JP2008297243A - Therapeutic or prophylactic agent for pruritic dermatosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new means capable of carrying out therapy or prophylaxis of pruritic dermatoses such as atopic dermatitis. <P>SOLUTION: A therapeutic or a prophylactic agent for the pruritic dermatoses contains semaphorin 3A protein known as a repulsive neuroaxonal guidance molecule or a specific derivative thereof etc., as an active ingredient. Since the therapeutic or the prophylactic agent for the pruritic dermatoses has a mechanism different from that of a steroidal agent etc., which are conventional standard therapeutic agents, the therapeutic or the prophylactic agent for the pruritic dermatoses may be effective against even the atopic dermatitis which becomes resistant to the conventional steroidal agent and is changed into an intractable state. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a therapeutic or prophylactic agent for pruritic skin diseases such as atopic dermatitis.

  Atopic dermatitis (AD) is a disease having a prevalence of about 10% in Japan. AD has been on the rise in recent years and is an important disease in the dermatological field that imposes many mental and physical pains on patients.

  As a method for treating AD, it is fundamental to use a steroid external preparation, a tacrolimus external preparation, an antihistamine / allergy internal medicine, or, in the most severe case, a steroid internal medicine (Non-patent Document 1).

  The mechanism of action of steroids and antihistamines / allergic drugs is to suppress various inflammatory and inflammatory substances released as a result of allergic reactions. That is, treatment methods using these drugs are symptomatic treatments, not fundamental treatment methods. Among AD patients, there are many intractable AD patients for whom the above-mentioned conventional standard treatment drugs are ineffective. In AD, it is known that C fibers, which are the nerves that transmit itch, extend to the stratum corneum of the skin surface and are hypersensitive to itch, but steroids and antihistamine / allergic drugs are known. Conventional drugs such as cannot fundamentally improve this hypersensitivity state. Although establishment of a novel method for treating AD that is different from conventional methods using drugs is desired, no satisfactory method for treating AD is known so far.

Atopic Dermatitis Treatment Guidelines 2005 (FY 1996 Long-term Chronic Diseases Comprehensive Research Project Allergy Comprehensive Research and 1997-2004 Welfare Labor Science Research)

  Accordingly, an object of the present invention is to provide a novel means capable of treating or preventing pruritic skin diseases such as atopic dermatitis.

  As a result of intensive research, the inventors of the present application have improved rash and further reduced scratch marks when semaphorin 3A protein known as a repulsive nerve axon guidance molecule is administered to NC / Nga, an AD model mouse. The present invention was completed by finding out that it reduces alleviation.

  That is, the present invention includes pruritic skin containing a polypeptide region of any of the following (a) to (c) and containing as an active ingredient a polypeptide having a therapeutic or prophylactic activity for pruritic skin diseases: A therapeutic or prophylactic agent for a disease is provided: (a) a polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 4 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing (B) a polypeptide comprising 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and comprising a region of 166aa to 235aa in SEQ ID NO: 2, (c) (a) or ( A polypeptide having a sequence homology of 80% or more with the polypeptide of b).

  The present invention provides a novel therapeutic or prophylactic agent capable of treating or preventing pruritic skin diseases such as atopic dermatitis, which are different from conventional standard therapeutic agents. As specifically shown in the Examples below, administration of the above polypeptide to the skin area of an atopic dermatitis model mouse improves the skin rash and reduces scratch marks, that is, reduces pruritus. Therefore, the therapeutic or prophylactic agent of the present invention is useful for the treatment or prevention of pruritic skin diseases such as atopic dermatitis. Since the therapeutic or prophylactic agent of the present invention has a different mechanism from conventional steroids and the like, it may be effective against atopic dermatitis that has become resistant to intractable conventional steroids.

The polypeptide contained as an active ingredient in the therapeutic or prophylactic agent for pruritic skin disease of the present invention is a polypeptide having therapeutic or prophylactic activity for pruritic skin disease. Specifically, it is a polypeptide comprising a region consisting of any of the following polypeptides (a) to (c) and having a therapeutic or prophylactic activity for pruritic skin diseases.
(a) Consists of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and means 166aa to 235aa in SEQ ID NO: 4 (meaning "166th amino acid to 235th amino acid") A polypeptide comprising the region of
(b) A polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
(c) A polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b).

