Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.     

Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC₅₀) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC₅₀s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC₅₀, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC₅₀s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC₅₀. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC₅₀s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.     

Development of a New Cartridge Radioimmunoassay for Determination of Intracellular Levels of Lamivudine Triphosphate in the Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus-Infected Patients

A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/10⁶ cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/10⁶ cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0.01 to 1.76 and 0.01 to 0.07 pmol/10⁶ cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 μM, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 μM. This is the first study to determine the intracellular levels of the active metabolites in HIV-infected subjects treated with this combination. These methods should prove very useful for in vivo pharmacodynamic studies of combination therapy.     

Efficacy of the Carbocyclic 2′-Deoxyguanosine Nucleoside BMS-200475 in the Woodchuck Model of Hepatitis B Virus Infection

Daily oral treatment with the cyclopentyl 2′-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10⁷- to 10⁸-fold, down to levels as low as 10² to 10³ virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.     

Design,synthesis, in silico docking,ADMET and anticancer evaluations of thiazolidine-2,4-diones bearing heterocyclic rings as dual VEGFR-2/EGFRT790M tyrosine kinase inhibitors

Fourteen recent thiazolidine-2,4-diones bearing furan and/or thiophene heterocyclic rings have been designed, synthesized and assessed for their anticancer activities against four human tumor cell lines HepG2, A549, MCF-7 and HCT-116 targeting both VEGFR-2 and EGFR tyrosine kinases. Molecular design was carried out to investigate the binding mode of the proposed compounds with VEGFR-2 and EGFR receptors. HepG2 was the most susceptible cell line to the influence of our derivatives. Compounds 5g and 4g revealed the highest activities against HepG2 (IC₅₀ = 3.86 and 6.22 μM), A549 (IC₅₀ = 7.55 and 12.92 μM), MCF-7 (IC₅₀ = 10.65 and 10.66 μM) and HCT116 (IC₅₀ = 9.04 and 11.17 μM) tumor cell lines. Sorafenib (IC₅₀ = 4.00, 4.04, 5.58 and 5.05 μM) and elotinib (IC₅₀ = 7.73, 5.49, 8.20 and 13.91 μM) were used as reference standards. Furthermore, the most active cytotoxic compounds 4d, 4e, 4f, 4g, 5d, 5e, 5f and 5g were selected to assess their VEGFR-2 inhibitory effects. Derivatives 5g, 4g and 4f were observed to be the highest effective derivatives that inhibited VEGFR-2 at the submicromolar level (IC₅₀ = 0.080, 0.083 and 0.095 μM respectively) in comparison to sorafenib (IC₅₀ = 0.084 μM). As well, compounds 4d, 4e, 4f, 4g, 5d, 5e, 5f and 5g were additionally assessed for their inhibitory activities against mutant EGFR^T790M. Compounds 5g and 4g could interfere with the EGFR^T790M activity exhibiting stronger activities than elotinib with IC₅₀ = 0.14 and 0.23 μM respectively. Finally, our derivatives 4g, 5f and 5g showed a good in silico calculated ADMET profile. The obtained results showed that our compounds could be useful as a template for future design, optimization, adaptation and investigation to produce more potent and selective dual VEGFR-2/EGFR^T790M inhibitors with higher anticancer activity.

Fourteen recent thiazolidine-2,4-diones bearing furan and/or thiophene heterocyclic rings have been designed, synthesized and assessed for their anticancer activities against four human tumor cell lines HepG2, A549, MCF-7 and HCT-116 targeting both VEGFR-2 and EGFR tyrosine kinases.     

