WO2016194024A1

WO2016194024A1 - Antigiardial agent

Info

Publication number: WO2016194024A1
Application number: PCT/JP2015/004847
Authority: WO
Inventors: Takaaki Masuda; Yoshiyuki Miyata
Original assignee: Pola Pharma Inc.; Nihon Nohyaku Co., Ltd.
Priority date: 2015-06-05
Filing date: 2015-09-24
Publication date: 2016-12-08
Also published as: JP2017002023A; JP5951864B1

Abstract

The present invention aims to provide an agent for the treatment of giardiasis. Provided is an antigiardial agent that includes a compound represented by general formula (1) and/or a salt thereof. The antigiardial agent is preferably for the treatment of infection with giardia, which parasites inside intestines, and the compound represented by general formula (1) and/or the salt thereof are preferably grounded. The grounded compound and/or the salt thereof preferably have an average particle size of from 1 to 1000 μm. General Formula (1) (In the formula, R1 and R2 each independently represent a hydrogen atom or a halogen atom, provided that at least one of R1 and R2 is a halogen atom.)

Description

ANTIGIARDIAL AGENT

The present invention relates to an antigiardial agent and more particularly to an antigiardial agent that includes a powdered compound represented by general formula (1) and/or a powdered salt thereof.

Protozoans of the genus Giardia includes species such as G. lamblia and G. duodenalis and parasites inside stomachs or intestines, causing diarrhea. Giardia not only causes diarrhea, but also is associated with a high prevalence of co-infection with protozoans such as Criptsporidium sp., Ascaris sp., and Helicobacter pylori, which may induce severe gastrointestinal dysfunction such as ulceration (see, for example, Non-Patent Document 1). It is thus desirable to treat Giardia infection early, while metronidazole is the only agent effective against Giardia. Although metronidazole is effective against protozoans in stomachs and duodena, metronidazole cannot be expected to be effective against protozoans in small intestines and intestinal tracts below the small intestines. Therefore, it has been desired to develop a novel antigiardial agent. Such situation has occurred mainly because giardiasis has been rarely present in Japan, and thus adequate measures have not been taken. However, in the present situation where the number of travelers from foreign countries where giardiasis is common is growing and where imports of pets are increasing, outbreak of giardiasis can be triggered by the travelers and the pets at any time.

Moreover, compounds represented by general formula (1) and/or salts thereof are known to have a strong antimycotic effect and a growth inhibitory effect on Trichomonas vaginalis, Chlamydia trychomastis, and the like (see, for example, Patent Document 1 and Patent Document 2). However, nothing has been known about an effect of the compounds and salts thereof on Giardia.

(In the formula, R1 and R2 each independently represent a hydrogen atom or a halogen atom, provided that at least one of R1 and R2 is a halogen atom.)

It has also been less well known that the effect of administered antimicrobial agents on pathogenic microorganisms in intestinal tracts is affected by their average particle size.

Patent Literature 1: WO/2014/115488
Patent Literature 2: WO/2014/115487

Non Patent Literature 1: James A. Cotton et. al.; PLOS ONE, 2014, 9 (10), e109087-

The present invention has been made in view of the above circumstances and is directed to providing a novel agent for the treatment of giardiasis.

As a result of intense research to find an agent capable of treating giardiasis in view of the forgoing, the inventors of the present invention have found that a compound represented by the above general formula (1) and/or a salt thereof have the above capability, thereby achieving the invention. The present invention includes the following aspects:

<1> An antigiardial agent comprising a compound represented by the following general formula (1) and/or a salt thereof:

<2> The antigiardial agent according to <1>, wherein the agent is for the treatment of infection with giardia, which parasites inside intestines.
<3> The antigiardial agent according to <1> or <2>, wherein the compound represented by general formula (1) and/or the salt thereof are powder.
<4> The antigiardial agent according to any of <1> to <3>, wherein the compound represented by general formula (1) and/or the salt thereof have an average particle size of from 1 to 1000 μm.
<5> An antigiardial pharmaceutical formulation comprising the antigiardial agent according to any of <1> to <4> as an active ingredient.
<6> A compound represented by the following general formula (1) and/or a salt thereof, wherein the compound and/or the salt thereof are powder:

(In the formula, R1 and R2 each independently represent a hydrogen atom or a halogen atom, provided that at least one of R1 and R2 is a halogen atom.)
<7> The compound represented by general formula (1) and/or the salt thereof according to <6>, wherein the powder has an average particle size of from 1 to 1000 μm.
<8> The compound represented by general formula (1) and/or the salt thereof according to <6> or <7>, wherein the compound and/or the salt thereof are for an antigiardial agent.

The present invention can provide an agent for the treatment of giardiasis.

The antigiardial agent of the present invention includes a compound represented by general formula (1) and/or a salt thereof as an active ingredient.
In general formula (1), the groups represented by R1 and R2 are a hydrogen atom or a halogen atom. Suitable examples of the halogen atom can include a chlorine atom, a bromine atom, a fluorine atom, and an iodine atom. Particularly preferably, the groups represented by R1 and R2 are a hydrogen atom or a chlorine atom.

Preferred examples of the compound represented by general formula (1) and/or the salt thereof are luliconazole (R1 = R2 = Cl; (R)-(-)-(E)-[4-(2,4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-1-imidazolylacetonitrile) and lanoconazole (R1 = H, R2 = Cl; 4-(2-chlorophenyl)-1,3-dithiolan-2-yliden-1-imidazolylacetonitrile). Luliconazole is particularly preferred.

The compound can be synthesized according to, for example, the method described in Japanese Unexamined Patent Application Publication No. S60-218387. More particularly, the compound represented by general formula (I) can be prepared, as described below, by reacting 1-cyanomethylimidazole with carbon disulfide to give a compound (III) and reacting the compound (III) with a compound that is represented by general formula (II) and that has leaving groups. Examples of the leaving groups can include a methanesulfonyloxy group, a benzenesulfonyloxy group, a p-toluenesulfonyloxy group, and a halogen atom.

In the scheme, R represents a hydrogen atom or a halogen atom, X represents a halogen atom, Y and Y' represent a leaving group such as a methanesulfonyloxy group, a benzenesulfonyloxy group, a p-toluenesulfonyloxy group, or a halogen atom, and M represents an alkali metal.

Any salt of the compound represented by general formula (I) can be used as long as the salt is pharmaceutically acceptable. Examples of the salt can include mineral acid salts such as hydrochlorides, nitrates, sulfates, and phosphates, organic acid salts such as citrates, oxalates, lactates, and acetates, and sulfuric acid salts such as mesylates and tosylates. The hydrochlorides are more preferred in terms of safety and solubility.

The compound represented by general formula (1) and/or the salt thereof may be recrystallized after the preparation.
The compound or the salt may be recrystallized with an alcohol, such as a lower alcohol having about 1-4 carbon atoms, that may contain 10-80% of water or may be recrystallized using an antisolvent. Use of an antisolvent means addition of water, in an amount sufficient to deposit crystals, to a solution of luliconazole in alcohol.
The compound and the salt thereof can be recrystallized according to routine procedures.

When the compound represented by general formula (1) and/or the salt thereof are used as an antigiardial agent, the compound and/or the salt thereof are used preferably in solid form and particularly preferably in the form of fine particles. Typically, a lower limit on a particle size is about 1 μm in the industry, while the compound represented by general formula (1) and/or the salt thereof can have a lower limit on the particle size that is smaller than about 1 μm, when possible. Even when the compound represented by general formula (1) and/or the salt thereof that do not have a lower limit on the average particle size as small as 1 μm, which is the typical lower limit in the industry, the compound and/or the salt can have their effect. The compound represented by general formula (1) and/or the salt thereof preferably have a lower limit on the average particle size of about 3 μm or 5 μm and preferably have a lower limit on the average particle size up to 20 μm or less. The compound represented by general formula (1) and/or the salt thereof preferably have an upper limit on the average particle size up to 1000 μm, which reduces the difference in efficacy between the particles and the crystals, more preferably 500 μm, which increases the difference in efficacy compared with the case of 1000 μm, and still more preferably 200 μm, which increases the difference in efficacy compared with the case of 500 μm. A lower upper limit is more preferred. More preferably, the upper limit is 100 μm or 50 μm, when possible. Comprehensively, the compound represented by general formula (1) and/or the salt thereof are used preferably in the form of powder having an average particle size of from 1 to 1000 μm, more preferably from 1 to 150 μm, still more preferably from 1 to 100 μm, and most preferably from 1 to 70 μm, as the compound and/or the salt thereof in such form have a particularly superior antigiardial effect.

