U.S. patent application number 11/194346 was filed with the patent office on 2005-11-24 for cross-linked gels of chemically activated carboxypolysaccharides and polyalkylene oxides.
This patent application is currently assigned to FZIOMED, INC.. Invention is credited to Berg, Richard, Liu, Lin-Shu.
Application Number | 20050260188 11/194346 |
Document ID | / |
Family ID | 29249386 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260188 |
Kind Code |
A1 |
Liu, Lin-Shu ; et
al. |
November 24, 2005 |
Cross-linked gels of chemically activated carboxypolysaccharides
and polyalkylene oxides
Abstract
Carboxypolysaccharides (CPS) including carboxymethyl cellulose
and their derivatives are provided that can be made into sponges,
gels, membranes, particulates and other forms, for a variety of
antiadhesion, antithrombogenic, drug delivery and/or hemostatic
applications during surgery and pharmacological therapeutics. CPSs
derivatized with primary amines can be used alone or in combination
with poly(ethylene glycol) and poly(ethylene oxides) and other
poly(alkylene oxides) to form materials having improved drug
delivery, antiadhesion, and hemostatic uses. Applications include
other types of chemical modifications of CPS to provide hydrogen,
ionic, Van der Walls interactions and/or covalent bonding with
drugs, biologicals and other therapeutic or diagnostic
purposes.
Inventors: |
Liu, Lin-Shu; (Wyncote,
PA) ; Berg, Richard; (Arroyo Grande, CA) |
Correspondence
Address: |
FLIESLER MEYER, LLP
FOUR EMBARCADERO CENTER
SUITE 400
SAN FRANCISCO
CA
94111
US
|
Assignee: |
FZIOMED, INC.
SAN LUIS OBISPO
CA
|
Family ID: |
29249386 |
Appl. No.: |
11/194346 |
Filed: |
August 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11194346 |
Aug 1, 2005 |
|
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10135133 |
Apr 30, 2002 |
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6923961 |
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Current U.S.
Class: |
424/94.64 |
Current CPC
Class: |
C08L 1/286 20130101;
C08L 2666/14 20130101; A61K 31/715 20130101; A61K 38/4833 20130101;
C08B 15/005 20130101; C08L 1/286 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/094.64 |
International
Class: |
A61K 038/48 |
Claims
We claim:
1. A covalently cross-linked gel, comprising: a derivatized
carboxypolysaccharide (CPS); and a derivatized polyalkylene oxide
(PAO).
2. The composition of claim 1, further comprising underivatized
CPS.
3. The composition of claim 1, wherein said derivatized CPS is
CMC-N.
4. The composition of claim 1, wherein said PAO is polyethylene
oxide (PEO).
5. The composition of claim 1, wherein said derivatized PAO is
selected from the group consisting of methoxy-PAO-SPA,
N-hydroxysuccinimide PEG, monomethoxy PEG (mPEG), di-SG-PEG and
di-SE-PEG.
6. The composition of claim 1, further comprising a drug.
7. The composition of claim 6, wherein the drug is a hemostatic
drug.
8. The composition of claim 7, wherein said hemostatic drug is
thrombin.
9. The composition of claim 6, wherein said hemostatic drug is a
vasoconstrictor.
10. The composition of claim 9, wherein said vasoconstrictor is
selected from the group consisting of norepinephrine and
epinephrine.
11. The composition of claim 6, wherein said drug is selected from
the group consisting of hormones, clotting factors,
antiinflammatory agents, steroids, antibiotics and
vasoconstrictors.
12. The composition of claim 11, wherein said hormone is selected
from the group consisting of growth factors, peptide hormones,
steroid hormones and protein hormones.
13. The composition of claim 6, wherein said drug is a nucleic
acid.
14. The composition of claim 13, wherein said nucleic acid is a
DNA.
15. The composition of claim 1, wherein said derivatized CPS
comprises a CPS and one or more members of the group consisting of
primary amines, diamines, sulfonyl chlorides, tresyl chlorides,
aldehydes, vinyl sulfones, carbodiimides, active esters, active
aldehydes, triflurorethanesulfonyl chlorides, periodates and
carbonylating agents.
16. The composition of claim 15, wherein said carbodiimide is
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC).
17. The composition of claim 1, wherein said composition is dried
to form a membrane.
18. The composition of claim 1, wherein said composition is a
sponge.
19. A cross-linked composition, comprising a CPS cross-linked with
a cross-linking agent selected from the group consisting of
glutaraldehyde, carbodiimides and disuccinimidyl suberate.
Description
CLAIM OF PRIORITY
[0001] This application is a Continuation of U.S. patent
application Ser. No. 10/135,133 filed Apr. 30, 2002 titled
"CHEMICALLY ACTIVATED CARBOXYPOLYSACCHARIDES AND METHODS FOR USE TO
INHIBIT ADHESION FORMATION AND PROMOTE HEMOSTASIS," Inventors:
Lin-Shu Liu and Richard Berg (Attorney Docket No: FZIO 6610 US0),
incorporated herein fully by reference.
FIELD OF THE INVENTION
[0002] This invention relates to derivatized carboxypolysaccharides
(CPS). Specifically, this invention relates to derivatized CPS and
uses in manufacturing chemically cross-linked gels and films
incorporation polyethylene oxide (PEO) for drug delivery and for
antiadhesion preparations. More specifically, this invention
relates to anti-adhesion and hemostatic compositions comprising
composites of activated CMC and PEO.
BACKGROUND OF THE INVENTION
[0003] Bleeding during any surgical operation is a major concern.
It delays surgical procedure and prolongs operation time.
Significant bleeding also obstructs a surgeon's view of the
surgical field. Blood transfusions or the use of blood salvage
devices may be required to compensate for blood lost during and
after surgery.
[0004] It is routine to tie off large bleeding vessels, press
bleeding crevices with direct pressure, use electrocautery, or
block punctures with sutures. These methods are moderately
successful. More recently, new methods and compositions have been
devised to stop bleeding. These include matrices derived from
collagen, collagen-derived materials such as Angio-Seal.RTM.
(Kensey Nash Corporation) and VasoSeal.RTM. (Datascope, Inc.);
Flowseal.TM. (Fusion Medical) and CoStasis.TM. (Cohesion Tech.,
Inc); an the combination of thrombin with collagen or
fibrinogen.
[0005] Collagen is a major structural protein in the human body.
Through interaction of peptide sequences comprising the three amino
acids, Arg-Gly-Asp (RDG) in the triplex polypeptide fibers of
collagen with surface receptors on platelet membranes,
collagen-based hemostatic reagents can activate platelets and
contribute to fibrin clot formation.
[0006] Polyethylene glycol (PEG)-derived matrices, such as
functionally active PEG including FocalSeal.TM. (Focal, Inc.) are
designed to form a three-dimensional hydrogel at the bleeding site,
which prevents fluid loss and seals punctures. Both collagen and
PEG based matrices demonstrated effective in situations of
diffusion bleeding.
[0007] Thrombin triggers a cascading set of chemical reactions
leading to blood clot formation. However, the use of thrombin alone
is of limited efficacy in hemostasis, primarily due to a lack of a
framework to which a clot can adhere. Thus, a combination of
thrombin with collagen matrices can accelerate the intrinsic
clotting mechanism by significantly concentration coagulation
factors at the bleeding site, thereby increasing efficacy at
controlling aggressive bleeding. Examples of such products are
Proceed.TM. (Fusion Medical Technology) and Gelfoam.TM. (Pharmacia
and Upjohn). However, to provide desirable coagulation activity,
these require mixing of thrombin with the matrix immediately prior
to use in the operating room.
[0008] Carboxymethylcellulose (CMC) is a water soluble,
biocompatible and bioresorbable semi-synthesized polysaccharide.
The safety of commercially available CMC having high purity has
been identified and approved by the Food and Drug Administration
(FDA) for incorporation into many products. CMC is able to react
with various polymers by way of electrostatic interaction, ionic
cross-linking, hydrogen bonding, Van der Waals interactions, and
physical interpenetration. Because of its safety, convenience and
diversity of physico-chemical properties, CMC has demonstrated wide
applications in the pharmaceutical, food and cosmetic
industries.
[0009] CMC is in a larger group of polymers termed
"carboxypolysaccharides- " (CPS), which include, but are not
limited to alginate, hyaluronic acid, carboxyethylcellulose,
chitin, and the like. CPS are used in the manufacture of
compositions useful for drug delivery and decreasing surgical
adhesions. Schwartz (U.S. Pat. No. 5,906,997), discloses
compositions and methods for decreasing post surgical adhesions
using films of CPS and poly(ethylene oxide) ("PEO"). Schwartz (U.S.
Pat. No. 6,017,301) discloses hydrogels of CPS and PEO, their
methods of manufacture and use for decreasing adhesion formation.
Schwartz (U.S. Pat. No. 6,034,140) discloses association complexes
of CPS and PEO and their use in decreasing adhesions. Schwartz
(U.S. Pat. No. 6,133,325) discloses antiadhesion membranes made of
association complexes of CPS and PEO.
[0010] Miller (U.S. Pat. No. 6,174,999) describes methods of
preparing water insoluble derivatives of polyanionic
polysaccharides, which require one or more polysaccharides, a
nucleophile, and an activating agent to crosslink the
polysaccharide to itself and the nucleophile to the polysaccharide.
The reaction is performed in the presence of hyaluronate or
carboxymethyl cellulose (CMC), 1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride ("EDC"), and a nucleophile. This patent
does not describe or suggest a primary amine-derivatized
polyanionic polymer as a water-soluble product, nor a diamine
derivatized polysaccharide. The methods described result in water
insoluble forms, because all of the components are mixed together
at the same time. Thus, the disclosed compositions would not trap
polyethylene oxides (PEO).
[0011] Burns (U.S. Pat. No. 6,030,958) describes crosslinking a
polysaccharide, and U.S. Pat. No. 5,527,893 describes incorporating
an acylureaderivative of hyaluronic acid (HA).
[0012] Goldberg et al (U.S. Pat. No. 6,010,692) describes methods
for decreasing surgical adhesions, by which tissue surfaces and
surgical articles involved in the surgery are coated with
hydrophilic solutions containing hyaluronic acid before the
operation.
[0013] Burns (U.S. Pat. No. 5,585,361) describes methods for
reducing or inhibiting platelet aggregation and adhesion by
administering pharmaceutical composition containing hyaluronic
acid.
[0014] Cook (U.S. Pat. Nos. 6,172,208 and 6,017,895) describe
conjugation of saccharides with an oligonucleotide.
[0015] Greenawalt U.S. Pat. No. 6,056,970) describes a hemostatic
composition consisting of a bioabsorbable polymer and a hemostatic
compound which is prepared in a nonaqueous solvent.
[0016] Liu (U.S. Pat. Nos. 5,972,385 and 5,866,165) disclose
methods of crosslinking polysaccharides by oxidizing them to
aldehydes and reacting them with proteins.
[0017] Berg (U.S. Pat. Nos. 5,470,911, 5,476,666 and 5,510,418)
describes methods for crosslinking glycosaminoglycans with
activated hydrophilic polymers. These patents also described
crosslinking collagen to derivatized hyaluronic acid using
activated hydrophilic polymers.
[0018] Liu (U.S. Pat. No. 6,096,344) described polymeric
polysaccharides ionically crosslinked into spheres for drug
delivery.
SUMMARY OF THE INVENTION
[0019] However, there are no easily manufactured CPS or CMC
derivatives that can form covalent, ionic, or other bonds with
other molecules and be biocompatible and/or bioresorbable, and be
useful for a variety of therapeutic uses.
[0020] Likewise, there are no methods for manufacturing
compositions having derivatized CMC or other CPS that are easy and
to carry out and result in biocompatible, bioresorbable
compositions.
[0021] Furthermore, there are no methods for using derivatized CMC
or other CPS in polymers useful for drug delivery, hemostasis
and/or adhesion prevention.
[0022] Therefore, this invention includes new types of CMC or CPS
derivatives carrying active functional groups, including side chain
primary amines, active aldehydes, sulfonyl groups, vinyl groups,
tresyl groups, and the like. The derivatized CPSs can be
manufactured using synthetic methods suited to the particular type
of derivative desired. Once manufactured, derivatized CPSs can be
mixed with other molecules, including unmodified CPSs or additional
polymers such as polyalkylene oxides (PAOs) including polyethylene
oxide (PEO), and/or pharmaceutical agents suitable for treating
disorders in patients. In certain embodiments, the derivatized CPS
may form bonds with the other polymer components to form a
cross-linked structure which may hold drugs, and/or may have longer
biological half-lives than non-covalently bonded structures. The
cross-linked structures may be incorporated into materials
including membranes, gels, fibers, non-woven films, sponges, woven
membranes, powders, particles or other physical forms.
[0023] When placed near a surgical site, the derivatized
CPS-containing structures can provide a barrier function,
decreasing the tendency of scars or adhesions to form at the
site.
[0024] Compositions of this invention can be provided with one or
more pharmaceutical agents, such as drugs or biological agents. The
types of agents are not limited, and include vasoactive agents,
hormones, nucleic acids, vectors, antiinflammatory agents and the
like. In embodiments for drug delivery, derivatized CPS-containing
compositions may release the drug in amore sustained fashion,
thereby diminishing adverse effects of rapid alterations in the
concentration of the drug.
[0025] Derivatized CPS containing compositions can be used as gels,
liquids or dried as membranes, sponges or spheres. Upon application
to a moist tissue, a membrane, sponge or sphere preparation can
take up water, becoming gel-like. By using a higher ratio of
derivatizing moiety, one can produce more highly cross-linked
structures and by using a lower ratio of derivatizing moiety, one
can produce structures having less cross-linking. Compositions made
of derivatized CPSs can have half-lives that can be controlled,
with more highly cross-linked structures having a longer half-life
and less cross-linked structures having a shorter half-life.
Moreover, the use of longer linkers can permit the formation of a
composition having larger pores than with the use of shorter
linkers. By selecting the type of derivatizing agent (e.g., amine,
tresyl, aldehyde, etc.) the ratio of derivatizing agent to CPS
active site (e.g., COOH residues) and the length of linkers, the
physical and biological properties of derivatized CPS can be
controlled to suit a particular purpose, whether antiadhesion,
antithrombogenesis, and/or hemostatic. By selecting the type and
size of PAO, one can provide compositions that have controllable
tissue adherence, platelet adherence and/or platelet aggregation
behavior. By selecting a drug for incorporation into a matrix, or
added to a matrix, one can provide additional, pharmacological
means for affecting adhesion formation, blood flow, bleeding or
other property.
[0026] The properties of the compositions can be varied by varying
the pH of the compositions. Many preparations can be desirably used
having a neutral pH (i.e., a pH of about 7). However, if desired,
pHs of the compositions can be higher or lower. Additionally,
derivatized CPS having positively charged groups can be used to
associate with negatively charged components (e.g., drugs,
negatively charged proteins and the like). Similarly, CPSs having
negative charges can be used to associate with positively charged
components (e.g, drugs, positively charged proteins and the
like).
[0027] The sites of delivery of drugs using the compositions of
this invention include, without limitation, skin, wounds, mucosa,
internal organs, endothelium, mesothelium, epithelium. In certain
embodiments, buccal, optical, nasal, intestinal, anal, vaginal
applications using compositions of this invention can be used.
Furthermore, the compositions of this invention are suitable for
placement between adjacent tissues for diminishing the formation of
unwanted adhesions.
[0028] We also provide novel hemostatic reagents comprising
conjugates of CPS derivatives having primary amine groups, for
example, (CMC-N), sulfonyl groups, other charged groups and PAOs.
Thrombin can be pre-loaded into the CPS-N/PAO matrices. These
compositions have hemostatic activity that is greater than that for
either matrices having no thrombin or thrombin alone.
BRIEF DESCRIPTION OF THE FIGURES
[0029] The invention will be described with respect to the
particular embodiments thereof. Other objects, features, and
advantages of the invention will become apparent with reference to
the specification and drawings in which:
[0030] FIG. 1 depicts NMR spectra of methoxy-polyethylene oxide
conjugated carboxymethylcellulose carrying primary amine groups
(CMC-N/PEO) The indicated peaks a-e refer to the hydrogen atoms
indicated on the insert (a, 3.379 ppm; b, 3.701 ppm; c, 3.52 ppm;
d, 3.166 ppm; e, 2.862 ppm).
[0031] FIG. 2 depicts a scanning electron micrographs of external
structures A, B and C and internal structures D, E and F of
matrices prepared in microtiter plates. Matrices shown in panels A
and D are made from CMC, matrices shown in panels B and E are made
from CMC-N, and matrices shown in panels C and F are made from
CMC-N/PEO. Magnification, 40.times. (A, B and C); 200.times.(D, E
and F).
[0032] FIG. 3 depicts results of studies of platelet aggregation
induced by matrix materials (25 .mu.g/ml) in the presence (top
graph A) and absence (bottom graph B) of thrombin (4 U/ml): CMC
(.circle-solid.), CMC-N (.box-solid.), CMC-N/PEO
(.tangle-solidup.), and control (.largecircle.). Matrices were
prepared in microtiter plates. Platelets were prepared from
citrated whole bovine blood at a concentration of
30-35.times.10.sup.4/.mu.l.
[0033] FIG. 4 depicts results of studies of activation of platelets
adhered on plates coated with matrix materials as indicated in the
different panels A, B, C and D. Panel A: CMC, panel B: CMC-N, panel
C: CMC-N/PEO, and panel D: control. Plates (diameter, 18 mm) cast
with each matrix were incubated with 2.0 ml of platelet rich plasma
having a platelet concentration of 30-35.times.10.sup.4/.mu.l at
37.degree. C. for 20 min.
[0034] FIG. 5 is a schematic representation of a spurting bleeding
model for studying hemostasis. The apparatus consists of a
reservoir connected through a tube to a receiver over which porcine
skin is stretched.
[0035] FIG. 6 depicts a graph of results of studies of resistance
to spurting bleeding (expressed as mm Hg) of various
matrix/thrombin formulations. Data are represented as three groups
of three bars each. The left group of three bars represents results
obtained using a CMC matrix, the middle group represents results
obtained using a CMC-N matrix and the right group represents
results obtained using CMC-N/PEO matrices. Within each group, the
left bars represent results obtained using matrices alone, the
middle bars represent results obtained using matrices with thrombin
loaded on site, and the right bars represent results obtained using
matrices pre-loaded with thrombin. Specimen of matrix materials
used in each test was 100 mg. Thrombin content in each type of
matrices was 2 U/mg. Each test was repeated for 5 times, the data
expressed as mean.+-.standard deviation. Resistance expressed by
using thrombin solution was 15 mm Hg.
DETAILED DESCRIPTION
[0036] This invention includes a variety of derivatized CPSs,
including CMCs that can interact with biologically active
substances under mild conditions of pH, body or room temperatures,
and/or in aqueous solutions. CMC is a polymer composed of sugar
residues linked together, and each of which may have a carboxyl
residue attached to the sugar moiety. There are three (3) potential
sites for carboxylation on each sugar residue of CMC. Because a
carboxyl residue can be chemically reactive, those locations on CMC
are potential sites for derivatization. By controlling the degree
of substitution (DS) of the CMC, the number of active groups on the
derivatized CMC can be controlled. Derivatized CPSs and CMCs of
this invention can be used for one or more of the following:
[0037] (1) as delivery vehicles for controlled release of bioactive
substances, such as growth factors, active peptides, genes, cells,
clotting factors such as thrombin, and antibiotics hormones
including epinephrine, steroids, antiinflammatory agents and the
like, and vasoconstrictors such as norepinephine and the like;
[0038] (2) as delivery vehicles for the localized release of
bioactive substances, such as growth factors, active peptides,
genes, cells, clotting factors such as thrombin, and antibiotics,
hormones including epinephrine, steroids, antiinflammatory agents
and the like, and vasoconstrictors such as norepinephine and the
like;
[0039] (3) as cross-linkers for artificial extracellular matrix
(ECM) construction;
[0040] (4) as binders for protein coupling and fatty absorption in
both tissue engineering and food industries; and
[0041] (5) as additives in food industries to produce value-added
milk products. However, it can be readily appreciated that the
derivatized CPSs and CMCs of this invention can be used for a
variety of purposes in which one or more physico-chemical
properties are desired. Those properties include, but are not
limited to bioadhesion, bioresorbability, antiadhesion, viscosity,
and physical interpenetration.
[0042] II Side Chain Modification of CMC
[0043] A CPSs and CMCs Having Primary Amines
[0044] Primary amines can be introduced to the side chains of a CPS
or CMC by covalent modification of the carboxylic acids in the
polysaccharide with short compounds containing primary amines at
either end (e.g., diamines, such as ethylenediamine), to form an
amide linkage with the carboxyl residue, leaving a free primary
amine at the other end of the linker. The length of the linker can
be between bout 2 and about 10 atoms, with certain embodiments
having between about 3 to about 8 atoms, in alternative embodiments
of between about 5 and 7 atoms, and in further embodiments, about 6
atoms. The length of the linker can be selected to provide a
"loose" structure, in which relatively long linkers are used, or
alternatively, a "tight" structure, in which relatively shorter
linkers are used. Long linkers and loose structures may be
desirable if the viscoelasticity of the composition is desired to
be relatively low, where a large, biologically active agent (e.g.,
a protein or a gene) is to be incorporated, or in which the
biological half-life is desired to be relatively short.
Alternatively, short linkers and tight structures may be desirable
if the viscoelasticity is desired to be relatively high (e.g.,
certain membranes and other solid structures), where a relatively
small biologically active molecules is desired (e.g., an ion, amino
acid, vitamin or pharmaceutical agent), or in which the biological
half-life of the structure is desired to be relatively long. It can
be appreciated that those of skill in the art can perform studies
to determine the optimum length of a linker to suit a particular
purpose.
[0045] To ensure that only one end of the linker is coupled to the
CPS strand, one can use a molar excess (based on the degree of
substitution of the CPS) of the linker. For example, one can use a
molar ratio of active carboxylic acid groups to linker in the range
of about 20-about 50 to provide a high degree of non-cross-linked
CPS or CMC. Alternatively, by using a lower molar ratio, relatively
more cross-linking between CPS molecules can be achieved. In
embodiments in which a highly-cross-linked CPS is desired, one can
use a relatively low (e.g., from less than about 1 to about 20)
molar ratio of carboxyl residues to linkers. It can be appreciated
that using a molar excess of linker molecules in a solution
containing non-constrained CPS molecules (e.g., a relatively dilute
solution of CPS) can promote derivatization of CPS with little
cross-linking. However, in situations in which CPS molecules are
constrained (e.g., high CPS concentrations) or are tightly packed
together, there may be an increased tendency for cross-links to
form between different CPS chains. It can be readily appreciated
that workers of skill in the art can select a molar ratio of
carboxyl residues to linker molecules to produce a desired degree
of cross-linked CPSs.
[0046] Amide bonds can be formed using any desired chemical
interactions, including carbodiimide mediated coupling, active
ester intermediates, and the use of carbonylating compounds. For
some applications, it may be desirable to use
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC).
[0047] B. CPSs Having Active Aldehydes
[0048] In other embodiments of this invention, aldehyde groups on a
CPS molecule can be produced by oxidation of the polysaccharide. In
certain embodiments, it can be desirable to use a periodate such as
sodium periodate. A reaction can occur between two adjacent
secondary hydroxyl residues to cleave a carbon-carbon bond between
them and to create two terminal aldehyde groups. The
aldehyde-modified CPS can then be recovered by lyophilization and
stored in a desiccator at a temperature of, for example, about
4.degree. C. in the dark.
[0049] C. CPSs Having Active Tresyl Groups
[0050] Hydroxyl groups of polysaccharides may also be activated by
certain compounds to form intermediate reactive derivatives
containing leaving groups suitable for nucleophilic substitution
reactions. However, hydroxyl groups of polysaccharides are only
mildly nucleophilic, having a nucleophilicity about equal to that
of water. To avoid hydrolysis of the active groups by cross-linking
reagents, the resulting bond should desirably be stable in aqueous
conditions. Sulfonyl chlorides, such as trifluoroethanesulfonyl
chloride (tresyl chloride), are well suited for hydroxyl
modification. The resulting CPS-sulfonyl chloride derivatives can
be used for protein coupling reactions or for cross-linking.
[0051] D. CPS Having Active Vinyl Sulfone Groups
[0052] Divinyl sulfone (DVS) can be effectively used to modify
hydroxyl groups of CMC and other polysaccharides. To avoid
disfavored intra- and intermolecular cross-linking, DVS should be
used in excess. As with the diamine linkers discussed above, the
molar ratio of hydroxyl resides to linker should be chosen to
provide a desired degree of non-cross-linked CPS such as a molar
ratio of vinyl sulfone to hydroxyl of 30 to 1.
[0053] E. Usefulness of the Invention
[0054] Effects of the modification of CPSs such as CMC with PEO or
other polyalkylene oxide (PAO) on the activation of coagulation
components were evaluated by determining their effect on
stimulating the intrinsic clotting pathway. The APTT procedure was
used to evaluate the intrinsic clotting pathway because it is a
clinically relevant test for the detection of deficiencies of
coagulation factors or abnormalities in the intrinsic pathway
(Imanishi, Ito et al. 1988). In certain embodiments, the presence
of CMC-N matrix without the modification of PEO was most active
towards the blood intrinsic coagulation system, as demonstrated by
decreasing APTT values. In other embodiments, the matrix composed
of CMC-N/PEO prolonged the APTT, indicating that grafted PEO
suppressed the activation of coagulation factors in the intrinsic
clotting pathway. These examples are illustrative of the effects
derivatized CPS and PAOs. In addition to effects on platelets,
proteins and clotting, PAOs can affect adherence of compositions to
tissues, physical properties, including stiffness, viscosity,
strength, and biological half-life, among others. By varying the
composition of derivatized CPS/PAO.
[0055] It has been documented to use a PEO coating on a surface to
prevent proteins or cells from depositing on the surface (Gombotz
1992). The mechanism of the effective repulsion of proteins from
PEO grafted materials surface is not known with certainty, but
according to one theory, the degree of conformational freedom of
proteins is reduced when they are close to the PEO layer, and
consequently, an entropic repulsion between the PEO and the
proteins may occur (Karlstrom 1997). Other theories may account for
the observation, and this invention is not intended to be limited
to any particular theory. In certain embodiments, the PEO modified
CMC matrices were less active toward the intrinsic coagulation
system (Table 3) and platelets (FIGS. 3, 4 and Table 3) than the
unmodified CMC and CMC-N matrices. The PEO modified CMC matrices
were also demonstrated to better preserve thrombin in pre-loaded
matrices from deactivation during the freezing and lyophilization
process (Table 4). Although the mechanisms are not well understood,
according to one theory, PEO may preserve thrombin activity or
alternatively, it may facilitate the release of thrombin from the
matrices when contacted with fluid. Because of the desirable
property of PEO on limiting protein interaction, CMC-N/PEO may be
considered an anti-coagulant; however, the additional property of
delivering of thrombin, matrices of CMC-N/PEO with exogenous
thrombin permits a new approach to treating bleeding by providing
antithrombogenic, hemostatic compositions.
[0056] The derivatized CPS-containing compositions of this
invention can be made which have a wide range of physical and
chemical properties. In addition to being able to vary charge,
water uptake and drug association, compositions having cross-linked
CPS can be formed, which can have prolonged residence times,
compared to non-cross-linked compositions. Cross-linked
preparations can permit the manufacture and use of the compositions
for a wide variety of hemostatic and drug delivery
applications.
[0057] The results of the current experiments reflect the
summarization of the various parameters of the matrix, which
include the hydration characteristics, the activity towards blood
intrinsic coagulation cascade, the interaction with exogenous
thrombin, and the mechanical strength in attaching to bleeding
surfaces. Taking balance of all considerations, thrombin-loaded CMC
derivatives grafting with primary amine or PEO demonstrated
excellent hemostasis. In addition, the results suggest that the
delivery of thrombin seems an effective strategy in the development
of hemostasis.
[0058] The discoveries of this invention will be useful for drug
delivery generally. CPSs at pHs above the pK of dissociation of the
hydroxyl hydrogen atom, yield moieties which contain negatively
charged or partially negatively charged carboxyl residues
(--COO.sup.-), which can form electrostatic interactions with
positively charged portions of drugs or proteins. For example, the
hemostatic protein thrombin is positively charged, and can form
ionically associated structures with CPS to form a delivery form of
the protein. Similarly, other proteins containing positively
charged amino acids, (e.g., lysine, arginine and the like) on an
exterior portion of the protein can also be electrostatically bound
to carboxyl groups. Even if a positively charged amino acid or
other moiety on a protein is not at the surface, if sufficient
positive charge is present at the surface to interact with a
negatively charged derivatized CPS, such molecules can be
associated with the CPS. Certain proteins contain positively
charged carbohydrate residues, such as N-acetylglucosamine, which,
at physiological pH ranges can bind a hydrogen ion to produce a
positively charged amino group. Such positively charged groups can
be a site of association with a negatively charged derivatized or
underivatized CPS.
[0059] Conversely, drugs, or proteins comprising negatively charged
residues (e.g., aspartic acid, glutamic acid and the like) can
become electrostatically attracted to positively charged moieties
on derivatized CPSs including CMC. Certain therapeutically useful
proteins, such as heparin, are glycoproteins, meaning that
carbohydrate moieties are attached to the amino acid core of the
protein molecule. Many of the carbohydrate moieties of
glycoproteins are negatively charged, and include sialic acid,
byway of illustration only. Moreover, certain glycoproteins have
sulfate residues (--SO.sub.4), which at many physiological pH
ranges are negatively charged. Such protein/derivatized CPS
formulations, either with or without added PAO can be used for
direct injection of the protein to a desired site.
[0060] Nucleic acids have numerous positively charged residues on
the nucleotide bases, arginine, thymine, guanine, cytosine or
uracil. Thus, DNAs and RNAs may be delivered using CPS and/or
derivatized CPSs. In certain embodiments, negatively charged
derivatized CPSs can be advantageously used. By way of example
only, sulfonyl groups, tresyl groups and the like can be used.
Moreover, underivatized CPSs at pHs at which carboxyl groups are at
least partially dissociated have negatively charged moieties which
can associate with positively charged nucleic acids. Such nucleic
acid delivery can be useful for gene therapy, antisense nucleotide
therapy, vector transfection, and viral transfection of cells in
vitro.
[0061] For gene transfection, the nucleic acid may comprise a
vector having a promoter region, an enhancer region and a coding
region. Many such nucleic acids are known in the art, and will not
be described herein further. Nucleic acids used for antisense
therapeutics include DNAs or RNAs having sequences complementary to
a mRNA encoding a protein whose translation is not desired.
Examples include RNAs directed against viral or cellular gene
sequences, as described in U.S. Pat. Nos. 5,858,998 and 6,291,438,
incorporated herein fully by reference. Additional antisense
nucleotides are known in the art and will not be discussed
further.
[0062] It can be readily appreciated that PAOs, including PEO can
be added to such delivery vehicles and thereby confer desirable
properties of antithrombogenesis, decreased platelet adhesion and
activation and other properties of the PAOs.
[0063] Moreover, certain drug-associated CPS can be formed and then
mixed with derivatized CPS. For example, an underivatized CPS
having negatively charged carboxyl moieties can be used to
associate with a positively charged drug for delivery (e.g.,
thrombin). Addition of this material to a composition comprising a
derivatized CPS (e.g., CMC-N) can provide a composition in which
the drug for delivery is associated with one of the CPSs and the
other CPS can confer desirable properties to the mixture (e.g,
increased or decreased viscosity), which can increase the half-life
of the delivered drug. It can be readily appreciated that forming
an association of a derivatized CPS with a drug, and then adding
underivatized CPS can provide a composition that has both desirable
drug-binding features and desirable physicochemical features (e.g.,
increased or decreased viscosity). Furthermore, one can use
different types of derivatized CPSs which can be associated with
different drugs for co-delivery. For example, a negatively charged,
derivatized CPS can be used to associate with a positively charged
drug, and a positively charged, derivatized CPS can be used to
associate with a negatively charged drug. By mixing the two
combinations together, one can create compositions which provide
desired pharmacodynamic properties (e.g., desired pharmaceutical
effects) as well as desirable pharmacokinetic properties (e.g.,
tissue half-life). Thus, using mixtures of derivatized and
un-derivatized CPS can provide a greater degree of flexibility in
formulating drug delivery compositions.
[0064] The types of drugs or biological agents that can be
advantageously delivered using the compositions of this invention
are not limited. Any agent that can be used for diagnosis or
treatment of a disease or condition can be delivered using the
compositions of the invention, so long as the efficacy of the agent
is not so reduced by association with the compositions as to render
them unsuitable for their intended purposes. For example, drugs
include vasoactive agents including vasodilators and
vasoconstrictors, hormones, chemotherapeutic agents, growth
factors, clotting factors, antibiotics, antiinflammatory
agents.
[0065] It can be appreciated that the above descriptions are not
intended to be limiting to the scope of the invention. Rather, they
are intended to be representative of the many different embodiments
of the invention.
EXAMPLES
[0066] The following examples are presented to illustrate certain
embodiments of this invention, and are not intended to limit the
scope to the embodiments so illustrated. Rather, workers of skill
in the art can modify or adapt the teachings of this invention to
make and use other variations without undue experimentation. All of
those embodiments are considered to be part of this invention.
Example 1
Preparation of CMC Having Primary Amine Groups
[0067] Primary amines can be introduced to the side chains of CMC
by covalent modification of the carboxylic acids (carboxylate
chains) in the polysaccharide with compounds containing primary
amines at either end, such as ethylendianine (EDA), to form amide
linkages. To ensure that only one end of the compound coupled to
each carboxylate and does not cross-link the macromolecules being
modified, the diamine should be used in excess. Amide bond
formation may be accomplished by several methods including
carbodiimide mediated coupling, active ester intermediates, and the
use of carbonylating compounds. In this study, a water soluble
carbodiimide, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC), was used. A protocol is described in the
following paragraph.
[0068] Materials:
[0069] CMC (degree of substitution, "DS", 1.19), 3.0 g in 300 ml
MES buffered saline (pH 4.7), EDA, 30.6 g in 45 ml MES; and EDC,
2.92 g in 5 ml MES.
[0070] Procedure:
[0071] To the CMC solution added with the EDC solution under
constant stirring. The reaction solution was stirred at room
temperature for 30 min followed by the addition of EDA solution.
After reaction for 48 h, the reaction solution was transferred into
a dialysis membrane tube (Spectro/Por.RTM., MWCO, 12-14,000) and
dialyzed against 4.0 L NaCl for 16 h, then against large volume of
running de-ionized water for an additional 24 h. The dialyzed
reaction solution was lyophilized to obtain dry materials. The dry
product thus formed stored in desiccators at room 4.degree. C. for
further application.
[0072] Polymer Identification
[0073] The introduction of primary amine onto CMC and the formation
of CMC-N and PEO conjugate were confirmed by NMR using a Varian 400
spectrometer. Signals were referenced to tetramethyl silane (TMS).
The amount of functional groups of --COOH and --NH.sub.2 on CMC,
CMC-N, and CMC-N/PEO was quantified by colorimetric methods using
rhodamine 6G (Liu, Ito et al. 1991) and TNBS (Ito, Liu et al.
1991), respectively.
[0074] FIG. 1 depicts the NMR analysis of a modified CMC-N of this
invention. The spectrum shown indicates peaks a, b, c, d, and e
along with a structure of derivatized CMC. Individual portions of
the derivatized CMC are indicated, corresponding to the peaks in
the NMR spectrum.
[0075] Table 1 shows the calculated and measured amounts of various
moieties in matrices containing derivatized or underivatized
CMC.
1TABLE 1 Determination of Active Groups in CMC-Derivatized Matrices
Calculated (.mu.mole/mg) Determined (.mu.mole/mg) Samples --COOH
--NH.sub.2 --COOH --NH.sub.2 CMC 4.63 0 4.74 .+-. 0.14 NF* CMC-N
4.23 NF 4.55 .+-. 0.09 CMC-N/PEO 4.02** NF 4.18 .+-. 0.11 *Not
found; **Calculated based on the mass ratio of PEO and CMC-N.
Determinations performed, n = 3.
Example 2
Viscosity of CMC-N and CMC-N/PEO Composites
[0076] Primary amine carried CMC derivatives (CMC-N) prepared from
CMCs with various DS (0.8 and 1.2) and molecular weight (700 K and
250 K Dalton) were firstly characterized for viscosity in various
formulations: (1) composite gel with PEO (CMC-N/PEO); (2) in the
presence (3) in the absence of calcium ion; and (4) as a coacervate
with CMC. Experiment was performed at ambient temperature using a
Brookfield Digital Viscometer (Model VD-II, Brookfield Engineering
Laboratory, Inc.; Stoughton, Mass.) at the shear rate of 05 and
spindle #29. The pH of these materials was about 7. Results are
shown in Table 2 below.
2TABLE 2 Viscosity of PEO/CMC Composites Solution composition (%)
CMC CMC-NH.sub.2 PEO Viscosity (cps) 1.0 0 0 0.3 .times. 10.sup.3
2.0 0 0 1.1 .times. 10.sup.3 .sup.a)2.0 0 0 3.4 .times. 10.sup.4
.sup.b)2.0 0 0 1.6 .times. 10.sup.3 0 1.0 0 0.3 .times. 10.sup.3 0
.sup.a)1.0 0 1.2 .times. 10.sup.3 0 2.0 0 0.7 .times. 10.sup.3 1.0
0 1.0 0.4 .times. 10.sup.3 0 1.0 1.0 0.7 .times. 10.sup.3 0
.sup.a)1.0 1.0 1.7 .times. 10.sup.3 1.0 1.0 0 2.3 .times. 10.sup.3*
.sup.a)1.0 .sup.a)1.0 0 0.9 .times. 10.sup.6* Unless indicated, CMC
used to prepare CMC-N derivative and for the viscosity test is that
with the DS of 1.19; MW, 250,000. .sup.a)CMC with the DS of 0.8;
MW, 700,000. .sup.b)Ca.sup.++ in present. *The CMC/CMC-NH.sub.2
mixture was prepared by mixing equal volume of 2% CMC and
CMC-NH.sub.2 solutions. The mixture was allowed to stand at room
temperature for 5 minutes prior to test.
