Note: Descriptions are shown in the official language in which they were submitted.
W O 95/25748 - 2 1 6 2 9 9 7 PCTAUS95/03451
,
-1 -
TOPICAL FIBRINOGEN COMPLEX
This ar~plic~lion is a continuation-in-part of Inl~r"atio"al Application
PCT/US92/07493, which was filed as a continuation-in-part of U.S. Serial
No. 755,156, filed Se~le,),ber 5, 1991, now abandoned.
BACK~;ROUND OF THE INVENTION
Field of The Invention
The present invention relates to a fibrinogen composition and its method
of preparation, wherein the composition can be used for wound closure
in conjunction with thrombin and calcium.
Related Art
The attempt to use fibrinogen to achieve topical i ,e" ,ostasis was
investigated as far back as the early 20th Century when fibrinogen
patches for hemostasis were used in cer~br~l surgery. Later, mixtures of
plasma and ll ,n)l "l,in were used for skin grafting and intracavity injections
in the therapy of tuberculosis. However, these early allelll,,~s had two
major drawbacks: since the source of fibrinogen was plasma, the
concentration of fibrinogen was low which resulted in a fibrin film of
insufficient strength; and it was not possible to inhibit the normal
physiologic process of fibrinolysis such that the fibrin film was degraded
relatively quickly.
Prior attempts to develop an effective fibrin sealant have also been
hampered by the fact that most of these preparations contain high levels
- of plasminogen which required these compositions to additionally contain
an anti-fibrinolytic agent in order to prevent premature degradation of the
fibrin seal. Because anti-fibrinolytic agents are typically derived from a
non-human source the possibility of a patient having an adverse reaction
to such foreign proteins is significant, especially upon multiple exposure
to these agents. Although Rose, et al., (U.S. 4,627,879) report the
production of a fibrin adhesive which does not necessarily require the
W O 95/25748 2 1 6 2 9 9 7 PCTrUS95/03451
-2-
presence of an anti-fibrinolytic additive, the composition disclosed in this
reference does not deal with another major drawback of these prior fibrin
sealing compositions, namely, the possible presence of i"rectious agents,
such as He~ ilis B or Hl\~ in the plas",a. As a conseq.lence, the Rose
reference requires that the co",positions described therein be derived
from a single donor, in order to avoid the l,d"s",ission of infectious
agents which might be associated with pooled plasma.
Thus, there is considerable need for a fibrin sealant which can be derived
from pooled plasma and which is free of anti-fibrinolytic compounds,
animal prole;ns, and infectious agents such as viruses. The present
invention addlt:sses these needs by providing such cor~,lJosilions.
2 1 62997
WO 95/25748 - . ` t, , PCTtUS95tO3451
.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that pooled plasma,
even when subst~ntially depleted of Factor Vlll, can be processed to
prpduce a fibrinogen p,~pa,dlion which, when reacted with lhr~mL,in and
calcium, will produce a fibrin sealant that can be used to promote
he" losl~is.
In detail, the invention provides a fibrinogen composition which, in addition
to being essentially free of Factor Vlll and plasminogen, does not require
the use of an anti-fibrinolytic agent and has been treated to eliminate the
presence of infectious agents, such as lipid enveloped viruses. A further
advantage of the composition is that essentially all of the prolc"1s present
in the composition are of human origin.
The composition of the invention, through its transient in vivo presence,
provides a matrix which persists for a period of time sufficient to achieve
a medical effect, essentially lacks host toxicity upon degradation, and
provides mechanical strength to promote hemostasis.
WO 95/25748 ~ 2 ~ 6 2 9 9 7 PCT/US95/03451
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 Schematic representation for prepa,~lion of Topical Fibrinogen
Complex.
FIGURE 2 Effect of calcium ion concer,l,dlion on fibrin polymer formation.
Fibrinogen (90 mg total protein/ml) and Ihr~",L,i" (500 U/ml) were mixed
and allowed to incubate for 10 minutes. Lane A: MW markers; Lane B:
Control fibrinogen; Lane C: OmM Ca++; Lane D: 1mM Ca++; Lane E:
3mM Ca++; Lane F: 6mM Ca++; Lane G: 10mM Ca++; Lane H: 20mM
Ca++; Lane l: 30mM Ca++; Lane J: MW markers.
FIGURE 3 Effect of calcium ion concel~ lion on fibrin polymer formation.
Fibrinogen (130 mg total protein/ml) and ll ,ro" Ibil~ (500 U/ml) were mixed
and allowed to incllbate for 10 minutes. Lane A: MW markers; Lane B:
Control fibrinogen; Lane C: 0 mM Ca++; Lane D: 1mM Ca++; Lane E:
3mM Ca++; Lane F: 6mM Ca++; Lane G: 10mM Ca++; Lane H: 20mM
Ca++; Lane l: 30mM Ca++; Lane J: MW markers.
FIGURE 4 Rate of fibrin polymeri~alion. Fibrinogen (130 mg total
protein/ml) and thrombin (500 U/ml) were mixed in the presence of
calcium ion (40mM CaCI2). Lane A: MW markers; Lane B: 0 min; Lane C:
1 min; Lane D: 3 min; Lane E: 5 min; Lane F: 10 min; Lane G: 30 min;
Lane H: 60 min; Lane l: 2 hr; Lane J: 4 hr; Lane K: 8 hr; Lane L: 24 hr;
Lane M: fibrinogen control; Lane N: MW markers.
woss/2s74s ; 2 1 62 9 9 7 PCT/US95/03451
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The inventors have devised compositions which, when reacted with
thrombin, will produce a fibrin sealant that represents a significant
improvement over the prior art CGlllpOSitiGllS inle"de~ to accGnl,~lisll this
effect. These ColllposiliGIls (collectively, "Topical FiL,ri,-Ggen Complex
(TFC)"), are advdnl~geolJs because, like prior art cG""~osili~ns, they will
effectively induce hemostasis in human tissue but, unlike prior art
compositions, can do so without risk of causing an immunologically
adverse response in the treated person to non-human proteins and
without substantial risk of viral infection.
r,efer,~d blood fractions for producing the compositions of the invention
are plasma, cryopreci,citdle, and/or Factor Vlll-depleted cold-precipitate.
Because the pre~er,~d blood fraction for use as a sldllil19 material is
human plasma, the starting ",alerial will her~a~ler be referred to as
plasma, although it will be understood by those of skill in the art that the
compositions of the invention can be produced by sla,li"g with any
human blood-derived fraction which has not been significantly depleted of
fibrinogen. Generally, the process involves the formation of a cryoprecipi-
tate from plasma which is high in Factor Xlll (F Xlll) and fibrinogen. This
step may (and pr~feral,ly will) be followed by cold-precipitation of pr~tei"s
from the cryoprec;pi1ale. The product of the cold-precipitation process,
if any, (i.e, the cold-p,~c;,.~ilale), will typically CGI ,lain a high concentration
of fibrinogen and very low levels of F Vlll.
More specifically, the ,urt:~r,~d method for producing the TFC
compositions of the present invention uses frozen human plasma from
one or more donors as a starting material. Brefeldbly, the plasma to be
used will have been screened using con\,~nliol ~al assay techniques for the
presence of infectious viral cGnldl~;.1ants, such as hepalilis B and human
immunodeficiency virus (HIV) to eliminate plasma for use as a starting
material which CGnlai"S detectable levels of such cGI ~Idminants.
