DE4011375A1 - New anticoagulant hirudin-polyvinyl lactam conjugates - for treating e.g. myocardial infarction, peripheral arterial occlusion disorders, deep venous thrombosis and pulmonary embolism - Google Patents

Abstract

Conjugates of formula (I), made from polyvinyl lactams and hirudin, desulphatohirudin or coagulation-inhibiting hirudin muteins are new. A = H, OH or CH3. n = 2-4; m = 50-500. B = -CO-, -CH=, -CH2-, -CH2-X-CO-, -CH2-X-Z-CO-, -CH2-X-CO-NH-Z-NH-CO-, -CH2-X-CH2-(1,4-Ph)-N2-, -CH2-X-CH2-CH(OH)-CH2-(1,4-Ph)-N2-, -CH2-X-CO-CH2-CH2-CO or -CH2-X-(4-W-(1,3,5-triazin-2,6-yl)). X = -S-, -O-, -NH-, -N(CH3)- or -N(C2H5)-. Z = 1-6C alkylene or p-phenylene. W = H, Cl or OH. Hir = the hirudin, desulphatohirudin or hirudin mutein, which is bound to B via the serine, threonine or lysine side chain(s). USE/ADVANTAGE - For the treatment and prevention of thromboembolic disorders (e.g. myocardial infarction), peripheral arterial occlusion disorders, deep venous thrombosis and pulmonary embolism. (I) can be used to prevent reocclusion after re-opening of arterial blood vessels by mechanical methods of lysis. Administration is i.v. or i.m. (I) have longer in vivo half life (1-100 hours) than hirudin alone (50 minutes) or even than hirudin-dextran conjugates (7 hours).

Description

The invention relates to new Hirudinpolyvinyllactamkonjugate, the Her position and use for combating diseases.

Hirudin is a natural product with anticoagulant properties (Natural Sciences 42, 537 (1955), Hoppe-Seylers Z. Biol. Chem. 366, 379 (1985)). The naturally occurring hirudin is sulfated at the amino acid tyrosine in position 63, which does not apply to the recombinantly produced hirudin. Hirudin is eliminated from the blood relatively quickly (t _1/2 ∼50 min) (Thromb. Haemost. 47, 226 (1982), Pharmazie 43, 202 (1988)). The half-life of hirudin can be extended to over 7 hours by binding to dextran (Pharmazie 44, 72 (1989)).

It is also known that the half-lives in the blood vary from one Let series of proteins be extended by binding to polyethylene glycol (Polymer Reprints 20, 357 (1979); Enzyms as Drugs 1981, pages 367-383; US 41 79 337).

It has been found that conjugates of the formula I are derived from polyvinyl lactam Derivatives with hirudin, desulfatohirudin or anticoagulant Hirudin muteins

wherein
A is a hydrogen atom or a hydroxyl or methyl group,
n is the number 2, 3 or 4
m is a number from 50 to 500
B one of the groups -CO-, -CH =, -CH₂-, -CH₂-X-CO-, CH₂-XZ-CO-

or  

(with X in the meaning of -S-, -O-, -NH-, -N (CH₃) - or -N (C₂H₅) -, Z in the meaning of C₁-C₆ alkylene or p-phenylene and W in the meaning of H, Cl or OH)
p is the number 1, 2, 3 or 4 and
Hir one via the serine, threonine or lysine side chain (s) to the rest (s) of formula II

bound hirudin, desulfatohirudin or hirudin mutein residue mean have better properties.

The hirudin, desulfatohirudin or hirudin mutein residues are preferred to the rest (s) via the lysine side chains

coupled. Compounds which have only one are particularly preferred Lysine residue by a residue

have derivatized.  

The term hirudin means natural or recombinant hirudin Polypeptide consisting of 65 amino acids with that of Dodt (Hirudine, the Thrombin inhibitors of the leech Hirudo medicinalis. Dissertation, Ludwig Maximilian University, Munich, 1984) published sequence or one of the natural or recombinant isohirudins (Biol. Chem. Hoppe-Seyler 367: 803 (1986); Folia Haematol. 115, (1988)). Hirudin muteins are the result of deletion, addition or exchange one or more amino acids of hirudin obtained with polypeptides Hirudin effect.

The new conjugates can be prepared by

• a) a compound of formula III wherein
  A, m and n have the meaning given and
  E represents one of the radicals -CH₂Cl, -CH₂Br, -CH₂I, -CHO, -COY or -CH₂-XZ-COY (with Y meaning a leaving group and X and Z in the meaning given), with hirudin, resulfatohirudin or one Hirudin mutein, or
• b) a compound of formula IV wherein A, m and n have the meanings given and T represents one of the radicals -SH, -OH, -NH₂, -NHCH₃ or -NHC₂H₅, after reaction with a linker compound of the formula where Y and Z have the meanings given,

can react with hirudin, desulfatohirudin or a hirudin mutein.

The implementation of III with hirudin, desulfatohirudin or one Hirudin mutein happens as follows:

The compound of formula III is in stoichiometric amounts or with an excess in a suitable buffer (pH 7-10), in water, if necessary with the addition of an auxiliary base such as sodium or potassium carbonate or -hydrogen carbonate, alkali hydroxide, triethylamine, H-methylmorpholine, Diisopropylamine or pyridine in an organic solvent (methanol, Ethanol, isopropanol, acetonitrile, dimethylformamide, N-methylpyrrolidone, Dichloromethane, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, toluene) or in mixtures of the solvents mentioned at temperatures between -20 ° C. and + 100 ° C, preferably at temperatures between 0 ° C and + 60 ° C with Hirudin, desulfatohirudin or a hirudin mutein. The resulting conjugates are isolated and with those in protein chemistry usual methods cleaned and characterized.

