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Purpose We assessed the differential effect of clarithromycin, a strong inhibitor of cytochrome P450 (CYP) 3A4 and P-glycoprotein, on the pharmacokinetics of a regular dose of edoxaban and on a microdose cocktail of factor Xa inhibitors (FXaI). Concurrently, CYP3A activity was determined with a midazolam microdose. Methods In an open-label fixed-sequence trial in 12 healthy volunteers, the pharmacokinetics of a microdosed FXaI cocktail (μ-FXaI; 25 μg apixaban, 50 μg edoxaban, and 25 μg rivaroxaban) and of 60 mg edoxaban before and during clarithromycin (2 x 500 mg/d) dosed to steady-state was evaluated. Plasma concentrations of study drugs were quantified using validated ultra-performance liquid chromatography–tandem mass spectrometry methods. Results Therapeutic clarithromycin doses increased the exposure of a therapeutic 60 mg dose of edoxaban with a geometric mean ratio (GMR) of the area under the plasma concentration-time curve (AUC) of 1.53 (90 % CI: 1.37–1.70; p < 0.0001). Clarithromycin also increased the GMR (90% CI) of the exposure of microdosed FXaI apixaban to 1.38 (1.26–1.51), edoxaban to 2.03 (1.84–2.24), and rivaroxaban to 1.44 (1.27–1.63). AUC changes observed for the therapeutic edoxaban dose were significantly smaller than those observed with the microdose (p < 0.001). Conclusion Clarithromycin increases FXaI exposure. However, the magnitude of this drug interaction is not expected to be clinically relevant. The edoxaban microdose overestimates the extent of the drug interaction with the therapeutic dose, whereas AUC ratios for apixaban and rivaroxaban were comparable to the interaction with therapeutic doses as reported in the literature. Trial Registration EudraCT Number: 2018-002490-22

Purpose The use of two-dimensional (2D) printing technologies of drugs on orodispersible films (ODF) can promote dose individualization and facilitate drug delivery in vulnerable patients, including children. We investigated midazolam pharmacokinetics after the administration of 2D-printed ODF. Methods Midazolam doses of 0.03 and 3 mg were printed on an ODF using a 2D drug printer. We investigated the bioavailability of the two midazolam doses with ODF swallowed immediately (ODF-IS) or delayed after 2 min (ODF-DS) by comparing their pharmacokinetics with intravenous and oral midazolam solution in 12 healthy volunteers. Results The relative bioavailability of ODF-IS 0.03 mg was 102% (90% confidence interval: 89.4–116) compared to oral solution and for 3 mg 101% (86.8–116). Cmax of ODF-IS 0.03 mg was 95.5% (83.2–110) compared to oral solution and 94.3% (78.2–114) after 3 mg. Absolute bioavailability of ODF-IS 0.03 mg was 24.9% (21.2–29.2) and for 3 mg 28.1% (23.4–33.8) (oral solution: 0.03 mg: 24.4% (22.0–27.1); 3 mg: 28.0% (25.0–31.2)). Absolute bioavailability of ODF-DS was significantly larger than for ODF-IS (0.03 mg: 61.4%; 3 mg: 44.1%; both p Conclusion This trial demonstrates the tolerability and unchanged bioavailability of midazolam printed on ODF over a 100-fold dose range, proving the suitability of ODF for dose individualization. Midazolam ODF-IS AUC0–∞ in both doses was bioequivalent to the administration of an oral solution. However, Cmax of the therapeutic dose of ODF-IS missed bioequivalence by a clinically not relevant extent. Prolonged mucosal exposure increased bioavailability. (Trial Registration EudraCT: 2020–003984-24, August 10, 2020).

Transdermal fentanyl is widely used to control pain in cancer patients. The high pharmacokinetic variability of fentanyl is assumed to be due to cytochrome P450 3A-mediated (CYP3A) N-dealkylation to norfentanyl in humans. However, recently published clinical studies question the importance of the described metabolic pathway. In this small study in palliative cancer patients under real-life clinical conditions, the influence of CYP3A on fentanyl variability was investigated. In addition to the determination of midazolam plasma concentration to reveal CYP3A activity, plasma concentrations of fentanyl and its metabolite, norfentanyl, were measured in identical blood samples of 20 patients who participated in an ongoing trial and had been on transdermal fentanyl. Fentanyl, norfentanyl, midazolam, and 1′-OH-midazolam were quantified by liquid chromatography/tandem mass spectrometry. Plasma concentrations of fentanyl and norfentanyl exhibited a large variability. Mean estimated total clearance of fentanyl and mean metabolic clearance of midazolam (as a marker of CYP3A activity) were 75.5 and 36.3 L/h. Both clearances showed a weak correlation and hence a minimal influence of CYP3A on fentanyl elimination.

Background: Cytochrome P450 3A is the most relevant drug-metabolizing enzyme in humans as it is involved in the elimination of 50% of marketed drugs. Nothing is known about the activity of cytochrome P450 3A in palliative care patients who have complicated symptoms often associated with a terminal illness. Aim: In order to improve drug dosing in end-of-life care and to avoid drug interactions, cytochrome P450 3A activity was determined in patients of a palliative care unit under real-life clinical conditions. Design: As midazolam is an established marker substance for cytochrome P450 3A activity, this single-arm prospective trial was designed to obtain a 4-h pharmacokinetic profile of midazolam after oral administration of a 10-µg dose from each enrolled patient. Plasma concentrations of midazolam and its primary metabolite 1′-hydroxy-midazolam were quantified by mass spectrometry techniques. Cytochrome P450 3A activity was calculated as partial metabolic clearance from a limited sampling area under the curve. All other drugs taken by the participating patients were considered, as well as recent blood test results and patients’ diagnoses. The trial was registered at German Clinical Trials Register ( www.drks.de ): DRKS00011753. Setting/participants: The trial was carried out at a university palliative care unit under real-life clinical conditions. Every patient admitted to the ward was screened for possible participation, independent of the individual performance status. Results: Partial metabolic clearance of midazolam in palliative care patients was 31.7 ± 32.1 L/h. This was a highly significant 40% reduction ( p Conclusion: Dosing of cytochrome P450 3A substrate drugs (e.g. macrolide antibiotics, benzodiazepines, calcium channel blockers) needs to be adjusted in palliative care patients; otherwise, escalation of debilitating symptoms due to drug interactions might occur.

