Safety and Tolerability of Nicotinamide Riboside in Heart Failure With Reduced Ejection Fraction.

The mitochondrial dysfunction characteristic of heart failure (HF) is associated with changes in intracellular nicotinamide adenine dinucleotide (NAD+) and NADH levels. Raising NAD+ levels with the NAD+ precursor, nicotinamide riboside (NR), may represent a novel HF treatment. In this 30-participant trial of patients with clinically stable HF with reduced ejection fraction, NR, at a dose of 1,000 mg twice daily, appeared to be safe and well tolerated, and approximately doubled whole blood NAD+ levels. Intraindividual NAD+ increases in response to NR correlated with increases in peripheral blood mononuclear cell basal (R 2 = 0.413, P = 0.003) and maximal (R 2 = 0.434, P = 0.002) respiration, and with decreased NLRP3 expression (R 2 = 0.330, P = 0.020). (Nicotinamide Riboside in Systolic Heart Failure; NCT03423342).

Novel Long-Acting Drug Combination Nanoparticles Composed of Gemcitabine and Paclitaxel Enhance Localization of Both Drugs in Metastatic Breast Cancer Nodules.

PURPOSE: To develop drug-combination nanoparticles (DcNPs) composed of hydrophilic gemcitabine (G) and hydrophobic paclitaxel (T) and deliver both drugs to metastatic cancer cells. METHODS: GT DcNPs were evaluated based on particle size and drug association efficiency (AE%). The effect of DcNP on GT plasma time-course and tissue distribution was characterized in mice and a pharmacokinetic model was developed. A GT distribution study into cancer nodules (derived from 4 T1 cells) was performed. RESULTS: An optimized GT DcNP composition (d = 59.2 nm ±9.2 nm) was found to be suitable for IV formulation. Plasma exposure of G and T were enhanced 61-fold and 3.8-fold when given in DcNP form compared to the conventional formulation, respectively. Mechanism based pharmacokinetic modeling and simulation show that both G and T remain highly associated to DcNPs in vivo (G: 98%, T:75%). GT DcNPs have minimal distribution to healthy organs with selective distribution and retention in tumor burdened tissue. Tumor bearing lungs had a 5-fold higher tissue-to-plasma ratio of gemcitabine in GT DcNPs compared to healthy lungs. CONCLUSIONS: DcNPs can deliver hydrophilic G and hydrophobic T together to cancer nodules and produce long acting exposure, likely due to stable GT association to DcNPs in vivo.

The Impact of Proximal Roux-en-Y Gastric Bypass Surgery on Acetaminophen Absorption and Metabolism.

BACKGROUND: Roux-en-Y gastric bypass (RYGBS), a surgery that creates a smaller stomach pouch and reduces the length of small intestine, is one of the most common medical interventions for the treatment of obesity. AIM: The aim of this study was to determine how RYGBS affects the absorption and metabolism of acetaminophen. MATERIALS AND METHODS: Ten morbidly obese patients received 1.5 g of liquid acetaminophen (APAP) orally on three separate pharmacokinetic study days (i.e., pre-RYGBS baseline and 3 and 12 months post-RYGBS). Plasma was collected at pre-specified timepoints over 24 hours, and the samples were analyzed using liquid chromatography-mass spectrometry for APAP, APAPglucuronide (APAP-gluc), APAP-sulfate (APAP-sulf), APAP-cysteine (APAP-cys), and APAP-Nacetylcysteine (APAP-nac). RESULT: Following RYGBS, peak APAP concentrations at the 3-month and 12-month visits increased by 2.0-fold compared to baseline (p=0.0039 and p=0.0078, respectively) and the median time to peak concentration decreased from 35 to 10 minutes. In contrast, peak concentrations of APAP-gluc, APAP-sulf, APAP-cys, and APAP-nac were unchanged following RYGBS. The apparent oral clearance of APAP and the ratios of metabolite area under the curve (AUC)-to-APAP AUC for all four metabolites decreased at 3 and 12 months post-RYGBS compared to the presurgical baseline. In a simulation of expected steady-state plasma concentrations following multiple dosing of 650 mg APAP every 4 hours, post-RYGBS patients had higher steady-state peak APAP concentrations compared to healthy individuals and obese pre-RYGBS patients, though APAP exposure was unchanged compared to healthy individuals. CONCLUSION: Following RYGBS, the rate and extent of APAP absorption increased and decreased formation of APAP metabolites was observed, possibly due to downregulation of Phase II and cytochrome P450 2E1 enzymes.

Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system

The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysio­logical systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.

Identification of Intestinal UDP-Glucuronosyltransferase Inhibitors in Green Tea (Camellia sinensis) Using a Biochemometric Approach: Application to Raloxifene as a Test Drug via In Vitro to In Vivo Extrapolation.

Green tea (Camellia sinensis) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 µg/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC50 values (105 and 59 µM, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 µM, respectively). Using the clinical intestinal UGT substrate raloxifene, the Ki values were ∼1.0 and 2.0 µM, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing.

Development of a microphysiological model of human kidney proximal tubule function.

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and is also a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function in vivo. This microphysiological system can serve as an ideal platform for ex vivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.

Proximal Roux-en-Y gastric bypass alters drug absorption pattern but not systemic exposure of CYP3A4 and P-glycoprotein substrates.

STUDY OBJECTIVES: To evaluate the effect of Roux-en-Y gastric bypass surgery (RYGB) on the pharmacokinetics of midazolam (a CYP3A4 substrate) and digoxin (a P-glycoprotein substrate). DESIGN: Prospective, nonblinded, longitudinal, single-dose pharmacokinetic study in three phases: presurgery baseline and postoperative assessments at 3 and 12 months. PATIENTS: Twelve obese patients meeting current standards for bariatric surgery. MEASUREMENTS AND MAIN RESULTS: At each study visit, patients received a single dose of oral digoxin and midazolam at 8 a.m. Blood samples were collected at regular intervals for 24 hours after dosing. Continuous 12-lead electrocardiogram (EKG), heart rate, blood pressure, and respiratory rate were monitored, and pharmacokinetic parameters from the three visits were compared. The peak plasma concentration (Cmax ) of midazolam increased by 66% and 71% at 3- and 12-month post-RYGB (p=0.017 and p=0.001, respectively), whereas the median time to peak concentration (Tmax ) was reduced by 50%. The mean Cmax for 1'-hydroxymidazolam increased by 87% and 80% at 3 and 12 months (p=0.001 and p<0.001, respectively). However, neither the area under the concentration-time curve (AUC) for midazolam nor the metabolite-to-parent AUC ratio changed significantly over time. For digoxin, the median Tmax decreased from 40 minutes at baseline to 30 and 20 minutes at 3 and 12 months, respectively. The mean AUC for digoxin, heart rate, and EKG patterns were similar across the three study phases. CONCLUSION: Contemporary proximal RYGB increases the rate of drug absorption without significantly changing the overall exposure to midazolam and digoxin. The Cmax of a CYP3A4 substrate with a high extraction ratio was substantially increased after RYGB.

Specialty supplement use and biologic measures of oxidative stress and DNA damage.

BACKGROUND: Oxidative stress and resulting cellular damage have been suggested to play a role in the etiology of several chronic diseases, including cancer and cardiovascular disease. Identifying factors associated with reduced oxidative stress and resulting damage may guide future disease-prevention strategies. METHODS: In the VITamins And Lifestyle (VITAL) biomarker study of 209 persons living in the Seattle area, we examined the association between current use of several specialty supplements and oxidative stress, DNA damage, and DNA repair capacity. Use of glucosamine, chondroitin, fish oil, methylsulfonylmethane (MSM), coenzyme Q10 (CoQ10), ginseng, ginkgo, and saw palmetto was ascertained by a supplement inventory/interview, whereas the use of fiber supplements was ascertained by questionnaire. Supplements used by more than 30 persons (glucosamine and chondroitin) were evaluated as the trend across number of pills/week (non-use, <14 pills/week, 14+ pills/week), whereas less commonly used supplements were evaluated as use/non-use. Oxidative stress was measured by urinary 8-isoprostane and PGF2α concentrations using enzyme immunoassays (EIA), whereas lymphocyte DNA damage and DNA repair capacity were measured using the Comet assay. Multivariate-adjusted linear regression was used to model the associations between supplement use and oxidative stress/DNA damage. RESULTS: Use of glucosamine (Ptrend: 0.01), chondroitin (Ptrend: 0.003), and fiber supplements (P: 0.01) was associated with reduced PGF2α concentrations, whereas CoQ10 supplementation was associated with reduced baseline DNA damage (P: 0.003). CONCLUSIONS: Use of certain specialty supplements may be associated with reduced oxidative stress and DNA damage. IMPACT: Further research is needed to evaluate the association between specialty supplement use and markers of oxidative stress and DNA damage.

Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system.

Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4ß,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4ß,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4ß,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4ß-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Use of glucosamine and chondroitin in relation to mortality.

Glucosamine and chondroitin are products commonly used by older adults in the US and Europe. There is limited evidence that they have anti-inflammatory properties, which could provide risk reduction of several diseases. However, data on their long-term health effects is lacking. To evaluate whether use of glucosamine and chondroitin are associated with cause-specific and total mortality. Participants (n = 77,510) were members of a cohort study of Washington State (US) residents aged 50-76 years who entered the cohort in 2000-2002 by completing a baseline questionnaire that included questions on glucosamine and chondroitin use. Participants were followed for mortality through 2008 (n = 5,362 deaths). Hazard ratios (HR) for death adjusted for multiple covariates were estimated using Cox models. Current (baseline) glucosamine and chondroitin use were associated with a decreased risk of total mortality compared to never use. The adjusted HR associated with current use of glucosamine (with or without chondroitin) was 0.82 (95 % CI 0.75-0.90) and 0.86 (95 % CI 0.78-0.96) for chondroitin (included in two-thirds of glucosamine supplements). Current use of glucosamine was associated with a significant decreased risk of death from cancer (HR 0.87 95 % CI 0.76-0.98) and with a large risk reduction for death from respiratory diseases (HR 0.59 95 % CI 0.41-0.83). Use of glucosamine with or without chondroitin was associated with reduced total mortality and with reductions of several broad causes of death. Although bias cannot be ruled out, these results suggest that glucosamine may provide some mortality benefit.

Is ethnicity associated with morphine's side effects in children? Morphine pharmacokinetics, analgesic response, and side effects in children having tonsillectomy.

OBJECTIVES/AIMS: To examine whether morphine pharmacokinetics (PK) and/or genetic polymorphisms in opioid-related genes, underlie differences in analgesic response and side effects to morphine in Latino (L) vs non-Latino Caucasian (NL) children. BACKGROUND: Morphine has high interindividual variability in its analgesic response and side effects profile. Earlier studies suggest that morphine response may vary by race and ethnicity. METHODS: Prospective cohort study in L and NL children, 3-17 years of age comparing pain scores, occurrence of side effects, plasma morphine, morphine-6- and morphine-3-glucuronide concentrations measured after a single morphine IV bolus administration. Noncompartmental pharmacokinetic analysis and genotyping for 28 polymorphisms in eight genes (UGT1A8, UGT2B7, ABCB1, COMT, STAT6, MC1R, OPRM1, and ARRB2) were performed. RESULTS: We enrolled 68 children (33 L, 35 NL). There were no differences in pain scores or need for rescue analgesia. Statistically significant differences in the occurrence of side effects were documented: While 58% of L children experienced at least one side effect only 20% of NL did (P = 0.001). Pruritus was four times (P = 0.006) and emesis seven times (P = 0.025) more frequent in L compared with NL. PK parameters were similar between groups. None of the assessed polymorphisms mediated the association between ethnicity and side effects. CONCLUSIONS: We found statistically significant differences in the occurrence of side effects after morphine administration between L and NL children. Neither differences in morphine or metabolite concentrations, nor the genetic polymorphisms examined explain these findings. Studies are needed to further investigate reasons for the increase in morphine side effects by Latino ethnicity.

Pulmonary administration of a water-soluble curcumin complex reduces severity of acute lung injury.

Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin's association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.

Prospective open-label trial of etanercept as adjunctive therapy for kawasaki disease.

OBJECTIVE: To determine the safety and pharmacokinetics of etanercept (Amgen, Thousand Oaks, California) a tumor necrosis factor-α receptor blocker, in children with acute Kawasaki disease (KD). Standard therapy of acute KD includes intravenous immunoglobulin (IVIG) and high-dose aspirin, but a substantial number of patients are refractory and require additional treatment. Tumor necrosis factor-α levels are elevated in children with KD, suggesting a role for etanercept in treatment. STUDY DESIGN: We performed a prospective open-label trial of etanercept in patients with KD (age range, 6 months-5 years; n = 17) meeting clinical criteria and with fever ≤ 10 days. All received IVIG and high-dose aspirin. They received etanercept immediately after IVIG infusion and then weekly two times. For the initial safety evaluation, the first 5 patients received 0.4 mg/kg/dose. Subsequent subjects received 0.8 mg/kg/dose. RESULTS: Fifteen patients completed the study. The pharmacokinetics were similar to that in older children in published series. No serious adverse events related to etanercept occurred. No patient demonstrated prolonged or recrudescent fever requiring re-treatment with IVIG. No patient showed an increase in coronary artery diameter or new coronary artery dilation/cardiac dysfunction. CONCLUSION: Etanercept appears to be safe and well tolerated in children with KD. The data support performance of a placebo-controlled trial.

