Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1985-03-18
1986-08-05
Schain, Howard E.
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
435183, 435184, 530380, A61K 3748, C07K 308, C12N 996, C12N 999
Patent
active
046042857
DESCRIPTION:
BRIEF SUMMARY
This invention relates to enzyme derivatives, and in particular to derivatives of Protein C which have anti-coagulant activity useful in treating thrombotic disorders, such as venous thrombosis. Protein C is a vitamin K dependent plasma protein consisting of two disulphide-linked polypeptide chains, and in order to function as an anticoagulant it must first be activated, according to known methods, to `activated Protein C`, hereinafter referred to as Protein Ca. A disadvantage of Protein Ca as an anticoagulant is that it has a short lifetime in the circulation due to inhibition by an endogenous plasma inhibitor protein.
It has now been discovered that the disadvantages of Protein Ca can be overcome or reduced by acylating the protein to form a derivative in which the anticoagulant activity is masked, but which will gradually hydrolyse in vivo to regenerate the active enzyme. Such acylation will thereby achieve a sustained release action for Protein Ca and chemical control over the kinetics of the anticoagulation process.
Accordingly, the invention provides an enzyme derivative comprising Protein Ca in which the active site essential for anti-coagulant activity is blocked by an acyl group which is removable in vivo by enzymatic hydrolysis to provide a sustained and controlled release of Protein Ca. Preferred acyl groups are optionally substituted benzoyl or acryloyl groups.
Suitable substituted benzoyl groups include those substituted with halogen, amino, C.sub.1-6 alkylamino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.2-7 alkanoyloxy and/or C.sub.2-7 alkanoylamino (RCONH-) or alkanoylhydrazino (RCONHNH-). Examples include 4-fluorobenzoyl, 2-, 3-, or 4-toluoyl, 2-, or 4-methoxybenzoyl (i.e. anisoyl), 2-, or 4-ethoxybenzoyl, 2,4-dimethoxybenzoyl, 3,4-dimethylbenzoyl, 4-butylbenzoyl, 3-methyl-4-methoxybenzoyl, 2-acetoxybenzoyl (i.e. acetylsalicyloyl), 2- or 4-aminobenzoyl, and 4-acetamidobenzoyl. Suitable optionally substituted acryloyl groups include C.sub.1-6 alkyl-acryloyl, furyl-acryloyl, cinnamoyl and C.sub.1-6 alkyl-cinnamoyl.
The derivatives of the invention may be prepared by reacting Protein Ca with an acylating agent site, and B is a acyl group preferably a benzoyl group or acryloyl group as defined above.
Examples of the group A include 4-amidinophenyl and 4-acetamidinophenyl or structurally similar substituted phenyl groups containing a positively charged moiety in the 3- or 4-position.
Examples of suitable acylating agents are: 4-amidinophenyl 4'fluorobenzoate, 4-amidinophenyl 4'-toluate, 4-amidinophenyl 4'anisate, 4-amidinophenyl benzoate, 4-amidinophenyl cinnamate, 4-amidinophenyl 3-(2-furyl)-acrylate, 4-amidinophenyl 2-naphthoate, 4-amidinophenyl 3,3-dimethylacrylate, 4-amidinophenyl 4't-butyl benzoate, 4-amidinophenyl 2',4'-dimethoxybenzoate, 4-amidinophenyl acetylsalicylate, 4-amidinophenyl 4'-ethoxybenzoate, 4-acetamidinophenyl 4'anisate, 4-amidinophenyl 2'-toluate, 4-amidinophenyl 2'anisate, 4-amidinophenyl 3',4'-dimethylbenzoate, 4-amidinophenyl 3'-methyl-4'-methoxy benzoate, 4-amidinophenyl 4'-aminobenzoate, and 4-amidinophenyl 4'-acetamidobanzoate.
The acylating reactions are suitably carried out in aqueous buffered media at a pH range which is not detrimental to the Protein Ca, acylating agent or product, e.g. from pH 6 to pH 9 and preferably at approximately pH 7.
The reaction is generally carried out by mixing the acylating agent with Protein Ca at a moderate temperature. The concentration of acylating agent is preferably 0.05-1.0 mM.
The time for which the reaction is allowed to proceed depends upon the acylating agent employed, and the temperature at which the reaction is carried out. A convenient time is about 0.5 to 1 hour at 0.degree. C. but the reaction may be allowed to continue for longer.
After the reaction is complete the derivative is purified by standard methods such as dialysis, affinity chromatography, and ultrafiltration, and thereafter recovered by standard methods such as freeze-drying from aqueous media. Where necessary the material may be adapted, for example by ster
REFERENCES:
patent: 4285932 (1981-08-01), Smith
patent: 4507283 (1985-03-01), Smith
J. Clin. Invest. 74, 200-204 (Jul. 1984), Colucci et al.
Blood, 59, No. 5, 1067-1072, (1982), Marlar et al.
Biological Abstracts, vol. 73, 1982, Comp et al, No. 44211.
Biological Abstracts, vol. 74, 1982, Marlar et al, No. 66170.
Cassels Robert
Smith Richard A.
Beecham Group p.l.c.
Schain Howard E.
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