Supplementary Online Material
In vitro enzymology study details
Triplicate incubations were made up in 100 mM potassium phosphate bufferat pH 7.4 and contained 10 µM 14C-bitopertin, 0.80 mg/ml human liver microsomes, 2 µl test inhibitor in dimethyl sulfoxide (DMSO) and 1 mMnicotinamide adenine dinucleotide phosphate (NADPH). Total incubation volumes were 500 µl. Finalinhibitor concentrations are shown in Figure 2. 100% and 0% activity control incubations contained an equal volume of DMSO without inhibitor (0.4% v/v final). These were incubated in the same way as the other samples, with the exception that phosphate buffer was added to the 0% activity control incubations instead of NADPH solution. Incubations were prewarmed for 10 minutes to 37 °C before addition of NADPH. After 20 minutes incubation time the reactions were stopped by addition of 500 µl cold acetonitrile. Samples were chilled on ice for 30 minutes before centrifugation (20,000x g for 10 minutes). The supernatant was removed and dried down. Samples were re-dissolved in 150 µl 90% eluent A / 10% eluent B and 50 µl analysed by radio-HPLC. The 21–32 minute high-performance liquid chromatogram regions were fractionated into 7 ml scintillation vials in 1 ml aliquots. Each aliquot was then combined with 3.5 ml Ultima Gold liquid scintillation fluid and radioactivity determined by liquid scintillation counting.
Table 1.Key input data for PBPK modelling of bitopertin drug interaction.
The input data and assumptions for the basic PBPK model for bitopertin has been previously describeda,b.To allow for the modelling of drug–drug interactions it was necessary to assign the clearance of bitopertin to specific enzymes and this was done assuming that CYP3A4 is the only enzyme responsible for metabolism in the liver and gut. The model parameters used for bitopertin intestinal metabolism and for the inhibition of CYP3A4 enzyme by ketoconazole and erythromycin are given below.
Bitopertin / Ketoconazole / ErythromycinFraction escaping gut, first-pass / 0.97
Competitive: Ki, unbound, μMc / 0.0015
Time dependentd: Kinact, min-1, and Ki, μM / 0.024 and 13
a Parrott N, Hainzl D, Alberati D, Hofmann C, Robson R, Boutouyrie B et al. Physiologically based pharmacokinetic modelling to predict single- and multiple-dose human pharmacokinetics of bitopertin. Clinical pharmacokinetics. 2013;52(8):673-83.
b Parrott N, Hainzl D, Scheubel E, Krimmer S, Boetsch C, Guerini E et al. Physiologically based absorption modelling to predict the impact of drug properties on pharmacokinetics of bitopertin. The AAPS journal. 2014;16(5):1077-84.
c KatoM, Shitara Y, Sato H, Yoshisue K, Hirano M, Ikeda T, et al. The quantitative prediction of CYP-mediated drug interaction by physiologically based pharmacokinetic modeling. Pharm Res. 2008;25(8):1891–1901.
dGhanbari F, Rowland-Yeo K, Bloomer JC, Clarke SE, Lennard MS, Tucker GT, et al. A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation. Curr Drug Metabol. 2006;7:315–334.
Figure 1.14C-Labelled bitopertin metabolism by recombinantly expressed individual human drug metabolising CYP isoforms.
Figure 2.Effect of CYP-selective chemical inhibitors on human liver microsomal metabolism of bitopertin.
Figure 3.Individual erythromycin a)Cmax and b) AUC0–12 hour values following 500 mg twice daily erythromycin alone or concurrently with 30 mg bitopertin.
(a)(b)
Software used: MS Office Excel
Figure 4.Simulated and observed bitopertin profiles for the a)ketoconazole and b) erythromycin drug-interaction studies.
(a)
(b)
Table 2.Pharmacokinetic Parameters of metabolites after a singledose of bitopertinadministered with and without 400 mg qdketoconazole.
Subject / Treatment / Analyte / Cmax(nmol/L) / AUC0-312
(h x nmol/L) / Metabolic ratios
AUC0-312 (%) / Cmax (%)
104 / Bitopertin / Bitopertin / 120.33 / 8171.27 / -- / --
RO5008459 / 15.35 / 408.71 / 5 / 12.76
RO5008582 / 6.63 / 74.46 / 0.91 / 5.51
RO5200126 / 2.48 / 58.82 / 0.72 / 2.06
104 / Bitopertin + Keto / Bitopertin / 176.82 / 19776.09 / -- / --
RO5008459 / 2.46 / 643.96 / 3.26 / 1.39
107 / Bitopertin / Bitopertin / 122.54 / 3262.84 / -- / --
RO5008459 / 10.94 / 93.59 / 2.87 / 8.93
RO5008582 / 12.98 / 96.55 / 2.96 / 10.59
RO5200126 / 5.08 / 122.03 / 3.74 / 4.14
107 / Bitopertin + Keto / Bitopertin / 145.72 / 15618.96 / -- / --
RO5008459 / 2.74 / 669.97 / 4.29 / 1.88
108 / Bitopertin / Bitopertin / 105.24 / 3353.03 / -- / --
RO5008459 / 12.91 / 74.66 / 2.23 / 12.27
RO5008582 / 18.06 / 111.28 / 3.32 / 17.16
RO5200126 / 5.52 / 122.55 / 3.65 / 5.25
108 / Bitopertin + Keto / Bitopertin / 125.48 / 11274.91 / -- / --
RO5008459 / 2.83 / 654.59 / 5.81 / 2.26
112 / Bitopertin / Bitopertin / 124.93 / 8764.48 / -- / --
RO5008459 / 9.74 / 273.08 / 3.12 / 7.8
RO5008582 / 7.54 / 110.31 / 1.26 / 6.04
RO5200126 / 3.25 / 56.93 / 0.65 / 2.6
112 / Bitopertin + Keto / Bitopertin / 130.08 / 21184.64 / -- / --
RO5008459 / 2.37 / 459.77 / 2.17 / 1.82
Table 3.Pharmacokinetic Parameters of metabolites after a singledose of bitopertinadministered with and without 500 mg bid erythromycin.
Treatment / Analyte / Cmax(ng/mL) / AUC0-∞
(h x ng/mL) / Metabolic ratios
AUC0-∞(%) / Cmax (%)
Bitopertin / Bitopertin / 214 (12.7%) / 6570 (21.9%) / -- / --
RO5008459 / 22.7 (15.8%) / 610 (23.5%) / 9.30 (21.3%) / 10.6 (19.9%)
RO5008582 / 23.3 (18.9%) / 740 (22.0%) / 11.3 (21.8%) / 10.9 (19.1%)
RO5200126 / 9.34 (21.0%) / 823 (23.3%) / 12.5 (34.2%) / 4.36 (19.0%)
Bitopertin + Erythromycin / Bitopertin / 265 (16.1%) / 13600 (25.6%) / -- / --
RO5008459 / 13.6 (21.5%) / 985 (34.5%) / 7.24 (21.3%) / 5.14 (23.7%)
RO5008582 / 10.2 (19.5%) / 1120 (27.8%) / 8.20 (21.6%) / 3.84 (23.9%)
RO5200126 / 4.79 (22.1%) / 800 (18.4%) / 5.88 (25.4%) / 1.81 (31.9%)
Note: No correction for their respective molecular weight as all molecular mass were very close; Geometric mean with CV% are provided