U.S. patent number RE38,558 [Application Number 09/460,298] was granted by the patent office on 2004-07-20 for polyoxypropylene/polyoxyethylene copolymers with improved biological activity.
This patent grant is currently assigned to CytRx Corporation. Invention is credited to Paula H. Culbreth, R. Martin Emanuele, Robert L. Hunter.
United States Patent |
RE38,558 |
Emanuele , et al. |
July 20, 2004 |
Polyoxypropylene/polyoxyethylene copolymers with improved
biological activity
Abstract
The present invention comprises novel preparations of
polyoxypropylene/polyoxyethylene copolymers which retain the
therapeutic activity of the commercial preparations, but are
substantially free from the undesirable effects which are inherent
in the prior art preparations. Because the preparations of
polyoxypropylene/polyoxyethylene copolymers which comprise the
present invention are a less polydisperse population of molecules
than the prior art polyoxypropylene/polyoxyethylene copolymers, the
biological activity of the copolymers is better defined and more
predictable.
Inventors: |
Emanuele; R. Martin
(Alpharetta, GA), Hunter; Robert L. (Bellaire, TX),
Culbreth; Paula H. (Loganville, GA) |
Assignee: |
CytRx Corporation (Los Angeles,
CA)
|
Family
ID: |
32686215 |
Appl.
No.: |
09/460,298 |
Filed: |
December 9, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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087136 |
Jul 2, 1993 |
5523492 |
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847874 |
Mar 13, 1992 |
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673289 |
Mar 19, 1991 |
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Reissue of: |
460192 |
Jun 2, 1995 |
05696298 |
Dec 9, 1997 |
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Current U.S.
Class: |
568/623;
568/624 |
Current CPC
Class: |
A61K
31/765 (20130101); A61K 31/77 (20130101); A61K
45/06 (20130101); C08G 65/08 (20130101); A61K
47/56 (20170801); C08G 65/2624 (20130101); C08G
65/30 (20130101); A61K 47/60 (20170801); C08G
65/2609 (20130101) |
Current International
Class: |
A61K
31/74 (20060101); A61K 31/765 (20060101); A61K
31/77 (20060101); A61K 45/00 (20060101); A61K
45/06 (20060101); A61K 47/48 (20060101); C08G
65/26 (20060101); C08G 65/08 (20060101); C08G
65/30 (20060101); C08G 65/00 (20060101); C07C
043/02 (); C07C 043/04 (); C07C 043/11 () |
Field of
Search: |
;568/623,624 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0000704 |
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Feb 1979 |
|
EP |
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0003399 |
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Sep 1979 |
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EP |
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0049422 |
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Apr 1982 |
|
EP |
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0098110 |
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Jan 1984 |
|
EP |
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33193/70 |
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Oct 1970 |
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JP |
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5094 |
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Jan 1979 |
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JP |
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206763/88 |
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Aug 1988 |
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JP |
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1183112 |
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Oct 1985 |
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SU |
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WO 87/06831 |
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Nov 1987 |
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WO |
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WO 87/06836 |
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Nov 1987 |
|
WO |
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WO 88/06038 |
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Aug 1988 |
|
WO |
|
WO 90/07336 |
|
Jul 1990 |
|
WO |
|
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|
Primary Examiner: Shaver; Paul F.
Attorney, Agent or Firm: Arismendi, Jr.; Andy
Parent Case Text
This is a division of application Ser. No. 08/087,136 filed Jul. 2,
1993, now U.S. Pat No. 5,523,492, which is a con of Ser. No.
07/847,874 Mar. 13, 1992, abandoned, which is a
continuation-in-part of U.S. patent application Ser.No. 07/673,289,
filed Mar. 19, 1991, now abandoned.
Claims
We claim:
1. A method of preparing a .[.non-toxic.]. surface-active copolymer
.Iadd.composition .Iaddend.including first condensing propylene
oxide with a base compound containing a plurality of reactive
hydrogen atoms to produce .Iadd.a first composition containing a
plurality of .Iaddend.polyoxypropylene .[.polymer.]. .Iadd.polymers
.Iaddend.and then condensing ethylene oxide with the
.Iadd.plurality of .Iaddend.polyoxypropylene .[.polymer.].
.Iadd.polymers .Iaddend.to produce a
polyoxypropylene/polyoxyethylene block copolymer .Iadd.composition,
said block copolymer composition consisting essentially of block
copolymers .Iaddend.with .Iadd.each of the block copolymers having
.Iaddend.the following general formula:
2. The method of claim 1, wherein the polydispersity value .[.of
the polyoxypropylene/polyoxyethylene copolymer.]. is less than
approximately 1.05.
3. The method of claim 1, wherein the polydispersity value .[.of
the polyoxypropylene/polyoxyethylene copolymer.]. is less than
approximately 1.03.
4. The method of claim 1, wherein the .[.polyoxypropylene
polymer.]. .Iadd.first composition .Iaddend.is purified by gel
permeation chromatography..[.
5. The method of claim 1, wherein the copolymer is substantially
unsaturated..].
6. .[.A method of preparing a non-toxic surface-active copolymer
including first condensing propylene oxide with a base compound
containing a plurality of reactive hydrogen atoms to produce
polyoxypropylene polymer and then condensing ethylene oxide with
the polyoxypropylene polymer to produce a
polyoxypropylene/polyoxyethylene block copolymer with the following
formula:
.Iadd.The method of claim 1, .Iaddend.wherein .Iadd."a" is an
integer such that .Iaddend.the molecular weight .[.of the
hydrophobe (C.sub.3 H.sub.6 O).]. .Iadd.represented by the
polyoxypropylene portion of the respective block copolymer
.Iaddend.is approximately 1750 daltons and the total molecular
weight of the .[.compound.]. .Iadd.respective block copolymer
.Iaddend.is approximately 8400 daltons.[., the improvement being
the purification of the polyoxypropylene polymer before the step of
condensing ethylene oxide with the polyoxypropylene polymer thereby
providing a polyoxypropylene/polyoxyethylene block copolymer
preparation with a polydispersity value of less than approximately
1.07.]. .
7. The method of claim 6, wherein the polydispersity value .[.of
the polyoxypropylene/polyoxyethylene copolymer.]. is less than
approximately 1.05.
8. The method of claim 6, wherein the polydispersity value .[.of
the polyoxypropylene/polyoxyethylene copolymer.]. is less than
approximately 1.03.
9. The method of claim 6, wherein the .[.polyoxypropylene
polymer.]. .Iadd.first composition .Iaddend.is purified by gel
permeation chromatography..[.
10. The method of claim 6, wherein the copolymer is substantially
unsaturated..].
11. A method of preparing a .[.non-toxic surface-active
copolymer.]. .Iadd.polyoxypropylene/polyoxyethylene block copolymer
composition .Iaddend.including first condensing propylene oxide
with a base compound containing a plurality of reactive hydrogen
atoms to produce .Iadd.a first composition containing a plurality
of .Iaddend.polyoxypropylene .[.polymer.]. .Iadd.polymers
.Iaddend.and then condensing ethylene oxide with the
.Iadd.plurality of .Iaddend.polyoxypropylene .[.polymer.].
.Iadd.polymers .Iaddend.to produce a
polyoxypropylene/polyoxyethylene block copolymer .Iadd.composition,
said block copolymer composition consisting essentially of block
copolymers .Iaddend.with .Iadd.each of the block copolymers having
.Iaddend.the following .Iadd.general .Iaddend.formula:
12. The method of claim 11, wherein the .Iadd.respective block
.Iaddend.copolymer has a molecular weight range of between
approximately 1,200 and 6500 daltons.
13. The method of claim 11, wherein polyoxyethylene portion of the
.Iadd.respective block .Iaddend.copolymer constitutes between
approximately 10% and 90% of the .Iadd.respective block
.Iaddend.copolymer.
14. The method of claim 11, wherein the copolymer is substantially
.[.unsaturated.]. .Iadd.free of unsaturation.Iaddend...Iadd.
15. A method for preparing a surface active composition including
first condensing propylene oxide with a base compound containing a
plurality of reactive hydrogen atoms to produce a first composition
containing a plurality of polyoxypropylene polymers and then
condensing ethylene oxide with the plurality of polyoxypropylene
polymers to produce a polyoxypropylene/polyoxyethylene block
copolymer composition, said block copolymer composition comprising
an impurity containing unsaturation and block copolymers with each
of the block copolymers having the following general formula:
16. The method of claim 15, wherein the fractionation is performed
by gel permeation chromatography. .Iaddend..Iadd.
17. The method of claim 15, wherein the polydispersity value is
less than approximately 1.05. .Iaddend..Iadd.
18. The method of claim 15, wherein the polydispersity value is
less than approximately 1.03. .Iaddend.
Description
TECHNICAL FIELD
The present invention relates to a preparation of
polyoxypropylene/polyoxyethylene copolymer which has an improved
toxicity and efficacy profile. The present invention also includes
polyoxypropylene/polyoxyethylene block copolymers with a
polydispersity value of less than approximately 1.05.
