Scientific Posters

Human monoclonal antibody stability in rat cell cultures using surrogate peptide LC-MS/MS

Published:  04 June 2019

Evaluating stability of human monoclonal antibody in rat cell cultures using a surrogate peptide LC-MS/MS approach

Nadya Galeva, Reed Murbach, Krystal Gilligan, Kevin Westland, Seema Muranjan
Sekisui XenoTech, LLC, Kansas City, KS, USA

In vitro evaluations of monoclonal antibody (mAb) in drug development can be both useful and challenging. Cytotoxicity and stability of protein therapeutic candidates at relevant concentrations in cell culture are of interest in pharmaceutical research.  Bioanalytical strategies for mAb LC-MS based analysis involve using either intact or digested mAb. Our approach for determining the stability of a human mAb in cultures of primary rat hepatocytes was to use enzymatic protein digestion followed by LC-MS/MS quantitation of five surrogate peptides...
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Assessment of in vitro inhibition of CYPs, UGTs and transporters by oligonucleotides

Published:  28 May 2019

An assessment of the in vitro inhibition of cytochrome P450 enzymes (CYP), UDP-glucuronsyltransferases (UGT) and transporters by phosphodiester- or phosphorothioate-linked oligonucleotides

Dr. Brian W. Ogilvie
Sekisui XenoTech, LLC, Kansas City, KS, USA
What is already known about this subject?
  • Antisense oligonucleotides (ASOs) are increasingly being developed for many indications and are often modified with phosphorothioate linkages in lieu of phosphodiester linkages.
  • Few in vitro DDI studies with ASOs have been performed, including the investigational imetelstat, volanesorsen, and ISIS 681257.
  • No clinical DDIs have been reported.
What this study adds:
  • This study evaluated the inhibitory effects of two non‑therapeutic oligonucleotides with either phosphodiester (PD-GP and PD-Ac) or phosphorothioate (PT-GP and PT-Ac) linkages on the major drug-metabolizing CYPs and UGTs in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHH), and toward select transporters in expression systems.
Regulatory guidance documents do not have specific recommendations for ASO DDI evaluation, but these molecules do fall under the scope of overall DDI testing.
  1. Evaluate different ASOs as inhibitors of CYPs, UGTs and transporters in vitro.
  2. Determine if in vitro inhibition of CYPs and UGTs in HLM by phosphorothioate ASOs is an artefact not observed in CHH...
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CYP1A2 Enzyme Localization in Normal and Diseased Pediatric Livers

Published:  13 March 2019

B. W. Ogilvie1, M. Czerwinski1, A. Kats2, M. T. Pritchard3,4, and S. E. Tague5.
1Sekisui XenoTech LLC, Kansas City, KS; 2Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO; 3Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 4The Liver Center, University of Kansas Medical Center, Kansas City, KS; and 5Kansas Intellectual and Developmental Disabilities Research Center, University of Kansas Medical Center, Kansas City, KS

CYP1A2 is a Phase 1 drug metabolizing enzyme whose expression begins between birth and 4 weeks of age, and gradually increases to about half of the adult levels by 6 years of age. The enzyme is expressed predominantly in liver where it constitutes 4 - 16% of the total hepatic CYP pool, and is a major determinant of the biotransformation of ~9% of clinically used drugs. Interestingly, CYP1A2 activity is decreased in adults with non-alcoholic fatty liver disease (NAFLD). Considering the increase in the number of medications given to children, as well as the rise in childhood obesity and NAFLD, this study utilized a pediatric liver tissue microarray (TMA) to determine if CYP1A2 abundance and localization was influenced by donor age and disease...

As this was selected to be a 20-min. Xenobiotic Disposition in Disease and Toxicities Topic platform presentation at the Society of Toxicology (SOT) Meeting & ToxExpo, no poster was presented. However, you may request information from the presentation or a webinar on the topic.

Patterns of Hyaluronan Deposition in Normal and Diseased Pediatric Livers

Published:  13 March 2019

Presented at the March 2019 Annual Society of Toxicology (SOT) Meeting in Baltimore, MD.
Maciej Czerwiński1, Alexander Kats2, Jordan Surgnier3, Sarah E. Tague4, and Michele T. Pritchard3,5
Sekisui XenoTech, Kansas City, KS., 2Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO., 3Department of Pharmacology, Toxicology and Therapeutics, 4Kansas Intellectual and Developmental Disabilities Research Center, 5The Liver Center, University of Kansas Medical Center, Kansas City, KS

The incidence of obesity and non-alcoholic fatty liver disease (NAFLD) is growing in children. Hyaluronan (HA), an extracellular matrix (ECM) glycosaminoglycan, is increased in liver and blood from adults with advanced liver disease. HA accumulation is also associated with inflammation and fibrosis in extrahepatic tissues. HA levels have not been examined in healthy or diseased livers from pediatric patients. Here, we sought to determine if hepatic HA deposition was associated with donor age and/or disease by using a pediatric liver tissue microarray (TMA)...

Alcohol Consumption Uniquely Increases Hepatic Hyaluronan Content in Patients with Liver Steatosis

Published:  12 November 2018

History of Alcohol Consumption Uniquely Increases Hepatic Hyaluronan Content in Patients with Liver Steatosis

Dr. Maciej Czerwinski1, Jordan Surgnier2, Dr. Sarah E. Tague3 and Dr. Michele T. Pritchard2
 Sekisui XenoTech LLC; 2 Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center; 3 Kansas Intellectual and Developmental Disabilities Research Center, University of Kansas Medical Center

Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is increased in plasma from donors with advanced liver disease and is used as a biomarker to assess liver disease severity. Extrahepatic HA content and its fragmentation are associated with inflammation and/or fibrosis.

In chronic, persistent hepatitis, as compared to normal tissue, HA was increased in fibrous areas around the portal tracts and the central vein, but it was rarely detected in the sinusoidal walls. In chronic, aggressive hepatitis, HA was present in periportal sinusoidal walls in addition to fibrous tissues of the portal tract (Ichida, 1996).

In cirrhotic tissues, as compared to normal, HA was significantly increased in fibrous areas of the portal tract but to a lesser extent in the sinusoidal walls around periportal areas (Ichida, 1996).

In alcoholic cirrhosis, HA was also observed in the sinusoidal wall and fibrous areas around the portal tract and central vein and its abundance in these locations diminished upon alcohol abstinence (Urashima, 1999).

Since hepatic levels and patterns of distribution of HA have not been examined in early liver disease (steatosis), we utilized liver tissue microarrays (TMAs) and histochemical staining with a hyaluronan binding protein (HABP) to determine if hepatic HA increases in donors with steatosis and if HA content is affected by history of alcohol consumption.

Cytokine-Mediated Suppression of CYP Enzymes By a Toll-like Receptor 9 Agonist in Hepatocytes

Published:  07 November 2018

Cytokine-Mediated Suppression of CYP Enzymes By the Toll-like Receptor 9 Agonist, IMO-2125, in Cultured Human Hepatocytes

Paul Tarantino1, Tim Sullivan1, Brian Ogilvie2, Maciej Czerwiński2
1 Idera Pharmaceuticals, Inc., 167 Sidney Street, Cambridge, MA 02139;
2 Sekisui XenoTech LLC, 1101 W. Cambridge Cir. Drive, Kansas City, KS 66103

The innate immune response to some drugs may involve the release of proinflammatorycytokines, such as IL-6, which have the ability to suppress xenobiotic-metabolizing CYP enzymes and affect the pharmacokinetics of co-administered small molecules. The 2012 FDA Drug-Drug Interaction (DDI) guidance recommended the evaluation of therapeutic proteins such as cytokines or cytokine modulators for their ability to modulate the expression of CYP enzymes or transporters (Food and Drug Administration (US), 2012). Although not a peptide drug, it is possible that an immuno-modulator, such as the oligonucleotide, Tilsotolimod(IMO-2125), an investigational agonist of toll-like receptor 9 designed to enhance T-cell responses to tumor antigens, could precipitate DDIs. Tilsotolimodalters the tumor microenvironment by improving antigen presentation by dendritic cells and macrophages with subsequent proliferation of antigen-specific cytotoxic T lymphocytes (CD8+ T cells) in both injected and distant tumors resulting in tumor cell death (Figure 1). In this study, we investigated the potential of Tilsotolimodto to cause direct and cytokine-mediated effects on CYP mRNA expression and enzyme activity in primary human hepatocytes according to a published in vitro method. The method comprised stimulation of human blood with a drug or a therapeutic protein followed by separation and subsequent incubation of human hepatocytes co-cultured with Kupffer cells with the drug-treated plasma to assess the potential of the drug to alter the expression of drug metabolizing enzymes and transporters through its effect on cytokines. All cultures of hepatocytes used in this study were co-cultures of hepatocytes and Kupffer cells isolated from the same donors in routine perfusion and plating procedures. The test system accounts for the combined effects of all cytokines stimulated by the drug in blood on CYP expression in primary cultures of human hepatocytes...

Inhibitory effect of typical CYP inhibitors under long term incubation in human liver microsomes

Published:  05 October 2018

Sho Nishinoaki, Ryo Fujino, Kenta Hashizume, Tsutomu Negama
Drug Development Solutions Center, Drug Development Solutions Division, Sekisui Medical Co., Ltd. 2117 Muramatsu, Tokai, Ibaraki 319-1182, Japan

Cytochrome P450 (CYP) isoforms are responsible for the metabolism of the majority of drugs in human. In case of the metabolic enzyme identification of CYP isoforms for the low intrinsic clearance (CLint) candidate compounds, it is necessary to perform metabolic reaction with long term incubation. In addition, when performing the metabolic enzyme identification with long term incubation, the specificity and sustainability of a typical inhibitor are important. In this research, we evaluated the inhibitory effect of typical inhibitor for each CYP isoform in human liver microsomes (HLM) under long term incubation (incubation time of CYP typical inhibitors was 60 minutes, final concentration of HLM was 1 mg protein/mL) to clarify an optimal concentration of the CYP typical inhibitors under long term incubation. In this presentation, we have confirmed specificity and sustainability of each typical inhibitor for CYP isoform under long term incubation in HLM.