Background The impact of major liver resection (LR) on the detoxifying function of the remaining liver tissue as represented by CYP3A activity has yet to be assessed. Therefore, this study evaluates the changes in CYP3A activity between preoperative values and after liver resection. Material and methods To determine CYP3A activity, midazolam (MDZ) was used as a marker substance, 3 μg were applied intravenously one day before surgery and on the 3rd day after surgery. Subsequently blood was withdrawn at 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4 and 6 h post application of the study drug. Plasma MDZ and 1-OH-MDZ concentration was assessed using a LC-MS/MS method. Volumetric analysis of the resected liver was done by syngo.CT liver analysis software (Siemens Healthineers) using preoperative multidetector computed tomography. Results N = 13 (8 male/5 female) patients were included in this study and received preoperative evaluation, 11 patients were studied also after liver resection. The mean age was 62 (±15.3) years with a mean BMI of 23.6 ± 4.8 kg/m2. No patient suffered from acute liver dysfunction postoperatively. None of the pharmacokinetic parameters assessed were significantly altered by liver resection. CYP3A activity over time was not significantly reduced by major liver resection. Conclusion This study gives first time data on the impact of major liver resection on CYP3A activity. It was shown that MDZ clearance representing in vivo CYP3A activity is not altered by major liver resection. This suggests no dose adjustment of commonly applied drugs which are CYP3A substrates needs to be carried out.

Purpose Efavirenz-based combination antiretroviral therapy (cART) is associated with neuropsychiatric adverse events. We investigated the time to onset, duration, clinical implications, impact of pharmacogenetic variations, and anti-tuberculosis co-treatment on efavirenz-associated neuropsychiatric manifestations. Methods Prospective cohort study of cART naïve HIV patients with or without tuberculosis (HIV-TB) co-infection treated with efavirenz-based cART. Rifampicin-based anti-tuberculosis therapy was initiated 4 weeks prior to efavirenz-based cART in HIV-TB patients. Data on demographic, clinical, laboratory, and a 29-item questionnaire on neuropsychiatric manifestations were collected for 16 weeks after cART initiation. Genotyping for CYP2B6, CYP3A5, SLCO1B1, and ABCB1 and quantification of efavirenz plasma concentration were done on the 4th and 16th week. Results Data from 458 patients (243 HIV-only and 215 HIV-TB) were analyzed. Overall incidence of neuropsychiatric manifestations was 57.6% being higher in HIV-only (66.7%) compared to HIV-TB patients (47.4%) (p

AIMS To test the in vivo activity of Cytochrome P450 (CYP) 2E1 in obese children vs. nonobese children, aged 11-18 years. Secondly, whether the activity of CYP2E1 in these patients is associated with NALFD, diabetes or hyperlipidaemia. METHODS Seventy children were divided into groups by body mass index (BMI) standard deviation score (SDS). All children received 250 mg oral chlorzoxazone (CLZ) as probe for CYP2E1 activity. Thirteen blood samples and 20-h urine samples were collected per participant. RESULTS Obese children had an increased oral clearance and distribution of CLZ, indicating increased CYP2E1 activity, similar to obese adults. The mean AUC0-∞ value of CLZ was decreased by 46% in obese children compared to nonobese children. The F was was increased twofold in obese children compared to nonobese children, P

Myrcludex B acts as a hepatitis B and D virus entry inhibitor blocking the sodium taurocholate cotransporting polypeptide (SLC10A1). We investigated the effects of myrcludex B on plasma bile acid disposition, tenofovir pharmacokinetics, and perpetrator characteristics on cytochrome P450 (CYP) 3A. Twelve healthy volunteers received 300 mg tenofovir disoproxil fumarate orally and 10 mg subcutaneous myrcludex B. Myrcludex B increased total plasma bile acid exposure 19.2-fold without signs of cholestasis. The rise in conjugated bile acids was up to 124-fold (taurocholic acid). Coadministration of tenofovir with myrcludex B revealed no relevant changes in tenofovir pharmacokinetics. CYP3A activity slightly but significantly decreased by 29% during combination therapy. Myrcludex B caused an asymptomatic but distinct rise in plasma bile acid concentrations and had no relevant impact on tenofovir pharmacokinetics. Changes in CYP3A activity might be due to alterations in bile acid signaling. Long-term effects of elevated bile acids will require critical evaluation.

Objective We sought to assess whether pharmacological inhibition of hypoxia-inducible transcription factor (HIF)-prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) is a suitable strategy to stimulate liver regeneration after partial hepatectomy for colorectal liver metastases (CRLM). Background Liver regeneration occurs in a hypoxic environment. PHD1 to PHD3 are molecular oxygen sensors and increasingly considered as putative therapeutic targets. However, little is known about the effect of pharmacological PHD inhibition on tumor expansion, and on liver regeneration after surgical resection. Methods Various mouse models of liver regeneration after extended partial hepatectomy and portal vein ligation for multiple bilobar CRLM were applied to assess the effect of the small molecule pan-PHD inhibitor ethyl-3,4-dihydroxybenzoate (EDHB) on liver regeneration and metastatic tumor growth. Metabolism and biodistribution of EDHB were analyzed using liquid chromatography coupled to tandem mass spectrometry. Results EDHB selectively augmented liver regeneration after partial hepatectomy and portal vein ligation, and increased the expression of cell cycle-promoting cyclin proteins, without enhancing metastatic tumor growth. Systemically administered EDHB and its active metabolite 3,4-dihydroxybenzoic acid accumulated in the liver to selectively induce hepatoprotective effects in the liver, but not in tumor tissue, without humoral adverse effects. Conclusions Pharmacological inhibition of PHDs using EDHB might represent a novel and safe strategy in the treatment of multiple bilobar CRLM.

Cytochrome P450 3A (CYP3A) is the most relevant drug-metabolizing enzyme in human beings involved in the elimination of about 50% of the marketed drugs. Comprehensive in vivo data of CYP3A activity in palliative patients with haematological diseases are missing. Therefore, CYP3A activity was determined under real-life clinical conditions in patients to gain knowledge about dose adjustments for supportive therapies and symptom management in haematology. The single-arm, prospective trial obtained a 4-hours pharmacokinetic profile of midazolam after oral administration of a microdose as marker substance from each enrolled patient. Plasma concentrations of midazolam and its primary metabolite 1'-hydroxy-midazolam were quantified by mass spectrometry techniques. CYP3A activity was calculated as partial metabolic clearance from an established limited sampling area under the curve. All other drugs taken by the participating patients were considered as well as recent laboratory test results and the patients' diagnoses. Partial metabolic clearance of midazolam in patients with haematological diseases was highly variable (36.9 ± 52.7 L/h). In comparison with the CYP3A activity of healthy individuals, this was a highly significant 30% reduction of activity (P < 0.0001). Dosing of major CYP3A substrate drugs needs to be reduced in palliative patients with haematological diseases, otherwise escalation of debilitating symptoms due to drug interactions might occur.

Aims Erythromycin is a macrolide antibiotic, which is frequently used as a topical formulation for the treatment of acne. It is also known as an inhibitor of the cytochrome P450 (CYP) isoenzyme 3A4. In this study, the systemic availability of topical erythromycin, hence the influence on the activity of CYP3A, is evaluated in comparison to orally administered erythromycin.

BackgroundMidazolam is a first-line drug for treatment of status epilepticus,1 2 both by buccal and intravenous administration. In children with obesity, the midazolam pharmacokinetics may be altered, and the current dosing guidelines may therefore be insufficient.The aim of the study was to investigate the pharmacokinetics of midazolam, after intravenous administration, in obese and non-obese children, aged 11–18 years.MethodsTrial subjects were divided into groups by Body Mass Standard Deviation Score (SDS). All children received 1 µg midazolam administered an intravenous bolus dose. Thirteen blood samples were collected per participant at prespecified timepoints over 9 hours. Plasma concentration-time data was fitted to pharmacokinetic models using non-linear mixed effects modelling (NONMEM, 7.4).ResultsSeventy-two children were enrolled in the study, of these 67 children were included in the analysis. The pharmacokinetics of midazolam was best described with a two-compartment model. The changes in pharmacokinetics in children with obesity were best described with a linear function of BMI SDS on inter-compartmental clearance and peripheral volume. Thus, the rate of distribution was faster, and the peripheral volume of distribution was larger in children with obesity as compared to non-obese children. Simulations revealed that long-term infusions based on total body weight, could lead to high plasma concentrations in children with obesity. Furthermore, simulated plasma concentrations after a fixed buccal dose showed that children with obesity may be at risk of subtherapeutic plasma concentrations.ConclusionBMI SDS was shown to have a significant influence on the peripheral volume of distribution and the inter-compartmental clearance of midazolam. The current Danish dosing guidelines for status epilepticus (http://www.paediatri.dk), where midazolam dose is adjusted to total body weight or age, may lead to both supra- and subtherapeutic doses respectively, in children with obesity. However, confirmatory studies are needed.ReferencesSmith R, Brown J. Midazolam for status epilepticus. Aust Prescr. februar 2017;40(1):23–5.Ulvi H, Yoldas T, Müngen B, Yigiter R. Continuous infusion of midazolam in the treatment of refractory generalized convulsive status epilepticus. Neurol Sci Off J Ital Neurol Soc Ital Soc Clin Neurophysiol oktober 2002;23(4):177–82.Disclosure(s)Nothing to disclose

Aims The aim of this study was to assess the effect of the cytochrome P450 (CYP) 3A4 and organic anion-transporting polypeptide (OATP) 1B1 inhibitor clarithromycin on the pharmacokinetics of bosentan. We also aimed to evaluate the impact of CYP2C9 and SLCO1B1 (encoding for OATP1B1) genotypes and their combination. Methods We assessed the effect of the OATP and CYP3A inhibitor clarithromycin on bosentan pharmacokinetics at steady state and concurrently quantified changes of CYP3A activity using midazolam as a probe drug. Sixteen healthy volunteers received therapeutic doses of bosentan (125 mg twice daily) for 14 days and clarithromycin (500 mg twice daily) concomitantly for the last 4 days, and bosentan pharmacokinetics was assessed on days 1, 10 and 14. Results Clarithromycin significantly increased bosentan area under the plasma concentration–time curve of the dosing interval 3.7-fold and peak concentration 3.8-fold in all participants irrespective of the genotype. Clarithromycin also reduced CYP3A activity (midazolam clearance) in all participants; however, these changes were not correlated to the changes of bosentan clearance. Conclusions Clarithromycin substantially increases the exposure to bosentan, suggesting that dose reductions may be necessary.