MDR1 (ABCB1) G1199A (Ser400Asn) polymorphism alters transepithelial permeability and sensitivity to anticancer agents.

PURPOSE: P-glycoprotein (P-gp), encoded by MDR1 (or ABCB1), is important in anticancer drug delivery and resistance. We evaluated alterations in P-gp-mediated transport of anticancer agents due to the MDR1 G1199A polymorphism. METHODS: Using stable recombinant epithelial cells expressing wild-type (MDR1 (wt)) or G1199A (MDR1 (1199A)), anticancer drug sensitivity and transepithelial permeability were evaluated. RESULTS: The recombinant cells MDR1 (wt) and MDR1 ( 1199A ) displayed comparable doxorubicin resistance. However, MDR1 (1199A) cells displayed greater resistance to vinblastine, vincristine, paclitaxel, and VP-16 (11-, 2.9-, 1.9-, and 2.9-fold, respectively). Alterations in transepithelial permeability paralleled these changes. Efflux of doxorubicin was similar between MDR1 wt - and MDR1 (1199A)-expressing cells, while P-gp-mediated transport was greater for vinblastine and vincristine in MDR1 (1199A) cells (2.9- and 2.0-fold, respectively). CONCLUSIONS: The occurrence and magnitude of the MDR1 G1199A effect is drug specific. Overall, the MDR1 G1199A polymorphism may impact anticancer efficacy through modulation of drug distribution and delivery to target tumor cells.

Real-time dose adjustment of cyclophosphamide in a preparative regimen for hematopoietic cell transplant: a Bayesian pharmacokinetic approach.

PURPOSE: Dose-related toxicity of cyclophosphamide may be reduced and therapeutic efficacy may be improved by pharmacokinetic sampling and dose adjustment to achieve a target area under the curve (AUC) for two of its metabolites, hydroxycyclophosphamide (HCY) and carboxyethylphosphoramide mustard (CEPM). To facilitate real-time dose adjustment, we developed open-source code within the statistical software R that incorporates individual data into a population pharmacokinetic model. EXPERIMENTAL DESIGN: Dosage prediction performance was compared to that obtained with nonlinear mixed-effects modeling using NONMEM in 20 cancer patients receiving cyclophosphamide. Bayesian estimation of individual pharmacokinetic parameters was accomplished from limited (i.e., five samples over 0-16 hours) sampling of plasma HCY and CEPM after the initial cyclophosphamide dose. Conditional on individual pharmacokinetics, simulations of the AUC of both HCY and CEPM were provided for a range of second doses (i.e., 0-100 mg/kg cyclophosphamide). RESULTS: The results compared favorably with NONMEM and returned accurate predictions for AUCs of HCY and CEPM with comparable mean absolute prediction error and root mean square prediction error. With our method, the mean absolute prediction error and root mean square prediction error of AUC CEPM were 11.0% and 12.8% and AUC HCY were 31.7% and 44.8%, respectively. CONCLUSIONS: We developed dose adjustment software that potentially can be used to adjust cyclophosphamide dosing in a clinical setting, thus expanding the opportunity for pharmacokinetic individualization of cyclophosphamide. The software is simple to use (requiring no programming experience), reads individual patient data directly from an Excel spreadsheet, and runs in less than 5 minutes on a desktop PC.

MDR1 G1199A polymorphism alters permeability of HIV protease inhibitors across P-glycoprotein-expressing epithelial cells.

OBJECTIVE: To evaluate the impact of the human multidrug resistance gene (MDR1) G1199A polymorphism (amino acid change Ser400Asn) on P-glycoprotein (P-gp)-dependent transepithelial permeability and uptake kinetics of HIV protease inhibitors (PI), by using recombinant epithelial cells expressing wild-type MDR1 (MDR1wt) or the G1199A variant (MDR1(1199A)). METHODS: Using a recombinant expression system developed previously, the transepithelial permeability and uptake kinetic parameters of five PI, amprenavir, indinavir, lopinavir, ritonavir, and saquinavir were estimated across polarized epithelial cells. RESULTS: For all PI, the transepithelial permeability ratio (basolateral-to-apical transport divided by apical-to-basolateral transport) was significantly greater in MDR1(1199A) cells than MDR1wt cells: amprenavir (1.7-fold), indinavir (1.8-fold), lopinavir (1.5-fold), ritonavir (2.8-fold), and saquinavir (2.1-fold). However, the impact of G1199A on P-gp activity appeared to primarily influence drug permeability in the apical-to-basolateral direction. Kinetic analysis of ritonavir and saquinavir uptake by MDR1wt- and MDR1(1199A)-expressing cells showed that Vmax was similar, while uptake Km was significantly higher in cells expressing the G1199A variant suggesting that alterations in P-gp-dependent efflux mediated by G1199A were due to changes in transporter affinity. CONCLUSIONS: Alterations in transepithelial permeability of HIV PI due to the G1199A polymorphism may impact oral bioavailability of PI and penetration into cells and tissues of the lymphoid and central nervous systems.

Valproic acid, but not lamotrigine, suppresses seizure-induced c-fos and c-Jun mRNA expression.

Seizure-induced activity has been shown to increase the expression of immediate early genes (IEGs) c-fos and c-Jun in the CNS. Anti-epileptic drugs (AEDs) can suppress the induction of a seizure, but it is unknown if AEDs affect the expression of seizure-induced IEGs. We found that valproic acid (VPA), but not lamotrigine (LTG), was capable of suppressing seizure-induced c-fos and c-Jun mRNA expression in rats despite a similar anticonvulsant effect. LTG in some regions of the CNS enhanced seizure-induced IEG expression. These studies indicate that the older AED (VPA), as compared to the newer AED (LTG), can suppress seizure-induced IEG expression. The consequence of this suppression of IEGs following a generalized seizure may be viewed either as a neuroprotective or detrimental effect upon the brain.

Contribution of CYP3A5 to hepatic and renal ifosfamide N-dechloroethylation.

Ifosfamide nephrotoxicity is attributed to the formation of a toxic metabolite, chloroacetaldehyde, via N-dechloroethylation, a reaction that is purportedly catalyzed by CYP3A and CYP2B6. Because allelic variants of CYP3A5 are associated with polymorphic expression of microsomal CYP3A5 in human liver and kidneys, we hypothesized that ifosfamide N-dechloroethylation depends on CYP3A5 genotype. We compared ifosfamide N-dechloroethylation activity in cDNA-expressed CYP3A4 and CYP3A5. Ifosfamide N-dechloroethylation was also assessed in liver (N = 20) and kidney (N = 21) microsomes from human donors with different CYP3A5 genotypes. Ifosfamide N-dechloroethylation was catalyzed by recombinant CYP3A5 at a rate comparable with recombinant CYP3A4. In human liver microsomes matched for CYP3A4 protein content, N-dechloroethylation was more than 2-fold higher in that from donors carrying CYP3A5*1 allele that express CYP3A5 relative to that from donors homozygous for the mutant CYP3A5*3. Correlation analysis revealed that ifosfamide N-dechloroethylation was significantly associated with CYP3A4 and CYP3A5 protein concentration but not with age, sex, or CYP2B6 protein concentration. In hepatic microsomes not expressing CYP3A5 protein, ifosfamide N-dechloroethylation was inhibited 53 to 61% and 0 to 3% by monoclonal antibodies specific for CYP3A4/5 or CYP2B6, respectively. Ifosfamide N-dechloroethylation was not detected in renal microsomes obtained from CYP3A5*3/*3 donors. In contrast, it was readily measurable in microsomes isolated from four kidneys of CYP3A5*1 carriers, which was almost completely inhibited by the CYP3A inhibitor ketoconazole. CYP2B6 protein could not be detected in this panel of human renal microsomes. In conclusion, CYP3A5*1 genotype is associated with higher rates of ifosfamide N-dechloroethylation in human liver and kidneys.

Development and characterization of a recombinant Madin-Darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1).

Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells. The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors. The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated. The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport. Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs.