LU87007A1 - UROKINASE-BASED PHARMACEUTICAL COMPOSITIONS - Google Patents

Description

'' ~ r y * = 7 ~, Λ, "J GRAND-DUCHÉ DE LUXEMBOURG

• Patent N · Ö, U iU ^ Mister Minister of ......... c λ ......... h. Economy and the Middle Classes _ ,, ÎbfrJSÊ. Intellectual Property Service

Ί TOredeh, rt ........................-.............. I gg) LUXEMBOURG

f '** Patent Application "(_____________ (1) I. Request

The so-called company: SANDOZ SA, Lichtstrasse 35, CH-4002 Basel, Switzerland, ^ represented by Mr Louis ÉMRÎNGER, lawyer, residing in Luxembourg, acting in suit of MayTdatli ^ ~ ......... .................................................. ....---------------------------------------------- ------- (3) depositor) this ....... first October 1900-four-vinqt-sept ____ (4) at IJLûo- heures, at the Ministry of Economy and Middle Classes, in Luxembourg : 1. the present request for obtaining a patent for an invention concerning: .......... Pharmaceutical compositions ..... ... based on urokinase ..__ (5) 2. the description in English ___________ ^ ran9aise__________________ of the invention in triplicate; 3 ............................... ........ / .. / ---------------- drawing plates, in triplicate; 4. the receipt of fees paid to the Registration Office at Luxembourg, L-JJÖL-JL9 87________; 5. the delegation of power, dated ^ a ^ e ___________________________________ on „23 „^ .E.p. t.a [abj: .Ê..JL-98.7; 6. the successor document (authorization); claims (s) for the above patent application the priority of one (s) request (s) of (7) ........................ .................................................. ....... filed in (8) ___________ on (9) 3_ ° ctobre 1986 under n °, 8623823, on December 2, 1986 under n °. 8628741, 2§88§qg3NE {? 9) ..1§ 3Q March 1987 under no. 8707551 and le 3 jnil lof 1QR7 ςηπς] ρ n ° p. 8715678 in the name of the applicant _____________________: ..............

elect (elect) domicile for him / her and, if designated, for his / her representative, in Luxembourg 1.4.L., .. r..U..e _Al ber t .. Unden, 2652 ..... Luxembourg ________ m __________________ ( 12) request) the issue of a patent for the subject described and represented in the abovementioned appendices, with postponement of this issue to _____________________________________months. (13)

The depositor / agent: ----------------------------- (“> Π. Minutes of Deposit

The above patent application has been filed with the Ministry of the Economy and the Middle Classes, Property Service InteUpt ^ Hè- irihj | & nbourg, dated: //. footasikNN .i. · / * \ Pr. the Minister of Economy and the Middle Classes, at „AsLSfl! ____ hours I ξ / '; jpl Jpd.

i'S I 'The head of the intellectual property department, fU K A6atW7_ A A_ A J EXPLANATIONS RELATING TO THE DEPOSIT FORM. ΈΓ "Ί / f \ J (1) if applicable" Request for certificate of addition to the main patent, at the patent application prindpaTNo ............ from .... .......- (2) enter the nenn, first name, profession, * ^ 'address of the applicant, when the latter is an individual or company name, legal form, address of head office, when the applicant is a legal person - (3) register Λ CLAIMING THE PRIORITY of the patent application In Great Britain From October 3, 1986 and from March 30, 1987 DESCRIPTIVE MEMORY filed in support of a request for

PATENT

at

LUXEMBOURG

Case 118-6968 in the name of: SANDOZ S.A.

for: Pharmaceutical compositions based on urokinase.

The present invention relates to the therapeutic lysis of fibrin clots in human patients.

It is known that enzymes of natural origin participate in the lysis of fibrin clots and have been used in therapeutic therapy for the lysis of clots in patients, for example patients suffering from coronary affections, in which they have formed. life-threatening clots. Two of these enzymes are pro-urokinase and urokinase. The latter appears to be synthesized in vivo as a single chain pro-enzyme (pro-urokinase) which is transformed into a double chain form by proteolytic cleavage (urokinase). Pro-urokinase is also called urine plasminogen activator, single chain (scu-PA).

The two enzymes, urokinase and pro-urokinase, can be used therapeutically as thrombolytic agents during fresh myocardial infarction to limit or even prevent total myocardial necrosis. For this purpose, urokinase is used at doses greater than 1,000,000 IU, the intravenous doses preferably being between 2,000,000 and 3,000,000 IU per application. The use of pro-urokinase for this indication is still under study, and results to date have shown that doses greater than 6,500,000 IU are necessary to obtain good results.

