Inhibition of Human Hepatitis B Virus Replication by AT-61, a Phenylpropenamide Derivative,Alone and in Combination with (?)β-l-2′,3′-Dideoxy-3′-Thiacytidine

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 μM. AT-61 (27 μM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 μM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (−)-β-l-2′,3′-dideoxy-3′ thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 μM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.Hepatitis B virus (HBV) is estimated to chronically infect approximately 300 million people worldwide. These individuals are at increased risk for the development of liver failure, cirrhosis, and hepatocellular carcinoma (3, 23). In addition, it is estimated that of those chronically infected, approximately 1 million die annually from HBV-induced liver disease (19).At the present, interferon (IFN) is the only available treatment for chronic hepatitis in the United States. However, its efficacy is partial and of limited duration, with less than 30% of the chronic carriers being treated with IFN responding to treatment. In addition, approximately 50% of those who initially respond to IFN therapy experience a recurrence of viremia after the cessation of treatment (6, 29). In clinical trials, two nucleoside analogs, lamivudine [(−)-β-l-2′,3′-dideoxy-3′-thiacytidine; 3TC] and ganciclovir, have proven to be effective in decreasing the levels of HBV DNA in the serum of chronically infected patients (4, 7–9). However, many patients relapsed shortly after the cessation of therapy. In addition, there are now reports of the isolation of 3TC-resistant variants of HBV from the serum of immunosuppressed patients undergoing 3TC therapy (2, 20, 30).Here we report that AT-61, a member of a class of phenylpropenamide derivatives with antiviral activity against HBV replication (22), is a potent inhibitor of the replication of both wild-type and 3TC-resistant HBV in HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cell lines. This compound does not inhibit the replication of duck HBV (DHBV), woodchuck HBV (WHBV), human immunodeficiency virus (HIV) type 1 (HIV-1), herpes simplex virus (HSV) type 1, (HSV-1), vesicular stomatitis virus (VSV), or Newcastle disease virus (NDV) and has very low toxicity in a number of cell lines. Moreover, when used in combination with 3TC, AT-61 acted synergistically to inhibit HBV replication in HepAD38 cells. Finally, the data suggest that this compound may exert its antiviral effect by interfering with the packaging of the pregenomic RNA into the immature core particle.     

Dose-Dependent Uptake of 3′-deoxy-3′-[18 F]Fluorothymidine by the Bowel after Total-Body Irradiation

Purpose

The aim of this study is to non-invasively assess early, irradiation-induced normal tissue alterations via metabolic imaging with 3′-deoxy-3′-[¹⁸?F]fluorothymidine ([¹⁸?F]FLT).

Procedures

Twenty-nine male C57BL/6 mice were investigated by [¹⁸?F]FLT positron emission tomography for 7 days after total body irradiation (1, 4, and 8 Gy) versus ‘sham’ irradiation (0 Gy). Target/background ratios were determined. The imaging results were validated by histology and immunohistochemistry (Thymidine kinase 1, Ki-67).

Results

[¹⁸?F]FLT demonstrated a dose-dependent intestinal accumulation post irradiation. Mean target/background ratio (±standard error) 0 Gy: 1.4 (0.2), 1 Gy: 1.7 (0.1), 4 Gy: 3.1 (0.3), 8 Gy: 4.2 (0.6). Receiver operating characteristic analysis (area under the curve, p value): 0 vs. 1 Gy: 0.81, 0.049; 0 vs. 4 Gy: 1.0, 0.0016; and 0 vs. 8 Gy: 1.0, 0.0020. Immunohistochemistry confirmed the results.

Conclusions

[¹⁸?F]FLT seems to provide dose-dependent information on radiation-induced proliferation in the bowel. This opens the perspective for monitoring therapy-related side-effects as well as assessing, e.g., radiation accident victims.     

