Ken Grime | AstraZeneca - Academia.edu (original) (raw)

Papers by Ken Grime

Research paper thumbnail of CHAPTER 8. Prediction of Human Pharmacokinetics, Exposure and Therapeutic Dose in Drug Discovery

The Handbook of Medicinal Chemistry, 2015

Research paper thumbnail of Species Differences in Biliary Clearance and Possible Relevance of Hepatic Uptake and Efflux Transporters Involvement

Drug Metabolism and Disposition, 2013

From a search of the available literature, a database of 22 drugs of all charge types and several... more From a search of the available literature, a database of 22 drugs of all charge types and several different therapeutic classes was compiled to compare rat and human biliary clearance data. Dog biliary excretion data were also found for nine of the drugs. For 19 of the 22 drugs (86%), rat unbound biliary clearance values, when normalized for body weight, exceeded those for humans by factors ranging from 9 to over 2500-fold, whereas human/dog differences were much less dramatic. It was possible to define hepatic uptake and efflux transporter involvement for many of the drugs. On the basis of the findings, it is postulated that regardless of the biliary efflux transporters implicated, when drugs do not require active hepatic uptake to access the liver there may be fairly insignificant differences in rat, dog, and human biliary clearance. Conversely, when the organic anion-transporting polypeptide drug transporters are involved, one may expect at least a 10-fold discrepancy in rat to human biliary clearance normalized for body weight and corrected for plasma protein binding.

Research paper thumbnail of Determination of Human Hepatocyte Intrinsic Clearance for Slowly Metabolized Compounds: Comparison of a Primary Hepatocyte/Stromal Cell Co-culture with Plated Primary Hepatocytes and HepaRG

Drug metabolism and disposition: the biological fate of chemicals, 2016

A key requirement in drug discovery is to accurately define intrinsic clearance (CLint) values of... more A key requirement in drug discovery is to accurately define intrinsic clearance (CLint) values of less than 1 µl/min/10(6) hepatocytes, which requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHHs), and the HµREL human hepatocyte/stromal cell co-cultures for determination of low CLint values. The investigation demonstrated that the systems were capable of providing statistically significant CLint estimations down to 0.2 µl/min/10(6) cells. The HµREL assay provided a higher level of reproducibility, with repeat significant CLint values being defined in a minimum of triplicate consecutive assays for six of seven of the low CLint compounds compared with four of seven for PHHs and two of seven for HepaRG. The assays were also compared with a suspension assay using drugs with higher CLint values and diverse enzymology. The CLint values from the PHH...

Research paper thumbnail of A novel lung pharmacokinetic model predicts lung exposure and supports dose selection for clinical study of AZD2115, a dual muscarinic receptor antagonist/beta2 adrenoceptor agonist (MABA)

European Respiratory Journal, 2015

Research paper thumbnail of The Development of a Cocktail CYP2B6, CYP2C8, and CYP3A5 Inhibition Assay and a Preliminary Assessment of Utility in a Drug Discovery Setting

Drug Metab Disposition, 2006

Tools for studying the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have recently becom... more Tools for studying the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have recently become available. The level of interest in these enzymes has been elevated because investigations have revealed substrate promiscuity and/or polymorphic expression. In this study, we aimed to develop a single cocktail inhibition assay for the three enzymes and assess its utility in drug discovery. Bupropion hydroxylation, amodiaquine N-deethylation, and midazolam 1'-hydroxylation were chosen as probe reactions for CYP2B6, CYP2C8, and CYP3A5 and were analyzed using liquid chromatography-tandem mass spectrometry. Kinetic analyses were performed to establish suitable conditions for inhibition assays, which were subsequently automated. CYP2B6, CYP2C8, and CYP3A5 IC(50) values were determined for marketed drugs and almost 200 AstraZeneca discovery compounds from 16 separate discovery projects. For the marketed drugs, results obtained were comparable with literature values. Data were also compared with IC(50) values determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. In this dataset, the majority of compounds were more potent inhibitors of CYP2C9, CYP2C19, CYP2D6, and CYP3A4 than of CYP2B6, CYP2C8, or CYP3A5. The potential impact of these findings on a cytochrome P450 inhibition strategy is discussed.

