Toward highly sensitive and reproducible LC-MS/MS analysis of MK-8591 phosphorylated anabolites in human peripheral blood mononuclear cells.

Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.

Discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity and pharmacokinetics.

Selective inhibition of Kv1.5, which underlies the ultra-rapid delayed rectifier current, IKur, has been pursued as a treatment for atrial fibrillation. Here we describe the discovery of MK-1832, a Kv1.5 inhibitor with improved selectivity versus the off-target current IKs, whose inhibition has been associated with ventricular proarrhythmia. MK-1832 exhibits improved selectivity for IKur over IKs (>3000-fold versus 70-fold for MK-0448), consistent with an observed larger window between atrial and ventricular effects in vivo (>1800-fold versus 210-fold for MK-0448). MK-1832 also exhibits an improved preclinical pharmacokinetic profile consistent with projected once daily dosing in humans.

Adenosine analogue inhibitors of S-adenosylhomocysteine hydrolase.

Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer's disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.

Quantitation of physiological and biochemical barriers to siRNA liver delivery via lipid nanoparticle platform.

Effective delivery of small interfering RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in lipid nanoparticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14- or C18-anchored PEG-lipid(s), cationic lipid(s), and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED50's ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag-fluorescently labeled siRNA. One hour after dosing a majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small subcellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution, and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.

Benzimidazole CB2 agonists: design, synthesis and SAR.

A new series of CB2-selective agonists containing a benzimidazole core is reported. Design, synthesis, SAR and pharmacokinetic data for selected compounds are described.

Liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel profiling tool for drug distribution and metabolism analysis: the terfenadine example.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a novel surface profiling technique that combines micro-liquid extraction from a solid surface with nano-electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA-MS can provide drug- and metabolite-specific distribution information. Here, we evaluate LESA-MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI-MSI. The spatial resolution of LESA-MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also enabling drug profiling within a single organ. LESA-MS can readily identify the biotransformation of terfenadine to its major, active metabolite fexofenadine. Relative quantification can confirm the rapid absorption of terfendine after oral dosage, its extensive first pass metabolism and the distribution of both compounds into systemic tissues such as muscle, spleen and kidney. The elimination appears to be consistent with biliary excretion and only trace levels of fexofenadine could be confirmed in brain. We found LESA-MS to be more informative in terms of drug distribution than a comparable MALDI-MS imaging study, likely due to its favorable overall sensitivity due to the larger surface area sampled. LESA-MS appears to be a useful new profiling tool for examining the distribution of drugs and their metabolites in tissue sections.

Decahydroquinoline amides as highly selective CB2 agonists: role of selectivity on in vivo efficacy in a rodent model of analgesia.

A novel series of decahydroquinoline CB2 agonists is described. Optimization of the amide substituent led to improvements in CB2/CB1 selectivity as well as physical properties. Two key compounds were examined in the rat CFA model of acute inflammatory pain. A moderately selective CB2 agonist was active in this model. A CB2 agonist lacking functional CB1 activity was inactive in this model despite high in vivo exposure both peripherally and centrally.

Imidazopyridine CB2 agonists: optimization of CB2/CB1 selectivity and implications for in vivo analgesic efficacy.

A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1 selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both centrally and peripherally. Administration of a highly selective CB2 agonist in a rat model of analgesia was ineffective despite substantial CNS exposure, while administration of a moderately selective CB2/CB1 agonist exhibited significant analgesic effects.

Induction of Alzheimer's-like changes in brain of mice expressing mutant APP fed excess methionine.

Elevated plasma homocysteine, a risk factor for Alzheimer's disease, could result from increased production from methionine or by inefficient clearance by folate- and B-vitamin-dependent pathways. Understanding the relative contributions of these processes to pathogenesis is important for therapeutic strategies designed to lower homocysteine. To assess these alternatives, we elevated plasma homocysteine by feeding mutant amyloid precursor protein (APP)-expressing mice diets with either high methionine (HM) or deficient in B-vitamins and folate (B Def). Mutant APP mice fed HM demonstrated increased brain beta amyloid. Interestingly, this increase was not observed in mutant APP mice fed B Def diet, nor was it observed in C57Bl6 or YAC-APP mice fed HM. Furthermore, HM, but not B Def, produced a prolonged increase in brain homocysteine only in mutant APP mice but not wild-type mice. These changes were time-dependent over 10 weeks. Further, by 10 weeks HM increased brain cholesterol and phosphorylated tau in mutant APP mice. Transcriptional profiling experiments revealed robust differences in RNA expression between C57Bl6 and mutant APP mice. The HM diet in C57Bl6 mice transiently induced a transcriptional profile similar to mutant APP cortex, peaking at 2 weeks , following a time course comparable to brain homocysteine changes. Together, these data suggest a link between APP and methionine metabolism.

Discovery of triarylethanolamine inhibitors of the Kv1.5 potassium channel.

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure-activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.

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.

Potent antagonists of the Kv1.5 potassium channel: synthesis and evaluation of analogous N,N-diisopropyl-2-(pyridine-3-yl)acetamides.

This letter describes the discovery of a novel series of potent Kv1.5 ion channel antagonists based on a diisopropyl amide scaffold. Structure-activity relationships of functionalized analogs are discussed. Key compound 1-(3-(diisopropylcarbamoyl)-2-phenyl-3-(pyridin-3-yl)propyl)-3-(2-fluorobenzyl)urea (10) exhibits significant atrial-selective effects in an in vivo model.