Lucidone Suppresses Hepatitis C Virus Replication by Nrf2-Mediated Heme Oxygenase-1 Induction

Upon screening of plant-derived natural products against hepatitis C virus (HCV) in the replicon system, we demonstrate that lucidone, a phytocompound, isolated from the fruits of Lindera erythrocarpa Makino, significantly suppressed HCV RNA levels with 50% effective concentrations of 15 ± 0.5 μM and 20 ± 1.1 μM in HCV replicon and JFH-1 infectious assays, respectively. There was no significant cytotoxicity observed at high concentrations, with a 50% cytotoxic concentration of 620 ± 5 μM. In addition, lucidone significantly induced heme oxygenase-1 (HO-1) production and led to the increase of its product biliverdin for inducing antiviral interferon response and inhibiting HCV NS3/4A protease activity. Conversely, the anti-HCV activity of lucidone was abrogated by blocking HO-1 activity or silencing gene expression of HO-1 or NF-E2-related factor 2 (Nrf2) in the presence of lucidone, indicating that the anti-HCV action of lucidone was due to the stimulation of Nrf-2-mediated HO-1 expression. Moreover, the combination of lucidone and alpha interferon, the protease inhibitor telaprevir, the NS5A inhibitor BMS-790052, or the NS5B polymerase inhibitor PSI-7977, synergistically suppressed HCV RNA replication. These findings suggest that lucidone could be a potential lead or supplement for the development of new anti-HCV agent in the future.     

In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen,a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122

Miravirsen is a β-d-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC₅₀) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2′-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5′ untranslated region (UTR) from cells passaged in the presence of up to 20 μM (40-fold the miravirsen EC₅₀ concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5′UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01200420","term_id":"NCT01200420"}}NCT01200420).     

Inhibition of Human Hepatitis B Virus Replication by AT-61, a Phenylpropenamide Derivative,Alone and in Combination with (?)β-l-2′,3′-Dideoxy-3′-Thiacytidine

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 μM. AT-61 (27 μM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 μM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (−)-β-l-2′,3′-dideoxy-3′ thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 μM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.Hepatitis B virus (HBV) is estimated to chronically infect approximately 300 million people worldwide. These individuals are at increased risk for the development of liver failure, cirrhosis, and hepatocellular carcinoma (3, 23). In addition, it is estimated that of those chronically infected, approximately 1 million die annually from HBV-induced liver disease (19).At the present, interferon (IFN) is the only available treatment for chronic hepatitis in the United States. However, its efficacy is partial and of limited duration, with less than 30% of the chronic carriers being treated with IFN responding to treatment. In addition, approximately 50% of those who initially respond to IFN therapy experience a recurrence of viremia after the cessation of treatment (6, 29). In clinical trials, two nucleoside analogs, lamivudine [(−)-β-l-2′,3′-dideoxy-3′-thiacytidine; 3TC] and ganciclovir, have proven to be effective in decreasing the levels of HBV DNA in the serum of chronically infected patients (4, 7–9). However, many patients relapsed shortly after the cessation of therapy. In addition, there are now reports of the isolation of 3TC-resistant variants of HBV from the serum of immunosuppressed patients undergoing 3TC therapy (2, 20, 30).Here we report that AT-61, a member of a class of phenylpropenamide derivatives with antiviral activity against HBV replication (22), is a potent inhibitor of the replication of both wild-type and 3TC-resistant HBV in HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cell lines. This compound does not inhibit the replication of duck HBV (DHBV), woodchuck HBV (WHBV), human immunodeficiency virus (HIV) type 1 (HIV-1), herpes simplex virus (HSV) type 1, (HSV-1), vesicular stomatitis virus (VSV), or Newcastle disease virus (NDV) and has very low toxicity in a number of cell lines. Moreover, when used in combination with 3TC, AT-61 acted synergistically to inhibit HBV replication in HepAD38 cells. Finally, the data suggest that this compound may exert its antiviral effect by interfering with the packaging of the pregenomic RNA into the immature core particle.     

