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Purpose Variability in cytochrome P450 3A4 (CYP3A4) metabolism is mainly caused by non-genetic factors, hence providing a need for accurate phenotype biomarkers. Although 4β-hydroxycholesterol (4βOHC) is a promising endogenous CYP3A4 biomarker, additional investigations are required to evaluate its ability to predict CYP3A4 activity. This study investigated the correlations between 4βOHC concentrations and hepatic and intestinal CYP3A4 protein expression and ex vivo microsomal activity in paired liver and jejunum samples, as well as in vivo CYP3A4 phenotyping (midazolam) in patients with a wide body weight range. Methods The patients (n = 96; 78 with obesity and 18 normal or overweight individuals) were included from the COCKTAIL-study (NCT02386917). Plasma samples for analysis of 4βOHC and midazolam concentrations, and liver (n = 56) and jejunal (n = 38) biopsies were obtained. The biopsies for determination of CYP3A4 protein concentration and microsomal activity were obtained during gastric bypass or cholecystectomy. In vivo CYP3A4 phenotyping was performed using semi-simultaneous oral (1.5 mg) and intravenous (1.0 mg) midazolam. Results 4βOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity (ρ = 0.53, p ρ = 0.30, p = 0.027), but not with intestinal CYP3A4 concentrations (ρ = 0.18, p = 0.28) or intestinal microsomal CYP3A4 activity (ρ = 0.15, p = 0.53). 4βOHC concentrations correlated weakly with midazolam absolute bioavailability (ρ = − 0.23, p = 0.027) and apparent oral clearance (ρ = 0.28, p = 0.008), but not with systemic clearance (ρ = − 0.03, p = 0.81). Conclusion These findings suggest that 4βOHC concentrations reflect hepatic, but not intestinal, CYP3A4 activity. Further studies should investigate the potential value of 4βOHC as an endogenous biomarker for individual dose requirements of intravenously administered CYP3A4 substrate drugs. Trial registration Clinical.Trials.gov identifier: NCT02386917.

Introduction Rosuvastatin pharmacokinetics is mainly dependent on the activity of hepatic uptake transporter OATP1B1. In this study, we aimed to investigate and disentangle the effect of Roux-en-Y gastric bypass (RYGB) and weight loss on oral clearance (CL/F) of rosuvastatin as a measure of OATP1B1-activity. Methods Patients with severe obesity preparing for RYGB (n = 40) or diet-induced weight loss (n = 40) were included and followed for 2 years, with four 24-hour pharmacokinetic investigations. Both groups underwent a 3-week low-energy diet (LED; < 1200 kcal/day), followed by RYGB or a 6-week very-low-energy diet (VLED; < 800 kcal/day). Results A total of 80 patients were included in the RYGB group (40 patients) and diet-group (40 patients). The weight loss was similar between the groups following LED and RYGB. The LED induced a similar (mean [95% CI]) decrease in CL/F in both intervention groups (RYGB: 16% [0, 31], diet: 23% [8, 38]), but neither induced VLED resulted in any further changes in CL/F. At Year 2, CL/F had increased by 21% from baseline in the RYGB group, while it was unaltered in the diet group. Patients expressing the reduced function SLCO1B1 variants (c.521TC/CC) showed similar changes in CL/F over time compared with patients expressing the wild-type variant. Conclusions Neither body weight, weight loss nor RYGB per se seem to affect OATP1B1 activity to a clinically relevant degree. Overall, the observed changes in rosuvastatin pharmacokinetics were minor, and unlikely to be of clinical relevance.

Obesity is associated with comorbidities of which pharmacological treatment is needed. Physiological changes associated with obesity may influence the pharmacokinetics of drugs, but the effect of body weight on drug metabolism capacity remains uncertain. The aim of this study was to investigate ex vivo activities of hepatic drug metabolizing CYP enzymes in patients covering a wide range of body weight. Liver biopsies from 36 individuals with a body mass index (BMI) ranging from 18 to 63 kg/m2 were obtained. Individual hepatic microsomes were prepared and activities of CYP3A, CYP2B6, CYP2C8, CYP2D6, CYP2C9, CYP2C19 and CYP1A2 were determined. The unbound intrinsic clearance (CLint,u) values for CYP3A correlated negatively with body weight (r = −0.43, p < 0.01), waist circumference (r = −0.47, p < 0.01), hip circumference (r = −0.51, p < 0.01), fat percent (r = −0.41, p < 0.05), fat mass (r = −0.48, p < 0.01) and BMI (r = −0.46, p < 0.01). Linear regression analysis showed that CLint,u values for CYP3A decreased with 5% with each 10% increase in body weight (r2 = 0.12, β = −0.558, p < 0.05). There were no correlations between body weight measures and CLint,u values for the other CYP enzymes investigated. These results indicate reduced hepatic metabolizing capacity of CYP3A substrates in patients with increasing body weight.

Aim: Roux-en-Y gastric bypass (RYGB) may influence drug disposition due to surgery-induced gastrointestinal alterations and/or subsequent weight loss. The objective was to compare short- and long-term effects of RYGB and diet on the metabolic ratios of paraxanthine/caffeine (cytochrome P450 [CYP] 1A2 activity), 5-hydroxyomeprazole/omeprazole (CYP2C19 activity) and losartan/losartan carboxylic acid (CYP2C9 activity), and cross-sectionally compare these CYP-activities with normal-to-overweight controls. Methods: This trial included patients with severe obesity preparing for RYGB (n = 40) or diet-induced (n = 41) weight loss, and controls (n = 18). Both weight loss groups underwent a 3-week low-energy diet (

Because several steroid hormones are metabolized to their respective 6β-hydroxy forms by CYP3A4 and CYP3A5, these isoenzymes have been assumed to metabolize the immunosuppressive drug prednisolone, with conflicting results in the literature with respect to their relative importance. A direct study of the metabolism of prednisolone by microsomal CYP3A4 and CYP3A5 is missing. The aim of this in vitro study was to investigate the relative importance of recombinant CYP3A4 and recombinant CYP3A5 in the metabolism of prednisolone and to compare the extent of formation of 6β-OH-prednisolone by the two enzymes. Through in vitro incubations using rCYP3A4 and rCYP3A5 enzymes, intrinsic clearance (CLint ) of prednisolone was determined by the substrate depletion approach. Formation of the metabolite 6β-OH-prednisolone by rCYP3A4 and rCYP3A5, respectively, was compared. Prednisolone concentrations were measured, and its metabolite 6β-OH-prednisolone was identified using a HPLC-MS/MS in-house method. CLint for prednisolone by rCYP3A5 was less than 26% relative to rCYP3A4. Formation of 6β-OH-prednisolone by rCYP3A5 was less than 11% relative to rCYP3A4. The study indicates that 6β-hydroxylation of prednisolone assessed in vitro in recombinant CYP enzymes depends on rCYP3A4 rather than rCYP3A5 and that CYP3A5 may be responsible for the formation of other prednisolone metabolite(s) in addition to 6β-OH-prednisolone.

