JP2019038749A - Histone deacetylase inhibitor having antiprotozoal action - Google Patents

Abstract

To provide an agent that acts on an Apicomplexa protozoan while having low toxicity on a host cell.SOLUTION: The present invention provides an agent for treatment and/or prevention of Apicomplexa protozoan infection, containing a compound represented by the formula (I), an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture of them as an active ingredient.EFFECT: The anti-Apicomplexa protozoan agent can be used for prevention and/or treatment of toxoplasmosis, malaria, cryptosporidiosis.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an antiprotozoal agent comprising a known null script as an active ingredient as an analog compound of a scriptoid that is a histone deacetylase inhibitor.

  Malaria parasites, Toxoplasma, and Cryptosporidium belonging to Apicomplexa are protozoa that have caused tremendous damage to the world.

  In Plasmodium falciparum, one of the world's three major infectious diseases, the emergence of strains resistant to the conventionally used drug chloroquine has become a problem. In recent years, the emergence of artemisinin-resistant strains, which are first-line drugs, in Southeast Asia has been confirmed, and when these appear and spread in endemic areas, it is thought to have a significant effect on malaria control, and new drugs are required.

  Toxoplasmosis is caused by eating meat that has been latently infected with Toxoplasma gondii in an uncooked state, or by oral protozoa that form in the feces of cats infected with cats that are end hosts. It is a protozoal infection caused by ingestion. About 30% of the world population is estimated to be latently infected, but the symptoms are minor in healthy individuals. However, immunosuppressed patients may become severely fatal, and if they are infected for the first time during pregnancy, fetal brain growth failure and the risk of blindness become a problem.

  Cryptosporidium parvum is a common protozoan parasite that causes severe diarrhea. Large amounts of oocysts excreted from feces are chlorine-resistant and often cause large outbreaks. However, there are no drugs approved in Japan. In addition, nitazoxanide, a salicylanilide derivative that has been approved in the United States and used as an antiprotozoal drug, is not effective when administered with 100 mg / kg of nitazoxanide in a cryptosporidium-infected animal test using immunodeficient mice (Non-patent Document 1). Since the effects of nitazoxanide administration on immunocompromised patients have not been proven, it is fatal in immunocompromised patients. In addition, calves infected with this protozoan disease cause productivity loss and drowning. From the above, this protozoan disease is a serious problem in livestock and public health.

  Since apicomplexa parasites have a complex life cycle, it is known that modification of genes and histones not accompanied by DNA changes plays an important role in the stage conversion. Accordingly, drugs focused on protozoan epigenetics, for example, inhibitors of histone deacetylase (HDAC), have been attracting attention, but many are said to be toxic to the host as well.

Theodos C.I. et al. , Antimicrob Agents Chemother. 1998, 42 (8): 1959-1965

  An object of this invention is to provide the chemical | medical agent which can be utilized as an antiprotozoal agent.

  As a result of experiments, the present inventors have found that Nullscript (Chemical Formula 1), which is a related compound of HDAC inhibitor and scriptoid, has low toxicity to the host and has a medicinal effect on protozoa. Nullscript is an inactive analog of scriptaide and is used as a negative control for scriptaid and does not inhibit host HDAC.

(1) For the treatment and / or prevention of apicomplexa protozoa infections comprising the compound nullscript represented by the formula (I), isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof as active ingredients Drugs.
(2) The drug for treatment and / or prevention according to (1), wherein the apicomplexa protozoan is Cryptosporidium.
(3) The therapeutic and / or prophylactic agent according to (1), wherein the apicomplexa protozoa is malaria.
(4) The drug for treatment and / or prevention according to (1), wherein the apicomplexa protozoa is Toxoplasma protozoa (5) treatment and / or cryptosporidiosis comprising the anti-cryptospodium agent according to (2) Or medicine for prevention.
(6) A medicament for treatment and / or prevention of malaria, comprising the antimalarial agent according to (3).
(7) A medicament for treatment and / or prevention of Toxoplasma gondii disease comprising the anti-Toxoplasma agent according to (4).

