JP2008261849A - Screening method and evaluation method for antipruritic substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method easily performable in a short time, and detecting an antipruritic substance with excellent reproducibility; and a method for evaluating the itching soothing effect of the antipruritic substance. <P>SOLUTION: (1) This screening method for detecting the antipruritic substance or a method for evaluating the itching soothing effect is characterized by applying an itching scratching causing substance to an experiment animal for causing itching scratching behavior. (2) The screening method for detecting the antipruritic substance or the method for evaluating the itching soothing effect of the antipruritic substance includes the following processes : (a) a process of applying the itching scratching causing substance to the experiment animal, (b) a process of prescribing an examining substance to the experiment animal, and (c) a process of investigating how the scratching behavior caused by applying the itching scratching causing substance to the experiment animal changes with the existence of prescriptions of the examining substance. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a screening method for finding an antipruritic substance and an evaluation method for the antipruritic effect of the antipruritic substance.

  Conventionally, it has been reported that an antipruritic effect is evaluated using as an index the scratching behavior induced by subcutaneously administering compound 48/80, a pruritus-inducing substance, to mice (for example, Non-Patent Documents 1 and 2). reference.). However, the number of scratches by this method is about 60 to 90 times in 30 minutes, and since the variation is large, it is difficult to evaluate the antipruritic effect. Moreover, although subcutaneous administration used in this method is an embodiment of systemic administration, itching occurs on the skin surface, and it is difficult to say that it is optimal as a method for evaluating the antipruritic effect. Furthermore, since the administration site needs to be shaved before subcutaneous administration, the experimental operation becomes complicated.

  Although the administration method etc. is slightly different from the above, it is reported that the antipruritic effect of an external preparation is evaluated using the scratching behavior induced by intradermal administration of histamine and compound 48/80, which are pruritus, to mice. (For example, refer to Patent Document 1). However, the number of scratches by this method is about 70 to 110 times (histamine) or about 80 to 130 times (compound 48/80) in 30 minutes, and since the variation is large, it is difficult to evaluate the antipruritic effect. In this method, the animal to be used must be shaved under ether anesthesia on the previous day, and then the pruritus-inducing substance must be administered intradermally. Therefore, the experiment operation becomes complicated.

In addition, it has been reported that the antipruritic effect of an external preparation is evaluated using as an index the scratching behavior induced by intradermal administration of serotonin, a pruritus-inducing substance, to mice (see, for example, Patent Document 2). However, the number of scratches by this method is about 110 to 170 times in 30 minutes, and since the variation is also large, it is difficult to evaluate the antipruritic effect. In this method, the test substance is subjected to a 24-hour closed patch, and then the scratching behavior caused by intradermal injection of serotonin is examined, which cannot be performed in a short time. Even high technology is required. Furthermore, before the intradermal administration, the administration site needs to be shaved, and the experimental operation becomes complicated.
JP 2001-324495 A JP-A-8-217677 Kuraishi Y, et al, Eur. J. et al. Pharmacol. , 275, 229-233 (1995) Ishiguro K, et al, Journal of Natural Products, 61 (9), 1126-1129 (1998)

  An object of the present invention is to provide a screening method for finding an antipruritic substance that is simple, can be performed in a short time, and has good reproducibility, and a method for evaluating the antipruritic effect of the antipruritic substance.

  The present inventor has found that scratching behavior is induced only by applying a pruritus-inducing substance to a laboratory animal, and as a result of further research based on such knowledge, has found that the above object can be solved. Completed the invention.

Examples of the present invention include the following.
(1) A screening method for finding an antipruritic substance characterized by applying a pruritus-inducing substance to a laboratory animal to induce scratching behavior. (2) Initiated by applying a pruritus-inducing substance to a laboratory animal. (3) The screening method of (1) above, which comprises examining how the scratching behavior changes depending on the presence or absence of administration of the test substance. A screening method for finding an antipruritic substance, comprising the steps of (1) the screening method (4), which comprises measuring the number of times the vicinity is stroked using the forelimbs or hindlimbs (the number of scratches). (A) Itching Applying an inducer to a laboratory animal,
(B) a step of administering a test substance to an experimental animal; and (c) examining how the scratching behavior induced by applying the pruritus-inducing substance to the experimental animal changes depending on whether or not the test substance is administered. Step (5) The screening method of (4) above, comprising the step of measuring the number of times that the experimental animal in which the scratching behavior has been induced strokes the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or hindlimbs (number of scratches) ( 6) A method for evaluating the antipruritic effect of an antipruritic substance, characterized by applying an pruritus-inducing substance to an experimental animal in order to induce scratching behavior. (7) Initiated by applying an pruritus-inducing substance to an experimental animal. (8) The evaluation method of (6) above, which comprises examining how the scratching behavior changes depending on whether or not the antipruritic substance is administered. near A method for evaluating the antipruritic effect of an antipruritic substance, comprising the following steps: (9) comprising measuring the number of strokes using the forelimbs or hindlimbs (the number of scratches). Applying a pruritus-inducing substance to a laboratory animal
(B) a step of administering an antipruritic substance to an experimental animal, and (c) investigating how the scratching behavior induced by applying the pruritus-inducing substance to the experimental animal changes with or without administration of the antipruritic substance. Step (10) The evaluation method according to (9) above, comprising the step of measuring the number of times (the number of scratches) that the experimental animal in which the scratching behavior has been induced strokes the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or hindlimbs

  The present invention is described in detail below.

  In the present invention, “scratching behavior” refers to behavior in which an experimental animal scratches the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or hind limbs. The degree of such scratching behavior can be determined, for example, by measuring the number of times the experimental animal strokes using the forelimbs or hind limbs (the number of scratches), measuring the range (area) the experimental animal scratches using the forelimbs or hind limbs, or It can be evaluated by measuring the total time of scratching. Among these evaluation methods, it is preferable to measure the number of strokes (the number of scratches) using the forelimbs or hindlimbs to evaluate the degree of scratching behavior.

  In the present invention, the “pruritus-inducing substance” refers to a substance that can cause itching, and examples thereof include serotonin or a salt thereof, histamine, and compound 40/80. The pruritus-inducing substance that can be used in the present invention is not limited to these, but serotonin or a salt thereof (for example, serotonin hydrochloride) is particularly preferable.

  The experimental animal that can be used in the present invention is not particularly limited as long as it can carry out the present invention. Specific examples include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs and monkeys. Of these, mice, rats and guinea pigs are preferred, and mice that are small, easy to handle and inexpensive are more preferred. Among the mice, ddY mice, C57BL / 6J mice, and ICR mice are particularly preferable.

  The “applying site” of the pruritus-inducing substance is not particularly limited, but when measuring the number of strokes (number of scratches), the site where the forelimb or hindlimb of the experimental animal reaches and the head and neck of the experimental animal cannot reach Is preferred, and the back of the neck is more preferred.

The method for “applying a pruritus-inducing substance to a laboratory animal” is not particularly limited as long as it can be effectively applied. For example, a method of directly applying a pruritus-inducing substance as it is, a method of dissolving pruritus-inducing substance in an appropriate solvent, or Examples thereof include a method of applying (dropping) a suspended liquid using an appropriate instrument (eg, pipette) and a method of applying a lotion or ointment containing a pruritus-inducing substance. Among these application methods, a method of applying (dropping) a solution obtained by dissolving or suspending a pruritus-inducing substance in an appropriate solvent using an appropriate instrument (eg, pipette) is preferable.
The solvent for dissolving or suspending the pruritus-inducing substance is not particularly limited, but it is preferably a solvent that dissolves the pruritus-inducing substance and has low irritation to the skin, such as ethanol or water (purified water, distilled water). Water, physiological saline, tap water, etc.).