BACKGROUND. Methodology for estimation of cerebrospinal fluid (CSF) tracer clearance could have wide clinical application in predicting excretion of intrathecal drugs and metabolic solutes from brain metabolism and for diagnostic workup of CSF disturbances. METHODS. The MRI contrast agent gadobutrol (Gadovist) was used as a CSF tracer and injected into the lumbar CSF. Gadobutrol is contained outside blood vessels of the CNS and is eliminated along extravascular pathways, analogous to many CNS metabolites and intrathecal drugs. Tracer enrichment was verified and assessed in CSF by MRI at the level of the cisterna magna in parallel with obtaining blood samples through 48 hours. RESULTS. In a reference patient cohort (n = 29), both enrichment within CSF and blood coincided in time. Blood concentration profiles of gadobutrol through 48 hours varied between patients diagnosed with CSF leakage (n = 4), idiopathic normal pressure hydrocephalus dementia (n = 7), pineal cysts (n = 8), and idiopathic intracranial hypertension (n = 4). CONCLUSION. Assessment of CSF tracer clearance is clinically feasible and may provide a way to predict extravascular clearance of intrathecal drugs and endogenous metabolites from the CNS. The peak concentration in blood (at about 10 hours) was preceded by far peak tracer enhancement at MRI in extracranial lymphatic structures (at about 24 hours), as shown in previous studies, indicating a major role of the spinal canal in CSF clearance capacity. FUNDING. The work was supported by the Department of Neurosurgery, Oslo University Hospital; the Norwegian Institute for Air Research; and the University of Oslo.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion-CYP2B6, diclofenac-CYP2C9, omeprazole-CYP2C19, bufuralol-CYP2D6, and midazolam-CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

It remains uncertain whether pharmacokinetic changes following Roux‐en‐Y gastric bypass (RYGB) can be attributed to surgery‐induced gastrointestinal alterations per se and/or the subsequent weight loss. The aim was to compare short‐ and long‐term effects of RYGB and calorie restriction on CYP3A‐activity, and cross‐sectionally compare CYP3A‐activity with normal weight to overweight controls using midazolam as probe drug. This three‐armed controlled trial included patients with severe obesity preparing for RYGB (n = 41) or diet‐induced (n = 41) weight‐loss, and controls (n = 18). Both weight‐loss groups underwent a 3‐week low‐energy‐diet (

Tacrolimus is currently the cornerstone of immunosuppressive protocols for renal transplant recipients. Despite therapeutic whole blood monitoring, tacrolimus is associated with nephrotoxicity and it has been hypothesized that intrarenal accumulation of tacrolimus and/or its metabolites are involved. As tacrolimus is a substrate of P-glycoprotein (P-gp), the expression and activity of this efflux transporter could influence the levels of tacrolimus in renal tissue. The primary aim of this study was to develop and validate a method for quantification of tacrolimus in tissue homogenates from single human renal core biopsies. The secondary aim was to provide measures of P-gp expression and of the demethylated metabolites of tacrolimus in the same renal biopsy. Human renal tissue, with and without clinical tacrolimus exposure, was used for method development and validation. Homogenates were prepared with bead-beating, and concentrations of tacrolimus and its demethylated metabolites were analyzed with liquid chromatography tandem mass spectrometry after protein precipitation. A Western blot method was used for semi-quantification of P-gp expression in the homogenates. The final methods were applied to renal core biopsies from two transplant patients. The tacrolimus assay showed within- and between-run mean accuracy between 99.7 % and 107 % and coefficients of variation ≤6.7 %. Matrix effects were non-significant and samples were stable for three months pre-analytically when stored at -80 °C. Tacrolimus concentrations in the renal core biopsies were 62.6 and 43.7 pg/mg tissue. The methods for measurement of desmethyl-tacrolimus and P-gp expression were suitable for semi-quantification in homogenates from renal core biopsies. These methods may be valuable for the elucidation of the pharmacokinetic mechanisms behind tacrolimus-induced nephrotoxicity in renal transplant recipients.

The liver and small intestine restrict oral bioavailability of drugs and constitute the main sites of pharmacokinetic drug-drug interactions. Hence, detailed data on hepatic and intestinal activities of drug metabolizing enzymes is important for modeling drug disposition and optimizing pharmacotherapy in different patient populations. The aim of this study was to determine the activities of seven cytochrome P450 (P450) enzymes in paired liver and small intestinal samples from patients with obesity. Biopsies were obtained from 20 patients who underwent Roux-en-Y gastric bypass surgery following a 3-week low-energy diet. Individual hepatic and intestinal microsomes were prepared and specific probe substrates in combined incubations were used for determination of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A activities. The activities of CYP2C8, CYP2C9, CYP2D6, and CYP3A were quantified in both human liver microsomes (HLM) and human intestinal microsomes (HIM), while the activities of CYP1A2, CYP2B6, and CYP2C19 were only quantifiable in HLM. Considerable interindividual variability was present in both HLM (9- to 23-fold) and HIM (5- to 55-fold). The median metabolic HLM/HIM ratios varied from 1.5 for CYP3A to 252 for CYP2C8. The activities of CYP2C9 in paired HLM and HIM were positively correlated (r = 0.74, P 0.05). Small intestinal CYP3A activities were higher in females compared with males (P < 0.05). Hepatic CYP2B6 activity correlated negatively with body mass index (r = -0.72, P < 0.001). These data may be useful for further in vitro-in vivo predictions of drug disposition in patients with obesity. SIGNIFICANCE STATEMENT: Hepatic and intestinal drug metabolism is the key determinant of oral drug bioavailability. In this study, paired liver and jejunum samples were obtained from 20 patients with obesity undergoing gastric bypass surgery following a 3-week low-energy diet. We determined the hepatic and small intestinal activities of clinically important P450 enzymes and provide detailed enzyme kinetic data relevant for predicting in vivo disposition of P450 substrates in this patient population.

