Minimally invasive atopic dermatitis examination method using microneedle patch and minimally invasive atopic examination kit including microneedle patch

Abstract

The present invention relates to a minimally invasive atopic dermatitis inspection method. The minimally invasive atopic dermatitis inspection method of one embodiment of the present invention includes: a step of applying a microneedle patch to the skin of a subject, the microneedle patch including a plurality of microneedles formed of a biodegradable polymer hyaluronic acid and having a solid structure and a bottom layer that becomes a substrate for forming the plurality of microneedles, the plurality of microneedles being formed of a biodegradable polymer hyaluronic acid, and the bottom layer forming the plurality of microneedles; a step of maintaining the microneedle patch attached to the skin of the subject for a predetermined time; after the predetermined time, separating the microneedle patch from the skin of the subject and subjecting it to a quantitative inspection; a step of reading the amount of interleukin-4 and interleukin-13 adsorbed on the microneedle surface of the microneedle patch in the quantitative inspection step; a step of evaluating the activity of atopic dermatitis based on the reading of the amount of interleukin-4 and interleukin-13.

Description

Technical Field

[0001] The invention relates to a minimally invasive atopic dermatitis examination method using a microneedle patch and a minimally invasive atopic examination kit comprising the microneedle patch. Background Art

[0002] Although many drugs and physiologically active substances have been developed for the treatment of diseases, in the process of delivering drugs and physiologically active substances into the body, problems such as passing biological barriers (such as skin, oral mucosa and cerebrovascular barrier) and the efficiency of drug delivery still need to be improved.

[0003] Although drugs and physiologically active substances are usually orally administered in tablet or capsule dosage forms, many drugs cannot be effectively delivered by the above-mentioned administration methods alone because they are digested or absorbed in the gastrointestinal tract or disappear by the mechanism of the liver. In addition, some drugs cannot be effectively diffused through the intestinal mucosa. For example, for seriously ill patients who need to take drugs at specific time intervals or cannot take drugs, patient compliance is also a problem.

[0004] Another common technique for delivering drugs and bioactive substances is to use a conventional needle. This method is more effective than oral administration, but has the problem of causing pain at the injection site, local skin damage, bleeding, and infection at the injection site.

[0005] In order to solve the above problems of oral administration and subcutaneous injection, a transdermal administration method using a patch is used. Transdermal administration using a patch has few side effects, high patient compliance, and is easy to maintain a predetermined blood concentration of the drug.

[0006] In order to solve the above-mentioned problems, various microstructures including microneedles have been developed. As materials for microneedles, metals and various polymers are used. Recently, biodegradable polymers have attracted much attention as materials for microneedles.

[0007] A typical method for making a microstructure of a biodegradable material is to use a mold. When a semiconductor manufacturing process is applied, a mold is first made by engraving the shape of the microstructure to be formed, and the microstructure material is poured into the mold and solidified and then removed to form a microstructure, i.e., a microneedle.

[0008] As a new method for making microstructures that can replace this molding method, the applicant has obtained multiple patents at home and abroad for the manufacturing method of the air supply stretching method. The air supply stretching method is briefly described as follows: after positioning a viscous bioaffinity polymer substance on the bottom layer of a patch located on a substrate or the bottom layer before combining with the patch, the same bioaffinity polymer substance is positioned on the bottom layer of a patch located on another substrate or the bottom layer before combining with the patch or omitted, and the above-mentioned other substrate is reversed to make it close to the above-mentioned bioaffinity polymer substance, and finally contact, and then the relative distance between the two substrates is separated to stretch the contacting viscous bioaffinity polymer substance, and after the stretching process is completed, air is supplied to fix the shape in the stretched state, and the middle part is cut to form the same microstructure on two different substrates.

[0009] The above briefly describes the technical meaning of the microstructure as a drug and physiologically active substance delivery unit and the method for making the microstructure. The applicant proposes to use the microstructure for a new purpose other than the delivery of drugs and physiologically active substances in order to realize the present invention. The microstructure, i.e., a patch including microneedles, is used in minimally invasive atopic dermatitis examination.

[0010] Atopic dermatitis is an inflammatory skin disease accompanied by itching. It is the most representative allergic disease along with allergic rhinitis and bronchial asthma. The prevalence rate in children is 10-30%. It mostly occurs in infants and children, but recently, a lot of atopic dermatitis in adults has increased. The main pathological mechanisms of atopic dermatitis involve abnormalities in the skin barrier, immunological abnormalities, genetic factors, environmental factors, etc. The abnormality of the skin barrier is caused by the reduction of many biological factors related to the differentiation of skin keratinocytes such as filaggrin and the synthesis of ceramide, which is an important factor of skin barrier lipids, causing abnormalities in the skin barrier, thereby causing dry skin and pruritus. Immunological abnormalities are caused by the combined effects of innate and adaptive immune responses of multiple cells such as dendritic cells, T cells, and other innate immune cells (mast cells, eosinophils, basophils, and neutrophils). Multiple cytokines such as TSLP, IL-4, and IL-13, and multiple inflammatory response substances such as chemokines are secreted to cause immune responses. Among them, IL-4 and IL-13 are one of the most core cytokines for the cause of the disease. Recently, many biological drugs targeting these markers have been developed and are widely used as new therapeutic agents for refractory atopic dermatitis, showing very excellent effects.

