Application of alarms S100B in prediction of curative effect of JAK inhibitor on alopecia areata

Abstract

The invention relates to application of seroalarm as a biomarker in predicting the curative effect of a JAK inhibitor on alopecia areata. Particularly, S100A8 / A9 and S100B are the most preferable biomarkers, and the curative effect of the JAK inhibitor on alopecia areata can be effectively predicted by detecting the concentration of the biomarkers in the serum of an alopecia areata patient, so that valuable reference is provided for selection of an alopecia areata treatment scheme.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular diagnosis, and specifically relates to the application of serum alarmin as a biomarker in predicting the efficacy of JAK inhibitors in treating alopecia areata. Background Art

[0002] Alopecia areata (AA) is a common inflammatory non-scarring hair loss. It is a local immune response of hair follicles induced by the external environment under a certain genetic background. Alopecia areata is mainly caused by CD8+ / NKG2D+T cells attacking their own hair follicles. Foreign studies have shown that the lifetime prevalence of the population is about 2%, and about half of the patients develop the disease before the age of 30. The specific pathogenesis of alopecia areata has not yet been clarified. Existing studies have shown that it is mainly related to the loss of local immune exemption of hair follicles, recognition of cytotoxic immune cells, and immune response of hair follicles. Other factors, including mental and psychological factors, are also involved in the onset of alopecia areata. Alopecia totalis and alopecia universalis are severe types of alopecia areata, which are often difficult to treat and are prone to relapse after improvement. There is currently no specific treatment drug.

[0003] In recent years, the recognition of the JAK / STAT pathway in the pathogenesis of alopecia areata has gradually brought JAK inhibitors into the public eye. The JAK / STAT signaling pathway is widely present in the innate and adaptive immune systems and is involved in many important biological processes, including cell proliferation, differentiation, apoptosis, and immune regulation. JAK inhibitors have broad, relatively nonspecific anti-inflammatory activity, can block a variety of signaling pathways including type 1 and type 2 cytokines, and significantly reduce the number and activity of T cells, dendritic cells, and NK cells. The mechanism of JAK inhibitors in treating alopecia areata is mainly to inhibit the production of inflammatory factors by immune cells, and may also directly act on hair follicle epithelial cells to promote the hair follicle cycle into the growth phase.

[0004] Compared with traditional immunosuppressive drugs, JAK inhibitors have a narrower target and therefore fewer side effects. Baricitinib (JAK1 / 2 inhibitor) is the first drug approved by the FDA for the treatment of severe alopecia areata, and was also approved in my country in March 2023 for the treatment of severe alopecia areata in adults. Tofacitinib (JAK1 / 3 inhibitor) has been reported in many case reports and clinical studies at home and abroad for the treatment of alopecia areata in children and adults, with no serious adverse reactions.

[0005] Alarmins are a class of endogenous, constitutively expressed proteins or peptides released by cells that are infected or stimulated by chemical, thermal, or physical damage during the immune response, such as IL-1α, IL-33, the S100 protein family, ATP, high-mobility group protein B1 (HMGB1), and defensins. As an intercellular signal defense, alarmins stimulate immune cells to participate in host defense by interacting with chemotactic and pattern recognition receptors (PRRs). They are considered to be endogenous damage-associated molecular patterns (DAMPs) that can induce sterile inflammation. Alarmin family molecules are universal markers of damage and early warning signals of the immune system. They participate in processes such as leukocyte chemotaxis, activation and proliferation of various immune cells, and thus play a key role in determining the type of immune response.

[0006] In skin immunity, alarmin cytokines mediate complex intercellular communication between keratinocytes and immune cells to regulate skin immune responses. Therefore, alarmins are increased in affected organs and systems in patients with autoimmune skin diseases, such as systemic lupus erythematosus, dermatomyositis, vitiligo, psoriasis, pemphigus, etc. Some alarmin factors can serve as valuable biomarkers for disease diagnosis, activity, and prediction of treatment response, and modulation of alarmin-related pathways may have potential therapeutic applicability.