The average particle size of the compound represented by general formula (1) and/or the salt thereof can be obtained as a number average particle size. The number average particle size can be determined by analysis of microscope images. For example, the number average particle size is determined in the following manner: First, the powdered compound represented by general formula (1) and/or the powdered salt are observed using a Diaphot inverted microscope from Nikon Corp. Then, some of the particles are selected and measured for particle size. In the measurement, 100 or more particles shall be measured. The average particle size of the compound represented by general formula (1) and/or the salt thereof can also be determined using a laser diffraction particle size distribution analyzer.

To produce such powder form, for example, the crystals of the compound represented by general formula (1) and/or the salt thereof that are produced by recrystallization may be wet- or dry-milled according to routine procedures. Preferred examples of milling machines can include jet mills, dynomills, coball mills, grinders, and ball mills. When the crystals are dry-milled with a jet mill, it is preferred to previously give the crystals a coarse grind with a speed mill or the like. In the coarse grind, an excipient such as starch or cellulose, a lubricant such as magnesium stearate, and/or the like may be added. When the crystals are wet-milled with a media mill such as a dynomill or a coball mill, it is preferable that the crystals are milled using zirconia or titanium media in an aqueous media that may include alcohol and/or the like, the solid milling media is removed by, for example, filtration, and then the liquid media is removed by, for example, distillation under reduced pressure. In some cases, a surfactant can be added to provide improved wettability with the liquid media to the compound represented by general formula (1) and/or the salt thereof. When a grinder is used, the grinder is preferably fitted with an agate mortar and pestle. Needless to say, the crystals may be manually ground with a mortar and pestle. A ball mill may be used for wet-milling or dry-milling.

When the antigiardial agent of the present invention is used for treatment, the antigiardial agent can be formulated into a pharmaceutical formulation using optional pharmaceutical ingredients commonly used in pharmaceutical formulations. Suitable examples of the optional ingredients include excipients such as lactose, croscarmellose, cellulose, and starch; binders such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, and gum arabic; lubricants such as magnesium stearate; coating agent such as sucrose, hydroxypropyl cellulose caprylate, and shellac; surfactants such as sorbitan fatty acid esters that may have a polyoxyethylene group, including POE sorbitan stearate, POE sorbitan laurate, POE sorbitan oleate, sorbitan stearate, sorbitan laurate, and sorbitan oleate, polyoxyethylene fatty acid esters including POE stearate, POE laurate, and POE oleate, and polyoxyethylene alkyl (alkenyl) ethers including POE lauryl ether, POE cetyl ether, POE stearyl ether, POE oleyl ether, and POE lanolin alcohol ether; salts such as carbonates, oxalates, and phosphates; acids excluding lactic acid such as citric acid, oxalic acid, and phosphoric acid; inorganic powder such as silica and alumina; flavoring agents; colorants; and antioxidants. These agents and ingredients can be processed according to routine procedures to produce the pharmaceutical formulation.
Preferably, the pharmaceutical formulation is designed to be compatible with its intended route of administration. Particularly preferably, the pharmaceutical formulation is designed so that the antigiardial agent of the present invention can take the form of powder having a particle size of from 1 to 1000 μm, which effectively provide the effects of the agent. Examples of suitable dosage forms can include effervescent tablets that provide the above powder form after administration. The pharmaceutical formulation preferably includes the antigiardial agent of the present invention in an amount of 10% by mass or more based on the total weight of the pharmaceutical formulation, and the pharmaceutical formulation may be composed of the antigiardial agent only (the pharmaceutical formulation may include the antigiardial agent in an amount of 100% by mass based on the total weight of the pharmaceutical formulation). The pharmaceutical formulation more preferably include the antigiardial agent in an amount of from 20 to 80% by mass and still more preferably from 30 to 70% by mass based on the total weight of the pharmaceutical formulation.

Suitable examples of a route of administration of the pharmaceutical formulation can include oral, intravenous, subcutaneous, intradermal, and rectal administration. Preferable is the route of administration which ensures that the antigiardial agent of the present invention in the formulation remains in the form of powder having an average particle size of from 1 to 1000 μm, which ensures that the antigiardial agent has a great effect. More particularly, oral or rectal administration is preferred. Thus, preferred dosage forms are tablets, capsules, granules, powders, and suppositories.

Although a preferred dosage of the antigiardial agent of the present invention differs depending on the age and body weight of the subject, the administration route, and the purpose, the compound represented by general formula (1) and/or the salt thereof are preferably administered at, for example, a daily dosage of from 0.1 to 10 g in single or divided doses.

Now, the present invention will be described in more detail with reference to examples, although the present invention is not limited to the examples without departing from the spirit of the invention.

