JP2021120360A - Therapeutic and/or preventive agent of schistosomiasis - Google Patents

Abstract

To provide a therapeutic and/or preventive agent of Schistosomiasis.SOLUTION: A compound shown by the following formula (I) (in the formula, R1 denotes hydrogen atom or acyl group, R2 denotes hydrogen atom, a straight chain or branched alkyl group, cycloalkyl group, aryl group or aralkyl group) or salts thereof.SELECTED DRAWING: None

Description

The present invention relates to a therapeutic and / or prophylactic agent for schistosomiasis, and more particularly, a therapeutic and / or prophylactic agent for schistosomiasis containing 5-aminolevulinic acid (5-ALA) or a derivative thereof or a salt thereof. Regarding the treatment and / or prevention of schistosomiasis using.

Schistosomiasis is a disease that causes chronic health problems and is transmitted by people's contact with water (fresh water) contaminated with the larval form (cercariae) of vascular parasitic trematodes known as schistosomiasis. Adults live in the urinary tract and veins that are excreted from the intestines. Most of the eggs they lay are trapped in tissues, and the body's reaction to this can cause great damage. Schistosomiasis affects about 240 million people worldwide, with more than 700 million people living in areas where schistosomiasis is endemic. The infection is endemic in tropical and subtropical areas of poor communities without drinking water and adequate sanitation. Schistosomiasis vulgaris includes Birhardz schistosomiasis (pathogen is Schistosoma schistosomiasis), Manson schistosomiasis belonging to intestinal schistosomiasis, S. japonicum, Japan. Five types are known: S. schistosomiasis and S. intercalatum (Non-Patent Documents 1 and 2).

Schistosomiasis eggs leave the human body through urine (in the case of urinary schistosomiasis) or stool (in the case of intestinal schistosomiasis) and hatch in water to release larvae (miracidium). The larvae invade the freshwater snail host, and after several weeks of growth and replication, the larval form (cercariae) emerges from the snail. When humans come into contact with contaminated water (playing in water, swimming, washing, planting rice), cercariae invade human skin, transform and then migrate from the lungs to the liver to mature into adults. .. Mature adults migrate to veins in the abdominal cavity or urinary tract. Most of the eggs produced are trapped in tissues, but some are excreted from the intestine or bladder.

Schistosomiasis is primarily caused by eggs laid by adults in blood vessels around the bladder or intestinal tract. The classic sign of urogenital schistosomiasis is hematuria (blood in the urine), and in women, urogenital schistosomiasis is a cervical and vaginal lesion, vaginal bleeding, pain during sexual intercourse, vulva. It may present with various signs and symptoms such as nodules. In areas where genitourinary schistosomiasis is endemic, the majority of women may suffer from female genital schistosomiasis (FGS). The WHO Working Group in recent years has concluded that female genital schistosomiasis is a risk factor for HIV transmission in women, with biological relevance. Genital schistosomiasis also affects men, causing pathology of the seminal vesicles, prostate, and other organs. The disease can also have other long-term irreversible consequences, including infertility. In addition, bladder and ureteral fibrosis and hydronephrosis are common symptoms in advanced cases, and bladder cancer may also be a late complication (Non-Patent Documents 1 to 3).

Intestinal schistosomiasis is associated with nonspecific symptoms such as abdominal pain, diarrhea, and bloody stools. In severe cases, liver hypertrophy is a common symptom, often associated with ascites and other signs of portal hypertension, and may be accompanied by splenomegaly. Other major symptoms of schistosomiasis are dermatitis, urticaria, hepatosplenomegaly, fever, stomach pain, diarrhea, liver and brain inflammation, liver fibrosis, and cirrhosis. In addition, worm-egg embolization of blood vessels in the brain may cause various neuroses such as nest symptoms similar to brain tumors. Furthermore, it is also known that symptoms similar to brain tumors other than neurological symptoms, spinal cord compression symptoms due to spinal cord lesions, and cauda equina syndrome occur (Non-Patent Documents 1 to 4).

