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.     

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).     

Ollier′s病2例

1　病例报告　　例 1:男 ,2 8岁。因肢体畸形 ,活动受限而来诊。诉自幼两腿弯曲畸形 ,随年龄增长畸形加重 ,近年较稳定 ,家族无疑似病例。查体见身材低矮呈侏儒状 ;脊椎略有侧弯 ,两下肢与躯干比例失调 ,并严重扭曲畸形 ,手足粗大 ,全身肌肉、肌力尚正常 ;神经系统检查及血液、尿液检查均未见明显异常。 X线检查见脊柱各椎体仅见有代偿性侧变及扭曲 ;肋骨末段、左髂前上棘处、左侧桡骨远端、右尺骨下段、左手第 2、3掌骨及拇指、右手第 3、4掌骨、左侧股骨中段、左胫骨远近端、右股骨下段、右胫腓骨远近端、跖骨远端及趾骨等处均膨大 ,呈囊…     

Pharmacokinetics of 4′-cyano-2′-deoxyguanosine,a novel nucleoside analog inhibitor of the resistant hepatitis B virus,in a rat model of chronic kidney disease

IntroductionThe novel nucleoside analog, 4′-cyano-2′-deoxyguanosine (CdG), possesses inhibitory activity against both the wild-type and resistant hepatitis B virus. Since the dosage of the currently available nucleoside analog preparations needs to be adjusted, depending on renal function, we investigated the effect of renal dysfunction on the pharmacokinetics of CdG in a rat model of chronic kidney disease (CKD).MethodsCKD model rats were either intravenously or orally administered CdG at a dose of 1 mg/kg. The concentration of CdG in plasma, organs (liver and kidney) and urine samples were determined by means of a UPLC system interfaced with a TOF-MS system.ResultsFollowing intravenous administration, the plasma retention of CdG was prolonged in CKD model rats compared to healthy rats. In addition, the clearance of CdG was well correlated with plasma creatinine levels in CKD model rats. Similar to the results for intravenous administration, the plasma concentration profiles of CdG after oral administration were also found to be much higher in CKD model rats than in healthy rats. However, the results for the organ distribution and urinary excretion of CdG, the profiles of which were similar to that of healthy rats, indicated that CdG did not accumulate to a significant extent in the body.ConclusionThe extent of renal dysfunction has a direct influence on the pharmacokinetics (plasma retention) of CdG without a significant accumulation, indicating that the dosage of CdG will be dependent on the extent of renal function. .     

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.)     

Metabolism in Human Cells of the d and l Enantiomers of the Carbocyclic Analog of 2′-Deoxyguanosine: Substrate Activity with Deoxycytidine Kinase,Mitochondrial Deoxyguanosine Kinase,and 5′-Nucleotidase

The carbocyclic analog of 2′-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the l enantiomer rather than, as expected, with the d enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5′-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the l enantiomer was the better substrate. Even though the d enantiomer was the preferred substrate with 5′-nucleotidase, the rate of phosphorylation of the l enantiomer was substantial. The phosphorylation of d-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of d-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5′-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. d-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of d-CdG and related analogs and in the design of new biologically active analogs.d-CdG is an analog of CdG that has broad-spectrum antiviral activity (27–29). Until recently, the biological activity of a nucleoside analog was assumed to be due only to the “natural” β-d form. However, there are now numerous observations of antiviral activity associated with nucleosides in l configurations (for reviews, see references 10 and 26) and one report of antitumor activity associated with a β-l enantiomer (11). Even though l-CdG is much less active against HSV (4) and HCMV (unpublished results), it is essential that the metabolism of both enantiomers be examined to thoroughly understand the mechanism of action of a nucleoside analog. We have reported earlier that both enantiomers of CdG are extensively phosphorylated in cells infected with HSV-1 and that the enantiomers had equal activities as substrates for the virus-encoded kinase (4, 5). It was also observed that d-CdG was converted to its triphosphate (but to a much lesser extent) in noninfected cells and that more than one enzyme appeared to be involved in the initial phosphorylation. Since cellular enzymes presumably are responsible for the activation of CdG in cells infected with HBV (which is not known to code for a kinase) and may also be important for the activation of CdG in cells infected with HCMV (which induces cellular kinases [6, 8, 21, 23, 32, 44], in addition to encoding a ganciclovir-phosphorylating enzyme [22, 35]), we have evaluated the enantiomers of CdG as substrates for the three cellular enzymes known to catalyze phosphorylation of deoxyguanosine (2): dCyd kinase, mitochondrial dGuo kinase, and 5′-nucleotidase. We also report here observations on the metabolism of d-CdG in cells infected with HCMV and in duck hepatocytes infected with DHBV.(Some of these results have been presented elsewhere in preliminary form [1].)     

Evaluation of 2-Deoxy-2-[18F]Fluoro-D-glucose- and 3′-Deoxy-3′-[18F]Fluorothymidine–Positron Emission Tomography as Biomarkers of Therapy Response in Platinum-Resistant Ovarian Cancer

Purpose

We evaluated whether 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and 3??-deoxy-3??-[¹⁸F]fluorothymidine ([¹⁸F]FLT) positron emission tomography (PET) could be used as imaging biomarkers of platinum resensitization in ovarian cancer.

Procedures

Paired platinum-sensitive and platinum-resistant ovarian cancer cells from the same patient, PEO1 and PEO4, grown as tumor xenografts in nude mice, were assessed by PET.

Results

The AKT inhibitor, API-2, resensitized platinum-resistant PEO4 tumors to cisplatin, leading to a markedly lower Ki67 labeling index (p????0.006, n?=?6 per group). [¹⁸F]FDG-PET and [¹⁸F]FLT-PET imaging variables were lower after combination treatment compared with vehicle treatment (p????0.006, n?=?6 per group). No changes were seen with either drug alone. PRAS40 phosphorylation status was a sensitive biochemical marker of pathway inhibition, whereas reductions thymidine kinase 1 expression defined the [¹⁸F]FLT response.

Conclusions

Therapeutic inhibition of AKT activation in acquired platinum-resistant disease can be imaged noninvasively by [¹⁸F]FDG-PET and [¹⁸F]FLT-PET warranting further assessment.     

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.     

HCV基因型与2′- 5′寡腺苷酸合成酶活性的相关性研究

目的 了解丙型肝炎病毒基因型在不同人群中的分布规律与流行优势型及其与2′—5′寡腺苷酸合成酶(2-5AS)的相关性。方法 分别对门诊体检、住院病人和血液透析患者抗-HCVIgG阳性标本,采用RT-nested-PCR方法检测HCV RNA;用5′端非编码区(5′NCR)1、2、3、1b型特异性引物进行扩增和基因分型;用PEI-cellulose薄板层析法检测外周血单个核细胞的2-5AS。结果 三组人群HCV RNA感染率分别为1．21％、2．66％、38．84％;HCV基因型以1b亚型为主,1b及1b的相关型占91．67％。以ATP转化率表示2-5AS活性水平,1b型和1b相关的混合感染2-5AS活性显著升高。而其他基因型与健康对照组无明显差别。结论 2-5AS活性的基础水平显著升高可能是HCV1b亚型对干扰素反应低下的原因。     

2-(8′-羟基喹啉-5′-磺酸-7′-偶氮)-变色酸测定血清镁

目的建立简便、灵敏、试剂稳定性好的２－（８′－羟基喹啉－５′－磺酸－７′－偶氮）－变色酸（８Ｑ５ＳＡＣ）血清镁测定法。方法用国内合成的８Ｑ５ＳＡＣ作显色剂，ＥＧＴＡ掩蔽钙，在甘氨酸－ＮａＯＨ缓冲体系中以分光光度法测定血清镁。结果该法线性０～３ｍｍｏｌ／Ｌ，平均回收率９８．１％，变异系数１．９２％～２．９８％。与Ｃａｌｍａｇｉｔｅ法对照，ｒ＝０．９９６，Ｐ＞０．２；与ＭＴＢ法对照，ｒ＝０．９８３，Ｐ＞０．２；测试灵敏度分别是上述方法的２．５倍和４．３倍。结论该法简便、灵敏、试剂稳定性明显优于ＭＴＢ法和Ｃａｌ－ｍａｇｉｔｅ法，适于血清镁的常规手工分析和自动分析。     

2—（8′—羟基喹啉—5′—磺酸—7′—偶氮）—变色酸测定血清镁

目的 建立简便，灵敏，试剂稳定性好的２－（８′－羟基喹啉－５′－磺酸－７′－偶氮）－变色酸（８Ｑ５ＳＡＣ）血清镁测定法，方法 用国内合成的８Ｑ５ＳＡＣ作显色剂，ＥＧＴＡ掩蔽钙，在甘氨酸－ＮａＯＨ缓冲体系中以分光光度法测定血清镁。结果 该法线性０～３ｍｍｏｌ／Ｌ，平均回收率９８．１％，变异系数１．９２％～２．９８％，与Ｃａｌｍａｇｉｔｅ法对照，ｒ＝０．９９６，Ｐ〉０．２；与ＭＴＢ法对照，ｒ＝０．９８     

Inhibition of Herpesvirus Replication by 5-Substituted 4′-Thiopyrimidine Nucleosides

A series of 4′-thionucleosides were synthesized and evaluated for activities against orthopoxviruses and herpesviruses. We reported previously that one analog, 5-iodo-4′-thio-2′-deoxyuridine (4′-thioIDU), exhibits good activity both in vitro and in vivo against two orthopoxviruses. This compound also has good activity in cell culture against many of the herpesviruses. It inhibited the replication of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus with 50% effective concentrations (EC₅₀s) of 0.1, 0.5, and 2 μM, respectively. It also inhibited the replication of human cytomegalovirus (HCMV) with an EC₅₀ of 5.9 μM but did not selectively inhibit Epstein-Barr virus, human herpesvirus 6, or human herpesvirus 8. While acyclovir-resistant strains of HSV-1 and HSV-2 were comparatively resistant to 4′-thioIDU, it retained modest activity (EC₅₀s of 4 to 12 μM) against these strains. Some ganciclovir-resistant strains of HCMV also exhibited reduced susceptibilities to the compound, which appeared to be related to the specific mutations in the DNA polymerase, consistent with the observed incorporation of the compound into viral DNA. The activity of 4′-thioIDU was also evaluated using mice infected intranasally with the MS strain of HSV-2. Although there was no decrease in final mortality rates, the mean length of survival after inoculation increased significantly (P < 0.05) for all animals receiving 4′-thioIDU. The findings from the studies presented here suggest that 4′-thioIDU is a good inhibitor of some herpesviruses, as well as orthopoxviruses, and this class of compounds warrants further study as a therapy for infections with these viruses.Iterative medicinal chemistry studies can optimize molecules for the inhibition of viral enzymes and yield molecules that are highly effective and remarkably specific. This approach, as well as others, has led to the development of highly selective therapies for the treatment of viral infections and has been reviewed previously (5, 9, 20). While these therapeutic agents have excellent activities against the target enzymes from specific viruses, they can have reduced or poor efficacy against other closely related viruses. Thus, it is important to assess the activities of candidate molecules against an array of viruses to determine their spectra of activity. The development of compounds with activities against many viruses is challenging, yet essential, given the vast array of viral pathogens that cause human disease. A broad spectrum of activity is particularly important in the development of therapies for infections caused by emerging pathogens.Nucleoside phosphonate antiviral agents have been approved for the treatment of infections with hepatitis B virus and human immunodeficiency virus and are under development for infections caused by hepatitis C virus and DNA viruses (6). Cidofovir (CDV) is one such compound that has activity against many viruses, and recently, alkoxyalkyl ester prodrug forms of CDV have been reported to be active at concentrations up to 3 orders of magnitude lower than the active concentrations of the parent compound (36). The compound CMX001 (hexadecyloxypropyl CDV) has excellent activity against adenoviruses, orthopoxviruses, and polyomaviruses, as well as the herpesviruses (13, 18, 32, 36). Equally important is the increased oral bioavailability conferred by the alkoxyalkyl substituent (1, 3, 17, 30), which appears to reduce drug exposure in the mouse kidney and may reduce the potential of the drug for renal toxicity (4). While the discovery of truly broad spectrum antiviral drugs will be difficult, the development of CMX001 represents a significant advance.Previously, the efficacies of a series of nucleoside analogs against vaccinia and cowpox viruses were evaluated and a number of thymidine analogs that have good activities against these viruses were identified (8, 26). This result was intriguing since the orthopoxviruses encode thymidine kinase (TK) homologs that are closely related to the human cytosolic TK (TK1) (15). The TK homologs encoded by the orthopoxviruses and the human cytosolic TK are both type II TK homologs, which are characterized by a number of features including their homotetrameric structures and their comparatively narrow substrate specificities (2). Some herpesviruses encode distinct type I TK enzymes, which are active as dimers and have broader substrate specificities that include thymidine, cytidine, and even some purine analogs such as acyclovir (ACV) (16). Results from genetic studies with orthopoxvirus and herpesvirus TK mutants suggested that these enzymes could confer sensitivity to a few thymidine analogs, presumably through selective phosphorylation to the level of the monophosphate (26, 29). This result was confirmed subsequently by the findings of enzymatic studies, which showed that the orthopoxvirus and herpesvirus TK enzymes share the ability to catalyze the phosphorylation of certain thymidine analogs, some of which are not substrates for the cellular cytosolic TK (25). This group of analogs includes a few 2′-deoxyuridine analogs with large substituents in the 5 position (25). The carbocyclic analog N-methanocarbathymidine has also been reported previously to exhibit good activity against both the orthopoxvirus and herpesvirus families (26) and is selectively phosphorylated by each of the TK homologs encoded by these viruses (data not presented). The unexpected finding that orthopoxvirus TK homologs possess broader substrate specificities than the human cytosolic TK homolog was interesting and suggested a potential avenue for the development of therapies for infections with these viruses. Importantly, the overlapping specificities of vaccinia virus TK and herpes simplex virus (HSV) TK also suggested that it might be possible to identify additional thymidine analogs with spectra of activity that included both the orthopoxviruses and some of the human herpesviruses (HHVs). Compounds with this property would be desirable since therapies for herpesvirus infections are more viable economically than those for orthopoxvirus infections, which may help drive the continued development of these compounds. Ideally, they should also retain activity against drug-resistant strains of these viruses.A series of 2′-deoxy-4′-thiopyrimidine nucleosides were synthesized previously and reported to be active against human cytomegalovirus (HCMV) (33). Other related analogs identified by Rahim and colleagues were also reported to have good activities against varicella-zoster virus (VZV), as well as HSV (31). One analog in this series, 5-iodo-4′-thio-2′-deoxyuridine (4′-thioIDU), also exhibited activity against HCMV. Recently, we described the antiviral activities of this and related molecules against vaccinia virus and cowpox virus (19). The most active analog was 4′-thioIDU, which inhibited viral replication in vitro at submicromolar concentrations. This compound also significantly reduced the mortality of mice infected with cowpox virus when administered orally at concentrations of 5 mg/kg of body weight and when therapy was initiated as late as 4 days after infection. The activity of the compound was shown to be largely dependent on the orthopoxvirus TK homologs, so we examined the activities of this and related compounds against HSV-2 and confirmed that 4′-thioIDU had the highest activity. The spectrum of activity of this compound also included HSV-1, VZV, and cytomegalovirus, but selective activity against the other HHVs was not observed. Immunofluorescence studies showed that the compound was specifically phosphorylated in infected cells, incorporated into viral DNA, and dependent on viral TK activity. However, in contrast to previous results that indicated good in vivo efficacy against two orthopoxviruses, the findings of the present study revealed that the compound did not significantly reduce the mortality of mice infected intranasally with HSV-2.