Patent Application
20210401821
The present invention relates to a prophylactic or therapeutic agent and a medicinal composition for an IL-31-mediated disease.
Priority is claimed on Japanese Patent Application No. 2018-215017, filed on Nov. 15, 2018 and Japanese Patent Application No. 2019-144913, filed on Aug. 6, 2019, the contents of which are incorporated herein by references.
Interleukin-31 (IL-31) is a cytokine expressed in various human tissues, and it has been reported that interleukin-31 is associated with a chronically pruritic skin disorder such as atopic dermatitis (for example, refer to Non Patent Documents 1 and 2).
Atopic dermatitis is a chronic disease with itchy eczema that worsens and relieves repeatedly. Many patients have an enhanced response to allergens. In recent years, the number of patients with atopic dermatitis has been increasing, and since atopic dermatitis significantly impairs the quality of life of patients, there is an urgent need to develop a therapeutic method for atopic dermatitis.
Furthermore, intradermal administration of IL-31 in mice induces scratching behavior. The scratching behavior in mice can be used as a model of scratching behavior induced by itching in atopic dermatitis.
The severity of atopic dermatitis is known to correlate with serum IL-31 levels. It was also reported that administration of an anti-IL-31 receptor antibody alleviates pruritus in patients with atopic dermatitis. Front these findings, it is considered that the causative substance that induces itching in atopic dermatitis is IL-31.
It has also been reported that IL-31 mediates the pathophysiology of various diseases by inducing the expression of other inflammatory cytokines in addition to the induction of pruritus. IL-31-mediated diseases such as atopic dermatitis, pruritus in atopic dermatitis, dermatomyositis, pruritus in dermatomyositis, chronic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, pruritus in cutaneous T cell lymphoma, prurigo nodularis, prurigo chronica multiformis, urticaria pigmentosa, and bullous pemphigoid have been reported (for example, refer to Non Patent Documents 1 to 8). Dermatomyositis is an autoimmune inflammatory myopathy with a rash, in which the severity of a skin symptom correlates with itching, and it has been reported that the expression of IL-31 and IL-31 receptors is enhanced in lesioned skin with itching (Non Patent Document 3). Cutaneous T cell lymphoma (CTCL) is a group of malignant lymphomas that occur in the skin, and is a disease in which cells from which tumors are derived are T cells. Regarding CTCL, it has been reported that disease progression and severity of pruritus correlate with IL-31 and IL-31 receptor expression levels in lesioned skin and serum IL-31 levels (Non Patent Document 4). Regarding prurigo nodularis and allergic contact dermatitis it has been reported that the enhanced expression of IL-31 is observed in skin tissues (Non Patent Documents 5 and 6), and regarding urticaria pigmentosa, it has been reported that serum IL-31 levels correlate with the severity of disease and pruritus (Non Patent Document 7). Furthermore, it has been reported that IL-31 levels in serum and tissues were increased in patients with dermatitis herpetiformis or bullous pemphigoid, and many IL-31-positive cells in the skin tissues at lesion sites of these patients were observed (Non Patent Document 8).
The inventors have previously clarified that the production of IL-31 from CD4+ T cells was enhanced in mice deficient for the Dedicator of cytokinesis 8 (Dock8) gene and transgenic for a rearranged T cell receptor (TCR) gene (for example, refer to Non Patent Document 9).
Here, AND can be used as the rearranged TCR. The AND is the TCR that recognizes a peptide (SEQ ID NO: 1) consisting of 88th to 103rd amino acids of moth cytochrome c (MCC), which forms a complex with an MHC class II I-Eκ molecule. On the other hand, AND-expressed T cells normally differentiate and mature in the thymus of mice that express the 1-Ab molecule, but AND TCR shows relatively high autoreactivity to the I-Ab molecule.
Regarding the genetic background of C57BL/6 mice expressing the I-Ab molecule, Dock8 knockout and AND transgenic (Dock8−/− AND Tg) mice show enhanced scratching behaviors and symptoms of dermatitis. Thus, Dock 8−/− AND Tg mice can be used as a non-human animal model of IL-31-mediated disease such as atopic dermatitis, such as for atopic dermatitis.
As described above, IL-31 is considered to be a typical factor that induces itching in inflammatory diseases such a atopic dermatitis. However, the mechanism by which IL-31 induces itching has not been fully elucidated. Therefore, an object of the present invention is to clarify a molecular mechanism by which IL-31 induces itching, and to provide a prophylactic or therapeutic technique for an IL-31-mediated disease such as atopic dermatitis.
The present invention includes the following aspects.
[1] A prophylactic or therapeutic agent for an IL-31-mediated disease comprising a neurokinin B signal blocker.
[2] The prophylactic or therapeutic agent according to [1], in which the IL-31-mediated disease includes topic dermatitis, pruritus in topic dermatitis, dermatomyositis, pruritus in dermatomyositis, chronic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, pruritus in cutaneous T cell lymphoma, prurigo nodularis, prurigo chronica multiformis, urticaria pigmentosa, or bullous pemphigoid.
[3] The prophylactic or therapeutic agent according to [1], in which the IL-31-mediated disease is atopic dermatitis.
[4] The prophylactic or therapeutic agent according to any one of [1] to [3], in which the neurokinin B signal blocker is a neurokinin 3 receptor antagonist.
[5] The prophylactic or therapeutic agent according to [4], in which the antagonist is a compound represented by General Formula (1), (2), or (3) or a salt thereof, or a solvate thereof,
[in Formula (1),
Ar represents a piperidinyl group, a pyridin-2-yl group, a phenyl group, or a phenyl group substituted with a halogen atom, a methyl group, or an alkoxy group having 1 to 4 carbon atoms;
R1 represents a methyl group and R11 represents a hydrogen atom, or R1 and R11 together represent a —(CH2)3— group or a —(CH2)2O— group;
R2 represents a hydroxyl group, a C1-7 alkoxy group, a C1-7 acyloxy group, a cyano group, a —NR6R4 group, a —NR3COR4 group, a —NR3COOR8 group, a —NR3SO2R9 group, a —NR3CONR10R12 group, a C1-7 acyl group, a C1-7 alkoxycarbonyl group, a —CONR10R12 group, a CH2OH group, a C1-7 alkoxymethyl group, a C1-7 acyloxymethyl group, a C1-7 alkylaminocarbonyloxymethyl group, a —CH2NR13—R14 group, a —CH2NR3COR4 group, a —CH2NR3COOR8 group, a —CH2NR3SO2R9 group, or a —CH2NR3CONR10R12 group, R2 constitutes a double bond between the carbon atom to which it is attached and the adjacent carbon atom of the piperidine ring, or Ar and R2 together with a piperidine ring to which Ar and R2 are bonded form a group represented by Formula (1a) or (1b),
where R3 represents a hydrogen atom or a C1-4 alkyl group and R4 represents a hydrogen atom, a C1-7 alkyl group, a phenyl group, a benzyl group, a pyridyl group, an unsubstituted C3-7 cycloalkyl group, or a C3-7 cycloalkyl group substituted with one or more methyl groups, or R3 and R4 together represent a —(CH2)n— group (where n is 3 or 4);
T represents a methylene group, a carbonyl group, a —COO— group, or a —CONR5— group and A represents a single bond, a methylene group, an ethylene group, a propylene group, or a vinylene group, or -T-A- represents a —SO2— group; and
Z represents a phenyl group or a phenyl group substituted with one or more of a halogen atom, a C1-4 alkyl group, a C1-4 alkoxy group, and a nitro group.
where R5 represents a hydrogen atom or a C1-4 alkyl group,
R6 represents a hydrogen atom or a C1-7 alkyl group and R7 represents a hydrogen atom, a C1-7 alkyl group, a C3-7 cycloalkylmethyl group, a benzyl group, or a phenyl group, or R6 and R7 together with a nitrogen atom to which R6 and R7 are bonded form a heterocycle selected from the group consisting of an azetidine group, a pyrrolidine group, a piperidine group, a morpholine group, a thiomorpholine group, and a perhydroazepine group.
