Karthick Vishwanathan

Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases.

With the diminishing effectiveness of current antibacterial therapies, it is critically important to discover agents that operate by a mechanism that circumvents existing resistance. ETX0914, the first of a new class of antibacterial agent targeted for the treatment of gonorrhea, operates by a novel mode-of-inhibition against bacterial type II topoisomerases. Incorporating an oxazolidinone on the scaffold mitigated toxicological issues often seen with topoisomerase inhibitors. Organisms resistant to other topoisomerase inhibitors were not cross-resistant with ETX0914 nor were spontaneous resistant mutants to ETX0914 cross-resistant with other topoisomerase inhibitor classes, including the widely used fluoroquinolone class. Preclinical evaluation of ETX0914 pharmacokinetics and pharmacodynamics showed distribution into vascular tissues and efficacy in a murine Staphylococcus aureus infection model that served as a surrogate for predicting efficacious exposures for the treatment of Neisseria gonorrhoeae infections. A wide safety margin to the efficacious exposure in toxicological evaluations supported progression to Phase 1. Dosing ETX0914 in human volunteers showed sufficient exposure and minimal adverse effects to expect a highly efficacious anti-gonorrhea therapy.

Discovery of Novel DNA Gyrase Inhibiting Spiropyrimidinetriones: Benzisoxazole Fusion with N-Linked Oxazolidinone Substituents Leading to a Clinical Candidate (ETX0914).

A novel class of bacterial type-II topoisomerase inhibitor displaying a spiropyrimidinetrione architecture fused to a benzisoxazole scaffold shows potent activity against Gram-positive and fastidious Gram-negative bacteria. Here, we describe a series of N-linked oxazolidinone substituents on the benzisoxazole that improve upon the antibacterial activity of initially described compounds of the class, show favorable PK properties, and demonstrate efficacy in an in vivo Staphylococcus aureus infection model. Inhibition of the topoisomerases DNA gyrase and topoisomerase IV from both Gram-positive and a Gram-negative organisms was demonstrated. Compounds showed a clean in vitro toxicity profile, including no genotoxicity and no bone marrow toxicity at the highest evaluated concentrations or other issues that have been problematic for some fluoroquinolones. Compound 1u was identified for advancement into human clinical trials for treatment of uncomplicated gonorrhea based on a variety of beneficial attributes including the potent activity and the favorable safety profile.

Assessment of the Mass Balance Recovery and Metabolite Profile of Avibactam in Humans and In Vitro Drug-Drug Interaction Potential

Avibactam, a novel non-β-lactam β-lactamase inhibitor with activity against Ambler class A, class C, and some class D enzymes is being evaluated in combination with various β-lactam antibiotics to treat serious bacterial infections. The in vivo mass balance recovery and metabolite profile of [14C] avibactam (500 mg/1-h infusion) was assessed in six healthy male subjects, and a series of in vitro experiments evaluated the metabolism and drug-drug interaction potential of avibactam. In the mass balance study, measurement of plasma avibactam (using a validated liquid chromatography-tandem mass spectrometry method) and total radioactivity in plasma, whole blood, urine, and feces (using liquid scintillation counting) indicated that most of the avibactam was excreted unchanged in urine within 12 hours, with recovery complete (>97% of the administered dose) within 96 hours. Geometric mean avibactam renal clearance (158 ml/min) was greater than the product of unbound fraction of drug and glomerular filtration rate (109.5 ml/min), suggesting that active tubular secretion accounted for some renal elimination. There was no evidence of metabolism in plasma and urine, with unchanged avibactam the major component in both matrices. Avibactam demonstrated in vitro substrate potential for organic anion transporters 1 and 3 (OAT1 and OAT3) proteins expressed in human embryonic kidney 293 cells (Km > 1000 μM; >10-fold the Cmax of a therapeutic dose), which could account for the active tubular secretion observed in vivo. Avibactam uptake by OAT1 and OAT3 was inhibited by probenecid, a potent OAT1/OAT3 inhibitor. Avibactam did not interact with various other membrane transport proteins or cytochrome P450 enzymes in vitro, suggesting it has limited propensity for drug–drug interactions involving cytochrome P450 enzymes.

