Supplementary Material I.Pharmacokinetic Parameters in Minipig Compared to Those in Human

Supplementary material I.Pharmacokinetic parameters in minipig compared to those in human and other preclinical species

Compound / Parameter / Minipig, male / Minipig,
female / Human / Monkey / Dog / Rat
Antipyrine / CL (ml/min/kg) / 1.5 ± 0.9 (58) / 4.8 ± 1.1 (24) / 0.6 / 6.9 / 9.3 / 5.2
Vss (L/kg) / 0.8 ± 0.3 (41) / 0.7 ± 0.2 (28) / 0.6 / 1 / 0.7 / 0.9
T1/2 (h) / 9.3 ± 5.8 (62) / 1.7 ± 0.4 (23) / 12 / 2.1 / 1.5 / 2.2
F (%) / 36.3 ± 10 (28) / 31.1 ± 5.3 (17) / 100 / 120 / 97
Sources / [1-3] / [4-6] / [5, 7, 8] / [9, 10]
Atenolol / CL (ml/min/kg) / 5.8 ± 1.7 (29) / 10.1 ± 2 (20) / 2 / 7.5 / 4.4 / 31.2
Vss (L/kg) / 1.5 ± 0.1 (8) / 1.5 ± 0.1 (6) / 1.2 / 1.6 / 5.1
T1/2 (h) / 5.3 ± 0.3 (7) / 3.5 ± 1.7 (48) / 6.5 / 5.35 / 1.9
F (%) / 21.8 / 55.8 / 55.1 / 57 / 32.9
Sources / [11-15] / [11, 16] / [15, 17, 18] / [10]
Cimetidine / CL (ml/min/kg) / 31.8 ± 2.2 (7) / 43 ± 8.2 (19) / 8.3 / 17.2 / 11.6 / 38.2
Vss (L/kg) / 1.3 ± 0.1 (4) / 1.6 ± 0.3 (16) / 1.1 / 2 / 1.9 / 3.25
T1/2 (h) / 0.8 ± 0.0 (3) / 0.7 ± 0.2 (24) / 2 / 1.7 / 1.6 / 1.9
F (%) / 32.4 / 32.9 / 60 / 75
Sources / [19, 20] / [21] / [22] / [23]
Diazepam / CL (ml/min/kg) / 9.3 ± 0.7 (8) / 9.9 ±1.5 (15) / 0.4 / 24.5 / 35.4 / 61.8
Vss (L/kg) / 0.8 ± 0.3 (40) / 0.9 ± 0.3 (41) / 1.1 / 0.9 / 4.7 / 10.9
T1/2 (h) / 1.5 ± 0.5 (34) / 1.9 ± 0.4 (22) / 35.5 / 0.8 / 4.4 / 3.5
F (%) / 141.2 / 147.5 / 95 / 10 / 7.6
Sources / [24-26] / [5] / [5, 26, 27] / [26, 28]
Hydrochlorothiazide / CL (ml/min/kg) / 11.8 ± 1.5 (13) / 11.8 ± 1.9 (16) / 3 / 5.9
Vss (L/kg) / 2.8 ± 0.8 (27) / 3.6 ± 0.8 (22)
T1/2 (h) / 4.5 ± 0.6 (14) / 5.9 ± 1.2 (20) / 8 / 7.4 / 8.8
F (%) / 58.8 ± 6.9 (12) / 65.5 ± 4.1 (6) / 61.6 / 30.7
Source / [3, 29, 30] / [31] / [32] / [33]
Midazolam / CL (ml/min/kg) / 29.2 ± 6.1 (21) / 15.4 ± 1.9 (13) / 5.2 / 12.9 / 35.2 / 79.5
Vss (L/kg) / 1.5 ± 0.6 (37) / 1 ± 0.2 (19) / 0.8 / 1.4 / 1.1 / 1.4
T1/2 (h) / 0.9 ± 0.5 (60) / 1.0 ± 0.2 (24) / 3.1 / 2 / 0.6 / 0.6
F (%) / 19.4 / 4.6 / 34 / 1 / 15.1 / 14.9
source / [2, 34] / [4, 35] / [36] / [3, 37, 38]
Theophylline / CL (ml/min/kg) / 0.9 ± 0.3 (29) / 1.4 ± 0.1 (9) / 0.8 / 2.4 / 1.97 / 1.9
Vss (L/kg) / 1.0 ± 0.1 (8) / 0.9 ± 0 (0) / 0.4 / 0.6 / 0.8 / 0.4
T1/2 (h) / 15.6 ± 3.1 (20) / 8.2 ± 0.5 (7) / 7.1 / 1.7 / 4.8 / 2.7
F (%) / 97.3 / 127.3 / 96 / 86.3 / 91 / 86.2
Sources / In house / In house / [39, 40] / [41, 42] / [43-45] / [46, 47]

