JP2001519356A - Use of neuronal and lymphocyte potassium channel inhibitors for treating neurological diseases of immune origin - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of neuronal and lymphocyte potassium channel inhibitors for the preparation of a medicament for the treatment of neurological diseases involving the immune component, which blocks neurotransmission in humans and animals. The present invention also relates to novel pharmaceutical compositions, novel potassium channel inhibitors, and uses thereof for treating the above-mentioned conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of potassium channel inhibitors (or blockers) as therapeutics in neurological disorders involving the immune component, where blockage of neurotransmission occurs, and especially in multiple sclerosis.

[Prior art]

[0002] Multiple sclerosis (MS) is the most well-known nervous system disease of human nerve demyelination, which occurs on average at a rate of 5-300 per 100,000. It costs adolescents and tends to be 1.5 to 2 times more likely to be female. Its prevalence increases with latitude in the order of three regions: low, medium and high risk, and the geographic distribution of MS is not uniform. The peak morbidity is located in the area between 45 ° and 60 ° north latitude, and thus especially in northern Europe.

As the disease progresses, intermittent neuronal damage occurs that can affect motor and sensory functions, vision, balance, and sphincter control.
Fifty to sixty percent of patients enter a progressive phase of paralysis after about ten years, while 15% of patients have a direct manifestation of this advanced condition.

[0004] Due to the relatively high morbidity of the disease, accompanied by painful and disabling advanced conditions, the scientific community is working to improve the lives of patients. Therefore, between 1993 and 1996, as many as 2,400 articles related to MS were published in professional journals.

[0005] Clinical and anatomical details of MS are as follows.

[0006] MS is due to the progressive destruction of the myelin sheath of the central nervous system axons without glial scarring without axonal degeneration, which together form multiple sclerosis.
Damaged myelin is not regenerated at all or little by glial cells (oligodendrocytes). This speeds up the progress of the disease. Histopathological examination reveals the presence of an inflammatory infiltrate in white matter, and the spread of damage indicates a variety of symptoms. This inflammation is accompanied by disrupted or blocked neurotransmission, which causes loss of nerve function.

During the active phase of the disease, the presence of an abnormal number of T lymphocytes and a small amount of B lymphocytes in cephalorachidien fluid, the concentration of immunoglobulins (IgG) against different viral antigens Elevated and, in certain patients, antibodies to oligodendrocytes and basic myelin protein are seen. To date, commonly used drugs have not appeared in a systematic way.

[0008] The damage is caused by monocyte infiltration, mainly by B and T lymphocyte infiltration and demyelination. T lymphocytes contain interleukin 2 and H, evidence of cell proliferation.
Receptors for LA molecules are mainly shown. Capillary resident cells, astrocytes, microglial cells, and endothelial cells are also class II HLs that show an inflammatory response in the brain.
A molecule is shown.

[0009] Inflammation is particularly harmful in the central nervous system because it causes squeezing, demyelination damage, and disrupted neurotransmission. One consequence of the worsening of demyelination is the disruption of transmission due to disrupted ion channel activity responsible for neurotransmission in axons.

[0010] Current therapies for MS are treatments applied to a variety of clinical allergic encephalomyelitis (EAE), which are the main models of MS, and cancer, tissue transplantation and other inflammatory processes From various anti-inflammatory, immunosuppressive or immunomodulating agents used in These are based on the hypothesis that MS is an autoimmune disease of the CNS against myelin. Thus, these therapies require treatment with interferons or corticosteroids, which first suppress the immune system, for example to limit or prevent the inflammatory process in the CNS (reviewed by Jacob et al., 1994). Treatment of symptoms aimed at regenerating or improving the defective neurological function is slow progressing or inefficient. With the exception of aminopyridine, these treatments do not take into account the role of potassium channels in blocking the transmission of demyelinating nerves, which is the main cause of sensory and motor impairment.

[0011] Potassium channel inhibitors and especially aminopyridines (4-AP and 3,4
-DAP) has been described as an effective clinical effect in multiple sclerosis since 1983 (see literature: Noseworthy, 1993: Polemann and Hartung, 1995). It has been recognized that the use of aminopyridines can inhibit neuronal potassium channels and thus enhance axonal transmission. In fact, according to clinical trials,
AP and, in particular, 3,4-DAP have been shown to significantly improve the symptoms of some patients.

[0012]

[Problems to be solved by the invention]

However, aminopyridines are less selective and affect many potential-dependent potassium channels, and among these, those present at synaptic terminals (Sherrat, Bostock, And Se
ars et al., 1980). It is not only the Kv channels Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, but also channels of other genetic families, Kv3.
1 and Kv3.4 channel. Blocking these channels promotes excitatory neurotransmitter release and causes a number of related peripheral and central effects (Bever, 1994). Prolonged administration of 4-AP to patients with MS has shown good effects, as well as severe neurological effects with induction of local or global epileptic discharges. (Pollman et al., 1994). Furthermore, the effect of aminopyridine on the Tv lymphocyte Kv channel is:
Relatively weak (Ic50 = 200 μM). This indicated that 4-AP (as well as quinidine) was unable to cause significant changes in the progression of EAE in Lewis rats (Utidehaag et al., 1994).

[0013] Aminopyridine-based therapies are aimed at treating neurological conditions. At the concentrations used, due to the duration of treatment, they caused secondary effects due to lack of specificity.