Whether or not the plasma has been previously screened, the
cryop-~c;pilale or cold-prec;,~ilale product of the plasma to be used in the
WO 95125748 2 1 6 2 9 9 7 PCT/US95/03451
compositions of the invention will be treated as further described below
to reduce the viral activity therein to undetectable levels. For purposes of
this disclosure, the phrase "undetectable levels" will refer to levels of viral
activity which can be detected by viral assay protocols which are well
known to those of ordinary skill in the art, such as delectiG" of plaque
forming units in infected tissue or cells (~e, e.g., Example 5). A redlJction
in the known viral activity level in a given composition will be
conventionally desc, il~ed herein as the "log,0 redlJction factor". Generally,
using the available techniques for reducing viral activity in a protein
containing composition which are known in the art and/or described
herein (~e, e.g., Example 5), it can be expected that the compositions of
the inventions will have a log,0 reduction factor for lipid enveloped viruses
of at least 4 logs (pr~ferably at least 6 logs) and a lesser reduction factor
for other viral pathogens.
AS described in Exal l l~!e 1, to process the frozen plasma to a
cryoprec;~ e, the plasma is thawed in a controlled environment. The
resulting cryoprecipitate may be further processed to TFC without first
being converted to a cold-precipitate. However, to limit the total protein
con~enl of the compositions of the invention to fibrinogen, residu~l
amounts of fibronectin and any added protein components (such as
albumin), the cryoprecipitate will ~.referdbly be further processed to a cold-
precipit~te.
To that end, a cryoprecipitate can be produced by freezing the blood
fraction (for example, plasma) which is thered~ler warmed to a
temperature not exceeding about +6C. The cryoprecipitate is dissolved
in distilled water at about 20C-35C. Calcium chloride is added at a
conce"L,dle from about 1,uM to about 1000 IlM. rreferdbly, calcium
chloride is added at this step at a concen~rdtion of about 40 ~lM and the
pH adjusted to 6.8 + 0.3 to enhance the precipitation of fibrinogen.
The dissolved cryoprecipitate is then cooled to about 10C with mixing,
whereupon the cold-precipitate forms. The precipitate is removed from
solution by centrifugation at, for example, 5600 9 to 7200 9. The
precipitate may be stored at -60C or lower if desired. Where cold-
WO 95/25748 . . ' ~ 2 1 6 2 9 9 7 PCTtUS95/03451
pr~c;,~ dlion is used, the addition of a calcium ion source during the
process will enhance the preci,c ildlion of fibrinogen, as well as fibronectin,
thereby inc,easin~ the co,1cel,lralion of these subsL~nces in the cold-
,ur~-,;pi1~le. The fibrinogen can also be further concenl,~dle-~ by the
acldilion of polyethylene glycol (PEG) to the cold-prec;pita~e.
The cryoprec;pilale or cold-prec;pilale will usually be dissol~d in buffer
with mixing and the pH of the cold-prec;pitdle adjusted to about 7.2,
preferably to 7.2 + 0.1. To prevent interaction between the fibrinogen of
the cryoprecipitate or cold-precipitate and any lhlol~lbil) which may be
present therein, the cryo- or cold-precipitate will ,urererdbly be resuspen-
ded in the presence of a protease inhibitor, such as PPACK (D-Phe-L-Pro-
L-Arg-chloromethylketone), heparin cofactor ll, hirudin or anti-thrombin lll
(ATIII) to inhibit lhr~n,bin which may be pr~sen~. Most pre~n~d as
thrombin inhibitor is PPACK at a concenl,dlion from about 0.75 ~M to
about 1.75 ~M. The product of a composition of the invention which has
been treated with an effective amount of a thrombin inhibitor will be
considered to be essentially free of active lhlo~ . If used, the thrombin
inhibitor will be removed from the compositions of the invention before
Iyoph~ lio,) by, ~r~ferdbly, PEG precipitation and, optionally, also the
DEAE column step of the procedure ~e, FIGURE 1 and Example 1).
Those of skill in the art will know of other protease inlliLJitol~ of thrombin
suitable for use in the compositions of the invention and their effective
concenl~lions.
To remove any ,c,roll,r~mbin complex which may be present in the cryo-
or cold-precipitate, the precipitate suspension is ~ns~r~d to a buffer
solution containing a salt such as tri-calcium phosphate. Exposure to the
salt-con~ lil lg buffer will minimize the likelihood of ,uroll ,rombin
conversion to thrombin which, if such a reaction were to occur, could lead
to the conversion of fibrinogen to fibrin. In this state, the resulting
composition can be considered to be essentially free of prolhrumbin
complex. The calcium phosphate, in turn, is removed from the process
by centri~ugation and/or filtration. Additional techniques for removal of
plulhr~ bin are described by Murano (Prothrombin and Other Vitamin K
Proteins, Vol. Il, Seegers and Walz, eds., CRC Press, Inc. Boca Raton, Fl.,
WO 95125748 ' 2 1 6 2 9 9 7 PCT/US95103451
1986); Heystek, et a/., (V~ Sang., 25:113, 1973); Banowcliffe, et al., (V~
Sang., 25:426, 1973); Chandra, et a/., (V~K Sang., 41:257, 1981); and
Chanas, et a/., (US. 4,465,623), incor~.ordled herein by reference. Thus,
by removing prull,rur,,bi,, and inhibiting Ihru,,lL,in in the compositions of
the invention, the final compositions will be essentially free of fibrin
molecules; i.e., all of the fibrinogen therein will be unreacted until
se,ua,~dlely ex~ose.l to thrombin (~Jrt:rerdbly in vivo) to produce a fibrin
sealanl.
In a pre~er,~d embodiment, the dissolved cryo- or cold-prec;pil~te is
warmed to about 23-27C and contacted with a Iysine affinity column,
such as the matrix column product sold commercially under the
tradename Iysine-Sepharose 4B. Residu~l plasminogen present in the
cryo- or cold-precipitate will be adsorbed by the matrix while fibrinogen will
not, thus rendering the resulting solution essentially plasminogen free.
For example, using the Iysine-Sepharose ",alerial referred to above, the
resulting final TFC composition will contain no more than 10~9
plasminogen/milliliter of TFC.
The removal of plas"linogen is important for two reasons. First,
plasi" ,i, logen, when converted to plasmin, will break down ~ibri,1ogen and
fibrin molecules. The latter are formed from interaction between fibrinogen
and thrombin in the fibrin sealant to be produced from the compositions
of the invention. Second, although fairly effective plasminogen inhibitors
are known in the art, their use requires additional manufacturing steps (to
deter" ,ine the concer,l,clio,1 of plasminogen ,uresenl and concenlralion of
inhibitor needed). More importantly, presently commercially available
plasminogen inhibitors are of non-human origin and therefore pose the
risk of immunologically adverse reactions to composilions which contain
them, particularly on repeated ~I,plic~lions thereof. This risk is avoided
by removal of plasminogen from the compositions of the invention.
The solution collected from the Iysine matrix will preferably be
concel,l,~led by, for example, the addition of polyethylene glycol (PEG).
