ISSX 2013 - Aldehyde Oxidase

Published:  29 September 2013

Selection of human liver S9 and cytosol fractions for evaluating clearance by aldehyde oxidase (AO): The impact of low versus high AO activity lots.

Aldehyde oxidase (AO) is a cytosolic enzyme present in the liver of humans and other mammals that catalyzes various oxidation and reduction reactions.  Biotransformation by AO is an important clearance mechanism for many drugs and drug candidates, with increasing importance in certain chemical spaces, and in some cases, such as zaleplon, Aldehyde oxidase metabolism leads to rapid in vivo clearance. Several publications have demonstrated the under-prediction of in vivo human clearance from in vitro clearance data, which are typically conducted with human liver subcellular fractions, such as S9 or cytosol.  Zientek and colleagues (2010)1 described a rank order approach, or ‘yard-stick’ approach, to categorize known AO substrates into low, medium or high clearance categories based on in vivo data.  With this approach, new drugs candidates can be evaluated in vitro in S9 or cytosol and the predicted in vivo clearance can be qualitatively ranked from low- to high-clearance.  These subcellular fractions, S9 and cytosol, are commercially available from multiple sources and in many formats (individuals and pools of various sizes), which causes variation in AO activity. 
Because of the necessity to scale AO clearance with a rank-order approach, the present study set forth to determine which human liver S9 and cytosol lots (individual or pooled) can be utilized to predict in vivo AO clearance once threshold values are determined with appropriate probe drugs.  Therefore, this study evaluates the impact of low versus high AO activity in human liver S9 and cytosol preparations on the prediction of scaled clearance for AO substrates with the ‘yard-stick’ approach.

ISSX 2013 - BCRP Transport Inhibition

Published:  29 September 2013

System-dependent inhibition of BCRP transport by high- and low-permeable compounds in bi-directional MDCKII cells and membrane vesicles

Breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter that blocks absorption at the apical membrane of enterocytes in the intestine, blood-testis barrier, blood–brain barrier, mammary gland and other cells. At the apical membranes of hepatocytes and renal proximal tubule cells, BCRP enhances excretion and elimination of xenobiotics. Inhibition of BCRP by drugs or xenobiotics has the potential to cause pharmacokinetic drug-drug interactions whereby increasing the exposure of co-administered compounds typically excreted from cells by BCRP.  For example, inhibition of BCRP in the intestine upon oral administration of GF120918 caused a 2.4-fold increase in the AUC of topotecan, a BCRP substrate.
Therefore, the recent FDA and EMA Guidance recommends evaluation of BCRP inhibition by new drug candidates, which is typically conducted in one of two test systems: 1) Bi-directional cell-based transport assays (e.g. MDCKII-BCRP cells) or 2) BCRP-expressing inverted membrane vesicles.  In the case of cell-based assays, BCRP function is evaluated by measuring active efflux across the cell monolayer in the apical to basal direction inasmuch as the substrate binding site(s) is located inside the cell.  Conversely, BCRP function in inverted membrane vesicles is evaluated by monitoring the uptake of a probe substrate into the vesicles inasmuch as the substrate binding domain is located on the outside of the vesicle and is exposed to free drug available in the incubation medium.  Because of this difference in substrate or inhibitor exposure to the substrate binding domain of BCRP, compounds with various intrinsic permeabilities may cause test system-dependent inhibition of BCRP in vitro

In this study, we evaluated the system-dependent effects of both a high- and low-permeability compounds on BCRP-mediated efflux, namely Ko143 and sulfasalazine, in MDCKII-BCRP cells and BCRP-expressing membrane vesicles.   Ko143 is a potent and specific inhibitor of BCRP with moderate permeability (predicted logD7.4 >2 and total polar surface area (TPSA) of ~98 Å2) as referenced in Table 1. Sulfasalazine, an anti-inflammatory agent, was reported to cause potent inhibition of BCRP transport (IC50 ~ 1 µM) in BCRP-expressing oocytes.  However, sulfasalazine, with a predicted logD7.4 ~ 0 and TPSA of >140 Å2 (Table 1), is poorly absorbed with limited permeability across physiological membranes.  We hypothesize that, due to limited membrane permeability, sulfasalazine would cause significantly less inhibition of BCRP transport in cell-based assays.