[0007] There are few studies on alarmins in alopecia areata, and the alarmins involved include granulysin, IL-31, IL-1α, HMGB1, etc. Ono and Oba found that the level of granulysin in the serum of patients with alopecia areata was significantly increased, and there was a correlation between the concentration of granulysin and the size of the hair loss area. Granysin may mediate immune attack on hair follicles and serve as a marker of disease activity in the acute phase. IL-1α is a key mediator of hair loss in alopecia areata. Studies have shown that the serum IL-1α level of AA patients is significantly higher than that of the control group, and the IL-1α gene is closely related to the onset of hair loss. Bain et al. found that the IL-31 level of patients with alopecia areata was also significantly higher than that of healthy individuals. Saleem et al., through a review and analysis of 61 articles, concluded that IL-31 overexpression may cause hair loss. In addition, other authors have also studied the role of HMGB1 in alopecia areata. In subjects with alopecia areata, it was observed that the serum level of HMGB1 increased immediately at the onset of the disease, and its level continued to increase in adverse responders, indicating a negative correlation between serum levels and treatment response.

[0008] So far, the research on alarmin in alopecia areata is still very limited, especially the lack of research on the correlation between alarmin levels and the course, severity, and efficacy of alopecia areata, which has led to its inability to be applied in the diagnosis and treatment of alopecia areata. Summary of the invention

[0009] The present invention determines candidate biomarkers based on the role of alarmins in serum in the development of alopecia areata. By exploring the difference in expression of alarmins in alopecia areata and healthy people, further correlation analysis is performed between alarmins and the efficacy of JAK inhibitors, thereby determining biomarkers that can be used to predict the efficacy of JAK inhibitors in treating alopecia areata.

[0010] The first aspect of the present invention provides biomarkers that can be used to predict the efficacy of JAK inhibitors in treating alopecia areata: S100A8 / A9, S100B, and it is found that the combined use of multiple biomarkers can achieve better prediction results. Therefore, the present invention further provides the following biomarker combination:

[0011] (1) S100A8 / A9+S100B;

[0012] (2)S100A8 / A9+S100B+IgE.

[0013] In a preferred embodiment, the JAK inhibitor is baricitinib or tofacitinib.

[0014] The second aspect of the present invention provides a reagent for detecting the concentration of the above-mentioned biomarker in a sample, wherein the reagent can predict the efficacy of JAK inhibitors in treating alopecia areata by detecting the level of the biomarker.

[0015] The sample includes various biological samples derived from a subject, preferably blood, and more preferably serum.

[0016] The reagent can measure the concentration of the biomarker by various conventional detection methods, and the measurement can be performed at the protein level, mRNA level, gene expression (methylation) level, etc. For example, the measurement can be performed by enzyme-linked immunosorbent assay, quantitative PCR, methylation PCR, etc., preferably by enzyme-linked immunosorbent assay.

[0017] The third aspect of the present invention provides a kit for predicting the efficacy of JAK inhibitors in treating alopecia areata, which comprises a reagent for detecting the concentration of the above-mentioned biomarker in a sample. The biomarker can be a single biomarker, or a combination thereof. That is, the kit can contain one or more reagents for detecting different biomarkers. For example, it contains a reagent for detecting S100A8 / A9, a reagent for detecting S100B, two reagents for detecting S100A8 / A9 and S100B, or three reagents for detecting S100A8 / A9, S100B and IgE.

[0018] The kit can be a test kit for determining the concentration of biomarkers in a sample by various conventional detection methods, and accordingly contains other necessary reagents required for the detection method. Preferably, the kit is an enzyme-linked immunosorbent assay kit, and contains other necessary reagents for enzyme-linked immunosorbent assay.

[0019] The positive progressive effect of the present invention is that the present invention can simply measure the concentration of specific biomarkers in the serum of patients with alopecia areata, and use this to predict the efficacy of using JAK inhibitors to treat alopecia areata in the patients, thereby providing great reference value for the treatment of alopecia areata, and further providing great help for patients to reasonably choose treatment plans and save treatment time and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The research design process of the present invention.

[0021] Figure 2 The SALT score measures area.

[0022] Figure 3 Scatter plot of correlation between baseline SALT and serum IgE and alarmin.

[0023] Figure 4 ROC curve of S100A8 / A9 predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0024] Figure 5 ROC curve of S100B predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0025] Figure 6 ROC curve of HMGB1 predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0026] Figure 7 ROC curve of α-defensins predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0027] Figure 8 ROC curve of IgE predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0028] Fig. 9 ROC curve of S100A8 / A9+S100B combined to predict the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0029] Fig.10 ROC curve of S100A8 / A9+S100B+IgE combined to predict the efficacy of JAK inhibitors in the treatment of alopecia areata. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the present invention is not limited thereto. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0031] 1. Identify candidate biomarkers

[0032] As we all know, the prognosis of alopecia areata is closely related to factors such as the course of the disease, severity, and age of onset. In other treatments for alopecia areata, some studies have shown that the treatment effect and adverse reactions can be predicted by some pre-treatment laboratory test items, such as baseline serum IgE levels can predict the efficacy of Dabituo and are related to the incidence of adverse reactions during DPCP treatment; the alopecia areata prediction score based on trichoscope can predict the probability of hair regeneration in patchy alopecia areata. At present, no research has found relevant biomarkers that can predict the treatment effect of JAK inhibitors. JAK inhibitors are expensive, and their efficacy and safety are still inconclusive. Therefore, early detection and confirmation of factors closely related to the treatment effect of JAK inhibitors can provide a reference for alopecia areata patients to choose treatment options, saving patients time and treatment costs.

[0033] Epidermal-derived alarmin cytokines mediate complex intercellular communication between epidermal keratinocytes and immune cells, thereby regulating immune surveillance in the skin. Alarmins are endogenous molecules that act as danger signals and are rapidly released into the extracellular environment upon tissue damage, triggering defensive immune responses. An increasing number of studies have shown that alarmins can also be used as biomarkers to monitor the therapeutic effects of autoimmune diseases. In patients with rheumatoid arthritis, serum S100A8 / A9 levels decreased after infliximab or anti-TNF-α treatment; serum S100A8 / S100A9 concentrations significantly decreased in patients with juvenile idiopathic arthritis after intra-articular injection of triamcinolone acetonide; serum HMGB1 levels decreased in patients with granulomatosis with polyangiitis after statins or prednisolone treatment. Serum alarmins can also predict patient prognosis and relapse. Relapse of ANCA-associated vasculitis is closely associated with S100A8 / A9 levels.

[0034] S100A8 and S100A9 are the most abundant alarmins in many inflammatory diseases and they belong to the S100 calcium-binding protein family. Members of the S100 protein family have tissue or cell type-specific expression patterns. All S100 proteins have two so-called EF-hand-type calcium-binding sites. Binding of calcium to these calcium-binding sites induces conformational changes, leading to the interaction of S100 proteins with different ligands or binding to specific receptors. The typical feature of most S100 proteins is the formation of homodimers, heterodimers and / or higher oligomers. For example, S100A8 and S100A9 usually exist in the form of heterodimers (S100A8 / A9), which are called calprotectins. Intracellular S100 proteins are involved in many processes, including cell cycle control, proliferation, differentiation, migration, metabolism, cell dynamics, signal transduction and cell death. High expression and release of different S100 proteins are closely associated with disease activity and are found in many inflammatory diseases, such as rheumatoid arthritis (RA), inflammatory bowel or lung diseases, as well as in Alzheimer's disease, cardiovascular diseases and cancer.

[0035] S100 proteins are highly upregulated in different forms of childhood arthritis and autoimmune diseases. The specific secretion of S100 proteins by activated phagocytes at local sites of inflammation makes these molecules useful markers for monitoring disease activity. For the early diagnosis of systemic juvenile idiopathic arthritis, S100A8 / S100A9 is currently the most specific biomarker. In addition, it is also used in clinical studies to predict disease flares in patients with JIA or RA after drug treatment is discontinued or reduced after clinical remission. S100B was found in a recent study to be useful for the diagnosis of vitiligo with a sensitivity of up to 92.31%. Multivariate logistic regression analysis showed that S100B (OR, 1.019; 95% CI, 1.002-1.038; P = 0.03), S100A9 (OR 1.002; 95% CI, 1.001-1.003; 0.001), and HMGB1 (OR, 1.915; 95% CI, 1.186-3.091; P = 0.008) were significantly associated with vitiligo activity.

[0036] Recent studies have shown that high-mobility group protein B1 (HMGB1) is expressed in alopecia areata skin and serum samples, and serum HMGB1 levels are significantly increased in patients with acute onset and poor treatment response, indicating that HMGB1 can be used as a marker to assess the severity of AA, suggesting that it may play a role in the pathogenesis of AA. HMGB1 was originally identified as a nuclear DNA-binding protein, but during inflammation, it can be secreted by cells and act as a proinflammatory cytokine. HMGB1 has been reported to be elevated in several autoimmune diseases such as rheumatoid arthritis, SLE, and systemic sclerosis. Recent studies have revealed a new mechanism for the release of HMGB1 during inflammation: human hair follicle outer root sheath (ORS) cells constitutively express inflammasome proteins; dsRNA activates NLRP3 inflammasomes, leading to the release of IL-1β and HMGB1 from ORS cells, and proves that inflammasomes play a key role in mediating the release of HMGB1 from immune cells.