[0011] Observing the changes in many biological substances related to the pathogenesis of atopic dermatitis as described above is related to the development of new therapeutic drugs. In order to discover the new pathogenesis of atopic dermatitis, determine the therapeutic effect, perform prognosis prediction tests, and discover new therapeutic agents, the most common method is to biopsy the lesions of atopic dermatitis and analyze the biological factors related to atopic dermatitis in the tissue. Figure 1 This type of biopsy is indicated. However, biopsy requires the skilled technique of the surgeon. In addition, there is a disadvantage that the patient suffers pain and aversion, and a scar is left at the biopsy site after the test. Due to this reason, it is actually difficult to detect this factor one by one for the human body, which becomes an obstacle to the development of new technologies or new drugs. In order to improve this disadvantage, a non-invasive stripping method of a tape is proposed, which utilizes the attachment / removal of the tape at the lesion site to analyze the skin test object adhered to the tape. However, there is a disadvantage that the substance adhered to the tape is limited to the stratum corneum. On the contrary, atopic dermatitis is a skin disease caused by abnormalities in the immune system in the stratum corneum, epidermis, and dermis, so it is particularly important to collect tissues in the epidermis and dermis as in a biopsy. Therefore, the tape stripping method can be used as a part of an auxiliary method for the development of new drugs or the study of the pathogenesis of atopic dermatitis, but it is difficult to obtain complete results. In this regard, (multiple) inventors of the present invention used skin biopsy, tape stripping and various microstructure patches to obtain skin samples, and confirmed whether skin samples that can replace skin biopsy were obtained by stripping with tape or microstructure patches, thereby confirming the usefulness of the collection method that can replace skin biopsy.

[0012] There are three types of microstructured patches used in this study, namely, blank patches without microstructures, hollow microstructured patches, and dissolving microstructured patches as the control groups of the microstructured patches. The blank patches used as the control group are usually composed only of hydrocolloid patches mainly used for wound healing. The hollow microstructured patch has the advantage of being able to deliver drugs while minimizing the risk of pain and infection through holes with tiny cross-sectional areas inside the microstructure. In the dissolving microstructured patch, microneedles of hyaluronic acid components of pharmaceutical grade are formed on the hydrocolloid patch. The manufacturing methods of the microstructured patch include the above-mentioned mold method and stretching method, etc. Regardless of the manufacturing method, microneedles of hyaluronic acid components are formed on the patch. When this microstructured patch is applied to the skin, such as Figure 2 As shown, the microstructure penetrates the stratum corneum into the skin. The inventors attached a patch for a sufficient time when multiple factors located in the epidermis and dermis can be adsorbed on the microstructure that enters the skin, and then a skin test object is obtained from the removed patch. Afterwards, the inventors proposed a new minimally invasive atopic dermatitis examination method that can replace tissue biopsy through comparative analysis between tissue biopsy, tape stripping, and three microstructure patch methods, which is used to detect biomarkers important to the cause of the disease or measure the expression level. Summary of the invention

[0013] Technical issues

[0014] The present invention aims to solve the problems of the conventional tissue biopsy and tape stripping methods for atopic dermatitis examination.

[0015] Existing tissue biopsy has the disadvantages of requiring the skilled techniques of the surgeon, causing discomfort to the patient, and leaving scars at the biopsy site after the test.

[0016] Existing tape stripping has the disadvantage that the material adhered to the tape is confined to the stratum corneum and can only be used as an auxiliary inspection method and cannot provide complete results.

[0017] The purpose of the present invention is to provide a new minimally invasive atopic dermatitis examination method and a minimally invasive atopic dermatitis examination kit, which are almost painless and leave no scars compared to existing tissue biopsy, can improve the patient's convenience of use while collecting skin factors inside the stratum corneum, and can improve the reliability of existing tape stripping comparative analysis results.

[0018] Solutions to the problem

[0019] The minimally invasive atopic dermatitis inspection method of one embodiment of the present invention includes: a step of applying a microneedle patch to the skin of a subject, wherein the microneedle patch includes a plurality of microneedles formed of a biodegradable polymer hyaluronic acid and having a solid structure and a bottom layer that serves as a base for forming the plurality of microneedles; a step of maintaining the microneedle patch attached to the skin of the subject for a specified time; a step of separating the microneedle patch from the skin of the subject after the specified time has passed and subjecting the patch to a quantitative inspection; a step of reading the amounts of interleukin-4 and interleukin-13 adsorbed on the microneedle surfaces of the microneedle patch in the quantitative inspection step; and a step of determining the activity of the atopic dermatitis being treated based on the read amounts of interleukin-4 and interleukin-13.

[0020] The minimally invasive atopic dermatitis test kit of one embodiment of the present invention includes: a device capable of quantitatively analyzing the amount of protein extracted from the skin of a subject; a microneedle patch, comprising a plurality of microneedles formed of a biodegradable polymer hyaluronic acid and having a solid structure and a bottom layer that serves as a base for forming the plurality of microneedles. The above-mentioned microneedle patch is applied to the skin of the subject, and the above-mentioned device is used to quantitatively analyze the protein of the subject adsorbed on the surface of the microneedles of the above-mentioned microneedle patch in a separated state after maintaining for a specified time. The above-mentioned device reads the amount of interleukin-4 and interleukin-13 adsorbed on the surface of the microneedles of the above-mentioned microneedle patch. The activity of atopic dermatitis is evaluated based on the reading of the above-mentioned interleukin-4 and interleukin-13.

[0021] Furthermore, additional structures may be provided to the minimally invasive atopic dermatitis examination method or minimally invasive atopic dermatitis examination kit of the present invention.

[0022] Effects of the Invention

[0023] According to the present invention, the problems of the conventional tissue biopsy and tape stripping methods for examining atopic dermatitis can be solved.

[0024] Existing tissue biopsy has the disadvantages of requiring the skilled techniques of the surgeon, causing discomfort to the patient, and leaving scars at the biopsy site after the test.

[0025] Existing tape stripping has the disadvantage that the material adhered to the tape is confined to the stratum corneum and can only be used as an auxiliary inspection method and cannot provide complete results.

[0026] According to the present invention, a new minimally invasive atopic dermatitis examination method and a minimally invasive atopic dermatitis examination kit can be provided, which are almost painless and leave no scars compared to existing tissue biopsy, can improve the patient's convenience of use while collecting skin factors inside the stratum corneum, and can improve the reliability of existing tape stripping comparison analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A diagram showing a skin biopsy method in the prior art.

[0028] Figure 2 This is a diagram conceptually showing the skin biopsy method of the present invention.