R8 represents a C1-7 alkyl group or a phenyl group,
R9 represents a C1-7 alkyl group, an amino group or an amino group substituted with one or two C1-7 alkyl groups, a phenyl group or a phenyl group substituted with one or more groups selected from the group consisting of a halogen atom, a C1-7 alkyl group, a trifluoromethyl group, a hydroxyl group, a C1-7 alkoxy group, a carboxyl group, a C1-7 alkoxycarbonyl group, a C1-7 alkylcarbonyloxy group, a cyano group, a nitro group, an amino group, and an amino group substituted with one or two C1-7 alkyl groups (where, in a case where a plurality of groups are selected, the plurality of groups are the same as or different from each other),
R10 represents a hydrogen atom or a C1-7 alkyl group and R12 represents a hydrogen atom, a C1-7 alkyl group, a C3-7 cycloalkyl group, a C3-7 cycloalkylmethyl group, a hydroxyl group, a C1-4 alkoxy group, a benzyl group, or a phenyl group, or R10 and R12 together with a nitrogen atom to which R10 and R12 are bonded form a heterocycle selected from the group consisting of an azetidine group, a pyrrolidine group, a piperidine group, a morpholine group, a thiomorpholine group, and a perhydroazepine group,
R13 represents a hydrogen atom or a C1-7 alkyl group,
R14 represents a hydrogen atom, a C1-7 alkyl group, a C3-7 cycloalkylmethyl group, or a benzyl group,
n3 is 2 or 3, and
in a case where Ar is a phenyl group, R2 is a hydroxyl group, and -T-A-Z is a benzoyl group, R1 is not a methyl group; in a case where Ar is a phenyl group, R2 is a —NHCOCH3 group, and -T-A-Z is a benzoyl group, R1 and R11 together do not form a —(CH2)3— group; in a case where Ar is a phenyl group, R2 is a hydroxyl group, and -T-A-Z is a 3-methoxybenzyl group, R1 and R11 together do not form a —(CH2)3— group; and in a case where Ar is a phenyl group, R2 is a —NHCOCH3 group, and -T-A-Z is a benzyloxycarbonyl group, R1 is not a methyl group],
[in Formula (2),
Y is a group represented by Formula (2a) or (2b),
where Ar2 represents a phenyl group, a naphthyl group, or a C5-7 cycloalkadienyl group, which is optionally substituted, or represents an optionally substituted single or condensed heterocyclic group having aromatic properties, having 5 to 12 ring atoms, and containing up to 4 heteroatoms selected from sulfur atoms, oxygen atoms, and nitrogen atoms in a ring or in each ring,
R100 represents a linear or branched C1-8 alkyl group, a C3-7 cycloalkyl group, a C4-7 cycloalkylalkyl group, an optionally substituted phenyl group or a phenyl C1-6 alkyl group, an optionally substituted 5-membered heteroaromatic ring containing up to 4 heteroatoms selected from oxygen atoms and nitrogen atoms, a hydroxy C1-6 alkyl group, an amino C1-6 alkyl group, a C1-6 alkylaminoalkyl group, a di C1-6 alkylaminoalkyl group, a C1-6 acylaminoalkyl group, a C1-6 alkoxyalkyl group, a C1-6 alkylcarbonyl group, a carboxy group, a C1-6 alkoxycarbonyl group, a C1-6 alkoxycarbonyl C1-6 alkyl group, an aminocarbonyl group, a C1-6 alkylaminocarbonyl group, a di C1-6 alkylaminocarbonyl group, or a halogeno C1-6 alkyl group, or in a case of forming a ring with Ar2, R100 forms a group-(CH2)p— (where p is 2 or 3),
R101 and R102 are the same as or different from each other, and each independently represents a hydrogen atom, a C1-6 linear chain or branched alkyl group, or together forms a —(CH2)n1— group (where n1 is 3, 4, or 5), or R101 and R100 together form a group-(CH2)q— (where q is 2, 3, 4, or 5).
R110 is R103 or (R405)q1,
R111 is R104 or a group represented by Formula (2c) or (2g),
R112 is R105 or a group represented by Formula (2d),
R401 is selected from a hydrogen atom, a C1-4 alkyl group, a C3-6 cycloalkyl group, and a C1-4 alkylOC(O)—,
A2 is a phenyl group or a C3-7 a cycloalkyl group,
each R402 is independently selected from a hydrogen atom, —OH, —NH2. —CN, a halogen atom, a C1-6 alkyl group, a C3-7 cycloalkyl group, a C1-6 alkoxy group, and a C1-6 alkoxy C1-6 alkyl group,
n2 is 1, 2, or 3,
each R403 is independently selected from a hydrogen atom. —OH, —NH2, —NO2. —CN, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a C1-6 alkoxy C1-6 alkyl group,
m is 1, 2, or 3,
r1 is 0, 1, 2, or 3,
r2 is 1, 2, or 3,
R404 and R409 are selected from a C1-4 alkyl group, a C1-6 alkoxy C1-6 alkyl group, a C3-7 cycloalkyl group, and E-(CH2δ)b—, wherein E is —NR406R407, —SR406, a —SOC1-6 alkyl group, a —SO2C1-6 alkyl group. N+(O−)R407, —NR406SO2R407, an aryl group, and a N- or C-bonded 5- or 6-membered aromatic or non-aromatic heterocyclic ring having 1, 2, 3, or 4 nitrogen atoms or a N-oxide thereof, and b is 0, 1, 2, 3, 4, or 5,
each R405 is independently selected from a hydrogen atom, —OH, —CN, a halogen, —R406, —OR406, —NR406R407, —SR406, —SOR406, and —SO2R406,
q1 is 1, 2, or 3.
R406 and R407 are each independently selected from a hydrogen atom, a C1-6 linear or branched alkyl group, a C2-6 linear or branched alkenyl group or alkynyl group, and a C3-7 carbocyclic group having 0, 1, or 2 double or triple bonds, where the groups are unsubstituted or substituted with one or more groups selected from —OH, ═O, —NH2, —CN, a halogen atom, an aryl, and a C1-3 alkoxy group.
R408 is selected from a hydrogen atom, a C1-5 linear or branched alkyl group, and a C3-5 cycloalkyl group, where the groups are unsubstituted or substituted with one or more groups selected from —OH, ═O, —NH2, —CN, a halogen, an aryl group, and a C1-3 alkoxy group,
in a case where R404 is E-(CH2)b—, and E is a N- or C-bonded 5- or 6-membered aromatic or non-aromatic heterocyclic ring or a N-oxide thereof, E is unsubstituted or independently selected from —OH, ═O, —NH2, —CN, a halogen atom, a C1-4 alkyl group, a C1-4 alkoxy group, a C1-4 alkyl-CO—, —NR406R407, an aryl, and a N- or C-bonded 5- or 6-membered aromatic or non-aromatic heterocyclic ring having 1, 2, 3, or 4 nitrogen atoms or a N-oxide thereof,
in a case where R401, R402, R403, or R404 is an alkyl group, a cycloalkyl group, an alkoxy group, or an alkoxyalkyl group, the group is unsubstituted or has 1, 2, 3, 4, or 5 substituents each of which is independently selected from —OH, —NH2, —CN, phenyl, and a halogen,
R103 and R104 are the same as or different from each other, and are independently selected from a hydrogen atom, a C1-6 linear or branched alkyl group, a C1-6 alkenyl group, an aryl group, a C1-6 alkoxy group, a hydroxy group, a halogen atom, a nitro group, a cyano group, a carboxy group, a carboxyamide group, a sulfonamide group, a C1-6 alkoxycarbonyl group, a trifluoromethyl group, an acyloxy group, a phthalimide group, an amino group, a mono- or di-C1-6 alkylamino group, an —O(CH2)r—NW2 group (where r is 2, 3, or 4, and W is a hydrogen atom or a C1-6 alkyl group or forms a group
with nitrogen adjacent thereto [in Formula (2e) or (2f), V and V1 independently represent a hydrogen atom or an oxygen atom, and u is 0, 1, or 2]),
an —O(CH2)t—OE2 group (where t is 2, 3, or 4, and E is a hydrogen atom or a C1-6 alkyl group), a hydroxyalkyl group, an aminoalkyl group, a mono- or di-alkylaminoalkyl group, an acylamino group, an alkylsulfonylamino group, an aminoacylamino group, and a mono- or di-alkylaminoacylamino group, up to 4 R103 substituents are present in a quinoline nucleus, or R104 forms a —(CH2)e— group (where e is 1, 2, or 3) in a case of forming a ring with R105 as an aryl,
R105 represents a branched or linear C1-6 alkyl group, a C3-7 cycloalkyl group, a C4-7 cycloalkylalkyl group, an optionally substituted aryl group, or an optionally substituted single or condensed heterocyclic group having aromatic properties, having 5 to 12 ring Moms, and containing up to 4 heteroatoms selected from sulfur atoms, oxygen atoms, and nitrogen atoms in a ring or in each ring, and
X represents an oxygen atom, a sulfur atom, or ═N—C≡N], and
in Formula (3).
R301 is a hydrogen atom, a fluorine atom, or a methyl group,
R301′ is a hydrogen atom,
R302 is a hydrogen atom, a fluorine atom, a chlorine atom, or a methoxy group,
R302′ is a hydrogen atom or a fluorine atom,
R303 is a hydrogen atom, a fluorine atom, a chlorine atom, a methyl group, a trifluromethyl group, or a nitrile group,
R304 is methyl, ethyl, n-propyl, hydroxyethyl, methoxyethyl, trifluoromethyl, difluoromethyl, or fluoromethyl,
R305 is methyl, ethyl, methoxymethyl, trifluoromethyl, difluoromethyl, fluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, or 2,2,2-trifluoroethyl,
X1 is a nitrogen atom and X2 is a sulfur atom or an oxygen atom, or X1 is a sulfur atom and X2 is a nitrogen atom.
represents a single bond or a double bond, depending on X1 and X2, and
represents an (R)-enantiomer or racemic compound of the compound of Formula (3)].
[6] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (I) is osanetant.
[7] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (1) is SSR-146977
[8] The prophylactic or therapeutic agent according to [5], wherein the compound represented by Formula (1) is SSR-241586.
[9] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (1) is CS-003.
[10] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (2) is talnetant.
[11] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (2) is pavinetant.
[12] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (2) is SB-235375.
[13] The prophylactic or therapeutic agent according to [5], in which the compound represented by Formula (3) is fezolinetant.
[14] The prophylactic or therapeutic agent according to any one of [1] to [3], in which the neurokinin B signal blocker is an inhibitor of a tachykinin processing enzyme or a decomposition accelerator of a tachykinin processing enzyme.
[15] The prophylactic or therapeutic agent according to [14], in which the inhibitor is an inhibitor of proprotein convertase subtilisin/kexin 1, proprotein convertase subtilisin/kexin 2, carboxypeptidase H, or peptidyl-glycine alpha-amidating monooxygenase.
[16] The prophylactic or therapeutic agent according to any one of [1] to [3], in which the neurokinin B signal blocker is an expression inhibitor of neurokinin B (NKB), a neurokinin 3 receptor, a tachykinin processing enzyme, gastrin-releasing peptide (GRP), or a GRP receptor.
[17] The prophylactic or therapeutic agent according to any one of [1] to [3], in which the neurokinin B signal blocker is a removing agent for neurons expressing a neurokinin 3 receptor or a GRP receptor.
[18] The prophylactic or therapeutic agent according to [17], in which the removing agent is GRP, a specific binding substance to a GRP receptor, NKB, or a specific binding substance to a neurokinin 3 receptor, to which a cytotoxic substance is bound.
[19] The prophylactic or therapeutic agent according to [18], in which the cytotoxic substance is a ribosome-inactivating protein or a diphtheria toxin.
[20] The prophylactic or therapeutic agent according to [19], in which the ribosome-inactivating protein is saporin, ricin, or abrin.
[21] A prophylactic or therapeutic medicinal composition for an IL-31-mediated disease comprising the prophylactic or therapeutic agent according to any one of [1] to
[20] and a pharmaceutically acceptable carrier.
[22] A prophylactic or therapeutic medicinal composition for atopic dermatitis comprising the prophylactic or therapeutic agent according to any one of [1] to [20] and a pharmaceutically acceptable carrier.
According to the present invention, it is possible to provide a prophylactic or therapeutic technique for an IL-31-mediated disease such as atopic dermatitis.
In one embodiment of the present invention, a prophylactic or therapeutic agent including a neurokinin B signal blocker for an IL-31-mediated disease is provided. In one embodiment of the present invention, the prophylactic or therapeutic agent for an IL-31-mediated disease including a neurokinin B signal blocker as an active ingredient may be provided.
In the present invention, an IL-31-mediated disease is a disease in which IL-31 is involved in the onset or progression. The term “IL-31-mediated” indicates not only that IL-31 is involved in the onset or progression of a disease through direct action, but also that, as a result of the action of IL-31 on body tissues or somatic cells, new biomolecules are produced, expressed, or released or activated, so that a disease develops or progresses due to the biomolecules.
Examples of the IL-31-mediated disease include atopic dermatitis, pruritus in atopic dermatitis, dermatomyositis, pruritus in dermatomyositis, chronic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, pruritus in cutaneous T cell lymphoma, prurigo nodularis, prurigo chronica multiformis, urticaria pigmentosa, bullous pemphigoid, and the like, but are not limited thereto. In these diseases, it has been reported that the expression of IL-31 and IL-31 receptors in a lesioned tissue is enhanced, a serum IL-31 level is higher than that in healthy subjects, and these phenomena correlate with the onset of a disease.
It is possible to prevent or treat the IL-31-mediated disease or symptoms associated with the disease by suppressing the IL-31 production in vivo or by inhibiting the action of IL-31 after the IL-31 production. IL-31 exerts its activity through the binding of IL-31 to its receptor, intracellularly transmitting the stimulation signal. Therefore, the action of IL-31 can be suppressed by blocking the binding of IL-31 to its receptor. For that purpose, for example, an anti-IL-31 antibody or an anti-IL-31 receptor antibody can be used. A heterodimer consisted of an IL-31 receptor A and an oncostatin M receptor is formed to function as the IL-31 receptor, and it has been reported that an anti-IL-31 receptor A antibody is used to suppress atopic dermatitis and pruritus in atopic dermatitis (Kabashima K, et al. Nemolizumab in patients with moderate-to-severe atopic dermatitis: Randomized, phase 11, long-term extension study. J Allergy Clin Immunol. 142:1121-1130, 2018).
Suppression of the action of IL-31 is also achieved by inhibiting the production, expression or release, or activation of biomolecules involved in the IL-31-mediated disease, induced by the stimulation of the IL-31 receptor. Such activation includes a case where inactive or low-activity biomolecules we activated or highly activated to enhance the function of the biomolecules.
So far, the present inventors have clarified that Dock 8−/− AND Tg mice show enhanced scratching behavior, and a concentration of IL-31 in blood is increased. In addition, as detailed in examples, the present inventors have clarified that an expression level of Tachykinin2 (Tac2) gene increases by a factor of about 23 in dorsal root ganglia of these mice.
The Tac2 gene encodes preprotachykinin-3 (PPT-3) that is a precursor of a neuropeptide. PPT-3 is post-translationally modified by a processing enzyme, and then becomes mature neurokinin B (NKB). NKB binds to a neurokinin receptor 3 (NK3R).
The present inventors have clarified that an expression level of NKB increases as the concentration of IL-31 increases, NKB activates neurons expressing NK3R, neurons expressing gastrin-releasing peptide (GRP) we activated, and neurons expressing GRP receptors are activated, so that scratching behavior of mice is induced.
In the present specification, molecules and neural circuits that function in a process of NKB inducing scratching behavior are referred to as a neurokinin B signal.
The scratching behavior induced by the increased concentration of IL-31 can be suppressed by blocking neurokinin B signal described above. In addition, the IL-31-mediated disease can be prevented or treated by blocking neurokinin B signal.