Schistosoma japonicum (S. japonicum) is the cause of Schistosoma japonicum, which is one of the main schistosoma japonicum as described above, and parasitizes the portal vein of humans. Its intermediate host is Oncomelania hupensis nosophora, and its final host is humans, cats, dogs and cattle. The female Schistosoma japonicum has a dark brown body color, is elongated and has a body length of 15 to 25 mm. Males have a pale body color, are short in shape and have a body length of 9-18 mm. The nutrient source for Schistosoma japonicum is red blood cells. The most practical method for diagnosing Schistosoma japonicum is to identify eggs in the stool.

Praziquantel is used as the only recommended therapeutic agent for schistosomiasis, but praziquantel has no preventive effect (Non-Patent Document 2). The drug is also known to have hepatotoxicity and central nervous system toxicity.

Therefore, there is still a need for the development of further therapeutic agents that are effective for the treatment or prevention of schistosomiasis.

Non-Patent Document 1: World Health Organization (WHO) Home Page: "Schistosomiasis", URL: https://www. who. int / schistosomiasis / epidemiology / en /, Access date: November 21, 2019 Non-Patent Document 2: National Institute of Infectious Diseases (NIID) Homepage: "What is schistosomiasis?", URL: https://www. niid. go. jp / niid / ja / kansennohanashi / 413-schistosoma. html, access date: November 25, 2019 Non-Patent Document 3: Frontiers in Immunology, 2018, Vol. 9, doi: 10.3389 / fimmu. 2019.02492
Non-Patent Document 4: Journal of Japanese Society of Gastroenterology, 2010, Vol. 43, No. 5, pp. 572-577

An object of the present invention is to provide a new therapeutic and / or preventive agent for schistosomiasis. More specifically, it is to provide a therapeutic and / or preventive agent for schistosomiasis, which comprises 5-ALA or a derivative thereof or a salt thereof.

As a result of intensive research aimed at solving the above problems, the present inventors, surprisingly, act as a metabolite, Heme, to suppress schistosomiasis spawning and live. The present invention has been completed by finding that it treats or prevents schistosomiasis.

5-ALA is a common precursor of heme compounds produced in intracellular mitochondria. It has been known that 5-ALA has an anti-inflammatory effect on specific inflammatory diseases, but 5-ALA and its in vivo metabolites suppress the spawning of schistosomiasis and treat or treat schistosomiasis. It was not known that it could be prevented.
5-ALA is metabolized to porphobilinogen (EC 4.2.1.24) by porphobilinogen synthase in mammals, and further hydroxymethylbilane-> uroporphyrinogen III-> coproporphyrinogen. III-> Protoporphyrinogen IX-> Protoporphyrin IX. Protoporphyrin becomes a heme by coordinating iron ions.
Heme is a trivial name for a complex consisting of a porphyrin and an iron ion, and includes a complex having a different porphyrin structure. The divalent complex is called ferrohem, the trivalent complex is called ferrihem, and the ferrihem with one Cl ⁻ or OH ⁻ coordinated is called hemin and hematin, respectively. It has been.
It is known that application of 5-ALA to cells increases the intracellular heme concentration. Furthermore, it is also known that the effect of increasing the heme concentration is increased by co-administration of a metal-containing compound such as SFC (Biochemistry and Biopharmacology Reports, 2017, Vol. 11, pp. 105-111, European Journal). of Pharmacology, 2018, Vol. 833, pp. 25-33).
5-ALA administered externally to the host animal, or 5-ALA whose synthesis is enhanced in the host animal, is metabolized in the animal cell to produce heme and acts on the blood-flukes parasitizing the body.

Therefore, the present invention newly provides a medicament containing 5-ALA and its use for treating or preventing schistosomiasis.

（式中、Ｒ^１は、水素原子又はアシル基を表し、Ｒ^２は、水素原子、直鎖若しくは分岐状アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す。）
で示される化合物又はその塩を含有する住血吸虫症の治療及び／又は予防剤。
[項目２]
Ｒ^１及びＲ^２が水素原子であることを特徴とする、項目１に記載の住血吸虫症の治療及び／又は予防剤。
[項目３]
さらに、一種又は二種以上の金属含有化合物を含有することを特徴とする項目１又は２記載の住血吸虫症の治療及び／又は予防剤。
[項目４]
金属含有化合物が、鉄、マグネシウム、亜鉛、ニッケル、バナジウム、銅、クロム、モリブデン又はコバルトを含有する化合物であることを特徴とする項目３記載の住血吸虫症の治療及び／又は予防剤。
[項目５]
金属含有化合物が、鉄、マグネシウム又は亜鉛を含有する化合物であることを特徴とする項目３記載の住血吸虫症の治療及び／又は予防剤。
[項目６]
金属含有化合物が、鉄を含有する化合物であることを特徴とする項目３記載の住血吸虫症の治療及び／又は予防剤。
[項目７]
さらに、他の住血吸虫症の治療又は予防剤と併用される、項目１〜６のいずれか一項に記載の住血吸虫症の治療及び／又は予防剤。
[項目８]
（１）下記式（Ｉ）

（式中、Ｒ^１は、水素原子又はアシル基を表し、Ｒ^２は、水素原子、直鎖若しくは分岐状アルキル基、シクロアルキル基、アリール基又はアラルキル基を表す。）
で示される化合物又はその塩を、薬学的許容される賦形剤と共に投与することを含む、住血吸虫症の治療及び／又は予防方法。 That is, the present invention provides the following.
[Item 1]
(1) The following formula (I)

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group.)
A therapeutic and / or preventive agent for schistosomiasis containing the compound indicated by or a salt thereof.
[Item 2]
The therapeutic and / or prophylactic agent for schistosomiasis according to item 1, wherein R ¹ and R ^{2 are hydrogen atoms.}
[Item 3]
The therapeutic and / or preventive agent for schistosomiasis according to item 1 or 2, which further comprises one or more metal-containing compounds.
[Item 4]
The treatment and / or preventive agent for schistosomiasis according to item 3, wherein the metal-containing compound is a compound containing iron, magnesium, zinc, nickel, vanadium, copper, chromium, molybdenum or cobalt.
[Item 5]
The therapeutic and / or preventive agent for schistosomiasis according to item 3, wherein the metal-containing compound is a compound containing iron, magnesium or zinc.
[Item 6]
The therapeutic and / or preventive agent for schistosomiasis according to item 3, wherein the metal-containing compound is an iron-containing compound.
[Item 7]
The treatment and / or preventive agent for schistosomiasis according to any one of items 1 to 6, which is used in combination with another treatment or preventive agent for schistosomiasis.
[Item 8]
(1) The following formula (I)

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group.)
A method for treating and / or preventing schistosomiasis, which comprises administering the compound shown in (1) or a salt thereof together with a pharmaceutically acceptable excipient.

According to the treatment and / or preventive agent for schistosomiasis of the present invention, an excellent schistosomiasis treatment / prevention effect can be obtained.

FIG. 1 is a diagram showing the state of each well of the 24-well plate used in the examples. FIG. 2 is a diagram showing the effect of hemin on the activity of adults. FIG. 3 is a diagram showing the effect of hemin on adult pairing. FIG. 4 is a diagram showing the effect of hemin on spawning.

(Definition)
In the present specification, when a range of a plurality of numerical values is indicated, a range consisting of a combination of arbitrary lower limit values and upper limit values of the plurality of ranges is also meant in the same manner.

(Active ingredient of the therapeutic and / or preventive agent for schistosomiasis of the present invention)
The compound used as an active ingredient of the therapeutic and / or preventive agent for schistosomiasis of the present invention is a compound represented by the formula (I) or a salt thereof (hereinafter, these may be collectively referred to as "ALAs"). Can be exemplified as. 5-ALA, which is also called δ-aminolevulinic acid, is ^{a case where R 1} and R ² of the formula (I) are both hydrogen atoms, and is one of the amino acids. As the 5-ALA derivative, R ¹ of the formula (I) is a hydrogen atom or an acyl group, and R ² of the formula (I) is a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl. Compounds other than the underlying 5-ALA can be mentioned.

Examples of the acyl group in the formula (I) include linear or branched alkanoyl groups having 1 to 8 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl and benzylcarbonyl groups. , Benzoyl, 1-naphthyl, 2-naphthyl group and other aloyl groups having 7 to 14 carbon atoms can be mentioned.

Examples of the alkyl group in the formula (I) include linear or branched groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl and octyl groups. Alkyl groups having 1 to 8 carbon atoms can be mentioned.

As the cycloalkyl group in the formula (I), a saturated or partially unsaturated bond such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, or 1-cyclohexenyl group may be present. , Cycloalkyl groups having 3 to 8 carbon atoms can be mentioned.

Examples of the aryl group in the formula (I) include an aryl group having 6 to 14 carbon atoms such as a phenyl, naphthyl, anthryl, and phenanthryl group.

As the aralkyl group in the formula (I), the aryl moiety can be exemplified in the same manner as the above aryl group, and the alkyl moiety can be exemplified in the same manner as the above alkyl group. Specifically, benzyl, phenethyl, phenylpropyl, phenylbutyl and benzhydryl can be exemplified. , Trityl, naphthylmethyl, naphthylethyl group and other aralkyl groups having 7 to 15 carbon atoms can be mentioned.

As the 5-ALA derivative ^{, a compound in which R 1} is a formyl, acetyl, propionyl, butyryl group or the like, or a compound in which R ² is a methyl, ethyl, propyl, butyl, pentyl group or the like is preferable, and the above R ^{Compounds in which the combination of 1} and R ^{2 is} a combination of formyl and methyl, acetyl and methyl, propionyl and methyl, butyryl and methyl, formyl and ethyl, acetyl and ethyl, propionyl and ethyl, and butyryl and ethyl can be preferably mentioned. can.

The 5-ALAs may act as an active ingredient in the state of 5-ALA of the formula (I) or a derivative thereof in vivo, and various salts, esters, etc. for increasing the solubility may be used depending on the form of administration. Alternatively, it may be administered as a prodrug (precursor) that is decomposed by an enzyme in the living body. For example, examples of the salt of 5-ALA and its derivative include a pharmacologically acceptable acid addition salt, metal salt, ammonium salt, organic amine addition salt and the like. Examples of the acid addition salt include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate and other inorganic acid salts, formate, acetate, propionate and toluenesulfonic acid. Salt, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Organic acid addition salts can be exemplified. Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, and metal salts such as aluminum and zinc. Examples of the ammonium salt include alkylammonium salts such as ammonium salt and tetramethylammonium salt. Examples of the organic amine salt include triethylamine salt, piperidine salt, morpholine salt, toluidine salt and the like. These salts can also be used as a solution at the time of use.

Among the above ALAs, desirable ones are 5-ALA and various esters such as 5-ALA methyl ester, 5-ALA ethyl ester, 5-ALA propyl ester, 5-ALA butyl ester and 5-ALA pentyl ester. , And these hydrochlorides, phosphates, sulfates, and ALA hydrochlorides, 5-ALA phosphates can be particularly preferably exemplified.

The above ALAs can be produced by any known method of chemical synthesis, production by microorganisms, and production by enzymes. In addition, the above ALAs may form a hydrate or a solvate, and either one may be used alone or two or more thereof may be used in combination as appropriate.

The treatment and / or preventive agent for sickness of the present invention preferably contains a metal-containing compound within a range that does not cause excess disease, and the metal portion of the metal-containing compound includes iron, magnesium, zinc, and the like. Nickel, vanadium, cobalt, copper, chromium and molybdenum can be mentioned, but iron, magnesium and zinc are preferable, and iron can be preferably exemplified.

