Preliminary concept paper

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*Recommend use of 2 structurally unrelated CYP3A4/5 substrates for evaluation of in vitro CYP3A inhibition. If the drug inhibits at least one CYP3A substrate in vitro, then in vivo evaluation is warranted.

2. Design considerations for in vitro CYP inhibition studies

a. Typical kinetic experiments for determining IC50 or Ki involve incubating varying concentrations of substrate and inhibitor with fixed amounts of enzyme for a constant period of time. The substrate and inhibitor concentrations used should cover the range above and below the Km and Ki, respectively.
b. Microsomal protein concentration usually ranges from 0.01 to 0.5 mg/ml.
c. Because buffer strength, type, and pH can all significantly affect Vmax and Km, standardized assay conditions are recommended.
d. Preferably no more than 10% substrate or inhibitor depletion should occur. However, with low Km substrates, it may be difficult to avoid >10% substrate depletion at low substrate concentrations.
e. The relationship between time and amount of product formed should be linear.
f. The relationship between amount of enzyme and product formation should be linear.
g. Any solvents should be used at low concentrations (<1% (v/v) and preferably <0.1%). Some of the solvents inhibit or induce enzymes. The experiment may include a no-solvent control and a solvent control.
h. Use of an active control (known inhibitor) is optional
3. Determining whether an NME is a reversible inhibitor
Theoretically, significant enzyme inhibition occurs when the concentration of the inhibitor present at the active site is comparable to or in excess of the Ki. In theory, the degree of interaction (R, expressed as percent change in AUC) can be estimated by the following equation: R = 1+ [I]/Ki, where [I] is the concentration of inhibitor exposed to the active site of the enzyme and Ki is the inhibition constant.
Although the [I]/Ki ratio is used to predict the likelihood of inhibitory drug interactions, there are factors that affect selection of the relevant [I] and Ki. Factors that affect [I] include uncertainty regarding the concentration that best represents concentration at the enzyme binding site and uncertainty regarding the impact of first-pass exposure. Factors that affect Ki include substrate specificity, binding to components of incubation system, substrate and inhibitor depletion.
Current recommended approach-

Due to the concerns listed above, the use of [I]/Ki to predict the potential for inhibitory drug interactions needs to be further evaluated. Thus, we use a conservative approach to determine the likelihood of an in vivo interaction, based on in vitro data. Calculate [I]/Ki, where [I] represents the mean steady-state Cmax value for total drug (bound plus unbound) following administration of the highest proposed clinical dose. As the ratio increases, the likelihood of an interaction increases. If the ratio is <0.02, the likelihood of an interaction is remote, and an in vivo metabolism-based drug-drug interaction study is not needed. Quantitative predictions of the magnitude of an in vivo interaction, based on in vitro data, are not possible at this time. Although quantitative predictions of in vivo drug-drug interactions from in vitro studies are not possible, rank order across the different CYP enzymes for the same drug may help prioritize in vivo drug-drug interaction evaluations.

4. Determining whether an NME is a mechanism based inhibitor
Time-dependent inhibition should be examined in standard in vitro screening protocols, because the phenomenon cannot be predicted with complete confidence from chemical structure. A 30 minute pre-incubation of a potential inhibitor, prior to addition of substrate, is recommended. Any time-dependent and concentration-dependent loss of initial product formation rate indicates mechanism based inhibition. For compounds containing amines, metabolic intermediate complex formation can be followed spectroscopically. Detection of time-dependent inhibition kinetics in vitro should be followed up with in vivo studies in humans (or possibly in a human hepatocyte study).

Appendix C. Evaluation of CYP induction

A drug that induces a drug-metabolizing enzyme can increase the rate of metabolic clearance of a co-administered drug that is a substrate of the induced pathway. A potential consequence of this type of drug-drug interaction is sub-therapeutic blood concentrations. Alternatively, the induced metabolic pathway could lead to increased formation of an active compound resulting in an adverse event.
1. Chemical inducers as a positive control
If one is evaluating the potential for a drug to induce a specific CYP enzyme, the experiment should include an acceptable enzyme inducer as a control such as those listed in Table 4. The use of a positive control helps quantify enzyme catalytic activity. The positive controls should be potent inducers (> 2 fold increase in enzyme activity of probe substrate at inducer concentrations < 500 µM). The selection of test drug probes is discussed in Section A.
Table 4. chemical inducers for in vitro experiment(1)


Inducer (1)







Inducer (1)





















































none identified


none identified






















2.9- 6.9

  1. Except for the cases noted below, the following test substrates were used: CYP1A2, 7-ethoxyresorufin; CYP 2A6, coumarin; CYP2C9, tolbutamide, CYP2C19, S-mephenytoin; CYP3A4, testosterone.

  2. CYP1A2: 1 of 4 references for -naphthoflavone used phenacetin

  3. CYP3A4: 2 of 13 references for rifampin and 1 of 3 references for phenobarbital used midazolam

  4. CYP3A4: 1 of the 4 references for dexamethasone used nifedipine

2. Design of drug induction studies in vitro

Presently, the most reliable method to study a drug’s induction potential is to quantify the enzyme activity of primary hepatocyte cultures following treatments including the potential inducer drug, a probe inducer drug (positive control, see Table 4), and non treated hepatocytes (negative control), respectively. Either freshly isolated hepatocyte cultures or cryopreserved hepatocytes that can be thawed and cultured are acceptable for these studies.

a) Test drug concentrations should be utilized based on the expected human plasma drug concentrations. At least three concentrations spanning the therapeutic range should be studied, including at least one concentration that is an order of magnitude greater than the average expected plasma drug concentration. If this information is not available, concentrations ranging over at least two orders of magnitude should be studied.
b) Following treatment of hepatocytes for 3-4 days, the resulting enzyme activities should be determined using appropriate CYP-specific probe drugs (see Table 3). Either whole cell monolayers or isolated microsomes can be utilized to monitor drug-induced enzyme changes, however, the former tissue is the simplest and most direct method,
c) When conducting experiments to determine enzyme activity, the experimental conditions listed in section B.2 are relevant.
d) Based on inter-individual differences in induction potential, experiments should be conducted with hepatocytes prepared from at least three individual donor livers.

