Assessment Under Initial Rate Conditions

Published:  23 October 2014

Assessment Under Initial Rate Conditions of the Selectivity and Time Course of Cytochrome P450 Inactivation in Pooled Human Liver Microsomes and Hepatocytes: Optimization of Inhibitor Conditions Used for Reaction Phenotyping Studies

The identification of the metabolic pathways (reaction phenotyping) for new drug candidates is an essential component of drug development, as these studies increase the understanding of how a drug is cleared and the potential for the drug to be subject to drug-drug interactions with co-administrated medications. One approach used to identify which cytochrome P450 (CYP) enzymes are responsible for the metabolism of a drug is the use of specific chemical inhibitors in test systems such as pooled human liver microsomes (HLM) or pooled cryopreserved human hepatocytes (CHH). The design of these studies is critical because factors such as metabolic depletion of the inhibitor, protein binding, and insufficient enzyme inactivation can produce convoluted and misleading results. In the present study, we examined the selectivity and duration of CYP inactivation, with a range of protein concentrations and incubation times, for a variety of commonly used CYP inhibitors all under initial rate probe substrate conditions. Briefly, NADPH-fortified pooled HLM (n = 200) at 0.1, 0.5 and 1 mg/mL or pooled CHH (n = 100) at 1 million cells/mL, were pre-incubated for 30 min at 37°C with various CYP inhibitors, namely furafylline (10 μM), phencyclidine (up to 30 μM), gemfibrozil glucuronide (100 μM), tienilic acid (20 μM), esomeprazole (10 μM), quinidine (5 μM), paroxetine (1 or 5 μM), ketoconazole (1 or 4 μM), CYP3cide (2.5 μM), mibefradil (1 μM), and troleandomycin (50 μM). Following the pre-incubation step, marker substrate (» Km) incubations were performed for up to 5 min (10 min for CHH) at three post pre-incubation time points (0, 30, 120 min; simulating the time course of an unknown drug) to determine CYP1A2 (phenacetin), CYP2B6 (bupropion), CYP2C8 (amodiaquine), CYP2C9 (diclofenac), CYP2C19 (S‑mephenytoin), CYP2D6 (dextromethorphan), CYP2E1 (chlorzoxazone) and CYP3A4/5 (midazolam) residual activities. Metabolite formation was determined by LC-MS/MS analysis. Results indicated that, in both HLM and CHH, inhibition of specific CYP enzymes can be accomplished over the duration of 120 minutes after pre-incubation with an inhibitor.

Effects of Various Media

Published:  23 October 2014

The Effect of Buffer Ionic Strength or Various Media on the In Vitro Metabolism of Cytochrome P450 Substrates in Pooled Human Liver Microsomes and Cryopreserved Human Hepatocytes

In our previous work, we reported that neither cofactor availability nor membrane permeability accounted for the much slower in vitro clearance of midazolam in suspended cryopreserved human hepatocytes (CHH) compared with human liver microsomes (HLM). We posited the difference was possibly an effect of ionic strength (Kazmi et al., 2013a; Kazmi et al., 2013b). In the present study we evaluated the effects of buffer ionic strength and various media on CYP1A1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and multiple CYP3A4/5 activities in human liver microsomes and cryopreserved human hepatocytes.  CYP activities were measured at three ionic strengths (5, 50 and 200 mM phosphate buffer) and/or in five commonly used cell culture media (KHB, MCM+, DMEM+HEPES, Waymouth’s and William’s E+HEPES) in both human liver microsomes and cryopreserved human hepatocytes. NADPH-fortified pooled HLM (n = 200) at 0.1mg/mL were incubated for 5 min with phenacetin, coumarin, bupropion, amodiaquine, diclofenac (tolbutamide for CHH), S‑mephenytoin, dextromethorphan, chlorzoxazone, midazolam, nifedipine, alfentanil, verapamil, testosterone and atorvastatin at their approximate Km values. In the case of pooled CHH (n = 50), incubations were conducted at 1 million cells/mL for 10-60 min. Reactions were terminated with an equal volume of organic solvent containing internal standard, followed by protein precipitation and analysis by LC/MS/MS. In human liver microsomes, phenacetin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), and dextromethorphan (CYP2D6) activities all were highest at 50 mM phosphate buffer. For chlorzoxazone (CYP2E1) and midazolam (CYP3A4/5), the enzymatic activities were highest at 200 mM phosphate buffer. When incubations in human liver microsomes were conducted with various media, MCM+ medium was found to support the highest activity of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19 and CYP2D6.

