JP2009221113A - Therapeutic agent of protozoan disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new treating agent of protozoan infectious diseases and a method for treating the same. <P>SOLUTION: This treating agent of the protozoan infectious diseases and the method for treating the same are disclosed by using a compound having a structure expressed by formula (I), or its salt. The compound of formula (I) or its salt is considered to inhibit the propagation of the protozoa by bonding with a retinoid receptor and inhibiting the up take of cholesterol in host cells. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for protozoan diseases such as toxoplasmosis and neosporosis.

  In research and development of therapeutic agents for conventional protozoan infections, starting from experience, the mechanism of action of the drug was deciphered, and then there were many approaches to synthesize new similar compounds. However, only a few antiprotozoal drugs have been clarified in their mechanism of action, and no effective drug has been developed. In recent years, research aimed at deciphering protozoan-specific metabolic mechanisms and host immune mechanisms against protozoa has progressed, and protozoa-specific therapeutic agents are being developed. For example, Toxoplasma takes in para-aminobenzoic acid and synthesizes an enzyme (CoF) essential for glycolysis, but pyrimethamine and sulfadiazine are protozoa because they have similar chemical structures to para-aminobenzoic acid and folic acid It is taken up by and inhibits CoF synthesis in an antagonistic manner. There are also efforts to develop antiprotozoal drugs targeting nucleic acid metabolism. However, if the therapeutic agent for these protozoal infections exceeds a certain amount, it acts negatively on the human body, and side effects such as leukopenia and vomiting appear after long-term use.

  Furthermore, the emergence of drug-resistant protozoa is another cause that makes it difficult to develop antiprotozoal drugs. The therapeutic concept of antiprotozoal drugs that have been developed so far targets enzymes that control the metabolism of the protozoa. However, since protozoa have a mutation mechanism by regulating protein expression and gene recombination, the morphology and antigenicity can be easily changed. Due to such a special avoidance mechanism of protozoa, drug target molecules in the protozoa are changed, and drug-resistant protozoa appear. As a result, current antiprotozoal drugs that have been developed targeting protozoan-specific metabolic systems may eventually become ineffective.

Under such circumstances, development of new therapeutic agents and treatment methods for protozoal infections is required.
Nishikawa et al., Cell Microbiol. 7 (6), 849-867, 2005

  An object of this invention is to provide the new therapeutic agent and treatment method with respect to a protozoan infection.

The inventors have surprisingly found that tamibarotene, a retinoic acid receptor agonist, is effective in treating protozoal infections. Specifically, the present invention has the following configuration.
(1) The following formula (I):
A therapeutic agent for protozoan infections, comprising as an active ingredient a compound having the structure represented by:
(2) The therapeutic agent according to (1), wherein the protozoal infection is toxoplasmosis or neosporosis;
(3) The therapeutic agent according to (1) or (2), wherein the animal to be treated is human or livestock;
(4) The therapeutic agent according to any one of (1) to (3), which is an injection or an oral preparation;
(5) A method for treating a protozoal infection, comprising administering or orally administering the therapeutic agent of any one of (1) to (4) to a mammal other than a human.

The compound of formula (I) which is an active ingredient of the therapeutic agent of the present invention has the following structure:
A retinoid having the chemical name (chemical name 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) carbamoyl] benzoic acid) and retinoic acid receptor RARα / β It is known that it specifically binds to the retinoid X receptor and does not bind to the retinoid X receptor. This compound is marketed under the generic name tamibarotene as a therapeutic agent for acute promyelocytic leukemia, and is said to have an immunomodulatory action and angiogenesis-inhibiting action. There is no knowledge that there is.

  Toxoplasma is a protozoan that infects mammals including humans and causes toxoplasmosis. Toxoplasma has been shown to grow using host cell-derived cholesterol (Nishikawa et al., Cell Microbiol. 7 (6), 849-867, 2005). The mechanism of uptake and changes in lipid metabolism in host cells during Toxoplasma infection have not yet been elucidated. The present inventors currently believe that the compound of formula (I) inhibits the proliferation of toxoplasma by directly acting on the retinoid receptor of the host cell and suppressing the host cell cholesterol uptake. ing. Neospora is an intracellular protozoan that is closely related to Toxoplasma and can suppress its growth according to the method of the present invention. Examples of protozoa whose growth can be suppressed by the same mechanism include malaria parasites, trypanosoma and leishmania.

  As shown in the Examples below, mammalian cells (J774) were pretreated with a compound of formula (I) for 6 hours, infected with Toxoplasma or Neospora in the presence of the drug, and protozoan growth was measured after 20 hours. However, it was revealed that tamibarotene has an action of suppressing the growth of Toxoplasma and Neospora. If it becomes possible to control protozoal infections by regulating cholesterol uptake of host cells, it will lead to the development of therapeutic agents and treatments for new protozoal infections in humans and livestock.

  The protozoan disease therapeutic agent of the present invention can be formulated by methods known to those skilled in the art. For example, the compound of formula (I) is mixed with pharmaceutically acceptable carriers or excipients well known in the art, and emulsifiers, suspending agents, surfactants, stabilizers, bulking agents, thickening agents. Tablets, pills, dragees, powders, granules for oral or parenteral administration, formulated with appropriate combinations with agents, binders, disintegrants, lubricants, penetrants, flavoring agents, coloring agents, preservatives, etc. , Capsules, solutions, emulsions, gels, syrups, slurries, suspensions, inhalants and the like.

