Antidote for hirudin and synthetic thrombin inhibitors and method of use

The invention relates to an antidote for hirudin and synthetic thrombin inhibitors; the antidote contains a compound that splits prothrombin into meizothrombin, a prothrombin intermediate, a pharmacologically acceptable salt thereof or a mixture of these compounds, together with conventional vehicles and/or diluents. The present invention also relates to the application of a compound that splits prothrombin into meizothrombin, a prothrombin intermediate, or a pharmacologically acceptable salt thereof or a mixture of these compounds, together with conventional vehicles and/or diluents as an antidote for hirudin and synthetic thrombin inhibitors or for the preparation of an antidote for hirudin and synthetic thrombin inhibitors.

BACKGROUND OF THE INVENTION 
The invention relates to an antidote for hirudin and synthetic thrombin 
inhibitors, the application of a compound that splits prothrombin into 
meizothrombin, a prothrombin intermediate or a pharmacologically 
acceptable salt thereof as the antidote for hirudin and synthetic thrombin 
inhibitors or for the preparation of an antidote for hirudin and synthetic 
thrombin inhibitors. 
Anticoagulants, such as heparin or coumarins, are often used today for 
therapy and prophylaxis of thromboembolic diseases, in particular for the 
treatment of coronary infarctions, arteriosclerosis, furthermore during 
blood transfusions and following operations. They can also be used as 
agents to prevent death from exposure, since they prevent the blood from 
coagulating at extremely cold temperature; otherwise the blood vessels 
would partially clog and the frozen organs would be cut off from the blood 
circulation. 
Hirudin, which is obtained from the salivary gland of the Hirudo 
medicinalis (leech), is an anticoagulant, whose effect is based on the 
formation of a chemical compound with thrombin, whereby its catalytic 
action is inhibited. Hirudin is a miniprotein comprising 65 amino acids 
with a molecular weight of 7 kD. Owing to its strong affinity for thrombin 
(k.sub.i values of 10.sup.-12 mol/l) and its direct mechanism of action, 
it is of great interest. Its clinical application was extremely limited in 
the past, since hirudin was not easily accessible in the standardized form 
and no antidote was available. Today hirudin can be produced through 
genetic engineering; and, therefore, its clinical application can be 
expected in the near future. 
For example, pharmaceutical preparations for oral administration are 
described in the EP-A-0 468 327; said preparations contain recombinant 
hirudin. 
Recently hirudin has been intensively investigated pharmacologically; and 
the pharmacological data were acquired from experimental animals and 
humans. Hirudin is not metabolized in the liver, but rather eliminated in 
an unchanged form through the kidneys. Hirudin has an elimination 
half-life of about 1 to 2 hours and is distributed into the extracellular 
fluid spaces of the body. Analogous to heparin, hirudin is not resorbed 
orally. Past investigations have demonstrated that hirudin is active in 
almost all models of thrombosis, thus even during endotoxic shock and 
during experimental cardiac infarction and during prevention of 
reocclusion following thrombolysis. No immunological reactions were 
detected in the clinical-pharmacological investigations. During clinical 
investigations hirudin has proven to be superior to heparin as an 
anticoagulant and antithrombotic agent. 
Recently synthetic thrombin inhibitors have gained in importance. Currently 
many research laboratories are working world-wide on synthesizing such 
synthetic inhibitors. Investigations with derivatives of benzamidines, 
such as the small molecular synthetic thrombin inhibitor NAPAP 
(N.alpha.-(2-naphthylsulforyl-glycyl)-D,L-amidinophenylalanine-piperidide) 
and with so called tripeptides have made the most progress. All synthetic 
thrombin inhibitors are currently in preclinical research. Their effects 
can be equated qualitatively with those of hirudin. However, the 
metabolism of the synthetic thrombin inhibitors differs from that of 
hirudin. Usually the thrombin inhibitors are metabolized in the liver or 
in the blood. It is anticipated that such substances will be available 
soon for clinical testing. The advantage over hirudin lies predominantly 
in the fact that the compounds can be administered orally. 
