FI95998B - t-PA solubilization - Google Patents

Abstract

The invention relates to a pharmaceutical preparation of a protein with t-PA activity, with an enzymatic activity of at least 0.25 MU/ml and a pH of 4.5 to 9, containing a substance from the group citric acid, ascorbic acid, 2-oxoglutaric acid, fumaric acid, tris and EDTA in a concentration of at least 0.2 mol/l, and to a drug based on a protein with t-PA activity and a process for producing it.

Description

95998 t-PA leaching - t-PA solubilization

The tissue activator of human plasminogen, t-PA, is therapeutically very important in the dissolution of blood clots, for example in the case of cardiac thrombosis. t-PA affects the coagulation cascade by activating plasminogen to plasmin. Plasmin, in turn, dissolves fibrin, the major protein that forms the backbone of clotted blood.

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Natural t-PA consists of several functional regions: F, G, ΚΙ, K2, and P. Region P contains a proteolytically active center that affects the distribution of plasminogen into plasmin. The production of t-PA or various t-PA muteins with one of the regions F, G, K1 and K2 deleted by genetic engineering in eukaryotic or prokaryotic cells is known. In contrast to natural t-PA, t-PA derivatives prepared in prokaryotic cells are in a non-glycosylated form.

Proteins with t-PA activity are poorly soluble in buffers commonly used to dissolve proteins, e.g. 50 mmol / l Na citrate, 50 • 25 mmol / l phosphate or physiological NaCl solution. However, the enzyme activity of the t-PA active protein used as a therapeutic agent should be at least 0.25 MU / ml, e.g. 0.25 to 10 MU / ml.

EP-A-0217379 discloses a method for increasing the solubility of t-PA using neutral or slightly temporal arginine compositions. However, one disadvantage of the process is the poor stability of highly concentrated t-PA solutions under neutral or slightly alkaline conditions.

U.S. Patent No. 2,95,998 discloses a pharmaceutical composition comprising human t-PA and a pharmaceutically acceptable buffer containing arginium ions at a chloride ion content of up to 0.3 mol / L. EP-A-0156169, EP-5 A-0303351 and EP-A-0297294, in turn, discuss the possibility of promoting the solubility of t-PA active proteins in buffers by using amino acids, their salts, derivatives or homologues. t-PA can also be stabilized by the addition of gelatin (EP-A-0123304), the addition of Albu-10 (EP-A-0112940) or by the addition of polysulphuric acid esters or sulphated sugars of an saccharide (EP-A-0198321). PCT / US88 / 04402 discloses a method for increasing the solubility of t-PA using an aqueous medium and a basic amino acid, especially arginine, at a concentration of 0.02 to 0.2 mol / L and a citric acid group at a concentration of 0.02 to 0 .08 mol / l, pH 5-8.

However, these different combinations are not suitable for all proteins that exhibit t-PA activity. It was particularly clear that unglycosylated t-PA, muteins of unglycolyzed t-PA, and glycosylated t-PA all dissolve in very different ways.

; The object of the invention was to provide a pharmaceutical preparation containing glycosylated and non-glycosylated t-PA or t-PA muteins with an activity of more than 0.25 MU / ml and in which the t-PA remains stable for a longer period of time.

This was achieved by preparing a pharmaceutical composition comprising t-PA active protein at a concentration of at least 0.4 MU / ml at pH 4.5 to 9 and a substance selected from the group consisting of citric acid, ascorbic acid, 2-oxoglutaric acid, fumaric acid , Tris and EDTA in a concentration of 0,2 mol / l or more.

<1: IMI mil II ill 3 95998

Preferably the concentration of this substance should be 0.2-1 mol / l, even more preferably 0.3-1 mol / l.

A suitable pH for the compound is 4.5 to 9, particularly preferred is 6 to 7.5.

In the invention, t-PA-active proteins are meant unmodified t-PA from prokaryotic or eukaryotic organisms as well as all t-PA mute-10s. Examples of t-PA muteins are described, for example, by Harris (Protein Engineering 1, 1987, 449-458).

