JP2014156433A - Soluble microneedle for allergy test - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diagnostic formulation with less burden on a subject and high accuracy, which has been required because delivery to a skin surface site with immune cells being widely distributed requires high skills and involves a pain when a liquid formulation containing an allergen is administered by intradermal injection.SOLUTION: In a soluble microneedle/array/chip formulation, a water soluble and thread-forming polymer substance is used in a base and a polymer substance or a fine particle which will be an allergen is kept at the tip part.

Description

The present invention relates to a skin allergy test preparation excellent in long-term stability that enables direct delivery of allergens to immune system cells localized in the epidermis layer and upper dermis of the skin. is there. It is applicable to allergens such as protein, saccharides, and particulate allergens, and is based on a water-soluble leaky polymer. The tip of the soluble microneedle can be inserted into the skin surface. By localizing the allergen, it can be administered painlessly with a small amount of allergen and clinically required allergy diagnosis can be performed.

Allergy is a kind of biological immune reaction to allergens, and various substances such as proteins and sugars contained in foods become allergens. In order to establish immunity, it is necessary to deliver a sufficient amount of allergen to induce an immune response in the body where immune system cells are present, and to induce an immune response by the action of immune system cells.

It is known that an immune response can occur by intradermal injection of allergens and administration from the transmucosal route such as oral and nasal. Patch tests are also used clinically, and inventions relating to patch test sheets and tools have been made (Patent Documents 1 and 2). However, intradermal injection is painful because of the use of a syringe. In addition, since the skin and mucous membrane have a barrier function to exclude foreign substances from the outside world, there are cases in which the effect of allergen administration by patch test from this route is uncertain, and to obtain a clinically sufficient immune response May require large amounts of allergens.

JP 2002-255781 A JP-A-6-238008 Japanese Patent No. 4913030 WO 2009/066763 (PCT / JP2008 / 71224) Japanese Patent Application 2010-240609

  Recent advances in immunology have revealed that immunocompetent cells are abundantly distributed in the surface layer of the skin (the epidermis and the upper layer of the dermis). It has been pointed out that it can be triggered. Further, since nerve endings relating to pain sensation are not distributed in the epidermis, allergen administration to the skin surface layer can be performed by a minimally invasive method with little pain or pain. However, the conventional system using an injection needle and a syringe barrel has a problem that it is difficult to administer allergen to the skin surface layer easily and reliably.

The allergen in the present invention may be purified to a single allergen, but it is sufficient that the allergen is purified to the extent considered necessary from the viewpoint of diagnosis. An object of the present invention is to provide a microneedle for allergy diagnosis that can be easily administered to the surface layer of a skin that can withstand long-term storage at high temperatures and can elicit an immune response equivalent to or superior to that of conventional intradermal injection. • To provide array patches.

  The inventor first invented a microneedle-like or thread-like percutaneous absorption preparation, a percutaneous absorption preparation-holding sheet, and a percutaneous absorption preparation-holding device that dissolve and release the active ingredient when inserted into the surface layer of the skin. (Patent Document 3). Furthermore, a microneedle-like percutaneous absorption preparation having a multilayer structure containing an active ingredient only at the tip was invented (Patent Document 4). Furthermore, a three-layer soluble microneedle array chip for skin epidermis delivery of vaccine antigens was invented (Patent Document 5). In order to solve the above-mentioned problems, we have earnestly researched, using a water-soluble leaky polymer substance as a base, and a soluble microneedle array containing allergen at the tip inserted into the skin surface layer. The present invention has been completed regarding an allergic diagnostic preparation capable of delivering an allergen to the skin surface layer simply by preparing an arrayed patch preparation and pressing it against the skin.

  The soluble microneedle array patch preparation of the present invention is a microneedle array patch that retains an allergen at the tip portion inserted into the surface layer of the skin and is based on a water-soluble leaky polymer substance. It is a formulation.

The allergen is various proteins, saccharides, particles and the like generally used for allergy diagnosis.

Examples of the allergen include indoor dust, mites, tree pollen, grass pollen, weed pollen, fungi and bacteria, animals, insects, parasites, and foods.

The water-soluble leaky polymer substance is at least one substance selected from the group consisting of polysaccharides, polyvinyl alcohol, carboxyvinyl polymers, and sodium polyacrylate. In addition, about these polymer substances, only one may be used and you may use in combination of multiple types.

The leaky polysaccharide is at least one substance selected from dextran, sodium chondroitin sulfate, dextran sulfate, sodium hyaluronate, cyclodextrin, hydroxypropylcellulose, alginic acid, agarose, and pullulan and their derivatives.

