Evaluation of the drug-drug interaction potential for trazpiroben (TAK-906), a D2/D3 receptor antagonist for gastroparesis, towards cytochrome P450s and transporters.

Trazpiroben (TAK-906), a peripherally selective dopamine D2/D3 receptor antagonist, is being developed for the treatment of patients with gastroparesis. The potential of trazpiroben to act as a perpetrator or a victim for cytochrome P450 (CYP)- or transporter- mediated drug-drug interactions (DDIs) was evaluated following the latest regulatory guidelines.In vitro studies revealed that trazpiroben is metabolised mainly through a non-CYP pathway (56.7%) by multiple cytosolic, NADPH-dependent reductase, such as aldo-keto reductase and short-chain dehydrogenase/reductase including carbonyl reductases. Remaining metabolism occurs through CYP3A4 and CYP2C8 (43.3%). Trazpiroben is neither an inhibitor nor an inducer of major CYP enzymes at a clinically relevant dose. It is a substrate of P-glycoprotein (P-gp) and organic anion transporting polypeptide (OATP) 1B1/1B3, but is not an inhibitor of transporters listed in the DDI guidelines at a clinically relevant dose. This is consistent with findings from CYP3A and P-gp-based clinical assessment showing no substantial change (≤2-fold) in trazpiroben exposure when co-administered with itraconazole.Collectively, trazpiroben has low potential of enzyme-mediated DDIs and is unlikely to act as a perpetrator of transporter-mediated DDIs but there may be a potential to act as a victim of OATP1B1/1B3 DDI that will be evaluated clinically.

Biotransformation Pathways and Metabolite Profiles of Oral [14C]Alisertib (MLN8237), an Investigational Aurora A Kinase Inhibitor, in Patients with Advanced Solid Tumors.

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [14C]alisertib oral solution (∼80 µCi) in three patients with advanced malignancies. On average, 87.8% and 2.7% of the administered dose was recovered in feces and urine, respectively, for a total recovery of 90.5% by 14 days postdose. Unchanged [14C]alisertib was the predominant drug-related component in plasma, followed by O-desmethyl alisertib (M2), and alisertib acyl glucuronide (M1), which were present at 47.8%, 34.6%, and 12.0% of total plasma radioactivity. In urine, of the 2.7% of the dose excreted, unchanged [14C]alisertib was a negligible component (trace), with M1 (0.84% of dose) and glucuronide conjugate of hydroxy alisertib (M9; 0.66% of dose) representing the primary drug-related components in urine. Hydroxy alisertib (M3; 20.8% of the dose administered) and unchanged [14C]alisertib (26.3% of the dose administered) were the major drug-related components in feces. In vitro, oxidative metabolism of alisertib was primarily mediated by CYP3A. The acyl glucuronidation of alisertib was primarily mediated by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, and 1A8 and was stable in 0.1 M phosphate buffer and in plasma and urine. Further in vitro evaluation of alisertib and its metabolites M1 and M2 for cytochrome P450-based drug-drug interaction (DDI) showed minimal potential for perpetrating DDI with coadministered drugs. Overall, renal elimination played an insignificant role in the disposition of alisertib, and metabolites resulting from phase 1 oxidative pathways contributed to >58% of the alisertib dose recovered in urine and feces over 192 hours postdose. SIGNIFICANCE STATEMENT: This study describes the primary clearance pathways of alisertib and illustrates the value of timely conduct of human absorption, distribution, metabolism, and excretion studies in providing guidance to the clinical pharmacology development program for oncology drugs, for which a careful understanding of sources of exposure variability is crucial to inform risk management for drug-drug interactions given the generally limited therapeutic window for anticancer drugs and polypharmacy that is common in cancer patients.

Considerations from the IQ Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Focus on Downregulation, CYP2C Induction, and CYP2B6 Positive Control.