Preferably, the composition of the invention should contain native glycosylated t-PA from e.g. CHO cells. If the preparation according to the invention contains native glycosylated t-PA, its enzymatic activity should preferably be at least 1.4 MU / ml.

Unit U is a defined unit of t-PA activity (WHO, 20 National Institute for Biological Standards and Control).

It is further preferred to use non-glycosylated prokaryotic t-PA (pro-t-PA) which can be obtained according to DE-3537708. Pro-t-PA means t-PA starting from amino acid -3 (Gly) to amino acid +1 (Ser) and ending at 527 (Pro) (Harris, Protein Engineering 1, 1987, 449 -458). If the preparation according to the invention contains pro-t-PA, its enzymatic activity must be at least 0.25 MU / ml.

30

Further preferred is a non-glycosylated t-PA mutein having the regions K1, K2 and P, denoted pro-KlK2P (preparation as in DE-39233391.1). pro-KlK2P starts at amino acids 85-92 and ends at position 527 (Pro). If the preparation of the invention contains pro-KlK2P, its enzymatic activity should be at least 0.4 MU / ml.

95998 4

Also preferred is a non-glycosylated t-PA mutein with regions K2 and P, designated pro-K2P (preparation as in EP-A-0382174). pro-K2P starts at amino acids 174-179 and ends at position 527 (Pro). Both K2P and pro-KlK2P may contain 5 partially or completely amino acids -3 (Gly) to amino acid +5 (Ile) (Harris, supra). If the preparation according to the invention contains pro-K2P, its enzymatic activity must be at least 1.4 MU / ml. However, all other prokaryotic or eukaryotic t-PA muteins are suitable.

10

The following are some particularly preferred compositions of the preparations of the invention.

One of them contains 300 mmol / l citric acid / NaOH, pH 6.

Another contains 300 mmol / l ascorbic acid, pH 6.

One contains 300 mmol / l 2-oxoglutaric acid, pH 6. One contains 300 mmol / l EDTA, pH 6. One contains 300 mmol / l fumaric acid / NaOH, pH 6. One contains 1 mol / l Tris / HCl, pH 7, 2.

20

Another object of the invention is a medicament based on the t-PA active protein as active ingredient in solution or as a lyophilisate with active ingredients, optionally in addition to other suitable pharmaceutically acceptable excipients, auxiliaries, carriers and excipients.

The pharmaceutical preparations according to the invention can be used, for example, in solutions for injection or infusion. This can be accomplished by preparing a ready-to-inject solution containing a composition of the invention. However, it is possible to use the composition according to the invention in the form of lyophilisates. They, in turn, can be reconstructed using known injection media or solutions. Of the injection media, the most preferred is water, which also contains other common additives such as stabilizers, solvent media, buffers and isotonic additives (e.g. physiological NaCl concentrate). Such additives include, for example, mannitol, tartrate or citrate buffer, ethanol, complexing agents such as ethylenediaminetetraacetic acid and their non-toxic salts, e.g. high molecular weight polymers such as liquid polyethylene oxide for viscosity control. Liquid carriers for injection should be sterile and should preferably be enclosed in ampoules.

The present invention further relates to the use of glycosylated t-PA active proteins for the preparation of the pharmaceutical preparations according to the invention.

The following examples illustrate the concrete implementation of the invention.

Example 1

Effect of different substances on the solubility of non-glycosylated t-PA mute-20ins (regional composition K2P)

The example describes the solubility of pro-K2P (preparation as in EP-A-0382174) in various buffers. As can be seen from Table 1, pro-K2P is soluble in the tested: 25 buffer solutions with an activity well above 1.4 MU / ml.

170 ml of purified pro-K2P (dissolved, 0.5 mol / l arginine / phosphoric acid, pH 7.2) was ultrafiltered, using an Amicon YM-10 membrane for concentration. 1 ml of concentrate was dialyzed against each of the buffer waves listed in Table 1. After centrifugation of the test batches, the enzymatic activity of the clear surface was measured.

