端粒酶抑制剂
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.。
端粒酶在临床诊断和治疗中的应用
端粒酶在临床诊断和治疗中的应用摘要：端粒的长短和稳定性决定了细胞的寿命 ,并与细胞的衰老和癌变密切相关。
端粒酶在细胞中负责端粒的延长，能以自身携带的 RNA 为模板 , 不断合成新的端粒 DNA 序列添加到染色体末端, 弥补端粒丢失阻止端粒缩短。
端粒酶在正常人体组织中的活性被抑制，其激活与细胞的癌变密切相关。
它是所有癌症亚类细胞, 包括癌干细胞, 永生化所必需的成分。
在正常和肿瘤组织之间,端粒酶表达、端粒长度和细胞动力学存在明显的差异。
关键词：端粒酶;诊断;治疗;癌症前言：近年来, 端粒及端粒酶的研究已成为生物学热点, 随着对端粒酶的研究深入,应用靶向端粒酶的方法治疗肿瘤成为热点 ,并有较多的新进展, 是人类抗肿瘤药物研究的新“靶点”。
随着人们对端粒及端粒酶结构和功能认识的加深 ,针对端粒酶的抗肿瘤治疗研究也不断深入, 从早期抑制肿瘤细胞端粒酶活性 ,发展到新近与免疫治疗和基因治疗相结合端粒酶在临床上应用更广阔。
端粒酶是在细胞中负责端粒的延长的一种酶，能以自身携带的 RNA 为模板 , 不断合成新的端粒 DNA 序列添加到染色体末端, 弥补端粒丢失阻止端粒缩短。
端粒酶在正常人体组织中的活性被抑制，其激活与细胞的癌变密切相关。
端粒在不同物种细胞中对于保持染色体稳定性和细胞活性有重要作用，端粒酶能延长缩短端粒（缩短的端粒其细胞复制能力受限），从而增强体外细胞的增殖能力。
端粒酶在保持端粒稳定、基因组完整、细胞长期的活性和潜在的继续增殖能力等方面有重要作用。
人类端粒酶是一种由RNA和相关蛋白质组成的核酸蛋白质复合物，由与端粒DNA互补的RNA 模板(human telomerase RNA,hTR)、端粒酶相关蛋白(TEP1)和端粒酶催化亚单位(端粒酶逆转录酶 human telomerase reverse transcriptase, hTERT)三个亚基构成【1】。
其中以hTERT和hTR组成的端粒酶最小核心结构对维持端粒酶的活性有重要作用。
端粒酶
端粒酶抑制剂用于肿瘤治疗的研究新进展近年来，肿瘤细胞永生化的“端粒—端粒酶学说”已为越来越多的研究结果所证实。
端粒酶是唯一的一种以自身RNA序列为模板将端粒重复序列复制添加到染色体3′末端的核蛋白酶。
许多研究结果表明：人类85%～90%的肿瘤细胞的端粒酶活性呈阳性，端粒酶的激活及端粒长度的稳定与肿瘤、衰老的发生和发展有密切关系，针对端粒酶基因的调控可以通过抑制端粒酶的活性来抑制肿瘤的生长并促进其凋亡，具有对肿瘤细胞特异性的作用，是肿瘤靶向治疗的一个新的研究方向。
本文中对端粒、端粒酶的结构和功能，端粒酶活性的调控，端粒酶与肿瘤和衰老的关系，端粒酶抑制剂的最新研究进展以及在肿瘤等疾病治疗中的应用前景进行综述和分析，为进一步开发天然来源的端粒酶抑制剂、研发肿瘤靶向治疗新药提供参考。
1.端粒与端粒酶1.1端粒端粒（T elomers）为真核细胞染色体末端的一种特殊结构，由端粒DNA和端粒蛋白质组成，其DNA 含有大量的TTAGGG n串联重复结构，其功能是完成染色体末端的复制，防止染色体融合、重组和降解，起着保护染色体末段的作用。
端粒是线性DNA，它的末端会随周期性的复制而逐渐缩短。
Muller1于1938年首次发现这一结构并将其命名为端粒（T elomers）。
由于末端复制问题，细胞每分裂1次，端粒就缩短50～100，当端粒缩短到临界长度时，细胞就会出现衰老以致死亡，因此在正常真核细胞中，端粒可被看成是“生命的时钟”或“有丝分裂的计数器”2。
1.2端粒酶端粒酶(T elomerase) 是一种由RNA和蛋白质组成的特异核糖核酸蛋白复合体，具有逆转录的酶活性，能以自身的RNA为模板5’-CUAACCCUAAC-3’通过逆转录合成端粒重复序列并连接到染色体末端以补偿细胞分裂时端粒的缩短，使细胞获得无限增殖能力3。
端粒酶的主要功能是：①通过自身的RNA 模板、催化亚单位和辅助蛋白将端粒DNA 添加到染色体的末端；②维持和平衡端粒序列长度；③修复断裂的染色体末端4。
端粒酶抑制剂在恶性肿瘤治疗中的作用
端粒酶抑制剂在恶性肿瘤治疗中的作用【摘要】端粒酶作为一种负责延长端粒的核蛋白逆转录酶，对于细胞染色体的稳定性和细胞活性的维持有重要作用。
