关于三氧化二砷（ As2O3 ）治疗早幼粒细胞白血病（ APL ）研究史实的参考资料 哈尔滨医科大学第一临床医学院 张 鹏 一． 一篇不易查到的、较早年（ 1999 ）的综述，其包括历史、临床、作用机制及作用的生物学途径等方面的内容。 二． 三氧化二砷主要是通过诱导 APL 细胞分化和细胞凋亡（肿瘤凋亡治疗学）机制发挥治疗 APL 的作用。 关于细胞分化和凋亡的血细胞形态学所见。 已在我血液科九五到九六年发表的三篇文章中描述，但因当时彩照难以在杂志中刊登而只好在本文中选登。此后的同道们的一些相关报道，又用分子生物学方法予以证明。 三． 一篇由于“苛求”内容“全面完整”，作者未按《 BLOOD 》编委（ 2008 年）的要求进行删减（ 4000 字左右 ）修稿，但作者认为今天本文可能仍然不失其对“史实”的研究和和临床实践有所裨益。 On Arsenic Trioxide in theClinical Treatment of Acute Promyelocytic Leukemia (Review) Peng Zhang The Weihai MunicipalHospital,China Abstract Arsenic isgenerally considered hypertoxic. However,it has been used in traditionalChinese medicine since ancient times, to treat serious illnesses. Recently, asingle dose of arsenic trioxide (As 2 O 3 ) has been found especially effective intreating acute promyelocytic leukemia (APL). Generally speaking, As 2 O 3 is a moreeffective treatment of APL than other, newer medications and has less severeadverse reactions and greater safety. Key Words Arsenic;Trioxide; Treatment ; Acute Promylocytic Leukmia Mailing address: 7 0 ＃ Heping Road, The Weihai MunicipalHospital, Weihai, China 264200. E-mail: zpeng1941@163.com Arsenic is widelydispersed throughout nature,and its toxic effect in humans ,which focus mainly on somatic cells ,as well known. Epidemiological researchand in vitro testinghaveshown that long-term contact with arsenic can induce theformation of various neoplasms via cellular aberrationor mutation, either directly or synergistically with other carcinogenic factors .Despite arsenic’s hypertoxicity, it is necessary for growth and reproduction inhumans and lower animals ,and it has been used as a traditionalChinese medicine to treat of seriousillness . Even in Western countries, arsenic has long beenincluded in the medical armamentarium for the treatment of. tumors .Since the first report published in 1995on the clinical outcome andmechanisms of arsenic trioxide (As 2 O 3 ) given as a single dose for the treatment ofacute promyelocytic leukemia (APL) , As 2 O 3 come into wideuse in clinical treatment of APL in china. Indicationsfor an As 2 O 3 Regimen The following are clinical situations in which the use ofan As 2 O 3 regimen may be indicated : 1) Previously untreated (or newly diagnosed) APLespecially in patients who are positive for t(15;17) or the PML/RARα/PML-fusiongene, a key feature in more than 90% of such patients; 2) APL that isrefractory to all-trans retinoic acid (RA) or combined chemotherapy, recurrent disease,or relapsed after bone marrowtransplantation; 3) APL in patients for whom RA and combined chemotherapy areintolerable or inadvisable; 4) Maintenance treatment after CR from APL; and 5) CGL and certain acute nonlymphocytic leukemiasubtypesas well as those with myelodysplastic syndromes (MDS), if these are accompaniedby an excessive increase in the number ofpromyelocytes. As 2 O 3 treatmentis not suitable for a first choice for someAPL patients , such as positivity for either t(11;17),t(5;17) or for the PLZF/RARαfusion gene, moderate to severe liver or kidney dysfunction caused byconditions other than leukemia, relapse during continuous As 2 O 3 maintenance treatment or long-term arsenic exposure . Methods of Treatment Induction ofRemission In adults with APL, a dailyinjection of 10 ml of As 2 O 3 (1 g/L) is administered after over in 250-500 ml of glucose solution (50 g/L) or normal saline for intravenous over during 3 to4 hours. In children with APL, the daily dose is 6 mg/m 2 (approximately0.16mg/kg). The single treatment course spans 4 weeks, sometimes with a 5- to 7-daybreak at the midpoint. Peripheralhyperleukocytosis (HLT) can be prevented by administering oral hydroxyurea (1.0-8.0 g/d in divided doses ), or a small dose of homoharringtonine cytarabine,or both (by intravenousdrip) when the white blood cell (WBC) count ≥10×10 9 /L before treatment or after As 2 O 3 treatmen .fatal bleeding may be contraled by infusion of activated factor7(novoseven) which stopped hemorrhage . Treatment After Remission The amountand type of consolidation therapy necessary for an individual APL patient mayremain something of an open question and require risk-adapted protocols. Ingeneral, the author treats patients after remission in the following ways: Consolidation treatmentwith As 2 O 3 . The routine doseof As 2 O 3 is used for 2 to 3 weeks in a treatment course,with break of 1 month between courses in the first year, 2 months in the secondyear, 3 to 4 months in the third year, and 6 months after 3 years. Use of As 2 O 3 and chemotherapy alternatively. HA, DA, or Ara-Cplusmitoxantrone or etoposide or another similar drug are used inrotation, with break between courses asdescribed for the consolidation treatment. In consolidation treatment, Ara-C 1.0 g/day × x3 can be added to increase efficacy. Because As 2 O 3 is far less effective than conventional chemotherapy. for inhibiting APL cellproliferation, the author recommends the alternating use of As 2 O 3 and conventionalchemotherapy in tatients after remission . Pharmacokinetics of As 2 O 3 Shen et al . administered As 2 O 3 intravenously