35 6 95998

Enzymatic activity is expressed as volume activity MU / ml and total activity MU.

t-PA activity can be measured in the usual manner by degrading 5 using a chromogenic substrate (H. Lill, ZGIMAL 42, (1987), 478-486). Unit U is defined as t-PA activity (WHO, National Institute for Biological Standards and Control).

10 TABLE 1

Buffer Agent Activity

MU / ml MU

15 _ 0.3 mol / l citric acid / NaOH, pH 6 2.23 2.05 0.3 mol / l ascorbic acid / NaOH, pH 6 1.68 1.95 1 mol / l Tris / HCl, pH 7.2 8.76 5.8 0.3 mol / l EDTA / NaOH, pH 6 1.95 1.95 20 0.3 mol / l 2-oxoglutaric acid, pH 6 2.64 3.17 0.3 mol / l fumaric acid / NaOH, pH 6 1.85 4.08

Example 2 Solubility of 25-KLC2P

Purified pro-KLC2P (dissolved, 0.5 M arginine / H 3 PO 4, pH 7.2) was ultrafiltered using an Amicon YM-10 membrane. 1 ml of concentrate (activity 3.5 MU / ml) was dialyzed against each of the buffers listed in Table 2 t. After centrifugation of the test batches, the enzymatic activity of the clear surface was measured.

35 Enzymatic activity is expressed as volume activity MU / ml and total activity MU.

7 95998 t-PA activity can be measured in the usual manner by digestion using a chromogenic substrate (H. Lill, ZGIMAL 42, (1987), 478-486). Unit U is the defined activity of t-PA (WHO, National Institute for Biological Standards 5 and control).

TABLE 2 10 Buffer Agent Activity

MU / ml MU

1 mol / l Tris / HCl, pH 7.2 1.65 1.07 15

Example 3 Solubility of pro-tPA

Purified pro-tPA (dissolved, 0.5 mol / L arginine-20 μl / H 3 PO 4) was ultrafiltered, concentrated using

Amicon YM-10 film. 1 ml of concentrate (activity 2.4 MU / ml) was dialyzed against each of the buffer waves listed in Table 3. After centrifugation of the test batches, the enzymatic activity of the clear surface was measured.

Enzymatic activity is expressed as volume activity MU / ml and total activity MU.

30 t-PA activity can be measured in the usual manner by degradation using a chromogenic substrate (H. Lill, ZGIMAL

42, (1987), 478-486). Unit U is defined as t-PA activity (WHO, National Institute for Biological Standards and Control).

35 95998 8 TABLE 3 5 Your Buffering Activity

MU / ml MU

0.3 mol / l citric acid / NaOH, pH 6 0.29 0.16 1 mol / l Tris / HCl, pH 7.2 2.15 1.07 10

Example 4 Solubility of CHO-tPA 15

Purified CHO-tPA (dissolved, 0.5 mol / L arginine / H 3 PO 3) was ultrafiltered, using an Amicon YM-10 membrane for concentration. 1 ml of concentrate (activity 6.6 MU / ml) was dialyzed against each of the 20 buffer waves listed in Table 3. After centrifugation of the test batches, the enzymatic activity of the clear surface was measured.

Enzymatic activity is expressed as volume activity MU / ml and total activity MU.

t-PA activity can be measured in the usual manner by degradation using a chromogenic substrate (H. Lill, ZGIMAL 42, (1987), 478-486). Unit U is defined as t-PA Akti-30 activity (WHO, National Institute for Biological Standards and Control).

il: m i mu 1.1 x αι 35 9 95998 TABLE 4

Buffer Agent Activity

5 MU / ml MU

1 mol / l Tris / HCl, pH 7.2 6.16 6.49 0.3 mol / l citric acid / NaOH, pH 6 4.44 4.44

Claims (15)

