Evaluation of non-responsiveness in IBD patients

Abstract

Non-responsiveness assessment in IBD patients The present invention relates to an in vitro method for predicting the response of patients with inflammatory bowel disease (IBD) to treatment with intracellular immunosuppressants. In this method, samples are collected from IBD patients in the early stages of treatment with an immunosuppressant of interest, and these samples contain effector mononuclear cells. Responsiveness is predicted based on the difference between the activity level of the multidrug-resistant ABC transporter and the activity level of the reference transporter in effector mononuclear cells in the sample. This method is useful in the treatment of IBD and can be used, for example, to monitor disease progression or to determine whether to switch from the initial treatment to another medication (e.g., csDMARD or tsDMARD).

Description

[Technical Field]

[0001] The inventors have found that the responsiveness of IBD patients to intracellular immunosuppressants can be evaluated by measuring ABC transporter activity levels, preferably transport activity levels.

[0002] This invention relates to a method for predicting the response of IBD patients to treatment with intracellular immunosuppressants. The method is performed using biological samples collected from IBD patients before treatment with the immunosuppressant in question, or within four months after the start of treatment. The samples contain effector mononuclear cells, and the patient's treatment response is predicted by measuring the transport activity by multidrug-resistant ABC transporters. [Background technology]

[0003] inflammatory bowel disease Crohn's disease (CD) and ulcerative colitis (UC) are collectively known as inflammatory bowel disease (IBD), a group of chronic diseases characterized by relapses and remissions, which place a lifelong burden on a large portion of the growing population. In the early 21st century, the incidence of inflammatory bowel disease accelerated even in newly industrialized countries, becoming a global disease in increasingly Westernized societies. Although the incidence rate has stabilized in Western countries, the prevalence rate remains above 0.3%, resulting in a significant healthcare burden. Medical expenses after patients develop CD or UC are also high [Ng SC et al., 2017]. CD can affect almost the entire gastrointestinal tract and multiple layers of tissue, with the terminal ileum and colon being the most frequent sites. On the other hand, UC tends to be localized mainly to the superficial mucosa of the colon, especially the part including the proximal end of the rectum [Torres, J et al., 2017; Ungaro, R et al., 2017; D'Haens G et al., 2008].

[0004] Although the etiology of IBD remains unclear despite intensive research, an inappropriate mucosal immune response to the gut microbiota is widely recognized as a major causative phenomenon [Xu XR et al., 2014; Lamas B et al., 2016]. Despite differing clinical presentations, the two major classifications of IBD, UC and CD, share common characteristics such as infiltration of inflammatory effector cells into the intestinal wall, elevated inflammatory biomarkers in the blood and stool, and a pattern of persistent intestinal mucosal inflammation.

[0005] Both the innate and adaptive immune systems play crucial roles in intestinal inflammation. T lymphocytes, comprised of diversely differentiated T cell subsets, play a major role in both the regulation and effector phases of the immune response. Several innate immune cells have been shown to contribute to the pathogenesis of IBD. Neutrophils perpetuate colitis through impaired epithelial barrier function and the release of multiple inflammatory mediators [de Souza HS, and Fiocchi C, 2016]. Dendritic cells (DCs) regulate crosstalk between innate and adaptive immunity and maintain homeostasis [Bani Ahluwalia et al., 2018].

[0006] Numerous studies have investigated differences in immune effector mechanisms between the two subtypes of IBD. The neutrophil response is prominent in UC [Wysoczanski R et al., 2019], while in CD, dysfunction is observed, including reduced neutrophil migration, superoxide production, and phagocytic function [Hayee B et al., 2011; Segal AW, 2019]. Furthermore, there is ample evidence that defects in mucosal immunomodulation, including abnormal changes in T cells, B cells, granulocytes, macrophages, and the cytokines and chemokines produced by these cells, play a major role in the pathogenesis of UC [Kaur A et al., 2020].

[0007] The diversity of treatments and pharmacological mechanisms of action indicates the heterogeneity of the disease and the existence of multiple pathways involved in molecular and cellular pathology [Bani Ahluwalia et al., 2018].

[0008] Biomarkers in IBD Early diagnosis and immediate, effective treatment are crucial to preventing disease progression, functional impairment, and a decline in quality of life. Despite extensive research, there is currently no ideal serum biomarker for IBD. Research into serum profiling and non-coding RNAs is finally beginning to bear fruit, showing great potential for future clinical applications.

[0009] In recent years, serum biomarkers have become a major focus of IBD research and have made significant progress due to their non-invasive nature, convenience, and relatively low cost compared to biopsy tissue, stool, breath, and other fluid markers [Chen P et al., 2020]. Appropriate monitoring is essential for identifying disease relapses and providing timely treatment. Treatment should be individualized and adjusted throughout the disease course based on disease severity, extent of lesions, clinical behavior of the disease, and response to medication. The response to treatment needs to be closely monitored to assess effectiveness and prevent disease complications.

[0010] Biomarkers for IBD have been identified in colorectal tissue, blood, stool, urine, and breath. Blood-derived biomarkers are non-invasive, easy to collect, have a low risk of contamination, and are the most widely used. An ideal biomarker would be non-invasive, highly sensitive, disease-specific, easy to administer, and cost-effective [Vermeire S., 2006]. However, an ideal biomarker that possesses all these characteristics and can accurately diagnose IBD, differentiate IBD subtypes, and monitor disease activity does not currently exist.

[0011] Current Treatment Options for IBD Existing IBD treatment algorithms rely on sequential treatment, where different therapeutic classes of drugs are applied stepwise if initial treatment fails. As a result, many patients receive expensive and ineffective treatments over extended periods. Achieving sustainable global IBD care requires the ability to more precisely match patients with treatments, avoid unnecessary drug use, and provide cost-effective care. Research has consistently shown that immediate and effective treatment in the early stages of the disease is more effective than step-up treatment [D'Haens G et al., 2008].

[0012] Current treatment approaches focus on suppressing abnormal intestinal inflammation and immune responses. In recent years, treatment options for IBD have expanded, and traditional treatment strategies aim to achieve the best possible quality of life for patients by inducing and maintaining clinical remission, and by focusing on mucosal healing and prevention of long-term complications [Bani Ahluwalia et al., 2008].

[0013] Traditional treatment for IBD (inflammation of ulcerative colitis) primarily involves drug therapy, including aminosalicylic acid preparations, corticosteroids (CSs), immunomodulators, and biological agents, combined with general therapeutic measures and surgical resection as needed to control symptoms. Advances in treatment, particularly the emergence of biological agents, have revolutionized the paradigm of IBD treatment and completely changed the way we view it. While treatment effectiveness was previously evaluated mainly by clinical symptom scores, now, based on a "treat-to-target" approach with clearly defined endpoints, disease activity can be assessed using objective indicators such as endoscopic findings and biomarkers [Nakase H et al., 2021]. Treatment goals include inducing and maintaining symptom remission, preventing and treating complications, and achieving mucosal and histological healing.

[0014] Despite the existence of advanced treatment options, primary inefficacy, loss of efficacy, or the emergence of side effects affect more than 50% of all patients, indicating the need for additional treatment methods. Small-Molecule Drugs (SMD) are one of the most promising new approaches in IBD treatment development. SMD have the advantages of a shorter half-life, a lower immunogenic risk compared to biologics, and the ability to be administered orally, thus significantly improving patient medication compliance and quality of life.

[0015] Despite the availability of multiple drugs for IBD treatment, many patients do not respond to treatment or lose their response. Approximately 30% of CD patients do not respond to Infliximab or Adalimumab (both anti-TNF antibodies), and the annual risk of losing response to Infliximab is approximately 13% per patient [Adegbola S.O. et al., 2018].

[0016] Recent advances in novel IBD treatments have provided new treatment options for these patients. Gut-specific α4β7 integrin antibodies (Vedolizumab, Etrolizumab) and IL-12 / IL-23 inhibitors (Ustekinumab) have shown efficacy in patients with refractory IBD or those who have lost response to anti-TNF treatment [Hazel K. and O'Connor A., 2020; Liefferinckx C. et al., 2019]. Furthermore, as new treatments such as S1P receptor inhibitors (Ozanimod, Etrazimod) are clinically introduced, precision medicine in IBD treatment becomes an extremely important issue. This includes accurate patient stratification, the use of effective and reliable biomarkers, and the determination of the optimal clinical pathway for individual patients [Chen P. et al., 2020].

[0017] After biological agents have achieved success in the clinical management of IBD, research on alternative effective anti-cytokine therapies has been actively pursued. Tofacitinib (CP-690,550) is the first orally administered pan-Janus kinase (JAK) inhibitor and is known to be effective and safe for patients with moderate to severe UC [Weisshof R. et al., 2018].

[0018] Multidrug-resistant transporters in healthy individuals and in diseased individuals Multidrug resistance (MDR-ABC) transporters (MDR1 / P-gp / ABCB1, MRP1 / ABCC1, BCRP / ABCG2) are known to play important roles in the development of drug resistance in malignancies [Gottesman, M.M. et al., 2002] and autoimmune diseases such as rheumatoid arthritis (RA) [Marki-Zay, J. et al., 2013]. Major classical systemic disease-modifying anti-rheumatic drugs (csDMARDs; including Methotrexate (MTX), Sulfasalazine, Leflunomide, Hydroxychloroquine) are all substrates of MDR proteins.

[0019] Regarding IBD, research on multidrug resistance transporters is limited compared to RA. Existing studies mainly focus on genetic factors involved in IBD susceptibility [Petryszyn, P., 2021; Cao Y., 2015] and the role of transporter proteins in glucocorticoid resistance [De Iudicibus S., 2011; Farrell RJ., 2000]. Despite research into the role of MDR1 in the clinically observed multidrug resistance phenomenon with conventional chemotherapy and DMARD therapy in RA, there are few reports in IBD. Several csDMARDs, frequently used as first-line treatments for IBD, are known to be substrates of MDR1, and there is growing evidence supporting the transport of newly approved small molecular weight drugs (Jakinibs) by MDR1 or BCRP [European Medicines Agency Public Assessment Reports EMA / CHMP / 853224 / 2016, EMA / 13493 / 2017, EMA / 424374 / 2020, EMA / 608624 / 2019 Corr., EMA / 705612 / 2020, EMA / 647846 / 2021].

[0020] Cario, Elke [Cario, Elke, 2017] states that the multidrug-resistant transporter ABCB1 (also known as MDR1 or P-glycoprotein, P-gp) plays a crucial role in protecting the intestinal barrier from the accumulation of foreign substances. Altered ABCB1 expression may contribute to the development and persistence of chronic colitis in IBD. Decreased P-gp efflux activity may increase disease susceptibility and drug toxicity, while increased efflux activity may lead to decreased drug responsiveness and resistance to therapeutic agents in IBD. However, the potential of molecularly targeted therapies for MDR1 requires further investigation.

[0021] Drozdzik, Marek et al. [Drozdzik, Marek et al., 2020] demonstrated that gastrointestinal (e.g., IBD) and systemic pathological conditions may influence the expression and function of membrane transporters in the gastrointestinal tract. In the case of MDR1 (ABCB1), changes in P-gp levels may affect the outcome of IBD treatment. P-gp expression may be involved in modulating drug responses to 5-aminosalicylic acid (5-ASA), immunosuppressants, and corticosteroids. In patients who received 5-ASA and did not respond to the drug, a significant decrease in P-gp expression was observed in inflamed mucosa compared to non-inflammatory sites. Therefore, measurements were performed on samples derived from inflamed sites. Some glucocorticoids are known to be substrates of P-gp, and changes in their expression may affect drug responses. Another MDR transporter, BCRP (also known as ABCG2), has also been reported to potentially affect responses to 5-ASA, corticosteroids, and immunosuppressants. In patients who did not respond to 5-ASA, a significant decrease in BCRP expression was observed at the inflammatory site compared to the non-inflammatory site. In this review, transporter levels were primarily measured as mRNA expression levels.

[0022] Chen, Yan et al. (2022) compared the effects of aminosalicylic acids, glucocorticoids, and immunosuppressants on the expression levels of multidrug resistance-related genes in UC patients, aiming to provide a theoretical and therapeutic basis for the diagnosis, treatment, and prevention of UC. They quantified the mRNA expression levels of the MDR1 gene before and after treatment. Their results showed that 5-ASA administration did not correlate with MDR1 expression in UC, while glucocorticoid and immunosuppressant administration showed a positive correlation with the MDR1 expression profile. In patients who did not respond to glucocorticoids or immunosuppressants, the expression levels of MDR1 mRNA and its product P-gp were significantly elevated. Conversely, 5-ASA did not affect the expression levels of MDR1 and P-gp in UC patients. This result is noteworthy as it contradicts the report by Drozdzik et al. (2020). Chen et al. also suggested that the use of glucocorticoids and immunosuppressants may increase MDR1 expression. RNA expression was measured in colonic mucosal tissue specimens, endoscopic pathological mucosal tissue, or postoperative pathological biopsies (all samples derived from inflammatory sites).