In one certain form, the microneedle according to claim 1 has a total length of 30 to 500 micrometers, a bottom diameter of 20 to 300 micrometers, and a length of the tip part holding the allergen is 10 to 200 micrometers and 1 square centimeter. It exists on the base chip at a density of 100 to 300 per unit.

In one certain form, 0.01 microgram-1.0 milligrams of allergen are contained in the front-end | tip part of the microneedle of Claim 1.

  According to the present invention, allergens, which are a kind of immune reaction, are efficiently induced by delivering allergens easily and reliably to the surface layer of the skin (the epidermis and the upper layer of the dermis), and allergy diagnosis can be easily performed without pain. Can be done. Since allergens are delivered directly to the skin surface layer where immune system cells are abundantly distributed, a sufficient immune response can be obtained with a low dose of allergens as compared with conventional intradermal injection methods. Since the administration site is limited to the surface layer of the skin, a minimally invasive diagnosis with little pain or pain is possible. In addition, self-administration is possible because the patch preparation can be administered by a simple operation simply by pressing it against the skin. Furthermore, since the patch preparation is a solid preparation, the allergen stability can be ensured and it can withstand long-term storage.

  The microneedle array chip used in the present invention is based on a water-soluble leaky polymer and has a structure in which allergens are localized at the tip corresponding to the portion inserted into the surface layer of the skin when administered. It has a microneedle array on a chip that is a base as a diagnostic drug formulation.

  Further, the base used in the present invention is at least one substance selected from the group consisting of water-soluble leaky polymer substances, that is, polysaccharides, polyvinyl alcohol, carboxyvinyl polymers, and sodium polyacrylate. . In addition, about these polymer substances, only one may be used and you may use in combination of multiple types.

  Preferably, the leaky polysaccharide is at least one selected from dextran, sodium chondroitin sulfate, dextran sulfate, hyaluronic acid, cyclodextrin, hydroxypropylcellulose, alginic acid, agarose, pullulan, glycogen and derivatives thereof. Is one substance.

The allergen used here is not particularly limited as long as it has immunogenicity. For example, proteins derived from various pathogenic microorganisms generally used for vaccination (tetanus toxin and its toxoid, diphtheria toxin and its toxoid, pertussis toxin and its detoxified product, influenza virus hemagglutinin, hepatitis B virus HB s Allergens), polysaccharides (such as pneumococcal capsular polysaccharides, hemophilus influenza b type capsular polysaccharides), particles (inactivated hepatitis A virus particles, human papillomavirus virus-like particles, etc.), various lifestyles Related allergens (pollen such as cedar pollen, bee venom, mite allergen, house dust, etc.), cancer allergens and the like.

The life-related allergens include indoor dust (house dust), mites, tree pollen (cedar, cypress, juniper, alder, hippopotamus, Japanese oak, beech, pine, elm, willow, maple, walnut, mulberry, acacia, olive and Its genus), gramineous pollen (camodia, blue whale, hargaya, gypsy moth, giant moss teppo, sorghum sorghum, barley, nagahagusa, hirohashinokegusa, reed, wheat, suzumee, konukagusa and swine pollen Redwood, redwood, mugwort, green mugwort, french chrysanthemum, dandelion, canamgras, hera plantain, white mosquito, nettle, dromedary and its genus, fungi and bacteria (alternaria, candida, aspergillus, cladosporium, Nicosium, Mucor, Helmintosporium, Malassezia (genus), Pityrosporum (Malacethia), Trichophyton, Staphylococcus aureus enterotoxin A, Staphylococcus aureus enterotoxin B), animal (cat, dog, molomote, hamster, mouse, Rat, rabbit, cow, pig, pig, goat, sheep, budgerigar, chicken, duck, goose, pigeon dung), insect (roach, chironomid, wasp, honeybee, wasp, aedes, moth), parasite (Anixus) , Roundworm, worm, occupational allergen (silk, cotton, latex, psyllium seed, isocyanate MDI, isocyanate HDI, phthalic anhydride, ethylene oxide, formalin), food (milk, egg white, egg yolk, ovomucoid (heat-resistant egg protein) ), Rice, buckwheat, wheat, ω-5 gliadin, o Wheat, oats, rye, millet, millet, millet, corn, soy, green beans, peas, peanuts, almonds, walnuts, cashews, coconuts, brazil nuts, hazel, strawberries, apples, peaches, bananas, melons, oranges, grapefruits, kiwis , Mango, avocado, pear, tomato, celery, parsley, onion, watermelon, carrot, yam, potato, sweet potato, pumpkin, spinach, bamboo shoot, garlic, sesame, mustard, malt, beer yeast, cacao, cheese, mold cheese , Α-lactalbumin, β-lactoglobulin, casein, gluten, beef, pork, chicken, lamb, shrimp, lobster, crab, mussel, clams, oysters, scallops, squid, octopus, mackerel, horse mackerel, sardines, Cod , Salmon, tuna, and salmon roe, and the like.

