WO1994002153A1

WO1994002153A1 - Use of sulphur containing phospholipids for treating leishmaniasis

Info

Publication number: WO1994002153A1
Application number: PCT/EP1992/001674
Authority: WO
Inventors: Simon L. Croft; Dieter Herrmann
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1992-07-22
Filing date: 1992-07-22
Publication date: 1994-02-03

Abstract

Use of sulphur containing phospholipids having general formula (I), in wich X signifies -i.a.- a valency bond, oxygen, sulphur, the sulphinyl or sulphonyl group, a cycloalkylene with 3 to 8 carbon atoms or phenylene radical, Y oxygen or sulphur, R1 hydrogen, a saturated or unsaturated alkyl radical with 1 to 18 carbons atoms, which is possibly substituted, R2 a saturated or unsaturated alkylene chain with 1 to 18 carbon atoms, which is possibly substituted, R3 a saturated or unsaturated alkylene chain with 2 to 8 carbon atoms, which is possibly substituted, R4 alkylene chain with 2 to 4 carbon atoms, R5 hydrogen or a lower alkyl group and n the numbers 0, 1 or 2, as well as their pharmacologically acceptable salts for the treatment of leishmaniasis and pharmaceutical compositions containing at leat one of said compounds.

Description

Use of Sulphur Containing Phoεpholipids for Treating Leishmaniasis

subjects of the present invention are the use of sulphur containing phospholipids and pharmacological acceptable salts thereof for treating leishmaniasis, especially visceral and cutaneous leishmaniasis, and pharmaceutical compositions containing these substances for the treatment of leishmaniasis .

The usual drugs for the clinical treataent of visceral and cutaneous leishmaniasis are the pentavalent antimonialε sodium stibogluconate ( ' Pentostam' ) and megluπtine antinoniate ( 'Glucantime' ) which require the use of high doses, long courses of treatment and parenteral administration (see the WHO Technical Report Series,701. The Leiεhmaniases 1984). Although there have been several important advances in the chemotherapy of leishmaniasis, both in the identification of new drugs which exploit the boichemical differences between host and parasite and the reformulation or administration of established drugs (see the articles "Chemotherapy for leishmaniasis: biochemical mechanisms, clinical efficacy, and future srategies" of J.D.Berman, Re .Infec .Dis . , 10, 560- 585,1988 and "Recent developments in the chemotherapy of leishmaniasis" of S.L.Croft, Trends Pharmacol.Sci. ,3 , 376- 381,1988), there is still a need for a drug that is orally effective against all forms of leishmaniasis .

One line of study in this search for new anti-leiεhmanial drugs has indicated that some O-alkyl lyεophospholipids (ALPs) show selective toxicity against Leishmania. Previous studies revealed that ether phospholipids are cytotoxic to the promastigotes of Leishmania donovani (see the article "Cytotoxicity of ester and ether lysophoεpholipids on Leishmania donovani promastigotes" of V.Achterberg and G.Gercken, Mol.Biochem.Parasit. ,23,117-122,1987) , Leishmania major, Leishmania mexicana, and Leishmania panamensis (see the article "Anti-leishmanial activity of ether analogues of lysophospholipids" of N.P.Fiavey and B.Z.Ngwenya, Am.J.Trop. Med.Hyg. ,143,119-120,1991) and that a serieε of O-alkyl phosphorylcholines were selectively toxic to Leishmania donavani a astigoteε in macrophageε in vitro and in BALB/c mouse (see the article "The activity of alkyl phosphoryl¬ cholines and related derivatives against Leishmania donovani" of S.L.Croft et al. , Biochem.Pharmacol.,36,2633-2636, 1987) . The major area of interest of ALPs has been their anti-tumour activity where some have reached the stage of clinical trials (see the article "Clinical phase I pilot study of the alkyl lysophospholipid ET-18-0CH₃" of W.E.Berdel et al. , Lipids,22,967-969, 1987).

It could be shown now that alkanol phosphoric acid monoammonium alkyl esters, which are substituted by an alkylated thio, sulphinyl or sulphonyl group, are especially and surprisingly active against leishmaniasis.

Such sulphur containing phospholipids are known for their anti-tumoral properties. They are described in the European Patent No. 0.050.327 and in the EP Application No. 90103661.6-.

According to the present invention compounds of the general formula I

0

II -

R₁-X-R₂-S(0)„-R₃-Y-P-0-R4-N(R_β)₃ (I)

I o^θ

in which X signifies a valency bond, oxygen, sulphur, the sulphinyl or sulphonyl group, the -NHCO-, -CONH- or -NHCONH- group, a cycloalkylene with 3 to 8 carbon atoms or phenylene radical, Y oxygen or sulphur, R-. hydrogen, a straight-chained or branched, saturated or unsaturated alkyl radical with 1 to 18 carbon atoms, which is possibly substituted one or more times by aryl, halogen, a lower alkoxy, alkylmercapto, alkoxycarbonyl, alkaneεulphinyl or alkanesulphonyl group, R_z a straight-chained or branched, saturated or unsaturated alkylene chain with 1 to 18 carbon atoms, which is possibly substituted one or more times by aryl, halogen, a lower alkoxy, alkylmercapto, alkoxycarbonyl, alkanesulphinyl or alkanesulphonyl group, R₃ a straight-chained or branched, saturated or unsaturated alkylene chain with 2 to 8 carbon atoms, which can also be part of a cyclopentane, cyclohexane or cycloheptane ring, possibly substituted by a lower alkyl group, and is possibly substituted one or more times by hydroxyl, halogen, nitrile, a cycloalkyl, phenyl, alkoxycarbonyl, optionally substituted alkylated carbamoyl, alkylmercapto, alkanesulphinyl, alkanesulphonyl, optionally acylated a ino group or by alkoxy which, in turn, can be substituted by aryl, alkylmercapto, alkanesulphinyl, alkanesulphonyl, optionally acylated NH₂ alkoxycarbonyl, nitrile, hydroxyl, alkoxy or optionally alkylated carbamoyl, R-_ a straight-chained or branched alkylene chain with 2 to 4 carbon atoms, R₅ hydrogen or a lower alkyl group and n the numbers 0, 1 or 2, with the proviso that the group Rι-X-R₂ , for the case in which it represents an alkyl group (X = valency), has up to 20 carbon atoms, as well as their pharmacologically acceptable salts are used for the treatment of leishmaniasis.

The group R_.-R₂ is preferably straight-chained or branched ond optionally substituted and contains up to 20 carbon atoms.

The group R₃ consists preferably of a -CH₂-CH₂-CH₂- group the middle carbon atom of which is once or twice substituted by ydroxyl, lower alkyl or an alkoxy group, where both groups can, in turn, be substituted by alkoxy. The group R_Λ and the group R₅ signify preferably the -CH₂-CH_a- group or methyl respectively.

Preferred compounds used in accordance with the present application are the following esters:

(3-octadecylthiopropyl)-(2-trimethylammonioethyl) phosphate, [3-(2-methoxycarbonyl)heptadecylthio-2-methylpropyl]-(2- trimethylammonioethyl) phosphate,

(3-hexadecanesulphinyl-2-methylpropyl)-( (2-trimethylammonio¬ ethyl) phosphate,

(3-heptadecylthio-2-methylpropyl)-(2-trimethylammonioethyl) phosphate,

(3-hexadecylthio-2-hexadecyloxypropyl)-(2-trimethylammonio- ethyl) phosphate,

(3-methylthio-2-hexadecyloxypropyl)-( 2-trimethylammonio¬ ethyl) phosphate,

(3-hexadecylthio-2,2-bis-methoxymethylpropyl)-(2- trimethylammonioethyl) phosphate, (3-hexadecansulphinyl-2-methoxymethylpropyl)-(2- trimethylammonioethyl) phosphate, (3-hexadecanesulphonyl-2-methoxymethylpropyl)-(2- trimethylammonioethyl) phosphate,

(3-hexadecanesulphonyl-2,2-bis-methoxymethylpropyl)-(2- trimethylammonioethyl) phosphate and

(3-hexadecylthio-2-methoxymethylpropyl)-( 2-trimethylammonio¬ ethyl) phosphate.

Of these compounds most preferred as drugs for treating leishmaniasis are Ilmofosine having the formula (3-hexa- decylthio-2-methoxymethylpropyl)-(2-trimethylammonioethyl) phosphate and (3-hexadecanesulphonyl-2-methoxymethylpropyl)- (2-trimethylammonioethyl) phosphate. Ilmofosine is currently in Phase II clinical trials for efficacy against malignant tumours (see the articles "Ilmofosine (BM41.440) a new cytotoxic etherphospholipid" of D.B.J.Herrmann and U.Bicker, Drugε of the Future,13,543-554 , 1988 and "Short- and long-term tolerability study of the thioether phoεpholipid derivative Ilmofosine (BM41.440) in cancer patients" of D.B.J.Herrmann et al., in Contributions to Oncology: New Drugs in Oncology, (Eckardt,S. ,Holzner,J.H. and Nagel,G.A. ,Eds) , Karger, Basel., pp.236-247,1989)

In the following the invention is described in more detail taking as an example the activity of Ilmofosine against Leishmania donovani and the antimoy-resistant line of Leishmania infantum. It is however pointed out that though said drug and its application to said parasites are preferred, the inventive concept also works with other compounds being enclosed by the general formula I and is applicable against other types of Leishmanias.

Leishmania donovani (MHOM/ET/67/L82) was routinely maintained in male golden hamsters (Wright's strain) by passage every 6 to 8 weeks. The antimony-resistant line of Leishmania infantum was developed by V.Waits and W.L.Hanson (University of Georgia) and was maintained in hamsters (see the article "Reversal of drug resistance in Trypanosoma cruzi and Leishmania donovani by verapamil" of R.A.Neal et al., Transactions of the Royal Society of Tropical Medicine and Hygiene,83,197-198,1989) .

Ilmofosine, molecular weight 525, was formulated in medium for the in vitro studies and in 0.25% cellacol for the in vivo studies.

In vitro studies

Mouε- eritoneal acrophages were isolated from outbred CD1 mice (Charles Rivers Ltd.) und cultured in Labtek 8 chamber slides in RPMI 1640 medium with 10% heat inactivated foetal calf serum at 37^βC in a 5% C0₂-air mixture. Macrophageε were infected with amastigotes derived from hamster spleen 24 hours prior to drug treatment following the procedure described in the article "An in vitro system for determining the activity of compounds against the intracellular amastigote form of Leishmania donovani" of R.A.Neal and S.L.Croft, published in J.Anti ic.Chemoth. , 14,463-475,1984. Infected cultures were maintained in medium containing Ilmofosine for 7 days including two changes of fresh medium with drug, in a three-fold dilution series from 30 μM with quadruplicate cultures at each concentration. After the 7 day exposure the proportion of infected acrophages in Giemsa stained preparations of each culture was determined. ED_So values were calculated by linear regression analysis.

Ilmofosine showed high activity against intracellular Leish¬ mania donovani amastigotes, clearing 90% of macrophages at 10 -M and having an ED_so value of 3.74 -<-M as shown in table 1.

Table 1. The in vitro activity of Ilmofosine against Leishmania donovani and Leishmania infantum - Sb""" resistant amastigotes

macrophages cleared of ED_ε0 values (μM) amastigoteε (P95 fiducial li¬

30μM 10μM 3μM lμM mits)

L.donovani 100 (T) 94.4 31.5 1.8 3.73 (3 4.26)

L. infantum 100 (T) 97.4 43.7 3.46 36 - 4.39) Sb^v resist.

Although all parasites appeared to be killed at 30μM there was also toxicity to the macrophages at this concentration with a reduction in host cell number and damage to those that remained. A similar pattern of activity and toxicity was determined in tests on the antimony-resistant line of Leish¬ mania infantum (see table 1). In the same model the ED_so value of the standard anti-leishmanial sodium stibogluconate is around 5 mg Sb^v /ml for Leishmania donovani (see the article of Neal and Croft (1984) cited above) and 90 mg Sb^v /ml for the resistant line of Leishmania infantum (see the article of Neal at al. (1989) cited above).

In vivo studies

Male BALB/c mice (Olac Ltd) were infected with 5 x 10⁶ amastigotes by the tail vein and randomly assorted into groups of five. Starting one week after infection mice were dosed with Ilmofosine as formulated in 0.25% cellacol by either the oral or the subcutaneous route once/day for 5 consecutive days and sacrificed 4 days after the completion of the treatment. Drug activity was determined by counting the number of amastigotes/liver cell in smears prepared from weighed livers of treated and untreated mice. ED_so values were determined by linear regression analysis.

In three experiments Ilmofosine showed high levels of activity against Leishmania donovani liver infections in BALB/c mice following administration by both the subcutaneous and oral routes. A top dose of 50 mg/kg x 5 consistently reduced the liver amastigote load by 80-90% (see table 2). At this top dose of the drug there was evidence of some toxicity with a 10% weight loss/mouse observed. In a full dose range study an ED₅o value of 10.55 mg/kg x 5 was determined following oral dosing. In the same model the EDso value of sodium stibogluconate is 10-15 mg Sb^v/kg. Table 2. The activity of Ilmofosine against Leishmania do¬ novani in BALB/c mice.

*) Liver load = No. of amastigotes/500 liver cell x liver weight in mg x 10^s, mean of 5 animals/group ± SEM) . ^α= EDso value, *^"= EDso value

In the only study comparable to the one outlined above the in vitro and in vivo efficacy of the most active of a series of O-alkyl phosphorylcholine analogues showed also a considerable efficacy against leishmania donovani amastigotes (see the article of Croft et al . (1987) cited above). However a particular advantage of Ilmofosine is its anti- leishmanial activity following delivery by the oral route. In pharmacokinetic studies in rats a single oral dose of 15 mg/kg produced a peak serum concentration of 1.7 μg/ml after 7 hours with a half-life of 7 hours, while after daily administration for 5 weeks a mean serum concentration of - ? -

3.3 μg/ml was measured (see for example the article "Pharmacokinetics of the thioether phospholipid analogue BM41.440 in rats" of D.B.J.Herrmann et al. , Lipids,22,952- 954,1987). Ilmofosine is also preferable, because there are already extensive data on the pharmacological and toxicological properties of Ilmofosine in experimental animals and men gathered during the development of this compound as an anti-cancer drug (see for example the article "In vivo antitumour activity of Ilmofosine" of D.B.J.Herrmann et al., Cancer Treat.Rev. ,17,247-252,1990 and the article of D.B.J.Herrmann et al.(1989) cited above, especially page 237) .

The preparation of the compounds of the general formula I and of the pharmacologically acceptable salts thereof is described in European Patent No. 0.050.327 and in EP Application No. 90103661.6-.

The compounds of the general formula I can be administered enterally or parenterally in liquid or solid form. For this purpose use can be made of all forms of administration, for example, tablets, capsules, drageeε, syrups, solutions and the like. As injection medium, it is preferred to use water which contains the additives usual in the case of injection solutions, such as stabilizing agents and/or buffers. Additives of this kind include, for example, tartrate and citrate buffers, ethanol, complex formers (such as ethylene- diamine-tetraacetic acid and the non-toxic salts thereof) and high molecular weight polymers (such as liquid poly-ethylene oxide) for viscosity regulation. Liquid carrier materials for injection solutions must be sterile and are preferably placed into ampoules. Solid carrier materials include, for example, starch, lactose, mannitol, methylcellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid), gelatine, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats and solid high molecular polymers (such as polyethylene glycols) . Compositions suitable for oral administration can, if desired, contain flavouring and/or sweetening agents.

The dosage used depends upon various factors, such as the mode of administration, species, age and/or individual conditions. The dosage to be administered daily is usually from about 0.05 to 100 mg/kg and preferably 1 to 25 mg/kg of body weight, where for long-term applications the preferred maximum tolerated daily oral dose in patients is about 5 mg/kg of body weight.

Claims

- I I -Claims

1. Use of sulphur containing phospholipids having the general formula I

0

in which X signifies a valency bond, oxygen, sulphur, the sulphinyl or sulphonyl group, the -NHCO-, -CONH- or -NHCONH- group, a cycloalkylene with 3 to 8 carbon atoms or phenylene radical, Y oxygen or sulphur, R_α hydrogen, a straight-chained or branched, saturated or unsaturated alkyl radical with 1 to 18 carbon atoms, which is possibly substituted one or more times by aryl, halogen, a lower alkoxy, alkylmercapto, alkoxycarbonyl, alkanesulphinyl or alkanesulphonyl group, R₂ a straight-chained or branched, saturated or unsaturated alkylene chain with 1 to 18 carbon atoms, which is possibly substituted one or more times by aryl, halogen, a lower alkoxy, alkylmercapto, alkoxycarbonyl, alkanesulphinyl or alkanesulphonyl group, R₃ a straight-chained or branched , saturated or unsaturated alkylene chain with 2 to 8 carbon atoms, which can also be part of a cyclopentane, cyclohexane or cycloheptane ring, possibly substituted by a lower alkyl group, and is possibly substituted one or more times by hydroxyl, halogen, nitrile, a cycloalkyl, phenyl, alkoxycarbonyl, optionally substituted alkylated carbamoyl, alkylmercapto, alkanesulphinyl, alkanesulphonyl, optionally acylated amino group or by alkoxy which, in turn, can be substituted by aryl, alkylmercapto, alkanesulphinyl, alkanesulphonyl, optionally acylated NH₂ alkoxycarbonyl, nitrile, hydroxyl, alkoxy or optionally alkylated carbamoyl, R_* a straight-chained or branched alkylene chain with 2 to 4 - ι ~> -

carbon atoms, R_s hydrogen or a lower alkyl group and n the numbers 0, 1 or 2, with the proviso that the group R-.-X-R₂ , for the case in which it represents an alkyl group (X = valency), has up to 20 carbon atoms, as well as their pharmacologically acceptable saltε for the preparation of medicaments for the treatment of leishmaniasis.

2. Use according to claim 1 of compounds of the formula (I) as defined in claim 1, in which R_Λ represents a -CH₂-CH₂- chain and R₅ the methyl group.

3. Use according to claim 1 of compounds of the formula (I) as defined in claim 2 in which Y represents oxygen and R_α and R₂ together have 16 to 18 carbon atoms and X represents a valency bond or oxygen.

4. Use according to claim 1 of compounds of the formula (I) as defined in claim 3 in which R₃ represents a -CH₂-CH₂-CH₂- chain the middle carbon atom of which can be substituted by hydroxyl, lower alkyl or an alkoxy group, where both groups can, in turn, be substituted by alkoxy.

5. Use according to claim 1 of Ilmofosine or its pharmacologically acceptable salts.

6. Use according to claim 1 of (3-hexadecanesulphonyl-2- methoxymethylpropyl)-(2-trimethylammonioethyl) phosphate or its pharmacologically acceptable salts.

7. Use according to any one of claims 1 to 6 for the treatment of leishmaniaεiε being caused by leishmania donovani or leishmania infantum.

8. Use according to claims 1 to 7, wherein the active agent is administered orally.

9. Use according to any one of claims 1 to 8, wherein the active agent is administered daily in an amount of about 0.05 to about 100 mg/kg of body weight.

10. Use according to claim 9, wherein the amount of the active agent is about 1 to about 25 mg/kg of body weight.

11. Use according to claim 9, wherein for long-term applications the amount of the active agent is about 1 to about 5 mg/kg of body weight.

12. Pharmaceutical composition for the treatment of leishmaniasis containing at least one substance as defined in any one of claims 1 to 6 as active agent as well as conventional carrier and auxiliary materials.

13. Pharmaceutical composition according to claim 12 containing Ilmofosine or a pharmacologically acceptable salt thereof.

14. Pharmaceutical composition according to claim 12 containing (3-hexadecanesulphonyl-2-methoxymethylpropyl)-(2- trimethylammonioethyl) phoεphate or a pharmacologically acceptable εalt thereof.