U.S. patent application number 16/937246 was filed with the patent office on 2020-11-12 for water soluble nitric oxide-releasing polyglucosamines and uses thereof.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Yuan Lu, Mark H. Schoenfisch.
Application Number | 20200354480 16/937246 |
Document ID | / |
Family ID | 1000004975211 |
Filed Date | 2020-11-12 |
View All Diagrams
United States Patent
Application |
20200354480 |
Kind Code |
A1 |
Schoenfisch; Mark H. ; et
al. |
November 12, 2020 |
WATER SOLUBLE NITRIC OXIDE-RELEASING POLYGLUCOSAMINES AND USES
THEREOF
Abstract
The presently disclosed subject matter provides nitric
oxide-releasing polysaccharides and oligosaccharides, in
particular, polyglucosamines, and their use in biomedical and
pharmaceutical applications. More particularly, in some
embodiments, the presently disclosed subject matter provides nitric
oxide-releasing polysaccharides and oligosaccharides that release
nitric oxide in a controlled and targeted manner, thereby
prolonging the therapeutic effects of nitric oxide and improving
the specificity of nitric oxide delivery to targeted cells and/or
tissues.
Inventors: |
Schoenfisch; Mark H.;
(Chapel Hill, NC) ; Lu; Yuan; (Chapel Hill,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
1000004975211 |
Appl. No.: |
16/937246 |
Filed: |
July 23, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15836121 |
Dec 8, 2017 |
10759877 |
|
|
16937246 |
|
|
|
|
14421525 |
Feb 13, 2015 |
9850322 |
|
|
PCT/US2013/055360 |
Aug 16, 2013 |
|
|
|
15836121 |
|
|
|
|
61684373 |
Aug 17, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08B 37/00 20130101;
A61K 47/60 20170801; C08L 5/08 20130101; C08B 37/003 20130101 |
International
Class: |
C08B 37/08 20060101
C08B037/08; C08B 37/00 20060101 C08B037/00; A61K 47/60 20060101
A61K047/60; C08L 5/08 20060101 C08L005/08 |
Goverment Interests
GOVERNMENT INTEREST
[0001] This invention was made with government support under
EB000708 awarded by The National. Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A polyglucosamine comprising, at least one structural unit:
##STR00037## and optionally, at least one structural unit:
##STR00038## wherein, R.sub.1, R.sub.2, R.sub.3 and R.sub.4, if
present, are each independently selected from the group consisting
of hydrogen, C.sub.1-5 alkyl(C.dbd.O)-- and C.sub.1-5 alkyl; in
each instance, is a single or double bond, wherein in each instance
where it is a double bond, R.sub.1, R.sub.2, R.sub.3 or R.sub.4
attached to the double bond-O is absent; when R.sub.1 is absent,
R.sub.5 is hydrogen, hydroxyl, C.sub.1-5 alkyl or C.sub.1-5 alkoxy;
when R.sub.3 is absent, R.sub.6 is hydrogen, hydroxyl, C.sub.1-5
alkyl or C.sub.1-5 alkoxy; wherein in each instance where it is a
single bond, R.sub.1, R.sub.2, R.sub.3 or R.sub.4 attached to the
double bond-O is present; when R.sub.1 is present, R.sub.5 is
hydrogen; when R.sub.3 is present, R.sub.6 is hydrogen; Q is
--(CR.sub.cR.sub.d).sub.v--; wherein R.sub.c and R.sub.d are
independently hydrogen or C.sub.1-5 alkyl; and v is an integer from
2 to 6; p is an integer from 1 to 10; A is ##STR00039## wherein, L
is S, O or N; and G, in each instance, is independently, hydrogen,
or is taken together with L to form a nitric oxide donor or is
absent; X is hydrogen, C.sub.1-5 alkyl or is taken together with N
to form a nitric oxide donor; B is one hydrogen or --Y--Z, wherein
Y is a spacer and Z is a polymer or a terminus; D is
NR.sub.aR.sub.b, wherein R.sub.a and R.sub.b are independently
selected from the group consisting of hydrogen, formyl, C.sub.1-5
alkyl(C.dbd.O)--, C.sub.1-5 alkyl and C.sub.1-5 alkyl ester; or D
is ##STR00040##
2. The polyglucosamine of claim 1, wherein at least one of said X
and G is taken together with the atom on the polyglucosamine to
which it is bound to form a nitric oxide donor.
3. The polyglucosamine of claim 1, comprising the structural unit:
##STR00041## wherein, m is an integer from 1 to 10,000.
4. The polyglucosamine of claim 1, wherein said nitric oxide donor
is taken together with the atom on the polyglucosamine to which it
is bound is selected from the group consisting of a
diazeniumdiolate, nitrosothiol, a nitrosamine, a hydroxyl
nitrosamine, a hydroxyl amine, a hydroxyurea, and combination
thereof.
5. The polyglucosamine of claim 4, wherein said nitric oxide donor
is diazeniumdiolate.
6. The polyglucosamine of claim 3, wherein m is an integer from 1
to 50.
7. The polyglucosamine of claim 3, wherein m is an integer from 1
to 10.
8. The polyglucosamine of claim 1, comprising at least one
structural unit: ##STR00042## wherein, D is --NR.sub.aR.sub.b,
wherein R.sub.a and R.sub.b are independently selected from the
group consisting of hydrogen, formyl, C.sub.1-5 alkyl(C.dbd.O)--,
C.sub.1-5 alkyl and C.sub.1-5 alkyl ester.
9. The polyglucosamine of claim 8, wherein , in each instance, is a
single bond R.sub.1, R.sub.2, R.sub.3 and R.sub.4, are each
hydrogen, and R.sub.5 and R.sub.6 are each hydrogen.
10. The polyglucosamine of claim 9, comprising at least one
structural unit: ##STR00043##
11. The polyglucosamine of claim 10, wherein B is hydrogen.
12. The polyglucosamine of claim 11, wherein B is --Y--Z.
13. The polyglucosamine of claim 12, wherein B is --Y--Z, wherein Z
has the structure: ##STR00044## wherein j, in each instance, is an
integer from 1 to 100.
14. The polyglucosamine of claim 12, wherein Y has the structure:
##STR00045## wherein, R.sub.p, R.sub.q, R.sub.s and R.sub.t, in
each instance, are independently, hydrogen or hydroxyl; and k is an
integer from 1 to 20.
15. The polyglucosamine of claim 1, comprising the structural unit:
##STR00046## wherein, D is ##STR00047##
16. The polyglucosamine of claim 15, wherein , in each instance, is
a single bond, and R.sub.1, R.sub.2, R.sub.3 and R.sub.4, are each
hydrogen.
17. The polyglucosamine of claim 1, wherein B is --Y--Z, wherein Z
has the structure: ##STR00048## wherein j, in each instance, is an
integer from 1 to 100.
18. The polyglucosamine of claim 17, wherein j is an integer from 1
to 50.
19. The polyglucosamine of claim 17, wherein j is an integer from 1
to 15.
20. The polyglucosamine of claim 1, wherein A is ##STR00049##
wherein G is hydrogen, or is taken together with N to form a nitric
oxide donor or is absent; and B is hydrogen.
21. The polyglucosamine of claim 1, comprising the structural unit:
##STR00050## wherein, m is an integer from 1 to 1,000, and n is an
integer from 1 to 1,000.
22. The polyglucosamine of claim 21, wherein m and n are each
independently selected from an integer of 1 to 50.
23. The polyglucosamine of claim 21, comprising the structural
unit: ##STR00051##
24. The polyglucosamine of claim 20, wherein X is hydrogen or is
taken together with N to form a diazeniumdiolate; and A is
##STR00052## wherein G is hydrogen or is taken together with N to
form a diazeniumdiolate.
25. The polyglucosamine of claim 21, wherein B is --Y--Z, wherein Z
has the structure: ##STR00053## wherein j, in each instance, is an
integer from 1 to 100.
26. The polyglucosamine of claim 22, wherein Y has the structure:
##STR00054## wherein, R.sub.p, R.sub.q, R.sub.s and R.sub.t, in
each instance, are independently, hydrogen or hydroxyl; and k is an
integer from 1 to 20.
27. The polyglucosamine of claim 1, wherein A is N.
28. The polyglucosamine of claim 1, wherein A is S.
29. The polyglucosamine of claim 1, wherein R.sub.c and R.sub.d are
independently hydrogen or methyl; and v is 2.
30. A method of delivering nitric oxide to a subject, comprising:
administering an effective amount of said polyglucosamine of claim
1 to said subject.
31. A method of treating a disease state, comprising: administering
an effective amount of said polyglucosamine of claim 1 to a subject
in need thereof, wherein said disease state is selected from the
group consisting of a cancer, a cardiovascular disease, a microbial
infection; platelet aggregation and platelet adhesion caused by the
exposure of blood to a medical device; pathological conditions
resulting from abnormal cell proliferation; transplantation
rejections, autoimmune diseases, inflammation, vascular diseases;
scar tissue; wound contraction, restenosis, pain, fever,
gastrointestinal disorders, respiratory disorders, sexual
dysfunctions, and sexually transmitted diseases.
32. The method of claim 31, wherein said disease state is cystic
fibrosis.
33. A pharmaceutical formulation comprising: iii. said
polyglucosamine of claim 1; and iv. a pharmaceutically acceptable
carrier.
34. The pharmaceutical formulation of claim 33, wherein said
polyglucosamine is water-soluble.
35. The polyglucosamine of claim 1, wherein said polyglucosamine is
water soluble.
Description
FIELD OF THE INVENTION
[0002] The presently disclosed subject matter provides nitric
oxide-releasing polysaccharides and oligosaccharides, in
particular, polyglucosamines, and their use in biomedical and
pharmaceutical applications. More particularly, in some
embodiments, the presently disclosed subject matter provides nitric
oxide-releasing oligosaccharides that release nitric oxide in a
controlled and targeted manner, thereby prolonging the therapeutic
effects of nitric oxide and improving the specificity of nitric
oxide delivery to targeted cells and/or tissues.
BACKGROUND OF THE INVENTION
[0003] The discovery of the multifaceted role of nitric oxide (NO)
in biology, physiology, and pathophysiology, see Marietta, M. A.,
at al., BioFactors, 2, 219-225 (1990), has led to the search for
nitric oxide donors capable of controlled nitric oxide release. See
Keefer, L. K., Chemtech, 28, 30-35 (1998). To date, researchers
have discovered that NO regulates a range of biological processes
in the cardiovascular, gastrointestinal, genitourinary,
respiratory, and central and peripheral nervous systems. See
Ignarro, L. J., Nitric Oxide: Biology and Pathobiology; Academic
Press: San Diego, 2000; and Ignarro, L. J. et al., Proc. Natl.
Acad. Sci., USA., 84, 9265-9269 (1987). Furthermore, the discovery
of NO as a vasodilator and its identification as both an antibiotic
and a tumoricidal factor have made NO an attractive pharmaceutical
candidate. See, for example, Radomski, M. W., et al., Br. J.
Pharmacol., 92, 639-646 (1987); Albina, J. E., and Reichner, J. S.;
Canc. Mews. Rev., 17, 19-53 (1998); Nablo, B. J., at al., J. Am.
Chem. Soc., 123, 9712-9713 (2001); Cobbs, C. S., et al., Cancer
Res., 55, 727-730 (1995); Jenkins, D. C., at al., Proc. Natl. Acad.
Sci., USA., 92, 4392-4396 (1995); and Thomsen, L. L., et al., Br.
J. Cancer., 72, 41-44 (1995).
Several nitric oxide donors have been reported, the most notable
being N-diazeniumdiolates. Generally, N-diazeniumdiolate NO donors
are small molecules synthesized by the reaction of amines with NO
at elevated pressure and have been used, for example, to
spontaneously generate NO in aqueous solution. See Hrabie, J. A.
and Keefer, L. K., Chem. Rev., 102, 1135-1154 (2002).
[0004] Therapeutic strategies to explore the activities of nitric
oxide donors, for example, to kill tumor cells, are problematic in
part because the nitric oxide delivery systems known in the art
release or donate nitric oxide indiscriminately. Thus, there is a
need in the art for a nitric oxide delivery system that releases or
donates nitric oxide in a controlled and/or targeted manner to
facilitate an improved understanding of the function of NO in
physiology and to provide for the development of NO-associated
therapies.
SUMMARY OF THE INVENTION
[0005] In embodiments, the subject matter disclosed herein is
directed to a polyglucosamine that contains a covalently bound
nitric oxide releasing moiety, i.e. a NO donor. At least one
structural unit in the polyglucosamine backbone contains the
structural unit of formula I. Optionally, at least one structural
unit of the polyglucosamine further comprises the structural unit
of formula II. Advantageously, the polyglucosamine described herein
is water soluble and is capable of delivering NO to a target.
[0006] In embodiments, the subject matter disclosed herein is
directed to methods of delivering or releasing NO to a subject
comprising administering a polyglucosamine comprising at least one
structural unit of formula I and optionally, further comprising at
least one structural unit of formula II.
[0007] In embodiments, the subject matter disclosed herein is
directed to methods of treating a disease state in a subject
comprising administering a polyglucosamine comprising at least one
structural unit of formula I and optionally, further comprising at
least one structural unit of formula II.
[0008] In embodiments, the subject matter disclosed herein is
directed to a pharmaceutical composition comprising a
polyglucosamine containing at least one structural unit of formula
I and optionally, further comprising at least one structural unit
of formula II and a pharmaceutically acceptable carrier, excipient
or diluent.
[0009] In embodiments, the subject matter disclosed herein is
directed to methods of preparing a polyglucosamine comprising at
least one structural unit of formula I and optionally, further
comprising at least one structural unit of formula II.
[0010] In another embodiment, the subject matter disclosed herein
is directed to a method of disrupting, eradicating or preventing a
biofilm by employing a polyglucosamine comprising at least one
structural unit of formula I and optionally, further comprising at
least one structural unit of formula II.
