Marker group for evaluating curative effect of treating leucoderma by combining baricitinib with narrow-spectrum medium-wave ultraviolet rays, kit and application

Abstract

The invention discloses a marker group for evaluating sensitivity of bicitinib combined with narrow-spectrum medium-wave ultraviolet rays in treatment of leucoderma, a substance for detecting the marker group, a kit and an evaluation method. The marker group contains a plasma biomarker P selectin (SELP), an apolipoprotein A4 (APOA4), a mannan binding lectin associated serine protease 1 (MASP1), and a urine biomarker immune globulin J chain (IGJ) and an interleukin 18 binding protein (IL-18BP). By detecting the expression change of the group of markers in a sample before and after treatment and combining with a vitiligo area scoring index (VASI), the treatment sensitivity of a patient can be accurately evaluated, an individualized scheme can be made in an assisted manner, the blank of the combination therapy curative effect evaluation marker is filled, and the combination therapy curative effect evaluation marker has a relatively high clinical transformation value.

Description

Technical Field

[0001] This invention relates to a combined detection biomarker set, specifically a biomarker set, kit, and application for evaluating the efficacy of baricitinib combined with narrowband UVB therapy for vitiligo. Background Technology

[0002] Vitiligo is a common acquired depigmenting skin disease with a global incidence of approximately 0.5% to 2%. It is characterized by being "easy to diagnose but difficult to treat," not only causing obvious skin abnormalities but also easily triggering psychological problems such as anxiety and depression, severely impacting patients' quality of life. Currently, there is no specific treatment plan, and treatment outcomes often vary from person to person, making it difficult to meet clinical needs.

[0003] Existing research has clearly established that the IFN-γ / JAK / STAT signaling pathway plays a central role in the pathogenesis of vitiligo. Abnormal activation of this pathway can mediate melanocyte damage and apoptosis, ultimately leading to skin depigmentation. Based on this mechanism, various topical and oral JAK inhibitors (such as baricitinib) have been proven effective in controlling vitiligo progression and promoting repigmentation in recent years. Further research has found that combining JAK inhibitors with narrow-band ultraviolet B (NB-UVB) can significantly improve treatment efficacy by synergistically regulating the immune microenvironment and enhancing melanocyte activity. Prospective controlled clinical trials have validated that baricitinib combined with NB-UVB is not only effective but also safe for treating progressive non-segmental vitiligo, providing a new and effective treatment option for this disease.

[0004] However, current clinical practice lacks effective biomarkers for evaluating the efficacy and sensitivity of the "baricitinib combined with NB-UVB" combination therapy. This makes it impossible to predict patient treatment sensitivity using objective indicators or dynamically monitor treatment effects, hindering clinicians' ability to accurately assess treatment response and adjust treatment plans in a timely manner, thus restricting the implementation of personalized treatment. Therefore, screening for differentially expressed molecules in bodily fluids (such as blood and urine) before and after treatment and identifying biomarkers for efficacy evaluation is of significant clinical value for improving the precision of vitiligo treatment and optimizing personalized treatment strategies.

[0005] As direct executors of biological functions, proteins' expression levels directly reflect dynamic changes in downstream biological processes, making them ideal sources of biomarkers. Mass spectrometry (MS) is a core tool in proteomics research. Data-independent acquisition (DIA) tandem mass spectrometry, in particular, enables continuous and unbiased acquisition of all secondary mass spectrometry information. It boasts advantages such as broad detection coverage, high quantitative accuracy, and good reproducibility, making it one of the most widely used techniques in clinical proteomics research. Furthermore, bodily fluid samples such as blood and urine are convenient to collect, minimally invasive, and have high patient compliance, making their research results easier to translate into clinical applications.

[0006] Unfortunately, proteomics research in the field of vitiligo is still relatively scarce, especially the lack of research on screening biomarkers related to the efficacy evaluation and treatment sensitivity assessment of "JAK inhibitor combined with phototherapy" using blood and urine proteomics technology. There is an urgent need to carry out relevant explorations to fill this gap. Summary of the Invention

[0007] The primary technical problem to be solved by this invention is to provide an application of a new set of biomarkers, which can be used in the preparation of substances for evaluating, screening and dynamically monitoring the therapeutic effect and sensitivity of baricitinib combined with narrowband UVB for vitiligo.

[0008] Another technical problem to be solved by the present invention is to provide a kit for detecting a new set of biomarkers, which can be used to evaluate, screen and dynamically monitor the therapeutic effect and sensitivity of baricitinib combined with narrowband UVB for vitiligo.

[0009] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: An application of a substance for detecting a group of biomarkers includes one or more of the following applications: A1) Use in the preparation of products for evaluating the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients; A2) Application in the preparation of products for detecting the sensitivity of vitiligo patients to baricitinib combined with narrowband UVB therapy; A3) Application in the preparation of products for screening the sensitivity of vitiligo to baricitinib combined with narrow-band UVB therapy; A4) Application in the preparation of products for dynamically monitoring the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients.

[0010] The biomarker set includes P-selectin (SELP), apolipoprotein A4 (APOA4), mannan-binding lectin-associated serine protease 1 (MASP-1), immunoglobulin J chain (IGJ), and interleukin-18 binding protein (IL-18BP).

[0011] Preferably, the substance is a reagent for detecting the expression level of a biomarker, a reagent for specifically recognizing and binding to a biomarker, or a reagent for detecting the content of a biomarker.

[0012] Preferably, the substance is selected from mass spectrometry identification reagents, monoclonal antibodies, polyclonal antibodies, or antigen-binding fragments of antibodies.

[0013] Preferably, the substance used to detect the group of markers is any one of the following a), b), or c): a) Reagents for detecting the expression levels of the biomarker group; b) A reagent group containing the reagent described in a); c) A kit containing either the reagent of a) or the reagent group of b).

[0014] Preferably, the detection sample for the biomarkers P-selectin (SELP), apolipoprotein A4 (APOA4), and mannan-binding lectin-associated serine protease 1 (MASP-1) is plasma; and the detection sample for the biomarkers immunoglobulin J chain (IGJ) and interleukin-18 binding protein (IL-18BP) is urine.

