JP4460709B2 - New malaria prevention and treatment - Google Patents

Description

で表わされるキジマイシンまたはその製薬学的に許容できる塩を有効成分として含有することを特徴とする、抗マラリア予防または治療薬が提供される。
【０００７】
キジマイシンの塩の例としては、第４級アンモニウム塩などの、製薬学的に許容できる無機塩との塩、ならびに製薬学的に許容できる各種金属との塩、例えばアルカリ金属塩およびアルカリ土類金属塩、更に有機塩基との塩、例えばアミンがある。
キジマイシンは、これを医薬の有効成分として使用する場合には経口的または非経口的にも投与できる。
【０００８】
第１の本発明によるマラリア疾患の治療および予防薬では、有効成分としてのキジマイシンまたはその塩を、単独に、あるいは賦型剤と混合した組成物の形に製剤できる。そして注射剤、経口剤、軟膏剤、坐剤等として製剤化できる。組成物に配合できる賦型剤は製剤学的に許容される液体状の担体、例えばグリセリン、オリーブ油、水、エタノールなど、あるいは固体担体、例えば乳糖、タルク、ペクチン、スターチなどであることが出来る。担体の種類および組成は投与経路や投与方法によって適宜選択する。
キジマイシンまたはその塩は、その投与量として0.2mgから1,500mg程度を経口的あるいは非経口的（例えば注射剤により）に投与できる。
【０００９】
第２の本発明においては、式(I)のキジマイシンまたはその製薬学的に許容できる塩を有効成分として含む殺マラリア原虫剤が提供される。
【００１０】
次に、本発明で有効成分として用いるキジマイシンが、抗マラリア活性を示すことを試験例１〜２で例証し、またキジマイシンの急性毒性等の安全性について試験例３〜４で例証する。
試験例１（in vitro抗マラリア活性試験）
本試験では、キジマイシンおよび供試の比較化合物の各々につき、抗マラリア活性の試験、および哺乳動物の培養細胞を用いた細胞毒性の試験を平行して実施した。供試化合物の抗マラリア活性と細胞毒性との活性比から、その供試化合物の抗マラリア剤としての有用性を判定した。
【００１１】
１．培養熱帯熱マラリア原虫の培養
本試験には、プラスモジウム属の培養熱帯熱マラリア原虫（Plasmodium falciparum）FCR-3(ATCC 30932)株を被検マラリア原虫として用いた。一方、マラリア原虫用の培地は各種の濃度で供試化合物を含む濾過滅菌したPRMI1640培地で、pHを7.4に調整後、赤血球を含む人血清を10%となるように添加した。マラリア原虫の培養は、酸素濃度5.0%、炭酸ガス濃度5.0%、窒素濃度90%の環境下で、36.5℃の温度で行った。ヘマトクリット値（赤血球浮遊液中に占める赤血球の体積の割合）は、5%に調整してから使用した。培養開始時の熱帯熱マラリア原虫の初期感染率は0.1%とした。24穴培養プレートを用いて上記の環境下で赤血球と共にマラリア原虫を培養し、培地は毎日交換し、マラリア原虫による赤血球の感染率4%の時期に継代を行った。感染率は赤血球の薄層塗末標本を作成し、ギムザ染色或いはDiff-Qick染色を行った後に、顕微鏡（油浸、1,000×）下でマラリア原虫による感染赤血球の個数を計測して下記の計算式で算出した。

【００１２】
２．マラリア原虫の増殖阻害の試験方法
培養マラリア原虫に感染した赤血球を遠心で集め、血清を含む培地で洗浄を行った後、非感染赤血球を加え、初期感染率を0.3%とした。この時のヘマトクリット値は3%である。実験に用いる供試化合物は滅菌水、ジメチルホルムアミド(DMF)、あるいはジメチルスルホキシド(DMFO)に溶解した溶液としてあらかじめ用意しておく。
24穴培養プレートに供試化合物の溶液を5〜10μlずつ加え、試験は、duplicateあるいはtriplicateで行った。対照区は滅菌水、DMF、あるいはDMSOを10μlずつウエルに加えた。
【００１３】
次に、あらかじめ用意しておいた熱帯熱マラリア原虫培養液を各ウエルに990〜995μlずつ加え、静かにピペッティングを行い培地に一様に懸濁させる。
【００１４】
培養プレートはCO₂−O₂−N₂インキュベーター中で72時間培養した後、それぞれのウエルについて、薄層塗末標本を作成した。赤血球を染色した後、顕微鏡下で観察し、供試化合物を加えた本発明区の感染率、及び対照区の感染率を算出する。
【００１５】
求めた感染率から下記の計算式によって増殖率を算出する。
後記の表１に、既存の抗マラリア剤（比較の供試化合物）及びキジマイシンについて得られた試験結果を示す。

但しａ：感染率（Zero時間の）
ｂ：供試化合物添加区の感染率
ｃ：対照区の感染率
【００１６】
３．マウス乳癌由来FM3A細胞に対する供試化合物の毒性の評価
マウス乳癌由来FM3A細胞の野生株であるF28-7株を供試細胞として用いた。培地は、ES培地に非働化した胎児牛血清を2%となるように添加したものであり、CO₂濃度5%、37℃で培養した。この条件下でのマウス乳癌由来FM3A細胞の倍加時間は約12時間である。
【００１７】
前培養を行い、対数増殖期に入った供試細胞を、5×10⁴cells/mlになるように培地で希釈する。
【００１８】
供試化合物の溶液は、マラリア原虫の抗マラリア活性測定時に調製した溶液を用いる。24穴培養プレートに供試化合物溶液を5〜10μlずつ加える（培地等を加えると最終濃度は1×10^−４〜1×10^−６Mとなる）。
【００１９】
試験はduplicateあるいはtriplicateで行った。対照区として滅菌水、DMF、あるいはDMSOを10μl加えたウエルも同時に用意した。次に、用意しておいた培養細胞浮遊液を990〜995μlずつ加え、静かにピペッティングを行い培地に一様に懸濁させた。これを前記の培養環境下でFM3A細胞を48時間培養した後、それぞれのウエルについて細胞数をcell counter(CC-108, Toa Medical Electronics)で計数する。
【００２０】
FM3A細胞増殖に対する阻害活性は、供試化合物を添加したウエルの細胞数及び対照区の細胞数から、次式で算出する。これより供試化合物の細胞毒性を評価する。

但しＡ：細胞数（Zero時間の）
Ｂ：対照区：48時間培養後の細胞数
Ｃ：供試化合物添加区：48時間培養後の細胞数
【００２１】
マラリア原虫に対する供試化合物の薬効判定は、下記の選択毒性を基準にして行う。ここで選択毒性とは、下記のことである。

【００２２】
上記のEC₅₀値とは、マラリア原虫、あるいはFM3A細胞の培地に供試化合物を添加していない対照区のマラリア原虫感染率、あるいは増殖率を100%とみなして、供試化合物の添加によって、対照区のマラリア原虫またはFM3A細胞の増殖を50%阻害する供試化合物濃度のことである（モル濃度で表示する）。

【００２３】
試験例２（in vivo抗マラリア活性試験）
１．使用マウスおよびマラリア原虫
マウスは、ICR系の体重26〜31g、５週令の雄を１群５匹用いる。実験にはネズミマラリア原虫（Plasmodium berghei NK65株)を用いる。本マラリア原虫は、マウスに感染すると急激に増殖し、感染マウスをことごとく死亡させる猛毒株として知られている。
２．試験方法
ネズミマラリア原虫（Plasmodium berghei NK65株)に感染したICRマウスの心臓から採血し、赤血球に対するネズミマラリア原虫の感染率を測定した。次いで採血した血液サンプルの１mlの赤血球浮遊液あたり５×10⁶匹の原虫となるように調整し、非感染マウスの尾静脈に0.2ml接種する。この原虫接種の時を０日として、それぞれ２時間、１日、２日、３日後に、DMSO(100μm)に溶解させた供試化合物を腹腔内に投与した。４日目に、マウス尾部静脈より血液を採取し、血液の塗抹標本を作成し、顕微鏡下で観察した。無処理区と処理区との感染率を測定した。
【００２４】
この結果、キジマイシンは５mg/kgの濃度で70％の率で原虫の増殖を抑制する高い活性を示した。一方、同時に行った実験において、現在臨床に用いられている比較化合物としてのアルテミシニン（５mg/kg）の投与区では、45％の率で原虫増殖を抑制した。
【００２５】
試験例３（マウスにおける急性経口毒性試験）
ICR系雄性マウス(５週令）を用いて、キジマイシン（モノNa塩)の経口投与による急性毒性試験を行った。経口投与時のキジマイシンのLD₅₀値は180.3mg/kgを示した。従って、本発明によるキジマイシンの急性経口毒性は、モネンシン[mice(経口投与)でLD₅₀ 43.8±5.2mg/kg]およびサリノマイシン[mice(経口投与)でLD₅₀ 50mg/kg]と比較して３倍以上も低毒性であった。
【００２６】
【発明の効果】
本発明によれば、新規な抗マラリア剤として、新規な化学構造を有ししかも安全性の高いキジマイシンまたはその塩を有効成分として含有するマラリア疾患の予防・治療薬が提供される。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a therapeutic and prophylactic agent for malaria disease comprising kijimycin or a salt thereof as an active ingredient.
[0002]
[Prior art]
According to the World Health Report of WHO published in 1996, malaria was an infection that had already been considered to be overcome, along with tuberculosis and diphtheria, but reemerging infections as malaria appears to show signs of an epidemic. Has been pointed out. In 1995, 2.1 million people died of malaria worldwide, and 270 million people were infected with malaria. Due to global warming, malaria-producing areas are expanding to temperate regions that have been rarely identified so far, and there are concerns about the epidemic in Honshu in Japan in the near future. In addition, malaria parasites have acquired resistance to various drugs, making them difficult to eradicate. Under these circumstances, the development of vaccines to eradicate malaria, elucidation of the ecophysiology of mosquitoes that mediate, the elucidation of the physiological mechanisms of malaria parasites, the analysis of human immune responses, the analysis of pathophysiology, the analysis of new antimalarial drugs Research is being done from all directions, including development. In particular, the development of new antimalarial agents is eagerly desired to eradicate malaria.
[0003]
Quinine, chloroquine, pyrimethamine, mefloquine, artemisinin, etc. are known as drugs currently used for the treatment of malaria, but in terms of their effects, toxicity to humans, side effects, and the development of resistant malaria parasites, etc. Therefore, it is not always a satisfactory treatment for malaria.
[0004]
In addition, the polyether antibiotics monensin, nigericin (“Life Sci.” Vol. 59 (20) pp. 309-315 (1996)) and salinomycin (“Zentralbl. Bakteriol., Mikrobiol. Hyg.,”) Ser A 256 (3), pp. 305-313 (1984)] is known as a substance exhibiting antimalarial activity produced by microorganisms. However, monensin, nigericin and salinomycin all have high acute oral toxicity as compared to pheasymycin according to the present invention, and the antimalarial effect is not stable and low, so that they have not been put into practical use until now.
[0005]
The polyether antibiotic MI215-NF3 is a known antibiotic described in Japanese Patent No. 2780828, and is produced by culturing Actinomadura actinomycetes (FERM P-2675). It was classified as a polyether antibiotic due to its structural characteristics. This MI215-NF3 substance has now been renamed Kijimycin. Kijimycin is a substance that clearly differs in structure from the currently used antimalarials. In this study, it was found that KIJYMYCIN exhibits stable and high effects in the anti-malarial activity test of KIJYMYCIN using animals. Moreover, it has low toxicity. Further, it is presumed that kijimycin has a slightly different mechanism of action from that of conventional polyether antibiotics because it has an effective anti-coccidial activity for polyether antibiotic-resistant coccidium. Therefore, kijimycin can be expected as an antimalarial preventive or therapeutic agent. Based on these findings, the present invention has been completed.
[0006]
Therefore, according to the first aspect of the present invention, the following formula (I)

