U.S. patent application number 14/743754 was filed with the patent office on 2015-10-08 for polymers for reversing heparin-based anticoagulation.
The applicant listed for this patent is University of British Columbia. Invention is credited to Donald E. Brooks, Cedric J. Carter, Jayachandran N. Kizhakkedathu, Rajesh A. Shenoi.
Application Number | 20150283171 14/743754 |
Document ID | / |
Family ID | 47258202 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150283171 |
Kind Code |
A1 |
Kizhakkedathu; Jayachandran N. ;
et al. |
October 8, 2015 |
POLYMERS FOR REVERSING HEPARIN-BASED ANTICOAGULATION
Abstract
Embodiments presented herein relate to various polymers. Some of
the polymer embodiments are heparin binding polymers. Some
embodiments of the heparin binding polymers can be employed to bind
to heparin for methods such as separating, purifying, removing,
and/or isolating heparin and heparin like molecules.
Inventors: |
Kizhakkedathu; Jayachandran N.;
(New Westminster, CA) ; Shenoi; Rajesh A.;
(Vancouver, CA) ; Carter; Cedric J.; (Vancouver,
CA) ; Brooks; Donald E.; (Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of British Columbia |
Vancouver |
|
CA |
|
|
Family ID: |
47258202 |
Appl. No.: |
14/743754 |
Filed: |
June 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14098184 |
Dec 5, 2013 |
9095666 |
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14743754 |
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13458899 |
Apr 27, 2012 |
8637008 |
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14098184 |
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13504841 |
Apr 27, 2012 |
8519189 |
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PCT/CA2011/050603 |
Sep 27, 2011 |
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13458899 |
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61492299 |
Jun 1, 2011 |
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Current U.S.
Class: |
424/78.3 ;
525/409 |
Current CPC
Class: |
A61K 31/785 20130101;
C08G 2340/00 20130101; A61M 2202/0478 20130101; A61K 47/34
20130101; A61K 31/765 20130101; C08L 71/02 20130101; C08G 73/024
20130101; A61K 31/795 20130101; B01D 15/36 20130101; A61M 2202/0413
20130101; A61M 1/3679 20130101; A61M 2202/0021 20130101; A61M
1/3675 20130101; A61K 9/06 20130101; A61K 9/0014 20130101 |
International
Class: |
A61K 31/795 20060101
A61K031/795; C08G 73/02 20060101 C08G073/02; A61M 1/36 20060101
A61M001/36; A61K 31/785 20060101 A61K031/785 |
Claims
1-111. (canceled)
112. A heparin binding polymer, the polymer comprising: a first
dendritic polyol; and one or more cationic moieties attached to the
first dendritic polyol, wherein the polymer comprises either: a)
the structure of Formula (I): ##STR00010## wherein L.sub.1 is the
first dendritic polyol, L.sub.2 is a second dendritic polyol,
L.sub.3 is a third dendritic polyol, L.sub.4 is a fourth dendritic
polyol, and L.sub.5 is a fifth dendritic polyol, or b) the
structure of Formula (II): ##STR00011## wherein L.sub.1 is the
first dendritic polyol and L.sub.2 is a second dendritic polyol,
and wherein L.sub.3 is a third dendritic polyol, and L.sub.4 is a
fourth dendritic polyol.
113. The heparin binding polymer of claim 112, wherein the polymer
comprises the structure of Formula (I): ##STR00012##
114. The heparin binding polymer of claim 113, wherein at least the
first dendritic polyol comprises a polyether polyol, and wherein
the polymer is immobilized on a support.
115. The heparin binding polymer of claim 113, wherein the cationic
moiety comprises Formula (VI): ##STR00013## and wherein the heparin
binding polymer comprises an average of 4 to 100 cationic
moieties.
116. The heparin binding polymer of claim 113, further comprising a
protective moiety covalently attached to the first dendritic
polyol.
117. The heparin binding polymer of claim 116, wherein the
protective moiety comprises polyethylene glycol.
118. The heparin binding polymer of claim 117, wherein the polymer
has a polyethelene glycol content of about 75% by weight.
119. The heparin binding polymer of claim 113, wherein the one or
more cationic moieties comprises an amine group.
120. The heparin binding polymer of claim 119, wherein the amine
group is a tetra-amine group, hexa-amine group, or a deca-amine
group.
121. The heparin binding polymer of claim 119, wherein the amine
group comprises a methylated amine.
122. The heparin binding polymer of claim 119, wherein the amine
group comprises a methylated tetra-amine group, a methylated
hexa-amine group, or a methylated deca-amine group.
123. The heparin binding polymer of claim 113, wherein the first
dendritic polyol comprises a polyether polyol.
124. The heparin binding polymer of claim 123, wherein the
polyether polyol is hyperbranched.
125. The heparin binding polymer of claim 123, wherein the
polyether polyol has a degree of branching in the range of 0.05 to
0.95.
126. The heparin binding polymer of claim 125, wherein the degree
of branching in the range of 0.40-0.65.
127. The heparin binding polymer of claim 112, wherein the polymer
comprises the structure of Formula (II): ##STR00014## wherein
L.sub.1 is the first dendritic polyol and L.sub.2 is a second
dendritic polyol, and wherein L.sub.3 is a third dendritic polyol,
and L.sub.4 is a fourth dendritic polyol.
128. The heparin binding polymer of claim 127, wherein at least the
first dendritic polyol comprises a polyether polyol, and wherein
the polymer is immobilized on a support.
129. The heparin binding polymer of claim 127, wherein the cationic
moiety comprises Formula (VI): ##STR00015## and wherein the heparin
binding polymer comprises an average of 4 to 100 cationic
moieties.
130. The heparin binding polymer of claim 127, further comprising a
protective moiety covalently attached to the first dendritic
polyol.
131. The heparin binding polymer of claim 130, wherein the
protective moiety comprises polyethylene glycol.
132. The heparin binding polymer of claim 131, wherein the polymer
has a polyethelene glycol content of about 75% by weight.
133. The heparin binding polymer of claim 127, wherein the one or
more cationic moieties comprises an amine group.
134. The heparin binding polymer of claim 133, wherein the amine
group is a tetra-amine group, hexa-amine group, or a deca-amine
group.
135. The heparin binding polymer of claim 133, wherein the amine
group comprises a methylated amine.
136. The heparin binding polymer of claim 133, wherein the amine
group comprises a methylated tetra-amine group, a methylated
hexa-amine group, or a methylated deca-amine group.
137. The heparin binding polymer of claim 127, wherein the first
dendritic polyol comprises a polyether polyol.
138. The heparin binding polymer of claim 137, wherein the
polyether polyol is hyperbranched.
139. The heparin binding polymer of claim 137, wherein the
polyether polyol has a degree of branching in the range of 0.05 to
0.95.
140. The heparin binding polymer of claim 139, wherein the degree
of branching in the range of 0.40-0.65.
141. The heparin binding polymer of claim 127, wherein the heparin
binding polymer binds to heparin selected from at least one of:
unfractionated heparin (UFH), low molecular weight heparin (LMWH),
ultra low molecular weight heparin (ULMWH), fondaparinux,
idraparinux enoxaparin, dalteparin, tinzaparin, semuloparin, or
heparinoid.
142. The heparin binding polymer of claim 141, wherein the heparin
binding polymer binds to fondaparinux.
143. The heparin binding polymer of claim 127, wherein the heparin
binding polymer binds to heparin selected from at least one of:
unfractionated heparin (UFH), low molecular weight heparin (LMWH),
ultra low molecular weight heparin (ULMWH), fondaparinux,
idraparinux enoxaparin, dalteparin, tinzaparin, semuloparin, or
heparinoid.
144. The heparin binding polymer of claim 143, wherein the heparin
binding polymer binds to fondaparinux.
145. A method of counteracting heparin in a subject, the method
comprising administering a heparin binding polymer of claim 112 to
a subject, wherein the heparin binding polymer binds to heparin and
thereby counteracts heparin in the subject.
146. The method of claim 145, wherein the heparin binding polymer
further comprises polyethylene glycol covalently attached to the
heparin binding polymer.
147. The method of claim 146, wherein the cationic moiety is an
amine group.
148. The method of claim 147, wherein the amine group is a
tetra-amine group, a hexa-amine group, or a deca-amine group.
149. The method of claim 146, further comprising identifying a
subject who has received an excess amount of heparin, wherein the
identifying occurs before the heparin binding polymer is
administered to the subject.
150. The method of claim 146, wherein an excess amount of heparin
was administered to the subject.
151. The method of claim 150, wherein the excess amount of heparin
was administered for the maintenance of an intravenous
catheter.
152. The method of claim 150, wherein the excess amount of heparin
was administered to treat one or more of the following: acute
coronary syndrome, atrial fibrilllation, cardiac bypass,
extracorporeal membrane oxygenation, pancreatitis, hemofiltration,
or burns.
153. The method of claim 145, further comprising identifying a
subject that would benefit from reducing a level of exogenous
heparin that is present in the subject's blood.
154. The method of claim 153, wherein the subject will benefit from
an accelerated removal of exogenous heparin from the subject.
155. The method of claim 146, wherein the method is performed in
vivo.
156. The method of claim 146, wherein the method is performed ex
vivo.
157. The method of claim 145, wherein the subject is a human.
158. The method of claim 145, wherein the subject is a dog, a cat,
a horse, a cow, a pig, a mouse, a rat, a rabbit, a monkey, or a
guinea pig.
159. The method of claim 145, wherein the heparin binding polymer
is administered subcutaneously.
160. The method of claim 145, wherein the heparin binding polymer
is administered intravenously.
161. The method of claim 145, wherein the heparin binding polymer
binds to heparin selected from at least one of: unfractionated
heparin (UFH), low molecular weight heparin (LMWH), ultra low
molecular weight heparin (ULMWH), fondaparinux, idraparinux
enoxaparin, dalteparin, tinzaparin, semuloparin, or heparinoid.
162. The method of claim 161, wherein the heparin binding polymer
binds to fondaparinux.
163. The method of claim 150, wherein the excess amount of heparin
is selected from selected from at least one of: unfractionated
heparin (UFH), low molecular weight heparin (LMWH), ultra low
molecular weight heparin (ULMWH), fondaparinux, idraparinux
enoxaparin, dalteparin, tinzaparin, semuloparin, or heparinoid.
164. The method of claim 163, wherein the excess amount of heparin
is fondaparinux.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/492,299, filed Jun. 1, 2011, hereby
incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present embodiments relates to novel polymers that bind
to heparin, heparin derivatives and heparinoids and methods for
making such polymers.
BACKGROUND
[0003] Heparin is an anionic compound involved in a variety of
biological processes including blood coagulation. Heparin
derivatives used in current clinical anticoagulation therapy
include unfractionated heparin (UFH), low molecular weight heparin
(LMWH), ultra low molecular weight heparin (ULMWH) and the
synthetic pentasccharide derivatives fondaparunix and
idraparinux.
[0004] Heparinoids can be naturally occurring and synthetic
highly-sulfated polysaccharides of similar structure to heparin.
Heparinoid preparations have been used for a wide range of
applications including as anticoagulant and anti-inflammatories and
they have been claimed to have hypolipidemic properties.
[0005] Heparin neutralization, which can be desired when a subject
is given too much heparin, can be achieved, for example, via
protamine or through filtration of the blood through an
extracorporeal device.
SUMMARY
[0006] Some embodiments provided herein relate to heparin binding
polymers. In some embodiments, the polymer has a first dendritic
polyol and one or more cationic moieties attached to the first
dendritic polyol.
[0007] In some embodiments, a heparin binding device is provided.
The device can have a support and a heparin binding polymer
immobilized on the support. The heparin binding polymer can have a
first dendritic polyol and one or more cationic moieties attached
to the first dendritic polyol.
[0008] In some embodiments, a method of making a heparin binding
polymer is provided. The method can include combining a glycidol
and a PEG-epoxide, methylating the glycidol and the PEG-epoxide to
form a hyperbranched polyglycerol, tosylating the hyperbranched
polyglycerol to form a tosylated hyperbranched polyglycerol, and
coupling the tosylated hyperbranched polyglycerol with at least one
amine group, thereby making a heparin binding polymer.
[0009] In some embodiments, a method of counteracting heparin in a
subject is provided. The method can include administering a heparin
binding polymer to a subject. The heparin binding polymer can
include a first dendritic polyol and one or more cationic moieties
attached to the first dendritic polyol. The heparin binding protein
binds to heparin and thereby counteracts heparin in the
subject.
[0010] In some embodiments, a method of processing a subject's
blood is provided. The method can include providing a heparin
binding device. The device can include a support and a heparin
binding polymer immobilized on the support. The heparin binding
polymer can include a first dendritic polyol and one or more
cationic moieties attached to the first dendritic polyol. The
method can further include withdrawing blood from a subject,
contacting the blood to the heparin binding polymer, and returning
at least part of the blood to the subject, thereby processing the
subject's blood.
[0011] In some embodiments, a method of concentrating heparin is
provided. The method can include providing a support and a heparin
binding polymer immobilized on the support. The heparin binding
polymer can include a first dendritic polyol and one or more
cationic moieties attached to the first dendritic polyol. The
method can further include contacting a first fluid including
heparin with the heparin binding polymer and flowing the fluid off
of the heparin binding polymer. This can thereby concentrate
heparin.
[0012] In some embodiments, a heparin binding composition is
provided. The composition can include a first heparin binding
polymer having a first dendritic polyol and one or more cationic
moieties attached to the first dendritic polyol. The composition
can further include a pharmaceutically acceptable carrier.
[0013] In some embodiments, a heparin binding macromolecule is
provided. The macromolecule can include a hyperbranched
polyglycidol core, at least one polyvalent cation attached to the
hyperbranched polyglycidol core, and at least one protective moiety
attached to the hyperbranched polyglcidol core.
[0014] In some embodiments, a heparin composition is provided. The
composition can include heparin and a heparin binding polymer. The
heparin binding polymer can include a first dendritic polyol and
one or more cationic moieties attached to the first dendritic
polyol.
[0015] In some embodiments, a kit is provided. The kit can include
a first container including heparin and a second container
including a heparin binding polymer. The heparin binding polymer
can include a first dendritic polyol and one or more cationic
moieties attached to the first dendritic polyol.
[0016] In some embodiments, the heparin binding polymer can be used
for binding and drug delivery of anionic drug molecules such as
heparins, synthetic pentasaccharide anticoagulants, low and ultra
low molecular weight heparins or methatrexate or similar compounds.
The foregoing summary is illustrative only and is not intended to
be in any way limiting. In addition to the illustrative aspects,
embodiments, and features described above, further aspects,
embodiments, and features will become apparent by reference to the
drawings and the following detailed description.
[0017] In some embodiments, a controlled release delivery device is
provided. The device can include a surface and a heparin binding
polymer attached to the surface. The heparin binging polymer can
include a first dendritic polyol and one or more cationic moieties
attached to the first dendritic polyol. The controlled release
delivery device can also include heparin. In some embodiments, the
heparin is bound to the heparin binding polymer.
[0018] In some embodiments, a controlled release heparin
composition is provided. The composition can include a heparin
binding polymer. The heparin binding polymer can include a first
dendritic polyol and one or more cationic moieties attached to the
first dendritic polyol. The controlled release heparin composition
can also include heparin. In some embodiments, the heparin is bound
to the heparin binding polymer.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1A depicts a schematic representation of some
embodiments of a heparin binding synthetic polyvalent cationic
macromolecule with different amine groups (R=tetra-amine (4
nitrogens), hexa-amine (6 nitrogens) and deca-amine (10
nitrogens).
[0020] FIG. 1B depicts various forms of heparin.
[0021] FIG. 2A is a depiction of some embodiments for the synthesis
of heparin and heparin-derivative binding polymers with high charge
density.
[0022] FIG. 2B is a depiction of some embodiments of the synthesis
of heparin and heparin-derivative binding polymer with high charge
density. The embodiment employs MPEG-400 and an amine with four
nitrogen atoms.
[0023] FIG. 3 is a depiction of an embodiment of the design of
heparin binding synthetic polyvalent cationic macromolecule
(HBSPCM).
[0024] FIGS. 4A and 4B are graphs demonstrating heparin binding
synthetic polyvalent cationic macromolecule JNK-1 neutralization of
A) unfractionated heparin (UFH) B) LMWH (Tinzaparin) in human blood
under in vitro conditions. Protamine neutralization is given as a
comparison.
[0025] FIGS. 5A-5D are graphs depicting the percentage of
unfractionated heparin (UFH) and low molecular weight heparin
(LMWH) neutralization calculated from an activated partial
thromboplastin time (APTT) assay by heparin binding synthetic
polyvalent cationic macromolecule JNK-1 and protamine.
[0026] FIGS. 6A-6D are graphs depicting heparin binding synthetic
polyvalent cationic macromolecule JNK-1 neutralization of
unfractionated heparin (UFH) and low molecular weight heparin
(LMWH) Tinzaparin using a thromboelastograph (TEG) as measured by a
thromboelastograph in human whole blood.
[0027] FIGS. 7A-7B are graphs depicting heparin binding synthetic
polyvalent cationic macromolecule JNK-1 neutralization of
unfractionated heparin (UFH) and low molecular weight heparin
(LMWH) Tinzaparin using thromboelastograph (TEG) in terms of
coagulation time and clot strength in human whole blood.