[0015] A kit for detecting a group of biomarkers, the kit comprising one or more of the following applications: A1) Use in the preparation of products for evaluating the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients; A2) Application in the preparation of products for detecting the sensitivity of vitiligo patients to baricitinib combined with narrowband UVB therapy; A3) Application in the preparation of products for screening the sensitivity of vitiligo to baricitinib combined with narrow-band UVB therapy; A4) Application in the preparation of products for dynamically monitoring the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients.

[0016] The biomarker group includes plasma biomarkers and urine biomarkers; the plasma biomarkers are P-selectin (SELP), apolipoprotein A4 (APOA4), and mannan-binding lectin-associated serine protease 1 (MASP-1); the urine biomarkers are immunoglobulin J chain (IGJ) and interleukin-18 binding protein (IL-18BP).

[0017] Preferably, the kit contains reagents for detecting the expression levels of the biomarker group; including specific antibodies against SELP, APOA4, MASP1, IGJ, and IL-18BP.

[0018] Preferably, the kit further comprises at least three of the following: sample processing reagents, standards, quality control products, diluent, washing solution, enzyme-labeled antibody, substrate solution, and stop solution.

[0019] A method for evaluating the sensitivity of patients with progressive non-segmental vitiligo to baricitinib combined with NB-UVB therapy as a non-disease diagnostic and treatment method includes the following steps: (1) Biomarker detection: The above kits were used to detect the expression levels of SELP, APOA4 and MASP1 in plasma samples before and after treatment, and the expression levels of IGJ and IL-18BP in urine samples before and after treatment. (2) Result determination: If the expression levels of SELP and MASP1 in plasma are significantly downregulated after treatment compared with those before treatment (P<0.05), and the expression level of APOA4 in plasma is significantly upregulated compared with those before treatment (P<0.05), and the expression levels of IGJ and IL-18BP in urine are significantly downregulated compared with those before treatment (P<0.05), then the patient is determined to have high sensitivity to baricitinib combined with NB-UVB treatment.

[0020] This invention can also be used for dynamic monitoring of treatment effects and to assist in the development of personalized treatment plans. Monitoring method: The Vitiligo Area Score Index (VASI) is used for auxiliary verification. If the patient's T-VASI is ≥50 after 3 months of treatment and meets the characteristics of biomarker expression changes in step (2), then the patient's high sensitivity to the combined treatment is further confirmed; if the patient's T-VASI is <50 after 3 months of treatment and does not meet the characteristics of biomarker expression changes in step (2), then the patient's low sensitivity to the combined treatment is determined. Based on the above treatment effects and sensitivity assessment, personalized treatment plans can be customized for patients.

[0021] Using the kit provided by this invention, the expression levels of the biomarkers SELP, APOA4, and MASP1 in the peripheral blood of subjects, as well as the expression levels of IGJ and IL-18BP in the urine of subjects, can be detected. For patients with progressive non-segmental vitiligo who are sensitive to baricitinib combined with NB-UVB therapy, after treatment, the expression levels of SELP and MASP1 in plasma were significantly downregulated compared to before treatment (P < 0.05), the expression level of APOA4 in plasma was significantly upregulated compared to before treatment (P < 0.05), and the expression levels of IGJ and IL-18BP in urine were significantly downregulated compared to before treatment (P < 0.05). Based on these information of upregulation or downregulation of protein expression, the sensitivity and therapeutic effect of baricitinib combined with narrowband UVB therapy for vitiligo can be further determined, thereby enabling the evaluation, screening, and formulation of treatment plans for the efficacy of baricitinib combined with narrowband UVB therapy for vitiligo.

[0022] The kit provided by this invention may include appropriate packaging and instructions for use in the methods disclosed herein. The kit contains reagents for identifying the expression levels of the aforementioned biomarker proteins, which may be mass spectrometry reagents, antibodies, or antigen-binding fragments thereof. The kit may also be a chip immobilized with the aforementioned protein identification reagents.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The biomarker screening is highly accurate and has been experimentally verified to be reliable. This invention uses patients with progressive non-segmental vitiligo as the research subjects. Data-independent (DIA) tandem mass spectrometry is used to perform proteomics analysis on plasma and urine samples before and after treatment. Three differentially expressed proteins, SELP, APOA4, and MASP1, were screened from plasma, and two differentially expressed proteins, IGJ and IL-18BP, were screened from urine. Furthermore, ELISA experiments in the validation cohort confirmed that after treatment, the levels of SELP and MASP1 in plasma of patients with sensitive and superior treatment efficacy were significantly lower than before treatment (P<0.05), the level of APOA4 was significantly higher than before treatment (P<0.05), and the levels of IGJ and IL-18BP in urine were significantly lower than before treatment (P<0.05), which is completely consistent with the trend of mass spectrometry screening results, ensuring the reliability and accuracy of the biomarkers.

[0024] (2) The specificity and accuracy of efficacy assessment showed that the biomarker group had good efficacy assessment value: the AUC values ​​of SELP, APOA4 and MASP1 in plasma were 0.715, 0.750 and 0.648, respectively, and the AUC values ​​of IGJ and IL-18BP in urine were 0.668 and 0.699, respectively; when the five biomarkers were analyzed together, the AUC value increased to 0.807, which was significantly higher than the assessment efficacy of the individual biomarkers. It can more accurately distinguish the sensitivity of patients to baricitinib combined with NB-UVB for the treatment of vitiligo, and provide a highly specific and accurate objective indicator for clinical efficacy assessment.

[0025] (3) Sample collection is convenient and patient compliance is high, which is conducive to clinical translation. This invention selects plasma and urine as test samples. Only 4 ml of peripheral blood is required for plasma samples and only 12 ml of clean-fast midstream urine is required for urine samples. Both have the advantages of minimal invasiveness and convenient operation. Moreover, the sample processing procedures are mature (such as using a high-depth blood proteomics sample preparation kit for plasma de-abundance processing and using an EasyPeptide Micro pretreatment kit for urine pretreatment). No complicated invasive operations are required, and patient acceptance and compliance are high. The research results are more easily translated into routine clinical testing applications.

[0026] (4) It enables dynamic monitoring of treatment effects and individualized treatment guidance. This invention can dynamically assess treatment effects by comparing changes in the expression of biomarkers before and after treatment, combined with the Vitiligo Area Score Index (VASI): if after 3 months of treatment, the patient's T-VASI ≥ 50, and plasma SELP and MASP1 are downregulated, APOA4 is upregulated, and urine IGJ and IL-18BP are downregulated, the patient can be judged as treatment-sensitive (fast response group); if T-VASI < 50 and there are no significant changes in biomarkers, the patient is judged as treatment-insensitive (slow response group). This assessment method can help clinicians identify treatment response in a timely manner, providing a basis for maintaining the original treatment plan for sensitive patients and adjusting the dosage of baricitinib or the frequency of NB-UVB irradiation for insensitive patients, significantly improving the accuracy of individualized treatment for vitiligo.

[0027] (5) Filling the gap in biomarkers for evaluating the efficacy of "JAK inhibitor combined with phototherapy" in vitiligo. This invention screened and verified specific body fluid biomarkers for this combined therapy and established a "plasma + urine" combined evaluation model. This not only enriched the data on vitiligo proteomics research, but also solved the problem of lacking objective efficacy evaluation indicators in clinical practice. It provides a reference for the technical ideas and experimental basis for monitoring the efficacy of similar treatment regimens in the future. Attached Figure Description

[0028] Figure 1A Volcano plot of differentially expressed plasma proteins before and after treatment; Figure 1B Volcano plot of differentially expressed plasma proteins in the rapid response group before and after treatment; Figure 2A A graph showing the correlation between the percentage change in SELP quantitative values ​​and the percentage change in patients' VASI scores; Figure 2B Here is the ROC curve for SELP; Figure 2C A graph showing the correlation between the percentage change in MASP1 quantitative values ​​and the percentage change in patients' VASI scores; Figure 2D The ROC curve for MASP1; Figure 2E A graph showing the correlation between the percentage change in APOA4 quantitative values ​​and the percentage change in patients' VASI scores; Figure 2F Here is the ROC curve for APOA4; Figure 3A Volcano plot of differentially expressed proteins in urine before and after treatment; Figure 3B Volcano plot of differential protein in urine after treatment in the rapid response group and before treatment in the rapid response group; Figure 4A IGJ ROC curve for candidate biomarkers of urinary efficacy assessment; Figure 4B ROC curve of IL-18BP, a candidate biomarker for evaluating the efficacy of urinary therapy; Figure 5A A graph showing the plasma SELP levels before and after treatment with baricitinib combined with NB-UVB, measured by ELISA. Figure 5B A graph showing the plasma APOA4 levels before and after treatment with baricitinib combined with NB-UVB, measured by ELISA. Figure 5C A graph showing plasma MASP1 levels before and after treatment with baricitinib combined with NB-UVB, measured by ELISA. Figure 6A The image shows the urinary IGJ levels before and after treatment with baricitinib combined with NB-UVB, measured by ELISA. ; Figure 6B The image shows the IL-18BP levels in urine before and after treatment with baricitinib combined with NB-UVB, measured by ELISA. ; Figure 7 ROC curves of combined plasma and urine biomarkers for evaluating the efficacy of treatment before and after treatment. Detailed Implementation

[0029] The technical content of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0030] Example 1: Screening of biomarkers to evaluate the sensitivity of baricitinib combined with narrowband UVB (NB-UVB) therapy for vitiligo. I. Biomarker Screening Methods 1. Research Subjects Sixteen patients who received baricitinib in combination with NB-UVB were included in this section. This section included 16 adult patients with non-segmental progressive vitiligo, including 13 males and 3 females, with a mean age of 32.1 ± 7.4 years, a mean disease duration of 11.3 ± 7.9 years, and a mean VASI score of 14.2 ± 10.9 before treatment.

[0031] 1.1 Specimen Collection Peripheral blood samples (4 ml, EDTA anticoagulated) were collected from patients before treatment and at follow-up points. The samples were gently inverted to mix, centrifuged at 1600g for 10 min at 4°C, and the supernatant plasma was collected. Simultaneously, 12 ml of clean-fasting midstream urine was collected from the patients. The plasma and urine samples were numbered, aliquoted, and stored at -80°C for use in plasma and urine proteomics studies.

[0032] 1.2 Clinical efficacy evaluation The clinical assessment of patient treatment effectiveness was conducted using the Vitiligo Area Scoring Index (VASI). The scoring details are as follows: The body surface was divided into six regions for calculating the area of ​​pigment loss: face and neck, hands, upper limbs (excluding hands), trunk, lower limbs (excluding feet), and feet. The area of ​​pigment loss was estimated using the palm method, where one palm unit (including the palm and the palmar surface of all fingers) is approximately 1% of the body surface area (BSA), and one thumb unit is approximately 0.1% of the BSA.

[0033] The degree of pigment loss is divided into 7 levels, namely 0 (no pigment loss), 10% (only punctate pigment loss), 25% (the area of ​​pigment loss is smaller than the area of ​​no pigment loss), 50% (the area of ​​pigment loss is equal to the area of ​​no pigment loss), 75% (the area of ​​pigment loss is larger than the area of ​​no pigment loss), 90% (a small amount of pigment spots remain) and 100% (complete pigment loss).

[0034] VASI is calculated by multiplying the area of ​​pigment loss in the corresponding region by the degree of pigment loss, and then summing the values ​​for all areas.

[0035] Based on whether patients in the combination therapy group achieved T-VSAI 50 after 3 months of treatment with baricitinib combined with NB-UVB, patients were divided into a fast-response group and a slow-response group.

