TW201006489A - S/O type transdermal immunizing agent - Google Patents

Abstract

The invention provides a non-invasive transdermal immunizing technology, in which the antibody production in the serum can be enhanced without causing inflammation or lumps on the skin as the existing transdermal immunization method. The S/O type transdermal immunizing agent comprising a solid antigen-surfactant complex, in which an antigen is coated with a surfactant, and an oily phase, where the solid antigen-surfactant complex is dissolved or dispersed in the oily phase.

Description

201006489 IX. Description of the invention: , [Technical field to which the invention pertains] The present invention relates to an s/o type transdermal immunizing agent, in particular, to prevent inflammation of the skin due to percutaneous immunity and to improve antibody production. s / ο type transdermal immunization agent. [Prior Art] In addition to being a physical and chemical barrier capable of invading a variety of Φ-infected cells containing bacteria or viruses, the skin tissue has a physico-chemical barrier that prevents a part of the pathogen from being administered subcutaneously. The role of so-called immune function specificity in the production of antibodies. However, transdermal immunization with subcutaneous injection requires not only a high-purity antigen produced by aseptic means, but also a strong side effect, a secondary infection due to acupuncture, and the like, and there is inflammation in the tissue administered subcutaneously. , the problem of generating hard blocks.

Rain, in place of the subcutaneous immunization method of subcutaneous administration of an injection needle, it has been revealed that the stratum corneum located at the outermost layer of the epidermal tissue is dissolved in some way (patent document, perforation of tiny protruding needles) (Patent Document 2), scratching (Patent Document 2), etc. However, these methods are the same as using the injection needle 'as for subcutaneous injection, and (4) is for the physical invader of the stratum corneum with barrier action. In the case of the above-mentioned non-invasive percutaneous immunization method in which the antigen is not physically invaded by the skin, a method of using the poison of the pathogenic microorganism is disclosed (for example, Patent Documents 4 to 7, Non-Patent Documents i to 3, etc.) ), however, in the use of toxins derived from pathogenic microorganisms, there is a question about safety 2226-9899-PF 5 201006489

On the other hand, in the case of a preparation which mainly enhances the absorption of an enzyme or a physiologically active peptide or a hydrophilic agent, a preparation of s/〇 (oil-packed solid, s〇i id h οι 1) is known. The S/0 type preparation is a preparation in which a drug coated with a surfactant is dispersed and dissolved in an oily substrate (oil phase) such as soybean oil, and the drug is completely dissolved in the oil phase. / 〇 type preparation, or S / 〇 type preparation in which the drug is dispersed in the suspended state of the oil phase. - Figure 1 shows a schematic view of the composition of the S/o type preparation 10. As shown in Fig. J, the drug 1 is present in the form of a drug-containing (tetra) complex (solid phase) 3 coated with a molecule of interfacial 胄2, and the drug-containing complex 3 is dispersed in an oily substrate (oil phase). The state of 4 exists. Such an s/〇 type preparation can be obtained by a squatting method: a W/Q type emulsion obtained by mixing an aqueous solution containing a drug and an organic solvent solution containing a surfactant, and drying, to obtain a drug interface. The drug-containing complex coated with the active agent 2 (the solid solution reads and dissolves in the oil phase. The S/G type preparation, the phase (4) emulsion (four) or the microlipid body has the following advantages: (4) because the drug is in a solid phase form If it exists, it is stable in hydrolysis, (b) excellent in thermal stability, (C) can be stored at room temperature for a long period of time, (d) can be formulated into a patch, etc. Long-term / so far, it has been proposed that if the above 5/〇 is used According to the chemical technology, the hydrophilic agent can be dissolved in the oily substrate, and the drug (leakage) can be prevented from becoming a s/() agent which is suppressed by the leakage of the hydrophilic agent. The inventor of the present invention is also interested in SS/0 type preparation. Among them, the specially recorded white matter composition or iridium dihydrochloride ". contains the egg irinotecan (irinotecaii iiCl) and other eucalyptus

2226-9899-PF 6 201006489 (camptcrthecine) A s/〇 type preparation of a drug of a derivative, or a s/〇/w type preparation obtained by further dispersing the s/〇 type preparation in an aqueous phase. Further, Patent Document 9 discloses that a mixture of a hydrophilic low molecular weight drug and a hydrophilic drug leakage inhibiting 14 protein and/or a drug leakage inhibiting polysaccharide (stabilizing agent) is coated with a surfactant. / Deuteration technology, in which the leakage of low molecular weight agents in a strong acid environment is significantly reduced, in contact with intestinal tubes in a weakly acidic to neutral environment, low molecular weight agents

The release is significantly promoted by the 1S/Q technique, especially for water-soluble non-steroidal anti-inflammatory analgesics (hereinafter referred to as "NSAIDs"), such as CDielofenac S〇dium (DFNa) or aspirin. It is extremely effective to have a serious gastric dysfunction disorder such as a gastric ulcer. Further, Patent Document 1 discloses an external preparation for improving the skin penetration of a hydrophilic drug such as NSAID. Patent Document 1 Patent Document 2 Patent Document 3 Patent Document 4 Patent Document 5 Patent Document 6 Patent Document 7 Patent Document 8 曰本特表20 02-504522 曰本本表 2005-334594号曰本本表2005-51 Japanese Laid-Open Patent Publication No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei.

Patent Document 9 · International Publication No. 2005/094789 Booklet Patent Document ίο · International Publication No. 2006/025583 Booklet Non-Patent Document 1. F. Mawas et al., The JDuriml of Infection 2226-9899-PF 7 201006489

Disease, 190; 1177-1182.2 004 Non-Patent Document 2: M. Guebre-Xabler et al_, J〇urnai 0f Virology, 78: 7640-7618, 2004 Non-Patent Document 3. GM Glennetal·, Nature. Med. 6 (12 ): 1403-1406, 2000 [Summary of the Invention] [The subject to be solved by the invention]

SUMMARY OF THE INVENTION An object of the present invention is to provide a non-invasive percutaneous immunological technique which does not cause inflammation or lumps of the skin such as the stratum corneum by conventional percutaneous immunization using subcutaneous administration, and can improve the production of antibodies in serum. In particular, it is an object of the present invention to provide a novel transdermal immunization technique which does not dissolve the stratum corneum which acts as a physicochemical barrier in the outermost layer of epidermal tissue, as disclosed in the prior art. The use of physical invasive methods to destroy, without the use of toxins derived from pathogenic microorganisms as a high-safety non-invasive light percutaneous immunization method, because of the excellent antibody production ability, can effectively perform transcutaneous immunization. [Means for Solving the Problem] The present invention can solve the above-mentioned problems. The Q/n*丨丨α. 4 I-supplemented S/〇-type transdermal immunizing agent mainly consists of containing the antigen through the interface. a solid antigen-surfactant complex formed by coating with a surfactant, and a saponin and an oil phase, and the solid antigen-surfactant complex is dissolved or dispersed in an oil phase . In a preferred embodiment, the bacterium that is poisoned or inactivated in the preferred embodiment and/or the antigen is a protein or a peptide. Disease The above antigen is a living bacterium and/or virus. 2226-9899-ρρ 8 201006489 In a preferred embodiment, the antigen is an animal source or a plant source. In a preferred embodiment, the hlb value of the above surfactant is i 〇 or less. In a preferred embodiment, the oil phase is at least one selected from the group consisting of vegetable oils, animal oils, neutral lipids, and long-chain fatty acid esters. The present invention also encompasses the S/0/W type transdermal immunizing agent which is obtained by dispersing the above s/o type transdermal immunizing agent in an aqueous phase. [Effects of the Invention] According to the present invention, not only can the antibody production be improved in the same manner as the conventional percutaneous injection method using subcutaneous injection, but also problems caused by subcutaneous injection (inflammation of the skin tissue, occurrence of acupuncture accident, physicochemical property) The dissolution or destruction of the barrier stratum corneum, etc.) is completely eliminated. Therefore, the transdermal immunizing agent of the present invention is very useful as a highly safe non-invasive immunogenic preparation or vaccine. The transdermal immunizing agent of the present invention is useful not only for the defense against pathogenic bacteria or viruses but also for the treatment of elimination from the body, and is also used in the treatment of inducing immune tolerance by dividing a small amount of antigen into several transdermal administrations. it works. [Embodiment] The transdermal immunizing agent of the present invention is characterized in that the antigen s is deuterated by the S/0 technique described in the above Patent Document 7. The antigen of a hydrophilic polymer represented by a protein or the like is originally very low in skin penetration, but in the present invention, 'the antigen is soluble in an oily substrate by the s/o technique', so the skin penetration of the antigen Sexual improvement, taking into account the inhibition of skin inflammation, with 2226-9899-PF 9 201006489 can be considered to play a good antibody production capacity. Specifically, as shown in the examples below, the lysozyme (by lysin) is used as the antigen to coat the lysozyme antigen with a surfactant to form a w/〇 type emulsion to change the antigen to hydrophobicity. The S/O-derived transdermal immunosuppressant (s/o antigen) dispersed in an oil phase (dispersion medium) such as vegetable oil is applied to the auricle or back of the rabbit to perform transcutaneous immunization. Compared to the conventional subcutaneous immunization method, no inflammation or lumps occur under the skin, and an antibody specific for chicken lysogenase is produced in the serum. The s/o type transdermal immunizing agent of the present invention comprises a solid antigen-surfactant complex (A) obtained by coating an antigen (A_n by a surfactant (A-2), and an oil phase (B) Solid antigen-surfactant complex (A), dissolved or dispersed in the oil phase (preferably, antigen-surfactant complex (A), containing hydrophilic protein and/or hydrophilicity The saccharide (A-3) is used as a stabilizer. The preparation of the present invention is substantially the same as the S described in the above Patent Document 7, except that the s/〇 type I towel described in the above Patent Document 8 is replaced with an antigen. The composition of the /0 type is the same. Hereinafter, each constituent element will be specifically described. '(A) Antigen-surfactant complex antigen-surfactant complex (hereinafter sometimes referred to as an antigen-containing complex) containing an antigen and The surfactant is a main constituent component. The hydrophilic portion of the surfactant is concentrated on the antigen and surrounds the periphery. The antigen-surfactant complex may be composed only of an antigen and a surfactant in order to prevent the particles from being excessively large. Can also contain It is described as 2226-9899, PF 10 201006489 An adjuvant (an adjuvant, as described later) for use as a stabilizer or an antibody production enhancer, and may also contain a formulation component (additional component, as will be described later) which is acceptable in a pharmaceutical preparation. A-1) Antigen The antigen used in the present invention is a substance that causes an immune response to the skin, and is not particularly limited as long as it can induce immunogenicity (antibody production), and an antigen or vaccine used in conventional transcutaneous immunization or the like can be used. An immunogenic substance, etc., for example, a protein or a peptide such as a physiologically active substance (for example, a lysozyme derived from chicken protein, an antigenic peptide of cedar pollen, etc.), a half of a binding protein or a peptide An antigenic substance, a living or inactivated (dead) bacteria or virus (all) or a partial decomposition product thereof, and a cancer cell (all) or a partial decomposition product thereof, a nucleic acid, etc. which are deactivated (dead), etc. A person (for example, a saccharide, a lipid, an inorganic substance, or the like) can be used as an antigen by a method such as haptenation, or can be used as an antigen. The antigen can be used as an animal source or a plant = for example, the antigen can be home. For poultry, livestock antigens, pets, for cattle antigens, difficult antigens, pig antigens, or for the source of the snails. Also, the antigen may be the source of food. (A - 2) Surfactant Restriction ::Γ: As long as it is acceptable in medical preparations, there is no special: nonionic surfactant, anionic surfactant, cationic interfacial acid salt. w扪 Injectable surfactant, bile nonionic Surfactant, ester, ten: from t, from condensed ricinoleic acid (deCaglyCeri ester), glycerol fatty acid ester, thin

