Article Figures & Data
Figures
- Fig. 1.
Elution of hydroxypimozide isomers and DHPBI with products of CYP2D6- and CYP3A4-mediated metabolism. Chromatograms for combined standards of 50 nM DHPBI (m/z 218.10 → 84.00) (A) and 5-/6-hydroxypimozide metabolites (m/z 478.30 → 328.10) (B) are provided for reference. Chromatograms from incubations of therapeutically relevant concentrations (10 nM) of pimozide in 2 pmol/ml recombinant CYP2D6, m/z 218.10 → 84.00 (C) and m/z 478.30 → 328.10 (D), and CYP3A4, m/z 218.10 → 84.00 (E) and m/z 478.30 → 328.10 (F), are also displayed. Retention times are listed in blue above the respective peaks.
- Fig. 2.
Kinetics of pimozide biotransformation to DHPBI and 5-hydroxypimozide (5-OH-Pimozide) by recombinant CYP3A4 (left) and CYP2D6 (right), respectively. The displayed results are from a representative experiment. Individual data points represent the means of technical duplicates at a given pimozide concentration from the same experimental replicate, and the solid line reflects the fit of a single-enzyme Michaelis-Menten model to the data. Mean ± S.D. for parameter (Km and Vmax) estimates from n = 3 experiments using each isoform appear as text at the top of the respective graphs.
- Fig. 3.
Kinetics of pimozide biotransformation to DHPBI (left) and 5-hydroxypimozide (5-OH-Pimozide) (right) in pooled human liver microsomes. The displayed results are from a representative experiment. Individual data points represent the means of technical duplicates at a given pimozide concentration from the same experimental replicate, and the solid line reflects the fit of a single-enzyme Michaelis-Menten model to the data. Mean ± S.D. for parameter (Km and Vmax) estimates from n = 3 experiments appear as text at the bottom of the respective graphs for each reaction.
- Fig. 4.
Correlation of CYP2D6 protein abundance and the rate of biotransformation of pimozide to 5-hydroxypimozide (5-OH-Pimozide) (left) and 6-hydroxypimozide (6-OH-Pimozide) (right) in HLMs isolated from individual donors (n = 7). Data points represent the means of separate experiments (n = 3), and error bars reflect the S.D.; error bars are not visible for many points because of low interexperimental variability. The r2 corresponding to the fit of a simple linear regression model for each metabolite is listed at the top of each respective graph. Expression of CYP2D6 protein was determined via MS/MS analysis. Data points were color coded to reflect activity score assignments for CYP2D6 genotypes: 0 (red), 0.5 (yellow), 1.0 (green), 2.0 (blue), and >2.25 (purple) (Caudle et al., 2020).
- Fig. 5.
Correlation of CYP3A4 protein abundance and the rate of pimozide N-dealkylation to DHPBI in HLMs isolated from individual donors (n = 7). Data points represent the means of separate experiments (n = 3), and error bars reflect the S.D.; error bars are not visible for most points because of low interexperimental variability. The r2 corresponding to the fit of a simple linear regression model for each metabolite is listed at the top of each respective graph. Expression of CYP3A4 protein was determined via MS/MS analysis.
- Fig. 6.
Revised pathway of pimozide biotransformation. The principle products of CYP2D6-mediated pimozide metabolism are 5- and 6-hydroxypimozide, and the major product of CYP3A4-mediated metabolism of pimozide is DHPBI. Pimozide and metabolite structures were made using ChemDoodle Web Components 2D Sketcher (iChemLabs, Chesterfield, VA).
Tables
- TABLE 1
Donor demographics, CYP2D6 genotype, and cytochrome P450 protein content in single-donor HLMs
All single-donor HLMs obtained for Sekisui Xenotech. Activity scores were assigned according to the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines (Caudle et al., 2020). Protein abundances for CYP2D6 and CYP3A4 were determined via MS/MS analysis.
Donor Product no. Lot no. Sex Age (y) CYP2D6 Genotype CYP2D6 Activity Score CYP2D6 (pmol/mg HLM protein) CYP3A4 (pmol/mg HLM protein) H0289 0710505 F 60 1/*2x2 >2.25a 11.93 71.71 H0439 0710037 F 78 *1/*4 1.0b 3.31 4.97 H0446 0710044 M 21 *4/*41 0.5b 0.80 48.85 H0455 0710053 F 37 *1/*4 1.0b 4.25 35.53 H0485 0710139 M 10 *1/*2 2.0c 4.20 2.41 H0486 0710140 F 49 *4/*5 0.0d <LOQ 44.44 H0493 0710147 F 64 *1/*1 2.0c 7.49 34.94 - TABLE 2
Molar balance of pimozide metabolism in pooled HLMs
Mean (S.D.) amounts (picomoles) of pimozide molecules depleted and pimozide metabolites formed by pooled (n = 200 donors) human liver microsomes incubated with pimozide (10 pmol starting amount, 50 nM starting concentration) for 20 min. Percentage standardization of metabolites formed relative to pimozide molecules depleted is also provided.