The neurokinin B signal blocker may be an agent that suppresses functions of PPT-3, a processing enzyme, NKB, NK3R, GRP, GRP receptors, and the like.
The neurokinin B signal blocker may be an agent that suppresses expression levels of PPT-3, a processing enzyme, NKB, NK3R, GRP, GRP receptors, and the like, or an agent that removes neurons expressing NK3R receptors or GRP receptors.
The neurokinin B signal blocker may be an antagonist (antagonistic drug) of NK3R. The neurokinin B signal blocker can also be referred to as an NK3R signal blocker. The NK3R antagonist can also be referred to as an NK3R blocker (blocking drug).
NKB has the highest affinity for NK3R, and NK3R is activated mainly by the binding of NKB.
As will be described later in examples, the present inventors have clarified that the administration of IL-31 increases the expression of NKB to induce scratching behavior. The inventors have also clarified that this scratching behavior is suppressed by the NK3R antagonists such as osanetant, fezolinetant, talnetant, pavinetant, SSR-146977, SB-235375. SSR-241586, and CS-003.
That is, the scratching behavior induced by an increased concentration of IL-31 can be suppressed by inhibiting the activation of NK3R. In addition, the IL-31-mediated disease can be prevented or treated by inhibiting the activation of NK3R.
Factors that inhibit the activation of NK3R include low molecular weight compounds, peptides, an antibody against NK3R, and the like.
In one embodiment of the present invention, in the prophylactic or therapeutic agent for an IL-31-mediated disease, the above described NK3R antagonist is a compound represented by Formula (1), (2), or (3) or a salt thereof, or a solvate thereof.
In Formula (1), an alkyl group or an alkoxy group may be linear or branched. A halogen atom is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a fluorine atom, a chlorine atom, or an iodine atom.
An acyl group may be a formyl group or an alkylcarbonyl group having 1 to 6 carbon atoms.
The salt of the compound represented by Formula (1), (2), or (3) is an inorganic acid salt or an organic acid salt, and exemplary examples thereof include salts that are suitable for separation or crystallization of the compound represented by Formula (1), (2), or (3). More specifically, exemplary examples of the salt of the compound represented by Formula (1) include a picrate and an oxal salt: a salt with an optically active acid, for example, mandelate and camphor sulfonate; and a pharmaceutically acceptable salt.
Exemplary examples of the pharmaceutically acceptable salt of the compound represented by Formula (1), (2), or (3) include a hydrochloride, a bromate, a sulfate, a hydrogen sulfate, a dihydrogen phosphate, a methanesulfonate, a methylsulfate, a maleate, a fumarate, a 2-naphthalene sulfonate, a benzenesulfonate, a glycolate, a gluconate, a citrate, an isethionate, a para-toluenesulfonate, and the like.
The compound represented by Formula (1), (2), or (3) may be a racemate or a pure enantiomer.
The compound represented by Formula (1) may be osanetant. Osanetant is the international nonproprietary name (INN) of N-[1-[3-[(3R)-1-benzoyl-3-(3,4-dichlorophenyl)piperidine-3-yl)propyl)-4-phenylpiperidine-4-yl]-N-methyl acetamide, and is a compound having a chemical structure represented by Formula (4). The CAS number of the compound of Formula (4) is 160492-56-8.
The compound represented by Formula (1) may be SSR-146977. SSR-146977 is N-[1-(3-[1-benzoyl-3 (R)-(3,4-dichlorophenyl)piperidine-3-yl]propyl]-4-phenylpiperidine-4-yl]-N′,N′-dimethylurea, and is a compound having a chemical structure represented by Formula (5). The CAS number of the compound of Formula (5) is 264618-44-2.
The compound represented by Formula (1) may be SSR-241586. SSR-241586 is (+)-1′-[2-[4-benzoyl-2(R)-(3,4-dichlorophenyl)morpholine-2-yl]ethyl]-N,N-dimethyl-1,4′-bipiperidin-4′-carboxamide, and is a compound having a chemical structure represented by Formula (6). The CAS number of the compound of Formula (6) is 1239279-30-1.
The compound represented by Formula (1) may be CS-003. CS-003 is 1′-[2-[2-(R)-(3,4-dichlorophenyl)-4-(3,4,5-)trimethoxybenzoyl]morpholine-2-yl]ethyl]spiro[benzo[c]thiophene-1(3H)4′-piperidine]2(S)-oxide, and is a compound having a chemical structure represented by Formula (7). The CAS number of the compound of Formula (7) is 191672-52-3.
The compound represented by Formula (2) may be talnetant. Talnetant is N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide, and is a compound having a chemical structure represented by Formula (8). The CAS number of the compound represented by Formula (8) is 174636-32-9.
The compound represented by Formula (2) may be pavinetant. Pavinetant is 3-(methanesulfonamide)-2-phenyl-N-[1S]-1-phenylpropyl]quinoline-4-carboxamide, and is a compound represented by Formula (9). The CAS number of the compound represented by Formula (9) is 941690-55-7.
The compound represented by Formula (2) may be SB-235375. SB-235375 is 2-[2-phenyl-4-[N-[1 (S)-phenylpropyl]carbamoyl]quinoline-3-yloxy]acetic acid, and is a compound represented by Formula (10). The CAS number of the compound represented by Formula (10) is 224961-34-6.
The compound represented by Formula (3) may be fezolinetant. Fezolinetant is (4-fluorophenyl)-[(8R)-8-methyl-3-(3-methyl-1,2,4-thiadiazole-5-yl)-6,8-dihydro-5H-[1,2,4]triazolo[4,3-a]pyrazine-7-yl]-methanone, and is a compound represented by Formula (11). The CAS number of the compound represented by Formula (11) is 1629229-37-3.
A mouse Tac2 gene encodes preprotachykinin-3 (PPT-3). PPT-3 is post-translationally modified, and then neurokinin B (NKB) is synthesized.
It is considered that this post-translational modification proceeds as follows. The precursor PPT-3 is cleaved at a position where two consecutive lysine residues or arginine residues are present. This cleavage is catalyzed by a prohormone converterase (PC).
PCs constitute a proprotein convertase subtilisin/kexin (PCSK) family. The PCSK family is composed of PCSK1 to -9. Among these. PCSK1 and PCSK2 ae major enzymes that process in vivo peptides.
In neural tissues of mice, in which expression levels of PCSK1 and PCSK2 ae decreased, a concentration of neurokinin A is decreased.
The lysine residue and arginine residue at the C-terminus of the cleaved peptide are removed by carboxypeptidase. Among these, an enzyme mainly involved in this reaction is carboxypeptidase E (CPE).
A peptide from which a basic peptide has been removed contains a glycine residue at the C-terminus. An α-position amino group of this C-terminus glycine residue is amidated by a peptidyl-glycine alpha-amidating monooxygenase (PAM).
In humans, PAM is encoded by a PAM gene. The PAM is composed of a peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) domain.
The PHM domain hydroxylates the glycine residue at the C-terminus of a peptide. The PAL domain subsequently removes the hydroxylated glycine residue. With a series of reactions, glycine is cleaved, and the C-terminus of a peptide is amidated.
Human PPT-3 and mouse PPT-3 have an amino acid sequence of NKB shown in SEQ ID NO: 2. In addition, human PT-3 and mouse PPT-3 have the above described basic amino acid residue and glycine residue involved in processing.
Thus, it is considered that post-translational modifications of human PPT-3 and mouse PPT-3 proceed by the same mechanism.
Cleavage with PCSK and CPE and amidation with PAM re required for NKB to have activity. That is, the action of NKB can be suppressed by inhibiting the activity of these enzymes.
The neurokinin B signal blocker is an inhibitor of a tachykinin processing enzyme or a decomposition accelerator of a tachykinin processing enzyme. The inhibitor of a tachykinin processing enzyme or the decomposition accelerator of a tachykinin processing enzyme can suppress the action of NKB to suppress the scratching behavior induced by an increased concentration of IL-31. Therefore, the inhibitor of a tachykinin processing enzyme can be used to prevent or treat the IL-31-mediated disease.
