AbstractThe induction of drug metabolising enzymes, particularly cytochromes P450 (CYP), can have significant pharmacological and toxicological consequences. The use of monolayer cultures of human hepatocytes is established as the definitive model for the prediction of CYP induction in vitro, with earlier difficulties in the procurement of fresh human liver largely ameliorated through the commercial availability of plateable cryopreserved cells. Covance has been performing in vitro enzyme induction studies for many years and, as such, a significant amount of data on the effects of positive control inducers in hepatocyte cultures has been generated. In this presentation, noteworthy trends in Covance historical data generated using freshly isolated and commercially sourced cryopreserved hepatocytes are discussed, including the effect of donor demographics and the impact of basal enzyme activity on the extent of induction elicited. In addition, recent FDA/EMA regulatory guidance has advocated the use of CYP mRNA expression as the preferred endpoint for the quantification of induction1, 2. Through the use of RT-PCR and Taqman® methodology, the effects of prototypical inducers on CYP1A2, CYP2B6 and CYP3A4 mRNA expression in hepatocytes from multiple human donors are presented, together with the corresponding enzyme activity data from the same cells. The extent of induction elicited using both endpoints are directly compared and discussed in relation to regulatory guidance.

Materials and MethodsHuman HepatocytesFresh human hepatocytes were supplied by commercial vendors or isolated at Covance from ethically-sourced liver samples using a two-step collagenase perfusion technique. All cells were isolated from surgical waste tissue from patients undergoing hepatic resection. No multi-organ donor or cadaveric tissue was used and the time from resection to plating was as short as practically possible. Cryopreserved plateable human hepatocytes were purchased from various commercial vendors.

IncubationsAll procedures generally followed regulatory/industry recommendations. Hepatocytes were suspended in an appropriate supplemented incubation medium and cell viability was assessed by trypan blue exclusion (only those preparations with viabilities of >70% were used). The cells were seeded onto 24 or 48-well collagen coated plates and, after a 2-4 hour attachment period with a subsequent switch to serum-free medium and application of a Matrigel overlay, the cells were incubated for a further ca. 36 hours. The hepatocytes were then treated with omeprazole (CYP1A2 inducer, 30-50 µM), phenobarbital (CYP2B6 inducer, 1 mM) or rifampicin (CYP2C/CYP3A4 inducer, 20-50 µM) for a further 48-72 hours prior to assessment of enzyme induction as follows:

CYP enzyme activity: the incubation medium was replaced with fresh medium containing phenacetin (CYP1A2 probe, 100-150 µM), bupropion (CYP2B6 probe, 500 µM) or midazolam/testosterone (CYP3A4 probes, 50-150 µM). After further incubation for 1 hour, the cells were scraped and metabolite formation quantified using validated UPLC-MS/MS methods.

CYP mRNA expression: after cell washing and lysis, RNA was collected using a Qiagen RNeasy® Mini Kit. RNA concentrations were quantified and purity determined by UV spectroscopy. Relative concentrations of mRNA for CYP1A2, CYP2B6, and CYP3A4 were determined by Real Time PCR using an Applied Biosciences 7900HT instrument, TaqMan® RNA-to-CT 1-Step kits (Applied Biosciences), and isoenzyme-specific probes. CYP mRNA was normalized to the mRNA of the endogenous control (18S rRNA) in each sample.

ResultsEffect of Prototypical Inducers on Enzyme ActivitiesThe effects of the prototypical inducers omeprazole, phenobarbital and rifampicin on CYP-mediated enzyme activities were investigated in freshly isolated hepatocytes from up to 135 donors and in 14 batches of plateable cryopreserved cells, the findings of which are summarised in Table 1. The effects of inducer treatment on CYP1A2, CYP2B6, CYP2C19 and CYP3A4-mediated activities were generally consistent with published data. Enzyme activities mediated by CYP2C9 were not markedly increased, although it is apparent from the findings of other workers that this particular isoform does not significantly respond to inducer treatment in vitro and these results are therefore not unusual3,4.

