WO1995012407A1

WO1995012407A1 - A pharmaceutical composition for the treatment of coronary thrombosis

Info

Publication number: WO1995012407A1
Application number: PCT/DK1994/000414
Authority: WO
Inventors: Claus Bregengaard
Original assignee: Novo Nordisk A/S
Priority date: 1993-11-05
Filing date: 1994-11-03
Publication date: 1995-05-11
Also published as: DK125693D0; IL111501A0; ZA948721B; AU8058194A

Abstract

A method of treating coronary thrombosis, the method comprising administering, to a patient in need of such treatment, an effective amount of lys-plasminogen and, simultaneously or sequentially, an effective amount of tissue plasminogen activator.

Description

A pharmaceutical composition for the treatment of coronary thrombosis

FIELD OF INVENTION

The present invention relates to a method of treating coronary thrombosis, a method of preventing reocclusion of coronary arteries, as well as a pharmaceutical composition for use in the methods.

BACKGROUND OF THE INVENTION

The use of plasminogen activators such as urokinase, streptokinase and tissue plasminogen activator as the sole fibrinolytic agents in the treatment of thrombosis is well known, for instance as reviewed in E.J. Topol, "Advances in Thrombolytic Therapy for Acute Myocardial Infarction", J. Clin. Pharmacol.27, 1987, pp. 735-745; N.J. Marder and S. Sherry, "Thrombolytic Therapy: Current Status". New England J. Med. 318. 1988, pp. 1585-1595; J. Loscalzo, "Thrombolysis in the Management of Acute Myocardial Infarction and Unstable Angina Pectoris", Drugs 37, 1989, pp. 191-204; and S. Sherry, J. Int. Med. 229. 1991, pp. 113-116. The use of lys-plasminogen as a thrombolytic agent has been proposed previously by K. Anderle et al., Haemostasis 18, 1988, pp. 165-175, reviewing studies of the systemic use for the treatment of deep venous thrombosis of lys-plasminogen activated by urokinase and streptokinase. Lys-plasminogen in combination with urokinase is also proposed for the treatment of pulmonary embolism.

S.F. Badylak et al., Haemostasis 21, 1991, pp. 278-285, and S.F. Badylak et al., Thrombosis Research 62, 1991, pp. 115-126 report the enhancement of the thrombolytic effect of urokinase by lys-plasminogen in a dog model of arterial thrombosis.

SUMMARY OF THE INVENTION

The combination of lys-plasminogen with tissue plasminogen activator for the treatment of coronary thrombosis is believed to be novel. Accordingly, the present invention relates to a method of treating coronary thrombosis, the method comprising administering, to a patient in need of such treatment, an effective amount of lys-plasminogen and, simultaneously or sequentially, an effective amount of tissue plaminogen activator (t-PA).

It was surprisingly found (vide the study described below in Example 1) that when lys- plasminogen was administered in combination with t-PA, the combined treatment not only reduced the time to restoration of flow, but also prevented reocclusion of the coronary artery. Reocclusion of coronary arteries represents a major problem in thrombolytic therapy. Although early and complete restoration of coronary patency may result in substantial salvage of myocardium during infarction, this initial benefit may be attenuated by subsequent reocclusion. Reocclusion rates vary from 10 to 15% (cf. D. Collem et al, Drugs 38, 1989, 346-388)., and patients with reoccluded arteries frequently suffer from reinfarction (cf. S.H. Schaer et al., Circulation 76 (Suppl. II), 1987, pp. 57- 62. Also, the time to reperfusion seems to be highly important for the outcome of thrombolytic therapy. Thus, accelerated t-PA treatment resulting in a more rapid infarction-artery patency during thrombolytic therapy is associated with a lower mortality rate (cf. The GUSTO investigators. An Internal Randomized Trial Comparing four Thrombolytic Strategies for Acute Myocardial Infarction, New England J. Med. 329, 1993, pp. 673-682). Thus, the finding that the combined administration of lys- plasminogen and t-PA is effective to decrease the time to reperfusion and prevent reocclusion of the coronary artery is of vital importance for the efficient and lasting treatment of coronary thrombosis.

