CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims the priority of U.S. Provisional Patent Application No. 63/599,511, filed on November 15, 2023, which claims the benefit of the Korean Patent Application No. 10-2022-0152832, filed on November 15, 2022, which are hereby incorporated by reference in their entirety.

BACKGROUND

Field of the Disclosure

[2] The present disclosure relates to biomarkers that are useful in predicting the efficacy of a composition containing a GPCR19 agonist for treating atopic dermatitis (AD) in subjects, as well as methods of treating atopic dermatitis by identifying subjects who exhibit threshold levels of these biomarkers.

Description of the Background

[3] AD is a recurring chronic skin condition characterized by intense itching. AD is characterized by a highly heterogeneous clinical phenotype resulting from genetic and environmental factors/interactions. The global incidence of AD is rapidly rising, primarily due to increased exposure to environmental pollutants, with a prevalence reaching 20% of the total population. Individuals suffering from AD experience a diminished quality of life due to limitations in their daily activities, and there is a growing economic burden associated with its treatment. Consequently, there is an urgent requirement for effective measures to manage AD.

[4] AD commonly starts during infancy or childhood and is often associated with a family history of the disease. Symptoms typically begin in infancy, particularly around the age of 2 months, with approximately 50% of cases occurring before the age of 2 years. Most cases manifest before the age of 5 years, whereas the occurrence of initial symptoms in adulthood is rare. In some patients, symptoms improve or resolve spontaneously as they grow older, and more than half of those with symptoms in infancy show improvement by the age of 2 years.

[5] The presentation and distribution of skin lesions in AD exhibit specific characteristics and are accompanied by itching (pruritus), dry skin, and distinctive eczema. In infants, eczema primarily emerges on the face and extremities, while as the child grows, it tends to appear in the flexural areas of the arms and behind the knees. In adults, a process known as lichenifi cation, characterized by thickening of the skin due to repetitive scratching, often occurs in skin folds. Eczema can manifest on the face, chest, nape, as well as the extremities, differing from the pattern observed in children and infants.

[6] AD serves as a precursor to an allergic progression, including conditions like allergic asthma and rhinitis. However, due to the lack of a precise understanding regarding the exact cause and mechanism of AD, a definitive curative treatment has not yet been developed. Presently, therapies for AD encompass the use of steroids, PDE4 inhibitors, JAK inhibitors, and antibody treatments. Anti-inflammatory analgesics and steroid-based immunosuppressants are primarily employed to alleviate inflammation and modulate immune responses. While these treatments exhibit the advantage of delivering rapid improvement, the symptoms may rapidly worsen if their usage is reduced or discontinued. Furthermore, prolonged use of these treatments may lead to secondary adrenal insufficiency, diabetes, digestive problems, and systemic side effects such as ulcers, hirsutism, alopecia, and pigmentation disorders. Notably, cataracts may occur in children. Steroid ointments can induce severe side effects, including skin thinning or atrophy, redness due to blood vessel dilation, and folliculitis. Non-steroidal immunomodulators such as pimecrolimus cream and tacrolimus ointment have been developed as alternatives to steroid ointments, demonstrating less side effects associated with conventional steroid ointments even with long-term use. These non-steroidal options are often utilized on sensitive skin areas such as the neck and have rapidly gained popularity, capturing around 30% of the entire atopic dermatitis market. Nevertheless, concerns regarding the potential carcinogenic effects of calcineurin inhibitors have arisen, leading to a decline in sales. Consequently, only low-concentration usage is recommended for patients under the age of 16, and the use of low-concentration formulations is not approved for children under 2 years old.

[7] The PDE4 inhibitor, crisaborole which was approved by the FDA in 2016 for patients with mild to moderate severity, demonstrated some improvement compared to a placebo; however, it was not sufficient to fill-up medical unmet needs. Moreover, many patients reported avoiding prescriptions due to the burning sensation associated with the drug. JAK inhibitors are among the most extensively studied to treat atopic dermatitis. Currently, ruxolitinib, abrocitinib, upadacitinib and baricitinib had FDA approval. However, safety concerns have emerged during clinical trials for this class of drugs, including severe infections, fatalities, cancers, major adverse cardiovascular events, and blood clots. Some JAK inhibitors carry a boxed warning that limits their use, and certain drugs are still awaiting review and approval. Due to these safety’ concerns, the FDA has expressed reservations regarding the entire class of JAK inhibitors and recommends their use only for selected patients. Specifically, in September 2021, the FDA decided to include information about serious side effects and deaths on the labels of JAK inhibitors from specific manufacturers. Consequently, many experts suggest restricting the use of JAK inhibitors in the treatment of atopic dermatitis which is a non-lifethreatening condition.

[8] Meanwhile, in the case of monoclonal antibody treatment, in 2017, dupilumab received FDA approval for the first time in patients with moderate to severe atopic dermatitis. Dupilumab controls the symptoms of severe allergic diseases such as atopy and asthma. However, more than 10% of dupilumab users reported a gradual decrease in efficacy and redness at the injection site due to an increase in blood concentration of antibodies to the drug. In addition, insomnia, oral viral infection, gastritis, toothache, eosinophilia, herpes infection, conjunctivitis, sore throat, and arthralgia were observed in 1-10% of users of dupilumab, and in less than 1%, hypersensitivity reaction, dry ⁷ eye, and eosinophilic side effects of granulosa, eosinophilic pneumonia, and erythema nodosum were identified. Due to the above safety ⁷ and efficacy issues, use in children and adolescents is not recommended. In addition, along with high drug prices, dupilumab is only covered by insurance for the treatment of patients who do not respond to steroids, calcineurin inhibitors, crisaborole, and systemic treatments. As a result, patients' access to dupilumab treatment is very ⁷ limited.

[9] In other atopic treatments, antihistamines and, in severe cases, taking short-term adrenocortical hormones and applying external agents and skin UV treatment or interferon treatment are being implemented. However, in most cases, only temporary improvement is induced, and AD recurs when the medication is stopped. Therefore, most patients with atopic dermatitis still desperately need the development of therapeutic and preventive drugs with fewer side effects than conventional treatments and high efficacy.

[10] Accordingly, there is a need in the art for improved methods of treating atopic dermatitis, especially treatments that are effective and cause fewer adverse effects.

SUMMARY

[11] Accordingly, the present disclosure is directed to a biomarkers for predicting for the treatment efficacy of GPCR19 agonists in the treatment of atopic dermatitis and methods for treating subjects with such biomarkers that substantially obviate one or more of problems due to limitations and disadvantages described above.

[12] Additional features and advantages of the disclosure will be set forth in the description which follows and in part will be apparent from the description, or may be learned by practice of the disclosure. Other advantages of the present disclosure will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

[13] To achieve these and other advantages and in accordance with the present disclosure, as embodied and broadly described, a method of predicting the efficacy of a composition comprising a GPCR19 agonist for treating an atopic dermatitis in a subject, comprising providing a concentration of each of one or more biomarkers in a sample from the subject and comparing each concentration to a threshold level, wherein the concentration of at least one of the one or more biomarkers at the threshold level is indicative of the composition being more likely to be effective in treating the atopic dermatitis, and wherein the one or more biomarkers are selected from the group consisting of IGHA2, ENTP6, SMOC1, ENPL, and CRK.

[14] In another aspect of the present disclosure, a method of predicting the efficacy of a composition comprising a GPCR19 agonist for treating an atopic dermatitis in a subject, comprises obtaining a sample from the subject; quantifying a concentration of each of one or more biomarkers in the sample; and comparing each concentration to a threshold level, wherein if the concentration of at least one of the one or more biomarkers is at the threshold level, then the composition is more likely to be effective in treating the atopic dermatitis; and wherein the one or more biomarkers are selected from the group consisting of IGHA2, ENTP6, SMOC1, ENPL, and CRK.

[15] In another aspect of the present disclosure, a composition comprising TDCA for use in treating atopic dermatitis in a subject, wherein the subject has Type A or Type A’ atopy.

[16] In a further aspect of the present disclosure, use of a composition comprising TDCA in the manufacture of a medicament for treating atopic dermatitis in a subject, wherein the subject has Type A atopy or Type A atopy.

[17] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[18] The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of the disclosure, illustrate aspects of the disclosure and together with the description serve to explain the principle of the disclosure.

[19] In the drawings : [20] FIG. 1 shows a schematic of the enrollment, randomization, and inclusion in the primary analysis of a clinical trial on the GPCR19 agonist taurodeoxy cholic acid (TDCA) disclosed herein (a formulation of which is referred to herein as NUGEL®).

