FR2673945A1 - Polypeptides derived from the venum of Dendroaspis polylepis and their use as medicinal product - Google Patents

Abstract

The subject of the invention is polypeptides of 60 amino acid residues cross-linked via disulphide bridges, whose linear amino acid sequence corresponds to the following formula: X Arg Ile Cys Tyr Ile His Lys Ala Ser Leu Pro Arg Ala Thr Lys Thr Cys Val Glu Asg Thr Cys Tyr Lys Met Phe Ile Arg Thr Gln Arg Glu Tyr Ile Ser Glu Arg Gly Cys Gly Cys Pro Thr Ala Met Trp Pro Tyr Gln Thr Glu Cys Cys Lys Gly Asp Arg Cys Asn Lys Y (I> in which X represents hydrogen or a group blocking the terminal amino group and Y represents a hydroxyl group or a group blocking the terminal carboxyl group. The polypeptides can be used in therapy as inhibitors of calcium channels.

Description

POLYPEPTIDES FROM POLYLEPIS DENDROASPIS VENOM AND THEIR USE AS
DRUG
The subject of the present invention is new polypeptides that inhibit calcium channels, the corresponding antibodies and the use of said polypeptides as medicaments.

 L-type calcium channels are located in the outer membrane of muscle cells. They allow the entry of calcium into the cells and participate in a fundamental way in the regulation of the intracellular concentration of calcium which determines in particular the operations of contraction or relaxation of the vascular smooth muscle and of the heart muscle.

 Calcium channel blockers are therefore particularly used in therapy in the treatment of cardiovascular pathologies such as coronary insufficiency and arterial hypertension. The best known of these are molecules of the 1,4 dihydropyridine family such as nifedipine or nitrendipine, molecules of the phenylalkalamine family such as verapamil or molecules of the benzothiazepine family such as diltiazem .

 While none of these molecules is peptide in nature, we have now discovered a polypeptide extracted from the venom of Dendroaspis polylepis and having an inhibitory activity on the calcium channels involved in the excitation-contraction coupling of smooth and cardiac muscle cells; this inhibition naturally leads to vascular relaxation, analogous to that observed with molecules of the 1,4 dihydropyridine family.

 The venom of Dendroaspis polylepis is known to contain dendrotoxins which are small neurotoxins of 57 to 60 amino acid residues that facilitate the release of brain neurotransmitters.

H. SCHWEITZ et al., In TOXICON Vol. 28, No. 7, pp.

847-856, 1990, described the purification of 28 peptides extracted from Dendroaspis polylepis, half of which belongs to the family of dendrotoxins.

 The research carried out by the inventors has made it possible to highlight, among the 14 peptides extracted from Dendroaspis polylepis and not belonging to the dendrotoxin family, a new polypeptide inhibitor of calcium channels type L.

The subject of the present invention is therefore this new polypeptide consisting of 60 amino acid residues crosslinked by disulfide bridges and whose linear amino acid sequence corresponds to the following formula (I):
X Arg Ile Cys Tyr Ile His Lys Ala Ser Leu
Pro Arg Ala Thr Lys Thr Cys Val Glu Asn Thr Cys Tyr Lys
Met Phe Ile Arg Thr Gln Arg Glu Tyr Ile Ser Glu Arg Gly
Cys Gly Cys Pro Thr Ala Met Trp Pro Tyr Gln Thr Glu Cys
Cys Lys Gly Asp Arg Cys Asn Lys Y (I)
wherein X represents hydrogen or a group blocking the terminal amino group and Y represents a hydroxy group or a group blocking the terminal carboxyl group.

 the groups blocking the amino and terminal carboxyl groups are advantageously usual protective groups compatible with pharmaceutical use. Among the groups protecting the amino terminal function, there may be mentioned an acyl (for example acetyl) or hydrocarbyloxycarbonyl (in particular aralkoxycarbonyl or alkoxycarbonyl, for example benzyloxycarbonyl, tert-butyloxycarbonyl, etc.) group; mention will also be made of the terminal carboxyl groups protected in the form of an amide, of an ester (alkyl ester, benzyl ester, p-nitrobenzyl ester, etc.) or of dthydrazine (carbobenzoxyhydrazide, t-butyloxy-carbonylhydrazide, trityl- hydrazide, etc.).