Theoretically, accelarated thrombolysis and prevention of reocclusion may furthermore result in decreased secondary restenosis in the vessels due to shorter exposure to the vessel wall to inducers of cell proliferation known to be produced by the thrombi (for instance PDGF, thrombin, etc.).

In another aspect, the present invention therefore relates to a method of preventing reocclusion of coronary arteries following treatment of coronary thrombosis, the method comprising administering, to a patient in need of such treatment, an effective amount of lys-plasminogen and, simultaneously or sequentially, an effective amount of tissue plaminogen activator (t-PA).

In a further aspect, the invention relates to a pharmaceutical composition for the treatment of coronary thrombosis, the composition comprising, in separate containers, lys-plasminogen and t-PA, each together with a pharmaceutically acceptable carrier or diluent.

In a still further aspect, the invention relates to the use of lys-plasminogen in combination with t-PA for the preparation of a medicament for the treatment of coronary thrombosis as well as for the prevention of reocclusion of coronary arteries following such treatment.

DETAILED DESCRIPTION OF THE INVENTION

Lys-plasminogen for use according to the present invention may be prepared from native plasminogen (glu-plasminogen) by brief exposure of glu-plasminogen to plasmin resulting in cleavage of 77 amino-terminal amino acids and the formation of lys- plasminogen (named for the N-terminal lysine).

Native human plasminogen may be purified from plasma by several methods. Recent methods of purification from human plasma are based on affinity chromatography as described by D.G. Deutsch and E.T.Mertz, Fed. Proc. 29, 1979, p. 647, and Science 170. 1970, pp. 1095-1096. An affinity matrix (lysine-Sepharose^® ) is prepared by covalent coupling of the α-amino group of L-lysine to Sepharose^®. Plasma diluted with water is passed through a lysine-Sepharose^® column equilibrated with phosphate buffer (pH7.4) at room temperature after which the column is washed with phosphate buffer. Plasminogen is then eluted with 0.2 M e -amino caproic acid (pH 7.4). The e -amino caproic acid is then removed from plasminogen in the cold, by gel filtration on Sephadex^® equilibrated with phosphate buffer. The use of human proteins purified from human plasma involves a certain risk of viral infection. In particular, the risk of HIV and hepatitis infections has become a major concern in dealing with plasma-derived human proteins. To avoid such risk, it is therefore preferable to produce plasminogen by recombinant DNA techiques. cDNA 5 sequences encoding plasminogen and lys-plasminogen as well as the preparation thereof are described in EP 319 944 (Zymogenetics).

Likewise, it is preferred to produce the t-PA to be used according to the present invention by recombinant DNA techniques. cDNA encoding t-PA has been described by Pennica et al., Nature 301. 1983, pp. 214-221; Kaufman et al., Mol. Cell. Biol. 5,

10 1985, pp. 1750-1759; US 4,766,075; and Verheijen et al, EMBO J. 5, 1986, pp. 3525- 3530. In stead of native t-PA, a t-PA analogue may be used according to the invention. Examples of t-PA analogues are described in, e.g. D.L. Higgins and W.F. Bennett, Ann. Rev. Pharmacol. Toxicol. 30, 1990, pp. 91-121; L. Pierard and A. Bollen, Biotechnology 15, 1990, pp. 283-304; and H. Pannekoek et al., Fibrinolysis 2, 1988, pp.

15 123-132.

The DNA coding for the desired polypeptide (in casu plasminogen, lys-plasminogen, t-PA or t-PA analogue) may be introduced into a suitable recombinant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which 20 it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

5 In the vector, the DNA sequence encoding the desired polypeptide should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the desired polypeptide in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter. A suitable promoter for use in insect cells is the polyhedrin promoter (Vasuvedan et al., FEBS Lett. 311, (1992) 7 - 11). Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPIl (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters. Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpjA promoter.

The DNA sequence encoding the desired polypeptide may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPIl (Alber and Kawasaki, op_^ cit.) or ADH3 (McKnight et al., op. cit.) terminators. The vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate.

The procedures used to ligate the DNA sequences coding for the desired polypeptide, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

The host cell into which the expression vector is introduced may be any cell which is capable of producing the desired polypeptide and may suitably be a eukaryotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells. Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841 - 845.

Alternatively, fungal cells (including yeast cells) may be used as host cells of the invention. Examples of suitable yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae. Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.

The desired polypeptide may then be produced by a method which comprises culturing a cell as described above in a suitable nutrient medium under conditions which are conducive to the expression of the desired polypeptide and recovering the resulting polypeptide from the culture.

The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).

Attempts to produce plasminogen in transfected mammalian cells have, however, shown that intracellular plasminogen activation and subsequent degradation provides an obstacle to the production of intact recombinant plasminogen in reasonable yields. The problem has been solved by coexpression of plasminogen with a protease inhibitor capable of inhibiting plasminogen activation, substantially as described in EP 319 944.

Thus, lys-plasminogen or glu-plasminogen may be produced by coexpression of lys- or glu-plasminogen with α-2-plasmin inhibitor or α-1-antitrypsin (AAT) in transfected BHK cells. Coexpression with AAT causes inhibition of urokinase produced by the BHK cells. Activation of plasminogen by urokinase and plasmin-mediated proteolytic cleavage is thereby greatly reduced and consequently, high levels of intact plasminogen is obtained.

The polypeptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.

The pharmaceutical composition of the invention may be compounded in any form suitable for parenteral administration (i.e. for intravenous injection or infusion), for instance by dissolving or suspending lys-plasminogen and t-PA, respectively, in sterile water or isotonic saline. In order to obtain the desired thrombolytic effect of the composition, it is contemplated that the suitable dosage of lys-plasminogen may be in the range of 0.1-3.0 mg/kg body weight, preferably 0.3-1.5 mg/kg body weight, more preferably about 1 mg/kg body weight, dependent on the severity of the condition, the age and general health of the patient to whom the composition is administered, etc. A corresponding suitable dosage of t-PA is one which is capable of activating the lys- plasminogen administered to the patient. Thus, the dosage of t-PA co-administered with lys-plasminogen may suitably be in the range of 0.2-2.0 mg/kg body weight, preferably 0.3-1.5 mg/kg body weight, more preferably about 1 mg/kg body weight. This means that the unit dosage of lys-plasminogen in the composition will typically be in the range of 20-100 mg, and the unit dosage of t-PA will typically be in the range of 20-100 mg.

In one embodiment of the method of the invention, the administration of lys- plasminogen may be substantially simultaneous with the administration of t-PA. This may, for instance, be effected by providing lys-plasminogen and t-PA in a device which makes it possible to administer two substances simultaneously. Alternatively, either lys- plasminogen or t-PA may be administered first and the other component may be administered immediately after that ("immediately" meaning any period of time up to one minute after administration of the first component).

In another embodiment, t-PA may be administered before the administration of lys- plasminogen.

In a further, currently preferred embodiment, lys-plasminogen may be administered before the administration of t-PA. To obtain the desired activation of lys-plasminogen by t-PA, it is contemplated that the t-PA may be adminstered 5-20 minutes, preferably about 10 minutes, after the administration of lys-plasminogen.

In a still further aspect, the present invention relates to the use of lys-plasminogen in combination with t-PA for the preparation of a medicament for the treatment of coronary thrombosis. For this purpose, the lys-plasminogen and t-PA are provided in separate containers in a form adapted to the simultaneous or sequential co- administration of lys-plasminogen and t-PA.

The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed. Example 1

In vivo study of the thrombolytic effect of lys-plasminogen on t-PA-induced thrombolysis

Recombinant human lys-plasminogen was expressed in BHK cells substantially as described in EP 319 944 and purified by the method described in PCT/DK93/00206. The purified product was freeze-dried by conventional methods. The freeze-dried lys- plasminogen was stored in vials containing 50 mg protein per vial.

T-PA was obtained from Boehringer Ingelheim.

15 dogs with a mean body weight of 20 kg were included in the study. Formation of coronary thrombi was induced electrically in the LAD coronary artery. After the thrombi had been stable for 20 minutes, the dogs were treated with either saline (n=10) or 2 mg/kg lys-plasminogen (n=5). Lys-plasminogen or saline did not induce reflow in any of the dogs. Ten minutes later, t-PA (1 mg/kg over 20 minutes) was infused and blood flow characteristics were observed over 2 hours.

Time to restoration of flow was less in the lys-plasminogen-treated dogs (14 +/-4 vs. 58 +/-19 minutes in saline-treated dogs, mean +/- SD P< 0.001), and the peak restored flow was greater in the lys-plasminogen-treated dogs (73 47-22 vs. 47 +/-28 ml/minute).

The restored flow persisted in all lys-plasminogen-treated dogs and in only 2 out of 10 saline-treated dogs over the two-hour period (reocclusion rates 0 vs. 80%, P< 0.001).

Myocardial shortening fraction at 2 hours after t-PA infusion in the LAD supplied region in lys-plasminogen-treated dogs was +1 +/-1% (vs. -10 +/-5% in saline-treated dogs, P<0.01). Plasma fibrinogen levels were lower in lys-plasminogen-treated dogs (0.3

+/-0.1 vs. 1.5 +/-0.0 g/1, P<0.01) at 2 hours after t-PA infusion. The α-2-antiplasmin levels were also lower (29 +/-5 vs. 44 +1-6% of the control).

To conclude, lys-plasminogen administered before t-PA reduces the time to restoration of flow, sustains the restored flow and preserves myocardial function.

Claims

1. A method of treating coronary thrombosis, the method comprising administering, to a patient in need of such treatment, an effective amount of lys-plasminogen and, simultaneously or sequentially, an effective amount of tissue plaminogen activator (t- PA).

2. A method according to claim 1, wherein the effective amount of lys-plasminogen is in the range of 0.1-3.0 mg/kg body weight, preferably 0.3-1.5 mg/kg body weight, more preferably about 1 mg/kg body weight.

3. A method according to claim 1 or 2, wherein the effective amount of t-PA is in the range of 0.2-2.0 mg/kg body weight, preferably 0.3-1.5 mg/kg body weight, more preferably about 1 mg/kg body weight.

4. A method according to claim 1, wherein the administration of lys-plasminogen is followed, after a suitable interval, by administration of t-PA.

5. A method according to claim 4, wherein said suitable interval is in the range of 0-120 minutes, preferably about 5 minutes.

6. A pharmaceutical composition for the treatment of coronary thrombosis, the composition comprising, in separate containers, lys-plasminogen and t-PA, each together with a pharmaceutically acceptable carrier or diluent.

7. A pharmaceutical composition according to claim 6, which contains 20-100 mg of lys- plasminogen.

8. A pharmaceutical composition according to claim 6, which contains 20-100 mg t-PA.

9. Use of lys-plasminogen in combination with t-PA for the preparation of a medicament for the treatment of coronary thrombosis.

10. Use according to claim 9, wherein the lys-plasminogen and t-PA are provided in separate containers in a form adapted to the simultaneous or sequential co- administration of lys-plasminogen and t-PA.

11. Use according to claim 10, wherein the lys-plasminogen is present in an amount of 5 20-100 mg, and wherein the t-PA is present in an amount of 20-100 mg.

12. A method of preventing reocclusion of coronary arteries following treatment of coronary thrombosis, the method comprising administering, to a patient in need of such treatment, an effective amount of lys-plasminogen and, simultaneously or sequentially, an effective amount of tissue plaminogen activator (t-PA).

10 13. A method according to claim 12, wherein the effective amount of lys-plasminogen is in the range of 0.1-3.0 mg/kg body weight, preferably 0.3-1.5 mg/kg body weight, more preferably about 1 mg/kg body weight.

14. A method according to claim 12 or 13, wherein the effective amount of t-PA is in the range of 0.2-2.0 mg/kg body weight, preferably 0.3-1.5 mg/kg body weight, more

15 preferably about 1 mg kg body weight.

15. A method according to claim 12, wherein the administration of lys-plasminogen is followed, after a suitable interval, by administration of t-PA.

16. A method according to claim 15, wherein said suitable interval is in the range of 0- 120 minutes, preferably about 5 minutes.

20 17. Use of lys-plasminogen in combination with t-PA for the preparation of a medicament for the prevention of reocclusion of coronary arteries following treatment of coronary thrombosis.

18. Use according to claim 17, wherein the lys-plasminogen and t-PA are provided in separate containers in a form adapted to the simultaneous or sequential co- administration of lys-plasminogen and t-PA.

19. Use according to claim 18, wherein the lys-plasminogen is present in an amount of 20-100 mg, and wherein the t-PA is present in an amount of 20-100 mg.