HK1152892B - Combination of a bis-thiazolium salt or a precursor thereof and artemisinin or a derivative thereof for treating acute malaria - Google Patents

Description

Combination of salts of Chinese pulsatilla thiazolicola or precursors thereof and artemisinin or derivatives thereof for the treatment of acute malaria

Technical Field

The invention relates to a novel combination of active ingredients against malaria, namely dithiazolesA combination of a salt or a precursor thereof and artemisinin or a derivative thereof and to a pharmaceutical composition comprising such a combination for the treatment and/or prevention of malaria.

Background

Malaria (malaria) is one of the primary causes of infectious death worldwide and affects more than 5 million individuals each year, with 150 to 300 million individuals dying each year. Such mishaps occur primarily in sub-Saharan Africa, southeast asia and latin america.

Malaria is transmitted by four types of parasites of the genus Plasmodium (Plasmodium) transported by Anopheles mosquitoes (Anopheles mosquitoes) (p.falciparum), Plasmodium malariae (p.malariae), Plasmodium vivax (p.vivax) and Plasmodium ovale (p.ovale)). Plasmodium falciparum (plasmodium falciparum), which is widespread in africa, is the most deadly parasite and responsible for most of the fatal forms of the disease.

The strong prevalence of disease observed in recent years has been attributed to several factors, including the resistance of many plasmodium falciparum strains to currently used drugs, such as chloroquine, mefloquine, amodiaquine, or antifolates (antifolates) and antifolates (antifolates), such as pyrimethamine and sulfadoxine.

Many individuals with malaria present with acute infections with few signs other than mild anemia and increased spleen volume (splenomegaly). Symptoms depend on the type of malaria. However, the general clinical features are in particular: discomfort (feeling unwell), hyperthermia (fever), headache, digestive problems such as nausea, vomiting and/or abdominal pain, diarrhea, weakness, muscle pain, joint pain (artralgia), and jaundice.

Treatment is usually carried out by administration of chloroquine (or basic quinine hydrochloride if resistance to chloroquine occurs) until improvement is obtained and the parasite disappears in the blood (usually between 3 and 5 days). The patient then typically receives a single dose of a drug consisting of 1.5g of sulfadoxine and 75mg of pyrimethamine.

Other antifolate and antifolate-based therapies or alternatively malarone-based therapies are also used.

Severe malaria caused by plasmodium falciparum is cerebral malaria (cerebral malaria) with substantial elevated temperature (40 ℃) and coma (coma) which, even treated, has a poor prognosis and whose mortality rate sometimes rises to 20% in adults and 15% in children. Severe malaria manifests itself as either gradual or sudden. It occurs after transient and transient twitching of one or more muscles, followed by decontractions. They are located or distributed throughout the body. This type of malaria is accompanied by nystagmus (the eye beating endlessly from side to side), sometimes with neck rigidity and reflex disturbances. Severe malaria can also be accompanied by retinal hemorrhage, hypoglycemia, pulmonary edema, renal involvement (kidney involvements), anemia, and/or hematemesis (blood reflux from damaged organs, blood swallowed and then reappeared as vomitus with bleeding).

In complex malaria or cerebral malaria and other severe manifestations caused by plasmodium falciparum, it is a medical emergency when 1% of red blood cells (red blood cells) or more are parasitic in individuals who have not yet been immunized against the disease.

In the event of severe malaria, the preferred treatment is to reduce parasitemia (parasitemia) largely and rapidly (within 24 to 48 hours after treatment initiation) to rule out a fatal prognosis. Once parasitemia is controlled and an extremely important prognosis is confirmed, more conventional anti-malarial treatments, such as chloroquine or quinine hydrochloride, for example, may be administered.

Treatment of severe malaria is difficult to administer orally or rectally because patients often suffer vomiting with a large amount of diarrhea.

Artemisinin, which was isolated in 1972 from the plant artemisinin (artemia annua) and has been used in china for centuries, has potent antimalarial activity. Derivatives with improved pharmacological properties such as artemether, arteether and artesunate are also commercially available.

Artemisinin and its derivatives, in particular artesunate, are nowadays part of the most potent active ingredient against plasmodium falciparum. However, these compounds have difficulty in completely restoring the disease and a flooding of the disease is noted. Thus, the use of artemisinin or its derivatives in monotherapy may be a causal factor in the selection of anti-parasitic strains.