Furthermore, a molecule derived from scorpion venom, margotox
in) has been described as an immunosuppressant that can be used in the sense of treating autoimmune diseases or preventing transplant rejection (US Patent No. 5.494.895). The immunosuppressive effect of margotoxin results from its ability to inhibit the Kv1.3 channel in lymphocytes. However, no neuronal Kv channel inhibitory effect is described or suggested in this US patent.

[0015] The present invention provides compounds that act on a limited number of neuronal Kv channels, while allowing the immune system to cause the secondary effects seen in the clinical trials of prior art compounds without causing effects. It is intended to ensure an effective effect on the neurological pathology in which the involvement and the blockage of neurotransmission occur.

The present invention also provides novel pharmaceutical compositions for use in the treatment of neurological conditions relating to the immune component, in particular MS, which are associated with a blockade of neurotransmission. Aim.

[0017]

[Means for Solving the Problems]

The present invention relates to the preparation of a drug, Kv, for the preparation of a medicament for the treatment of a neurological disease relating to an immune component in a human or animal, in which the blockage of neurotransmission occurs.
1.1, use of compounds that inhibit Kv1.2 neuronal potassium channel and Kv1.3 lymphocyte potassium channel.

The compounds used in the sense of the present invention are potential-dependent Kv1.1 and Kv1.2 neuronal potassium channels located at the Ranvier ring (the neuronal channels comprising: In particular, it is described in the following article: a potassium channel that has an inhibitory effect on McNamara et al., 1996; Wang et al., 1994) and is dependent on the Kv1.3 potential found in lymphocytes (the lymphocytes). The channels are further characterized in that they have an inhibitory effect on, inter alia, Chandy and Gutman, 1994; Grissmer et al., 1994).

Above and below, by potential-dependent potassium channel is meant a membrane protein that is selectively permeable to potassium ions, and
The permeability of this ion depends on the cell potential.

The flow of potassium ions through the channel constitutes the flow of potassium. Characteristics of the relationship between potassium flow and cell potential are Kv1.1, Kv1.2, and Kv
The 1.3 stream was described by Stuhmer et al. (1989). The sequences of the main protein subunits (subunit a) that make up the potassium channels Kv1.1, Kv1.2 and Kv1.3 are likewise described in this document. Potential-dependent classification of potassium channels is further described in Chandy and Gutman.

[0021] Above and below, mean any molecule capable of restricting the flow of potassium through a potassium channel by acting directly on the channel by an inhibitor (or blocker) of a potential-dependent potassium channel. . In the special case of kaliotoxin and other peptide (or pseudopeptide) compounds described below, inhibition is effected by closing portions outside the channel. The determination of the inhibition of potassium channels by the compounds of the invention can be performed by the potent method, as described in the above-mentioned article by Crest et al., 1992.

Among the neurological conditions, in particular those relating to the immune component, which are dealt with in the sense of the present invention, include:-inflammatory with respect to cerebral and medulla localization Central nervous conditions, especially multiple sclerosis and related diseases (baro, devic, hemorrhagic and acute sporadic encephalitis, optic neuropathy alone):-Related or inflammatory peripheral conditions, especially: acute And chronic polyradiculoneuritis Neuropathy associated with infectious attacks and collagen diseases. -Degenerative neurological conditions, in particular amyotrophic lateral sclerosis, neuropathy with blockade of transmission,-primary or secondary tumor neuropathy with disturbances of the immune system

The present invention likewise relates to complications predominant in systemic diseases, in particular circulatory diseases such as, for example, Beheet, Sarcoidosis, and other vascular diseases, In the treatment of other central cerebral and medulla attacks, particularly metabolic conditions, arterial ischemic vascular conditions, venous and hemorrhagic, traumatic conditions, It concerns the use described above.

The present invention is particularly directed to the use of scorpion venom or marine anemone venom (except for the use of margotoxin, in the sense that it treats neurological diseases related to the immune component, such as MS, for that purpose). A compound derived from a sea anemone venom that inhibits Kv1.1, Kv1.2 neuronal potassium channels, and Kv1.3 lymphocyte potassium channels, as described above, For the preparation of a medicament intended for treating the above diseases.

The present invention particularly relates to a peptide compound derived from a scorpion venom or a compound derived from a scorpion venom, ie, the following general formula (I):

[0026]

Embedded image

(Wherein n1 and n2 independently represent 0 or 1; AA _{9 to} AA ₁₃ , AA _{15 to} AA ₁₇ , AA _{19 to} AA ₂₈ , AA _{31 to} AA ₃₃ and AA _{3 6,} independently of one another, a compound or derivative comprising an amino acid corresponding to represent natural or unnatural amino acids), relates to the use as described above.

The present invention particularly relates, for its purposes, to formula (I) where n1 = n2 = 1,
And the following equation (II):

[0029]

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(Wherein, AA ₂ , AA ₃ , AA ₁₂ , AA ₁₅ , AA ₁₆ , AA _{20 to} AA ₂₂ , AA ₂₄ , AA ₂₅ , A
A ₂₈ , AA ₃₁ , AA ₃₉ and AA ₄₀ independently represent a natural or unnatural amino acid.) A compound comprising the amino acid sequence of｝ is used as described above.

The present invention particularly relates to the compound of formula (II) comprising noxiustoxin corresponding to the amino acid sequence SEQ ID NO 1 (eg derived from scorpion toxin named Centruoides noxius) Related to using.

