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Transporters contribute to renal elimination of drugs; therefore drug disposition can be impacted if transporters are inhibited by comedicant drugs. Regulatory agencies have provided guidelines to assess potential drug-drug interaction (DDI) risk for renal organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 and 2-K (MATE1/2-K) transporters. Despite this, there are challenges with translating in vitro data using currently available tools to obtain a quantitative assessment of DDI risk in the clinic. Given the high number of drugs and new molecular entities showing in vitro inhibition toward OCT2 and/or MATE1/2-K and the lack of translation to clinically significant effects, it is reasonable to question whether the current in vitro assay design and modeling practice has led to unnecessary clinical evaluation. The aim of this review is to assess and discuss available in vitro and clinical data along with prediction models intended to provide clinical context of risk, including static models proposed by regulatory agencies and physiologically-based pharmacokinetic models, in order to identify best practices and areas of future opportunity. This analysis highlights that different in vitro assay designs, including substrate and cell systems used, strongly influence the derived concentration of drug producing 50% inhibition values and contribute to high variability observed across laboratories. Furthermore, the lack of sensitive index substrates coupled with specific inhibitors for individual transporters necessitates the use of complex models to evaluate clinical DDI risk.

An acceptable human pharmacokinetic profile increases the probability of a promising candidate drug becoming a successful therapy. The development of a mechanistic understanding of the disposition of candidate drugs towards confident predictions of key properties associated with human drug absorption, metabolism, distribution and excretion (ADME) is critical. This is achievable from a consideration of physicochemical properties, data from in vitro assays with human-derived material and from in vivo animal studies. This chapter is targeted at medicinal chemists and drug design teams and provides general definitions of the fundamental PK parameters together with best practice characterization, optimization and prediction strategies employed in the authors' laboratory.

The International Consortium for Innovation and Quality (IQ) Physiologically Based Pharmacokinetic (PBPK) Modeling Induction Working Group (IWG) conducted a survey across participating companies around general strategies for PBPK modeling of induction, including experience with its utility to address various questions, regulatory interactions, and regulatory acceptance. The results highlight areas where PBPK modeling is used with high confidence and identifies opportunities where confidence is lower and further evaluation is needed. To enhance the survey results, the PBPK-IWG also collected case studies and analyzed recent literature examples where PBPK models were applied to predict CYP3A induction-mediated drug-drug interactions. PBPK modeling of induction has evolved and progressed significantly, proving to have great potential to accelerate drug discovery and development. With the aim of enabling optimal use for new molecular entities that are either substrates and/or inducers of CYP3A, the PBPK-IWG proposes initial workflows for PBPK application, discusses future trends, and identifies gaps that need to be addressed.

A physiologically based pharmacokinetic model was developed to describe the tissue distribution kinetics of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) in plasma, liver, spleen, and tumors. Tumor growth data from MV-4-11 tumor-bearing mice were incorporated to investigate the exposure/efficacy relationship. The nanoparticle demonstrated improved antitumor activity compared to the conventional API formulation, owing to the extended released API concentrations at the site of action. Model simulations further enabled the identification of critical parameters that influence API exposure in tumors and downstream efficacy outcomes upon nanoparticle administration. The model was utilized to explore a range of dosing schedules and their effect on tumor growth kinetics, demonstrating the improved antitumor activity of nanoparticles with less frequent dosing compared to the same dose of naked APIs in conventional formulations.

