Christoph Markert

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.

Determining the Time Course of CYP3A Inhibition by Potent Reversible and Irreversible CYP3A Inhibitors Using A Limited Sampling Strategy

We established a new limited sampling strategy to assess CYP3A activity and evaluated the time course of reversible (voriconazole) and irreversible (ritonavir) CYP3A inhibition. In this randomized trial, two groups, each with eight healthy participants, received CYP3A inhibitors voriconazole or ritonavir orally for 9 days, with 3 mg midazolam (MDZ) administered before the inhibitor treatment, on days 1, 2, 3, 5, 8, and 9 during inhibitor treatment, and on days 10, 11, and 12 (3 days) after discontinuation. Plasma MDZ area under the curve (AUC) between 2 and 4 h after oral administration in the form of a solution strongly correlated with MDZ clearance. Using this parameter, maximum inhibition of voriconazole and ritonavir was calculated to have occurred only 48 h after starting of the inhibitor (percentage of baseline MDZ clearance, voriconazole: 10.6%; ritonavir: 8.4%). Recovery of CYP3A activity occurred with a half‐life of 24 h after voriconazole, whereas ritonavir inhibition was still strong 3 days after discontinuation. These findings underscore the substantial and gradual alterations in dose requirements in the first days of and after such combination therapies.

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.

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

OBJECTIVE We assessed the effect of St. Johns 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.

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. Johns 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.

CYP2C9 polymorphism is not a major determinant of bosentan exposure in healthy volunteers.

In their recent article, “Association of CYP2C9*2 With Bosentan-Induced Liver Injury,”1 Markova et al. report a study in which they assessed the relationships of polymorphisms in cytochrome P450 2C9 (CYP2C9), adenosine triphosphate (ATP)-binding cassette members B11 and C2 (ABCB11 and ABCC2), and organic anion–transporting polypeptide 1B (OATP1B) with hepatotoxicity during bosentan treatment, and the only associated polymorphic allele was CYP2C9*2. In their in vitro analysis of bosentan metabolism, CYP2C9*3 was less active than *2, and both were less active than the wild-type cytochrome, suggesting that bosentan accumulates in these patients and causes hepatotoxicity. In a recent series of drug interaction studies2 (Eudra-CT: 2010-021392-93, 2010-022328-64, and 2010-023690-19, approved by the responsible ethics committee), we administered therapeutic doses of bosentan (125 mg b.i.d.) to 36 healthy volunteers until steady state was reached. Twenty-eight of the subjects were CYP2C9 extensive metabolizers (EMs) and eight were poor metabolizers (PMs). Interindividual variability of bosentan pharmacokinetics was considerable (Figure 1) and independent of the CYP2C9 genotype (geometric mean of the area under the plasma concentration–time curve (AUC) (95% confidence interval) for EMs: 7,100 h·ng/ml (6,000, 8,390); for PMs: 5,570 h·ng/ml (4,430, 7,000); P = 0.12, Mann–Whitney U-test). However, the AUC of the active main metabolite hydroxy bosentan (RO48-5033), which is partly formed by CYP2C9,3 was 68.7% higher (P = 0.02) in the EM group (568 h·ng/ml (465, 693)) than in the PM group (337 h·ng/ml (231, 491)). Accordingly, the molar metabolic ratio of bosentan/hydroxy bosentan in plasma was 30.7% higher in the PM group (P = 0.01). The AUC of the primary phenol metabolite (RO47-8634), which in vitro is exclusively formed by CYP3A4,3 was similar in EMs (109 h·ng/ ml, (92.8, 127)) and PMs (121 h·ng/ml, (87.7, 166); P = 0.39), but in EMs, the corresponding metabolic ratio was 44% higher (EMs: 64.1 (57.7, 71.3); PMs: 44.4 (34.4, 57.3); P = 0.008). Finally, the molar metabolic ratio of bosentan divided by the sum of its three metabolites was similar (EMs: 6.03 (5.57, 6.53); PMs: 6.49 (4.87,8.64); P = 0.48). These findings suggest that in healthy volunteers plasma bosentan is not substantially modulated by CYP2C9 genotype; it was found that metabolic changes were present but substantially smaller than expected.1 The involvement of CYP2C9 in bosentan metabolism has only been investigated in vitro,3 and its contribution to the metabolism in vivo is unknown. In addition, whatever the number may be, it refers only to noninduced conditions and makes any extrapolation to chronic treatment conditions difficult. Bosentan induces CYP3A, as shown by a 3.2-fold increase in midazolam clearance,2 and it also induces CYP2C9, with a 1.4-fold increase in warfarin clearance. Because polymorphic CYP2C9 is inducible,4 at steady state, metabolic differences might be mitigated. Finally, CYP2C9-dependent intrahepatic changes in concentrations of bosentan and its metabolites might contribute to the occurrence of hepatotoxicity via inhibition of ABCB11,5 but these might not be closely mirrored in plasma concentrations. Therefore, exposure to bosentan and its metabolites should now be evaluated Clinical Pharmacology & Therapeutics 10.1038/clpt.2013.188

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.

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

Abstract 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 1.5 pg/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.