[0029] Figure 3 The figure shows four control groups in an experiment (hereinafter referred to as "Basic Experiment 1") performed by (multiple) inventors for a minimally invasive skin biopsy method closely related to the concept of the present invention before clinical trials related to the examination method of atopic dermatitis, showing 1) a blank patch without microneedles, 2) solid microneedles made of metal material, 3) a low molecular weight hyaluronic acid microneedle patch, and 4) a high molecular weight hyaluronic acid microneedle patch.

[0030] Figure 4 This is a table showing the results of a procollagen 1 detection test performed on a patient group in their 30s and a patient group in their 60s using the four control groups of Basic Experiment 1.

[0031] Figure 5 This is a table showing the results of measuring the length changes of the low molecular weight hyaluronic acid microneedle patches and the high molecular weight hyaluronic acid microneedle patches before and after biopsy application in the four control groups of Basic Experiment 1.

[0032] Figure 6This is a table showing the results of another basic experiment (hereinafter referred to as "Basic Experiment 2"), in which the control groups used were 1) a blank patch without microneedles, 2) a low molecular weight hyaluronic acid microneedle patch, and 3) a high molecular weight hyaluronic acid microneedle patch. In each control group, the skin of a group of patients in their 20s and a group of patients in their 60s was applied for 5 seconds and 60 seconds, then removed, and the results of pro-collagen 1 detection were measured.

[0033] Figure 7 This table shows the results of measuring the procollagen 1 detection results after applying the product to the skin of a group of patients in their twenties and a group of patients in their sixties for 10 seconds and 30 seconds respectively and then removing the product.

[0034] Figure 8 This is a table showing the results of another basic experiment (hereinafter referred to as "Basic Experiment 3"), in which the control groups used were 1) low molecular weight chitosan microneedle patch, 2) high molecular weight chitosan microneedle patch. In each control group, the skin was applied for 5 seconds, 60 seconds and 300 seconds and then removed, and the procollagen (Pro-Collagen) 1 detection results were measured.

[0035] Fig. 9 These are multiple images of three types of patches used in a clinical trial to verify the effects of the present invention.

[0036] Fig.10 This is a table showing the progress schedule of clinical trials.

[0037] Fig.11 A table that organizes information related to clinical trial subjects.

[0038] Fig.12 This is a table showing the amount of protein attached to the four skin sample acquisition units: tape, blank patch, HA microneedle, and hollow needle.

[0039] Fig.13 and Fig.14 The graphs show the results of confirming the expression levels of IL-13 and IFN-γ (gamma) using tape and microneedles, respectively.

[0040] Fig.15 The table shows the expression of IL-4 in skin samples obtained from multiple clinical trial subjects using HA microneedles as determined by enzyme-linked immunosorbent assay (ELISA).

[0041] Fig.16 The table shows the expression of IL-13 in skin samples obtained from multiple clinical trial subjects using HA microneedles as determined by enzyme-linked immunosorbent assay (ELISA).

[0042] Fig.17 This is a table showing the expression of IFN-γ (gamma) in skin samples obtained from multiple clinical trial subjects using HA microneedles as determined by enzyme-linked immunosorbent assay (ELISA).

[0043] Fig.18 The results of confirming the correlation between the expression of IL-4 in skin samples obtained from multiple clinical trial subjects using HA microneedles and the clinical course of atopic dermatitis by statistical analysis (SPSS) are presented.

[0044] Fig.19 The present invention shows the results of confirming the correlation between the expression of IL-13 in skin samples obtained from multiple clinical trial subjects using HA microneedles and the clinical course of atopic dermatitis by statistical analysis (SPSS).

[0045] Fig. 20 The results show the results of confirming the expression of IFN-γ (gamma) in skin samples obtained from multiple clinical trial subjects using HA microneedles and the correlation with the clinical course of atopic dermatitis through statistical analysis (SPSS). DETAILED DESCRIPTION

[0046] The detailed description of the invention described below refers to the accompanying drawings that show as examples specific embodiments in which the invention may be practiced. Such embodiments are described in detail so that a person skilled in the art of the invention can fully practice the invention. It should be understood that the various embodiments of the invention, although different, are not necessarily mutually exclusive. The detailed description described below is not intended to be limiting, and the scope of the invention includes the scope requested in the multiple claims of the claims and all scopes equivalent thereto.

[0047] Hereinafter, various preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings in order to enable those skilled in the art to easily implement the present invention.

[0048] Results of basic experiments related to minimally invasive skin biopsy methods

[0049] Figure 2 This is a diagram conceptually showing the skin biopsy method of the present invention.

[0050] Figure 2 The upper left column of the figure shows a microneedle patch on the market with the product name "Therapass". This product is a product on the market and sold by the applicant company, and is a high molecular weight hyaluronic acid microneedle patch with a molecular weight of 1000 kDa. Figure 2 The lower left section shows the state in which the microneedle patch is applied to the skin, and more specifically, shows the state in which the microneedles of the patch penetrate into the dermis layer of the skin. Figure 2The right side of the figure conceptually shows the representative technical idea of ​​the present invention that a microneedle patch is applied to a dermatitis patient and then a biopsy is performed using the proteins in the skin of the microneedles attached to the patch. The term bio-mining used here means mining proteins in the skin of a subject for biopsy.

[0051] Figure 3 It represents four control groups of basic experiment 1 performed by (multiple) inventors for the minimally invasive skin biopsy method closely related to the concept of the present invention before the clinical trial related to the inspection method of atopic dermatitis. The first group of these four control groups is a blank patch without microneedles. The second group is a solid microneedle made of metal material. The third group is a low molecular weight hyaluronic acid microneedle patch. The molecular weight of hyaluronic acid used for the low molecular weight hyaluronic acid microneedle patch is 110kDa. The fourth group is a high molecular weight hyaluronic acid microneedle patch. The molecular weight of hyaluronic acid used for the high molecular weight hyaluronic acid microneedle patch is 1000kDa.

