U.S. patent application number 11/058489 was filed with the patent office on 2005-06-23 for methods of using therapeutic fibrinogen compositions.
Invention is credited to Pines, Eli, White, William J..
Application Number | 20050136046 11/058489 |
Document ID | / |
Family ID | 21818974 |
Filed Date | 2005-06-23 |
United States Patent
Application |
20050136046 |
Kind Code |
A1 |
Pines, Eli ; et al. |
June 23, 2005 |
Methods of using therapeutic fibrinogen compositions
Abstract
A therapeutic composition effective on contact with thrombin at
a site of treatment in a patient as a tissue adhesive, hemostat or
sealant, said composition comprising non-autologous, non-single
donor mammalian fibrinogen that is capable of polymerizing when
provided in solution at said site at a concentration of about 30
mg/ml thereof or less, to a fibrin network having therapeutically
effective strength, wherein said composition contains less than
about 30% (w/w), based on total protein mass present therein, of
proteins other than fibrinogen, and further comprises a sufficient
amount of one or more low molecular weight
physiologically-compatible solutes such that said composition, if
formulated as a lyophilized material, can be reconstituted
therefrom at room temperature in sterile water for injection in
about 30 minutes or less, at about 25 mg/ml of said fibrinogen.
Additionally, methods for producing and maintaining said
composition, and methods for the use thereof.
Inventors: |
Pines, Eli; (Watchung,
NJ) ; White, William J.; (Wayne, PA) |
Correspondence
Address: |
E. Victor Donahue, Esq.
Kenyon & Kenyon
1025 Connecticut Ave., N.W.
Washington
DC
20036
US
|
Family ID: |
21818974 |
Appl. No.: |
11/058489 |
Filed: |
February 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11058489 |
Feb 14, 2005 |
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09338221 |
Jun 22, 1999 |
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09338221 |
Jun 22, 1999 |
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08805703 |
Feb 25, 1997 |
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08805703 |
Feb 25, 1997 |
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08225853 |
Apr 8, 1994 |
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5605887 |
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08225853 |
Apr 8, 1994 |
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08024121 |
Mar 1, 1993 |
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5330974 |
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Current U.S.
Class: |
424/94.64 ;
514/13.6; 514/13.7; 514/15.2 |
Current CPC
Class: |
C07K 14/75 20130101;
A61K 38/00 20130101; A61L 24/106 20130101 |
Class at
Publication: |
424/094.64 ;
514/002 |
International
Class: |
A61K 038/48 |
Claims
1-17. (canceled)
18. A method for inducing tissue adhesion, sealing of tissue, or
hemostatis in a mammalian patient at a site of treatment therein
comprising contacting said site with a therapeutically effective
amount of a therapeutic composition effective on contact with
thrombin at a site of treatment in a patient as a tissue adhesive,
hemostat or sealant, said composition comprising non-autologous,
non-single donor mammalian, clottable fibrinogen recovered from a
process comprising precipitating fibrinogen from a sample of
non-human, mammalian blood plasma with polyethylene glycol 1000 and
reprecipitating said fibrinogen with glycine, wherein precipitation
of said fibrinogen with polyethylene glycol is performed only once,
such that at least about 90% of the fibrinogen present in said
sample is recovered, wherein said recovered fibrinogen polymerizes
when provided in solution at said site at a therapeutically
effective fibrinogen concentration of about 30 mg/ml thereof or
less, to a fibrin network having therapeutically effective
strength, wherein said therapeutically effective fibrinogen
concentration at said site is about 30 mg/ml or less, wherein said
composition contains less than about 30% (w/w), based on total
protein mass present therein, of proteins other than fibrinogen,
and said composition further comprises a sufficient amount of one
or more low molecular weight physiologically-compatible solutes
such that said composition, if formulated as a lyophilized
material, can be reconstituted therefrom at room temperature in
sterile water for injection in about 30 minutes or less, at about
25 mg/ml of said fibrinogen.
19. A method according to claim 18 further comprising contacting
said site or said composition with an amount of thrombin effective
to convert fibrinogen of said composition to a fibrin network
having therapeutically effective strength.
20. A method according to claim 19 wherein the amount of thrombin
utilized therein is from about 0.1 NIH unit up to about 1000 NIH
units thereof per milliliter of fibrinogen-containing therapeutic
composition utilized therein.
21. A method according to claim 20 wherein said amount of thrombin
is from about 1.0 NIH unit up to about 300 NIH units thereof.
22. A method according to claim 19 wherein said thrombin and said
fibrinogen-containing therapeutic composition are applied
separately to said site of treatment.
23. A method according to claim 19 wherein said thrombin and said
fibrinogen-containing therapeutic composition are applied
concurrently to said site of treatment.
24. A method according to claim 18 wherein said thrombin and said
fibrinogen-containing therapeutic composition are first combined
and then applied to said site of treatment.
25. A method according to claim 18 wherein said
fibrinogen-containing therapeutic composition that contacts said
site of treatment is in the form of a dry lyophilized powder.
26. A method for inducing tissue adhesion, sealing of tissue, or
hemostatis in a mammalian patient at a site of treatment therein
comprising contacting said site with a therapeutically effective
amount of a therapeutic composition comprising, as percent by
weight of total protein contained therein, clottable fibrinogen, at
about 56% or greater; serum albumin, at less than about 20%; gamma
globulin, at less than about 10%; plasminogen, at less than about
1%; and plasma fibronectin, at less than about 3%.
Description
FIELD OF THE INVENTION
[0001] This invention relates to therapeutic compositions
comprising non-autologous non-single donor fibrinogen. More
particularly, the invention is directed to the provision of a
fibrinogen-containing composition effective as a tissue adhesive,
hemostat, or sealant.
REPORTED DEVELOPMENTS
[0002] There is a recognized need for therapeutic compositions of
physiological origin that are effective as tissue adhesives, as
tissue sealants, or as hemostatic agents. Although there are
available well-known synthetic materials for such therapeutic use,
disadvantages have been identified with the use thereof. For
example, the use of cyanoacrylate glue following surgery as a
sealant or adhesive has been determined to cause toxic effects in
tissues contacted therewith resulting in tissue necrosis and
foreign body immune reactions. See, for example, Epstein, G. H. et
al., Ann. Otol. Rhinol, Larynaol., 95, 40-45 (1986). Similarly, the
use of synthetic suture materials has been reported to result in
tissue ischemia and necrosis.
[0003] It is known that therapeutic compositions for use as tissue
adhesives, sealants or hemostatic agents can be made using the
proteins fibrinogen and thrombin, Cronkite, E. P. et al., J.A.M.A.,
124, 976 (1944), Tidrick, R. T. and Warner, E. D., Surgery, 15, 90
(1944). Fibrinogen is a soluble protein found in the blood plasma
of all vertebrates that when contacted by thrombin (another plasma
protein) becomes polymerized to an insoluble gel-like network. In
polymerized form, the fibrinogen is referred to as fibrin. The
conversion of fibrinogen to fibrin is crucial to normal hemostasis
in vertebrates.
[0004] There are numerous potential advantages, relative to the use
of synthetic materials, associated with the use of fibrinogen as an
adhesive, sealant or hemostatic agent. For example, when applied to
a wound, polymerized fibrinogen (fibrin) forms a network or
scaffolding through which it is more likely that immunologically
active cells (to defend against invading pathogens) and also
epithelial cells (for tissue regeneration and repair) can migrate.
Additionally, fibrin materials may be dissolved gradually by the
body (a process termed fibrinolysis) after treatment leading to
more normal appearance of the healed site.
