Note: Descriptions are shown in the official language in which they were submitted.
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THROMBIN PREPARATION AND PRODUCTS AND FIBRIN SEALANT METHODS
EMPLOYING SAME
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the preparation of thrombin material products, and
the use of the
products inter alia in the production of fibrin sealants. Fibrin sealants are
sealant compositions
useful for sealing tissue and other medical and surgical purposes, which
generally comprise two
fluid components having active agents derived from blood plasma namely a
polymerizable , ,
fibrinogen component and a thrombin activator. When mixed, the components cure
to provide a
solid film or deposit of fibrin. If the plasma used to prepare one or another
components of the
sealant is obtained from a subject to be treated with the sealant, the sealant
is said to be
"autologous". The invention also relates to novel fibrin sealants wherein both
the thrombin and
the fibrinogen components are autologous and to their preparation .
2. Description of Related Art Including Information Disclosed under 37 CFR
1.97 and 37
CFR 1.98
Customarily, in prior art fibrin sealant compositions, one component contains
fibrinogen which
becomes insoluble fibrin when polymerized and cross-linked by an activator and
the other
component comprises an activation mixture which generally includes thrombin.
Alternatives to
thrombin, such as thrombin analogs and reptile-sourced coagulants, have also
been proposed.
s~
Depending upon the ingredients present and their strengths, the mixed sealant
components will
rapidly gel and eventually form a tough insoluble clot. Fibrin sealants are
uniquely valuable for
certain specialized surgical procedures, for example in otology, and offer
advantages in the
treatment of a wide range of wound conditions by virtue of their
biocompatibility, their fostering
of the wound healing process and the fact that no foreign object or material
remains on or in the
tissue to effect wound closure or hemostasis.
Patient autologous fibrin sealants were developed in the early 1980s as a
response to the lack of
availability of homologous fibrin sealant in the United States pursuant to FDA
regulations
proscribing use of sealants prepared from pooled human sera. Known methods of
preparing
autologous fibrin sealant compositions have focused exclusively on the
production of the first
component comprising fibrinogen in admixture with Factor XIII. Factor XIII, or
fibrin
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stabilizing factor, ("FSF'~, is a fibrin cross-linking agent which is
activated by thrombin in the
presence of calcium ions, Ca++. The latter components, thrombin and Ca++ are
accordingly
usually provided in the second component.
Examples of the preparation of such two-component fibrin sealants may be found
inter olio in
Epstein U. S. patent number 5,226,877 ("Epstein '877" herein) and Sierra U. S.
patent number
5,290,552. Many others exist.
"Epstein '877" teaches a process and apparatus for one-step preparation of
fibrinogen adhesive
by polyethylene glycol-mediated precipitation from plasma. The Epstein '877
process begins
with the withdrawal of whole blood from e.g. autologous donor in the presence
of an
anticoagulant, such as citrate, and separation of plasma from the red blood
cell fraction.
Typically, the separation can be effected by centrifugation; a suitable
protocol involves spinning
at about 2000-5000 g for about 5-10 minutes. After the plasma is separated
from the red blood
cells, it is treated directly, at ambient temperature, without prior treatment
to remove thrombin,
with a physiologically acceptable nontoxic precipitant notably polyethylene
glycol in a
molecular range of 200-8000 supplied as a concentrated solution. For example,
an
approximately 60% solution of PEG of molecular weight 1000 in saline at pH
7.4, can be added
to the plasma in sufficient volume (typically 10% w/v of precipitant
solution/final volume) to
obtain a resulting concentration of 8-15% w/v PEG in the volume of the final
mixture so that the
plasma itself is, only slightly diluted. The precipitation of fibrinogen-rich
material is complete
essentially instantaneously. The Epstein '877 fibrinogen precipitation process
can be used as an
o tional relimi i -
p p nary step in practicing the invention described hereinbelow.
Epstein does not teach preparation of a thrombin component. Epstein '87Ts
autologous
fibrinogen adhesive preparation is intended for admixture with known
commercial thrombin
preparations. Such preparations would commonly have been of bovine origin.
Thrombin is readily available commercially as an off the-shelf product
produced from bovine
plasma and is commonly indicated for use as a topical hemostatic agent for
treatment of diffuse
capillary bleeding. In the past little concern has been focused on the use of
bovine thrombin, but
in recent times theoretical, psychological and case report problems have
emerged.
Thus, recent investigations implicate thrombin in the etiology of various
coagulopathies that
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occur in some individuals given repeated doses of thrombin. Also, thrombin
impurities, for
example Factor V, being a relatively high molecular weight protein of bovine
origin, can cause
an immunological response in humans. A still more serious problem is that
antibodies formed
against bovine Factor V may in taro react with the host's own Factor V,
disrupting normal
hemostasis, leading to severe hemorrhage and even death. Theoretically, these
problems might
be ameliorated by using higher purity thrombin, but the applicant is not aware
of enabling
teachings regarding such an approach.
A still further drawback arises from recent widespread publicity regarding
possible transmission
of bovine spongioform encephalopathy (BSE) to humans from beef products. It is
believed that
this adventitious agent, an infectious protein known as a "priors", may in
turn mutate in the host
to produce a Cruetzfield-Jakob-like disease state. This is a terminal,
incurable
neurodegenerative disease and there are currently no known effective
treatments for most
bovine-sourced products to inactivate or filter the causative priors.
Another approach has been to eliminate the need for thrombin and its
associated problems by
employing an alternative pathway to produce fibrin. In one such approach,
patient autologous
plasma is reacted with a snake venom enzyme having some similarities to
thrombin, such as
batroxobin, under acidic conditions to form a non-aggregating fibrin-1
"prepolymer". The
batroxobin is then removed from the plasma by chromatographic means and the
fibrin
prepolymer isolated and concentrated. Gelation occurs when a buffer is mixed
with the fibrin
preparation, bringing the pH back to neutrality. In addition to the
difficulties associated with
obtaining the batroxobin, the end product sealant may lack adequate strength.
The nature of the
process is such that no Factor XIII is present in the mixture to strengthen
the resultant clot.
Furthermore, the fibrinogen is subject to proteolytic cleavage by such snake
venoms and the
resultant fibrin may lack adequate strength owing to failure to activate all
the aggregation sites.
There is accordingly a need for a thrombin product that can be used as an
activator in fibrin
sealant compositions and which is not subject to these drawbacks.
Theoretically, recombinant
human thrombin might offer a potential solution but expectations in early 1998
are that such a
product is at least five years away. Also the efficacy of such a hypothetical
synthetic product,
absent natural biological co-factors, is unknown.
There is accordingly still a need for a thrombin product which is an effective
activator in a multi-
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component fibrin sealant composition, which can be quickly prepared, and which
provides a low
risk of inducing an immunologic response or disease. Autologous thrombin,
thrombin derived
from the subject to be treated, meets the requirements of low immunogenicity
and infectivity and
is a desirable product. However, there are diffculties in obtaining autologous
thrombin since
the subject's blood plasma is the only possible source of the desired thrombin
product. Any
method of preparing autologous thrombin should be quick, easily effected in
proximity to a
surgical environment, without needing special equipment, and capable of
producing an effective
fibrinogen coagulation activator from a small sample of blood.
Cochrum U. S. patent 5,510,102, and others, teach use of biocompatible
polymers such as
alginates, poly-L-amino acids, chitosan and chitin in a hemostatic adhesive
agent comprising a
possibly autologous platelet-rich plasma concentrate having up to 10 times the
concentration of
normal plasma of , said concentrate containing 5 to 10 times higher
concentration of fibrinogen
and other plasma proteins than normal plasma. However, Cochrum provides no
teaching of a
thrombin material that can be mixed with a fibrinogen material to provide a
fibrin sealant for
surgical use.