Research paper thumbnail of Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

Journal of Chromatography B: Biomedical Sciences and Applications, 2001

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of... more A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C18 analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic

Research paper thumbnail of Montelukast Disposition: No Indication of Transporter-Mediated Uptake in OATP2B1 and OATP1B1 Expressing HEK293 Cells

Pharmaceutics, 2015

Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent ab... more Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast.

Research paper thumbnail of The Application Of An OATP1B1 Inhibition Assay Within Drug Discovery: Use Of In vitro and In silico Approaches To Aid Drug Design

The Application Of An OATP1B1 Inhibition Assay Within Drug Discovery: Use Of In vitro and In sili... more The Application Of An OATP1B1 Inhibition Assay Within Drug Discovery: Use Of In vitro and In silico Approaches To Aid Drug Design. The organic anion transporting polypeptides (OATPs), and more specifically OATP1B1 have been shown to transport a large range of anionic drugs, most notably statins. It is therefore imperative that any potential drug-drug interactions mediated via OATP1B1 are assessed. Consequently an OATP1B1 inhibition assay using pitavastatin as a non-radiolabelled substrate has been developed. The selection of an appropriate substrate has been shown to be critical in producing accurate assessment of inhibition potential. For example the IC50 determined for simvastatin using either pitavastatin or estradiol-17-glucuronide as a substrate was 6 M whereas a value of 40 M was obtained using estrone-3-sulfate. The screening of over 200 inhibitors from both the literature and in house chemistry has allowed the development of an in silico model which has subsequently been use...

Research paper thumbnail of HepaRG cells as human-relevant in vitro model to study the effects of inflammatory stimuli on cytochrome P450 isoenzymes

Drug metabolism and disposition: the biological fate of chemicals, 2015

The suppression of hepatic cytochrome P450 (P450) expression during inflammatory and infectious d... more The suppression of hepatic cytochrome P450 (P450) expression during inflammatory and infectious diseases and the relief of this suppression by successful disease treatment have been previously demonstrated to impact drug disposition. To address this clinically relevant phenomenon preclinically, the effect of proinflammatory cytokines on P450 isoenzymes in human hepatocytes has been examined by several researchers. In the present study we used the human hepatoma cell line (HepaRG) and cryopreserved primary human hepatocytes to investigate the effects of various inflammatory stimuli on P450 levels with the aim of further characterizing HepaRG cells as a useful surrogate for primary hepatocytes. In this study, HepaRG cells were exposed to bacterial lipopolysaccharide (LPS), interleukin-6 (IL-6), and interleukin-18 (IL-18) for 48 or 72 hours. The effects on CYP1A2, CYP2B6, and CYP3A4 mRNA and catalytic activity (phenacetin-O-deethylase, bupropion-hydroxylase, and midazolam-1'-hydrox...

Research paper thumbnail of Metabolic screening in vitro: metabolic stability, CYP inhibition and induction

Drug discovery today. Technologies, 2004

Automated, miniaturised assays are now commonplace within Discovery DMPK (drug metabolism and pha... more Automated, miniaturised assays are now commonplace within Discovery DMPK (drug metabolism and pharmacokinetics). These have evolved considerably since their inception around a decade ago, in-line with both technology and the desire to provide quality data comparable to more traditional analyses. Several formats exist for routine screening of metabolic stability and cyp (cytochrome P450; see Glossary) inhibition and induction, with the major focus being on in vitro systems using human-derived material. Data from other species remain valuable in assessing in vitro-in vivo projections and are pivotal to support safety assessment studies.:

Research paper thumbnail of Pharmacokinetic consequences of time-dependent inhibition using the isolated perfused rat liver model

Xenobiotica; the fate of foreign compounds in biological systems, 2006

Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibi... more Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibition (TDI) and these have been reported to predict accurately for several marketed drugs. However, their robustness using in-depth, carefully controlled pre-clinical studies has yet to be established. In the current study, the isolated perfused rat liver was employed to investigate the effects of TDI under carefully controlled conditions. A five-compartmental model was used to describe the observed data and bring context to in vitro TDI data (kinact and KI). Co-administration of midazolam with troleandomycin, mifepristone, erythromycin and the discovery compound, AZ-X, increased midazolam area under the curve (AUC) 3.2-, 2.5-, 1.6- and 1.0-fold, respectively, compared with AUC increases of 1.8-, 1.4-, 1.2- and 1.1-fold predicted by the model. These experimental findings, whilst modest in overall effect, support the use of this model in the rat and it is proposed that projections can be m...

Research paper thumbnail of Utilization of In Vitro Cytochrome P450 Inhibition Data for Projecting Clinical Drug-Drug Interactions

Drug Discovery, Development, and Manufacturing, 2010

Research paper thumbnail of Discovery and Evaluation of a Novel Monocyclic Series of CXCR2 Antagonists

Bioorganic & Medicinal Chemistry Letters, 2015

Antagonism of the chemokine receptor CXCR2 has been proposed as a strategy for the treatment of i... more Antagonism of the chemokine receptor CXCR2 has been proposed as a strategy for the treatment of inflammatory diseases such as arthritis, chronic obstructive pulmonary disease and asthma. Earlier series of bicyclic CXCR2 antagonists discovered at AstraZeneca were shown to have low solubility and poor oral bioavailability. In this Letter we describe the design, synthesis and characterisation of a new series of monocyclic CXCR2 antagonists with improved solubility and good pharmacokinetic profiles.

Research paper thumbnail of Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

Journal of Chromatography B: Biomedical Sciences and Applications, 2001

Research paper thumbnail of Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5

Xenobiotica, 2006

To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate se... more To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL(int) estimates using CYP3A5 compared with CYP3A4 including midazolam (CL(int) = 3.4 versus 3.3 microl min(-1) pmol(-1)). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL(int) = 10.3 versus 2.4 microl min(-1) pmol(-1)), and ritonavir (CL(int) = 0.76 versus 0.47 microl min(-1) pmol(-1)). The majority of compounds studied showed a greater inhibitory potential (IC(50)) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.

Research paper thumbnail of Efficient assessment of the utility of immortalized Fa2N-4 cells for cytochrome P450 (CYP) induction studies using multiplex quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and substrate cassette methodologies

Xenobiotica, 2008

Induction of cytochrome P450 (CYP) 1A2, CYP2B6, and CYP3A4 by 22 prototypical inducers was evalua... more Induction of cytochrome P450 (CYP) 1A2, CYP2B6, and CYP3A4 by 22 prototypical inducers was evaluated in the Fa2N-4 immortalized human hepatic cell line. To facilitate this a duplex one-step quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for CYP1A2 and CYP3A4 and a substrate cassette allowing simultaneous monitoring of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activity were developed. CYP1A2 messenger RNA (mRNA) and activity were induced by the prototypical aryl hydrocarbon receptor (AhR) ligand beta-naphthoflavone (E(max) = 217- and 11-fold, respectively, and EC(50) = 8 microM). CYP3A4 mRNA and activity were induced by the prototypical pregnane X receptor (PXR) ligands, rifampicin (E(max) = 36- and 6-fold, respectively, and EC(50) = 4 microM) and phenobarbital (E(max) = 12- and 4-fold, respectively, and EC(50) = 205 microM). No induction of CYP2B6 was detected with several prototypical constitutive androstane receptor (CAR) ligands. A large mRNA-activity E(max) ratio was observed for some time-dependent inhibitors of CYP3A4, whereas EC(50) determinations appeared to be independent of the endpoint. In conclusion, Fa2N-4 cells are a good surrogate for primary human hepatocytes for assessing AhR and PXR-mediated CYP1A2 and CYP3A4 induction, respectively, but not for CAR-mediated CYP2B6 induction. The sensitive and selective methodologies presented in this paper afford maximal data generation and enhanced throughput capability and are readily transferable to primary human hepatocytes or alternate cellular systems.

Research paper thumbnail of A novel matrix for the short-term storage of cells: utility in drug metabolism and drug transporter studies with rat, dog and human hepatocytes

Xenobiotica, 2013

1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hep... more 1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hepatocyte cellular function and offers the potential use of primary cells "as fresh" for several days post isolation. In the study presented, the maintenance of several key phase I and II drug metabolizing enzyme and drug transporter activities is demonstrated with rat and dog hepatocytes preserved for up to 7 days after cell isolation. 2. Intrinsic clearance values were determined for 60 new chemical entities using rat hepatocytes freshly isolated at AstraZeneca and rat hepatocytes prepared at the facilities of Abcellute Ltd (SureTran purveyors), stored and incubated 24 hours after isolation. A very good correspondence in the intrinsic clearance values underlines the utility of the cell maintenance matrix. 3. For human hepatocytes many of the enzyme activities assayed were well maintained for 7 days of storage but some declined to below 50% of initial values between day 4 and 7 of storage. Human OATP1B1 activity was only determined with one batch and declined to 51% of the initial test value by day 4 and further down to 35% by day 7.

