Patent Application
20080039430
Protozoans belonging to the Trypanosomatidae family account for numerous pathologies afflicting man or animals.
Thus, among protozoans of the Trypanosoma genus, T. brucei and T. cruzi are for instance the etiological agents of sleep disease and Chagas disease.
Protozoans of the Leishmania genus, such as L. aethiopica, L. donovani, L. infantum, L. major, L. mexicana or L. tropica are responsible for leishmaniasis (also named leishmaniosis). Infections by these parasites are endemic in more than 88 countries. WHO estimates that more than 12 millions individuals are infected by these parasites and more than 350 millions would be exposed to infections daily. Three major forms of leishmaniosis are documented, among which the most dangerous form, visceral leishmaniosis, can have a lethal outcome in absence of treatment. This situation has worsened since the occurrence of HIV, because these infections are more frequently found as opportunistic infections in individuals afflicted by the acquired immunodeficiency syndrome (AIDS), in particular in South-West Europe. Parasites take advantage of the immunosuppressed status of the host to establish themselves or to reactivate.
Current leishmaniosis treatments are based on drugs difficult to handle, such as amphotericin B or drugs belonging to the antimonial family, which have serious side effects.
Niacin is the generic name for 2 compounds: nicotinamide (NAm) and nicotinic acid. Both were first used clinically in 1937, when these compounds were each shown to act as <<pellagra-preventive>> factor. High dose of NAm and its acid derivative nicotinic acid, are often used interchangeably to treat a number of conditions including anxiety, osteoarthritis, and psychosis. Furthermore, NAm is currently in trials as therapy to prevent cancer recurrence and insulin-dependent (Type I) diabetes (4). Beside this, activity of NAm has been evaluated in anti-mycobacterium tuberculosis studies performed during 1945-1961 and in anti-HIV studies performed from 1991 to the present (reviewed in 7).
It is an object of the present invention to provide new medicaments, lacking the drawbacks of the currently used medicaments, for the treatment of protozoan parasitic diseases, such as leishmaniosis.
Thus, the present invention relates to the use of at least one compound of the following general formula (I):
wherein R represents OH, NH2,
or of precursors or derivatives thereof,
or of the pharmaceutically acceptable salts of said compound or of its precursors or derivatives,
for inhibiting the SIR2 protein expressed by parasites, in particular by protozoan parasites, more particularly by Leishmania, under their respective intracellular or extracellular forms.
As intended herein “precursors or derivatives” of compounds of formula (I) represent compounds which are liable to yield compounds of formula (I) in vivo or compounds which are derived from compounds of formula (I) by means of chemical modifications.
“SIR2 protein” stands for Silent Information Regulatory (SIR2) protein. SIR2 is a class III NAD-dependent deacetylase protein. It is in particular defined in Marmorstein (2004) Biochem. Transac. Society 32:904-909 or in Blander & Guarente (2004) Annu. Rev. Biochem. 73:417-435.
The expression “parasites” relates to unicellular eukaryotic organisms which are able to infect mammals and to survive and/or multiply in the infected mammal.
The present invention also relates to the use of at least one compound of the following general formula (I):
wherein R represents OH or NH2,
or of precursors or derivatives thereof,
or of the pharmaceutically acceptable salts of said compound or of its precursors or derivatives,
for the manufacture of a medicament intended for the prevention or the treatment of parasitic diseases, in particular of protozoan parasitic diseases, more particularly of leishmaniosis, and especially for the prevention or the treatment of parasitic diseases occurring in immunodepressed patients.
As intended herein “parasitic diseases” relate to diseases caused by parasites as defined above.
Advantageously, the use of compounds of formula (I) for the prevention or the treatment of parasitic diseases is sound, since numerous bioavailability studies have assessed that high plasma concentrations of these compounds, e.g. 2.3 mM, could be achieved without serious side effects.
In a preferred embodiment of the above defined use of a compound of formula (I), R represents OH, said compound corresponding to niacin (vitamin B3), of the following formula (II):
In another preferred embodiment of the above defined use of a compound of formula (I), R represents NH2, said compound corresponding to nicotinamide, of the following formula (III):
According to another preferred embodiment of the above defined use, the medicament is suitable for an administration of the compound of formula (I) by oral, intravenous, topical or intralesional route.
As intended herein “intralesional” means that the medicament is suitable to be administered at the sites of parasite-caused skin lesions of patients, in particular in case of Leishmania infections.
According to a particularly preferred embodiment of the above defined use, the medicament is suitable for an administration of the compound of formula (I) at a unit dose of about 10 mg to about 10 g, in particular of about 1 g to about 6 g.
According to another particularly preferred embodiment of the above defined use, the medicament is suitable for an administration of the compound of formula (I) at a dosage of about 5 mg/m2/day to about 5 g/m2/day, in particular of about 500 mg/m2/day to about 3 g/m2/day.
In another preferred embodiment of the above defined use, the compound of formula (I) in association with at least one anti-parasitic compound, such as a compound selected from:
miltefosin, antimonials, amphotericin B, benznidazol, nifurtimox, paromomycin, pentamidin and its derivatives, arsenic derivatives, melarsopol and difluoromethylornithin.
Advantageously, the association of a compound of formula (I) with an anti-parasitic compound has additive or synergic effects which enables a diminished administration of said anti-parasitic compound and thus diminished side effects.
The present invention also relates to a pharmaceutical composition comprising as active substances:
at least one compound of the following general formula (I):
wherein R represents OH or NH2,
or precursors or derivatives thereof, or of the pharmaceutically acceptable salts of said compound or of its precursors or derivatives, and
at least one anti-parasitic compound, such as a compound selected from:
miltefosin, antimonials, amphotericin B, benznidazol, nifurtimox, paromomycin, pentamidin and its derivatives, arsenic derivatives, melarsopol and difluoromethylornithin,
in association with a pharmaceutically acceptable carrier.
In a particular embodiment of the above defined pharmaceutical composition, R represents OH, the compound of formula (I) hence corresponding to niacin (vitamin B3).
In another particular embodiment of the above defined pharmaceutical composition, R represents NH2, the compound of formula (I) hence corresponding to nicotinamide.
According to a preferred embodiment, the above defined pharmaceutical composition is suitable for an administration by oral intravenous, topical or intralesional route.
According to another preferred embodiment, the above defined pharmaceutical composition is suitable for the administration of the compound of formula (I) at a unit dose of about 10 mg to about 10 g, in particular of about 1 g to about 6 g.
According to yet another preferred embodiment, the above defined pharmaceutical composition is suitable for the administration of the compound of formula (I) at a dosage of about 5 mg/m2/day to about 5 g/m2/day, in particular of about 500 mg/m2/day to about 3 g/m2/day.
The present invention also relates to products containing:
at least one compound of the following general formula (I):
wherein R represents OH or NH2,
or precursors or derivatives thereof,
or the pharmaceutically acceptable salts of said compound or of its precursors or derivatives, in association with
at least one anti-parasitic compound, such as a compound selected from:
miltefosin, antimonials, amphotericin B, benznidazol, nifurtimox, paromomycin, pentamidin and its derivatives, arsenic derivatives, melarsopol and difluoromethylornithin, as a combined preparation for simultaneous, separate or sequential use in the prevention or the treatment of parasitic diseases, in particular of protozoan parasitic diseases, more particularly of leishmaniosis, and especially for the prevention or the treatment of parasitic diseases occurring in immuno-depressed patients.
In a preferred embodiment of the above defined products, R represents OH, the compound of formula (I) hence corresponding to niacin (vitamin B3).
In another preferred embodiment of the above defined product, R represents NH2, the compound of formula (I) hence corresponding to nicotinamide.
The present invention also relates to a method for the prevention or the treatment of parasitic diseases, in particular of protozoan parasitic diseases, more particularly of leishmaniosis, and especially for the prevention or the treatment of parasitic diseases occurring in immuno-depressed patients, characterized in that at therapeutically effective amount of at least one compound of the following general formula (I):
wherein R represents OH, NH2,
or of precursors or derivatives thereof,
or of the pharmaceutically acceptable salts of said compound or of its precursors or derivatives,
is administered to a patient in need thereof.
In a preferred embodiment of the above defined method, R represents OH, the compound of formula (I) hence corresponding to niacin (vitamin B3).
In another preferred embodiment of the above defined method, R represents NH2, the compound of formula (I) hence corresponding to nicotinamide.
According to a particular embodiment of the above defined method, the compound of formula (I) is administered by oral intravenous, topical or intralesional route.
According to another particular embodiment of the above defined method, the compound of formula (I) is administrated at a unit dose of about 10 mg to about 10 g, in particular of about 1 g to about 6 g,
According to yet another particular embodiment of the above defined method, the compound of formula (I) is administered at a dosage of about 5 mg/m2/day to about 5 g/m2/day, in particular of about 500 mg/m2/day to about 3 g/m2/day.
In another preferred embodiment of the above defined method, the compound of formula (I) is administered in association with at least one anti-parasitic compound, such as a compound selected from:
miltefosin, antimonials, amphotericin B, benznidazol, nifurtimox, paromomycin, pentamidin and its derivatives, arsenic derivatives, melarsopol and difluoromethylornithin.
The growth of Leishmania amastigotes and promastigotes was followed in axenic culture conditions in the presence or absence of NAm.
A cloned line of L. infantum (MHOM/MA/67/ITMAP-263) was used in all experiments. Each subculture was initiated at 5×105 parasites/ml of medium. Axenically grown amastigote forms of L. infantum were maintained at 37° C. with 5% CO2 by weekly subpassages in a cell-free medium called MAA/20 (medium for axenically grown amastigotes) in 25-ml flasks, as previously described (10). Promastigote forms were maintained at 26° C. by weekly subpassage in SDM 79 medium supplemented with 10% foetal calf serum (FCS) and 100 units/ml penicillin and 100 μg/ml streptomycin. Nicotinamide (SIGMA, St Louis) was added at the appropriate concentration and the mean number of viable parasites determined using FACs analysis, as previously described (11).
As shown in
The nature of NAm-induced amastigotes growth arrest was then investigated
Cells were seeded at 5×105 parasites/ml and NAm was added at various concentrations ranging from 25 to 100 mM. After 24, 48 and 72 hours of incubation aliquots (106 parasites) were collected, washed and incubated for 10 min with 10 μM of YOPRO-1, an apoptotic cell marker (Molecular probes). The mean percentage of YOPRO-1 positive cells was determined as previously described (9). At concentrations higher than 25 mM, NAm exerted a strong dose-dependent leishmanicidal activity against axenic amastigote, as demonstrated by the occurrence of YOPRO-1 positive cells. Maximal effect was observed after 3 days of culture in the presence of 100 mM of NAm (
Having observed that NAm induced axenic amastigotes death, it was of interest to examine its effect on intracellular amastigotes proliferation.
In a first series of experiments, THP-1 monocytes were incubated during 3 days with various concentrations of NAm and the growth and viability of cells were recorded. Up to 10 mM of NAm, no effect on cell growth and viability was observed. In contrast, 20 mM NAm inhibited the proliferation of THP-1 monocyte by about 45% in agreement with the values recorded for other cell types: SupT1 and PBLs cells (6).
Thus, THP-1 differentiated macrophages were infected with stationary phase amastigotes at a host cell-parasite ratio of 5:1. After 4 hours, non adherent parasites were removed and nicotinamide was added to the medium at the appropriate concentration. After 3 days of incubation time, cells were fixed with methanol and stained with giemsa. Parasitic index PI (mean percentage of infected macrophages X number of amastigotes per macrophage) was determined. As shown in
It has been recently demonstrated that NAm is a substrate of sir2-like enzymes in vitro (5). Therefore, complementary experiments were conducted in order to examine whether NAm could interfere with Leishmania deacetylase activity in vitro. To test this possibility, a commercially available “cyclex SIR2 assay kit” and SIRT1 as a standard enzyme (MBL, Japan) were used.
As shown in
Having established a standard inhibitory assay, the effect of NAm was then examined on the NAD-dependent deacetylase activity contained in Leishmania extracts from mutant parasites carrying extra copies of LmSIR2 gene (pTEX-LmSIR2) or empty plasmid DNA (pTEX) (11).
Briefly, 2 105 parasites were collected and washed two times with PBS 0.01M pH 7.2 and incubated in a lysis solution (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP40, 5 μM Trichostatin A, pH 8.8), cells were then centrifuged for 20 min at 10 000 rpm at 4° C. Deacetylase activity in the presence or absence of 200 μM NAD was measured. Results are expressed as relative F355/F460 counts=F355/F460 counts in the presence of NAD—F355/F460 counts in the absence of NAD. This allowed to discriminate between fluorescence due to the action of LmSIR2 to fluorescence due to the presence of compounds which could interfere with the test. As shown in
In yeast and C. elegans, SIR2 is a limiting component of longevity (reviewed in 3) and NAm is able to accelerate yeast ageing by inhibiting SIR2 in vivo (2). In the protozoan parasite L. infantum, amastigotes carrying extracopies of LmSIR2 (LiSIR2) gene, when maintained under normal axenic culture conditions, showed striking increase in the survival due to an inherent resistance to apoptosis-like death, leading to a longer stationary phase of growth (11).
To further examine the possible correlation between the level of SIR2 expression and the sensitivity/resistance to NAm-induced Leishmania amastigotes death, NAm was added to cultures of mutant L. infantum amastigotes which overexpress LmSIR2 or carrying the empty pTEX plasmid as controls.
As shown in
The microbicidal mechanism of action of NAm is not currently known. Its activity may come to be understood as that of an indirect antimicrobial that has primarily a prohost effect. Among the reasons to suggest effect is the body of literature that reports an immunodulatory role for nicotinamide in a wide variety of experimental systems (8, 7). Moreover, antioxydant and cryoprotective effect of NAm is well documented (12).
Thus, the present invention represents the first report showing the anti-parasitic activity of NAm. Furthermore, although NAm could inhibit the NAD-dependent deacetylase activity of SIR2-like enzymes, its main target in Leishmania seems not to be LmSIR2. In fact Leishmania possesses two other SIR2 related members whose function and localization are currently unknown. Implication of this protein family in the survival of Leishmania parasite has to be investigated. It can be hypothesized that one or all of them are essential for the parasite survival, and that their inhibition leads to the parasite death. Alternatively, other essential physiological functions would be the targets for NAm. The concentration of NAm and Nicotinic acid found to inhibit the intracellular growth of Leishmania infantum (IC50 inferior to 2.5 mM) are far higher than those found in whole blood (about 45 μM) but is closer to the plasmatic concentration of nicotinamide achievable (0.7 to 2.3 mM) in patient treated with accelerated radiotherapy for head and Neck cancer (1).
In conclusion, nicotinamide is an inexpensive and orally available agent without significant side effects. Since nicotinamide and its derivatives are potentially beneficial components, leishmaniasis will benefit from therapeutic use of such components, optionally in combination with anti-parasitic drugs.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/07715 | 7/15/2005 | WO | 4/19/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60588802 | Jul 2004 | US |
