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.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Bioengineering of bacterial pathogens for noninvasive imaging and in vivo evaluation of therapeutics.

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2'deoxy-2'-fluoro-ß-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [18F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [18F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥107 bacteria per infection site. This was further verified by administering [18F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.

Spectrum of activity and mechanisms of resistance of various nucleoside derivatives against gammaherpesviruses.

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-ß-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase.

Noncompetitive inhibition of hepatitis B virus reverse transcriptase protein priming and DNA synthesis by the nucleoside analog clevudine.

All currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.

Enhancing the efficacy of Ara-C through conjugation with PAMAM dendrimer and linear PEG: a comparative study.

1ß-d-Arabinofuranosylcytosine (Cytarabine, Ara-C) is a key drug in the treatment of acute myeloid leukemia. Ara-C has a number of limitations such as a rapid deactivation by cytidine deaminase leading to the formation of a biologically inactive metabolite, Ara-U (1ß-d-arabinofuranosyluracil), a low lipophilicity, and fast clearance from the body. To address these problems, we developed a conjugate in which hydroxyl-terminated PAMAM dendrimer, G4-OH ["D"] and PEG were used as carriers for the drug (Ara-C). The conjugates were synthesized using an efficient multistep protection/deprotection method resulting in the formation of a covalent bond between the primary hydroxyl group of Ara-C and dendrimer/PEG. The structure, physicochemical properties, and drug release kinetics were characterized extensively. (1)H NMR and MALDI-TOF mass spectrometry suggested covalent attachment of 10 Ara-C molecules to the dendrimer. The release profile of Ara-C in human plasma and in PBS buffer (pH 7.4) showed that the conjugates released the drug over 14 days in PBS, with the release sped up in plasma. In PBS, while most of the drug is released from PEG-Ara-C, the dendrimer continues to release the drug in a sustained fashion. The results also suggested that the formation of the inactive form of Ara-C (Ara-U) was delayed upon conjugation of Ara-C to the polymers. The inhibition of cancer growth by the dendrimer-Ara-C and PEG-Ara-C conjugates was evaluated in A549 human adenocarcinoma epithelial cells. Both dendrimer- and PEG-Ara-C conjugates were 4-fold more effective in inhibition of A549 cells compared to free Ara-C after 72 h of treatment.

High-dose clevudine impairs mitochondrial function and glucose-stimulated insulin secretion in INS-1E cells.

BACKGROUND: Clevudine is a nucleoside analog reverse transcriptase inhibitor that exhibits potent antiviral activity against hepatitis B virus (HBV) without serious side effects. However, mitochondrial myopathy has been observed in patients with chronic HBV infection taking clevudine. Moreover, the development of diabetes was recently reported in patients receiving long-term treatment with clevudine. In this study, we investigated the effects of clevudine on mitochondrial function and insulin release in a rat clonal ß-cell line, INS-1E. METHODS: The mitochondrial DNA (mtDNA) copy number and the mRNA levels were measured by using quantitative PCR. MTT analysis, ATP/lactate measurements, and insulin assay were performed. RESULTS: Both INS-1E cells and HepG2 cells, which originated from human hepatoma, showed dose-dependent decreases in mtDNA copy number and cytochrome c oxidase-1 (Cox-1) mRNA level following culture with clevudine (10 µM-1 mM) for 4 weeks. INS-1E cells treated with clevudine had reduced total mitochondrial activities, lower cytosolic ATP contents, enhanced lactate production, and more lipid accumulation. Insulin release in response to glucose application was markedly decreased in clevudine-treated INS-1E cells, which might be a consequence of mitochondrial dysfunction. CONCLUSIONS: Our data suggest that high-dose treatment with clevudine induces mitochondrial defects associated with mtDNA depletion and impairs glucose-stimulated insulin secretion in insulin-releasing cells. These findings partly explain the development of diabetes in patients receiving clevudine who might have a high susceptibility to mitochondrial toxicity.

