The Future of In Vitro Systems for the Assessment of Induction and Suppression of Enzymes and Transporters AAPS, San Diego November 6 th, 2014 Andrew Parkinson, XPD Consulting Lisa Almond, Simcyp-Certara Jane Kenny, Genentech Harma Ellens, GSK 1
Induction and Suppression Induction Can increase clearance and cause loss of therapeutic action (e.g., OCS) Xenosensor FDA CYP Control Inducer AhR CYP1A2 Omeprazole Direct agonist CITCO Direct agonist CAR CYP2B6 Phenobarbital Indirect agonist PXR CYP3A4 Rifampin Direct agonist Suppression Can decrease clearance Activators of other nuclear receptors Cytokines (e.g., TNF, IL-6) NF- B activation xenosensor inactivation (and vice versa) Biologics may act directly on hepatocytes/kupffer cells or release cytokines from PBMC Inducers tend to be small drug molecules Suppressors tend to be biologics (therapeutic proteins) but there are exceptions 2
3 Induction studies before and after the FDA 2012 DDI Guidance 3 Induction assay Before 2012 After 2012 Endpoints Positive attribute Negative attribute CYP activity Fold induction Percent of positive control Endpoint = net effect of induction & irreversible inhibition CYP inhibition could mask induction CYP mrna EC 50 and E max Endpoint = induction only (inhibition examined separately) Assumes induction is always due to increased transcription (not so for CYP2C19 or 2E1) control criteria > 2 fold > 6 fold Test article (TA) concentration Up to10 x plasma C max In vitro E max TA treatment 3 days 2 or 3 days IVIVE >2 fold (or >1.5 fold) >40% (or >20%) control R 3 < 0.9
CYP3A4 induction (E max ) : mrna versus activity Predictive utility of in vitro rifampin induction data generated in fresh and cryopreserved human hepatocytes, Fa2N-4, and HepaRG cells. Templeton IE, Houston JB, Galetin A. Drug Metab Dispos 39: 1921-1929, 2011 Relatively large Relatively small 4
CYP3A4 induction (EC 50 ) : mrna versus activity Predictive utility of in vitro rifampin induction data generated in fresh and cryopreserved human hepatocytes, Fa2N-4, and HepaRG cells. Templeton IE, Houston JB, Galetin A. Drug Metab Dispos 39: 1921-1929, 2011 Comparable but Fa2N-4 EC 50 values are high 5
CYP3A4 mrna induction: EC 50 in different cells Comparison of immortalized Fa2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction. Hariparsad N, Carr BA, Evers R, Chu X. Drug Metab Dispos 36: 1046-1055, 2008 Fa2N-4 cells EC 50 = 8 µm Human hepatocytes EC 50 = 0.8 µm 6 Levels of PXR mrna in Fa2N-4 cells human hepatocytes but CAR mrna is <1% Rifampicin is an OATP substrate Levels of OATP1B1 and OAPT1B3 mrna levels in Fa2N-4 cells <1% of hepatocytes
[Drug] Measuring the in vitro concentration of the inducing drug seems like a good idea but... where is the drug? Whole-system loss In many cases most of the drug moves from the medium to the cells after only a few minutes Medium loss 0 5 10 15 20 Time (min) 7
Measuring the in vitro concentration of the inducing drug seems like a good idea but... where is the drug? For certain basic drugs, they undergo extensive partitioning into phospholipid membranes (not shown) or ion-partitioning into the acidic lysosomes Total cell concentration >> unbound intracellular concentration 8
Measuring the in vitro concentration of the inducing drug seems like a good idea but... where is the drug? 9 For certain acidic/zwitterion drugs, they undergo transporter-mediated uptake and efflux into the bile canaliculi Total cell concentration >> unbound intracellular concentration
Should we correct for variation in EC 50? 10 Perfect results in all 3 human hepatocyte preparations. Correction? None
Should we correct for variation in EC 50? 11 A high EC 50 value observed for both the positive control and test article in 1 of 3 preparations of human hepatocyte preparations. Correction? Probably YES
