Efavirenz Is Predicted To Accumulate in Brain Tissue: an In Silico, In Vitro, and In Vivo Investigation.

Adequate concentrations of efavirenz in the central nervous system (CNS) are necessary to suppress viral replication, but high concentrations may increase the likelihood of CNS adverse drug reactions. The aim of this investigation was to evaluate the efavirenz distribution in the cerebrospinal fluid (CSF) and the brain by using a physiologically based pharmacokinetic (PBPK) simulation for comparison with rodent and human data. The efavirenz CNS distribution was calculated using a permeability-limited model on a virtual cohort of 100 patients receiving efavirenz (600 mg once daily). Simulation data were then compared with human data from the literature and with rodent data. Wistar rats were administered efavirenz (10 mg kg of body weight-1) once daily over 5 weeks. Plasma and brain tissue were collected for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median maximum concentrations of drug (Cmax) were predicted to be 3,184 ng ml-1 (interquartile range [IQR], 2,219 to 4,851 ng ml-1), 49.9 ng ml-1 (IQR, 36.6 to 69.7 ng ml-1), and 50,343 ng ml-1 (IQR, 38,351 to 65,799 ng ml-1) in plasma, CSF, and brain tissue, respectively, giving a tissue-to-plasma ratio of 15.8. Following 5 weeks of oral dosing of efavirenz (10 mg kg-1), the median plasma and brain tissue concentrations in rats were 69.7 ng ml-1 (IQR, 44.9 to 130.6 ng ml-1) and 702.9 ng ml-1 (IQR, 475.5 to 1,018.0 ng ml-1), respectively, and the median tissue-to-plasma ratio was 9.5 (IQR, 7.0 to 10.9). Although it is useful, measurement of CSF concentrations may give an underestimation of the penetration of antiretrovirals into the brain. The limitations associated with obtaining tissue biopsy specimens and paired plasma and CSF samples from patients make PBPK modeling an attractive tool for probing drug distribution.

Simulating Intestinal Transporter and Enzyme Activity in a Physiologically Based Pharmacokinetic Model for Tenofovir Disoproxil Fumarate.

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0-24; 3,045 ng · h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0-24 (3,036 ng · h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies.

A multisystem investigation of raltegravir association with intestinal tissue: implications for pre-exposure prophylaxis and eradication.

OBJECTIVES: Recent clinical data have suggested high raltegravir concentrations in gut tissue after oral administration, with implications for treatment and prevention. We have used in silico, in vitro, ex vivo and in vivo models to further investigate the accumulation of raltegravir in gut tissue. METHODS: Affinity of raltegravir for gut tissue was assessed in silico (Poulin-Theil method), in vitro (Caco-2 accumulation) and ex vivo (rat intestine) and compared with the lipophilic drug lopinavir. Finally, raltegravir concentrations in plasma, gut contents, small intestine and large intestine were determined after oral dosing to Wistar rats 1 and 4 h post-dose. Samples were analysed using LC-MS/MS and scintillation counting. RESULTS: Gut tissue accumulation of raltegravir was less than for lopinavir in silico, in vitro and ex vivo (P < 0.05). After oral administration to rats, raltegravir concentrations 4 h post-dose were lower in plasma (0.05 µM) compared with small intestine (0.47 µM, P = 0.06) and large intestine (1.36 µM, P < 0.05). However, raltegravir concentrations in the contents of both small intestine (4.0 µM) and large intestine (40.6 µM) were also high. CONCLUSIONS: In silico, in vitro and ex vivo data suggest low raltegravir accumulation in intestinal tissue. In contrast, in vivo animal data suggest raltegravir concentrates in intestinal tissue even when plasma concentrations are minimal. However, high raltegravir concentrations in gut contents are the likely driving factor behind this observation, rather than blood-to-tissue drug distribution. The methods described can be combined with clinical investigations to provide a complete strategy for selection of drugs with high gut accumulation.

Rilpivirine inhibits drug transporters ABCB1, SLC22A1, and SLC22A2 in vitro.

Rilpivirine is a nonnucleoside reverse transcriptase inhibitor approved for treatment of HIV-1 infection in antiretroviral-naive adult patients. Potential interactions with drug transporters have not been fully investigated. Transport by and inhibition of drug transporters by rilpivirine were analyzed to further understand the mechanisms governing rilpivirine exposure and determine the potential for transporter-mediated drug-drug interactions. The ability of rilpivirine to inhibit or be transported by ABCB1 was determined using ABCB1-overexpressing CEMVBL100 cells and Caco-2 cell monolayers. The Xenopus laevis oocyte heterologous protein expression system was used to clarify if rilpivirine was either transported by or inhibited the function of influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A2, SLC22A6, and SLC22A8. The ability of rilpivirine to inhibit or be transported by SLC22A1 was determined using SLC22A1-expressing KCL22 cells. Rilpivirine showed higher accumulation in SLC22A1-overexpressing KCL22 cells than control cells (27% increase, P = 0.03) and inhibited the functionality of SLC22A1 and SLC22A2 transport with 50% inhibitory concentrations (IC50s) of 28.5 µM and 5.13 µM, respectively. Inhibition of ABCB1-mediated digoxin transport was determined for rilpivirine, which inhibited digoxin transport in the B-to-A direction with an IC50 of 4.48 µM. The maximum rilpivirine concentration in plasma in patients following a standard 25-mg dosing regimen is around 0.43 µM, lower than that necessary to substantially inhibit ABCB1, SLC22A1, or SLC22A2 in vitro. However, these data indicate that SLC22A1 may contribute to variability in rilpivirine exposure and that interactions of rilpivirine with substrates of SLC22A1, SLC22A2, or ABCB1 may be possible.

Predicting intestinal absorption of raltegravir using a population-based ADME simulation.

OBJECTIVES: Raltegravir pharmacokinetics (PK) show high intra- and inter-patient variability and are also influenced by co-administered substances that alter the gastrointestinal tract environment, such as pH-altering or metal-containing agents. The aim of this investigation was to develop a population-based absorption, distribution, metabolism and excretion (ADME) model to investigate the effects of gastrointestinal pH and ingested magnesium on raltegravir PK. METHODS: In vitro data describing the disposition of raltegravir were obtained from literature sources or generated by standard methods. Raltegravir (400 mg single dose) PK were simulated in healthy volunteers (50 subjects per group, 20-50 years old, 0.5 proportion female subjects) over a 12 h period. RESULTS: Simulated raltegravir PK correlated well with data from clinical trials, with a mean deviation in C(max), AUC(0-12) and C(trough) of <20%. Solubility of raltegravir in the gastrointestinal tract was increased at higher luminal pH. Increased intestinal pH and transit time both correlated with higher raltegravir absorption (P<0.001). Magnesium ingestion reduced raltegravir exposure in simulated subjects, with mean C(trough) reduced by 32% (P<0.001). CONCLUSIONS: The in vitro-in vivo extrapolation model developed in this study predicted raltegravir PK in virtual individuals with different gastrointestinal pH profiles. The main PK variables were predicted with good accuracy compared with reference data, and both luminal pH and magnesium were able to influence drug absorption. This modelling system provides a tool for investigating the absorption of other drugs, including HIV integrase inhibitors currently in development, which have also shown interactions with food and metal-containing products.

Antimalarial pharmacology and therapeutics of atovaquone.

Atovaquone is used as a fixed-dose combination with proguanil (Malarone) for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travellers. Indeed, in the USA, between 2009 and 2011, Malarone prescriptions accounted for 70% of all antimalarial pre-travel prescriptions. In 2013 the patent for Malarone will expire, potentially resulting in a wave of low-cost generics. Furthermore, the malaria scientific community has a number of antimalarial quinolones with a related pharmacophore to atovaquone at various stages of pre-clinical development. With this in mind, it is timely here to review the current knowledge of atovaquone, with the purpose of aiding the decision making of clinicians and drug developers involved in the future use of atovaquone generics or atovaquone derivatives.

Antitubercular pharmacodynamics of phenothiazines.

OBJECTIVES: Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development. METHODS: Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O2-limited conditions, time-kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc1. RESULTS: Steady-state kinetic analyses revealed a substrate preference for coenzyme Q2 and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time-kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD(+) couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded. CONCLUSIONS: Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.

Divalent metals and pH alter raltegravir disposition in vitro.

Raltegravir shows marked pharmacokinetic variability in patients, with gastrointestinal pH and divalent-metal binding being potential factors. We investigated raltegravir solubility, lipophilicity, pK(a), and permeativity in vitro to elucidate known interactions with omeprazole, antacids, and food, all of which increase gastric pH. Solubility of raltegravir was determined at pH 1 to 8. Lipophilicity of raltegravir was determined using octanol-water partition. Raltegravir pK(a) was determined using UV spectroscopy. The effects of pH, metal salts, and omeprazole on the cellular permeativity of raltegravir were determined using Caco-2 monolayers. Cellular accumulation studies were used to determine the effect of interplay between pH and ABCB1 transport on raltegravir accumulation. Samples were analyzed using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) or scintillation counting. Raltegravir at 10 mM was partly insoluble at pH 6.6 and below. Raltegravir lipophilicity was pH dependent and was reduced as pH was increased from 5 to 9. The pK(a) of raltegravir was 6.7. Raltegravir cellular permeativity was heavily influenced by changes in extracellular pH, where apical-to-basolateral permeativity was reduced 9-fold (P < 0.05) when apical pH was increased from 5 to 8.5. Raltegravir cellular permeativity was also reduced in the presence of magnesium and calcium. Omeprazole did not alter raltegravir cellular permeativity. Cellular accumulation of raltegravir was increased independently by inhibiting ABCB1 and by lowering extracellular pH from pH 8 to 5. Gastrointestinal pH and polyvalent metals can potentially alter the pharmacokinetic properties of raltegravir, and these data provide an explanation for the variability in raltegravir exposure in patients. The evaluation of how divalent-metal-containing products, such as multivitamins, that do not affect gastric pH alter raltegravir pharmacokinetics in patients is now justified.

Raltegravir is a substrate for SLC22A6: a putative mechanism for the interaction between raltegravir and tenofovir.

The identification of transporters of the HIV integrase inhibitor raltegravir could be a factor in an understanding of the pharmacokinetic-pharmacodynamic relationship and reported drug interactions of raltegravir. Here we determined whether raltegravir was a substrate for ABCB1 or the influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A1, SLC22A6, SLC10A1, SLC15A1, and SLC15A2. Raltegravir transport by ABCB1 was studied with CEM, CEM(VBL100), and Caco-2 cells. Transport by uptake transporters was assessed by using a Xenopus laevis oocyte expression system, peripheral blood mononuclear cells, and primary renal cells. The kinetics of raltegravir transport and competition between raltegravir and tenofovir were also investigated using SLC22A6-expressing oocytes. Raltegravir was confirmed to be an ABCB1 substrate in CEM, CEM(VBL100), and Caco-2 cells. Raltegravir was also transported by SLC22A6 and SLC15A1 in oocyte expression systems but not by other transporters studied. The K(m) and V(max) for SLC22A6 transport were 150 µM and 36 pmol/oocyte/h, respectively. Tenofovir and raltegravir competed for SLC22A6 transport in a concentration-dependent manner. Raltegravir inhibited 1 µM tenofovir with a 50% inhibitory concentration (IC(50)) of 14.0 µM, and tenofovir inhibited 1 µM raltegravir with an IC(50) of 27.3 µM. Raltegravir concentrations were not altered by transporter inhibitors in peripheral blood mononuclear cells or primary renal cells. Raltegravir is a substrate for SLC22A6 and SLC15A1 in the oocyte expression system. However, transport was limited compared to endogenous controls, and these transporters are unlikely to have a great impact on raltegravir pharmacokinetics.

Dietary geranylgeraniol can limit the activity of pitavastatin as a potential treatment for drug-resistant ovarian cancer.

Pre-clinical and retrospective studies of patients using statins to reduce plasma cholesterol have suggested that statins may be useful to treat cancer. However, prospective clinical trials have yet to demonstrate significant efficacy. We have previously shown that this is in part because a hydrophobic statin with a long half-life is necessary. Pitavastatin, the only statin with this profile, has not undergone clinical evaluation in oncology. The target of pitavastatin, hydroxymethylglutarate coenzyme-A reductase (HMGCR), was found to be over-expressed in all ovarian cancer cell lines examined and upregulated by mutated TP53, a gene commonly altered in ovarian cancer. Pitavastatin-induced apoptosis was blocked by geranylgeraniol and mevalonate, products of the HMGCR pathway, confirming that pitavastatin causes cell death through inhibition of HMGCR. Solvent extracts of human and mouse food were also able to block pitavastatin-induced apoptosis, suggesting diet might influence the outcome of clinical trials. When nude mice were maintained on a diet lacking geranylgeraniol, oral pitavastatin caused regression of Ovcar-4 tumour xenografts. However, when the animal diet was supplemented with geranylgeraniol, pitavastatin failed to prevent tumour growth. This suggests that a diet containing geranylgeraniol can limit the anti-tumour activity of pitavastatin and diet should be controlled in clinical trials of statins.

Development and validation of an LC-MS/MS assay for the quantification of efavirenz in different biological matrices.

AIM: The non-nucleoside reverse transcriptase inhibitor efavirenz is one of the most prescribed antiretroviral therapeutics. Efavirenz-containing therapy has become associated with the occurrence of CNS side effects, including sleep disturbances, depression and even psychosis. RESULTS: The investigation of efavirenz distribution required the development of a versatile and sensitive method. In addition to plasma, quantification was required in brain tissue and phosphate-buffered saline. The assay presented here was linear from 1.9 to 500 ng/ml. Accuracy and precision ranged between 93.7 and 99.5%, and 1.5 and 5.6%, respectively. DISCUSSION: The method developed here represents a versatile, sensitive and easy-to-use assay. The assay has been applied to in vitro and in vivo samples demonstrating reliable efavirenz quantification in multiple matrices.

Corrigendum: The role of drug transporters in the kidney: lessons from tenofovir.

[This corrects the article on p. 248 in vol. 5, PMID: 25426075.].

Interactions of antiretroviral drugs with the SLC22A1 (OCT1) drug transporter.

The SLC22A1 influx transporter is expressed on the basolateral membrane of hepatocytes and is involved in the excretion of numerous cations. Inhibition of SLC22A1 by several antiretrovirals, such as the protease inhibitor darunavir, has not previously been determined. In order to better understand and predict drug-SLC22A1 interactions, a range of antiretrovirals were screened for SLC22A1-associated inhibition and transport. Stable SLC22A1-expressing KCL22 cells were produced previously by nucleofection. Control KCL22 cells were transfected with the empty vector pcDNA3.1. Accumulation of tetraethylammonium (5.5 µM, 30 min) was determined in SLC22A1-expressing and mock-transfected cells with and without 50 µM of SLC22A1 inhibitor prazosin, or 50 µM of each antiretroviral drug. SLC22A1 IC50 values for efavirenz, darunavir, and prazosin were determined. Cellular accumulation of efavirenz and darunavir was also assessed in SLC22A1-expressing KCL22 cells and reversibility of this accumulation was assessed using prazosin. Tetraethylammonium accumulation was higher in SLC22A1-expressing cells compared to mock-transfected cells (10.6 ± 0.8 µM vs. 0.3 ± 0.004 µM, p = 0.009) and was significantly reduced in SLC22A1-expressing cells when co-incubated with all antiretrovirals tested except atazanavir, lamivudine, tenofovir, zidovudine, and raltegravir. Particularly noticeable was the predominance of SLC22A1 inhibitors in the protease inhibitor and non-nucleoside reverse transcriptase inhibitor classes. Absolute SLC22A1 IC50 values for efavirenz, darunavir, and prazosin were 21.8, 46.2, and 2.8 µM, respectively. Efavirenz accumulation was higher in SLC22A1-expressing cells compared to mock-transfected cells (17% higher, p = 0.009) which was reversed using prazosin, whereas no difference was observed for darunavir (p = 0.86). These data inform the mechanistic basis for disposition, drug-drug interactions and pharmacogenetic candidate gene selection for antiretroviral drugs.

The role of drug transporters in the kidney: lessons from tenofovir.

Tenofovir disoproxil fumarate, the prodrug of nucleotide reverse transcriptase inhibitor tenofovir, shows high efficacy and relatively low toxicity in HIV patients. However, long-term kidney toxicity is now acknowledged as a modest but significant risk for tenofovir-containing regimens, and continuous use of tenofovir in HIV therapy is currently under question by practitioners and researchers. Co-morbidities (hepatitis C, diabetes), low body weight, older age, concomitant administration of potentially nephrotoxic drugs, low CD4 count, and duration of therapy are all risk factors associated with tenofovir-associated tubular dysfunction. Tenofovir is predominantly eliminated via the proximal tubules of the kidney, therefore drug transporters expressed in renal proximal tubule cells are believed to influence tenofovir plasma concentration and toxicity in the kidney. We review here the current evidence that the actions, pharmacogenetics, and drug interactions of drug transporters are relevant factors for tenofovir-associated tubular dysfunction. The use of creatinine and novel biomarkers for kidney damage, and the role that drug transporters play in biomarker disposition, are discussed. The lessons learnt from investigating the role of transporters in tenofovir kidney elimination and toxicity can be utilized for future drug development and clinical management programs.