Anti-Hepatitis B Virus Activity and Metabolism of 2′,3′-Dideoxy-2′,3′-Didehydro-β-l(?)-5-Fluorocytidine

2′,3′-Dideoxy-2′,3′-didehydro-β-l(−)-5-fluorocytidine [l(−)Fd4C] was found to be at least 10 times more potent than β-l-2′,3′-dideoxy-3′-thiacytidine [l(−)SddC; also called 3TC, or lamivudine]against hepatitis B virus (HBV) in culture. Its cytotoxicity against HepG2 growth in culture was also greater than that of l(−)SddC (3TC). There was no activity of this compound against mitochondrial DNA synthesis in cells at concentrations up to 10 μM. The dynamics of recovery of virus from the medium of cells pretreated with equal drug concentrations were slower with l(−)Fd4C than with l(−)SddC (3TC). l(−)Fd4C could be metabolized to mono-, di-, and triphosphate forms. The degree of l(−)Fd4C phosphorylation to the 5′-triphosphate metabolite was higher than the degree of l(−)SddC (3TC) phosphorylation when equal extracellular concentrations of the two drugs were used. The apparent K_m of l(−)Fd4C phosphorylated metabolites formed intracellularly was higher than that for l(−)SddC (3TC). This may be due in part to a difference in the behavior of l(−)Fd4C and l(−)SddC (3TC) towards cytosolic deoxycytidine kinase. Furthermore, l(−)Fd4C 5′-triphosphate was retained longer within cells than l(−)SddC (3TC) 5′-triphosphate. l(−)Fd4C 5′-triphosphate inhibited HBV DNA polymerase in competition with dCTP with a K_i of 0.069 ± 0.015 μM. Given the antiviral potency and unique pharmacodynamic properties of l(−)Fd4C, this compound should be considered for development as an expanded-spectrum anti-HBV drug.Hepatitis B virus (HBV) infection is one of the most serious health issues in the world today (1, 3). β-l(−)-2′,3′-Dideoxy-3′-thiacytidine [l(−)SddC; also called 3TC, or lamivudine] (Fig. ​(Fig.1)1) is the first β-l(−) nucleoside analog identified by us and others to have potent activity against HBV in culture (4, 8, 12, 17). This drug exerts its action by inhibiting HBV DNA synthesis due to the preferential interaction of the l(−)SddC (3TC) 5′-triphosphate metabolite with HBV DNA polymerase (4). Unlike dideoxycytidine (ddC, or zalcitabine), a β-d(+) nucleoside analog with the natural nucleoside conformation in DNA and RNA, l(−)SddC (3TC) does not have potent activity against mitochondrial DNA (mtDNA) synthesis, which is important for maintaining the function of cells (4). Clinical trials of l(−)SddC (3TC) for the treatment of chronic HBV infection are ongoing and look promising (2, 7, 13, 16, 18, 19). Its potential value for HBV-infected patients undergoing liver transplantation is also being evaluated, since l(−)SddC (3TC) can suppress HBV DNA serum levels in these patients. However, apparent l(−)SddC (3TC)-resistant HBV emerged in some patients upon long-term treatment (13, 16, 18). The HBV-resistant genotype appears to be associated with the mutation of methionine to valine or isoleucine in the YMDD motif of HBV DNA polymerase (13, 16, 18). This mutation was previously demonstrated and could render duck HBV resistant to l(−)SddC (3TC) (11). It is not clear if this mutation alone can lead to resistance and, if so, to what degree HBV resistance to l(−)SddC (3TC) develops. Open in a separate windowFIG. 1Structures of l(−)deoxycytidine analogs.One approach to overcoming clinical drug resistance is to use higher dosages of l(−)SddC (3TC) given the therapeutic index of the compound in vitro. However, the potency of l(−)SddC (3TC) against HBV in the clinic could be a limiting factor given the dosage application. The antiviral potency is determined not only by its antiviral activity but also by the pharmacodynamic nature of its active metabolite, l(−)SddC (3TC) 5′-triphosphate, in vivo. A more potent compound with more favorable pharmacodynamic behavior of intracellularly active metabolites would be worth exploring.In the studies reported herein, we describe the anti-HBV activity, metabolism, and pharmacodynamic properties of 2′,3′-dideoxy-2′,3′-didehydro-β-l(−)-5-fluorocytidine [l(−)Fd4C](Fig. 1) in comparison with those of l(−)SddC (3TC), including its behavior toward deoxycytidine kinase and the interaction of l(−)Fd4C 5′-triphosphate with virion-associated HBV DNA polymerase. Preliminary studies of the anti-HBV and anti-human immunodeficiency virus (HIV) activities of l(−)Fd4C were previously reported by us and others (10, 15).     