Table 1: pharmacokinetic parameters (presented as mean ± standard deviation and (coefficient ofvariation)) obtained by non-compartmental analysis from the plasma concentration-time profiles of seven reference compounds dosed intravenously and orally in male and female Göttingen minipig. Comparison to mean values of the corresponding parameters in human, monkey, dog and rat as reported in the literature.

Male Minipig / Female minipig
Compound / Parameter / Mean / SD / CV (%) / Mean / SD / CV (%)
Antipyrine / AUC / Dose (ng. h/mL) / (mg/kg) / 4886.8 / 3530.4 / 72 / 1415.8 / 535.1 / 38
Cmax / Dose (ng/ml) / 550.7 / 137.1 / 25 / 371.5 / 205.0 / 55
Tmax (h) / 2.4 / 1.3 / 53 / 0.7 / 0.4 / 55
t1/2(h) / 3.7 / 1.9 / 52 / 3.0 / 2.2 / 71
Atenolol / AUC / Dose(ng. h/mL) / (mg/kg) / 662.6 / 58.9 / 9 / 951.3 / 53.4 / 6
Cmax / Dose (ng/ml) / 94.4 / 12.1 / 13 / 200.2 / 78.9 / 39
Tmax (h) / 1.5 / 0.6 / 38 / 1.8 / 0.5 / 29
t1/2(h) / 5.2 / 0.7 / 14 / 4.6 / 0.5 / 10
Cimetidine / AUC / Dose (ng. h/mL) / (mg/kg) / 170.5 / 70.6 / 41 / 131.2 / 5.5 / 4
Cmax / Dose (ng/ml) / 30.3 / 16.0 / 53 / 34.0 / 17.0 / 50
Tmax (h) / 2.0 / 1.3 / 68 / 1.5 / 0.6 / 38
t1/2(h) / 9.3 / 7.3 / 79 / 7.7 / 2.0 / 25
Diazepam / AUC / Dose (ng. h/mL) / (mg/kg) / 2406.9 / 206.5 / 9 / 2523.3 / 1055.5 / 42
Cmax / Dose (ng/ml) / 324.7 / 163.2 / 50 / 316.1 / 195.3 / 62
Tmax (h) / 0.7 / 0.3 / 43 / 1.1 / 0.6 / 56
t1/2(h) / 12.0 / 3.5 / 29 / 13.3 / 3.5 / 26
Hydrochlorothiazide / AUC / Dose (ng. h/mL) / (mg/kg) / 838.4 / 97.9 / 12 / 938.4 / 201.9 / 22
Cmax / Dose (ng/ml) / 83.5 / 25.1 / 30 / 91.7 / 24.4 / 27
Tmax (h) / 1.4 / 0.8 / 54 / 3.3 / 1.5 / 46
t1/2(h) / 6.1 / 1.8 / 29 / 6.0 / 1.3 / 22
Midazolam / AUC / Dose (ng. h/mL) / (mg/kg) / 92.2 / 40.6 / 44.0 / 45.8 / 32.4 / 71
Cmax / Dose (ng/ml) / 10.0 / 4.8 / 47.4 / 14.5 / 12.4 / 86
Tmax (h) / 1.5 / 0.6 / 41.2 / 2.1 / 1.6 / 76
t1/2(h) / 10.0 / 6.2 / 61.8 / 4.3 / 2.7 / 63
Theophylline / AUC / Dose (ng. h/mL) / (mg/kg) / 20112.3 / 4669.0 / 23 / 14964.7 / 2608.9 / 17
Cmax / Dose (ng/ml) / 1185.4 / 119.1 / 10 / 855.5 / 169.2 / 20
Tmax (h) / 1.8 / 0.5 / 29 / 3.5 / 1.0 / 29
t1/2(h) / 11.6 / 2.7 / 23 / 11.6 / 2.4 / 20

