METHOD OF TREATING BACTERIAL INFECTIONS AND PHARMACEUTICAL COMPOSITION FOR TREATING BACTERIAL INFECTIONS

Abstract

The invention relates to a compound having the general formula (I), or one of the pharmaceutically acceptable salts thereof, for it use as a medicament, in particular for treating a bacterial infection in a subject:

##STR00001##

In particular, this compound is capable of inhibiting the Mfd activity of bacteria.

Claims

1. A method of treating a subject, comprising administering to the subject a therapeutically-effective amount of a compound having the general formula (I): ##STR00008## wherein m is an integer between 0 and 2, n is equal to 0 or 1, o is equal to 0 or 1, p is an integer between 0 and 2, p′ is an integer between 0 and 2, either the double line represents a single bond and X is selected from the group consisting of an oxygen atom, CH.sub.2, NH, N—R.sub.5 and —NCO—R.sub.5, wherein R.sub.5 represents a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, and Y represents a methylene group, a carbonyl group, a thiocarbonyl group or a C═N—R.sub.9 group wherein R.sub.9 represents a hydrogen atom or an alkyl or aryl group, or the double line represents a double bond and X represents a nitrogen atom and Y represents a methine group, Z represents a covalent bond, a methylene group, a sulfonyl group, an amide group, an ester group, an ether group or a carbonyl group, provided that Z does not represent a carbonyl group when p is equal to 0, W represents a nitrogen atom or a C—R.sub.10 group wherein R.sub.10 represents a hydrogen atom or a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, R.sub.1 is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a primary amine group, a linear or branched and/or cyclic saturated or unsaturated carbon-based radical, optionally aromatic, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, a —O—R.sub.6, —NH—R.sub.6 or —NH—CO—R.sub.6 group wherein R.sub.6 represents a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, and an —NR.sub.7R.sub.8 group wherein R.sub.7 and R.sub.8, which may be identical or different, each represent a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, and R.sub.2 is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a sulfonic acid group, a primary amine group, and a linear, branched and/or cyclic, saturated or unsaturated, carbon-based radical, optionally substituted, optionally aromatic, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, or one of the pharmaceutically acceptable salts thereof.

2. The method of claim 1, wherein X is selected from the group consisting of CH.sub.2, NH, N-alkyl and NCO-alkyl.

3. The method of claim 1, wherein R.sub.1 is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a carbonitrile group, a trifluoromethyl group, a carboxyl group, a primary amine group and an alkyl, alkoxy, aryl, arylalkyl, acyl, —CO—O-alkyl, —NH-alkyl or —NH—CO-alkyl group, all of which may optionally be substituted.

4. The method of claim 1, wherein R.sub.2 represents a —SO.sub.2—NR.sub.3R.sub.4 radical, wherein R.sub.3 and R.sub.4 are identical or different linear or branched alkyl groups.

5. The method of claim 1, wherein the compound has the formula (Ia): ##STR00009## or is one of the pharmaceutically acceptable salts thereof.

6. The method of claim 1, which is for treating a bacterial infection in said subject.

7. The method of claim 6, which is for inhibiting the Mfd activity of a pathogenic bacteria infecting said subject.

8. The method of claim 6, wherein said bacterial infection is caused by Gram-negative and/or Gram-positive bacteria.

9. The method of claim 1, wherein said compound is administered by oral, parenteral, topical, intranasal, rectal or pulmonary route to said subject.

10. The method of claim 1, wherein said compound is administered once daily to said subject.

11. A pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, a compound having the general formula (I): ##STR00010## wherein m is an integer between 0 and 2, n is equal to 0 or 1, o is equal to 0 or 1, p is an integer between 0 and 2, p′ is an integer between 0 and 2, either the double line represents a single bond and X is selected from the group consisting of an oxygen atom, CH.sub.2, NH, N—R.sub.5 and —NCO—R wherein R.sub.5 represents a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, and Y represents a methylene group, a carbonyl group, a thiocarbonyl group or a C=N−R.sub.9 group wherein R.sub.9 represents a hydrogen atom or an alkyl or aryl group, or the double line represents a double bond and X represents a nitrogen atom and Y represents a methine group, Z represents a covalent bond, a methylene group, a sulfonyl group, an amide group, an ester group, an ether group or a carbonyl group, provided that Z does not represent a carbonyl group when p is equal to 0, W represents a nitrogen atom or a C—R.sub.10 group wherein R.sub.10 represents a hydrogen atom or a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, R.sub.1 is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a primary amine group, a linear or branched and/or cyclic saturated or unsaturated carbon-based radical, optionally aromatic, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, a —O—R.sub.6, —NH—R.sub.6 or —NH—CO—R.sub.6 group wherein R.sub.6 represents a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, and an —NR.sub.7R.sub.8 group wherein R.sub.7 and R.sub.8, which may be identical or different, each represent a linear, branched and/or cyclic, saturated or unsaturated, hydrocarbon-based radical, optionally substituted, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, and R.sub.2 is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a sulfonic acid group, a primary amine group, and a linear, branched and/or cyclic, saturated or unsaturated, carbon-based radical, optionally substituted, optionally aromatic, optionally comprising one or several heteroatoms and/or one or more groups containing one or more heteroatoms, or one of the pharmaceutically acceptable salts thereof.

12. The pharmaceutical composition of claim 11, which is suitable for administration by oral, parenteral, topical, intranasal, rectal or pulmonary route.

13. The pharmaceutical composition of claim 11, wherein said compound of formula (I) is encapsulated in nanoparticles.

14-15. (canceled)

16. The method of claim 1, wherein said compound is administered twice daily to said subject.

17. The method of claim 1, wherein said compound is comprised in a pharmaceutical composition, in a pharmaceutically acceptable vehicle.

18. The method of claim 17, wherein said pharmaceutical composition is suitable for administration by oral, parenteral, topical, intranasal, rectal or pulmonary route.

19. The method of claim 1, wherein said compound is encapsulated in nanoparticles.

20. The method of claim 4, wherein R.sub.3 and R.sub.4 are methyl groups.

21. The pharmaceutical composition of claim 11, wherein X is selected from the group consisting of CH.sub.2, NH, N-alkyl and NCO-alkyl.

22. The pharmaceutical composition of claim 11, wherein R.sub.1 is selected from the group consisting of a hydrogen atom, a halogen atom, a hydroxyl group, a carbonitrile group, a trifluoromethyl group, a carboxyl group, a primary amine group and an alkyl, alkoxy, aryl, arylalkyl, acyl, —CO—O-alkyl, —NH-alkyl or —NH—CO-alkyl group, all of which may optionally be substituted.

23. The pharmaceutical composition of claim 11, wherein R.sub.2 represents a —SO.sub.2—NR.sub.3R.sub.4 radical wherein R.sub.3 and R.sub.4 are identical or different linear or branched alkyl groups.

24. The pharmaceutical composition of claim 23, wherein R.sub.3 and R.sub.4 are methyl groups.

Description

[0089] The features and advantages of the invention will emerge more clearly in the light of the following examples of implementation, provided for illustrative purposes only and in no way limitative of the invention, with the support of FIGS. 1 to 8, in which:

[0090] FIG. 1 shows a bar graph representing the relative inhibition of E. coli Mfd ATPase activity in vitro, with respect to the vehicle (“DMSO”), by 100 μM of compounds according to the invention (Ia) and (Ib) and by comparative compounds (IIa) and (IIb), respectively for two different ATP concentrations in the medium (0.035 mM and 0.070 mM);

[0091] FIG. 2 is a graph showing in vitro E. coli Mfd ATPase activity, expressed in terms of phosphate release (nmol) as a function of ATP concentration, in the presence of different doses of a compound according to the invention (Ia);

[0092] FIG. 3 shows a bar graph representing the relative survival rate of B. cereus after 4 h of culture under nitric oxide stress in the presence of different concentrations of a compound according to the invention (Ia), with respect to the vehicle alone (0 μM of (Ia));

[0093] FIG. 4 shows a bar graph representing the relative survival rate of K. pneumoniae after 4 h of culture under nitric oxide stress in the presence of different concentrations of a compound according to the invention (Ia), with respect to the vehicle alone (0 μM of (Ia));

[0094] FIG. 5 shows a bar graph representing the relative survival rate of A. baumanii after 4 h of culture under nitric oxide stress in the presence of different concentrations of a compound according to the invention (Ia), with respect to the vehicle alone (0 μM of (1a));

[0095] FIG. 6 shows a graph representing the rate of survival of insects of the species Bombyx mori infected with bacteria of the species B. cereus, as a function of the administrated dose of a compound (Ia) according to the invention;

[0096] FIG. 7 shows a graph representing the rate of survival of insects of the species Bombyx mori infected with bacteria of the species P. aeruginosa, as a function of the administrated dose of a compound (Ia) according to the invention;

[0097] and FIG. 8 shows graphs representing the bacterial load in the tissues of various organs of a model mouse infected by B. cereus, respectively the lung (a/), the liver (b/) and the spleen (c/), after administration of a dose of 12 mg/kg of a compound according to the invention (Ia).

[0098] A/ Inhibition of Mfd ATPase Activity In Vitro

[0099] For this in vitro assay, the following compounds were used: [0100] compound (Ia) according to the invention, having the formula:

##STR00005## [0101] compound (Ib) according to the invention, having the formula:

##STR00006## [0102] and comparative compounds (IIa) and (IIb), the chemical formulae of which do not fall within general formula (I), but are close to this general formula:

##STR00007##

[0103] Mfd enzyme activity was evaluated by measuring the quantity of inorganic phosphate (PO.sub.4i) released using BIOMOL® Green reagent microtiter-plate assay (Enzo Life Sciences). Mfd from E. coli (0.35 μM) was incubated with DMSO or with the tested compounds at a concentration of 100 μM in DMSO, for 10 min at 37° C. ATPase reaction was measured in Tris pH8 0.05M; NaCl 0.3M; DTT 0.002M; MgCl2 0.0025M; DMSO 2%; containing ATP (at different concentrations between 0.0022 and 0.279 mM) for 30 min at 37° C. 50 μL of each reaction medium was transferred into clear, flat-bottom 96-well plates and the reaction was terminated by the addition of 100 μl of BIOMOL® Green reagent. The absorbance at 620 nm was measured in a microplate reader (Tecan). The absorbance values were then transformed into nmols of PO.sub.4i released based on a PO.sub.4i standard curve prepared as recommended by the supplier.

[0104] The potency of each compound is calculated relative to the vehicle DMSO control. The results are shown in FIG. 1. Each point is the mean of duplicate experiments of N=2+/−SEM (Standard error of the Mean).

[0105] For both ATP concentrations, it can be observed that the compounds of the invention (Ia) and (Ib) exhibit a much higher capacity of inhibition of the Mfd ATPase activity than the comparative compounds, which do not inhibit Mfd ATPase activity. Compound (Ia) is more efficient than compound (Ib).

[0106] Mfd ATPase activity at ATP concentrations ranging from 0 to 0.279 mM was measured in the same conditions as described above, without and with presence of various doses of compound (Ia): 0 μM, 3 μM, 10 μM, 30 μM, 65 μM and 100 μM. Curves were fit to the Michaelis-Menten equation with the GraphPad PRISM software. Results are the mean of duplicate experiment of N=3-10+/−SEM.

[0107] The results obtained are shown on FIG. 2. They show that compound (Ia) of the invention works as a competitive inhibitor of Mfd ATPase activity.

[0108] The Ki of compound (Ia) was calculated at a value of 27.32 μM.

[0109] These results demonstrate the high capability of compound (Ia) of the invention to inhibit Mfd ATPase activity.

[0110] B/ Antimicrobial Activity During Nitric Oxide Stress

[0111] The following assay was carried out in order to evaluate whether the compound of the invention (Ia) can inhibit Mfd function in the bacterial resistance against nitric oxide stress.

[0112] The power of compound (Ia) to inhibit bacterial growth, specifically in the presence of in vitro-produced nitric oxide, is measured for different bacterial species: Bacillus cereus and two antibiotherapy challenging bacteria, regarding medical interest and therapeutic needs, namely Klebsiella pneumoniae and Acinetobacter baumannii.

[0113] Gram-positive B. cereus (Bc 407) and Gram-negative K. pneumoniae (CIP700603) and A. baumannii (CIP7034) strains were grown to exponential phase in Luria-Bertani (LB) medium. Bacteria solution were prepared in Roswell Park Memorial Institute (RPMI) medium and 150 μL were dispatched into 96-wells plate. To measure effect of compound (Ia), bacteria were exposed to 50 μL of NO at a concentration that induces a survival below 90% compared to the condition without NO, in absence (0 μM) or presence (at a dose ranging from 10 to 200 μM) of compound (Ia).

[0114] Bacteria survival rate was quantified after 4 h at 37° C. by normalizing bacterial load in (Ia)-treated samples against that in DMSO-treated samples (0 μM of (Ia)).

[0115] The results obtained are shown in FIG. 3 for B. cereus, in FIG. 4 for K. pneumoniae and in FIG. 5 for A. baumanii. In these bar graphs the data represent the mean of duplicate experiment of N=3/12+/−SEM. Two-tailed t-student tests are employed to investigate statistical differences, using GraphPad Prism.

[0116] These results show that compound (Ia) of the invention reduces bacteria resistance to NO in vitro for Gram-positive (B. cereus) as well as for Gram-negative (K. pneumoniae and A. baumanii) bacteria. Compound (Ia) reduces bacterial survival during nitric oxide stress.

[0117] C/ Therapeutic Efficiency In Vivo for Treating Bacterial Infection in Insects

[0118] The following experiment was carried out in order to evaluate whether compound (Ia) of the invention can decrease the bacteria virulence in vivo, in silkworm larvae.

[0119] Silkworm larvae were infected with Gram-positive Bacillus cereus (Bc 407) or Gram-negative Pseudomonas aeruginosa (CIP27853) bacteria, as follows.

[0120] Bombyx mori larvae at the 3rd-4th instar stage (having a weight between 0.7 and 1 g) were used. Larvae were starved for 5 h before the experiment.

[0121] B. cereus bacteria (1×10.sup.2CFU) or P. aeruginosa bacteria (1×10.sup.4CFU) were injected into the larvae (n=10) in absence (0 μM) or in presence of compound (Ia) of the invention, which was injected concomitantly with the bacteria, at different doses ranging between 0.3 and 5.5 μg/g of larvae. The numbers of surviving larvae after 24 h at 27° C. were measured.

[0122] The results are shown in FIG. 6 for B. cereus and in FIG. 7 for P. aeruginosa. In these graphs, the data represent the mean of duplicate experiment of N=2/10+/−SEM. Two-tailed t-student tests are employed to investigate statistical differences, using GraphPad Prism.

[0123] Compound (Ia) of the invention shows no toxicity towards the insects and, as can be seen on the figures, it provides a significant protective effect against infection with B. cereus, as well as against infection with P. aeruginosa at higher doses. These results demonstrate the in vivo innocuity of the compound of the invention and its efficacy for preventing bacteria from killing silkworms.

[0124] D/ Efficiency In Vivo for the Treatment of an Infection by B. cereus in a Mouse Model

[0125] The efficiency of the compound of the invention (Ia) against a bacterial pathogen was assessed in a mouse model of infection (systemic infection model).

[0126] Eight weeks old female C57BL/6JRj (Janvier Labs) mice were infected with B. cereus (Bc 407) (4×10.sup.6 CFU) by intraperitoneal injection (100 μL). 100 μl of compound of the invention (Ia) in DMSO at a dose of 12 mg/kg, or of the vehicle alone (DMSO), were further injected to the mice by intraperitoneal injection.

[0127] 6 h after the infection mice were sacrificed by cervical dislocation. Mice organs were collected and homogenized in cold Phosphate Buffered Saline (PBS). Bacteria burdens in organs were determined by plating serially dilution of the homogenate on LB agar media.

[0128] The results obtained are shown on FIG. 8 in a/ for the lung, in b/ for the liver and in c/ for the spleen. In these figures, the data are represented as mean+/−SEM from 3 different experiments with 6-8 mice per group. Each point represents one animal.

[0129] It is observed that compound (Ia) of the invention decreases the bacterial load in all these organs following infection by B. cereus.