at a dose of 10mg/day for the treatment of 8 patients withrelapsed APL. The arsenic content was measured by gas-phase chromatography. Themaximal plasma concentration was 0.94±0.37 mg/L, the time to peak concentration was 4 hours, the plasma distributionhalf-time was 0.89±0.29 hours,the eliminationhalf-time was 12.13±3.31 hours, the a apparent distribution volume was 3.83±0.45 L, the system clearance was 1.43±0.17 L/hour, and the area under the curvewas 7.25±0.97 L/hour. Thecontinuous administration of As 2 O 3 did not alter itspharmacokinetic behavior. During As 2 O 3 treatment, the 24-hourarsenic content in urine accounted for 1 to 8% of the daily dose. The arsenicaccumulation in the hair and nails increased continuously, with a peakconcentration rose 5- to 7-fold higher than pretreatment levels. Importantly,the arsenic content of urine, hair, and nails declined gradually after drugwithdrawal. No bone marrow suppression or severe organimpairment were observed.The researchers concluded that As 2 O 3 is a relatively safe and effective for the treatment ofpatients with relapsed APL, despite the arsenic accumulation in some tissues. Hu et al . found that arsenic content in the cerebrospinalfluid was 4.8±0.4 μg/L in 40 healthy people,comparatively, the content inpatients before and 12 hours after treatment with a routine dose of As 2 O 3 was 4.8±0.3 μg/L and 5.2±0.1 μg/L, respectively. Similarly, in 46patients with APL, no significant difference was found between these groups (p .05). However, 12hours after treatment, the arsenic content in peripheral blood (30.0±5.0 μg/L)was significantly higher than that of cerebrospinal fluid (p .01), suggestingthat it is inadvisable to use intravenous As 2 O 3 therapy for patients with central nervous system (CNS) leukemia. ARetrospective Study of As 2 O 3 Therapy for APL: Efficacyand Course Study group The comparativeeffectiveness of As 2 O 3 therapy was evaluated in 242patients with APL treated at HMU Hospital. The patientswere divided into 4 groups, as follows: 1) previously untreated patients, 2)relapse group A (previously treated with As 2 O 3 ) ,3)relapse group B (previouslytreatedwith chemotherapy or other standard methods), and 4) the refractory group （ Table1 ） . The response rates for previouslyuntreated children and adults are listed in Table 2, and the average number oftreatment days and total As 2 O 3 doses used to achieve CRin each of the four groups are listed in Table 3 . Inour review of reports from other hospitals in China, CR was 89.7% (183/204) inpatiets with previously untreated (ornewly diagnosed) APL, and 84.2% (287/341) in patients with relapsed APL afterinduction RA, chemotherapy, or both ,or during maintenance therapy . Camacho et al .used As2O 3 for remission induction in 26 patientswith relapsed or refractory APL at daily doses that ranged from 0.06 to 0.17mg/kg, and 23 patients (88.5%) achieved CR. Elsewhere. 12 patients with APL that had relapsed after extensive priortherapy were treated with As 2 O 3 , and 11 of them had CR . Eight of 11 patients who were initially foundto be positive for the PML/RAR α -fusiontranscript by the a reverse transcriptase polymerase chain reaction(RT-PCR) assaylater tested negative; 3 other patients who persistently tested positive hadearly relapses . Shigeno et al .used As2O 3 to trteat 34 patients whose disease had relapsed, or had become refractory to RA and conventionalchemotherapy ， and 31 (91.2%) had CR.Eighteen of 25 patients who achieved CR also lostthe previously evident PML/RARα-fusion transcript, as shown by RT-PCR assay.Additionally,10(90.9%)of 11 children with hypergranular type of APL achieved hematological remissionafter a mean duration of 48 days with all 10 patients achieving molecularremission after a median duration of 81days .Ghavamzadeh et al .reportedthat CR were achieved in 82 (86.3%) patients of 94 new cases of APL ,and in13(76.5%) of 17 patients with relapse APL by As 2 O 3 treatment. 44cases of 48patients who werehematological remission found to be negative for the PML/RAR α -fusiontranscript; 3 cases of 4 other patients who tested positive had relapse inclinical expressions after persistent CR for one year.Recently,Mothews et al .observed that 62(86.1%)of 72 patientswith newly diagnosed cases of APL achieved hematologic CR after As 2 O 3 treatment.RT-PCR analysis for the PML/RAR α -fusion transcript wasavailable in 54 patients, and 11cases(20.4%) were negative at the end ofinduction.Of the 43 who were positive 30(69.8%) became negative after adrug-free interval 4 weeks. Shen et al . reported on a low-dose(0.08 mg/kg·d -1 , for 28 days) As 2 O 3 treatmentfor relapsed APL. Of 20 patients treated, 16 (80.0%) achieved CR. The estimated2-year OS and relapse-free survival were 61.6±15.8% and 49.1±15. 1%,respectively, and there was nodifference compared with those values in patients treated with a conventionaldose. The authors concluded that low-dose As 2 O 3 had thesame effect as the conventional dose, and the mechanism of low-dose arsenic seemed to be, primarily, the induction ofdifferentiation in APL cells. Relapse Inour series of 86 APL patients who were on post-CR maintenance treatment with As 2 O 3 ,5 (5.8%)had relapses within 1 year after CR,12(14.0%)within 2 years, and23(26.7%)within 3years (range ,of 5 to 37 months, average of 25.4±12.9 months).The relapse rate within 3 years of CRwas significantly higher in male patients (19/57 or 33.3%) than in females(4/29 or 13.8%) . A test positive for the PML/RAR α -fusion transcriptis a best method to judge CR patients with APL to be relapse in the nearfuture. Overall Survival Of 136 patients followed up after As 2 O 3 treatment at HMU Hospital , 11 died within5 years and 125 have lived for 5 or moreyears (i.e., a 5-year overall survival of 91.9%) . Factors Relatedto Therapeutic Effectiveness Therapeuticeffectiveness and prognosis have a positive correlation with the band-cellcount of peripheral WBC, hemoglobin content, platelet count, and bone marrownormoblast count. We found a negative correlation with WBC count, peripheraljuvenile blood cell count, myeloproliferation degree, and lactic dehydrogenase(LDH) activity . Drug Resistance Inclinical practice, there is no cross – drug-resistance between As 2 O 3 and RA or other antileukemic agents. This may be explained by the non-liposolublenature of As 2 O 3 , its small molecular weight, and itsdistinctive modes of action . In our observation, primary As 2 O 3 resistance was found in 35.9% of refractory patients, 15.3% of patients whosedisease relapsed after nonarsenicinduction and consolidation, and in 5.7% of newly diagnosed APL patients; therate of acquired resistance to As 2 O 3 was 26.7% (23/86).In the latter instance, an increased dose of As 2 O 3 enabled more than half of resistantpatients to regain CR, presumably through overcoming an “inertia” in arsenicreceptor or signaling pathways . Geng et al . foundcross – drug-resistance between As 2 O 3 and cisplatin. An increased As 2 O 3 dose or action timeresulted in a decrease of multidrug resistance protein expression and reversedthe resistance. Toxicity andSide Effects Hyperleukocytosis Of 242 APLpatients studied, hyperleukocytosis (HLS) occurred in 183 (75.6%). Most of theincreased WBC count consisted of transitional cells (i.e., those betweenabnormal promyelocytes and myelocytes and fairly mature granulocytes) ,theseresult from As 2 O 3 induced differentiation of APL cells .Of the 242 patients, 2 developedhydrothorax and ascites, with a WBC count of 198.5×10 9 /L (myelocyte0.90, promyelocyte 0.05) in one, and 123.0×10 9 /L in the other. Fourother patients in this group had significantly elevated aspartateaminotransferase (AST) and alanine aminotransferase levels(ALT); 2 had elevated blood urea nitrogen,and 3 had peripheral WBC counts ≥50×10 9 /L .Chen et al . observedthat 1 APL patient developed RA syndrome and anapparently related HLS during As 2 O 3 treatment forrelapse, after an RA-induced CR. Roberts et al . observed that a patient with relapsed APLmanifested a markedly increased WBC count and CNSinfarction relative to HLS during induction therapy with As 2 O 3 .Moreover, after As 2 O 3 treatment for APL, HLS with a WBCcount ≥10×10 9 /L occurred in 58.1% (25/43) of patients with relapsed andin 74.1% (43/58) of those with newly diagnosed disease . Of26 patients with relapsed or refractory APL who were treated with As 2 O 3 to induce remission, 15 (57.7%) showed HLS, which resolved in all cases withoutthe use of other cytotoxic therapy . In studies byother investigators, HLS occurred in 58.6% to100% of APL patients treated withAs 2 O 3 . In ourobservation, HLS ≥50×10 9 /L influenced the treatment of disseminatedintravascular coagulation During a singledose of As 2 O 3 treatment, a fewpatients with APL occurred APL differentiation syndrome(APLDS) becauseexcessive HLS plus patient ` s constitution factors.APLDS(ATRAsyndrome be longs also to APLDS) usually maifestde as palpitation ,chestdepression, accelerated respiration, oedema of the whole body hydrothorax , ascites,hydropericarditis and even respiratory distress or fate terminated in pulmonaryhemorrhage and resqiratory failure. Control HlS is emphasis on the preventionof APLDS development by plus chemotherapy when WBC count is on the increase, near10 × 10 9 /L.Thetreatment of APLDS may use higher dose of dexamethosone plus chemotherapy andthe treatment of some symptomses after As 2 O 3 stopped. Myelosuppression and Changes in Red Cells We foundmild and transient myelosuppression in 2 children and 1 adult who were given alarger-than-usual dose of As 2 O 3 . Of the 242 patientswho received conventional treatment, 15 (6.2%) after a mean 18.4 days, depressedhemogram and myelogram readings , includingdecreases in bone marrow karyocytes, peripheral leukopenia (with the lowestcount of 0.2×10 9 /L in some cases), and less significantoligochromemia and thrombocytopenia. The inhibition resolved after a mean 21.4days, generally without requiring drug withdrawal and with only occasionally useof granulocyte colony-stimulating or granalocyte-macrophage colony-stimulatingfaclor. The red cells