Table 2: pharmacokinetic parameters (mean, standard deviation, and coefficient of variation) obtained by non-compartmental analysis from the plasma concentration-time profiles of seven reference compounds dosed orally in male and female Göttingen minipigs.

1.Danhof, M., et al., Studies of the different metabolic pathways of antipyrine in man. Oral versus i.v. administration and the influence of urinary collection time. Eur J Clin Pharmacol, 1982. 21(5): p. 433-41.

2.Pentikainen, P.J., et al., Pharmacokinetics of midazolam following intravenous and oral administration in patients with c honic liver disease and in healthy subjects. J Clin Pharmacol, 1989. 29(3): p. 272-7.

3.Musther, H., et al., Animal versus human oral drug bioavailability: do they correlate? Eur J Pharm Sci, 2014. 57: p. 280-91.

4.Takahashi, M., et al., The species differences of intestinal drug absorption and first-pass metabolism between cynomolgus monkeys and humans. J Pharm Sci, 2009. 98(11): p. 4343-53.

5.Koyanagi, T., et al., Age-related pharmacokinetic changes of acetaminophen, antipyrine, diazepam, diphenhydramine, and ofloxacin in male cynomolgus monkeys and beagle dogs. Xenobiotica, 2014.

6.Doyle, E. and L.F. Chasseaud, Comparative pharmacokinetics of antipyrine (phenazone) in the baboon, cynomolgus monkey and rhesus monkey. Toxicology, 1981. 19(2): p. 159-68.

7.KuKanich, B., et al., Comparative disposition of pharmacologic markers for cytoc home P-450 mediated metabolism, glomerular filtration rate, and extracellular and total body fluid volume of Greyhound and Beagle dogs. J Vet Pharmacol Ther, 2007. 30(4): p. 314-9.

8.Abramson, F.P., The effect of induction with phenobarbital on the kinetics and bioavailability of antipyrine in the dog. Eur J Drug Metab Pharmacokinet, 1988. 13(2): p. 123-7.

9.Witkamp, R.F., et al., Species- and sex-related differences in the plasma clearance and metabolite formation of antipyrine. A comparative study in four animal species: cattle, goat, rat and rabbit. Xenobiotica, 1991. 21(11): p. 1483-92.

10.Belpaire, F.M., et al., Effect of aging on the pharmcokinetics of atenolol, metoprolol and propranolol in the rat. J Pharmacol Exp Ther, 1990. 254(1): p. 116-22.

11.Takahashi, M., et al., Characterization of gastrointestinal drug absorption in cynomolgus monkeys. Mol Pharm, 2008. 5(2): p. 340-8.

12.Kirch, W., et al., Pharmacokinetics of atenolol in relation to renal function. Eur J Clin Pharmacol, 1981. 19(1): p. 65-71.

13.Reeves, P.R., et al., Metabolism of atenolol in man. Xenobiotica, 1978. 8(5): p. 313-20.

14.Mason, W.D., et al., Kinetics and absolute bioavailability of atenolol. Clin Pharmacol Ther, 1979. 25(4): p. 408-15.

15.Kvetina, J., et al., Experimental Goettingen minipig and beagle dog as two species used in bioequivalence studies for clinical pharmacology (5-aminosalicylic acid and atenolol as model drugs). Gen Physiol Biophys, 1999. 18 Spec No: p. 80-5.

16.Reeves, P.R., et al., Disposition and metabolism of atenolol in animals. Xenobiotica, 1978. 8(5): p. 305-11.

17.McAinsh, J. and B.F. Holmes, Pharmacokinetic studies with atenolol in the dog. Biopharm Drug Dispos, 1983. 4(3): p. 249-61.