The correspondences between the amino acids and the abbreviations used in formula (I) are as follows
Ala alanine
Cysteine
Asp aspartic acid
Glu glutamic acid
Phe phenylalanine
Gly glycine
His histidine
Isoleucine island
Lysine lily
Leu leucine
Met methionine
Asn asparagine
Pro proline
Gln glutamine
Arg arginine
Ser serine
Thr threonine
Val valine
Trp tryptophan
Tyr tyrosine
The invention also relates to the fragments of the polypeptide of formula (I) capable of possessing an activity which inhibits calcium channels. Those skilled in the art have the means to identify those of the polypeptides of shorter sequences which fall within the scope of the invention. As a general means allowing him to carry out this identification, mention may be made of the processing of the polypeptide of formula (I) with a protease cleaving the polypeptide in a chosen peptide site, after separation the fragments thus obtained are tested for their inhibitory activity on calcium channels of type L.

The above polypeptides can be modified as long as it retains the pharmacological activity defined above. Consequently, the invention also encompasses variants of said polypeptides which differ from it, for example by insertion and / or deletion and / or substitution of one or more amino acids. By variants is also meant the polypeptides in which the peptide bond (-CO
NH-) is replaced by structures -CO-N (CH3) -, -CH2
CH2-, -CO-CH2-, or alternatively, those in which the peptide backbone has one or more intercalated groups such as the groups -CH2-, -NH-, -O-. the present invention also relates to the polypeptides in which the amino acids having an asymmetric carbon are in D or L form.

 The polypeptides of the invention comprise several cysteine residues capable of forming disulfide bridges between them, these disulfide bridges give the polypetides a crosslinked structure. Thus, the inventors have demonstrated four disulfide bridges in the peptide of formula (I).

 The polypeptides of the invention can be obtained by purification from the biological material which constitutes the venom of Dendroaspis polylepis by chromatography or precipitation techniques. The polypeptides of the invention or its fragments can also be obtained by genetic engineering techniques according to techniques analogous to those used for example for obtaining human insulin, or also be prepared in a conventional manner by chemical synthesis. chemical synthesis consists, for example, in condensing several fragments previously formed by successive couplings of the different amino acid residues in the appropriate order, taking care to protect beforehand all the reactive functions carried by the amino acid residues or the fragments, except the amine and carboxyl functions involved in the peptide bond formed during the condensation.

 The present invention also relates to antibodies formed against the polypeptides of the invention. Antibodies which may be polyclonal, or monoclonal and then produced by any hybridoma prepared according to conventional methods of cell fusion between spleen cells, activated in vitro by a polypeptide of the invention or originating from an animal immunized against said polypeptide, and cells of a myeloma cell line. The preceding monoclonal antibodies can be advantageously used to select from the polypeptides of the invention those which exhibit inhibitory activity on calcium channels of type L.

 The polypeptide of formula (I) is distinguished by interesting pharmacological properties and can therefore be used therapeutically as a medicament.

 The invention therefore also relates to the use of the polypeptide of formula (I) or of any suitable peptide fragment or variant derived from the latter, as an inhibitor of type L calcium channels. The polypeptides of the invention exhibit a powerful effect on the cardiac and vascular calcium channels, but have no effect on the calcium channels of neurons and striated muscle, and consequently has neither neurotoxicity nor myototoxicity.

 The intracellular variations of calcium play a major role in the process of muscle contraction but also in the processes of cellular proliferation, thus a hyperproliferation of the smooth muscle cells and endothelial cells is at the origin of the processes arléroséroslérose.

