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.

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).

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.

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.