  In the present invention, the “antipruritic substance” is, for example, atopic dermatitis, urticaria, psoriasis, ringworm, psoriasis, vulgaris vulgaris, local skin pruritus caused by insect excretion / secretory, nodular urticaria , Renal dialysis, diabetes, blood disease, liver disease, kidney disease, endocrine / metabolic disorder, visceral malignancy, hyperthyroidism, autoimmune disease, multiple sclerosis, neurological disease, psychoneuropathy, allergic conjunctivitis, spring It refers to a substance that can suppress itchiness caused by catarrhal, atopic keratoconjunctivitis or excessive use of luxury goods / drugs.

The method of “administering a test substance to an experimental animal” and the method of “administering an antipruritic substance to an experimental animal” are not particularly limited as long as the test substance or the antipruritic substance can be administered to the experimental animal. Examples thereof include transdermal administration, oral administration, intravenous administration, subcutaneous administration, intraperitoneal administration, and intradermal administration. Specifically, a method of instilling a solution obtained by dissolving or suspending a test substance or an antipruritic substance in an appropriate solvent, a method of applying the test substance or an antipruritic substance as it is, an agent for causing pruritus dissolved or suspended in an appropriate solvent Applying (dropping) the liquid using an appropriate device (eg, pipette), applying a lotion or ointment containing the test substance or antipruritic substance, or containing the test substance or antipruritic substance as it is Examples thereof include a method of giving a drug from the mouth and a method of injecting a solution obtained by dissolving or suspending a test substance or an antipruritic substance in an appropriate solvent into a vein, intraperitoneally, subcutaneously or intradermally. Among these administration methods, transdermal administration and oral administration are preferred, and among transdermal administration, the application method is preferred. In the case of the method by application, it is preferable to apply by the same method as the method of applying the pruritus-inducing substance.
The solvent for dissolving or suspending the test substance or the antipruritic substance is not particularly limited, but the same solvent as that for dissolving or suspending the pruritus-inducing substance is preferable.

The screening method or evaluation method according to the present invention is performed using as an index the scratching behavior caused by applying a pruritus-inducing substance to an experimental animal. Specifically, for example, a screening method or an evaluation method including the following steps (a) to (d) can be given.
(A) a step of applying an ethanol solution containing serotonin or a salt thereof to the back of the neck of a mouse;
(B) a step of applying an ethanol solution containing a test substance to the application site or a step of applying ethanol not containing a test substance in the case of control;
(C) a step of measuring the number of times that the mouse strokes the vicinity of the application site of an ethanol solution containing serotonin or a salt thereof using the forelimb or hindlimb (the number of scratches); and (d) an ethanol solution containing a test substance or an antipruritic substance. Comparing the number of scratches of a mouse coated with ethanol and the number of scratches of a mouse coated with ethanol containing neither a test substance nor an antipruritic substance

  The amount of the pruritus-inducing substance applied varies depending on the type of pruritus-inducing substance used, the type and age of the experimental animal, the dose or type of test substance or antipruritic substance, etc., preferably 1 μL / animal to 1000 μL / animal, 10 μL / More preferably, the animal is 300 μL / animal. More preferably, it is 100 μL / animal.

  The dose of the test substance or antipruritic substance varies depending on the degree of the antipruritic action exhibited by the test substance or antipruritic substance used and its administration method, the type and age of the experimental animal, the type and amount of the pruritus-inducing substance, etc. When the test substance or antipruritic substance is dissolved in ethanol and administered by application, 1 μL / animal to 1000 μL / animal is preferable, and 10 μL / animal to 300 μL / animal is more preferable. More preferably, it is 100 μL / animal. Moreover, when orally administering a test substance or an antipruritic substance, 0.01 mg / kg to 100 mg / kg is preferable, and 0.3 mg / kg to 30 mg / kg is more preferable. More preferably, it is 10 mg / kg.

  The order of administration and time difference between the application of pruritus-inducing substance and the administration of test substance or antipruritic substance are the kind of pruritus-inducing substance, application amount and application method, type of test substance or antipruritic substance, dosage and administration method, and Depending on the type, for example, when administering a test substance or antipruritic substance by application, apply the test substance or antipruritic substance immediately after applying the pruritus-inducing substance, or conversely apply the test substance or antipruritic substance. It is preferable to apply a pruritus-inducing substance immediately thereafter. In addition, when a test substance or an antipruritic substance is orally administered, it is preferable to orally administer the test substance or an antipruritic substance 10 minutes to 1 hour before applying the pruritus-inducing substance, and 20 to 40 minutes before the test substance. Or it is more preferable to administer an antipruritic substance orally.

    When measuring the number of scratches, the time to measure the scratching behavior caused by applying a pruritus-inducing substance to a laboratory animal starts observation immediately after application of the pruritus-inducing substance or administration of a test substance or antipruritic substance. Then, it is preferably measured continuously for 5 minutes to 60 minutes, more preferably measured for 10 minutes to 30 minutes.

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

Test example 1
A pruritus-inducing substance dissolved in physiological saline was applied (100 μL), subcutaneously (100 μL) or intradermally (50 μL) on the back of the neck of 4-6 week old male ICR mice (Japan SLC Co., Ltd.) The number of scratches in the vicinity of the back of the neck caused by the case (the number of strokes of the forelimb or hindlimb) was measured for 30 minutes immediately after application of the pruritus-inducing substance. As pruritus-inducing substance, serotonin hydrochloride (1% by weight, manufactured by Nacalai Tesque, hereinafter referred to as serotonin) is used in the case of administration by application, and compound 48/80 (manufactured by Nacalai Tesque, 5 μg) is used in the case of subcutaneous administration. In the case of intradermal administration, compound 48/80 (20 μg), serotonin (100 nmol), or histamine (100 nmol) was used. The result is shown in FIG.
As shown in FIG. 1, the number of scratches in the serotonin application group was the largest, with an average of 245.8 times (standard error 18.4). The number of scratches by other methods was 76.7 to 139.0 on average, which was less than that of the serotonin application group, and the standard error representing the variation of the test system was as large as 21.0 to 27.1.
From these results, the scratching behavior induced by serotonin application is superior to other methods in terms of the number of scratching behaviors and small standard error, and is the best test system for evaluating the inhibitory effect of scratching behavior. It is clear that

Test example 2
Immediately after 100 μL of 0.1 wt% serotonin hydrochloride dissolved in physiological saline was applied to the back of the neck of 4-6 week old male ICR mice, compound A / 4-[((1S, 2R ) -2-{[Amino (imino) methyl] amino} cyclohexyl) amino] -N-isobutyl-6-methylquinazoline-2-carboxamide dihydrochloride, 100 μL of its ethanol solution (0.01 wt. 1% by weight) was applied to the back of the neck. Immediately after the application of Compound A, the number of times that the mouse strokes the back of the neck using the forelimbs or hindlimbs (the number of scratches) was measured for 15 minutes. A group of mice coated with 100 μl of ethanol not containing Compound A was used as a control. The result is shown in FIG.
As shown in FIG. 2, the average number of scratches in the control group was 222.3 (standard error 35.0), but when the antipruritic substance was applied, the average was 135.3 when 0.01% by weight was applied. In the case of 0.1% by weight (standard error 20.8), an average of 82.0 times (standard error 12.7) was suppressed in a dose-dependent manner.
“*” In FIG. 2 means that the significant difference level from the control group by Dunnett test is P <0.05, and “**” means that the significant difference level is P <0.01. means.