Cochrum provides an extensive discussion of the complex blood coagulation
cascade that yields
wound-protecting clots. As explained by Cochnun, the essential reaction in
coagulation of the
blood is the enzymatic conversion of the soluble protein fibrinogen into the
insoluble protein
fibrin by thrombin. Fibrinogen exists in the circulating blood as such.
Thrombin is formed from
an inactive circulating precursor, prothrombin, as a result of tissue injury,
bleeding or blood loss.
s_
The activation of prothrombin depends on the presence of calcium ions and on
the presence of
thromboplastin, factor III, which is released or derived from the damaged
tissues, from the
disintegrating platelets or from the plasma itself.
Antanavich U.S. patent number 5,585,007 teaches a method of preparing a
concentrate for use as
a tissue sealant by contacting plasma with a concentrator that absorbs water,
electrolytes and
small proteins (column 12, lines 29-33) to yield a platelet-rich plasma
product. The concentrator
is a solid water-absorbent material, for example beads or discs of materials
such as
polyacrylamide, dextranomer, silica gel or starch. At column 21, lines 7-9,
Antanavich teaches
that in a further embodiment, the activator can be autologous thrombin and
platelets. So far as
can be ascertained in the lengthy patent specification, it appears that the
only method Antanavich
teaches for providing sutologous thrombin is to use a needle tip to disrupt
platelets, enabling a
4
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single-component concentrate to be applied (as a sealant), without use of
bovine thrombin, see
column 24, lines 32-35. However, no data is given as to the efficacy of such a
single-component
concentrate. Again, Antanavich does not appear to provide a thrombin material
that can be
mixed with a fibrinogen material to provide a fibrin sealant for surgical use.
Toward the objective of a fully autologous fibrin sealant wherein both
fibrinogen and thrombin
are sourced from the intended patient or other single donor, Hirsh U. S.
patent number
5,643,192, teaches that the serum supernatant available after an initial
precipitation of fibrinogen
from a sample of the intended patient's plasma can be mixed with a calcium
chloride solution.
Residual fibrin that forms is skimmed.out. After at least 15 or more minutes,
a dilute (<
20U/ml) thrombin solution is ready. Drawbacks of this process are that it may
be unduly time-
consuming and that the resultant thrombin solution is weaker and more dilute
than is desirable
and may yield a sealant which is an ineffective treatment for many of the
major conditions for
which a fibrin sealant is indicated, notably hemostasis, fluid-tight sealing
and microsurgical
adhesion.
Cederholm-Williams U.S. patent number 5,795,571 teaches use of a possibly
autologous
thrombin fraction for treatment of hemostasis, prevention of blood loss, in
animals, including
humans. Cederholm-Williams lowers the ionic strength and pH of a blood plasma
sample from
which cells and platelets have been removed to yield a precipitate described
as a "euglobulin
fraction." The euglobulin fraction contains prothrombin, fibrinogen and many
other blood
proteins, but is substantially free of antithrombin III a plasma protein which
may prevent the
r~
conversion of prothrombin to thrombin or may inactivate thrombin. The
prothrombin of the
euglobulin fraction is then redissolved and converted to thrombin to form the
desired thrombin
blood fraction, for example by solubilizing the euglobulin fraction in saline,
buffered to a neutral
pH. However, Cederholm-Williams's method does not provide a separate
fibrinogen-
containing material to serve as the other component of an autologous fibrin
sealant.
Cederholm-Williams provides an extensive bibliography of literature in the
field, including a
number of references regarding thrombin preparation.
There is accordingly a need for a method of producing thrombin which can
quickly and easily
provide the thrombin component of an autologous fibrin sealant
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SUMMARY OF THE INVENTION
The invention, as claimed, is intended to provide a remedy. It solves the
problem of providing
an effective thrombin activator for a mufti-component fibrin sealant
composition, which can be
quickly prepared and which, if desired, can be prepared autologously along
with an autologous
fibrinogen component, from the same blood sampie, to provide a fully
autologous fibrin sealant.