[0052] Figure 4 This is a table showing the results of a procollagen 1 detection test conducted on a patient group in their 30s and a patient group in their 60s using the above four control groups.

[0053] Figure 4 The x-axis of the graph shows a blank patch, a low molecular weight hyaluronic acid patch, and a high molecular weight hyaluronic acid patch. The mass per unit volume of procollagen 1 detected on the y-axis is expressed in picograms per milliliter. The solid microneedles made of metal in the four control groups are not shown. This is because protein quantification itself was not performed at all. Figure 4 The "enzyme-linked immunosorbent assay (ELISA)" shown is a method for quantitatively measuring the intensity and amount of antigen-antibody reaction by measuring the activity of the enzyme for antibody or antigen labeling enzyme, and refers to a method used in modern life engineering. This method is used in the entire experiment of the present invention.

[0054] Figure 4 What needs to be carefully observed in the table is whether there is a substantial difference from the blank patch as the first control group. This is because the blank patch is a prior art that existed before the application of the present invention. When the test results are not substantially different from the blank patch, the present invention does not help to improve the biological detection effect. Figure 4The results showed that the amount of procollagen 1 detected in the low molecular weight (110 kDa) hyaluronic acid patch was not substantially different from that in the blank patch. However, it was found that the amount of procollagen 1 detected in the high molecular weight (1000 kDa) hyaluronic acid patch was significantly increased compared to the blank patch and the low molecular weight hyaluronic acid patch.

[0055] The inventors of the present invention have been continuously experimenting on the problem of how to present such significant differences in results according to the molecular weight of the hyaluronic acid that is a component of the microneedle patch, analyzing the reasons, and finding out that the reason is the dissolution rate into the skin according to the difference in molecular weight. The microneedle components of the current commercialized microneedle patch are biocompatible and biodegradable polymers, which is the general trend. "Biocompatible substances" means substances that are non-toxic and chemically inert to the human body. And, "biodegradable substances" means substances that can be decomposed in vivo using body fluids, enzymes or microorganisms. And, it is well known that in a variety of biodegradable substances, the smaller the molecular weight, the faster the dissolution rate into the organism, and the larger the molecular weight, the slower the dissolution rate into the organism. On the other hand, the substances known as biocompatible polymers are as follows.

[0056] Hyaluronic acid (HA), gelatin, chitosan, collagen, alginate, pectin, carrageenan, chondroitin sulfate, dextran sulfate, polylysine, carboxymethyl chitin, fibrin, agarose, pullulan, cellulose, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose, polyols, gum arabic, alginate, cyclodextrin, dextrin, glucose, fructose, starch, trehalose, dextrose, maltose, lactose, lactulose, levulose, turanose, raffinose, melezitose, dextran, sorbitol, xylitol, palakinite, polylactic acid, polyglycolic acid acid), polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, polymaleic acid, polyethylene glycol-polyester copolymer (poly(ethyleneglycol) / polyester), chitosan / glycerol phosphate, polyphosphazene, polycaprolactone, polycarbonate, polyethylene glycol-polypropylene glycol (Poly(ethylene glycol) / poly(propylene glycol)), polycyanoacrylate, polyorthoester, poly(N-(2-hydroxyethyl)methacrylamide-lactate), poly(propylenephosphate), etc.

[0057] When the microneedle patch is attached to the skin, the protein can be attached to the biocompatible microneedle structure and extracted during the process of the microneedles of the patch entering the dermis of the skin and maintaining the microneedles after entering. However, for low molecular weight hyaluronic acid microneedles, even if the protein of the organism adheres to its surface, the dissolution rate is relatively fast. When attached to the skin for a long time, as it dissolves itself, the protein attached to the surface may be lost. Analysis shows that the reason for this is Figure 4 The experimental results supporting this are shown in Figure 6 middle.

[0058] Figure 5This is a table showing the results of measuring the length changes of the low molecular weight hyaluronic acid microneedle patches and the high molecular weight hyaluronic acid microneedle patches before and after biopsy application in the above four control groups.

[0059] according to Figure 5 As a result, the microneedles of the 110kDa low molecular weight hyaluronic acid microneedle patch showed a length reduction of approximately 30% during the period of attachment to the human skin. On the other hand, the microneedles of the 1000kDa high molecular weight hyaluronic acid microneedle patch showed only a length reduction of approximately 10% during the period of attachment to the skin. Even if the low molecular weight hyaluronic acid microneedles have biological proteins attached to their surface, the dissolution rate is relatively fast. When attached to the skin for a long time, as the microneedles themselves dissolve, the proteins attached to the surface may be lost. This is Figure 4 The reason for the experimental results shown is that this experimental result supports this point. Figures 4 to 5 In the experiment of the first embodiment shown, the skin attachment time of each control group was 5 minutes. In response to this, the inventors found that it was necessary to shorten the attachment time to the skin to less than 5 minutes, perform experiments during different time lengths, and plan an experiment (experiment 2) different from the above-mentioned basic experiment (experiment 1).

[0060] Figure 6 Table showing the results of basic experiment 2 which is different from the above-mentioned basic experiment 1, wherein the control groups used are 1) blank patches without microneedles, 2) low molecular weight hyaluronic acid microneedle patches, and 3) high molecular weight hyaluronic acid microneedle patches. In each control group, the skin of the patient group in their 20s and the patient group in their 60s was applied for 5 seconds and 60 seconds before being removed, and the results of pro-collagen 1 were determined. The molecular weight of the low molecular weight hyaluronic acid and the molecular weight of the high molecular weight hyaluronic acid are the same as those in basic experiment 1. As the low molecular weight hyaluronic acid microneedle patch, the microneedle patch for beauty use in the market of the applicant was used, and as the high molecular weight hyaluronic acid microneedle patch, the microneedle patch for medical equipment use in the market of the applicant (trade name: "Therapass") was used. According to the experimental results of basic experiment 1, the biological protein extraction performance of the solid microneedle made of metal material is far from sufficient, so it is not used as a control group in this embodiment.