  Here, “therapeutic or preventive activity for pruritic skin disease” means an activity for improving the symptoms of skin lesions of pruritic skin diseases (for example, atopic dermatitis, etc.) or preventing exacerbation of the symptoms. To do. Whether or not the polypeptide has the activity is determined by, for example, administering the polypeptide to the animal developing atopic dermatitis (AD) by intradermal injection or the like and improving the symptom of the lesion. It can be examined by evaluating. As an AD developing animal, for example, an AD model animal such as an NC / Nga mouse described in Examples below can be used. NC / Nga mice are widely used in the art as animal models of atopic skin diseases. When reared under normal breeding conditions, the atopy is applied to the face, ears, upper back, etc. from about 7 weeks of age. Develops dermatitis similar to dermatitis. Evaluation of therapeutic or prophylactic activity of pruritic skin diseases using NC / Nga mice can be carried out by, for example, intradermal injection into the skin lesions of NC / Nga mice for a certain period of time, as specifically described in the Examples below. This can be done by administering a polypeptide and evaluating the presence or absence of improvement in the symptoms of the lesion. The presence or absence of improvement of symptoms in the skin lesion area is determined, for example, as described in the Examples below, as a clinical skin score (Leung, Clin Exp Immunol 107 (Suppl. 1), 25-30, 1997; Suto et al. , Int Arch Allergy Immunol 120 (Suppl. 1), 70-75, 1999). The clinical skin score is divided into 4 categories of rash, erythema / bleeding, edema, erosion, desquamation / dryness, and 0 to 3 for each item (0 (none), 1 (mild), 2 (Medium), 3 (severe)), the total is calculated, the maximum score is 12 points. For example, when the mean value of the clinical skin score of the AD-onset individual group administered with the polypeptide for a certain period of time is significantly lower than the average value of the clinical skin score of the untreated AD-onset individual group, or, for example, the polypeptide If the mean score of the administration group is about 2/3 or less, preferably about 1/2 or less than the mean score of the untreated population, the polypeptide may be used to treat pruritic skin disease or It can be judged that it has preventive activity.

In addition, prior to the evaluation of the above-mentioned activity using AD-developed animals, the polypeptide of interest was analyzed in vitro using a dorsal root ganglion (DRG) growth cone (Luo et al., Cell). vol.75, 217-227, 1993). The regression assay can confirm whether the polypeptide has an activity similar to the physiological activity of Sema3A protein, but Sema3A protein is a symptom of skin lesions in AD, which is a pruritic skin disease, as shown in the Examples below. Therefore, according to the regression assay, it is possible to examine whether the polypeptide has a therapeutic or prophylactic activity for pruritic skin diseases. Specifically, the assay can be performed, for example, as described in the examples below. That is, for example, DRG is extracted from an egg embryo such as a chicken on the seventh day of embryonicity, and the DRG is placed on a glass bottom containing a medium containing nerve growth factor (NGF) and cultured at 37 ° C. for about 24 hours. Thereby extending NGF-sensitive DRG neurons. Polypeptide solutions of various dilution ratios were administered to this, and static culture was performed at 37 ° C for about 30 minutes. After fixing, the regression of DRG was observed, and the growth cone of all NGF-sensitive DRG neurons was retracted. Calculate the percentage of neurons. Polypeptides that regress many neurons at lower concentrations (high dilution factor) are expected to have higher therapeutic or prophylactic activity for pruritic skin diseases. In the present invention, the titer of the polypeptide is XU / ml when the dilution ratio of the most diluted polypeptide solution in which 50% or more neuronal regression is observed is X-fold dilution. In the regression assay, although not particularly limited, when a growth cone derived from the same species as the species from which the polypeptide to be examined for the therapeutic or prophylactic activity of pruritic skin disease is used, the accuracy of the evaluation of the therapeutic or preventive action described above is used. For example, when examining the therapeutic or prophylactic activity of a chicken Sema3A protein fragment (that is, an example of the polypeptide of (a) above), it is preferable to use a chicken growth cone. When the therapeutic or prophylactic activity of the polypeptide (a) is examined by a regression assay, for example, the 50% regression concentration (EC ₅₀ ) of the DRG growth cone of the polypeptide (a) is measured, The activity is evaluated based on the concentration. At this time, it is desirable to use a chicken Sema3A protein consisting of the full length of the amino acid sequence shown in SEQ ID NO: 4 as a positive control. It is not particularly limited, but is usually within 5 times the EC ₅₀ for EC ₅₀ of a positive control for the subject polypeptides, especially if within twice with, the polypeptide may have a therapeutic or prophylactic activity of pruritic skin diseases Is expensive.

  In the present invention, “polypeptide” refers to a molecule formed by peptide bonding of a plurality of (two or more) amino acids, and includes not only high molecular weight molecules having a large number of amino acids but also the number of amino acids. Low molecular weight molecules (oligopeptides) and proteins are also included.

  In the present invention, “having an amino acid sequence” means that amino acid residues are arranged in such an order. Thus, for example, “a polypeptide having the amino acid sequence represented by SEQ ID NO: 2” means Met Gly Trp Leu (abbreviated) ... 771 amino acid residues having the amino acid sequence of Pro Arg Ser Val. Means polypeptide.