The present invention likewise provides, for that purpose, compounds of formula (I) where n2 = 0 and the compound of formula (III):

[0033]

Embedded image

Wherein: AA ₆ , AA ₇ , AA ₉ , AA ₁₀ , AA ₁₂ , AA ₁₃ , AA ₁₅ , AA ₁₆ , AA ₁₇ , AA ₂₂ , AA ₂₄ , AA ₃₁ , AA ₃₃ , AA ₃₆ , AA ₃₈ and AA ₃₉ independently of one another represent a natural or unnatural amino acid, and n1 represents 0 or 1). Related to using.

The present invention is particularly directed to the use of kaliotoxin or a peptide or pseudopeptide derivative of KTX as a compound comprising the sequence represented by formula (III) as defined above. is there.

Caryotoxin is a 38 amino acid peptide whose amino acid sequence is shown below: GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK (again, a sequence corresponding to the sequence SEQ ID NO 2 shown below)

This sequence was first introduced in 1992 by Crest et al., J. of Biological Chemist.
ry, 1992, vol. 267, pp. 1640-1670.

[0038] Addition or deletion of one or several amino acids by a peptide or pseudopeptide derivative of KTX, or substitution of one or several amino acids by natural or unnatural amino acids By KTX means a compound derived from KTX, wherein the caryotoxin derivative has the latter property of inhibiting the Kv1.3 lymphocyte potassium channel as well as the Kv1.1 and Kv1.2 neuron potassium channels. Have.

The present invention especially has for its purpose the above-defined formula (IV)
)

[0040]

Embedded image

(Wherein: na, nb and n1 independently represent 0 or 1; AA ₃ , AA ₅ , AA ₇ , AA ₉ , AA ₁₀ , AA ₁₂ , AA ₁₃ , AA ₁₅ , AA ₁₆ , A
A ₁₇ , AA ₂₂ , AA ₂₄ , AA ₃₁ , AA ₃₃ and AA ₃₆ independently of one another represent any natural or unnatural amino acid), as described above.

The invention relates in particular to the formula (IV) as defined above, wherein n1 = 0 and: nb = 1, in particular the following peptide compounds: the amino acid sequence corresponding to SEQ ID NO 3 Caryotoxin 2 and, in contrast, those where na = 0, nb = 1, caryotoxin 3 corresponding to the amino acid sequence SEQ ID NO 4 (wherein, instead of KTX, AA ₂ AA ₅ represents proline, AA ₇ represents serine, AA ₉ represents lysine, AA ₁₀ represents histidine, AA ₁₂ represents glycine, and AA ₁₆ represents isoleucine), and On the other hand, na = 0
Agiotoxin 2 corresponding to the amino acid sequence SEQ ID NO 5 and, whereas, na = nb = 1, or nb = 0, especially the amino acid sequence SEQ ID NO KTX (6
-37), and, on the other hand, those compounds in which na = nb = 0, as described above.

The present invention provides, for that purpose, KTX derivatives comprising the above formula (III) and, in particular, a formula (IV) as defined above, wherein n1 represents 0 or 1, and At least one of AA ₁₂ or AA ₁₇ represents aminobutyric acid (Abu); and / or at least one of AA ₂₄ or AA ₃₁ represents a norisoleucine residue) It is to be used like.

The present invention therefore particularly relates to compounds of formula (IV) as defined above, wherein n1 is 0 or 1, and AA ₁₂ represents aminobutyric acid (Abu) or lysine, and And / or AA ₁₇ represents aminobutyric acid (Abu) or valine, and / or AA ₂₄ and AA ₃₁ independently of one another represent methionine or norleucine). .

The present invention particularly relates to the compounds of formula (IV) as defined above, wherein n1 represents 0 or 1, and at least one of AA ₁₂ or AA ₁₇ is aminobutyric acid (Abu), in particular compounds of formula (IV) where n1 = 0, na = nb = 1, and AA ₁₂ and A
A ₁₇ represents Abu, for example represented by SEQ ID NO 7, (P12 / Abu, P17 / Ab
u) at least one of a KTX derivative, named KTX,-and / or AA ₂₄ or AA ₃₁ represents a norleucine residue, in particular:-a formula (IV) wherein n1 = 0, na = nb = 0, AA ₁₂ and AA ₁₇ then represent Abu, and AA ₂₄ and AA ₃₁ represent norleucine, eg, SEQ ID N
A compound of KTX represented by O 8 and termed Kaline) or a KTX derivative of formula (IV) (where n1 = 1, na = nb = 1) and a compound of formula (IV) V):

[0046]

Embedded image

Wherein AA _{1 to} AA ₇ , AA ₉ , AA ₁₀ , AA ₁₅ , AA ₃₃ , AA ₃₆ and AA _{38 to} AA ₄₀ independently of one another represent any natural or unnatural amino acid. The KTX derivative corresponding to｝ is used as described above.

The invention likewise provides, in particular, for its purposes, KTX comprising the above formula (III) and, in particular, formula (IV) as defined above where n1 = 1, in particular na = N
those wherein b = 1, for example: in KTX derivative (here having a modified helix represented by SEQ SEQ ID NO 9, not as KTX, AA12 represents lysine, AA ₁₃ represents glutamic acid, AA ₁₅ represents tryptophan, AA ₁₆ represents serine, AA ₁₇ represents valine, and AA ₂₂ represents tyrosine), corresponding to the amino acid sequence SEQ ID NO 10 (P12 / K, P17 / V, F22 A) a KTX derivative named KTX (wherein, like KTX, A ₁₂ represents lysine;
A ₁₇ represents valine and AA ₂₂ represents phenylalanine)｝) is used as described above.

