JP2012090767A - Soluble three-layer microneedle array patch for vaccine antigen epidermis delivery - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve a problem of a multilayer microneedle array for percutaneous vaccine, wherein an optimum arrangement portion of vaccine antigens remains to be solved.SOLUTION: In a multilayer soluble microneedle using a water-soluble stringy polymer as a base, a three-layer microneedle array wherein the antigens are disposed in the second layer corresponding to a portion 160-220 μm distant from the tip can deliver the antigens to the epidermal layer wherein Langerhans cells are distributed after administration to the skin as compared to a two-layer microneedle array wherein the antigens are disposed in a tip part, so that increase of antibody titer in circulation blood that is an immune response can be more efficiently obtained.

Description

  The present invention relates to a structure of a soluble three-layer microneedle array for obtaining an antibody titer with high efficiency.

  Microneedles are finely sized needles that do not feel pain when stabbed into the skin, and physically protect the stratum corneum that is located on the outermost side of the skin and protects the body from the entry of bacteria and viruses from the outside world. It is a formulation technique that improves the absorption rate of drugs, cosmetic compounds, or drugs with a very low absorption rate, which cannot be absorbed even when administered transdermally by conventional techniques. As the material of the microneedle, silica or the like is used in addition to the same metal as the conventional injection needle (Patent Documents 1 to 52).

  The microneedle array has a hollow structure similar to that of an injection needle and is a type for injecting a chemical solution or a microneedle array made of a biodegradable polymer such as polylactic acid. Furthermore, dissolution type microneedles based on water-soluble substances have also been developed. That is, the target substance can be administered transcutaneously by holding the target substance in a base of a water-soluble substance and inserting it into the skin, and then the base dissolves with moisture in the skin. For example, microneedles based on maltose are disclosed (Patent Documents 1 to 3, 40 to 43). Many patents relating to devices, instruments, or microneedle materials, shapes, manufacturing methods, etc. for injecting drugs via microneedles are also known (Patent Document 4, Patent Documents 7 to 50).

  The present inventor has filed a patent application for a microneedle based on a water-soluble leaky polymer substance as a microneedle overcoming the disadvantages of the microneedle based on maltose (Patent Document 5). Since the invention has low skin permeability such as genetically modified protein drugs, vaccines, high molecular weight drugs such as genetic DNA, water-soluble difficult / low transdermal absorbable drugs, sufficient effects cannot be expected with conventional transdermal administration. Increases skin permeability of drugs.

  Furthermore, the inventor has invented an industrial manufacturing method of microneedles based on chondroitin sulfate, dextran, hyaluronic acid and the like, which are water-soluble leaky polymer substances, and filed a patent application (Patent Document 6). . Patents relating to microneedle devices and microneedle delivery devices have also been devised (Patent Document 9). Furthermore, the inventor has devised a patch having a multilayer microneedle array formed on the surface (Patent Document 51).

  In addition, the present inventor uses macromolecules such as vaccine antigens and peptide / protein drugs such as insulin as target substances and uses chondroitin sulfate, dextran, hyaluronic acid, etc. as a base, and has a length of about 500 μm and a bottom diameter of about A manufacturing method of a microneedle array was constructed in which 100 300 μm soluble microneedles were constructed in 10 rows and 10 columns on, for example, a 1 cm 2 patch (Patent Document 52). Furthermore, a porous base suitable for constructing a microneedle array was invented (Patent Document 53). A patent was also filed regarding a sealed package administration system integrated with an administration device for inserting a microneedle array patch perpendicular to the skin (Patent Document 54).