The European Medicines Agency (EMA), the Pharmaceutical and Medical Devices Agency (PMDA), and the Food and Drug Administration (FDA) have issued guidelines for the conduct of drug-drug interaction studies. To examine the applicability of these regulatory recommendations specifically for induction, a group of scientists, under the auspices of the Drug Metabolism Leadership Group of the Innovation and Quality (IQ) Consortium, formed the Induction Working Group (IWG). A team of 19 scientists, from 16 of the 39 pharmaceutical companies that are members of the IQ Consortium and two Contract Research Organizations reviewed the recommendations, focusing initially on the current EMA guidelines. Questions were collated from IQ member companies as to which aspects of the guidelines require further evaluation. The EMA was then approached to provide insights into their recommendations on the following: 1) evaluation of downregulation, 2) in vitro assessment of CYP2C induction, 3) the use of CITCO as the positive control for CYP2B6 induction by CAR, 4) data interpretation (a 2-fold increase in mRNA as evidence of induction), and 5) the duration of incubation of hepatocytes with test article. The IWG conducted an anonymous survey among IQ member companies to query current practices, focusing specifically on the aforementioned key points. Responses were received from 19 companies. All data and information were blinded before being shared with the IWG. The results of the survey are presented, together with consensus recommendations on downregulation, CYP2C induction, and CYP2B6 positive control. Results and recommendations related to data interpretation and induction time course will be reported in subsequent articles.

Contribution of metabolites to P450 inhibition-based drug-drug interactions: scholarship from the drug metabolism leadership group of the innovation and quality consortium metabolite group.

Recent European Medicines Agency (final) and US Food and Drug Administration (draft) drug interaction guidances proposed that human circulating metabolites should be investigated in vitro for their drug-drug interaction (DDI) potential if present at ≥ 25% of the parent area under the time-concentration curve (AUC) (US Food and Drug Administration) or ≥ 25% of the parent and ≥ 10% of the total drug-related AUC (European Medicines Agency). To examine the application of these regulatory recommendations, a group of scientists, representing 18 pharmaceutical companies of the Drug Metabolism Leadership Group of the Innovation and Quality Consortium, conducted a scholarship to assess the risk of contributions by metabolites to cytochrome P450 (P450) inhibition-based DDIs. The group assessed the risk of having a metabolite as the sole contributor to DDI based on literature data and analysis of the 137 most frequently prescribed drugs, defined structural alerts associated with P450 inhibition/inactivation by metabolites, and analyzed current approaches to trigger in vitro DDI studies for metabolites. The group concluded that the risk of P450 inhibition caused by a metabolite alone is low. Only metabolites from 5 of 137 drugs were likely the sole contributor to the in vivo P450 inhibition-based DDIs. Two recommendations were provided when assessing the need to conduct in vitro P450 inhibition studies for metabolites: 1) consider structural alerts that suggest P450 inhibition potential, and 2) use multiple approaches (e.g., a metabolite cut-off value of 100% of the parent AUC and the R(met) strategy) to predict P450 inhibition-based DDIs caused by metabolites in the clinic.

Mutagenicity and tumorigenicity of the four enantiopure bay-region 3,4-diol-1,2-epoxide isomers of dibenz[a,h]anthracene.