Rapid Ganciclovir Susceptibility Assay Using Flow Cytometry for Human Cytomegalovirus Clinical Isolates

Rapid, quantitative, and objective determination of the susceptibilities of human cytomegalovirus (HCMV) clinical isolates to ganciclovir has been assessed by an assay that uses a fluorochrome-labeled monoclonal antibody to an HCMV immediate-early antigen and flow cytometry. Analysis of the ganciclovir susceptibilities of 25 phenotypically characterized clinical isolates by flow cytometry demonstrated that the 50% inhibitory concentrations (IC₅₀s) of ganciclovir for 19 of the isolates were between 1.14 and 6.66 μM, with a mean of 4.32 μM (±1.93) (sensitive; IC₅₀ less than 7 μM), the IC₅₀s for 2 isolates were 8.48 and 9.79 μM (partially resistant), and the IC₅₀s for 4 isolates were greater than 96 μM (resistant). Comparative analysis of the drug susceptibilities of these clinical isolates by the plaque reduction assay gave IC₅₀s of less than 6 μM, with a mean of 2.88 μM (±1.40) for the 19 drug-sensitive isolates, IC₅₀s of 6 to 8 μM for the partially resistant isolates, and IC₅₀s of greater than 12 μM for the four resistant clinical isolates. Comparison of the IC₅₀s for the drug-susceptible and partially resistant clinical isolates obtained by the flow cytometry assay with the IC₅₀s obtained by the plaque reduction assay showed an acceptable correlation (r² = 0.473; P = 0.001), suggesting that the flow cytometry assay could substitute for the more labor-intensive, subjective, and time-consuming plaque reduction assay.     

Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus-Infected Cells

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised patients. The current standard of therapy for the treatment of HCMV infections is ganciclovir (GCV). However, high incidence rates of adverse effects are prevalent and limit the use of this drug. Cyclopropavir (CPV) is 10-fold more effective against HCMV in vitro than GCV (50% effective concentrations [EC₅₀s] = 0.46 and 4.1 μM, respectively) without any observed increase in cytotoxicity (S. Zhou, J. M. Breitenbach, K. Z. Borysko, J. C. Drach, E. R. Kern, E. Gullen, Y. C. Cheng, and J. Zemlicka, J. Med. Chem. 47:566–575, 2004, doi:10.1021/jm030316s). We have previously determined that the viral protein kinase pUL97 and endogenous cellular kinases are responsible for the conversion of CPV to a triphosphate (TP), the active compound responsible for inhibiting viral DNA synthesis and viral replication. However, this conversion has not been observed in HCMV-infected cells. To that end, we subjected HCMV-infected cells to equivalently effective concentrations (∼5 times the EC₅₀) of either CPV or GCV and observed a time-dependent increase in triphosphate levels for both compounds (CPV-TP = 121 ± 11 pmol/10⁶ cells; GCV-TP = 43.7 ± 0.4 pmol/10⁶ cells). A longer half-life was observed for GCV-TP (48.2 ± 5.7 h) than for CPV-TP (23.8 ± 5.1 h). The area under the curve for CPV-TP produced from incubation with 2.5 μM CPV was 8,680 ± 930 pmol · h/10⁶ cells, approximately 2-fold greater than the area under the curve for GCV-TP of 4,520 ± 420 pmol · h/10⁶ cells produced from incubation with 25 μM GCV. We therefore conclude that the exposure of HCMV-infected cells to CPV-TP is greater than that of GCV-TP under these experimental conditions.     

Efficacy of pyrvinium pamoate against Cryptosporidium parvum infection in vitro and in a neonatal mouse model