[0061] from Figure 6The following facts can be seen from the experimental results. First, at an attachment time of 5 seconds, no significant improvement in the results of the blank patch compared to the microneedle patch can be expected. Second, at an attachment time of 60 seconds, multiple microneedle patches (low molecular weight and high molecular weight) showed significantly outstanding biological protein extraction performance compared to the blank patch. Third, at an attachment time of 60 seconds, the biological protein extraction performance of the high molecular weight microneedle patch was also outstanding compared to the biological protein extraction performance of the low molecular weight microneedle patch. In order to Figure 6 The experimental results were additionally conducted for the time period of adhesion greater than 5 seconds and less than 60 seconds. Figure 7 experiment.

[0062] Figure 7 This table shows the results of measuring pro-collagen 1 by applying the product to the skin of a group of patients in their twenties and a group of patients in their sixties in the control group of basic experiment 2 for 10 seconds and 30 seconds respectively, then removing the product.

[0063] For the blank patch, there was no significant change in the results when attached for either 10 seconds or 30 seconds. Figure 7 Only the results when attached for 30 seconds are marked in the table. Figure 7 The facts that can be known from the results are as follows. First, with only 10 seconds of attachment time, it can be confirmed that the biological protein extraction performance of multiple microneedle patches (low molecular weight and high molecular weight) is greatly improved compared to the blank patch. Second, at an attachment time of 10 seconds, the protein extraction performance of the low molecular weight microneedle patch and the high molecular weight microneedle patch is similar. Third, at an attachment time of 30 seconds, the protein extraction performance of the high molecular weight microneedle patch far exceeds the protein extraction performance of the low molecular weight microneedle patch.

[0064] It was confirmed that the biopsy performance of the microneedle patch can be meaningfully exerted when the attachment time is maintained for more than 10 seconds. Before the low molecular weight microneedles dissolve in the skin, that is, during the attachment time of about 10 seconds, the low molecular weight microneedle patch shows a biopsy performance similar to that of the high molecular weight microneedle patch, which is evaluated as having a significant effect on this. Figure 7 The technical significance of the experiment.

[0065] Finally, in order to understand whether microneedle patches made of other biocompatible polymers besides hyaluronic acid are suitable for biopsy purposes, several inventors used chitosan microneedle patches to conduct Fig. 9Experiment (Basic Experiment 3). Chitosan is a biocompatible polymer like hyaluronic acid, but it is not biodegradable. It is well known that chitosan is a substance made by processing chitin contained in crustaceans into a substance that is easily absorbed by the human body. It has the effect of activating aged cells to inhibit aging and enhance immunity. Its effect has also been proven in the activation of skin cells. It is widely used as a cosmetic ingredient and also as a component of microneedle patches for skin beauty purposes.

[0066] Figure 8 This is a table showing the results of Basic Experiment 3, in which the control groups used were 1) low molecular weight chitosan microneedle patch, 2) high molecular weight chitosan microneedle patch. In each control group, the skin was applied for 5 seconds, 60 seconds and 300 seconds and then removed, and the procollagen (Pro-Collagen) 1 detection results were measured.

[0067] exist Figure 8 In the experiment, the molecular weight of low molecular weight chitosan 1 was 50kDa to 100kDa. The molecular weight of high molecular weight chitosan 2 was 250kDa to 350kDa. After attaching the chitosan to the patient group in their 20s for 5 seconds, 1 minute, and 5 minutes, the expression of procollagen I was confirmed. Figure 8 In the experimental results of Example 3 shown, the present inventors confirmed that chitosan, one of the biocompatible polymers, can also play the same protein extraction function for biopsy as hyaluronic acid. In addition to hyaluronic acid and chitosan exemplified in this specification, a variety of other biocompatible polymers are also likely to become candidates for microneedle patch materials for minimally invasive skin biopsy.

[0068] Results of research related to new atopic dermatitis examination method (implementation of clinical trials)

[0069] first, Fig. 9 These are multiple images of three types of patches used in a clinical trial to verify the effects of the present invention.

[0070] Fig. 9 The blank patch on the left side is composed only of a hydrocolloid patch, on which no needle-like structure is formed. The hollow patch in the middle is a microneedle commercially marketed by INCYTO, which is a structure in which a hole with a small cross-sectional area is formed in the center of a slender metal needle. The hyaluronic acid (HA) microneedle patch on the right side represents a microneedle patch marketed under the product name "Therapass". This product is a product listed and sold by the applicant company, and is a high molecular weight hyaluronic acid microneedle patch with a molecular weight of 1000kDa.

[0071] The target number of subjects for this clinical trial is 20 patients with atopic dermatitis (forearm). The study period is 5 months after IRB approval. The study method is as follows.

[0072] 1) Week 0

[0073] -Tissue inspection: 3mm tissue inspection

[0074] -Tape stripping: 15 times using D-Squame disk tape stripping

[0075] -Protein extraction: Sodium dodecyl sulfate (SDS) is used to extract proteins from the tape or microneedle patch

[0076] 2) Week 2

[0077] -Three types of microneedle patches inspection: Attach the three types of microneedle patches for 5 minutes and then remove them

[0078] -Protein extraction: Use SDS to extract proteins from tape or microneedle patches

[0079] In this way, the subjects were examined at week 0 and week 2, and the test results and clinical course were compared to confirm the clinical usefulness of the test method. In this confirmation, protein quantification was performed by BCA assay, and ELISA was performed to quantify IL-4, IL-13, and IFN-γ (gamma). SPSS was used as a statistical analysis technique.

[0080] Fig.10 Table showing the schedule for advancing clinical trials. As mentioned above, the clinical trial will be conducted for 5 months after IRB. After recruiting test subjects, tissue biopsy, D-squame tape stripping, blank patches, HA microneedles, and hollow needle patches are applied to multiple subjects in visit week 0, and proteins are separated from the detection units, respectively. In visit week 2, blank patches, HA microneedles, and hollow needle patches are applied to multiple subjects, and proteins are separated from the detection units, respectively. Finally, the amount of separated protein is quantified, and enzyme-linked immunosorbent assay (ELISA) is performed on IL-4, IL-13, and IFN-γ (gamma), and the data is analyzed.