The scientific organisation currently recommends the use of combinations of active ingredients, and in particular artemisinin or its derivatives with other antimalarial active ingredients. These multidrug therapies, named ACT (artemisinin based combination therapy), have been recognized by the World Health Organization (WHO) since 2002. They offer multiple advantages: improved therapeutic efficacy against drug-resistant strains, mutual protection of the two active ingredients against the manifestation of drug resistance, reduction of disease transmission and reduction of drug resistance transmission.

Several combinations of this type are known.

For example, have been proposed toThe name of (1) is marketed as a combination between artemether and lumefantrine, just like artesunate and amodiaquineAs in combinations of (a).

The combination of ferrocenequine (ferroquinone) with artemisinin derivatives is described in document WO 2006/111647.

However, the beneficial effects of ACT multidrug therapy are neither obvious nor predictable. For example, the publication Experimental parasitity, 115(2007), 296-300 to C.Snyder et al discloses that the combination of piperaquine with the compound OZ277 shows promise over the combination of piperaquine with artemisinin derivatives such as artemesinol methyl ether.

Similarly, the combination of chloroquine and artesunate did not achieve a satisfactory level of efficacy (am.J. trop. Med. Hyg., 2003, 69(1), 19-25 and Transactions of the Royal Society of clinical and Hygiene, 2003, 97, 429-.

Thus, even if the ACT strategy is approved by the WHO, the search for new combinations of antimalarial active ingredients is still not easy and should continue.

Disclosure of Invention

The invention relates to dithiazolesCombinations of salts (bisthiazolium salt) and artemisinin or derivatives thereof, the dithiazolesA salt is a compound of formula (VI) as defined below or a precursor thereof. Among the artemisinin derivatives used in the present invention, mention may be made of, for example, artesunate, artemether, arteether, hydroartemisinin or dihydroartemisinin.

The combination shows advantageous and unexpected synergistic effects and is therefore useful for use in the prevention and/or treatment of malaria, in particular severe malaria.

The compounds of formula (VI) and their precursors for the purposes of the present invention are described in the patent publication EP 1196371. For the purposes of the present invention, the dithiazoles of the formula (VI)The precursors of the salts correspond to formulae (I), (II), (III), (IV) and (V) of EP 1196371. In fact this patent describes prodrugs having an antimalarial effect, characterized in that they are products capable of producing diquaternary ammonium salts and they correspond to general formula (I):

wherein

-a and a' are identical to or different from each other and represent:

groups A which are each of the formula₁And A'₁：

Wherein n is an integer from 2 to 4; r'₁Represents a hydrogen atom, C₁To C₅Alkyl optionally substituted with aryl (particularly phenyl), hydroxy, alkoxy, whereinAlkyl groups containing 1 to 5 carbon atoms, or aryloxy groups (specifically phenoxy groups);

and W represents a halogen atom selected from chlorine, bromine or iodine, or a nucleofugic group such as tosylCH₃-C₆H₄-SO₃-, methylsulfonyl CH₃-SO₃-、CF₃-SO₃-group or NO₂-C₆H₄-SO₃-a group of,

or a radical A₂Which represents formyl-CHO or acetyl-CO-CH₃，

-B and B' are identical to or different from each other and represent:

if A and A' each represent A₁And A'₁Each is a group B₁And B'₁，B₁And B'₁Represents a group R₁It has R 'as above'₁But not a hydrogen atom,

or if A and A' represent A₂Are respectively B₂And B'₂Then B is₂Or B'₂Is a group R as defined above₁Or a group of the formula:

wherein-Ra represents an RS-or RCO-group, wherein R is a linear, branched or cyclic C₁To C₆Especially C₁To C₅Alkyl, optionally substituted with one or more hydroxy, alkoxy or aryloxy groups, or amino and/or-COOH or COOM groups, where M is C₁To C₃An alkyl group; phenyl or benzyl, wherein the phenyl is optionally substituted by at least one C₁To C₅Alkyl or alkoxy, said radicals being optionally substituted by amino groups or nitrogen-or oxygen-containing heterocyclyl radicals, -COOHA group or a-COOM group; or a group-CH₂-heterocyclyl, wherein said heterocyclyl has 5 or 6 atoms and is nitrogen-and/or oxygen-containing (oxygenated);

R₂represents a hydrogen atom, C₁To C₅Alkyl or-CH₂-COO(C₁To C₅) An alkyl group;

and R is₃Represents a hydrogen atom; c₁To C₅Alkyl or alkenyl, said C₁To C₅Alkyl or alkenyl groups, where appropriate substituted by-OH, phosphate group, alkoxy, wherein the alkyl group is C₁To C₃Alkyl, or aryloxy; or an alkyl (or aryl) carbonyloxy group;

or R₂And R₃Together form a ring containing 5 or 6 carbon atoms; r and R₃Can be linked to form a ring containing 5-to 7 atoms (carbon, oxygen, sulfur);