Cisplatin (diaminodichloroplatinum) is the favored platinum (Pt) drug for the treatment of head and neck squamous cell carcinoma (HNSCC). However, Pt drug alternatives such as carboplatin (diaminoplatinum-cyclobutan-1,1-dicarboxylate) or oxaliplatin [oxalato[(1R,2R)-cyclohexanediamino]platinum] have not been comprehensively investigated in HNSCC. Moreover, little data reveal the decisive efficacy determinant and whether Pt drug efficacy is truly concentration-dependent. Using five human HNSCC cell lines, we determined the concentrations of cisplatin, carboplatin, and oxaliplatin leading to 50% inhibition of cell proliferation (IC(50)). Concurrently we quantified cellular drug uptake by liquid chromatography coupled to tandem mass spectrometry and evaluated mRNA expression of drug transporters involved in Pt drug uptake by quantitative real-time polymerase chain reaction. Mean IC(50) among the five cell lines was 6.2 ± 1.9 μM for cisplatin and 11.6 ± 4.2 μM for oxaliplatin, whereas carboplatin showed significantly lower proliferation inhibition (IC(50) 107.5 ± 21.2 μM). In agreement with this finding carboplatin poorly accumulated in HNSCC cells, compared with cisplatin and oxaliplatin. HNSCC cell lines expressed Pt drug transporters. Taken together, the results demonstrate: 1) carboplatin was less effective and was poorly taken up; 2) a high individuality among cell lines was found concerning the accumulation of cisplatin and oxaliplatin despite similar in vitro efficacy; and 3) distinct expression of SLC22A2 and ABCC2 accompanies strong uptake and cytotoxicity of Pt drugs. In conclusion, we demonstrate that in vitro efficacy of cisplatin and oxaliplatin in HNSCC is concentration-independent because they exhibited different uptake characteristics but similar efficacies, suggesting oxaliplatin as a promising alternative against HNSCC that needs further evaluation in clinical trials.

Background— Pulmonary hypertension as a frequent complication of left heart disease (PH-LHD) is characterized by lung endothelial dysfunction and vascular remodeling. Although PH-LHD contributes to morbidity and mortality in heart failure, established therapies for PH-LHD are lacking. We tested the effect of chronic sildenafil treatment in an experimental model of PH-LHD. Methods and Results— In Sprague-Dawley rats, PH-LHD was induced by supracoronary aortic banding. Oral sildenafil treatment (60 mg/kg daily) was initiated after 7 days, and lung endothelial function (n=5), vascular remodeling, and right ventricular function (n=11 each) were analyzed 9 weeks after banding. As compared with sham-operated controls, aortic banding induced pulmonary hypertension and lung endothelial dysfunction evident as lack of endothelial nitric oxide production and endothelium-dependent vasodilation. These changes were associated with an increased pulmonary vascular resistance, medial thickening, and biventricular cardiac hypertrophy. Sildenafil treatment largely attenuated these pathological changes and was not associated with detectable adverse effects pertinent to lung vascular barrier function, edema formation, or systemic hemodynamics. Conclusions— Our data identify sildenafil as a promising therapy for PH-LHD. In light of its documented protective effects at the myocardial level in heart failure, sildenafil presents a particularly attractive strategy in that it simultaneously targets cardiac remodeling and secondary PH-LHD.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Nausea and vomiting are the most distressing side-effects of a high dose melphalan regimen. • Aprepitant in addition to an antiemetic standard regimen has been reported to improve significantly both acute and delayed chemotherapy-induced nausea and vomiting. WHAT THIS STUDY ADDS • Anti-emetic regimens including aprepitant have no clinically relevant effect on the pharmacokinetics of the anticancer agent melphalan when administered 1 h before high dose melphalan infusion. AIMS The objective of this investigation was to assess the effect of aprepitant on the pharmacokinetics of high-dose melphalan used as conditioning therapy before blood stem cell transplantation in multiple myeloma. METHODS Aprepitant (125 mg) or placebo was administered 1 h before melphalan therapy (1 h infusion of 100 mg m−2). Eleven plasma samples were obtained over 8 h and melphalan was quantified using an LC/MS/MS method. Standard pharmacokinetic parameters were calculated and nonparametric testing was applied to assess the differences between aprepitant and placebo treatment. RESULTS Twenty patients received placebo and 10 patients aprepitant treatment. There were no differences observed for Cmax at the end of melphalan infusion (placebo 3431 ± 608 ng ml−1vs. aprepitant 3269 ± 660 ng ml−1). In addition, AUC and terminal elimination half-life were not changed by aprepitant. Total clearance of melphalan was 304 ± 58 ml min−1 m−2 (placebo) which was not influenced by aprepitant (288 ± 78 ml min−1 m−2). CONCLUSIONS The administration of the NK1 receptor antagonist aprepitant 1 h before a high-dose chemotherapy does not influence the exposure and the elimination of melphalan. Therefore, oral administration of 125 mg aprepitant 1 h before melphalan infusion does not alter the disposition of intravenously administered melphalan.

OBJECTIVE To assess the efficacy of methylene blue (MB) monotherapy in semi-immune adults with uncomplicated malaria in Burkina Faso. METHODS In an open-label controlled phase II study with 60 semi-immune adults with uncomplicated falciparum malaria in Nouna, north-western Burkina Faso, MB monotherapy (390 mg twice daily) was given sequentially to groups of 20 adults for 7 days (MB7), 5 days (MB5) and 3 days (MB3), respectively. The primary outcome was the rate of adequate clinical and parasitological response (ACPR) on day 28 of follow-up. RESULTS Of the study population, 27/58 (47%) and 5/51 (10%) patients still had parasites on days 2 and 3, respectively, of follow-up resulting in 9/58 (16%) early treatment failures. By day 14, no recrudescence was observed but in 4/19 (MB5) and 2/20 (MB3) individuals by day 28. The PCR-corrected rate of ACPR was 72%, 58% and 85% in groups 7, 5 and 3, respectively, by per protocol analysis. Self-limiting dysuria was the most frequent adverse event. CONCLUSIONS MB acts slowly against the blood stages of P. falciparum. MB alone needs to be given for at least 7 days to be efficacious in the treatment of falciparum malaria but should be used in combination with a fast acting antimalarial.