Each enantiomer of the diastereomeric pair of bay-region dibenz[a,h]anthracene 3,4-diol-1,2-epoxides in which the benzylic 4-hydroxyl group and epoxide oxygen are either cis (isomer 1) or trans (isomer 2) were evaluated for mutagenic activity. In strains TA 98 and TA 100 of Salmonella typhimurium, the diol epoxide with (1S,2R,3S,4R) absolute configuration [(-)-diol epoxide-1] had the highest mutagenic activity. In Chinese hamster V-79 cells, the diol epoxide with (1R,2S,3S,4R) absolute configuration [(+)-diol epoxide-2] had the highest mutagenic activity. The (1R,2S,3R,4S) diol epoxide [(+)-diol epoxide-1] also had appreciable activity, whereas the other two bay-region diol epoxide enantiomers had very low activity. In tumor studies, the (1R,2S,3S,4R) enantiomer was the only diol epoxide isomer tested that had strong activity as a tumor initiator on mouse skin and in causing lung and liver tumors when injected into newborn mice. This stereoisomer was about one-third as active as the parent hydrocarbon, dibenz[a,h]anthracene as a tumor initiator on mouse skin; it was several-fold more active than dibenz[a,h]anthracene as a lung and liver carcinogen when injected into newborn mice. (-)-(3R,4R)-3ß,4α-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(-)-3,4-dihydrodiol] was slightly more active than dibenz[a,h]anthracene as a tumor initiator on mouse skin, whereas (+)-(3S,4S)-3α,4ß-dihydroxy-3,4-dihydro-dibenz[a,h]anthracene [(+)-3,4-dihydrodiol] had only very weak activity. The present investigation and previous studies with the corresponding four possible enantiopure bay-region diol epoxide enantiomers/diastereomers of benzo[a]pyrene, benz[a]anthracene, chrysene, benzo[c]phenanthrene, dibenz[c,h]acridine, dibenz[a,h]acridine and dibenz[a,h]anthracene indicate that the bay-region diol epoxide enantiomer with [R,S,S,R] absolute stereochemistry has high tumorigenic activity on mouse skin and in newborn mice.

Evaluation of the Pharmacokinetics of Felcisetrag (TAK-954), a 5-HT4 Receptor Agonist, in the Presence and Absence of Itraconazole, a Potent CYP3A4 Inhibitor.

The 5-hydroxytryptamine type-4 receptor agonist felcisetrag (TAK-954) is being investigated for improving gastrointestinal motility in postoperative gastrointestinal dysfunction. Polypharmacy often occurs in this setting, and as in vitro data indicate, felcisetrag is primarily metabolized by cytochrome P450 (CYP) 3A4, its CYP3A4-mediated drug-drug interaction potential requires consideration. This phase 1, fixed-sequence, open-label, crossover trial (ClinicalTrials.gov identifier NCT03173170) investigated the effect of itraconazole, a potent CYP3A4 inhibitor, on felcisetrag pharmacokinetics in healthy adults. Over 2 study periods (period 1, 6 days; period 2, 9 days), participants received a single felcisetrag 0.2-mg intravenous dose (day 1, period 1; and day 4, period 2), and once-daily oral itraconazole 200-mg doses (days 1-8, period 2). For felcisetrag alone, felcisetrag total systemic exposure was lower than with itraconazole coadministration. The geometric mean ratio for area under the plasma concentration-time curve from time 0 to infinity of felcisetrag plus itraconazole: felcisetrag alone was 1.49 (90% confidence interval, 1.39-1.60). Peak exposure was similar between regimens (geometric mean ratio, 1.06; 90% confidence interval, 0.96-1.18), and both treatments were well tolerated. These data suggest limited CYP3A4-mediated drug-drug interaction inhibition for felcisetrag.

Clinical and Nonclinical Disposition and In Vitro Drug-Drug Interaction Potential of Felcisetrag, a Highly Selective and Potent 5-HT4 Receptor Agonist.

BACKGROUND AND OBJECTIVE: Felcisetrag (previously TAK-954 or TD-8954) is a highly selective and potent 5-HT4 receptor agonist in clinical development for prophylaxis and treatment of postoperative gastrointestinal dysfunction (POGD). The rat, dog, and human absorption, distribution, metabolism, and excretion (ADME) properties of felcisetrag were investigated. METHODS: The metabolism and victim and perpetrator drug interaction potentials towards cytochrome P450s (CYP) and transporters were determined using in vitro models. The excretion, metabolite profile, and pharmacokinetics were determined during unlabeled and radiolabeled ADME studies in rat and dog for comparison with human. Due to a low clinical dose (0.5 mg) and radioactivity (~ 1.5 µCi), a combination of liquid scintillation counting and accelerator mass spectrometry was used for analysis of samples in this study. RESULTS: The ADME properties, including metabolite profile, for felcisetrag are generally conserved across species. Felcisetrag is primarily cleared through renal excretion (0.443) and metabolism in humans (0.420), with intact parent as the predominant species in circulation. There are multiple metabolites, each representing < 10% of the circulating radioactivity, confirming no metabolites in safety testing (MIST) liabilities. Metabolites were also detected in animals. The potential for major CYP- and transporter-based drug-drug interaction (DDI) of felcisetrag as a victim or perpetrator is considered to be low. CONCLUSIONS: Felcisetrag is primarily cleared in humans through renal excretion. Although the metabolism of felcisetrag is primarily through CYP3A, the potential for clinically relevant DDI as a victim is significantly reduced as metabolism plays a minor role in the overall clearance.

