Peptide substrates for the identification of factor Xa

Details Peptide substrates for the identification of factor Xa Peptide substrates for the identification of factor Xa

1991-10-18

2000-09-19

Woodward, Michael P.

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

530331, 435 13, A61K 3800, A61K 3806, A61K 3836

Patent

active

061212393

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates, by way of novel industrial products, to tri- and tetra-peptide compounds. These novel products are particularly useful as substrates for the identification and assay of Factor Xa (i.e. activated Factor X) involved in the mechanisms of hemostasis. The invention further relates to the method of preparing these novel products and to the method of assaying and/or identifying Factor Xa by means of said products.


PRIOR ART

The enzymes belonging to the class E.C. 3.4.21 [as defined in the work "Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam 1973, pages 238 et seq. (former nomenclature: class E.C. 3.4.4)] are known to be substances which cleave the amide linkages of the protein or peptide backbone at the carboxyl group of Arg, Lys, Orn and His residues. This cleavage mechanism is well known to those skilled in the art and is amply exemplified in the documents of the prior art cited below.
The currently used substrates for enzymes belonging to said class are essentially tri- or tetra-peptide compounds whose N-terminal end is generally substituted by a blocking group such as benzoyl, benzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, tosyl, acetyl or the like and whose CO-terminal end is amidated by an aminated group which can be a radioactive radical or a radical, especially a p-nitroanilino group, capable of imparting coloration or fluorescence before or (preferably) after cleavage. Reference is made in this connection to the following patent documents: FR-A-2 372 798, EP-A-0 004 256, U.S. Pat. Nos. 4,508,644, 4,448,715, FR-A-2 471 411, FR-A-2 317 280 and FR-A-2 459 226.
It is found that the peptide derivatives in the patent documents cited above have little affinity for water. They have a low solubility or dispersibility in water; consequently, it is sometimes necessary to add an organic solvent to make them usable. The systems comprising mixed solvents are scarcely compatible, in general terms, with biological media: they cause either a decrease in the activity of the substrate or, in certain cases, a deterioration in the enzyme which it is desired to assay. Furthermore, the low affinity of these substrates for water causes a decrease in the sensitivity of the enzyme assay methods.
To improve the solubility of enzyme substrates in water, EP-A-0 025 190 has disclosed a first technical solution which consists in substituting, on said N-terminal end, a polyethylene glycol residue monoetherified by an alkyl group, for example Me--O(CH.sub.2 CH.sub.2 O).sub.x --CO (in which X is an integer such that the polyethylene glycol residue has an average molecular weight of about 600), the amino acid residue containing the CO-terminal end of the peptide chain being other than the Arg and Lys residues which are present according to the invention, as will be seen below; also, EP-A-0 280 610 (to the Applicant) has disclosed another technical solution which consists in attaching an alkoxycarbonylmethylenecarbonyl radical, such as the methoxymalonyl radical (i.e. Me--O--CO--CH.sub.2 --CO, abbreviated to MM), to the N-terminal end of a dipeptide.
Furthermore, according to document EP-A-0 280 610 cited above, dipeptide substrates containing said methoxymalonyl radical on said N-terminal end are generally presented as being more sensitive towards enzymes belonging to the class E.C. 3.4.21 than tri- and tetra-peptide substrates containing said methoxymalonyl radical. However, according to EP-A-0 280 610, these dipeptide substrates are not particularly specific towards Factor Xa and do not permit an effective assay of said Factor Xa.
Factor Xa, which belongs to the subclass E.C. 3.4.21.6, is known to be one of the important components involved in the mechanisms of hemostasis. The activation of Factor X leads to the formation of Factor Xa, which is a serine protease responsible for the conversion of prothrombin to thrombin. It is therefore of very great interest, from the diagnostic point of view, to have peptide substrates which are specific for Fac

REFERENCES:
patent: 4508644 (1985-04-01), Heber et al.
Morrison & Boyd, Organic Chemistry (3rd Ed.).
Allyn & Bacon Inc. (1973) 1147-1149.

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