ISSX 2013 - Clearance of CYP2D6 and CYP3A4

Published:  29 September 2013

Test system-dependent clearance of CYP2D6 and CYP3A4/5 substrates: A comparison of human liver microsomes and cryopreserved human hepatocytes

The in vitro to in vivo extrapolation (IVIVE) of drug clearance involves the determination of intrinsic clearance in vitro (CLint), based on in vitro measurements of Vmax/Km or half-life (t1/2) in human liver microsomes or cryopreserved human hepatocytes, which are then scaled to predict hepatic clearance in vivo (CLH,int). Although in vitro values of CLint often underpredict in vivo values of CLH,int, the values of CLint determined with human liver microsomes would be expected to match those determined in hepatocytes for drugs predominantly cleared by cytochrome P450 (CYP).  However, in the case of drugs rapidly cleared by CYP3A4, there are reports, showing that CLint values determined in microsomes are much greater than those determined in hepatocytes whereas the opposite has been observed with drugs that are slowly cleared by CYP2D6 1, 2.  In the present study we examined the clearance of the CYP3A4/5 substrate midazolam (high intrinsic clearance) and the CYP2D6 substrate dextromethorphan (low intrinsic clearance) in human liver microsomes and cryopreserved human hepatocytes to confirm the findings of previous reports and investigate why hepatocytes cannot support the same high rates of drug clearance supported by human liver microsomes.

ISSX 2013 - CYP Induction Time Course

Published:  29 September 2013

Time-course of Cytochrome P450 (CYP450) induction in cultured human hepatocytes: Evaluation of activity and mRNA expression profiles for six inducible CYP450 enzymes 

Enzyme induction potential for new drug candidates is typically evaluated in vitro with cultured human hepatocytes with a demonstrated utility for the quantitative prediction of drug interactions involving cytochrome P450 (CYP450) enzymes, particularly CYP3A4.  Recently, many researchers have begun to measure CYP450 mRNA expression in vitro for prediction of clinical outcomes for CYP3A4 with relative success. Furthermore, recent regulatory guidance from the FDA and EMA now recommends CYP450 mRNA expression as the primary in vitro endpoint as opposed to CYP450 activity. 
The purpose of this study was to evaluate the time-course of CYP450 induction in cultured human hepatocytes to determine the adequate treatment period and establish CYP450 mRNA expression or enzymatic activity as reasonable endpoints for each of the six inducible hepatic CYP450 enzymes, namely CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4.

ISSX 2013 - Comparing Ki and IC50 Values

Published:  29 September 2013

Comparison of Ki and IC50 values for prototypical and clinically-relevant probe substrates of the hepatic transporters OATP1B1 and OATP1B3

Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1, SLCO1B1 and OATP1B3, SLCO1B3) are transmembrane proteins expressed in the sinusoidal membrane of human hepatocytes and are capable of transporting a wide range of hydrophilic molecules from the blood into the hepatocyte for subsequent metabolism and/or elimination.  Inhibition of OATP1B1 and/or OATP1B3 by xenobiotics can lead to pharmacokinetic drug-drug interactions whereby the exposure (AUC) of a co-administered victim drug (substrate) is increased in the presence of the inhibitory drug.  For example, orally administered cyclosporin inhibits OATP-mediated uptake of pravastatin-, a prototypical in-vivo probe substrate of OATP, which causes a 9.9-fold increase in AUC. The increase in systemic exposure to narrow safety margin drugs (e.g. statins) results in undesired side-effects and dose-limiting toxicities.  Therefore, the FDA and EMEA recommend the evaluation of new drug candidates for the potential to inhibit OATP1B1 and OATP1B3 which is often first evaluated with in vitro test systems, typically IC50 experiments.

The recent EMEA Guideline on the Investigation of Drug Interactions (2012) recommends the calculation of Ki values when evaluating the inhibition of a transporter by investigational drugs. Although Ki values have been reported for inhibitors of various uptake transporters, the difficulty of experimental design coupled with vigorous mathematical extrapolation can make the determination of intrinsic Ki values difficult and oftentimes reverts to the determination of extrinsic IC50 values. Additionally, it is important to consider clinically relevant substrates rather than prototypical probe substrates when evaluating potential DDIs of investigational drugs at the transporter level. In this study, Ki and IC50 values were determined for one prototypical (estradiol-17β-glucoronide (E2G)) and one clinically-relevant (pravastatin) substrate of the hepatic uptake transporters OATP1B1 and OATP1B3 with an experimental design incorporating multiple substrate and inhibitor concentrations.

