WO2013005603A1

WO2013005603A1 - Agent for prevention and/or treatment of allergic inflammation in conjunctiva

Info

Publication number: WO2013005603A1
Application number: PCT/JP2012/066207
Authority: WO
Inventors: 純美田中; 信久水木; 良郎五嶋
Original assignee: 公立大学法人横浜市立大学
Priority date: 2011-07-01
Filing date: 2012-06-26
Publication date: 2013-01-10
Also published as: WO2013005603A9

Abstract

Provided are eye drops, and a subconjunctival injection drug for refractory allergic conjunctival disease, foremost for seasonal allergic conjunctivitis. This agent for prevention and/or treatment of allergic inflammation in the conjunctiva contains semaphorin-3A as an active component.

Description

Preventive and / or therapeutic agent for allergic inflammation in conjunctiva

The present invention relates to a preventive and / or therapeutic agent for allergic inflammation in the conjunctiva.

In recent years, allergic diseases represented by hay fever have increased, and this has become a major social problem involving about 30% of the people involved in multiple organs. About 85% of patients with allergic conjunctival diseases are estimated to be pollen, and severe cases exist at a rate of 1% or more (Non-patent Document 1). It has been pointed out that it is related to air pollutants as a factor in the increase of these allergic diseases.

Currently, there are nine types of antiallergic eye drops that can be prescribed, and antihistamine eye drops are the mainstream. However, non-steroidal eye drops are not effective enough. Allergic conjunctival disease is a general term for conjunctival diseases involving type I allergic reactions. Anti-allergic eye drops alone cannot suppress various lesions, and steroid eye drops have been prescribed (non-patent literature). 2).

On the other hand, although steroid eye drops have strong effects, they have strong side effects such as increased intraocular pressure in the eye, cataract, retinopathy, induction and exacerbation of infection, vascular abnormalities, skin symptoms, and hirsutism. It is not possible. For this reason, non-steroidal eye drops are being used as alternative treatments, and eye drops that are more effective than steroids and avoid side effects such as keratoconjunctival peripheral nerve sensory disturbance and conjunctival vascular abnormalities have been developed.

A typical semaphorin molecule Sema3A 機能 whose function in the immune system has been clarified has been energetically analyzed around the world for its function as a guidance factor in the nervous system (Non-patent Document 3), but immune cell migration The inhibitory effect has also been reported recently (Non-patent Document 4). Suppression of T cell receptor (TCR) signal pathway and MAP kinase signal pathway or inhibition of actin accumulation suppresses activation, and NP-1NP / pelexin-A4 complex is negative for Temacell immune response as a receptor for Sema3A (Non-patent Documents 5 and 6).

However, there is no report that the semaphorin molecule Sema3A is effective for allergic conjunctival diseases.

An object of the present invention is to provide eye drops and subconjunctival injections for intractable allergic conjunctival diseases including seasonal allergic conjunctivitis.

The present inventors have demonstrated that semaphorin 3A (Sema3A) molecule is effective as a novel therapeutic agent for severe and refractory allergic conjunctival diseases including seasonal allergic conjunctivitis due to its unprecedented pharmacological action in the ophthalmic field. As a result, the present invention has been completed. Sema3A is thought to be effective in preventing and / or treating allergic inflammation in the conjunctiva.

The gist of the present invention is as follows.
(1) A preventive and / or therapeutic agent for allergic inflammation in the conjunctiva, containing semaphorin 3A as an active ingredient.
(2) The preventive and / or therapeutic agent according to (1), wherein the allergic inflammation in the conjunctiva is seasonal allergic conjunctivitis.
(3) The preventive and / or therapeutic agent according to (2), wherein the seasonal allergic conjunctivitis is hay fever.
(4) A method for preventing and / or treating allergic inflammation in the conjunctiva, comprising administering to a subject a pharmaceutically effective amount of semaphorin 3A.
(5) Use of semaphorin 3A for prevention and / or treatment of allergic inflammation in the conjunctiva.
(6) Semaphorin 3A for use in a method for preventing and / or treating allergic inflammation in the conjunctiva.