[0005] By way of background, restriction of the flow of blood in
response to a wound involves a complex series of physical steps and
biochemical reactions that are divided broadly into two major
processes, primary and secondary hemostasis. Primary hemostasis
involves the formation of a soft clot composed primarily of
platelets, non-nucleated blood cells approximately 5 microns in
diameter. Primary hemostasis is accomplished when platelets attach
to adhesive macromolecules exposed on damaged vascular
endothelium.
[0006] Secondary hemostasis refers to the reinforcement of the soft
platelet clot. This secondary process is initiated by coagulation
factors, enzymes that circulate in the plasma in inactive form but
that become activated during primary hemostasis. The sequential
activation of these enzymes results ultimately in the production of
the coagulation factor thrombin from its inactive precursor form
known as prothrombin. Thrombin then acts to polymerize fibrinogen
into the insoluble polymeric matrix known as fibrin. As described
below, an additional protein factor, factor XIII, is involved in
stabilizing the fibrin network. The fibrinogen molecule has a
molecular weight of about 340,000 and is a rod or ellipsoid-shaped
particle. It has been determined that fibrinogen, in circulating
form, consists of a dimer of 2 identical units each consisting of 3
polypeptides known as A.alpha., B.beta., and .gamma.. The
polypeptides contain numerous binding sites important to the final
assembly of the fibrin network. For a detailed review of fibrinogen
structure see Blomback, B., "Fibrinogen and Fibrin Formation and
its Role in Fibrinolysis", Chapter 11, pp. 225-269, in Goldstein,
J. ed., Biotechnology of Blood, Butterworth-Heinemann, Boston,
Mass. 1991.
[0007] Although the use of fibrinogen as an adhesive is known, the
physical or chemical properties (for example, solubility) of the
protein limit substantially its use. As noted in U.S. Pat. No.
4,650,678, at Column 1 thereof, difficulty is encountered in
reconstituting fibrinogen from lyophilized material (the form of
fibrinogen preferred for long term storage for clinical use). The
'678 patent discloses also that fibrinogen solutions, to be
effective as adhesive compositions, are generally believed to
require a concentration of clottable fibrinogen therein of about 80
mg/ml or more (which may then be diluted 1:1, for example, at the
time of use and at the treatment site with thrombin solution).
[0008] Additional fibrinogen-containing adhesive compositions and
methods for the preparation thereof are provided in U.S. Pat. No.
4,298,598, No. 4,362,567, No. 4,377,572, and No. 4,414,976.
Therapeutic adhesive fibrinogen compositions disclosed therein are
stated to require concentrations of fibrinogen of at least about 70
mg/ml (which may again be diluted 1:1 at the treatment site by
contact with a thrombin-containing solution).
[0009] The present invention relates to fibrinogen-containing
compositions that have surprising clinical (medical) utility as
adhesives, sealants, or hemostatic agents, and that provide
therapeutically effective strength at fibrinogen concentrations at
the treatment site of, for example, only about 10 mg/ml. The more
dilute and less viscous nature of the therapeutic compositions
provided according to the practice of the present invention
decreases substantially the time necessary to resuspend such
compositions from the lyophilized form, an important advantage in,
for example, the hospital emergency room. Filtration of the
fibrinogen during processing is also facilitated. In preferred form
the fibrinogen used in the therapeutic compositions of the
invention is of non-human mammalian origin, eliminating risk of
contamination of product with human viruses.
SUMMARY OF THE INVENTION
[0010] Broadly stated, this invention provides for a therapeutic
fibrinogen composition effective as a tissue adhesive, hemostat, or
sealant, and that is more effective, per concentration of
fibrinogen contained therein, than presently available
compositions. Accordingly, there is provided a therapeutic
composition effective on contact with thrombin at a site of
treatment in a patient as a tissue adhesive, hemostat or sealant,
said composition comprising non-autologous, non-single donor
mammalian fibrinogen that is capable of polymerizing when provided
in solution at said site at a concentration of about 10 mg/ml
thereof or less, to a fibrin network having therapeutically
effective strength, and further comprising a sufficient amount of
one or more physiologically-compatible solutes such that said
composition, if formulated as a lyophilized material, can be
reconstituted therefrom at room temperature in sterile water for
injection in about 30 minutes or less, at about 25 mg/ml of said
fibrinogen.
[0011] There is provided also a therapeutic composition effective
on contact with thrombin at a site of treatment in a patient as a
tissue adhesive, hemostat or sealant, said composition comprising
non-autologous, non-single donor mammalian fibrinogen that is
capable of polymerizing when provided in solution at said site at a
concentration of about 30 mg/ml thereof or less, to a fibrin
network having therapeutically effective strength, wherein said
composition contains less than about 30% (w/w), based on total
protein mass present therein, of proteins other than fibrinogen,
and further comprises a sufficient amount of one or more low
molecular weight physiologically-compatible solutes such that said
composition, if formulated as a lyophilized material, can be
reconstituted therefrom at room temperature in sterile water for
injection in about 30 minutes or less, at about 25 mg/ml of said
fibrinogen.
[0012] The fibrinogen-containing compositions of the invention,
effective at low protein concentration, are of importance in a
clinical setting as they are more rapidly resuspended from the
lyophilized state such as, for example, for emergency use in a
hospital.
[0013] Still another aspect of the invention provides a method for
producing a therapeutic fibrinogen composition comprising three or
more steps including at least the steps of:
[0014] (A) precipitating fibrinogen from a sample of mammalian
blood plasma with polyethylene glycol 1000;
[0015] (B) resuspending said fibrinogen in solution; and
[0016] (C) reprecipitating said fibrinogen with glycine; wherein
precipitation of said fibrinogen with polyethylene glycol is
performed only once.
[0017] The yield of purified fibrinogen derived from samples of
mammalian blood plasma according to this process is typically about
90% or greater.
[0018] There are provided also methods for inducing tissue adhesion
or hemostasis in a mammalian patient, or sealing a tissue in said
patient, at a site of treatment therein, comprising contacting said
site with a therapeutically effective amount of a composition of
the invention.
[0019] Additional embodiments of the invention and the clinical
importance thereof are described in connection with the following
detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Introduction
[0021] This invention provides for fibrinogen-containing
therapeutic compositions effective as tissue adhesives, tissue
sealants or hemostatic agents. Although a therapeutic composition
of the invention may perform only one of the above functions at a
particular site of treatment in a patient, all the compositions
retain, nonetheless, the capacity to perform all three of the
aforementioned functions. Additionally, there are clinical
(medical) indications, such as the treatment of burns, plastic or
reconstructive surgery, tissue grafting, or the treatment of
anastomotic sites for which the compositions may perform
simultaneously two or even all three of the aforementioned
functions, that is, as adhesive, sealant and hemostatic agent.
[0022] As described below, important aspects of the invention
include new methods for the purification of fibrinogen suitable for
the formulation of the aforementioned compositions, and methods for
the use thereof.
[0023] The importance of the present development is based upon the
discovery that fibrinogen compositions provided according to the
practice of the invention possess greater adhesive strength than
that of prior art compositions. Without being limited as to theory,
it is likely that the particular combinations of steps in the
fibrinogen purification procedures described below facilitate rapid
isolation of samples of clottable fibrinogen containing therein
only small amounts of irreversibly aggregated or inactive protein
molecules. Additionally, fibrinogen produced according to the
practice of the invention and prepared in lyophilized form, is
provided in intimate contact with particular
physiologically-compatible solutes (proteins and/or low molecular
weight solutes), the presence of which stabilizes, and facilitates
resuspension of, the fibrinogen.
[0024] As will be described in detail below, the novel compositions
of the present invention have important clinical benefits. In
particular, since only a low concentration of fibrinogen is
contained therein, and said fibrinogen is of a soluble and
clottable character, the compositions can be resuspended from the
lyophilized state for clinical use very rapidly, thereby
facilitating emergency treatments and/or minimizing the time needed
to complete surgical procedures. Filtration of fibrinogen solutions
during processing (such as through a 0.22 micron filter) is also
facilitated thereby.