18.Lombardo, F., et al., Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 2: clearance. J Clin Pharmacol, 2013. 53(2): p. 178-91.

19.Obach, R.S., F. Lombardo, and N.J. Waters, Trend analysis of a database of intravenous pharmacokinetic parameters in humans for 670 drug compounds. Drug metabolism and disposition: the biological fate of chemicals, 2008. 36: p. 1385-1405.

20.Somogyi, A. and R. Gugler, Clinical pharmacokinetics of cimetidine. Clin Pharmacokinet, 1983. 8(6): p. 463-95.

21.Tahara, H., et al., Is the monkey an appropriate animal model to examine drug-drug interactions involving renal clearance? Effect of probenecid on the renal elimination of H2 receptor antagonists. J Pharmacol Exp Ther, 2006. 316(3): p. 1187-94.

22.Le Traon, G., S. Burgaud, and L.J. Horspool, Pharmacokinetics of cimetidine in dogs after oral administration of cimetidine tablets. J Vet Pharmacol Ther, 2009. 32(3): p. 213-8.

23.Kaka, J.S., M.O. Tanira, and K.I. al-Khamis, Disposition kinetics of cimetidine and ranitidine in endotoxin pretreated rats. Drug Chem Toxicol, 1989. 12(1): p. 49-59.

24.Andersson, T., et al., Effect of omeprazole and cimetidine on plasma diazepam levels. Eur J Clin Pharmacol, 1990. 39(1): p. 51-4.

25.Divoll, M., et al., Absolute bioavailability of oral and intramuscular diazepam: effects of age and sex. Anesth Analg, 1983. 62(1): p. 1-8.

26.Klotz, U., K.H. Antonin, and P.R. Bieck, Pharmacokinetics and plasma binding of diazepam in man, dog, rabbit, guinea pig and rat. J Pharmacol Exp Ther, 1976. 199(1): p. 67-73.

27.Loscher, W. and H.H. Frey, Pharmacokinetics of diazepam in the dog. Arch Int Pharmacodyn Ther, 1981. 254(2): p. 180-95.

28.Tsang, C.F. and G.R. Wilkinson, Diazepam disposition in mature and aged rabbits and rats. Drug Metab Dispos, 1982. 10(4): p. 413-6.

29.Patel, R.B., et al., Bioavailability of hydrochlorothiazide from tablets and suspensions. J Pharm Sci, 1984. 73(3): p. 359-61.

30.Barbhaiya, R.H., et al., Pharmacokinetics of hydrochlorothiazide in fasted and nonfasted subjects: a comparison of plasma level and urinary excretion methods. J Pharm Sci, 1982. 71(2): p. 245-8.

31.Akabane, T., et al., A comparison of pharmacokinetics between humans and monkeys. Drug Metab Dispos, 2010. 38(2): p. 308-16.

32.Huang, X.H., et al., Pharmacokinetic and pharmacodynamic interaction between irbesartan and hydrochlorothiazide in renal hypertensive dogs. J Cardiovasc Pharmacol, 2005. 46(6): p. 863-9.

33.Asdaq, S.M. and M.N. Inamdar, The potential for interaction of hydrochlorothiazide with garlic in rats. Chem Biol Interact, 2009. 181(3): p. 472-9.

34.Thummel, K.E., et al., Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clin Pharmacol Ther, 1996. 59(5): p. 491-502.

35.Sakuda, S., T. Akabane, and T. Teramura, Marked species differences in the bioavailability of midazolam in cynomolgus monkeys and humans. Xenobiotica, 2006. 36(4): p. 331-40.

36.Kuroha, M., et al., Effect of multiple dosing of ketoconazole on pharmacokinetics of midazolam, a cytoc home P-450 3A substrate in beagle dogs. Drug Metab Dispos, 2002. 30(1): p. 63-8.

37.Hovinga, S., et al., Pharmacokinetic-EEG effect relationship of midazolam in aging BN/BiRij rats. Br J Pharmacol, 1992. 107(1): p. 171-7.