Background Chloroquine (CQ) resistance has reached high levels in Africa in recent years. Little is known about variations of resistance between urban and rural areas. Objectives To compare the rates of in vivo resistance to CQ and the prevalences of the main molecular marker for CQ resistance among young children from urban and rural areas in Burkina Faso. Methods The current analysis used the frame of a randomized controlled trial (ISRCTN27290841) on the combination CQ–methylene blue (MB) ( n = 177) compared to CQ alone ( n = 45) in young children with uncomplicated malaria. We examined clinical and parasitological failure rates as well as the prevalence of the Plasmodium falciparum chloroquine resistance transporter gene ( pfcrt ) T76 mutation. Results Clinical and parasitological failure rates of CQ–MB differed significantly between urban (70%) and rural areas (29%, p pfcrt T76 was more frequently seen among parasite strains from urban areas (81%) when compared to rural ones (64%, p = 0.01). In the presence of parasites exhibiting pfcrt T76, the odds of overall clinical failure were increased to 2.6-fold ([1.33, 5.16], p LR = 0.005). CQ was detected at baseline in 21% and 2% of children from the urban and the rural study area, respectively ( p Chi = 0.002). Conclusion Even within circumscribed geographical areas, CQ efficacy can vary dramatically. The differences in the prevalence of pfcrt T76 and in CQ failure rates are probably explained by a higher drug pressure in the urban area compared to the rural study area. This finding has important implications for national malaria policies.

The aim of this study was to compare the bioavailability of cyclosporine (CyA) from the generic dispersion formulation Cicloral (CIC) with the microemulsion formulation Neoral (NEO) and the original Sandimmune (SIM) capsules after single doses of 100, 300, or 600 mg of drug, respectively. The study was performed according to an open 3-period cross-over design with 12 young healthy male volunteers for each dosage. The concentrations of CyA and its main metabolites were determined by high performance liquid chromatography in whole blood and urine up to 48 hours postdosing. Peak concentrations and area under the time-concentration curve were greater for the NEO and CIC formulations compared with SIM, and the mean bioavailability of CIC was significantly (P

1557 Background: Epidemiological studies consistently observed an association between metformin use in diabetic patients and decreased colon cancer incidence and mortality at clinical doses (1500-2250 mg day), corresponding to plasma levels of 0.003-0.045 mM. Preclinical models report much higher doses of metformin, ranging between 1 and 40 mM for in vitro and 0.006-0.145 mM for in vivo studies. A recent trial using a low dose of metformin (250 mg/day, Higurashi T et al. Lancet 2016;17:475) showed a 40% reduction of colorectal adenoma recurrence. We performed a pilot study to measure metformin in plasma and colonic tissue of subjects with diabetes or metabolic syndrome and to explore their correlation with different tissue biomarkers, including Ki-67, pS6k, a mTOR effector protein, and EGFR expression. Methods: We used LC-MS/MS to quantify metformin in plasma and colonic tissues from 13 volunteers with diabetes or metabolic syndrome undergoing screening colonoscopy. All patients had received metformin at 1000-1850 mg/day for a median of 6.5 yrs (range 2-15), except for one who served as untreated control. Tissue quantifications were performed on specimens derived from right and left colon biopsies with or without washing to exclude a potential contamination due to lumen accumulation by the drug. Biomarker assessment was performed by standard IHC assay. Results: The mean plasma metformin was 0.025 mM/L (range 5x10-5-0.017), whereas the colonic tissue was ~100 fold higher in right [0.39 mM/Kg (range 0.034-1.75)] and left colon [0.46 mM/Kg (range 5x10-3-1.86)]. There was no significant difference between washed and unwashed biopsies. No significant correlations were detected between metformin levels in plasma/tissue and tissue biomarkers. Conclusions: Metformin can attain ~100 fold higher colonic tissue levels than in plasma. These levels are in the range of a direct antitumor effect observed in in vivo preclinical models. A two-by-two clinical trial of metformin and aspirin in colon cancer patients is underway by our group to further elucidate independent and synergistic antitumor effects.

Background Since 2006 the national malaria control program in Mali recommended artemether-lumefantrine (AL) as the first-line treatment of uncomplicated malaria. The role of lumefantrine in this combination is to eliminate remaining parasites after the action of artemether and to protect the patient against a new blood infection. Some studies showed a correlation between lumefantrine9s day 7 concentration and the efficacy of AL after treatment of a single episode of malaria. The objective of this work is to validate this observation after repetitive treatment of uncomplicated malaria patients with AL. Methods During a phase IIIb/IV comparative, randomised, multicentre, clinical study of artemisinin-based combination therapies, we collected plasma on Day 7 from patients treated with standard dose of AL in Sotuba, Bougoula Hameau, and Kolle (Mali). The age of the patients enrolled in this study was from 6 months old. The plasma samples were kept at – 80°C until lumefantrine analysis using high performance liquid chromatography was performed. Results We included 1076 subjects, of which 595 were females and a mean age of 12 years old in this analysis. The median concentration was 66% higher (p vs 372.5 (255.7–538.4; n=157). Day 7 concentrations increased with age; the difference between age group was statistically significant: 305.9 (207.3–491.5, n=140), 447 (290.7–622.9, n=399), 544.7 (383.9–738.5, n=254) and 571.1 (378.8–850.9), n=283) in patients under 5 years old, 5–9 years old, 10–14 years old and 15 years old and older, respectively. Girls under 5 years old had a lower lumefantrine concentration at day 7 compared to other age groups of 223.3 ng/ml (159.7–425.6, n=37). Conclusions We found a lower concentration of lumefantrine in patients with recurrent parasitaemia at day 28.

