Note: Descriptions are shown in the official language in which they were submitted.
20'~9~'~~
"Im roved tissue glue prepared by using cryoprecipitate"
This invention relates to a tissue glue comprising two
components A and B, a process for preparing the tissue
glue, the use of a high amount of aprotinin and the use
of a snake venom proteolytic enzyme for preparing a tissue
glue.
Improvement of local hemostasis at the site of a surgical
wound by application of plasma proteins is a well-known
concept. Thus, fibrin patches for hemostasis in cerebral
surgery have been used. Blood plasma and thrombin were
used to produce a fibrin film over the surgical wound. In
the last 20 years there are a lot of publications de-
scribing applications of "fibrin glue" or !'fibrin ad-
hesive" or "fibrin sealant" for most surgical disciplines.
In the last 10 years commercial preparations of "glue"
are used widely in Europe. The "glue" is composed of two
components, whereas a mixture of these components produce
a clot. The first component is a fibrinogen concentrate.
This concentrate also contains fibronectin and factor
XIII which are important for clot stabilization and
strength. The second component is thrombin, an active
enzyme that converts fibrinogen, the last component of
the normal coagulation system into a fibrin clot. This
process bypasses most of the steps of normal coagulation
and mimicks its last phase. Some manufacturers add plas-
minogen which is an enzyme that will induce clot lysis
after some time whereas others add aprotinin which is an
inhibitor of proteases for preventing clot lysis.
Although, these products give satisfactory results in
patients although with mild bleeding disorders, but
i
2~79~'~7
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patients suffering severe bleeding disorders such as hemo-
phi3ia A or B, still have a very high risk of postopera-
tive bleeding. Sometimes a delayed bleeding complications
after an average of days from the surgery occur. Also
patients who are treated with anticoagulation factors
cannot be treated with the tissue glue of the prior art.
Another severe disadvantage of the commerical concentrates
are the high producing costs.
WO 86/01814 discloses a method of preparing a cryopreci-
pitated suspension containing fibrinogen and Factor VIII
. useful as a precursor in the preparation of a fibrin glue
which involves (a) freezing fresh frozen plasma from a
single donor such as a human or other animal which has
been screened for blood transmitted diseases at about
-80°C for at least abount six hours; (b) raising the
temperature of the frozen plasma, e.g. to between about
0°C and room temperature, so as to form a supernatant and
a cryoprecipitated suspension containing fibrinogen and
Facot VIII: and (c) recovering the cryoprecipitated sus-
pension. There is also disclosed a method of preparing a
fribrin glue useful in surgical procedures which com-
prises: (a) preparing .a cryoprecipitated suspension as
described abovet (b) applying a defined volume of the
suspension to a desired site; and (c) applying a compo-
sition containing a sufficient amount of thrombin to the
site so as to cause the fibrinogen in the suspension to
be converted to the fibrin glue which then solidifies.
EP-A-0 341 007 discloses a surgical adhesive comprising,
in an aqueous composition, patient autogenous plasma,
collagen, thrombin, and optionally, an antifibrinolytic
agent. The present adhesive is formed from the patient's
plasma without the use of any added reagents for concen-
tration or isolation of the fibrinogen. Conveniently, the
adhesive is formulated as a two-part composition which is
mixed together just prior to use.
~0~~0~7.
- 3 -
EP-A-0 253 198 discloses a one-component-tissue glue
having an aqueous solution of fibrinogen, Factor VIII,
a thrombin inhibitor, prothrombin factors, calcium
ions and optionally a plasmin inhibitor. The tissue
glue can be reconstituted from a lyophylized sample by
adding water. The tissue glue may contain all active
substances in a pateurizated form in order to avoid
hepatitis and HTLV III transference.
An object of the present invention is to provide a
tissue glue which is also suitable for patients with
severe blood coagulation disorders like hemophilia A
or B. A further object of the present invention is to
provide a tissue glue which can also be used for
patients which have already developed antibodies
against bovine thrombin which is the active factor of
the component B. Still another object of the present
invention is to provide a tissue glue for patients who
are treated with anticoagulation factors like heparin.
Because of the risk of transferring viral diseases
with the components of the tissue glue it must be
ensured that fractions of the tissue glue are virus
inactivated.