Research paper thumbnail of Application of In Silico, In Vitro and Preclinical Pharmacokinetic Data for the Effective and Efficient Prediction of Human Pharmacokinetics

Molecular Pharmaceutics, 2013

In the present age of pharmaceutical research and development, focused delivery of decision makin... more In the present age of pharmaceutical research and development, focused delivery of decision making data is more imperative than ever before. Resulting from several years' success, failure and consequential learning, this article also proffers advice and guidance on which in vitro and in vivo experiments to perform to facilitate efficient and cost-effective pursuit of candidate drugs with acceptable human pharmacokinetic profiles. Predictive in silico models are important in directing design toward compounds with the highest probability of having suitable DMPK properties rather than in predicting human pharmacokinetics per se, and the value and utility of such approaches are reviewed with the intention of providing direction to DMPK scientists. Relating to absorption, distribution, elimination and effective half-life, strategies are described to provide direction in commonly encountered scenarios.

Research paper thumbnail of Time-dependent CYP inhibition

Expert Opinion on Drug Metabolism & Toxicology, 2007

Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubatio... more Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubation or dosing period in vivo. Potential mechanisms include the formation of inhibitory metabolites and mechanism-based inhibition (MBI). In vitro experiments are configured to assess TDI and MBI is inferred, at least initially. MBI is more profound after multiple-dosing and the recovery period is independent of continued drug exposure. Advances in in vitro-in vivo extrapolations for competitive inhibition and the potential relationship between MBI and reactive metabolite-mediated toxicity, have redirected emphasis to CYP TDI. In contrast, with reversible inhibition, strategies for projecting the risks from TDI are less developed and the traditional I/K(i) model often yields a dramatic underprediction. This review explores the contribution of TDI to drug-drug interactions and idiosyncratic drug toxicity.

Research paper thumbnail of Exploring concepts of in vitro time-dependent CYP inhibition assays

Expert Opinion on Drug Metabolism & Toxicology, 2014

Introduction: Evaluation of time-dependent inhibition (TDI) properties in drug candidates is gene... more Introduction: Evaluation of time-dependent inhibition (TDI) properties in drug candidates is generally required for any compound entering development. Methods to evaluate TDI, particularly in abbreviated formats, differ widely among laboratories and there appears to be lack of consensus how to address certain assay shortcomings. Areas covered: As a first objective of this work, we provide commentary on experimental and theoretical considerations in the conduct of abbreviated TDI testing. Methods considered are the single K obs , the progress curve, the '2 + 2' method, the measurement of partition ratios and the IC 50 shift assay. The merits of multiple experimental variations in the IC 50 shift assay, including in depth discussion on the use of a dilution step are explored. Growing evidence suggests that the use of hepatocytes provides certain advantages over liver microsomes. Therefore, a second major objective of this work is to consider merits of the use of hepatocytes in TDI testing. Expert opinion: An in-depth technical understanding of methods to evaluate TDI is critical to enable a selection of an assay aimed at efficiency while minimizing erroneous classification of TDI properties.

Research paper thumbnail of CHAPTER 8. Prediction of Human Pharmacokinetics, Exposure and Therapeutic Dose in Drug Discovery

The Handbook of Medicinal Chemistry, 2015

Research paper thumbnail of Species Differences in Biliary Clearance and Possible Relevance of Hepatic Uptake and Efflux Transporters Involvement

Drug Metabolism and Disposition, 2013

From a search of the available literature, a database of 22 drugs of all charge types and several... more From a search of the available literature, a database of 22 drugs of all charge types and several different therapeutic classes was compiled to compare rat and human biliary clearance data. Dog biliary excretion data were also found for nine of the drugs. For 19 of the 22 drugs (86%), rat unbound biliary clearance values, when normalized for body weight, exceeded those for humans by factors ranging from 9 to over 2500-fold, whereas human/dog differences were much less dramatic. It was possible to define hepatic uptake and efflux transporter involvement for many of the drugs. On the basis of the findings, it is postulated that regardless of the biliary efflux transporters implicated, when drugs do not require active hepatic uptake to access the liver there may be fairly insignificant differences in rat, dog, and human biliary clearance. Conversely, when the organic anion-transporting polypeptide drug transporters are involved, one may expect at least a 10-fold discrepancy in rat to human biliary clearance normalized for body weight and corrected for plasma protein binding.

