Evaluation of the probable synergistic toxicity of selected potentiated antiretroviral and antibiotics on some aquatic biomarker organisms.

Environmental effects of active pharmaceutical compounds (APCs) in the environment are not well characterized, hence the need for comprehensive evaluation. This study employed three bioassays using three organisms, namely, Allium cepa, Daphnia magna, and Salmonella typhimurium, in the ecotoxicity study of lone and a mixture of selected APCs, namely, lamivudine (L), an antiretroviral, and ciprofloxacin (C) and sulfamethoxazole (S), antibiotics, at a concentration range between 10 and 100 ppb, in order to evaluate the potential of the lone and ternary mixture to exert synergistic toxicity. Study results from exposure to lone APCs showed that the L, C, and S trio individually had fatal impacts on daphnids, with mortality rates of 100, 75, and 95%, respectively, after 48 h. Sulfamethoxazole showed a mutagenic tendency, with a mutation ratio (background/sample ratio) of 2.0. Lamivudine showed a lethal impact on the root length of A. cepa (p > 0.05, p = 3.60E-3). Further microscopic examination of the A. cepa root tip revealed chromosomal aberrations on exposure to each compound. The LCS-mix ecotoxicology bioassays indicated a synergistic effect on the daphnids, probably due to potentiation. Although the LCS mix had a cytotoxic effect (evidenced by the absence of bacteria colonies) on exposed TA 98 P450 Salmonella typhimurium strain, this effect was not observed in other bacterial strains. Microscopic examination of A. cepa exposed to the LCS-mix revealed an aberration in the mitotic stage of the cell. The impact of combination of the pharmaceuticals in aqueous ecosystems was greater than when exposed to the tested individual pharmaceutical compounds. Study result showed that these compounds have tendencies to pose a higher risk to exposed living entities when in combined/potentiated forms, and this could lead to distortion of the regular functioning of the ecosystem, particularly bacterial and other microbial populations that are listed among primary producers of the aquatic food web.

In Vitro and In Vivo Renoprotective Effects of Telbivudine in Chronic Hepatitis B Patients Receiving Nucleotide Analogue.

AIM: Renal toxicity of adefovir disoproxil (ADV) and tenofovir disoproxil fumarate (TDF) is a significant concern in chronic hepatitis B (CHB) patients. Early observational clinical data suggested that telbivudine (LdT) might have renoprotective effects. METHODS: In this prospective study, consecutive CHB patients on combined lamivudine (LAM) + ADV/TDF were switched to LdT + ADV/TDF at recruitment and were followed up for 24 months. Estimated glomerular filtration rate (eGFR) was calculated with the modification of diet in renal disease equation. The effects of LdT on cell viability and expression of kidney injury or apoptotic biomarkers were investigated in cultured renal tubular epithelial cell line HK-2. RESULTS: Thirty-one patients (median age 55 years, 90.3% male) were recruited (54.8% TDF: 45.2% ADV). Serum HBV DNA was undetectable at all time points. Median eGFR was 70.2 (IQR 62.6-77.9) and 81.5 (IQR 63.6-99.1) mL/min/1.73 m2 at baseline and 24 months, respectively (p < 0.001). Downstaging of chronic kidney disease was observed in eight (25.8%) patients and was more common in ADV-treated compared to TDF-treated patients (7/8 vs. 1/17, p = 0.011; OR 16, 95% CI 1.643-155.766, p = 0.017). In vitro data showed that adding LdT to ADV or TDF was associated with improved cell viability and lower expression of injury and apoptotic biomarkers compared with ADV or TDF alone. Treatment was prematurely discontinued in four(12.9%) patients due to myalgia. CONCLUSIONS: Clinical and in vitro data suggest that LdT has renoprotective effects in patients on long-term ADV/TDF treatment. LdT may be considered as an adjuvant therapy in this special group of patients with renal impairment (NCT03778567).

Effect of co-administration of Hypoxis hemerocallidea extract and antiretroviral therapy (HAART) on the histomorphology and seminal parameters in Sprague Dawley rats.

