Randomized, double-blind, multicenter safety and efficacy study of rifalazil compared with azithromycin for treatment of uncomplicated genital Chlamydia trachomatis infection in women.

A randomized, double-blind study comparing single-dose chlamydia therapies of oral rifalazil (25 mg) and azithromycin (1 g) was conducted in 82 women with uncomplicated genital Chlamydia trachomatis infection. The microbiologic cure rate of C. trachomatis with rifalazil (n = 33) was 84.8% at the visit on day 22 to 26 (test-of-cure visit), versus 92.1% with azithromycin (n = 38), and the number of treatment failures in each group was 5 and 3, respectively. The difference in cure rate was -7.3%, with a lower limit of the 95% confidence interval (95% CI) of -22.5, and thus, noninferiority was not established at the prespecified margin (lower limit of CI of -15%). The overall treatment-emergent adverse event (TEAE) and treatment-related TEAE rates were lower in the rifalazil group (68% and 55%) than in the azithromycin group (71% and 62%), respectively. Subjects classified as treatment failures at day 22 to 26 had a lower mean plasma concentration of rifalazil at the visit on day 8 to 12 than those classified as treatment cures, but this difference was not significant; however, the levels were similar for both groups at the visit on day 22 to 26. A single 25-mg dose of rifalazil was well tolerated and eradicated C. trachomatis in most of these women with uncomplicated genital C. trachomatis infection. (The study was registered at clinicaltrials.gov under registration no. NCT01631201).

Phenolics with antiviral activity from Millettia erythrocalyx and Artocarpus lakoocha.

From the leaves of Millettia erythrocalyx, a new flavone named 3',5'-dimethoxy-[2",3": 7,8]-furanoflavone and three known compounds were isolated. Assays for anti-herpes simplex virus activity (HSV-1 and HSV-2) were performed on 24 phenolic compounds obtained from M. erythrocalyx and Artocarpus lakoocha. It was found that the flavones ovalifolin, pongol methyl ether and millettocalyxin A, and the stilbene oxyresveratrol possessed moderate activity against both types of HSV. In addition, oxyresveratrol was evaluated for potential anti-HIV activity against a wild-type human immunodeficiency virus type 1 (HIV-1/LAI) isolate and was found to be a modest inhibitor of HIV (EC50 28.2 microM), showing no toxicity in PBM, CEM and Vero cells at 100 microM. The heartwood of A. lakoocha, which contains a large amount of oxyresveratrol, could be considered as a source of starting material for the development of new natural product-based anti-HSV and anti-HIV agents.

Synthesis, anti-HIV activity, and molecular mechanism of drug resistance of L-2',3'-didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides.

beta-l-2',3'-Didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides (beta-l-2'-F-4'-S-d4Ns) have been synthesized and evaluated against HIV-1 in primary human lymphocytes. The key intermediate 8, which was prepared from 2,3-O-isopropylidene-l-glyceraldehyde 1 in 13 steps, was condensed with various pyrimidine and purine bases followed by elimination and deprotection to give the target compounds, beta-l-2'-F-4'-S-d4Ns (17-20 and 27-30). The antiviral activity of the newly synthesized compounds was evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which the cytosine 17, 5-fluorocytosine 18, and adenine 27 derivatives showed potent anti-HIV activities (EC(50) = 0.12, 0.15, and 1.74 microM, respectively) without significant cytotoxicity up to 100 microM in human PBM, CEM, and Vero cells. The cytosine derivative 17 (beta-l-2'-F-4'-S-d4C), however, showed cross-resistance to a 3TC-resistant variant (HIV-1(M184V)). Molecular modeling studies suggest that the pattern of antiviral activity, similar to that of beta-l-2'-F-d4N, stemmed from their conformational and structural similarities. The isosteric substitution of sulfur for 4'-oxygen was well tolerated in the catalytic site of HIV-1 reverse transcriptase in the wild-type virus. However, the steric hindrance between the sugar moiety of the unnatural l-nucleoside and the side chains of Val184 of M184V RT in 3TC-resistant mutant HIV strains destabilizes the RT-nucleoside triphosphate complex, which causes the cross-resistance to 3TC (M184V mutant).

l-2',3'-Didehydro-2',3'-dideoxy-3'-fluoronucleosides: synthesis, anti-HIV activity, chemical and enzymatic stability, and mechanism of resistance.