JP 2003-238347 A JP 2005-154321 A JP 2005-152180 A Special table 2006-500973 public information International Publication No. 2006/080508 Pamphlet Japanese Patent Application No. 2007-150574 JP-T-2006-513811 (WO2005 / 044364) Japanese translation of PCT publication No. 2006-502831 (WO 2004/035105) JP 2005-533625 A (WO 2004/009172) Japanese translation of PCT publication No. 2005-514179 (WO2003 / 059431) JP 2005-503194 A (WO2002 / 100474) JP-T-2005-501615 (WO2003 / 020359) Japanese translation of PCT publication No. 2004-532698 (WO2002 / 100476) Japanese translation of PCT publication No. 2004-507371 (WO2002 / 017985) Japanese translation of PCT publication No. 2004-504120 (WO2002 / 007813) Japanese translation of PCT publication No. 2002-526273 (WO00 / 16833) JP 2008-46507 A JP 2008-29710 A JP 2008-29559 A JP 2008-6178 A JP 2007-130030 A JP 2006-335754 A JP 2005-246595 A Japanese translation of PCT publication No. 2007-523771 (WO2005 / 082596) Japanese translation of PCT publication 2007-511318 (WO2005 / 049107) WO2007 / 030477 WO2004 / 000389 WO2007 / 091608 WO2007 / 116959 publication JP 2008-011959 A JP 2008-074763 A JP 2008-125864 A JP 2008-237675 A JP 2008-296037 A JP 2009-062121 A JP 2009-072270 A JP 2009-078074 A Japanese translation of PCT publication No. 2009-501066 (WO2008 / 010681) Japanese translation of PCT publication No. 2009-502261 (WO2007 / 012144) JP 2009-254756 A JP 2009-238772 A JP 2009-201956 A JP 2009-237772 A JP 2009-233170 A JP 2009-241357 A JP 2009-241358 A JP 2009-254876 A JP-T 2009-507573 (WO2007 / 030477) JP 2009-533197 A (WO2008 / 004781) JP-T 2009-540984 Publication (WO2007 / 147671) WO2009 / 066763 JP 2009-195583 A Japanese Patent Application No. 2010-031317 Japanese Patent Application No. 2010-201875 Ghartey-Tagoe Esi et al., United States Patent Application 20090155330 J. C. Birchall, Stratum corneum bypassed or removed.In Enhancement in Drug Delivery, ed by E. Touitou and B. W. Barry, CRC Press, 2007, pp.337-351. Sparber, F. et al., Immunobiology, 215, 770-779, 2010. Parton, M. et al., Vaccine, 28, 6104-6113, 2010. Romani, N. et al., Immunol. Cell Biol., 88, 424-430, 2010. Furmanov, K. et al., J. Immunol., 184, 4889-4897, 2010. Flacher, V. et al., J. Inves. Derm., 130, 755-762, 2010. Rozis, G. et al., Eur. J. Immunol., 35, 2617-2626, 2005

Numerous studies have been conducted on the size of microneedles, and the present inventor has also prepared soluble microneedles of various lengths using chondroitin sulfate and dextran, which are water-soluble leaky polymer substances. However, in view of operability and strength at the time of piercing the skin, it was concluded that a microneedle array having a length of 500 μm and a bottom diameter of 300 μm is practically optimal. As the physiological structure of the skin, it is said that the thickness of the human stratum corneum is 10-20 μm and the thickness of the epidermis layer is 50-150 μm (Non-patent Documents 1 and 2). The dermis layer exists to a depth of several millimeters from the skin surface.

It has been reported that the thickness of the epidermis layer of experimental animals is 11.58 ± 1.02 μm in rats and 22.47 ± 2.40 μm in dogs (Non-patent Document 3). However, even if the microneedle array patch is pressed with a finger and stabbed into the skin, the entire length of the 500 μm microneedle cannot be inserted into the skin. In an experiment in which hairless and depigmented rats were administered to the skin, approximately half of the tip was inserted into the skin. On the other hand, in the case of transdermal vaccine, regarding the problem of which immune system cells of Langerhans cells distributed a lot in the epidermis layer and dendritic cells distributed abundantly in the dermis layer mainly recognize the antigen protein administered transdermally Despite numerous studies in the field of immunology, no conclusion has yet been reached (Non-Patent Documents 2 to 7). A patent on the transdermal delivery of a vaccine using a multilayer microneedle array has been filed (Patent Document 55), but the antigen is arranged at a high concentration at the tip of the microneedle which is the first layer, and the second layer is It is a technology that mainly uses a base. In this patent document, the specific lengths of the first layer and the second layer are not specified.