The present invention also provides peptide compounds derived from sea anemones, or peptide or pseudopeptide derivatives of these compounds (ie, by adding or deleting one or several amino acids, or A compound derived from marine anemone by substituting one or several amino acids with an unnatural amino acid) as described above, wherein the derivative comprises only the Kv1.3 lymphocyte potassium channel described above. And have the properties of marine anemone-derived compounds that inhibit Kv1.1 and Kv1.2 neuronal potassium channels, and these compounds or derivatives have the following general formula (VI):

[0050]

Embedded image

[0051] (wherein: - N1 to N8 are independently of one another, 0 or _{1, - AA 2 -AA 2 -AA} 4 -AA 6 ~AA 11, AA 22, AA 23, AA 25, AA _{28 to} A
A ₃₀ , AA ₃₂ , AA ₃₆ , and AA ₃₇ independently of one another represent any natural or unnatural amino acid).

The present invention particularly provides, for that purpose, BgK (eg Bunodosoma granulife) corresponding to the amino acid sequence SEQ ID NO 11:
from the sea anemones called ra),-ShK corresponding to the amino acid sequence SEQ ID NO 12 (eg Stichodactyla helian
derived from a sea anemone called this) or kaliseptine corresponding to the amino acid sequence SEQ ID NO 13 (eg Anemonia sulcat
a) derived from a sea anemone, referred to as a), is used as described above.

The invention likewise is characterized in that it comprises, for example, one or several peptide compounds as defined above, together with a pharmaceutically acceptable medium. Any pharmaceutical composition.

Preferably, the pharmaceutical composition of the present invention is provided in a form to be administered intravenously, intramuscularly, anal or subcutaneously, in particular from about 0.05 mg / kg to about 1 mg / kg or about 0.5 mg / kg. / kg to about 1 mg / kg, provided on a 1-3 dose per day basis.

Preferred pharmaceutical compositions are those comprising caryotoxin or a derivative thereof, especially caryotoxin 3, in the sense of treating MS.

Another preferred pharmaceutical composition is (P12 / Abu, P17
/ Abu) containing a KTX compound.

The present invention also provides a derivative of KTX, as defined above, and the following formula (IV):

[0058]

Embedded image

Wherein na, nb and n1 independently represent 0 or 1; and AA ₃ , AA ₅ , AA ₇ , AA ₉ , AA ₁₀ , AA ₁₂ , AA ₁₃ , AA _15, AA _16, A
A ₁₇ , AA ₂₂ , AA ₂₄ , AA ₃₁ , AA ₃₃ and AA ₃₆ independently of one another represent any natural or unnatural amino acid, provided that when n1 = 0:-nb = 0,-or AA ₂ represents glycine, AA ₅ represents proline, AA ₇ is table serine, AA ₉ represents lysine, AA ₁₀ represents histidine, AA ₁₂ is table glycine, and AA ₁₆ is isoleucine Represents, and / or at least one of AA ₁₂ or AA ₁₇ is aminobutyric acid (Ab
u),-and / or at least one of AA ₂₄ or AA ₃₁ relates to those corresponding to｝ representing norleucine residues.

The present invention particularly relates to the compounds of formula (IV), as defined above,
n1 = 0, and: na = 0, nb = 1, AA_TwoRepresents glycine; AA_FiveRepresents proline and AA ₇ Represents serine and AA₉Represents lysine, AA_TenRepresents histidine, and AA₁₂Is
Represents glycine, and AA₁₆Represents isoleucine, in particular, the amino acid sequence SEQ
Caryotoxin 3 corresponding to ID NO 4, or-a KTX derivative of nb = 0, especially KTX (6-33) 33 represented by SEQ ID NO 6.

The present invention particularly relates to a compound of formula (IV) as defined above, wherein n1 is 0 or 1, and at least one of AA ₁₂ or AA ₁₇ is aminobutyric acid A compound of formula (IV), which represents (Abu), in particular of the formula (IV): wherein n1 = 0, na = nb = 1, AA ₁₂ and AA ₁₇ represent aminobutyric acid (Abu), for example SEQ ID NO 7 derivatives represented by (P12 / Abu, P17 / Abu ) KTX, - and / or at least one of AA ₂₄ or AA ₃₁ represents a norleucine residue, in particular: - formula (IV) (wherein, n1 = 0, na = nb = 0, AA ₁₂ and AA ₁₇ represent aminobutyric acid (Abu) and AA ₂₄ and AA ₃₁ represent norleucine residues, for example, SEQ ID
A compound of Kailine represented by DNO 8, or a KTX derivative of formula (IV) (where n1 = 1, na = nb = 1) and the following formula (V):

[0062]

Embedded image

Wherein AA _{1 to} AA ₇ , AA ₉ , AA ₁₀ , AA ₁₅ , AA ₃₃ , AA ₃₆ and AA _{38 to} AA ₄₀ independently of one another represent natural or unnatural amino acids. KT of the equivalent
X derivatives.

The present invention particularly relates to a compound of the formula (IV) ｛wherein, as defined above,
The following compounds in which n1 = 1, in particular, na = nb = 1, for example: KTX derivatives with a modified helix represented by the sequence SEQ ID NO 9 (here, AA but not KTX) ₁₂ represents lysine, AA ₁₃ represents glutamic acid, AA ₁₅ represents tryptophan, AA ₁₆ represents serine, AA ₁₇ represents valine, and AA ₂₂ represents tyrosine), the amino acid sequence SEQ ID NO 10 A KTX derivative called (P12 / K, P17 / V, F22) KTX, where AA ₁₂ represents lysine and not like KTX,
A ₁₇ represents valine and AA ₂₂ represents phenylalanine)｝ is a KTX derivative.