By using the above ALAs in combination with a metal-containing compound, an excellent therapeutic and / or preventive effect on schistosomiasis can be obtained even when a lower concentration of ALAs is used.

The iron compound may be an organic salt or an inorganic salt, and examples of the inorganic salt include ferric chloride, iron sesquioxide, iron sulfate, and ferrous pyrophosphate, and examples of the organic salt are carboxylates. For example, citrates such as ferrous citrate, sodium iron citrate, sodium ferrous citrate (SFC), ammonium iron citrate, which are hydroxycarboxylates, and ferric pyrophosphate. , Hem iron, dextran iron, iron lactate, ferrous gluconate, sodium diethylenetriamine pentaacetate, ammonium diethylenetriamine pentaacetate, sodium ethylenediamine tetraacetate, ammonium ethylenediamine pentaacetate, sodium dicarboxymethylglutamate, dicarboxymethyl Organic acid salts such as ammonium iron glutamine, ferrous fumarate, iron acetate, iron oxalate, ferrous succinate, sodium iron citrate succinate, iron triethylenetetraamine, iron lactoferrin, iron transferase, iron chlorophyllin Examples thereof include sodium, ferritin iron, sugar-containing iron oxide, and ferric glycine sulfate.

Examples of the magnesium compound include magnesium citrate, magnesium benzoate, magnesium acetate, magnesium oxide, magnesium chloride, magnesium hydroxide, magnesium carbonate, magnesium sulfate, magnesium silicate, magnesium nitrate, diethylenetriamine pentamagnesium diammonate, and ethylenediamine tetraacetate. Examples include magnesium disodium and magnesium protoporphyrin.

Examples of the zinc compound include zinc chloride, zinc oxide, zinc nitrate, zinc carbonate, zinc sulfate, diethylenetriamine diammonium pentaacetate, disodium ethylenediamine tetraacetate, zinc protoporphyrin, and zinc-containing yeast.

As the metal-containing compound, one type or two or more types can be used, respectively, and the dose of the metal-containing compound may be 0 to 100 times the molar ratio of 5-ALA, which is 0. It is preferably 0.01 to 10 times, more preferably 0.1 to 8 times.

The therapeutic and / or prophylactic agent for schistosomiasis of the present invention can be used in combination with other therapeutic or prophylactic agents for schistosomiasis as long as it does not cause excess disease. As other concomitant agents, praziquantel and artemether (Artemether, Trop.Med.Infect.Dis.2018, Vol.3, no.125, doi: 10.3390 / tropicalmed3040125) can be used, but are limited thereto. Not done.
The dose of these other agents and the ratio to the dose of 5-ALA can be appropriately adjusted by those skilled in the art.

(Method of administration of therapeutic and / or preventive agent of the present invention)
The ALAs and the metal-containing compound or other agents contained in the therapeutic and / or preventive agent for schistosomiasis of the present invention may be a composition containing any two or more of them, or each of them alone. It can also be administered as a composition contained in. When a composition containing ALAs and a metal-containing compound or another drug is used alone, these may be administered at the same time or separately. Preferably, the ALAs and the metal-containing compound or other drug can be administered in combination so as to have an additive effect, preferably a synergistic effect. When the compositions containing the ALAs and the metal-containing compound or other drug alone are administered separately, the administration intervals thereof are 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 1 hour. It can be set to, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc., but is not limited thereto.

The route of administration of the therapeutic and / or prophylactic agent for blood-sucking insect disease of the present invention is oral administration including sublingual administration, intravenous administration by inhalation administration, injection, infusion, etc., suppository, gel agent, lotion agent, etc. Examples thereof include transdermal administration, suppositories, and parenteral administration such as administration by a forced enteral nutrition method using a nasal gastrointestinal tract, a nasal intestinal tract, a gastric fistula tube, or an intestinal fistula tube.