For the implementation of polyvinyl lactam carboxylic acid

with the lysine side chain (s) of hirudin, desulfatohirudin or a hirudin mutein can be one of the methods commonly used in peptide chemistry be used to form an amide bond, the activated Polyvinyl lactam carboxylic acid

can be isolated or generated in situ.

Chemical methods for amide bond formation are discussed in detail at Müller, Methods of Organic Chemistry Vol XV / 2, pp 1-364,  Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid Phase Peptides Synthesis, pp 31-34, 71-82, Pierce Chemical Company, Rockford, 1984; Bodanszy, Klausner, Ondetti, Peptide Synthesis, pp 85-128, John Wiley & Sons, New York, 1976 and other standard works of peptide chemistry. The azide method, the symmetrical and mixed anhydride method, in situ generated or preformed active esters and amide bond formation using coupling reagents (Activators), especially dicyclohexylcarbodiimide (DDc), diisopropyl carbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), N, N-bis (2-oxo-3-oxazolidinyl) amido phosphoric acid chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), Castro’s Reagent (BOP), O-benzotriazolyl-N, N, N ′, N′-tetramethyluronium salts (HBTU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich reagent; HOTDO) and 1,1′-carbonyl-diimidazole (CDI). The coupling reagents can alone or in combination with additives such as N, N'-dimethyl-4-aminopyridine (DMAP), N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine can be used.

After the implementation of the polyvinyllactamaldehydes

with the lysine side chain (s) the resulting imine sensibly reduced to the secondary amine (cf. Walsmann et al; Pharmazia 47, 72 (1969)).

The implementation of IV with hirudin, desulfatohirudin or a hirudin mutein with the interposition of a linker is as follows guided:

The compound of formula IV is with the stoichiometric amount or with implemented an excess of the linker connection, possibly with the addition of a Base such as B. sodium or potassium carbonate or hydrocarbonate, alkali hydroxide, triethylamine, N-methylmorpholine, diisopropylethylamine or Pyridine at temperatures between -20 ° C and + 160 ° C, but preferably at Temperatures between 0 ° C and 100 ° C in an inert solvent, preferably water, methanol, ethanol, isopropanol, acetonitrile, Dichloromethane, dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide,  Tetrahydrofuran, 1,4-dioxane, toluene or in mixtures of these solvents, and the reaction product in water or a suitable buffer (e.g. 0.1 M Borax pH 9.4, 0.1 M Phosphate pH 8 or 0.1 Tris pH 8) if necessary with the addition of one of the above organic solvents in stoichiometric amounts or with 0.1 to 10 equivalents of excess Hirudin, desulfatohirudin or a hirudin mutein. The resulting conjugates are isolated and with those in protein chemistry usual methods cleaned and characterized. The reaction product from the compound of formula IV and chloric, bromic, iodoacetic acid, Succinic anhydride or one of the two nitro compounds must be added before Reaction with hirudin, desulfatohirudin or a hirudin mutein again be activated, which is analogous to the carboxylic acids described above described polyvinyl lactam carboxylic acids can happen and in the Nitro compounds by reduction to the amine and subsequent diazotization he follows.

The new compounds have an extended half-life with a high one anticoagulant activity and show very low antigenicity. The half-life can be determined via the molecular weight of the conjugated polyvinyl lactam residues between 1 and 100 hours to adjust.

The new compounds can be used in human and veterinary medicine Use treatment and prophylaxis of the following diseases: thromo-embolic Diseases such as myocardial infarction, peripheral arterial occlusive disease, deep vein thrombosis and pulmonary embolism. In addition, you can to prevent reocclusion after arterial reopening Vessels can be used by mechanical methods or lysis. you will be usually i. v. or I. m. in the usual forms of application turned.

Claims (4)

1. Conjugates of the formula I from polyvinyl lactam derivatives with hirudin, desulfatohirudin or hirudin muteins which inhibit blood clotting wherein
A is a hydrogen atom or a hydroxyl or methyl group,
n is the number 2, 3 or 4
m is a number from 50 to 500
B one of the groups -CO-, -CH =, -CH₂-, -CH₂-X-CO-, CH₂-XZ-CO- (with X in the meaning of -S-, -O-, -NH-, -N (CH₃) - or -N (C₂H₅) -, Z in the meaning of C₁-C₆ alkylene or p-phenylene and W in the meaning of H, Cl or OH)
p is the number 1, 2, 3 or 4 and
Hir one via the serine, threonine or lysine side chain (s) to the rest (s) of formula II bound hirudin, desulfatohirudin or hirudin mutein residue
mean. 2. Conjugates of formula I according to claim 1 for use in loading fighting diseases. 3. A process for the preparation of the conjugates of the formula I according to claim 1, characterized in that

• a) a compound of formula III wherein
  A, m and n have the meaning given and
  E represents one of the radicals -CH₂Cl, -CH₂Br, -CH₂I, -CHO, -COY or -CH₂-XZ-COY (with Y in the meaning of a leaving group and X and Z in the meaning given), or
• b) a compound of formula IV wherein A, m and n have the meanings given and T represents one of the radicals -SH, -OH, -NH₂, -NHCH₃ or -NHC₂H₅, after reaction with a linker compound of the formula where Y and Z have the meanings given,

react with hirudin, desulfatohirudin or a hirudin mutein leaves.