The biologics of the present invention are greatly different from conventional antiallergic eye drops as follows.
・ It is an advantage that it is as safe and safe as other anti-allergic eye drops.
・ Maintain normal corneal tissue and corneal epithelium.
・ As a therapeutic agent, it does not cause side effects such as keratoconjunctival peripheral nerve sensory disturbance and conjunctival vascular abnormalities.
・ Maintains normal conjunctival epithelial and goblet cell function.
-Prevention and / or treatment of eosinophilic conjunctival vasculitis.
・ Can be used with other antiallergic eye drops.
・ Prevents dryness.
・ There are almost no side effects and immediate effects can be expected.
-Expected to be effective with Sema3A alone for severe allergic conjunctival disease.
・ Sema3A can be expected to be effective 2 times / day for 4 to 6 times / day of antiallergic eye drops.
• Initial therapy is effective twice a day.
・ No abnormalities in the anterior chamber. No abnormalities in intraocular tissues such as cornea, ciliary body, and lens. Does not cause secondary ciliary dysfunction or secondary glaucoma.
・ Does not cause secondary cataract than steroids.
-Less damage to intraocular tissues.
・ Because it contains no preservatives, there is little burden on the eyes even with frequent and relatively long-term instillation.
・ Can be used without worrying about side effects such as sleepiness.
・ It can be expected that the patient's QOL will be better than allergy medicine.

The biologic of the present invention has a pharmacological action that cannot be achieved with chemical substances.

This specification includes the contents described in the specification and / or drawings of the Japanese patent application, Japanese Patent Application No. 2011-147038, which is the basis of the priority of the present application.

Change in total clinical findings score. Change in eosinophil infiltration score. Immediate phase score (above) and late phase score (below). Eye drops a: 0.025% levocabastine hydrochloride ophthalmic solution, eye drops b: 0.02% fluorometholone ophthalmic solution, eye drops c: 0.5% tranilast ophthalmic solution, eye drops d: 0.1% olopatadine hydrochloride ophthalmic solution, eye drops e: 0.01 % Ibudilast ophthalmic solution, ophthalmic solution f: Semaphorin-3A 1000 U / ml ophthalmic solution. Results of T cell proliferation assay.

Hereinafter, the present invention will be described in detail.

The present invention provides a preventive and / or therapeutic agent for allergic inflammation in the conjunctiva, containing semaphorin 3A as an active ingredient.

The active ingredient semaphorin 3A may be a natural type or a mutant. Specific examples of natural semaphorin 3A or semaphorin 3A mutant include a region consisting of any of the following polypeptides (a) to (c), and have an activity of preventing and / or treating allergic inflammation in the conjunctiva: The polypeptide which has can be mentioned.
(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 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, “preventive and / or therapeutic activity of allergic inflammation in the conjunctiva” means an activity that improves or prevents the symptoms of the conjunctival lesion of allergic inflammation (eg, allergic conjunctivitis) in the conjunctiva. Means. Whether or not the polypeptide has the activity is evaluated by, for example, administering the polypeptide to the animal developing allergic conjunctivitis by instillation, subconjunctival administration, etc., and improving the symptoms of the lesion. Can be examined. As an animal having an allergic conjunctivitis, for example, a model animal such as a mouse having an allergic conjunctivitis described in Examples below can be used. This mouse was sensitized by administering ragweed pollen as an antigen, and caused allergic conjunctivitis by inducing antigen instillation. In addition to clinical symptoms such as tears, eyes, and hyperemia and edema, Eosinophils that have infiltrated into are observed. The evaluation of the prevention and / or treatment activity of allergic inflammation in the conjunctiva using this mouse can be carried out by, for example, applying polymorphism for a certain period of time by instillation and subconjunctival administration to the conjunctival lesion of the mouse, as specifically described in the following examples. This can be done by administering the peptide and evaluating the presence or absence of improvement in the symptoms of the lesion. The presence or absence of improvement of symptoms in the conjunctival disease area, for example, as described in the following examples, evaluate clinical symptoms such as hyperemia and edema, the number of eosinophils infiltrated in the conjunctiva as an index of conjunctivitis Can do. For example, compared to the average score value of the conjunctivitis symptoms of the allergic conjunctivitis onset group of the untreated allergic conjunctivitis, the average score value of the conjunctivitis symptoms of the allergic conjunctivitis onset group administered the polypeptide for a certain period significantly decreased, Alternatively, for example, when the average score of the polypeptide administration group is about two-thirds or less, preferably about one-half or less of the average score of the untreated individuals, the polypeptide is allergenic. It can be judged that it has the activity of preventing and / or treating conjunctivitis.