Exemplary examples of tachykinin processing enzymes include proprotein convertase subtilisin/kexin 1 (PCSK1), proprotein convertase subtilisin/kexin 2 (PCSK2), carboxypeptidase E (CPE), peptidyl-glycine alpha-amidating monooxygenase (PAM), and the like, but are not limited thereto, and any enzyme may be used as long as the enzyme is involved in the processing of tachykinin.
The inhibitor of the tachykinin processing enzyme may be a low molecular weight compound, may be a peptide, or may be an antibody against the tachykinin processing enzyme, or the like.
In one embodiment of the present invention, the prophylactic or therapeutic agent for an IL-31-mediated disease, in which the neurokinin B signal blocker is an expression inhibitor of neurokinin B, a neurokinin 3 receptor, a tachykinin processing enzyme, GRP, or a GRP receptor, is provided.
Exemplary examples of the expression inhibitor include siRNA, shRNA, and the like. An expression inhibitor for a gene encoding a protein involved in the NKB signal is administered to reduce the expression level of any of the above factors, so that the NKB signal can be blocked. As a result, the scratching behavior induced by an increased concentration of IL-31 can be suppressed. In addition, the IL-31-mediated disease can be prevented or treated by administering these expression inhibitors.
In one embodiment of the present invention, the prophylactic or therapeutic agent for an IL-31-mediated disease as a removing agent for neurons expressing a neurokinin 3 receptor or a GRP receptor is provided.
The removing agent for neurons may be GRP, a specific binding substance to a GRP receptor, NKB, or a specific binding substance to a neurokinin 3 receptor, to which a cytotoxic substance is bound.
Examples of a cytotoxic substance include a ribosome-inactivating protein (RIP), a diphtheria toxin, and the like. Exemplary examples of the RIP include saporin, ricin, abrin, and the like.
The GRP bound to a cytotoxic substance is captured by GRP receptor-expressing neurons and taken up into cells by endocytosis. Similarly, neurokinin B bound to a cytotoxic substance is captured by neurokinin 3 receptor (NK3R)-expressing neurons and taken up into cells by endocytosis. Subsequently, the neurons that have taken up the cytotoxic substance are killed and denervated.
As will be described later in examples, the present inventors denervated the neurons by administering GRP-saporin obtained by conjugating saporin to GRP to mice to induce cell death in neurons expressing the GRP receptor. As a result, it was clarified that the scratching behavior induced by the administration of IL-31 and NKB was suppressed in the mice from which the neurons expressing the GRP receptor were denervated.
That is, the neurokinin B signal can be blocked by denervating the neurons expressing the GRP receptor or NK3R. As a result, the scratching behavior induced by an increased concentration of IL-31 can be suppressed.
In addition, the above described removing agent for neurons may be an agent in which a cytotoxic substance is bound to proteins specifically captured on surfaces of target neurons. The proteins captured on the surfaces of the target neurons may be, for example, ligands bound to a receptor that is specifically expressed on the target neurons, or a specific binding substance expressed on the target neurons and specifically bound to proteins that are present on cell surfaces. Exemplary examples of the specific binding substances include antibodies, antibody fragments, aptamers, and the like. Exemplary examples of the antibodies include an anti-GRP receptor antibody, an anti-NK3R antibody, and the like.
In one embodiment of the present invention, a prophylactic or therapeutic medicinal composition for an IL-31-mediated disease including the above described prophylactic or therapeutic agent for an IL-31-mediated disease and a pharmaceutically acceptable carrier is provided.
The medicinal composition of the present embodiment may be in a dosage form used orally or in a dosage form used parenterally. Exemplary examples of the dosage form used orally include tablets, capsules, elixirs, microcapsules, and the like. Exemplary examples of the dosage form used parenterally include injections, inhalants, suppositories, topical skin preparations, patches, and the like.
Exemplary examples of the pharmaceutically acceptable carrier include solvents such as sterilized water and physiological saline; binders such as gelatin, cornstarch, tragant gum, and arabic rubber; excipients such as crystalline cellulose; and swelling agents such as alginic acid.
The medicinal composition may further contain additives. Exemplary examples of the additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; flavors such as peppermint and Gaultheria adenothrix oil; stabilizers of benzyl alcohol and phenol; buffers such as phosphate and sodium acetate; dissolution adjuvants such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.
The medicinal composition can be formulated by appropriately combining and mixing the above carriers and additives in a unit dose form required for generally accepted pharmaceutical practice.
Administration methods to patients known to those skilled in the art, such as intramedullary injection, intraarterial injection, intravenous injection, subcutaneous injection, as well as intranasal, transbronchial, intramuscular, percutaneous, or oral administration, can be adopted. A dose varies depending on the weight and age of a patient, the patient's symptoms, an administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
A dose of the prophylactic or therapeutic agent for an IL-31-mediated disease such as atopic dermatitis varies depending on the type of the prophylactic or therapeutic agent for atopic dermatitis, the patient's symptoms, or the like. However, in a case of oral administration, it is considered that in general adults (assuming a weight of 60 kg), a dose of about 0.1 to 500 mg per day, preferably about 1.0 to 250 mg per day, or more preferably about 1.0 to 100 mg per day is appropriately administered once a day or in several divided doses throughout a day.
In a case of parenteral administration, a dose thereof varies depending on the type of the prophylactic or therapeutic agent for an IL-31-mediated disease such as atopic dermatitis, the patient's symptoms, an administration site, an administration method, or the like. However, in a case of systemic administration, it is considered that in general adults (assuming a weight of 60 kg), a dose of about 0.1 to 500 mg per day, preferably about 1.0 to 250 mg per day, or more preferably about 1.0 to 100 mg per day is appropriately administered once a day or in several divided doses throughout a day. In a case of topical administration, it is considered that in general adults (assuming a weight of 60 kg), a dose of about 0.001 to 10 mg per day, preferably about 0.01 to 5 mg per day, or more preferably about 0.02 to 2 mg per day is appropriately administered once a day or in several divided doses throughout a day.
In one embodiment of the present invention, a prophylactic or therapeutic method for an IL-31-mediated disease including administration of an effective amount of a neurokinin B signal blocker to a patient in need of treatment is provided.
In one embodiment of the present invention, a neurokinin B signal blocker for prevention or treatment of an IL-31-mediated disease is provided.
In one embodiment of the present invention, a neurokinin B signal blocker for producing a prophylactic or therapeutic agent for an IL-31-mediated disease is provided.
In each of the above embodiments, the neurokinin B signal blocker may be a neurokinin 3 receptor antagonist. The neurokinin B signal blocker is an inhibitor of a tachykinin processing enzyme or a decomposition accelerator of a tachykinin processing enzyme. The neurokinin B signal blocker may be an expression inhibitor of neurokinin B (NKB), a neurokinin 3 receptor, a tachykinin processing enzyme, a gastrin-releasing peptide (GRP), or a GRP receptor. The neurokinin B signal blocker may be a removing agent for neurons expressing a neurokinin 3 receptor or a GRP receptor.
The neurokinin 3 receptor antagonist may be a compound represented by General Formula (1), (2), or (3) or a salt thereof, or a solvate thereof. The compound represented by General Formula (1) may be a compound represented by Formula (4), (5). (6), or (7), the compound represented by General Formula (2) may be a compound represented by Formula (8), (9), or (10), and the compound represented by General Formula (3) may be a compound represented by Formula (11).
The inhibitor may be an inhibitor of proprotein convertase subtilisin/kexin 1, proprotein convertase subtilisin/kexin 2, carboxypeptidase E, or peptidylglycine alpha-amidating monooxygenase.
The removing agent may be GRP, a specific binding substance to a GRP receptor, NKB, or a specific binding substance to a neurokinin 3 receptor, to which a cytotoxic substance is bound. The cytotoxic substance may be a ribosome-inactivating protein or a diphtheria toxin. The ribosome-inactivating protein may be saporin, ricin, or abrin.
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.