38.Mandema, J.W., et al., Pharmacokinetic-pharmacodynamic modeling of the electroencephalographic effects of benzodiazepines. Correlation with receptor binding and anticonvulsant activity. J Pharmacol Exp Ther, 1991. 257(1): p. 472-8.

39.Hendeles, L., M. Weinberger, and L. Bighley, Absolute bioavailability of oral theophylline. Am J Hosp Pharm, 1977. 34(5): p. 525-7.

40.Kaumeier, H.S., et al., A cross-over study of oral and intravenous administration of theophylline in male volunteers. Absolute bioavailability of theophylline tablets. Arzneimittelforschung, 1984. 34(1): p. 92-5.

41.Nishimura, T., et al., Species difference in intestinal absorption mechanism of etoposide and digoxin between cynomolgus monkey and rat. Pharm Res, 2008. 25(11): p. 2467-76.

42.Wong, H., et al., The chimpanzee (Pan troglodytes) as a pharmacokinetic model for selection of drug candidates: model characterization and application. Drug Metab Dispos, 2004. 32(12): p. 1359-69.

43.Shiu, G.K., et al., The effect of food on the absorption of controlled-release theophylline in mini-swine. Pharm Res, 1988. 5(1): p. 48-52.

44.McKiernan, B.C., et al., Pharmacokinetic studies of theophylline in dogs. J Vet Pharmacol Ther, 1981. 4(2): p. 103-10.

45.Kawai, H., et al., Pharmacokinetic study of theophylline in dogs after intravenous administration with and without ethylenediamine. Methods Find Exp Clin Pharmacol, 2000. 22(3): p. 179-84.

46.Nam, B.H., et al., Effect of hepatic cirrhosis on the pharmacokinetics of theophylline in rats. Arch Pharm Res, 1997. 20(4): p. 318-23.

47.Matsunaga, N., et al., Simultaneous assessment of the in vivo amount of CYP1A2 and CYP3A2 by the PKCYP-test using theophylline in rats. Drug Metab Pharmacokinet, 2002. 17(3): p. 190-8.

Supplementary material II.In vitro – in vivo correlation for hepatic intrinsic clearance in Göttingen Minipig hepatocytes.

Computation of the hepatic intrinsic clearance from in vivo data.

Calculation of the in vitro – in vivo correlation should ideally take into account the protein binding in in plasma and in the in vitro incubations, the blood to plasma partitioning and the liver blood flow. However in practicein our laboratory these binding measurements are not typically available when investigating potential animal models for toxicity testing. Therefore, a rough IVIVC is performed by scaling the intrinsic clearance measured in hepatocytes Clint using the hepatocellularity (HPGL) the liver weight (LW) and the body weight (BW) (eq. 1). To evaluate this method for prediction of in vivo hepatic metabolism the obtained Clint,h,pred can be compared to the intrinsic hepatic clearance Clint,h,mes estimated from the plasma clearance measured in vivo as expressed in equation 2. This correlation assumes that the binding in blood and in vitro are equivalent and is the approach chosen in this paper with a view to our future use in early clearance predictions.

For completeness we have also estimated in vivo intrinsic hepatic clearances taking into account all the measured plasma and blood binding as in equation 3 [1]. This estimated in vivo intrinsic hepatic clearance is then compared to the value scaled from in vitro measurements also accounting for the measured in vitro bindingin the incubation medium (fu_inc) as in equation 4

/ (1)
/ (2)
/ (3)
/ (4)

Table 1: Hepatic clearances measured of reference compounds measured from intravenous dosing in male and female Göttingen minipigs, and calculation of the corresponding intrinsic hepatic clearance using two different equations.

