Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Drug metabolite identification using ultrahigh-performance liquid chromatography-ultraviolet spectroscopy and parallelized scans on a tribrid Orbitrap mass spectrometer.

RATIONALE: To capture all metabolites in metabolite identification studies, MS/MS information is required in both positive and negative ionization mode, usually involving several sample injections to gain all information about samples. A high-resolution and high mass accuracy quadrupole/linear trap/Orbitrap tribrid instrument was used to gain this information in a novel single injection 'capture-all' approach to metabolite identification. METHODS: Diclofenac, a model compound, was incubated in human and rat hepatocytes. These incubated samples were run using an ultrahigh-performance liquid chromatography/ultraviolet (UHPLC-UV) system coupled to a Thermo Fusion tribrid mass spectrometer. Five parallel scans were used: positive and negative ion full scan, data-dependent MS/MS, both high energy dissociation and collision-induced dissociation, and data-independent all ion fragmentation (AIF) spectra were collected in positive and negative ion mode. RESULTS: Nine metabolites were identified; a metabolite observed in the UV trace, but not positive ion full scan MS, was detected in the same sample injection by negative ion full scan MS. This was identified as a sulphate metabolite, and the corresponding negative ion AIF allowed for some structural elucidation. The use of a photo-diode array (PDA) detector allowed for spectral assessment in case of changes in absorbance spectra, and the subsequent semi-quantification of metabolites. CONCLUSIONS: This method provided good-quality MS/MS data across the m/z range in both positive and negative ion mode. The addition of both negative ion full scan MS and negative ion MS/MS allowed for the detection and structural elucidation of metabolites not observed in positive ion mode. The use of the PDA detector allowed for the semi-quantification of metabolites.

An Investigation into the Prediction of the Plasma Concentration-Time Profile and Its Interindividual Variability for a Range of Flavin-Containing Monooxygenase Substrates Using a Physiologically Based Pharmacokinetic Modeling Approach.

Our recent paper demonstrated the ability to predict in vivo clearance of flavin-containing monooxygenase (FMO) drug substrates using in vitro human hepatocyte and human liver microsomal intrinsic clearance with standard scaling approaches. In this paper, we apply a physiologically based pharmacokinetic (PBPK) modeling and simulation approach (M&S) to predict the clearance, area under the curve (AUC), and Cmax values together with the plasma profile of a range of drugs from the original study. The human physiologic parameters for FMO, such as enzyme abundance in liver, kidney, and gut, were derived from in vitro data and clinical pharmacogenetics studies. The drugs investigated include itopride, benzydamine, tozasertib, tamoxifen, moclobemide, imipramine, clozapine, ranitidine, and olanzapine. The fraction metabolized by FMO for these drugs ranged from 21% to 96%. The developed PBPK models were verified with data from multiple clinical studies. An attempt was made to estimate the scaling factor for recombinant FMO (rFMO) using a parameter estimation approach and automated sensitivity analysis within the PBPK platform. Simulated oral clearance using in vitro hepatocyte data and associated extrahepatic FMO data predicts the observed in vivo plasma concentration profile reasonably well and predicts the AUC for all of the FMO substrates within 2-fold of the observed clinical data; seven of the nine compounds fell within 2-fold when human liver microsomal data were used. rFMO overpredicted the AUC by approximately 2.5-fold for three of the nine compounds. Applying a calculated intersystem extrapolation scalar or tissue-specific scalar for the rFMO data resulted in better prediction of clinical data. The PBPK M&S results from this study demonstrate that human hepatocytes and human liver microsomes can be used along with our standard scaling approaches to predict human in vivo pharmacokinetic parameters for FMO substrates.

An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CLint) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CLint u) for each compound. This was compared with observed unbound intrinsic clearance (observed CLint u) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CLint u using hepatocytes (R2 = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained (R2 = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates.

CYP-Mediated Sulfoximine Deimination of AZD6738.