Although the successful introduction and rollout of antiretroviral therapy has impacted positively on morbidity and mortality of HIV-positive patients, its interaction with plant-based adjuvants remain sparsely investigated. We report the interaction and effects of adjuvant treatment with highly active antiretroviral therapy (HAART) and Hypoxis hemeocallidea (HH) extracts on testicular structure of rats. A total of 63 pathogen-free adult male Sprague Dawley rats were divided into nine groups and treated according to protocols. HAART cocktail predisposed to significant negative testicular parameters of sperm count, motility and seminiferous tubular epithelial height (quantitatively) (p < .03) and also altered the histomorphology of tubules with diffuse hypoplasia in seminiferous tubules. The higher dose of HH showed a better ability to mitigate the altered parameters and compares favourably with vitamin C in this protocol. While HH did not show any deleterious impact on morphometric data, its role as adjuvant did not significantly reduce the negative impact of HAART on morphometric indices especially with the lower dosage. Further investigations are warranted on the interactions between HAART and Hypoxis.

Chronic action of lamivudine and ritonavir on maternal and fetal liver and kidney of albino pregnant rats (Rattus norvegicus albinus, Rodentia, Mammalia): morphological and biochemical aspects.

PURPOSE: To investigate the morphological and biochemical effects of lamivudine associated with ritonavir on maternal and fetal livers and kidneys throughout the pregnancy of albino rats. MATERIALS AND METHODS: Forty pregnant rats were divided into four numerically equal groups: control (C), experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3). Only distilled water was given to the control group, while groups E1, E2, and E3 received, respectively, 5, 15 and 45 mg/kg of lamivudine associated with 20, 60, and 180 mg/kg of ritonavir, per day, throughout the pregnancy. On the 20th day of the pregnancy, the histological structure of the maternal and fetal livers and kidneys was analyzed by means of optical microscopy, along with the blood concentrations of AST, ALT, urea, and matrix creatinine. The numerical variables were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison test. RESULTS: The histological alterations occurred in both the maternal livers and the maternal kidneys, particularly in group E3, which received the greatest therapeutic dosage (nine times). The blood levels ofALT in group E3 were significantly lower than in the other groups (p = 0.0037). The urea and creatinine levels in the blood were significantly lower in group E1 (p = 0.0420 andp = 0.0108, respectively). CONCLUSIONS: rhe association of lamivudine and ritonavir affected the histological structure of the kidneys of the matrices of group E3. There was a significant decrease in the blood values of urea e creatinine in group El.

Inhibitory effect of Phyllanthus urinaria L. extract on the replication of lamivudine-resistant hepatitis B virus in vitro.

BACKGROUND: Long-term treatment of chronic hepatitis B (CHB) with nucleos(t)ide analogs results in the emergence of drug-resistant hepatitis B virus (HBV) harboring mutations in the polymerase (P) gene. The Phyllanthus extract has anti-HBV activity; however, its antiviral activity against lamivudine (LMV)-resistant mutants has not been examined. METHODS: HBV harboring LMV-resistant mutations (rtM204I, rtM204V, and rtM204S) in the P gene at the YMDD ((203)tyrosine-methionine-aspartate-aspartate(206)) reverse transcriptase (RT) active site were generated and their sensitivity to Phyllanthus urinaria koreanis extract examined. Southern blotting and real-time PCR were used to determine the concentration of plant extract required to inhibit HBV DNA synthesis by 50 and 90% (EC50 and EC90, respectively). An enzyme-linked immunosorbent assay was used to measure the EC50 of HBV surface antigen (HBsAg) and HBV core antigen (HBcAg) secretion, and the 50% cytotoxic concentration of the extract was measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Real-time RT-PCR was used to measure mRNA expression levels. RESULTS: The expression of intracellular HBV DNAs in HBV WT- or mutant-transfected HepG2 cells decreased upon treatment with Phyllanthus extract. The secretion of HBsAg and HBcAg also fell in a dose-dependent manner. Phyllanthus extract induced interferon-beta (IFN-ß), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) mRNA expression in HBV WT-transfected HepG2 cells, possibly via activation of extracellular signal-regulated kinases and c-jun N-terminal kinases and the induction of retinoic acid inducible gene-I, toll-like receptor 3, myeloid differentiation primary response gene 88, and/or tumor necrosis factor receptor-associated factor 6 gene expression. HBV transfection in the absence of extract or exposure of cells to extract alone did not trigger these signaling cascades. CONCLUSIONS: Phyllanthus extract inhibited HBV DNA synthesis and HBsAg and HBcAg secretion by replicating cells harboring HBV wild-type and LMV-resistant mutants, likely by inducing the expression of IFN-ß, COX-2, and IL-6. These data indicate that Phyllanthus extract may be useful as an alternative therapeutic agent for the treatment of drug-resistant CHB patients.