Table 1. Summary of Historical Data

Evaluation of Cytochrome P450 Enzyme Induction In Vitro: Trend Analysis and Correlation of Enzyme Activity Data with mRNA ExpressionDavid Wilkinson1, Sarah Andrews1, Gang Luo2, Heidi Thorson2, Anthony Glazier1 and John Kendrick11Covance Laboratories Ltd., Harrogate, UK; 2Covance Laboratories Inc., Madison, WI, USA

Presented at the 13th European ISSX Meeting 2015

Effect of Donor Gender and Age on CYP InductionThe effects of donor gender on CYP induction in freshly isolated hepatocytes are summarised in Figure 1. There were no notable differences in the maximum extent of induction of the CYP enzymes under investigation using hepatocytes from either male or female donors. The influence of donor age on CYP induction is summarised in Figure 2. These data were arranged into five groups: <40 years, 41-50 years, 51-60 years, 61-70 years and >70 years. Although the number of data points in some of these arbitrarily assigned groups were low, these data nevertheless indicated that donor age had no significant impact on the extent of induction elicited.

Figure 1. Effect of donor gender on CYP induction in freshly isolated hepatocytes.

Figure 2. Effect of donor age on CYP induction in freshly isolated hepatocytes.

Effect of Cell Viability on CYP InductionFigure 3 represents the relationship between initial cell viability, as measured by trypan blue exclusion, and the extent of CYP induction following exposure of freshly isolated hepatocytes to inducers. Although no statistically significant effects were evident, in most cases the induction effect did not appear to correlate to cell viability, i.e. batches of cells which had the highest uptake of trypan blue typically did not produce an induction effect which was weaker in comparison with cells which displayed the highest initial viability.

Figure 3. Effect of initial cell viability on CYP induction in freshly isolated hepatocytes.

Effect of Basal Enzyme Activities on the Extent of CYP InductionIt has been suggested that the dynamic range of the response to inducer treatment is related to basal enzyme activity, i.e. cells with high basal enzyme activity may reach a maximal induction threshold during the course of an experiment and will therefore produce a low ‘fold induction’ value when this is used as an endpoint. Conversely, cells with low basal activity can appear to produce a more marked induction effect on a fold induction basis5. To test this hypothesis, trends in the CYP1A2, CYP2B6 and CYP3A4 induction data from freshly isolated cells were analysed, the results of which are presented in Figure 4. When data points were placed in rank order in terms of control enzyme activities, a general trend was indeed apparent in that the magnitude of the CYP1A2, CYP3A4 and, to a lesser extent, CYP2B6 induction effect did appear to inversely correlate to basal enzyme activity. Overall, these findings indicate that, in isolation, the traditional ‘fold induction’ endpoint may not be a reliable representation of the potential for a substance to up regulate drug metabolising enzymes and lends support to the regulatory guidance recommendations which advocate the use of alternative markers, i.e. mRNA levels.

Figure 4. Effect of basal enzyme activity in freshly isolated hepatocytes on the extent of CYP induction elicited by inducers.

Enzyme Activities in Freshly Isolated and Cryopreserved HepatocytesFigure 5 represents the mean fold induction elicited by positive control inducers of CYP1A2, CYP2B6 and CYP3A4-mediated enzyme activity in the freshly isolated and cryopreserved hepatocytes from which the dataset in Table 1 was produced. These data indicated that notably higher levels of CYP1A2 and CYP3A4 induction were produced in cryopreserved cells, which was perhaps a corollary of the generally lower basal enzyme activities. This same trend was not evident from the CYP2B6 data.

Figure 5. Mean fold induction values in fresh and cryopreserved hepatocytes.

Enzyme Activities and mRNA ExpressionCurrent regulatory guidance stipulates mRNA expression as the preferred endpoint for the in vitro assessment of CYP enzyme induction, as this method is more informative in cases where a drug is a concurrent enzyme inhibitor. Uncertainties remain, however, regarding threshold values for the classification of a notable effect, the possibility of false positives etc. As such, co-analysis with catalytic activity may be considered prudent. The data presented in Figure 6 represent CYP1A2, CYP2B6 and CYP3A4 catalytic activity and corresponding mRNA expression in multiple batches of cryopreserved hepatocytes, with both parameters measured within the same experiment. Generally, the maximum fold increase in mRNA levels was greater than the corresponding value for enzyme activity and displayed a greater dynamic range. The rank order of increases in mRNA as a response to inducer treatment was CYP1A2>CYP3A4>CYP2B6 and, generally, these data were consistent with the findings of other workers6. This indicated that the mRNA endpoint was a more sensitive marker for induction. However, in some instances increases in the enzyme activity were greater and overall a marked variation in responses between lots was apparent. In all experiments, however, both endpoints produced Emax values consistent with a notable induction effect. These data confirmed that the mRNA endpoint is an appropriate marker for enzyme induction and demonstrated that increases are typically translated into an increase in functional catalytic activity. The data did, however, reinforce the requirement for investigations in hepatocytes from multiple donors given the marked inter-batch variability.

Figure 6. Comparison of catalytic activity and mRNA levels in cryopreserved hepatocytes following treatment with inducers.

Conclusions▶ There were no significant effects on the extent of CYP induction in freshly

isolated hepatocytes as a function of donor gender, age or initial cell viability.

▶ The extent of CYP1A2 and CYP3A4 induction elicited was related to the basal enzyme activities in untreated cells, i.e. cells with the highest control activities typically produced the lowest ‘fold induction’ values and vice versa. These findings suggest that the latter, in isolation, may not be the most informative endpoint for the evaluation of in vitro CYP induction data.

▶ Plateable cryopreserved hepatocytes were shown to respond as well, or better, than freshly isolated cells and confirmed that this test system was a viable alternative for the conduct of CYP induction studies.

▶ Determination of mRNA levels was found to be a more sensitive marker for CYP induction than catalytic activity with a wider dynamic range. Moreover, this endpoint is not susceptible to false negatives due to, e.g. concomitant inhibition or low responses due to high basal enzyme activities. The use of enzyme activities as an adjunct to mRNA determination is nevertheless recommended.

Cel

l typ

e

Gen

der

Mea

n ag

e (y

)

Ran

ge (y

)

Mea

n ce

ll vi

abili

ty (%

)

Mea

n co

ntro

l act

ivity

*

Mea

n in

duce

d A

ctiv

ity*

Mea

n fo

ld in

crea

se**

Ran

ge

n

CYP1A2

Fresh M 64 33-88 82.8 0.735 10.6 17.5 3.8-82.0 63

F 59 24-89 83.7 0.739 11.9 21.0 2.5-79.3 57

Cryo M 34 2-69 92.7 1.44 46.5 34.2 19.4-54.9 7

F 45 18-62 94.3 2.90 146 52.4 32.9-97.0 5

CYP2B6

Fresh M 68 45-88 81.9 17.8 116 6.7 1.6-15.8 42

F 58 27-89 83.7 17.4 111 6.3 1.7-14.1 39

Cryo M 34 2-69 92.7 1.75 8.13 5.57 1.2-10.5 7

F 45 18-62 94.3 5.03 29.3 7.96 4.3-12.8 5

CYP2C9

Fresh M 64 45-79 82.1 318 475 1.8 1.0-3.5 17

F 55 27-79 83.7 307 461 1.8 0.9-3.7 22

Cryo M 28 13-46 93.7 82.1 261 2.9 2.0-3.7 3

F 40 18-62 94.0 34.0 153 4.2 3.5-4.9 2

CYP2C19

Fresh M 62 33-79 82.9 16.7 54.2 3.2 1.8-6.6 27

F 58 27-76 82.8 20.7 52.5 2.3 0.8-4.9 23

Cryo M 28 13-46 93.7 4.18 21.0 6.3 5.0-7.7 2

F 40 18-62 94.0 0.193 2.14 13.1 9.6-16.7 2

CYP3A4

Fresh M 64 28-88 83.3 117 481 4.6 0.9-20.2 68

F 60 27-89 84.0 176 555 3.8 1.5-12.8 53

Cryo M 34 2-69 92.7 55.5 437 11.6 4.23-16.5 7

F 45 18-62 94.3 109 661 11.0 3.8-32.5 5

*pmoles/min/million cells**relative to appropriate control

0

5

10

15

20

25

30

35

40

CYP1A2 CYP2B6 CYP2C9 CYP2C19 CYP3A4M

ean

fold

indu

ction

ove

r con

trol

Male Female

0

10

20

30

40

50

60

70

CYP1A2 CYP2B6 CYP3A4

Mea

n fo

ld in

ducti

on o

ver c

ontr

ol Fresh Cryopreserved

0

5

10

15

20

25

30

35

40

CYP1A2 CYP2B6 CYP2C19 CYP3A4

Mea

n fo

ld in

ducti

on o

ver c

ontr

ol

<40 years 41-50 years 51-60 years 61-70 years >70 years

n=6n=17

n=26

n=35

n=35

n=5

n=10n=16 n=23 n=27

n=3 n=5 n=16 n=15n=9

n=6

n=17 n=25n=35 n=38

0

5

10

15

20

25

30

35

40

45

CYP1A2 CYP2B6 CYP2C19 CYP3A4

Mea

n fo

ld in

ducti

on o

ver c

ontr

ol

70-80% 81-90% 91-100%

n=46 n=48

n=22

n=35n=31 n=17

n=17 n=24 n=6

n=44

n=51 n=25

0

10

20

30

40

50

60

70

80

90

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

Fold

indu

ction

ove

r con

trol

Activ

ity (p

mol

/min

/10e

6 ce

lls)

Rank order of basal enzyme activity

CYP1A2/Omperazole

0

2

4

6

8

10

12

14

16

18

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70 80

Fold

indu

ction

ove

r con

trol

Activ

ity (p

mol

/min

/10e

6 ce

lls)

Rank order of basal enzyme activity

CYP2B6/Phenobarbital

0

2

4

6

8

10

12

14

0

100

200

300

400

500

600

700

800

0 20 40 60 80 100 120

Fold

indu

ction

ove

r con

trol

Activ

ity (p

mol

/min

/10e

6 ce

lls)

Rank order of basal enzyme activity

CYP3A4/Rifampicin

0

50

100

150

200

250

300

350

400

450

500

550

600

HUM4011 HUM4021HUM4016 GC4003

GC4008 OGFCDP

BHLXVN

HUM4017Hu8110

Hu8114Hu8130

YJM

Mea

n fo

ld in

ducti

on o

ver c

ontr

ol

CYP1A2/Omeprazole

Enzyme activity mRNA

1.2

2.84.0

10.4

0.5

11.4

1.6

1.210.2

0.7

6.0

1.70.7

13.6

Mean Emax (catalytic activity): 37Mean Emax (mRNA): 155Mean ratio ± SD (mRNA vs. enzyme activity): 4.7 ± 4.7

0

10

20

30

40

50

60

70

80

90

HUM4011HUM4021

HUM4016 GC4003GC4008 OGF

CDPBHL

XVNHUM4017

Hu8110Hu8114

Hu8130YJM

Mea

n fo

ld in

ducti

on o

ver c

ontr

ol

CYP2B6/Phenobarbital

Mean Emax (catalytic activity): 6.4Mean Emax (mRNA): 25.4Mean ratio ± SD (mRNA vs. enzyme activity): 6.1 ± 7.8

1.11.9

4.54.6

1.0

27.1

20.0

2.72.4

0.7 2.6

2.66.6 7.4

Enzyme activity mRNA

0

20

40

60

80

100

120

140

160

HUM4011HUM4021

HUM4016 GC4003GC4008 OGF

CDPBHL

XVNHUM4017

Hu8110Hu8114

Hu8130YJM

Mea

n fo

ld in

ducti

on o

ver c

ontr

ol

CYP3A4/Rifampicin

Mean Emax (catalytic activity): 12.3Mean Emax (mRNA): 37.0Mean ratio ± SD (mRNA vs. enzyme activity): 3.1 ± 4.5

0.6

5.43.9

1.30.4 0.6

0.4

4.7

3.2

0.5 1.0

2.1

2.0

17.8Enzyme activity mRNA

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LeCluyse, E.L., Zech, K., Robertson, P., Koch, P., Antonian, L., Wagner, G., Yu, L. and Parkinson, A. (2003) Drug Met. Disp., 31, 421.5. Hewitt, N.J., de Kanter, R. and LeCluyse, E. (2007) Chem.-Biol. Interactions, 168, 51.6. Fahmi, O., Kish, M., Boldt, S., Obach, R.S. (2010) Drug Met. Disp., 38, 1605.