In hepatic S9 and human liver microsomes (HLMs) the sulfoximine moiety of the ATR inhibitor AZD6738 is metabolized to its corresponding sulfoxide (AZ8982) and sulfone (AZ0002). The initial deimination to AZ8982 is nominally a reductive reaction, but in HLMs it required both NADPH and oxygen and also was inhibited by 1-aminobenzotriazole at a concentration of 1 mM. Studies conducted in a panel of 11 members of the cytochrome P450 (P450) family (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 CYP2J2, CYP3A4, and CYP3A5) confirmed that deimination was an oxidative process that was mediated largely by CYP2C8 with some CYP2J2 involvement, whereas the subsequent oxidation to sulfone was carried out largely by CYP2J2, CYP3A4, and CYP3A5. There was no measureable metabolism in flavin-containing monooxygenase (FMO) enzymes FMO3, FMO5 or NADPH cytochrome C reductase. Studies using Silensomes, a commercially available HLM in which specific members of the P450 family have been inhibited by selective mechanism-based inhibitors, showed that when CYP2C8 was inhibited, the rate of deimination was reduced by 95%, suggesting that CYP2J2 is only playing a minor role in HLMs. When CYP3A4 was inhibited, the rate increased by 58% due to the inhibition of the subsequent sulfone formation. Correlation studies conducted in HLM samples from different individuals confirmed the role of CYP2C8 in the deimination over CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. Hence, although nominally a reduction, the deimination of AZD6738 to its sulfoxide metabolite AZ8982 is an oxidation mediated by CYP2C8, and this metabolite is subsequently oxidized to the sulfone (AZ0002) largely by CYP3A.

Managing the Risk of CYP3A Induction in Drug Development: A Strategic Approach.

Induction of cytochrome P450 (P450) can impact the efficacy and safety of drug molecules upon multiple dosing with coadministered drugs. This strategy is focused on CYP3A since the majority of clinically relevant cases of P450 induction are related to these enzymes. However, the in vitro evaluation of induction is applicable to other P450 enzymes; however, the in vivo relevance cannot be assessed because the scarcity of relevant clinical data. In the preclinical phase, compounds are screened using pregnane X receptor reporter gene assay, and if necessary structure-activity relationships (SAR) are developed. When projects progress toward the clinical phase, induction studies in a hepatocyte-derived model using HepaRG cells will generate enough robust data to assess the compound's induction liability in vivo. The sensitive CYP3A biomarker 4ß-hydroxycholesterol is built into the early clinical phase I studies for all candidates since rare cases of in vivo induction have been found without any induction alerts from the currently used in vitro methods. Using this model, the AstraZeneca induction strategy integrates in vitro assays and in vivo studies to make a comprehensive assessment of the induction potential of new chemical entities. Convincing data that support the validity of both the in vitro models and the use of the biomarker can be found in the scientific literature. However, regulatory authorities recommend the use of primary human hepatocytes and do not advise the use of sensitive biomarkers. Therefore, primary human hepatocytes and midazolam studies will be conducted during the clinical program as required for regulatory submission.

Exploring concepts of in vitro time-dependent CYP inhibition assays.

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 IC50 shift assay. The merits of multiple experimental variations in the IC50 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.

Application of PBPK modelling in drug discovery and development at Pfizer.

Early prediction of human pharmacokinetics (PK) and drug-drug interactions (DDI) in drug discovery and development allows for more informed decision making. Physiologically based pharmacokinetic (PBPK) modelling can be used to answer a number of questions throughout the process of drug discovery and development and is thus becoming a very popular tool. PBPK models provide the opportunity to integrate key input parameters from different sources to not only estimate PK parameters and plasma concentration-time profiles, but also to gain mechanistic insight into compound properties. Using examples from the literature and our own company, we have shown how PBPK techniques can be utilized through the stages of drug discovery and development to increase efficiency, reduce the need for animal studies, replace clinical trials and to increase PK understanding. Given the mechanistic nature of these models, the future use of PBPK modelling in drug discovery and development is promising, however, some limitations need to be addressed to realize its application and utility more broadly.

Simulation of human intravenous and oral pharmacokinetics of 21 diverse compounds using physiologically based pharmacokinetic modelling.

BACKGROUND: The importance of predicting human pharmacokinetics during compound selection has been recognized in the pharmaceutical industry. To this end there are many different approaches that are applied. METHODS: In this study we compared the accuracy of physiologically based pharmacokinetic (PBPK) methodologies implemented in GastroPlus™ with the one-compartment approach routinely used at Pfizer for human pharmacokinetic plasma concentration-time profile prediction. Twenty-one Pfizer compounds were selected based on the availability of relevant preclinical and clinical data. Intravenous and oral human simulations were performed for each compound. To understand any mispredictions, simulations were also performed using the observed clearance (CL) value as input into the model. RESULTS: The simulation results using PBPK were shown to be superior to those obtained via traditional one-compartment analyses. In many cases, this difference was statistically significant. Specifically, the results showed that the PBPK approach was able to accurately predict passive distribution and absorption processes. Some issues and limitations remain with respect to the prediction of CL and active transport processes and these need to be improved to further increase the utility of PBPK modelling. A particular advantage of the PBPK approach is its ability to accurately predict the multiphasic shape of the pharmacokinetic profiles for many of the compounds tested. CONCLUSION: The results from this evaluation demonstrate the utility of PBPK methodology for the prediction of human pharmacokinetics. This methodology can be applied at different stages to enhance the understanding of the compounds in a particular chemical series, guide experiments, aid candidate selection and inform clinical trial design.

An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point.

The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.

Induction of cytochrome P450: assessment in an immortalized human hepatocyte cell line (Fa2N4) using a novel higher throughput cocktail assay.

Over recent years the application of cocktail studies to measure biological markers has become increasingly popular. The current study investigated a novel approach in assessing cytochrome P450 (P450) enzyme induction in an immortalized cell line using a cocktail of five P450 substrate probes compared with the traditional single-probe approach. The findings reported herein support use of a cocktail approach to assess the induction of the major P450s, namely, CYP3A4, CYP1A2, and CYP2C9. CYP2C19 and CYP2D6 could also be followed as part of the cocktail approach reported. Response to prototypical inducers did not differ to those observed in the presence of the specific probes alone. Consequently, this approach requires significantly fewer sample numbers if screening the induction potential of more than one P450. Moreover, these studies highlight the utility of the immortalized cell line Fa2N4 as a robust model system for induction studies. In conclusion, the current experimental setup is an improvement on current approaches used to assess P450 induction, significantly increasing sample throughput.

CYP3A5 genotype has significant effect on quinine 3-hydroxylation in Tanzanians, who have lower total CYP3A activity than a Swedish population.

OBJECTIVES: To study the correlation between CYP3A5 genotype and quinine 3-hydroxylation in black Tanzanian and Swedish Caucasians as well as to investigate the interethnic differences in CYP3A activity between the two populations. METHODS: Tanzanian (n=144) and Swedish (n=136) healthy study participants were given a single oral 250 mg dose of quinine hydrochloride and a 16-h post-dose blood sample was collected. The metabolic ratio of quinine/3-hydroxyquinine was determined in plasma by high-performance liquid chromatography. All the participants were genotyped for the known mutations of CYP3A5, which are relevant for the respective population. Correlation between quinine metabolic ratio and CYP3A5 genotype as well as the interethnic difference in CYP3A activity between the two populations was studied. RESULTS: Tanzanians had significantly higher (P<0.0001) mean quinine metabolic ratio (9.5+/-3.5) than Swedes (7.6+/-3.1). As expected, the frequency of high CYP3A5 expression alleles was higher in Tanzanians (51%) than in Swedes (7%). The mean+/-SD quinine metabolic ratio (10.7+/-3.9) in Tanzanians homozygous for low CYP3A5 expression gene was significantly higher than the corresponding mean metabolic ratio in participants heterozygous (9.5+/-3.3; P=0.02) or homozygous (8.1+/-3.1; P=0.002) for high expression CYP3A5 alleles, respectively. A tendency to higher quinine metabolic ratio in Swedes with low expression alleles compared with those with one or two high expression alleles was observed. Tanzanians homozygous for low CYP3A5 expression gene (i.e. only CYP3A4 is expressed) had significantly (P<0.0001) higher quinine metabolic ratio (10.7+/-3.9) than corresponding Swedes (7.7+/-3.1). CONCLUSIONS: Clear interethnic differences were observed in the activity of CYP3A between Tanzanians and Swedes. A significant association is noted between CYP3A5 genotype and quinine 3-hydroxylation in Tanzanians, indicating a significant contribution of CYP3A5 to total 3A activity. The CYP3A4 catalyzed hydroxylation of quinine (two low CYP3A5 expression alleles) was lower in Tanzanians than in Swedes.

Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios.

Glucuronidation is a listed clearance mechanism for 1 in 10 of the top 200 prescribed drugs. The objective of this article is to encourage those studying ligand interactions with UDP-glucuronosyltransferases (UGTs) to adequately consider the potential consequences of in vitro UGT inhibition in humans. Spurred on by interest in developing potent and selective inhibitors for improved confidence around UGT reaction phenotyping, and the increased availability of recombinant forms of human UGTs, several recent studies have reported in vitro inhibition of UGT enzymes. In some cases, the observed potency of UGT inhibitors in vitro has been interpreted as having potential relevance in humans via pharmacokinetic drug-drug interactions. Although there are reported examples of clinically relevant drug-drug interactions for UGT substrates, exposure increases of the aglycone are rarely greater than 100% in the presence of an inhibitor relative to its absence (i.e., AUCi/AUC < or = 2). This small magnitude in change is in contrast to drugs primarily cleared by cytochrome P450 enzymes, where exposures have been reported to increase as much as 35-fold on coadministration with an inhibitor (e.g., ketoconazole inhibition of CYP3A4-catalyzed terfenadine metabolism). In this article the evidence for purported clinical relevance of potent in vitro inhibition of UGT enzymes will be assessed, taking the following into account: in vitro data on the enzymology of glucuronide formation from aglycone, pharmacokinetic principles based on empirical data for inhibition of metabolism, and clinical data on the pharmacokinetic drug-drug interactions of drugs primarily cleared by glucuronidation.

Reaction phenotyping in drug discovery: moving forward with confidence?

For the pharmaceutical industry, one of the challenges in evaluating the risk of future compound attrition at the discovery stage is the successful prediction of the major routes of clearance in humans. For compounds cleared by metabolism, such information will help to avoid the development of compounds that will exhibit large interpatient differences in pharmacokinetics via 1). routes of metabolism catalyzed by functionally polymorphic enzymes and/or 2). clinically significant metabolic drug-drug interactions, in the later stages of development. The degree of intersubject variability that is acceptable for a drug candidate is uncertain in the discovery stage where knowledge of other important factors is limited or unavailable (i.e. therapeutic index, pharmacodynamic variability, etc). Reaction phenotyping is the semi-quantitative in vitro estimation of the relative contributions of specific drug-metabolizing enzymes to the metabolism of a test compound. However, reaction phenotyping in the discovery stage of drug development is complicated by the absence of radiolabelled parent compound or metabolite bioanalytical standards relative to later stages of development. In this commentary, some of the approaches, based on published data, which can be taken to overcome these challenges are discussed. In addition, knowledge of the molecular structure (i.e. specific chemical substituents), physicochemical properties, and routes of clearance in animals can all help in making a successful prediction for the routes of clearance in humans. In combination, the objective of these studies should be to reduce to a minimum the risk of finding significant inter-patient differences in pharmacokinetics at a later stage in development due to significant metabolism by polymorphic enzymes or drug-drug interactions. Consequently, this data should be used to avoid costly late stage attrition.

In vitro inhibitory effect of 1-aminobenzotriazole on drug oxidations catalyzed by human cytochrome P450 enzymes: a comparison with SKF-525A and ketoconazole.

1-Aminobenzotriazole (ABT) is widely used as a non-specific inhibitor of animal cytochrome P450 (CYP). In the present study, the inhibitory effect of ABT was investigated on drug oxidations catalyzed by human CYP isoforms. This inhibitory effect was compared with that of SKF-525A, another non-specific inhibitor, and ketoconazole, a potent inhibitor of CYP3A. Bacurovirus-expressed recombinant human CYP isoforms were used as an enzyme source. The specific activities for human CYP isoforms are: phenacetin O-deethylation, for CYP1A2; diclofenac 4'-hydroxylation, for CYP2C9; S-mephenytoin 4'-hydroxylation, for CYP2C19; bufuralol 1'-hydroxylation, for CYP2D6; chlorzoxazone 6-hydroxylation, for CYP2E1; testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 1'-hydroxylation, for CYP3A4. ABT inhibited both CYP1A2-dependent activity (Ki=330 microM) and CYP2E1-dependent activity (Ki=8.7 microM). In contrast, SKF-525A weakly inhibited CYP1A2-dependent activities (46% inhibition at 1200 microM) and CYP2E1-dependent activities (65% inhibition at 1000 microM). ABT exhibited the highest Ki value for CYP2C9-dependent diclofenac 4'-hydroxylation among those determined by this assay (Ki=3500 microM). Moreover, SKF-525A showed strong inhibition of CYP2D6-dependent bufuralol 1'-hydroxylation (Ki=0.043 microM). Ketoconazole inhibited all tested drug oxidations, however, its inhibitory effect on CYP1A2-dependent activities was very weak (50% inhibition at 120 microM). ABT, SKF-525A, and ketoconazole showed different selectivity and had a wide range of Ki values for the drug oxidations catalyzed by human CYP enzymes. Therefore, we conclude that inhibitory studies designed to predict the contribution of CYP enzymes to the metabolism of certain compounds should be performed using multiple CYP inhibitors, such as ABT, SKF-525A, and ketoconazole.