Ultrasensitive quantitation assay of oligonucleotide therapeutics based on the PALSAR method

Published:  05 October 2018

Akane Omori, Koichi Shibusawa, Kazuhiro Takeuchi, Akira Ideno
Tokai Research & Development Center, Research & Development, SEKISUI MEDICAL CO., LTD. 2117 Muramatsu, Tokai, Ibaraki 319- 1182, Japan

Recently, development of oligonucleotide therapeutics has accelerated in the pharmaceutical industry, and the needs for ultrasensitive quantitation methods have greatly increased. Conventional approaches, such as hybridization, LC-MS/MS, PCR are used in the analysis. Hybridization is commonly used, yet sensitivity is insufficient. Sensitivity of LC-MS/MS is improved by instrumental advancement, but still not enough. PCR and qRT-PCR are also known as sensitive detection methods; however, the high variability of measured values is a problem. To solve these problems, we aimed to develop an ultrasensitive hybridization assay method without nucleic acid amplification procedure.

Microsomal Cytochrome P450 Enzyme Activities in Nonalcoholic Steatohepatitis Livers

Published:  16 July 2018

Maciej Czerwinski1, Brian Oberheide1, Nicholas Hatfield1, Bill Ewy1, Christopher Seib1, Eva Gatineau2, Frederique Yiannikouris2, Brian Ogilvie1
1 Sekisui XenoTech, LLC
2 University of Kentucky, Department of Pharmacology and Nutritional Sciences

The prevalence of nonalcoholic steatohepatitis (NASH), a chronic liver disease, has increased drastically in parallel with the raise of obesity in the US. This condition affects hepatic drug metabolism and has potential to impact drug-drug interactions. Our study aimed to evaluate microsomal CYP enzyme activities, organ fibrosis and microvesicular steatosis in NASH tissues deposited in the Sekisui XenoTech Biobank, and to establish whether these tissues have application as an in vitro test system for the study of NASH impact on metabolism of xenobiotics. NASH tissues were identified based on the examination of hematoxylin and eosin (H&E), Masson’s trichrome staining and donor history of alcohol consumption. All samples had intra-lobular inflammation, ballooning necrosis and macrovesicular fat >5%. Fibrosis stage was assigned based on Brunt et al. The degree of microvesicular steatosis, evaluated by H&E staining, did not correlate with the body mass index (BMI) nor with progressive stages of fatty liver disease in 51 donors. However, the extent of microvesicular steatosis correlated positively with triglyceride contents (R2=0.66), but not with total cholesterol levels. Microsomal protein yield was weakly negatively correlated with microvesicular fat content (R2=0.11) but did not correlate with BMI. The majority of average microsomal CYP activities in NASH livers were lower than in the general population of liver donors by 6 to 65%. In contrast, the average CYP2E1 microsomal activity in NASH livers was 3.5% higher than in the general population of liver donors in the Biobank. These observations were in agreement with published clinical data. A microsomal pool of five NASH donors and tissue micro arrays containing NASH and fatty livers from donors with and without history of alcohol consumption were prepared to assist in disease evaluation. The NASH pattern of CYP enzyme activities seen in the patients and in the microsomes prepared from non-transplantable NASH livers suggest that the subcellular fraction is an appropriate test system for analysis of CYP-mediated xenobiotic metabolism associated with the disease state.

Current In Vitro DDI Regulatory Guidance Reference Poster

Published:  29 May 2018

Brian W. Ogilvie1 and Andrew Parkinson2
1Sekisui XenoTech, LLC, Kansas City, KS, USA and 2XPD Consulting, Shawnee, KS, USA

In September, 2017, the Japan PMDA revised its 2014 guideline and released it for comments. In October, 2017, the US FDA revised and split its 2012 draft guidance for industry on in vitro drug-drug interaction (DDI) studies, into one document for in vitro DDI studies, and another for clinical DDI studies. An overview of the major changes, a comparison of each agency’s equations and cut-off values (including the most recently released 2013 EMA DDI guidelines), and a comparison of experimental details will be highlighted. This poster will also highlight strategies to harmonize the design of in vitro DDI studies to meet the expectations of both agencies.

Evaluation of interspecies metabolic activities for Non-CYP enzymes, suitable incubation conditions

Published:  01 December 2017

Evaluation of interspecies metabolic activities for Non-CYP enzymes and into a suitable incubation condition for human
Recently, lead compound screenings has become widely used in drug development. This has led to the development of increasing range of lead compound structures with metabolic stability for Cytochrome P450 (CYP). However, because of the unpredictable metabolic reaction and species differences by non-CYP enzymes, there have been cases that the test compound’s blood concentration was found to be significantly lower than expected in the clinical phase, resulting in the discontinuation in its development. Therefore, it is necessary to establish an in vitro evaluation system of metabolic activities for non-CYP enzymes. We evaluated interspecies differences in metabolic activities for non-CYP enzymes (aldehyde oxidase (AO), aldo-keto reductase (AKR) and carbonyl reductase (CR)) in liver cytosol (human, monkey, rat, mouse and dog). We also evaluated the inhibitory effect of typical inhibitor for each non-CYP enzyme (AO, xanthine oxidase (XO), AKR and CR). And, we tested the metabolic activities of non-CYP enzymes in anaerobic conditions using human liver hepatocytes. In this presentation, we have established an evaluation method for metabolic activities of non-CYP enzymes (AO, XO, AKR, CR).

Comparison of two hepatic uptake assay methods

Published:  01 December 2017

Comparison of two hepatic uptake assay methods, plated and oil layer methods, using human and preclinical species cryopreserved hepatocytes
The prediction of in vivo human hepatic clearance (CL) of drug from in vitro data is important for drug development. Primary human cryopreserved hepatocytes are widely used as an in vitro tool for the prediction of in vivo CL since a variety of hepatic uptake transporters are expressing in them. The oil layer method is a current major hepatic uptake assay method. On the other hand, the plated method has come to be used to evaluate hepatic uptake recently. In this study, we compared the difference between the plated method and the oil layer method using human cryopreserved hepatocytes in order to show the usefulness of the plated method. Also, we examined whether or not the plated method is useful for the evaluation of species difference of hepatic uptake using preclinical species (monkey, dog, and rat) cryopreserved hepatocytes.

Further Exploration into the Drug-Drug Interaction Between Gemfibrozil and Repaglinide in Rats

Published:  27 September 2017

Further Exploration into the Drug-Drug Interaction Between Gemfibrozil and Repaglinide in Rats: Uptake Transport
Chase I. McCoy, Forrest A. Stanley, Chandra Kollu, Seema Muranjan, Krystal M. Green and Joanna E. Barbara

The 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 (P450) 3A4 and 2C8 and UGT1A1 and 1A3. Gemfibrozil and its glucuronide metabolite inhibit CYP2C8 and UGT1A1, as well as the hepatic uptake transporter OATP1B1. These factors have been implicated in the clinical interaction. A gemfibrozil / repaglinide DDI resulting in increased repaglinide and metabolite plasma exposure (>3-fold) and a vectoral shift in elimination pathways from biliary to urinary excretion following administration of gemfibrozil was previously established in male Sprague-Dawley rats. In that study, negligible effects on liver enzyme activity were observed following gemfibrozil treatment, suggesting that the DDI in rats was unlikely to be caused by P450 or UGT inhibition. In the present study, the potential role of uptake transporter proteins in the interaction was explored in vitro.

The Effects of Organ Preservation Solution on Aldehyde Oxidase and Xanthine Oxidase Activity in...

Published:  27 June 2017

The Effects of Organ Preservation Solution on Aldehyde Oxidase and Xanthine Oxidase Activity in Pooled Human Liver S9

Non-transplant quality human livers donated for research are a common source of both cellular and subcellular material utilized in in vitro drug metabolism studies. At the time of organ recovery, livers are flushed with an ice-cold perfusion and storage solution that preserves the tissue during delivery from the recovery site to the research facility. The two most commonly used cold storage solutions are UW (University of Wisconsin) solution and Custodiol HTK (histidine tryptophan ketoglutarate) solution. One notable difference between the two solutions is the presence of 1 mM allopurinol in UW solution. It has been previously shown that1 mM allopurinol inhibits xanthine oxidase activity, but not aldehyde oxidase in human liver cytosol (Barr et al., 2014). Allopurinol exposure to human livers during cold storage could lead to underestimation of the contribution of xanthine oxidase in the metabolism of new chemical entities. Here, we hypothesized that preparing a pool of human liver S9 prepared exclusively from Custodiol HTK-preserved livers will have significantly higher xanthine oxidase activity compared to pools of liver S9 prepared from livers preserved primarily in UW solution. In this study, we compared AO and XO activity in pooled human liver S9 made from 20 donors preserved in HTK with those in two commercially available pools of S9 (n = 50 and 200) produced by Sekisui XenoTech from livers preserved primarily in UW solution. Xanthine oxidase activity was determined by measuring the oxidation of 6-ntiroquinazolinone to 6-nitroquinazolinedione. Aldehyde oxidase activity was determined by measuring the oxidation of phthalazine to 1-phthalazinone, p-vanillin to vanillic acid and the zaleplon to 5-oxo-zaleplon. To determine whether UW and HTK affected other drug metabolizing enzymes, we looked at the metabolism of appropriate marker substrates for the major cytochrome P450 (CYP) enzymes. S9 prepared from HTK-only livers had a 539% and a 264 to 359% higher xanthine oxidase and aldehyde oxidase than S9 prepared from primarily UW donors. Activities of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were not statistically different between the HTK and UW pools. These data indicate that a preparation of pooled human liver S9 prepared from tissue that is cold-preserved in HTK solution has increased xanthine oxidase and aldehyde oxidase activity compared to S9 prepared from livers preserved in UW solution.

