Identification of 1-Methylnicotinamide as a specific biomarker for the progression of cirrhosis to hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a prevalent malignant tumor, often arising from hepatitis induced by the hepatitis B virus (HBV) in China. However, effective biomarkers for early diagnosis are lacking, leading to a 5-year overall survival rate of less than 20% among patients with advanced HCC. This study aims to identify serum biomarkers for early HCC diagnosis to enhance patient survival rates. METHODS: We established an independent cohort comprising 27 healthy individuals, 13 patients with HBV-induced cirrhosis, 13 patients with hepatitis B-type HCC, and 8 patients who progressed from cirrhosis to hepatocellular carcinoma during follow-up. Serum metabolic abnormalities during the progression from cirrhosis to HCC were studied using untargeted metabolomics. Liquid chromatography-mass spectrometry-based metabolomics methods characterized the subjects' serum metabolic profiles. Partial least squares discriminant analysis (PLS-DA) was employed to elucidate metabolic profile changes during the progression from cirrhosis to HCC. Differentially expressed metabolites (DEMs) between cirrhosis and HCC groups were identified using the LIMMA package in the R language. Two machine learning algorithms, Least Absolute Shrinkage and Selection Operator (LASSO), and Random Forest Classifier (RF), were used to identify key metabolic biomarkers involved in the progression from cirrhosis to HCC. Key metabolic biomarkers were further validated using targeted metabolomics in a new independent validation cohort comprising 25 healthy individuals and 25 patients with early-stage hepatocellular carcinoma. RESULTS: A total of 155 serum metabolites were identified, of which 21/54 metabolites exhibited significant changes in HCC patients compared with cirrhosis patients and healthy individuals, respectively. PLS-DA clustering results demonstrated a significant change trend in the serum metabolic profile of patients with HBV-induced cirrhosis during the progression to HCC. Utilizing LASSO regression and RF algorithms, we confirmed 10 key metabolic biomarkers. Notably, 1-Methylnicotinamide (1-MNAM) exhibited a persistent and significant decrease in healthy individuals, cirrhosis, and HCC patients. Moreover, 1-MNAM levels in developing patients were significantly higher during the cirrhosis stage than in the HCC stage. Targeted metabolomic validation in an external cohort further confirmed the good diagnostic performance of 1-MNAM in early HCC detection. CONCLUSION: Our findings imply that 1-MNAM may be a specific biomarker for the progression of cirrhosis to HCC.

Pharmacokinetics and apparent Michaelis constant for metabolite conversion of sorafenib in healthy and hepatocellular carcinoma-bearing rats.

Aim: Investigation of the pharmacokinetics of sorafenib (SRF) in rats with hepatocellular carcinoma (HCC). Methods: A reproducible ultra-HPLC-MS method for simultaneous determination of serum SRF, N-hydroxymethyl sorafenib and N-demethylation sorafenib. Results: Both the maximum serum concentrations (2.5-times) and the area under the serum concentration-time curve from 0 h to infinity (4.5-times) of SRF were observed to be significantly higher, with a greater than 3.0-fold decrease in the clearance rate in the HCC-bearing rats compared with these values in healthy animals. Further study revealed approximately 3.8- and 3.2-times increases in the apparent Michaelis constant for N-hydroxymethyl sorafenib and N-demethylation sorafenib conversions in the HCC-bearing rats. Conclusion: The low efficiency for the SRF conversions was a key contributor to the increased serum concentrations of SRF.

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Simultaneous determination of nicotinamide and N1 -methylnicotinamide in human serum by LC-MS/MS to associate their serum concentrations with obesity.