[0037] Defensins are a class of cationic host defense peptides that are mainly synthesized by Paneth cells, neutrophils, and epithelial cells and participate in host defense. Based on amino acid homology and cysteine ​​residue linkage, mammalian defensins are divided into α-defensins (α-DF), β-defensins (β-DF), and θ-defensins (θ-DF). However, humans only produce α-defensins and β-defensins. Defensins play a role in many biological processes, including showing immunomodulatory and chemotactic activities, maintaining mucosal barrier function, balancing intestinal flora, regulating organ development, and cell death. Therefore, defensins are gradually considered to be innate immune factors. In the past two decades, it has been found that defensins regulate the chemotaxis of immune cells and are involved in regulating sperm activity, male infertility, thrombosis, melanin deposition, and other important biological functions. In addition, defensins have been shown to induce the host's innate immune response, enhance the host's adaptive immune response, and promote the activation of immune cells such as T cells and macrophages.

[0038] Based on a large number of literature reviews, this study identified the levels of alarmins S100A8 / A9, S100B, HMGB1, α-defensin and IgE in serum as candidate biomarkers for predicting the prognosis and recurrence of alopecia areata treated with JAK inhibitors. The above alarmin molecules are all known in the art, for example, they can be human S100A8, human S100A9, human S100B, human HMGB1, and human α1-defensin with GenBank Gene IDs of 6279, 6280, 6285, 3146, and 1667, respectively, and the structure of IgE is well known in the art.

[0039] 2. Research subjects

[0040] 2.1 Case Source

[0041] This study was a case-control study in accordance with the ethical guidelines of the Declaration of Helsinki, approved by the Ethics Committee of the General Hospital of the Eastern Theater Command, and informed consent was signed by the patients or their guardians. The case group consisted of patients with alopecia areata aged 7 years and above who visited the dermatology outpatient clinic of the General Hospital of the Eastern Theater Command from October 2022 to December 2023. All patients with alopecia areata were diagnosed by two experienced dermatologists based on the clinical manifestations, dermoscopy, and scalp biopsy pathology (if necessary) listed in the Guidelines for the Diagnosis and Treatment of Alopecia Areata in China (2019); the control group consisted of the general population who underwent a physical examination at the physical examination center of our hospital during the same period and were excluded from the diagnosis of alopecia areata. Finally, 35 cases in the case group and 32 cases in the control group were included, of which 18 patients in the case group met the diagnostic criteria for moderate to severe or refractory alopecia areata, and the patients and their guardians agreed to use JAK inhibitors for treatment. The demographic and clinical characteristics of the subjects in each group are shown in Table 1. The average age of the case group and the control group was 27.63±12.4 years and 25.65±4.76 years, respectively. There was no significant difference in age and gender among the groups (P>0.05).

[0042] Table 1 Baseline characteristics of the study population

[0043]

[0044]

[0045] 2.2 Inclusion criteria for JAK inhibitor treatment group

[0046] (1) Aged 7 to 60 years old, regardless of gender;

[0047] (2) meet the diagnostic criteria for moderate to severe alopecia areata (SALT score > 50%, or combined dermoscopy and skin histopathology diagnosis);

[0048] (3) Meet the diagnostic criteria for refractory alopecia areata: The patient has not recovered after at least three or more other treatments or has not responded to local treatment for 6 months.

[0049] Conditions (1)(2) or (1)(3) must be met simultaneously.

[0050] 2.3 Exclusion criteria for JAK inhibitor treatment group

[0051] (1) Lymphocyte count (ALC) less than 0.5×10 9 / L; neutrophil count (ANC) less than 1×10 9 / L; hemoglobin value is less than 8g / dL; platelet count is less than 50×10 9 / L;

[0052] (2) Active tuberculosis;

[0053] (3) renal impairment; creatinine clearance <30 mL / min;

[0054] (4) Severe liver damage;

[0055] (5) Active hepatitis B or hepatitis C infection;

[0056] (6) Allergic reaction to the active substance or any excipients;

[0057] (7) severe active infection;

[0058] (8) Coagulation disorders;

[0059] (9) pregnancy or breastfeeding;

[0060] (10) Unwilling to sign the informed consent form.

[0061] 2.4 Exit criteria:

[0062] (1) Serious adverse events or patient death occurred during treatment;

[0063] (2) Those who receive other treatment methods or drugs during treatment;

[0064] (3) Those who voluntarily withdraw from the treatment or fail to follow the prescribed treatment plan, resulting in incomplete data and inability to accurately judge the efficacy of the treatment.

[0065] 3. Research Methods

[0066] 3.1 Research indicators

[0067] Physical examinations were performed on patients with alopecia areata, and detailed inquiries were made and records were kept of the age of onset, disease progression, number of attacks, area of ​​hair loss, subjective symptoms, nail lesions, and previous treatment regimens. The severity of the disease was assessed by two experienced dermatologists using the Scoring of Alopecia Tool (SALT). The treatment effect was evaluated using the clinical efficacy evaluation scale.

[0068] 3.1.1 SALT score

[0069] The SALT score is calculated by measuring the percentage of hair loss in each of the four scalp areas: right side (18% of the total scalp area), left side (18%), top (40%), and occipital (24%) (see Figure 2 ) and using the weighted sum of the percentage of hair loss in the four areas, a composite score, the SALT score, is obtained. A SALT score of 0 indicates no hair loss, while a SALT score of 100 indicates complete hair loss.

[0070] 3.1.2 Clinical efficacy evaluation level

[0071] The clinical efficacy evaluation grades are divided into the following 4 levels:

[0072] Cured: The hair scalp coverage area is 90% < n ≤ 100%; all new hairs have grown out, densely distributed, and the thickness and color of the hairs are the same as normal hair, and the hair pulling test is (-);

[0073] Markedly effective: 50% ≤ n < 90%; many vellus hairs have become thick hairs, and the hair pulling test is (-);

[0074] Improved: 10% ≤ n < 50%; new hairs grow, but the growth is slow, and the hair pulling test is (±);

[0075] Ineffective: 0 ≤ n < 10%; no new hairs grow or there are only a small amount of vellus hairs and white hairs, and the hair pulling test is (+).

[0076] Total effective rate = (number of cured cases + number of markedly effective cases + number of improved cases) / total number of cases in this group × 100%

[0077] 3.2 Specimen collection and processing

[0078] 3.2.1 Specimen collection and detection

[0079] All subjects took 3.5 ml of fasting venous blood in the early morning using a separation gel vacuum blood collection tube (BD, USA). After collecting the blood, the serum and red blood cells were quickly and carefully separated by centrifuging at 3000 revolutions for 10 minutes using a low-speed centrifuge. The serum was aliquoted into sterile EP tubes and stored frozen at -80°C and tested within 1 year. The alarmin S100A8 / A9, S100B, HMGB1, and α-defensin in the serum were determined by enzyme-linked immunosorbent assay, and the serum IgE was detected at the hospital testing center.

[0080] 3.2.2 Equipment and reagents

[0081] Human S100 calcium-binding protein A8 / A9 complex (S100A8 / A9) ELISA scientific research kit, purchased from Shanghai Kexing Trading Co., Ltd.;

[0082] Human S100B protein ELISA scientific research kit, purchased from Shanghai Kexing Trading Co., Ltd.;

[0083] Human α-defensin ELISA scientific research kit, purchased from Shanghai Kexing Trading Co., Ltd.;

[0084] Human HMGB1 ELISA kit, purchased from Wuhan Huamei Bio-Engineering Co., Ltd.;

[0085] Low-speed centrifuge, purchased from Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.;

[0086] Ultra-low temperature (-80°C) refrigerator, purchased from Qingdao Haier Special Electric Appliance Co., Ltd.;

[0087] The microplate reader was purchased from Thermo Fisher, USA.