For use in humans, the PEG used to concenl~le the eluate will have a
molecular weight in a range which is not toxic to humans (e.g., 3000-
WO 9512S748 2 ~ 6 2 9 q 7 PCT/US95/03451
.
g
6000) and will be added to a final conce~ ,l,dlion from about 3% to about
7%, pr~ferdl)ly about 4% (w/w). Most ,c,ererdLly the resulting PEG
pl~c;pil~e will be dissol~d in a buffer solution, filtered and the pH thereof
adjusted to an alkaline level; e.g., about 8.6, ,~r~terdbly 8.6 + 0.1. In an
alternate embodiment of the invention, it is also possible to perform the
PEG prec;pil~lion step prior to adsor~lion with the Iysine matrix.
In all embodiments, the corrlpositisns (pr~r~rdbly the concentrate
solution) will be Irt:aled with an effective amount of a viral activity reducingagent, such as a detergent, which, typically, acts by disrupting the lipid
envelope of such viruses as Hepatitis B, Hl\! and HTL\C The term
"effective amount of viral activity reducing" agent means that the
conce~lr~lio,1 of viral activity reducing agent added to the composition is
sufficient to reduce the viral activity in the compositions of the invention
to undetectable levels. Of course, the conce"l,dlion of viral activity
reducing agent should not significantly inhibit the ability of the composition
to form a fibrin sealant in the presence of thrombin; i.e., the viral activity
reducing agent used will not denature fibrinogen.
To some extent, viral contaminants may be removed from the
compositions of the invention by virtue of process steps which do not
involve the addition of a viral activity reducing agent to the composition.
Because viral activity reducing agents typically affect only lipid enveloped
viruses (by disrupting the inl~y, ily of the lipid envelope), the process steps
will likely be the principal means given the current state of the art by which
any viruses present which lack a lipid envelope will be removed from the
compositions of the invention. Such process steps will be known to, or
can readily be ascertained by, one of ordinary skill in the art and include
viral partitioning and adsorption/filtration with calcium phosphate.
Nondenaturing detergents which are useful as such agents can be
selected by one of ordinary skill in the art from such recognized groups
as anionic, cationic, and non-ionic detergents. Examples include sulfated
WO 9S125748 2 ~ 6 2 9 9 7 PCT/US95/03451
-10-
alcohols and sodium acid salts such as sulfated oxyethylated alkylphenol
(sold co",r"e,~ially under the tradenames "Triton W-30" and "Triton X-
100") sodium dodecylbe"~e"sulrul,dle (sold col"",er~ially under the
tradename "Nacconol NR") sodium 2-sulfoethyl oleate (sold co""~,er~;ally
underthetradename "Igepon A') sodium cholale sodium deoxycholate
sodium dodecylsulfo"ale dodecyldimethylbenzylan "~ ,onium chloride (sold
col"",ercially under the tradename "Triton K-60"), oxyethylated amines
(sold commercially under the tradename "Ethomeen") N-dodecylamino-
ethanesulfonic acid ethylene oxide-propylene oxide conde"saLes
("Pluronic" copolymers) polyoxyethylated derivatives of esters (sold
commercially under the tradenames "Tween 80" and "Polysorbate 80")
polyoxyethylene fatty alcohol ethers (sold commercially under the
tradenames "Br3 35") tetramethylsutylphenyl ethers of polyethylene
glycols (sold commercially as "octoxynols") as well as detergents sold
commercially under the tradenames "nonidet P-40" and "Lubrox PX".
Those of skill in the art will know of other suitable nonionic detergents for
use as all or part of the viral activity reducing agent and may wish to refer
for examples to U.S. Patent Nos. 4481189 4 540 573 4 591 505,
4314997 and 4315919.
In the preferred embodiment the viral activity reducing agent will consist
of an organic solvent ,urt:fer~bly tri-n-butyl phosphate (TNBP) mixed with
nonionic delerye"ls ,c,r~f~rdL,ly polysorbate 80 and octoxynol 9. As
desc, iL,ed in Examples 1 and 5 undenatured compositio"s of the
invention in which viral activity is at undeteclable levels can be produced
using a ~ fer,~d viral activity reducing agent co",~rised of TNBP
(concentration 0.03-0.3%), polysorbate 80 (conce,lt,~lion 0.03-0.3%) and
octoxynol 9 (concent,alion 0.1-1.0%). In addition chaotropic agents
may also be utilized to inactivate viruses providing the agent does not
denature fibrinogen.
Alternatively the concenlrclion of organic solvent and detergent used in
the practice of the prefel,t:d embodiments of the invention can vary
depending upon the composition to be treated and upon the solvent or
delergenl selected. The alkyl phosphates can be used in concenl-alions
from about 0.10 mg/ml of mixture treated to 1.0 mg/ml pr~ferdbly
WO95125748 ; ` ` 2 1 62 9 97 PCT/US95/034~1
-11-
between about 0.1 mg/ml to about 10 mg/ml. The amount of detergent
or wetting agent utilized is not crucial since its function is to improve the
conlact between the organic solvent and the virus. For most of the
nonionic " ,ale, ials which are useful, the wetting agent can vary from about
0.001% to 10%"ur~erdbly from about 0.01% to about 2% of the aqueous
mixture, depending upon the amount of fatty malerial in the treated
aqueous mixture.
Whatever viral activity reducing agent is used, it will be removed after it is
conlacLed with the compositions of the invention for a period of time
sufficient to reduce the viral activity in the composition to undetectable
levels (at least one minute). In the preferred embodiment, DEAE
diethylaminoethyl cellulose (sold commercially under the tradename "DE
52") is the matrix utilized for the removal of the solvent/detergent from the
fibrinogen composition. The fibrinogen binds to the diethylaminoethyl
cellulose and, after thorough washing to remove unbound material and
deleryenl~ is eluted with, for example, 0.3M NaCI. Other ion exchange
male,ials which can be utilized for removal of the solvent/detergent
include virtually any of the commercially avn ;~ 'Q anion e;cchange mal, ices
including, but not limited to, cellulose and agarose ~al~ices. The specific
parameters for binding and eluting from these various ion exchange
r"alerials are known to those of skill in the art, or can be readily
asce, lai, led without undue experimentation.
The stability of the TFC co"".osilions of the invention may be enhanced
through the use of such excipienls as human serum albumin (HSA),
hydroxyethyl starch, deAl,an, or combinations thereof. The solubility of
the compositions may also be enhanced by the addition of a
nondenaturing nonionic detergent, such as polysorbate 80. Suitable
concer,L,dLions of these compounds for use in the compositions of the
invention will be known to those of skill in the art, or can be readily
ascertained without undue experimentation. The compositions of the
invention are, however, sufficiently stable to be stored and used without
the use of a st~hi~i~er. Thus, to minimize the non-fibrinogen protein
content of the compositions, the most ,ur~er,ed embodiment of the
WO 95125748 2 1 6 2 9 9 7 PCT/US95/03451
-12-
compositions will either contain no added stabilizer or will contain a
nGnprc"ei. ,aceous stabilizer.