An integrated strategy for in vivo metabolite profiling using high-resolution mass spectrometry based data processing techniques

An ongoing challenge of drug metabolite profiling is to detect and identify unknown or low-level metabolites in complex biological matrices. Here we present a generic strategy for metabolite detection using multiple accurate-mass-based data processing tools via the analysis of rat samples of two model drug candidates, AZD6280 and AZ12488024. First, the function of isotopic pattern recognition was proved to be highly effective in the detection of metabolites derived from [(14)C]-AZD6280 that possesses a distinct isotopic pattern. The metabolites revealed using this approach were in excellent qualitative correlation to those observed in radiochromatograms. Second, the effectiveness of accurate mass based untargeted data mining tools such as background subtraction, mass defect filtering, or a data mining package (MZmine) used for metabolomic analysis in detection of metabolites of [(14)C]-AZ12488024 in rat urine, feces, bile and plasma samples was examined and a total of 33 metabolites of AZ12488024 were detected. Among them, at least 16 metabolites were only detected by the aid of the data mining packages and not via radiochromatograms. New metabolic pathways such as S-oxidation and thiomethylation reactions occurring on the thiazole ring were proposed based on the processed data. The results of these experiments also demonstrated that accurate mass-based mass defect filtering (MDF) and data mining techniques used in metabolomics are complementary and can be valuable tools for delineating low-level metabolites in complex matrices. Furthermore, the application of distinct multiple data-mining algorithms in parallel, or in tandem, can be effective for rapidly profiling in vivo drug metabolites.

Application of Quantitative NMR in Pharmacological Evaluation of Biologically Generated Metabolites: Implications in Drug Discovery

It is important to gain an understanding of the pharmacological activities of metabolite(s) of compounds in development, especially if they are found in systemic circulation in humans. Pharmacological evaluation of metabolites is normally conducted with synthetic standards, which become available during various stages of drug development. However, the synthesis of metabolite standards may be protracted, taking anywhere from several weeks to months to be completed. This often slows down early pharmacological evaluation of metabolites. Once a metabolite(s) is found to possess comparable (or greater) pharmacological activity than the parent compound, additional studies are performed to better understand the implications of circulating pharmacologically active metabolite(s). To conduct some of these studies as early as possible without slowing the progression of a compound in development is important, especially if critical go or no-go decisions impinge on the outcomes from these studies. Early pharmacological evaluation of significant metabolites is hereby proposed to be conducted in the drug discovery stage so that all pertinent studies and information can be gathered in a timely manner for decision-making. It is suggested that these major metabolites be isolated, either from biological or chemical sources, and quantified appropriately. For biologically generated metabolites, NMR is proposed as the tool of choice to quantitate these metabolites before their evaluation in pharmacological assays. For metabolites that have the same UV characteristics as the parent compound, quantitation can be conducted using UV spectroscopy instead of NMR. In this article, we propose a strategy that could be used to determine the pharmacological activities of metabolites isolated in submilligram quantities.

Abstract B153: Effect of food and gastric pH modifiers on the pharmacokinetics of AZD9291

