JPH06157308A - Antimalarial agent - Google Patents

Abstract

PURPOSE:To provide a new antimalarial agent containing a specified tetrapyrrole derivative or its salt having a remarkable antimalarial activity as the active component, and useful, e.g. for prevention and treatment of malaria. CONSTITUTION:An antimalarial agent containing a tetrapyrrole derivative having a basic structure of the formula (R<1> and R<2> are each OH or a substituted hydroxyl group) which may be substituted at 2, 3, 7, 8, 12, 13, 17 and 18 positions or its pharmacetically permissible salt (Biliverdin, bilirubin or salts thereof are especially preferable) as the active component. The tetrapyrrol derivative of the formula or its salt as the active component of the antimalarial agent can prevent merozoite from invading the red blood cell and exhibit an inhibitory effect on growth of a material growing in the red blood cell, e.g. ring or trophozoite. The tetrapyrrole derivative of the formula can be collected from a natural component such as bile pigment and also can be synthesized. Bile pigment itself also can be used. This compound is excellent in activity and free from remarkable side effects since this compound is a normal metabolite.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antimalarial agent, and more particularly to an antimalarial agent containing a tetrapyrrole derivative as an active ingredient.

[0002]

Malaria is an infectious disease transmitted by mosquitoes, which is still widespread in a wide range of tropical and subtropical regions, with 800 million infected people, and more than 100 million people per year. Is a highly infectious disease that causes 1.2 million people and 1.2 million deaths. Although there is no epidemic of malaria in our country at present, the number of cases of illness is increasing locally or after returning to Japan as the number of opportunities to go abroad has increased recently. Malaria is roughly classified into falciparum malaria, vivax malaria, vivax malaria, and egg malaria depending on the type of the pathogen, the protozoa. Clinically, falciparum malaria is the most aggressive, causing fulminant and cerebral malaria,
The mortality rate is also high.

As mentioned above, malaria is a mosquito-borne infectious disease. In the body of the mosquito, it sexually reproduces and performs postfertilization sporulation. The formed spores (sporozoites) gradually gather in the salivary glands and enter the human body through the snout during blood feeding. Sporozoites that have entered the human body are carried by the bloodstream to the liver,
Invades hepatocytes. Schizonts in hepatocytes
It develops, becomes many merozoites, appears by destroying hepatocytes, and then invades red blood cells. In erythrocytes, it progresses from ring (ring body, early vegetative body) to trophozoite (late vegetative body) and shiizont, breaks erythrocyte membrane, and many merozoites appear again, which invade new red blood cells and repeat the same cycle. To proliferate. During repeated development in red blood cells, some protozoa become reproductive mothers, which, when sucked by mosquitoes, carry out the aforementioned sexual reproduction,
If not sucked, it will die sooner or later.

[0004]

Regarding the above-mentioned therapeutic agent for malaria, at present, there is no known drug that kills sporozoites, and there is no true infection preventive drug. Therefore, a drug such as a ring or trophozoite that kills or suppresses the growth of red blood cells is used.
Chloroquine, fancidal, quinine, mefloquine,
Primakin and others are known. However, all of these drugs are relatively highly toxic and have side effects such as gastrointestinal injury, headache and fever. Regarding chloroquine, which is commonly used for P. falciparum malaria, which exhibits the most serious symptoms, drug-resistant strains are appearing in various places, and chloroquine cannot be used in these regions. There is a problem that it cannot be used for pregnant women and newborns, and new antimalarial agents are desired.

The present invention has been made in view of the above problems, and as a result of researching a novel and biosafe antimalarial agent, the present inventors have found a substance having a remarkable antimalarial activity. That is, the present inventors have already found that the tetrapyrrole derivative acts on the complement system and has an inhibitory effect, and as an action, it inhibits the reaction by binding to properdin in the second pathway (alternative pathway). It was speculated that On the other hand, propargin has been reported to be similar in amino acid sequence to the proteins of the malaria parasite sporozoites and merozoites (Nature, 335; 79-82,
1988). From such findings, the present inventors have conducted various studies on the antimalarial action of the tetrapyrrole derivative, and have completed the present invention by finding that the specific tetrapyrrole derivative has a remarkable antimalarial activity, The present invention aims to provide a novel antimalarial agent.

