WO1998029445A1

WO1998029445A1 - α-AMINO-BUTYRATE DERIVATIVES OF VENOMS

Info

Publication number: WO1998029445A1
Application number: PCT/EP1997/007333
Authority: WO
Inventors: Jean-Marc Sabatier; Modhammed El-Ayeb; Jurphaas Van Rietschoten; Hervé Rochat
Original assignee: Armel S.A.
Priority date: 1996-12-31
Filing date: 1997-12-30
Publication date: 1998-07-09
Also published as: GB2335923A; GB9627115D0; GB9915067D0; AU5861198A; GB2335923B

Abstract

Synthetic peptides have amino acid sequences homologous or substantially homologous to those of naturally occurring peptide animal or plant venoms save that each cystine residue present in the venom has been replaced in the synthetic peptide by two α-amino-butyric acid residues. The synthetic peptides are non-toxic but retain the antigenic properties of the venom. They can therefore be used to raise vaccines against the venom, and also for treatment of persons already envenomated.

Description

TITLE

c -Amino-Butyrate Derivatives of Venoms

DESCRIPTION

The invention relates to synthetic peptides which have amino acid sequences derived from naturally occurring animal or plant venoms but which incorporate α-amino-butyric acid residues, and to preparations containing the said synthetic peptides. Such synthetic peptides may have prophylactic and therapeutic uses, and these uses are included within the scope of the invention.

Venomous animals have dangerous or lethal toxins against other species. Toxin inoculation, essentially neurotropic, is responsible for envenomation which exerts a neurotoxic activity altering the action potential of nerves and muscles.

Scorpions, toxic to human beings and other mammals, are essentially represented by the following four species: Androctonus, Buthus, Tityus and Centruroides . Every year, world wide, there are approximately 150,000 incidences of human beings being stung by scorpions. 1,200 of these result in fatalities. In North Africa, scorpions of the species Androctonus are responsible for 80% of the incidences and 95% of the deaths. The scorpion neurotoxins which are lethal to mammals are basic peptides made up from 60 to 70 amino acid residues cross-linked by four disulphide bridges. Their target is the sodium potential depending channel of excitable cell membranes. There is a structural homology in toxins which determines a protein with specificities. However there is an antigenic polymorphism mainly among the proteins of this family.

Classic treatment of envenomation is based on a poly specific serotherapy, the specific antibodies of which are obtained by a mixture of appropriate venoms of several scorpion species living in the same geographic area. Sometimes, in order to improve this serotherapy, a venom fraction enriched with toxins can be used. However, this serotherapy is limited, because the toxins are so effective that the neutralising levels are low and the serum volume which must be administered is high. This serum antivenom, usually of horse origin, is sometimes responsible for severe and even lethal anaphy lactic shocks.

The invention provides a synthetic peptide having an amino acid sequence homologous or substantially homologous to that of a naturally occurring peptide animal or plant venom or to an active part thereof save that each cystine residue present in the venom or the active part thereof has been replaced in the synthetic peptide by two o.-amino-butyric acid residues, the synthetic peptide being non-toxic but retaining the antigenic properties of the venom.

The use of o.-amino-butyric acid residues (Abu) to substitute each half of the cystine residues, which provide the disulphide bridges which cross-link the natural venoms, leads to non-toxic molecules which nevertheless retain the immunogenic and binding properties of the natural venoms. The synthetic peptides obtained can thus be used to raise specific sera in animals without adverse effects, and also to counteract recent envenomation by administration of non-toxic competitive agents. The invention thus opens routes to vaccines against animal or plant venoms, and treatments for those persons already envenomated.

The following Example illustrates the invention.

EXAMPLE

Two anatoxins of scorpion Androctonus australis Hector venom, named by us 8(Abu)-AaH I and 8(Abu)-AaH II, respectively derivatives of toxin I and toxin II from scorpion Androctonus australis Hector, were obtained by solid phase synthesis following classical strategies. Stepwise assembly of the peptide was carried out automatically on 4-oxymethyl- -phenylacetamidomethyl copoly(styrene-l % divinylbenzene) for 8(Abu)-AaH I and on p-methyl-benzhydrylamine for 8(Abu)-AaH II. The Boc/benzyl strategy was used, including a systematic double coupling scheme with hydroxybenzotriazole active esters (Boc-AA-OBt). Final cleavage from the resin was effected with anhydrous hydrogen fluoride (1 hour at 0°C). The peptides were washed with diethyl ether and solubilised in water. After lyophilisation, the synthetic anatoxins were characterised by reverse phase analytical high pressure liquid chromatography (C18), the analysis of amino acids being performed after acid hydrolysis (6N hydrochloric acid, 115°C, 24 hours) and circular dichroism.

8(Abu)-AaH I contained 63 amino acid residues, its primary structure being KRDGYIVYPNN(Abu)VYH(Abu)VPP(Abu)DGL(Abu)KKNGGSSGS(Abu)SFLVPSGLA(A bu)W(Abu)KDLPDNVPIKDTSRK(Abu)T (SEQ ID NO. 1).