Comparison of Ki and IC50 Values for Prototypical Inhibitors of the Major Drug Uptake Transporters

Published:  20 October 2014

Comparison of Ki and IC50 Values for Prototypical Inhibitors of the Major Drug Uptake Transporters

We previously reported on the similarities and differences in Ki and IC50 values for various inhibitors of hepatic uptake transporters OATP1B1 and OATP1B3 when in vitro experiments were conducted with either clinically-relevant or prototypical (non-drug) substrates (Tibbets et al., 2013). In the current study, we further characterized Ki and IC50 values for various inhibitors of, not only OATP1B1 and OATP1B3, but also OAT1, OAT3, OCT1 and OCT2 with in vitro experiments in stably transfected HEK293 cell lines. Predicted Ki values were also mathematically derived from the experimentally determined IC50 values with the Cheng-Prusoff equation that incorporates the marker substrate concentration in relation to its Km value (Cheng et al., 1973). Prior to inhibition experiments, Km values were experimentally determined with each probe substrate: pravastatin (OATP1B1 and OATP1B3), estradiol glucuronide (OATP1B1 and OATP1B3), p-aminohippuric acid (OAT1), estrone sulfate (OAT3), tetraethylammonium bromide (OCT1) and metformin (OCT2). IC50 and Ki were experimentally determined for the prototypical inhibitors rifampin (OATP1B1 and OATP1B3), probenecid (OAT1 and OAT3) and quinidine (OCT1 and OCT2). IC50 experiments were conducted with probe substrate concentrations at or below the experimentally determined Km values. Ki experiments were conducted with a range of probe substrate concentrations (typically one-third to 3-fold Km). Experiments with OATP1B1, OATP1B3, OAT1 and OCT2 yielded IC50 and Ki values within two-fold. However, experiments with OAT3 and OCT1 yielded IC50 and Ki values that differed by more than two-fold.  In the case of OAT3, Ki values were approximately 10-fold above the IC50 value. In the case of OCT1 the converse was true; the IC50 value was five-fold higher than the Ki value. OATP1B1 and OATP1B3 were also evaluated with the clinically-relevant probe substrate pravastatin. Experiments with OATP1B1 resulted in Ki and IC50 values for pravastatin with less than a two-fold difference. However, the Ki and IC50 values for estradiol glucuronide were four- to five-fold higher than the Ki and IC50 values determined with pravastatin demonstrating substrate-dependent inhibition.

Pooled Plated Cryopreserved Human Hepatocytes

Published:  20 October 2014

The Use of Pooled Plated Cryopreserved Human Hepatocytes for the Determination of Metabolic Clearance, Cytochrome P450 Enzyme Induction and Uptake Transporter Studies