from bone marrow had abnormalities such as binuclearearly erythroblasts, megaloblasts, petal-shaped nuclei, Howell-Jolly bodies,basophilic stippled erythrocytes, karyokinesis, and irregular-sized mature redcells . Other Adverse Events As 2 O 3 generally causes less severe adverse reactions (e.g; hemorrhage), and isconsidered comparatively safe for the treatment of APL. In the 242 patients described above, other adverse events, in order offrequency, included gastrointestinal such as, anorexia, abdominal discomfort,nausea, vomiting, diarrhea(24.0%); skin lesions, such as xerosis cutis,pigmentation, erythema(22.7%); changes of liver function (14.1%):elevated AST,ALT, alkaline phosphatase, gamma-glutamy1 transpeptidase and blood bilirubin. Infrequent manifestations included facial and limb edema, ulcerativestomatitis, headache, changes in cardiac activity (e,g;sinus tachycardia and changes in the STsegment and T wave on electrocardiogrophy), prolongation of the partial remission interval, toothache,hydrothorax, ascites, elevation of blood urea nitrogen , nosebleed, gingivalbleeding, persecution complex, and agnosia . Rust and Soignet reportedthat a multicenter trial in the United States of As 2 O 3 in 40 patients with APLthat relapsed after conventionaltherapy, confirmed the positive safety and efficacy findings fromof a smaller12-patient pilot study. Common adverse events inceuded HLS, APL differentiationsyndrome, a prolonged QT interval on elecirocardiography, skin rash, andhyperglycemia. The occurrence of some toxic events including gastrointestinal disturbance, facial edema, andcardiac toxicity seemed less severe in the group given low-dose As 2 O 3 ,than in the standard-dose group . As of July 2002, in clinicaltrials with Trisenox ,an intravenous formulation of As 2 O 3 ,522 patients (224 with APL, 298 with other hematologic malignancies) have beentreated in the United states and the Europe.The adverse events noted in postmarketing use of As 2 O 3 are generally similar to those observed in clinical trials, and no deaths dueto As 2 O 3 related cardiac arrhythmia have been reported.This experience appears to confirm that As 2 O 3 isgenerally well tolerated and that the observed adverse effects are manageable and reversible . However, when 7 patients with refractory orrelapsed APL were treated with As 2 O 3 , 6 noted waterretention (shown by weight gain, pleural and pericardial exudates); 2 of 3patients on As 2 O 3 maintenance therapy showedpolyneuropathy related to chronic arsenic poisoning, and 1 of those 2 patientssuffered myoatrophy of a limb end . Individual differences in the respionse of APLpatients to As 2 O 3 treatment are related to the amount ofarsenic accumulation, detoxification and excretion, susceptibility and tolerance,or distinctive interactions between the patient’s physical condition and thetoxic effects of arsenic . Although arsenic, in consideration of its proposed mutagenicity, issuspected of having the capacity to induce a second tumor, few relevant clinicalreports exist. As 2 O 3 and RA: Modes of Actionand Combined Use As 2 O 3 treatmentwas recently proposed as an alternative therapy for APL, because it can induceCR in patients with either RA-sensitive or RA-resistant APL.Intriguingly, As 2 O 3 was also induced degradation of PML/RARα chimeras and to reorganize PML nuclear bodies （ PML-NBs ） . In APLpatients, RA triggers differentiation, whereas As 2 O 3 induces both a partial differentiation and apoptosis. Although their mechanismsof action are believed to be distinct, both drugs induce catabolism of the oncogenic PML/RARα-fusion protein.Although APL cell lines resistant to one of these agents are sensitive to theother, the benefit of combining RA and arsenic in cell culture remainscontroversial. Shao et al . believe that As 2 O 3 and RA inhibit each other ， s therapeutic effects. Lallemand et al .used syngeneic grafts of leukemic blasts from PML/RARα transgenic rats as amodel for APL to establish that RA and As 2 O 3 actsynergistically in vivo , and encouraged using this combination for APLpatients. This exemplifies how murine models of human leukemia can be used todesign or optimize therapies. RA and As 2 O 3 together alsoprolonged the survival of recipients mare than did either drug alone. In contrast, neither in promyelocyticzinc finger protein (PLZF)-RARα transgenic rats nor in nude rats that received transplanted of PML/RARα cellsdid any of the 3 regimens induceCR . However, in a clinical trial,RA combined with As 2 O 3 for de novo APL treatment, achieved CR in 29/31 (93.5%) patients. ThePML/RARα-fusion gene that was positive in all 29 patients before treatmentturned negative in only 3 of them (10.3%) by the time of CR, and 10/13 patients(76.9%) who were PML/RARα positive became negative after the consolidationtreatment. However, the results were not significant compared with those for As 2 O 3 and RA usage or chemotherapy at a single dosage to treat APL . Inthe author ， s clinical practice, single-agent As 2 O 3 or RA administration, rather than the combination, is used for inductiontherapy in newly diagnosed APL cases to prevent a possible aggravation of APLDS,or other adverse events. Nevertheless, As 2 O 3 and RA in combination has been adopted in someinstances involving refractory or resistant disease or multiple relapses.Clearly, both drugs are highly effective;they do not cause cross – drug-resistance, and theyshare the same principal mode of action, specifically the induction ofdifferentiation . 