Background AZD9291 is a potent, oral, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) selective for sensitizing (EGFRm) and T790M resistance mutations. Clinical activity has been demonstrated in patients with advanced EGFR T790M positive non-small cell lung cancer (NSCLC). We report results from two Phase I clinical studies, one investigating the effect of food, and another on the impact of increased gastric pH, on the pharmacokinetics (PK) of AZD9291. Design The studies were conducted in accordance with ICH/GCP guidance; protocols were reviewed and approved by IEC and IRB prior to implementation. The likely commercial formulation of AZD9291 80 mg film-coated tablet was used. The Food effect study (NCT02163733) had two parts: A and B. Part A was an open-label randomized two-period crossover design where the primary objective was to evaluate the effect of food on AZD9291 PK following oral dosing of 80 mg tablet in patients with EGFRm+ NSCLC, whose disease had progressed following treatment with a prior EGFR-TKI. Part B is an ongoing extension phase where patients may receive continuous AZD9291 80 mg dosing, if they continue to derive benefit, with safety monitoring conducted for up to 1 year. The Gastric pH interaction study (NCT02224053) was an open-label, two-fixed-period study with primary objective to evaluate the effect of omeprazole 40 mg on AZD9291 exposure in healthy, male volunteers. In Period 1, subjects received omeprazole on Days 1-4 and on Day 5 they received AZD9291 and omeprazole after a 10-hour fast. In Period 2, after a washout of at least 21 days, subjects received AZD9291 alone after a 10-hour fast. After Period 1 the AZD9291 AUC 0-72 exposure was assessed and subjects with exposures exceeding set limits were withdrawn and replaced. In both studies, blood samples were taken serially post dose in each treatment period to determine concentrations of AZD9291 and its active metabolites (AZ5104 and AZ7550). The safety and tolerability of AZD9291 was a secondary objective in the Food effect (fed and fasted state) and Gastric pH interaction studies (AZD9291±omeprazole). Results Food effect study (Part A only): 38 patients were assigned to treatment. Geometric LS mean ratios comparing fed to fasted treatments for AZD9291 C max and AUC 0-72 were 92.75% (81.40, 105.68) and 106.05% (94.82, 118.60), respectively and 90% confidence intervals were contained within the predefined equivalence limits of 70% to 143%. Gastric pH interaction study: 68 subjects were assigned to treatment and 47 subjects completed both study periods. The geometric LS mean ratios comparing AZD9291 C max and AUC when given with omeprazole compared to administration of AZD9291 alone were 101.65% (94.65, 109.16) and 106.66% (100.26, 113.46), respectively and 90% confidence intervals were within the equivalence limits of 80% to 125%. In addition, neither food nor co-administration of AZD9291 with omeprazole had a clinically relevant effect on exposure (C max and AUC) for the AZD9291 metabolites AZ5104 and AZ7550. Oral administration of 80 mg AZD9291 was well tolerated, with no deaths or discontinuations due to AE in either study. Conclusions These studies suggest that AZD9291 can be administered without regard to food and no restrictions are necessary when administered with gastric pH modifying agents. AZD9291 was well tolerated under fed or fasted conditions, and when co-dosed with omeprazole. Citation Format: Karthick Vishwanathan, Paul A. Dickinson, Khanh Bui, Doris K. Weilert, Karen So, Karen Thomas, Eleanor A. Lisbon, Ruth Plummer. Effect of food and gastric pH modifiers on the pharmacokinetics of AZD9291. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B153.

Harnessing Meta‐analysis to Refine an Oncology Patient Population for Physiology‐Based Pharmacokinetic Modeling of Drugs

Certain oncology compounds exhibit fundamental pharmacokinetic (PK) disparities between healthy and malignant conditions. Given the effects of tumor‐associated inflammation on enzyme and transporter expression, we performed a meta‐analysis of CYP‐ and transporter‐sensitive substrate clinical PK to quantitatively compare enzyme and transporter abundances between healthy volunteers (HV) and cancer patients (CP). Hepatic and intestinal CYP1A2, CYP2C19, and CYP3A4 abundance were subsequently adjusted via Simcyps sensitivity analysis tool. Of the 11 substrates we investigated, seven displayed marked exposure differences >1.25‐fold between CP and HV. Although CP studies are limited, meta‐analysis‐based reduction in CYP1A2, CYP2C19, and CYP3A4 enzyme abundances in a virtual oncology population effectively captures CP‐PK for caffeine, theophylline, midazolam, simvastatin, omeprazole, and a subset of oncology compounds. These changes allow extrapolation from HV to CP, enhancing predictive capability; therefore, conducting simulations in this CYP‐modified oncology (MOD‐CP) population provides a more relevant characterization of CP‐PK.

Development, Verification, and Prediction of Osimertinib Drug–Drug Interactions Using PBPK Modeling Approach to Inform Drug Label

Osimertinib is a potent, highly selective, irreversible inhibitor of epidermal growth factor receptor (EGFR) and T790M resistance mutation. In vitro metabolism data suggested osimertinib is a substrate of cytochrome P450 (CYP)3A4/5, a weak inducer of CYP3A, and an inhibitor of breast cancer resistance protein (BCRP). A combination of in vitro data, clinical pharmacokinetic data, and drug‐drug interaction (DDI) data of osimertinib in oncology patients were used to develop the physiologically based pharmacokinetic (PBPK) model and verify the DDI data of osimertinib. The model predicted the observed monotherapy concentration profile of osimertinib within 1.1‐fold, and showed good predictability (within 1.7‐fold) to the observed peak plasma concentration (Cmax) and area under the curve (AUC) DDI ratio changes, when co‐administered with rifampicin, itraconazole, and simvastatin, but not with rosuvastatin. Based on observed clinical data and PBPK simulations, the recommended dose of osimertinib when dosed with strong CYP3A inducers is 160 mg once daily. PBPK modeling suggested no dose adjustment with moderate and weak CYP3A inducers.

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 4β-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.

Novel diversity-oriented synthesis-derived respiratory syncytial virus inhibitors identified via a high throughput replicon-based screen

Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns.