WO1995033471A1

WO1995033471A1 - Intravasal thrombolysis

Info

Publication number: WO1995033471A1
Application number: PCT/SE1994/000550
Authority: WO
Inventors: D Serv Europe S.A. M
Original assignee: Hellgren, Lars; Mohr, Viggo; Vincent, Jan; Vincent, Josef; KARLSTAM, Björn
Priority date: 1994-06-07
Filing date: 1994-06-07
Publication date: 1995-12-14
Also published as: AU7278094A

Abstract

Use of one or more enzymes derived from crustacea of the order Euphausiaceae in particular the genus Euphausia (krill enzymes) for the manufacture of an intravasal pharmaceutical composition for thrombolysis in a mammal host including man, said enzyme(s) preferably being administered locally or as genetically modified to prevent inhibition thereof. A method for intravasal administration of an enzyme composition to dissolve intravasal thromboses in a mammal host having such thromboses, said method comprising administering in a blood vessel of said host an effective amount of one or more enzymes derived from crustaceans of the order Euphausiaceae in particular the genus Euphausia, in a pharmaceutically acceptable carrier suitable for intravasal administration. A composition for such purpose.

Description

INTRAVASAL THROMBOLYSIS

DESCRIPTION

BACKGROUND ART

Damage of the arteriosclerotic blood vessels, resulting in stenosis, predispose thrombosis or embolies and may lead to heart infarction or ischemia. Thrombolytic treatment has been developed from local to systemic administration. Thrombolytic treatment with enzymes e.g. Strepto inas^® (Glaxo, Boehringer Ingelheim) Kabikinas^® (Pharmacia) and Urokinas^® (Abbott,

Boehringer Ingelheim) have attained an important role in the last years in the therapeutic arsenal to clear the blood vessels, dissolve thrombus and normalize blood flow. Several extensive controlled studies from Europe and the U.S. have shown that e.g. patients treated with streptokinase have significantly longer survival time compared to their controls. As a consequence, at least 90% of the U.S. emergency hospitals actually use streptokinase as a thrombolytic agent in acute heart infarction. Another enzyme for thrombolytic treatment is urokinase and lately r-TPA

(tissue plasminogen activator). EP-A-0170115 (EP 85108526-6, Kao Corporation) discloses an aqueous extract of krill for dissolution of thromboses. Although alleging a more general scope, the disclosure is limited to oral administration and intrapetitoneal D5_Q in the rat. The present inventors have verified the expected fact that the gastro-intestinal liquids of mammals will completely digest and inactivate krill enzymes.

DESCRIPTION OF THE INVENTION

Below, proteolytic enzymes derived from Crustacea of the order Euphausiaceae in particular the genus Euphausia are occasionally referred to as krill enzymes. Krill enzymes were demonstrated to possess unique thrombolytic properties in vitro, indicating their potential as new thrombolytic agents. Krill enzymes, both partially and highly purified, have shown excellent thrombolytic effect in vitro. Their effects are significantly more pronounced than that of streptokinase and r-TPA.

Accordingly, the present invention provides use of one or more enzymes derived from Crustacea of the order Euphausiaceae in particular the genus Euphausia for the manufacture of an intravasal pharmaceutical composition for thro bolysis in an mammal host including man.

In the in vivo situation, due to the presence of natural protective mechanisms in the living tissue, enzyme inhibitors counteract directly acting enzymes such as krill enzymes. Thus, as expected, krill enzymes are inactivated when applied in vivo. To overcome this problem, two solutions are provided by preferred embodiments of this invention:

To achieve the full effect in vivo, a special catheter is introduced close to the thrombus (local thrombolysis) . This technique using the existing perfusion catheters and intra- coronary balloon catheters with systemic heparinisation is now routinely used. In the same way krill enzymes may be allowed to act locally e.g. using a double balloon catheter introduced and inflated with its balloons enclosing the thrombus, thus mimicking the in vitro situation.

Another approach to avoid enzyme inhibition in vivo is to modify krill enzymes via protein engineering in order to change their specificity and this way eliminate the risk that serum inhibitors recognize the enzyme(s) i.e. systemic thrombolysis. For systemic thrombolysis, heparinisation is recommended to the same extent as with r-TPA, to prevent an early or immediate re-thrombosis.

In another aspect the present invention provides a method for intravasal administration of an enzyme composition to dissolve intravasal thromboses in a mammal host having such thromboses, said method comprising administering in a blood vessel of said host an effective amount of one or more enzymes derived from crustaceans of the order Euphausiaceae in particular the genus Euphausia , in a pharmaceutically acceptable carrier suitable for intravasal administration.

In a further aspect the invention provides an enzyme composi¬ tion for intravasal administration to dissolve thromboses in a mammal host, said composition comprising an effective amount of one or more enzymes derived from crustaceans of the order Euphausiaceae in particular the genus Euphausia , in a pharmaceutically acceptable carrier suitable for intravasal administration.

