Utilizing a Dual Human Transporter MDCKII-MDR1-BCRP Cell Line to Assess Efflux at the Blood Brain Barrier.

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB), a Madin-Darby canine kidney (MDCK) cell line was established that over expresses both P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters [BCRP substrate dantrolene efflux ratio (ER) 16.

A PKPD Case Study: Achieving Clinically Relevant Exposures of AZD5991 in Oncology Mouse Models.

Capturing human equivalent drug exposures preclinically is a key challenge in the translational process. Motivated by the need to recapitulate the pharmacokinetic (PK) profile of the clinical stage Mcl-1 inhibitor AZD5991 in mice, we describe the methodology used to develop a refined mathematical model relating clinically relevant concentration profiles to efficacy. Administration routes were explored to achieve target exposures matching the clinical exposure of AZD5991.

Interspecies evaluation of a physiologically based pharmacokinetic model to predict the biodistribution dynamics of dendritic nanoparticles.

The exposure of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) was determined in mouse, rat and dog, with the aim of investigating interspecies differences facilitating clinical translation. Plasma area under the curves (AUCs) were found to be dose proportional across species, while dose normalized concentration time course profiles in plasma, liver and spleen were superimposable in mouse, rat and dog. A physiologically based pharmacokinetic (PBPK) model, previously developed for mouse, was evaluated as a suitable framework to prospectively capture concentration dynamics in rat and dog.

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CL) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (P > 5 × 10 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined.

ADME and DMPK considerations for the discovery and development of antibody drug conjugates (ADCs).

 The therapeutic concept of antibody drug conjugates (ADCs) is to selectively target tumour cells with small molecule cytotoxic drugs to maximise cell kill benefit and minimise healthy tissue toxicity.An ADC generally consists of an antibody that targets a protein on the surface of tumour cells chemically linked to a warhead small molecule cytotoxic drug.To deliver the warhead to the tumour cell, the antibody must bind to the target protein and in general be internalised into the cell.

Challenges and Opportunities for Improved Drug-Drug Interaction Predictions for Renal OCT2 and MATE1/2-K Transporters.

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.

Quantitative Evaluation of Dendritic Nanoparticles in Mice: Biodistribution Dynamics and Downstream Tumor Efficacy Outcomes.

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.

Mechanistic physiology-based pharmacokinetic modeling to elucidate vincristine-induced peripheral neuropathy following treatment with novel kinase inhibitors.

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.

Discovery of AZD9833, a Potent and Orally Bioavailable Selective Estrogen Receptor Degrader and Antagonist.

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 -[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6,8)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3-pyrazolo[4,3-]isoquinolin-6-yl]pyridin-3-amine (). 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.

Evaluation of the Disconnect between Hepatocyte and Microsome Intrinsic Clearance and In Vitro In Vivo Extrapolation Performance.

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.

Optimising proteolysis-targeting chimeras (PROTACs) for oral drug delivery: a drug metabolism and pharmacokinetics perspective.

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.

Improving the Accuracy of Predicted Human Pharmacokinetics: Lessons Learned from the AstraZeneca Drug Pipeline Over Two Decades.

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.

Further Considerations Towards an Effective and Efficient Oncology Drug Discovery DMPK Strategy.

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.

Building on the success of osimertinib: achieving CNS exposure in oncology drug discovery.

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.

Discovery of AZD-2098 and AZD-1678, Two Potent and Bioavailable CCR4 Receptor Antagonists.

-(5-Bromo-3-methoxypyrazin-2-yl)-5-chlorothiophene-2-sulfonamide 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.

Application of in silico, in vitro and preclinical pharmacokinetic data for the effective and efficient prediction of human pharmacokinetics.

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.

Prediction of human renal clearance from preclinical species for a diverse set of drugs that exhibit both active secretion and net reabsorption.

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.

Evaluation of multiple in vitro systems for assessment of CYP3A4 induction in drug discovery: human hepatocytes, pregnane X receptor reporter gene, and Fa2N-4 and HepaRG cells.

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.

Integrated in vitro analysis for the in vivo prediction of cytochrome P450-mediated drug-drug interactions.

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.

From ATP to AZD6140: the discovery of an orally active reversible P2Y12 receptor antagonist for the prevention of thrombosis.

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.

The pivotal role of hepatocytes in drug discovery.

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).

Evaluation of time-dependent cytochrome P450 inhibition using cultured human hepatocytes.

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.

Prediction of CYP2C9-mediated drug-drug interactions: a comparison using data from recombinant enzymes and human hepatocytes.

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, p < 0.