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of nicotinamide and its metabolite N1 -methylnicotinamide in human serum. Serum samples were prepared by protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Spherisorb S5 CN microbore column (2.0 × 100 mm, 5 µm) with gradient elution within 7 min. Acetonitrile and 5 mm ammonium formate aqueous solution (containing 0.1% formic acid) were used as mobile phases. Nicotinamide, N1 -methylnicotinamide and N'-methylnicotinamide (internal standard) were detected with a triple-quadrupole tandem mass spectrometer in the positive ion mode. Multiple reaction monitoring was used to monitor precursor to product ion transitions of m/z 123.1 → 80.1 for nicotinamide, m/z 137.1 → 94.1 for N1 -methylnicotinamide and m/z 137.1 → 80.1 for the internal standard. The linear ranges of nicotinamide and N1 -methylnicotinamide were 5.000-160.0 and 2.500-80.00 ng/ml, respectively. The intra- and inter-day precisions (RSD) of both analytes were within 6.90%. The recoveries were >88%. The analytes were proven to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the concentrations of nicotinamide and N1 -methylnicotinamide in human serum to investigate the association between their concentrations and obesity in 1160 Chinese subjects.

Metabolomic Profiling of Poor Ovarian Response Identifies Potential Predictive Biomarkers.

Objective: To characterize the serum metabolomic profile and its role in the prediction of poor ovarian response (POR). Patients: Twenty-five women with normal ovarian reserve (24-33 years, antral follicle count [AFC] ≥5, anti-Müllerian hormone [AMH] ≥1.2 ng/ml) as the control group and another twenty-five women with POR (19-35 years, AFC <5, AMH < 1.2 ng/ml) as the study group were collected in our study. The serum levels of the women in both groups were determined from their whole blood by untargeted liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analysis and cell signal pathways analysis were used to reveal the results. Results: A total of 538 different metabolites were finally identified in the two groups. Tetracosanoic acid, 2-arachidonoylglycerol, lidocaine, cortexolone, prostaglandin H2,1-naphthylamine, 5-hydroxymethyl-2-furancarboxaldehyde, 2,4-dinitrophenol, and D-erythrulose1-phosphate in POR were significantly different from control as were most important metabolites in support vector machines (p <0.05). Metabolomic profiling, together with support vector machines and pathway analysis found that the nicotinate and nicotinamide metabolism pathway, including L-aspartic acid, 6-hydroxynicotinate, maleic acid, and succinic acid semialdehyde, was identified to have significant differences in POR women compared to control women, which may be associated with ovarian reserve. Conclusion: This study indicated that LC-MS-based untargeted metabolomics analysis of serum provided biological markers for women with POR. The nicotinate and nicotinamide metabolism pathway may offer new insight into the complementary prediction and therapeutic potential of POR. The functional associations of these metabolites need further investigation.

A plasma metabolomic signature of Leber hereditary optic neuropathy showing taurine and nicotinamide deficiencies.

Leber's hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e-05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.

The specific metabolome profiling of patients infected by SARS-COV-2 supports the key role of tryptophan-nicotinamide pathway and cytosine metabolism.

The biological mechanisms involved in SARS-CoV-2 infection are only partially understood. Thus we explored the plasma metabolome of patients infected with SARS-CoV-2 to search for diagnostic and/or prognostic biomarkers and to improve the knowledge of metabolic disturbance in this infection. We analyzed the plasma metabolome of 55 patients infected with SARS-CoV-2 and 45 controls by LC-HRMS at the time of viral diagnosis (D0). We first evaluated the ability to predict the diagnosis from the metabotype at D0 in an independent population. Next, we assessed the feasibility of predicting the disease evolution at the 7th and 15th day. Plasma metabolome allowed us to generate a discriminant multivariate model to predict the diagnosis of SARS-CoV-2 in an independent population (accuracy > 74%, sensitivity, specificity > 75%). We identified the role of the cytosine and tryptophan-nicotinamide pathways in this discrimination. However, metabolomic exploration modestly explained the disease evolution. Here, we present the first metabolomic study in SARS-CoV-2 patients which showed a high reliable prediction of early diagnosis. We have highlighted the role of the tryptophan-nicotinamide pathway clearly linked to inflammatory signals and microbiota, and the involvement of cytosine, previously described as a coordinator of cell metabolism in SARS-CoV-2. These findings could open new therapeutic perspectives as indirect targets.

Significantly Elevated Levels of Plasma Nicotinamide, Pyridoxal, and Pyridoxamine Phosphate Levels in Obese Emirati Population: A Cross-Sectional Study.