OBJECTIVES To assess the effect of the major efavirenz metabolizing enzyme (CYP2B6) genotype and the effects of rifampicin co-treatment on induction of CYP3A by efavirenz. PATIENTS AND METHODS Two study arms (arm 1, n = 41 and arm 2, n = 21) were recruited into this study. In arm 1, cholesterol and 4β-hydroxycholesterol were measured in HIV treatment-naive patients at baseline and then at 4 and 16 weeks after initiation of efavirenz-based antiretroviral therapy. In arm 2, cholesterol and 4β-hydroxycholesterol were measured among patients taking efavirenz during rifampicin-based tuberculosis (TB) treatment (efavirenz/rifampicin) just before completion of TB treatment and then serially following completion of TB treatment (efavirenz alone). Non-linear mixed-effect modelling was performed. RESULTS A one-compartment, enzyme turnover model described 4β-hydroxycholesterol kinetics adequately. Efavirenz treatment in arm 1 resulted in 1.74 (relative standard error = 15%), 3.3 (relative standard error = 33.1%) and 4.0 (relative standard error = 37.1%) average fold induction of CYP3A for extensive (CYP2B6*1/*1), intermediate (CYP2B6*1/*6) and slow (CYP2B6*6/*6) efavirenz metabolizers, respectively. The rate constant of 4β-hydroxycholesterol formation [mean (95% CI)] just before completion of TB treatment [efavirenz/rifampicin co-treatment, 7.40 × 10(-7) h(-1) (5.5 × 10(-7)-1.0 × 10(-6))] was significantly higher than that calculated 8 weeks after completion [efavirenz alone, 4.50 × 10(-7) h(-1) (4.40 × 10(-7)-4.52 × 10(-7))]. The CYP3A induction dropped to 62% of its maximum by week 8 of completion. CONCLUSIONS Our results indicate that efavirenz induction of CYP3A is influenced by CYP2B6 genetic polymorphisms and that efavirenz/rifampicin co-treatment results in higher induction than efavirenz alone.

Erythromycin is a macrolide antibiotic, which is frequently used as a topical formulation for the treatment of acne. It is also known as an inhibitor of the cytochrome P450 (CYP) isoenzyme 3A4. In this study, the systemic availability of topical erythromycin, hence the influence on the activity of CYP3A, is evaluated in comparison to orally administered erythromycin.Sixteen healthy volunteers received consecutively topical (two applications of 800 mg) and oral erythromycin (two dose groups, 250 and 1000 mg, with n = 8) to assess erythromycin pharmacokinetics. A microdose of midazolam (3 μg orally) was used to determine the effect on CYP3A activity.After topical administration, erythromycin was detected in the plasma of every participant without causing a statistically significant alteration of CYP3A activity. After oral administration, the dose-normalized erythromycin exposure (AUC∞ ) was 1335 h ng ml(-1) after 250 mg and 3-fold higher after the 1000 mg dose (4051 h ng ml(-1); P0.01), suggesting nonlinear pharmacokinetics of erythromycin. Both oral doses inhibited CYP3A activity; midazolam clearance was decreased to 61% (250 mg) and 21% (1000 mg). The relationship between erythromycin exposure and CYP3A activity (Hill equation) revealed a 50% reduction of CYP3A activity by an erythromycin AUC∞ of 2106 h ng ml(-1).Topical erythromycin did not cause clinically relevant CYP3A alterations, although low systemic availability of erythromycin was observed. This supports the assumption that treatment with topical erythromycin is not critical in terms of CYP3A inhibition. Furthermore, substantial nonlinearity of erythromycin pharmacokinetics after two different oral doses was observed, possibly due to autoinhibition.

Objectives: We evaluated the importance of ethnicity and pharmacogenetic variations in determining efavirenz pharmacokinetics, auto-induction and immunological outcomes in two African populations. Methods: ART nao ¨ve HIV patients from Ethiopia (n = 285) and Tanzania (n = 209) were prospectively enrolled in parallel to start efavirenz based HAART. CD4+ cell counts were determined at baseline, 12, 24 and 48 weeks. Plasma and intracellular efavirenz and 8-hydroxyefvairenz concentrations were determined at week 4 and 16. Genotyping for common functional CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 variant alleles were done. Result: Patient country, CYP2B6*6 and ABCB1 c.4036A.G (rs3842A.G) genotype were significant predictors of plasma and intracellular efavirenz concentration. CYP2B6*6 and ABCB1 c.4036A.G (rs3842) genotype were significantly associated with higher plasma efavirenz concentration and their allele frequencies were significantly higher in Tanzanians than Ethiopians. Tanzanians displayed significantly higher efavirenz plasma concentration at week 4 (p,0.0002) and week 16 (p = 0.006) compared to Ethiopians. Efavirenz plasma concentrations remained significantly higher in Tanzanians even after controlling for the effect of CYP2B6*6 and ABCB1 c.4036A.G genotype. Within country analyses indicated a significant decrease in the mean plasma efavirenz concentration by week 16 compared to week 4 in Tanzanians (p = 0.006), whereas no significant differences in plasma concentration over time was observed in Ethiopians (p = 0.84). Intracellular efavirenz concentration and patient country were significant predictors of CD4 gain during HAART. Conclusion: We report substantial differences in efavirenz pharmacokinetics, extent of auto-induction and immunologic recovery between Ethiopian and Tanzanian HIV patients, partly but not solely, due to pharmacogenetic variations. The observed inter-ethnic variations in efavirenz plasma exposure may possibly result in varying clinical treatment outcome or adverse event profiles between populations.