- α represents:

when A and A ' represent A1 and A ' 1, or when A and A ' represent A₂I.e. -CHO or-COCH₃A group, and B₂And B'₂Represents:

when the temperature of the water is higher than the set temperature,

alpha represents a single bond

Or when A and A' represent A₂And B₂And B'₂Represents R₁When, α represents a group of the formula:

or a group of the formula:

wherein (a) represents a bond to Z and (b) a bond to a nitrogen atom,

-Z represents C₆To C₂₁In particular C₁₂Or C₁₃To C₂₁Alkyl, where appropriate interrupted by one or more multiple bonds and/or one or more heteroatoms O and/or S, and/or one or more aromatic rings,

and pharmaceutically acceptable salts of these compounds, with the proviso that when n ═ 3 or 4, R₁Is represented by C₁To C₄Alkyl and Z represents C₆To C₁₀Alkyl is R'₁Not representing H or C₁Or C₂An alkyl group.

EP 1196371 also describes a subgroup of precursor compounds as described above, corresponding to the general formula (II):

wherein R is₁、R′₁W, n and Z are as defined above.

EP 1196371 also describes a subgroup of precursor compounds, including precursors as defined above, wherein Z represents C₁₃To C₂₁An alkyl group.

EP 1196371 also describes a subgroup of precursor compounds, including the precursors described in the preceding subgroup, in which Z represents- (CH)₂)₁₆-a group.

EP 1196371 describes in particular precursors selected from N, N '-dimethyl-N, N' - (5-chloropentyl) -1, 16-hexadecanediamine dihydrochloride or N, N '-dimethyl-N, N' - (4-chloropentyl) -1, 16-hexadecanediamine dihydrochloride.

EP 1196371 also describes the following group of precursors, corresponding to general formula (III):

or corresponds to general formula (IV):

or corresponds to general formula (V):

wherein Ra, R₁、R₂、R₃And Z is as defined above.

EP 1196371 describes in particular precursor compounds of formulae (III), (IV) and (V) as defined above, selected from:

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-S-sulfanylbenzoyl-4-methoxybut-1-enyl ] -1, 12-diaminododecane (TE4c),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-S- (p-diethylaminomethylphenylcarboxy) thio-4-methoxybut-1-enyl ] -1, 12-diaminododecane (TE4f),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-S- (p-morpholinomethylphenylcarboxy) -sulfanyl-4-methoxybut-1-enyl ] -1, 12-diaminododecane (TE4g),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-S-sulfanylbenzoyl-4-methoxybut-1-enyl ] -1, 16-diaminohexadecane (TE8),

-N, N '-diformyl-N, N' -bis [1- (2-oxo-4, 5-dihydro-1, 3-oxathian-4-ylidene) ethyl ]1, 12-diaminododecane (TE 3);

or is selected from:

-N, N '-diformyl-N, N' -bis [ 1-methyltetrahydrofurfuryl-2-ylmethyldithio-4-hydroxybut-1-enyl ] -1, 12-diaminododecane (TS3a),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-propyldithio-4-hydroxybut-1-enyl ] -1, 12-diaminododecane (TS3b),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-benzyldithio-4-hydroxybut-1-enyl ] -1, 12-diaminododecane (TS3c),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2- (2-hydroxyethyl) dithio-4-hydroxybut-1-enyl ] -1, 12-diaminododecane (TS3d),

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-propyldithio-4-methoxybut-1-enyl ] -1, 12-diaminododecane (TS3d) and

-N, N '-diformyl-N, N' -bis [ 1-methyl-2-propyldithiovinyl ] -1, 12-diaminododecane (TS6b),

or is selected from:

-2, 17- (N, N '-diformyl-N, N' -dimethyl) diamino-3, 16-S-sulfanyl p-methoxybenzoyl-6, 13-dioxaoctadeca-2, 16-diene (TE9),

-2, 17- (N, N '-diformyl-N, N' -dibenzyl) diamino-3, 16-S-sulfanyl p-methoxybenzoyl-6, 13-dioxaoctadeca-2, 16-diene (TE10),

-ethyl 3, 18- (N, N '-diformyl-N, N' -dimethyldiamino-4, 17-S-sulfanylbenzoyl-eicosa-3, 17-dienoate (TE12),

-ethyl 3, 18- (N, N '-diformyl-N, N' -dibenzyl) diamino-4, 17-S-sulfanylbenzoyl-eicosa-3, 17-dienedioate (TE13),

or finally selected from:

-2, 15- (N, N '-diformyl-N, N' -dimethyl) diamino-1, 16-S-sulfanylbenzoylhexadeca-1, 15-diene (TE15),

-2, 15- (N, N '-diformyl-N, N' -dibenzyl) diamino-1, 16-S-sulfanylbenzoylhexadeca-1, 15-diene (TE 16).