ISSX 2013 - Cytokines in Kupffer Cells

Published:  29 September 2013

Endotoxin up-regulates the proinflammatory cytokines TNF-α and IL-6 in freshly-isolated human Kupffer cells.

Kupffer cells, macrophages endogenous to the liver, can modulate hepatic inflammation and injury associated with various pathophysiologies and toxicities.  Pro-inflammatory cytokines released by activated Kupffer cells, such as TNF-α and IL-6, are associated with up-regulation of acute-phase response proteins and suppression of CYP enzymes.  For new biological entities (NBE), particularly modulators of the immune system, evaluating the potential for Kupffer cell activation is an emerging concept in preclinical development.  As testing of NBE develops, robust and predictable test systems are necessary, thus the need to evaluate Kupffer cell isolation, culturing and stimulation for in vitro applications.  In the present study, Kupffer cells were isolated from non-transplantable human livers followed by expansion in culture and evaluation of the cytokine response to endotoxin (E. coli lipopolysaccharide, LPS). 

ISSX 2013 - S9 Long-Term Storage

Published:  29 September 2013

Human liver S9 fractions stored at -70°C maintain high phase I and phase II enzymatic activities over multiple freeze/thaw cycles and for at least 10 years 

Expanded interest in the biotransformation of xenobiotics by phase II enzymes and other various cytosolic enzymes has led to an increase in the use of S9 fractions in metabolic stability, clearance, and phenotyping studies. To ensure the integrity of an in vitro test system, it is important to understand the stability of metabolic enzymes during long-term storage and throughout multiple freeze/thaw cycles.  Data regarding the stability of human liver microsomes have been previously published by multiple authors (Pearce et al, 1996, Yamazaki et al, 1996).  However, metabolic enzyme stability for S9 stored for long periods and/or subjected to multiple freeze/thaw cycles has not been thoroughly investigated.
The objectives of this study were to evaluate the effects of long-term storage at -70°C or below and multiple freeze/thaw cycles on the enzymatic activities in human liver S9 fractions.  Nine lots of pooled (n = 16 to 200) human liver S9 samples were prepared over a ten year period and stored at -70°C or below for up to 10 years.  These S9 fractions were analyzed for their ability to catalyze reactions for various phase I and phase II enzymes including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), sulfotransferase (SULT), aldehyde oxidase (AO), and N-acetyltransferase (NAT). Additionally, a single lot of pooled (n = 200) human liver S9 samples were subjected to up to 10 freeze/thaw cycles and further evaluated for potential loss of enzymatic activities with increasing freeze/thaw cycles.

ISSX 2013 - Midazolam Clearance

Published:  29 September 2013

Midazolam clearance in human hepatocytes is restricted compared with human liver microsomes but not by cell permeability or cofactor availability

In our previous work (see accompanying poster) we confirmed previous reports that the clearance of midazolam, but not dextromethorphan, in cryopreserved human hepatocytes (n = 50) was an order of magnitude less than that in pooled human liver microsomes (n = 200). We also demonstrated that this was not due to low levels or activity of CYP3A4/5 in human hepatocytes because CYP3A4/5 activity in microsomes isolated from the pooled human hepatocytes was comparable to that in pooled human liver microsomes (within 20%). Previous investigators have proposed that the restricted clearance of midazolam (and other high clearance drugs metabolized by CYP3A4) in hepatocytes may be due a limitation imposed by membrane permeability or the availability of cofactor (NADPH); two factors that would not impact midazolam clearance by human liver microsomes. In the present study, we investigated if these factors were involved in the greatly reduced clearance of midazolam in human hepatocytes relative to human liver microsomes. Furthermore, the influence of ionic strength on CYP3A4/5 activity was also investigated, and was identified as a potential cause for the observed differences in CYP3A4/5 activity in microsomes versus hepatocytes.

