Piericidin A_线粒体复合体I抑制剂_2738-64-9_Apexbio
端粒酶抑制剂伊美司他治疗血小板增多IF56
T h e ne w engl a nd jour na l o f medicineFrom the Department of Hematology, University Hospital of B ern and Univer-sity of B ern, B ern, Switzerland (G.M.B., E.O.L., M.D.); the Department of Hema-tology, School of Medicine, Cardiff Uni-versity, Cardiff, United Kingdom (O.G.O.); Upstate Oncology Associates, Greenville, SC (G.S.); the Section of Hematology and Oncology, University of Chicago, Chica-go (O.O.); the Divisions of Hematologic Malignancies and Hematology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Med-icine, B altimore (M.A.M.); the Depart-ment of Hematology, University Hospital Essen, Essen, Germany (A.R.); and Geron, Menlo Park (B.B., M.S.), and the Depart-ment of Hematology and Hematopoietic Cell Transplantation, City of Hope, Gehr Family Center for Leukemia Research, Duarte (D.S.S.) — both in California. Ad-dress reprint requests to Dr. Baerlocher at the Department of Hematology, Stem Cell and Molecular Diagnostics Labora-tory, Freiburgstr. 4, Inselspital, University Hospital of Bern, CH-3010 Bern, Switzer-land,or at g abriela.b aerlocher@i nsel.c h. N Engl J Med 2015;373:920-8.DOI: 10.1056/NEJMoa1503479Copyright © 2015 Massachusetts Medical Society.BACKGROUNDImetelstat, a 13-mer oligonucleotide that is covalently modified with lipid exten-sions, competitively inhibits telomerase enzymatic activity. It has been shown to inhibit megakaryocytic proliferation in vitro in cells obtained from patients with essential thrombocythemia. In this phase 2 study, we investigated whether ime-telstat could elicit hematologic and molecular responses in patients with essential thrombocythemia who had not had a response to or who had had unacceptable side effects from prior therapies.METHODSA total of 18 patients in two sequential cohorts received an initial dose of 7.5 or9.4 mg of imetelstat per kilogram of body weight intravenously once a week until attainment of a platelet count of approximately 250,000 to 300,000 per cubic milli-meter. The primary end point was the best hematologic response.RESULTSImetelstat induced hematologic responses in all 18 patients, and 16 patients (89%) had a complete hematologic response. At the time of the primary analysis, 10 pa-tients were still receiving treatment, with a median follow-up of 17 months (range, 7 to 32 [ongoing]). Molecular responses were seen in 7 of 8 patients who were positive for the JAK2 V617F mutation (88%; 95% confidence interval, 47 to 100). CALR and MPL mutant allele burdens were also reduced by 15 to 66%. The most common adverse events during treatment were mild to moderate in severity; neu-tropenia of grade 3 or higher occurred in 4 of the 18 patients (22%) and anemia, headache, and syncope of grade 3 or higher each occurred in 2 patients (11%). All the patients had at least one abnormal liver-function value; all persistent elevations were grade 1 or 2 in severity.CONCLUSIONSRapid and durable hematologic and molecular responses were observed in patients with essential thrombocythemia who received imetelstat. (Funded by Geron; number, NCT01243073.)ABSTR ACTTelomerase Inhibitor Imetelstat in Patients with Essential ThrombocythemiaGabriela M. Baerlocher, M.D., Elisabeth Oppliger Leibundgut, Pharm.D.,Oliver G. Ottmann, M.D., Gary Spitzer, M.D., Olatoyosi Odenike, M.D., Michael A. McDevitt, M.D., Ph.D., Alexander Röth, M.D., Michael Daskalakis, M.D., Bart Burington, Ph.D., Monic Stuart, M.D.,and David S. Snyder, M.D.Imetelstat in Essential ThrombocythemiaE ssential thrombocythemia, a myelo-proliferative neoplasm, is a clonal disorderof a multipotent hematopoietic progenitor cell.1,2 The disease is associated with an in-creased risk of thrombotic complications, hemor-rhagic complications, or both, and can evolve into myelofibrosis or, in rare cases, can trans-form to acute leukemia.3 Common mutations associated with essential thrombocythemia are found in the Janus kinase 2 (JAK2) gene, the gene encoding the thrombopoietin receptor (MPL), and the calreticulin (CALR) gene.4-8 Cur-rent standard therapies for high-risk patients with essential thrombocythemia induce nonspe-cific reductions in platelet counts but do not typically eliminate or alter the biologic charac-teristics of the disease.9-12We have reported that telomerase activity in malignant cells obtained from patients with es-sential thrombocythemia and induced telomerase activity in cells isolated from healthy donors were inhibited by the telomerase inhibitor imetelstat.13 However, imetelstat inhibited spontaneous pro-liferation of megakaryocytic colonies obtained from patients with essential thrombocythemia but did not inhibit cytokine-induced megakaryo-cytic colonies from healthy donors. These find-ings suggest an intrinsic sensitivity of essential-thrombocythemia megakaryocytes to telomerase inhibition.13Imetelstat, a 13-mer thiophosphoramidate oli-gonucleotide that is covalently modified with lipid extensions, inhibits telomerase enzymatic activity14 (see the Supplementary Background sec-tion in the Supplementary Appendix, available with the full text of this article at ). In this phase 2 study, we