The objective of this study was to assess the incidence, timing and identify pharmacogenetic, efavirenz (EFV) pharmacokinetic and biochemical predictors of EFV-based antiretroviral therapy (ART) drug-induced liver injury (DILI). ART-naive HIV patients (n = 285) were prospectively enrolled. Pretreatment laboratory evaluations included hepatitis B surface antigen and C antibody, CD4 count and viral load. Liver tests were done at baseline, 1st, 2nd, 4th, 8th, 12th, 24th and 48th weeks during ART. Plasma EFV and 8-hydroxyefvairenz concentration was determined at week 4 using liquid chromatography-mass spectrometry. CYP2B6, CYP3A5, ABCB1 3435C/T and UGT2B7*2 genotyping was done using Taqman genotyping assay. Data were analyzed using survival analysis and Cox proportional hazards model. The incidence of DILI was 15.7% or 27.9 per 100 person-years and that of severe injury was 3.4% or 6.13 per 100 person-years. The median time for the development of DILI and severe injury was 2 and 4 weeks after initiation of ART, respectively. There was significant association of DILI with lower baseline platelet, albumin, log plasma viral load and CD4 count (P = 0.031, 0.037, 0.06 and 0.019, respectively). Elevated baseline alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, plasma EFV level and CYP2B6*6 were good predictors for the development of DILI (P = 0.03, 0.01, 0.016, 0.017 and 0.04, respectively). We report for the first time CYP2B6*6 as a putative genetic marker and high plasma EFV concentration as intermediate biomarker for vulnerability to EFV-induced liver injury in HIV patients. CYP2B6 genotyping and/or regular monitoring of EFV and lever enzymes level during early therapy is advised for early diagnosis and management of DILI.

The objective of this investigation was to assess the effect of aprepitant on the pharmacokinetics of high-dose melphalan used as conditioning therapy before blood stem cell transplantation in multiple myeloma.Aprepitant (125 mg) or placebo was administered 1 h before melphalan therapy (1 h infusion of 100 mg m⁻²). Eleven plasma samples were obtained over 8 h and melphalan was quantified using an LC/MS/MS method. Standard pharmacokinetic parameters were calculated and nonparametric testing was applied to assess the differences between aprepitant and placebo treatment.Twenty patients received placebo and 10 patients aprepitant treatment. There were no differences observed for C(max) at the end of melphalan infusion (placebo 3431 ± 608 ng ml⁻¹ vs. aprepitant 3269 ± 660 ng ml⁻¹). In addition, AUC and terminal elimination half-life were not changed by aprepitant. Total clearance of melphalan was 304 ± 58 ml min⁻¹ m⁻² (placebo) which was not influenced by aprepitant (288 ± 78 ml min⁻¹ m⁻²).The administration of the NK₁ receptor antagonist aprepitant 1 h before a high-dose chemotherapy does not influence the exposure and the elimination of melphalan. Therefore, oral administration of 125 mg aprepitant 1 h before melphalan infusion does not alter the disposition of intravenously administered melphalan.

Methylene blue (MB) has recently been reevaluated for malaria treatment. With the aim of excluding treatment failures due to low bioavailability, we have investigated the absolute bioavailability of MB given as an aqueous oral formulation and its interaction with chloroquine (CQ).A phase I study in 16 healthy individuals was performed as a monocenter prospective open randomized intra-individual cross-over comparison of MB single doses [50 mg intravenous (i.v.), 500 mg orally, separated by a 1-week wash-out]. After a second week, the group was split for a randomized parallel group comparison of CQ 750 mg administered orally alone or combined with 500 mg MB orally.Mean MB plasma area under the substrate concentration-time curve (AUC 0-infinity) was 7,639 +/- 3,384 ng/mL*h and 51,171 +/- 17,147 ng/mL*h after i.v. and oral administration, respectively (dosage 1:10), and 76,897 +/- 46,037 ng/mL*h after MB combined with CQ. The absolute bioavailability was 72.3 +/- 23.9%. Co-administration with CQ significantly increased MB plasma concentrations (por= 0.016); CQ kinetics remained unaffected.The absolute bioavailability of MB is high. Co-administration of MB and CQ increases plasma, but not whole blood MB concentrations.

Statin/fibrate combinations are frequently used to treat mixed dyslipidemia. However, these combinations may cause life-threatening drug interactions (e.g. rhabdomyolysis) possibly induced by modifications of cytochrome P450 isozyme activities. Some statins are also transported by P-glycoprotein (Pgp) and may act as inhibitors of this drug efflux pump. So far, nothing is known about possible Pgp modulating effects of fibrates. We tested whether gemfibrozil, fenofibrate, fenofibric acid, and bezafibrate inhibit Pgp in vitro using a calcein acetoxymethylester (calcein-AM) uptake assay and confocal laser scanning microscopy with bodipy-verapamil as substrate in L-MDR1 cells, which overexpress human Pgp. In uptake assays in cells with (L-MDR1) and without (LLC-PK1) human Pgp we also investigated whether these compounds are transported by Pgp. Intracellular concentrations were measured by liquid chromatography tandem mass spectrometry. Of the tested fibrates, only fenofibrate increased calcein-AM uptake into cells indicating an inhibition of Pgp mediated transport by this compound. The potency of fenofibrate (mean+/-SD: 7.1+/-3.2 microM), evaluated by calculating the concentration needed to double baseline fluorescence (f2), was similar to that of simvastatin (5.8+/-1.5 microM), lovastatin (10.1+/-1.0), and verapamil (4.7+/-0.8 microM). For simvastatin and fenofibrate Pgp inhibition was confirmed with confocal laser scanning microscopy. Fenofibrate, fenofibric acid, gemfibrozil, and bezafibrate showed no difference in the cellular uptake between LLC-PK1 and L-MDR1, indicating that the tested fibrates are not Pgp substrates. In conclusion, this study demonstrates that fenofibrate inhibits Pgp in vitro with a potency similar to simvastatin.