EP 1196371 also describes the synthesis of thiazolesSalt precursors give rise to cyclized derivatives which have good antimalarial activity and correspond to the general formula (VI):

wherein

Rb represents R₁Or T, T represents a group of formula:

provided that when Rc, Rd, R₂And R₃When representing a methyl group, or when Rc and Rd together form an aromatic ring containing 6 carbon atoms on the one hand, and R on the other hand₂And R₃When taken together to form an aromatic ring containing 6 carbon atoms, Z does not represent C₆To C₈An alkyl group, a carboxyl group,

rd represents R₂Or P, P represents a group of formula:

rc represents R₃Or U, U represents a group of formula:

R₁、R₂、R₃and Z is as defined above, and wherein,

it is understood that if Rc ═ R₃And Rd ═ R₂Then Rb is T; if Rc ═ R₃And Rb ═ R₁Then Rd ═ P; and if Rb is R₁And Rd ═ R₂And Rc ═ U.

According to the invention, the compound of formula (VI) may be in the form of a free base, a salt, a hydrate or a solvate.

In particular, among the cyclized derivatives described above, the derivatives mentioned in EP 1196371 are selected from:

-1, 12-dodecamethylenebis [ 4-methyl-5- (2-hydroxyethyl) thiazole]Dibromide (T3);

-1, 12-dodecamethylenebis [ 4-methyl-5- (2-methoxyethyl) thiazole]Diiodide (T4);

-1, 12-dodecamethylenebis (4-methylthiazole)) Diiodide (T6);

-1, 16-hexadecamethylenedi [ 4-methyl-5- (2-methoxyethyl) thiazole]Diiodide (T8);

-3, 10-dioxadodecamethylenebis [5- (1, 4-dimethyl) thiazole]Diiodide (T9);

-3, 10-dioxadodecamethylenebis [5- (1-benzyl-4-methyl) thiazole]Dibromide (T10);

dodecamethylenebis [5- (1-methyl-4-ethoxycarbonylethyl) thiazole]Diiodide (T12);

dodecamethylenebis [5- (1-methyl-4-ethoxycarbonylethyl) thiazole]Dibromide (T13);

-dodecamethylenebis [4- (1-methyl) thiazole]Diiodide (T15);

-dodecamethylenebis [4- (1-benzyl) thiazoleDibromide (T16).

Finally, EP 1196371 describes the obtaining of the above-mentioned dithiazolesSalts and dithiazolesMethods for precursors, in particular the synthesis of compounds of formula (VI) such as T3, T4, T6, T8, T9, T10, T12, T13, T15 and T16. In particular, the synthesis of T3 is described on page 18 of EP 1196371. Similarly, the antimalarial activity of the T3 compound is described in table 13, page 28 of the same document.

The invention therefore relates to combinations comprising as active ingredients dithiazolesSalts and artemisinin or its derivatives, said dithiazolesThe salt is a compound of formula (VI) or a precursor thereof.

According to a second subject, the invention relates to dithiazolesCombinations of salts and esters of artesunic acid, said dithiazolesThe salt is a compound of formula (VI) or a precursor thereof.

According to a third subject, the invention relates to dithiazolesCombinations of salts and esters of artesunic acid, said dithiazolesA salt is a compound of formula (VI) selected from:

-1, 12-dodecamethylenebis [ 4-methyl-5- (2-hydroxyethyl) thiazole]Dibromide (T3);

-1, 12-dodecamethylenebis [ 4-methyl-5- (2-methoxyethyl) thiazole]Diiodide (T4);

-1, 12-dodecamethylenebis (4-methylthiazole)) Diiodide (T6);

-1, 16-hexadecamethylenedi [ 4-methyl-5- (2-methoxyethyl) thiazole]Diiodide (T8);

-3, 10-dioxadodecamethylenebis [5- (1, 4-dimethyl) thiazole]Diiodide (T9);

-3, 10-dioxadodecamethylenebis [5- (1-benzyl-4-methyl) thiazole]Dibromide (T10);

dodecamethylenebis [5- (1-methyl-4-ethoxycarbonylethyl) thiazole]Diiodide (T12);

dodecamethylenebis [5- (1-methyl-4-ethoxycarbonylethyl) thiazole]Dibromide (T13);

-dodecamethylenebis [4- (1-methyl) thiazole]Diiodide (T15);

-dodecamethylenebis [4- (1-benzyl) thiazole]Dibromide (T16).

