A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1.

Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.

Ultra-sensitive and selective quantification of endothelin-1 in human plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry.

Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor peptide and the plasma concentrations are commonly quantified by immunoassays such as enzyme-linked immuno-sorbent assays (ELISA) with the disadvantage of possible cross-reactivity with closely related endothelin derivatives. The aim of this study was to develop and validate an ultra-sensitive and selective assay for the quantification of ET-1 in human plasma, using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) after solid phase extraction. The assay fulfilled the requirements of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines for assay validation, with a lower limit of quantification of 1.5pg/mL for ET-1. Recovery rates from plasma ranged between 80.8% and 93.6%, and matrix effect varied between 121% and 135%. The assay was successfully applied to assess the time course of plasma ET-1 concentrations in two human volunteers after co-administration of bosentan and clarithromycin. In this trial, the concentrations measured by UPLC-MS/MS were slightly lower than those measured by ELISA, with a strong positive correlation between the two methods. Our novel UPLC-MS/MS method is applicable to the clinical setting and may have better selectivity for ET-1 than ELISA.

First-in-human application of the novel hepatitis B and hepatitis D virus entry inhibitor myrcludex B.

BACKGROUND & AIMS: Myrcludex B is a first-in-class compound, which blocks entry of hepatitis B and D virus into hepatocytes in vitro and in animal models. Based on the required preclinical data we aimed to translate this compound into the first application in humans. METHODS: Single ascending doses of myrcludex B, a 47 amino acid peptide, were administered up to 20mg intravenously and 10mg subcutaneously in a prospective open first-in-human, phase I clinical trial to 36 healthy volunteers. Safety, tolerability and plasma concentrations of myrcludex B were assessed and a pharmacokinetic model was derived. RESULTS: Myrcludex B was well tolerated and no serious or relevant AEs representing off-target effects, and no immunogenic effects were observed up to the highest applied dose of 20mg (intravenously). Myrcludex B showed dose-dependent pharmacokinetics, best described by a 2-compartment target-mediated drug disposition model. Bioavailability of the subcutaneous application was large (85%). Interindividual variability was moderate. The pharmacokinetic model suggested that subcutaneous doses of 10mg and above reach a target saturation of over 80% for at least 15h. CONCLUSIONS: Myrcludex B showed excellent tolerability up to high doses. Pharmacologic properties followed a 2-compartment target-mediated drug disposition model. These findings are vital for planning of further multiple dose efficacy trials in patients. LAY SUMMARY: After showing antiviral activity in cell culture and animal models, myrcludex B, a new drug intended for the treatment of hepatitis B and D, has been administered the first time in humans. Healthy volunteers received the drug intravenously and subcutaneously up to high doses (20mg). The drug was well tolerated and the characteristics of the drug determining its way in the human body could be described. These results will allow testing myrcludex B in hepatitis B and D patients.

The effect of induction of CYP3A4 by St John's wort on ambrisentan plasma pharmacokinetics in volunteers of known CYP2C19 genotype.

To evaluate the impact of CYP2C19 polymorphisms on ambrisentan exposure and to assess its modification by St. John's wort (SJW), 20 healthy volunteers (10 CYP2C19 extensive, four poor and six ultrarapid metabolizers) received therapeutic doses of ambrisentan (5 mg qd po) for 20 days and concomitantly SJW (300 mg tid po) for the last 10 days. To quantify changes of CYP3A4 activity, midazolam (3 mg po) as a probe drug was used. Ambrisentan pharmacokinetics was assessed on days 1, 10 and 20, and midazolam pharmacokinetics before and on days 1, 10, 17 and 20. At steady state, ambrisentan exposure was similar in extensive and ultrarapid metabolizers but 43% larger in poor metabolizers (p < 0.01). In all volunteers, SJW reduced ambrisentan exposure and the relative change (17-26%) was similar in all genotype groups. The extent of this interaction did not correlate with the changes in CYP3A activity (midazolam clearance) (rs = 0.23, p = 0.34). Ambrisentan had no effect on midazolam pharmacokinetics. In conclusion, SJW significantly reduced exposure with ambrisentan irrespective of the CYP2C19 genotype. The extent of this interaction was small and thus likely without clinical relevance.

Pre-systemic elimination of tilidine: localization and consequences for the formation of the active metabolite nortilidine.