During early drug discovery and development, conventional test systems such as subcellular hepatic fractions or pooled, suspended cryopreserved human hepatocytes are used to determine metabolic clearance (intrinsic clearance; CLint) or in the case of cytochrome P450 (CYP) induction, individual cryopreserved or fresh plated primary human hepatocytes. Conventional test systems often fail to predict CLint and half-life (t1/2) of low turnover drugs due to limitations in test system stability (≤ 4 hours), leading to inaccurate scaling of in vitro to in vivo CLint. In the case of CYP induction screening, the use of individual plated primary human hepatocytes can lead to inter-individual variability in the induction response or susceptibility to cytotoxicity. Lastly, hepatic uptake experiments are frequently conducted in pooled suspended hepatocytes but typically only with individual plated hepatocytes.  In the present study we evaluated the utility of pooled plated cryopreserved human hepatocytes to determine metabolic clearance, CYP induction, and uptake transporter functionality. Briefly, individual plateable primary human hepatocytes were pooled (n = 5) using a proprietary single-freeze pooling process. In vitro metabolic clearance of a range of low to high clearance drugs (typically 1 µM) was evaluated with pooled plated hepatocytes cultured in 48‑well plates for 4 hours followed by incubations lasting up to 48 hours. Loss of parent drug was determined by LC/MS/MS analysis. For CYP induction, pooled human hepatocytes were cultured in 48-well plates and treated once daily for 3 days with MCM+ medium containing either 0.1% DMSO, rifampin (0.1- 20 μM), omeprazole (50 μM), or phenobarbital (750 μM).

In Vitro System-Dependent Inhibition

Published:  20 October 2014

In Vitro System-Dependent Inhibition of Cytochrome P450 Enzymes (CYP), UDP-Glucuronosyltransferases (UGT) and Transporters by Oligonucleotides

We previously examined the cytochrome P450 (CYP) inhibition potential of oligonucleotides and reported that the phosphorothioate, but not phosphodiester, backboned molecules caused potent inhibition of CYP1A2 and CYP2C8 in human liver microsomes (HLM) but not in cryopreserved human hepatocytes (CHH) (Buckley et al., 2009). In this study we expanded upon our previous work to include direct, time- (TDI) and metabolism-dependent inhibition (MDI) of 7 CYP and 8 UGT enzymes in pooled human liver microsomes and pooled cryopreserved human hepatocytes. Additionally, inhibition of OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, Pgp and BCRP was also examined. Briefly, two oligonucleotides with either phosphodiester (oligo # 1 and #2) or phosphorothioate (oligo #3 and #4) backbones were evaluated in human liver microsomes to determine IC50 values without a pre-incubation (direct inhibition) or with a 30 min pre-incubation +/- NADPH (MDI and TDI evaluation respectively). For UGTs, UDPGA was added to HLM and inhibition potential was assessed with a single concentration of oligos (30 μM). For cryopreserved human hepatocytes assays, MDI was assessed with 30 and 90 min pre-incubations, and 4‑methylumbelliferone (4-MU) was used as a marker of overall UGT activity. Marker substrate incubations with CYP and UGT enzyme probes were performed for 5-10 min and residual activity was assessed by LC/MS/MS analysis. For uptake transporter assays, oligos were first pre-incubated for 15 min with transporter transfected HEK cells followed by incubation of substrate-oligo mix for 2 min. Uptake of radiolabeled substrates was measured by scintillation counting. Inhibition of efflux transporters was evaluated with either Caco-2 or MDCK cells in bi-directional assays. The results indicated that oligos #1 and #2 with the phosphodiester backbone had little to no inhibitory effect on all CYP and UGT enzymes in human liver microsomes and cryopreserved human hepatocytes with the exception of oligo #2 in HLM for CYP2C19. Conversely, the oligos with the phosphorothioate backbone caused direct inhibition of CYP and UGT enzymes. The rank order of direct inhibition in human liver microsomes for oligo #3 was CYP1A2 > CYP2C8 > UGT1A1 > UGT2B17 > CYP2B6 > UGT1A9 > CYP2C19 > UGT2B10 > UGT2B15 > UGT1A3 > UGT1A6 > CYP2D6 > CYP2C9. Similar results were observed for oligo #4: CYP1A2 > UGT1A1> CYP2C8 > UGT2B17 > CYP2B6 > UGT2B10 > UGT1A9 > UGT2B15 > UGT1A3 > UGT1A6 > CYP2C19. There was evidence of NADPH-independent inhibition of CYP enzymes with oligo #3 and #4 for CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP3A4/5. In contrast to human liver microsomes, we observed little to no direct inhibition by any oligo in cryopreserved human hepatocytes (with the exception of oligo #2 with CYP2C19 and TDI by oligo #3 with CYP2C8), demonstrating system-dependent outcomes. Inhibition was observed for all uptake but not efflux transporters by the phosphorothioate oligos. Overall this study has implications on the design of in vitro DDI studies for oligonucleotide based therapeutics based upon test system-dependent differences.