According to a fourth subject matter, the invention relates to dithiazoles of formula (VI)A combination of a salt and artesunate, wherein formula (VI) is 1, 12-dodecamethylenebis [ 4-methyl-5- (2-hydroxyethyl) thiazoleDibromide (T3).

In these subjects, each active ingredient is used at a concentration that reflects their respective efficacy in treating infections. The respective amounts of these compounds in each combination are determined by their active dosages.

1, 12-dodecamethylenebis [ 4-methyl-5- (2-hydroxyethyl) thiazole present in the combinations according to the invention]The dibromide (T3) has the following structure:

the artesunate present in the combination of the invention has the following structure:

the subject of the invention is also a pharmaceutical composition comprising, as active ingredient, a dithiazole of formula (VI) as defined aboveA combination of a salt or a precursor thereof, in particular T3, and artemisinin or a derivative thereof, in particular artesunate.

The pharmaceutical composition contains a therapeutically effective amount of dithiazoleSalt and artemisinin or its derivatives, and at least one pharmaceutically acceptable excipient, said dithiazoleThe salt is a compound of formula (VI) or a precursor thereof. The excipients are selected from conventional excipients known to those skilled in the art, depending on the pharmaceutical dosage form and the desired method of administration. The range of ratios of each compound is defined in terms of its dose, which exerts antimalarial activity in monotherapy and reflects their respective efficacy in treating infections.

Suitable unit dosage forms include oral dosage forms such as tablets, soft or hard gelatin capsules, powders, granules and oral solutions or suspensions, sublingual dosage forms, buccal (baccal) dosage forms, intratracheal dosage forms, intraocular dosage forms or intranasal dosage forms, dosage forms for administration by inhalation, topical administration, transdermal administration, subcutaneous administration, intramuscular administration or intravenous administration (bolus injection or infusion), rectal dosage forms and implants. For topical administration, the compounds of the present invention may be used in creams, gels, ointments or lotions.

The routes of administration envisaged are the oral, rectal and parenteral routes, in particular the intramuscular, intrarectal and intravenous routes, especially via the drip (clip) route.

For parenteral administration, aqueous suspensions, isotonic saline or sterile and injectable solutions may be used, containing pharmacologically compatible dispersing and/or wetting agents, such as propylene glycol or butylene glycol. If necessary, the suspension may also contain anti-oxidant adjuvants, or the active ingredient of the invention may be diluted in a suitable solution, such as glucose or NaCl, for subsequent administration by instillation.

For parenteral administration, and in particular administration by instillation, each preferred daily dose of the combination of the two active ingredients of the invention is as indicated above:

dithiazoles of formula (VI)Salts, in particular T3: the dosage is between 0.01 and 3 mg/kg;

artemisinin or its derivatives, in particular artesunate: the dosage is between 1 and 5 mg/kg.

Special cases for high or low doses are also included; such dosages do not depart from the scope of the present invention. The dosage appropriate for each patient is determined by a physician according to the method of administration and the weight and responsiveness of the patient in question, according to routine practice.

The combination of the invention is intended for continuous administration for 1 to 4 days, with one or more intakes of each of the two active ingredients per day, preferably 1 to 3 intakes per day (oral or rectal) or as a bolus. The treatment time is preferably limited to 4 days or less, which is particularly advantageous for the massive and rapid reduction of parasitemia and allows the following self-treatment of malaria in male or female patients after avoiding their fatal prognosis. The subsequent treatment can be by monotherapy with artemisinin derivatives, any other known method of treating malaria and method of administering the combination of the invention.

An example of a dose and method of administration may be the first 24 hours of treatment (D1) of a patient presenting with severe malaria: 2.4 mg/kg/day artesunate and 1 mg/kg/day T3, followed by the next 48 hours of treatment (D2 and D3): 1.2 mg/kg/day artesunate and 0.8 mg/kg/day T3, artesunate and T3 were administered by drip in a single drip bag (drip bag) at D1 and in separate drip bags at D2 and D3.

Administration of each of the two active ingredients can be carried out simultaneously or separately over time (continuous administration).

When administered simultaneously, the two active ingredients may be combined in a single pharmaceutical dosage form (fixed combination), such as a tablet or gel suitable for oral administration, or combined in the same infusion bag, or administered in a formulation suitable for rectal administration.

The administration of the two active ingredients of the combination of the invention, whether administered simultaneously or not, may be in different pharmaceutical forms. To achieve this effect, the combination of the invention can also be in the form of a kit comprising, on the one hand, a compound of formula (VI) such as T3 and artesunate in separate compartments and intended for administration simultaneously, separately or in a temporally dispersed (sequential) manner, or a precursor thereof, in particular T3 or a salt, hydrate or solvate thereof, and, on the other hand, artemisinin or a derivative thereof, in particular artesunate.

By way of example, a unit dosage form of T3 in the form of a tablet comprises the following ingredients:

T3 6mg

mannitol 224mg

Croscarmellose sodium 6mg

Corn starch 15mg

Hydroxypropyl methylcellulose 2mg

Magnesium stearate 3mg

Likewise, by way of example, unit dosage forms of artesunate in tablet form may comprise 50 or 100mg artesunate and conventional excipients, such as lactose, croscarmellose, anhydrous colloidal silicon dioxide, microcrystalline cellulose and magnesium stearate.

Likewise, by way of example, a unit dosage form comprising a combination of artesunate and T3 may comprise 1.5mg of T3 and 50mg of artesunate, and also conventional excipients such as those mentioned above, to achieve a daily dose for an adult of 4 intakes one tablet at a time or 4 tablets at a time.

The same dose of T3, artesunate, or a combination of T3 and artesunate was used in unit form containing excipients known to those skilled in the art for rectal administration.

For the method of administration by parenteral injection, the unit dosage form of T3 may comprise, for example, 1mg of T3 in a vial designed for 10kg of body weight, together with conventional excipients known to those skilled in the art, such as glycerol and phosphate buffer.

Likewise, by way of example, the unit dosage form of artesunate and T3 in vials administered at a rate of one vial per 10kg body weight may comprise 24mg artesunate and 1mg T3, together with conventional excipients such as those mentioned above.

The subject of the present invention is also a method for the treatment and/or prevention of malaria, which comprises administering to a patient a therapeutically effective dose of at least one thiazoleSalts and therapeutically effective amounts of artemisinin or its derivatives, in particular artesunate, said thiazolesA salt is a compound of formula (VI) or a precursor thereof, particularly T3, for simultaneous or sequential administration to said patient as hereinbefore described.

The invention also relates to a method as defined above for the treatment and/or prevention of malaria, in particular severe malaria.

The subject of the present invention is also the use of a combination comprising, as active principle, dithiazoles for the preparation of a medicament for the treatment or prevention of malaria, in particular severe malariaSalts and artemisinin or its derivatives, in particular artesunate, said dithiazolesA salt is a compound of formula (VI) as described above or a precursor thereof.

Finally, the subject of the invention is

Kit for the treatment or prevention of malaria, comprising, on the one hand, at least one dithiazoleA salt which is a compound of formula (VI) or a precursor thereof, and on the other hand comprises artemisinin or a derivative thereof, in particular artesunate, said compound/precursor of formula (VI) and artemisinin/derivative being in different drip bags or in compartments and intended for simultaneous or sequential administration, and

-a kit as defined above, characterized in that it comprises, on the one hand, at least one 1, 12-dodecamethylenebis [ 4-methyl-5- (2-hydroxyethyl) thiazole]Dibromide, and in another aspect at least one artesunate.

The combinations of the invention are the subject of in vivo pharmacological and biochemical tests in mice infected with Plasmodium falciparum (Plasmodium vinckepeteri) strain) of the Plasmodium falciparum type, and can be used to demonstrate the efficacy and synergy they provide for the treatment of malaria.

Para-1, 12-dodecamethylenebis [ 4-methyl-5- (2-hydroxyethyl) thiazole in mice infected with P.venomous ]In vivo measurement of antimalarial Activity of combinations of dibromides (T3) with artesunate

Definition of terms used in this test:

-ED₅₀the dose at which 50% reduction in parasitemia was observed in infected mice (mg/kg/day),

-ED₉₀the dose at which a 90% reduction in parasitemia was observed in infected mice (mg/kg/day),

-iD is administered intraperitoneally,

-iv is administered intravenously,

-ir ═ rectal administration.