(Dosage form of therapeutic and / or preventive agent of the present invention)
The dosage form of the therapeutic and / or preventive agent for schistosomiasis of the present invention can be appropriately determined according to the above-mentioned administration route, but injections, infusions, tablets, capsules, fine granules, powders, etc. Examples thereof include liquid preparations, liquid preparations dissolved in syrup and the like, poultices, suppositories and the like.

Pharmacologically acceptable carriers, excipients, diluents, additives, disintegrants, binders, coatings, as needed, to prepare therapeutic and / or prophylactic agents for schistosomiasis of the invention. Agents, lubricants, gliding agents, lubricants, flavoring agents, sweeteners, solubilizers, solvents, gelling agents, nutrients, etc. can be added, and those skilled in the art can add each specific ingredient according to the purpose. Can be selected.

(Dose of therapeutic and / or prophylactic agent of the present invention)
The amount, frequency, and duration of administration of the treatment and / or preventive agent for schistosomiasis of the present invention vary depending on the age, weight, symptoms, etc. of the schistosomiasis patient and the person who intends to prevent schistosomiasis, but ALA The dose of the class is 0.01 mmol to 25 mmol / day, preferably 0.025 mmol to 15 mmol / day, more preferably 0.25 mmol to 10 mmol / day, still more preferably 0.25 mmol / day, per adult in terms of 5-ALA mol. It can be 0.75 mmol to 7.5 mmol / day, and it is desirable to continuously ingest a low dose, especially when used as a prophylactic agent. The frequency of administration may be exemplified by administration once to a plurality of times a day or continuous administration by infusion or the like. The duration of administration as a therapeutic or prophylactic agent can be determined by a method known to pharmacologists and clinicians in the art based on indicators of the status of schistosomiasis.

(Applicable Symptoms of Therapeutic and / or Preventive Agents of the Present Invention)
The therapeutic and / or prophylactic agent for schistosomiasis of the present invention can be applied to the treatment and prevention of various symptoms caused by the infestation of schistosomiasis. Such symptoms include abdominal pain, diarrhea, bloody stools, cirrhosis, ascites, portal hypertension, splenoma, dermatitis, urticaria, hepatic splenoma, cold, fever, stomach pain, diarrhea, liver inflammation, brain inflammation, Examples include, but are not limited to, liver fibrosis, cirrhosis, brain tumor-like symptoms due to ascites embolization of intracerebral blood vessels, spinal cord compression due to spinal cord lesions, and horse tail syndrome.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