[0081] Fig.11 A table that organizes information related to clinical trial subjects. Fig.11The table shows the gender, age, and symptom course of the subjects participating in this clinical trial after 2 weeks (for the course of patient No. 10, the "slimproved" marked on the table means slightly improved, i.e., a slightly better course). In addition to the failure (Fail), a total of 20 subjects were registered and studied. Fig.11 The subjects No. 4, No. 5, No. 7, No. 16, No. 20 and No. 26, which were shown in a manner distinguishable from the other subjects in the table, refused to attach / remove the patch a second time and were excluded from the test subjects.

[0082] Skin samples were obtained using forearm biopsy, tape stripping, and three microneedle patches at week 0, and three microneedle patches at week 2. The obtained samples were placed in a 60 mm dish, and proteins were immediately extracted using SDS solution in the laboratory and stored at -80 degrees Celsius.

[0083] Fig.12 Table showing the amount of protein attached to the four skin samples of tape, blank patch, HA microneedle, and hollow needle. The obtained skin test material was quantified by BCAassay. In week 0, tape, blank patch, hyaluronic acid microneedle, and hollow needle were all applied to multiple patients. After 2 weeks, blank patch, hyaluronic acid microneedle and hollow needle were provided to multiple patients in addition to tape. For normal people, tape, blank patch, and hyaluronic acid microneedle were applied in addition to hollow needle.

[0084] The amount of protein quantified can be Fig.12 confirmed in the chart (normal people are not included in the IRB). Fig.12 The NET optical density (OD) (absorbance) value as the y-axis in the graph was obtained by "OD value of protein obtained from the skin test material" - "OD value of each patch in the blank state". The protein quantification results confirmed that almost no protein was extracted in the blank patch or hollow needle patch compared to the tape or hyaluronic acid microneedle patch. This confirmed that blank patches or hollow needles are not suitable for target factor detection using non-invasive methods.

[0085] The expression levels of IL-4, IL-13 and IFN-γ (gamma) were confirmed in the test samples obtained from normal people and atopic dermatitis patients by tissue biopsy, tape and microneedle. In the tissue biopsy, ribonucleic acid (RNA) was collected and the expression levels of IL-4, IL-13 and IFN-γ (gamma) were confirmed by real-time fluorescence quantitative PCR (qRT-PCR) test. Unlike the samples of atopic dermatitis patients, the expression of the above-mentioned multiple targets was not confirmed in normal people. Then, in the tape and microneedle, the protein was collected and the expression levels of IL-13 and IFN-γ (gamma) were confirmed by enzyme-linked immunosorbent assay (ELISA) test. Fig.13 and Fig.14 As shown, in the test sample using the tape, the expression of the above-mentioned multiple targets was not confirmed in both normal people and atopic dermatitis patients, but in the test sample using the microneedle patch, higher expression levels of IL-13 and IFN-γ (gamma) were confirmed in atopic dermatitis patients than in normal people.

[0086] The expression of IL-4, IL-13, and IFN-γ (gamma) in the obtained test material was measured by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figures 15 to 17 middle. Fig.15 The table shows the expression of IL-4 in skin samples obtained from multiple clinical trial subjects using HA microneedles as determined by enzyme-linked immunosorbent assay (ELISA). Fig.16 The table shows the expression of IL-13 in skin samples obtained from multiple clinical trial subjects using HA microneedles as determined by enzyme-linked immunosorbent assay (ELISA). Fig.17 This is a table showing the expression of IFN-γ (gamma) in skin samples obtained from multiple clinical trial subjects using HA microneedles as determined by enzyme-linked immunosorbent assay (ELISA).

[0087] pass Figures 15 to 17 The measurement results confirmed the changes in the target protein amount in the skin test sample obtained using the hyaluronic acid microneedle patch when visiting atopic dermatitis patients at week 0 and week 2.

[0088] More specifically, the following describes Fig.15 The measurement results.

[0089] The OD value of IL-4 in patient 1 was 0.021 at week 0 and 0.044 at week 2.

[0090] The OD value of IL-4 in patient 2 was 0.029 at week 0 and 0.012 at week 2.

[0091] The OD value of IL-4 in patient 3 was 0.026 at week 0 and 0.017 at week 2.

[0092] The OD value of IL-4 in patient 6 was 0.053 at week 0 and 0.043 at week 2.

[0093] The OD value of IL-4 in patient 8 was 0.025 at week 0 and 0.021 at week 2.

[0094] The OD value of IL-4 in patient No. 9 was 0.072 at week 0 and 0.141 at week 2.

[0095] The OD value of IL-4 in patient 10 was 0.065 at week 0 and 0.093 at week 2.

[0096] The OD value of IL-4 in patient 11 was 0.012 at week 0 and 0.098 at week 2.

[0097] The OD value of IL-4 in patient No. 12 was 0.053 at week 0 and 0.095 at week 2.

[0098] The OD value of IL-4 in patient 13 was 0.026 at week 0 and 0.022 at week 2.

[0099] The OD value of IL-4 in patient 14 was 0.029 at week 0 and 0.027 at week 2.

[0100] The OD value of IL-4 in patient 15 was 0.034 at week 0 and 0.025 at week 2.

[0101] The OD value of IL-4 in patient 17 was 0.036 at week 0 and 0.027 at week 2.

[0102] The OD value of IL-4 in patient 18 was 0.038 at week 0 and 0.025 at week 2.

[0103] The OD value of IL-4 in patient 19 was 0.023 at week 0 and 0.021 at week 2.

[0104] The OD value of IL-4 in patient 21 was 0.026 at week 0 and 0.052 at week 2.

[0105] The OD value of IL-4 in patient 22 was 0.012 at week 0 and 0.038 at week 2.

[0106] The OD value of IL-4 in patient 23 was 0.031 at week 0 and 0.017 at week 2.