Krill enzymes can also be used alone or together with other anticoagulants such as heparin, dicumarol, warfarin, rheo- logical agents (dextran) , thrombocyte aggregation inhibiting agents (ASA, dipyramidol) or other thrombolytic agents (streptokinase, urokinase or r-TPA) . This kind of combination therapy may be beneficial to achieve different targeting against thrombi, something which could contribute in an additive, synergistic way to the efficacy of krill enzymes. The invention thus encompasses all therapeutic thrombolytic applications of krill enzymes, partially or highly purified, alone or in combination with other therapeutics in this field.

Biochemical Characteristics of Krill Enzymes,

Enzymes from krill, represent a naturally occurring enzyme mixture prepared as an extract of Antarctic krill (Euphausia superba) using modern separation technology, including membrane ultrafiltration and gel chro atography. The final product is well characterized with respect to its proteolytic activities, batch-to-batch variations and uniformity. The extract is defined as a mixture of acidic endopeptidases (trypsin- and chymotrypsin-like enzymes) and exopeptidases (carboxypeptidase A and B) in the molecular weight range of 20-40 kD. Standardized levels of enzyme activities as well as batch-to-batch variations, are determined by means of advanced biochemical and immunochemical methods.

As mentioned, krill enzymes comprise both endo- and exopeptidases assuring a two-level breakdown of proteinaceous material (polypeptides) , where endopeptidases first attack the peptide bonds in intra-structural parts of a polypeptide chain. This leads to an number of peptides with different sizes, which are subsequently cleaved by the exopeptidases into small peptides and free amino acids. In this manner, an effective and complete degradation of proteins is achieved without autodigestion of participating enzymes.

Experimental

The invention will be described in detail with reference to the appended drawings, wherein

Fig. 1 is a graphic representation of the fibrinolytic activity of krill enzymes dissolved and diluted in 0.1 M phosphate buffer pH 7.4 ( ♦ ) or in human plasma (□) . The fibrinolytic activity is expressed as lysed area (cirr) .

Fig. 2 is a graphic representation of the fibrinolytic activity of krill enzymes in human plasma, measured in Chandler loops.

Fig. 3 shows a dose-response curve for krill enzymes. Data are taken from Fig. 2 after a fixed lysis time of 3 hours.

Fig. 4 is a graphic representation of per cent clot lysis versus lysis time for krill enzymes and purified trypsin-like krill enzymes, respectively. Example l; Preparation of krill enzymes

Krill enzymes are extracted from the raw material, Antarctic krill (Euphausia superba) according to standard procedures (EP-A 107,634, EP-A 177,605). In short, krill is frozen immediately after caught and stored at -30°C before prepara¬ tion. The thawed krill is mixed with water, homogenised and ceritrifuged in the cold. Such an aqueous crude extract is defatted and further purified by gel filtration. Fractions containing substances with molecular weights of 20-40 kD are pooled and concentrated by ultrafiltration. The purified extract is subjected to an aseptic manufacturing process including sterile filtration, filling in glass vials and freeze-drying.

The purification process can also be continued in order to achieve a highly purified preparation containing either individual proteases or mixtures thereof. In such a procedure, conventional purification technology such as gel or affinity chromatography combined with e.g. more sophisticated methods like FPLC (fast protein liquid chromatography) may be used. Furthermore, modern or emerging technologies such as genetic engineering can also be applied to clone and manufacture the enzymes, followed by sterile purification methods as above.

Example 2: Intravenous infusion of krill enzymes in mammals (tolerance test)

Mice were injected with 0.5 ml 1% krill enzymes in a physio¬ logical solution. No toxic or allergenic symptoms are observed. Thus krill enzymes are well tolerated.

Example 3 : Pre-weighed samples of fibrin were introduced in test tubes and incubated with different concentrations of krill enzymes (water bath at 33 °C) . The break-down was registered at different time periods and the results are exemplified in Table 1.

TABLE 1

The effect of krill enzymes and Streptokinas^®, respectively, on human fibrin. Data are expressed as dry weight of the initial values according to the ranking scale below:

Scale: weight decrease 76-100 weight decrease 51-75

— weight decrease 26-50 % - weight decrease 1-25 % 0 no change 0 %

Enzymes Concentration Incubation time

1 h 2 h

0.1 % (-)

Krill 0.5 % 1.0 % (-)

Streptokinas^® 1:5 ml -(-) 1:1 ml — --(-)

One vial of Streptokinas^® (100000 IU) was dissolved in 5 and 1 ml water, respectively.

From Table 1 it is quite obvious that krill enzymes have a high fibrinolytic activity compared to Streptokinas^®.

Example 4 : Effect on blood clots

Human blood clots are weighed and the thrombolytic effect of krill enzymes (10 mg/ml) is evaluated. The clot weight is dramatically reduced (75-100 %) within one hour.

