EP2459188A1

EP2459188A1 - Use of rosmarinic acid and the derivatives thereof to treat ciguatera

Info

Publication number: EP2459188A1
Application number: EP10747916A
Authority: EP
Inventors: Dominique Laurent; Serge Pauillac; Mireille Chinain; Taiana Darius; Shilpa Kumar-Roine
Original assignee: Institut de Recherche pour le Developpement IRD
Current assignee: INSTITUT LOUIS MALARDE; Institut de Recherche pour le Developpement IRD
Priority date: 2009-07-31
Filing date: 2010-07-21
Publication date: 2012-06-06
Also published as: FR2948567A1; US20120196929A1; AU2016273875A1; JP2013500953A; WO2011012780A1; AU2010277458B2; JP5995235B2; FR2948567B1; US9060985B2; AU2010277458A1

Abstract

The present invention relates to rosmarinic acid and to the derivatives thereof, expressed by the compounds of general formula (I), for the use thereof to prevent and/or treat disorders caused by ciguatoxin poisoning and for preparing a pharmaceutical composition for preventing and/or treating disorders caused by ciguatoxin poisoning.

Description

Use of rosmarinic acid and its derivatives to treat ciguatera

The present invention relates to the use of rosmarinic acid and its derivatives for the manufacture of a medicament for the treatment of fish poisoning by food, in particular ciguatera.

Ciguatera is a form of ichthyosarcotoxism, that is, food poisoning by the flesh of fish contaminated with ciguatoxins (CTX) of the Gambierdiscus spp. Benthic microalgae found in coral reefs.

It is one of the most common fish poisoning forms in the Pacific, Indian Ocean and Caribbean regions.

Ciguatoxins enter the food chain through coral grazing fish and herbivores grazing algae on which Gambierdiscus toxicus is attached. These fish are then the prey of omnivorous and carnivorous fish; toxins are accumulated along the food chain. Among the 400 potentially infected species, large predatory fish such as barracuda, moray eels, grouper or jacks are the most likely to cause poisoning.

Ciguatoxins are liposoluble polyethers, thermostable, low molecular weight, they are among the most powerful marine biotoxins since it is barely a microgram to kill a man.

The purification and detection techniques by chromatography made it possible to isolate 39 different ciguatoxins. They vary according to their geographical origin (23 Pacific ciguatoxins or P-CTX, 12 Caribbean ciguatoxins or C-CTX and 4 Indian Ocean ciguatoxins or I-CTX), according to the fish species and according to their position in the food chain . Their molecular structures are not all yet elucidated.

Ciguatoxins are neurotoxins whose action on potential-dependent sodium channels is responsible for the clinical manifestations observed during intoxication. CTXs act by keeping the sodium channels open at potential values where normally they should be closed, resulting in spontaneous and repetitive onset of action potentials. The opening of the sodium channels is responsible for a massive influx of sodium ions into the cell, causing swelling; this phenomenon is particularly visible at the Ranvier nodes and at the axonal endings.

Nerve manifestations result from this action of toxins, both on the motor and sensory nerves and on the neuro-motor junctions. The Cardiac and digestive manifestations of the disease are also dependent on the action of ciguatoxins on the sympathetic and parasympathetic nervous system.

By ciguatoxins, is meant in particular, ciguatoxins of the Pacific or P-CTX-I to 23 of type 1 (Murata et al., 1989. J. Am Chem Soc., 111: 8929-31):

and type 2 (Satake et al., 1993. Tetrahedron Letter 34: 7P75-75):

ciguatoxins from the Caribbean or C-CTX-I to 12 (Lewis et al., 1998. J. Am Chem 120 S: 5914-20):

and ciguatoxins from the Indian Ocean or I-CTX-1 to 4 (Hamilton et al., 2002, Toxicon 40: 685-93). To date there is no cure for ciguatera; Western medicine offering only medications relieving the symptoms: anti-emetic and anti-diarrhea in cases of gastrointestinal disorders; Antihistamines against pruritus and analgesics in musculoskeletal disorders.

Mannitol is a compound that has been studied for the treatment of ciguatera; administered 48 to 72 hours after intoxication, mannitol is described as being able to relieve neurological and muscular disorders (Palafox et al, 1988, JAMA 259, 2740-2742, Blythe et al, 1994, Mem. , 465-470). However, blind clinical trials with mannitol and saline could not demonstrate a beneficial effect of mannitol on ciguatera symptoms after 24 hours (Schnorf et al., 2002, Neurol 58, 1155-1163). ); its effectiveness is therefore controversial.

Two products are currently being studied for the treatment of ciguatera:

two monoclonal antibodies anticiguatoxin whose simultaneous administration neutralizes in vitro and in vivo the action of ciguatoxin C-TX3C (Inoue et al, 2009, Toxicon doi: 10.1016 / j.toxicon.2009.02.17);

the brevenal, isolated from the dinoflagellate producing the Karenia brevis patentoxins (Bourdelais et al., 2005, J. Nat Prod., 68 (1), 2-6):

brevenal

It is a potent inhibitor of the neurotoxic effects induced by ciguatoxins and a potential treatment for ciguatera (Bourdelais et al, 2004 Molecular Neurobiology, 24 (4), 553-563, Mattei et al, 2008, cells, PLoS ONE 3 ( 10) 1-9). This compound is, however, difficult to synthesize and can only be obtained in small amounts from Karenia brevis cultures.

The traditional pharmacopoeia is richer in remedies than Western medicine: in the Pacific, many traditional herbal remedies are used to treat ciguatera.

Ethnopharmacological surveys conducted mainly in New Caledonia and Vanuatu have made it possible to draw up a list of nearly a hundred plants used in traditional medicine (Bourdy et al, 1992, J. Ethnopharmacology 36, 163-174; Laurent et al, 1993, ORSTOM Editions, Paris, pp. 51-116). In New Caledonia, the use of these remedies is extremely popular and the majority of the population prefers to go to healers instead of consulting doctors. Among these remedies, the decoction of leaves Heliotropium foertherianum Diana & Hilger is widespread in many islands of the South Pacific (New Caledonia, French Polynesia, Vanuatu, Tonga, Micronesia) and even in the Ryukyu Islands of Japan. According to a survey conducted among the population of Nouméa, more than 40% of the 500 people surveyed use this plant for treatment (Amade & Laurent, 1992, eds Gopalakrishnakone P. and Tan CK, 2, 503-508).

Widely distributed in Polynesia, Micronesia and Melanesia and also present in the Indian Ocean, Heliotr opium foertherianum is a coastal plant of the family Boraginaceae. Originally named Tournefortia argentea Lf, it was later named Argusia argentea (Lf) Heine and remained under this name until recently when it was renamed Tournefortia before being called Heliotropium in 2003 after molecular studies. It has also been listed under the scientific names Messerschmidia argentea (Lf) Johnston, T. arborea Blanco, T. sarmentosa sensu Non Christian Lam. and T. sericea Charn _¬ In vivo and in vitro studies on leaf extracts of Heliotropium foertherianum have demonstrated the effectiveness of this plant in controlling the symptoms of ciguatera. Evaluated on a weight loss test of the mouse poisoned by murine liver containing eiguatoxins, the leaf extract ά ⁷ H. foertherianum caused a decrease in induced body reduction (Amade & Laurent, 1992). This extract also showed identical effects to mannitol on isolated frog axons treated with Pacific ciguatoxin. Indeed, this extract inhibits the discharges of spontaneous and repetitive action potentials caused by the blocking of sodium channels in the open position by the eiguatoxins and the resulting sodium re-entry. It also inhibits the swelling of the Ranvier nodes of the myelin axons caused by the return of water responding to the osmotic change (Benoit et al., 2000, Cybium 24, 33-40). H. foertherianum also inhibits the swelling of human erythrocytes or frogs caused by ciguatoxin (Boydron et al, 2001, Meetings in Toxinology: Exploring, Exploiting Toxins and Controlling Producing Organisms, Scientific and Medical Editions Elsevier, Paris, pp. 63 -6 and Boydron et al, 2002, Meetings in Toxinology: Toxins and Biomedical Research, Elsevier Scientific and Medical Publications, Paris, pp. 101-104). Subsequently, using murine neuroblastomas (neuro-2a), leaf extracts from this plant were shown to exert a protective effect against the cytotoxic action of ciguatoxins and Brevetoxins (Boydron-Le Garrec et al., 2005). Toxicon 46, 625-634). Brevetoxins belong to another class of marine biotoxins that resemble ciguatoxins by their polyetheric structure and that bind to the same site of voltage-dependent sodium channels.