  According to the present invention, it is possible to provide an anti-cryptospodium agent, an anti-toxoplasma agent, and an antimalarial agent that are highly safe for the host.

The antitoxoplasma activity of Epigenetics Library43 compound and the toxicity evaluation to a host cell are shown. Shows the rate of entry of Toxoplasma into the host cells in the presence or absence of Nullscript. The number of protozoan divisions in host cells 24 hours after toxoplasma infection in the presence or absence of null script is shown. The number of daughter insects divided within a single Toxoplasma parasite after 24 hours of Toxoplasma infection in the presence or absence of Nullscript is shown. The results are shown in which 5-week-old immunodeficient (SCID) mice were infected with cryptosporidium by oral administration, null-script was orally administered, and the number of cryptosporidium oocysts excreted in feces was measured.

  Hereinafter, preferred embodiments for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly. Moreover, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

The first aspect of the present invention is an anti-cryptospodium or anti-malarial agent comprising as an active ingredient a compound represented by the following formula (I), an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof. , Relating to anti-toxoplasma agents

  The compound represented by the formula (I) can be synthesized by a general organic chemical method.

  “Isomers” means enantiomers, diastereomers (including cis-trans isomers), conformers, cis-trans isomers in double bonds, and the like that may exist in the compound represented by formula (I). To do.

  Pharmaceutically acceptable salts of the compounds of formula (I) include salts with alkali metals such as sodium and potassium; salts with alkaline earth metal salts such as calcium and magnesium; ammonium salts; and trimethylamine , Triethylamine, tributylamine, pyridine, N, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine and N, Examples thereof include salts with nitrogen-containing organic bases such as N′-dibenzylethylenediamine.

  In the context of the present invention, anti-cryptospodium, anti-malarial, and anti-toxoplasma are: cryptosporidium, malaria parasite, toxoplasma growth inhibition and killing, host cell infection inhibition, host protozoa control The agent used for the suppression, reduction, stop, etc. of the number of each protozoa and / or its activity.

  The compound represented by the formula (I) may be used as it is as an anti-cryptospodium agent, an antimalarial agent, an anti-toxoplasma agent, and further, an excipient, a binder, a disintegrant, a disintegration inhibitor, a caking / adhesion agent Inhibitors, lubricants, absorption / adsorption carriers, solvents, extenders, tonicity agents, solubilizers, emulsifiers, suspending agents, thickeners, coating agents, absorption accelerators, gelation / coagulation accelerators , Light stabilizer, preservative, moisture-proofing agent, emulsification / suspension / dispersion stabilizer, anti-coloring agent, deoxygenation / antioxidant, flavoring / flavoring agent, coloring agent, foaming agent, antifoaming agent, painless Various additives such as an agent, an antistatic agent, and a buffer / pH adjuster may be blended and used as an anti-cryptospodium agent, an antimalarial agent, an anti-toxoplasma agent, or a medicine or pharmaceutical composition containing the same.

  The second aspect of the present invention relates to a medicament for the treatment and / or prevention of cryptosporidiosis, malariasis, and toxoplasmosis, including the anti-cryptospodium agent, antimalarial agent, and anti-toxoplasma agent of the first aspect.

  Cryptosporidiosis in the context of the present invention refers to Cryptosporidium infection or any symptom or condition caused by the infection. The medicament of the present invention is used by being administered to a homeothermic animal for the cure, remission, amelioration or prevention of such infection or the symptoms or conditions associated therewith.

  In the context of the present invention, malaria refers to an infection of a malaria parasite or any symptom or condition caused by the infection. The medicament of the present invention is used by being administered to a homeothermic animal for the cure, remission, amelioration or prevention of such infection or the symptoms or conditions associated therewith.

  Toxoplasmosis in the context of the present invention refers to an infection of Toxoplasma gondii or any symptom or condition caused by the infection. The medicament of the present invention is used by being administered to a homeothermic animal for the cure, remission, amelioration or prevention of such infection or the symptoms or conditions associated therewith.

  The medicament of the present invention can be applied to all thermostatic animals that can be infected with cryptosporidium, malaria parasite, and toxoplasma. Examples of such thermostats include birds such as chickens, turkeys, quails, ducks, cows, horses, pigs, sheep, goats, dogs, cats, rabbits, monkeys and other non-human mammals and humans. it can.