3. Endpoints for subsequent prediction of enzyme induction
When analyzing the results of experiments to determine enzyme activity, the following issues are relevant.
a) The simplest and most frequently used endpoints to identify enzyme induction are the fold induction activity:
fold induction = (activity of test drug treated cells) / (activity of negative control)
or percent of positive control activity:
% positive control = (activity of test drug treated cells x 100) / (activity of positive control)

b) An alternative endpoint is the use of an EC50 (effective concentration at which 50% maximal induction occurs) value, which represents a potency index that can be used to compare the potency of different compounds.

c) A drug that produces a > 2 fold increase in probe drug enzyme activity or the fold-change that is more than 40% of the positive control can be considered as an enzyme inducer in vitro and in vivo evaluation is warranted.
4. Other methods proposed for identifying enzyme induction in vitro
Although the most reliable method for quantifying a drug’s induction potential involves measurement of enzyme activities after incubation of the drug in primary cultures of human hepatocytes, other methods are being evaluated. Several of these methods are described briefly below.
a) Western immunoblotting or immunoprecipitation probed with specific polyclonal antibodies. Relative quantification of specific P450 enzyme protein requires that the electrophoretic system clearly resolve the individual enzymes and/or the primary antibodies be specific for the enzyme quantified. Enzyme antibody preparations are highly variable.
b) Measurement of mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR can quantify mRNA expression for a specific CYP enzyme but is not necessarily informative of enzyme activities. The measurement of mRNA levels are helpful when both enzyme inhibition and induction are operative.
c) Receptor gene assays for receptors mediating induction of P450 enzymes. Cell receptors mediating CYP1A, CYP2B and CYP3A induction have been identified. Higher throughput AhR (aromatic hydrocarbon receptor) and PXR (pregnane X receptor) binding assays and cell-based reporter gene assays have been developed and utilized to screen for compounds that have CYP1A and CYP3A induction potential. However, correlation of receptor binding and activation with in vivo CYP enzyme induction requires additional validation.
d) Enzyme activity in immortal cell lines. Differential expression of the individual CYP450 enzymes and corresponding regulatory factors (e.g., nuclear receptors and associated cofactors) over time in culture suggests that this model system is not reflective of in vivo profiles. Although negative results from this method cannot rule out an induction effect, positive results can indicate a need for further clinical evaluation.

VII. References:

  1. Tucker G, Houston JB, and Huang S-M, Optimizing drug development: strategies to assess drug metabolism/transporter interaction potential- toward a consensus, Clin Pharmacol Ther. 2001 Aug;70(2):103-14; Br J Clin Pharmacol. 2001 Jul;52(1):107-17; , Eur J Pharm Sci. 2001 Jul;13(4):417-28; Pharm Res. 2001 Aug;18(8):1071-80

  2. Bjornsson TD, Callaghan JT, Einolf HJ, et al, The conduct of in vitro and in vivo drug-drug interaction studies, A PhRMA perspectives, J Clin Pharmacol, 43: 443-469, 2003

  3. Yuan R, Madani S, Wei X, Reynolds K, and Huang S-M, Evaluation of P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos. 2002 Dec;30(12):1311-9

  4. Huang, S-M, Hall, SD, Watkins, P, Love, LA, Serabjit-Singh, C, Betz, JM, Hoffman, FA, Honig, P, Coates, PM, Bull, J, Chen, ST, Kearns, GL, Murray, MD, Drug interactions with herbal products & grapefruit juice: a conference report, Clin Pharmacol Ther 2004; 75:1-12

  5. Huang S-M, Lesko, LJ, Drug-drug, drug-dietary supplement, and drug-citrus fruit and other food interactions- what have we learned? J Clin Pharmacol 2004; 44:559-569

  6. ACPS-CPS advisory committee meeting minutes April 20-21, 2003 (CYP3A classification and P-gp) , Nov 17-18, 2003 (CYP2B6 and CYP2C8)

1 CDER/CBER guidance for industry “Population pharmacokinetics”, February 1999

1 CDER/CBER guidance for industry “Exposure-response relationships- study design, data analysis and regulatory applications” April 2003

2 ICH E14 step 2 document, “The Clinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential for Non-antiarrhythmic Drugs”

3 Comment is requested on the use of multiple inhibitors or multiple impaired conditions to achieve maximum inhibition of the investigational drug’s clearance pathway.

4 Draft guidance for industry ”voluntary pharmacogenomic data submission”, November 2003

3 3 Schuirmann, D.J., "A Comparison of the Two One-Sided Tests Procedure and the Power Approach for Assessing the Bioequivalence of Average Bioavailability," J. Pharmacokin. and Biopharm., 15:657-80, 1987.

Topic 2A_Concept_paper_drug interactions_Oct_1_2004_Huang_v1

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