexed. DMAB (Dimethyl
aminobenzaldehyde) reagent was made by adding 70 mls of glacial
acetic acid and 10 ml of 11.6N (concentrated) HCl to 8 grams of
p-dimethyl aminobenzaldehyde. Samples were then incubated at
37.degree. C. for 20 minutes.
[0319] To prepare a standard curve, the following procedure was
utilized. A GlcNac stock solution was diluted to 0.1 mg/ml with
0.010M sodium acetate buffer (pH 4.5), and 0 .mu.l, 20 .mu.l, 30
.mu.l, 90 .mu.l or 120 .mu.l of the diluted GlcNac solution was
added to a set of test tubes. This was followed by the addition of
150 .mu.l, 130 .mu.l, 60 .mu.l or 30 .mu.l, respectively, of 0.010M
sodium acetate buffer (pH 4.5) to the test tubes. Next, 25 d of
0.25M potassium phosphate buffer (pH 7.1) and 35 .mu.l of 0.8M
potassium borate buffer (pH 9.8) were added to each test tube. A
duplicate set of test tubes is prepared by the same procedure.
[0320] The test tubes are capped and boiled at 100.degree. C. for
exactly 3 minutes. The tubes are then immersed in an ice bath. The
tubes are removed from the ice bath and 1 ml of DMAB reagent,
freshly prepared according to the method described above, is added
to each tube.
[0321] The tubes are incubated at 37.degree. C. for 20 minutes. The
absorbance of the contents of each tube is read at 585 nM.
Absorbance should be read as quickly as possible. The standard
curve is plotted on graph paper and used to determine the
concentration of N-acetyl glucosamine in the reaction samples. A
typical standard curve is shown in FIG. 18.
[0322] 15.2. Results
[0323] The in-vitro biodegradability of p-GlcNac materials was
studied in experiments which assayed the relative susceptibility of
p-GlcNac membrane materials to degradation by lysozyme. p-GlcNac
membranes were exposed to an excess of lysozyme in a 10 mM acetate
buffer, and the subsequent release of N-acetyl glucosamine was
determined using the assay described, above, in Section 15.1.
[0324] The results of these experiments indicated that partially
deacetylated membranes are more susceptible to digestion by
lysozyme (see FIG. 19) and, further, that the rate of lysozyme
degradation is directly related to the extent of deacetylation (see
FIG. 20, which compares the degradation rates of a 50% to a 25%
deacetylated p-GlcNac membrane).
[0325] p-GlcNac In Vivo Degradation
[0326] Experiments were performed which addressed the in-vivo
biodegradability of p-GlcNac materials. Such experiments utilized
an abdominal implantation model. Three p-GlcNac materials, as
listed below, were tested.
[0327] p-GlcNac materials tested: [0328] 1) p-GlcNac, fully
acetylated (designated prototype 1); [0329] 2) partially
deacetylated p-GlcNac membrane (designated prototype 3A); and
[0330] 3) lyophilized and partially deacetylated p-GlcNac membrane
(designated prototype 4).
Results
[0331] The fully acetylated p-GlcNac (prototype 1) was resorbed
within 21 days, as shown in FIGS. 21A-21C. The partially
deacetylated p-GlcNac membrane (prototype 3A) was completely
resorbed within 14 days, as shown in FIGS. 21D-21E. The lyophilized
and partially deacetylated p-GlcNac membrane (prototype 4) had not
yet been completely resorbed after 21 days post-implantation.
[0332] Histopathology analyses showed that once the p-GlcNac
material has been resorbed there were no histological differences
detectable between tissue samples obtained from the treated and
from the control animals.
16. EXAMPLE
p-GlcNac Stimulation of Endothelin-1 Secretion and Induction of
Arterial Vasoconstriction
[0333] This example demonstrates that p-GlcNac of the present
invention can be used to stimulate endothelin-1 release and to
induce arterial vasoconstriction in vivo.
[0334] 16.1. Treatment and Analysis of Aortic Incisions; Materials
and Methods
[0335] ANIMALS. This study was conducted in immature female
Yorkshire White swine weighing between 25 and 30 kg (average 27.5
kg). The following protocol was used in every case.
