Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-03-29
1997-08-26
Jagannathan, Vasu S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
530300, 530324, 530345, 514 15, 514 12, C07K 14815, C12P 2106, A61K 3803
Patent
active
056610017
DESCRIPTION:
BRIEF SUMMARY
The invention pertains to the field of thrombin inhibitors and describes the production of modified hirudins more especially desulphatohirudin muteins, with the aid of genetic engineering. It is a further object of the invention to provide a method for the preparation of biologically active high molecular weight hirudin by combining two to four monomers of said hirudins.
The hirudins are anticoagulant agents that occur naturally in leeches (e.g. in medicinal leech Hirudo medicinalis). The hirudins are equally acting polypeptides having an accumulation of hydrophobic amino acids at the N-terminus and of polar amino acids at the C-terminus, three disulfide bridges and the anticoagulant activity in common. A characteristic feature of most natural hirudins is the presence of a tyrosine sulphate residue at the C-terminal part (Tyr.sup.63) of the molecules. Apart from the well-known hirudin variants HV1, HV2 and HV3 additional hirudins have been reported to exist in nature, see, for example, M. Scharf et al. FEBS Lett. 255, 105-110 (1989), supporting the concept of hirudins as a family of isoinhibitors.
The hirudins, for example hirudin variant 1 (HV1), are the most potent and most specific known inhibitors of thrombin, the serine protease that catalyzes the final step (the conversion of the zymogen fibrinogen in clottable fibrin) in blood coagulation. Other enzymes of the blood coagulation cascade are not inhibited by hirudins. In contrast to heparin which is the preferred anticoagulant in conventional anticoagulation therapy, the hirudins exert their inhibiting action directly on thrombin and, unlike the former, do not act through antithrombin III. The only pharmacologically detectable effect of purified hirudins is the inhibition of blood coagulation and the prophylaxis of thrombosis. No side effects, such as effects on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and hemoglobin, have been observed after intravenous administration of hirudins to dogs, even in high doses. In a series of animal models hirudins have proved effective in experimental thrombosis (induced either by stasis or by the injection of thrombin), in endotoxin shock, and also in DIC (disseminated intravascular coagulation). Whenever direct comparison tests have been carried out, hirudins have proved to be superior to heparin.
In recent years cDNAs and synthetic genes coding for hirudin variants have been cloned and expressed in microbial hosts, such as Escherichia coli and, in particular, Saccharomyces cerevisiae. Although the expression products lack the sulphate monoester group at Tyr.sup.63 - and were therefore designated "desulphatohirudins"--they turned out to exhibit essentially the same biological activity as the natural sulphated hirudins.
A characteristic of the therapeutic application of recombinant desulphatohirudin is its half-life in the circulation of about 50 min. and therefore, the rapid excretion from the human body. The reason for the rapid excretion is the filtration of the glomerulum in the kidney for substances having a molecular weight below 70 000. Because of this rapid excretion, the daily dose of desulphatohirudin is administered normally in two or more separate portions. One strategy to obtain longer acting thrombin inhibitors is to synthesize high molecular weight hirudins. In WO 91/08229 the conjugation of hirudin with polyalkylenglycol is described. As the polyalkylenglycols are normally very heterogeneous in molecular size and weight, their combination with hirudin leads to a heterogeneous mixture and the administration of a defined dose of hirudin is difficult. In another approach the hirudin is crosslinked with other proteins like albumin (WO 92/05748). These kinds of conjugates show diminished activities and increased immunogenic properties and are therefore unsuitable for long term administration.
In WO 91/09125 relatively inactive fusion proteins are described that have to be activated by enzymes of the clotting cascade to have fibrinolytic or clot formation inhibition activity.
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REFERENCES:
patent: 4572798 (1986-02-01), Koths et al.
patent: 4620948 (1986-11-01), Builder et al.
patent: 5236898 (1993-08-01), Krstenansky et al.
Steiner, V. 1988 Diploma Thesis. University of Basel, Switzerland.
Krstenansky et al 1988 Biochimica et Biophysica Acta 957:53-59.
Talbot et al 1989 Thrombosis & Haemostasis 61(1): 77-80.
Maraganore et al 1990 Biochemistry 29:7095-7101.
Altman, J.D., et al., "Intracellular Expression of BPTI Fusion Proteins and Single Column Cleavage/Affinity Purification by Chymotrypsin", Protein Engineering, 4(5):593-600 (1991).