The present inventor has intensively studied to solve the above problems and completed the present invention. That is, as described above, practical microneedles from the viewpoints of operability and strength are concentrated into microneedles having a length of about 500 μm and a bottom diameter of about 300 μm. If the present inventor has applied for a hermetic microneedle array patch sealed package administration system (Patent Document 54) based on a water-soluble, leaky polymer substance previously applied for, The microneedle array patch can be administered to the skin with pressure, and a portion of the same length from the tip of the microneedle can be inserted into the skin with good reproducibility. Therefore, the microneedle array has a three-layer structure, and the first layer, which is the most advanced, is formed of a water-soluble, leaky polymer substance that does not contain any antigen, and the second part from the tip. Succeeded in obtaining a vaccine effect with higher efficiency by placing a vaccine antigen for delivery to the skin epidermis layer where many Langerhans cells are distributed, and placing a base at the root part corresponding to the third. Solved the above problem.

More specifically, the microneedle of the present invention is
It is a soluble microneedle array patch consisting of a three-layer structure for efficiently delivering a vaccine antigen to the skin epidermis layer.

The base used in the soluble three-layer microneedle array is a soluble microneedle array patch which is a water-soluble leaky polymer substance.

In the three-layer microneedle array patch, the first layer corresponding to the most distal portion of the needle is a soluble microneedle array formed by a water-soluble leaky polymer substance as a base. It is a patch.

The three-layer microneedle array patch is a soluble microneedle array patch in which a vaccine antigen is arranged in the second layer from the tip.

In the three-layer microneedle array patch, the third layer from the tip is a soluble microneedle array patch formed of a water-soluble leaky polymer substance as a base.

In the three-layer microneedle array patch, when the size of the microneedle is 500 μm in length and the diameter of the bottom is 300 μm, the length of the first layer, which is the most advanced, is 160 μm from the tip, and the second The third layer is a soluble microneedle array patch corresponding to a portion 160 to 220 μm from the tip, and the third layer is a portion 220 to 500 μm from the tip. These numerical values are values obtained in the verification experiment using rats, and there are slight fluctuations in the case of humans.

When a soluble microneedle array having a three-layer structure is pierced into the skin, the most advanced part enters the dermis of the skin while dissolving, so the second part from the tip of the three-layer microneedle array By placing the vaccine antigen in the cell, it is possible to achieve delivery to Langerhans cells, which are antigen-recognizing cells widely distributed in the skin epidermis layer.

Since the third layer from the tip of the microneedle array is a portion that does not pierce the skin, there is no need to place an antigen.

In the case of a microneedle having a length of about 500 μm and a bottom diameter of about 300 μm, specifically, the first layer from the tip to 160 μm is formed using a water-soluble leaky polymer material as a base. The second layer corresponding to a length from the tip of 160 to 220 μm can be efficiently delivered to the skin epidermis layer by arranging the vaccine antigen. The root portion of the microneedle, which is the third layer having a length of 220 μm or more from the tip, is molded using a water-soluble leaky polymer material in the same manner as the first layer. By using a patch in which such a microneedle array having a three-layer structure is constructed, the above problem can be solved. In addition, the numerical value regarding said length is based on the result of the verification experiment in a rat, and when applying to human skin, there are some fluctuations.

Specific embodiments will be described below with reference to examples. Of course, the present invention is not limited to the following examples.