[0025] Additionally, and as described further below, although
addition of solubilizing agents to fibrinogen compositions in order
to facilitate the resuspension thereof has been described
previously, most such agents (at least at the resultant
concentrations thereof) are non-physiological, limiting the types
of therapeutic treatments that may be performed with the resultant
compositions at target tissue sites. For example, non-physiological
concentrations of a salt in a fibrinogen adhesive may interfere,
osmotically, with communication of vital tissue fluids between a
grafted tissue and the graft bed. Accordingly, the presence in such
compositions of high concentrations of solute sets a limit on the
clinical utility thereof.
[0026] The therapeutic compositions of the invention comprise
non-autologous, non-single donor mammalian fibrinogen, that is,
they comprise fibrinogen derived (pooled) from multiple mammalian
donors. Preferred donors are mammals other than the human. The
preparation of autologous or of single donor human fibrinogen for
use as an adhesive sealant or hemostatic agent is well known in the
art. See, for example, Dresdale, A. et al., Surgery, 97(6), 750-754
(1985); Siedentop, K. H. et al., Laryngoscope, 95, 1074-1075
(1985); Epstein, G. H. et al., Ann. Otol. Rhinol. Laryngol, 95,
40-45 (1986). An advantage associated with autologous fibrinogen
preparations is that use thereof obviates the concern for
transmission of human viruses. Disadvantages associated with the
use thereof include unpredictable adhesive strength, and that the
product may be available only in limited quantities and not be
available on demand. Additionally, in a clinical setting, emergency
room personnel would need to be diverted to the production thereof
at a time when available personnel may be limited.
[0027] Fibrinogen-based adhesives (fibrin glues) are accepted for
therapeutic use in Europe. It is noted however that such
compositions, if made from pooled human donor plasma, are not
approved for therapeutic use in the United States because of the
risk of transmission of viral disease such as AIDS and hepatitis B
and C. Numerous incidents of infection have been reported.
Accordingly, practitioners of the art have sought to provide the
aforementioned autologous or single donor fibrinogen compositions
to minimize also risk of viral infection. As aforementioned, there
is however substantial variation in the fibrinogen content of such
preparations owing to individual patient (donor) variability.
Accordingly, a further disadvantage associated with the use of such
preparations is the difficulty in predicting, accurately, the
clinically effective dose thereof.
[0028] An alternate resolution to the above-mentioned risk of viral
infection, characteristic of human plasma-derived therapeutic
products, is to provide fibrinogen from a mammalian source other
than from humans. Fibrinogen compositions that could be provided
from mammalian species other than the human are disclosed, for
example, in U.S. Pat. Nos. 4,377,572 and 4,362,567. However, the
therapeutic compositions defined therein are stated to contain at
least about 70 mg/ml or more of fibrinogen (prior to any dilution
at the site of treatment) leading potentially to the presence also
therein of a substantial amount of additional and antigenic protein
impurities, there resulting an associated risk of severe immune
response. For example, rejection of grafted tissue triggered by
immune response to the adhesive used at the graft-host interface
may be more likely to occur. Such immune responses are predicted to
affect, most severely, patients requiring numerous repeated
treatments such as, for example, in response to severe burn injury,
scarring or wounding.
[0029] In contrast, a principal advantage of the therapeutic
compositions of the present invention is that they provide
therapeutically effective strength at a treatment site when the
fibrinogen thereof comes to be present at a concentration of only
about 10 mg/ml, or even lower. Additionally, up to about 95%, or
greater, of the total protein present therein is fibrinogen.
Accordingly, the likelihood of adverse immune response is
minimized.
[0030] It is noted that fibrinogen itself has an amino acid
sequence and also a tertiary structure that is conserved
substantially between, for example, the cow and the human forms, so
that by proper selection of a donor mammalian species, risk of
potential antigenicity to a patient can be very significantly
minimized. In this regard, alternate preferred sources of mammalian
fibrinogen include swine, goats and horses.
[0031] Use of highly purified bovine fibrinogen compositions for
the treatment of human patients is preferred, therefore, since the
product may be applied, efficaciously, at a dose that presents to
the patient's immune system very little foreign antigen (derived
also from the total composition of other serum proteins).
Additional advantages of the therapeutic compositions of the
invention will be apparent from the following further discussion of
the properties thereof.
[0032] Therapeutic compositions of the invention can be formulated
as lyophilized materials, or in the form of solutions or as frozen
solutions. It is most preferred, if long term storage of product is
required, that the compositions be formulated as lyophilized
materials. Storage in the form of frozen solutions is preferred
also in which case storage at or below -20.degree. C. is most
preferred. Therapeutic compositions of the invention prepared in
the form of aqueous solutions are best used within about 4 hours of
being prepared in solution form.
[0033] In connection with administrating the therapeutic
fibrinogen-containing compositions at a treatment site in a
patient, the fibrinogen composition (typically a solution thereof
reconstituted from the lyophilized state) is normally administered
in conjunction with an additional thrombin-containing solution,
there resulting dilution of the fibrinogen to a final lower
concentration. Typical protocols in the art call for equal volumes
of therapeutic fibrinogen-containing composition (as a solution)
and of an additional thrombin composition (also typically a
solution). The use of numerous fibrinogen-containing compositions
known in the art has been stated to require the presence therein of
a minimum of about 70 mg/ml of fibrinogen, there being derived
therefrom, fibrinogen, of at least about 35 mg/ml, at the treatment
site. The fibrinogen-containing therapeutic compositions of the
present invention are effective, however, even when the final
concentration of fibrinogen derived therefrom at the treatment site
(taking into account the volume of any thrombin solution applied
therewith) is only about 10 mg/ml or lower.
[0034] Therapeutic Compositions of the Invention
[0035] A characteristic and novel feature of the
fibrinogen-containing therapeutic compositions of the invention is
that from such compositions fibrinogen can be provided that is
capable of polymerizing, at a site of treatment in a patient, at a
concentration of about 30 mg/ml or less, to a fibrin network having
therapeutically effective strength. Provided also according to the
practice of the invention are therapeutic compositions wherein said
therapeutic strength is achieved at a fibrinogen concentration of
about 5 to about 10 mg/ml.
[0036] As described below the fibrinogen-containing compositions of
the invention are mixed with an additional thrombin containing
composition to provide, at the treatment site, a reactive
therapeutic composition containing fibrinogen having the
aforementioned efficacy. In connection with the use of the
fibrinogen-containing therapeutic compositions of the invention,
the following considerations are of note. The therapeutic
compositions of the invention can of course be used to provide
efficacious fibrinogen at a site of treatment in a patient wherein
the concentration thereof is greater than about 30 mg/ml, as long
as the fibrinogen so provided is effective (as measured by the
procedure of Example 2) at below about 30 mg/ml. Additionally, the
concentration (and therefore volume) of a fibrinogen-containing
therapeutic composition can be adjusted to accommodate the
concentration (and therefore volume) of the additional
thrombin-containing composition to provide a volume of reactive
therapeutic composition appropriate to the course of treatment and
as the clinical practitioner selects.
[0037] Preferable in the practice of the invention are those
fibrinogen-containing therapeutic compositions from which
fibrinogen can be effectively provided, in solution, at a site of
treatment in a patient at a final concentration of between about
7.5 mg/ml and about 25 mg/ml. Highly preferred are compositions
from which fibrinogen can be so provided at a final concentration
between about 10 mg/ml and about 20 mg/ml.