~~~~t~~~
- 3a -
The tissue glue according to the invention comprises a
component A which comprises a concentrated
cryoprecipitate of whole blood and an amount of a
protease inhibitor corresponding to 3,000 to 5,000
KIU/ml units of aprotinin, preferably aprotinin, and a
component B, comprising a proteolytic enzyme being
capable of cleaving specifically fibrinogen present in
component A and causing the formation of a fibrin
polymer.
Commercially available cryoprecipitate can be used for
preparation of the tissue glue of the invention.
However, it can be advantageous to concentrate the
cryoprecipitate between a factor 2 and 5, preferably
f actor 3 .
The addition of a protease inhibitor in sufficient
con-
207907
"~" - 4 -
centration corresponding to an amount of 3,000 to 5,000
KIU/ml units of aprotinin.to the cryoprecipitate makes the
tissue glue of the invention suitable for use in patients
with severe bleeding disorders. The preferred protease
inhibitor is aprotinin, which is commerically available
under the trademark TrasylolR or AntagosanR.
The cryoprecipitate can be obtained from the patient him-
self by donating an autologous blood unit prior to the
operation. This approach prevents the risk of transmission
of viral infections by blood derivatives. However, in
order to. have a proper commercial product, the cryopreci-
pitate has to become virus-inactivated. A procedure for
virus-inactivation is described in PCT/EP 91/00503. The
basic principle is treatment of the cryoprecipitate with
special detergents and removing the detergent lateron
from the cryoprecipitate.
The second component, component B, of the tissue glue of
the present invention is prepared by a solution of a pro-
teolytic enzyme being capable of cleaving specifically
fibrinogen. Usually thrombin has been used which was iso-
lated from plasma of human beings or mamals such as
bovine. This thrombin can be delivered in a lyophilized
form. The reconstitution of thrombin occurs with a 40
mmol solution of calcium chloride. The preferred concen-
tration of thrombin is 50 to 200 u/ml.
For preparing a fast tissue glue the thrombin solution of
roughly 100 u/ml of calcium chloride will be prepared.
For preparing a slow glue for example by filing of
cavities, i. e. tooth extraction or sealing the cavity of
transphenoided hypophisectomy the thrombin will be further
dissolved to a concentration of 25 u/ml with the appro-
priate calcium chloride solution.
Another embodiment of the improved tissue glue of the
1
''~..' _ 5 _
invention comprises as component B a proteolytic enzyme
which is isolated from snake venom. This embodiment is
advantageous because also patients having developed anti-
bodies against thrombin can be treated. Moreover, patients
which are pretreated with heparin can be treated with the
tissue glue according to the invention, because heparin
does not influence the reaction of the snake venom enzyme.
In a very preferred embodiment of the present invention
there is used the snake venom enzyme batroxobin which can
be isolated from the South American pit viper Bothrpos
mou eni. Preferably component B contains 0.5 to 10 u/ml
of the respective proteolytic enzyme of snake venom.
Chemically batroxobin is a single chain glycopeptide with
a molecular weight of approximately 36,000. DefibraseR
causes cleavage of a 16 Arg/17 Gly bound in fibrinogen
which causes the release of fibrinopeptide A and the
formation of monomeric fibrin I.
When aprotinin is used in the amounts of the invention,
also the tissue glues comprising purified fibrinogen,
fibronectin and factor XIII can be used as component A.
The risk of after-bleeding is then dramatically reduced.
When proteolytic proteases from snake venom are used for
the preparation of component B, also "conventional" com-
ponents A having fibrinogen, fibronectin and factor XIII
can be used. The use of high amounts of aprotinin ac-
cording to the invention is preferred. A very preferred
'embodiment is the combination of the component A of the
invention derived from cryoprecipitate with or without
high amounts of aprotinin and the component B of the
invention having the proteolytic enzyme isolated from
snake venom.
The process for preparing the fibrin glue of the invention
comprises the steps of manufacturing component A com-
20'~~0'~'~
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prising the steps of preparing a cryosolution from cryo-
precipitate, '
- a virus inactivation,
- the removal of virucidal agent,
- the addition of the protease inhibitor and
- preparing a appropiate protease-solution.
Preferably a cryopaste is prethawed over night at 4 to l0
°C. The cryopaste is dissolved in a buffer containing
sodiumchlorid trisodiumcitrate and glycin and having a pH
of 7.0 to 7.2 and than heated to 30 to 35°C. The cryopaste
should dissolve readily otherwise it is not suitable~for
the preparation. The dissolution can be speeded up by
cutting the cryopaste in small pieces after thawing. After
cooling the solution to almost room temperature and ad-
justing the pH to a value of 7.0 to 7.2 aluminiumhydroxid
is added under stirring. The precipitate is centrifuged
and discarded. Optionally a filtration step is carried
out. Than calcium chloride is added up to the desired
final concentration of calcium chloride.