ISSX 2013 - HIV Protease Inhibitors

Published:  29 September 2013

Substrate-Specific Inactivation of CYP3A by the HIV Protease Inhibitors Ritonavir, Saquinavir and Amprenavir

HIV protease inhibitors (PIs), such as ritonavir, saquinavir, and amprenavir, produce profound and clinically significant drug-drug interactions (DDIs) by time-dependent inactivation of CYP3A enzymes. Therefore, it is surprising that these PIs occasionally do not produce a clinically significant DDI with some CYP3A substrates when one is expected. For example, chronic administration of ritonavir significantly increases midazolam AUC but has no effect on alprazolam AUC and this has been shown not to be due to CYP3A4 induction.  Since CYP3A4 has multiple binding sites, we hypothesized that the PIs inactivate CYP3A enzymes in a substrate-dependent manner. Therefore, in the present study, we evaluated the in vitro CYP3A inactivation kinetics of ritonavir, saquinavir or amprenavir with several model CYP3A probe substrates, namely alprazolam, testosterone, nifedipine, alfentanil, or midazolam. Inactivation of CYP3A enzymes in human liver microsomes or rCYP3A4 was quantified by determining the maximum inactivation rate constant (kinact) and the inactivation constant (KI).

ISSX 2013 - Repaglinide Case Study

Published:  29 September 2013

High-resolution mass spectrometry for rapid metabolite characterization and reaction phenotyping: a case study with repaglinide 

Assessing the involvement of specific cytochrome P450 (P450) enzymes in biotransformation of a drug is an important step in evaluating overall disposition and victim potential for clinically-relevant drug-drug interactions. Current methods for biotransformation pathway identification (reaction phenotyping) rely on prior knowledge of metabolites, biotransformation routes, and availability of metabolite reference standards or radioisotopically-labeled drug. Synthesis of metabolite standards and radiolabeled compounds is time-consuming and expensive and the material is typically not available in early preclinical testing. Reaction phenotyping can be achieved by monitoring loss of the parent drug, but that approach suffers from a lack of specificity. Consequently, it is preferable to monitor the formation of one or more metabolites to obtain a comprehensive biotransformation map for the drug of interest. In general, this requires radiolabeled drug or metabolite reference standard material, potentially adding several months to the drug development timeline before biotransformation pathway assignments can be made.
High-resolution mass spectrometry (HRMS) is a powerful tool for a priori metabolite profiling and characterization because complex data sets comprising information on all of the components in a sample, within a specified mass range, are acquired. HRMS data can be employed for relative quantitation of all detected components and have potential for derivation of earlier stage reaction phenotyping information without the need for metabolite reference standards. In the present study, the prandial glucose regulator repaglinide was incubated with various in vitro reaction phenotyping test systems, and the samples were analyzed by HRMS metabolite profiling approaches to characterize all of the repaglinide metabolites formed. Relative amounts of each individual metabolite detected in different incubation test systems were compared to establish relationships between specific P450 enzymes and repaglinide metabolites. In the absence of metabolite reference standards, the abundance values were not compared across different metabolites since ionization efficiency for each metabolite is unknown. The approach was assessed for its suitability for rapid determination of specific biotransformation pathways for a drug in development without the additional time and cost associated with generating radiolabeled drug or metabolite reference standards.

AAPS Transporter 2013 - Uptake and Efflux Transporters

Published:  05 March 2013

Comparison of IC50 Values for Prototypical and Clinically-Relevant Probe Substrates and Inhibitors of Selected Uptake and Efflux Transporters

The EMEA Guideline on the Investigation of Drug Interactions (2012) recommends the incorporation of more than one positive control inhibitor into experiments to evaluate the inhibitory potential of investigational drugs on drug transporters. Therefore, the present study was designed to compare the effect of compounds that are prototypical inhibitors or that cause clinically-significant transporter interactions on the transport of compounds that represent either prototypical and/or clinically-relevant transporter substrates with in vitro, cell-based test systems.

AAPS Transporter 2013 - Inhibitors of Uptake Transporters

Published:  05 March 2013

Comparison of Ki and IC50 Values for Prototypical Inhibitors of Uptake Transporters

Ki values were determined for prototypical inhibitors of uptake transporters with an experimental design incorporating multiple substrate and inhibitor concentrations. The EMEA’s Guideline on the Investigation of Drug Interactions (2012) recommends that Ki values be calculated for the inhibition of a transporter by investigational drugs and IC50 values used only when a Ki value is not possible. Although Ki values have been reported for inhibitors of various uptake transporters, the methodology differs between labs and oftentimes relies on evaluation of a single concentration of the probe substrate combined with a mathematical extrapolation of the Ki value. The present study was designed to compare IC50 values to Ki values experimentally determined with four concentrations of both probe substrate and inhibitor.