The part where the allergen is localized at the tip of the microneedle array is preferably 200 micrometers or less from the tip of the microneedle. More preferably, it is 100 micrometers or less from the tip.

There is no particular limitation on the method for retaining the target substance in the base, and various methods can be applied. For example, a microneedle array can be made by inserting a target substance into a female mold while the target substance is retained in the base by containing the target substance in a base, and then drying and curing. Can do. As another example, a microneedle array can be removed by injecting into a female mold in a suspended state by adding it into a dissolved base material, and then drying and curing. .

  The total length of the microneedles constituting the microneedle array is not particularly limited, but is preferably about 20 to 700 micrometers, more preferably about 30 to 500 micrometers. The diameter of the bottom part is preferably 20 to 300 micrometers, and the length of the tip part holding the allergen is preferably about 10 to 200 micrometers.

  The area of the base is not particularly limited, but is preferably about 10 square centimeters, more preferably about 5.0 square centimeters, and even more preferably about 2.0 to 1.0 square centimeters. Further, the thickness of the substrate is not particularly limited, but is preferably about 5 mm, more preferably about 3 to 1 mm. As the density, for example, it is constructed with a density of about 100 to 300 on a chip of 1 square centimeter.

  The following examples, which are not intended to limit the present invention, are further illustrated.

Example 1
On the surface of a resin substrate having a bottom surface of 1 cm in length and 1 cm in width, 225 inverted conical pores having a depth of about 500 micrometers and an opening diameter of about 300 micrometers were provided. This substrate was used as a mold for producing microneedles. Poly L-arginine hydrochloride (Sigma) was used as a model allergen. Add 20 microliters of purified water to 1.0 mg of poly L-arginine, mix well, then add 9.0 mg of polymer dextran (Nacalai Tesque) as a base, mix well, and add a viscous concentrate (solution A) was prepared. Further, 30 μL of purified water was added to 30 mg of chondroitin sulfate C sodium salt and dissolved and mixed well to prepare a concentrated solution (solution B). Solution A was applied while being applied to each pore of the mold, and the surface was covered with a wrapping film (Saran Wrap (registered trademark); Asahi Kasei Co., Ltd.). The mold was set in a tabletop centrifuge (KUBOTA 4000) and centrifuged at 3000 rpm for 5 minutes. The wrapping film was removed, and the residue on the mold surface was removed. About 0.5 g of a mixture of cellulose acetate and hydroxypropyl cellulose 100: 10 is put into a mortar of a single tableting machine (Ichibashi Seiki, HANDTAB 100), and the diameter is 1.5 cm and the thickness is about 2. with a tableting pressure of about 10 kN. A 0 mm base chip (support) was prepared. Solution B was packed while being applied to each pore of the mold, and the base chip was placed over the entire surface of the mold. After drying, the base chip was slowly peeled off to obtain a patch agent holding the microneedle array.
(Example 2)
In the same manner as in Example 1, 225 reverse conical pores having a depth of about 500 micrometers and an opening diameter of about 300 micrometers were provided on the surface of the resin substrate. Add 20 microliters of purified water to 2.5 mg of poly-L-arginine and mix well, then add 7.5 mg of polymer dextran as a base and mix well to prepare a thick viscous solution (solution A) did. Further, 30 μL of purified water was added to 30 mg of chondroitin sulfate C sodium salt and dissolved and mixed well to prepare a concentrated solution (solution B). Solution A was applied to each pore of the mold while being packed, and the surface was covered with a wrapping film. The mold was set in a tabletop centrifuge and centrifuged at 3000 rpm for 5 minutes. The wrapping film was removed, and the solution B was packed while being applied to each pore of the mold, and the same chip for the substrate (support) as in Example 1 was placed over the entire surface of the mold. After drying, the base chip was slowly peeled off to obtain a patch agent holding the microneedle array.

(Example 3)
In the same manner as in Example 1, 225 reverse conical pores having a depth of about 500 micrometers and an opening diameter of about 300 micrometers were provided on the surface of the resin substrate. Add 20 microliters of purified water to 5.0 mg of poly L-arginine and mix well, then add 5.0 mg of polymer dextran as a base and mix well to prepare a thick viscous solution (Solution A) did. Further, 30 μL of purified water was added to 30 mg of chondroitin sulfate C sodium salt and dissolved and mixed well to prepare a concentrated solution (solution B). Solution A was applied to each pore of the mold while being packed, and the surface was covered with a wrapping film. The mold was set in a tabletop centrifuge and centrifuged at 3000 rpm for 5 minutes. The wrapping film was removed, and the solution B was packed while being applied to each pore of the mold, and the same chip for the substrate (support) as in Example 1 was placed over the entire surface of the mold. After drying, the base chip was slowly peeled off to obtain a patch agent holding the microneedle array.

Example 4
Poly L-arginine was extracted from the microneedle array chip preparations prepared in Examples 1 to 3 using isotonic phosphate buffer, and the concentration was measured by the Bradford method.

(Example 5)
The contents of poly L-arginine in the microneedle array chips prepared in Examples 1 to 3 were 9.3 ± 2.0 μg, 31.1 ± 2.9 μg, and 61.9 ± 9.3 (SD) μg.

(Example 6)
A Wistar male rat weighing about 300 g was fixed to the operating table under pentobarbital anesthesia.
The rat's back was depilated to expose the skin. The next day, 5 rats were used in the experiment as a group. The poly L-arginine-containing microneedle array chip prepared in Examples 1 to 3 was pressed against the rat's back hair removal skin, and the state where the microneedle was punctured into the skin was maintained for 3 minutes. By this operation, the tip portion of the microneedle inserted into the skin was dissolved, and the contained poly L-arginine was delivered to the skin surface layer. As a positive control, an aqueous solution of poly L-arginine was prepared and administered to rats consisting of 5 animals per group by subcutaneous injection. The dosage was 9.3 micrograms, 31.1 micrograms and 61.9 micrograms. After administration of poly L-arginine, a model allergen, the behavior of the rats was observed for 30 minutes, and the number of times the administration site was scratched with the foot during that time was recorded. A microneedle array chip containing no poly L-arginine was prepared and used in the same experiment as a negative control. In that case, the number of scratches was zero. The average number of scratches of the preparation of Example 1 is 9.8 ± 7.6 (standard deviation), the average number of scratches at the time of subcutaneous injection of 9.3 micrograms of poly L-arginine is 0.4 ± 0.9, and the average number of scratches of the preparation of Example 2 60.4 ± 18.6 times, poly L-arginine 31.1 micrograms, the average number of scratches when subcutaneously injected was 5.0 ± 3.1 times, the average scratch number of the preparation of Example 3 was 95.7 ± 26.0 times, poly L-arginine 61.9 The average number of scratches during microgram subcutaneous injection was 20.5 ± 9.1.

  In view of its effectiveness and long-term stability at high temperatures, the soluble microneedle array patch that retains allergens at the tip corresponding to the skin insertion part replaces allergic tests by conventional intradermal injection. May be widely used. In addition, since self-administration is possible, it is expected to be used in developing countries where medical infrastructure is not fully prepared.

  According to the present invention, it is possible to provide a microneedle array patch preparation for an allergy test that is not painful, is stable for a long time even under high temperature conditions, and is convenient for use such as self-administration.

Claims (7)

A microneedle array patch preparation for skin allergy testing based on a water-soluble leaky polymer substance with an allergen held at the tip inserted into the surface layer of the skin. The microneedle array patch preparation for skin allergy test according to claim 1, wherein the allergen is various proteins, saccharides, particles and the like generally used for allergy tests. 3. The microneedle array for allergy testing according to claim 2, wherein the allergen is indoor dust, mites, tree pollen, grass pollen, weed pollen, fungi and bacteria, animals, insects, parasites, foods, etc. -Patch formulation. The water-soluble leaky polymer substance is at least one substance selected from the group consisting of polysaccharides, polyvinyl alcohol, carboxyvinyl polymers, and sodium polyacrylate. In addition, about these polymer substances, only one may be used and you may use in combination of multiple types. The leaky polysaccharide is at least one substance selected from dextran, sodium chondroitin sulfate, dextran sulfate, sodium hyaluronate, cyclodextrin, hydroxypropylcellulose, alginic acid, agarose, and pullulan and their derivatives. In one certain form, the microneedle according to claim 1 has a total length of 30 to 500 micrometers, a bottom diameter of 20 to 300 micrometers, and a length of the tip part holding the allergen of 10 to 200 micrometers and 1 square centimeter. It exists on the base chip at a density of 100 to 300 per unit. In one certain form, 0.01 microgram-1.0 milligrams of allergen are contained in the front-end | tip part of the microneedle of Claim 1.