The invention likewise relates to any peptide derivative described above, in particular a peptide derivative by substituting, deleting or adding one or several amino acids, or a peptide derivative described above. Any fragment, wherein the peptide derivative or fragment is capable of inhibiting the Kv1.3 lymphocyte potassium channel as well as the Kv1.1 and Kv1.2 neuronal potassium channels.

The present invention also relates to the use of these peptides, peptide derivatives or fragments in the sense of preparing them, as described above, as well as the transformation of host cells with vectors comprising the nucleotide sequences. Thus, the present invention relates to a nucleotide sequence encoding a peptide or peptide derivative or a fragment of the latter by culturing the host cell thus transformed and collecting the produced peptide or peptide derivative.

The invention is further illustrated by examples of the synthesis of the compounds according to the invention and by the effect in the example of MS experimental animals.

[0068]

【Example】

I-Synthetic Techniques Caryotoxin and its different analogs have been identified by Ro
Boc chemistry according to the protocol described in Mi et al., 1993, J. of Biological Chemistry, Vol. 268, pp. 26302-26309, or Kharrat et al., 199.
6 years, Eur. J. of Biochemistry, Vol. 42, pp. 491-498, by solid-phase synthesis by using any of the Fmoc chemistry following the protocol described for comparable peptides. Synthesized. The resins used are based on PAM resin, paramethyl-BHA resin, HMB resin, or polystyrene 1% divinyl-benzene derived from LINK resin. Protected chains of amino acids are: cyclohexyl or t-butyl, Ser and T for Asp and Glu.
benzyl or also t-butyl for hr, carbobenzoxy for His,
Tosyl or trityl, p-methylbenzyl, Acm, or trityl for Cys, xanthyl or trityl for Asn and Gln, tosyl or Pmc for Arg, and 2Cl-CbZ or Boc for Lys. It is. Amino acid synthesis was performed in the form of HBOt-ester or by coupling with HBTU reagent. Amino acid side chain cleavage and deprotection was performed in HF media in the presence of TFA with either a different radical group separator: p-cresol, ethanedithiol, thioanisole, or phenol. Peptides were reoxidized in the presence of Tris-Hcl buffer on contact with air. After disulfide bond formation and molecular reconstitution, HPLC was performed. Oxidized peptides are purified by preparative HPLC and identified by analytical HPLC, amino acid analysis, mass spectrometry, and other biological tests: electrophysiology, toxicity in mice, fixation to their receptors Was done.

The physicochemical properties are as follows: amorphous white, lyophilized powder, high solubility in water (> 10 mg / ml), at 4 ° C. in lyophilized state and in aqueous solution And stored at -20 ° C. Fast denaturation in oxidation and reduction media, fast denaturation in basic and highly acidic media.

II Study of the Effect of the Compounds of the Invention on Animal Models of MS

A) Methodology

1) Affinity of Caryotoxin for Kv-Type Potassium Channel The affinity of caryotoxin for Kv-type potassium channel was determined by electrophysiology (fixed potential) after expression of ARN in heterologous egg cells (Kushner). Et al., 1989).

2) Blocking of Kv channels in lymphocytes by KTX

The activity of potassium channels was determined by recording the overall flow of lymphocytes with the aid of the patch-clamp technique in global cell placement.

3) In vitro effects of caryotoxin on the growth of T lymphocyte lines

T lymphocytes were placed in culture in a growth medium containing 10% T cell growth factor (TCGF) by alternately repeating the antigen activation cycle and the amplification cycle. 2 × 10 ⁶ antigen host cells, basic protein myelin (BPM) (10 mg / ml) /
The antigen was activated by culturing 3 × 10 ⁴ LcT in 200 ml of the stimulation medium / microplate well. At the end of 48 hours, a radioactive precursor of DNA (H3-Tdr. 1 mCi / well) was added: 18 hours later, cells were collected and lysed,
Then, the radioactive substance incorporated in the DNA was calculated.

Inhibitors were added prior to antigen activation to maintain the culture overall. Four measurements were made for each concentration of inhibitor. The results were expressed as the mean of 4 replicates ± standard deviation of the sample.

4) Effect of Caryotoxin on Experimental Allergic Encephalomyelitis (EAE) Induced in Rats, an Animal Model of MS

EAE was induced by selectively transferring specific T lymphocytes (LcT) of myelin basic protein (MBP). The lymphocytes were from the Lewis rat TCD4 +, Th1, "PAS" lymphocyte line: they were activated by MBP for 48 hours before injection into normal syngeneic subjects.

[0080] Consisting of 5 200-gram Lewis rats (Charles River), 10 weeks old,
Five groups (AE) received 2-5 million specific LcT (strain TPAS) from basic myelin protein MBP via the intraperitoneal route on day 0. In groups B and C, KTX (0.8 and 8 μg / 0.1 ml PBS / rat) (PBS,
Phosphate buffer) was injected subcutaneously on the same day and daily until day 6 (D6). For the control group (Group A), 1 ml of PBS was given instead of KTX. Group D rats received 8 μg KTX after clinical signs of EAE were seen. Group E rats received on day 0 LcT (100 μg / ml) pre-treated with KTX in vitro and 8 μg KTX daily until D6. The rats in the sixth group (F) only received KTX (8 μg) from D0 to D6.