IntroductionRoux-en-Y gastric bypass (GBP) is associated with changes in cardiometabolic risk factors and bioavailability of drugs, but whether these changes are induced by calorie restriction, the weight loss or surgery per se, remains uncertain. The COCKTAIL study was designed to disentangle the short-term (6 weeks) metabolic and pharmacokinetic effects of GBP and a very low energy diet (VLED) by inducing a similar weight loss in the two groups.Methods and analysisThis open, non-randomised, three-armed, single-centre study is performed at a tertiary care centre in Norway. It aims to compare the short-term (6 weeks) and long-term (2 years) effects of GBP and VLED on, first, bioavailability and pharmacokinetics (24 hours) of probe drugs and biomarkers and, second, their effects on metabolism, cardiometabolic risk factors and biomarkers. The primary outcomes will be measured as changes in: (1) all six probe drugs by absolute bioavailability area under the curve (AUCoral/AUCiv) of midazolam (CYP3A4 probe), systemic exposure (AUCoral) of digoxin and rosuvastatin and drug:metabolite ratios for omeprazole, losartan and caffeine, levels of endogenous CYP3A biomarkers and genotypic variation, changes in the expression and activity data of the drug-metabolising, drug transport and drug regulatory proteins in biopsies from various organs and (2) body composition, cardiometabolic risk factors and metabolic biomarkers.Ethics and disseminationThe COCKTAIL protocol was reviewed and approved by the Regional Committee for Medical and Health Research Ethics (Ref: 2013/2379/REK sørøst A). The results will be disseminated to academic and health professional audiences and the public via presentations at conferences, publications in peer-reviewed journals and press releases and provided to all participants.Trial registration numberNCT02386917.

Everolimus (EVE) is an immunosuppressive drug dosed according to therapeutic drug monitoring in renal transplant recipients. The primary site of action is within activated lymphocytes. EVE is a substrate of the efflux transporter ABCB1 also known as P-glycoprotein. Limited data exist regarding a possible association between whole blood and intralymphocyte concentrations of EVE and the potential influence of ABCB1.Twelve renal transplant recipients (5 men and 7 women) treated with EVE underwent a pharmacokinetic investigation where EVE concentrations in whole blood and in peripheral blood mononuclear cells (PBMC) were determined within a dosing interval. In addition, the activity of ABCB1 in PBMC was determined using the Rhodamine123 efflux assay and the patients' genotypes of ABCB1 were determined.There was a significant correlation between EVE AUC0-12 in whole blood and in PBMC (r = 0.90, P0.01), and no association was demonstrated between the ABCB1 activity and EVE PBMC/whole blood ratio of trough concentrations (r = 0.23, P = 0.76). Furthermore, ABCB1 1236CT, 3435CT, and 2677GT/A polymorphism did not influence EVE AUC0-12 PBMC/whole blood ratio.The results revealed a significant association between EVE whole blood and PBMC concentrations, suggesting that ABCB1-mediated efflux from PBMC to be of minor importance for the distribution of EVE.

Individual variability in expression and function of organic anion-transporting polypeptide 1B1 (OATP1B1), multidrug resistance protein 1 (MDR1), and/or cytochrome P450 3A4 (CYP3A4) may impact the clinical response of many drugs. We investigated the correlation between expression of these proteins and pharmacokinetics of atorvastatin, a substrate of all three, in 21 obese patients with paired biopsies from liver and intestinal segments. The patients were also screened for the SLCO1B1 c.521T→C variant alleles. Approximately 30% (r(2) = 0.28) of the variation in oral clearance (CL/F) of atorvastatin was explained by hepatic OATP1B1 protein expression (P = 0.041). Patients carrying the SLCO1B1 c.521C variant allele (homozygous, n = 4; heterozygous, n = 2) exhibited 45% lower CL/F of atorvastatin than the c.521TT carriers (P = 0.067). No association between hepatic and intestinal expression of MDR1 or CYP3A4 and atorvastatin pharmacokinetics was found (P > 0.149). In conclusion, this study suggests that OATP1B1 phenotype is more important than CYP3A4 and MDR1 phenotypes for the individual pharmacokinetic variability of atorvastatin.

Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin.

The aim of this study was to investigate the potential of calcineurin inhibitors [cyclosporine A (CsA) and tacrolimus (Tac)] to inhibit cellular uptake of atorvastatin mediated by the liver-specific organic anion-transporting polypeptide 1B1 (OATP1B1) in vitro. Patients with solid organ transplants are frequently treated with HMG-CoA reductase inhibitors (statins). CsA increases atorvastatin systemic exposure severalfold, an effect not observed with Tac. The effect of CsA and Tac on atorvastatin transport via OATP1B1 was investigated in transfected human embryonic kidney 293 cells. An in vitro-in vivo extrapolation (IVIVE) was performed to estimate the clinical potential for CsA and Tac to inhibit OATP1B1-mediated transport. CsA inhibited OATP1B1-mediated uptake of atorvastatin approximately 90-fold more efficiently than Tac, with half-maximal inhibitory concentration (IC(50)) values of 0.021 +/- 0.004 and 1.99 +/- 0.42 muM, respectively. Coincubation compared with preincubation with CsA showed a 20-fold lower inhibitory capacity, with an IC(50) value of 0.47 +/- 0.34 muM. The IVIVE showed that clinically obtainable concentrations of CsA, but not Tac, inhibit OATP1B1 transport of atorvastatin. CsA inhibition ranged from 28 to 77% within a dosing interval, whereas it was less than 1% for Tac, considering free concentrations and assuming competitive inhibition. This does not fully explain the clinically observed interaction with CsA, suggesting that a more complex inhibitory mechanism may be present. This is also supported by the decreased IC(50) value of CsA after preincubation. This study provides evidence that OATP1B1 inhibition is a relevant mechanism for the interaction observed between CsA and atorvastatin.