In one aspect the invention provides a method of preparing a thrombin material
suitable for use
as an autologous fibrin sealant component, the method comprising exposing
prothrombin-
containing material to an extended surface area material capable of activating
conversion of
prothrombin to thrombin in an aqueous medium. The extended surface area
material is
., -
preferably covered with polar groups of a nature found on the exterior
surfaces of activated
platelets or endothelial cells. Preferably, the polar groups comprise
phosphatidyl groups and
basic nitrogen groups, and the extended surface area material comprises
phospholipid liposomes.
Also, to facilitate the conversion, it is desirable that the aqueous medium
comprises factor V,
factor X and calcium ions.
Use of such an extended surface area material facilitates the conversion of
prothrombin to
thrombin, providing an excellent yield of thrombin. It is also desirable, but
optional, that the
aqueous medium comprise a reduced proportion of antithrombin III relative to
prothrombin as
compared with the proportions in blood plasma.
It is furthermore preferable that the prothrombin material comprises a
precipitate obtained from
t~
fibrinogen- and factor XIII-depleted plasma. The fibrinogen- and factor XIII-
depleted plasma
can be prepared by precipitating fibrinogen and factor XIII from a blood
plasma sample from
which the cellular components have been removed. The fibrinogen and factor
XIII precipitate
can be used to prepare the fibrinogen component of a fibrin sealant, and by
then using the
residual plasma for preparation of the thrombin component, a fully autologous
fibrin sealant can
be obtained by taking the blood sample from the subject to be treated with
fibrin sealant.
In another aspect the invention provides a method of preparing a thrombin
material suitable for
use as an autologous sealant component, the method comprising:
a) treating a blood plasma sample obtained by removal of cellular and platelet
components
from a whole blood sample to yield a fibrinogen precipitate containing
fibrinogen and
factor XIII and to provide fibrinogen- and factor XIII-depleted plasma;
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b) treating the fibrinogen- and factor XIII-depleted plasma to yield a
prothrombin-
containing precipitate; and
c) treating the prothrombin-containing precipitate to yield thrombin.
Preferably, the treatment of the prothrombin-containing precipitate in step c)
comprises
dissolving the prothrombin-containing precipitate in an aqueous reagent and
exposing the
dissolved prothrombin-containing material to an extended surface area
substrate capable of
converting prothrombin to thrombin.
As stated above, the extended surface area material comprises a suspension of
phosphatidyl
Iiposomes, preferably with a major proportion of phosphatidyl choline and a
minor proportion of
phosphatidyl serine.
In a further aspect the invention provides a method of preparing an autologous
fibrin
composition comprising separate fibrinogen and thrombin components mixable to
provide a
sealant, the method comprising the steps of:
a) removal of cellular and platelet components from a sample of patient
autologous blood to
yield a plasma supernatant; and
b) generating a precipitate containing fibrinogen and Factor XIII from the
plasma
supernatant for reconstitution as the fibrinogen component of the fibrin
sealant;
V
c) treating the plasma supernatant from step b) to yield a prothrombin-
containing
precipitate; and
d) separating the prothrombin-containing precipitate from seep c) and
dissolving the
prothrombin-containing precipitate in a sufficient quantity for solution of a
tissue-
compatible aqueous activation reagent comprising:
i} a sufficient quantity of calcium chloride, or an equivalent thereof, to be
effective
in the tissue sealant; and
ii) an effective quantity of a dispersed, tissue-compatible prothrombin-
converting
particulate substrate;
to provide a thrombin-containing component.
The particulate substrate can comprise phosphatidyl liposomes having a major
proportion of
phosphatidyl choline and a minor proportion of phosphatidyl serine while the
treatment of step
c) comprises reducing the ionic strength and the pH of the solution to the
isoelectric point of
7
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prothrombin.
