EP1021461A1

EP1021461A1 - Use of neuronal and lymphocytic potassium canal inhibitors for treating neurological diseases of immune origin

Info

Publication number: EP1021461A1
Application number: EP98949027A
Authority: EP
Inventors: Marcel Crest; Maurice Gola; Evelyne Beraud; Jurphaas Van Rietschoten
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 1997-10-06
Filing date: 1998-10-06
Publication date: 2000-07-26
Also published as: CA2305786A1; FR2769226B1; JP2001519356A; WO1999018123A1; FR2769226A1; AU9544298A

Abstract

The invention concerns the use of neuronal and lymphocytic potassium canal inhibitors for preparing medicines for treating neurological diseases with immune component in human beings or animals which block nervous conduction. The invention also concerns novel pharmaceutical compositions, and novel potassium canal inhibitors, and their use for treating said pathologies.

Description

USE OF POTASSIUM NEURAL AND LYMPHOCYT AIR BLOCKERS IN THE TREATMENT OF NEUROLOGICAL DISEASES OF IMMUNE ORIGIN

The present invention relates to the use of inhibitors (or blockers) of potassium channels as therapeutic agents for neurological diseases with an immune component in which a blockage of nerve conduction occurs, and in particular multiple sclerosis (MS). Multiple sclerosis (MS) is the most common human demyelinating disease of the nervous system with an average prevalence of 5 to 300 / 100,000 inhabitants. It affects young adults and 1.5 to 2 times more often women. The geographic distribution of MS is not uniform since its prevalence increases with latitude, according to three areas of low, medium and high risk. The peak of prevalence is located in regions between 45 ° and 60 ° north latitude, therefore in particular in Northern Europe.

The progression of the disease occurs in relapses, with the appearance of neuronal lesions which can impair motor and sensitive functions, vision, balance and control of the sphincters. In 50 to 60% of cases, an increasingly disabling progressive phase sets in after ten years, but these progressive forms also appear immediately in 15% of cases.

The relatively high prevalence of this disease, combined with its painful and debilitating progressive nature, explain the efforts of the scientific community to improve the lives of patients. Between 1993 and 1996, 2400 references relating to MS were published by specialized journals.

The clinical and pathological data for MS are as follows.

MS is due to a progressive destruction of the myelin sheaths of the axons of the central nervous system, without axonal degeneration, accompanied by a glial scar. the whole forming the sclerosis plaque The injured myelin is not or little reconstituted by glial cells (oligodendrocytes), which accentuates the progression of the disease. Histopalhological examination reveals the existence of inflammatory infiltrates in the white matter and the dissemination of lesions gives an account of the polymorphism of the symptoms. These inflammations are accompanied by disorders of nerve conduction, the conduction blocks, which are at the origin. of the loss of (nei vuscs anointings

SUBSTITUTE SHEET (REGI-E 26) During the active phases of the disease, we observe in the cerebrospinal fluid the presence of an abnormal number of T lymphocytes and to a lesser degree, B lymphocytes, an increase in the concentration of immunoglobulins (IgG) directed against different antigens viral, and especially in certain cases of the antibodies directed against the oligodendrocytes and against the basic protein myelin. To date, no infectious agent has been formally and systematically involved.

The lesions are formed by infiltration of monocytes, B and T lymphocytes (majority) and by demyelination plates. T lymphocytes predominantly express receptors for interleukin 2 and HLA molecules, witnesses of cell proliferation. Resident cells, astrocytes, microglial cells and capillary endothelial cells, also express HLA class II molecules, which indicates an inflammatory reaction in the brain. Inflammation is particularly harmful in the central nervous system, generating compressions, demyelination lesions, disturbances of the nerve impulse. One of the aggravating consequences of demyelination is the appearance of conduction blocks due to the disruption of the activity of the ion channels responsible for nerve impulses in the axons. Current MS therapies derive from treatments applied to various forms of experimental allergic encephalomyelitis (EAE) which have become the essential model of MS, and from anti-inflammatory, immunosuppressive or immunomodulatory drugs used in cancers, transplantations. organs and other inflammatory processes They are based on the assumption that MS is an autoimmune disease against myelin CNS. Therefore, these therapies involve treatments, with interferon or corticosteroids for example, which primarily interfere with the immune system to limit or prevent inflammatory processes in the CNS (Review by Jacobs et al, 1994) Symptomatic treatments aimed at restoring or improving deficient neurological functions are poorly developed and ineffective, with the exception of aminopyπdines. these treatments do not take into consideration the role of potassium channels in the conduction blocks of demyhnized nerves. main causes of sensory and motor insufficiency Beneficial clinical effects of potassium channel blockers and in particular of aminopyπdines (4-AP and 3,4-DAP) in multiple sclerosis have been described since 1983 (see i cvues Noseworthy 1993. Polman and Hartung. 1995) The use of πdιnes is justified for their ability to block neuronal potassium channels and thus to facilitate axonal conduction De tait, clinical trials have shown that 4-AP and especially 3.4-DAP significantly improve symptoms in certain patients

However, the aminopyπdines are not very selective and touch a large number of potassium channels dependent on the potential and inter alia, those present in the synaptic terminations (Sherrat, Bostock and Sears, 1980) These are Kv channels of Kvl type. l, Kvl .2, Kvl 3, Kvl 4, Kvl 5 but also channels of another genetic family, channels Kv3 1 and v3 4 Blocking these channels facilitates the release of excitatory neuromediators and induces numerous peripheral side effects and central (Bever, 1994)

The sustained administration of 4-AP to patients with MS has beneficial effects, but also serious neurological effects with induction of local or generalized epileptiform discharges (Polman et al., 1994) Furthermore, the efficacy of aminopyπdines on the Kv channels of T lymphocytes is relatively low (Ic50 = 200 μM). This explains why 4-AP (as well as qumidine) proved incapable of inducing significant changes in the evolution of EAE in the Lewis rat (Uitdehaag et al, 1994)

Aminopyπdines-based therapies aim to treat neurological symptoms At the concentrations used and because of the duration of the treatments, they induce side effects due to their lack of specificity

Furthermore, margatoxin, a molecule derived from scorpion venom, is described as an immunosuppressant capable of being able to be used in the treatment of autoimmune diseases, or the prevention of rejection of transplants (US Patent 5,494,895) This immunosuppressive effect of margatoxin tesulteiait of its action of blocking the Kvl 3 channels of the lymphocytes However, no effect of blocking of the neural Kv channels is described or suggested in this US patent

The present invention aims to provide compounds acting on a limited number of neural Kv channels, while ensuring high efficiency in neurological pathologies in which the immune system is involved and a blockage of nerve conduction occurs, without for as much engendered the side effects observed within the framework of thei apeutic tests of the compounds of aerial art The present invention also aims to provide new pharmaceutical compositions which can be used in the context of ti alternate pathologies immunological components associated with a blockage of neon conduction, in particular of MS The subject of the present invention is the use of compounds which inhibit the neural potassium channels Kvl. l, Kv l 2, and lymphocyte potassium channels Kv l. 3, for the preparation of medicaments intended for the treatment of neurological diseases with an immune component in humans or animals, in which blockage of nerve conduction occurs.