2226-9899-PF 11 201006489 Glycerol fatty acid ester, polyoxyethylene bar. Glycerol fatty acid ester, sorbitan phthalate, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene linseed fat / by • Hardening Blue sesame oil, sucrose fatty acid ester (sucrose stearate, sucrose, sucrose myristate, sucrose oleate, sucrose laurate: cane acid erucate, sucrose mixed fatty acid ester), etc. . One type of these may be used, or a mixture of two or more types may be used. The nonionic surfactant is preferably erucic acid or a vinegar compound derived from an unsaturated fatty acid such as an acid, more preferably a sucrose sorrel, a sucrose oleate or a sucrose mixed fatty acid. ester. Or, using glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene glycerin fatty acid ester, mountain = sugar anhydride fatty acid ester, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester, One or more of the group consisting of polyoxyethylene castor oil and hardened castor oil. Further, the surfactant is preferably used by HLB for 1 〇 夕 古 # # # 〈 疏 疏. The reason is that the antigen-containing complex is easily dissolved or dispersed in the oil phase. The antigen-containing complex (A) of the present invention has a structure in which the hydrophilic agent (A-i) is coated with the surfactant (A_2) as described above.

Further, it may further contain a hydrophilic protein and/or a hydrophilic polysaccharide (A-3) as a stabilizer D (A-3) stabilizer, and is a hydrophilic protein and/or a hydrophilic polysaccharide. The stabilizer for use in the present invention can improve the stability of the antigen-containing complex by breaking the surfactant with the antigen at the same time, and in particular, has the effect of preventing the antigen in the transdermal agent from leaking out to the antigen-containing complex. . The above-mentioned 2226-9899*-Pf 12 201006489 stabilizes the κ hydrophilic protein and/or polysaccharide, and is not particularly required as long as it is acceptable as a component of a medical preparation. However, in order to effectively exert the drug leakage inhibiting action, it is preferred to use a protein having an average molecular weight of about 10,000 or more. "Medium hydrophilic j. raw protein, such as serum albumin (molecular weight: about 67, 〇〇〇), ie albumin (molecular weight: about 45, _, casein (molecular weight about 19, _ or more), lysozyme (molecular weight ' About 14,0〇〇 or more), lipolytic φ enzyme (molecular weight: about 45, _, etc., w may be selected from these, or two or more types may be used in combination. Preferably, the use is selected from serum albumin, A group of two or more species consisting of ovalbumin and complexin. Also, 'hydrophilic polysaccharides, such as maltose, sugar, lactose, trehalose fiber-sugar, polydextrose (puUuian), pectin, dragon pectin , propyl methacrylate phthalic acid vinegar, heparin, alginic acid, and a few methyl cellulose, etc., may be selected from the above, or may be selected and used in combination. Preferably, S, Read_Lian.» saccharide trehalose, polytriglucose, LM pectin, • HM fruit 谬 and propyl methacrylate phthalate phthalate phthalate Or more than two kinds. In the antigen-containing complex of the present invention, the weight ratio of the stabilizer to the antigen, The range of 〇.〇1~100 is better. If it is less than 〇〇1, the inhibitory effect of the drug in the transdermal agent may not be fully exerted, and if it exceeds 10:, the ratio of the agent to the complex changes. Less, sometimes unable to play within the range. 10 is more than 0.5~5, and the weight ratio of surfactant to antigen and stabilizer is

2226-9899-PF 201006489 0. The range of 5 to 100 is better, more preferably in the range of 5 ,, and even more preferably in the range of 2 to 25. (A-4) Other

The antigen-containing complex (A) of the present invention has a structure in which an antigen (Α-Ϊ) is coated with a surfactant (A-2) as described above, but may contain an adjuvant in order to enhance the ability to produce antibodies. The adjuvant acts as a regulatory factor that assists in inducing an immune response, and is usually administered together with an antigen, but in the present invention, the adjuvant is an optional component and is not an essential component. As shown in the examples described later, it has been confirmed that when the S/0 type transdermal immunizing agent of the present invention is used, antibody production can be enhanced without the addition of an adjuvant. The adjuvant to be used in the present invention is not particularly limited as long as it is a general user such as subcutaneous injection, and is representative of: a mineral oil developed by Freund, a surfactant, and a mixture of heated tuberculosis bacteria (Froide Complete Adjuvant) , FCA), cholera toxin or toxin-producing coliform fungi. Further, a variant non-toxic cholera toxin, or a variant non-toxicated thermotoxin, which is a non-toxic and adjuvant-active ADP-ribosyltransferase activity, is also preferred. When an adjuvant is used to prepare the S/0 type transdermal immunizing agent of the present invention, the (4) adjuvant may be added to the antigen, and the antigen-adjuvant-surfactant complex may be obtained according to the method described later, and then dissolved or dispersed. The oil phase substrate or (8) prepared by synthesizing the surfactant complex with the antigen and the surfactant complex, and then dissolving or dispersing the antigen-surfactant complex U body in the oil phase according to the method described later. In the substrate. (B) Oil phase 2226-9899-PF 14 201006489 = Inventive s/o type transdermal immunizing agent, which is a solution or suspension of the aforementioned antigen-containing complex/disintegrated or dispersed in an oil phase. It becomes a solution or a suspension depending on the type of surfactant or oil phase, the presence or absence of ultrasonic treatment, and the like. The oil phase used in the present invention is not particularly limited as long as it is acceptable in the pharmaceutical preparation. For example, 'vegetable oil, animal oil, neutral lipid (monosubstituted or disubstituted glyceride), long chain fatty acid ester, synthetic oil, sterol derivative. Specifically, soybean oil, cottonseed oil, rapeseed oil, sesame oil, corn oil, chemical oil, safflower oil, saffl〇wer 〇il, sunflower oil, olive oil, vegetable oil, perilla oil, fennel oil, cocoa butter , vegetable oils such as cinnamon oil, peppermint oil, bergamot oil, etc.; animal oils such as tallow, lard, fish oil, etc., medium-chain fatty acid diglyceride, triolein, tri-palm Tripa 1 mi tin), tristeostin, trimyristin, triarachidoin and other neutral lipids; synthetic lipids such as area; cholesterol oleate, cholesterol linoleic acid S, cholesterol Sterol derivatives such as myristate, cholesterol palmitate, cholesterol arachidate; myristic acid myristate, octyl cinnabar myristate, cetyl myristate, ethyl oleate, linseed oil Long-chain fatty acid vinegar such as ethyl acetate, linolenic acid isopropyl vinegar, palmitic acid isopropyl ester, butyl stearate; lactate such as ethyl lactate and cetyl lactate; triethyl citrate Diisopropyl acid, diethyl sebacate, hydrazine Diisopropyl polycarboxylic like vinegar; other esters of 2-ethylhexanoic acid cetyl ester; petrolatum, paraffin hydrocarbons such as squalene burning; Shi Xi ketone. One of these can be selected, or 2226-9899-PF 15 201006489 can be selected and used in combination. Preferably, one or more of the group consisting of: soybean oil, sesame oil, olive oil, safflower oil (saff l〇wer 〇i), sunflower oil, vegetable oil and purple oil are used. It is particularly preferable to use triglyceride or a vegetable oil as a main component thereof, and practically, soybean oil is preferred. In particular, soybean oil which is purified by high purity is preferred. Further, a neutral lipid or a long-chain fatty acid ester is also suitable, and a long-chain fatty acid ester is more preferable, and an isopropyl myristate (IPM) is more preferable. • The ratio of the oil phase to the percutaneous immunizing agent of the present invention varies depending on the type of the oil component or other constituent components, but is preferably in the range of 5 〇 to 99 5 w/v%, and is preferably '60 to 90 w/v%. Better in the range. The S/0/W type preparation of the present invention is one in which the above s/〇 type transdermal immunizing agent is further dispersed in an aqueous phase. This preparation has the characteristics of easy administration and high convenience. The aqueous phase is acceptable as long as it is a pharmaceutical preparation, and for example, pure water, purified water, steamed water, and physiological saline can be used. φ buffer, etc. Next, a method for producing the s/〇-type transdermal immunizing agent of the present invention will be described. The preparation method of the preparation of the present invention comprises the following steps: (1) mixing an antigen with an interface active agent, and an organic solvent solution containing an adjuvant or a stabilizer to prepare a W/0 type emulsion; 2) the above w/ The type 0 emulsion is dried to prepare a complex σ body of the 3 antigen '3) The above antigen-containing complex is dissolved or dispersed in the oil phase. '(l) W/0 type emulsion preparation step First, an aqueous solution of an antigen was prepared. Add adjuvant as needed