Research paper thumbnail of Determination of Human Hepatocyte Intrinsic Clearance for Slowly Metabolized Compounds: Comparison of a Primary Hepatocyte/Stromal Cell Co-culture with Plated Primary Hepatocytes and HepaRG

Drug metabolism and disposition: the biological fate of chemicals, 2016

A key requirement in drug discovery is to accurately define intrinsic clearance (CLint) values of... more A key requirement in drug discovery is to accurately define intrinsic clearance (CLint) values of less than 1 µl/min/10(6) hepatocytes, which requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHHs), and the HµREL human hepatocyte/stromal cell co-cultures for determination of low CLint values. The investigation demonstrated that the systems were capable of providing statistically significant CLint estimations down to 0.2 µl/min/10(6) cells. The HµREL assay provided a higher level of reproducibility, with repeat significant CLint values being defined in a minimum of triplicate consecutive assays for six of seven of the low CLint compounds compared with four of seven for PHHs and two of seven for HepaRG. The assays were also compared with a suspension assay using drugs with higher CLint values and diverse enzymology. The CLint values from the PHH...

Research paper thumbnail of A novel lung pharmacokinetic model predicts lung exposure and supports dose selection for clinical study of AZD2115, a dual muscarinic receptor antagonist/beta2 adrenoceptor agonist (MABA)

European Respiratory Journal, 2015

Research paper thumbnail of The Development of a Cocktail CYP2B6, CYP2C8, and CYP3A5 Inhibition Assay and a Preliminary Assessment of Utility in a Drug Discovery Setting

Drug Metab Disposition, 2006

Tools for studying the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have recently becom... more Tools for studying the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have recently become available. The level of interest in these enzymes has been elevated because investigations have revealed substrate promiscuity and/or polymorphic expression. In this study, we aimed to develop a single cocktail inhibition assay for the three enzymes and assess its utility in drug discovery. Bupropion hydroxylation, amodiaquine N-deethylation, and midazolam 1'-hydroxylation were chosen as probe reactions for CYP2B6, CYP2C8, and CYP3A5 and were analyzed using liquid chromatography-tandem mass spectrometry. Kinetic analyses were performed to establish suitable conditions for inhibition assays, which were subsequently automated. CYP2B6, CYP2C8, and CYP3A5 IC(50) values were determined for marketed drugs and almost 200 AstraZeneca discovery compounds from 16 separate discovery projects. For the marketed drugs, results obtained were comparable with literature values. Data were also compared with IC(50) values determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. In this dataset, the majority of compounds were more potent inhibitors of CYP2C9, CYP2C19, CYP2D6, and CYP3A4 than of CYP2B6, CYP2C8, or CYP3A5. The potential impact of these findings on a cytochrome P450 inhibition strategy is discussed.

Research paper thumbnail of Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

Journal of Chromatography B: Biomedical Sciences and Applications, 2001

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of... more A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C18 analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic

Research paper thumbnail of Montelukast Disposition: No Indication of Transporter-Mediated Uptake in OATP2B1 and OATP1B1 Expressing HEK293 Cells

Pharmaceutics, 2015

Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent ab... more Clinical studies with montelukast show variability in effect and polymorphic OATP2B1-dependent absorption has previously been implicated as a possible cause. This claim has been challenged with conflicting data and here we used OATP2B1-transfected HEK293 cells to clarify the mechanisms involved. For montelukast, no significant difference in cell uptake between HEK-OATP2B1 and empty vector cell lines was observed at pH 6.5 or pH 7.4, and no concentration-dependent uptake was detected. Montelukast is a carboxylic acid, a relatively potent inhibitor of OATP1B1, OATP1B3, and OATP2B1, and has previously been postulated to be actively transported into human hepatocytes. Using OATP1B1-transfected HEK293 cells and primary human hepatocytes in the presence of OATP inhibitors we demonstrate for the first time that active OATP-dependent transport is unlikely to play a significant role in the human disposition of montelukast.