[0038] In connection with selecting an appropriate concentration of
mammalian fibrinogen (for example, bovine fibrinogen) produced
according to the practice of the invention for contact at the
treatment site, the following factors are among those to be
considered: (A) minimizing the concentration of fibrinogen will
result also in limiting the amount of contaminating (bovine) plasma
proteins that may cause an immune response in the patient, (B)
minimizing the concentration of fibrinogen limits the time
necessary to resuspend a lyophilized starting material, (C)
minimizing the concentration of fibrinogen reduces the viscosity of
the fibrinogen solution thereby improving delivery characteristics
in a clinical setting, and (D) providing fibrinogen of the highest
purity. Additionally, it is also necessary to provide sufficient
therapeutically effective fibrinogen in a short period of time so
that the polymerized fibrin so formed is of sufficient therapeutic
strength.
[0039] For many clinical indications, contacting the treatment site
with a solution of therapeutic composition providing fibrinogen at
a resultant final concentration of about 10 mg/ml to about 20 mg/ml
thereof is most appropriate. Routine experimentation, however, as
is known in practice of the present art can be used to optimize an
appropriate concentration (whether higher or lower) of fibrinogen
for any particular clinical application. This may be accomplished
by monitoring the treatment site to determine, for example, if
hemostasis has been stably achieved, whether a graft has adhered
properly, or whether site of anastomosis has been sealed. In the
event the result is not yet satisfactory, additional composition
may be applied accordingly to the methodology of the clinical art.
It is important to note again that the clinical use of fibrinogen
at a final concentration thereof that is higher than the
concentrations referred to above in connection with describing the
capabilities of fibrinogen compositions of the present invention
is, nonetheless, within the practice of the invention if the
fibrinogen, or therapeutic compositions containing same, express
the appropriate properties that are used herein to define the
fibrinogen-containing therapeutic compositions of the
invention.
[0040] In the preferred practice of the invention, at least about
80% of the fibrinogen present in a therapeutic composition of the
present invention will be clottable, that is, polymerizable to
fibrin in the presence of thrombin. There are numerous reasons
(such as denaturation) why at least a portion of the fibrinogen
molecules derived from a purification process therefor may not be
clottable. As described below (see Example 1) fibrinogen that is
90% clottable, or higher, is produced readily according to the
practice of the invention.
[0041] However, and without being limited as to theory, it is
believed that the residual amounts (and types) of blood plasma
proteins that are present in the therapeutic compositions of the
present invention stabilize fibrinogen, preserving its clottability
and facilitating also resuspension thereof from the lyophilized
form. As discussed in greater detail below, such blood plasma
proteins are referred to in the practice of the invention as
physiologically-compatible solutes.
[0042] Determination of the percent of fibrinogen contained in a
sample that is clottable can be determined following, generally,
any of several standard assay procedures. See, for example,
Blombck, B. and Blombck, M., Arkiv Kemi., 10, 415 (1956) and
Jacobson, K., Scand. J. Clin. Lab Invest., 7(Suppl. 14), 1 (1955).
Clottability determinations were performed, according to the
practice of the present invention, using the method described in
Example 3 below.
[0043] The therapeutic compositions of the present invention
contain a population of fibrinogen molecules that is capable of
polymerizing when provided in solution at a site of treatment in a
patient at a concentration of about 30 mg/ml thereof or less, to a
fibrin network having therapeutically effective strength. The term
"therapeutically effective strength" is defined specially herein,
according to the methodology of Example 2 below, to mean that a
liquid sample of the therapeutic fibrinogen-containing composition
provided for testing (as 0.8 ml of a 20 to 21 mg/ml fibrinogen
solution prepared by reconstition from a lyophilized powder)
demonstrates an average adhesive strength of at least about 900
grams, based on multiple trials. Therapeutic compositions preferred
according to the practice of the invention have average adhesive
(breaking) strengths, based on multiple trials according to the
procedure of Example 2, of at least about 1100 grams, with an
average of at least about 1200 grams, or higher, being most
preferred.
[0044] Example 2 below makes reference also to numerous other
testing procedures that have been used in the art to test,
perpendicularly, horizontally or at an intermediate angle, the
effective strength of fibrin networks. Although the absolute value
of the adhesive strength of the therapeutic compositions of the
present invention will vary, as will that of the prior art
compositions depending, necessarily, on the specific test procedure
employed, the relative values of adhesive strength of the
compositions of the invention, in relation to those of the prior
art, per equivalent amount of fibrinogen per sample, are
substantially maintained.
[0045] As aforementioned, it is preferred that at least about 80%
of the fibrinogen in the therapeutic compositions of the invention
be clottable. It is preferred also that the compositions contain
less than about 30% (w/w) based on total protein mass present
therein, of proteins other than fibrinogen. According to the
practice of the invention (see Example 1 below), purification of
fibrinogen may leave in contact therewith amounts of other protein
species, notably serum albumin, gamma globulin, plasminogen, plasma
fibronectin, and also factor XIII. Preferably, therapeutic
fibrinogen compositions within the practice of the invention
comprise, as percent by weight of total protein contained therein,
clottable fibrinogen of at least about 56%, about 14% or less of
non-clottable fibrinogen, serum albumin at less than about 20%,
gamma globulin at less than about 10%, plasminogen at less than
about 1%, and plasma fibronectin at less than about 3%. The
concentration of serum albumin is most preferably less than about 4
to 5%.
[0046] A highly preferred therapeutic composition which is produced
routinely according to the process described in Example 1 below
comprises (expressed as percent (w/w) of total protein contained
therein) bovine fibrinogen at about 95%, and of which about 90%
thereof is clottable; serum albumin at about 0.8%; gamma globulin
at about 0.02%; plasma fibronectin at less than 0.5%; plasminogen
at less than 0.02%; and also factor XIII at about 0.1 unit/mg
fibrinogen.
[0047] Fibrinogen concentrations of up to about 95%, expressed as
(w/w) of total protein, are achieved according to the process of
Example 1 of the invention as described below.
[0048] The presence of coagulation factor XIII (fibrin-stabilizing
factor) in the therapeutic compositions of the invention is highly
preferred in that factor XIII acts to further stabilize the fibrin
network with covalent crosslinking bonds. Preferred amounts of
factor XIII in the compositions of the invention range from about
0.05 to about 0.2 units/mg fibrinogen. It is noted, however, that
no special procedure need be followed in order that at least a
sufficient amount of factor XIII be present in the therapeutic
compositions of the invention, since sufficient factor XIII
copurifies, invariably, with the product fibrinogen.
[0049] As described above, the therapeutic compositions of the
invention contain fibrinogen molecules that provide
therapeutically-effective adhesive strength at a fibrinogen
concentration, at the treatment site, of only about 10 mg/ml.
Without being limited as to theory, it is believed that the small
quantities of the copurifying plasma proteins present, for example,
in the highly preferred therapeutic composition defined directly
above, are representative of a range of concentrations (preferably
from about 1 to about 10%) of copurifying protein (expressed as %
w/w of protein) that when present in conjunction with low molecular
weight physiologically-compatible solutes (defined and described
directly below) facilitate solubilization (reconstitution) of
fibrinogen from the lyophilized state without interfering with the
polymerization of the fibrinogen (fibrin) lattice. The
below-described low molecular weight physiologically-compatible
solutes are also effective in this regard when the total
concentration of the copurifying plasma proteins is below about
1%.
[0050] In addition to the aforementioned copurifying proteins, the
therapeutic compositions of the invention comprise also a
sufficient amount of one or more low molecular weight
physiologically-compatible solutes such that said resultant
compositions, if formulated as lyophilized materials, can be
reconstituted therefrom, at room temperature and in sterile water
for injection, in about 30 minutes or less at a concentration of
about 25 mg/ml of fibrinogen.