The therapeutic activity of tilidine, an opioid analgesic, is mainly related to its active metabolite nortilidine. Nortilidine formation mainly occurs during the high intestinal first-pass metabolism of tilidine by N-demethylation. Elimination of the active nortilidine to the inactive bisnortilidine is also mediated by N-demethylation and is supposed to take place in the liver, probably at a smaller rate. The aim of this study was the investigation of the pre-systemic elimination of tilidine using grapefruit juice (GFJ) as an intestinal CYP3A4 inhibitor and efavirenz (EFV) as a CYP3A4 activator. A randomized, open, placebo-controlled, cross-over study was conducted in 12 healthy volunteers using 100 mg tilidine solution p.o., regular strength GFJ 250 mL (3 times at 12-hr intervals) and EFV 400 mg (12 hr before tilidine administration). Tilidine, nortilidine and bisnortilidine in plasma and urine were quantified by a validated LC/MS/MS analysis. GFJ did not change any pharmacokinetic parameter of tilidine and its metabolites, which suggests that intestinal CYP3A4 does not contribute to the first-pass metabolism of tilidine. No effect of EFV on the pharmacokinetics of the active nortilidine was observed except a significant reduction of the terminal elimination half-life by 15%. Overall elimination (renal and metabolic clearances) was unaffected by every treatment. CYP3A4 does not seem to play a major role in tilidine first-pass and overall metabolism. Other unknown metabolites and their enzymes responsible for their formation have to be investigated as they account for the majority of renally excreted metabolites.

Lack of a clinically significant interaction of grapefruit juice with ambrisentan and bosentan in healthy adults.

OBJECTIVE: We assessed the effect of 1 x 300 mL/d and 3 x 300 mL/d grapefruit juice (GFJ) on ambrisentan and bosentan pharmacokinetics in healthy volunteers. METHODS: In the ambrisentan study, 12 healthy extensive metabolizers (EM) of CYP2C19 received therapeutic doses of ambrisentan (5 mg q.d. on study days 1 - 11) and GFJ (1 x 300 mL/d on study days 6 - 8 and 3 x 300 mL/d on study days 9 - 11). Ambrisentan pharmacokinetics was assessed on study days 5, 8, and 11. In the bosentan study, 16 healthy EM of CYP2C9, who were stratified into two groups (CYP3A5 expressors (n = 8) and CYP3A5 non-expressors (n = 8)), received bosentan (125 mg b.i.d. on study days 1 - 13) and GFJ (1 x 300 mL/d on study days 8 - 10 and 3 x 300 mL/d on study days 11 - 13). Bosentan pharmacokinetics was assessed on study days 7, 10, and 13. RESULTS: Whereas 1 x 300 mL/d GFJ had no effect on the pharmacokinetics of ambrisentan and its metabolite, 3 x 300 mL/d GFJ increased the exposure with ambrisentan by 8% and the molar plasma metabolic ratio by 22% (both p < 0.05). Correspondingly, the apparent oral clearance of ambrisentan decreased to 92% (p < 0.05). GFJ slightly prolonged t(max) of bosentan and increased the metabolic ratio of bosentan/hydroxy-bosentan by 19% (p < 0.05). CONCLUSION: GFJ had no clinically relevant effect on the pharmacokinetics or safety profile of ambrisentan and bosentan. Therefore, no dose adjustments are needed, and GFJ can be safely co-administered even at very high doses.

Influence of St. John's wort on the steady-state pharmacokinetics and metabolism of bosentan.