Tac2−/− mice were prepared by genome editing with a CRISPR/Cas9 system. A target site was selected within exon 4 of a mouse Tac2 gene using a CHOPCHOP web design tool (https://chopchop.rc/fas.harvard.edu/). Oligonucleotides shown in SEQ ID NO: 3 and SEQ ID NO: 4 and BbsI ligation adapter were synthesized. The sequences shown by SEQ ID NO: 5 and SEQ ID NO: 6 are guide sequences. In order to co-express a sgRNA and a Cas9 protein, two nucleotides thereof were synthesized, annealed, and then inserted into a BbsI-digested px330 vector. The prepared px330 vector (concentration of 5 ng/μL. Dalbecco's PBS) was injected into a pronucleus of a fertilized C57BL/6 mouse ovum that was fertilized in vitro in M2 medium (Sigma-Aldrich, Inc.). The injected germ cells were grown in CZB medium in an environment of 37° C. and 5% of CO2 until embryos in the 2-cell stage matured. Subsequently, 24 to 36 embryos were transplanted into oviducts of female ICR mice. The genotypes of mouse pups were identified by PCR using the primers shown in Sequence 6 and Sequence 7, and by TA cloning and DNA sequencing. Mouse pups with desired mutations (Δ4 and Δ15) were mated with C57BL/6 mice or Dock8−/− AND Tg mice.
Regarding the preparation of IL31−/− mice, firstly, based on a pNT1.1 vector, an enhanced green fluorescent protein (EGFP) and a flippase regression target (FRT)-franked neomycin resistant cassette (neo) were inserted just behind an initiation codon to prepare a targeting vector. This targeting vector was introduced into embryonic stem cells (ES cells) by electroporation. The ES clone into which the vector was correctly introduced was injected into a blastocyst by microinjection, and the obtained male chimeric individuals were mated with female C57BL/6 mice. Mutant heterozygous mice were mated with CAG-FLPe transgenic mice (RBRC01834) to remove neo. Neo-removed mutant mice were backcrossed with C57BL/6 mice 5 times or more before mating with Dock8′4 AND Tg mice or Dock8+/− Osmr+/− mice. Osmr is a gene encoding an Oncostatin M receptor (OSMR), which forms a heterodimer with the IL-31 receptor a and transmits the IL-31 signal intracellularly.
All mice were bred in a special and pathogen-free environment within the animal facility of Kyushu University. As a control of the experiment, litters of the same age and gender were used. The methods of animal experiments were approved by the Animal Experiment Ethics Committee of Kyushu University.
GRP-saporin, Nppb-saporin, and saporin only (2 μg/5 μL in each, Advanced Targeting Systems) were intrathecally administered to the L3/4 site to denerve spinal cord neurons that expressed a GRP receptor and a Nppb receptor. Two weeks after the toxic agent injection, the mice were used in experiments.
Prior to the experiments, mice were placed in an acrylic cage (11 cm×14 cm×20 cm) for at least 1 hour to acclimatize to the environment. Subsequently, 50 μL of a pruritus-inducing substance dissolved in sterilized physiological saline was injected intradermally into shoulders of the mice, and the behavior of these mice was videotaped. The total number of scratching behavior within a specified time was determined by playing the video. During the scratching behavior of a mouse, the mouse extends its hind leg to the itchy area, tilts its head toward the hind leg, quickly turns the hind leg several times, and returns the hind leg to the ground. These series of actions were counted as one scratching behavior. As pruritus-inducing substances. IL-31 (1 μg/50 μl; PeproTech Inc.). SLIGRL-NH2 (100 μg/50 μl; Bachem Holding AG), chloroquine (100 μg/50 μl; Wako Pure Chemical Industries.), and histamine (100 μg/50 μl; Wako Pure Chemical Industries.) were used.
In some experiments, the actions of NK3R antagonists on the scratching behavior of the mice were investigated. As compounds having activity as an NK3R antagonist or an NK3R antagonist, osanetant (Axon Medchem.), talnetant (NAMIKI SHOJI Co., Ltd.), pavinetant (NAMIKI SHOJI Co., Ltd.), fezolinetant (Haoyuan Chemexpress Co., Ltd.). SSR-146977 (Tocris Bioscience.), SSR-241586 (WuXi AppTec New Drug Development Co. Ltd.), SB-235375 (WuXi AppTec New Drug Development Co. Ltd.), and CS-003 (WuXi AppTec New Drug Development Co. Ltd.) were used. These compounds were administered to the mice in a predetermined amount per weight of each mouse 45 minutes before the intradermal injection of the pruritus-inducing substance. Osanetant was dissolved in phosphate buffered saline (PBS) containing 0.1% Tween-20 and intraperitoneally injected at 5 mg/kg. Talnetant and pavinetant were dissolved in a mixture of 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300, 5% Tween-80, and 45% saline and were intraperitoneally injected at 30 mg/kg. Fezolinetant was dissolved in PBS containing 10% DMSO and orally administered at 10 mg/kg. SSR-146977 was dissolved in PBS containing 0.1% ethanol and intraperitoneally injected at 0.3 mg/kg. SSR-241586 was dissolved in a 0.01% Tween-80 aqueous solution and intraperitoneally injected at 3 mg/kg. SB-235375 was dissolved in PBS and orally administered at 3 mg/kg. CS-003 was dissolved in PBS containing 5% glucose and intravenously injected at 3 mg/kg.
Skin tissues were fixed with 4% (w/v) paraformaldehyde and embedded in a paraffin block. Sections with a thickness of 3 μm were stained with hematoxylin and eosin, and observed with an optical microscope. In immunohistochemical analysis of dorsal root ganglia (DRG) and spinal cords, mice were anesthetized with isoflurane, and PBS and 4% paraformaldehyde were sequentially refluxed from the heart. The DRG and spinal cord tissues were collected, fixed overnight at 4° C., and then immersed in 30% sucrose-PBS to prevent tissue frost damage. Tissue samples were embedded with an OCT compound (Sakura Finetech Japan Co., Ltd.) and frozen on dry ice. Sections with a thickness of 10 μm were prepared using a cryostat, the sections were blocked by G-block (Genostaff, GB-01) for 30 minutes at room temperature, and incubated overnight at 4° C. with primary antibodies. The primary antibodies were detected with secondary antibodies (Thermo Fisher Scientific K.K.) conjugated with a fluorescent dye. DAPI (1:5000, DOJINDO LABORATORIES) was used for each staining. All images were acquired using a confocal laser scanning microscope (FV3000, Olympus Group). As the primary antibodies, rabbit anti-neurokinin B antibodies (1:500; Novus Biologicals, NB300-201), rabbit anti-NK3R antibodies (1:50; Novus Biologicals, NB300-102), rabbit anti-TRPV1 antibodies (1:500; Millipore Corporation, AB9554), anti-IL-31RA antibodies (1:100; R & D Systems. AF2107), mouse anti-c-fos antibodies (1:1000; EnCor Biotechnology Inc., MCA-2H2), rabbit anti-GRPR antibodies (1:50; LifeSpan BioSciences, Inc., LS-A831-50), and rabbit anti-NPRA antibodies (1:50; LifeSpan BioSciences, Inc., LS-C164506) were used.
Total RNAs were separated using ISOGEN (NIPPON GENE CO., LTD.), and cRNA was amplified and labeled using Low Input Quick Amp Labeling Kit (Agilent Technologies, Inc.). The cRNA was hybridized to 44K 60-mer oligomicroarray (Whole Mouse Genome oligo DNA Microarray Kit Ver 2.0; Agilent Technologies, Inc.). Hybridized microarray slides were scanned using Agilent scanner. A relative intensity of hybridization and a background value of hybridization were calculated using Feature Extraction Software version 9.5.1.1 (Agilent Technologies. Inc.). Raw signal intensity data and flag values of probes wee calculated from the hybridization intensity and a spot location according to the approach recommended by Agilent Technologies, Inc. In order to identify genes whose expression levels were increased or decreased in the test samples. Z-scores and ratios were calculated based on the normalized signal intensity of each probe. Genes with a Z-score greater than 2.0 and a ratio greater than 1.5 were considered to have an increased expression level, and genes with a Z-score smaller than −2.0 and a ratio smaller than 0.66 were considered to have a decreased expression level.
