First-line Management Strategy And Mechanism Of Corticosteroids Resistance Mediated By P-gp In Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune syndrome and account for approximately one third of the clinical bleeding diseases. Classic pathogenesis of ITP is over-destruction of platelet mediated by platelet-specific autoantibody. Recent studies indicate that direct lysis of platelet by cytotoxic T cell and hepatic clearance of desialylated platelet are also critical approaches to thrombocytopenia in ITP. Besides, both platelet-specific autoantibody and cytotoxic T cell can suppress maturation and apoptosis of megakaryocytes in bone marrow, resulting in impaired platelet production.Pathogenesis of ITP involves complicated upstream autoimmune abnormalities under a mechanism of the loss of autoimmune tolerance. Corticosteroids broadly regulate the autoimmune disorders in ITP. Apoptosis of autoreactive T and B cells are decreased in ITP, which leads to abnormal prolonged survival and can be reversed by dexamethasone via suppression of BAFF in vitro. There are polarizations in auto-reactive subsets and protective subsets of immunocytes, such as imbalanced Th1/Th2 and Th1/Th17/Treg ratio. Dexamethasone restores the balance of Thl/Th2 and normal quantity and function of Thl7 and Tregs. It also promotes the function of protective subsets CD8+ Tregs and CD5+ Bregs. Besides, dexamethasone regulates the balance of the surface molecule groups of antigen presenting cells and reconstitutes the autoimmune tolerance in ITP.Corticosteroids are the recommended first-line treatment strategy for newly-diagnosed adult ITP by practice guidelines. Approximately two thirds of the patients respond to standard prednisone treatment. Long-term outcomes are limited and adverse events are frequent due to continuing administration of the medication. Single arm studies show that high-dose dexamethasone (HD-DXM) achieves over 80% incidence of initial response with few adverse events. It is possible to shorten the duration and reduce the adverse effects upon ever better efficacy. However, none of these studies included PDN as a control and randomized evidence is still needed to clarify which of the corticosteroid regimens is superior.Proportions of patients respond poorly to corticosteroids or relapse within a short interval, which is defined as corticosteroid-resistance. It is of great value to avoid this effect and improve the efficacy of corticosteroids in first-line management of ITP. P-glycoprotein is encoded by multi-drug resistance gene MDR-1, and is a type of ATP-dependent transport pump. Over-expression or upregulated function of P-gp conduct corticosteroid-resistance in multiple autoimmune diseases. Expression of P-gp is increased in peripheral blood mononuclear cells (PBMC) of patients with systemic erythematous lupus and rheumatoid arthritis, especially in those with poor response to dexamethasone. Moreover, uptake of dexamethasone is reduced in these patients, which can be inhibited by Ciclosporin or Tacrolimus. The effects and mechanisms of P-gp in corticosteroid-resistance in ITP deserve to be investigated.This prospective, multicenter, randomized controlled study compares the efficacy and safety between HD-DXM and conventional prednisone, and provides high level evidence for clarification of the preferred corticosteroids strategy in first-line management of ITP. Besides, expression of P-gp and uptake of dexamethasone by PBMCs are detected and analyzed with a stratification of efficacy. Cyclosporine is attempted to inhibit P-gp function and restore the dexamethasone uptaking.Part I:Comparison of High-dose Dexamethasone versus Conventional Prednisone for First-line Management of Adult Primary Immune Thrombocytopenia:a Prospective, Multicenter, Randomized Controlled StudyObjectiveTo compare the incidence of initial response and time to response between HD-DXM and conventional prednisone for management of adult primary ITP; to compare the incidence of sustained response and the Kaplan-Meier curve between the two agents; to evaluate the adverse events and tolerability of patients between the two agents, and compare the safety; to evaluate the outcomes and prognostic value of bleeding; to evaluate the prognostic value of platelet-specific autoantibodies. Provide evidence for the optimization of first-line strategy and identify prognostic factors in corticosteroids management.Methods1. Eligible patients were randomly assigned 1:1 to receive either HD-DXM or standard dose prednisone.2. Dexamethasone was administered orally at 40 mg daily for 4 consecutive days and then stopped. An additional 4-day course of HD-DXM (40 mg daily) was given after day 10. Patients received PDN orally at 1.0mg/kg body weight daily for 4 consecutive weeks. In responders, the medication was tapered gradually to a maintenance dose of less than 15 mg daily or complete termination, and prednisone was rapidly tapered to termination in nonresponders after 4 weeks.3. The primary end points are initial response and sustained response, evaluation of initial response is according to guidelines of ITP. Sustained response is defined as platelet count maintained 30x109/L with an absence