Water-soluble vitamins like B3 (nicotinamide), B6 (pyridoxine), and B9 (folic acid) are of utmost importance in human health and disease, as they are involved in numerous critical metabolic reactions. Not surprisingly, deficiencies of these vitamins have been linked to various disease states. Unfortunately, not much is known about the physiological levels of B6 vitamers and vitamin B3 in an ethnically isolated group (such as an Emirati population), as well as their relationship with obesity. The aim of the present study was to quantify various B6 vitamers, as well as B3, in the plasma of obese and healthy Emirati populations and to examine their correlation with obesity. A sensitive and robust HPLC-MS/MS-based method was developed for the simultaneous quantitation of five physiologically relevant forms of vitamin B6, namely pyridoxal, pyridoxine, pyridoxamine, pyridoxamine phosphate, and pyridoxal phosphate, as well as nicotinamide, in human plasma. This method was used to quantify the concentrations of these vitamers in the plasma of 57 healthy and 57 obese Emirati volunteers. Our analysis showed that the plasma concentrations of nicotinamide, pyridoxal, and pyridoxamine phosphate in the obese Emirati population were significantly higher than those in healthy volunteers (p < 0.0001, p = 0.0006, and p = 0.002, respectively). No significant differences were observed for the plasma concentrations of pyridoxine and pyridoxal phosphate. Furthermore, the concentrations of some of these vitamers in healthy Emirati volunteers were significantly different than those published in the literature for Western populations, such as American and European volunteers. This initial study underscores the need to quantify micronutrients in distinct ethnic groups, as well as people suffering from chronic metabolic disorders.

Effect of niacin monotherapy on high density lipoprotein composition and function.

BACKGROUND: Niacin has modest but overall favorable effects on plasma lipids by increasing high density lipoprotein cholesterol (HDL-C) and lowering triglycerides. Clinical trials, however, evaluating niacin therapy for prevention of cardiovascular outcomes have returned mixed results. Recent evidence suggests that the HDL proteome may be a better indicator of HDL's cardioprotective function than HDL-C. The objective of this study was to evaluate the effect of niacin monotherapy on HDL protein composition and function. METHODS: A 20-week investigational study was performed with 11 participants receiving extended-release niacin (target dose = 2 g/day) for 16-weeks followed by a 4-week washout period. HDL was isolated from participants at weeks: 0, 16, and 20. The HDL proteome was analyzed at each time point by mass spectrometry and relative protein quantification was performed by label-free precursor ion intensity measurement. RESULTS: In this cohort, niacin therapy had typical effects on routine clinical lipids (HDL-C + 16%, q < 0.01; LDL-C - 20%, q < 0.01; and triglyceride - 15%, q = 0.1). HDL proteomics revealed significant effects of niacin on 5 proteins: serum amyloid A (SAA), angiotensinogen (AGT), apolipoprotein A-II (APOA2), clusterin (CLUS), and apolipoprotein L1 (APOL1). SAA was the most prominently affected protein, increasing 3-fold in response to niacin (q = 0.008). Cholesterol efflux capacity was not significantly affected by niacin compared to baseline, however, stopping niacin resulted in a 9% increase in efflux (q < 0.05). Niacin did not impact HDL's ability to influence endothelial function. CONCLUSION: Extended-release niacin therapy, in the absence of other lipid-modifying medications, can increase HDL-associated SAA, an acute phase protein associated with HDL dysfunction.

Absorption, Pharmacokinetics, and Urinary Excretion of Pyridines After Consumption of Coffee and Cocoa-Based Products Containing Coffee in a Repeated Dose, Crossover Human Intervention Study.

