JP5789827B2 - Malaria parasite infection treatment and prevention agent - Google Patents

Description

  The present invention relates to a Plasmodium falciparum, a Plasmodium falciparum, a Plasmodium falciparum, a Plasmodium falciparum, an oval malaria parasite and a simian malaria parasite which contain the aminopyrimidine compound nilotinib free base or a salt thereof as an active ingredient. The present invention relates to a human infectious malaria parasite growth inhibitor and a malaria parasite infection therapeutic agent and preventive agent.

  Malaria parasites that parasitize humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium species of Plasmodium 4 More recently, it was revealed that Plasmodium knowlesi infects humans. Among these, the most troublesome is P. falciparum, which accounts for 80% of those infected with malaria, and in severe cases it becomes cerebral malaria and is fatal.

  As an existing antimalarial agent against these malaria parasites, chloroquine and funcidal (combination of pyrimesamine and sulfadoxine), which are chemical synthetic drugs developed mainly in 1930-1960, which are called classic drugs, etc. Artemisinin, which is an active ingredient of herbal medicine Qingdao, which has been developed since 1980, which is called a new drug, has been used. However, at present, drug-resistant malaria parasites against chloroquine and fancidar are widespread in malaria-endemic areas, and multidrug-resistant strains that exhibit drug resistance against both drugs have also emerged. Usefulness is significantly reduced in malaria endemic areas. Artemisinin is fast acting as an action and has attracted attention as a temporary therapeutic agent, but there is a problem that it is not completely cured and is easy to relapse.

  As described above, drug-resistant strains against existing antimalarial drugs are also one of the reasons that malaria is prevalent as a re-emerging infectious disease, and the development of antimalarial drugs effective for drug-resistant strains has been desired. The epidemic area of Plasmodium falciparum is particularly widespread in the tropics and subtropics, and malaria epidemics are a very serious problem in developing countries belonging to these areas. According to malaria. Furthermore, due to recent global warming, the epidemic area of malaria parasites is expanding to developed countries including not only developing countries but also temperate regions. It is thought to increase.

  In the development of antimalarial drugs, even if the effect is confirmed in vitro, there are many cases where the effect cannot be confirmed in vivo or the toxicity is confirmed, which is effective for drug-resistant strains and the effect can be confirmed in vivo. Few drugs have been found.

  Nilotinib is a known compound known as an anticancer agent. For example, Patent Document 1 reported a production method and properties of nilotinib, and disclosed that it has an inhibitory action on tyrosine kinases among protein kinases. However, according to the genome analysis of the malaria parasite, it has been reported that there is no tyrosine kinase in the malaria parasite (Non-Patent Document 1), and the relationship between nilotinib and malaria has not been known.

International Publication No. 2004/005281 Pamphlet

Doering, C; Billker, T.W. Haysted, P.M. Sharma, A .; B. Tobin & N. C. Waters: Protein kinases of malaria paragraphs: an update. Trends in Parasitology 24: 570-576, 2008

  Therefore, the object of the present invention is to treat infection and suppress the growth of human infectious malaria parasites such as Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasite and Salmonaria parasite. It is to provide a novel chemotherapeutic agent. Another object of the present invention is to provide an infectious agent for treating human infectious malaria parasites that is effective not only in vitro but also in vivo and is clinically applicable. In particular, an object of the present invention is to provide an infectious agent for treating human infectious malaria parasites that is effective against malaria parasites that are resistant to existing antimalarial agents such as chloroquine and funcidal. .

  The present inventors are effective in both in vitro and in vivo against malaria parasites that are resistant to existing antimalarial agents such as chloroquine and fancidar in order to solve the various problems in the antimalarial drugs described above. As a result of intensive research on such compounds, it was found that nilotinib has excellent efficacy for inhibiting the growth of drug-resistant malaria parasites. In particular, the present inventors have found that nilotinib exhibits an excellent therapeutic effect and safety in a malaria parasite-infected animal model, and has completed the present invention.

  As described above, since the inhibitory action of tyrosine kinase of nilotinib has been reported, the present inventors have determined whether or not other tyrosine kinase inhibitors have antimalarial effects as well. When confirmed by an activity evaluation system, it was found that erlotinib and imatinib, which is an aminopyrimidine-related compound of nilotinib, show almost no antimalarial activity. Therefore, the present inventors confirmed that the antimalarial activity by nilotinib is not due to the tyrosine kinase inhibitory action. This result was consistent with the absence of tyrosine kinase in the malaria parasite in the above report of the genome analysis of the malaria parasite. From these results, the present inventors have found that the antimalarial effect of nilotinib (especially the growth inhibitory effect of drug-resistant malaria parasites) is due to other activities different from the tyrosine kinase inhibitory activity of nilotinib. I found it.

Therefore, the present invention relates to human infectious malaria parasites such as P. falciparum, P. falciparum malaria, P. falciparum malaria parasite, oval malaria parasite and sal malaria parasite (especially chloroquine and funcidal). Nilotinib free base or a salt thereof is provided as a novel chemotherapeutic agent for treating infection and suppressing growth of malaria parasites resistant to existing antimalarial agents such as
Specifically, the present invention relates to the following formula (I)

で表されるニロチニブ遊離塩基又はその塩を有効成分として含有する、マラリア原虫類の増殖抑制剤に関する。また、本発明は前記式（Ｉ）で表わされるニロチニブ遊離塩基又はその塩を投与することを特徴とするマラリア原虫類の増殖抑制方法に関する。

The antiproliferative agent of the malaria parasite which contains the nilotinib free base represented by these, or its salt as an active ingredient. The present invention also relates to a method for inhibiting the growth of malaria parasites, characterized by administering nilotinib free base represented by the formula (I) or a salt thereof.

  The present invention also provides the following formula (I)

で表されるニロチニブ遊離塩基又はその塩を有効成分として含有する、マラリア原虫類の感染治療剤及び予防剤に関する。また、本発明は前記式（Ｉ）で表わされるニロチニブ遊離塩基又はその塩を投与することを特徴とするマラリア原虫類の感染の治療方法及び予防方法に関する。

The present invention relates to a prophylactic and preventive agent for malaria parasites, which contains nilotinib free base represented by the formula (I) or a salt thereof as an active ingredient. The present invention also relates to a method for treating and preventing malaria parasite infection, characterized by administering nilotinib free base represented by the formula (I) or a salt thereof.

In this specification, nilotinib or nilotinib free base has the chemical name 4-methyl-N- [3- (4-methylimidazol-1-yl) -5- (trifluoromethyl) phenyl] -3-[( 4-pyridin-3-yl-pyrimidin-2-yl) amino] benzamide, which is an aminopyrimidine-based compound having a molecular weight of 529.52 and represented by the molecular formula C ₂₈ H ₂₂ F ₃ N ₇ O.

  In addition, since nilotinib of the present invention has an amine group, it can react with an acid to form a salt. The malaria growth inhibitor, therapeutic agent, and prophylactic agent of the present invention may contain such a salt as an active ingredient. In the present specification, the type of nilotinib salt is not particularly limited as long as it is pharmaceutically acceptable. For example, salts with inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, boric acid, and formic acid, Mention may be made of salts with organic acids such as acetic acid, lactic acid, citric acid, fumaric acid, maleic acid and tartaric acid.

  In the present specification, “protozoan malaria” is a protozoan belonging to the order of the apicomplexa spore genus Coccidia. The malaria parasite is preferably a human infectious malaria parasite, and includes Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasite and Salmonaria parasite.

  Since the compound of the present invention is effective against both drug-resistant malaria parasites and drug-sensitive malaria parasites, in the present specification, the malaria parasites are preferably drug-resistant malaria parasites, and more preferably drugs. Resistant human infectious malaria parasites, more preferably, drug-resistant P. falciparum, drug-resistant Plasmodium falciparum, drug-resistant Plasmodium falciparum, drug-resistant oval malaria, and drug-resistant plasmodium is there. As used herein, drug resistance means resistance to an existing antimalarial drug, and particularly resistance to chloroquine and / or fancidar.

  Nilotinib free base, which is an aminopyrimidine compound represented by the above formula (I), can be produced according to the method described in WO 2004/005281. Nilotinib free base can also be purchased from commercial products (eg, LC laboratories, USA). Nilotinib salts can be prepared by methods well known to those skilled in the art.

The agent for treating and preventing malaria parasite infection of the present invention can be used in an oral dosage form or a parenteral dosage form such as an injection or infusion. 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. The dose varies depending on the degree of symptoms, age, type of disease, etc.
Administer mg to 500 mg divided into 1 to several times a day.

  The agent for treating and preventing malaria parasite infection 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 add solvents, granules, powders, capsules, etc. by conventional methods. 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.

Example 1 In Vitro Antimalarial Activity Test of Nilotinib Hydrochloride Nilotinib hydrochloride was prepared according to a conventional method using nilotinib free base (LC laboratories, USA). That is, nilotinib free base 49.6
mg was dissolved in 0.5 mL of 1,4-dioxane (Kanto Chemical Co., Japan), and 2 mL of 1,4-dioxane (Watanabe Chemical Co., Ltd., Japan) containing 4N-hydrochloric acid was added dropwise. The resulting yellow precipitate was filtered off, and the obtained precipitate was washed with 1 mL of 1,4-dioxane three times and dried to thereby obtain 44.9 nilotinib hydrochloride.
mg was obtained.

  Using the K1 strain, which is a drug-resistant strain of Plasmodium falciparum, and the FCR3 strain, which is a drug-sensitive strain, distributed by Prof. Kiyoshi Kita of the Graduate School of Medicine, the University of Tokyo, The anti-malarial activity of nilotinib hydrochloride in vitro was determined by the method of Otoguro et al. (Otoguro, K., Kohana, A., Manabe, C., Shiyama, A., Ui, H., Shiomi, K., Yamada, H. et al.). & Omura, S .: Potent animalial activity of polyether antibiotic, X-206. J. Antibiot., 54: 658-663, (2001)).

  For the culture of the test protozoa, the method of Trager and Jensen (Trager, W and Jensen, J .: Human maria parasites in continuous culture, Science, 193: 673-677, (1976)) was maintained, passaged. What was performed was used. Specifically, protozoa-infected erythrocytes passaged using RPMI 1640 medium supplemented with 10% human plasma and fresh human erythrocytes were diluted in a culture dish (hematocrit value: 2 to 5%, protozoa-infected erythrocyte rate: 0.25). Incubation was performed at 37 ° C. under a mixed gas of 3% O 2 -4% CO 2 -93% N 2 and continuous culture was performed every 2 to 3 days by adding medium and adding fresh erythrocytes.

  The drug susceptibility test was performed according to the method of Desjardins et al. (Desjardins, RE, Canfield, CJ, Haynes, DE and Chulray, JD: Quantitative assessment of antigenic activity. Antimicrob. Agents Chemother., 16: 710-718 (1979)). As test compounds, in addition to nilotinib prepared by the above-mentioned method, nilotinib-related compounds imatinib, erlotinib, and artemisinin (Aldrich, USA), artesunate (existing antimalarial agent for cultured Plasmodium falciparum) Cerbios Pharma (Switzerland) and chloroquine (Sigma, USA) were used. Specifically, the protozoan suspension (hematocrit value: 2%, protozoa-infected erythrocyte rate: 0.5 or 1%) pre-cultured in each well of a 96-well plate has a final concentration of 100 to 0.0001 μg / mL. 10 μL of the test compound solution (50% ethanol solution) diluted in such a concentration step was added, mixed, and cultured for 72 hours under the above-mentioned mixed gas.

  The measurement of protozoa growth is the method of Makler et al. (Makler, MT, Rise, JM, Williams, JA, Bancroft, JE, Piper, RC, Gibbins, B.L. And Hinrichs, D.J .: Parasite lactate dehydrogenase as an Assay for Plasmodium falciparum drug sensitivity, Am. J. Med. Hyg., 48: 739 at 74 (M). ) Was used for the colorimetric determination of protozoan lactate dehydrogenase (p-LDH).

  That is, after culturing for 72 hours, the 96-well plate was directly frozen at −20 ° C. for 18 hours and then thawed at 37 ° C. to hemolyze protozoa-infected erythrocytes and destroy the protozoa to prepare a crude enzyme solution. Add 100 μL of Malstat reagent and 20 μL of crude enzyme solution to each well of a new 96-well plate, mix, react for 15 minutes at room temperature, then nitroblue tetrazolium 2 mg / mL: phenazine etosulphate 0 20 mg of a 1 mg / mL = 1: 1 solution was added to each well and allowed to react at room temperature for 2 hours under light-shielding conditions.

  The presence or absence of protozoa was determined colorimetrically by measuring the absorbance of blue formazan product produced by the reaction at a measurement wavelength of 655 nm using a microplate reader (Labossystems, Finland). . The 50% protozoan growth inhibitory concentration (IC50 value) of the compound was determined from the compound concentration action curve. The antimalarial activity of cultured nilotinib hydrochloride, its related compounds and known antimalarial agents used in the present invention against cultured Plasmodium falciparum was as shown below.

Nilotinib hydrochloride has an IC ₅₀ value of 1.22 μg / mL against K1 strain, which is a drug-resistant strain of Plasmodium falciparum, and is 1/2 of artemisinin and artesunate, which are existing antimalarials. The antimalarial activity was about 244-1203 times that of chloroquine and 1 / 6.6 times that of chloroquine. Furthermore, it has an IC ₅₀ value of 0.64 μg / mL for the drug-sensitive FCR3 strain, exhibits antimalarial activity about 1/640 to 1/107 times that of artemisinin and artesunate, and 1/42. It showed 7-fold antimalarial activity.

  Nilotinib hydrochloride showed the same activity against the drug-resistant strain K1 and the drug-sensitive FCR3 strain, and the difference in activity between the two strains was not seen as with artemisinin. On the other hand, chloroquine showed a difference in activity with respect to both strains, and a difference of about 12 times with respect to the K1 strain was observed compared with the FCR3 strain. This indicates that nilotinib hydrochloride has antimalarial activity with a mechanism of action different from that of chloroquine in drug-resistant malaria parasites. On the other hand, imatinib and erlotinib, which are related compounds of nilotinib, showed almost no antimalarial activity.

(Example 2) Cytotoxicity test of nilotinib hydrochloride The cytotoxicity test of nilotinib hydrochloride was performed according to the method of Otoguro et al. (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 (50% ethanol solution) diluted stepwise to a final concentration of 100 to 0.1 μg / mL are added to each well. Cultivation was carried out for 7 days. 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 results were as follows.

  The cytotoxicity (IC50 value) of nilotinib hydrochloride against human fetal lung-derived normal fibroblast MRC-5 was 89.07 μg / mL. The selective toxicity ratio of nilotinib hydrochloride to antimalarial activity (IC50 value of cytotoxicity / IC50 value of antimalarial activity) is 73 and 139 for drug resistant K1 strain and drug sensitive FCR3 strain, respectively, and high malaria selective toxicity showed that.

(Example 3) In vivo anti-malarial activity test of nilotinib hydrochloride. The therapeutic effect in vivo on the infection test model of berghei N strain (drug-sensitive strain) was examined by the method of Otoguro et al. (2001) and Peters et al. (Peters, W., Portus, JH and Robinson, B. et al. XXII. Measured with modification. Murine malaria parasite berghei N strain is a strain of Dr. W. Dispensation from Peters (Northwick Park Institute for Medical Research, Medlexex, UK).

As test animals, males of ICR mice (Nippon Charles River), 5 groups of 18 to 20 g in weight were used. 2 × 10 ⁶ parasite-infected erythrocytes were prepared from the protozoa maintained and passaged by in vivo passage, and infected by tail vein inoculation. The treatment experiment was conducted in a suppressive test for 4 days. On day 0 of infection, the compound solution (10% dimethyl sulfoxide aqueous solution-Tween 80) was administered intraperitoneally (ip) or orally (po) 2 hours after the infection. 3 consecutive days (1st to 3rd day), blood smear prepared from tail vein on 4th day, observed protozoa infected erythrocyte rate (parasitaemia), treatment effect than compound non-administered group infection rate (% Inhibition) was determined.

  As a result of the intraperitoneal administration test, nilotinib hydrochloride was found to have a protozoa-infected erythrocyte rate of 83.3% compared to the control group to which no drug was added at a dose of 60 mg / kg, compared to the experimental model of murine malaria parasite P. berghei N Inhibition effect was observed and infection treatment effect was observed. Artesunate, an existing antimalarial agent used as a positive control, showed an inhibitory effect on the erythrocyte-infected erythrocyte rate of 86.7% at a dose of 10 mg / kg. Furthermore, in the oral administration group, the nilotinib hydrochloride 60 mg / kg administration group had a suppression of the erythrocyte-infected erythrocyte rate of 75.8% compared to the control group without any drug, and an infection treatment effect was observed. In the artesunate-administered group used as a positive control, 79.4% suppression of the protozoa-infected erythrocyte rate was observed at a dose of 10 mg / kg. From this, it was shown that nilotinib hydrochloride has an equivalent therapeutic effect at a dose about 6 times that of artesunate by intraperitoneal administration or oral administration. Since artesunate is easily metabolized in vivo, combination therapy with other agents is performed. The present invention has also found the possibility of combination therapy with artesunate.

  As described above, nilotinib free base or a salt thereof used in the present invention exhibits antimalarial activity against human infectious Plasmodium falciparum and exhibits a therapeutic effect against malaria parasite infection model, It is expected to be clinically applicable as an antimalarial agent.

Claims (6)

Formula (I)

A drug for suppressing the growth of protozoan parasites, comprising a nilotinib free base represented by the formula (1) or a salt thereof , and a pharmaceutically acceptable carrier . Formula (I)

A drug for treating and preventing malaria parasite infection , characterized by suppressing the growth of the malaria parasite species, comprising a nilotinib free base represented by the formula (1) or a salt thereof and a pharmaceutically acceptable carrier . The drug according to claim 1 or 2 , wherein the malaria parasite is a human infectious malaria parasite. 3. The drug according to claim 1 or 2 , wherein the malaria parasite is a Plasmodium falciparum, a Plasmodium falciparum, a Plasmodium falciparum, an oval malaria parasite or a simian malaria parasite. The drug according to any one of claims 1 to 4 , wherein the malaria parasite is a drug-resistant malaria parasite. The drug according to any one of claims 1 to 5 , which is an oral dosage form or a parenteral dosage form.