investigated whether im-etelstat could elicit hematologic and molecular responses in patients with essential thrombocy-themia who had disease that was refractory to prior therapies or who had had unacceptable side-effects from previous therapies.MethodsEligibility CriteriaPatients who were 18 years of age or older and had received a diagnosis of essential thrombocy-themia defined according to World Health Orga-nization criteria15 (Table S1 in the Supplemen-tary Appendix) were eligible for enrollment if they required cytoreduction because of a platelet count higher than 600,000 per cubic millimeter and if they had not had a response to or had had unacceptable side effects from at least one prior therapy or if they declined to receive standard therapy. Additional inclusion and exclusion cri-teria are described in the Supplementary Ap-pendix.Study DesignThis was a phase 2, open-label study that was conducted at seven sites in the United States, Germany, and Switzerland; enrollment occurred during the period from January 2010 through January 2013. Patients were enrolled into se-quential cohorts that received weekly doses of 7.5 or 9.4 mg of imetelstat per kilogram of body weight, intravenously, until a platelet count of approximately 250,000 to 300,000 per cubic milli-meter was achieved or toxic effects occurred. Maintenance dosing at a reduced frequency be-gan after a patient had a complete or partial hematologic response (Table S2 in the Supple-mentary Appendix), with doses (7.5 to 11.7 mg per kilogram) adjusted according to the patient’s response and the side-effect profile of the drug. Additional details regarding the study design are provided in the Supplementary Appendix.The primary end point of the study was the best overall hematologic response, which was defined according to the platelet response com-ponent of the 2009 European LeukemiaNet (ELN) response criteria16 (Table S2 in the Supplemen-tary Appendix). Secondary end points included the frequency and severity of adverse events, the duration of hematologic response, the ELN “clin-icohematologic” response, and the molecular response in patients with JAK2 V617F mutations (Table S2 in the Supplementary Appendix), MPL W515L or MPL W515K mutations, or CALR mu-tations.All patients were assessed for JAK2 V617F, MPL W515L, MPL W515K, and CALR mutations at baseline and were reassessed every 12 weeks (±4 weeks) (see the Supplementary Methods sec-tion in the Supplementary Appendix). Patients who were enrolled in Europe underwent testing of spontaneous growth inhibition of mega-karyocyte colony-forming units (CFUs) (see the Supplementary Methods section in the Supple-mentary Appendix).T h e ne w engl a nd jour na l o f medicineSafety AssessmentData were collected on the extent of imetelstat exposure and on all adverse events. Adverse events were summarized according to terms used in the Medical Dictionary for Regulatory Activities, and severity was assessed with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE),17 version 4.03. For events with varying severity, the maxi-mum reported grade was used in summaries. Laboratory abnormalities were defined accord-ing to normal laboratory ranges from each insti-tution and converted to CTCAE grades.Study OversightThis phase 2 study was approved by the institu-tional review board at each participating site. The study was conducted in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines. All patients provid-ed written informed consent.The sponsor, Geron, provided the study drug and in collaboration with the authors designed the study and analyzed the data. The first author wrote the initial draft, and subsequent drafts were written by all authors with assistance from medical writers paid by Janssen Research and Development. All the authors made the decision to submit the manuscript for publication and vouch for the accuracy and completeness of the data and analyses and for adherence to the study protocol, which is available at . Clini-cal-trial agreements between the sponsor and the authors’ institutions included confidentiality provisions.Statistical AnalysisThe analysis of the primary end point of hema-tologic response included all patients who re-ceived at least one dose of imetelstat. The analy-sis of the duration of response included all patients who had a response. The analysis of molecular response included patients who re-ceived at least one dose of imetelstat and who had a JAK2 V617F, MPL W515L, MPL W515K, or CALR mutant allele burden at baseline. For re-sponse end points, exact 95% confidence inter-vals were calculated. The duration of hemato-logic response was estimated with the use of the Kaplan–Meier method. Patients who were lost to follow-up, discontinued the study, or began to receive other essential thrombocythemia–directed therapy before a hematologic response was documented and confirmed were counted as not having had a response. Safety analyses included all patients who received at least one dose of imetelstat.R esultsPatientsA total of 18 patients were enrolled in the study, and all had received one or more previous treat-ments; 17 had received hydroxyurea (94%), 13 had received anagrelide (72%), and 4 had received interferon (22%) (Table 1). Nine of the 18 pa-tients (50%) had disease that was resistant to at least one prior therapy, and 14 (78%) had had unacceptable side effects from prior therapy. The median time from the initial diagnosis ofe ssential thrombocythemia to enrollment was7.2 years (range, 0.3 to 24.9), and the median baseline platelet count was 788,000 per cubic millimeter (range, 521,000 to 1,359,000). Fifteen patients had baseline mutant allele burdens (8 with JAK2 V617F mutations, 2 with MPL W515L or MPL W515K mutations, and 5 with CALR mu-tations) (Table 1). Table S3 in the Supplementary Appendix describes additional baseline charac-teristics of the patients.Primary Efficacy End Point and Key Secondary Efficacy End PointOf the 18 patients who received imetelstat, 7 re-ceived an initial dose of 7.5 mg per kilogram and 11 received 9.4 mg per kilogram intravenously. The overall rate of hematologic response was 100% (95% confidence interval [CI], 83 to 100), and 16 of the 18 patients (89%) had a complete hematologic response (95% CI, 65 to 99) (Fig. 1A). In the 2 patients with a partial hematologic re-sponse, the lowest platelet counts were 261,000 per cubic millimeter at 23 weeks and 56,000 per cubic millimeter at 18 weeks, but neither response was sustained. Among the 16 patients who had a complete hematologic response, the median time to a complete response was 6.1 weeks (range, 5.1 to 12.1); the median time to a com-plete response was similar in JAK2 V617F–posi-tive and JAK2 V617F–negative patients. A trend was observed toward a more rapid response in patients in whom the initial dose was 9.4 mg per kilogram than in those in whom the initial dose was 7.5 mg per kilogram (median, 6.1 vs. 12.1Imetelstat in Essential Thrombocythemia weeks), although the difference was not signifi-cant (P = 0.71).During maintenance dosing, intermittent dos-ing was attempted in most patients and thefrequency of dosing generally decreased withtime; all the patients who had a complete hema-tologic response received a dose every 2 weeksor less frequently. At the time of the primaryanalysis, with a median follow-up of 17 months,10 of the 16 patients with a complete hemato-logic response (62%) were still receiving treat-ment, and the median duration of responsehad not been reached (follow-up range, 5 to 30months).While receiving the study treatment, one pa-tient had grade 2 bilateral retinal ischemia andwas recovering, and another had grade 2 lefthemiparesis and recovered; these thromboem-bolic events were assessed by the study investi-gators as being potentially related to underlyingessential thrombocythemia. The durations ofresponse ended at the reported onset of theseconditions, no further clinical follow-up infor-mation was obtained, and neither patient dis-continued imetelstat. Three patients had diseaseprogression from essential thrombocythemia tosecondary myelofibrosis, either during the studytreatment period or during the safety follow-upperiod.Other Secondary Efficacy End PointsMolecular ResponseA partial molecular response was detected inseven of eight patients with JAK2 V617F mutationsat baseline (88%; 95% CI, 47 to 100) (Fig. S1 inthe Supplementary Appendix). The median JAK2V617F mutant allele burden was reduced by 71%at month 3 after the initiation of treatment andremained reduced by 59% at month 12 despiteless frequent maintenance dosing (Fig. 1B).MPL W515L, MPL W515K, and CALR mutantallele burdens were also reduced, by 15 to 66%. Further information is provided in Table S4 in the Supplementary Appendix.Inhibition of ProliferationA megakaryocyte CFU assay was performed in five patients. It showed substantial inhibition of spontaneous proliferation at 1 month after ini-tiation of imetelstat treatment, with a median 92% reduction from baseline (Table S5 in the Supplementary Appendix).ELN “Clinicohematologic” ResponseResults with respect to the secondary end pointof a “clinicohematologic” response according toELN criteria16 (see Table S2 of the Supplementary Appendix) were similar to those of the primaryend point. A complete ELN clinicohematologic response was observed in 17 of the 18 patients (94%), and a partial response was observed in1 patient (6%). In 1 patient, splenomegaly thatwas palpable at 3 cm at baseline became nonpal-pable after the first cycle of treatment. None of* O ne patient did not meet the inclusion criteria for the minimum baseline platelet count, but the investigator thought that cytoreductive therapy was clinically indicated.