Metabolomics and Lipidomics Analyses Aid Model Classification of Type 2 Diabetes in Non-Human Primates.

Type 2 diabetes (T2D) is a global public health issue characterized by excess weight, abdominal obesity, dyslipidemia, hyperglycemia, and a progressive increase in insulin resistance. Human population studies of T2D development and its effects on systemic metabolism are confounded by many factors that cannot be controlled, complicating the interpretation of results and the identification of early biomarkers. Aged, sedentary, and overweight/obese non-human primates (NHPs) are one of the best animal models to mimic spontaneous T2D development in humans. We sought to identify and distinguish a set of plasma and/or fecal metabolite biomarkers, that have earlier disease onset predictability, and that could be evaluated for their predictability in subsequent T2D studies in human cohorts. In this study, a single plasma and fecal sample was collected from each animal in a colony of 57 healthy and dysmetabolic NHPs and analyzed for metabolomics and lipidomics. The samples were comprehensively analyzed using untargeted and targeted LC/MS/MS. The changes in each animal's disease phenotype were monitored using IVGTT, HbA1c, and other clinical metrics, and correlated with their metabolic profile. The plasma and fecal lipids, as well as bile acid profiles, from Healthy, Dysmetabolic (Dys), and Diabetic (Dia) animals were compared. Following univariate and multivariate analyses, including adjustments for weight, age, and sex, several plasma lipid species were identified to be significantly different between these animal groups. Medium and long-chain plasma phosphatidylcholines (PCs) ranked highest at distinguishing Healthy from Dys animals, whereas plasma triglycerides (TG) primarily distinguished Dia from Dys animals. Random Forest (RF) analysis of fecal bile acids showed a reduction in the secondary bile acid glycoconjugate, GCDCA, in diseased animals (AUC 0.76[0.64, 0.89]). Moreover, metagenomics results revealed several bacterial species, belonging to the genera Roseburia, Ruminococcus, Clostridium, and Streptococcus, to be both significantly enriched in non-healthy animals and associated with secondary bile acid levels. In summary, our results highlight the detection of several elevated circulating plasma PCs and microbial species associated with fecal secondary bile acids in NHP dysmetabolic states. The lipids and metabolites we have identified may help researchers to differentiate individual NHPs more precisely between dysmetabolic and overtly diabetic states. This could help assign animals to study groups that are more likely to respond to potential therapies where a difference in efficacy might be anticipated between early vs. advanced disease.

Investigating Performance of the SLIM-Based High Resolution Ion Mobility Platform for Separation of Isomeric Phosphatidylcholine Species.

Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.

Mass spectrometry imaging of diclofenac and its metabolites in tissues using nanospray desorption electrospray ionization.

Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, previous attempts using traditional mass spectrometry imaging (MSI) techniques have completely or partially failed and therefore little is known about their localization in tissues. Herein, we use nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI), an ambient liquid extraction-based ionization technique, as a viable alternative to other MSI techniques to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. MSI data acquired over a broad m/z range showed low signals of the drug and its metabolites resulting from the low ionization efficiency and substantial signal suppression on the tissue. Significant improvements in the signal-to-noise were obtained using selected ion monitoring (SIM) with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide and hydroxydiclofenac are localized to the inner medulla and cortex of the kidney, respectively, which is consistent with the previously reported localization of enzymes that process diclofenac into its respective metabolites. In contrast, a uniform distribution of diclofenac and its metabolites was observed in the liver tissue. Concentration ratios of diclofenac and hydroxydiclofenac calculated from nano-DESI MSI data are generally in agreement to those obtained using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collectively, our results demonstrate that nano-DESI MSI can be successfully used to image diclofenac and its primary metabolites and derive relative quantitative data from different tissue regions. Our approach will enable a better understanding of metabolic processes associated with diclofenac and other drugs that are difficult to analyze using commercially available MSI platforms.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

Simultaneous multielement imaging of liver tissue using laser ablation inductively coupled plasma mass spectrometry.

Analysis of the spatial distribution of metals, metalloids, and non-metals in biological tissues is of significant interest in the life sciences, helping to illuminate the function and roles these elements play within various biological pathways. Chemical imaging methods are commonly employed to address biological questions and reveal individual spatial distributions of analytes of interest. Elucidation of these spatial distributions can help determine key elemental and molecular information within the respective biological specimens. However, traditionally utilized imaging methods prove challenging for certain biological tissue analysis, especially with respect to applications that require high spatial resolution or depth profiling. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been shown to be effective for direct elemental analysis of solid materials with high levels of precision. In this work, chemical imaging using LA-ICP-MS has been applied as a powerful analytical methodology for the analysis of liver tissue samples. The proposed analytical methodology successfully produced both qualitative and quantitative information regarding specific elemental distributions within images of thin tissue sections with high levels of sensitivity and spatial resolution. The spatial resolution of the analytical methodology was innovatively enhanced, helping to broaden applicability of this technique to applications requiring significantly high spatial resolutions. This information can be used to further understand the role these elements play within biological systems and impacts dysregulation may have.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

Synthesis and optimization of N-heterocyclic pyridinones as catechol-O-methyltransferase (COMT) inhibitors.

A series of N-heterocyclic pyridinone catechol-O-methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and clogP, were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

Antiviral efficacy upon administration of a HepDirect prodrug of 2'-C-methylcytidine to hepatitis C virus-infected chimpanzees.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide, and the current standard of care, a combination of pegylated interferon alpha and ribavirin, is efficacious in achieving sustained viral response in ~50% of treated patients. Novel therapies under investigation include the use of nucleoside analog inhibitors of the viral RNA-dependent RNA polymerase. NM283, a 3'-valyl ester prodrug of 2'-C-methylcytidine, has demonstrated antiviral efficacy in HCV-infected patients (N. Afdhal et al., J. Hepatol. 46[Suppl. 1]:S5, 2007; N. Afdhal et al., J. Hepatol. 44[Suppl. 2]:S19, 2006). One approach to increase the antiviral efficacy of 2'-C-methylcytidine is to increase the concentration of the active inhibitory species, the 5'-triphosphate, in infected hepatocytes. HepDirect prodrug technology can increase intracellular concentrations of a nucleoside triphosphate in hepatocytes by introducing the nucleoside monophosphate into the cell, bypassing the initial kinase step that is often rate limiting. Screening for 2'-C-methylcytidine triphosphate levels in rat liver after oral dosing identified 1-[3,5-difluorophenyl]-1,3-propandiol as an efficient prodrug modification. To determine antiviral efficacy in vivo, the prodrug was administered separately via oral and intravenous dosing to two HCV-infected chimpanzees. Circulating viral loads declined by ~1.4 log(10) IU/ml and by >3.6 log(10) IU/ml after oral and intravenous dosing, respectively. The viral loads rebounded after the end of dosing to predose levels. The results indicate that a robust antiviral response can be achieved upon administration of the prodrug.

Sustained viral response in a hepatitis C virus-infected chimpanzee via a combination of direct-acting antiviral agents.

Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

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.

Robust antiviral efficacy upon administration of a nucleoside analog to hepatitis C virus-infected chimpanzees.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2'-C-methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC(50)) of 0.3 microM (EC(90) = 1.3 microM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log(10) IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log(10), and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.