Article Figures & Data
Figures
- Fig. 1.
Proposed metabolism of VU238 to major metabolites M1 and M2 in hepatic S9 (absent NADPH) of the human, monkey, rat, mouse, guinea pig, and minipig. M2 was not detected in extracts from incubations of VU238 with human and minipig S9 but was detected only in extracts from direct incubations with M1 (VU922).
- Fig. 2.
LC-MS/MS spectra of (A) VU238 and metabolites (B) M1 (VU922) and (C) M2 obtained from either synthetic standard solutions (A and B) or extract from male cynomolgus monkey hepatic S9 incubation with VU238 (C).
- Fig. 3.
LC-UV chromatograms and mass spectra of VU238, VU180, M1, and M2 obtained from extracts of incubations with VU238 or VU180 (20 μM) in male cynomolgus monkey hepatic S9 (5 mg/ml). (A) LC-UV chromatograms obtained from extracts of incubations with VU238 (top) or VU180 (bottom). (B) Mass spectra of VU238 (top) or VU180 (bottom). (C) Mass spectra of M1 obtained from extracts of incubations with VU238 (top) or VU180 (bottom). (D) Mass spectra of M2 obtained from extracts of incubations with VU238 (top) or VU180 (bottom).
- Fig. 4.
Extracted ion chromatograms (XICs) obtained from extracts of incubations with VU238 (5 μM) in (A) male cynomolgus monkey hepatic S9 (2 mg/ml) and (B) male rat hepatic S9 (2 mg/ml) demonstrating the formation of M1 and M2 in the presence or absence of the XO inhibitor allopurinol (100 μM) or the AO inhibitor hydralazine (50 μM).
- Fig. 5.
Extracted ion chromatograms (XICs) obtained from extracts of incubations with M1 (VU922, 5 μM) in (A) male cynomolgus monkey hepatic S9 (2 mg/ml) and (B) male rat hepatic S9 (2 mg/ml) demonstrating formation of M2 in the presence or absence of the XO inhibitor allopurinol (100 μM) or the AO inhibitor hydralazine (50 μM).
- Fig. 6.
Extracted ion chromatograms (XICs) obtained from extracts of incubations with (A) VU238 (5 μM) and (B) M1 (VU922, 5 μM) in multiple species’ hepatic S9 (2 mg/ml). Human data were obtained using mixed-gender S9. All other data were obtained using male S9.
- Fig. 7.
Extracted ion chromatograms (XICs) obtained from extracts of incubations with VU238 (5 μM) in (A) female cynomolgus monkey hepatic S9 (2 mg/ml) and (B) female rat hepatic S9 (2 mg/ml) demonstrating the formation of M1 and M2 in the presence or absence of the XO inhibitor allopurinol (100 μM) or the AO inhibitor hydralazine (50 μM).
- Fig. 8.
Extracted ion chromatograms (XICs) obtained from extracts of incubations with M1 (VU922, 5 μM) in (A) female cynomolgus monkey hepatic S9 (2 mg/ml) and (B) female rat hepatic S9 (2 mg/ml) demonstrating formation of M2 in the presence or absence of the XO inhibitor allopurinol (100 μM) or the AO inhibitor hydralazine (50 μM).
- Fig. 9.
Extracted ion chromatograms (XICs) obtained from extracts of incubations with BIBX1382 (5 μM) in (A) male cynomolgus monkey hepatic S9 (2 mg/ml) and (B) male rat hepatic S9 (2 mg/ml) demonstrating formation of M1b and M2b in the presence or absence of the XO inhibitor allopurinol (100 μM) or the AO inhibitor hydralazine (50 μM). (C) Summary of BIBX1382 hepatic S9 metabolism in humans, monkeys, rats, mice, guinea pigs, and minipigs. Formation of M1b and M2b in incubations of BIBX1382 with hepatic S9 was inhibited by hydralazine in all species. C, cynomolgus monkey; G, guinea pig; H, human; M, mouse; MP, minipig; R, rat; Rh, rhesus monkey.
Tables
- TABLE 1
Proposed predominant metabolic pathway mediating the formation of M1 or M2 in multispecies’ hepatic S9 (2 mg/ml) determined from incubations of VU238 (5 μM) or M1 (VU922, 5 μM) with the AO inhibitor hydralazine (50 μM) or the XO inhibitor allopurinol (100 μM)
Designations of AO or XO indicate that formation of the metabolite was inhibited predominantly by either hydralazine or allopurinol, respectively.
M1 M2 Humana AO n.d. Cynomolgus AO AO Rhesus AO AO Rat AO XO Mouse AO XO Guinea pig AO AO Minipig AO n.d. Female cynomolgus AO n.d. Female rat AO XO n.d., not determined.