† T hree patients had no mutation.T h e ne w engl a nd jour na l o f medicinethe 18 patients had constitutional symptoms at baseline.Telomerase Activity, Telomere Length, and Bone Marrow ResponseFigure S3 in the Supplementary Appendix shows telomerase activity. Assessments of telomere length and bone marrow response are described in the text of the Supplementary Appendix.S afetyThe most frequent adverse events that occurred during treatment (those that occurred in 50% or more of the patients and selected clinically sig-nificant adverse events) are listed in Table 2. All adverse events are listed in Table S6 in the Sup-plementary Appendix. Adverse events were re-ported in all patients, and 15 of the 18 patients (83%) reported at least one event of grade 3 or higher. Eighteen events of grade 3 or higher were attributed to imetelstat by the investigators, including neutropenia, headache, anemia, and a syncopal episode due to an infusion reaction (a second syncopal episode in the same patient was deemed to be unrelated to imetelstat). One grade 4 adverse event (a femoral-neck fracture) was reported by the treating clinician as being unrelated to imetelstat (Table S6 in the Supple-mentary Appendix). Eight of the 18 patients (44%) discontinued the study (Table S7 in the Supplementary Appendix).The most common nonlaboratory adverse events attributed to imetelstat were fatigue (in 15 of the 18 patients), nausea (in 12), diarrhea (in 11), and headache (in 8) (Table S8 in the Supplemen-tary Appendix). Infections occurred during treat-ment in 17 of the 18 patients (94%); 15 of these 17 patients (88%) had infections of grade 2 or lower. The most common infections were upper respiratory infections (in 39% of the patients), urinary tract infections (in 22%), and influenza (diagnosed on the basis of clinical and labora-tory findings), nasopharyngitis, and rash (each in 17%). The infections lasted less than 3 weeks in 81% of the patients and were not typically as-sociated with cumulative imetelstat exposure or with concurrent neutropenia or lymphopenia.At least one increase in grade, from baseline, in a liver-function value was observed in all 18 patients (Table 3). The majority of patients had grade 1 increases in alanine aminotransferase levels, with accompanying grade 1 increases in aspartate aminotransferase levels (the latter per-Figure 1. Hematologic and Molecular Responses in Patients Who Received Imetelstat.Panel A shows the hematologic response according to mutation status (cal-reticulin [CALR ], Janus kinase 2 [JAK2] V617F, MPL W515L or MPL W515K mutations, or no mutation). A complete hematologic response was observed in 16 of the 18 patients in the study (89%), and a partial hematologic response (“no complete response”) was observed in 2 patients (11%). The median time to a complete hematologic response was 1.4 months. Percentages shown are the best percent change in the mutant allele burden from base-line. Panel B shows the molecular response in JAK2 V617F–positive patients after treatment with imetelstat. The median JAK2 V617F mutant allele bur-den was reduced by 71% at month 3 and by 59% at month 12 despite less frequent maintenance dosing. I bars indicate 95% confidence intervals. The percent change shown is the median percent change in the JAK2 V617F mutant allele burden from baseline.CALR−31−36−48−55−15JAK2 V617FNo mutation04628121026181614282220243230MonthsB Molecular ResponseA Hematologic ResponseTime to first platelet count of 400×103/mm 3Time to complete responseNocomplete responseTreatment continuedM e d i a n J A K 2 V 617F (p e r c e n t c h a n g e f r o m b a s e l i n e )C h a n g e f r o m B a s e l i n e (%)−40−20−60−80−100Baseline 3691215MonthsP=0.001No. at Risk Percent Change88−718−688−727−595−52Discontinuation of treatment−96−82MPL−93−24−90−72−96−82−66−16−50−25Imetelstat in Essential Thrombocythemia sisted with ongoing imetelstat treatment). With-in 4 weeks after the initiation of imetelstat treat-aminotransferase level of 5 to 7 times the stan-dard upper limit of the normal range (grade 3)and concurrent increases in the aspartate ami-notransferase level of 2 to 6 times the standardupper limit of the normal range (grades 1 to 3)with dose reduction and did not recur with con-tinued treatment. With a longer duration oftreatment, persistent (≥6 weeks) grade 1 in-creases in alkaline phosphatase levels were ob-served in 14 of the 18 patients; these increaseswere associated with grade 1 or 2 unconjugated hyperbilirubinemia in 4 patients (Table 3).In post-study follow-up safety assessments,among the 14 patients who had persistent (≥6weeks) abnormalities in liver-function values, 11(79%) had values that reversed to normal orbaseline values. In 3 patients, the liver-functionvalues did not return to the upper limit of thenormal range; all hepatic biochemical abnor-malities in 2 of these patients improved, withcomplete resolution of the abnormal values, andthe 1 remaining patient whose condition wasnot improving at the end of follow-up had pro-patients who underwent extended follow-up forpersistent (≥6 weeks) abnormalities on liver-function tests, the median time to resolutionafter discontinuation of treatment was 12 weeks.The observed resolutions of these abnormalitiesfollowed permanent discontinuation of ime-telstat in all patients as the result of a full clini-cal hold issued by the Food and Drug Adminis-tration (FDA) (see the Discussion section).After the primary analysis was performed inOctober 2013, one patient who had received im-etelstat for approximately 3 years died frommonths after discontinuation of treatment. Cir-rhosis, secondary to hepatic steatosis, was thesuspected underlying disease. Although the pa-tient’s age (83 years), multiple coexisting condi-tions (including a history of exposure to hepati-tis B), and concomitant medications confoundedassessment, imetelstat could not be conclusivelyexcluded as a contributory agent.Hematologic toxic effects, defined according to changes in laboratory results from baseline values, are listed in Table 4. Thrombocytopenia of less than grade 3 was seen in 9 of the 18 pa-tients (50%). Neutropenia was observed in 15 pa-tients (83%), including 7 with grade 3 neutrope-nia (39%) and 3 with grade 4 neutropenia (17%).* I ncluded are the adverse events that occurred in 50% or more of the patients and selected clinically significant adverse events, regardless of whether they were deemed to be related to the study drug. The grade of adverse event was defined on the basis of the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE).* L aboratory abnormalities were defined on the basis of normal laboratory rang-es at each institution and were graded according to the CTCAE. The maximum grade was calculated for patients who had an increase in grade, as compared with the baseline grade. There were no grade 4 increases.na l o f medicineNo cases of febrile neutropenia were reported,and no patients with grade 4 neutropenia had a concurrent infection. Lymphopenia was reported in 6 patients (33%), with one grade 3 event. Fif-teen patients (83%) had anemia, and 3 (17%) had grade 3 anemia. Three patients required red-cell transfusions, including a patient who had a postsurgical hemorrhage. Three reported cases of cytopenia led to dose reductions: one for grade 1 thrombocytopenia, one for grade 3 ane-mia in a patient who required a red-cell transfu-sion, and one for grade 3 neutropenia; all three cases resolved.DiscussionIn this study, imetelstat rapidly induced hemato-logic responses in patients with essential throm-bocythemia who had disease that was refractory to conventional therapies or who had had unac-ceptable side effects from conventional therapies. All the patients had a hematologic response, and 89% had a complete hematologic response, a rate that exceeded rates observed with hydroxyurea, anagrelide, and interferon treatment.12,18-21 Else-where in this issue of the Journal , Tefferi et al. report the results of a pilot study of imetelstat therapy showing the induction of complete or partial remissions, as well as molecular remis-sions, in a subgroup of patients with myelofi-brosis.22JAK2 V617F may provide a useful marker of neoplastic proliferation, and current therapies elicit molecular responses in only a minority of patients with this mutation.12,20,23,24 In contrast,a rapid, substantial reduction in mutant allele burden was observed in all JAK2 V617F–positive patients in this study; 88% of patients had a partial molecular response. Attenuation and loss of a molecular response later during therapy may be due in part to reduced-frequency maintenance dosing; this suggests that the duration of ime-telstat treatment was insufficient to eliminate all mutated committed and clonal primitive hema-topoietic progenitors. The decrease in the CALR and MPL mutant allele burdens provides support for the hypothesis that imetelstat can reduce the various malignant clones observed in myelopro-liferative neoplasms. The decrease in the mutant allele burden appeared to be more pronounced for the JAK2 V617F mutations than for the CALR mutations, possibly because of activation of telomerase reverse transcriptase by the Janus kinase–signal transducer and activator of tran-scription (JAK-STAT) signaling pathway in pa-tients with essential thrombocythemia who had JAK2 V617F mutations.25,26Megakaryocyte CFU assays that were used to more directly measure inhibition of proliferation showed more than 90% inhibition of baseline proliferation activity within 1 month after the initiation of treatment in four of the five patients tested. These data suggest that imetelstat may have a selective inhibitory effect on the growth of the neoplastic clone or clones that drive es-sential thrombocythemia.Although all the patients had hematologic responses, there were subsequent fluctuations above normal platelet levels in all patients. Re-sponses were considered to be durable if inter-mittent dosing restored and maintained the platelet count (≤600,000 per cubic millimeter for a partial response or ≤400,000 per cubic milli-meter for a complete response). Once an initial response was achieved, patients with platelet counts that were not controlled by maintenance dosing (i.e., platelet counts that were >600,000 per cubic millimeter in patients with a partial response or >400,000 per cubic millimeter in patients with a complete response) for at least 8 weeks were considered to have a loss of re-sponse. The reported complete hematologic re-sponses were durable but required ongoing ime-telstat therapy; interpatient variability was noted in the frequency of dosing required to manage platelet levels (every 2 weeks to every 4 weeks or more) (Fig. S2 in the Supplementary Appendix).