Purpose Limited information is available regarding the drug–drug interaction (DDI) potential of molecular targeted agents and rituximab plus cyclophosphamide, doxorubicin (hydroxydaunorubicin), vincristine (Oncovin), and prednisone (R-CHOP) therapy. The addition of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib to R-CHOP therapy results in increased toxicity versus R-CHOP alone, including higher incidence of peripheral neuropathy. Vincristine is a substrate of P-glycoprotein (P-gp, ABCB1); drugs that inhibit P-gp could potentially cause increased toxicity when co-administered with vincristine through DDI. While the combination of the BTK inhibitor acalabrutinib and R-CHOP is being explored clinically, the DDI potential between these therapies is unknown. Methods A human mechanistic physiology-based pharmacokinetic (PBPK) model of vincristine following intravenous dosing was developed to predict potential DDI interactions with combination therapy. In vitro absorption, distribution, metabolism, and excretion and in vivo clinical PK parameters informed PBPK model development, which was verified by comparing simulated vincristine concentrations with observed clinical data. Results While simulations suggested no DDI between vincristine and ibrutinib or acalabrutinib in plasma, simulated vincristine exposure in muscle tissue was increased in the presence of ibrutinib but not acalabrutinib. Extrapolation of the vincristine mechanistic PBPK model to other P-gp substrates further suggested DDI risk when ibrutinib (area under the concentration–time curve [AUC] ratio: 1.8), but not acalabrutinib (AUC ratio: 0.92), was given orally with venetoclax or digoxin. Conclusion Overall, these data suggest low DDI risk between acalabrutinib and P-gp substrates with negligible increase in the potential risk of vincristine-induced peripheral neuropathy when acalabrutinib is added to R-CHOP therapy.

Proteolysis-targeting chimeras (PROTACs) are an emerging therapeutic modality with the potential to open target space not accessible to conventional small molecules via a degradation-based mechanism; however, their bifunctional nature can result in physicochemical properties that breach commonly accepted limits for small-molecule oral drugs. We offer a drug metabolism and pharmacokinetics (DMPK) perspective on the optimisation of oral PROTACs across a diverse set of projects within Oncology R&D at AstraZeneca, highlighting some of the challenges that they have presented to our established screening cascade. Furthermore, we challenge some of the perceptions and dogma surrounding the feasibility of oral PROTACS and demonstrate that acceptable oral PK properties for this modality can be regularly achievable despite the physicochemical property challenges they present.

The use of in vitro in vivo extrapolation (IVIVE) from human hepatocyte (HH) and human liver microsome (HLM) stability assays is a widely accepted predictive methodology for human metabolic clearance (CLmet). However, a systematic underprediction of CLmet from both matrices appears to be universally apparent, which can be corrected for via an empirical regression offset. After physiological scaling, intrinsic clearance (CLint) for compounds metabolized via the same enzymatic pathway should be equivalent for both matrices. Compounds demonstrating significantly higher HLM CLint relative to HH CLint have been encountered, raising questions regarding how to predict CLmet for such compounds. Here, we determined the HLM:HH CLint ratio for 140 marketed drugs/compounds, compared this ratio as a function of physiochemical properties and drug metabolism enzyme dependence, and examined methodologies to predict CLmet from both matrices. The majority (78%) of compounds displaying a high HLM:HH CLint ratio were CYP3A substrates. Using HH CLint for CYP3A substrates, the current IVIVE regression offset approach remains an appropriate strategy to predict CLmet (% compounds overpredicted/correctly predicted/underpredicted 27/62/11, respectively). However, using the same approach for HLM significantly overpredicts CLmet for CYP3A substrates (% compounds overpredicted/correctly predicted/underpredicted 56/33/11, respectively), highlighting that a different IVIVE offset is required for CYP3A substrates using HLM. This work furthers the understanding of compound properties associated with a disproportionately high HLM:HH CLint ratio and outlines a successful IVIVE approach for such compounds. SIGNIFICANCE STATEMENT: Oral drug discovery programs typically strive for low clearance compounds to ensure sufficient target engagement. Human liver microsomes and isolated human hepatocytes are used to optimize and predict human hepatic metabolic clearance. After physiological scaling, intrinsic clearance for compounds of the same metabolic pathway should be equivalent between matrices. However, a disconnect in intrinsic clearance is sometimes apparent. The work described attempts to further understand this phenomenon, and by achieving a mechanistic understanding, improvements in clearance predictions may be realized.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

During drug discovery and prior to the first human dose of a novel candidate drug, the pharmacokinetic (PK) behavior of the drug in humans is predicted from preclinical data. This helps to inform the likelihood of achieving therapeutic exposures in early clinical development. Once clinical data are available, the observed human PK are compared with predictions, providing an opportunity to assess and refine prediction methods. Application of best practice in experimental data generation and predictive methodologies, and a focus on robust mechanistic understanding of the candidate drug disposition properties before nomination to clinical development, have led to maximizing the probability of successful PK predictions so that 83% of AstraZeneca drug development projects progress in the clinic with no PK issues; and 71% of key PK parameter predictions [64% of area under the curve (AUC) predictions; 78% of maximum concentration (Cmax) predictions; and 70% of half-life predictions] are accurate to within twofold. Here, we discuss methods to predict human PK used by AstraZeneca, how these predictions are assessed and what can be learned from evaluating the predictions for 116 candidate drugs.

Background: DMPK data and knowledge are critical in maximising the probability of developing successful drugs via the application of in silico, in vitro and in vivo approaches in drug discovery. Methods: The evaluation, optimisation and prediction of human pharmacokinetics is now a mainstay within drug discovery. These elements are at the heart of the ‘right tissue’ component of AstraZeneca’s ‘5Rs framework’ which, since its adoption, has resulted in increased success of Phase III clinical trials. With the plethora of DMPK related assays and models available, there is a need to continually refine and improve the effectiveness and efficiency of approaches best to facilitate the progression of quality compounds for human clinical testing. Results: This article builds on previously published strategies from our laboratories, highlighting recent discoveries and successes, that brings our AstraZeneca Oncology DMPK strategy up to date. We review the core aspects of DMPK in Oncology drug discovery and highlight data recently generated in our laboratories that have influenced our screening cascade and experimental design. We present data and our experiences of employing cassette animal PK, as well as re-evaluating in vitro assay design for metabolic stability assessments and expanding our use of freshly excised animal and human tissue to best inform first time in human dosing and dose escalation studies. Conclusion: Application of our updated drug-drug interaction and central nervous system drug exposure strategies are exemplified, as is the impact of physiologically based pharmacokinetic and pharmacokinetic-pharmacodynamic modelling for human predictions.

AZD9833 is a potent, orally delivered, non-steroidal selective estrogen receptor degrader (SERD) that both antagonizes and degrades ERα. It is currently in clinical testing for the treatment of ER+ metastatic breast cancer (SERENA-1; NCT03616587). While ER is a clinically validated target, sustained inhibition of the target via oral delivery has proven an elusive goal. Despite intensive research, no oral SERD options are currently approved. Poor pharmacokinetic (PK) properties and/or short half-life (t ½) are often underlying features that limit oral SERD candidates from once or twice daily dose regimens. AZD9833 is a clinical stage next generation oral SERD that may overcome these challenges. The pre-clinical PK properties of AZD9833 are consistent with once daily oral dosing in patients and are reported herein. AZD9833 is a low molecular weight ( Citation Format: Eric T. Gangl, Roshini Markandu, Pradeep Sharma, Andy Sykes, Petar Pop-Damkov, Pablo Morentin Gutierrez, James S. Scott, Dermot F. McGinnity, Adrian J. Fretland, Teresa Klinowska. Preclinical pharmacokinetic and metabolic characterization of the next generation oral SERD AZD9833 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1042.

Due to the blood-brain barrier (BBB) limiting the exposure of therapeutics to the central nervous system (CNS), patients with brain malignancies are challenging to treat, typically have poor prognoses, and represent a significant unmet medical need. Preclinical data report osimertinib to have significant BBB penetration and emerging clinical data demonstrate encouraging activity against CNS malignancies. Here, we discuss the oncology drug candidates AZD3759 and AZD1390 as case examples of discovery projects designing in BBB penetrance. We demonstrate how these innovative kinase inhibitors were recognized as brain penetrant and outline our view of experimental approaches and strategies that can facilitate the discovery of new brain-penetrant therapies for the treatment of primary and secondary CNS malignancies as well as other CNS disorders.

N-(5-Bromo-3-methoxypyrazin-2-yl)-5-chlorothiophene-2-sulfonamide 1 was identified as a hit in a CCR4 receptor antagonist high-throughput screen (HTS) of a subset of the AstraZeneca compound bank. As a hit with a lead-like profile, it was an excellent starting point for a CCR4 receptor antagonist program and enabled the rapid progression through the Lead Identification and Lead Optimization phases resulting in the discovery of two bioavailable CCR4 receptor antagonist candidate drugs.

