Robust Translation of γ-Secretase Modulator Pharmacology across Preclinical Species and Human Subjects.

The amyloid-ß peptide (Aß)-in particular, the 42-amino acid form, Aß1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aß synthesis or increase the clearance of Aß have entered clinical trials, including γ-secretase inhibitors, anti-Aß antibodies, and amyloid-ß precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aß1-42 production, and may also decrease Aß1-40 while simultaneously increasing one or more shorter Aß peptides, such as Aß1-38 and Aß1-37. GSMs are particularly attractive because they do not alter the total amount of Aß peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, ß-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aß1-42 and Aß1-40 levels while increasing Aß1-38 and Aß1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

Phosphocholine conjugation: an unexpected in vivo conjugation pathway associated with hepatitis c ns5b inhibitors featuring a bicyclo[1.1.1]pentane.

During a medicinal chemistry campaign to identify inhibitors of the hepatitis C virus nonstructural protein 5B (RNA-dependent RNA polymerase), a bicyclo[1.1.1]pentane was introduced into the chemical scaffold to improve metabolic stability. The inhibitors bearing this feature, 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)-4-fluorophenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (1) and 5-(3-(bicyclo[1.1.1]pentan-1-ylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide (2), exhibited low turnover in incubations with liver S9 or hepatocytes (rat, human), with hydroxylation of the bicyclic moiety being the only metabolic pathway observed. In subsequent disposition studies using bile-duct-cannulated rats, the metabolite profiles of bile samples revealed, in addition to multiple products of bicyclopentane-oxidation, unexpected metabolites characterized by molecular masses that were 181 Da greater than those of 1 or 2. Further LC/MSn and NMR analysis of the isolated metabolite of 1 demonstrated the presence of a phosphocholine (POPC) moiety bound to the methine carbon of the bicyclic moiety through an ester bond. The POPC conjugate of the NS5B inhibitors was assumed to result from two sequential reactions: hydroxylation of the bicyclic methine to a tertiary alcohol and addition of POPC by CDP-choline: 1,2-diacylglycerol cholinephosphotransferase, an enzyme responsible for the final step in the biosynthesis of phosphatidylcholine. However, this pathway could not be recapitulated using CDP-choline-supplemented liver S9 or hepatocytes due to inadequate formation of the hydroxylation product in vitro. The observation of this unexpected pathway prompted concerns about the possibility that 1 and 2 might interfere with routine phospholipid synthesis. These results demonstrate the participation in xenobiotic metabolism of a process whose function is ordinarily limited to the synthesis of endogenous compounds.

Labware additives identified to be selective monoamine oxidase-B inhibitors.

Plastic labware is used in all processes of modern pharmaceutical research, including compound storage and biological assays. The use of these plastics has created vast increases in productivity and cost savings as experiments moved from glass test tubes and capillary pipettes to plastic microplates and multichannel liquid handlers. One consequence of the use of plastic labware, however, is the potential release of contaminants and their resultant effects on biological assays. We report herein the identification of biologically active substances released from a commonly used plastic microplate. The active contaminants were identified by gas chromatography-mass spectroscopy as dodecan-1-ol, dodecyl 3-(3-dodecoxy-3-oxopropyl)sulfanylpropanoate, and dodecanoic acid, and they were found to be selective monoamine oxidase-B 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.

Cytotoxic xanthones from Psorospermum molluscum from the Madagascar rain forest.

Two new cytotoxic xanthones were isolated from extracts of the Madagascar rain forest plant Psorospermum cf. molluscum using bioassay-guided fractionation with the Escherichia coli SOS chromotest. The structures of the new dihydrofuranoxanthones, designated 3',4'-deoxy-4'-chloropsoroxanthin-(3',5'-diol) ( 1) and psoroxanthin ( 4), were determined on the basis of 2D-NMR, MS, and UV spectroscopic data and are structurally related to the psorospermins, a known class of plant antitumor agents. A new hydroxyprenylated xanthone ( 5) is also described. Xanthones 1 and 4 showed selective in vitro cytotoxicity against ABAE cells (bovine endothelial cell line).

An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates.

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.