Despite the beneficial effects, hirudin has not been used to date 
clinically or its use has been quite limited, since, as aforementioned 
there is no antidote, as, for example, in the case of heparin there is 
protamine sulfate. The presence of an antidote is absolutely necessary for 
a thrombin inhibitor, because in the case of accidental overdosing or for 
patients with kidney function disorders, for whom there is the risk of 
bleeding complications, an antidote must be on hand immediately, should 
overdosing be determined. Such bleeding complications occur, for example, 
as hemorrhagic side effects, above all in the vascular regions of the 
peritoneum, the pleura, the pericardium and the pia mater, but also in 
wounds from surgical incision, as observed in animals with extremely high 
blood levels of a thrombin inhibitor. 
In the past different antidote principles for hirudin have been 
experimentally researched; thus the use of gamma thrombin preparations, 
like DFP thrombin or benzoyl thrombin (Bruggener, E., Walsmann, P., 
Markwardt, F. "Neutralization of Hirudin Anticoagulant Action by 
DIP-Thrombin." Pharmazie 1989; 44: 648-9). These preparations have not 
been successful in practice and they are too toxic. Even activated plasma 
fractions, like FEIBA or autoplex preparations, are unsuitable owing to 
the thromboplastin-similar activity (Fareed, J., Walenga, J.M., "Do we 
need to neutralize hirudin's anticoagulant effects to minimize bleeding?" 
Fed Proc 1989; 3: A328; Walenga, J. M, Piffarre, R., Hoppensteadt, D. A., 
Fareed: "Development of recombinant hirudin as a therapeutic anticoagulant 
and antithrombotic agent. Some objective considerations." Sem Thromb 
Hemost 1989; 15: 316-33). 
SUMMARY OF THE INVENTION 
The present invention is based on the problem of providing an antidote for 
hirudin and synthetic thrombin inhibitors. The antidote shall be quite 
effective, i.e. exhibit an adequate affinity for hirudin and synthetic 
thrombin inhibitors; its dosing shall be simple and it shall be readily 
accessible. 
The subject matter of the invention is an antidote for hirudin and 
synthetic thrombin inhibitors, which is characterized in that it contains 
a compound that splits prothrombin into meizothrombin, a prothrombin 
intermediate, a pharmacologically acceptable salt thereof or a mixture of 
these compounds, together with conventional vehicles and/or diluents, 
whereby it contains as the prothrombin intermediate meizothrombin, PIVKA 
prothrombin, meizothrombin-des-fragment-1 or a compound containing 
meizothrombin. 
The subject matter of the invention is also the application of a compound 
that splits the prothrombin into meizothrombin, a prothrombin intermediate 
defined above, a pharmacologically acceptable salt thereof or a mixture of 
these compounds together with the conventional vehicles and/or diluents as 
the antidote for hirudin and synthetic thrombin inhibitors or for the 
preparation of an antidote for hirudin and synthetic thrombin inhibitors. 
Suitable thrombin inhibitors are, according to the invention, hirudin and 
synthetic, preferably small molecular, thrombin inhibitors. Examples of 
synthetic thrombin inhibitors are NAPAP 
(N.alpha.-(2-naphthylsulforyl-glycyl)-D,L-amidinophenylalanine-piperidide) 
; moreover, the derivatives of tripeptide phe-pro-arg, like boric acid 
derivatives, argininals, chloromethylketone derivatives and amino 
acid-modified derivatives and benzamidine derivatives and also socalled 
hirologs, i.e. synthetic hirudin-analogous partial sequences. For all of 
these compounds the antidote principle is the same as for hirudin.

DETAILED DESCRIPTION OF THE INVENTION 
The antidote according to the invention exists in a suitable form for 
parenteral administration, i.e. a form that is suitable for the 
subcutaneous, intramuscular or intravenous administration. Intravenous 
administration is preferred. Optionally the antidote according to the 
invention can also be administered as continuous infusion. 
According to the invention, snake venom is used as the compound that splits 
prothrombin into meizothrombin. Examples of snake venom are Ecarin and 
poisons from Dispholidus, Rhabdophis, Bothrops, Notechis, Oxyuranus and 
Russel viper types. Preferred is Ecarin, a purified poison fraction from 
Echis carinatus toxin. All of the snake venoms can be acquired as 
biochemicals, for example, at Sigma Chemie GmbH, 8024 Deisenhofen. 
Purified fractions of individual, listed snake venoms can be acquired upon 
inquiry at Pentapharm company in Switzerland. The snake venoms, especially 
Ecarin and immobilized Ecarin, can also be purchased from the Pentapharm 
company in Switzerland. 
The poisons are available as a dry substance, usually freeze-dried, in 
amounts ranging from 5 mg to 1 g. All poisons are readily water soluble 
and should be absorbed with 0.9% saline solution. The amount of the dose 
can be easily determined by the expert. The dose is a function of the body 
weight, the hirudin content and the method of administration. The amounts 
used range for one person weighing 70 kg from 0.5 to 5 mg. 
According to the invention, meizothrombin, meizothrombin-des-fragment-1, or 
PIVKA prothrombin or a compound containing meizothrombin is used as the 
prothrombin intermediate. Meizothrombin is commercially available and can 
also be obtained from the aforementioned Pentapharm company. However, 
meizothrombin, PIVKA-prothrombin, meizothrombin-des-fragment-1, or other 
prothrombin intermediates can also be formed in vitro. 
As shown in the following diagram, four factors of the coagulation 
system--factor II (prothrombin), factor VII, factor IX and factor X--are 
characterized in that they contain gamma-carboxyglutamic acid groups. This 
gamma-carboxylation at the glutamic acid does not take place until after 
the ribosomal synthesis of the "acarboxy factor" in the liver with the aid 
of an enzyme system, which requires vitamin K as the cofactor. 
Therapeutic Mechanism of Vitamin K and Coumarins 
##STR1## 
The gamma-carboxyglutamic acid groups are essential for the coagulation 
action. They represent the necessary bonding valences for calcium ions. 
For treatment with indirect anticoagulants of the Dicumarol type ("vitamin 
K antagonists"), the postribosomal gamma-carboxylation cannot take place; 
and the blood exhibits incomplete coagulation factors or acarboxy factors, 
because they lack the calcium-binding gamma-carboxy groups. These 
coagulation factors are also called PIVKA factors (PIVKA=proteins induced 
by vitamin K antagonists). 
When Ecarin is added to the plasma of patients treated with such 
anticoagulants of the Dicumarol type, PIVKA meizothrombin is produced in 
this plasma from the PIVKA prothrombin in the same manner through a 
limited proteolysis, as is also the case in normal plasma samples with 
prothrombin. 
This PIVKA meizothrombin or other PIVKA intermediates have retained their 
ability to bond with hirudin, but they have no or significantly less 
effect on other factors of the coagulation cascade (platelets, fibrinogen, 
thrombomodulin etc.). According to the invention, meizothrombin, PIVKA 
meizothrombin, their intermediates and PIVKA intermediates from PIVKA 
prothrombin can be used as the antidote. They can originate from humans or 
from other mammals. 
To prepare meizothrombin immobilized Ecarin (product of Pentapharm AG, 
Basel) can be packed, for example, in mini columns ranging in size from 
2-4 cm.sup.3 for example. Ecarin immobolizate is afforded in the swollen 
state, suspended in an aqueous solution of sodium chloride 0.15 M, sodium 
acetate 0.02 M, Prionex (R) (trademark of Pentapharm AG from a 
protein-stabilizing polypeptide fraction from cleaned pig skin collagen) 
0.2% and trichloroisobutanol 0.3%, pH 5.5. One gram of swollen Ecarin 
immobilizate produces from barium citrate eluate at 37.degree. C., pH 8.4, 
within 30 minutes 500 to 700 U amidolytic activity (1 U=123 NIH units), 
measured at tos-gly-pro-arg-pNA (Chromozym (R) TH). 
Then purified prothrombin fractions are put on these columns; and the 
formed meizothrombin, optionally following stabilization with heparin, is 
subsequently freeze-dried. The freeze-dried material can be packed into 
ampoules and then reconstituted with a suitable solvent, preferably with 
sterilized sodium chloride solution, which is suitable for intravenous 
injection, for application as an antidote. 
To prepare meizothrombin-des-fragment-1, the same process as for 
meizothrombin is used. In the batch process only a longer reaction time 
(3-4 hours) has to be planned. Meizothrombin-desfragment-1 is a product 
following the activation of meizothrombin. 
For parenteral administration the antidote can be formulated for the 
injection, like the intravascular, e.g. intravenous, intramuscular or 
subcutaneous, injection. The intravascular administration is preferred. 