OBJECTIVE: We assessed the effect of St. John's wort (SJW) on bosentan pharmacokinetics at steady-state in different CYP2C9 genotypes in healthy volunteers. METHODS: Nine healthy extensive metabolizers of CYP2C9 and 4 poor metabolizers received therapeutic doses of bosentan (125 mg q.d. on study day 1; 62.5 mg b.i.d. on study day 2, 125 mg b.i.d. on study days 3 - 20) for 20 days and SJW (300 mg t.i.d.) concomitantly for the last 10 days. Bosentan pharmacokinetics was assessed on days 1, 10, and 20. Concurrently, we repeatedly quantified changes of CYP3A activity using low dosed midazolam (3 mg p.o.) as a probe drug. RESULTS: Due to auto-induction of its metabolism, Cl/F increased by 67%, thus significantly lowering bosentan exposure (AUC) to 60% after 10 days of bosentan administration (n = 13, p < 0.05). Concurrently, midazolam clearance (CYP3A activity) increased by 224% (n = 13, p < 0.05) and further increased after SJW by 374% compared to baseline (n = 13, p < 0.05). SJW increased midazolam clearance by 47% (n = 13, p < 0.05) but failed to alter bosentan exposure and clearance consistently. No significant differences in bosentan exposure and clearance changes were observed in CYP2C9 poor metabolizers. CONCLUSION: SJW increased CYP3A activity but had no consistent effect on bosentan clearance. However, inter-individual changes of the interaction were large, suggesting that close monitoring of bosentan effects may be advisable. The contribution of CYP2C9 to this interaction seems to be minor.

Clarithromycin substantially increases steady-state bosentan exposure in healthy volunteers.

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.

Interaction of ambrisentan with clarithromycin and its modulation by polymorphic SLCO1B1.

PURPOSE: We assessed the effect of cytochrome P450 (CYP) 3A4 and the OATP1B1 inhibitor clarithromycin on ambrisentan steady-state kinetics and its relationship to the SLCO1B1 15 haplotype in healthy volunteers. METHODS: In this open-label, monocenter, one-sequence crossover clinical trial ten male healthy participants were stratified according to CYP2C19 and SLCO1B1 (encoding for OATP1B1) genotype into two groups: group 1 (n = 6), with CYP2C19 1/1 (extensive metabolizer, EM) and SLCO1B1 wild-type; group 2 (n = 4), with CYP2C19 EM and homozygous (n = 3) or heterozygous for SLCO1B1 15 (n = 1). The participants were administered a once-daily oral dose of 5 mg ambrisentan on study days 1 and days 3-14 and twice-daily oral doses of 500 mg clarithromycin on study days 11-14. To monitor CYP3A activity 3 mg midazolam was given orally 1 day before the first ambrisentan administration and on days 1, 10, and 14 of ambrisentan treatment. Ambrisentan plasma kinetics was assessed on days 1 (single dose), 10 (steady-state), and 14 (CYP3A4/OATP1B1 inhibition by clarithromycin). RESULTS: Consistent with the expectation that ambrisentan does not induce its own metabolism, ambrisentan exposure and peak concentration (Cmax) were similar after the first dose and at steady-state. Clarithromycin increased the area under the plasma concentration-time curve of ambrisentan by 41 % and Cmax by 27 % (n = 10, both p < 0.05). No contribution of SLCO1B1*15 to the extent of this interaction was observed. CONCLUSIONS: Clarithromycin increased ambrisentan exposure to a similar extent to ketoconazole, namely, clinically minor and likely irrelevant.

Effect of the CYP3A inhibitor ketoconazole on the PXR-mediated induction of CYP3A activity.

PURPOSE: The aim of this clinical study was to investigate a previously proposed mechanism of ketoconazole-mediated inhibition of cytochrome P450 3A (CYP3A) induction. METHODS: A two-phase, randomized, cross-over, open, mono-centre trial was carried out. Participants received ketoconazole and St John's wort for 8 days to study the proposed suppression of St John's wort-mediated induction of CYP3A at the transcriptional level. In the second phase, we studied the inhibitory effect of a single dose of ketoconazole directly at the enzyme level during CYP3A induction by St John's wort. Midazolam served as a marker substance of CYP3A activity using an established limited sampling strategy. RESULTS: After 8 days of simultaneous ketoconazole and St John's wort administration, CYP3A-mediated midazolam metabolism was strongly inhibited (81 % decrease in clearance). Following the induction of CYP3A with St John's wort (6.6-fold increase in clearance on day 8), a single dose of ketoconazole strongly inhibited midazolam metabolism to the same degree (82 % decrease in clearance in relation to baseline). An induction of midazolam metabolism was observed after discontinuation of both drugs in both study phases. These results apparently contradict the in vitro results where ketoconazole showed an inhibitory effect on the transcription of CYP3A genes. CONCLUSIONS: Ketoconazole is a strong inhibitor of CYP3A, also when used concomitantly with St John's wort. In therapeutic doses it does not inhibit pregnane X receptor-mediated induction of CYP3A in vivo.