The number of eosinophils infiltrated into the conjunctiva is determined by removing the conjunctival tissue from animals with allergic conjunctivitis, preparing a specimen, confirming eosinophil infiltration with an optical microscope, and calculating the number. It can be carried out. For example, the average value of the number of eosinophils in the allergic conjunctivitis-onset population administered with the polypeptide for a certain period was significantly lower than the average value of the number of eosinophils in the untreated allergic conjunctivitis-onset population. Or, for example, when the average eosinophil count of the polypeptide administration group is about 2/3 or less, preferably about 1/2 or less of the average eosinophil count of the untreated population It can be determined that the polypeptide has a prophylactic and / or therapeutic activity for allergic conjunctivitis.

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 a Met７７Gly Trp Leu ... (omitted) ... Pro Arg Ser Val amino acid sequence of 771 amino acid residues Means polypeptide.

The amino acid sequences shown in SEQ ID NOs: 2 and 4 are semaphorin 3A protein sequences (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 of ~ 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-inhibiting action is a region of 70 amino acids corresponding to the 166th to 235th of the sema domain (Koppel et al., Neuron , Vol. 19, 531-537, 1997). The 70 amino acid region (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 a polypeptide contained as an active ingredient in the preventive and / or therapeutic agent of the present invention includes a region comprising the polypeptide of (a) and has an activity of preventing and / or treating allergic inflammation in the conjunctiva. (Hereinafter referred to as “active ingredient polypeptide a” for convenience). The active ingredient polypeptide a may be composed only of the polypeptide (a) as long as it has preventive and / or therapeutic activity for allergic inflammation in the conjunctiva, and may be one end of the polypeptide (a). Alternatively, an amino acid or polypeptide added at both ends may be used.

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 preventive and / or therapeutic activity for allergic inflammation in the conjunctiva is as specifically described in the following examples. In addition, even if a fragment lacking a part of the N-terminus or C-terminus of chicken Sema3A protein has the activity of preventing and / or treating allergic inflammation in the conjunctiva, it is within the scope of the present invention as an active ingredient polypeptide a. Is included.