Test example 3
Naltrexone 30 mg / kg, an opioid antagonist (antipruritic substance), was orally administered to 4-6 week old male ICR mice. Thirty minutes later, 100 μL of 1 wt% serotonin hydrochloride dissolved in physiological saline was applied to the back of the neck of the mouse, and the number of scratches (number of strokes of the forelimb or hindlimb) that was induced was applied to the mouse. Measurement was performed for 15 minutes immediately after. In addition, the mouse | mouth group which orally administered the distilled water which does not contain naltrexone was set as the control. The result is shown in FIG.
As shown in FIG. 3, the average number of scratches in the control group was 222.8 (standard error 48.5), but when naltrexone was administered, the average was 145.7 times (standard error 21.0). Suppression was observed.
As a result of the t test, the level of significant difference from the control group was P = 0.094.
Naltrexone suppresses itching in the clinic (Lancet, 1996, 348 (9041), 1552-1554, Gastroenterology, 1997, 113 (4), 1264-1269, J. Am. Acad. Dermatol., 1999, 41 (4 ), 533-539) and the activity of suppressing itchiness associated with atopic dermatitis (Exp. Dermatol., 2002, 11 (5), 448-455) have been reported.

Test example 4
4-6 week old male ICR mice were orally administered with TRK-820 (nalflavin), an opioid κ receptor agonist (antipruritic substance). Thirty minutes later, 100 μL of 0.1% by weight serotonin hydrochloride dissolved in physiological saline was applied to the back of the neck of the mouse, and the number of scratches in the vicinity of the back of the neck caused (the number of strokes of the forelimb or hindlimb) was calculated. Measurements were taken for 15 minutes immediately after serotonin application. In addition, the mouse | mouth group which orally administered the distilled water which does not contain TRK-820 was set as the control. The result is shown in FIG.
As shown in FIG. 4, the average number of scratches in the control group was 144.0 (standard error 19.9), but when TRK-820 was administered at 30 μg / kg, the average was 118.3 (standard error 27). .8), an average of 51.8 times (standard error 8.4) when administered at 100 μg / kg, and an average of 7.8 times (standard error of 7.0) when administered at 300 μg / kg. Suppression of scratching behavior was observed.
TRK-820 is a compound currently under development as an anti-pruritic agent and has been reported to suppress itching in renal dialysis in clinical trials (Eur. J. Pharmacol., 2002, 453, 259-264, J. Biol. Am. Soc. Nephrol., 2005, 16, 3742-3747).
“**” in FIG. 4 means that the level of significant difference from the control group by Dunnett test is P <0.01.

  As described above, according to the present invention, frequent scratching behavior is induced in a short time, and there is little variation in the total time for performing the number of scratches. Further, according to the present invention, the scratching behavior is induced by applying a pruritus-inducing substance, but there is no need for shaving of the administration site required in the conventional method of subcutaneously or intradermally administering the pruritus-inducing substance, Experimental operation is easy. Further, since it is not necessary to perform injection, the antipruritic effect can be accurately evaluated without stimulation by the injection needle.

  According to the present invention, antipruritic substances can be screened easily and accurately compared to conventional methods, and the antipruritic effect of antipruritic substances can be evaluated.

FIG. 1 shows the number of scratches when the pruritus-inducing substance is administered by different methods. The vertical axis represents the number of scratches (average value ± standard error) for 30 minutes. The black column shows the results of applying serotonin, and the white column shows the results of subcutaneous administration of compound 48/80, intradermal administration of compound 48/80, intradermal administration of setronin, and intradermal administration of histamine in order from the left. FIG. 2 shows the number of scratches when Compound A is applied. The vertical axis represents the number of scratches (average value ± standard error) for 15 minutes. The white column shows the result of applying (control) the solvent, and the black column shows the result of applying Compound A. FIG. 3 shows the number of scratches when naltrexone was orally administered. The vertical axis represents the number of scratches (average value ± standard error) for 15 minutes. The white column shows the result of oral administration (control) of the solvent, and the black column shows the result of oral administration of naltrexone. FIG. 4 shows the number of scratches when TRK-820 was orally administered. The vertical axis represents the number of scratches (average value ± standard error) for 15 minutes. The white column shows the result of oral administration (control) of the solvent, and the black column shows the result of oral administration of TRK-820.

