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.

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.

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.

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.

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.

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.

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.

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.

Development of a validated liquid chromatographic method for quantification of sorafenib tosylate in the presence of stress-induced degradation products and in biological matrix employing analytical quality by design approach.

The current research work envisages an analytical quality by design-enabled development of a simple, rapid, sensitive, specific, robust and cost-effective stability-indicating reversed-phase high-performance liquid chromatographic method for determining stress-induced forced-degradation products of sorafenib tosylate (SFN). An Ishikawa fishbone diagram was constructed to embark upon analytical target profile and critical analytical attributes, i.e. peak area, theoretical plates, retention time and peak tailing. Factor screening using Taguchi orthogonal arrays and quality risk assessment studies carried out using failure mode effect analysis aided the selection of critical method parameters, i.e. mobile phase ratio and flow rate potentially affecting the chosen critical analytical attributes. Systematic optimization using response surface methodology of the chosen critical method parameters was carried out employing a two-factor-three-level-13-run, face-centered cubic design. A method operable design region was earmarked providing optimum method performance using numerical and graphical optimization. The optimum method employed a mobile phase composition consisting of acetonitrile and water (containing orthophosphoric acid, pH 4.1) at 65:35 v/v at a flow rate of 0.8 mL/min with UV detection at 265 nm using a C18 column. Response surface methodology validation studies confirmed good efficiency and sensitivity of the developed method for analysis of SFN in mobile phase as well as in human plasma matrix. The forced degradation studies were conducted under different recommended stress conditions as per ICH Q1A (R2). Mass spectroscopy studies showed that SFN degrades in strongly acidic, alkaline and oxidative hydrolytic conditions at elevated temperature, while the drug was per se found to be photostable. Oxidative hydrolysis using 30% H2 O2 showed maximum degradation with products at retention times of 3.35, 3.65, 4.20 and 5.67 min. The absence of any significant change in the retention time of SFN and degradation products, formed under different stress conditions, ratified selectivity and specificity of the systematically developed method.

Utility of Cremophor RH 40 as a micellar improvement for spectrofluorimetric estimation of sorafenib in pure form, commercial preparation, and human plasma.

An easy, quick, simple and accurate spectrofluorimetric method was recognized and validated for evaluation of sorafenib (SOR) in pure form and biologically in plasma. Cremophor RH 40 (Cr RH 40) used for enhancing the fluorescence activity of SOR in phosphate buffer (pH 7). Cr RH 40 improved the native fluorescence of SOR remarkably in water. The fluorescence spectrum of SOR was observed at 405 nm after excitation at 265 nm. The linearity appeared to be in the range of 5 to 600 ng ml-1 for pure and from 9 to 500 ng ml-1 for plasma using the protein precipitation (ppt) method while from 10 to 500 ng ml-1 for plasma using liquid-liquid extraction method. The precisions and the accuracy of the estimated method gave satisfactory results. The recommended method was effectively applied for determination of SOR in human plasma with high recovery values. The results of some compounds that are possibly found in plasma were studied. The proposed method was also focused on real volunteers and a drug dissolution test.

Pregnancy Increases the Renal Secretion of N1-methylnicotinamide, an Endogenous Probe for Renal Cation Transporters, in Patients Prescribed Metformin.

N1-methylnicotinamide (1-NMN) has been investigated as an endogenous probe for the renal transporter activity of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2-K (MATE1 and MATE2-K). As pregnancy increased the renal secretion of metformin, a substrate for OCT2, MATE1, and MATE2-K, we hypothesized that the renal secretion of 1-NMN would be similarly affected. Blood and urine samples collected from women prescribed metformin for type 2 diabetes, gestational diabetes, and polycystic ovarian syndrome during early, mid, and late pregnancy (n = 34 visits) and postpartum (n = 14 visits) were analyzed for 1-NMN using liquid chromatography-mass spectrometry. The renal clearance and secretion clearance, using creatinine clearance to correct for glomerular filtration, were estimated for 1-NMN and correlated with metformin renal clearance. 1-NMN renal clearance was higher in both mid (504 ± 293 ml/min, P < 0.01) and late pregnancy (557 ± 305 ml/min, P < 0.01) compared with postpartum (240 ± 106 ml/min). The renal secretion of 1-NMN was 3.5-fold higher in mid pregnancy (269± 267, P < 0.05) and 4.5-fold higher in late pregnancy compared with postpartum (342 ± 283 versus 76 ± 92 ml/min, P < 0.01). Because creatinine is also a substrate of OCT2, MATE1, and MATE2-K, creatinine clearance likely overestimates filtration clearance, whereas the calculated 1-NMN secretion clearance is likely underestimated. Metformin renal clearance and 1-NMN renal clearance were positively correlated (rs = 0.68, P < 0.0001). 1-NMN renal clearance increases during pregnancy due to increased glomerular filtration and net secretion by renal transporters.

Metabolomic Endotype of Asthma.