For the virus inactivation the solution is heated up to
30°C. Than the detergents are added. Other stirring for
some time the solution is transferred into a virus free
container and left at slightly elevated temperatures for
several hours without stirring.
The virucidal agents are removed by adding an amount of
ricine oil and gently stirring for several minutes. When
the oil-/water-phases have been seperated the solution is
cooled to room temperature. The aqueous layer is withdrawn
in a virussafe container and~the oillayer is discarded.
The aqueous layer is clarified by filtration. The pH must
be checked to be 7.0 to 7.2. Then the protein solution is
pumped through a reversed phase column at ambient tem-
perature. After having measured the protein content (in
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-~_
the range of 10 to 60 mg/ml the eluate is concentrated by
diafiltration to a protein content of 60 to 100 mg/ml and-
dialysed against a buffer which is identical to the buffer
mentioned above but having additionally a relatively high
concentration of calcium chloride. Then the protease in-
hibitor is added. A sterile filtration is carried out and
the sample is filled and deep frozen in suitable con-
tainers.
Component B is preferably a freeze dried protease. Parti-
cularly preferred ,is lyophilized thrombin or lyophilized
fraction of the South American pip viper Bothrpos moujeni.
The proteolyic enzyme is known under the tradename Repti-
lase and is the enzyme batroxobin.
The proteolytic enzymes are dissolved in a calcium
chloride buffer.
The application of the two components A and B is performed
using a double syringe technique for example through a
plastic connector. Upon mixing of the two components a
clot will be formed. The application can occur via a
canula or may be sprayed to a three lumen catheter. Each
one of the two components is injected into a separate
i lumen and an air pressure source in the range of some
atmospheres is connected to the third lumen in order to
spray the mixture.
The tissue glue of the invention is advantageous because
it can be used with patients having severe blood coagu-
lation disorders and being still cheaper than the known
tissue glues. Patients with. severe hemophilia can sub-
sequently, for example undergo tooth extractions without
preventive infusions of factor VIII concentrates with a
success rate of over 80%. This means only about one fifth
of the patients need infusions due to post extraction
bleeding. Moreover, such patients who are pretreated with
.. 20'0077
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heparin can be treated with the tissue glue of the in-
vention. Another advantage is that people who raised anti-
bodies against thrombin the second component of the tissue
glue can be treated with a tissue glue according to the
invention wherein thrombin is substituted by a protease
from snake venom especially DefibraseR which is the serine
protease batroxobin isolated from the venum of the South
American pit viper Bothrpos moujeni.
The invention is further disclosed in the following
examples which are non limiting.
Human fibrinogen (grade L) was from Kabi (Stockholm),
bovine thrombin from Merz-Dade. Chromogenic substrate
N-a-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and analy-
tic grade reagents were from Sigma (St. Louis, MO).
Reagents and salts were diluted with 0.015 M Tris, 0.15 M
NaCl, with pH 7.4. Fibrinogen was dialyzed in Tris buffer
with concentration determined from Abs280 using a con-
version factor of E1°280 = 15.
Bovine thrombin was from commercial sources (Merz-Dade or
Parke Davis) with activity rating by the manufacturer.
ReptilaseR, a snake venom which only releases FPA, was
from Pentapharm (Basel). The proteolytic activity of
ReptilaseR was normalized to that of thrombin by comparing
their rates of proteolysis of a non-specific chromogenic
substrate BAPNA (0.25 mM) at 37°C, in Tris/saline, pH
8.0, monitored at 405 nm for 15 minutes.
On the basis of their esterolytic activity, the unit
activity of the reptilase was normalized to that of
thrombin.
Fibrin glue was essentially generated by a dual syringe
method with pure or cryoprecipitate fibrinogen substrate
in one syringe, and reptilase (20 U/ml) or thrombin with
CaCl2 (20 mM) in the other.
-
Clotting time (CT) was determined with a Research Model
300-R ACL Coagulation Analyzer (IL, Milan). Viscoelasti-
city (TEG) was determined on a 3-channel Heiliger Thrombo-
elastograph at 37°C. Breaking strength (BS) of glues (in
grams) was determined by mixing the glue components
between two pieces of coarse weaved, synthetic fiber (0.5
x 1 cm), allowing the formation of gel totally interweaved
between the two pieces of coarse mesh and after 2 hours
at 24°C the ensemble of mesh-glue-mesh pulled apart using
an Accuforce Cadet Tensionometer (AMATEK, Mansfield &
Greene, USA).