The invention also comprises thrombin material produced by any of the methods
of the
invention and autologous fibrin sealant compositions produced by an inventive
method.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
One or more embodiments of the invention and of making and using the
invention, as well as the
best mode contemplated of carrying out the invention, are described in detail
below, by way of
example, with reference to the accompanying drawings, in which:-
Figure 1 is a block flow diagram of one method of preparing thrombin,
according to the
invention; and
Figure 2 is a block flow diagram of a method of preparing thrombin, according
to a
preferred embodiment of the invention, which embodiment is described more
fully hereinbelow in connection with Example 2.
IS
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring now to Figure 1, the invention provides, in one embodiment, a method
of preparing an
optionally sutologous thrombin composition suitable for use in a fibrin
sealant comprising the
steps of
a) collecting a suitable amount of patient autologous blood, for example about
100 ml or
less, in a citrate solution, or equivalent aqueous medium;
b) removing cellular and platelet components from the solution, for example by
t_
centrifugation or filtration, to yield a plasma supernatant;
c) generating a precipitate containing fibrinogen and Factor XIII from the
plasma
supernatant for reconstitution as a fibrinogen component of the fibrin
sealant;
d) treating the serum supernatant from step c) to yield a prothrombin-
containing precipitate;
e) separating the prothrombin-containing precipitate from step d), for example
by
centrifugation or filtration;
f) dissolving the prothrombin-containing precipitate from step e) in a
sufficient quantity for
solution of a tissue-compatible aqueous activation reagent comprising:
i) a sufficient quantity of calcium chloride, or an equivalent thereof, to be
effective
in the tissue sealant; and
ii) an effective quantity of a dispersed, tissue-compatible prothrombin-
converting
particulate substrate;
8
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to provide the desired thrombin-containing component.
Preferably, the prothrombin-converting particulate substrate comprises
liposomes having bipolar
external moieties, for example, such as provided by phospholipids, for example
lecithin-based
phospholipids. Preferably also, the liposome particles are relatively large in
size, being of the
order of 0.1 to 5 mm in diameter, more preferably about 1 or 2 mm.
Altenlatively, the
particulate substrate can comprise platelets collected and isolated from the
patient's blood or
from a screened donor, for example a close relative. Alternatively rather than
particles a lipid
emulsion may be used. A suitable quantity of substrate particles is from about
1 to about 500
mg, preferably from about 5 to about 100 mg and more preferably about 10 to
about SO mg per
100 ml. of original blood volume. The substrate material can be incorporated
in from about 0.5
to about 100m1, preferably about 1 to about 20 ml, and more preferably about 2
to about 20 ml
of saline, optionally with calcium chloride.
Preferably, the pH is also raised back to about 7 and extraneous fibrin is
removed as the Factor
11 prothrombin converts to thrombin precipitating residual dissolved
fibrinogen as fibrin.
After a short time, less than 10 minutes, for example about 3 minutes, the
thrombin-containing
component is ready for use as the second, activation component of the fibrin
sealant. The first
sealant component is preferably the product of step c), although, as will be
understood by those
skilled in the art, the novel thrombin product of step f) could be used with
other non-autologous
fibrinogen components, or for other purposes, if desired.
e_
The thrombin-containing component can be obtained as a concentrated thrombin
solution with a
yield of about 2 ml from a 100 ml blood sample. In a preferred embodiment the
concentration is
sufficient for a gelation time of about 5 seconds when the thrombin-containing
component is
mixed in a I : I volume ratio with the fibrinogen component 1 of step c). The
invention thus
provides an autologous thrombin fibrin sealant component capable of effecting
gelling in less
than one minute, preferably less than 30 seconds and still more preferably,
when desired, in
about 10 seconds or less.
The entire process of producing the two sealant components can be compieted in
about 20-30
minutes. The majority of the processing time is taken up with centrifugation
rather than
attention-requiring precipitation and activation steps. This short time and
the lack of
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requirements for special equipment suit the process to be carried out in
conjunction with an
surgical theater environment where centrifuges are usually available.