Structure-activity relationships of 1-alkyl-5-(3,4-dichlorophenyl)- 5-[2-[(3-substituted)-1-azetidinyl]ethyl]-2-piperidones. 1. Selective antagonists of the neurokinin-2 receptor.

The design, synthesis, and pharmacological evaluation of a novel class of neurokinin-2 (NK2) antagonists 1-alkyl-5-(3,4-dichlorophenyl)-5-[2-[(3-substituted)-1-azetidinyl]ethyl]-2-piperidones (5-44) are described. These compounds are formally derived from 2 by incorporating the metabolically vulnerable N-methylamide function into a more stable six-membered ring lactam 4, resulting in increased stability in human liver microsome (HLM) preparations relative to 2 (T1/2(HLM) of 30 min vs <10 min for 2). This series was further optimized by replacing the 4,4-disubstituted piperidine functionality found in 4 with simple 3-substituted azetidines. This series, exemplified by 1-benzyl-5-(3,4-dichlorophenyl)-5-[2-[3-(4-morpholinyl)-1-azetidinyl]ethyl]-2-piperidone 5, was found to possess excellent functional potency for the NK2 receptor in the Rabbit pulmonary artery (RPA) assay (pA2 = 9.3) and increased in vitro metabolic stability (T1/2(HLM) = 70 min) relative to 4. Metabolic route identification studies revealed that N-benzyl oxidation was a major route in this relatively lipophilic lead (log D = 3.2). Further exploration of the N-lactam substituent SAR targeting reduced lipophilicity to attenuate P-450 metabolism revealed that incorporation of a cyclopropylmethyl group in this region of the molecule gave a balance of good potency and high metabolic stability. For example, the significantly less lipophilic analogue 29 (log D = 2.3) possessed both good functional potency (RPA, pA2 = 8.1) and high in vitro metabolic stability (T1/2(HLM) = 120 min). Optimization in this N-cyclopropylmethyllactam series by modification of the nature of the azetidine 3-substituent as a strategy to further increase potency and moderate log D led to the identification of sulfamide analogue 33, which possessed both excellent metabolic stability in vitro (T1/2(HLM) >120 min) and high potency in both RPA (pA2 = 8.9) and human bladder smooth muscle (pK(b) = 8.9) functional assays. In addition, NK2 antagonist 33 (IC50 = 4 nM) showed excellent selectivity over both the related human neurokinin receptors h-NK1 (IC50 = 7.9 microM) and h-NK3 (IC50 = 1.8 microM) in radioligand binding studies.

Development of a combined protein and pharmacophore model for cytochrome P450 2C9.

A combined protein and pharmacophore model for cytochrome P450 2C9 (CYP2C9) has been derived using various computational chemistry techniques. A combination of pharmacophore modeling (using 31 metabolic pathways for 27 substrates), protein modeling (using the rabbit CYP2C5/3 crystal structure), and molecular orbital calculations was used to derive a model that incorporated steric, electronic, and chemical stability properties. The initial pharmacophore model (based on a subset of 17 metabolic pathways for 16 substrates) and the protein model used to construct the combined model were derived independently and showed a large degree of complementarity. The combined model is in agreement with experimental results concerning the substrates used to derive the model and with site-directed mutagenesis data available for CYP2C9. The model has been successfully used to predict the metabolism of substrates not used to construct the model, of which four examples are discussed in detail. The model has also been successful in explaining the differences in substrate specificity between CYP2C9 and CYP2C19.