2226-9899-PF 16 201006489 or stabilizers ^ v points. For use herein: A pharmaceutical preparation can be added as water, for example, pure water, purified water, distilled water, physiological saline, or a buffer. i love 亜, ^ organic solvents. However, it is necessary to pay attention to the addition of water such as ethanol. In Taixi, sometimes it is difficult to form an emulsion. When the agent is in the middle, the antigen concentration (further addition of adjuvant or stabilization is not particularly limited to the brakes, and 蚝 蚝 蚝 蚝 实质 实质 实质 实质 ' ' ' ' ' 例如 例如 例如 例如 例如 例如 例如 例如 例如; 〇 ing / mLe organic SI 'preparation of the organic solvent solution of the surfactant. The organic solvent used here, only ® ~ ~ such as β ~ ι with <, that is not particularly limited to the agent and can be spread in the next step The aromatic hydrocarbon is removed, such as a toluene or the like, such as an alcohol such as methanol or ethanol; a fat such as a hexagram, or the like; (4) an aliphatic hydrocarbon, etc. Further, a solvent of a diazide is used, and a solvent of dichloromethane is about H. mass%. There is also no particular limitation on the use of an aqueous solution of the above-mentioned antigen (sometimes containing an adjuvant or a stabilizer) and an organic solvent solution of a surfactant, such as a sputum, for example, using a twisted tree, W ff/Ο type ^ s . /V = machine for idle stirring, or use propeller mixing

Mixer with mixer or disperser, or W, can use #,° °纟 to illuminate the ultrasonic wave. Wo Membrane membrane emulsification 'Preparation of W-type emulsion. A porous membrane, for example, a commercially available 鞒> k 孔 齐 J · · · 水性 水性 水性 Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi Shi — The glazed glaze film (SPG, U 丨 in the porous glazing film, and the porous film, which has a particle size composite which can obtain a pore diameter corresponding to the glass film). (2) Drying step 2226-9899-pf 17 201006489 Here, the W/0 type emulsion obtained in the above step "1 is dried, m, the antigen-containing complex. Drying the soil; (4) drying The method of L is not particularly limited, and it can be dried under cold decompression, and can be dried or frozen.

It is also expected that coal is better at the end of the day. The specific conditions 'can be used according to the usual law. Further, in this step, it is preferable to remove water and an organic solvent. The reason is that the residual factor of the remaining eight cockroaches can be easily caused by the hydrolysis of proteins or antigens, and the organic solvent, which is adversely affected. (5) Broken ^, but it is caused by the living body, and ^, for example, the moisture content measured by the Karl Fischer method is about 1% or less. Buy 忐 忐 疋 疋, (3) Dispersion step Here, the antigen-containing phase obtained in the above step (2) is dispersed in the oil phase, and the s/f) boundary 曰 骚 办 solution or chemistry. Specifically, the homogenizer is used for the agitation, or the propeller is used to select the arbitrarily, the net is selected, and the amount of the oil phase used in the above steps can be used, depending on the compatibility of the surfactant, etc. Different, for example, acne * a person's cheek ❿ antigen complex "body per kg, can be about 1 ~ 10mL 〇 can also be obtained in this way S / mourning j Gong 丨, 忖 q b The /o type preparation was further dispersed in an aqueous phase according to a usual method to prepare an S/0/w type preparation. The above-mentioned > trough liquid or suspension can be applied to the site where the skin such as the inner side of the wrist is thin and well coated from the state of J, and it is preferable to add other additive components which are usually added as medicines. Formulation. Shangcheng Wo '4 eight on iL $ plus ingredients, such as excipients (such as sugar and other sugars; dextrin and other hoppers from the hopper. Seeking uncle Yang He bio, thiol cellulose sodium (. Ze (4) S〇diUm) Such as cellulose derivatives; water-soluble polymers such as Sanxian gum, etc.), colorants, binders (such as the aforementioned agents or 肫) 2226-9899-PF 18 201006489, etc.), emulsifiers, tackifiers, wet Agent (for example, glycerin, etc.); stabilizer (for example, methylparaben, propylparaben, p-hydroxybenzoic acid sulfonium, such as gas butanol, benzyl alcohol, ethanol of ethanol; hydroxyl Benzalkonium chloride; phenol such as phenol or cresol; thimerosal; acetic anhydride; sorbic acid, etc., preservative ♦ (for example, water, glycerol, ethanol or oleyl alcohol) Higher alcohols, etc.). A dissolution aid, a suspending agent (for example, carmel lose sodium, etc.), a buffer, a pH adjuster, a base (for example, polyethylene glycol, crotamiton (cr〇tamit〇) n), diethyl sebacate, petrolatum), petrolatum or microcrystalline wax and other hydrocarbons, jojoba oil (j〇j〇ba