  The amino acid sequences shown in SEQ ID NOs: 2 and 4 are the sequences of semaphorin 3A protein (human and chicken, respectively), and are registered in GenBank as accession numbers NM006080 and U02528, respectively. Semaphorins are proteins that have been identified as molecules that guide nerve axon elongation. It is also called collapsin because it was discovered based on the activity of collapsing the growth cone at the tip of the neurite. Semaphorins constitute a secreted or transmembrane protein family, and more than 30 members have been identified to date from invertebrates such as nematodes to vertebrates such as humans. Semaphorin 3A (Sema3A) is a secreted protein member of semaphorin and is a protein molecule known as a repulsive nerve axon guidance molecule. Secreted semaphorins such as Sema3A have three structural domains: a semaphorin (sema) domain with a ~ 500aa amino terminus, a C-2 type immunoglobulin (Ig) domain, and a basic terminal domain. It is known that the region necessary for Sema3A to exert physiological activities such as nerve elongation inhibitory activity is a region of 70 amino acids, particularly the 166th to 235th regions of the sema domain (Koppel et al., Neuron). , Vol. 19, 531-537, 1997). The region of 70 amino acids (hereinafter referred to as “70aa region”) is a region of 166aa to 235aa in both SEQ ID NO: 2 and SEQ ID NO: 4. Sema3A has been identified from various animals such as humans, chickens, mice and rats. The homology of these Sema3A is as high as about 85% when compared as a whole protein, and is even higher as about 95% or more when compared only with the above 70 amino acid region. In the chicken Sema3A having the amino acid sequence of SEQ ID NO: 4, the sema domain is 61aa to 567aa, the Ig domain is 592aa to 655aa, and the basic terminus is 726aa to 772aa (Feiner et al., Neuron, Vol. 19, 539 -545, 1997). In human Sema3A having the amino acid sequence of SEQ ID NO: 2, the sema domain is 61aa to 567aa, the Ig domain is 591aa to 654aa, and the basic terminal domain is 726aa to 771aa.

  The polypeptide (a) is a polypeptide comprising a region containing at least the 70aa region of the chicken Sema3A protein having the amino acid sequence shown in SEQ ID NO: 4. Specifically, from the full length of SEQ ID NO: 4 Or a fragment thereof containing the 70aa region. One example of the polypeptide contained as an active ingredient in the therapeutic or prophylactic agent of the present invention includes a region comprising the polypeptide of (a) and has a therapeutic or prophylactic activity for pruritic skin diseases (Hereinafter referred to as “active ingredient polypeptide a” for convenience). The active ingredient polypeptide a may be composed only of the polypeptide of (a) as long as it has therapeutic or prophylactic activity for pruritic skin diseases, and may be one or both ends of the polypeptide of (a). An amino acid or a polypeptide may be added.

  Specific examples of the active ingredient polypeptide a consisting only of the polypeptide (a) are not particularly limited, and examples thereof include a polypeptide consisting of the entire amino acid sequence shown in SEQ ID NO: 4 (ie, chicken Sema3A protein). . The fact that chicken Sema3A protein has a therapeutic or prophylactic activity for pruritic skin diseases is as specifically described in the following examples. Further, even a fragment lacking a part of the N-terminal or C-terminal of chicken Sema3A protein is included in the scope of the present invention as an active ingredient polypeptide a as long as it has therapeutic or prophylactic activity for pruritic skin diseases. Is done.

  In addition, specific examples of the active ingredient polypeptide a in which a polypeptide or the like is added to the polypeptide of (a) are not particularly limited. For example, the 70aa region of SEQ ID NO: 4 corresponds to a secreted semaphorin other than Sema3A. Examples include chimeric proteins constructed by replacing the regions, and polypeptides in which various tags such as His tags, FLAG tags, and myc tags are added to the polypeptide (a). Examples of secretory semaphorins other than Sema3A include, but are not limited to, collapsin 2, collapsin 3, and collapsin 5. The amino acid sequences of collapsin 2, collapsin 3 and collapsin 5 are known as described in Feiner et al., Neuron, vol. 19, 539-545 (1997). As disclosed in the above paper by Koppel et al., The chimeric protein constructed by replacing the region containing at least the 70aa region of Sema3A with the corresponding region of collapsin 2 retains the DRG retraction activity of Sema3A, and is similar to Sema3A. Demonstrate physiological activity. Accordingly, such a chimeric protein is also included in the scope of the present invention as an active ingredient polypeptide a because it has a therapeutic or prophylactic activity for pruritic skin diseases. Furthermore, Sema3A lacking the Ig domain also has DRG regression activity (Koppel et al., 1997), but such a polypeptide is also present in Sema3A in the region containing 70aa N-terminal to the Ig domain. A polypeptide to which a basic domain is added is included in the active ingredient polypeptide a and is within the scope of the present invention. The specific examples described above are merely examples, and any amino acid or polypeptide added to the polypeptide (a) may be used as long as it has therapeutic or prophylactic activity for pruritic skin diseases. Polypeptide a is included within the scope of the present invention.