Example 5: Fibrinolytic effect on fibrin plates

The fibrinolytic effect of krill enzymes was tested on fibrin plates prepared as follows: 45 ml of a 1% agarose solution was heated to 54 °C on a water bath and then mixed with 5 ml of a human fibrinogen solution (15 mg/ml) and 50 μl of human thro bin (30 NIH/ml) . After mixing the solution was poured on to a glass plate and allowed to congeal. Fibrinogen will then, in the presence of thrombin, form a thin opaque film layer in the gel, which can serve as a substrate for com¬ pounds with fibrinolytic activity. Krill enzymes were diluted in buffer and human or rabbit plasma to concentrations ranging from 0.125 to 3 CU/ml (casein units/ml). 10 μl out of each sample was applied to the fibrin plate and incubated over night at 37 °C. During diffusion through the gel, the test compounds will lyse fibrin and form a circular clear area which is proportional to the fibrinolytic effect. Fig 1 shows the results from the fibrin plate assay. Krill enzymes show, when diluted in buffer, a strong fibrinolytic effect, which is directly proportional to the enzyme concentrations. In plasma, however, this effect is inhibited and a significant lysis can only be observed at concentrations higher than 2 CU/ml.

Example 6: Chandler loop assay

The Chandler loop assay is an excellent system to study the rate at which different compounds exert their fibrinolytic activity in circulating plasma. The assay contains 1.5 ml of human plasma, which is put into a plastic tube and mixed with trace amounts of ^liiJI-labelled human fibrinogen. After addition of Ca²⁺ to the system, a radio-labelled plasma clot is formed within 1 hour. After addition of fibrinolytic compounds, the clot lysis can be continuously be measured as

1 c , the release of ^l^I-labelled fibrin degradation products from the clot into the surrounding plasma. Krill enzymes were introduced into the loops in final concentrations ranging from 0.25 to 10 CU/ml. In agreement with the fibrinolytic assay, the fibrinolytic activity of krill enzymes was very low at concentrations below 2 CU/ml (Figs 2 and 3) , while higher concentrations showed a very good and rapid clot lysis. In fact, doses of 4-10 CU/ml showed the most rapid lysis ever observed in this system (in comparison with TPA and streptokinase) . When the purified trypsin-like krill enzymes were tested in Chandler loops, a two-fold increase in fibrinolytic activity was observed, i.e. 1 and 2 CU/ml of krill enzymes had the same fibrinolytic activity as 0.5 and 1 CU/ml of the purified enzymes (Fig. 4) .

Example 7: Thrombolysis in vivo

Based on the above experiments in vitro, the activity of krill enzymes was studied in vivo (rabbit) . It was demon¬ strated that krill enzymes were effectively inactivated by plasma inhibitors, thus avoiding the most important risk factor of streptokinase - bleeding - thus contributing to the safe administration of krill enzymes. The optimal dose for systemic heparinisation together with modified krill enzymes in prevention of immediate re-thrombosis can be established by routine experimental work.

Claims

1. Use of one or more enzymes derived from Crustacea of the order Euphausiaceae in particular the genus Euphausia for the manufacture of an intravasal pharmaceutical composition for thrombolysis in an mammal host including man.

2. Use as claimed in claim 1 for the manufacture of a pharmaceutical composition for local intravasal thrombolysis.

3. Use as claimed in claim 2 for the manufacture of a pharmaceutical composition for lysis of a thrombus isolated from the blood circulation of said host.

4. Use as claimed in claim 1 of one or more enzymes, genetically modified to prevent inhibition by serine inhibitors, for the manufacture of a pharmaceutical composition for systemic intravasal thrombolysis.

5. A method for intravasal administration of an enzyme composition to dissolve intravasal thromboses in a mammal host having such thromboses, said method comprising adminis¬ tering in a blood vessel of said host an effective amount of one or more enzymes derived from crustaceans of the order Euphausiaceae in particular the genus Euphausia , in a pharmaceutically acceptable carrier suitable for intravasal administration.

6. A method for intravasal administration of an enzyme composition as claimed in claim 5, comprising administering said enzyme composition locally in said blood vessel.

7. A method for intravasal administration of an enzyme composition as claimed in claim 6, comprising administering said enzyme composition locally in said blood vessel to a thrombus isolated from the blood circulation of said host.

8. A method for intravasal administration of an enzyme composition as claimed in claim 5, comprising systemic intravasal thrombolysis by administering an enzyme, genetically modified to prevent inhibition thereof.

9. An enzyme composition for intravasal administration to dissolve thromboses in a mammal host, said composition comprising an effective amount of one or more enzymes derived from crustaceans of the order Euphausiaceae in particular the genus Euphausia , in a pharmaceutically acceptable carrier suitable for intravasal administration.