Proposal of a new limited sampling strategy to predict CYP3A activity using a partial AUC of midazolam.

PURPOSE: Midazolam metabolic clearance to 1'-hydroxymidazolam is an accurate measure of CYP3A activity which requires extensive plasma and urine sampling. The objective of this study was to find a new limited sampling strategy (LSS) to predict midazolam metabolic clearance to 1'-hydroxymidazolam and subsequently CYP3A activity in an easy and reliable way, reducing costs and labour. METHODS: The development of this LSS is based on data from an in vivo drug-drug interaction study carried out in our clinical research unit. Various partial AUCs of midazolam were calculated and correlated with metabolic clearance of midazolam to 1'-hydroxymidazolam by non-linear regression. The correlation with highest r (2) values was chosen to predict the midazolam metabolic clearance. Statistical significance of this method was verified by calculating the 95% confidence interval of the differences (%) between predicted and measured metabolic clearance of midazolam to 1'-hydroxymidazolam. RESULTS: The midazolam partial AUC from 2 to 4 h after oral administration correlated best with metabolic clearance of midazolam to 1'-hydroxymidazolam with r (2) = 0.9816. This partial AUC comprised four midazolam concentrations at 2, 2.5, 3 and 4 h after oral administration of a midazolam solution. The 95% confidence interval of the differences between predicted metabolic clearance and measured metabolic clearance of midazolam to 1'-hydroxymidazolam was -0.97 to +13.2. CONCLUSION: The determination of the partial AUC using four plasma samples from 2 to 4 h after oral administration of a midazolam solution is proposed to be an easy and reliable CYP3A phenotyping measure which of course needs to be validated in prospective clinical trials.

Pharmacokinetics and pharmacodynamics of antimicrobial drugs.

BACKGROUND: Antimicrobial drugs exhibit different characteristics in their correlation between antimicrobial drug concentrations and effects on microorganisms. These correlations have been studied using different approaches including in vitro analyses with constant and fluctuating concentrations and in vivo analyses involving animals and humans. Mathematical analysis includes correlation of pharmacokinetic-pharmacodynamic (PK-PD) indices to an outcome parameter. Further insight can be gained by mechanism-based modelling of antimicrobial drug effects. METHODS AND RESULTS: This review aims to provide an overview on the various approaches used to analyse antimicrobial pharmacodynamics, to discuss the limitations of these approaches, to indicate recent developments and to summarise the current knowledge on PK-PD target values as derived from human studies. CONCLUSION: It is expected that PK-PD analysis of antimicrobial drug effects will lead to a more efficient and possibly also less toxic antimicrobial drug therapy.

Tryptophan hydroxylase-2 gene variation influences personality traits and disorders related to emotional dysregulation.

Variation in the tryptophan hydroxylase-2 gene (TPH2) coding for the rate-limiting enzyme of serotonin (5-HT) synthesis in the brain modulates responses of limbic circuits to emotional stimuli and has been linked to a spectrum of clinical populations characterized by emotional dysregulation. Here, we tested a set of common single nucleotide polymorphisms (SNPs) in and downstream of the transcriptional control region of TPH2 for association with personality traits and with risk for personality disorders in two cohorts comprising of 336 healthy individuals and 420 patients with personality disorders. Personality dimensions were assessed by the Tridimensional Personality Questionnaire (TPQ) and the revised NEO Personality Inventory (NEO-PI-R). Personality disorders were diagnosed with the Structured Clinical Interview of DSM-IV and were allocated to clusters A, B, and C. Individual SNP and haplotype analyses revealed significant differences in genotype frequencies between controls and cluster B as well as cluster C patients, respectively. In both patient groups, we observed overrepresentation of T allele carriers of a functional polymorphism in the upstream regulatory region of TPH2 (SNP G-703T, rs4570625) which was previously shown to bias responsiveness of the amygdala, a structure critically involved in emotionality. Furthermore, significant effects of TPH2 variants on anxiety-related traits defined primarily by the TPQ Harm Avoidance were found in healthy individuals. The results link potentially functional TPH2 variants to personality traits related to emotional instability as well as to cluster B and cluster C personality disorders. These findings implicate alterations of 5-HT synthesis in emotion regulation and confirm TPH2 as a susceptibility and/or modifier gene of affective spectrum disorders.