FR2834637A1 - USE OF A NEW CLASS OF MEDICAMENTS FOR THE TREATMENT OF PARASITIC DISEASES - Google Patents

Abstract

The invention relates to the use of at least one benzodiazepine peripheral receptor ligand molecule (PBR) for the manufacture of medicaments for the treatment of parasitic diseases.

Description

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 Use of a new class of drugs for the treatment of parasitic diseases.

The invention relates to the use of a new class of drugs for the treatment of parasitic diseases. The new class of medicaments contemplated by the invention relates more particularly to molecules of the ligands of the peripheral benzodiazepine receptor (PBR).

As part of the fight against parasitic diseases and more specifically against malaria and toxoplasmosis, it has become essential to identify new molecules that could lead to the development of new and effective therapeutic pathways.

In recent years, resistance to anti-infective agents in malaria treatments has intensified and spread to many countries. Resistance to chloroquine, a classic medicine for the treatment of malaria, has been found in almost all countries affected by malaria: Southeast Asia, Africa, South America and Oceania. In addition, many areas of Southeast Asia have developed resistance to all currently known drugs, leading to an upsurge in the spread of the disease.

On the other hand, there are currently no drugs against quiescent cystic forms of T. gondii. However, it turns out that this particular form of T. gondii is likely to wake up and to cause fatal toxoplasmic encephalitis in immunodepressed patients (cancer, grafts, AIDS, ...). These findings led the inventors to search for molecules with anti-parasitic activity, using a

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 original therapeutics to complement the arsenal of molecules available today.

The present invention therefore relates to the use of at least one peripheral benzodiazepine receptor ligand molecule (PBR) for the manufacture of medicaments for the treatment of parasitic diseases.

PBR is a dynamic receptor consisting of three subunits: the 18 kD isoquinoline receptor (PK11195), the 32 kD voltage-gated anion channel that binds benzodiazepines and the purine transporter.

This family of PBR ligand molecules has never been considered for the treatment of parasitic diseases. The main advantage of the invention is to provide not only a new family of drugs but also to open the way to other families of drugs. Indeed, the therapeutic route used by the drugs according to the invention is new and thus constitutes a new direction of research.

Moreover, some of these molecules may have neurological adverse effects, which means that this type of molecule is endowed with a particularly interesting neurological tropism, given the peculiarities of certain parasitic diseases such as toxoplasmosis or malaria. Indeed, cystic forms of T. gondii and mature forms of P. falciparum (pernicious form, cerebral malaria) can be localized in the brain.

More particularly, the present invention relates to the use of peripheral benzodiazepine receptor (PBR) ligand molecules of general formula (I):

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où X est un atome d' halogène choisi parmi F, Cl, Br ou I, un atome d' hydrogène, OH, COOH, SH, NH2, OR, COOR, SR, NHR ou
NRR', où R et R' identiques ou différents, représentent une chaîne alkyle de 1 à 4 C,
R1 est un atome d'hydrogène, une chaîne alkyle de 1 à 4 C ou forme avec R4 une liaison de type C-N,
R2 est un atome d'hydrogène ou une chaîne hydrocarbonée de
1 à 6 C, comportant le cas échéant un groupement NHR ou
NRR', où R et R' identiques ou différents, représentent une chaîne alkyle de 1 à 4 C,
R3 est un atome d'hydrogène ou une chaîne hydrocarbonée de
1 à 2 C formant un cycle avec R4,
R4 forme avec R1 une liaison C-N, une chaîne hydrocarbonée de 1 à 2 C formant un cycle avec R3 ou un atome d'hydrogène.

where X is a halogen atom selected from F, Cl, Br or I, a hydrogen atom, OH, COOH, SH, NH2, OR, COOR, SR, NHR or
NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C,
R1 is a hydrogen atom, an alkyl chain of 1 to 4 C or form with R4 a CN type bond,
R2 is a hydrogen atom or a hydrocarbon chain of
1 to 6 C, optionally comprising an NHR group or
NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C,
R3 is a hydrogen atom or a hydrocarbon chain of
1 to 2 C forming a ring with R4,
R4 forms with R1 a CN bond, a hydrocarbon chain of 1 to 2 C forming a ring with R3 or a hydrogen atom.

où X est un atome d' halogène choisi parmi F, Cl, Br ou I, un atome d'hydrogène, OH, COOH, SH, NH2, OR, COOR, SR, NHR ou
NRR', où R et R' identiques ou différents, représentent une chaîne alkyle de 1 à 4 C,
R1 est un atome d'hydrogène, une chaîne alkyle de 1 à 4 C, Preferably, the compound corresponds to formula (II):

where X is a halogen atom selected from F, Cl, Br or I, a hydrogen atom, OH, COOH, SH, NH2, OR, COOR, SR, NHR or
NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C,
R1 is a hydrogen atom, an alkyl chain of 1 to 4 C,

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où X est un atome d'halogène choisi parmi F, Cl, Br ou I, un atome d' hydrogène, OH, COOH, SH, NH2, OR, COOR, SR, NHR ou
NRR', où R et R' identiques ou différents, représentent une chaîne alkyle de 1 à 4 C,
R2 est un atome d'hydrogène ou une chaîne hydrocarbonée de
1 à 6 C, comportant le cas échéant un groupement NHR ou
NRR', où R et R' identiques ou différents, représentent une chaîne alkyle de 1 à 4 C. R2 is a hydrogen atom or a hydrocarbon chain of
1 to 6 C, optionally comprising an NHR group or
NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C, or formula (III):

where X is a halogen atom selected from F, Cl, Br or I, a hydrogen atom, OH, COOH, SH, NH 2, OR, COOR, SR, NHR or
NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C,
R2 is a hydrogen atom or a hydrocarbon chain of
1 to 6 C, optionally comprising an NHR group or
NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C.

à savoir le 1-(2-chlorophényl)-N-méthyl-N-(1-méthylpropyl)-3-isoquinoléinecarboxamide, The present invention more specifically targets two compounds, respectively, of formula (IV):

that is 1- (2-chlorophenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinolinecarboxamide,

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à savoir le 7-chloro-l-(2-diéthylamino-éthyl)-5-(2-fluoro- phényl)-1,3-dihydro-benzo[e][1,4]diazepin-2-one dont la dénomination commune internationale est le flurazépam. hereinafter referred to as isoquinoline, or of formula (V):

namely 7-chloro-1- (2-diethylaminoethyl) -5- (2-fluorophenyl) -1,3-dihydro-benzo [e] [1,4] diazepin-2-one, the denomination of which is international common is flurazepam.

The parasitic diseases targeted by the invention are preferably due to pathogens of the Apicomplexa family, in particular Toxoplasma gondii and Plasmodium falciparum.

The medicaments according to the invention exhibit a dose-dependent inhibitory effect with respect to the parasite (s). This inhibitory effect will be better understood on reading Example 1.

They can also be advantageously used in combination therapy, together and / or in supplements of anti-parasitic drugs, preferably chloroquine and / or mefloquine. By HAART, we mean a therapeutic indication proposing several active ingredients that will act synergistically. Example 1 provides an example of dual therapy: chloroquine is co-administered with isoquinoline. Since the mechanism of action of

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 The medicaments according to the invention are different from the mechanisms of action currently known for the treatment of parasitic diseases, there is no competition between the various known active ingredients and those of the invention.

In addition, isoquinoline is active on multidrug-resistant Plasmodium strains, particularly the Plasmodium falciparum strain Dd2, while flurazepam has the particularity of exhibiting activity on all parasitic stages of Plasmodium Falciparum. A multidrug-resistant strain is considered when treatment with drugs such as chloroquine or mefloquine produces no improvement in the patient's condition.

Advantageously, the drug is formulated to allow oral or intravenous administration.

Suitably, the doses administered orally are between 10 g and 0.5 mg / kg.

The present invention also relates to the use of the peripheral benzodiazepine receptor (PBR) as a ligand for the screening of molecules with anti-parasitic activity.

The medicaments according to the invention open up new research possibilities in the field of parasitic diseases.

As such, the screening of molecules using PBR is a particularly advantageous method.

It furthermore proposes a test for inhibiting the antiparasitic activity, characterized in that it comprises the following steps: incubation of the cells with variable concentrations of parasites and / or inhibitors to be tested,

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 and then bringing the incubated medium into contact with the molecules as used in claims 1 to 6 and optionally inhibitors.

This inhibition test makes it possible: either to test new applications of the molecules (new parasites that can be envisaged) or to test new molecules, obtained for example after screening, or to test new drug combinations.

Other characteristics and advantages of the invention will become apparent in the examples which follow, with reference to FIGS. 1 to 7, which represent, respectively: FIGS. 1A, 1B, 2A, 2B, 3A and 3B show the dose-inhibiting effect -dependent on isoquinoline and flurazepam on T. gondii (Fig. A) and P. falciparum (Fig. B), alone (Fig. 1 and 2) or in combination (Fig. 3).

FIGS. 4A and 4B show the inhibitory activity of chloroquine and isoquinoline on the Dd2 strain of
P. falciparum.

 FIGS. 5A and 5B show the dose-dependent effect of chloroquine alone or in combination with isoquinoline on P. falciparum strain Hb3.

 FIGS. 6A to 6D show the modifications undergone by trophozoites of P. falciparum in the presence of the medicaments according to the invention.

 - Figure 7 shows the anti-parasitic activity of flurazepam on the different parasitic stages of P. falciparum.

Example 1: Testing of antimicrobial agents 1. Materials and methods

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 In vitro culture of P. falciparum.

The tests were carried out with two strains adapted to Plasmodium falciparum culture: the chloroquine-sensitive strain HB3 and the mefloquinochloroquine-resistant strain Dd2. The parasites were maintained on 0+ human erythrocytes in RPMI 1640 culture medium (GibcoBRL) supplemented with 27.5 mM NaHC03, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) ( pH 7.4), in 11 mM glucose and 7.5% (v / v) heat-inactivated human AB + serum, under 5% CO 2, 5% O 2 and 90% N 2 at 37 C (18). The tests were carried out on 96-well plates. Different drug concentrations were added to asynchronous or synchronous parasite cultures (0.5% parasitaemia and 18% hematocrits) in the presence of 0.5 Ci of [3H] hypoxanthine (Amersham, 1 mCi / ml) per well. . After incubation for 48 hours at 37 ° C. under a control atmosphere, the cells were collected in each well by means of an automatic device (Microbeta ™ 1450) on glass fiber filters. The dried filters containing the radioactivity bound to the parasites were measured using a scintillation counter. All drug concentrations were tested two or three times in triplicate in each trial.

Growth inhibition for each concentration was determined by comparing the radioactivity incorporated in the treated culture with that incorporated into control (drug-free) cultures present on the same plate.

In vitro culture of T. gondii.

Tachyzoites of Toxoplasma gondii strain 76K were cultured in human foreskin fibroblasts (HFF) in Dulbecco-Eagle medium (DMEM, Biowhittaker, Belgium) supplemented with 10% fetal calf serum (Dutscher), 2 mM glutamine (Sigma) and 0.05 mg / ml of gentamycin (Sigma). The tests were carried out on 6-well plates. The

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 HFF cell monolayer was infected with 2 x 105 parasites per well and 6 hours later the drugs were added in the concentration ranges described above, alone or in combination with each other. After another 48 hours of culture (5% CO 2, 37 C), the intracellular tachyzoites were pulsed with 2uCi [3H] uracil (Amersham) per well for 6 hours. In this test, only the parasites were labeled due to the absence of phosphoribosyltransferase in the host cells, whereas this rescue enzyme is present in T. gondii tachyzoites (1). After labeling, the infected cells were lysed with detergent and the radioactively labeled nucleic acids were precipitated by TCA and recovered on glass fiber filters. The radioactivity related to the parasites was measured using a scintillation counter (2). All drugs were tested two or three times in triplicate in each trial.

Antimicrobial agents.

The following antimicrobial agents (supplied by the manufacturers indicated) were used: chloroquine (CQ), flurazepam (Fz), 1- (2-chlorophenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinoline-carboxamide (IsoQ) (Sigma Chemical Co., St. Louis, Mo) and mefloquine (Pasteur Institute of Lille, France). IsoQ was initially dissolved in dimethylsulfoxide (DMSO) at a concentration of 3.3 mg / ml and Fz in Dulbecco's modified Eagle's medium (DMEM, Biowhittaker, Belgium) supplemented with 10% fetal calf serum inactivated by heat (FCS, Dutscher), 2 mM glutamine (Sigma) and 0.05 mg / ml gentamycin (Sigma), at a concentration of 5 mg / ml. The culture medium described above (DMEN-FCS) was used for the intracellular culture of T. gondii tachyzoites. CQ and MQ were initially dissolved in 70% methanol at concentrations of 5 mg / ml, DMSO containing DMEN-FCS or RPMI-FCS and methanol needed to dissolve

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 drugs tested and showed no function on T. gondii and P. falciparum, respectively. Dilutions were prepared immediately before each run. For P. falciparum, the following concentrations of each agent (alone or in combination) were tested: CQ and MQ at 9.19, 37.75 and 150 ng / ml). For T. gondii, Fz and IsoQ were tested alone or in combination at the following concentrations: 5.10, 25.40, 50.70 and 80 μg / ml.

 2. Results The experiments described above made it possible to demonstrate the dose-dependent inhibitory effect of the peripheral benzodiazepine receptor (PBR) ligand molecules on the multiplication and growth of Plasmodium falciparum and Toxoplasma gondii strains.

Figures 1A, 1B, 2A, 2B, 3A and 3C show the results in graph form. Concentrations corresponding to 50% inhibition (IC50) were determined graphically.

They are summarized in the following table:

<Tb>
<tb> Isoquinoline <SEP> Flurazepam
<tb> T. <SEP> gondii <SEP> Between <SEP> 10 <SEP> and <SEP> 20 <SEP> g / ml <SEP> Between <SEP> 25 <SEP> and <SEP> 50 <SEP> g / ml
<tb> (Fig. <SEP> 1A) <SEP> (Fig. <SEP> 2A)
<tb> P. <SEP> falciparum <SEP> Between <SEP> 20 <SEP> and <SEP> 25 <SEP> g / ml <SEP> Between <SEP> 10 <SEP> and <SEP> 20 <SEP> g / ml
<tb> (Fig. <SEP> 1B) <SEP> (Fig. <SEP> 2B)
<Tb>

Table I: IC50 of isoquinoline and flurazepam on T. gondii and P. falciparum.

A comparison of FIGS. 3A and 3B with FIGS. 1A to 2B shows that the drugs exert an additive effect.

In addition, isoquinoline retains inhibitory activity on a chloroquine- and mefloquine-resistant strain of P.falciparum (strain Dd2) (Fig. 4A and 4B). The IC 50 of isoquinoline is then 25 g / ml.

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Finally, the use of PBR ligand molecules does not alter the anti-parasitic effects of already known anti-malarial drugs such as chloroquine or mefloquine. FIGS. 5A and 5B show the chloroquine-dependent effect alone or in combination with isoquinoline on P. falciparum strain Hb3. It can be seen that a significant decrease in parasite activity is obtained for a concentration of between 9.08 and 18.2 nM chloroquine when it is in combination with isoquinoline, whereas this same decrease is only observed when between 36.3 and 72.7 nM chloroquine when used alone.

Example 2 Evaluation of the Morphology of P. falciparum and T. gondii, Development and Replication
1. Materials and methods Morphology, development and replication of asynchronous or synchronous P. falciparum cultures were evaluated in cultures by light microscopic observation of Giemsa-stained fine blood smears. Smears from the drug-free cultures were used each time as controls. Parasitaemia was measured by counting 1000 red blood cells and the value obtained is indicated as the percentage of all parasitized erythrocytes. Morphologically normal and abnormal parasites are included in the measurements.

For transmission electron microscopy, T. gondii tachyzoites (strain 76K) grown in HFF cells or P. falciparum infected red cells (strain Hb3) were treated with Fz or IsoQ for two days. Treated intracellular parasites were fixed in 2.5% glutaraldehyde prepared in a buffer

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 0.1M cacodylate and subsequently set in 1% OsO.sub.4 in the same buffer. After dehydration with ethanol, the pellet was included in Epon. A series of thin sections was cut using an ultramicrotome and collected on longitudinal bar grids. After staining with 2% uranyl acetate prepared in 50% ethanol and incubation with lead citrate solution, the sections were observed using a Hitachi H-600 electron microscope. .

 2. Results For P. falciparum, the images demonstrate, at the ultrastructural level, a profound alteration of the endoplasmic reticulum and the digestive vacuole. This vacuole, necessary for the survival of the parasite, is sometimes absent when the parasites are subjected to the drug. When present, it is characterized by a decrease in its volume and by the significant reduction of intravacuolar hemozoine crystal usually observed in the mature stages of the parasite (heme nucleus polymer compound from hemoglobin or ferriprotoporphyrin IX). This is similar to the toxic inhibition of heme polymerization in P. falciparum described for chloroquine and mefloquine.

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References: (1) Lin L. et al. (1999) Phenolic aporphine-benzylisoquinoline alkaloids from Thalictrum faberi.

 Phytochemistry 50: 829-834.

(2) François G. et al. (1996) Naphtylisoquinoline alkaloids exhibit strong growth-inhibiting activities against
Plasmodium falciparum and P. berghei in vitro structure activity relationships of dionophylin C. Ann. Too much.

 Med. Parasitol. 90: 115-123.

Claims (15)

1. Use of at least one ligand molecule of the peripheral benzodiazepine receptor (PBR) for the manufacture of medicaments for the treatment of parasitic diseases. 2. Use according to claim 1, characterized in that the ligand molecules of the peripheral benzodiazepine receptor (PBR) are compounds of formula (I):

 where X is a halogen atom selected from F, Cl, Br or I, a hydrogen atom, OH, COOH, SH, NH 2, OR, COOR, SR, NHR or NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C, R1 is a hydrogen atom, an alkyl chain of 1 to 4 C or form with R4 a C-N type bond, R2 is a hydrogen atom or a hydrocarbon chain of 1 to 6 C, optionally comprising an NHR group or NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C, R3 is a hydrogen atom or a hydrocarbon chain of 1 to 2 C forming a ring with R4, R4 forms with Ri a C-N bond, a hydrocarbon chain of 1 to 2 C forming a ring with R3 or a hydrogen atom. 3. Use according to claim 2, characterized in that the molecules correspond to formula (II): <Desc / Clms Page number 15>

 where X is a halogen atom selected from F, Cl, Br or I, a hydrogen atom, OH, COOH, SH, NH 2, OR, COOR, SR, NHR or NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C, R1 is a hydrogen atom, an alkyl chain of 1 to 4 C, R2 is a hydrogen atom or a hydrocarbon chain of 1 to 6 C, optionally comprising an NHR group or NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C. 4. Use according to claim 3, characterized in that the compound corresponds to formula (IV):

 5. Use according to claim 2, characterized in that the compound corresponds to formula (III): <Desc / Clms Page number 16>

 where X is a halogen atom selected from F, Cl, Br or I, a hydrogen atom, OH, COOH, SH, NH 2, OR, COOR, SR, NHR or NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C, R2 is a hydrogen atom or a hydrocarbon chain of 1 to 6 C, optionally comprising an NHR group or NRR ', where R and R' are identical or different, represent an alkyl chain of 1 to 4 C. 6. Use according to claim 5, characterized in that the compound corresponds to formula (V):

 7. Use according to any one of the preceding claims, characterized in that the parasitic diseases are due to pathogens of the Apicomplexa family. 8. Use according to any one of the preceding claims, characterized in that the pathogens are <Desc / Clms Page number 17>  Toxoplasma and Plasmodium, in particular Toxoplasma gondii and Plasmodium Falciparum. 9. Use according to any one of the preceding claims, characterized in that the drugs have a dose-dependent inhibitory effect vis-à-vis the parasite (s). 11. Use according to any one of the preceding claims, characterized in that the drugs are used in combination therapy, jointly and / or in supplements of anti-parasitic drugs, preferably chloroquine and / or mefloquine. 12. Use according to claim 3 or 4, characterized in that the compound is active on multi-drug resistant Plasmodium strains, in particular the strain Dd2 of Plasmodium falciparum. 13. Use according to claim 5 or 6, characterized in that the compound is active on all parasitic stages of Plasmodium falciparum. 14. Use according to any one of the preceding claims, characterized in that the drug is formulated to allow oral or intravenous administration. 15. Use according to any one of the preceding claims, characterized in that the doses administered orally are between 10 g and 0.5 mg / kg. 16. Use of the peripheral benzodiazepine receptor (PBR) as a ligand for the screening of molecules with anti-parasitic activity. <Desc / Clms Page number 18>  17. An inhibition test of the anti-parasitic activity, characterized in that it comprises the following steps: incubation of the cells with variable concentrations of parasites and / or inhibitors to be tested, then contacting the medium incubated with the molecules as used in any one of claims 1 to 6.