[0336] Protocol [0337] 1. After standard premedication, anesthetize
animal by inhalation with 100% O.sub.2 and 1-2% Halothane. [0338]
2. Draw control blood sample for CBC and platelet count. [0339] 3.
Expose abdominal aorta. [0340] 4. With ties in place, make 1 cm
vertical wound in aorta. [0341] 5. Release ties while applying test
article. [0342] 6. Compress for one minute [0343] 7. Remove
compression, observe for bleeding. [0344] 8. If bleeding, repeat
steps 4 and 5. [0345] 9. Test article fails if 15 one minute
compressions fail to stop bleeding. [0346] 10. Collect tissues for
pathology
[0347] 16.2 Treatment and Analysis of Splenic Incisions; Materials
and Methods
[0348] ANIMALS. This study was conducted in four immature female
Yorkshire White swine weighing between 34 and 37 kg. The following
protocol was used in every case.
[0349] Protocol
[0350] 1. After standard premedication, anesthetize animal by
inhalation with 100% O.sub.2 and 1-2% Halothane. Draw control blood
sample for CBC and platelet count
[0351] 2. Deliver spleen through midline abdominal incision using
electrocautery to maintain absolute hemostasis.
[0352] 3. Isolate spleen with sponges.
[0353] 4. Create a 2 cm.times.2 cm area of capsular stripping on
the surface of the spleen to a depth of 3 mm.
[0354] 5. Allow wound to bleed freely for 10 seconds.
[0355] 6. Remove accumulated blood with Surgical sponge.
[0356] 7. Apply test agent
[0357] 8. Apply gentle pressure for 1 minute.
[0358] 9. Remove pressure, observe for bleeding for 2 minutes.
[0359] 10. If wound bleeds, repeat 5 and 6.
[0360] 11. Record the number of compressions needed to control
bleeding and the time to hemostasis.
[0361] 12. Document if complete cessation of bleeding was achieved.
(Defined as no rebleeding for two minutes after cessation of
bleeding.)
[0362] 13. Collect the tissues for pathology
[0363] 16.3 Spleen Immunostaining Protocol
[0364] Immunostaining was performed using the ET-1 Staining Kit
from Peninsula Laboratories, Inc. (Cat. #HIS-6901) with minor
modifications.
[0365] Slide Preparation and Staining Procedure
[0366] 1. Spleen tissue is sampled and preserved by embedding the
samples in paraffin, on slides, using standard methods. Paraffin is
subsequently removed from the by incubating them for 10 minutes in
100% xylene. Rehydrate the slides in a graded series of 100%
Ethanol, 95% Ethanol, and then in tap water by dipping them 5 times
in each solution. Circumscribe tissue samples with an 1 mm Edge
waterproof pen (Vector Laboratories Cat. #H-4000). Store slides in
PBS pH 7.4 solution in a coplin jar.
[0367] 2. Dilute Antigen Unmasking solution (Vector Laboratories
Cat. #H-3300) 100.times. and heat for 30-45 seconds in another
coplin jar. Transfer the slides to this solution and incubate for
20 minutes. Make sure there is enough solution to cover the tissue
samples to prevent drying out. Rinse slides well with PBS pH 7.4
solution for 2 minutes; repeat twice. Drain or blot the slides to
remove excess solution.
[0368] 3. Add 2 drops or 100 .mu.L of Normal Goat Serum Blocking
Solution to each slide. Incubate for 20 minutes at room
temperature. Drain or blot excess solution from the slides. Do not
rinse.
[0369] 4. Reconstitute the lyophilized primary antibody with 32 L
of PBS pH 7.4 solution. From this stock solution, dilute the
primary antibody by a dilution factor of 400. Add 2 drops or 100
.mu.L of diluted primary antibody to each slide. Place slides
horizontally on wooden sticks in a moisture chamber and incubate
overnight at 4.degree. C. Rinse well with PBS pH 7.4 solution for 2
minutes; repeat twice.
[0370] 5. Add 2 drops or 100 .mu.L of Biotinylated secondary
antibody to each slide. Incubate for 30 minutes at room
temperature. Rinse well with PBS pH 7.4 solution for 2 minutes;
repeat twice.
