AMPK activation is sufficient to increase skeletal muscle glucose uptake and glycogen synthesis but is not required for contraction-mediated increases in glucose metabolism.

The AMP-activated protein kinase (AMPK) is a cellular sensor of energetics and when activated in skeletal muscle during contraction can impart changes in skeletal muscle metabolism. Therapeutics that selectively activate AMPK have been developed to lower glucose levels through increased glucose disposal rates as an approach to abrogate the hyperglycemic state of diabetes; however, the metabolic fate of glucose following AMPK activation remains unclear. We have used a combination of in vivo evaluation of glucose homeostasis and ex vivo skeletal muscle incubation to systematically evaluate metabolism following pharmacological activation of AMPK with PF-739, comparing this with AMPK activation through sustained intermittent electrical stimulation of contraction. These methods to activate AMPK result in increased glucose uptake but divergent metabolism of glucose: pharmacological activation results in increased glycogen accumulation while contraction-induced glucose uptake results in increased lactate formation and glucose oxidation. These results provide additional evidence to support a role for AMPK in control of skeletal muscle metabolism and additional insight into the potential for AMPK stimulation with small molecule direct activators.

A quantitative LC-MS/MS approach for monitoring 2'-fluoro-2'-deoxy-D-glucose uptake in tumor tissue.

Purpose: Develop a quantitative LC-MS/MS method for FDG, FDG-monophosphate, glucose and glucose-monophosphate in mouse tumor models to assist in validating the use of [18F]FDG-positron emission tomography (PET) imaging for anticancer therapies in a clinical setting. Methodology/results: Analytes were isolated from tumors by protein precipitation and detected on a Sciex API-5500 mass spectrometer. Improved assay robustness and selectivity were achieved through chromatographic separation of FDG-monophosphate from glucose-monophosphate, selection of a unique ion transition and incorporation of stable isotope labeled internal standards. In a mouse JIMT-1 tumor model, FDG-monophosphate levels measured by LC-MS/MS correlated with [18F]FDG-PET imaging results. Conclusion: LC-MS/MS analysis of FDG-monophosphate accumulation in tumors is a cost-effective tool to gauge the translational potential of [18F]FDG-PET imaging as a noninvasive biomarker in clinical studies.

A Microfluidic Perfusion Platform for In Vitro Analysis of Drug Pharmacokinetic-Pharmacodynamic (PK-PD) Relationships.

Static in vitro cell culture studies cannot capture the dynamic concentration profiles of drugs, nutrients, and other factors that cells experience in physiological systems. This limits the confidence in the translational relevance of in vitro experiments and increases the reliance on empirical testing of exposure-response relationships and dose optimization in animal models during preclinical drug development, introducing additional challenges owing to species-specific differences in drug pharmacokinetics (PK) and pharmacodynamics (PD). Here, we describe the development of a microfluidic cell culture device that enables perfusion of cells under 2D or 3D culture conditions with temporally programmable concentration profiles. Proof-of-concept studies using doxorubicin and gemcitabine demonstrated the ability of the microfluidic PK-PD device to examine dose- and time-dependent effects of doxorubicin as well as schedule-dependent effects of doxorubicin and gemcitabine combination therapy on cell viability using both step-wise drug concentration profiles and species-specific (i.e., mouse, human) drug PK profiles. The results demonstrate the importance of including physiologically relevant dynamic drug exposure profiles during in vitro drug testing to more accurately mimic in vivo drug effects, thereby improving translatability across nonclinical studies and reducing the reliance on animal models during drug development.

Development of an In Vivo Retrodialysis Calibration Method Using Stable Isotope Labeling to Monitor Metabolic Pathways in the Tumor Microenvironment via Microdialysis.

Microdialysis is a technique that utilizes a semipermeable membrane to sample analytes present within tissue interstitial fluid. Analyte-specific calibration is required for quantitative microdialysis, but these calibration methods are tedious, require significant technical skill, and often cannot be performed jointly with the experimental measurements. Here, we describe a method using retrodialysis with stable-isotope-labeled analytes that enables simultaneous calibration and quantification for in vivo tumor microdialysis. Isotope-labeled amino acids relevant to immuno-metabolism in the tumor microenvironment (tryptophan, kynurenine, glutamine, and glutamate) were added to the microdialysis perfusate, and microdialysis probes were inserted in subcutaneous CT26 and MC38 tumors in mice. The levels of both the endogenous and isotope-labeled amino acids in the perfusate outlet were quantified using LC-MS/MS. Plasma and tumor tissue samples were also collected from the same mice and amino acid levels quantified using LC-MS/MS. Amino acids which showed statistically significant differences between the CT26-bearing and MC38-bearing mice in tumor lysate (tryptophan, kynurenine, and glutamine) and plasma (glutamate) were not the same as those identified as significantly different in tumor interstitial fluid (kynurenine and glutamate), underscoring how microdialysis can provide unique and complementary insights into tumor and immune metabolism within the tumor microenvironment.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

Identification and Evaluation of Novel MicroRNA Biomarkers in Plasma and Feces Associated with Drug-induced Intestinal Toxicity.

Gastrointestinal toxicity is dose limiting with many therapeutic and anticancer agents. Real-time, noninvasive detection of markers of toxicity in biofluids is advantageous. Ongoing research has revealed microRNAs as potential diagnostic and predictive biomarkers for the detection of select organ toxicities. To study the potential utility of microRNA biomarkers of intestinal injury in a preclinical toxicology species, we evaluated 3 rodent models of drug-induced intestinal toxicity, each with a distinct mechanism of toxicity. MiR-215 and miR-194 were identified as putative intestinal toxicity biomarkers. Both were evaluated in plasma and feces and compared to plasma citrulline, an established intestinal injury biomarker. Following intestinal toxicant dosing, microRNA changes in feces and plasma were detected noninvasively and correlated with histologic evidence of intestinal injury. Fecal miR-215 and miR-194 levels increased, and plasma miR-215 decreased in a dose- and time-dependent manner. Dose-dependent decreases in plasma miR-215 levels also preceded and correlated positively with plasma citrulline modulation, suggesting miR-215 is a more sensitive biomarker. Moreover, during the drug-free recovery phase, plasma miR-215 returned to predose levels, supporting a corresponding recovery of histologic lesions. Despite limitations, this study provides preliminary evidence that select microRNAs have the potential to act as noninvasive, sensitive, and quantitative biomarkers of intestinal injury.

Liquid chromatography-tandem mass spectrometry method for quantification of thymidine kinase activity in human serum by monitoring the conversion of 3'-deoxy-3'-fluorothymidine to 3'-deoxy-3'-fluorothymidine monophosphate.

Thymidine kinase 1 (TK1) is an enzyme involved in DNA synthesis whose activity in serum is indicative of tumor proliferation and the severity of blood malignancies. 3'-deoxy-3'-fluorothymidine (FLT), a specific exogenous substrate for TK1, is phosphorylated by TK1 in the presence of a phosphorylating buffer, therefore the conversion of FLT to 3'-deoxy-3'-fluorothymidine monophosphate (FLT-MP) can be measured to assess serum TK1 activity. Here we describe a liquid chromatography-MS/MS (LC-MS/MS) method for quantification of FLT and FLT-MP from serum using protein precipitation and column switching followed by detection on an Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. The method was linear over the range of 0.5-500 ng/mL for FLT and 2.5-2000 ng/mL for FLT-MP with a mean correlation coefficient of 0.9964 and 0.9935 for FLT and FLT-MP, respectively. The lower limit of quantification was 0.5 ng/mL for FLT and 2.5 ng/mL for FLT-MP. Intra-assay accuracy and inter-assay accuracy was within ±12% for both FLT and FLT-MP. Intra-assay precision was 2.8% to 7.7% for FLT and 3.3% to 5.8% for FLT-MP. Inter-assay precision was 4.6% to 14.9% for FLT and 4.9% to 14.6% for FLT-MP. Serum TK1 activity was measured in serum from hepatocellular carcinoma patients and age-matched controls under standardized conditions. Elevated TK1 activity was detected in 26.3% of hepatocellular carcinoma samples compared to controls. This method provides a robust alternative to radiometric and immunochemical assays of serum TK1 activity.