Aripiprazole, a relatively new antipsychotic drug, is metabolized by cytochrome P450 3A4 (CYP3A4) and CYP2D6 to an active metabolite, dehydroaripiprazole. As studies on pharmacokinetic drug interactions with aripiprazole are so far limited, the aim of the present study was to investigate the impact of comedication on serum concentrations of aripiprazole and dehydroaripiprazole in psychiatric patients in a clinical setting. A therapeutic drug monitoring database was screened for patients receiving aripiprazole tablets as part of their treatment. Of the 361 samples included, 78% were from patients receiving comedication. The remaining 79 samples constituted the control group. Steady-state dose-adjusted serum concentrations (concentration to dose ratios, C:D ratios) of aripiprazole, dehydroaripiprazole and the sum of aripiprazole and dehydroaripiprazole, and the metabolic ratio (dehydroaripiprazole/aripiprazole) in the different comedication groups were compared with controls. Coadministration of a CYP3A4 inducer resulted in approximately 60% lower mean C:D ratios of aripiprazole, dehydroaripiprazole, and the sum of aripiprazole and dehydroaripiprazole (P < 0.05, P < 0.01, and P < 0.01, respectively). Combination with a CYP2D6 inhibitor resulted in a 45% higher mean C:D ratio of aripiprazole (P < 0.05), with no effect on the C:D ratio of dehydroaripiprazole. When aripiprazole was coadministered with alimemazine or lithium, a 56% (P < 0.01) and 43% (P = 0.05) higher mean C:D ratio of aripiprazole, respectively, was observed. Olanzapine, risperidone injections, escitalopram, or lamotrigine also had statistically significant effects on aripiprazole disposition but to a lesser extent. In conclusion, concurrent treatment with CYP3A4 inducers, CYP2D6 inhibitors, alimemazine, or lithium resulted in changes in the systemic exposure of aripiprazole between 40% and 60%. This is of such a magnitude that dose adjustments of aripiprazole may be required.

In vitro drug metabolism techniques with human CYP c-DNA expressed systems are frequently used to predict human drug metabolism in vivo. The aim of this study was to compare midazolam enzyme kinetics in recombinant expressed CYP3A4 microsomes from human and insect cells. The amounts of 1'- hydroxymidazolam and 4-hydroxymidazolam formed in CYP3A4 microsomes from transfected human liver epithelial cells (T5-3A4 microsomes) and baculovirus-infected insect cells (with and without coexpressed cytochrome b(5)) were analysed by LC-MS. Enzyme kinetic parameters were estimated by nonlinear regression. Mean K(m) for the formation of 1'-hydroxymidazolam was 3- and 4-fold higher in T5-3A4 microsomes than in insect microsomes (p

The effect of changes in microsomal incubation conditions (NADPH, Mg(2+), Cl(-), NADPH-regenerating system and pH) on the formation of the CYP3A4 metabolites AM1 and AM9 from CsA were studied by application of a fractional factorial design. Metabolism was studied in microsomes of transfected human liver epithelial (THLE) cells specifically expressing CYP3A4. Within the conditions tested, a 3-4-fold difference in formation of CsA metabolites was observed. Formation of both AM1 and AM9 was favoured by a low Mg(2+) concentration (0.5 mM) and no addition of Cl(-) to the incubation matrix. However, while a high NADPH concentration (1.75 mM) was the single most important factor for the formation of AM1, changes in NADPH concentration between 0.25 and 1.75 mM had no influence on AM9 formation. Formation of the two metabolites also differed in their influence by pH changes, as a change in pH from 7.2 to 7.5 significantly increased the formation of AM9, while formation of AM1 was unaffected by this change. The present study showed that relatively small changes in the incubation matrix had a significant influence on the microsomal CYP3A4-mediated metabolism of CsA. Systematic studies on microsomal incubation conditions could be a key to improve metabolic in vitro-in vivo extrapolations in drug development.

The oral availability of many drugs metabolised by the enzyme cytochrome P(450) 3A4 (CYP3A4) is increased if co-administered with grapefruit juice. Extracts from grapefruit peel have also demonstrated inhibitory activity and, during commercial manufacturing of grapefruit juice, inhibitory components might be squeezed into the juice from the peel. Thus, the aim of this in vitro study was to identify CYP3A4 inhibitors in grapefruit peel.Grapefruit peel was extracted with diethyl ether, and the extract was further fractionated by normal-phase chromatography. Fractions demonstrating significant CYP3A4 inhibitory activity, as measured by the relative reduction in N-demethylation of diltiazem in transfected human liver epithelial cells, were subsequently separated by preparative thin-layer chromatography. Constituents of the fractions and isolated compounds were identified by nuclear magnetic resonance spectroscopy. Analysis of diltiazem and N-demethyl-diltiazem was performed using high-performance liquid chromatography.Of the identified components in grapefruit peel, only epoxybergamottin demonstrated a concentration-dependent inhibition of the CYP3A4-mediated N-demethylation of diltiazem. The IC(50) value was calculated to be 4.2+/-1.1 micro M. Coumarins without the furan ring and flavonoids isolated from grapefruit peel did not interfere with the metabolism of diltiazem. The results indicated the presence of other CYP3A4 inhibitors in grapefruit peel, but these agents were lost during the purification process excluding their identification.The furanocoumarin epoxybergamottin, present in grapefruit peel, is an inhibitor of CYP3A4. In commercial manufacturing of grapefruit juice, epoxybergamottin is possibly distributed into the juice. During manufacturing, however, epoxybergamottin may be hydrolysed to 6',7'-dihydroxybergamottin, which has been suggested as an important CYP3A4 inhibitor in grapefruit juice.

The extensive use of routine monitoring of cyclosporin A (INN, ciclosporin) whole blood levels of patients undergoing such therapy has resulted in a wide variety of chromatographic conditions for analysing this drug. The aim of this study was to evaluate the importance of essential parameters in the chromatographic determination of cyclosporin A and its main metabolites, AM1, AM9 and AM4N. A D-optimal design was used to evaluate the effect of type and amount of organic modifier, temperature, flow rate, pH and gradient steepness. The optimal chromatographic conditions were determined by multi-linear regression. In the final chromatographic method separation of the compounds was carried out on a reversed phase C(8) column maintained at 80 degrees C. The mobile phase consisted of a linear gradient with two mobile phases containing acetonitrile and water. The flow rate was set at 0.8 ml/min. UV detection was carried out at 214 nm. Validation of the analytical method showed linearity over the range 25-1000 ng/ml (r>0.997). The detection limits of cyclosporin A, AM1, AM9 and AM4N were 1.3 pmol on column. The within-day and between-day relative standard deviations were