[0088] 3.3 Data Analysis

[0089] SPSS 27.0 data classification software was used for data processing and statistical analysis. An Excel database was established to enter baseline information and clinical data. The normality test of continuous variables was performed using the Shapiro-Wilk test (sample size ≤ 50) or the Kolmogorov-Smimov test (sample size > 50), and the variance homogeneity test was performed using the Levene test. The mean of normally distributed data was expressed as mean ± standard deviation, and the mean of skewed distributed data was expressed as median (interquartile range). For continuous variables that followed normal distribution and variance homogeneity, the independent sample t test was used to compare the two groups; for continuous variables that did not meet normality or variance homogeneity, the Mann-Whitney U test was used to compare the two groups. Categorical variables were expressed as case numbers and percentages, and unordered categorical variables were compared between the two groups using the chi-square test or Fisher's exact test. When the overall sample size was less than 40 or the theoretical frequency was greater than 1 and less than 5, the continuity-corrected chi-square test was selected. For ordered categorical variables, the Kruskal-Wallis H test was used to compare the two groups.

[0090] The test level was 0.05, and the difference was statistically significant when p<0.05.

[0091] Example 1 Analysis of serum biomarkers in patients with alopecia areata

[0092] According to the above method, the serum S100A8 / A9 (ng / mL), S100B (pg / mL), HMGB1 (pg / mL), α-defensin (pg / mL), and IgE (ng / mL) levels of 35 cases in the case group (moderate to severe alopecia areata patient group A1-A19, mild alopecia areata patient group B1-B16) and 32 cases in the control group (C1-C32) were measured and statistical analysis was performed in groups.

[0093] 1. Comparison of serum S100A8 / A9, S100B, HMGB1, α-defensin, and IgE levels between the case group and the control group

[0094] The serum levels of S100A8 / A9, S100B, HMGB1, α-defensin, and IgE in the case group were 3008.43±1016.21, 482.07 (402.52, 536.70), 59.49 (0, 536.70), 233.79 (199.38, 250.99), and 67.6 (34.9, 215.5), respectively, while those in the control group were 2176.93±1194.23, 388.06 (0, 521.02), 0 (0, 84.70), 215.87 (78.80, 269.61), and 43.8 (27.6, 79.15), respectively. The baseline serum S100A8 / A9, S100B, and HMGB1 levels in the alopecia areata case group were higher than those in the control group, and the difference was statistically significant (P < 0.05). There was no statistically significant difference in serum α-defensin and IgE levels between the case group and the control group (P > 0.05).

[0095] Table 2 Comparison of serum biomarker levels between case group and control group

[0096]

[0097]

[0098] 2. Comparison of serum S100A8 / A9, S100B, HMGB1, α-defensin, and IgE levels among patients with different severity

[0099] The serum S100A8 / A9, S100B, HMGB1, α-defensin, and IgE levels of patients with moderate to severe alopecia areata were 3036.26±1241.32, 535.25 (477.18, 587.52), 103.58 (0, 503.50), 229.88 (199.97, 259.03), and 93.8 (35.1, 230), respectively, while those of patients with mild alopecia areata were 2975.37±699.68, 424.86 (250.20, 465.39), 42.95 (0, 165.18), 237.08 (197.55, 248.86), and 47.85 (36.25, 168), respectively. The serum S100B level in patients with moderate to severe alopecia areata was significantly higher than that in the mild alopecia areata group, and the difference was statistically significant (P=0.041). There was no statistical difference in the serum S100A8 / A9, HMGB1, α-defensin, and IgE levels between patients with mild, moderate to severe alopecia areata.

[0100] Table 3 Comparison of serum biomarker levels among different severity levels of alopecia areata

[0101]

[0102] There was no significant correlation between baseline SALT scores and serum biomarkers in patients with alopecia areata (see Figure 3 ).

[0103] Example 2 Clinical study on the treatment of moderate to severe alopecia areata with JAK inhibitors

[0104] A total of 18 eligible patients with moderate to severe or refractory alopecia areata were included in this study, with an age range of 8 to 43 (25.11±11.75) years, a male to female ratio of 2.6:1, and a disease course of 6 months to 19 years. All 18 patients took oral JAK inhibitor tofacitinib 10 mg / d as the initial dose and were followed up once a month.

[0105] The SALT score of 18 patients before treatment was 70.97±33.80. There were 18 patients with a treatment time of ≥12 weeks. At the 12th week, the SALT score was 60.59±33.23, and the effective rate was 66.7%. There were 7 patients with a treatment time of ≥24 weeks. At the 24th week, the SALT score was 25.81±20.94, and the effective rate was 100%. There were 4 patients with a treatment time of ≥36 weeks. At the 36th week, the SALT score was 7.5±10.95, and the effective rate was 100%. At the 12th, 24th, and 36th weeks of treatment, the SALT scores before and after treatment were all lower than those before treatment, and the changes were statistically significant (P<0.05).