Chi) or recording it; tallow or eucalyptus oil, such as triglycerin, menthol, etc., a solubilizing accelerator, an isotonic agent, an organic solvent, a surfactant, etc., which can be compounded in a usual amount. The ointment, lotion, aerosol, plaster, aqueous porridge dressing, cream, ointment can be prepared by mixing the above solution or suspension with other additions into a knife according to the usual method of each dosage form. , gel, hard plaster (Ρΐ-6Γ), storage type patch, matrix type patch, patch, etc., but are not limited thereto. The administration amount of the transdermal immunizing agent of the present invention is appropriately adjusted depending on the type or amount of antigen to be visually matched and the age or symptom of the patient. For example, in the case of a transdermal immunizing agent containing no antigen and only an antigen, usually one adult is owed, and the approximate (10)' range is called g, more preferably roughly _ circumference, spanning weeks to months, at least ^ In the case of both adjuvants, 1 is preferred. X, containing antigen, small monthly, usually 1 adult per adult, about 5〇

2226-9899-PF 19 201006489 The range of 50〇eg, more preferably approximately 5〇~2〇〇#g range, preferably at least 2 times over several weeks to several months. EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples and test examples, but the scope of the invention is not limited thereto. Lysozyme f rom hen egg white (HEL) is used as a model antigen for s/0 transdermal immunization, and φ is used for transcutaneous immunization with S/0 percutaneous immunosuppressant containing HEL. It does not cause skin irritation as in the conventional subcutaneous immunization method, and the antibody production ability is also excellent. The HEL solution is added to the solution containing the HEL, and the solution containing HEL is added to the solution containing HEL. 5.0% by weight of a cyclohexane solution of erucic acid ester (manufactured by Mitsubishi Chemical Foods Co., Ltd., 290, erucic acid, 90% by weight, HLB: 2), and homogenized at a high speed machine for 2 minutes at 26,00 rpm. w/0 type emulsion. The emulsion was freeze-dried overnight to obtain a surfactant _HEL complex. To the composite, 4 mL of isopropyl myristate (IPM) was added and dispersed to obtain a suspension of the S/0 type complex of the present invention (the preparation of the present invention). Wherein, HEL containing 〇. 5 mg/mL. Fig. 2 shows the particle size distribution (frequency of cloth) of the hel-containing composite obtained by the above method. The average particle diameter of the composite containing HEL was measured by a laser scattering method (using a nanozs apparatus manufactured by sysmex) and found to be 458.7 nm. 2226-9899-PF 20 201006489 (Comparison of preparation of comparative preparations) *· For the positive control, 'Comparative preparation 1 is prepared by emulsifying HEL antigen and Freund's complete adjuvant (FCA) in the same amount, and only Comparative preparation 2 containing hel antigen. In detail, 'Solve HEL2.0mg in 2mL phosphate buffered saline (?35, ?117.4) to form a solution containing 1^, in this solution containing 11豇, add the same amount (2mL) FCA (stock solution) Comparative preparation j was prepared by emulsification. ❶ And Comparative Preparation 2 was prepared in the same manner as Comparative Preparation j except that FCA was not added. Using the preparation of the present invention and the comparative preparations 丨, 2 obtained in this manner, the following experiment was conducted to investigate the effects on the amount of antibody produced and the site to be administered (observation of the naked eye and examination of pathological tissues). Test Example 1 Antibody Production of Jinxi Earthworm 4 male rabbits (Newzealand White species) weighing about 2 kg were prepared, and divided into 2 positive control groups (for comparison preparation i '2) and 2 experimental groups (for φ preparations of the present invention) The administration was carried out as follows. (positive control group) The back skin of rabbits (2 rats) of the positive control group was shaved with an electric razor 'and will be compared " (addition of adjuvant), comparative preparation 2 (without adjuvant), for each Subcutaneous injection was performed at 1 m 1 . (Experimental group) In the two rabbits used in the preparation experiment group, one of the back skins was bristled with the electric bayonet in the same manner as above, and the sample was coated with 1 mL in an area of about 1 inch (four) square. Invention and production! (back coating). for