HBV carrying drug-resistance mutations in chronically infected treatment-naive patients.

BACKGROUND: Nucleoside/nucleotide analogue (NA) treatment causes selection pressure for HBV strains carrying mutations conferring NA resistance. Drug-resistance mutations occur in the reverse transcriptase (RT) region of the HBV polymerase gene and spontaneously arise during viral replication. These mutations can also alter the hepatitis B surface (HBs) protein and in some cases reduce binding to HBs antibodies. The spread of NA-resistant HBV may impact the efficacy of antiviral treatment and hepatitis B immunization programmes. In this study, we used direct sequencing to assess the occurrence of HBV carrying known mutations that confer NA resistance in the largest cohort of treatment-naive patients with chronic hepatitis B (CHB) to date. METHODS: HBV DNA samples isolated from 702 patients were sequenced and the RT region subjected to mutational analysis. RESULTS: There was high genetic variability among the HBV samples analysed: A1 (63.7%), D3 (14.5%), A2 (3.3%), A3 (0.1%), B1 (0.1%), B2 (0.1%), C2 (0.9%), D1 (0.9%), D2 (4.6%), D4 (5.1%), D unclassified subgenotype (0.7%), E (0.6%), F2a (4.6%), F4 (0.4%) and G (0.4%). HBV strains harbouring mutations conferring NA resistance alone or combined with compensatory mutations were identified in 1.6% (11/702) of the patients. CONCLUSIONS: HBV strains harbouring resistance mutations can comprise the major population of HBV quasispecies in treatment-naive patients. In Brazil, there is a very low frequency of untreated patients who are infected with these strains. These findings suggest that the spread and natural selection of drug-resistant HBV is an uncommon event and/or most of these strains remain unstable in the absence of NA selective pressure.

A new antiviral: chimeric 3TC-AZT phosphonate efficiently inhibits HIV-1 in human tissues ex vivo.

Although more-recently developed antivirals target different molecules in the HIV-1 replication cycle, nucleoside reverse transcriptase inhibitors (NRTIs) remain central for HIV-1 therapy. Here, we test the anti-HIV activity of a phosphonate chimera of two well-known NRTIs, namely AZT and 3TC. We show that this newly synthesized compound suppressed HIV-1 infection in lymphoid tissue ex vivo more efficiently than did other phosphonates of NRTIs. Moreover, the new compound was not toxic for tissue cells, thus making the chimeric phosphonate strategy a valid approach for the development of anti HIV-1 compound heterodimers.

Testicular histomorphologic and stereological alterations following short-term treatment with highly active antiretroviral drugs (HAART) in an experimental animal model.

