Dieter M. Drexler

An empirical process for the design of high-throughput screening deck filters

A process for objective identification and filtering of undesirable compounds that contribute to high-throughput screening (HTS) deck promiscuity is described. Two methods of mapping hit promiscuity have been developed linking SMARTS-based structural queries with historical primary HTS data. The first compares an expected assay hit rate to actual hit rates. The second examines the propensity of an individual compound to hit multiple assays. Statistical evaluation of the data indicates a correlation between the resultant functional group filters and compound promiscuity. These data corroborate a number of commonly applied filters as well as producing some unexpected results. Application of these models to HTS collection triage reduced the number of in-house compounds considered for screening by 12%. The implications of these findings are further discussed in the context of the HTS screening set and combinatorial library design as well as compound acquisition.

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed.

P-Glycoprotein Efflux and Other Factors Limit Brain Amyloid β Reduction by β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitors in Mice

Alzheimers disease (AD) is a progressive neurodegenerative disease. Amyloid β (Aβ) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Aβ. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Aβ formation in cultured cells with IC50 values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Aβ peptides by mass spectrometry showed that these inhibitors decreased Aβ by inhibiting BACE1. An assay for Aβ1–40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Aβ1–40, but not brain Aβ1–40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Aβ1–40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Aβ1–40 and brain Aβ1–40 dose responses for these three compounds revealed differences in relative ED50 values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

An Automated Liquid Chromatography-Mass Spectrometry Process to Determine Metabolic Stability Half-Life and Intrinsic Clearance of Drug Candidates by Substrate Depletion

An automated process is described for the detailed assessment of the in vitro metabolic stability properties of drug candidates in support of pharmaceutical property profiling. Compounds are incubated with liver microsomes using a robotic liquid handler. Aliquots are taken at various time points, and the resulting samples are quantitatively analyzed by liquid chromatography-mass spectrometry utilizing ion trap mass spectrometers to determine the amount of compound remaining. From these data metabolism rates can be calculated. A high degree of automation is achieved through custom software, which is employed for instrument setup, data processing, and results reporting. The assay setup is highly configurable, allowing for any combination of up to six user-selected time points, variable substrate concentration, and microsomes or other biologically active media. The data, based on relative substrate depletion, affords an estimate of metabolic stability through the calculation of half-life (t(1/2)) and intrinsic clearance, which are used to differentiate and rank order drug leads. In general, t(1/2) is the time necessary for the metabolism, following first-order kinetics, of 50% of the initial compound. Intrinsic clearance is the proportionality constant between rate of metabolism of a compound and its concentration at the enzyme site. Described here is the setup of the assay, and data from assay test compounds are presented.

Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.e. selected reaction monitoring, SRM). Due to the difference in CID processes, triple quadrupoles and ion traps often generate significantly different fragmentation spectra of product ion species and intensities. The large peptidic analytes investigated here generated fewer fragments with higher relative abundance on the ion trap as compared to those generated on the triple quadrupole, resulting in lower limits of detection on the ion trap.

Utility of quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI) in the assessment of ocular distribution of drugs.

INTRODUCTION Assessment of drug candidate properties and potential liabilities can greatly benefit from issue driven studies that are designed to address specific toxicological effects such as ocular phototoxicity. If a compound absorbs light in the wavelength range of 290-700 nm (UV-A, UV-B, and visible light) and generates a positive response in a standard in vitro neutral red uptake phototoxicity assay in Balb/c 3T3 mouse fibroblasts, a single-dose in vivo study may be conducted to assess the potential for drug-induced phototoxicity in the eyes and skin of pigmented Long-Evans rats. Critical to ocular phototoxicity assessment is the hypothesis that the drug or drug-related material must be present in the affected substructures such as the uveal tract, retina, lens, or cornea. For compounds that induce a positive ocular response in the in vivo phototoxicity assay, data on distribution patterns to substructures of the eye can inform decisions regarding the nature of the ocular findings and possibly influence compound advancement. METHODS Quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer employing higher order mass spectrometric scanning functions were utilized for localization of dosed drug or metabolites in eye substructures. RESULTS In investigative studies designed to simulate an in vivo phototoxicity study, rats were administered radio-labeled test article for QWBA analysis and un-labeled test article for IMS analysis. Autoradiograms from the QWBA study indicated that the radio-labeled analyte(s) preferentially distributed to the uveal tract and not the cornea. However, QWBA did not provide information on the nature of the detected analyte(s); i.e. intact parent drug versus potential metabolites or degradants. Multistage MS experiments performed directly on tissue sections demonstrated semi-quantitative localization in the uveal tract and unequivocal identification of the analyte as the dosed parent drug; no potential metabolites were detected. DISCUSSION Image analysis by QWBA and IMS by MALDI proved complementary in the localization and identification of small molecule drug distribution within the eye.

Extraction, Identification, and Functional Characterization of a Bioactive Substance From Automated Compound-Handling Plastic Tips

Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-unsaturated fatty acid amide commonly used in plastics manufacturing, by gas chromatography/mass spectroscopy analysis of the DMSO-extracted material. Tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a functional bioassay of a known G-protein-coupled fatty acid receptor. A sample of a different disposable tip product from the same vendor did not release detectable erucamide following solvent extraction, and DMSO extracts prepared from this product were inactive in the receptor functional assay. These results demonstrate that solvent-extractable contaminants from some plastic labware used in the contemporary pharmaceutical research and development (R&D) environment can be introduced into physical and biological assays during routine compound management liquid-handling processes. These contaminants may further possess biological activity and are therefore a potential source of assay-specific confounding artifacts.

A high-throughput bioanalytical platform using automated infusion for tandem mass spectrometric method optimization and its application in a metabolic stability screen

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate-based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high-quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan 2.0, a unique high-throughput solution featuring MS/MS method optimization by automated infusion, with a customized in-house software tool in support of a Metstab screen. In this platform, a dual-column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%.

Automated identification of isotopically labeled pesticides and metabolites by intelligent ‘real time’ liquid chromatography tandem mass spectrometry using a bench-top ion trap mass spectrometer

A screening method was developed to specifically detect and identify isotopically labeled compounds automatically in real-time using a HPLC/ion trap mass spectrometer utilizing newly developed isotopic pattern recognition software. The ion trap mass spectrometer specifically detected isotopically labeled compounds in complex matrices based on isotopic pattern recognition utilizing full scan MS and full scan MS/MS. The resultant data included MS and MS/MS structural information for each detected compound containing the appropriate isotopic response, which was to be used for structural confirmation.

Cassette incubation followed by bioanalysis using high-resolution MS for in vitro ADME screening assays

BACKGROUND High-resolution MS (HRMS) has recently received a considerable interest in quantitative bioanalysis using full-scan acquisition mode. The benefits include complete elimination of compound-specific MS method development, and simultaneous collection of mass spectral data on both targeted and non-targeted components. One additional advantage that has not been widely discussed is its suitability for simultaneous quantitation of, theoretically, an unlimited number of compounds, which is not possible with selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. MATERIALS & METHODS We took advantage of this unique bioanalytical capability of HRMS and developed a novel in vitro ADME workflow of cassette incubation of as many as 32 compounds, followed by quantitative bioanalysis using full-scan acquisition on an Orbitrap HRMS. The workflow was evaluated for a serum protein-binding assay and a parallel artificial membrane permeability (PAMPA) assay. RESULTS The bioanalytical assay displayed acceptable sensitivity, selectivity and linearity for all compounds in the cassettes, and the biological results obtained using this approach were similar to those from discrete incubation and analysis, demonstrating the feasibility of the workflow. Additional benefits of this platform include a saving of analysis time due to the reduced sample numbers from the cassette approach, as well as cost saving due to the reduction in the required assay reagents. CONCLUSION Cassette incubation with bioanalysis using HRMS is a feasible approach for high-throughput in vitro ADME assays evaluated in this study.