[21] FIG. 2A-B show analyses of Eczema Area and Severity Index (EASI) and Validated Investigator Global Assessment (IGA) for AD patients with a baseline blood concentration of SMOC1 of > 30 pM. FIG. 2A. The primary endpoint was defined as percent change from baseline in EASI (Change EASI). FIG. 2B. The secondary endpo2int was defined as percent change from baseline in IGA (Change IGA). Change EASI and Change IGA of Placebo group, 0.3% taurodeoxycholic acid (TDCA) gel group and 0.5% TDCA gel group were compared. The subgroup of patients with baseline concentration of blood SMOC 1 > 30 pM were analyzed. The number of patient (n) and % of patients analyzed in each group (%) was Placebo, n=12 (46%); 0.3% TDCA gel, n=12 (50%), 0.5% TDCAgel, n=8 (40%). The boxes represent median and interquartile (first and third quartile) range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. P values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Difif) of outcomes between placebo and 0.5% TDCA gel.

[22] FIG. 3A-B show analyses of EASI and IGA for AD patients with a baseline blood concentration of CRK < 4518.2 pM. FIG. 3A. The primary endpoint was defined as Change EASI. FIG. 3B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup of patients with baseline concentration of blood CRK < 4518.2 pM were analyzed. The number (n) and ratio (%) of patients analyzed in each group was Placebo, n=18 (69%); 0.3% TDCAgel. n=12 (50%), 0.5% TDCA gel, n=6 (30%). fP values were determined by ANOVA test. iP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[23] FIG. 4A-B show EASI and IGA analyses for AD patients with a baseline blood concentration of ENTP6 < 650 pM. FIG. 4A. The primary endpoint was defined as Change EASI, FIG. 4B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The subgroup of patients with a baseline concentration of blood ENTP6 < 650 pM were analyzed. The number (n) and ratio (%) of patients analyzed was Placebo, n=12 (46%); 0.3% TDCA gel, n=13 (54%), 0.5% TDCA gel, n=5 (25%). The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. |P values were determined by ANOVA test. J P values were determined by Rank Sum test and mean difference (Mean Diff. ) of outcomes between placebo and 0.5% TDCA gel.

[24] FIG. 5A-B show EASI and IGA analyses for AD patients having a baseline blood concentration of IGHA2 > 149,579 pM. FIG. 5A. The primary endpoint was defined as Change EASI. FIG. 5B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup of patients with a baseline concentration of blood IGHA2 > 149,579 pM were analyzed. Number (n) and ratio (%) of patients analyzed was Placebo, n=l 1 (42%); 0.3% TDCA gel, n=16 (67%), 0.5% TDCA gel, n=ll (55%). P values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[25] FIG. 6A-B show EASI and IGA analyses for AD patients having a baseline blood concentration of ENPL < 148.8 pM. FIG. 6A. The primary endpoint was defined as Change EASI. FIG. 6B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup of patients with a baseline concentration of blood ENPL < 148.8 pM. The number (n) and ratio (%) of patients analyzed was Placebo, n=7 (29%); 0.3% TDCA gel, n=8 (34%), 0.5% TDCA gel, n=6 (30%). fP values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[26] FIG. 7 shows Venn diagrams showing the classification criteria of atopic dermatitis patients classified by blood biomarkers.

[27] FIG. 8A-B shows EASI and IGA analyses for AD patients classified as having Type A atopy. FIG. 8A. The primary endpoint was defined as Change EASI. FIG. 8B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup with baseline concentrations of blood SMOC1 > 30 pM or CRK < 4518.2 pM were analyzed. The number (n) and ratio (%) of patients analyzed was Placebo, n=20 (77%); 0.3% TDCA gel, n=21 (88%), 0.5% TDCA gel, n=10 (50%). fP values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[28] FIG. 9A-B show EASI and IGA analyses for AD patients classified as having Type A’ atopy. FIG. 9A. The primary endpoint was defined as Change EASI. FIG. 9B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup with a baseline concentration of blood ENTP6 < 650 pM in Type A atopy criteria were analyzed. The number (n) and ratio (%) of patients analyzed was Placebo, n=9 (35%); 0.3% TDCA gel, n=12 (50%), 0.5% TDCA gel. n=5 (25%). fP values were determined by ANOVA test. iP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[29] FIG. 10A-B show EASI and IGA analyses for AD patients with baseline blood concentrations of SMOC1 > 30 pM or ENTP6 < 650 pM. FIG. 10A. The primary endpoint was defined as Change EASI. FIG. 10B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup of patients with baseline concentrations of blood SMOC1 > 30 pM or ENTP6 < 650 pM were analyzed. The number (n) and ratio (%) of patients analyzed was Placebo, n=l 8 (69%); 0.3% TDCA gel, n=19 (79%), 0.5% TDCA gel, n=9 (45%). fP values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[30] FIG. 11A-B show EASI and IGA analyses for AD patients having baseline blood concentrations of ENTP6 < 650 pM or CRK < 4518.2 pM. FIG. 11 A. The primary endpoint was defined as Change EASI, FIG. 11B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup of patients with baseline concentrations of blood ENTP6 < 650 pM or CRK < 4518.2 pM were analyzed. The number (n) and ratio (%) of patients analyzed was Placebo. n=21 (81%); 0.3% TDCAgel, n=16 (67%), 0.5% TDCA gel, n=8 (40%). fP values were determined by ANOVA test. P values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[31] FIG. 12A-B show EASI and IGA analyses of AD patients with baseline blood concentrations of SMOCl > 30 pM or ENTP6 < 650 pM or CRK < 4518.2 pM. FIG. 12A. The primary endpoint was defined as Change EASI. FIG. 12B. The secondary ⁷ endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The subgroup of patients with baseline concentrations of blood SMOC1 > 30 pM or ENTP6 < 650 pM or CRK < 4518.2 pM were analyzed. The number (n) and ratio (%) of patients analyzed was Placebo. n=23 (88%); 0.3% TDCA gel, n=22 (92%); 0.5% TDCA gel. n=10 (50%). fP values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[32] FIG. 13A-B show EASI and IGA analyses of AD patients with a baseline blood concentration of FAS. FIG. 13 A. The primary endpoint was defined as Change EASI. FIG. 13B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The number (n) was Placebo, n=27 ; 0.3% TDCA gel, n=27; 0.5% TDCA gel, n=25 |P values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

[33] FIG. 14A-B show EASI and IGA analyses of PP. FIG. 14A. The primary endpoint was defined as Change EASI. FIG. 14B. The secondary endpoint was defined as Change IGA. Change EASI and Change IGA of Placebo group, 0.3% TDCA gel group and 0.5% TDCA gel group were compared. The boxes represent median and interquartile range; the whiskers extend to the highest and lowest values within 1.5 x the interquartile range. The number (n) and ratio (%) was Placebo, n=24 (89%); 0.3% TDCA gel, n=20 (74%); 0.5% TDCA gel, n=19 (73%). fP values were determined by ANOVA test. JP values were determined by Rank Sum test and mean difference (Mean Diff.) of outcomes between placebo and 0.5% TDCA gel.

DETAILED DESCRIPTION

[34] As of 2022, there are 149 biomarker-accompanied diagnostic treatment products approved by the FDA, and the target indications are all cancerous diseases, except for two, obesity and non-transfusion-dependent thalassemia (NTDT). Except for one imaging diagnostic product, the rest are limited to genetic testing or intratissue protein staining. Biomarkers and companion diagnostic treatments using them are important concepts from the perspective of precision medicine and are in the limelight to increase the accuracy, effectiveness, and safety of treatment and are recommended or preferred by health regulatory- agencies around the world. As a representative example, Iressa. which received conditional approval in 2003 as a third-line non-small cell cancer treatment, was withdrawn in 2005 due to lack of efficacy but was reapproved as a first-line treatment in 2015 in recognition of its effectiveness along with EGFR mutation biomarker diagnostics. In addition, Atezolizumab also had no clear efficacy compared to existing treatments for all patients in non-small cell lung cancer clinical trials, but an increase in efficacy was confirmed when combined with PD-L1 in vitro diagnostics and was approved as a companion diagnostic treatment.

[35] Biomarkers are being used for different purposes in medicine, especially as a diagnostic tool. Notably, atopic dermatitis diagnosis and treatment, unlike other chronic diseases, relies completely on clinical scores rather than biochemical markers. Therefore, having a reliable biomarker could reduce observatory differences and bring a great outcome to precision medicine approaches to improve AD management. Furthermore, biomarkers may have various implications for prevention approach and provide useful strategies for development of upcoming new drugs.

[36] Since atopic dermatitis has a clinical phenotype that is affected by various heterogeneous factors, such as various environments, genetics, metabolism, and diseases, there is no treatment that is excellent in terms of ease of use, economic feasibility, and efficacy at the same time. There have been studies exploring biomarkers, but there are no clinically valid results yet. According to the International Eczema Council (IEC), in which more than 100 atopic skin disease research experts participate, atopic dermatitis is a polymorphic- heterogeneous disease in which at least three phenotypes are combined. The IEC formally stated a need to improve patient care and treatment by stratifying phenotype and using it as a predictor for treatment response.

[37] The inventors have developed biomarkers that are surprisingly capable of selecting patients who respond significantly to G protein-coupled receptor 19 (GPCR19) agonists, including taurodeoxycholic acid (TDCA). Specifically, the inventors have developed biomarkers that can identify atopic dermatitis patients who respond effectively to TDCA. More specifically, the inventors have developed the methods to select atopic dermatitis patients who respond effectively to TDCA based on blood protein baseline values of SMOC1, ENTP6, and CRK. These biomarkers can be used as a companion diagnostic composition that can predict before treatment and improve the treatment effect in atopic dermatitis patients with GPCR19 agonists.

1. Definitions.

[38] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms ‘'a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[39] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

[40] “Treatment” or “treating,” when referring to protection of a subject from a disease, means suppressing, repressing, or eliminating the disease. Preventing the disease involves administering a composition described herein to a subject prior to onset of the disease. Suppressing the disease involves administering the composition to a subject after induction of the disease but before its clinical appearance. Repressing the disease involves administering the composition to a subject after clinical appearance of the disease.

2. GPCR19 Agonists

[41] Provided herein is a compound that may be effective in treating atopic dermatitis. The compound may be a G coupled protein receptor 19 (GPCR19) agonist, a famesoid X receptor agonist, or an anti-inflammatory agent. In one example, the compound is a GPCR19 agonist. The GPCR19 agonist may comprise taurodeoxy cholic acid (TDCA), a derivative thereof, or a pharmaceutically acceptable salt of the foregoing. The TDCA may have a chemical structure represented by Formula I.

Formula I

[42] The TDCA may also have a chemical structure represented by Formula II.

Formula II

[43] In one example, the TDCA is sodium taurodeoxycholate. Embodiments of the TDCA and compositions thereof may disclosed in U.S. Patent No. 9,855,283, the contents of which are incorporated herein by reference.

[44] Also provided is a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient. The pharmaceutical composition may comprise about 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 or 5.0 wt/wt% of the compound, which in one example is TDCA. In one example, the pharmaceutical composition comprises 0.3 or 0.5% TDCA. In one example, the pharmaceutical composition is intended for topical use. The pharmaceutical composition may be applied to skin affected by atopic dermatitis, which may be a lesion. In one example, the pharmaceutical composition is administered 1, 2, 3, 4, or 5 times a day. In one example the pharmaceutical composition is administered 2 times a day. The pharmaceutical composition may be administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In one example, the pharmaceutical composition is administered for at least about 3 weeks.

[45] In one example, pharmaceutical composition comprises a solvent. The solvent may comprise polyethylene glycol (PEG), which may be PEG 400. The solvent may also comprise ethanol. In one example, the pharmaceutical composition comprises 70% PEG 400 and 30% ethanol in distilled water.

[46] The pharmaceutical composition may also comprise gel. cream, ointments, tablets, or injectables. In one example, the pharmaceutical composition is hyaluronic acid gel. The gel may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% hyaluronic acid gel. In one example, the pharmaceutical composition comprises 5% hyaluronic acid gel. In one example, 0.5 g sodium taurodeoxy cholic acid is mixed with 20 ml distilled water and dissolved. 80 mg chlorobutanol and 70 ml distilled water may be mixed together with the 25% sodium taurodeoxy cholic acid solution. One gram of hyaluronic acid may be added and mixed together with the solution. Distilled water may be added to make up 100 ml. In one example, the pharmaceutical composition is NUGEL® (Shaperon Inc., Seoul, South Korea). In another example, 0.5 g, 1 g, 2 g, 4 g sodium taurodeoxycholic acid is mixed with 0.5 g Carbomer 940, 0.3 g diethanolamine, 0.15 g methylparaben, 50 mg prophyparaben, and 55 mg ethanol. Distilled water may be added to make up 100 ml. In another example, 0.5 g, 1g, 2g, 4g sodium taurodeoxycholic acid is mixed with 50 mg polysorbate 80, 1 g sodium hyaluronate, 0.2 g methylparaben. Distilled water may be added to make up 100 ml. In another example, 0.5 g, 1 g, 2 g, 4 g sodium taurodeoxycholic acid is mixed with 1.5 g Carbomer 940, 1 g glycerin, 1.5 g triethanolamine, 0.15 g methylparaben, 50 mg propylparaben, and 1 g ethanol. Distilled water may be added to make up 100 ml.

3. Markers of GPCR19 Agonist Efficacy

[47] Provided herein are biomarkers and their use in predicting the efficacy of a composition described herein for treating atopic dermatitis. Further provided herein is a method of predicting the efficacy of the composition for treating atopic dermatitis, which may comprise determining or quantifying the concentration of each one or more of the biomarkers in a sample from the subject. The concentration of at least one of the one or more biomarkers, which may be at a threshold level, may indicate that the composition is more likely to be effective in treating atopic dermatitis in the subject. The increased likelihood of efficacy in the subject may be relative to a population of humans with atopic dermatitis for whom at least one of, or all of, the one or more biomarkers is at the threshold level.

[48] In one example, the sample is a blood sample, which may be serum or plasma. The subject may be of any ethnicity, but in one example the subject is of Asian descent, and in a more specific example the subject is of Korean descent. In other examples, the subject is of American Indian descent. Alaska Native descent, Black American descent. Black British descent, Black Caribbean descent, African descent, Hispanic or Latino descent, native Hawaiian or other Pacific Islander descent, Middle Eastern descent, or White European descent

[49] The biomarkers may comprise one or of IGHA2 (Immunoglobulin Heavy Constant Alpha 2; UniProt accession no. P01877; SEQ ID NO: 1); ENTP6 (ectonucleoside Triphosphate Diphosphohydrolase 6; UniProt accession no. 075354; SEQ ID NO: 2) or an isoform thereof; SMOC1 (SPARC -related modular calcium-binding protein 1; UniProt accession no. Q9H4F8; SEQ ID NO: 3) or an isoform thereof; ENPL (Endoplasmin or Heat shock protein 90 kDa beta member 1; UniProt accession no. P14625; SEQ ID NO: 4); and CRK (adapter molecule crk or proto-oncogene c-Crk; UniProt accession no. P46108; SEQ ID NO: 5) or an isoform thereof. The sequences of these biomarkers are known in the art, as are those of their respective isoforms. Each biomarker may comprise a complete sequence of one of the foregoing proteins, a fragment thereof, or a protein having sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing.

[50] The concentration of each biomarker in a patient sample that is predictive of the efficacy of the composition for treating atopic dermatitis may be a threshold level. Being at a threshold level may mean that a concentration is at or above, or at or below a set concentration, as described herein. The threshold level of IGHA2 may be a concentration greater than or equal to about 130,000; 135,000; 140,000; 141,000; 142,000, 143,000; 144,000; 145,000; 146,000; 147,000; 148,000; 149,000; 150,000; 151,000; 152,000; 153,000; 154,000; 155,000; 156,000; 157,000; 158,000; 159,000; or 160,000 pM. In one example, the threshold level of IGHA2 is a concentration greater than or equal to about 149,579 pM. The threshold level of ENTP6 may be a concentration less than or equal to about 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 pM. In one example, the threshold level of ENTP6 is a concentration less than or equal to about 650 pM. The threshold level of SMOC1 may be a concentration greater than or equal to about 20, 21, 22, 23, 24, 25. 26. 27. 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pM. In one example, the threshold level of SMOC1 is a concentration greater than or equal to about 30 pM. The threshold level of ENPL may be a concentration less than or equal to about 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 pM. In one example, the threshold level of ENPL is a concentration less than equal to about 148.8 pM. The threshold level of CRK may be a concentration less than or equal to about 4300, 4310, 4320, 4330, 4340, 4350, 4400, 4450, 4500, 4550, 4600, 4650, or 4700 pM. In one example, the threshold level of CRK is a concentration less than or equal to about 4518.2 pM. As used for a threshold concentration, “about’ ⁷ may mean a value within 0.5, 1. 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15. 16. 17, 18, 19, or 20% of a stated concentration.

[51] If a concentration of a biomarker in a sample is at a threshold level, then the sample or subject may be referred to as being “positive” for the biomarker, and may indicate that the composition is more likely to be effective in treating atopic dermatitis in the subject. In one example, the one or more biomarkers comprise one or more of SMOC1, ENPT6, and CRK. In a further example, one or more of a threshold level of SMOC1 about >30 pM, a threshold level of ENTP6 about <650 pM, and a threshold level of CRK about <4518.2 pM is indicative that the composition is more likely to be effective against atopic dermatitis.