Research paper thumbnail of The Application Of An OATP1B1 Inhibition Assay Within Drug Discovery: Use Of In vitro and In silico Approaches To Aid Drug Design

The Application Of An OATP1B1 Inhibition Assay Within Drug Discovery: Use Of In vitro and In sili... more The Application Of An OATP1B1 Inhibition Assay Within Drug Discovery: Use Of In vitro and In silico Approaches To Aid Drug Design. The organic anion transporting polypeptides (OATPs), and more specifically OATP1B1 have been shown to transport a large range of anionic drugs, most notably statins. It is therefore imperative that any potential drug-drug interactions mediated via OATP1B1 are assessed. Consequently an OATP1B1 inhibition assay using pitavastatin as a non-radiolabelled substrate has been developed. The selection of an appropriate substrate has been shown to be critical in producing accurate assessment of inhibition potential. For example the IC50 determined for simvastatin using either pitavastatin or estradiol-17-glucuronide as a substrate was 6 M whereas a value of 40 M was obtained using estrone-3-sulfate. The screening of over 200 inhibitors from both the literature and in house chemistry has allowed the development of an in silico model which has subsequently been use...

Research paper thumbnail of HepaRG cells as human-relevant in vitro model to study the effects of inflammatory stimuli on cytochrome P450 isoenzymes

Drug metabolism and disposition: the biological fate of chemicals, 2015

The suppression of hepatic cytochrome P450 (P450) expression during inflammatory and infectious d... more The suppression of hepatic cytochrome P450 (P450) expression during inflammatory and infectious diseases and the relief of this suppression by successful disease treatment have been previously demonstrated to impact drug disposition. To address this clinically relevant phenomenon preclinically, the effect of proinflammatory cytokines on P450 isoenzymes in human hepatocytes has been examined by several researchers. In the present study we used the human hepatoma cell line (HepaRG) and cryopreserved primary human hepatocytes to investigate the effects of various inflammatory stimuli on P450 levels with the aim of further characterizing HepaRG cells as a useful surrogate for primary hepatocytes. In this study, HepaRG cells were exposed to bacterial lipopolysaccharide (LPS), interleukin-6 (IL-6), and interleukin-18 (IL-18) for 48 or 72 hours. The effects on CYP1A2, CYP2B6, and CYP3A4 mRNA and catalytic activity (phenacetin-O-deethylase, bupropion-hydroxylase, and midazolam-1'-hydrox...

Research paper thumbnail of Metabolic screening in vitro: metabolic stability, CYP inhibition and induction

Drug discovery today. Technologies, 2004

Automated, miniaturised assays are now commonplace within Discovery DMPK (drug metabolism and pha... more Automated, miniaturised assays are now commonplace within Discovery DMPK (drug metabolism and pharmacokinetics). These have evolved considerably since their inception around a decade ago, in-line with both technology and the desire to provide quality data comparable to more traditional analyses. Several formats exist for routine screening of metabolic stability and cyp (cytochrome P450; see Glossary) inhibition and induction, with the major focus being on in vitro systems using human-derived material. Data from other species remain valuable in assessing in vitro-in vivo projections and are pivotal to support safety assessment studies.:

Research paper thumbnail of Pharmacokinetic consequences of time-dependent inhibition using the isolated perfused rat liver model

Xenobiotica; the fate of foreign compounds in biological systems, 2006

Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibi... more Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibition (TDI) and these have been reported to predict accurately for several marketed drugs. However, their robustness using in-depth, carefully controlled pre-clinical studies has yet to be established. In the current study, the isolated perfused rat liver was employed to investigate the effects of TDI under carefully controlled conditions. A five-compartmental model was used to describe the observed data and bring context to in vitro TDI data (kinact and KI). Co-administration of midazolam with troleandomycin, mifepristone, erythromycin and the discovery compound, AZ-X, increased midazolam area under the curve (AUC) 3.2-, 2.5-, 1.6- and 1.0-fold, respectively, compared with AUC increases of 1.8-, 1.4-, 1.2- and 1.1-fold predicted by the model. These experimental findings, whilst modest in overall effect, support the use of this model in the rat and it is proposed that projections can be m...