Example 1
Prepared by adding 700 μl of purified water to 375 mg of sodium C chondroitin sulfate (Nacalai Tesque) on a silicon substrate having an inverted conical pore with a depth of about 500 microns per square centimeter and an opening diameter of about 300 microns. A viscous liquid was applied. It was covered with Saran Wrap (Kureha Chemical), and the resin base was rotated at 3500 rpm for 15 minutes using a desktop centrifuge (Kubota 4000), and filled under load using centrifugal force. Saran wrap was removed, and drying was performed by rotating under the same conditions for 30 minutes. Next, a viscous concentrated solution prepared by adding 170 μl of purified water to 20 mg of ovalbumin (Sigma, USA), a small amount of Evans Blue (Nacalai Tesque) and 35 mg of chondroitin sulfate C sodium salt as a model vaccine antigen was applied. The resin substrate was rotated at 3500 rpm for 20 minutes in a table centrifuge and filled under load using centrifugal force. The Saran wrap was removed, and it was rotated for 30 minutes under the same conditions in a desktop centrifuge and dried. After removing the residue on the resin substrate, a viscous liquid prepared by adding 1 ml of purified water to 1 g of chondroitin sulfate C sodium was applied to the hole on the resin substrate. The resin substrate was rotated at 3,500 rpm for 30 minutes in a table centrifuge and filled under load using centrifugal force. A circular substrate coated with chondroitin sulfate C sodium viscous liquid was placed on the resin substrate and dried under load. A three-layer microneedle array patch was obtained by pulling off the circular substrate after 6 hours. Ten of the prepared three-layer microneedle array patches are taken out and observed with a video microscope (VH-Z, manufactured by Keyence) to measure the length of the transparent first layer and the second layer colored blue. As a result, the following results were obtained.
First layer, 160.2 ± 7.2 μm
Second layer, 58.7 ± 4.2 μm

(Example 2)
On a resin base similar to that used in Example 1, a viscous concentrated solution prepared by adding 170 μl of purified water to 20 mg of ovalbumin, a small amount of Evans Blue, and 35 mg of chondroitin sulfate C sodium salt was applied. It was covered with Saran wrap, rotated at 3,500 rpm for 45 minutes with the resin substrate in a desktop centrifuge, and filled under load using centrifugal force. The Saran wrap was removed and dried by rotating for 15 minutes under the same conditions. After removing the residue on the resin substrate, a viscous liquid prepared by adding 1 ml of purified water to 1 g of chondroitin sulfate C sodium was applied to the hole on the resin substrate. The silicon resin substrate was rotated at 3,500 rpm for 30 minutes in a table centrifuge and filled under load using centrifugal force. A circular substrate coated with chondroitin sulfate C sodium viscous liquid was placed on the resin substrate and dried under load. A double layer microneedle array patch was obtained by pulling off the circular substrate after 6 hours. Ten of the prepared two-layer microneedle array patches were taken out, observed with a video microscope, and the length of the first layer colored blue was measured. The following results were obtained.
First layer, 153.7 ± 7.4 μm

(Example 3)
The ovalbumin content in the microneedle array patches prepared in Examples 1 and 2 was measured as the amount of protein using the Lawry method. as a result,
Three-layer microneedle array patch of Example 1, 211.2 ± 31.1 μg
Two-layer microneedle array patch of Example 2, 226.9 ± 25.6 μg

Example 4
Male rats weighing approximately 300 grams were anesthetized with pentobarbital and fixed on their back on the operating table. After removing the abdominal body hair, it was fixed to the operating table. About 0.2 mL of blood was collected from the jugular vein, and then administered by pressing the transdermal vaccine patch containing ovalbumin, which is a model antigen prepared in Examples 1 and 2, against the abdomen of the depilated rat. As a positive control, an ovalbumin injection solution was prepared and administered to rats by subcutaneous injection at a dose of 1000 μg. Two weeks after administration, blood was collected from rats under anesthesia with pentobarbital, and a second transdermal vaccine patch containing ovalbumin and an ovalbumin injection solution were boosted. When the content of ovalbumin in the microneedle array patch collected after the first and second administrations was measured as a residual amount by the Lawry method, the following results were obtained.
Three-layer microneedle array patch of Example 1, 7.0 ± 2.6 μg
Two-layer microneedle array patch of Example 2, 7.1 ± 2.0 μg
Therefore, when the amount of ovalbumin administered transdermally was calculated, the following results were obtained.
Three-layer microneedle array patch of Example 1, 204.2 ± 2.6 μg
Two-layer microneedle array patch of Example 2, 219.8 ± 2.0 μg