No effective approved drug therapy exists for Cryptosporidium infection of immunocompromised patients. Here we investigated the nonabsorbed anthelmintic drug pyrvinium pamoate for inhibition of the growth of the intestinal protozoan parasite Cryptosporidium parvum. The concentration of pyrvinium that effected 50% growth inhibition in human enterocytic HCT-8 cells by a quantitative alkaline phosphatase immunoassay was 354 nM. For comparison, in the same assay, 50% growth inhibition was obtained with 711 μM paromomycin or 27 μM chloroquine. We used a neonatal mouse model to measure the anti-Cryptosporidium activity of pyrvinium pamoate in vivo. Beginning 3 days after infection, pyrvinium at 5 or 12.5 mg/kg of body weight/day was administered to the treatment group mice for 4 or 6 consecutive days. Nine days after infection, the mice were sacrificed, and drug efficacy was determined by comparing the numbers of oocysts in the fecal smears of treated versus untreated mice. The intensities of trophozoite infection in the ileocecal intestinal regions were also compared using hematoxylin-and-eosin-stained histological slides. We observed a >90% reduction in infection intensity in pyrvinium-treated mice relative to that in untreated controls, along with a substantial reduction in tissue pathology. Based on these results, pyrvinium pamoate is a potential drug candidate for the treatment of cryptosporidiosis in both immunocompetent and immunocompromised individuals.     

Discovery of dihydrooxazolo[2,3-a]isoquinoliniums as highly specific inhibitors of hCE2

Human carboxylesterase 2 (hCE2) is one of the most abundant esterases distributed in human small intestine and colon, which participates in the hydrolysis of a variety of ester-bearing drugs and thereby affects the efficacy of these drugs. Herein, a new compound (23o) with a novel skeleton of dihydrooxazolo[2,3-a]isoquinolinium has been discovered with strong inhibition on hCE2 (IC₅₀ = 1.19 μM, K_i = 0.84 μM) and more than 83.89 fold selectivity over hCE1 (IC₅₀ > 100 μM). Furthermore, 23o can inhibit hCE2 activity in living HepG2 cells with the IC₅₀ value of 2.29 μM, indicating that this compound has remarkable cell-membrane permeability and is capable for inhibiting intracellular hCE2. The SAR (structure–activity relationship) analysis and molecular docking results demonstrate that the novel skeleton of oxazolinium is essential for hCEs inhibitory activity and the benzyloxy moiety mainly contributes to the selectivity of hCE2 over hCE1.

Novel oxazoliniums are highly specific inhibitors of hCE2 over hCE1 and have good cell-membrane permeability for inhibiting intracellular hCE2.     

Pharmacokinetics and Distribution over the Blood-Brain Barrier of Zalcitabine (2′,3′-Dideoxycytidine) and BEA005 (2′,3′-Dideoxy-3′-Hydroxymethylcytidine) in Rats, Studied by Microdialysis

Microdialysis was applied to sample the unbound drug concentration in the extracellular fluid in brain and muscle of rats given zalcitabine (2′,3′-dideoxycytidine; n = 4) or BEA005 (2′,3′-dideoxy-3′-hydroxymethylcytidine; n = 4) (50 mg/kg of body weight given subcutaneously). Zalcitabine and BEA005 were analyzed by high-pressure liquid chromatography with UV detection. The maximum concentration of zalcitabine in the dialysate (C_max) was 31.4 ± 5.1 μM (mean ± standard error of the mean) for the brain and 238.3 ± 48.1 μM for muscle. The time to C_max was found to be from 30 to 45 min for the brain and from 15 to 30 min for muscle. Zalcitabine was eliminated from the brain and muscle with half-lives 1.28 ± 0.64 and 0.85 ± 0.13 h, respectively. The ratio of the area under the concentration-time curve (AUC) (from 0 to 180 min) for the brain and the AUC for muscle (AUC ratio) was 0.191 ± 0.037. The concentrations of BEA005 attained in the brain and muscle were lower than those of zalcitabine, with C_maxs of 5.7 ± 1.4 μM in the brain and 61.3 ± 12.0 μM in the muscle. The peak concentration in the brain was attained 50 to 70 min after injection, and that in muscle was achieved 30 to 50 min after injection. The half-lives of BEA005 in the brain and muscle were 5.51 ± 1.45 and 0.64 ± 0.06 h, respectively. The AUC ratio (from 0 to 180 min) between brain and muscle was 0.162 ± 0.026. The log octanol/water partition coefficients were found to be −1.19 ± 0.04 and −1.47 ± 0.01 for zalcitabine and BEA005, respectively. The degrees of plasma protein binding of zalcitabine (11% ± 4%) and BEA005 (18% ± 2%) were measured by microdialysis in vitro. The differences between zalcitabine and BEA005 with respect to the AUC ratio (P = 0.481), half-life in muscle (P = 0.279), and level of protein binding (P = 0.174) were not statistically significant. The differences were statistically significant in the case of the half-life in the brain (P = 0.032), clearance (P = 0.046), volume of distribution (P = 0.027) in muscle, and octanol/water partition coefficient (P = 0.019).     