As antiviral nucleosides containing a 2',3'-unsaturated sugar moiety with 2'-fluoro substitution are endowed with increased stabilization of the glycosyl bond, it was of interest to investigate the influence of the fluorine atom at the 3'-position. Various pyrimidine and purine L-3'-fluoro-2',3'-unsaturated nucleosides were synthesized from their precursors, L-3',3'-difluoro-2',3'-dideoxy nucleosides, by elimination of hydrogen fluoride. In the L-3',3'-difluoro-2',3'-dideoxy nucleoside series, cytidine 16 and 5-fluorocytidine 18 analogues showed modest antiviral activity (EC(50) 11.5 and 8.8 microM, respectively) when evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells. In the 2',3'-unsaturated series, L-3'-fluoro-2',3'-didehydro-2',3'-dideoxycytidine 24 and 5-fluorocytidine 26 showed highly potent antiviral activity (EC(50) 0.089 and 0.018 microM, respectively) without significant cytotoxicity. The guanosine analogue 48 showed only marginal anti-HIV activity with some cytotoxicity (EC(50) 38.5 microM, and IC(50) 17.4, 58.4, 36.5 microM in PBM, CEM, and Vero cells, respectively). The cytidine 24 and 5-fluorocytidine 26 analogues, however, showed significantly decreased antiviral activity against the clinically important lamivudine-resistant variants (HIV-1(M184V)). Molecular modeling studies demonstrated that the 3'-fluoro atom of the L-3'-fluoro-2',3'-unsaturated nucleoside is within the hydrogen bonding distance with the amide backbone of Asp185, which favors the binding of the nucleoside triphosphate to the wild-type RT. This favorable binding mode, however, cannot be maintained when the triphosphate of 3'-fluoro 2',3'-unsaturated nucleoside binds to the active site of M184V RT because the bulky side chain of Val184 occupies the space needed for the nucleotide. The biological results suggest that, in addition to the sugar conformation, the base moiety may also play a role in their interaction with the M184V RT.

Stereoselective synthesis and antiviral activity of D-2',3'-didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides.

As 2',3'-didehydro-2',3'-dideoxy-2'-fluoronucleosides have exhibited interesting antiviral effects against HIV-1 as well as HBV, it is of interest to synthesize the isosterically substituted 4'-thionucleosides in which 4'-oxygen is replaced by a sulfur atom. To study structure-activity relationships, various pyrimidine and purine nucleosides were synthesized from the key intermediate (2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-2-phenylselenyl-4-thio-beta-D-ribofuranoside 8, which was prepared from the 2,3-O-isopropylidene-D-glyceraldehyde 1 in 13 steps. The antiviral activity of the synthesized compounds were evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which cytidine 17, 5-fluorocytidine 18, adenosine 24, and 2-fluoroadenosine 32 showed moderate to potent anti-HIV activities (EC(50) 1.3, 11.6, 8.1, and 1.2 microM, respectively). It is noteworthy that 2-fluoroadenosine analogue 32 showed antiviral potency as well as high cytotoxicity (IC(50) 1.5, 1.1, and 7.6 microM for PBM, CEM, and Vero, respectively) whereas no other compound showed cytotoxicity up to 100 microM. The cytidine 17 and 5-fluorocytidine 18 analogues showed significantly decreased antiviral activity against the clinically important lamivudine-resistant variants (HIV-1(M184V)), whereas the corresponding D-2'-Fd4 nucleosides showed limited cross-resistance. Molecular modeling studies demonstrated that the larger van der Waals radius as well as the close proximity to Met184 of the 4'-sulfur atom of D-2'-F-4'-Sd4C (17) may be the reasons for the decreased antiviral potency of synthesized 4'-thio nucleosides against the lamivudine-resistant variants (HIV-1(M184V)).

N4-acyl-modified D-2',3'-dideoxy-5-fluorocytidine nucleoside analogues with improved antiviral activity.

A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.

Chutes and ladders in hepatitis C nucleoside drug development.