Objective: The aim of the present study was to investigate the effect of grapefruit juice on the pharmacokinetics of cyclosporin A (CsA), as Sandimmun Neoral, and its main metabolites, Ml, M9 and M4N, in renal transplant recipients. Methods: Ten renal transplant recipients, on CsA-based immunosuppressive therapy, were included in this open, randomized crossover study. Patients were given their individualized morning dose of CsA, administered with either 250 ml water or 250 ml grapefruit juice and 12-hour CsA pharmacokinetic investigations were performed. The 2 investigation days were separated by at least 7 days. Results: Administration of CsA with grapefruit juice compared with water significantly increased the area under the whole blood concentration versus time curve in the interval from 0 - 12 hours (AUC 0-12 ) of CsA, by an average of 25 ± 19% (p = 0.002). Intake of grapefruit juice did not have any significant influence on maximum whole blood concentration (C max ) or time to C max (t max ) of CsA. AUC (0-12) and C max of M9 decreased significantly with intake of grapefruit juice, on average 22 ± 11% (p = 0.0007) and 36 ± 6% (p = 0.0001), respectively. AUC (0-12 ) of M1, however, was on average 13 ± 14% (p = 0.02) higher upon co-administration of CsA with grapefruit juice as compared with water. The level of M4N was below the limit of quantification in most samples, and an effect of co-administration of CsA with grapefruit juice could not be determined for this metabolite. Conclusion: The present study shows that co-administration of grapefruit juice with CsA compared with water affects the formation and/or elimination of the 2 metabolites M1 and M9 differently. In addition, administration of CsA with grapefruit juice compared with water induced a moderate, but significant increase in systemic exposure of CsA in renal transplant recipients.

Through the last decades it has become increasingly evident that disease-states involving cytokines affect the pharmacokinetics of drugs through regulation of expression and activity of drug metabolizing enzymes, and more recently also drug transporters. The clinical implication is however difficult to predict, since these effects are dependent on the degree of inflammation and may be changed when the diseases are treated. This article will give an overview of the present understanding of the effects of cytokines on cytochrome P450 enzymes and drug transporters, and highlight the importance of considering these issues in regard to increasing use of the relatively new class of drugs, namely therapeutic proteins.

It has earlier been shown that the isoenzymes CYP2D6 and CYP3A4 are involved in O- and N-demethylation of diltiazem (DTZ), respectively. Apparently, CYP3A4 plays a more prominent role than CYP2D6 in the overall metabolism of DTZ. However, previous observations indicate that the opposite might be true for the pharmacologically active metabolite desacetyl-DTZ (M1). Thus, the aim of the present in vitro investigation was to study the relative affinity of M1 to CYP2D6 and CYP3A4. Immortalized human liver epithelial cells transfected with either CYP2D6 or CYP3A4 were used as a model system, and the presence of M1 and its metabolites in the cell culture medium was analyzed by high-performance liquid chromatography/UV detection both before and following 90 min of incubation. The estimated K(m) value for the CYP2D6-mediated O-demethylation of M1 was approximately 5 microM. In comparison, the affinity of M1 to CYP3A4 (N-demethylation) was about 100 times lower (K(m), approximately 540 microM) than to CYP2D6. These in vitro data suggest that M1 metabolism via CYP2D6, in contrast to the parent drug, probably is the preferred pathway in vivo. Metabolism mediated through CYP2D6 is associated with a substantial interindividual variability, and since M1 expresses pharmacological activity, individual CYP2D6 metabolic capacity might be an aspect to consider when using DTZ.

Cyclosporine A (CsA) and tacrolimus (Tac) are immunosuppressive drugs used in the majority of patients with solid organ transplants, generally in combination with a wide range of drugs. CsA and Tac seem not only to be substrates of CYP3A but have also been described as inhibitors of CYP3A. For CsA, in particular, inhibition of CYP3A has been suggested as the main mechanism of interactions seen clinically with various drugs. The aim of this study was to investigate the inhibitory effect and inhibition characteristics of CsA and Tac on CYP3A4 and CYP3A5 in vitro and to evaluate its clinical relevance. Inhibition by CsA and Tac was studied using midazolam as the probe substrate in coincubation and preincubation investigations using human liver microsomes (HLMs) as well as specific CYP3A4- and CYP3A5-expressing insect microsomes (Supersomes). In vitro-in vivo extrapolations (IVIVEs) were performed to evaluate the clinical relevance of the inhibition. Both CsA and Tac competitively inhibited CYP3A in HLMs, showing inhibition constants (K(i)) of 0.98 and 0.61 μM, respectively. Experiments in Supersomes revealed that Tac inhibited both CYP3A4 and CYP3A5, whereas CsA only inhibited CYP3A4. In contrast to the HLM experiments, studies in Supersomes showed inhibition by Tac to be NADPH- and time-dependent, with a 5-fold reduction in IC(50) after preincubation, supporting a time-dependent inhibition mechanism in recombinant microsomes. By application of HLM data, IVIVE estimated the area under the concentration versus time curve of midazolam to increase by 73 and 27% with CsA and Tac, respectively. The inhibitory effect was predominantly on the intestinal level, whereas hepatic intrinsic clearance seemed unaffected.

Data from in vitro drug metabolism studies with recombinant enzyme systems are frequently used to predict human drug metabolism in vivo. However, for the CYP3A probe substrate midazolam (MDZ), considerable variability in enzyme kinetic parameters has been observed in different in vitro studies. The aim of this study was to explore the effect of varying activities of the electron donor NADPH-cytochrome P450 reductase (CPR) on CYP3A5-mediated metabolism of MDZ. Microsomes with similar levels of CYP3A5 but 12-fold difference in CPR activity showed a 30-fold difference in intrinsic clearance for the formation of 1'-OH-MDZ. Significantly higher K(m) and lower V(max) for the formation of 1'-OH-MDZ were found in microsomes with low CPR activity compared with microsomes with higher CPR activity (P = 0.024 and 0.001). In the microsomes with lowest CPR activity, the formation of 1'-OH-MDZ displayed Michaelis-Menten kinetics, whereas substrate inhibition was observed in the two preparations with higher CPR activity. The present study shows that the CPR activity in different recombinant enzyme preparations is crucial for in vitro CYP3A5-mediated clearance of MDZ. This suggests that the CPR activity of enzyme preparations could be an important factor for the ability of in vitro data to predict human drug metabolism in vivo.