The compounds used in the context of the present invention are further characterized in that they have an inhibitory effect on the neuronal potassium channels Kvl. l and Kvl .2 potential dependent, located in Ranvier nodes (said neural channels being described in particular in the following articles: McNamara et al., 1996; Wang et al., 1994), and an inhibitory effect on potassium dependent channels of the Kvl .3 potential that is found in lymphocytes (said lymphocyte channels being described in particular in Chandy and Gutman, 1994; Grissmer et al., 1994).

By potassium channels dependent on the potential, in what precedes and what follows, one understands membrane proteins selectively permeable to potassium ions and whose permeability to this ion depends on the potential of the cell. The flow of potassium ions through the channel constitutes a potassium current. The characteristics of the relationship between potassium current and cell potential have been described by Stϋhmer et al. (1989) for the currents Kv l. l, Kv l .2 and Kvl .3. The sequence of the main protein subunit (α subunit) constituting the Kvl potassium channels. l, Kvl .2 and Kvl .3 is also described in this article. The classification of potential-dependent potassium channels is also explained in the review by Chandy and Gutman (1994). By inhibitors (or blockers) of potassium channels dependent on the potential, in what precedes and what follows, one understands any molecule able to limit the potassium current through a potassium channel by a direct action on the channel. In the particular case of kaliotoxin, and of the other peptide (or pseudopeptide) compounds described below, the blocking is carried out by obturation of the external part of the channel. The measurement of blockage of potassium channels by the compounds of the invention can be carried out by the method of the imposed potential such as that described in the article Crest et al. 1992, mentioned above

Among the neurological pathologies, in particular with an immune component, which can be treated in the context of the present invention, there may be mentioned the central neurological pathologies with inflammatory inflammation of the brain and spinal cord, in particular multiple sclerosis and diseases related (Balo, Devic. acute disseminated encephalitis and hemorrhagic, isolated optic neuropathies);

- peripheral inflammatory or related pathologies, in particular: acute and chronic polyradiculoneuritis, neuropathies linked to infectious diseases and coliagenoses, degenerative neurological pathologies, in particular amyotrophic lateral sclerosis, neuropathies with conduction block, tumor pathologies, primary or secondary, associated with a dysregulation of the immune system.

The invention also relates to the aforementioned use of the compounds described above in the context of the treatment of other central cerebral and spinal cord injuries, in particular pathologies of metabolic origin, ischemic arterial, venous and hemorrhagic vascular pathologies, traumatic pathologies, as well as central complications of systemic diseases, especially circulatory, such as Beheet, Sarcoidosis, and other vasculitides.

The invention more particularly relates to the use of peptide compounds as described above, inhibiting the neuronal potassium channels, Kvl. l, Kvl .2, and the lymphocyte channels Kvl .3, said compounds being derived from scorpion venoms or sea anemones, for the preparation of medicaments intended for the treatment of neurological diseases with component mentioned above, with exclusion of the use of margatoxin in the treatment of neurological diseases with an immune component, such as MS.

The invention relates more particularly to the aforementioned use of peptide compounds derived from scorpion venoms, or of compounds derived from the latter, these compounds or derivatives comprising the amino acid sequence corresponding to the following general formula (I) below:

Cys- AA9- AA i QA A ι \ - AA \ 2- AA [3-Cys- AA 15-AA \ - AA \ 7-Cys- AA 19- A A20, - A A21 - (A22) n ^AA 23 " ^AA 24 " ^AA 25" ^AA 26 " ^AA 7" ( ^AA 28) n2 "Lys-Cys- A31 - AA32-AA33-Lys-Cys-AA36-Cys (I)

in which :

- ni, and n2. independently of each other, represent zero or 1. - AA9 a ΛA 13, AA15 to AA 17, AA 19 to AA284 ^AA 3 1 <> AA33, e AA3, independently of each other, represent any amino acid, natural or not.

A more particular subject of the invention is the aforementioned use of compounds comprising the amino acid sequence of formula (I) in which ni = n2 = 1, and represented by the following formula (II)

AA2-AA3-Ile-Asn-Val-Lys-Cys-Thr-Ser-Pro-AAi2-Gln-Cys-AAi5-AAi6-Pro- Cys-Lys-AA20 "AA21 -AA22-Gly-AA24-AA25-Ala- Gly-AA28 "Lys-Cy S-AA3 - Asn-Gly-Lys-Cys-Lys-Cys-Tyr-AA39-AA4o (II)

in which AA2, AA3, AA12, AA15, AA ^, AA20 to AA22. AA24,

AA25, AA28, AA31, AA39, and AA40, independently of each other, represent any natural or unnatural amino acid. The invention relates more particularly to the use, as compound of formula (II), noxiustoxin (such as derived from the scorpion venom called

Centrudoides noxius) corresponding to the amino acid sequence SEQ ID NO

1.

A subject of the invention is also the above-mentioned use of compounds, comprising the amino acid sequence of formula (I), in which n2 = 0, said compounds comprising the sequence represented by the following formula (III):

AA6-AA7-Cys-AA9-AA 10-Ser-AA 12-AA 13-Cys-AA 15-AA 15- AA 17-Cys-Lys- Asp-Ala- (AA22) nr ^Glv -AA24-Arg-Phe- Gly-Lys-Cys-AA3 i-Asn-AΛ33-Lys-

Cys-AA36-Cys-AA38-AA39 (III)

in which

- AAg, AA7, AA9, AAJO, AA12, AA1, AA15, AA 16, ΛA 17, ΛA22- AA24, AA31, AA33, AA35, AA38 and AA39, independently of each other, represent any natural amino acid or not,

- ni represents zero or 1

The invention more particularly relates to the abovementioned use, as compounds comprising the sequence represented by the mule law (111) defined above, of kaliotoxin (KTX), or of peptide or pseudopeptide derivatives of KTX

Kaliotoxin is a 38-amino acid peptide with the following amino acid sequence GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK (still corresponding to the sequence SEQ ID NO 2 shown below) This sequence was for the first time published in 1992 in the article, Crest et al, 1992, J of Biological Chemistry, vol. 267, pl640- 1647 By peptide or pseudopeptide derivatives of KTX, we mean compounds deriving from KTX by addition or deletion of one or more amino acids, or by replacement of one or more amino acids with natural amino acids or no, said derivatives of kaliotoxin possessing the property of the latter of inhibiting the neuronal potassium channels Kvl. l and Kvl.2, as well as the above-mentioned potassium channels Kvl .3.