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.

Unbound IC(50) (IC(50,u)) values of 15 drugs were determined in eight recombinantly expressed human cytochromes P450 (P450s) and human hepatocytes, and the data were used to simulate clinical area under the plasma concentration-time curve changes (deltaAUC) on coadministration with prototypic CYP2D6 substrates. Significant differences in IC(50,u) values between enzyme sources were observed for quinidine (0.02 microM in recombinant CYP2D6 versus 0.5 microM in hepatocytes) and propafenone (0.02 versus 4.1 microM). The relative contribution of individual P450s toward the oxidative metabolism of clinical probes desipramine, imipramine, tolterodine, propranolol, and metoprolol was estimated via determinations of intrinsic clearance using recombinant P450s (rP450s). Simulated deltaAUC were compared with those observed in vivo via the ratios of unbound inhibitor concentration at the entrance to the liver to inhibition constants determined against rP450s ([I](in,u)/K(i)) and incorporating parallel substrate elimination pathways. For this dataset, there were 20% false negatives (observed deltaAUC >or= 2, predicted deltaAUC < 2), 77% correct predictions, and 3% false positives. Thus, the [I](in,u)/K(i) approach appears relatively successful at estimating the degree of clinical interactions and can be incorporated into drug discovery strategies. Using a Simcyp ADME (absorption, metabolism, distribution, elimination) simulator (Simcyp Ltd., Sheffield, UK), there were 3% false negatives, 94% correct simulations, and 3% false positives. False-negative predictions were rationalized as a result of mechanism-based inhibition, production of inhibitory metabolites, and/or hepatic uptake. Integrating inhibition and reaction phenotyping data from automated rP450 screens have shown applicability to predict the occurrence and degree of in vivo drug-drug interactions, and such data may identify the clinical consequences for candidate drugs as both "perpetrators" and "victims" of P450-mediated interactions.

This review promotes the value of isolated hepatocytes in modern Drug Discovery programmes and outlines how increased understanding, particularly in the area of in vitro-in vivo extrapolation (IVIVE), has led to more widespread use. The importance of in vitro metabolic intrinsic clearance data for predicting in vivo clearance has been acknowledged for several years and the greater utility of hepatocytes, compared with hepatic microsomes and liver slices, for this application is discussed. The application of hepatocytes in predicting drug-drug interactions (DDIs) resulting from reversible and irreversible (time-dependent) inhibition is relatively novel but affords the potential to study both phase I and phase II processes together with any impact of drug efflux and/or uptake (cellular accumulation). Progress in this area is reviewed along with current opinions on the comparative use of primary hepatocytes and higher throughput reporter gene-based systems for studying cytochrome P450 (CYP) induction. The appreciation of the role of transporter proteins in drug disposition continues to evolve. The study of hepatic uptake using isolated hepatocytes and the interplay between drug transport and metabolism with respect to both clearance and DDIs and subsequent IVIVE is also considered.

Based on a robust understanding of how physicochemical properties drive PK, relatively little experimental data is required establish a drug discovery optimisation strategy and gain confidence that clinical candidates are selected with a high likelihood of achieving an acceptable clinical PK profile. This chapter is designed to be used by the practicing medicinal chemist and attempts to provide both the context and some specific advice to apply best practice for PK and efficacious dose prediction. Hints and tips are suggested throughout the chapter and methods to predict human PK parameters outlined. Worked examples of both proprietary candidate and approved drugs are used to highlight particular themes. This chapter can be used in conjunction with the associated Dose to Man iPad app which allows medicinal chemists to simulate therapeutic doses and exposures in Man for their drug discovery projects via the approaches and equations presented herein. ‘Dose to man’ approach integrates information to rank and select compounds, which as a minimum, is a sophisticated scoring function, and at best, is an effective tool for assessing clinical utility.