Bergmeyer (ed), "Samples, Reagents, Assessment of Results", Meth. in Enzym. Analysis, vol. II, pp. 314-316 (1983), Verlag Chemie, Weinheim (FRG).
Bornsen, K.O., et al., "Matrix-Assisted Laser Desorption and Ionization Mass Spectrometry and Its Applications in Chemistry", Chimia, 44:412-416 (1990).
Botstein, D., et al., "Strategies and Applications of In Vitro Mutagenesis", Science, 229:1193-1201 (1985).
Braun, P.J., et al., "Use of Site-Directed Mutagenesis to Investigate the Basis for the Specificity of Hirudin", Biochemistry, 27:6517-6522 (1988).
Chang, J-Y, "Production, Properties, and Thrombin Inhibitory Mechanism of Hirudin Amino-terminal Core Fragments", J. Biol. Chem., 265(36):22159-22166 (1990).
Chang, J-Y, et al., "Direct Analysis of the Disulfide Content of Proteins: Methods for Monitoring the Stability and Refolding Process of Cystine-Containing Proteins", Anal. Biochem., 197:52-58 (1991).
Chang, J-Y, et al., "Production of Disulfide-linked Hirudin Dimer by In Vitro Folding", FEBS Letters, 336(1):53-56 (1993).
Chatrenet, B., et al., "The Folding of Hirudin Adopts a Mechanism of Trial and Error", J. Biol. Chem., 267(5):3038-3043 (1992).
Dodt, J., et al., "The Complete Amino Acid Sequence of Hirudin, A Thrombin Specific Inhibitor", FEBS Letters, 165(2):180-184 (1984).
Gardella, T.J., et al., "Expression of Human Parathyroid Hormone-(1-84) in Escherichia coli as a Factor X-cleavable Fusion Protein", J. Biol. Chem., 265(26):15854-15859 (1990).
Grutter, M.G., et al., "Crystal Structure of the Thrombin-hirudin Complex: A Novel Mode of Serine Protease Inhibition":, The EMBO Journal, 9(8):2361-2365 (1990).
Halenbeck, R., et al., "Renaturation and Pruification of Biologically Active Recombinant Human Macrophage Colony-Stimulating Factor Expressed in E. coli", Biotechnology, 7:710-715 (1989).
Hinnen, A., "Transformation of Yeast", PNAS, 75(4):1929-1933 (1978).
Markwardt, F., et al., "Pharmacological Studies on the Antithrombotic Action of Hirudin in Experimental Animals", Thromb. Haemost., 47(3):226-229 (1982).
Meyhack, B., et al., "Desulfatchirudin, a Specific Thrombin Inhibitor: Expression and Secretion in Yeast", Thromb. Res. Suppl., VII:33 (1987).
Narang, S.A., "DNA Synthesis", Tetrahedron, 39(1):3-22 (1983).
Nishikawa, S. et al., "Efficient Cleavage by .alpha.-thrombin of a Recombinant Fused Protein which Contains Insulin-like Growth Factor I", Protein Engineering, 1(6):487-492 (1987).
Norris, K., et al., "Efficient Site-directed Mutagenesis by Simultaneous Use of Two Primers", Nucleic Acids Research, 11(15):5103-5112 (1983).
Sabin, E.A., et al., "High-level Expression and In Vivo Processing of Chimeric Ubiquitin Fusion Proteins in Saccharomyces cerevisiae", Biotechnology, 7:705-709 (1989).
Scharf, M., et al., "Primary Structures of New Iso-hirudins", FEBS Letters, 255(1):105-110 (1989).
Steiner, V., et al., "Primary Structure and Function of Novel O-Glycosylated Hirudins from the Leech", Biochemistry, 31:2294-2298 (1992).
Stone, S.R., et al., "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25:4622-4628 (1986).
Wallace, A., et al., "Contribution of the N-Terminal Region of Hirudin to its Interaction with Thrombin", Biochemistry, 28:10079-10084 (1989).
Zoller, M.J., et al., "Oligonucleotide-Directed Mutagenesis of DNA Fragments Cloned into M13 Vectors", Methods Enzymol., 100:468-500 (1983).
Chang Jui Yoa
Grossenbacher Hugo
Marki Walter
Carlson Karen Cochrane
Ciba-Geigy Corporation
Ferraro Gregory D.
Jagannathan Vasu S.
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