[0371] 6. Quench the slides in 3% Certified Hydrogen Peroxide
(Fisher Cat. #H 312-500) for 30 minutes at room temperature in a
coplin jar. Rinse well with PBS pH 7.4 for 2 minutes; repeat
twice.
[0372] 7. Add 2 drops or 100 L of Streptavadin-HRP conjugate to
each slide and incubate for 30 minutes at room temperature. Rinse
well with PBS pH 7.4 solution for 2 minutes; repeat twice.
[0373] 8. Make DAB Chromagen-Solution (Vector Laboratories Cat.
#sk-41067) by adding 5.0 mL of distilled water to a glass
scintillation vial. Add 2 drops of Buffer Stock Solution and mix
well. Then, add 4 drops of DAB stock solution and mix well.
Finally, add 2 drops of Hydrogen Peroxide solution and mix well.
Add 200 .mu.L of DAB Chromagen-Solution to each slide. Incubate for
3 minutes at room temperature. Rinse well with distilled water and
blot.
[0374] 9. Counterstain the slides with a stock solution of 0.2%
Working Light Green Solution (Sigma Cat. #L 5382) with a dilution
factor of 6. Dip the slides 3 times in Working Light Green solution
and then dip the slides 5 times each in a dehydrating series of
distilled water, then 95% Ethanol, then 100% Ethanol, and finally
in 100% xylene. Drain or blot the slides to remove excess
xylene.
[0375] 10. Add 2 drops of Cytoseal XYL mounting solution (Stephens
Scientific Cat. #8312-4) and mount the slide.
[0376] 16.4 Artery Immunostaining Protocol
[0377] Immunostaining of arterial tissues was performed using an
ET-1 Staining Kit from Peninsula Laboratories, Inc. (Cat.
#HIS-6901) with some modifications.
[0378] Slide Preparation [0379] 1. Pulmonary arteries are excised
from deer obtained commercially. [0380] 2. Place the arteries in
100 mL of RPMI media and place on ice. [0381] 3. Make an incision
in the artery with a scalpel. [0382] 4. Place a 1 cm.times.1 cm
square membrane consisting of fully acetylated p-GlcNac fibers,
over the incision for 15 minutes. [0383] 5. Make cross section
slices of the artery at the membrane application site, for
histology. [0384] 6. Place the sections in 9% Formaldehyde. Prepare
the slides with Paraffin.
[0385] Staining Procedure
[0386] 1. Deparaffinize the slides by incubating them for 10
minutes in 100% xylene. Rehydrate the slides in a graded series of
100% Ethanol, 95% Ethanol, and then in tap water by dipping them 5
times in each solution. Circumscribe tissue samples with an 1 mm
Edge waterproof pen (Vector Laboratories Cat. #H-4000). Store
slides in PBS pH 7.4 solution in a coplin jar.
[0387] 2. Dilute Antigen Unmasking solution (Vector Laboratories
Cat. #H-3300) 100-fold and heat for 30-45 seconds in another coplin
jar. Transfer the slides to this solution and incubate for 20
minutes. Make sure there is enough solution to cover the tissue
samples to prevent drying out. Rinse slides well with PBS pH 7.4
solution for 2 minutes; repeat twice. Drain or blot the slides to
remove excess solution.
[0388] 3. Add 2 drops or 100 .mu.L of Normal Goat Serum Blocking
Solution to each slide. Incubate for 20 minutes at room
temperature. Drain or blot excess solution from the slides. Do not
rinse.
[0389] 4. Reconstitute the lyophilized primary antibody with 32
.mu.L of PBS pH 7.4 solution. From this stock solution, dilute the
primary antibody by a dilution factor of 100. Add 2 drops or 100
.mu.L of diluted primary antibody to each slide. Place slides
horizontally on wooden sticks in a moisture chamber and incubate
overnight at 4.degree. C. Rinse well with PBS pH 7.4 solution for 2
minutes; repeat twice.
[0390] 5. Add 2 drops or 100 .mu.L of Blotinylated secondary
antibody to each slide. Incubate for 30 minutes at room
temperature. Rinse well with PBS pH 7.4 solution for 2 minutes;
repeat twice.
[0391] 6. Quench the slides in 3% Certified Hydrogen Peroxide
(Fisher Cat. #H 312-500) for 30 minutes at room temperature in a
coplin jar. Rinse well with PBS pH 7.4 for 2 minutes; repeat
twice.
[0392] 7. Add 2 drops or 100 .mu.L of Streptavadin-HRP conjugate to
each slide and incubate for 30 minutes at room temperature. Rinse
well with PBS pH 7.4 solution for 2 minutes; repeat twice.
[0393] 8. Make DAB Chromagen-Solution (Vector Laboratories Cat.
#sk-41067) by adding 5.0 mL of distilled water to a glass
scintillation vial. Add 2 drops of Buffer Stock Solution and mix
well. Then, add 4 drops of DAB stock solution and mix well.
Finally, add 2 drops of Hydrogen Peroxide solution and mix well.
Add 200 .mu.L of DAB Chromagen-Solution to each slide. Incubate for
3 minutes at room temperature. Rinse well with distilled water and
blot.
[0394] 9. Counterstain the slides with a stock solution of 0.2%
Working Light Green Solution (Sigma Cat. #L 5382) with a dilution
factor of 6. Dip the slides 3 times in Working Light Green solution
and then dip the slides 5 times each in a dehydrating series of
distilled water, then 95% Ethanol, then 100% Ethanol, and finally
in 100% xylene. Drain or blot the slides to remove excess
xylene.
[0395] 10. Add 2 drops of Cytoseal XYL mounting solution (Stephens
Scientific Cat. #8312-4) and mount the slide.
[0396] Results
[0397] Histological and immunological examination of the arterial
tissue treated with a membrane consisting of fully acetylated
p-GlcNac fibers stimulated immediate vasoconstriction at the
contact site of injured artery tissue and the p-GlcNac polymer. The
vasoconstriction induced by application of the p-GlcNac membrane
was more easily seen, histologically, with the larger the
experimental animals. Constriction of arterial tissue is more
pronounced on the side of the artery to which the p-GlcNac membrane
was applied. The results of these analyses are depicted in FIG. 23
and FIG. 24. Sixty minutes after application of a gauze dressing to
porcine artery (FIG. 23 (A), and FIG. 24, sample A), comparable
values for arterial wall thickness were obtained, whether the wall
was measured at the point of contact with the gauze, (1), or at a
point on the side opposite from the point where the gauze dressing
was applied. In contrast, application of a membrane formulated with
semi-crystalline p-GlcNac to porcine artery (FIG. 23 (B), FIG. 24,
sample B), induced a marked thickening of the wall at the area of
contact (1), which was apparent 15 minutes after application of the
membrane. After 60 minutes of contact, the thickness of the
arterial wall, as measured at the area of contact with the p-GlcNac
membrane (1), had returned to a level comparable to that measured
at point on the opposite side of the artery (2).
[0398] Immunostaining experiments with antibodies to endothelin-1
showed secretion of endothelin-1 in the site of contact between the
p-GlcNac membrane and living tissue. The in vitro experiment with
deer pulmonary artery showed presence of endothelin-1 only on the
contact surface of the artery with the p-GlcNac membrane. In vivo
experiments showed substantially greater endothelin-1 release, not
only on the contact surface between the treated tissue and the
p-GlcNac membrane, but also in deeper layers of tissue. Within the
first 15 minutes after application of the p-GlcNac membrane, more
endothelin-1 secreted was detected than in the comparable analysis
performed only after 60 minutes of contact between the treated
tissue and the p-GlcNac membrane. Nevertheless, the constriction
effect was stronger than other samples examined.
[0399] The same endothelin-1 immunostaining was observed on slides
with other samples, but it was much lower than with the p-GlcNac
membrane. Histological and immunological analysis of spleen tissue
contacted with the p-GlcNac membrane revealed a similar enhancement
of endothelin-1 release. Again, within the first 15 minutes after
application of the experimental membranes, endothelin-1 was
observed only in those samples to which the p-GlcNac membrane had
been applied. After 60 minutes of contact between the experimental
membranes and the treated tissues, all samples revealed comparable
levels of endothelin-1.
