JP5576086B2 - Infection treatment for malaria parasites - Google Patents

Description

  The present invention relates to the growth of human infectious malaria parasites, for example, P. falciparum, P. vivax, P. vivax, and oval malaria, using the known tropolone antibiotic puberulic acid. The present invention relates to an infection treatment agent for malaria parasites that is effective for treatment of infection with malaria parasites.

  There are four types of malaria parasites that parasitize humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. being classified. Of these, the most troublesome is P. falciparum, which accounts for 80% of those infected with malaria and, in severe cases, becomes cerebral malaria and is fatal.

  The existing antimalarial agents against these malaria parasites are called classical drugs and are mainly chemically synthesized drugs developed in the 1930s and 1960s, such as chloroquine and funcidal (a combination of pyrimesamine and sulfadoxine). , Etc., and artemisinin, which is an active ingredient of the herbal medicine Qingdao, which was called a new drug and was developed after 1980, was used. However, drug-resistant malaria parasites to chloroquine and fancidar are now widely spread in malaria-endemic areas, and multidrug-resistant strains of both have emerged. Their usefulness as antimalarial agents is remarkably observed in malaria-endemic areas. It is falling. 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.

  Thus, drug-resistant strains against existing anti-malarial drugs are also one of the causes that malaria is prevalent as a re-emerging infection, and the development of anti-malarial drugs effective for drug-resistant strains is desired on a global scale. The epidemic area of Plasmodium falciparum is particularly widespread in the tropics and subtropics, and it is a very serious problem in developing countries belonging to these areas. Malaria is considered to be the leading cause of death from parasitic infections. ing. In addition, due to the recent global warming, the epidemic area of malaria parasites is showing a tendency to expand not only to developing countries but also to developed countries including temperate regions.

The present inventors have intensively studied to search for effective compounds in vitro and in vivo for malaria parasites resistant to existing antimalarial agents such as chloroquine and fancidar, and surprisingly, Gram-positive It has been found that puberulic acid, which has an effect on fungi, has an excellent effectiveness for suppressing the growth of malaria parasites.

  The puberulic acid produced by Penicillium sp is already known. For example, Biochemical Journal (Biochem. J.) Vol. 26, pp. 441-453 (1932), Vol. 28, p. 11-15 (1934) reported manufacturing methods, properties, and structural analysis by derivatization. Although it has been disclosed that it has an antibacterial action against certain gram-positive bacteria, the effectiveness as an antimalarial agent has not been disclosed.

  Therefore, the present inventors have extensively searched for physiologically active substances exhibiting the activity in microbial metabolites in order to solve various problems as described above, and as a result, the filamentous fungus FKI-4410 strain collected from soil has been found. It was found that the substance to be produced has antimalarial activity. Therefore, when the physicochemical properties of this substance were examined, this substance was found to be a tropolone antibiotic, puerulic acid (Journal Chemical Society Transaction 1 (J. Chem. Soc. Perkin Trans 1), Vol. 16, pp. 1913-1920 (1993) / total synthesis study), the compound was identified and the present invention was completed.

  It is an object of the present invention to treat infection and suppress growth of human infectious malaria parasites, such as Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum and Oval malaria parasites by chemotherapy. acid) to provide a clinically effective therapeutic agent for infection with human infectious malaria parasites.

  In order to solve the problems as described above, the present inventors have extensively searched for bioactive substances exhibiting the activity in microbial metabolites, and as a result, the present invention has been found. That is, the following formula

で表されるプベルリン酸 (puberulic acid) 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に関する。

The present invention relates to an infection treatment agent for malaria parasites, which contains puberulic acid represented by the formula:

  The present invention further relates to an agent for treating malaria parasite infection which comprises suppressing the growth of the malaria parasite species, wherein the malaria parasite is a human infectious malaria parasite, including P. falciparum, S. falciparum, It is one selected from the group of P. falciparum and egg-shaped malaria, and the malaria parasite infection therapeutic agent is used in oral administration forms or parenteral administration forms such as injections and infusions.

  The present invention further provides that the therapeutic agent for infection is effective against drug-resistant malaria parasites and drug-sensitive malaria parasites, and also against Plasmodium falciparum, Plasmodium falciparum, Plasmodium falciparum and Plasmodium falciparum. The present invention relates to an infection treatment agent for malaria parasites which is used in the manufacture of a medicament for infection treatment of a disease selected from the group.

  The puberulic acid represented by the above formula is a substance produced by the filamentous fungus FKI-4410, and its production method and NMR spectrum data are Birkinshaw, JH & Raistrick H .: Studies in the biochemistry of micro- organisms: Puberulic acid C8H6O6 and an acid C8H4O6, new products of the metabolism of glucose by Penicllium puberulum bainler and Penicillium aurantiovirens biourge, with an appendix on certain dihydroxybenzenecarboxylic acids. Biochem. J., 26: 441-453, 1932, Barger, J & Dorrer, O .: Chemical properties of puberulic acid, C8H6O6, and a yellow acid, C8H4O6. Biochem. J., 28: 11-15, 1934 and Martin, GB; Maree, PC; Maureen, FM & Sharon, L / R .: cis-Dihydrocatechols as precursors to highly oxygenated troponoids. Part 2. Regiocontrolled syntheses of stipitatic and puberulic acids. J. Chem. Soc. Perkin Trans 1, 16: 1913-1920, 1993. Its action has been disclosed as an antibacterial agent.

  Explaining how to produce puberic acid, inoculate 100 mL of seed medium containing carbon source, nitrogen source, inorganic salts, etc. in an appropriate amount with puberulic acid producing bacteria at 27 ° C for 72 hours. Shake culture and use as seed. 1% of this seed is inoculated into 10 ml of 10 mL 500 mL Erlenmeyer flasks in a puberulic acid production liquid medium, cultured at 27 ° C for 72 hours with shaking, and then incubated for 240 hours To do. Extraction is performed by adding 1 L of ethanol to 1 L of the culture solution thus obtained, followed by filtration under reduced pressure. After distilling off ethanol from the filtrate, the aqueous solution obtained is adjusted to pH 6 and extracted with ethyl acetate. The extract is concentrated under reduced pressure to obtain an extract.

  The above extract is purified by ODS gel column chromatography using acetonitrile-water as an elution solvent, and the elution fraction containing puerulic acid is concentrated to obtain a crude product. The crude product is separated by high performance liquid column chromatography using a reverse phase column, and the corresponding peak is concentrated under reduced pressure to obtain puberulic acid.

  As the carbon source used in the above, for example, glucose, sucrose, molasses, starch, dextrin, cellulose, glycerin and the like are used alone or in combination, and as the nitrogen source, for example, peptone, meat extract, yeast extract, soybean flour, Corn, steep liquor, cottonseed meal, casein, etc. are used alone or in combination. Further, examples of inorganic salts include sodium salts, potassium salts, magnesium salts, phosphates, etc. Added.

  When this compound is administered as a therapeutic agent for various diseases, it may be administered orally as tablets, powders, granules, syrups, etc., or parenterally as injections or drops. The dosage 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.

  In formulating, it is produced by a conventional method using an ordinary pharmaceutical 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, and a subcutaneous or intravenous injection is prepared 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.
Example 1

  Seed medium 100 mL (2% glucose, 0.5% polypeptone, 0.2% yeast extract, 0.2% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.1% agar, adjusted to pH 6 before autoclaving) Puberic acid producing filamentous fungus FKI-4410 strain was inoculated and cultured at 27 ° C. for 72 hours with shaking as a seed mother. Puberic acid production liquid medium (100% each) in 10 500 mL Erlenmeyer flasks (3% sucrose, 3% water-soluble starch, 1% malt extract, 0.3% Ebios, phosphorus Potassium dihydrogen 0.5%, magnesium sulfate heptahydrate 0.05%, adjusted to pH 6 before autoclaving) was inoculated 1% at a time, cultured at 27 ° C. for 72 hours with shaking, and then allowed to stand for 240 hours.

  1 L of ethanol was added to 1 L of the culture broth thus obtained for extraction, followed by filtration under reduced pressure. After ethanol was distilled off from the filtrate, the resulting aqueous solution was adjusted to pH 6 and extracted with ethyl acetate. This extract was concentrated under reduced pressure to obtain 760 mg of an extract.

  The above extract was purified by ODS gel column chromatography using acetonitrile-water as an elution solvent, and a 20% acetonitrile aqueous solution fraction containing puberulic acid was concentrated to obtain 70 mg of a purified product. This purified product is retained by high performance liquid column chromatography (column: Waters XBridge C18 (10f x 250 mm), mobile phase: 20% methanol / 0.1% aqueous trifluoroacetic acid, flow rate: 4 mL / min, detection: 270 nm) The peak at 8 minutes was collected and concentrated under reduced pressure to obtain 5 mg of puberulic acid.

K1 strain resistant to Plasmodium falciparum (available from Professor Kitayoshi, Graduate School of Medicine, the University of Tokyo) and FCR3 strain sensitive to drugs (Professor Kitayoshi, Graduate School of Medicine, the University of Tokyo) measurements of antimalarial activity of Puberurin acid in in vitro against dispensable) (puberulic acid) is Otoguro et al method [Otoguro, K., Kohana, A. , Manabe, C., Ishiyama, A., Ui, H., Shiomi, K., Yamada, H. & Omura, S .: Potent antimalarial activity of polyether antibiotic, X-206. J. Antibiot., 54: 658-663, (2001)].

As a test protozoa, the method of Trager and Jensen [Trager, W and Jensen, J .: Human malaria parasites in continuous culture, Science, 193: 673-677, (1976)] was slightly modified and maintained and passaged. A thing was used. That is, dilute protozoa-infected erythrocytes passaged using RPMI1640 medium supplemented with 10% human plasma and fresh human erythrocytes in a culture dish (hematocrit value: 2-5%, protozoa-infected erythrocyte rate: 0.25-1 ) At 37 ° C. under a mixed gas of 3% O ₂ -4% CO ₂ -93% N ₂ , and continuously cultured by changing the medium and adding fresh erythrocytes every 2-3 days .

  The drug susceptibility test was performed by the method of Desjardins et al. [Desjardins, RE, Canfield, CJ, Haynes, DE and Chulay, JD: Quantitative assessment of antimalarial activity invitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother., 16: 710-718 ( 1979)] was modified and pre-cultured in each well of a 96-well plate (hematocrit value: 2%, protozoa-infected erythrocyte rate: 0.5 or 1%) 190 μl and compound solution (50% ethanol solution) After adding 10 μl and mixing, the cells were cultured for 72 hours under the mixed gas described above.

  The measurement of protozoan growth is the method of Makler et al. [Makler, MT, Rise, JM, Williams, JA, Bancroft, JE, Piper, RC, Gibbins, BL and Hinrichs, DJ: Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity, Am. J. Med. Hyg., 48: 739-741 (1993)] and colorimetric determination of protozoan lactate dehydrogenase (p-LDH) with Malstat reagent (Flow, USA) I went there.

  That is, a 96-well plate that had been cultured for 72 hours was directly frozen at −20 ° C. for 18 hours and then thawed at 37 ° C., whereby protozoa-infected erythrocytes were hemolyzed and protozoa were destroyed 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 ethosulfate 20 μl of a 0.1 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 blue formazan product produced by the reaction was colorimetrically quantified by measuring the absorbance at a measurement wavelength of 655 nm with a microplate reader (Labosystems, Finland). . The 50% protozoan growth inhibitory concentration (IC ₅₀ value) of the compound was determined from the compound concentration action curve. The antimalarial activity of the puberulic acid and known antimalarial agents used in the present invention against cultured Plasmodium falciparum was as shown below.

  Artemisinin (Aldrich, USA), artesunate (Cerbios Pharma, Switzerland), and chloroquine (Sigma, USA) were used as known antimalarial agents against cultured Plasmodium falciparum.

─────────────────────────────────────
IC ₅₀ value ng / ml)
Compound K1 stock FCR3 stock ─────────────────────────────────────
Puerulic acid 10.0 10.0
Artemisinin 6.0 6.0
Artesunate 4.2 1.0
Chloroquine 300.0 20.0
─────────────────────────────────────

  The puberic acid used in the present invention is effective on the order of 10 ng / ml against the drug-resistant K1 strain of Plasmodium falciparum, and the existing antimalarial agents artemisinin and artesunate are effective. The antimalarial activity was about 1 / 2.4 to 1 / 1.7 times, and the antimalarial activity was 30 times that of chloroquine. It is also effective against the drug-sensitive FCR3 strain on the order of 10 ng / ml, exhibits antimalarial activity about 1 / 2.4 to 1/10 times that of artemisinin and artesunate, and twice that of chloroquine. It showed malaria activity.

Thus, puberulic acid has the same level of activity against the drug-resistant K1 strain and the drug-sensitive FCR3 strain, and also exhibits a strong activity almost as high as the existing malaria drug artemisinin, Puberic acid indicates that antimalarial activity acts on both K1 and FCR3 strains by a mechanism different from that of chloroquine in drug-resistant malaria parasites.
Example 2

  The cytotoxicity test for puberulic acid used in the present invention was performed by the method of Otoguro et al. [Otoguro, K., Kohana, A., Manabe, C., Ishiyama, A., Ui, H., Shiomi, K. , Yamada, H. and Omura, S .: Potent antimalarial activity of polyether antibiotic, X-206. J. Antibiot., 54: 658-663, (2001)]. That is, human fetal lung-derived normal fibroblast MRC-5 cells [can be dispensed from Dr. L. Maes (Tibotec NV, Mechelen, Belgium)] as a host cell model with 10% fetal calf serum (FCS) and antibiotics What was maintained and subcultured in an added MEM medium was used.

Prepare human fetal lung-derived normal fibroblast MRC-5 cells with 10% FCS-MEM to adjust the suspension to 1 x 103 cells / well, add 100 μl to a 96-well plate, mix, and then 37 ° C After incubating under 5% CO ₂ -95% air for 24 hours, add 90 μl of 10% FCS-MEM and 10 μl of the compound solution (50% ethanol solution) to each well. Cultivation 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% protozoan growth inhibitory concentration (IC ₅₀ value) of the compound was determined from the compound concentration action curve. The results were as follows.

─────────────────────────────────
Cytotoxicity of puberulic acid on cultured human cells
IC ₅₀ value (ng / ml)
Compound MRC-5 cells ───────────────────────────────
Puberulic acid 57,200
─────────────────────────────────

The cytotoxicity (IC ₅₀ value) of puberulic acid used in the present invention to human fetal lung-derived normal fibroblast MRC-5 cells is 57,200 ng / ml, and the selective toxicity ratio with antimalarial activity is cytotoxic. (IC ₅₀ value / anti-malarial activity IC ₅₀ value) was 5,720 for the drug-resistant K1 strain and the drug-sensitive FCR3 strain, indicating high selective toxicity.
Example 3

The puberic acid used in the present invention, P. berghei N strain (drug-sensitive strain) [Dr. W. Peters (Northwick Park Institute for Medical Research, Meddlesex, UK)] measurements of the therapeutic effect of the in vivo for model Otoguro et al method [Otoguro, K., Kohana, A. , Manabe, C., Ishiyama, A., Ui, H., Shiomi, K., Yamada, H. and Mura, S .: Potent antimalarial activity of polyether antibiotic, X-206. J. Antibiotics, 54: 658-663, (2001)] and Peters et al. [Peters, W .; Portus, JH and Robinson, BL: XXII. The value of drug-resistant strains of P. berghei in screening for blood schizonticidal activity. Ann. Trop. Med. Parasitol., 69: 155-171, (1975)] It was.

In other words, ICR mice (Charles River Japan) males were used as test animals, and each group consisted of 5 animals with a body weight of 18-20g. 2 x 106 parasitic infections were maintained and passed through in vivo passage. Red blood cells were prepared and infected by tail vein inoculation. The treatment experiment was conducted with a 4 days suppressive test. Assuming that the day of infection is day 0, a compound solution (10% dimethyl sulfoxide solution-Tween 80) is administered subcutaneously (sc) 2 hours after infection, and then administered continuously once a day for 3 days (days 1 to 3) On day 4, blood smears were prepared from the tail vein, the protozoa-infected erythrocyte rate (parasitaemia) was observed, and the therapeutic effect (inhibition%) was determined from the infection rate in the non-compound group.

────────────────────────────────────
Therapeutic effect of subcutaneous administration of puberulic acid
Compound Dose Therapeutic effect (Inhibition%)
────────────────────────────────────
Puberulic acid 2 mg / kg x 4 69.0%
Artesunate 10 mg / kg x 4 90.6%
Artesunate 1 mg / kg x 4 36.7%
────────────────────────────────────

When puberulic acid used in the present invention was administered subcutaneously, puberulic acid was not added at a low dose of 2 mg / kg to the experimental model of P. berghei N strain infection in murine malaria parasites. Compared with the control group, 69.0% of the protozoa-infected erythrocyte rate was suppressed, and an infection treatment effect was observed. Artesunate, an existing antimalarial drug used as a control for drug addition, showed a 36.7-90.6% suppression of protozoa-infected erythrocyte rate at doses of 1-10 mg / kg, and 50% effective concentration of artesunate (ED ₅₀ ) Was 1.7 mg / kg. This indicates that puberic acid has therapeutic effects at low doses similar to artesunate.

  As described above, the puberic acid used in the present invention exhibits antimalarial activity against human infectious Plasmodium falciparum, exhibits therapeutic effects against malaria parasite infection models, and exhibits antimalarial activity. It is expected to be clinically applicable as an agent.

Claims (7)

Following formula

An agent for treating malaria parasites, which contains puberulic acid represented by the formula: The agent for treating infection with malaria parasites according to claim 1, which comprises suppressing the growth of malaria parasites. 3. The malaria parasite is a human infectious malaria parasite, and is one selected from P. falciparum, T. falciparum malaria, T. fever malaria parasite, and oval malaria parasite. For treating malaria parasites. The malaria parasite infection therapeutic agent according to any one of claims 1 to 3, wherein the malaria parasite infection therapeutic agent is effective against drug-resistant malaria parasites. The malaria parasite infection therapeutic agent according to any one of claims 1 to 3, wherein the malaria parasite infection therapeutic agent is effective against drug-sensitive malaria parasites. The malaria parasite infection therapeutic agent according to any one of claims 1 to 5, wherein the malaria parasite infection therapeutic agent is an oral administration form or a parenteral administration form. A human infectious Plasmodium, P. falciparum, P. vivax, claim used in the manufacture of a medicament for the infection treatment of diseases selected from malariae parasite and P. ovale 1 7. The antimalarial infection treatment agent according to any one of 6 to 6 .