Chutes and Ladders is an exciting up-and-down-again game in which players race to be the first to the top of the board. Along the way, they will find ladders to help them advance, and chutes that will cause them to move backwards. The development of nucleoside analogs for clinical treatment of hepatitis C presents a similar scenario in which taking shortcuts may help quickly advance a program, but there is always a tremendous risk of being sent backwards as one competes for the finish line. In recent years the treatment options for chronic hepatitis C virus (HCV) infection have expand due to the development of a replicon based in vitro evaluation system, allowing for the identification of multiple drugable viral targets along with a concerted and substantial drug discovery effort. Three major drug targets have reached clinical study for chronic HCV infection: the NS3/4A serine protease, the large phosphoprotein NS5A, and the NS5B RNA-dependent RNA polymerase. Recently, two oral HCV protease inhibitors were approved by the FDA and were the first direct acting anti-HCV agents to result from the substantial research in this area. There are currently many new chemical entities from several different target classes that are being evaluated worldwide in clinical trials for their effectiveness at achieving a sustained virologic response (SVR) (Pham et al., 2004; Radkowski et al., 2005). Clearly the goal is to develop therapies leading to a cure that are safe, widely accessible and available, and effective against all HCV genotypes (GT), and all stages of the disease. Nucleoside analogs that target the HCV NS5B polymerase that have reached human clinical trials is the focus of this review as they have demonstrated significant advantages in the clinic with broader activity against the various HCV GT and a higher barrier to the development of resistant viruses when compared to all other classes of HCV inhibitors.

Antiviral activity and tolerability of amdoxovir with zidovudine in a randomized double-blind placebo-controlled study in HIV-1-infected individuals.

BACKGROUND: Amdoxovir acts synergistically with zidovudine in vitro and the combination prevents or delays the selection of thymidine analogue and K65R mutations. In silico studies have shown that a reduced dose of zidovudine (200 mg) results in decreased zidovudine-monophosphate levels, associated with toxicity, while maintaining zidovudine-triphosphate levels, which are associated with antiviral effects. Here, we aimed to assess the short-term tolerability and antiviral activity of amdoxovir in combination with reduced and standard doses of zidovudine. METHODS: The study was a double-blind, placebo-controlled study in HIV-1-infected patients not receiving antiretroviral therapy and with plasma HIV-1 RNA > or =5,000 copies/ml. Patients were randomized to 10 days of twice-daily treatment with 200 mg zidovudine, 300 mg zidovudine, 500 mg amdoxovir, 500 mg amdoxovir plus 200 mg zidovudine or 500 mg amdoxovir plus 300 mg zidovudine. The mean change in viral load (VL) log(10) and area under the virus depletion curve (AUC(VL)) from baseline to day 10 were determined. Laboratory and clinical safety monitoring were performed. RESULTS: Twenty-four patients were enrolled. The mean VL log(10) change was 0.10 with placebo, -0.69 with zidovudine 200 mg, -0.55 with zidovudine 300 mg, -1.09 with amdoxovir, -2.00 with amdoxovir plus zidovudine (200 mg) and -1.69 with amdoxovir plus zidovudine (300 mg). Amdoxovir plus zidovudine (200 mg) was significantly more potent than amdoxovir monotherapy in AUC(VL) and mean VL decline (P=0.019 and P=0.021, respectively), suggesting synergy. There was markedly decreased VL variability with the combination compared with amdoxovir alone. All adverse events were mild to moderate. CONCLUSION: The combination of amdoxovir plus zidovudine appeared synergistic with reduced VL variability. This combined therapy, including the use of a lower zidovudine dosage, warrants further development for the therapy of HIV infection.

Antiviral and cellular metabolism interactions between Dexelvucitabine and lamivudine.