PSI-7851, a Pronucleotide of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine Monophosphate,Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine-5′-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ± 0.050 μM (mean ± standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 μM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.Hepatitis C virus (HCV) currently affects more than 170 million people worldwide. Approximately 70% of infected individuals develop chronic hepatitis, among whom about 20% will develop liver cirrhosis and fibrosis and up to 5% will progress to hepatocellular carcinoma (2). The current standard of care (SOC), which combines pegylated alpha interferon (PegIFN-α) and ribavirin (RBV), has limited efficacy in providing a sustained virological response (SVR), especially in individuals with HCV genotype 1 (∼50%), the most prevalent genotype in Western countries (8, 11, 35). The impact of genetic diversity of HCV in patients receiving SOC therapy has been reviewed (26): SVR rates are higher in patients infected with genotype 2 or 3 (∼80%), patients infected with genotype 4 appear to have a slightly better SVR rate (∼60%) than patients infected with genotype 1, and patients infected with genotypes 5 and 6 may achieve an SVR at a level between those of genotypes 1 and 2/3. In addition to the variability in efficacy, the lengthy treatment (24 to 48 weeks) with SOC is frequently associated with undesirable side effects that may include anemia, fatigue, and depression (7). There is an urgent medical need to develop anti-HCV therapies that are safer and more effective. Direct-acting antivirals (DAAs) are compounds that target a specific viral protein. Currently, four major classes of DAAs are being investigated in phase II or III clinical trials: NS3 protease inhibitors, NS5A inhibitors, allosteric nonnucleoside NS5B polymerase inhibitors, and nucleoside/-tide NS5B polymerase inhibitors (21, 27, 46). Challenges for these DAAs include safety, pan-genotypic activity, and/or emergence of resistant viruses. An effective antiviral therapy against hepatitis C should encompass a broad spectrum of activity against all HCV genotypes, shorten treatment duration, have minimal side effects, and have a high barrier to resistance.The HCV NS5B RNA-dependent RNA polymerase (Pol) is a critical component of the replicase complex and is responsible for initiating and catalyzing viral RNA synthesis (16, 32, 58). There is no human homolog of this protein, and it is absolutely required for viral infectivity (19). As a result, the HCV NS5B is an attractive target for the development of antiviral compounds. There are two major classes of NS5B inhibitors: nucleoside analogs, which are anabolized to their active triphosphates and act as alternative substrates for the polymerase, and nonnucleoside inhibitors (NNIs), which bind to allosteric regions on the protein. Two major drawbacks associated with NNIs are that the activity appears to vary significantly among different HCV genotypes and even subtypes (15, 33) and that there is a relatively low barrier for resistance as evidenced by the numerous naturally occurring resistant variants reported in the literature (18). In contrast, nucleoside analogs are similarly active across HCV genotypes (13, 15, 33) and have a higher barrier of resistance compared to the NNIs and NS3 protease inhibitors (36). To date only two amino acid changes within the NS5B polymerase that confer resistance to nucleoside inhibitors have been identified: S96T and S282T (1, 29). The S96T mutation confers resistance to 4′-azidocytidine (R1479), while the S282T mutation is resistant to a number of 2′-C-methyl-modified nucleoside inhibitors (1, 29, 38, 43).In order for nucleoside analogs to be active as alternative substrates, they must first be phosphorylated by cellular kinases to their corresponding 5′-triphosphates, which are active alternative substrate inhibitors for the NS5B polymerase. The efficiency of these metabolic steps, the stability of the triphosphates, and the affinity of the triphosphates for the NS5B polymerase are all important factors in determining the antiviral activities of nucleoside inhibitors. PSI-6130, 2′-F-2′-C-methylcytidine, was previously shown to be a specific inhibitor of HCV RNA replication in the replicon assay system (52). However, when the uridine analog, 2′-F-2′-C-methyluridine (referred to as PSI-6206), was tested in the replicon assay, it failed to inhibit HCV RNA synthesis due to the inability of cellular enzymes to metabolize PSI-6206 to its triphosphate, PSI-7409 (5, 34, 42). Biochemical studies with PSI-7409 showed that this compound was able to inhibit RNA synthesis mediated by the HCV replicase complex and by purified recombinant HCV NS5B polymerase (34, 42). Furthermore, in vitro stability studies using primary human hepatocytes demonstrated that PSI-7409 has a significantly longer half-life (t_1/2, 38 h) than PSI-6130-TP (t_1/2, 4.7 h), which could be a desirable pharmacologic benefit (34).In order to bypass the initial nonproductive phosphorylation step of PSI-6206, the phosphoramidate prodrug methodology was explored as an approach to deliver 2′-F-2′-C-methyluridine monophosphate (47, 48). An extensive series of phosphoramidate prodrugs were synthesized, and PSI-7851 demonstrated the desired characteristics with regard to activity and in vitro toxicity. Herein we present the results of in vitro studies characterizing PSI-7851, a potent and specific anti-HCV compound with pan-genotype activity.     