Hepatocytes Are the Preferred Test System to Evaluate Oligonucleotide-CYP Interactions In Vitro

Published:  02 March 2017

Sekisui XenoTech previously examined the potential of oligonucleotides to inhibit cytochrome P450 (CYP) 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 HLM and pooled CHH. Briefly, oligonucleotides with either phosphodiester (oligo # 1 and #3) or phosphorothioate (oligo #2 and #4) backbones were evaluated in HLM 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 was assessed with a single concentration of oligos (30 mM). For CHH assays, MDI was assessed with 30 and 90 min pre-incubations, and 4-methylumbelliferone (4-MU) was used as a marker substrate of overall UGT activity. Incubations with CYP and UGT marker substrates were performed for 5–10 min and residual activity was assessed by LC-MS/MS analysis. The results indicated that the phosphodiester-backbone oligonucleotides had little to no inhibitory effect on CYP and UGT enzymes in HLM and CHH with the exception of oligo #2 in HLM for CYP2C19. Conversely, the phosphorothioate-backbone oligonucleotides caused direct inhibition of CYP and UGT enzymes. CYPs 1A2, 2B6, 2C8, and UGTs 1A1 and 2B17 were most highly inhibited by both phosphorothioate-backbone oligonucleotides. There was evidence of NADPH-independent inhibition of CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP3A4/5with oligo #2 and #4. In contrast to HLM, we observed little to no direct inhibition by any oligo in CHH (with the exception of oligo #2 with CYP2C19 and TDI by oligo #3 with CYP2C8), demonstrating test system-dependent outcomes. Overall this study indicates that hepatocytes, a test system physiologically more relevant than isolated microsomes, are the preferred model to evaluate oligonucleotide-CYP interactions in vitro.

Research Collection of Variants of Normal and Fatty Disease Human Livers

Published:  28 October 2016

Sekisui XenoTech has developed a Research Biobank that is a collection of normal, steatosis and steatohepatitis tissue samples gathered and characterized to facilitate the study of human liver disease with an emphasis on the progression of fatty liver disease. A portion of each of the human livers, which were harvested with the intent of transplantation but subsequently rejected for this purpose, and were obtained through partnerships with non-profit Organ Procurement Organizations (OPO) which are members of the United Network for Organ Sharing (UNOS), is saved in the Research Biobank. Donor-specific data provided by the organ procurement organizations includes demographics, cause of death, BMI, and alcohol and diabetes history. Pathologist’s review of the H&E slides includes classification of the samples into normal, steatosis or steatohepatitis categories and quantification of macrovesicular fat, inflammation, ballooning hepatocytes and fibrosis. Presence of fibrosis is confirmed with Masson's Trichrome staining. Tissues deposited in the bank are flash frozen in liquid nitrogen and stored at -80°C. Cells isolated from multiple tissues deposited in the bank are available as cryopreserved hepatocytes for culture in suspension or as an attached cell monolayer. These cells are suitable for an array of studies including in vivo in vitro correlation of drug metabolism and biomarker expression that characterize fatty liver disease. The bank contains normal, steatosis and steatohepatitis specimens, with and without a history of alcohol use. Photomicrographs of H&E slides of each tissue are available. The levels of CYP2A6, CYP2C19 and CYP3A4 mRNA expression in normal (10), steatosis (19) and steatohepatitis (11) specimens were analyzed by RT-PCR. The relative quantification of each of the enzymes was based on the ΔΔCT with GAPDH serving as an endogenous control. The relative quantification of the mRNAs was not affected by tissue pathology (ANOVA). In conclusion, we have established a Research Biobank, derived from transplantation-rejected organs, that is focused on alcoholic and non-alcoholic fatty liver disease.

Evaluation method for mitochondrial toxicity using Crabtree effect

Published:  15 October 2016

Mitochondria plays a crucial role in living organisms, one of which being the synthesis of ATP. Mitochondrial toxicity is widely tested as part of the compound’s toxicity screening and within FDA’s registered drug with a ‘black box warning’, roughly 80% of drugs are reported with mitochondrial toxicity. The aim of this research is to devise a simple method to evaluate mitochondrial toxicity across different toxicological mechanisms using human liver derived cells.

Evaluation of the CYP metabolic activities in the presence of albumin in human liver microsomes

Published:  15 October 2016

Cytochrom P450 (CYP) isoforms are responsible for the metabolism of the majority of drugs in human. An intrinsic clearance (CLint) determined from in vitro intrinsic clearance can be used to predict the in vivo hepatic metabolic clearance (CLH) of drugs in humans. However, in vitro-in vivo extrapolation (IV-IVE) commonly underestimates the in vivo CLint and CLH of drugs metabolized by CYP isoforms. Recently, it was reported that the addition of albumin to the in vitro incubation system increased the metabolic activities of some CYP isoforms due to inhibition of the effect of polyunsaturated long-chain fatty acids (PUFAs). Therefore, we evaluated the effect of bovine serum albumin (BSA) on the metabolic activities of CYP isoforms in human liver microsomes (HLM). We found that some CYP isoforms were observed to show enhancement of CLu,met in the presence of albumin. In this presentation, we have summerised the effect of albumin on the metabolic activity of each CYP isoform in HLM.

In Vitro Evaluation of Ketoconazole and its Alternatives as Non-CYP Inhibitors

Published:  13 June 2016

The In Vitro Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Inhibitors (Ritonavir, Clarithromycin and Itraconazole) as Inhibitors of Non-CYP Enzymes

Ketoconazole is a potent CYP3A4/5 inhibitor, and until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as a “strong” CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and EMA recommended suspension of ketoconazole use in DDI studies in 2013. FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors for use in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of non-CYP drug metabolizing enzyme activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto- and N-desalkyl itraconazole) towards several drug metabolizing enzymes were systematically assessed in human subcellular fractions (microsomes, S9 or cytosol) from hepatic or extrahepatic tissues or recombinant enzyme systems. The enzyme families or metabolic reactions tested for inhibition include UGTs (13), sulfonation, GST, CES1/2, AO, XO, FMO, MAO-A and B, NAT1 and 2, and COMT. Several enzymes were not markedly inhibited (< 20% inhibition) by any of the compounds tested, including XO, FMO, sulfonation, CES1, GST, NAT1 and COMT. Two enzymes, namely CES2 and NAT2 were moderately inhibited (20-40% inhibition) by one or more compounds tested.  AO was inhibited by ketoconazole, ritonavir and clarithromycin, but not itraconazole with IC50 values of 3, 29 and 64 µM, respectively. MAO-A was inhibited by only ketoconazole (IC50 values » 8 µM) and MAO-B by N-desalkyl-itraconazole and ketoconazole (IC50 values » 6 µM). One or more UGT enzymes was potently inhibited (IC50 values < 10 µM) by ketoconazole, ritonavir and itraconazole or its metabolites; however, clarithromycin did not cause potent inhibition (≥ 43 µM) of the UGT enzymes tested. Ketoconzole inhibited 12 of 13 UGT enzymes tested with IC50 values less than 10 µM for 6 of those enzymes.  Similarly, ritonavir and itraconazole and/or its metabolites potently inhibited 5/13 and 4/13 UGT enzymes, respectively, with IC50 values less than 10 µM in these cases.  The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin and itraconazole each have unique enzyme inhibition profiles. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when non-CYP enzymes are potentially involved in a victim drug’s pharmacokinetics.

Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Drug Transport Inhibitors

Published:  29 May 2016

Updated: Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

Ketoconazole is an orally available, synthetic, broad spectrum, antifungal agent. Approved in 1982 by the FDA for use in fungal infections, it is a known substrate and strong inhibitor of cytochrome P450 (CYP) 3A4 and 3A5.

Previously, a high dose of ketoconazole was considered the gold standard for use in clinical drug-drug interaction (DDI) as a strong CYP3A4/5 inhibitor. By 2013, ketoconazole use in clinical studies had been banned due in part to evidence demonstrating the potential for liver injury following long dosing periods. Typically, patients would exhibit asymptomatic, reversible liver function test abnormalities. As early as 1984, Van Tyle demonstrated evidence of DILI in approximately 0.1 to 1.0% of patients, with results indicating that there was no association with the dose, but with the duration of dosing. In later estimates, studies showed that ~134 per 100,00 persons, 4.9 cases per 10,000 patients, and 3.6 to 4.2% demonstrated liver abnormalities.

After ketoconazole was banned in clinical study use, the FDA recommended clarithromycin or itraconazole as an alternative, but indicating that other drugs may be used. Ritonavir was suggested by some as an alternative CYP3A4/5 inhibitor. Following an extensive study by Ke et al. where inhibitors were systematically evaluated, only itraconazole and clarithromycin were considered acceptable. Exclusion criteria included, the drug not being approved in the U.S., known non-specific inhibition of CYPs, safety issues, exclusive use with ritonavir, or only moderate information of CYP3A4/5.

There is minimal drug transporter data in the literature; which includes ketoconazole and ritonavir inhibition of P-gp and OATP1B1. The goal of this study was to allow for a more informed choice of a strong CYP3A4/5 inhibitor for clinical DDI studies involving a drug candidate known to be a substrate of one or more of the transporters and to help reduce confounding DDI results.

Published paper and webinar also available.

Automated High Content Drug Transport Screening with Transwell Plates for Drug Discovery

Published:  25 January 2016

  • The purpose of this study was to develop a method to use bidirectional intestinal permeability and drug transport to identify substrates and inhibitors of MDR1 or BCRP.
  • The alteration of drug transporters potentially affects clearance and efficacy.
  • Cells grown on transwell permeable membranes were washed, treated, and samples were collected with an automated liquid handler.
  • Data showed the automated method as consistent with in-house historic values and within and between assays.

Measuring bidirectional intestinal permeability and drug transport using Caco-2 and MDCKII can identify potential substrates or inhibitors of MDR1 or BCRP and identify the permeability class of potential new drugs. Two recent FDA documents, 2012’s Drug Interaction draft guidance and 2015’s Waiver of In Vivo Bioavailability and Bioequivalence, show the increasing interest and significance in transporters and permeability, particularly in MDR1 (P-gp) and BCRP. The alteration of drug transporters potentially affects clearance and efficacy. Early assessment of this potential leads to better, safer drugs and reduced development costs. A method utilizing a Tecan liquid handler and Caco-2 or MDCKII cells grown on permeable membranes (Figure 1) was created to measure bidirectional intestinal permeability and drug transport. The screen evaluates up to 14 compounds in one assay and measures permeability and efflux. Read more about Drug Transporter Screening.

In vitro characterization of human liver lysosomes isolated from fresh tissue

Published:  18 January 2016

Evaluation of lysosomal catabolism is an integral part of the development of biologic drugs including antibody-drug conjugates (ADC). Biologics can enter the cell by fluid-phase or cell surface antigen-mediated endocytosis and can be degraded by multiple catabolic enzymes found in lysosomes. Purified rat liver tritosomes or human lysosomes are convenient test systems for an in vitro evaluation of lysosomal stability of antibodies, linkers and small molecule drugs comprising ADC. Hepatic lysosomes were isolated from fresh human liver donors according to the discontinuous density ultracentrifugation method based on Wang, et al (2012). Twelve individual fractions collected from the OptiPrep density gradient were characterized for acid phosphatase, cathepsin B, and cytochrome c oxidase activity in order to confirm separation of the lysosome- from the mitochondria-specific proteins. Acid phosphatase activity was distributed among multiple fractions while cathepsin B was higher in lighter lysosomal fractions in contrast to the heavier fractions containing more cytochrome c oxidase. Fractions were further characterized with Western blotting for distribution of lysosomal-associated membrane protein 1 (LAMP-1, CD107a) and microsomal cytochrome c oxidase subunit 4 (COX4). In agreement with the enzyme activity data, the blots demonstrated separation of the LAMP-1 signal, detected in the lighter lysosomal fractions from that of the COX4 signal found in the heavier lysosomal fractions of the OptiPrep density gradient. Expected enrichment of lysosome-specific cathepsins L and S in isolated lysosomes was demonstrated with sandwiched ELISA Human Protease Array (R&D Systems). This work provides a characterization of isolated human liver lysosomes that constitutes a test system for an in vitro assessment of catabolic stability of biologics drugs entering the cell by the endosomal–lysosomal pathway.

Related Webinar: Investigation of freshly purified rat tritosomes and human hepatic lysosomes as an in vitro tool for characterization of biologic drugs

Efficient use of chimeric mice with humanized livers for drug development support

Published:  14 November 2015

Chimeric mice with humanized livers can be used to obtain supportive data for selecting the best candidate compound for new drug development. To utilize this costly animal model efficiently, we tried to verify that consecutive experiments could be conducted with the same animals to generate predictive human ADME.

Evaluation of Chemical Inhibitors for UDP-glucuronosyltransferase UGT Reaction Phenotyping Assays

Published:  21 October 2015

Evaluation of Chemical Inhibitors for UDP-glucuronosyltransferase (UGT) Reaction Phenotyping Assays in Human Liver Microsomes

In the development of a new chemical entity (NCE) reaction phenotyping studies are carried out to determine specific enzymes involved in the metabolism of the new drug candidate. These studies increase the understanding of how a drug is cleared and it’s potential to be subject to drug-drug interactions (DDI) with co-administrated medications. Polymorphisms and DDIs involving non-CYP metabolism routes (e.g., UDP-glucuronosyltransferases [UGT]) are becoming more widely recognized by regulatory agencies and therefore a need for further investigation exists (Harper et al., 2008, Rowland et al., 2013).

One approach to identify which UGTs are responsible for the metabolism of a drug is the use of chemical inhibitors in an in vitro test system utilizing pooled human liver microsomes (HLM), or pooled cryopreserved human hepatocytes (CHH) (Ogilvie et al., 2008). Factors such as cross-reactivity of inhibitors, protein binding, and depletion of the cofactor, etc. must be considered when designing these studies. Additionally, in vitro metabolism incubations should be conducted under initial rate conditions to obtain an accurate assessment of inhibition (Parkinson et al., 2011).

In the present study, we examined the selectivity of UGT inhibition with a range of protein concentrations and incubation times in HLM, for a variety of commonly used UGT inhibitors with the goal of identifying selective chemical inhibitors.

The Impact of Various Solvents and Solvent Concentrations on In Vitro Enzyme Induction Assessment

Published:  21 October 2015

The Impact of Various Solvents and Solvent Concentrations on In Vitro Enzyme Induction Assessment in Cultured Human Hepatocytes

Induction of cytochrome P450 (CYP) enzymes is one of the principal mechanisms of drug-drug interactions inasmuch as regulators recommend that new drug candidates be evaluated for their ability to induce CYP enzymes. The FDA (US), EMA (Europe) and PMDA (Japan) recently revised the guidelines for evaluating CYP induction (FDA, 2012). According to this new guidance document, a new chemical entity (NCE) should be evaluated at very high concentrations in vitro (e.g., ((0.1*dose)/250 mL) or 50x the total Cmax,ss) (EMA, 2013). Achieving these concentrations in vitro can be problematic if the test article is insoluble in preferred solvents (e.g., 0.1% v/v DMSO) and/or cell culture medium. Consequently, test articles are evaluated for solubility in other solvents; however, potential cytotoxic effects and effects on CYP induction of these chemicals are often unknown.

In the present study, the effects of various solvents on CYP induction and cytotoxicity were evaluated in three preparations of sandwich-cultured cryopreserved human hepatocytes. These solvents include DMSO, ascorbic acid, acetic acid, acetonitrile, dimethylacetamide, ethyl acetate, and methanol. Cytotoxicity and enzyme induction potential was evaluated by in situ marker substrate incubations and mRNA expression.

UGT Inhibition Studies in the Presence or Absence of Alamethicin Evaluation of UGT1A1 and UGT2B7

Published:  21 October 2015

UGT Inhibition Studies in the Presence or Absence of Alamethicin: Evaluation of UGT1A1 and UGT2B7 Inhibition in Human Liver Microsomes and Recombinant Enzymes

The importance of evaluating new drug candidates for the potential to inhibit
UDP-glucuronosyltransferase (UGT) enzymes
has recently garnered scientific and regulatory interest. To date, several published studies have demonstrated the importance of various membrane disruptors (e.g., alamethicin or CHAPS) and other exogenous protein sources (e.g., BSA or FABP) for improvement of the in vitro to in vivo prediction of metabolic clearance due to glucuronidation (Fisher et al., 2000, Rowland et al., 2007, Walsky et al., 2012). The active site of UGTs faces the lumen of the endoplasmic reticulum, and typically a detergent is used to disrupt the membrane allowing for maximal enzyme activity. However, there is little data available to evaluate the impact of these exogenous factors on evaluating inhibition of UGT enzymes in vitro

In the present study, we examined the effect of alamethicin, a pore forming agent, on UGT1A1 and UGT2B7 inhibition (IC50) in Human Liver Microsomes (HLM) and recombinant UGTs by a variety of commonly used UGT inhibitors with the goal of optimizing assays conditions.

Comparative Investigation of Benzoapyrene, Procaine and Nevirapine Metabolism in Human Hepatocytes

Published:  21 October 2015

A Comparative Investigation of Benzo[a]pyrene, Procaine and Nevirapine Metabolism in Human Hepatocytes and a 3D Skin Tissue Model

Skin metabolism is an important contributor to the safety and efficacy of dermally applied drugs, despite the fact that drug metabolizing enzyme activity is typically lower in the skin than in other organs more commonly associated with metabolism (Oesch et al., 2007). Therefore in vitro skin metabolism assays are necessary for development of topically-applied compounds. Knowledge of the drug metabolizing enzymes present in skin is incomplete, but several cytochrome P450 enzymes, flavin-dependent monooxygenases, alcohol and aldehyde dehydrogenases, various hydrolytic enzymes, and conjugation enzymes including glutathione S-transferase, glucuronosyltransferase, sulfotransferase, acetyltransferases and methyl transferase have been characterized in mammalian skin (Oesch et al., 2007; van Eijl et al., 2012). Consequently, skin metabolism can be as diverse as liver metabolism and needs careful experimentation to fully understand exposure profiles. The European animal testing ban of 2013 recently further reinforced the need for simple and reliable skin metabolism modeling assays for cosmetics safety testing. Procuring and working with skin-based test systems can be challenging. Viable whole skin for short-term culture is difficult to obtain and expensive apparatus are needed. Skin has high collagen content, which makes it difficult to process to subcellular fractions. Drug metabolizing enzymes found in skin are generally localized to keratinocytes in the epidermis making them a viable option, but they are difficult to isolate and culture, and culture conditions can strongly affect the drug metabolizing capabilities of the cells (Oesch, et al., 2007). The use of normal human 3D skin tissue models for genotoxicity and sensitization assays is rapidly increasing as an alternative approach (Brinkmann et al., 2013). In the present study, the commercially-available 3D skin model EpiDermTM (MatTek, Ashland, MA) was evaluated for metabolism of the three diverse test compounds benzo[a]pyrene, procaine and nevirapine and was compared to the well-established liver metabolism model cryopreserved human hepatocytes. EpiDermTM is a tissue culture model comprising reconstructed human epidermal cells differentiated into keratinocytes, resulting in a close representation of the human epidermis (Boelsma et al., 2000).

Comparison of Ki and IC50 Values for Prototypical Inhibitors of the Human ABC Transporters P-gp and

Published:  21 October 2015

Comparison of Ki and IC50 Values for Prototypical Inhibitors of the Human ABC Transporters P-gp and BCRP in Membrane Vesicles

P-gp (ABCB1/MDR1) and BCRP (ABCG2) are members of the ATP-binding cassette (ABC) superfamily of transporters that use ATP to actively transport compounds across a cell membrane. P-gp and BCRP are expressed on the luminal membrane of enterocytes, endothelial cells in the brain, the brush border membrane of renal proximal tubules and the canalicular membrane of hepatocytes where they limit intestinal absorption, blood-brain barrier penetration and facilitate excretion into the bile and urine. Compounds that inhibit these transporters may be perpetrators of drug-drug interactions [1,2]. While the FDA Draft Guidance for Industry (2012) recommends IC50 values for evaluating the inhibitory potential of transporters, the EMA’s Guideline on the Investigation of Drug Interactions (2013) recommends the determination of Ki values [3,4]. The EMA recommends the use of IC50 values only when Ki determinations are not possible.