A refined cytochrome P540 IC50 shift assay for reliably identifying CYP3A time-dependent inhibitors.

A refined cytochrome P450 (P450) enzyme IC50 shift assay for more accurately screening CYP3A time-dependent inhibitors (TDIs) is presented. In contrast to the regular IC50 shift assay, in which only one pair of P450 inhibition curves is generated, this modified method generates two pairs of inhibition curves; one pair of curves is created from human liver microsomal incubations with the test article in the presence or absence of NADPH (curves 1 and 2) (same as the traditional assay), and the other pair is created from new microsomal incubations with extract (compound/metabolites) of previous incubations (curves 3 and 4). To assess the true CYP3A time-dependent inhibition, we propose a new parameter, the vertical IC50 curve shift (VICS), represented by vertical shift difference between the two sets of curves divided by inhibitor concentration at which maximal vertical shift of curves 1 and 2 is observed. A shift in the curves 1 and 2 could mean a time-dependent inhibition or formation of a more active inhibitory metabolite(s). The new method provides more reliable characterization of the shift as a result of a true TDI- or metabolite-mediated reversible inhibition. Nine known TDI drugs were evaluated using this refined shift assay. The derived VICS values correlated well with the reported k(inact)/K(I) values derived via the conventional dilution assay method. Thus, the refined assay can be used to identify a true TDI and quantitatively assess the inactivation potential of TDIs in a high-throughput fashion. This assay can be invaluable to screen for true P450 TDIs in the early drug discovery.

Quantitative prediction and clinical observation of a CYP3A inhibitor-based drug-drug interactions with MLN3897, a potent C-C chemokine receptor-1 antagonist.

A novel in vitro model was recently developed in our laboratories for the prediction of magnitude of clinical pharmacokinetic drug-drug interactions (DDIs), based on reversible hepatic cytochrome P450 (P450) inhibition. This approach, using inhibition data from human hepatocytes incubated in human plasma, and quantitative P450 phenotyping data from hepatic microsomal incubations, successfully predicted DDIs for 15 marketed drugs with ketoconazole, a strong competitive inhibitor of CYP3A4/5, generally used to demonstrate a "worst-case scenario" for CYP3A inhibition. In addition, this approach was successfully extended to DDI predictions with the moderate competitive CYP3A inhibitor fluconazole for nine marketed drugs. In the current report, the general applicability of the model has been demonstrated by prospectively predicting the degree of inhibition and then conducting DDI studies in the clinic for an investigational CCR1 antagonist MLN3897, which is cleared predominantly by CYP3A. The clinical studies involved treatment of healthy volunteers (n = 17-20), in a crossover design, with ketoconazole (200 mg b.i.d.) or fluconazole (400 mg once a day), while receiving MLN3897. Administration of MLN3897 and ketoconazole led to an average 8.28-fold increase in area under the curve of plasma concentration-time plot (AUC) of MLN3897 at steady state, compared with the 8.33-fold increase predicted from the in vitro data. Similarly for fluconazole, an average increase of 3.93-fold in AUC was observed for MLN3897 in comparison with a predicted value of 3.26-fold. Thus, our model reliably predicted the exposure changes for MLN3897 in interaction studies with competitive CYP3A inhibitors in humans, further strengthening the utility of our in vitro model.