  The pharmaceutical dosage form of the present invention includes oral preparations (tablets, capsules, powders, granules, fine granules, pills, suspensions, emulsions, solutions, syrups, etc.), injections, suppositories, and external use. Agents (ointments, patches, etc.) and aerosols.

  Oral solid preparations such as tablets, powders and granules include, for example, lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, crystalline cellulose, anhydrous dicalcium phosphate, partially pregelatinized starch, cone Excipients such as thio and alginic acid; simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethyl cellulose, shellac, methyl cellulose, ethyl cellulose Binders such as sodium alginate, gum arabic, hydroxypropyl methylcellulose, hydroxypropyl cellulose, water and ethanol; dry starch, alginic acid, agar powder, starch, crosslinked polyvinylpyrrolidone, crosslinked carboxymethylcellulose Sodium, mosquito Disintegrating agents such as boxymethylcellulose calcium and sodium starch glycolate; disintegrating inhibitors such as stearyl alcohol, stearic acid, cocoa butter and hydrogenated oil; aluminum silicate, calcium hydrogen phosphate, magnesium oxide , Talc, silicic anhydride and other anti-caking and anti-caking agents; carnauba wax, light anhydrous silicic acid, aluminum silicate, magnesium silicate, hardened oil, hardened vegetable oil derivative, sesame oil, white beeswax, titanium oxide, dry aluminum hydroxide Lubricants such as gels, stearic acid, calcium stearate, magnesium stearate, talc, calcium hydrogen phosphate, sodium lauryl sulfate and polyethylene glycol; enhanced absorption of quaternary ammonium salts, sodium lauryl sulfate, urea and enzymes Agent; Demp , Lactose, kaolin, bentonite, silicic acid anhydride, using hydrated silicon dioxide, the solid formulation pharmaceutical additives such as absorption and adsorption carrier such as magnesium aluminometasilicate and colloidal silicic acid, may be prepared according to a conventional method.

  Further, the tablets can be made into tablets with ordinary coatings, for example, sugar-coated tablets, gelatin-encapsulated tablets, gastric-coated tablets, enteric-coated tablets, and water-soluble film-coated tablets as necessary.

  Capsules are prepared by filling the various medicines exemplified above into hard gelatin capsules and soft capsules.

  In addition, using the various liquid formulation additives described above such as solvents, extenders, tonicity agents, solubilizers, emulsifiers, suspending agents, thickeners, etc. Oily suspensions, solutions, syrups and elixirs may be used.

  Injections include, for example, diluents such as water, ethyl alcohol, macrogol, propylene glycol, citric acid, acetic acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid and sodium hydroxide; sodium citrate, sodium acetate And pH adjusting agents and buffers such as sodium phosphate; stabilizers such as sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid and thiolactic acid; isotonic agents such as sodium chloride, glucose, mannitol or glycerin; Solubilizers such as sodium carboxymethyl cellulose, propylene glycol, sodium benzoate, benzyl benzoate, urethane, ethanolamine, glycerin; painless such as calcium gluconate, chlorobutanol, glucose, benzyl alcohol An agent; and a pharmaceutical for liquid formulation such as a local anesthetic With additives, it may be prepared according to a conventional method

  The method for administering the medicament of the present invention is not particularly limited, but is appropriately determined according to the form of the preparation. For example, in the case of a parenteral preparation, intravascular administration (preferably intravenous administration), intraperitoneal administration, intestinal administration, subcutaneous administration and the like can be mentioned. In one preferred embodiment, the anti-cryptospodium agent, anti-malarial agent, and anti-toxoplasma agent of the present invention are administered to a living body by oral administration or intravenous administration.

  The dose of the medicament of the present invention may be an amount that exhibits a therapeutic and / or prophylactic effect in the administered subject, that is, an effective amount. The effective amount is appropriately selected according to the type of animal to be examined, the degree of symptoms, and in the case of humans, the age, sex, form of disease, and other conditions of the patient. It is -2000 micrograms, Preferably it is 50 micrograms-1000 micrograms, More preferably, it is 100 micrograms-500 micrograms, and this can be divided into once or several times a day, or can be administered intermittently.

  A third aspect of the present invention relates to a method for treating and / or preventing cryptosporidiosis, malariasis, toxoplasmosis, comprising administering an effective amount of the medicament of the second aspect to a subject.

  The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

<Experimental material>
(1) Compound Epigenetics Library (histone deacetylase inhibitor 43 compound) is provided and purchased from Kanazawa University Cancer Research Institute, and Nitazoxanide is purchased from Tokyo Chemical Industry Co., Ltd. and dissolved in DMSO. Used in the following experiments.

(2) Host cells Human foreskin fibroblasts (HFF cells) and human colon cancer-derived cells (HCT-8) were obtained from ATCC and used. HFF cells and HCT-8 cells were maintained in DMEM and RPMI 1640 medium containing 10% FBS.

(3) Toxoplasma Recombinant RH / 2F strain (ATCC50839), which has low ability to differentiate into latent infectious parasites and highly expresses bacterial β-galactosidase, was screened for anti-toxoplasma activity, IC ₅₀ confirmation, host cell Used for confirmation of invasion and mitotic inhibition, tachyzoites were prepared according to the method described by Sugi et al. (Anal. Biochem., 2014, 464, 9-11).

(4) Plasmodium falciparum 3D7 strain was used for the growth effect suppression test and the like.

(5) Cryptosporidium Cryptosporidium HNJ-1 strain was used for the growth effect suppression test and administration experiment using mice.

<Example 1> Screening evaluation of antiprotozoal activity (1) Evaluation of anti-toxoplasma activity HFF cells are seeded in a 96-well plate so as to be confluent. Aspirate the medium and add the medium in which the test substance and DMSO are dissolved to a final concentration of 2.5 μM. Thereafter, 1 × 10 3 cubed tachyzoites were inoculated and cultured in a CO ₂ incubator at 37 ° C. for 48 hours. After culturing, the decrease in β-galactosidase activity was measured using a luminometer using Beta-Glo Assay System (Promega) as compared with the DMSO group as a solvent control.

(2) Evaluation of anti-cryptospodium activity HCT-8 cells are seeded in a 96-well plate so as to be confluent. The medium of the cells is removed by suction, and a medium in which the test substance and DMSO are dissolved is added to a final concentration of 10 μM. Thereafter, 1 × 10 4 of the cryptosporidium sporozoites were inoculated, and cultured in a CO ₂ incubator at 37 ° C. for 48 hours. After culture, the number of protozoa in the cells was counted using sporo-glo (a polyclonal antibody against Cryptosporidium).

(3) Evaluation of toxicity to host cells HFF cells or HCT-8 cells are seeded in a 96-well plate so as to be confluent. The cell culture medium was removed by aspiration, and a test substance having a concentration in the range of <100 μM and a medium in which DMSO was dissolved were added and cultured in a CO ₂ incubator at 37 ° C. for 48 hours. Thereafter, endogenous ATP was quantified using CellTiter-Glo Kit (Promega), and the number of surviving cells was measured.

  As a result of the evaluation of the antiprotozoal activity, it was confirmed that null script, which is an analog of scriptatide among 43 compounds, inhibits protozoan growth and has low toxicity to host cells.

<Example 2> Measurement of IC ₅₀ for each protozoan of null script (1) Measurement of IC ₅₀ for toxoplasma HFF cells are seeded in a 96-well plate so as to be confluent. The cell culture medium is removed by aspiration, and a null script that has prepared a dilution series and a medium in which DMSO is dissolved are added. Thereafter, 1 × 10 3 cubed tachyzoites were inoculated and cultured in a CO ₂ incubator at 37 ° C. for 48 hours. After culturing, the decrease in β-galactosidase activity was measured using a luminometer using Beta-Glo Assay System (Promega) and compared with the DMSO group as a solvent control, and IC ₅₀ was measured.

(2) Measurement of IC ₅₀ against malaria parasites Red blood cells infected with malaria parasites were cultured with the drug for 96 hours, and the decrease in the number of parasites when compared with the DMSO group as a solvent control was determined by counting using Giemsa staining. It was measured.