The aim of this study was to evaluate a unified method for predicting human in vivo intrinsic clearance (CL(int, in vivo)) and hepatic clearance (CL(h)) from in vitro data in hepatocytes and microsomes by applying the unbound fraction in blood (fu(b)) and in vitro incubations (fu(inc)). Human CL(int, in vivo) was projected using in vitro data together with biological scaling factors and compared with the unbound intrinsic clearance (CL(int, ub, in vivo)) estimated from clinical data using liver models with and without the various fu terms. For incubations conducted with fetal calf serum (n=14), the observed CL(int, in vivo) was modeled well assuming fu(inc) and fu(b) were equivalent. CL(int, ub, in vivo) was predicted best using both fu(b) and fu(inc) for other hepatocyte data (n=56; r(2)=0.78, p=3.3 x 10(-19), average fold error=5.2). A similar model for CL(int, ub, in vivo) was established for microsomal data (n=37; r(2)=0.77, p=1.2 x 10(-12), average fold error=6.1). Using the model for CL(int, ub, in vivo) (including a further empirical scaling factor), the CL(h) in humans was also calculated according to the well stirred liver model for the most extensive dataset. CL(int, in vivo) and CL(h) were both predicted well using in vitro human data from several laboratories for acidic, basic, and neutral drugs. The direct use of this model using only in vitro human data to predict the metabolic component of CL(h) is attractive, as it does not require extra information from preclinical studies in animals.

The intrinsic clearances (CLint) of 50 neutral and basic marketed drugs were determined in fresh human hepatocytes and the data used to predict human in vivo hepatic metabolic clearance (CLmet). A statistically significant correlation between scaled CLmet and actual CLmet was observed (r2 = 0.48, p < 0.05), and for 73% of the drugs studied, scaled clearances were within 2-fold of the actual clearance. These data have shown that CLint data generated in human hepatocytes can be used to provide estimates of human hepatic CLmet for both phase I and phase II processes. In addition, the utility of commercial and in-house cryopreserved hepatocytes was assessed by comparing with data derived from fresh cells. A set of 14 drugs metabolized by the major human cytochromes P450 (P450s) (CYP1A2, 2C9, 2C19, 2D6, and 3A4) and uridine diphosphate glucuronosyltransferases (UGT1A1, 1A4, 1A9, and 2B7) have been used to characterize the activity of freshly isolated and cryopreserved human and dog hepatocytes. The cryopreserved human and dog cells retained on average 94% and 81%, respectively, of the CLint determined in fresh cells. Cryopreserved hepatocytes retain their full activity for more than 1 year in liquid N2 and are thus a flexible resource of hepatocytes for in vitro assays. In summary, this laboratory has successfully cryopreserved human and dog hepatocytes as assessed by the turnover of prototypic P450 and UGT substrates, and both fresh and cryopreserved human hepatocytes may be used for the prediction of human hepatic CLmet.

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis - or trans -butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO 2 H) 3+ and oxenoid-iron (FeO) 3+ , can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.

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.

Identifying any extrahepatic excretion phenomenon in preclinical species is crucial for an accurate prediction of the pharmacokinetics in man. This understanding is particularly key for drugs with a small volume of distribution, because they require an especially low total clearance to be suitable for a once-a-day dosing regimen in man. In this study, three animal scaling techniques were applied for the prediction of the human renal clearance of 36 diverse drugs that show active secretion or net reabsorption: 1) direct correlations between renal clearance in man and each of the two main preclinical species (rat and dog); 2) simple allometry; and 3) Mahmood's renal clearance scaling method. The results show clearly that the predictions to man for the methods are improved significantly when corrections are made for species differences in plasma protein binding. Overall, the most accurate predictions were obtained by using a direct correlation with the dog renal clearance after correcting for differences in plasma protein binding and kidney blood flow (r² = 0.84), where predictions, on average, were within 2-fold of the observed renal clearance values in human.