BACKGROUND OF THE INVENTION
Certain polyoxypropylene/polyoxyethylene copolymers have been found
to have beneficial biological effects when administered to a human
or animal. These beneficial biological effects are summarized as
follows:
Polyoxypropylene/polyoxyethylene Copolymers as Rheologic Agents
The copolymers can be used for treating circulatory diseases either
alone or in combination with other compounds, including but not
limited to, fibrinolytic enzymes, anticoagulants, free radical
scavengers, antiinflammatory agents, antibiotics, membrane
stabilizers and/or perfusion media. These activities have been
described in U.S. Pat. Nos. 4,801,452, 4,873,083, 4,879,109,
4,837,014, 4,897,263, 5,064,643; 5,028,599; 5,047,236; 5,089,260;
5,017,370; 5,078,995; 5,032,394; 5,041,288; 5,071,649; 5,039,520;
5,030,448; 4,997,644; 4,937,070; 5,080,894; and 4,937,070, all of
which are incorporated herein by reference.
The polyoxypropylene/polyoxyethylene copolymers have been shown to
have quite extraordinary therapeutic activities. The surface-active
copolymers are useful for treating pathologic hydrophobic
interactions in blood and other biological fluids of humans and
animals. This includes the use of a surface-active copolymer for
treatment of diseases and conditions in which resistance to blood
flow is pathologically increased by injury due to the presence of
adhesive hydrophobic proteins or damaged membranes. This adhesion
is produced by pathological hydrophobic interactions and does not
require the interaction of specific ligands with their receptors.
Such proteins and/or damaged membranes increase resistance in the
microvasculature by increasing friction and reducing the effective
radius of the blood vessel. It is believed that the most important
of these proteins is soluble fibrin.
Pathological hydrophobic interactions can be treated by
administering to the animal or human suffering from a condition
caused by a pathological hydrophobic interaction an effective
amount of a surface-active copolymer. The surface-active copolymer
may be administered as a solution by itself or it may by
administered with another agent, including, but not limited to, a
fibrinolytic enzyme, an anticoagulant, or an oxygen radical
scavenger.
The method described in the foregoing patents comprises
administering to an animal or human an effective amount of a
surface-active copolymer with the following general formula:
A preferred surface-active copolymer is a copolymer having the
following formula:
The surface-active copolymer is effective in any condition where
there is a pathological hydrophobic interaction between cells
and/or molecules. These interactions are believed to be caused by
1) a higher than normal concentration of fibrinogen, 2) generation
of intravascular or local soluble fibrin, especially high molecular
weight fibrin, 3) increased friction in the microvasculature, or 4)
mechanical or chemical trauma to blood components. All of these
conditions cause an increase in pathological hydrophobic
interactions of blood components such as cells and molecules.
It is believed that fibrin, especially soluble fibrin, increases
adhesion of cells to one another, markedly increases friction in
small blood vessels and increases viscosity of the blood,
especially at low shear rates. The effects of the surface-active
copolymer are believed to be essentially lubrication effects
because they reduce the friction caused by the adhesion.
Although not wanting to be bound by the following hypothesis, it is
believed that the surface-active copolymer acts according to the
following mechanism: Hydrophobic interactions are crucial
determinants of biologic structure. They hold the phospholipids
together in membranes and protein molecules in their native
configurations. An understanding of the biology of the
surface-active copolymer is necessary to appreciate the biologic
activities of the compound. Water is a strongly hydrogen bonding
liquid which, in its fluid state, forms bonds in all directions
with surrounding molecules. Exposure of a hydrophobic surface,
defined as any surface which forms insufficient bonds with water,
produces a surface tension or lack of balance in the hydrogen
bonding of water molecules. This force can be exceedingly strong.
The surface tension of pure water is approximately 82 dynes/cm.
This translates into a force of several hundred thousand pounds per
square inch on the surface molecules.
As two molecules or particles with hydrophobic surfaces approach,
they adhere avidly. This adhesion is driven by the reduction in
free energy which occurs when water molecules transfer from the
stressed non-hydrogen bonding hydrophobic surface to the
non-stressed bulk liquid phase. The energy holding such surfaces
together, the work of adhesion, is a direct function of the surface
tension of the particles:.sup.1
where W.sub.AB =work of adhesion or the energy necessary to
separate one square centimeter of particle interface AB into two
separate particles, .gamma..sub.A and .gamma..sub.B are the surface
tensions of particle A and particle B, .gamma..sub.AB the
interfacial tension between them.
Consequently, any particles or molecules in the circulation which
develop significant surface tensions win adhere to one another
spontaneously. Such adhesion within membranes and macromolecules is
necessary to maintain their integrity. We use the term "normal
hydrophobic interaction" to describe such forces. Under normal
circumstances, all cells and molecules in the circulation have
hydrophilic non-adhesive surfaces. Receptors and ligands which
modulate cell and molecular interactions are generally located on
the most hydrophilic exposed surfaces of cells and molecules where
they are free to move about in the aqueous media and to interact
with one another. Special carrier molecules are necessary to
transport lipids and other hydrophobic substances in the
circulation. In body fluids such as blood, nonspecific adhesive
forces between mobile elements are extremely undesirable. These
forces are defined as "pathologic hydrophobic interactions" because
they restrict movement of normally mobile elements and promote
inappropriate adhesion of cells and molecules.
In damaged tissue, hydrophobic domains normally located on the
interior of cells and molecules may become exposed and produce
pathologic adhesive surfaces whose interaction compounds the
damage. Fibrin deposited along vessel walls also provide an
adhesive surface. Such adhesive surfaces appear to be
characteristic of damaged tissue. It is believed that the ability
of the surface-active copolymer to bind to adhesive hydrophobic
surfaces and convert them to non-adhesive hydrated surfaces closely
resembling those of normal tissues underlies its potential
therapeutic activities in diverse disease conditions.
Adhesion due to surface tension described above is different from
the adhesion commonly studied in biology. The commonly studied
adhesion is due to specific receptor ligand interactions. In
particular, it is different from the receptor-mediated adhesion of
the fibrinogen--von Willibrands factor family of
proteins..sup.2
Both the hydrophilic and hydrophobic chains of the surface-active
copolymer have unique properties which contribute to biologic
activity. The hydrophilic chains of polyoxyethylene (POE) are
longer than those of most surfactants and they are flexible. They
bind water avidly by hydrogen bond acceptor interactions with
ether-linked oxygens. These long, strongly hydrated flexible chains
are relatively incompressible and form a barrier to hydrophobic
surfaces approaching one another. The hydroxyl moieties at the ends
of the molecule are the only groups capable of serving as hydrogen
bond donors. There are no charged groups.
This extremely limited repertoire of binding capabilities probably
explains the inability of the molecule to activate host mediator
and inflammatory mechanisms. The POE chains are not necessarily
inert, however. Polyoxyethylene can bind cations by ion-dipole
interactions with oxygen groups. The crown polyethers and reverse
octablock copolymer ionophores are examples of such cation
binding..sup.3 It is possible that the flexible POE chains form
configurations which bind and modulate calcium and other cation
movements in the vicinity of damaged membranes or other hydrophobic
structures.
The hydrophobic component of the surface-active copolymer is large,
weak and flexible. The energy with which it binds to a cell
membrane or protein molecule is less than the energy which holds
the membrane phospholipids together or maintains the tertiary
conformation of the protein. Consequently, unlike common detergents
which dissolve membrane lipids and proteins, the surface-active
copolymer adheres to damaged spots on membranes and prevents
propagation of the injury.
The ability of the surface-active copolymer to block adhesion of
fibrinogen to hydrophobic surfaces and the subsequent adhesion of
platelets and red blood cells is readily demonstrated in vitro.
Most surfactants prevent adhesion of hydrophobic particles to one
another, however, the surface-active copolymer has a unique balance
of properties which optimize the anti-adhesive activity while
minimizing toxicity. Thus, the surface-active copolymer is not
routinely used by biochemists who use nonionic suffactants to lyse
cells or dissolve membrane proteins. The surface-active copolymer
protects cells from lysis. The hydrophobe effectively competes with
damaged cells and molecules to prevent pathologic hydrophobic
interactions, but cannot disrupt the much stronger normal
hydrophobic interactions which maintain structural integrity.
The viscosity of blood is generally assumed to be the dominant
determinant of flow through vessels with a constant pressure and
geometry. In the smallest vessels, such as those in damaged tissue,
other factors become significant. When the diameter of the vessel
is less than that of the cell, the blood cell must deform in order
to enter the vessel and then must slide along the vessel wall
producing friction. The deformability of blood cells entering small
vessels has been extensively studied.sup.4 but the adhesive or
frictional component has not. The adhesion of cells to vessel walls
is generally attributed to specific interactions with von
Willebrand's factor and other specific adhesive molecules..sup.5
Our data suggests that in pathologic situations, friction resulting
from nonspecific physicochemical adhesion between the cell and the
vessel wall becomes a major determinant of flow.
