Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities

Impact of the Herbal Medicine Sophora flavescens on the Oral Pharmacokinetics of Indinavir in Rats: The Involvement of CYP3A and P-Glycoprotein

Sophora flavescens is a Chinese medicinal herb used for the treatment of gastrointestinal hemorrhage, skin diseases, pyretic stranguria and viral hepatitis. In this study the herb-drug interactions between S. flavescens and indinavir, a protease inhibitor for HIV treatment, were evaluated in rats. Concomitant oral administration of Sophora extract (0.158 g/kg or 0.63 g/kg, p.o.) and indinavir (40 mg/kg, p.o.) in rats twice a day for 7 days resulted in a dose-dependent decrease of plasma indinavir concentrations, with 55%–83% decrease in AUC0-∞ and 38%–78% reduction in Cmax. The CL (Clearance)/F (fraction of dose available in the systemic circulation) increased up to 7.4-fold in Sophora-treated rats. Oxymatrine treatment (45 mg/kg, p.o.) also decreased indinavir concentrations, while the ethyl acetate fraction of Sophora extract had no effect. Urinary indinavir (24-h) was reduced, while the fraction of indinavir in faeces was increased after Sophora treatment. Compared to the controls, multiple dosing of Sophora extract elevated both mRNA and protein levels of P-gp in the small intestine and liver. In addition, Sophora treatment increased intestinal and hepatic mRNA expression of CYP3A1, but had less effect on CYP3A2 expression. Although protein levels of CYP3A1 and CYP3A2 were not altered by Sophora treatment, hepatic CYP3A activity increased in the Sophora-treated rats. All available data demonstrated that Sophora flavescens reduced plasma indinavir concentration after multiple concomitant doses, possibly through hepatic CYP3A activity and induction of intestinal and hepatic P-gp. The animal study would be useful for predicting potential interactions between natural products and oral pharmaceutics and understanding the mechanisms prior to human studies. Results in the current study suggest that patients using indinavir might be cautioned in the use of S. flavescens extract or Sophora-derived products

Detection of Epstein-Barr virus (EBV) in human lymphoma tissue by a novel microbial detection array

BACKGROUND: Infectious agents are estimated to play a causative role in approximately 20% of cancers worldwide. Viruses, notably the Epstein-Barr virus (EBV), are associated with 10-15% of B-cell lymphomas and are found at a higher frequency in immunosuppressed patients. In this study, we screened human lymphoma tissues using a novel Lawrence Livermore Microbial Detection Array (LLMDA), a comprehensive detection system that contains probes for all sequenced viruses and bacteria. This technology has been applied to identify pathogen-associated diseases. RESULTS: We evaluated samples from 58 cases with various lymphoid tissue disorders using LLMDA. These included 30 B-cell lymphomas (9 indolent and 21 aggressive type), 2 T-cell lymphomas and 2 NK/T cell lymphomas, 4 plasmacytomas as well as 8 specimens of benign lymphoid tissue. Five of 21 high-grade B-cell lymphomas were positive for Epstein-Barr virus-encoded small RNA (EBER+), while all the indolent B-cell lymphomas were EBER-. Similarly, both NK/T cell lymphomas were EBER+, and the benign tissues were EBER-. We also screened 10 cases of post-transplant lymphoproliferative disorder (PTLD). Five of these cases (4 B-cell lymphomas and 1 NK/T cell lymphoma) were EBER+, and the remaining five cases were EBER-. CONCLUSIONS: We have confirmed the reliability of the LLMDA methods by detecting EBV in EBV-positive lymphomas while observing no false-positive results in EBV-negative lymphomas. The LLMDA technique provides a sensitive and alternative method for identifying known viral pathogen associated with tumors and may prove useful for future clinical identification of novel cancer-associated viral pathogens

Pharmacokinetic Modelling of Efavirenz, Atazanavir, Lamivudine and Tenofovir in the Female Genital Tract of HIV-Infected Pre-Menopausal Women

BACKGROUND AND OBJECTIVES: A previously published study of antiretroviral pharmacokinetics in the female genital tract of HIV-infected women demonstrated differing degrees of female genital tract penetration among antiretrovirals. These blood plasma (BP) and cervicovaginal fluid (CVF) data were co-modelled for four antiretrovirals with varying CVF exposures. METHODS: Six paired BP and CVF samples were collected over 24 h, and antiretroviral concentrations determined using validated liquid chromatography (LC) with UV detection or LC-mass spectrometry analytical methods. For each antiretroviral, a BP model was fit using Bayesian estimation (ADAPT5), followed by addition of a CVF model. The final model was chosen based on graphical and statistical output, and then non-linear mixed-effects modelling using S-ADAPT was performed. Population mean parameters and their variability are reported. Model-predicated area under the concentration-time curve during the dosing interval (AUC(τ)) and exposure ratios of CVF AUC(τ):BP AUC(τ) were calculated for each drug. RESULTS: The base model uses first-order absorption with a lag time, a two-compartment model, and a series of transit compartments that transfer the drug from BP to CVF. Protein-unbound drug transfers into CVF for efavirenz and atazanavir; total drug transfers for lamivudine and tenofovir. CVF follows a one-compartment model for efavirenz and atazanavir, and a two-compartment model for lamivudine and tenofovir. As expected, inter-individual variability was high. Model-predicted CVF AUC(τ):BP AUC(τ) ratios are consistent with published results. CONCLUSIONS: This is the first pharmacokinetic modelling of antiretroviral disposition in BP and CVF. These models will be further refined with tissue data, and used in clinical trials simulations to inform future studies of HIV pre-exposure prophylaxis in women