1. Description of in vivo testing used

Infection of mice

On day 0 (D0), female "Swiss" mice (OF1, 22-26g) (Charles river laboratories France, 59rue de la Paix, 76410Saint-Aubin-les-Elbeuf) were vaccinated with 10 by injection in the caudal vein⁸Parasite-infected erythrocytes suspended in 200. mu.l NaCl (0.9%). The parasite used was of the plasmodium venomorpha type (strain 279 BY, supplied BY dr.i Landau, Paris, France).

Mice were acclimated two weeks in advance. Allowing them to eat and intake water at will.

Injection 10 on day one (D1)⁸Individual parasite-infected erythrocytes caused parasitemia levels between 5% and 10% (7.2% ± 0.2%). Number of Plasmodium strains of Venezueli mice were infected by 10 weekly in mice⁷To 10⁸Individual parasite-infected erythrocytes (infected by intraperitoneal administration) suspended in phosphate buffer (0.9%) were maintained.

-Preparation of T3 and artesunate solution

The product was dissolved in 0.3M phosphate buffer (pH 8.1). Depending on the dose, the final concentration of the T3 solution was between 40 and 200 mg/l. Depending on the dose, the final concentration of the artesunate solution is between 100 and 500 mg/l.

The product was administered intraperitoneally. The volume administered was of the order of 100. mu.l, but was determined by the body weight of the mice.

-treatment of

Mice were treated once daily for 4 days, at days D1, D2, D3 and D4 (pets, W., J.H.Portus, and B.L.Robinson.1975.the chemotherapy of cadent malacia.XXII.the value of drug-resistant strains of P.berghei In screening for pulmonary activity.Ann.trop.D.Parasitol.69: 155. 171; and: Ancellin ML, Calas.A., Bonhure A., Herbute S.and visual H., In viral activity of bacteria and bis of nanoparticles In vivo efficacy of bacteria and tissue culture of microorganism with strain of bacteria, 2003. 2598). Four mice were used per dose.

Animals were dosed intraperitoneally with either T3, artesunate, or a solubilized mixture of the two active ingredients as appropriate.

Measurement of parasitemia

On the fifth day, several drops of blood were taken from the mouse tail to determine parasitemia by FACS (fluorescence-activated cell sorter) and to perform blood smear (bloodsmear). Parasitemia was first determined by FACS on 20000 cells. The removed erythrocytes for FACS were fixed with glutaraldehyde and then with a fluorescent dyeA marker, said fluorochrome labeling the DNA thus labeling only the parasite-infected cells (Barkan, D., Ginsburg, H., and Goenser, J.2000. optimization of flow cytometric measurement of parasitaemia in plasma-induced microorganism. int.J.Parasitol.30: 649-. Subsequently, less than 15% of parasitemia was recalculated on the smear. The smear was fixed with methanol and then stained with Giemsa stain (Giemsa stain). The blood cells infected with the parasite were counted under a microscope. Parasitemia is expressed as the percentage of infected red blood cells present in the specimen, with the sample being 2000 cells. ED is determined at D5₅₀And ED₉₀The value is obtained. Parasitemia was determined in the treatment from day one to day ten, then day 15, day 22 and day 47.

Mice showing no parasite traces at the smear of D5 were observed at least one more month after the end of treatment to detect any possible parasite growth. The curative dose is the product dose that will ensure survival of the entire batch of treated animals after one month.

ED ₅₀ Determination of a value

0% inhibition corresponds to the mean value of parasitemia observed in untreated infected mice. 100% inhibition corresponds to very weak or no parasitemia, less than 0.01%. ED (electronic device)₅₀Values were determined by linear interpolation of the dose response curve expressed in log concentration.

2. In vivo measurement of antimalarial Activity against T3 and Artemisia annua esters on P.venomosus parasite strains

To demonstrate the synergistic effect between two compounds, it is important to know the activity when they are administered alone, in order to approach their ED₅₀The dosage of (c) is combined. The situation of parenteral administration corresponds to a clinical situation of severe malaria. In this case, it is important to reduce parasitemia rapidly within the first few days of treatment. At the doses tested, artesunate alone failed to reduce parasitemia the first day and a similar event followed in the next few days (fig. 1). T3 was effective in reducing parasitemia after 4 days of treatment at a dose of 1mg/kg/d, but did not prevent an increase in parasitemia after the first day of treatment (FIG. 2).

The obtained ED₅₀The values are shown in table 1 below:

TABLE 1

	ED50(mg/kg/day)
		T3	0.55
Artemisinin	＞25

ED₅₀The lower the activity, the better. Thus, T3(0.55 mg/kg/day) alone showed the phaseBetter activity against parasitemia with artesunate alone (> 2.5 mg/kg/day). In addition, T3 at a dose of 0.5 mg/kg/day alone achieved complete recovery after one month (results not shown), whereas artesunate at a dose of 2.5 mg/kg/day alone failed to achieve complete recovery.