Measured hepatic clearances in vivo (ml/min/kg) / Intrinsic hepatic clearance eq. 2 (ml/min/kg) / Intrinsic hepatic clearance eq. 3 (ml/min/kg)
Mean / SD / Mean / SD / Mean / SD
Antipyrine / 3.2 / 2.1 / 3.5 / 2.2 / 3.6 / 2.3
Atenolol / 5.2 / 2.7 / 6.1 / 3.0 / 6.1 / 3.0
Cimetidine / 29.8 / 8.2 / 128.1 / 10.4 / 181.2 / 11.3
Diazepam / 9.6 / 1.1 / 12.8 / 1.1 / 441.0 / 30.8
Midazolam / 22.3 / 8.6 / 52.3 / 11.0 / 5367.7 / 194.2
Theophylline / 1.0 / 0.3 / 1.0 / 0.3 / 1.2 / 0.4
Hydrochlorothiazide / 7.2 / 1.6 / 8.9 / 1.7 / 18.1 / 3.3

Table 2: In vitro measurements of intrinsic clearance of reference compounds in human and minipig hepatocytes

Compound / Clint in human
(µl/min/Mcells) / Clint in minipig
(µl/min/Mcells) / Clint,h,pred computed using eq. 1 / Clint,h,pred computed using eq. 4
Antipyrine / 0.5 / 0.7 ± 2.4 / 1.4 ± 4.8 / 1.8± 6.1
Atenolol / 0.7 / 1.6 ± 1.8 / 3.2 ± 3.6 / 3.2± 3.6
Cimetidine / 0.95 ± 0.55 / 5.65 ± 0.05 / 11.3 ± 0.1 / 13.2± 0.1
Diazepam / 1.3 ± 0.2 / 4.05 ± 0.75 / 8.1 ± 1.5 / 17.3± 3.2
Midazolam / 19.8 ± 5.2 / 10.8 ± 2.2 / 21.6 ± 4.4 / 135.2± 27.5
Theophylline / 0.75 ± 0.15 / 0.95 ± 0.95 / 1.9 ± 1.9 / 2.6± 2.6
HCTZ / 1.2 ± 1.7 / 2.4 ± 3.4 / 3.0± 4.3

Figure 1:intrinsic hepatic clearances computed using eq. 3 from in vivo measurements of hepatic clearances, versus intrinsic hepatic clearances predicted from measurements in hepatocytes using eq. 4, for 7 reference compounds in the Göttingen minipig. ATP: antipyrine, ATE: atenolol, CIM: cimetidine, DZP: diazepam, HCTZ: hydrochlorothiazide, MDZ: midazolam, THP: theophylline. The straight line is the line of unity while the dashed line and dotted line correspond to 2 and 3 fold differences respectively.

1.Yang, J., et al., Misuse of the well-stirred model of hepatic drug clearance. Drug Metab Dispos, 2007. 35(3): p. 501-2.

Supplementary material III Determination of the minipig hepatocellularity

Table 1: HPGL derivation from DNA quantification

DNA content of liver
mg/g liver / DNA content of hepatocytes
µg/106 cells / HPGL
106 cells/g liver
Literature / Measured / Literature / Measured / Literature / Measured
Rat / 1.09 (1)
2.7 ± 0.3 (2)
2.5 ± 0.07 (3)
2.51 ± 0.3 (4)
4.7 (3) / 3.4 ± 0.3 / 19.3 (4)
23.7 ± 2.8 (2) / 14.5 ± 3 / 85 (2)
128 ± 7 (4) / 130± 19
Minipig / 2.9 ± 0.6 / 17.5 (5) / 17.1 ± 0.8 / 119 ± 15

Table 2: HPGL derivation from protein quantification

protein content of liver
mg/g liver / protein content of hepatocytes
mg/106 cells / HPGL
106 cells/g liver
Literature / Measured / Literature / Measured / Literature / Measured
Rat / 112 ± 10 (6)
163.5 ± 8 (2) / 139.8 ± 1.8 / 0.99 ± 0.21 (6)
1.39 ± 0.16 (7)
1.5 ± 0.2 (2) / 1.3 ± 0.12 / 109 (2)(4)
117 (6)
120 (8) / 110 ± 6
Minipig / 127.9 ± 13 / 0.34 (5) / 0.97± 0.1 / 129 ± 13

References

1.Lowe CU, Rand RN. The effect of cortisone on DNA content of rat hepatocytes. J Biophys Biochem Cytol. 1956;2(6):711-24.