An antimalarial prophylactic or therapeutic agent is provided, which comprises an effective ingredient of pheymycin represented by the formula (I) or a pharmaceutically acceptable salt thereof.
[0007]
Examples of salts of kijimycin include salts with pharmaceutically acceptable inorganic salts, such as quaternary ammonium salts, and salts with various pharmaceutically acceptable metals such as alkali metal salts and alkaline earth metals. There are also salts with organic bases, such as amines.
Kijimycin can be administered orally or parenterally when it is used as an active ingredient of a medicament.
[0008]
In the therapeutic and prophylactic agent for malaria disease according to the first aspect of the present invention, kijimycin or a salt thereof as an active ingredient can be formulated alone or in the form of a composition mixed with an excipient. And it can be formulated as injections, oral preparations, ointments, suppositories and the like. The excipients that can be incorporated into the composition can be pharmaceutically acceptable liquid carriers such as glycerin, olive oil, water, ethanol, or solid carriers such as lactose, talc, pectin, starch and the like. The type and composition of the carrier are appropriately selected depending on the administration route and administration method.
Kidimycin or a salt thereof can be administered orally or parenterally (for example, by injection) in a dosage of about 0.2 mg to 1,500 mg.
[0009]
According to a second aspect of the present invention, there is provided a malarial protozoan agent comprising pheizycin of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
[0010]
Next, Test Examples 1 and 2 illustrate that pheasymycin used as an active ingredient in the present invention exhibits antimalarial activity, and tests 3 and 4 illustrate the safety of acute toxicity and the like of pheasymycin.
Test Example 1 ( In vitro antimalarial activity test)
In this test, a test for antimalarial activity and a test for cytotoxicity using cultured mammalian cells were performed in parallel for each of kijimycin and the comparative compound of the test. From the activity ratio between the antimalarial activity and cytotoxicity of the test compound, the usefulness of the test compound as an antimalarial agent was determined.
[0011]
1. Cultivation of cultured Plasmodium falciparum In this test, Plasmodium cultivated Plasmodium falciparum FCR-3 (ATCC 30932) was used as a test malaria parasite. On the other hand, the culture medium for Plasmodium was PRMI1640 medium sterilized by filtration containing the test compound at various concentrations, and after adjusting the pH to 7.4, human serum containing red blood cells was added to 10%. The malaria parasite was cultured in an environment with an oxygen concentration of 5.0%, carbon dioxide concentration of 5.0% and nitrogen concentration of 90% at a temperature of 36.5 ° C. The hematocrit value (the ratio of the volume of red blood cells in the red blood cell suspension) was adjusted to 5% before use. The initial infection rate of P. falciparum at the start of culture was 0.1%. Plasmodium was cultured with erythrocytes in a 24-well culture plate in the above environment, the medium was changed every day, and subculture was performed at a time when the infection rate of erythrocytes by malaria parasites was 4%. The infection rate is calculated as follows by preparing a thin layer of red blood cell sample, performing Giemsa staining or Diff-Qick staining, and measuring the number of infected red blood cells by malaria parasite under a microscope (oil immersion, 1,000 ×). Calculated by the formula.

[0012]
2. Test method for inhibition of malaria parasite growth Erythrocytes infected with cultured malaria parasites were collected by centrifugation, washed with a medium containing serum, and non-infected erythrocytes were added to an initial infection rate of 0.3%. The hematocrit value at this time is 3%. The test compound used in the experiment is prepared in advance as a solution dissolved in sterilized water, dimethylformamide (DMF), or dimethyl sulfoxide (DMFO).
The test compound solution was added to each 24-well culture plate in an amount of 5 to 10 μl, and the test was performed in duplicate or triplicate. In the control group, 10 μl of sterilized water, DMF, or DMSO was added to each well.
[0013]
Next, 990-995 μl of the P. falciparum culture solution prepared in advance is added to each well and gently pipetted to suspend uniformly in the medium.
[0014]
The culture plate was cultured for 72 hours in a CO ₂ —O ₂ —N ₂ incubator, and then a thin-layered paint specimen was prepared for each well. After staining erythrocytes, it is observed under a microscope, and the infection rate of the present invention group to which the test compound is added and the infection rate of the control group are calculated.
[0015]
The proliferation rate is calculated from the obtained infection rate by the following formula.
Table 1 below shows the test results obtained for the existing antimalarial agent (comparative test compound) and pheizycin.

Where a: infection rate (zero time)
b: Infection rate of test compound added group c: Infection rate of control group
3. Evaluation of toxicity of test compound to mouse breast cancer-derived FM3A cells F28-7 strain, a wild strain of mouse breast cancer-derived FM3A cells, was used as a test cell. The medium was obtained by adding fetal bovine serum inactivated to ES medium to 2%, and cultured at a CO ₂ concentration of 5% at 37 ° C. Under these conditions, the doubling time of mouse breast cancer-derived FM3A cells is about 12 hours.
[0017]
Pre-culture is performed, and the test cells that have entered the logarithmic growth phase are diluted with a medium to 5 × 10 ⁴ cells / ml.
[0018]
As the solution of the test compound, a solution prepared at the time of measuring the antimalarial activity of the malaria parasite is used. Add 5 to 10 μl of the test compound solution to a 24-well culture plate (addition of a medium or the like results in a final concentration of 1 × 10 ⁻⁴ to 1 × 10 ⁻⁶ M).
[0019]
The test was conducted in duplicate or triplicate. As a control, a well containing 10 μl of sterilized water, DMF, or DMSO was also prepared. Next, the prepared cultured cell suspension was added in an amount of 990 to 995 μl, and gently pipetted to be uniformly suspended in the medium. After culturing FM3A cells for 48 hours in the above culture environment, the number of cells in each well is counted with a cell counter (CC-108, Toa Medical Electronics).
[0020]
The inhibitory activity against FM3A cell proliferation is calculated by the following formula from the number of cells in the well to which the test compound is added and the number of cells in the control group. Based on this, the cytotoxicity of the test compound is evaluated.

A: Number of cells (Zero time)
B: Control group: Number of cells after 48 hours of culturing C: Test compound addition group: Number of cells after 48 hours of culturing
Judgment of the efficacy of test compounds against Plasmodium is based on the following selective toxicity. Here, the selective toxicity is as follows.

[0022]
The above EC ₅₀ value is considered to be malaria parasite or malaria parasite infection rate in the control group to which the test compound is not added to the medium of FM3A cells, or the growth rate is 100%, and by adding the test compound, This is the concentration of the test compound that inhibits the growth of malaria parasites or FM3A cells in the control group by 50% (expressed in molar concentration).

[0023]
Test Example 2 (In vivo antimalarial activity test)
1. As the mice used and the Plasmodium mice, 5 males having a body weight of 26 to 31 g of ICR system and 5 weeks old are used. A murine malaria parasite (Plasmodium berghei NK65 strain) is used in the experiment. This malaria parasite is known as a highly toxic strain that grows rapidly when infected with a mouse and causes all infected mice to die.
2. Test method Blood was collected from the heart of an ICR mouse infected with a rat malaria parasite (Plasmodium berghei NK65 strain), and the infection rate of the rat malaria parasite to erythrocytes was measured. Then, the blood sample collected is adjusted to 5 × 10 ⁶ protozoa per 1 ml erythrocyte suspension, and 0.2 ml is inoculated into the tail vein of uninfected mice. The test compound dissolved in DMSO (100 μm) was intraperitoneally administered 2 hours, 1 day, 2 days and 3 days after the protozoa inoculation as day 0, respectively. On the fourth day, blood was collected from the tail vein of the mouse, a blood smear was prepared and observed under a microscope. The infection rate between untreated and treated areas was measured.
[0024]
As a result, Kijimycin showed a high activity of inhibiting the growth of protozoa at a rate of 70% at a concentration of 5 mg / kg. On the other hand, in the experiment conducted at the same time, protozoa growth was suppressed at a rate of 45% in the administration group of artemisinin (5 mg / kg) as a comparative compound currently used in clinical practice.
[0025]
Test Example 3 (Acute oral toxicity test in mice)
An acute toxicity test was conducted by oral administration of Kijimycin (mono Na salt) using ICR male mice (5 weeks old). The LD ₅₀ value of pheasymycin at the time of oral administration was 180.3 mg / kg. Therefore, the acute oral toxicity of pheasymycin according to the present invention is three times that of monensin [LD ₅₀ 43.8 ± 5.2 mg / kg for mice (oral administration)] and salinomycin [LD _{50 50} mg / kg for mice (oral administration)]. These were also low toxicity.
[0026]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the prophylactic / therapeutic agent of the malaria disease which has a novel chemical structure, and has high safety | security kijimycin or its salt as an active ingredient as a novel antimalarial agent is provided.

Claims (2)

Formula (I)

A therapeutic and prophylactic agent for malaria disease, which comprises, as an active ingredient, kijimicin represented by the formula (I) or a pharmaceutically acceptable salt thereof: Formula (I)

A protozoan protozoan characterized by containing kijimycin represented by the formula: or a pharmaceutically acceptable salt thereof as an active ingredient.