[0028] FIGS. 8A-8C are graphs depicting heparin binding synthetic
polyvalent cationic macromolecule JNK-1 neutralization of
unfractionated heparin (UFH) and low molecular weight heparin
(LMWH) Tinzaparin in rats.
[0029] FIG. 8D is a graph depicting in vivo UFH and LMWH (by
HBSPCM-3) neutralization in rats.
[0030] FIG. 9 is a graph depicting electrostatic charge
neutralization of unfractionated heparin (UFH) and low molecular
weight heparin (LMWH) by heparin binding synthetic polyvalent
cationic macromolecule JNK-1.
[0031] FIG. 10A is a bar graph depicting complement activation upon
interaction of heparin binding synthetic polyvalent cationic
macromolecule JNK-1 with platelet poor plasma.
[0032] FIG. 10B is a bar graph depicting platelet activation upon
interaction of JNK-1 with platelet rich plasma at 37 C.
[0033] FIGS. 11A and 11B are bar graphs depicting the effect of
heparin binding synthetic polyvalent cationic macromolecule JNK-1
on prothrombin time (PT) and activated partial thromboplastin time
(APTT) in platelet poor plasma (PPP).
[0034] FIGS. 12A-12D depict optical micrographs of human red blood
cells after 1 hour incubation with heparin binding synthetic
polyvalent cationic macromolecule JNK-1 at different concentrations
in whole blood at 37.degree. C.
[0035] FIG. 13 is a synthetic scheme for disulfide-containing
heparin binding synthetic polyvalent cationic macromolecule
(HBSPCM).
[0036] FIG. 14 is a depiction of a structure of a ketal
group-containing heparin binding synthetic polyvalent cationic
macromolecule (HBSPCM).
[0037] FIG. 15 is a bar graph depicting a blood compatibility
analysis (activated partial thromboplastin time (APTT)
biocompatibility) of HBSPCM polymers JNK-3 through JNK-8-control as
compared to protamine and saline.
[0038] FIG. 16 is a graph depicting blood compatibility analysis
using thromboelastograph (TEG) in terms of coagulation time and
kinetics parameter of HBSPCM polymers JNK-3 through JNK-8-control
as compared to protamine and saline.
[0039] FIGS. 17A and 17B are photos and bar charts depicting red
blood cell lysis after incubation with HBSPCM polymers JNK-3
through JNK-8 as compared to protamine and saline.
[0040] FIGS. 18A-18C are graphs depicting the neutralization of
unfractionated heparin (FIG. 18A), Tinzaparin (FIG. 18B) and
Enoxaparin (FIG. 18C) by HBSPCM polymers JNK-1 through JNK-8 as
compared to protamine in human blood in vitro.
[0041] FIG. 19 is a graph depicting the neutralization of low
molecular weight heparin using a thromboelastograph (TEG) in human
whole blood by HBSCPM polymers JNK-5 and JNK-6.
[0042] FIG. 20 is a bar chart depicting complement activation by
the HBSPCM polymers JNK-3 to JNK-8 in comparison to protamine and
saline.
[0043] FIGS. 21A and 21B are graphs depicting the ability of HBSPCM
polymer JNK-3 to neutralize unfractionated heparin (FIG. 21A) and
Exonaparin (FIG. 21B) in living rats.
[0044] FIG. 22 is a graph plotting body weight over time (0 to 29
days post injection) of female BALB/c mice treated with HBSPCM
polymer JNK-3 by bolus intravenous injection.
[0045] FIG. 23 is a graph depicting LDH activity in serum of female
BALB/c mice treated with HBSPCM polymer JNK-3 after a bolus
intravenous injection.
[0046] FIGS. 24A-24C are graphs depicting neutralization of
unfractionated heparin (UFH) (FIG. 24A), Tinzaparin (FIG. 24B) and
Enoxaparin (FIG. 24C) by HBSPCM polymers JNK-9, JNK-10 and JNK-11
as compared to protamine in human plasma in vitro.
[0047] FIG. 25 is a bar graph depicting blood compatibility
(complement activation) of HBSPCM and the effect of the number of
charges on the polymer.
[0048] FIG. 26 is a bar graph depicting blood compatibility
(platelet activation) of HBSPCM and the effect of the number of
charges on the polymer.
[0049] FIG. 27 is a graph depicting the tolerance of mice to HBSPCM
(JNK-1)
[0050] FIG. 28 is a graph depicting the tolerance of mice to
protamine.
[0051] FIG. 29A and FIG. 29B are graphs depicting the
bio-distribution and pharmacokinetics of HBSPCM-3 in BALB/c
mice.
[0052] FIGS. 30A, 30B, 30C, and 30D are graphs depicting the
bio-distribution and pharmacokinetics of HBSPCM-3 in BALB/c
mice.
[0053] FIGS. 31A, 31B, 31C, and 31D are graphs depicting the
bio-distribution and pharmacokinetics of HBSPCM-1 in BALB/c
mice.
[0054] FIGS. 32A and 32B are graphs depicting the bio-distribution
and pharmacokinetics of HBSPCM-1 in BALB/c mice.
DETAILED DESCRIPTION
[0055] A complication and health concern associated with the use of
heparin and its derivatives for anticoagulation therapy is
bleeding. Antidotes are needed to reverse the effects of heparin or
its derivatives to avoid bleeding complications while maintaining
sufficient levels of the anticoagulant to prevent thrombosis. Apart
from bleeding complications, neutralization of anticoagulant drugs
following major surgical procedures can be useful to prevent
postoperative complications.
[0056] The only antidote available for UFH approved by the U.S.
Food and Drug Administration (FDA) is protamine. There are numerous
disadvantages to using protamine as a heparin antidote, including
its narrow window for therapeutic dosage, poorer outcomes for blood
transfusions and the lack of a reliable method for the
quantification of protamine in blood. Protamine is associated with
a number of anaphylactic adverse drug reactions (Horrow et al. 1985
Anesth Analg. 64:348-61; Kimmel et al. 1998 J Clin Epidemiol.
51:1-10).
[0057] Biospecific polybases, including polypeptides, synthetic
peptides and cationic polymers, can bind and neutralize heparin and
heparin derivatives to overcome the disadvantages of protamine and
other antidotes. As disclosed herein, the efficiency of
neutralization of heparin and heparin derivatives depends on the
net charge, charge density and molecular structure of
polybases.
[0058] Provided herein are various molecules and compositions that
can be used for binding to heparin. It has been discovered that a
class of biocompatible hyperbranched polyol polymers have the
ability to bind to heparin and heparin derivatives and, in some
embodiments, to act as an antidote to heparin-based anticoagulation
therapy. In some embodiments, the molecules are hyperbranched
polyether polyol polymers. It is understood by the present
inventors that this new use is distinct from other uses of
hyperbranched polyether polyols, including their use as carriers,
drug delivery vehicles or modulating an energy substrate. In some
embodiments, the molecules include a dendritic polyol attached to
one or more cationic moieties. As described in greater detail
below, in some embodiments, such molecules can be employed to
purify and/or isolate heparin, among other uses.
[0059] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. The illustrative embodiments
described in the detailed description, drawings, and claims are not
meant to be limiting. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented herein. It will be readily understood
that the aspects of the present disclosure, as generally described
herein, and illustrated in the Figures, can be arranged,
substituted, combined, separated, and designed in a wide variety of
different configurations, all of which are explicitly contemplated
herein.
[0060] The present disclosure provides a general set of definitions
(which can include various embodiments) of terms that are relevant
to some of the embodiments. The disclosure then provides a detailed
set of embodiments and examples. While various sections of the
disclosure include headings, these headings are not to be
interpreted in a limiting manner, and are present merely for the
sake of convenience.
DEFINITIONS AND EMBODIMENTS
[0061] Any terms not directly defined herein shall be understood to
have all the meanings commonly associated with them as understood
within the art. Certain terms are discussed below, or elsewhere in
the specification, to provide additional guidance to the
practitioner in describing the devices, methods and the like of
various embodiments, and how to make or use them. It will be
appreciated that the same thing may be said in more than one way.
Consequently, alternative language and synonyms may be used for any
one or more of the terms discussed herein. No significance is to be
placed upon whether or not a term is elaborated or discussed
herein. Some synonyms or substitutable methods, materials and the
like are provided. Recital of one or a few synonyms or equivalents
does not exclude use of other synonyms or equivalents, unless it is
explicitly stated. Use of examples in the specification, including
examples of terms, is for illustrative purposes only and does not
limit the scope and meaning of the embodiments herein.
[0062] As used herein, a "HPG polymer" refers to a hyperbranched
polyether polyol, including hyperbranched polyglycerol (HPG) and
heparin-binding synthetic polyvalent cationic macromolecules
(HBSPCMs). Various methods for synthesizing and modifying HPG
polymers are described herein, and in the literature. Initiating
species can include polyols and polyamine. In some embodiments, HPG
polymers can be further derivatized with alkyl groups, polyethylene
glycol groups, amine groups, sulfate groups, carbohydrates,
peptides, amino acids and the like.
[0063] As used herein, a "HBSPCM polymer" or "HBSPCM" refers to a
heparin-binding synthetic polyvalent cationic macromolecule.
Various methods for synthesizing and modifying HBSPCM polymers are
described herein. In some embodiments, the core of the HBSPCM
polymer can include a HPG polymer. The core of the HBSPCM can be
capped with short chain polyethylene glycols (PEGs) to create a
macromolecule.
[0064] The term "heparin binding polymer" is generic to both "HPG
polymer" and "HBSPCM polymer".
[0065] "Subject", as used herein, refers to an entity to receive
the molecule, compound, treatment, etc. In some embodiments, the
subject is a human patient, human test subject, a primate, or other
mammal, such as a rat, mouse, dog, cat, cow, pig, sheep, monkey or
the like.
[0066] As used herein, a "pharmaceutically acceptable excipient"
includes any and all solvents, dispersion media, coatings,
antibacterial, antimicrobial or antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. The excipient may be suitable for intravenous or
intraarterial administration. The excipient can include sterile
aqueous solutions or dispersions for extemporaneous preparation of
sterile injectable solutions or dispersion. Examples of sterile
aqueous solutions include saline, Ringer's lactate or other
solutions as may be known in the art. Use of such media in the
preparation of a medicament is known in the art.
[0067] As used herein, a "pharmacologically effective amount" of a
medicament refers to using an amount of a medicament present in
such a concentration to result in a therapeutic level of drug
delivered over the term that the drug is used. This can be
dependent on mode of delivery, time period of the dosage, age,
weight, general health, sex and diet of the subject receiving the
medicament.
[0068] In some embodiments, HPG polymers can be administered to a
subject to neutralize the effect of heparin or heparin derivatives
on the subject. As used herein, the term "neutralize" includes, in
its various grammatical forms (e.g., "neutralized",
"neutralization", "neutralizing", etc.), binding to in any
capacity, immobilize, counteract, change, alter, modify.
[0069] The term "heparin" as used herein is generic to both
traditional, naturally occurring forms of heparin, as well as
heparin derivatives and/or artificial forms of heparin. Heparin is
a glycosaminoglycan and acts as an anticoagulant. The molecule has
a negative charge density. Some forms of heparin have average
molecular weights from 2 kDa to 30 kDa, such as between 12 kDa and
15 kDa. FIG. 1B depicts some embodiments of heparin. Low molecular
weight embodiments of heparin include enoxaparin, dalteparin,
fondaparinux, tinzaparin and ultra low molecular weight embodiments
of heparin include semuloparin. Heparin sulfate is included within
the genus of the term "heparin".
[0070] LMWH is enzymatically or chemically modified UFH with high
bioavailability, predictable pharmacokinetics and dose-response and
therefore has a lower risk of bleeding complications. Fondaparinux
and idraparinux are synthetic versions of the pentasaccharide
moiety responsible for anti-factor Xa activity in UFH and LMWH.
However, the anticoagulant activity of LMWHs, fondaparinux and
idraparinux cannot be fully neutralized with a currently available
antidote (Makris et al. 2000 Br J Haematol. 108:884-5). Ultra low
molecular weight heparin such as semuloparin has high anti-factor
Xa and residual anti-factor II a activities and is currently under
development for treatment of venous thromboembolism (VTE) and
Cancer therapy.
[0071] The term "polyol" as used herein denotes an alcohol
including two or more hydroxyl groups. This includes diols, triols,
tetrols, etc.
[0072] The term "dendritic" when used in regard to polymers denotes
a branching, tree-like structure (see, e.g., FIG. 1A or FIG. 2A).
In some embodiments, dendritic polymers can have a large number of
end functional groups and have low melt and solution
viscosities.
[0073] The term "cationic moiety" denotes a chemical moiety that
has a positive charge.
[0074] The term "protective moiety" denotes a chemical moiety that
can assist in reducing non-specific interactions between a
polyglycerol core and/or polyvalent cationic groups and chemicals
other than heparin. Examples include, for example, polyethylene
glycol.
[0075] The term "polyethylene glycol" or "PEG" denotes an oligomer
and/or polymer of ethylene oxide and encompasses, for example,
polyethylene oxide (PEO) and polyoxyethylene (POE).
[0076] The term "polyether polyol" denotes a polyol that has the
ether functional group in the chain.
[0077] The term "hyperbranched" in reference to polymers denotes a
polymer having 3 or more chain ends. In some embodiments,
hyperbranched polymers are not perfectly branched. In some
embodiments, hyperbranched polymers can be prepared in a one-step
procedure. In some embodiments, hyperbranched polymers are
molecular constructs having a branched structure, generally around
a core. In some embodiments, their structure generally lacks
symmetry, the base units or monomers used to construct the
hyperbranched polymer can be of diverse nature and their
distribution is non-uniform. In some embodiments, the branches of
the polymer can be of different natures and lengths. In some
embodiments, the number of base units, or monomers, can be
different depending on the different branching. In some
embodiments, while at the same time being asymmetrical,
hyperbranched polymers can have: a branched structure, around a
core, successive generations or layers of branching, and a layer of
end chains. In some embodiments, the chain ends can be connected to
cationic moieties and/or protective moieties. In some embodiments,
there are three to 100,000 chain ends, e.g., 3-50,000, 3-20,000,
3-10,000, 3-5,000, or 100-1000.
[0078] The term "cleavage site" denotes a bond that can be
selectively cleaved over the remaining section of the polymer
backbone. Cleavage sites include, for example, disulfide bonds and
ketal groups (see, e.g., FIG. 13. and FIG. 14).
[0079] The term "ketal" group denotes a compound that includes the
general Formula: Ia:
##STR00001##
R.sup.1 and R.sup.2 can be an alkyl or aryl.
[0080] An "amine" denotes an organic compound and/or functional
group that include a basic nitrogen with a lone pair.
[0081] The term "alkyl," as used herein, means any unbranched or
branched, substituted or unsubstituted, saturated hydrocarbon, with
C.sub.1-C.sub.12 unbranched, saturated, unsubstituted hydrocarbons,
including, e.g., methyl, ethyl, isobutyl, and tert-butylpropyl, and
pentyl.
[0082] The term "alkoxy" refers to any unbranched, or branched,
substituted or unsubstituted, saturated or unsaturated ether,
including C.sub.1-C.sub.6 unbranched, saturated, unsubstituted
ethers. Dimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl
ethers also included. The term "cycloalkoxy" refers to any
non-aromatic hydrocarbon ring, including those having five to
twelve atoms in the ring.
[0083] The "number average molecular weight" or "Mn" is the
ordinary arithmetic mean or average of the molecular weights of the
individual macromolecules. It can be determined by measuring the
molecular weight of n polymer molecules, summing the weights, and
dividing by n.
[0084] The phrase "degree of pegylation" denotes the fraction of
the total end-functional (hydroxyl) groups of the macromolecule
that has been modified with polyethylene glycol (PEG).
[0085] A glycidol is an organic compound that includes both an
epoxide and alcohol functional groups linked by a methylene
(CH.sub.2) group or (CH.sub.2).sub.n where n=2-10.
[0086] The term "hydrodynamic radius" denotes a theoretical
hydrodynamic radius R.sub.hyd. R.sub.hyd is defined by:
1 R hyd = def 1 N 2 i .noteq. j 1 r ij ##EQU00001##
[0087] where r.sub.ij is the distance between subparticles i and j,
and where the angular brackets . . . represent an ensemble
average.
General Embodiments
Heparin Binding Molecules
[0088] In some embodiments, the heparin binding molecule is a
heparin binding polymer. In some embodiments, the heparin binding
polymer can include a first dendritic polyol and one or more
cationic moieties attached to the first dendritic polyol. In some
embodiments the heparin binding polymer can include a first linear
polyol and one or more cationic moieties attached to the first
linear polyol.