 As an inhibitor of the cardiac and vascular calcium channels, the polypeptides of the invention are particularly suitable for the treatment of cardiovascular pathologies such as hypertension, as well as for the treatment of arthritis sclerosis.

 For the previous indications, the dose depends on the mode of administration and the desired treatment.

In general, satisfactory results are obtained when the polypeptide is administered at a daily dose of between 0.6 mg / kg and about 6 mg / kg.

In humans, administration is carried out for example intravenously, in divided doses up to 3 times a day, in the form of unit doses containing from 0.2 mg / kg to approximately 2 mg / kg of active substance.

 The polypeptides of the invention can be administered in free form or in dl form, a pharmaceutically acceptable salt. Such salts have the same order of activity as the free forms and can be easily prepared according to conventional methods.

 The invention therefore relates to the polypeptides in free form or in the form of a pharmaceutically acceptable salt, for use as medicaments.

 As a medicament, the polypeptides are administered in the form of a pharmaceutical composition comprising a polypeptide of the invention, in free form or in the form of a pharmaceutically acceptable salt, in combination with a pharmaceutically acceptable vehicle or diluent. Such compositions, which also form part of the invention, can be presented for administration by the enteral route, for example in the form of tablets, or for administration by the parenteral route, for example in the form of solutions or suspensions injectable by the intravenous or muscular, or for administration in the form of a nasal spray.

 Other characteristics and advantages of the invention will appear on reading the examples which follow and which are illustrated by the appended figures, it being understood that these examples should not be interpreted as tending to reduce the scope of the claims.

I - Purification of the polypeptide of formula (I)
500 mg of Dendroapis polylepis venom also known as black mamba is dissolved in 7.5 ml of 1% acetic acid, then loaded onto a sephadex G50 column (3 x 39 cm) -
The biological material is eluted in 4 peaks with the same solvent at a flow rate of 72 ml / hour; the third peak corresponds to the peptide fraction and contains 85% of the biological material by dry weight. The optical density of the eluate is recorded at 280 nm.

This peptide fraction is combined and a third is loaded directly onto an HPLC column of TSK type.
SP 5 PW (21.5 x 150 mm) sold by the Company
BECKMANN) previously balanced with 1% acetic acid adjusted to pH 3 with ammonia (solvent A). FIG. 1 represents the elution of the peptide fractions A to N, by a linear gradient from solvent A to solvent B consisting of sodium acetate 1
M, in 100 minutes at a flow rate of 8 ml / minute.

Fraction E is then directly loaded onto a column of RP18 type (25 × 250 mm), 7, sold by the company MERCK. FIG. 2 represents the elution of the peptide fractions El, E2 and
E3, by a linear gradient of 10 to 40% acetonitrile in water containing 0.5% trifluoroacetic acid and 0.9% triethylamine, in 75 minutes at a flow rate of 8 ml / minute. The polypeptide of formula (I) corresponds to the E3 fraction.

The HPLC purifications were carried out with a Waters device (pump 501, U6K injector, automatic gradient former and spectrophotometer 481) equipped with a Shimadzu integrator-recorder.
CR3A. The E3 fraction was lyophilized and desalted on a column of trisacryl GF05M (2.5 x 20 cm) with 1% acetic acid as eluent, and re-freeze-dried to give the pure polypeptide of formula (I) which is preserved and stored dried at - 200 C.

 The polypeptide of formula (I) represents 2.8% of the venom by dry weight, its molecular mass is 7043 and its extinction coefficient at 280 nm is 11510.

II - Pharmacological study
The results of pharmacological studies showing the properties of the polypeptide of formula (I) are given below.