A more particular subject of the invention is the aforementioned use of derivatives of KTX defined above, and corresponding to the following formula (IV):

[( ^G1 y) na- ^v al-AA3-Ile-AA ₅ ] _nb -Val-AA7-Cys-AA9-AA ₁ o-Ser-AA ₁ 2-AA ₁ 3-Cys- AAi5-AAi6-AAi7-Cys -Lys-Asp-Ala- (AA22) _n ^G1 y-AA24-Arg-Phe-Gly-Lys-

Cys-AA _{3 1} -Asn-AA ₃ 3-Lys-Cys-AA36-Cys-Thr-Pro-Lys (IV)

in which :

- na, nb, and ni, independently of each other, represent 0 or 1, - AA3, AA5, AA7, AA9, AA10 _" AA12, AA13, AA15, AA16, AA17,

AA22 _> AA24, AA31, AA33, and AA36, independently of each other, represent any natural or unnatural amino acid

The invention relates more particularly to the above-mentioned use of compounds of formula (IV) defined above in which ni = 0, and - nb = 1, in particular the following peptide compounds

. kaliotoxin 2 corresponding to the amino acid sequence SEQ ID NO 3, and for which na = 0, nb = 1, kaliotoxin 3 corresponding to the amino acid sequence

SEQ ID NO 4, in which, unlike KTX, AA2 represents a glycine, AA5 represents a proline, AA7 represents a seine, AA9 i represents a lysine, AA J Q represents a histidine. AA [2 lepiesente glycine, and AAjg represents an isoleucine, and for which na = 0 nb = 1 agitoxin 2 corresponding to the amino acid sequence SEQ ID NO 5, and for which na = nb = 1, - or nb = 0, in particular the KTX (6-37) lepiesentee pai SEQ ID NO 6 ci for which na = nb = 0

The invention also relates to the aforementioned use of KTX drugs comprising the aforementioned (ormule (III) and more particularly corresponding to formula (IV) defined above, in which ni represents 0 or 1, and

- at least one of AA12 ^or ^ ^e A 17 represents aminobutyπque acid (Abu), - and / or, at least one of AA24 or AA31 represents a norleucine residue

As such, the invention relates more particularly to the aforementioned use of the KTX derivatives of formula (IV) defined above, in which ni = 0 or 1, and - AA12 represents aminobutyπque acid (Abu) or the lysine, and / or

AA17 represents ammobutyπque acid (Abu) or valine, and / or

- AA24 and AA31 represent, independently of one another, methiomne or norleucine.

A more particular subject of the invention is the aforementioned use of derivatives of the KTX of formula (IV) defined above, in which ni represents 0 or 1, and

- at least one of AA12 or AA - represents aminobutyπque acid (Abu), in particular the compounds of formula (IV) in which ni = 0, na = nb = 1, and AA12 and AA17 represent Abu, such that the KTX drift designates (P12 / Abu, P17 / Abu) KTX represents by SEQ ID NO 7,

- And / or at least one of AA24 or AA31 represents a norleucine residue, in particular the compounds of formula (IV) in which ni = 0, na = nb - 0, AA12 ^and AA17 represent Abu, and AA24 and AA31 represent a norleucine residue, such as the derivative of KTX denotes Kaline, said derivative being represented by SEQ ID NO 8, or the derivatives of KTX of formula (IV) in which ni = 1. na = nb = 1, and responding to the following formula (V)

AAι -AA2-AA3-AA4-AA5-AA ₍ 3-AA7-Cys-AA9-AA ιo-Seι -Abu-Gln-Cys-AA i5-

Lys-Abu-Cys-Lys-Asp-Ala-Gly-Nle-Arg-Phe Gly-Lys-Cys-Nle Asn AA33-LVS Cys-AA3 (3-Cys-AA3g-AA39-AA40 (V)

in which AA j has AA7 AA9, AA J Q ΛA 15 AA33 AA3 and ΛA38 <ι A40 independently of each other, they represent any natural or unnatural amino acid

The invention also more particularly relates to the aforementioned use of dei nes of the <KTX ι compi enant the mule (III) above and more particularly corresponding to the formula (IV) defined above, in which ni = 1, in particular those for which na = nb = 1, such as

- the KTX derivative with a modified α helix represents by the sequence SEQ ID NO 9, in which, unlike KTX. AA ^ 2 represents a lysine, AA 13 represents a glutamic acid, AA15 represents a tryptophan, AA _j g represents a serine, AA17 represents a valine, and AA22 represents a tyrosine,

- the KTX derivative designated (P12 / K, P17 / V, F22) KTX corresponding to the amino acid sequence SEQ ID NO 10, in which, unlike KTX, AA 2 represents a lysine, AA JJ represents a valme , and AA22 represents a phenylalamne

The invention also relates to the aforementioned use of peptide compounds derived from sea anemones, or peptide or pseudopeptide derivatives of these compounds (namely compounds derived from the latter by addition or removal of one or more amine acids, or by replacing one or more amino acids with natural or unnatural amino acids), said derivatives having the property of compounds derived from sea anemones of inhibiting the neuronal potassium channels Kvl 1 and Kvl 2, as well as the lymphocyte potassium channels Kvl 3 above, these compounds or derivatives comprising the amino acid sequence corresponding to the following general formula (VI) below

(AA 1) _n ι - (AA2) n2-Cys-AA4-Asp-AA6-AA7-AAg-AA9-AA \ o-AA γ \ -Cys- ( ^AA 13) n3-AA ₁ 4- (AA ₁₅ ) _n4 -AA ₁₆ - (AA ₁ 7) _n-AA 5 ₁₈ - (AA _I 9) _n6 -AA20-Cys- AA22- (AA23) AA25 n7-Ser-Lys-Tyr-AA ₂ 8- (AA29) n8 - ^AA 30- ^c y ^s - ^AA 32- ^L y ^s

Thr-Cys-AA3 ₆ -AA ₃₇ -Cys (VI)

in which

- neither at n8, independently of each other, represent zero or 1 - AA2, AA2, AA4, AA <3 a AAn, AA22 ^AA 23> ^AA 25 _* ^AA 28

AA30. AA32, ^AA 36 and AA37, independently of each other, there is any natural or unnatural amino acid

A more particular subject of the invention is the above-mentioned use of the peptide compounds of formula (VI) following BgK coπ espondant to the amino acid sequence SLQ ID NO 1 1 (such as derived from the sea anemone designated Bunodosoma gian lifeia) the SK corresponding to the amino acid sequence SCQ ID NO 12 (as derived from the met anemone designated Sluhυchu txla eliantlms) kaliseptin corresponding to the amino acid sequence SEQ ID NO 13 (such as from the sea anemone designated Anemonia sulcata)

The subject of the invention is also any pharmaceutical composition, characterized in that it comprises one or more peptide compounds as defined above, in combination with a pharmaceutically acceptable vehicle.

