Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice.

BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Discovery of a potent nicotinic Acid receptor agonist for the treatment of dyslipidemia.

Nicotinic acid has been used clinically for decades to control serum lipoproteins. Nicotinic acid lowers very low-density lipoprotein (VLDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and lipoprotein-a (LPa), and it is also effective in raising high-density lipoprotein (HDL)-cholesterol. However, nicotinic acid has several side effects in clinical use. The most notable is intense cutaneous vasodilation "flushing" on the upper body and face. We discovered a pyranopyrimidinedione series to be nicotinic acid receptor agonists. A potent nicotinic acid receptor agonist from this series {5-(3-cyclopropylpropyl)-2-(difluoromethyl)-3H-pyrano[2,3-d]pyrimidine-4,7-dione}with reduced flushing side effect in dogs was identified.

Development of a biomarker assay for 3-indoxyl sulfate in mouse plasma and brain by liquid chromatography/tandem mass spectrometry.

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric method for the determination of 3-indoxyl sulfate (3-IS), an endogenous compound in mammals, in mouse plasma and brain samples. The analytical method involves direct dilution of samples with water and protein precipitation with acetonitrile containing an internal standard, followed by separation of 3-IS on a MonoChrom C(18) column and detected by selected reaction monitoring (SRM) in negative ionization mode using turbo ion-spray ionization. Due to high endogenous levels of 3-IS in control mouse plasma and brain, blank guinea pig plasma and brain were used for the preparation of standard curves and quality controls (QCs). The compound of interest was well separated from interference peaks from the matrices with a total runtime of 2.7 min under a gradient condition. The method was partially validated. The linear concentration range was 0.1 to 100 microg/mL in mouse plasma and 10 to 10,000 ng/g in mouse brain. Inter-assay mean bias and relative standard deviation (RSD) for plasma were in the range of -4.8% to 3.1% and 2.5% to 3.2%, respectively. Intra-assay mean bias and RSD for plasma were in the range of -3.3% to 1.4% and 1.9% to 2.8%, respectively. Inter-assay mean bias and RSD for brain were in the range of -1.8% to 3.5% and 1.7% to 8.1%, respectively. Intra-assay mean bias and RSD for brain were in the range of -1.7% to 3.9% and 4.1% to 7.3%, respectively. The lower limit of quantitation (LLOQ) for this assay was 0.1 microg/mL for plasma and 10 ng/g for brain. The matrix effect was not observed in both guinea pig plasma and mouse plasma.

Ultra-performance liquid chromatography/tandem mass spectrometric determination of diastereomers of SCH 503034 in monkey plasma.

This paper describes the development and qualification of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of diastereomers of SCH 503034 in monkey plasma. The analytical method involves direct protein precipitation with a mixture of methanol/acetonitrile (10/90) containing an internal standard, followed by separation of the stereoisomers on an Acquity UPLC C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using atmospheric pressure chemical ionization (APCI). The effects of ion-pairing agents on separation and ionization efficiency were investigated. The two diastereomers were well separated (R=1.3) with a runtime of 5 min under an isocratic condition. The method was qualified. The linear concentration range was 1-2500 ng/ml for the both stereoisomers. Inter-assay mean bias and relative standard deviation (R.S.D.) were in the range of -1.2% to 3.6% and 2.8-10%, respectively. Intra-assay mean bias and R.S.D. were in the range of -1.3% to 5.5% and 2.3-7.8%, respectively. Recoveries of the stereoisomers at concentration levels of 2.5, 50 and 1000 ng/ml were 87.2-90.0%, 89.1-90.4% and 92.3-94.3%, respectively. The LLOQ for this assay was 1 ng/ml. No matrix interferences were observed in six different sources of blank monkey plasma.

Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples.

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.

A study of common discovery dosing formulation components and their potential for causing time-dependent matrix effects in high-performance liquid chromatography tandem mass spectrometry assays.

Hydroxyproyl-beta-cyclodextran (HPBCD), methyl cellulose (MC), Tween 80 and PEG400 are commonly used in dosing formulations in pharmacokinetic (PK) studies during the early drug discovery stage. A series of studies was designed to evaluate the potential matrix effects of these dosing vehicles when the samples are assayed by high-performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS/MS). These dosing vehicles were dosed into the rats via either an intravenous (IV) or an oral route (PO) and plasma samples were collected for a 24-h post-dose period. Five test compounds with CLog P values ranging from 0.9 to 5.4 were spiked into the collected rat plasma. After protein precipitation, these samples were analyzed using a generic fast-gradient HPLC/MS/MS method. Three popular mass spectrometers (Thermo-Finnigan Quantum with ESI and APCI, AB-Sciex API 3000 with ESI and APCI, and Waters-Micromass Quattro Ultima with ESI) were used to test these plasma samples. Results indicated that there was no observed matrix effect for all five compounds when 20% HPBCD or 0.4% MC was used as the vehicle in either the IV or the PO route, respectively. In addition, 0.1% Tween 80 dosed either IV or PO caused significant ion suppression (50-80%, compared to results obtained from plasma samples free from vehicles) for compounds that eluted at the beginning of the chromatogram. Also, PEG400 when used in an oral formulation caused significant ion suppression (30-50%) for early eluting compounds. These matrix effects were not only ionization mode (ESI or APCI) dependent, but also source design (Thermo-Finnigan, AB-Sciex or Waters-Micromass) dependent. Overall, the APCI mode proved to be less vulnerable to matrix effects than the ESI mode. Some possible mechanisms of these matrix effects are proposed and simple strategies to avoid these matrix effects are discussed.

Comparison of atmospheric pressure chemical ionization, electrospray ionization, and atmospheric pressure photoionization for the determination of cyclosporin A in rat plasma.

Atmospheric pressure chemical ionization was compared with electrospray ionization and atmospheric pressure photoionization (APPI) as an interface of high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) for the determination of cyclosporin A (CsA) in biological fluids in support of in vivo pharmacodynamic studies. These ion sources were investigated in terms of their suitability and sensitivity for the detection of CsA. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the photoionization efficiency of CsA in the positive ion mode under normal-phase HPLC conditions were explored. The ionization mechanism in the APPI environment with and without the use of the dopant was studied using two test compounds and a few solvent systems employed for normal-phase chromatography. The test compounds were observed to be ionized mainly by proton transfer with the self-protonated solvent molecules produced through photon irradiation. Furthermore, ion suppression due to sample matrix interference in the normal-phase HPLC-APPI-MS/MS system was monitored by the postcolumn infusion technique. The applicability of these proposed HPLC-API-MS/MS approaches for the determination of CsA at low nanogram per milliliter levels in rat plasma was examined. These proposed methods were then compared with respect to specificity, linearity, detection limit, and accuracy.

Reduced cardiovascular effects of methamphetamine following treatment with selegiline.

Selegiline is a specific MAO-B inhibitor. As MAO-B has been shown to be significantly involved in the metabolism of dopamine in certain regions of the primate brain, selegiline has been proposed for use in the treatment of drug addiction. Selegiline is also metabolized in vivo to l-methamphetamine. Therefore, when given in combination with psychostimulants such as d-methamphetamine, there is the potential for adverse effects. To study this possibility, squirrel monkeys were treated with chronic selegiline and tested with two doses of d-methamphetamine (0.1 and 1.0 mg/kg, i.v.). Following at least 7 days of treatment with once daily 0.3 mg/kg i.m. selegiline, the effects of methamphetamine on blood pressure and heart rate were no different than the effects of methamphetamine observed prior to selegiline treatment. However, following at least 10 days of treatment with 1.0 mg/kg i.m. selegiline, the effects of methamphetamine on blood pressure and heart rate were significantly reduced. Both methamphetamine and amphetamine were detected in plasma following chronic selegiline treatment. When monkeys were given an acute selegiline injection prior to methamphetamine, reduced cardiovascular effects were also seen. These results indicate that selegiline can be used safely even in combination with methamphetamine, as the cardiovascular effects of the drug combination were no greater than either drug alone, and were actually reduced at the higher selegiline dose.

Simultaneous fast HPLC-MS/MS analysis of drug candidates and hydroxyl metabolites in plasma.

A rapid bioanalytical method was evaluated for the simultaneous determination of drug discovery compounds and their potential metabolites in plasma samples within 1 min run time by fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The fast HPLC-MS/MS system is achieved by using mini-column HPLC coupled to tandem mass spectrometer which is advantageous over regular HPLC-MS/MS systems, such as a shorter chromatographic region of ionization suppression, less solvent consumption and higher throughput. Matrix ionization suppression effect of the test compounds in plasma samples when using fast HPLC-MS/MS method was examined by a post-column infusion technique. In the described example, the proposed approach has been successfully employed to determine the plasma concentration of the test compound and its hydroxyl metabolite (M+16) in monkey in the low ng/ml region. The monkey pharmacokinetic results obtained by the proposed fast HPLC-MS/MS method were in good agreement within 20% error with those obtained by the regular HPLC-MS/MS method based on the same sample preparation procedure.

High-performance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometric analysis for small molecules in plasma.

A generic high-performance liquid chromatography (HPLC) system interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer was developed for the quantitative determination of small molecules in plasma in support of exploratory in vivo pharmacokinetics. This report summarizes the effects of variations in reversed-phase mode HPLC conditions such as mobile-phase flow rate, solvent composition, organic modifier content, and nebulizer temperature on the photoionization efficiency of both clozapine and lonafarnib. The matrix ionization suppression effect on this method was investigated using the postcolumn infusion technique. The procedure was used to quantitate plasma levels following oral administration of 42 drug discovery compounds to rats. The pharmacokinetic results of 42 drug discovery compounds in rats evaluated by both APPI and atmospheric pressure chemical ionization interfaces were found to be well correlated.

Zirconia-based column high-performance liquid chromatography/atmospheric pressure photoionization tandem mass spectrometric analyses of drug molecules in rat plasma.

A method using zirconia-based column high-performance liquid chromatography (HPLC) interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer (MS/MS) was developed for the quantitative determination of new chemical entities in rat plasma in support of pharmacokinetics studies. The ionization suppression resulting from endogenous components of the biological matrices on the quantitative zirconia-based column HPLC/APPI-MS/MS method was investigated using the post-column infusion technique. The analytical results for 'rapid rat pharmacokinetics' for 12 drug discovery compounds, obtained by both silica-based phase (S-phase) and zirconia-based phase (Z-phase) chromatographic separation, are in good agreement in terms of accuracy. The application of a Z-phase column for high-temperature fast HPLC/MS/MS methods was explored to reduce the analysis time from 3 min to 30 s for column temperatures of 25-110 degrees C, respectively. The chromatographic retention times and peak responses of all analytes were found to be reproducible under high-temperature conditions following 100 continuous injections, with %CV less than 0.4 and 5, respectively.

Direct plasma analysis of drug compounds using monolithic column liquid chromatography and tandem mass spectrometry.

A monolithic silica column high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the high-speed direct simultaneous determination of a drug discovery compound and its major circulating metabolite (M-72) in rat plasma. This methodology makes use of flow programming and an alkyl-bonded silica rod column for fast macromolecule removal and chromatographic separation without the need for significant sample preparation. The matrix ionization suppression effect on the monolithic column HPLC-MS/MS system was investigated using the postcolumn infusion technique. After 200 plasma injections on a 50 x 4.6 mm monolithic silica column, consistent column efficiency of close to 39,000 theoretical plates/m and reproducible retention times for the analytes were observed. The apparent on-column recoveries of 12 test compounds in rat plasma samples were greater than 90%. The proposed fast direct plasma injection method was tested over a 3-day period with the interday coefficient of variation less than 15% for both analytes.