Increase of Serum Kallikrein-8 Level After Long-term Telbivudine Treatment.

BACKGROUND/AIM: Our previous cDNA microarray study revealed increased cellular mRNA levels of a panel of genes, including kallikrein-8 (KLK8), after long-term telbivudine treatment in chronic hepatitis B patients. The aim of this study was to verify whether serum protein levels of KLK8, a cancer-related enzyme, are indeed increased after telbivudine treatment. PATIENTS AND METHODS: A total of 83 chronic hepatitis B patients receiving telbivudine for >2 years were retrospectively analyzed. Serum KLK8 protein and estimated glomerular filtration rate (eGFR) changes were compared before and after treatment. RESULTS: Both serum KLK8 protein and eGFR increased significantly after long-term telbivudine treatment (paired t-test: KLK8, p<0.001; eGFR, p=0.001). No direct correlation was found between KLK8 increase and eGFR change. However, eGFR change was positively associated with post-treatment KLK8 levels following adjustment for body height (p<0.001). CONCLUSION: Telbivudine treatment resulted in increased levels of serum KLK8 protein. Furthermore, eGFR increase was associated with body height-adjusted, post-treatment KLK8 levels.

Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), a mini-chromosome essential for HBV replication, is supposed to be resistant to nucleos(t)ide analogue treatment. We investigated the effect of long-term nucleos(t)ide analogue treatment on cccDNA. METHODS: Among 129 patients who had been enrolled in previous international nucleos(t)ide analogue clinical trials and had liver biopsies at baseline and one year after treatment, we recruited 43 patients on long-term continuous treatment for 72 to 145months for a third liver biopsy. Serum HBV DNA, hepatitis B surface antigen (HBsAg) levels, total intrahepatic HBV DNA (ihHBV DNA), cccDNA, HBV pregenomic RNA (pgRNA) as well as histologic changes were examined. RESULTS: At the time of the third biopsy, serum HBV DNA levels were undetectable in all but one patient. The median levels of HBsAg, ihHBV DNA, and cccDNA were 2.88logIU/ml, 0.03copies/cell, and 0.01copies/cell, respectively. Compared to baseline levels, there was reduction of HBsAg levels by 0.54log (71.46%), ihHBV DNA levels by 2.81log (99.84%), and cccDNA levels by 2.94log (99.89%), with 49% having cccDNA levels below the detection limit. One patient had undetectable HBsAg. The median pgRNA level, measured only in the third biopsy, was 0.021copies/cell, with 40% of patients having undetectable pgRNA. CONCLUSIONS: Long-term nucleos(t)ide analogue treatment induced marked depletion of cccDNA in the majority of patients while serum HBsAg levels, though reduced, were detectable in all but one patient. Whether cccDNA depletion is sustained and associated with better patient outcome requires further study. LAY SUMMARY: It is generally presumed that a form of hepatitis B virus DNA, called covalently closed circular DNA (cccDNA), which hides inside the nuclei of liver cells of patients with chronic hepatitis B, cannot be reduced by antiviral treatment. The present study showed that with prolonged treatment (median period 126months), cccDNA can be markedly reduced, with 49% of liver biopsies having undetectable cccDNA. This suggests that viral replication capacity would be very low after prolonged antiviral treatment.

Telbivudine therapy for chronic hepatitis B: A journey to identify super-responders and to optimize treatment using the roadmap model.

Hepatitis B virus (HBV) infection is one of the most serious health problems worldwide with a high risk for cirrhosis and liver cancer. Several antiviral agents have been approved for the treatment of chronic hepatitis B, leading to a rapid reduction in HBV DNA and normalization of serum alanine aminotransferase levels. Telbivudine, a potent inhibitor of HBV replication, has been shown to be well tolerated. Because of the emergence of drug resistance, optimization strategies for telbivudine therapy have been shown to improve patient responses. Optimal baseline characteristics in so-called super-responders have been used to predict the virological response. Baseline HBV DNA levels < 9 log10 copies/mL (2 × 108 IU/mL) or alanine aminotransferase levels of more than or equal to twofold the upper limit of normal in HBeAg-positive patients and HBV DNA < 7 log10 copies/mL (2 × 106 IU/mL) in HBeAg-negative patients were strong predictors for virological response. In addition, the roadmap model, based on early virological response at week 24 of therapy, is considered as a powerful tool to identify patients at risk of treatment failure (HBV DNA ≥ 300 copies/mL, i.e. 60 IU/mL) and to reduce the risk of antiviral resistance. When considering pre-treatment characteristics and on-treatment responses, telbivudine may provide physicians with a wide choice of options to effectively treat patients with chronic hepatitis B, especially those with or at risk of renal impairment, or women of childbearing age.

Inhibition of Nucleotide Synthesis Targets Brain Tumor Stem Cells in a Subset of Glioblastoma.

Inhibition of both the de novo (DNP) and salvage (NSP) pathways of nucleoside synthesis has been demonstrated to impair leukemia cells. We endeavored to determine whether this approach would be efficacious in glioblastoma. To diminish nucleoside biosynthesis, we utilized compound DI-39, which selectively targets NSP, in combination with thymidine (dT), which selectively targets DNP. We employed in vitro and ex vivo models to determine the effects of pretreatment with dT + DI-39 on brain tumor stem cells (BTSC). Here, we demonstrate that this combinatorial therapy elicits a differential response across a spectrum of human patient-derived glioblastoma cultures. As determined by apoptotic markers, most cultures were relatively resistant to treatment, although a subset was highly sensitive. Sensitivity was unrelated to S-phase delay and to DNA damage induced by treatment. Bioinformatics analysis indicated that response across cultures was associated with the transcription factor PAX3 (associated with resistance) and with canonical pathways, including the nucleotide excision repair pathway, PTEN (associated with resistance), PI3K/AKT (associated with sensitivity), and ErbB2-ErbB3. Our in vitro assays demonstrated that, in sensitive cultures, clonal sphere formation was reduced upon removal from pretreatment. In contrast, in a resistant culture, clonal sphere formation was slightly increased upon removal from pretreatment. Moreover, in an intracranial xenograft model, pretreatment of a sensitive culture caused significantly smaller and fewer tumors. In a resistant culture, tumors were equivalent irrespective of pretreatment. These results indicate that, in the subset of sensitive glioblastoma, BTSCs are targeted by inhibition of pyrimidine synthesis. Mol Cancer Ther; 15(6); 1271-8. ©2016 AACR.

Efficacy, safety, bone and metabolic effects of HIV nucleoside reverse transcriptase inhibitor BMS-986001 (AI467003): a phase 2b randomised, controlled, partly blinded trial.

BACKGROUND: BMS-986001 is a thymidine analogue nucleoside reverse transcriptase inhibitor (NRTI) designed to maintain in-vitro antiviral activity while minimising off-target effects. We assessed the efficacy and safety of BMS-986001 versus tenofovir disoproxil fumarate in treatment-naive patients with HIV-1. METHODS: In this phase 2b, randomised, active-controlled trial (AI467003), we recruited treatment-naive (no current or previous exposure to an antiretroviral drug for >1 week) adults (aged at least 18 years) with HIV-1 from 47 sites across Asia, Australia, Europe, North America, South Africa, and South America. Patients with plasma HIV-1 RNA greater than 5000 copies per mL and CD4 counts greater than 200 cells per µL were randomly assigned (2:2:2:3) to receive BMS-986001 100 mg, 200 mg, or 400 mg once a day or to receive tenofovir disoproxil fumarate 300 mg once a day; each allocation was given with efavirenz 600 mg once a day and lamivudine 300 mg once a day. Both patients and investigators were masked to BMS-986001 dose (achieved with similar looking placebo tablets), but not allocation up to and including week 48. The primary endpoints were the proportion of patients with plasma HIV-1 RNA less than 50 copies per mL and safety events (serious adverse events and adverse events leading to discontinuation) through week 24; the main analysis was with a modified intention-to-treat population. Resistance analysis was a secondary endpoint, and additional safety parameters were exploratory endpoints. This trial is registered with ClinicalTrials.gov, number NCT01489046, and the European Clinical Trials Database, number EudraCT 2011-003329-89. FINDINGS: Patients were recruited between Jan 25, 2012, and Oct 3, 2012; 757 patients were assessed for eligibility and 301 were randomly assigned to receive either BMS-986001 once a day (67 patients to 100 mg, 67 to 200 mg, and 66 to 400 mg) or tenofovir disoproxil fumarate (n=101). 297 patients received at least one dose of study drug. At week 24, 57 (88%) of 65 patients for whom there were data in the 100 mg group, 54 (81%) of 67 in the 200 mg group, 62 (94%) of 66 in the 400 mg group achieved HIV-1 RNA less than 50 copies per mL, compared with 88 (89%) of 99 in the tenofovir disoproxil fumarate group (modified intention-to-treat population). BMS-986001 was generally well tolerated through week 48. Two patients had BMS-986001-related serious adverse events (atypical drug eruption and thrombocytopenia) and two in the tenofovir disoproxil fumarate group had study drug-related serious adverse events (potential drug-induced liver injury and depression or lipodystrophy) that led to discontinuation. NRTI resistance-associated mutations were reported in four (2%) of 198 patients, and non-NRTI mutations in 17 (9%) of 198 patients receiving BMS-986001 versus none of 99 and one (1%) of 99 patients receiving tenofovir disoproxil fumarate, respectively. Compared with tenofovir disoproxil fumarate, individuals in the BMS-986001 groups showed a smaller decrease in lumbar spine and hip bone mineral density but greater accumulation of limb and trunk fat, subcutaneous and visceral adipose tissue, and increased total cholesterol. INTERPRETATION: BMS-986001 had similar efficacy to that of tenofovir disoproxil fumarate and was associated with a smaller decrease in bone mineral density; however, greater resistance and gains in both peripheral and central fat accumulation were recorded for the investigational drug. Bristol-Myers Squibb has discontinued its involvement in the development of BMS-986001, and future decisions on development will be made by Oncolys BioPharma. FUNDING: Bristol-Myers Squibb.

Comparison of renal safety and efficacy of telbivudine, entecavir and tenofovir treatment in chronic hepatitis B patients: real world experience.

This study aims to assess the nephrotoxicity and efficacy of tenofovir disoproxil fumarate (tenofovir), telbivudine and entecavir. A retrospective study of 587 patients with chronic hepatitis B treated with tenofovir (n = 170), telbivudine (n = 184) and entecavir (n = 233) for at least 1 year. Renal function and efficacy were assessed. The estimated glomerular filtration rate (eGFR) decreased significantly in the tenofovir group after a mean of 17 months treatment (from 92.2 to 85.6 mL/min/1.73 m(2), p < 0.001), but increased in the telbivudine group after a mean of 32 months of treatment (from 86.1 to 95 mL/min/1.73 m(2), p < 0.001). There was no significant change in eGFR in the entecavir group after a mean of 44 months. By multivariate analysis, pre-existing renal insufficiency (p = 0.003), tenofovir (p = 0.007) and diuretic treatment (p = 0.001) were independent predictors for renal function deterioration. Cumulative virological breakthrough was 0% in tenofovir after 2 years, 3.4% in entecavir after 7 years and 22.9% in telbivudine after 5 years. Liver cirrhosis (p = 0.008) and virological breakthrough (p = 0.040) were independently associated with increased risk of hepatocellular carcinoma development. Tenofovir may lead to deterioration in renal function as assessed by serial eGFR measurements. Although telbivudine appeared to be associated with an improvement in eGFR, it was associated with high rates of virological breakthrough, which was an independent risk factor for HCC development. With low rates of virological breakthrough and preservation of renal function, entecavir could be the best choice among these three agents.

Monotherapy for hepatitis B infection: a review of treatment options.

Chronic hepatitis B (CHB) is a global health problem, causing liver failure, cirrhosis and hepatocellular carcinoma. CHB treatment aims to prevent liver-related complication. The treatment of CHB infection includes monotherapy with either interferons (IFNs) or nucleos(t)ide (NUC) analogs. IFNs have moderate antiviral effects, and their use is limited by side effects. With the availability of NUCs, IFN-intolerant and decompensated cirrhotic patients began to be treated. Lamivudine and telbivudine, nucleoside analogs, have low genetic barrier to resistance. Adefovir, a nucleotide analog, has moderate potency and potential nephrotoxicity. Entecavir and tenofovir, with their high potency, high genetic barrier to resistance and favorable safety profile are the standard of care in CHB treatment. Long-term use of NUCs with maintained viral suppression results in a decrease in liver-related complications.

Long-Term Telbivudine Treatment Results in Resolution of Liver Inflammation and Fibrosis in Patients with Chronic Hepatitis B.

INTRODUCTION: The long-term goal of chronic hepatitis B (CHB) treatment is improvement of liver disease and prevention of cirrhosis. The aim of this study was to assess whether prolonged telbivudine treatment improves liver inflammation and fibrosis. The primary objective was to evaluate the proportion of patients with absence/minimal inflammation (Knodell necroinflammatory score ≤3) on liver biopsy at Year 5. METHODS: Fifty-seven patients aged 16-70 years with a clinical history of CHB and active viral replication (38 hepatitis B e antigen [HBeAg] positive and 19 HBeAg negative) were followed for 6 years: 33 received telbivudine 600 mg/day continuously for 5 years; 24 received lamivudine 100 mg/day for 2 years and then telbivudine for 3 years. Liver biopsies were taken pre-treatment and after 5 years of treatment. RESULTS: At baseline, mean (standard deviation) serum hepatitis B virus (HBV) DNA load was 8.5 (1.7) log10 copies/mL, Knodell necroinflammatory score was 7.6 (2.9), and Ishak fibrosis score was 2.2 (1.1). After antiviral treatment (median duration: 261 weeks), liver histology improved with increased proportions of patients with absence/minimal liver inflammation (Knodell necroinflammatory score ≤3), from 16% (9/57) at baseline to 98% (56/57), and absence/minimal fibrosis (Ishak score ≤1), from 25% (14/57) at baseline to 84% (48/57). At Year 5, HBV DNA load was <300 copies/mL for all patients; cumulative HBeAg loss and seroconversion rates were 88% and 77%, respectively. At Year 6, 95% of patients with abnormal baseline glomerular filtration rate (60-90 mL/min/1.73 m(2)) improved to normal GFR (>90 mL/min/1.73 m(2)). CONCLUSION: Long-term telbivudine treatment with profound and durable viral suppression significantly improved liver histology, thus achieving the long-term goals of CHB treatment. FibroScan(®) results after 5 and 6 years of treatment (in almost 20% of patients) were consistent with this information. FUNDING: Novartis and National Science and Technology Major Project (2012ZX10002003). TRIAL REGISTRATION: ClinicalTrials.gov # NCT00877149.

Evaluation of TK1 targeting carboranyl thymidine analogs as potential delivery agents for neutron capture therapy of brain tumors.

In this report we describe studies with N5-2OH, a carboranyl thymidine analog (CTA), which is a substrate for thymidine kinase 1 (TK1), using the F98 rat glioma model. In vivo BNCT studies have demonstrated that intracerebral (i.c.) osmotic pump infusion of N5-2OH yielded survival data equivalent to those obtained with i.v. administration of boronophenylalanine (BPA). The combination of N5-2OH and BPA resulted in a modest increase in MST of F98 glioma bearing rats compared to a statistically significant increase with the RG2 glioma model, as has been previously reported by us (Barth et al., 2008). This had lead us to synthesize a second generation of CTAs that have improved in vitro enzyme kinetics and in vivo tumor uptake (Agarwal et al., 2015).

Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection

Background: Research has shown that hepatitis B virus (HBV) genotypes are closely linked to the clinical manifestations, treatment, and prognosis of the disease. Objective: To study the association between genotype and drug-resistant HBV mutations in 620 Chinese patients with chronic HBV infection. Methods: HBV DNA levels were determined using real-time quantitative PCR in plasma samples. Microarrays were performed for the simultaneous detection of HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations. A portion of the samples analyzed using microarrays was selected randomly and the data were confirmed using direct DNA sequencing. Results: Most samples were genotype C (471/620; 76.0%), followed by genotype B (149/620; 24.0%). Among the 620 patient samples, 17 (2.7%) had nucleotide analogs (NA) resistance-related mutations. Of these, nine and eight patients carried lamivudine (LAM)-/telbivudine (LdT)-resistance mutations (rtL180M, rtM204I/V) and adefovir (ADV)-resistance mutations (rtA181T/V, rtN236T), respectively. No patients had both lamivudine (LAM)- and either ade-fovir (ADV) or entecavir (ETV) resistance mutations. Additionally, out of the 620 patient samples, 64.0% (397/620) were also detected with the precore stop-codon mutation (G1896A) by microarray assay. Conclusion: The results of the current study revealed that the prevalence of nucleotide analogs (NA)-resistance in Chinese hospitalized HBV-positive patients was so low that intensive nucleotide analogs (NA)-resistance testing before nucleotide analog (NA) treatment might not be required. In addition, the present study suggests that chronic HBV patients with genotype C were infected with fitter viruses and had an increased prevalence of nucleotide analogs (NA)-resistance mutations compared to genotype B virus. .

Maternal dietary uridine causes, and deoxyuridine prevents, neural tube closure defects in a mouse model of folate-responsive neural tube defects.

BACKGROUND: Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure. OBJECTIVE: We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model. DESIGN: Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA. RESULTS: Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations. CONCLUSIONS: Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.

Is there an association of hepatitis B virus infection with minimal change disease of nephrotic syndrome? A clinical observational report.

The rate of hepatitis B virus (HBV) infection is high in the Chinese population, and the implications of HBV infection are widely recognized, and membranous nephropathy is the most common renal lesion to be associated with HBV infection. Minimal change disease (MCD) is one of the most important histopathological characteristics in patients with nephrotic syndrome. There is no any study to report that HBV infection is associated with the etiology of MCD. Herein, we report four MCD patients with HBV infection and speculate that there is an association of HBV infection with the pathological type of MCD. In this study, we also reported the treatment schedule for these four MCD patients, and found that the anti-virus alone and combination of anti-virus with immunosuppressive agent could obtain a benefit for MCD patients with HBV infection. However, a well-designed study should be performed to confirm this association.

Growth and development of children prenatally exposed to telbivudine administered for the treatment of chronic hepatitis B in their mothers.

OBJECTIVES: We studied the growth and development of children prenatally exposed to telbivudine used to treat chronic hepatitis B virus (HBV) infection in their mothers. METHODS: Maternal abnormalities during pregnancy and delivery and infant congenital anomalies, physical development status, developmental quotient (DQ), HBV vertical transmission status, and HBV vaccination outcomes of 54 infants were evaluated (2010-2013). RESULTS: No fetal abnormalities were observed during pregnancy or delivery. Postpartum, three infants (5.56%) had abnormalities: ankyloglossia, cutaneous hemangioma, and vaginal canal leak. Height and weight were within the normal range at birth and at 6 weeks, but were higher than the reference at 12 months (p<0.05). Body mass index increased gradually with age (p<0.05). DQ scores were normal (84.81%, 229/270) in 37 children (68.52%), abnormal or suspicious for a developmental delay (15.19%, 41/270) in 17 children (31.48%), and indicated a developmental delay (4.07%, 11/270) in seven children (12.96%). There were no significant differences in developmental delay between children prenatally exposed to telbivudine and controls (p>0.05). HBV vertical transmission was successfully blocked in all infants. The effective HBV vaccination rate was 98.15% (53/54). CONCLUSIONS: The growth and development of children prenatally exposed to telbivudine was normal, indicating that telbivudine treatment during pregnancy is safe and effective.

Comparing the efficacy and clinical outcome of telbivudine and entecavir naïve patients with hepatitis B virus-related compensated cirrhosis.

BACKGROUND AND AIM: There is limited data on the efficacy and outcome of telbivudine (LdT) therapy in patients with chronic hepatitis B and compensated cirrhosis. We evaluated LdT as first-line therapy in these patients and compared with those treated with entecavir (ETV). METHODS: We consecutively enrolled 88 chronic hepatitis B patients with compensated cirrhosis primarily treated with LdT at least for 2 years or less than 2 years but developed resistance, and evaluated the efficacy and clinical outcomes. Meanwhile, we matched a control group who treated with ETV for comparison. RESULTS: In LdT group, alanine aminotransferase normalization (65.8%), hepatitis B e antigen seroconversion (39.8%), hepatitis B virus (HBV) DNA undetectablility (71.6%), and virologic resistance (23.9%) were noted after 2 years treatment. Compared with ETV group, there were significant difference in HBV DNA undetectablility (P < 0.001) and virologic resistance (P < 0.001). In addition, the decline of serum hepatitis B surface antigen levels, hepatocellular carcinoma development, mortality, disease progression, and the change of renal function were similar. Cox regression analysis showed that pretreatment low albumin level and high model for end-stage liver disease scores were risk factors for disease progression. CONCLUSIONS: These results indicated that although LdT and ETV are similar in clinical outcomes for patients with HBV-related compensated cirrhosis, LdT still had lower HBV undetectablility and higher resistant rate after 2 years treatment, which was a challenge for being as first-line therapy in these patients who need lifelong therapy.

A comparison of efficacy and safety of 2-year telbivudine and entecavir treatment in patients with chronic hepatitis B: a match-control study.