17. EXAMPLE
p-GlcNac Induction of Vasoconstriction and Endothelin Release in
the Absence of Blood Products
[0400] This example demonstrates that the fully acetylated,
semi-crystalline p-GlcNac of the present invention induces arterial
vasoconstriction, in the absence of blood. More specifically, this
example demonstrates that fully acetylated p-GlcNac significantly
contracts isolated rat aortic rings via an endothelium-dependent
mechanism, partly by endothelin-1 release from endothelial cells,
in the absence of any of the components of the clotting
cascade.
[0401] 17.1 Materials and Methods
[0402] Aortic rings were obtained from Male Sprague-Dawley rats
weighing 275-300 g. The rats were anesthetized with pentobarbital
sodium (60 mg/kg) injected intraperitoneally. The aorta and the SMA
were rapidly removed from rats and suspended in a warmed
Krebs-Henseleit (KH) buffer consisting of (in mmol/l): 118 NaCl,
4.75 KCl, 2.54 CaCI.sub.2.2H.sub.2O, 1.19 KH.sub.2 PO.sub.4,
1.19MgSO.sub.4.7H.sub.2O, 12.5 NaHCO.sub.3, and 10.0 glucose.
Isolated vessels were carefully freed of connective tissue and cut
into rings 2-3 mm in length. The rings were then mounted on
stainless steel hooks, suspended in a 10-ml tissue bath, and
connected to FT-03 force displacement transducers (Grass
Instrument, Quincy, Mass.) to record changes in force on a Grass
model 7 oscillographic recorder. The baths were filled with KH
buffer and aerated at 37.degree. C. with 95% O.sub.2+5% CO.sub.2. A
resting force of 0.5 g was applied to the SMA rings, and then the
rings were equilibrated for 90 minutes. During this period, the
buffer in the tissue bath was replaced every 15-20 minutes, and the
resting force of the vascular rings was adjusted until 0.5 g of
pre-load was maintained. After 90 to 120 minutes of equilibration,
the rings were exposed to 100 nM U-46619
(9,11-dideoxy-9.alpha.-11.alpha.-methaneepoxy-prostagalandin
F.sub.2 .alpha., Biomol Research Laboratories, Plymouth Meeting,
Pa.), a thromboxane A.sub.2 mimetic, to generate 1.0 g of developed
force. Once a stable contraction was obtained, acetylcholine, a
typical endothelium-dependent vasodilator, was added to the bath in
cumulative concentrations of 0.1, 1, 10, and 100 nM to assess the
integrity of endothelium. After the cumulative response was
stabilized, the rings were washed and again allowed to equilibrate
to baseline.
[0403] The procedure was repeated with U-46619 followed by
p-GlcNAc. p-GlcNAc produced a concentration-dependent
vasocontraction from 14 to 140 g/ml, as indicated in FIG. 23. At a
developed concentration of 140 g/ml, p-GlcNAc significantly
contracted aortic rings by 218.+-.21 mg of developed force
(p<0.01). De-endothelialized (i.e. endothelium was removed by
gently rolling the aortic rings over a twisted stainless steel wire
covered with cotton) aortic rings were contracted by only 33.+-.12
mg of developed force. Pretreatment with an endothelin EtA receptor
antagonist, JKC-301
(Cyclo[.sub.D-Asp-Pro-.sub.D-Ile-Leu-.sub.D-Trp]), Sigma
Biochemicals and Reagents, St. Louis, Mo.) (0.5 and 1 M),
significantly diminished p-GlcNac-induced vasoconstriction by 57 to
61% (p<0.01).
[0404] The procedure was repeated with U-46619 followed by 70%
deacetylated p-GlcNAc. Substitution of 70% deacetylated p-GlcNac
for the fully-acetylated, semi-crystalline p-GlcNac used above,
failed to demonstrate vasoconstriction in this blood-free model
system, at all concentrations tested.
[0405] It is apparent that many modifications and variations of
this invention as set forth here may be made without departing from
the spirit and scope thereof. The specific embodiments described
above are given by way of example only, and the invention is
limited only by the terms of the appended claims.
[0406] Various publications are cite herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *