WO2010087260A1 - Anti-tuberculosis agent and use thereof - Google Patents

Abstract

Disclosed are a novel anti-tuberculosis agent and a use thereof. The anti-tuberculosis agent comprises disulfiram, diethyldithiocarbamate, or a pharmaceutically acceptable salt of disulfiram or diethyldithiocarbamate as an active ingredient.

Description

Antituberculosis drugs and their uses

The present invention relates to an antituberculosis drug and its use (prevention or treatment of tuberculosis). This application claims priority based on Japanese Patent Application No. 2009-21026 filed on Jan. 31, 2009, the entire contents of which are incorporated by reference.

1993 Following the WHO Tuberculosis Emergency Declaration in 1993, the Ministry of Health, Labor and Welfare issued a tuberculosis emergency declaration in July 1999, and tuberculosis is recognized in Japan as an infectious disease that needs to be addressed. After World War II, effective tuberculosis treatments such as streptomycin (SM), isoniazid (INH) and rifampicin (RFP) were developed, and the number of tuberculosis patients was steadily decreasing. Currently, more than 1.8 million people have died, and since the mid-1980s, the trend has gradually increased, especially in urban areas. The cause of this is refractory anti-tuberculosis such as multidrug-resistant tuberculosis (MDR-TB) resulting from chemotherapy failure and non-tuberculous mycobacteria M. avium-intracellulare complex (MAC), which is a chronic disease caused by opportunistic infections. This is a major reason why acid mycosis turned to an increasing trend. For MDR-TB, the DOTS strategy launched in 1994 has achieved remarkable results worldwide. On the other hand, in developing countries such as South Africa, the emergence of extensive drug-resistant Mycobacterium tuberculosis (Extensively Drug-Resistant TB; XDR-TB), in which 90% die within a month after infection due to double infection with HIV, has been confirmed. In Japan, 11 types of drugs have been used in the past.However, fluoroquinolone (FQ) is not approved for insurance, and capreomycin (CPM) is removed from the drug price standard. ing. In tuberculosis medicine for which development of new drugs is delayed, development of new drugs with new chemical structural formulas and new mechanisms of action is desired.

Under the above background, an object of the present invention is to provide a novel antituberculosis drug and its use.

The present inventors paid attention to the drug disulfiram used as an anti-alcohol drug, and examined its action against Mycobacterium tuberculosis. As a result, it was revealed that disulfiram and its metabolite diethyldithiocarbamate (DDC) have a strong antibacterial activity against Mycobacterium tuberculosis H ₃₇ Rv (MTB). The strength was intermediate between the first-line drugs and the second-line drugs, and the in vitro MIC was 0.78 to 1.56 μg / ml, which is equivalent to the existing second-line drugs for antituberculosis drugs. Disulfiram was reported as an aldehyde dehydrogenase inhibitory effect by Erik Jacobsen et al. In 1948 and is now widely used as a drug with indications for chronic alcohol intoxication, so far, Methicillin resistant Staphylococcus aureus aureus (Non-patent Document 1), Giardia (Non-patent Document 2), Metronidazole-resistant Trichomonas vaginalis (Metronidazole) -resistant Trichomonas vaginalis) (non-patent document 3) and fungi such as Aspergillus spp (non-patent document 4) have been reported to have antibacterial activity.

As a result of further investigation, significant and interesting findings have been obtained regarding the antibacterial activity, intracellular translocation, cytotoxicity, and primary action point of disulfiram and DDC, and these compounds are extremely effective as active ingredients for antituberculosis drugs. It has been shown. In particular, the very high selectivity for Mycobacterium tuberculosis and the antibacterial activity against drug-resistant bacteria strongly support the effectiveness of the compound.

Based on the above knowledge, the following invention is provided.
[1] An antituberculosis drug containing disulfiram or diethyldithiocarbamate or a pharmaceutically acceptable salt thereof as an active ingredient.
[2] an antituberculosis drug containing isoniazid, rifampicin, streptomycin, kanamycin, amikacin, ethambutol, or para-aminosalicylate as an active ingredient The antituberculosis drug according to [1], which is administered in combination.
[3] A method for preventing or treating tuberculosis, comprising a step of administering the antituberculous drug according to [1] to a subject.
[4] An antituberculosis drug containing isoniazid, ryfampicin, streptomycin, kanamycine, amikacin, ethambutol, or paraaminosalicylate (p-aminosalicylate) as an active ingredient The method for preventing or treating tuberculosis according to [3], which is administered in combination.
[5] Use of disulfiram or diethyldithiocarbamate or a pharmaceutically acceptable salt thereof for the manufacture of an antituberculosis drug.

Antibacterial activity against various acid-fast bacilli, Staphylococcus aureus and Escherichia coli of existing drugs by antibacterial activity measurement method (BDT method) using the concentration of bacteria in liquid medium as an index The minimum inhibitory concentrations (MIC) of various antibacterial drugs are summarized in the table. Antibacterial activity of disulfiram and DDC against various acid-fast bacteria, Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations (MIC) of disulfiram and DDC are summarized in the table. INH: isoniazid, RFP: rifampicin, SM: streptomycin, EB: ethambutol, PAS: paraaminosalicylic acid, AMK: amikacin, KM: kanamycin, GM: gentamicin, TC: tetracycline, DOXY: doxycycline, CAM: clarithromycin, CPFX: cystein Profloxacin, VCM: Vancomycin, PCG: Benzylpenicillin, ABPC: Ampicillin The graph which shows the cytotoxicity of disulfiram and DDC. Cytotoxicity was compared with existing antituberculosis drugs. A: Toxicity to A-549 cells. B: Toxicity to THP-1 cells. C: Toxicity to THP-1 cells. The horizontal axis is the drug concentration (logarithm), and the vertical axis is the ratio (%) to the control. The table | surface which shows the nontoxic area | region of the disulfiram with respect to THP-1 cell. Compared with existing drugs. INH: isoniazid, RFP: rifampicin, SM: streptomycin, EB: ethambutol, DDC: diethyldithiocarbamate. The graph which shows the antibacterial activity with respect to the tubercle bacillus parasitizing the host cell of disulfiram and DDC. A: Activity in THP-1 cells. B: Activity in A549 cells. The horizontal axis represents the drug concentration, and the vertical axis represents the amount of decrease in the number of colonies (logarithm). The table | surface which shows the pH dependence of the antituberculous action of disulfiram and DDC. CPFX: Ciprofloxacin, DDC: Diethyldithiocarbamate. Examination of the mode of action (bactericidal or bacteriostatic) of disulfiram on M. bovis BCG. The results of the drug sensitivity test are summarized in a graph. The activity of the drug was evaluated by the number of colonies of Mycobacterium bovis BCG. A: sensitivity to isoniazid, B: sensitivity to ethambutol, C: sensitivity to disulfiram, D: sensitivity to DDC, E: sensitivity to disulfiram, D: sensitivity to DDC. The horizontal axis is the number of days after drug addition. The vertical axis of A to D is the absorbance at 530 nm. The vertical axis of E and F is the number of colonies (CFU). Examination of the mode of action (bactericidal or bacteriostatic) of disulfiram against Staphylococcus aureus. The results of the drug sensitivity test are summarized in a graph. The activity of the drug was evaluated by the number of colonies of Staphylococcus aureus. A: sensitivity to disulfiram, B: sensitivity to DDC, C: sensitivity to CPFX. The horizontal axis is the time after drug addition. The vertical axis is the absorbance at 530 nm. Examination of mode of action (bactericidal or bacteriostatic) of disulfiram against M. smegmatis. The results of the drug sensitivity test are summarized in a graph. The activity of the drug was evaluated by the number of colonies of Mycobacterium smegmatis. A: sensitivity to disulfiram, B: sensitivity to DDC, C: sensitivity to lysozyme. The horizontal axis is the time after drug addition. The vertical axis is the absorbance at 530 nm. Graph (A) showing cholinesterase inhibitory action of disulfiram and DDC and IC ₅₀ (B) of each drug. The horizontal axis of A is the drug concentration, and the vertical axis is the residual activity (%). The table | surface which shows the antibacterial action with respect to drug resistance M. * smegmatis of a disulfiram and DDC. The result of measuring MIC (minimum inhibitory concentration) of each drug by 7H11 agar plate method. INH: isoniazid, RFP: rifampicin, SM: streptomycin, EB: ethambutol, KM: kanamycin, PAS: paraaminosalicylic acid, CPFX: ciprofloxacin, DSF: disulfiram, DDC: diethyldithiocarbamate. The table | surface which shows the antimicrobial activity of a disulfiram related compound. DSF: Disulfiram, DDC: Diethyldithiocarbamate. Table showing antibacterial activity of disulfiram and DDC against clinical isolates (drug-sensitive strains). Table showing antibacterial activity of disulfiram and DDC against clinical isolates (drug resistant strains). INH: isoniazid, RFP: rifampicin, EB: ethambutol, LVFX: levofloxacin, SPFX: spafloxacin, CPFX: ciprofloxacin, KM: kanamycin, SM: streptomycin, PAS: paraaminosalicylic acid, TH: ethionamide, PZA: pyrazinamide, DSF: Disulfiram, DDC: diethyldithiocarbamate. Table showing drug resistance frequency for disulfiram and DDC. INH: isoniazid, RFP: rifampicin, SM: streptomycin, EB: ethambutol, KM: kanamycin, CPFX: ciprofloxacin, PAS: paraaminosalicylic acid, DSF: disulfiram, DDC: diethyldithiocarbamate. Outline of experimental system using tuberculosis chronic infection model mouse. RFP: rifampicin, DSF: disulfiram. The graph which shows the weight change of a tuberculosis chronic infection model mouse. A: Rifampicillin was administered at 20 mg / kg (R20), 10 mg / kg (R10) or 5 mg / kg (R5), and changes in body weight were recorded. B: Disulfiram was administered at 160 mg / kg (D160), 80 mg / kg (D80) or 40 mg / kg (D40), and changes in body weight were recorded. The graph which shows the infection protective effect of disulfiram. Disulfiram (40 mg / kg (D40), 80 mg / kg (D80) or 160 mg / kg (D160)), rifampicin (5 mg / kg (R5), 10 mg / kg (R10) or 20 mg / kg (R20)) or 5% gum arabic (control group) was administered, and then the lung, spleen and liver were excised and subjected to a colony assay. Mean values (n = 5) of logarithmic bacterial counts (logCFU) of lung (left) and spleen (right) are shown. Error bars indicate mean ± standard deviation (n = 5). Evaluated by Student's t-test. *, P <0.05 (vs. control group), **, p <0.01 (vs. control group), ***, p <0.001 (vs. control group). The graph of the result of having examined the toxicity of disulfiram. Disulfiram (40 mg / kg (D40), 80 mg / kg (D80) or 160 mg / kg (D160)), rifampicin (5 mg / kg (R5), 10 mg / kg (R10) or After administration of 20 mg / kg (R20)) or 5% gum arabic (control group), liver injury markers in the liver and serum were detected. The relative activities of GOT (left) and GPT (right) are shown. Error bars indicate mean ± standard deviation (n = 5). Evaluated by Student's t-test. *, P <0.05 (vs. control group), **, p <0.01 (vs. control group), ***, p <0.001 (vs. control group). NS is not significantly different. The graph of the result of having examined the toxicity of disulfiram. Disulfiram (40 mg / kg (D40), 80 mg / kg (D80) or 160 mg / kg (D160)), rifampicin (5 mg / kg (R5), 10 mg / kg (R10) or After administration of 20 mg / kg (R20)) or 5% gum arabic (control group), liver injury markers in the liver and serum were detected. ALP level (left) and ChE inhibitory effect (right) are shown. Error bars indicate mean ± standard deviation (n = 5). Evaluated by Student's t-test. *, P <0.05 (vs. control group), **, p <0.01 (vs. control group), ***, p <0.001 (vs. control group). NS is not significantly different.

The first aspect of the present invention relates to an antituberculosis drug. In the antituberculosis drug of the present invention, disulfiram (1- (diethylthiocarbamoyldisulfanyl) -N, N-diethyl-methanethioamide) or its metabolite diethyldithiocarbamate or a salt thereof is used as an active ingredient. The salt here is not particularly limited as long as it is pharmaceutically acceptable, and salts (inorganic acid salts) with hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, boric acid, formic acid, acetic acid, lactic acid, fumaric acid Examples thereof include salts (organic acid salts) with maleic acid, tartaric acid, citric acid and the like. These salts can be prepared by conventional means.

Disulfiram was reported as an aldehyde dehydrogenase inhibitory effect by Erik Jacobsen et al. In 1948 and is now widely used as a drug with indications for chronic alcoholism. The structure of disulfiram is shown below.

Disulfiram is known to be metabolized in vivo as follows.

Disulfiram is used clinically as an anti-alcohol drug and is readily available. For example, Mitsubishi Tanabe Pharma Co., Ltd., Nacalai Tesque Co., Ltd., Wako Pure Chemicals Co., Ltd., and SIGNA-ALORICHORI Group sell disulfiram. Diethyldithiocarbamate is available from Nacalai Tesque.

The active ingredient can be formulated according to a conventional method. In the case of formulation, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited, and can be prepared, for example, as tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories. The drug of the present invention thus formulated can be applied to a subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal injection, etc.) depending on the form. The “subject” here is not particularly limited, but preferably a human. The content of the active ingredient in the antituberculosis drug of the present invention generally varies depending on the dosage form, but is, for example, about 0.001% by weight to about 90% by weight so as to achieve a desired dose.

¡In addition to the above active ingredients, anti-tuberculosis drugs including existing anti-tuberculosis drugs may be used. If drugs with different action points are combined, a multifaceted antituberculosis action can be exerted. When the same or similar drugs are combined, enhancement of antituberculous action can be expected. Examples of existing anti-tuberculosis agents include Isoniazid, Rifampicin, Streptomycin, Kanamycin, Amikacin, Ethambutol, and para-aminosalicylate.

In another aspect of the present invention, a method for preventing or treating tuberculosis using the above antituberculosis drug (hereinafter, these two methods are collectively referred to as “treatment method” for convenience) is provided. The treatment method of the present invention includes a step of administering the antituberculosis drug of the present invention to the living body. The administration route is not particularly limited, and examples thereof include oral, intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, and transmucosal. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). In addition, oral administration is preferable because administration is easy.

The dose of the antituberculosis drug varies depending on the symptom, age of the subject, sex, weight, etc., but a person skilled in the art can appropriately set an appropriate dose. For example, the dose can be set so that the amount of the active ingredient per day is about 100 mg to about 1000 mg, preferably about 250 mg to about 500 mg for an adult (body weight: about 60 kg). As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In setting the administration schedule, it is possible to consider the condition of the administration subject, the duration of effect of the drug, and the like.

The antituberculosis drug of the present invention is administered alone or in combination with other antituberculosis drugs. Examples of other anti-tuberculosis drugs include Isoniazid, Rifampicin, Streptomycin, Kanamycin, Amikacin, Ethambutol, or para-aminosalicylate It is an anti-tuberculosis drug.

1. Antibacterial activity of existing anti-tuberculosis drugs and disulfiram against Mycobacterium tuberculosis (1) Method (BDT method)
A 96-well plate was prepared so that the drug solution was a 2-fold dilution series. A certain amount of a bacterial solution (100-fold dilution of OD = 0.1) was added thereto and cultured in the presence of 37 ° C. and 5% CO ₂ . Visually, the minimum drug concentration at which no bacteria were growing was defined as the minimum inhibitory concentration (MIC). In addition, this was used as a specimen, and 10 μl each was plated on 7H11 solid medium to determine the minimum bactericidal concentration (MBC). The experiment was performed twice independently, and the average value was calculated.

(2) Results The results are shown in FIGS. It was confirmed that the existing antituberculosis drugs INH, SM, EB, PAS, etc. have antibacterial activity specific to mycobacteria. The TC system has broad spectrum activity, and β-lactams PCG and ABPC are not effective against Mycobacterium tuberculosis, but are effective against other Gram-positive and Gram-negative bacteria. CPFX, a MAC infection remedy, has strong antibacterial activity against Mycobacterium tuberculosis, but because of its broad spectrum, there is concern about its impact on resident bacteria.

Disulfiram had a minimum inhibitory concentration of 0.78 to 1.56 μg / ml against M. tuberculosis® H37Rv. Disulfiram is metabolized to diethyldithiocarbamate (DDC) by glutathione reductase in the blood, excreted in the urine after hepatic metabolism. DDC was shown to be a narrow-spectrum compound with no antibacterial activity in M.Msmegmatis, Gram-positive bacteria S. aureus, and Gram-negative bacteria E. coli even in the same mycobacteria group . The MBC / MIC values of disulfiram and DDC were both 1, suggesting the possibility of bactericidal action (data not shown).

2. Cytotoxicity test of disulfiram (1) Method Seed each cell suspension in a 96-well plate, add a pre-prepared drug dilution, incubate at 37 ° C for 3 days, stain with crystal violet, and absorb at 595 nm It was measured. The experiment was performed three times independently, and the average value was calculated. THP-1 (1.0 × 10 ⁶ cells / ml), MRC-5 (1.5 × 10 ⁵ cells / ml), and A-549 (3.0 × 10 ⁵ cells / ml) were used as cells.

(2) Results The results are shown in FIG. The minimum concentrations at which cytotoxicity did not appear were 80 μg / ml for disulfiram and 300 μg / ml for DDC. From FIG. 4, it was found that the TD ₅₀ value of disulfiram corresponds to that of RFP. However, because RFP has a low MIC, the therapeutic range is wide. On the other hand, the therapeutic range of disulfiram was suggested to be narrow compared with existing drugs. In addition, according to past reports, safety has been established for the pharmacokinetics of disulfiram after oral administration.

3. Intracellular and pH-dependent changes in drug susceptibility Mycobacterium tuberculosis is one of the host body parasites. It is a latent infection in the phagosome of human Mφs and relapses with aging and immunity, causing pulmonary tuberculosis. It is a fungus. The phagosomes in the Mφ are slightly acidic (pH 6.1-6.5) and are in a restricted environment. With existing drugs, CAM, CPFX, INH, etc. have been found to decrease in activity under acidic conditions. Therefore, human Mφ-like cells were used to examine the intracellular migration of disulfiram, and the antibacterial activity of disulfiram under acidic conditions was examined.

3-1. Examination of intracellular translocation of disulfiram (1) Method THP-1 was treated with 100 ng / ml of PMA for 2 days, and spread in a 24-well plate at 1.0 × 10 ⁶ cells / ml. After incubation for 1 day and infection with 5.0 × 10 ⁷ cfu / ml of H37Rv for 4 hours, the cells were washed twice with HBSS and treated with SM for 20 hours. After washing twice with HBSS, a drug dilution series was made and added and treated for 4 hours. After washing twice with HBSS and lysing the cells with 0.1% SDS solution, a colony assay was performed. The cells were cultured for 14 days and evaluated by the number of colonies. The experiment was performed three times independently, and the average value was calculated.

(2) Results The results are shown in FIG. In human Mφ-like cell THP-1 and type II alveolar epithelial cell line A549, it was confirmed that INH and RFP significantly reduced the viable cells in the cells. SM, PAS, etc. were not effective against intracellular bacteria. Disulfiram has been shown to be effective against intracellular live bacteria at 10 μg / ml and above. As a titer, 30 μg / ml of disulfiram was shown to correspond to 1 μg / ml of RFP. On the other hand, DDCNa did not show a concentration-dependent bactericidal action, but was suggested to be effective against intracellular bacteria. Although disulfiram was inferior to the first-line drugs INH and RFP, it was suggested that it could move into cells and Mφ and kill intracellular parasites.

3-2. Study on changes in pH-dependent drug sensitivity (1) Method (BDT method)
A 7H9 medium (pH 6.6, pH 6.2) containing 10% ADC and 0.02% Tyroxapol and a modified Kirchner medium (pH 6.8, pH 5.6) were prepared. The experiment was performed three times independently, and antibacterial activity against MTB, M. bovis BCG, and M. smegmatis was measured.

(2) Results The results are shown in FIG. Disulfiram and DDCNa were shown to have no decrease in antibacterial activity even under acidic conditions of pH 6.2 and pH 5.4. Dimethyldithiocarbamate, a related carbamate, has been reported to become unstable under acidic conditions and decompose into DMA and CS ₂ (Owens, R 1964, Boyce Thompson Institute, 1964, Vol. 22, p. 241 -257). The pH in the human phagosome Mφ is around 6.8. When Mφ is activated and PL fusion (phagosome-lysosome fusion) occurs, the pH is lowered to around 6.2, and finally, it is considered that the pH is lowered to 4.5 under the influence of acidic sterilizing substances in the lysosome. From this result and the result of FIG. 5, it was suggested that disulfiram may be effective in Mφ.

4). Drug sensitivity test (1) Method (M. bovis BCG)
The bacterial solution cultured in 7H9 liquid medium until the early log phase was dispensed in 3.6 ml aliquots. The drug was prepared so that the concentrations were 1 MIC, 10 MIC, and 50 MIC, 400 μl was added to a test tube containing the bacterial solution, and cultured at 37 ° C. Absorbance measurement and colony assay were performed on days 2, 4, 7, 10, 14, and 21 with day 0 as the day when the drug was added. The number of colonies after 14 days of culture was evaluated.

(2) Results The results are shown in FIG. It has been clarified that M. bovis BCG and MTB have a genetic homology of 99.5% or more. In addition, since a correlation was observed in proliferation, a drug sensitivity test was performed using M. bovis BCG. Previous reports have shown that disulfiram has a bactericidal action against other bacteria. From FIGS. 7E and 7F, it was suggested that disulfiram and DDC have a bactericidal action on the bacterial solution in the logarithmic growth phase as well as other bacteria. Interestingly, it was suggested that disulfiram and DDC have a lytic action (FIGS. 7C and D). A lytic action is defined as the death of a dead cell with cell wall collapse, leaving no dead cells. Compounds having such activity include lysozyme and penicillin G (PCG). However, the MIC of lysozyme against BCG is 50 μg / ml. Under this concentration, the medium is suspended and concentration-dependent visible lysis cannot be confirmed. On the other hand, it goes without saying that PCG is not effective against acid-fast bacteria. Therefore, it was examined whether other bacteria were lysed. From FIG. 8C, it was confirmed that S. aureus was lysed at a high CPFX concentration, but lysis did not occur with disulfiram or DDC. Similar results were obtained with M. smegmatis (Figure 9). From these results, it was suggested that disulfiram and DDC have lytic activity specific to human and M. tuberculosis. In addition, since no other existing drugs have lytic activity, a synergistic effect with existing drugs can be expected.

5). Study on cholinesterase inhibitory action (1) Method (DTNB method)
To a 96-well plate, 150 μl of 0.01% DTNB (5,5′-dithiobis-2-nitrobenzoic acid) solution was sprinkled, and 5 μl of 5% acetylthiocholine iodide was added. A dilution series of each drug was prepared in advance using FBS (non-immobilized) as a solvent. 2 μl of this solution was added to the plate, cultured at 37 ° C., and after 10 minutes, the wavelength at an absorbance of 405 nm was measured. The cholinesterase inhibitory effect was evaluated by IC ₅₀ . Neostigmine bromide was used as a positive control.

(2) Results The results are shown in FIG. Organic sulfur carbamates are roughly classified into insecticides, fungicides and herbicides. Among these, those having a cholinesterase inhibitory effect are included as harmful effects affecting the human body. As a result of examination by the DTNB method, disulfiram and DDC did not show ChE inhibitory action at low concentrations. It has been reported in the past that disulfiram has no ChE inhibitory action.

6. Measurement of MIC by 7H11 agar plate method Shishido Y (2007 Dec; 11 (12): 1334-8. Anti-tuberculosis drug susceptibility testing of Mycobacterium bovis BCG Tokyo strain.), Maurice L. Cohn (Am Rev Respir Dis. 1968 Aug; 98 (2): 295-6.The 7H11 medium for the cultivation of mycobacteria.), Using 7H11 OADC-enhanced agar medium, the minimum inhibitory concentration against M. bovis BCG Tokyo172 strain ( MIC) was measured.
(1) Method 10 μl of 105-106 CFU / ml of bacterial solution was seeded on 7H11 agar, and the number of colonies after 10-14 days of culture was counted. Negative colonies were 5 or less, and positive were more than 5. The bacterial solution used was cultured in a 7H9-glycerol-6.6 liquid medium until the logarithmic growth phase. The experiment was performed three times in duplicate.

(2) Results Although INH, SM, and EB showed values equivalent to the previous reports, RFP showed a slightly stronger effect (Fig. 12). Disulfiram had the same antibacterial activity as kanamycin.

7). Antibacterial activity of disulfiram and DDC analogs Dialkyldithiocarbamates have been reported to exhibit antibacterial activity against some acid-fast bacteria such as M. leprae (Vadim, 2006 J Antimicrob Chemother. 2006 Jun; 57 (6): 1134-8. Epub 2006 Apr 4. Synthesis and antileprosy activity of some dialkyldithiocarbamates.). In this study, MICs against acid-fast bacteria were measured for tetraalkylthiuram disulfides, thiuram and disulfiram, and several dialkyldithiocarbamates (dimethyldithiocarbamate, diethyldithiocarbamate, dibutyldithiocarbamate, dibenzyldithiocarbamate).
(1) Method A 96-well plate was prepared so that the drug solution was in a 2-fold dilution series. A certain amount of a bacterial solution (O.D. = 100-fold dilution of 0.1) was added thereto and cultured at 37 ° C. in the presence of 5% CO ₂ . The minimum drug concentration at which bacteria did not grow visually was defined as the minimum inhibitory concentration (MIC). The experiment was performed in duplicate, and the average value was calculated.

(2) Results It was shown that the MIC was increased by extending the alkyl chain of dialkyldithiocarbamate (FIG. 13). Moreover, when it had an aromatic ring, antibacterial activity further decreased. On the other hand, it was suggested that thiuram has stronger antibacterial activity than disulfiram. However, the ADI for sterilized plastic dressings (containing 60 mg per can) and thiuram used as a pesticide is 8.4 μg / kg / day. In an adult patient weighing 60 kg, the dose is 504 μg / day and cannot be administered to humans. Therefore, at this stage, disulfiram and DDC, which have been administered to humans, can be said to be promising drugs.

8). Measurement of antibacterial activity against clinical isolates DS-TB and MDR-TB MIC against M. tuberculosis detected from patients with pulmonary tuberculosis was measured.
(1) Method After thawing drug-sensitive strains (20 strains) and drug-resistant strains (22 strains) from freeze bank, they were cultured in MycoBroth (trade name, Kyokuto Pharmaceutical Co., Ltd.) for 7 to 10 days, and subcultured once (1) 7-10 days) was used. The bacterial solution was diluted to a turbidity (0.16-0.20) corresponding to McFarland No. 1, and further seeded to a 50-fold dilution. The experiment was performed twice in duplicate. For other procedures, see 7. The method was followed.

Regarding the drug-sensitive strain (20 strains), the MIC of disulfiram was 0.78 to 1.56 μg / ml, and the MIC of DDC was 1.56 to 3.13 μg / ml (FIG. 14). On the other hand, the MIC of disulfiram for the drug-resistant strain (22 strains) was 0.78 to 1.56 μg / ml, and the MIC of DDC was 1.56 to 3.13 μg / ml (FIG. 15). The MIC for drug-sensitive strains and the MIC for drug-resistant strains were equivalent, suggesting that disulfiram and DDC do not have cross-resistance with existing drugs. It was also shown that disulfiram and DDC have antibacterial activity against quinolone (MXFX, FXGFLX, LVFX) resistant strains, which are new drug candidate compounds.

9. Calculation of drug resistance frequency of disulfiram and DDC G. Canetti et al. (Bull World Health Organ. 1963; 29: 565-78. MYCOBACTERIA: LABORATORY METHODS FOR TESTING DRUG SENSITIVITY AND RESISTANCE.) The drug resistance frequency was examined.
(1) Method 7H11 agar (X-plate) containing 20 μg / ml was prepared for each of disulfiram, DDC, SM, EB, and KM. Moreover, 7H11 agar (X-plate) containing 1 μg / ml was prepared for each of INH, RFP, CPFX and PAS. A dilution series of 1.8 × 10 ⁸ CFU / ml bacterial solution was prepared and 100 μl was added to each drug plate. The resistance frequency was determined from the number of colonies after 2 weeks of culture. The experiment was performed twice in duplicate. Further, the obtained resistant bacteria were subcultured 3 times (6 weeks), and 10 strains were stored.

(2) Results The resistance frequency of disulfiram was similar to that of existing drugs (FIG. 16). In addition, it was suggested that disulfiram is more resistant to resistant bacteria than DDC.

10. 10. Examination of in vivo protective effect of disulfiram 10-1. Recording of changes in body weight of mice (1) Method A healthy female mouse of 5 weeks old was infected with human tuberculosis H37Rv and reared for 28 days (FIG. 17). Thereafter, each drug (RFP and disulfiram) was orally administered repeatedly for 28 days, and body weight changes for 49 days were recorded every week.
(2) Results No significant weight change was observed for either RFP (FIG. 18A) or disulfiram (FIG. 18B).

10-2. Infectious protective effect of disulfiram (1) Method Five-week-old female healthy mice were infected with human tuberculosis H37Rv and reared for 28 days (FIG. 17). Thereafter, each drug (RFP and disulfiram) was orally administered repeatedly for 28 days. Body weight (g) was measured every other week. On day 57 of infection, lungs, spleen and liver were removed, and each homogenate was subjected to a colony assay (FIG. 17).
(2) Results RFP significantly reduced pulmonary parasitic bacteria at a dose of 20 mg / kg (FIG. 19). Disulfiram significantly reduced pulmonary parasitic bacteria at doses of 80-160 mg / kg (FIG. 19). It was shown that disulfiram 80 mg / kg corresponds to RFP 10 mg / kg in terms of protective effect.

10-3. Examination of Toxicity of Disulfiram (1) Method Five-week-old female healthy mice were infected with human tuberculosis H37Rv and reared for 28 days (FIG. 17). Thereafter, each drug (RFP and disulfiram) was orally administered repeatedly for 28 days. And the expression of the liver damage marker in the extracted liver homogenate and serum was measured according to the reported method. Also, it has cholinesterase (ChE) inhibitory action. It investigated according to the method of.

(2) Results The level of each marker when disulfiram was administered was equal to or less than that when RFP was administered (FIG. 20, FIG. 21 left). In particular, the low dose was equivalent to the control, suggesting that disulfiram is not highly hepatotoxic. The cholinesterase inhibitory effect (FIG. 21 right) also showed that the toxicity of disulfiram was not high.