Another experiment was performed by administering different doses of KTX: 0.8,
8, 16, and 32 μg / rat / day. For the highest dose (32 μg), use 0.
One or two injections were made with 1 or 1 ml of PBS. The route of administration was subcutaneous: during a single experiment, it was compared to intravenous administration. The clinical observation of the animals is 1 per day,
Performed two or four times.

The rats were observed daily and weighed. The clinical symptoms of EAE (same as those obtained by aggressive provocation) were recorded between 0 and 4 as follows (Bourdoulous et al., 1995): 0 = neurological No symptoms 1 = flaccid tail 2 = posterior quarter paresis with tremors 3 = posterior quarter flaccid paralysis with incontinence, eye and nose bleeding 4 = moribund condition (forefoot) Dragging: dyspnea)

[0083] The clinical average of EAE was obtained by adding the strongest clinical symptoms in each rat: it was scored as zero, including rats showing no symptoms. For weight loss, the minimum and maximum body weight of each animal was examined during the 6 days of treatment.

5) Release of transmission block by caryotoxin

[0085] Two tests were performed. Rat optic nerve in vitro A nerve of the central nervous system that has been myelinated by oligodendrocytes associated with astrocytes. In Lewis rats, the optic nerve was removed and placed in a perfused container, oxygen was injected, and the temperature was controlled at 36 ° C. Recording of the overall activity of axons was performed with the aid of suction electrodes, which served as support for nerve endings. A second same electrode, placed at the other end, helped stimulate the nerve (maximum response: 0.1 ms,
Stimulation giving 10-30V). Controls performed were prepared in the presence of lymphocytes that reacted to myelin basic protein (2 × 10 ⁵ cells / ml) placed in DMEM culture medium.

Evoked potential (potential)

The functional status of the ascending fibers of the brainstem could be assessed by detecting the CNS evoked potential due to the stimulus applied to the hind paws of the rat. Detection was at the thalamus level. Under stereotactic control, metal electrodes were constantly implanted, two in the posterior abdominal lateral thalamic nucleus and the other two in the scalp. For animals anesthetized with chloral (0.1 g / rat), hind paw irritation was performed with the help of a bracelet surrounding the paw and containing two conductors. Stimulus threshold (2 ms, 25-60
V) was visually detected by contractile reflex. The evoked potential was obtained by averaging about 50 stimuli repeated every 5 seconds.

B) Results

1) Affinity of Caryotoxin for Kv Channel

According to the dose-response curve, channels Kv1.1 (IC50 = 1.5 nM), Kv1
.2 (IC50 = 24 nM) and Kv1.3 (IC50 = 0.1 nM) showed high sensitivity to KTX. Channel Kv3.1 was not sensitive. (IC50> 1 μM). In addition, by other authors, neuron channels Kv1.5 and Kv1.5
3.1 was shown to be insensitive to KTX (Grissmer et al., 1994).

Conclusion: This activity profile is dependent on the charge plan (char
ge plan) corresponded ideally: high affinity for lymphocyte Kv1.3 channels; high and selective for Kv1.2 and Kv1.1 axonal Kv channels compared to other neuronal channels. Affinity.

This activity profile is ideal for linking efficient immunosuppressive effects with neurological effects on axonal transmission, which allows to avoid the effects of aminopyridines to induce epilepsy. It is.

2) Blocking of Kv lymphocyte channels by KTX

Results: In the various lymphocyte lines tested, the sensitivity of the endogenous Kv channel, either structurally expressed or expressed after antigen activation, has a dissociation constant Kd of 1-2 nM.
Characterized by

Conclusion: Although weak, this sensitivity was close to the data obtained in the Kv1.3 endogenous channel expressed in heterologous egg cells.

3) In vitro effect of caryotoxin on proliferation of encephalitis-inducing T lymphocyte line

Results: A typical experiment is shown in Appendix 1. In four consecutive experiments, 1
0-50 nM KTX has been shown to inhibit uptake of radioactive precursors of DNA by up to 60%, and thus inhibit cell proliferation.

Conclusion: This data confirms the major role of the Kv1.3 channel in the proliferative role of T lymphocytes and the immunosuppressive effect of KTX.

4) Effect of KTX on EAE induced in rats

Results: According to the analysis, from a clinical point of view, treating rats with systemic administration of KTX (8 and 0.8 μg) from day 0 to day 6 indicates that the severity of the disease is not significant. And mortality has been avoided. The recovery was complete within 10 days. The results of Group D treated with 8 μg KTX for only 2 days after the onset of clinical symptoms were even more significant. The clinical symptoms did not exceed moderate severity of 1.1 and disappeared rapidly.

Conclusions: Treatment of Lewis rats with KTX improved the severity of EAE and weight loss from the day selected and transferred to the experiment. Efficient treatment of EAE could be achieved by treatment with KTX for only days 1 to 3 after the onset of clinical symptoms (see Table 1).

In the sense of these experiments, the optimal conditions to be observed are: 32 μg KTX / rat / day: the dose at which this result is most reproducible once daily subcutaneous injection of KTX in 1 ml PBS once daily animals Body weight measurement and clinical examination

[0103]

[Table 1]

EAE was induced in Lewis rats on day 0 by intraperitoneal injection of 2-5 million "PAS" T lymphocytes activated by MBP.

KTX was injected subcutaneously in PBS between days 1 and 3 after clinical symptoms were observed. By adding the maximal clinical symptom intensity of each rat, as assessed by the scale of Sommer et al. (Nature Med. 1995, Vol. 1, p. 247), the EA
A clinical average of E was obtained.

“N” represents the number of rats in each group. In the statistical analysis, Mann and
Performed by Whitney's test.

The data confirm the anti-inflammatory and immunosuppressive effects of caryotoxin in animals recognized as representatives of multiple sclerosis. In addition, the healing of slowly treated Group D animals, as described above, indirectly demonstrated the characteristic effects of KTX on the CNS Kv channel. This conclusion was confirmed by the following two tests.

5) Release of blockade of transmission by caryotoxin

Rat optic nerve in vitro

Results: In conjunction with the data of Yarom et al. (1983), incubation of rat optic nerve with T lymphocytes preactivated against myelin's basic protein in 1.5-2 hours. , The total potential (potential) derived by the suction electrode
Was able to block almost entirely. Therefore, after removing the lymphocytes,
In 2-3 hours, a partial recovery of the control activity could be obtained. On the other hand, K
Addition of TX (100 nM) was able to restore nerve transmission capacity quickly (15-20 minutes) even in the presence of lymphocytes.

Conclusion: This experiment demonstrates that transmission blockade in demyelinating central fibers can be relieved by acting on neuronal potassium channels. This also showed that caryotoxin also showed its effect at nanomolar concentrations due to its specificity.

Evoked potential (evoked potential)

Results: In animals where the clinical symptoms of EAE reached level 2 (hindral paralysis), the potential elicited by electrically stimulating the hind paws was performed prior to inducing EAE. As compared with the control record, it was suppressed by about 80 to 90% later. Subcutaneous injection of KTX (10 μg) resulted in significant recovery of evoked potential amplitude and latency. The recovery was complete in 90 minutes.

Conclusions: This experiment, performed in vivo, abolished the control of transmission resulting from the inflammatory process of fibers located in the lumbar nerve of the brainstem by early injection of KTX. The results confirmed the hypothesis about the positive neurological effects of KTX. These directly lead to the inhibitor slowly blocking the meningeal barrier,
Probably at the site of inflammation, it was shown to be able to pass.

This last conclusion is significant with respect to the possibility of restoring sufficient axonal transmission that can restore degenerated motor function.

[0116]

[Table 2]

[0117]

[Table 3]

[0118]

[Sequence list]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Gora, Maurice F-13400 Auvergne, France, Les Jardins Dewool 17 (72) Inventor Belo, Evelyn France, F-13400 Auvergne, Les Solans, Travels Garnier (72) Inventor Van Ritciyotan, Julfas France, E-13100 Aix-en-Provence, Avne des・ Ecole Mi Retail 48, Val de la Torres Aar 2 F term (reference) 4C084 AA02 BA01 BA08 BA19 BA23 ZA012 ZA332 ZB052 ZB072 ZB112 ZC412 4C087 AA02 BB18 BB21 CA16 MA01 NA14 ZA01 ZA3 3 ZB05 ZB07 ZB11 4H045 AA30 BA18 BA19 DA83 EA21 EA22

Claims (16)