[0023] Patent document WO2017 / 223409A1 discloses a method for monitoring MDR1 transporter function in human IBD patients, a method for patient stratification in bile acid-related therapy, and a treatment method integrating stratification and bile acid adsorption therapy. This method quantifies MDR1 activity in human T cells in a biological sample. T cells are CD4 + Effector / Memory Cell, CD8 + Naive cells, CD8 + Effector / Memory Cell, CD8 + Selected from short-lived effector / memory cells. Preferably, MDR1 function is MDR1 in the T cell subset. + It is evaluated by measuring cell frequency. Alternatively, in IBD patients, MDR1 is compared to healthy controls. + If a significant decrease in cell frequency is observed, the patient is deemed eligible for bile acid adsorption therapy. In other words, the detection of MDR1 function loss in a patient indicates eligibility for this therapy.

[0024] CN104535774 discloses a novel detection index for drug sensitivity in inflammatory bowel disease (IBD) and its application to the design of drug therapy. While elevated P-gp expression in peripheral blood lymphocytes may indicate drug resistance, this resistance can be reversed with P-gp-specific inhibitors. Furthermore, P-gp expression levels in peripheral blood lymphocytes can be used to assess drug resistance in IBD patients.

[0025] US2009258848A1 discloses a method for determining the inflammatory bowel disease (IBD) status in subjects and a method for identifying biomarkers for IBD diagnosis. According to this invention, approximately 400 biomarkers are suitable for determining the IBD status, one example being the ABCB1 gene, which is downexpressed in IBD patients. Despite the abundance of research in this field, there is still a lack of appropriate biomarkers for accurately diagnosing IBD, differentiating subtypes, and monitoring disease activity. Furthermore, much remains unclear about the role of MDR transporters in IBD diagnosis and whether they are suitable as predictive biomarkers for IBD treatment effectiveness.

[0026] The present inventors' method is useful as an in vitro diagnostic method for predicting non-responsiveness to immunosuppressants in IBD patients, and as an in vitro evaluation method for assessing the condition of IBD patients treated with immunosuppressants, thereby enabling support and implementation of IBD treatment. [Overview of the Initiative]

[0027] 1. The present invention relates to an in vitro method for predicting the response of IBD patients to treatment with intracellular immunosuppressants, and comprises the following steps: ● A step of providing a biological sample collected from an IBD patient during the initial stages of treatment with the target immunosuppressant (preferably before the start of treatment or within 4 months after the start of treatment). The sample comprises effector mononuclear cells (preferably lymphocytes and / or monocytes and / or NK cells) derived from the patient. ●A step of measuring the transport activity of multidrug ABC transporters (preferably selected from ABC transporters of group B, group C, and group G, more preferably selected from group B and group C, or group B and group G) in effector mononuclear cells in the sample, and obtaining the transporter activity level. ● A step of comparing the obtained transporter activity level with the reference transporter activity level. ● A step in determining that a patient is a **non-responder** to intracellular immunosuppressants if the measured transporter activity level is significantly different from the corresponding reference transporter activity level.

[0028] The present invention also relates to an in vitro method for predicting the response of IBD patients, comprising the above steps, and comprising the following: ● A process of collecting biological samples from IBD patients before treatment with the target immunosuppressant or within 4 months after the start of treatment, and providing the samples. The sample contains effector mononuclear cells (preferably lymphocytes and / or monocytes and / or NK cells). ●A step of measuring the transport activity by one or more multidrug-resistant ABC transporters (selected from group B, group C, and group G; preferably group B and group C, or group B and group G) in effector mononuclear cells in the sample, and obtaining **one or more transporter activity values ​​(transporter activity values)**. ● A step of comparing one or more obtained transporter activity values ​​with the corresponding **reference transporter activity values ​​(s)**. ● A step in determining a patient as a **non-responder** if one or more measured transporter activity values ​​differ significantly from the corresponding reference values.

[0029] In a preferred embodiment, this method is a diagnostic method. More preferably, the method is a diagnostic method that combines treatment with the target immunosuppressant. Furthermore, the method may also be a treatment method using an intracellular immunosuppressant, but only if the patient responds to the immunosuppressant. In another preferred embodiment, the process further includes administering treatment if the patient is not determined to be unresponsive to the immunosuppressant.

[0030] In a particularly preferred embodiment, the present invention relates to the process of sample collection: ●If the patient is receiving treatment with an intracellular immunosuppressant, or / and ●If the patient is **naive** to the immunosuppressant, This relates to an in vitro response prediction method applicable to this method.

[0031] In another preferred embodiment, the present invention provides an in vitro diagnostic method comprising the following steps: ● A process of preparing multiple biological samples collected from IBD patients at multiple time points in time, and providing each sample. ● A step of obtaining the activity values ​​of one or more transporters from each sample. ● A step of comparing each sample with the reference transporter activity value corresponding to the acquisition time. ● A step of determining that a patient is unresponsive to an intracellular immunosuppressant if, in at least one, preferably all, samples, the transporter activity value is significantly different from the reference value.

[0032] 2. The present invention also relates to the in vitro diagnostic method of paragraph 1, and is a method for evaluating the condition of an IBD patient who has previously been treated with an intracellular immunosuppressant, and predicting whether or not the patient is unresponsive to an intracellular immunosuppressant of interest, comprising the following steps: ● A step of providing a biological sample from the IBD patient, wherein the sample comprises effector mononuclear cells (preferably lymphocytes and / or monocytes and / or NK cells) derived from the patient. ●A step of measuring the transport activity by a multidrug-resistant ABC transporter (or one or more multidrug-resistant ABC transporters) in effector mononuclear cells in the sample and obtaining the transporter activity level (or the activity value of one or more transporters), wherein the transporter is selected from ABC transporter groups B, C, and G. ● A step of comparing the obtained transporter activity level (or one or more transporter activity values) with a reference transporter activity level (or one or more reference transporter activity values). ● A step of determining a patient to be unresponsive to intracellular immunosuppressant therapy of interest when the transporter activity level (or one or more transporter activity values) is significantly different from the corresponding reference transporter activity level (or one or more reference transporter activity values).

[0033] 3. Preferably, the present invention also relates to the in vitro method described in any of paragraphs 1 to 2, wherein the biological sample is a blood-derived sample.

[0034] 4. Preferably, the present invention also relates to the in vitro method described in paragraph 3, wherein the effector mononuclear cell is selected from the group consisting of lymphocytes, monocytes, and NK cells, preferably CD3+ T lymphocytes, CD14+ monocytes, CD16+ NK cells, or neutrophil granulocytes.

[0035] 5. In the in vitro method described in any of paragraphs 1 to 4, The aforementioned intracellular immunosuppressants are **small-molecule drugs** used in IBD, and are selected from the following: ● Glucocorticoids, optionally used in combination with conventional disease-modifying agents (DMARDs (cDMARDs)) ● Conventional disease-modifying drugs (DMARDs (cDMARDs)) (without concomitant use of glucocorticoids), ●Steroids, ●tsDMARD, Furthermore, the ABC transporter is selected from groups B and G, preferably ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (BCRP).

[0036] In embodiments, the steroid is a systemic steroid, preferably selected from **prednisolone, methylprednisolone, prednisone, and hydrocortisone**.

[0037] 6. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 2 to 5, wherein the aforementioned intracellular immunosuppressant is a small molecular drug used in IBD, and is an anti-inflammatory agent, preferably a steroidal anti-inflammatory drug or a nonsteroidal anti-inflammatory drug (NSAID). Preferably, the steroid is a systemic steroid, preferably selected from **prednisolone, methylprednisolone, prednisone, and hydrocortisone**.

[0038] 7. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 2 to 4, wherein the aforementioned intracellular immunosuppressant is a small molecular drug used in IBD, ● Glucocorticoids (optionally used in combination with DMARDs (cDMARDs)), or ● Conventional disease-modifying drugs (DMARDs (cDMARDs)) (without concomitant use of glucocorticoids) That is the case.

[0039] 8. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 1 to 7, preferably paragraph 6 or 7, wherein the intracellular immunosuppressant of interest is a cDMARD.

[0040] 9. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 1 to 8, preferably any of paragraphs 2 to 4 and 6 to 8, wherein the ABC transporter is selected from group B and group G, preferably **ABCB1 (MDR1) and ABCG2 (BCRP)**.

[0041] 10. Preferably, the present invention also relates to the in vitro method described in paragraph 7, wherein the intracellular immunosuppressant of interest is different from a steroid and also different from an NSAID. Preferably, the second intracellular immunosuppressant is a small molecular anti-IBD drug. Preferably, the second intracellular immunosuppressant is a tsDMARD, and more preferably a selective or non-selective (pan) Janus kinase (JAK) inhibitor.

[0042] 11. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 1 to 10, or any of paragraphs 2 to 4 and 6 to 10, wherein the patient is **naive** to the intracellular immunosuppressant of interest before or at the time of the initiation of such treatment.

[0043] 12. An in vitro diagnostic method described in paragraphs 1-7 or 9-11, wherein the intracellular immunosuppressant of interest is a tsDMARD, preferably selected from a JAK inhibitor, an S1PR agonist, or a PDE4 inhibitor, and preferably a JAK inhibitor.

[0044] 13. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 1 to 7 or paragraphs 10 to 12, wherein the ABC transporter is selected from group B and group C, preferably **ABCB1(MDR1) and ABCC1(MRP1)**.

[0045] 14. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 1 to 14, preferably any of paragraphs 1 to 10 and 12 to 14, in which case the patient is being treated for a predetermined period of time with an intracellular immunosuppressant of interest. Preferably, the period is selected from at least 2 weeks, 4 weeks, 1 month, 2 months, or 3 months.

[0046] 15. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 1 to 14, wherein the method is a diagnostic method. Preferably, the method is a diagnostic method combined with treatment with an intracellular immunosuppressant of interest. More preferably, the method is a treatment method with an intracellular immunosuppressant of interest when the patient is responsive to the drug. Preferably, the treatment also includes the use of the drug if the patient has not been determined to be unresponsive to the drug.

[0047] 16. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 10 to 15. The cells are lymphocytes, preferably CD3+ / CD8+ lymphocytes, and the MDR1 transport activity is measured to obtain the MDR1 transport activity value. ●Compare the MDR1 transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the MDR1 transport activity value obtained from the sample is significantly lower than one or more of the above reference values, the patient will be determined to be a non-responder to the JAK inhibitor therapy. Preferably, the cells are lymphocytes, preferably CD3+ / CD8+ lymphocytes, and the MDR1 transport activity is measured to obtain the MDR1 transport activity value. ●Compare the MDR1 transport activity value with one or more reference values, The reference values ​​in question are values ​​obtained (or have been obtained) from patients who are naive to the treatment in question. ●If the MDR1 transport activity value obtained from the sample is significantly lower than one or more of the above reference values, the patient will be determined to be a non-responder to the JAK inhibitor therapy.

[0048] 17. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 10 to 15. The cells are monocytes, preferably CD14+ monocytes, and the MDR1 transport activity is measured to obtain the MDR1 transport activity value. ●Compare the MDR1 transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the MDR1 transport activity value obtained from the sample is significantly higher than one or more of the above reference values, the patient will be determined to be a non-responder to the JAK inhibitor therapy.

[0049] 18. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 10 to 15. The cells are monocytes, preferably CD14+ monocytes, and the MRP1 transport activity is measured to obtain the MRP1 transport activity value. ●Compare the MRP1 transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the MRP1 transport activity value obtained from the sample is significantly higher than one or more of the above reference values, the patient will be determined to be a non-responder to the JAK inhibitor therapy. Preferably, the cells are monocytes, preferably CD14+ monocytes, and the MRP1 transport activity is measured to obtain the MRP1 transport activity value. ●Compare the MDR1 transport activity value with one or more reference values, The reference values ​​in question are values ​​obtained (or have been obtained) from patients who are naive to the treatment in question. ●If the MRP1 transport activity value obtained from the sample is significantly higher than one or more of the above reference values, the patient will be determined to be a non-responder to the JAK inhibitor therapy.

[0050] 19. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 15 to 18, wherein the JAK inhibitor is **tofacitinib**.

[0051] 20. Preferably, the present invention also relates to a method for carrying out the in vitro method described in any of paragraphs 15 to 19. The IBD patient is a responder to another JAK inhibitor, and / or Propose and / or administer treatment with another JAK inhibitor to the IBD patient. Preferably, the JAK inhibitor is a JAK1 / JAK3 inhibitor.

[0052] 21. The present invention also relates to a method for carrying out the in vitro method described in any of paragraphs 15 to 19, The IBD patient is unresponsive to the tsDMARD, and treatment with another anti-IBD drug (e.g., another tsDMARD) is suggested and / or implemented, and / or Propose or implement bDMARD therapy.

[0053] 22. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 1 to 20, wherein IBD is UC. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 1 to 21, or any of paragraphs 2 to 4 and 6 to 9, wherein the patient is **naive** to an intracellular immunosuppressant of interest prior to the initiation of such treatment.

[0054] 23. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 5 to 9, IBD patients are being administered steroids as intracellular immunosuppressants of interest. The cells are lymphocytes, preferably CD3+ lymphocytes. ●Measure the combined MDR1+MRP1 transport activity and obtain the combined MDR1+MRP1 transport activity value. ●Compare the combined MDR1+MRP1 transport activity value with one or more reference values. These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the combined MDR1+MRP1 transport activity value is significantly higher than one or more of the above reference values, the patient is determined to be unresponsive to the steroid therapy. Preferably, the value is higher than the reference value obtained from responders of the same therapy, and preferably, transport activity is measured at least 2 weeks, 1 month, or 1 to 3 months after the start of steroid treatment, and the value measured at **week 0** is optionally used as the reference value.

[0055] 24. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 5 to 9, IBD patients are being treated with small molecular anti-IBD drugs as intracellular immunosuppressants of interest. The cells are lymphocytes, preferably CD3+ lymphocytes, and the combined MDR1+MRP1 transport activity is measured to obtain the combined MDR1+MRP1 transport activity value. ●Compare the combined MDR1+MRP1 transport activity value with one or more reference values. These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the combined MDR1+MRP1 transport activity value is significantly higher than one or more of the above reference values, the patient is determined to be unresponsive to the steroid therapy. In a preferred embodiment, values ​​are also obtained for responders of the same therapy. Preferably, transport activity is measured at least 2 weeks, 1 month, or 1 to 3 months after the start of steroid treatment, with the week 0 value optionally used as the baseline. Preferably, the small molecular anti-IBD drug is selected from steroids, azathioprine, and 5-ASA.

[0056] 25. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 5 to 9. IBD patients are being administered steroids as intracellular immunosuppressants of interest. The cells are lymphocytes, preferably CD3+ lymphocytes. ●Measure MDR1 transport activity and obtain the MDR1 transport activity value. ●Compare the MDR1 transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the MDR1 transport activity value is significantly lower than one or more of the above reference values, the patient shall be determined to be a non-responder to the steroid therapy (preferably lower than the reference value obtained from responders of the same therapy; preferably transport activity is measured at **week 0**), and / or ●If the BCRP transport activity value is higher than the reference value obtained from responders of the same therapy, the patient is determined to be a non-responder to the steroid therapy (preferably, the transport activity is measured 1 to 3 months after the start of steroid treatment, and the value at that time is arbitrarily used as the reference).

[0057] 26. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 5 to 9. ●Measure MDR1 transport activity and obtain the MDR1 transport activity value. ●Compare the MDR1 transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the MDR1 transport activity value is significantly higher than one or more of the above reference values, the patient is determined to be a non-responder to the steroid therapy (preferably higher than the values ​​obtained from responders of the same therapy). Transport activity is measured at least 2 weeks, 1 month, or 1 to 3 months after the start of steroid treatment, and the value at that time or week 0 is optionally used as the reference. Preferably, the small molecular anti-IBD drug is selected from steroids, azathioprine, and 5-ASA.

[0058] 27. Preferably, the present invention also relates to an in vitro method described in any of paragraphs 5 to 9, IBD patients are being administered steroids as intracellular immunosuppressants of interest. The cells are lymphocytes, preferably CD3+ lymphocytes. ● Measure BCRP transport activity and obtain BCRP transport activity values. ●Compare the BCRP transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the BCRP transport activity value is significantly lower or higher than one or more of the above reference values, the patient is determined to be a non-responder to the steroid therapy (preferably lower than the reference value obtained from responders of the same therapy, preferably measured at **week 0**), and / or Preferably, ●If the BCRP transport activity level is lower than the reference level obtained from non-responders of the same therapy, the patient is determined to be a responder to the steroid therapy (preferably measured at **week 0**), and / or ●If the BCRP transport activity value is higher than the reference value obtained from responders of the same therapy, the patient will be classified as a non-responder to the steroid therapy (transport activity is measured 1 to 3 months after the start of steroid treatment, and the value at that point in time is arbitrarily used as the reference).

[0059] 28. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 5 to 9. IBD patients are being treated with small molecular anti-IBD drugs as intracellular immunosuppressants of interest. ● Measure BCRP transport activity and obtain BCRP transport activity values. ●Compare the BCRP transport activity value with one or more reference values, These reference values ​​are obtained (or are obtained) from patients with low levels of inflammation. ●If the BCRP transport activity value is significantly lower or higher than one or more of the above reference values, the patient is determined to be a non-responder to the small molecular anti-IBD drug therapy (preferably lower than the reference value obtained from responders of the same therapy, preferably measured at **week 0**), and / or Preferably, ●If the BCRP transport activity level is lower than the reference level obtained from non-responders of the same therapy, the patient is determined to be a responder to the small molecular anti-IBD therapy (preferably measured at **week 0**), and / or ●If the BCRP transport activity value is higher than the reference value obtained from responders of the same therapy, the patient will be classified as a non-responder to the small molecular anti-IBD drug therapy (transport activity will be measured 1 to 3 months after the start of the small molecular treatment, and the value at that time will be arbitrarily used as the reference). Preferably, the small molecular anti-IBD drug is selected from steroids, azathioprine, and 5-ASA. The present invention also relates to a method for carrying out the method described in any of paragraphs 23 to 28, wherein the IBD patient is unresponsive to the small molecular anti-IBD drug, preferably the anti-IBD drug is selected from a steroid, azathioprine, or 5-ASA, and the treatment for the patient is as follows: ● Suggest and / or administer treatment with another small molecular anti-IBD drug, and / or ● Propose or administer treatment with a cDMARD or csDMARD.

[0060] 29. Preferably, the present invention also relates to an in vitro diagnostic method described in any of paragraphs 1 to 28, wherein one or more reference transporter activity values ​​are selected from the following. ● This involves comparing the activity levels of one or more transporters with one or more predetermined threshold transporter activity levels. Each threshold transporter activity level is the threshold for the transport activity of one or more multidrug-resistant transporters, determined using the same one or more substrates.

[0061] 30. Preferably, the threshold transporter activity level is determined using one or more substrates by the following method. ●In known responder patient groups and known non-responder patient groups, measure or quantify the transport activity of one or more multidrug resistance transporters in the effector mononuclear cells. ● Analyze the transport activity values ​​measured in the responder group and the non-responder group (preferably statistically analyze them as a distribution) to find a threshold level to distinguish between the transport activity values ​​of responders and those of non-responders.

[0062] 31. In a highly preferred embodiment, the substrate is a fluorescent substrate. In a highly preferred embodiment, the substrate is a compound whose transport properties and / or fluorescence spectrum are converted to a different form by an intracellular enzyme. Preferably, the compound is an ester that is hydrolyzed by an esterase. In a preferred embodiment, the substrate is a calcein ester, preferably calcein AM. In a preferred embodiment, the substrate is an ester compound of fluorescein and / or a rhodamine derivative, and preferably includes a phenanthrene derivative moiety. In a highly preferred embodiment, when the substrate is a fluorescein derivative ester compound, it is a PhenGreen compound. In a highly preferred embodiment, the activity is quantified as a multidrug activity factor (MAF).

[0063] 32. Preferably, the patient has a record of cDMARD therapy failure or drug intolerance, and therefore requires a switch or modification of cDMARD therapy, and a switch to tsDMARD therapy is required.

[0064] 33. In a preferred embodiment, the method is a therapeutic method, wherein the IBD patient is treated with an intracellular immunosuppressant of interest. However, if the patient If a person is not determined to be a non-responder to the drug by the method of the present invention, or This applies only if the person is determined to be a responder to the drug by the method of the present invention.

[0065] 34. As a preferred alternative variation, the present invention relates to an in vitro diagnostic method for monitoring the IBD status of a patient under treatment with an intracellular immunosuppressant of interest, comprising the following steps: ● A step of providing a biological sample from the IBD patient, wherein the sample comprises effector mononuclear cells (preferably lymphocytes and / or monocytes and / or NK cells) derived from the patient. ● A step of measuring the transport activity of multidrug-resistant ABC transporters (one or more selected from groups B, C, and G) in effector mononuclear cells in the sample, and obtaining the initial transporter activity level (activity value of one or more transporters). Similarly, the process involves measuring the transport activity of the multidrug-resistant ABC transporters (one or more selected from groups B, C, and G) in effector mononuclear cells in the sample to obtain follow-up transporter activity levels (values ​​of one or more transporters). ● A step of comparing the tracking transporter activity level (or one or more transporter activity values) with the initial transporter activity level (or one or more reference transporter activity values). ● A step to evaluate the progression or improvement of the patient's condition when the tracking transporter activity level (or the activity value of one or more transporters or each individual transporter) is significantly different from the initial transporter activity level (or the activity value of one or more reference transporters).

[0066] definition "Measuring" or "measurement" as used herein means the quantitative characterization of a physical object or entity, or a number (group or multiple) thereof, or their functions, or a physical or chemical process, and includes the act of assigning a quantity (e.g., a numerical value or a number representing the characteristics of the object, etc.) of such object or entity, or its multiples, functions, or processes, by comparison with units and with other objects, entities, multiples, functions, or processes. Preferably, the measurement is consistent with methods known in the art or with international guidelines of metrology. Magnitude is usually a numerical characterization obtained by a appropriately selected measuring instrument, and **unit** is a mathematical weighting factor assigned to the magnitude as a ratio of the properties of artifacts used as standard units, or physical quantities in nature, when used as units.

[0067] "Quantifying," "quantification," or "quantitation" in this specification means the assignment of physical quantities to a physical object or entity, or a number (group or group) of such objects or entities, or their functions, or to a physical or chemical process, expressed in numerical values ​​and units, and including comparison with other objects or entities. Preferably, "quantifying" or "quantification" is the measurement itself or an essential component of the measurement.

[0068] Measurements are inherently subject to uncertainty, which can represent random and systematic errors in the measurement procedure. Those skilled in the art understand this and can handle such errors in light of the measurements or quantifications performed.

[0069] In this specification, "comparing" two levels, preferably two activity levels, includes comparing numerically represented quantities that characterize those levels to determine whether they are high or low, calculating the difference, determining the ratio of those levels or the value derived therefrom, and adding other mathematical procedures as appropriate, depending on the quantification or calculation method.

[0070] A "membrane transporter" is a membrane-integrated protein that is permanently fixed to a membrane, has a transmembrane region, and has structures on both sides of the membrane, and while integrated within the membrane, has the ability to perform transport (excretion, push-out, uptake) actively or passively. The entities being transported are, for example, molecules or molecular ions, and are preferably fluorescent.

[0071] "ABC transporter" is an abbreviation for ATP-binding cassette transporters and refers to a superfamily of membrane transporters that utilize the energy from adenosine triphosphate (ATP) hydrolysis to perform biological processes, including the transport of entities across membranes. The names and subfamilies of ABC transporters follow the designations of the HUGO Gene Nomenclature Committee (HGNC). In this specification, for example, membrane transporters of the "ABCG family" belong to the G subfamily, which consists of half-transporters that oligomerize for functional transporter formation.

[0072] "Multidrug transporter" in this specification refers to a type of ABC transporter, also called an ABC multidrug transporter, that is capable of transporting multiple, preferably diverse, chemical compounds from the cell membrane in which it exists.

[0073] As used herein, “Multidrug resistance” refers to the ability of cells to acquire resistance to a wide range of structurally or functionally unrelated drugs through a multidrug transporter. Preferably, “multi-drug resistance” refers to a state that depends on the expression or overexpression of MDR1, MRP1 or related homologs, and / or amplification of the gene encoding the multidrug transporter protein.

[0074] "ABC transporter activity," or "activity" of the ABC transporter protein, refers to all activities exhibited by the transporter, including, for example, its biological function, i.e., the transport activity of drugs across the membrane carried by the protein, or ATPase activity (insofar as it serves as an indicator of transport activity, such as substrate-stimulated ATPase activity). Preferably, the activity measured in the present invention is one that characterizes, relates to, or correlates with the transport activity of a multidrug-resistant transporter.

[0075] **"Substrate" of ABC transporters** refers to compounds that can be transported out of cells by the ABC transporter-mediated active transport mechanism. In a preferred embodiment, the ABC multidrug resistance transporter whose activity is measured in the present invention is selected from the following: ●ABCB1(MDR1): A subfamily of ABC transporters belonging to the ABCB family. ●ABCC1 (MRP1): A Multidrug Resistance Protein (MRP), belonging to the ABCC family, and part of the C subfamily of ABC transporters. ●A preferred embodiment is ABCG2 (also known as BRCP, MXR1, CDw338, etc.): a membrane transporter consisting of half-transporters belonging to the ABCG family, i.e., the G subfamily, which forms a functional transporter through oligomerization.

[0076] "Measuring transport activity of a multidrug transporter" means measuring and / or quantifying the transport activity in cells or cell populations where the multidrug transporter is present or presumed to be present, and preferably refers to the overall or combined transport activity of one or more multidrug transporters. In a preferred embodiment, if the expression of the transporter increases, the activity also increases. In general, any physical quantity that quantitatively characterizes the transport activity of a multidrug transporter is applicable to the present invention. In a preferred embodiment, the physical quantity is obtained by comparing a value obtained for cells in which the transporter is active with a value obtained for cells in which the transporter is inhibited.

[0077] In preferred embodiments, activity is measured via, or using, a substrate compound that can permeate the cell membrane and be transported from the cell in which the transporter resides. In a particularly preferred embodiment, the activity is measured via, or using, a substrate compound that is a derivative of a “detectable fluorescent compound” and can be transported across the cell membrane from the cell in which the transporter resides. Within the cell, the derivative is converted into a detectable fluorescent compound that is membrane-impermeable, preferably hydrophilic or charged, and preferably not a substrate of the transporter protein.