[0077] We observed that the solutions of underivatized and
derivatized CMC had substantially the same viscosity. However, we
unexpectedly found that the addition of PEO to either the CMC or
CMC-N preparations had different effects. Adding PEO to CMC altered
viscosity slightly (from 0.3 to 0.4 10.sup.3 cps), whereas adding
PEO to CMC-N increased viscosity substantially more (from 0.3 to
0.7.times.10.sup.3 cps). Thus, one can vary the composition of a
matrix to provide a desired viscosity, depending upon the
particular drug delivery or hemostatic needs.
Example 3
Tissue Adhesiveness of CMC-N/PEO Composites
[0078] The tissue adhesive property of gels was determined by
measuring the force needed to detach the gels from the membrane
using a modified Tape Loop Tack Tester (Model LT-100;
ChemiInstruments, Fairfield, Ohio) equipped with a digital force
meter (Chatillon Model DFM; Greensboro, N. J.). Membranes of
porcine intestine were used as the receiver. The membranes were
mounted onto each surface of both the test panel and specimen jaw,
which was attached to the tension head by means of an yok and a
release pin. The gap between the two membranes was adjusted to
2.+-.1 mm by releasing and tightening the release pin. 5.0.+-.0.1
ml of the gel were applied on the membrane bound to the test panel.
All measurements were performed on settings as:
[0079] Specimen jaw lowering speed: 9 mm/s
[0080] Contact time: 3 minutes
[0081] Contact area: 5.31 cm.sup.2
[0082] Specimen jaw withdrawal rate: 9 mm/s
[0083] Withdrawal height: 4.5 cm
[0084] Each experiment was carried out five times. The force (N)
needed to detach the gel was recorded and represented as the mean
value with standard deviation. Results are shown in Table 3
below.
3TABLE 3 Tissue Adhesive Properties of PEO/CMC Composites Solution
composition (%) Peak detachment force CMC CMC-NH.sub.2 PEO (N) 1.0
0 0 0.12 .+-. 0.01 2.0 0 0 0.33 .+-. 0.01 .sup.a)2.0 0 0 0.65 .+-.
0.02 .sup.b)2.0 0 0 0.49 .+-. 0.01 0 1.0 0 0.19 .+-. 0.04 0
.sup.a)1.0 0 0.19 .+-. 0.07 0 2.0 0 0.46 .+-. 0.13 1.0 0 1.0 0.15
.+-. 0.03 0 1.0 1.0 0.24 .+-. 0.04 0 .sup.a)1.0 1.0 0.37 .+-. 0.01
1.0 1.0 0 0.76 .+-. 0.14* .sup.a)1.0 .sup.a)1.0 0 1.12 .+-. 0.09*
Unless notice, CMC used to prepare CMC-N derivative and for the
tissue adhesion test is that with the DS of 1.19; MW, 250,000.
.sup.a)CMC with the DS of 0.8; MW, 700,000. .sup.b)Ca.sup.++ in
present. *The CMC/CMC-NH.sub.2 mixture was prepared by mixing equal
volume of 2% CMC and CMC-NH.sub.2 solutions. The mixture was
allowed to stand at room temperature for 5 minutes prior to
test.
[0085] As shown in Table 3, these results show that adding CMC and
CMC-N together increase tissue adhesiveness more than expected
based on the individual tissue adhesivenesses. This indicates
synergistic actions between CMC and CMC-N. One possible theory for
this interaction is that the CMC and the CMC-N form a coascervate,
thereby stabilizing the composition, and can therefore increase the
time needed for the composition to dissolve in body fluids.
Example 4
PEO and CMC Released from CMC-N/PEO Composites
[0086] Studies on the release of CMC and PEO from gels were
performed using PBS as release medium. Membranes of porcine
intestine were mounted on the bottoms of a petri dish (g=50 mm)
using double side adhesive tape. An aliquot of 5.0 ml of each gel
was evenly spread over the surface of the membrane. PBS, 10 ml, was
carefully loaded on the top of the gel layer followed by incubation
at room temperature under gentle shaking. At the time periods of 3,
10, 20 min, 1 h and 2 h, the dish was tipped to one side and 1.0 ml
of the incubation solution was pipetted from the solution above the
gel and analyzed for the amount of PEO and CMC released.
[0087] The released PEO was quantified by measuring the absorbance
of the fluorescein moiety attached at the PEG chain at 500 nm. CMC
amount in the incubation solution was determined by measuring the
absorbance at 480 nm after incubating with phenol and sulfuric acid
at 30.degree. C. for 20 minutes. Results are shown in Table 4
below.
4TABLE 4 Amounts of Gel Components Released Into Medium Incubation
PEO (mg) released from CMC (mg) released from time (min) CMC/PEO
CMC-N/PEO CMC/PEO CMC-N/PEO 3.0 0.22 .+-. 0.11 0.28 .+-. 0.15 ND*
ND 10.0 0.30 .+-. 0.10 0.28 .+-. 0.17 ND ND 20.0 1.04 .+-. 0.18
0.59 .+-. 0.09 ND ND 60.0 5.07 .+-. 0.69 2.24 .+-. 0.21 4.03 .+-.
0.21 2.15 .+-. 0.19 *ND, Not detectable.
Example 5
Stability of CMC/CMC-N Polyelectrolyte Gels
[0088] CMC/CMC-NH.sub.2 coacervate gel was prepared by mixing equal
volume of 2% CMC and CMC-NH.sub.2 solutions under vigorous stirring
at room temperature. Upon mixture, a white precipitate was
observed, indicating the formation of coacervate. The precipitate
was allowed to grow and stabilize at room temperature for 5 min.
followed by centrifugation at 1,500 rpm for additional 5 min. The
precipitate thus formed was incubated with D.I. H.sub.2O, PBS, and
tissue culture medium, Dulbecco's Modified Eagle's Medium (DMEM,
Sigma Chemical, San Luis) at ambient temperature for two weeks. CMC
with different DS and the gel of CMC cross-linked with calcium were
used as control. The stability of each sample was judged by eye
observation and subjectively graded on the scale of 1 (dissociate
easily), 2 (partially dissociate in two weeks), and 3 (stable for
two weeks). Results are shown in Table 5 below.
5TABLE 5 Stability of CMC/CMC-N Composites Media .sup.a)CMC/Ca++
.sup.a)CMC .sup.b)CMC .sup.b)CMC/CMC-N D.I. H.sub.2O 1 1 1 3 PBS 1
1 1 2 DMEM 1 1 1 2 .sup.a)CMC with the DS of 0.8; MW, 700,000.
.sup.b)CMC with the DS of 1.19; MW, 250,000.
Example 6
Preparation of CMC-N Cross-Linked Membranes
[0089] CMC-N, 3.0 g dissolved in 15.0 ml D.I. water. The solution
was cast on a peri dish (100.times.15 mm) and placed in a hood at
room temperature for 2 days to air-dry. The membrane thus obtained
was placed in an isopropyl alcohol/H.sub.2O solution containing
0.5% glutaraldehyde, shaken gently for 6 h followed by washing with
3.times.5 0 ml D.I. water, then air-dry. The cross-linked CMC-N
membrane is stable in D.I. water, PBS, 1.0 N NaCl, and tissue
culture medium.
Example 7
Preparation of CMC-N Cross-Linked Sponge
[0090] CMC-N, 3.0 g dissolved in 15.0 ml D.I. water. The solution
was cast on a peri dish (100.times.15 mm) and submitted to
freeze-dry. The dry product thus obtained is a sponge-like porous
matrix. The matrix was placed in an isopropyl alcohol/H.sub.2O
solution containing 0.5% glutaraldehyde, shaken gently for 6 h
followed by washing with 3.times.50 ml D.I. water, then
re-lyophilized. The cross-linked CMC-N porous matrix is stable in
D.I. water, PBS, 1.0 N NaCl, and tissue culture medium.
Example 8
Preparation of CMC-N Cross-Linked Particles
[0091] CMC-N, 3.0 g dissolved in 15.0 ml D.I. water. To the
solution added with 0.1 g of disuccinimidyl suberate in 2.0 ml
dimethyl sulfoxide (DSS/DMSO) under vigorous stirring. Precipitates
appeared shortly after mixing. The reaction was carried out under
stirring for 6 h, then standing on bench for 24 h. The supernatant
was discarded and the solid was washed for three times with large
volume of D. I. Water. CMC-N particles cross-linked with DSS thus
formed is stable in water, saline, and tissue culture medium.
[0092] II. Hemostatic Derivatized CMC/PEO Composites
[0093] Derivatized CMCs of this invention can be very desirable as
hemostatic agents. CMC/PEO composites can be manufactured as
described above and used as is, or can have hemostatic factors
incorporated therein. In certain embodiments, thrombin can be
advantageously incorporated into hemostatic products to slow
aggressive bleeding and promote clot formation.
Example 9
Modification of CMC with Ethylenediamine
[0094] Materials:
[0095] Carboxymethyl cellulose (CMC) having an average molecular
weight of 250 kdaltons (kdal) and the degree of substitution of
1.19 was obtained from Hercules Inc. (Wilmington, Del.).
1-Ethyl-3-(3-dimethylaminopropyl)c- arbodiimide (EDC),
ethylenediamine dihydrochloride (EDA), activated partial
thromboplastin time reagent (APTT), and thrombin were purchased
from Sigma Chemical Co. (St. Louis, Mo.). Polyethylene glycol
derivative, methoxy-PEO-SPA (mPEO-SPA, M.W. 5 kD) was obtained from
Shearwater Polymers (Huntsville, Ala.). Fresh porcine skin was from
a local market.