(3) Measurement of IC ₅₀ against Cryptosporidium HCT-8 cells are seeded in an 8 well chamber plate to be confluent. The cell culture medium is removed by suction, and a medium in which the drug and DMSO are dissolved is added at a concentration of 1 to 10 μM. Thereafter, 1 × 10 5 of the power of Cryptosporidium sporozoite was inoculated and cultured in a CO ₂ incubator at 37 ° C. for 48 hours. After culture, the number of protozoa in the cells was counted using sporo-glo (a polyclonal antibody against Cryptosporidium).

The 50% inhibitory concentrations of Nullscript against protozoa and hosts are summarized in Table 1. Nullscript had antiprotozoal activity in three species of protozoa, but particularly suppressed the growth of Toxoplasma and Cryptosporidium at low concentrations. Cryptosporidium was confirmed to be less toxic to host cells than nitazoxanide currently used. In addition, since the scriptide which is a histone deacetylase inhibitor is too toxic to the host, a 50% inhibitory concentration of protozoa could not be determined.

Example 3 Evaluation of Host Cell Invasion and Intracellular Growth Inhibitory Effect of Nullscript against Toxoplasma (1) Invasion Inhibitory Effect HFF cells are cultured to become confluent in glass coverslip. Thereafter, Toxoplasma was suspended in null script (2 μM) or DMSO (control group) to give MOI5 and inoculated into host cells. After culturing for 1 hour at the same time, the relative invasion rate of protozoa was determined by counting with IMC7 antibody. Measured and confirmed the effect of invasion inhibition.

(2) Host cell growth inhibitory effect HFF cells are cultured to become confluent in glass coverslip. In the presence or absence (DMSO) of Nullscript 2 μM, Toxoplasma was infected to MOI5, and the number of daughter insects divided (parasite number / PV) in one worm was measured 24 hours after infection.

  As a result, in the presence of Nullscript 2 μM, a decrease in the rate of entry of Toxoplasma into the host cells was confirmed as compared to the absence (FIG. 2). In addition, the number of protozoan divisions 24 hours after the infection was significantly reduced (FIG. 3), and the number of daughter insect divisions in one Toxoplasma parasite was reduced (FIG. 4). Therefore, it has been clarified that Nullscript not only has an effect on preventing invasion of Toxoplasma but also works mainly on preventing protozoa from dividing in host cells.

<Example 4> Administration test using SCID mice Cryptosporidium was orally administered to 5 week-old immunodeficient (SCID) mice (female) at the power of 1x10 5 and nullscript 10 mg / kg. As comparative controls, Cryptosporidium was administered 5 × 1 × 10 5 and DMSO was orally administered. Six mice in each group, a total of 12 mice, were used in the experiment. After the protozoa administration, null script was orally administered at 10 mg / kg / day, and DMSO was orally administered in the comparison group. Feces were collected every day until day 9 after protozoan inoculation, and the number of oocysts discharged was measured using the sucrose suspension method. As a result, in the Nullscript administration group, the oocyst discharge was delayed by one day and the oocyst discharge amount was significantly reduced as compared with the DMSO administration group as a comparative control (FIG. 5).

Claims (7)

A drug for the treatment and / or prevention of apicomplexa protozoa infection comprising a compound nullscript represented by the following formula (I), an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof as an active ingredient .

  The agent for treatment and / or prevention according to claim 1, wherein the apicomplexa protozoan is Cryptosporidium.   The agent for treatment and / or prevention according to claim 1, wherein the apicomplexa protozoa is malaria.   The therapeutic and / or prophylactic agent according to claim 1, wherein the apicomplexa protozoan is Toxoplasma gondii.   A medicament for the treatment and / or prevention of cryptosporidiosis comprising the anti-cryptospodium agent according to claim 2.   A medicament for treatment and / or prevention of malaria, comprising the antimalarial agent according to claim 3.   A medicament for the treatment and / or prevention of Toxoplasma gondii disease comprising the anti-Toxoplasma agent according to claim 4.

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