Long-term treatment outcomes of clevudine in antiviral-naive patients with chronic hepatitis B.

AIM: To evaluate the treatment outcomes of clevudine compared with entecavir in antiviral-naive patients with chronic hepatitis B (CHB). METHODS: We retrospectively analyzed the clinical data of CHB patients treated with clevudine 30 mg/d and compared their clinical outcomes with patients treated with entecavir 0.5 mg/d. The biochemical response, as assessed by serum alanine aminotransferase (ALT) activity, virologic response, as assessed by serum hepatitis B virus DNA (HBV DNA) titer, serologic response, as assessed by hepatitis B e antigen (HBeAg) status, and virologic breakthrough with genotypic mutations were assessed. RESULTS: Two-hundred and fifty-four patients [clevudine (n = 118) vs entecavir (n = 136)] were enrolled. In clevudine-treated patients, the cumulative rates of serum ALT normalization were 83.9% at week 48 and 91.5% at week 96 (80.9% and 91.2% in the entecavir group, respectively), the mean titer changes in serum HBV DNA were -6.03 and -6.55 log(10) copies/mL (-6.35 and -6.86 log(10) copies/mL, respectively, in the entecavir group), and the cumulative non-detection rates of serum HBV DNA were 72.6% and 83.1% (74.4% and 83.8%, respectively, in the entecavir group). These results were similar to those of entecavir-treated patients. The cumulative rates of HBeAg seroconversion were 21.8% at week 48 and 25.0% at week 96 in patients treated with clevudine, which was similar to patients treated with entecavir (22.8% and 27.7%, respectively). The virologic breakthrough in the clevudine group occurred in 9 (7.6%) patients at weeks 48 and 15 (12.7%) patients at week 96, which primarily corresponded to genotypic mutations of rtM204I and/or rtL180M. There was no virologic breakthrough in the entecavir group. CONCLUSION: In antiviral-naive CHB patients, long-term treatment outcomes of clevudine were not inferior to those of entecavir, except for virologic breakthrough.

Evaluation of the in vitro anti-HBV activity of clevudine in combination with other nucleoside/nucleotide inhibitors.

BACKGROUND: To reduce the incidence of drug resistance and to maintain viral suppression, patients chronically infected with HBV might require combination therapy using two or more drugs with different resistance profiles. We investigated the activity of clevudine (CLV) in combination with other nucleoside/nucleotide analogues to determine if these combinations were compatible in vitro. METHODS: Using the HepAD38 cell line, which expresses wild-type HBV, and a real-time PCR assay, we tested the anti-HBV activity of CLV in combination with entecavir, lamivudine, adefovir, tenofovir and telbivudine (TBV). We evaluated the uptake and phosphorylation of CLV in the presence of TBV, using HepAD38 cells and primary hepatocytes to determine the effect of TBV on the phosphorylation of CLV and vice versa. Phosphorylation of TBV and CLV to their corresponding monophosphate by deoxycytidine kinase, thymidine kinase-1 and thymidine kinase-2, and the phosphorylation of TBV monophosphate and CLV monophosphate by thymidylate kinase was evaluated and compared. RESULTS: When CLV was combined with entecavir, lamivudine, adefovir or tenofovir, a synergistic antiviral effect was observed; however, the combination of CLV and TBV gave an antagonistic antiviral response. The results of in vitro metabolism and enzyme studies suggest that the antagonism observed with the CLV/TBV combination involves competition for uptake and phosphorylation. CONCLUSIONS: The results of our studies demonstrate that combination treatments can provide enhanced antiviral activity and, when used in conjunction with appropriate metabolic investigations, provide a rational basis for the design and development of combination regimens for treating chronic HBV infection.

The design, synthesis and antiviral evaluation of a series of 5-trimethylsilyl-1-beta-D-(arabinofuranosyl)uracil phosphoramidate ProTides.