端粒酶的活性在正常组织中被抑制，而在恶性肿瘤细胞中其阳性率可达84%~95%。
故而端粒酶的恶性转化，对于恶性肿瘤的发生和发展有着潜在的关联。
针对这一现象，并结合端粒酶本身的特点，人们开发出端粒酶抑制剂以治疗恶性肿瘤。
【关键词】端粒酶抑制剂；染色体；恶性肿瘤端粒酶抑制剂对于恶性癌症的临床作用，通过近几年来的研究，正逐渐为人所熟悉。
其作用机制各有不同，但最终的效用都能够抑制恶性肿瘤细胞的生长与分裂［1］。
此类物质包括：反义寡核苷酸,逆转录抑制剂，G-四连体稳定剂，蛋白激酶C抑制剂，化疗药物。
1 反义寡核苷酸(antisense oligodexoynuclectide, ASODN)反义寡核苷酸能够与靶RNA或DNA特异性互补，能够使得恶性肿瘤细胞端粒酶活性降低，进而其增殖能力下降，甚至调亡。
应用这一原理，人工的合成针对模板序列的反义核苷酸抑制剂在研究中已取得很大突破。
例如Mete等［2］使用硫代反义核苷酸(PAS-ODN)对Burkitt s瘤中淋巴瘤细胞系OMA-BLI生长有明显的压制作用，导致细胞死亡。
此外夏雪梅等［3］对凋亡抑制基因survivin反义寡核苷酸的研究表面，通过使用bcl-2反义寡核苷酸对肺癌细胞株的抑制作用十分显著，可出现肺癌细胞生长抑制以至凋亡，细胞survivin mRNA明显下降。
肺癌细胞株survivin基因表达受到survivin反义寡核苷酸的有效抑制。
并诱导细胞凋亡和生长延缓，同时也证明联合bcl-2反义寡核苷酸对提升抗癌能力有明显功用。
2 逆转录酶抑制剂(reserve transcripitase inhabitors)3-叠氮-3-脱氧胸腺核苷(3-azido-2,3-dideoxythymidine, AZT)作为逆转录抑制剂中最为人所关注的一种，其临床价值得到广泛肯定。
端粒酶抑制剂
端粒酶抑制剂在肿瘤治疗中的最新研究进展摘要：端粒酶是一种特殊的逆转录酶，能以自身的 RNA为模板，反转录成端粒的重复单元TT AGGG加到人染色体末端，阻止端粒随细胞分裂而缩短，使细胞绕过衰老途径成为永生化细胞，导致人类肿瘤的发生。
以端粒酶为靶点，可以有多种治疗途径，本文主要介绍了端粒酶抑制剂的研究现状及新进展，重点对新型G ‐四联体稳定剂类端粒酶抑制剂、逆转录酶抑制剂及其他新型端粒酶抑制剂的研究进展进行介绍。
关键词：端粒酶抑制，G‐四联体，逆转录酶抑制剂，肿瘤，研究进展The newest research progress of the telomerase inhibitorsin cancer therapyKeywords:telomerase inhibitors，G-quadrplex DNA，Reverse transcriptase inhibitors，Tumor，research progress引言：端粒酶作为一种负责延长端粒的核蛋白逆转录酶，对于细胞染色体的稳定性和细胞活性的维持有重要作用，端粒酶的活性在正常组织中被抑制，而在恶性肿瘤细胞中其阳性率可达 84% ～95%，人体绝大部分恶性肿瘤的发生发展过程与端粒酶活性有非常紧密的联系，针对这一现象，并结合端粒酶本身的特点，人们开发出端粒酶抑制剂，应用不同端粒酶抑制剂针对端粒酶的不同组分及作用途径进行破坏或阻断，从而抑制端粒酶活性最终限制肿瘤的生长及发展，这是近年来国内外学者积极探索的一个方向。
1端粒与端粒酶概述端粒是位于真核细胞染色体末端的一种特殊结构，由DNA片段和蛋白组成，其主要功能是维护染色体的完整性，端粒长度随着有丝分裂逐渐缩短，当缩短至不能维护染色体稳定时，则导致细胞凋亡。
人端粒是染色体末端的一段富含GC 的重复序列，其生物学功能主要有：①保护染色体末端完整性；②参与染色体的定位和复制，保证细胞的正常分化与繁殖；③与细胞凋亡和永生化关系密切[1]，在细胞分裂过程中，染色体末端逐渐缩短(图 1、2)，当端粒缩短至l隔界值(4kb)以下时细胞趋向凋亡。
端粒酶与肿瘤的研究进展
' 第 26卷 第 2期 黄 石 理 工 学 院 学 报V o.l 26 N o. 2 2010年 4月JOURNA L O F HUANG SH I INST ITUT E OF TECHNOLOGYA pr. 2010do:i 10. 3969 / .j issn. 1008- 8245. 2010. 02. 013端粒酶与肿瘤的研究进展陈玉华(黄石理工学院 医学院, 湖北 黄石 435003)摘要: 端 粒及端粒 酶已成为生 命科学和医 学的研究 热点, 端粒酶在癌 症发生发 展中起重要 的作用。