Sterile cryoprecipitate (cryo) was prepared from frozen
(-30°C) human plasma which_ was thawed at 4°C and the
supernatant plasma removed. Five such units were pooled
to determine protein and fibrinogen concentrations was
determined by the Buiret method before and after clotting
the cryoprecipitate (diluted 1 . 5) with 2 U/mL thrombin.
Factor XIII was determined by measuring [3H~-putrescine
incorporation into dimethylated casein after activation
of the samples with 4 U/mL bovine thrombin, 10 min, 22°C.
A notable feature of the CT-fibrinogen curve is that it
is biphasic for a fixed level of thrombin or reptilase
(i.e. 1 U/ml, figure lA) and reaches a minimum in the 1 -
8 mM fibrinogen range. This differs somewhat from the
maximal turbidity (after 10 min) which peaks in the range
20 to 40 mM fibrinogen. A converse experiment shows the
dependency of CT on either thrombin or reptilase levels.
This curve shows a near linear inverse dependency of
gelling rate at low enzyme levels (less than 2 U/mL),
which plateaus above at higher levels.
The development of viscoelasticity of pure fibrin is some-
what slower than its turbidity. Ca(II) is a major cofactor
in gel reinforcement through factor XIIIa-induced covalent
interlocking of protein chains. Such gel crosslinking is
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a major source of mechanical strength of the gel, which
plateaus after 20 min.
A note about. the ability of reptilase to induce factor
XIIIa activity seems appropriate.
Protein Levels of pooled cryoprecipitate:
Pooled cryo prepared from 5 units, gave the following
mean values:
Protein: 75 mg/mL
Fibrinogen: 36 mg/mL
Factor XIII: 4.10 U/mL
Coagulation rates:
The clotting time (CT) of cryo is linearly dependent on
thrombin or reptilase levels. However, above 3 U/ml, in-
creasing enzyme levels exert little effect on CT. For a
fixed level of enzyme, serial dilution of cryo, gives a
biphasic CT-curve equivalent to the fibrinogen-dependency
noted in the pure fibrin system.
Viscoelasticity (TEG) and Breaking Strength (BS) of Cryo
Glues.
The development of viscoelasticity of cryo glues was in-
vestigated with either thrombin or reptilase. This para-
meter takes much longer to develop than turbidity. How-
ever, cryo glues prepared with excess of CaCl2 and either
thrombin or reptilase achieve equivalent TEG values in
roughly the same time frame. It seems that after the ini-
tial onset of gelation, factor XIIIa-induced cross-linking
bolsters the gel fiber structure, so that the TEG values
for both glues converge within 1 hour. Similarly with the
final BS of both cryo glues formed with an excess of
CaCl2. Both cryo glues break at 50 to 60g. These experi-
- 11 -
ments indicate that the gel fibers within the glue become
reinforced by factor XIIIa-induced, covalent cross-
linking.
Preparation of a cryo-solution. Commerically cryopaste is
prethawed~ over night at 4 to 10 ° C. One kilo of the cryo
is dissolved in two liters of buffer A (120 mM/1 NaCl, 10
mM/1 trisodiumcitrate, 120 mM/1 glycin and pH 7.0 to 7.2)
and preheated to 30 to 35°C. The cryopaste should dissolve
readily otherwise it is not suitable for the preparation.
In order to speed up the dissolution, cut the cryopaste
in small pieces after thawing. Then the solution is
cooled to 20°C to 22°C and the pH is checked. Optionally
it must be adjusted to pH 7.0 to 7.2 by adding diluted
sodiumhydroxid or acidic acid. 100 ml aluminiumhydroxid
is added and stirred for another 30 minutes. The precipi-
tated is centrifuged and discarded. The supernatant is
filtrated using a 1 ~.m filter. 0.1 M/1 CaCl2 is added to
render a final concentration of Ca2+ of 1 mM/1. Again the
pH must be checked.
Virus inactivation.
The solution is heated up to 30°C. 1% w/v TNBP and 1% w/v
Triton X 100 is added. The mixture is gently stirred for
1/2 hour. The solution is than transferred into a virus-
free container and left at 30°C for 3 1/2 hours without
stirring.