Typically, after formulation the bulk is concel,l,dted from about 20% to
about 50% of its original eluate volume, then diluted to the pre-
conce,lt,dlion eluate volume. The bulk may then be conc6"l,aled to a
final total protein cGncenl~dlion of about 4g + 1g/dL CGIl~positio~, (w/v)
before sterile ~.,ucessi,lg and Iyophilization. As noted above, a pre:rer,~d
composition of the invention is one which, when r~consli1, ~ed, will CGn5i51
essentially of fibrinogen; i.e., the ,cr~teins in the composition will be
fibrinogen, no more than a residual amount of fibronectin (i.e., 20 mg/ml
or less, ~r~fe,dbly 10 ~lg/ml or less), no more than a residual amount of
plasminogen (no more than 10 ~g/ml"ur~rerdbly no more than 5 ~g/ml)
and from about 1 to 40 Units/ml of Factor Xlll (,cr~fer~L,ly more than 10
Units/ml). However, in aller"alive embodiments of the invention, the
composition may also contain components such as a protein stabilizer;
e.g., human serum albumin. Thus, the fibrinogen component of the
composition may comprise from about 50% to 100% of the total protein
in a TFC composition of the invention (w/v), and ,ult:rerdbly will comprise
at least 75% of the composition (w/v).
In the prt:rerlt:d embodiment, concenl,dliGn of the protein(s) in the
composition is accomplished by ullldrillldlion using a mell,l)rdl1e with a
molecular weight exclusion large enough to allow NaCI to be removed, but
small enough to retain protein molecules. This filtration is most pr~ferdbly
performed using a mel"brd,1e with a 30,000 MW exclusion. When the
TFC c~m~osition of the invention is Iyopl~ ed, the pre-lyophili~aliG"
voiume is usually y,~a~er than the volume to which the Iyophilizate is
resuspended at time of use.
If desired, the compositions of the invention can be modified to include
non-proteinaceous as well as proteinaceous drugs. The term "non-
proteinaceous drugs" encompasses compounds which are cl~-ssically
referred to as drugs, such as mitomycin C, daunorubicin, and vinblastine,
as well as antibiotics.
WO 95/25748 ` .: ` 2 1 6 2 9 9 7 PCT/US95/03451
-13-
The proteinaceous drugs which can be added to the fibrinogen
comrositions of the invention include immunomo~ tors and other
bicl-g;c-' r~spOnSe modifiers. The term "biological ,~spo"se modifiers"
is meant to enco",,uass suL~slances which are involved in modifying a
- 5 biological response, such as the immune ,t:s,uo"se or tissue growth and
repair, in a manner which enhances a particular lesi, ~.1 therapeutic effect,
for example, the cytolysis of bacterial cells or the growth of epidermal
cells. Examples of response modifiers include such compounds as
Iymphokines. Examples of Iymphokines include tumor necrosis factor, the
interleukins, Iymphotoxin, macrophage activating factors, mig,dlion
inhibition factor, colony stimulating factors, and the inlel rer~ns. In
addition, peptide or polysaccharide fragments derived from these
proteinaceous drugs, or independently produced, can also be
incorporated into the fibrinogen compositions of the invention. Those of
skill in the art will know, or can readily ascertain, other substances which
can act as proteinaceous or non-prulei, ,aceous drugs.
The compositions of the invention can also be modified to incorporate a
diagnostic agent, such as a radiop~-llJe agent. The presence of such
agents allow the physician to monitor the progression of wound healing
occurring internally, such as at the liver, gall bladder, urinary tract,
bronchi, lungs, heart, blood vessels, and spinal canal. Such compounds
include barium sulfate as well as various organic compounds containing
iodine. Examples of these latter compounds include iocetamic acid,
iodipamide, iodoxamate meglumine, iopanoic acid, as well as dial,i~oate
derivatives, such as dial,i~oate sodium. Other contrast agents which can
be utilized in the compositions of the invention can be readily asce, lail1ed
by those of skill in the art.
The concer,L,dlion of drug or diagnostic agent in the composition will vary
with the nature of the compound, its physiological role, and desired
therapeutic or diagnostic effect. The term "therapeutically effective
amount" means that the therapeutic agent is present in a sufficient
concenlfdlion to minimize toxicity, but display the desired effect. Thus, for
example, the co"ce"l,~dlion of an antibiotic used in providing a cytolytic
therapeutic effect will likely be clif~t:r~nl from the concenl,dlion of an
WO 95/25748 2 1 6 2 9 9 7 PCT/US95/03451
immune r~spo"se modulator where the therapeutic effect is to stimulate
the ,ur~lif~,dli~n of immune cells at the site of application of the fibrinogen
complex. The term "diagnostically effective amount" denotes that
co"cenl,dlion of diagnostic agent which is effective in allowing the fibrin
glue to be n,o"ito,ed, while minimizing pote,ltial toxicity. In any event, the
c~esi,~d conce~lt~dlioll in a particular instance for a particular compound
is readily ascel lainable by one of skill in the art.
The above ~iscloslJre generally describes the pr~:senl invention. A further
unde,~lan.ling can be obtained by reference to the following specific
exar"ples which are provided herein for purposes of illusLIdliGn only, and
are not intended to be limiting unless otherwise specified.
WO 9S/25748 ~` 2 ~ 6 2 9 9 7 PCT/US9S/034Sl
-15-
EXAMPLE 1
PREPARATION OF TOPICAL FIBRINOGEN COMPLEX
Topical fibrinogen complex (TFC) was pror~uce~ by the initial ~ r~lJa~lio"
of a cr~ Jr~c;~;t~e of plasma. The cryoprec;pilate for such use was
prepared by two difrer~"l techniques depending upon the physical form
of the plasma.
In one technique, sealed plastic bottles of frozen plasma were thawed in
a controlled env;,~"ment by contact with a heat exc~ange medium, such
as air or water. The thaw was col ,I,."ed by programming the
temperature and flow of the heat-exchange medium so that the maximum
te",,uerdlure of the plas",a did not exceed +6C. The containers were
then opened and the contents pooled into a jacketed stainless steel
thawing tank. In the thawing tank, the plasma was gently warmed (while
being mixed) to melt the remaining ice. The thawed plasma was then
pumped directly to a centrifuge or into a jacketed stainless steel holding
tank where it was maintained at 2.5C + 3.5C. The plasma was
centrifuged to remove the cryoprecipitate. The cryoprecipit~te, so
pr~par~d, may be stored at or below -25C or immediately processed to
antihemophilic factor. The cryo-poor plasma was collected in a jacketed
-~lain'~ss steel reaction tank.
Alternatively, cryoprec;~,ilale was prepared by placing sealed plastic bags
of frozen ~.lasma in a liquid nitrogen bath for several seconds. The bags
were removed from the bath and the crisp, cracked bags were stripped
from the plasma. The ,ulasma was then placed into a jacketed stainless
steel thawing tank. Alteratively, sealed plastic bags of frozen plasma were
arranged so as to warm the bags so that the frozen plasma would break
away from the plastic. The containers were then opened and the contents
pooled into a jacketed stainless steel thawing tank. In the thawing tank
the plasma was gently warmed, while being mixed, to melt the remaining
ice. The thawed plasma was pumped directly to a centrifuge or into a
stainless steel holding tank where it was maintained at 2.5C + 3.5C.
The plasma was centrifuged to remove the cryoprecipitate. The
cryoprecipitate, so prepared, may be stored at or below -25C, or
WO 95125748 - 2 1 6 2 9 9 7 PCT/US95/03451
-16-
immediately be processed to anlihe" lophilic factor. The cryo-poor plasl "a
was collected in a jacketed stainless steel reactioi1 tank.
After the cryoprec;pil~e was prepared, it was dissol~d in distilled water
at 20C to 35C. This part of the prolocol is illustrated schematically in
FIGURE 1. Sumcient calcium chloride was added to obtain a minimum
calcium conce, Itldliol ~ of about 40 ~M and the pH adjusted to 6.8 + 0.3.