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Table 1.Curative effects of AS2O3 in 242patients Patients N CR (%) PR (%) NR (%) PU Group 124 109(87.9) 8(6.5) 7(5.6) Relapse Group A 20 12(60.0) 1(5.0) 7(35.0) Relapse Group B 59 41(69.5) 9(15.3) 9(15.3) Refractory Group 39 19(48.7) 6(15.4) 14(35.9) T otal 242 181(74.8) 24(9.9) 37(15.3) Group A: Relapsed APL treated with As2O3 aspost-CR consolidation treatment Group B: Relapsed APL treated withchemotherapeutic or other medicines as post-CR consolidation treatment. * PU = previously untreated; CR= complete remission; PR = partialremission; NR = no remission. Table 2. Comparison of curative effects between childrenand adults in PU Group Patients N CR(%) PR(%) NR(%) ChildrenGroup 23 16(69.6) 4(17.4) 3(13.0) Adult Group 101 93(92.1) 4(4.0) 4(4.0) P Value ＜ 0.01 ＜ 0.05 ＞ 0.05 * CR = completeremission; PR = partial remission ; NR = no remission. Table 3. Days to achieve CR and the total dose of As 2 O 3 used Patients N Days to Total Dose of AchieveCR As 2 O 3 used (mg) X ± SD X ± SD PU Group 93 30.26 ± 7.4 302.6 ± 74 Relapse Group A 12 37.65 ± 22.2 376.5 ± 222 Relapse Group B 41 32.08 ± 10.26 320.8 ± 102.6 Refractory Group 19 31.22 ± 17.99 31.22 ± 17.99 * PU = previously untreated; CR = complete remission
我国在砒霜治疗急性早幼粒型白血病方面的开创性研究 砒霜是中国古代传统的“毒物”。明代《本草纲目》记载了利用砒霜以毒攻毒的一些应用：“砒石解毒治壅（yōng）、烂肉，蚀瘀腐、瘰疬（luǒ lì）”。 二十世纪70年代开始，哈尔滨医学院第一附属医院借鉴民间方子，制备了含有由砒霜、氯化亚汞的“癌灵1号”注射液 。在临床实践中, 哈医大一院的医生们逐步发现“癌灵1号”可单独治疗白血病，且对急性早幼粒型白血病（Acute Promyelocytic Leukemia，APL）效果最好 。在90年代初，又去除了有毒副作用的氯化亚汞，明确指出砒霜（即三氧化二砷）是“癌灵1号”的有效成分，单独使用三氧化二砷（以下简称ATO）注射液即可治疗急性早幼粒白血病 。 1994-1996年，上海瑞金医院与哈医大一院展开合作研究，应用ATO治疗对反式维甲酸和化疗不敏感的APL病人，取得良好治疗效果 。相关研究结果发表在1997年的《Blood》杂志，引发国际关注。 中国的知识产权保护情况 1995年8月23日，哈尔滨医学院第一附属医院以张亭栋为发明人申请了中国专利，1999年8月11日拿到授权，专利号为ZL95108768.1 。该专利只有一个独立权利要求： “1.一种抗白血病、肝癌、淋巴瘤注射液，其组成包括：注射用水，其特征是：所述的注射用水中溶有三氧化二砷1-10克，氧化钠8克，共计1000毫升。” 这是一个典型的注射液组分专利。由于“癌灵”的成分早已公开，1995年专利的新颖性主要在于去除了轻粉（氯化亚汞）。该专利存在以下几点严重的缺陷： 1. 发明点遗漏 该专利所称的“注射用水”其实指的治疗药品的起始配制液，而并不是注射入人体时的组分浓度或治疗病人每日所需的剂量。这样的权利要求完全没有保护到这一科技成果的核心价值点，严重的影响了技术的价值。 2. 权利要求范围太窄 注射用水液组分保护范围狭窄，ATO或氯化钠的含量稍多或稍少于权利要求所述的范围，就可以轻易绕过该专利。如溶解0.5g ATO于1L水中配制起始溶液，稀释成最终注射液时稀释比例减半，也可以达到相同的效果而不侵权。 3. 权利要求没有上位 在权利要求中提到的适应症包括白血病、肝癌、淋巴瘤。一般情况下，专利中一种药物能治疗三种以上的肿瘤，原则上能拿到这种药物治疗所有肿瘤的权利要求。但是此专利中并没有对ATO适应症进行上位。更为重要的是，虽然李元善与张亭栋早在1988年就已经用ATO对白血病外的癌症进行了研究 ，但是该专利的实施例中完全没有提及ATO治疗肝癌、淋巴瘤的内容。因此，即使是ATO治疗肝癌、淋巴瘤的这两种适应症，被无效的风险也很高。 值得注意的是，1996年，张亭栋也在美国申请了专利，申请号08702011，并要求了中国的优先权，但是在美国审查并不顺利 。直至2004年4月11日，该专利终于在美国拿到授权，即US6720011 。美国专利的授权权利要求范围相较于中国专利没有实质性的突破。其权利要求1的范围与中国授权专利的主要区别在于：1.将中国专利的“注射用水中溶有三氧化二砷1-10克，氧化钠8克，共计1000毫升”换算成了组分百分比。2.药物的适应症仅针对白血病，去除了无实施例支持的肝癌与淋巴瘤适应症，3.增加了10%葡萄糖的组分。独立权利要求3保护的范围稍大，要求了0.1%-1% ATO溶液治疗白血病的用途。美国专利的权利要求依然存在重大缺陷，作为一个注射液组分的专利，只要求了稀释使用前的ATO起始配制液的浓度，没有要求最终注入人体的ATO浓度，也没有要求ATO治疗病人的日剂量，致使发明无法得到有效保护。2012年6月5日，这一专利在美国因不交专利费而无权。 美国的跟进研究和知识产权运作 1997年，美国Memorial Sloan-Kettering Cancer Center（以下简称SK中心）的Raymond P. Warrell, Jr.博士在获知中国这一科研成果后，迅速开始了对这一药物的布局。首先，他在美国组织了用ATO治疗了12例APL的1期临床试验，效果良好；随后组织了包括40个病人的多中心参与的2期临床试验，获得巨大成功 。 在美国的临床实验中所用剂量参考了中国的临床实验数据，后来由于受试病人中的儿童对固定剂量的ATO有强烈副作用反应，Raymond等根据病人体重进行了剂量改进，最终确定0.15mg/kg的ATO每日治疗剂量。据此，1997年11月10日，SK中心申请了ATO治疗白血病的剂量方面的临时专利，申请号60064655 。 SK中心的工作受到了资本的青睐，1997年3月，派拉蒙投资公司（Paramount Capital）出资成立了PolaRx Biopharmaceuticals Inc.（以下简称PolaRx公司）。公司的核心成员包括Raymond P. Warrell, Jr.博士与Fred Mermelstein博士这样精通的早期药物研发与专利布局的资深人士 。PolaRx公司成立之后，为了使得ATO能顺利上市，开展了一系列的知识产权运作。 首先， 1998年2月9日，PolaRx公司许可了SK中心的60064,655专利 。PolaRx公司许可SK中心的专利后，利用美国专利申请规则，多次提出continuation，至今在全球已经申请了54个同族专利，其中进入FDA Orange Book列表的专利有六个。这一系列专利要求了ATO按照体重使用的剂量每人每天0.15 mg/kg治疗APL的方法，并对ATO所治疗APL的具体表征、使用剂量微调、首次治疗范围、治疗周期等方面进行了详细的定义 。 1998年3月3日，凭借Raymond组织的ATO治疗APL的临床二期试验，PolaRx公司获得了美国孤儿药指定权（Orphan drug designation），编号97-1096 。所谓孤儿病，是指每百万人口中的患者少于1000人的罕见病。因为病人数量少，市场小，医药公司缺乏开发相关药物的热情。为了激励孤儿病药物开发，美国规定“在治疗孤儿病的任何药物问世后的7年之内，如果再出现治疗同一种病的其他新药，卫生部不对其发放许可证 。”因此PolaRx公司得到了ATO治疗APL除专利外的另一层保护。 接着，PolaRx公司许可了中国的两个专利。1999年5月24日，PolaRx公司通过Samuel Waxman Cancer Research Foundation（以下简称Waxman基金）许可获得了哈医大一院张亭栋在美专利，也就是ATO起始配置液的组分专利 。1998年8月5日，PolaRx公司又从北京人民医院血液病研究所获得了雄黄治疗血癌的专利的全球独占许可 。值得一提的是，从北京人民医院血液病研究所2002年《Blood》杂志发表的临床实验结果来看，雄黄治疗APL也具有很好疗效，而且相较ATO具有可口服、毒性低等优势 。很显然，PolaRx公司许可雄黄专利的目的是防止竞争产品上市、为ATO上市并取得垄断创造条件。在雄黄的许可合同中，由于中方缺乏对PolaRx公司的关键性约束条款，如缺乏产品开发时间和专利回收的约定，导致该专利被束之高阁，雪藏至今。 美国三氧化二砷药物Trisenox®上市 2000年3月，美国Cell Therapeutics的全资子公司CTI Technologies, Inc. (CTIT)并购PolaRx公司，总价5百万股（市价约3400万美元）+1400万美元+2%销售提成，获得TRISENOX的全球权利 。2000年9月25日，美国FDA批准Trisenox® 上市，现在售价约为300美元支天。而ARSENIC TRIOXIDE的orange book编号为N021248 。PolaRx公司作为一家仅成立3年的小微型公司，通过小规模的临床2期试验与知识产权运营，挣得了 超过4千8百万美元的收益 ，并创造在美从临床试验到药品上市只用30个月的行业纪录！ 值得注意的是，Trisenox®在销售中使用小安瓿（ampule），10mL一瓿，ATO浓度为 1 mg/mL。仅从专利角度，这一浓度落入了张亭栋专利中起始ATO 浓度1-10 mg/mL的保护范围。