Removal of Virucidal Agents.
150 ml Ricine oil is added to the mixture prepared as
described above and stirred gently for 30 minutes. While
waiting"for the oil/water separation (30 to 45 minutes)
the solution is cooled to 20°C. The aqueous layer is with-
drawn into a virussafe container whereas the oillayer is
discarded. The aqueous layer is clarified by filtration
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on 1 um/0.45 ~.m filter cascade. The protein solution is
than pumped through a reversed phase column (C-18-Column)
at a rate of 3 liter/h at ambient temperature. The through-
put is monitored by UV and collected until the absorbance
has returned to 50%. The fraction contains roughly 40
mg/ml as measured in a protein assay.
The eluate is concentrated by diafiltration to a protein
content of 70 to 80 mg/ml and dialyse against sufficient
amount of a buffer B (same ingredients as buffer A but
additionally 1 mM/1 calcium chloride). Then 4 mio. KIU
aprotinin per liter solution is added. Afterwards a
sterile filtration carried out using a 0.45 ~.m + 0.2 um
cascade. The solution is than filled and deep frozen in
plastic bags, optionally lyophilized.
Preparation of a thrombin solution
Lyophilized thrombin is dissolved in a solution of 40
mM/L calcium chloride. The amount of thrombin is 100 U/ml
in the glue. For a fast working glue, for example for
spraying of the glue to the area of the wound, a thrombin
solution of 100 U/ml in calcium chloride will be suffi-
cient. For a slow glue, for example filling of cavities
during a tooth extraction or sealing the cavity of trans-
phenoided hypophisectomy the thrombin will be further
dissolved to a final concentration of 25 U/ml by adding
great amounts of CaCl2.
The preparation of reptilase is similar to that of
thrombin. However, the amount of reptilase is roughly 2
U/ml.
Clinical case report
The patient from the age of 21, MY (a 21 year old male)
suffered from severe bleeding diathesis due to acquired
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inhibitor against thrombin. No background disease (i.e.
- tumor, or autoimmune disease) could explain this problem.
Laboratory test, confirmed by two outside laboratories
indicated that MY had high levels of anti-thrombin IgG
anitbody. In the last year he suffered repeated attacks
of renal colic due to a large stone in his~left kidney
pelvis. Elective lithotripsy by ultrasound~was planned.
Based on the technique that IgG binds to protein-A affini-
ty columns, the patient was placed on immunosuppressive
therapy combined with extra-corporal immuno-adsorption.
After 8 treatments, in which 60 L of the patient's plasma
was processed through passage on the protein-A column,
the inhibitor titer decreased by 98%. this was determined
by measuring the thrombin time (TT) of normal pooled
plasma, with pre- and post-affinity purified MJ plasma.
Nevertheless, the TT as well as PT and APTT values were
prolonged. At this time, the kidney stone moved to the
urethra, causing complete blockage of the kidney accompa-
nied by hydronephrosis. The patient received 10 more
immuno-adsorption treatments (roughly 80 liter plasma)
followed by intensive plasmapheresis (roughly 50 liter)
and high doese immunoglobulin infusion (2 g/kg). At this
point, the thrombin-inhibitor level dereased to 0.5 %.
PTT was decreased to 47" (vs. 85 - 90" pretreatment) and
the TT was 35" (vs. 90" pre-treatment and 27" normal
control). It was decided to remove the stone by surgery,
using biological adhesive (cryo glue) made up from cryo-
precipitate and high levels (200 U/mL) of thrombin. With
this mix, the cryo gelled immediately upon being sprayed.
However, in the patient gelling did not occur and local
hemostasis was achieved by suturing. At the end of surgery
the wound looked "dry". Nevertheless, six hours later,
the patient was bleeding from surgical drains. Immuno-
absorption of 10 liter plasma was carried out, but with
no effect on bleeding which actually increased.
The patient was re-operated to find the source of
2Q'~~a'~'~
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bleeding. Though no surgical bleeding was found. Diffuse
-bleeding was observed from the entire wound surface areas.
This time, a mix of cryo and reptilase (2 U/mL: Defibrase)
was sprayed onto the wound. The spray clotted immediately,
the wound surface appeared turbid and bleeding stopped.
The patient continued to receive daily immuno-adsorption
therapy for another 5 days, with no bleeding. This demon-
strates the advantage of using the snake proteasis as
component B of the tissue glue of the invention.