[0011] The subject matter is described fully in the drawings herein
and in the specification set forth below.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 describes an advantageous property of the presently
disclosed functionalized polyglucosamines.
[0013] FIG. 2 shows the percent of C, H and N found in certain
embodiments.
[0014] FIG. 3 is a .sup.1H NMR of certain embodiments.
[0015] FIG. 4A depicts the release profile of certain chitosan
oligosaccharides CSO 2-NO in different NO conjugation solvents.
Methanol:H.sub.2O 7:3 resulted in total NO storage of 0.87
.mu.mol/mg; FIG. 4B depicts specific data points for the nitric
oxide release profiles of Chitosan 2/NO-5k in methanol (solid
square), methanol/water 9:1 (solid circle), 8:2 (open triangle),
7:3 (solid triangle), and 6:4 v/v (open square).
[0016] FIGS. 5A & 5B depicts (A) Real time NO release profiles
for certain NO-releasing chitosan oligosaccharides; and (B) plot of
t[NO] vs time for certain NO-releasing chitosan
oligosaccharides.
[0017] FIG. 6 shows the t[NO], [NO].sub.max, and t.sub.1/2 of
certain embodiments.
[0018] FIG. 7 depicts the data showing NO-releasing CSO led to
5-log reduction of P. aeruginosa bacteria with biofilms.
Anti-biofilm efficacy of NO-releasing (solid symbols) and control
(open symbols) chitosan oligosaccharides (Chitosan 1-5k; CSO 1-NO
(sphere), Chitosan 2-5k; CSO 2-NO (square), and Chitosan 3-5k; CSO
3-NO (triangle)) against established P. aeruginosa biofilms.
Control chitosan oligosaccharides resulted in no significant
reduction in bacteria viability.
[0019] FIGS. 8 A and B depict cytotoxicity in L929 mouse
fibroblast. The viability of L929 mouse fibroblasts exposed to
control and NO-releasing chitosan oligosaccharides at concentration
for 5-log bacteria viability reduction (MBC) against P. aeruginosa
biofilms. Each parameter was analyzed with multiple replicates
(n=3).
[0020] FIG. 9A-F depict confocal fluorescence images of
RITC-labeled chitosan oligosaccharide association with P.
aeruginosa in biofilms (A. Chitosan 2/NO-5k, B. Chitosan 3/NO-5k,
C. Chitosan 2-10k) and images of syto 9 labeled biofilms incubated
with D) Chitosan 2/NO-5k, E) Chitosan 3/NO-5k and F) Chitosan
2/NO-10k. Green fluorescence of syto 9 indicates the P. aeruginosa
bacteria embedded in the biofilms. Red fluorescence of RITC
indicates the association of RITC-labeled chitosan oligosaccharides
with P. aeruginosa in biofilms. Scale bar: 40 .mu.m.
[0021] FIG. 10A-H depict Bright field and fluorescent images of
RITC-modified Chitosan 2/NO-5k at A) 24, B) 28, C) 42 min and
Chitosan 3/NO-5k at D) 82, E) 86, F) 110, H) 120 min (150 .mu.g
mL.sub.-1) association with P. aeruginosa. Overlay images of P.
aeruginosa incubated with G) Chitosan 2/NO-5k at 44 min and H)
Chitosan 2/NO-5k at 120 min.
DETAILED DESCRIPTION
[0022] The presently disclosed nitric oxide (NO) releasing
polyglucosamines, also referred to in embodiments as NO-releasing
chitosan oligosaccharides, are advantageously water soluble and
tunable. These properties contribute to the usefulness of the
presently disclosed polyglucosamines in therapeutics and disease
states where water soluble therapeutics are advantageous, for
example, in the treatment of cystic fibrosis. Other advantages over
known NO releasing particles that the presently disclosed
functionalized NO releasing polyglucosamines possess include: 1.
Distinct synthesis routes and chemical composition by grafting
secondary amine-containing oligomer chains onto chitosan
oligosaccharides; 2. Tunability of NO storage and NO-release
kinetics is an advantage. By tuning the number of secondary amines
on the aziridine oligomer side chains, NO storage can be
controlled. Further functionalization of the amines on the
resulting materials by compounds of different
hydrophobicity/hydrophilicity enables the control over NO-release
kinetics. Indeed, much larger NO storage was yielded by the
presently disclosed functionalized polyglucosamines; and 3. In
contrast to particles, the functionalized polyglucosamines
described herein are water soluble, facilitating a wider range of
applications including biomedical application where
water-solubility is desired. A previously disclosed NO-releasing
chitosan (U.S. Pat. No. 6,451,337) is not water soluble. This
highlights another advantage that the presently disclosed water
soluble functionalized can readily penetrate and eradicate
biofilms.
[0023] The present invention can be embodied in different forms and
should not be construed as limited to the embodiments set forth
herein. Rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the invention to those skilled in the art. For example,
features illustrated with respect to one embodiment can be
incorporated into other embodiments, and features illustrated with
respect to a particular embodiment can be deleted from that
embodiment. In addition, numerous variations and additions to the
embodiments suggested herein will be apparent to those skilled in
the art in light of the instant disclosure, which do not depart
from the instant invention.
[0024] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0025] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference herein in
their entirety.
[0026] As used herein, "a," "an," or "the" can mean one or more
than one. For example, "a" NO releasing moiety can mean a single or
a multiplicity.
[0027] Also as used herein, "and/or" refers to and encompasses any
and all possible combinations of one or more of the associated
listed items, as well as the lack of combinations when interpreted
in the alternative ("or").
[0028] Furthermore, the term "about," as used herein when referring
to a measurable value such as an amount of a compound or agent of
this invention, dose, time, temperature, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, .+-.0.5%,
or even .+-.0.1% of the specified amount.
[0029] The term "consists essentially of" (and grammatical
variants), as applied to the compositions of this invention, means
the composition can contain additional components as long as the
additional components do not materially alter the composition.
[0030] The term "treatment effective amount" or "effective amount,"
as used herein, refers to that amount of a functionalized
polyglucosamine that imparts a modulating effect, which, for
example, can be a beneficial effect, to a subject afflicted with a
disorder, disease or illness, including improvement in the
condition of the subject (e.g., in one or more symptoms), delay or
reduction in the progression of the condition, prevention or delay
of the onset of the disorder, and/or change in clinical parameters,
disease or illness, etc., as would be well known in the art. For
example, a therapeutically effective amount or effective amount can
refer to the amount of a composition, compound, or agent that
improves a condition in a subject by at least 5%, e.g., at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or at least
100%.
[0031] "Treat" or "treating" or "treatment" refers to any type of
action that imparts a modulating effect, which, for example, can be
a beneficial effect, to a subject afflicted with a disorder,
disease or illness, including improvement in the condition of the
subject (e.g., in one or more symptoms), delay or reduction in the
progression of the condition, and/or change in clinical parameters,
disease or illness, etc., as would be well known in the art.
[0032] The terms "disrupting" and "eradicating" refer to the
ability of the presently disclosed functionalized polyglucosamines
to combat biofilms. The biofilms may be partially eradicated or
disrupted, meaning that the cells no longer attach to one another
or to a surface. The biofilm may be completely eradicated, meaning
that the biofilm is no longer an interconnected, cohesive or
continuous network of cells to a substantial degree.
[0033] As used herein, the term "water soluble" means that the
functionalized polyglucosamine is more soluble in water at room
temperature than the polyglucosamine before functionalization.
Preferably, water soluble functionalized polyglucosamines disclosed
herein are soluble such that >50 mg of functionalized
polyglucosamine dissolves per mL of water. More preferably, water
soluble functionalized polyglucosamines disclosed herein are
soluble such that >75 mg of functionalized polyglucosamine
dissolves per mL of water. Most preferably, water soluble
functionalized polyglucosamines disclosed herein are soluble such
that >100 mg of functionalized polyglucosamine dissolves per mL
of water.
[0034] The terms "nitric oxide donor" or "NO donor" refer to
species that donate, release and/or directly or indirectly transfer
a nitric oxide species, and/or stimulate the endogenous production
of nitric oxide in vivo and/or elevate endogenous levels of nitric
oxide in vivo such that the biological activity of the nitric oxide
species is expressed at the intended site of action.
[0035] The terms "nitric oxide releasing" or "nitric oxide
donating" refer to methods of donating, releasing and/or directly
or indirectly transferring any of the three redox forms of nitrogen
monoxide (NO+, NO.sup.-, NO). In some cases, the nitric oxide
releasing or donating is accomplished such that the biological
activity of the nitrogen monoxide species is expressed at the
intended site of action.
[0036] The term "microbial infection" as used herein refers to
bacterial, fungal, viral, and yeast infections.
[0037] The "patient" or "subject" treated in the many embodiments
disclosed herein is desirably a human patient, although it is to be
understood that the principles of the presently disclosed subject
matter indicate that the presently disclosed subject matter is
effective with respect to all vertebrate species, including
mammals, which are intended to be included in the terms "subject"
and "patient." Suitable subjects are generally mammalian subjects.
The present invention finds use in research as well as veterinary
and medical applications. The term "mammal" as used herein
includes, but is not limited to, humans, non-human primates,
cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents
(e.g., rats or mice), etc. Human subjects include neonates,
infants, juveniles, adults and geriatric subjects.
[0038] The subject can be a subject "in need of" the methods
disclosed herein can be a subject that is experiencing a disease
state and/or is anticipated to experience a disease state, and the
methods and compositions of the invention are used for therapeutic
and/or prophylactic treatment.
[0039] The oligosaccharide described herein are polyglucosamines.
Polyglucosamines and derivatives thereof are known in the as
chitosans and derivatives thereof. Particularly useful
polyglucosamines are polymers that can range in size 100 to 20,000
g/mol. Smaller polyglucosamines having molecular weights below 100
g/mol and larger ones having molecular weights above 20,000 g/mol
are also contemplated. Chitosans having a molecular weight above
20,000 g/mol may need to be further functionalized to be water
soluble. An important feature of useful polyglucosamines is an
available nitrogen on the carbohydrate backbone that is derivatized
according to the methods described herein to form a NO-releasing
polyglucosamine. Advantageously, the polyglucosamines disclosed
herein are water soluble.
[0040] Chitosan is a linear polysaccharide composed of randomly
distributed .beta.-(1-4)-linked D-glucosamine (deacetylated unit)
and N-acetyl-D-glucosamine (acetylated unit). It is a
polyglucosamine. Chitosan is derived from chitin, a polysaccharide
found in the exoskeleton of shellfish such as shrimp, lobster, and
or crabs. It has the following structure:
##STR00001##
Chitosan is biodegradable and biocompatible. Chitosan itself is
only soluble under acidic conditions. Chitosan polysaccharides are
insoluble under physiological conditions. Additionally, their NO
storage is rather modest (.about.0.2 .mu.mol/mg) likely due to poor
solubility of polysaccharides in basic solutions necessary for NO
donor formation. Valmikinathan, C. M.; Mukhatyar, V. J.; Jain, A.;
Karumbaiah, L.; Dasari, M.; Bellamkonda, R. V. Photocrosslinkable
chitosan based hydrogels for neural tissue engineering. Soft Matter
2012, 8, 1964-1976; Zhang, J. L.; Xia, W. S.; Liu, P.; Cheng, Q.
Y.; Tahirou, T.; Gu, W. X.; Li, B. Chitosan Modification and
Pharmaceutical/Biomedical Applications. Mar. Drugs 2010, 8,
1962-1987; Wan, A.; Gao, Q.; Li, H. L. Effects of molecular weight
and degree of acetylation on the release of nitric oxide from
chitosan-nitric oxide adducts. J. Appl. Polym. Sci. 2010, 117,
2183-2188. To synthesize N-diazeniumdiolate-functionalized chitosan
derivatives with improved NO storage, we prepared water-soluble
chitosan oligosaccharides by the degradation of chitosan
polysaccharides in hydrogen peroxide. A benefit of the chitosan
oligosaccharides described herein are relatively low-molecular
weight from 100 to 20,000 g/mol, in particular about 10,000 g/mol
or less; or about 8000 g/mol or less; or about 5000 g/mol or less;
or about 2,500 g/mol or less and their ability to readily penetrate
biofilms. Maghami, G. G.; Roberts, G. A. F. Evaluation of the
viscometric constants for chitosan. Makromol. Chem. 1988, 189,
195-200; Porporatto, C.; Bianco, I. D.; Riera, C. M.; Correa, S.
G., Chitosan induces different L-arginine metabolic pathways in
resting and inflammatory macrophages. Biochem. Biophy. Res. Comm.
2003, 304, 266-272. Chitosan oligosaccharides described herein have
greater NO storage of up to about 8.7 .mu.mol/mg and are also
soluble under neutral and basic conditions.
[0041] The primary amino groups on the backbone of chitosan are
chemical handles for the preparation of the NO-releasing
oligosaccharides disclosed herein. As shown in the schemes below,
secondary amino groups are prepared from the primary amino
groups.
[0042] Useful agents to form the secondary amino groups are
selected from the group consisting of aziridines, in particular
2-methyl aziridine, and thiiranes and the like.