[0107] The OD value of IL-4 in patient No. 24 was 0.035 at week 0 and 0.028 at week 2.

[0108] The OD value of IL-4 in patient No. 25 was 0.025 at week 0 and 0.024 at week 2.

[0109] More specifically, the following describes Fig.16 The measurement results.

[0110] The Net OD value of IL-13 in patient 1 was 0.0027 at week 0 and 0.0017 at week 2.

[0111] The Net OD value of IL-13 in patient 2 was 0.0072 at week 0. The Net OD value of IL-13 in week 2 was 0.0002.

[0112] The Net OD value of IL-13 in patient 3 was 0.0072 at week 0 and 0.0017 at week 2.

[0113] The Net OD value of IL-13 in patient 6 at week 0 was 0.0132, and the Net OD value of IL-13 at week 2 was 0.0077.

[0114] The Net OD value of IL-13 in patient 8 was 0.0062 at week 0 and 0.0059 at week 2.

[0115] The Net OD value of IL-13 in patient No. 9 at week 0 was 0.0059. The Net OD value of IL-13 at week 2 was 0.0069.

[0116] The Net OD value of IL-13 in patient 10 was 0.0052 at week 0 and 0.0059 at week 2.

[0117] The Net OD value of IL-13 in patient No. 11 at week 0 was 0.0052. The Net OD value of IL-13 at week 2 was 0.0052.

[0118] The Net OD value of IL-13 in patient 12 was 0.0022 at week 0 and 0.0062 at week 2.

[0119] The Net OD value of IL-13 in patient 13 was 0.0132 at week 0 and 0.0067 at week 2.

[0120] The Net OD value of IL-13 in patient No. 14 was 0.0072 at week 0 and 0.0069 at week 2.

[0121] The Net OD value of IL-13 in patient No. 15 was 0.0037 at week 0 and 0.0042 at week 2.

[0122] The Net OD value of IL-13 in patient 17 was 0.0042 at week 0 and 0.0037 at week 2.

[0123] The Net OD value of IL-13 in patient No. 18 was 0.0027 at week 0 and 0.0032 at week 2.

[0124] The Net OD value of IL-13 in patient No. 19 at week 0 was 0.0059, and the Net OD value of IL-13 at week 2 was 0.0049.

[0125] The Net OD value of IL-13 in patient No. 21 at week 0 was 0.0065, and the Net OD value of IL-13 at week 2 was 0.0082.

[0126] The Net OD value of IL-13 in patient No. 22 at week 0 was 0.0102. The Net OD value of IL-13 at week 2 was 0.0102.

[0127] The Net OD value of IL-13 in patient No. 23 was 0.0079 at week 0. The Net OD value of IL-13 in week 2 was 0.0079.

[0128] The Net OD value of IL-13 in patient No. 24 at week 0 was 0.0137, and the Net OD value of IL-13 at week 2 was 0.0092.

[0129] The Net OD value of IL-13 in patient No. 25 at week 0 was 0.0502, and the Net OD value of IL-13 at week 2 was 0.0082.

[0130] More specifically, the following describes Fig.17 The measurement results.

[0131] The Net OD value of IFN-γ(gamma) in patient 1 at week 0 was 0.0220. The Net OD value of IFN-γ(gamma) in week 2 was 0.0380.

[0132] The Net OD value of IFN-γ(gamma) in patient 2 at week 0 was -0.0005. The Net OD value of IFN-γ(gamma) at week 2 was 0.0410.

[0133] The Net OD value of IFN-γ(gamma) in patient 3 at week 0 was 0.0015. The Net OD value of IFN-γ(gamma) at week 2 was -0.0045.

[0134] The Net OD value of IFN-γ(gamma) in patient 6 at week 0 was 0.0350. The Net OD value of IFN-γ(gamma) at week 2 was 0.0140.

[0135] The Net OD value of IFN-γ(gamma) in patient 8 at week 0 was 0.0395. The Net OD value of IFN-γ(gamma) at week 2 was 0.0340.

[0136] The Net OD value of IFN-γ(gamma) in patient No. 9 at week 0 was 0.0345. The Net OD value of IFN-γ(gamma) at week 2 was 0.0640.

[0137] The Net OD value of IFN-γ(gamma) in patient No. 10 at week 0 was 0.0145. The Net OD value of IFN-γ(gamma) at week 2 was 0.0320.

[0138] The Net OD value of IFN-γ(gamma) in patient 11 at week 0 was 0.0515. The Net OD value of IFN-γ(gamma) at week 2 was 0.0465.

[0139] The Net OD value of IFN-γ(gamma) in patient 12 at week 0 was 0.0295. The Net OD value of IFN-γ(gamma) at week 2 was 0.0340.

[0140] The Net OD value of IFN-γ(gamma) in patient 13 at week 0 was 0.0410. The Net OD value of IFN-γ(gamma) at week 2 was 0.0340.

[0141] The Net OD value of IFN-γ(gamma) in patient No. 14 at week 0 was 0.0380, and the Net OD value of IFN-γ(gamma) at week 2 was 0.0615.

[0142] The Net OD value of IFN-γ(gamma) in patient 15 at week 0 was 0.0455. The Net OD value of IFN-γ(gamma) at week 2 was 0.0560.

[0143] The Net OD value of IFN-γ(gamma) in patient 17 at week 0 was 0.0310, and the Net OD value of IFN-γ(gamma) at week 2 was 0.0390.

[0144] The Net OD value of IFN-γ(gamma) in patient 18 at week 0 was 0.0405. The Net OD value of IFN-γ(gamma) at week 2 was 0.0275.

[0145] The Net OD value of IFN-γ(gamma) in patient No. 19 at week 0 was 0.0510. The Net OD value of IFN-γ(gamma) at week 2 was 0.0415.

[0146] The Net OD value of IFN-γ(gamma) in patient No. 21 at week 0 was 0.0285. The Net OD value of IFN-γ(gamma) at week 2 was 0.0000.