[0036] 2. Main experimental reagents and equipment 2.1 The main experimental instruments are shown in Table 1.

[0037] Table 1 2.2 Main reagents and consumables are shown in Table 2.

[0038] Table 2 3. Experimental Methods 3.1 Sample Preparation 3.1.1 De-abundance treatment of plasma samples This part was performed strictly in accordance with the instructions for the high-depth blood proteomics sample preparation kit.

[0039] (1) Protein preparation ① Remove the plasma sample from the -80℃ freezer and place it at room temperature; ② Mix the nano-magnetic beads by pipetting, transfer 50 μL to a new EP tube, place it on a magnetic rack until clear, remove the preservation solution, add 200 μL of Resuspension Buffer to resuspend the nano-magnetic beads, mix by pipetting, and sonicate in a water bath for 10 min. Place the EP tube on a magnetic rack and let it stand for 3-5 min, discard the supernatant, and repeat once; ③ Add 100 μL of Resuspension Buffer to the magnetic beads, resuspend the magnetic beads, sonicate in a water bath for 10 min, then add 100 μL of plasma, mix well by pipetting, and incubate at 37°C for 1 h at 1000 rpm. ④ Instantaneous separation (rotation speed not exceeding 1000 rpm): Place the tube on a magnetic rack for 3–5 minutes and remove the supernatant. Add 500 μL of washing buffer, mix by pipetting, and vortex at 1000 rpm for 5 minutes. Use a magnetic rack to assist in discarding the supernatant, and repeat the washing process twice more. (2) Proteolytic enzyme digestion: ① Mix the Lysis Buffer thoroughly, then add 50 μL of Lysis Buffer to the magnetic bead precipitate; ②Incubate in a metal bath at 95℃ and 1000rpm for 10 minutes; ③ Allow the sample to cool naturally to room temperature, then briefly centrifuge (rotation speed not exceeding 1000 rpm). Add 2.5 μL of Digestion Buffer, incubate in a metal bath at 37°C, 1000 rpm, for 2 hours. ④ Immediately centrifuge the sample, add 5 μL of Stop Buffer, and mix well. This will terminate the enzymatic reaction, producing a large amount of precipitate. Centrifuge at 16000g for 5 minutes, then aspirate the supernatant for subsequent desalting.

[0040] 3.1.2 Urine protein extraction and pretreatment Some of the reagents used in this process came from the EasyPeptide Micro pretreatment kit, as shown in Table 3.

[0041] Table 3 ① Take the urine sample out of the -80℃ freezer, warm it in a 37℃ water bath, vortex it evenly, centrifuge it at 5000g for 10min, take 500μL of urine supernatant, add 3 times the volume of pre-cooled acetone, vortex it evenly, and place it in the -20℃ freezer to precipitate overnight. ② Centrifuge at 14000g for 15 minutes, discard the acetone, and allow the precipitate to evaporate in a ventilated place until there is no acetone odor. Add 30μL of 20mM Tris buffer (AMRESCO, C4H) 11 NO3 (purity ≥99%), after dissolving, add 1.2 μL of reagent B and mix well; ③ Heat in a 95℃ metal bath for 5 minutes. After returning to room temperature, add 2μL of reagent C and 5μL of reagent D respectively, mix well, and incubate overnight at 37℃ for enzymatic hydrolysis. ④ After the enzymatic hydrolysis is complete, add 3 μL of reagent E, mix well, terminate the enzymatic hydrolysis, centrifuge at 14000g for 5 min, and then aspirate the supernatant for subsequent desalting.

[0042] 3.1.3 C 18 solid phase extraction ①C 18 Solid-phase extraction column activation: Pipette 100 µL of 100% ACN to activate the extraction column; ②C 18Solid-phase extraction column equilibration: Pipette 100 µL of 50% ACN 0.1% FA to wash away any residual ACN in the extraction column; ③C 18 Solid-phase extraction column equilibration: Pipette 100 µL of 2% ACN 0.1% FA and equilibrate the extraction column; ④ Sample loading: Pipette the enzyme-digested peptide solution into the extraction column and repeat 3 times; ⑤ Washing and desalting: Take 100 µL of 2% ACN 0.1% FA to wash away the salt in the sample, repeat twice; ⑥ Elution: Take 50 µL of 50% ACN 0.1% FA, repeat twice, and collect the eluent using an EP tube; ⑦ Place the collected eluent in a rotary vacuum dryer, and after vacuum drying without heating, store it in a -80℃ refrigerator for later use.

[0043] 3.2 LC-MS / MS Analysis 3.2.1 Liquid Chromatography Analysis ① Redissolve the polypeptide component in 10 µL of 0.1% FA solution; ② After passing through U3000 liquid chromatography, nanoViper C 18 chromatographic column ( Separation was performed using a 0.1% FA aqueous solution in phase A and an 80% acetonitrile and 0.1% FA aqueous solution in phase B. The effective elution gradient was 1%–35%, the total elution time was 90 min, and the flow rate was 1.2 µL / min. The liquid phase conditions were as follows: Table 4 Low pH reversed-phase chromatography gradient 3.2.2 Mass Spectrometry Analysis The isolated peptides were injected into an NSI ion source (2.1 kV) for ionization and then identified using a Q-Exactive HF mass spectrometer.

[0044] A spectral library was established in DDA mode. MS parameters were set as follows: full scan at a resolution of 60,000 m / z in the range of 350–1500 m / z; cycle time of 3 s; automatic gain control (AGC) of 1e6; injection time <50 ms; and HCD collision energy of 32%. Precursors were screened for charge states using a 30-second dynamic exclusion duration and +2 to +6 charge states. Precursor separation was performed using a quadrupole with a 1.6 m / z separation window, with the strongest ion in each measurement scan used for fragmentation. The resulting fragments were analyzed using a Q-Exactive HF analyzer. The number of precursor ions in each isolation window was balanced based on the m / z distribution of precursor ions in the mixed sample.

[0045] For DIA analysis, to maintain retention time stability, the LC settings were the same as in the DDA experiments. Additionally, an iRT kit was added to all samples to correct for retention times between samples. MS parameters were set as follows: full scan at 120,000 resolution in the range of 350–1500 m / z; cycle time of 3 s; AGC of 3e6; injection time <100 ms; and HCD collision energy of 32%. Precursors were selected for charge state screening with a dynamic exclusion duration of 10 seconds and +2 to +6 charge states.

[0046] 3.3 Data Analysis 3.3.1 DIA Data Analysis Direct-DIA database search was performed using Spectronaut Pulsar X software (Biognosys, AG, Schlieren, Switzerland) with default settings. The database was: uniprot-human-81803-20230327.fasta. The optimal XIC extraction window was determined based on the iRT calibration strategy. The quality tolerance strategy was set to dynamic based on large-scale quality calibration. Cross-normalization was set to local normalization based on local regression. The total peak area of ​​fragment ions in MS2 was used to quantify peptide intensity. Search parameters included: protein database of the corresponding species, trypsin digestion, a maximum of two missed cleavage sites, precursor ion and fragment ion quality errors of 10 ppm and 0.02 Da, respectively, fixed modification as Carbamidomethyl (C), and variable modifications as Oxidation (M) and Acetyl (N-terminal). The false discovery rate (FDR) for peptides and proteins was <1.0%, and at least one specific peptide was identified for each protein.

[0047] The missing protein values ​​were filled using the KNN nearest neighbor method on the Wukong Cloud Platform (https: / / www.omicsolution.com / wkomics / main / ).

[0048] 3.3.2 Bioinformatics Analysis Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using SIMCA software (version 14.1, Umetrics, Sweden) to visualize the grouping.

[0049] The ratio of the means of all quantitative values ​​for each protein in the comparison sample pair was defined as the fold change (FC). All protein quantitative values ​​were log2 transformed, and a t-test was used to assess the significance of protein expression between groups. The corresponding P-value was used as the significance index. The criteria for screening differentially expressed proteins (DEPs) were: FC ≥ 1.5 or FC ≤ 1 / 1.5, P < 0.05.

[0050] 3.3.3 Correlation analysis between differentially expressed proteins and clinical indicators Correlation analysis was performed between the quantitative values ​​of DEPs and the percentage change in patients' VASI scores. The percentage change in VASI scores was calculated using the formula: (Post-treatment VASI score - Pre-treatment VASI score) / Pre-treatment VASI score. The criteria for screening correlated proteins were: correlation coefficient r > 0.4, P < 0.05. The predictive ability of target proteins was assessed using ROC curve analysis. Correlation analysis, graphing, and ROC curve plotting were all performed using R language (version 4.2.2).

[0051] II. Results of the Biomarker Screening Phase 1. Plasma sample like Figure 1A and Figure 1B As shown, Figure 1A Volcano plot of differentially expressed plasma proteins before and after treatment. Figure 1A The graph uses log2 (Fold Change) as the x-axis (representing the fold change in protein expression, with positive values ​​indicating upregulation and negative values ​​indicating downregulation), and -log... 10 The ordinate (P-value) represents the significance of the difference; a larger value indicates higher significance. This plot shows the distribution of differentially expressed proteins in 32 plasma samples (16 before treatment and 16 after treatment) from 16 patients with progressive non-segmental vitiligo, analyzed by DIA tandem mass spectrometry. The results identified 370 differentially expressed proteins, of which 86 were upregulated (red dots on the right side of the figure), 284 were downregulated (blue dots on the left side of the figure), and proteins with no significant difference were represented by gray dots. The dashed line in the figure represents the differential protein screening threshold (FC≥1.5 or FC≤1 / 1.5, P<0.05), used to define significantly differentially expressed proteins.

[0052] Figure 1B This is a plasma differential protein volcano plot showing the results before and after treatment in the rapid-response group. The coordinates of this plot are defined as follows: Figure 1AConsistent with the results, this paper presents the distribution of differentially expressed proteins in 16 plasma samples (8 before treatment and 8 after treatment) from 8 patients in the rapid response group, analyzed by DIA tandem mass spectrometry. The results showed that a total of 465 differentially expressed proteins were identified, of which 139 proteins were upregulated (red dots on the right side of the figure), 326 proteins were downregulated (blue dots on the left side of the figure), and proteins with no significant difference were represented by gray dots. The dashed line represents the differential protein screening threshold (FC≥1.5 or FC≤1 / 1.5, P<0.05), which can visually distinguish significantly different proteins from non-differential proteins.

[0053] The union of the differentially expressed proteins between the two groups was taken. A total of 581 differentially expressed proteins were identified in the plasma of the combined treatment group before and after treatment. Plasma biomarkers that can be used to evaluate the efficacy of the combined treatment group will be screened from these proteins in the future.

[0054] Correlation analysis was performed on the percentage changes in the quantitative values ​​of all differentially expressed proteins before and after treatment and the percentage changes in VASI scores of 16 patients. After removing 109 duplicate proteins, 187 differentially expressed proteins correlated with the percentage changes in VASI scores were identified. Further literature review, combined with the biological functions and AUC values ​​of the differentially expressed proteins, selected P-selectin (SELP), apolipoprotein A-IV (APOA4), and mannan-binding lectin serine protease 1 (MASP1) as candidate plasma biomarkers for efficacy assessment. SELP and MASP1 were downregulated after treatment, with correlation coefficients of 0.58 and 0.66, respectively, while APOA4 was upregulated after treatment, with a correlation coefficient of -0.59. The AUC values ​​of the three proteins were 0.715, 0.648, and 0.750, respectively.

