ASSESSING RESPONSIVENESS OF RHEUMATOID ARTHRITIS PATIENTS TO BIOLOGICAL TREATMENT
20210382049 · 2021-12-09
Inventors
- Gergely TOLDI (Budapest, HU)
- Kata FILKOR (Salgótarján, HU)
- Péter SZERÉMY (Szeged, HU)
- András APJOK (Szeged, HU)
- Zoltán SZEKANECZ (Debrecen, HU)
Cpc classification
G01N21/6428
PHYSICS
G01N33/564
PHYSICS
G01N2800/102
PHYSICS
G01N2800/52
PHYSICS
International classification
G01N33/564
PHYSICS
Abstract
The relates to the field of diagnosis and treatment of Rheumatoid Arthritis, in particular of assessing responsiveness of rheumatoid arthritis patients to biological treatment. In particular the it has been found that measurement of MDR1 and/or MRP1 transport activities in the early phase of or before a bDMARD treatment is appropriate to provide a prediction on the effectiveness or success of bDMARD therapy once csDMARD therapy has failed. Thus, the invention relates to an in vitro diagnostic method for assessing the responsiveness of a sDMARD treated RA patient to bDMARD therapy, wherein preferably the patient is in need of a switch or modification of the sDMARD therapy by measuring transport activities of the above-mention transporters or their composite activities. The invention also relates to use of kits for the methods of the invention and methods for treatment comprising the diagnosis or prediction of the invention.
Claims
1-17. (canceled)
18. A method for treating an RA patient by assessing the responsiveness of a (synthetic disease-modifying antirheumatic drug (sDMARD) treated RA patient to biological synthetic disease-modifying antirheumatic drug therapy (bDMARD therapy), wherein the patient is in need of a switch or modification of the sDMARD therapy, said method comprising the steps of providing a biological sample of said sDMARD treated RA patient, said sample comprising CD3.sup.+ T-lymphocytes from said patient, obtaining one or more transporter activity value(s) by measuring transport activity by MDR1 (ABCB1) in the CD3.sup.+ T-lymphocytes of said sDMARD treated RA patient, before or at an initial phase of a bDMARD therapy, by using one or more detectable, fluorescent substrate(s) of MDR1, said substrate(s) being taken up by CD3.sup.+ T-lymphocytes once contacted with them in a biological sample, comparing the one or more transporter activity value(s) with one or more pre-determined threshold transporter activity level(s), wherein each pre-determined threshold transporter activity level is a threshold value for the transport activity of said one or more multidrug transporters and which has been determined using the same one or more substrates, carrying out a bDMARD therapy when the level of each transporter activity value is not higher than the respective threshold level.
19. (canceled)
20. The method of claim 18, wherein the transporter activity value is obtained by measuring transport activity of one or more multidrug transporters comprising MDR1 and MRP1, in the CD3.sup.+ T-lymphocytes of said sDMARD treated RA patient before a bDMARD therapy or in an initial phase thereof, and the transporter activity value is compared with a pre-determined threshold transporter activity level, wherein said pre-determined threshold transporter activity level has been determined using the same substrate, carrying out a bDMARD therapy when the level of each transporter activity value is not higher than the respective threshold level.
21. The method of claim 20 wherein said MDR1 and MRP1 activity is measured with a substrate of both MDR1 and MRP1, the transporter activity comprising activities of both MDR1 and MRP1 and the two transporters are not differentiated by inhibition, whereby a composite transporter MDR1-MRP1 activity value is obtained.
22. The method of claim 18 wherein said threshold transporter activity level has been determined by measuring the transport activity of said one or more multidrug transporters in the CD3.sup.+ T-lymphocytes in a reference patient group known to be responder to the bDMARD therapy and a further reference patient group known to be non-responder to the bDMARD therapy, and the transport activity values measured in the responder and non-responder patient groups are statistically analysed as distributions to find a threshold level which differentiates between responder transport activity values and non-responder transport activity values.
23. The method of claim 20 wherein measuring the transporter activity comprises contacting at least the CD3.sup.+ T-lymphocytes in the biological sample with the one or more transporter substrate(s), said substrate being a derivative of a detectable fluorescent compound, and wherein said derivative is taken up by at least the CD3.sup.+ T-lymphocytes and is hydrolyzed into said fluorescent compound in the cells, wherein said fluorescent compound gets trapped inside said T-lymphocytes, and measuring fluorescence in the CD3.sup.+ T-lymphocytes, obtaining the transport activity value from the fluorescence in the CD3.sup.+ T-lymphocytes.
24. The method of claim 18 wherein the substrate is a detectable fluorescent ester compound, and the activity is quantified as a multidrug activity factor (MAF), wherein the fluorescent ester compound is a calcein ester.
25-31. (canceled)
32. The method of claim 18, wherein said bDMARD therapy is selected from the group consisting of anti-TNF therapy, T-cell activation inhibitor therapy, B lymphocyte depletion therapy, anti-IL6 therapy, preferably anti-TNF therapy and T-cell activation inhibitor therapy.
33. The method of claim 18, wherein measuring the transporter activity comprises contacting at least the CD3.sup.+ T-lymphocytes in the biological sample with the one or more transporter substrate(s), said substrate being a derivative of a detectable fluorescent compound, and wherein said derivative is taken up by at least the CD3.sup.+ T-lymphocytes and is hydrolyzed into said fluorescent compound in the cells, wherein said fluorescent compound gets trapped inside said T-lymphocites, and measuring fluorescence in the CD3.sup.+ T-lymphocytes, obtaining the transport activity value from the fluorescence in the CD3.sup.+ T-lymphocytes.
34. The method of claim 18, wherein the sDMARD therapy is a “csDMARD” therapy (“classic synthetic” or “conventional synthetic disease-modifying antirheumatic drug” therapy), optionally selected from the group of compounds consisting of azathioprine, cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine and glucocorticoids, preferably selected from methotrexate, chloroquine and salazopryne.
35. The method of claim 18 wherein said threshold transporter activity level has been determined by measuring or quantifying the transport activity of said MDR1 in the CD3.sup.+ T-lymphocytes in a reference patient group known to be responder to the bDMARD therapy and a further reference patient group known to be non-responder to the bDMARD therapy, and the transport activity values measured in the responder and non-responder patient groups are statistically analysed as distributions to find a threshold level which differentiates between responder transport activity values and non-responder transport activity values.
36. The method of claim 18, wherein said method comprises carrying out an alternative therapy when the level of said one or more transporter activity value(s) measured is above said respective threshold level, said alternative therapy being preferably a targeted synthetic disease-modifying antirheumatic drug (tsDMARD) therapy.
37. An in vitro diagnostic method for assessing the responsiveness of a synthetic disease-modifying antirheumatic drug (sDMARD) treated RA patient to biological synthetic disease-modifying antirheumatic drug therapy (bDMARD therapy), said method comprising the steps of providing a biological sample of said sDMARD treated RA patient, said sample comprising CD3.sup.+ T-lymphocytes from said patient, obtaining one or more transporter activity value(s) by measuring transport activity by MDR1 (ABCB1) in the CD3.sup.+ T-lymphocytes of said sDMARD treated RA patient, before or at an initial phase of a bDMARD therapy, by using one or more detectable, fluorescent substrate(s) of MDR1, said substrate(s) being taken up by CD3.sup.+ T-lymphocytes once contacted with them in a biological sample, comparing the one or more transporter activity value(s) with one or more pre-determined threshold transporter activity level(s), wherein each pre-determined threshold transporter activity level is a threshold value for the transport activity of said one or more multidrug transporters and which has been determined using the same one or more substrates, considering said RA patient as a non-responder to the bDMARD therapy when the level of said one or more transporter activity value(s) measured is above said threshold level, and considering said RA patient as a responder to the bDMARD therapy when the level of each transporter activity value is not higher than said threshold level.
38. The in vitro method of claim 37, wherein said bDMARD therapy is selected from the group consisting of anti-TNF therapy, T-cell activation inhibitor therapy, B lymphocyte depletion therapy, anti-IL6 therapy, preferably anti-TNF therapy and T-cell activation inhibitor therapy.
39. The in vitro method of claim 37, wherein measuring the transporter activity comprises contacting at least the CD3.sup.+ T-lymphocytes in the biological sample with the one or more transporter substrate(s), said substrate being a derivative of a detectable fluorescent compound, and wherein said derivative is taken up by at least the CD3.sup.+ T-lymphocytes and is hydrolyzed into said fluorescent compound in the cells, wherein said fluorescent compound gets trapped inside said T-lymphocites, and measuring fluorescence in the CD3.sup.+ T-lymphocytes, obtaining the transport activity value from the fluorescence in the CD3.sup.+ T-lymphocytes.
40. The in vitro method of claim 37, wherein the one or more transporter activity value is/are obtained by measuring transport activity by both MDR1 and MRP1 in the CD3.sup.+ T-lymphocytes of said sDMARD treated RA patient before a bDMARD therapy or in an initial phase thereof, and the transporter activity value is compared with a pre-determined threshold transporter activity level, wherein said pre-determined threshold transporter activity level has been determined using the same substrate, considering said RA patient as a non-responder to the bDMARD therapy when the level of the MDR1 and MRP1 transporter activity value is above said threshold level, and considering said RA patient as a responder to the bDMARD therapy when the level of the MDR1 and MRP1 transporter activity value is not higher than said threshold level.
41. The in vitro method of claim 37 wherein said threshold transporter activity level has been determined by measuring the transport activity of said one or more multidrug transporters in the CD3.sup.+ T-lymphocytes in a reference patient group known to be responder to the bDMARD therapy and a further reference patient group known to be non-responder to the bDMARD therapy, and the transport activity values measured in the responder and non-responder patient groups are statistically analysed as distributions to find a threshold level which differentiates between responder transport activity values and non-responder transport activity values.
42. The in vitro diagnostic method of claim 37 wherein the substrate is a detectable fluorescent ester compound, and the activity is quantified as a multidrug activity factor (MAF), wherein preferably the fluorescent ester compound is a calcein ester.
43. The method of claim 37, wherein said method comprises carrying out an alternative therapy, when the level of said one or more transporter activity value(s) measured is above said respective threshold level, said alternative therapy being preferably a targeted synthetic disease-modifying antirheumatic drug (tsDMARD) therapy.
44. Method for use of a kit for assessing the responsiveness of a synthetic disease-modifying antirheumatic drug (sDMARD) treated RA patient to biological synthetic disease-modifying antirheumatic drug therapy (bDMARD therapy) before or at an initial phase of the bDMARD therapy, or for a purpose as defined herein, by obtaining one or more transporter activity value(s) by measuring transport activity by MDR1 (ABCB1) in the CD3.sup.+ T-lymphocytes of said sDMARD treated RA patient, wherein said RA patient is considered as a non-responder to the bDMARD therapy when the level of each transporter activity value is above a respective threshold level, and considering said RA patient as a responder to the bDMARD therapy when the level of each transporter activity value is not higher than a respective threshold level, said kit comprising one or more substrate(s) of MDR1 for the measuring of the respective transporter activity, said substrate being taken up by CD3.sup.+ T-lymphocytes once contacted with them in a biological sample, wherein said substrate is a detectable, fluorescent substrate, optionally a label for CD3.sup.+ T-lymphocytes.
45. The method of claim 44, wherein the one or more transporter activity value(s) is/are obtained by measuring transport activity by both MDR1 and MRP1 in the CD3.sup.+ T-lymphocytes of said sDMARD treated RA patient before a bDMARD therapy or in an initial phase thereof, wherein said kit comprises, a substrate for MDR1 and MRP1 for the measuring MDR1 and MRP1 transport activities or a composite MDR1 and MRP1 transport activity, said substrate being taken up by leukocytes, once the CD3.sup.+ T-lymphocytes are contacted with the reagents in a biological sample, wherein said substrate is detectable, fluorescent substrate; label for CD3.sup.+ T-lymphocytes, and preferably inhibitor for MDR1, MRP1 and/or inhibitor for one or more other multidrug transporter.
46. The method of claim 44, wherein measuring the transporter activity comprises contacting at least the CD3.sup.+ T-lymphocytes in the biological sample with the one or more transporter substrate(s), said substrate being a derivative of a detectable fluorescent compound, and wherein said derivative is taken up by at least the CD3.sup.+ T-lymphocytes and is hydrolyzed into said fluorescent compound in the cells, wherein said fluorescent compound gets trapped inside said T-lymphocites, measuring fluorescence in the CD3.sup.+ T-lymphocytes, and obtaining the transport activity value from the fluorescence in the CD3.sup.+ T-lymphocytes.
47. The method of claim 44, wherein the substrate is a detectable fluorescent ester compound, and the activity is quantified as a multidrug activity factor (MAF), wherein preferably the fluorescent ester compound is a calcein ester, preferably calcein AM.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0213]
[0214]
[0215]
[0216]
[0217] The curves demonstrate cut-off values based on various sensitivity and specificity values. The closer we are to the upper left corner of the graph, the more specific and sensitive the cut-off value is. Since no test with perfect specificity and sensitivity exists in real life, a compromise needs to be made against variable specificity and sensitivity values. In our calculations, these values were chosen to be above 60-70% where possible.
DETAILED DESCRIPTION
[0218] Current Treatment Recommendations in RA
[0219] RA is a common inflammatory rheumatic disease which causes persistent pain, stiffness and joint damage resulting in significant disability, loss of quality of life and employment. The disease mostly affects women and it appears at the 5.sup.th decade of the life.
[0220] Based on current guidelines, treatment aims to induce clinical and radiological remission for optimizing physical function, improving the quality of life and work capacity and reducing the risk of comorbidities (Linde, Sorensen et al. 2010; Provan, Semb et al. 2011; van der Heijde 2012; Kavanaugh, Fleischmann et al. 2013; Thiele, Huscher et al. 2013; Radner, Smolen et al. 2014). Current treatment guidelines recommend treatment with csDMARD, in particular, MTX eventually in combination with glucocorticoids to be administered for newly diagnosed RA patients which is applicable in several cases. If the first line MTX therapy does not improve symptoms the next step may be either to switch to another csDMARD (e.g., sulfasalazine, leflunomide, hydroxychloroquine) or to add a biological DMARD (bDMARD) to csDMARD e.g. MTX therapy.
[0221] The activity of RA in patients is characterized or quantified by a combined index called Disease Activity Score (DAS28) (Fransen and van Riel 2005). It has been extensively validated for its use in clinical trials in combination with the European League Against Rheumatism (EULAR) response criteria. The DAS28 score is based on the examination of 28 joints.
[0222] There are a wide range of measures of disease activity in RA including: examination of the joints for swelling and tenderness, applying a global score of pain and overall status, possibly in the form of questionnaires, measuring blood markers of inflammation (e.g. ESR and CRP), the presence of anti-citrullinated antibodies (ACPA) measurement by X-rays or other, possibly newer imaging techniques such as ultrasound and MRI, however, an RA specific biomarker to determine prognosis and/or treatment response has not characterized yet.
[0223] Evaluation of response to a treatment can be made much easier and more objective using the DAS or DAS28. The DAS will provide a number between 0 and 10, indicating how active the RA is at this moment, however, DAS28 serves as a real-time data, it does not reflect to the possible disease outcome.
[0224] MDR protein function may also predict patient response to csDMARD treatment as well as biological treatment helping the physician to tailor the therapy. However, switching to biologicals (bDMARDs), including the case when csDMARD treatment is continued in parallel, is often challenging due to unpredictable drug susceptibility and high costs, especially in patients with mildly elevated DAS28 scores.
[0225] Earlier results of the present inventors and others by measuring MDR1, MRP1 and BCRP activities with the SOLVO MDQ Kit™, with cell surface staining applied to differentiate CD3.sup.+, CD4.sup.+ and CD19.sup.+ cells suggested that low BCRP and MRP1 MAF activities on CD3.sup.+ cells may predict the need to start biological therapy in RA patients whose symptoms do not improve on csDMARD treatment. In this setting DAS28 scores, CRP, IL-6, aCCP and RF values were also recorded. It has been suggested that further decrease of CD3.sup.+ BCRP and increase in CD3.sup.+ MRP1 MAF upon follow-up may indicate a good therapeutic response to biological therapy.
[0226] To date, although the role of MDR transporter activity in the prediction of response to MTX has been characterized to some extent in RA (see the Background Art chapter above), little is known about the relation of MDR proteins to therapeutic success of biologicals. In contrast to MTX and other csDMARDs, these molecules do not enter the cell, and are therefore not substrates of MDR proteins. However, the cytokines they target are known to interact with these transporters which may provide an indirect effect on these transporter, hitherto largely unknown.
[0227] In the prior art clear guidance was not provided as to how to predict the effectiveness of a bDMARD therapy, in particular, anti-TNF therapy, after a csDMARD therapy has been found insufficient before biological therapy is started, in particular not by measuring multidrug transporter activity.
[0228] The present inventors have unexpectedly recognized that measuring at least MDR1 or MRP1 activity or MDR1 and MRP1 composite (MAF.sub.C) activity in CD3.sup.+ cells in early stage of bDMARD treatment or even before bDMARD treatment a prediction can be made on the effectiveness of bDMARD therapy. Further measurements may also help further the reliability as disclosed herein.
[0229] The results of the present inventors indicate that the determination of MAF.sub.C values in CD3.sup.+ cells of RA patients is of predictive value prior to the initiation of biological therapy to establish whether the patient will demonstrate sufficient therapeutic response to a biological therapy, in particular anti-TNF therapy or anti-T-cell therapy. Moreover, it has been found that determination of MAF.sub.MDR1 values in CD3.sup.+ cells of RA patients is also appropriate to find a threshold which is predictive prior to the initiation of biological therapy to decide whether the patient will be respondent or non-respondent to biological therapy. demonstrate sufficient therapeutic response. A similar tendency could be observed with MRP1 at 0 weeks of the bDMARD treatment, however, in the experiments the distributions of the MAF.sub.MDR1 values for responders and non-responders could be separated to a lesser extent. Nevertheless, a threshold value can plausibly be found in this type of measurement as well which may predict non-responders e.g. with a sufficient sensitivity or at least positive predictive value.
[0230] It is of particular advantage of both MAF.sub.MDR1 and MAF.sub.C values are determined and both of them is above the given pre-determined threshold whereby the patient can be considered as a non-responder, or not higher than the threshold wherein the patient is an expectable responder to the bDMARD treatment. Additionally, determining MAF.sub.MDR1 values will contribute to the reliability of the test.
[0231] It is of high importance that the present method is appropriate to provide predictors before the start of the bDMARD therapy as in this way applying an expensive therapy, with special danger of side-effects and loss of time by using an ineffective therapy. The results provide an option to decide about further treatment before symptoms reflect the success of the therapy. As in RA the window of opportunity to apply therapy is limited, the invention is useful to find the appropriate treatment in time.
[0232] Nevertheless, the method can also be applied in an initial state of the bDMARD therapy for example within the first two months the latest or preferably earlier, e.g. in the first 6 weeks or the first 4 weeks or the first 2 weeks of the therapy. Certain predictive values can even be more pronounced in this stages.
[0233] Thus, if bDMARD therapy has already been started, it makes sense to run the method of the invention as early as possible. Moreover, if the first measurement resulted in a result which allowed to try and initiate bDMARD therapy it is or may be advisable to repeat the measurement at a later though still relatively early stage, e.g. at 4 to 7 weeks of the bDMARD therapy. Measuring MAF.sub.C, MAF.sub.MRP and MAF.sub.MDR values in CD3.sup.+ cells at 4 to 7 weeks after the start of biological treatment further improves the accuracy of prediction as to whether adequate therapeutic response may be expected. This knowledge provides help the physician to individually tailor the patient's therapy in a timely manner resulting in positive implications with regards to the cost of treatment and the spectrum of side effects.
[0234] The skilled person will understand that MAF values report on transport activity of the substrate applied, and other methods to measure or quantify transport activities in the CD3.sup.+ T-lymphocytes can be used in the present invention. Also using a substrate which is transportable by both MRP1 and MDR1 is preferred as when a composite activity value is to be obtained this can be done simply and reliably in a single measurement. The individual activity values for MDR1 and MRP1 can be obtained by using specific inhibitors in this setting.
[0235] It is of particular advantage is the substrate is reportable, preferably fluorescent and the results can be obtained and quantified by flow cytometry. This is particularly advantageous as in the present setting the transport activity is to be measured in CD3.sup.+ T-lymphocytes only.
[0236] The skilled person will also understand that applying the MAF values to report on activities is preferred as this way of quantification of the results reliably reports the activity in a manner which is highly independent from conditions of the measurement, like flow cytometry parameters, and rather sensitive to the cell type which is in line with the nature of this inventive method.
[0237] In the present invention when patient samples are measured and responsiveness is assessed no control samples are necessary as pre-determined threshold (cut-off) values are applied.
The Utility of csDMARDs and bDMARDs in RA
[0238] Today, recommendations for RA treatment are based on the current EULAR guideline (Smolen, Landewe et al. 2017). The most important feature of this guideline is that the decision making should be shared between the patient and the rheumatologist, however, the aim of the therapy is to achieve the treatment goal of remission or at least low disease activity within the time frame of 6 months, at least 50% clinical improvement within 3 months is desirable (Aletaha, Alasti et al. 2016). To achieve this goal Therapy should be started as soon as possible, preferably at the time of diagnosis. Importantly, therapy success should be monitored regularly especially in active disease (every 1-3 moths) and, if there is no improvement, or the goal is not reached by 6 moths, therapy should be modified. If the therapy goals are achieved, the dose of the respective medicine could be declined, or, in complete remission, terminated.
[0239] For checking treatment success, the determination of DAS28 levels together with the measurement of rheuma factor (RF), CRP, ACPA and erythrocyte sedimentation rate (ESR) are widely used and a person skilled in the art is able to apply these methods to the present invention.
[0240] csDMARDs, especially MTX together with lefluonomide, sulfasalazine and hydroxychloroquine, or in some cases, glucocorticoids, serve as Phase I therapy. Although these agents sometimes have poorly tolerated side effects, MTX should be the first medication in present EULAR recommendations. Importantly, when MTX is contraindicated, or its side effects are poorly tolerated, the patient should be switched to leflunomide, or bDMARD.
[0241] Anti-TNF agents (infliximab, trade name: Remicade; etanercept, trade name: Embrel, adalimumab, trade name Humira, golimumab, trade name: Simponi and certolizumab pegol, trade name: Cimzia) serve as the first-line biological originator (bo)DMARDs, since biosimilar (bs)DMARDs are also available. The first suggested anti-TNF agent is infliximab (IFX), however, it is a chimeric monoclonal, thus, anti-drug antibodies may develop which drastically cuts down the efficiency of this expensive therapy. When first-line anti-TNF agent (bo or bs, respectively) is not successful, another anti-TNF antibody should be used. Importantly, drugs altering immune response should carefully be applied, as in some cases, it may lead infections, moreover, to cancer development (Bongartz, Sutton et al. 2006). Importantly, these Phase II therapies should be given in parallel with csDMARDs or glucocorticoids to make therapy more effective (Nurmohamed and Dijkmans 2008).
[0242] When the treatment goal was not reached by using anti-TNF agents, other bDMARDs should be used. These drugs are targeting costimulation (T cell activation, abatacept, trade name: Orencia), causing B cell depletion (rituximab, trade name: Rituxan), blocking IL-6 receptor (tocilizumab, trade name: Actemra, sarilumab, trade name: Kevzara), IL-6 inhibitors (clazakizumab, sirukumab), or blocking IL-1 receptor (anakinra, trade name: Kineret). These treatments, together with tsDMARDS serve as Phase III therapy. tsDMARDs are inhibiting JAK kinases (tofacitinib, trade name: Xeljanz, or baricitinib, trade name: Olumiant). Importantly, Phase II and Phase III therapies should be given in parallel with csDMARDs and it is based on the patient's necessities.
[0243] It is then up to the medical personnel guiding the treatment that in case of non-responsiveness for a patient treated or to be treated by anti-TNF therapy or anti T cell activation therapy should be switched to a tsDMARD therapy or at first other bDMARD therapy of different target, like B-cell depletion or IL-6 inhibitors or blocking IL-6 receptor or IL-1 receptor should be applied. Advisably the contemporary EULAR guidance or its national variant should be observed.
MDRs in Health and Disease
[0244] Transport of compounds between the intra- and extracellular compartments is an essential physiologic phenomenon. For this, several transmembrane pumps evolved, showing strong sequence homology between different species.
[0245] The core functional unit of ABC transporters contains two membrane-spanning domains, each of which typically contains 6 transmembrane (TM) helicases. In the intracellular compartment, 2 nucleotide-binding domains (NBDs) are localized which contain Walker A and Walker B domains, that are necessary for ATP binding and hydrolysis (Deeley, Westlake et al. 2006; Silva, Almeida et al. 2015),
[0246] As ABC transporters originally involved in the detoxification of the organism, the members of the ABC transporter family are expressed on a wide variety of tissues and organs, like intestine, lung, liver, testes, placenta, skeletal and cardiac muscle and on the endothelial surface of the blood-brain barrier (Flens, Zaman et al. 1996; St-Pierre, Serrano et al. 2000; Wijnholds, deLange et al. 2000; Mercier, Masseguin et al. 2004; Castilho-Martins, Canuto et al. 2015). The majority of ABC transporters are expressed on the apical and basolateral surface of polarized cells (Hipfner, Gauldie et al. 1994; Evers, Zaman et al. 1996), however, in special cases, i.e. in drug selected cell lines, MDR1 is shown to be localized in the Golgi complex as well (Cole, Bhardwaj et al. 1992).
[0247] Beside their crucial role in the maintenance of homeostasis, ABC transporters are also involved in the phenomenon, called multidrug resistance (MDR), which makes therapy ineffective by removing drugs from target cells. Since MDR is the principal mechanism by which many tumours develop resistance to chemotherapeutics or immunosuppressant drugs administered in different types of leukaemia, solid tumours and autoimmune diseases and to patients who underwent transplantation. Conventional anticancer drugs (doxorubicin, gefitinib, irinotecan, methotrexate, paclitaxel, tamoxiphen, topotecan, etc.) are substrates of MDR transporters. Moreover, MDR transporters play distinct role in the fine tuning of the immune response.
[0248] qRT-PCR, immunohistochemistry and Western blots are the most frequently used methods to determine the MDR transporter status in clinical samples. More recently, mass spectrometry based methods have been described to quantify transporter expression (Prasad, Lai et al. 2013). On the other hand, several polymorphisms affecting transporter functions have been reported (Porcelli, Lemos et al. 2009; Lee, Chau et al. 2010). Therefore, relevance of even protein levels as solitary pieces of data is questionable. Some of the genetic variants affect transporter trafficking, and, thus, FACS-based determination of cell surface expression of MDR transporters is a significant progress (Damiani, Tiribelli et al. 2006). However, antibodies recognizing the extracellular MDR1 (Georges, Tsuruo et al. 1993; Vasudevan, Tsuruo et al. 1998) and BCRP (Telbisz, Hegedus et al. 2012) epitopes are conformation sensitive, making their determination challenging.
[0249] Such methods can be applied in and to the present invention, however, adaptation is needed. Using fluorescent substrates and flow cytometry and quantifying the results as MAF values as shown herein is advantageous due to reliability and simplicity. Using a calcein ester which is substrate of both MDR1 and MRP1 and which is trapped in the CD3.sup.+ T-lymphocytes once cleaved are of particular advantage as explained more specifically below.
The Determination of Transporter Activities
[0250] The transporter activity can be among other measured by a kit designed for functional quantitative measurement of drug resistance in live cells. The procedure is preferably fast, sensitive, and quantitative. The procedure should preferably measure the drug transport activity of at least two subfamilies of multidrug resistance proteins: MDR1 and MRP1. MDR1 and MRP1 are ATP-dependent trans-membrane proteins that remove hydrophobic xenobiotic compounds (typically environmental toxins) from the cell. A preferred kit utilizes calcein-AM, a non-fluorescent hydrophobic compound that enters all cells by passive diffusion via the plasmamembrane. Calcein-AM is an excellent substrate for targeted extrusion by multi-drug transporters. If MDR1 and MRP1 are active, the hydrophobic calcein-AM will be removed intact before it can be hydrolyzed. If MDR1 and MRP1 are not active, enzymatic cleavage of the calcein-AM by endogenous esterases results in the fluorescent hydrophilic free-acid, calcein, which is retained within the cytoplasm. Normal, drug sensitive cells will fluoresce when exposed to calcein-AM. The degree of fluorescence observed in test cells is inversely proportional to MDR1 and MRP1 activity. An example for such a kit is The SOLVO MDQ Kit which has CE-IVD certification (available from MDQuest, Szeged, Hungary).
[0251] Quantitation of this fluorescence is possible through the development of the MDR Activity Factor (MAF). The dye efflux activity of the MDR transporter is measured as the difference between the amount of the dye accumulated in the presence and absence of inhibitors.
[0252] The fluorescence measurement in the presence of an inhibitor specific to both MDR1 and MRP1 constitutes the maximal potential fluorescence with the given cell population when the multidrug transporters are rendered nonfunctional. This represents a standardization method, which eliminates unknown cell type-specific variables that influence cellular calcein accumulation, such as esterase activity, cell size, etc. This, in turn, allows for intra- and interlaboratory comparison of test results and MAF values. The transport activity of MDR1 and MRP1 can be easily distinguished with inhibitors specific to one of these proteins.
[0253] Inhibitors, which are known to those skilled in the art, preferably includeverapamil, and also included, but are not limited to, e.g., verapamil, indomethacin, oligomycin, or cyclosporin.
[0254] The kit has been optimized for and its preferred use is in flow cytometry, but can be adapted for use in other cell-based assay formats such as fluorescence microscopy, spectrophotometry, or 96 well plate assays. If these applications are utilized it is necessary to consider the following: [0255] Heterogeneous cell populations accumulate calcein at different rates, which cannot be resolved by fluorometry (cuvette or plate reader). [0256] Homogeneous cell population can be easily tested in the above-mentioned formats. [0257] For consistency and reproducibility, adequate mixing of cell suspensions and temperature control are necessary. [0258] Protocol adaptation for other formats will be necessary.
[0259] A more detailed description of a particular kit is provided in the Examples.
Practical Aspects in Carrying Out Transport Activity Measurements
[0260] In particular, for determining transporter functions in RA patients with commercially available detection kits, application of any internal and external controls are not required, since the cut-off values for each transporters in all time points are clearly defined.
[0261] For testing the performance of the preferred kit, cell lines overexpressing of MDR1, MRP1 and BCRP could be used.
[0262] For checking the flow cytometry equipment, commercially available fluorescent microbeads are recommended.
[0263] In one embodiment of the method of detecting multi-drug resistance in a biological sample, the control cells can be a portion of the biological sample itself, the method further including exposing the control cells to an inhibitor of multi-drug resistance. By using portions of the same biological sample, or by controlling the temporal sequence by which the components are added, the control acts as an internal, or “self”, control.
[0264] The MAF values of healthy adults on CD3.sup.+ T lymphocytes have already been determined according to the CLSI guideline C28-A2. In that study, 120 healthy adults (age between 18 and 74 years) were enrolled. In parallel with measuring MAF values, CD4/CD8 ratio, blood cell count, liver and kidney function were determined. For performing transporter activity measurements, 6 mls of K.sub.3EDTA anticoagulated peripheral blood samples were collected from each individual. PBMCs were separated by using Ficoll Histopaque density gradient centrifugation according to the manufacturer's instructions. The applied assay was performed as it was described by the instructions for users. After running assay, CD3.sup.+ cells were labelled with PerCP or FITC conjugated anti-CD3 antibodies. The measurements were carried out on a BD FACSCalibur flow cytometer equipped with 488 nm argon and 635 nm red diode lasers. The calculation of MAF values were performed as it was described previously. Importantly, no statistical significance was determined between men (n=62) and women (n=58). Interestingly, the age of the individual had no impact on MAF values in case of MRP1 and BCRP, however, in case of MDR1 and MAF.sub.C values, a negative correlation was determined between the values and the age of the studied individuals. Based on the previous facts, the cut-off values for transporter activities, which can be considered as an average for the healthy European adult population, are the following: MAF.sub.C: 16.5; MAF.sub.MDR1: 12.9; MAF.sub.MRP1: 2.5; MAF.sub.BCRP: 3.4.
Finding an Activity Threshold Value to Distinguish Between Responders and Non-Responders.
[0265] The receiver operator curve (ROC) is a fundamental tool for diagnostic test evaluation. When the results of a diagnostic test are considered to discriminate between two populations (eg. responders versus non-responders), a perfect separation between the two groups is rarely observed. For every possible cut-off point selected to discriminate between the two populations, there will be some cases with the responder status correctly classified as responder (True Positive), but some responders will be classified into the non-responder group (False Negative). On the other hand, the majority of non-responders will be correctly classified as non-responders (True Negative), but some will be classified as responders (False Positive). In a ROC curve the true positive rate (Sensitivity: calculated as the True Positive/(True Positive+False Negative)) is plotted against the false positive rate (100-Specificity; wherein specificity is calculated as the False Positive/(False Positive+True Negative), i.e. 100-Specificity is the True Negative/(False Positive+True Negative)), demonstrating different cut-off points of a parameter. Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. The area under the ROC curve (AUC) is a measure of how well a parameter can distinguish between two diagnostic groups (Zweig and Campbell 1993). While the ROC analysis is widely applied, the skilled person will understand that any means preferably mathematical statistical means for finding a threshold to separate the two overlapping distributions may be applied in the present invention.