端粒酶是一种逆转录酶, 它的存在解决了染色体复制末端缩短 问题, 同 时认为端 粒酶的过度 表达与细 胞的永生化 和癌变直接相关。
随着人们对端 粒及端粒酶结构和功能认识的深入, 在临床上以端粒 酶为靶标治疗肿瘤已取 得积极进展。
文章综述了端粒酶 与肿瘤的关系, 以及端粒酶在治疗肿瘤方面的应用。
关键词: 端粒; 端粒 酶; 肿瘤中图分类号: R73 文献标识码: A 文章编号: 1008- 8245( 2010) 02- 0043- 03Advance in Research of Telomerase and TumorCHEN Yuhua( Schoo l o fM edic ine, H uangshi Institu te of Techno logy, H uangsh iH ube i 435003)Abstrac t: T elom ere and te lm om erase have becom e the focus of bio log ica l and m edica l study. Th e telom erase plays an im po rtant role in the deve lopm ent o f cancer. T e lom erase is a k ind of reve rse transcriptnpl enzym e that can so lve the end- replication problem. The over expression of te lom erase is directly re lated to ce ll imm orta liz ation and canceration. W ith people s' increas ing unde rstand ing o f the function and struc ture of te lom ere and telom erase, prog ress has been a ch ieved in c linical trea tm ent o f tumo r by te lom erase. Th is paper has rev iewed the c lose re la tionsh ip betw een tum or and telom erase and the applica tion o f telom erase in the trea tm en t of tumo r.K ey word s: telom ere; te lome rase; tum or为靶 标的肿 瘤 诊 断 治疗 已 取 得 进 展 , 本 文 就 端 粒0 引言临床 上恶 性肿瘤 已 成 为威 胁 人 类生 命 和 健康 的 常见 病之 一。
端粒酶的发展word资料7页
抗癌新靶点：端粒及端粒酶抑制剂研究进展端粒酶在绝大多数恶性肿瘤中特异性表达，这使得人们对肿瘤的抗端粒酶疗法产生了特别的关注。
设想以端粒、端粒酶为靶点，通过抑制癌细胞端粒酶活性或直接抑制端粒延长、稳定，而使细胞无法连续增殖，继而进入衰老途径，直至死亡。
同时端粒、端粒酶在肿瘤细胞与正常体组织之间的差别又可以减少端粒、端粒酶抑制剂对机体的毒副作用。
现对端粒、端粒酶抑制剂研究进展予以分类介绍。
1 控制端粒延长靶点的物质目前对端粒的研究表明，端粒是真核生物染色体末端的特殊结构，包含若干的DNA双链重复序列，其末端为含多个G的单链DNA．不同物种端粒的重复序列和长度是不一样的，但每种生物体有其特定的序列和平均长度［如人的端粒为（TTAGGG）n，大约在15kb左右］。
此外，端粒DNA上还结合有蛋白质，有两种结合形式：一种是结合于单链DNA；另一种则与端粒双链进行结合。
前者在端粒末端提供帽状结构以稳定端粒；后者可能直接参与端粒长度平衡的维持，是端粒延伸的负调节因子［1］。
目前所分离到的人端粒结合蛋白hTRF是一种双链结合蛋白，它参与维持端粒长度的稳定［2］。
改变端粒结构和功能的抑制剂介绍如下。
1．1改变端粒结构导致的端粒酶活性失常端粒是由大量串联的重复序列组成的，其中一条链富含G，另一条富含C。