- The polypeptide of formula (I) blocks at concentrations of 10-6 to 10-8 M the contractions of different smooth muscles, spontaneous contractions (portal vein, uterus) or contractians induced by depolarization by potassium (aorta) or by Bay K 8644 which is an activator of the slow calcium channel. FIG. 3 shows the relaxation by the polypeptide of formula (I) of a rat aorta previously contracted with 40 mM K + (o) or with 300 nM Bay K 8644

- The polypeptide of formula (I) blocks at concentrations of 10-6 to 10-9 M the spontaneous contractions induced by electrical stimulation or by
Bay K 8644. FIG. 4 shows the negative inotropic effect of the polypeptide of formula (I) on a rat left atrium which is only electrically stimulated (o) or electrically stimulated and by the
Bay K 8644

 - Injection of the polypeptide of formula (I) into genetically hypertensive rats produces a very significant drop in blood pressure with only little effect on the heart rate.

 - The polypeptide of formula (I) has no effect on the calcium channels of neurons and striated muscle and therefore has neither neurotoxicity nor myotoxicity (LD 50> 20 fg / g in mice).

 The polypeptides of the invention are the first calcium channel blockers of peptide nature described to date.

 The peptides that are present in venoms most of the time have their hormonal equivalent in mammals. Sarafotoxin, a peptide extracted from snake venom Atractaspis Engaddensis, is a newly described vasoconstrictor peptide (C.

TAKASEKI et al. (1988) TOXICON, 26, 543-548; C. TAKASEKI et al. (1988) NATURE, 335, 303); there is in mammals and in humans in particular, a structural and functional equivalent of this toxin called endothelin (M. YANAGISAWA et at. (1988) NATURE 332, 411415). Endothelin is a vasoconstrictor hormone produced by vascular endothelial cells and involved in the contraction of the corresponding smooth muscle. it is therefore probable that there exists in humans a vasodilating hormone of structure analogous to the polypeptide of formula (I) and which is involved in the physiological regulation of vasorelaxation. Such a hormone could be demonstrated by testing the antibodies of the invention on human biological samples.

Claims (5)

RENDICATIONS  1) Polypeptide having an inhibitory activity on type L calcium channels consisting of all or part of the following amino acid sequence  X Arg Ile Cys Tyr Ile His Lys Ala Ser Leu Pro Arg Ala Thr Lys Thr Cys Val Glu Asn Thr Cys Tyr Lys Met Phe Ile Arg Thr Gln Arg Glu Tyr Ile Ser Glu Arg Gly Cys Gly Cys Pro Thr Ala Met Trp Pro Tyr Gln Thr Glu Cys Cys Lys Gly Asp Arg Cys Asn Lys Y  wherein X represents hydrogen or a group blocking the terminal amino group and Y represents a hydroxy group or a group blocking the terminal carboxyl group.  2) Polypeptide according to claim 1, characterized in that it comprises 60 amino acid residues crosslinked by four disulfide bridges whose linear amino acid sequence corresponds to the following formula  X Arg Ile Cys Tyr Ile His Lys Ala Ser Leu Pro Arg Ala Thr Lys Thr Cys Val Glu Asn Thr Cys Tyr Lys Met Phe Ile Arg Thr Gln Arg Glu Tyr Ile Ser Glu Arg Gly Cys Gly Cys Pro Thr Ala Met Trp Pro Tyr Gln Thr Glu Cys Cys Lys Gly Asp Arg Cys Asn Lys Y (I)  wherein X represents hydrogen or a group blocking the terminal amino group and Y represents a hydroxy group or a group blocking the terminal carboxyl group.  3) Monoclonal antibody characterized in that it recognizes one or more polypeptides immunologically according to one of claims 1 to 2.  4) Polypeptide according to one of claims 1 to 2, in free form or in the form of a pharmaceutically acceptable salt, for use as a medicament.  5) Pharmaceutical composition, characterized in that it comprises a polypeptide according to any one of claims 1 to 2, in free form or in the form of a pharmaceutically acceptable salt in combination with a pharmaceutically acceptable vehicle.