[Claims] Claims 1. To prepare a medicament for the treatment of a neurological disease relating to an immune component in a human or animal, in which a block in neurotransmission occurs, use of Kv1.1, Kv1.2 neuronal potassium channels, and Kv1. .3 Use of compounds that inhibit lymphocyte potassium channels 2. The following neurological diseases relating to the immune component:-Inflammatory central neurological conditions relating to cerebral and medulla localization, in particular multiple sclerosis and related diseases (Varo, Devic, Hemorrhagic and acute sporadic encephalitis, optic neuropathy alone):-Inflammatory peripheral or related pathologies, especially:-Acute and chronic multiradiculoneuritis,-Infectious attacks and nerves associated with collagen disease. Disorders,-degenerative neurological disorders, especially amyotrophic lateral sclerosis, neuropathy with blockade of transmission,-tumor neuropathy with disturbances in the immune system: Use according to claim 1 for the preparation. 3. A scorpion venom-derived peptide compound or a compound derived from a scorpion venom, that is, the following general formula (I): (Wherein: - n1 and n2, independently of one another, 0 or _{1, - AA 9 ~AA 13,} AA 15 ~AA 17, AA 19 ~AA 28, AA 31 ~AA 33 and AA _{3 6} is , Independently of one another, representing a natural or unnatural amino acid). 4. A compound of the following formula (II), for example noxiustoxin represented by SEQ ID NO 1: (Wherein, AA ₂ , AA ₃ , AA ₁₂ , AA ₁₅ , AA ₁₆ , AA _{20 to} AA ₂₂ , AA ₂₄ , AA ₂₅ , A
A ₂₈ , AA ₃₁ , AA ₃₉ and AA ₄₀ independently of one another represent a natural or unnatural amino acid). 5. The following formula (III): (Wherein: AA ₆ , AA ₇ , AA ₉ , AA ₁₀ , AA ₁₂ , AA ₁₃ , AA ₁₅ , AA ₁₆ , AA ₁₇ , AA ₂₂ , AA ₂₄ , AA ₃₁ , AA ₃₃ , AA ₃₆ , AA ₃₈ , And AA ₃₉ independently of one another represents any natural or unnatural amino acid; -n1 represents 0 or 1). 6. A caryotoxin represented by SEQ ID NO 2 or the following formula (IV): (Wherein: na, nb and n1 independently represent 0 or 1;-AA ₃ , AA ₅ , AA ₇ , AA ₉ , AA ₁₀ , AA ₁₂ , AA ₁₃ , AA ₁₅ , AA ₁₆ , A
_{A 17, AA 22, AA 24} , AA 31, AA 33 and AA ₃₆ are independently of each other, the KTX derivatives corresponding to the representative) to any natural or unnatural amino acids, the use of claim 5, wherein. 7. A compound of formula (IV): wherein n1 = 0 and: nb = 1, for example the following peptide compounds: caryotoxin 2, SEQ ID NO 4 represented by SEQ ID NO 3 A compound such as caryotoxin 3 represented by the formula or agiotoxin 2 represented by SEQ ID NO 5 or-or nb = 0, such as KTX (6.37) represented by SEQ ID NO 6) The use according to claim 6, 8. A compound of formula (IV) wherein n1 represents 0 or 1, and:-for example, a (P12 / Avbu, P17 / Abu) KTX derivative represented by SEQ ID NO 7 At least one of AA ₁₂ or AA ₁₇ are either an amino acid (Abu), and / or, - for example, such as quince (Kaline) represented by SEQ ID NO 8, the AA ₂₄ young properly AA ₃₁ At least one represents a norisoleucine residue) or a KTX derivative of the following formula (V): Wherein AA _{1 to} AA ₇ , AA ₉ , AA ₁₀ , AA ₁₅ , AA ₃₃ , AA ₃₆ and AA _{38 to} AA ₄₀ independently of one another represent any natural or unnatural amino acid.
7. Use according to claim 6, of an X derivative. 9. A compound of formula (IV) wherein n1 = 1, in particular na = nb = 1, for example: a KTX derivative having a modified helix represented by the sequence SEQ ID NO 9, or Use according to claim 6, of a KTX derivative of (p12 / K, p17 / V, F22) KTX derivative represented by SEQ ID NO10. 10. A peptide compound derived from sea anemones or a compound derived from sea anemones, that is, a compound represented by the following general formula (VI): (Wherein: - N1 to N8 are independently of one another, 0 or _{1, - AA 2 -AA 2 -AA} 4 -AA 6 ~AA 11, AA 22, AA 23, AA 25, AA 28 ~A
A ₃₀ , AA ₃₂ , AA ₃₆ , and AA ₃₇ independently of one another represent any natural or unnatural amino acid). use. 11. The following peptide compounds:-Bgk corresponding to the amino acid sequence SEQ ID NO 11;-ShK corresponding to the amino acid sequence SEQ ID NO 12; or-Kaliseptine corresponding to the amino acid sequence SEQ ID NO 13: The use according to claim 10. 12. A composition comprising one or several peptide compounds as defined in one of claims 1 to 13 together with a pharmaceutically acceptable medium. Pharmaceutical composition. 13. The following formula (IV): (Wherein, - na, nb, and n1 are, independently of one another, denote 0 or _{1, - AA 3, AA 5} , AA 7, AA 9, AA 10, AA 12, AA 13, AA 15, AA ₁₆ , A
A ₁₇ , AA ₂₂ , AA ₂₄ , AA ₃₁ , AA ₃₃ and AA ₃₆ independently of one another represent any natural or unnatural amino acid, provided that when n1 = 0:-nb = 0 or Or AA ₂ represents glycine, AA ₅ represents proline, AA ₇ represents serine, AA ₉ represents lysine, AA ₁₀ represents histidine, AA ₁₂ represents glycine, and AA ₁₆ represents isoleucine; and / or at least one of AA ₁₂ or AA ₁₇ is aminobutyric acid (Ab
and / or at least one of AA ₂₄ or AA ₃₁ represents a norleucine residue). 14. Formula (IV): where n1 = 0 and: na = 0, nb = 1, AA_TwoRepresents glycine; AA_FiveRepresents proline and AA ₇ Represents serine and AA₉Represents lysine, AA_TenRepresents histidine, and AA₁₂Is
Stands for glycine and AA₁₆Stands for isoleucine, in particular for caryotoxin 3, represented by SEQ ID NO 4, or for nb = 0, in particular for KTX (6.37) 7 represented by SEQ ID NO 6, KTX according to claim 13 Derivatives. 15. A formula (IV) {wherein, n1 is 0 or 1, and: - at least one of AA ₁₂ or AA ₁₇ is amino acid (Abu), in particular, formula (IV) : (wherein, n1 = 0, na = nb = 1, AA 12 and AA ₁₇ represents an amino acid (Abu), for example, represented by SEQ ID NO 7 (P12 / Abu , P17 / Abu) KTX induction And / or at least one of AA ₂₄ or AA ₃₁ represents a norleucine residue, in particular of the formula (IV) wherein n 1 = 0, na = nb = 0, AA ₁₂ and A KTX derivative according to claim 13, wherein AA ₁₇ represents aminobutyric acid (Abu) and AA ₂₄ and AA ₃₁ represent norleucine residues, for example, a kaline compound represented by SEQ ID NO 8). Or the following formula (V): Wherein the AA _{1 to} AA ₇ , AA ₉ , AA ₁₀ , AA ₁₅ , AA ₃₃ , AA ₃₆ , and AA _{38 to} AA ₄₀ independently represent a natural or unnatural amino acid. 16. The formula (IV) ｛where n1 = 1, and particularly, na = nb =
The following compound which is 1, for example:-a KTX derivative with a modified helix, represented by the sequence SEQ ID NO 9,-a KTX represented by SEQ ID NO 10, (p12 / K, P17 / V, F22) The KTX derivative according to claim 13, which is a derivative｝.