但奇怪的是，据知情人士透露，哈尔滨医学院第一附属医院最终没能获得药物在美国市场销售的相关收益。推测原因可能有以下几点： 1. 美方对ATO最终使用浓度进行了改进。Trisenox® 在ATO注入人体浓度上采用0.038-0.091 mg/ml。而张亭栋专利中1-10 mg/mL的保护范围仅是限定在起始安瓿的浓度，而没有在权利要求中明确表述最终施用的浓度，根据专利说明书能算出ATO最终滴注浓度为0.020 mg/mL 。 2. 张亭栋的专利在2000年Trisenox®上市时还没有在美国授权，2004年授权后也没有进入FDA orange book的列表。 3. 张亭栋专利的国外开发权首先被Waxman基金以极低的价格取得，后又被PolaRx转手获得，其中的许可合同条款很可能存在隐藏的不平等条款，规避了PolaRx公司的支付责任。 ATO药物虽然在美国、欧洲、日本等地相继上市，但没能在国际上为中国挣得应有的利益；而雄黄（四硫化四砷）治疗白血病的药物更是被完全雪藏，至今没有药品上市，中方自然也无法获得收益。 PolaRx公司目光长远的专利布局 在PolaRx公司成立之初，该公司即开始在 砷化物治疗肿瘤整个领域 进行布局。除了上述介绍的通过专利运作促使Trisenox®成功上市用于治疗白血病，PolaRx公司还持续进行砷化物治疗实体瘤领域的专利布局。 1997年10月15日，在没有开展任何实质性研究的情况下，PolaRx公司提出了砷化物治疗实体瘤的美国临时专利申请（Provisional patent application），一举抢占了该领域的优先权。利用美国continuation-in-part（CIP）的申请规则，不断分案，补充数据，获得了砷化物治疗多种实体瘤的专利，如今已经在世界各国申请了70个同族专利 。 值得注意的是，仅是在美国FDA clinical trials官方备案进行的砷化物治疗肿瘤的临床研究就多达121个。 PolaRx公司的专利作为基础专利，dominate了该领域，为其后续收益奠定了基础。 启示 由于缺乏知识产权质量管理与交易管理， 中国的医生与科学家们辛勤研究了30多年的伟大科技成果最终转化为了美国公司的经济利益 ，这一教训值得深思。经过这些年的发展，中国已经初步逐步具有了产生世界级科研成果的潜力，然而 知识产权管理和运作能力 依然落后、专业化国际化 人才 严重缺失。这一短板将严重影响我国科技成果的有效转化和国家竞争力的进一步提升，如不改善，将有更多的技术流失。 本文意在抛砖引玉，如有错漏之处请多批评指教。 附注：中国的三氧化二砷药品 1998年，哈尔滨医大药业有限公司成立。1999年，公司拿到生产批文：国药准字H19990191，商品名伊泰达。这一成果因还为效果突出被破格提升为二类中药。2008年，北京双鹭药业拿到生产批文。如今伊泰达国内售价在一支130-200元之间。具体用法为亚砷酸注射液(10mg)加入250～500ml生理盐水或5%葡萄糖溶液（0.020-0.038 mg/ml）中每日一次静脉滴注3～4h滴完 。 参考文献 1.张亭栋, et al., “癌灵注射液”治疗6例白血病初步临床观察. 黑龙江医药, 1973. 3 p. 66-67. 2.哈医大一院中医科 and 哈医大一院检验科, 癌灵1号注射液与辨证论治对17例白血病的疗效观察. 哈医大学报, 1974. 2 p. 25-30. 3.张亭栋 and 荣福祥, 癌灵一号注射液与辩证论治治疗急性粒细胞型白血病. 黑龙江医药, 1979. 4 p. 7-11. 4.孙鸿德, et al., 癌灵1号结合中医辨证治疗急性早幼粒白血病32例. 中国中西医结合杂志, 1992. 12 p. 170-171. 5.Shen, Z.-X., et al., Use of Arsenic Trioxide (As2O3) in the Treatment of Acute Promyelocytic Leukemia (APL) II. 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三氧化二砷（As 2 O 3 ）是中药砒霜的有效成分，正如青蒿素是中药青蒿（黄花蒿）的有效成分一样。在中国，关于谁最先发现三氧化二砷是治疗白血病的有效成分以及青蒿素是抗疟疾的有效成分，似乎有着十分相似的故事，并且都存在很大的争议。 然而，青蒿及青蒿素抗疟疾的历史从头至尾都是由中国人书写的。早在1500多年前，晋代葛洪就在他的《肘后备急方》中写道：“青蒿一握，以水二升渍，绞取汁，尽服之”。此方专治“打摆子”（实为疟疾）。 其实，中国人将青蒿入药的历史更加久远。据考证，1972年湖南长沙马王堆出土的汉墓已有2000多年的历史，随墓出土的一块丝绸织物上写着《五十二病方》，其中就提到青蒿可用来治疗痔疮，青蒿二字清晰可见。 近代，无论是从青蒿中提取出青蒿素，还是测定出青蒿素的结构，都无一例外是中国人的功劳。就连为了解决青蒿素不溶于水而研制的青蒿琥酯和蒿甲醚，也是中国人的“独创”，而外国人仿此研制的蒿乙醚只能算“跟踪”！ 可以说，青蒿素是唯一让外国人心服口服的中国原创，因为在他们祖先留下的文明史上这一块就是空白。在中国人完成并报道了一系列“划时代”、“里程碑”工作之前，外国人对于青蒿及青蒿素抗疟的“神奇”简直闻所未闻！ 相反，西方人将三氧化二砷入药的历史可以追溯到公元前400年的古希腊希波克拉底时代，而且他们配制的Fowler氏溶液（含亚砷酸钾）最先用于白血病治疗，这些事实都留下了足够让他们“自豪”的空间。 我仅列举3篇有关三氧化二砷的国外文献，让我们看看外国人是如何书写该药的历史的，以免我们不断并长期陷入“坐井观天，夜郎自大”的窘境。但是，有人可能要问：究竟是中国人的研究成果不入洋人的“法眼”，还是他们捏造事实呢？ 第一篇文献是美国哥伦比亚大学教授Karen H. Antman专门为2000年7月19日在纽约召开的“The Promise of Trisenox: Charting an Appropriate Scientific and Clinical Course”会议所写的“开场白”，全文发表在2001年4月《癌学家》（Oncologists）特刊上（ http://theoncologist.alphamedpress.org/content/6/suppl_2/1.full ）。 他在该文的摘要中写下了这样一段话： Although arsenic can be poisonous, and chronic arsenic exposure from industrial or natural sources can cause serious toxicity, arsenic has been used therapeutically for more than 2,400 years. Thomas Fowler's potassium bicarbonate-based solution of arsenic trioxide (As 2 O 3 ) was used empirically to treat a variety of disorders, and in 1878, was reported to reduce white blood cell counts in two normal individuals and one with “leucocythemia.” Salvarsan, an organic arsenical for treating syphilis and trypanosomiasis, was developed in 1910 by Paul Ehrlich . In the 1930s, arsenic was reported to be effective in chronic myelogenous leukemia. After a decline in the use of arsenic during the mid-20th century, reports from China described a high proportion of hematologic responses in patients with acute promyelocytic leukemia (APL) who were treated with arsenic trioxide. Randomized clinical trials in the U.S. led to FDA approval of arsenic trioxide for relapsed or refractory APL in September 2000. 他说，三氧化二砷作为药用已有2400年的历史。早在1878年，Fowler氏溶液就用来减少两位正常人及一名白血病人血液中的白血球数目。1910年，有机砷剂Salvarsan（洒尔佛散）已被用来治疗梅毒和锥虫病。1930年，砷剂就被证明治疗急性髓性白血病有效。中国人到了20世纪中期才报道用三氧化二砷治疗急性早幼粒细胞性白血病（APL）。2000年9月，美国食品药品监督管理局正式批准三氧化二砷用于复发的或难治性APL的临床治疗。 由此可见，中国人在三氧化二砷用于白血病的治疗上，既不是原创，也不是首创，只是从中药砒霜治病绕到砒霜有效成分三氧化二砷治疗白血病，而外国人早在1930年就直接用三氧化二砷治疗白血病。在外国人眼中，中国人用砷剂治病仅仅发生在砷剂的药用在世界范围内式微之时（After decline in the use of arsenic during the mid-20th century）！ 第二篇文献是美加两国多名学者撰写的一篇综述，题目是“三氧化二砷的作用机理”，全文发表在2002年7月15日出版的《癌症研究》（Cancer Research）上（ http://cancerres.aacrjournals.org/content/62/14/3893.full ）。 在这篇文章中，他们以表格的形式列举了三氧化二砷药物研究的历史： 由上表可见，早在公元前400年的古希腊希波克拉底时代，砷剂（二硫化二砷、三硫化二砷）就被用来医治溃疡。公元50年希腊医生迪奥斯克利德斯就用三硫化二砷脱毛。14世纪用砷剂抗瘟疫。16世纪用砷剂作发汗药。17世纪用砷剂治疗疟疾。18世纪砷剂已有多种药用。19世纪用亚砷酸钾治疗哮喘、舞蹈病、湿疹、霍奇金病（淋巴瘤）、天疱疹、恶性贫血、牛皮癣。20世纪用有机砷治疗梅毒和锥虫病。 虽然表中提到中国人用砷剂治病已有几百年，但并不包括白血病，并且现在仍然只治疗一些无关紧要或可用其他药物代替的小病，如蛀齿去腐、牛皮癣、风湿病和梅毒。显然，这篇文章故意回避中国在1970年代用三氧化二砷治疗白血病的事实，也不是一种求真务实的态度。 