The precipitate resulting from step c) can be isolated by filtration or
centrifugation to provide the
first component of a fibrin sealant, yielding about 2 ml. A variety of methods
of performing step
c) is known to the art and may be used, some of which are described in the
literature cited herein,
for example, polyethylene glycol ("PEG"citrate precipitation,
cryoprecipitation, ammonium
sulfate precipitation and ethanol precipitation.
The prothombin precipitating treatment of step d) can be effected with a
mildly acidic aqueous
medium, or other precipitating agent. for example a buf~'ered polyethylene
glycol solution.
Preferably, the aqueous diluent used in step d), which can be water, is used
in a proportion of
from about 1:1 to about 20:1 to the quantity of blood collected, more
preferably from about 2:1 to
about 10:1, and still more preferably, from about 3:1 to about 5:1. The
acidity is preferably in a
I S pH range of about 4 to about 6, more preferably about 5. Preferably also
antithrombin III is
inactivated, for example by such acidification.
In step e) the prothrombin-containing precipitate is preferably isolated and
concentrated and the
supernatant can be discarded.
In a preferred embodiment, in step f) and the aqueous component of the reagent
comprises isotonic
saline, calcium chloride and the phospholipid liposomes.
It will be understood that the above-mentioned quantities are relative to an
in initial blood sample
aliquot of about 100 ml, and are simply indicative of relative proportions
that may be used,
depending upon the size of the aliquot.
While not being bound by any particular theory, the invention can be further
described in the
following terms. In step c), fibrinogen and Factor XIII are precipitated out
of the collected plasma
while the remaining coagulation factors, specifically II, V and X are
subsequently precipitated in
step d), for example by altering the pH of the serum supernatant, so that
fibrinogen and factor FIII
attain their isoelectric points. Selectivity for these particular proteins can
be enhanced by reducing
the ionic strength of the serum by dilution with purified water, for example
to from two to ten
times the original blood sample volume, preferably about four or five times.
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In order to convert the zymogen Factor II, prothrombin, into its active form,
thrombin, several
components can be employed, specifically, Factor V, Factor X, Ca" and the
substrate particles,
preferably phospholipid liposomes. The presence or absence of any of these
components can
directly impact the rate of thrombin production. Factors V and X become
activated when
calcium ion as a cofactor becomes available. These three entities are believed
to form a non-
covalently bound complex in which Factor 11 undergoes protealytic cleavage to
form thrombin.
The present inventor has discovered that the use of a synthetic activator
presenting a
phospholipid surface for this complex to bind to can dramatically increase the
rate of thrombin
production compared with what would otherwise be obtainable, for example by
several orders of
magnitude. A preferred composition comprises a major proportion of
phosphatidyl choline (PC)
and a minor proportion of phosphatidyl serine (PS) in a ratio of from about I
:1 to about 5:1,
preferably about 7:3. Such a composition is believed to provide an external
surface with
similarities to that of activated platelets or endothelial cells. While, as
has been conventional
phospholipid of this composition can be obtained from brain extracts of
rabbit, sheep or bovine
origin it is preferred for the purposes of this invention, that, liposomes
preferably having a mean
diameter of at least 1 mm are produced from a synthetically derived lipid
preparation (e.g.
"Synthetic Phosphoiipid Blend", Avanti Polar-Lipids, Inc. Alabaster. AL),
"PCPS" hereinafter.
This composition apparently closely approximates platelets and endothelial
cells in composition
and physical configuration and may contribute to reaction efficiency.