Should we correct for variation in EC 50? 12 A high EC 50 value observed for ONLY the positive control in 1 of 3 preparations of human hepatocyte preparations. Correction? Case-by-case
In vitro to in vivo extrapolation (IVIVE) A comparison of 5 models of CYP3A4 induction Evaluation of various static and dynamic modeling methods to predict clinical CYP3A induction using in vitro CYP3A4 mrna induction data. Einolf HJ, Chen L, Fahmi OA, Gibson CR, Obach RS, Shebley M, Silva J, Sinz MW, Unadkat JD, Zhang L, Zhao P. Clin Pharmacol Ther 95: 179-188, 2014 13
In vitro to in vivo extrapolation (IVIVE) A comparison of 5 models of CYP3A4 induction Correlation approach = C max EC 50 Relative induction score Basic static model (R 3 ) 14
In vitro to in vivo extrapolation (IVIVE) A comparison of 5 models of CYP3A4 induction Mechanistic static model (AUC Ratio) Liver Intestine where A, B and C are terms for TDI, induction, and reversible inhibition in the liver, respectively and X, Y and Z are terms for TDI, induction, and reversible inhibition in the intestine, respectively Mechanistic dynamic model (modeling & simulation) 15 AUCR + PBPK modeling. Incorporates such variables such as time-dependent changes in inducer concentration, which is complex when the drug is an auto-inducer
In vitro to in vivo extrapolation (IVIVE) A comparison of 5 models of CYP3A4 induction Model False negative False positive True negative True positive Within 2 fold Correlation 0 3 3 22 23 RIS 0 2 4 22 19 R 3 (d = 1) 0 3 3 22 17 Mechanistic Static 2 (0) 0 (3) 6 (3) 20 (22) 19 (20) Mechanistic Dynamic 2 (0) 1 (3) 5 (3) 20 (22) 11 (14) 16 [I] = total plasma C max (bound + unbound) Values in parentheses are without the time-dependent inhibition component, which tends to be overestimated for CYP3A4
The FDA s R 3 Raising the negative result bar If we assume d=1 and use plasma C max as the in vivo inducer concentration, then the equation for R 3 is: 1 R 3 = 1 + E max + C max EC 50 + C max The bare minimum cutoff value of is R 3 is 0.8999 (marginally below 0.9). This value is obtained when: E max + C max EC 50 + C max = 0.1112 Accordingly, EC 50 C max = E max 0.1112 1 To fall just below the FDA s cutoff of 0.9, EC 50 (as a multiple of C max ) increases almost linearly with E max. 17
The FDA s R 3 Raising the negative result bar This graph shows the relationship between EC 50 (as a multiple of C max ) and E max when the value of R 3 is 0.899 (marginally below the FDA s cutoff of 0.9) Note the near linear relationship General Rule You are safe if EC 50 is greater than C max x 10 E max 18
The FDA s R 3 Raising the negative result bar This graph shows the dividing line between a positive and negative result for CYP induction when E max = 10 (the induced activity was 11 times control) Note that the EC 50 value is 89 times C max 19
The FDA s R 3 Raising the negative result bar This graph shows a series positive results for CYP induction when E max = 10. R 3 values range from 0.9 (safe) to 0.167, which is achieved when EC 50 = C max. Note that when the in vitro concentration of inducer equals C max (or even 10 x C max ), the degree of induction is far less than 40% of E max in the majority of cases 20
The FDA s R 3 Raising the negative result bar This graph shows the dividing line between a positive and negative result for CYP induction when E max ranges from 1 to 10 (the induced activity was 2 to 11 times control) Note that for ALL these safe curves, negligible induction is observed when the in vitro concentration of inducer equals C max regardless of the value of E max 21
The FDA s R 3 Raising the negative result bar To be sure of a negative result (defined as an R 3 of 0.9 or greater), it is necessary to test concentrations of test article that exceed C max As E max increases (as it does when mrna levels are measured) so must the concentration of test article (as a multiple of C max ) to demonstrate no potential for clinical induction 22