[52] The subject may be SMOC l positive or CRK positive, and may be classified as having Type A atopy. In one example, the subject may be classified as having Type A atopy and is more likely to be responsive to the composition for treating atopic dermatitis. In one example, the subject is classified as having Type A atopy and is also ENTP6 positive, and is classified as having Type A’ atopy. In one example, the subject with Type A’ atopy is SMOC1 positive and ENTP6 positive. In another example, the subject with Type A’ atopy is CRK positive and ENTP6 positive. In one example, the subject may be classified as having Type A atopy and is more likely to be responsive to the composition for treating atopic dermatitis.

153] Methods of detecting and quantifying the concentration of protein in a sample are known in the art. The concentration of the one or more biomarkers may be quantified by using mass spectrometry', antibodies, enzy me-linked immunoassay, capillary' immunoassay (WES), western blotting, aptamers, peptide, peptide mimetics, oligonucleotides or probes. In one example, the concentration of the one or more biomarkers is measured by using mass spectrometry. The mass spectrometry (MS) may be multiple reaction monitoring MS (MRM- MS). A threshold level of a biomarker may be determined by measuring a maximum area under the curve value and comparing clinical reactivity w ith a receiver operating characteristic curve.

[54] Further provided herein are kits comprising reagents and/or labware suitable for quantifying the concentration of the one or more biomarkers. Kits may comprise one or more of positive and negative controls. Kits may ⁷ also comprise reagents and/or labware for collecting subject samples. Kits may also comprise reagents and/or labware for pretreat subject samples. The kits may further comprise instructions for carrying out quantification and/or sample collection methods.

[55] The efficacy of the composition in treating atopic dermatitis may be measured by any method known in the art. In one example, the method is a clinical diagnosis. In another example, efficacy is measured by calculating an Eczema Area and Severity' Index (EASI) score, whereby a decrease in EASI score measured at a later time point as compared to an earlier time point indicates improvement. Calculating the EASI score may comprise assessing clinical findings in 4 stages (0 = none, 1 = mild, 2 = moderate, 3 = severe) of the severity' of erythema, edema/induration, abrasion, and lichenification on the subject's head/neck, trunk, and lower extremities, and the clinical findings may be scored by body part area lesion area score (0 = none, 1 = <10%, 2 = 10-29%, 3 = 30-49%, 4 = 50-69%, 5 = 70-89%, 6 = 90-100%). The scores for each body part may be multiplied by 0.1 for the head/neck, 0.2 for the upper extremities, 0.3 for the trunk, and 0.4 for the lower extremities and then added together to calculate the EASI score.

[56] In a further example, efficacy is measured by calculating a Validated Investigator Global Assessment (IGA) score, whereby a decrease in IGA score measured at a later time point as compared to an earlier time point indicates improvement. The IGA score may be based on a 5-point scale ranging from 0 to 4 (0 = Clear; No inflammatory signs of atopic dermatitis, 1 = Almost Clear; Just perceptible erythema and just perceptible papulation induration; 2 = Mild; Mild erythema and mild papulation induration, No oozing or crusting; 3 = Moderate, Moderate erythema and moderate papulation induration, Oozing and crusting may be present; 4= Severe, Severe erythema and severe papulation induration, Oozing and crusting is present).

4. Methods of Treating Atopic Dermatitis

[57] Provided herein is a method of treating atopic dermatitis in a subject in need thereof. The method may comprise administering a composition comprising a GPCR19 agonist to the subject. The composition may comprise a GPCR19 agonist, particularly TDCA, a derivative thereof, or a pharmaceutically acceptable salt of the foregoing. The composition may be a pharmaceutical composition. Also provided are use of the composition in treating atopic dermatitis and use of the composition in the manufacture of a medicament for treating atopic dermatitis. In one example, the composition is administered topically or is intended for topical administration.

[58] The subject may have been identified as being more likely to be effectively treated by the composition as determined by a method of predicting the efficacy of the composition in treating atopic dermatitis as described herein. In one example, the method comprises administering the composition to a subject who has been identified as being more likely to be effectively treated according to a predictive method described herein. The subject may be positive (as described above) for at least one of IGHA2, ENTP6, SMOC1, ENPL, and CRK. The subject may have Type A atopy and be positive for SMOC 1 or CRK. The subject may have Type A atopy and be positive for ENTP6 and at least one of SMOC1 and CRK.

[59] In another example, the method comprises providing a concentration of each of one or more biomarkers in a sample from the subject, comparing each concentration to a threshold level, and administering the composition to the subject if at least one of the one or more biomarkers is at its respective threshold level. In one example, the method comprises obtaining or providing the sample from the subject and quantifying the concentration of each of the one or more biomarkers, thereby providing the concentration of each of the one or more biomarkers.

[60] The method may comprise administering the composition to the subject, wherein the subject is positive for at least one of IGHA2, ENTP6, SMOC1, ENPL, and CRK. In one example, the subject is positive for at least one of ENTP6, SMOC1, and CRK. In one example, the method comprises administering to the subject the composition, wherein the subject has Type A atopy. In another example, the method comprises administering to the subject the composition, wherein the subject has Type A atopy. The method may comprise a step of determining whether the subject is positive for one or more biomarkers before administering the composition, or identifying the subject as being positive for one or more biomarkers before administering the composition.

[61] The present invention has multiple aspects, illustrated by the following non-limiting examples.

Example 1

Biomarkers can be used to identify atopic dermatitis patients who respond to GPCR19 agonists

[62] Atopic dermatitis (AD) is the most common chronic inflammatory skin disorder, impacting up to 25% of children and 8% of adults worldwide. Current traditional AD treatment strategies, such as corticosteroids, calcineurin inhibitors, PDE4 inhibitors, JAK inhibitors, UV phototherapy and antibodies targeting the IL-4/IL-3 pathway, focus on epidermal barrier function, abnormal Th2/17/22 responses, and Thl-mediated pathogenesis in later stages. Unfortunately, none of the approaches could fulfill the therapeutic unmet need or be recognized as one size fits all. In addition to serious unfavorable adverse events, an estimated 20% of patients with AD comprise a subset called treatment-resistant AD, for which traditional therapeutics and management strategies are unsuccessful. Novel AD therapy applying precision medicine ''right treatment to the right patient” could solve the current unmet need for AD patients. This example demonstrates that GPCR19 colocalized with P2X7R, and NUGEL®, a TDCA pharmaceutical composition described herein, inhibited the activation of P2X7R. Noncistronically, NUGEL® inhibited NF-kB activation via the adenylyl cyclase-PKA pathway, and BzATP-mediated Ca ²⁺ mobilization. Cistronically, NUGEL® suppressed the expression of P2X7R and NLRP3 inflammasome (N3I) components in keratinocytes. NLRP3 oligomerization and production of mature IL-1 (3 and IL-18 were suppressed by NUGEL® treatment in keratinocy tes. Topical treatment with NUGEL® ameliorated atopic inflammatory features in different AD mouse models. Furthermore, using biomarkers as a patient-selecting strategy in adults with mild to moderate AD, we evaluated the therapeutic efficacy of NUGEL® for AD in a phase 2, double-blind, randomized, placebo-controlled trial. In the screening stage during patient recruitment, blood samples were analyzed, and only biomarker-positive patients were evaluated. Patients were randomly assigned to receive placebo, NUGEL® 0.3% and 0.5% twice daily for 8 weeks. In this study, patients were classified as type A and type A’ based on the biomarkers applied. In patients who were received NUGEL® 0.5% with type A and type A’ atopy, improvement in Eczema Area and Severity Index (EASI) was achieved by 53% (p = 0.002) and 83% (p = 0.008) compared to the placebo group, respectively. Validated Investigator Global Assessment (IGA) improvement from the patients’ baseline was achieved by 33% (p = 0.007) and 53% (p = 0.005) in type A and type A’, respectively. No adverse events were reported in this study. The results indicate that, surprisingly, using NUGEL®, a GPCR19 agonist with a favorable safety profile, together with the use of molecular markers to identify patients for whom the drug is more efficacious, can overcome the cunent unmet need for AD patient treatment.

Methods

Study Design

[63] To evaluate the efficacy of the treatment in recruited clinical trial patients, doubleblinded, placebo-controlled, parallel, and multicenter clinical trials were conducted (ClinicalTrials.gov no.: NCT04530643). In this study, NUGEL® (Shaperon, Korea), the composition containing taurodeoxycholic acid (TDCA) as an active ingredient, was used as a therapeutic agent. Enrollment began in August 2020 and ended in July 2021.

