Research On Treatment Of TPO Receptor Agonists In Primary Immune Thrombocytopenia

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Cytokine changes in response to TPO receptor agonist treatment in primary immune thrombocytopeniaBackgroundPrimary immune thrombocytopenia(ITP)is an acquired immune-mediated bleeding disorder characterized by autoantibody-mediated platelet destruction and impaired megakaryocyte maturation with reduced platelet production.More recently,it has become obvious that ITP is a more complex disorder in which T cell abnormalities play important roles in platelet destruction.Antiplatelet autoantibody production is under the control of T helper(Th)cells,and elevated antiplatelet T-cell reactivity in ITP has been observed.Th cell polarization in ITP has been attributed to increased Th1 and Th17 cells,decreased Th2 cells,and reduced or impaired CD4+CD25+Foxp3+ regulatory T cells(Tregs).Moreover,enhanced cytotoxic T lymphocyte-mediated platelet destruction has been reported in ITP.The precise reason for these abnormalities remains to be clarified.Thrombopoietin(TPO)is the principal hematopoietic cytokine that stimulates thrombopoiesis by activating the cell through TPO-receptor(TPO-R),c-MPL.As insufficient TPO has been found to contribute to the decreased platelet production in ITP,a series of thrombopoietic agents have been developed.Two types of TPO receptor agonists(TPO-RAs),eltrombopag and romiplostim,have been approved by the US Food and Drug Administration as second-line options for the management of ITP.In addition,recombinant human TPO(rhTPO)has been used for the treatment of corticosteroid-resistant and relapsed ITP patients in China.A rapid response can often be achieved during TPO-RA treatment.Nevertheless,platelet counts usually return to pretreatment levels once the regimen is withdrawn,making it difficult to achieve a long-term drug-independent remission.Cytokine-mediated immunity plays an important role in the pathogenesis of various autoimmune disorders.Aberrant cytokine profiles have been correlated with the loss of immune tolerance in ITP patients.Moreover,response to different therapeutic strategies,such as high-dose dexamethasone,splenectomy,rituximab,and Helicobacter pylori eradication,is often associated with correction of the cytokine abnormalities in ITP.Even though TPO-RAs have been used to treat ITP patients for several years,their roles in cytokine modulation remain largely unknown.Objective1.To identify the curative effect and advise effect of TPO-RAs treatment in ITP2.To evaluate the effect of TPO-RAs on cytokine modulation in ITPMethod1.A total of 26 corticosteroid-resistant/relapsed chronic ITP patients,who are all treated with single Eltrombopag or rhTPO were enrolled.Eltrombopag was administered orally with an initial dose of 25 mg once daily.The dose could be increased to 50 or 75 mg once daily to maintain platelet counts P50×109/L.rhTPO was administered subcutaneously at a daily dose of 300 U/kg initially.The dose frequencies could be adjusted to every other day once platelet counts ascended above 100×109/L.2.After 6 weeks treatment,the platelet counts and the liver and kidney funtions were evaluated.According the platelet counts and if there is bleeding,the patients are classified into complete response(CR),response(R)and no response(NR).3.Plasma levels of IL-2,IL-4,IL-17A and TGF-?1 of the patients pre-and post-treatment together with the healthy controls were measured by enzyme-linked immunosorbent assay(ELISA).4.Total RNA was extracted from PBMCs of the patients pre-and post-treatment as well as the healthy controls.And the mRNA expression of IL-2.IFN-?,IL-4,IL-17A,TGF-?1,RORyt,and GAPDH(endogenous control)were quantified by real-time PCR.Evaluated the difference between the patients and the healthy contols,the patients pre-and post-treatment,the pre-and post-treatment responders or non-responders.Results1.Response was achieved in 19(73.08%)patients,including 7 with CR(26.92%).NR was found in 7(26.92%)of the 26 patients.Two patients received platelet transfusions during the study(both are because the platelets<10×109/L at the first two weeks during the eltrombopag treatment)Eltrombopag and rhTPO therapy were well tolerated in most patients.Liver transaminase increased transiently in 3 patients(2 treated with eltrombopag,1 with rhTPO)and returned to normal levels after liver-protecting therapy.Four patients reported headaches(3 with eltrombopag,1 with rhTPO),two patients fatigue(1 with eltrombopag,1 with rhTPO),and two patients reported nausea and diarrhea(1 with eltrombopag,1 with rhTPO)2.Plasma concentrations of IL-2 and IFN-y were significantly higher in untreated ITP patients than in healthy controls(HCs),By contrast,plasma IL-4 and TGF-?1 levels were lower in untreated ITP patients than in HCs.No statistically significant difference was found in plasma IL-17A levels between untreated ITP patients and HCs.We did not observe any significant change in plasma IL-2,IFN-y,IL-4,or IL-17A levels after TPO-RA treatment,whereas plasma levels of TGF-?1 increased significantly(P = 0.008).Plasma concentration of TGF-?1 was not statistically different between TPO-RA-treated ITP patients and healthy controls.3.Significantly higher IL-2,IFN-c,IL-17A,and RORyt together with remarkably lower IL-4 and TGF-?1 mRNA levels were found in untreated ITP patients compared with HCs.Consistent with the plasma cytokine findings,there was no significant change in mRNA expression levels of IL-2,IFNy,IL-4,IL-17A,and RORyt in PBMCs.whereas mRNA expression level of TGF-?1 increased significantly after TPO-RA treatment(P= 0.039).4.There was no significant change in plasma levels or mRNA expression of IL-2,IFN-?,IL-4,and IL-17A in TPO-RA responders compared with their pre-treatment levels,neither was