（６）寄生虫成虫ペアの準備：
寄生虫に感染したオンコメラニア属貝からセルカリアを回収した。マウスの尻尾を、セルカリア３０隻を入れた水（試験管）に浸漬して、寄生虫に経皮感染させた。感染後、マウスを４−５頭ずつケージに入れて実験動物施設にて飼育した。感染６週（３８日）目に１２頭のマウスを解剖した。腸間膜静脈を生理食塩水で灌流して、日本住血吸虫の成虫ペアを合計５０ペア回収した。
（７）寄生虫成虫ペアの培養（薬剤への暴露）実験
（７−１）成虫ペア５０ペアを、培養液１ｍｌの入った ２４−ｗｅｌｌ Ｐｌａｔｅ（Ｃｏｒｎｉｎｇ Ｃｅｌｌ Ｃｕｌｔｕｒｅ Ｐｌａｔｅ ２４ ｗｅｌｌ：３５２６）の各ウエルに１ペアずつ入れて、５％ＣＯ_２培養器（３６．５℃）で２４時間予備培養した。
（７−２）翌日に成虫ペアを観察して、まだペアを形成していて産卵の程度が同じものを、３２ペア選択して実験に供した。
（７−３）図１のように調整した２４−ｗｅｌｌ Ｐｌａｔｅの各ウエルに成虫ペアを１ペアずつ入れて薬剤に暴露した。各ウエルに添加した培養液の量は１ｍｌとした。図中の番号は上記「（５）実験区」の処置群の番号に対応する。薬剤への暴露は２４時間として、その後は、虫体を新鮮培地入りのウエルに移して培養した。培養液は２４時間毎に交換した。
（７−４）薬剤への暴露および培地の交換は、各ペアを、別プレートに準備した薬剤入りウエルあるいは新鮮培地入りのウエルに、ピンセットを用いて移動して行った。
（７−５）薬剤への暴露後２４、４８および７２時間後に実体顕微鏡で各ウエルを観察して、成虫の活性、成虫ペアの状況、および産卵の状況を評価した。
（８）評価基準：
（８−１）成虫の活性は０−３のスケールで評価した（Ｐａｒａｓｉｔ Ｖｅｃｔｏｒｓ，２０１９，Ｖｏｌ．１２，ａｒｔｉｃｌｅ ｎｏ．１９９（ｄｏｉ：１０．１１８６／ｓ１３０７１−０１９−３４４２−７）参照）。
０：死滅虫体
１：部分的な運動を呈する白濁した虫体
２：全身運動を呈するが動きが鈍くまた遅い透明な虫体
３：活発な全身運動を呈する透明な虫体
（８−２）成虫ペアの状況は０−１のスケールで評価した。
０：雌雄が分離した虫体
１：雌雄ペア虫体
（８−３）産卵の状況は０−２のスケールで評価した。
０：産卵なし
１：わずかな産卵（虫卵数 ２０以下）
２：産卵あり（虫卵数 ２０より多い） Parasite activity inhibition test of PPIX and Hemin (1) Experimental animals:
Twenty-one mice (ICR male: 4 weeks old 17-20 g) were used in the experiment.
(2) Parasites:
Schistosoma japonicum S. Japanicum (Yamanashi stream) was used.
(3) Culture solution:
A culture solution was prepared by adding a mixture of Penicillin and Streptomycin (Sigma P4333) to NCTC135 (Sigma N3264) medium so that the final concentrations were 50 U / ml and 50 μg / ml, respectively (Tropical Medicine and Health, 2014). , Vol. 42: pp. 87-92)
(4) Reagent:
Dimethyl sulfoxide (Culture Sure ^R DMSO: WAKO 031-24051), derived from Hemin pig (Hemin from Porcine: FUJIFILM 089-10321) Protoporphyrin IX (Protoporphyrin 9: chem Praziquantel) BAYER) was used.
(5) Experimental area:
The following eight experimental plots were set up.

(6) Preparation of adult parasite pair:
Cercaria was recovered from the parasite-infected Oncomelania mussel. The tail of the mouse was immersed in water (test tube) containing 30 cercariae to transdermally infect the parasite. After infection, 4-5 mice were placed in cages and bred in a laboratory animal facility. Twelve mice were dissected 6 weeks (38 days) after infection. The mesenteric vein was perfused with physiological saline, and a total of 50 adult pairs of Schistosoma japonicum were collected.
(7) Culture (exposure to drug) experiment of adult parasite pair (7-1) Each well of 24-well Plate (Corning Cell Culture Plate 24 well: 3526) containing 1 ml of culture solution containing 50 pairs of adult worms. The cells were _{pre-cultured in a 5% CO 2} incubator (36.5 ° C.) for 24 hours.
(7-2) The next day, the adult pairs were observed, and 32 pairs that were still paired and had the same degree of spawning were selected and subjected to the experiment.
(7-3) One adult pair was placed in each well of the 24-well plate prepared as shown in FIG. 1 and exposed to the drug. The amount of the culture solution added to each well was 1 ml. The numbers in the figure correspond to the numbers of the treatment groups in the above "(5) Experimental group". Exposure to the drug was allowed for 24 hours, after which the insect bodies were transferred to wells containing fresh medium for culturing. The culture medium was changed every 24 hours.
(7-4) Exposure to the drug and exchange of the medium were carried out by moving each pair to a well containing the drug or a well containing fresh medium prepared on a separate plate using tweezers.
(7-5) 24, 48 and 72 hours after exposure to the drug, each well was observed with a stereomicroscope to evaluate adult activity, adult pair status, and spawning status.
(8) Evaluation criteria:
(8-1) Adult activity was evaluated on a scale of 0-3 (see Parasit Vectors, 2019, Vol. 12, article no. 199 (doi: 10.1186 / s13071-019-3442-7)).
0: Dead insect body 1: Cloudy insect body exhibiting partial movement 2: Transparent insect body exhibiting whole body movement but slow and slow movement 3: Transparent insect body exhibiting active whole body movement (8-2) The status of adult pairs was evaluated on a 0-1 scale.
0: Insects separated by males and females 1: Male-female pair insects (8-3) The spawning status was evaluated on a scale of 0-2.
0: No spawning 1: Slight spawning (20 or less insect eggs)
2: With spawning (more than 20 insects)