The inventors continued research on H. foertherianum with the aim of identifying the substance capable of combating the symptoms of ciguatoxins.

From a preparation of the remedy according to the method usually used (Laurent et al, 1993): 200 grams of yellowed leaves are boiled in 1 liter of water for 30 minutes, the inventors isolated by chemical fractionation bioguide the principle active ά 'H. foertherianum.

Structural analysis of the product is carried out by proton and carbon NMR and mass spectrometry; it allowed us to identify rosmarinic acid.

The present invention thus relates to compounds of general formula

(I):

in which :

R ₁ to R ₁₀ represent, independently of one another:

a hydrogen atom or a halogen atom;

o alkyl Ci-C ₆ acyl, C ₂ -C ₆ alkyl or an aryl radical of C ₃ -C _10;

o -O-Z with Z ₁ selected from the group consisting of hydrogen, alkyl Ci-C ₆ acyl, C ₂ -C ₆ alkyl, aryl C ₃ - Ci ₀ and a glycosyl radical;

a radical -SZ ₂ with Z ₂ selected from the group consisting of a hydrogen atom and a C ₁ -C ₆ alkyl radical; o a radical with Z ₃ and Z ₄ selected, independently of one another, from the group consisting of a hydrogen atom, an oxygen atom, a nitrogen atom, a C ₁ -C ₆ alkyl radical, a acyl, C ₂ -C ₆ alkyl, aryl C ₃ -cj ₀ and a glycosyl radical;

o a radical -COOZ ₅ or a radical with Z ₅ and Z ₆ selected, independently of one another, from a hydrogen atom and a C 1 -C ₆ alkyl radical; o a -CN function;

it being understood that two adjacent radicals selected from R 1 to R ₅ or from R ₆ to R ₁₀ may together form a ring selected from the group consisting of phenyl, naphthyl, furanyl, thiophenyl, pyrrolyl and triazolyl;

Xi to Xi ₂ represent, independently of one another, a carbon atom or a nitrogen atom;

Yi and Y ₂ represent, independently of one another:

a hydrogen atom or a halogen atom;

a C ₁ -C ₆ alkyl radical or a C ₃ -C ₁₀ aryl radical,

o a O-Z'i Radical with Z'i selected from the group consisting of hydrogen, alkyl CpC _6, a C ₂ -C ₆ acyl radical and an aryl radical C ₃ -CiO ;

o a radical with Z ' ₂ and Z' ₃ selected, independently of each other, from the group consisting of a hydrogen atom, an oxygen atom, a nitrogen atom, a C ₁ -C ₆ alkyl radical a C ₂ -C ₆ acyl radical and a C ₃ -C ₁₀ aryl radical;

o a radical -COOZ ' ₄ or a radical with Z ' ₄ selected from the group consisting of a hydrogen atom and a C ₂ -C ₆ acyl radical and Z' ₅ selected from the group consisting of a hydrogen atom, a C ₂ -C ₆ acyl radical; and a C ₃ -C ₁₀ aryl radical;

o a -CN function; Z represents an oxygen atom or a chain -NZ ₇ -, with Z ₇ selected from the group consisting of a hydrogen atom, a C ₁ -C ₆ alkyl radical and an aryl radical;

W ₁ and W ₂ represent, independently of one another:

o a hydrogen atom;

o a function -NO ₂ , -N ₃ or -CN;

o a radical -COOZ " ₁ or a radical with Z "j selected from the group consisting of hydrogen and an alkyl radical in C₁-C ₆ and Z" ₂ selected from the group consisting of a hydrogen atom, an alkyl radical in C ₁ -C ₆ and a C ₃ -C ₁₀ aryl radical;

their enantiomers (R, S) and their pharmaceutically acceptable salts,

for their use for the prevention and / or treatment of disorders caused by poisoning with ciguatoxins; the present invention also relates to the use of the compounds as defined above for the preparation of a pharmaceutical composition intended for the prevention and / or treatment of disorders caused by poisoning with ciguatoxins.

The compounds of general formula (I) that are the subject of the present invention are especially used after having been isolated, for example, from plant extracts; they can also be obtained isolated after a chemical synthesis.

Halogen atom means the chemical elements of group VII of the Periodic Table of Elements, in particular fluorine, chlorine, bromine and iodine.

The term "C₁-C alkyl-alkyl radical" means a linear or branched hydrogen-carbon radical of 1 to 6 carbon atoms.

O

By C ₂ -C ₆ acyl radical is meant a radical ^R JL ^R ', comprising 2 to 6 carbon atoms, R being an alkyl chain with m carbon atoms, m between 0 and 4, and R' a radical alkyl with n carbon atoms, n between 1 and 5, and such that m + n is between 1 and 5. By chain or alkyl radical is meant respectively a linear or branched chain or a hydrogenocarbon radical.

By C ₃ -C ₁₀ aryl radical is meant a mono- or bicyclic aromatic radical of 3 to 10 carbon atoms. Preferably the aryl radicals in C ₃ -C _O are selected from the group consisting of pyrrole, thiophene, furan, oxazole, isoxazole, thiazole, imidazole, pyrazole pyridinyl, pyrimidinyl, guanyl, naphthyl, quinolinyl, indolyl, benzofuranyl, benzothiophenyl.

By glycosyl radical is meant a monomer or dimer of residues glycosyl, mannosyl, glucuronyl, glucosamine and mannosamine.

Preferably, the compounds of general formula (I) are such that:

- Xi to Xj ₂ represent a carbon atom; and or

Y ₁ and Y ₂ represent a hydrogen atom.

A compound of general formula (I) which is preferred according to the invention is rosmarinic acid.

Rosmarinic acid ((R) -a - [[3- (3,4-Dihydroxyphenyl) -1-oxo-2E-propenyl] oxy] -3,4-dihydroxybenzenepropanoic acid, molecular weight 360 and crude chemical formula Ci _S Hi ₆ O ₈ ) is a polyphenolic compound of structure:

Rosmarinic acid

The chemical synthesis of rosmarinic acid has been described by Zinsmeister (Zinsmeister, H.D. et al, 1991, Angew Chem.103, 134-151).

Rosmarinic acid can also be produced by a biotechnological plant cell culture method (Hippolyte et al., 1992, 1. Plant Cell Rep.11, 109-112) or be isolated from plants (Lu et al., 1999 Phytochemistry 51, 91-94, Zgorka et al., J. J. Pharmaceut, Biomed 26, 79-87.).

This compound is known to present biological activities of interest: antiviral, antibacterial, anti-inflammatory and antioxidant (Petersen et al, 2003,

Phytochemistry 62, 121-125); It is commonly used in the food and cosmetics industries. However, it has never been described as likely to have a beneficial effect on food poisoning such as ciguatera.

The compounds of general formula (I) can be synthesized by peptide coupling (obtaining ester and amide analogues) between carboxylic acid units and alcohol / amine units as described by See-Hyoung Park et al. (Bioorganic and Medicinal Chemistry Letters 2003, 12 (20), 3455-3459). Disorders caused by poisoning with ciguatoxins are of several types:

- Digestive disorders:

They are the first to appear; they may be isolated or immediately associated with nervous signs. The most common symptoms are diffuse abdominal pain, nausea and vomiting and diarrhea, mostly watery. Other symptoms may occur such as tenesmus and hiccups.

- Disorders of the central and / or peripheral nervous system:

They appear in a few days, or even hours, they are the signs that can persist the longest (a few weeks, sometimes months). They can affect the entire nervous system.

Neurological signs that specifically target the central nervous system include cerebellar syndromes (cerebellar ataxia may be observed with disturbances of coordination and balance), central nervous system involvement, chronic fatigue (asthenia, lethargy, sleep disturbances with nightmares, early awakenings, intolerance to exercise, lack of motivation, various pains, etc.), headaches, dizziness, dizziness, visual or auditory hallucinations

Those targeting the peripheral nervous system most commonly include paresthesia (tingling, numbness, tingling) that are observed mainly in the extremities, the face (perioral, labial, lingual, pharyngeal); dysaesthesia, which are also frequent and considered as one of the most suggestive signs of ciguatera: the patient complains of burning sensations or electric shocks when in contact with cold objects; Ataxia can also be mentioned.

Other signs related to neurological disorders can be observed: swallowing disorders, convulsions, disorders of consciousness, coma, paralysis of the respiratory function.

Cardiovascular disorders:

They are usually considered to be the gravity of intoxication. When present, they occur early but rarely last more than a week. The most common findings are: sinus bradycardia less than 60 / min, hypotension, orthostatic hypotension lasting up to four weeks.

- Musculoskeletal disorders: Among them, can be mentioned: myalgia, arthralgia (especially large joints, especially the knees, ankles, shoulders and elbows).

- Skin disorders:

Pruritus is one of the main symptoms of ciguatera, so much so that it gave its name to the disease in New Caledonia: "the guitar". It is the palms of the hands and the soles of the feet that are most often affected. Pruritus can sometimes be generalized, especially after ingestion of alcohol or following a significant physical exercise, ie when the cutaneous blood flow is increased. The itching can be very severe and cause scratching lesions with their lot of complications: excoriations, abscesses, cellulitis, scars, lichenification.

Other skin disorders can be observed such as rashes: non-specific erythematous-papular eruptive lesions, sometimes with desquamation during the healing phase.

Thus, the compounds and / or the pharmaceutical compositions according to the invention are more particularly intended to treat diffuse abdominal pain and / or nausea and vomiting and / or diarrhea and / or cerebellar syndrome and / or chronic fatigue and / or headache and / or vertigo and / or dizziness and / or visual or auditory hallucinations and / or paresthesia and / or dysesthesia and / or disorders of swallowing and / or convulsions and / or disturbances of consciousness and / or coma and / or respiratory function paralysis and / or sinus bradycardia less than 60 / min and / or arterial hypotension and / or orthostatic hypotension and / or myalgia and / or arthralgia and / or pruritus and / or rash.

The amount of compound of general formula (I) and in particular rosmarinic acid to be administered to the mammal depends in particular on the severity of the pathology and symptoms to be treated, resulting in particular from the amount of ciguatoxin absorbed and the route by which it has been, it finally depends on the age and weight of the individual to be treated.

As rosmarinic acid is not very toxic, it can be administered in high concentration. By way of example and without limitation, the daily dose of rosmarinic acid or its derivative is between 0.1 mg and 1 g, preferably between 5 and 500 mg, taken in 1 to 3 times.

The pharmaceutical composition comprising a compound of general formula (I) may be suitable for oral, parenteral, pulmonary, ocular, nasal administration. The present invention will be better understood using the additional description which follows, which refers to examples of isolation and characterization of rosmarinic acid as the active ingredient of H. foertherianum and the evaluation of its activity against the effects of ciguatoxins; and the appended figures in which:

FIGURES

FIG. 1 is a graph showing the cellular viability of neuroblastomas in the presence of ciguatoxins contained in samples of toxic giant clam (GPS59) at 100 μg / ml with or without rosmarinic acid (in a range of 10 to 1000 μg / ml).

Figure 2 is the competition curve between radioligand ([ ³ H] -PbTx-

3) and rosmarinic acid on the sodium channels of a preparation of rat brain membranes. This curve shows the displacement of labeled brevetoxin ^[3 H] PbTx-3 its site of action by rosmarinic acid.

It goes without saying, however, that these examples are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

EXAMPLE

I- Isolation of the product

The active ingredient of / f. foertherianum was isolated by bioguided chemical fractionation.

Yellified leaves to H. foertherianum were harvested at the foot of a tree in the coastal region of New Caledonia (Anse Vata, Nouméa). The remedy is prepared according to the method usually used (Laurent et al, 1993). Briefly, 200 g of yellowing leaves are boiled in 1 liter of water for 30 minutes. The solution (about 750 ml) is then filtered and lyophilized to give 7.934 g of crude extract. 7.8 g are then redissolved in water and partitioned with n-butanol. 500 mg of the butanol phase are chromatographed on a silica column and eluted with 6 mixtures of dichloromethane and methanol of increasing polarity. The active principle (approximately 360 mg) is eluted mainly (90%) at CH ₂ Cl ₂ MeOH.

II- Analysis of the product

The structural analysis of the product is carried out by proton and carbon NMR using a Brucker ARX400 spectrometer. The mass spectrum is obtained on a Perkin Elmer API Sciex triple quadrupole spectrophotometer equipped with electrospray. The compound was identified as rosmarinic acid by comparison of mass spectral and NMR data with those of a control compound and those of the literature.

III- Toxicity of rosmarinic acid

The LD50 of rosmarinic acid was determined after injection of intravenous (iv) solution at 531 mg / kg in mice (Sigma-Aldrich record, derived from Petersen, 2003. Reference: Parnham, MJ, Kesselring, K., 1985. Rosmarinic Acid Drugs of the Future 10, 756-757.

These results show that at this concentration, rosmarinic acid does not show any toxicity.

IV- Activity of rosmarinic acid

IV.1. Inhibition of toxic toxicity caused by toxic loach extracts or toxic clams on cell cultures of neuroblastomas

The salmon lamb sample (Refl5), supplied by the Louis Malardé Institute, was collected at Mururoa (Tuamotu Archipelago, French Polynesia). The giant clam sample (GPS59) was collected at Raivavae (Austral Islands, French Polynesia). The toxicity of these two samples was previously confirmed on mouse, by a cytotoxicity test on neuroblastomas and radioligand tests with P-CTX-3.

The samples are stored at 4 ° C. Rosmarinic acid is from Sigma.

96-well plates are prepared with a number of 50,000 cells in 100 μl of medium per well. Outer wells are not used, to avoid variations in response due to edge effects. The cells are incubated for 24 hours at 37 ° C. with 5% CO ₂ to give them time to adhere.

A mixture Ouabaine (cardiotonic glycoside which intervenes in cell permeability by inhibiting the pump Na / K) and veratridine (polycyclic alkaloid which causes a persistent activation of the sodium channels), hereinafter designated O / V, in which the two assets are present respectively at a final concentration of 0.33 and 0.033 mM in 20 μl, is added to the wells.

Ouabain is an inhibitor of Na / K ATPase, and veratridine, an activator of sodium channels. The joint action of these two products induces a high intracellular level of sodium and causes an alteration of the morphology of the cells which significantly reduces their viability. The induction by these two compounds of sensitization to the toxins acting on the sodium channels makes it possible to visualize the cytotoxicity of the toxins. Are then added to extracts of loach (LST) or clam (GSP) predissolved in ₅ the culture medium to the desired concentration (final concentration per well of 250, 125 and 100 mcg / ml), and finally, the acid rosmarinique is added at different concentrations (final concentration per well between 10 and 1000 μg / ml). Each well is supplemented with the culture medium in order to obtain a final volume of 200 μl.

All plates have standard medium control (cells only) and control with cells in the presence of the O / V mixture. Rosmarinic acid is tested alone with the O / V mixture to record effects of cytotoxicity or cell proliferation, as well as possible staining interferences due to high product concentrations, which may lead to false positives in spectrophotometer reading. .

Each sample is tested in 3 replicates with and without rosmarinic acid but always in the presence of the O / V mixture.

The cells are thus incubated at 37 ° C. and 5% CO ₂ for 18 h.

After the incubation, the wells are emptied of their medium and the plate is allowed to dry on absorbent paper in order to carry out the MethylThiazolyldiphenyl-Tetrazolium (MTT) bromide test.

The MTT assay is based on the ability of viable cells (mitochondrial succinate dehydrogenase) enzymes to convert the yellow tetrazolium salt to PBS insoluble formazan crystals. An MTT solution to

0.8 mg / ml is prepared in PBS (phosphate buffered saline); 50 μl of this solution is added to each of the wells.

After incubation for one and a half hours, the formed formazan crystals are dissolved by means of 150 μl of DMSO (dimethylsulfoxide) for each well. After homogenization, the plates are read with a UV plate reader at 490 nm. Then data is transferred and processed in Excel® and analyzed with GraphPad software

Prism® (version 4).

The graph of FIG. 1 shows the cytotoxicity measured by the cellular viability of a toxic fatty clam extract (GPS59) at 100 μg / ml in the presence or absence of rosmarinic acid at different concentrations (10 to 1000 μg / ml). This graph clearly shows the protective effect of rosmarinic acid, the effect of this compound significantly increases cell viability from 50 μg / ml. Similar results were observed with toxic salmoneal loach extract. On the basis of these results, it can be estimated that the IC50 of rosmarinic acid (concentration of rosmarinic acid to reduce by 50% the cell mortality caused by ciguatoxins and associated toxins) is between 0.5 and 1 mg / ml. IV.2. Displacement of the ³ H ^" | PbTx-3-labeled Brevetoxin from its site of action (voltage-gated sodium channels)

Rat brain synaptosomes are prepared as previously described by Dechraoui et al. (1999, Toxicon 37 (1): 125-43). The protein concentration of the synaptosomal preparations is determined in duplicate by the Bradford method with bovine serum albumin as standard (Bradford, M.M., 1976, Anal Biochem 72: 248-254).

The final concentration of protein is of the order of 120 μg / ml.

After incubation of these preparations with [ ³ H] PbTx-3 tritinated streptoxin, a maximum of 10% of the total radioactivity is observed.

The ligand-receptor interaction detection test is carried out in a tube with a final concentration of 1,1 nM tritiated [ ³ H] PbTx-3, according to the protocol of Darius et al. (Darius et al, 2007 Toxicon 50 (5), 612-626). Radioactivity is determined using a Perkin Elmer Microbeta Trilux 1450 scintillation counter in 2 ml counting vials of a Betaplate scintillation liquid (Perkin Elmer).

The non-specific interaction is evaluated by measuring the radioactivity in the presence of a saturating [ ³ H] PbTx-3 (0.67 mM) concentration and subtracted from the total interaction to lead to the specific interaction.

Ciguatoxin P-CTX-3C (obtained from a clonal culture of Gambierdiscus polynesiensis) is known to have a better affinity to the site of action than Brevetoxins; it has been used as an internal standard for sample calibration (Chinain et al, 1999, Darius et al, 2007).

Two quality control tubes of radioligand experiments are used at each experiment; they are composed of a toxic extract of Gambierdiscus polynesiensis with a known concentration of P-CTX-3C ciguatoxin equivalent to 3.1 μg.

Eight dilutions in duplicate were tested from 2 concentrations of rosmarinic acid at 5 and 20 mg / ml.

The sigmoidal curves and IC ₅₀ values are obtained using the Graphpad Prism v4.0 software. L ⁵ IC ₅₀ is defined as the concentration of extract capable to induce 50% inhibition of the interaction with [ ³ H] PbTx-3 and is expressed in equivalent mg / ml of extract.

Figure 2 shows the competition curve between the radioligand ([ ³ H] -

PbTx-3) and rosmarinic acid on the sodium channels of a rat brain membrane preparation. In the presence of rosmarinic acid at 5 and 20 mg / ml, there is a decrease in the measured radioactivity indicating a replacement of the tritiated toxoxine on the sodium channels with rosmarinic acid.

It is thus possible to calculate the value of the IC50: 0.88 mg / ml and the value of Ki at 1.43 × 10 ^-3 M

V- Estimation of the amount of active ingredient ingested by the man during a treatment with the traditional remedy

The level of rosmarinic acid is estimated at 1.2 mg / g of fresh leaves of Heliotropium foertherianum; for leaves of about 10 grams, a dozen mg of rosmarinic acid and for a remedy based on a decoction of 4 to 5 leaves, about fifty mg of this product.

This is an indicative value, the dosage of a traditional remedy being very variable; moreover, the content of the active substance of the remedy may also vary according to whether the plants are used at sea or inland.

Claims

1. Isolated Compounds of General Formula (I):

in which :

R ₁ to R ₁₀ represent, independently of one another:

a hydrogen atom or a halogen atom;

o alkyl Ci-C _6, a radical acyl, C ₂ -C ₆ alkyl or an aryl radical C3-C10;

a radical-O-Z ₁ with Z ₁ selected from the group consisting of a hydrogen atom, a C ₁ -C ₆ alkyl radical, a C ₂ -C ₆₅ acyl radical and a C ₃ -C ₁₀ aryl radical; and a glycosyl radical;

a radical -SZ ₂ with Z ₂ selected from the group consisting of a hydrogen atom and a C ₁ -C ₆ alkyl radical;

o a radical with Z ₃ and Z ₄ selected, independently of one another, from the group consisting of a hydrogen atom, an oxygen atom, a nitrogen atom, a C ₁ -C ₆ alkyl radical, a acyl, C ₂ -C ₆ alkyl, aryl C ₃ -C ₀ and a glycosyl radical;

o with Z ₅ and Z ₆ selected, independently of one another, from a hydrogen atom and a C ₁ -C ₆ alkyl radical; o a -CN function;

Xi to X ₁₂ independently of one another, a carbon atom or a nitrogen atom; Yi and Y ₂ represent, independently of one another:

a hydrogen atom or a halogen atom;

a C ₁ -C ₆ alkyl radical or a C ₃ -C ₁₀ aryl radical,

o -O-Z'i with Z'j selected from the group consisting of hydrogen, Cj-C ₆ alkyl radical, an acyl radical C ₂ -C ₆ alkyl and aryl C ₃ -CiO;

o a radical and Z ' ₃ selected, independently of one another, from the group consisting of a hydrogen atom, an oxygen atom, a nitrogen atom, a C ₁ -C ₆ alkyl radical, a radical acyl, C ₂ -C ₆ alkyl and aryl C ₃ -C _(O;

o a -CN function;

Z represents an oxygen atom or a -NZ ₇ - chain, with Z ₇ selected from the group consisting of a hydrogen atom, a C ₁ -C ₆ alkyl radical and a C ₃ -C ₁₀ aryl radical;

Wi and W ₂ represent, independently of one another:

o a hydrogen atom;

o a function -NO ₂ , -N ₃ or -CN;

o a radical -COOZ "1 or a radical with Z "1 selected from the group consisting of a hydrogen atom and a C ₁ -C ₆ alkyl radical and Z" ₂ selected from the group consisting of a hydrogen atom, a C ₁ -C ₆ alkyl radical and a C ₃ -C ₁₀ aryl radical;

their enantiomers (R ₃ S) and their pharmaceutically acceptable salts,

for their use for the prevention and / or treatment of disorders caused by poisoning with ciguatoxins.

2. Compound according to claim 1, characterized in that it is such that: X ₁ to Xi ₂ represent a carbon atom and / or Y ₁ and Y ₂ represent a hydrogen atom.

3. Compound according to claim 1 or claim 2, characterized in that said compound is rosmarinic acid.

4. Compound according to any one of the preceding claims, characterized in that the ciguatoxins are selected from the Pacific ciguatoxins or P-CTX-I to 23, the ciguatoxins of the Caribbean or C-CTX-I to 12 and the ciguatoxins of the Indian Ocean or I-CTX-1 to 4.

5. Compound according to any one of the preceding claims, characterized in that said disorders caused by poisoning with ciguatoxins are digestive disorders and / or disorders of the central and / or peripheral nervous systems and / or cardiovascular disorders and / or musculoskeletal disorders and / or skin disorders.

6. Compound according to claim 5, characterized in that said disorders are diffuse abdominal pain and / or nausea and vomiting and / or diarrhea and / or cerebellar syndrome and / or chronic fatigue and / or headache and / or or dizziness and / or dizziness and / or visual or auditory hallucinations and / or paresthesia and / or dysesthesia and / or swallowing disorders and / or convulsions and / or disturbances of consciousness and / or coma and / or paralysis of respiratory function and / or sinus bradycardia less than 60 / min and / or arterial hypotension and / or orthostatic hypotension and / or myalgia and / or arthralgia and / or pruritus and / or or rashes.

7. Compound according to any one of the preceding claims, characterized in that its use is such that its daily dose is between 0.1 mg and 1 g per intake in 1 to 3 times.

8. A compound according to any one of the preceding claims, characterized in that it is used for the preparation of a pharmaceutical composition adapted for oral, parenteral, pulmonary, ocular, nasal administration.