there in TPO-RA non-responders.By contrast,both plasma concentration and mRNA expression of TGF-?1 increased significantly in TPO-RA responders(P<0.05),but we did not observe any significant change in non-responders after TPO-RA treatment.ConclusionTPO-RAs promoted the elevation of platelet counts effectively and were well tolerated in corticosteroid-resistant and relapsed ITP patients.However,monotherapy of TPO-RAs did not correct the aberrant cytokine profiles,which is an important direction to the combined therapy.Part II Study of Recombinant Human Thrombopoietin in Management of Immune Thrombocytopenia in Pregnancy using a mouse modelBackgroundThrombocytopenia in pregnancy,defined as platelet count of less than 150 x 109/L,is common in pregnancies.It can result from a variety of physiologic or pathologic conditions,among of which,ITP accounts for about 4.1%of cases.The incidence of ITP in pregnant women is 1/1000-1/10000.Because maternal immunoglobulin G antiplatelet antibodies can cross the placenta,placing the fetus and neonate at risk of thrombocytopenia,so ITP in pregnancy is a serious medical disorder that has the potential for maternal and fetal morbidity.The management of ITP in pregnancy is complex,and to reach optimum requires collaboration among the obstetrician,the hematologist and the neonatologist.Primary treatment options for ITP in pregnancy are similar to those for other adult ITP patients.Corticosteroids,intravenous immunoglobulin(IVIG),or both are the first-line treatments for maternal ITP,But choices are limited if the patients have no response to first-line therapy.A series of domestic research has confirmed the efficacy and safety of recombinant human thrombopoietin(rhTPO)in management of chronic ITP.And there is some related report on rhTPO in management of pregnant women with ITP,which demonstrated it is effective,fast-onset and no side effects on mother and fetus.But the efficacy and safety of rhTPO in management of ITP in pregnancy still need the reliable supportive by study on murine model.Objective1.To develop an ideal mouse model that simulates human ITP in pregnancy.2.To evaluate the effect and safety of rhTPO in management of ITP in pregnancy using the murine model3.To further identify the changes of Tregs and TGF-?1 after rhTPO treatment using the murine model.Method1.Establishment of a murine model of ITP in pregnancy:female and male SPF C57 mice were mated(2:1).then the pregnancy mice,defined as day 0,were intraperitoneal injected with MWReg30 antibody at a dose that was increased depending on the platelet counts or PBS for the control group.The injection was oprerated every other day.The platelet counts were measured on day 0,day 1,day 3,day 7,day 10 and day 14.2.The murine model of ITP in pregnancy were randomly divided into NS group,Low-dose rhTPO group(L),Media-dose rhTPO group(M)and High-dose rhTPO group(H),which were separately subcutaneously injected 100ul normal saline,150U/kg rhTPO,1500U/kg rhTPO,and 15000U/kgTPO for consecutive 14 days.The platelet counts were measured on day 0,1,3,7,14 and after delivery.After treatment,the serum of each group was acquired for biochemistry detection.After delivery,half the maternal mice of each group sacrificed and bone narrow were acquired for smear and reticular fiber staining.The platelets of the fetus mice bred by the sacrificed maternal mouse were analysized.The left half cages of maternal and fetus mice were observed at least 3 weeks to assess their vital signs.3.The serum level of TGF-?1 after delivery was detected using ELISA,and the propotion of CD4+C25+FOXP3+ Treg cells in peripheral blood and spleen from the sacrificed mice are analyzed using FCM.Result1.The murine model of ITP in pregnancy:1 day later after the injection of MWReg30 antibody,the platelet count of the mice reduced to the lowest level.Then the platelet count gradually recovered,but it was still significantly lower than the control group until Day 10.So the murine model was recognized successfully developed.2.The general condition,appetite and mental condition of the mice in the 4 groups had no obvious change during the rhTPO injection as well as the withdrawal period.There is no redness and swelling at the local injection site.Dystocia occurred in 8 mice(3 of NS group,2 of M group and and 1 of Mid-dose group and 2 of High-dose group),among which 2 were dead and the other 6 alive after timely treatment.There was no apparent association between dystocia and rhTPO.3.The platelet counts of mice in rhTPO treatment group were significantly elevated than those in NS group.The effect of increasing the platelet was enhanced with the increased rhTPO dosage,but no statistical significance was shown.4.There was no significant difference in the results of biochemistry and the marrow fibosis between the NS group and the three rhTPO groups after delivery.rhTPO had no statistical effect on the average amount of fetus.We didn't observed abnormalitis on the fetus mice during 3 weeks after birth.5.Consistent with the result of the vitro result in the first part of our study,The serum level of TGF-?1 together with CD4+C25+FOXP3+ Tregs in splenocytes were significantly higher in the rhTPO treated groups than the NS group,But we didn't find statistical difference in the frequency of Tregs in peripheral blood from the 4 group mice.ConclusionThe mouse model of ITP in pregnancy can simulate the process of ITP induced by anti-platelet antibody in a certain degree.And the reduced platelet didn't disturb the process of delivery.RhTPO can increase the platelet of ITP mice in pregnancy effectively without significant side effects.rhTPO treatment has additional tolerance-inducing effects associated with Tregs stimulation.However,the long-term safety and efficacy of rhTPO in treatment of pregnancy with ITP...