[0147] The Net OD value of IFN-γ(gamma) in patient No. 22 at week 0 was 0.0160. The Net OD value of IFN-γ(gamma) at week 2 was 0.0255.

[0148] The Net OD value of IFN-γ(gamma) in patient No. 23 at week 0 was 0.0140. The Net OD value of IFN-γ(gamma) at week 2 was 0.0260.

[0149] The Net OD value of IFN-γ(gamma) in patient No. 24 at week 0 was 0.0230. The Net OD value of IFN-γ(gamma) at week 2 was 0.0320.

[0150] The Net OD value of IFN-γ(gamma) in patient No. 25 at week 0 was 0.0365. The Net OD value of IFN-γ(gamma) at week 2 was 0.0285.

[0151] The inventors confirmed the correlation between the expression of target proteins in skin samples obtained using hyaluronic acid microneedle patches and the clinical course of atopic dermatitis through statistical analysis (SPSS). The results are shown in Figures 18 to 20 middle.

[0152] For the statistical analysis of the above correlation, the inventors assigned different scores according to the clinical course of the lesions. Patients No. 12 and No. 15, whose symptoms were clinically judged to be significantly worse or slightly worse, were assigned -1 point, and all patients except No. 15 were assigned 1 point. Fig.11 The clinical course after 2 weeks shown on the far right of the table shows that patient No. 12 showed symptoms of "neither good nor bad (so so) + urticaria", and the clinical judgment was that the symptoms had worsened. Patient No. 15 showed "aggravated", and the clinical judgment was that the symptoms had significantly worsened. The remaining patients were clinically judged to have maintained their symptoms or improved.

[0153] Figures 18 to 20 The X-axis of the graph shown in the upper part represents the clinical course. The Y-axis of these graphs respectively represents the changes in IL-4, IL-13 and IFN-γ(gamma) ("target expression level in week 2" - "target expression level in week 0"). According to the statistical analysis results, a significant correlation was confirmed between the changes in IL-4 and IL-13 in the clinical course of atopic dermatitis (p<0.05). In addition, according to the statistical analysis results, it was confirmed that there was no correlation between the clinical course of atopic dermatitis and the change in IFN-γ(gamma). This verifies the clinical usefulness of target factor detection using a non-invasive method (application of hyaluronic acid microneedle patch). Through sorting, it was confirmed that the detection levels of interleukin-4 and interleukin-13 decreased in patients whose atopic symptoms were relieved, and the detection levels of interleukin-4 and interleukin-13 increased in patients whose atopic symptoms worsened. Therefore, in the case of non-invasive surgery such as tissue biopsy, after the biodegradable polymer hyaluronic acid microneedles are attached to the human body and removed, the expression level changes of biomarkers important for the cause of the disease are measured, and the activity of atopic dermatitis can be reliably measured without the intervention of clinical judgment of medical staff including doctors.

[0154] Moreover, when using a minimally invasive atopic dermatitis test kit of one embodiment of the present invention, which is composed of a microneedle patch formed of biodegradable polymer hyaluronic acid and a device that can achieve quantitative analysis of proteins obtained from human skin, the activity of atopic dermatitis can be easily and accurately evaluated by simply comparing a predetermined value with the interleukin-4 and interleukin-13 amount-related values ​​obtained by the quantitative analysis device without relying on the medical knowledge or experience of medical personnel. The above-mentioned atopic dermatitis activity evaluation can be automatically performed by the functional unit that can be included in the atopic dermatitis test kit of one embodiment of the present invention. In this case, the functional unit may include: a storage unit for storing data related to the correlation between the interleukin-4 and interleukin-13 quantity-related data obtained from the quantitative analysis device and the atopic dermatitis activity; an interface unit for transmitting data from the quantitative analysis device; a comparison and judgment unit for comparing and judging the data stored in the storage unit and the data transmitted from the quantitative analysis device to generate information related to the atopic dermatitis activity; and a display unit for displaying the information generated in the comparison and judgment unit.

[0155] The present invention is described above using a number of specific matters such as specific structural elements and limited embodiments and figures, but these are provided only to help to more generally understand the present invention. The present invention is not limited to the multiple embodiments described above, and a person of ordinary skill in the technical field to which the present invention belongs can implement various modifications and variations from such descriptions.

[0156] Therefore, the concept of the present invention should not be limited to the above-described embodiments, and not only the attached claims but also all contents equivalent to or equivalently modified to the claims should fall within the scope of the concept of the present invention.

Claims

1. A minimally invasive atopic dermatitis examination kit, characterized in that: include: a device capable of quantitatively analyzing the amount of protein extracted from the skin of a subject; A microneedle patch includes a plurality of microneedles formed of biodegradable polymer hyaluronic acid and having a solid structure and a bottom layer serving as a base for forming the plurality of microneedles. The microneedle patch is applied to the skin of a subject, and the device is used to quantitatively analyze the proteins of the subject adsorbed on the surface of the microneedles of the microneedle patch in a separated state after a specified time. The device reads the amounts of interleukin-4 and interleukin-13 adsorbed on the surface of the microneedles of the microneedle patch, and evaluates the activity of atopic dermatitis based on the read amounts of interleukin-4 and interleukin-13.

2. The minimally invasive atopic dermatitis test kit according to claim 1, characterized in that: The device is used to implement enzyme-linked immunosorbent assay.

3. The minimally invasive atopic dermatitis test kit according to claim 2, characterized in that: The amount of IFN-gamma detected is not considered in the activity assessment of atopic dermatitis.

4. The minimally invasive atopic dermatitis test kit according to claim 3, characterized in that: It is considered that the predetermined time is predetermined according to the dissolution rate of the molecular weight of the hyaluronic acid constituting the microneedles.

5. The minimally invasive atopic dermatitis examination kit according to claim 4, characterized in that: As the molecular weight of the hyaluronic acid constituting the microneedle increases from a low molecular weight to a high molecular weight, the microneedle can be attached to the skin of the subject for a longer time, thereby improving the biological detection performance.

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