<Summary>
Tuberculosis is a chronic respiratory infection, and new anti-tuberculosis drugs are required to be orally administrable and cross-resistant with existing drugs, and have excellent pharmacokinetics in cells and lungs. Furthermore, it has a narrow spectrum that shows bactericidal activity against M. tuberculosis in both the logarithmic growth phase and the mitotic resting phase. The results indicate that disulfiram and DDC meet these conditions. Moreover, since it was effective also against drug resistant M. smegmatis (FIG. 11), it was suggested that an action point differs from the existing drug (data not shown). It was also found to have a lytic action specific to human and bovine tuberculosis. Since none of the existing anti-tuberculosis drugs have a lytic action, when disulfiram or DDC is used in combination with an existing anti-tuberculosis drug, a synergistic effect can be greatly expected. By the way, there is an interaction with INH due to competition of Dopamine-β-hydroxylase as a point to be discussed about the combined use of disulfiram during tuberculosis treatment period. However, with increased compliance with the DOTS strategy started in 1994 and DOTS-Plus protocol therapy for MDR-TB in 2006, interaction with INH is currently negative. In fact, there are reports that no central side effects were observed (W. J. Burman, 2002, The international journal of tuberculosis and lung disease, 2002, vol. 6, No. 9, p. 839-842). As a result of examining toxicity by oral administration, it was shown that the toxicity of disulfiram was not high (FIGS. 18 to 21). The effect of ChE inhibition comes to mind when you hear carbamate. Disulfiram is a kind of carbamate, but unlike its use as an insecticide, its inhibitory effect on ChE has been denied. In addition, FIG. 2 suggested that disulfiram may be metabolized to DDC in the body so that the fungal substituting phenomenon is less likely to occur.

Currently, the mainstream treatment for tuberculosis is 2RHZ + 4RH multi-drug combination therapy, which takes 6-9 months. MDR-TB spends more than two years. TB-Alliance and WHO Stop TB Partnership have proposed a strategic program to shorten the standard treatment period for tuberculosis from March to April by 2010-2015 based on the concept of “simultaneous clinical introduction of two or more new drugs” Yes. Clinical trials of various antituberculosis drugs including Nitroimidazole (OPC-67683, PA-824) and Quinolone (MXFX) are underway as candidates.

Since its launch, disulfiram (antabuse) has been used as a treatment for chronic alcoholism. In recent years, antibacterial activity against pathogenic bacteria, parasitic protists, and fungi has been reported. It has also been proven effective in vivo against Trichomonas muris and Candida albicans. However, there are no reports that disulfiram is effective against Mycobacterium tuberculosis.

It has been reported that disulfiram has zinc finger activity (Zinc-finger-activity) and exhibits the action of detaching Zn ²⁺ from the NCp7 protein of HIV-1 and inhibiting viral replication (Hathout, 1996, Drug Metabolism and disposition, 1996, vol. 24, No. 12, p. 1395-1400). Pulmonary tuberculosis is also important as a complication of HIV. The emergence of XDR-TB, mainly in South Africa, occurs mainly in HIV endemic countries (SCIENCE, 2008, vol 319, No. 15 p. 894-895). The death rate of HIV patients infected with XDR-TB exceeds 90%, and it is reported that death occurs approximately 2 weeks after onset. In addition, the use of antituberculosis drugs is restricted for HIV patients infected with TB. As a result of the study by the present inventors, a compound (disulfiram, DDC) having a lytic action against Mycobacterium tuberculosis in the logarithmic growth phase was found. Elucidation of the mechanism of action of the compound may contribute to the development of AIDS treatment as well as tuberculosis medicine.

The anti-tuberculosis drug of the present invention comprises a compound having characteristics such as (1) high selectivity for M. tuberculosis and (2) bactericidal action, and is extremely useful for the treatment or prevention of tuberculosis. It is. In addition, since the compound of the active ingredient showed antibacterial activity against drug-resistant bacteria, it can be highly expected to be effective against drug-resistant tuberculosis bacteria. It is also possible to use the antituberculous drug of the present invention in combination with other antituberculous drugs.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (5)

1. An antituberculosis drug containing disulfiram or diethyldithiocarbamate or a pharmaceutically acceptable salt thereof as an active ingredient.
2. Isoniazid, Ryfampicin, Streptmycin, Kanamycine, Amikacin, Ethambutol, or paraaminosalicylate (p-aminosalicylate) as an active ingredient in combination The antituberculosis drug according to claim 1, wherein
3. A method for preventing or treating tuberculosis, comprising the step of administering the antituberculous drug according to claim 1 to a subject.
4. Concomitant administration of anti-tuberculosis drugs with isoniazid, ryfampicin, streptmycin, kanamycine, amikacin, ethambutol, or paraaminosalicylate (p-aminosalicylate) as active ingredients The method for preventing or treating tuberculosis according to claim 3, wherein:
5. Use of disulfiram or diethyldithiocarbamate or a pharmaceutically acceptable salt thereof for the manufacture of an antituberculosis drug.

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