第三篇文献就是“维基百科”（Wikipedia）中的Arsenic Trioxide词条： Despite the well known toxicity of arsenic, arsenic trioxide has long been of biomedical interest, dating to traditional Chinese medicine , where it is known as Pi Shuang and is still used to treat cancer and other conditions, and to homeopathy , where it is called arsenicum album . Some discredited patent medicines , e.g., Fowler's solution , contained derivatives of arsenic oxide. Arsenic trioxide under the trade name Trisenox (manufacturer: Cephalon) is a chemotheraputic agent of idiopathic function used to treat leukemia that is unresponsive to "first line" agents. It is suspected that arsenic trioxide induces cancer cells to undergo apoptosis . Due to the toxic nature of arsenic, this drug carries significant risks. Use as a cytostatic in the treatment of refractory promyelocytic (M3) subtype of acute myeloid leukemia . The combination therapy of arsenic trioxide and all-trans retinoic acid (ATRA) has been approved by the U.S. Food and Drug Administration (FDA) for treatment of certain leukemias. 虽然该词条中提到三氧化二砷是中药砒霜的成分，但并没有说中国人首先使用砷剂治病，所列举的药物仅有国外的Fowler氏溶液和Trisenox（Cephalon公司产品）。还提到全反式维甲酸与三氧化二砷的联合治疗方案被美国食品药品监督管理局批准用于某些白血病的治疗。 综上所述，三氧化二砷并非中国人首创，三氧化二砷用于治疗白血病也不是中国人原创，它在新药研发历史上的地位根本无法与青蒿素的创制及青蒿素高效抗疟疾效果的发现媲美。青蒿素及青蒿素药用价值的研究才是令外国人不得不服的世界一流成果，不仅够得上拉斯克奖，也完全有资格获诺贝尔奖。 当然，由于文革时代信息封闭，不排除我国本土科学家不了解国外三氧化二砷的研究情况，可能是根据自己的创意“首次”发现三氧化二砷可治疗白血病的“奇效”。这样的成果在那个年代完全可以评上“国家发明奖”，但当今不可能为它追溯一个新的国内奖项，更不用奢谈具有世界科学视野的诺贝尔奖了！ 附：中医古籍对“砒霜”治病的描述 1、《本草衍义》：砒霜，疟家或用，才过剂，则吐泻兼作，须浓研绿豆汁，仍兼冷水饮。 2、《医学折衷》：疟丹多用砒霜大毒之药，《本草》谓主诸疟风痰在胸膈，可作吐药，盖以其性之至燥，大能燥痰也。 3、《本草经疏》：砒霜，禀火之毒气、复经煅炼，《开宝本草》虽云味苦酸，而其气则大热，性有大毒也。酸苦涌泄，故能吐诸疟风痰在胸膈间。大热大毒之物，故不可久服，能伤人也。砒黄既已有毒，见火则毒愈甚，而世人多用砒霜以治疟，不知《内经》云夏伤于暑，秋必疡疟，法当清暑，益气，健脾，是为正治，岂宜用此大热大毒之药。如果元气壮实，有痰者服之，必大吐，虽暂获安，而所损真气实多矣。 4、《本经逢原》：砒霜疟家常用，入口吐利兼作，吐后大渴，则与绿豆汤饮之。砒性大毒，误食必死。然狂痴之病，又所必需，胜金丹用之无不应者。枯痔散与白矾同用，七日痔枯白落，取热毒之性以枯歹肉也。 5、《日华子本草》：治妇人血气冲心痛，落胎；“治疟疾、肾气。带辟蚤虱。” 6、《开宝本草》：主诸疟，风痰在胸膈，可作吐药。 7、《本草蒙筌》：截疟除吼，膈上风痰可吐；溃坚磨积，腹内宿食能消。 8、《医学入门》：主恶疮瘰疬，腐肉，和诸药敷之，自然蚀落。又治蛇尿着人手足，肿痛肉烂，指节脱落。为末，以胶清调涂。 9、《纲目》：蚀痈疽败肉，枯痔，杀虫。 。“除齁喘积痢，烂肉，蚀瘀腐瘰疬。”又“蚀痈疽败肉，枯痔杀虫。” 10、《玉揪药解》：治寒痰冷癖，久疟积痢，疗痔漏瘰疬，心疼齁喘，蚀痈疽腐肉，平走马牙疳。
http://news.sciencenet.cn/htmlnews/2010/12/241037.shtm NASA发现生命新可能 砷元素或能形成生命体 http://www.gopubmed.org/web/gopubmed/1?WEB012jamrtwdn90cIoI1I00h001000j10040001rl 16,795 documents semantically analyzed 1 2 3 ... 7 Top Years Publications 2010 1,201 2009 1,188 2007 1,108 2008 1,104 2006 952 2005 857 2004 789 2002 688 2003 683 2001 490 2000 378 1999 299 1998 252 1996 218 1997 200 1992 197 1989 193 1977 188 1972 188 1993 185 1 2 3 ... 7 1 2 3 ... 7 Top Countries Publications USA 3,348 China 1,307 India 640 Japan 624 Taiwan 467 Germany 455 United Kingdom 426 Spain 395 Canada 350 France 337 Italy 252 Australia 227 South Korea 211 Mexico 171 Sweden 168 Belgium 137 Brazil 103 Poland 88 Netherlands 75 Bangladesh 75 1 2 3 ... 7 1 2 3 ... 65 Top Cities Publications Beijing, China 305 Taipei 244 New York City 230 Kolkata 184 Shanghai, China 178 Tucson 124 Seoul, South Korea 94 Boston 86 Kao-hsiung 83 London 82 Chicago 80 Tokyo 80 Detroit 80 Cincinnati 76 Madrid 76 Harbin 75 Paris 74 Berkeley 71 Houston 71 Hong Kong, Hong Kong 67 1 2 3 ... 65 1 2 3 ... 121 Top Journals Publications Environ Sci Technol 445 Sci Total Environ 403 Environ Health Persp 306 Talanta 278 J Hazard Mater 249 Chemosphere 235 Toxicol Appl Pharm 224 Environ Pollut 216 J Biol Chem 177 B Environ Contam Tox 168 Mutat Res-fund Mol M 134 Water Res 131 Environ Res 117 Toxicol Sci 111 Biochim Biophys Acta 109 Environ Monit Assess 108 Toxicol Lett 108 J Environ Sci Heal A 107 Anal Chem 105 Arch Environ Con Tox 105 1 2 3 ... 121 1 2 3 ... 1037 Top Terms Publications Arsenic 8,453 Humans 6,737 Animals 4,632 Arsenicals 3,455 Oxides 3,374 Arsenites 2,377 Arsenic Poisoning 2,314 Adult 1,992 Evaluation Studies as Topic 1,806 Water Pollutants, Chemical 1,765 Proteins 1,724 Cadmium 1,559 Middle Aged 1,424 Hydrogen-Ion Concentration 1,354 Patients 1,343 Environmental Monitoring 1,298 Mercury 1,238 Sodium 1,225 Metals, Heavy 1,224 Tissues 1,187 1 2 3 ... 1037 1 2 3 ... 2197 Top Authors Publications Rosen B 78 Waalkes M 64 Chen C 51 Aposhian H 48 Chen Z 44 Vahter M 42 Del Razo L 40 Flora S 39 Cullen W 39 Buchet J 39 Meharg A 38 Cebrin M 38 Burger J 37 Stblo M 37 Francesconi K 35 Ahsan H 35 Hsueh Y 35 Lee T 35 Thomas D 34 Gossler W 34 1 2 3 ... 2197
http://www.gopubmed.org/web/gopubmed/WEB1mOWEB10O00d000j10020001000f01000j100300.y Arsenic trioxide and acute promyelocytic leukemia 723 documents semantically analyzed Top Years Publications 2008 75 2002 71 2009 69 2007 67 2001 61 2006 60 2005 59 2004 56 2003 55 2000 45 1999 41 2010 26 1998 23 1997 10 1996 3 1 2 Top Countries Publications USA 173 China 160 Japan 55 France 49 Italy 25 Taiwan 24 Canada 23 Germany 19 Hong Kong S.A.R., China 15 South Korea 14 India 10 Spain 8 Iran 7 United Kingdom 7 Sweden 7 Australia 6 Lebanon 5 Austria 5 Brazil 4 Netherlands 2 1 2 1 2 3 ... 9 Top Cities Publications Shanghai 62 New York 32 Paris 29 Harbin 25 Chicago 25 Houston 18 Taipei 16 Montreal 15 Hong Kong 15 Hamamatsu 12 Rome 11 Beijing 11 Tokyo 9 Tehran 7 Berlin 7 Seoul 7 Tianjin 6 Vellore 6 Wuhan 6 London 6 1 2 3 ... 9 1 2 3 ... 13 Top Journals Publications Blood 44 Leukemia 37 Haematologica 21 Leukemia Lymphoma 20 Zhonghua Xue Ye Xue Za Zhi 17 Leukemia Res 16 Ann Hematol 15 Brit J Haematol 15 Int J Hematol 11 Chinese Med J-peking 11 J Clin Oncol 10 Cancer Res 10 Oncogene 10 Oncologist 10 J Biol Chem 9 Semin Hematol 9 Rinsho Ketsueki 9 Cancer Chemoth Pharm 9 P Natl Acad Sci Usa 8 Int J Oncol 8 1 2 3 ... 13 1 2 3 ... 