[0106] The total effective rate of JAK inhibitors in treating patients with moderate to severe alopecia areata was 66.7%. Among all patients receiving JAK inhibitors, 2 were cured, 3 were significantly effective, 7 were improved, and 6 were ineffective (see Table 4).

[0107] Table 4 Treatment effect

[0108]

[0109] Table 5 Changes in SALT scores during treatment

[0110]

[0111] No serious adverse reactions were reported by any patient during treatment. Five patients reported mild adverse reactions, including upper respiratory tract infection (n=3, 16.67%), acne (n=3, 16.67%), and mild elevation of liver transaminases (n=1, 5.56%).

[0112] Example 3 Study on the prediction of the efficacy of JAK inhibitors in treating alopecia areata

[0113] The serum S100A8 / A9, S100B, HMGB1, α-defensin and IgE levels of all 18 patients treated with JAK inhibitors before treatment were counted (see Table 6). All patients were divided into groups according to whether the efficacy was (cured + markedly effective + improved) or not (ineffective). ROC curves were constructed using various biomarkers alone and in combination ( Figures 4 to 10 ), and the analysis results are summarized in Table 7.

[0114] Table 6 Serum biomarkers and efficacy of patients in the treatment group

[0115]

[0116]

[0117] Table 7 Prediction effect of different markers

[0118]

[0119] As can be seen from Table 7, the AUC value of α-defensin as a detection marker for predicting the efficacy of JAK inhibitors in the treatment of alopecia areata is less than 0.5, which does not conform to the theoretical design, indicating that there are errors in the data, and it is discarded; the AUC value of HMGB1 is 0.604, indicating that HMGB1 has a low predictive value for the efficacy of JAK inhibitors in the treatment of alopecia areata, and it is discarded; the AUC values ​​of S100A8 / A9, S100B, and IgE are 0.740, 0.753, and 0.721, respectively, indicating that they have a high predictive value for the efficacy of JAK inhibitors in the treatment of alopecia areata; however, the detection sensitivity corresponding to the optimal cutoff value of IgE is 0.455, which lacks practical value and is also discarded. In summary, S100A8 / A9 and S100B are biomarkers with greater predictive value for the efficacy of JAK inhibitors in the treatment of alopecia areata.

[0120] When S100A8 / A9 and S100B were combined to predict the efficacy of JAK inhibitors in the treatment of alopecia areata, the AUC value further increased to 0.805, and the optimal cut-off value was 0.766. At this time, the sensitivity and specificity were 0.909 and 0.857, respectively, and the diagnostic value was further increased.

[0121] When S100A8 / A9, S100B and IgE were combined to predict the efficacy of JAK inhibitors in the treatment of alopecia areata, the AUC value further increased to 0.896, and the optimal cut-off value was 0.766. At this time, the sensitivity and specificity were 0.909 and 0.857, respectively, and the diagnostic value was further increased.

[0122] The above specific implementation methods are only for the purpose of illustrating the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Technical solutions recorded in the implementation but not claimed in the claims should not be construed as a waiver of the patent rights.

Claims

1. Application of S100B in the preparation of reagents for predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

2. Application of S100B and S100A8 / A9 in the preparation of reagents for predicting the efficacy of JAK inhibitors in the treatment of alopecia areata.

3. Use of reagent A in the preparation of a kit for predicting the efficacy of JAK inhibitors in treating alopecia areata, wherein: The reagent A is used to determine the concentration of S100B in the serum of a subject.

4. The use according to claim 3, wherein: The kit also contains a reagent B for determining the concentration of S100A8 / A9 in the serum of a subject.

5. The use according to claim 3 or 4, wherein: The concentrations of S100B and / or S100A8 / A9 in the serum of the subjects were measured by enzyme-linked immunosorbent assay.

6. A kit for predicting the efficacy of JAK inhibitors in treating alopecia areata, wherein: The kit comprises a reagent A for determining the concentration of S100B in the serum of a subject.

7. The kit according to claim 6, further comprising a reagent B for determining the concentration of S100A8 / A9 in the serum of a subject.

8. The kit according to claim 6 or 7, which is used for determining the concentration of S100B and / or S100A8 / A9 in the serum of a subject by enzyme-linked immunosorbent assay.

9. The use according to claims 1-5 or the kit according to claims 6-8, wherein: The JAK inhibitor is baricitinib or tofacitinib.

Images