[0043] In an embodiment, the subject matter disclosed herein is
directed to a polyglucosamine, e.g., a chitosan oligosaccharide
comprising at least one structural unit of formula I:
##STR00002##
[0044] and optionally, at least one structural unit of formula
II:
##STR00003##
[0045] wherein, [0046] R.sub.1, R.sub.2, R.sub.3 and R.sub.4, if
present, are each independently selected from the group consisting
of hydrogen; C.sub.1-5 alkyl(C.dbd.O)--, when the C.sub.1-5 alkyl
is methyl, Me(C.dbd.O)-- is an acyl, Ac; and C.sub.1-5 alkyl;
[0047] in each instance, is a single or double bond, [0048] wherein
in each instance where it is a double bond, R.sub.1, R.sub.2,
R.sub.3 or R.sub.4 attached to the double bond-O is absent; when
R.sub.1 is absent, R.sub.5 is hydrogen, hydroxyl, C.sub.1-5 alkyl
or C.sub.1-5 alkoxy; when R.sub.3 is absent, R.sub.6 is hydrogen,
hydroxyl, C.sub.1-5 alkyl or C.sub.1-5 alkoxy; [0049] wherein in
each instance where it is a single bond, R.sub.1, R.sub.2, R.sub.3
or R.sub.4 attached to the double bond-O is present; when R.sub.1
is present, R.sub.5 is hydrogen; when R.sub.3 is present, R.sub.6
is hydrogen; [0050] Q is --(CR.sub.cR.sub.d).sub.v--; [0051]
wherein R.sub.c and R.sub.d are independently hydrogen or C.sub.1-5
alkyl, such as methyl, ethyl, n-propyl, isopropyl, t-butyl,
n-butyl, isobutyl and pentyl. Preferably, R.sub.c and R.sub.d are
independently hydrogen, methyl or ethyl; and v is an integer from 2
to 6; preferably, v is 2; [0052] p is an integer from 1 to 100,
preferably 1 to 50; more preferably 1 to 25; most preferably 1 to
10; [0053] A is
[0053] ##STR00004## [0054] wherein, L is S, O or N; and [0055] G,
in each instance, is independently, hydrogen, or is taken together
with L to form a nitric oxide donor; [0056] X is hydrogen,
C.sub.1-5 alkyl or is taken together with N to form a nitric oxide
donor; [0057] B is hydrogen or --Y--Z, wherein Y is a spacer and Z
is a polymer or a terminus group; or B is absent; [0058] D is
--NR.sub.aR.sub.b, wherein R.sub.a and R.sub.b are independently
selected from the group consisting of hydrogen, formyl, C.sub.1-5
alkyl(C.dbd.O)--, when the C.sub.1-5 alkyl is methyl, Me(C.dbd.O)--
is an acyl, Ac, C.sub.1-5 alkyl and C.sub.1-5 alkyl ester; [0059]
or D is
##STR00005##
[0060] Useful values of R.sub.1, R.sub.2, R.sub.3 and R.sub.4, if
present, are each independently selected from the group consisting
of hydrogen and C.sub.1-5 alkyl. When one or more of R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 is C.sub.1-5 alkyl, it is selected
from methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl
and pentyl. Preferably, R.sub.1, R.sub.2, R.sub.3 and R.sub.4, if
present, is hydrogen or methyl. Most preferably, R.sub.1, R.sub.2,
R.sub.3 and R.sub.4, if present, is hydrogen.
[0061] In all embodiments, , in each instance, is a single or
double bond. Preferably, it is a single bond in all instances.
[0062] Q is --(CR.sub.cR.sub.d).sub.v--; wherein R.sub.c and
R.sub.d are independently hydrogen or C.sub.1-5 alkyl, such as
methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl and
pentyl. Preferably, R.sub.c and R.sub.d are independently hydrogen,
methyl or ethyl. Useful values of v are integers from 2 to 6.
Preferably, v is 2.
[0063] Useful values of p include any integer from 1 to 100.
Preferably p is an integer from 1 to 50. More preferably, p is an
integer from 1 to 25. Most preferably, p is an integer from 1 to
10, such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
[0064] Useful values of L are N, S and O. Preferably, L is N or S.
In each instance that G occurs, it is independently hydrogen or a
nitric oxide donor. Since the nitric oxide donor contributes to the
amount of available NO on the polyglucosamine, it is preferable
that G is a nitric oxide donor. In preferred embodiments, at least
30% of G present on a polyglucosamine is a nitric oxide donor. More
preferably, at least 50% of G present on a polyglucosamine is a
nitric oxide donor. Even more preferably, at least 90% of G present
on a polyglucosamine is a nitric oxide donor. Most preferably, at
least 95% of G present on a polyglucosamine is a nitric oxide
donor.
[0065] Useful values of X are hydrogen, C.sub.1-5 alkyl or a nitric
oxide donor. Since the nitric oxide donor contributes to the amount
of available NO on the polyglucosamine, it is preferable that X is
a nitric oxide donor. In preferred embodiments, at least 30% of X
present on a polyglucosamine is a nitric oxide donor. More
preferably, at least 50% of X present on a polyglucosamine is a
nitric oxide donor. Even more preferably, at least 90% of X present
on a polyglucosamine is a nitric oxide donor. Most preferably, at
least 95% of X present on a polyglucosamine is a nitric oxide
donor.
[0066] Useful values of B are hydrogen, --Y--Z, wherein Y is a
spacer and Z is a monomer or polymer, or B is a terminus group. B
may also be absent when L is O or S. As used herein, a terminus
group is any end-capping group at the terminus of a polymer or
monomer. These groups are known in the art. Preferably, when B is a
terminus group, it is hydrogen, hydroxyl or C.sub.1-5 alkyl.
[0067] Useful values of Z include monomers and polymers known in
the art, especially those used in active pharmaceutical
ingredients. Particularly useful polymers or monomers include:
##STR00006##
wherein j, in each instance, is an integer from 1 to 100.
[0068] Useful spacers, Y, in the formulae disclosed herein include
spacers or linkers known in the art, especially those used in
active pharmaceutical ingredients. Particularly useful spacers
include the following:
##STR00007##
wherein, R.sub.p, R.sub.q, R.sub.s and R.sub.t, in each instance,
are independently, hydrogen or hydroxyl; and k is an integer from 1
to 20.
[0069] Using the strategies disclosed herein, any secondary amino
group present on the oligosaccharide can be modified as described
herein to form a NO-releasing oligosaccharide. The secondary amino
groups attached directly to the sugar backbone moieties or
secondary amino groups pendant on the backbone sugar moieties can
be functionalized with a NO releasing moiety. As disclosed fully
herein in the synthetic routes, primary amines are modified to
secondary amines. This modification can be facilitated by
aziridines, thiiranes and the like.
[0070] Useful NO releasing moieties include any NO releasing group
known in the art. Particularly useful are residues of NO releasing
groups, i.e. NO donors, are covalently bound to N, S or O on the
polyglucosamine. The NO donor is taken together with the atom on
the polyglucosamine to which it is bound to form a moiety selected
from the group consisting of a diazeniumdiolate, --NO as part of a
nitrosothiol group for example, a nitrosamine, a hydroxyl
nitrosamine, a hydroxyl amine, a hydroxyurea, and combination
thereof. Preferably, the NO releasing moiety is a diazeniumdiolate.
These groups may be present in the form of a salt.
[0071] In some embodiments, the NO donor is a N-diazeniumdiolate
(i.e., a 1-amino-substituted deazen-1-lum-1,2-diolate),
N-Diazeniumdiolates are particularly attractive as NO donors due to
their ability to generate NO spontaneously under biological
conditions. See Hrabie, J. A. and Keefer, L. K., Chem. Rev., 102,
1135-1154 (2002); and Napoli, C. and Ianarro, L. J., Annu. Rev.
Pharmacol. Toxicol., 43, 97-123 (2003). Several N-diazeniumdiolate
compounds have been synthesized using a range of nucleophilic
residues that encompass primary and secondary amines, polyamines,
and secondary amino acids, See Hrabie, J. A., and Keefer L. K.,
Chem. Rev., 102, 1135-1154 (2002). In the formation of the
N-diazeniumdiolate, one equivalent of amine reacts with two
equivalents of nitric oxide under elevated pressure. A base (e.g.,
an alkoxide like methoxide) removes a proton from the amine
nitrogen to create the anionic, stabilized [N(O)NO] group. While
stable under ambient conditions, N-diazeniumdiolates decompose
spontaneously in aqueous media to generate NO at rates dependent
upon pH, temperature, and/or the structure of the amine moiety. For
example, N-diazeniumdiolate-modified proline (PROLI/NO),
2-(dimethylamino)-ethylputreamlne (DMAEP/NO),
N,N-dimethylhexanediamine (DMHD/NO), and diethylenetriamine
(DETA/NO) have been developed as small molecule NO donors with
diverse NO release half-lives ranging from 2 seconds to 20 hours at
pH 7.4 and 37.degree. C. See Hrabie, J. A., and Keefer, L. K.,
Chem. Rev., 102, 1135-1154 (2002); and Keefer, L. K., Annu, Rev.
Pharmacol. Toxicol 43, 585-607 (2003).
[0072] The secondary amine functional group of the polyglucosamine
is converted in high yields to a nitric oxide donor in the presence
of a strong base and gaseous nitric oxide. As provided herein, the
solvent system can affect the charging of the polyglucosamine with
NO.
[0073] In an embodiment, when the polyglucosamine is specifically
functionalized with an aziridine as described herein, the
functionalized polyglucosamine comprises at least one structural
unit of formula Ia:
##STR00008##
and optionally, at least one structural unit of formula IIa:
##STR00009##
wherein, the definitions of the variables are as defined above with
the exception of D. In this embodiment, D is --NR.sub.aR.sub.b,
wherein R.sub.a and R.sub.b are independently selected from the
group consisting of hydrogen, formyl, C.sub.1-5 alkyl(C.dbd.O)--,
C.sub.1-5 alkyl and C.sub.1-5 alkyl ester; or D has the
structure:
##STR00010##
wherein, X, Q, p and B are as described above.
[0074] In an aspect of this embodiment, when the polyglucosamine is
specifically functionalized with an aziridine as described herein,
the functionalized polyglucosamine comprises at least one
structural unit of formula VIII:
##STR00011##
wherein, the variables are as described above for this
embodiment.
[0075] The term "amino" and "amine" refer to nitrogen-containing
groups such as NR.sub.3, NH.sub.3, NHR.sub.2, and NH.sub.2R,
wherein R can be as described elsewhere herein. Thus, "amino" as
used herein can refer to a primary amine, a secondary amine, or a
tertiary amine. In some embodiments, one R of an amino group can be
a diazeniumdiolate (i.e., NONO).
[0076] The terms "cationic amine" and "quaternary amine" refer to
an amino group having an additional (i.e., a fourth) group, for
example a hydrogen or an alkyl group bonded to the nitrogen. Thus,
cationic and quaternary amines carry a positive charge.
[0077] The term "alkyl" denotes a straight or branched hydrocarbon
chain containing 1-24 carbon atoms, e.g., 1-12 carbon atoms.
Examples of alkyl group include methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, tert-butyl, and the like.
[0078] The term "alkoxy" is used herein to mean a straight or
branched chain alkyl radical, as defined above, unless the chain
length is limited thereto, bonded to an oxygen atom, including, but
not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the
like. Preferably the alkoxy chain is 1 to 5 carbon atoms in length,
more preferably 1-3 carbon atoms in length.
[0079] The following specific embodiments are disclosed: [0080] 1.
A polyglucosamine (chitosan oligosaccharide) comprising, [0081] at
least one structural unit:
[0081] ##STR00012## [0082] and optionally, at least one structural
unit:
[0082] ##STR00013## [0083] wherein, [0084] R.sub.1, R.sub.2,
R.sub.3 and R.sub.4, if present, are each independently selected
from the group consisting of hydrogen, C.sub.1-5 alkyl(C.dbd.O)--
and C.sub.1-5 alkyl; [0085] in each instance, is a single or double
bond, [0086] wherein in each instance where it is a double bond,
R.sub.1, R.sub.2, R.sub.3 or R.sub.4 attached to the double bond-O
is absent; when R.sub.1 is absent, R.sub.5 is hydrogen, hydroxyl,
C.sub.1-5 alkyl or C.sub.1-5 alkoxy; when R.sub.3 is absent,
R.sub.6 is hydrogen, hydroxyl, C.sub.1-5 alkyl or C.sub.1-5 alkoxy;
[0087] wherein in each instance where it is a single bond, R.sub.1,
R.sub.2, R.sub.3 or R.sub.4 attached to the double bond-O is
present; when R.sub.1 is present, R.sub.5 is hydrogen; when R.sub.3
is present, R.sub.6 is hydrogen; [0088] Q is
--(CR.sub.cR.sub.d).sub.v--; [0089] wherein R.sub.c and R.sub.d are
independently hydrogen or C.sub.1-5 alkyl; and v is an integer from
2 to 6; [0090] p is an integer from 1 to 10; [0091] A is
[0091] ##STR00014## [0092] wherein, L is S, O or N; and [0093] G,
in each instance, is independently, hydrogen, or is taken together
with L to form a nitric oxide donor or is absent; [0094] X is
hydrogen, C.sub.1-5 alkyl or is taken together with N to form a
nitric oxide donor; [0095] B is one hydrogen or --Y--Z, wherein Y
is a spacer and Z is a polymer or a terminus; [0096] D is
--NR.sub.aR.sub.b, wherein R.sub.a and R.sub.b are independently
selected from the group consisting of hydrogen, formyl, C.sub.1-5
alkyl(C.dbd.O)--, C.sub.1-5alkyl and C.sub.1-5 alkyl ester; [0097]
or D is
[0097] ##STR00015## [0098] 2. The polyglucosamine of embodiment 1,
wherein at least one of the X and G is taken together with the atom
on the polyglucosamine to which it is bound to form a nitric oxide
donor. [0099] 3. The polyglucosamine of embodiment 1, comprising
the structural unit: 4.
[0099] ##STR00016## [0100] wherein, [0101] m is an integer from 1
to 10,000. [0102] 5. The polyglucosamine of embodiment 1, wherein
the nitric oxide donor is taken together with the atom on the
polyglucosamine to which it is bound is selected from the group
consisting of a diazeniumdiolate, nitrosothiol, a nitrosamine, a
hydroxyl nitrosamine, a hydroxyl amine, a hydroxyurea, and
combination thereof. [0103] 5. The polyglucosamine of embodiment 4,
wherein the nitric oxide donor is diazeniumdiolate. [0104] 6. The
polyglucosamine of embodiment 3, wherein in is an integer from 1 to
50. [0105] 7. The polyglucosamine of embodiment 3, wherein m is an
integer from 1 to 10. [0106] 8. The polyglucosamine of embodiment
1, comprising at least one structural unit:
[0106] ##STR00017## [0107] wherein, [0108] D is --NR.sub.aR.sub.b,
wherein R.sub.a and R.sub.b are independently selected from the
group consisting of hydrogen, formyl, C.sub.1-5 alkyl(C.dbd.O)--,
C.sub.1-5 alkyl and C.sub.1-5 alkyl ester. [0109] 9. The
polyglucosamine of embodiment 8, wherein [0110] in each instance,
is a single bond [0111] R.sub.1, R.sub.2, R.sub.3 and R.sub.4, are
each hydrogen, and [0112] R.sub.5 and R.sub.6 are each hydrogen.