ELISA method (Rat) using plasma samples collected from rats after 2 weeks and 4 weeks
Anti-ovalbumin Ig ELISA kit (ALPHA DIAGNOSTICS) was used to measure the ovalbumin antibody titer in plasma, and the following results were obtained.
Plasma sample after 2 weeks Three-layer microneedle array patch administration group of Example 1, 54.99 ± 7.08 units / ml
Double-layer microneedle array patch administration group of Example 2, 8.93 ± 2.78 units / ml
Administration group by subcutaneous injection, 98.34 ± 3.82 units / ml
Plasma samples after 4 weeks Three-layer microneedle array patch administration group of Example 1, 1578.57 ± 182.59 units / ml
Double-layer microneedle array patch administration group of Example 2, 351.27 ± 156.52 units / ml
Administration group by subcutaneous injection, 655.70 ± 118.01 units / ml
From the above, the three-layer microneedle array patch of the present invention containing ovalbumin as a model antigen protein in the second layer from the tip has a higher antibody titer in circulating plasma than the two-layer microneedle array patch. Induced and demonstrated superiority as a transdermal vaccine.

(Example 5)
Three-layer and two-layer microneedle array patches containing ovalbumin fluorescently labeled with fluorescein isothiocyanate (FITC) by the method shown in Examples 1 and 2 were prepared. After administration to the hair removal skin of the male rat abdomen under pentobarbital anesthesia, the skin section obtained by excision with time was used to photograph with a video microscope under fluorescence irradiation. After the three-layer microneedle array patch prepared in Example 1 was administered to the rat skin, strong fluorescence was observed in the epidermis layer close to the surface of the skin 30 seconds later. Even after 2 minutes, strong fluorescence was observed in the epidermal layer, and delivery of FITC ovalbumin to the epidermal layer was confirmed. After administration of the two-layer microneedle array patch prepared in Example 2 to the skin of a rat, after 30 seconds and 2 minutes, strong luminescence was observed in the dermis layer away from the skin surface, and ovalbumin Delivery to the dermis layer has been demonstrated.

In order to obtain a transdermal vaccine effect with higher efficiency, whether the vaccine antigen delivery site should be a skin epidermis layer in which Langerhans cells as immune system cells are mainly distributed or a dermis layer in which dendritic cells are mainly distributed The question of which part is the optimal part has been controversial for many years. Therefore, when a soluble microneedle array patch is used for delivery of transdermal vaccine, it is a multi-layer microneedle array patch and solves the problem of which layer of vaccine antigen is best placed There was a need. According to the present invention, if a vaccine antigen is placed in a portion 160 to 220 μm from the tip corresponding to the second layer from the tip of a microneedle having a length of about 500 μm and a bottom diameter of about 300 μm, the vaccine antigen can be delivered to the epidermis layer. It has been clarified that it can be achieved and a more effective pharmacological effect can be derived.

Claims (6)

A soluble microneedle array patch consisting of a three-layer structure for efficiently delivering vaccine antigens to the skin epidermis layer.       The dissolvable microneedle array patch, wherein the base used in the dissolvable three-layer microneedle array according to claim 1 is a water-soluble leaky polymer substance.       The three-layer microneedle array patch according to claim 1, wherein the first layer corresponding to the most distal portion of the needle is formed of a water-soluble leaky polymer substance as a base. ·patch.       The three-layer microneedle array patch according to claim 1, wherein the vaccine antigen is arranged in the second layer from the tip. The three-layer microneedle array patch according to claim 1, wherein the third layer from the tip is formed of a water-soluble leaky polymer substance as a base. When the size of the microneedle is 500 μm in length and the bottom diameter is 300 μm in the three-layer microneedle array patch according to claim 1, the length of the first layer which is the most advanced is 160 μm from the tip, and the second The layer is a soluble microneedle array patch corresponding to a portion 160 to 220 μm from the tip, and the third layer is a portion 220 to 500 μm from the tip. In addition, these figures are those in rat experiments, and there are some fluctuations in the case of humans.