Protecting-group-free synthesis of clevudine (L-FMAU), a treatment of the hepatitis B virus.

Unnatural nucleoside analogues are valuable research and clinical tools as antiproliferative, antibacterial or antiviral agents. In this context, clevudine (L-FMAU), a reverse transcriptase inhibitor, is currently used for the treatment of the hepatitis B virus. Herein, we describe a new strategy for the preparation of clevudine. Starting from 2-deoxy-2-fluoro-D-galactopyranose, we developed the shortest and highest yield synthesis of this unnatural L-nucleoside. Key steps involve an iodine-promoted cyclization and oxidative cleavage to access the L-arabinofuranosyl scaffold.

Clevudine attenuates bleomycin-induced early pulmonary fibrosis via regulating M2 macrophage polarization.

Pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. It is a growing clinical problem which can result in breathlessness or respiratory failure and has an average life expectancy of 3 years from diagnosis. Predominantly accumulation of M2 macrophages accelerates fibrosis progression by secreting multiple cytokines that promote fibroblast to myofibroblast transition and aberrant wound healing of epithelial cells. Targeting activated macrophages to inhibit the pro-fibrotic phenotype is considered as an approach for the potential treatment of PF. Clevudine is s a purine nucleoside analogue which in an oral formulation is approved for treatment of patients with hepatitis B virus (HBV). Here, we found that clevudine is capable of suppressing pro-fibrotic phenotype (i.e., CD206, Arg1 and YM1) of M2 macrophages while enhancing anti-fibrotic phenotype (i.e., CD86, IL-6 and IL-10) by inhibiting PI3K/Akt signaling pathway. This effect further alleviates M2-induced myofibroblast activation and epithelial-to-mesenchymal transition (EMT), thus resulting in a decline of collagen deposition, pro-fibrotic cytokines secretion, with a concomitant recover ofpulmonary functions in vivo. Less infiltration of M2 macrophages between α-SMA + cells was also found in clevudine treated mice. Our findings indicate a potential anti-fibrotic effect of clevudine by regulating macrophage polarization and might be meaningful in clinical settings.

Simultaneous determination of cytosine arabinoside and its metabolite uracil arabinoside in human plasma by LC-MS/MS: Application to pharmacokinetics-pharmacogenetics pilot study in AML patients.

Purine analogs like aracytine (AraC) are a mainstay for treating acute myeloid leukemia (AML). There are marked differences in drug dosing and scheduling depending on the protocols when treating AML patients with AraC. Large inter-patient pharmacokinetics variability has been reported, and genetic polymorphisms affecting cytidine deaminase (CDA), the liver enzyme responsible for the conversion of Ara-C to inactive uracil arabinoside (AraU) could be a culprit for either life-threatening toxicities or poor efficacy related to substantial changes in plasma exposure levels among patients. The quantitative determination of Ara-C in plasma is challenging due the required sensitivity because of the short half-life of this drug (i.e., <10 min) and the metabolic instability in biological matrix upon sampling possibly resulting in erratic values. We developed and validated a liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) for the simultaneous determination of Ara-C and Ara-U metabolite in human plasma. After simple and rapid precipitation, analytes were successfully separated and quantitated over a 1-500 ng/ml range for Ara-C and 250-7500 ng/ml range for AraU. The performance and reliability of this method was tested as part of an investigational study in AML patients treated with low dose cytarabine and confirmed marked differences in drug exposure levels and metabolic ratio, depending on the CDA status of the patients. Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in AML patients with respect to their CDA phenotypes.

Journey of 2'-deoxy-2'-fluoro-5-methyl-1-ß-D-arabinofuranosyluracil (FMAU): from Antiviral Drug to PET Imaging Agent.