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. Therefore, such a chimeric protein also has the activity of preventing and / or treating allergic inflammation in the conjunctiva, and thus is included in the scope of the present invention as the active ingredient polypeptide a. Furthermore, Sema3A lacking the Ig domain also has DRG regression activity (Koppel et al., 1997), but such a polypeptide is also present in the region of Sema3A containing 70aa at the N-terminal side of 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) can be used as long as it has preventive and / or therapeutic activity for allergic inflammation in the conjunctiva. The active ingredient polypeptide a is included in 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 a polypeptide contained as an active ingredient in the preventive and / or therapeutic agent of the present invention includes a region consisting of the polypeptide (b), and prevents and / or treats allergic inflammation in the conjunctiva. A polypeptide having activity (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 allergic conjunctivitis in mice that are not chickens, so chicken sema3A can be used to treat allergic conjunctivitis other than chickens and mice such as humans. It is also possible to prevent. Therefore, the active ingredient polypeptide b such as human Sema3A protein is also useful for the preparation of a preventive and / or therapeutic agent for allergic inflammation in the conjunctiva such as allergic conjunctivitis, like the above active ingredient polypeptide a such as chicken Sema3A protein. It is. 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 preventive and / or therapeutic activity for allergic inflammation in the conjunctiva. In addition, 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), allergic inflammation in the conjunctiva, is not particularly limited. Examples thereof include fragments thereof having prophylactic and / or therapeutic activity, chimeric proteins according to the above embodiment with secreted 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) can be used as long as it has the activity of preventing and / or treating allergic inflammation in the conjunctiva. The active ingredient polypeptide b is included in 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 consisting 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, it includes the region consisting of the polypeptide of (c) above, and is allergic in the conjunctiva. A polypeptide having preventive and / or therapeutic activity for inflammation (hereinafter referred to as “active ingredient polypeptide c” for convenience) is also prevented and / or treated according to the present invention in the same manner as the active ingredient polypeptides a and b. Useful for the preparation of agents.

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), 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 amino acid residues in the polypeptide of (a) or (b) above, by substituting between these groups, allergic inflammation in the conjunctiva of the polypeptide used as the active ingredient The possibility of maintaining preventive and / or therapeutic 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 (c), and the conjunctiva A polypeptide having an activity of preventing and / or treating allergic inflammation in 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 preventive and / or therapeutic activity for allergic inflammation in the conjunctiva, 80% or more sequence homology with a region containing 70aa region Examples include chimeric proteins in which a region and other regions of secreted 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) may have any activity for preventing and / or treating allergic inflammation in the conjunctiva. The active ingredient polypeptide c is included in the scope of the present invention.

The active ingredient polypeptide c is particularly preferably a polypeptide whose homology in the 70aa region is higher than the homology of the entire region comprising the polypeptide (c). 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 preventive and / or therapeutic 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 used in the present invention may be subjected to these known modifications for in vivo stabilization as long as it has preventive and / or therapeutic activity for allergic inflammation in the conjunctiva. The term “polypeptide” in the claims and in the claims is used to include those 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 sialylolulins displaying 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. Ullbricht K, Bucha E, Poschel KA, Stein G, Wolf G, Nowak G., '' The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting influence on clotting of the extracorporeal system and hemostatic parameters. ", Clin Nephrol. 2006 Mar; 65 (3): 180-90., Dharap SS, Wang Y, Chandna P, Khandare JJ, Qiu B, Gunaseelan S, Sinko 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 3000 to 7000. Degrees, preferably those of about 5000 is used.

A method in which at least a part of amino acids constituting a polypeptide is D-form is also 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 and Wilkemeyer MF, Chen SY, Menkari CE, Sulik KK, Charness ME.," Ethanol antagonist peptides: structural specificity without "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

Semaphorin 3A 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, a Sema3A gene cDNA is prepared by RT-PCR from RNA extracted from a tissue expressing the Sema3A gene, and the full length or a desired part of the cDNA is incorporated into an expression vector and introduced into a host cell. The desired polypeptide can be obtained. RNA extraction, RT-PCR, cDNA integration 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.

Semaphorin 3A, which is an active ingredient in the preventive and / or therapeutic agent of the present invention, is effective in treating and / or preventing allergic inflammation in the conjunctiva. Allergic inflammation in the conjunctiva includes eosinophilic conjunctival disease, allergic conjunctivitis (including seasonal allergic conjunctivitis such as pollen conjunctivitis), allergic keratoconjunctivitis, allergic blepharitis, hay fever, atopic keratoconjunctivitis Semaphorin 3A is useful for preventive initial treatment (therapy) of these diseases, pre-seasonal administration and / or therapeutic agent conservative treatment, maintenance therapy / induction therapy. is there.