Cellular Pharmacology of the Anti-Hepatitis B Virus Agent β-l-2′,3′-Didehydro-2′,3′-Dideoxy-N4-Hydroxycytidine: Relevance for Activation in HepG2 Cells

ß-l-2′,3′-Didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM; 50% cytotoxic dose [CD₅₀] = 2,500 μM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[³H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5′-mono-, 5′-di-, and 5′-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ß-d-N⁴-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5′-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 μM l-[³H]Hyd4C for 24 h, the 5′-triphosphate accumulated to 19.4 ± 2.7 pmol/10⁶ cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/10⁶ cells. The intracellular half-life of the 5′-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5′-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5′-diphosphate during a drug-free incubation. The high 5′-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.A large number of nucleoside analogues have been described as inhibitors of hepatitis B virus (HBV) and HIV replication. Recently l-nucleoside analogues in particular have gained increasing interest. They are characterized by an opposite configuration from that of the natural d-nucleoside analogues and represent one of the most attractive groups of antiretroviral compounds, including ß-l-2′,3′-dideoxy-3-thiacytidine (3TC) and its 5-fluoro derivative (FTC), ß-l-2′,3′-didehydro-2′,3′-dideoxy-cytidine (l-d4C) and its 5-fluoro derivative (l-d4FC), ß-l-thymidine, ß-l-fluoroarabinosylyluracil (l-FMAU), and ß-l-2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-cytidine (l-2′Fd4C) (3, 5, 22).Some of them not only have been found to be more potent than their corresponding d-nucleosides but seem to exhibit lower cytotoxicity and have been proved to be effective and selective agents for the treatment of chronic hepatitis B virus infections (4). However, only long-term therapy with a single nucleoside for several years was shown to be able to completely suppress HBV DNA in serum of patients and to reverse the progression of the disease. The disadvantage connected with such therapy regimens is the development of drug-resistant HBV strains (22). Therefore, the challenge will be to develop more-efficient drugs for shorter treatment regimens and to combine them to reach synergistic or at least additive drug action. This approach has been described not only as being highly efficient for the treatment of HIV infections but also as preventing the development of resistant mutants. Therefore, AIDS therapy is considered a model for future therapy of chronic HBV infections (17).Recently we described a series of new ß-l-N⁴-hydroxydeoxycytidine and ß-l-5-methyl-deoxycytidine derivatives as inhibitors of HBV replication. Between them, ß-l-2′,3′-didehydro-2′,3′-dideoxy-N⁴-hydroxycytidine (l-Hyd4C) (Fig. ​(Fig.1)1) emerged as the most effective in suppression of virus production in HepG2.2.15 cells (50% effective dose [ED₅₀] = 0.03 μM), displaying an extremely low cytotoxicity (50% cytotoxic dose [CD₅₀] for HepG2 cells = 2,500 μM) (12).Open in a separate windowFIG. 1.Structure of l-Hyd4C and possible metabolites formed by reduction (l-d4C) or by deamination (l-d4U).These encouraging features have prompted us to investigate the cellular pharmacology of l-Hyd4C in a hepatic cell line. This included the activation of this unnatural l-deoxycytidine nucleoside to its 5′-mono-, 5′-di-, and 5′-triphosphate, the search for other metabolites, and the estimation of the intracellular half-lives (t_1/2) of the 5′-di- and 5′-triphosphate of l-Hyd4C.(This work was presented in part at BIT''s 5th Anniversary Congress of International Drug Discovery Science and Technology, 7 to 13 November 2007, Xi''an and Beijing, China.)     