Biliopancreatic diversion with duodenal switch is a combined restrictive and malabsorptive surgical weight loss procedure. Given that this procedure introduces a bypass of the proximal small intestine, it is a suitable model for investigating the influence of the proximal intestine on drug bioavailability. Eight-hour pharmacokinetic profiles were obtained the day before surgery and again after surgery at (median) 6 weeks (range, 4–8 weeks) in 10 morbidly obese patients who were receiving treatment with 20–80 mg atorvastatin each morning. The bioavailability of atorvastatin acid was significantly increased, with a mean twofold higher AUC0–8 after surgery (range 1.0–4.2, P = 0.001). Time to maximum plasma concentration (Cmax) increased from 1.2 h before surgery to 2.3 h after surgery (P = 0.03). The results emphasize the protective nature of the proximal small intestine against ingested exogenous compounds. Consequently, retitration to the lowest effective dose should be considered after biliopancreatic diversion with duodenal switch in the case of drugs with a high degree of intestinal first pass metabolism and a narrow therapeutic window. Clinical Pharmacology & Therapeutics (2010) 87 6, 699–705. doi: 10.1038/clpt.2010.32

Synaptic vesicles have been isolated from rat spinal cord-medulla at different postnatal ages, and the ontogeny of the uptake of glycine, γ-aminobutyric acid (GABA) and glutamate has been investigated. The accumulation of the 3 amino acids increased with increasing time after birth reaching adult level at about postnatal day 30. This developmental increase probably parallels the synaptogenesis and suggests a functional role of the uptake of the amino acids into synaptic vesicles in the nerve terminals. The developmental time course for these vesicular uptake systems was totally different from those of the corresponding plasma membrane uptakes.

The antipsychotic drug quetiapine is extensively metabolized by CYP3A4, but little is known about the possible influence of the polymorphic enzyme CYP3A5. This in vitro study investigated the relative importance of CYP3A4 and CYP3A5 in the metabolism of quetiapine and compared the metabolic pattern by the two enzymes, in the presence or absence of cytochrome b(5). Intrinsic clearance (CL(int)) of quetiapine was determined by the substrate depletion approach in CYP3A4 and CYP3A5 insect cell microsomes with or without coexpressed cytochrome b(5). Formation of the metabolites quetiapine sulfoxide, N-desalkylquetiapine, O-desalkylquetiapine, and 7-hydroxyquetiapine by CYP3A4 and CYP3A5 were compared in the different microsomal preparations. CL(int) of quetiapine by CYP3A5 was less than 35% relative to CYP3A4. CL(int) was higher (3-fold) in CYP3A4 microsomes without cytochrome b(5) compared with CYP3A4 microsomes with coexpressed cytochrome b(5), whereas in CYP3A5 microsomes CL(int) was similar for both microsomal preparations. Metabolism of quetiapine by CYP3A5 revealed a different metabolic pattern compared with CYP3A4. The results indicated that O-desalkylquetiapine constituted a higher proportion of the formed metabolites by CYP3A5 compared with CYP3A4. In conclusion, the present study indicates that CYP3A5 is of minor importance for the overall metabolism of quetiapine, regardless of the presence of cytochrome b(5). However, a different metabolic pattern by CYP3A5 compared with CYP3A4 could possibly result in different pharmacological and/or toxicological effects of quetiapine in patients expressing CYP3A5.

Although displacement from plasma protein binding (dPB) is usually of little clinical significance, it should be taken into account when interpreting changes in total plasma concentrations of drugs subject to metabolically based drug-drug interactions (mDDI). The aim of this study was to develop an approach to predict changes in the free fractions (fu) of pairs of drugs that compete for plasma binding, knowing their binding affinity constants, and to consider the implications of associated concentration- and time-dependence of such changes with respect to drug exposure. Experimental fu values of valproic acid and phenytoin in the presence of ibuprofen, diflunisal, or naproxen were predicted successfully (within 0.99- to 1.36-fold) by the model. In addition, the simulation of time-dependent changes in fu of valproic acid following administration of ibuprofen indicated different extents of dPB during 'first-pass' through the liver after oral absorption and on systemic recirculation. To understand the impact of the time-dependent change in fu, a full physiologically based pharmacokinetic model, that accounts for concentration-time profile of displacee and displacer and their mutual effect on each other, is required. The approach developed in this study is a first step towards the development of such a model.

The aim of the present study was to develop a chromatographic method for the analysis of atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human plasma after administration of atorvastatin at the lowest registered dose (10 mg) in clinical studies. Sample preparation was performed by solid-phase extraction and was followed by separation of the analytes on an HPLC system with a linear gradient and a mobile phase consisting of acetonitrile, water and formic acid. Detection was achieved by tandem mass spectrometry operated in the electrospray positive ion mode. Validation of the method for the compounds for which reference compounds were available (acid forms of atorvastatin, o- and p-hydroxyatorvastatin) showed linearity within the concentration range (0.2-30 ng/ml for atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5-30 ng/ml for o-hydroxyatorvastatin acid) (r2 > or = 0.99, n = 5 for all analytes). Accuracy and precision (evaluated at 0.5, 3 and 30 ng/ml for atorvastatin, p-hydroxyatorvastatin and 1, 3 and 30 ng/ml for o-hydroxyatorvastatin) were both satisfactory. The detection limit was 0.06 ng/ml for atorvastatin and p-hydroxyatorvastatin, and 0.15 ng/ml for o-hydroxyatorvastatin. The method has been successfully applied in a clinical study where atorvastatin, o- and p-hydroxyatorvastatin (both acid and lactone forms) could be detected in a 24-h sampling interval after administration of the lowest registered dose of atorvastatin (10 mg) for one week.

The aim of the present study was to evaluate the possible influence of atorvastatin on the pharmacokinetics of cyclosporine (INN ciclosporin) and its main metabolites, AM1 and AM9, in renal transplant recipients.Whole blood samples from 18 renal transplanted patients on cyclosporine-based immunosuppressive therapy were collected prior to and after 4 weeks of treatment with atorvastatin (10 mg/day) and analysed with regard to both cyclosporine and its main metabolites, AM1 and AM9, using a specific chromatographic method with ultraviolet detection.On average, AUC(0-12) [area under the whole blood concentration versus time curve in the dosing interval (0-12 h)] of cyclosporine was 5% (-16, 5) (90% confidence interval) lower upon co-administration with atorvastatin. No statistically significant changes in any of the calculated pharmacokinetic variables [AUC(0-12), maximum whole blood concentration (C(max)), whole blood concentration 12 h post dose (C(12)), time to C(max) (t(max)), terminal half-life (t(1/2))] for cyclosporine or the two metabolites, AM1 and AM9, upon atorvastatin treatment were observed. On average, atorvastatin did not affect the ratio between the CYP3A4-mediated metabolite AM9 and cyclosporine, suggesting that atorvastatin does not affect the CYP3A4 metabolism of cyclosporine to any significant extent. However, the influence of atorvastatin on the ratio between AM9 and cyclosporine showed large interindividual variability.The results of this study indicate that atorvastatin does not, on average, affect cyclosporine pharmacokinetics in renal transplant recipients.