[64] This clinical trial was designed as a random assignment, and subjects who finally met the inclusion criteria but did not meet the exclusion criteria were assigned to 0.3% TDCA gel treatment group, 0.5% TDCA gel treatment group, and placebo group in a 1: 1: 1 ratio. This clinical trial is conducted in a randomized way to minimize the influence and bias of baseline variables on the test results. This trial was designed as a double-blind study, so both investigators and subjects were blinded. Prior to the end of the study, neither the test subjects nor the placebo subjects were aware of the type of drug being administered. Although placebo administration in the placebo group has the disadvantage of causing unnecessary administration, double blinding was applied considering that the subjective judgment of the subjects or investigators may be involved in the clinical evaluations. Patients are considered to have mild to moderate atopic dermatitis. Dosage design was repeated transdermal administration. Patients applied an appropriate amount of gel to the lesion twice a day in the morning and evening.

Participants

[65] This clinical trial was conducted on male and female patients with atopic dermatitis. Those who agreed to participate in this clinical trial and met the inclusion criteria but did not meet the exclusion criteria were enrolled. The investigator carefully reviewed the validity of the subject registration and fully considered the purpose of the test and the subject's rights and health conditions. Those who could not voluntarily sign the consent form were not selected for this study.

[66] Participants who met the following inclusion criteria were included in the study: age of at least 19 years, who have a clinical diagnosis of atopic dermatitis according to the criteria of Hanifin and Rajka, IGA of 2 or 3 at baseline visit (Day 1), BSA covered with AD of at least 5% and no more than 40% at baseline visit.

[67] Exclusion criteria were as follows: Treatment with steroids, oral antibiotics, body photochemotherapy, immunosuppressive drug before the baseline visit. Those who have AST/ALT or creatinine values more than two times of the upper limit of normal range at screening test.

[68] During the clinical period, the use of prescription moisturizers and moisturizers containing additives such as ceramide, hyaluronic acid, urea or filaggrin was prohibited. In addition, immune function modulators, cortical hormones, antihistamines, photochemotherapy, existing atopic dermatitis treatments, and other drugs that are thought to affect immune function were prohibited during the clinical period.

[69] Among the concomitant medications that the subject had been taking before participating in this clinical trial, concomitant medications that were not expected to affect the interpretation of the results of this clinical trial were allowed under the judgment of the clinician. Drugs used transiently for the purpose of treating other diseases or adverse events were concurrently administered through consultation with the doctor in charge.

Study protocol and measurements

[70] From day -21 to 0 day, the patients were screened to assess eligibility for inclusion and exclusion and enrolled into any of the three treatment arms at day 1. During the treatment period, the patient received treatment with 0.3% TDCA gel, 0.5% TDCA gel, or placebo. The safety of test drugs was monitored by laboratory tests according to the discretion of the primary investigators. The routine laboratory tests were performed at a central laboratory of each trial site.

Endpoints

[71] The primary outcome is percent change from baseline in EASI clinical score. A negative change from baseline indicates improvement. On the starting day (baseline), during the 2-week and 4-week visits, the investigator measured the patient's EASI clinical score. The same examiner scored clinical findings in 4 stages (0 = none, 1 = mild, 2 = moderate, 3 = severe) of the severity’ of erythema, edema/induration, abrasion, and lichenification on the subject's head/neck, trunk, and lower extremities, and the clinical findings score by body part area lesion area score (0 = none, 1 = <10%, 2 = 10-29%, 3 = 30-49%, 4 = 50-69%, 5 = 70-89%, 6 = 90-100%). Afterwards, the scores for each body part were multiplied by 0.1 for the head/neck, 0.2 for the upper extremities, 0.3 for the trunk, and 0.4 for the lower extremities and then added together to calculate the total EASI score.

[72] The secondary ⁷ outcome is percent change from baseline in IGA clinical score. A negative change from baseline indicates improvement. On the starting day (baseline), during the 2-week, and the 4-week visits, the investigator measured the patient's IGA clinical score. The IGA is a rating scale used in clinical trials to measure the severity ⁷ of atopy and the clinical response to treatment based on a 5-point scale ranging from 0 to 4 (0 = Clear; No inflammatory ⁷ signs of atopic dermatitis, 1 = Almost Clear; Just perceptible erythema and just perceptible papulation induration, 2 = Mild; Mild erythema and mild papulation induration. No oozing or crusting, 3 = Moderate; Moderate erythema and moderate papulation induration. Oozing and crusting may be present, 4= Severe; Severe erythema and severe papulation induration. Oozing and crusting is present).

Proteomic analysis

[73] To identify potential biomarkers for predicting therapeutic drug responses, we conducted a quantitative analysis of 802 proteins in blood plasma samples from patients using MRM-MS. These samples were collected before drug administration.

[74] A total of 802 target proteins, detectable in blood plasma, were selected based on previous research and expert opinions. These proteins consist of disease biomarkers approved by FDA (Food and Drug Administration)/LDT (Laboratory Developed Test) and biomarker candidates identified in-house by our research team. To identify a biomarker candidate group that can be analyzed by MRM-MS with 6495 QQQ mass spectrometers among 802 candidate proteins, each pooling sample for each biomarker candidate protein, an internal standard peptide (Stable isotope-23 labeled synthetic (SlS)-peptide, SIS peptide) was analyzed together to confirm the detected marker.

[75] We excluded targets with intensities below 100 counts when analyzed solely with SIS, targets with no co-elution with SIS in blood plasma, and targets with intensities below 100 for both endogenous peptides and SIS. The final 502 proteins/peptides were selected as the target biomarker candidates for MRM-MS analysis of individual samples. The amount of SIS for the analysis was determined by analyzing the pooled plasma endogenous peptides and SIS, and selecting the amount that is closest to 1 while satisfying the range of 0.1 to 10 for the peak area ratio between the two (= amount of endogenous peptides/amount of SIS). This amount was then used for the analysis of individual blood plasma samples. For the final results, blood plasma samples were first pre-treated by tryptic digestion. Subsequent analyses were conducted on blood plasma samples of 26 patients from the placebo group, 24 patients from the 0.3% TDCAgel treatment group, and 20 patients from the 0.5% TDCAgel treatment group. To prevent experimental bias, we used block randomization to set up random pre-treatment batches. Each batch contained 23 or 24 randomly selected samples. For target proteomic analysis, plasma samples were thawed on ice and centrifuged at 4°C and 10,000 * g for 10 minutes, and the supernatants were centrifuged through a 0.22-pm filter (12,000 g, room temperature). The centrifuged samples were transferred to fresh tubes. The proteins of the samples were then quantified by bicinchoninic acid assay (BCA assay) using the Pierce™ BC A Protein Assay Kit (Thermo Scientific).

[76] Two hundred micrograms of proteins from each crude plasma sample were digested with RapiGest surfactant and trypsin. More specifically, 40 pL of 0.2% RapiGest, 20 mM dithiothreitol (DTT), and 100 mM ABC buffer, pH 8.0 were added to the 200 pg plasma proteins, of which volume was adjusted with HPLC-grade water to 40 pL. After 1 hour incubation at 60°C, 20 pL of 100 mM iodoacetamide (IAA) was added, and the samples were incubated in the dark for 30 minutes further at room temperature.. Trypsin, dissolved in 50 mM ABC buffer (pH 8.0), was added to the samples, and incubated at 37 °C for 4 hours. To stop the enzy matic reaction, 10% formic acid solution was added to the samples to achieve final concentration of 1%. Then, the samples were incubated at 37 °C for 30 minutes to hydrolyze the RapiGest surfactant. After centrifugation at 15.000 rpm and 4°C for 1 hour to precipitate the cleaved RapiGest surfactant, the supernatant was transferred to a new tube. Finally, crude stable isotope-labeled internal standard (SIS) peptides were added to the plasma peptide samples.