SCOPE: The present study assesses the absorption, pharmacokinetics, and urinary excretion of coffee pyridines and their metabolites after daily regular exposure to specific dosages of coffee or cocoa-based products containing coffee (CBPCC), considering different patterns of consumption. METHODS AND RESULTS: In a three-arm, crossover, randomized trial, 21 volunteers are requested to randomly consume for 1 month: one cup of espresso coffee per day, three cups of espresso coffee per day, or one cup of espresso coffee plus two CBPCC twice per day. The last day of the one-month treatment, blood and urine samples are collected for 24 h. Trigonelline, N-methylpyridinium, N-methylnicotinamide, and N-methyl-4-pyridone-5-carboxamide are quantified. Trigonelline and N-methylpyridinium absorption curves and 24-h urinary excretion reflect the daily consumption of different servings of coffee or CBPCC, showing also significant differences in main pharmacokinetic parameters. Moreover, inter-subject variability due to sex and smoking is assessed, showing sex-related differences in the metabolism of trigonelline and smoking-related ones for N-methylpyridinium. CONCLUSION: The daily exposure to coffee pyridines after consumption of different coffee dosages in a real-life setting is established. This data will be useful for future studies aiming at evaluating the bioactivity of coffee-derived circulating metabolites in cell experiments, mimicking more realistic experimental conditions.

The acute effect of metabolic cofactor supplementation: a potential therapeutic strategy against non-alcoholic fatty liver disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD) continues to increase dramatically, and there is no approved medication for its treatment. Recently, we predicted the underlying molecular mechanisms involved in the progression of NAFLD using network analysis and identified metabolic cofactors that might be beneficial as supplements to decrease human liver fat. Here, we first assessed the tolerability of the combined metabolic cofactors including l-serine, N-acetyl-l-cysteine (NAC), nicotinamide riboside (NR), and l-carnitine by performing a 7-day rat toxicology study. Second, we performed a human calibration study by supplementing combined metabolic cofactors and a control study to study the kinetics of these metabolites in the plasma of healthy subjects with and without supplementation. We measured clinical parameters and observed no immediate side effects. Next, we generated plasma metabolomics and inflammatory protein markers data to reveal the acute changes associated with the supplementation of the metabolic cofactors. We also integrated metabolomics data using personalized genome-scale metabolic modeling and observed that such supplementation significantly affects the global human lipid, amino acid, and antioxidant metabolism. Finally, we predicted blood concentrations of these compounds during daily long-term supplementation by generating an ordinary differential equation model and liver concentrations of serine by generating a pharmacokinetic model and finally adjusted the doses of individual metabolic cofactors for future human clinical trials.

Determination of KD025 (SLx-2119), a Selective ROCK2 Inhibitor, in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry and its Pharmacokinetic Application.

KD025 (SLx-2119), the first specific Rho-associated protein kinase 2 (ROCK2) inhibitor, is a potential new drug candidate currently undergoing several phase 2 clinical trials for psoriasis, idiopathic pulmonary fibrosis, chronic graft-versus-host disease, and systemic sclerosis. In this study, a bio-analytical method was developed and fully validated for the quantification of KD025 in rat plasma and for application in pharmacokinetic studies. KD025 and GSK429286A (the internal standard) in rat plasma samples were analyzed by high-performance liquid chromatography-tandem mass spectrometry with m/z transition values of 453.10 → 366.10 and 433.00 → 178.00, respectively. The method was fully validated according to the United State Food and Drug Administration guidelines in terms of selectivity, linearity, accuracy, precision, sensitivity, matrix effects, extraction recovery, and stability. The method enabled the quantification of KD025 levels in rat plasma following oral administration of 5 mg/kg KD025 and intravenous administration of 2 mg/kg KD025 to rats, respectively. Our findings suggest that the developed method is practical and reliable for pharmacokinetic studies of KD025 in preclinical animals.

Pellagra, an Almost-Forgotten Differential Diagnosis of Chronic Diarrhea: More Prevalent Than We Think.

Pellagra, caused by vitamin B3 (niacin) deficiency, is traditionally described as dermatitis, diarrhea, dementia (3D), and even death (4D) syndrome if not recognized and treated promptly. Although full-blown pellagra with all 3D features has become rare, pellagra still exists, especially in high-risk populations, which is actually more prevalent than we think. We report that a recently treated patient with the full spectrum of 3D clinical features of pellagra presents as chronic diarrhea of unknown etiology for 1 year. It reminds us that keeping a high index of suspicion and maintaining a broad differential diagnosis are critical for recognition and management of this potentially fatal but treatable condition.

Peripheral tryptophan, serotonin, kynurenine, and their metabolites in major depression: A case-control study.

AIM: Tryptophan is the sole precursor of both peripherally and centrally produced serotonin and kynurenine. In depressed patients, tryptophan, serotonin, kynurenine, and their metabolite levels remain unclear. Therefore, peripheral tryptophan and metabolites of serotonin and kynurenine were investigated extensively in 173 patients suffering from a current major depressive episode (MDE) and compared to 214 healthy controls (HC). METHODS: Fasting plasma levels of 11 peripheral metabolites were quantified: tryptophan, serotonin pathway (serotonin, its precursor 5-hydroxytryptophan and its metabolite 5-hydroxyindoleacetic acid), and kynurenine pathway (kynurenine and six of its metabolites: anthranilic acid, kynurenic acid, nicotinamide, picolinic acid, xanthurenic acid, and 3-hydroxyanthranilic acid). RESULTS: Sixty (34.7%) patients were antidepressant-drug free. Tryptophan levels did not differ between MDE patients and HC. Serotonin and its precursor (5-hydroxytryptophan) levels were lower in MDE patients than in HC, whereas, its metabolite (5-hydroxyindoleacetic acid) levels were within the standard range. Kynurenine and four of its metabolites (kynurenic acid, nicotinamide, picolinic acid, and xanthurenic acid) were lower in MDE patients. CONCLUSION: Whilst the results of this study demonstrate an association between the metabolites studied and depression, conclusions about causality cannot be made. This study uses the largest ever sample of MDE patients, with an extensive assessment of peripheral tryptophan metabolism in plasma. These findings provide new insights into the peripheral signature of MDE. The reasons for these changes should be further investigated. These results might suggest new antidepressant therapeutic strategies.

Increased fatty acid oxidation and mitochondrial proliferation in liver are associated with increased plasma kynurenine metabolites and nicotinamide levels in normolipidemic and carnitine-depleted rats.

Dysregulation of the tryptophan (Trp)-NAD+ pathway has been related to several pathological conditions, and the metabolites in this pathway are known to influence mitochondrial respiration and redox status. The aim of this project was to investigate if stimulation of beta-oxidation and mitochondrial proliferation by the mitochondrial-targeted compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) would influence metabolites of the Trp-Kyn-NAD+ pathway. We wished to investigate how carnitine depletion by meldonium-treatment influenced these metabolites. After dietary treatment of male Wistar rats with 1-triple TTA for three weeks, increased hepatic mitochondrial- and peroxisomal fatty acid oxidation resulted. The plasma content of total carnitines decreased compared to control animals, whereas hepatic genes involved in CoA biosynthesis were upregulated by 1-triple TTA treatment. The plasma Trp level and individual metabolites in the kynurenine pathway were increased by 1-triple TTA, associated with decreased hepatic gene expression of indoleamine2,3-dioxygenase. 1-triple TTA treatment increased conversion of Trp to nicotinamide (Nam) as the plasma content of quinolinic acid, Nam and N1-methylnicotinamide (mNam) increased, accompanied with suppression of hepatic gene expression of α-amino-α-carboxymuconate-ε-semialdehyde decarboxylase. A positive correlation between mitochondrial fatty acid oxidation and Trp-derivatives was found. Almost identical results were obtained by 1-triple TTA in the presence of meldonium, which alone exerted minor effects. Moreover, the plasma Kyn:Trp ratio (KTR) correlated negatively to mitochondrial function. Whether increased flux through the Trp-NAD+ pathway increased redox status and lowered inflammation locally and systemically should be considered.

Sake cake (sake-kasu) ingestion increases branched-chain amino acids in the plasma, muscles, and brains of senescence-accelerated mice prone 8.

To examine metabolic effects of sake cake ingestion, plasma and tissues were analyzed in senescence-accelerated mice prone 8 (SAMP8) fed a sake cake diet. As a result, branched-chain amino acids (BCAA) were found to be significantly higher in the plasma, gastrocnemius muscles and brains of the sake cake group than in the control group. Mice in the sake cake group showed stronger grip strength than the control group. High levels of circulating BCAA have been reported to be associated with pathological states, such as metabolic diseases, but the parameters of glucose and lipid metabolism were not affected between the two groups. Otherwise, pyridoxal was significantly higher and nicotinamide as well as 1-methylnicotinamide showed a tendency to be higher in the plasma of the sake cake group than in the control group. These findings indicate that intake of sake cake increases the levels of BCAA, vitamin B6, and vitamin B3. Abbreviation: CE-TOFMS: capillary electrophoresis time-of-flight mass spectrometry.

Pharmacokinetics of the SABRE agent 4,6-d2-nicotinamide and also nicotinamide in rats following oral and intravenous administration.

To prepare the way for using the isotopically labelled SABRE hyperpolarized 4,6-d2-nicotinamide as an MRI agent in humans we have performed an in-vivo study to measure its pharmacokinetics in the plasma of healthy rats after intravenous and oral administration. Male Han Wistar rats were dosed with either 4,6-d2-nicotinamide or the corresponding control, non-labelled nicotinamide, and plasma samples were obtained at eight time points for up to 24 h after administration. Pharmacokinetic parameters were determined from agent concentration-versus-time data for both 4,6-d2-nicotinamide and nicotinamide. 4,6-d2-Nicotinamide proved to be well tolerated regardless of route of administration at the concentrations used (20, 80 and 120 mg/kg). Pharmacokinetic parameters were similar after oral and intravenous administration and similar to those obtained for nicotinamide. Analysis of nicotinamide plasma concentrations after dosing 4,6-d2-nicotinamide intravenously demonstrates a reversible exchange of endogenous nicotinamide by this labelled agent over the time-course of our assays. Supported by a large body of evidence for the safety of nicotinamide when dosed orally in humans, we conclude that 4,6-d2-nicotinamide can also be safely administered intravenously, which will provide significant benefit when using this agent for planned imaging studies in humans.

Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies.

4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function. The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma (n = 48) and control group (n = 100). The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099 ± 0.009 vs. 0.066 ± 0.006 µmol/L and 1.10 ± 0.08 vs. 1.97 ± 0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253 ± 17 vs. 369 ± 19) as well as plasma MNA (0.057 ± 0.004 vs. 0.069 ± 0.003 µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116 ± 0.019 vs. 0.131 ± 0.014 and 0.102 ± 0.006 vs. 0.113 ± 0.011, respectively). In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.

Development and validation of a UPLC-MS/MS method for determination of motesanib in plasma: Application to metabolic stability and pharmacokinetic studies in rats.

Motesanib is a potent angiokinase inhibitor, has shown potential therapeutic effects against various cancers. An accurate, reproducible, rapid, specific, sensitive, and valid ultraperformance liquid chromatography-tandem mass spectrometry method was established to quantify motesanib in rat plasma. Motesanib and linifanib (used as an internal standard; IS) were extracted from plasma by liquid-liquid extraction using tert-butyl methyl ether as extracting agent. Chromatographic separation was performed on Acquity™ UPLC BEH™ C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters Corp., USA) using a mobile phase comprising of 0.1% formic acid acetonitrile: ammonium acetate (90:10 v/v) eluted at a flow rate of 0.25 mL/min. The electrospray ionization in the positive-mode was used for sample ionization. In the multiple reaction monitoring mode, motesanib and the IS were quantified using precursor-to-product ion transitions of m/z 374.03 → 212.02 and m/z 376.05 → 251.05, respectively. The ranges of the calibration curves were 5.0-1000.0 ng/mL with coefficient of determination of ≥0.998. The method was validated by following recently implemented USFDA guideline for bioanalytical method validation. The lower limit of quantification was 5.0 ng/mL, whereas the intra-day and inter-day accuracies of quality controls (QCs) samples were ranged between 88.91% to 95.65% and 90.20% to 102.17%, respectively. In addition, the linearity, recovery, precision, and stability parameters were found to be within the acceptable range. The method was applied successfully to in vitro microsomal metabolic stability and preliminary oral pharmacokinetic studies in rats. The applied UPLC/MS/MS method was found to be adequately sensitive and therefore suitable for application in routine motesanib pharmacokinetic studies.