2226-9899-PF 21 201006489 The present invention 'painting... For both the experimental group and the positive control group, in the reading = two 28th (2nd immunization), 42nd (3rd immunization) (the 4 times immunization), the same administration as above was carried out. The first time of the immunization ~ the fourth immunization before the administration of each ear [from the ear (in the aurant coating of the formulation of the present invention, the group of the ear is not the ear of the other side of the ear) Blood sampling... The USA method checks the amount of anti-mouse antibody in the sample. The details of the ELISA method are as follows (1) to (5), and the washing operation of each step is sufficiently carried out using phosphate buffered saline (PBS), PBST [pBS + Tween 2® (hydrophilic surfactant)], or 1 M NaCl. Further, the experiment was carried out three times in total. (1) Add HEL phosphate buffered saline (PBS, pH 74) (protein amount: 5 mg/mL) to a 96-well microplate, and make 2 〇 (u ^ well, let stand for 1 night, let HEL adsorb In the micro-plate. (2) In order to prevent non-specific adsorption, in the case of blockers, use bovine serum

A PBS solution (containing BSA 5 mg/ml) of albumin (Bovine Serum Albumin; BSA) was added to the above-mentioned microplate plate to which fjEL was adsorbed, and blocked at 200 yL/well (37 ° C, 2 hours). (3) The sample was added to a well of 200; /L/, and an antigen-antibody reaction (37 ° C, 2 hours) occurred between the HEL adsorbed on the microplate and the anti-HEL antibody in the sample. (4) After removing the blocking agent, carefully rinse the microplate with PBS containing 2% Tween 80, and add a goat-derived anti-rabbit IgG antibody labeled with alkaline phosphatase (AP). Diluted to 5,000 times with 0.1% BSA, 222 6-98 99-PF 22 201006489 The antigen-antibody reaction was fully reacted (3 7 °c, 2 hours). (5) Free AP-labeled goat-derived anti-rabbit igG antibody was washed thoroughly after washing. Add 1. mM mM p-nitrophenyl phosphate (dissolved in mM)