The increased accessibility of antiretroviral therapy continues to positively drive the reduction in viral load and survival of patients despite the attendant reproductive toxicities. We propose that testicular damage caused by highly active antiretroviral therapy (HAART) can be attenuated by antioxidant treatment by investigating the testicular histomorphologic and stereological effects of antiretroviral drugs and its interaction with antioxidants using an experimental animal model. Sprague-Dawley rats were divided into seven groups of six rats per group (A, B... G) using simple random sampling and treated orally with 0.9% normal saline as placebo, a HAART cocktail of stavudine, lamivudine and nevirapine using the adjusted human therapeutic doses of 200, 600 and 350-400 mg/day, respectively, and antioxidants ascorbic acid (vitamin C) and I.M α-tocopherol (vitamin E). Animals were killed after 4 weeks and testicular tissue harvested and processed for light microscopy and stereological evaluations. The results were interpreted by a Veterinary pathologist blinded to the study. No animal died during the experimental period. The histopathological assessment of the testis of animals treated with placebo, ascorbic acid alone and α-tocopherol alone as well as vitamin E + HAART displayed normal testicular microanatomy. Groups treated with HAART alone, HAART + vitamin C + vitamin E and vitamins C + HAART showed extensive seminiferous tubular atrophy, necrosis and hypocellularity in the histoarchitectural patterns. While testicular cross-sectional area of seminiferous tubules remained unaffected by HAART, epithelial heights significantly decreased (p < 0.05) when compared with controls. There was marked (p < 0.05) increased in testicular-body weight ratio in HAART group. The results show that vitamin E could be useful in protecting testicular tissue from toxicities of HAART regimes as these results mirrors stereological data for the groups. HAART presents with deleterious histopathological changes in the testes causing tubular atrophy with altered morphometric indices. Supplementation with vitamin E appears to be a better adjuvant antioxidant that ameliorates these deleterious effects.

Preclinical characterization of GLS4, an inhibitor of hepatitis B virus core particle assembly.

Hepatitis B virus (HBV)-associated chronic liver diseases are treated with nucleoside analogs that target the virus polymerase. While these analogs are potent, drugs are needed to target other virus-encoded gene products to better block the virus replication cycle and chronic liver disease. This work further characterized GLS4 and compared it to the related BAY 41-4109, both of which trigger aberrant HBV core particle assembly, where the virus replication cycle occurs. This was done in HepAD38 cells, which replicate HBV to high levels. In vitro, GLS4 was significantly less toxic for primary human hepatocytes (P < 0.01 up to 100 µM), inhibited virus accumulation in the supernantant of HepAD38 cells (P < 0.02 up to 100 nM), inhibited HBV replicative forms in the liver with a significantly lower 50% effective concentration (EC50) (P < 0.02), and more strongly inhibited core gene expression (P < 0.001 at 100 to 200 nM) compared to BAY 41-4109. In vivo characterization was performed in nude mice inoculated with HepAD38 cells, which grew out as tumors, resulting in viremia. Treatment of mice with GLS4 and BAY 41-4109 showed strong and sustained suppression of virus DNA to about the same extents both during and after treatment. Both drugs reduced the levels of intracellular core antigen in the tumors. Alanine aminotransferase levels were normal. Tumor and total body weights were not affected by treatment. Thus, GLS4 was as potent as the prototype, BAY 41-4109, and was superior to lamivudine, in that there was little virus relapse after the end of treatment and no indication of toxicity.

Characterization of antiviral activity of benzamide derivative AH0109 against HIV-1 infection.

In the absence of an effective vaccine against HIV-1 infection, anti-HIV-1 strategies play a major role in disease control. However, the rapid emergence of drug resistance against all currently used anti-HIV-1 molecules necessitates the development of new antiviral molecules and/or strategies against HIV-1 infection. In this study, we have identified a benzamide derivative named AH0109 that exhibits potent anti-HIV-1 activity at an 50% effective concentration of 0.7 µM in HIV-1-susceptible CD4(+) C8166 T cells. Mechanistic analysis revealed that AH0109 significantly inhibits both HIV-1 reverse transcription and viral cDNA nuclear import. Furthermore, our infection experiments indicated that AH0109 is capable of disrupting the replication of HIV-1 strains that are resistant to the routinely used anti-HIV-1 drugs zidovudine, lamivudine, nevirapine, and raltegravir. Together, these findings provide evidence for a newly identified antiviral molecule that can potentially be developed as an anti-HIV-1 agent.

Vitamin D attenuates nucleoside reverse transcriptase inhibitor induced human skeletal muscle mitochondria DNA depletion.

OBJECTIVE: To evaluate the impact of the active metabolite of vitamin D, 1α,25-dihydroxycholecalciferol (1,25D3), on nucleoside reverse transcriptase inhibitor (NRTI) induced mitochondrial DNA (mtDNA) depletion in human skeletal muscle myoblasts and myotubes. DESIGN: mtDNA was quantified in human skeletal muscle myoblasts and myotubes following 1,25D3 and NRTI treatment using real-time PCR. METHODS: Human skeletal muscle myoblasts and myotubes were treated with didanosine (ddI), stavudine (d4T), zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) alone or in combination either in the presence or absence of 1,25D3 for 5 days. Cells were harvested, DNA extracted and mtDNA quantified. RESULTS: ddI and ddI-d4T significantly decreased both myoblast and myotube mtDNA in the absence of 1,25D3 compared with untreated controls (P≤0.029). In addition, the ZDV-3TC combination resulted in a 47% decrease in myotube mtDNA (P=0.005). 1,25D3 increased myotube mtDNA levels in ddI, ZDV, 3TC, ABC, ddI-d4T, d4T-3TC, ZDV-3TC, ZDV-ABC and ZDV-3TC-ABC-containing regimens and myoblast mtDNA levels in ddI, d4T, ZDV, 3TC, ddI-d4T, ZDV-3TC and ZDV-ABC-containing regimens. Of note, 1,25D3 protected against myotube mtDNA depletion following ZDV-3TC treatment, rendering them similar to 1,25D3 untreated controls (P=0.62), and increased both myotube and myoblast mtDNA two to three-fold in ddI-containing regimens (P<0.05). CONCLUSION: 1,25D3 confers a protective effect against NRTI-induced mitochondrial toxicity in skeletal muscle myoblasts and myotubes. These findings support a protective role for vitamin D in preventing mitochondrial toxicity and suggest that supplemental vitamin D may protect against NRTI-associated mitochondrial toxicity.

Synergistic antivirus effect of combined administration of Combivir with Angelica polysaccharide sulfate.

This study is to investigate the synergistic effect of Anglica polysaccharide sulfate (APS-1) and Combivir, an anti-AIDS drug, on murine leukemia virus in vivo. As the results shown, the virus replication was significantly decreased by the combination of APS-1 and Combivir, which tended to be further decreased (58% inhibition) when compared with that of Combivir alone (51% inhibition). Furthermore, both the percentage of CD4(+) cells and CD4(+)/CD8(+) ratio in peripheral blood cells were significantly enhanced by this combined administration, while the CD4(+) cells was only slightly increased and CD4(+)/CD8(+) ratio was not affected by Combivir alone. Additionally, combination of APS-1 and Combivir also alleviated the toxicity of Combivir. APS-1 not only increased the survival rate of mice administered with LD(50) dose of Combivir, but also reduced the hematologic toxicity induced by Combivir, RBC, HGB and PLT were restored to normal level. These results suggest that APS-1 had synergistic effect with Combivir, which provided new insight into the potential clinical use of polysaccharide sulfate in anti-AIDS field.

Antiviral activity of nucleoside analogues against norovirus.

BACKGROUND: Norovirus (NoV) is the leading cause of epidemic gastroenteritis worldwide. The lack of a cell culture has significantly hampered the development of effective therapies against human NoV. Clinically approved nucleoside and non-nucleoside analogues have been used successfully against RNA viruses. METHODS: In this study, we evaluated the efficacy of four nucleoside analogues (2'-C-MeC, 2'-F-2'-C-MeC, ß-D-N(4)-hydroxycytidine [NHC] and lamivudine) on Norwalk virus (NV) RNA levels and protein expression in NV replicon-harbouring cells (HG23 cells), and their efficacy in blocking murine norovirus (MNV) replication in RAW 264.7 cells. RESULTS: 2'-C-MeC and 2'-F-2'-C-MeC reduced MNV RNA levels and infectivity in RAW 264.7 cells in a concentration- and time-dependent manner. The median effective concentrations (EC(50)) of 2'-C-MeC and 2'-F-2'-C-MeC were 6.9 µM and 12.7 µM, respectively. 2'-C-MeC, 2'-F-2'-C-MeC and NHC reduced NV RNA levels and protein expression in HG23 cells. For the NV replicon, the EC(50) of 2'-C-MeC (1.3 µM) was comparable to the antiviral activity of NHC (1.5 µM) and twofold more potent than 2'-F-2'-C-MeC (3.2 µM). The combination of 2'-C-MeC/ribavirin resulted in modest synergistic activity, whereas NHC/ribavirin was antagonistic for NV replication in HG23 cells. CONCLUSIONS: The antiviral activity of 2'-C-MeC against strains of two different NoV genogroups and the low EC(50) suggest that this nucleoside analogue may be effective against the more prevalent GII NoVs. In the absence of a vaccine, antiviral agents could be an effective intervention to control the spread of human NoV in populations at a high risk for NoV disease.

Anti-HBV activities of Streblus asper and constituents of its roots.

The extracts from leaves, heartwood, barks and roots of the Streblus asper were investigated for anti-HBV activities, separately. The results suggested that the MeOH extracts of the heartwood, barks, and roots exhibited good anti-HBV activities. Further investigations displayed that ethyl acetate and n-butanol soluble parts of their MeOH extracts showed more significant anti-HBV activities. Moreover, a new lignan, together with 11 known compounds, was isolated from n-butanol-soluble part of MeOH extract of the roots of S. asper. The structures were elucidated by spectroscopic methods, including 1D NMR ((1)H NMR, (13)C NMR), 2D NMR (HMQC, HMBC) and HR-EI-MS experiments. Compounds 1-3 were evaluated for their anti-HBV activities. Honokiol showed significant anti-HBV activity with IC(50) values of 3.14µM and 4.74µM for HBsAg and HBeAg with no cytotoxicity respectively, while lamivudine (3TC, positive control) exhibited weak anti-HBV activity with IC(50) values of 11.81µM and 25.80µM for HBsAg and HBeAg respectively.

High-resolution melting and real-time PCR for quantification and detection of drug-resistant HBV mutants in a single amplicon.

BACKGROUND: Antiviral therapy by nucleoside/nucleotide analogues (NAs) effectively reduces HBV replication in chronic hepatitis B (CHB) patients. Because long-term NA treatments will eventually select for drug-resistant mutants, early detection of mutants and frequent monitoring of viral loads is crucial for successful NA therapy. Because no efficient test for one-tube quantification and qualification of various HBV-resistant mutants exists, we propose to use high-resolution melting (HRM) analysis in combination with real-time PCR to achieve this unmet need. METHODS: We developed a single amplicon for detecting HBV mutants resistant to lamivudine (LMV), adefovir (ADV) and entecavir (ETV), which are commonly used for CHB treatment. Our design consists of two steps: real-time PCR for viral quantification, and hybridization probe HRM analysis for detection of specific drug-resistant mutants. RESULTS: Assay quantification was accurate (R=0.98) for viral loads from 10(3) to 10(9) copies/ml. HRM analysis produced distinct melting temperatures that clearly distinguished the mutants, rtM204V/I (LMV), rtA181V and rtN236T (ADV), and rtT184G and rtM250V (ETV), from their respective wild types. The assay detected mutants at only 10-25% of the HBV population. The clinical applicability of this assay was tested in a pilot study with serial samples from patients receiving LMV treatment. CONCLUSIONS: Flexibility, speed and cost-efficiency are additional benefits unique to our assay. The clinical sample results further support the feasibility of applying our design to frequent and long-term monitoring of CHB patients receiving NA treatments in the clinical setting.

Measuring enzymatic HIV-1 susceptibility to two reverse transcriptase inhibitors as a rapid and simple approach to HIV-1 drug-resistance testing.

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC-resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive "Amp-RT" assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings.

5'-phosphonate derivatives of 2',3'-dideoxy-3'-thiacytidine as new anti-HIV prodrugs.

Two new phosphonate 3TC prodrugs were synthesized and studied in MT-4 cells as inhibitors of HIV replication. Their pharmacokinetic parameters were evaluated following intragastric administration in rabbits and oral administration in dogs. Both compounds were much less toxic than parent 3TC in cell cultures and could generate the active nucleoside in laboratory animals.

Anti-hepatitis B virus activities of cinobufacini and its active components bufalin and cinobufagin in HepG2.2.15 cells.

Cinobufacini (Huachansu) is a Chinese medicine prepared from the skin of Bufo bufo gargarizans Cantor (Bufonidae), which has long been used in traditional Chinese medicine (TCM). The aim of present study was to examine the anti-hepatitis B virus (HBV) activities of cinobufacini and its active components bufalin and cinobufagin in the human HBV-transfected cell line HepG2.2.15. The hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) concentrations in cell culture medium were determined by chemiluminescent enzyme immunoassay after HepG2.2.15 cells were respectively treated with different concentrations of cinobufacini, bufalin, and cinobufagin for 3 or 6 d. HBV DNA and mRNA were determined using transcription-mediated amplification and real-time polymerase chain reaction (PCR), respectively. On d 3, cinobufacini at a concentration of 1 µg/ml had no activity against HBV virological markers. However, on d 6, cinobufacini at 1 µg/ml effectively inhibited the secretion of HBsAg, HBeAg, and HBcrAg by 29.58, 32.87, and 42.52%. It was more potent than the positive control lamivudine (100 µg/ml). Bufalin and cinobufagin slightly inhibited HBV antigen secretion. Treatment with cinobufacini, bufalin, or cinobufagin had no anti-HBV effect on DNA in cell culture medium. Consistent with the HBV antigen reduction, HBV mRNA expression was markedly inhibited in comparison to the control when HepG2.2.15 cells were treated with cinobufacini, bufalin, or cinobufagin. Results suggested that cinobufacini had more potent activity against HBV antigen secretion than its components bufalin and cinobufagin and this inhibitory role was attributed to the specific inhibition of HBV mRNA expression.

Scutellariae radix suppresses hepatitis B virus production in human hepatoma cells.

The traditional Chinese medicine Xiao-Chai-Hu-Tang (HD-7) has been widely used to treat liver diseases in China and Japan. HD-7 consists of seven different ingredients, but which one provides the therapeutic benefits is still unknown. Here, we identified the "Minister herb" Scutellariae radix (HD-1S), but not the "King herb" Bupleuri radix (HD-1B) in HD-7, as the active component that suppresses HBV gene expression and virus production in human hepatoma cells. We have found that an aqueous extract of HD-1S not only suppressed wild-type virus but also lamivudine-resistant HBV mutant in human hepatoma cells. We show that HD-1S selectively suppresses HBV core promoter activity. Electrophoretic mobility shift assay analysis has revealed that HD-1S treatment decreases the DNA-binding activity of nuclear extract of HepA2 cells to a specific cis-element of the HBV core promoter, which includes the peroxisome proliferator-activated receptor binding site and HNF4. Furthermore, ectopic expression of PGC-1 abolished the suppression of HD-1S on HBV core promoter activity suggesting that HD-1S may act through modulating hepatic transcriptional machinery to suppress HBV viral gene expression and virus production.

Inhibitory effect of emodin and Astragalus polysaccharide on the replication of HBV.

AIM: To evaluate the anti-viral effect of emodin plus Astragalus polysaccharide (APS) in hepatitis B virus (HBV) transgenic mice. METHODS: Sixty HBV transgenic mice (HBV TGM) whose weight varied between 18 and 24 g were randomly divided into 3 groups, with 20 mice in each group. Group A was the normal control, where the mice were treated with physiological saline; group B was the positive control where the mice were treated with lamivudine solution (100 mL/kg per day). Group C was the experimental group where the mice were treated with physiological saline containing emodin and APS (57.59 mg/kg per day and 287.95 mg/kg per day, respectively). The mice were treated daily for 3 wk. After 1 wk recovery time, the mice were sacrificed and serum as well as liver tissues were collected for ELISA and histological examination. RESULTS: After 21 d treatment, HBV DNA levels in group B and group C significantly declined when compared with group A (P < 0.05). However, a significant increase in HBV DNA content was observed in group B, whereas this phenomenon was not observed in group C. A reduction in the contents of HBsAg, HBeAg and HBcAg in the mice from group B and C was observed when compared with group A. CONCLUSION: Emodin and APS have a weak but persistent inhibitory effect on HBV replication in vivo, which may function as a supplementary modality in the treatment of hepatitis B infection.