In vitro absorption, distribution, metabolism and elimination (ADME) assays are widely used for profiling compounds in pharmaceutical drug discovery programs. Many compounds are screened in metabolic stability assays, using liver microsomes as a model of intrinsic hepatic clearance. Analysis of metabolic stability assays has relied on high throughput LC-MS/MS techniques to keep up with automated assays and compound profiling needs. An experimental alternative to sample analysis via fast chromatography employs an open port interface (OPI) which dilutes and directs acoustically-ejected droplets from microtiter plates to a conventional electrospray ion source for ionization and introduction into a mass spectrometer. Metabolic stability assays of 37 commercial drug compounds using in human, dog, rat and mouse liver microsomes (LMs), were analyzed by LC-MS/MS and an experimental breadboard version of an ADE-OPI-MS/MS system. Results from the experiments comparing intrinsic clearance (CLint) generated with ADE-OPI-MS/MS vs fast LC-MS/MS for all compounds showed ≥86% of CLint values were within a factor of two with R2 ≥ 0.86 using 25 nL and 5 nL sample ejection volumes on the ADE-OPI-MS/MS instrument. Throughput with the experimental ADE-OPI-MS/MS system used in this study was more than ten-fold faster than analysis by the fast LC-MS/MS at 1.3 s/sample versus 17.2 s/sample, respectively.

Electrospray ionization (ESI) in-source fragmentation (ISF) has traditionally been minimized to promote precursor molecular ion formation, and therefore its value in molecular identification is underappreciated. In-source annotation algorithms have been shown to increase confidence in putative identifications by using ubiquitous in-source fragments. However, these in-source annotation algorithms are limited by ESI sources that are generally designed to minimize ISF. In this study, enhanced in-source fragmentation annotation (eISA) was created by tuning the ISF conditions to generate in-source fragmentation patterns comparable with higher energy fragments generated at higher collision energies as deposited in the METLIN MS/MS library, without compromising the intensity of precursor ions (median loss ≤10% in both positive and negative ionization modes). The analysis of 50 molecules was used to validate the approach in comparison to MS/MS spectra produced via data dependent acquisition (DDA) and data independent acquisition (DIA) mode with quadrupole time-of-flight mass spectrometry (QTOF-MS). Enhanced ISF as compared to QTOF DDA enabled higher peak intensities for the precursor ions (median: 18 times in negative mode and 210 times in positive mode), with the eISA fragmentation patterns consistent with METLIN for over 90% of the molecules with respect to fragment relative intensity and m/z. eISA also provides higher peak intensity as opposed to QTOF DIA for over 60% of the precursor ions in negative mode (median increase: 20%) and for 88% of the precursor ions in positive mode (median increase: 80%). Molecular identification with eISA was also successfully validated from the analysis of a metabolic extract from macrophages. An interesting side benefit of enhanced ISF is that it significantly improved molecular identification confidence with low resolution single quadrupole mass-spectrometry-based untargeted LC/MS experiments. Overall, enhanced ISF allowed for eISA to be used as a more sensitive alternative to other QTOF DIA and DDA approaches, and further, it enabled the acquisition of ESI TOF and ESI single quadrupole mass spectrometry instrumentation spectra with improved molecular identification confidence.

Accurate determination of the free fraction of a drug in plasma can be challenging when it falls below 1% and even more so when below 0.1%. Equilibrium dialysis with diluted plasma has been used to determine unbound fraction below 1%, but some analytes are not amenable to this method. One robust alternative for accurately measuring very highly bound compounds is equilibrium gel filtration; however, radiolabeled compounds have been used with this technique to quantify the low analyte concentrations. This report examined results obtained using radiolabeled compounds with liquid scintillation detection and those obtained using their nonradiolabeled analogs with liquid chromatography-tandem mass spectrometry detection. The 2 methods provided comparable results over the range of 0.005%-4% free, with a slope of 1.0 and a R-2 = 0.93. These results demonstrate that equilibrium gel filtration with liquid chromatography-tandem mass spectrometry detection can be used earlier in the drug discovery process to determine the unbound fraction of highly bound drugs and may help obviate the need for radiolabeled compound. (c) 2019 The Authors. Published by Elsevier Inc. on behalf of the American Pharmacists Association (R).

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

Successful drug discovery relies on the selection of drug candidates with good in vivo pharmacokinetic (PK) properties as well as appropriate preclinical efficacy and safety profiles. In vivo PK profiling is often a bottleneck in the discovery process. In this review, we focus on the tiered in vivo PK approaches implemented at the Genomics Institute of the Novartis Research Foundation (GNF), which includes snapshot PK, rapid PK and full PK studies. These in vivo PK approaches are well integrated within discovery research, allow tremendous flexibility and are highly efficient in supporting the diverse needs and increasing demand for in vivo profiling. The tiered in vivo PK studies expedite compound profiling and help guide the selection of more desirable compounds into efficacy models and for progression into development.

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

With the extensive use of different strains of mice and rats in in vivo efficacy models, lack of relevant metabolic clearance data among strains has been a concern. Metabolic clearance is an important parameter impacting drug discovery, and it is often used as a compound selection filter. Metabolically stable compounds are often preferred, and will have a better chance to achieve the desired exposure in vivo. The present study examined strain differences in mouse and rat, using 96 compounds which spanned a wide range of intrinsic clearances. The in vitro clearances were determined using liver microsomes from commonly used strains of mouse (BALB/c, C57BL/6J, and CD-1) and of rat (Sprague-Dawley, Fischer, and Wistar Han). There were few discrepancies in the interpretation of the in vitro intrinsic clearance results within species for the 96 compounds tested in mouse and rat liver microsomes. This data gives us confidence that the phase I hepatic clearance can be determined using only one strain of mouse or rat liver microsomes.

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.

Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.

Described in this article are strategies implemented to increase the throughput of in vivo rodent pharmacokinetic (PK) studies using the snapshot PK study design and automated methods for compound submission, sample processing, data analysis and reporting. Applying snapshot PK studies to categorize the oral exposure of >1300 discovery compounds as low, moderate or high resulted in an attrition rate of 86%. The follow up full PK studies on the remaining compounds found that 98% of the compounds were predicted in the correct (69%) or adjacent (29%) oral exposure category by the snapshot PK studies. These results demonstrate that the snapshot PK screen in rodents can serve as an effective and efficient in vivo tool in the compound selection process in drug discovery.

ADVERTISEMENT RETURN TO ISSUEPREVPerspectiveNEXTChanging Requirements of Purification as Drug Discovery Programs Evolve from Hit DiscoveryJohn Isbell*View Author Information Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, California 92121* E-mail: [email protected]Cite this: J. Comb. Chem. 2008, 10, 2, 150–157Publication Date (Web):January 9, 2008Publication History Received17 September 2007Published online9 January 2008Published inissue 1 March 2008https://pubs.acs.org/doi/10.1021/cc700152vhttps://doi.org/10.1021/cc700152vreview-articleACS PublicationsCopyright © 2008 American Chemical SocietyRequest reuse permissionsArticle Views618Altmetric-Citations24LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access options SUBJECTS:Drug discovery,High-performance liquid chromatography,Purification,Salts,Screening assays Get e-Alerts

The 1,536-well microplate format has widely supplanted the 384-well microplate format for high-throughput screening and for IC(50) assays. Previously, liquid chromatography/mass spectrometry (LC/MS) analyses of such samples required manual transfers of the wells of interest from a 1,536-well plate into a 384-well plate. Because this manual transfer introduced a source of potential error, it became clear that a more appropriate solution would be to sample directly from the 1,536-well plates. Currently, commercially available 1,536-well plate auto samplers are not compatible with Waters LC/MS systems. The authors have modified their CTC PAL autosampler to support injection from up to twenty-four 1,536-well plates. This allows them to cherry-pick any sample from up to 36,864 wells on the autosampler. Because of its success at this Institute, sampling from 1,536-well plates has not only become the preferred method for LC/MS analysis from IC(50) plates but also become the standard format used for the handling of and the sampling from large combinatorial libraries.

In the quest for novel pharmaceuticals to treat important diseases, biologists often use in vitro assays to assess apparent activity. Thus, when testing novel synthetic compounds in search of a new class of drugs, it is important to know the solubility of the compound under the assay conditions used so that the activity data may be interpreted correctly. As the screening throughput increases, so do the demands on solubility determinations and the reliance on this data to aid in the selection of the more promising compounds for further studies. Since many compounds synthesized in drug discovery compounds contain UV chromophores, solubility determinations using UV plate readers are widely applicable to common druglike compounds. A more analytically rigorous approach calculates the solubility using a calibration curve rather than a ratio to a reference concentration. Rather than use the λ max of the analyte for quantification, an automated method is used for intelligent selection of the wavelength most appropriate for quantification. Using a BioMek FX and Peak Seeker, our visual basic application written in-house, 128 compounds may be assayed at a single pH within 5 h using MultiScreen Solubility plates.