JP2016164125A - Therapeutic agent and prophylactic agent of babesiosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a growth inhibitor, a therapeutic agent, and a prophylactic agent of canine babesia, bovine babesia, equine babesia, and rodent babesia as a human and livestock infectious babesia of a species of Protozoa mediated by Ixodidae mite.SOLUTION: The invention provides a drug for growth inhibition of babesias, such as canine babesia, equine babesia, bovine babesia, and rodent babesia, which contains clofazimine, a free base of clofazimine, or a pharmacologically acceptable salt thereof as an active ingredient; and a therapeutic or prophylactic agent and the like of the infection of babesias in a human and livestock; and the like. Clofazimine is 2-(4-chloroanilino)-3-isopropylimino-5-(4-chlorophenyl)3,5-dihydrophenazine, a phenazine compound.SELECTED DRAWING: None

Description

  The present invention relates to a therapeutic and prophylactic agent for babesiosis in humans and domestic animals containing the phenazine-based compound clofazimine as an active ingredient.

  Babesia is a Babesia infection that primarily affects livestock animals. Babies (Babesia gibsoni), bovine babesia (Babesia bovis, Babesia bigemina), horse babesia (Babesia caballi), babies (Theeliaia equii), ticks (Besia (Theileria) quasi) Infected by Babesia repetitively grows in erythrocytes, and thus exhibits fever, splenomegaly, anemia, hemoglobinuria, and the mortality rate is 10 to 50%. Robe babesia has also been reported to infect humans and is recognized as a zoonotic disease. In the case of human babesiosis, death may occur, particularly in elderly people, splenectomized patients, and AIDS patients. In addition, there are cases of subclinical infection, and there are concerns about infection to others by blood transfusion.

  Ganazek (diminazen acetate), imidocarb, phenamidine, and trypan blue are used for babesiosis for livestock, but it is difficult to completely kill protozoa, and hepatotoxicity In addition, there are serious side effects such as the above, and the persistence is a problem. Furthermore, the emergence of Babesia resistant to these drugs has been reported. A combination of azithromycin and atovacon or quinine and clindamycin is used to treat human babesiosis, but no new therapeutic agent has been developed.

  Thus, development of a novel anti-babesia drug that overcomes the shortcomings of existing anti-babesia drugs is desired. Especially in large-scale livestock management in Latin America, Africa, Asia, Oceania, etc., there is a concern that livestock babesiosis will lead to a decline in productivity and lead to food economy problems. In addition, the recent global changes in the environment have caused the Babesia endemic areas to expand further. Therefore, it is considered that there is an extremely high need for the development of new anti-babesia drugs that have a high protozoan effect and low toxicity to the host.

  In the development of anti-babesial drugs, some of which have been confirmed to be effective in vitro have been reported, but the effect cannot be confirmed in vivo, toxicity has been confirmed, and there is no effect on some Babesia. However, no safe drug has been found.

  Clofazimine has been reported to have antituberculous action, anti-inflammatory action, and immunosuppressive action, and is a compound known as a therapeutic drug for leprosy (Patent Document 1 and Non-Patent Document 1). For example, Patent Document 1 discloses a production method and properties of clofazimine. However, the relationship between clofazimine and Babesia was not known.

US Patent 2,948,726

M.M. C. Cholo, H.C. C. Steel, P.M. B. Fouria W. A. Germishuizen & R. Anderson: Clofazimine: current status and future prospects. Journal of Antimicrobial Chemotherapy 29: 290-298, 2012

  Accordingly, it is an object of the present invention to provide a novel pharmaceutical composition for the treatment and prevention of babesiosis in humans and livestock caused by infection with Babesias, such as dog Babesia, bovine Babesia, equine Babesia, rodent Babesia. . Another object of the present invention is to provide a therapeutic and prophylactic agent for human and veterinary babesiosis that is effective not only in vitro but also in vivo, has low toxicity, and is clinically applicable.

  In order to solve various problems in the above-described existing anti-Babesia drugs, the present inventors have conducted intensive studies on compounds effective against both various in vitro and in vivo, and clofazimine has been found to be Babesia. It was found that the compound is excellent in inhibiting the growth of, and has low toxicity. In particular, the present inventors have found that clofazimine exhibits an excellent therapeutic effect and safety in a Babesia-infected animal model, and has completed the present invention.

Thus, the present invention provides a novel pharmaceutical composition for the treatment and prevention of infectious diseases in humans and livestock such as Babesias, for example, Dog Babesia, Bovine Babesia, Horse Babesia, and Rodent Babesia, and inhibition of the growth of Babesias. , Providing clofazimine.
Specifically, the present invention relates to a Babesia growth inhibitor containing clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof as an active ingredient. The present invention also relates to a method for inhibiting the growth of Babesia, comprising administering clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof.

  The present invention also relates to an agent for treating and preventing Babesia infections containing clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof as an active ingredient. The present invention also relates to a method for treating and preventing Babesia infections comprising administering clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof.

In this specification, “clofazimine” is represented by the following formula, and has a chemical name of 2- (4-chloroanilino) -3-isopropylimino-5- (4-chlorophenyl) 3,5-dihydrophenazine, It is a phenazine-based compound having a molecular weight of 473.40 represented by C ₂₇ H ₂₂ Cl ₂ N ₄ .

  Since clofazimine has a secondary or tertiary amine group, protons can coordinate to form a base in an aqueous solution or the like. In this specification, clofazimine includes such (free) base of clofazimine. Similarly, clofazimine in the present specification includes a salt formed by combining such a clofazimine base and an inorganic or organic acid, and includes a salt that can be administered to the body as a medicine. . The “pharmacologically acceptable salt” is a salt formed by combining clofazimine with an inorganic or organic base or acid, and acceptable for administration to the body as a medicine. . Such salts are described, for example, by Berge et al. Pharm. Sci. 66: 1-19 (1977). Examples of the salt include salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, tartaric acid, fumaric acid, Salts with organic acids such as maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid; or asparagine Examples thereof include salts with acidic amino acids such as acid and glutamic acid. In addition, clofazimine hydrate or solvate and clofazimine salt hydrate or solvate are also included in clofazimine of the present invention.

  In the present specification, “Babecias” are protozoa belonging to the order Papiplasma of Apicomplexa sporeworm. In this specification, Babesias having the ability to infect humans or livestock (human or livestock infectious Babesia) are preferable as Babesias. Whether or not Babesias are infectious to humans or livestock is determined by known literature or by contacting the test Babesias with red blood cells derived from the test animals and measuring whether or not the Babesias are proliferating. Can be examined by determining that the test animal is infectious. Babesias include: Babesia gibbsoni, Bobesia bovis, Babesia bigenimina, Babesia caballi, and Tyleria (Baileia, Thiberia, tiB), Tebia, and Tebia. The Babesias are preferably dog Babesia (Babesia gibsoni), Thelelia (Babesia (Theileria) equi), and rodent Babesia microti. Further, in the present specification, the Babesias are preferably drug resistant Babesias. The drug resistant drug babesia is not particularly limited as long as it is a drug that can be used or can be used as an anti-babesial drug, for example, Ganazek (Diminazen Acetate), Mention may be made of imidocarb, phenamidine or trypan blue. Whether or not the test Babesia is a drug-resistant Babesia can be known from known information regarding the Babesia. Alternatively, whether or not the test Babesias are drug-resistant Babesias is determined by contacting the test Babesias with erythrocytes derived from animals together with the anti-Babesia drug at a concentration capable of exerting an anti-Babesia action, so that the Babesias grow. When this is measured and proliferates, the test Babesias can be examined by determining that they are drug resistant Babesias.

It is a graph which shows the result of the anti-babessia activity test in vivo of clofazimine. The vertical axis indicates the protozoa-infected erythrocyte rate (Parasisitemia) (%), and the horizontal axis indicates the number of days elapsed after Babesia administration (Days post-inoculation). Black circles indicate a negative control group with no drug added, white circles indicate a group in which clofazimine was orally administered at 20 mg / kg, triangles indicate groups in which clofazimine was administered intraperitoneally at 20 mg / kg, and inverted triangles indicate ganazek at 25 mg / kg. Shows the group administered subcutaneously.

  Clofazimine, which is a phenazine-based compound represented by the formula (I), can be produced according to the method described in US Patent 2,948,726. Clofazimine can also be purchased as a commercial product (eg, Sigma, USA).

  The pharmaceutical composition of the present invention can be used in an oral dosage form or a parenteral dosage form such as an injection (intramuscular injection, intravenous injection, subcutaneous injection), a drip infusion or the like. The method for inhibiting the growth of Babesias according to the present invention and the method for treating and preventing Babesia infections are those requiring an effective amount of clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof. Administration. As a subject to be treated or prevented with clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof, mammals are preferable, and particularly humans and domestic animals (dogs, cats, horses, sheep, goats, cattle, Mammals such as pigs, horses, horses, alpaca, guinea pigs, rabbits, minks, rats, and mice), more preferably humans, cows, horses, dogs, and rodents, most preferably humans, Cattle and horses. When this compound is administered to mammals or the like, it may be administered orally as tablets, powders, granules, syrups, etc., or may be administered parenterally as injections or drops. Moreover, when administering to livestock etc., it can administer in the form mixed with feed. The dose varies depending on the degree of symptoms, age, type of disease, etc., but usually 50 mg to 500 mg per day for an adult is divided into 1 to several times a day.

  The Babesia infection treatment and prevention agent of the present invention can be formulated by a conventional method using a normal pharmaceutically acceptable carrier. When preparing a solid preparation for oral administration, add excipients to the active ingredient and, if necessary, binders, disintegrants, lubricants, etc., and then use solvents, granules, powders, capsules, etc. And When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. are added to the main drug as necessary to obtain a subcutaneous or intravenous injection by a conventional method.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

In vitro anti-Babesia growth activity of clofazimine Using incubate of Babesicia (Babesia gibsoni) possessed by Kitasato University School of Veterinary Medicine, in vitro anti-Babecia activity of clofazimine against Matsuba et al. (Matsuu, A., Kosida, Y., Kawahara, M., Inoue, K., Ikadai, H., Hikasa, Y., Okano, S., Higuchi, S., Efficacy of avaquane agin in babies in ab. 124: 9-18. (2004)).
Using the bovine babesia (Babesia bogemina), horse babesia (Babesia caballi, Babesia equui) possessed by the Research Center for Protozoan Diseases in Obihiro University of Agriculture and Veterinary Medicine, the in vitro activity of clofazimine against these babesia in vitro Method (Bork, S., Yokoyama, N., Ikehara, Y., Kumar, S., Sugimoto, C., Igarashi, I., Growth in the effect of heparin a bet. -241 (2004)).

  For the culture of Babesia gibsoni, the method of Matsuu (Matsuu, A., Kosida, Y., Kawahara, M., Inoue, K., Ikadai, H., Hikasa, Y., Okano, S., Higuchi, S., Higuchi). , Efficacy of atovaquone agains Babes gibsoni in vivo and in vitro., Vet Parasitol., 124: 9-18 (2004)) was used. That is, protozoa-infected erythrocytes subcultured using RPMI1640 culture medium supplemented with 20% canine serum and fresh dog erythrocytes in a 24-well culture plate were diluted (hematocrit value: 5-10%, protozoa-infected erythrocyte rate: The culture was carried out at 37 ° C. under 5% CO 2 -95% air, followed by continuous culture by daily medium exchange and addition of fresh erythrocytes every 4 to 5 days.

Babesia bovis, Babesia bigemina, Babesia caballi, for the culture of Babesia equi, respectively Levy and Ristic methods (Levy, MG and Ristic, M., Babesia bovis:. Continuouscultivation in a microaerophilous stationary phase culture, Science, 207:. 1218 -1220, (1985)), Vega et al. (Vega, CA,. Buening, GM., Green, TJ., Carson, CA., In vitro culture of Babesia bigemina,. American Journal of Venture. ry Research., 46: 416-420 (1985)), the method of Holman et al. (Holman, PJ., Frerichs, WM., Chieves, L., Wagner, GG, Culture confirmation of the carrier-the-carrier in the carrier. horses., Journal of Clinical Microbiology., 31: 698-701 (1993), Holman et al. (Holman, PJ., Chieves, L., Frerichs, WM., Olson, G., Wagner, G., Wagner, G., Wagner, G. erythrocytic stage continuously cut in an en acid medium., Journal of Parasitology., 80: 232-236 (1994)), which was slightly modified, maintained and passaged, ie, 40% bovine or equine plasma in a 24-well culture plate 199 (Babesia bovis, Babesia bigemina, Babesia equi) or RPMI1640 (Babesia caballi) culture medium supplemented with CHO and diluted with protozoa-infected erythrocytes passaged using fresh bovine or horse erythrocytes (hematocrit value: 5-10%, (Protozoa-infected erythrocyte rate: 0.5 to 1%), cultured at 37 ° C. in a mixed gas of 5% O ₂ -5% CO ₂ -90% N ₂ , daily medium exchange and every 4 to 5 days Fresh erythrocytes were added to the cells for continuous culture.

  Babesia gibsoni was isolated from a naturally infected Aomori Tosa dog and maintained in continuous culture. Babesia bovis and Babesie bigmina were professor Guy Palmer, Washington State University Veterinary Medicine, and Babesia cabali and Babesia equi were distributed by the Japanese horse racing world race horse research institute Tochigi branch Takumi Kanemi.

  The drug susceptibility test was performed according to the method of Matsuu (Matsuu, A., Yamasaki, M., Xuan, X., Ikadai, H., Hikasa, Y., In vitro evaluation of the growth in vitro activity. Amori strain)., Vet Parasitol., 157: 1-8 (2008) and the method of Bork et al. (Bork, S., Yokoyama, N., Ikehara, Y., Kumar, S., Sugimoto, C., Igarashi, I., Growth inhibitory effect of heparin on Babesia paras Ites., Antimicrobial Agents and Chemotherapy., 48: 236-241 (2004)) The test compound used was clofazimine, Ganazec (Diminazendiaceturate, Novartis), an existing anti-babesial agent. Specifically, in the case of Babesia gibsoni, 200 μL of cultured Babesia-infected erythrocytes (infection rate: 1 to 3%) in each well of a 96-well plate was serially diluted to a final concentration of 0.0001 to 1 μM. Culturing was carried out under conditions containing 20% plasma culture medium (RPMI1640) containing the test compound, and the culture medium containing the test compound was replaced every 24 hours: Babesia bovis, Babesia bigemina, Babesia ca. In the case of balli and Babesia equi, 40% plasma containing 20 μL of Babesia-infected erythrocytes (infection rate: 1%) cultured in each well of a 96-well plate and a test compound diluted stepwise to a final concentration of 0.05 to 200 μM 200 μL of an additional culture solution (RPMI 1640 or 199 culture solution) was mixed and cultured for 96 hours under the above-mentioned mixed gas, and the culture solution containing the test compound was replaced every 24 hours.

  The measurement of protozoa growth is based on the method of Matsuu (Matsuu, A., Yamasaki, M., Xuan, X., Ikadai, H., Hikasa, Y., In vitro evaluation of the growth in vitro activity. strain)., Vet Parasitol., 157: 1-8 (2008) and the method of Bork et al. (Bork, S., Yokoyama, N., Ikehara, Y., Kumar, S., Sugimoto, C., Igarashi, I , Growth inhibitory effect of heparin on Babesia parasites. Antimicrobial Agents and Chemotherapy, 48:. 236-241 (2004)) was used to change slightly.

  That is, when changing the culture medium every day, a smear was prepared using a part of the infected red blood cells, and fixed with methanol, followed by Giemsa staining. Thereafter, about 1,000 red blood cells were observed, the number of red blood cells infected with Babesia was determined, and the infection rate was calculated. The 50% protozoan growth inhibitory concentration (IC50 value) of the compound was determined from the compound concentration action curve using the Babesia infection rate on the 3rd day of culture.

  Table 1 shows the anti-babesial activity of the clofazimine compound used in the present invention and the known anti-babesial agent against cultured Babesia.

Clofazimine has an IC ₅₀ value of 0.74 μM4.5, 4 M3, M3, 4 M3, 4 M3, 4 M3, 4 M3, 4 M3, 4 M3, 4 M3, 4 M, 4 M, 4 M, respectively. 0.29 μM, showing anti-babessia activity about 1/477 to 0.46 times that of ganazek, which is an existing anti-babesia agent, and the effect of inhibiting proliferation particularly against Babesia equi was remarkable.

Clofazimine cytotoxicity test The clofazimine cytotoxicity test was performed by the method of Otoguro et al. (Otoguro, K., Kohana, A., Manabe, C., Ishiyama, A., Ui, H., Shiomi, K., Yamada, H. et al.). & Omura, S .: Potential axial activity of polyetherantiotic, X-206. J. Antibiot., 54: 658-663, (2001)). That is, Dr. L. Human fetal lung-derived normal fibroblasts MRC-5 cells, a model of host cells, distributed from Maes (Tibotec NV, Mechelen, Belgium) in 10% fetal calf serum (FCS) and antibiotic-added MEM medium Those subjected to maintenance and subculture were used.

100 μL of human fetal lung-derived normal fibroblast MRC-5 cell suspension adjusted to 1 × 10 ³ cells / well with 10% FCS-MEM was added to and mixed with a 96-well plate at 37 ° C. Culturing was performed under 5% CO2-95% air for 24 hours. Thereafter, 90 μL of 10% FCS-MEM and 10 μL of a test compound solution (5% dimethyl sulfoxide aqueous solution) diluted stepwise to a final concentration of 250 to 0.1 μg / mL were added to each well. Culture was performed for 7 days under gas. The presence or absence of proliferation of MRC-5 cells was colorimetrically determined by the MTT method. The 50% cell growth inhibitory concentration (IC50 value) of the compound was determined from the compound concentration action curve. The selective toxicity ratio (SI) was calculated by (IC50 value of cytotoxicity) / (IC50 value of anti-babesial activity).

  Table 2 shows the cytotoxicity of clofazimine used in the present invention and existing anti-babessia agents.

  The cytotoxicity (IC50 value) of clofazimine against human fetal lung-derived normal fibroblast MRC-5 was 211.24 μM. Further, the cytotoxicity value of Ganazec, which is an existing drug, was 18.40 μM. The cytotoxicity of clofazimine was as weak as about 11.5 times that of Ganazek.

  Table 3 shows the selective toxicity ratio (SI) between clofazimine used in the present invention and the existing anti-babessia agent.

The selective toxicity ratio (SI) was calculated by (IC50 value of cytotoxicity) / (IC50 value of anti-babesial activity).
Clofazimine was SI: 285, 47, 70, 49, 7 against Babesia (Babesia gibsoni), Bovine babesia (Babesia bovisina, Babesia bigigena), and Babesia balbesia (Babesia caballi, Babesia equii), respectively.

In vivo anti-babesial activity test of clofazimine The in vivo treatment effect of clofazimine on murine Babesia (Babesia microti Munich strain experimental model) (AbouLaila, M., Munkargar, U., Munkargar, U.S. , A., Nakano, Y., Yokoyama, M., Yoshinary, T., Nagano, D., Katayama, K., El-Bahy, N., Yokoyama, N., Ia and i. inhibit the growth of Babesia parasites. obial Agents and Chemotherapy, 56: 3196-3206 (2012)) was measured using a.
The murine Babesia Bebesia microti Munich strain is available from Dr. O. Dispensing from Heydorn (Free University of Berlin, Germany).

As test animals, 8 week old BALB / c mice (CLEA Japan, Inc.) females, 5 to 19 g of body weight were used. B. maintained and passaged in vivo passage. Microti 1 × 10 ⁷ infected erythrocytes were infected by intraperitoneal inoculation. When the infection day is day 0, clofazimine dissolved in 10% aqueous dimethyl sulfoxide (DMSO) is injected intraperitoneally (ip) when the erythrocyte infection rate reaches 1% (day 1 after infection) or Oral (po) and ganazek are administered subcutaneously (sc), then once a day for 5 consecutive days (1-5 days), and blood smears are prepared from the tail vein every day. The protozoa-infected red blood cell rate (parasitemia) was measured.

  The result of the therapeutic effect in the infection experiment model of clofazimine used in the present invention and the existing anti-babessia agent is shown in FIG.

In the control group to which no drug was added, the ratio of the protozoa-infected erythrocyte in Babesia microti reached the highest value of 45.9% 7 days after the infection, and then gradually decreased and disappeared from the peripheral blood 20 days after the infection. With intraperitoneal administration of clofazimine at 20 mg / kg, a maximum parasitism rate of 8.8% was observed 7 days after infection, and the protozoa-infected erythrocyte rate was suppressed by 80.8% compared to the control group, and a therapeutic effect was observed. . Moreover, oral administration of clofazimine at 20 mg / kg did not show the highest parasitism rate from 5.3% 5 days after infection, and showed an inhibitory effect on the erythrocyte-infected erythrocyte rate of 88.5% compared to the control group. A therapeutic effect was observed. On the other hand, Ganazek, which is an existing anti-babesial agent used as a positive control, reached a maximum parasitic ratio of 5.4% 6 days after infection when administered subcutaneously at 25 mg / kg. .2% protozoa-infected erythrocyte rate suppression effect was observed. This indicates that clofazimine has the same therapeutic effect at the same dose as ganazec by intraperitoneal or oral administration. Ganazec has strong side effects in vivo, but the present invention has also found the possibility of reducing the dose of Ganazec in combination with clofazimine.
The LD50 for oral administration of clofazimine is> 13,300 mg / kg for rats, 8,400 mg / kg for rats, 4,400 mg / kg for guinea pigs, and 1,500 mg / kg for rabbits (reference: Japanese standard product classification number). 87239 pharmaceutical interview form). On the other hand, LD50 in the subcutaneous administration of Ganazec is 258 mg / kg for mice and is more toxic than clofazimine. (Bauer, F ,. Arzneimtel-forsching. 6: 674-677 (1956))

  As described above, clofazimine used in the present invention exhibits anti-babesial activity in vitro and in vivo against Babesias, and thus is used for clinical application of humans and domestic animals as a therapeutic and preventive agent for Babesia. be able to.

Claims (7)

An agent for inhibiting the growth of Babesia, comprising clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof as an active ingredient. An agent for treating or preventing Babesia infections, comprising clofazimine, clofazimine free base or a pharmacologically acceptable salt thereof as an active ingredient.   The therapeutic or prophylactic agent according to claim 2 for treating or preventing Babesia infections in humans or livestock animals.   The medicine according to any one of claims 1 to 3, wherein the Babesias are human and livestock infectious Babesia.   The medicine according to any one of claims 1 to 3, wherein the Babesia is Inubabesia, Umababesia, Bovine Babesia, or Rodent Babesia.   The medicine according to any one of claims 1 to 5, wherein the Babesia is drug-resistant Babesia.   The drug according to any one of claims 1 to 6, which is an oral dosage form or a parenteral dosage form.