166 Top Terms Publications Leukemia, Promyelocytic, Acute 691 Granulocyte Precursor Cells 661 Humans 642 Leukemia 613 Arsenicals 595 Oxides 590 Antineoplastic Agents 437 Patients 386 Tretinoin 314 Apoptosis 301 apoptosis 276 Recurrence 265 retinoic acid metabolic process 225 retinoic acid catabolic process 225 response to retinoic acid 225 induction of apoptosis 202 Proteins 190 Cell Line 179 Therapeutics 169 Remission Induction 160 1 2 3 ... 166 1 2 3 ... 136 Top Authors Publications Chen Z 37 Chen S 28 Wang Z 27 Tallman M 26 Chen G 23 Waxman S 21 De Th H 21 Au W 20 Kwong Y 20 Lo Coco F 17 Ohnishi K 16 Shen Z 16 Naoe T 14 Takeshita A 14 Zhu J 13 Miller W 13 Yang B 13 Ohno R 13 Kantarjian H 12 Naito K 12 1 2 3 ... 136 最新研究进展 《科学》：我国白血病研究又获重大突破 发现抗急性早幼粒细胞白血病药物三氧化二砷的作用靶点 上海交通大学医学院附属瑞金医院上海血液学研究所/医学基因组学国家重点实验室4月9日在国际权威杂志《科学》（ Science ）上发表了三氧化二砷治疗急性早幼粒细胞性白血病（APL）分子机制的最新研究成果，该研究揭示了癌蛋白PML-RAR 是砷剂治疗APL的直接药物靶点。他们发现三氧化二砷直接与癌蛋白PML端的锌指结构中的半胱氨酸结合，诱导蛋白质发生构象变化和多聚化，继而发生SUMO化、泛素化修饰而被蛋白酶体降解。癌蛋白的降解最终导致白血病细胞走向分化和凋亡。使APL成为人类急性白血病分子靶向治疗取得临床治愈的成功范例。这一成果丰富了APL靶向治疗的理论，对于推动其它类型白血病和实体瘤的分子靶向治疗研究也具有十分重要的指导意义。 两院院士张杰、陈亚珠、王恩多、陆道培、王振义、王红阳、丁健和有关方面领导对此深感兴奋，专门到瑞金医院上海血液学研究所，向陈赛娟等科研人员表示祝贺。 APL是一种特殊类型的急性髓细胞性白血病，具有特征性的PML-RAR 癌蛋白。APL曾经被认为是最为凶险的白血病之一，很多患者在发病早期即死于严重出血而缺乏有效的治疗方法。1985年，上海血研所王振义教授在国际上率先应用全反式维甲酸（ATRA）治疗APL患者取得成功，80%以上患者可以完全缓解，但短期内容易复发。上世纪90年代，在哈尔滨医科大学张庭栋教授等应用传统中药三氧化二砷治疗APL患者取得疗效的基础上，上海血研所陈竺、陈赛娟研究员等用三氧化二砷成功治疗全反式维甲酸耐药复发的APL患者，并发现砷剂诱导白血病细胞分化和凋亡的双重药理学机制。他们经过进一步临床实践证明全反式维甲酸和三氧化二砷联合应用可以使约90%的APL患者达到5年无病生存，且未见明显长期毒性作用，从而使APL成为第一种基本可以被治愈的急性髓细胞性白血病。追本溯源，揭示临床现象所蕴涵的科学本质，阐释药物的分子靶点和机制，对于认识恶性肿瘤的发病机制和探索治疗理论的创新都具有重要意义。全反式维甲酸通过靶向结合到癌蛋白PML-RAR 的维甲酸受体（RAR ）结构域，重新启动髓系细胞的分化基因调控网络，诱导白血病细胞分化成熟继而凋亡。 但三氧化二砷的直接分子靶点和分子机理是什么？这个问题长期困扰着研究人员。基于对前期研究结果的分析和对重要科学问题的敏感性，陈竺、陈赛娟首先提出：砷剂很可能直接靶向PML-RAR癌蛋白，发挥特异性治疗作用。药物分子靶点的研究首先需要解决的难题是对药物进行标记和示踪。三氧化二砷是一种小分子无机化合物，难以标记。研究人员巧妙地借助两种有机砷一种用生物素加以标记，另一种则在与蛋白相邻巯基结合后可以发出红色荧光信号，证实了砷剂在细胞内可以直接结合癌蛋白PML-RAR，而未标记的三氧化二砷则能竞争性抑制此种结合。进一步的追踪则将砷的结合部位定位到癌蛋白上的PML结构域。随后，研究人员利用生物技术合成PML结构域蛋白，通过多种质谱和光谱学分析证实砷通过与半胱氨酸形成砷硫配位共价键结合到PML结构域上。他们又与其他课题组合作，借助结构生物学和生物物理学的方法解析了砷与PML蛋白结合的配位模式和局部结构。PML-RAR 癌蛋白的PML部分含有锌指结构域，生理条件下与锌结合，研究者通过核磁共振技术发现砷在较高浓度下可以竞争性替换锌与蛋白的结合。接着，他们又解析了砷的结合如何决定该癌蛋白的命运这一问题，通过细胞内、外的实验发现砷剂结合PML结构域后诱导蛋白质构象变化和多聚化，促进其与一种介导翻译后修饰的酶UBC9之间的相互作用增强，使癌蛋白更容易被一种类泛素样蛋白SUMO修饰，继而发生泛素化修饰而被蛋白酶体降解。癌蛋白的降解最终导致白血病细胞走向分化和凋亡，使APL成为人类急性白血病分子靶向治疗取得临床治愈的成功范例。 这一研究成果不仅阐释了三氧化二砷治疗APL的药物分子靶点和分子机制，上海药物所所长丁健院士点评该成果时认为，其更为深远的意义在于：全反式维甲酸和砷剂分别靶向结合到同一癌蛋白的不同区域而发挥协同作用，诱导白血病细胞分化凋亡，为肿瘤治疗的新策略提供了理论和实践依据；砷剂是一种具有2000多年历史记载的古老中药，用现代科学手段揭示中药的分子作用机制，将促进我国科学家对祖国传统医学宝库进行深入探索和挖掘。 陈赛娟院士强调：这一研究成果是多学科多研究领域的整合和交叉的结果，为临床科学研究开拓了新的途径。据了解，该项研究所取得的突破是以上海血研所老中青三代科研队伍为核心，联合了国内外多家科研机构，包括中科院上海生命科学院生化细胞所和上海药物所，中科院高能物理所，中国科学技术大学生命科学院，合肥国家同步辐射实验室，中科院动物所，中法生命科学与基因组研究中心，法国巴黎第七大学等多家单位的课题组精诚合作、联合攻关的结晶。 该项研究得到了国家863计划，973计划，国家自然科学基金，上海市重大科技专项基金等的资助。 更多阅读 《科学》发表论文摘要（英文） PNAS：中国治疗急性白血病方案安全可靠 陈竺夫人陈赛娟：成立中国的NIH是陈竺最大的愿望 绚丽的生命风景线：记陈竺、陈赛娟院士 http://www.sciencemag.org/cgi/content/abstract/328/5975/240 Science 9 April 2010: Vol. 328. no. 5975, pp. 240 - 243 DOI: 10.1126/science.1183424 Prev | Table of Contents | Next Reports Arsenic Trioxide Controls the Fate of the PML-RAR Oncoprotein by Directly Binding PML Xiao-Wei Zhang, 1 ,* Xiao-Jing Yan, 1 ,* Zi-Ren Zhou, 2 Fei-Fei Yang, 3 Zi-Yu Wu, 3 Hong-Bin Sun, 4 Wen-Xue Liang, 1 Ai-Xin Song, 2 Valrie Lallemand-Breitenbach, 5 Marion Jeanne, 5 Qun-Ye Zhang, 1 Huai-Yu Yang, 6 Qiu-Hua Huang, 1 Guang-Biao Zhou, 7 Jian-Hua Tong, 1 Yan Zhang, 1 Ji-Hui Wu, 4 Hong-Yu Hu, 2 Hugues de Th, 5 ,8 Sai-Juan Chen, 1 ,8 , Zhu Chen 1 ,8 , Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As 2 O 3 ) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RAR (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RAR degradation is triggered by their SUMOylation, but the mechanism by which As 2 O 3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RAR and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As 2 O 3 provides new insights into the drugs mechanism of action and its specificity for APL. 1 State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, 197 Rui Jin Road II, Shanghai 200025, China. 2 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS), Shanghai 200031, China. 3 National Synchrotron Radiation Laboratory, University of Science and Technology of China and Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, CAS, Beijing 10004, China. 4 Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China. 5 Universit de Paris 7/INSERM/CNRS UMR 944/7151, Equipe Labellise No. 11 Ligue Nationale Contre le Cancer, Hpital St. Louis, Avenue C. Vellefaux, 75475 Paris CEDEX 10, France. 6 Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, CAS, Shanghai 201203, China. 7 Laboratory of Molecular Carcinogenesis and Targeted Therapy for Cancer, State Key Laboratory of Biomembrane and Membrane Biotechnology, and Key Laboratory of Stem Cell Development, Institute of Zoology, CAS, Beijing, China. 8 The Ple Sino-Franais de gnomique et de Sciences du vivant de lHpital Rui-Jin, 197 Rui-Jin Road II, Shanghai, China. * These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: zchen@stn.sh.cn
标签: 三氧化二砷