端粒合成时先由端粒酶将端粒重复序列加到富G链上去，再由DNA聚合酶合成富C链。
不同生物的端粒G链一般都采用紧实结构。
而在所有的结构中，G-四联体是理论上最稳定的结构，它除了可在两条DNA分子间形成外，还可以在含四段重复端粒序列单链DNA中形成。
Zahleretal［3］考察了端粒DNA 的不同折叠方式作为引物时对四膜虫端粒酶的影响。
他们发现端粒G－四联体结构启动端粒酶延伸端粒的效率最差，不能作为端粒酶的引物，另外，他们的进一步研究发现，端粒酶所需的引物可能不应有任何折叠。
折叠的端粒DNA结构由于无法作为引物与端粒酶RNA组分碱基配对、结合，或者改变了端粒引物从端粒酶解离的速度而影响端粒的延伸。
端粒酶抑制剂的研究进展
强有力的端粒酶抑制剂可导致端粒逐渐缩短，最终导致肿瘤 细胞死亡。接下来将对合成类和天然类端粒酶抑制剂近几年的 研究进行概述。 2.1 合成类端粒酶抑制剂 2.1.1 伊美司他
伊 美 司 他 (imetelstat) 是 一 种 血 液 肿 瘤 治 疗 药 物，由 美 国 Geron 公司研发，作为靶向端粒酶 RNA 的寡核苷酸药物，其结构 中 特 有 的 硫 代 氨 基 磷 酸 酯 寡 核 苷 酸 化 学 骨 架 ，可 以 使 肿 瘤 细 胞 端粒酶 RNA 上的 5′ -CUAACCCUAAC-3′序列沉默，从而抑 制端粒酶的活性 [5]。骨髓纤维化是一种骨髓增殖性肿瘤 (MPN)，
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World Latest Medicine Information (Electronic Version) 2019 Vo1.19 No.06
·综述·
端粒酶抑制剂的研究进展
严雯雯，陶毅明 *
( 桂林医学院，广西 桂林 )
摘要：端粒酶是一种具有逆转录酶活性的核糖核蛋白复合体，在 90% 的肿瘤细胞中特异性表达，能合成重复序列添至端粒末端，维持端粒长
Advances in Research on Telomerase Inhibitors YAN Wen-wen, TAO Yi-ming*
(Guilin Medical College, Guilin Guangxi)
ABSTRACT: Telomerase is a ribonucleoprotein polymer with reverse transcriptase activity, specifically expressed in 90% of tumor cells, which can synthesize repeat sequences to the end of telomere and maintain telomere length.Telomerase can be used as an important target of anti-tumor drugs.In recent years, telomerase inhibitors as a potential anti-tumor drug, provide new prospects for the treatment of malignant tumors. Therefore, this article reviews the relevant literatures in recent years, discusses the latest research progress of telomerase inhibitors, and provides a reference for the further research of anti-tumor drugs. KEY WORDS: Telomere; Telomerase; Inhibitor; Anti-tumor drug
《锁阳多糖延缓衰老的作用及端粒酶调控机制研究》范文
《锁阳多糖延缓衰老的作用及端粒酶调控机制研究》篇一一、引言随着人类寿命的延长，抗衰老问题成为了重要的科学研究方向。
自然界的植物成分在抗衰老领域表现出了独特的效果。
其中，锁阳作为一种具有悠久历史的中药材，其多糖成分被广泛研究并认为具有延缓衰老的作用。
本文旨在研究锁阳多糖的抗衰老作用及其与端粒酶的调控机制之间的关系。
二、锁阳多糖的概述锁阳，为中药名，其内含多种活性成分，包括多糖、黄酮类等化合物。
多糖类成分具有明显的生物活性，在保护机体细胞、抗氧化的过程中起到关键作用。
研究发现，锁阳多糖能增强免疫力，抑制体内自由基的产生，从而在延缓衰老方面表现出良好的效果。
三、锁阳多糖延缓衰老的作用1. 抗氧化作用：锁阳多糖能够清除体内的自由基，减少自由基对细胞的损伤，从而起到抗氧化的作用。
2. 保护细胞：锁阳多糖能够保护细胞免受外界有害物质的侵害，维持细胞的正常功能。
3. 调节免疫系统：锁阳多糖能够增强机体的免疫力，提高机体的抗病能力，从而延缓衰老。
四、端粒酶调控机制研究端粒酶是一种能够维持染色体端粒长度的酶，与细胞的寿命密切相关。
研究表明，端粒酶的活性与机体的衰老程度呈负相关关系。
锁阳多糖可能通过调控端粒酶的活性来延缓衰老。
具体机制可能包括：1. 促进端粒酶的表达：锁阳多糖能够促进端粒酶的基因表达，从而提高端粒酶的活性。
2. 维持端粒稳定性：锁阳多糖能够保护端粒免受损伤，维持其稳定性，从而延长细胞的寿命。
3. 抑制细胞凋亡：锁阳多糖能够抑制细胞凋亡，保护细胞免受程序性死亡的影响，从而延缓衰老。
五、实验研究为进一步探究锁阳多糖的抗衰老作用及端粒酶的调控机制，我们进行了以下实验：1. 细胞实验：采用不同浓度的锁阳多糖处理细胞，观察其对细胞生长、抗氧化能力、端粒酶活性等指标的影响。
2. 动物实验：以动物为模型，观察长期摄入锁阳多糖对动物寿命、抗氧化能力、端粒酶活性等指标的影响。
通过实验，我们发现锁阳多糖能够显著提高细胞的抗氧化能力，增强端粒酶的活性，延长细胞的寿命。
端粒酶抑制剂研究进展
端粒酶抑制剂研究进展作者：左利娟李洪雪来源：《中国实用医药》2010年第05期【摘要】端粒酶是一种特殊的逆转录酶,能以自身的RNA为模板,反转录成端粒的重复单元TTAGGG加到人染色体末端,阻止端粒随细胞分裂而缩短,使细胞绕过衰老途径成为永生化细胞,导致人类肿瘤的发生。
以端粒酶为靶点,可以有多种治疗途径,本文主要介绍了端粒酶抑制剂的研究现状及进展,重点对最新型的寡核苷酸类及G四联体稳定剂类端粒酶抑制剂进行介绍。
【关键词】端粒;端粒酶; G四联体;寡核苷酸近年来,端粒及端粒酶的研究已成为生物学热点,也是人类抗肿瘤药物研究的新“靶点”。
根据目前已经检测出的肿瘤组织标本的统计学分析,恶性肿瘤组织端粒酶活性的阳性率达到85%~95%,而良性肿瘤和正常组织的端粒酶活性检出率仅为4%左右[4]。
因此,端粒酶抑制剂的开发成为了抗肿瘤药物研究的又一热点。
1 端粒及其在DNA复制过程中的作用端粒(Telomere)是由位于真核细胞染色体末端的DNA及蛋白质构成的天然末端,是由富含鸟嘌呤(G)的核苷酸经5’3’方向串联组成。
不同物种端粒的重复序列和长度不一样,但每种生物体都有其特定的序列和平均长度。
人类端粒重复序列是5’TTAGGG3’,端粒长度在5~15Kb之间[5]。
端粒有维持染色体稳定和基因组完整的功能,防止染色体末端被化学修饰或被核酸降解、端端融合和重排,并参与染色体在核内的定位及基因表达调控[6]。
研究证明,端粒与细胞的寿命密切相关。
细胞分裂时,胚系细胞可以重建与延长端粒DNA,而体细胞则随着细胞的分裂,端粒不断缩短,缩短速度为50~200 bp/次分裂。
当其长度减小到一定的临界值时,细胞即趋向于衰老、死亡,因此,端粒被认为是绝大多数体细胞的“生物钟”[7]。
2 端粒酶及其与肿瘤发生的关系端粒酶(Telomerase)是位于细胞核内的一种逆转录酶[9],由人类端粒酶RNA(hTR)、端粒酶相关蛋白1(hTP1)和端粒酶逆转录酶(hTERT)三部分构成。
端粒酶逆转录酶的结构分析和抑制剂发现
端粒酶逆转录酶的结构分析和抑制剂发现端粒酶逆转录酶（Telomerase Reverse Transcriptase, TERT）是一种独特的酶，它包含有催化功能和RNA模板区两个重要部分。
它的生物学功能主要是维持染色体末端的长度稳定性，在几个生命过程中都有重要作用，比如在某些细胞里参与细胞的增殖和肿瘤的生长，还有可能延长人的寿命。
TERT在细胞核中形成凹槽型三级结构，由四个不同的区域组成：催化区（CAT）、RNA模板区（TR）、催化区与TR之间的连接区（CR4/5）和N端蛋白质结构域（N）。
结构域的存在是为了提供TERT活性所必须的配体分子，其中，TR和CAT区域是最重要的部分。