Advantageously, the pharmaceutical compositions of the invention are in a form which can be administered by intravenous, intramuscular, anal or subcutaneous routes, in particular at a rate of approximately 0.05 to approximately 1 mg / kg, or approximately 0.5 mg / kg to about 1 mg / kg, 1 to 3 times a day.

Preferred pharmaceutical compositions are those containing kaliotoxin or its derivatives, in particular kaliotoxin 3, in the context of the treatment of MS. Other preferred pharmaceutical compositions are those containing the compound (P12 / Abu, P17 / Abu) KTX, in the context of the treatment of MS.

The invention also relates to the KTX derivatives, as defined above, and corresponding to the following formula (IV):

[(Gly) _na -Val-AA3-Ile-AA ₅ ] _n b-Val-AA7-Cys-AA9-AA ₁ o-Ser-AA ₁₂ -AA ₁ 3-Cys-

AA 15- AA 16-AA \ 7-Cys-Lys- Asp-Ala- (AA22) n 1 -Gly-AA24- Arg-Phe-Gly-Lys- Cys-AA3 i-Asn-AA33-Lys-Cys-AA36 -Cys-Thr-Pro-Lys (IV)

in which . - na, nb, and ni, independently of each other, represent 0 or 1.

- AA3, AA5, AA7, AA9, AAio- ^AA 12 _' ^AA 13 _' A 15, AA ι ₀ . AA 17, AA22, AA24, AA31, AA33, and AA36, independently of each other, represent any amino acid, natural or not, provided that when ni = 0, then ^• - nb = 0,

- or AA2 represents a glycine, AA5 represents a proline. AA7 represents a sequence, AA9 represents a lysine, AA IQ represents an histidine. AA d? represents a glycine, and AA _j represents an isoleucine.

- and / or at least one of AA or AA 12 17 l epresente ^the .immobutyπque acid (Abu).

- and / or, at least one of AA24 or AA31 i represents a norleucine residue A more particular subject of the invention is the KTX derivatives of formula (IV), as defined above, in which ni = 0, and:

- na = 0, nb = 1, AA2 represents a glycine, AA5 represents a proline, AA7 represents a serine, AA9 represents a lysine. AAI Q represents a histidine. AA ^ 2 represents a glycine, and AA JÔ represents an isoleucine, in particular kaliotoxin 3 corresponding to the amino acid sequence SEQ ID NO 4, or

- nb = 0, in particular KTX (6-37) represented by SEQ ID NO 6. The invention relates more particularly to the KTX derivatives of formula (IV), as defined above, in which ni = 0 or 1, and:

at least one of AA12 or AA17 represents aminobutyric acid (Abu), in particular the compounds of formula (IV) in which ni = 0, na = nb = 1, and AA12 and AAi7 represent aminobutyric acid ( Abu), such as the derivative (P12 / Abu, P17 / Abu) KTX represented by SEQ ID NO 7, - and / or, at least one of AA24 or AA31 represents a norleucine residue, in particular:

. the compounds of formula (IV) in which ni = 0, na = nb = 0, AA12 and AA17 represent aminobutyric acid (Abu), AA24 and AA31 represent a norleucine residue, such as the Kaline represented by SEQ ID NO 8, or

. the KTX derivatives of formula (IV) in which ni = 1, na = nb = 1, and corresponding to the following formula (V):

AA 1 -AA2-AA3-AA4-AA5-AA6-AA7-Cys-AA9-AA 1 g-Ser- Abu-Gln-Cys-AA 15- Lys-Abu-Cys-Lys-Asp-Ala-Gly-Nle- Arg-Phe-Gly-Lys-Cys-Nle-Asn-AA33-Lys-

Cys-AA36-Cys-AA3g-AA39-AA40 (V) in which AA1 to AA7, AA9, AAI Q, AA15, AA33, AA3.5, and AA3 to AA40, independently of each other, represent any natural amino acid or not. A more particular subject of the invention is the KTX derivatives of formula (IV), as defined above, in which ni = 1. in particular the following compounds for which na = nb = 1, such as:

- the modified α-helix KTX derivative represented by the sequence SEQ ID

NO 9, in which, unlike KTX, AA _j 2 represents a lysine, AA 13 represents a glutamic acid, AA15 represents a tryptophan, AA [6 represents a serine. AA17 represents a valine. and AA22 represents a tvrosine. the derivative of KTX designated (P12 / K, P17 / V, F22) KTX corresponding to the amino acid sequence SEQ ID NO 10, in which, unlike KTX, AA12 represents a lysine, AA17 represents a valine, and AA22 represents a phenylalamne. The invention also relates to any peptide derived from those described above, in particular by substitution, deletion or addition of one or more amino acids, or any fragment of the peptides described above, said derivative peptides or fragments being capable of blocking the neural potassium channels Kvl. l and Kvl.2, as well as the above-mentioned Kvl.3 lymphocyte potassium channels.

The invention also relates to the nucleotide sequences coding for the peptides, or derived peptides, or fragments thereof, described above, as well as their use in the context of the preparation of said peptides, derived peptides, or fragments, by transformation of host cells with vectors containing said nucleotide sequences, culturing the host cells thus transformed, and recovery of the peptides, derived peptides produced

The invention will be further illustrated with the aid of the examples of synthesis of the compounds of the invention, and of their effects on an animal experimental model of MS.

I - Synthesis techniques

Kaliotoxin and its various analogues were synthesized by the solid phase synthesis method using, for the protection of amino acids, either Boc chemistry according to the protocol explained in the article Romi et al., 1993, J of Biological Chemistry , vol 268, p 26302-26309. either chemistry

Fmoc according to the protocol explained for peptides comparable in the article

Kharrat et al., 1996, Eur J of Biochemistry 42, p 491-498 The resins used are based on polystyrene 1% divinyl-benzene denvatise in PAM-isine, paramethyl-BHA resin, HMB resin, or RINK resin Protective chains amino acids are cyclohexyl or t-butyl for Asp and Glu, benzyl or t-butyl for Ser and Thr, carbobenzoxy. tosyl or tutyl for His, p-methylbenzyl, Acm, or tπtyl for Cys, or xantyl tπtyl poui Asn and Gln, tosyl or Pmc for Arg and 2Cl-Cbz or Boc for Lys ^The assembly of the amino acids was constructed as of HOBt-ester or by coupling with the HBTU reagent

The cleavage and deprotection of the side chains of the amino acids were carried out either in HF medium or in the presence of TFA ditteients traps a p-cresol, ethanedithiol, thioanisole or phenol radicals The peptides were placed in reoxidation in the presence of a Tπs-HCl buffer in contact with air. The constitution of the disulfide bridges and the restructuring of the molecules were monitored by HPLC. The oxidized peptides were purified by preparative HPLC and characterized by: analytical HPLC, analysis of amino acids, mass spectrometry, and by various biological tests: electrophysiology, toxicity in mice, fixation on their receptors.

The physico-chemical characteristics are as follows. amorphous white lyophilized powder, of great solubility in water (> 10 mg / ml), storage at 4 ° C in the lyophilized state and at -20 ° C in solution in water.

Rapid degradation in oxidizing and reducing media, rapid degradation in basic and extreme acid media (<1).

II - Study of the effect of the compounds of the invention on an animal model of MS

A) Methodology

1) Affinity of kaliotoxin for Kv-type potassium channels The affinity of kaliotoxin (KTX) for Kv-type potassium channels was determined by electrophysiology (imposed potential) after expression of RNA in the xenopus oocyte ( Kushner er coll., 1989).

2) Blockage of lymphocyte Kv channels by KTX The activity of potassium channels is determined by recording the overall current of a lymphocyte using the patch-clamp technique in the whole cell configuration.

3) In vitro effects of kaliotoxin on the proliferation of a T lymphocyte line T lymphocytes are maintained in culture by alternating cycles of antigen activation and amplification cycles in growth medium containing 10% of "T cell growth factoi s "(TCGF) The antigenic activation is carried out by cultivating 3x10 ⁴ LcT with 2x10 ^ cells presenting the antigen, the basic protein of myelin (PBM) (10 mg / ml) / 200 ml stimulation medium / microplate well After 48 hrs, a radioactive DNA precursor (H3-Tdr. lmCi / well) is added, 18 hrs later, the cells are harvested, lysed and the ladioactive material incorporated into the DNA is counted. The blockers are added at the start of antigen activation for the entire duration of the culture. Each blocker concentration is tested in quadreplicate. The results are expressed as an average of cpm of the 4 samples ± standard deviation.

4) Effects of kaliotoxin on an animal model of MS, experimental allergic encephalomyelitis (EAE) induced in rats

EAE is induced by adoptive transfer of T lymphocytes (LcT) specific for the basic myelin protein (PBM). These lymphocytes come from a CD4 + T cell line, Thl, "PAS", derived from Lewis rats; they are activated by PBM 48 hours before being injected into normal syngeneic recipients. Five groups (A to E) of 5 Lewis rats aged 10 weeks, 200 g (Charles River) receive this 2 to 5 million LcT (T PAS line) specific for the basic myelin protein PBM intraperitoneally on day 0 (D0). In groups B and C, KTX (0.8 and 8 μg / 0.1 ml PBS / rat)

(PBS, phosphate buffer) is injected subcutaneously on the same day and daily until the 6th day (D6). A control group (group A) receives instead of KTX, 1 ml of PBS. Group D rats receive 8 μg of KTX after the appearance of clinical signs of EAE. The rats of group E receive on day 0 LcT pretreated with KTX in vitro (100 ng / ml) and daily 8 μg of KTX until D6. The rats of the sixth group (F) receive only KTX (8 μg) from D0 to D6.

Another experiment was carried out by testing KTX at different doses: 0.8, 8, 16 and 32 μg / rat / day. The highest dose (32 μg) was injected once or twice in 0.1 or 1 ml of PBS. The route of administration was subcutaneous; it was compared to the intravenous route in one experiment. The clinical observation of the animals was carried out 1, 2 or 4 times a day.

The rats are observed and weighed daily. The clinical signs of EAE (identical to those obtained by active induction) are noted from 0 to 4. as follows (after Bourdoulous et al., 1995) -

0 = absence of neurological signs.

1 = flaccid tail,

2 = paresis of the hind limbs which may be accompanied by tremors,

3 = paralysis flaccid hind limb s may ^accompany incontinence, ocular and nasal bleeding. 4 = moribund state (paralysis of the upper limbs, respiratory disorders)

The average clinical EAE is obtained by adding together the clinical signs of maximum intensity of each rat; rats showing no signs are included and scored 0 Weight loss takes into account the minimum and maximum weight of each rat during the 6 days of treatment

5) Lifting of conduction blocks with kaliotoxin Two tests were selected In vitro rat optic nerve

It is a nerve of the central nervous system, myehnized by oligodendrocytes coupled to astrocytes. The optic nerve is removed from Lewis rats and placed in a perfused, oxygenated and thermostated chamber at 36 ° C. The recording of the overall activity of the axons is carried out using a suction electrode serving to contain a end of the nerve A second identical electrode, placed at the other end, is used to stimulate the nerve (stimulation giving a maximum response 0 1 ms, 10 to 30 V) The controls carried out, the preparation is put in the presence of lymphocytes reactive against myelin basic protein (2 10 ^ cells / ml) maintained in a DMEM culture medium.

Evoked potentials

The detection in the CNS of the evoked potentials by stimulation applied to a hind paw of a rat makes it possible to evaluate the functional state of the ascending fibers of the brainstem. The detection is done at the thalamic level. The metal electrodes are implanted permanently under stereotaxic control. , two in the ventro-postero-lateial thalamic nucleus and two others on the scalp. The animal being anesthetized with chloral (0.1 g / rat), the stimulation of the right hind paw is carried out using a bracelet encircling the leg and containing two conductors The threshold stimulation (2 ms. 25 to 60 V) is detected visually by a reflex contraction The evoked potentials are obtained by means of around fifty stimulations repeated every 5s

B) Results

1) Affinity of kaliotoxin for Kv channels

The dose-response curves show a high sensitivity of the channels Kv l 1 (IC50 = 1 5 nM), Kvl 2 (IC50 = 24 nM) and Kv l (IC50 = 0 1 nM) at KTX. The Kv3.1 channel is insensitive (IC50> 1 μ). In addition, other authors have shown that the neural channels Kvl .5 and Kv 3.1 are insensitive to KTX (Grissmer et al, 1994).