The subject of administration of the preventive and / or therapeutic agent of the present invention is a mammal, and examples thereof include humans, dogs, cats, rabbits, hamsters and the like. The use of semaphorin 3A derived from the same species as the patient to be prevented or treated is considered to have a higher prevention or treatment effect, and is also desirable from the viewpoint of safety in clinical application. Therefore, for example, when the subject to which the preventive and / or therapeutic agent of the present invention is administered is a human, the prophylactic and / or therapeutic agent containing the active ingredient polypeptide b as an active ingredient is particularly preferable.

The preventive and / or therapeutic agent of the present invention may consist of semaphorin 3A alone, or a pharmaceutically acceptable excipient, stabilizer, preservative, buffer, suitable for each dosage form, Additives such as solubilizers, emulsifiers, diluents, tonicity agents and the like can be mixed as appropriate to prepare a preparation. Formulation methods and usable additives are well known in the field of pharmaceutical preparations, and any method and additive can be used.

The preventive and / or therapeutic agent of the present invention is used by administering to the conjunctival part to be prevented or the conjunctival lesion part to be treated. Examples of the administration method include local administration (instillation on the conjunctiva, administration by subconjunctival injection, etc.) and the like. Examples of the dosage form include eye ointments, injections, eye drops and the like. The dosage is appropriately selected according to symptoms, age, body weight, administration method and the like, and is not particularly limited. In the case of eye drops, the amount of active ingredient is usually about 6000 to 60000 U per day for the target animal, preferably In the case of subconjunctival injection amount, the amount of active ingredient is usually about 500-5000 U per day, preferably about 500-5000 U, and divided into 1 to several times. Administered. 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. Here, “1 U” is a semaphorin titer that collapses a growth cone of 50% of NGF-sensitive DRG neurons with a stock solution of a purified polypeptide solution obtained by a conventional method. collapse assay) (Luoet al., Cell vol.75, 217-227, 1993).

The preventive and / or therapeutic agent of the present invention may be used alone or in combination with other prophylactic and / or therapeutic agents. The prophylactic and / or therapeutic agent of the present invention can be used in combination with anti-histamine eye drops, non-steroid eye drops and steroid eye drops, which are conventional anti-allergic eye drops.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[Example 1]
(Materials and methods)
Ragweed pollen (short ragweed pollen: RW) was used as an antigen and an emulsion with aluminum hydroxide (Alum) was prepared and administered intraperitoneally twice to BALB / c mice at 10-day intervals. Instillation of RW after sensitization induces allergic conjunctivitis (EC) in mice, and the immediate phase observes and evaluates clinical scores 30 minutes after instillation, collects conjunctiva 24 hours after instillation, and infiltrate eosinophils Numbers were counted. As a control, normal mice were instilled with PBS. The late phase was sampled one week after the start of instillation. The number of infiltrating eosinophils was counted.

1. For animal experiments, BALB / cCrSlc female mice purchased from Japan SLC Co., Ltd. (Hamamatsu, Japan) at the age of 5 weeks were used. Mice are raised in a constant temperature and humidity chamber at room temperature 25 ± 1 ° C and humidity 55 ± 5% under light and dark conditions with a 12-hour cycle, and with standard mouse food (solid feed: 5LJ1 (PMI FEEDS) and sterile water) There were 6 mice per group, and a total of 120 mice were used.

2. Induction of allergic conjunctivitis Induction of allergic conjunctivitis (EC) by active immunity. Ragweed pollen (RW) was induced in the abdominal cavity by active sensitization by administration twice in total at 10-day intervals and by instillation challenge with an antigen solution.
・ Allergic conjunctival disease induction method: Alum and emulsion were prepared using RW as an antigen. RW was purchased from Polysciences (Warrington, Pennsylvania, USA). Aluminum hydroxide was purchased from Sigma (St. Louis, Missouri, USA).
-Whole body sensitization was performed by intraperitoneal injection of ragweed pollen (RW). Active sensitization by administration twice at a 10-day interval in the abdominal cavity, antigen RW was dissolved in PBS 10 days after systemic sensitization and challenged by instillation to induce allergic conjunctivitis. The tissue sample was taken after 24 hours. The late phase was sampled one week after challenge. Conjunctival inflammation such as hyperemia, edema, tears, and eyes was observed for 30 minutes after instillation and evaluated.