[0089] In some embodiments, the heparin binding polymer can further
include a protective moiety covalently attached to the first
dendritic polyol. In some embodiments, the protective moiety
includes polyethylene glycol (e.g., as shown in FIG. 1A). In some
embodiments, a new layer of polyglycerol can be a protective
moiety. In some embodiments, the protective moiety can be a
biocompatible polymer such as polyvinyl pyrolidone, carbohydrate
polymers, polypeptides, linear polyglycerol, polymers and
copolymers of PEG acrylates and methacrylates, PEG acrylamide and
methacrylamides
[0090] In some embodiments, the heparin binding polymer is a
polyether polyol. In some embodiments, the heparin binding polymer
is a carbohydrate polymers, linear polyglycerol, polypeptides,
branched or linear polymers and copolymers of PEG acrylates and
methacrylates, PEG acrylamide and methacrylamides.
[0091] In some embodiments, the polyether polyol can be
hyperbranched. In some embodiments, the degree of branching in the
range of 0.0 to 0.95, for example, in the range of 0.0, 0.05, 0.1
to 0.9, 0.2-0.7, 0.4-0.65, or about 0.5. In some embodiments, the
heparin binding polymer is a hyperbranched polyglycerol or a linear
polyglycerol.
[0092] In some embodiments, the heparin binding polymer can include
a polyether polyol, which can be attached to a support. In some
embodiments, the heparin binding polymer is attached to the support
via a disulfide bond. In some embodiments, the polymer is attached
to the support via an acetal bond or a ketal bond. The support can
be any material to which the molecule can be attached. In some
embodiments, the support can be a bead or a flat surface.
[0093] In some embodiments, the heparin binding polymer includes a
core unit that includes a C.sub.1-18 alkyl substituted with two or
more of --OR.sup.1. In some embodiments, each R.sup.1 is
independently selected from at least one of: a hydrogen, a cationic
moiety, and a polymer segment having monomer units represented by
Formula (III):
##STR00002##
n can be an integer from 1 to 10,000. Each R.sup.2 can be
independently selected from: a hydrogen, carbon, a cationic moiety,
R.sup.1, and a polymer segment represented by Formula (IV):
##STR00003##
m can be an integer from 1 to 10,000
[0094] Each R.sup.3 can be independently selected from: an oxygen
cationic moiety, a hydroxyl, and a polymer segment represented by
Formula (IV). m can be an integer from 1 to 10,000 and each R.sup.4
can be a C.sub.1-6 alkoxy.
[0095] In some embodiments, the core unit is represented by Formula
(V):
##STR00004##
[0096] In some embodiments, the cationic moiety includes Formula
(VI):
##STR00005##
wherein the heparin binding polymer includes an average of 4 to 250
cationic moieties. In some embodiments, the heparin binding polymer
includes an average of 1, 2, 4, 5, 16, 20, 24, 23, 8, 11, 16, or 36
cationic moieties. In some embodiments, the polymer segment can be
represented by Formula (IV):
##STR00006##
and includes a polyethylene glycol having a size of 400 Da.
[0097] In some embodiments, the number average molecular weight
(Mn) of the polymer segment represented by Formula (IV) is about
400 Da. In some embodiments, the average molecular weight (Mn) of
the polymer segment represented by Formula (IV) is between 50 and
40,000 Da, for example 100 to 800, 200 to 700, 300 to 600, 300 to
500, or 350 to 450.
[0098] In some embodiments, the heparin binding polymer has a
degree of PEGylation that is from about 5% to about 95%. In some
embodiments, the polymer has a degree of PEGylation that is from
about 20% to about 40%.
[0099] In some embodiments, the cationic moiety is selected from at
least one of the group of: Formula (VI), Formula (VII), and Formula
(VIII)
##STR00007##
[0100] In some embodiments, the heparin binding polymer is at least
one of: JNK-4, JNK-5, JNK-6, JNK-3, JNK-7, JNK-8, JNK-9, JNK-10,
JNK-11, and any combination thereof
[0101] In some embodiments, the heparin binding molecule is a
hyperbranched polyether polyol, which can be a hyperbranched
polyglycerol (HPG).
[0102] In some embodiments, the hyperbranched polyether polyol
polymer structure is a synthetic polycationic dendritic polymer
based on HPG capped with short chain polyethylene glycols (PEGs) to
create a macromolecule.
[0103] In some embodiments, the hyperbranched polyether polyol can
further include one or more tetra (4 nitrogens) amine groups. In
some embodiments, the hyperbranched polyether polyol can include
one or more hexa (6 nitrogens), octa (8 nitrogens), or deca (10
nitrogens) amine groups.
[0104] In some embodiments, the hyperbranched polyether polyol can
be of varying molecular weights of varying charge to optimize the
reversal of anticoagulation due to a specific heparin species, such
as low molecular weight heparin, or to otherwise optimize the
administration of the polymer to a subject.
[0105] In some embodiments, the heparin binding polymer is the
polymer shown in FIG. 1A. In some embodiments, the heparin binding
polymer shown in FIG. 1A lacks the PEG groups in FIG. 1A (and thus
would not be a "macromolecule" as the term is used herein). In some
embodiments, one can employ a polymer that lacks PEG for heparin
neutralization. In some embodiments, the heparin binding polymer is
part of a macromolecule (as shown in FIG. 1A and FIG. 3).
[0106] In some embodiments, the heparin binding polymer has one or
more R groups that includes 4, 6, 8, or 10 nitrogens. In some
embodiments, each R group of the heparin binding polymer has 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
22, 25, 30 or more nitrogens, including any range defined between
any two of the preceding values and any range greater than any one
of the preceding values.
[0107] In some embodiments, all of the R groups for a particular
heparin binding polymer are the same. In some embodiments, a single
heparin binding polymer can have different types and/or sizes of R
groups. In some embodiments, a single heparin binding polymer can
have two or more of 4N, 6N, 8N, and 10N R groups. In some
embodiments, the R groups are cationic groups. In some embodiments,
the spacer between the nitrogen atoms in the R groups can be
(CH.sub.2).sub.n where n=1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10) or the spacer can have a cyclic aliphatic structure.
[0108] In some embodiments, heparin binding polymers and/or
hyperbranched polyglycidols (HPGs) can be synthesized using various
methods known in the art (Kainthan et al. 2006 Biomacromolecules.
7(3):703-709; Kainthan et al. 2007 Biomaterials. 28(32):
4779-4787). In some embodiments, the heparin binding polymers
and/or hyperbranched polyglycidol polymer is a heparin-binding
synthetic polyvalent cationic macromolecule (HBSPCM), e.g., as
shown in FIG. 1A. In such embodiments, the HBSPCM can incorporate
multiple high binding cationic units (R) attached on a dendritic
HPG core, and can be capped with short chain polyethylene glycols
(PEGs) to generate a polyvalent cationic macromolecule, as shown in
FIG. 1A. In some embodiments, the cationic unit (R) is a methylated
tetramine with four cationic amine groups per cationic units (R=4N)
and will produce high binding affinity for heparin and heparin
derivatives. FIG. 3 is a graphical interpretation of one embodiment
with a HPG core on which polyvalent cationic groups are attached
(C) and capped with a protective barrier to non-specific
interactions. In some embodiments, the protective barrier is
composed of short chain PEGs of approximately 400 Da each. The
presence of a protective layer of short chain PEGs on the HBSPCM
makes it highly biocompatible.
[0109] In some embodiments, the hydroxyl groups on the dendritic
HPG core will be used for adding cationic units to the HBSPCM. The
number of hydroxyl group is equal to degree of polymerization;
e.g., each 74 Da molecular weight will have one hydroxyl group. The
design of HBSPCM enables multiple embodiments including, but not
limited to, changing the number of cationic units, changing the
size of the HPG core (molecular weight) while keeping the PEG chain
length constant, and changing the number of PEG chains attached to
HPG core.
[0110] In some embodiments, the HPG core capped with PEGs is
modified in a controlled manner by the covalent attachment of
multi-valent cationic groups through a three step synthetic method
involving tosylation of hydroxyl groups, coupling with amines (or
other cationic groups), and methylation as shown in FIG. 2A and/or
2B. Possible amine groups include, but are not limited to,
tetra-amine, hexa-amine, octa-amine, deca-amine groups, and any
other amine groups disclosed herein. The HBSPCM properties such as,
but not limited to, molecular weight, molecular weight
distribution, PEG content, charge density and plasma residence time
can be controlled by methods known in the literature, such as, but
not limited to, the anionic ring opening polymerization of glycidol
and MPEG-epoxide and similar methods (Kainthan et al. 2006
Biomacromolecules. 7(3):703-709; Nair et al. 2007 Prog Polymer Sci.
32(8-9):762-798; Nisha et al. 2004 Langmuir. 20(6):2386-2396; Nisha
et al. 2004 Langmuir. 20(20):8468-8475; Kizhakkedathu et al. 2005
Macromol. Biosci. 5(6):549-558).
[0111] Examples of synthesized HBSPCMs are shown in Tables 1, 2,
and 3. Two embodiments, JNK-1 (116 000 Da) and JNK-2 (48 000 Da),
have 69 and 28 cationic groups (R; 4 charges/cationic group),
respectively. The hydrodynamic radii of these molecules are in the
range of 4 to 10 nm with a PEG content of approximately 75% by
weight.
[0112] In some embodiments, the interactions between cationic and
anionic charges are maximized due to the dendritic nature of the
HPG core and PEG caps (maximum surface exposure of charges and
multivalent effect). This characteristic has advantages over linear
polymers, which cannot maximize surface exposure of charges.
[0113] In some embodiments of the technology, biodegradable HBSPCMs
are synthesized for optimal clearance of HBSPCM and its complexes
from the vascular system and from the body. These HBSPCMs will have
precise degradation points. Degradation points can be constructed
from one or more elements from the following categories including,
but not limited to, disulphide bonds (FIG. 13) and ketal groups
(FIG. 14). In one embodiment, the disulfide initiator is prepared
by reaction of octa-amine and 2 hydroxy, 2'-carboxy ethylene
disulfide (FIG. 14). Since the disulfde bonds are cleaved by
glutathione inside the cell, it will stable in vascular system. In
another embodiment, ketal groups are cleaved by the pH in endosomes
in the cell. Since ketal groups are stable at physiological pH and
above, this embodiment of the HBSPCM will be stable in blood as
well as during the chemical modification.
TABLE-US-00001 TABLE 1 CHARACTERISTICS OF JNK-1 AND JNK-2 HBSPCM
POLYMERS No. cationic Molecular units (R) of Average Zeta Kb C50
Blood weight 4 amines no. of potential (M-1) (nM) Rh compatibility
Sample (Da) each charges (mV) [1] [1] (nm) at 1 mg/ml JNK-1 116 700
69 276 11.4 .+-. 2.9 1 108 10 10 excellent JNK-2 48 000 28 112 11.1
.+-. 2.1 ND ND 4 excellent Protamine ~4500 -- 21 ND ND ND ND poor
[3] Tetramine 146 -- 4 ND 3.6 105 2788 ND poor (spermine) PEG-based
1.8 106 -- ND ND 1 104 100000 ND ND linear cationic polymer [4] PEG
= polyethylene glycols. Rh = hydrodynamic radius. ND = no data. [1]
Binding affinity of polymers for ctDNA (a highly anionic
bio-macromolecule) in buffered water (pH 7.2) in the presence of 50
mM NaCl at 25.degree. C. [3] Showed complement activation, platelet
activation, behaved like an anticoagulant, showed massive
hemolysis. [4] Nisha et al. 2004 Langmuir. 20(6): 2386-2396.
[0114] In some embodiments, the binding affinity and charge density
of HBSPCMs can be increased by incorporating hexa amine cationic
units with six nitrogens per unit instead of tetramine cationic
units (FIG. 1). In some embodiments deca amine cationic units with
10 nitrogens per unit instead of tetramine units (FIG. 1) are
provided. In some embodiments, the HBSPCM structure is optimized
using tosylation of HPG hydroxyl groups for the neutralization of
heparin derivatives with a smaller number of negative charges than
LMWH or UFH. In some embodiments, the HBSPCM structure is optimized
using reductive amination of HPG for the neutralization of heparin
derivatives.
[0115] In some embodiments, the number of cationic units (R) is
lower and varies, each having four cationic amine groups per
cationic unit and giving a total charge per polymer of 4.times.R.
For example, the number of cationic units (R) can range from 4 to
24, giving a range of 16 to 96 for charges per polymer. Table 2
lists the characteristics of such polymers, each with a molecular
weight of 23 kDa, that have been synthesized in some embodiments
with different numbers of R groups, and hence charge per polymer,
as well as different amine contents. In some situations, JNK-8 can
be considered a control because it is a HBSPCM polymer without
methylation of the amines, whereas in the other HBSPCM polymers in
Table 2 the primary amine groups are dimethylated.
TABLE-US-00002 TABLE 2 CHARACTERISTICS OF HBSPCMS AT 23,000 G/MOL
WITH DIFFERENT CHARGES No. cationic Average no. Average units (R)
of of charges amine content 4 amines each per polymer (% N) JNK-4 4
16 3.0 JNK-5 5 20 5.0 JNK-6 16 64 13 JNK-3 20 80 17 JNK-7 24 96 21
JNK-8 (Control) 23 92 17.5
[0116] In some embodiments, the molecular weight of the HBSPCMs is
varied in addition to the number of charges per polymer. For
example, Table 3 lists three additional polymers (JNK-9, JNK-10 and
JNK-11) with molecular weights of 9, 10, and 16 kDa, respectively,
and number of charges per polymer of 32, 44 and 64,
respectively.
TABLE-US-00003 TABLE 3 CHARACTERISTICS OF HBSPCMS WITH DIFFERENT
MOLECULAR WEIGHTS Molecular Average No. Average no. Average weight
cationic units (R) of charges amine content Sample (Da) of 4 amines
each per polymer (% N) JNK-9 9 000 8 32 16.4 JNK-10 10 000 11 44
20.9 JNK-11 16 000 16 64 19.6
[0117] Cationic Groups
[0118] In some embodiments, the heparin binding polymer further
includes a cationic moiety. In some embodiments, the cationic
moiety includes an amine group. In some embodiments, the cationic
moiety can be arginine, secondary amine and primary amine groups,
and/or N-methylated lysine. In some embodiments, the amine is
covalently attached to the polyol. In some embodiments, the
cationic moiety includes an arginine. In some embodiments, the
amine group is a tetra-amine group, hexa-amine group, and/or a
deca-amine group.
[0119] In some embodiments, the heparin binding polymer includes
1-300 cationic moieties, for example 2-200, 3-150, 4-100, 5-50,
6-40, or 7-30. In some embodiments, the heparin binding polymer has
1 to 100 cationic moieties, for example, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 99 charges,
including any amount between any of the two preceding values. In
some embodiments, the polymer has 4 to 23 cationic moieties. In
some embodiments, the polymer has 16 to 92 cationic charges.
[0120] Cleavage Sites
[0121] In some embodiments the heparin binding polymer can include
one or more cleavage site within the polymer. In some embodiments,
there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,
60, 80, 100, or 200, including any range greater than any of the
preceding values and any range defined between any two of the
preceding values. In some embodiments, there are 1-10 cleavage
sites. In some embodiments, the heparin binding polymer includes a
second dendritic polyol, where the at least one cleavage site links
the first dendritic polyol to the second dendritic polyol.
[0122] In some embodiments, the cleavage site can be a disulfide
bond. In some embodiments the heparin binding polymer includes at
least two sulfurs that form a disulfide bond. In some embodiments,
the two sulfurs are part of the structure of Formula (I):
##STR00008##
where L.sub.1 can be a first dendritic polyol, L.sub.2 can be a
second dendritic polyol, L.sub.3 can be a third dendritic polyol,
L.sub.4 can be a fourth dendritic polyol, and L.sub.5 can be a
fifth dendritic polyol.
[0123] In some embodiments, the heparin binding polymer can include
at least one ketal group. In some embodiments there are 1, 2, 3, 4,
5, 6 7, 8, 9, 10, 12, 15, 20, 25, 30, 50, 80, 100 or more ketal
groups and/or disulfide bonds in the heparin binding polymer. In
some embodiments, the ketal group is part of the structure of
Formula (II):
##STR00009##
L.sub.1 can be a first dendritic polyol. L.sub.2 can be a second
dendritic polyol. L.sub.3 can be a third dendritic polyol. L.sub.4
can be a fourth dendritic polyol.
[0124] In some embodiments, the hyperbranched polyether polyol can
further include one or more ketal groups. In some embodiments, the
hyperbranched polyether polyol containing one or more ketal groups
is degraded to form smaller polymers of lower molecular weight. In
some embodiments, the cleavage group can be that shown in FIG. 13.
In some embodiments, the cleavage group can be that shown in FIG.
14.
[0125] In some embodiments, the hyperbranched polyether polyol can
include one or more disulphide bonds. In some aspects, the
hyperbranched polyether polyol containing one or more disulphide
bonds is degraded to form smaller polymers of lower molecular
weight.
[0126] Compositions and Pharmaceutical Formulations of Heparin
Binding Polymers
[0127] In some embodiments, compositions including at least one HPG
or heparin binding polymer can be administered at a
pharmacologically effective amount in a pharmaceutically acceptable
excipient to a subject previously administered with heparin,
unfractionated heparin and/or a heparin derivative. Examples of
heparin derivatives include, but are not limited to, unfractionated
heparin, low molecular weight heparins, ultra low molecular weight
heparins, fondaparinux, idraparinux, heparinoids and the like.