[77] For MRM-MS analysis, we used an Agilent 6495 triple quadrupole mass spectrometer coupled to an Agilent 1260 Infinity HPLC system. Solvents A and B for the HPLC consisted of 0.1% formic acid in water (v/v) and 0.1% formic acid in acetonitrile (v/v), respectively. A total of 40 pl of digested sample was injected into a guard column (2.1 x 15.0 mm, 1.8 pm, 80 A, Agilent). Online desalting was performed directing the effluent to waste at 5 pL/min for 10 minutes in 3% solvent B at 40°C. Then the desalted sample was transferred to the analytical column (0.5 x 35.0 mm, 3.5 pm, 80 A, Agilent) in 3% solvent B at a flow rate of 40 pL/min for 5 minutes. The analytical column was heated and maintained at 40 °C by an oven. The total run time per MRM-MS analysis was 140 min. Approximately 10 pg of digested peptides was inj ected per MRM-MS run. The peptides were separated on the column and eluted with a linear gradient of 3% to 40% acetonitrile with 0.1% formic acid (FA) for 125 min at a flow rate of 40 pL/min. The separated peptides were monitored in scheduled MRM mode for each protein transition using a mass spectrometer. Peak integration was performed using the Skyline tool, an MRM-MS data preprocessing program. The peak integration process is a process of deriving a peak area ratio value, and the value obtained by multiplying the peak area ratio value of each target by the amount of the internal standard injected was determined as the final quantitative value. To identify targets suitable for stable quantification, we initially screened 502 targets from 70 individual samples before dosing. Targets exhibiting unstable peak shapes (skewed peaks) or intensities below 100 for the internal standard and endogenous peptide were excluded. As a result, 33 proteins were excluded, and 469 proteins were finally selected as the final targets for statistical analysis. The dynamic range for the quantitative values (fimol) of all 469 target proteins was approximately 7 orders of magnitude. Approximately 90% of the protein targets had a dynamic range of 5 orders of magnitude, while the top 5% of protein targets, including the highly abundant protein human albumin, had a dynamic range of 2.5 orders of magnitude. The bottom 5% of low abundant proteins had a dynamic range of approximately 1 order of magnitude. Considering that the dynamic range of the 6495 QQQ equipment used for the analysis is 6 orders of magnitude, our optimized MRM-MS analysis method was able to stably quantify 469 target proteins in blood samples. Since a total of 70 blood plasma samples were collected from 5 centers before administration, the distribution of protein quantification values by institution was checked to determine whether technical variability that may occur between hospitals affects the quantification value of protein targets. It was confirmed that the distribution of protein quantification values did not show statistically significant differences between institutions, and it was confirmed that samples were evenly distributed between hospitals in the clustering analysis (PCA). Thus, we concluded that the influence of inter-hospital technical variability on protein target quantitative values is negligible.

[78] Subsequently, a stratified analysis was conducted to explore the correlation between baseline blood plasma protein levels and the therapeutic efficacy of the drug, as indicated by EASI and IGA scores. An effective cutoff value, pivotal for determining potential biomarker candidates and drug responsiveness, was established. For population with positive biomarker levels, TDCA treatment group showed a statistically higher therapeutic efficacy than placebo treatment group. Receiver operating characteristic (ROC) analysis was used to determine whether protein levels correlated with treatment responsiveness. Statistical analysis

[79] The Full Analysis Set (FAS) consists of subjects whose EASI value, which is the primary endpoint, was measured at baseline and within a period of 4 weeks after administration of the investigational drug among subjects who had been administered at least once (n=79). The Per-Protocol (PP) included patients from FAS without any major protocol deviations described in the approved synopsis (n=63). The Biomarker Analysis Set (BAS) consists of subjects who have completed the final schedule and for whom baseline biomarkers were measured in the blood plasma, excluding those who stopped the clinical trial among the subjects included in the FAS analysis group (n=70). In this analysis set, biomarker is used for stratification factor with clinical efficacy analysis.

[80] The mean and SEM of the efficacy evaluation variables were determined for each group. Differences in efficacy between the groups were assessed using ANOVA. In addition, a post hoc analysis was performed to determine if there was a significant difference between the groups using the rank sum test. Statistical analysis of this clinical trial was performed using SAS version 9.4.

[81] For the safety analysis, adverse events were categorized and analyzed according to treatment group, affected organ, and preferred term, encompassing adverse events, adverse drug reactions, serious adverse events, and adverse events that caused participant dropout. Differences in the incidence of these adverse events between treatment groups were summarized as frequencies and percentages. The descriptive statistics of the laboratory results before treatment (screening) and after the end of administration of the investigational drug or placebo were presented for each treatment group. In addition, for each laboratory’ result, the frequency and ratio of subjects who had normal/ clinically nonsignificant abnormalities before treatment (screening) but changed to clinically significant abnormalities after completion of administration of investigational drugs were presented by treatment group. For vital signs (blood pressure, heart rate, body temperature), descriptive statistics (number of subjects, mean, standard deviation, minimum value, median value, maximum value) were calculated and presented for each treatment group. The physical examination results for each treatment group were summarized by frequency and percentage to identify changes caused by the administration of the investigational drug. The electrocardiogram (ECG) results were compared before treatment (screening) and after administering the test drug, and the observed changes were summarized by treatment group. The percentage of each treatment group was presented. All reported adverse events were reported using the MedDRA Ver.24.0. Results

Study population

[82] Of the 94 patients who were assessed for eligibility, 80 underwent randomization, of which 27 were assigned to the 0.3% TDCA gel group, 26 were assigned to the 0.5% TDCA gel group, and 27 were assigned to the placebo group (Fig. 1). The median age (years) of the patients was 28 in 0.3% TDCA gel. 26 in 0.5% TDCA gel, and 26 in placebo. 37% in 0.3% TDCA gel, 56% in 0.5% TDCA gel, and 52% in placebo were male (Table 1).

Table 1 - Baseline Demographic Information

[83] The baseline EASI score (mean ± SD) was 7.41 ± 3.77 in 0.3% TDCA gel, 8.96 ± 4.47 in 0.5% TDCA gel, and 8.50 ± 4.80 in placebo. The baseline IGA score (mean ± SD) was 2.30 ± 0.47 in 0.3% TDCA gel, 2.56 ± 0.51 in 0.5% TDCA gel, and 2.37 ± 0.49 in placebo (Table 2).

Table 2 - Baseline clinical score of disease Biomarker Exploration

[84] To explore specific blood biomarkers related to the prediction of the drug response of NUGEL® for atopic dermatitis patients, NUGEL®, a gel-type clinical drug of TDCA, a GPCR19 agonist, was used. In a phase 2 clinical trial, the correlation between the blood protein baseline level of patients prescribed high-dose treatment (0.5% TDCA gel) and the treatment response after 4 weeks of drug treatment was analyzed.

[85] Specifically, ROC curve analysis was performed to determine whether atopic symptoms were improved, as assessed by blood quantitative values of each protein before treatment and EASI clinical improvement in the patient group prescribed high-dose treatment. Afterwards, 42 proteins with sensitivity /specificity with AUC (area under an ROC curve) > 0.7 were selected, and stratification analysis standard values (cut-off) were expected (Table 3).

Table 3 - Biomarker candidates

Biomarker-stratified analysis

[86] For the FAS population, the mean ± SEM of EASI change rate, which is the primary outcome, was, -12.2 ± 8.8%, -11.9 ± 11.1% and -2.91 ± 9.0% in the 0.3% TDCA gel, 0.5% TDCA gel and placebo groups, respectively. The difference between placebo vs. 0.5% TDCA gel was not significant (p=0.531) by Rank-sum test. The mean ± SEM of IGA change rate, which was the secondary outcome, was also -1.9 ± 4.7% in the placebo and -9.3 ± 5.0% in the 0.5% TDCA gel treatment group. The difference between placebo vs. 0.5% TDCA gel was not significant (p = 0.299) by Rank-sum test (Fig. 13).

[87] For the PP population, the mean ± SEM of EASI change rate was, -18.0 ± 8.9%, -9.4 ± 14.1% and -3.0 ± 10.2% in the 0.3% TDCA gel, 0.5% TDCA gel, and placebo groups, respectively. The difference between placebo vs. 0.5% TDCAgel was not significant (p =0.669) by Rank-sum test. The mean ± SEM of IGA change rate, which was the secondary outcome, was also -2. 1 ± 5.3% in the placebo and -7.9 ± 6.0% in the 0.5% TDCA gel treatment groups. The difference between placebo vs. 0.5% TDCA gel was not significant (p = 0.506) by Ranksum test (Fig 14).

[88] Surprisingly, five protein biomarkers (IGHA2, ENTP6, SMOC1, ENPL, and CRK) had significant ROC response with EASI and/or IGA clinical improvement (Table 4). Based on the cut-off value of each biomarker concentration, SMOC1 -positive (defined as >30 pM), CRK-positive (defined as <4518.2 pM). ENTP6-positive (defined as <650 pM), IGHA2- positive (defined as >149,579 pM) and ENPL-positive (defined as <148.8 pM) patients were filtered.

Table 4 - Biomarker-stratified status in group

[89] The capability of each biomarker predicting efficacy of the treatment was evaluated. Stratified analysis was conducted on patients at threshold level of SM0C1. As shown in Fig. 2, the therapeutic efficacy of 0.5% NUGEL® was statistically higher than the placebo. The validity of biomarker-based treatment effect prediction was confirmed. Specifically, the number of SMOC1 -positive patients per group (n) and the ratio (%) to the total number of patients analyzed w ere as follows: n=12 (46%) in the placebo group; n=12 (50%) in the 0.3% TDCA gel treatment group; and n = 8 (40%) in the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was 2.1 ± 10.7% for the placebo group and -51.3 ± 10.9% for the 0.5% TDCA gel treatment group, thus showing a difference in treatment effect with a significant treatment synergistic effect of 53.4% (p=0.005). The IGA change rate (mean ± SEM) was also 1.4 ± 8.1% in the placebo group and -33.3 ± 8.9% in the 0.5% TDCA gel treatment group, showing a significant therapeutic synergistic effect with a difference in treatment effect of 34.7% (p = 0.014).