Rapid quantitation of multiple ions released from HeLa cells during emodin induced apoptosis by low-cost capillary electrophoresis with capacitively coupled contactless conductivity detection

Change in cation concentration, including that of potassium and sodium, is characteristic of apoptosis, therefore it is significant to detect cation concentration changes. In this work a rapid, sensitive, and practical method was developed for the determination of Na⁺ and K⁺ concentration in HeLa cells during emodin induced apoptosis by a low-cost capillary electrophoresis device with capacitively coupled contactless conductivity detection (CE-C⁴D). Under the optimized conditions, both ions were baseline separated in 4 min with 40 mM MES/40 mM His containing 1 mM 18-crown-6 as the separation buffer at pH 6.0. The limit of detections (LODs) and limit of quantifications (LOQs) were 0.47–1.15 μM and 1.58–3.86 μM, respectively. The precision for migration times and peak areas was below 0.56% and 3.74%, respectively. The data proved that the concentration of cations in cells can be accurately quantified. It was found that the K⁺ concentration decreased from 82.2 μM to 52.7 μM, and the Na⁺ concentration increased from 62.4 μM to 127.2 μM during the process of apoptosis when the cell density was 1 × 10⁵ cells per mL. The low-cost CE-C⁴D provides a convenient way to decipher the interaction of Na⁺ and K⁺ in the regulation of cell apoptosis.

Change in cation concentration, including that of potassium and sodium, is characteristic of apoptosis, therefore it is significant to detect cation concentration changes.     

Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

Antimicrobial resistance among Acinetobacter baumannii is increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates of A. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 μg/ml). Time-kill studies were performed over 24 h using approximately 10⁶ CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without polymyxin B (0.5 μg/ml). The in vivo efficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (≥4×) was observed in the presence of PAβN. The intracellular concentration and in vitro bactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocycline-susceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.     

Cembrane-type diterpenoids from the gum resin of Boswellia carterii and their biological activities

Eight new cembrane-type diterpenoids, boscartins AH–AK (1–8), along with two known ones (9-10), were isolated from the gum resin of Boswellia carterii. Compounds 1–3 were characteristic of high oxidation assignable to three epoxy groups, while compounds 4–8 were characteristic of two epoxy groups. Spectroscopic examination was used to elucidate their structures. All isolates were evaluated for antiproliferative activity against HCT-116 human colon cancer cells, anti-inflammatory activity against nitric oxide (NO) production, and hepatoprotective activity in vitro. All of them showed weak antiproliferative activity (IC₅₀ > 100 μM), 8 exhibited potent inhibitory effects on NO production (IC₅₀ of 14.8 μM), with the others showing weak anti-inflammatory activity (IC₅₀ > 30 μM), and 1 exhibited more potent hepatoprotective activity than the positive control, bicyclol, at 10 μM against the damage induced by paracetamol in HepG2 cells.

Cembrane-type diterpenoids from the gum resin of Boswellia carterii.     

Inhibition of L5178Y Cells in Culture by Methotrexate and Antibiotics

Methotrexate, tetracycline, gentamicin, streptomycin, and penicillin inhibited the growth of L5178Y murine leukemia cells in culture with I₅₀ (concentration of drug that caused a 50% inhibition of growth at 72 h) values of 0.0028 μg/ml (6.2 × 10⁻⁹ M), 7.9 μg/ml, 200 μg/ml, 1,700 μg/ml, and 3,000 μg/ml (5,000 U/ml), respectively. At concentrations achieved clinically or utilized in the laboratory, the antibiotics did not alter the I₅₀ of methotrexate.