2.Carlile DJ, Zomorodi K, Houston JB. Scaling factors to relate drug metabolic clearance in hepatic microsomes, isolated hepatocytes, and the intact liver: studies with induced livers involving diazepam. Drug Metab Dispos. 1997;25(8):903-11.

3.Fiszer-Szafarz B, Szafarz D, Guevara de Murillo A. A general, fast, and sensitive micromethod for DNA determination application to rat and mouse liver, rat hepatoma, human leukocytes, chicken fibroblasts, and yeast cells. Anal Biochem. 1981;110(1):165-70.

4.Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29-83.

5.te Velde AA, Ladiges NC, Flendrig LM, Chamuleau RA. Functional activity of isolated pig hepatocytes attached to different extracellular matrix substrates. Implication for application of pig hepatocytes in a bioartificial liver. J Hepatol. 1995;23(2):184-92.

6.Sohlenius-Sternbeck AK. Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements. Toxicol In Vitro. 2006;20(8):1582-6.

7.Bayliss MK, Bell JA, Jenner WN, Park GR, Wilson K. Utility of hepatocytes to model species differences in the metabolism of loxtidine and to predict pharmacokinetic parameters in rat, dog and man. Xenobiotica. 1999;29(3):253-68.

8.Zahlten RN, Stratman FW. The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique. Arch Biochem Biophys. 1974;163(2):600-8.

Supplementary material IV.Metabolism of Midazolam and Diazepam

Proposed metabolic pathway for the major metabolites of midazolam detected following incubation in human and minipig hepatocytes at 10µM, and in minipig plasma following 1 mg/kg iv dose.

Relative abundance of Midazolam and its detected metabolites in hepatocytes and minipig plasma

% Drug Related Materiala
Hepatocytes / Minipig Plasma
Component / RT / Human / Minipig / 0.5h / 2h
Midazolam / 5.81 / 49 / 63 / 48 / 29
4-Hydroxymidazolam / 5.33 / 6.6 / 8.6 / 2.2 / 1.8
1-Hydroxymidazolam / 5.65 / 31 / 6.1 / - / -
HydroxyMidazolam / 6.17 / - / 1.3 / - / -
1,4-Dihydroxymidazolam / 5.17 / 1.8 / 0.6 / 0.7 / -
Midazolam N-glucuronide / 5.25 / 3.2 / 1.4 / - / -
OH-Glucuronide / 4.65 / - / 0.8 / 2.0 / 4.4
4-Hydroxymidazolam glucuronide / 4.78 / - / 9.2 / 31 / 47
1- Hydroxymidazolam glucuronide / 5.03 / 8.4 / 9.0 / 9.2 / 9.3
1,4-Dihydroxymidazolam glucuronide / 5.07 / - / - / 6.1 / 8.6

aThe peak area of metabolites was estimated by comparison of peak areas of MS ion intensities. This semi-quantitative approach is based on several simplified assumptions (equimolar response of different analytes, no matrix effect, etc.) and consequently, it cannot be excluded that in some cases the semi-quantitative data will be different to data generated by validated bioanalytical methods using individual reference compounds or by structure independent radioactivity measurements.

Proposed metabolic pathway for the metabolites of diazapam detected following incubation in human and minipig hepatocytes at 10µM, and in minipig plasma following 1 mg/kg iv dose

Relative abundance of diazepam and its detected metabolites in hepatocytes and minipig plasma

% Drug Related Materiala
Hepatocytes / Minipig Plasma
Component / RT / Human / Minipig / 0.5h / 2h
Diazepam / 8.23 / 92 / 76 / 81 / 63
N-Desmethyldiazepam / 7.10 / 4.3 / 13 / 2.6 / 6.7
3-Hydroxydiazepam / 7.82 / 3.3 / 4.7 / 3.1 / 3.8
Hydroxydiazepam / 6.33 / - / 1.4 / - / 3.0
Hydroxydiazepam glucuronide / 4.15 / - / 0.4 / 1.8 / 1.5
Hydroxydiazepam glucuronide / 6.00 / - / 0.5 / 5.9 / 9.6
Hydroxydiazepam glucuronide / 6.33 / trace / 4.6 / 5.8 / 12