This solution was cooled to 10C + 2C while mixing. The prec~ le
which forms was removed by centrifugation (56009-72009). The
prt:c;~.ilaLe may be stored at or below -60C or processed directly to TFC.
The precipitate was then suspended in Process Solution I at a ratio of four
liters of Process Solution I per kg of prec;pil~le. Process Solution I
comprises: (a) 0.5M glycine, 0.5M sodium chloride, and 0.1M sodium
citrate; pH ~djusted to 7.2 + 0.1 with NaOH, (b) protease inhibitor: 0.75-
1.75 ~M PPACK (D-phe-L-pro-L-arg-chloromethyl ketone) or equivalent,
and (c) 0.6 + 0.1 U/ml heparin. The temperature was adjusted to
24-32C and the suspension stirred for approximately one hour.
After the precipitate was suspended in Process Solution 1, the suspension
was l,~ns~er,ed into a tank containing 200 1 of process Solution ll a
10-15C and stirred for at least 30 minutes. Process Solution ll
co~,prises: 7 + 1 mM sodium phosphate monobasic monohydrate, 18
+ 2 mM sodium phosphate dibasic heptahydrate, calcium phosphate
Il ibasic 0.25% (w/v). The suspension was then allowed to settle
undisturbed for at least 30 minutes. The suspension may be centrifuged
(56009-72009) at this step to remove some precipitate. The suspension
was then clarified by filtration first through a 0.45~ filter, then through a
filter of at least 0.2~1 in pore size.
The filtrate was warmed to 23-27C and applied to a Lysine Sepharose 4B
column or equivalent. The gel was packed in a chromatography column
and equilibrated with 5 column volumes of Process Solution lll (25 mM
phosphate buffer: 7 + 1 mM sodium phosphate monobasic monohydrate,
18 + 2 mM sodium phosphate dibasic heptahydrate).
WO 95125748 . - : ` 2 1 6 2 9 9 7 PCT/US95/03451
The unbound ",alerial was collected in a tank. The column was washed
with at least two gel volumes of Process Solution lll. The unbound
f~d~ions were pooled. Next, the temperature of the bulk containing the
pooled unbound ~,actions was adjusted to 14 + 4C. Polyethylene glycol
3350 was added to a final concer,L,dlion of 4% (w/w). The suspension
was mixed for at least 30 minutes and the pr~zc;f il~le removed by
centrifugation (87009) at 10-18C. The resulting PEG ~r~c;r.ilale was
dissolved in Process Solution IV (39 mM tris~ hos~l ~dle with pH adjusted
to 8.6 + 0.1 with phosphoric acid) approximately 15 I/kg precipitate. The
protein conce"l,~lion of the suspension was adjusted to 0.6 + 0.2 9%
(w/v), then the suspension was clarified by filtration. A mixture of Triton
X-100, tri (N-butyl) phosphate (TNBP) and Polysorbate-80 was added to
the solution to a final concentration of 1.0% (v/v), 0.3% (v/v), and 0.3%
(v/v), respectively. The protein-detergent solution was mixed for 1 hour.
The bulk solution was then applied to a c~"on,alography column
conlaining DE 52 ion exchange cellulose resin or equivalent. The resin
was packed in a chromatography column and regenerated with 3 column
volumes of 1.0M NaCI, then 3 column volumes of 0.5M HCI, 3 column
volumes of 0.9% saline, and then 3 column volumes of 0.5M NaOH, and
equilibrated with 3 column volumes of 0.5M tris-phosphate buffer (pH
adjusted to 8.6), and 3 column volumes of Process Solution IV.
The ion exchange column was then washed with a minimum of 20 column
volumes of Process Solution V (0.02M histidine, 0.01 M NaCI, pH adjusted
to 7.0 + 0.1 with 6N HCI). The fibrinogen was eluted with Process
Solution Vl (0.02M histidine, 0.3M NaCI, pH adjusted to 7.0 + 0.1 with 6N
HCI).
The fibrinogen was then supplemented with human serum albumin (5%
or 25% human albumin released for therapeutic use) to a concentration
of about 80 mg of albumin or less per gram of protein. Polysorbate-80
was added to a final concentration of 15 mg per gram of protein.
W095/25748 2 1 6 2 9 9 7 PCT/US95tO3451
-18-
Finally, the fibrinogen was co"cet,lrdled to about 25% of the original
volume by ult~ ldtion using a 30,000 MW cut-off ,,e,,~Lrcl, ,e, then diluted
to its preconcenl,~lion volume with Process Solution Vll (0.02M histidine,
pH ~d3usterl to 7.0 + 0.1). The bulk was again concer,l,dled by
ultl~fill,alion to achieve a final protein conce"l,dlio" of 4 + 1 9% (w/v).
The bulk was sterile filtered (0.2~), aseptically filled into sterile final
containers, Iyophilized under aseptic cor,clilions, and closed with sterile
closures.
EXAMPLE 2
TFC in vftro STUDIES
A. Clotting Evaluation
Studies were done to determine the TFC composition which will produce
fast clotting in the presence of thrombin (i.e., on formation of a fibrin
sealant (FS)). Fast cl~lil ,y was arbil,drily defined as 1-2 seconds.
Shorter time periods generally result in clotting within the delivery device
and longer times lead to a loose mixture of the components that flows
with ill-defined direction.
In performing these experiments, a clean pyrex glass plate was positioned
at ~30 from the l,ori~onlal axis. A 2" line was drawn on the underside to
define the area of FS application.
Human fibrinogen was tested at 50-130 mg/ml total protein with bovine
ll"on,bin (Armour Pharmaceutical Co.) at 100-1000 NIH U/ml without
Ca+ +. For the purpose of this study the concentration of fibrinogen in the
TFC composition was considered to equal the value of total proteins,
although it is likely that a residual amount of fibronectin was also present.
The effect of adding [Ca++] to a fibrin sealant (FS, TFC, lhrur,,bin and
calcium salt) was studied at 10, 20, 40, and 60 mM.
The FS was delivered using an experimental dual syringe device (Fenwal)
following the 2" line described above, starting at the upper end and
3û moving downward.
W O 95/25748 : . ` ' ;- 2 1 6 2 9 9 7 p~lr/lJSg5l0345l
-1 9-
Table 1 summarizes the data obtained by testing fibrinogen at 50-130
mg/ml with II,ru,nL,in at 100-1000 NIH U/ml in the absence of calcium.
- Each data point is the average of four determinations.
TA B L E 1
AV E R A G E C L~JI IIN G TIM E S IN S t~Ol~D S ~ S D)
Tl,,u,,,b n in NIH U / m l
Total PrDtein ( m g/ ml) 100 250 500 1000
1 0
5.3+ 1.3 2.5+ 0.4 2.3+ 0.41.2(N = 1)
3. a 0.4 2.4_ 0.4 1.9~ 0.41.4(N = 1)
110 5.3+ 1.6 2.2+ 0.5 1.6~ 0.1 *
130 3.6_ 0.6 1.8+ 0.2 0.8(N = 1 ) *
* Not Determined - clotting occurred too fast for time measurement.
~N~ refers to the mean of the 4 m easu,e",erlt~ made)
As Table 1 shows clulling times at the lower thrombin concel ,l,dlion (e.g.