* L aboratory abnormalities were defined on the basis of normal laboratory rang-es at each institution and were graded according to the CTCAE. The maxi-mum grade was calculated for patients who had an increase in grade, as com-pared with the baseline grade.Imetelstat in Essential ThrombocythemiaAfter discontinuation of treatment, increases in platelet counts were consistent with the main-tenance interval during treatment. Patients with longer treatment intervals had more gradual increases after discontinuation of treatment, whereas patients who required more frequent dosing had more rapid increases.At the time of the primary analysis, 10 pa-tients were still receiving treatment, with a median follow-up of 17 months (range, 7 to 32 [ongoing]). Of the adverse events attributed to imetelstat, most were mild to moderate; 18 events were of grade 3 or higher (Table S8 in the Sup-plementary Appendix). Upper respiratory infec-tions, which were reported in 6 of 18 patients (33%), were mostly mild to moderate and were not associated with myelosuppression or disease progression. Three patients had disease progres-sion from essential thrombocythemia to second-ary myelofibrosis, either during the study treat-ment period or during the safety follow-up period, and JAK inhibitor therapies were initiat-ed. Among these 3 patients, one had an unrelated grade 2 upper respiratory tract infection and grade 3 osteomyelitis and another had grade 2 influenza.In a study involving 1104 patients, the rates of progression from essential thrombocythemia and from early or prefibrotic primary myelofi-brosis to overt myelofibrosis were 0.8% and 12.3%, respectively, at 10 years and 9.3% and 16.9%, respectively, at 15 years.26 The prognostic variables associated with death or progression to leukemia or myelofibrosis were bone marrow histologic features (early or prefibrotic primary myelofibrosis vs. essential thrombocythemia), age (>60 years), history of thrombosis, leukocy-tosis, and anemia.27 The 3 patients in our study who had disease that progressed to myelofibro-sis had essential thrombocythemia for 12, 13, and 21 years and were 67, 61, and 56 years old, respectively, when myelofibrosis was diagnosed. Two of the 3 patients received the diagnosis near the date of their last dose of imetelstat, and 1 received the diagnosis 6 months after the last dose, during extended follow-up of liver func-tion. At baseline, 2 of the 3 patients had 1+ and 2+ reticulin fibrosis in the bone marrow, and the hemoglobin level in all 3 patients was lower than 12 g per deciliter. The rate of progression to myelofibrosis in the current study (17%; 3 of 18 patients) is higher than historical estimates; however, the small number of patients in this study and the presence of prognostic factors make cross comparisons difficult.Abnormal results of liver-function tests were observed frequently but were mostly mild in grade; one fatal event of bleeding esophageal varices occurred after the primary-analysis time point. On March 11, 2014, the FDA issued a full clinical hold on imetelstat, citing a lack of evi-dence of reversibility of hepatotoxicity, concern regarding a risk of chronic liver injury, and a lack of adequate follow-up in patients who had hepatotoxic effects. All the patients who were still receiving the study medication permanently discontinued active treatment and were observed for safety. Follow-up safety data showed that treatment-related abnormalities on liver-function tests resolved in most patients (Table S9 in the Supplementary Appendix), and the FDA lifted the clinical hold on October 31, 2014.In conclusion, imetelstat, a telomerase inhibi-tor, had a clinically significant effect on disease burden in patients with essential thrombocythe-mia who had not had a response to previous treatment or who had had unacceptable side ef-fects from conventional therapies. Neutropenia and abnormal liver-function tests were the main adverse events. Furthermore, molecular respons-es were observed in patients with mutated JAK2 and CALR molecular signatures; this finding suggests therapeutic activity in the malignant clones that are the underlying source of essential thrombocythemia.Supported by Geron.Disclosure forms provided by the authors are available with the full text of this article at .We thank the patients who volunteered to participate in this study and the study site staff who cared for them: Renata Bünter, Ursina Sager, Regula Jäggi, and Dr. Stephan Reichenbach at the Clinical Trials Unit of the University of Bern, Switzerland; Drs. Alexandre Theocharides, Gabi Vetsch, Giuseppe Colucci, and other members of the Clinical Hematology Team of the Univer-sity Hospital of Bern; Dr. Monika Haubitz, Dr. Meike Dahlhaus, Ingrid Helsen, Barbara Hügli, Elisabeth Ischi, and other mem-bers of the Experimental Hematology, Molecular Diagnostics, and Stem Cell Laboratory of Hematology teams of the University of Bern and University Hospital of Bern; the staff of Geron who were involved in data collection and analyses, including Dr. Joi Ninomoto for contributions to study design, and Ted Shih, Neeru Batra, Dianne Morfeld, and Raheela Kauser Steiner of Geron for their contributions to data acquisition, review, and interpreta-tion in previous studies and throughout the span of the trial; and Dr. Tracy T. Cao of Source One Technical Solutions and Dr. Namit Ghildyal of Janssen Research and Development, for edi-torial assistance with an earlier version of the manuscript.。