Mathematically, both the strength of adhesion between two particles
and the friction force which resists sliding of one along the other
are direct functions of their surface tensions which are largely
determined by their degree of hydrophobic interaction. The friction
of a cell sliding through a small vessel consists of an adhesion
component and a deformation component.sup.6 which are in practice
difficult to separate:
where F is the friction of cells, Fa is the adhesion component and
Fd is the deformation component.
The deformation component within a vessel differs from that
required for entry into the vessel. It may be similar to that which
occurs in larger vessels with blood flowing at a high rate of
shear..sup.7 Friction within blood vessels has been studied very
little, but undoubtedly involves the same principles which apply to
polymer systems in which the friction force correlates directly
with the work of adhesion:.sup.8
where Fa is the adhesional component of the friction force, WA the
work of adhesion, and k and c constants which pertain to the
particular system studied. Many lubricants act as thin films which
separate the two surfaces and reduce adhesion..sup.9
The effects of the surface-active copolymer on microvascular blood
flow were evaluated in several models ranging from artificial in
vitro systems where critical variables could be rigidly controlled
to in vivo systems mimicking human disease. First, the
surface-active copolymer can be an effective lubricant when used at
therapeutic concentrations in a model designed to simulate movement
of large cells through small vessels. It markedly reduced the
adhesive component of friction, but had no detectable effect on the
deformation component of friction. Second, the surface-active
copolymer greatly accelerates the flow through the narrow channels
formed by the thrombogenic surfaces of glass and air. A drop of
blood was placed on a cover slip and viewed under a microscope with
cinemicroscopy during the time it took the blood to flow to the
edges of the cover slip in response to gentle pressure. The
surface-active copolymer inhibited the adhesion of platelets to the
glass and maintained the flexibility of red cells which enabled
them to pass through the microscopic channels. While the
surface-active copolymer did not inhibit the formation of rouleaux
by red cells, it did cause the rouleaux to be more flexible and
more easily disrupted. Third, the surface-active copolymer
increases the flow of blood through tortuous capillary-sized
fibrin-lined channels by over 20-fold. It decreased viscosity of
the blood by an amount (10%) far too small to account for the
increased flow.
In a more physiologic model, the surface-active copolymer increased
coronary blood flow by a similar amount in isolated rat hearts
perfused with human red blood cells at a 30% hematocrit following
ischemic damage.
In an in vivo model of stroke produced by ligature of the middle
cerebral artery of rabbits, the surface-active copolymer increases
blood flow to ischemic brain tissue. As much as a two-fold increase
was measured by a hydrogen washout technique. In each of these
models, there were controls for hemodilution and there was no
measurable effect on viscosity at any shear rate measured.
It is believed that available data suggests that the surface-active
copolymer acts as a lubricant to increase blood flow through
damaged tissues. It blocks adhesion of hydrophobic surfaces to one
another and thereby reduces friction and increases flow. This
hypothesis is strengthened by the observation that the
surface-active copolymer has little effect on blood flow in normal
tissues where such frictional forces are small..sup.10
The surface-active copolymers are not metabolized by the body and
are quickly eliminated from the blood. The half-life of the
copolymer in the blood is believed to be approximately two hours.
It is to be understood that the surface-active copolymer in the
improved fibrinolytic composition is not covalently bound to any of
the other components in the composition nor is it covalently bound
to any proteins.
The surface-active copolymer can be administered with a
fibrinolytic enzyme, a free radical scavenger, or it can be
administered alone for treatment of certain circulatory conditions
which either are caused by or cause pathological hydrophobic
interactions of blood components. These conditions include, but not
limited to, myocardial infarction, stroke, bowel or other tissue
infarctions, malignancies, adult respiratory distress syndrome
(ARDS), disseminated intravascular coagulation (DIC), diabetes,
unstable angina pectoris, hemolytic uremic syndrome, red cell
fragmentation syndrome, heat stroke, retained fetus, eclampsia,
malignant hypertension, burns, crush injuries, fractures, trauma
producing shock, major surgery, sepsis, bacterial, parasitic, viral
and rickettsial infections which promote activation of the
coagulation system, central nervous system trauma, and during and
immediately after any major surgery. It is believed that treatment
of the pathological hydrophobic interactions in the blood that
occurs in these conditions significantly reduces microvascular and
other complications that are commonly observed.
The surface-active copolymer is also effective in increasing the
collateral circulation to undamaged tissues with compromised blood
supply. Such tissues are frequently adjacent to areas of vascular
occlusion. The mechanism appears to be reducing pathological
hydrophobic interactions in small blood vessels. Circulatory
conditions where the surface-active copolymers are effective
include, but are not limited to, cerebral thrombosis, cerebral
embolus, myocardial infarction, unstable angina pectoris, transient
cerebral ischemic attacks, intermittent claudication of the legs,
plastic and reconstructive surgery, balloon angioplasty, peripheral
vascular surgery, and orthopedic surgery, especially when using a
tourniquet.
The surface-active copolymer has little effect on the viscosity of
normal blood at shear rates ranging from 2.3 sec.sup.-1 (low) to 90
sec.sup.-1 (high). However, it markedly reduces the abnormally high
viscosity found in postoperative patients and in those with certain
pathologic conditions. This observation posed two questions: 1)
what caused the elevated whole blood viscosity in these patients
and, 2) by what mechanisms did the surface-active copolymer, which
has only minor effects on the blood viscosity of healthy persons,
normalize pathologic elevations in viscosity?
It is generally accepted that hematocrit and plasma fibrinogen
levels are the major determinants of whole blood viscosity. This
has been confirmed in normal individuals and in many patients with
inflammatory conditions. However, these factors could not explain
the changes that were observed. In patients having coronary artery
cardiac bypass surgery, it was found that hematocrit fell an
average of 23.+-.4% and fibrinogen fell 48.+-.9% within six hours
after surgery. The viscosity did not decrease as expected, but
increased from a mean of 23.+-.2 to 38.+-.4 centipoise (at a shear
rate of 2.3 sec.sup.-1). Viscosities in excess of 100 were found in
some patients. The abnormally high viscosity of blood was
associated with circulating high molecular weight polymers of
soluble fibrin..sup.11 The soluble fibrin levels rose from 19.+-.5
.mu.g/ml to 43.+-.6 .mu.g/ml during surgery. These studies utilized
a colorimetric enzymatic assay for soluble fibrin.sup.12 and
Western blotting procedures with SDS agarose gels to determine the
molecular weight of the large protein polymers..sup.13
In the absence of specific receptors, cells and molecules in the
circulation adhere to one another if the adherence reduces the free
energy or surface tension between them. An assessment of the
surface tension of various components of the blood can be made by
measuring contact angles.
Red blood cells, lymphocytes, platelets, neutrophils all have
contact angles in the range of 14 to 17 degrees. Peripheral blood
proteins, such as albumin, .alpha..sub.2 macroglobulin, and Hageman
factor have contact angles in the slightly lower range of 12-15.
This means that these proteins have no adhesive energy for the
cells. In contrast, fibrinogen has a contact angle of 24 degrees
and soluble fibrin of 31. Consequently, fibrinogen adheres weakly
to red blood cells and other cells in the circulation promoting
rouleaux formation. Fibrin promotes a very much stronger adhesion
than fibrinogen because of its elevated contact angle and its
tendency to form polymers with fibrinogen. Soluble fibrin in the
circulation produces the increased adhesion which results in a very
markedly increased viscosity at low shear rates. This adhesion also
involves the endothelial walls of the blood vessels. If the
adhesive forces are insufficient to slow movement of cells, they
produce an increased friction. This is especially important in the
very small blood vessels and capillaries whose diameters are equal
to or less than that of the circulating cells. The friction of
cells sliding through these small vessels is significant. The
surface-active copolymer blocks the adhesion of fibrinogen and
fibrin to hydrophobic surfaces of cells and endothelial cells. This
prevents their adhesion and lubricates them so there is a greatly
reduced resistance to flow. This can be measured only partially by
measurements of viscosity.
Whether a certain fibrinogen level is sufficient to cause a problem
in circulation is dependent upon several parameters of the
individual patient. High hematocrits and high levels of fibrinogen
are widely regarded as the primary contributors to increased
viscosity. However, elevated fibrinogen levels are frequently
associated with elevated soluble fibrin in the circulation. Careful
studies have demonstrated that the fibrin is frequently responsible
for the most severe changes. The normal level of fibrinogen is
200-400 .mu.g/ml. It has been determined that, in most patients,
fibrinogen levels of greater than approximately 800 .mu.g/ml will
cause the high blood viscosity at the low shear rates mentioned
hereinabove. The normal level of soluble fibrin has been reported
to be approximately 9.2.+-.1.9..sup.14 Using the Wiman and
R.ang.nby assay, viscosity at low shear rates was unacceptably high
above about 15 .mu.g/ml. It must be understood that soluble fibrin
means molecular species that have a molecular weight of from about
600,000 to several million.