Claims (20)

A screening method for finding an antipruritic substance, which comprises applying a pruritus-inducing substance to an experimental animal in order to induce scratching behavior. The screening method according to claim 1, comprising examining how the scratching behavior induced by applying the pruritus-inducing substance to a laboratory animal changes depending on the presence or absence of administration of the test substance. The screening method according to claim 1, comprising measuring the number of times (the number of scratches) that the experimental animal in which the scratching behavior has been induced strokes the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or hindlimbs. A screening method for finding an antipruritic substance, comprising the following steps.
(A) applying a pruritus-inducing substance to a laboratory animal,
(B) a step of administering a test substance to an experimental animal, and (c) examining how the scratching behavior induced by applying the pruritus-inducing substance to the experimental animal changes depending on whether or not the test substance is administered. Process. The screening method according to claim 4, which comprises the step of measuring the number of times (the number of scratches) that the experimental animal in which scratching behavior has been induced strokes the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or hindlimbs. The screening method according to claim 1, wherein the pruritus-inducing substance is serotonin or a salt thereof. The screening method according to any one of claims 1 to 5, wherein the experimental animal is a mouse, a rat or a guinea pig. The screening method according to claim 7, wherein the mouse is an ICR mouse. The screening method according to any one of claims 1 to 5, wherein the site of applying the pruritus-inducing substance is the back of the neck of a laboratory animal. The screening method in any one of Claims 1-5 or 9 including the following processes.
(A) a step of applying an ethanol solution containing serotonin or a salt thereof to the back of the neck of a mouse;
(B) a step of applying an ethanol solution containing a test substance to the application site or a step of applying ethanol not containing a test substance in the case of control;
(C) a step of measuring the number of times that the mouse strokes the vicinity of the application site of the ethanol solution containing serotonin or a salt thereof using the forelimbs or hind limbs (the number of scratches); and (d) an ethanol solution containing the test substance is applied. A step of comparing the number of scratches of a mouse with the number of scratches of a mouse to which ethanol containing no test substance is applied. A method for evaluating the antipruritic effect of an antipruritic substance, which comprises applying a pruritus-inducing substance to an experimental animal in order to induce scratching behavior. The evaluation method according to claim 11, comprising examining how the scratching behavior caused by applying the pruritus-inducing substance to a test animal changes depending on whether or not the antipruritic substance is administered. The evaluation method according to claim 11, comprising measuring the number of times (the number of scratches) that the experimental animal in which the scratching behavior is induced strokes the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or the hindlimbs. A method for evaluating the antipruritic effect of an antipruritic substance, comprising the following steps.
(A) applying a pruritus-inducing substance to a laboratory animal,
(B) A step of administering an antipruritic substance to an experimental animal, and (c) Examining how the scratching behavior caused by applying the pruritus-inducing substance to the experimental animal changes with or without administration of the antipruritic substance. Process The evaluation method according to claim 14, comprising the step of measuring the number of times (the number of scratches) that the experimental animal in which the scratching behavior has been induced strokes the vicinity of the site where the pruritus-inducing substance is applied using the forelimbs or hindlimbs. The evaluation method according to any one of claims 11 to 15, wherein the pruritus-inducing substance is serotonin or a salt thereof. The evaluation method according to claim 11, wherein the experimental animal is a mouse, a rat, or a guinea pig. The evaluation method according to claim 17, wherein the mouse is an ICR mouse. The evaluation method according to any one of claims 11 to 15, wherein the application site of the pruritus-inducing substance is the back of the neck of a laboratory animal. The evaluation method according to claim 11, which includes the following steps.
(A) a step of applying an ethanol solution containing serotonin or a salt thereof to the back of the neck of a mouse;
(B) a step of applying an ethanol solution containing a test substance to the application site or a step of applying ethanol not containing a test substance in the case of control;
(C) a step of measuring the number of times that the mouse strokes using the forelimbs or hind limbs of the vicinity of the application site of the ethanol solution containing serotonin or a salt thereof (the number of scratches); and (d) an ethanol solution containing an antipruritic substance was applied. A step of comparing the number of scratches of a mouse with the number of scratches of a mouse to which ethanol containing no antipruritic substance is applied.

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