[0266] Our ROC analysis revealed that the assessment of multidrug activity of peripheral blood lymphocytes carries predictive value for response to bDMARD treatment in RA patients at the start of therapy. Patients with MAF values above the cut-off thresholds are likely to be Non-responders to treatment. Of note, these cut-off values are all below the respective reference ranges in healthy individuals established in our earlier study.
Additional Criteria to Distinguish Between Responders and Non-Responders
[0267] MDR-ABC transporters transport a variety of endogenic molecules, such as cytokines and chemokines that play an important role in the pathogenesis of RA and therefore may be used as biomarkers to monitor disease progression in RA. They may also be used as a predictive tool to establish responsiveness to biological therapy. In this multicenter clinical trial, we aimed to assess the predictive value of flow-cytometry based multidrug resistance activity measurement of three clinically relevant MDR proteins (MDR1, MRP1, BCRP) for biological therapeutic response in rheumatoid arthritis in CD3.sup.+ and CD19.sup.+ lymphocytes before as well as 4 to 6 and 12 weeks after the initiation of biological therapy.
Examples
Methods
Measurement of MDR1 and MRP1 Activities by the Calcein Assay
[0268] Quantitative measurement of MDR1 and MRP1 activities in viable cells is carried out using the calcein-assay technology (see U.S. Pat. No. 5,872,014A). As a preferred kit the SOLVO MDQ Kit was used. This method has several advantages against other fluorescent dye accumulation tests: it is quick, quantitative, selective for MDR1 and MRP1 transporters and it has validated internal standard. This assay utilizes the fluorogenic dye calcein-acetoxymethyl ester (calcein-AM) a hydrophobic compound that readily penetrates the cell membrane. After entering into the living cell, the non-fluorescent calcein-AM is rapidly hydrolysed by endogenous esterases to form a highly fluorescent free acid derivative of the dye which becomes trapped in the cytoplasm due to its high hydrophilicity. Another advantage of calcein is the relative insensitivity to changes of various cellular parameters, including intracellular pH, Ca.sup.2+ and Mg.sup.2+ concentrations.
[0269] As calcein-AM is an excellent substrate of both MDR1 and MRP1, activity of these efflux transporters results in lower cellular accumulation of the fluorescent calcein. Consequently, the more MDR proteins are active in the cell membrane, the less calcein is accumulated intracellularly. In MDR expressing cells, the addition of selective inhibitors of MDR1 and MRP1 blocks the dye exclusion activity of the relevant transporter and increases calcein accumulation in the cells. In the absence of significant MDR transporter activity, the lack of transporter mediated efflux means that the net calcein accumulation is faster in the cells, which, in turn, is not influenced by the presence of an MDR transporter inhibitor or substrate.
[0270] Respective activities of MDRs are reflected by the difference between the amount of calcein accumulated in the presence or absence of selective inhibitors. When calculating the MAF values, this accumulation difference is normalized to the dye uptake measured in the presence or the absence of the inhibitor and the results of the assay are expressed in MDR activity factor (MAF) values. Thus, the result of the test is independent from factors influencing the cellular accumulation of Calcein other than the activity of the multidrug transporters. Such factors involve the difference in cellular properties (membrane lipid composition, intracellular esterase activity, cell size, cell surface, etc) and the methodological differences (i.e.: using different equipment, amplification and individual variables). Since the influence of these non-MDR transporter mediated factors are reduced by the normalization approach mentioned above, this facilitate intra- and interlaboratory comparison of MAF values.
[0271] Selective inhibitors can be used to distinguish between the transport activity of MDR1 and MRP1. The pan-MDR1/MRP1 inhibitor blocks both MDR1 and MRP1 mediated dye effluxes, providing dye accumulation rate that can be used for standardization, while MRP1 blocker helps to determine MDR1 and MRP1 activity. After a short, simple calculation, separate measurement of multidrug resistance for both MDR1 and MRP1 activity can be obtained.
[0272] BCRP activity is measured using a similar principle: intracellular accumulation of the fluorescent BCRP specific probe substrate is measured in the presence or the absence of selective BCRP inhibitor. However, in this case, the BCRP specific probe substrate is direct fluorescent and does not require cleavage by intracellular enzymes.
[0273] It is possible to perform MDR activity measurement on a dedicated cell population of interest by labelling them with fluorochrome-conjugated antibodies after running assay procedure. The assay-compatible fluorochromes are listed in Table 1.
TABLE-US-00001 TABLE 1 Examples for assay compatible fluorescent conjugates Compatible Transporter(s) Dye/substrate Channel fluorochrome MDR1, MRP1 calcein ~515 nm PerCP; PerCP-5.5 BCRP mitoxanthrone ~684 nm FITC, PE
Use of the SOLVO MDQ Kit™
[0274] The SOLVO MDQ Kit™ was used strictly following the manufacturer's instructions. PBMCs were loaded with fluorescent MDR activity reporter substrates (Calcein-AM for MDR1 and MRP1, em: 515 nm and mitoxantrone for BCRP, em: 684 nm, respectively) and treated with MDR protein specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively) to obtain multidrug activity factor (MAF) values.
[0275] Cell surface staining was applied to select CD3.sup.+ T lymphocytes using anti-human CD3-PerCP monoclonal antibodies in case of Calcein-AM stained cells and anti-human CD3-FITC monoclonal antibodies in case of mitoxantrone stained cells according to the manufacturer's instructions.
[0276] MAF values were calculated from the difference between the geometric mean fluorescent intensity (MFI) of cells with and without the specific inhibitors, respectively.