of bleeding symptoms or no requirement for additional ITP-modifying treatment of 6 consecutive months following achievement of initial response. Secondary end points included bleeding scores, time to response, duration of response, and adverse events.4. Baseline anti-GPⅡb/Ⅲa and anti-GPⅠb/Ⅸ autoantibodies were tested in all patients by a modified monoclonal antibody-specific immobilization of platelet antigen assay.Results1. HD-DXM demonstrates higher incidence of overall response and complete response, with a shorter time to response, compared with prednisone. The additional course of HD-DXM significantly increased the incidence of overall response, although patients responding to the additional course were less likely to achieve CR than those who responded to the initial course.2. Patients whose baseline platelet count was 10×109/L or less presented with significantly more severe bleeding manifestations. Bleeding was more effectively controlled in the HD-DXM arm, with fewer bleeding events and lower bleeding scores. A baseline bleeding score$8 had a negative impact on the initial response.3. The incidence of sustained response showed no significant difference between the two arms. Initial CR was the only definite positive indicator associated with an increased incidence of sustained response in the initial responders. Overall duration of response was similar between the two arms, estimated by the Kaplan-Meier analysis. The Kaplan-Meier cumulative incidence of loss of response was significantly lower among initial CR responders.4. Presence of anti-GPⅡb/Ⅲa autoantibody was associated with both lower baseline platelet count and more severe bleeding manifestations. Any presence of antiplatelet autoantibodies significantly indicated reduced opportunity to achieve SR.5. The frequency of adverse eventswas higher in the prednisone arm.Conclusion1. One or two courses of HD-DXM provide a more effective and more rapid response as initial treatment of ITP, with at least comparable long-term prognosis when compared with conventional prednisone.2. HD-DXM is better tolerated and enables patients to avoid the burden of long-term corticosteroids and adverse effects without loss of long-termoutcomes.3. HD-DXM could become a preferred corticosteroid approach for first-line management of adult primary ITP.Mechanism of Corticosteroids Resistance Mediated by P-gp in Immune ThrombocytopeniaObjectiveTo detect expression of P-gp on the surface of PBMCs, peripheral CD4+T cells and CD19+B cells in ITP patients stratified by the efficacy of HD-DXM treatment; to establish the methodology of ultra-performance liquid chromatography (UPLC) detecting the dexamethasone concentration in cell culture fluid, and evaluate the absorption of dexamethasone in culture fluid by PBMC; to investigate the inhibition of cyclosporine to PBMC in vitro. Elucidate the mechanism of P-gp quantity and function in corticosteroid-resistance, and investigate potential reversing approach.Methods1. Collect peripheral blood from newly-diagnosed ITP patients who were planned to receive HD-DXM treatment and healthy volunteers, and isolate PBMCs.2. PBMCs were stained by mouse anti-human PE-conjugated CD4, APC-conjugated CD 19 and FITC-conjugated P-gp. The expression of P-gp on PBMC, CD4+T cells and CD19+B cells from ITP patients and healthy controls is analyzed by flow cytometry.3. Co-culture the PBMC from ITP patients and healthy controls with dexamethasone only or in combination with cyclosporine in vitro, and collect the PBMCs and supernate.4. Specify the chromatographic condition of UPLC and sample extraction method. Test assay linearity, sensitivity, intra-and inter-day precision, recovery of the extraction, and stability with series diluted standard solution,5. The expression of P-gp on PBMC, CD4+T cells and CD19+B cells from ITP patients and healthy controls after culture is analyzed by flow cytometry. Residue concentration of dexamethasone in supernate of culture fluid is tested by UPLC.Results1. Expression of P-gp on PBMC, CD4+T cells and CD19+B cells from ITP patients is significantly higher than healthy controls. It is also higher in non-responders to HD-DXM than in responders.2. Expression of P-gp demonstrated no significant difference before and after co-culture with dexamethasone and/or Cyclosporine.3. Residue concentration of dexamethasone in supernate of culture fluid is higher in ITP patients, and non-responders to HD-DXM show significantly more dexamethasone residue than responders.4. Cyclosporine significantly decreases residue concentration of dexamethasone in supernate of culture fluid. Addition of cyclosporine lowers the residue concentration of dexamethasone in supernate of culture fluid of ITP patients to a level equal to healthy controls.Conclusions1. Expression of P-gp on PBMC, CD4+T cells and CD19+B cells from non-responders to HD-DXM in ITP. The ability of PBMCs to uptake dexamethasone decreases. Upregulation of quantity and function of P-gp is an important mechanism of corticosteroid-resistance in ITP.2. Cyclosporine inhibits P-gp function and restores the uptake of dexamethasone, which could be a potential approach to reverse corticosteroid-resistance in ITP.