[0006]

The antimalarial agent of the present invention, which has been made to solve the above-mentioned problems, has a basic structure represented by the following structural formula (1).
1st, 3rd, 7th, 8th, 12th, 13th, 17th and 1st
The active ingredient is a tetrapyrrole derivative which may have a substituent at the 8-position or a pharmacologically acceptable salt thereof.

[0007]

[Chemical 2]

(In the formula, R ¹ and R ² each represent a hydroxyl group or a substituted hydroxyl group, and a single bond or a double bond is present between the carbon at the 10th position and the carbon at the 11th position.) The above structural formula (1)
In the above, for convenience, symbols A to D are attached in order from the left ring. Further, in the A ring and D ring at both ends of the structural formula (1), when the group R ¹ and / or R ² is a hydroxyl group, the ring is an enol-keto represented by the following partial structural formulas (a) and (b). It is widely known to those skilled in the art that tautomerism of In the present specification, such a tautomer is represented by the partial structural formula (a) for convenience.

[0009]

[Chemical 3]

The active ingredient of the antimalarial agent of the present invention has a basic structure represented by the above structural formula (1), and the 2-position, 3-position, 7-position, 8-position, 12-position of the structural formula: It is a tetrapyrrole derivative which may have a substituent at the 13th, 17th and 18th positions, or a pharmacologically acceptable salt thereof. The pharmacologically acceptable salts include inorganic metal salts such as alkali metal salts (eg sodium salt, potassium salt etc.) and alkaline earth metal salts (magnesium salt, calcium salt etc.), ammonium salts, organic bases. Salts (eg, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, etc.), organic acid salts (eg, formate salt, acetate salt, maleate salt, tartrate salt, methanesulfonate salt, benzenesulfonate salt, toluenesulfonic acid salt) Salts, etc., inorganic acid salts (eg, hydrochloride, hydrobromide, sulfate, phosphate, etc.)
Etc.

Further, in the structural formula (1), the 2-position, 3-position, 7-position,
Examples of the group capable of substituting at the 8-position, 12-position, 13-position, 17-position and 18-position include various substituents, and preferably, for example, a lower alkyl group (eg methyl, ethyl, propyl, butyl, Isobutyl, tertiary butyl, pentyl,
Hexyl), alkenyl groups (eg vinyl, allyl, 1-propenyl, 3-butenyl, 4-pentenyl,
5-hexenyl, phytyl, farnesyl, etc.), carboxyalkyl groups and esterified carboxyalkyl groups (eg, carboxymethyl, carboxyethyl, methoxycarbonylmethyl, ethoxycarbonylethyl, glucuronide carbonylethyl, etc.) and the like. Examples of the substituted hydroxyl group of R ¹ and R ² include an acyloxy group (eg, acetoxy, propionyloxy, benzoyloxy, etc.), an alkoxy group (eg, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tertiary butoxy). , Pentyloxy, hexyloxy, etc.) and the like.

The tetrapyrrole derivative, which is the active ingredient of the antimalarial agent of the present invention, can be collected from natural ingredients such as bile pigment, or may be artificially synthesized, and the bile pigment itself is used. You can also Particularly preferred compounds include biliverdin (hereinafter referred to as BV) and bilirubin, and these compounds are advantageous in that they are excellent in action, are normal metabolites, and have few side effects, and BV is particularly preferable.

The antimalarial agent of the present invention can be used in either oral or parenteral administration form.
Upon administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and then administered in the form of a conventional pharmaceutical preparation. Examples of such a preparation include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. It can be prepared by conventional means. Examples of the nontoxic carrier for pharmaceutical use include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin. , Water, physiological saline and the like. If necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents and the like can be added as appropriate. More specifically, in the case of an oral preparation, for example, the active ingredient is made into a suspension using a dispersant such as a nonionic surfactant, and sugar, sugar alcohol, silicic acid anhydride, nonionic surface After adding a disintegrating agent such as an active agent, freeze-dry it to powder, and according to the usual method, make it into any dosage form (eg, capsule, powder, coarse granule, granule, tablet, liquid, etc.). A liposome preparation can be obtained by using the liposome-forming method obtained by formulating, and described in JP-A-60-208910 and JP-A-61-27965. In the case of an intravenous preparation, the active ingredient is obtained by formulating it using a conventional means such as emulsification with a surfactant, inclusion with cyclodextrin, liposome formation, or fat emulsion formation. To be In the antimalarial agent of the present invention, the dose of the active ingredient is the patient's age, body weight, symptoms, degree of disease, administration route, administration schedule,
Depending on the formulation form, etc., it is appropriately selected and determined.
Oral administration generally 0.5-300 mg / day
It is said to be about kg body weight.

[0014]

INDUSTRIAL APPLICABILITY The tetrapyrrole derivative and its salt, which are the active ingredients of the antimalarial agent of the present invention, have the effects of inhibiting the invasion of merozoites into erythrocytes and suppressing the growth of erythrocyte growth bodies such as rings and trophozoites. Therefore, the antimalarial agent of the present invention is useful for preventing and treating malaria.

[0015]

The present invention will be described in more detail based on the following examples and test examples, but the present invention is not limited to these examples. Example 1 BV (20 g) was suspended in 50 ml of a 5 W / V% polyoxyethylene polyoxypropylene glycol aqueous solution, and wet pulverized using glass beads. Then, 30 g of sucrose fatty acid ester was added to 50 ml of the obtained pulverized liquid,
After freeze-drying in a dry ice / methanol bath, it was dried to prepare a freeze-dried preparation.

Test Example In order to investigate the effect of the antimalarial agent of the present invention, BV, which is one of the active ingredients, was tested for its antimalarial effect.
The method and test results are shown below. The materials used and their preparation methods are as follows. (1) Medium All the basic medium and antibiotics were purchased from GIBCO BRL. The human plasma and red blood cells used were the packs of the Japanese Red Cross Society. Hepes buffer RPMI16
40 medium 500 ml plus penicillin and streptomycin (10,000 units and 10,000 μg / ml) 5 ml, gentamicin (50 mg / ml) 0.1 ml was used as an incomplete medium and further human plasma (A ⁺ , inactivated) 5
The medium to which 0 ml was added was used as a complete medium. Human A ⁺ red blood cells were collected from human whole blood by centrifugation at 2500 rpm for 5 minutes,
After washing twice with the incomplete medium, the complete medium containing the same volume of red blood cells as 50% RBC was refrigerated and used within 1 week.

(2) Culture method Malaria infects red blood cells, grows and proliferates therein, and then
It proliferates in a cycle of jumping out and invading nearby blood cells again. The time required for one cycle is about 48 hours. The MAD20 strain of human falciparum malaria (M
cBride, JS et al, J. Exp. Med., 161; 160-180, 19
85) and M4 strain. The thawed and washed malaria-infected erythrocytes were placed in a petri dish together with fresh 50% RBC, and a complete medium was added to start culture. The total amount of red blood cells was about 6% of the medium, and 50% RBC was added as needed depending on hemolysis. It is necessary to add fresh RBC at least once a week even when malaria growth is poor or only maintained. Since the petri dish is cultivated in the anoxic state, put it in a desiccator together with two candles that have been ignited, and seal it.
The temperature was kept at 5 to 37 ° C. The medium was exchanged once a day by sucking only the supernatant while leaving the red blood cells that had settled on the bottom still, and adding more new medium so that more than half of the medium was replaced.

(3) Method of arranging developmental stages In order to prevent the effects from varying depending on the developmental stage at the start of treatment, all experiments were performed using plasma gel (Cellular Products).
Inc. USA) was used to align the stages of development of malaria parasites, and the treatment of the drug was started from both the ring and trophozoite stages. That is, the erythrocytes in the culture were collected in a centrifuge tube, washed once with an incomplete medium, added with an incomplete medium in an amount 3 times that of blood cells and 4 times the amount of plasma gel, mixed well, and then placed in a 37 ° C constant temperature bath. I let it stand. Since it separated into two layers in 30 to 60 minutes, the interface was not disturbed and only the upper layer was transferred to another centrifuge tube. Trophozoites (hereinafter referred to as T) are gathered in the upper layer, and rings (hereinafter referred to as R) are gathered in the lower layer. The cells were centrifuged, washed once with an incomplete medium and once with a complete medium, and then cultured at an appropriate concentration.

(4) Method of thawing BV It was thawed to be 10 times the final concentration, dispensed in single doses, and stored frozen. However, since it does not dissolve directly in water,
First, dissolve in DMSO at 4-40 mg / ml, then add RP
It was diluted with MI medium and sterilized by filtration with a 0.2 μm filter.

(5) Determination of Infection Rate A small amount of blood cells were collected from the dish in culture together with the medium and transferred to a tube. The blood cells were collected by centrifugation and smeared on a slide glass. After air-drying, add 100% methanol to 1
After fixing for 30 minutes, Giemsa staining was performed for 30 minutes. Gimbsa liquid is PB
It was diluted to about 2% with S (-) and remade every day to use a new one. A 100 × oil immersion objective lens was used for observation.
The total number of red blood cells and the number of infected red blood cells were counted for each visual field, and several visual fields were observed until the total blood cell count exceeded 1000. Infected erythrocytes are ring (R), trophozoite (T), shiizant (S) depending on the developmental stage of malaria parasite.
The number of each of the three stages was counted. In many cases, one red blood cell contains several protozoa due to the characteristics of Plasmodium falciparum. In that case, the number of insects was counted. The infection rate was calculated by the following formula. Infection rate (%) = [(R + T + S) / total red blood cell count] × 10
0

Test Example 1 8 day administration test of BV M4 strain and MAD20 which had the same developmental stages as plasma gel
About the ring of the strain, cultured using the following test medium,
The infection rate was calculated. Test medium Control 1; complete medium only Control 2; DMSO 0.5% added BV 10; DMSO 0.5%, BV 10μ
g / ml addition BV 20; DMSO 0.5%, BV 20μ
g / ml addition BV 50; DMSO 0.5%, BV 50μ
g / ml addition BV 100; DMSO 0.5%, BV 100μ
g / ml addition That is, the drug treatment was carried out for 8 days, and sampling for checking the infection rate was performed every day. The blood cells remaining on the 8th day were transferred to a normal medium of a new petri dish, and 50% RBC was also added. Sampling was performed 1 week after the transfer to the normal medium. The results are shown in Table 1. In the table, Table 1-A is MAD2.
0 (R), Table 1-B shows the case of M4 (R). As shown in Table 1, when cultured in a medium containing BV, it was found that the proportion of infected red blood cells was low and the growth of malaria parasite was suppressed.

[0022]

[Table 1]

Test Example 2 Two week administration test of BV The amount of DMSO was reduced by half to 0.25%, and the same treatment as in Test Example 1 was repeated, and administration for two weeks was tried. However, since the administration period was long, 1 ml of 50% RBC was added to all experimental plots on the 7th day (after completion of sampling). The sampling was performed immediately before drug administration, on the 8th day (the day after RBC addition), and 1 week after the transfer to the normal medium. The results are shown in Table 2. In the table, Table 2-A shows the case of MAD20 (R) and Table 2
-B shows the case of M4 (R). As shown in Table 2, it was found that when BV was administered for 2 weeks, the proportion of infected red blood cells was low even at low concentrations, and the growth after transfer to normal medium was suppressed.

[0024]

[Table 2]

Test Example 3 BV life-prolonging effect test by in vivo system ICR mice (6 weeks old, female) were treated with murine malaria (Pla).
Infected by intraperitoneal administration of infected erythrocytes of S. berghei (mice die in about 1 week).
The malaria-infected mice were divided into 3 groups (10 mice in each group),
For groups 1 and 2, BV dispersed in physiological saline was 100 m each for 6 days from the day after malaria infection.
g / kg body weight (BV100), 200 mg / kg body weight
(BV200) was administered intraperitoneally. The third group served as a control and was administered with physiological saline. Then, the survival rate of each group was observed over time. The results are shown in Table 3. As shown in Table 3, all the subjects died by the 9th day in the control, but by the 10th day in the BV100, 1 in the BV200.
There were surviving individuals by day 5.

[0026]

[Table 3]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Noriko Nanami 3-3 Midorigahara, Tsukuba-shi, Ibaraki Central Research Laboratory, Nippon Ham Co., Ltd. (72) Inventor Tatsuyoshi Nakagami 3-3 Midorigahara, Tsukuba-shi, Ibaraki Japan (72) Inventor Shigekubo 3-3 Midorigahara, Tsukuba City, Ibaraki Prefecture

Claims (2)

[Claims] 1. The following general formula: (In the formula, R ¹ and R ² each represent a hydroxyl group or a substituted hydroxyl group, and a carbon atom at the 10-position and a carbon atom at the 11-position represent a single bond or a double bond). 2-position, 3-position, 7-position, 8-position, 12-position, 13-position, 1-position of the structural formula
An antimalarial agent comprising a tetrapyrrole derivative which may have a substituent at the 7- and 18-positions or a pharmacologically acceptable salt thereof as an active ingredient. 2. The antimalarial agent according to claim 1, wherein the active ingredient is biliverdin or a pharmacologically acceptable salt thereof.