Inhibition of hepatic organic anion-transporting polypeptide by RNA interference in sandwich-cultured human hepatocytes: an in vitro model to assess transporter-mediated drug-drug interactions.

Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo- and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.

Prediction of pharmacokinetic drug-drug interactions using human hepatocyte suspension in plasma and cytochrome P450 phenotypic data. III. In vitro-in vivo correlation with fluconazole.

Whereas ketoconazole is often used to study the worst-case scenario for clinical pharmacokinetic drug-drug interactions (DDIs) for drugs that are primarily metabolized by CYP3A4, fluconazole is considered to be a moderate inhibitor of CYP3A4, providing assessment of the moderate-case scenario of CYP3A-based DDIs. Fluconazole is also a moderate inhibitor of CYP2C9 and CYP2C19. For predicting clinical DDIs using conventional approaches, determining the in vivo inhibitor concentration at the enzymatic site [I], a critical parameter, is still not practical. In our previous study, a novel method involving hepatocyte suspension in plasma was used to circumvent the need to determine the elusive [I] value. In this study, the CYP1A2, 2C9, 2C19, 2D6, and 3A4 activities remaining in the presence of fluconazole were determined in human hepatocytes suspended in human plasma, covering a range of fluconazole clinical plasma concentrations (C(avg) and C(max)). Because the protein-binding effect of fluconazole is expected to be close to that in vivo, the inhibition observed in vitro will be similar to that in vivo. This inhibition information was then applied to the cytochrome P450 (P450) phenotypic data to predict DDIs. Using the available P450 phenotypic information on theophylline, tolbutamide, omeprazole, S-warfarin, phenytoin, cyclosporine, and midazolam and that determined in this study for sirolimus and tacrolimus, we found that the predictions for area under the curve increases for most of these drugs in the presence of fluconazole were remarkably similar (within 35%) to the observed clinical values. This study proves the general applicability of our approach using human hepatocyte incubation in human plasma to predict DDIs.

Prediction of pharmacokinetic drug-drug interactions using human hepatocyte suspension in plasma and cytochrome P450 phenotypic data. II. In vitro-in vivo correlation with ketoconazole.

Traditional cytochrome P450 (P450) based drug-drug interaction (DDI) predictions are based on the ratio of an inhibitor's physiological concentration [I] and its inhibition constant K(i). Determining [I] at the enzymatic site, although critical for predicting clinical DDIs, remains a technical challenge. In our previous study, a novel approach using cryopreserved human hepatocytes suspended in human plasma was investigated to mimic the in vivo concentration of ketoconazole at the enzymatic site (Lu et al., 2007), effectively eliminating the estimation of the elusive [I] value. P450 inhibition in this system appears to model that in vivo. Using the ketoconazole inhibition information in a human hepatocyte-plasma suspension together with quantitative P450 phenotypic information, we successfully predicted the pharmacokinetic DDIs for a small set of drugs, such as theophylline, tolbutamide, omeprazole, desipramine, midazolam, loratadine, cyclosporine, and alprazolam, as well as an investigational compound. For the applicability of this model on a wider scale the in vitro-in vivo correlation data set needed to be expanded. However, for most drugs in the literature there is not enough quantitative information on the involvement of individual P450s to predict DDIs retrospectively. To facilitate that, in this study we determined quantitative P450 phenotyping for seven marketed drugs: budesonide, buprenorphine, loratadine, sirolimus, tacrolimus, docetaxel, and methylprednisolone. Augmentation of the new data set with the one generated previously produced broader a database that provided further support for the wider applicability of this approach using ketoconazole as a potent CYP3A inhibitor. This application is predicted to be equally effective with other P450 inhibitors that are not substrates of efflux pumps.

CYP Suppression in Human Hepatocytes by Monomethyl Auristatin E, the Payload in Brentuximab Vedotin (Adcetris®), is Associated with Microtubule Disruption.

BACKGROUND AND OBJECTIVES: Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. METHODS: MMAE was incubated at 1-1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-ß-tubulin antibody and imaging. RESULTS: MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of ß-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. CONCLUSIONS: MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (< 10 nM) explains the lack of pharmacokinetic drug interaction between brentuximab vedotin and midazolam, a sensitive CYP3A substrate, reported in patients.

Metabolism, Excretion and Pharmacokinetics of MLN3897, a CCR1 Antagonist, in Humans.

UNLABELLED: MLN3897 is a small molecule antagonist of the C-C chemokine receptor-1. Since preclinical studies showed that the molecule was metabolized into two halves, the metabolism, excretion, and pharmacokinetics of MLN3897 were investigated in humans using MLN3897 14C-radiolabeled either on the chlorophenyl (CP) or the tricyclic (TC) half of MLN3897 after an oral dose. OBJECTIVE: To evaluate the mass balance, metabolism and pharmacokinetics of MLN3897 in two cohorts of six randomized healthy subjects. METHOD: After receiving informed consent, subjects were dosed after an overnight fast of 10-hours followed by at least 4- hours after dosing on day-1. Each cohort received a single 29 mg oral dose of either the CP or the TC as an oral solution in water. Serial blood samples, urine and feces were collected over a 10-day period post-dose. RESULTS: For both radiolabeled moieties, 55-59% of the dose was recovered in feces and 32% recovered in urine. MLN3897 was metabolized extensively in humans, with minor amounts of intact MLN3897 detected in the urine and feces. N-oxidation of the tricyclic moiety (M28) and N-dealkylation of the piperidinyl moiety were the primary metabolic pathways leading to further formation of the carboxylic acid metabolite (M19) and the (4-(4-chlorophenyl)-3,3- dimethylpiperidin-4-ol) metabolite (M40). Oxidative metabolites M11, M19, M28, M44 were present at >10% of the total circulating radioactivity and also at >25% of MLN3897 exposure. Metabolites resulting from the chlorophenyl-labeled moiety (M40) had significantly more systemic exposure compared to the tricyclic-labeled moiety (M19).

Strategy of utilizing in vitro and in vivo ADME tools for lead optimization and drug candidate selection.

The high-throughput screening in drug discovery for absorption, distribution, metabolism and excretion (ADME) properties has become the norm in the industry. Only a few years ago it was ADME properties that were attributed to more failure of drugs than efficacy or safety in the clinic trials. With the realization of new techniques and refinement of existing techniques better projections for the pharmacokinetic properties of compounds in humans are being made, shifting the drug failure attributes more to the safety and efficacy properties of drug candidates. There are a tremendous number of tools available to discovery scientists to screen compounds for optimization of ADME properties and selection of better candidates. However, the use of these tools has generally been to characterize these compounds rather than to select among them. This report discusses applications of the available ADME tools to better understand the clinical implication of these properties, and to optimize these properties. It also provides tracts for timing of studies with respect to the stage of the compound during discovery, by means of a discovery assay by stage (DABS) paradigm. The DABS provide the team with a rationale for the types of studies to be done during hit-to-lead, early and late lead optimization stages of discovery, as well as outlining the deliverables (objectives) at those stages. DABS has proven to be optimal for efficient utilization of resources and helped the discovery team to track the progress of compounds and projects.

MLN8054 and Alisertib (MLN8237): Discovery of Selective Oral Aurora A Inhibitors.

The Aurora kinases are essential for cell mitosis, and the dysregulation of Aurora A and B have been linked to the etiology of human cancers. Investigational agents MLN8054 (8) and alisertib (MLN8237, 10) have been identified as high affinity, selective, orally bioavailable inhibitors of Aurora A that have advanced into human clinical trials. Alisertib (10) is currently being evaluated in multiple Phase II and III clinical trials in hematological malignancies and solid tumors.

Preclinical drug metabolism and pharmacokinetics, and prediction of human pharmacokinetics and efficacious dose of the investigational Aurora A kinase inhibitor alisertib (MLN8237).