[0051] A low molecular weight physiologically-compatible solute, as
defined functionally according to the practice of the invention,
represents any solute of less than about 5000 daltons (preferrably
less than about 1000 to 2000 daltons) that, at the particular
concentration thereof in solution that is selected for use (or at a
particular amount thereof present in a lyophilized
fibrinogen-containing composition), facilitates reconstitution of
fibrinogen as aforementioned, but does not interfere with the
potential range of therapeutic functions of the fibrinogen
composition so reconstituted. Additionally, a low molecular weight
physiologically-compatible solute is one that is approved, or comes
to be approved, by the United States Food and Drug Administration
for use at the intended concentration, for clinical indications
involving adhesives, sealants or hemostatic agents in humans. It is
understood that a particular solute may qualify as physiologically
compatible at one concentration (NaCl at 140 mM) but be
incompatible at another concentration (for example, 1000 mM).
[0052] One example of how a low molecular weight solute can produce
a physiologically incompatible effect has been mentioned, that is,
by creating at the concentration thereof that is proposed to be
used, an osmotic imbalance between a grafted tissue and a graft bed
site interfering therefore with adhesion and/or healing at the site
of treatment. Additionally, the substance may cause other
deleterious effects at a treatment site, or at a site remote
therefrom, such as to affect adversely wound healing, or to act as
a barrier to physiological processes such as cell migration
associated with tissue repair. The existence of such effects, for
each potential solute, is well known in the art. Representative of
solutes known to the biochemical art as effective solubilizing
agents for fibrinogen protein but that are incompatible (at
fibrinogen-solubilizing concentrations thereof) with clinical use
are urea at about 500 mM or more; and sodium dodecyl sulfate at
about 1% (w/v) or more.
[0053] Low molecular weight physiologically-compatible solutes
representative of those which satisfy the functional criteria
stated above include, at appropriate (fibrinogen-solubilizing)
concentrations or amounts thereof, sodium chloride, sodium
phosphate, the amino acids histidine, arginine, leucine and
glycine, guanidine compounds, sodium citrate, and fatty acids.
Concentrations (or amounts) of solutes that are appropriate to the
practice of the invention may be determined for each particular
solute by characterizing the properties of fibrinogen product
(percent clottability, post-lyophilization stability and rate of
solubilization) made according to the general protocol of Example
1.
[0054] Particularly preferred solutes that stabilize fibrinogen in
lyophilized form, and that facilitate reconstitution therefrom, are
sodium citrate incorporated into lyophilized fibrinogen-containing
compositions at between about 0.10 mg and about 0.50 mg thereof per
mg of fibrinogen; sodium phosphate similarly incorporated at
between about 0.05 mg and about 0.5 mg per mg of fibrinogen; sodium
chloride at between about 0.10 mg and about 1.0 mg per mg of
fibrinogen; and epsilon-aminocaproic acid (added also as a
fibrinolysis inhibitor) at between about 0.075 mg and about 1.0 mg
per mg of fibrinogen. Other efficacious amounts of each of the
aforementioned solutes, or of other solutes, whether present
separately, or in combination, can be determined.
[0055] Example 1 below provides for the preparation of a highly
preferred therapeutic fibrinogen composition that, in lyophilized
form, contains a combination of the above low molecular weight
solutes, in intimate contact with said fibrinogen, whereinby
reconstitution of the therapeutic composition (at about 25 mg/ml of
fibrinogen in 30 minutes or less, with sterile water for injection
used as diluent at room temperature) is facilitated. Amounts of the
above-identified solutes present in contact with the fibrinogen in
the lyophilized preparation thereof were, approximately, and per mg
of fibrinogen therein, 0.14 mg (NaCl), 0.12 mg (sodium phosphate),
and 0.21 mg (sodium citrate), and 0.21 mg (epsilon-aminocaproic
acid).
[0056] As described in detail below, the therapeutic compositions
of the invention (including the lyophilized forms thereof for
resuspension) are preferably formulated at (or for use at) a pH of
about 6.5 to 8.5. Most preferably the pH of formulation is
maintained at from about 7.5 to about 8.5.
[0057] Preparation of the Therapeutic Compositions of the
Invention
[0058] The therapeutic compositions of the invention are prepared
using a process that comprises, generally, the steps of (A)
precipitating fibrinogen from mammalian blood plasma with
polyethylene glycol 1000; and then (B) resuspending said fibrinogen
in solution; and then (C) reprecipitating said fibrinogen with
glycine; wherein precipitation of said fibrinogen with said
polyethylene glycol was performed only once. More specifically,
therapeutic fibrinogen-containing compositions of the invention
were prepared following the procedure described in Example 1 below.
Although the above-described effective methods are novel and
nonobvious when viewed as a whole, individual steps thereof provide
also important contributions to the clinical utility of fibrinogen
product derived therefrom.
[0059] First, although samples of polyethylene glycol ("PEG")
polymer having average molecular weights that vary through a wide
range (including, for example, samples having average molecular
weights of 5000 and 8000, "PEG-5000" and "PEG-8000" respectively)
are useful in the practice of the invention, it is PEG 1000 that is
preferred.
[0060] Precipitation of a pellet of fibrinogen with PEG-1000 (as
specified according to the procedure of Example 1) leads to a
cohesive fibrinogen precipitate that is more readily collected, for
resuspension, than fibrinogen precipitate resulting from contact
with, for example, PEG-8000. Accordingly, use of low molecular
weight PEG (such as PEG-1000) facilitates recovery of clottable
fibrinogen.
[0061] An additional advantage associated with the purification
processes of the invention is that they are adaptable to the
insertion of one or more additional steps, known in the art and
approved in relation to the manufacture of clinical products, for
the neutralization of mammalian viruses.
[0062] A further additional advantage that is derived according to
the practice of the invention is that the substance
epsilon-aminocaproic acid, added for its art-recognized role as
fibrinolysis inhibitor, functions also as a low molecular weight
physiologically-compatible solute that stabilizes fibrinogen,
facilitating reconstitution thereof in therapeutically effective
form from lyophilized material.
[0063] With respect to the use of low molecular weight
physiologically-compatible solutes other than the species thereof
utilized according to the specific procedure of Example 1 below,
for the purpose of stabilizing fibrinogen and/or facilitating
reconstitution thereof from the lyophilized form, appropriate
solutions containing such solutes can be added (substituted) at
particular points in the purification process such as, for example,
when the fibrinogen, present as a glycine precipitate, is
resuspended. Alternatively, the aforementioned fibrinogen
precipitate may be resuspended with the buffer defined in Example
1, and then diafiltered against a solution containing other low
molecular weight physiologically-compatible solutes useful in the
practice of the invention.
[0064] Use of the Therapeutic Compositions of the Invention
[0065] The therapeutic compositions of the invention are useful in
any of the clinical applications recognized in the art for which
fibrinogen-based adhesives, sealants or hemostatic compositions can
be used. In connection with the description of the invention
herein, the terms adhesive, sealant, and hemostatic agent
(hemostat) are defined broadly and used as these terms are
understood in the art.
[0066] Tissue adhesion, sealing of tissue or hemostasis are induced
in a mammalian patient at a site of treatment therein, according to
the practice of the invention, by contacting the treatment site
with a therapeutically effective amount of fibrinogen composition.
According to the practice of the invention, such effective amounts
need not be equivalent to amounts that cause complete or permanent
adhesion of tissue, that effect, for example, total sealing of
tissue boundary, or arrest completely bleeding or loss of tissue
fluid from a tissue or tissue boundary. Rather, such compositions
are within the scope of the invention, if the use thereof provides
at least a partial effect that is of benefit to the patient in the
course of a treatment.