Preparations for the injection can be on hand as one dose, for example in 
ampoules, or in multiple dose containers with added preservative. The 
preparations can exist as suspensions, solutions or emulsions in oily or 
aqueous carriers, and contain preparation aids, such as suspending, 
stabilizing and/or dispersing agents, and/or agents for adjusting the 
tonicity of the solution. As an alternative the active ingredient can be 
present as a powder for constituting with a suitable carrier, for example 
sterilized pyrogen-free water, prior to application. 
In ampoules and multiple dose containers for intravascular application, the 
product, e.g. the meizothrombin, should exist preferably in the 
freeze-dried state. In this form it is completely soluble in sterilized 
water, physiological saline solution or buffer solutions, which contain 
Ca.sup.++ ions and are adjusted to a pH value ranging from 6.5 to 7. The 
ampoules should contain 0.5; 1; 2 or 5 mg of meizothrombin; multiple dose 
containers should contain 10; 20 or 50 mg. A short-term storage (days-1 
month) is also possible in the deep-frozen state (-25.degree. C.). For 
extravascular parenteral application mixtures with 1% Dimeticon 
suspensions, 5% erythrocyte membrane fragments or barium sulfate emulsions 
are suitable. Even a liposome adsorption for extravascular application is 
suitable. The content of the single formulation of meizothrombin is 
equivalent in these preparations to the filling into ampoules and multiple 
doses as for intravascular application. 
According to the invention, preferably Ecarin, a highly purified fraction 
of Echis carinatus toxin, is used as the snake venom. Ecarin splits a 
peptide bond at arginine 323 of the prothrombin, producing the 
intermediate meizothrombin. Normally the additional reaction occurs 
through autocatalysis or through thrombin acceleration. When hirudin or 
synthetic thrombin inhibitors are present in the blood, the meizothrombin 
and the inhibitor interact. In contrast, heparin cannot react with 
meizothrombin. The attached FIG. 1 shows these actions. 
It could be demonstrated that in highly diluted human plasma due to Ecarin 
the prothrombin activation is induced. The thrombin/meizothrombin activity 
was measured with Chromozym (R) TH. FIG. 2 shows the results that were 
obtained. The dose independent residual activity that could be detected 
with amounts of heparin ranging from 2.5-35 IE/ml corresponds to the 
degree to which meizothrombin is formed following the effect of Ecarin. As 
a function of the dose, hirudin totally inhibits the formation of 
meizothrombin/thrombin. 
To confirm the effect of the antidote, various pharmacological tests were 
conducted. The antidote effect of the antidote according to the invention 
was proven in rat tests. FIG. 3 shows the results that were obtained. Rat 
citrate plasma was made to coagulate with thrombin. The controls have 
coagulation times of 17 seconds on average. If 0.1 .mu.g hirudin/ml are 
added to the test batch, the thrombin time is extended to 30 seconds. If 
the batch with 0.25 mU of Ecarin/ml is preincubated, the thrombin time 
drops to 27.5 seconds, following a preincubation period of 20 seconds, 
owing to the consumption of hirudin in the plasma. At a 50 second 
preincubation the thrombin time matches the control value (without 
addition of hirudin). The Ecarin concentration itself that was used does 
not cause the coagulation to accelerate in the test batch in this time 
range. 
To confirm the antidote action, constant blood levels of hirudin in the 
range of 3.5 to 4.2 .mu.g/ml following intravenous application of 1 mg/kg 
hirudin were also produced in nephrectomized rats. During the infusion of 
50 .mu.g of Ecarin/kg h.sup.-1, the hirudin level drops rapidly and is 
significantly reduced to 2.1 .mu.g/ml just after 30 minutes following the 
start of application. At the end of the toxin infusion the blood level has 
decreased to 1.2 .mu.g/ml. Rebound phenomena are not observed. The finding 
that in these tests the platelet count and the fibrinogen level remained 
virtually unchanged has to be evaluated as especially important. Even when 
the Ecarin infusion duration was reduced to 30 or 15 minutes, and thus the 
dose was reduced by half or to a fourth, this drop in the hirudin level 
can also be proven in a similar manner (cf. FIG. 4). 
In another series of tests, the antidote mechanism was confirmed with a 
bleeding model. To this end, nephrectomized rats were administered 
intravenously 5 mg/kg of hirudin. After 2 hours a constant blood level of 
18 .mu.g/ml of hirudin was reached. At this instant a bleeding time of 
longer than 100 minutes was measured. If the rats are infused with 
Echis-carinatus toxin (1 mg/kg h.sup.-1), then the bleeding ceases after 
90 minutes. The blood loss from the experimental incised cut is 
significantly reduced; the hirudin blood level has fallen to values 
ranging from 1 to 3 .mu.g/ml. The following table shows the results that 
were obtained. 
Ecarin-Induced Meizothrombin Formation as Antagonism Against Toxic Hirudin 
Blood Levels in Rats; 
______________________________________ 
Ecarin-Induced Meizothrombin Formation as Antagonism Against 
Toxic Hirudin Blood Levels in Rats 
plasma level 
bleeding 
time of hirudin time* blood 
(h) test log (.mu.g/ml) (min.) loss 
______________________________________ 
0 bilateral 
nephrectomy 
2 0 2.52 - 
2 hirudin i.v. 
(5 mg/kg) 
4 17.8 &gt;30 +++ 
4 E. carinatus toxin 
infusion (1 mg/kg .multidot. h.sup.-1) 
prothrombin substitution 
5 5.9 8.17 (+) 
6 3.5 6.40 - 
______________________________________ 
(* incision into the abdominal wall) 
"-" means no bleeding 
"(+)" means minor bleeding 
"+++" means intensive bleeding 
It is clear from the above tests that Ecarin, acts as an indirect antidote 
against hirudin intoxications. The Ecarin transforms prothrombin in the 
plasma to meizothrombin, which is the direct antidote. Similar 
pharmacological results were obtained with other prothrombin 
intermediates. 
The following examples explain the invention. 
EXAMPLE 1 
Preparation Of Meizothrombin In Batch Process 500 ml Of Prothrombin 
Solution: 
Prothrombin is precipitated by means of BaSO.sub.4 from 2 liters of oxalate 
plasma and subsequently washed with 0.1 M of sodium oxalate and 0.006 M of 
sodium citrate at pH 7.5. Following elution of the prothrombin with 0.15 M 
of sodium citrate and pH adjustment to 7.5, an alcohol precipitation (19%) 
is conducted at -5.degree. C. The supernatant is adjusted to pH 5.5. and 
the alcohol concentration is increased to 25%. The precipitate is 
dissolved and heated to 50.degree. C. for 5 minutes, centrifuged at 6000 g 
and then filled to 500 ml with acetate buffer. 
50 mg of human prothrombin dissolved in 500 ml of acetate buffer at pH 5.5 
are stirred with 10 g of immobilized Ecarin for 60 minutes at 20.degree. 
C. Following centrifugation (15 minutes at 6000 g) the supernatant is 
calibrated with the aid of a calibration curve, which was obtained with a 
meizothrombin standard, to 1 mg/ml, filled into 10 ml ampoules, 
freeze-dried and heat-sealed. The filling was conducted with simultaneous 
sterilization under aseptic conditions. Following freeze-drying, the 
ampoules are sealed and stored at 4.degree. C. In this form the 
preparations can be stored for at least 12 months without any activity 
loss. 
EXAMPLE 2 
Preparation Of PIVKA Meizothrombin In The Column Process 
250 ml of acetic acid buffer solution (pH 5.5.), containing 25 mg of PIVKA 
prothrombin (preparation according to example 1, starting material 1 liter 
of oxalate plasma, obtained by pooling the plasma of patients treated with 
Dicumarol) are eluted over 40 cm.sup.3 columns (50-75 cm length), packed 
with 10 g of swollen Ecarin immobilizate. The eluted volume is limited to 
500 ml, subsequently sterilized by filtration (with sterilized filters) 
and then filled into ampoules in 10 ml portions. Following freeze-drying, 
the ampoules are sealed and stored in the same manner as described in 
example 1. 
EXAMPLE 3 
The lyophilized 1 mg ampoules are dissolved with 10 ml of 0.9% NaCl and 
administered in this form. For intravenous infusion multiple dose 
containers, containing 10 mg of meizothrombin, are dissolved with 0.9% 
NaCl and administered in 500 ml of 0.9% Nacl infusion solution each. The 
rate of infusion should be about 1000 ml/h. The plasma hirudin content 
must be controlled continuously with a bedside method. 
Variations of the invention will be apparent to the skilled artisan.