Although Ki values have been reported for inhibitors of various transporters, the methodology differs between labs and oftentimes relies on mathematical extrapolation (e.g., using the IC50 value to determine the Ki value with the Cheng-Prusoff equation). In this study the inhibitory potential of various chemical inhibitors was evaluated with in vitro experiments in transporter-expressing membrane vesicles to determine both Ki and IC50 values. Prior to the conduct of inhibition experiments, Km values were experimentally determined with each probe substrate, namely N-methylquinidine (NMQ) for P-gp and estrone-3-sulfate (E3S) for BCRP. Subsequently, IC50 and Ki values were experimentally determined for the prototypical inhibitors verapamil (P-gp) and Ko143 (BCRP).

Effects of Albumin-Fused Human Growth Hormone TV-1106 on CYP Enzyme Expression in Human Hepatocytes

Published:  22 June 2015

Direct and Cytokine-Mediated Effects of Albumin-Fused Human Growth Hormone, TV-1106, on CYP Enzyme Expression in Human Hepatocytes In Vitro
Drug-drug interactions involving therapeutic proteins that can modulate effects of cytokines and potentially impact cytochrome P450 (CYP) enzymes have been of increased interest to regulatory agencies and pharmaceutical industry sponsors in recent years. The well-documented therapeutic protein DDI mechanism involves pro-inflammatory, cytokine-mediated changes in drug-metabolizing enzymes. Multiple in vitro and a number of in vivo human studies have demonstrated the effect of individual cytokines and their modulators on P450s and transporters (Evers et al., 2013).
TV-1106 is recombinant human albumin (rhA), genetically fused at its C-terminus to recombinant human growth hormone (rhGH) to prolong systemic circulation of rhGH and improve its therapeutic activity (Osborn et al., 2002; Sleep, 2014). Phase 1 clinical trial of TV-1106 demonstrated that the drug is well tolerated, has a prolonged half-life in the circulation, and is biologically active in adults with GH deficiency.
GH also enhanced pro-inflammatory cytokines IL1-alpha, IL-6 and TNF-alpha production by lipopolysaccharide (LPS)-activated monocytes in whole blood and its administration at high doses to critically ill adults was associated with an increase in morbidity and mortality (Uronen-Hansson et al., 2003). GH has been shown to be a major determinant of hepatic CYP expression in rats (Morgan et al., 1998). An in vitro study showed an increased CYP3A4 gene expression in cultured human hepatocytes after exposure to GH (Liddle et al., 1998). In healthy elderly men GH induces CYP1A2 and, to a lesser extent, inhibits CYP2C19, but it exerts no effects on CYP2D6 and CYP3A4 enzymes (Jurgens et al., 2002).
An evaluation of TV-1106 effects on plasma cytokines and subsequently on hepatic drug metabolism is warranted by the fact that rhGH is known to modulate plasma cytokines in GH-deficient and normal children of short stature (Bozzola et al., 2003; Pagani et al., 2005).
An in vitro test system to examine direct- and cytokine-mediated effects of therapeutic proteins on hepatic drug metabolism that is based on treatment of whole blood with the drug, followed by separation of plasma and application of that plasma to cultured hepatocytes, has been recently developed (Czerwinski et al., 2015). Here we evaluated the ability of TV-1106 to stimulate cytokine release in whole blood and the effect of up to 50% plasma from TV-1106-treated blood on CYP expression in cultured human hepatocytes and compared the direct effects of treating primary cultures of human hepatocytes with TV-1106 and rhGH on the expression of CYP enzymes.

In Vitro Assessment of the Drug-Drug Interaction of Rasagiline and Its Metabolite Aminoindan

Published:  22 June 2015

In Vitro Assessment of the Pharmacokinetic Drug-Drug Interaction Potential of Rasagiline and Its Major Metabolite Aminoindan
Rasagiline mesylate (PAI) is the active pharmaceutical ingredient of the anti-Parkinson’s drug Azilect, marketed by Teva Neuroscience, Inc. Rasagiline (R-N-2-Propynyl-1-indanamine) is a second generation, selective and irreversible inhibitor of monoamine oxidase (MAO)-B. Prior to FDA approval in 2006, the metabolism of rasagiline to its major metabolite aminoindan (AI) by CYP1A2 was characterized in vitro by traditional reaction phenotyping approaches and in a follow-up clinical study where the AUC of rasagiline (2 mg/day) increased by 83% when co-dosed with the strong CYP1A2 inhibitor ciprofloxacin (500 mg b.i.d.) (Azilect label). Additionally, the potential for rasagiline to cause direct and/or metabolism-dependent inhibition of cytochrome P450 (CYP450) enzymes was evaluated; rasagiline did not inhibit any of the CYP450 enzymes tested (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5 and CYP4A11).
In the current study, we further evaluated rasagiline and its major human metabolite, aminoindan (Figure 1), for the potential to be the perpetrator or victim of pharmacokinetic based drug-drug interactions. The experimental procedures were based on the recommendations and scientific principles described in the FDA DDI draft guidance for industry (2012), the EMA guideline on the investigation of drug interactions (2013) and in the 2014 Japanese MHLW DDI draft guidance. Rasagiline and aminoindan were tested for the potential to cause induction of CYP450 enzymes (CYP1A2, CYP2B6, CYP3A4/5) and direct and/or metabolism-dependent inhibition of CYP450 enzymes (rasagiline, CYP2B6, CYP2C8 and CYP3A4/5; aminoindan, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5). Furthermore, the potential of rasagiline to be a substrate (P-gp and BCRP) and rasagiline and aminoindan to be inhibitors (P-gp, BCRP, OATP1B1, OATP1B3, OCT2, OAT1, OAT3, MATE1 and MATE2-K) of drug transporters was evaluated in vitro.

Evaluation of BSEP Inhibitors Using B-CLEAR® Human Hepatocytes to Predict Inhibition and Cholestasis

Published:  22 June 2015

Evaluation of Clinically Relevant Inhibitors of BSEP Using B-CLEAR® Human Sandwich-Cultured Hepatocytes to Better Predict Inhibition and Cholestasis

Drug-induced liver injury (DILI) is characterized as liver injury due to intake of medications or xenobiotics, which leads to liver abnormalities or dysfunction. There are a large number of drugs that have been withdrawn from the market due to acute DILI; including, troglitazone (antidiabetic and anti-inflammatory), benzbromarone (gout), and sitaxsentan (pulmonary arterial hypertension). The mechanism behind DILI is multifaceted and can be exacerbated by unpredictable metabolism and bodily response, as well as the complex relationship between an individual’s genetic makeup and environmental risk factors. Furthermore, several factors may contribute to hepatocellular injury and cholestasis; including, acute hepatocyte necrosis, the production of reactive metabolites (and oxidative stress) during cytochrome P450 metabolism of the parent drug, activation of stress signaling, and mitochondrial dysfunction.
Bile acids are water soluble end products of cholesterol metabolism which are highly regulated by metabolism, excretion, absorption, and feedback mechanisms, in order to limit their intracellular accumulation. It is hypothesized that dysfunction to the bile salt efflux pump (BSEP) may contribute to the mechanism of action behind DILI. Current preclinical, in vitro models of BSEP inhibition may not accurately predict the potential for DILI. Therefore, an in vitro model which better reflects in vivo transporter expression and activity may better predict the potential for novel xenobiotics to cause DILI in the clinic. The purpose of this work was to evaluate the use of B-CLEAR® human sandwich-cultured hepatocytes to investigate the hepatobiliary disposition of taurocholic acid (TCA) in the presence of cholestatic agents.

Comparison of Ki and IC50 Values for Prototypical Inhibitors

Published:  20 April 2015

Comparison of Ki and IC50 Values for Prototypical Inhibitors of ABC Transporters P-gp and BCRP

Ki values were determined for prototypical inhibitors of ABC transporters P-gp and BCRP with an experimental design incorporating multiple substrate and inhibitor concentrations in vesicles. The EMA’s Guideline on the Investigation of Drug Interactions (2012) recommends Ki values for evaluating transporter inhibition. The use of IC50 values is recommended only when Ki determinations are not possible. Although Ki values have been reported for inhibitors of various transporters, the methodology differs between labs and oftentimes relies on evaluation of a single probe substrate concentration combined with mathematical extrapolation. These experiments were conducted to compare IC50 and Ki values determined in transporter-expressing membrane vesicles.

Accumulation of Taurocholic Acid in Hepatocytes

Published:  16 April 2015

Accumulation of Taurocholic Acid in B-CLEAR® Human Sandwich-Cultured Hepatocytes in the Presence of Clinically Relevant Cholestatic Agents

Drug induced liver injury (DILI) has led to the withdrawal of drugs from the market. One postulated mechanism of DILI is cholestasis caused by inhibition of the bile salt efflux pump (BSEP). Inhibition of BSEP can be measured in vitro with BSEP expressing vesicles. However, not all drugs identified as BSEP inhibitors cause DILI as there are many pathways involved in cholestatic toxicity. An in vitro system which models in vivo transporter expression and functionality more completely may better predict the potential of drugs to cause cholestasis or DILI. We evaluated the hepatobiliary disposition of taurocholic acid (TCA) in the presence of cholestatic agents using B-CLEAR® human sandwich-cultured hepatocytes.

Automated Plasma Protein Binding Using Rapid Equilibrium Dialysis

Published:  06 February 2015

High Content Automated Plasma Protein Binding Screening and Definitive Assays Using Rapid Equilibrium Dialysis for Drug Development

A pharmacokinetic/pharmacodynamic principle important in drug development is that it is the free (unbound) and not the total dosed drug that exerts a pharmacological effect or is available for distribution, metabolism and clearance from the body. Plasma proteins such as albumin have a high propensity to bind drugs that are typically responsible for the bulk of non-specific in vivo drug binding. Therefore the extent of plasma protein binding (PPB) is a critical parameter to determine during drug development as it can influence efficacy factors such as receptor occupancy and disposition factors such as metabolic clearance. In the present study we developed automated methods for PPB screening and definitive PPB assessment on a Tecan liquid handler with endpoints measuring fraction unbound (fu), mass balance (% recovery) and plasma stability (% remaining). Rapid equilibrium dialysis was selected as the methodology of choice for PPB assessment as it is commonly used in the industry and amenable to automation.

Rapid mRNA Induction Screen

Published:  06 February 2015

A Rapid High Content Cytochrome P450 (CYP) mRNA Induction Screen for Early Drug Development

Cytochrome P450 (CYP) enzymes play an important role in the oxidative metabolism of many drugs. Consequently, inhibition or induction of these enzymes by perpetrator drugs can result in alterations in the clearance of a victim drug that is metabolized by CYP pathways (i.e. drug-drug interactions; DDIs). The induction of CYP enzymes, which results in elevated CYP expression levels, can lead to an increase in the clearance of a victim drug resulting in potential loss of drug efficacy. Thus, characterizing a new drug's CYP induction potential early in drug development can lead to better and safer drug design. In the present study, we developed a rapid CYP induction screen evaluating up to 10 compounds in one assay (at three concentrations, with an additional positive control) with the fold change of CYP3A4 mRNA expression levels measured as an endpoint.

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.

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 - 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 - 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 - 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 - 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 - 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 - 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.

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.

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.

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.

ISSX 2011, Poster 145

Published:  14 September 2011

Esomeprazole, omeprazole sulfone, 5-O-desmethyl omeprazole and 5-hydroxylansoprazole are in vitro metabolism-dependent inhibitors of CYP2C19

The intensively researched interaction between clopidogrel and proton pump inhibitors (PPIs) and the impact of CYP2C19 poor metabolizer phenotype on clopidogrel efficacy have prompted warnings from the FDA and EMA in recent years (2010). The FDA specifically warns against coadministration of clopidogrel and omeprazole and further specifically suggests that pantoprazole may be a safer alternative (2010).

ISSX 2011, Poster 209

Published:  14 September 2011

Evaluating the potential for lysosomal trapping in immortalized human hepatocytes (Fa2N-4 cells)

Sequestration of high concentrations of lipophilic amines (a.k.a cationic amphiphilic drugs or CADs) in the acidic (pH 4-5) environment of lysosomes contributes to their presystemic clearance (by lysosomal trapping in liver and lung), their large volume of distribution (Vd) and their propensity to cause phospholipidosis (Hanumegowda et al, 2010; Daniel and Wojcikowski, 1999). In a related study with Lysotracker Red, a fluorescent probe that, by virtue of being a lipophilic amine, accumulates in lysosomes, we demonstrated that Fa2N-4 cells (immortalized human hepatocytes) contain functional lysosomes, similar to those observed in non-transformed human hepatocytes (Kazmi et al, 2011).

ISSX 2011, Poster 140

Published:  14 September 2011

Can Ki values for direct inhibition of CYP enzymes be reliably estimated from IC50 values?

Regulatory agencies recommend that the potential for a drug candidate to cause clinically relevant, direct inhibition of cytochrome P450 (CYP) enzymes be estimated based on the ratio of [I]/Ki (or 1+[I]/Ki) where [I] is the in vivo concentration of drug candidate and Ki is the dissociation constant for the enzyme-inhibitor complex for direct inhibition (US FDA, 2006). Typically, inhibition of CYP enzymes by a drug candidate is first evaluated in vitro by determining the concentration of drug candidate that causes 50% inhibition of a specific CYP enzyme activity (IC50) using a marker substrate concentration approximately equal to Km for the marker substrate reaction. Determining the mechanism of direct inhibition (competitive, uncompetitive, noncompetitive and mixed) and measuring the Ki value requires an in vitro evaluation of the effects of multiple concentrations of the drug candidate versus multiple concentrations of CYP probe substrate (the former spanning Ki and later spannign Km).

ISSX 2011, Poster 112

Published:  14 September 2011

An in vitro test system to evaluate drug-drug interactions with biologics

Inflammation, infection, vaccination, and some marketed therapeutic proteins (biologics) are associated with cytokine-mediated suppression (down-regulation) of drug-metabolizing enzymes (DME). Biologics, such as monoclonal antibodies, can trigger the release of pro-inflammatory cytokines (e.g. IL-1ß. IL-6, INFy and TNFα); extreme cases of which are known as a cytokine storm. Protein therapeutics may change the clearance of small molecule drugs (SMDs) by affecting DME expression and thereby precipitate drug-drug interactions (DDIs). Potential of biologics to cause DDI is a safety concern recognized by the FDA (Lee et al). In the present study, we developed an in vitro method to evaluate the potential of biologics to elicit DDIs with SMDs via alteration of DME expression. This method involves treating fresh human blood with a biologic to stimulate the release of pro-inflammatory cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to primary human hepatocytes co-cultured with Kupffer cells to evaluate effects of biologics on cytochrome P450 (CYP) enzyme expression. E. coli lipopolysaccharide (LPS) and murine anti-CD28 monoclonal antibody were evaluated for their potential to perpetrate DDI with SMDs.

ISSX 2011, Poster 147

Published:  14 September 2011

Evaluation of dilution, dialysis and ultracentrifugation methods to assess the reversibility of metabolism-dependent inhibitors (MDIs) of cytochrome P450 (CYP) enzymes

Metabolism-dependent inhibition (MDI) of P450 enzymes is a well-recognized cause of clinically significant drug-drug interactions (DDI). For this reason, the US Food & Drug Administration (FDA) and the European Medicines Agency (EMA) have both published draft guidance documents on DDI that require an in vitro assesment of the ability of drug candidates to cause MDI of the major drug-metabolizing P450 enzymes (2006, 2010). The most recent PhRMA publication and EMA draft guidance discuss in vitro experiments, to not only identify the potential for a drug candidate to cause MDI. (e.g. IC50 shift experiments), but also to evaluate whether MDI involves reversible or irreversible inhibition (Grimm et al., 2009).

ISSX 2011, Poster 203

Published:  14 September 2011

A robust method to identify compounds that undergo intracellular lysosomal sequestration

Lysosomes are acidic organelles (pH 4-5) that play a key role in various metabolic processes such as turnover of phospholipids, the breakdown of waste products (including bacteria and viruses) and apoptosis. Lipophilic and amphiphilic drugs (a.k.a. cationic amphiphilic amines or CADs) with ionizable amines (pKa>6) can accumulate in lysosomes (a process known as lysosomal trapping), which contributes to presystemic clearance in lysosome-rich organs (such as liver and lung) and, together with the binding of lipophilic amines to phospholipids, is associated with a large volume of distribution of numerous cardiovascular and CNS drugs (MacIntyre and Cutler, 1988; Daniel and Wojcikowski, 1999; Houston and Halifax, 2007). The prolonged accumulation of lipophilic amines in lysosomes and their binding to phospholipids, both of which disrupt lysosomal function, have been implicated as the major cause of phospholipidosis, where an excessive accumulation of phospholipids occurs in various tissues (Hanumegowda et al., 2010). Furthermore, elevated levels of CADs in lysosomes can lead to high organ to blood ratios of drugs that can be mistaken for active drug transport.

ISSX 2011, Poster 170

Published:  14 September 2011

Temporal changes in CYP3A4 mRNA and activity following treatment of cultured human hepatocytes with interleukin-6 (IL-6): Implications for study design and endpoint selection

In recent years the focus of pharmaceutical drug development (once dominated by small molecule (NCE) therapies) has shifted and is now shared with a significant number of new therapies emerging from biological (New Biological Entities or NBE's) development. Since the approval of the first biological treatment in the United States (recombinant insulin, 1982), more than 250 biologics have reached the market, representing roughly one-quarter of all new drugs approved by U.S. and European Union authorities (Trusheim et al, 2010). Biologics include a broad range of therapies including (but not limited to) vaccines, cell or gene therapies, therapeutic protein hormones, cytokines and tissue growth factors, and monoclonal antibodies.

ISSX 2010, Poster 146

Published:  01 September 2010

In vitro inhibition and induction of human liver cytochrome P450 enzymes by NTBC and its metabolism in human liver microsomes

2-(2-Nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC, also known as nitisinone and marketed as Orfadin®) is an inhibitor of 4-hydroxyphenylpyruvate dioxgenase (HPPD) that is used to prevent the liver and kidney toxicity associated with tyrosinemia type 1, a metabolic disorder in the tyrosine catabolism caused by fumarylacetoacetate hydrolase (FAH) deficiency. Genetically modified mice deficient in FAH [Fah-/-/II2rg-/-Rag2-/- (FRG) mouse strain] can be repopulated with human hepatocytes to support, among other applications, studies of drug metabolism and disposition. To sustain mouse hepatocellular function prior to (and during) the repopulation with human hepatocytes, FRG mice are treated with NTBC to inhibit the formation of hepatotoxic levels of fumarylacetoacetate (FAA). In the present study, we investigated the metabolism of NTBC in human liver microsomes (HLM) and the potential for NTBC to inhibit or induce human cytochrome P450 (CYP) enzymes. NTBC (1, 10 and 100 μM) was incubated with multiple concentrations of NADPH-fortified pooled HLM (0.5, 1 and 2 mg /mL) for multiple incubation times (0, 30, 60, 120 and 240 min). Little-to-no loss of NTBC was observed, suggesting that NTBC undergoes little or no oxidative metabolism by human liver CYP enzymes and little-to-no ketone reduction by microsomal carbonyl reductase. These results are consistent with the long clinical plasma half-life (t1/2 ~ 52 –54 h) reported for NTBC. In CYP inhibition experiments, performed with pooled HLM (0.1 mg/mL), NTBC caused direct inhibition of CYP2C9 (IC50 11 μM). NTBC caused no direct inhibition of CYP1A2, 2B6, CYP2C8, 2C19, 2D6 and 3A4/5. Furthermore, NTBC caused no metabolism dependent inhibition of any of the CYP enzymes evaluated...