100 NIH U/ml) and low protein content (e.g. 50 mg/ml) were long. The
mixture was also observed to be runny. Higher conce"lr~lions of
ll,ru,,,bin (e.g. 1000 NIH U/ml) generally clotted within the delivery device
and therefore were considered unsuitable. Addition of CaCI2 improved the
appearance of the clot and generally shortened the clolli"g time.
Table 2 shows the effect of [CA++] in the range of 0-60 mM. CaCI2
solution was used to reco~slilute the thrombin so the final [Ca++] in the
1:1 mixture of FS is half that reported in the Table.
WO 95/25748 . - 2 1 6 2 9 9 7 PCT/US95/03451
-20-
TABLE 2
EFFECT OF lCa++] ON TIME TO CLOT
Thrombin in NIH U/ml
Total Prot~i., mg/ml 100 250 500
1. [Protein]=50 mg/ml
[calcium ion]
0 mM 4.3+0.7 2.6+0.2 2.0+0.6
10 mM 2.4+0.3 1.8+0.2 *
20 mM 2.3+0.2 1.2+0.5 *
40 mM 2.6+0.2 * *
60 mM 3.0_0.5 * *
2. [Protein]=90 mg/ml
[calcium ion]
0 mM 4.1 +1.1 2.2+0.2 1.8_0.3
10 mM 2.9+0.3 2 8+0 3 *
20 mM 2.0+0.4 2 6+01 *
40 mM 2.5+0.2 1.9_0.1 *
60 mM 3.2+0.1 * *
3. [Protein] = 110 mg/ml
[calcium ion]
0 mM 4.8_1.8 2.4+0.5 1.7+0.3
10 mM 2.9_0.4 2.4+1.2 *
20 mM 2.6+0.2 * *
40 mM 2.6+0.7 * *
4. [Protein]=130 mg/ml
[calcium ion]
0 mM 3.1 +0.8 1.5+0.1 *
* Not Determined - clotting occurred too fast for time measurement
[Protein] refers to the conce"L,~lion of total protein in mg/ml TFC.
Based on the above results it was concluded that a protein range of
90-130 mg/ml and a Ll"~o",bin concentration of 250-500 NIH U/ml are
appropriate for further studies. It was aiso apparent that additional
WO 95125748 : ; 2 1 6 2 9 9 7 PCT/US95103451
calcium ion was needed to enhance the clot. Consequently, Ca++
co"ce"l,dlion was incl~Jded as a variable in later c\,~lu~tions.
B. Rate of Cross-Linking
These studies focused on deler,~ ing the role of Ca ions and the effect
of [Ca+ +] on the extent of fibrin polymeri~aLion as well as the rate of cross
linking with time due to fibrin polymerization.
The cross-linking reaction of fibrin was tested in a system under reduced
conditions which utilized SDS/PAGE. The resolving gels at 7.5% and the
stacking gels at 3.75% were cast as described by Schwartz, et al.,
(Journal of Clinical Investigation, 50: 1506, 1971).
Thrombin (Armour Pharmaceutical) was reconstituted with or without
CaCI2 solution at the desired molarity, i.e., 0, 2, 6, 12, 20, 40, or 60 mM.
Fibrinogen was reconstituted with water, quickly mixed with the thrombin
in a 12 x 75 mm test tube and sampled at the appropriate time periods for
the studies. The clots were rinsed with 0.15M NaCI then dissolved in
three times the clot volume of 9M urea co,llaini.1g 3% SDS and 3%
13-mercaptoethanol by boiling in a water bath at 95 ~ 5C. The dissolved
clot solutions were then stored at 5C until the gel electrophoresis was
performed.
The effect of CaCI2 conce,1l,dlio,- was tested with thrombin at 500 NIH
U/ml and fibrinogen at 90 mg/ml and 130 mg/ml after 10 minutes clotting
time. The effect of Ca++ conceut,dlion on the rate of disalJpearance of
the y-band to form the y-y dimer is shown in FIGURES 2 and 3.
As illustrated in FIGURES 2 and 3, the presence of Ca+ + is necessary for
complete fibrin polymerization. As calcium ion in the range of 20-60 mM
gives comparable results, a midpoint concentration of 40 mM was chosen
to ensure optimal polymerization. Also, there was no si5y~ icanl difference
in fibrin polymerization between the two fibrinogen concentrations (90
mg/ml and 130 mg/ml) when measured at the time point studied (10
minutes~.
WO 95/25748 ` 2 1 6 2 9 9 7 PCT/US95/0345l
-22-
Four co",l)inaLions of Ll"o~"bin at 250 NIH U/ml or 500 NIH U/ml in 40
mM CaCI2 and ~ibri"oye" at 90 mg/ml or 130 mg/ml were tested to study
the effect of varying the concenL,aLions of the co",ponenls. Samples were
taken for gel electrophoresis after 10 minutes of clotting time. Gel
electrophGr~sis of the four combi~aLiol~s of Ll,r~mL)in (in 40 mM CaCI2)
and fibrinogen did not reflect any significant dif~r~"ces at the time point
studied (10 minutes).
The time study was conducted and sampled over 24 hours. FIGURE 4
shows the fibrin polymerization reaction as it progresses through a 24
hour period. Formation of the y-y dimer occurs very rapidly in the
presence of Ca++ (within one minute) as is shown in FIGURE 4. The
polymer is not cleLecLal,le by this system until 10 minutes incubation time.
As the a polymer increases with increasing incubation time (up to 24
hours) the a monomer band shows a cor,~sponding decrease in inLel IsiLy.
To summarize the time study demonstrates that initial polyme, i~aLion (y ~y
dimer) occurs almost insLanLa,1eously as the reactants are mixed with the
a polymers forming more slowly. From this study it can be concluded
that the presence of Ca ion is necessary for polymeri~aLio,1 and that the
results are similar to those previously I~JOl Led in the literature (T. Seelich
J.Head&NeckPathol.,3:65-69 1982;M.Schwartz etal. J.ClinicalInu,
50:1506-1513 1971).
C. Tensile St~
The tensile strength of the fibrin sealant was cv~luated by applying strain
to the clot until rupture of the bulk material was observed and measuring
the force needed in a tensile stress-strain system. In addition the change
in rupture stress as a function of varying the components in the
polymerization mixture used to produce the sealant was studied.
WO 95/25748 . : :` 2 ~ 6 2 9 9 7 PCT/US95/03451
-23-
To study the tensile sl,~nylh of the FS, a mold was designed based on
that descriL,ed by Nowotny, et al., (Biomaterials, 2:55, 1981) with some
~odi~icalions. The newly designed mold was ~aL,ricated of transparent
plastic to facilitate visual inspection of clot rO m~aLio~ ,. Clotting was allowed
to proceed in disposable clot holders for ease of cleaning.
The clot holders were obtained by cutting plastic disposable l,~"srer
pipettes (SAMCO, San Fernando Mfg. Co.). Two small pieces of
moistened sponge were used to anchor the clotting mixture at both ends.
Dispos~hle clot holders with the sponges in place were inserted through
the end holders and into the mold, (end holders were included in the
mold). A te"sior"eter instrument (T10, Monsanto) was used to measure
and record the peak rupture stress of the clots. A~apte,~ for the T10
grippers were fabricated to hold the end holders.
The clots were formed by injecting equal volumes of fibrinogen and
thrombin (with or without CaCI2) using a dual syringe acJn,inisl,dlion
device (Fenwal) and a 3 inch 22 gauge needle. All bubbles were removed
prior to placing the syringes in their holder. The needle was inserted
through one sponge "top", through the mold and into the other sponge
"bottom". Parafilm (American Can Co.) placed under the entire mold
prevented leakage of excess mixture. As the clotting mixture filled the
mold, the needle was withdrawn.