The CYP family of enzymes are the primary facilitators of oxidative biotransformation in the liver and are responsible for the metabolism of the majority of human drugs. The CYP family metabolize a very broad range of substrates and play a central role in the mediation of clinically relevant drug–drug interactions (DDIs). This article summarizes the current knowledge base and the complexities of CYP inhibition and provides a guide for embarking on prediction of clinical DDIs with particular emphasis on the drug discovery setting. Keywords: drug–drug interactions (DDIs); inhibition; cytochrome P450; substrate selection; inhibitors

Prototypic CYP3A4 inducers were tested in a pregnane X receptor (PXR) reporter gene assay, Fa2N-4 cells, HepaRG cells, and primary human hepatocytes, along with negative controls, using CYP3A4 mRNA and activity endpoints, where appropriate. Over half of the compounds tested (14 of 24) were identified as time-dependent inhibitors of CYP3A4 and high mRNA/activity ratios (>10) were consistent with CYP3A4 time-dependent inhibition for compounds such as troleandomycin, ritonavir, and verapamil. Induction response was compared between two human donors; there was an excellent correlation in the EC(50) estimates (r(2) = 0.89, p < 0.001), and a weak but statistically significant correlation was noted for maximum observed induction at an optimum concentration (E(max)) (r(2) = 0.38, p = 0.001). E(max) and EC(50) estimates determined from the PXR reporter gene assay and Fa2N-4 and HepaRG cells were compared with those from hepatocytes. Overall, EC(50) values generated using hepatocytes agreed with those generated in the PXR reporter gene assay (r(2) = 0.85, p < 0.001) and Fa2N-4 (r(2) = 0.65, p < 0.001) and HepaRG (r(2) = 0.99, p < 0.001) cells. However, E(max) values generated in hepatocytes were only significantly correlated to those determined in Fa2N-4 (r(2) = 0.33, p = 0.005) and HepaRG cells (r(2) = 0.79, p < 0.001). "Gold standard" cytochrome P450 induction data can be generated using primary human hepatocytes, but a restricted, erratic supply and interdonor variability somewhat restrict routine application within a drug discovery setting. HepaRG cells are a valuable recent addition to the armory of in vitro tools for assessing CYP3A4 induction and seem to be an excellent surrogate of primary cells.

In this article approaches to predict human pharmacokinetics (PK) are discussed and the capability of the exemplified methodologies to estimate individual PK parameters and therapeutic dose for a set of marketed oral drugs has been assessed. For a set of 63 drugs where the minimum efficacious concentration (MEC) and human PK were known, the clinical dose was shown to be well predicted or in some cases over-estimated using a simple one-compartment oral PK model. For a subset of these drugs, in vitro potency against the primary human targets was gathered, and compared to the observed MEC. When corrected for plasma protein binding, the MEC of the majority of compounds wasor=3 fold over the respective in vitro target potency value. A series of in vitro and in vivo experiments were conducted to predict the human PK parameters. Metabolic clearance was generally predicted well from human hepatocytes. Interestingly, for this compound set, allometry or glomerular filtration rate (GFR) ratio methods appeared to be applicable for renal CL even where CL(renal)GFR. For approximately 90% of compounds studied, the predicted CL using in vitro-in vivo (IVIV) extrapolation together with a CL(renal) estimate, where appropriate, was within 2-fold of that observed clinically. Encouragingly volume of distribution at steady state (V(ss)) estimated in preclinical species (rat and dog) when corrected for plasma protein binding, predicted human V(ss) successfully on the majority of occasions--73% of compounds within 2-fold. In this laboratory, absorption estimated from oral rat PK studies was lower than the observed human absorption for most drugs, even when solubility and permeability appeared not to be limiting. Preliminary data indicate absorption in the dog may be more representative of human for compounds absorbed via the transcellular pathway. Using predicted PK and MEC values estimated from in vitro potency assays there was a good correlation between predicted and observed dose. This analysis suggests that for oral therapies, human PK parameters and clinical dose can be estimated from a consideration of data obtained from in vitro screens using human derived material and in vivo animal studies. The benefits and limitations of this holistic approach to PK and dose prediction within the drug discovery process are exemplified and discussed.

Starting from adenosine triphosphate (ATP), the identification of a novel series of P2Y(12) receptor antagonists and exploitation of their SAR is described. Modifications of the acidic side chain and the purine core and investigation of hydrophobic substituents led to a series of neutral molecules. The leading compound, 17 (AZD6140), is currently in a large phase III clinical trial for the treatment of acute coronary syndromes and prevention of thromboembolic clinical sequelae.

Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters kinact and KI) which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4'-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1'-hydroxylation in a time- and concentration-dependent manner. Fluoxetine also inhibited CYP2C19-dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLMs, and recombinant CYP2C19 (KI 0.4 microM and kinact 0.5 min(-1)). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, a proprietary AstraZeneca compound; KI 30 microM and kinact 0.02 min(-1)) effectively abolished CYP2C9 activity over 24 h at low (micromolar) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time-dependent inhibitors, which may have dramatic inhibitory effects on P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate.

The IC50 values of 14 drugs were determined in recombinantly expressed CYP2C9 (rCYP2C9) and human hepatocytes and the data used to simulate clinical area under the plasma concentration-time curve (AUC) changes upon coadministration with prototypic CYP2C9 substrates. There was an excellent correlation between IC(50, apparent) values determined using diclofenac and naproxen as CYP2C9 substrates (r2 = 0.82, p0.0001), with values being generally higher in the naproxen assay. After correcting for nonspecific binding, the IC(50, unbound) values were similar between the assays, for the majority of compounds. Two compounds, amiodarone and benzbromarone, demonstrated substrate-specific differences, activating naproxen O-demethylase to approximately 250% of control activity at 1 mM and 1 microM, respectively, while inhibiting diclofenac 4'-hydroxylation with IC(50, apparent) values of 3 microM and 0.04 microM, respectively. CYP2C9 IC(50, apparent) values generated in human hepatocytes were systematically higher than those determined with rCYP2C9. After correcting for nonspecific binding, there was an excellent correlation of IC(50, unbound) values generated in the different milieu (r2 = 0.88, p0.0001). The ratio of inhibitor concentration at the entrance to the liver to the inhibition constant ([I]in/Ki) was used to simulate clinical deltaAUC changes and compared with that observed in vivo. Where [I]in, total/Ki, apparent) was used, there were zero false negatives (observed deltaAUCor=2, predicted deltaAUC2), eight correct assignations, and seven false positives (observed deltaAUCor=2, predicted deltaAUC2. Where [I]in, unbound/Ki, unbound was used, there was one false negative, 14 correct assignations, and zero false positives. In summary, the data presented here suggest that for CYP2C9 interactions, the use of total liver inhibitor concentrations may indeed avoid false negatives, but more realistic predictions may be achieved using unbound liver inhibitor concentrations and unbound in vitro inhibition parameters.

The amino acid sequence of the diheme cytochrome c peroxidase from Paracoccus denitrificans has been determined as the result of sequence analysis of peptides generated by chemical and enzymatic cleavages of the apoprotein. The sequence shows 60% similarity to the cytochrome c peroxidase from Pseudomonas aeruginosa, 39% similarity to an open reading frame encoding a putative triheme c-type cytochrome in Escherichia coli, and remote similarity to the MauG proteins from two methylotrophic bacteria. It is proposed, on the basis of the pattern of conserved residues in the sequences, that a change in iron coordination in the N-terminal heme domain may accompany reduction to the active mixed valence state, a change which may be accompanied by conformational adjustments in the highly conserved interface between the N- and C-terminal domains. These conformational adjustments may also lead to the appearance of a second Ca2+ binding site in the mixed valence enzyme. The exposed edge of the heme in the C-terminal domain is surrounded by several different patterns of charged residues in the Paracoccus and Pseudomonas enzymes, and this is consistent with the interaction of the former with the highly positively charged front face of the donor cytochrome c-550.

The binding of Ca'+ to the dihaem cytochrome-c. peroxidasc from 1'urur.oc:c~us dmitrificans was analyscd by following perturbations in the visiblc and 'H-NMR spectra of both haem groups. The enzyine contains at least two types of Ca' ' -binding sitc. Site I is occupied in the isolated enzynic, binds Ca' with a redox-state-independent Kcl of 1.2 pM and acconiinodates neither Me" nor Mn' . Site I1 is unoccupied in dilute solutions of the isolated oxidised enzyme and hinds Ca" cooperatively with a K