The Impact of Incomplete Dose-Response Curves on EC50 and Emax Determinations in Enzyme Induction

Published:  20 October 2014

The Impact of Incomplete Dose-Response Curves on EC50 and Emax Determinations in Enzyme Induction Assessment

In evaluating the enzyme induction potential of a new drug candidate, it is common practice to calculate EC50 and Emax values to aid the prediction of drug-drug interactions. Typically, EC50 and Emax values are calculated when an increase of two-fold or higher is observed and these values are used to assess clinical induction potential. In literature, there are two common approaches, but not limited to, the sigmoid 3-parameter and Hill 3-parameter equations. In general, the sigmoid 3-parameter equation forces the calculated Emax towards the maximal experimentally observed value regardless of whether a ‘true’ Emax has been reached. Conversely, the Hill 3-parameter equation extrapolates the data set to calculate Emax and EC50 values that are closer to those obtained with a comprehensive data set. Therefore, these equations can generate disparate Emax, and therefore EC50 values with data sets where maximal induction is not reached.

Exploring the Drug-Drug Interaction Between Gemfibrozil and Repaglinide in Rats

Published:  20 October 2014

Exploring the Drug-Drug Interaction Between Gemfibrozil and Repaglinide in Rats: Metabolism and Transport

A clinically-relevant drug-drug interaction (DDI) between the dyslipidemia drug gemfibrozil and the antidiabetic repaglinide is well-documented throughout the literature. In humans, repaglinide is predominantly cleared by hepatic metabolism involving cytochrome P450 (CYP) 3A4 and 2C8 and UGT1A1 and 1A3. Gemfibrozil and its glucuronide metabolite inhibit CYP2C8 (irreversibly) and UGT1A1, which has been proposed as a key cause of the clinical interaction. Additionally, gemfibrozil and gemfibrozil glucuronide are established inhibitors of the hepatic uptake transporter OATP1B1, proposed as a confounding factor. A mechanistic assessment of the gemfibrozil/repaglinide DDI was undertaken in male Sprague-Dawley rats. Subjects (n = 3/group) received 200 mg/kg/day gemfibrozil or vehicle control by oral gavage for 2 days. On day 3, rats were fasted for 12 h and dosed once orally with 1 mg/kg repaglinide coadministered with gemfibrozil or vehicle. Blood, bile and urine were collected for 12 h following repaglinide administration. Livers were extracted for microsome preparation. In-life work was performed by Xenometrics LLC (Stilwell, KS). Rat plasma time-points, Hamilton-pooled plasma, and 0-12 h urine and bile pools were analyzed by LC-MS/MS for pharmacokinetics (PK) and metabolite profiling. P450 and UGT enzyme activities in liver microsomes were analyzed by LC-MS/MS. Gemfibrozil treatment greatly reduced repaglinide clearance in rats. Gemfibrozil-treated rats exhibited a 4-fold higher repaglinide Cmax and a 3.5-fold greater AUC0-12­ than control rats, but the tmax (1.2 and 1.7 h) and t­1/2 (2.6 h) values were similar. The Vd,obs  and plasma CLobs were approximately two-thirds lower in gemfibrozil-treated rats than control rats.