  The polypeptide of (b) above is a polypeptide comprising a region containing at least the 70aa region of the human Sema3A protein having the amino acid sequence shown in SEQ ID NO: 2, specifically, from the full length of SEQ ID NO: 2. Or a fragment thereof containing the 70aa region. Another example of the polypeptide contained as an active ingredient in the therapeutic or prophylactic agent of the present invention is a polypeptide comprising the region consisting of the polypeptide (b) and having a therapeutic or prophylactic activity for pruritic skin diseases. It is a peptide (hereinafter referred to as “active ingredient polypeptide b” for convenience). The homology between chicken Sema3A (SEQ ID NO: 4) and human Sema3A (SEQ ID NO: 2) is 86% over the entire protein length, and is even higher at 95% when compared only with the 70aa region important for the physiological activity of Sema3A. As described in the Examples below, chicken Sema3A can treat or prevent AD in mice that are not chickens, so chicken Sema3A can also be used to treat or prevent chickens such as humans and AD other than mice. Is possible. Therefore, the active ingredient polypeptide b such as human Sema3A protein is also useful for the preparation of a therapeutic or prophylactic agent for pruritic skin diseases such as AD, like the active ingredient polypeptide a such as chicken Sema3A protein. Similarly to the active ingredient polypeptide a described above, the active ingredient polypeptide b may be composed only of the polypeptide of (b) as long as it has a therapeutic or prophylactic activity for pruritic skin diseases. An amino acid or polypeptide may be added to one or both ends of the polypeptide (b). Specific examples of such a polypeptide are the same as those of the active ingredient polypeptide a. For example, the polypeptide consisting of the full length of SEQ ID NO: 2 (ie, human Sema3A protein), pruritic skin disease Examples thereof include fragments thereof having therapeutic or prophylactic activity, chimeric proteins according to the above embodiment with secretory semaphorins other than Sema3A, polypeptides obtained by adding various tags to the polypeptide (b), and the like. These specific examples are also merely examples, and any amino acid or polypeptide added to the polypeptide of (b) is an active ingredient as long as it has therapeutic or prophylactic activity for pruritic skin diseases. Polypeptide b is included within the scope of the present invention.

  The polypeptide (c) is a polypeptide in which a small number (preferably 1 to several) amino acid residues of the polypeptide (a) or (b) are substituted, deleted and / or inserted. Peptide, which is a polypeptide having homology with the original sequence of 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more. In general, a protein may have almost the same function as the original protein even when a small number of amino acid residues in the amino acid sequence of the protein are substituted, deleted, or inserted. Are widely known to those skilled in the art. Therefore, since the region composed of the polypeptide of (c) can also bring about the physiological activity of the Sema3A protein in the same manner as the 70aa region described above, the region composed of the polypeptide of (c) above is included, and itchy skin A polypeptide having a therapeutic or prophylactic activity for a disease (hereinafter referred to as “active ingredient polypeptide c” for convenience) is also used for the preparation of the therapeutic or prophylactic agent of the present invention, similar to the active ingredient polypeptides a and b. Useful.

  Here, the “homology” of the amino acid sequences means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and the number of matched amino acid residues is defined as the total number of amino acid residues. It is the percentage divided by the number. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the homology (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing. The 20 amino acids constituting the natural protein are neutral amino acids having low side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), and neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) It is known that the properties of a polypeptide often do not change as long as it can be grouped and the substitution between them. Therefore, when substituting an amino acid residue in the polypeptide of (a) or (b) above, by substituting between each of these groups, the polypeptide used as an active ingredient is resistant to pruritic skin diseases. The possibility of maintaining therapeutic or prophylactic activity is increased.