Studies on cellular drug interactions with antiretroviral agents prior to clinical trials are critical to detect possible drug interactions. Herein, we demonstrated that two 2'-deoxycytidine antiretroviral agents, dexelvucitabine (known as beta-d-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, DFC, d-d4FC, or RVT) and lamivudine (3TC), combined in primary human peripheral blood mononuclear (PBM) cells infected with human immunodeficiency virus 1 strain LAI (HIV-1(LAI)), resulted in additive-to-synergistic effects. The cellular metabolism of DFC and 3TC was studied in human T-cell lymphoma (CEM) and in primary human PBM cells to determine whether this combination caused any reduction in active nucleoside triphosphate (NTP) levels, which could decrease with their antiviral potency. Competition studies were conducted by coincubation of either radiolabeled DFC with different concentrations of 3TC or radiolabeled 3TC with different concentrations of DFC. Coincubation of radiolabeled 3TC with DFC at concentrations up to 33.3 microM did not cause any marked reduction in 3TC-triphosphate (TP) or any 3TC metabolites. However, a reduction in the level of DFC metabolites was noted at high concentrations of 3TC with radiolabeled DFC. DFC-TP levels in CEM and primary human PBM cells decreased by 88% and 94%, respectively, when high concentrations of 3TC (33.3 and 100 microM) were added, which may influence the effectiveness of DFC-5'-TP on the HIV-1 polymerase. The NTP levels remained well above the median (50%) inhibitory concentration for HIV-1 reverse transcriptase. These results suggest that both beta-d- and beta-l-2'-deoxycytidine analogs, DFC and 3TC, respectively, substrates of 2'-deoxycytidine kinase, could be used in a combined therapeutic modality. However, it may be necessary to decrease the dose of 3TC for this combination to prove effective.

Mechanism of anti-human immunodeficiency virus activity of beta-D-6-cyclopropylamino-2',3'-didehydro-2',3'-dideoxyguanosine.

To better understand the importance of the oxygen in the ribose ring of planar unsaturated nucleoside analogs that target human immunodeficiency virus (HIV), a 6-cyclopropyl-substituted prodrug of 2',3'-didehydro-2',3'-dideoxyguanosine (cyclo-d4G) was synthesized, and its cellular metabolism, antiviral activity, and pharmacokinetic behavior were studied. Cyclo-d4G had selective anti-HIV activity in primary blood mononuclear cells (PBMCs), effectively inhibiting the LAI strain of HIV-1 by 50% at 1.1 +/- 0.1 microM while showing 50% inhibition of cell viability at 84.5 microM. The antiviral activity in PBMCs was not markedly affected by mutations of methionine to valine at position 184 or by thymidine-associated mutations in the viral reverse transcriptase. Mutations of leucine 74 to valine and of lysine 65 to arginine had mild to moderate resistance (as high as fivefold). Studies to delineate the mechanism of cellular metabolism and activation of cyclo-d4G showed reduced potency in inhibiting viral replication in the presence of the adenosine/adenylate deaminase inhibitor 2'-deoxycoformycin, implying that the antiviral activity is due to its metabolism to the 2'-dGTP analog d4GTP. Intracellular formation of sugar catabolites illustrates the chemical and potentially enzymatic instability of the glycosidic linkage in d4G. Further studies suggest that cyclo-d4G has a novel intracellular phosphorylation pathway. Cyclo-d4G had a lower potential to cause mitochondrial toxicity than 2',3'-dideoxycytidine and 2',3'-didehydro-3'-deoxythymidine in neuronal cells. Also, cyclo-d4G had advantageous synergism with many currently used anti-HIV drugs. Poor oral bioavailability observed in rhesus monkeys may be due to the labile glycosidic bond, and special formulation may be necessary for oral delivery.

2('),3(')-didehydro-2('),3(')-dideoxynucleosides are degraded to furfuryl alcohol under acidic conditions.

2('),3(')-Didehydro-2('),3(')-dideoxynucleosides are clinically relevant antiviral agents. These nucleosides could be degraded under acidic conditions. Acidic stability studies showed the D4N had the following increasing stability order: D4G

Antiviral activities and cellular toxicities of modified 2',3'-dideoxy-2',3'-didehydrocytidine analogues.

The antiviral efficacies and cytotoxicities of 2',3'- and 4'-substituted 2',3'-didehydro-2',3'-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC(90)s], 0.14 to 5.2 micro M), but marked increases in EC(90)s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC(90)s, 53 to >100 micro M). The beta-L-enantiomers of both classes of compounds were more potent than the corresponding beta-D-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC(90), 0.25 micro M). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 micro M. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4'-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC(90), 0.08 micro M) and lamivudine-resistant HIV-1 (EC(90) = 0.15 micro M) is markedly reduced by introduction of a 3'-fluorine in the sugar (EC(90)s of compound 2a, 37.5 and 494 micro M, respectively).