[90] In Fig. 3 the validity' of predicting the biomarker-based treatment effect was confirmed by comparing and analyzing the treatment effect of 0.5% TDCA gel compared to placebo by performing stratified analysis on patients with a positive baseline blood level of CRK. Specifically, the number of CRK-positive patients per group (n) and the ratio (%) to the total number of patients analyzed w ere n=18 (69%) in the placebo group; n=12 (50%) in the 0.3% TDCA gel treatment group; and n = 6 (30%) in the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was 6.4 ± 11.0% in the placebo group and -52.6±9.9% in the 0.5% TDCA gel treatment group, showing a significant therapeutic synergistic effect with a difference in treatment effect of 59.1 % (p=0.005). The IGA change rate (mean ± SEM) was also 4.6 ± 6.8% in the placebo group and -38.9 ± 10.3% in the 0.5% TDCA gel treatment group, showing a significant treatment synergistic effect with a difference in treatment effect of 43.5% (p = 0.005).

[91] In Fig. 4, the validity of the biomarker-based treatment effect prediction was confirmed by comparing and analyzing the treatment effect of 0.5% TDCA gel compared to placebo by performing stratified analysis on patients with a positive baseline blood level of ENTP6. Specifically, the number of ENTP 6-positive patients per group (n) and the ratio (%) to the total number of analyzed patients were n=12 (46%) in the placebo group; n=13 (54%) in the 0.3% TDCA gel treatment group; and n = 5 (25%) in the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was 8.5 ± 15.1% in the placebo group and -61.3 ± 11.2% in the 0.5% TDCA gel treatment group, showing a difference in treatment effect with a significant treatment synergistic effect of 69.8% (p=0.007). The IGA change rate (mean ± SEM) was also 9.7 ± 7.5% in the placebo group and -40.0 ± 6.7% in the 0.5% TDCA gel treatment group, showing a significant therapeutic synergistic effect with a difference in treatment effect of 49.7% (p = 0.002).

[92] In Fig. 5, the efficacy of biomarker-based treatment effect prediction was confirmed by comparing and analyzing the treatment effect of 0.5% TDCA gel compared to placebo by performing stratified analysis on patients with a positive baseline blood level of IGHA2. Specifically, the number of IGHA2 -positive patients per group (n) and the ratio (%) to the total number of analyzed patients were n=ll (42%) in the placebo group; n=16 (67%) in the 0.3% TDCA gel treatment group; and n=ll (55%) in the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was -5.0 ± 13.0% in the placebo group and -45.4 ± 9.6% in the 0.5% TDCA gel treatment group. The effect difference was 40.4% (p=0.036), showing a significant treatment synergistic effect. The IGA change rate (mean ± SEM) was also 6.1 ± 7.2% for the placebo and -24.2 ± 9.1% for 0.5% in the TDCA gel treatment, with a difference in treatment effect of 30.3% (p = 0.017), showing a significant therapeutic synergistic effect.

[93] In Fig. 6, the validity of predicting the biomarker-based treatment effect was confirmed by comparing and analyzing the treatment effect of 0.5% TDCA gel compared to placebo by performing stratified analysis on patients with a positive baseline blood level of ENPL. Specifically, the number of ENPL-positive patients per group (n) and the ratio (%) to the total number of patients analyzed were n=7 (29%) in the placebo group; n=8 (34%) in the 0.3% TDCA gel treatment group; and n = 6 (30%) in the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was 39.6 ± 20.3% in the placebo group and -53.3 ± 11.6% in the 0.5% TDCA gel treatment group, showing a difference in treatment effect with a significant treatment synergistic effect of 92.9% (p=0.005). The IGA change rate (mean ± SEM) was also 16.7 ± 12.6% in the placebo and -33.3 ± 12.2% in the 0.5% TDCA gel treatment group, showing a significant treatment synergistic effect with a difference in treatment effect of 50.0% (p = 0.035).

Additional classification and analysis

[94] The results above indicate that the biomarkers can be used to predict the efficacy of TDCA treatment of AD, including the 0.5% TDCA gel. All of the blood protein baseline levels described above have useful potential for companion diagnostic treatment using TDCA gel. In particular, SMOC 1 , ENTP6, and CRK are considered to be the most useful biomarkers, because patient recruitment is easy because the filtering stringency is not high, and it is a statistically significant drug response stratification factor. Therefore, it was confirmed that 0.5% TDCA gel showed excellent efficacy for atopic dermatitis patients whose blood baseline values were SMOC 1 -positive, CRK-positive, or ENTP6-positive.

[95] More specifically, we classified a patient group that is positive for either SMOC1 or CRK and has a high response to TDCA gel therapy as ‘“Type A atopy.” We also classified a patient group that is ENTP6 positive in Type A as “Type A’ atopy,” which responds best to TDCA gel treatment (Fig. 7). In addition, the accompanying diagnostic value for predicting the drug efficacy of the biomarkers increased not only when the biomarkers were individually reflected but also when two or more were combined.

[96] Specifically, the number (n) of SMOC 1 -positive or CRK-positive “Type A atopy” patients per group and the ratio (%) to the total number of patients analyzed were n=20 (77%) in the placebo group; n=21 (88%) in the 0.3% TDCA gel treatment group; and n=ll (55%) in the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean±SEM) was 2.7 ± 10.4% for the placebo group and -50.0 ± 8.7% for 0.5% TDCA gel treatment group. The difference in treatment effect was 52.7% (p=0.002), showing a significant therapeutic synergistic effect. The IGA change rate (mean ± SEM) was also 2.5 ± 6.4% in the placebo group and -30.0 ± 7.8% in the 0.5% TDCA gel treatment group, showing a significant therapeutic synergistic effect with a difference in treatment effect of 32.5% (p = 0.007) (Fig. 8). [97] The number of patients per group (n) and the ratio (%) to the total number of patients subject to analysis corresponding to "T pe A' atopy" who were ENTP6 positive in Type A at the same time was n= 9 (35%) for the placebo group; n=12 (50%) for the 0.3% TDCA gel treatment group; and n=5 (25%) for the 0.5% TDCA gel treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was 22.1 ± 18.0 for the placebo group and -61.3 ± 11.2% for the 0.5% TDCA gel treatment group. The difference in treatment effect was 83.4% (p=0.008), showing a significant therapeutic synergistic effect. The IGA change rate (mean ± SEM) was also 13.0 ± 9.9% for the placebo group and -40.0 ± 6.7% for the 0.5% TDCA gel treatment group, showing a significant therapeutic synergistic effect with a difference in treatment effect of 53% (p = 0.005) (Fig. 9).

[98] Stratification analysis was performed on SMOC1 -positive or ENTP-positive patients to compare and analyze the treatment effect of TDCA gel 0.5% compared to placebo, thereby confirming the effectiveness of prediction of treatment effect based on the combination of two biomarkers. Specifically, the number of SMOC1 -positive or ENTP-positive patients per group (n) and the ratio (%) to the total number of patents analyzed were n=18 (69%) in the placebo group; n=19 (79%) for the TDCA gel 0.3% treatment group; and n=9 (45%) for the TDCA gel 0.5% treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was -1.2 ± 10.9% for the placebo group and -51.4 ± 9.6% for the TDCA gel 0.5% treatment group. The difference in treatment effect was 50.2% (p=0.006), showing a significant therapeutic synergistic effect. The IGA change rate (mean ± SEM) was also 1.9 ± 6.3% in the placebo group and -33.3 ± 7.9% for the TDCA gel 0.5% treatment group, showing a significant therapeutic synergistic effect with a difference in treatment effect of 35.2% (p = 0.003) (Fig. 10).

[99] The effectiveness of biomarker-based treatment effect prediction was confirmed by comparing the treatment effect of 0.5% TDCA gel to placebo in patients stratified by ENTP6- positive or CRK-positive status Specifically, the number of ENTP6-positive or CRK-positive patients per group (n) and the ratio (%) to the total number of patients analyzed were n=21 (81%) in the placebo group; n=16 (67%) for the TDCA gel 0.3% treatment group; and n=8 (40%) for the TDCA gel 0.5% treatment group. The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was 0.9 ± 9.9% for the placebo group and -50.9 ± 8.5% for the TDCA gel 0.5% treatment group. The difference in treatment effect was 51.8% (p=0.003), showing a significant therapeutic synergistic effect. The IGA change rate (mean ± SEM) was 4.0 ± 5.9% for the placebo group and -37.5 ± 7.6% for the TDCA gel 0.5% treatment groups, showing a significant treatment synergistic effect with a difference in treatment effect of 41.5 % (p<0.001) (Fig. 11).

[100] The efficacy of biomarker-based treatment effect prediction was confirmed by comparing the treatment effect of 0.5% TDCA gel to placebo in patients stratified by SMOC 1- positive, ENTP6-positive, or CRK-positive status. Specifically, the number of SMOC1- positive, ENTP6-positive, or CRK-positive patients per group and the ratio (%) to the total number of analyzed patients were n=23 (88%) in the placebo group. The TDCA gel 0.3% treatment group was n=22 (92%) and the TDCA gel 0.5% treatment group was n=10 (50%). The primary efficacy evaluation variable (EASI change rate; mean ± SEM) was -1.8 ± 9.3% for the placebo group and -50.0 ± 8.7% for the TDCA gel 0.5% treatment group. The difference in treatment effect was 48.1% (p=0.003), showing a significant therapeutic synergistic effect. The IGA change rate (mean ± SEM) was 2.2 ± 5.6% for the placebo group and -30.0 ± 7.8% for the TDCA gel 0.5% treatment groups, showing a significant treatment synergistic effect with a difference in treatment effect of 32.2% (p = 0.004) (Fig 12).

[101] Through biomarker-based phase 2 clinical efficacy analysis, it was confirmed that the clinical efficacy of TDCA gel could be predicted by the baseline levels of biomarker proteins, more specifically, SMOC1, ENTP6, and CRK.

Safety Outcomes

[102] In the FAS population, which includes all 80 patients, there were no serious side effects caused by 0.3% TDCA gel and 0.5% TDCA gel administration during the clinical trial, and adverse drug reactions, adverse events that caused permanent administration discontinuation, and dropout were not reported. There were few side effects, and there was no difference compared to the placebo group. The side effects that occurred were very mild and were temporary symptoms that all recovered during the clinical trial. In addition, the causal relationship of all adverse events was evaluated as 'not related' by the clinical doctor. Through this, the safety and excellent tolerability of TDCA gel, the treatment of this experiment, were confirmed. (Table 5). Table 5 - Adverse Events

[103] Furthermore, compared with existing licensed treatments such as crisaborole, a PDE4 inhibitor, ruxolitinib. a JAK inhibitor, and topical steroids, as shown in the table below. TDCA was targeted at patients with "type A atopy,” which accounted for 73% of patients with mild or moderate atopic dermatitis. 0.5% TDCA gel has an equal or superior therapeutic effect compared to existing drugs. And for patients with “A' type atopy,” who accounted for 37% of the most responsive drugs, 0.5% TDCA gel shows much better efficacy than existing drugs and at the same time is highly safe.

Table 6 - Efficacy and safety' of TDCA gel 0.5% for atopic dermatitis treatment compared to conventional drugs

[104] In this small multicenter phase II trial involving 80 hospitalized patients, the differences in primary' endpoint between groups did not reach statistical significance at a threshold of 5%. However, after biomarker-stratified analysis the significance of clinical outcome was higher in the 0.5% TDCA gel group compared with the placebo group. This suggests a higher probability of clinical improvement by TDCA gel. when biomarkers are used as companion diagnostics. Looking at the field of cancer treatment, where companion diagnosis is firmly established, triple-negative breast cancer (TNBC) is the cancer type with the poorest treatment prognosis among the ty pes of breast cancer. Triple-negative breast cancer is a breast cancer in which all estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor (HER2) are negative, and the three receptor biomarkers are currently used to predict prognosis and drug response to anticancer drugs. Based on the above precedent, it is judged that the three biomarkers of SMOC1, CRK, and ENTP6 are screened before atopy treatment and used to predict treatment prognosis and drug response have sufficient validity. Therefore, it is possible to establish a diagnostic system for classifying atopic patients using the above three protein biomarker baseline values in the screening stage prior to treatment of patients with atopic dermatitis.

[105] The present disclosure is about biomarkers predicting the response to TDCA treatment for atopic dermatitis. It relates to a companion diagnostic composition based on the baseline levels of SMOC 1, CRK and/or ENTP6 in the blood plasma, predicting patients group who are likely to respond to TDCA treatment for atopy dermatitis. , This invention confirms that these biomarker can be usefully used as a companion diagnostic composition capable of predicting atopy dermatitis patients who respond better to TDCA.

[106] It will be apparent to those skilled in the art that various modifications and variations can be made in the biomarkers for predicting for the treatment efficacy of GPCR19 agonists in the treatment of atopic dermatitis and methods for treating subjects with such biomarkers of the present disclosure without departing from the spirit or scope of the aspects of the present disclosure. Thus, it is intended that the present disclosure covers the modifications and variations of the aspects provided they come within the scope of the appended claims and their equivalents.

SEQUENCE LISTING

SEQ ID NO: 1 (IGHA2)

ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQ DASGDLYTTSSQLTLPATQCPDGKSV

TCHVKHYTNSSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLR DASGATFTWTPSSGKSAVQGPPERDL

CGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPP SEELALNELVTLTCLARGFSPKDVLV RWLQGSQELPREKYLTWASRQEPSQGTTTYAVTSILRVAAEDWKKGETFSCMVGHEALPL AFTQKTIDRMAGKPTHINVSV VMAEADGTCY

SEQ ID NO: 2 (ENTP6)

MKKGIRYETSRKTSYIFQQPQHGPWQTRMRKISNHGSLRVAKVAYPLGLCVGVFIYV AYIKWHRATATQAFFSITRAAPGAR

WGQQAHSPLGTAADGHEVFYGIMFDAGSTGTRVHVFQFTRPPRETPTLTHETFKALK PGLSAYADDVEKSAQGIRELLDVA

KQDIPFDFWKATPLVLKATAGLRLLPGEKAQKLLQKVKKVFKASPFLVGDDCVSIMN GTDEGVSAWITINFLTGSLKTPGGS SVGMLDLGGGSTQIAFLPRVEGTLQASPPGYLTALRMFNRTYKLYSYSYLGLGLMSARLA ILGGVEGQPAKDGKELVSPCLS PSFKGEWEHAEVTYRVSGQKAAASLHELCAARVSEVLQNRVHRTEEVKHVDFYAFSYYYD LAAGVGLIDAEKGGSLVVGD FEIAAKYVCRTLETQPQSSPFSCMDLTYVSLLLQEFGFPRSKVLKLTRKIDNVETSWALG AIFHYIDSLNRQKSPAS

SEQ ID NO: 3 (SMOC1)

MLPARCARLLTPHLLLVLVQLSPARGHRTTGPRFLISDRDPQCNLHCSRTQPKPICA SDGRSYESMCEYQRAKCRDPTLGVV

HRGRCKDAGQSKCRLERAQALEQAKKPQEAVFVPECGEDGSFTQVQCHTYTGYCWCV TPDGKPISGSSVQNKTPVCSGSVT

DKPLSQGNSGRKDDGSKPTPTMETQPVFDGDEITAPTLWIKHLVIKDSKLNNTNIRN SEKVYSCDQERQSALEEAQQNPREGI

VIPECAPGGLYKPVQCHQSTGYCWCVLVDTGRPLPGTSTRYVMPSCESDARAKTTEA DDPFKDRELPGCPEGKKMEFITSLL

DALTTDMVQAINSAAPTGGGRFSEPDPSHTLEERVVHWYFSQLDSNSSNDINKREMK PFKRYVKKKAKPKKCARRFTDYCD

LNKDKVISLPELKGCLGVSKEGRLV

SEQ ID NO; 4 (ENPL)

MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQ LDGLNASQIRELREKSEKFAFQAE

VNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKC DKEKNLLHVTDTGVGMTREELVKNLG

TIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQH IWESDSNEFSVIADPRGNTLGRGTTIT

LVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEE SDDEAAVEEEEEEKKPKTKKVEKTV

WDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSIL FVPTSAPRGLFDEYGSKKSDYIKLYV

RRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLD MIKKIADDKYNDTFWKEFGTNIKL

GVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAES SPFVERLLKKGYEVIYLTEPVDEYCI

QALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKA VVSQRLTESPCALVASQYGWSGN

MERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLA VVLFETATLRSGYLLPDTKAYGDRI

ERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKES TAEKDEL

SEQ ID NO: 5 (CRK)

MAGNFDSEERSSWYWGRLSRQEAVALLQGQRHGVFLVRDSSTSPGDYVLSVSENSRV SHYIINSSGPRPPVPPSPAQPPPGVS

PSRLRIGDQEFDSLPALLEFYKIHYLDTTTLIEPVSRSRQGSGVILRQEEAEYVRAL FDFNGNDEEDLPFKKGDILRIRDKPEEQ

WWNAEDSEGKRGMIPVPYVEKYRPASASVSALIGGNQEGSHPQPLGGPEPGPYAQPS VNTPLPNLQNGPIYARVIQKRVPNA

YDKTALALEVGELVKVTKINVSGQWEGECNGKRGHFPFTHVRLLDQQNPDEDFS