[0113] 10. The polyglucosamine of embodiment 9, comprising at least
one structural unit:
[0113] ##STR00018## [0114] 11. The polyglucosamine of embodiment
10, wherein B is hydrogen. [0115] 12. The polyglucosamine of
embodiment 11, wherein B is --Y--Z. [0116] 13. The polyglucosamine
of embodiment 12, wherein B is --Y--Z, wherein Z has the
structure:
[0116] ##STR00019## [0117] wherein j, in each instance, is an
integer from 1 to 100. [0118] 14. The polyglucosamine of embodiment
12, wherein Y has the structure:
[0118] ##STR00020## [0119] wherein, [0120] R.sub.p, R.sub.q,
R.sub.s and R.sub.t, in each instance, are independently, hydrogen
or hydroxyl; and [0121] k is an integer from 1 to 20. [0122] 15.
The polyglucosamine of embodiment 1, comprising the structural
unit:
[0122] ##STR00021## [0123] wherein, [0124] D is
[0124] ##STR00022## [0125] 16. The polyglucosamine of embodiment
15, wherein [0126] , in each instance, is a single bond, and [0127]
R.sub.1, R.sub.2, R.sub.3 and R.sub.4, are each hydrogen. [0128]
17. The polyglucosamine of embodiment 1, wherein [0129] B is
--Y--Z, wherein Z has the structure:
[0129] ##STR00023## [0130] wherein j, in each instance, is an
integer from 1 to 100. [0131] 18. The polyglucosamine of embodiment
17, wherein j is an integer from 1 to 50. [0132] 19. The
polyglucosamine of embodiment 17, wherein j is an integer from 1 to
15. [0133] 20. The polyglucosamine of embodiment 1, wherein [0134]
A is
[0134] ##STR00024## [0135] wherein G is hydrogen, or is taken
together with N to form a nitric oxide donor or is absent; and
[0136] B is hydrogen. [0137] 21. The polyglucosamine of embodiment
1, comprising the structural unit:
[0137] ##STR00025## [0138] wherein, [0139] m is an integer from 1
to 1,000, and [0140] n is an integer from 1 to 1,000. [0141] 22.
The polyglucosamine of embodiment 21, wherein m and n are each
independently selected from an integer of 1 to 50. [0142] 23. The
polyglucosamine of embodiment 21, comprising the structural unit:
24.
[0142] ##STR00026## [0143] 25. The polyglucosamine of embodiment
20, wherein [0144] X is hydrogen or is taken together with N to
form a diazeniumdiolate; and [0145] A is
[0145] ##STR00027## [0146] wherein G is hydrogen or is taken
together with N to form a diazeniumdiolate. [0147] 26. The
polyglucosamine of embodiment 21, wherein [0148] B is --Y--Z,
wherein Z has the structure:
[0148] ##STR00028## [0149] wherein j, in each instance, is an
integer from 1 to 100. [0150] 27. The polyglucosamine of embodiment
22, wherein Y has the structure: 28.
[0150] ##STR00029## [0151] wherein, [0152] R.sub.p, R.sub.q,
R.sub.s and R.sub.t, in each instance, are independently, hydrogen
or hydroxyl; and [0153] k is an integer from 1 to 20. [0154] 29.
The polyglucosamine of embodiment 1, wherein A is N. [0155] 30. The
polyglucosamine of embodiment 1, wherein A is S. [0156] 31. The
polyglucosamine of embodiment 1, wherein [0157] R.sub.c and R.sub.d
are independently hydrogen or methyl; and [0158] v is 2. [0159] 32.
A method of delivering nitric oxide to a subject, comprising:
[0160] administering an effective amount of the polyglucosamine of
claim 1 to the subject. [0161] 33. A method of treating a disease
state, comprising: [0162] administering an effective amount of the
polyglucosamine of embodiment 1 to a subject in need thereof,
wherein the disease state is selected from the group consisting of
a cancer, a cardiovascular disease, a microbial infection; platelet
aggregation and platelet adhesion caused by the exposure of blood
to a medical device; pathological conditions resulting from
abnormal cell proliferation; transplantation rejections, autoimmune
diseases, inflammation, vascular diseases; scar tissue; wound
contraction, restenosis, pain, fever, gastrointestinal disorders,
respiratory disorders, sexual dysfunctions, and sexually
transmitted diseases. [0163] 34. The method of embodiment 31,
wherein said disease state is cystic fibrosis. [0164] 35. A
pharmaceutical formulation comprising: [0165] i. the
polyglucosamine of claim 1; and [0166] ii. a pharmaceutically
acceptable carrier. [0167] 36. The pharmaceutical formulation of
embodiment 33, wherein the polyglucosamine is water-soluble. [0168]
35. The polyglucosamine of embodiment 1, wherein the
polyglucosamine is water soluble.
[0169] In all embodiments, combinations of substituents and/or
variables are permissible only if such combinations result in
compounds that conform to a known valence for each atom.
[0170] Specific functionalized polyglucosamines include:
##STR00030##
In each of the above structures of formula I and embodiments
therein (i.e., Formulae III, VI and VII), when present, m is an
integer from 1 to 10,000, preferably 1 to 1000 or 1 to 500; more
preferably 1 to 200 or 1 to 100; and most preferably 1 to 50; 1 to
20 or 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n is an
integer from 1 to 10,000, preferably 1 to 1000 or 1 to 500; more
preferably 1 to 200 or 1 to 100; and most preferably 1 to 50; 1 to
20 or 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and 1 is an
integer from 1 to 10,000, preferably 1 to 1000 or 1 to 500; more
preferably 1 to 200 or 1 to 100; and most preferably 1 to 50; 1 to
20 or 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0171] In an embodiment, the subject matter disclosed herein is
directed to a method of delivering nitric oxide to a subject,
comprising administering an effective amount of a functionalized
polyglucosamine to a subject.
[0172] In another embodiment, the subject matter disclosed herein
is directed to a method of treating a disease state, comprising
administering an effective amount of said polyglucosamine of claim
1 to a subject in need thereof, wherein said disease state is
selected from the group consisting of a cancer, a cardiovascular
disease, a microbial infection; platelet aggregation and platelet
adhesion caused by the exposure of blood to a medical device;
pathological conditions resulting from abnormal cell proliferation;
transplantation rejections, autoimmune diseases, inflammation,
vascular diseases; scar tissue; wound contraction, restenosis,
pain, fever, gastrointestinal disorders, respiratory disorders,
sexual dysfunctions, and sexually transmitted diseases. Preferably,
the disease state is cystic fibrosis.
[0173] In another embodiment, the subject matter disclosed herein
is directed to a method of disrupting, eradicating or preventing a
biofilm. This method comprises contacting a surface or area that
contains a biofilm or is susceptible to a biofilm forming or
occupying some or all of the surface or area with a functionalized
polyglucosamine as described herein. The term "biofilm" is intended
to mean an aggregate of one or more microorganisms in which cells
adhere to each other, usually on a surface. Most any free-floating
microorganisms can form a biofilm and/or attach to a surface.
Microorganisms can adhere to a surface or each other through weak,
reversible adhesion via van der Waals forces. The microorganisms
can more permanently anchor using cell adhesion or structures such
as pili.
[0174] In yet another embodiment, the subject matter disclosed
herein is directed to a pharmaceutical formulation comprising a
functionalized polyglucosamine and a pharmaceutically acceptable
carrier. Preferably, the functionalized polyglucosamine is
water-soluble as described throughout the present disclosure.
[0175] "Pharmaceutically acceptable," as used herein, means a
material that is not biologically or otherwise undesirable, i.e.,
the material can be administered to an individual along with the
compositions of this invention, without causing substantial
deleterious biological effects or interacting in a deleterious
manner with any of the other components of the composition in which
it is contained. The material would naturally be selected to
minimize any degradation of the active ingredient and to minimize
any adverse side effects in the subject, as would be well known to
one of skill in the art (see, e.g., Remington's Pharmaceutical
Science; 20 ed. 2005). Exemplary pharmaceutically acceptable
carriers for the compositions of this invention include, but are
not limited to, sterile pyrogen-free water and sterile pyrogen-free
physiological saline solution.
[0176] The presently disclosed therapeutic compositions, in some
embodiments, comprise a composition that includes a presently
disclosed nitric oxide-releasing polyglucosamine and a
pharmaceutically acceptable carrier. Suitable compositions include
aqueous and non-aqueous sterile injection solutions that can
contain antioxidants, buffers, bacteriostats, bactericidal
antibiotics and solutes that render the formulation isotonic with
the bodily fluids of the intended recipient; and aqueous and
non-aqueous sterile suspensions, which can include suspending
agents and thickening agents.
[0177] The compositions used in the presently disclosed methods can
take such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and can contain formulatory agents, such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0178] The therapeutic compositions can be presented in unit-dose
or multi-dose containers, for example sealed ampoules and vials,
and can be stored in a frozen or freeze-dried (lyophilized)
condition requiring only the addition of sterile liquid carrier
immediately prior to use.
[0179] For oral administration, the compositions can take the form
of, for example, tablets or capsules prepared by a conventional
technique with pharmaceutically acceptable excipients, such as
binding agents (e.g., pregelatinized maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycollate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods known in the art. For example, a therapeutic
agent can be formulated in combination with hydrochlorothiazide,
and as a pH stabilized core having an enteric or delayed release
coating which protects the therapeutic agent until it reaches the
target organ.
[0180] Liquid preparations for oral administration can take the
form of, for example, solutions, syrups or suspensions, or they can
be presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations can be
prepared by conventional techniques with pharmaceutically
acceptable additives, such as suspending agents (e.g., sorbitol
syrup, cellulose derivatives or hydrogenated edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles
(e.g., almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations also
can contain buffer salts, flavoring, coloring and sweetening agents
as appropriate. Preparations for oral administration can be
suitably formulated to give controlled release of the active
compound. For buccal administration the compositions can take the
form of tablets or lozenges formulated in conventional manner.
[0181] The compounds also can be formulated as a preparation for
implantation or injection. Thus, for example, the compounds can be
formulated with suitable polymeric or hydrophobic materials (e.g.,
as an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble salt).
The compounds also can be formulated in rectal compositions (e.g.,
suppositories or retention enemas containing conventional
suppository bases, such as cocoa butter or other glycerides),
creams or lotions, or transdermal patches.
[0182] Pharmaceutical formulations also are provided which are
suitable for administration as an aerosol by inhalation.
Preferably, the functionalized polyglucosamines described herein
are formulated in solution and/or aerosol form. These formulations
comprise a solution or suspension of a NO-releasing polyglucosamine
described herein. The desired formulation can be placed in a small
chamber and nebulized. Nebulization can be accomplished by
compressed air or by ultrasonic energy to form a plurality of
liquid droplets or solid particles comprising the NO-releasing
polyglucosamine. For example, the presently disclosed NO-releasing
polyglucosamine can be administered via inhalation to treat
bacterial infections related to cystic fibrosis. Cystic
fibrosis-related bacterial infections include, but are not limited
to stenotrophomonis, mybacterium avium intracellulaire and M.
abcessus, burkhoderia cepacia and Pseudomonas aeruginosa (P.
aeruginosa) infections.
[0183] The term "effective amount" is used herein to refer to an
amount of the therapeutic composition (e.g., a composition
comprising a nitric oxide-releasing polyglucosamine) sufficient to
produce a measurable biological response. Actual dosage levels of
active ingredients in an active composition of the presently
disclosed subject matter can be varied so as to administer an
amount of the active compound(s) that is effective to achieve the
desired response for a particular subject and/or application. The
selected dosage level will depend upon a variety of factors
including the activity of the composition, formulation, the route
of administration, combination with other drugs or treatments,
severity of the condition being treated, and the physical condition
and prior medical history of the subject being treated. Preferably,
a minimal dose is administered, and dose is escalated in the
absence of dose-limiting toxicity to a minimally effective amount.
Determination and adjustment of an effective dose, as well as
evaluation of when and how to make such adjustments, are known to
those of ordinary skill in the art of medicine.
[0184] For administration of a composition as disclosed herein,
conventional methods of extrapolating human dosage based on doses
administered to a murine animal model can be carried out using the
conversion factor for converting the mouse dosage to human dosage:
Dose Human per kg=Dose Mouse per kg.times.12. See Freireich et al.,
Cancer Chemother Rep. 50, 219-244 (1966). Drug doses also can be
given in milligrams per square meter of body surface area because
this method rather than body weight achieves a good correlation to
certain metabolic and excretionary functions. Moreover, body
surface area can be used as a common denominator for drug dosage in
adults and children as well as in different animal species. See
Freireich et al., Cancer Chemother Rep. 50, 219-244 (1966).
Briefly, to express a mg/kg dose in any given species as the
equivalent mg/sq in dose, multiply the dose by the appropriate km
factor. In an adult human, 100 mg/kg is equivalent to 100
mg/kg.times.37 kg/sq m=3700 mg/m.sup.2.
[0185] For additional guidance regarding formulation and dose, see
U.S. Pat. Nos. 5,326,902; 5,234,933; PCT International Publication
No. WO 93/25521; Berkow et al., The Merck Manual of Medical
Information, Home ed., Merck Research Laboratories: Whitehouse
Station, N.J. (1997); Goodman et al., Goodman &Gilman's the
Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health
Professions Division: New York (1996); Ebadi, CRC Desk Reference of
Clinical Pharmacology, CRC Press, Boca Raton, Fla. (1998); Katzunq,
Basic &Clinical Pharmacology, 8th ed. Lange Medical
Books/McGraw-Hill Medical Pub. Division: New York (2001); Remington
et al., Remington's Pharmaceutical Sciences, 15th ed. Mack Pub.