Evaluation of potential gastrointestinal biomarkers in a PAK4 inhibitor-treated preclinical toxicity model to address unmonitorable gastrointestinal toxicity.

Although gastrointestinal (GI) toxicity is a significant dose-limiting safety concern noted in multiple therapeutic areas, there are no GI biomarkers that can accurately track, precede, or reliably correlate with histologic evidence of injury. While significant efforts have been made within the pharmaceutical industry, academia, and consortia to address the biomarker gaps in other target organs such as liver, kidney, and muscle (cardiac and skeletal), there have been no concerted efforts in the area of GI biomarkers. Using PAK4 inhibitor as a preclinical rat model of gastric toxicity, selected candidate biomarkers from literature were evaluated to test their usefulness as gastric injury biomarkers in this study. Biomarkers selected in this study include plasma diamino oxidase and citrulline, fecal calprotectin, bile acids, and miRNA. Based on the results, L-citrulline and miR-194 results appear to correlate well with histopathology findings. Although these biomarkers will need additional assay validation and qualification to test if they truly predict the injury prior to histopathology, the results provide promise for further testing using additional GI toxicants. In addition, this article highlights important gaps in GI biomarkers and provides substrate and rationale for additional investments either for further testing of already available biomarkers or to pursue extensive biomarker discovery approaches.

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution.

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.

The validation of a simple LC/MS/MS method for determining the level of mevalonic acid in human plasma.

A simple plasma extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous mevalonic acid (MVA), a biomarker indicative of the rate of cholesterol biosynthesis, in human plasma samples. The analyte was extracted from the plasma matrix using a straightforward liquid-liquid sample preparation procedure. The extract supernatants were evaporated, reconstituted in aqueous solvent and injected into the LC/MS/MS system without further processing. The chromatographic separation was achieved on a reverse-phase high-performance liquid chromatography column. The accuracy and precision of the method was determined over the concentration range 0.25-25 ng/mL MVA from human plasma extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%RE) of the quality control samples were ≤7.00% (at lower limit quality control) and ≤6.10%, respectively. The sensitivity and throughput of this assay was significantly improved relative to previously published methods, resulting in smaller sample requirements and shorter analysis time. Assay results from a clinical study following the oral administration of an exploratory statin demonstrate that this procedure could potentially be used in the investigation of therapies associated with hypercholesterolemia.

Quantitative sphingosine measurement as a surrogate for total ceramide concentration-preclinical and potential translational applications.

Biomarkers are an increasingly important constituent of the drug development process, offering the potential of increased efficiency through reduced compound attrition and earlier proof of mechanism and/or efficacy. Assays developed for compound screening that can be directly translated for clinical trials are especially valuable, but their successful adoption requires a careful balance between assay performance and implementation costs. One such 'fit-for-purpose' biomarker assay, the indirect measurement of pharmacological modulation of sphingolipid biosynthesis and disposition, is presented here. Among sphingolipids, numerous ceramide species are readily detectable in different lipoprotein fractions of mammalian plasma, but their parallel quantification can be prohibitively expensive and time consuming. Ceramides differ in their fatty acid moiety, which is readily removed by hydrolysis, yielding a common sphingosine derivative, the measurement of which serves as an indicator of total ceramide. When followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) for detection, robust analyte quantification becomes relatively straightforward. The practical utility of a method developed to be fit for the purpose of rapidly and quantitatively measuring treatment-induced variations in total ceramide from hamster plasma and individual lipoprotein fractions is described. With a linear calibration range from 0.003 to 33.4 microm sphingosine, precision and accuracy error in plasma-based quality controls spiked with ceramides was less than 15%. The specificity of the assay for ceramides was also assessed. The simplicity of the method would allow for its potential translation to other preclinical species, as well as for clinical applications in later-stage drug development.

Creating a fatty acid methyl ester database for lipid profiling in a single drop of human blood using high resolution capillary gas chromatography and mass spectrometry.

Capillary gas chromatography (CGC) in combination with mass spectrometry (MS) was optimized for the separation and detection of the fatty acids occurring in the lipid fraction of blood. A fingertip blood sample (ca. 50 microL) was transesterified into the methyl esters and analyzed on a 100 m x 0.25 mm ID column coated with a biscyanopropyl polysiloxane (HP-88) stationary phase. The method was retention time locked. Programmed temperature vaporization injection (PTV) in the solvent venting mode was applied to minimize the sample size, while maintaining high sensitivity. The total analysis time was ca. 60 min. Retention times and both electron impact (EI) and positive chemical ionization (PCI) mass spectrometry were combined to elucidate the fatty acids according to alkyl chain, degree of unsaturation and position of the double bonds. Using extracted ion chromatograms about 100 fatty acids and related compounds were detected in blood samples and most of them were identified. This work resulted in a very large fatty acid methyl esters database, containing retention time and mass spectral information that will be applied to metabolomic studies.

Serine palmitoyltransferase inhibitor myriocin induces the regression of atherosclerotic plaques in hyperlipidemic ApoE-deficient mice.

Myriocin, a potent inhibitor of serine palmitoyltransferase (SPT), has been shown to reduce plasma sphingolipids, cholesterol and triglycerides in hyperlipidemic apolipoprotein E knockout (apoE KO) mice. We hypothesized that the inhibition of sphingolipid biosynthesis modulates the composition of atherosclerotic plaque via its lipid-lowering effects. To test this hypothesis, the effect of myriocin on plasma lipids, sphingolipids and atherosclerosis progression, regression and lesion composition was investigated in apoE KO mice. Myriocin was administered to 24-week-old male apoE KO mice for 12 weeks. Myriocin-treated apoE KO mice had significant reductions in plasma total cholesterol, triglycerides, VLDL-cholesterol, ceramide, sphinganine and sphingomyelin (SM) compared to 24- and 36-week-old control mice. The ratio of SM to phosphatidylcholine (SM/PC), an independent risk factor for coronary artery disease, was also reduced by myriocin. Compared to 24- and 36-week controls, atherosclerotic lesion area and macrophage content in the aortic root and brachiocephalic arteries of myriocin-treated ApoE KO mice were reduced but there was only a slight increase in smooth muscle content. However, the content of collagen within aortic root lesions was increased in myriocin-treated apoE KO mice. In summary, the inhibition of SPT lowers plasma sphingolipids and atherogenic plasma lipids leading to the regression of pre-existing atherosclerotic lesions and to the formation of a stable plaque phenotype.

Modulation of lipoprotein metabolism by inhibition of sphingomyelin synthesis in ApoE knockout mice.

Plasma sphingomyelin (SM) has been suggested as a risk factor for coronary heart disease independent of cholesterol levels. A decrease of SM in lipoproteins is known to improve the activities of lecithin:cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) in vitro. Inhibition of SM biosynthesis may reduce lipoprotein SM content and thus improve cholesterol distribution in lipoproteins by enhancing reverse cholesterol transport and clearance of triglyceride-rich lipoproteins. To examine this hypothesis, ApoE KO mice were fed a western diet and treated for 4 weeks with various concentrations of myriocin, a specific inhibitor of serine palmitoyltransferase. Myriocin treatment lowered plasma cholesterol and TG levels in a dose-dependent manner. In addition, myriocin treatment reduced cholesterol contents in VLDL and LDL and elevated HDL-cholesterol. Observed lipid-lowering effects of myriocin were associated with suppression of HMG CoA reductase and fatty acid synthase via reduced levels of SREBP-1 RNA and protein. Induction of apoAI and lecithin:cholesterol acytransferase (LCAT) in the liver by myriocin was associated with an increased HDL. Lesion area and macrophage area were also diminished in the cuffed femoral artery of ApoE KO mice. In conclusion, inhibition of sphingolipid biosynthesis can be a novel therapeutic target for dyslipidemia and atherosclerosis.

A validated LC/MS/MS method for the quantification of pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies.

A novel skin tissue extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous pyrrole-2,3,5-tricarboxylic acid (PTCA), a eumelanin specific biomarker, in human skin punch biopsies. The analyte is extracted from the matrix (2 mm skin punch biopsies) using a simple oxidative degradation procedure. The extract supernatants are evaporated, reconstituted in mobile phase solvent, and injected into the LC/MS/MS system without further derivatization. The chromatographic separation is achieved on a reverse phase high performance liquid chromatography (HPLC) column. The accuracy and precision of the method was determined over the concentration range of 1-1000 ng/mL PTCA from human skin extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%R.E.) of the quality control samples were

Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice.

BACKGROUND: In clinical studies, sphingomyelin (SM) plasma levels correlated with the occurrence of coronary heart disease independently of plasma cholesterol levels. We hypothesized that inhibition of SM synthesis would have antiatherogenic effects. To test this hypothesis, apolipoprotein E (apoE)-knockout (KO) mice were treated with myriocin, a potent inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in SM biosynthesis. METHODS AND RESULTS: Diet-admix treatment of apoE-KO mice with myriocin in Western diet for 12 weeks lowered SM and sphinganine plasma levels. Decreases in sphinganine and SM concentrations were also observed in the liver and aorta of myriocin-treated animals compared with controls. Inhibition of de novo sphingolipid biosynthesis reduced total cholesterol and triglyceride plasma levels. Cholesterol distribution in lipoproteins demonstrated a decrease in beta-VLDL and LDL cholesterol and an increase in HDL cholesterol. Oil red O staining of total aortas demonstrated reduction of atherosclerotic lesion coverage in the myriocin-treated group. Atherosclerotic plaque area was also reduced in the aortic root and brachiocephalic artery. CONCLUSIONS: Inhibition of de novo SM biosynthesis in apoE-KO mice lowers plasma cholesterol and triglyceride levels, raises HDL cholesterol, and prevents development of atherosclerotic lesions.

Development and validation of an LC/MS/MS procedure for the quantification of endogenous myo-inositol concentrations in rat brain tissue homogenates.

myo-Inositol is being investigated as a biomarker to monitor disease states involving the central nervous system. We have developed and validated a quantitative method to study endogenous myo-inositol metabolism in rat brain tissue. Tissue samples were homogenized, and their myo-inositol content was determined using spiked calibration curves and mass spectrometry. The assay was validated on an LC/MS/MS platform, and specificity was evaluated using accurate mass measurements. A novel chiral LC/MS/MS method was also developed to resolve myo-inositol from other endogenous inositol epimers and confirm the selectivity of the quantitative procedure. The validated method is selective, convenient, precise (<15% RSD), accurate (<15% RE), and sensitive over a linear range of 0.100-100 microg/mL. This method could potentially be used as an instrument for monitoring pathological conditions related to psychotherapeutics, as well as a tool for screening curative pharmaceuticals for efficacy.

Determination of hydroxyproline in plasma and tissue using electrospray mass spectrometry.

A simple and highly specific method that was developed for the determination of hydroxyproline in biological samples is described. This method could potentially be used for monitoring pathological conditions related to collagen degradation, as well as for screening remedial pharmaceuticals for efficacy. Tissue or plasma samples were prepared by hydrolysis and their hydroxyproline content was determined using spiked calibration curves and LC/MS/MS. Specificity of the method was evaluated using an API Time-Of-Flight (TOF) LC/MS to expose potential interferences. The method proposed here appears to be selective, convenient, precise (<10% R.S.D.), accurate (<10% RE), and sensitive over a linear range of 0.010-10 microg/ml.

Quantitative bioanalysis of enantiomeric drugs using capillary electrophoresis and electrospray mass spectrometry.

A novel assay method for an enantiomeric pair of drugs has been developed using a combination of capillary electrophoresis and electrospray tandem mass spectrometry connected with a homemade interface. Accurate quantification was demonstrated in plasma from 0.25 to 50 microg/ml. A liquid-liquid sample preparation technique allowed improvement in the quantitation limit to 10 ng/ml. Variables for the enantiomeric separation, including chiral selective reagent, organic solvents, buffer and acid concentration as well as injection technique, were optimized. This assay proved adequate for analysis of neat, spiked plasma, and plasma from a pharmacological study of the drug enantiomers.