Approximately 2 to 5 minutes before testing, the clot was removed from
the mold and placed in the T10 grippers. At testing time, the clot was
stretched at a rate of 100 mm/min. The gauge length was set (somewhat
arbitldrily) at 6.0 cm and the cross sectional area of the clot was 0.049
cm2. The T10 reported the stress values in Kgf/cm2.
W095/25748 2 1 6 2 9 9 7 PCT/USg5/03451
-24-
TABLE 3
EFFECT OF CaC~ ON TENSILE STRENGTH OF FIBRIN SEALANT
Tensile St~e~ tl~ KgF/cm2
Fgn.* Conc. CaC~ ~ 250 NIH U/ml ~? 500 NIH U/ml
mg~ml mMol Tl--u~ . - (N) Tl.. u,.. ~ln (N)
go 0 0.93+ 0.136 (9) 1.23_ 0.165 (8)
110 0 1.07+ 0.150 (10) 1.29~0.237 (9)
130 0 0.94+0.157 (14) 1.36+0.177 (16)
1.6~:0.380 (12) 1.92~0.350 (8)
110 10 2.6t~0.540 (8) 2.8~0.610 (8)
130 10 2.3~0.760 (11) 3.24_1.170 (8)
1.65+ 0.406 (8) 2.1 ~ 0.540 (8)
110 20 2.26~0.530 (8) 4.11+1.080 (8)
130 20 2.23+ 0.489 (8) 3.54i 1.030 (8)
go 40 1.74+0.410 (8) 2.36+0.390 (11)
110 40 2.36+ 0.660 (8) 3.79~ 0.626 (8)
130 40 2.63+0.670 (12) 4.0 +0.940 (9)
2.1 Q~ 0.38 (8) 2.29~ 0.450 (8)
110 60 3.0Q~0.690 (8) 4.13+0.896 (8)
130 60 3.69~0.787 (10) 3.57+1.160 (9)
Fibrinogen Lot #2830R129
Measurements of tensile strength (peak stress) were taken 10 minutes
after injection of the clotting mixture. The results in Table 3 show the
effect of varying the CaCI2 concentration when II,r~i"bin is 250 NIH U/ml
or 500 NIH U/ml. Three concenl~dlions of fibrinogen (90 110 and 130
mg total protein/ml) were tested. Each measurement of peak stress was
the average of N determinations. A minimum of 8 readings were taken
per point.
As shown in Table 3 in the absence of Ca++ the clots had the lowest
peak stress values at both thrombin concentrations and at all fibrinogen
levels. Addition of calcium ions at 10-60 mM increased the tensile
sl,~nylh for all thrombin/fibrinogen concentrations. Generally higher
WO 95/25748 2 1 6 2 9 9 7 PCT/US95/03451
.
-25-
values were observed at the higher thrombin concer,l,~lion, i.e., 500 NIH
U/ml of thrombin, which also gave somewhat similar values for [Ca+ +] in
the range of 20-60 mM of calcium ions. The lowest standard deviation
(SD) values were observed at 40 mM CaCI2.
A second lot of human fibrinogen was tested to confi"" these findings
and the data were compared at 40 and 60 mM CaCI2 and 500 NIH U/ml
ll"oi,lbin. The results of testing a second lot of fibrinogen showed
generally simiiar values of peak stress particularly at the higher fibrinogen
concentrations of 110 and 130 mg/ml and also showed higher values at
90 mg/ml.
Time studies of the clot tensile strength were performed over a 24 hour
time period using fibrinogen at 90 mg/ml and 130 mg/ml with thrombin
concentration at 500 NIH U/ml and CaCI2 at 40 mM. Over a 24 hour
period, the tensile strength showed no signi~icanl decrease in value. A
gradual increase in peak stress was expected to occur as cross linking
continued with time.
D. Clot Lysis Studies
The length of time that a fibrin clot will remain solid when incubated at
37C under sterile, moist conditions, with and without a plasminogen
activator, was determined. Also tested was the effect on the clot longevity
of adding protease inhibitor (Apr~tini,)) to the reaction mixture.
Sterile human fibrinogen solution was prepared using one of the following
diluents:
a. sterile water (i.e., zero KlU/ml Aprotinin)
b. Aprotinin solution at 1000 KlU/ml
c. Aprotinin solution at 3000 KlU/ml
to yield one of three concentrations; 90, 110, or 130 mg/ml of total
protein.
WO 9S125748 PCT/US95/03451
21 62997
Thrombin was prepared by reconstituting with a 40 mM CaCI2 solution to
produce either a 250 or 500 NIH U/ml. Thus, six combinations of
ll,,u,,,bin and fibrinogen were tested. Urokinase (Abbott) was prepared
at 5 U/ml in normal saline.
Fibrin clots were tOI " ,ed by mixing equal volumes of fibrinogen (in H20 or
Aprotinin) and thrombin (in CaCI2 solution) in cylindrical silicone tubing (5
mm inner diameter). The mixture was delivered using a dual syringe
administration device (Fenwal). All the delivery devices and the silicone
tubing were slerili~ed by autoclaving.
A 10 cm length of silicone tubing was sealed at one end using parafilm.
Holding the tubing about 10 from vertical, the Fenwal device was used
to inject the fibrinogen and Ihror"bin rapidly into the tubing with the (22 9)
needle tip barely penetrating the parafilm. After the clot had solidified for
20 minutes, the 10 cm silicone tubing was cut into 3 cm lengths to yield
a clot volume of 590 ~I. Each 3 cm segment was cut in half and the two
halves were placed in one well of a sterile 24-well plate (Corning). Any
segment that was found to contain air bubbles was discarded. The clot
was extruded from the tubing by gently squeezing the tube at one end.
It was rinsed with 1 ml of sterile saline then 1 ml of either urokinase or
saline was added to the well and the plate was placed in a sterile, moist,
37C incubator. Every 24 hours, the super~ala,1ls from each well were
removed for testing using the Fibrin(ogen) Degradation Products
agglutination kit (Baxter Dade).
The urokinase and saline solutions were replaced daily with fresh reagents
before returning the plate to the 37C incubator. All preparations and
sampling of supernatants were performed under sterile conditions. The
clots were visually inspected and their appearance noted. After 14 days
the experiment was terminated. Total number of conditions tested was 36
and all conditions were performed in duplicate.
W O 95/25748 2 1 6 2 9 q 7 PCT~US95/03451
-27-
TABLE 4
CLQT IYSIS TIMES
~+ UROKINASE. NO APROTININI
CONDITION TIME CLOT LOST CYLINDRICAL SHAPE
130 mg/ml riLri"ogen
+ 500 U/ml Illlu~ I Day 10
130 mg/ml ri~ril,ogen
+ 250 U/ml Illlu~ , Day 8
110 mg/ml fibrinogen
+ 500 U/ml Illlulll~.l Day 7
110 mg/ml ~iL.,i"ogen
+ 250 U/ml Ill~nl ~, Day 11
90 mg/ml tiL.ri"ogen
+ 500 U/ml thrombin Day 8
1~ 90 mg/ml ~il,ri"ogel~
+ 250 U/ml Ll llul I l~, Day 8
Table 4 sl""",a,i~es the clot Iysis time (defined as the time clots lost their
cylindrical shape) in the presence of urokinase when no Aprotinin was
included. The observed range was 7-11 days with a mean of 8.66 +
1.5d. Measurements of Fibrin(ogen) Degradation Products (FDP) showed
a peak in activity that generally con~sponded to or soon followed the time
when the clots lost their well-defined shape.