[0067] The amount of sealant, hemostat or adhesive necessary to
perform clinical procedures varies widely depending on, for
example, the size of the treatment site in the patient, the nature
of the condition in need of treatment and such factors as may be
unique to each patient.
[0068] Additionally, in the context of many clinical applications,
determination of an effective amount of therapeutic composition for
use in connection therewith may depend on experimentation or
"titration" of the treatment site with, for example, therapeutic
samples that deliver increasing amounts of clottable fibrinogen to
the treatment site. It is accepted in the art and well within the
skill of clinical practitioners to determine for each patient and
for each clinical indication, amounts (including, for example,
volumes, concentrations, and number of any multiple layerings
thereof that may come to be needed) of therapeutic fibrinogen
composition that are effective.
[0069] The therapeutic compositions of the invention become
effective, for use at a site of treatment in a patient, on contact
with thrombin. Generally thrombin, at any effective amount thereof,
may be provided to polymerize the fibrinogen of the compositions
according to any method (whether generally or narrowly applicable)
known to be effective in the art. Included therefore in the
practice of the invention is the use of therapeutic fibrinogen
compositions in which polymerization of the fibrinogen, in whole or
part, is accomplished by endogenous thrombin, that is, thrombin of
the patient that is or comes naturally to be present at the
treatment site. Generally speaking, however, in the practice of the
invention, thrombin is provided in the form of an additional
composition, whether liquid or solid, (for example, see Example 2),
provision of which is through any of numerous specific procedures
that are, or may come to be, known in the art.
[0070] According to the practice of the invention, the
aforementioned additional composition comprising thrombin is
applied, usually, at the site of treatment in such a manner that
from about 0.10 NIH unit up to about 1000 NIH units thereof are
provided (preferably from a thrombin-containing solution) for each
milliliter of fibrinogen-containing therapeutic composition
utilized. However, greater or lesser amounts of thrombin may be
used by the clinician depending on the circumstance of each patient
according to good medical practice. Generally, it is preferred that
from about 1.0 to about 300 NIH units of thrombin be provided for
each milliliter of composition, however, the amount thereof may be
varied according to the circumstance again following good medical
practice.
[0071] The fibrinogen-containing therapeutic composition and the
additional thrombin composition can be applied separately, or
concurrently, to the site of the treatment, or they may be first
combined and then applied rapidly (within a time frame generally
known in the art or subject to determination by routine
experimentation) to the site of treatment. In order that good
mixing be achieved and for most clinical applications, it is
preferred that the thrombin be applied to the treatment site
concurrently with the fibrinogen solution, as a solution having a
volume approximately equal to that of the said fibrinogen solution.
It is within the practice of the invention to deliver the thrombin
and fibrinogen-containing solutions at any combination of relative
volumes, although certain combinations of volumes are likely, in
most circumstances, to lead to the formation of less effective
"reactive therapeutic compositions" as that term is defined below.
For example, 1 .mu.l of thrombin solution added to 2 ml of
fibrinogen solution will likely lead to a composition of
non-uniform strength due to time constraints on mixing and
non-homogeneity of the formed matrix.
[0072] The thrombin and fibrinogen can be delivered to the
treatment site through, for example, syringes including dual barrel
syringes, or by other devices or means which allow for on-contact
mixing. Examples of suitable delivery devices are described in U.S.
Pat. No. 5,104,375, No. 4,359,049, No. 4,631,055, and No.
4,874,368. Other of suitable delivery devices include pipettes.
[0073] The mixture formed from a fibrinogen-containing therapeutic
composition and an additional thrombin composition define,
according to the practice of the invention, a "reactive therapeutic
composition", which contains, typically, and per milliliter thereof
in contact with a site of treatment in a patient, between about
0.05 and 500 NIH units of thrombin and between about 7.5 and about
30 mg of fibrinogen. The selection of the particular concentration
of fibrinogen and of thrombin present in a reactive therapeutic
composition to be used in a particular clinical application, and
for a particular patient, is guided by factors well known in the
medical art including the size of the treatment site and the nature
of the procedure to be effected. For example, if the
fibrinogen-containing composition is used to effect rapid
hemostasis at a large wound, it is generally appropriate to use a
high concentration of thrombin in relation to fibrinogen (for
example, about 500 units per ml of solution containing about 25 mg
of fibrinogen) such that the fibrin clot is formed "immediately",
that is, within seconds. Additionally, use of up to about 2000 to
3000 units of thrombin per ml of a fibrinogen-containing solution
is known and is within the practice of the invention. However, if
adhesion of a grafted tissue is being performed, wherein careful
placement thereof and follow-up manipulation are required, a lower
rate of polymerization may be effected using, for example, about 10
units of thrombin per ml of solution containing about 25 mg of
fibrinogen.
[0074] In connection with the use of the therapeutic compositions
of the invention, the following additional considerations are also
noteworthy.
[0075] Lyophilized fibrinogen compositions of the invention can
also be used directly in powder form as a sealant, adhesive or
local hemostatic agent. If the lyophilized preparation is to be
used in this fashion, it may be sprinkled directly, for example,
onto a wound site or surgical incision where it reacts with
endogenous thrombin to effect a seal or hemostasis. This is
typically useful when the site (for example, a vessel or wound) to
be closed is small, and blood loss is not rapid.
[0076] Additionally, the fibrinogen composition and/or thrombin may
be applied, for example, to a wound or surgical incision by
incorporation into a gauze pad, sponge, collagen or gel-type matrix
or into a similar device and treating the area to initiate
hemostasis or adhesion as necessary.
[0077] The fibrinogen-based compositions described herein afford a
number of significant advantages over conventional surgical
techniques (e.g., suturing) whether used alone as a means of
surgical closure or used in combination with other techniques. The
therapeutic compositions of the invention provide, potentially, a
matrix for platelet adhesion and cell migration. Additionally, when
the preparation is applied topically to an actively bleeding site,
the fibrin matrix so formed may effectively trap platelets,
triggering hemostasis-associated morphological changes in the
platelets, and through a series of reactions involving both primary
and secondary hemostasis, contribute further to effective clot
formation.
[0078] Additionally, the fibrin matrix provides a compatible medium
for the growth of contiguous cellular tissue. In this manner, cells
from surrounding "like" tissue can infiltrate the matrix, promoting
healing and facilitating replacement of damaged cells. The
architecture of the affected tissue may therefore be restored
substantially to that of the neighboring tissue by cells migrating
from surrounding tissue sites. Similarly, production of scar tissue
is minimized.
[0079] The therapeutic composition may be placed in any of
pharmaceutically acceptable containers (such as packets, vials or
bottles) depending on the intended clinical use. The containers may
also be sized to accommodate small or large quantities of
therapeutic composition depending upon the expected (or unexpected)
needs of the physician. Preferably the containers are light
resistant, and are stored also at or below about 5.degree. C. prior
to use.
[0080] Numerous of "inert" additives (substances such as
preservatives, dispersants or additional diluents) known in the art
can be added to the therapeutic compositions of the invention, with
the understanding that such substances must be physiologically
compatible as that term has been defined previously.
[0081] As aforementioned, the therapeutic compositions and methods
defined by the present invention are useful in connection with any
of the clinical applications where adhesives, sealants, and
hemostatic agents can be used. In order that the clinical
importance of the present invention be further illustrated, there
follows hereafter mention of but some of the diverse clinical
applications for which compositions and methods of the invention
are very useful. Provision of high quality fibrinogen for use in
conjunction with laser tissue welding, Oz, C. M. et al., J.
Vascular Surgery, 11(5), 718-725, (1990); treatment and
preservation of the ruptured spleen, Brands, W. et al., World J.