[0078] Preferably, the derivative is an ester derivative. In preferred embodiments, the substrate is an ester compound of fluorescein and / or a rhodamine derivative, preferably containing a phenanthrene derivative moiety. Preferably, detectable fluorescent compounds are detected within cells. In measuring or quantifying transport activity (or using synonymous expressions), in preferred embodiments, the activity is quantitatively characterized by a coefficient, which relates to or is proportional to the difference between the fluorescence of a detectable fluorescent compound measured in a cell in the presence of a membrane transporter inhibitor and the fluorescence measured in the absence of the inhibitor (i.e., when the transporter is active). When the transporter is active, the intracellular fluorescence is lower and the difference is larger in the absence of the inhibitor because the compound (or derivative, if a derivative is used) is transported.

[0079] "Detectable fluorescent compound derivatives" or the measurement of fluorescent compounds means measuring the amount of the compound accumulated inside the cell, which means it serves as an inverse indicator of the amount of the compound expelled from the cell by multidrug transporter proteins. Techniques for measuring intracellular calcein include, but are not limited to, flow cytometry, fluorimetry, and cell imaging.

[0080] In a preferred embodiment, the transport activity of the MDR transporter is measured as the difference in dye accumulation in the presence or absence of the inhibitor, i.e., via the transport (excretion) of derivative compounds. Fluorescence measurement in the presence of the inhibitor yields the maximum (latent) fluorescence (Fmax) in the cell population when the multidrug transporter is dysfunctional. Fluorescence measurement in the absence of the inhibitor yields the minimum fluorescence (F0) in the cell population when the multidrug transporter is functioning. This corresponds to a standardization method that eliminates unknown cell type-specific variability factors such as esterase activity and cell size.

[0081] In a preferred embodiment, the **quantification** of this fluorescence is performed by introducing the MDR Activity Factor (MAF), which is calculated by the following formula: MAF = (Fmax - Fo) / Fmax Alternatively, when expressed as a percentage, MAF = 100 × (Fmax - Fo) / Fmax. MAF, expressed as a percentage, is often written as MAF%; however, unless otherwise specified, MAF values ​​in this specification are given as percentages. Even when the compound is a substrate for multiple multidrug transporters, selective inhibitors can easily identify the transport activity of each transporter. Other membrane transporters can also be inhibited as needed.

[0082] In this specification, **"transporter activity level"** means a quantified value representing the measurement result of the transporter activity described above. The transporter activity level is preferably a physical quantity in a sample that characterizes substrate transport activity, and preferably characterizes transporter activity in a biological sample taken from a subject or patient, or transporter activity in the body of a subject. Preferably, the transporter activity level is related to substrate transport activity and can be obtained, for example, by direct or indirect measurement of substrate transport. The "transporter activity level" is expressed as a mathematical / physical value ("transporter activity value") suitable for characterizing the activity, and can be represented, for example, by the MAF value or by an index based on other well-known methods. The "transporter activity level" or "transporter activity value" can be expressed as a single value or as a set of values ​​("transporter activity value(s)") (e.g., statistical distribution, range, threshold (which may mean a range of values ​​above / below the threshold)). It can also be expressed as an interval or a value with a standard error** (e.g., confidence interval).

[0083] In this specification, the reference transporter activity level (value) refers to a transporter activity level (value) that characterizes transporter activity in a patient or group of patients (subject group) and is used as a comparison criterion for the IBD patient group that is the target of prediction in this invention. The reference value may characterize responder patients (group), depending on the embodiment, healthy subjects (patients) or groups thereof, or drug-naive patients (group). Depending on the embodiment, multiple reference activity values ​​may be used, the measured activity value may be compared with multiple reference values, and the responsiveness may be predicted based on the multiple comparisons. The reference transporter activity level (value) should be expressed as a mathematical / physical quantity and should be comparable to the transporter activity value (level) measured by the method of the present invention. For example, if the activity value obtained by the method of the present invention is an MAF value (indicating the activity level in a subject or patient), then the reference activity value should also be an MAF value obtained for the same transporter. A single transporter can be a source of multiple activity values ​​and may represent a composite activity value.

[0084] Where applicable, the reference activity value is determined in the patient or patient group providing the comparison standard by the same method used in the present invention. Optionally, a reliable reference value is derived from multiple measurements (preferably from multiple patients) by mathematical (e.g., statistical) methods. In a particular embodiment, the reference activity value is obtained as follows: ● Provide a biological sample from the aforementioned patient(s), and the sample contains effector mononuclear cells (preferably lymphocytes and / or monocytes and / or NK cells), ●In the effector mononuclear cells in the sample, the transport activity of one or more ABC multidrug resistance transporters selected from groups B, C, and G is measured to obtain one or more activity values ​​(preferably for the same transporter as the target of measurement in the present invention), thereby obtaining a reference value.

[0085] Depending on the embodiment, the reference activation value may include a range or a value with a standard error (e.g., confidence interval). Depending on the embodiment, the reference activation value may be a threshold (level). In embodiments, comparing one or more activity values ​​to one or more reference activity values ​​and / or considering a patient as a non-responder to intracellular immunosuppressant therapy of interest when one or more activity values ​​or each activity value differs significantly from the corresponding one or more reference activity values ​​may involve mathematical (e.g., statistical) methods that draw conclusions with a predetermined probability. Furthermore, the determination of significance depends on the mathematical (statistical) method applied. Those skilled in the art can select appropriate methods that provide reliable results in the domain.

[0086] In an embodiment, "level" is expressed as **intensive quantity**. Comparing two levels (e.g., as values ​​or sets of values) can involve determining high or low, calculating differences, calculating ratios, or calculating values ​​derived from the levels, and may include other mathematical procedures (calculations) required by the measurement method as needed. In embodiments, the comparison may include calculating the difference between the two levels (e.g., subtraction of levels after normalization or baseline correction). In embodiments, the comparison may include transformations to both levels (e.g., logarithmic transformation or other functions). In embodiments, the comparison may include creating statistics, calculating errors and / or means, and assessing statistical significance.

[0087] A threshold can be the upper or lower limit of a normal or typical range defined for the purpose of the measurement.

[0088] "Kit" refers to a collection of materials consisting of multiple substances contained in multiple containers, intended for use in a certain method, preferably the method (assay) of the present invention. Preferably, the kit includes the procedure for performing the method. "Instructions" means a list of procedures or a description of the invention for instructing a practitioner (e.g., a clinical laboratory technician) to perform the assay of the present invention, and may take the form of paper, electronic (in silico), audio, video, or a link to a website.

[0089] A "biological sample" refers to a sample obtained from a mammal and containing optionally processed viable immune cells. A biological sample may be isolated from the mammal as a body fluid (preferably blood or synovial fluid). Preferably, the biological sample is a blood sample. Preferred immune cells are at least T lymphocytes and / or a subset of T lymphocytes, and optionally / additionally include B cells.

[0090] "Subject" refers to a vertebrate individual, preferably a mammal, especially a primate, hominin, or human. "Patient" refers to an object that is under or will be under medical or veterinary care (observation, supervision, diagnosis, treatment). Preferably, the patient is a primate, hominin, or human.

[0091] "Treatment" refers to any procedure, action, application, or therapy performed under medical or veterinary support with the aim of directly or indirectly improving the condition of the subject or patient.

[0092] "Therapy" refers to a treatment method that involves administering a specific medicine or pharmaceutical composition to a patient for a certain period of time with the aim of improving the patient's condition. In this specification, "disease-modifying drug" refers to a pharmaceutical product that is useful for treating IBD. In IBD, some of the same active ingredients as those used in rheumatoid arthritis are sometimes employed. These compounds generally act on immune processes (e.g., immune cell division induced by methotrexate and thiopurine) to reduce inflammation; therefore, in this specification, the term DMARD is also used for compounds with anti-IBD activity. However, there are differences in scope of application and usage. For example, 5-ASA is frequently used in IBD but rarely in RA, and although there is some overlap in scope, this terminology is used. Therefore, "sDMARD" stands for "synthetic disease-modifying antirheumatic drug," which is a synthetic chemical compound originally defined for use in rheumatoid arthritis (RA), but in this specification also includes its use in IBD, and slows disease progression by targeting the immune system or its pathways through mechanisms other than simple reduction of inflammation.

[0093] In one embodiment, csDMARDs are different from NSAIDs (non-steroidal anti-inflammatory drugs).

[0094] In specific embodiments, NSAIDs are anti-inflammatory compounds that act by inhibiting COX enzymes (e.g., Algopyrin, Aspirin, Panadol, Algoflex, Nurofen, etc.) and are generally used for antipyretic, analgesic, and anti-inflammatory purposes.

[0095] From one perspective, 5-ASA can be considered an NSAID in terms of its structure. From another perspective, 5-ASA is not considered an NSAID because it is not commonly used as a general antipyretic, analgesic, or anti-inflammatory drug.

[0096] "csDMARDs" are defined as "classic synthetic" or "conventional synthetic disease-modifying antirheumatic drugs," which are preferably synthetic chemical compounds with broad-spectrum effects. They are defined as drugs that target the immune system to slow disease progression in rheumatoid arthritis (RA) and are not developed to inhibit JAK or target specific intracellular pathways. Therefore, csDMARDs are a subgroup of sDMARDs and are listed in the EULAR recommendations (Smolen, Landewe et al., 2020). In preferred embodiments, the csDMARD is selected from azathioprine, cyclophosphamide (for refractory cases such as lupus renal impairment), cyclosporine (for lupus refractory to other therapies, etc.), hydroxychloroquine sulfate (an antimalarial drug), leflunomide (as an alternative or in combination for methotrexate intolerance), methotrexate, mycophenolate mofetil (for RA refractory to other therapies), and sulfasalazine (triple therapy; methotrexate + sulfasalazine + hydroxychloroquine), and is preferably selected from methotrexate, chloroquine, sulfasalazine, and optionally from glucocorticoids.

[0097] In a preferred embodiment, the definition of "csDMARD" does not include glucocorticoids. "tsDMARD" stands for "targeted synthetic disease-modifying antirheumatic drug," which is a synthetic chemical compound that targets specific sites of the immune system involved in inflammation in the intestines and other organs to reduce inflammation. In one embodiment, tsDMARD is a Janus Kinase (JAK) inhibitor. JAK inhibitors are developed to specifically target the JAK kinase pathway. JAK inhibitors can be classified into several overlapping classes: immunomodulatory agents, disease-modulating agents (DMARDs), and subclasses of tyrosine kinase inhibitors. They modulate the immune system by inhibiting cytokine activity.

[0098] Preferably, the JAK inhibitor of the present invention is an immunomodulator. Preferably, it is a JAK1 kinase inhibitor and has selectivity for JAK2. In preferred embodiments, the JAK inhibitor is selected from tofacitinib (e.g., Xeljanz(R)), upadacitinib (e.g., Rinvoq(R)), and baricitinib (e.g., Olumiant(R)). In one embodiment, tsDMARD is a sphingosine 1-phosphate (S1P) receptor modulator.

[0099] In a preferred embodiment, the S1P receptor modulator is ozanimod (Zeposia®), an FDA-approved oral small molecular drug for the treatment of moderate to severe ulcerative colitis (UC) in adults. The S1P receptor modulator suppresses inflammation in UC by blocking the migration of immune cells from the lymph nodes to the intestinal tract.

[0100] In a preferred embodiment, the tsDMARD is selected from baricitinib and **tofacitinib (Xeljanz)**.

[0101] A "low molecular weight compound" is preferably an anti-IBD compound, typically a compound with a molecular weight of less than 1000 Da, preferably less than 900 Da, and can be prepared by chemical synthesis (total synthesis or semi-synthesis). In preferred embodiments, the low molecular weight compound refers to a compound that can permeate the cell membrane due to the hydrophobicity of the molecule itself without using cell surface receptors, and is particularly less than 1000 Da, preferably less than 900 Da. "Switching a therapy" refers to a change in treatment that involves administering a drug or pharmaceutical composition that has not been previously administered to the patient.

[0102] In one embodiment, this includes discontinuing a previously administered drug or composition. In another embodiment, this includes continuing administration of a previously administered drug or composition (with or without changes in dosage or regimen). Preferably, the target disease is IBD, preferably UC. In particular, "switch of the therapy" or "alteration of the therapy" as used herein refers to the initial treatment of IBD, preferably UC, and optionally includes a change from the initial anti-inflammatory drug, including a modification of treatment with a DMARD different from the initial drug.

[0103] In this embodiment, the initial anti-inflammatory agent is a steroid. In this embodiment, the initial anti-inflammatory drug is a cDMARD. In this embodiment, the initial anti-inflammatory drug is a csDMARD. In this embodiment, the DMARD that is different from the initial drug is the csDMARD, and the initial drug is different from this. In this embodiment, the DMARD that is different from the initial drug is the bDMARD. In this embodiment, the DMARD that is different from the initial drug is the tsDMARD. In this embodiment, a DMARD different from the initial drug is the csDMARD. In this embodiment, the DMARD that is different from the initial drug is the tsDMARD.

[0104] "Predicting the responsiveness" (or "assessing the success or outcome of a treatment or therapy") as used herein means a diagnostic method that provides a quantitative result, predicts whether a particular treatment is effective for a particular patient, and contributes to future treatment decisions, i.e., continuation or modification. The assessment typically involves measurement (including calculation) and preferably includes reviewing the results and / or deriving conclusions. In this specification, even in the singular form, the indefinite articles "a" and "an" (and the referential "the," to the extent the context allows) encompass the plural, meaning "one or more" (unless the context explicitly states otherwise).

[0105] The words "comprises," "comprising," and "including" should be interpreted in a non-limiting sense, allowing for the addition of further features, processes, or components to the listed features, processes, or components.

[0106] The expressions "consisting essentially of" or "comprising substantially" mean substantially consisting of the essential features, processes, or components enumerated (for example, in a claim), but allow for the additional inclusion of other features, processes, or components that do not substantially affect the essential characteristics of the use, method, composition, or other subject matter. In this specification, where necessary, "comprises," "comprising," and "including" may be replaced with "consisting essentially of" or "comprising substantially" without adding new material.

[0107] [Table A] [Brief explanation of the drawing]

[0108] Figures 1-9 show the results for the first patient cohort treated with conventional disease-modifying agents (cDMARDs and GCs). Figures 1 and 2 show combined (MDR1 + MRP1) activity (abbreviated as MAFC). Figures 3 and 4 show MDR1 activity (abbreviated as MAFMDR1). Figures 5 and 6 show MRP1 activity (abbreviated as MAFMRP1). Figures 7 and 8 show BCRP activity (abbreviated as MAFBCRP). Figures 10-14 show the results for the second patient cohort treated with targeted synthetic disease-modifying anti-diarrheal medications (tsDMARDs). [Figure 1] MAFC values ​​in IBD patients receiving steroid therapy. At diagnosis (Week 0, A) and 8 weeks after the start of steroid treatment (B), CD3+ lymphocyte MAFC values ​​were moderately elevated in steroid non-responders and higher compared to responders. In responders, a slight decrease in MAFC was detected at 8 weeks after the start of treatment compared to Week 0 (C). In contrast, non-responders showed a slight increase compared to Week 0 (D). The cutoff value at diagnosis was determined with a sensitivity of 75% and a specificity of 72.72% (E, AUC=0.6136). Sensitivity reached 100% at 8 weeks (F, AUC=0.7955). (Bars are mean ± SEM, * indicates p≦0.005, Student's unpaired t-test). Table 3 shows the baseline patient data. [Figure 2] MAFC values ​​of IBD patients treated with small molecular drug therapy. MAFC values ​​in non-responders before treatment initiation were slightly higher than those of responders (A), and the difference became statistically significant at follow-up (B). In responders, small molecular drug therapy did not affect MAFC values ​​(C), but a significant increase was observed in non-responders at follow-up (D). Cutoff values ​​were determined at Week 0 (E) and follow-up week (F), with sensitivity at Week 0 being 66, 67% and sensitivity at follow-up being 100%, and specificity at 76, 92%. (Bars are mean ± SEM, * indicates p ≤ 0.005, Student's unpaired t-test). Table 5 shows baseline patient data. Follow-up period: steroids 8 weeks; **5-ASA, azathioprine** 12 weeks. [Figure 3] MAFMDR1 activity (MAFMDR1) in IBD patients receiving steroid therapy. After 8 weeks, MAFMDR1 levels in non-responders were significantly higher than in responders. At diagnosis, MAFMDR1 levels in non-responders were slightly lower than in responders (A). Compared to diagnosis, there was a significant increase in non-responders after 8 weeks (B). In responders, steroid therapy resulted in a moderate decrease (C), while in non-responders, it increased (D). The cutoff value at 8 weeks (F) was significantly lower than at diagnosis (E). (Bars are mean ± SEM, * indicates p ≤ 0.005, Student's unpaired t-test). See Table 3. [Figure 4] MDR1 activity (MAFMDR1) in IBD patients treated with small molecular drug therapy. MAFMDR1 in CD3+ lymphocytes has strong predictive power for small molecular therapy. In non-responders, MAFMDR1 at Week 0 was slightly lower than in responders (A). At follow-up, it was significantly higher in non-responders (B). A significant decrease was observed in successful treatments (C). In non-responders, there was a significant increase compared to Week 0 (D). Cutoff values ​​were determined for Week 0 (E) and follow-up (F), with follow-up showing higher sensitivity and specificity. (Bars are mean ± SEM, * indicates p ≤ 0.005, Student's unpaired t-test). See Table 5. Follow-up period: Steroids 8 weeks; **5-ASA, Azathioprine** 12 weeks. [Figure 5] MAFMRP1 activity in IBD patients receiving steroid therapy. Steroid treatment did not significantly affect MAFMRP1. Measured in CD3+ lymphocytes at the start of treatment and at 8 weeks. Non-responders showed slightly higher levels at Week 0 (A), but no significant difference was observed between groups at 8 weeks (B). Responders showed a mild increase compared to Week 0 (C), while non-responders showed a moderate decrease (D). The cutoff values ​​were the same at the start (E) and at 8 weeks (F), but the predictive ability was higher at Week 0. (Mean ± SEM, * indicates p ≤ 0.005, unpaired t). See Table 3. [Figure 6]MRP1 activity (MAFMRP1) in IBD patients treated with small molecular drug therapy. Treatment did not significantly affect MAFMRP1. Measured with CD3+ lymphocytes at diagnosis and after check-up. At diagnosis, non-responders showed slightly higher levels than responders (A). At follow-up, levels were moderately elevated compared to responders (B). Responders showed a moderate increase (C), and non-responders showed a moderate decrease (D). Cutoff values ​​were determined at Week 0 (E) and follow-up (F), with Week 0 showing higher predictive ability. (Mean ± SEM, * indicates p ≤ 0.005, unpaired t). See Table 5. Follow-up period: Steroids 8 weeks; **5-ASA, Azathioprine** 12 weeks. [Figure 7] BCRP activity (MAFBCRP) in IBD patients receiving steroid therapy. Steroid therapy resulted in elevated MAFBCRP in non-responders. At diagnosis, MAFBCRP levels in non-responders were significantly lower than in responders (A), but after 8 weeks, levels were higher in non-responders (B). MAFBCRP decreased in responders (C) and increased in non-responders (D). Cutoffs were set at Week 0 (E) and Week 8 (F). Sensitivity at 8 weeks was 100% (F, AUC=0.7955). (Mean ± SEM, * indicates p≦0.005, unpaired t). See Table 3. [Figure 8] BCRP activity (MAFBCRP) in IBD patients treated with small molecular drug therapy. MAFBCRP significantly decreased with treatment. At Week 0, non-responders had significantly lower values ​​than responders (A). At follow-up, the pattern reversed, with non-responders having significantly higher values ​​(B). Responders showed a significant decrease (C), while non-responders showed a marked increase, but the difference was not statistically significant (D). Cutoff values ​​were determined pre-treatment (E) and follow-up (F), with high sensitivity at Week 0 and high specificity at follow-up. (Mean ± SEM, * indicates p ≤ 0.005, unpaired t). See Table 5. Follow-up period: Steroids 8 weeks; **5-ASA, Azathioprine** 12 weeks. [Figure 9]Disease activity scores of IBD patients treated with small molecular drug therapy. In responders, the activity score decreased with treatment. Mayo score (A; ulcerative colitis) and CDAI (B; Crohn's disease) were recorded at diagnosis (Week 0, black) and check-up (follow-up week, red). In patients with good response, both Mayo and CDAI decreased. In unresponsive ulcerative colitis, Mayo increased. On the other hand, in Crohn's disease with poor treatment response, CDAI decreased significantly. (Mean ± SEM, * indicates p ≤ 0.005, unpaired t). See Table 5. Follow-up period: Steroids 8 weeks; **5-ASA, Azathioprine** 12 weeks. [Figure 10] MAFMDR1 activity in CD3+ / CD8+ lymphocytes of IBD patients treated with tofacitinib. The tofacitinib treatment group and the tofacitinib non-responder group were compared. MAFMDR1 in CD8+ lymphocytes showed strong predictive ability for tofacitinib treatment. MAFMDR1 levels were significantly lower in non-responders than in the treatment group (A). (Bars represent mean ± SD, * indicates p ≤ 0.05, unpaired t). ROC analysis showed sensitivity 88, 89% and specificity 58, 82%. [Figure 11] MAFMDR1 activity in CD14+ monocytes of IBD patients treated with tofacitinib. The treatment group and the non-responder group were compared. MAFMDR1 in CD14+ cells showed strong predictive power and was significantly higher in the non-responder group (A). (Mean ± SD, * indicates p ≤ 0.05, unpaired t). ROC analysis: Sensitivity 100% / Specificity 66.67%. [Figure 12] MAFMRP1 activity in CD14+ monocytes of IBD patients treated with tofacitinib. The treatment group and the non-responder group were compared. MAFMRP1 in CD14+ monocytes showed strong predictive power and was significantly higher in non-responders (A). (Mean ± SD, ** indicates p ≤ 0.005, unpaired t). ROC analysis: Sensitivity 100% / Specificity 80%. [Figure 13]MAFMDR1 activity in CD3+ / CD8+ lymphocytes of IBD patients treated with tofacitinib. Comparison of the tofacitinib-naive group and the non-responder group. CD8+ MAFMDR1 showed strong predictive ability and was significantly lower in non-responders than in the naive group (A). (Mean ± SD, * indicates p ≤ 0.05, unpaired t). ROC analysis: Sensitivity 77, 78% / Specificity 78, 57%. [Figure 14] MAFMRP1 activity in CD14+ monocytes of IBD patients treated with tofacitinib. Comparison of the tofacitinib-naive group and the non-responder group. MAFMRP1 in CD14+ cells showed strong predictive ability and was significantly higher in non-responders than in the naive group (A). (Mean ± SD, * indicates p ≤ 0.05, unpaired t). ROC analysis: Sensitivity 100% / Specificity 63, 64%. [Figure 15] This document outlines IBD treatment schemes and the possibility of transitioning from one treatment scheme to another. [Modes for carrying out the invention]

[0109] Detailed description of the invention The inventors have surprisingly discovered that the responsiveness of IBD patients to intracellular immunosuppressants can be evaluated by measuring ABC-transporter activity levels, preferably transport activity levels. Furthermore, the method of the present invention can also be used to monitor the progression or improvement of the patient's condition.

[0110] In the prior art, there was no proposal that measuring the transport activity of MDR transporters is appropriate for predicting the efficacy or success of conventional or targeted synthetic (cDMARD or tsDMARD, respectively) therapies in IBD patients. Given the multilayered molecular-level complexity of immunodysregulation described in the background of the invention, and the diverse immune cells and local tissue cells involved in the pathogenesis, neither the exact cell population nor the specific molecule, structure, or analyte that can meet the requirements of an appropriate biomarker is obvious. Activity evaluation aimed at elucidating the pharmacokinetic interaction characteristics of multidrug resistance transporters (P-gp / MDR1 / ABCB1, MRP1 / ABCC1, BCRP / ABCG2) measured in this study unexpectedly revealed a correlation between transporter activity and therapeutic response in various mononuclear immune cells (i.e., CD3+ lymphocytes, more specifically their CD8+ subtype, and CD14+ peripheral monocytes).

[0111] These cells are not directly related to the site of inflammation in the intestinal tract, and the association between the transport activity of multidrug-resistant transporters and the patient's response to various anti-IBD therapies in the early stages of treatment is a very unexpected discovery by the inventors.

[0112] Therefore, by measuring the transport activity of multidrug-resistant transporters, it becomes possible to assess responsiveness early, and if there is no response, a switch to another drug can be implemented early. This may make it possible to avoid surgical intervention, more specifically, the inflamed site or total colectomy.

[0113] Each measurement in this study was performed early (Week 0, and 8 or 12 weeks as a follow-up) within 4 months of the start of the treatment regimen. The difference between specific transporter activity in non-responders and reference transporter activity in the reference patient group (e.g., a treatment-naive group, a responder group, or more generally, a group including multiple types of patients with disease activity, from which mean values ​​etc. can be obtained), i.e., a deviation of the responder's value from the reference level (background), suggests and can predict non-response.

[0114] While there are certain commonalities, observational results can vary depending on the transporter, drug type, and even the time of observation. The first patient cohort included patients with moderate to severe IBD (n=35; CD n=17, UC n=18). Patients were treated with steroids alone or in combination with conventional disease-modifying agents (5-ASA, azathioprine) (i.e., systemic steroids (methylprednisolone 32-64 mg) or topical glucocorticoids (budesonide 9 mg)). For statistical analysis, different types of steroids were not distinguished and were treated collectively as "steroids". Follow-up blood samples were taken at 8 weeks for patients treated with steroids alone, and at 12 weeks for patients treated with conventional disease-modifying agents.