BACKGROUND: Nucleoside analogues always require phosphorylation to be active. This appears to be a particular limitation for uridine-based nucleosides. Our ProTide method allows the direct use of masked membrane-soluble preformed nucleoside phosphates, bypassing the need for the initial phosphorylation step. We herein applied it to some novel 5-trimethylsilyl arabinosyl uridines. METHODS: 5-Trimethylsilyl-1-beta-D-(arabinofuranosyl)uracil was prepared in six steps starting from uridine, and five phosphoramidate ProTide derivatives were synthesized. These compounds were investigated for activity against a range of DNA and RNA viruses, including herpes simplex virus type-1 and type-2, vaccinia virus and HIV. RESULTS: Overall, these compounds did not show significant antiviral activity against any of the viruses tested. CONCLUSIONS: The inactivity of the ProTides of this nucleoside could correspond with poor ProTide activation in vitro, poor onward metabolism or low activity of the putative monophosphate metabolite.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism.

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Identification and characterization of clevudine-resistant mutants of hepatitis B virus isolated from chronic hepatitis B patients.

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV) infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to in vitro drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common. In vitro phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.

Clevudine for hepatitis B.

Clevudine is distinguished from other oral agents by its sustained suppression of HBV DNA for several months after cessation of therapy, according to a comprehensive review of hepatitis B in the 2 October 2008 issue of the New England Journal of Medicine. Clevudine is differentiated by i) an unusual activation pathway to the biochemically active triphosphate; ii) a mechanism of action of clevudine triphosphate that inhibits multiple steps of the hepatitis B virus (HBV) intracellular life cycle; iii) a long half-life and iv) significant reduction of covalently closed circular DNA (cccDNA) in animal models. Clevudine was approved and is marketed in South Korea based on two 24-week phase III trials vs. placebo. In these studies with treatment-naïve patients, 59% of 248 HBeAg-positive patients had undetectable HBV DNA after 24 weeks of treatment compared with 92% of 89 HBeAg-negative patients, while the percentage of patients with normal liver enzymes was 68% in the HBeAg-positive patients and 75% in the HBeAg-negative patients (all statistically significant versus placebo). Follow-up studies include trials vs. lamivudine as well as a phase IV study of long-term clevudine. Larger and longer phase III trials in the United States, European Union, Asia and South America of clevudine vs. adefovir are ongoing. An ANRS-sponsored trial of clevudine vs. tenofovir vs. the combination of the two agents is poised to begin. Literature published through November 2008 and presentations from the 59th annual meeting of the American Association for the Study of Liver Diseases held 31 October to 4 November 2008 are included.

New drugs for chronic hepatitis B: a review.

Three nucleotide/nucleoside analogs are used for chronic hepatitis B (HBV): lamivudine, adefovir dipivoxil, and entecavir. Lamivudine and adefovir are advantageous for oral administration and safety but induce a sustained response after withdrawal of therapy in only a minority of patients. Thus, the treatment should be given in trials in a majority of patients for a long period of time. In addition, the long-term efficacy of lamivudine is limited by the frequent emergence of drug-resistant HBV mutants. Adefovir is associated with a low frequency of resistance, but its antiviral effect is not optimal. Entecavir, a cyclopentyl guanosine analog, is a potent inhibitor of HBV-DNA polymerase and it inhibits both priming and elongation steps of viral DNA replication. In phase II and III clinical trials, entecavir was found to be superior to lamivudine for all primary end points evaluated in both nucleoside-naive and lamivudine-resistant patients, and it was effective in both HBeAg-positive and HBeAg-negative nucleoside-naive patients. Only one trial has shown cases of viral resistance to this drug. The approved dosage in treatment-naive patients is 0.5 mg per day orally, whereas in patients who have failed lamivudine therapy or who are known to harbor lamivudine-resistant mutants, the approved dosage is 1.0 mg per day. Recent preliminary results show that clevudine, telbivudine, and emtricitabine may be potent analogs available for the treatment of HBV. Further studies are being conducted to assess the long-term efficacy and safety of these drugs.