It was originally expected that the effects of pro-urokinase and urokinase would, at best, be strictly additional. However, the Applicant has now surprisingly found that urokinase exerts a synergistic effect on the therapeutic activity of pro-urokinase. In particular, it has been found that after a rapid initial injection of urokinase, the subsequent administration of pro-urokinase is much more effective, which makes it possible to considerably reduce the total dose required in order to obtain a beneficial therapeutic effect. .

The Applicant has also found that this synergistic effect is not limited to the case where the administration of urokinase 5 is followed by the administration of pro-urokinase, but also occurs when the pro-urokinase is administered first. , then urokinase, or when pro-urokinase and urokinase are administered simultaneously by separate infusions or injections, by single injection with a double-compartment syringe, or by single injection or infusion of a mixture of pro- urokinase and urokinase.

A subject of the invention is therefore the use of urokinase together with pro-urokinase, in the form of a free or fixed association, as a thrombolytic agent. The invention also relates to the use of urokinase for the preparation of an agent intended to potentiate the thrombolytic effect of pro-urokinase.

By "synergistic effect" is meant a lysing activity of a fibrin clot, greater (either in terms of increased lysis rate or greater lysis, or both) than that resulting from the simple addition of the effects of each substance administered separately.

Urokinase can be used in a low molecular weight form (molecular weight / y 33,000 daltons) or in a high molecular weight form (molecular weight / y 55,000 daltons), the high molecular weight form being preferred. Urokinase can be obtained from natural sources (eg urine) or by genetic recombination techniques including cleavage of the originally formed single chain form (pro-urokinase), as described by W.E. Holmes et al. in Bio / Technology 3_ 923 30 (1985). The high molecular weight urokinase is available commercially under the brand name Ukidan or Actosolv, and the low molecular weight urokinase product under the brand name Abbokinase.

3

Pro-urokinase can also be obtained from urine as described in European Patent No. 40238, or from cultures of natural cell lines or cell lines transformed by genetic recombination techniques, in varying forms in the degree of glucosylation or in other aspects.

The pro-urokinase used according to the present invention will normally be a pro-urokinase of human origin having the no-acid friend sequence as described for the pro-urokinase in FIG. A of European patent application No. 92182 , without taking into account the sequence -20 to -1. However, the pro-urokinase used according to the present invention may differ from this structure by substitution, deletion or addition of one or more amino acid residues, provided that it retains essentially the same biological activity. Thus, for example, a pro-urokinase of non-human origin or a compound as described in international PCT application WO 86/04351 having another amino acid in place of Lys-135 and / or Phe-157, or as described in European patent application No. 200 451 having Phe-157 or Lys-158 deleted .20 or substituted by another amino acid, fall under the definition of "pro-urokinase" provided that these compounds retain biological activity. In addition, truncated forms of pro-urokinase, such as those described for example by Rijken et al. in Thrombosis Research / tee p.749-760 and 761-768 are included in the definition of "pro-urokinase" provided that they retain their biological activity. Such molecules may have their amino terminal at Lys-136, corresponding to the low-chain single molecular weight urokinase, or at any other suitable site, for example Ala-132, Lys-144 and Glu-150.

Other forms of pro-urokinase having additional amino acids, for example an initial methionine, are also included, provided they are active.

4 The urokinase used according to the present invention may also differ correspondingly from one of the two normal molecules of human urokinase (high molecular weight urokinase and low molecular weight urokinase), provided that it retains its activity organic.

The invention is particularly suitable for patients who have had a recent operation, those who have recently had a myocardial infarction caused by clots and those suffering from deep vein thrombosis. Pro-urokinase and urokinase, separately or in combination, are formulated with a pharmaceutically acceptable carrier, for example physiological saline, and administered parenterally either intravenously or by. injection into the affected arteries of the heart. Intravenous administration is preferred, and can be by infusion, rapid injection, or a combination of the two.

Preferably the patient is administered heparin, for example a rapid injection of 5000 IU of heparin before the administration of pro-urokinase or urokinase, optionally followed by an infusion of 1000 IU / hour heparin. Heparin can also be administered as a mixture with pro-urokinase or urokinase, or as a mixture of these three compounds.

The preferred amount of urokinase administered according to the invention is from 100,000 IU to 300,000 IU, more preferably from 150,000 IU to 250,000 IU, in particular 200,000 IU The preferred amount of pro-urokinase administered is less than 6,500,000 IU, more preferably between 2,000,000 and 4,000,000 IU

5

The amount of urokinase mentioned above is most effective when administered by rapid injection 15 minutes or less before the administration of pro-urokinase. However, to facilitate administration, it may be preferable to administer a 5 mixture of pro-urokinase and urokinase in a single injection or infusion, preferably in a single rapid injection. According to another aspect, the invention therefore also relates to a pharmaceutical composition comprising a synergistic mixture of urokinase and pro-urokinase in combination with a pharmaceutically acceptable diluent or vehicle, or a synergistic mixture of urokinase and pro urokinase in pure lyophilized form.

The components of the synergistic mixture of the invention can also be presented in the form of a combined packaging, that is to say in a single packaging containing separate unit doses of urokinase and pro-urokinase as well as the instructions. for concomitant administration.

The composition is preferably essentially pure, i.e. it does not contain a significant amount of any pharmaceutically active substance other than urokinase and pro-urokinase. The composition is preferably analytically pure, that is to say that it contains no protein other than urokinase and pro-urokinase, in an amount detectable by analytical methods available on the date of the present application. When a diluent or vehicle is present, it is preferably sterile water for injection or sterile isotonic physiological solution.

The weight ratio of urokinase to pro-urokinase in the composition according to the invention is preferably between 1:40 and 1: 6.7, more preferably between 1:20 and 1:10, in particular between 1 : 15 and 1:10. The composition is preferably presented in the form of unit doses, for example vials for injection or bottles for infusion, each containing from 100,000 to 300,000 IU of urokinase and from 2,000,000 to 4,000,000 IU of pro-urokinase , more preferably 200,000 IU of urokinase and between 2,000,000 and 3,000,000 IU of pro-urokinase.

6

Such a unit dose contains a combined total dose of urokinase and pro-urokinas of between 2,100,000 and 4,300,000 IU, corresponding to approximately 21-43 mg of protein. The amounts of each compound are thus lower than the amount of each of them used for optimal lysis of the clot when administered in the absence of the other for the same therapeutic indication.

According to another aspect of the invention, the patient receives a rapid injection of urokinase and a rapid injection of pro-urokinase followed by an infusion of pro-urokinase. The amount of urokinase administered by rapid injection is as indicated above, that is to say preferably between 100,000 and 300,000 IU, more preferably between 150,000 and 250,000 IU, particularly 200,000 IU The amount of pro-urokinase 15 administered by rapid injection is preferably between 300,000 and 1,000,000 IU, more preferably between 400,000 and 600,000 IU, in particular 500,000 IU These amounts can be administered in two separate rapid injections , delivered in any order, or using a double-compartment syringe, or in a single rapid injection of a mixture of urokinase and pro-urokinase ^ which represents another composition of l 'invention.

The weight ratio of urokinase to pro-urokinase in this composition is preferably between 1:10 and 1: 1, more preferably between 1: 4 and 1: 1.6, in particular 1: 2.5 . However, urokinase is preferably given before pro-urokinase.

It may also be desirable to administer heparin concurrently with the rapid injection of urokinase and / or pro-urokinase. The amount of heparin to be added to the amounts indicated above of urokinase, pro-urokinase or mixture of urokinase and pro-urokinase, is preferably between 1000 and 10,000 IU, more preferably around 5,000 IU.

7

The quantity of pro-urokinase administered subsequently by infusion is preferably approximately 100,000 IU / minute, which can be administered for approximately 40 minutes, for a total of 4,000,000 IU. In many cases, the artery is cleared. Obstructed blood pressure can occur after 30 minutes or before, and the infusion can be stopped after this time if artery clearance is noted, for example by coronary angiography.

For use according to this aspect, the invention relates to a kit comprising a unit dose of urokinase for injection, a unit dose of pro-urokinase for injection, and a unit dose of pro-urokinase for infusion. The kit is preferably presented in a single package, containing the instructions for administration. The unit doses for injection may be in the form of sterile urokinase or pro-urokinase solutions in pure water or in physiological saline solution, or in a freeze-dried or freeze-dried solid form which is may add sterile water or physiological saline solution before injection. The unit dose of pro-urokinase for infusion may be a solution of pro-urokinase in physiological saline or other sterile aqueous medium for infusion, or a freeze-dried or freeze-dried solid or a liquid concentrate from which prepare a solution for infusion. The solution for injection or infusion may contain other components, for example buffer salts such as Na2HPO4 / NaH2PO4 and preservatives, for example mannitol and human albumin, these compounds may also be in a lyophilized solid form or freeze dried.

The amount of urokinase and pro-urokinase in the unit doses for injection is preferably as described above, in particular 200,000 IU of urokinase and 500,000 IU of pro-urokinase; the amount of pro-urokinase in the unit dose for infusion is preferably between 30,000 and 5,000,000 I.U., more preferably 4,000,000 I.U.

8 The combination of low-dose urokinase (200,000 IU) and low-dose pro-urokinase (3,000,000 IU) allows effective thrombo-lysis in most cases of myocardial infarction. To achieve the same success rate using urokinase alone, 5 doses between 2,000,000 and 2,500,000 IU would be required, and at these doses, side effects such as systemic fibrinogenolysis and loss of plasminogen appear. . To achieve the same results with pro-urokinase administered alone, doses well above 6,500,000 IU, for example 10.9 million IU, may be necessary. These results indicate a synergistic interaction between urokinase and pro-urokinase in thrombolysis.

The amount of each compound is expressed below either by weight (mg) or in IU according to "The Intenational Reference 15 Preparation for Urokinase" of the National Institute for Biological Standards and Control, London, determined on a standardized fibrin plate (Brakman, Fibrinolysis , A Standardized Fibrin Plate Method and a Fibrinolytic Assay of Plasminogen, Scheltema and Holkema NV, Amsterdam, 1967, p 1-24). Pro-urokinase is tested after activation by plasmin as described by Gurewich et al., In J. Clin. Invest. (1984) 21 ”P 1731-1739. Urokinase (both high molecular weight and low molecular weight forms) and pro-urokinase showed similar specific activities (approximately 100,000 IU / mg), which compared the concentrations of 25 protein (1 IU = 10 ng protein).

Whereas urokinase is available in ampoules containing 250,000 IU, a dose of 250,000 IU of urokinase can replace 200,000 IU in the description and examples. The use of a dose of 250,000 IU avoids the difficulty of evaluating 4/5 of an ampoule, and gives the same beneficial effects as the dose of 200,000 IU.

Example 1

A 58-year-old woman with a myocardial infarction received a rapid injection of 200,000 IU of urokinase intravenously, followed by a rapid injection of 1,000,000 IU of pro-urokinase and an infusion of pro-urokinase rate of 5.5 million IU / hour. The partial perfusion was reached after 15 minutes and the full perfusion after 30 minutes, that is to say after administration of a total of 3,250,000 IU of pro-urokinase. 24 hours after administration, the blood circulation was still restored.

Example 2

A man with myocardial infarction received a mixture of 200,000 IU of urokinase and 2,500,000 IU of pro-urokinase by infusion for 15 minutes. Blood circulation was restored at the end of this period.

Example 3 Formulations, a) Mixture for injection 200,000 IU of urokinase and 2,000,000 IU of pro-urokinase are dissolved in 20 ml of sterile isotonic physiological solution and packaged in a vial for injection.

b) Freeze-dried mixture

200,000 IU of urokinase and 3,000,000 IU of pro-urokinase are lyophilized and the solid residue is sealed in a vial to which physiological saline solution for injection can be added.

c) Double compartment syringe

One compartment of the prepackaged syringe contains 200,000 IU of urokinase in 10 ml of sterile isotonic physiological solution, and the other contains 3,000,000 IU of pro-urokinase in 10 ml of sterile isotonic physiological solution.

d) Twin packaging

A paired package includes a vial containing 200,000 IU of urokinase in lyophilized form and a vial containing 3,000,000 IU of pro-urokinase in lyophilized form, with instructions for co-administration.

Example 4

Formulations are also prepared as described in Example 3 using 500,000 IU of pro-urokinase instead of 2,000,000 or 3,000,000 IU of pro-urokinase.

10

Example 5

In a prospective, multi-center, open, non-comparative clinical trial, the following protocol was used:

Participation criteria 5 1) pain lasting at least 30 minutes, characteristic of myocardial infarction, 2) onset of symptoms less than 5 hours before the start of lytic therapy 3) characteristic signs on ECG myocardial infarction 4) informed consent of the patient.

Exclusion criteria 1) coronary angiography contraindications 2) lytic therapy contraindications 15 a) initiation of heparin or coumarin therapy b) venipuncture or arterial puncture in the last 8 days c) stroke in both last month 20 d) active bleeding e) gastric or duodenal ulcer in the past year f) surgery in the last 8 days g) hemorrhagic diathesis 25 3) complete obstruction of a coronary artery (as indicated by angiography coronary) 4) partial or complete restoration of blood supply after administration of nitroglycerin 5) cardiac coma 30 6) severe valvular or cardiac diseases, hypertrophic cardio myopathy 7) serious renal or hepatic diseases, which can disturb the excretion of pro-urokinase .

8) use of oral contraceptives.

• 11 Method

5000 IU of heparin is administered to patients meeting the above criteria, and coronary angiography is performed. If total arterial obstruction is observed, between 100 and 200 µg of nitroglycerin is administered intracoronally, and angiography of the obstructed vessel is performed.

If no clearance is observed after administration of nitroglycerin, the patient is administered 200,000 IU of high molecular weight urokinase by rapid intravenous injection, followed by 500,000 IU of pro-urokinase by intravenous injection by rapid injection within 3 minutes. Immediately after an intravenous infusion of 4,000,000 IU of pro-urokinase is performed within 40 minutes. A coronary angiography is carried out every 15 minutes to check the evolution of lysis of the 15 clot, and the same is done 60 minutes after the success of the lysis, and also 24 hours and 3 weeks after the treatment, in order to rule out any danger of further obstruction.

Results

Out of 19 patients treated according to this protocol, 20 were observed in 14 (74¾) of them clearing or clearing the obstructed artery after 40xminutes of infusion. Among these patients, two had a new obstruction after 24 hours.

Claims (20)

12 1. The use of urokinase in combination with pro-urokinase, in the form of a free or fixed association, as a thrombolytic agent. 5 2. The use according to claim 1, characterized in that the dose of urokinase is between 100,000 and 300,000 IU and the dose of pro-urokinase is less than 6,500,000 IU. 3. The use according to claim 2, characterized in that the dose of urokinase is between 150,000 and 250,000 4. Use according to any one of the preceding claims, characterized in that the urokinase is administered by rapid injection before the injection or infusion of pro-urokinase. 5. The use of urokinase for the preparation of an agent intended to potentiate the thrombolytic effect of pro-urokinase. 6. A twin pack, characterized in that it contains separate unit doses of urokinase and pro-urokinase as well as the instructions for co-administration. 7. A twin package according to claim 5, characterized in that the unit dose of urokinase is between 100,000 and 300,000 IU and the unit dose of pro-urokinase is less than 6,500,000 IU. 8. A twin package according to claim 7, characterized in that the unit dose of urokinase is between 150,000 and 250,000 IU and the unit dose of pro-urokinase is between 2,000,000 and 4,000,000 IU. 9. A kit comprising a unit dose of urokinase for injection, a unit dose of pro-urokinase for injection and a unit dose of pro-urokinase for infusion. 13 10. A kit according to claim 9, characterized in that the unit dose of urokinase for injection is between 150,000 and 250,000 IU, the unit dose of pro-urokinase for injection is between 400,000 and 600,000 IU and the unit dose 5 of pro-urokinase for infusion is between 3,000,000 and 5,000,000 IU 10 IU and the dose of pro-urokinase is between 2,000,000 and 4,000,000 IU. 11. A twin pack or a kit according to any one of claims 6 to 10, characterized in that at least one of the unit doses of urokinase or pro-urokinase also contains 10 from 1,000 to 10,000 IU heparin. 12. A pharmaceutical composition, characterized in that it comprises a synergistic mixture of urokinase and pro-urokinase in combination with a pharmaceutically acceptable diluent or vehicle, or a synergistic mixture of urokinase and pro-urokinase in lyophilized form pure. 13. A pharmaceutical composition according to claim 12, characterized in that it does not contain a significant amount of any pharmaceutically active substance other than urokinase and pro-urokinase. 14. A pharmaceutical composition according to claim 13, characterized in that it contains no protein other than \ urokinase and pro-urokinase in an amount detectable according to analytical methods. 15. A pharmaceutical composition according to any one of claims 12 to 14, characterized in that the weight ratio of urokinase to pro-urokinase is between 1:40 and 1: 6.7. 16. A pharmaceutical composition according to claim 15, characterized in that the weight ratio of urokinase to pro-urokinase is between 1:15 and 1:10. 17. A unit dose of the pharmaceutical composition according to claim 15, characterized in that it contains from 100,000 to 300,000 IU of urokinase and from 2,000,000 to 4,000,000 IU of pro-urokinase. Λ 14 18. A pharmaceutical composition according to any one of claims 12 to 14, characterized in that the weight ratio of urokinase to pro-urokinase is between 1: 4 and 1: 1.6. 19. A unit dose of the pharmaceutical composition according to claim 18, characterized in that it contains from 150,000 to 250,000 IU of urokinase and from 400,000 to 600,000 IU of pro-urokinase. 20. A unit dose according to claim 19, 10 characterized in that it also contains 1000 to 10 000 IU of heparin. \