Research paper thumbnail of Utilization of In Vitro Cytochrome P450 Inhibition Data for Projecting Clinical Drug-Drug Interactions

Drug Discovery, Development, and Manufacturing, 2010

Research paper thumbnail of Discovery and Evaluation of a Novel Monocyclic Series of CXCR2 Antagonists

Bioorganic & Medicinal Chemistry Letters, 2015

Antagonism of the chemokine receptor CXCR2 has been proposed as a strategy for the treatment of i... more Antagonism of the chemokine receptor CXCR2 has been proposed as a strategy for the treatment of inflammatory diseases such as arthritis, chronic obstructive pulmonary disease and asthma. Earlier series of bicyclic CXCR2 antagonists discovered at AstraZeneca were shown to have low solubility and poor oral bioavailability. In this Letter we describe the design, synthesis and characterisation of a new series of monocyclic CXCR2 antagonists with improved solubility and good pharmacokinetic profiles.

Research paper thumbnail of Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

Journal of Chromatography B: Biomedical Sciences and Applications, 2001

Research paper thumbnail of Comparative analysis of substrate and inhibitor interactions with CYP3A4 and CYP3A5

Xenobiotica, 2006

To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate se... more To evaluate the role that cytochrome (CYP) 3A5 plays in hepatic drug metabolism, the substrate selectivity and inhibitory potential of over 60 compounds towards CYP3A4 and CYP3A5 were assessed using Escherichia coli recombinant cell lines. CYP3A4-mediated metabolism predominated for many of the compounds studied. However, a number of drugs gave similar CL(int) estimates using CYP3A5 compared with CYP3A4 including midazolam (CL(int) = 3.4 versus 3.3 microl min(-1) pmol(-1)). Significant CYP3A5-mediated metabolism was also observed for several drugs including mifepristone (CL(int) = 10.3 versus 2.4 microl min(-1) pmol(-1)), and ritonavir (CL(int) = 0.76 versus 0.47 microl min(-1) pmol(-1)). The majority of compounds studied showed a greater inhibitory potential (IC(50)) towards CYP3A4 compared with CYP3A5 (eightfold lower on average). A greater degree of time-dependent inhibition was also observed with CYP3A4 compared with CYP3A5. The range of compounds investigated in the present study extends significantly previous work and suggests that CYP3A5 may have a significant role in drug metabolism particularly in populations expressing high levels of CYP3A5 and/or on co-medications known to inhibit CYP3A4.

Research paper thumbnail of Efficient assessment of the utility of immortalized Fa2N-4 cells for cytochrome P450 (CYP) induction studies using multiplex quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and substrate cassette methodologies

Xenobiotica, 2008

Induction of cytochrome P450 (CYP) 1A2, CYP2B6, and CYP3A4 by 22 prototypical inducers was evalua... more Induction of cytochrome P450 (CYP) 1A2, CYP2B6, and CYP3A4 by 22 prototypical inducers was evaluated in the Fa2N-4 immortalized human hepatic cell line. To facilitate this a duplex one-step quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for CYP1A2 and CYP3A4 and a substrate cassette allowing simultaneous monitoring of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activity were developed. CYP1A2 messenger RNA (mRNA) and activity were induced by the prototypical aryl hydrocarbon receptor (AhR) ligand beta-naphthoflavone (E(max) = 217- and 11-fold, respectively, and EC(50) = 8 microM). CYP3A4 mRNA and activity were induced by the prototypical pregnane X receptor (PXR) ligands, rifampicin (E(max) = 36- and 6-fold, respectively, and EC(50) = 4 microM) and phenobarbital (E(max) = 12- and 4-fold, respectively, and EC(50) = 205 microM). No induction of CYP2B6 was detected with several prototypical constitutive androstane receptor (CAR) ligands. A large mRNA-activity E(max) ratio was observed for some time-dependent inhibitors of CYP3A4, whereas EC(50) determinations appeared to be independent of the endpoint. In conclusion, Fa2N-4 cells are a good surrogate for primary human hepatocytes for assessing AhR and PXR-mediated CYP1A2 and CYP3A4 induction, respectively, but not for CAR-mediated CYP2B6 induction. The sensitive and selective methodologies presented in this paper afford maximal data generation and enhanced throughput capability and are readily transferable to primary human hepatocytes or alternate cellular systems.

Research paper thumbnail of A novel matrix for the short-term storage of cells: utility in drug metabolism and drug transporter studies with rat, dog and human hepatocytes

Xenobiotica, 2013

1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hep... more 1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hepatocyte cellular function and offers the potential use of primary cells "as fresh" for several days post isolation. In the study presented, the maintenance of several key phase I and II drug metabolizing enzyme and drug transporter activities is demonstrated with rat and dog hepatocytes preserved for up to 7 days after cell isolation. 2. Intrinsic clearance values were determined for 60 new chemical entities using rat hepatocytes freshly isolated at AstraZeneca and rat hepatocytes prepared at the facilities of Abcellute Ltd (SureTran purveyors), stored and incubated 24 hours after isolation. A very good correspondence in the intrinsic clearance values underlines the utility of the cell maintenance matrix. 3. For human hepatocytes many of the enzyme activities assayed were well maintained for 7 days of storage but some declined to below 50% of initial values between day 4 and 7 of storage. Human OATP1B1 activity was only determined with one batch and declined to 51% of the initial test value by day 4 and further down to 35% by day 7.

Research paper thumbnail of Application of In Silico, In Vitro and Preclinical Pharmacokinetic Data for the Effective and Efficient Prediction of Human Pharmacokinetics

Molecular Pharmaceutics, 2013

In the present age of pharmaceutical research and development, focused delivery of decision makin... more In the present age of pharmaceutical research and development, focused delivery of decision making data is more imperative than ever before. Resulting from several years' success, failure and consequential learning, this article also proffers advice and guidance on which in vitro and in vivo experiments to perform to facilitate efficient and cost-effective pursuit of candidate drugs with acceptable human pharmacokinetic profiles. Predictive in silico models are important in directing design toward compounds with the highest probability of having suitable DMPK properties rather than in predicting human pharmacokinetics per se, and the value and utility of such approaches are reviewed with the intention of providing direction to DMPK scientists. Relating to absorption, distribution, elimination and effective half-life, strategies are described to provide direction in commonly encountered scenarios.

Research paper thumbnail of Time-dependent CYP inhibition

Expert Opinion on Drug Metabolism & Toxicology, 2007

Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubatio... more Time-dependent inhibition (TDI) of CYP refers to a change in potency during an in vitro incubation or dosing period in vivo. Potential mechanisms include the formation of inhibitory metabolites and mechanism-based inhibition (MBI). In vitro experiments are configured to assess TDI and MBI is inferred, at least initially. MBI is more profound after multiple-dosing and the recovery period is independent of continued drug exposure. Advances in in vitro-in vivo extrapolations for competitive inhibition and the potential relationship between MBI and reactive metabolite-mediated toxicity, have redirected emphasis to CYP TDI. In contrast, with reversible inhibition, strategies for projecting the risks from TDI are less developed and the traditional I/K(i) model often yields a dramatic underprediction. This review explores the contribution of TDI to drug-drug interactions and idiosyncratic drug toxicity.

Research paper thumbnail of Exploring concepts of in vitro time-dependent CYP inhibition assays

Expert Opinion on Drug Metabolism & Toxicology, 2014

Introduction: Evaluation of time-dependent inhibition (TDI) properties in drug candidates is gene... more Introduction: Evaluation of time-dependent inhibition (TDI) properties in drug candidates is generally required for any compound entering development. Methods to evaluate TDI, particularly in abbreviated formats, differ widely among laboratories and there appears to be lack of consensus how to address certain assay shortcomings. Areas covered: As a first objective of this work, we provide commentary on experimental and theoretical considerations in the conduct of abbreviated TDI testing. Methods considered are the single K obs , the progress curve, the '2 + 2' method, the measurement of partition ratios and the IC 50 shift assay. The merits of multiple experimental variations in the IC 50 shift assay, including in depth discussion on the use of a dilution step are explored. Growing evidence suggests that the use of hepatocytes provides certain advantages over liver microsomes. Therefore, a second major objective of this work is to consider merits of the use of hepatocytes in TDI testing. Expert opinion: An in-depth technical understanding of methods to evaluate TDI is critical to enable a selection of an assay aimed at efficiency while minimizing erroneous classification of TDI properties.