Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency.

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8-/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.

Evaluation of a six-probe cocktail (caffeine, tolbutamide, omeprazole, dextromethorphan, midazolam, and digoxin) approach to estimate hepatic drug detoxification capability and dosage requirements after a single oral dosing in healthy Chinese volunteers.

The primary objectives of this study were to investigate the suitability of a 6-probe cocktail (caffeine, tolbutamide, omeprazole, dextromethorphan, midazolam, and digoxin) to be used as a tool for assessing the activity of drug metabolizing enzymes and transporters, and examine differences in the way drugs are handled among groups with different genetic regulation of these processes. This was a single-center, open-label, phase I clinical study involving 20 young, healthy Chinese volunteers (equal gender distribution). The subjects were administered a single, oral dose of the 6-probe cocktail and serum samples were collected to assess the disposition of the different probe substrates and produced metabolites. The serum samples were analyzed using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry technology. The DNA samples were subjected to whole exome sequencing. Nineteen healthy volunteers completed the study. The 6-probe cocktail was safe and well-tolerated by all the subjects. The parent substrates and metabolites-caffeine (paraxanthine), dextromethorphan (dextrorphan), digoxin, midazolam (1-hydroxy-midazolam), omeprazole (5-hydroxy-omeprazole), and tolbutamide (4-hydroxy-tolbutamide)-were within the detectable window. Genetic variations known to alter drug metabolism (CYP2D6*10, CYP2C19*2, CYP2C19*3, and CYP2C9*3) were identified and generally correlated with phenotypic status. The 6-probe cocktail appeared to be suitable for assessing drug metabolizing activities. This, in conjunction with individual genetics, will pave the way for the implementation of personalized medicine in clinical practice. This will hopefully improve efficacy and reduce the incidence of adverse drug reactions.

Population Pharmacokinetic Model of Plasma and Cellular Mycophenolic Acid in Kidney Transplant Patients from the CIMTRE Study.

BACKGROUND AND OBJECTIVE: Mycophenolate mofetil is widely used in kidney transplant recipients. Mycophenolate mofetil is hydrolysed by blood esterases to mycophenolic acid (MPA), the active drug. Although MPA therapeutic drug monitoring has been recommended to optimise the treatment efficacy by the area under the plasma concentration vs time curve, little is known regarding MPA concentrations in peripheral blood mononuclear cells, where MPA inhibits inosine monophosphate dehydrogenase. This study aimed to build a pharmacokinetic model using a population approach to describe MPA total and unbound concentrations in plasma and into peripheral blood mononuclear cells in 78 adult kidney transplant recipients receiving mycophenolate mofetil therapy combined with tacrolimus and prednisone. METHODS: Total and unbound plasma concentrations and peripheral blood mononuclear cell concentrations were assayed. A three-compartment model, two for plasma MPA and one for peripheral blood mononuclear cell MPA, with a zero-order absorption and a first-order elimination was used to describe the data. RESULTS: Mycophenolic acid average concentrations in peripheral blood mononuclear cells were well above half-maximal effective concentration for inosine monophosphate dehydrogenase and no relationship was found with the occurrence of graft rejection. Three covariates affected unbound and intracellular MPA pharmacokinetics: creatinine clearance, which has an effect on unbound MPA clearance, human serum albumin, which influences fraction unbound MPA and the ABCB1 3435 C>T (rs1045642) genetic polymorphism, which has an effect on MPA efflux transport from peripheral blood mononuclear cells. CONCLUSION: This population pharmacokinetic model demonstrated the intracellular accumulation of MPA, the efflux of MPA out of the cells being dependent on P-glycoprotein transporters. Nevertheless, further studies are warranted to investigate the relevance of MPA concentrations in peripheral blood mononuclear cells to dosing regimen optimisation.

Design optimisation for pharmacokinetic modeling of a cocktail of phenotyping drugs.

