Evaluation of the human prediction of clearance from hepatocyte and microsome intrinsic clearance for 52 drug compounds.

We compare three different approaches to scale clearance (CL) from human hepatocyte and microsome CL(int) (intrinsic CL) for 52 drug compounds. By using the well-stirred model with protein binding included only 11% and 30% of the compounds were predicted within 2-fold and the average absolute fold errors (AAFE) for the predictions were 5.9 and 4.1 for hepatocytes and microsomes, respectively. When predictions were performed without protein binding, 59% of the compounds were predicted within 2-fold using either hepatocytes or microsomes and the AAFE was 2.2 and 2.3, respectively. For hepatocytes and microsomes there were significant correlations (P = 8.7 x 10(-13), R(2) = 0.72; P = 2.8 x 10(-9), R(2) = 0.61) between predicted CL(int in vivo) (obtained from in vitro CL(int)) and measured CL(int in vivo) (obtained using the well-stirred model). When CL was calculated from the regression, 76% and 70% of the compounds were predicted within 2-fold and the AAFE was 1.6 and 1.8 for hepatocytes and microsomes, respectively. We demonstrate that microsomes and hepatocytes are in many cases comparable when scaling of CL is performed from regression. By using the hepatocyte regression, CL for 82% of the compounds in an independent test set (n = 11) were predicted within 2-fold (AAFE 1.4). We suggest that a regression line that adjusts for systematic under-predictions should be the first-hand choice for scaling of CL.

Comparison of intrinsic metabolic clearance in fresh and cryopreserved human hepatocytes.

1. We compared the intrinsic clearance (CL(int)) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors. 2. CL(int) values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9 +/- 3.4, 18 +/- 7.2, 5.1 +/- 4.9, 6.3 +/- 3.3, 9.8 +/- 5.8 and 22 +/- 14 microl min(-1)/10(6) cells, respectively, and they correlated well with corresponding CL(int) values using cryopreserved hepatocytes from 25 different donors. 3. CL(int) values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL(int) in fresh and cryopreserved hepatocytes for each of the three livers (p < 0.002) and the geometric mean of the ratio of fresh to cryopreserved CL(int) values was 1.03. 4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

The sensitivity of hepatic CYP2C gene expression to baseline growth hormone (GH) bioactivity in dwarf rats: effects of GH-binding protein in vivo.

Hepatic mRNA transcripts for the steroid-metabolizing enzymes cytochrome P4502C11 (male specific) and P4502C12 (female specific) differ in abundance by 10- to 20-fold in male and female rats and are regulated by their different patterns of GH secretion. This sex difference is also found in dwarf rats with low GH secretion, implying that these transcripts may be very sensitive to low level GH exposure. This has now been characterized in normal and dwarf rats. Continuous i.v. infusion of recombinant human (h) GH (0, 3, 12, and 48 micrograms/day) in both dwarf and normal male rats caused a dose-dependent decrease in P4502C11 and an increase in P4502C12, so that the 2C11/2C12 ratio fell from 17.9 +/- 1.3 to 1.5 +/- 1.0 in normal males and from 6.5 +/- 0.9 to 0.4 +/- 0.3 in dwarf males (0 vs. 48 micrograms hGH/day); over this dose range of hGH, body weight gain, total hepatic insulin-like growth factor-I mRNA levels, and plasma GHBP levels were largely unaffected. These effects of hGH were pattern dependent. The 2C11/2C12 ratio in dwarf males was feminized (from 11.9 +/- 1.3 to 0.08 +/- 0.03) by continuous infusion of hGH (36 micrograms/day), whereas a pulsatile infusion (3-min pulses every 3 h) of the same daily hGH dose was much less effective. Neither continuous nor pulsatile hGH affected P4502C11 or P4502C12 transcripts in dwarf females, although pulsatile hGH infusion caused a significant weight gain. To test whether baseline GH levels could be modified by circulating GH-binding protein (GHBP), hGH infusions were given with and without recombinant hGHBP in different patterns. Pulsatile infusions of recombinant hGHBP (42 micrograms/day, i.v.) did not prevent the feminizing effect of continuously infused hGH (36 micrograms/day, sc) in dwarf males (2C11/2C12 ratios were 0.08 +/- 0.01 and 0.09 +/- 0.01 for hGH vs. hGH plus hGHBP, respectively). This suggested that intermittent complex formation with GHBP did not prevent continuous access of hGH to the hepatic GH receptors. Furthermore, pulses of hGH complexed with GHBP significantly reduced the 2C11/2C12 ratio in dwarf males (from 21.5 +/- 3.9 with pulsatile hGH alone to 9.2 +/- 2.5 with pulses of hGH plus hGHBP), indicating that GHBP prolongs the exposure to hGH. Thus, 2C11/2C12 expression is very sensitive to basal GH levels in dwarf rats, and GHBP can alter hepatic gene expression by modifying the pattern of GH exposure.