The amount of anti-hEL antibody in rabbit serum was determined by measuring the absorbance at λ = 410 nm in Tris-HCl buffer, pH 7.5. The above results are organized in Figure 3. Figure 3 shows the results of the positive control group and the experimental group showing 'from the left side: Comparative preparation 2 (no φ adjuvant, subcutaneous injection), comparative preparation 丨 (with adjuvant, subcutaneous injection), the present invention The result of the formulation (auricular coating) and the formulation of the invention (back coating). From Figure 3' can be considered as follows. First, the comparison of transdermal immunization using subcutaneous injection as in the conventional method was carried out in both the first immunization and the second immunization, and an increase in the amount of the anti-HEL antibody was observed. In particular, in the comparative preparation 1 in which an adjuvant was added, an apparent increase in the amount of the antibody was observed in the first immunization. On the other hand, the preparation of the present invention in which S/deuteration was carried out was carried out in the case of performing auricular coating φ cloth and back coating, and the amount of the antibody was considered to be increased in the first immunization and the second immunization. In particular, 'the auricle coating example in which the skin tissue is thin and the percutaneous absorption is high is significantly thicker than the subcutaneous tissue and the back coating example having a lower transdermal absorbability than the auricle. , to the extent that 制剂 compares formulation 2 to approximately the same extent. Test Example 2__Important visual observation of the administration site The administration site of each rabbit of the experimental group and the positive control group was visually observed at the time of the first immunization. The result 'Comparative preparation for subcutaneous injection of rabbit back and ratio 2226-9899-PF 23 201006489 • Compared with the preparation 2, obvious nodular lumps occurred from the first immunization, especially, subcutaneous injection of 3 adjuvants In the comparative preparation 1 of the antigen, the degree is more remarkable (not shown). C is relative to this, and the preparation of the present invention is administered, and the back coating example is self-contained, so that the irritating sputum is sharp. The boat & vortex was coated, and no swelling or congestion of the blood vessels was considered, and no skin irritation was observed at all. Test Example 3__Clinical_The histological examination of apricot φ Here, hematoxylin and eosin (HE) staining was performed on the skin of each rabbit on the 66th day after the administration, and the pathological tissue specimen was prepared and observed by an optical microscope (magnification 80 times). , 200 times, 400 times), check the inflammation of the skin. Fig. 4 to Fig. 7 each show a pathological photograph of the preparation of the present invention (auricle coating), the preparation of the present invention (back coating), Comparative Formulation 1, and Comparative Formulation 2. First, the preparation of the present invention is considered. Fig. 4 is a photograph showing the pathological structure of the preparation (aurous coating) of the present invention, and Fig. 4A is an enlargement photograph of 80 times and Fig. 4B is 400 times. As shown in Fig. 4A and Fig. 4B, the skin subjected to auricle coating was not inflamed at all, and a subcutaneous tissue containing a normal epidermis, a dermis layer, and fat was observed. Fig. 5 is a photograph showing the pathological structure of the preparation of the present invention (back coating), and Fig. 5A is an enlargement photograph of 80 times and Fig. 5B is 400 times. As shown in Fig. 5A to Fig. 5B, the skin on which the back coating was applied was not found to be inflamed, and the subcutaneous tissue containing the normal epidermis, the dermis layer, and the fat was observed. As described above, from the case of transdermal immunization using the preparation of the present invention from Fig. 4 and Fig. 5, no skin irritation was observed in either the auricle coating or the back coating. 2226-9899-PF 24 201006489 Secondly, consider comparing formulations. Fig. 6 is a photograph showing the pathological structure of Comparative Formulation 1 (FCA addition), and Fig. 6A is an enlarged photograph of 80 times, Fig. 6B is 200 times, and Fig. 6C is 400 times. From the figure, it is considered that a large cyst (see Fig. 6A) is observed in the dermis layer, and a necrotic tissue is considered to be present around the large cyst (see Fig. 6B). Further, as shown in Fig. 6C, inflammation was widely observed from the dermis layer to the subcutaneous tissue, and it was markedly hypertrophic. In these sites, there was a large vacuole or foamy cytoplasm and an expanded giant phagosome group was observed, and cell infiltration was observed between them. Fig. 7 is a photograph of a pathological tissue of a comparative preparation (without adding FCA), and Fig. 7A is an enlarged photograph of 80 times, Fig. 7 is 200 times, and Fig. 7C is 400 times. As shown in the figures, a portion of the epidermis (left end of Figures 7A, 7B, and 7C) is slightly hypertrophic, and in the lower dermis layer, densely weak fibroblasts travel with a slight cellular infiltration. From the above results, it can be considered that the skin infection is inflamed and inflamed by the conventional subcutaneous injection of the subcutaneous immunization method, and the comparative preparation containing the FCA is more noticeably observed than the comparative preparation 2 without the FCA. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the constitution of an S/Ο type preparation. Figure 2 shows the particle size distribution of the formulation of the present invention. Fig. 3 is a graph showing the temporal change of the amount of the antibody in each of the administered groups in Test Example 1. Fig. 4A shows a pathological photograph (8 倍) of the preparation of the present invention (auricular coating). Fig. 4B shows a pathological photograph (400 times) of the preparation of the present invention (auricle coating). 2226-9899-PF 25 201006489 times) 襕5A shows the pathological photo of the preparation of the present invention (back coating) (8〇 Figure 5B shows the pathological photo of the preparation of the present invention (back coating) (400 times). 6A shows a photo of the pathological tissue of the comparative preparation (addition of FCA) (80 times). Figure 6B shows a photo of the pathological tissue of the comparative preparation 添加 (addition of FCA) (2 〇〇 Figure 6C shows the pathological photo of the comparative preparation 丨 (addition of FCA) (4) Figure 7A shows a photograph of the pathological tissue (80 times) of Comparative Formulation 2 (without addition of FCA). The figure shows a photograph of the pathological tissue (200 times) comparing Formulation 2 (without adding FCA). Figure C shows Comparative Formulation 2 ( Photograph of pathological tissue without added FCA) (400 times) f main component symbol description] 1~ drug; 2~ surfactant; 3~ complex containing drug (solid phase); 4~ oily substrate (oil phase) ; 10~S/0 type preparation 2226-9899-PF 26

Claims (1)

201006489 X. Patent application scope: 1. An s/o type transdermal immunization agent comprising a solid antigen-surfactant complex formed by coating an antigen by a surfactant, and an oil phase, and the solid antigen The surfactant active compound system is dissolved or dispersed in the oil phase. 2. The s/o type transdermal immunizing agent according to item 1 of the patent application, wherein the antigen is a protein or a peptide. 3. The S/o type transdermal immunizing agent according to claim 1, wherein the antigen is a live or inactivated bacterium and/or virus. 4. The S/0 type transdermal immunizing agent according to claim 1, wherein the source of the antigen is an animal or a plant. The S/〇-type transdermal immunizing agent according to any one of claims 1 to 4, wherein the surfactant is a nonionic surfactant. 6. The S/〇 type transdermal immunizing agent according to any one of claims 1 to 5, wherein the surfactant has an HLB price of 丨〇 or less. ® 7. The S/〇 type transdermal immunizing agent according to any one of claims 1 to 6 wherein the oil phase is selected from at least one of the following groups: vegetable oil, animal oil, neutral Lipids, and long chain fatty acid esters. 8. An S/0/W type transdermal immunizing agent characterized by dispersing a s/〇-type transdermal immunizing agent according to any one of the claims of claim 41 in the aqueous phase. 2226-9899-PF 27