[0115] The inventors were able to demonstrate that in IBD patients treated with steroid therapy, the transporter activity of MDR1 and MRP1 (expressed as MAFC values) was moderately elevated in non-responders with CD3+ lymphocytes from the time of diagnosis (Week 0). Similarly, in patients treated with steroids / azathioprine / 5-ASA, MAFC values ​​were slightly higher in non-responders compared to responders (see Figures 1 and 2).

[0116] In patients receiving steroid therapy, MAFMDR1 levels alone were evaluated, and an increase was observed 8 weeks after diagnosis (moderate decrease in responders). Similar results were observed in patients receiving small molecular drug therapy. Therefore, MAFMDR1 in CD3+ lymphocytes has a strong predictive ability for small molecular drug therapy, which is particularly pronounced after follow-up. MRP1 activity alone showed relatively small increases in both the steroid treatment group and the small molecular therapy group at the time of diagnosis, but a decrease in MAFMRP1 was shown in non-responders after follow-up. BCRP activity showed a significant decrease at diagnosis in non-responders, and a significant increase was observed in non-responders in both the small molecular therapy group and the steroid monotherapy group during follow-up.

[0117] Based on the above, the diagnostic value of MDR1 activity and BCRP activity is solid, and although the contribution of MRP1 is relatively weak, it may be useful as an additional marker based on these results. In the second patient cohort, based on data from IBD patients who were unresponsive to the JAK inhibitor (tofacitinib), we were able to demonstrate significant differences between the active and non-responder groups in MDR1 activity in CD8+ cells, and in MDR1 and MRP1 activity in CD14+ cells. Although this cohort did not distinguish between initial and follow-up samples, significant differences were observed even in mixed samples collected at different time points after the start of treatment. Furthermore, we were able to demonstrate significant differences in MDR1 activity in CD8+ cells and MRP1 activity in CD14+ cells between the tofacitinib-naive group and the non-responder group.

[0118] These results illustrate that the transporter activity values ​​obtained using the Theresa kit presented here can be used to assess the effectiveness of ongoing treatments. Activity measurement from blood samples is less invasive than conventional endoscopy and yields results in a relatively short time. For significant results, ROC curves were constructed to illustrate the sensitivity-specificity relationship. Based on the ROC, some measurements showed relatively weak sensitivity to specificity. However, the sample size in each comparison was relatively small, and sample size estimation suggests that sensitivity could be significantly improved by increasing the number of enrolled patients.

[0119] In another embodiment, patient status can be monitored using the method of the present invention. As shown in Figure 9, small molecular drug therapy resulted in a decrease in disease activity scores in responders. Mayo and CDAI scores were recorded at diagnosis (Week 0) and check-up (follow-up week), and both scores decreased in patients who showed a good treatment response. Mayo scores increased in unresponsive ulcerative colitis, while CDAI scores decreased significantly after treatment in Crohn's disease with poor treatment response. Therefore, these results support the identification of responders and non-responders and can be observed in parallel with ABC-transporter activity measurements shown in Figures 1 to 8. Based on the results of the method of the present invention, decisions regarding the initiation and / or continuation of treatment can be made at an early stage.

[0120] For example, and not limited to, a decision to switch from initial glucocorticoid or NSAID therapy to steroid therapy or small molecular IBD drug combination therapy is supported if the patient's responsiveness is predicted or not determined to be unresponsive according to the present invention. Therefore, if responsiveness to small molecular combination therapy is confirmed by this method, the treatment can be initiated or continued.

[0121] As a further example, though not limited to this, a switch from cDMARD therapy to tsDMARD therapy may be decided when the patient's responsiveness to the tsDMARD can be predicted by this method. Alternatively, it may be decided even if the patient is not determined to be unresponsive to the tsDMARD in question. Other variations are as mentioned in the explanation.

[0122] The treatment regimen is well-known in this field. For example, the use of immunosuppression in combination with other therapies is described in D'Haens G et al. (2008). Further methods for the diagnosis and management of Crohn's disease and ulcerative colitis are well described in consensus papers (Gomollon F et al., 2017, Harbord M. et al., 2022). Novel therapies for UC are proposed in Kaur A and Goggolidou P (2020) and Hazel K and O'Connor A (2020).

[0123] The European Medicines Agency (EMA) Public Assessment Reports also provide educational information on the various therapies mentioned above (Xeljanz Public Assessment Report, EMA / CHMP / 853224 / 2016; Olumiant Public Assessment Report EMA / 13493 / 2017, 15 Dec. 2016; Jyseleca Public Assessment Report EMA / 424374 / 2020; Rinvoq Public Assessment Report EMA / 608624 / 2019 Corr.; Inrebic Public Assessment Report EMA / 705612 / 2020; Cibingo Public Assessment Report EMA / 647846 / 2021). Further examples of how changes in ABC-transporter levels demonstrate responsiveness are shown in the **Examples** chapter. [Examples]

[0124] Example 1: Materials and Method patient treatment All participating patients were enrolled in this study after obtaining written informed consent from the University of Szeged Albert Szent-Gyorgyi Medical School, Ist dept. of Medicine. This study was conducted in accordance with authorizations issued by the relevant national regulatory authorities (OGYEI, reg Nr 42432-4 / 2019 and ETT-TUKEB, BMEU / 728-1 / 2022 / EKU). Participants were broadly divided into two main groups. In the first cohort, patients with moderate to severe IBD (n=35; Crohn's disease (CD) n=17, ulcerative colitis (UC) n=18) were enrolled and treated with conventional disease-modifying agents (5-ASA, azathioprine) in combination with systemic glucocorticoids [methylprednisolone 32-64 mg].

[0125] The second cohort included patients with severe IBD (UC only) who had received tofacitinib treatment. Patients were randomized to a treatment group based on their administered medications. Patients in the tofacitinib-naive group had never taken tofacitinib before the initial blood draw and started tofacitinib treatment on the day of the blood draw. The active treatment group consisted of patients who had previously started tofacitinib treatment and were continuing to take it at the time of the blood draw. This group also included patients at the 12-week control visit who had not discontinued tofacitinib early for any reason. Patients in the non-responder group had previously discontinued tofacitinib due to lack of efficacy and still had high disease activity and severity. Peripheral blood samples anticoagulated with K3-EDTA were collected from all patients.

[0126] cell Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of IBD and UC patients using Ficoll-Hypaque density gradient centrifugation following a standard procedure. Blood was collected from each patient once or twice under sterile conditions using a blood collection tube (Vacuette, Greiner-BioOne) containing tripotassium EDTA (K3-EDTA). For some patients, it was not possible to perform a **second follow-up blood sample**. This included cases where surgical intervention (e.g., colectomy) was performed after the initial blood sample. In the first cohort, pre-treatment samples for small molecule drug therapy were typically collected before the patient began taking the medication (before treatment initiation), and a second blood sample was taken at the end of the follow-up period (FU). The follow-up period varied depending on the treatment. ●In the steroid treatment group, for 8 weeks ●The treatment period was 12 weeks in the 5-ASA or azathioprine treatment group. For the second cohort, samples were collected from the JAK inhibitor (tofacitinib) treatment group at the following two time points. ●At the time of the first blood draw: Before the patient has started taking tofacitinib (before treatment begins) ●Second blood draw: After continuing tofacitinib treatment for 12 weeks (during a regular follow-up visit)

[0127] Teresa Kit Reagents [Table 1]

[0128] antibody To measure MDR activity in isolated cell populations, fluorescently labeled population marker antibodies were used according to the Theresa protocol (see Table 1). [Table 2]

[0129] Flow cytometry in IBD patients treated with steroids Flow cytometry measurements were performed using the Solvo MDQ kit. Reagents were prepared according to the Instructions for Use (IFU) included with the Solvo MDQ kit. In measuring MDR1 activity, CD3 + Lymphocytes were loaded with calcein-AM**, and their transporter activity was inhibited with verapamil. For MRP1 activity measurement, a reagent pair consisting of calcein (fluorescent probe) and indomethacin (MRP1-specific inhibitor) was used, while for BCRP activity measurement, mitoxantrone (fluorescent probe) and KO-134 (inhibitor)** were used. The isolated peripheral blood mononuclear cells (PBMCs) were first washed twice with Hanks' Balanced Salt Solution (HBSS) (300g, centrifugation for 5 minutes twice). Then, 5 × 10⁶ cells were extracted from each sample. 5 Each PBMC pellet was processed according to the IFU (Instructional Facility). The cell suspension was kept in a constant temperature water bath at 37±5°C both before and during processing, and stored on ice in the dark until measurement.

[0130] Flow cytometry using the Teresa kit Sample preparation The following dilution conditions were calculated for MDR activity measurement in a single donor. For multiple donors, multiply the following dilutions by the number of donors to obtain the required amount of reagent. Transporter inhibitors are **ready-to-use** and do not require dilution. To prepare a 100 μM K3-EDTA-containing HBSS solution, 47.5 μl of Reagent 6 stock solution was diluted with 18.95 ml of HBSS solution. Calcein-AM activating solution was prepared by diluting 5 μl of Reagent 1 stock solution with 995 μl of 100 μM EDTA-HBSS activating solution. This calcein solution must be used within 1 hour of preparation. The working solution of Phen Green-SK diacetate was prepared by diluting 5 μl of the stock solution of Reagent 4 with 695 μl of 100 μM EDTA-HBSS working solution, and this working solution should be used within 15 minutes after preparation. The isolated PBMCs were first washed twice with HBSS (Capricorn) (centrifuged at 300 g for 5 minutes, twice). Then, 5×10 5 PBMC pellets per sample were suspended in 500 μl of HBSS containing 100 μM Reagent 6. The cell suspension was maintained in a constant temperature water bath at 37 ± 5°C both before and during the treatment.

[0131] Treatment with Teresa reagent For the inhibition of MDR transporter, the cell suspension was treated with the following inhibitors at 37 ± 5°C for 5 minutes: ● Tariquidar (2.02 μM, Reagent 2), 5 μl ● Indomethacin (2.53 mM, Reagent 3), 5 μl ● KO143 (hydrate, 101 μM, Reagent 5), 5 μl

[0132] To the control samples, 5 μl of HBSS containing 100 μM Reagent 6 without inhibitors was added. Without washing, 100 μl of Calcein-AM (0.605 μM, Reagent 1) or Phen Green (1.51 μM, Reagent 4) diluted to the working concentration in advance was added, and the cells were treated at 37 ± 5°C for 10 minutes in the dark. Immediately after centrifuging at 1000 g for 1 minute, the supernatant was removed to collect the cells. For labeling with population marker antibodies, the supernatant was completely removed and kept on ice as soon as possible for the purpose of minimizing the MDR transporter activity.

[0133] antibody labeling Primary cells include monocytes, CD4+ Lymphocytes and CD8 + To identify lymphocytes, they were stained with anti-human CD14 antibody, anti-human CD4 antibody, and anti-human CD8 antibody, respectively. The antibody labeling conditions are shown in Table 2. After incubation, unbound antibodies were washed with 1 ml of ice-cold HBSS without Reagent 6 and removed by centrifugation at 1000 g for 1 minute. Measurements were performed on a Partec / Sysmex Cyflow Space flow cytometer, and data analysis was carried out using FlowJo V10 software.

[0134] statistical analysis Statistical analysis was performed using **TIBCO Statistica (Windows version, version 14.0)**. A p-value < 0.05 was considered statistically significant. The specific statistical tests applied are described in the **Brief Description of the Figures**. Data collected from patients who discontinued treatment early or who missed the 12-week follow-up visit for any reason were excluded from the analysis due to the lack of longitudinal evaluation.

[0135] Example 2: Results from the first patient cohort treated with conventional disease-modifying drugs (cDMARDs and GCs) The initial patient cohort enrolled **patients with moderate to severe inflammatory bowel disease (IBD) (n=35)**, including both **Crohn's disease (CD, n=17) and ulcerative colitis (UC, n=18)**. These patients received **conventional disease-modifying drugs (cDMARDs)**, specifically 5-aminosalicylic acid (5-ASA) and azathioprine**, in combination with **systemic glucocorticoids (methylprednisolone, 32–64 mg) or topical glucocorticoids (budesonide, 9 mg)**.

[0136] Combined (MDR1 and MRP1) activity In IBD patients receiving steroid therapy, the CD3 count of steroid-non-responder patients was higher at **diagnosis (Week 0, Figure 1A)** and 8 weeks after the start of steroid therapy (Figure 1B)**. + A moderate elevation in MAFC levels in lymphocytes was observed compared to **responders**. In responsive patients, a mild decrease in MAFC levels (downregulation) was detected 8 weeks after the start of steroid therapy compared to Week 0 (Figure 1C). In contrast, non-responsive patients showed a **mild increase in MAFC levels (up-regulation)** compared to Week 0, 8 weeks after the start of steroid therapy (Figure 1D). The **cut-off value** at the time of diagnosis was set with **sensitivity of 75% and specificity of 72.72%** (Figure 1E, AUC = 0.6136). At 8 weeks after the start of treatment, the **cutoff sensitivity** reached 100% (Figure 1F, AUC = 0.7955).

[0137] [Table 3]

[0138] [Table 4]

[0139] Abbreviation: CRP: C-reactive protein WBC: White blood cell count Htk: Hematocrit value Hgb: Hemoglobin Plt: Platelet count Statistical notes: a: Comparison of Week 0 and Week 8 in responders. b: Comparison of Week 0 and Week 8 in non-responder patients. c: Comparison of reactive and non-reactive patients (Week 0) d: Comparison of reactive and non-reactive patients (Week 8) Statistical methods: If p ≤ 0.05, statistical significance was determined using Student's paired t-test. In patients treated with steroids, azathioprine, and 5-aminosalicylic acid (5-ASA), MAFC levels were slightly elevated in non-responders compared to responders before the initiation of small molecule drug therapy (Fig. 2A). This difference became more pronounced at the follow-up time (Fig. 2B) and was confirmed to be statistically significant. In responsive patients, small molecule drug therapy did not affect MAFC levels (Fig. 2C). On the other hand, in non-responsive patients, a significant increase in MAFC levels was observed during the follow-up week (Fig. 2D). Furthermore, the cut-off values ​​for **Week 0 (Fig. 2E) and the follow-up week (Fig. 2F)** were set with **specificity of 100% and sensitivity of 76.92%**.

[0140] [Table 5]

[0141] [Table 6]

[0142] Abbreviation: CRP: C-reactive protein WBC: White blood cell count Htk: Hematocrit value Hgb: Hemoglobin Plt: Platelet count Statistical notes: a: Comparison of Week 0 and Week 8 in responders. b: Comparison of Week 0 and Week 8 in non-responder patients. c: Comparison of reactive and non-reactive patients (Week 0) d: Comparison of reactive and non-reactive patients (Week 8) Statistical methods: If p ≤ 0.05, statistical significance was determined using Student's paired t-test.

[0143] MDR1 activity Eight weeks after the initiation of steroid therapy, MAFMDR1 levels in steroid-non-responder patients were significantly elevated (up-regulation) compared to steroid-responder patients. At the time of diagnosis, MAFMDR1 levels in non-responsive patients were slightly lower than those in responsive patients (Fig. 3A). However, a statistically significant increase was observed in non-responsive patients 8 weeks after the start of steroid therapy (Fig. 3B). On the other hand, in responsive patients, steroid therapy resulted in a moderate decrease (down-regulation) of MAFMDR1 levels (Fig. 3C), while in non-responsive patients, an increase in MAFMDR1 levels was observed (Fig. 3D). Interestingly, the **cutoff value (Fig. 3F)** 8 weeks after the start of steroid therapy was significantly lower than that at the time of diagnosis (Fig. 3E). CD3 +MAFMDR1 levels in lymphocytes showed high predictive value as a predictor of response to small molecule drug treatment in IBD patients. In non-responsive patients, pre-treatment MAFMDR1 levels were slightly lower than in responsive patients (Fig. 4A), but at follow-up, they were significantly higher than in responsive patients (Fig. 4B). In responsive patients, a significant decrease in MAFMDR1 levels was observed with successful treatment (Fig. 4C). On the other hand, in non-responsive patients, a significant increase (up-regulation) was detected after small molecule drug treatment compared to Week 0 (Fig. 4D). The cutoff values ​​for MAFMDR1 were determined at Week 0 (Fig. 4E) and the follow-up week (Fig. 4F)**, with higher sensitivity and specificity observed at the follow-up.

[0144] MRP1 activity Steroid therapy had some effect on MAFMRP1 levels, but no significant changes were observed. CD3 + MAFMRP1 levels in lymphocytes were measured at the start of steroid therapy and 8 weeks later. In Week 0, MAFMRP1 levels were slightly higher in non-responsive patients than in responsive patients (Fig. 5A). However, no significant difference was observed between the two groups after 8 weeks of steroid therapy (Fig. 5B). Interestingly, steroid therapy resulted in a mild increase in MAFMRP1 levels in reactive patients (Fig. 5C), while a moderate decrease was observed in non-reactive patients (Fig. 5D). The cutoff values ​​were set at **initiation (Fig. 5E)** and 8 weeks later (Fig. 5F)**, but the values ​​were the same at both time points, with Week 0 showing higher predictive sensitivity and specificity. Small molecule drug therapy also had a mild effect on MAFMRP1 levels, but no significant changes were observed. CD3 +MAFMRP1 levels in lymphocytes were measured at the time of diagnosis and during follow-up. At the time of diagnosis, MAFMRP1 levels in non-responsive patients were slightly higher than those in responsive patients (Fig. 6A). In contrast, a moderate elevation was observed in non-responsive patients during the follow-up week (Fig. 6B). In responsive patients, small molecule drug therapy resulted in a moderate increase in MAFMRP1 levels (Fig. 6C), while in non-responsive patients showed a moderate decrease (Fig. 6D). The cutoff values ​​were set for **Week 0 (Fig. 6E) and the follow-up week (Fig. 6F)**, but Week 0 showed higher predictive sensitivity and specificity.

[0145] BCRP activity Steroid therapy caused an increase in MAFBCRP levels (up-regulation) in non-responsive patients. At the time of diagnosis, MAFBCRP levels in non-responsive patients were significantly lower than those in responsive patients (Fig. 7A). However, eight weeks after the start of steroid therapy, MAFBCRP levels were higher in non-responsive patients than in responsive patients (Fig. 7B). In responsive patients, a decrease in MAFBCRP levels was observed with steroid therapy (Fig. 7C), while an increase was observed in non-responsive patients (Fig. 7D). The cutoff values ​​for BCRP functional activity were set for **Week 0 (Fig. 7E) and Week 8 (Fig. 7F)**, and **100% sensitivity (AUC = 0.7955)** was observed in Week 8. A significant decrease in MAFBCRP levels (down-regulation) was observed with small molecule drug therapy. In Week 0, MAFBCRP levels in non-responsive patients were significantly lower than those in responsive patients (Fig. 8A). However, at follow-up, this pattern was completely reversed, with MAFBCRP levels significantly higher in non-responsive patients than in responsive patients (Fig. 8B). In responsive patients, a significant decrease in MAFBCRP levels was observed with treatment (Fig. 8C), while in non-responsive patients, a significant increase was observed without statistical significance (Fig. 8D). Cutoff values ​​were set **before treatment (Fig. 8E) and at follow-up (Fig. 8F)**, with high sensitivity at Week 0 and higher specificity at the follow-up week.

[0146] Disease activity score Small molecule drug therapy resulted in **down-regulation** of disease activity scores in responsive patients. **The Mayo score (applicable to ulcerative colitis, Fig. 9A) and the CDAI score (applicable to Crohn's disease, Fig. 9B)** were measured at diagnosis (Week 0, black bars) and at follow-up (white bars). In responsive patients, both scores showed a clear decrease with treatment. On the other hand, non-responsive patients with ulcerative colitis showed higher Mayo scores than responsive patients. Furthermore, a significant decrease in CDAI scores was observed after treatment in Crohn's disease patients with poor treatment response.

[0147] Example 3: Results from a second patient cohort treated with targeted synthetic disease-modifying drugs (tsDMARDs). The second cohort included **patients with severe inflammatory bowel disease (IBD) (ulcerative colitis [UC] only)** who received treatment with **tofacitinib, a JAK inhibitor**. Patients were classified into the following treatment groups based on their administration status. (i) Tofacitinib-naive group ("Naive" group): Patients in this group had never taken tofacitinib before the initial blood draw and started treatment on the day of the blood draw. (ii) Tofacitinib treatment group ("Active" group): This group includes patients who previously started tofacitinib treatment and are still taking tofacitinib at the time of blood sampling. This group also includes patients who have reached the 12-week control visit without interrupting tofacitinib treatment. (iii) Non-responder group: Patients who had the tofacitinib discontinued early due to lack of treatment effect, including those with still high disease activity. Blood samples with K3-EDTA anticoagulant added were collected from all patients. Cell processing, antibody labeling, and flow cytometry measurements using the Theresa kit were performed according to the procedures described in Example 1 of "Materials and Methods". The measured **Raw measured data** are shown in Tables 7.1, 7.2, and 7.3. In the statistical analysis, since the number of patients was relatively small and only a very small number of patients were able to have samples collected at both Week 0 and 12-week follow-up, the control period and the follow-up collection period were not analyzed individually. Nevertheless, useful markers suggesting treatment responsiveness were unexpectedly clearly identified.

[0148] [Table 7-1]

[0149] [Table 7-2]

[0150] [Table 7-3]

[0151] Notes and Considerations Negative values obtained in the measurement were regarded as having no detectable transporter activity (MAF = 0). As a practical treatment, these values that were substantially equal to zero were input into the program as 0.1. This is because the statistical analysis software STATISTICA can more easily process the value of 0.1 than the zero value. Therefore, a value of MAF = 0.1 is considered to substantially indicate a lack of transporter activity. Therefore, data masking is not performed in this method, and it is methodologically sound.

[0152] Differences between the "Active" group and the "Non-responder" group Based on data from a limited number of IBD patients, the inventors confirmed the following statistically significant differences: ● CD8 + MDR1 transporter activity in lymphocytes ● CD14 + MDR1 and MRP1 transporter activities in monocytes

[0153] This result indicates that the measurement of transporter activity, particularly MDR1 and MRP1 activities, can be used to evaluate the effectiveness of ongoing treatment. Compared with conventional endoscopy, the measurement of transporter activity from blood samples has the advantages of being less invasive and providing results in a shorter time. Furthermore, the inventors found that there were also significant differences between the "Naive" group and the "Non-responder" group in the MDR1 activity of CD8 + cells and the MRP1 activity of CD14 + cells. This suggests the possibility of predicting non-responsive patients before the start of treatment. Receiver Operating Characteristic (ROC) curve analysis showed the relationship between sensitivity and specificity. Based on the analysis results based on the ROC curve and the sample size estimation, it was suggested that the sensitivity of this method may further improve with an increase in the number of patients.

[0154] Additional insights Unexpectedly, no statistically significant differences were observed between the "Naive" group and the "Active" group in any cell type. Furthermore, CD4+ Measurements targeting cells have proven to be of low utility. CD4 + This is because no significant difference was observed in the transporter activity obtained from the cells between the Responder group and the Non-responder group. This result is CD4 + While cells play a central and diverse role in regulating T cell-dependent adaptive and innate immune responses, their high degree of heterogeneity in cellular function suggests they are unsuitable for predicting therapeutic response. Comparison of tofacitinib-treated patients (active) and non-responsive patients (non-responders) In this experimental group, the results of **tofacitinib-unresponsive patients (non-responders)** were compared with those of **patients continuing treatment (active)**. The Active group was treated as a single group without further subdivision.

[0155] MDR1 activity / MDR1 activity CD3 + / CD8 + MAFMDR1 levels in lymphocytes were confirmed to be a strong predictor of tofacitinib treatment response in IBD patients. In non-responsive patients, MAFMDR1 levels were significantly lower than in the active group (Fig. 10). Also, CD14 + In monocytes, MDR1 transporter activity was shown to be a strong predictor of tofacitinib treatment response. In this case, MAFMDR1 levels were significantly higher in non-responsive patients than in the active group (Fig. 11).

[0156] MRP1 activity / MRP1 activity CD14 + MAFMRP1 levels in monocytes were also shown to be a strong predictor of tofacitinib treatment response. In non-responsive patients, MAFMRP1 levels were significantly higher than in the active group, which was consistent with the results for MDR1 in the same cell population (Fig. 12).

[0157] Comparison of tofacitinib-naive patients (naive) and non-responder patients (non-responder). In this experimental group, the results of patients who were unresponsive to tofacitinib were compared to those of **patients who did not receive tofacitinib (naive)**.

[0158] MDR1 activity / MDR1 activity CD3 + / CD8 + MAFMDR1 levels in lymphocytes were found to be a strong predictor of tofacitinib treatment response in IBD patients. In non-responsive patients, MAFMDR1 levels were significantly lower than in the naive group (Fig. 13). These results are consistent with the comparison between non-responsive patients and the active group, indicating that the reference value can be derived from either the naive or active group. Note that the naive group may include patients who have received prior treatment with steroids or other small molecule drugs.

[0159] MRP1 activity / MRP1 activity CD14 + MAFMRP1 levels in monocytes were also confirmed to be a strong predictor of tofacitinib treatment response. In non-responsive patients, MAFMRP1 levels were significantly higher than in the naive group (Fig. 14). These results were consistent with the MDR1 and MRP1 results in the comparison between non-responsive patients and the active group.

[0160] Summary These results were further confirmed by the fact that there was no significant difference in MAF values ​​for MDR1 and MRP1 between the Naive and Active groups (data not shown). These findings support that **transporter activity (especially MDR1 and MRP1)** is useful in predicting responsiveness to targeted synthetic DMARD therapy including tofacitinib and evaluating treatment efficacy.

Industrial Applicability

[0161] The inventors have found that the **treatment responsiveness of IBD patients to intracellularly acting immunosuppressive agents** can be evaluated by measuring the **ABC transporter activity level (especially the transport activity level)**. Furthermore, by the method of the present invention, it is also possible to monitor the progression or improvement of the patient's condition over time. Each measurement shown in this specification was carried out at the **initial stage of the treatment regimen (within 4 months from the start of treatment)**, and the measurements were taken at the start of treatment (Week 0) and at the follow-up after 8 or 12 weeks depending on the treatment content. Therefore, the method of the present invention is useful in assisting clinical decision-making regarding IBD treatment and further contributes to the establishment of new or improved treatment methods for IBD.