Our paper proposes a methodological strategy to select optimal sampling designs for phenotyping studies including a cocktail of drugs. A cocktail approach is of high interest to determine the simultaneous activity of enzymes responsible for drug metabolism and pharmacokinetics, therefore useful in anticipating drug-drug interactions and in personalized medicine. Phenotyping indexes, which are area under the concentration-time curves, can be derived from a few samples using nonlinear mixed effect models and maximum a posteriori estimation. Because of clinical constraints in phenotyping studies, the number of samples that can be collected in individuals is limited and the sampling times must be as flexible as possible. Therefore to optimize joint design for several drugs (i.e., to determine a compromise between informative times that best characterize each drug's kinetics), we proposed to use a compound optimality criterion based on the expected population Fisher information matrix in nonlinear mixed effect models. This criterion allows weighting different models, which might be useful to take into account the importance accorded to each target in a phenotyping test. We also computed windows around the optimal times based on recursive random sampling and Monte-Carlo simulation while maintaining a reasonable level of efficiency for parameter estimation. We illustrated this strategy for two drugs often included in phenotyping cocktails, midazolam (probe for CYP3A) and digoxin (P-glycoprotein), based on the data of a previous study, and were able to find a sparse and flexible design. The obtained design was evaluated by clinical trial simulations and shown to be efficient for the estimation of population and individual parameters.

A limited sampling strategy based on maximum a posteriori Bayesian estimation for a five-probe phenotyping cocktail.

PURPOSE: Cocktail approach using a combination of probes to phenotype several cytochromes P450 or transporters is of high interest in anticipating drug­drug interactions and personalized medicine. Its clinical use remains however limited by the intensive sampling scheme required to obtain phenotyping indexes (PI) which consists in calculating the area under the concentration­time curves. We proposed to use maximum a posteriori Bayesian estimation (MAPBE) that incorporates available information from the whole population to derive PI from a few individual observations. The performance of a limited sampling strategy (LSS) based on MAPBE was evaluated for a five-probe cocktail. METHODS: The studied cocktail included midazolam, tolbutamide, caffeine, dextromethorphan, omeprazole and their relevant metabolites. Prior information for MAPBE was obtained by nonlinear mixed-effect modelling of data from a pilot study. Sampling times were chosen based on optimal design theory using the Bayesian Fisher information matrix. Through a simulation study, we investigated the predicted PI in terms of bias and imprecision for varying number and timing of samples. RESULTS: Some three-point Bayesian designs gave mean prediction errors in [−5 %, 5 %], root mean square errors below 30 % for all probes, except dextromethorphan whose model should be consolidated further with additional data. This approach gave overall less outlier predicted values than single-point metrics and was more flexible to the timing of the latest sampling. CONCLUSIONS: MAPBE is accurate for predicting simultaneously several PI while being flexible in terms of integrating clinical constraints. Therefore, LSS based on MAPBE could help reduce the time of presence in hospital for individuals to be phenotyped.

Safety and pharmacokinetics of the CIME combination of drugs and their metabolites after a single oral dosing in healthy volunteers.

This phase I, pilot clinical study was designed to evaluate the safety and the pharmacokinetic (PK) profiles of the CIME (Metabolic Identity Card) combination of ten drugs, with a view to its use as a phenotyping cocktail. Ten healthy Caucasian subjects were orally dosed with the CIME combination (caffeine-CYP1A2, repaglinide-CYP2C8, tolbutamide-CYP2C9, omeprazole-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A, acetaminophen-UGT1A1, 6&9 and 2B15, digoxin-P-gp, rosuvastatin-OATP1B1&3 and memantine-active renal transport). Blood was collected over 3 days and on day 7. CIME probes and relevant metabolites were assayed by LC-MS/MS and PK parameters were calculated. Main results were: (1) good safety with reversible mild or moderate adverse effects, (2) an analytical method able to quantify simultaneously the 10 probes and the major metabolites, (3) calculation of PK parameters for all probes in general agreed with published values, and (4) identification of the low CYP2D6 metabolizer. This pilot study showed that the CIME combination was well tolerated and that its pharmacokinetics could be accurately measured in healthy volunteers. This combination can now confidently be checked for sensitivity and specificity and for lack of interaction to be validated as a phenotyping cocktail.

Evaluation of seven drug metabolisms and clearances by cryopreserved human primary hepatocytes cultivated in microfluidic biochips.