Conclusion: This activity profile ideally corresponds to the load plan set in the context of the present invention: high affinity for the Kvl .3 lymphocyte channel; high and selective affinity for Kvl axonal Kvl channels. l and Kvl.2, compared to other neural channels.

This activity profile is ideal for combining an effective immunosuppressive effect and a neurological effect oriented on axonal conduction which may make it possible to avoid the epileptogenic effects of aminopyridines.

2) Blockage of lymphocyte Kv channels by kaliotoxin Results: On the various lymphocyte lines tested, the sensitivity of endogenous Kv channels expressing themselves constitutively or after antigenic activation is characterized by a dissociation coefficient Kd of 1 to 2 nM.

Conclusion: Although lower, this sensitivity is in agreement with the data obtained on the exogenous Kvl .3 channels expressed in the xenopic oocyte.

3) In vitro effects of kaliotoxin on the proliferation of an encephalitogenic T lymphocyte line.

Results: Those of a typical experiment are presented in appendix 1. Four series of experiments indicate that 10 to 50 nM of KTX inhibits by 60% the incorporation of the radioactive precursor of DNA and therefore, cell proliferation.

Conclusion: These data confirm the major role of the Kvl .3 channels in the proliferative phases of T lymphocytes and the effectiveness of the immunosuppressive effect of KTX.

4) Effects of KTX on EAE Induced in the Rat

Results: Analysis shows that. From a clinical point of view, the treatment of rats systemically with KTX (8 and 0.8 μg) from day 0 to day 6 considerably reduces the severity of the disease and abolishes mortality. The remission is complete in less than 10 days. Even more significant are the results of group D treated after the appearance of clinical signs with 8 μg of KTX and only for 2 days. The clinical signs do not exceed an average severity of 1.1 and regress rapidly. Conclusion: Treatment of Lewis rats with KTX from the day of the adoptive transfer reduced the severity of EAE and weight loss. Treatment with KTX as soon as clinical signs appear, for only one to three days, constitutes an effective treatment for EAE (see Table 1).

The optimal conditions to retain in the context of these experiments are:

- 32 μg of KTX / rat / day: dose at which the results are the most reproducible,

- injection of KTX in 1 ml PBS, 1 time per day, by subcutaneous route,

- weighing and clinical examination of animals only once / day.

Table 1. Treatment of adoptive EAE by KTX

¹¹

EAE 1 Clinical Score

PBS KTX control (32 μg), experiment 1 3 ± 1.8 1.2 ± 0.7 1 n = 5 n = 5 experiment 2 2.5 ± 1.0 1.1 ± 0.35 1

1 n = 7 n = 8 experience 3 3.6 ± 0.4 2.51 ± 1.5 n = 7 n = 8 experience 4 2.3 ± 1 1 6 ± 1.3 n = 5 n = 5

EAE is induced in Lewis rats by intraperitoneal injection of 2 to 5 million “PAS” T lymphocytes activated by PBM on day 0.

KTX is injected subcutaneously in PBS as soon as clinical signs appear, for 1 to 3 days. The average clinical EAE is obtained by adding together the clinical signs of maximum intensity of each rat evaluated according to the scale of Sommer et al. (Nature Med. 1995, 1: 247).

"n" represents the number of rats per group. Statistical analysis is performed by the Mann and Whitney test.

These data are a confirmation, on a recognized animal model representative of multiple sclerosis, of the anti-inflammatory and immunosuppressive action of kaliotoxin. In addition, the recovery of animals from The aforementioned group D treated late is an indirect argument in favor of a symptomatic action of KTX on the Kv channels of the CNS. This conclusion is confirmed by the following two tests.

5) Lifting of conduction blocks with kaliotoxin

In vitro rat optic nerve

Results: In agreement with the data of Yarom et al. (1983), the incubation of a rat optic nerve with T lymphocytes pre-activated against the basic protein of myelin leads, in 1 h 30-2 h, an almost total block of the global potential derived by a suction electrode. A partial restoration of the control activity can then be obtained 2 to 3 hours after removing the lymphocytes. On the other hand, the addition of KTX (100 nM) quickly restores (15-20 min) the nerve conduction capacity even in the presence of lymphocytes.

Conclusion: These experiments validate the hypothesis that the conduction blocks in the demyelinated central fibers can be lifted by acting on the neuronal potassium channels. They also show that kaliotoxin, by its specificity, exerts this effect at nanomolar concentrations.

Potentials evoked Results: On an animal whose clinical signs of EAE reach level 2 (paralysis of the hindquarters), the potentials evoked by electrical stimulation of the hind paw are delayed and depressed by 80 to 90% compared to the control recordings. performed before induction of EAE. A subcutaneous injection of KTX (10 μg) is followed by a progressive recovery of the amplitude and the latency of the evoked potentials. Recovery becomes complete in 90 minutes.

Conclusion: This in vivo experiment shows that the conduction blocks consecutive to the inflammatory process of the fibers located in the lumbar part of the brainstem are lifted by an acute injection of KTX. The results confirm our hypothesis of a positive neurological effect of KTX. They directly show that the blocker can slowly pass the blood-brain barrier, probably at the sites of inflammation. This last conclusion is of importance with regard to the possibility of restoring a satisfactory axon conduction capable of restoring an impaired motor function. Bibliographical references

Bever CT, (1994), The current status of studies of aminopyridines in patients with multiple sclerosis. Ann Neurol. 36; SI 18-121.

Bourdoulous et al., (1995), Eur. J. Immunol .. 25, p 1176.

Chandy K.G. and Gutman G. (1994), Voltage-gated K + genes. In Handbook of

Receptors and Channels, A. North, ed., CRC Press, Boca Raton, FL, p 1.71.

Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilo icz MJ, Auperin DD and Chandy KG, (1994) Pharmacological characterization of five cloned voltage-gated K ⁺ channels, types Kvl. l, 1.2,

1.3, 1.5 and 3.1, stably expressed in mammalian cell Unes. Molec. Pharmacol. 45; p. 1227.

Jacobs L, Goodkin DE, Rudick RA and Herndorn R, (1994) Advances in specifies therapy for multiple sclerosis. Cur. Opin. Neurol. 7; p. 250.