(Effect on adult activity)
As shown in FIG. 2, a strong inhibitory effect on adult activity was obtained in the hemin (Hemin) 50 μM group and the hemin (Hemin) 500 μM group. In addition, although the effect was slightly inferior, the effect of suppressing adult activity was similarly obtained in the protoporphyrin IX (PPIX) 50 μM group and the protoporphyrin IX (PPIX) 500 μM group.

(Effect on adult pairing)
As shown in FIG. 3, DMSO used as a solvent showed an effect of inhibiting adult pairing at 5%, but did not show an inhibitory effect at 0.5%. On the other hand, in the hemin (Hemin) 50 μM group to which a drug containing hemin (Hemin) 50 μM in 0.5% DMSO was applied, a strong adult pairing inhibitory effect was obtained. Further, in the hemin (Hemin) 500 μM group to which a drug containing hemin (Hemin) 500 μM in 5% DMSO was applied, more strong adult pairing was performed as compared with the solvent control 5% group in which only 5% DMSO was provided. An inhibitory effect was obtained. The effect of suppressing adult pairing by the hemin was stronger than the effect of 100 μMPQZ.

(Effect on spawning)
As shown in FIG. 4, a strong effect of suppressing spawning was obtained in the hemin 500 μM group. In addition, although the effect was slightly inferior, the same effect of suppressing egg laying was obtained in the protoporphyrin IX (PPIX) 500 μM group.

As shown above, the treatment and / or preventive agent for schistosomiasis containing 5-aminolevulinic acid (ALA) or a derivative thereof or a salt thereof of the present invention and the method for treating and / or preventing schistosomiasis using the same are By strongly suppressing the spawning of schistosomiasis, various symptoms of schistosomiasis can be treated and / or prevented.

Claims (7)

(1) The following formula (I)

(In the formula, R ¹ represents a hydrogen atom or an acyl group, and R ² represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group.)
A therapeutic and / or preventive agent for schistosomiasis containing the compound indicated by or a salt thereof. The therapeutic and / or preventive agent for schistosomiasis according to claim 1, wherein R ¹ and R ^{2 are hydrogen atoms.} The therapeutic and / or preventive agent for schistosomiasis according to claim 1 or 2, further comprising one or more metal-containing compounds. The treatment and / or preventive agent for schistosomiasis according to claim 3, wherein the metal-containing compound is a compound containing iron, magnesium, zinc, nickel, vanadium, copper, chromium, molybdenum or cobalt. The therapeutic and / or preventive agent for schistosomiasis according to claim 3, wherein the metal-containing compound is a compound containing iron, magnesium or zinc. The therapeutic and / or preventive agent for schistosomiasis according to claim 3, wherein the metal-containing compound is an iron-containing compound. The treatment and / or preventive agent for schistosomiasis according to any one of claims 1 to 6, which is used in combination with another treatment or preventive agent for schistosomiasis.

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