[0128] In some embodiments, a composition including at least two
HPG polymers can be administered to a subject at a
pharmacologically effective amount in a pharmaceutically acceptable
excipient previously administered with heparin or a heparin
derivative. The HPG polymers can have different molecular weights,
different functional groups, different PEG group sizes, different
charge density and the like, as described herein and known in the
art. Additionally, other agents can be coadministered with at least
one HPG polymer. Examples of such agents may include protamine,
protamine derivatives, pharmaceutical excipients, and the like.
[0129] In some embodiments, a heparin binding composition is
provided. The composition can include a first heparin binding
polymer and a pharmaceutically acceptable carrier. In some
embodiments, the composition can include a first heparin binding
polymer and an anionic therapeutic agent (e.g. heparins,
methetrexate, insulin) for the controlled drug delivery. The
heparin binding polymer can be any of the heparin binding polymer
embodiments disclosed herein. In some embodiments, the heparin
binding polymer includes a first dendritic polyol and one or more
cationic moieties attached to the first dendritic polyol. In some
embodiments, the composition further includes a second heparin
binding polymer. The second heparin binding polymer can be
different from the first heparin binding polymer. In some
embodiments, the composition can include a protamine, a protamine
derivative, or a combination thereof
[0130] In some embodiments, the first heparin binding polymer is
present in a unit dose. In some embodiments, the unit dose of the
first heparin binding polymer is between 10 mg per kg of subject to
be treated and 200 mg per kg of subject to be treated. In some
embodiments, the subject weighs between 1 kg and 200 kg, for
example, between 5 kg and 150 kg, between 10 kg and 100 kg, or
between 20 kg and 90 kg. In some embodiments, the composition
includes between 100 mg and 40 g of the first heparin binding
polymer, for example, between 500 mg and 20 g, between 1 g and 15
g, between 2 g and 10 g, or between 2 g and 5 g of the first
heparin binding polymer.
[0131] In some embodiments, the heparin binding polymer can be
prepared in a mixture with a pharmaceutically acceptable carrier
and/or excipient. Therapeutic compositions can be administered
intravenously, subcutaneously, topically or via catheters When
administered systemically, therapeutic compositions can be sterile,
pyrogen-free and in a parenterally acceptable solution having due
regard for pH, isotonicity, and stability. These conditions are
known to those skilled in the art. In some embodiments, dosage
formulations of the compounds are prepared for storage or
administration by mixing the compound having the desired degree of
purity with physiologically acceptable carriers, excipients, or
stabilizers. Such materials are non-toxic to the recipients at the
dosages and concentrations employed, and include buffers such as
TRIS HCl, phosphate, citrate, acetate and other organic acid salts;
antioxidants such as ascorbic acid; low molecular weight (less than
about ten residues) peptides such as polyarginine, proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidinone; amino acids such as glycine,
glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as mannitol or sorbitol; counterions
such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS
or polyethyleneglycol.
[0132] Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice as described in
Remington's Pharmaceutical Sciences (18.sup.th ed, Mack Publishing
Company, Easton, Pa., 1990). For example, dissolution or suspension
of the active compound in a vehicle such as water or naturally
occurring vegetable oil like sesame, peanut, or cottonseed oil or a
synthetic fatty vehicle like ethyl oleate or the like can be
employed. Buffers, preservatives, antioxidants and the like can be
incorporated according to accepted pharmaceutical practice.
[0133] In some embodiments, a heparin composition is provided. The
heparin composition can include heparin and a heparin binding
polymer. The heparin binding polymer can include a first dendritic
polyol and one or more cationic moieties attached to the first
dendritic polyol. In some embodiments, the heparin composition
further includes blood or plasma. In some embodiments, the blood is
human blood or the plasma is human plasma. In some embodiments, the
heparin is selected from the group of low molecular weight heparin,
fondaparinux, idraparinux, heparinoid, and any combination
thereof
[0134] In some embodiments, a method of making a heparin binding
polymer is provided. The method can include polymerizing a glycidol
and a methylated PEG-epoxide, to form a hyperbranched polyglycerol,
tosylating the hyperbranched polyglycerol to form a tosylated
hyperbranched polyglycerol, and coupling the tosylated
hyperbranched polyglycerol with at least one amine group, thereby
making a heparin binding polymer.
[0135] In some embodiments, the amine group is coupled to the
polymer through a primary amino group. In some embodiments,
following the coupling, the amino groups are methylated. In some
embodiments, the amine group is a tetra-amine group, a hexa-amine
group, or a deca-amine group. In some embodiments, the tosylating
process occurs in the presence of pyridine and at room temperature.
In some embodiments, it occurs between 20 and 30.degree. C. In some
embodiments, the polymerization process occurs at about 95 degrees
C. In some embodiments, it occurs between about 95.degree. C. to
120.degree. C. and employs potassium methoxide. In some
embodiments, it can employ potassium tert-butoxide, sodium hydride,
or sodium napthalide instead of potassium tert-butoxide, for
example.
[0136] In some embodiments, molecules having cleavable bonds
(HBSPCM polymer with biodegradable disulfide linkages) from an
initiator with disulfide linkages. This disulfide containing
initiator can be prepared, in some embodiments, by the reaction of
the octamine with 2-hydroxy, 2'-carboxy ethylene disulfide. HBSPCM
polymer with biodegradable ketal linkages can be prepared, in some
embodiments, by using an initiator containing ketal groups. This
initiator can be prepared, in some embodiments by first reacting
ethylene diamine with 4 equivalents of glycidol, followed by
reaction with 4-hydroxy-2-butanone.
[0137] Macromolecule
[0138] The term "macromolecule" as used herein denotes the presence
of a protective substance associated with the polyol core and/or
cationic group. The term heparin binding polymer encompasses
various macromolecules and is a broader genus to the possible types
of macromolecules that can employ heparin binding polymers.
Examples of macromolecules are shown in FIG. 1A and FIG. 3.
[0139] In some embodiments, the macromolecule includes a protective
barrier (although it need not actually surround or completely
surround the core and/or cationic groups in all embodiments.
[0140] In some embodiments, the heparin binding macromolecule
includes a hyperbranched polyglycidol core, at least one polyvalent
cation attached to the hyperbranched polyglycidol core, and at
least one protective moiety attached to the hyperbranched
polyglcidol core. In some embodiments, this protective moiety
includes polyethylene glycol.
[0141] In some embodiments, the macromolecule has a polyethelene
glycol content between 50 and 95% by weight, e.g., 60-80%. In some
embodiments the macromolecule has a polyethelene glycol content
between 55% and 90%, 60% and 85%, 65% and 80%, 70% or 80% by
weight. In some embodiments the macromolecule has a polyethelene
glycol content of about 75% by weight.
[0142] In some embodiments, the polyvalent cation includes an amine
group (such as one or more of those shown in FIG. 1A). In some
embodiments, the amine group includes a methylated amine. In some
embodiments, the amine can include an alkylated amine. In some
embodiments, the amine group includes a methylated tetra-amine
group, a methylated hexa-amine group, or a methylated deca-amine
group. In some embodiments, the macromolecule has a hydrodynamic
radius of about 4 nm and about 10 nm.
[0143] Any of the other embodiments disclosed herein (such as the
presence of particular cationic groups, cleavage sites, etc.), can
be used in combination with any of the macromolecule
embodiments.
[0144] Devices, Kits, Compositions, and Ex Vivo Techniques
[0145] In some embodiments, a heparin binding device is provided.
The device can include a support and a heparin binding polymer
immobilized on the support. In some embodiments, the heparin
binding polymer is any of the heparin binding polymers and/or
macromolecules described herein. In some embodiments, the heparin
binding polymer includes a first dendritic polyol and one or more
cationic moieties attached to the first dendritic polyol.
[0146] In some embodiments, the support includes a surface of one
or more beads. In some embodiments, the support includes metal,
plastic, glass, or ceramic. In some embodiments, the support is a
material that is biocompatible. In some embodiments, the material
can be polyurethane, PVC, silicone, etc.
[0147] In some embodiments, the device also includes a fluid flow
path configured to allow a fluid to flow through the fluid flow
path while coming in contact with the heparin binding polymer on
the support.
[0148] In some embodiments, the device also includes a pump
configured to pump blood through the fluid flow path. In some
embodiments, the pump is a peristaltic pump.
[0149] In some embodiments, the support upon or in which the
heparin binding polymer is associated is, or is part of, a
removable cartridge. In some embodiments, the removable cartridge
allows for the replacement of one set of heparin binding polymers
with a different set. In some embodiments, the cartridge allows for
the ready replacement of a used set of heparin binding polymers
with a new set of polymers.
[0150] In some embodiments, the support is part of the removable
cartridge. In some embodiments, the support in the cartridge is
configured so as to allow the support to be exposed to a fluid in
the fluid flow path. In some embodiments, the heparin binding
polymer is attached to a surface of the cartridge. In some
embodiments, the removable cartridge, when engaged with the device,
forms part of the flow path or an outer surface of the flow path.
In some embodiments, the cartridge is configured such that the
fluid flows over the support section of the cartridge. In some
embodiments, the cartridge is configured such that the fluid flows
through the support section of the cartridge (e.g., the support can
be a filter or screen). In some embodiments, the cartridge is
configured such that the fluid flows around and/or to the side of
the support section of the cartridge (e.g., the support can be a
structure that is inserted into the flow path itself).
[0151] In some embodiments, the device will include blood when in
use. In some embodiments, the blood is mammalian, such as human,
pig, horse, cow, mouse, etc.
[0152] In some embodiments, the fluid flow path includes a first
inlet, through which unprocessed blood can enter the flow path, and
an outlet, through which processed blood can exit the heparin
binding device.
[0153] In some embodiments, the device includes a reservoir in
fluid communication with a second inlet. In some embodiments, the
reservoir includes one or more plasma constituents. In some
embodiments, the reservoir includes one or more blood constituents.
In some embodiments, the plasma constituents or the blood
constituents include a non-heparin molecule that binds to the
heparin binding polymer. In some embodiments, the reservoir
contains a medicine for the treatment of at least one of the
following: acute coronary syndrome, atrial fibrillation, cardiac
bypass, extracorporeal membrane oxygenation, pancreatitis, wounds,
hemofiltration, inflammation, cancer and burns.
[0154] In some embodiments, the device includes tubing, or is the
tubing itself, and the inside of the tubing is coated inside with a
heparin binding polymer.
[0155] In some embodiments, HPG polymers can be used in an
extracorporeal circuit having heparin or heparin derivatives
induced into the blood for reuse in a subject, where the
extracorporeal device contains HPG polymers to neutralize the
heparin or heparin derivatives in the blood before returning it to
the subject. In some embodiments, the HPG polymers are HBSPCM
polymers attached to the part of the extracorporeal device coming
into contact with blood.
[0156] In some embodiments, a kit is provided. The kit can include
a first container that includes heparin and a second container that
includes a heparin binding polymer. In some embodiments, the
heparin binding polymer includes a first dendritic polyol and one
or more cationic moieties attached to the first dendritic polyol.
In some embodiments, the amount of heparin binding polymer included
is sufficient to bind to the amount of heparin included. In some
embodiments, the heparin binding polymer is divided into unit
dosage amounts, such that the amount of heparin binding polymer in
any single dose amount is sufficient to bind to a standard dose of
heparin. In some embodiments, the kit can include a needle and/or
tubing for withdrawing blood from a subject. In some embodiments,
the kit can include cartridges and/or a device as described herein.
In some embodiments, the kit can include one or more syringes. In
some embodiments, the syringe is sized so as to be capable of
accepting a unit dose of heparin and/or the heparin binding
polymer. In some embodiments, a unit dose of the heparin binding
polymer is prefilled into the syringe. In some embodiments, the kit
includes components for intravenous administration of the polymer.
In some embodiments, material for measuring thrombin and/or fXa
activity can also be included.
[0157] In some embodiments, a controlled release delivery device is
provided. The device can include a surface and a heparin binding
polymer attached to the surface. The heparin binging polymer can
include a first dendritic polyol and one or more cationic moieties
attached to the first dendritic polyol. In some embodiments, any of
the polymers described herein can be employed. In some embodiments,
the controlled release delivery device can also include heparin. In
some embodiments, the heparin is bound to the heparin binding
polymer. In some embodiments, the type of heparin can be any of the
types described herein. In some embodiments, the amount of heparin
is adequate for a specific therapeutic use. In some embodiments,
the delivery device is adequately sterile for the use of the device
in a subject, such as a human or mammal. In some embodiments, the
controlled release delivery device allows for an extended release
of heparin in a subject. In some embodiments, the controlled
release delivery device allows for a lower amount of heparin to be
administered to a subject. In some embodiments, the controlled
release delivery device allows for relatively localized delivery of
heparin. In some embodiments, instead of heparin, an anionic drug,
such as methotrexate, phenoxymethyl pencillin, insulin,
indomethacin, diclofenac, etc. can be used, thereby providing a
controlled release anionic delivery device.
[0158] In some embodiments, a controlled release heparin
composition is provided. In some embodiments, the composition can
include a heparin binding polymer. In some embodiments, the heparin
binding polymer can include a first dendritic polyol and one or
more cationic moieties attached to the first dendritic polyol. In
some embodiments, the controlled release heparin composition can
also include heparin. In some embodiments, the heparin is bound to
the heparin binding polymer. In some embodiments, the type of
heparin can be any of the types described herein. In some
embodiments, the amount of heparin is adequate for a specific
therapeutic use. In some embodiments, the composition is adequately
sterile for its application to a subject, such as a human or
mammal. In some embodiments, the controlled release heparin binding
composition allows for an extended release of heparin in a subject.
In some embodiments, the controlled release heparin binding
composition allows for a lower amount of heparin to be administered
to a subject. In some embodiments, the controlled release heparin
binding composition allows for relatively localized delivery of
heparin. In some embodiments, instead of heparin, an anionic drug,
such as methotrexate, phenoxymethyl pencillin, insulin,
indomethacin, diclofenac, etc. can be used, thereby providing a
controlled release anionic drug composition.
[0159] Methods of Using Heparin Binding Polymers
[0160] In some embodiments, methods are provided for neutralizing
heparin, heparin derivatives, and/or heparinoids. The method can
include administering a composition having at least one species of
heparin binding polymer and/or hyperbranched polyether polyol
polymers to a subject. The subject can have had a previous
administration of heparin or heparin derivative.
[0161] In some embodiments, there is provided a method for
neutralizing heparin or heparin derivatives, the method including
directing the blood from a subject through an extracorporeal
circuit that contains at least one species of heparin binding
polymer and/or hyperbranched polyether polyol polymer before
returning the blood to the subject for reuse. The subject may have
had a previous administration of heparin, heparin derivative and/or
heparinoids.
[0162] In some embodiments, a method of counteracting heparin in a
subject is provided. In some embodiments, the method includes
administering a heparin binding polymer to a subject. In some
embodiments, the heparin binding polymer includes a first dendritic
polyol and one or more cationic moieties attached to the first
dendritic polyol. In some embodiments, the heparin binding protein
binds to heparin and thereby counteracts heparin in the subject. In
some embodiments, the heparin binding polymer further includes
polyethylene glycol covalently attached to the heparin binding
polymer. In some embodiments, the cationic moiety is an amine
group. In some embodiments, the amine group is a tetra-amine group,
a hexa-amine group, or a deca-amine group. In some embodiments, the
heparing binding polymer is any one or more of the heparing binding
polymers and/or macromolecules provided herein.
[0163] In some embodiments, the method can include identifying a
subject who has received an excess amount of heparin, wherein the
identifying occurs before the heparin binding polymer is
administered to the subject. In some embodiments, this can be done
by identifying a subject who has received heparin in a clinical
setting. In some embodiments, the excess amount of heparin was
administered to the subject. In some embodiments, the excess amount
of heparin was administered for the maintenance of an intravenous
catheter or other hospital or medical care related treatment or
service. In some embodiments, the excess amount of heparin was
administered to treat one or more of the following: acute coronary
syndrome, atrial fibrilllation, cardiac bypass, extracorporeal
membrane oxygenation, pancreatitis, hemofiltration, cancer therapy,
or burns.
[0164] In some embodiments, the method includes identifying a
subject that would benefit from reducing a level of exogenous
heparin that is present in the subject's blood. In some
embodiments, the subject will benefit from an accelerated removal
of exogenous heparin from the subject.
[0165] In some embodiments, the method is performed in vivo. In
some embodiments, the method is performed ex vivo. In some
embodiments, blood is taken from a subject and heparin is removed
from the blood and/or plasma via the heparin binding polymer. In
some embodiments, the subject is a human. In some embodiments, the
subject is not a human. In some embodiments, the subject is a dog,
a cat, a horse, a cow, a pig, a mouse, a rat, a rabbit, a monkey,
or a guinea pig.
[0166] In some embodiments, the heparin binding polymer can be
administered subcutaneously. In some embodiments, the heparin
binding polymer can be administered intravenously.