Kushner L, Lerma J, Bennet MVL and Zukin RS, (1989) Using the xenopus oocyte System for expression and cloning of neuroreceptors and channels. In, Methods in Neurosciences. Flight. 1; p. 3. Académie Press Inc.

McNamara N.M.C., Averill S., Wilkin G.P., Dolly J.O., Priestley J.V., (1996), Ultrastructural localization of a voltage-gated K channel subunit (Kvl .2) in the rat cerabellum, Eur. J. of Neurosciences, 8 p. 688-699

Noseworthy JH (1993) Clinical trials in multiple sclerosis Cu r. Opin.

Neurosurg. 6; 209-215.

Polman CH, Bertelsman FW, van Loenen AC and Koetsier JC, (1994), 4-aminopyridine in the treatment of patients with multiple sclerosis. Long-term efficacy and safety. Arch. Neurol. 51; p. 292. Polman CH and Hartung HP, (1995) The treatment of multiple sclerosis: current and future. Curr .Opin. Neurol. 8; p. 200.

Sherratt RM, Bostock H and Sears TA, (1980) Effects of 4-aminopyridine on normal and demyelinated mammalian nerve fibers. Nature. 283; p. 570.

Stϋhmer W., Ruppesberg JP, Schroter KH, Sakmann B., Stocker M., Giese KP, Perschke A., Baumann A., Pongs O., (1989), Molecular basis of functional diversity of voltage-gated potassium channel in mammalian brain, The EMBO Journal, 8: p. 3235-3244.

Uitdehaag BM, Polman CH, de Groot CJ and Dijkstra CD, (1994), Effect of K ⁺ channel blockers on the clinical course and histological features of experimental allergy encephalomyelitis. Acta. Neurol. Scand. 90; p. 299.

Wang H., Kunkel DD, Sch artzkroin PA, Tempel BL, (1994), Localization of Kvl.l and Kvl.2, two K channel protein, to synaptic terminais, somata, and dendrites in the mouse brain, The J. of Neuroscience, 14: p. 4588-

4599.

Yarom Y, Naparstek Y, Lev-Ram V, Holoshitz J, Ben-Nun A and Cohen

IR, (1983) Immunospecific inhibition of nerve conduction by T lymphocytes reactive to basic protein of myelin. Nature. 303; p 246.

Claims

1. Use of compounds that inhibit the neural potassium channels Kvl. l, Kvl.2, and potassium lymphocyte channels Kvl .3, for the preparation of medicaments intended for the treatment of neurological diseases with an immune component in humans or animals, in which a blockage of nerve conduction occurs.

2. Use according to claim 1, for the preparation of medicaments intended for the treatment of neurological diseases with immune component following: central inflammatory neurological pathologies with cerebral and medullary localization, in particular multiple sclerosis and related diseases (Balo, Devic, acute encephalitis disseminated and hemorrhagic, isolated optic neuropathies); peripheral inflammatory or related pathologies, in particular:

. acute and chronic polyradiculoneuritis,. neuropathies linked to infectious diseases and collagenoses, degenerative neurological pathologies, in particular amyotrophic lateral sclerosis, neuropathies with conduction block, tumor pathologies with disruption of the immune system.

3. Use according to claim 1 or 2. of peptide compounds derived from scorpion venoms, or of compounds derived from the latter, these compounds or derivatives comprising the amino acid sequence corresponding to the following general formula (I) below:

Cys-AA9-AAιo-AAn-AAi2-AAi3-Cys-AAi5-AAi6-AAi7-Cys-AAi9-AA2θ-

AA2i- (AA22) nl-AA23-AA24-AA25-AA26-AA27- (AA ₂ 8) n2- ^L y ^s - ^c y ^s -AA3 i- AA32-AA33-Lys-Cys-AA36-Cys (I)

in which: - ni, and n2. independently of each other, represent zero or 1,

- AA9 to AA13, AA15 to AA17, AA19 to AA28. AA31 to AA33, and AA3.5, independently of each other, represent any amino acid, natural or not.

4. Use according to claim 3, of peptide compounds of formula (II) below:

AA2-AA3-Ile-Asn-Val-Lys-Cys-Thr-Ser-Pro-AAi2-Gln-Cys-AAi5-AAi6-Pro-

Cys-Lys-AA20-AA2i-AA22-Gly-AA24-AA25-Ala-Gly-AA28-Lys-Cys-AA3i-

Asn-Gly-Lys-Cys-Lys-Cys-Tyr-AA39-AA4o (II)

in which AA2, AA3, AA12, AA15, AAiβ, AA20 to AA22, AA24 _, AA25, AA28, AA31, AA39, and AA40, independently of each other, represent any natural or unnatural amino acid, such as the noxiustoxin represented by SEQ ID NO 1.

5. Use according to claim 3, of peptide compounds comprising the sequence of formula (III) below:

AA6-AA7-Cy S-AA9-AA iQ-Ser-AA 12- AA \ 3-Cys-A A 15- AA γ £ -AA 17-Cys-Ly s- Asp-Ala- (AA22) nl- ^G1 y -AA24-Arg-Phe-Gly-Lys-Cys-AA3 i -Asn-AA33-Lys- Cys-AA36-Cys-AA38-AA39 (III)

in which :

- AAg, AA7, AA9, AA10, AA12, AA13, AA15, AA16, AA17, AA22> AA24, AA31, AA33, AA36, AA38 and AA39, independently of each other, represent any natural amino acid or not. - ni represents zero or 1.

6. Use according to claim 5, of kaliotoxin represented by SEQ ID NO 2, or of derivatives of KTX corresponding to the following formula (IV):

[(Gly) _na -Val-AA3-Ile-AA ₅ ] _nb -Val-AA7-Cys-AA9-AA ₁ o-Ser-AA _l 2-AA ₁ 3-Cys-

AAi5-AAi6- ^AA 17-Cys-Lys-Asp-Ala- (AA22) n ^G1 y- ^AA 24- ^Ar S- ^phe - ^G1 y ^"L y ^s" Cys-AA31 -Asn-AA33-Lys-Cys-AA36 -Cys-Thr-Pro-Lys (IV)

in which: - na, nb, and ni, independently of each other, represent 0 or 1,

- AA3, AA5, AA7, AA9, AAJQ, AA124 A13, AA 15. AA [6, AA17. AA22- AA24, AA31, AA33, and AA36, independently of each other, represent any amino acid, natural or not.