脑脊液蛋白定量α,α′-联吡啶测定法

脑脊液（CSF）蛋白定量常用浊度法，仅缩脲试剂比色法［1］和考马斯蓝（CBG）染料结合法［2］等。我们参考有关文献［3，4］用α，α′-联吡啶作CSF蛋白定量测定，方法简便，精密度高，重复性好。1材料和方法1．1原理在酸性溶液中，蛋白质还原Fe为Fe，后者与α，α′-联吡啶结合生成红色复合物，颜色深浅与蛋白含量成正比．1．2试剂1．2．1pH4．00．25mol／L醋酸盐缓冲液。1．224．0g／LFeCl3溶液。1．2．3显色剂α，α′-联吡啶200mg，溶于5ml无水乙醇，加醋酸盐缓冲液至200ml，置棕色瓶，冰箱保存，可长期使用。1．2．4蛋白标准液…     

Validity of Simplified 3′-Deoxy-3′-[18F]Fluorothymidine Uptake Measures for Monitoring Response to Chemotherapy in Locally Advanced Breast Cancer

Purpose

Positron emission tomography using 3??-deoxy-3??-[¹⁸F]fluorothymidine ([¹⁸F]FLT) has been suggested as a means for monitoring response to chemotherapy. The aim of this study was to evaluate the validity of simplified uptake measures for assessing response to chemotherapy using [¹⁸F]FLT in locally advanced breast cancer (LABC).

Procedures

Fifteen LABC patients underwent dynamic [¹⁸F]FLT scans both prior to and after the first cycle of chemotherapy with fluorouracil, epirubicin or doxorubicin, and cyclophosphamide. The net uptake rate constant of [¹⁸F]FLT, K _i , determined by non-linear regression (NLR) of an irreversible two-tissue compartment model was used as the gold standard. In addition to Patlak graphical analysis, standardised uptake values (SUV) and tumour-to-whole blood ratio (TBR) were used for analysing [¹⁸F]FLT data. Correlations and relationships between simplified uptake measures and NLR before and after chemotherapy were assessed using regression analysis.

Results

No significant differences in both pre- and post-chemotherapy relationships between any of the simplified uptake measures and NLR were found. However, changes in SUV between baseline and post-therapy scans showed a significant negative bias and slope less than one, while TBR did not.

Conclusions

In LABC, TBR instead of SUV may be preferred for monitoring response to chemotherapy with [¹⁸F]FLT.     

