NO305584B1 - Process for preparing a pharmaceutical surface preparation of a non-glycosylated t-PA derivative K1K2P pro - Google Patents

Abstract

The invention relates to a pharmaceutical preparation of a non-glycosylized t-PA derivative, K1K2P pro, with an enzymatic activity of at least 0.4 MU/ml and a pH of 4.5 to 6, containing citrate and at least one compound from the following group: a) ascorbic acid, b) EDTA, c) amino compounds of formula R?1R?2N -R - X, where X = SO3?H, CH(NH2?)-CO2?H, CO2?H, H, NH2? or OH, R = C1?-C9? alkylene, C3?-C6? cycloalkylene or benzylidene and R?1 and R?2, independently of each other, are H or C1?-C3? alkyl, d) guianidinobutyric acid, e) dimethylbiguanide, f) arginine, g) glucosamine, fructose, h) pyrimidine nucleosides and pyrimidine nucleotides, i) carboxylic acids substituted with one or more hydroxyl, keto and/or further carboxyl groups. The invention also relates to a drug containing the t-PA derivative K1K2P pro as the active ingredient and a process for producing it.

Description

The present invention relates to a method for producing a pharmaceutical preparation of a non-glycosylated t-PA derivative K1K2P pro.

Human tissue plasminogen activator (t-PA) is of great therapeutic importance in dissolving blood clots, e.g. in case of heart attack. t-PA causes the dissolution of blood clots through the activation of plasminogen to plasmin. Plasmin further dissolves fibrin, which is the main component of the protein matrix of coagulated blood.

Natural t-PA is composed of several functional domains F, E, K1, K2 and P. The domain P has the protolytically active center which causes the cleavage of plasminogen into plasmin. The genetic engineering production of t-PA or different t-PA muteins, in which one or more of the domains F, E, K1 and K2 are deleted, is already known in eukaryotic and prokaryotic cells. Here, t-PA derivatives from prokaryotes are synthesized in relation to natural t-PA in non-glycosylated form.

It is also known that the proportion of sugar has a major impact on the solubility and aggregation of proteins (J. Biol. Chem. 263 (1988), 8832-8837). It was now found that non-glycosylated t-PA derivative K1K2P pro is significantly less soluble than glycosylated t-PA.

K1K2P pro does not dissolve in the buffers usually used for dissolving proteins, such as 50 mmol/1 Na citrate, 50 mmol/1 phosphate or the physiological Na-Cl solution. For use as a therapeutic agent, however, K1K2P pro should be present with a higher enzymatic activity of at least 0.4 MU/ml, preferably 0.4 MU/ml to 10 MU/ml. The unit U is thus defined according to the WHO, National Institute for Biological Standards and Control (vgl. H. Lill, ZGIMAL 42

(1987), 478-486).

From EP-A 0.217.379 it is known to increase the solubility of t-PA from prokaryotic organisms (t-PA pro) through neutral or slightly alkaline arginine formulations. A disadvantage of this method, however, is that good solubility of t-PA pro is only achieved with very high arginine concentrations.

The purpose of the invention was thus to develop pharmaceutical preparations containing K1K2P pro with an activity greater than 0.4 MU/ml, where the t-PA derivative must be stable over a longer period of time.

The task that formed the basis of the invention was solved through a method for the production of a pharmaceutical preparation of a non-glycosylated t-PA derivative K1K2P pro, characterized by dissolving the non-glycosylated t-PA derivative KK2P pro in a pharmaceutically acceptable buffer containing citrate and at least one compound from the group consisting of

a) ascorbic acid,

b) EDTA,

c) amino compounds of formula

there

X SO 3 H, CH(NH 2 )-CO 2 H, CO 2 H, H, NH 2 or OH,

R = C₁-C₆-alkylene, C₆-C₆-cycloalkylene or benzylidene and

r! and R<2> is independently H or C 1 -C 2 -alkyl,

d) guanidinobutyric acid,

e) dimethylbiguanide,

f) arginine,

g) glucosamine, fructose,

h) pyrimidine nucleosides and pyrimidine nucleotides,

i) with one or more hydroxy, keto or/and further

carboxy group-substituted carboxylic acids and which exhibit a pH value of 4.5 to 6.5, until it comprises an enzymatic activity of at least 0.4 MU/ml, as well as common pharmaceutical additives, auxiliary or/and carrier substances

and transferring it to a suitable pharmaceutical administration form such as an injection solution or a lyophilisate; and which optionally contains one or more hydroxy-, keto- or/and further carboxy group substituted carboxylic acid, malic acid, lactic acid, fumaric acid or 2-oxoglutaric acid, or in addition optionally contains one or more amino acids and chloride ions.