Rates of metabolism of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil by fresh and cryopreserved rat liver slices, and some comparisons with microsomes.

In the present study we have investigated the disappearance of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil from the medium in which fresh and cryopreserved rat liver slices were incubated. These compounds are all substrates of major isoforms of cytochrome P450 expressed in the liver. The metabolism of five of these compounds in microsomes from rat liver was also examined. Determinations of the concentrations of the compounds were performed employing LC/MS. Intrinsic clearance values (CL(ints)) were calculated on the basis of the concentration-vs.-time curves. No significant differences in the CL(int) values obtained with fresh and cryopreserved rat liver slices were observed for any of the compounds. The highest CL(int) value estimated with liver slices was observed for testosterone and the lowest values were with chlorzoxazone and 7-ethoxycoumarin. The total CL(int) values for 7-ethoxycoumarin and imipramine, calculated using scaling factors, were similar for liver slices and microsomes. In the case of testosterone, this total CL(int) was approximately 3.7-fold lower, whereas for dextromethorphan and quinidine it was 2.5- and 8.5-fold higher, respectively, with liver slices than with microromes. In conclusion, the rate of metabolism of the seven compounds tested with rat liver slices was not affected by cryopreservation. This finding adds further support to the general conclusion that the major activities involved in drug metabolism are not affected by cryopreservation of rat liver slices.

High conservation of both phase I and II drug-metabolizing activities in cryopreserved rat liver slices.

1. Xenobiotic-metabolizing enzymes, including both cytochrome P450 and phase II-conjugating systems, have been characterized in rat liver slices cryopreserved in 12 or 18% dimethylsulphoxide (DMSO). 2. Several cytochrome P450 isoforms in rat liver slices metabolized testosterone to a variety of hydroxylated products. The rates of formation of these same products were well maintained during cryopreservation of the slices in both 12 or 18% DMSO. 3. After cryopreservation of rat liver slices in 18% DMSO, the rates of metabolism of ropivacaine to 3-hydroxyropivacaine, 4-hydroxyropivacaine and PPX (all catalysed by different cytochrome P450 isoforms) were approximately 94, 79 and 82% respectively of the corresponding rates observed with fresh slices. 4. The rates of conjugation of 7-hydroxycoumarin and 1-naphthol by rat liver slices were significantly decreased after cryopreservation in 12% DMSO, but they were maintained when the concentration of this cryopreservant was increased to 18% 5. After cryopreservation in 12% DMSO, the mitochondrial reduction of the tetrazolium salt MTT by rat liver slices was significantly lowered. In contrast, slices cryopreserved in 18% DMSO demonstrated no significant decrease in their capacity to reduce MTT. 6. Thus, in agreement with previous studies, it was found that cytochrome P450-dependent activities are retained after cryopreservation of liver slices. Although phase II-conjugating enzyme activities are more sensitive to cryopreservation, it was shown that increasing the concentration of DMSO present during cryopreservation could circumvent the problem. This modification improves the usefulness of cryopreserved rat liver slices as a tool in drug metabolism studies.

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations.

A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.

Characterization of testosterone metabolism and 7-hydroxycoumarin conjugation by rat and human liver slices after storage in liquid nitrogen for 1 h up to 6 months.

1. Slices of human and rat liver were cryopreserved in 18% dimethyl sulphoxide (DMSO) and subsequently stored in liquid nitrogen for periods up to as long as 6 months. After thawing, the metabolism of testosterone to hydroxylated products and conjugation of 7-hydroxycoumarin were investigated. 2. Rat liver slices stored in liquid nitrogen for 6 months exhibited rates of formation of 7alpha-, 6beta- 16alpha- and 2alpha-hydroxytestosterone, and of androstenedione that did not differ significantly from those observed with fresh slices. 3. No formation of 2alpha-hydroxytestosterone was detected with slices of human liver. However, in contrast with the rat, human slices produced 2beta-hydroxytestosterone. The rates of formation of 7alpha-, 6beta-, 16alpha- and 2beta-hydroxytestosterone and of androstenedione by human liver slices after 6 months of storage in liquid nitrogen were 82, 71, 236, 66 and 92%, respectively, of the corresponding rates by fresh slices. 4. The rates of sulphation and glucuronidation of 7-hydroxycoumarin by slices from rat liver were 97 and 119%, respectively, of the corresponding fresh values after 6 months of storage in liquid nitrogen. 5. 7-Hydroxycoumarin glucuronidation by human liver slices was 53% of the corresponding fresh values after 6 months of storage. However, human slices showed little or no capacity to conjugate 7-hydroxycoumarin with sulphate. 6. It was demonstrated that slices of both human and rat liver can be cryopreserved and stored in liquid nitrogen for at least 6 months without major changes in their rates of metabolism of testosterone to its hydroxylated products and of 7-hydroxycoumarin conjugation. These findings further emphasize that cryopreservation of liver slices can be an effective tool in the use of biological material of limited availability.