Numerous methods have been used for demonstrating soluble fibrin.
These include cryoprecipitation especially cryofibrinogen. Heparin
has been used to augment the precipitate formation. Ethanol and
protamine also precipitate fibrin from plasma. Modem techniques
have demonstrated that the soluble fibrin in the circulation is
generally complexed with solubilizing agents. These are most
frequently fibrinogen or fibrin degradation products. Des AA fibrin
in which only the fibrin of peptide A moieties have been cleaved,
tends to form relatively small aggregates consisting of one
molecule of fibrin with two of fibrinogen. If both the A and B
peptides have been cleaved to produce des AABB fibrin, then much
larger aggregates are produced in the circulation. Fibrin
degradation products can polymerize with fibrin to produce varying
size aggregates depending upon the particular product involved.
Soluble fibrin in the circulation can markedly increase blood
viscosity, especially at low shear rates. However, the relevance of
this for clinical situations remains unclear. Viscosity assesses
primarily the aggregation of red blood cells which is only one of
many factors which determine in vivo circulation. Other factors
affected by soluble fibrin are the endothelial cells, white blood
cells and platelets. Soluble fibrin is chemotactic for endothelial
cells, adheres to them avidly and causes their disorganization. It
also has stimulatory effects for white blood cells, especially
macrophages. Some of the effects of soluble fibrin may be mediated
by specific receptors on various types of cells. However, since the
free energy, as measured by contact angles of soluble fibrin, is
less than that of any other plasma protein, it adheres avidly by a
nonspecific hydrophobic interactions to virtually all formed
elements in the blood.
Circulating soluble fibrin is normally cleared by macrophages and
fibrinolytic mechanisms without producing damage. However, if the
production of soluble fibrin is too great or if the clearance
mechanisms have been compromised or if complicating disease factors
are present, then soluble fibrin can induce deleterious
reactions.
Soluble fibrin is produced in damaged or inflamed tissues.
Consequently, its effects are most pronounced in these tissues
where it coats endothelial cells and circulating blood cells in a
fashion which markedly reduces perfusion. The largest effects are
in the small blood vessels where soluble fibrin coating the
endothelial cells and white blood cells produces a severe increase
in friction to the movement of white cells through the small
vessels. Friction appears to be a much more severe problem with
white blood cells and red blood cells because they are larger and
much more rigid.
If production of soluble fibrin is sufficient, then effects are
noticed in other areas. The best studied is the adult respiratory
distress syndrome where soluble fibrin produced in areas of damaged
tissue produces microthrombi and other processes in the lungs which
can cause pulmonary failure. However, lesser degrees of vascular
compromise can be demonstrated in many other organs.
Soluble fibrin, either alone or in complex with fibrinogen and
other materials, is now recognized as being a major contributor to
the pathogenesis of a diverse range of vascular diseases ranging
from coronary thrombosis through trauma, bums, reperfusion injury
following transplantation or any other condition where there has
been localized or generalized activation of coagulation. A recent
study demonstrated that virtually all patients with acute
myocardial infarction or unstable angina pectoris have markedly
elevated levels of soluble fibrin in their circulation.
An example of the effects of soluble fibrin has been shown in
studies using dogs. A normal dog is subjected to a hysterectomy.
Then, while the animal is still under anesthesia, the external
jugular vein is carefully dissected. Alternatively, the vein may be
occluded by gentle pressure with the fingers for seven minutes. It
is examined by scanning electron microscopy for adhesion of fibrin,
red blood cells and other formed elements.
One finds that very few cells adhere to the endothelia of veins
from dogs which had not undergone hysterectomy, whether or not
there had been stasis produced by seven minutes occlusion.
Similarly, there was only a small increase in adhesion of red blood
cells to the endothelium of the jugular vein in animals who had
undergone hysterectomy. If, however, the animals had a hysterectomy
in addition to mild seven minute occlusion of the veins, then there
was a striking increase in adhesion of formed elements of blood to
the endothelial surfaces in some cases producing frank mural
thrombi. Both red blood cells and fibrin were visibly adherent to
the endothelial surfaces. In addition, there was disruption of the
normal endothelial architecture. All of the animals had elevated
levels of soluble fibrin after the surgery. This model demonstrates
the effects of soluble fibrin produced by relatively localized
surgery to produce a greatly increased risk of deep vein thrombosis
at a distant site.
The surface-active copolymer addresses the problems of fibrin and
fibrinogen in the blood by inhibiting the adhesion of fibrin,
fibrinogen, platelets, red blood cells and other detectable
elements of the blood stream. It blocks the formation of a thrombus
on a surface. The surface-active copolymer has no effect on the
viscosity of water or plasma. However, it markedly increases the
rate of flow of water and plasma in small segments through tubes.
The presence of air interfaces at the end of the columns or air
bubbles which provide a significant surface tension produce a
friction along the walls of the tubes. The surface-active copolymer
reduces this surface tension and the friction and improves flow.
This is an example whereby the surface-active copolymer improves
flow of fluid through tissues through a tube even though it has no
effect on the viscosity of the fluid as usually measured.
The surface-active copolymer has only a small effect on the
viscosity of whole blood from normal individuals. It has little
effect on the increase that occurs with high hematocrit. However,
it has an effect on the very large increase in viscosity at low
shear rates thought to be caused by soluble fibrin and fibrinogen
polymers.
Recent studies demonstrate that the surface-active copolymer also
has the ability to protect myocardial and other cells from a
variety of noxious insults. During prolonged ischemia, myocardial
cells undergo "irreversible injury." Cells which sustain
irreversible injury are morphologically intact but are unable to
survive when returned to a normal environment. Within minutes of
reperfusion with oxygenated blood, cells containing such occult
lesions develop swelling and contraction bands and die.
Irreversibly injured myocardial cells have mechanical and osmotic
fragility and latent activation of lipases, proteases and other
enzymes. Reperfusion initiates a series of events including calcium
loading, cell swelling, mechanical membrane rupture and the
formation of oxygen free radicals which rapidly destroy the cell.
The surface-active copolymer retards such injury in the isolated
perfused rat heart model. The mechanisms probably include osmotic
stabilization and increased mechanical resistance in a fashion
similar to that known for red blood cells.
The protective effects of the surface-active copolymer on the
myocardium are not limited to the myocardial cells. It also
protects the endothelial cells of the microvasculature as assessed
morphologically. By maintaining the integrity of such cells and
helping to restore and maintain non-adhesive surfaces, the
surface-active copolymer tends to reduce the adhesion of
macromolecules and cells in the microvasculature, to reduce
coronary vascular resistance and to retard development of the no
reflow phenomenon.
Examples of conditions where the surface-active copolymer can be
used is in the treatment of sickle cell disease and preservation of
organs for transplantation. In both of these embodiments, blood
flow is reduced because of pathologic hydrophobic interactions.
During a sickle cell crisis, sickled red blood cells aggregate
because of the abnormal shape of the cells. In many cases, there
are high concentrations of soluble fibrin due to disseminated
intravascular coagulation. This results in pathological hydrophobic
interactions between blood cells, cells lining the blood vessels
and soluble fibrin and fibrinogen. By administering to the patient
the surface-active copolymer, blood flow is increased and tissue
damage is thereby reduced. The surface-active copolymer may be
given prior to a sickle cell crisis to prevent onset of the crisis.
In addition, the solution with the effective amount of
surface-active copolymer may also contain an effective amount of
anticoagulant.
In organs that have been removed from a donor for transplantation,
the tissue is damaged due to ischemia and lack of blood.
Preferably, the surface-active copolymer is mixed with a perfusion
medium. The perfusion media that can be used with the
surface-active copolymer are well known to those of ordinary skill
in the art. The perfusion media can also be whole blood or plasma.
The solution can be perfused through the organ thereby reducing the
damage to the tissue. Because the tissue damage is reduced by
perfusing the organ with the surface-active copolymer solution, the
time the organ is viable and therefore the time the organ can be
transplanted is increased.
Because the surface-active copolymer improves flow of blood through
diseased or damaged tissue with minimal effect on blood flow in
normal tissue, it is contemplated that the surface-active copolymer
includes a method for delivering drugs to damaged tissue comprising
the step of administering to the animal or human a solution
containing an effective amount of a drug, and an effective amount
of the surface-active copolymer.
Any drug that has an activity in diseased or damaged tissue is
suitable for use with the surface-active copolymer. These drugs
include: 1. antimicrobial drugs antibiotics antifungal drugs
antiviral drugs antiparasitic drugs; 2. antifungal drugs; 3.
chemotherapeutic drugs for treating cancers and certain infections;
4. free radical scavenger drugs, including those drugs that prevent
the production of free radicals; 5. fibrinolytic drugs; 6.
perfusion media; 7. anti-inflammatories, including, but not limited
to, both steroids and nonsteroid antiinflammatory drugs; 8.
membrane stabilizers, such as dilantin; 9. anticoagulants; 10.
ionotropic drugs, such as calcium channel blockers; 11. autonomic
nervous system modulators.
Polyoxypropylene/polyoxyethylene Copolymers as Adjuvants
Other polyoxypropylene/polyoxyethylene copolymers are also useful
as an adjuvant and a vaccine which is comprised of an antigen and
an improved adjuvant. In one embodiment, the antigen is admixed
with an effective amount of a surface-active copolymer having the
following general formula:
The improved vaccine also comprises an antigen and an adjuvant
wherein the adjuvant comprises a surface-active copolymer with the
following general formula:
The improved vaccine also comprises an antigen and an adjuvant
wherein the adjuvant comprises a surface-active copolymer with the
following general formula:
HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sup.6 O).sub.a (C.sub.2
H.sub.4 O).sub.b H wherein the molecular weight of the hydrophobe
(C.sub.3 H.sub.6 O) is between approximately 3000 to 5500 daltons
and the percentage of hydrophile (C.sub.2 H.sub.4 O) is between
approximately 5% and 15% by weight, and a lipopolysaccharide (LPS)
derivative. The adjuvant comprising a combination of LPS and
surface-active copolymer produces a synergy of effects in terms of
peak titer, time to reach peak titer and length of time of
response. In addition, the combination tends to increase the
protective IgG2 isotypes.
The adjuvants also comprise an octablock copolymer (poloxamine)
with the following general formula: ##STR1##
wherein: the molecular weight of the hydrophobe portion of the
octablock copolymer consisting of (C.sub.3 H.sub.6 O) is between
approximately 5000 and 7000 daltons; a is a number such that the
hydrophile portion represented by (C.sub.2 H.sub.4 O) constitutes
between approximately 10% and 40% of the total molecular weight of
the compound; b is a number such that the (C.sub.3 H.sub.6 O)
portion of the octablock copolymer constitute between approximately
60% and 90% of the compound and a lipopolysaccharide
derivative.
The (C.sub.3 H.sub.6 O) portion of the copolymer can constitute up
to 95% of the compound. The (C.sub.2 H.sub.4 O) portion of the
copolymer can constitute as low as 5% of the compound.
The combination of lipid conjugated polysaccharide with copolymer
and an immunomodulating agent such as monophosphoryl lipid A,
induces the production of a strong IgG response in which all of the
subclasses of IgG are present. In particular, the IgG2 and IgG3
subclasses which are protective against pneumococcal infections are
predominant. This is an unexpected finding because there is no
protein or peptide in the immunogen preparation. It is believed
that peptide moieties are essential for stimulating T cells which
are required for production of these isotypes. Others have reported
that polysaccharides are incapable of stimulating T cells.
Nevertheless, the combination of copolymer, lipid conjugated
polysaccharide and immunomodulating agent is able to produce such a
response. The adjuvant activity of the poloxamers and the
poloxamines is described in detail in copending U.S. patent
application Ser. No. 07/544,831, which is incorporated herein by
reference.
Polyoxypropylene/polyoxyethylene Copolymers as Antiinfective
Agents
Another group of polyoxypropylene/polyoxyethylene copolymers
inhibit the growth of bacteria and viruses. For example, these
surface-active copolymers have been shown to inhibit HIV viruses,
Mycobacteria species and Toxoplasma gondii.
The surface-active copolymers are effective in treating a viral
infection in a human or animal including infections caused by the
HIV virus or related strains. The present invention provides a
composition that can be administered to patients who are infected
with HIV viruses or similar viruses. The surface-active copolymer
is effective in inhibiting or suppressing the replication of the
HIV virus and related virus strains in cells.
The surface-active copolymers are useful for treating infections
caused by microorganisms when used alone or with a conventional
antibiotic. Several conventional antibiotics that can be used with
the surface-active copolymer include, but are not limited to,
rifampin, isoniazid, ethambutol, gentamicin, tetracycline, and
erythromycin.
The surface-active copolymer has the following general formula:
The antiinfective activity of the poloxamers is described in detail
in copending U.S. patent application Ser. No. 07/760,808, which is
incorporated herein by reference.
Polyoxypropylene/polyoxyethylene Copolymers as Growth Stimulators
and Immune Stimulators
Certain of the polyoxypropylene/polyoxyethylene copolymers are
capable of effecting biological systems in several different ways.
The biologically-active copolymers are capable of stimulating the
growth of an organism, stimulating the motor activity of an
organism, stimulating the production of T-cells in the thymus,
peripheral lymphoid tissue, and bone marrow cells of an animal, and
stimulating immune responsiveness of poultry.
The biologically-active copolymers also have a wide variety of
effects on individual cells. These compounds have ionophoric
activity, i.e., they cause certain ions to be transported across
cell membranes. The compounds can cause non-cytolytic mast cell
degranulation with subsequent histamine release. In addition, it
has been found that certain members of this class of
biologically-active copolymers are capable of specifically killing
certain cancer cell lines.
Certain of the biologically-active copolymers can be administered
orally to animals to stimulate the growth of food animals such as
chickens and swine. These and other biological activities are
discussed in detail in copending U.S. patent application Ser. Nos.
07/107,358 and 07/610,417, which are incorporated herein by
reference.
Polyoxypropylene/polyoxyethylene Copolymer Structure
The surface-active copolymer blocks are formed by condensation of
ethylene oxide and propylene oxide at elevated temperature and
pressure in the presence of a basic catalyst. However, there is
statistical variation in the number of monomer units which combine
to form a polymer chain in each copolymer. The molecular weights
given are approximations of the average weight of copolymer
molecule in each preparation. A more detailed discussion of the
preparation of these compounds is found in U.S. Pat. No. 2,674,619,
which is incorporated herein by reference. A more general
discussion of the structure of poloxamers and poloxamine block
copolymers can be found in Schmolka, I. R., "A Review of Block
Polymer Surfactants", J. AM. OIL CHEMISTS' SOC., 54:110-116 (1977),
which is incorporated herein by reference.
It has been determined that the commercially available preparations
of polyoxypropylene/polyoxyethylene copolymers vary widely relative
to the size and configuration of the constituent molecules. For
example, the preparation of poloxamer 188 that is purchased from
BASF (Parsippany, N.J.) has a published structure of a molecular
weight of the hydrophobe (C.sub.3 H.sub.6 O) of approximately 1750
daltons and the total molecular weight of the compound of
approximately 8400 daltons. In reality, the compound is composed of
molecules which range from a molecular weight of less than 3,000
daltons to over 20,000 daltons. The molecular diversity and
distribution of molecules of commercial poloxamer 188 is
illustrated by broad primary and secondary peaks detected using gel
permeation chromatography.
In addition to the wide variation in polymer size in the poloxamer
preparations currently available, it has been further determined
that these fractions contain significant amounts of unsaturation.
It is believed that this unsaturation in the polymer molecule is
responsible, at least in part, for the toxicity and variable
biological activities of the available poloxamer preparations.
Thus, the wide diversity of molecules which are present in the
commercially available polyoxypropylene/polyoxyethylene copolymers
make prediction of the biological activity difficult. In addition,
as is shown in the poloxamer 188 preparations, the presence of
other molecular species in the preparation can lead to unwanted
biological activities.
The surface-active copolymer poloxamer 188 has been used as an
emulsifier for an artificial blood preparation containing
perfluorocarbons. It has been reported that patients receiving the
artificial blood preparations have exhibited toxic reactions. The
toxic reactions included activation of complement.sup.15, paralysis
of phagocyte migration.sup.16, and cytotoxicity to human and animal
cells in tissue culture.sup.17. Efforts using supercritical fluid
fractionalion to reduce the toxicity of the copolymers proved only
partially successful..sup.18 In addition, in toxicological studies
in beagle dogs, infusion of poloxamer 188 was shown to result in
elevated liver enzymes, (SCOT) and increased organ weights
(kidney). Histologic evaluation of the kidney demonstrated a dose
related cytoplasmic vacuolation of the proximal tubular epithelial
cells.
The enormous variation that can occur in biological activity when
only small changes are made in chain length in the poloxamer
copolymers is illustrated in Hunter, et al..sup.19 The authors show
that a difference of 10% in the chain length of the polyoxyethylene
portions of the poloxamer polymer can mean the difference between
an excellent adjuvant and no adjuvant activity at all. Poloxamer
121 has a molecular weight of approximately 4400 daltons and
contains approximately 10% by weight of polyoxyethelene. Poloxamer
122 has a molecular weight of approximately 5000 daltons and
contains approximately 20% by weight of polyoxyethelene. The amount
of polyoxypropylene in each molecule is approximately the same. As
shown in Hunter, et al., when poloxamer 121 was used as an adjuvant
with bovine serum albumin, the antibody titers were 67,814.+-.5916.
When poloxamer 122 was used as an adjuvant with bovine serum
albumin under the same conditions, the antibody titer against BSA
was 184.+-.45. The control titer without any adjuvant was <100.
Thus, a relatively small change in the chain length of the
poloxamer can result in enormous changes in biological
activity.
Because the commercially available sources of the
polyoxypropylene/polyoxyethylene copolymers have been reported to
exhibit toxicity as well as variation in biological activity, what
is needed is a preparation of polyoxypropylene/polyoxyethylene
copolymers which retain the therapeutic activities of the
commercial preparations but are free from their other biological
activities such as toxicity. In addition, what is needed is a
preparation of polyoxypropylene/polyoxyethylene copolymers which is
less polydisperse in molecular weight and contains less
unsaturation and therefore is more efficacious.
SUMMARY OF THE INVENTION
The present invention comprises novel preparations of
polyoxypropylene/polyoxyethylene copolymers which retain the
therapeutic activity of the commercial preparations, but are free
from the undesirable effects which are inherent in the prior art
preparations. Because the polyoxypropylene/polyoxyethylene
copolymers which comprise the present invention are a less
polydisperse population of molecules than the prior art
polyoxypropylene/polyoxyethylene copolymers, the biological
activity of the copolymers is better defined and more predictable.
In addition, the polyoxypropylene/polyoxyethylene copolymers which
comprise the present invention are substantially free of
unsaturation.
The present invention also comprises a
polyoxypropylene/polyoxyethylene copolymer which has the following
formula:
It has been determined that the toxicity exhibited by the
commercially available surface-active copolymer poloxamer 188 is
primarily due to the small amounts of high and low molecular weight
molecules that are present as a result of the manufacturing
process. The high molecular weight molecules (those greater than
15,000 daltons) are probably responsible for activation of the
complement system. The low molecular weight molecules (those lower
than 5,000 daltons) have detergent-like physical properties which
can be toxic to cells in culture. In addition, the low molecular
weight molecules have unsaturated polymers present in the
population.
The optimal rheologic molecules of poloxamer 188 are approximately
8,400 to 9400 daltons. It has also been determined that poloxamer
188 molecules above 15,000 and below 5,000 daltons are less
effective rheologic agents and exhibit unwanted side effects. A
preparation containing molecules between 5,000 and 15,000 daltons
is a more efficient rheologic agent.
The present invention also includes a method of preparing
polyoxypropylene/polyoxyethylene block copolymers with
polydispersity values of less than 1.05. The method of preparing a
non-toxic surface-active copolymer includes first condensing
propylene oxide with a base compound containing a plurality of
reactive hydrogen atoms to produce polyoxypropylene polymer and
then condensing ethylene oxide with the polyoxypropylene polymer to
produce a polyoxypropylene/polyoxyethylene block copolymer with the
following general formula:
Accordingly, it is an object of the present invention to provide a
surface-active copolymer with a higher proportion of
therapeutically active molecules while also eliminating molecules
responsible for toxic effects.
It is another object of the present invention to provide a more
homogeneous polyoxypropylene/polyoxyethylene copolymer relative to
the molecular weight range.
It is another object of the present invention to provide a
preparation of polyoxyethylene/polyoxypropylene block copolymer
with a polydispersity value of less than 1.05.
It is another object of the present invention to provide a
preparation of polyoxyethylene/polyoxypropylene block copolymer
with substantially no unsaturation.
It is another object of the present invention to provide a
surface-active copolymer with the therapeutic activity of poloxamer
188 that will not activate complement.
It is yet another object of the present invention to provide a
purified poloxamer 188 that can be used safely in both humans and
animals in treating tissue that has been damaged by ischemia.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals in treating tissue that has been damaged by reperfusion
injury.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals as a vaccine adjuvant.
It is another object of the present invention to provide a
surface-active copolymer with the therapeutic activity of poloxamer
188 that is not cytotoxic.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals in treating stroke.
It is yet another object of the present invention to provide a
surface-active copolymer which has less renal toxicity and less
detergent-like activity.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals as an antimicrobial agent.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals as an antibacterial, an antiviral, an antifungal and an
antiprotozoa agent.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals in treating myocardial damage.
It is yet another object of the present invention to provide a
surface-active copolymer that can be used safely in both humans and
animals in treating adult respiratory distress syndrome.
These and other objects, features and advantages of the present
invention will become apparent after a review of the following
detailed description of the disclosed embodiments and the appended
claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a poloxamer grid for naming poloxmer compounds.
FIG. 2 is a chromatogram of commercially available poloxamer 188
subjected to gel permeation chromatography.
FIG. 3 is a chromatogram of fraction 1 of the poloxamer 188
collected from the chromatographic run described in Example I.
FIG. 4 is a chromatogram of fraction 2 of the poloxamer 188
collected from the chromatographic run described in Example I.
FIG. 5 is a chromatogram of fraction 3 of the poloxamer 188
collected from the chromatographic run described in Example I.
FIG. 6 is a chromatogram of fraction 4 of the poloxamer 188
collected from the chromatographic run described in Example I.
FIG. 7 is a chromatogram of fraction 5 of the poloxamer 188
collected from the chromatographic run described in Example I.
FIG. 8 is a chromatogram of fraction 6 of the poloxamer 188
collected from the chromatographic run described in Example I.
FIGS. 9A through 9C are gel permeation chromatograms of
unfractionated and fractionated poloxamer 760.5.
FIGS. 10A through 10C are nuclear magnetic spectra of the fractions
represented in FIGS. 9A through 9C.
FIGS. 11A through 11C are gel permeation chromatograms of three
fractions of poloxamer 188.
FIGS. 12A through 12C are gel permeation chromatograms of
unfractionated and fractionated poloxamer 331.
DETAILED DESCRIPTION
Although the prior art preparations of
polyoxypropylene/polyoxyethylene block copolymers may have been
suitable for industrial uses, it has been determined that the newly
discovered uses for the copolymers as therapeutic agents require
less polydisperse populations of molecules in the preparations.
The present invention comprises polyoxypropylene/polyoxyethylene
copolymers that have a polydisperse value of less than 1.05. The
novel copolymers can be prepared by removing disparate molecules
from the prior art preparation or by preparing the copolymer
according to the method that is contemplated as part of the present
invention. The method of preparation of the copolymers of the
present invention is the purification of the polyoxypropylene block
of the polyoxypropylene/polyoxyethylene copolymer before the
polyoxyethylene blocks are added to the molecule. In this way, the
partially polymerized polyoxypropylene polymers are removed before
the addition of polyoxyethylene polymers to the molecule. This
results in a block copolymer that is within the physical parameters
which are contemplated as the present invention.
The present invention also comprises a
polyoxypropylene/polyoxyethylene block copolymer which has the
following formula:
wherein the molecular weight represented by the polyoxypropylene
portion of the copolymer is between approximately 900 and 15000
daltons with a more preferred molecular weight of between 1,200 and
6500 daltons and the molecular weight represented by the
polyoxyethylene portion of the copolymer constitutes between
approximately 5% and 95% of the copolymer with a more preferred
range of between approximately 10% and 90% of the copolymer and the
polydispersity value is less than approximately 1.07.
The present invention also comprises a
polyoxypropylene/polyoxyethylene block copolymer which has the
following formula:
HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2
H.sub.4 O).sub.b H
wherein the molecular weight of the hydrophobe (C.sub.3 H.sub.6 O)
is approximately 1750 daltons and the average molecular weight of
the compound is approximately 8300 to 9400 daltons. The compound
has a molecular weight distribution ranging from approximately
5,000 to 15,000 daltons with a preferred molecular weight range of
between approximately 7,000 to 12,000 daltons. In addition, the
copolymer has substantially no unsaturation as measured by nuclear
magnetic resonance.
The nomenclature of the poloxamer compounds is based on a poloxamer
grid (FIG. 1). The poloxamer grid is the relationship between
nomenclature and composition of the various polymer members. The
hydrophobe (polyoxypropylene) molecular weights are given as
approximate midpoints of ranges. The first two digits of a
poloxamer number on the grid, multiplied by 100, gives the
approximate molecular weight of the hydrophobe. The last digit,
times 10, gives the approximate weight percent of the hydrophile
(polyoxyethylene) content of the surfactant.sup.20. For example,
poloxamer 407, shown in the upper right hand quadrant of the grid
(FIG. 1), is derived from a 4000 molecular weight hydrophobe with
the hydrophile comprising 70% of the total molecular weight of the
copolymer. Another example is poloxmer 760.5 which has a hydrophobe
with a molecular weight of 7600 daltons and has a hydrophile which
comprises 5% of the total molecular weight of the copolymer.
The representative poloxamers that are described in this patent
application along with their Pluronic.RTM. numbers are shown in
Table I.