7. Use according to claim 6, of compounds of formula (IV) in which ni = 0, and:

- nb = 1, such as the following peptide compounds:

. kaliotoxin 2 represented by SEQ ID NO 3,. kaliotoxin 3 represented by SEQ ID NO 4,

. agitoxin 2 represented by SEQ ID NO 5,

- or nb = 0, such as the KTX (6-37) represented by SEQ ID NO 6.

8. Use according to claim 6, of KTX derivatives of formula (IV), in which ni represents 0 or 1, and:

- at least one of AAχ2 or AA1 represents aminobutyric acid (Abu), such as the derivative (P12 / Abu, P17 / Abu) KTX represented by SEQ ID NO 7, and / or

at least one of AA24 or AA31 represents a norleucine residue such as Kaline represented by SEQ ID NO 8, or the KTX derivatives of formula (V) below:

AAι-AA2-AA3-AA4-AA5-AA6-AA7-Cys-AA9-AAi ₍₎ -Ser-Abu-Gln-Cys-AAi5- Lys-Abu-Cys-Lys-Asp-Ala-Gly-Nle-Arg- Phe-Gly-Lys-Cys-Nle-Asn-AA33-Lys- Cys-AA3 ₆ -Cys-AA ₃₈ -AA39-AA40 (V)

in which AAj to AA7, AA9, AAIQ, AA15, AA33, AA3.5, and AA3 to AA40, independently of each other, represent any amino acid, natural or not.

9. Use according to claim 6, of KTX derivatives of formula (IV) in which ni = 1, in particular those for which na = nb = 1, such as:

- the derivative of the modified α-helix KTX represented by the sequence SEQ ID NO 9,

- the derivative (P12 / K. P17 / V, F22) KTX represented by SEQ ID NO 10.

10. Use according to claim 1 or 2. of peptide compounds derived from sea anemones, or of compounds derived from the latter, these compounds or derivatives comprising the amino acid sequence corresponding to the following general formula (VI) below: (AA ι) _n ι - (AA2) n2 ~ y S-AA4-ASP-AA6- AA7-A A8- AA9-AA 10-AA \ \ -Cy s- (AAi3) _n 3-AA ₁₄ - (AA ₁₅ ) _n 4-AA ₁₆ - (AA ₁₇₎ _n5 -AA ₁ 8- (AA ₁ 9) _n6 -AA20-Cys- AA22- (AA23) _n 7-Ser-AA25-Lys-Tyr-AA28- (AA29) n8 -AA30-Cys-AA32-Lys- Thr-Cys-AA36-AA ₃₇ -Cys (VI)

in which :

- nor at n8, independently of each other, represent zero or 1,

- AA2, AA2, AA4, AAg to AAn, AA22- AA23, AA25, AA28 to AA30, AA32, AA36 and AA37, independently of each other, represent any amino acid natural or not.

11. Use according to claim 10, of the following peptide compounds:

- the BgK corresponding to the amino acid sequence SEQ ID NO 11, - the ShK corresponding to the amino acid sequence SEQ ID NO 12,

- kaliseptine corresponding to the amino acid sequence SEQ ID NO 13.

12. Pharmaceutical composition, characterized in that it comprises one or more peptide compounds defined in one of claims 1 to 13, in combination with a pharmaceutically acceptable vehicle.

13. KTX derivatives, corresponding to the following formula (IV):

[( ^G1 y) na- ^Val -AA3-Ile-AA ₅ ] _nb -Val-AA7-Cys-AA9-AA ₁₀ -Ser-AA ₁ 2-AA ₁ 3-Cys- AAi5-AAi6-AAi7-Cys-Lys -Asp-Ala- (AA22) nr ^G 'y "AA24-Arg-Phe-Gly-Lys-

Cy S-AA31 -Asn-AA33-Lys-Cys-A A36-Cys-Thr-Pro-Ly s (IV)

in which :

- na, nb, and ni, independently of each other, represent 0 or 1, - AA3, AA5, AA7, AA9, AAI Q, AAi2- A13, AA15, AA ^ AA17,

AA22 _* AA24, AA31, AA33, and AA36, independently of each other, represent any amino acid, natural or not. provided that when ni = 0, then:

- nb = 0, - or AA2 represents a glycine, AA5 represents a proline. AA7 represents a serine, AA9 represents a lysine. AA JQ represents a histidine. AAj2 represents a glycine, and AAjg represents an isoleucine. - and / or at least one of AAj2 or AA17 represents aminobutyric acid (Abu),

- And / or, at least one of AA24 or AA31 represents a norleucine residue.

14. KTX derivatives according to claim 13, of formula (IV) in which ni = 0, and:

- na = 0, nb = 1, AA2 represents a glycine, AA5 represents a proline, AA7 represents a serine, AA9 represents a lysine, AAI Q represents a histidine, AA12 represents a glycine, and AA16 represents an isoleucine, in particular kaliotoxin 3 represented by SEQ ID NO 4,

- or nb = 0, in particular the KTX (6-37) represented by SEQ ID NO 6.

15. KTX derivatives according to claim 13, of formula (IV) in which ni = 0 or 1, and:

- at least one of AA12 or AA17 represents aminobutyric acid (Abu), in particular the compounds of formula (IV) in which ni = 0, na = nb = 1, AA12 and AA17 represent aminobutyric acid (Abu ), such as the derivative (P12 / Abu, P17 / Abu) KTX represented by SEQ ID NO 7, - and / or, at least one of AA24 or AA31 represents a norleucine residue, in particular the compounds of formula (IV ) in which ni = 0, na = nb = 0, AA12 and AA17 represent aminobutyric acid (Abu), AA24 and AA31 represent a norleucine residue, such as the Kaline represented by SEQ ID NO 8, or the KTX derivatives of the following formula (V):

AAι-AA2-AA3-AA4-AA5-AA6-AA7-Cys-AA9-AAιo-Ser-Abu-Gln-Cys-AAi 5- Lys-Abu-Cys-Lys-Asp-Ala-Gly-Nle-Arg-Phe -Gly-Lys-Cys-Nle-Asn-AA33-Lys- Cys-AA36-Cys-AA3g-AA39-AA40 (V) in which AAj to AA7, AA9, AA ^ AA15, AA33, AA36, and AA38 to AA40, independently of one another, represent any natural or unnatural amino acid.

16. KTX derivatives according to claim 13. of formula (IV) in which ni = 1, in particular the following compounds for which na = nb = 1. such as:

- the modified propeller KTX derivative represented by the sequence SEQ ID NO 9,

- the derivative (P12 / K. P17 / V, F22) KTX represented by SEQ ID NO 10.