<Example 1>
Luliconazole was recrystallized from 80% water-containing ethanol to give crystals, which were then ground in an agate mortar for a minute and 5 minutes. The average particle size of the ground powders was determined. The ground powders had a particle size of 1000 μm or less as determined under a microscope (a Diaphot inverted microscope from Nikon Corp.).

Giardia lamblia (a clinical isolate, isolated and maintained by Department of Infectious Diseases, Keio University School of Medicine) was precultured in Diamond's TYI-S-33 medium (Diamond LS, et.al.; J. Eukaryot. Microbiol. 1995; 42 (3): 277-8) and diluted with the medium to 10,000 protozoans per 1 mL (hereinafter referred to as "culture"). Four test tubes were provided. To each of the test tubes, 5 mL of the medium was added, and then 1 mg of the crystals, the crystals ground for a minute, or the crystals ground for 5 minutes was added. Then, 100 μL of the culture was added. The test tube that contains only the medium and the culture was used as a control. The test tubes were incubated at 37°C, and after 24 hours and 96 hours, protozoans were counted under an inverted microscope. 10 fields of view were examined, and the total number of protozoans found in these fields was added. The results are shown in Table 1. The results show that the compound of general formula (1) has an antigiardial effect and that the ground compound has a much greater antigiardial effect.

<Example 2>
2.5 μg of luliconazole as a 2% solution in 100 μL of methanol was added to 5mL of the medium, and 2.5 μg of luliconazole ground in an agate mortar for 5 minutes was added to 5mL of the medium. To each of the solution and the ground crystals, the culture was added in the same manner as in Example 1. As a control, a test tube that contains only 5 mL of the medium and the culture was used. After 24 hours from addition of the culture, counts were made for 10 fields of view under an inverted microscope, and the total number of protozoans found in the 10 fields was determined. A t-test was performed for each of the values determined by counting the 10 fields of view. The results are shown in Table 2. The results show that addition of the compound in solid form has a greater effect compared with addition of a solution of the compound.

<Example 3>
To determine the boundary between the powder prepared by grinding the crystals for 3 minutes and the powder prepared by grinding the crystals for 5 minutes, the powders were prepared by grinding the crystals for a period of from 3 to 5 minutes in the same manner as in Example 1 and evaluated in the same manner as in Example 1 except that the powders were observed after 48 hours from addition of the culture to clarify the differences. A table below shows the total numbers of viable protozoans found in 10 fields of view after 48 hours from addition of the culture. The results show that the crystals ground for 5 minutes and the crystals ground for 4 minutes have a significant difference in efficacy. Thus, the results also show that the crystals ground for 5 minutes is far preferable to the crystals ground for 4 minutes.

<Example 4>
The average particle size of the unground crystals, the crystals ground for a minute, the crystals ground for 3 minutes, the crystals ground for 4 minutes, and the crystals ground for 5 minutes was determined using a laser diffraction particle size distribution analyzer. The results are shown in a table below. The results show that the average particle size is preferably 200 μm or less, more preferably 150 μm or less, still more preferably 100 μm or less, and most preferably 70 μm or less.

The present invention can be used in pharmaceutical applications.

Claims

An antigiardial agent comprising a compound represented by the following general formula (1) and/or a salt thereof:

wherein R1 and R2 each independently represent a hydrogen atom or a halogen atom, provided that at least one of R1 and R2 is a halogen atom.
The antigiardial agent according to claim 1, wherein the agent is for the treatment of infection with giardia, which parasites inside intestines.
The antigiardial agent according to claim 1 or 2, wherein the compound represented by the general formula (1) and/or the salt thereof are powder.
The antigiardial agent according to any one of claims 1 to 3, wherein the compound represented by general formula (1) and/or the salt thereof have an average particle size of from 1 to 1000 μm.
An antigiardial pharmaceutical formulation comprising the antigiardial agent according to any one of claims 1 to 4 as an active ingredient.
A compound represented by the following general formula (1) and/or a salt thereof, wherein the compound and/or the salt thereof are powder:

wherein R1 and R2 each independently represent a hydrogen atom or a halogen atom, provided that at least one of R1 and R2 is a halogen atom.
The compound represented by general formula (1) and/or the salt thereof according to claim 6, wherein the powder has an average particle size of from 1 to 1000 μm.
The compound represented by general formula (1) and/or the salt thereof according to claim 6 or 7, wherein the compound and/or the salt thereof are for an antigiardial agent.