5-Hydroxypimozide 6-Hydroxypimozide DHPBI Sum of Metabolitesa Pimozide Depletedb Amount (pmol) 0.27 (0.040) 0.11 (0.035) 2.4 (0.20) 2.8 (0.17) 3.1 (0.80) % Depleted Accountedc 8.7 (1.3) 3.5 (1.1) 78 (6.5) 90 (5.5) N/A N/A, not applicable.
↵a Denotes sum of amounts of DHPBI, 5-hydroxypimozide, and 6-hydroxypimozide formed during each incubation.
↵b Denotes the difference between pimozide amounts measured at time 0 and 20 min in each incubation.
↵c Sum of metabolite amounts divided by amount of pimozide depleted over the course of the incubation, expressed as a percentage (see eq. 1).
- TABLE 3
Comparison of pimozide metabolite concentrations produced in incubations of recombinant cytochrome P450 isoforms
Recombinant human cytochrome P450 isoforms (2 pmol/ml) were incubated with 2500 nM pimozide for 30 min. Numbers represent the mean (S.D.) from triplicate experiments for the concentrations (nanomolars) of the specific metabolites observed after quenching of incubations. A “—” denotes a cytochrome P450 isoform that was not observed to produce detectable amounts of the listed metabolites. The lower LOQs for this experiment were 1 nM for 5-/6-hydroxypimozide and 5 nM for DHPBI. Experimental conditions were not confirmed to be linear with respect to time.
Cytochrome P450 Isoform 1A1 1A2 2A6 2B6 2C8 2C9 2C18 2C19 2D6 2E1 2J2 3A4 3A5 3A7 DHPBI — — — — — — — — — — — 78 (6.5) <LOQ 14 (1.5) 5-OH-Pimozide <LOQ <LOQ — — — — <LOQ — 35 (11) — <LOQ 2.4 (0.24) 2.5 (0.22) <LOQ 6-OH-Pimozide <LOQ <LOQ — — — — <LOQ — 5.8 (1.2) — <LOQ 1.7 (0.33) 1.4 (0.16) <LOQ LOQ, limit of quantification. 5-OH, 5-hydroxy. 6-OH, 6-hydroxy.
Data Supplement
- Supplemental Data -
Supplementary Table 1 - Cone Voltage and Collision Energy for Analysis of Analyte Fragmentation on XEVO-TQD.
Supplementary Table 2 - Cone Voltage and Collision Energy for Analyte Quantification on XEVO-TQ-XS.
Supplementary Figure 1 - Daughter ions produced from pimozide (top) and the hydroxypimozide (bottom)
metabolite(s) suggest the benzimidazolone ring is a site of ring hydroxylation in human liver microsomes isolated
from a donor genotyped as a CYP2D6 ultra rapid metabolizer (UM) and incubated with 10 μM pimozide.Supplementary Figure 2 - SmartCYP predictions for sites of metabolism by CYP2D6. Likelihood of being the principle site of site of oxidation decreases with increasing rank number.
Supplementary Figure 3 - Elution of hydroxypimozide isomers and DHPBI with products of CYP3A4-mediated
metabolism at supratherapeutic concentrations of pimozide.Supplementary Figure 4 - Correlation of CYP2D6 protein abundance and the rate of dextromethorphan O-demethylation to dextrorphan in HLMs isolated from individual donors (n = 7).
Supplementary Figure 5 - Correlation of CYP3A4 protein abundance and the rate of pimozide 5-hydroxylation (left)
and 6-hydroxylation (right) in HLMs isolated from individual donors (n = 7).Supplementary Figure 6 - Correlation of CYP3A4 protein abundance and the rate of midazolam 1’-hydroxylation
(left) and testosterone 6β-hydroxylation (right) in HLMs isolated from individual donors (n = 7).Supplementary Figure 7 - Correlation of CYP2D6 protein abundance and the rate of DHPBI formation in HLMs
isolated from individual donors (n = 7).Supplementary Figure 8 - Correlation of CYP3A4 and CYP2D6 protein abundance.
- Supplemental Data -