3. In vivo measurement of antimalarial Activity on T3/Artemisia annua ester combination on P.wengii parasite strains

For this combination study, a dose of 2.5mg/kg/d artesunate (slightly lower but close to ED) was chosen₅₀) The dose of 0.5mg/kg/d was completely inactive against parasitemia for 4 days. T3 was evaluated in combination at lower doses, close to and at ED₅₀Above, i.e., 0.2, 0.5 and 1 mg/kg/d.

Surprisingly, as shown in figures 3, 4 and 5, a combination comprising T3 (at all the above mentioned doses) and artesunate present at a dose of 2.5 mg/kg/day significantly reduced the level of parasitemia, as compared to separate administration of T3 alone or artesunate alone. In particular, it is important to note that a reduction in parasitemia was observed in mice 24 hours after the first dose (day 2) and regardless of the dose of T3 administered, whereas at this time point the product administered alone was inactive. Table II (and figures 3, 4 and 5) shows the mean values of parasitemia recorded at this day 2 (4 animals per dose). It should be noted that there was a statistical synergy after the first administration (day 2) of T3 at doses 0.2, 0.5 and 1mg/kg in combination with artesunate at a dose of 2.5mg/kg for 24 hours. This very significant synergy, after the first day of treatment, is particularly important in the treatment of severe malaria situations where clinicians precisely seek out large and rapid reductions in parasitemia in patients to avoid a fatal prognosis.

TABLE II

Starting with T3 at a dose of 0.5 mg/kg/day in combination with artesunate at a dose of 2.5 mg/kg/day, parasitemia remained at its initial value before and throughout the treatment period, whereas administration of each product alone failed to achieve this effect (Table II, FIGS. 3, 4 and 5).

The T3 compound is important for the main effect of complete recovery of mice after one month. Co-administration of artesunate at a dose of 2.5 mg/kg/day did not improve the cure dose obtained with T3 alone.

Table III summarizes the results discussed above.

TABLE III

Thus, when 2 active ingredients were administered in combination, the parasitemia was significantly reduced within the first 4 days of treatment. The effect of the combination is much greater at comparable or even lower doses than when each of the two active ingredients is administered alone at the dosages defined.

In particular, when T3 was administered in combination with artesunate at a dose of at least 2.5 mg/kg/day, a beneficial effect was observed in the reduction of parasitemia. It will thus be appreciated that the combination of the two active ingredients is particularly effective over the therapeutic window, which is defined by the minimum dose of one of the compounds and the proportion of one of the compounds compared to the other compounds within the therapeutic window.

The results obtained in mice infected with P.venomous demonstrate that the combination of the invention comprising artemisinin or its derivatives (in particular artesunate), and bithiazole is advantageous for the treatment of malaria, especially severe malariaSalts of the dithiazolesSalts are compounds of formula (VI), in particular T3.

Claims (8)

1. Composition for treating and/or preventing severe malaria comprising dithiazole as active ingredientSalts, and artemisinin derivatives, said dithiazolesA salt is a compound of formula (VI) selected from:

-1, 12-dodecamethylenebis [ 4-methyl- ]5- (2-hydroxyethyl) thiazole]Dibromide;

the artemisinin derivative is selected from artesunate.

2. A composition according to claim 1, characterized in that each active ingredient is administered simultaneously or sequentially.

3. Pharmaceutical composition for the treatment and/or prevention of severe malaria comprising a therapeutically effective dose of artesunate and at least one bithiazoleSalts of the dithiazolesA salt is a compound of formula (VI) as defined in claim 1, together with at least one pharmaceutically acceptable excipient.

4. Pharmaceutical composition according to claim 3, characterized in that it is suitable for oral administration, rectal administration or administration by injection.

5. Pharmaceutical composition according to claim 3, characterized in that it is suitable for intravenous injection.

6. Pharmaceutical composition according to claim 3, characterized in that it is suitable for intravenous injection in the form of a drip.

7. Use of a composition according to claim 1 in the manufacture of a medicament for the treatment or prevention of severe malaria.

8. Kit for the treatment or prevention of severe malaria, in one aspect the kit comprisesOne less dithiazoleA salt which is a compound of formula (VI) as defined in claim 1, and in another aspect the kit comprises artesunate, said compound of formula (VI) and artesunate being administered in separate compartments and simultaneously or sequentially.