Article Figures & Data
Figures
- Fig. 1.
Total cytochrome P450 in liver microsomes from vehicle and TCDD-treated WT, hCYP1A1/1A2, and Cyp1a KO mice. Liver microsomes were prepared from vehicle (corn oil) and TCDD-treated hCYP1A1/1A2, Cyp1a KO, and WT mice (n = 4), and total cytochrome P450 content was measured as detailed in Materials and Methods. Data are presented as the mean ± S.D.; **significantly different from corresponding corn oil–treated group (unpaired t test; two-tailed P value; P < 0.01).
- Fig. 2.
Basal and TCDD-inducible expression of Cyp1a/CYP1A in tissues from WT, Cyp1a KO, and hCYP1A1/1A2 mice. Microsomes were prepared from vehicle (corn oil) and TCDD-treated hCYP1A1/1A2, Cyp1a KO, and WT mice from the tissues shown and immunoblotted for hCYP1A1 (A and D), human CYP1A2 (B), and mouse Cyp1a (C) as detailed in Materials and Methods. Standards: CYP1A1 (0.36 pmol/lane) and CYP1A2 (1 pmol/lane) expressed in bacterial membranes.
- Fig. 3.
EROD and MROD activities in hepatic and extrahepatic tissues from WT, Cyp1a KO, and hCYP1A1/1A2 mice. Liver microsomes were prepared from vehicle (corn oil) and TCDD-treated hCYP1A1/1A2, Cyp1a KO, and WT mice for the tissues shown, and EROD (A) and MROD (B) activities were measured as detailed in Materials and Methods. Microsomes from extrahepatic tissues of TCDD-treated hCYP1A1/1A2 and WT mice were prepared, and EROD (C) and MROD (D) activities were measured. Data are presented as the mean ± S.D. (n = 4); *significantly different from corresponding WT group (unpaired t test; two-tailed P value; **P < 0.01; ***P < 0.001; ****P < 0.0001). Heart, lung, spleen, kidney, brain, and testis microsomes were prepared from pooled organs of each experimental group. Their activities are presented as the mean ± S.D. of three measurements of the pooled sample. No test for statistical significance was performed on these data. HLM, pooled human liver microsomes.
- Fig. 4.
Effect of quinidine on MROD activity catalyzed by recombinant human CYP1A1 and CYP1A2. Recombinant human CYP1A1 (closed circles) and CYP1A2 (open circles) were coexpressed with human P450 reductase in bacterial microsomes, and activity was measured as detailed in Materials and Methods.
- Fig. 5.
Effect of quinidine on EROD and MROD activities catalyzed by recombinant human CYP1A1, CYP1A2, and liver microsomes from Cyp1a KO mice at different substrate concentrations. EROD (A, C, and E) and MROD (B, D, and F) catalyzed by recombinant human CYP1A1 (A and B), CYP1A2 (C and D), and liver microsomes from TCDD-treated Cyp1a KO mice (E and F) at different concentrations of substrate and quinidine, as detailed in Materials and Methods. Symbols are the measured reaction rates. Lines are nonlinear regression analysis of the data using eqs. 1–6 (for details, see Data Analysis in the Supplemental Materials).
- Fig. 6.
EROD and MROD activities measured in the presence or absence of quinidine in liver microsomes from TCDD-treated hCYP1A1/1A2 humanized mice. EROD (A) and MROD (B) with and without quinidine catalyzed by liver microsomes from TCDD-treated hCYP1A1/1A2 humanized mice. Symbols are the measured reaction rates. Lines are nonlinear regression analysis of the data using eqs. 7 and 8, respectively (for details, see Data Analysis in the Supplemental Materials). Contribution of CYP1A1, CYP1A2, and non-CYP1A components of the reactions were calculated using eqs. 9–14 (for details, see Data Analysis in the Supplemental Materials) for EROD (C and E) and MROD (D and F) with (E and F) and without (C and D) quinidine (200 μM).
- Fig. 7.
EROD and MROD activities catalyzed by liver microsomes from vehicle-treated hCYP1A1/1A2 humanized mice in the presence or absence of quinidine. EROD (A) and MROD (B) with and without quinidine catalyzed by liver microsomes from vehicle-treated (corn oil) hCYP1A1/1A2 humanized mice. Symbols are the measured reaction rates, and the line is derived from nonlinear regression analysis of the MROD in the absence of quinidine using eq. 5 (for details see Data Analysis in the Supplemental Materials).
- Fig. 8.
Microsomal stability and in vitro clearance of ramelteon and tacrine. Microsomal stability (A and B) and in vitro clearance (C and D) of human CYP1A2 substrates ramelteon (A and C) and tacrine (B and D) measured in liver microsomes from WT, Cyp1a KO, and hCYP1A1/1A2 mice and human donors (human liver microsomes). Symbols are the measured concentrations of the compounds. A no-NADPH control was also run. Lines are nonlinear regression analysis using the equation for double-exponential (tacrine with Cyp1a KO microsomes) or monoexponential (all other incubations) decay in the software package GraFit 7.0.3. (Erithacus, West Sussex, UK).
- Fig. 9.
Caffeine pharmacokinetics in WT, Cyp1a KO, hCYP1A1/1A2, and HRN mice. Symbols are caffeine concentrations measured in mouse whole blood. All data are expressed as the mean ± S.D. (n = 4 mice per treatment group).
- Fig. 10.
Caffeine pharmacokinetics in hCYP1A1/1A2, Cyp1a KO, and HRN mice extrapolated to humans. Caffeine pharmacokinetics in hCYP1A1/1A2, Cyp1a KO, and HRN mice extrapolated to humans using the complex Dedrick plot approach (see Materials and Methods for details). The caffeine concentration time course in placebo- or fluvoxamine-treated healthy subjects (Culm-Merdek et al., 2005) was corrected for mean caffeine concentration in predose. Symbols are the corrected caffeine concentrations measured in human plasma or extrapolated from mouse whole blood. All data are expressed as mean values (n = 4 mice per treatment group; n = 7 healthy subjects per treatment group).
- Fig. 11.
Effect of fraction metabolized on AUC ratios of “substrate drug.” Simulation of AUC ratios of a substrate drug with inhibitor to that without inhibitor as a function of inhibitor concentration for different contributions of the inhibited enzyme [fraction metabolized (fm)] to the substrate drug elimination. The enzyme contribution is expressed as part of total clearance. Horizontal lines separate areas with strong (AUC ratio >5-fold), moderate (2 < AUC ratio < 5), weak (1.25 < AUC ratio < 2), and “no effect” (AUC ratio <1.25-fold) inhibition.
Tables
Microsomes Origin Ks1 ± S.E. Ks2 ± S.E. Ki ± S.E. α Quinidine Inhibition Type μM μM μM EROD rCYP1A1 0.09 ± 0.008 11 ± 1 3.3 ± 0.35 31 ± 8 Mixed rCYP1A2 (Km) 1.2 ± 0.2 NA NA NA NA Cyp1a KO 0.83 ± 0.04 13 ± 1.5 422 ± 18 NA Noncompetitive MROD rCYP1A1 0.47 ± 0.07 1.3 ± 0.18 2.2 ± 0.18 NA Competitive rCYP1A2 0.58 ± 0.08 12 ± 3.5 NA NA NA Cyp1a KO 0.96 ± 0.18 3.4 ± 0.8 272 ± 28 NA Noncompetitive Ki, dissociation constant of the enzyme-inhibitor complex; Ks1, dissociation constant of the productive enzyme-substrate complex; Ks2, dissociation constant of the inhibitory enzyme-substrate complex; NA, not applicable; rCYP1A1, recombinant CYP1A1; rCYP1A2, recombinant CYP1A2; α, parameter describing the effect of inhibitor binding on the binding of the substrate and vice versa.
- TABLE 2
Caffeine pharmacokinetic parameters in hCYP1A1/1A2, Cyp1a KO, WT, and HRN mice Data are the mean ± S.D. (% mean of C57BL/6J ± %S.D.); n = 4.
Mouse Line Cmax V/F(obs) HL AUCinf(obs) CL/F(obs) ng/ml ml/kg h h*ng/ml ml/h/kg C57BL/6J 2383 ± 628 (100 ± 26) 1253 ± 494 (100 ± 39) 0.94 ± 0.25 (100 ± 27) 5704 ± 1265 (100 ± 22) 905 ± 170 (100 ± 19) Cyp1a KO 3991 ± 472** (167 ± 20) 992 ± 188 (79 ± 15) 2.39 ± 0.2*** (253 ± 22) 17651 ± 2073**** (309 ± 36) 286 ± 35*** (32 ± 4) hCYP1A1/1A2 3082 ± 194 (129 ± 8) 1111 ± 55 (89 ± 4) 1.3 ± 0.33 (138 ± 35) 8397 ± 1797* (147 ± 32) 615 ± 121* (68 ± 13) HRN 3744 ± 794* (157 ± 33) 1057 ± 82 (84 ± 7) 8.4 ± 0.8**** (891 ± 85) 57,271 ± 2583**** (1004 ± 45) 87 ± 3.8**** (9.7 ± 0.42) AUCinf(obs), area under the curve from dosing time extrapolated to infinity from the last observed caffeine concentration; CL/F(obs), clearance calculated using AUCinf(obs); Cmax, maximum observed concentration; HL, terminal half-life; V/F(obs), volume of distribution calculated using AUCinf(obs).
↵* Significantly different from C57BL/6J (unpaired t test; two-tailed P values; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
- TABLE 3
Contribution of CYP1A2 to caffeine systemic clearance calculated from published mouse studies The difference between caffeine systemic clearance in the defined mouse strain and that in the Cyp1a KO mouse line was divided by the value of the systemic clearance in the defined mouse strain and then multiplied by 100% to obtain the contribution of CYP1A2. The clearance value measured in CYP1A2−/− mice was used to calculate CYP1A2 contribution to caffeine systemic clearance in C57BL/6N mice reported by Buters et al. (1996).
Mouse Line Dose Route Clearance Reference CYP1A2 Contribution mg/kg ml/(h*kg) % CYP1A2−/− 2 i.p. 276 Buters et al., 1996 0 C57BL/6N 2 i.p. 2268 Buters et al., 1996 88 Cyp1a KO 5 PO 286 This study 0 C57BL/6J 5 PO 905 This study 68 hCYP1A1/1A2 5 PO 614 This study 53 C57BL/6J 5 PO 472a Li et al., 2017 39 Swiss 20 PO 311b Samojlik et al., 2016 8 Swiss 20 i.p. 398b Samojlik et al., 2016 28 C57BL/6J 5 PO 726 Scheer et al., 2014 61 CD-COBS 1 i.v. 732 Walton et al., 2001 61 CD-1 20 i.p. 640 Kaplan et al., 1990 55 CD-1 40 i.p. 380 Kaplan et al., 1990 25
Data Supplement
- Supplemental Data -
Supplemental methods, 1 scheme, 1 table, and 1 figure.
- Supplemental Data -