[0096] Citrated bovine blood was prepared by mixing one part of
sodium citrate solution with nine parts of whole blood from a
healthy adult bull (courtesy of Dr. William Plummer of the Animal
Science Department, California Polytechnic State University, San
Luis Obispo, Calif.).
[0097] Polymer Modification:
[0098] A. Modification of CMC with Ethylenediamine
[0099] Introduction of primary amine groups into CMC was conducted
according to published methods (Liu, 1991). Briefly, to a CMC
solution, 0.30 ml of a solution of MES buffer, pH 4.7, was added
with EDC (0.29 gm) in 2.0 ml MES, under constant stirring. The
reaction solution was stirred at room temperature for 30 minutes,
followed be the addition of EDA (3.0 gm) in 8.o ml MES buffer.
After 48 hours, the reaction solution was transferred into a
dialysis membrane tube (Spectro/Por.RTM., M.W. cutoff:
12,000-14,000 daltons) and was dialyzed against 1 N NaCl (4.0 L)
for 16 hours, then against a large volume of running de-ionized
(DI) water for an additional 24 hours. The dialyzed solution was
lyophilized to obtain a dry preparation of CMC derivative and was
processed for the quantification of introduced primary amines to
form aminated CMC (CMC-N). The dry product thus formed was stored
in a dessicator at 4.degree. C. for the following experiments.
Example 10
Conjugation of CMC-N with mPEO-SPA
[0100] PEO was grafted onto the side chains of CMC-N by the method
described previously (Rhee W 1997), where mPEO-SPA, 0.5 g in 5.0 ml
of 30.0 mM HCl, was mixed with 100.0 ml of CMC-N solution
containing 1.50 g of CMC-N in 10.0 mM NaOH (mole ratio of
succinimidyl to primary amine: 1.5/100) under vigorous stirring at
room temperature for 24 h. The reaction solution was dialyzed
against running deionized water for 24 h using Spectro/Por.RTM.
dialysis membrane tube (MW cut off, 12-14,000), then lyophilized to
produce CMC-N and PEO conjugate, CMC-N/PEO.
[0101] Collagen materials were cross-linked by two different
difunctional polyethylene glycols (PEGs) for a variety of
applications. Cross-linking was mediated by carbamation of primary
amines on collagen with N-hydroxysuccinimide ester end-groups on
the PEG crosslinkers. One crosslinker, disuccinimidyl glutarate PEG
(di-SG-PEG), contained internal ester linkages which were capable
of hydrolyzing and hence produced degradable networks. The other
crosslinker, disuccinimidyl propionate PEG (di-SE-PEG), contained
hydrolytically stable internal ether linkages and hence produced a
more durable network. In vitro experiments verified that di-SE-PEG
networks were more hydrolytically stable (Rhee W. 1997). In vivo
experiments (rat subcutaneous model) revealed similar degradation
rates for both networks indicating that other degradation
mechanisms (probably enzymatic) also influence degradation in
vivo.
[0102] Previous work on PEG modified collagen has involved
conjugating monomethoxy-PEG (mPEG) to collagen. In order to
covalently bind PEG to collagen, hydroxyl end groups of PEG must
first be activated with a suitable reagent (Rhee, W.; Wallace, D.
G.; Michaels, A.; Burns, R.; Fires, L.; Delustro, F.; Bentz, H.,
Collagen-Polymer Conjugates, U.S. Pat. No. 5,162,430, 1992).
N-hydroxysuccinimide (NHS) ester modifications of PEG are
particularly useful since they react under mild conditions within a
relatively short period of time (30 min, pH <7.8, 25.degree. C.
(Zalipsky, S.; Seltzer, R.; Menon-Rudolph, S. S. Biotechno. Appl.
Biochem. 1992, 15, 100-114) (Rhee, W. 1997).
[0103] PEG-N-succinimidyl glutarate and PEG-N-succinimidyl
propionate were prepared as described by A. Abuchowski et. al. with
minor modification (Abuchowski, A.; Kazo, G. M.; Verhoest, C. R.,
Jr.; van Es, T.; Kafkewitz, D.; Nucci, M. L.; Viau, A. T.; Davis,
F. F. Cancer Biochem. Biophys., 1984,7,175-186). PEG was converted
to the active ester in two chemical steps. In the first step,
PEG-diacid was prepared from the PEG (3400 daltons). The second
step was dicyclohexylcarbodiimide-mediated condensation of the
PE-diacid with N-hydroxysuccinimide. (Rhee W. 1997).
Example 11
Matrix Fabrication
[0104] Matrices of CMC, CMC-N and CMC-N/PEO composites were
fabricated by loading solutions of the polymers (1.0% weight/volume
in DI water) into a 96-well tissue culture plate (100 .mu.L/well),
and were frozen at a temperature of -10.degree. C.
[0105] To determine activated partial thrombin time (APPT),
matrices were prepared by casing the polymer solutions of CMC,
CMC-N and CMC-N/PEO composites in glass tubes (16.times.125 mm; 4.0
ml per tube). The tubes were placed in a vacuum-oven and dried at a
temperature of 37.degree. C. at a pressure of 1.times.10.sup.-2
Torr for 72 hours.
[0106] To study platelet adhesion and aggregation, matrix materials
were cast onto micro cover glass plates (Van Waters & Rogers,
18 mm diameter, 2.0 ml/cm.sup.2) and then air dried. To evaluate
water uptake and the potential for stopping spurting bleeding,
matrices were prepared by freezing each polymer solution (5.0 ml,
1% weight/volume in DI water) in a 15 ml polypropylene tube and
then freeze dried.
[0107] Thrombin was incorporated by pipetting 80 .mu.L of
reconstituted thrombin solution (500 U/ml, in Tris-HCl buffer, pH
5.0) onto dried matrices. The process was performed at 4.degree. C.
to minimize loss of thrombin activity. Thrombin-loaded matrices
were then lyophilized and stored desiccated at a temperature of
-4.degree. C. for further application.
Example 12
Evaluation of Structure of CMC/PEO Composites
[0108] To evaluate the structures of CMC/PEO composites of this
invention, CMC, CMC-N and CMC-N/PEO matrices were studied using a
scanning electron microscope (SEM; model S-806, Hitachi Ltd.,
Tokyo, Japan). Dried matrix specimens were coated with a thin layer
of platinum (Pt; 15 nm) using an ion coated (Polaron SEM coating
system, Tousimis Research Corporation, Rockville, Md.) with
settings as follows: pressure: 0.5 mbar; current: 20 mA; coating
period: 60 sec. The coated specimens were examined at 15 K V
accelerating voltage.
[0109] To determine internal structure of matrices, dried matrices
were also frozen and fractured at a temperature of -78.degree. C.,
coated with Pt, then examined by SEM as described above.
[0110] Despite differences in the chemical compositions and methods
of fabrication of the matrices, all CMC-derived materials had
similar general external and internal surface morphology. They were
highly porous, having a open pore structure. All pores were
channeled with each other, resulting in a sponge-like network of
sinuses within the matrices. Structural characteristics were
created by controlling the lyophilizing conditions, because
matrices were pre-swelled in a continuous aqueous phase, which was
removed during the freeze-drying process. Although the general
morphology of the matrices have similarities, there are
differences, which may account for different properties of the
matrices.
[0111] An example of a scanning electron micrograph of a Pt-coated
matrix is shown in FIGS. 2A-2F. In FIG. 2A, the surface of a CMC
matrix has numerous pores (Magnification: 400.times.). The surface
of an CMC-N matrix is shown in FIG. 2B. In contrast, FIG. 2C shows
that the surface of a CMC-N/PEO matrix has large and irregularly
shaped pores. One theory which may account for this observation is
that there are weaker interactions between the polymers of the
CMC-N/PEO matrix compared to the relatively stronger interactions
between CMC and CMC-N polymers in those matrices without PEO (e.g.,
FIGS. 2A and 2B).
[0112] The internal structure of a CMC matrix is shown in FIG. 2D,
at a magnification of 200.times.. FIG. 2E shows the internal
structure of an CMC-N matrix, and has larger internal pores than
the corresponding CMC matrix. FIG. 2F shows the internal structure
of CMC-N/PEO matrix, which has large, irregularly shaped open
areas.
[0113] Of particular and unexpected interest was the finding that
the pure CMC matrices have the smallest overall pore size (e.g.,
see FIG. 2D), with the CMC-N matrices having an intermediate pore
size (e.g., FIG. 2E), and the CMC-N/PEO matrix having the largest
pore size.
[0114] These results indicate that matrices having smaller pore
sizes can bind materials within the matrix more tightly than
matrices having larger pore sizes. Because it one can alter pore
size by selecting conditions of polymer concentration, type of
polymer, extent of cross-linking and other factors, one can select
a matrix type that best suits the needs of the particular
application.
Example 13
Water Uptake
[0115] Swelling behavior of matrices of various compositions was
evaluated by measuring the speed and amount of water uptake. Matrix
specimens were immersed in DI water, and the time required for the
swelling to reach a steady state was recorded, and the water uptake
by each type of specimen was measured according to methods
described previously (Liu 1999). Prior to experiments, matrix
specimens were dried at 102.degree. C. at a pressure of
1.times.10.sup.-2 Torr in a vacuum-oven for 72 hours, and the
weight, Wd, was determined using an analytical balance. After
incubation in water, the adherent water was removed by placing the
wet specimens on a glass plate, tipping the plate at an angle of
60.degree. for 2 min, tapping the specimens with tissue pledgets
(Kimwipes.TM.), and the weight of the wet matrix, Ww, was recorded.
The water content was calculated and expressed as (Ww-Wd)/Ww, and
swellability, Ws/Wd.