  The polypeptide (c) is specifically a polypeptide having a sequence homology of 80% or more with a polypeptide consisting of the full length of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 (ie, Sema3A protein) Or a polypeptide having a sequence homology of 80% or more with a Sema3A protein fragment containing a 70aa region. Therefore, the active ingredient polypeptide c is a polypeptide consisting only of these polypeptides, or an amino acid or polypeptide added to one or both ends of the polypeptide of (c), and It is a polypeptide having therapeutic or prophylactic activity for erosive skin diseases. Specific examples thereof are the same as those of the active ingredient polypeptides a and b, and are not particularly limited. For example, the polypeptide having the full length of SEQ ID NO: 2 or 4 (ie, Sema3A protein) has a sequence homology of 80% or more. A polypeptide having 80% or more sequence homology with a Sema3A protein fragment having a therapeutic or prophylactic activity for pruritic skin disease, a region containing 70aa region and a region having 80% or more sequence homology And chimeric proteins in which other regions of secretory semaphorins other than Sema3A are fused, polypeptides in which various tags are added to the polypeptide of (c), and the like. These specific examples are also merely examples, and any amino acid or polypeptide added to the polypeptide of (c) is an active ingredient as long as it has therapeutic or prophylactic activity for pruritic skin diseases. Polypeptide c is included within the scope of the present invention.

  As the active ingredient polypeptide c, a polypeptide whose homology in the 70aa region is higher than the homology of the entire region comprising the polypeptide (c) is particularly preferable. For example, when the active ingredient polypeptide c is a polypeptide having 80% homology with the polypeptide having the amino acid sequence of SEQ ID NO: 2, those having a homology in the 70aa region of more than 80% are preferable. Polypeptides having homology in the region of 90% or more, more preferably 95% or more, further preferably 98% or more, and more preferably 70aa region are identical. Further, for example, the active ingredient polypeptide c is a chimeric protein in which a region having 80% sequence homology with the sema domain of human Sema3A protein and a region other than the sema domain of secreted semaphorin other than Sema3A are fused. In some cases, the homology in the 70aa region in the sema domain is preferably more than 80%, in particular, the homology in the 70aa region is 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably Chimeric proteins having the same sequence in the 70aa region are preferred.

  Among the active ingredient polypeptides a to c described above, in particular, 80% or more, preferably 90% or more, more preferably 95% or more, and further preferably, a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Is preferably a polypeptide having a homology of 98% or more. Among them, as described above, a polypeptide having a homology in the 70aa region higher than the homology of the entire polypeptide is preferable. Most preferably, the polypeptide contained as an active ingredient in the therapeutic or prophylactic agent of the present invention is a polypeptide having the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.

  In general, in a pharmaceutical comprising a polypeptide, a sugar chain or a polyethylene glycol (PEG) chain is added to the polypeptide or at least a part of the amino acids constituting the polypeptide in order to increase the stability of the polypeptide in vivo. A technique using a D-form amino acid is widely known and used. By adding sugar chains or PEG chains, or by using D-form amino acids as at least part of the amino acids that make up the polypeptide, it is less susceptible to degradation by peptidases in vivo, reducing the polypeptide in vivo by half. The period becomes longer. The polypeptide of the present invention may have these known modifications for in vivo stabilization as long as it has therapeutic or prophylactic activity for pruritic skin diseases. The term “polypeptide” in the scope is used in the sense to include those that have been modified for in vivo stabilization, unless the context clearly indicates otherwise.

  Glycosylation to polypeptides is well known, for example, Sato M, Furuike T, Sadamoto R, Fujitani N, Nakahara T, Niikura K, Monde K, Kondo H, Nishimura S., "Glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity. ", J Am Chem Soc. 2004 Nov 3; 126 (43): 14013-22, Sato M, Sadamoto R, Niikura K, Monde K, Kondo H, Nishimura S," Site- specific introduction of sialic acid into insulin. ", Angew Chem Int Ed Engl. 2004 Mar 12; 43 (12): 1516-20. The sugar chain can be bound to the N-terminus, C-terminus, or an amino acid therebetween, but is preferably bound to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide. The number of sugar chains to be added is preferably 1 or 2, and preferably 1. The sugar chain is preferably monosaccharide to tetrasaccharide, more preferably disaccharide or trisaccharide. The sugar chain can be bound to a free amino group or carboxyl group of the polypeptide directly or via a spacer structure such as a methylene chain having about 1 to 10 carbon atoms.

  Addition of PEG chains to polypeptides is also well known, e.g. Ulbricht K, Bucha E, Poschel KA, Stein G, Wolf G, Nowak G., "The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting Time: influence on clotting of the extracorporeal system and hemostatic parameters. ", Clin Nephrol. 2006 Mar; 65 (3): 180-90. and Dharap SS, Wang Y, Chandna P, Khandare JJ, Qiu B, Gunaseelan S, Sinko PJ, Stein S, Farmanfarmaian A, Minko T., "Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide.", Proc Natl Acad Sci USA. 2005 Sep 6; 102 (36): 12962-7. The PEG chain can be attached to the N-terminus, C-terminus, or the amino acid between them, and usually one or two PEG chains are attached to a free amino group or carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited, but is usually about 3000 to 7000, preferably about 5000.

