Senescence and oxidative stress toxicities induced by lamivudine and tenofovir in Drosophila melanogaster.

BACKGROUND: Lamivudine and tenofovir disoproxil fumarate act against the replication of hepatitis B and human immunodeficiency viruses via inhibition of the reverse transcriptase enzyme activity, thereby preventing the synthesis of viral DNA. Chronic administration of these drugs has been associated with toxicities, including senescence, oxidative stress and premature death. A study of these toxicities in Drosophila melanogaster, which share 75% genomic similarity with humans could help to develop a pharmacologic intervention. METHODS: Susceptibility of D. melanogaster for lamivudine and tenofovir-induced toxicities were investigated. First, flies (≤3 days old) were fed with drugs-supplemented diet at varying concentrations (1mg to 300mg/10-gram diet) or distilled water for seven days to determine LD50. Secondly, five groups of 60 flies were fed with four concentrations of test drugs: 2.9mg, 5.82mg, 11.64mg and 23.28mg each per 10-gram diet for 28 days survival and lifespan assays. Then 5-day treatment plan was utilized to determine drugs toxicities on climbing ability and some biomarkers of oxidative stress. Finally, molecular docking was carried out using the Auto-dock vina mode to predict the biological interactions between the test drugs and D. melanogaster acetylcholinesterase (AChE) or glutathione-S-transferase (GST). RESULTS: The LD50 of lamivudine or tenofovir was 47.07 or 43.95mg/10g diet, respectively. Each drug significantly (P<0.05) reduced the survival rate, longevity and climbing performance of the flies dose-dependently. These drugs also altered levels of biochemical parameters: AChE, GST, superoxide dismutase (SOD), catalase (CAT), total thiol (T-SH), and malondialdehyde (MDA) of the flies significantly (P<0.05). In silico molecular analysis showed that the test drugs interacted with significantly (P<0.05) higher binding affinities at the same catalytic sites of D. melanogaster GST and AChE compared with substrates (glutathione or acetylcholine). CONCLUSION: The significant lamivudine and tenofovir-induced toxicities observed as increased mortality, climbing deficits and compromised antioxidant defence in D. melanogaster demands further research for possible pharmacological intervention.

The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts.

Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC50 values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 µmol·L-1, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC0-∞) and maximal plasma concentration (Cmax) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.

In vitro screening of nucleoside analog combinations for potential use in anti-HIV therapy.

With the results from the Delta and ACTG 175 clinical trials clearly showing an increased benefit of two drugs over monotherapy, combination nucleoside analog therapy looks set to play a major role in the battle against HIV. It is therefore essential that suitable combinations of drugs are used in clinical trials. We investigated the intracellular activation of zidovudine (ZDV), zalcitabine (ddC), and lamivudine (3TC) in MOLT-4 cells in two- and three-drug combinations at clinically achieved concentrations. The phosphorylation of ZDV and 3TC to their active triphosphate anabolites was not affected by the presence of the other drugs studied. However, the phosphorylation of ddC was significantly inhibited when incubated with 3TC, resulting in levels of ddC triphosphate (ddC-TP) less than 50% of control values. This can be explained by the requirement of both nucleoside analogs for the enzyme deoxycytidine kinase to carry out the initial step in their phosphorylation pathways, and by the comparatively low plasma concentrations of ddC achieved in vivo. These results suggest that regimens containing nucleoside analogs should be designed taking into account potential interactions affecting phosphorylation.