Alisertib (MLN8237) is an investigational potent Aurora A kinase inhibitor currently under clinical trials for hematological and nonhematological malignancies. Nonclinical investigation showed that alisertib is a highly permeable compound with high plasma protein binding, low plasma clearance, and moderate volume of distribution in rats, dogs, monkeys and chimpanzees. Consistent with the above properties, the oral bioavailability in animals was greater than 82%. The predicted human oral pharmacokinetic (PK) profile was constructed using allometric scaling of plasma clearance and volume of distribution in the terminal phase from animals. The chimpanzee PK profiles were extremely useful to model absorption rate constant, which was assumed to be similar to that in humans, based on the fact that chimpanzees are phylogenetically closest to humans. The human plasma clearance was projected to be low of 0.12 L/hr/kg, with half-life of approximately 10 hr. For human efficacious dose estimation, the tumor growth inhibition as a measure of efficacy (E) was assessed in HCT116 xenograft mice at several oral QD or BID dose levels. Additionally, subcutaneous mini-pump infusion studies were conducted to assess mitotic index in tumor samples as a pharmacodynamic (PD) marker. PK/PD/E modeling showed that for optimal efficacy and PD in the xenograft mice maintaining a plasma concentration exceeding 1 µM for at least 8-12 hr would be required. These values in conjunction with the projected human PK profile estimated the optimal oral dose of approximately 103 mg QD or 62.4 mg BID in humans. Notably, the recommended Phase 2 dose being pursued in the clinic is close to the projected BID dose.

Preclinical pharmacokinetic/pharmacodynamic/efficacy relationships for alisertib, an investigational small-molecule inhibitor of Aurora A kinase.

PURPOSE: Alisertib (MLN8237) is an investigational inhibitor of Aurora A kinase (AAK). Aurora A plays an essential role in the regulation of spindle assembly and chromosome alignment during mitosis. Inhibition of Aurora A by alisertib in tissue culture has previously been demonstrated to lead to improper chromosomal alignment and disruption of spindle organization, resulting in a transient mitotic delay. The spindle organization defects induced by alisertib have been used to develop a pharmacodynamic (PD) assay for Aurora A inhibition based on the percentage of mitotic cells with proper chromosomal alignment at the metaphase plate (% aligned spindles, abbreviated as AS). The transient mitotic delay that occurs with AAK inhibition permits the use of the mitotic index (the fraction of cells in the population currently undergoing mitosis, abbreviated as MI) as an additional PD assay. When the two PD assays were used in Phase I clinical trials, the reduction in AS was strongly correlated with dose levels and exposures in patients from single time point PD measurements; however, MI failed to show any correlation. To further understand this clinical finding, we constructed PK/PD/efficacy models for AS and MI that can precisely capture the temporal dynamics of the PD markers from in vivo xenograft studies. METHODS: A PK/PD study was conducted using a single oral dose of alisertib at 3, 10, and 20 mg/kg in HCT-116 xenografts implanted subcutaneously in mice. An extravascular, two-compartmental pharmacokinetic (PK) model was used to describe the drug kinetics. Consistent with the mechanistic hypothesis for AAK inhibition, the PD biomarkers such as AS and MI were fitted to PK using a direct response inhibitory sigmoid model and an indirect response turnover model, respectively. The antitumor activity of alisertib dosed orally for 21 days with different dose levels and schedules was evaluated. RESULTS: The PK/PD models showed a fast, sustained response for AS after alisertib administration, whereas MI exhibited a slow, transient response. The PK/efficacy relationship for alisertib in HCT-116 xenografts closely corresponds to the PK/PD relationship for the PD markers, with all three IC50s in close agreement (303, 270, and 280 nM, respectively). CONCLUSION: The PK/PD and PK/efficacy models show that both AS and MI are equally relevant as mechanism-based PD markers to capture drug activity. However, of the two PD markers, the fast, sustained response of AS makes it the only clinically viable PD marker for defining a dose-response relationship, as its maximal effect can be captured from a wider time window with a single PD sampling; while the window to capture dose-related MI response is narrower.