BACKGROUND: Developed as an antiviral drug in the 1960s and 1970s, the thymidine analogue 2'-deoxy-2'-fluoro-5-methyl-1-ß-D-arabinofuranosyluracil (FMAU) was translated to clinical application for treatment of herpes simplex virus infection. In phase I clinical trial of FMAU; however, patients experienced neurotoxicity at the pharmacological dose, and FMAU was withdrawn from the trial. More recently, FMAU has been developed as a tracer for positron emission tomography (PET) imaging in early detection of cancer through its binding to human thymidine kinase, which is upregulated in cancer cells. FMAU radiolabeled with 11C or 18F has been examined for PET imaging of tumor cell proliferation and DNA synthesis. Although many reports have been partially published on FMAU, systematic reviews outlining the historic development and imaging probe are lacking. This review is focused on the identification of kinases, the chemistry of FAMU and its application in cancer diagnosis and therapy assessment. OBJECTIVE: The aim of this study was to review the historic development of FMAU, from its synthetic development and antiviral activity studies to its radiolabeling and evaluate it as a PET imaging probe for the early detection of cancer and assessment of treatment response, including published reports on the clinical utility of 18F-FMAU. CONCLUSION: While FMAU was not successful as an antiviral agent, 18F-FMAU is a suitable radiotracer for early detection of cancer and assessment of response to therapy by PET. The process of clinical grade 18F-FMAU production requires further improvement. 18F-FMAU has high potential for clinical application, but further extensive studies are needed to establish this tracer in the diagnosis of various cancers and assessment of their response to therapy.

Clevudine Induced Mitochondrial Myopathy.

Clevudine was approved as an antiviral agent for hepatitis B virus, which showed marked, rapid inhibition of virus replication without significant toxicity. However, several studies have reported myopathy associated with clevudine therapy. Also, we experienced seven patients who suffered from myopathy during clevudine therapy. To characterize clevudine-induced myopathy, we collected previously reported cases of clevudine myopathy and analyzed all the cases including our cases. We searched electronic databases that were published in English or Korean using PubMed and KoreaMed. Ninety-five cases with clevudine myopathy, including our seven cases, were selected and analyzed for the demographic data, clinical features, and pathologic findings. The 95 patients with clevudine-induced myopathy comprised 52 women and 43 men aged 48.9 years (27-76 years). The patients received clevudine therapy for about 14.2 months (5-24 months) before the development of symptoms. Weakness mainly involved proximal extremities, especially in the lower extremities, and bulbar and neck weakness were observed in some cases (13.7%). Creatine kinase was elevated in the majority of patients (97.9%). Myopathic patterns on electromyography were observed in most patients examined (98.1%). Muscle biopsy presented patterns compatible with mitochondrial myopathy in the majority (90.2%). The weakness usually improved within about 3 months after the discontinuation of clevudine. Though clevudine has been known to be safe in a 6-month clinical trial, longer clevudine therapy for about 14 months may cause reversible mitochondrial myopathy. Careful clinical attention should be paid to patients with long-term clevudine therapy.

Efficacy of Nucleot(s)ide Analogs Therapy in Patients with Unresectable HBV-Related Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis.

Aim. To determine whether nucleot(s)ide analogs therapy has survival benefit for patients with HBV-related HCC after unresectable treatment. Method. A systematic search was conducted through seven electronic databases including PubMed, OVID, EMBASE, Cochrane Databases, Elsevier, Wiley Online Library, and BMJ Best Practice. All studies comparing NA combined with unresectable treatment versus unresectable treatment alone were considered for inclusion. The primary outcome was the overall survival (OS) after unresectable treatment for patients with HBV-related HCC. The secondary outcome was the progression-free survival (PFS). Results were expressed as hazard ratio (HR) for survival with 95% confidence intervals. Results. We included six studies with 994 patients: 409 patients in nucleot(s)ide analogs therapy group and 585 patients without antiviral therapy in control group. There were significant improvements for the overall survival (HR = 0.57; 95% CI = 0.47-0.70; p < 0.001) and progression-free survival (HR = 0.84; 95% CI = 0.71-0.99; p = 0.034) in the NA-treated group compared with the control group. Funnel plot showed that there was no significant publication bias in these studies. When it comes to antiviral drugs and operation method, it also showed benefit in NA-treated group. At the same time, overall mortality as well as mortality secondary to liver failure in NA-treated group was obviously lesser. Sensitivity analyses confirmed the robustness of the results. Conclusions. Nucleot(s)ide analogs therapy after unresectable treatment has potential beneficial effects in terms of overall survival and progression-free survival. NA therapy should be considered in clinical practice.

Management of Clevudine-Resistant Chronic Hepatitis B: A Multicenter Cohort Study.

BACKGROUND/AIMS: Data are lacking regarding the management of chronic hepatitis B (CHB) with resistance to clevudine (CLV). This study evaluated the efficacy of different rescue therapies for CLV-resistant CHB. METHODS: Patients with CLV-resistant CHB were enrolled in the cohort, and all patients developed virologic breakthrough during CLV therapy and had confirmed-genotypic resistance to CLV (rtM204I mutation) before enrollment. RESULTS: Of the 107 patients, 12 received adefovir (ADV), 21 received a CLV plus ADV combination (CLV+ADV), 34 received a lamivudine plus ADV combination (LAM+ADV), and 40 received entecavir (ETV) therapy for 48 weeks. The CLV+ADV group had the lowest hepatitis B virus (HBV) DNA level (p<0.0001) and showed the greatest reduction of HBV DNA levels from baseline compared to all other groups (p=0.004) at week 48. HBV DNA was undetectable (<70 IU/mL) in 0%, 57.1%, 21.2%, and 27.5% (p=0.003) of the patients in each group, respectively, at week 48. At the end of the study, the mean alanine transaminase (ALT) level, rate of ALT normalization, and rate of hepatitis B envelope antigen loss or seroconversion did not differ between groups. CONCLUSIONS: CLV+ADV combination therapy in patients with CLV-resistant CHB more effectively suppresses HBV replication than ETV, ADV, or LAM+ADV therapy.

Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Off-treatment virologic relapse and outcomes of re-treatment in chronic hepatitis B patients who achieved complete viral suppression with oral nucleos(t)ide analogs.

BACKGROUND: The durability of off-treatment virologic responses has not been fully elucidated in chronic hepatitis B (CHB) patients who have previously achieved complete virologic suppression with nucleos(t)ide analog (NA) therapy. This study aimed to assess off-treatment virologic relapse rates and to characterize the outcomes of subsequent re-treatment in CHB patients who have discontinued oral NA following complete virologic suppression. METHODS: Ninety-five CHB patients who showed complete virologic suppression were withdrawn from NAs: entecavir, lamivudine, and clevudine in 67, 15, and 13 patients, respectively. Consolidation therapy was given for 6 and 12 months for HBeAg-positive and -negative CHB, respectively, before cessation. Virologic relapse was managed with the same NA that had induced complete virologic response before discontinuation. RESULTS: The cumulative rates of virologic relapse at 12 and 24 months were 73.8% and 87.1%, respectively. The relapse rates were independent of HBeAg positivity, HBeAg seroconversion, and type of oral NA. In a multivariate analysis, duration of oral NA therapy was the only significant predicting factor associated with off-treatment virologic relapse. Although the majority of patients regained complete virologic suppression, some patients did not respond to re-treatment with the initial NA and developed genotypic resistance. CONCLUSIONS: NA consolidation therapy for 6 and 12 months is associated with high off-treatment virologic relapse in HBeAg-positive and -negative CHB patients, respectively. Drugs with high genetic barriers to resistance should be considered as a rescue therapy for off-treatment relapse in CHB.

Antiviral therapy of chronic hepatitis B.

Since the licensing of lamivudine in 1999, the treatment of chronic hepatitis B has been revolutionized by the introduction of oral nucleoside and nucleotide analogues (NAs), which act as inhibitors of the HBV polymerase. The effectiveness of the first of these substances was limited by incomplete response and resistance development in many patients, but today, highly potent substances are available that make a reliable and durable suppression of HBV replication, a reduction of necroinflammatory activity in the liver, and even a reversion of liver fibrosis achievable for almost all patients. Beyond that, NA treatment can prevent the development of hepatocellular carcinoma in many patients. HBeAg seroconversion appears in approximately 50% of all HBeAg-positive patients during NA treatment. However, the ideal treatment endpoint, the serologic loss of HBsAg, remains a rare event almost exclusively achievable for HBeAg-positive patients. After cessation of the treatment, HBV replication tends to relapse in most patients, which is why the duration of NA treatment is indefinite. Future treatment strategies should aim at tailoring individual NA treatment regimens to increase HBs loss rates and optimize treatment duration.

Patients with chronic hepatitis B treated with oral antiviral therapy retain a higher risk for HCC compared with patients with inactive stage disease.

BACKGROUND: It is generally stated that oral antiviral therapy in patients with chronic hepatitis B (CHB) decreases the risk of developing hepatocellular carcinoma (HCC). Although oral nucleos(t)ide analogues (NUCs) may induce a state similar to inactive stage CHB, the long-term risk for HCC in patients treated with NUCs compared with inactive CHB is unclear. METHODS: A total of 1378 patients who were treatment naïve and started NUC therapy and 1014 patients with inactive stage CHB who were HBeAg-negative and continuously had hepatitis B DNA <2000 IU/mL during follow-up were enrolled. The NUC group was divided into two groups by continuous viral suppression: NUC complete responder (CR) group and NUC incomplete responder (IR) group. Cumulative HCC incidence rates were compared between the groups. RESULTS: The risk of developing HCC was significantly higher in the NUC CR group compared with the inactive CHB group, regardless of the presence of baseline liver cirrhosis (p<0.001). Risk factors associated with the development of HCC were treatment groups (p<0.001), age (p<0.001), sex (p<0.001) and the presence of liver cirrhosis at baseline (p=0.005). Of the NUC group, the cumulative incidence of HCC in the NUC IR group was significantly higher compared with the NUC CR group (p=0.028). CONCLUSIONS: The use of potent oral antiviral therapy can effectively suppress HBV replication in patients with CHB. However, the risk of HCC development in patients treated with oral antiviral agent is still significantly higher than patients with inactive stage CHB.

The human genome: its modifications and interactions with those of the microbiome, and the practice of genomic medicine.

In genomic medicine, the nuclear genome is usually the focus of discussion. However, recent developments in genomics show that our interaction with the biological world at large alters our susceptibility to disease and our response to drugs. For example, in addition to causing infection, the trillions of microbial cells that inhabit our bodies (our microbiome), are now known to shape our immune system and our metabolic health. Moreover, mitochondria, the long-assimilated symbionts, are the focus of considerable current genomic research that is making possible, interventions that were at one time in the realms of science fiction. Furthermore, genomics research of the cancer cell is sufficiently refined to enable us to define its genome as a distinct entity suitable for selective drug-targeting. In this contribution we discuss, within their historical contexts, some of these seemingly disparate scientific strands to highlight the importance of embracing a broader multiple-genomic vista when dealing with disease causation, prevention, and management.

[Efficacy of entecavir switching therapy in chronic hepatitis B patients with clevudine-induced myopathy].

