N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

INTRODUCTION Hepatitis B virus (HBV) infects more than 350 million people worldwide and is a leading cause of end-stage liver disease and of hepatocellular carcinoma[1]. HBV is non- cytopathic for hepatocytes; however, most newly HBV-infected adult patien…     

大剂量阿糖胞苷治疗儿童急性淋巴细胞白血病的疗效观察

目的:观察大剂量阿糖胞苷(Ara-C)治疗儿童急性淋巴细胞白血病(ALL)的临床疗效。方法:选择我院2003年11月-2006年11月收治的50例ALL患者,均分为治疗组和对照组。在常规诱导方案联合化疗达到完全缓解后,治疗组给予HD(H:高三尖杉酯碱;D:柔红霉素)联合大剂量Ara-C巩固治疗,对照组仅给予HD巩固治疗。观察、比较2组的临床疗效,患者化疗结束后1、3、5年内的无病生存率及不良反应。结果:治疗组总有效率为88.0%,显著高于对照组(44.0%),2组比较差异有统计学意义(χ2=4.399,P<0.05);治疗组1、3、5年的无病生存率分别为88.0%、64.0%、32.0%,对照组分别为48.0%、20.0%、0,治疗组均显著高于对照组(χ2分别为4.116、5.439、6.821,P<0.05);2组在治疗过程中均出现不同程度的骨髓抑制和胃肠道反应,2组不良反应发生率比较差异无统计学意义(P>0.05)。结论:大剂量Ara-C方案巩固治疗儿童ALL临床疗效及安全性较好,可以显著延长患者的无病生存期。     

Disposition and metabolism of almotriptan in rats,dogs and monkeys

Almotriptan is a new highly potent selective 5-HT_1B/1D receptor agonist developed for the treatment of migraine, and the disposition of almotriptan in different animal species is now addressed in the current study. Almotriptan was well absorbed in rats (69.1%) and dogs (100%) following oral treatment. The absolute bioavailability was variable reflecting different degrees of absorption and first-pass metabolism (18.7–79.6%). The elimination half-life was short and ranged between 0.7 and 3?h. The main route of elimination of almotriptan was urine with 75.6% and 80.4% of the dose recovered over a 168-h period in rats and dogs, respectively. The γ-aminobutyric acid metabolite formed by oxidation of the pyrrolidine ring was the main metabolite found in urine, faeces, bile, and plasma of rats and in monkey urine. By contrast, the unchanged drug, the indole acetic acid metabolite formed by oxidative deamination of the dimethylaminoethyl group, and the N-oxide metabolite were the main metabolites in dog.     

Metabolism,pharmacokinetics and excretion of the GABAA receptor partial agonist [14C]CP-409,092 in rats

1. The metabolism and excretion of a GABA_A partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [¹⁴C]CP-409,092.

2. The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15?mg kg^?1, respectively, were characterized by high clearance of 169?±?18?ml min^?1 kg^?1, a volume of distribution of 8.99?±?1.46 l kg^?1, and an oral bioavailability of 2.9% ± 3%.

3. Following oral administration of 100?mg kg^?1 [¹⁴C]CP-409,092, the total recovery was 89.1% ± 3.2% for male rats and 89.3% ± 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48?h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces.

4. The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation.

5. The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.

     

含胞嘧啶脱氨酶基因重组腺病毒的构建及其对肺癌抑制作用的研究

目的：构建含胞嘧啶脱氨酶基因（CD基因）重组腺病毒（AdE1CMVCD），并鉴定；用自杀基因治疗系统（AdE1CMVCD/5-FC）对肺癌进行抑瘤作用的实验研究。方法：体外实验：用不同稀释度的AdE1CMVCD)转染人肺癌细胞H460后分别加入不同浓度的5-氟胞嘧啶（5-FC），用MTT法检测OD570nm值，按公式计算细胞生长抑制率；体内实验：建立T739鼠肺腺癌荷瘤模型，瘤体内导入AdE1CMVCD，腹腔注射5-Fc，观察实验组及各对照组瘤体及生存期差异。结果：该系统转染的人肺癌H460细胞生长抑制率随前药5-FC浓度及感染重组病毒剂量的增加而增加，最大抑制率达61.29％，同时观察到了明显的旁杀伤作用；在体内实验中观察到AdE1CMVCD/5-FC对小鼠肺癌有明显的生长抑制作用，明显延长荷瘤小鼠生存期。结论：本文建立的AdE1CMVCD/5-FC系统体内外对肺癌均有明显的抑制作用，为临床肺癌基因治疗的应用奠定了基础。     

CD基因转移及与放射联合对宫颈癌细胞株作用的体外实验研究

目的：研究胞嘧啶脱氨酶（CD）基因转移联合5－氟胞嘧啶（5－Fc）在体外对人宫颈癌细胞株Hela生长的影响及放射增敏作用。方法：将CD基因转入Hela细胞，加入含5－Fc的培养基，联合放射，采用MTT法和克隆形成试验分别比较单一CD／5－Fc体系及与放射治疗联合对该细胞生长的影响。结果：5－Fc能显著抑制转入了CD基因的Hela细胞的生长，并能通过旁观者效应杀死基因转移细胞周围未转入该基因的细胞。CD／5－Fc体系联合放射作用于Hela细胞，其生长抑制强度与单一治疗相比显著增强，两者的联合作用具有协同效应（P＜0．05）。结论：CD基因转移联合5－Fc作用对Hela细胞具有显著的杀伤作用，对放射具有增敏作用。     

Ara-C differentially affects multiprotein forms of human cell DNA polymerase

Purpose: The antimetabolite 1-β-d-arabinofuranosylcytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia although the precise mechanism by which ara-C induces cytotoxicity remains unclear. Our laboratory has previously isolated from human cells a DNA replication complex, termed the DNA synthesome, which is fully competent to orchestrate, in vitro, all of the reactions required to efficiently and faithfully replicate DNA. Using this system and the active metabolite of ara-C, ara-CTP, we demonstrated that the human DNA synthesome can efficiently incorporate ara-CTP into internucleotide positions of newly replicated DNA in vitro mimicking results obtained using intact cells and isolated nuclei. We then hypothesized that DNA polymerase auxiliary proteins, present within the DNA synthesome, may aid in incorporating this nucleotide analog into DNA. Methods: To test this hypothesis, we utilized three distinct multiprotein complexes each of which contained human DNA polymerase α and examined with standard in vitro polymerase assays the effectiveness of ara-C in inhibiting various aspects of their polymerase function. Results and conclusion: These polymerase-mediated elongation assays, which included ara-CTP- or ara-C-containing primers in the reaction mixture, showed that the rate of DNA elongation in the presence of ara-CTP was significantly enhanced when the DNA polymerase was associated with its auxiliary proteins, and that the elongation resulted in the formation of internucleotide ara-CMP. Nevertheless, the enhanced activities resulting from the association of these auxiliary proteins with polymerase α did not fully account for the remarkable efficiency with which the DNA synthesome incorporated ara-C into internucleotide positions during DNA replication. Received: 22 June 1999 / Accepted: 4 January 2000     

Recombinant adenoviral vector containing tumor-specific l-plastin promoter fused to cytosine deaminase gene as a transcription unit: generation and functional test

The expression of therapeutic transgenes in recombinant adenoviral vectors is a major cause of toxicity in dividing cancer cells as well as non dividing normal cells. To solve the problem of toxicity to normal cells, we have reported on a recombinant adenoviral vector system (AdLP-) in which the expression of the transgene is directed by the tumor-specific L-plastin promoter (LP) (Chung et al., 1999). The object of this study was to generate a recombinant adenoviral vector system which would generate tumor cell specific expression of cytosine deaminase (CD) gene. We report the construction of a replication-incompetent adenoviral vector in which CD is driven by the L-plastin promoter (AdLPCD). Infection of 293 cells by AdLPCD generated the functional CD protein as measured by HPLC analysis for the conversion of 5-Fluorocytosine (5-FC) to 5-Fluorouracil (5-FU). HPLC analysis in conjunction with counting radioactivity for [6-3H]-5FC and [6-3H]-5FU demonstrated vector dose-dependent conversion of 5-FC to 5-FU in AdLPCD infected ovarian cancer cells. The results from present and previous studies (Peng et al., 2001; Akbulut et al., 2003) suggest that the use of the AdLPCD/5-FC system may be of value in the treatment of cancer including microscopic ovarian cancer in the peritoneal cavity.     

Low dose Ara-C for myelodysplastic syndromes: is it still a current therapy?

The myelodysplastic syndromes (MDS) are characterized by hemopoietic insufficiency associated with severe cytopenias, leading to serious morbidity, and acute leukemia development. MDS typically occur in elderly people, with a median age at diagnosis ranging between 60 and 75 years. The patients' prognosis, estimated according to the International Prognostic Scoring System, age and performance status should be considered before choosing among the various treatment options. A therapeutic dilemma exists in MDS, due to the multifactorial pathogenetic features of the disease, the heterogeneous stage and the elderly age of patients at diagnosis. This is underlined by the absence of a Food and Drug Administration-approved agent with an indication for this disease. The therapeutic end-points vary from symptom management (using low-intensity treatment with biological targeted agents, or only supportive therapy), to attempts to change the natural history of the disease (generally using high intensity treatment, including intensive chemotherapy and hemopoietic stem cell transplantation). The main goal of low-intensity therapies is generally to induce hematological improvements and is mainly used for low-risk disease. On the other hand, high-intensity therapies generally aims to alter the disease's natural history (improving survival, and decrease progression to acute myeloid leukemia), and are mainly used for high-risk disease. This review will focus on the current role of low-dose Ara-C therapy in the management of MDS. In fact, there is evidence that low-dose chemotherapy with Ara-C can induce responses in patients with MDS. In particular, the use in combinations with growth factors, such as G-CSF or M-CSF, looks promising, suggesting further investigations about this old - new therapeutic tool.     

组织特异性CD/5-FC系统对大肠癌的原位基因治疗

  目的 探讨癌胚抗原(carcinoembryonic antigen,CEA)组织特异性胞嘧啶脱氨基酶基因对不同分泌CEA大肠癌组织的靶向杀伤作用. 方法 脂质体法将CEA组织特异性逆转录病毒载体G1CEACDNa在PA317细胞中进行包装,大肠癌细胞LoVo和SW480分别接种到BALB/c裸鼠大腿皮下,成瘤2周后,瘤内多点注射法行原位基因转染,每天腹腔注射500mg/kg的5-FC(5-fluorocytosine),观察治疗效果. 结果 病毒滴度为5.6×106CFU/L.经多次注射法转染,目的基因在肿瘤组织中能有效表达,治疗21天后,基因治疗组有明显的抑瘤作用,但对LoVo细胞肿瘤的抑瘤作用明显大于对SW480细胞肿瘤. 结论 CEA组织特异性CD/5-FC系统对LoVo细胞肿瘤的抑瘤作用更明显.