[0167] In some embodiments, a method of processing a subject's
blood is provided. The method can include providing a heparin
binding device. The heparin binding device can be one described
herein. In some embodiments, the device can include a support and a
heparin binding polymer immobilized on the support. The heparin
binding polymer can include a first dendritic polyol and one or
more cationic moieties attached to the first dendritic polyol. The
method can further include withdrawing blood from a subject,
contacting the blood to the heparin binding polymer, and returning
at least part of the blood to the subject, thereby processing the
subject's blood. In some embodiments, the process is done with
and/or to the subject's plasma instead of blood. In some
embodiments, the method further includes isolating plasma from the
subject, treating the plasma to remove heparin, and then returning
the plasma to the same subject and/or a different subject. In some
embodiments, the subject's blood contains heparin. In some
embodiments, the heparin is exogenous heparin. In some embodiments,
the exogenous heparin binds to the heparin binding polymer and is
thereby at least partially removed from the subject's blood.
[0168] In some embodiments, the method further includes the step of
selecting a heparin binding polymer based upon the type of
exogenous heparin administered to the subject. In some embodiments,
the heparin binding polymer of a particular molecular weight is
selected based upon the type of exogenous heparin. In some
embodiments, this is done based upon the binding strength and
neutralization capacity of the exogenous heparin. In some
embodiments, a heparin binding polymer having a particular number
of cationic groups is selected based upon the type of exogenous
heparin. In some embodiments, the heparin binding polymer works for
a majority of heparins or all heparins. In some embodiments, the
heparin binding polymer works for negatively charged heparins,
including derivatives thereof
[0169] In some embodiments, returning at least part of the blood to
the subject further includes fortifying the blood with one or more
blood constituents that also bind to the heparin binding polymer.
In some embodiments, the fortifying is performed after the blood
has contacted the heparin binding polymer. In some embodiments, the
blood can be fortified with negatively charged coagulation factors
and/or proteins.
[0170] In some embodiments, a method of concentrating heparin is
provided. In some embodiments, the method includes providing a
support and a heparin binding polymer immobilized on the support.
The heparin binding polymer can be any of the heparin binding
polymers disclosed herein. In some embodiments, the heparin binding
polymer includes a first dendritic polyol and one or more cationic
moieties attached to the first dendritic polyol. In some
embodiments, the method also includes contacting a first fluid that
includes heparin with the heparin binding polymer and flowing the
fluid off of the heparin binding polymer. This can concentrate the
heparin.
[0171] In some embodiments, the first fluid includes blood. In some
embodiments, the blood is human. In some embodiments, the fluid
includes a cell lysate or other fluid from the manufacturing
process of manufacturing heparin.
[0172] In some embodiments, the method further includes the process
of removing the heparin from the heparin binding polymer. In some
embodiments, the heparin is removed by flowing a second fluid
including an ionic solution over the heparin binding polymer. In
some embodiments, the first support is part of an affinity
chromatography device. In some embodiments, the first fluid is
derived from mucosal tissue from a meat animal. In some
embodiments, the mucosal tissue from a meat animal is either
porcine intestine, bovine lung, from cell culture, or a combination
thereof
[0173] In some embodiments, flowing the fluid off of the heparin
binding polymer removes at least one impurity, thereby purifying
the heparin.
[0174] In some embodiments, heparin binding polymer is used for the
controlled delivery of anionic therapeutic agents such as heparins,
methetrixate, insulin or similar for the treatments of VTE, cancer,
inflammation or diabetes.
[0175] In some embodiments, the HBSPCM polymers are stable from
-80.degree. C. to 120.degree. C. In some embodiments, heparin
solutions are stable in the temperature range 4.degree. C. to body
temperature. In some embodiments, the methods can occur in part or
in whole within one or both of the above temperature ranges.
[0176] In some embodiments, a polymer pad is provided. The polymer
pad can be coated with any of the polymers disclosed herein. In
some embodiments, this can be used as a controlled delivery
platform for heparin based drugs or anionic drugs and/or for the
local delivery of heparin. In some embodiments, the heparin binding
polymer can be employed as a preventative, and thus can be applied
prophylactically. In some embodiments the heparin binding polymers
can be employed to treated heparin sulphate related disorders. For
example, as a treatment or prophylactic for subjects who
overexpress heparin sulphate.
EXAMPLES
Example 1
Synthesis of HPG-PEG-23K Precursor Polymer
[0177] The present example outlines a method of producing 23 kDa
HBSPCM polymers; the synthesis steps include the following.
Firstly, a HPG-PEG-23K precursor polymer was synthesized using the
following steps. A three-necked round bottomed flask was cooled
under vacuum and filled with argon. To this,
1,1,1-Tris(hydroxymethyl)propane (TMP, 0.480 g) and potassium
methylate (25 wt % solution in methanol, 0.440 mL) were added and
stirred for 30 minutes. Methanol was removed under vacuum for 4
hours. The flask was heated to 95.degree. C. and glycidol (10 mL)
was added over a period of 15 hours. After complete addition of
monomer, the reaction mixture was stirred for additional 3 hours.
MPEG-epoxide-400 (32 mL) was added over a period of 12 hours. The
reaction mixture was stirred for additional 4 hours. The polymer
was dissolved in methanol, passed through Amberlite IR-120H resin
to remove the potassium ions and twice precipitated from diethyl
ether. The polymer was dissolved in water and dialyzed against
water using MWCO-1000 membrane for 3 days with periodic changes in
water.
Example 2
Synthesis of JNK-5 Polymer
[0178] The JNK-5 polymer was synthesized from the HPG-PEG-23K
precursor polymer described in the previous paragraph as follows.
HPG-PEG-23K (0.5 g) was dissolved in 10 mL of pyridine. To this,
p-toluene sulfonyl chloride (0.5 g) was added and stirred at room
temperature for 24 hours. Pyridine was removed by rotary
evaporation; the polymer was dissolved in 0.1 N HCl and dialyzed
overnight. The polymer was isolated by freeze drying.
HPG-PEG-Tos-23K-1 (0.4 g; polymer from previous step) and
tris(2-aminoethylamine) (2 mL) were dissolved in 1,4-dioxane (10
mL) and refluxed for 24 hours. Dioxane was removed under vacuum,
the polymer was dissolved in minimum amount of methanol and
precipitated twice from diethyl ether. Polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 2
days. The resulting polymer solution was added to a mixture of
formaldehyde (3 mL) and formic acid (3 mL) at 0.degree. C. The
reaction mixture was refluxed overnight. After cooling to room
temperature, the pH of the solution was adjusted to 10 using NaOH
and the polymer was extracted with dichloromethane. Dichloromethane
was removed under vacuum. The polymer was dissolved in water and
dialyzed using MWCO-1000 membrane for 3 days. The yield of JNK-5
polymers was shown to be 0.3 g, and the amine content (by
conductometric titration) was 4.8 mol %.
Example 3
Synthesis of JNK-3 Polymer
[0179] The HPG-PEG-23k precursor polymer (0.5 g) was dissolved in
10 mL of pyridine. To this, p-toluene sulfonyl chloride (1 g) was
added and stirred at room temperature for 24 hours. Pyridine was
removed by rotary evaporation; the polymer was dissolved in 0.1 N
HCl and dialyzed overnight. The polymer was isolated by freeze
drying. HPG-PEG-Tos-23K-2 (0.5 g; polymer from previous step) and
tris(2-aminoethylamine) (4 mL) were dissolved in 1,4-dioxane (10
mL) and refluxed for 24 hours. Dioxane was removed under vacuum,
the polymer was dissolved in minimum amount of methanol and
precipitated twice from diethyl ether. Polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 2
days. The resulting polymer solution was added to a mixture of
formaldehyde (3 mL) and formic acid (3 mL) at 0.degree. C.
[0180] The reaction mixture was refluxed overnight. After cooling
to room temperature, the pH of the solution was adjusted to 10
using NaOH and the polymer was extracted with dichloromethane.
Dichloromethane was removed under vacuum; the polymer dissolved in
water and dialyzed using MWCO-1000 membrane for 3 days. The yield
of JNK-3 polymers was shown to be 0.3 g, and the amine content (by
conductometric titration) was 16.9 mol %.
Example 4
Synthesis of JNK-4 Polymer
[0181] The HPG-PEG-23k precursor polymer (0.5 g) was dissolved in
10 mL of pyridine. To this, p-toluene sulfonyl chloride (0.25 g)
was added and stirred at room temperature for 24 hours. Pyridine
was removed by rotary evaporation; the polymer was dissolved in 0.1
N HCl and dialyzed overnight. The polymer was isolated by freeze
drying. HPG-PEG-Tos-23K (0.4 g; polymer from previous step) and
tris(2-aminoethylamine) (2 mL) were dissolved in 1,4-dioxane (10
mL) and refluxed for 24 hours. Dioxane was removed under vacuum,
the polymer was dissolved in minimum amount of methanol and
precipitated twice from diethyl ether. Polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 2
days. The resulting polymer solution was added to a mixture of
formaldehyde (2 mL) and formic acid (2 mL) at 0.degree. C.
[0182] The reaction mixture was refluxed overnight. After cooling
to room temperature, the pH of the solution was adjusted to 10
using NaOH and the polymer was extracted with dichloromethane.
Dichloromethane was removed under vacuum; the polymer dissolved in
water and dialyzed using MWCO-1000 membrane for 3 days. The yield
of JNK-4 polymers was shown to be 0.25 g, and the amine content (by
conductometric titration) was 3 mol %.
Example 5
Synthesis of JNK-6 and JNK-7 Polymers
[0183] The HPG-PEG-23k precursor polymer was used as starting
polymer for the synthesis of the JNK-6 and JNK-7 polymers. To
change the amine content of the polymer, different amounts of
p-toluene sulfonyl chloride were used in the tosylation step. By
controlling the amount of tosyl groups incorporated, the amine
coupling was changed. All other experimental conditions for
synthesis were the same as for the JNK-3 polymer.
Example 6
Synthesis of JNK-8 Polymer
[0184] The HPG-PEG-23k precursor polymer was used as starting
polymer for the synthesis of the JNK-8 polymers. We followed
similar experimental conditions and reagents as for the JNK-3
polymer except the last dimethylation using formaldehyde and formic
acid, which was not performed. JNK-8 is a primary amine containing
polymer without the dimethylation of amines.
Example 7
Synthesis of JNK-9 Polymer
[0185] Firstly, a precursor polymer HpG-PEG-9K was synthesized as
follows. A three-necked round bottomed flask was cooled under
vacuum and filled with argon. To this,
1,1,1-Tris(hydroxymethyl)propane (TMP, 0.240 g) and potassium
methylate (25 wt % solution in methanol, 0.220 mL) were added and
stirred for 30 minutes. Methanol was removed under vacuum for 4
hours. The flask was heated to 95 C and glycidol (3 mL) was added
over a period of 15 hours. After complete addition of monomer, the
reaction mixture was stirred for additional 3 hours.
MPEG-epoxide-400 (10 mL) was added over a period of 12 hours. The
reaction mixture was stirred for additional 4 hours. The polymer
was dissolved in methanol, passed through Amberlite IR120H resin to
remove the potassium ions and twice precipitated from diethyl
ether. The polymer was fractional precipitated in methanol/ether
mixture (1:25 v/v) to obtain HPG-PEG-9k polymer. The polymer was
dissolved in water and dialyzed against water using MWCO-1000
membrane for 3 days with periodic changes in water.
[0186] Secondly, a precursor polymer HPG-PEG-Amine-9K was
synthesized as follows. HPG-PEG-9K (1.0 g) was dissolved in 20 mL
of pyridine. To this, p-toluene sulfonyl chloride (5.0 g) was added
and stirred at room temperature for 24 hours. Pyridine was removed
by rotary evaporation; the polymer was dissolved in 0.1 N HCl and
dialyzed overnight. The polymer was isolated by freeze drying.
HPG-PEG-Tos-9K (0.65 g; polymer from previous step) and
tris(2-aminoethylamine) (3 mL) were dissolved in 1,4-dioxane (25
mL) and refluxed for 24 hours. Dioxane was removed under vacuum;
the polymer was dissolved in minimum amount of methanol and
precipitated twice from diethyl ether. Polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 2
days.
[0187] The above polymer solution was added to a mixture of
formaldehyde (3 mL) and formic acid (3 mL) at 0.degree. C. The
reaction mixture was refluxed overnight. After cooling to room
temperature, the pH of the solution was adjusted to 10 using NaOH
and the polymer was extracted with dichloromethane. Dichloromethane
was removed under vacuum; the polymer dissolved in water and
dialyzed using MWCO-1000 membrane for 3 days. The yield was 0.4 g
and the amine content (by conductometric titration) was 16.35 mol
%.
Example 8
Synthesis of JNK-10 Polymer
[0188] Firstly, a precursor polymer HPG-PEG-10K was synthesized as
follows. A three-necked round bottomed flask was cooled under
vacuum and filled with argon. To this,
1,1,1-Tris(hydroxymethyl)propane (TMP, 0.240 g) and potassium
methylate (25 wt % solution in methanol, 0.220 mL) were added and
stirred for 30 minutes. Methanol was removed under vacuum for 4
hours. The flask was heated to 95.degree. C. and glycidol (2.5 mL)
was added over a period of 15 hours. After complete addition of
monomer, the reaction mixture was stirred for additional 3 hours.
MPEG-epoxide-400 (9 mL) was added over a period of 12 hours. The
reaction mixture was stirred for additional 4 hours. The polymer
was dissolved in methanol, passed through Amberlite IR-120H resin
to remove the potassium ions and twice precipitated from diethyl
ether. The polymer was fractional precipitated in methanol/ether
mixture (1:10 v/v) to obtain HPG-PEG-10k polymer. The polymer was
dissolved in water and dialyzed against water using MWCO-1000
membrane for 3 days with periodic changes in water.
[0189] Secondly, a precursor polymer HPG-PEG-Amine-10K was
synthesized as follows. HPG-PEG-10K (2.4 g) was dissolved in 25 mL
of pyridine. To this, p-toluene sulfonyl chloride (8.0 g) was added
and stirred at room temperature for 24 hours. Pyridine was removed
by rotary evaporation; the polymer was dissolved in 0.1 N HCl and
dialyzed overnight. The polymer was isolated by freeze drying.
HPG-PEG-Tos-10K (2.8 g; polymer from the previous step) and
tris(2-aminoethylamine) (8 mL) were dissolved in 1,4-dioxane (25
mL) and refluxed for 24 hours. Dioxane was removed under vacuum;
the polymer was dissolved in minimum amount of methanol and
precipitated twice from diethyl ether. Polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 2
days.
[0190] The above polymer solution was added to a mixture of
formaldehyde (6 mL) and formic acid (6 mL) at 0.degree. C. The
reaction mixture was refluxed overnight. After cooling to room
temperature, the pH of the solution was adjusted to 10 using NaOH
and the polymer was extracted with dichloromethane. Dichloromethane
was removed under vacuum; the polymer dissolved in water and
dialyzed using MWCO-1000 membrane for 3 days. The yield was 1 g and
the amine content (by conductometric titration) was 20.9 mol %.
Example 9
Synthesis of JNK-11 Polymer
[0191] Firstly, a precursor polymer HPG-PEG-16K was synthesized as
follows. A three-necked round bottomed flask was cooled under
vacuum and filled with argon. To this,
1,1,1-Tris(hydroxymethyl)propane (TMP, 0.240 g) and potassium
methylate (25 wt % solution in methanol, 0.220 mL) were added and
stirred for 30 minutes. Methanol was removed under vacuum for 4
hours. The flask was heated to 95 C and glycidol (3 mL) was added
over a period of 15 hours. After complete addition of monomer, the
reaction mixture was stirred for additional 3 hours.
MPEG-epoxide-400 (10 mL) was added over a period of 12 hours. The
reaction mixture was stirred for additional 4 hours. The polymer
was dissolved in methanol, passed through Amberlite IR-120H resin
to remove the potassium ions and twice precipitated from diethyl
ether. The polymer was fractional precipitated in methanol/ether
mixture (1:5 v/v) to obtain HPG-PEG-16k polymer. The polymer was
dissolved in water and dialyzed against water using MWCO-1000
membrane for 3 days with periodic changes in water.
[0192] Secondly, a precursor polymer HPG-PEG-Amine-16K was
synthesized as follows. HPG-PEG-16K (1.7 g) was dissolved in 20 mL
of pyridine. To this, p-toluene sulfonyl chloride (6.0 g) was added
and stirred at room temperature for 24 hours. Pyridine was removed
by rotary evaporation; the polymer was dissolved in 0.1 N HCl and
dialyzed overnight. The polymer was isolated by freeze drying.
HPG-PEG-Tos-16K (2 g; polymer from the previous step) and
tris(2-aminoethylamine) (6 mL) were dissolved in 1,4-dioxane (25
mL) and refluxed for 24 hours. Dioxane was removed under vacuum;
the polymer was dissolved in minimum amount of methanol and
precipitated twice from diethyl ether. Polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 2
days.
[0193] The above polymer solution was added to a mixture of
formaldehyde (6 mL) and formic acid (6 mL) at 0.degree. C. The
reaction mixture was refluxed overnight. After cooling to room
temperature, the pH of the solution was adjusted to 10 using NaOH
and the polymer was extracted with dichloromethane. Dichloromethane
was removed under vacuum; the polymer dissolved in water and
dialyzed using MWCO-1000 membrane for 3 days. The yield was 1.3 g
and the amine content (by conductometric titration) was 19.62 mol
%.
Example 10
Binding Studies with Heparin and Heparin Derivatives
[0194] To demonstrate the utility and performance of HBSPCMs for
neutralizing heparin, binding studies were performed with both
unfractionated heparin (UFH) and low molecular weight heparin
(LMWH) in in vitro human blood studies and in rats. HBSPCMs were
also characterized for their ability to induce anticoagulation and
clots in blood.
[0195] UFH and LMWHs are neutralized significantly more efficiently
with the HBSPCMs than with protamine. Furthermore, HBSPCMs do not
show any adverse effects on blood coagulation, platelet activity,
complement activation, hemolysis and cytotoxicity while protamine
and other cationic macromolecules do have adverse side effects.
[0196] Table 4 demonstrates a comparison of two embodiments of the
polymer (JNK-1 and JNK-2) in terms of their ability to neutralize
unfractionated heparin and low molecular weight heparin (LMWH), and
shows a comparison with equivalent values for protamine. Both JNK-1
and JNK-2 result in greater neutralization of unfractionated
heparin at two doses (50 and 100 .mu.g/ml), Tinzaparin (LMWH) and
Enoxaparin (LMWH).
TABLE-US-00004 TABLE 4 NEUTRALIZATION EFFECT OF JNK-1 AND JNK-2
POLYMERS Anti Xa Anti Xa Anti Xa Anti Xa Neutrali- Neutrali-
Neutrali- Neutrali- zation (%) zation (%) zation (%) zation (%) of
UFH of Tinzaparin of Enoxaparin of UFH at 100 .mu.g/ml at 100
.mu.g/ml at 160 .mu.g/ml at 50 .mu.g/ml Sample [1] [1] [1] [1]
JNK-1 89 .+-. 3 90 .+-. 2 81 .+-. 3 86 JNK-2 90 .+-. 2 92 .+-. 4 89
.+-. 3 90 Protamine 85 .+-. 0 77 .+-. 1 73 .+-. 4 84 UFH =
unfractionated heparin. [1] One IU UFH or LMWH (Enoxaparin or
Tinzaparin) per ml in platelet poor plasma.
Example 11
JNK-1 Neutralization of UFH
[0197] In one example, a heparin binding synthetic polyvalent
cationic macromolecule JNK-1 can neutralize UFH in human blood
under in vitro conditions (FIGS. 4A and 4B). Citrate plasma was
anti-coagulated with 1.0 U/ml UFH (UFH control) and was titrated
with different concentrations of JNK-1 polymers (0.009 to 8.69
nanomoles/ml) and protamine (0.2 to 222.2 nanomoles/ml). The
activated partial thromboplastin time (APTT) was measured for each
sample and is shown in FIGS. 4A and B. These results show that
JNK-1 polymers effectively neutralized UFH (FIG. 4A) and do not
cause anticoagulant effect even at high concentration, unlike
protamine (FIG. 4B). JNK-1 polymers showed greater UFH
neutralization capacity as compared to protamine.
Example 12
JNK-1 Neutralization of LMWH Tinzaparin
[0198] The present example demonstrates that JNK-1 polymers can
neutralize LMWH Tinzaparin in human blood in vitro (FIGS. 4A and
4B). Citrate plasma was anti-coagulated with 2.0 U/ml LMWH (LMWH
control) and was titrated with different concentrations of JNK-1
polymers (0.009 to 8.69 nanomoles/ml) (FIG. 4A) and protamine (0.2
to 222.2 nanomoles/ml) (FIG. 4B). The activated partial
thromboplastin time (APTT) was measured for each sample, as shown
in FIGS. 4A and 4B. Results show that JNK-1 effectively neutralizes
Tinzaparin and does not cause any anticoagulant effect at even
higher concentration, unlike protamine. Unlike protamine, JNK-1
polymers completely neutralized the anticoagulation of LMWH and
showed greater efficiency.
Example 13
Percentage of UFH and LMWH Tinzparin Neutralization
[0199] The present example demonstrates the percentage of UFH and
LMWH Tinzparin neutralization calculated from an activated partial
thromboplastin time (APTT) assay by JNK-1 or protamine. FIG. 5A
shows the JNK-1 polymer neutralization of UFH. FIG. 5B shows the
protamine neutralization of UFH. FIG. 5C shows the JNK-1 polymer
neutralization of LMWH. FIG. 5D shows the protamine neutralization
of LMWH. The IC.sub.50 of UFH neutralization by JNK-1 polymers is
0.1 nanomoles and of Tinzaparin is 0.2 nanomoles, which is a few
orders lower (more efficient) than protamine. JNK-1 completely
neutralizes UFH and Tinzaparin.
Example 14
Thromboelastography in the Presence of Various Heparin Binders
[0200] This example examines thromboelastography in the presence of
various heparin binders. Thromboelastography (TEG) provides an in
vitro method for monitoring of the evolution of the viscoelastic
properties and coagulation kinetics of blood as clotting proceeds.
The principle components of the TEG are a cylindrical cup and a bob
suspended in the cup from a weak torsion wire. The warmed cup
oscillates over a 10 second period through an angle of 4.degree.
45'. Blood is added and a torque is transmitted to the bob as the
gel forms. The rate of clot formation, the strength of the clot,
and the rate of clot lysis are reflected in the shape traced out by
the maxima in the bob oscillation as a function of time (the trace
of the TEG). The clot's physical properties, i.e. rate of clot
formation, clot strength, and stability are dependent on the
interaction of fibrinogen, platelets, and plasma proteins. TEG
studies yield the cumulative effect of several components of
coagulation (global homeostasis) as a function of time.
[0201] To carry out TEG experiments, citrate anti-coagulated whole
blood with heparin or heparin derivatives added are incubated with
buffered HBSPCM polymers (sodium citrate or saline) for 1 minute
before recalcification at 37.degree. C. Clot formation kinetics are
monitored in a TEG machine (350 .mu.l whole blood+35 .mu.l polymer
or control solution) at 37.degree. C.
[0202] JNK-1 polymers can neutralize UFH and LMWH Tinzaparin, as
demonstrated by using a TEG measurement of human whole blood (FIG.
6). TEG traces of different experiments are given. JNK-1 polymer
neutralization of UFH is shown in FIG. 6A. Protamine neutralization
of UFH is shown in FIG. 6B. JNK-1 polymer neutralization of
Tinzaparin is shown in FIG. 6C. Protamine neutralization of
Tinzaparin is shown in FIG. 6D. The results demonstrate the ability
of JNK-1 to neutralize UFH and Tinzaparin even at higher
concentration, unlike protamine. There was no clot formation
observed in presence of either UFH or Tinzaparin alone.
Example 15
JNK-1 Polymers can Neutralize UFH and LMWH Tinzaparin
[0203] The present example demonstrates that JNK-1 polymers can
neutralize UFH and LMWH Tinzaparin, as demonstrated by using a TEG
measurement of human whole blood (FIGS. 7A and 7B). The coagulation
time (FIG. 7A) and maximum clot strength, or maximum amplitude (MA)
(FIG. 7B) are shown. Citrated whole blood was added to UFH or
Tinzaparin (0.5 U/ml) and re-calcified at 37.degree. C. in a TEG
cup and monitored using a TEG machine. JNK-1 polymers or protamine
at different concentrations were used as neutralizing agents.
[0204] The results demonstrate that JNK-1 polymers effectively
neutralize anticoagulation effect of both UFH and Tinzaparin.
Protamine at a higher concentration (0.25 mg/ml) did not form a
stable clot (see maximum amplitude (MA) data) with Tinzaparin.
There was no clot formation observed in presence of either UFH or
Tinzaparin alone.
Example 16
Electrostatic Charge Neutralization of UFH and LMWH Tinzaparin by
JNK-1 Polymers
[0205] This example demonstrates the electrostatic charge
neutralization of UFH and LMWH Tinzaparin by JNK-1 polymers
(results shown in FIG. 9). The electrophoretic mobilities of
heparin/JNK-1 complexes formed by mixing the different ratios of
heparin and JNK-1 polymers were measured. JNK-1 polymers neutralize
UFH and Tinzaparin at similar concentrations. The results are
presented in FIG. 9.
Example 17
Complement Activation
[0206] The present example examines complement activation due to
heparin binding polymers. Complement activation is one of the major
adverse side effects associated with the clinical use of protamine
in the reversal of anticoagulation in cardiopulmonary bypass and
other surgical conditions (Shastri et al. 1997 Cardiovasc Surg.
114(3):482-8). Complement activation induced by various
biomaterials has been studied (Lamba et al 1999. Biomaterials
21:89; Kidane et al 1999. J Biomed Mate Res 48:640; Lim et al 1993.
Biomaterials 14:537; Payne et al 1987. J Biomed Mater Res 21:843;
Jantova et al 1991. Complement and Inflammation 8:61). Examples of
polymers which cause complement activation include dextran,
regenerated cellulose, sephadex, nylon, poly(methylmethacrylate),
poly(propylene), poly(acrylamide), poly(hydroxyethylmethacrylate),
plasticised PVC. Complement activation is not a property of all
polymers however. For example, poly(N-vinylpyrrolidone) does not
cause complement activation (Beillat et al 1984. ASAIO J 7:57).
[0207] To assess complement activation, the activation of the
complement component C3 was monitored by measuring the formation of
its activation peptides using a commercial C3a enzyme immunoassay
kit (Quidel, San Diego, Calif.), following manufacturer's
instructions. The C3a concentrations (in ng/ml) were calculated
using a standard curve with net absorbance values plotted on the
y-axis for each C3a concentration indicated on the x-axis.
[0208] Complement activation upon interaction of JNK-1 polymers
with platelet poor plasma (10 mg/ml and 1 mg/ml) is shown in FIG.
10A at 37.degree. C. for 1 hour at 9:1 dilution of platelet poor
plasma. Plasma incubated with saline as a control and insulin (a
potent complement activator) as positive control are also given.
Results show that JNK-1 polymers do not initiate complement
activation.
[0209] FIG. 20 demonstrates the complement activation by the HBSPCM
polymers described in Table 2 (JNK-3, JNK-4, JNK-5, JNK-6, JNK-7
and JNK-8-control) in comparison to protamine and a saline control.
Complement activation is shown upon interaction of the HBSPCM
polymers in platelet poor plasma (PPP) at 37.degree. C. for 1 hour
at a 9:1 dilution of PPP. At 0.5 mg/mL, the HBSPCM polymers all had
reduced complement activation as compared to protamine. At 0.05
mg/mL, all but one HBSPCM polymer (JNK-7) had reduced complement
activation as compared to protamine and similar activation to
saline.
Example 18
Platelet Activation
[0210] The present example examines platelet activation in the
presence of heparin binding polymers. Platelet activation leads to
aggregation, which can cause adverse effects such as thrombotic
complications or arterial embolization. To measure platelet
activation, blood was collected in sodium citrate anticoagulant and
the platelet rich plasma (PRP) isolated by centrifugation. Plasma
was then incubated at 37.degree. C. with JNK-1 at 10 mg/mL (9:1
dilution), polyethyleneimine (PEI) at 1 mg/ml, insulin (a potent
complement activator) as a positive control, saline, or PRP
incubated with adenosine diphosphate and thrombin as a positive
control for 30 minutes. Aliquots of the incubation were assessed
for activation state of the platelets using fluorescence flow
cytometry. Expression of the platelet activation marker CD62P was
detected using a Coulter Epics-XL (Miami, Fla.). Briefly, the
post-incubation polymer/platelet mix was diluted and incubated with
the monoclonal anti-CD62P-FITC antibody. Data is reported as the
percentage of platelets positive (activated) for CD62.
[0211] FIG. 10B demonstrates the platelet activation upon
interaction with JNK-1 polymers and controls. Results show that
JNK-1 does not induce platelet activation as compared to known
activators (PEI and positive control). JNK-1 polymers induced
activation that was no greater than that seen with saline alone;
thus, the low level CD62 expression seen for JNK-1 polymers can be
attributed to the choice of suspending medium for the polymer.
Example 19
Blood Coagulation
[0212] The present example examines blood coagulation in the
presence of a heparin binding polymer. JNK-1 polymers were tested
for blood compatibility using the activated partial thromboplastin
time (APTT) and the prothrombin time (PT) in fresh human plasma. PT
can be used to evaluate the extrinsic and common coagulation
pathway and the results are expressed in seconds required for a
fibrin clot to form after tissue thromboplastin (innovin) has been
added to the blood sample. APTT is used to evaluate the intrinsic
and common coagulation pathway. The results are expressed in
seconds required for a fibrin clot to form in the plasma sample
after a partial thromboplastin reagent (actin) and calcium chloride
have been added to the sample. Blood was mixed with a measured
amount of citrate and the plasma was obtained by centrifugation,
using methods known in the art.
[0213] The effect of JNK-1 polymers on PT and APTT in platelet poor
plasma is shown in FIG. 11A. A final JNK-1 concentration of 10
mg/ml is used at in a 9:1 dilution of plasma at 37.degree. C.
Control experiments were done adding identical volumes of saline
solution. JNK-1 polymer solutions were incubated with platelet poor
plasma for 5 minutes at 37.degree. C. before adding PT and APTT
reagents. The results show that JNK-1 polymers do not influence
blood coagulation on their own as there was no significant
difference between the JNK-1 polymers and the saline control.
[0214] Plasma re-calcification time can be calculated as the time
it takes to detect a clot after the addition of calcium ions. FIG.
11B shows the effect of JNK-1 polymers on plasma re-calcification
time in platelet poor plasma. A final JNK-1 polymer concentration
of 10 mg/ml was used in a 9:1 dilution of plasma at 37.degree. C.
Control experiments were done adding identical volumes of saline
solution. In this experiment no additional agent is added to induce
the coagulation except the calcium chloride solution
(recalcification). There was no significant difference between the
JNK-1 and plasma control. The results show that JNK-1 polymers do
not influence the coagulation on their own.
[0215] Optical micrographs are shown in FIGS. 12A-12D of human red
blood cells after one-hour incubation with JNK-1 polymers at
different concentrations in whole blood (9:1 dilution) at
37.degree. C. All images are at 400.times. magnification. FIG. 12A
is an incubation of JNK-1 polymers (10 mg/ml). FIG. 12B is an
incubation of JNK-1 polymers (1 mg/ml). FIG. 12C is an incubation
of polyethyleneimine (PEI) (1 mg/ml). FIG. 12D is an incubation of
saline control. Results show that JNK-1 polymers do not induce
adverse effects such as red cell aggregation or hemolysis, unlike
cationic polymers such as PEI (FIG. 12C).
Example 20
Heparin and Heparin Derivative Neutralization in Human Blood
[0216] Human plasma treated with sodium citrate was anti-coagulated
with unfractionated heparin (UFH) or low molecular weight (LMWH)
Tinzaparin. Samples subsequently mixed with JNK-1 and JNK-2
polymers showed complete neutralization for both UFH and Tinzaparin
and did not show anticoagulation at high doses.
[0217] Conversely, for protamine treated samples, complete
neutralization occurred at a higher dosage of protamine for UFH and
not at all for Tinzaparin. Furthermore, at high doses protamine had
an anticoagulant effect and was not an effective antidote.
[0218] These results were confirmed by analysis of the activated
partial thromboplastin time (APTT), which showed that JNK-1
polymers were capable of complete neutralization of UFH and LMWH
but that protamine was incapable of neutralizing LMWH and acted
with less efficiency for UFH within a smaller dose window. Overall,
JNK-1 polymers showed greater UFH and LMWH neutralization capacity
as compared to protamine in citrated plasma.
[0219] JNK-1 and JNK-2 polymers were also tested with human whole
blood for efficacy in neutralizing UFH and LMWH. Thromoelastography
measurements showed that human whole blood containing UFH or LMWH
was efficiently neutralized by the polymers JNK-1 and JNK-2.
Conversely, protamine was not effective at neutralizing UFH or LMWH
Tinzaparin and Enoxaparin in terms of the time to clot formation in
human whole blood. Although protamine was able to allow UFH treated
blood to regain the same clot strength, it only capable of enabling
LMWH Tinzaparin treated blood to regain the same clot strength at a
lower dose and not at all at a high dose.
[0220] This study in human citrated plasma and whole blood
indicates that the JNK-1 and JNK-2 polymers are effective for human
samples with UFH and LMWH. Furthermore, these studies provide
evidence that JNK-1 and JNK-2 polymers display properties that are
consistent with being useful for human clinical use.
[0221] The results in FIGS. 18A-18C demonstrate the neutralization
of different heparins: FIG. 18A: unfractionated heparin (UFH); FIG.
18B: LMWH Tinzaparin; and FIG. 18C: LMWH Enoxaparin by the HBSPCM
polymers described in Table 2 (JNK-3 to JNK-8) in comparison to
protamine in human blood in vitro.
[0222] Citrate plasma was anticoagulated with 2.0 U/ml UFH,
Enoxaparin and Tinzaparin and was treated with different
concentrations of the HBSPCM polymers and protamine (0.001 to 0.25
mg/ml). The APTT was measured for each sample. Results show that
the HBSPCM polymers, unlike protamine, effectively neutralize UFH
and LMWH (Tinzaparin and Enoxaparin) and do not cause any
anticoagulant effect at even higher concentration. HBSPCM polymers
can almost completely neutralize the anticoagulation of LMWH
(Tinzaparin and Enoxaparin) at certain doses and showed greater
efficiency.
[0223] The results in FIG. 19 also demonstrate that HBSPCM polymers
can effectively neutralize LMWH using a thromboelastograph (TEG) in
human whole blood. HBSCPM polymers JNK-5 and JNK-6 (0.1 mg/ml) were
added to the anticoagulated blood and compared to a control with no
Enoxaparin added (no anti-coagulation with Enoxaparin) and no
neutralization (Enoxaparin control). The results demonstrate the
ability of JNK-5 and JNK-6 to neutralize Enoxaparin in terms of the
TEG strength. The TEG trace of the enoxaparin neutralized blood is
almost identical to saline control, which was not coagulated.
Together with FIG. 18, these data demonstrate the efficiency of
HBSPCM polymers such as JNK-5 and JNK-6 to neutralize LMWH under in
vitro conditions.
[0224] The data in FIGS. 24A-24C demonstrate the neutralization of
different heparins ((FIG. 24A): unfractionated heparin (UFH); (FIG.
24B): LMWH Tinzaparin; and (FIG. 24C): LMWH Enoxaparin) by the
HBSPCM polymers described in Table 3 (JNK-9, JNK-10, JNK-11) in
comparison to protamine in human plasma in vitro. Citrate plasma
was anticoagulated with UFH (2.0 IU/mL), Enoxaparin (2.0 IU/mL) and
Tinzaparin (1.14 IU/mL) and was titrated with different
concentrations of the HBSPCM polymers and protamine (0.001 to 0.25
mg/ml). The APTT was measured for each sample. Results show that
the HBSPCM polymers, unlike protamine, effectively neutralize UFH
and LMWH (Tinzaparin and Enoxaparin) and do not cause any
anticoagulant effect at higher concentrations than protamine. The
HBSPCM polymers JNK-9, JNK-10 and JNK-11 can almost completely
neutralize the anticoagulation of LMWH (Tinzaparin and Enoxaparin)
at certain doses and showed greater efficiency.
Example 21
High Binding Affinity of Heparin to HBSPCM Polymer JNK-2
[0225] The present example examines the binding affinity of a
heparin binding polymer to heparin. The HBSPCM polymer JNK-2 was
tested for UFH, LWMH and fondaprinux binding by isothermal
titration calorimetry. Isothermal titration calorimetry (ITC) was
performed using a VP-ITC (MicroCal, Inc., Northampton Mass.).
Samples were in PBS buffer pH 7.2. Titrations were performed by
injecting consecutive 5-10 .mu.L aliquots of JNK-2 solution into
the ITC cell (volume=1.4 mL) containing heparin or heparin
derivatives. The ITC data were corrected for the heat of dilution
of the titrant by subtracting mixing enthalpies for 5-10 .mu.L
injections of JNK-2 solution into heparin- and heparin
derivative-free buffer. At least two independent titration
experiments were performed for each system at 25.degree. C. to
determine the binding constant of HBSPCM to heparin derivatives.
Binding stoichiometry, N, enthalpy, .DELTA.H.sub.b, entropy,
.DELTA.S, and equilibrium association constants, K.sub.a, were
determined by fitting the corrected data to a bimolecular
interaction model. This study demonstrated high binding affinity
and enthalpy of interaction of JNK-2 molecules to heparin
derivatives. Table 5 shows the summary of isothermal titration
calorimetry (ITC) results for JNK-2 titrations with unfractionated
heparin, Enoxoparin and Fondaparinux.
TABLE-US-00005 TABLE 5 SUMMARY OF JNK-2 TITRATIONS K.sub.a .DELTA.G
.DELTA.H T.DELTA.S Macromolecule N.sup.1 (M.sup.-I) (kcal/mol)
(kcal/mol) (kcal/mol) Unfrac- 0.9 (.+-.0.I) 1A (.+-.0.7) .times.
106 -8.3 (.+-.0.2) -94 (.+-.15) -85 (.+-.15) tionated heparin LMWH
2.95 (.+-.0.08) 1.3 (.+-.0.2) .times. 105 -7.0 (.+-.0.I) -34.8
(.+-.0.5) -27.8 (.+-.0A) (Enoxoparin) Fondaparinux 2.3 (.+-.0.0I)
1.5 (.+-.0.2) .times. 105 -7.1 (.+-.0.I) -17.9 (.+-.0.2) -10.9
(.+-.0.I) .sup.1N = number of heparin molecules that binds to one
JNK-2 (molecular weight = 48 kDa). Experiments performed at
25.degree. C. (errors represent the standard deviations of
replicate experiments). ~G, ~H, T.DELTA.S are calculated per mole
of macromolecule.
Example 22
Heparin and Heparin Derivative Neutralization in Living Rats
[0226] The present example examines heparin binding polymer
effectiveness in vivo. HBSPCM polymers were tested for their
ability to neutralize UFH and LMWH in living rats. Rats were first
injected with heparin or heparin derivative, and then injected with
the JNK-1 polymers, protamine or saline control to neutralize the
previously administered heparin or heparin derivative.
Neutralization was measured by detecting the percentage of factor
Xa using an anti-Xa assay as a measure of anticoagulant
activity.
[0227] The results are shown in FIGS. 8A-8D. The data in FIG. 8A-8D
demonstrates the ability of JNK-1 and JNK-5 (HBSPCM-3) polymers to
neutralize UFH and LMWH (Tinzaparin and Exonaparin) in rats. FIG.
8A shows data for protamine+UFH and UFH alone and FIG. 8B shows
data for protamine+Tinzaparin and Tinzaparin alone, were compared
to JNK-1 polymers+UFH (FIG. 8A) and JNK-1 polymers+Tinzaparin (FIG.
8B), respectively. In addition, Enoxaparin alone and JNK-1
polymers+Enoxaparin were compared (FIG. 8C). An anti-Xa assay was
used to measure heparin activity in rat blood. In this pilot study,
rats were injected with 25 U of UFH, Tinzaparin or Enoxaparin (0
minutes). Blood was collected after UFH, Tinzaparin, or Enoxaparin
infusion (5 minutes). Approximately 1 mg of JNK-1 polymers or
protamine (dose was 3 mg/kg) was injected at 7 minutes and the
blood was collected at 12 minutes after UFH/Tinzaparin/Enoxaparin
infusion (5 minutes after JNK-1 infusion), and 17 minutes (10
minutes after JNK-1 polymer infusion). For subjects receiving
Enoxaparin, blood was collected at additional time points (25
minutes and 30 minutes). Blood samples collected before the UFH,
Enoxaparin or Tinzaparin infusion were used as a base control.
JNK-1 polymers fully neutralize the anticoagulant activity of UFH,
Enoxaparin and Tinzaparin and factor Xa levels reach almost to
control levels after JNK-1 polymer infusion. Each data point shown
reflects an average and standard deviation from five rats. The
chronological order of events during the experiment is given on the
bottom portion of the graphs in FIGS. 8A-8C; arrows indicate the
time points.
[0228] The data in FIG. 8A show that for unfractionated heparin
(UFH), the JNK-1 polymers can completely neutralize the UFH within
at most five minutes after infusion, performed equally well as
protamine and outperformed the control. The data in FIG. 8B shows
that for low molecular weight heparin (LMWH), the JNK-1 polymers
can completely neutralize the LMWH within at most five minutes
after infusion, outperforming both protamine and the control, which
could not completely neutralize the LMWH.
[0229] The data in FIG. 8D shows the ability of JNK-5 polymer to
neutralize LMWH enoxaparin in rats. Rats were injected with 25 U of
Enoxaparin (0 minutes). Blood was collected after enoxaparin
infusion (5 minutes). Approximately 1 mg of JNK-5 polymers or
protamine (dose was 3 mg/kg) was injected at 7 minutes and the
blood was collected at 12 minutes after enoxaparin infusion (5
minutes after JNK-5 infusion), 17 minutes (10 minutes after JNK-5
polymer infusion) and 65 minutes (one hour after JNK-5 polymer
infusion). For subjects receiving enoxaparin, blood was collected
at additional time point (25 minutes). Blood samples collected
before the enoxaparin infusion were used as a base control. JNK-5
polymers fully neutralize the anticoagulant activity of enoxaparin
and factor Xa levels reach almost to control levels after JNK-5
polymer infusion. Each data point shown reflects an average and
standard deviation from five rats. The chronological order of
events during the experiment is given on the bottom portion of the
graphs in FIG. 8D; arrows indicate the time points.
Example 23
Heparin and Heparin Derivative Neutralization in Living Rats
[0230] The data in FIGS. 21A and 21B demonstrate the ability of
another embodiment, JNK-3 polymers, to neutralize UFH and LMWH
Enoxaparin in living rats. Unfractionated heparin (UFH) alone and
JNK-3 polymers+UFH were compared (FIG. 21A). Enoxaparin alone and
JNK-3 polymers+Enoxaparin were compared (FIG. 21B). An anti-Xa
assay was used to measure heparin activity in rat blood.
[0231] In this study, rats were injected with 25 U of UFH or
Enoxaparin (0 minutes). Blood was collected after UFH or Enoxaparin
infusion (3 minutes). 1 mg of JNK-3 polymers was injected at 5
minutes and the blood was collected at 7, 10, 15 and 35 minutes
after UFH/Enoxaparin infusion. For subjects receiving UFH infusion,
blood was also collected at 65 minutes. Blood samples collected
before the UFH or Enoxaparin infusion were used as a base control.
JNK-3 polymers fully neutralize the anticoagulant activity of UFH
and Enoxaparin and factor Xa levels reach almost to control levels.
The chronological order of event during the experiment is given on
the bottom portion of the graphs in FIG. 21; arrows indicate the
time points.
[0232] These in vivo studies indicate that heparin binding
polymers, including both JNK-1 and JNK-3 polymers, work effectively
on both UFH and LMWH in the intact living system of rat model,
providing evidence that the findings may be extrapolated to
systemic human use.
Example 24
Heparin and Heparin Derivative Neutralization in an Extracorporeal
Device
[0233] The present example is directed to an extracorporeal device
that can be used to remove and/or reduce heparin and derivatives
thereof. The blood from a subject previously administered with
heparin or heparin derivatives is directed into an extracorporeal
circuit bypass of a circulation system is filtered for the removal
of heparin or heparin derivatives for reuse by the subject.
[0234] The extracorporeal device can include a structure including,
but not limited to, a cylindrical cartridge, hollow tubing, a rigid
or flexible vesicle, and the like. HBSPCM polymers are attached to
some the part of the extracorporeal device coming into contact with
blood and are attached either directly, or through a means of
tethering. Tethering can include, but is not limited to, polymer
brushes or surface functionalities or by non-covalent complexation
(electrostatic).
[0235] As blood is moved through the extracorporeal device, after
it leaves the subject and before it is returned to the subject,
HBSPCM polymers attached to the device neutralize heparin or
heparin derivatives in the blood.
Example 25
Blood Compatibility Analysis
[0236] Blood compatibility of the polymers was assessed by several
methods (blood coagulation, platelet activation, complement
activation, red cell hemolysis and aggregation. The change in
activated partial thromboplastin time (APTT) was evaluated in
comparison to a saline control. A significant change in the APTT
shown by the heparin binding polymers as compared to a saline
control suggests that the HBSPCM polymers are not interfering with
blood coagulation proteins such as tissue factor and other
coagulation factors.
[0237] FIG. 15 shows an analysis of blood compatibility by
demonstrating the activated partial thromboplastin time (APTT)
assay by the HBSPCM polymers described in Table 2. Blood
coagulation is measured using APTT. The amine content changed the
APTT and protamine resulted in a different blood coagulation
profile.
[0238] FIG. 16 shows another analysis of blood compatibility using
thromboelastograph (TEG) in terms of coagulation time (R) and
kinetics parameter (K) at a polymer concentration of 0.5 mg/ml for
the polymers described in Table 2. TEG studies yielded the
cumulative effect of several components of coagulation (global
homeostasis) as a function of time. The polymers with higher amine
content and protamine resulted in a different blood coagulation
profile.
Example 26
Rate of Red Blood Cell Lysis
[0239] This example examines the rate of red blood cell lysis in
the presence of a heparin binding polymer.
[0240] Red blood aggregation in plasma/buffer is not easily
disrupted by shear forces and can result in high blood viscosity
and complications. Severe aggregation can cause cell damage and
hemolysis, leading to a variety of complications including anemia
and jaundice. Red blood cell hemolysis shows the toxicity of the
compounds tested.
[0241] In the present example, the degree of hemolysis was observed
by monitoring the degree of red color present in the supernatant
plasma or buffer upon centrifugation of the red blood cells after
incubation for 1 hour at 37.degree. C. with heparin binding
polymers. Red blood cell lysis was determined by measuring the
amount of extracellular hemoglobin against the total hemoglobin
concentration. Briefly, 50 microliters of red blood cell suspension
and 100 microliters of red blood cell supernatant were mixed with
Drabkin's reagent (1 ml each) and the absorbance was monitored at
540 nm running Drabkin's reagent as blank.
[0242] FIG. 17A and FIG. 17B demonstrate the results of a red blood
cell lysis assay, which measured the percent of red blood cells
that were lysed after incubating different HBSPCM polymers as
described in Table 2 with red blood cells at 37.degree. C. for 1
hour at two different concentrations: (FIG. 17A) 0.1 mg/ml; (FIG.
17B) 5 mg/ml. Shown on the left side of FIG. 17A and FIG. 17B are
photographs of red blood cell suspensions centrifuged after
incubation with the polymers.
Example 27
Tolerance of HBSPCM Polymers in Mice
[0243] The present example examines the degree of tolerance that
animals have to various heparin binding polymers, and to HBSPCMs in
particular.
[0244] FIG. 22 shows the body weights over time (0 to 29 days post
injection) of female BALB/c mice treated with JNK-3 polymers (23
kDa) by bolus intravenous injection of increasing concentration of
JNK-3 polymers (1 mg/kg to 50 mg/kg). Mice were randomly assigned
to dosing groups. All injections (200 .mu.L of HBSPCM in saline)
were performed as scheduled with no adverse reactions noted. No
significant decreases in body weight or behavioral changes were
noted following administration of JNK-3 polymers. All the mice were
terminated as per study protocol on the 29.sup.th day with no
notation on necropsy.
[0245] The results show that JNK-3 polymers can be safely dosed by
bolus intravenous administration in female BALB/c mice at a dose of
up to 50 mg/kg (maximum dosage given). The tolerated dose is at
least 16-fold higher as compared to protamine, which has a maximum
tolerated dose of 3 mg/kg. The study was carried out in the
Department of Advanced Therapeutics, British Columbia Cancer
Agency, Vancouver, under an approved UBC animal ethics
protocol.
Example 28
Cytotoxicity of HBSPCM Polymers
[0246] The present example examines the cytotoxicity of a heparin
binding polymer. The lactate dehydrogenase (LDH) assay demonstrates
the cytotoxicity present in a sample containing cells based on the
activity of LDH released from damaged cells. The LDH assay was used
to assess the cytotoxicity of a HBSPCM polymer, JNK-3.
[0247] FIG. 23 demonstrates the LDH activity in serum of female
BALB/c mice treated with JNK-3 polymers (23 kDa) after an
intravenously applied bolus given on day 0 of increasing
concentration (1 mg/kg to 50 mg/kg) as measured on the termination
day (29.sup.th day of post injection). The results demonstrate that
the LDH activity associated with all doses was either below or not
dramatically greater than the control activity level. Results
dramatically higher than the control activity level would have been
indicative of liver and cell toxicity associated with the HBSPCM
polymers (JNK-3). Base LDH activity levels for control mice were
around 100 IU/I (dotted line in FIG. 23).
Example 29
[0248] The present example outlines a method of making HBSPCM
polymers JNK-1 and JNK-2. The HBSPCM polymers JNK-1 and JNK-2 were
synthesized using similar protocols to that of polymers JNK-3 to
JNK-11. The average number of charge in JNK-1 and JNK-2 are 276 and
112 respectively. Synthetic procedure for INK-1 is given below.
Synthesis of Polymer JNK-1 (116 kDa HBSPCM Polymer)
[0249] The synthesis steps include the following. Firstly, a
HPG-PEG-116K precursor polymer was synthesized using the following
steps. A three-necked round bottomed flask was cooled under vacuum
and filled with argon. To this, 1,1,1-Tris(hydroxymethyl)propane
(TMP, 0.120 g) and potassium methylate (25 wt % solution in
methanol, 0.110 mL) were added and stirred for 30 minutes. Methanol
was removed under vacuum tier 4 hours. The flask was heated to
95.degree. C. and glycidol (6 mL) was added over a period of 15
hours. After complete addition of monomer, the reaction mixture was
stirred for additional 3 hours. MPEG-epoxide-400 (20 mL) was added
over a period of 12 hours. The reaction mixture was stirred for
additional 4 hours. The polymer was dissolved in methanol, passed
through Amberlite IRC-50 resin to remove the potassium ions and
twice precipitated from diethyl ether. The polymer was dissolved in
water and dialyzed against water using MWCO-1000 membrane for 3
days with periodic changes in water.
[0250] The JNK-1 polymer was synthesized from the HPG-PEG-116K
precursor polymer described in the previous paragraph as follows.
HPG-PEG-116K (16 g) was dissolved in 100 mL of pyridine. To this,
p-toluene sulfonyl chloride was added and stirred at room
temperature for 24 hours. Pyridine was removed by rotary
evaporation; the polymer was dissolved in 0.1 N HO and dialyzed
overnight. The polymer was isolated by freeze drying. The dried
polymer and tris(2-aminoethylamine) (2 mL) were dissolved in
1,4-dioxane (100 mL) and refluxed for 24 hours. Dioxane was removed
under vacuum. The polymer was dissolved in minimum amount of
methanol and precipitated twice from diethyl ether. Polymer was
dissolved in water and dialyzed against water using MWCO-1000
membrane for 2 days. The resulting polymer solution was added to a
mixture of formaldehyde (15 mL) and formic acid (15 mL) at
0.degree. C. The reaction mixture was refluxed overnight. After
cooling to room temperature, the pH of the solution was adjusted to
10 using NaOH and the polymer was extracted with dichloromethane.
Dichloromethane was removed under vacuum; the polymer dissolved in
water and dialyzed using MWCO-1000 membrane for 3 days. The yield
of JNK-1 polymer was 10 g, and the amine content (by conductometric
titration) was 5.4 mol %.
Synthesis of JNK-2 Polymer (48 kDa HBSPCM Polymer)
[0251] JNK-2 polymer was synthesized using similar procedure as
that for JNK-1. First, a HPG-PEG-48K precursor polymer was
synthesized and a portion of the hydroxyl groups were converted
into tertiary amino groups by a similar procedure. Amine content of
the polymer (by conductometric titration) was 7.6 mol %.
Example 30
Comparative Analysis of UFH Embodiments Vs. Tinzaparin and
Enoxaparin
[0252] This example was performed to present a comparative analysis
of UFH embodiments vs. Tinzaparin and Enoxaparin.
[0253] FIGS. 24A-24C show the ability of HBSPCM polymers JNK-9,
JNK-10 and JNK-11 to neutralize UFH and LMWH tinzaparin and
enoxaparin in comparison to protamine and saline control as
measured by activated partial thromboplastin time (APTT).
[0254] FIG. 24A shows the neutralization of UFH by JNK-9, JNK-10
and JNK-11.
[0255] FIG. 24B shows the neutralization of LMWH Tinzaparin by
JNK-9, JNK-10 and JNK-11.
[0256] FIG. 24C shows the neutralization of LMWH Enoxaparin by
JNK-9, JNK-10 and JNK-11.
[0257] Ability of HBSPCM polymers JNK-9, JNK-10 and JNK-11 was
studied for the range of concentration from 0.01 to 0.25 mg/mL
prepared in 0.15 M saline. Citrated blood was spun at 3000 RPM for
10 min to obtain platelet poor plasma (PPP). The PPP was then
heparinized by adding 45 .mu.L of heparin (UFH, LMWH etc.) to 2200
.mu.L of PPP--final concentration of 2.0 IU/mL for UFH and
Tinzaparin and 1.14 IU/mL for Enoxaparin. Normal control was
prepared by adding 25 .mu.L of 0.15 M saline to 250 .mu.L of PPP.
25 .mu.L of each antidote solution (polymer or protamine) was added
to 225 uL of heparinized PPP. 250 .mu.L of APTT reagent (actin FSL)
was then added and samples were incubated for 3 min at 37.degree.
C. 250 .mu.L of normal control was combined with 250 uL of APTT
reagent and also incubated for 3 min. 50 .mu.L of 0.025M CaCl.sub.2
was added to 100 .mu.L aliquots of normal control and sample (in
triplicate). A heparinized control containing 225 uL of PPP and 25
.mu.L of saline was also prepared and run similar to normal
control. The results indicate that HBSPCM polymers JNK-9, JNK-10
and JNK-11 effectively neutralize UFH and LMWH tinzaparin and
enoxaparin over a broader range of concentration, while protamine
neutralizes UFH and LMWH only in the narrow concentration range,
and at higher concentrations protamine possesses anticoagulant
activity.
Example 31
Blood Compatibility of Various HBSPCMs
[0258] This example examined blood compatibility of various
HBSPCMs, and in particular the effect of the charge on the
polymer.
[0259] FIG. 25 shows the complement activation of HBSPCM polymer
having a molecular weight of 23 kDa with different charge density
(e.g., number of amine groups). The level of complement activation
was measured by a CH50 sheep erythrocyte lysis assay. Ten
microlitre of each of the 0.5 and 5 mg/mL polymers or protamine was
mixed with 90 .mu.L of serum for 1 h at 37.degree. C. The final
concentration of polymer/protamine to serum was 0.05 and 0.5 mg/mL.
One mg/mL heat-activated human IgG and 5 mM EDTA, after mixing with
serum, were the positive and negative controls, respectively, for
the study. 60 .mu.L of post-incubation serum/polymer or protamine
mixture was diluted by 120 .mu.L of GVB.sup.2+ (CompTech).
Seventy-five microlitre of GVB.sup.2+ diluted serum/polymer or
protamine mixture was incubated for 1 h at 37.degree. C. with 75
.mu.L of Ab-sensitized sheep erythrocyte (CompTech). The reaction
was stopped by addition of 300 .mu.L cold GVB-EDTA to each sample.
The samples were centrifuged and the optical density of supernatant
was measured at 540 nm. One hundred percent lysis of the sheep
erythrocyte was done by dH.sub.2O. The percentage of complement
activation was calculated by 100-% lysis of sheep erythrocytes.
[0260] The results reveal that 23 kDa HBSPCM polymer with 16-96
charges per polymer molecular do not activate the complement
system, whereas the polymer control sample with 92 charges (where
the primary amino groups are not methylated) and protamine showed
significant complement activation.
[0261] FIG. 26 shows the platelet activation of 23 kDa HBSPCM
polymers having a different number of amine groups per polymer
molecule (16-80 amine groups/polymer molecule). The level of
platelet activation was quantified by flow cytometry. Ninety
microliter of PRP was incubated at 37.degree. C. with 10 .mu.L of 5
mg/mL of polymer or protamine (final concentration 0.5 mg/mL).
After 1 h, aliquots of the incubation mixtures were removed for
assessment of the platelet activation state. Five microliter of
post-incubation platelet/polymer or protamine mixture, diluted in
HEPES buffer, was incubated for 20 minutes in the dark with 5 .mu.L
of monoclonal anti-CD62-PE (Immunotech). The samples were then
stopped with 0.5 mL of phosphate-buffered saline solution. The
level of platelet activation was analyzed in a BD FACSCanto II flow
cytometer (Becton Dickinson) by gating platelets specific events
based on their FITC-CD42 fluorescence and light scattering profile.
Activation of platelets was expressed as the percentage of platelet
activation marker CD62-PE fluorescence detected in the 10,000 total
events counted. Duplicate measurements were done, the mean of which
was reported. Controls were done for the flow cytometric analysis.
One U/ml of bovine thrombin (Sigma) was used as a positive control,
and PE conjugated goat anti-mouse IgG polyclonal antibodies
(Immunotech) was used as the non-specific binding control.
[0262] The results indicate that 23 kDa HBSPCM polymers with 16-80
number of charges per polymer molecule do not activate platelets,
whereas protamine showed significant levels of platelet
activation.
Example 32
Tolerance of HBSPCM Vs. Protamine
[0263] This example examined the tolerance of HBSPCM vs. protamine
in mice.
[0264] FIGS. 27 and 28 show the single dose tolerability of HBSPCM
polymer JNK-1 and protamine respectively, compared to a vehicle
control (saline) in female Balb/cJ mice.
[0265] For the study, the mice were individually weighed and
injected with a prescribed dose (mg/Kg) based on their individual
weights (200 .mu.L/20 g) of JNK-1 (FIG. 27), protamine (FIG. 28)
and control saline. Mice were continually monitored for acute signs
of toxicity for the first two hours following administration and
for additional toxicities 28 days post administration. Body weights
of the mice were measured over a period of 29 days.
[0266] The results are shown in FIG. 27 (HBSPCM) and FIG. 28
(Protamine). As is clearly shown in FIG. 27, these heparin binding
polymers are not toxic in mice up to the maximal dose studied (200
mg/Kg). In contrast, as shown in FIG. 28, the maximum dose of
protamine tolerated in mice was 20 mg/kg, with mice dying at doses
above 20 mg/kg.
Example 33
Biodistribution of HBSPCM-3
[0267] The biodistribution of HBSPCM-3 (23 kDa) was studied. The
molecule was radio-labeled (tritium) and was intravenously injected
into BALB/c mice. Radio-activity in different organs and plasma was
measured.
[0268] FIG. 29 and FIG. 30 show the results of the study conducted
to determine the pharmacokinetics and biodistribution of HBSPCM-3
(23 kDa) polymer in female Balb/cJ mice following bolus intravenous
injection.
[0269] 32 female Balb/cJ mice were administered with HBSPCM-3
polymer at a dose of 20 mg/Kg. Blood was collected at 5 min, 30
min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h post intravenous single
bolus injection. Four mice were terminated at each time point by
CO.sub.2 inhalation and blood was collected by cardiac puncture.
1004 whole blood sample was placed in pre-weighed tubes and the
remaining volume was processed for plasma. Upon termination, the
liver, spleen, lung, kidney and heart were weighed and processed
for scintillation counting. Livers were further processed by adding
water to make a 20% homogenate solution.
[0270] As shown in the charts, there was no accumulation in the
major organs such as liver, kidney and spleen suggesting non-toxic
behavior of HBSPCM-3.
Example 34
Biodistribution of HBSPCM-1
[0271] The biodistribution of HBSPCM-1 (116k) was studied. The
molecule was radio-labeled (tritium) and was intravenously injected
into BALB/c mice. Radio-activity in different organs and plasma was
measured. FIG. 31 and FIG. 32 show the results of a study conducted
to determine the pharmacokinetics and biodistribution of HBSPCM-1
(116 kDa) polymer in female Balb/cJ mice following bolus
intravenous injection.
[0272] 36 female Balb/cJ mice were administered with HBSPCM-1
polymer at a dose of 20 mg/kg. Blood was collected at 5 min, 30
min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h post intravenous
single bolus injection. Four mice were terminated at each time
point by CO.sub.2 inhalation and blood was collected by cardiac
puncture. 1004 whole blood sample was placed in pre-weighed tubes
and the remaining volume was processed for plasma. Upon
termination, the liver, spleen, lung, kidney and heart were weighed
and processed for scintillation counting. Livers were further
processed by adding water to make a 20% homogenate solution
[0273] The results are presented in FIGS. 31A, 31B, 31C, 31D, 32A,
and 32B. As shown in the charts, there was no accumulation in the
liver and spleen suggesting non-toxic behavior of HBSPCM.
Example 35
HBP to Reduce the Level of Effective Heparin
[0274] The present example outlines how a heparin binding polymer
can be used to reduce the level of effective heparin in a
subject.
[0275] A subject receiving treatment for a burn is administered an
amount of heparin. The amount of heparin is deemed to be excessive
at some point and the subject is then administered an adequate
amount of HBSPCM-3, to counteract the amount excessive amount of
heparin.
[0276] As will be appreciated by those of skill in the art, in
light of the present disclosure, in some embodiments, the
polymer-bound heparin can be cleared from the body depending on the
molecular weight. In some embodiments, polymers of molecular weight
less than 50 kDa can be cleared through the kidney, while the
higher molecular weight polymers can be excreted through feces.
Example 36
Heparin Binding Polymer for Reducing Heparin Levels for a Subject
Ex Vivo
[0277] The present example outlines how a heparin binding polymer
can be used to reduce heparin levels for a subject ex vivo.
[0278] A subject receiving treatment for acute coronary syndrome is
administered an amount of heparin. At some point, it is desired to
reduce the amount of heparin in the subject, and thus, blood is
taken from the subject and run through a filtering device that
includes HBSPCM-1 immobilized on a support such that the blood
passes over the HBSPCM-1. This allows the polymer to bind to and
remove the heparin, thereby providing filtered blood. The filtered
blood is then returned to the subject.
Example 37
Heparin Binding Polymer for Heparin Isolation
[0279] The present example outlines how a heparin binding polymer
can be used to isolate heparin.
[0280] A solution containing heparin is provided. The solution can
be the resulting mixture from a process of heparin preparation. A
heparin binding polymer, which includes a polyglycerol core and
polyvalent cationic groups, is immobilized on a surface. The
solution is flowed across the surface at room temperature and the
heparin is allowed to bind to the heparin binding polymer. The
surface is then washed with water (in the alternative, a buffer
solution (0.15 M NaCl or PBS or other similar solutions) can be
used) and then the isolated heparin can be eluted from the polymer
with a salt solution (1 to 3 M salt solution), which can be
performed at room temperature, or alternatively, at body
temperature. Some of the salt can then be removed from the solution
to provide an isolated heparin composition in an osmotically
physiological salt solution.
Example 38
HBSPCM Polymers for Drug Delivery Applications
[0281] The present example outlines the use of HBSPCM polymers for
the delivery of anionic drugs such as methotrexate, phenoxymethyl
pencillin, insulin, indomethacin, diclofenac etc. The cationic
nature of HBSPCM polymers results in effective binding to these
anionic drug molecules which could be released at a controlled rate
once inside the body.
[0282] Other aspects and features various embodiments will become
apparent to those ordinarily skilled in the art upon review of the
following description of specific examples.
[0283] The present disclosure is not to be limited in terms of the
particular embodiments described in this application, which are
intended as illustrations of various aspects. Many modifications
and variations can be made without departing from its spirit and
scope, as will be apparent to those skilled in the art.
Functionally equivalent methods and apparatuses within the scope of
the disclosure, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing
descriptions. Such modifications and variations are intended to
fall within the scope of the appended claims. The present
disclosure is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled. It is to be understood that this disclosure is
not limited to particular methods, reagents, compounds,
compositions or biological systems, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting.
[0284] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity.
[0285] It will be understood by those within the art that, in
general, terms used herein, and especially in the appended claims
(e.g., bodies of the appended claims) are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.). It will be
further understood by those within the art that if a specific
number of an introduced claim recitation is intended, such an
intent will be explicitly recited in the claim, and in the absence
of such recitation no such intent is present. For example, as an
aid to understanding, the following appended claims may contain
usage of the introductory phrases "at least one" and "one or more"
to introduce claim recitations. However, the use of such phrases
should not be construed to imply that the introduction of a claim
recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
embodiments containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should be interpreted to mean "at least one" or "one or
more"); the same holds true for the use of definite articles used
to introduce claim recitations. In addition, even if a specific
number of an introduced claim recitation is explicitly recited,
those skilled in the art will recognize that such recitation should
be interpreted to mean at least the recited number (e.g., the bare
recitation of "two recitations," without other modifiers, means at
least two recitations, or two or more recitations). Furthermore, in
those instances where a convention analogous to "at least one of A,
B, and C, etc." is used, in general such a construction is intended
in the sense one having skill in the art would understand the
convention (e.g., "a system having at least one of A, B, and C"
would include but not be limited to systems that have A alone, B
alone, C alone, A and B together, A and C together, B and C
together, and/or A, B, and C together, etc.). In those instances
where a convention analogous to "at least one of A, B, or C, etc."
is used, in general such a construction is intended in the sense
one having skill in the art would understand the convention (e.g.,
"a system having at least one of A, B, or C" would include but not
be limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, and/or A, B, and C
together, etc.). It will be further understood by those within the
art that virtually any disjunctive word and/or phrase presenting
two or more alternative terms, whether in the description, claims,
or drawings, should be understood to contemplate the possibilities
of including one of the terms, either of the terms, or both terms.
For example, the phrase "A or B" will be understood to include the
possibilities of "A" or "B" or "A and B."
[0286] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0287] As will be understood by one skilled in the art, for any and
all purposes, such as in terms of providing a written description,
all ranges disclosed herein also encompass any and all possible
subranges and combinations of subranges thereof. Any listed range
can be easily recognized as sufficiently describing and enabling
the same range being broken down into at least equal halves,
thirds, quarters, fifths, tenths, etc. As a non-limiting example,
each range discussed herein can be readily broken down into a lower
third, middle third and upper third, etc. As will also be
understood by one skilled in the art all language such as "up to,"
"at least," and the like include the number recited and refer to
ranges which can be subsequently broken down into subranges as
discussed above. Finally, as will be understood by one skilled in
the art, a range includes each individual member. Thus, for
example, a group having 1-3 cells refers to groups having 1, 2, or
3 cells. Similarly, a group having 1-5 cells refers to groups
having 1, 2, 3, 4, or 5 cells, and so forth.
[0288] From the foregoing, it will be appreciated that various
embodiments of the present disclosure have been described herein
for purposes of illustration, and that various modifications may be
made without departing from the scope and spirit of the present
disclosure. Accordingly, the various embodiments disclosed herein
are not intended to be limiting, with the true scope and spirit
being indicated by the following claims.
* * * * *