  A method in which at least a part of amino acids constituting a polypeptide is in D form is also well known. For example, Brenneman DE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I., “Protective peptides that are orally active and mechanistically nonchiral. ", J Pharmacol Exp Ther. 2004 Jun; 309 (3): 1190-7, Wilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME.," Ethanol antagonist peptides: structural specificity without stereospecificity ., J Pharmacol Exp Ther. 2004 Jun; 309 (3): 1183-9. A part of the amino acids constituting the polypeptide may be D-form, but since the activity of the polypeptide is not inhibited as much as possible, all of the amino acids constituting the polypeptide are D-form amino acids rather than only a part of the amino acids. It is preferable that

  The above polypeptide used as an active ingredient in the present invention can be easily prepared by a conventional method using, for example, a commercially available peptide synthesizer. Moreover, it can prepare easily using a well-known genetic engineering method. For example, cDNA of Sema3A gene is prepared by RT-PCR from RNA extracted from tissue expressing Sema3A gene, and the full length or desired part of the cDNA is incorporated into an expression vector and introduced into a host cell. The desired polypeptide can be obtained. Extraction of RNA, RT-PCR, integration of cDNA into a vector, and introduction of a vector into a host cell can be performed by known methods. Further, vectors and host cells to be used are well known, and various types are commercially available. A method for producing a chimeric protein is also well known in this field. For example, a chimeric protein can be produced by the method described in the above-mentioned paper by Koppel et al. The stabilization modification can also be easily performed by a known method as described in each of the above documents.

  As shown in the Examples below, when Sema3A is administered to the skin area of AD model mice, the skin rash is improved and scratch marks are also reduced. In addition, at the histological level, improvement of epidermal thickening and reduction of infiltration of inflammatory cells such as lymphocytes and eosinophils are observed. Although the details of the mechanism of action of Sema3A on AD are unknown, the following may be considered. The pathological condition of AD is a complicated pathological condition including hyperimmunity, nervous system hypersensitivity, and abnormal skin growth / repair processes. In recent years, in AD, C-fibers are increased by nerve growth factor (NGF) produced from keratinocytes by scratching stimulation, resulting in a “itching irritability state” that induces further scratching and rash It is thought that the vicious circle that causes the worsening of hemorrhoids, that is, the itch-scratch circle, is caused, which causes chronic and intractable AD. Presuming that Sema3A is a repulsive nerve axon guidance molecule and a molecule involved in nerve axon elongation, Sema3A has some effect on the increase of C fibers and cuts off the itch-scratch circle This is considered to contribute to the treatment or prevention of AD. Such an action is different from the action of conventional steroids and antihistamines / allergic drugs, that is, the action of suppressing various inflammatory and inflammatory substances released as a result of allergic reaction. Therefore, the therapeutic or prophylactic agent of the present invention can be effective against refractory AD for which conventional standard drugs are ineffective. In recent years, it has been reported that Sema3A has a T cell inhibitory effect, and it is considered that allergic reaction may be suppressed by administration of Sema3A.

  In addition, itch-scratch circles are involved in most pruritic skin diseases in addition to AD, to varying degrees. Therefore, the above-mentioned polypeptide used as an active ingredient in the present invention is not limited to atopic dermatitis but also other pruritic skin diseases such as eczema / dermatitis group (contact dermatitis, sebum-deficient eczema, Vidal Lichen, etc.), prurigo group (eg, prurigo, pigmented prurigo), cutaneous pruritus, inflammatory keratosis with pruritus (eg, psoriasis vulgaris, lichen planus), systemic disease (endocrine metabolic disease (diabetes) It is also useful for the treatment or prevention of cutaneous pruritus associated with blood diseases, visceral malignant tumors, liver disorders, kidney disorders, psychosis, etc.). However, pruritic skin diseases caused by infectious diseases are mainly caused by infected pathogens, and the itch-scratch circle contributes little. Therefore, the pruritic skin disease targeted by the therapeutic or prophylactic agent of the present invention is a pruritic skin disease other than an infectious disease.

  The administration target of the therapeutic or prophylactic agent of the present invention is a mammal, and examples thereof include humans, dogs, cats, rabbits, and hamsters. The use of Sema3A derived from the same species as the patient to be treated or prevented is considered to have a high therapeutic or preventive effect, and is also desirable from the viewpoint of safety in clinical application. Therefore, for example, when the subject of administration of the therapeutic or prophylactic agent of the present invention is a human, the therapeutic or prophylactic agent containing the above-described active ingredient polypeptide b as an active ingredient is particularly preferable.