Statistical analysis was performed using GraphPad Prism. First, a Kolmogorov-Smirnov test was used to examine whether the data followed a normal distribution. Parametric data wee analyzed using a two-tailed unpaired Student's t-test in a case of comparing data from two populations. Parametric data were analyzed using one-way ANOVA and then a Bonferroni post hoc test in a case of comparing data from a plurality of populations. Nonparametric data were analyzed using a Mann-Whitney test in a case of comparing data from two populations. A P-value of 0.05 or smaller was considered significant.
Total RNAs were extracted from each tissue using ISOGEN (NIPPON GENE CO., LTD.). Total RNAs were treated with RNase-free DNase I (Thermo Fisher Scientific K.K.). This RNA sample was reverse transcribed using an oligo (dT) primer (v) and SuperScript III reverse transcriptase (Thermo Fisher Scientific K.K.), and amplified by PCR. Real-time PCR was performed by Cfx Connect™ Thermal Cycler (Bio-Rad Laboratories, Inc.) using SYBR Green PCR Master Mix (Applied Biosystems). The expression level of the target gene was normalized by an expression level of Hprt gene. For the analysis, CFX Manager (ver3.1) attached to a device was used.
A neural circuit that transmits a pruritus sensation that is activated by IL-31 was analyzed. Many itching-inducing substances transmit a pruritus sensation to spinal nerves through natriuretic polypeptide b (Nppb) or gastrin-releasing peptide (GRP).
In order to remove neurons expressing GRP receptors or Nppb receptors, each of saporin-conjugated GRP or Nppb (GRP-saporin or Nppb-saporin) was injected to medullary cavities of wild-type C57BL/6 mice. Two weeks later. IL-31 was administered to these mice, and the scratching behavior of each mouse was analyzed.
Saporin is a ribosome-inactivating protein (RIP).
Proteins conjugated with ligands of receptors expressed on target neurons and RIPs are captured by the target neurons and incorporated into cells by endocytosis. The RIP-conjugated proteins that are incorporated into cells inhibit translation and kill the target neurons.
From the result of the intramedullary injection experiment, it was clarified that the scratching behavior induced by IL-31 was suppressed due to the injection of GRP-saporin. However, in a case where Nppb-saporin was injected, the scratching behavior was not suppressed.
Next, an expression level of GRP receptor (GRPR) mRNA or NPP receptor A (NPRA) mRNA and an expression level of the protein were analyzed to confirm whether or not GRP-saporin and Nppb-saporin removed nerves expressing GRPR and NPRA, respectively.
The expression levels of GRPR gene mRNA and NPRA gene mRNA were analyzed as follows. First, spinal cords were excised from the mice after the injection, total RNAs were extracted from the excised spinal cords, and a reverse transcription reaction was performed using the total RNAs as a template. Subsequently, quantitative PCR was performed to analyze the expression level of each gene.
From the result of analyzing the expression levels of GRPR mRNA and NPRA mRNA, it was confirmed that the expression levels of GRPR mRNA and NPRA mRNA in the spinal cord were decreased by the injection of GRP-saporin or Nppb-saporin.
The expression levels of a GRPR protein and an NPRA protein were analyzed as follows. The spinal cords after the injection were excised from the mice, and sections were prepared from the excised spinal cords. The prepared sections were analyzed by immunohistochemistry using anti-GRPR antibodies and anti-NPRA antibodies.
From the result of analyzing the expression levels of the GRPR protein and the NPRA protein, it was confirmed that the expression levels of the GRPR protein and the NPRA protein in the spinal cord were decreased by the injection of GRP-saporin or Nppb-saporin.
Summarizing the above results, it was confirmed that neurons expressing GRPR and NPRA were denervated by the injection of GRP-saporin or Nppb-saporin. In addition, it was clarified that the scratching behavior induced by the administration of IL-31 is not mediated by Nppb, but is mediated by GRP.
In order to analyze the function of IL-31, mice lacking the IL-31 gene were prepared. As described above, a neo cassette sandwiched between FRTs was inserted immediately after exon 1 of the IL-31 gene.
In
The genotypes of wild-type C57BL/6 mice and neo-removed mice were determined by PCR using primers shown in SEQ ID NOs: 9, 10, and 11.
The length of the PCR product using a wild-type C57BL/6 mouse-derived genome as a template was 300 bp, and the length of the PCR product using a knock-in mouse-derived genome from which IL-31 gene was removed as a template was 550 base pairs. From this result, it was confirmed that the knock-in mice were obtained.
Dermatitis and scratching behavior of Dock8−/− AND Tg mice were analyzed. In addition, genes whose expression levels were increased in the dorsal root ganglia of Dock8−/− AND Tg mice were analyzed.
First, the skin tissues of AND Tg mice having the genotypes of Dock8+/− Osmr+/+ IL31+/+, Dock8−/− Osmr+/− IL31−/−, Dock8−/− Osmr−/− IL31−/−, and Dock8−/− Osmr+/+ IL31−/− were analyzed by HE staining.
From the result of analyzing the skin tissues, it was clarified that atopic dermatitis-like dermatitis was induced in AND Tg mice lacking the Dock8 gene, whereas this dermatitis disappeared due to deficiency of the Osmr or IL31 gene.
Next, the scratching behavior of AND Tg mice having the genotypes of Dock8+/− Osmr+/+ IL31+/+, Dock8−/− Osmr+/− IL31+/+, Dock8−/− Osmr−/− IL31+/+, and Dock8−/− Osmr+/+ IL31−/− was analyzed.
From the result of analyzing the scratching behavior, it was clarified that AND Tg mice lacking the Dock8 gene exhibited the scratching behavior, whereas this scratching behavior disappeared due to deficiency of the Osmr or IL31 gene.
Furthermore, a concentration of serum IL-31 AND Tg mice having the genotypes of Dock8+/− Osmr+/+ IL31+/+, Dock8−/− Osmr+/− IL31+/+, Dock8−/− Osmr−/− IL31+/+, and Dock8−/− Osmr−/− IL31−/− was analyzed.
From the result of analyzing the concentration of IL-31, the concentration of serum IL-31 of AND Tg mice lacking the Dock8 gene increased, whereas this increase in the concentration disappeared due to deficiency of the IL-31 gene.
Next, a gene whose expression level in the dorsal root ganglia (DRG) of Dock8−/− AND Tg mice is higher than the expression level in DRG of Dock8+/− AND Tg mice was analyzed using a microarray.
From the analysis results of the microarray, it was clarified that expression levels of known signal transduction factor mRNA and neurotransmitter mRNA were increased. One of them was Tac2 encoding neurokinin B.
Next, an expression level of Tac2 mRNA in the DRG of AND Tg mice having the-genotype of Dock8+/− Osmr+/+ IL31+/+, Dock8−/− Osmr+/− IL31+/+, Dock8−/− Osmr−/− IL31+/+, and Dock8−/− Osmr+/+ IL31−/− was analyzed.
The expression level of Tac2 mRNA was analyzed as follows. First, total RNAs were extracted from the DRG of mice in each genotype, and a reverse transcription reaction was performed using the total RNAs as a template. Subsequently, quantitative PCR was performed to analyze the expression level of Tac2 gene mRNA.
As a result, it was clarified that the expression level of Tac2 mRNA increases by a factor of about 23 in the DRG of AND TS mice lacking the Dock8 gene. In addition, it was clarified that this increase in the expression level disappeared due to deficiency of the Osmr or IL31 gene.
Summarizing the above results, in Dock8−/− AND Tg mice, it was clarified that since the production amount of an IL-31 protein was increased, the IL-31 protein increased the expression level of the Tac2 gene through an IL-31 receptor protein including OSMR.
(Changes in Gene Expression by Stimulation with IL-31 and NKB)
Changes in the expression levels of Tac2, Grp. and Nppb through administration of IL-31 or NKB were analyzed. First. DRG neurons isolated from the wild-type C57BL/6 mice were stimulated by the addition of the IL-31 protein in vitro, and the expression levels of Tac2 mRNA, Grp mRNA, and Nppb mRNA were analyzed.