The aim of the present work was to develop a high-performance liquid chromatography-mass spectrometry method for analysis of diltiazem (DTZ) and metabolites in human plasma after single dose administration (120 mg). Human plasma samples (1 ml) were cleaned up by a solid phase extraction procedure (C18 cartridges) using codeine as an internal standard. Reconstituted extracts were separated on a reversed-phase C8 column with a linear gradient mobile phase system. The run time per sample analysis was 11 min. Detection was performed using selected ion monitoring following atmospheric pressure chemical ionization. The lower limit of quantification was estimated to be 1 microg/l (in spiked plasma) for all available reference compounds (i.e. DTZ and five metabolites). Validation of the method showed good linearity, precision and accuracy for quantification of these six reference compounds. In addition, tandem MS analyses of human plasma sampled from healthy individuals after peroral intake of 120 mg DTZ revealed that the method enabled detection of six additional metabolites for which reference compounds were not available.

Objectives. Grapefruit juice has been reported to increase the bioavailability of several calcium-channel antagonists, i.e. the dihydropyridines and verapamil, which are cytochrome P 450 3A4 (CYP3A4) substrates. The objective of the present study was to investigate the effect of grapefruit juice on the pharmacokinetics of diltiazem and the metabolites N-demethyl-diltiazem (MA) and desacetyl-diltiazem (M1). Methods. Ten healthy male volunteers were included in a randomised, open, crossover study, comparing the effect of a single oral dose of non-retard formulated diltiazem (120 mg) administered with 250 ml grapefruit juice or water. The study was performed on two investigation days separated by 13–38 days (median 28 days). Plasma samples were collected for measurement of diltiazem and the metabolites MA and M1. Blood pressure and heart rate were monitored throughout the study. Results. Grapefruit juice intake resulted in a statistically significant average individual increase in the area under the plasma diltiazem concentration–time curve (AUC0–24) of 20±25% (P=0.02) compared with water. The average individual increase in peak plasma concentration (Cmax) of diltiazem after grapefruit juice administration was 22±37%, but this effect was not statistically significant (P=0.14). The time to Cmax (tmax) or terminal half-life was not affected by grapefruit juice. Considerable interindividual variability in the interaction was observed. There were no statistically significant differences in blood pressure and heart rate between the two treatments. Conclusion. The present study demonstrated that a single intake of grapefruit juice (250 ml) caused a slight but statistically significant increase in the systemic exposure of diltiazem. Inhibition of intestinal metabolism and/or P-glycoprotein efflux transport may be responsible for this effect.

Objective: In a previous study of diltiazem (DTZ) pharmacokinetics in renal transplant patients, we speculated that a polymorphic enzyme could be involved in O-demethylation of diltiazem. The aim of this in vitro study was to investigate whether O-demethylation of DTZ is mediated by cytochrome P 450-2D6 (CYP2D6). Methods: DTZ was incubated with transfected human liver epithelial (THLE) cells expressing CYP2D6 (T5-2D6 clone). Metabolism of DTZ was studied over a concentration range of 12.5–400 µM and in the presence of quinidine (a CYP2D6 inhibitor) or erythromycin (a CYP3A4 inhibitor). THLE cells lacking CYP2D6 activity (T5-neo clone) were used as control. The culture medium of the cells, in which DTZ was dissolved, was analysed for DTZ and metabolites prior to and after 8 h of incubation using high-performance liquid chromatography (HPLC, UV detection). Authentic O-demethyl-DTZ (Mx) was not available, and this metabolite was therefore not identifiable. Results: Desacetyl-O-demethyl-DTZ (M4) was exclusively produced during incubations of DTZ with THLE cells expressing CYP2D6. The rate of M4 formation was described using Michaelis Menten kinetics in the concentration range of DTZ used. Production of M4 was inhibited by quinidine, but not erythromycin. An unidentified chromatographic peak, which was interpreted to be Mx, showed the same pattern of formation as M4 both in absence and presence of inhibitors. N-demethylated metabolites, formed by CYP3A4, were not observed in any of the cell lines. Conclusion: Evidence was provided in vitro that O-demethylation of DTZ is mediated by the polymorphic isoenzyme CYP2D6. Involvement of CYP2D6 in the metabolism of DTZ may have clinical implications regarding pharmacokinetic variability and interactions.

Bilateral cyclosporin A (CsA) and diltiazem pharmacokinetic interactions have previously been investigated, however, not with the new microemulsion preconcentrate formulation of CsA (Sandimmun Neoral). In addition, the pharmacokinetic effects on the pharmacological active metabolites of diltiazem have not previously been investigated. We performed a pharmacokinetic interaction study in renal transplant recipients, measuring both unmetabolised CsA and diltiazem in addition to three of the main metabolites of diltiazem (MA, M1, M2).Nine CsA-treated renal transplant patients were treated with diltiazem, 90-120 mg b.i.d., for 4 weeks. Pharmacokinetic investigations were performed both before and at the end of the diltiazem treatment period. Six non-CsA-treated renal transplant patients served as controls of CsA interactions with diltiazem and its metabolites.Diltiazem treatment resulted in a significant mean increase in the area under the concentration time curve (AUC) for CsA of 51(8)% (P0.008) and a peak concentration (Cmax) of 34(8)% (P0.05), without altering time to peak concentration (tmax). CsA, however, did not significantly influence diltiazem pharmacokinetics, though two of the metabolites (M1 and M2) tended to be increased.Diltiazem interacts significantly with the pharmacokinetics of CsA in the new microemulsion formulation. Microemulsion-formulated CsA, however, did not show significant interaction with diltiazem pharmacokinetics.

A sensitive and specific reversed-phase high-performance liquid chromatographic method was developed for determination of the benzothiazepine calcium channel blocker diltiazem and three of its main metabolites in human plasma. A solid-phase extraction method (C18) is described for isolating diltiazem and the metabolites N-demethyl-diltiazem (M(A)), deacetyldiltiazem (M1) and N-demethyl-deacetyl-diltiazem (M2) from human plasma spiked with trans-diltiazem as internal standard. Chromatographic analysis of the eluate was accomplished using a reversed-phase column (C8) and a mobile phase consisting of acetonitrile, potassium dihydrogen phosphate-buffer (pH 2.9), triethylamine and methanol (280:598:2:90 v/v/v/v). The limit of detection was 5 ng/ml for diltiazem and 2.5 ng/ml for the metabolites (UV detection). Recoveries were > 75% for all substances. Precision analysis indicated a coefficient of variation below 5% for both diltiazem and metabolites at plasma concentrations ranging from 30 to 120 ng/ml for diltiazem and from 15 to 60 ng/ml for the metabolites. The accuracy was < 3% for diltiazem at all concentrations investigated. Metabolites showed an accuracy of < 7% at higher concentrations, however at low concentration the accuracy was < 16%. A pilot study has been performed in order to study whether the method was applicable to patients and volunteers. Plasma samples from renal transplanted patients treated with cyclosporin A and healthy volunteers did not show any interference by cyclosporin A with the determination of diltiazem and metabolites.