MAF.sub.C(composite MAF of MRP1 and MDR1)=100×(F.sub.max−F.sub.o)/F.sub.max
MAF.sub.BCRP(MAF of MRP1)=100×(F.sub.MX−F.sub.0)/F.sub.max
MAF.sub.MDR1(MAF of MDR1)=MAF.sub.C−MAF.sub.MRP1
MAF.sub.BCRP(MAF of BCRP)=100×(F.sub.mx−F.sub.0)/F.sub.MX
F.sub.max/F.sub.MX: Calcein/mitoxantrone fluorescence with verapamil or KO134, respectively
F.sub.o: fluorescence without inhibitor
F.sub.MRP1: Calcein fluorescence with indomethacin
Patient Recruitment
[0277] 39 RA patients were recruited at the outpatient clinics of the Department of Rheumatology, University of Debrecen, Hungary and the Department of Rheumatology and Clinical Immunology, Charité, Berlin, Germany. Patients were sampled before the start of biological treatment as well as between 4 and 7 weeks and at 12 weeks of treatment. DAS28 and CRP values were also recorded in parallel with MAF determination. Patients were regarded as non-responders (n=12) if DAS28 values showed a decrease of less than 25% between the start of biologicals and at 12 weeks of treatment. Patient characteristics as well as details of the therapy received are included in Table 2. Healthy controls (n=35) were sampled at the Department of Rheumatology, University of Debrecen, Hungary on a single occasion. They had a negative history of autoimmune disorders including RA and a negative status upon physical examination as well as no infectious symptoms within three weeks before sampling.
[0278] Exclusion criteria for all participants included chronic infectious diseases requiring systemic treatment, autoimmune diseases other than RA, immunodeficiencies, allergic diseases and hematological malignancies or solid tumors, age below 18 years. Written informed consent was obtained from all participants and the study adhered to the tenets of the most recent revision of the Declaration of Helsinki.
Peripheral Blood Mononuclear Cell (PBMC) Isolation
[0279] 6 mls of K.sub.3EDTA anticoagulated peripheral blood sample was collected. PBMCs were separated by density gradient centrifugation using Ficoll Histopaque-1077 (Cat. No: H8889, Sigma-Aldrich, St. Louis, Mo., USA) according to the manufacturer's instructions.
Flow Cytometry
[0280] Measurements were conducted on a BD FACSCalibur flow cytometer (BD Biosciences, San Diego, Calif., USA) equipped with 488 nm and 635 nm lasers or on a Miltenyi MACSQuant flow cytometer, equipped with 405 nm, 488 nm and 638 nm lasers, respectively.
[0281] The SOLVO MDQ Kit was used strictly following the manufacturer's instructions. In this assay, fluorescent reporter substrates are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors, allowing for quantitative, standardized assessment. PBMCs were loaded with fluorescent MDR activity reporter substrates (Calcein-AM for MDR1 and MRP1, em: 515 nm and mitoxantrone for BCRP, em: 684 nm, respectively) and treated with MDR protein specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively) to obtain multidrug activity factor (MAF) values.
[0282] Cell surface staining was applied to select CD3.sup.+ and CD19.sup.+ cells using anti-human CD3-PerCP and CD19-PE monoclonal antibodies (Cat. No: 345766 and 345789, respectively, both BD Biosciences) in case of Calcein-AM stained cells and anti-human CD3-FITC and CD19-PE monoclonal antibodies (Cat. No: 345764 and 345789, respectively, both BD Biosciences) in case of mitoxantrone stained cells according to the manufacturer's instructions. Assay-compatible fluorochromes are listed in Table 1.
Results
[0283] ROC analysis was performed to evaluate the predictive value of MAF for response to treatment in RA patients at the start of biological therapy and at 6 wk. Cut-off thresholds were calculated for MAF values with ROCs of adequate p and AUC values (
[0284] In our multicenter clinical trial, 39 RA patients were enrolled. For determining the functional activities of MDR1, MRP1 and BCRP, 6 mls of K.sub.3EDTA anticoagulated blood peripheral blood samples were collected. PBMCs were separated by using Ficoll Histopaque density gradient centrifugation according to the manufacturer's instructions. SOLVO MDQ Kit™ assay was performed as it is described in the instructions for users. After performing the assay, CD3.sup.+ T lymphocytes were labelled with PerCP or FITC-conjugated anti-CD3 antibodies for 30 minutes. After removing unbound antibodies, transporter activities were determined on CD3.sup.+ T lymphocytes by flow cytometry.
[0285] Clinical characteristics of patients are indicated in Table 2.
TABLE-US-00002 TABLE 2 Clinical characteristics of Responder and Non-responder RA patients as well as healthy controls. Healthy controls Responder Non-responder (n = 35) (n = 27) (n = 12) Age (years) 54 (42-62) 56 (49-61) 51 (39-61) Gender (male/female) 4/31 2/25 1/11 RA duration (years) — 10 (5-14) 8.5 (5-15) No. of patients receiving — 15 (56%) 6 (50%) MTX No. of patients receiving — 9 (33%) 5 (42%) prednisolone No. of patients receiving — 2 (7%) 1 (8%) adalimumab No. of patients receiving — 5 (19%) 3 (25%) certolizumab pegol No. of patients receiving — 7 (26%) 3 (25%) etanercept No. of patients receiving — 13 (48%) 5 (42%) abatacept Data are expressed as median (IQR) for continuous variables and as number (percentage) for categorical variables. MTX—methotrexate
[0286] Importantly, in parallel with collecting blood samples at the time of diagnosis (0 week) and during regular checkups (2, 6 and 12 weeks, respectively) DAS28 score was determined and the routinely used inflammatory markers (RF, CRP, ESR, ACPA) were also measured from peripheral blood. bDMARD treatment responsivity was determined on the alterations of DAS28 scores.
[0287] Regarding to treatment success (
[0288] ROC analysis was performed to evaluate the predictive value of MAF for response to treatment in RA patients at the start of biological therapy and at 6 wk. Cut-off thresholds were calculated for MAF values with ROCs of adequate p and AUC values (
[0289] MAF values of CD3.sup.+ T lymphocytes from RA patients showed the following values: at the time of diagnosis, MAF.sub.C values of responders were almost the same as compared with healthy individuals (18.9 vs 18.3), however, in case of non-responders, MAF.sub.C values on CD3.sup.+ T lymphocytes were significantly upregulated as compared with controls (23.5 vs 18.3). During bDMARD treatment in case of responders, a slight down regulation was detected 6 weeks after starting therapy, however, in later time points, MAF.sub.C value did not showed any alterations as compared with control samples and values at the time of diagnosis. Importantly, in case of responders, average MAF.sub.C values were below the cut-off values at the time of diagnosis and 6 weeks after starting bDMARD treatment. In contrary with responders, MAF.sub.C values of non-responders were significantly higher as compared with healthy controls at the time of diagnosis (23.5 vs 18.3). As same as responders, bDMARD treatment had no impact on MAF.sub.C values, however, MAF.sub.C of CD3.sup.+ T lymphocytes during bDMARD treatment were significantly higher as compared with healthy counterparts.
[0290] Although in case of MAF.sub.MDR1 cut-off value statistical significance was not detected at the time of diagnosis (17.4; 0 weeks), its prognostic value is still high, in particular together with the 6 weeks cut-off value data (13.9). At the time of diagnosis, responder values did not showed any alterations as compared with controls, however, MAF.sub.MDR1 values of non-responders were significantly above the control data (19.1 vs 14.6). Prolonged bDMARD treatment had no significant impact on MAF.sub.MDR1 values of responders. In case of non-responders, mild down regulation was detected after starting bDMARD treatment as compared with values at the time of diagnosis.