We present characterization of the metabolic performance of human cryopreserved hepatocytes cultivated in a platform of parallelized microfluidic biochips. The RTqPCR analysis revealed that the mRNA levels of the cytochromes P450 (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4) were reduced after the adhesion period (when compared to the post-thawing step). The microfluidic perfusion played a part in stabilizing and partially recovering the levels of the HNF4α, PXR, OAPT2, CYP 1A2, 2B6, 2C19 and 3A4 mRNA on contrary to non-perfused cultures. Fluorescein diacetate staining and P-gp mRNA level illustrated the hepatocytes' polarity in the biochips. Drug metabolism was assessed using midazolam, tolbutamide, caffeine, omeprazole, dextromethorphan, acetaminophen and repaglinide as probes. Metabolite detection and quantification revealed that CYP1A2 (via the detection of paraxanthine), CYP3A4 (via 1-OH-midazolam, and omeprazole sulfone detection), CYP2C8 (via hydroxyl-repaglinide detection), CYP2C19 (via hydroxy-omeprazole detection) and CYP2D6 (via dextrorphan detection) were functional in our microfluidic configurations. Furthermore, the RTqPCR analysis showed that the drugs acted as inductors leading to overexpression of mRNA levels when compared to post-thawing values (such as for HNF4α, PXR and CYP3A4 by dextromethorpahn and omeprazole). Finally, intrinsic in vitro biochip clearances were extracted using a PBPK model for predictions. The biochip predictions were compared to literature in vitro data and in vivo situations.

P-glycoprotein, breast cancer resistance protein, Organic Anion Transporter 3, and Transporting Peptide 1a4 during blood-brain barrier maturation: involvement of Wnt/ß-catenin and endothelin-1 signaling.

Our current knowledge about drug transporters in the maturational brain is very limited. In this study, we provide a comprehensive overview of the expression and activity profile of P-glycoprotein (P-gp), Breast Cancer Resistance Protein (bcrp), Organic Anion Transporter 3 (oat3), and Transporting Peptide 1a4 (oatp1a4) transporters during blood-brain barrier (BBB) maturation. Gene and protein expressions of the analyzed transporters increase as the brain matures, with no variation in their activity for P-gp and bcrp, while the transport activity of oat3 and oatp1a4 increases during brain maturation from preterm up to adulthood. For the first time, we illustrate a downregulation of nuclear ß-catenin expression in brain capillaries when bcrp, P-gp, oat3, and oatp1a4 transporters are at their highest expression levels. In vivo activation of ß-catenin in rat brains, by intracerebroventricular (ICV) injection of a GSK-3 inhibitor, enhances the activity of P-gp, bcrp, oat3, and oatp1a4. Interestingly, in an in vitro BBB model consisting of a coculture of primary endothelial brain cells with astrocytes or in vivo, activation of ß-catenin enhances the mRNA expression of ET-1. Interestingly, blocking the ETA receptor for endothelin-1 in vivo by ICV injection of a ETA antagonist decreases transporter activity mediated by the activation of ß-catenin. These findings shed light on the role of an interaction between ß-catenin and endothelin-1 signaling in the regulation of these transporters at the BBB.

Can a cocktail designed for phenotyping pharmacokinetics and metabolism enzymes in human be used efficiently in rat?

We recently designed the CIME cocktail consisting of 10 drugs to assess the activity of the major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A), a phase II enzyme (UGT1A1/6/9), two drug transporters (P-gp and OATP1B1) and a component of the renal function ( Videau et al. 2010 ). The present work aimed at studying the usefulness of the CIME cocktail in the rat.The CIME cocktail was given per os to three male and three female rats, or incubated with rat liver microsomes. Parent substrates and metabolites were quantified by LC-MS/MS in plasma, urine and hepatic microsomal media, and phenotyping index were subsequently calculated.The CIME cocktail could therefore be used in the rat to phenotype rapidly and simultaneously CYP3A1/2 with omeprazole/omeprazole-sulfone, midazolam/1'-hydroxymidazolam or 4-hydroxymidazolam and/or dextromethorphan/3-methoxymorphinan, CYP2C6/11 with tolbutamide/4-hydroxytolbutamide, CYP2D1/2 with omeprazole/5-hydroxyomeprazole or dextromethorphan/dextrorphan, and UGT1A6/7 with acetaminophen/acetaminophen-glucuronide. Our results confirmed also several known gender differences and brought new information on the urinary excretion of rosuvastatin. However, the major rat CYPs, CYP2C11 and CYP2C12, are not specifically assessed. An optimized version of the CIME cocktail should therefore be designed and would be of major importance to more largely phenotype DMPK enzymes in rats to study DMPK variability factors such as disease, age, or to exposure to inductors or inhibitors.

In vitro primary human and animal cell-based blood-brain barrier models as a screening tool in drug discovery.

Brain penetration is characterized by its extent and rate and is influenced by drug physicochemical properties, plasma exposure, plasma and brain protein binding and BBB permeability. This raises questions related to physiology, interspecies differences and in vitro/in vivo extrapolation. We herein discuss the use of in vitro human and animal BBB model as a tool to improve CNS compound selection. These cell-based BBB models are characterized by low paracellular permeation, well-developed tight junctions and functional efflux transporters. A study of twenty drugs shows similar compound ranking between rat and human models although with a 2-fold higher permeability in rat. cLogP < 5, PSA < 120 Å, MW < 450 were confirmed as essential for CNS drugs. An in vitro/in vivo correlation in rat (R² = 0.67; P = 2 × 10⁻4) was highlighted when in vitro permeability and efflux were considered together with plasma exposure and free fraction. The cell-based BBB model is suitable to optimize CNS-drug selection, to study interspecies differences and then to support human brain exposure prediction.

A cocktail of metabolic probes demonstrates the relevance of primary human hepatocyte cultures in a microfluidic biochip for pharmaceutical drug screening.

In this paper, we compare the biotransformation capacities of cryopreserved primary human hepatocytes cultivated in a liver microfluidic biochip and in plates. The hepatocytes were exposed to the CIME cocktail (Carte d'Identité MEtabolique), a mixture of seven probes (acetaminophen, amodiaquine, caffeine, dextromethorphan, midazolam, omeprazole and tolbutamide) for key enzymes involved in the xenobiotic metabolism and pharmacokinetics. The purpose of the cocktail was to give an overview of the metabolic profile of the hepatocytes due to concomitant exposure and a simultaneous mass spectrometric detection method of the metabolites. The results showed a greater activity for CYP1A2, CYP2C9, CYP2C19 CYP2D6, CYP3A and UGT1A1 after 4 h of incubation in the microfluidic biochip when compared to the plate cultures. Furthermore, the metabolic ratio time-course measured at 1 h, 3 h and 4 h indicated that the enzymatic activity increased when the hepatocytes were cultivated in the microfluidic biochip, in contrast with their response in the plate cultures. These results illustrated the functional relevance of liver culture in the PDMS microfluidic biochip. The original method based on a microfluidic culture coupled with CIME cocktail analysis allowed the maintenance and the evaluation of the metabolic performances of the primary human hepatocytes through a new rapid assay. This metabolic analysis can thus become the reference situation when parallel studies of drug metabolism and toxicities are planned with functional hepatocytes in biochips.

Effect of a short-term HAART on SIV load in macaque tissues is dependent on time of initiation and antiviral diffusion.

BACKGROUND: HIV reservoirs are rapidly established after infection, and the effect of HAART initiated very early during acute infection on HIV reservoirs remains poorly documented, particularly in tissue known to actively replicate the virus. In this context, we used the model of experimental infection of macaques with pathogenic SIV to assess in different tissues: (i) the effect of a short term HAART initiated at different stages during acute infection on viral dissemination and replication, and (ii) the local concentration of antiviral drugs. RESULTS: Here, we show that early treatment with AZT/3TC/IDV initiated either within 4 hours after intravenous infection of macaques with SIVmac251 (as a post exposure prophylaxis) or before viremia peak (7 days post-infection [pi]), had a strong impact on SIV production and dissemination in all tissues but did not prevent infection. When treatment was initiated after the viremia peak (14 days pi) or during early chronic infection (150 days pi), significant viral replication persists in the peripheral lymph nodes and the spleen of treated macaques despite a strong effect of treatment on viremia and gut associated lymphoid tissues. In these animals, the level of virus persistence in tissues was inversely correlated with local concentrations of 3TC: high concentrations of 3TC were measured in the gut whereas low concentrations were observed in the secondary lymphoid tissues. IDV, like 3TC, showed much higher concentration in the colon than in the spleen. AZT concentration was below the quantification threshold in all tissues studied. CONCLUSIONS: Our results suggest that limited antiviral drug diffusion in secondary lymphoid tissues may allow persistent viral replication in these tissues and could represent an obstacle to HIV prevention and eradication.

Biochemical and analytical development of the CIME cocktail for drug fate assessment in humans.

Phenotyping based on drug metabolism activity appears to be informative regarding mechanism-based interactions during drug development. We report here the first steps of the development of the innovative CIME cocktail. This cocktail is designed not only for the major cytochrome P450, with caffeine, amodiaquine, tolbutamide, omeprazole, dextromethorphan and midazolam as substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, but also phase II enzymes UGT 1A1/6/9 with acetaminophen, P-gp and OATP1B1 with digoxin and rosuvastatin, and renal function with memantine. An assay combining ultra-performance liquid chromatography using a 1.7 microm particle size column with tandem mass spectrometry (UPLC/MS/MS) was set up for the simultaneous quantification of the 20 substrates and metabolites after extraction from human plasma using solid-phase extraction. The method was validated in the spirit of the FDA guidelines. Mean accuracy ranged from 87.7 to 115%, the coefficient of variance (CV%) of intra- and inter-run from 1.7 to 16.4% and from 1.6 to 14.9%, respectively, and for the limit of quantification (LOQ) with ten lots of plasma, accuracy ranged from 84 to 115% and CV% precision was <16%. Short-term stability was evaluated in eluate (4 h, room temperature), plasma (24 h, room temperature), the autosampler (24 h, 4 degrees C) and in three freeze/thaw cycles in plasma. All except three analytes were stable under these conditions. For the three others a specific process can be followed. This robust, fast and sensitive assay in human plasma provides an analytical tool for ten-probe drugs of the CIME cocktail. Clinical samples will be assayed in the near future using this new assay method.

Metabolism evaluation of biomimetic prodrugs by in vitro models and mass spectrometry.

Glycerolipidic prodrug is an interesting concept to enhance lymphatic absorption of polar drugs intended to oral delivery such as didanosine (ddI). In order to improve ddI bioavailability, two didanosine glycerolipidic prodrugs, the phosphorylated (ProddIP) and the non-phosphorylated derivatives (ProddINP) were synthesized to follow triglyceride metabolism. The biomimetism approach of these prodrugs has been studied in vitro at two steps. First, liposomal formulation of each prodrug was incubated with a lipolysis model based on pancreatin and analysed using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). These experiments evidenced that both didanosine prodrugs were recognized by the lipases; as expected, they were cleaved at both positions sn-1 and sn-3 of glycerol. ProddIP was metabolised twice more rapidly than ProddINP suggesting an implication of some phospholipases in ProddIP degradation. Secondly, the detection of dideoxyadenosine triphosphate (ddA-TP) into HIV-1 infected cells after their incubation with ProddINP loaded liposomes evidenced their ability to release ddI that could penetrate into the cells and be metabolised by intracellular kinases. These results confirmed that the synthesized glycerolipidic prodrugs of didanosine could be investigated for a biomimetic approach with final aiming of increasing the drug oral bioavailability by enhancing intestinal absorption.

Prevention of vaginal simian immunodeficiency virus transmission in macaques by postexposure prophylaxis with zidovudine, lamivudine and indinavir.

OBJECTIVE: To evaluate the efficacy of postexposure prophylaxis with a combination of zidovudine (ZDV), lamivudine (3TC) and indinavir (IDV), after vaginal exposure to HIV. DESIGN: : Experimental intravaginal exposure of female cynomolgus macaques to SIVmac251. METHODS: ZDV/3TC/IDV treatment was initiated 4 h after exposure and continued for 28 days. Groups of six animals received a placebo or a combination of oral ZDV (4.5 mg/kg), 3TC (2.5 mg/kg) and IDV (20 mg/kg) twice daily or subcutaneous ZDV (4.5 mg/kg) and 3TC (2.5 mg/kg) twice daily, and a higher dose of IDV (60 mg/kg) administered orally twice daily. RESULTS: In the placebo group, all animals were infected. Antiretroviral association protected one of the six animals if all drugs were administered orally and four of the six animals if ZDV and 3TC were administered subcutaneously and IDV was given orally at triple dose. In infected animals, viremia was significantly delayed and lowered in treated animals than in animals given placebo, and high CD4 cell counts were maintained in the treated animals, at least in the medium term. Antiretroviral dosages made in macaques receiving the same treatments showed that protection efficacy could be linked to antiretroviral plasmatic concentration. CONCLUSION: This study shows, for the first time in macaques, that the postexposure prophylaxis recommended for humans may be effective after vaginal exposure. Improvements in pharmacokinetic parameters significantly increased treatment efficiency.

High levels of zidovudine (AZT) and its intracellular phosphate metabolites in AZT- and AZT-lamivudine-treated newborns of human immunodeficiency virus-infected mothers.

Newborns from human immunodeficiency virus-infected mothers are given antiretroviral prophylaxis against mother-to-child transmission, including predominantly nucleoside reverse transcriptase inhibitors. Pharmacological monitoring of these drugs in newborns has so far been limited to plasma and cord blood. In this study, samples from newborns (up to 45 days old) treated with zidovudine (AZT) alone (n = 29) or in combination with lamivudine (3TC) (n = 20) were analyzed for both intracellular concentrations of phosphate metabolites in peripheral blood mononuclear cells and levels of parent drugs in plasma. Plasma AZT and intracellular AZT-monophosphate and AZT-triphosphate (TP) concentrations were significantly higher during the first 15 days of life (199 versus 52.7 ng/ml [P < 0.0001], 732 versus 282 fmol/10(6) cells [P < 0.0001], and 170 versus 65.1 fmol/10(6) cells [P < 0.0001], respectively) and then became comparable to those of adults. No difference in intracellular AZT metabolite concentrations was found when AZT- and AZT-3TC-treated groups were compared. Plasma 3TC levels (lower limit of quantification [LLOQ], 1,157 ng/ml; median, 412.5 ng/ml) were not associated with the newborn's age, gender, or weight. Intracellular 3TC-TP concentrations (LLOQ, 40.4 pmol/10(6) cells; median, 18.9 pmol/10(6) cells) determined for newborns receiving the AZT-3TC combination were associated with neither the age nor weight of the newborns. Concentrations in females were significantly higher (1.8-fold [P = 0.0415]) than those in males. Unexpectedly, newborns on AZT monotherapy whose mothers' treatment included 3TC displayed residual plasma 3TC and intracellular 3TC-TP levels up to 1 week after birth.

Specificity enhancement with LC-positive ESI-MS/MS for the measurement of nucleotides: application to the quantitative determination of carbovir triphosphate, lamivudine triphosphate and tenofovir diphosphate in human peripheral blood mononuclear cells.

Our previous negative ESI-LC-MS/MS method developed for nucleoside reverse transcriptase inhibitor (NRTI) triphosphate (-TP) measurements in human peripheral blood mononuclear cells (PBMC) encountered some specificity problems for several NRTI-TP and simultaneous endogenous nucleotide triphosphates analysis. As LC-MS/MS offers several possibilities to circumvent such problems, we have investigated the contribution of the positive electrospray ionization mode in enhancing the specificity of the intracellular analyses of triphosphate metabolites of lamivudine, abacavir, and tenofovir. For intracellular NRTI-TP analysis, after disruption of PBMCs, concentrated supernatants were directly injected into the LC-MS/MS system, dimethylhexylamine being used as ion-pairing agent to resolve NRTI-TP. MS/MS detection was performed after positive electrospray ionization. Total run time was 12 min instead of 26 min for NRTI-TP analysis. The validation parameters of the method met the international requirements, and endogenous chromatographic interferences were eliminated. The use of positive ESI, offering a better specificity and a slightly better sensitivity than the negative ESI mode for these compounds, resulted in specificity enhancement and more robust assay methods.

Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells.

The intracellular metabolism of nucleoside reverse transcriptase inhibitors (NRTI) in mononuclear cells has been thoroughly studied, but that in red blood cells (RBC) has been disregarded. However, the phosphorylation of other analogous nucleosides (in particular, ribavirin) has been described previously. In this study, we investigated for the first time the phosphorylation of NRTI in human RBC. The presence of intracellular zidovudine (AZT) monophosphate, AZT triphosphate, lamivudine (3TC) triphosphate, and tenofovir (TFV) diphosphate, as well as endogenous dATP, dGTP, and dTTP, in RBC collected from human immunodeficiency virus-infected patients was examined. We observed evidence of a selective phosphorylation of 3TC, TFV, and endogenous purine deoxynucleosides to generate their triphosphate moieties. Conversely, no trace of AZT phosphate metabolites was found, and only faint dTTP signals were visible. A comparison of intracellular TFV diphosphate and 3TC triphosphate levels in RBC and peripheral blood mononuclear cells (PBMC) further highlighted the specificity of NRTI metabolism in each cell type. These findings raise the issue of RBC involvement in drug-drug interaction, drug pharmacokinetics, and drug-induced toxicity. Moreover, the typical preparation of PBMC samples by gradient density centrifugation does not prevent their contamination with RBC. We demonstrated that the presence of RBC within PBMC hampers an accurate determination of intracellular TFV diphosphate and dATP levels in clinical PBMC samples. Thus, we recommend removing RBC during PBMC preparation by using an ammonium chloride solution to enhance both the accuracy and the precision of intracellular drug monitoring.

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells.

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.