Under K1K2P pro in connection with the present invention is understood a non-glycosylated t-PA derivative starting with amino acids 85-92 and ending at 527 (Pro). K1K2P pro can also contain all or part of the amino acids -3(Gly) to +5(Ile). Thus, a protein is preferred which begins at amino acid 87 (Asp) and which optionally further contains from the range -3 to +5 the amino acids Ser, Tyr, Gin, Val and ILe (nomenclature according to Harris, Protein Engineering, Band 1 (1987) 449- 458). A t-PA derivative K1K2P pro can be obtained according to the method described in DE-39,233,391.

A citrate buffer is particularly suitable for stabilizing K1K2P pro. The concentration of the citrate ions must be at least 5 mmol/1, preferably 5 to 100 mmol/1, particularly preferred is a concentration of citrate ions of 50 mmol/1. Depending on the basicity of the compound used, the pH value is preferably set with HC1 or a base such as NaOH or KOH.

It was surprisingly established that the solubility of non-glycosylated K1K2P pro is significantly reduced in other buffer systems, e.g. phosphate buffer, at equal ionic strength and equal pH value. It has proven to be appropriate to adjust the pH value of alkaline citrate solutions with HC1, i.e. that the composition also contains chloride ions. In the presence of chloride ions, highly concentrated solutions of K1K2P pro are surprisingly significantly more stable than e.g. in the presence of phosphate ions. Acidic nitrate solutions are usually adjusted with NaOH to the current pH value.

Suitable for a composition according to the invention is a pH value between 4.5 and 6.5, and a pH value of 6 is preferred.

Preferably, taurine, 4-aminobutanol-1,

5-aminopentanol-1, 6-aminohexanol-1, 1,9-diaminononane, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-aminobutane, 1, 3-aminopropane,

lysine, ornithine, 8-aminooctanoic acid, 7-aminoheptanoic acid, E-aminocaproic acid, S-aminovaleric acid, "Y-aminobutyric acid, tranexamic acid or p-aminomethylbenzoic acid. Preferred concentration of taurine and analogous compounds is 0.1 to 0.5 mol/l, particularly preferred is 0.1 to 0.3 mol/l.

4-aminobutanol-1,

5-aminopentanol-1, 6-aminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminobutane or

1,3-diaminopropane is preferably used in 10 to 100 mmol/l. Lysine, ornithine, 8-aminooctanoic acid, 7-aminoheptanoic acid, E-aminocaproic acid, S-aminovaleric acid,

■Y-aminobutyric acid, tranexamic acid or p-aminomethylbenzoic acid are preferably used in 0.5 to 20 mmol/1, particularly preferred in 1 to 10 mmol/1.

As carboxylic acid, which is substituted with one or more hydroxy, keto or/and further carboxyl groups, e.g. malic acid, lactic acid, fumaric acid or oxoglutaric acid. These compounds are preferably used in 1 mmol/1 to 1000 mmol/1, particularly preferred is in 10 to 500 mmol/1.

Guanidinobutyric acid or arginine is preferably used in 10 to 200 mmol/1, particularly preferably with 50 to 100 mmol/1. For dimethylbiguanide, the concentration is 50 to 400 mmol/1, preferably 100 to 300 mmol/1.

EDTA is preferably used with 1 to 200 mmol/1, particularly preferably with 10 to 100 mmol/1. Ascorbic acid is preferably used in 0.1 to 1 mol/l, particularly preferred in 0.2 to 0.3 mol/l.

Glucosamine and fructose are preferably used in concentrations of 1 to 500 mmol/1, particularly preferred with 10 to 300 mmol/1.

As pyrimidine nucleosides and pyrimidine nucleotides are e.g. thymidine, cytosine or uridine respectively. corresponding nucleotides suitable. They are preferably used in concentrations of 1 to 300 mmol/1, particularly preferably 10 to 300 mmol/1.

An object according to the invention is furthermore a composition according to the invention which additionally contains one or more amino acids, especially histidine.

A number of particularly preferred preparations according to the present invention are listed below.

One formulation contains 50 mmol/l Na citrate, pH 6 and 0.1 to 0.3 mol/l taurine. A formulation with 50 mmol/l Na citrate, pH 6 and 0.2 to 0.3 mol/l ascorbic acid is also preferred.

Further preferred is a formulation with 50 mmol/l Na-citrate/HCl, pH 6 and 1 mmol/1 to 10 mmol/1 7-aminoheptanoic acid, 8-aminooctanoic acid, p-aminomethylbenzoic acid, E-aminocaproic acid, ω-aminovacleric acid, -y- aminobutyric acid, transexamic acid, lysine or ornithine.

Furthermore, formulations containing 50 mmol/1 Na citrate/HCl, pH 6.0 and 50 to 100 mmol/1 guanidinobutyric acid or arginine are also particularly preferred.

A formulation containing 50 mmol/1 Na citrate, pH 6 and 10 to 100 mmol/1 EDTA is also preferred. Furthermore, a further formulation contains 50 mmol/l Na citrate/HCl, pH 6 and 100 to 300 mmol/l dimethylbiguanide.

A further formulation contains 50 mmol/l Na citrate/HCl, pH 6 and 10 to 300 mmol/l thymidine, cytosine or uridine. A further formulation contains 50 mmol/1 Na citrate/- HCl, pH 6 and 10 to 100 mmol/1

4-aminobutanol-1, 5-aminopentanol-1, 6-aminohexanol-1, 1,9-diaminononane, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1, 4-diaminobutane or 1,3-diaminopropane. A further formulation contains 50 mmol/l Na citrate/HCl, pH 6 and 10 to 300 mmol/l fructose or glucosamine.

Finally, a further formulation contains 50 mmol/1 Na citrate, pH 6 and 10 to 500 mmol/1 malic acid, lactic acid, fumaric acid or 2-oxoglutaric acid.

Also combinations of several of the above-mentioned compounds with citrate cause a very good solubility of the proteins with t-PA activity.

The pharmaceutical preparations produced according to the invention are preferably used as injection and infusion solutions. This can be achieved by providing a ready-to-spray solution that contains the composition. However, it is also possible to make available the pharmaceutical preparations in the form of lyophilisates. These are then reconstituted with agents or solutions known per se, suitable for injection purposes. As an injection medium, water is preferably used which contains the usual additives for injection solutions such as stabilizer, dissolution agent, buffer and isotonic additives, for example a physiological NaCl concentration. These additives are, for example, mannitol, tartrate or citrate buffer, ethanol, complex formers such as e.g. ethylenediaminetetraacetic acid and non-toxic salts thereof, as well as high molecular weight polymers, such as liquid polyethylene oxide for viscosity regulation. Liquid carriers for injection solutions must be sterile and are preferably filled in ampoules.

The following examples explain concrete embodiments according to the invention.

EXAMPLE 1

Resolution of K1K2P pro

Purified K1K2P pro (dissolved in 0.5 mol/l arginine/H3PO4, pH 7.2, is concentrated by ultrafiltration over a YM 10 membrane (Amicon). 0.5 ml of the concentrate (activity: 3.5 MU/ml ) is dialyzed against the buffer listed in Table 1. After centrifugation of the probe, the enzymatic activity is measured in the clear supernatant.

The enzymatic activity is indicated as a volume unit in MU/ml and as total activity in MU.

Measurement of tPA activity can thus be determined in the usual way through cleavage of a chromogenic substrate, (H. Lill, ZGIMAL 42 (1987), 478-486). Unit I is an activity unit according to the definition of the WHO, National Institute for Biological Standards and Control.

Claims (14)

1. Process for the production of a pharmaceutical preparation of a non-glycosylated t-PA derivative K1K2P pro, characterized by dissolving non-glycosylated t-PA derivative KK2P pro in a pharmaceutically acceptable buffer containing citrate and at least one compound from the group consisting of a) ascorbic acid, b) EDTA, c) amino compounds of formula there X = SO 3 H, CH(NH 2 )-CO 2 H, CO 2 H, H, NH 2 or OH, R = C₁-C₆-alkylene, C₃-C₆-cycloalkylene or benzylidene and R<1> and R<2> are independently H or C₁-C₃-alkyl, d) guanidinobutyric acid, e) dimethylbiguanide, f ) arginine, g) glucosamine, fructose, h) pyrimidine nucleosides and pyrimidine nucleotides, i) with one or more hydroxy-, keto- or/and additional carboxy group-substituted carboxylic acids and which exhibit a pH value of 4.5 to 6.5, until it comprises an enzymatic activity of at least 0.4 MU/ml, as well as common pharmaceutical additives, auxiliary or/and carrier substances and transfers it to a suitable pharmaceutical administration form such as an injection solution or a lyophilisate; and which optionally contains one or more hydroxy-, keto- or/and further carboxy group substituted carboxylic acid, malic acid, lactic acid, fumaric acid or 2-oxoglutaric acid, or in addition optionally contains one or more amino acids and chloride ions. 2. Method for producing a pharmaceutical preparation according to claim 1, characterized in that the amino compound is taurine, 4-aminobutanol-1, 5-aminopentanol-1, 6-aminohexanol-1, 1,9-diaminononane, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminobutane, 1,3-diaminopropane, lysine, ornithine, 8-aminooctanoic acid, 7-aminoheptanoic acid, E-aminocaproic acid, S-aminovaleric acid, -Y-aminobutyric acid, tranexamic acid or p-aminomethylbenzoic acid. 3. Process for producing a pharmaceutical preparation according to one of claims 1-2, characterized in that the citrate concentration amounts to 5 to 100 mmol/1, preferably 50 mmol/1. 4. Method for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate, pH 6 and 0.1 to 1 mol/l, preferably 0.2 to 0.3 mol/l ascorbic acid. 5. Process for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate, pH 6 and 1 to 200 mmol/1, preferably 10 to 100 mmol/1 EDTA. 6. Process for producing a preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate, pH 6, and 0.1 to 0.5 mol/l, preferably 0.1 to 0.3 mol/l taurine. 7. Process for the production of a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate/HCl, pH 6 and 0.5 to 20 mmol/1 lysine, ornithine, 8-aminooctanoic acid, 7-aminoheptanoic acid, E-aminocaproic acid, S-aminovaleric acid, 7-aminobutyric acid, tranexamic acid or p-aminomethylbenzoic acid. 8. Process for the production of a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate/HCl, pH 6, and 10 to 100 mmol/1 4-aminobutanol-1, 5-aminopentanol-1, 6-aminohexanol -1, 1,3-diaminopropane, 1,4-diaminebutane, 1,5-diaminepentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane or 1,9-diaminononane. 9. Method for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate/HCl, pH 6, and 10 to 200 mmol/1, preferably 50 to 100 mmol/1 guanidinobutyric acid or arginine. 10. Process for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate/HCl and 50 to 400 mmol/1, preferably 100 to 300 mmol/1 dimethylbiguanide. 11. Method for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate/HCl, pH 6, and 1 to 500 mmol/1, preferably 10 to 300 mmol/1 glucosamine or fructose. 12. Method for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate/HCl, pH 6, and 1 to 300 mmol/1, preferably 10 to 300 mmol/1 thymidine, cytosine or uridine. 13. Process for the production of a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate, pH 6, and 0.001 to 1 mol/l, preferably 0.01 to 0.5 mol/l malic acid, lactic acid, fumaric acid or 2-oxoglutaric acid. 14. Method for producing a pharmaceutical preparation according to claim 1, characterized in that it contains 50 mmol/1 Na citrate, pH 6, and a combination of compounds from the group a) to i) according to claim 1.