When urokinase was deleted and the clots were incuh~ted in normal
saline only, in presence or absence of A,ur~lin;n, the clots maintained their
integrity during the entire observation period (i.e., 14 days). FDP
meas~"e,ne"ls confirmed the absence of significant clot Iysis.
This study shows that when the clot is not influenced by any plasminogen
activators in situ, it should be expected to last for at least 14 days. When
urokinase is present, the clots last a minimum of 7 days. These time
periods may be sufficient for the healing mechanism to play its natural
role. Thus, based on these results, it can be concluded that the trace
WO 95125748 PCTtUS95/03451
2~ 62997
-28-
levels of plasminogen in the fibrinogen ~ Jardlio"s do not adversely
affect the clot longevity and that the addition of a protease inhibitor, such
as Aprotinin, is not necessary.
EXAMPLE 3
TFC in vivo TESTING
Studies were done to cva'~ e the optimal concenl,~liG" of TFC using an
in vivo model. Swiss Webster mice (20-25 9) were ar,~nged in 10 groups
of five for testing. In the protocol which was utilized, each animal was
anesll,eLi~ed, weighed, and a small piece of skin was removed from the
back of the animal. The skin specimen was dipped in a saline solution
and attached to a Gottlob device. Equal volumes of TFC and thrombin
at various concel IlldliOl ls (Table 2) were then added simultaneously to the
wound, the skin replaced onto the animal, and held in place for
approximately two minutes.
The anesthetized animal was placed face down on a ,cldl~orm which was
then positioned on a tensiometer (Monsanto Company) and the Gottlob
device attached to the grippers. The tensiometer parameters were set to:
(1) area: 1.76 cm2; (2) speed: 10.0 mm/min.; (3) gauge: 1.0 cm; (4)
stress range: 500.0%. The force required to separate the piston (with the
skin specimen) from the back of the animal was recorded in g/cm2. The
data from these experiments were ~Idlislically evaluated using
RS1/Discover software (BBN Software Corp., Cal),bridge, MA). Analysis
of the results of this study indicated that TFC at 120-130 mg/mL and
thrombin at 250 U/mL gave maximal adhesion responses.
The ability of the clot to adhere to tissue in vivo is important in maintaining
hemostasis. In this experiment, a maximum adhesion response occurred
within the range of reagents tested, collri"~ling the in vitro findings of
Example 2.
wo gsl25748 2 1 6 2 9 9 7 PCTIUS95/03451
._ ' !
-29-
EXAMPLE 4
CHARACTERISTICS OF TFC
A TFC composition was prepared yener~lly according to the process
steps described in Example 1. The chara.:teri~lics of TFC identified below
were analyzed with the following results:
TABLE 5
TEST RESULTS
SOLUBILITY 1050 seconds
pH 7.2
STERILITY STERILE
PYROGEN NON-PYROGENIC
HEPATITIS B tHBsAg) NON-REACTIVE
TOTAL PROTEIN 10.4 g/dL
CLOTTABLE PROTEIN 86 %
F-XIII 32.4 U/ml
PLASMINOGEN <0.1 mg/dL
PEG 0.0 g/dL
TRITON X-100 0.4 PPM
TNBP 1.1 PPM
TWEEN-80 0.1 %
ALBUMIN 0.64 g/dL
HISTIDINE 55.9 mM
SODIUM 213 mg/L
W095125748 ~` 2 1 PCT/US95/03451
-30-
E)(AMPLE 5
REDUCTION OF VIRAL ACTIVITY IN
TFC PROCESS INTERMEDIATES TO UNDETECTABLE LEVELS
TFC process intermedidles ~e, Example 1) were assayed for activity of
5 lipid enveloped viruses. The lipid enveloped viruses assayed were:
Pseudorabies ("PRV", as a model for herpes viruses), HIV Types 1 and 2,
Sindbis ("SIN", as a model for he,ualiLis C virus), and Vesicular Stomatis
Virus ("VSV", as a model for RNA viruses). The process intermediates
were obtained after the resuspension of PEG pr~c;,~,~ale in tris-phosphate
buffer, pH 8.6 + 0.3 (without buffer, the viricidal activity of the viral activity
reducing agent would be reduced and an agent of greater concentration
would be used).
For purposes of the assay, the samples were separately spiked with four
lipid enveloped viruses in the presence of the following viral activity
reducing agent: a mixture of TNBP, octoxynol 9 and polysorbate 80, in
respective ratios of 0.3%:1%:0.3% (v/v).
PRV, VSV and SIN were assayed by incubation with a~u,ur~priale cell lines
(porcine kidney 13 ("PK-13"), buffalo green monkey kidney ("BGMK") and
Vero, respectively) to deler",i,1e plaque forming units ("pfu") before and
after treatment with the viral activity reducing agent. HIV-1 was assayed
by incubation with susceptible T cells (H9) to determine 50% of the tissue
culture infectious dose end point (TCID50) of the virus before and after
treatment with the viral activity reducing agent. The results of these
assays are tabulated below, which demonstrate that the use of the viral
activity reducing agent during the manufacture of TFC is very effective in
reducing the activity of lipid envelope viruses therein.
WO 95/25748 2 1 6 2 9 ~ 7 PCT/US95/03451
-31 -
TABLE 6
VIRUS RECOVERY
HIV-1 ID5~sv Lopg~vpfu/mL
Virus + TFC Solution 12.5 9.0 8.9 8.9
Virus + TFC Solution Negative <1.9 ~1.9 <1.9
After Incub2tion
LOg,n Reduction Factor 12.5 7.1 7.1 6.9
No plaques were detected. The theoretical detection point of the assay
was used to calculate the virus recovery. Reagent cytotoxicity due to
solvent/deterge"l mixture (i.e. the viral activity reducing agent) has been
taken into account for these calculations.
EXAMPLE 6
PYROGENICITY OF TFC
The pyrogenicity of the TFC composition of Exa~r"~le 4 was tested using
the well known rabbit pyrogen test. Three rabbits were injected with a
dose of 0.5 ml/kg and their body temperatures ~,o"itur~d over a course
of three hours following the injection. The results of this test are tabulated
below which strongly indicate that TFC is non-pyrogenic.
WO 95/25748 `:. 2 1 6 2 9 ~ 7 PCT/US95/03451
-32-
TABLE 7
Rabbit TEST TEMPERATURE (C)
Weight Dose Pre-injectionMaximum
# (kg) (ml) Temperature (C) 1 hr 2 hr 3 hr Rise (C)
3.23 1.7 39.2 39.1 39.1 39.1 0.0
2 2.70 1.4 39.1 39.0 39.0 39.1 0.0
103 3.11 1.6 39.6 39.5 39.5 39.5 0.0
Sum of Maximum Rise: 0.0
The preceding examples and description are provided to assist in
understanding the present invention and, as such, are intended to be
exemplary only, not limiting. Those of skill in the art will recognize that
other materials or methods may be used, depending on the
circumstances, and still remain within the spirit and scope of the present
invention.