Nature子刊：线粒体融合的增加对老年动物延长寿命至关重要
Nature子刊：线粒体融合的增加对老年动物延长寿命至关重要订阅号APExBIO线粒体是经历融合（fusion）和裂变（fission）的动态细胞器。
线粒体动力学对于维持线粒体活力和应对外界生物反应是至关重要的。
来自美国佐治亚大学(University of Georgia)细胞生物学的研究团队通过研究秀丽隐杆线虫发现，胰岛素信号和SCF LIN-23通路通过抑制线粒体蛋白酶SPG-7和PPGN-1的表达来调控线粒体融合。
该通路是线粒体融合响应于体力消耗以及相关寿命延长所必需的。
研究还发现多种长寿信号通路突变体表现出细长的线粒体（线粒体融合）水平增加，增加的线粒体融合对于这些突变体寿命的延长至关重要，线粒体融合被抑制后其寿命减少到野生型水平。
值得注意的是，增加的线粒体融合不是长寿的主要驱动因素，而是老年动物在延长寿命期间维持存活所必需的。
背景介绍线粒体在信号传导、生理和代谢中起着不同的作用。
线粒体动力学调节线粒体的形态、数量和功能，以适应细胞需求。
线粒体裂变（Mitochondrial fission）对于线粒体，线粒体与子细胞的有丝分裂分离，和线粒体的亚细胞定位（如神经元轴突）是必需的。
线粒体融合（Mitochondrial fusion）是维持线粒体膜电位和呼吸能力以及防止哺乳动物细胞凋亡所必需的。
细胞调节线粒体形态以协调细胞对能量的需求和对资源的利用。
延长的形态与ATP产生增加，活性氧（ROS）产生减少相关联，而碎片形态与ATP产生减少和线粒体解偶联相关。
线粒体融合和裂变被严格调控，需要进化保守的GTP酶的活性。
酵母，无脊椎动物和哺乳动物中的线粒体裂变需要dynamin相关蛋白DNML1（发芽酵母中的Dnm1和秀丽隐杆线虫中的DRP-1）。
线粒体融合需要线粒体融合蛋白（mitofusins）进行外膜融合（哺乳动物中的MFN1和MFN2，芽殖酵母中的Fzo1和秀丽隐杆线虫中的FZO-1）和内膜融合（哺乳动物中的OPA1，发芽酵母中的Mgm1和秀丽隐杆线虫中的EAT-3）。
htra2omi调控线粒体未折叠蛋白反应及在脑缺血再灌注损伤中的作用
中文摘要HtrA2/Omi调控线粒体未折叠蛋白反应及在脑缺血再灌注损伤中的作用研究背景：线粒体是真核细胞进行氧化还原反应的重要细胞器，是机体产生能量的源泉。
线粒体内稳态的平衡，是机体维持细胞正常运转，对应各种损伤刺激和缓解细胞死亡必不可少的。
当线粒体无法维持自身内稳态的平衡时，如线粒体质量控制蛋白的缺失、抗氧化防御系统受损、线粒体未折叠蛋白的大量堆积或线粒体生物合成的无法正常运转等，会造成严重的线粒体功能障碍，往往产生许多各种各样的疾病，像神经退行性病、癌症、老化、血管类疾病、糖尿病、心力衰竭等，严重威胁着人类的健康。
HtrA2/Omi（High temperature requirement factor A2，HtrA2）是一种进化保守的线粒体丝氨酸蛋白酶，由内质网合成，定位于线粒体膜间隙IMS（Mitochondrial intermembrane space，IMS）中。
HtrA2/Omi作为一种线粒体质量控制蛋白，能够识别错折叠蛋白暴露出来的疏水表面，发挥分子伴侣的功能，抵抗应激，具有细胞保护的作用。
但当线粒体受损，线粒体膜通透性增加时，HtrA2/Omi从线粒体释放并进入细胞质和（或）细胞核中，通过其AVPS结构域结合并抑制XIAP（X-linked inhibitor apoptosis protein，XIAP），引起下游Caspase9的活化，发挥促凋亡作用，最终使得细胞死亡。
此外，HtrA2/Omi还可通过其自身蛋白酶的作用直接介导Caspase-independent细胞凋亡，这也是其独特之处，可明显区别于其他线粒体促凋亡蛋白，如细胞色素C（Cytochrome C，CytC）、AIF（Apoptosis inducing factor，AIF）和Smac/DIABLO（Second mitochondria-derived activator of caspases，Smac/DIABLO）。
雷帕霉素对胶质瘤干细胞的特异性凋亡作用
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线粒体复合物在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用
线粒体复合物在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用张志宏;黄晓莉;贾莉;张旭光;王晓琳;吴坤【期刊名称】《癌变·畸变·突变》【年(卷),期】2012(024)006【摘要】OBJECTIVE: To study four key enzyme complexes in the mitochondria that contribute to vitamin E succinate (VES)-inhibited cell growth in human gastric cancer cells SGC-7901. METHODS: Gastric cancer cells SGC-7901 were grown in vitro. Cells were treated with rotenone(ROT), thenoyltrifluoroacetone(TTFA), antimycin A(AA) or sodium azide(SA) alone before treatment with VES for 2 h. Then cells were treated with VES at 20μ g/ml. MTT assay was used to evaluate cell growth. ROS was detected by confocal microscope. FITC/Annexin V was used to assess apoptosis. RESULTS: Cell growth inhibition induced by VES were obviously down-regulated after complex Ⅱ was inhibited by TTFA(5 (μg/ml)(P<0.05). ROS induced by VES was all down-regulated after four complexes were inhibited respectively. Percentage of apoptosis induced by VES was all up-regulated after four complexes were inhibited respectively (P<0.05). However, levels of ROS and apoptosis were obviously lower than other three groups after complex Ⅱ was inhibited by TTFA (5 μ g/ml) (all P<0.05). Cytochrome C and caspase- 3 induced by VES were all down-regulated after cells treated with ROT, TTFA, AA or SA, respectively (P<0.05).Cytochrome C and caspase-3 were obviously down-regulated compared with other three groups(P<0.05). CONCLUSION: Key enzyme of complex Ⅱ (succinate dehydrogenase, SDH) was affected by VES. SDH induced ROS retention in cells. This may be the mechanism that VES contributes to vitamin E succinate-induced apoptosis in human gastric cancercells.u0000ups(P%目的:探讨维生素E琥珀酸酯(vitamin E succinate,VES)诱导人胃癌细胞凋亡过程中线粒体4种复合物的作用,并寻找对肿瘤细胞有抑制作用的关键酶.方法:体外常规培养SGC-7901细胞,分别加入线粒体复合物Ⅰ、Ⅱ、Ⅲ和Ⅳ(complex Ⅰ ～Ⅳ,CⅠ～Ⅳ)的抑制剂鱼藤酮(rotenone,ROT)、噻吩甲酰三氟丙酮(thenoyhrifluoroacetone,TTFA)、抗霉素A(antimycin A,AA)和叠氮钠(sodium azide,SA)2 h后以20μg/ml VES诱导处理,并设阴性对照组(含0.1％乙醇的培养液),20μg/ml VES单独处理组；MTT法测定细胞增殖情况,共聚焦显微镜观察活性氧(reactive oxygen species,ROS)水平,流式细胞术检测细胞凋亡情况,Western blot法检测细胞中细胞色素C和Caspase-3表达情况.结果:与VES 对照组比较,在VES诱导SGC-7901细胞凋亡的过程中,添加5μg/mlTTFA抑制线粒体复合物Ⅱ酶活性后,细胞增殖抑制率水平显著降低(P＜0.05).4个线粒体复合物抑制剂处理组ROS水平均显著降低(P＜0.05),而细胞凋亡率显著升高(P＜0.05)；但5μg/ml TTFA处理组ROS及凋亡率的升高均显著低于其他3个处理组(P均＜0.05).分别抑制CⅠ～Ⅳ酶活后,VES上调细胞中细胞色素C和Caspase-3的能力均受到抑制,其中VES+TTFA组抑制程度明显高于另外3组(P均＜0.05).结论:VES 处理SGC-7901细胞,可影响线粒体复合物Ⅱ中琥珀酸脱氢酶活性,致使线粒体中ROS堆积,这可能是VES诱导人胃癌细胞凋亡的机制之一.【总页数】5页(P431-435)【作者】张志宏;黄晓莉;贾莉;张旭光;王晓琳;吴坤【作者单位】哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨 150081;哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨150081;哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨150081;哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨150081;哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨150081;哈尔滨医科大学公共卫生学院营养与食品卫生学教研室,黑龙江哈尔滨150081【正文语种】中文【中图分类】Q279【相关文献】1.TRAIL途径在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用 [J], 张海金;吴坤2.BaX在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用 [J], 张晓华;夏薇;吴坤3.Bcl-2、Bak及caspase-9、3在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用 [J], 苑林宏;张晓华;张岭;吴坤4.Bcl-2家族蛋白在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用 [J], 张晓华;苑林宏;单毓娟;张岭;吴坤5.维生素E琥珀酸酯诱导人胃癌细胞凋亡中线粒体的作用 [J], 吴坤;李贵昌;赵艳;张晓华因版权原因，仅展示原文概要，查看原文内容请购买。
普利莱基因技术线粒体提取试剂盒说明书
升级版线粒体提取试剂盒C0010描述：线粒体制备试剂盒(Mitochondria Isolation Kit)用于从组织或培养细胞中分离线粒体和细胞胞浆成分。
加入分离溶液，匀浆破碎组织细胞，经过数次800g和12000g离心，在60分钟内即可分离出完整的线粒体和胞浆成分。
制备的线粒体具有很高的生物学活性，可进行各种功能研究如酶学测定，更可用于Western Blot、2D-胶、线粒体蛋白或DNA提取、蛋白质组学等研究。
严格按照说明操作，总是能制备获得高纯度线粒体。
一篇方法学研究论文发现，用普利莱试剂盒制备线粒体的得率、活性、纯度优于蔗糖密度梯度离心法和Invotrogen/Pierce线粒体提取试剂盒方法。
适用：从组织、培养细胞制备高纯度线粒体，同时分离细胞胞浆成分。
组成：Mito Solution100ml for50次制备200ml for100次制备储存：−20ºC12个月有效操作步骤：以下所有操作均在4ºC进行1.组织匀浆：100~200mg新鲜组织如肝、脑、肾、心肌等，剪为0.5cm2碎块放入小容量玻璃匀浆器内。
估计组织块总体积。
加入1.5ml冰预冷的Mito Solution。
用间隙严紧的研杵上下研磨组织20次。
培养细胞匀浆：800×g5min离心收集细胞。
单次提取需2-5×107个细胞。
加入1.5ml冰预冷Mito Solution 重悬细胞，将细胞悬液转移到小容量玻璃匀浆器内，用间隙严密的研杵研磨细胞30次。
2.将匀浆液转移到离心管中，800×g，4ºC离心5min。
（胞核、膜碎片、未裂解细胞等在管底，弃去）3.收集上清液并转移到新的离心管。
再次800×g离心5min at4ºC，弃沉淀。
4.将上清液转移到新的离心管。
10,000×g离心10min4ºC。
线粒体沉淀在管底。
离心后的上清含胞浆成分，可收集用于对照实验。
新型抗癫痫药物吡仑帕奈相关问题解答
新型抗癫痫药物吡仑帕奈相关问题解答吡仑帕奈（perampanel）是由卫材制药公司（EISAIINC）开发的世界首例非竞争性α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）受体拮抗药，2012年7月和10月分别被欧洲药品监管局（EMA）和美国食品药品监督管理局（FDA）批准，用于对12岁及以上、伴有或不伴有继发性全身大发作的部分发作癫痫患者进行辅助治疗。
2015年6月22日，卫材公司宣布，美国FDA已批准扩大吡仑帕奈水合物（fycompa）的适应证，作为12岁及以上癫痫患者原发性全面强直-阵挛癫痫发作的辅助治疗用药。
吡仑帕奈为AMPA受体拮抗药，通过抑制突触后AMPA受体谷氨酸活性，减少神经元过度兴奋而发挥抗癫痫作用，是首个经FDA批准的AMPA受体拮抗剂类抗癫痫药物。
谷氨酸是中枢神经系统主要的兴奋性递质，涉及一系列过度兴奋的神经系统疾病。
谷氨酸受体分为两类：一类为离子型受体，包括N-甲基-D-天冬氨酸受体（NMDAR）、海人藻酸受体（KAR）和α-氨基-3羟基-5甲基4-异恶哇受体（AMPAR），它们与离子通道偶联，形成受体通道复合物，介导快信号传递；另一类属于代谢型受体（mGluRs），它与膜内G-蛋白偶联，这些受体被激活后通过G-蛋白效应酶、脑内第二信使等组成的信号转导系统起作用，产生较缓慢的生理反应。
吡仑帕奈的半衰期长达106小时，因此可每日服用一次，其中酶诱导剂可减少吡仑帕奈血浆浓度，降低其效力。
一项多中心的随机双盲安慰剂对照临床Ⅲ期试验已证实，吡仑帕奈作为辅助治疗可将局灶性癫痫发作减少23.3%～27.2%。
另一项临床Ⅲ期试验显示，吡仑帕奈可将全面强直-阵挛发作减少76.5%（安慰剂组为38.4%，P<0.0001），另外吡仑帕奈组患者13周无发作的比率为30.9%，远大于安慰剂组的12.3%。
口服吡仑帕奈后，药物迅速完全吸收，食物不会影响药物的吸收程度但会减慢吸收速度。
Cell子刊：FDA库新筛出一种小分子可诱导线粒体融合,发现了嘧啶合成与线粒体形态间的重要联系
Cell子刊：FDA库新筛出一种小分子可诱导线粒体融合，发现了嘧啶合成与线粒体形态间的重要联系订阅号APExBIO研究意义线粒体是具有融合（fusion）和分裂（fission）能力的动态细胞器，是细胞进行有氧呼吸的主要场所。
越来越多的病理与线粒体动力学的改变有关。
线粒体融合由线粒体融合蛋白（MFN1和MFN2）和OPA1介导。
线粒体融合蛋白的减少与代谢紊乱、神经变性和心血管疾病等有关。
因此，线粒体融合蛋白已经成为潜在的药理学对象。
在这里，Miret-Casals等人通过FDA化合物库的高通量筛选发现了一种小分子——leflunomide（来氟米特），是MFN1 / MFN2表达的激活剂并促进线粒体的延伸。
来氟米特的作用机制揭示了嘧啶（pyrimidine）代谢与线粒体动力学之间的新的联系。
重要的是，基于这种联系发现另几种小分子化合物也可以诱导线粒体融合蛋白的表达并促进线粒体融合。
总之，该机制的探讨为治疗与线粒体功能障碍有关的疾病提供了新的视角。
图1 阻断DHODH介导的嘧啶生物合成促进线粒体融合线粒体是细胞生成大部分ATP的动态细胞器，是细胞的“能量工厂”，参与许多重要的细胞功能，例如钙信号传导、细胞分化、细胞凋亡和生长等。
线粒体动力学的一个关键过程是线粒体融合。
线粒体融合（mitochondrial fusion）是一个复杂的过程，指相邻的两个线粒体实现外膜和内模的融合从而变成一个线粒体，从形态上看发生了延伸。
在这个融合的过程中，有两个重要的位于线粒体外膜中的融合蛋白，线粒体融合蛋白 2（MFN2）和线粒体融合蛋白1（MFN1），以及位于线粒体内膜的OPA1。
MFN1和MFN2介导外膜融合，OPA1介导随后的内膜融合。
线粒体融合和裂变（fission）之间的平衡使线粒体维持功能性动力学。
而动力学缺陷，如MFN2或OPA1突变会导致人类疾病。
MFN2突变导致Charcot-Marie-Tooth 神经病变，有时与视觉障碍有关。
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In vitro: Previous study found that piericidin A could inhibit the electron transport system at two sites. Piericidin A specifically reacted at very low concentrations at a site near the reduced NADH dehydrogenase. At high concentrations, piericidin A inhibited the succinic dehydrogenase system, and the inhibition could be partially reversed by coenzyme Q. Moreover, it was found that both the succinate and NADH oxidase system were inhibited at high levels of piericidin A [1]. In vivo: Animal study was performed to evaluate the effect of piericidin A and antagonistic effect of vitamin K3 on respiration, blood pressure and heart rate in rats. Results showed that 0.167 mg/kg of piericidin A increased the respiratory rate and the lowered blood pressure rapidly. Furthermore, vitamin K3 (10-40 mg/kg) could restore the responses to piericidin A in rats [2]. Clinical trial: Up to now, piericidin A is still in the preclinical development stage.
ApexBio Technology
Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request
lism
信号通路:
Dehydrogenase
产品描述:
特别声明
产品仅用于研究，
不针对患者销售，望谅解。
每个产品具体的储存和使用信息显示在产品说明书中。ApexBio 产品在推荐的条件下是稳定 的。产品会根据不同的推荐温度进行运输。许多产品短期运输是稳定的，运输温度不同于长 期储存的温度。我们确保我们的产品是在保持试剂质量的条件下运输的。收到产品后，按照 产品说明书上的要求进行储存。
Soluble in DMSO
Store at -20°C
For obtaining a higher solubility , please warm the tube at 37°C and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20°C for several months.
Piericidin A is a mitochondrial complex I inhibitor. Complex I, also known as NADH:ubiquinone oxidoreductase, catalyzes the transfer of electrons from NADH to ubiquinone as part of the respiratory chain resulting in ATP generation.
参考文献: [1] Jeng M, Hall C, Crane FL, Takahashi N, Tamura S, Folkers K. Inhibition of mitochondrial electron transport by piericidin A and related compounds. Biochemistry. 1968 Apr;7(4):1311-22. [2] Mitsui T et al. Studies on piericidin antagonistic effect of vitamin K3 on the respiratory chain of insects and mammals in the presence of piericidin A. Agricultural and Biological Chemistry Vol. 34 (1970) No. 7 P 1101-1109
产品说明书
化学性质
产品名: Piericidin A 修订日期: 6/30/2016
产品名: Cas No.: 分子量: 分子式: 别名:
化学名:
SMILES:
溶解性: 储存条件: 一般建议:
运输条件:
Piericidin A
2738-64-9
415.6
C25H37NO4
AR 054|Shaoguanmycin B|SN 198E
2-[(2E,5E,7E,11E)-10R-hydroxy-3,7,9R,11-tetramethyl-2,5,7,11-tridec atetraen-1-yl]-5,6-dimethoxy-3-methyl-4-pyridinol
COC1=C(O)C(C)=C(C/C=C(C)/C/C=C/C(C)=C/[C@H]([C@@H](O)/C(C) =C/C)C)N=C1OC