8(Abu)-AaH II contained 63 amino acid residues, its primary structure being VKDGYIVDDVN(Abu)TYF(Abu)GRNAY(Abu)NEE(Abu)TKLKGESGY(Abu)QWASPYG NA(Abu)Y(Abu)KLPDHVRTKGPGR(Abu)H-NH₂ (SEQ ID NO. 2). The synthetic anatoxins 8(Abu)-AaH I and 8(Abu)-AaH II were injected into mice by the intracerebro ventricular route at doses of 200 μg. No toxic effects were observed. Repeated subcutaneous injections of 8(Abu)-AaH II did not elicit any sign of toxicity in mice.

To support the protective effects of Abu peptides, mice were injected with 8(Abu)-AaH II. Five weeks after the first injection of the synthetic anatoxin, the mice were subcutaneously injected with five times the LD₅₀ of the natural toxin AaH II. No symptoms of toxicity were observed. After a rest period of 12 weeks, the mice were again injected subcutaneously with the natural toxin AaH II, this time in the amount of twelve times the LD₅₀. Again the treated animals did not present any clinical or biological sign of toxicity. The animals were thus immunised against Androctonus australis Hector (AaH II) natural toxin.

The Abu peptides according to the invention retain the antigenic properties of the natural toxins but do not present the neurotoxic properties observed with the natural venom proteins.

We have demonstrated the immunoreactivity between natural antitoxin (AaH II) polyclonal antibodies and the 8(Abu)-AaH II synthetic derivative. We have also demonstrated the in vitro neutralisation of the natural antitoxin (AaH II) by the anti 8(Abu)-AaH II synthetic derivative mice polyclonal antibodies.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Armel S.A.

(B) STREET: 50 rue Basse

(C) CITY: L-7307 Steinsel

(E) COUNTRY: Luxembourg

(F) POSTAL CODE (ZIP) : none

(ii) TITLE OF INVENTION: alpha-Amino-Butyrate Derivatives of Venoms (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9627115.0

(B) FILING DATE: 31-DEC-1997

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION :1..63

(D) OTHER INFORMATION :/note= "Each Xaa in the sequence represents Abu"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Lys Arg Asp Gly Tyr lie Val Tyr Pro Asn Asn Xaa Val Tyr His Xaa 1 5 10 15

Val Pro Pro Xaa Asp Gly Leu Xaa Lys Lys Asn Gly Gly Ser Ser Gly 20 25 30

Ser Xaa Ser Phe Leu Val Pro Ser Gly Leu Ala Xaa Trp Xaa Lys Asp 35 40 45

Leu Pro Asp Asn Val Pro lie Lys Asp Thr Ser Arg Lys Xaa Thr 50 55 60

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..63

(D) OTHER INFORMATION: /note= "Each Xaa in the sequence represents Abu"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Val Lys Asp Gly Tyr lie Val Asp Asp Val Asn Xaa Thr Tyr Phe Xaa 1 5 10 15

Gly Arg Asn Ala Tyr Xaa Asn Glu Glu Xaa Thr Lys Leu Lys Gly Glu 20 25 30

Ser Gly Tyr Xaa Gin Trp Ala Ser Pro Tyr Gly Asn Ala Xaa Tyr Xaa 35 40 45

Lys Leu Pro Asp His Val Arg Thr Lys Gly Pro Gly Arg Xaa His 50 55 60

Claims

1. A peptide having an amino acid sequence homologous or substantially homologous to that of a naturally occurring peptide animal or plant venom or to an active part thereof save that each cystine residue present in the venom or the active part thereof has been replaced in the synthetic peptide by two ╬▒-amino-butyric acid residues, the synthetic peptide being non-toxic but retaining the antigenic properties of the venom.

2. A peptide according to claim 1 being homologous or substantially homologous to a scorpion neurotoxin.

3. A peptide according to claim 1 or claim 2 being homologous or substantially homologous to a neurotoxin of a scorpion of the species Androctonus.

4. A peptide according to any preceding claim being homologous or substantially homologous to toxin I or toxin II of Androctonus australis Hector.

5. The peptide KRDGYIVYPNN(Abu)VYH(Abu) VPP(Abu)DGL(Abu)KKNGGSSGS (Abu)SFLVPSGLA(Abu)W(Abu)KDLPDNVPIKDTSRK(Abu)T.

6. The peptide VKDGYIVDDVN(Abu)TYF(Abu)GRNAY(Abu)NEE(Abu)TKLKGESGY (Abu)QWASPYGNA(Abu)Y(Abu)KLPDHVRTKGPGR(Abu)H-NH₂.

7. A pharmaceutical composition comprising a peptide according to any preceding claim in admixture with a pharmaceutically acceptable diluent or carrier.

8. A vaccine prepared using a peptide according to any of claims 1 to 6 or a composition according to claim 7.