There are limited data comparing the clinical outcomes between telbivudine and entecavir. We consecutively enrolled 115 telbivudine-naive and 115 entecavir-naive chronic hepatitis B patients, who were matched for age, sex, hepatitis B e antigen (HBeAg) status and cirrhosis, and treated for at least 2 years or less than 2 years but had developed resistance. Except for the rate of HBeAg seroconversion, which was similar, patients in the entecavir group had better clinical outcomes than those in the telbivudine group for alanine aminotransferase normalization (85.2% vs 78.4%, p <0.048), undetectable HBV DNA (96.5% vs 74.8%, p <0.001), and viral resistance (0.9% vs 21.7%, p <0.001) after 2 years of treatment, After applying roadmap or super-responders concepts, entecavir still had better outcomes than telbivudine in undetectable HBV DNA and viral resistance. The cumulative incidence of hepatocellular carcinoma development was similar between telbivudine-naive and entecavir-naive patients (p 0.565). In renal function analysis, there were significantly more patients with estimated glomerular filtration rate (eGFR) category improvement in both the telbivudine and entecavir groups at year 1 (p 0.006 and p 0.047, respectively). The rate of virological improvement was significantly higher with entecavir than with telbivudine after 2 years of treatment, whether applying the concepts of roadmap or super-responders. The incidence of hepatocellular carcinoma was similar between telbivudine and entecavir. Both telbivudine and entecavir were associated with eGFR improvement, especially in patients with renal insufficiency.

Cellular influx, efflux, and anabolism of 3-carboranyl thymidine analogs: potential boron delivery agents for neutron capture therapy.

3-[5-{2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]thymidine (N5-2OH) is a first generation 3-carboranyl thymidine analog (3CTA) that has been intensively studied as a boron-10 ((10)B) delivery agent for neutron capture therapy (NCT). N5-2OH is an excellent substrate of thymidine kinase 1 and its favorable biodistribution profile in rodents led to successful preclinical NCT of rats bearing intracerebral RG2 glioma. The present study explored cellular influx and efflux mechanisms of N5-2OH, as well as its intracellular anabolism beyond the monophosphate level. N5-2OH entered cultured human CCRF-CEM cells via passive diffusion, whereas the multidrug resistance-associated protein 4 appeared to be a major mediator of N5-2OH monophosphate efflux. N5-2OH was effectively monophosphorylated in cultured murine L929 [thymidine kinase 1 (TK1(+))] cells whereas formation of N5-2OH monophosphate was markedly lower in L929 (TK1(-)) cell variants. Further metabolism to the di- and triphosphate forms was not observed in any of the cell lines. Regardless of monophosphorylation, parental N5-2OH was the major intracellular component in both TK1(+) and TK1(-) cells. Phosphate transfer experiments with enzyme preparations showed that N5-2OH monophosphate, as well as the monophosphate of a second 3-carboranyl thymidine analog [3-[5-(o-carboran-1-yl)pentan-1-yl]thymidine (N5)], were not substrates of thymidine monophosphate kinase. Surprisingly, N5-diphosphate was phosphorylated by nucleoside diphosphate kinase although N5-triphosphate apparently was not a substrate of DNA polymerase. Our results provide valuable information on the cellular metabolism and pharmacokinetic profile of 3-carboranyl thymidine analogs.

Meta-analysis: the impact of oral anti-viral agents on the incidence of hepatocellular carcinoma in chronic hepatitis B.

BACKGROUND: Five oral nucleos(t)ide analogues are available to treat chronic hepatitis B (CHB). With the availability of newer agents, their efficacy on incidence of hepatocellular carcinoma (HCC) is not well described. AIM: To determine the efficacy of oral anti-viral agents in reducing HCC risk in relationship with other known factors. METHODS: Published studies of at least 20 CHB patients treated with an oral anti-viral agent and followed for >2 years were analysed for incidence of HCC per 100 person years follow-up. RESULTS: Pooled homogeneous data from six studies showed lamivudine (LAM) treatment (n = 3306) to reduce HCC risk by 51% compared with no treatment (n = 3585) (3.3 vs. 9.7 per 100 person years, P < 0.0001). Pooled data from 49 studies (23 with LAM; 16 with adefovir; and 10 with entecavir, tenofovir or telbivudine) of 10 025 treated patients showed HCC incidence of 1.3 per 100 person years, independent of the agent used. Patient age >50 years and hepatitis B virus-DNA detectability at HCC diagnosis increased risk of HCC by twofold with a 10-fold higher risk among patients with cirrhosis compared with chronic hepatitis. Meta-regression showed patient age, study location (Eastern vs. Western) and type of study (randomised or not) contributed to heterogeneity. CONCLUSIONS: Lamivudine treatment significantly reduces the incidence of HCC compared with no treatment. However, HCC still develops at a rate of 1.3 per 100 patient years in CHB patients receiving an oral anti-viral agent. This finding highlights the need for continued HCC surveillance, particularly in CHB patients with inadequate viral suppression, older age and cirrhosis.