↵a Human data obtained using mixed-gender S9. All other data were obtained using male S9 unless otherwise indicated.
- TABLE 2
Intrinsic clearance (CLint, microliters per minute per milligrams protein) obtained from incubations of VU238 (1 μM) or M1 (VU922, 1 μM) with hepatic S9 (2.5 mg/ml) of multiple species
Data represent means ± S.D. of triplicate determinations.
Species VU238 VU922 (M1) Human 1.48 ± 0.312 n.d. Cynomolgus 12.1 ± 0.643 2.00 ± 0.040 Rhesus 5.15 ± 0.537 1.44 ± 0.139 Rat 13.7 ± 0.265 2.19 ± 0.201 Mouse 4.76 ± 0.386 n.d. Guinea pig 1.89 ± 0.180 3.40 ± 0.069 Minipig 3.51 ± 0.306 n.d. n.d., not determined because of low substrate turnover.
Data Supplement
- Supplemental Data -
Supplemental Figure 1 - Proton NMR spectra for VU0652922 (M1)
Supplemental Figure 2 - Extracted ion chromatograms (XIC) obtained from extracts of incubations with VU238 (5 mu-M) in (A) mixed gender human hepatic S9 (2 mg/mL), (B) male rhesus monkey hepatic S9 (2 mg/mL), (C) male mouse hepatic S9 (2 mg/mL), (D) male guinea pig hepatic S9 (2 mg/mL), and (E) male minipig hepatic S9 (2mg/mL) demonstrating formation of M1 and M2 in the presence or absence of the XO inhibitor allopurinol (100 mu-M) or the AO inhibitor hydralazine (50 mu-M)
Supplemental Figure 3 - Extracted ion chromatograms (XIC) obtained from extracts of incubations with VU922 (5 mu-M) in (A) mixed gender human hepatic S9 (2 mg/mL), (B) male rhesus monkey hepatic S9 (2 mg/mL), (C) male mouse hepatic S9 (2 mg/mL), (D) male guinea pig hepatic S9 (2 mg/mL), and (E) male minipig hepatic S9 (2mg/mL) demonstrating formation of M2 in the presence or absence of the XO inhibitor allopurinol (100 mu-M) or the AO inhibitor hydralazine (50 mu-M)
Supplemental Figure 4 - Extracted ion chromatograms (XIC) obtained from extracts of incubations with BIBX1382 (5 mu-M) in (A) mixed gender human hepatic S9 (2 mg/mL), (B) male rhesus monkey hepatic S9 (2 mg/mL), (C) male mouse hepatic S9 (2 mg/mL), (D) male guinea pig hepatic S9 (2 mg/mL), and (E) male minipig hepatic S9 (2mg/mL) demonstrating formation of M1b and M2b in the presence or absence of the XO inhibitor allopurinol (100 mu-M) or the AO inhibitor hydralazine (50 mu-M)
Supplemental Table 1 - Percent total MS peak area of parent drug and metabolites from biotransformation experiments with VU238, VU922, or BIBX1382 (5 mu-M) in hepatic S9 of multiple species (2 mg/mL) in the presence or absence of the XO inhibitor allopurinol (100 mu-M) or the AO inhibitor hydralazine (50 mu-M)
Supplemental Figure 5 - UV chromatograms obtained from extracts of 60-minute incubations with 6-thioxanthine (6-TX, 20 mu-M) in (A) male cynomolgus monkey hepatic S9 (2 mg/mL), (B) male rat hepatic S9 (2 mg/mL), (C) mixed gender human hepatic S9 (2 mg/mL), (D) male rhesus monkey hepatic S9 (2 mg/mL), (E) male mouse hepatic S9 (2mg/mL), (F) male guinea pig hepatic S9 (2mg/mL), and (G) male minipig hepatic S9 (2mg/mL) demonstrating formation of 6-thiouric acid (6-TUA) in the presence or absence of the XO inhibitor allopurinol (100 mu-M) or the AO inhibitor hydralazine (50 mu-M)
Supplemental Table 2 - Percent total UV peak area of parent drug (6-TX) and metabolite (6-TUA) from biotransformation experiments (Supplemental Figure 5) with 6-TX (20 mu-M) in hepatic S9 of multiple species (2 mg/mL) in the presence or absence of the XO inhibitor allopurinol (100 mu-M) or the AO inhibitor hydralazine (50 mu-M). 6-TX, 6-thioxanthine; 6-TUA, 6-thiouric acid
- Supplemental Data -
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Species-Specific Metabolism by Molybdenum Hydroxylases