Surg., 6, 366-368, (1982); sealing of vascular protheses,
Walterbusch, G. et al., Thorac. cardiovasc. Surgeon, 30, 234-235,
(1982); sealing of vascular grafts prior to implantation, Kalmer,
P. et al., Thorac. cardiovasc. Surgeon, 30, 230-231, (1982);
sealing of microvascular anastomoses, Pearl, R. M. et al., Surgery,
Gynecology & Obstetrics, 144, 227-230, (1977); for repair of
middle ear defects, Epstein, G. H. et al., Ann. Otol. Rhinol.
Larvngol., 95, 40-45, (1986), and Silberstein, L. E. et al.,
Transfusion, 28(4), 319-321, (1988). A further important
application includes bonding of a corneal inlay into a recess
prepared to receive same in the cornea of a patient.
EXAMPLES
[0082] The following Examples are representative of the practice of
the invention.
Example 1
Preparation of a Lyophilized Fibrinogen Preparation Having High
Capacity for Resuspension as Clottable Material
[0083] Preparation of Solutions
[0084] A fifty liter volume of bovine blood (obtained as about 10
liters each from 5 sacrificed animals) was collected into five
ten-liter containers each containing 1.5 liter of anti-coagulant
acid citrate dextrose solution, "ACD", USP Formula A, Baxter Health
Care. The plasma fraction was separated by centrifugation at 3000
rpm (1700 g) for 20 minutes with the temperature held at
2-8.degree. C., according to standard practice.
[0085] A 67% solution (w/v) of polyethylene glycol was prepared by
dissolving 670 gm of PEG-1000 (product P-3515, of Sigma Chemical
Co., St. Louis, Mo., having mean molecular weight of about 1000
daltons) in sterile water for injection ("WFI") brought to 1 liter
volume. A 3.33 molar solution of glycine was prepared by providing
250 g of glycine (Sigma Chemical Co., USP grade) in a 1000 ml
beaker, brought to 1 liter volume with fibrinogen purification
buffer as defined directly below (1 liter of the resultant glycine
solution is required for each liter of plasma processed).
[0086] A fibrinogen purification buffer (hereinafter "buffer") was
prepared as follows. Sodium chloride, 52.6 gm, for final
concentration 150 mM; sodium citrate, 88.23 gm, for final
concentration 50 mM; dibasic sodium phosphate, 42.59 gm, for final
concentration 50 mM; and epsilon-aminocaproic acid "EACA", 78.71
gm, for final concentration 100 mM, were placed in a 6 liter
Erlenmeyer flask and dissolved in 3 liters of WFI. 5M HCl or NaOH
was then used to adjust the pH to 8.0, after which the volume was
brought to 6.0 liters with additional WFI.
[0087] Purification Procedure
[0088] The following purification procedure, leading to product
that is highly preferred according to the practice of the
invention, was then carried out using aseptic technique as
recognized in the art. ACD-treated bovine plasma (24.6 liters) was
poured into a 55 liter Nalgene tank and stirred magnetically at a
moderate speed so as not to cause foam to develop in the tank. A
first precipitation of fibrinogen-containing material was commenced
by adding slowly, and with continuous stirring, 4340 ml of the 67%
(w/v) PEG-1000 solution over about a 5 minute period. At the end of
the precipitation, plasma and precipitate were poured into sterile
one liter polypropylene centrifuge bottles and capped. The samples
were then centrifuged at 4000 rpm (2700 g) at a temperature of
2-8.degree. C. for five minutes.
[0089] The resultant supernatant was then decanted and the
precipitate scraped aseptically from the bottles and placed into a
55 liter Nalgene tank. The empty bottles were then washed with
buffer to recover as much of the remaining precipitate as
practicable. A total of 8.2 liters of buffer was added, with the
slurry resultant from the process having a final volume of ten
liters. A magnetic bar was added to the flask to provide continuous
stirring at moderate speed until the precipitate dissolved (about
three hours).
[0090] A second precipitation was performed by adding continuously,
over a 5 minute period with constant stirring, 25 liters of the
aforementioned 3.33 M glycine solution to the solution containing
the redissolved precipitate. The resultant precipitate and solution
were then transferred aseptically to one liter centrifuge bottles
for centrifugation at 4000 rpm (2700 g), again at a temperature
held to 2-8.degree. C. for five minutes. The resultant supernatant
was then decanted and this second precipitate was transferred to a
five liter Nalgene beaker. As much of the precipitate as
practicable was removed by washing with a small amount of buffer.
The recovered precipitate was then dissolved in 2.46 liters of
buffer to yield a final solution volume of 3.5 liters.
[0091] The dissolved precipitate was then diluted further with 14
liters of WFI to provide fibrinogen in the solution at a
concentration of about 5.7 mg/ml, as measured by a fibrometry
assay, and the product solution was filtered (at 4-5 psi of
nitrogen gas) through a 0.22 micron Sartobran PH filter, Sartorius
Co. The resultant filtered volume was aliquoted to yield about 60
to 75 mg of fibrinogen per vial. For use in clinical practice,
vials may contain such other amounts of fibrinogen as is
appropriate for any of clinical indications for which such
therapeutic materials are, or may be, used in the art.
[0092] The vials were then frozen at -45.degree. C. over a period
of about two hours time in a Revco Ultra-Low apparatus, and then
lyophilized. Lyophilization was carried out as follows. Frozen
vials were loaded in trays onto lyophilizer shelves. After cooling
the shelves to about -50.degree. C. (with product temperature
brought to about -35.degree. C.), the vials were then maintained at
this temperature for about 3 hours. The chamber was evacuated to a
pressure of about 300 milliTorr Hg, and shelf heating was initiated
until, after a 16 hour period, the shelf temperature had reached
about +38.degree. C. (product temperature about -4.degree. C.), and
the chamber pressure had been decreased to about 200 milliTorr Hg.
The temperature was held at (+).sub.32 to 40.degree. C. for about
24 hours, over which time the pressure was gradually decreased to
about 75 milliTorr Hg. Vials were then stoppered and sealed. At
least about 90% to 95% of the fibrinogen present in the sample of
mammalian blood plasma subject to processing is typically
recovered.
[0093] A representative analysis of the product derived from the
above-described process was, as % (w/w) of total protein contained
therein, fibrinogen 95%; serum albumin, 0.8%; gamma globulin, at
0.02%; plasma fibronectin, less than 0.5%; plasminogen, at less
than 0.02%; and factor XIII, at about 1%, which equals an amount
thereof such that if the lyophilized product were reconstituted to
a solution having 20 to 25 mg/ml of fibrinogen, then the factor
XIII would be present therein at about 0.1 unit/mg fibrinogen.
Assays to determine the concentration of the copurifying proteins
were performed as follows. For (bovine) fibrinogen, absorbance at
280 nm was determined based on a molar absorptivity of 1.55
ml/mg-cm at pH 7.0. Serum albumin, gamma globulin, plasma
fibronectin, and plasminogen were each determined by ELISA
methodology.
[0094] Factor XIII was determined by a functional assay based on
the insolubility of crosslinked fibrin clot in dilute acid. One
unit of factor XIII activity was defined as that present in 1 ml of
normal citrated bovine plasma. See Simmons, A., Factor XIII
Detection (Screening), in Hematology, A Combined Theoretical and
Technical Approach, W. B. Saunders, Philadelphia, 1989, pp.
296-297.
[0095] epsilon-Aminocaproic acid was present in the final product
lyophilized powder at about 13% (w/w), with fibrinogen making up
about 59% (w/w) of the final weight of the product powder. The
remaining weight percent of the product powder was provided by
sodium chloride (about 9%), sodium citrate (about 12%), and sodium
phosphate (about 7%).
[0096] Prior to use in adhesion testing (Example 2), the vial was
reconstituted at room temperature with sterile water for injection
(WFI) to yield a solution containing fibrinogen at 20-25 mg/ml,
with about 90% thereof being clottable.
Example 2
Determination of Therapeutically Effective Strength of Fibrin
Networks
[0097] The following adhesion testing procedure involves
determination of force (weight) necessary to detach a fibrin
network sample from mammalian skin and is representative of the
wide variety of similar procedures used by practitioners of the
art. The procedure of this Example is adapted in part, and with
modification, from the procedures of Burnouf-Radosevich, M. et al.,
Vox. Sang., 55, 77-84 (1990) and Lindner, A. et al., Wiener
Klinische Wochenschrift, 92 (suppl. 109), 1-9 (1980). Other
recognized procedures that test adhesion strength in a
perpendicular direction are described by U.S. Pat. No. 4,650,678;
Wan, H. L. et al., Transfusion, 29, 41(s) (1989); and Marshall, J.
Urology, 119, 310-311. Testing procedures based upon horizontal
shearing or response to force at an angle are described by Saltz,
R. et al., Plastic and Reconstructive Surgery, 88(6), 1005-1014
(1991), and by Siedentop, K. et al., Laryngoscope, 98, pp. 731-733
(1988) respectively.
[0098] Although use of different test procedures results,
necessarily, in variation as to absolute amounts of adhesive
strength measured (for example, weight held), the relative adhesive
strength of particular fibrinogen compositions will be maintained
through a series of such assays irrespective of the protocol
employed.
[0099] For the present procedure, a section of full thickness skin
(about 20 square inches) was removed from a laboratory guinea pig
immediately after sacrifice. A scalpel was used to scrape excess
fascia from the dermal side of the tissue. The testing procedure
involves using 2 patches of skin each being about 2 inches square.
The remaining portion of the skin sample can be frozen for future
use. Prior to use, the skin patches were kept moist by wrapping in
a damp sterilized cheesecloth impregnated with distilled water.
[0100] In order to perform the adhesion test, one piece of 2 inch
square skin tissue was wrapped (dermal side out) around a brass
contact plug having a 1 inch diameter and secured in place by a
tightly wrapped nylon thread. The second square of skin tissue was
secured (dermal side out) on a flat surface. A flat rubber O-ring,
11/4 inch I.D., was placed on the center of the second tissue
square. The O-ring defines a confined space to be loaded with the
fibrinogen test sample and also thrombin.
[0101] Accordingly, 0.8 ml of fibrinogen solution (prepared
according to the procedure of Example 1 and reconstituted as
described from the lyophilized powder prepared therein) was
resuspended to a fibrinogen concentration of about 20 to 21 mg/ml
(see below) using WFI. The thrombin solution was prepared by
reconstituting lyophilized thrombin (Thrombostat.RTM. of
Parke-Davis Co.) to about 10 units/ml in WFI. Equivalent thrombin
is available as the following products, Thrombinar.RTM. of Jones
Medical Co., and Thrombogen.RTM. of Gentrac Co.
[0102] While the O-ring was secured in place, 0.8 ml of the
fibrinogen solution and 0.8 ml of the thrombin solution were
delivered, simultaneously, to the center of the ring from separate
syringes with thorough mixing.
[0103] The assay timer was started upon delivery of the two
solutions. The skin-covered contact plug was then immediately
placed into the ring where it was held gently in place for ten
further seconds with care being taken to assure that the clot was
not squeezed out of the ring. The contact plug was then allowed to
stand on its own for 20 minutes anchored into the clot within the
rubber ring.
[0104] Adhesion strength was measured by increasing, in 20-gram
increments and at 30 second intervals, the weights placed in a cup
attached to a string, the other end of which was attached to the
contact plug (the weights are made to hang vertically by running
the string through a pair of pulleys). In this particular version
of the procedure, the first weight applied weighed 100 grams, with
30 second periods elapsing before the first of the 20 gram weights,
and then the subsequent 20 gram weights, were applied. To prevent
improper detachment, care was taken to support the weight bucket
while each of the weights was added, the downward force on the
polymerized composition being therefore relaxed and then reapplied
following each addition of weight. The adhesive strength of the
polymerized composition is reported herein as the value of the last
weight held successfully, prior to breaking of the contact.
[0105] Fibrinogen purified, lyophilized, and reconstituted
according to the procedure of Example 1 had a therapeutically
effective strength (using 0.8 ml samples of fibrinogen solution, at
about 20 to 21 mg/ml) of at least about 1100 to about 1200 grams
based on multiple trials.
[0106] A single lyophilized fibrinogen preparation made according
to the procedure of Example 1, was used to generate three separate
resuspended (in WFI) samples having the following fibrinogen
concentrations: Sample I (21.4 mg/ml), Sample II (20.2 mg/ml) and
Sample III (20.1 mg/ml). Adhesive strengths measured by multiple
trials (subsamples) for each of the samples were 880, 838, and 1154
gm (for Sample I); 660, 1150, >1218 and >1218 gm (for Sample
II); and >1218, >1218, >1218, >1218 and >1218 gm
(for Sample III). With respect to reported weight values that
appear not to be even multiples of 20 gm, it was determined that
some of the weights used had values (in grams) that were slightly
different than 20 gm or a multiple thereof. The symbol >1218
(for greater than 1218 gm) indicates that the adhesive strength was
found to be greater than the highest weight combination tested
(1220 gm, which had an actual weight of 1218 gm).
[0107] The guinea pig skin samples can be used for subsequent runs
if maintained moist in distilled water after being wiped free of
residual clot material with, for example, a cheesecloth moistened
with distilled water.
Example 3
Determination of Clottable Fibrinogen
[0108] Clottable fibrinogen can be determined following generally
any of several standard assay procedures such as Blombck, B. and
Blombck, M., Arkiv Kemi., 10, 415-441, (1956) or Jacobsson, K.
Scand. J. Clin. Lab Invest., 7(Suppl. 14), 1, (1955). In the
practice of the invention percent clottability was determined by
taking advantage of the capability of a solution of thrombin (1
IU/ml, Sigma Chemical Co., St. Louis, Mo.) to polymerize fibrinogen
with resultant loss of soluble absorbing material (at 280 nm) from
the test samples.
[0109] Two ml fibrinogen test samples were adjusted (diluted or
concentrated) to contain about 1 mg/ml of fibrinogen (containing
therein unknown amounts of clottable versus inactive material)
using the fibrometric procedure of Clauss, A., Acta Haematologia,
17, 237, (1957) to assay for total fibrinogen. This procedure is
based on conversion of fibrinogen to fibrin in the presence of
excess thrombin. The resultant normalized samples (2 ml) were mixed
with 1 ml samples of thrombin (at 1 IU/ml). Samples were then
incubated at 37.degree. C. in a water bath for 30 minutes.
Resultant clotted fibrin was removed by low speed
centrifugation.
[0110] Appropriate control and blank solutions were also prepared
consisting of: (A) unclotted fibrinogen, 2 ml of original
undetermined sample at about 1 mg/ml total fibrinogen, and 1 ml
buffer; (B) a thrombin zeroing blank (3 ml) containing 1 ml of the
thrombin (1 IU/ml) solution, and 2 ml of assay buffer; and (C) a 3
ml blank of appropriate buffer for zeroing.
[0111] Percent clottability was defined as 100 times a fraction
defined by the quantity [A.sub.280 (unclotted fibrinogen) minus
A.sub.280 (corrected value)] divided by A.sub.280 (unclotted
fibrinogen), wherein the corrected value used therein represented
the A.sub.280 of fibrinogen remaining in the clotted supernatant
corrected for (reduced by) the A.sub.280 component contributed by
the added thrombin.
* * * * *