An increasing prevalence of morbid obesity has led to dramatic increases in the number of bariatric surgeries performed. Altered gastrointestinal physiology following surgery can be associated with modified oral drug bioavailability (Foral). In the absence of clinical data, an indication of changes to Foral via systems pharmacology models would be of value in adjusting dose levels after surgery. A previously developed virtual “post-bariatric surgery” population was evaluated through mimicking clinical investigations on cyclosporine and atorvastatin after bariatric surgery. Cyclosporine simulations displayed a reduced fraction absorbed through gut wall (fa) and Foral after surgery, consistent with reported observations. Simulated atorvastatin Foral postsurgery was broadly reflective of observed data with indications of counteracting interplay between reduced fa and an increased fraction escaping gut wall metabolism (FG). Inability to fully recover observed atorvastatin exposure after biliopancreatic diversion with duodenal switch highlights the current gap regarding the knowledge of associated biological changes. CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e47; doi:10.1038/psp.2013.23; advance online publication 12 June 2013

The ontogeny of the uptake of glutamate, GABA and glycine into synaptic vesicles isolated from rat brain has been investigated. The vesicular uptake of the three amino acids increased with developmental age in parallel with synaptogenesis, indicating a functional role of uptake of the amino acids by synaptic vesicles in the nerve terminals. Uptake of the amino acids by plasma membrane particles (synaptosomes) in brain homogenate showed a somewhat different developmental profile. The uptake of glutamate increased markedly with developmental time, while the uptake of GABA showed only a slight increase. Uptake of glycine by plasma membrane particles was very low and therefore not registered. The observed developmental increase in uptake of glycine by synaptic vesicles isolated from brain, supports previous reports indicating that glycine can be taken up by vesicles from non-glycine terminals.

The substrate specificity of vesicular GABA and glycine uptake was studied in vesicle fractions from brain and spinal cord, respectively. Glycine, beta-alanine and gamma-vinyl-GABA were competitive inhibitors of the GABA uptake were competitive inhibitors of the GABA uptake by synaptic vesicles in brain. Likewise GABA and beta-alanine turned out to be competitive inhibitors of vesicular uptake of glycine in spinal cord. The apparent K1 values were in the same range as the respective Km values for the transport systems. Accumulation of different amino acids were examined, and the structurally related amino acids GABA, beta-alanine and glycine were all taken up by both vesicle fractions. In the present study, we suggest that there are similarities in the vesicular transporters for GABA and glycine, and the two amino acids are probably taken up into the same vesicle population. The key factor in differentiating between GABA and glycine as transmitters in the terminals could be the synthesis and the high-affinity synaptosomal uptake.

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.

Background A previous study of 22 patients undergoing either gastric bypass or duodenal switch showed increased systemic exposure of atorvastatin acid 3–8 weeks after surgery in the majority of patients. This study aimed to investigate the long-term effects on systemic exposure of atorvastatin acid in the same group of patients. Methods An 8-h pharmacokinetic investigation was performed a median of 27 months (range 21–45 months) after surgery. Systemic exposure was measured as the area under the plasma concentration versus the time curve from 0 to 8 h postdose (AUC0–8). Linear mixed models with AUC0–8 as the dependent variable were implemented to assess the effect of time, surgical procedure, and body mass index (BMI) as explanatory variables. Results The study enrolled 20 patients. The systemic exposure of atorvastatin acid changed significantly over time (p = 0.001), albeit there was substantial variation between subjects. The effect of time was attenuated but remained significant after adjustment for surgical procedure and BMI (p = 0.048). The initial AUC0–8 increase seen in the majority of patients 3–8 weeks after surgery was normalized long term, with 7 of the 12 gastric bypass patients and 6 of the 8 duodenal switch patients showing decreased AUC0–8 compared with preoperative values. Conclusions The systemic exposure of atorvastatin showed a significant change over time after bariatric surgery, albeit with large inter- and intraindividual variations. The findings indicate that patients using atorvastatin or drugs with similar pharmacokinetic properties should be monitored closely for both therapeutic effects and adverse events the first years after gastric bypass and duodenal switch. ClinicalTrial NCT00331565.

Purpose Tacrolimus (Tac) and cyclosporine (CsA) are mainly metabolized by CYP3A4 and CYP3A5. Several studies have demonstrated an association between the CYP3A5 genotype and Tac dose requirements. Recently, CYP3A4, PPARA, and POR gene variants have been shown to influence CYP3A metabolism. The present study investigated potential associations between CYP3A5*3, CYP3A4*22, PPARA c.209-1003G>A and c.208 + 3819A>G, and POR*28 alleles and dose-adjusted concentrations (C/D) of Tac and CsA in 177 renal transplant patients early post-transplant. Methods All patients (n = 177) were genotyped for CYP3A4*22, CYP3A5*3, POR*28, PPARA c.209-1003G>A, and PPARA c.208 + 3819A>G using real-time polymerase chain reaction (PCR) and melting curve analysis with allele-specific hybridization probes or PCR restriction fragment length polymorphisms (RFLP) methods. Drug concentrations and administered doses were retrospectively collected from patient charts at Oslo University Hospital, Rikshospitalet, Norway. One steady-state concentration was collected for each patient. Results We confirmed a significant impact of the CYP3A5*3 allele on Tac exposure. Patients with POR*28 and PPARA variant alleles demonstrated 15 % lower (P = 0.04) and 19 % higher (P = 0.01) Tac C0/D respectively. CsA C2/D was 53 % higher among CYP3A4*22 carriers (P = 0.03). Conclusion The results support the use of pre-transplant CYP3A5 genotyping to improve initial dosing of Tac, and suggest that Tac dosing may be further individualized by additional POR and PPARA genotyping. Furthermore, initial CsA dosing may be improved by pre-transplant CYP3A4*22 determination. Electronic supplementary material The online version of this article (doi:10.1007/s00228-014-1656-3) contains supplementary material, which is available to authorized users