A novel mutation pattern emerging during lamivudine treatment shows cross-resistance to adefovir dipivoxil treatment.

AIMS: This study was conducted to clarify the resistance profile of a novel mutation pattern emerging during lamivudine (3TC) therapy and showing cross-resistance to adefovir dipivoxil (ADV) in a patient with chronic hepatitis B. METHODS AND RESULTS: Successful suppression of hepatitis B virus (HBV) replication by sequential therapy of 9 MU thrice weekly interferon (IFN) and 3TC was followed by genotypical resistance detected at month 28 of therapy (month 19 of lamivudine treatment). ADV was added to 3TC therapy on month 44 of antiviral treatment. Neither alanine aminotransferase normalization nor a stable decrease in HBV viral load was observed, although ADV was used for more than 40 months. The HBV pol region was amplified from serum samples obtained before and after ADV treatment. The complete genome was cloned into a TA vector. PCR products and 7-10 clones from each cloned vector were sequenced. A novel mutation, A181S, in the reverse transcriptase gene leading to a conversion of W172C in the overlapping surface antigen gene was detected along with a M2041 mutation. The complete genome comprising the A181S+M2041 pattern was cloned into an expression vector and its in vitro susceptibility to 3TC, ADV, tenofovir (PMPA), clevudine (L-FMAU) and emtricitabine (FTC) were determined in transiently transfected Huh7 cells. This mutation pattern displayed more than 1000-fold resistance to the nucleoside analogues 3TC and FTC and approximately sixfold resistance to L-FMAU, while it confers 28.23- and 5.57-fold resistance for the nucleotide analogues ADV and PMPA, respectively. CONCLUSION: A new mutation pattern, A181S+M2041, arising under lamivudine treatment confers cross-resistance to ADV both in vivo and in vitro.

Clevudine is highly efficacious in hepatitis B e antigen-negative chronic hepatitis B with durable off-therapy viral suppression.

UNLABELLED: Clevudine is a pyrimidine analog with potent and sustained antiviral activity against HBV. In the present study, we evaluated the safety and efficacy of clevudine 30 mg daily for 24 weeks and assessed the durability of antiviral response for 24 weeks after cessation of dosing in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB). We randomized a total of 86 patients (3:1) to receive clevudine 30 mg (n = 63) or placebo (n = 23) daily for 24 weeks. We followed patients for an additional 24 weeks after withdrawal of treatment. The median changes in HBV DNA from baseline were -4.25 and -0.48 log(10) copies/mL at week 24 in the clevudine and placebo groups, respectively (P < 0.0001). Viral suppression in the clevudine group was sustained after withdrawal of therapy, with 3.11 log(10) reduction at week 48. At week 24 and week 48, 92.1% and 16.4% of patients in the clevudine group had undetectable serum HBV DNA levels by Amplicor PCR assay (<300 copies/mL). The proportion of patients who achieved ALT normalization was 74.6% and 33.3% in the clevudine and placebo groups at week 24, respectively (P = 0.0006). ALT normalization in the clevudine group was well-maintained during the post-treatment follow-up period. The incidence of adverse events was similar in the 2 groups. No resistance to clevudine was detected during treatment. CONCLUSION: A 24-week clevudine therapy was well-tolerated and showed potent and sustained antiviral effect without evidence of viral resistance in e-CHB patients. However, treatment for longer than 24 weeks would be needed to achieve durable remission.

Growth inhibition of Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium in vitro: effect of 1-beta-D-2'-arabinofuranosyl and 1-(2'-deoxy-2'-fluoro-beta-D-2'-ribofuranosyl) pyrimidine nucleoside analogs.