[0291] MAF.sub.MRP1 values has strong prognostic value 6 weeks after starting bDMARD treatment. At the time of diagnosis (0 weeks?), mild upregulation was detected in RA patients as compared with healthy controls. 6 weeks after starting bDMARD treatment, a mild down regulation was detected as compared with 0 weeks value. Importantly, opposed to responders, a significant upregulation was detected in non-responders (2.2 vs 8.4). [0292] Results are summarized in Table 3.
TABLE-US-00003 TABLE 3 Activity of various MDR transporters on CD3.sup.+ and CD19.sup.+ cells in RA patients and healthy controls. 0 wk 6 wk 12 wk Non- Non- Non- Control Responder responder Responder responder Responder responder DAS28 — 5.94 .sup. 4.65.sup.b .sup. 3.71.sup.c 3.93 .sup. 3.00.sup.c .sup. 3.90.sup.b (5.11-6.17) (3.33-5.23) (2.79-4.45) (3.14-4.50) (2.23-3.67) (2.81-4.90) CRP — 11.1 8.4 4.4 4.4 3.7 7.5 (2.6-16.6) (1.4-15.1) (1.3-7.9) (1.5-10.4) (2.1-5.6) (2.7-11.6) CD3 MAF.sub.C 18.3 18.9 23.5.sup.b 17.1 22.7.sup.b 18.3 25.2 (14.7-22.9) (14.0-25.2) (17.1-33.7) (12.3-22.6) (16.7-29.2) (15.7-24.2) (15.9-30.7) CD3 3.1 4.8 5.7 2.2 .sup. 8.4.sup.b 5.7 7.7.sup.a MAF.sub.MRP1 (1.2-5.7) (0.0-8.0) (2.2-8.0) (0.0-7.9) (2.1-11.3) (3.7-8.5) (4.0-11.6) CD3 14.6 12.9 19.1.sup.b 12.4 15.8.sup.b 12.5 13.6 MAF.sub.MDR1 (12.5-18.1) (11.0-16.7) (11.2-24.0) (11.2-15.4) (14.3-18.7) (9.2-17.5) (6.0-20.0) CD3 2.5 3.1 5.0 2.0 3.9 1.4 4.5 MAF.sub.BCRP (0.8-5.7) (0.0-4.4) (2.0-8.0) (0.0-5.5) (2.5-10.7) (0.0-4.3) (1.8-5.8) CD19 MAF.sub.C 12.8 15.1 20.6 13.2 17.6 17.4 17.6 (8.9-17.9) (8.1-22.1) (13.5-31.0) (9.3-20.4) (11.4-27.2) (13.1-22.3) (9.2-25.9) CD19 2.2 0.9 4.4 0.6 .sup. 6.8.sup.b 3.2 5.1 MAF.sub.MRP1 (0.0-6.3) (0.0-7.7) (0.0-5.8) (0.0-5.1) (0.5-9.6) (0.3-6.8) (1.9-10.9) CD19 9.9 11.1 15.7 11.4 13.6 14.0 8.8 MAF.sub.MDR1 (8.0-14.0) (6.0-16.3) (8.4-25.4) (5.3-14.8) (8.6-17.7) (7.1-17.7) (1.9-15.7) CD19 3.8 3.1 4.5 2.7 5.0 2.9 3.0 MAF.sub.BCRP (1.0-6.3) (0.7-7.0) (0.0-11.0) (0.0-5.2) (3.1-8.4) (1.3-5.1) (1.8-3.7) Data are expressed as median (IQR), p < 0.05 .sup.avs Control, .sup.bvs Responder, .sup.cvs 0 wk value. MAF.sub.C—composite multidrug activity factor (of MRP1 and MDR1 activity), MAF.sub.MRP1—multidrug activity factor of MRP1, MAF.sub.MDR1—multidrug activity factor of MDR1, MAF.sub.BCRP—multidrug activity factor of BCRP
Case Studies
[0293] Examples from our own clinical trial for predicting patient's response to bDMARD treatment: Patient 1: 53 years old women who received abatacept (T cell blocking agent). Her DAS28 values showed gradient down regulation during the monitored period (5.94; 5.22; 4.11; 3.36, respectively). Her MAF.sub.C and MAF.sub.MDR1 values at the time of diagnosis were 4.2 and 4.1, respectively, which are remarkably below the cut-off value (21.3 and 17.4, respectively). 6 weeks after starting abatacept treatment, her prognostic values are the following: MAF.sub.C: 20.1 (cut-off 20.3); MAF.sub.MRP1: 7.8 (cut-off 6.0) and MAF.sub.MDR1 12.3 (cut-off:13.9). During all chekups remarkable improvement was recorded regarding to her disease status.
[0294] Patient 2: 61 years old female patient with etanercept (anti-TNF) treatment. Her DAS28 values showed significant decrease during the clinical trial (5.59; 4.58; 2.3; 1.9, respectively). Her baseline MAF.sub.C value was 20.5, which is below the cut-off value. 6 weeks after starting anti-TNF therapy, her MAF.sub.C value was drastically declined (11.5 vs 20.5) which also suggests favorable treatment response. In the same time point, her MAF.sub.MRP1 and her MAF.sub.MDR1 values were also significantly below the respective reference values (0.0 and 11.5, respectively). Importantly, her physician also recorded favorable treatment response during the whole study period.
[0295] In contrary with Patients 1 and 2, Patient 3 (68 years old woman) showed poor response to abatacept treatment. Regarding to her DAS28 values, no difference was detected during the whole study period (3.06; 3.06; 3.03; 3.03, respectively). Her baseline MAF.sub.C value was remarkably over the cut-off value (22.8 vs 21.3). 6 weeks after starting abatacept treatment, her MAF.sub.C value showed a more robust elevation (24.9) which is over the cut-off value. The same tendency was detected in case of MAF.sub.MDR1, the 8.3 MAF.sub.MDR1 value increased to 14.3 which suggests unfavorable treatment outcome. In accordance with the previously mentioned values, her MAF.sub.MRP1 was also dramatically elevated as compared with the cut-off value (10.6 vs 6.0). In accordance with transporter activity data, no improvement was detected reading to her disease status, thus a tsDMARD treatment would highly be recommended to her.
INDUSTRIAL APPLICABILITY
[0296] The invention is useful to provide predictors before the start of the bDMARD therapy and thereby an option to decide about further treatment before symptoms reflect the success of the therapy. As in RA the window of opportunity to apply therapy is limited, the invention is useful to find the appropriate treatment in time.
REFERENCES
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