Comparison of XenoTech Hepatocytes to HepaRG® Cell Line

Published:  18 May 2014

Determination of Low Intrinsic Clearance Values using Primary Human Hepatocytes and the HepaRG® Cell Line - A Comparison of Methods
Svanberg, et al - AstraZeneca R&D

Oral drugs typically require effective half lives in the region of 10 – 20 h for once or twice daily dosing. For candidate drugs with low distribution volumes it is necessary to define intrinsic clearance (CLint) values of 0.1 - 1µL/min/million human hepatocytes (Grime et al., 2013). We wanted to compare in vitro systems that potentially can provide a solution to the problem of robustly defining low CLint values in human hepatocytes. Recently the HepatoPac™ Platform and a novel relay suspension method (Di et al., 2012) have shown promising results producing reliable low CLint values. XenoTech have made a well characterised plateable pool of cryopreserved human hepatocytes (5 donors) commercially available, which makes plated hepatocytes methods attractive to evaluate. Also of interest is the HepaRG® human hepatoma cell line, since it offers stable expression of drug metabolising enzymes (DMEs) (Kanebratt et al., 2008, Aninat et al., 2006). We are evaluating all four methods but this poster focuses on HepaRG & plated primary hepatocytes since data from Hepatopac and Relay at present are inconclusive.

SLAS 2014 - Automated Metabolic Stability

Published:  07 January 2014

High content automated metabolic stability and CYP inhibition cocktail screening assays for early drug development.

The assessment of metabolic stability and cytochrome P450 (CYP) inhibition are becoming important parameters to determine earlier in the drug development process and are now increasingly assessed during early drug discovery. The evaluation of metabolic stability is important because the parameters of half-life (t1/2) and the elimination rate constant (kel) can be used to determine in vitro intrinsic clearance (CLint) which can then be scaled to estimate in vivo intrinsic clearance (CLint,H). This information is important since the goal of the pharmaceutical industry is to develop drugs that require minimal dosing and maximum therapeutic exposure. Conversely, the assessment of CYP inhibition is important because CYP enzymes are typically the primary pathways of biotransformation for the majority of drugs. As many drugs are co‑administered simultaneously, the inhibition of any given CYP isoform by a perpetrator drug may result in the impaired clearance of a victim drug, leading to elevated exposures that may be toxic (i.e. drug-drug interactions; DDIs).

In the present study we developed high content screens using automated methods on a Tecan liquid handler to provide metabolic stability endpoints (i.e. t1/2, kel, CLint) and CYP inhibition endpoints (i.e. IC50 values). Briefly the method for metabolic stability determination was developed to screen at a single concentration up to 16 compounds incubated at 37°C with human liver hepatocytes, microsomes, or other subcellular fractions from up to six different species at four time points. Compounds such as alprazolam & warfarin (low CLint), diltiazem & propranolol (intermediate CLint), verapamil & midazolam (high CLint) among others were used to qualify the assay. The compounds were analyzed by LC/MS/MS and the data were processed by a LIMS. For the assessment of CYP inhibition, the method was developed to screen up to 12 compounds at seven concentrations (with a solvent control) incubated with human liver microsomes (0.1 mg/mL) at 37°C, with and without a 30 min preincubation (for the assessment of metabolism-dependent inhibition; MDI) followed by a 5 min seven-substrate cocktail incubation (with probe substrates specific for seven CYP isoforms, namely phenacetin - CYP1A2; bupropion - 2B6; paclitaxel - 2C8; diclofenac ‑ 2C9; S-mephenytoin - 2C19; dextromethorphan - 2D6 and midazolam ‑ 3A4/5). Specific CYP inhibitors as well as the pan-CYP inhibitor 1‑aminobenzotriazole (1-ABT) were used to generate IC50 values and qualify the assay. Samples were analyzed by LC/MS/MS and the data were processed using a LIMS.

Overall the results from both screens demonstrated the robustness of these assays for use in early drug discovery screening, allowing for earlier drug liability detection and lead optimization.