A randomized controlled trial of sequential pegylated interferon-α and telbivudine or vice versa for 48 weeks in hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND: Short-term treatment for hepatitis B e antigen (HBeAg)-negative chronic hepatitis B remains unsatisfactory. The aim of our study was to compare the efficacy and safety of two sequential regimens of pegylated interferon (PEG-IFN)-α and telbivudine (LdT). METHODS: Adult patients with biopsy-proven HBeAg-negative chronic hepatitis B, elevated alanine aminotransferase (ALT) and serum HBV DNA ≥ 2,000 IU/ml were randomized 1:1 at baseline to receive PEG-IFN 180 µg/week for 24 weeks followed by LdT 600 mg/day for 24 weeks (PEG-IFN first), or vice versa (LdT first), plus 24-week follow-up; individuals with HCV, HDV or HIV coinfections and lamivudine resistance were excluded. Primary end points were serum HBV DNA<2,000 IU/ml and normal ALT at week 72. RESULTS: A total of 30 patients (86% male, median age 48 years) were enrolled: mean ±sd baseline serum HBV DNA was 5.56 ± 1.4 log IU/ml and ALT was 2.9 ± 2.5× upper limit of normal. At end of follow-up (week 72), HBV DNA<2,000 IU/ml was achieved in 13.3% of participants in the PEG-IFN first group versus 46.7% of those in the LdT first group (P=0.046). Mean ±sd ALT levels were significantly lower in the LdT first group (1.3 ± 0.9 versus 3.2 ± 2.7× upper limit of normal; P=0.03). PEG-IFN dose was reduced in 2 (7%) patients and 1 (7%) patient dropped out due to myalgia. CONCLUSIONS: Sequential treatment with 24 weeks PEG-IFN followed or preceded by 24 weeks of LdT is safe. Virological response rate at week 72 was significantly higher in patients treated with LdT followed by PEG-IFN than vice versa. A sequential antiviral regimen of LdT followed by PEG-IFN, if confirmed in larger series, could improve response rates compared with standard PEG-IFN monotherapy.

Cell fate control gene therapy based on engineered variants of human deoxycytidine kinase.

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

Glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction: clinical and pharmacologic factors affecting outcome.

PURPOSE: To assess the role of the recombinant bacterial enzyme, glucarpidase (carboxypeptidase-G(2)), leucovorin, and thymidine in the management and outcome of patients with high-dose methotrexate (HDMTX) -induced nephrotoxicity. METHODS: Patients with HDMTX-induced nephrotoxicity received one to three doses of intravenous (IV) glucarpidase and leucovorin rescue. The initial cohort (n = 35) also received thymidine by continuous IV infusion. Subsequently, thymidine was restricted to patients with prolonged exposure (> 96 hours) to methotrexate (MTX) or with substantial MTX toxicity at study entry. Plasma MTX, leucovorin, and 5-methyltetrahydrofolate (5-mTHF) concentrations were measured pre- and postglucarpidase. Toxicities were monitored, and logistic regression analysis was used to assess the relationship of baseline characteristics to the development of severe toxicity and death. RESULTS: Glucarpidase was administered at a median of 96 hours (receiving thymidine, n = 44) and 66 hours (not receiving thymidine, n = 56) after the start of the MTX infusion. Plasma MTX concentrations decreased within 15 minutes of glucarpidase by 98.7%. Plasma 5-mTHF concentrations also decreased more than 98% after administration of glucarpidase. Of 12 deaths, six were directly attributed to irreversible MTX toxicity. Presence of grade 4 toxicity before administration of glucarpidase, inadequate initial increase in leucovorin dosing, and administration of glucarpidase more than 96 hours after the start of the MTX infusion were associated with development of grade 4 and 5 toxicity. CONCLUSION: Early intervention with the combination of leucovorin and glucarpidase is highly effective in patients who develop HDMTX-induced renal dysfunction. Severe toxicity and mortality occurred in patients in whom glucarpidase rescue was delayed and occurred despite thymidine administration.