[0055] like Figures 2A to 2F As shown, this is a correlation analysis and ROC curve of candidate biomarkers for plasma therapy assessment with clinical indicators. Figure 2AThe correlation between the percentage change in SELP quantitative values ​​and the percentage change in patients' VASI scores was analyzed. The graph uses "percentage change in SELP quantitative values" as the x-axis (representing the change in SELP protein expression after treatment compared to before treatment) and "percentage change in patients' VASI scores" as the y-axis (representing the change in vitiligo area score index after treatment compared to before treatment, calculated as: (VASI score after treatment - VASI score before treatment) / VASI score before treatment). A scatter plot was used to illustrate the correlation between the two. The results showed a positive correlation between the percentage change in SELP quantitative values ​​and the percentage change in VASI scores, with a correlation coefficient r = 0.58 and P = 0.017. This indicates that changes in SELP expression are significantly associated with the degree of improvement in patients' skin lesions and can serve as a potential indicator for evaluating treatment efficacy.

[0056] Figure 2B The figure shows the SELP ROC curve; the area under the curve (AUC) is 0.715, and the 95% confidence interval (95% CI) is 0.529–0.900, indicating that SELP has moderate assessment efficacy when used alone and can effectively distinguish between treatment-sensitive and treatment-insensitive patients.

[0057] Figure 2C Correlation analysis was performed between the percentage change in MASP1 quantitative values ​​and the percentage change in patients' VASI scores; the figure shows the correlation between the percentage change in MASP1 quantitative values ​​and the percentage change in VASI scores. The results showed a positive correlation between the two, with a correlation coefficient r = 0.66 and P = 0.0053, indicating that changes in MASP1 expression are more closely associated with patients' treatment response and are a better potential indicator for assessing efficacy.

[0058] Figure 2D The ROC curve for MASP1 is shown below; the area under the curve (AUC) is 0.648, and the 95% confidence interval (95% CI) is 0.450–0.847, indicating that MASP1 has certain evaluative efficacy when used alone and can help determine the patient's treatment sensitivity.

[0059] Figure 2E Correlation analysis was performed between the percentage change in APOA4 quantitative values ​​and the percentage change in patients' VASI scores; the figure shows the correlation between the percentage change in APOA4 quantitative values ​​and the percentage change in VASI scores. The results showed a negative correlation between the two, with a correlation coefficient r = -0.59 and P = 0.016, indicating that when APOA4 expression was upregulated after treatment, the decrease in patients' VASI scores was more significant (more obvious improvement in skin lesions), which is the opposite of the trend of SELP and MASP1 expression changes, but can still reflect the treatment effect.

[0060] Figure 2FThe APOA4 ROC curve is shown. The area under the curve (AUC) is 0.750, and the 95% confidence interval (95% CI) is 0.568–0.932. It has the highest efficacy among the three plasma biomarkers for individual assessment and can more accurately identify treatment-sensitive patients.

[0061] 2. Urine sample like Figure 3A As shown in the figure, this figure illustrates the distribution of differentially expressed proteins in 28 urine samples (14 before treatment and 14 after treatment) from 14 patients with progressive non-segmental vitiligo, analyzed by DIA tandem mass spectrometry. The results identified 132 differentially expressed proteins, of which 108 were upregulated (red dots on the right side of the figure), 24 were downregulated (blue dots on the left side of the figure), and proteins with no significant difference were represented by gray dots. The dashed line represents the differential protein screening threshold (FC≥1.5 or FC≤1 / 1.5, P<0.05), clearly defining the range of significantly differentially expressed proteins.

[0062] Figure 3B This is a volcano plot showing the differentially expressed proteins in urine before and after treatment in the rapid-response group. The plot illustrates the distribution of differentially expressed proteins in 14 urine samples (7 before and 7 after treatment) from 7 patients in the rapid-response group, analyzed by DIA tandem mass spectrometry. The results showed that 47 differentially expressed proteins were identified, with 30 proteins upregulated (red dots on the right side of the plot) and 17 proteins downregulated (blue dots on the left side of the plot). Proteins with no significant difference are represented by gray dots. The dashed line represents the differential protein screening threshold (FC≥1.5 or FC≤1 / 1.5, P<0.05), allowing for a direct observation of the differential changes in urine proteins before and after treatment in the rapid-response group.

[0063] Further literature review was conducted on the differentially expressed urinary proteins identified before and after treatment. Based on the biological functions and AUC values ​​of these proteins, immunoglobulin J chain (IGJ) and interleukin-18-binding protein (IL-18BP) were selected as candidate urinary biomarkers for efficacy assessment. Figure 4A and Figure 4B As shown, Figure 4A IGJ ROC curve for candidate biomarkers of urinary efficacy assessment; Figure 4B ROC curve of IL-18BP, a candidate biomarker for evaluating the efficacy of urinary therapy. Figure 4AThe area under the ROC curve (AUC) of IGJ was 0.668, and the 95% confidence interval (95% CI) was 0.457–0.880, indicating that IGJ has certain assessment efficacy when used alone and can help determine the sensitivity of patients to combination therapy. Figure 4B The ROC curve for IL-18BP shows an area under the curve (AUC) of 0.699 and a 95% confidence interval (95% CI) of 0.485–0.913. Its assessment efficacy is slightly higher than that of IGJ, making it a superior potential biomarker in urine.

[0064] Example 2: Validation phase of biomarkers obtained from Example 1 I. Methods for the Biomarker Validation Phase 1. Research Subjects This section includes 16 patients from the combination therapy group in Part I, and 14 newly enrolled patients with progressive non-segmental vitiligo who visited the Dermatology Clinic of Peking Union Medical College Hospital. All 14 patients and the 16 patients in the selection group received the combination therapy regimen, namely oral baricitinib 2 mg / day combined with NB-UVB three times a week. The 30 patients included 22 males and 8 females, with a mean age of 33.5 ± 8.3 years, a mean disease duration of 10.6 ± 8.1 years, and a mean pre-treatment VASI score of 12.6 ± 9.4.

[0065] 2. Main experimental reagents and equipment 2.1 The main experimental instruments are shown in Table 5.

[0066] Table 5 2.2 Main reagents and consumables are listed in Table 6.

[0067] Table 6 3. Experimental Methods 3.1 Sample and Reagent Preparation ① Sample preparation: Remove plasma and urine samples from the -80℃ freezer, thaw on ice, centrifuge at 6000-10000 rpm for 1.5 min, and dilute the samples to be tested to the corresponding concentration according to the preliminary experimental results.

[0068] Table 7 ② Serial dilution of standard: Take one standard from the kit and centrifuge at 6000–10000 rpm for 30 seconds. Dissolve it with 1 ml of sample diluent, and repeatedly pipette the solution from the bottom of the cryovial to aid dissolution. Mix thoroughly to obtain standard S7, and set aside for later use. Arrange seven 1.5 ml centrifuge tubes (S0-S6) sequentially, and add 250 µl of sample diluent to each. Transfer 250 µl of standard S7 to the first centrifuge tube (S6) and gently pipette to mix. Transfer 250 µl of standard S7 to the second EP tube (S5) and gently pipette to mix. Continue this serial dilution process for the standards. S0 is the sample diluent. ③ Dilute the concentrated washing solution with deionized water at a ratio of 1:25; ④ Dilute the biotin-labeled antibody solution with biotin-labeled antibody diluent at a ratio of 1:100, and prepare the solution within 10 minutes before use; ⑤ Dilute horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin dilution solution at a ratio of 1:100, and prepare the solution within 10 minutes before use. 3.2 Experimental Procedure The ELISA kit used in this section employs a double-antibody sandwich method. A specific antibody is coated onto the carrier surface, and the plasma or urine sample to be tested, which may contain the corresponding antigen, is added. After incubation and washing, the sample is incubated again with enzyme-labeled specific antibody. The coating antibody, the antigen to be tested, and the enzyme-labeled antibody form a sandwich complex. Unbound material is washed away, substrate is added for color development, and the antigen is quantitatively detected based on the presence or intensity of the color.

[0069] ① Move all reagents to room temperature (18-25℃) and equilibrate for at least 30 minutes to prepare the relevant reagents for later use; ② Sample addition: Set up separate wells for standard and test samples. Add 100 μL of standard or test sample to each well, gently shake to mix, cover with a plate label, and incubate at 37°C for 2 hours; ③ Discard the liquid, spin dry, no washing required; ④ Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour; ⑤ Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200 μL / well, then spin dry; ⑥ Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour; ⑦ Discard the liquid in the wells, spin dry, and wash the plate 5 times. Each time, soak for 2 minutes, 200 μL / well, then spin dry; ⑧ Add 90 μL of substrate solution to each well in sequence and develop color at 37°C in the dark for 15–30 minutes; ⑨ Add 50 μL of the stop solution to each well sequentially to terminate the reaction; ⑩ Within 5 minutes after the reaction is terminated, use an ELISA reader to measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm.

[0070] 3.3 Data Analysis Plotting the standard concentration on the ordinate and the OD value on the x-axis, a standard curve regression equation was calculated using Curve Expert software (version 1.4, Cusabio Technology, Wuhan, China). The OD values ​​of the samples were then substituted into the equation to calculate the concentration of each sample. If the samples were diluted before testing, they need to be multiplied by the corresponding dilution factor.

[0071] For comparing differences between groups before and after treatment, paired t-tests were used if the differences were normally or approximately normally distributed; paired Wilcoxon signed-rank tests were used if the differences were severely skewed. For comparing differences between the fast-response and slow-response groups before treatment, t-tests were used if the data were normally distributed and homogeneous in variance; Wilcoxon Mann-Whitney tests were used if the data were not normally distributed or had unequal variances. A p-value < 0.05 was considered statistically significant. Graphpad Prism software (version 9.0, San Diego, CA, USA) was used for data analysis and graphing.

[0072] The predictive ability of the target protein was determined by ROC curve analysis, and the ROC curve was plotted using R language (version 4.2.2).

[0073] II. Biomarker Validation Phase 1. Plasma sample In the validation cohort, the levels of SELP, APOA4, and MASP1 in plasma samples from enrolled patients before and after treatment were measured. Figure 5A , Figure 5B and Figure 5C The images show the plasma levels of SELP, APOA4, and MASP1 before and after treatment with baricitinib combined with NB-UVB, as determined by ELISA. , .

[0074] The results showed that Figure 5A This image shows a comparison of plasma SELP levels before and after treatment with baricitinib combined with NB-UVB, measured by ELISA. The results indicate that plasma SELP levels after treatment were significantly lower than before treatment, with statistical significance. The results were consistent with the mass spectrometry screening results, which identified SELP as a “post-treatment downregulated protein,” thus validating the reliability of SELP as a biomarker for efficacy assessment.

[0075] Figure 5B The comparison of plasma APOA4 levels before and after treatment with baricitinib combined with NB-UVB using ELISA showed that plasma APOA4 levels were significantly higher after treatment than before treatment, with statistical significance. This is consistent with the results of mass spectrometry screening, which identified APOA4 as a "protein upregulated after treatment," further validating the evaluative value of APOA4.

[0076] Figure 5C This graph shows a comparison of plasma MASP1 levels before and after treatment with baricitinib combined with NB-UVB using an ELISA method. The graph indicates that plasma MASP1 levels were significantly lower after treatment than before treatment, with statistical significance. This is consistent with the results of mass spectrometry screening, which identified MASP1 as a "post-treatment downregulated protein," confirming the effectiveness of MASP1.

[0077] The above results indicate that decreased levels of SELP and MASP1 and increased levels of APOA4 in the patient's plasma can be used to evaluate the therapeutic effect of baricitinib combined with NB-UVB therapy during treatment.

[0078] 2. Urine sample In the validation cohort, the levels of IGJ and IL-18BP in urine samples from enrolled patients before and after treatment were measured. Results are shown below. Figure 6A and Figure 6B . Figure 6A and Figure 6B The image shows the levels of IGJ and IL-18BP in urine before and after treatment with baricitinib combined with NB-UVB using ELISA. .

[0079] Figure 6A This image shows a comparison of urinary IGJ levels before and after treatment with baricitinib combined with NB-UVB using an ELISA method. The results showed that urinary IGJ levels were significantly lower after treatment than before treatment, with statistical significance. This is consistent with the results of IGJ being identified as "post-treatment downregulated protein" in the mass spectrometry screening, thus validating the assessment efficacy of IGJ.

[0080] Figure 6B The image shows a comparison of urinary IL-18BP levels before and after treatment with baricitinib combined with NB-UVB using ELISA. The results showed that urinary IL-18BP levels were significantly lower after treatment than before treatment, with statistical significance. This is consistent with the results of mass spectrometry screening, which identified IL-18BP as a "post-treatment downregulated protein," further confirming the reliability of IL-18BP.

[0081] The above results indicate that reduced levels of IGJ and IL-18BP in patients' urine can be used to evaluate the therapeutic effect of baricitinib combined with NB-UVB therapy during treatment.

[0082] 3. Combined analysis of plasma and urine samples Further analysis was performed using ROC curves to jointly analyze three differentially expressed proteins (SELP, APOA4, and MASP1) validated in plasma and two differentially expressed proteins (IGJ and IL-18BP) validated in urine. The predictive ability of the target proteins was determined by ROC curve analysis using a logistic regression model. The ROC curves were plotted using R language (version 4.2.2).

[0083] Analysis revealed that, compared to single differentially expressed proteins, the combination of multiple biomarkers showed a higher AUC value (AUC = 0.807), indicating a better assessment of the treatment efficacy in patients treated with baricitinib combined with NB-UVB. Results are as follows... Figure 7 As shown. Figure 7 The ROC curve of the combined plasma and urinary biomarkers for evaluating the efficacy of treatment is shown. The area under the curve (AUC) was 0.807, significantly higher than the AUC values ​​of the five biomarkers used alone (SELP: 0.715, APOA4: 0.750, MASP1: 0.648, IGJ: 0.668, IL-18BP: 0.699), indicating that the combined biomarker group has higher assessment accuracy and can more accurately evaluate the treatment effect of baricitinib combined with NB-UVB before and after treatment.

Claims

1. An application of a substance for detecting a group of markers, characterized in that... Including one or more of the following applications: A1) Use in the preparation of products for evaluating the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients; A2) Application in the preparation of products for detecting the sensitivity of vitiligo patients to baricitinib combined with narrowband UVB therapy; A3) Application in the preparation of products for screening the sensitivity of vitiligo to baricitinib combined with narrow-band UVB therapy; A4) Application in the preparation of products for dynamically monitoring the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients; The biomarker group includes plasma biomarkers and urine biomarkers; the plasma biomarkers are P-selectin, apolipoprotein A4, and mannan-binding lectin-associated serine protease 1; the urine biomarkers are immunoglobulin J chain and interleukin-18 binding protein.

2. The application as described in claim 1, characterized in that: The substance is a reagent for detecting the expression level of a biomarker, a reagent for specifically recognizing and binding to a biomarker, or a reagent for detecting the content of a biomarker.

3. The application as described in claim 2, characterized in that: The substance is selected from mass spectrometry identification reagents, monoclonal antibodies, polyclonal antibodies, or antigen-binding fragments of antibodies.

4. The application as described in claim 1, characterized in that... The substance used to detect the group of markers is any one of the following a), b), or c): a) Reagents for detecting the expression levels of the biomarker group; b) A reagent group containing the reagents described in a); c) A kit containing either the reagents of a) or the reagent group of b).

5. The application as described in any one of claims 1 to 4, characterized in that: The detection samples for the biomarkers P selectin, apolipoprotein A4, and mannan-binding lectin-associated serine protease 1 were plasma, while the detection samples for the biomarkers immunoglobulin J chain and interleukin-18 binding protein were urine.

6. A reagent kit for detecting a group of biomarkers, characterized in that... Including one or more of the following applications: A1) Use in the preparation of products for evaluating the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients; A2) Application in the preparation of products for detecting the sensitivity of vitiligo patients to baricitinib combined with narrowband UVB therapy; A3) Application in the preparation of products for screening the sensitivity of vitiligo to baricitinib combined with narrow-band UVB therapy; A4) Application in the preparation of products for dynamically monitoring the efficacy of baricitinib combined with narrowband UVB therapy in vitiligo patients; The kit includes substances for detecting a group of biomarkers, including plasma biomarkers and urine biomarkers; the plasma biomarkers are P-selectin, apolipoprotein A4, and mannan-binding lectin-associated serine protease 1; the urine biomarkers are immunoglobulin J chain and interleukin-18 binding protein.

7. The kit as described in claim 6, characterized in that: The kit contains reagents for detecting the expression levels of the biomarkers, including specific antibodies against P-selectin, specific antibodies against apolipoprotein A4, specific antibodies against mannan-binding lectin-associated serine protease 1, specific antibodies against immunoglobulin J chain, and specific antibodies against interleukin-18 binding protein.

8. The kit according to claim 7, characterized in that: The kit also contains at least three of the following: sample processing reagents, standards, quality control products, diluents, washing solutions, enzyme-labeled antibodies, substrate solutions, and stop solutions.

9. A method for evaluating the sensitivity of patients with progressive non-segmental vitiligo to baricitinib combined with narrowband UVB therapy, as a non-disease diagnostic and treatment method, characterized in that... Includes the following steps: (1) Biomarker detection: Using the kit described in claim 6, 7 or 8, the expression levels of P-selectin, apolipoprotein A4 in plasma samples before and after treatment, and the expression levels of immunoglobulin J chain and interleukin-18 binding protein in urine samples before and after treatment were detected respectively. (2) Result determination: If the expression levels of P-selectin and mannan-binding lectin-associated serine protease 1 in plasma are significantly downregulated after treatment compared with those before treatment (P < 0.05), the expression level of apolipoprotein A4 in plasma is significantly upregulated compared with those before treatment (P < 0.05), and the expression levels of immunoglobulin J chain and interleukin-18 binding protein in urine are significantly downregulated compared with those before treatment (P < 0.05), then the patient is determined to have high sensitivity to baricitinib combined with narrowband UVB therapy.

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