Reference (* Tomokazu Hosokawa et al. Effects of diesel exhaust exposure on allergic conjunctivitis induced by cedar pollen http://www.env.go.jp/chemi/report/h16-13/02_2_2.pdf)
(* Takamura et al. Allergy / Immunology Vol.15, No3,2008)

3. Sema3A administration
Semaphorin 3A was prepared as previously reported (Journal of Investigative Dermatology (2008) 128, 2842-2849). Briefly, a chicken semaphorin 3A stable expression strain was cultured, and 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 extracted 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 that NGF-sensitive DRG neurons are growing, the semaphorin 3A solution obtained by purification as described above is 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 that have collapsed growth cones, and determine the semaphorin 3A concentration (X-fold dilution) at which 50% collapse is observed. U / ml.

As a result of the regression assay, the titer of semaphorin 3A obtained above was 30,000 μU / mL.

Semaphorin-3A was instilled twice daily on both eyelids at a concentration of 10,100,1000 units. The antigen solution was made into a single eye drop of 10 μl μl. For subconjunctival administration, 2 μl was administered subconjunctivally to both eyes of the animal at intervals of 1 day.

4). In the control group, PBS was administered at the same use amount. The clinical score group was observed over 4 weeks.

5. Sampling was performed in PBS, eye drops, and subconjunctival administration groups for 6, 10, and 14 days, for a total of 3 times.

6). Confirmation of suppression of immune T cell proliferation The spleen was extracted from normal BALB / c mice. Prepare spleen cells and seed 100 mL of spleen cells of appropriate concentration (6.25 × 10 ⁵ cells / mL to 2.5 × 10 ⁶ cells / mL) onto a T cell plate coated with anti-CD3 antibody (BD Pharmingen). Semaphorin-3A was administered at a concentration of 10,100,1000 units, and the control group was administered PBS.

The cells were cultured in a CO ₂ incubator at 37 ° C. for 45 to 48 hours. (Use of BD Biocoat TM T cell activation plate (anti-mouse CD3) on plate (anti-mouse CD3))
The OD at a measurement wavelength of 490 nm was measured with a microplate spectrophotometer at a control wavelength of 700 nm. In vitro, Sema3A was confirmed to suppress T cell proliferation (FIG. 4). It was revealed that T cells induce eosinophils. (Yuki Fukushima: Clinical study of allergies 28 (4), 2008).

The semaphorin molecule group has heretofore been known as a nerve guidance factor, but recently, it has become clear that various semaphorin molecules are also involved in the immune system. It has long been known that the nervous system and the immune system have a lot in common. In particular, semaphorin molecules involved in antigen-presenting cell-T cell interactions in the immune system have been discovered. It has been confirmed in vitro that a semaphorin 3A molecule is coated with a T cell receptor complex antibody to induce T cell activation and suppress T cell proliferation.

Tordjman et al. Discovered that Neuropilin-1 was found in human dendritic cells and resting T cells, T cells bound to Neuropilin-1-expressing Cos7 cells, and Neuropilin-1 was CD3 at the interface between dendritic cells and naive T cells. And co-localize. Anti-Neuropilin-1 antibody also suppresses T cell proliferation by dendritic cells. The semaphorin 3A molecule, which is a glycan in the nervous system, has the effect of suppressing the migration of immune cells, but its action cannot be inhibited by an antibody against Neuropilin. Therefore, Sema3A suppresses the proliferation of T cells in the immune system. It is suggested that it is carried by different receptors.

7). Sema3A ophthalmic solution is confirmed to be fast-acting The effect on allergic conjunctivitis is Sema3A 1000 U / ml ophthalmic solution, histamine H1 receptor antagonist 0.1% olopatadine hydrochloride ophthalmic solution, 0.025% levocabastine hydrochloride ophthalmic solution, mediator release inhibition RW antigen solution was instilled 15 minutes after instillation of the drug 0.5% tranilast ophthalmic solution, 0.01% ibudilast ophthalmic solution, steroid drug 0.02% fluorometholone ophthalmic solution or PBS (control), and the conjunctivitis and conjunctivitis inhibitory effects were compared. Subsequent conjunctivitis symptoms due to antigen induction were observed at each measurement time point, and a conjunctivitis score was assigned.

Conjunctivitis symptoms such as hyperemia, edema, eyes, and tears were observed for 30 minutes after instillation, and evaluated according to the scores for conjunctivitis symptoms shown in Table 1. The amount of instillation was 1 10 μ, and the animals were instilled twice daily for 6 days in total.

In the immediate phase score, Sema3A was ± 0 for control 3+ (moderate), eye drops for hyperemia, edema, eyes and tears 1 to 2+, suggesting a therapeutic effect (upper figure 3).

In the late phase score, control 4+ (severe), eye drops were congested, edema, eyes and tears 2 to 3+, Sema3A was 2+, suggesting a therapeutic effect corresponding to 0.02% fluorometholone (Fig. 3) under).

8). Removal of the conjunctival tissue and histopathological analysis For eyes containing the conjunctiva, the hair around the eyelid is shaved off, the area around the eyelid is cut to a width of about 5 mm, and the tip of the eyelid is left behind to leave about 2 mm. It was. The entire eyelid and bulbar conjunctival lamina were fixed with 10% buffered formalin.

Giemsa staining Confirmed infiltration of eosinophils in the mucosa, sections were counted in two observers given blinded samples.

The count section was the range including the pupil and optic disc from the center. Data were examined as mean ± standard error of all mice, one-way analysis of variance and Tukey-Kramer multiple comparison test.

Fluorescent staining
Sema3A tissue storage confirmation.

Mouse conjunctiva was frozen in liquid nitrogen embedded in OCT compound (VWR, Sawanee, GA, USA). This was prepared and fixed with methanol.

Primary antibody (rabbit-derived polyclonal antibody) Anti-Semaphorin 3A
Manufacturer Abcam, Cambridge
Secondary antibody (derived from chicken) Rabbit antibody Alexa 488 fluorescent label (green)
Manufacturer Molecular Probes-Invitrogen
Nuclear staining
To-PRO-3 iodide (blue)
Manufacturer Molecular Probes-Invitrogen
Cytoskeleton (actin labeling)
Alexa Fluor 546 phalloidin (red)
Manufacturer Molecular Probes-Invitrogen

9. Statistical processing The experimental results were expressed as mean ± standard error (SE). All groups consisted of 6 animals. For statistical processing, multiple tests by Dunnett's method were performed.

(result)
Effect on mouse experimental allergic conjunctivitis model
-As a result of the above experiments, the inflammatory tissue image of the eye conjunctiva was found to have inflammatory cell infiltration, hemorrhagic tendency and lymphatic vessel dilation mainly consisting of eosinophils. An increase in the conjunctivitis score mainly due to inflammatory cell infiltration based on the allergic reaction and an increase in eosinophils and T cells in the conjunctiva were confirmed. The model was found to be similar to human allergic conjunctivitis.
・ Sema3A instillation and subconjunctival administration showed a clear therapeutic effect in the RW allergic conjunctivitis model, which suppressed the increase in conjunctivitis score and decreased the increase in eosinophils and T cells at the same site.
・ Scores for allergic conjunctivitis symptoms (Table 1) were statistically calculated using a total of 5 items. Differences in clinical symptom suppression were confirmed over the 4-week observation period. There was no significant change from week 3 to week 4 (FIG. 1).
・ Significant difference in eosinophil infiltration score was observed in Sema3A concentration difference. (Figure 2)
Furthermore, a significant difference between the ophthalmic group and the subconjunctival administration group was confirmed by the difference in the eosinophil infiltration score.
・ Fluorescence staining demonstrated that Sema3A has good conjunctival tissue migration.
-The immediate effect of Sema3A was confirmed by administration of antiallergic ophthalmic solution histamine H1 receptor antagonist and mediator release inhibitor, steroid eyedrops, and Sema3A (FIG. 3).
-In vitro, suppression of T cell proliferation by Sema3A was confirmed (Fig. 4).
Instillation and subconjunctival administration of Sema3A showed a clear therapeutic effect in the RW allergic conjunctivitis model.
・ Score for allergic conjunctivitis symptom (Table 1) was statistically made up of a total of 5 items, and improvement of clinical symptoms was confirmed in the observation period of 4 weeks (Fig. 1).