The resurgence of tuberculosis and the emergence of multiple-drug-resistant strains of Mycobacteria necessitate the search for new classes of antimycobacterial agents. We synthesized a series of 1-beta-D-2'-arabinofuranosyl and 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl) pyrimidine nucleosides possessing diverse sets of alkynyl, alkenyl, alkyl, and halo substituents at the C-5 position of the uracil and investigated their effect on activity against M. tuberculosis, M. bovis, and M. avium. Among these molecules, 5-alkynyl-substituted derivatives emerged as potent inhibitors of M. bovis, M. tuberculosis, and M. avium. Nucleosides 1-beta-D-2'-arabinofuranosyl-5-dodecynyluracil (5), 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)-5-dodecynyluracil (24), and 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)-5-tetradecynyluracil (25) showed the highest antimycobacterial potency against M. bovis and M. tuberculosis. The MIC90 exhibited by compounds 5, 24, and 25 was similar or close to that of the reference drug rifampicin. The most active compounds 5, 24, and 25 were also found to retain sensitivity against a rifampicin-resistant strain of M. tuberculosis H37Rv at similar concentrations. Some of these analogs also revealed in vitro antimicrobial effect against several other gram-positive pathogens.

In vivo evaluation of 2'-deoxy-2'-[(18)F]fluoro-5-iodo-1-beta-D-arabinofuranosyluracil ([18F]FIAU) and 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil ([18F]FEAU) as markers for suicide gene expression.

PURPOSE: FIAU and FEAU were evaluated in vitro and in vivo as markers for HSV1-tk gene expression. METHODS: In vitro and biodistribution studies were performed in wild type and transduced HT-29 cells using [14C]FIAU and [3H]FEAU. PET imaging was performed using [18F]FIAU and [18F]FEAU. RESULTS: In vitro uptake of [14C]FIAU in tk-positive cells was 39-fold, 49-fold, and 43-fold higher (p<0.001) than in wild type cells at 30, 60, and 120 min, respectively. Uptake of [3H]FEAU in transduced cells was 46-fold, 62-fold, and 121-fold higher (p<0.001) than in wild type cells at the same time points. In vivo uptake of [14C]FIAU at 2 h in HSV1-tk positive tumors was 15.48+/-3.94, 6.7-fold higher (p<0.001) than in wild type tumors. Uptake of [3H]FEAU in transduced tumors was 9.98+/-1.99, 5.0-fold higher (p<0.001) than in wild type tumors. Micro-PET images using [18F]FIAU and [18F]FEAU also showed very high uptake in HSV-tk tumors. CONCLUSION: [18F]FIAU and [18F]FEAU appear to be potential PET imaging agents for gene expression.

Clevudine: a potent inhibitor of hepatitis B virus in vitro and in vivo.

Clevudine (CLV) is a nucleoside analog of the unnatural L-configuration that has potent anti-hepatitis B virus (HBV) activity in vitro and in vivo with a favorable toxicity profile in all species tested. In cell culture, CLV is readily phosphorylated to the corresponding 5'-triphosphate form of the compound. The mechanism of action of CLV involves the inhibition of the HBV polymerase by CLV 5'-triphosphate. In vivo efficacy studies performed in the duck and woodchuck models showed marked, rapid inhibition of virus replication and no significant toxicity. In the woodchuck model, there was a dose-dependent delay in viral recrudescence and a reduction or loss of covalently closed circular DNA. In Phase II clinical studies, CLV was well tolerated and exhibited potent antiviral activity at all doses investigated. In Phase III studies in both hepatitis B e antigen (HBeAg)-positive and -negative patients, CLV 30 mg administered once daily demonstrated potent antiviral efficacy and significant biochemical improvement after only 24 weeks of therapy. These effects were sustained in a significant portion of the patients when therapy was stopped after 6 months with no viral rebound occurring in approximately 3 and 16% in HBeAg-positive and -negative patients, respectively. There have been no significant safety or tolerance issues associated with the drug in these studies. Future studies will investigate the safety and tolerance of CLV 30 mg given once daily over 48 weeks and longer.