[0116] We found significant differences among matrices having
different chemical compositions (Table 6).
6TABLE 6 Swellability of CMC-Derivatized Matrices Time required for
Matrices equilibrium (min.) (W.sub.w - W.sub.d)/W.sub.w .times.
100% W.sub.w/W.sub.d CMC 3-4 85.3 .+-. 2.6 7.1 CMC-N 2-3 89.3 .+-.
3.7 9.1 CMC-N/PEO <1 93.4 .+-. 4.3 15.1 Data presented as mean
value with standard deviation (n = 5).
[0117] Table 6 shows that matrices prepared from primary
amine-containing CMC (CMC-N) had a faster and higher water uptake
compared to matrices prepared from unmodified CMC. The increase in
swelling speed was even more pronounced for CMC-N/PEO composites,
indicating that the CMC-N/PEO composites behave better than the
others in terms of the extent of fluid uptake and the rate of fluid
uptake.
[0118] III. Hemostatic Properties of CMC-N/PEO Composites
[0119] Hemostatic properties of CMC-N/PEO composites were
determined using methods described below.
Example 14
Activated Partial Thromboplastin Time
[0120] Effects of matrices on intrinsic blood coagulation was
determined using the activated partial thromboplastin time (APTT)
test, using APTT reagent, Alexin.TM.. Samples of citrate-treated,
platelet poor plasma (PPP) (0.5 ml) each were placed in a glass
tube pre-coated with a matrix material, followed by incubation at
37.degree. C. for 3 min. To the plasma sample, we then added 0.5 ml
of the APTT reagent and the mixture incubated for an additional 3
minutes. Then, 0.5 ml of a solution of a 20 mM CaCl.sub.2 was added
to the mixture, and the time required for a clot to form was
recorded. Table 7 shows results of these studies.
7TABLE 7 Activated Partial Thromboplastin Time (APTT) Samples APTT
(sec.) CMC 32 .+-. 4 CMC-N 22 .+-. 5 CMC-N/PEO 43 .+-. 2 Glass 33
.+-. 2 Data presented as mean value with standard deviation (n =
5).
[0121] We found that CMC-N was more effective on the intrinsic
coagulation system than either CMC alone or CMC-N/PEO. Thus,
incorporation of PEO into a matrix suppressed activation of
intrinsic coagulation.
[0122] One theory to account for the results is that thrombin can
be held by the matrices, and was therefore can be unavailable to
participate in coagulation, which involves many different chemicals
and substrates in the liquid medium. By decreasing the availability
of thrombin (a protein) to the liquid medium outside the matrix,
the rates of clotting reactions may be slowed. CMC matrices contain
numerous carboxylic acid residues which may bind to thrombin. Thus,
according to this theory, with the reduction in the number of free
and available COOH groups on CMC, by either derivatization with
amines or with formation of complexes containing PEO or PEGs, less
thrombin binding can occur, thereby promoting the release of
thrombin into the liquid medium, thereby promoting clotting.
[0123] Another theory which may account for the observations is
that CMC matrices have smaller pores than those of either CMC-N or
CMC-N/PEO matrices. FIGS. 3a-3f show that PEO-containing matrices
have larger pores that can be less effective at trapping thrombin
or other molecules of similar size and physical characteristics as
thrombin.
Example 15
Effects of Matrix Materials on Platelet Aggregation, Adhesion and
Activation
[0124] Effects of matrix materials on platelet adhesion and
aggregation were determined using platelet rich plasma (PRP), which
was prepared from citrated whole bovine blood. Citrated blood was
centrifuged at 800-1000 revolutions per minute (rpm) for 10
minutes, the supernate was collected and further centrifuged at
3000 rpm for 5 minutes to obtain platelet pellet and platelet poor
plasma (PPP). The platelet pellet was dispersed in the PPP to yield
a platelet suspension containing platelets at a concentration of
30-35.times.10.sup.4/.mu.L.
[0125] Platelet aggregation initiated by matrix materials was
determined by measuring the time course of the optical density of
PRP, beginning at the time of addition of solutions of matrix
materials. Briefly, aliquots of CMC, CMC-N and CMC-N/PEG solutions
containing thrombin (100 .mu.L; 250 .mu.g; 4 U/ml PBS) was added to
2.5 ml PRP in a UV spectrophotometer tube, and the optical density
(OD) was monitored at a wavelength of 580 nm at room temperature
under constant stirring using a UV spectrometer (model 160U,
Shimadzu, Japan) equipped with a magnetic stirrer. In another
experiment, matrix materials and thrombin were tested separately
for their abilities to initiate platelet aggregation.
[0126] Platelet adhesion and activation of adherent platelets were
determined by counting the number of platelets adhered to the
surfaces of matrices and by analyzing the morphology of adherent
platelets using SEM. Plates cast from each matrix material were
incubated with PRP, one piece per 2.0 ml, in borosilicate glass
vials at a temperature of 37.degree. C. After 20 minutes, the
plates were washed with 0.1 M cacodylate buffer for a total of 3
times, then fixed by immersion in a cacodylate buffered solution
containing 2.0% glutaraldehyde for 3 hours. The fixed plates were
dehydrated in graded ethanol solutions, submitted to critical point
drying using liquid CO.sub.2 as a transition fluid, coated, then
examined by SEM for the numbers of adherent platelets and the
extent of activation.
[0127] FIGS. 3A and 3B depict results of studies of platelet
aggregation induced by matrix materials (25 .mu.g/ml) in the
presence (top graph A) and absence (bottom graph B) of thrombin (4
U/ml): CMC (.circle-solid.), CMC-N (.box-solid.), CMC-N/PEO
(.tangle-solidup.), and control (.largecircle.). Matrices were
prepared in microtiter plates. Platelets were prepared from
citrated whole bovine blood at a concentration of
30-35.times.10.sup.4/.mu.l. The vertical axis represents turbidity
of the solution, as measured by optical density measured at a
wavelength of 580 nm. As the platelets aggregate, the optical
density decreases.
[0128] FIGS. 4A-4D depict results of studies of activation of
platelets adhered on plates coated with matrix materials as
indicated in the different panels A, B, C and D. Panel A: CMC,
panel B: CMC-N, panel C: CMC-N/PEO, and panel D: control. Plates
(diameter, 18 mm) cast with each matrix were incubated with 2.0 ml
of platelet rich plasma having a platelet concentration of
30-35.times.10.sup.4/.mu.l at 37.degree. C. for 20 min.
[0129] FIGS. 4A-4D depict photomicrographs of platelets which
adhered to surfaces comprising CMC (FIG. 4A), CMC-N (FIG. 4B),
CMC-N/PEO (FIG. 4C) and control (FIG. 4D). FIG. 4D is a control and
shows a platelet having 4 or 5 pseudopods extending from the
platelet, indicating that the platelet adhered tightly to the
surface. FIG. 4A depicts a platelet adhered to a CMC matrix. As
with the control shown in FIG. 4D, this platelet has between 4 and
6 pseudopods. In contrast, the CMC-N adherent platelet (FIG. 4B)
has fewer pseudopods (2 or 3), and appear broader. Finally, the
platelet adhered to the CMC-N/PEO surface (FIG. 4C) has no
pseudopods, indicating that this platelet did not actively adhere
to the substrate. Results of these studies are shown in Table
8.
8TABLE 8 Adhesion and Activation of Platelets on CMC Derivatized
Matrices Samples Platelet adhesion (.times.10.sup.6/cm.sup.2)
Aggregation CMC 6.3 .+-. 0.7 yes CMC-N 6.9 .+-. 0.7 yes CMC-N/PEO
0.26 .+-. 0.08 no Glass 6.4 .+-. 1.7 yes Data presented as mean
value and standard deviation (n = 5).
[0130] We observed no significant differences in platelet adhesion
or activation between surfaces of glass and those pre-treated with
CMC or CMC-N. However, CMC-N/PEO compositions of this invention
showed both substantially decreased platelet adhesion and platelet
aggregation compared to CMC, CMC-N and glass alone. Additionally,
pseudopod formation by platelets was inhibited by the CMC-N/PEO
composites.
Example 16
Thrombin Activity
[0131] Thrombin activity was measured in both solution form and
after loading to matrices in solid form. To glass tubes containing
1.0 ml fibrinogen (3 mg/ml) in PBS, pH 7.0, we added either 100
.mu.L reconstituted thrombin (40 U) or a piece of
thrombin-preloaded matrix. The tubes were incubated at a
temperature of 37.degree. C., and the time for a fibrin gel to form
was measured according to the methods of Liu (Liu 1999).
Reconstituted thrombin solutions were freeze-dried under the same
conditions used to prepare thrombin-loaded matrices. The thrombin
thus treated was used as a control. Results of these experiments
are shown in Table 9.
9TABLE 9 Determination of Thrombin Activity in CMC and CMC-N
Matrices Clotting time (sec) Samples Fibrinogen/PBS Whole blood
Thrombin in the form received 36 .+-. 2 15 .+-. 6 Thrombin after
lyophilization 50 .+-. 11 23 .+-. 6 CMC pre-loaded with thrombin
201 .+-. 46 149 .+-. 32 CMC-N pre-loaded with thrombin 153 .+-. 7*
113 .+-. 11 CMC-N/PEO pre-loaded with thrombin 110 .+-. 18 107 .+-.
9 Data presented as mean value with standard deviation (n = 5); P
< 0.05; *P < 0.1 in comparing with CMC-N/PEO pre-loaded with
thrombin.
[0132] Table 9 shows that lyophilization decreased thrombin
activity slightly in aqueous solution, as reflected by the
increased clotting time compared to unprocessed thrombin. Thrombin
activity progressively decreased when it was in a matrix, with the
order of decrease: CMC-N>CMC-N/PEO>CMC.