95998 A process for the preparation of a pharmaceutical preparation containing a protein having t-PA activity, an enzyme activity of at least 0.25 MU / ml and a pH of 4.5 to 9, characterized in that the t-PA active the protein is dissolved in at least one of the following substances: citric acid, ascorbic acid, 2-oxoglutaric acid, fumaric acid, Tris and EDTA, at a concentration of at least 0.2 mol / l, and the solution is brought into pharmaceutical form. Process according to Claim 1, characterized in that the concentration of said substances is 0.2 to 1 mol / l. 3. A compound according to claim 1, which is used in a concentration of 0.3-1 mol / l. 15 Process according to Claim 1, characterized in that the concentration of said substances is 0.3 to 1 mol / l. 4. A method according to claims 1-3, wherein the t-PA-active protein is K2P, the glycosylate and the enzymatic activity are 1.4 MU / ml. 20 Process according to one of Claims 1 to 3, characterized in that the domain composition of the t-PA-active protein is K2P, it is non-glycosylated and has an enzymatic activity of at least 1.4 MU / ml. 5. A method according to claims 1-3, wherein the t-PA-active protein is K1K2P, which is glycosylated and has an enzymatic activity of 0.4 MU / ml. 25 Process according to one of Claims 1 to 3, characterized in that the t-PA-active protein has a domain composition of K1K2P, is non-glycosylated and has an enzymatic activity of at least 0.4 MU / ml. 6. A method according to claims 1-3 for converting t-PA-active protein to a glycosylated pro-t-PA and having an enzymatic activity of 0.25 MU / ml. 30 7. Preferably, the t-PA-active protein is glycosylated pro-t-PA and the enzymatic activity is less than 1.4 MU / ml. 35 8. The formulation of the present invention is limited to 300 mmol / l of citric acid / NaOH, pH 6. 95998 Process according to one of Claims 1 to 3, characterized in that the t-PA-active protein is non-glycosylated pro-t-PA and has an enzymatic activity of at least 0.25 MU / ml. Method according to one of Claims 1 to 3, characterized in that the t-PA-active protein is glycosylated t-PA and has an enzymatic activity of at least 35 1.4 MU / ml. 95998 Process according to one of the preceding claims, characterized in that 300 mmol / l citric acid / NaOH, pH 6 is used. 9. A formulation according to the invention, which comprises a mixture of 300 mmol / l of ascorbic acid / NaOH, pH 6.5. 2-oxoglutaryl / NaOH, pH 6. Process according to one of the preceding claims, characterized in that 300 mmol / l ascorbic acid / NaOH, pH 6 is used. Process according to one of the preceding claims, characterized in that 300 mmol / l of 2-oxoglutaric acid / NaOH, pH 6 are used. 11. A preferred embodiment of the present invention is 10 vol, which is converted to an amount of 300 ramol / l of EDTA / NaOH, pH 6. Process according to one of the preceding claims, characterized in that 300 mmol / l EDTA / NaOH, pH 6, are used. 12. A preferred embodiment of the present invention is selected from the group consisting of 300 mmol / l of fumaric acid / NaOH, pH 6. Process according to one of the preceding claims, characterized in that 300 mmol / l of fumaric acid / poOH / NaOH, pH 6. 13. A preferred embodiment of the present invention is selected from the group consisting of 1 mol / l TRIS / HCl, pH 7.2. 20 Process according to one of the preceding claims, characterized in that 1 mol / l Tris / HCl, pH 7.2, is used. Process according to one of Claims 1 to 13, characterized in that conventional additives, auxiliaries and / or carriers are additionally added to the composition. A method according to claim 1, characterized in that the pharmaceutical administration form is a solution for injection or a lyophilisate. 95998 L. For the preparation of a pharmaceutical preparation containing protein with t-PA activity, enzyme activity of at least 0.25 MU / ml and a pH of 4.5-9, a maximum of 5 of the t-PA -active protein is added to the acid and above: lemon syrup, ascorbic acid, 2-oxoglutaric acid, fumaric acid, tris and EDTA, halogenated 0.2 mol / l, and added bring in a pharmaceutical dosage form. 2. A preferred embodiment of claim 1, wherein the concentration is at a concentration of 0.2-1 mol / l. 14. The invention is set forth in claims 1-13, the invention contemplating the use of a conveyance method, including a method and / or a method. 25 15. A patent according to claim 1, comprising a pharmaceutical dosage form for injection or lyophilization. • ·