  The therapeutic or prophylactic agent of the present invention may consist of only the above-mentioned polypeptide, or a pharmacologically acceptable excipient, stabilizer, preservative, buffer, dissolution aid suitable for each dosage form. An additive such as an agent, an emulsifier, a diluent, an isotonic agent and the like can be appropriately mixed for preparation. Formulation methods and usable additives are well known in the field of pharmaceutical formulations, and any method and additive can be used.

The therapeutic or prophylactic agent of the present invention is used by local administration to the skin lesion to be treated or the skin to be prevented. Examples of the local administration method include intradermal administration using injections, drops, etc., and local administration using ointments, creams, patches, etc. Among them, intradermal administration by injection is preferable. The dose is appropriately selected according to symptoms, age, body weight, administration method and the like, and is not particularly limited. Usually, the amount of active ingredient for the target animal is about 1.5 U to 1.5 × 10 ⁶ U per day, particularly 1.5 ×. It is about 10 U to 1.5 × 10 ⁵ U, and is administered once or divided into several times. Here, “1 U” is a semaphorin titer that collapses 50% growth cones of NGF-sensitive DRG neurons with a stock solution of purified polypeptide solution obtained by a conventional method. It can be examined by assay. Preferably, depending on the degree of symptom improvement, it is administered once or several times daily for several days to several months, or regularly once or several times every other day.

  The therapeutic or prophylactic agent of the present invention may be used alone or in combination with other therapeutic or prophylactic agents for pruritic skin diseases. As described above, the therapeutic or prophylactic agent of the present invention can be used in combination with steroidal agents and antihistamine / allergic agents, which are conventional standard therapeutic agents, since they have different mechanisms of action.

  Hereinafter, the present invention will be described more specifically based on examples.

1. Materials and methods
(1) Animals Twenty-four NC / Nga mice (12 weeks old, male, Japan SLC) that developed dermatitis on the upper back were prepared, and all upper backs were shaved. This was divided into 4 groups so that the average severity of rash was uniform. Considering that atopic dermatitis is a disease involving not only constitution but also environmental factors, 24 animals were kept in the same cage over the whole experiment period in order to prepare a rearing environment. Individuals were identified by marking on the tail.

(2) Preparation of Semaphorin 3A A chicken semaphorin 3A stable expression strain was cultured, and the semaphorin 3A protein produced in the culture supernatant was purified by a conventional method. The semaphorin 3A stable expression strain is obtained by incorporating a chicken semaphorin 3A gene (SEQ ID NO: 3) into HEK293 cells.

  The activity of the obtained semaphorin 3A protein was measured by a collapse assay using the dorsal root ganglion of the chicken. The regression assay was performed by the following method.

  Dorsal root ganglia (DRG) were removed from embryonic day 7 chick egg embryos. The extracted DRG was placed on the bottom of a glass containing NGF-containing medium, and cultured in a static place for 24 hours in a 37 ° C. incubator. After confirming the growth of NGF-sensitive DRG neurons, the semaphorin 3A solution purified as described above was diluted 1-fold (stock solution) to 100,000-fold and administered at 37 ° C for 30 minutes. Fixed after ground culture. Under the microscope, calculate the percentage (%) of all NGF-sensitive DRG neurons whose growth cone is collapsed, and calculate the semaphorin 3A concentration (X-fold dilution) at which 50% collapse is observed. / ml.

  As a result of the regression assay, the semaphorin 3A titer obtained above was 30,000 U / mL. This semaphorin 3A was diluted 40 times with physiological saline to obtain a semaphorin 3A agent, which was used for administration to NC / Nga mice.

  The culture supernatant of HEK293T cells not transfected with semaphorin 3A was purified by the same procedure as described above. The purified product was subjected to a regression assay to confirm that there was no activity. The purified product was diluted 40-fold with physiological saline to serve as a control agent and used for administration to NC / Nga mice.

(3) Intradermal injection NC / Nga mice divided into 4 groups consisted of (i) no treatment group (n = 6), (ii) physiological saline administration group (n = 5), (iii) control administration group ( n = 7), and (iv) semaphorin 3A administration group (n = 6), and each of the groups in (ii), (iii) and (iv) had their physiology on the shaved upper back of the mouse. Saline 1 mL, control agent 1 mL, and semaphorin 3A agent 1 mL were injected intradermally. Intradermal injection was performed twice a day for 5 consecutive days. As described above, the semaphorin 3A agent was injected with 1 mL of a 40-fold diluted 30,000 U / mL semaphorin 3A as described above, so the amount of semaphorin 3A administered in one injection was about 750 U. .

  The day of the first intradermal injection was defined as the first day, and the upper back skin eruption was observed over the period from the first day to the eleventh day. The skin rash is a clinical skin score according to the method of Leung (Clin Exp Immunol 107 (Suppl. 1), 25-30, 1997), Suto et al. (Int Arch Allergy Immunol 120 (Suppl. 1), 70-75, 1999). It was evaluated with. That is, for each individual, the skin rash is divided into 4 items of erythema / bleeding, edema, erosion, desquamation / dryness, and each of the 4 levels from 0 to 3 (0 (none), 1 (mild), 2 (medium) Degree), 3 (severe)), and the total score was calculated. The average score was calculated for each group and used as the score for each group. In order to follow the progress of the eruption, the eruption was photographed with a camera once a day.

(4) Histological examination Histological examination was also conducted on the effect of semaphorin 3A. The skin tissue of the eruption was collected on the 11th day from each group of mice (i) to (iv) described above. As a non-onset skin tissue control, skin tissue was also collected from the upper back of NC / Nga mice that did not develop rash. The collected skin tissue was fixed in a 20% formalin solution for 24-48 hours and embedded in paraffin. This was cut to a thickness of 3 μm, stained with hematoxylin and eosin (HE), and observed with a microscope.

2. result
(1) Semaphorin 3A clinically improved rash in NC / Nga mice FIG. 1 shows the score evaluation results for the above four groups (i) to (iv). The cross symbol in FIG. 1 represents the death of the mouse. For example, in the untreated group, one individual died on the second day during the experimental period, and the number of individuals in the group reached 5 at the end of the observation.

  In the semaphorin 3A administration group (iv), improvement of the skin rash was observed immediately after administration. In contrast, in the other three groups, that is, the non-treated group (i), the physiological saline administration group (ii), and the control administration group (iii), no improvement of rash was observed. In addition, it was considered that the semaphorin 3A administration group had fewer scratch marks and less pruritus than the other groups. The effect of improving the eruption in the semaphorin 3A administration group lasted for about 3 days after the administration was completed on the fifth day, but from the eighth day, the eruption tended to slightly relapse. The effect of semaphorin 3A was local, and no change was observed in the skin eruptions on the face and auricle that were not injected intradermally.

(2) The semaphorin 3A administration group showed improvement even at the histological level. In the skin tissue of NC / Nga mice that did not develop rash, the epidermis was composed of 1 to 2 keratinocytes, with a slight dermal area. Cell infiltration was observed. In the untreated group, the physiological saline administration group, and the control administration group, thickening of the epidermis due to proliferation of keratinocytes and dense inflammatory cell infiltration into the dermis were observed. These findings were similar to the characteristic findings seen in human atopic dermatitis. On the other hand, in the semaphorin 3A administration group, the thickening of the epidermis was improved and the infiltration of inflammatory cells was also reduced. From the above, the effect of semaphorin 3A was confirmed also at the histological level.

The evaluation result of the clinical skin score about 4 groups of NC / Nga mice is shown. (i) is an untreated group, (ii) is a physiological saline administration group, (iii) is a control administration group, and (iv) is a semaphorin 3A administration group. The cross symbol in the figure represents the death of the mouse, and the letter attached to the cross symbol represents the group to which the dead mouse belongs.

Claims (7)

A therapeutic or prophylactic agent for pruritic skin disease comprising a region comprising the polypeptide of any of the following (a) to (c) and containing as an active ingredient a polypeptide having a therapeutic or prophylactic activity for pruritic skin disease .
(a) A polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 4 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
(b) A polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
(c) A polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b).   The therapeutic or prophylactic agent according to claim 1, wherein the polypeptide (c) is a polypeptide having 95% or more sequence homology with the polypeptide (a) or (b). The therapeutic or prophylactic agent according to claim 1, wherein the polypeptide having therapeutic or prophylactic activity for pruritic skin disease is the following polypeptide (a) or (b).
(a) A polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 4 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
(b) A polypeptide comprising a region of 166aa to 235aa in SEQ ID NO: 2 consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.   The polypeptide having the therapeutic or prophylactic activity for pruritic skin disease is a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing, and a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing The therapeutic or prophylactic agent according to any one of claims 1 to 3, which is a polypeptide having a sequence homology of 80% or more.   The polypeptide having the therapeutic or prophylactic activity for pruritic skin disease is a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing, and a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing The therapeutic or prophylactic agent according to claim 4, which is a polypeptide having a sequence homology of 95% or more with any of them.   The polypeptide having the therapeutic or prophylactic activity for pruritic skin disease is a polypeptide having the amino acid sequence shown by SEQ ID NO: 4 in the sequence listing, or a polypeptide having the amino acid sequence shown by SEQ ID NO: 2 in the sequence listing The therapeutic or prophylactic agent according to claim 5.   The therapeutic or prophylactic agent according to any one of claims 1 to 6, wherein the pruritic skin disease is atopic dermatitis.

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