[0133] This result indicates that thrombin is more readily adsorbed
onto CMC matrices, and is therefore less available in solution to
participate in blood clotting reactions. The CMC-N does not have as
great a binding to thrombin as CMC does, the CMC-N/PEO matrices
bind thrombin to a lesser degree. Therefore, using the compositions
of this invention, one can regulate the amount of an active agent
(e.g., thrombin or other drug) released into free solution by
selecting different compositions of a matrix.
Example 17
Whole Blood Coagulation Time
[0134] Hemostatic activity of derivatized CMC matrices were
determined by measuring the time required to form a blood clot
(thrombus time) in contact with whole, citrated bovine blood. To a
borosilicate glass tube containing 1100 L of the matrix, we added
5.0 ml citrate-treated whole blood, which was immediately followed
by the addition of 0.5 ml CaCl.sub.2 solution (0.25M), and stirred
gently, while incubated at a temperature of 37.degree. C. The time
required for a clot to form was measured. results of these studies
are shown in Table 9. As shown by Table 9, the clotting time for
each type of matrix was increased, compared to that of either
unprocessed (as received from supplier) or after lyophilization.
The order of clotting times, in increasing times was:
CMC-N>CMC-N/PEO>CMC. Thus, as with the results of thrombin
time shown in Table 8, the CMC and derivatized CMC matrices
retained thrombin compared to either of the non-matrix bound
thrombin preparations.
Example 18
Clotting of a Spurting Bleeding Model
[0135] Matrices were tested in vitro for the potential to stop
aggressive bleeding by evaluating the ability to form matrix/fibrin
networks and its ability to resist increasing pressure in a
spurting bleeding model 500 as shown in FIG. 5. Fibrinogen solution
(35 mg/ml, PBS) 506 was pre-incubated at 37.+-.2.degree. C., stored
in liquid reservoir 504, which was connected with a PVC tube (i.d.
3.5 mm) 508 through a three-way stopcock (not shown). The other end
of the tubing was attached to a circular test panel 512 with a hole
(8 mm diameter) 516 in the center. Hairless porcine skin (not
shown) was mounted on circular panel and double fastened by cable
ties (not shown). Another hole (2 mm diameter; not shown) was
created in the skin, positioned 3 mm away from the hole in the test
panel. Fibrinogen solution was released to moisten the surface
prior to each experiment. Gel sponges of matrices containing either
CMC, CMC-N, CMC-N/PEO or control, 100 mg/sponge, were moistened
with 200 .mu.l of reconstituted thrombin solution (1000 U/ml) and
placed on the top of the hole and the surrounding area. After 2
min, the stopcock was opened and the liquid reservoir was lifted
gently. The stopcock was switched back upon leakage of liquid from
the hole onto the skin, the height (h) from the exit of test panel
to the level of liquid reservoir was recorded and used to calculate
the hydrostatic pressure (expressed as mm Hg) applied to the gel
covering the hole in the porcine skin. In another experiment,
thrombin pre-loaded matrices were tested in the same method as
described above.
[0136] All experiments for the determination of the haemostatic
activity of matrices were carried out three or five times. Data
presented as mean value with standard deviation.
[0137] FIG. 6 shows the results of these experiments. FIG. 6 is a
graph of hydrostatic pressure (in mm Hg) on the vertical axis, as a
function of the type of matrix used to cover the pore in the skin.
Three sets of three bars each are shown. Each set of bars
represents results of studies using matrices containing CMC alone
(left group of bars), N-CMC (CMC-N; middle group of bars) and
N-CMC/PEO (CMC-N/PEO; right group of bars). Within each group of
bars, the left bars (open bars) represent matrix materials alone
(e.g., without thrombin). Horizontally hatched bars (middle of each
set) represent matrix materials that had been pre-loaded prior to
placing the matrix over the skin. Diagonally hatched bars (right of
each set) represent matrices onto which thrombin had been placed on
site.
[0138] In the absence of any matrix material, the thrombin solution
alone prevented bleeding to a degree (spurting threshold: about 15
mm Hg). The matrices alone (open bars) showed somewhat greater
ability to prevent spurting, having thresholds of about 45 mm HG
(CMC alone), 53 mm Hg (CMC-N alone) and about 52 mm HG (CMC-N/PEO
alone).
[0139] In contrast, the addition of thrombin to each matrix
improved the ability of that matrix to inhibit spurt bleeding.
Addition of thrombin pre-loaded into CMC matrices increased spurt
threshold to more than a factor of about 2 (e.g., to about 85 mm
Hg), and addition of thrombin in situ increased the threshold for
spurt bleeding by about 3 fold (to about 120 mm Hg). The results
were substantially greater for CMC-N matrices, with the pre-loaded
matrix increasing threshold to about 130 mm Hg and the in situ
loaded matrix having a threshold of nearly 200 mm Hg. The CMC-N/PEO
matrix increased the spurt bleeding threshold even more, with the
pre-loaded matrix having a threshold of about 170 mm Hg, and the in
situ loaded matrix having a threshold of over 200 mmHg.
[0140] These studies indicated that CMC matrices, whether
derivatized or derivatized and conjugated with PEO can increase the
hemostatic effects of thrombin substantially. Matrices of this
invention can be provided with drugs and can increase the efficacy
of hemostatic agents, either when pre-loaded into the matrix,
making their manufacture and use easy, or when loaded in situ
during surgery. Incorporation of PEO into a CMC matrix can decrease
platelet adherence and activation, and can provide a matrix from
which pharmacologically active agents may be released and have
increased local effects.
[0141] IV Drug Delivery Using Derivatized CPS
[0142] It can be readily appreciated that any number of drugs,
biologicals and other chemical agents can be delivered using the
derivatized CPS and PE composites of this invention. Certain agents
can be advantageously used for local delivery, providing desired
concentration at a desired site, but while decreasing undesirable,
systemic effects. Such agents include, but are not limited to
therapeutic proteins, such as thrombin to aid in attaining and
maintaining hemostasis, growth factors for bone, cartilage, skin
and other tissue and cell types. Some of these peptide and protein
growth factors include bone morphogenic protein (BMP), epidermal
growth factor (EGF), connective tissue growth factor (CTGF),
platelet derived growth factor (PDGF), angiotensin and related
peptides, and RGD-containing peptides.
[0143] Additionally, locally acting drugs include fungicides,
histamine, antihistamine, anti-inflammatory drugs (methotrexate),
local anesthetics, angiogenesis promoting drugs (e.g., to treat
cardiovascular disease, and anti-angiogenesis factors (e.g., to
treat tumors).
[0144] DNA-based therapeutics, including antisense DNA, gene
therapeutics and RNA-based therapeutics are also suitably delivered
using the compositions of this invention. These agents can be used
to either inhibit or promote transcription of endogenous genes, or
alternatively, can provide exogenous gene products to promote local
treatment.
[0145] Locally delivered chemotherapeutic agents can also be
delivered. These include, byway of example only, antibiotics to
treat microbial conditions, antifungal agents, antiparasitic
agents, anti-neoplastic agents including alkylating agents,
anti-metabolites and the like.
[0146] It can also be appreciated that various hormones and
steroids can be delivered, as can other, systemically acting drugs,
which can be delivered transmucosally or transdermally. These
include IgG, clotting factors and enzymes for treating
mucopolysaccharidosis or other conditions.
[0147] Cardiovascular drugs include vasodilators such as
.beta.-adrenoreceptor agonists including terbutaline and low-dose
epinephrine, .alpha.-adrenoreceptor antagonists including
norepinephrine, high-dose epinephrine and the like, and
vasodilators including nitroprusside and nitroglycerin.
[0148] Vaccines can be delivered transmucosally or
transdermally.
[0149] CMC binding therapeutics including proteins and
transcription factors), CMC-N binding therapeutics (DNA, cDNA) and
other materials capable of being associated with CMC and CMC-N and
then released from them. Lipid binding protein, lysosomal
encapsulated proteins or drugs can also be advantageously delivered
using the derivatized CPS of this invention.
REFERENCES
[0150] Liu L S, Ito Y and Imanishi Y. Synthesis and
antithrombogenicity of heparinized polyurethanes with intervening
space chains of various kinds. Biomaterials 12: 390-396, 1991.
[0151] Ito Y, Liu L S, Matsuo R and Imanishi Y. Synthesis and
nonthrombogenicity of polymer membrane with surface-grafted
polymers carrying thrombin inhibitor. Journal of Biomedical
Materials Research 26: 1065-1080, 1992.
[0152] Liu L S, Thompson A Y, Heidaran M A, Poser J W and Spiro R
C. An osteoconductive collagen/hyaluronate matrix for bone
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[0153] Rhee W, Rosenblatt J. Castro M, Schroeder J, Rao P R, Harner
C H and Berg R A. In vivo stability of poly(ethylene
glycol)-collagen composites. In: Harris J M and Zalipsky S,
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Applications. Washington, D.C.: ACS Press: 1997: pp 420-440.
[0154] All of the above cited references, and all other references
cited herein are fully incorporated by reference in their
entirety.
[0155] It can be appreciated that the above descriptions and
examples are only representative of the scope of this invention.
Other embodiments, variations and applications of the derivatized
CMCs and matrices can be used without departing from the intent and
scope of this invention. Further understanding of the scope of the
invention is found in the claims.
INDUSTRIAL APPLICABILITY
[0156] The compositions and methods of this invention are useful
for controlled drug delivery, hemostatasis and in minimizing
surgical adhesions. Derivatizing CMCs with primary amines and/or
other types of active moieties can provide improved structural
features, including interstitial pores, that can hold biologically
active materials and release them under controlled conditions.
* * * * *