TR位于核酮糖绳中，它的核酮糖环是一个典型的3'-OH端，可以通过磷酸二酯键连接反式嘌呤。
磷酸二酯键被短端序列（named as P6 and P7）固定。
催化区由独特的P-loop、封锁序列和四个高度保守的胺基酸组成，它们在CAT功能中起了至关重要的作用。
由于TERT在肿瘤中具有非常重要的功能，因此TERT已成为肿瘤生物学研究的一个研究热点。
因为如果我们能够寻找到具有选择性抑制TERT功能的小分子抑制剂，那么就会对治疗癌症提供帮助。
近年来，科学家们在TERT的结构理解和抑制剂发现方面取得了重大进展。
例如，一项基于核磁共振分析的研究揭示了人类TERT的结构（TOT1），并提供了TERT催化凹槽中TMS结合位点的谷氨酸残基的详细结构。
另一个研究组，使用物理方法提取了TERT蛋白质中的CAT域，然后结合孪生法发现了TERT催化凹槽与TMS的非共价相互作用。
除此之外，近几年来还发现了许多TERT抑制剂。
例如，模拟TR的人工RNA寡核苷酸可以选择性地抑制催化功能；quindoline作为一种TR抑制剂和TERT的催化抑制剂，表现出良好的细胞外抑制活性；quinacrine被证明可以抑制TERT的催化活性，但对其他整合层次的端粒酶活性不产生影响; Celastrol也可以靶向TERT，在肿瘤的体内和体外模型中表现出较强的抗癌活性。
端粒酶抑制剂及其使用方法[发明专利]
![端粒酶抑制剂及其使用方法[发明专利]](https://img.taocdn.com/s3/m/ab8f4a3b76eeaeaad0f3305f.png)
专利名称：端粒酶抑制剂及其使用方法
专利类型：发明专利
发明人：阿利森·C·钦,瑞安·霍尔库姆,米奇斯瓦夫·A·皮亚蒂什科,乌品德·辛格,理查德·L·托尔曼,赤间务,菅田丰,浅
井彰,山下义则,远藤薰,山口弘之
申请号：CN00801875.8
申请日：20000630
公开号：CN1321153A
公开日：
20011107
专利内容由知识产权出版社提供
摘要：本发明涉及体外抑制端粒酶活性并治疗端粒酶介导的病症或疾病的噻唑烷酮类化合物、组合物及方法。
本发明的方法、化合物和组合物可单独或与其它药物活性剂组合用于治疗由端粒酶介导的病症或疾病如癌症。
还公开了测试或筛选端粒酶活性抑制剂的新方法。
申请人：杰龙公司,协和发酵工业株式会社
地址：美国加利福尼亚
国籍：US
代理机构：永新专利商标代理有限公司
代理人：过晓东
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端粒酶抑制剂在肿瘤治疗中的最新研究进展摘要：端粒酶是一种特殊的逆转录酶，能以自身的RNA为模板，反转录成端粒的重复单元TT AGGG加到人染色体末端，阻止端粒随细胞分裂而缩短，使细胞绕过衰老途径成为永生化细胞，导致人类肿瘤的发生。
以端粒酶为靶点，可以有多种治疗途径，本文主要介绍了端粒酶抑制剂的研究现状及新进展，重点对新型G‐四联体稳定剂类端粒酶抑制剂、逆转录酶抑制剂及其他新型端粒酶抑制剂的研究进展进行介绍。
关键词：端粒酶抑制，G‐四联体，逆转录酶抑制剂，肿瘤，研究进展The newest research progress of the telomerase inhibitors incancer therapyKeywords:telomerase inhibitors，G-quadrplex DNA，Reverse transcriptase inhibitors，Tumor，research progress引言：端粒酶作为一种负责延长端粒的核蛋白逆转录酶，对于细胞染色体的稳定性和细胞活性的维持有重要作用，端粒酶的活性在正常组织中被抑制，而在恶性肿瘤细胞中其阳性率可达84% ～95%，人体绝大部分恶性肿瘤的发生发展过程与端粒酶活性有非常紧密的联系，针对这一现象，并结合端粒酶本身的特点，人们开发出端粒酶抑制剂，应用不同端粒酶抑制剂针对端粒酶的不同组分及作用途径进行破坏或阻断，从而抑制端粒酶活性最终限制肿瘤的生长及发展，这是近年来国内外学者积极探索的一个方向。
1端粒与端粒酶概述端粒是位于真核细胞染色体末端的一种特殊结构，由DNA片段和蛋白组成，其主要功能是维护染色体的完整性，端粒长度随着有丝分裂逐渐缩短，当缩短至不能维护染色体稳定时，则导致细胞凋亡。
人端粒是染色体末端的一段富含GC 的重复序列，其生物学功能主要有：①保护染色体末端完整性；②参与染色体的定位和复制，保证细胞的正常分化与繁殖；③与细胞凋亡和永生化关系密切[1]，在细胞分裂过程中，染色体末端逐渐缩短(图1、2)，当端粒缩短至l隔界值(4kb)以下时细胞趋向凋亡。
图1端粒缩短示意图图2 端粒与端粒酶示意图端粒酶是一种能延长端粒末端的核糖核酸蛋白酶，由端粒RNA和蛋白质组成,通过识别并结合于端粒末端，以自身的模板逆转录合成端粒。
自身携带模板是端粒酶的特征之一，其主要功能有：通过自身的RNA模板；催化亚单位和辅助蛋白将端粒DNA添加到染色体的末端；维持和平衡端粒序列长度；修复断裂的染色体末端。
人端粒酶(telomerase) 是一种由人端粒酶逆转录酶(telomerasereverse trantranseriptase，hTERT) 人端粒酶RNA组分(hTR) 以及人端粒酶相关蛋白(hTEPI等)组成的具有特殊逆转录活性的核糖蛋白复合物。
hTERT是端粒酶的催化亚基，是控制细胞端粒酶活性的限制性成分。
通过逆转录hTR模板序列，合成端粒DNA序列并添加到染色体末端，催化末端的端粒DNA复制和延长"端粒酶的生物活性机制已被认为是细胞获得永生的必要途径，是恶性转化的基础，与细胞的衰、肿瘤发生密切相关[2-3]。
近年来随着肿瘤分子生物学的不断发展，越来越多的研究表明绝大部分恶性肿瘤细胞的端粒酶长度较正常细胞缩短。
自1994年，kim应用TRAP(Telomeric Repeat Amplification Protocol)法来检测人体组织中的端粒酶活性，结果发现90%的恶性肿瘤组织中都存在端粒酶活性。
端粒酶活性与恶性肿瘤之间的高度相关性使端粒酶成为近年来肿瘤治疗研究的新热点，对端粒酶活性的抑制是治疗恶性肿瘤的可能新途径之一[4]。
2 端粒酶抑制剂在肿瘤中的应用2.1端粒酶抑制剂G‐四联体稳定因子的研究进展端粒是端粒酶作用的靶位，端粒酶要结合到端粒上才能起作用，端粒部位富含G，体外它能在K +存在下自发折叠形成(分子间或分子内) G‐四联体[5-6]。
研究表明G- 四联体在体外能抑制端粒酶的活性，因而能稳定G‐四联体的小分子药物就能干扰端粒酶参与的端粒复制，体内G‐四联体存在的直接证据还有待证实，但是已经发现结合蛋白质的G‐四联体结构，如双链DNA 能转化成G ‐四联体，螺旋酶解聚G‐四联体快于双链DNA等，这样G‐四联体就成了新的抗肿瘤药物靶位，几个研究组就在从事G‐四联体及其配体的药物设计和开发工作，并已取得几个先导化合物。
几种基于G‐四联体的端粒酶抑制剂：①胺基取代的蒽醌化合物；②阳离子卟啉类化合物：卟啉类化合物由于能富集在肿瘤细胞，早就在进行光动力癌症治疗研究，由于其芳香性平面促使研究者把该类化合物应用于G4-DN结合。
肖云娜等[7]为了提高G4-DNA的稳定性，打破传统的一个G4-DNA分子和一种化合物相互作用的两元模式，采用NMM和PRER共同和G4-DNA作用的三元模式，通过光谱学手段证实了三者的最佳结合比为1：1：1，并且能诱导G4-DNA从反平行结构转变为平行结构。
热稳定性数据显示，三者相互结合后能G4-DNA的解链温度升高27.2C，而单独的NMM或PIER都不能稳定四链螺旋结构，这也是首次发现G4- DNA稳定剂之间的协同作用，为将来新型端粒酶抑制剂的开发提供了一个新思路。
③芘类化合物；④香港大学[8]新近合成的一种platinum( II) 类化合物［Pt( dppz - COOH)( N/C )］CF3SO3它能够使端粒的G－四联体结构折叠293倍，其体外实验显示对人端粒酶的IC50 为760nM 细胞毒性试验显示，此化合物的IC50 为17－25 M，并且对多要耐药性细胞KB－V－1及顺铂( cisplatin) 耐药性细胞CNE1 有很好的活性，该化合物G－四联体的亲和力比对正常的双链DNA 高800 倍左右；⑤近年来发现不少中药也具有端粒酶抑制活性[9-10]比如：去氢紫堇碱是一种较好的G- 四链体DNA 结合分子，它具有G -四链体结合分子的一般特点，为设计合成新型G- 四链体识别分子提供了新的结构模型[11]。
2.1.1 该类型抑制剂的最新研究进展端粒酶负责不同表型的癌细胞，抑制端粒酶可能专门针对癌细胞增殖。
配体能够选择性地结合到G‐四端粒DNA被视为端粒酶抑制剂，往往从定量检测端粒酶活性（法）进行聚合酶链反应步骤推断其作用机制，不提供深入的机制抑制。
此外，G‐四联体配体也被证明发挥作用的影响端粒结合蛋白与端粒体系。
SDanilo D’Ambrosio等[12]使用水溶性苝酰亚胺（hpdis）直接定量检测端粒酶活性、评价强度和作用机制。
hpdis是含苝和不同数量的正电荷的侧链。
IC50 值的HPDIs 是在低micromolar（0.5–5μM）根据数量和侧链的功能范围。
HPDIs 具有四个侧链成为最佳的这一系列化合物。
Telospot的竞争实验证明单链端粒DNA 的HPDIs具有很高的特异性，能够形成G-四链，超过基因组双链或端粒的dsDNA。
他们的研究支持，HPDIs是有效的端粒酶抑制剂，他们能够通过诱导来稳定长单链端粒引物的单分子的G-四链的结构。
2.2 端粒酶抑制剂新型杂环唑类衍生物的研究新型杂环的三个系列的唑类衍生物，含有吡嗪的化合物已设计、合成，其结构也确定，作为潜在的端粒酶抑制剂Yan-Bin Zhang等[13]对它们的生物活性进行了评价。
在恶二唑衍生物中，化合物2 -（（2 - 溴苄基氧基）-5 -（吡嗪-2 -基）-1，3，4 - 恶二唑(5c）是对反对SW1116癌细胞株（IC50 = 2.46μM对SW1116和IC50 = 3.55 μM端粒酶的最有效的生物活性。
化合物中2 -（（4 - 溴苄基）硫代）-5 - （吡嗪-2 - 基）-1，3，4 - 噻二唑（8h）是对肝癌细胞表现最好的噻二唑衍生物（IC50 = 0.78μM，端粒酶IC50 = 1.24μM）。
而化合物2 -（5 -（（4 - 氟代苄基硫基-4 - 苯基- 4H-1，2，4 - 三唑-3 -基吡嗪（11F）表明是最有效的生物活性（IC50 = 4.12 μM对SW1116和IC50 = 15.03μM端粒酶之间的三唑衍生物。
对接模拟定位化合物5C，4，8和11F到端粒酶结构的活性部位进行了探索可能的结合模式。
细胞凋亡的结果表明，化合物8h HEPG2细胞株具有良好的抗肿瘤活性。
因此，在抑制肿瘤生长的抑制活性的化合物8h可能是一个潜在的抗肿瘤药物对肝癌细胞癌细胞。
因此，恶二唑，噻二唑和三唑结构的引入增强我们的化合物与该受体的结合，导致生物活性的增强。
为深入了解其结构–活性关系观察端粒酶，分子对接的强效抑制剂5，8，11层的结合位点的端粒酶进行的具有约束力的模型基础上的端粒酶复杂结构。
分析了三种化合物结合的构象表明，这些化合物arg194有稳定的氢键相互作用。
细胞凋亡检测表明该化合物是潜在的抗肿瘤剂。
结果端粒酶抑制剂作为抗癌药物的设计提供了宝贵的信息. 杂环唑类衍生物，含有吡嗪三个系列已被合成并评价其抗肿瘤活性。
化合物5c表现出强大的抑制活性，对HepG2细胞的生长受到抑制的IC50分别为2.46μM和SW1116细胞的生长受到抑制的IC50分别4.22μM。
而化合物11F证明有效的抑制HeLa 细胞生长的抑制活性，IC50分别为7.30μM和SW1116细胞的生长受到抑制的IC50分别为4.12μM 。
化合物8h展示了最有力的抑制活性，对HepG2细胞的生长受到抑制的IC50分别为0.78 μM和SW1116细胞的生长受到抑制的IC50为1.47 μM ，它也抑制端粒酶活性的IC 50为1.24μ米，它能比较积极的控制星形孢菌素。
2.3端粒酶抑制剂壳聚糖共聚物纳米粒子的研究反义寡核苷酸，2'-O-甲基-RNA（OMR），被称为有效的端粒酶抑制剂，用于治疗肺癌，但受限于细胞内吸收不良。
与OMR相比，壳聚糖被覆聚合物纳米颗粒与作为非病毒载体的脱乙酰壳多糖溶液。
这项研究调查了壳聚糖的性质和浓度的作用，在nanocarriers加载后，提高效率了人类肺癌细胞株的端粒酶抑制的活力。
一定浓度的壳聚糖纳米粒子的表面是必要的，显着提高细胞吸收。
然而，过度的壳聚糖负面影响转染效率。
与脱乙酰壳多糖聚合物的自组装nanoplexes 优先吸附到细胞膜，而不是被内在化。
因此，聚合物纳米颗粒证明是优越的阳离子聚合物作为反义寡核苷酸的载体。
不能被认为是提高了转染的背后的主要因素。
空中接口上的细胞培养进行模拟体内条件摄取研究支持正常的文化，示出增强nanoplexes超过裸寡核苷酸的摄取的结果。
这些数据是正在进行的研究对肺灌注模型和体内实验的先决条件。
在这种情况下，纳米载体质粒DNA和艾滋病服务有前途的基因传递系统，取得了很大的关注，是对新一代疗法的诊断，治疗和预防方面的承诺。