In vitro ADME & PK
Cytochrome P450 Induction
Background Information Protocol Test System Cryopreserved hepatocytes (3 donors recommended) • Induction of cytochrome P450 enzymes is HepaRGTM cells are also available on request associated with an increased prevalence of clinical drug-drug interactions. Test Article Concentration
6 concentrations (dependent upon unbound Cmax, • Cyprotex’s Cytochrome P450 induction dose, solubility and cytotoxicity) plus vehicle control, assay identifies the potential of test in triplicate (alternative number of concentrations may compounds to induce CYP1A2, CYP2B6 be available on request) or CYP3A4 in cultured human hepatocytes by evaluating mRNA levels and/or catalytic CYP Isoforms activity. Assays are designed to meet FDA1 CYP1A2, CYP2B6 and CYP3A4 and EMA2 guidelines. For CYP2C8, CYP2C9 & CYP2C19 please contact ‘Cultured hepatocytes... directly for information are the preferred in vitro • Test drug concentrations should be system for induction (and based on the expected human plasma Controls down-regulation) in vitro drug concentrations and dose. Solubility, Omeprazole (CYP1A2 positive control) cytotoxicity and plasma protein binding Phenobarbital (CYP2B6 positive control) studies.’ should also be taken into consideration. Rifampicin (CYP3A4 positive control) Flumazenil (negative control) 2EMA (2012) Guideline on the • Cyprotex’s Cytochrome P450 induction investigation of drug interactions assay delivers fold-induction data Test Article Requirements normalised to vehicle control which can be Dependent on top concentration (recommend 0.1% compared to positive control responses. DMSO in incubation) If appropriate, data is fit using non-linear regression analysis to four-parameter Exposure Period 72 hr (media changed every 24 hours) sigmoidal equation to produce Emax and
EC50 values. Probe Substrates for Catalytic Activity • The clinical consequences of induction Phenacetin (CYP1A2) may be therapeutic failure caused by Bupropion (CYP2B6) a decreased systemic exposure of the Midazolam (CYP3A4) drug itself or a co-administered therapy, or toxicity as a result of increased Analysis Method bioactivation. LC-MS/MS quantification of acetaminophen Supplementary Assays (CYP1A2), hydroxybupropion (CYP2B6) and 1-hydroxymidazolam (CYP3A4) Preliminary aqueous solubility qRT-PCR for relative mRNA expression levels assessment (CYP1A2, CYP2B6 and CYP3A4) Preliminary cytotoxicity assessment using MTT in primary human Data Delivery TM hepatocytes or HepaRG Excel sheet detailing mRNA levels, fold induction Measurement of parent drug on relative to vehicle control, concentration of metabolite final day of dosing of probe substrate, Emax, EC50 and F2 (concentration which leads to a 2-fold increase above E ) if Relative induction score (RIS) min analysis appropriate. Written report available on request
To find out more [email protected] ‘The sponsor should evaluate the potential of an investigational drug to induce CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A4... If the result from at least one donor exceeds the pre-defined threshold, the sponsor should consider the drug to have induction potential and conduct a follow-up evaluation.1’
35
Figure 1 30 Induction of CYP1A2 mRNA levels by omeprazole in cryopreserved human hepatocytes. 25
20
15 relative to vehicle 10
Fold increase in mRNA expression 5
0 0.1 1 10 100 1000 Concentration (µM)
18
Figure 2 16
Induction of CYP2B6 mRNA levels by phenobarbital 14 in cryopreserved human hepatocytes. 12
10
8
relative to vehicle 6
4 Fold increase in mRNA expression 2
0 1 10 100 1000 10000 Concentration (µM)
50
Figure 3 45
Induction of CYP3A4 mRNA levels by rifampicin in 40
cryopreserved human hepatocytes. 35
30
25
20 relative to vehicle 15
10 Fold increase in mRNA expression 5
0 0.01 0.1 1 10 100 Concentration (µM)
References 1 FDA (2020) Guidance for industry: drug interaction studies-study design, data analysis, implications for dosing, and labeling recommendations. 2 EMA (2012) Guideline on the investigation of drug interactions.