Prediction of Drug Clearance by Glucuronidation from in Vitro Data: Use of Combined Cytochrome P450 and UDP-Glucuronosyltransferase Cofactors in Alamethicin-Activated Human Liver Microsomes

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Fig. 1.
Intrinsic clearance for buprenorphine, raloxifene, and ketoprofen obtained in human liver microsomes using either individual (P450 and UGT) or combined (P450 + UGT) cofactor incubation conditions.
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Fig. 2.
Prediction of clearance from in vitro data obtained in the presence of combined P450 and UGT cofactors and in the absence (A) and presence (B) of 2% BSA. , buprenorphine; ▾, carvedilol; ▴, codeine; □, diclofenac; , gemfibrozil; ▪, ketoprofen; , midazolam; , naloxone; •, raloxifene; ○, zidovudine. Error bars indicate range of scaling factors on the y-axis from 13 to 54 mg/g liver (Barter et al., 2007) and a range of Q_H on the x-axis from 17 to 25.5 ml/min/kg (Kato et al., 2003).
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Fig. 3.
Comparison of the in vitro fm_UGT – BSA () and fm_UGT + BSA (▪) with the in vivo fm_UGT (□) obtained from renal excretion data for six compounds investigated.
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Fig. 4.
-Fold difference in CL_{int, u} for 10 compounds obtained in the presence and absence of 2% BSA. The CL_int was obtained in the presence of P450 and UGT cofactors in both cases. The solid line indicates the CL_{int, u} + BSA/CL_int – BSA ratio of 1.

TABLE 1

Clearance obtained for 10 drugs in the presence of individual P450 and UGT cofactors

Data are mean ± S.D. References for major UGT enzymes are available at http://www.pharmacy.manchester.ac.uk/capkr/.

Compound	Major P450	Major UGT	CL_{int, u}			In Vitro^a
Compound	Major P450	Major UGT				P450	UGT	fm_UGT	fm_CYP
			μl/min/mg
Buprenorphine	CYP3A4	UGT1A1	472 ± 40	279 ± 28	0.37	0.63
Carvedilol	CYP2D6	UGT1A1	252 ± 17.6	36.0 ± 9.8	0.13	0.87
Codeine	CYP3A4	UGT2B7	2.44 ± 0.06	2.53 ± 0.01	0.51	0.49
Diclofenac	CYP2C9	UGT2B7	129 ± 32.2	214 ± 46.1	0.62	0.38
Gemfibrozil	CYP3A4	UGT2B7	18.4 ± 9.3	70 ± 9.3	0.79	0.21
Ketoprofen	CYP2C9	UGT2B7	9.35 ± 3.6	9.35 ± 4.3	0.50	0.50
Midazolam	CYP3A4	UGT1A4	144 ± 41.8	8.83 ± 1.9	0.06	0.94
Naloxone		UGT2B7	5.52 ± 3.2	10.2 ± 2.9	0.65	0.35
Raloxifene		UGT1A9/1A1	197 ± 25.8	444 ± 86.4	0.69	0.31
Zidovudine		UGT2B7	3.84 ± 0.7	2.21 ± 1.4	0.37	0.63

↵ a In vitro fm_UGT and fm_CYP were calculated using eqs. 5 and 6, respectively.

TABLE 2

Clearance obtained for 10 drugs in the presence and absence of BSA using combined P450 and UGT cofactors and scaled to in vivo

Compound	CL_{int, u} (P450 + UGT)			-Fold Difference: Cl_{int, u} + BSA/CL_{int, u} - BSA	fu_mic			Scaled CL_{int, u}^a
Compound		-BSA	+BSA	-Fold Difference: Cl_{int, u} + BSA/CL_{int, u} - BSA		-BSA	+BSA	-BSA	+BSA
	μl/min/mg						ml/min/kg
Buprenorphine	688	577	0.84	0.10	0.17	588.9	494
Carvedilol	356	246	0.69	0.10	0.04	305	210
Codeine	3.4	16.9	5.0	0.96	0.99	2.9	14.5
Diclofenac	369	2143	5.8	0.87	0.008	316	1834
Gemfibrozil	97.3	1274	13.0	0.77	0.01	83.3	1090
Ketoprofen	26.0	115	4.4	0.92	0.11	22.2	98.5
Midazolam	159	241	1.5	0.54	0.04	137	207
Naloxone	14.4	35.1	2.4	0.87	0.99	12.3	30.1
Raloxifene	544	1812	3.3	0.08	0.009	465	1551
Zidovudine	2.8	13.2	4.7	0.60	0.49	2.4	11.3

↵ a CL_{int, u} was scaled using a human microsomal scaling factor of 40 mg protein/g liver (Ito and Houston, 2005; Barter et al., 2007) and an average liver weight of 21.4 g liver/kg.

TABLE 3

In vivo clearance values for 10 drugs investigated and the main parameters used in in vitro-in vivo extrapolation

References for plasma clearance, R_B, and fu_p are available at http://www.pharmacy.manchester.ac.uk/capkr/.

Compound	fu_p	R_B	fu_b	Observed CL
				ml/min/kg
Buprenorphine	0.04	0.60	0.07	19
Carvedilol	0.02	1.00	0.02	7.8
Codeine	0.70	1.00	0.70	15
Diclofenac	0.005	0.50	0.009	3.5
Gemfibrozil	0.03	0.55	0.05	1.7
Ketoprofen	0.008	0.55	0.01	1.6
Midazolam	0.017	0.53	0.03	5.3
Naloxone	0.54	1.22	0.44	23
Raloxifene	0.05	1.00	0.05	735
Zidovudine	0.8	0.98	0.82	25

TABLE 4

Fold difference and fm_UGT estimates obtained for 10 drugs obtained in the presence of BSA using individual P450 and UGT cofactors

Compound	-Fold Difference: Cl_{int, u} + BSA/CL_{int, u} - BSA			In Vitro fm_UGT + BSA^a	In Vivo fm_UGT^b
Compound		P450	UGT	In Vitro fm_UGT + BSA^a	In Vivo fm_UGT^b
Buprenorphine	0.7	0.9	0.44	0.50
Carvedilol	1.1	7.9	0.50	N.A.
Codeine	0.8	3.2	0.81	0.82
Diclofenac	4.1	4.7	0.65	N.A.
Gemfibrozil	7.4	12.1	0.86	0.40
Ketoprofen	1.8	4.5	0.71	0.90
Midazolam	3.7	N.D.	N.D.	N.A.
Naloxone	0.6	2.6	0.89	0.65
Raloxifene	15.7	5.4	0.44	N.A.
Zidovudine	0.8	4.8	0.78	0.85

N.D., data not obtained in vitro; N.A., fm_UGT was not available from renal excretion data.
↵ a In vitro fm_UGT was calculated using eq. 5. The fm_UGT estimate obtained in the absence of BSA is shown in Table 1.
↵ b In vivo fm_UGT values were estimated from renal excretion data.