Liposomes or "lipid bodies", sometimes called "vesicles" are structures formed
spontaneously
t.
by polar lipid molecules, or amphiphilic molecules, each having a polar head
and a long
hydrophobic tail, e.g. phospholipids such as lecithin. Structurally, liposomes
comprise an outer
shell of one or more membrane-like, bi-layers of the molecules arranged
concentrically around a
hollow interior, or 'bacuole" which can serve as a storage compartment for
active agents. In the
outer layer the polar heads of the molecules are oriented outwardly of the
liposome, while the
hydrophobic tail, e.g. palmitic or stearic acid, depends inwardly. Lecithin
liposomes composed
primarily of phosphatidyl choline can be expected to be coated or covered, or
to present an outer
layer which comprises an array of dipoles. Thus lecithin molecules have polar
heads which
align to the outside of the liposome particle, and each has twin hydrophobic,
lipophilic fatty acid
tails which align inwardly. The polar character of the head of each lecithin
molecule has the
nature of a dipole with a positive charge located in the vicinity of the
molecule's distal
quaternary nitrogen atom, and a negative charge in the vicinity of its
neighboring phosphatidyl
11
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WO 99145938 PGT/US99105034
group. Phosphatidyl serine has a dipolar head provided by amino and
phosphatidyl groups and a
mixture of phosphatidyl choline and phosphatidyl serine will provide a surface
of quaternary
nitrogen, amino and phosphatidyl groups.
Many suitable materials for and methods of preparation of liposomes are known
to the art, see
for example Unger et al. ("Unger") U.S. Patents 5,469,854 and 5,580,575 and
can be used to
provide the extended surface area material of the invention. Alternatively,
the extended surface
area material can comprise a continuous substrate, for example a column of
material such as
SEPHADEX (trademark). Preferably, the extended surface area material comprises
a surface
layer of polar groups providing dipoles, namely pairs of positively and
negatively charged
groups, more preferably basic nitrogen groups or other biologically available
positively charged
groups, for example chelated or otherwise complexed metallic groups such as
ferrous or ferric .
and phosphatidyl or carboxyl or other negatively charged groups.
V
1 S Example l: (Comparative Example) A Known Thrombin Production Method
In a beaker, 100 ml of citrated bovine plasma are diluted with 900 ml of
deionized water. The
pH is adjusted to 5.3 with 2% acetic acid. The solution is centrifuged for 10
minutes at 2000 X
g at 4°C. The supernatant is decanted and discarded. The pellets are
collected and resuspended
in 25 ml of saline and 3 ml of 0.25 M CaCl2. The pH is raised to 7 with 2%
Na,C03. The
mixture is incubated for 2 hours and the precipitating fibrin skimmed off and
discarded.
Acetone (25 ml) is added and the mixture centrifuged for 10 minutes at 2000 X
g at 4°C. Saline
(25 ml) is added to extract the thrombin and the precipitate discarded. When
mixed with
concentrated fibrinogen-FXI 11, no gelation was noted. When the above method
was repeated t '
with serum from PEG-citrate precipitated fibrinogen (using the Epstein '877
method), a gelation
time of 30 seconds was observed. The resultant geI was of a friable
consistency.
Example 2: Thrombin Production Method with PEG-Precipitated Fibrinogen Serum
Referring now to Figure 2, two 50 cc centrifuge tubes containing 33 ml each of
citrated plasma
are mixed with 17 ml each of PEG-citrate precipitation solution. The tubes are
centrifuged for
10 minutes at 3600 rpm at 4 °C and the senun supernatant decanted into
a beaker. The
fibrinogen-FXIII precipitate is collected and diluted 1:1 v/v with saline. To
the serum
supernatant, 400 mL of deionized water are added and the pH adjusted to 5.3
with 2% acetic
acid. The solution is centrifuged for 10 minutes at 2000 X g, and the
supernatant decanted and
discarded. The pellets are collected and dissolved in 5 mL of a suspension
consisting of isotonic
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WO 99/45938 PCTlUS99/05034
saline, 30 mM CaCI2 and 20 mg of PCPS (7:3) liposomes. This conversion reagent
was
prepared by adding 5 ml of the saline-CaCh solution to a vial containing 20 mg
of lyophilized
PCPS lipid and a magnetic stir bar and vortexing intermittently over the
course of at least 15
minutes, and placed on a stir plate between vortexing. Fibrin formation from
unprecipitated
fibrinogen occurs in about 90 seconds upon addition of the liposome
suspension. The fibrin is
removed and no further fonmation is observed after approximately 4 minutes. To
a glass culture
tube, 1 mL of the PEG-precipitated fibrinogen-FXIII solution is added to 1 mL
of the thrombin
solution. Gelation of the mixture was observed within 5 seconds. This
experiment
demonstrates one method of producing both fibrin sealant components from the
same relatively
small plasma sample within a short time (25 minutes).
Example 3: Thrombin Production Method with CryoSUpernatant (Serum)
100 mL of citrated bovine plasma are frozen at -20°C for 24 hours, then
slowly thawed for 24
hours at 4°C. The plasma-precipitate is centrifuged for 10 minutes at
3600 rpm also at 4 °C and
1 S the serum supernatant decanted. The cryoprecipitate is collected and set
aside for later use as the
fibrinogen component of a fibrin sealant. The serum supernatant (ca. 97 mL) is
diluted with 400
mL deionized water and the pH reduced to 5.3 with 2% acetic acid. The same
methodology as
described in Example 2 is used to prepare the thrombin solution. Onset of
fibrin formation in
the thrombin solution occurs in 50 seconds. A gelation time of 5 seconds of a
1:1 vlv mixture of
the thrombin and the cryoprecipitate was obtained. This experiment illustrates
that the improved
thrombin production method is compatible with fibrinogen preparation methods
other than PEG-
citrate.
t-
The method of the invention may be scaled down in terms of dilution volume
reduction,
however, the activation time of the thrombin becomes increased with a
decreasing water/serum
ratio. The method may be performed using a simple arrangement of vessels such
as syringes,
blood handling bags, stopcocks and/or extension tubes. Parts of, or the entire
assembly may be
placed in a clinical centrifuge. Alternatively, the entire procedure may be
done in an automated
machine with a drop-in cartridge containing the disposable blood contacting
surfaces and
solutions specific for that particular case.
The residual fibrinogen in the thrombin solution may be eliminated by either
improving the
efficiency of the fibrinogen precipitation reaction step or by using a means
of immobilizing the
resultant fibrin in the thrombin conversion vessel. These methods include for
example: coating
13
CA 02322741 2000-08-31
WO 99/45938 PCTNS99/OSt134
biocompatible or biodegradable particulate or microspheres with PCPS, then
centrifuging out
the fibrin bound to the particulate. This may be accomplished by having the
thrombin reaction
occurring under agitation to prevent gel formation. The microspheres or
particulate may be
magnetized, then when a magnetic field is applied, the fibrin-bound material
is separated from
the thrombin supernatant. The surface of the reaction vessel may be coated
with the PCPS lipid
and the vessel centrifuged or spun longitudinally to separate the fibrin. An
insert with vanes
coated with the lipid could be placed in the reaction vessel to increase
further the efficacy and
efficiency of the fibrin removal.
As an alternative product concept, a patient autologous or screened single
donor thrombin
solution for use as a topical hemostat could be conveniently prepared with the
patient's own ~ '
blood in the operating room or blood bank. Donor serum may be used as well.
Preparation of
this could be done in a closed system. Such a product could be useful in
patients known to be
sensitive to bovine products or with coagulopathy from previous dosages of
bovine thrombin.
INDUSTRIAL APPLICABILITY
The present invention is particularly suitable for application in the health
industry providing new
means of treating wounds and other tissue problems.
While some illustrative embodiments of the invention have been described
above, it is, of
course, understood that various modifications and equivalents of the described
embodiments
will be apparent to those of ordinary skill in the art. Some equivalents will
be readily
recognized by those of ordinary skill while others may require no more than
routine
experimentation. Such modifications and equivalents are within the spirit and
scope of the
invention, which is limited and defined only by the appended claims.
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