Suppression of HIV-1 Viral Replication by Inhibiting Drug Efflux Transporters in Activated Macrophages.

BACKGROUND: Ethanol has been shown to increase oxidative stress, drug efflux transporter expression, and promote HIV progression. Macrophages, which express drug efflux transporters, serve as an essential sanctuary site for HIV. The antiretroviral drug lopinavir, a protease inhibitor, is a substrate of the drug efflux transporters P-glycoprotein and multidrug resistance-associated protein 1. The NF-κB signaling pathway is associated with inflammation and drug efflux transporter expression. OBJECTIVE: To examine the effects of ethanol on drug efflux transporters and HIV replication of macrophages and develop strategies to increase the efficacy of the protease inhibitor. METHODS: The expression of PGP and MRP1 was examined with western blot. The NF- κB inhibition was assessed with nuclear western blot. LC-MS/MS and p24 ELISA were used to assess intracellular LPV and viral replication. RESULTS: Ethanol at 40mM slightly increased drug efflux transporter PGP and MRP1 expression in activated macrophages. IKK-16, an NF- κB inhibitor, counteracted the increased transporter expression caused by ethanol exposure. MK571, an MRP1 inhibitor, and IKK-16 significantly increased intracellular LPV concentration with or without ethanol treatment. MK571 significantly increased LPV efficacy in suppressing viral replication with or without ethanol treatment. A decreasing trend and a significant decrease were observed with IKK-16+LPV treatment compared with LPV alone in the no ethanol treatment and ethanol treatment groups, respectively. CONCLUSION: In activated macrophages, inhibiting drug efflux transporter MRP1 activity and reducing its expression may represent a promising approach to suppress viral replication by increasing intracellular antiretroviral concentrations. However, different strategies may be required for ethanolrelated vs. untreated groups.

Intracellular Tenofovir and Emtricitabine Concentrations in Younger and Older Women with HIV Receiving Tenofovir Disoproxil Fumarate/Emtricitabine.

The altered immune states of aging and HIV infection may affect intracellular metabolism of tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC); increased cellular senescence decreases FTC-triphosphate (FTCtp) concentrations. The effects of age and inflammation on the ratio of intracellular metabolites (IMs; tenofovir diphosphate [TFVdp] and FTCtp) to their endogenous nucleotides (ENs; dATP and dCTP), a potential treatment efficacy marker, were assessed among participants of the Women's Interagency HIV Study (WIHS), who ranged from 25 to 75 years. Samples from women receiving TDF-FTC with viral loads of <200 copies/ml were dichotomized by age at collection into two groups (≤45 years and ≥60 years). IM/EN concentrations were measured in peripheral blood mononuclear cell (PBMC) pellets; interleukin-6 (IL-6) and sCD163 were measured in plasma; senescent CD8+ T cells were measured in viable PBMCs. The TFVdp:dATP and FTCtp:dCTP ratios had statistically significantly different distributions in older and younger women (log-rank test, P = 0.0023 and P = 0.032, respectively); in general, IM and EN concentrations were higher in the older women. After adjusting for potential confounders, these findings were not significant. In women aged ≤45 years, TFVdp was negatively associated with IL-6 and sCD163, while FTCtp was positively associated with sCD163 and IL-6 in women aged ≥60 years. Body mass index (BMI) was positively associated with IL-6 in both age groups and negatively associated with TFVdp in women aged ≤45 years. After adjustment, age remained significant for sCD163, while black race, BMI, and renal function remained significant for several IMs and ENs, suggesting that factors associated with aging, but not age itself, govern intracellular TDF-FTC pharmacology.

Can We Improve Stavudine's Safety Profile in Children? Pharmacokinetics of Intracellular Stavudine Triphosphate with Reduced Dosing.

Stavudine remains a useful replacement option for treatment for HIV+ children. WHO reduced the adult dose to 30 mg twice daily, which maintains efficacy and lowers mitochondrial toxicity. We explored intracellular stavudine triphosphate levels in children receiving a reduced dose of 0.5 to 0.75 mg/kg of body weight twice daily to investigate whether a similar dose optimization can safely be made. A population pharmacokinetic model was developed to describe the pharmacokinetics of intracellular stavudine triphosphate in 23 HIV+ children and 24 HIV+ adults who received stavudine at 0.5 mg/kg and 20 mg twice daily for 7 days, respectively. Simulations were employed to optimize the pediatric dosing regimen to match exposures in adults receiving the current WHO-recommended dose of 30 mg twice daily. A biphasic disposition model with first-order appearance and disappearance described the pharmacokinetics of stavudine triphosphate. The use of allometric scaling with fat-free mass characterized well the pharmacokinetics in both adults and children, and no other significant effect could be detected. Simulations of 30 mg twice daily in adults predicted median (interquartile range [IQR]) stavudine triphosphate minimum drug concentration (Cmin) and maximum drug concentration (Cmax) values of 13 (10 to 19) and 45 (38 to 53) fmol/106 cells, respectively. Targeting this exposure, simulations in HIV+ children were used to identify a suitable weight-band dosing approach (0.5 to 0.75 mg/kg), which was predicted to achieve median (IQR) Cmin and Cmax values of 13 (9 to 18) and 49 (40 to 58) fmol/106 cells, respectively. Weight-band dosing using a stavudine dose of 0.5 to 0.75 mg/kg is proposed, and it shows comparable exposures to adults receiving the current WHO-recommended dose of 30 mg twice daily. Our pharmacokinetic results suggest that the decreased stavudine dose in children >2 years would have a reduced toxic effect while retaining antiretroviral efficacy.

Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells.

Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016-0.73 and in non-pigmented cells 0.017-1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, Fic, were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3-4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity.

Development and validation of a high-performance liquid chromatography method for the quantitation of intracellular PARP inhibitor Olaparib in cancer cells.

Olaparib is a potent PARP inhibitor in clinical use for cancer therapy. A bioanalytical assay was developed and validated for quantitation of intracellular level of olaparib in cells exposed to the drug. The assay involves an optimized and straightforward sample pretreatment with acetonitrile for olaparib solubilization, cell lysis and protein precipitation, and a high performance liquid chromatography (HPLC) method with ultraviolet detection. Several parameters in both the sample preparation and the detection steps were investigated. Optimal chromatographic conditions were achieved with a 5 µL injection on a Nova-Pak® C18 column (150 × 3.9 mm, 4 µm) using a mobile phase consisting of acetonitrile and ultra-pure water in gradient mode, at a constant 1.2 mL/min flow rate, at 35 °C. Detection was carried out at 254 nm and a diode array detector was used to insure purity of the olaparib peak. The method was validated according to Food and Drug Administration guidelines. Linearity, accuracy and precisions were satisfactory over the concentration range of 200-2000 ng/mL. Limits of detection and quantification for olaparib were 50 ng/mL and 200 ng/mL, respectively. Good stability was showed in three relevant analytical conditions. Finally, the validated analytical method was successfully used to estimate the intracellular level of olaparib in SUM1315 breast cancer cells. A significant difference was observed in intracellular drug level after 1 and 3 h incubations. This method permitting measurement of drug level in tumor cells would allow dosage optimization and improvement of treatment response predictions.

Pseudomonas aeruginosa develops Ciprofloxacin resistance from low to high level with distinctive proteome changes.

Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. Different Ciprofloxacin concentrations selected different low level resistant mutants, and high level resistant mutants emerged from low level resistant mutants if stressed further by Ciprofloxacin. A deep quantitative proteomic study of the Ciprofloxacin resistant mutants uncovered the cellular pathways that supported such resistances. The two low level resistant mutants had different molecular mechanisms. One was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, and the other was probably due to iron and polyamine uptake and DNA repair. High level of resistance involved the mexCD-oprJ efflux pump and the downregulation of PQS quorum sensing. Other pathways might also have contributed to high level resistance, like the arginine deiminase pathway, catalase, peroxidase, protein degradation and DNA repair. The intracellular Ciprofloxacin concentration assay indicated that only the mexCD-oprJ overexpressed mutants had low drug accumulation. This study provided a comprehensive overview of the proteomic landscape in the evolution of Ciprofloxacin resistance in P. aeruginosa, and might have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa. Data are available via ProteomeXchange with identifier PXD004560. BIOLOGICAL SIGNIFICANCE: Pseudomonas aeruginosa infection is difficult to treat because of its drug resistance, but how it develops drug resistance remains largely unknown. In this study we investigated Ciprofloxacin resistance development in P. aeruginosa. We found that Ciprofloxacin resistance developed from low to high level. Two different low levels resistant molecular mechanisms were discovered from different mutants selected by different Ciprofloxacin concentrations, one was mainly due to switching to anaerobic respiration and overexpression of catalase and peroxidase, the other was probably due to iron, polyamine, and DNA repair. High level of Ciprofloxacin resistance all involved the efflux pump, mexCD-oprJ, and the downregulation of quorum sensing. The findings of this study provided insights into the evolution of Ciprofloxacin resistance in P. aeruginosa and should have implications in diagnosis and treatment of Ciprofloxacin resistant P. aeruginosa.

Polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics.

Most drugs are intended to act on molecular targets residing within a specific tissue or cell type. Therefore, the drug concentration within the target tissue or cells is most relevant to its pharmacological effect. Increasing evidences suggest that drug transporters not only play a significant role in governing systemic drug levels, but are also an important gate keeper for intra-tissue and intracellular drug concentrations. This review focuses on polyspecific organic cation transporters, which include the organic cation transporters 1-3 (OCT1-3), the multidrug and toxin extrusion proteins 1-2 (MATE1-2) and the plasma membrane monoamine transporter (PMAT). Following an overview of the tissue distribution, transport mechanisms, and functional characteristics of these transporters, we highlight the studies demonstrating the ability of locally expressed OCTs to impact intracellular drug concentrations and directly influence their pharmacological and toxicological activities. Specifically, OCT1-mediated metformin access to its site of action in the liver is impacted by genetic polymorphisms and chemical inhibition of OCT1. The impact of renal OCT2 and MATE1/2-K in cisplatin intrarenal accumulation and nephrotoxicity is reviewed. New data demonstrating the role of OCT3 in salivary drug accumulation and secretion is discussed. Whenever possible, the pharmacodynamic response and toxicological effects is presented and discussed in light of intra-tissue and intracellular drug exposure. Current challenges, knowledge gaps, and future research directions are discussed. Understanding the impact of transporters on intra-tissue and intracellular drug concentrations has important implications for rational-based optimization of drug efficacy and safety.

Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

MAIN PROBLEM: Islet transplantation has become a promising treatment for type 1 diabetes. However, immunosuppressive drugs used today cause islet deterioration and modification strategies are necessary. But little is known about pharmacokinetics interactions and intracellular concentrations of immunosuppressive drugs in human islets. METHODS: We determined the pharmacokinetics of tacrolimus and sirolimus in islets by measuring intracellular concentration after exposure alone or in combination at two different doses up to 48 h. A quantification technique established in our laboratory using a Micromass Quattro micro API MS/MS-instrument with electrospray ionization was used. Islets function was measured by oxygen consumption rates. Presence of drug transporters OATP1B1 and ABCB1 and metabolizing enzyme CYP3A4 in islets were quantified using real-time quantitative PCR. RESULTS: Islets incubated with tacrolimus and sirolimus had a significant decrease in intracellular concentration of sirolimus compared to sirolimus alone. Reduced intracellular sirolimus concentration was followed by increased p70S6k phosphorylation suggesting preservation of the mTOR-signaling pathway. Drug transporters OATP1B1 and ABCB1 and enzyme CYP3A4 were expressed in human islets, but were not involved in the reduced sirolimus concentration by tacrolimus. CONCLUSION: These findings provide new knowledge of the drug interaction between tacrolimus and sirolimus, suggesting that tacrolimus has an inhibitory effect on the intracellular concentration of sirolimus in human islets.

Monitoring of imatinib targeted delivery in human leukocytes.

The success of imatinib therapy in chronic myeloid leukemia is highly influenced by its active transport into target cells. However, the methodology for analytical evaluation of intracellular drug concentration is rare and usually reliant upon the use of radioactively labeled drugs. More specifically, there is no published method available in the literature for the determination of imatinib concentration in granulocytes. To gain further insight into the intracellular drug uptake a very reliable two-stage sample concentration procedure was devised and coupled with a sensitive ultra-high performance liquid chromatography tandem mass spectrometry. The reliability of this sample preparation and sensitivity of the analysis was confirmed by a successful validation of all necessary method parameters to an impressive lower limit of quantification of 0.5 ng imatinib per 10(6) cells still at the signal to noise ratio of 670. The usefulness of the method is further improved with only 6 mL of blood being necessary for patient analysis. The method has been applied to blood samples of 13 CML patients treated with imatinib and all the measured intracellular drug concentrations were within the validated range. These and further measurements will enable the research of factors which may, besides blood plasma concentration, influence the individual's response to imatinib therapy. Furthermore, individualisation of dosing based on the directly measured targeted drug delivery could be possible.

Determination of Intracellular Ara-CTP Level by RP-HPLC / 中国药房

OBJECTIVE:To establish a simple and rapid ion-pair-RP-HPLC method for the determination of intracellular concentration of ara-CTP.METHODS:Mononuclearcells were separated from the whole blood,centrifugated and injected for determination on Symmetry C18 column with the mobile phase consisted of 0.1 mol?L-1 KDP buffer solution and 0.01 mol?L-1 tetrabutylammonium hydrogen sulfate(pH=2.7)at a flow rate of 1.0 mL?min-1 with the UV wavelength set at 278 nm.RESULTS:The linear range of ara-CTP was 0.35-9.12 ?g?mL-1(r=0.998 6)with its detection limit at 0.35 ?g?mL-1.The recoveries of ara-CTP at low,middle and high concentrations,and the intra-day and inter-day RSDs were all up to the standards.CONCLUSION:The established method is simple,rapid,stable,reproducible and applicable for the monitoring of intracellular concentration of ara-CTP.