From the result of analyzing the expression levels of Tac2 mRNA, Grp mRNA, and Nppb mRNA, it was clarified that the expression levels of Tac2 mRNA and Grp mRNA were increased by the addition of IL-31, whereas the expression level in Nppb was not increased.
Next, DRG neurons isolated from the wild-type C57BL/6 mice were stimulated by neurokinin B (NKB) in vitro, and the expression levels of Grp mRNA and Nppb mRNA were analyzed.
From the result of analyzing the expression levels of Grp mRNA and Nppb mRNA, it was clarified that the expression levels of Grp mRNA and Nppb mRNA were increased by the stimulation of NKB. From this result, it is considered that NKB functions upstream of GRP.
The expression of NKB and IL-31 receptor A (IL-31RA) in the DRG of Dock8−/− AND Tg mice was analyzed by immunostaining.
The DRG was isolated from the Dock8−/− AND Tg mice to prepare for microscope sections. The prepared sections were analyzed by immunohistochemistry using anti-NKB antibodies and anti-IL-31RA antibodies.
In
From the result of immunostaining, it was clarified that NKB is expressed in cells expressing the IL-31RA protein in the DRG of Dock8−/− AND Tg mice.
Next, the expressions of a NKB protein and a Transient recipient potential cation Channel vanilloid subtype 1 (TRPV1) protein in the DRG of Dock8−/− AND Tg mice were analyzed by immunostaining.
TRPV1 is expressed in sensory neurons that sense itching, and the sensory neurons extend axons to outer layer lamina I of a spinal cord dorsal born.
The expressions of NKB and TRPV1 were analyzed as follows. The DRG was isolated from the Dock8−/− AND Tg mice to prepare sections. The prepared sections were analyzed by immunohistochemistry using anti-NKB antibodies and anti-TRPV1 antibodies.
In
From the result of immunostaining, it was clarified that NKB is expressed in some of the cells expressing TRPV1 in the DRG of Dock8−/− AND Tg mice.
In order to analyze a function of a Tac2 gene, CRISPR/Cas9 nuclease was used to prepare mice lacking the Tac2 gene (Tac2−/−).
Two strains of mutant mice (Δ4 and Δ15) were prepared by the above described genome editing. In PCR, primers with the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 were used. Electrophoresis was performed using a MultiNA microchip electrophoresis system (MCE-202, Shimadzu Corporation).
The sequences of the transcribed mRNAs were determined as follows. First, total RNAs were extracted front the DRG neurons in each mutant mouse, and cDNAs were obtained by a reverse transcription reaction. These cDNA were amplified by PCR using the primers shown in SEQ ID NO: 12 and SEQ ID NO: 13. The amplification products were cloned by TA cloning and cDNA sequences were analyzed.
Both mutations have deletion mutations in exon 4 and cause a frameshift in the amino acid sequence. Therefore, it was clarified that the mutated genes do not encode the mature peptide sequences.
Tac2 mutant mice (Δ4 and Δ15) were used to analyze a male of the NKB protein encoded by the Tac2 gene in the transmission of itching sensation induced by the administration of IL-31.
Histamine, chloroquine, SLIGRL-NH2 (PAR2 agonist), or IL-31, which is the pruritus-inducing substance, was intradermally injected into Tac2−/− (Δ4) mice and Tac2+/− mice. The results are shown in
Administration of histamine, chloroquine, and PAR2 agonist (SLIGRL-N H2) induced the scratching behavior in Tac2−/− (Δ4) mice and Tac2+/− mice, as previously reported. It was clarified that the administration of IL-31 induced the scratching behavior in Tac2+/− mice, whereas it hardly induced the scratching behavior in Tac2−/− (Δ4) mice.
The same experiment as described above was performed on Tac2−/− (Δ15) mice and Tac2+/− mice. The result of analyzing the scratching behavior is shown in
As in the case of Tac2−/− (4) mice, it was clarified that the administration of histamine induced the scratching behavior in Tac2−/− (Δ15) mice, whereas the administration of IL-31 hardly induced the scratching behavior.
The Dock8−/− AND Tg mice that have expressed Tac2 or have not expressed Tac2 (Tac2−/− (Δ4)) were established by the Tac2−/− (Δ4) mice being mated with the Dock8−/− AND Tg mice, and the skin tissues, the scratching behavior, and the serum IL-31 levels were analyzed. The results am shown in
Next, in order to identify the site where NKB was functioning. NKB was injected into medullary cavities of Tac2−/− (Δ4) mice, and scratching behavior was analyzed.
It was clarified that the intramedullary injection of NKB protein into Tac2−/− (Δ4) mice induced the scratching behavior. This result indicates that NKB induces the scratching behavior without activating peripheral nerves.
Next, the neural circuit that induces the scratching behavior by the NKB administration was analyzed. GRP-saporin or Nppb-saporin was injected into the medullary cavities of wild-type C57BL/6 mice, and two weeks later, NKB was injected, and scratching behavior was analyzed.
Mice from which neurons expressing the GRP receptor were removed did not show the scratching behavior after the administration of NKB, whereas mice from which neurons expressing the Nppb receptor were removed showed the scratching behavior after the administration of NKB. From this result, it was clarified that NKB is a neurotransmitter that functions upstream of GRP and transmits itching sensation induced by IL-31.
The function and localization of N3R were analyzed.
The expression of NK3R and TRPV1 was analyzed as follows. The spinal cord dorsal horns were excised from the mice, and sections were prepared from the excised spinal cord dorsal horns. The prepared sections were analyzed by immunohistochemistry using anti-NK3R antibodies and anti-TRPV1 antibodies.
It was clarified that NK3R was mainly expressed in the neurons in the outer layer lamina I of the spinal cord dorsal horn.
Next, it was analyzed whether or not itching sensation induced by IL-31 was mediated by the Neurokinin 3 receptor (NK3R).
After the administration of IL-31, c-fos activation in NK3R-expressing neurons in the spinal cord dorsal horn was analyzed. The result is shown in
In
Next, osanetant, a selective antagonist of NK3R and an anti-psychotic drug, was intraperitoneally injected into wild-type C57BL/6 mice, and then the effect of the compound on the scratching behavior induced by IL-31, histamine, or chloroquine was analyzed.
As a result, it was clarified that the administration of osanetant decreased the frequency of scratching behavior by IL-31, whereas it did not decrease the frequency of scratching behavior by histamine and chloroquine.
Similar to osanetant, fezolinetant that is a selective antagonist of NK3R was orally administered to wild-type C57BL/6 mice, and the effect on the scratching behavior induced by IL-31, histamine, or chloroquine was analyzed.
As a result, it was clarified that the administration of fezolinetant decreased the frequency of scratching behavior by IL-31, whereas it did not decrease the frequency of scratching behavior by histamine and chloroquine, which is the same result as the case of the administration of osanetant.
Furthermore, talnetant, pavinetant, SSR-146977, and SB-235375 that are other NK3R selective antagonists were administered to wild-type C57BL/6 mice, and then the effects of the compounds on the scratching behavior induced by IL-31 or chloroquine were analyzed.
From the results shown in
Furthermore, CS-003 that is an antagonist to neurokinin 3 receptor (NK3R) and also an antagonist to neurokinin 1 receptor (NK1R) and neurokinin 2 receptor (NK2R), and SSR-241586 that is an antagonist to NK3R and NK2R were administered to wild-type C57BL/6 mice, and then the effects of the compounds on the scratching behavior induced by IL-31 or chloroquine were analyzed.
From the results shown in
In each of
In summary, it was clarified that the administration of IL-31 activates neurons expressing NK3R. In addition, it was clarified that the pharmacological inhibition of NK3R selectively suppresses the scratching behavior by IL-31.
According to the present invention, it is possible to provide the prophylactic or therapeutic agent for an IL-31-mediated disease including a neurokinin B signal blocker as an active ingredient.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-215017 | Nov 2018 | JP | national |
| 2019-144913 | Aug 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/044886 | 11/15/2019 | WO | 00 |