ISSX 2010, Poster LB43

Published:  01 September 2010

Accurate mass spectrometry elucidates a misleading metabonate formed from amine-containing drugs in reactive metabolite screening assays

The propensity of drug candidates to undergo cytochrome P450 (CYP)-dependent metabolic activation to reactive electrophilic meta-bolites is often evaluated with NADPH-fortified human liver microsomes (HLM) in the presence of a soft nucleophile (like glutathione, GSH) or a hard nucleophile (like cyanide, CN–) with analysis by liquid chromatography tandem mass spectrometry (LC/MS/MS). In the present study, diltiazem, nordiltiazem and di-nordiltiazem (100 μM), compounds containing a tertiary, secondary and primary amine, respectively, were incubated for 15 min with NADPH-fortified HLM (1 mg/mL) in the presence of KCN or GSH (1 mM). Zero-cofactor and zero-min incubations were included as negative controls. Samples were analyzed by UltraPerformance LC tandem accurate MS on a Synapt G2 quadrupole time-of-flight mass spectrometer and data were processed with compound-specific mass defect filtering (± 35 mDa) and optimal parameters for detecting trapped reactive metabolites. Under these conditions, all three compounds were converted to cyanide-trapped components, as summarized below.

ISSX 2010, Poster 181

Published:  01 September 2010

In situ evaluation of CYP1A2, CYP2B6 and CYP3A4/5 induction in cultured human hepatocytes with a cocktail of probe substrates: A comparison of percent positive control values

It has previously been demonstrated that a cocktail of probe substrates can be effectively used to screen simultaneously for induction of CYP1A2, CYP2B6 and CYP3A4/5 with in situ (cell-based) incubations based on the magnitude of induction relative to appropriate positive controls. Industry guidelines recommend that a compound be considered an inducer in vitro and therefore warrant further investigation in vivo if it elicits an increase in cytochrome P450 (CYP) enzyme activity that is equal to or greater than 40% of that caused by a suitable enzyme inducer (percent positive control). The objective of this study was to determine the percent of positive control value of known CYP inducers (at varying concentrations) compared with the US FDA-preferred positive controls, omeprazole (100 μM, CYP1A2), phenobarbital (750 μM, CYP2B6) and rifampin (10 μM, CYP3A4/5) based on in situ incubations of primary cultures of human hepatocytes with three marker substrates (namely, 100 μM phenacetin, 500 μM bupropion, and 100 μM midazolam) either individually or as a cocktail. Freshly isolated human hepatocytes (n=3) were cultured in a sandwich configuration and treated for three days with vehicle control (dimethyl sulfoxide, DMSO; 0.1%, v/v), the aforementioned positive controls or one of the following enzyme inducers: β-naphthoflavone (1-100 μM), lansoprazole (1-100 μM), dexamethasone (1-100 μM), phenytoin (1-100 μM), carbamazepine (1-100 μM) , and efavirenz (0.25-25 μM). CYP induction was measured by LC/MS/MS based on phenacetin O-dealkylation (CYP1A2), bupropion hydroxylation (CYP2B6) or midazolam 1´-hydroxylation (CYP3A4/5). The results indicate that...

ISSX 2010, Poster 247

Published:  01 September 2010

The effect of multiple cryopreservation cycles on drug-metabolizing enzymes in human hepatocytes

Cryopreserved hepatocytes provide a convenient in vitro test system to study the phase-1 and phase-2 metabolism of new chemical entities; however cryopreservation (freeze-thaw cycles) can damage the cells.
Cryoinjury is associated with rapid dehydration of cells (osmotic effects) and formation of intracellular ice during the freezing process, which in turn leads to a disruption of cellular membranes, changes in protein conformation and nucleic acid damage (single and double DNA strand breaks). This study characterized the effects of multiple cryopreservation cycles on the activities of drug-metabolizing enzymes in individual and pooled samples of human hepatocytes. Hepatocytes isolated from four donors were cryopreserved once, twice or three times according to a stepwise protocol. Pooled hepatocytes (n=5) were prepared by two protocols: one involving the thawing of individual lots of frozen hepatocytes, followed by pooling and re-freezing, and the other by pooling cryopreserved hepatocyte pellets (CryostaX™) without thawing or re-freezing the cells. All cells were stored in the vapor phase of liquid nitrogen and were thawed under the same conditions. Viable hepatocytes were separated from non-viable cells by Percoll® gradient centrifugation. The following enzyme activities were measured in situ: CYP3A4 (testosterone 6β-hydroxylation), CYP1A2 (phenacetin O-dealkylation), CYP2B6 (bupropion hydroxylation), FAD-containing monooxygenase (FMO, benzydamine N-oxidation), UDP-glucuronosyltransferase (UGT), and sulfonotransferase (SULT, 7-hydroxycoumarin sulfonation)...

ISSX 2010, Poster 203

Published:  01 September 2010

Pitfalls in the Design of Metabolism-Dependent CYP Inhibition (MDI) Experiments With a Dilution Step: Inhibitor Depletion by Metabolism and/or Microsomal Binding Leads to Underestimation of the Shifted IC50 Value

We previously demonstrated that, when a dilution step is used to assess MDI potential (i.e., IC50 shift experiments), IC50 values for direct-inhibition and MDI should be processed based on the final, post-dilution concentration and the initial, pre-dilution concentration, respectively (Paris et al., 2009). When processed appropriately, the "shifted IC50 values" (those determined following a 30-min preincubation of the drug candidate with NADPH-fortified HLM) were notably higher for several known MDIs in experiments conducted with a 10-fold dilution step than those determined by a non-dilution method, suggesting less MDI occurs at the higher concentration of HLM. More than a two-fold difference in shifted IC50 values was observed between the dilution and non-dilution methods for five of ten inhibitors commonly used positive controls in MDI experiments; namely ticlopidine (CYP2B6), tienillic acid (CYP2C9), paroxetine (CYP2D6), S-fluoxetine (CYP2C19) and azamulin (CYP3A4). Experiments were performed to determine whether these discrepancies in shifted IC50 values between are attributable to 1) decreased free inhibitor concentration (fuinc) and/or 2) extensive metabolism of the inhibitor. In the case of S-fluoxetine, microsomal binding, not extensive metabolism, leads to the discrepancy...

ISSX 2010, Poster 197

Published:  01 September 2010

Identification of a novel carbamoyl glucuronide as a metabolism-dependent inhibitior of CYP2C8

Glucuronidation is a major route of drug biotransformation and detoxification, whereby a drug is conjugated with glucuronic acid in a reaction catalyzed by UDP glucuronosyltransferases (UGTs). However, previous reports suggest glucuronide conjugates can be inhibitors of Phase I metabolism. Of particular note is gemfibrozil glucuronide, which is oxidized by CYP2C8, causing clinically relevant irreversible inactivation of this enzyme (Ogilvie et al., 2006). In the present study, we evaluated the ability of Lu AA34893 and its carbamoyl glucuronide to inhibit in vitro the major drug metabolizing cytochrome P450 (CYP) enzymes. In NADPH-fortified human liver microsomes (HLM), Lu AA34893 was found to directly inhibit CYP2C19...

Induction of Liver and Intestinal Cytochrome P450 (CYP) Enzymes in Male and Female Cynomolgus Monkey

Published:  15 October 2008

The objective of the present study was to determine the effects of various prototypical inducers on the activity and mRNA expression of multiple CYP enzymes in monkey liver and intestine.

Male and female Cynomolgus monkeys were dosed orally (by nasogastric intubation) once daily for four days with saline (vehicle control), β-naphthoflavone (BNF, 50 mg/kg/day), phenobarbital (PB, 25 mg/kg/day), rifampin (RIF, 25 mg/kg/day), pyrazole (200 mg/kg/day) or omeprazole (OMP, 50 mg/kg/day). Microsomal samples from liver and intestine were analyzed for total cytochrome P450 content, cytochrome b5 content, NADPH-cytochrome c reductase activity, 7-ethoxyresorufin O-dealkylation (CYP1A1/2), testosterone 16α-hydroxylation and testosterone 16β-hydroxylation (CYP2B17), 4-nitrophenol hydroxylase (CYP2E1), testosterone 2α-hydroxylation and testosterone 6β-hydroxylation (CYP3A8). CYP mRNA levels in liver were determined for CYP1A1, 1A2, 2A24, 2B6, 2C8, 2C43, 2C75, 2E1, 3A8 and 4A11 by RT-PCR. Given the amount of data collected on this project, the primary focus will be CYP mRNA data.