  Pharmaceutically acceptable carriers or excipients include, but are not limited to, sterile water, saline, Hank's solution, Ringer's solution, lactose, sucrose, mannitol, sorbitol, starch, talc, calcium carbonate, calcium phosphate, stearin Magnesium acid, vegetable oil, cocoa butter, liquid paraffin, ethanol, propylene glycol, polyethylene glycol, polyvinylpyrrolidone, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, sorbitol, dextran, polyvinylpyrrolidone, agar, alginic acid, sodium alginate, Arabic gum, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer Such as chromatography solution, and the like.

  Suitable administration routes of the protozoan disease therapeutic agent of the present invention are not limited, but are oral, rectal, transmucosal, intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, nasal cavity Inside is mentioned. The administration route can be appropriately selected in consideration of the animal species to be treated, the medical condition, and other drugs used in combination. When administered to an animal, it may be added to a formulated feed or drinking water. The protozoan disease treatment agent of the present invention may be administered alone or in combination with other protozoan disease treatment agents, and may also be administered in combination with other agents such as antibacterial agents and antifungal agents. .

  The therapeutically effective dose of the protozoan disease therapeutic agent of the present invention varies depending on the administration route, the species of animal to be treated, the body weight and the condition, but in the range of 0.01 mg to 100 mg / kg body weight per administration You can choose.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Cell viability
[Materials and methods]
Cell viability (%) of Toxoplasma and Neospora was measured as follows. J774 cells (1x10 ⁵ ) were treated with Tamibarotene (6.25μM, 12.5μM, 25μM, 50μM in Dulbecco's modified Eagle's medium) at 37 ° C for 6 hours, and then various protozoa (1x10 ⁵ ) were added in the presence of drugs. And incubated at 37 ° C. for 20 hours. Next, 1 μCi of [5,6- ³ H] uracil was added and cultured at 37 ° C. for 2 hours. Thereafter, cells were fixed with 10% trichloroacetic acid and treated with 0.2N NaOH at 37 ° C. for 30 minutes. Incorporation of protozoa-specific [5,6- ³ H] uracil was measured with a shoreline measuring device. Cell viability (%), the table as a percentage of the incorporation of [5,6- ³ H] uracil protozoa in the presence of the agent for [5,6- ³ H] incorporation of uracil protozoa in the drug absence did.

The cell viability (%) of J774 cells was measured as follows. J774 cells ( ⁴ × 2.5 × 10) were treated with tamibarotene at 37 ° C. for 26 hours, Cell Counting Kit-8 (manufactured by Dojin Laboratories) was added, and the mixture was cultured at 37 ° C. for 1 hour. Next, the absorbance at 450 nm of the culture solution was measured. The cell viability (%) was expressed as a ratio of the absorbance value of 450 nm of J774 cells in the presence of the drug to the absorbance value of 450 nm of J774 cells in the absence of the drug.

[result]
The results are shown in FIG. The growth of Toxoplasma and Neospora was inhibited depending on the concentration of Tamibarotene. On the other hand, there was no change in the growth of mammalian cells J774.

Measurement of intracellular cholesterol levels
[Materials and methods]
Intracellular cholesterol was measured as follows. The Tamibarotene in J774 cells (2.5 × 10 ⁵ cells) for 6 hours at 37 ° C., then added Toxoplasma (1x10 ⁵ cells), or not added, and cultured for 40 hours at 37 ° C. under drug present. Next, the cells were collected, lipids were extracted with chloroform-methanol (2: 1), and intracellular cholesterol was measured with cholesterol / cholesteryl ester quantitation kit (Calbiochem). The amount of protein in the collected cells was measured with Lowry Protein Assay Kit (Pierce). Intracellular cholesterol was expressed as the amount of intracellular cholesterol (ng) per 1 μg of cellular protein.

[result]
The results are shown in FIG. Tamibarotene reduced cholesterol in Toxoplasma infected cells in a concentration-dependent manner.
(*) When compared with P <0.05, value without adding Tamibarotene
(**) P <0.05, in the presence of tamibarotene at the same concentration, when comparing the values of Toxoplasma infected cells and uninfected cells

Mouse infection test
[Materials and methods]
BALB / c mice (female, 8 weeks old, N = 18) were given tamibarotene 1.0 mg / kg or EME medium intraperitoneally. One day later, Toxoplasma (1x10 ³ ) was inoculated intraperitoneally, and then tamibarotene 1.0 mg / kg or EMS (Eagle's minimum essential) medium was intraperitoneally administered for 7 consecutive days. Survival days after toxoplasma infection were measured and expressed in percent survival. Survival analysis was performed by Kaplan-Meier method and Log-rank test.

[result]
The results are shown in FIG. Administration of tamibarotene prolonged the survival rate of mice ((*) P <0.05). The median survival time was 19 days for the tamibarotene administration group and 15 days for the control group. The hazard ratio was 1.945, and the rate of death was 1.945 times faster in the control group than in the treated group.

FIG. 1 shows the effect of Tamibarotene on Toxoplasma and Neospora growth. FIG. 2 shows the effect of tamibarotene on cholesterol levels in Toxoplasma infected cells. FIG. 3 shows the effect of tamibarotene administration on the survival rate of Toxoplasma infected mice.

Claims (5)

Formula (I):
The therapeutic agent with respect to the protozoan infection which contains the compound which has the structure represented by these, or its salt as an active ingredient. The therapeutic agent according to claim 1, wherein the protozoal infection is toxoplasmosis or neosporosis. The therapeutic agent according to claim 1 or 2, wherein the animal to be treated is human or livestock. The therapeutic agent according to any one of claims 1 to 3, which is an injection or an oral agent. A method for treating a protozoal infection, comprising administering the therapeutic agent according to any one of claims 1 to 4 to a mammal other than a human by injection or oral administration.