3′-Me-DAB诱发大鼠肝癌模型的建立与病理研究

目的　建立诱发性大鼠肝癌模型 ,动态观察肝癌发生发展的病理过程。方法　Spreque Dawley(SD)大鼠 75只 ,随机分为诱癌组 (6 0只 )和对照组 (15只 )。诱癌组以 3′ 甲基 4 二甲基氨基偶氮苯 (3′ Me DAB)分量饲喂法诱导大鼠肝癌。于诱癌第 1、2、3、4、5个月分别取 3～ 5只鼠称体重、肝重、取肝组织、癌灶及癌周组织作病理组织学检查。结果　 2 2例 (36 6 7% )诱癌成功 ,其中 8例为肝细胞肝癌 ,14例为混合性肝癌 ,Ⅰ级 2例 ,Ⅱ级 14例 ,Ⅲ级 6例。结论　3′ Me DAB分量饲喂法诱导的大鼠肝癌模型能够较好地模拟人类肝癌发生发展的过程。方法简便 ,可重复性好。实验方法通过改良 ,降低了诱癌过程中的死亡率 ,提高了诱癌成功率 ,是动态研究肝癌发生发展的理想的动物模型。     

200例甲亢T3,T4,FT3,FT4,TSH检测临床分析

1.资料和方法1.1资料 ①病例组:系1994年12月～1996年5月来,我院就诊的200例甲亢患者,其中男79例,女121例,年龄在14岁～65岁,平均29岁.②对照组:为企事业单位健康体检,排除甲状腺、肝、肾疾病及妊娠者,共50例,其中男27例,女23例,年龄18岁～60岁.1.2方法 ①T_3、T_4、FT_3、FT_4、TSH试剂药盒由北京     

用3′bcr探针对慢性粒细胞白血病作分子学诊断

慢粒有染色体9,22易位,形成所谓的Ph~1染色体,造成bcr-abl基因的杂合,结果产生一个新的8.7kb mRNA和一个新的多肽(210kd),后者(P210bcr/abl)具有酪氨酸激酶的作用,在慢粒发生中起关键作用.约有10%慢粒未找到Ph染色体而需要进行bcr重组的分子分析.作者在30例慢性期慢粒(29例Ph阳性和1例阴性)用3′bcr探针进行Southern blot分析.结果28例Ph阳性者和1例阴性者均有bcr重组,仅1例Ph阳性者没有查出1.2kb bcr探针.作者认为对Ph阴     

FoLey′s尿管应用不当致尿道裂伤3例报告

FoLey′s尿管目前已广泛应用于临床。它具有组织相容性好、不易感染、水囊内固定不易脱落等优点,但如果应用不当可致严重并发症。我院自1998年使用FoLey′s尿管以来,至今已导致3例患者出现尿道裂伤,现报道如下。1临床资料病例1,男,26a,因车祸致脾脏破裂于我院外科住院手术治疗,术前置Foley′s尿管并向水囊内注入无菌生理盐水25ml,留置尿管后向患者及家属交待注意事项,嘱患者及家属不能自行拔掉尿管,术后第3天,患者于夜间未告知医务人员,自行强行拔导尿管,拔除后尿管水囊未破裂,患者下腹剧痛,尿道出血,不能自行排尿,重新插尿管受阻不成功,…     

黑色素瘤抗原基因MAGE-A3 5′端CpG岛异常甲基化检测

目的研究黑色素瘤抗原基因MAGE-A3 5′端CpG岛异常甲基化状态,探讨其作为肝细胞肝癌(HCC)肿瘤标志物的可行性。方法利用重亚硫酸盐测序聚合酶链反应(BSP)分析HCC细胞株HepG2和健康对照者来源白细胞基因组中MAGE-A3基因5′端CpG岛异常甲基化状态,寻找HCC细胞MAGE-A3 5′端CpG岛去甲基化位点。结果 HepG2细胞和健康对照者来源白细胞基因组MAGE-A3 5′端CpG岛存在不同的甲基化状态,前者存在特异性去甲基化位点。结论 MAGE-A3 5′端CpG岛去甲基化位点的检测可用于HCC的早期诊断。     

基于5′→3′核酸外切酶实时PCR技术检测染色体

外套细胞淋巴瘤 (MCL )是与染色体 t(11,14 ) (q13;q32 )高度关联的具有组织学和免疫表型特征的一类独特的恶性淋巴瘤 ,在 MCL病人中 ,染色体 t(11;14 ) (q13;q32 )易位与位于 11q13的细胞癌基因bcl-1重叠 ,该癌基因与位于 14 q32的免疫球蛋白重链基因增强子毗邻 ,导致原癌基因失调 ,细胞周期紊乱。 bcl-1主要易位簇 (MTC)裂点区与免疫球蛋白绞链 (JH)区外显子的 3′部分具有高度同源序列 ,可通过 PCR法检测。材料和方法　作者研究了 5 0例 MCL病人 ,2 7例其它型非何杰金 (NHL )病人 (13例滤泡型、4例弥漫性大细胞型、2例小淋巴细胞…     

间断发热3个月,胸痛3天

(病案讨论见本期第 58页 )　　患者 ,男性 ,36岁 ,农民。 3个月来无明显原因间断发热 ,伴咳嗽、四肢关节痛 (不呈游走性 )。自认为感冒服用抗生素及退热药后 3～ 5天热退 ,关节痛好转 ,但隔 3～ 5天后又再度发热 ,体温常在 38℃左右 ,偶尔达 4 0℃。发热以午后及夜间明显 ,伴乏力、盗汗及关节痛 ,未正规诊治。近 1个月发现下肢浮肿 ,常感活动性心悸、气短、胸憋闷。近半月常因胸闷、气短而不能平卧。曾按上呼吸道感染治疗 ,效果差。入院前 3天 ,无任何诱因突发心前区剧烈疼痛 ,呈钝痛 ,不放散 ,伴大汗、心慌、气短、不能平卧 ,恶心呕吐两次 …     

Preclinical Evaluation of the Antifolate QN254, 5-Chloro- N′6′-(2,5-Dimethoxy-Benzyl)-Quinazoline-2,4,6-Triamine,as an Antimalarial Drug Candidate

Drug resistance against dihydrofolate reductase (DHFR) inhibitors—such as pyrimethamine (PM)—has now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N′6′-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparum V1S strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparum clinical isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S) forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The drug was remarkably efficacious against Plasmodium berghei and could fully cure infected mice with three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative in vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent.Malaria control is a global public health priority that has been hampered by the rapid development and spread of resistance against antimalarials. As a consequence, the World Health Organization (WHO) recommends the use of artemisinin-containing combination therapies (ACTs) as a first-line treatment for malaria. Although ACTs are designed to reduce the chance of artemisinin drug resistance development, there are considerable concerns that this may already have occurred. For instance, there is now mounting evidence that the efficacy of artemisinin derivatives is reduced in Southeast Asia, where artemisinin derivatives have been used for a long time as monotherapies (7, 28, 53). This is a cause of concern since the spread of artemisinin resistance will compromise the usefulness of ACTs globally. Thus, there is an urgent need to discover and develop new alternative drugs.For several decades, dihydrofolate reductase (DHFR) has been targeted with different classes of chemical entities for the development of new therapies for a broad range of therapeutic indications, including several parasitic diseases (13). DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which is an essential cofactor in the biosynthesis of deoxythymidylate monophosphate (dTMP), a metabolite essential to DNA synthesis and cell replication.Pyrimethamine (PM) is a potent inhibitor of the Plasmodium DHFR enzyme, and this compound has been widely used in combination with the dihydropteroate synthetase (DHPS) inhibitor sulfadoxine. Unfortunately, PM resistance is now common, rendering this drug ineffective (30). One of the documented mechanisms of antifolate resistance is through the mutation of the target itself—the Plasmodium falciparum dhfr-ts (Pfdhfr-ts) gene, encoding for a bifunctional enzyme which possesses both DHFR and thymidylate synthase (TS) activities carried by two distinct subdomains (29). PM resistance is associated with the mutation of the amino acid Ser to Asn at codon 108 of DHFR (S108N). Ancillary mutations of N51I and C59R are associated with an increase in resistance, and the presence of the mutation I164L results in an even higher level of PM resistance (14, 47). The presence of these mutations significantly decreases the sensitivity of the PfDHFR enzyme to PM inhibition in a biochemical assay (48).The antifolate triazine WR99210 (3, 22) is potent against P. falciparum bearing quadruple mutations of DHFR at S108N, N51I, C59R, and I164L (QM PfDHFR) (20). However, WR99210 has shown limited efficacy in vivo due to poor oral bioavailability and displayed some gastrointestinal toxicity. Attempts have been made to circumvent these issues and a prodrug form of WR99210 known as PS-15 has been shown to be orally active (2). The resolution of the three-dimensional structure of wild-type (WT) and QM PfDHFR—with either PM or WR99210 bound to its active site—provided structural insights into DHFR PM resistance mechanisms, as well as some understanding of the structural features of WR99210 that allow this compound to retain affinity for QM PfDHFR (55, 56).The quinazoline pharmacophore has been successfully used to design drugs for the treatment of cancer and other human diseases. For example, the DHFR inhibitor, trimetrexate (5-methyl-6-[(3,4,5-trimethoxy-phenylamino)-methyl]-quinazoline-2,4-diamine) has been developed to treat various cancers in human patients (15, 32, 33). In a previous study, a series of quinazoline derivatives was tested against the highly PM-resistant P. falciparum strain (V1S) and the DHFR inhibitor 2,4-diamino-5-chloro-6-[N-(2,5-dimethoxybenzyl)-amino]quinazoline (or QN254 here and compound 1 in reference 34) was found to have potent activity—with an IC₅₀ (i.e., the inhibitory concentration that reduces parasite growth by 50% in vitro) of 9 nM (34). Considering the increasingly widespread PM drug resistance, we set out to perform the experiments described here with the goal of further assessing the potential of QN254 as a candidate antimalarial to replace the failing PM. Collectively, our data demonstrate that QN254 (i) binds and inhibits the QM PfDHFR enzyme, (ii) is active on drug-resistant clinical isolates, and (iii) displays pharmacological properties compatible with an oral antimalarial drug candidate. However, preliminary toxicological findings indicate that QN254 does not show a therapeutic window sufficiently large to warrant its progression to the next development stage.     

The Extragenic Spacer Length Between the 5′ and 3′ Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors

In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.     

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β-l-2′,3′-Didehydro-2′,3′-Dideoxy-N4-Hydroxycytidine: Relevance for Activation in HepG2 Cells

ß-l-2′,3′-Didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM; 50% cytotoxic dose [CD₅₀] = 2,500 μM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[³H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5′-mono-, 5′-di-, and 5′-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ß-d-N⁴-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5′-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 μM l-[³H]Hyd4C for 24 h, the 5′-triphosphate accumulated to 19.4 ± 2.7 pmol/10⁶ cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/10⁶ cells. The intracellular half-life of the 5′-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5′-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5′-diphosphate during a drug-free incubation. The high 5′-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.A large number of nucleoside analogues have been described as inhibitors of hepatitis B virus (HBV) and HIV replication. Recently l-nucleoside analogues in particular have gained increasing interest. They are characterized by an opposite configuration from that of the natural d-nucleoside analogues and represent one of the most attractive groups of antiretroviral compounds, including ß-l-2′,3′-dideoxy-3-thiacytidine (3TC) and its 5-fluoro derivative (FTC), ß-l-2′,3′-didehydro-2′,3′-dideoxy-cytidine (l-d4C) and its 5-fluoro derivative (l-d4FC), ß-l-thymidine, ß-l-fluoroarabinosylyluracil (l-FMAU), and ß-l-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-cytidine (l-2′Fd4C) (3, 5, 22).Some of them not only have been found to be more potent than their corresponding d-nucleosides but seem to exhibit lower cytotoxicity and have been proved to be effective and selective agents for the treatment of chronic hepatitis B virus infections (4). However, only long-term therapy with a single nucleoside for several years was shown to be able to completely suppress HBV DNA in serum of patients and to reverse the progression of the disease. The disadvantage connected with such therapy regimens is the development of drug-resistant HBV strains (22). Therefore, the challenge will be to develop more-efficient drugs for shorter treatment regimens and to combine them to reach synergistic or at least additive drug action. This approach has been described not only as being highly efficient for the treatment of HIV infections but also as preventing the development of resistant mutants. Therefore, AIDS therapy is considered a model for future therapy of chronic HBV infections (17).Recently we described a series of new ß-l-N⁴-hydroxydeoxycytidine and ß-l-5-methyl-deoxycytidine derivatives as inhibitors of HBV replication. Between them, ß-l-2′,3′-didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) (Fig. ​(Fig.1)1) emerged as the most effective in suppression of virus production in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM), displaying an extremely low cytotoxicity (50% cytotoxic dose [CD₅₀] for HepG2 cells = 2,500 μM) (12).Open in a separate windowFIG. 1.Structure of l-Hyd4C and possible metabolites formed by reduction (l-d4C) or by deamination (l-d4U).These encouraging features have prompted us to investigate the cellular pharmacology of l-Hyd4C in a hepatic cell line. This included the activation of this unnatural l-deoxycytidine nucleoside to its 5′-mono-, 5′-di-, and 5′-triphosphate, the search for other metabolites, and the estimation of the intracellular half-lives (t_1/2) of the 5′-di- and 5′-triphosphate of l-Hyd4C.(This work was presented in part at BIT''s 5th Anniversary Congress of International Drug Discovery Science and Technology, 7 to 13 November 2007, Xi''an and Beijing, China.)     

M3病人D—二聚体,3P,PT,APTT及fg测量结果分析

急性早幼粒细胞白血病易诱发ＤＩＣ发生广泛而严重的出血 ,是Ｍ3病人死亡的主要原因 ,因此对Ｍ3病人是否诱发ＤＩＣ的诊断很重要。我们对 2 2例Ｍ3病人进行了Ｄ二聚体、3Ｐ、ＰＴ、ＡＰＴＴ及ｆｇ的检测试验 ,现分析报告如下。1　材料及方法 :1.1　仪器　ＣＡ - 10 0半自动血凝仪1.2　标本　选自我院住院的 2 2例Ｍ3病人(经骨髓细胞形态及细胞化学染色确诊 )。其中Ｍ3ａ8例 ,Ｍ3ｂ 14例 ;男性 12例 ,女性 10例 ;最大年龄58岁 ,最小年龄 2 2岁。 2 2例Ｍ3病人均有不同程度的出血。1.3　试剂1.3.1　自配的 1%硫酸鱼精球蛋白。1.3.2　Ｄ二聚体…     

An Evaluation of 2-deoxy-2-[18F]Fluoro-D-Glucose and 3′-deoxy-3′-[18F]-Fluorothymidine Uptake in Human Tumor Xenograft Models

Purpose

The aim of this study is to assess the variability of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]-FDG) and 3??-deoxy-3??-[¹⁸F]-fluorothymidine ([¹⁸F]-FLT) uptake in pre-clinical tumor models and examine the relationship between imaging data and related histological biomarkers.

Procedures

[¹⁸F]-FDG and [¹⁸F]-FLT studies were carried out in nine human tumor xenograft models in mice. A selection of the models underwent histological analysis for endpoints relevant to radiotracer uptake. Comparisons were made between in vitro uptake, in vivo imaging, and ex vivo histopathology data using quantitative and semi-quantitative analysis.

Results

In vitro data revealed that [1-¹⁴C]-2-deoxy-d-glucose ([¹⁴C]-2DG) uptake in the tumor cell lines was variable. In vivo, [¹⁸F]-FDG and [¹⁸F]-FLT uptake was highly variable across tumor types and uptake of one tracer was not predictive for the other. [¹⁴C]-2DG uptake in vitro did not predict for [¹⁸F]-FDG uptake in vivo. [¹⁸F]-FDG SUV was inversely proportional to Ki67 and necrosis levels and positively correlated with HKI. [¹⁸F]-FLT uptake positively correlated with Ki67 and TK1.

Conclusion

When evaluating imaging biomarkers in response to therapy, the choice of tumor model should take into account in vivo baseline radiotracer uptake, which can vary significantly between models.