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Claims

1. An in vitro method for predicting the response of patients with inflammatory bowel disease (IBD) to treatment with intracellularly acting immunosuppressants, A step of providing a sample derived from a biological sample collected from the IBD patient before the start of treatment with the immunosuppressant or within four months after the start of treatment, the sample containing effector mononuclear cells, The process involves obtaining the transporter activity level by measuring the transport activity of a multidrug ABC transporter, preferably selected from classes B, C, and G, in effector mononuclear cells in the aforementioned sample. A step of comparing the transporter activity level with the reference transporter activity level, The step of considering the patient as unresponsive to treatment with the intracellular immunosuppressant if the transporter activity level is significantly different from the corresponding reference transporter activity level. A method that includes this.

2. An in vitro method according to claim 1 for evaluating the condition of an IBD patient who has previously been treated with an intracellular immunosuppressant, and for predicting whether the patient is unresponsive to the target intracellular immunosuppressant, A step of providing a biological sample of the IBD patient, which includes effector mononuclear cells derived from the patient, The steps include: measuring the transport activity of one or more multidrug ABC transporters in effector mononuclear cells in the sample to obtain transporter activity levels, preferably the activity values ​​of one or more transporters; A step of comparing the obtained transporter activity level, preferably one or more transporter activity values, with a reference transporter activity level, preferably one or more reference transporter activity values. The step of considering the patient as unresponsive to treatment with the intracellular immunosuppressant if the obtained transporter activity level, preferably one or more or the activity value of each transporter, is significantly different from the reference transporter activity level, preferably the reference transporter activity value. In vitro methods, including [specific methods].

3. The in vitro method according to any one of claims 1 to 2, wherein the biological sample is a blood-derived sample.

4. The in vitro method according to claim 3, wherein the effector mononuclear cell is a cell selected from the group consisting of lymphocytes, monocytes, and NK cells, preferably a CD3+ T lymphocyte, CD14+ monocyte, CD16+ NK cell, or neutrophil granulocyte.

5. The immunosuppressant acting within the cell is a small molecule drug used for IBD, and the small molecule drug is Glucocorticoids, optionally conventional disease-modifying agents, such as glucocorticoids used in combination with DMADs (cDMADs), Conventional disease-modifying drugs that do not contain glucocorticoids, for example, DMADs (cDMADs), Steroids, and tsdmadard, Selected from, The in vitro method according to any one of claims 1 to 4, wherein the ABC transporter is selected from B and G classes, preferably from ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (BCRP).

6. The in vitro method according to any one of claims 2 to 5, wherein the previously used intracellular immunosuppressant is a small molecule drug used for IBD, and the small molecule drug is an anti-inflammatory drug, preferably a steroidal anti-inflammatory drug or a nonsteroidal anti-inflammatory drug (NSAID).

7. The previously used intracellular immunosuppressant is a small molecule drug used for IBD, and the said small molecule drug is Glucocorticoids, optionally conventional disease-modifying agents, such as glucocorticoids used in combination with DMADs (cDMADs), or Conventional disease-modifying drugs that do not contain glucocorticoids, such as DMADs (cDMADs) The in vitro method according to any one of claims 2 to 4.

8. The in vitro method according to any one of claims 1 to 7, wherein the immunosuppressant acting within the cell is a cDMAD.

9. The in vitro method according to any one of claims 1 to 8, wherein the ABC transporter is selected from B and G classes, preferably from ABCB1 (MDR1) and ABCG2 (BCRP).

10. The in vitro method according to any one of claims 1 to 5, wherein the intracellular immunosuppressant is not a steroid or an NSAID, and preferably the second intracellular immunosuppressant is selected from small molecule anti-IBD drugs and tsDMARDs, and preferably from JAK1 / JAK3 inhibitors.

11. The in vitro method according to any one of claims 1 to 7 or 9 to 10, wherein the immunosuppressant acting within the cell is a tsDMARD.

12. The in vitro method according to claim 11, wherein the tsDMARD is selected from a JAK1 / JAK3 inhibitor, an S1PR agonist, and a PDE4 inhibitor, and is preferably a JAK1 / JAK3 inhibitor.

13. The method according to any one of claims 1 to 7, or preferably according to claims 11 to 12, wherein the ABC transporter is selected from B and C classes of ABC transporters, preferably from ABCB1 (MDR1) and ABCC1 (MRP1).

14. The in vitro method according to any one of claims 1 to 13, preferably any one of claims 1 to 10 and 12 to 13, wherein the patient is treated with an immunosuppressant that acts within the cells for a predetermined period, preferably the predetermined period is selected from at least two weeks, at least four weeks, at least one month, at least two months, or at least three months.

15. An in vitro method for diagnosis, as described in any one of claims 10 to 15.

16. The cells are lymphocytes, preferably CD3+ / CD8+ lymphocytes, and the MDR1 transport activity is measured in the sample to obtain the MDR1 transport activity value. The MDR1 transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state or obtained in the past. If the MDR1 transport activity value obtained from the sample is significantly lower than the reference value of 1 or more, the patient is considered a non-responder to the JAK1 / JAK3 inhibitor treatment. The in vitro method according to any one of claims 10 to 15.

17. The cells are monocytes, preferably CD14+ monocytes, and the MDR1 transport activity is measured in the sample to obtain the MDR1 transport activity value. The MDR1 transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state or obtained in the past. If the MDR1 transport activity value obtained from the sample is significantly higher than the reference value of 1 or more, the patient is considered a non-responder to the JAK1 / JAK3 inhibitor treatment. An in vitro diagnostic method according to any one of claims 10 to 15.

18. The cells are monocytes, preferably CD14+ monocytes, and the MRP1 transport activity in the sample is measured to obtain an MRP1 transport activity value. The MRP1 transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state or obtained in the past. If the MRP1 transport activity value obtained from the sample is significantly higher than the reference value of 1 or more, the patient is considered a non-responder to the JAK1 / JAK3 inhibitor treatment. An in vitro diagnostic method according to any one of claims 10 to 15.

19. The in vitro diagnostic method according to any one of claims 16 to 18, wherein the JAK1 / JAK3 inhibitor is tofacitinib.

20. The in vitro diagnostic method according to any one of claims 15 to 19, wherein the IBD patient is a responder to different JAK1 / JAK3 inhibitors.

21. The in vitro diagnostic method according to any one of claims 1 to 20, wherein the IBD is UC.

22. An in vitro diagnostic method according to any one of claims 1 to 21, wherein the patient is naive to the intracellular immunosuppressant at the time of or before the initiation of treatment with the intracellular immunosuppressant.

23. The aforementioned IBD patient received treatment with steroids as an immunosuppressant acting within cells. The cells are lymphocytes, preferably CD3+ lymphocytes. The combined MDR1 + MRP1 transport activity in the aforementioned sample is measured to obtain the combined MDR1 + MRP1 transport activity value. The aforementioned combined MDR1 + MRP1 transport activity value is compared with a reference value obtained from patients with a low-inflammatory state, or a value obtained in the past that is higher than that value. If the combined MDR1 + MRP1 transport activity value obtained from the aforementioned sample is significantly higher than the reference value of 1 or more, the patient shall be considered a non-responder to the steroid treatment. Preferably, the transport activity is higher than the reference value obtained from responders of the same treatment, and preferably, the transport activity is measured at least two weeks, one month, or one to three months after the start of steroid treatment, and optionally the reference value is the value measured at week 0. An in vitro diagnostic method according to any one of claims 5 to 9.

24. The aforementioned IBD patient receives treatment with a small molecule anti-IBD drug as an immunosuppressant that acts within the cell. The cells are lymphocytes, preferably CD3+ lymphocytes, and the combined MDR1+MRP1 transport activity in the sample is measured to obtain the combined MDR1+MRP1 transport activity value. The aforementioned combined MDR1 + MRP1 transport activity value is obtained from patients with a low-inflammatory state, or has been obtained in the past, and is compared with a reference value of 1 or more. If the combined MDR1 + MRP1 transport activity value obtained from the aforementioned sample is significantly higher than the reference value of 1 or more, the patient shall be considered a non-responder to the steroid treatment. Preferably, it is higher than the reference value obtained from respondents of the same treatment. Preferably, transport activity is measured at least two weeks, one month, or one to three months after the start of steroid treatment, with the reference value optionally set to the value measured at week 0, and optionally set to the value measured at the time of treatment or before treatment. Preferably, the low molecular weight anti-IBD drug is selected from a steroid, azathioprine, and 5-aminosalicylic acid (5-ASA), the in vitro diagnostic method according to any one of claims 5 to 9.

25. The aforementioned IBD patient received treatment with steroids as an immunosuppressant acting within cells. The cells are lymphocytes, preferably CD3+ lymphocytes. The MDR1 transport activity in the aforementioned sample is measured to obtain the MDR1 transport activity value. The MDR1 transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state, or obtained in the past. If the MDR1 transport activity value obtained from the sample is significantly lower than the reference value of 1 or more, the patient shall be considered a non-responder to the steroid treatment. Preferably, the transport activity is lower than the reference value obtained from responders of the same treatment, preferably measured at 0 weeks, and / or If the BCRP transport activity value obtained from the sample is preferably higher than the reference value obtained from a responder of the same treatment, the patient is considered a non-responder to the steroid treatment, and the transport activity is measured 1 to 3 months after the start of steroid treatment, and the reference value is optionally set to the value measured at that time. An in vitro diagnostic method according to any one of claims 5 to 9.

26. The aforementioned IBD patient receives treatment with a small molecule anti-IBD drug as an immunosuppressant that acts within the cell. The MDR1 transport activity was measured in the aforementioned sample to obtain the MDR1 transport activity value. The MDR1 transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state or obtained in the past. If the MDR1 transport activity value obtained from the sample is significantly higher than the reference value of 1 or more, the patient is considered a non-responder to the steroid treatment. Preferably, the transport activity is higher than the value obtained from responders of the same treatment, and is measured at least 2 weeks, 1 month, or 1 to 3 months after the start of steroid treatment, with the reference value optionally set to the value measured at the same time or week 0. Preferably, the low-molecular-weight anti-IBD drug is selected from a steroid, azathioprine, and 5-aminosalicylic acid (5-ASA). An in vitro diagnostic method according to any one of claims 5 to 9.

27. The aforementioned IBD patient received treatment with steroids as an intracellular immunosuppressant. The cells are lymphocytes, preferably CD3+ lymphocytes. The BCRP transport activity was measured in the aforementioned sample to obtain the BCRP transport activity value. The BCRP transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state or obtained in the past. If the BCRP transport activity value obtained from the sample is significantly lower or higher than the reference value of 1 or more, the patient shall be considered a non-responder to the steroid treatment. Preferably, the transport activity is lower than the reference value obtained from responders of the same treatment, preferably measured at 0 weeks, and / or preferably, If the BCRP transport activity value obtained from the sample is preferably lower than the reference value obtained from non-responders of the same treatment, the patient is considered a responder to the steroid treatment, and preferably the transport activity is measured at 0 weeks, and / or, If the BCRP transport activity value obtained from the sample is preferably higher than the reference value obtained from a responder of the same treatment, the patient is considered a non-responder to the steroid treatment, the transport activity is measured 1 to 3 months after the start of steroid treatment, and the reference value is optionally set to the value measured at that time. An in vitro diagnostic method according to any one of claims 5 to 9,

28. The aforementioned IBD patient received treatment with a small molecule anti-IBD drug as an intracellular immunosuppressant. The BCRP transport activity was measured in the aforementioned sample to obtain the BCRP transport activity value. The BCRP transport activity value is compared with one or more reference values ​​obtained from patients with a low-inflammatory state or obtained in the past. If the BCRP transport activity value obtained from the sample is significantly lower or higher than the reference value of 1 or more, the patient shall be considered a non-responder to the small molecule anti-IBD drug treatment. Preferably, the transport activity is lower than the reference value obtained from responders of the same treatment, preferably measured at 0 weeks, and / or preferably, If the BCRP transport activity value obtained from the sample is preferably lower than the reference value obtained from non-responders of the same treatment, the patient is considered a responder to the small molecule anti-IBD drug treatment, and preferably the transport activity is measured at 0 weeks, and / or If the BCRP transport activity value obtained from the sample is preferably higher than the reference value obtained from responders of the same treatment, the patient is considered a non-responder to the small molecule anti-IBD drug treatment, and the transport activity is measured 1 to 3 months after the start of the small molecule treatment. The reference value is optionally set to the value measured at the same time. Preferably, the low-molecular-weight anti-IBD drug is selected from a steroid, azathioprine, and 5-aminosalicylic acid (5-ASA). An in vitro diagnostic method according to any one of claims 5 to 9.

29. If the reference transporter activity value is 1 or higher, The above-mentioned one or more transporter activity values ​​are selected by comparing them with one or more predetermined threshold transporter activity levels. The in vitro diagnostic method according to any one of claims 1 to 28, wherein each predetermined threshold transporter activity level is a threshold for the transport activity of one or more multidrug transporters determined using the same one or more substrates.

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