Treatment of monkeys with BNF caused significant increases in liver CYP1A1 and CYP1A2 mRNA levels, and little or no increase (< 4-fold) in the mRNA levels of the other enzymes examined. Omeprazole caused increases in liver CYP1A1 and 1A2 mRNA levels, but as a CYP1A mRNA inducer OMP was less effective than BNF. Treatment with BNF caused an increase in CYP1A1 mRNA levels (66- and 240-fold in males and females) and CYP1A2 mRNA levels (51- and 1600-fold in males and females). Treatment with OMP caused an increase in CYP1A1 mRNA levels (2.2- and 5.8-fold in males and females) and CYP1A2 mRNA levels (14.9- and 173-fold in males and females). Similarly, BNF and OMP caused increases in liver microsomal CYP1A1/2 activity. Treatment with BNF caused significant increases in CYP1A activity in both liver and intestine. The increase in liver CYP1A activity by BNF was similar between male and female monkeys (11- and 9.4-fold, respectively) whereas induction of intestinal CYP1A activity by BNF was much lower in male than female monkeys (2.7- and 19-fold, respectively). Though to a smaller extent compared to BNF, OMP also caused increases in microsomal CYP1A1/2 activity (3.7- and 4.3-fold in the liver) and (1.5 and 4.3-fold in intestine) for male and female monkeys, respectively. Treatment with PB caused a large increase in liver CYP2B6 mRNA levels (21.6- and 52.5-fold in males and females, respectively), and caused less pronounced increases in CYP2A24 (5.6- and 7.3-fold), 2C8 (7.3- and 6.5-fold), 2C43 (6.4- and 5.7-fold), 2C75 (6.2- and 7.0-fold), and 3A8 (2.6- and 6.0-fold) mRNA levels in males and females, respectively. PB also caused an increase in liver microsomal CYP2B17 and CYP3A8 activity (3.2- and 2.2-fold, respectively). Rifampin treatment caused an increase in liver CYP2B6 (3.6- and 8.2-fold), 2C8 (8.2- and 7.5-fold), 2C43 (6.9- and 8.1-fold), 2C75 (6.7- and 8.6-fold) and 3A8 (5.9- and 5.8-fold) mRNA levels in males and females, respectively. Except for a small increase (3.5-fold in CYP2B6 and 2.7-fold in CYP4A mRNA levels), pyrazole did not cause an increase in the liver mRNA levels of any of the CYP enzymes examined. Results from this study indicate that BNF and OMP are in vivo inducers of CYP1A1 and CYP1A2 in monkeys, while PB and rifampin are inducers of CYP2B, 2C and CYP3A enzymes.

Effects of Gender, Age and Ethnicity on Human Cytochrome P450 Activity

Published:  21 November 2006

It has been well documented that the expression of CYP enzymes is influenced by endogenous factors, such as genetic polymorphisms and hormone levels, and by exogenous factors, such as diet, exposure to drugs, alcohol consumption, cigarette smoking and various environmental factors (Parkinson, et al., 2004). For this study, we examined the effects of three endogenous factors and two exogenous factors on individual CYP activities in over 300 samples of human liver microsomes. The aim of this analysis was to evaluate whether the age, gender, or ethnicity of the donor should influence the selection of human liver microsomes for drug metabolism studies, as well as whether cigarette smoking and alcohol consumption are reliable indicators of elevated CYP1A2 and CYP2E1 activity, respectively...

Microsomes and S9 Prepared from Renal Tissue Yield High CYP, FMO and UGT Activities that are Stable…

Published:  31 October 2005

Microsomes and S9 Prepared from Renal Tissue Yield High CYP, FMO and UGT Activities that are Stable over Multiple Freeze/Thaw Cycles

We have developed procedures to prepare microsomes and S9 fraction from the kidneys of humans, Cynomolgus monkeys, Beagle dogs and Sprague Dawley rats, and we have assessed selected CYP, FMO and UGT activity in pooled and, in the case of humans, individual preparations. These microsomal samples were analyzed for their ability to catalyze the 12-hydroxylation of lauric acid (a marker of CYP4A activity), the N-oxygenation of benzydamine (a marker of FMO activity), the N-demethylation of benzydamine (a marker of CYP3A activity) and the glucuronidation of 4-methylumbelliferone (a reaction catalyzed by several forms of UDP-glucuronosyltransferase). We have also established that renal microsomes and S9 fraction can be subjected to as many as 10 freeze/thaw cycles with little or no loss of CYP4A, UGT, or NADPH-cytochrome c reductase activity. These results suggest that our method for processing kidney tissue is well suited to preserving the enzymatic activity of renal microsomes and S9 fraction, such that these subcellular fractions can be used to assess the Phase 1 and Phase 2 metabolism of drugs in the kidney.

Microsomes Prepared from Eluted Enterocytes Yield High Cytochrome P450 and UGT Activities that are…

Published:  25 August 2004

Microsomes Prepared from Eluted Enterocytes Yield High Cytochrome P450 and UGT Activities that are Stable over Multiple Freeze/Thaw Cycles

Enterocytes in the upper region of the small intestine play a significant role in the first-pass metabolism (pre-systemic clearance) of many orally ingested xenobiotics. For this reason, functionally active and stable intestinal subcellular fractions are required to assess the first-pass metabolism of drugs by cytochromes P450, UDP-glucuronosyltransferases and other drug-metabolizing enzymes. The present study summarizes enzymatic activity data from individual and pooled animal and human intestinal microsomes that were prepared from fresh duodenum/jejunum based on an enterocyte elution method with EDTA and various protease inhibitors. All samples were analyzed for their ability to catalyze testosterone 6ß-hydroxylation, 4-methylumbelliferone glucuronidation, and NADPH-cytochrome c reduction. Microsomes prepared from chemically eluted enterocytes had substantially greater CYP3A activity than those prepared from small intestinal samples subjected to mechanical scraping. Freezing/thawing small intestinal microsomes for up to 5 cycles did not cause significant loss of CYP3A, NADPH-cytochrome c reductase or UDP-glucuronosyltransferases (UGTs) activity. These results suggest that our elution method for processing small intestines is well suited to preserving microsomal enzymatic activities.

The Use of Immortalized Hepatocytes in Induction Studies

Published:  25 August 2004

Primary cultures of human hepatocytes are widely used to evaluate the cytochrome P450 (CYP) enzyme-inducing potential and/or toxicity of drug candidates. However, the availability of human hepatocytes for this purpose is limited and erratic, and there are large inter-individual differences in the magnitude of induction (due to differences in both "control" and "induced" CYP activities). Immortalized human hepatocytes, Fa2N-4, developed by Multicell Technologies, (Warwick, RI) have the potential to overcome these limitations. The cells, immortalized through transformation of human hepatocytes with SV40 T antigen, display in vitro cell morphology that closely resembles primary human hepatocytes (Figure 1). The Fa2N-4 cells retain many of the characteristics of primary hepatocytes, including the inducibility of multiple CYP mRNAs and enzyme activities (Mills et al., 2002; Morris et al., 2003; Czerwinski et al., 2003). In this study, we further characterized the ability of these cells to respond to enzyme-inducing xenobiotics. Additionally, we examined the toxicity profile of multiple enzyme inducers in primary hepatocytes and Fa2N-4 cells.

Induction of UDP-Glucuronosyltransferases in Cultures of Fresh and Immortalized Human Hepatocytes

Published:  25 August 2004

Induction of UDP-Glucuronosyltransferases in Primary Cultures of Fresh and Immortalized Human Hepatocytes

We have examined the induction of UDP-glucuronosyltransferases (UGTs) in primary cultures of human hepatocytes and in immortalized human hepatocytes (Fa2N-4 cells) under conditions that result in the induction of cytochrome P450 (CYP) enzymes. Primary cultures of human hepatocytes were treated daily for three days with either vehicle (0.1% DMSO or saline) or one of several prototypical human CYP inducers, namely b-naphthoflavone, omeprazole, phenobarbital, rifampin and isoniazid. Microsomes prepared from the primary hepatocyte cultures were analyzed by LC/MS/MS for thyroxine (T4) and triiodothyronine (T3) glucuronidation, and cell lysates were analyzed for UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 mRNA expression by the branched DNA (bDNA) assay. UGT1A1 mRNA levels increased (> 2 fold) following treatment of cultures with b-naphthoflavone, omeprazole, phenobarbital, rifampin or isoniazid. UGT1A6 mRNA expression was induced by b-naphthoflavone and isoniazid. UGT1A9 mRNA expression was induced by rifampin. UGT2B4 mRNA expression was induced by b-naphthoflavone and rifampin. UGT2B7 mRNA expression was induced by b-naphthoflavone and phenobarbital.

Fa2N-4 cells were treated for 3 days with DMSO (vehicle), omeprazole, phenobarbital, rifampin or 3-methylcholanthrene. Cells were analyzed for UGT1A1, 1A6, 1A9, 2B4 and 2B7 mRNA expression. Omeprazole induced UGT1A1 mRNA (8.8 fold) and UGT1A9 mRNA (4.4 fold) whereas b-naphthoflavone caused a 4.7-fold increase in UGT1A1 mRNA levels. The other inducers had little or no effect on UGT mRNA expression in Fa2N-4 cells. Microsomal UGT activity toward triiodothyronine and thyroxine was not induced in immortalized human hepatocytes treated with b-naphthoflavone or rifampin. Although statistically different from samples treated with vehicle, treatment of cultured human hepatocytes with rifampin caused less than a 2-fold increase in UGT activity toward T3. These results suggest that human UGTs are not as highly inducible as certain human CYP enzymes, nor as inducible as certain UGTs in rats.

Cytochrome P450 3A4 and 1A2 Induction in Immortalized and Primary Cultures of Human Hepatocytes

Published:  25 August 2004

Cytochrome P450 3A4 and 1A2 Induction in the Immortalized Hepatocyte Line, Fa2N-4, and Comparison with Primary Cultures of Human Hepatocytes

Primary cultures of human hepatocytes are the gold standard for evaluating induction of cytochrome P450 (CYP) enzymes by new molecular entities. Recently, immortalized human hepatocytes, known as Fa2N-4 cells, have been shown to express CYP3A4 and CYP1A2. Additionally, these cells exhibited enzyme induction in response to treatment with agonists of nuclear receptors (Mills et al., 2004). To establish the Fa2N-4 cells as a tool for determining CYP3A4 and CYP1A2 induction, we measured the cells' response to drugs previously evaluated in multiple primary hepatocyte cultures (Luo et al., 2002). Midazolam 1'-hydroxylase and phenacetin O-dealkylase activity were monitored using LC/MS/MS methods to determine the effects of the drugs on expression of CYP3A4 and CYP1A2, respectively. Toxic effects of the compounds were monitored with lactate dehydrogenase (LDH) leakage into the culture media. The magnitude of CYP3A4 and CYP1A2 induction in Fa2N-4 cells agrees closely with primary cultures of human hepatocytes. The comparison of the CYP response in the two cell culture systems validates immortalized hepatocytes as a preclinical tool for assessment of CYP3A4 and CYP1A2 induction.

Additional Posters Are Available Upon Request

Published:  24 August 2004

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