Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1992-01-27
1994-05-17
Hill, Jr., Robert J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352402, 4352523, 4353201, 536 235, 530350, 530380, 530395, 514 8, C07K 1300, C12N 1515, A61K 3764
Patent
active
053127366
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to new protein analogues of the Extrinsic Pathway Inhibitor (EPI; also known as Tissue Factor Pathway Inhibitor, TFPI, or Lipoprotein-Associated Pathway Inhibitor, LACI). The invention also features a method of making the analogues and therapeutic preparations containing them.
BACKGROUND OF THE INVENTION
Blood coagulation is a complex process involving many activating and inactivating coagulation factors. Anticoagulant proteins are known to be important for regulation of the coagulation process (see B. Lammle and J. Griffin (Clinics in Haematology 14 (1985), 281-342) and anticoagulants are thus important in the treatment of a variety of diseases, e.g. thrombosis, myocardial infarction, disseminated intravascular coagulation etc.
Thus heparin is used clinically to increase the activity of antithrombin III and heparin cofactor II. Antithrombin III is used for the inhibition of factor Xa and thrombin. Hirudin is used for the inhibition of thrombin and protein C may be used for the inhibition of factor V and factor VIII.
Anticoagulant proteins may also be used in the treatment of cancer. Thus, antistatin has been shown to have anti-metastatic properties (J. H. Han et al., Gene 75 (1989), 47-57). Also heparin and warfarin have been shown to possess antimetastatic properties (G. J. Gasic et al., Int. Rev. Exp. Pathol. 29 (1985), 173-209).
Coagulation can be initiated through the extrinsic pathway by the exposure of tissue factor (TF) to the circulating blood (Y. Nemerson, Blood 71 (1988), 1-8). Tissue factor is a protein cofactor for factor VII/VIIa and binding of tissue factor enhances the enzymatic activity of factor VIIa (FVIIa) towards its substrates factor IX and factor X. Placenta anticoagulant protein is able to inhibit tissue factor activity, probably by interfering with TF/FVIIa-phospholipid interaction (S. Kondo et al., Thromb. Res. 48 (1987), 449-459).
Recently a new anticoagulant protein, the extrinsic pathway inhibitor (EPI) has been isolated (Broze et al., Proc. Natl. Acad. Sci. 84 (1987), 1886-1890).
On a molar basis EPI has been shown to be a potent inhibitor of TF/FVIIa induced coagulation (R. A. Gramzinski et al., Blood 73 (1989), 983-989). EPI binds and inhibits factor Xa (FXa) and the complex between EPI and FXa inhibits TF/FVIIa (Rapaport, Blood 73 (1989), 359-365). EPI is especially interesting as an anticoagulant/antimetastatic agent as many tumor cells express TF activity (T. Sakai et al., J. Biol. Chem. 264 (1989), 9980-9988) and because EPI shows anti-Xa activity like antistatin.
EPI has been recovered by Broze et al. (supra) from HepG2 hepatoma cells (Broze EP A 300988) and the gene for the protein has been cloned (Broze EP A 318451). A schematic diagram over the secondary structure of EPI is shown in FIG. 1 and the amino acid sequence of EPI is shown in FIG. 2 where the N-terminal amino acid Asp is given the number 1. The protein consists of 276 amino acid residues and has in addition to three inhibitor domains of the Kunitz type three potential glycosylation sites at position Asn117, Asn167 and Asn229. The molecular weight shows that some of these sites are glycosylated. Furthermore, it has been shown that the second Kunitz domain binds FXa while the first Kunitz domain binds FVIIa/TF (Girard et al., Nature 338 (1989), 518-520). EPI has also been isolated from HeLa cells (PCT/DK90/00016) and it was shown that HeLa EPI binds heparin.
Heparin binding is an important factor for the pharmacokinetics of substances for injection. It has been shown that platelet factor 4 (M. Prosdomi et al., Thromb. Res. 39 (1985), 541-547) and aprotinin with one Kunitz domain (Arzneimittelforschung, 33 (4) 479-94, 1983) has a short half life probably due to the heparin binding properties. Lowering of the heparin binding capacity of an anticoagulant would therefore seem to be advantageous. Furthermore, it might be advantageous to use a smaller molecule than EPI for the medical treatment.
It has according to the present invention surprisingly been foun
REFERENCES:
patent: 5106833 (1992-04-01), Broze, Jr. et al.
Girard et al., Nature, vol. 338, pp. 518-520 (1989).
Warn-Cramer et al., Thrombosis Research, vol. 48, pp. 11-22 (1987).
Broze, Jr. et al., Proc. Natl. Acad. Sci. USA, vol. 84, pp. 1886-1890 (1987).
Wun, T.-C., et al. (1988) J. Biol. Chem. 263: 6001-04.
Broze, G. J., Jr., et al. (1990) Biochemistry 29: 7539-46.
Nordfang Ole J.
Rasmussen Jesper S.
Fitzgerald David L.
Hill Jr. Robert J.
Lowney Karen A.
Novo Nordisk A S
Zelson Steve T.
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