Philip R. Tiller

Quantitation of small molecules using high-resolution accurate mass spectrometers - a different approach for analysis of biological samples.

The quantitative capabilities of a linear ion trap high-resolution mass spectrometer (LTQ-Orbitrap) were investigated using full scan mode bracketing the m/z range of the ions of interest and utilizing a mass resolution (mass/FWHM) of 15000. Extracted ion chromatograms using a mass window of +/-5-10 mmicro centering on the theoretical m/z of each analyte were generated and used for quantitation. The quantitative performance of the LTQ-Orbitrap was compared with that of a triple quadrupole (API 4000) operating using selected reaction monitoring (SRM) detection. Comparable assay precision, accuracy, linearity and sensitivity were observed for both approaches. The concentrations of actual study samples from 15 Merck drug candidates reported by the two methods were statistically equivalent. Unlike SRM being a tandem mass spectrometric (MS/MS)-based detection method, a high resolution mass spectrometer operated in full scan does not need MS/MS optimization. This approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. An example of the identification of a major circulating metabolite for a preclinical development study is demonstrated.

Automated strategies for obtaining standardized collisionally induced dissociation spectra on a benchtop ion trap mass spectrometer

An automated method for obtaining standardized collisionally induced dissociation (CID) spectra using two novel ion activation techniques on a quadrupole ion trap mass spectrometer is described. This strategy simultaneously produces optimal CID spectra “on-the-fly” and maximizes the amount of structurally specific fragment ions obtained in a single experiment, thus eliminating the need for individual tuning of specific mass-to-charge ratios and permitting fragmentation conditions to be more consistently reproduced. Copyright

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation.

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).

Metabolite identification of small interfering RNA duplex by high‐resolution accurate mass spectrometry

On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1 Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes.

Synthesis and biological evaluation of a library containing potentially 1600 amides / esters. A strategy for rapid compound generation and screening

A library of potentially 1600amide/ester dimers was prepared by reacting 40 acid chlorides with 40 nucleophiles. The whole library was presented for biological screening in 80 sample mixtures, with each of the possible products appearing in a unique pair of samples. The potential of this strategy for rapid identification of chemical leads was demostrated by the discovery of (1) with micromolar affinity for the NK3 receptor and (2), a weak inhibitor of the matrix metalloprotease MMP-1.

Design, Synthesis, and In Vivo Efficacy of Glycine Transporter-1 (GlyT1) Inhibitors Derived from a Series of [4-Phenyl-1-(propylsulfonyl)piperidin-4-yl]methyl Benzamides

Design, Synthesis, and In Vivo Efficacy of Glycine Transporter-1 (GlyT1) Inhibitors Derived from a Series of [4-Phenyl-1(propylsulfonyl)piperidin-4-yl]methyl Benzamides Craig W. Lindsley,* Zhijian Zhao, William H. Leister, Julie O’Brien, Wei Lemaire, David L. Williams, Jr. , Tsing-Bau Chen, Raymond S. L. Chang, Maryann Burno, Marlene A. Jacobson, Cyrille Sur, Gene G. Kinney, Douglas J. Pettibone, Philip R. Tiller, Sheri Smith, Nancy N. Tsou, Mark E. Duggan, P. Jeffrey Conn, e] and George D. Hartman

Fractional mass filtering as a means to assess circulating metabolites in early human clinical studies

Recent changes in the regulatory environment have led to a need for new methods to assess circulating human drug metabolites in early clinical studies with respect to their potential toxicological impact. The specific goals of such studies are to determine if the metabolites present in human plasma following administration of a drug candidate also are observed in plasma from the animal studies employed for preclinical toxicological evaluation, and to estimate corresponding exposure margins (animal:human) for the major metabolites. Until recently, the accepted best practice for the characterization of circulating drug metabolites utilized liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methodologies, in conjunction with authentic chemical standards, for the detection and quantitative analyses of metabolites predicted from both animal studies and experiments with human liver preparations in vitro. While this approach is satisfactory for anticipated biotransformation products, metabolites that were not expected to circulate in human plasma frequently escape detection. Current accurate mass instruments enable the use of the technique of fractional mass filtering to detect both expected and unexpected metabolites in a rapid, less resource-intensive and more robust manner. Application of this technology to several clinical development programs at Merck Research Laboratories has demonstrated the value of fractional mass filtering in the assessment of circulating drug metabolites in early clinical trials.

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.

Discovery of N-{[1-(propylsulfonyl)-4-pyridin-2-ylpiperidin-4-yl]methyl}benzamides as novel, selective and potent GlyT1 inhibitors.

Employing an iterative analogue library approach, novel potent and selective glycine transporter 1 (GlyT1) inhibitors containing a 4-pyridin-2-ylpiperidine sulfonamide have been discovered. These inhibitors are devoid of time-dependent CYP inhibition activity and exhibit improved aqueous solubility versus the corresponding 4-phenylpiperidine analogues.

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.