Co.: Easton, Pa. (1975); and Speight et al., Avery's Drug
Treatment: A Guide to the Properties, Choice, Therapeutic Use and
Economic Value of Drugs in Disease Management, 4th ed. Adis
International: Auckland/Philadelphia (1997); Dutch et al., Toxicol.
Leu., 100-101, 255-263 (1998).
[0186] Suitable methods for administering to a subject a
composition of the presently disclosed subject matter include, but
are not limited to, systemic administration, parenteral
administration (including intravascular, intramuscular,
intraarterial administration), oral delivery, buccal delivery,
subcutaneous administration, inhalation, intratracheal
installation, surgical implantation, transdermal delivery, local
injection, and hyper-velocity injection/bombardment. Where
applicable, continuous infusion can enhance drug accumulation at a
target site (see, e.g., U.S. Pat. No. 6,180,082).
[0187] The particular mode of drug administration used in
accordance with the methods of the presently disclosed subject
matter depends on various factors, including but not limited to the
agent and/or carrier employed, the severity of the condition to be
treated, and mechanisms for metabolism or removal of the active
agent following administration.
[0188] In some embodiments, one or more additional therapeutic
agents can be used in combination with the functionalized
polyglucosamine. Such additional agents can be part of a
formulation comprising the functionalized polyglucosamine or dosed
as a separate formulation prior to, after, or at the same time
(concurrently) as a formulation including the functionalized
polyglucosamine. Such additional therapeutic agents include, in
particular, anti-cancer therapeutics, anti-microbial agents, pain
relievers, anti-inflammatories, vasodialators, and
immune-suppressants, as well as any other known therapeutic agent
that could enhance the alleviation of the disease or condition
being treated. "Concurrently" means sufficiently close in time to
produce a combined effect (that is, concurrently can be
simultaneously, or it can be two or more events occurring within a
short time period before or after each other). In some embodiments,
the administration of two or more compounds "concurrently" means
that the two compounds are administered closely enough in time that
the presence of one alters the biological effects of the other. The
two compounds can be administered in the same or different
formulations or sequentially. Concurrent administration can be
carried out by mixing the compounds prior to administration, or by
administering the compounds in two different formulations, for
example, at the same point in time but at different anatomic sites
or using different routes of administration.
[0189] The choice of additional therapeutic agents to be used in
combination with an NO-releasing polyglucosamine will depend on
various factors including, but not limited to, the type of disease,
the age, and the general health of the subject, the aggressiveness
of disease progression, and the ability of the subject to tolerate
the agents that comprise the combination.
[0190] A variety of chemical compounds, also described as
"antineoplastic" agents or "chemotherapeutic agents" can be used in
combination with the presently disclosed NO-releasing
polyglucosamines used in the treatment of cancer. Such
chemotherapeutic compounds include, but are not limited to,
alkylating agents, DNA intercalators, protein synthesis inhibitors,
inhibitors of DNA or RNA synthesis, DNA base analogs, topoisomerase
inhibitors, anti-angiogenesis agents, and telomerase inhibitors or
telomeric DNA binding compounds. For example, suitable alkylating
agents include alkyl sulfonates, such as busulfan, improsulfan, and
piposulfan; aziridines, such as a benzodizepa, carboquone,
meturedepa, and uredepa; ethylenimines and methylmelamines, such as
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide, and trimethylolmelamine; nitrogen
mustards such as chlorambucil, chlornaphazine, cyclophosphamide,
estramustine, iphosphamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichine, phenesterine,
prednimustine, trofosfamide, and uracil mustard; nitroso ureas,
such as carmustine, chlorozotocin, fotemustine, lomustine,
nimustine, and ranimustine.
[0191] Antibiotics used in the treatment of cancer include
dactinomycin, daunorubicin, doxorubicin, idarubicin, bleomycin
sulfate, mytomycin, plicamycin, and streptozocin. Chemotherapeutic
antimetabolites include mercaptopurine, thioguanine, cladribine,
fludarabine phosphate, fluorouracil (5-FU), floxuridine,
cytarabine, pentostatin, methotrexate, and azathioprine, acyclovir,
adenine .beta.-1-D-arabinoside, amethopterin, aminopterin,
2-aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil,
2'-azido-2'-deoxynucleosides, 5-bromodeoxycytidine, cytosine
.beta.-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,
5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.
[0192] Chemotherapeutic protein synthesis inhibitors include abrin,
aurintricarboxylic acid, chloramphenicol, colicin E3,
cycloheximide, diphtheria toxin, edeine A, emetine, erythromycin,
ethionine, fluoride, 5-fluorotryptophan, fusidic acid, guanylyl
methylene diphosphonate and guanylyl imidodiphosphate, kanamycin,
kasugamycin, kirromycin, and O-methyl threonine. Additional protein
synthesis inhibitors include modeccin, neomycin, norvaline,
pactamycin, paromomycine, puromycin, ricin, shiga toxin,
showdomycin, sparsomycin, spectinomycin, streptomycin,
tetracycline, thiostrepton, and trimethoprim. Inhibitors of DNA
synthesis, including alkylating agents such as dimethyl sulfate,
mitomycin C, nitrogen and sulfur mustards, intercalating agents,
such as acridine dyes, actinomycins, adriamycin, anthracenes,
benzopyrene, ethidium bromide, propidium diiodide-intertwining, and
agents, such as distamycin and netropsin, can be used as part of
the presently disclosed cancer treatments. Topoisomerase
inhibitors, such as coumermycin, nalidixic acid, novobiocin, and
oxolinic acid, inhibitors of cell division, including colcemide,
colchicine, vinblastine, and vincristine; and RNA synthesis
inhibitors including actinomycin D, a-amanitine and other fungal
amatoxins, cordycepin (3'-deoxyadenosine), dichlororibofuranosyl
benzimidazole, rifampicine, streptovaricin, and streptolydigin also
can be combined with functionalized polyglucosamines to provide a
suitable cancer treatment.
[0193] Thus, current chemotherapeutic agents that can be used in
combination with the presently described NO-releasing
functionalized polyglucosamines include, adrimycin, 5-fluorouracil
(5FU), etoposide, camptothecin, actinomycin-D, mitomycin,
cisplatin, hydrogen peroxide, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, ifosfamide, melphalan,
chjlorambucil, bisulfan, nitrosurea, dactinomycin, duanorubicin,
doxorubicin, bleomycin, pilcomycin, tamoxifen, taxol,
transplatimun, vinblastin, and methotrexate, and the like.
[0194] As used herein, the term "antimicrobial agent" refers to any
agent that kills, inhibits the growth of, or prevents the growth of
a babteria, fungus, yeast, or virus. Suitable antimicrobial agents
that can be incorporated into the presently disclosed NO-releasing
functionalized polyglucosamines to aid in the treatment or
prevention of a microbial infection, include, but are not limited
to, antibiotics such as vancomycin, bleomycin, pentostatin,
mitoxantrone, mitomycin, dactinomycin, plicamycin and amikacin.
Other antimicrobial agents include antibacterial agents such as
2-p-sulfanilyanilinoethanol, 4,4'-sulfinyldianiline,
4-sulfanilamidosalicylic acid, acediasulfone, acetosulfone,
amikacin, amoxicillin, amphotericin B, ampicillin, apalcillin,
apicycline, apramycin, arbekacin, aspoxicillin, azidamfenicol,
azithromycin, aztreonam, bacitracin, bambermycin(s), biapenem,
brodimoprim, butirosin, capreomycin, carbenicillin, carbomycin,
carumonam, cefadroxil, cefamandole, cefatrizine, cefbuperazone,
cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime,
cefmenoxime, cefininox, cefodizime, cefonicid, cefoperazone,
ceforanide, cefotaxime, cefotetan, cefotiam, cefozopran,
cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine,
ceftazidime, cefteram, ceftibuten, ceftriaxone, cefuzonam,
cephalexin, cephaloglycin, cephalosporin C, cephradine,
chloramphenicol, chlortetracycline, ciprofloxacin, clarithromycin,
clinafloxacin, clindamycin, clindamycin phosphate, clomocycline,
colistin, cyclacillin, dapsone, demecicycline, diathymosulfone,
dibekacin, dihydrostreptomycin, dirithromycin, doxycycline,
enoxacin, enviomycin, epicillin, erythromycin, flomoxef,
fortimicin(s), gentamicin(s), glucosulfone solasulfone, gramicidin
S, gramicidin(s), grepafloxacin, guamecycline, hetacillin,
imipenem, isepamicin, josamycin, kanamycin(s), leucomycin(s),
lincomycin, lomefloxacin, lucensomycin, lymecycline, meclocycline,
meropenem, methacycline, micronomicin, midecamycin(s), minocycline,
moxalactam, mupirocin, nadifloxacin, natamycin, neomycin,
netilmicin, norfloxacin, oleandomycin, oxytetracycline,
p-sulfanilylbenzylamine, panipenem, paromomycin, pazufloxacin,
penicillin N, pipacycline, pipemidic acid, polymyxin, primycin,
quinacillin, ribostamycin, rifamide, rifampin, rifamycin SV,
rifapentine, rifaximin, ristocetin, ritipenem, rokitamycin,
rolitetracycline, rosaramycin, roxithromycin, salazosulfadimidine,
sancycline, sisomicin, sparfloxacin, spectinomycin, spiramycin,
streptomycin, succisulfone, sulfachrysoidine, sulfaloxic acid,
sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin,
temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol,
thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin,
tosufloxacin, trimethoprim, trospectomycin, trovafloxacin,
tuberactinomycin and vancomycin. Antimicrobial agents can also
include anti-fungals, such as amphotericin B, azaserine,
candicidin(s), chlorphenesin, dermostatin(s), filipin,
fungichromin, mepartricin, nystatin, oligomycin(s), perimycin A,
tubercidin, imidazoles, triazoles, and griesofulvin.
[0195] In some embodiments, the NO-releasing polyglucosamine can be
incorporated into polymeric films. Such incorporation can be
through physically embedding the polyglucosamine into polymer
surfaces, via electrostatic association of the polyglucosamine onto
polymeric surfaces, or by covalent attachment of functionalized
polyglucosamine onto reactive groups on the surface of a polymer.
Alternatively, the functionalized polyglucosamine can be mixed into
a solution of liquid polymer precursor, becoming entrapped in the
polymer matrix when the polymer is cured. Polymerizable groups can
also be used to further functionalize the functionalized
polyglucosamine, whereupon, the polyglucosamine can be
co-polymerized into a polymer during the polymerization process.
Suitable polymers into which the NO-releasing polyglucosamine can
be incorporated include polyolefins, such as polystyrene,
polypropylene, polyethylene, polytetrafluoroethylene, and
polyvinylidene, as well as polyesters, polyethers, polyurethanes,
and the like. In particular, polyurethanes can include medically
segmented polyurethanes. Medically segmented polyurethanes can also
include one or more expander moieties, such as alkylene chains,
that add additional length or weight to the polymer. Such
polyurethanes are also generally non-toxic. One example of a
medically segmented polyurethane is TECOFLEX.RTM..
[0196] Polymeric films containing NO-releasing polyglucosamines can
be used to coat a variety of articles, particularly surgical tools,
biological sensors, and medical implants to prevent platelet
adhesion, to prevent bacterial infection, to act as a vasodilator.
These articles can be of use in vascular medical devices,
urological medical devised, biliary medical devices,
gastrointestinal medical devices, medical devices adapted for
placement at surgical sites, and medical devices adapted for
placement on skin wounds or openings. Thus, the polymers can be
used to coat arterial stents, guide wires, catheters, trocar
needles, bone anchors, bone screws, protective platings, hip and
joint replacements, electrical leads, biosensors, probes, sutures,
surgical drapes, wound dressings, and bandages.
[0197] In some embodiments, the device being coated can have a
metallic surface, such as, for example, stainless steel, nickel,
titanium, aluminum, copper, gold, silver, platinum, and
combinations thereof. In some embodiments, the films or polymers
containing the NO-releasing polyglucosamine can be used to coat
non-metallic surfaces, such as glass or fiber (e.g., cloth or
paper).
[0198] Additionally, polymers containing NO-releasing
polyglucosamine can be used to form the devices, themselves. For
example, the polymers can be fashioned into storage bags for blood
or tissue or as wound dressings.
[0199] Surfaces that can be contacted with a functionalized
polyglucosamine to prevent or disrupt biofilms include those
selected from the group consisting of medical devices, plumbing
fixtures, condenser coils, optical surfaces, boat hulls and
aircrafts. Other non-limiting examples include counter tops,
windows, appliances, hard floors, rugs, tubs, showers, mirrors,
toilets, bidets, bathroom fixtures, sinks, refrigerators,
microwaves, small kitchen appliances, tables, chairs, cabinets,
drawers, sofas, love seats, benches, beds, stools, armoires,
chests, dressers, display cabinets, clocks, buffets, shades,
shutters, entertainment centers, arm rails, lamps, banisters,
libraries, cabinets, desks, doors, shelves, couches, carts, pianos,
statues and other art, racks, fans, light fixtures, pool tables,
ping pong tables, soccer tables, card tables, tools (e.g., hand
powered and/or hand held tools, electrical tools, air powered
tools, etc.), telephones, radios, televisions, stereo equipment, CD
and DVD players, analog and digital sound devices, palm computers,
laptop computers, desktop and tower computers, computer monitors,
mp3 players, memory storage devices, cameras, camcorders, vehicle
surfaces (e.g., windshield; tires; metal, fiberglass, composite
material and/or plastic outer surfaces; fabric and/or vinyl outer
surfaces; fabric, vinyl, and/or leather interior surfaces; metal,
plastic, wood and/or composite material interior surfaces, glass
interior surfaces, etc.), bicycles, snowmobiles, motorcycles,
off-road-vehicles, yard equipment, farm equipment, washing
equipment (e.g., power washers, etc.), painting equipment (e.g.,
electric and air powered painting equipment, etc.), medical and/or
dental equipment, marine equipment (e.g., sail boats, power boats,
rafts, sail board, canoe, row boats, etc.), toys, writing
implements, watches, framed pictures or paintings, books, and/or
the like. Any surface where it is desirable to cause one or more
types of liquids to run off of a surface, to not be absorbed into a
surface, and/or to not stain a surface, can be a substrate. For
example, a surface that is exposed to environmental conditions.
Also where the surface can become a locus for microbial adhesion
such as medical devices that contact bodily tissues or fluids is
particularly preferred.
[0200] Medical devices such as catheters, which are adapted for
movement through blood vessels or other body lumens, are typically
provided with low-friction outer surfaces. If the surfaces of the
medical devices are not low-friction surfaces, insertion of the
devices into and removal of the devices from the body lumens
becomes more difficult, and injury or inflammation of bodily tissue
may occur. Low friction surfaces are also beneficial for reducing
discomfort and injury that may arise as a result of movement
between certain long term devices (e.g., long term catheters) and
the surrounding tissue, for example, as a result of patient
activity. Medical devices include a variety of implantable and
insertable medical devices (also referred to herein as "internal
medical devices"). Examples of such medical devices include,
devices involving the delivery or removal of fluids (e.g., drug
containing fluids, pressurized fluids such as inflation fluids,
bodily fluids, contrast media, hot or cold media, etc.) as well as
devices for insertion into and/or through a wide range of body
lumens, including lumens of the cardiovascular system such as the
heart, arteries (e.g., coronary, femoral, aorta, iliac, carotid and
vertebro-basilar arteries) and veins, lumens of the genitourinary
system such as the urethra (including prostatic urethra), bladder,
ureters, vagina, uterus, spermatic and fallopian tubes, the
nasolacrimal duct, the eustachian tube, lumens of the respiratory
tract such as the trachea, bronchi, nasal passages and sinuses,
lumens of the gastrointestinal tract such as the esophagus, gut,
duodenum, small intestine, large intestine, rectum, biliary and
pancreatic duct systems, lumens of the lymphatic system, the major
body cavities (peritoneal, pleural, pericardial) and so forth.
Non-limiting, specific examples of internal medical devices include
vascular devices such as vascular catheters (e.g., balloon
catheters), including balloons and inflation tubing for the same,
hydrolyses catheters, guide wires, pullback sheaths, filters (e.g.,
vena cava filters), left ventricular assist devices, total
artificial hearts, injection needles, drug delivery tubing,
drainage tubing, gastroenteric and colonoscopic tubing, endoscopic
devices, endotracheal devices such as airway tubes, devices for the
urinary tract such as urinary catheters and ureteral stents, and
devices for the neural region such as catheters and wires, trocar
needles, bone anchors, bone screws, protective platings, joint
replacements, electrical leads, biosensors, probes, sutures,
surgical drapes, wound dressings, and bandages. Many devices in
accordance with the invention have one or more portions that are
cylindrical in shape, including both solid and hollow cylindrical
shapes.
[0201] Solid substrate materials can include organic materials
(e.g., materials containing 50 wt % or more organic species) such
as polymeric materials, and inorganic materials (e.g., materials
containing 50 wt % or more inorganic species), such as metallic
materials (e.g., metals and metal alloys) and non-metallic
materials (e.g., including carbon, semiconductors, glasses and
ceramics, which may contain various metal- and non-metal-oxides,
various metal- and non-metal-nitrides, various metal- and
non-metal-carbides, various metal- and non-metal-borides, various
metal- and non-metal-phosphates, and various metal- and
non-metal-sulfides, among others). Specific examples of
non-metallic inorganic materials can be materials containing one or
more of the following: metal oxides, including aluminum oxides and
transition metal oxides (e.g., oxides of titanium, zirconium,
hafnium, tantalum, molybdenum, tungsten, rhenium, and iridium);
silicon; silicon-based ceramics, such as those containing silicon
nitrides, silicon carbides and silicon oxides (sometimes referred
to as glass ceramics); calcium phosphate ceramics (e.g.,
hydroxyapatite); carbon; and carbon-based, ceramic-like materials
such as carbon nitrides.
[0202] Further, the NO-releasing polyglucosamine can be
incorporated into detergents, such as, but not limited to,
anti-microbial soaps. For example, NO-release from functionalized
polyglucosamine embedded in bar soaps can be triggered by contact
with water and/or a drop in pH upon use. As the outer surface of
the bar is eroded or dissolved, additional functionalized
polyglucosamine within the bar surface become exposed for
subsequent uses of the bar. NO-releasing polyglucosamine also can
be suspended in liquid soaps. Such soaps or detergents can be used
for personal hygiene or to provide anti-microbial treatments for
fibers. Such soaps or detergents can also be used to treat
household surfaces or any surface in a hospital or other medical
environment that may be exposed to microbes such as bacteria, fungi
or viruses.
[0203] The term "biocompatible" refers herein to organic solvents
that do not induce toxic or unwanted side effects when administered
to a patient in certain amounts.
[0204] The formulations include all pharmaceutically acceptable
salt forms thereof. Examples of such salts include those derived
from pharmaceutically acceptable inorganic and organic acids and
bases. Examples of suitable acid salts include, without limitation,
acetate, adipate, alginate, aspartate, benzoate, butyrate, citrate,
fumarate, glycolate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, malonate, methanesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, hydroxynaphthoate,
pivalate, propionate, salicylate, succinate, sulfate, tartrate,
thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,
while not in themselves pharmaceutically acceptable, can be
employed in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their pharmaceutically
acceptable acid addition salts.
[0205] Salts derived from appropriate bases include, without
limitation, alkali metal (e.g., sodium, potassium), alkaline earth
metal (e.g., magnesium and calcium), ammonium and
N-(alkyl).sub.4.sup.+ salts.
[0206] The functionalized polyglucosamines also include those
having quaternization of any basic nitrogen-containing group
therein.
[0207] The discussion herein is, for simplicity, provided without
reference to stereoisomerism. Those skilled in the art will
appreciate that the polyglucosamines described herein can contain
one or more asymmetric centers and thus occur as racemates and
racemic mixtures, single optical isomers, individual diastereomers,
and diastereomeric mixtures. All such isomeric forms of these
compounds are expressly included in the present invention.
[0208] The present invention is explained in greater detail in the
following non-limiting Examples.
Examples
1. Synthesis of NO-releasing Chitosan Oligosaccharides
[0209] Chitosan oligosaccharides were synthesized by the oxidation
(hydrogen peroxide) (Kim, S. K.; Rajapakse, N. Enzymatic production
and biological activities of chitosan oligosaccharides (COS): A
review. Carbohydr. Polym. 2005, 62, 357-368) or enzymatic
degradation of chitosan polysaccharides. The resulting water
soluble chitosan oligosaccharides were modified by a cationic ring
opening of aziridine compounds (including but not limited to
aziridine and 2-methyl aziridine) to impart secondary amine
moieties, followed by the reaction with high pressure NO under
basic condition ("charging") to yield water soluble NO-releasing
chitosan oligosaccharides.
Materials and Methods
[0210] Medium molecular weight chitosan, 2-methyl aziridine (MAz),
rhodamine B isothiocyanate (RITC), poly(ethylene glycol) methyl
ether acrylate (average Mn=480) (PEG), fetal bovine serum (FBS),
Dulbecco's Modified Eagle's Medium (DMEM), phenazine methosulfate
(PMS),
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium inner salt (MTS), trypsin, phosphate buffered
saline (PBS), and penicillin streptomycin (PS) were purchased from
the Aldrich Chemical Company (Milwaukee, Wis.). Pseudomonas
aeruginosa (ATCC #19143) was obtained from the American Type
Culture Collection (Manassas, Va.). Trypic soy broth (TSB) and
Tryptic soy agar (TSA) are purchased from Becton, Dickinson, and
Company (Franklin Lakes, N.J.). L929 mouse fibroblasts (ATCC
#CCL-1) were obtained from the University of North Carolina Tissue
Culture Facility (Chapel Hill, N.C.). Distilled water was purified
with a Millipore Milli-Q Gradient A-10 water purification system
(Bedford, Mass.). Syto 9 green fluorescent nucleic acid stain was
purchased from Life Technologies (Grand Island, N.Y.). Common
laboratory salts and solvents were purchased from Fisher Scientific
(Pittsburgh, Pa.). All materials were used as received without
further purification unless noted otherwise. Nuclear magnetic
resonance (NMR) spectra were recorded on a 400 MHz Bruker
instrument. Elemental (carbon, hydrogen, and nitrogen or CHN)
analysis was performed using a PerkinElmer Elemental Analyzer
Series 2400 instrument (Waltham, Mass.).
[0211] Chitosan oligosaccharides were prepared by oxidative
degradation using hydrogen peroxide. Medium molecular weight
chitosan (2.5 g) was suspended in a hydrogen peroxide solution (15
or 30 wt %) under stirring for 1 h at 65-85.degree. C. Following
the removal of undissolved chitosan by filtration, chitosan
oligosaccharides were precipitated from solution by adding acetone
to the filtrate. The precipitate was collected by centrifugation,
washed twice with ethanol, and dried under vacuum at room
temperature. The viscosity of the chitosan oligosaccharides was
measured in a solution of NaCl (0.20 M) and CH3COOH (0.10 M) at
25.degree. C. using an Ubbleohde capillary viscometer.
Oligosaccharide molecular weight was determined using the classic
Mark-Houwink equation (i.e., [.eta.]=1.81.times.10-3 M.sup.0.93).
Maghami (1988).
[0212] Control of the molecular weight (M.sub.w) was achieved by
varying the concentration of hydrogen peroxide and degradation
temperature. The viscosity of the chitosan oligosaccharides was
determined in a solution of sodium chloride (0.20 M) and acetic
acid (0.10 M) using an Ubbleohde capillary viscometer. Du, J.;
Hsieh, Y. L. Nanofibrous membranes from aqueous electrospinning of
carboxymethyl chitosan. Nanotechnology 2008, 19, 125707. In
combination with the Mark-Houwink equation (i.e.,
[.eta.]=1.81.times.10.sup.-3 M.sup.0.93), molecular weights were
determined as a function of processing conditions. Collectively,
larger concentrations of hydrogen peroxide and elevated degradation
temperatures led to lower molecular weight chitosan. As shown in
Table 1, chitosan oligosaccharides of .about.10 kD molecular weight
were prepared in 15 wt % hydrogen peroxide at 65.degree. C. for 1
h. Increasing the degradation temperature to 85.degree. C. resulted
in significantly smaller size (MW .about.5 kD).
Table 1.
TABLE-US-00001 [0213] TABLE 1 Degradation conditions and elemental
analysis of chitosan oligosaccharides of different molecular
weights. Chitosan [H.sub.2O.sub.2] oligosaccharides M.sub.v.sup.a T
(.degree. C.) (wt %) 2.5 k 2657 85 30 5 k 5370 85 15 10 k 10142 65
15 .sup.aviscosity average molecular weight as determined by
classic Mark-Houwink equation (i.e., [.eta.] = 1.81 .times.
10.sup.-3 M.sup.0.93).
[0214] When both a larger concentration of hydrogen peroxide (i.e.,
30 wt %) and elevated temperature (i.e., 85.degree. C.) were
adopted, the molecular weight of chitosan oligosaccharides
decreased further (.about.2.5 kD) were achieved. As shown in Table
2, the CHN elemental analysis of the oligosaccharides indicated an
overall nitrogen content of 6.3 wt %.
TABLE-US-00002 TABLE 2 Elemental (CHN) analysis of chitosan
oligosaccharides and secondary amine-function alized derivatives.
Materials C (%) H (%) N (%) Chitosan oligosaccharides.sup.a 42.2
.+-. 1.6 6.8 .+-. 0.1 6.3 .+-. 0.2 Chitosan 1-5 k 43.5 .+-. 1.2 7.7
.+-. 0.3 8.9 .+-. 0.1 Chitosan 2-5 k 44.7 .+-. 1.8 8.4 .+-. 0.2
10.8 .+-. 0.8 Chitosan 3-5 k 51.0 .+-. 0.2 9.0 .+-. 0.2 3.1 .+-.
0.1 Clutosan 2-2.5 k 43.7 .+-. 0.7 8.5 .+-. 0.2 10.9 .+-. 0.1
Chitosan 2-10 k 44.7 .+-. 1.8 8.4 .+-. 0.2 10.8 .+-. 0.8
.sup.achitosan oligosaccharides before the grafting of 2-methyl
aziridine. Each parameter was analyzed with multiple replicates (n
= 3).
[0215] 2-methyl aziridine (MAz) was grafted onto the chitosan
oligosaccharides at different feed ratios (i.e., 2:1 and 1:1) to
alter the secondary amine functionalization and NO storage.
Increasing the feed ratio of 2-methyl aziridine to primary amines
from 1:1 (CSO 1; Chitosan 1-5k) to 2:1 (CSO 2; Chitosan 2-5k)
resulted in greater NO storage (e.g., .about.0.30 to 0.87
.mu.mol/mg, respectively). As shown in FIGS. 5A & B, the NO
flux and storage of Chitosan 1/NO-5k were lower than Chitosan
2/NO-5k, a result that may be attributable to the smaller amine
concentration of Chitosan 1-5k (.about.8.9 wt %) compared to
Chitosan 2-5k (.about.10.8 wt %).
[0216] Schemes A and B depict routes for preparing NO-releasing
chitosan oligosaccharides described herein.
##STR00031## ##STR00032##
[0217] In Scheme A, CSO 1 (x=1); CSO 2 (x=2); CSO 1-NO (x=1); CSO
2-NO (x=2). In scheme B, CSO 3 (x=2); CSO 3-NO (x=2). Scheme A
above is a synthetic route for preparing secondary amine- and
diazeniumdiolate-functionalized chitosan oligosaccharides
derivatives involves grafting of 2-methyl aziridine onto primary
amines of chitosan oligosaccharides (CSO 1, 2) and
diazeniumdiolation of the resulting materials (CSO 1, 2-NO). Scheme
B above is a synthetic route for preparing secondary amine- and
diazeniumdiolate-functionalized chitosan oligosaccharides
derivatives involves PEGylation of 2-methyl
aziridine-grafted-chitosan oligosaccharide (CSO 3) and the
diazeniumdiolation of the resulting material (CSO 3-NO).
[0218] Schemes A' and B' depict a synthesis route for chitosan
oligosaccharides disclosed herein:
##STR00033##
##STR00034##
[0219] Reaction of the secondary amine-functionalized chitosan
oligosaccharides (Chitosan 1, Chitosan 2, and Chitosan 3) with NO
(10 atm under basic conditions) yielded N-diazeniumdiolate NO
donor-functionalized chitosan oligosaccharides (Chitosan 1/NO,
Chitosan 2/NO, and Chitosan 3/NO). The NO conjugation ("charging")
solvent affects the charging efficiency and potentially total NO
storage. Carpenter, A. W.; Slomberg, D. L.; Rao, K. S.;
Schoenfisch, M. H., Influence of scaffold size on bactericidal
activity of nitric oxide-releasing silica nanoparticles. ACS Nano
2012, 5, 7235-7244. Aqueous solutions were necessary in order to
adequately dissolve the chitosan oligosaccharides. To examine the
influence of water concentration on N-diazeniumdiolate conversion
efficiency, mixtures of methanol (a common charging solvent)
(Carpenter (2012); Stasko, N. A.; Schoenfisch, M. H. Dendrimers as
a scaffold for nitric oxide release. J. Am. Chem. Soc. 2006, 128,
8265-8271) and water were prepared (10:0, 9:1, 8:2, 7:3, and 6:4
v/v) and the pH was adjusted to above 10 by adding sodium
methoxide.
[0220] Scheme C depicts a route for preparing nitrosothiol
NO-releasing chitosan oligosaccharides as described herein.
##STR00035##
[0221] Scheme D depicts diazeniumdiolate conjugation and release of
2 NO from a conjugated diazeniumdiolate.
##STR00036##
[0222] Secondary Amine-Functionalized Chitosan
Oligosaccharides:
[0223] 2-methyl aziridine (MAz) grafted chitosan oligosaccharides
were synthesized following a previously reported procedure. Wong,
K.; Sun, G. B.; Zhang, X. Q.; Dai, H.; Liu, Y.; He, C. B.; Leong,
K. W. PEI-g-chitosan, a novel gene delivery system with
transfection efficiency comparable to polyethylenimine in vitro and
after liver administration in vivo. Bioconj. Chem. 2006, 17,
152-158. Briefly, a mixture of concentrated HCl (11 .mu.L), water
(100 .mu.L) and MAz with a 1:1 (CSO 1; Chitosan 1) or 2:1 (CSO 2;
Chitosan 2) molar ratio to primary amines on the chitosan
oligosaccharides was added dropwise to a solution of chitosan
oligosaccharides (100 mg) in deionized water (5 mL). The resulting
solution was stirred at room temperature for 5 d, and then at
75.degree. C. for 24 h. The product was purified by dialysis and
collected by lyophilization. Any high molecular weight
poly(2-methyl aziridine) in the product was removed by washing with
methanol, and the resulting material was dried under vacuum at room
temperature. Chitosan 2 was then dissolved in water at pH 10.0. The
primary amine on the chitosan oligosaccharides was functionalized
by adding poly(ethylene glycol) methyl ether acrylate to generate
Chitosan 3. The resulting PEG-functionalized chitosan
oligosaccharide derivative was purified by dialysis and collected
by lyophilization. 1H NMR data of Chitosan 1 and Chitosan 2 (400
MHz, CD.sub.3OD, .delta.): 0.8-1.1
(NH.sub.2CH(CH.sub.3)CH.sub.2NH), 1.9 (C7: CHNHCOCH.sub.3), 2.3-2.7
(NH.sub.2CH(CH.sub.3)CH.sub.2NHCH, C2:
NH.sub.2CH(CH.sub.3)CH.sub.2NHCH), 3.3-4.0 (C3, C4, C5, C6: OHCH,
OCHCH(OH)CH(NH.sub.2)CH, OHCH.sub.2CH, OHCH.sub.2CH), 4.4 (C1:
OCH(CHNH.sub.2)O). 1H NMR data of Chitosan 3 (400 MHz, CD.sub.3OD,
.delta.): 0.8-1.1 (NH.sub.2CH(CH.sub.3)CH.sub.2NH), 1.9 (C7:
CHNHCOCH.sub.3), 2.3-2.7 (NH.sub.2CH(CH.sub.3)CH.sub.2NHCH, C2:
NH.sub.2CH(CH.sub.3)CH.sub.2NHCH), 3.2
(OCH.sub.2CH.sub.2OCH.sub.3), 3.3-4.0 (OCH.sub.2CH.sub.2O and C3,
C4, C5, C6: OHCH, OCHCH(OH)CH(NH.sub.2)CH, OHCH.sub.2CH,
OHCH.sub.2CH), 4.4 (C1: OCH(CHNH.sub.2)O).
[0224] N-Diazeniumdiolate-Functionalized Chitosan
Oligosaccharides:
[0225] Secondary amine-functionalized chitosan oligosaccharides
(CSO 1, Chitosan 1, CSO 2, Chitosan 2, chitosan 3 and CSO 3) and
5.4 mM sodium methoxide (75 .mu.l) were added to a methanol/water
mixture (2 mL) of different v/v ratios (e.g., 10:0, 9:1, 8:2, 7:3,
6:4). The suspension was added to vials in a Parr hydrogenation
vessel, which was purged rapidly (5-10 s) with argon three times
followed by three longer argon purge cycles (10 min) to remove
residual oxygen from the solution. The Parr hydrogenation vessel
was then pressurized to 10 atm with NO gas purified over KOH
pellets (to remove NO degradation products) and maintained at 10
atm for 3 d. The same argon purging protocol was performed to
remove unreacted NO and degradation products from the solution
prior to removing the vials from the vessel.
[0226] Fluorescently-Labeled Chitosan Oligosaccharides:
[0227] These chitosan oligosaccharides were prepared following a
previously reported procedure. Tokura, S.; Ueno, K.; Miyazaki, S.;
Nishi, N., Molecular weight dependent antimicrobial activity by
chitosan. Macromol. Symp. 1997, 120, 1-9. Briefly, chitosan
oligosaccharides (50 mg) were dissolved in water (2 mL) at pH 9.0.
Rhodamine B isothiocyanate (RITC) was added to the solution in a
1:100 molar ratio to the primary amine of the chitosan
oligosaccharides prior to the grafting of 2-methyl aziridine. The
solution was stirred at room temperature for 3 d in the dark.
Subsequent dialysis and lyophilization yielded the RITC-labeled
chitosan oligosaccharides.
[0228] By tuning the ratio of MAz and primary amine (e.g., 1:1 CSO
1 and Chitosan 1, 2:1 CSO 2 and Chitosan 2), the number of MAz
repeating units grafted onto the chitosan oligosaccharides was
tunable (supporting NMR data), leading to a range of secondary
amine concentrations and NO storage. Acrylate-functionalized PEG
chains were conjugated to the primary amines on CSO 2 and Chitosan
2 by the Michael addition reaction to yield PEG-modified scaffolds
(e.g., CSO 3 and Chitosan 3, See, schemes B and B'). Grafting of
2-methyl aziridine to the oligosaccharides increased the
corresponding nitrogen content from 6.3 to 8.9 and 10.8 wt % for
CSO 1 and Chitosan 1 and for CSO 2 and Chitosan 2, respectively.
The PEGylation of CSO 2 and Chitosan 2 led to a corresponding
decrease in nitrogen content (3.1 wt %) (CSO 3 and Chitosan 3).
2. Nitric Oxide Charging, Storage and Release in Functionalized
Polyglucosamines
[0229] The subject matter disclosed herein describes the
optimization of charging condition for
secondary-amine-functionalized chitosan oligosaccharides (e.g., CSO
1, 2, 3). Mixtures of methanol and water were used as the charging
solvents. FIG. 4 shows the NO release profiles for CSO 2-NO charged
in different solvents. When the 7:3 methanol/water was used, the
maximum total NO storage (e.g., .about.0.87 .mu.mol/mg) was
yielded.
TABLE-US-00003 TABLE 3 Nitric oxide release characteristics in for
secondary amine-functionalized chitosan oligosaccharides (CSO 2;
Chitosan 2/NO-5 k) PBS (pH = 7.4) at 37.degree. C. MeOH/H.sub.2O
10:0 9:1 8:2 7:3 6:4 t[NO] (.mu.mol/mg) 0.58 .+-. 0.09 0.74 .+-.
0.12 0.81 .+-. 0.14 0.87 .+-. 0.16 0.75 .+-. 0.18 [NO].sub.max
(ppb/mg) 2648 .+-. 120 4150 .+-. 70 4350 .+-. 484 5500 .+-. 414
5000 .+-. 572 Half-life (h) 2.40 .+-. 0.13 2.25 .+-. 0.02 2.05 .+-.
0.07 2.20 .+-. 0.14 2.05 .+-. 0.25
[0230] The subject matter disclosed herein describes the control of
total NO storage by tuning the ratio of aziridine compounds to the
primary amines on the chitosan oligosaccharides. By increasing the
use of aziridine compounds, greater secondary amine content and
thus total NO storage can be achieved. For example, as shown in
FIGS. 5A and B and Tables 4A and B, the synthesis with 2-methyl
aziridine/primary amines 2:1 ratio yielded total storage around
0.87 .mu.mol/mg while the ratio 1:1 led to the NO storage of 0.30
.mu.mol/mg. Accordingly, further increase of aziridine compound
usage would likely result in more enhanced NO storage. The maximum
NO storage (using the 7:3 methanol/water charging solvent ratio)
was 0.87 .mu.mol/mg, roughly 4.times. larger than that for
previously reported chitosan polysaccharides (.about.0.2
.mu.mol/mg). Du, J.; Hsieh, Y. L. Nanofibrous membranes from
aqueous electrospinning of carboxymethyl chitosan. Nanotechnology
2008, 19, 125707; Kim, S. K.; Rajapakse, N. Enzymatic production
and biological activities of chitosan oligosaccharides (COS): A
review. Carbohydr. Polym. 2005, 62, 357-368; Maghami (1988).
[0231] The subject matter disclosed herein describes the control of
NO-release kinetics by functionalizing the amine moieties of the
secondary amine-functionalized chitosan oligosaccharides (CSO 1,
2). As shown in FIGS. 5A and B and Tables 4A and 4B, chitosan
oligosaccharides modified with hydrophilic PEG (CSO 3-NO) exhibited
a greater initial NO flux and shorter half-life compared to the
counterparts before PEG functionalization (CSO 2-NO).
TABLE-US-00004 TABLE 4A Nitric oxide-release properties of
N-diazeniumdiolate NO donor- functionalized chitosan
oligosaccharides. t[NO] [NO].sub.max (.mu.mol/mg) (ppb/mg)
t.sub.1/2 (h) CSO 1-NO 0.30 .+-. 0.04 1600 .+-. 215 3.60 .+-. 0.13
CSO 2-NO 0.87 .+-. 0.16 5500 .+-. 414 2.20 .+-. 0.14 CSO 3-NO 0.35
.+-. 0.02 12600 .+-. 2121 0.15 .+-. 0.01
Accordingly, the oligosaccharide units will be present in mole
ratios that are reflected in the NO release properties. In
embodiments, m is from about 0.4 to about 0.9, for example about
0.4, 0.5, 0.6, 0.7, 0.8 or 0.9; n is from about 0.1 to about 0.6,
for example about 0.1, 0.2, 0.3, 0.4, 0.5, or 0.6; wherein m and n
represent the mole fraction of each unit and the sum of m and n is
1.
TABLE-US-00005 TABLE 4B Nitric oxide-release properties of
different N-diazeniumdiolate NO donor-functionalized chitosan
oligosaccharides in PBS (pH = 7.4, 37.degree. C.) as measured using
a chemiluminescence NO analyzer. t[NO].sup.a t[NO].sup.b
[NO].sub.max Material (.mu.mol/g) (.mu.mol/g) (ppb/mg) t.sub.1/2
(h) Chitosan 1/NO-5 k 0.30 .+-. 0.04 0.16 .+-. 0.03 1600 .+-. 215
3.60 .+-. 0.13 Chitosan 2/NO-5 k 0.87 .+-. 0.16 0.52 .+-. 0.15 5500
.+-. 414 2.20 .+-. 0.14 Chitosan 3/NO-5 k 0.35 .+-. 0.02 0.29 .+-.
0.01 12600 .+-. 2121 0.15 .+-. 0.01 Chitosan 2/NO-2.5 k 0.84 .+-.
0.04 0.49 .+-. 0.02 7500 .+-. 550 2.06 .+-. 0.10 Clutosan 2/NO-10 k
0.81 .+-. 0.05 0.47 .+-. 0.03 7350 .+-. 672 2.04 .+-. 0.05
.sup.atotal NO released and .sup.bNO released over 24 and 4 h
(.mu.mol) per milligram of secondary amine-functionalized PPI Each
parameter was analyzed with multiple replicates (n = 3).
N-diazeniumdiolate-functionalized chitosan oligosaccharides (1 mg)
(CSO 1-NO, Chitosan 1/NO, CSO 2-NO, Chitosan 2/NO, CSO 3-NO,
Chitosan 3/NO) in the water/methanol mixture were added into a
sample vessel containing 30 mL deoxygenated phosphate buffered
saline (PBS) (10 mM, pH=7.4) at 37.degree. C., which initiated NO
release. To quantify the NO released, the solution was purged with
nitrogen at a flow rate of 70 mL/min to carry the liberated NO to
the analyzer. Additional nitrogen flow was supplied to the vessel
to match the collection rate of the instrument (200 mL/min). The
analysis of NO was terminated when the NO release levels fell to
below 10 ppb NO/mg chitosan oligosaccharides. Chemiluminescence
data for the NO-releasing chitosan oligosaccharides were
represented as: 1) total amount of NO release (t[NO], .mu.mol NO/mg
of secondary amine-functionalized chitosan oligosaccharides); 2)
the maximum flux of NO release ([NO]max, ppb/mg of secondary
amine-functionalized chitosan oligosaccharides); and 3) the
half-life of NO release (t.sub.1/2).
3. Mouse Fibroblast Viability Assay
[0232] L929 mouse fibroblasts were grown in DMEM supplemented with
10% (v/v) fetal bovine serum (FBS) and 1 wt %
penicillin/streptomycin, and incubated in 5% (v/v) CO.sub.2 under
humidified conditions at 37.degree. C. After reaching 80%
confluency, the cells were trypsinized, seeded onto tissue-culture
treated polystyrene 96-well plates at a density of 3.times.10.sup.4
cells/mL and incubated at 37.degree. C. for 48 h. The supernatant
was then aspirated prior to adding 200 .mu.L fresh DMEM and 50
.mu.L of a NO-releasing chitosan oligosaccharides solution in PBS
to each well. After incubation at 37.degree. C. for 24 h, the
supernatant was aspirated and 120 .mu.L mixture of DMEM/MTS/PMS
(105/20/1, v/v/v) was added to each well. The absorbance of the
resulting colored solution after 1.5 h incubation at 37.degree. C.
was quantified at 490 nm using a Thermoscientific Multiskan EX
plate reader. The mixture of DMEM/MTS/PMS and untreated cells were
used as blank and control, respectively. The cell viability was
calculated by equation 1.
Cell Viability = ( Absorbance treated cell - Absorbance blank ) (
Absorbance untreated cell - Absorbance blank ) Eq . 1
##EQU00001##
[0233] 4. The cytotoxicity of control and NO-releasing chitosan
oligosaccharides were compared by exposing mouse fibroblast cells
to the oligosaccharides at the MBCs against P. aeruginosa biofilms
noted above. The results of the normalized cell viabilities of
control and NO-releasing chitosan oligosaccharides after 24 h
incubation are shown in FIGS. 8A and B. Regardless of size (i.e.,
molecular weight), the control and NO-releasing chitosan
oligosaccharides were non-toxic against mouse fibroblast cells at
the MBCs for the NO-releasing scaffolds, indicating an advantage of
these materials as anti-biofilm agents compared to other
antibacterial agents. The NO-releasing chitosan oligosaccharides
exhibited lower cytotoxicity than the chitosan controls.
5. Bactericidal Assays Under Static Conditions
[0234] P. aeruginosa bacterial cultures were grown from a frozen
(-80.degree. C.) stock overnight in TSB at 37.degree. C. A 500
.mu.L aliquot of the resulting suspension was added into 50 mL
fresh TSB and incubated at 37.degree. C. for .about.2 h until the
concentration reached .about.1.times.10.sup.8 colony forming units
(CFU)/mL, as confirmed by the OD600, replicate plating and
enumeration on nutrient agar. A working bacterial stock was
generated by plating the bacterial suspension on TSA and incubating
at 37.degree. C. overnight. The TSA bacterial stocks were prepared
weekly and stored at 4.degree. C. For bactericidal assays, colonies
of P, aeruginosa were taken from the TSA plate, dispersed in 3 mL
TSB, and incubated at 37.degree. C. overnight. A 500 .mu.L aliquot
of culture was added to 50 mL fresh TSB and incubated to a
concentration of .about.1.times.10.sup.8 CFU/mL. The bacteria was
collected by centrifugation, resuspended in PBS, and diluted to
.about.1.times.10.sup.6 CFU/mL. The bactericidal efficacy of
NO-releasing chitosan oligosaccharides against P. aeruginosa was
evaluated by incubating the bacteria suspension with NO-releasing
chitosan oligosaccharides at 37.degree. C. At 4 h, 100 .mu.L
aliquots of the bacterial suspensions were removed, diluted 10-fold
in PBS, plated on TSA, and incubated overnight at 37.degree. C. The
minimum concentration of NO-releasing chitosan oligosaccharides
that resulted in a 3-log reduction of bacterial viability was
defined as the minimum bactericidal concentration (MBC) for
planktonic studies.
[0235] Bacterial viability assays were performed under static
conditions to determine the concentration of chitosan required to
reduce bacteria viability by 3 logs (i.e., 99.9% killing), which
hereafter will be referred to as the minimum bactericidal
concentration or MBC. The amount of NO delivered from NO-releasing
chitosan oligosaccharides (Table 4) over the time of the assay (4
h) was also examined to quantitatively assess the NO dose necessary
for 99.9% bacterial killing. Both MBCs and the bactericidal NO
doses required for the chitosan oligosaccharides are provided in
Table 5.
TABLE-US-00006 TABLE 5 Minimum bactericidal concentration (MBC) and
NO doses of NO-releasing chitosan oligosaccharides for 3-log
reduction in planktonic P. aeruginosa viability. MBC NO dose
Chitosans (.mu.g/mL) (.mu.mol/mL) Chitosan 1/NO-5 k 2000 0.32
Chitosan 2/NO-5 k 200 0.10 Chitosan 3/NO-5 k 1500 0.45 Chitosan
2/NO-2.5 k 250 0.12
Regardless of size (i.e., molecular weight of about 2.5, 5, or 10
kDa), each of the NO-releasing chitosan oligosaccharides (Chitosan
2/NO-2.5 k, Chitosan 2/NO-5 k, Chitosan 2/NO-10 k) exhibited
similar bactericidal NO concentrations (i.e., .about.10 .mu.mol
NO/mL) for 3-log killing (Table 5). Each of the NO-releasing
chitosan oligosaccharides studied (including CSO 2-NO-5k) resulted
in .gtoreq.99.9% killing of P. aeruginosa. At equivalent
concentrations, the control (non-NO-releasing) chitosan did not
lead to a significant reduction in bacterial viability, indicating
NO as the bactericidal agent (data not shown).
6. Treatment of P. aeruginosa Biofilms with NO-Releasing Chitosan
Oligosaccharides
[0236] A CDC bioreactor (Biosurface Technologies, Bozeman, MT) was
used to grow P. aeruginosa biofilms over a 48 h period. Briefly,
medical grade silicone rubber substrates were mounted in coupon
holders prior to assembling the reactor. The assembled reactor was
then autoclaved. The reactor effluent line was clamped, and 1%
(v/v) sterile TSB (500 mL) was added aseptically. P. aeruginosa was
then cultured in TSB to 108 CFU/mL. The reactor was inoculated with
an aliquot (1 mL) of this bacterial suspension at a final
concentration .about.2.times.105 CFU/mL. The reactor was incubated
at 37.degree. C. for 24 h with slow stirring (150 rpm). Following
this "batch phase" growth, the reactor media was refreshed
continuously with 0.33% (v/v) TSB at 6 mL/min for another 24 h
through the effluent line.
[0237] P. aeruginosa biofilms grown on silicone rubber substrates
were exposed to chitosan oligosaccharide in PBS with slight
agitation (37.degree. C., 24 h) to determine the minimum
bactericidal concentration (MBC) necessary to elicit a 5-log
reduction in viability. At 24 h, samples were then sonicated and
vortexed to disrupt the biofilm. Aliquots (100 .mu.L) of the
bacteria/chitosan suspensions were diluted and plated on TSA. After
incubating the TSA plates overnight at 37.degree. C., bacteria
viability was determined by counting observed colonies. Of note,
the limit of detection for this selected plate counting method is
2.5.times.103 CFU/mL. As such, biofilm growth conditions were
selected to accurately represent a 5-log reduction in viability for
biofilms.
[0238] To evaluate the anti-biofilm activity of NO-releasing
chitosan oligosaccharides (e.g., Chitosan 1/NO-5k, Chitosan
2/NO-5k, Chitosan 3/NO-5k), P. aeruginosa biofilms were exposed to
0.2-1.3 mg/mL NO-releasing chitosan oligosaccharides for 24 h
(corresponding to .about.0.17-0.46 .mu.mol NO/mL). After treatment,
the biofilms were removed from the silicone rubber substrates by
vortexing and sonication to enable viability quantification.
Salmon, D. J.; Torres de Holding, C. L.; Thomas, L.; Peterson, K.
V.; Goodman, G. P.; Saavedra, J. E.; Srinivasan, A.; Davies, K. M.;
Keefer, L. K.; Miranda, K. M. HNO and NO release from a primary
amine-based diazeniumdiolate as a function of pH. Inorg. Chem. 50,
3262-70. Control experiments were performed to confirm the growth
of P. aeruginosa biofilms using the selected protocol. As shown in
FIG. 7, the viability of P. aeruginosa in the biofilm was
.about.2.times.10.sup.8 CFU when exposed only to PBS. The chitosan
concentrations for 5-log reduction of biofilm bacteria viability
(MBC) were 400, 700, and 1000 .mu.g/mL for Chitosan 2/NO-5k,
Chitosan 1/NO-5k, and Chitosan 3/NO-5k, respectively. Chitosan
2/NO-5k exhibited the greatest anti-biofilm efficacy, a likely
result due to both increased NO storage/release and rapid
association with the negatively charged bacteria. Although Chitosan
1/NO-5k and Chitosan 3/NO-5k stored similar levels of NO
(.about.0.3 .mu.mol/mg), Chitosan 1/NO-5k was more effective at
eradicating the biofilm bacteria (MBC 700 .mu.g/mL) compared to
Chitosan 3/NO-5k (MBC 1000 .mu.g/mL). The association of Chitosan
2/NO-5k and Chitosan 3/NO-5k with P. aeruginosa biofilm was
evaluated using confocal microscope. As shown in FIG. 9A-F,
biofilms exposed to Chitosan 2/NO-5k exhibited more intense red
fluorescence compared to Chitosan 3/NO-5k, again confirming the
enhanced association of the positively charged Chitosan 2/NO-5k
with the bacteria.
7. Confocal Microscopy
[0239] P. aeruginosa was cultured in TSB to a concentration of
.about.1.times.10.sup.8 CFU/mL, collected via centrifugation
(3645.times.g for 10 min), resuspended in sterile PBS, and adjusted
to .about.1.times.10.sup.6 CFU/mL. Aliquots of the bacteria
solution were incubated in a glass bottom confocal dish for 1.5 h
at 37.degree. C. A Zeiss 510 Meta inverted laser scanning confocal
microscope with a 543 nm HeNe excitation laser and a LP 585 nm
filter was used to obtain fluorescence images of the rhodamine B
isothiocyanate (RITC)-modified chitosan oligosaccharides. The
bright field and fluorescence images were collected by a N.A. 1.2
C-apochromat water immersion lens with a 40.times. objective.
Solutions of RITC-labeled NO-releasing chitosan oligosaccharides in
PBS (1.5 mL) were added to the bacteria solution (1.5 mL) in the
glass confocal dish to achieve a final concentration of 150
.mu.g/mL. Images were collected every 2 min to characterize the
association, if any, of the chitosan oligosaccharides with P.
aeruginosa temporally. To observe the association of chitosan
oligosaccharides with bacteria within biofilms, Established
biofilms stained with syto 9 (10 .mu.M) were incubated with
RITC-labeled chitosan oligosaccharides (150 .mu.g/mL in PBS) for
2.5 h. Prior to imaging, samples were rinsed with PBS (3.times.). A
Zeiss 510 Meta inverted laser scanning confocal microscope with 488
nm Ar and 543 nm HeNe excitation lasers, and a BP 505-530 nm and LP
585 nm filters, respectively, was used to obtain all confocal
images. Fluorescence images were collected with a 20.times.
objective.
[0240] Confocal microscopy was utilized to compare the association
kinetics of Chitosan 3/NO-5k and Chitosan 2/NO-5k with bacteria.
Rhodamine B isothiocyanate (RITC)-labeled Chitosan 2/NO-5k and
Chitosan 3/NO-5k were synthesized. Maghami (1988). The potential
impact of RITC on chitosan-bacteria association was minimized by
using small concentration of RITC (i.e., in 1:100 molar ratio to
total primary amines). The degree of association of the
NO-releasing chitosan oligosaccharides with bacteria was then
followed by measuring red fluorescence surrounding the bacteria.
Chitosan 2/NO-5k associated with the bacteria more rapidly (within
24 min) than Chitosan 3/NO-5k (86 min) (FIG. 10). The fluorescence
from Chitosan 2/NO-5k at 42 min was significantly greater than that
of Chitosan 3/NO-5k at 110 min, further demonstrating that Chitosan
3/NO-5k associated with the bacteria at a much slower rate due to
the PEG (neutral) modification. Further inspection of Chitosan
2/NO-5k and Chitosan 3/NO-5k association with P. aeruginosa
revealed enhanced bacteria association for Chitosan 2/NO-5k (FIG.
10-G, H).
[0241] Although chitosan molecular weight was not observed to play
a significant role in planktonic killing, less effective bacteria
killing was observed when using Chitosan 2/NO-10k, the largest
chitosan oligosaccharides (600 .mu.g/mL vs. 400 .mu.g/mL for
Chitosan 2/NO-10k and Chitosan 2/NO-2.5k) against biofilms. The
efficient association of chitosan oligosaccharides with bacteria in
biofilms is advantageous in view of previously reported
NO-releasing polysaccharides which are insoluble under
physiological conditions. All documents cited or referenced in the
application cited documents, and all documents cited or referenced
herein ("herein cited documents"), and all documents cited or
referenced in herein cited documents, together with any
manufacturer's instructions, descriptions, product specifications,
and product sheets for any products mentioned herein or in any
document incorporated by reference herein, are hereby incorporated
herein by reference, and may be employed in the practice of the
invention.
[0242] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
* * * * *