9612 Background: Aprepitant (A) is a NK1-antagonist approved for prevention of chemotherapy-induced nausea and vomiting (CINV). Though melphalan (M) is regarded only moderately emetogenic, CINV after high-dose M conditioning in autologous TPX can severely interfere with patients’ (pts) quality of life. Here we present the results of our monocentric, randomized (1:1), placebo-controlled, double-blind phase IIIb trial of A, granisetrone (G) and dexamethasone (D) in this setting. Methods: Pts were treated with A 125 mg po day 1, A 80 mg po days 2-4, G 2 mg po days 1-4 and D 4 mg po day 1, D 2 mg po days 2-3 (arm A) or placebo (P) days 1-4, G 2 mg po days 1-4 and D 8 mg day 1, D 4 mg days 2-3 (arm B). Melphalan 100 mg/m² iv was administered days 1-2. Episodes of emesis, modified functional living index – emesis questionnaire (FLIE) and intensity of nausea by visual analogue scale (VAS) were recorded between days 1-6. Primary endpoint was defined as no episode of vomiting and no additional antiemetic therapy within 120h of M administration. Results: Between 07/05 and 01/12 a total of 362 pts were randomized. 361 subjects (arm A=180; arm B=181; male n=229; female n=132; median age arm A=59.5 years; median age arm B=58 years) were available for the efficacy analysis. There were no significant differences in gender distribution and previous experience of CINV. Significantly less pts receiving A failed the primary endpoint (42% vs. 59%, OR=0.53 [0.34; 0.82], p=.0046). This effect was stressed in women (57% vs. 82%, OR=0.30) and patients with previous CINV (48% vs. 71%, OR=0.38). Emesis within 120h occurred in 22% of pts receiving A and 35% receiving P (OR=0.5 [0.31; 0.80], p=.0036). Though differences in overall nausea (16% vs. 22%, OR=0.65 [0.83; 1.10], p=.11) did not reach statistical significance, major nausea (VAS >25mm) was reduced (6% vs. 12%, OR=0.42 [0.19; 0.92], p=.026). Difference in FLIE score was -8.2 (p=.0007). Study medication was well tolerated. PK analyses showed no interaction between M and A. Conclusions: The combination therapy with A resulted in significantly less CINV compared to placebo. This effect was pronounced in women and pts with previous CINV. Clinical trial information: NCT00571168.

Objectives To investigate the timing, incidence, clinical presentation, pharmacokinetics and pharmacogenetic predictors for antiretroviral and anti-tuberculosis drug induced liver injury (DILI) in HIV patients with or without TB co-infection. Methods and Findings A total of 473 treatment naïve HIV patients (253 HIV only and 220 with HIV-TB co-infection) were enrolled prospectively. Plasma efavirenz concentration and CYP2B6*6, CYP3A5*3, *6 and *7, ABCB1 3435C/T and SLCO1B1 genotypes were determined. Demographic, clinical and laboratory data were collected at baseline and up to 48 weeks of antiretroviral therapy. DILI case definition was according to Council for International Organizations of Medical Sciences (CIOMS). Incidence of DILI and identification of predictors was evaluated using Cox Proportional Hazards Model. The overall incidence of DILI was 7.8% (8.3 per 1000 person-week), being non-significantly higher among patients receiving concomitant anti-TB and HAART (10.0%, 10.7per 1000 person-week) than those receiving HAART alone (5.9%, 6.3 per 1000 person-week). Frequency of CYP2B6*6 allele (p = 0.03) and CYP2B6*6/*6 genotype (p = 0.06) was significantly higher in patients with DILI than those without. Multivariate cox regression model indicated that CYP2B6*6/*6 genotype and anti-HCV IgG antibody positive as significant predictors of DILI. Median time to DILI was 2 weeks after HAART initiation and no DILI onset was observed after 12 weeks. No severe DILI was seen and the gain in CD4 was similar in patients with or without DILI. Conclusions Antiretroviral and anti-tuberculosis DILI does occur in our setting, presenting early following HAART initiation. DILI seen is mild, transient and may not require treatment interruption. There is good tolerance to HAART and anti-TB with similar immunological outcomes. Genetic make-up mainly CYP2B6 genotype influences the development of efavirenz based HAART liver injury in Tanzanians.

10041^ Background: New treatment options for patients with metastatic soft tissue sarcoma (STS) are urgently needed. Preclinical studies suggest activity of vorinostat (V), a histone desacetylase (HDAC) inhibitor, in STS. Methods: We initiated a multicenter, open-label, non-randomized phase II trial (SAHA-I, NCT00918489) to investigate efficacy and safety of V in patients with metastatic STS failing 1°-line anthracycline-based CTX. Patients were treated with V 400 mg po QD for 28 days followed by a therapy-free period of 7 days. Blood samples for pharmacokinetic (PK) analyses were collected on day 7 of treatment cycle 1. Samples were acquired before and 30, 60, 90, 120, 240, 360, and 480 minutes after oral administration of V. Plasma- and intracellular concentration of V in peripheral blood mononuclear cells (PBMCs) was determined by mass spectrometry. Statistical differences were assessed by Wilcoxon matched-pairs signed rank test. This trial is ongoing. Results: Data on PK was available for n=8 subjects (male=4, female=4, median age=62 years). In plasma samples, mean Cmax (maximum concentration), tmax (time to reach max. concentration), AUC (area under the plasma-concentration time curve), t1/2 (elimination half-life) and Cl/F (apparent total clearance) were 350 ng/mL, 101 min, 71.1 min*µg/mL, 103 min and 5903 mL/min. The corresponding parameters in PBMCs were 558 ng/mL, 97.5 min, 208.4 min*µg/mL, 286 min and 2475 mL/min, respectively.The AUC plasma/PBMC ratio was 2.93, indicating accumulation of V in PBMCs. Differences in AUC (p=.008) and t1/2 (p=.01) reached statistical significance. Conclusions: Interestingly, significantly higher concentrations of V were achieved in PBMCs andelimination half-life was prolonged compared to plasma. These results suggest potent intracellular activity of V.

10535Background: In preclinical pediatric cancer models, the HDAC inhibitor vorinostat showed significant activity only at higher concentrations compared with those achieved with currently recommen...