1) As receptors for semaphorin molecules, molecular groups belonging to the Neuropilin and plexin families are known. Neuropilin-1 is one of the receptors for VascularelEndothelial Growth Factor (VEGF), and Sema3A has been reported to antagonistically inhibit angiogenesis, but Sema3A 1000 units are instilled and conjunctival function by subconjunctival administration in this study. Insufficiency, corneal damage caused by it, and new blood vessels were not observed during the experiment, so suppression of corneal new blood vessels is expected.

2) Although application of Sema3A has been reported, such as nerve guidance and atopic dermatitis, in fact, the incidence of eosinophilic inflammatory diseases in the mucous membrane is high in the United States and Europe, and the disease concept has been established, pathological analysis and treatment Efforts are being made to establish the law. Since the Sema3A molecule did not show a pathological change at the tissue level due to administration to the conjunctiva, a therapeutic effect is expected for the above-mentioned eosinophilic inflammatory mucosal disease.

(Discussion)
Currently, there are three types of anti-allergic eye drops that can be prescribed, including histamine H1 receptor antagonists (3 types), mediator release inhibitors (6 types), steroid drops, oral drugs, and immunomodulator drops. Often recombination is also required.
Non-steroidal eye drops are not effective enough. Prolonged use causes refractory corneal epithelial disorder and dry eye. Steroid eye drops cause increased corneal lesions due to increased intraocular pressure, secondary glaucoma, secondary cataracts, and susceptibility to infection, leading to the risk of corneal transplantation and the final surgery.
It is a chronic disease that repeats inflammation, the highest rate of all symptoms of allergic disease, more than 90%, and because there are many complications with other allergic diseases (nose, trachea), etc. It causes a major hindrance to life and lowers the patient's QOL.

In order to overcome the above risks, biologics have a pharmacological action that cannot be achieved with chemical substances, and are expected to become eye drops with a fast-acting effect because they can be presumed to have little damage to intraocular tissues. The
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

The present invention can be used for the prevention and / or treatment of allergic inflammation in the conjunctiva.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the DNA sequence of the human semaphorin 3A gene.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the amino acid sequence of human semaphorin 3A.
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the DNA sequence of the chicken semaphorin 3A gene.
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the amino acid sequence of chicken semaphorin 3A.

Claims

A prophylactic and / or therapeutic agent for allergic inflammation in the conjunctiva, comprising semaphorin 3A as an active ingredient.
The preventive and / or therapeutic agent according to claim 1, wherein the allergic inflammation in the conjunctiva is seasonal allergic conjunctivitis.
The preventive and / or therapeutic agent according to claim 2, wherein the seasonal allergic conjunctivitis is hay fever.
A method for preventing and / or treating allergic inflammation in the conjunctiva, comprising administering to a subject a pharmaceutically effective amount of semaphorin 3A.
Use of semaphorin 3A for the prevention and / or treatment of allergic inflammation in the conjunctiva.
Semaphorin 3A for use in a method for preventing and / or treating allergic inflammation in the conjunctiva.