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.

A method employing capillary electrophoresis (CE) with tandem mass spectrometry (MS) has been developed for the simultaneous determination, on one hand, of zidovudine (AZT) with stavudine (d4T), and on the other hand, of lamivudine (3TC) with a didanosine metabolite (ddA), four potent human immunodeficiency virus reverse transcriptase (RT-HIV) inhibitors. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) as well as the influence of magnesium cation upon electroosmotic flow, electrophoretic mobility and peak efficiency has been studied. The limit of detection (LOD) by this method is 2.5 ppb for AZT and 20 ppb for d4T, 2 ppb for ddA and 5 ppb for 3TC, respectively. This paper illustrates the current importance in CE-ESI/MS/MS technique as a complementary or substituted method to measure levels (at ng/mL) of anti-HIV drugs alone or in combination.

Background Polygonum chinense Linn. is a common medicinal plant in Southeast Asia and has been used in traditional medicine in Vietnam. The plant contains phytochemicals with various biological properties; however, its antiviral effect has not yet been demonstrated. This study was aimed to evaluate the anti-influenza virus activity of crude extracts of P. chinense, to characterize antiviral metabolites therefrom and to investigate their mechanisms of antiviral action.MethodsThe methanol (MeOH) extract and organic solvent layers of P. chinense were prepared by extraction and partition with relevant solvents. The ethyl acetate (EtOAc) layer showing antiviral activity was chromatographed repeatedly on SiO2 and Sephadex LH-20 columns to give eight pure metabolites. Their chemical structures were determined by NMR and MS spectral data. Anti-influenza virus activity of the eight metabolites against virus strains A/Puerto Rico/8/34 (H1N1, PR8), A/Hong Kong/8/68 (H3N2, HK) and B/Lee/40 (Lee) was evaluated on the basis of cytopathic effect (CPE) and plaque inhibition assays. Time-of-addition, confocal microscopy and neuraminidase inhibition assay were performed for mode-of-action studies of active ingredients.ResultsThe MeOH extract of P. chinense showed anti-influenza virus activity with EC50 values ranging from 38.4 to 55.5 μg/mL in a CPE inhibition assay. Among the eight pure metabolites isolated from P. chinense, ellagic acid (PC5), methyl gallate (PC7) and caffeic acid (PC8) significantly inhibited viral replication in a dose-dependent manner in both plaque inhibition and CPE inhibition assays with EC50 values ranging from 14.7 to 81.1 μg/mL and CC50 values higher than 300 μg/mL. Mode-of-action studies suggested that PC5 and PC7 suppress virus entry into or replication in cells, while PC8 targets influenza viral neuraminidase, even oseltamivir-resistant one.ConclusionThese results demonstrated that P. chinense and its metabolites possess effective anti-influenza virus activities. The botanical materials of P. chinense could be a promising multitargeted inhibitor of influenza A and B viruses and applied to development of a novel herbal medicine.

BACKGROUND In high income countries in Europe and North America, early identification and intervention for autism spectrum disorders (ASD) have been linked to improved long-term outcomes. However, in most low and middle income countries (LMIC) little is known about use or availability of such services, which limits the ability of policy and development planning. The purpose of the present study was assess the use of ASD services in Vietnam, an Asian LMIC, in order to identify areas within the field that should be specifically targeted to improve ASD services in Vietnam. METHODS Surveys were conducted in six different cities across Vietnam with 90 parents/caregivers of children with ASD, 115 professionals working with children with ASD, and 10 directors of agencies providing ASD services. Parents/caregivers’ survey assessed demographics and information regarding their child’s symptoms and services the child received. Professionals’ survey assessed their demographic and professional background, the ASD services they provide, and their perspective on the quality of ASD services at their agency. Directors’ survey included these same questions as well as additional questions regarding the operation of the agency. RESULTS Early identification and intervention ASD services are available in Vietnam, at least in major cities. However, there is a lack of well-trained professionals, the tools used for evaluation and diagnosis are limited, outdated and unstandardized, and the quality of services is questionable. Most importantly, a scientific evidence base for services is absent, and the country lacks an official governmental policy for supporting children with ASD. CONCLUSIONS Research directly assessing the quality of ASD services in Vietnam is needed. Most centrally, efforts at facilitating governmental policy and support advocacy are needed to increase the likelihood that families and children with ASD will receive appropriate and effective services.

No abstract