TABLE I Poloxamer No. Pluronic .RTM. No. % POE 188 F68 80% 331 L101
10% 760.5 L180.5 5% 1000.5 L331 5%
Although molecular weight averages are important and useful when
characterizing polymers in general, it is important to know the
molecular weight distribution of a polymer. Some processing and
end-use characteristics (melt flow, flex life, tensile strength,
etc.) are often predicted or understood by observing the values
and/or changes occurring in specific molecular weight averages.
These values can also be assigned to biological properties of the
polyoxypropylene/polyoxyethylene copolymers. A list of the
processing characteristics follows.
Molecular Weight Processing Averages Characteristics Mz Flex
life/stiffness Mn Brittleness; flow Mw Tensile strength
For example, the breadth of the distribution is known as the
polydispersity (D) and is usually defined as Mw/Mn. A monodisperse
sample is defined as one in which all molecules are identical. In
such a case, the polydispersity (Mw/Mn) is 1.0. Narrow molecular
weight standards have a value of D near 1 and a typical polymer has
a range of 2 to 5. Some polymers have a polydispersity in excess of
20.
The equations for expressing polydispersity are as follows:
##EQU1##
where:
Area.sub.1 =area of the ith slice
Mi=molecular weight of the ith slice
Thus, by calculating the parameters listed above, one can specify a
certain polydispersity that is acceptable for a pharmaceutical
preparation. A high polydispersity value indicates a wide variation
in size for the population of molecules in a given preparation
while a lower polydispersity value indicates less variation.
Because molecular size is an important determinant of biological
activity, it is important to restrict the dispersity of the
molecules in the preparation in order to achieve a more homogeneous
biological effect. Thus, the polydispersity measurement can be used
to measure the dispersity of molecules in a preparation and
correlates to that compound's potential for variation in biological
activity.
It is to be understood that the polydispersity values that are
described herein were determined from chromatograms which were
obtained using a Model 600E Powerline chromatographic system
equipped with a column heater module, a Model 410 refractive index
detector, Maxima 820 software package (all from Waters, Div. of
Millipore, Milford, Mass.), two LiChrogel PS-40 columns and a
LiChrogel PS-20 column in series (EM Science, Gibbstown, N.J.), and
polyethylene glycol molecular weight standards (Polymer
Laboratories, Inc., Amherst, Mass.). Polydispersity values obtained
using this system are relative to the chromatographic conditions,
the molecular weight standards and the size exclusion
characteristics of the gel permeation columns. Polydispersity
measurements using different separation principles may give
absolute polydispersity values which are different from those
described herein. However, one of ordinary skill in the an can
easily convert any polydispersity value that is obtained using a
different separation method to the values described herein simply
by running a single sample on both systems and then comparing the
polydispersity values from each chrommatogram.
In accordance with the present invention, a composition is provided
that is a polyoxypropylene/polyoxy. ethylene block copolymer that
has a polydispersity value of less than 1.07. Preferably, the
polydispersity value is less than approximately 1.05, with a most
preferable polydispersity value of 1.03. It is to be understood
that the present invention includes, but is not limited to,
poloxamer compounds and poloxamine compounds.
Also in accordance with the present invention, a composition is
provided that is a surface-active copolymer comprising a
polyoxypropylene/polyoxyethylene block copolymer with the following
general formula:
HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2
H.sub.4 O).sub.b H
wherein the total molecular weight of the copolymer is between
approximately 5,000 and 15,000 daltons, preferably a molecular
weight of between approximately 7,000 and 12,000 daltons and the
molecular weight represented by the polyoxyethylene portion of the
copolymer constitutes approximately 80% of the copolymer.
One embodiment of the present invention comprises a
polyoxypropylene/polyoxyethylene copolymer which has the following
formula:
wherein the molecular weight of the hydrophobe (C.sub.3 H.sub.6 O)
is approximately 1750 daltons and the average molecular weight of
the compound is approximately 8300 to 9400 daltons. The
polydispersity value is less than approximately 1.05. A block
copolymer corresponding to at least these physical parameters has
the beneficial biological effects of the prior art poloxamer 188
but does not exhibit the unwanted side effects which have been
reported for the prior art compound. By reducing the polydispersity
value of the surface-active copolymer, it has been found that the
toxicity associated with the prior art poloxamer 188 is
significantly reduced. However, the beneficial therapeutic activity
of the modified poloxamer 188 is retained.
The surface-active copolymers of the present invention can be
prepared in a number of ways. The polydispersity value can be
reduced by subjecting the prior art compounds to gel permeation
chromatography. In addition, the compounds can be subjected to
molecular sieving techniques that are known to those of ordinary
skill in the art.
The surface-active copolymer of the present invention can be
prepared in several ways. In the first method, commercially
available poloxamer 188 is subjected to gel permeation
chromatography. The chromatogram that is obtained from this
procedure is shown in FIG. 1.
As can be seen in FIG. 1, commercial poloxamer 188 is composed of a
broad distribution of molecules with a peak molecular weight of
approximately 7900 to 9500 daltons. This corresponds generally to
the published molecular weight for poloxamer 188 of 8400 daltons.
The published molecular weight for poloxamer 188 is determined by
the hydroxyl method. The end groups of polyether chains are
hydroxyl groups. The number averaged molecular weight can be
calculated from the analytically determined "OH Number" expressed
in mg KOH/g sample. It should be understood that the molecular
weight of a polydisperse compound can be different depending upon
the methodology used to determine the molecular weight.
FIG. 1 also shows small secondary peaks or shoulders lying to the
left and fight of the primary peak. These areas of the poloxamer
188 chromatogram represent the high and low molecular weight
molecules respectively. The high molecular weight species range in
size from approximately 24,000 to 15,000 daltons. It is believed
that these larger molecules have a greater capacity to activate
complement compared to the lower molecular weight species. The
shoulder on the fight or lower molecular weight side of the
chromatogram is composed of molecules between approximately 2,300
daltons and 5,000 daltons. This species represents compounds which
have more detergent-like properties and are cytotoxic to cells.
Using the gel permeation chromatography procedure, it has been
determined that a fraction of poloxamer 188 with molecules ranging
from approximately 5,000 daltons to 15,000 daltons, preferably
between approximately 6,000 daltons and 13,000 daltons, with a peak
at approximately 8,700 daltons, represents a population of
surface-active copolymers which are essentially devoid of toxic
activities while still retaining the beneficial therapeutic
activity of the commercially available poloxamer 188. This new
composition is a much more homogeneous preparation than those
currently available and unexpectedly has fewer side effects than
the prior art preparation.
It should be understood that the molecular weight range that is
described as the optimum range for the copolymer is to be
considered the outside range and that any population of molecules
that fall within that range are considered as embodiments of the
present invention.
The present invention also includes a novel method of preparing a
surface-active copolymer composition with the specifications
described herein. The novel method involves the preparation of a
uniform hydrophobic polyoxypropylene polymer and then proceed with
the addition of the hydrophilic polyoxyethylene as is normally
done. It is believed that the toxic copolymers that are the result
of the standard commercial method of preparing poloxamer 188 are
due to truncated polymer chains and to unsaturation in the
polymer.
In practicing the present invention, the hydrophobic
polyoxypropylene polymer is purified to obtain a substantially
uniform population of polyoxypropylene polymers. The purification
can be performed using gel permeation chromatography. However, any
method known to one of ordinary skill in the art which gives the
desired range of polyoxypropylene polymers can be used.
In preparing the improved rheologic reagent, the polyoxypropylene
polymer should have an average molecular weight of approximately
1750 daltons with an approximate molecular weight range between
1,000 and 2,600 daltons. The preferred molecular weight range is
between 1,200 and 2,400 daltons.
After the desired polyoxypropylene copolymer has been obtained, the
ethylene portion of the copolymer is added to both ends of the
molecule by standard methods well known to those of ordinary skill
in the art. The final polymer population should have a
polyoxyethylene composition of approximately 20% of the total
molecular weight of the molecule.
This invention is further illustrated by the following examples,
which are not to be construed in any way as imposing limitations
upon the scope thereof. On the contrary, it is to be clearly
understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the present invention
and/or the scope of the appended claims.
EXAMPLE I
Poloxamer 188 (BASF Corporation, Parsippany N.J.) is dissolved in
tetrahydrofuran at a concentration of 20 mg/mL. A Model 600E
Powerline chromatographic system equipped with a column heater
module, a Model 410 refractive index detector and Maxima 820
software package (all from Waters, Div. of Millipore, Mifford,
Mass.) is used to fractionate the commercially prepared poloxamer
188 copolymer. The chromatographic system is equipped with two
LiChrogel PS-40 columns and a LiChrogel PS-20 column in series (EM
Science, Gibbstown, N.J.). The LiChrogel PS-40 columns are 10 .mu.m
particle size and the LiChrogel PS-20 column is 5 .mu.m particle
size. All columns are 7 mm by 25 cm in size.
200 .mu.L (4 mg) of the poloxamer 188 in tetrahydrofuran is added
to the column and the sample is run with the columns and the
detector at 40.degree. C. The resulting chromatogram is shown in
FIG. 2.
EXAMPLE II
The sample that was collected in Example I was fractionated into
five fractions and each fraction was run on the column as described
in Example I. The chromatograms from the various chromatographic
runs are shown in FIGS. 3 through 8. The fraction that demonstrates
the least toxicity while retaining the therapeutic activity of the
poloxamer 188 is shown in FIG. 5. As can be clearly seen, the
shoulders on either side of the peak in FIG. 5 are absent.
The average molecular weight for each fraction is shown in Table
II. The chromatogram for each fraction is indicated in FIGS. 3
through 8.
TABLE II Time off Column Molecular Polydispersity Fraction FIG.
(Min) Wt. Value 1 3 11.5-12.0 17000 1.0400 2 4 12.0-12.5 10270
1.0474 3 5 12.5-13.0 8964 1.0280 4 6 13.0-13.5 8188 1.0332 5 7
13.5-14.0 5418 1.1103 6 8 14.0-14.5 3589 1.0459
The polydispersity value for the unfractionated poloxamer 188 is
1.0896. The fraction that most closely corresponds to poloxamer 188
is fraction 3 which has a polydispersity value of approximately
1.0280.
EXAMPLE III
In a one-liter 3 neck round bottom flask equipped with a mechanical
stirrer, reflux condenser, thermometer and propylene oxide feed
inlet, there is placed 57 grams (0.75 mol) of propylene glycol and
7.5 grams of anhydrous sodium hydroxide. The flask is purged with
nitrogen to remove air and heated to 120.degree. C. with stirring
until the sodium hydroxide is dissolved. Sufficient propylene oxide
is introduced into the mixture as fast as it reacts until the
product possesses a calculated molecular weight of approximately
1750 daltons. The product is cooled under nitrogen and the NaOH
catalyst is neutralized with sulfuric acid and the product is then
filtered. The final product is a water-insoluble polyoxypropylene
glycol.
EXAMPLE IV
The polyoxypropylene glycol from Example III is dissolved in
tetrahydrofuran at a concentration of 20 mg/mL. A Model 600E
Powerline chromatographic system equipped with a column heater
module, a Model 410 refractive index detector and Maxima 820
software package (all from Waters, Div. of Millipore, Milford,
Mass.) is used to fractionate the commercially prepared poloxamer
188 copolymer. The chromatographic system is equipped with two
LiChrogel PS-40 columns and a LiChrogel PS-20 column in series (EM
Science, Gibbstown, N.J.). The LiChrogel PS-40 columns are 10 .mu.m
particle size and the LiChrogel PS-20 column is 5 .mu.m particle
size. All columns are 7 mm by 25 cm in size.
200 .mu.L (4 mg) of the polyoxypropylene glycol in tetrahydrofuran
is added to the column and the sample is run with the columns and
the detector at 40.degree. C. The fraction which corresponded to an
average molecular weight of 1750 daltons with a molecular weight
distribution between 1,000 and 2,600 daltons was collected. Other
fractions were discarded.
EXAMPLE V
The purified polyoxypropylene glycol from Example IV was placed in
the same apparatus as described in Example III with an appropriate
amount of anhydrous sodium hydroxide. An appropriate amount of
ethylene oxide was added at an average temperature of 120.degree.
C. using the same technique described in Example III. The amount of
added ethylene oxide corresponded to 20% of the total weight of the
polyoxypropylene glycol base plus the weight of added ethylene
oxide.
This procedure results in a polyoxypropylene/polyoxyethylene block
copolymer composed of molecules which are far more homogeneous
relative to molecular size and configuration compared to commercial
preparations.
EXAMPLE VI
Fractions of poloxamer 760.5 prepared by gel permeation
chromatography and were analyzed for weight percent of oxyethylene
and for unsaturation by NMR analysis as follows: Poloxamer 760.5
(BASF Corporation, Parsippany N.J.) is dissolved in tetrahydrofuran
at a concentration of 20 mg/mL. A Model 600E Powerline
chromatographic system equipped with a column heater module, a
Model 410 refractive index detector and Maxima 820 software package
(all from Waters, Div. of Millipore, Milford, Mass.) is used to
fractionate the commercially prepared poloxamer 760.5 copolymer.
The chromatographic system is equipped with Ultrastyragel 10.sup.3
A and 500 A in series (Waters, Div. of Millipore, Milford, Mass.).
Column size is 7.8 mm internal diameter by 30 cm. Precolumn filters
#A-315 with removable 2 .mu.m fits (Upchurch Scientific, Oak
Harbor, Wash.) were used for protection of the columns. 200 .mu.L
(4 mg) of the poloxamer 760.5 in tetrahydrofuran is added to the
column and the sample is run with the columns at 40.degree. C. and
the detector at 45.degree. C.
Sample one is an unfractionated sample of the polaxamer 760.5 as
obtained from BASF Corporation (Parsipanny, N.J.) and is shown in
FIG. 9A. Fraction one is an early fraction from the chromatographic
system and is shown in FIG. 9B. Fraction two is a late fraction and
is shown in FIG. 9C. All proton NMR analyses were performed in
accordance with the NF procedure "Weight Percent Oxyethylene" on a
Bruker 300 MHz instrument.
The proton nuclear magnetic resonance spectra from FIGS. 9B and 9C
showed slight ban broadening in the spectra when compared to the
unfractionated sample. The late eluting fraction (Fraction 2)
contains the largest amount of unsaturation as noted by a doublet
signal at about 4.0 ppm. The proton spectra for the early eluting
peak (Fraction 1) showed no impurities except water.
The weight percent oxyethylene was calculated for the samples. As
can be seen from Table III, the early eluting fraction, which is
the purest fraction, has the lowest percentage of oxyethylene. This
fraction also showed no unsaturation as measured by nuclear
magnetic resonance. Using the poloxamer nomenclature system
described above, the various fractions have the following
characteristics and poloxamer number.
TABLE III Fraction % POE.sup.a MW.sup.b Poloxamer
Unsaturation.sup.c Unfractionated 5.5 8135 760.5 Yes Early Fraction
3.9 10856 104.4 No Late Fraction 7.5 3085 291 Yes .sup.a As
measured by NMR .sup.b Polyoxypropylene as measured by gel
permeation chromatography .sup.c As measured by NMR
EXAMPLE VII
Poloxamer 188 (Pluronic.RTM. F68) was fractionated on a gel
permeation chromatography system according to Example I. Three
fractions were collected. FIG. 11A shows Fraction 1, an early, high
molecular weight fraction. FIG. 11B shows Fraction II, which is the
major peak. FIG. 11C shows Fraction III, a late eluting, lower
molecular weight population of molecules. The percent oxyethylene
of each fraction was determined by proton NMR using a 200 MHz NMR
spectrophotometer. Approximately 10 mg of each sample was tested.
Samples were prepared by adding approximately 0.7 mL of CDCl.sub.3
to each vial. The solution was filtered and transferred to a 5-mm
NMR tube. One drop of D.sub.2 O was added, and the tube was shaken
prior to measurement.
TABLE IV Fraction % POE.sup.a MW.sup.b Poloxamer Early 85 16,500
258 Middle Fraction 82 8652 178 Late Fraction 90 3751 039 .sup.a As
measured by NMR .sup.b As measured by gel permeation
chromatography
As shown in Table IV, the early eluting, the large molecular weight
fraction had a high percentage of oxyethylene and corresponded to a
poloxamer 258. The middle fraction had the smallest percentage of
oxyethylene while the late eluting, small molecular weight fraction
had the highest percentage of oxyethylene. The middle fraction had
a calculated poloxamer number of 178 which corresponds closely to
the desired number of 188. The late fraction had a calculated
poloxamer number of 039. Thus, the commercially available poloxamer
preparation has a significant population of polymers which may be
harmful in a biological system.
EXAMPLE VIII
Poloxamer 331 (Pluronic.RTM. L101) was fractionated according to
the protocol in Example VI. The chromatographs for unfractionated
poloxamer 331, an early eluting fraction and a late eluting
fraction are shown in FIGS. 12A through 12C respectively. The NMR
spectra for each sample was then determined as in Example VI. The
results of these spectra and chromatograms are summarized in Table
V.
TABLE V Fraction % POE.sup.a MW.sup.b Poloxamer Unsaturation.sup.c
Unfractionated 17 4045 342 Yes Early Fraction 15 4452 381 No Late
Fraction 31 1466 103 Yes .sup.a As measured by NMR .sup.b As
measured by gel permeation chromatography .sup.c As measured by
NMR
When the poloxamer number for each fraction is calculated based on
the empirical data collected, it is seen that the late fraction
polymer is a very different poloxamer than the unfractionated
preparation. In addition, the unsaturated population of polymers
has been removed by the fractionation procedure.
It should be understood that the foregoing relates only to a
preferred embodiment of the present invention and that numerous
modifications or alterations may be made therein without departing
from the spirit and the scope of the invention as set forth in the
appended claims.
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