BACKGROUND/AIMS: Clevudine is a potent antiviral agent against HBV. However, long-term clevudine therapy may cause myopathy. This study was carried out to identify the efficacy of entecavir switching therapy in chronic hepatitis B patients experiencing clevudine-induced myopathy. METHODS: One hundred forty six patients with chronic hepatitis B treated with 30 mg of clevudine per day for 73 weeks (range, 36-132 weeks) were enrolled. Among them, clevudine-induced myopathy occurred in 21 patients (14.4%) which was diagnosed if the patients had symptoms related to myopathy with concurrent CK and AST elevation. All the patients who were diagnosed as clevudine-induced myopathy stopped the therapy, and 17 patients (81%) were switched to entecavir 0.5 mg. RESULTS: The patients with clevudine-induced myopathy were switched to entecavir 0.5 mg for median 68 weeks, and all of them showed disappearance of clinical myopathic symptoms and normalization of CK and AST level within median 2.2 months. Eight patients (47%) were HBeAg positive before entecavir treatment, and HBeAg seroconversion was achieved in 2 patients (25%). HBV DNA level was elevated in 3 patients (17.6%) at the time when the patients were diagnosed as myopathy, all of them achieved virological response with entecavir switching therapy. ALT level was elevated in 3 patients (17.6%) before entecavir treatment, all of them showed normalization of ALT level. During entecavir therapy, genotypic resistance to entecavir or virological breakthrough was not noted. CONCLUSIONS: In chronic hepatitis B patients experiencing clevudine-induced myopathy, switching to entecavir 0.5 mg per day showed a resolution of myopathy and adequate viral suppression.

Mathematical modeling of triphasic viral dynamics in patients with HBeAg-positive chronic hepatitis B showing response to 24-week clevudine therapy.

BACKGROUND: Modeling of short-term viral dynamics of hepatitis B with traditional biphasic model might be insufficient to explain long-term viral dynamics. The aim was to develop a novel method of mathematical modeling to shed light on the dissociation between early and long-term dynamics in previous studies. METHODS: We investigated the viral decay pattern in 50 patients from the phase III clinical trial of 24-week clevudine therapy, who showed virological response and HBsAg decline. Immune effectors were added as a new compartment in the model equations. We determined some parameter values in the model using the non-linear least square minimization method. RESULTS: Median baseline viral load was 8.526 Log(10)copies/mL, and on-treatment viral load decline was 5.683 Log(10)copies/mL. The median half-life of free virus was 24.89 hours. The median half-life of infected hepatocytes was 7.39 days. The viral decay patterns were visualized as triphasic curves with decreasing slopes over time: fastest decay in the first phase; slowest in the third phase; the second phase in between. CONCLUSIONS: In the present study, mathematical modeling of hepatitis B in patients with virological response and HBsAg decline during 24-week antiviral therapy showed triphasic viral dynamics with direct introduction of immune effectors as a new compartment, which was thought to reflect the reduction of clearance rate of infected cells over time. This modeling method seems more appropriate to describe long-term viral dynamics compared to the biphasic model, and needs further validation.

Experience of anti-viral therapy in hepatitis B-associated membranous nephropathy, including Lamivudine-resistant strains.

BACKGROUND/AIMS: Chronic hepatitis B infection is a common cause of secondary membranous nephropathy (MN) in endemic areas. Lamivudine treatment improves renal outcome in patients with hepatitis B virus-associated MN (HBV-MN), but prolonged use leads to the emergence of lamivudine-resistant variants. We describe our experience treating lamivudine-resistant and other strains of HBV-MN with new antiviral drugs. METHODS: Of the 89 patients biopsied and diagnosed with MN from 1996 to 2011, 10 positive for hepatitis B surface antigen were recruited for this study. We investigated the clinical courses, therapeutic responses, and prognoses of patients with HBV-MN. RESULTS: The incidence of HBV-MN among the original 89 patients was 11.2%. Of these patients, four were treated with supportive care and six with antiviral drugs. One of the four patients treated with supportive care had a spontaneous remission. Four of the six patients treated with antiviral drugs were given lamivudine, and the other two were given entecavir. Two of the four patients treated with lamivudine achieved complete remission with seroconversion (i.e., development of anti-hepatitis B e antigen antibodies), whereas the other two had lamivudine-resistant strains, which were detected at 22 and 23 months after lamivudine treatment, respectively. We added adefovir to the treatment regimen for one of these patients, and for the other patient we substituted clevudine for lamivudine. Both of these patients experienced complete remission, as did the two patients initially treated with entecavir, neither of whom showed resistance to the drug. CONCLUSIONS: New nucleoside analogues, such as entecavir, adefovir, and clevudine, can be effective for treatment of HBV-MN, including lamivudine-resistant strains.

[New drugs of treatment of patients with chronic hepatitis B].

There are five approved drugs for the treatment of chronic hepatitis B(lamivudine, adefovir dipivoxil [ADV], entecavir [ETV], as well as pegylated and standard interferon) in Japan. In this paper, the reported data of new four drugs(tenofovir disoproxil fumarate [TDF], telbivudine, emtricitabine [FTC], clevudine) were described. TDF demonstrated potent antiviral efficacy in naive patients. Moreover, TDF is efficacious in lamivudine- or ADV-refractory patients. TDF monotherapy and the combination of FTC and TDF had similar efficacy in patients with incomplete viral suppression after therapy with ADV; response was not influenced by the presence of baseline lamivudine- or ADV-associated mutations.

Comparison of clevudine and entecavir for treatment-naive patients with chronic hepatitis B virus infection: two-year follow-up data.

BACKGROUND/AIM: Clevudine and entecavir are highly potent antiviral agents being used in treatment of chronic hepatitis B. However, no data comparing clinical efficacy and safety of these 2 drugs over a long-term period is available. The aims of this study are to compare virologic, biochemical, and serologic response rates of clevudine and entecavir, as well as treatment failure rates up to 2 years. METHODS: Data of patients who started clevudine (n = 86) or entecavir (n = 159) as a primary treatment for chronic hepatitis B at Korea University Ansan or Guro Hospital between January 2007 and June 2008 were analyzed. RESULTS: Treatment responses were compared at 3-month intervals up to 24 months. Per protocol analysis showed no difference in virologic responses between the 2 groups at all time points, except at 18 months. When analyzed on intention-to-treat basis for virologic response at 24 months, the response rates were 45.3% in the clevudine group and 72.3% in the entecavir group, which are significantly different (P < 0.001). Rates of biochemical response and HBeAg seroconversion were not significantly different between the groups at all time points. Up to 24 months, antiviral resistance developed in 18 patients (24.4%) in the clevudine group. Clevudine was discontinued owing to muscle-related problems in 10 patients (11.6%). CONCLUSIONS: Although both drugs showed potent antiviral activity, entecavir showed better virologic response at 24 months, primarily owing to treatment failures in the clevudine group that were associated with development of drug resistance and muscle-related problems.

Clevudine-induced viral response, associated with continued reduction of HBsAg titer, was durable after the withdrawal of therapy.

BACKGROUND: This study was conducted to evaluate the durability of clevudine-induced viral response after the withdrawal of treatment. METHODS: Patients who showed a complete response [alanine aminotransferase (ALT) normalization and hepatitis B virus (HBV) DNA <4,700 copies/mL for hepatitis B envelope antigen (HBeAg)-negative patients; ALT normalization, HBV DNA <4,700 copies/mL, and HBeAg seroconversion for HBeAg-positive patients] in the previous clevudine phase III trials were followed for an additional 96 weeks without any treatment for hepatitis B. RESULTS: Of the 63 patients in the study cohort, 73% and 35% of the patients had HBV DNA <141,500 and <4,700 copies/mL, respectively, and 75% of the patients had normal ALT at the end of follow-up. HBeAg seroconversion was maintained in 81% of the patients and hepatitis B surface antigen (HBsAg) loss occurred in 3 patients. Continued HBsAg titer decrease (-0.5 log IU/mL) was observed in the sustained viral responders, suggesting the reduction of covalently closed circular DNA in hepatocytes. CONCLUSIONS: The clevudine-induced viral response was durable in the majority of patients for 2 years after the withdrawal of treatment.