Stabilized compositions containing non-glysylated human tissue t

Details Stabilized compositions containing non-glysylated human tissue t Stabilized compositions containing non-glysylated human tissue t

1991-08-02

1994-10-04

Robinson, Douglas W.

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

424 941, 424 943, 435188, A61K 3748, A61K 3762, A61K 37547, C12N 996

Patent

active

053524527

DESCRIPTION:

BRIEF SUMMARY
DESCRIPTION

Human tissue type plasminogen activator (t-PA) possesses a great therapeutic importance in the dissolution of blood coagula, for e.g. heart infarcts. t-PA brings about the dissolution of the blood coagula by the activation of plasminogen to plasmin. Plasmin in turn dissolves fibrin, 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 contains the proteolytically-active centre which brings about the cleavage of plasminogen to plasmin. The gene technological production of t-PA or of different t-PA mutants, in which some of the domains F, E, K1 and K2 are deleted, in eukaryotic and prokaryotic cells is already known. In contrast to natural t-PA, these recombinant t-PA derivatives are synthesized in non-glycosylated form.
Furthermore, it is known that the sugar portion has considerable influence on the solubility and aggregation of proteins (J. Biol. Chem. 263 (1988), 8832-8837). It has now been ascertained that a non-glycosylated t-PA mutein with the domain composition K2P possesses substantially poorer solubility than glycosylated t-PA derivatives. The non-glycosylated t-PA variant dissolves to only a small extent in the buffers usually employed for the solubilization of proteins, such as e.g. 50 mmol/l. Na citrate, pH 6, 50 mmol/l. phosphate buffer or physiological NaCl solution. However, for the use as a therapeutically active material, the non-glycosylated t-PA derivative K2P pro should be present with a distinctly higher enzymatic activity of at least 1.4 MU/ml., preferably of 1.4 to 10 MU/ml.
From EP-A-0 217 379, it is known to increase the solubility of t-PA produced by prokaryotes (t-PA pro) by means of neutral or slightly alkaline arginine formulations. However, a disadvantage of this process is that good solubilities of t-PA pro can only be achieved with very high arginine concentrations. Furthermore, the stability of the highly concentrated t-PA derivative K2P pro is low under neutral or slightly alkaline conditions.
Consequently, it is the aim of the invention to develop formulations which contain the non-glycosylated t-PA derivative K2P pro with an enzymatic activity of at least 1.4 MU/ml., whereby the stability of the t-PA derivative is to remain over a comparatively long period of time.
The aim according to the invention is achieved by a pharmaceutical preparation of a non-glycosylated t-PA derivative K2P pro with an enzymatic activity of at least 1.4 MU/ml. and a pH value of 4.5 to 6.5, whereby this composition contains at least one compound from the group consisting of
a) ascorbic acid,
b) EDTA,
c) a amino compound of the formula OH, R=C.sub.1 -C.sub.9 -alkylene, preferably C.sub.4 -C.sub.7 -alkylene, C.sub.3 -C.sub.6 -cycloalkylene or benzylidene and R.sup.1 is H or C.sub.1 -C.sub.3 alkyl and R.sup.2 is H or C.sub.1 -C.sub.3 -alkyl,
d) a guanidine analogue of the formula ##STR1## whereby Y=H.sub.2 N.sup.+ or O, Z=H or (CH.sub.2).sub.m V, (CH.sub.2).sub.m CH(NH.sub.2)--CO.sub.2 H, CH(CO.sub.2 H)--(CH.sub.2).sub.m CO.sub.2 H, wherein V=NH.sub.2 or CO.sub.2 H and m=1 to 4,
e) a carboxylic acid substituted with one or more hydroxyl, keto and/or further carboxyl groups,
f) dimethylbiguanide,
g) a pyrimidine nucleoside are pyrimidine nucleotide and,
h) trehalose, glucosemine, N-methylglucamine.


BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 Illustration of a t-PA molecule in the prior art.
By K2P pro according to the present invention, one understands a t-PA derivative which consists of the kringle 2- and of the protease domain and begins at any one of the amino acids 174-180 and ends with the amino acid 527. In addition, K2P pro can also contain part or all of the amino acids -3 (Gly) to +5 (Ile). A preferred protein is one which begins at amino acid 176 and, in front thereof, possibly also containing amino acids Ser, Tyr, Gln from the region -3 to +5. This designation follows the nomenclature given in T. J. R. Harris, Protein Engineering, Volume 1 (1987) 449-458 for t-PA. The production of s

REFERENCES:
patent: 4505893 (1985-03-01), Mori et al.
patent: 4568544 (1986-02-01), Hasegawa et al.
patent: 4837022 (1989-06-01), Kakimoto et al.
patent: 4980165 (1990-12-01), Isaacs et al.
patent: 4985245 (1991-01-01), Kakimoto et al.
patent: 5068106 (1991-11-01), Paques et al.
patent: 5149540 (1992-09-01), Kunihiro et al.
Larsen, G. R., et al., "Blood," vol. 73 (7), May 15, 1989, pp. 1842-1850.
Martin, V., et al., "Thrombosis Research," vol. 62 (3), pp. 137-146, 1991.
Kelley, R. F., et al., "Biochemistry," vol. 28, 1989, pp. 4047-4054.

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