Metabolomics, the quantification of small biochemicals in plasma and tissues, can provide insight into complex biochemical processes and enable the identification of biomarkers that may serve as therapeutic targets. We hypothesized that the plasma metabolome of asthma would reveal metabolic consequences of the specific immune and inflammatory responses unique to endotypes of asthma. The plasma metabolomic profiles of 20 asthmatic subjects and 10 healthy controls were examined using an untargeted global and focused metabolomic analysis. Individuals were classified based on clinical definitions of asthma severity or by levels of fraction of exhaled NO (FENO), a biomarker of airway inflammation. Of the 293 biochemicals identified in the plasma, 25 were significantly different among asthma and healthy controls (p < 0.05). Plasma levels of taurine, lathosterol, bile acids (taurocholate and glycodeoxycholate), nicotinamide, and adenosine-5-phosphate were significantly higher in asthmatics compared with healthy controls. Severe asthmatics had biochemical changes related to steroid and amino acid/protein metabolism. Asthmatics with high FENO, compared with those with low FENO, had higher levels of plasma branched-chain amino acids and bile acids. Asthmatics have a unique plasma metabolome that distinguishes them from healthy controls and points to activation of inflammatory and immune pathways. The severe asthmatic and high FENO asthmatic have unique endotypes that suggest changes in NO-associated taurine transport and bile acid metabolism.

Herb-Drug Interaction between the Traditional Hepatoprotective Formulation and Sorafenib on Hepatotoxicity, Histopathology and Pharmacokinetics in Rats.

Sorafenib has been used as a standard therapy for advanced hepatocellular carcinoma (HCC). In Asia, patients with HCC are potentially treated with the combination of sorafenib and Chinese herbal medicines to improve the efficiency and reduce the side effects of sorafenib. However, limited information about the herb-drug interactions is available. We hypothesize that the Chinese herbal medicine may exert hepatoprotective effects on the sorafenib-treated group. The aim of this study is to investigate the pharmacokinetic mechanism of drug-drug interactions of sorafenib including interacting with hepatoprotective formulation, Long-Dan-Xie-Gan-Tang formulation (LDXGT) and with two cytochrome P450 3A4 (CYP3A4) inhibitors, grapefruit juice and ketoconazole. Liver enzyme levels and histopathology of liver slices were used to evaluate sorafenib-induced hepatotoxicity and the potential hepatoprotective effects of the LDXGT formulation on subjects treated with the combination of sorafenib and the herbal medicine. In this study, a validated HPLC-photodiode array analytical system was developed for the pharmacokinetic study of sorafenib in rats. As the result of the pharmacokinetic data, pretreatment with the LDXGT formulation did not significantly interact with sorafenib compared with sorafenib oral administration alone. Furthermore, grapefruit juice and ketoconazole did not significantly affect sorafenib metabolism. Furthermore, pretreatment with variable, single or repeat doses of the LDXGT formulation did not suppress or exacerbate the sorafenib-induced hepatotoxicity and histopathological alterations. According to these results, the LDXGT formulation is safe, but has no beneficial effects on sorafenib-induced hepatotoxicity. A detailed clinical trial should be performed to further evaluate the efficacy or adverse effects of the LDXGT formulation in combination with sorafenib in humans.

[Simultaneous determination of donafenib and its N-oxide metabolite in human plasma by liquid chromatography-tandem mass spectrometry].

Donafenib is the deuterium derivative of sorafenib, and is an anti-tumor drug in clinical trials. An accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of donafenib and its N-oxide metabolite in human plasma. The analytes and internal standards (sorafenib and sorafenib N-oxide) were extracted from plasma by protein precipitation with acetonitrile, and separated on a Gemini C18 (50 mm × 2.0 mm, 5 µm) column using a gradient elution procedure. The mobile phase consisted of acetonitrile and 5 mmol ·L−1 ammonium acetate (0.2% formic acid) at a flow rate of 0.7 mL·min−1. The total run time was 5.0 min. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 468.2 → 273.2 for donafenib and m/z 465.2 → 270.2 for its internal standard sorafenib, m/z 484.2 → 289.2 for donafenib N-oxide and m/z 481.2 → 286.2 for its internal standard sorafenib N-oxide. The standard curves were linear in the range of 5.00−5 000 ng·mL−1 for donafenib, and 1.00−1 000 ng·mL−1 for donafenib N-oxide. The method was validated and successfully applied to the pharmacokinetics study of donafenib tosylate tablets in volunteers.

The effects of triptolide on the pharmacokinetics of sorafenib in rats and its potential mechanism.

CONTEXT: Combining sorafenib with triptolide could inhibit tumour growth with greater efficacy than single-agent treatment. However, their herb-drug interaction remains unknown. OBJECTIVE: This study investigates the herb-drug interaction between triptolide and sorafenib. MATERIALS AND METHODS: The effects of triptolide (10 mg/kg) on the pharmacokinetics of different doses of sorafenib (20, 50 and 100 mg/kg) in rats, and blood samples were collected within 48 h and evaluated using LC-MS/MS. The effects of triptolide on the absorption and metabolism of sorafenib were also investigated using Caco-2 cell monolayer model and rat liver microsome incubation systems. RESULTS: The results showed that the Cmax (low dose: 72.38 ± 8.76 versus 49.15 ± 5.46 ng/mL; medium dose: 178.65 ± 21.05 versus 109.31 ± 14.17 ng/mL; high dose: 332.81 ± 29.38 versus 230.86 ± 9.68 ng/mL) of sorafenib at different doses increased significantly with the pretreatment of triptolide, and while the oral clearance rate of sorafenib decreased. The t1/2 of sorafenib increased significant (p < 0.05) from 9.02 ± 1.16 to 12.17 ± 2.95 h at low dose with the pretreatment of triptolide. Triptolide has little effect on the absorption of sorafenib in Caco-2 cell transwell model. However, triptolide could significantly decrease the intrinsic clearance rate of sorafenib from 51.7 ± 6.37 to 32.4 ± 4.43 µL/min/mg protein in rat liver microsomes. DISCUSSION AND CONCLUSIONS: These results indicated that triptolide could change the pharmacokinetic profiles of sorafenib in rats; these effects might be exerted via decreasing the intrinsic clearance rate of sorafenib in rat liver.

Higher maternal serum concentrations of nicotinamide and related metabolites in late pregnancy are associated with a lower risk of offspring atopic eczema at age 12 months.

BACKGROUND: Evidence that atopic eczema partly originates in utero is increasing, with some studies linking the risk of developing the condition with aspects of maternal diet during pregnancy. Nicotinamide, a naturally occurring nutrient that is maintained through the dietary intakes of vitamin B3 and tryptophan, has been used in the treatment of some skin conditions including atopic eczema. OBJECTIVE: To examine the relation of maternal serum concentrations of nicotinamide and related tryptophan metabolites to the risk of atopic eczema in the offspring. METHODS: Within the UK Southampton Women Survey, infantile atopic eczema at ages 6 and 12 months was ascertained (modified UK Working Party Criteria for the Definition of Atopic Dermatitis). Maternal serum levels of kynurenine, kynurenic acid, anthranilic acid, tryptophan, nicotinamide and N1-methylnicotinamide were measured in late pregnancy by mass spectrometry (n = 497) and related to the odds ratio of infantile atopic eczema. RESULTS: Maternal nicotinamide and related metabolite concentrations were not associated with offspring atopic eczema at age 6 months. Higher concentrations of nicotinamide and anthranilic acid were, however, associated with a lower risk of eczema at age 12 months (odds ratios 0.69, 95% CI 0.53-0.91/SD change, P = 0.007 and 0.63, 0.48-0.83, P = 0.001, respectively). The associations were robust to adjustment for potentially confounding variables. CONCLUSION AND CLINICAL RELEVANCE: This is the first study linking maternal serum concentrations of nicotinamide and related metabolites to the risk of atopic eczema in the offspring. The findings point to potentially modifiable maternal influences on this complex and highly prevalent condition.

Activation of the nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (MNA) pathway in pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with inflammatory response but it is unknown whether it is associated with alterations in NNMT activity and MNA plasma concentration. Here we examined changes in NNMT-MNA pathway in PAH in rats and humans. METHODS: PAH in rats was induced by a single subcutaneous injection of MCT (60 mg/kg). Changes in NNMT activity in the lungs and liver (assessed as the rate of conversion of nicotinamide (NA) to MNA), changes in plasma concentration of MNA and its metabolites (analyzed by LC/MS) were analyzed in relation to PAH progression. PAH was characterized by right ventricular hypertrophy (gross morphology), cardiac dysfunction (by MRI), lung histopathology, lung ultrastructure, and ET-1 concentration in plasma. NO-dependent and PGI2-dependent function in isolated lungs was analyzed. In naive patients with idiopathic pulmonary hypertension (IPAH) characterized by hemodynamic and biochemical parameters MNA and its metabolites in plasma were also measured. RESULTS: MCT-injected rats developed hypertrophy and functional impairment of the right ventricle, hypertrophy of the pulmonary arteries, endothelial ultrastructural defects and a progressive increase in ET-1 plasma concentration-findings all consistent with PAH development. In isolated lung, NO-dependent regulation of hypoxic pulmonary vasoconstriction was impaired, while PGI2 production (6-keto-PGF1α) was increased. NNMT activity increased progressively in the liver and in the lungs following MCT injection, and NNMT response was associated with an increase in MNA and 6-keto-PGF1α concentration in plasma. In IPAH patients plasma concentration of MNA was elevated as compared with healthy controls. CONCLUSIONS: Progression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.

Pimasertib, a selective oral MEK1/2 inhibitor: absolute bioavailability, mass balance, elimination route, and metabolite profile in cancer patients.

AIM: This trial (NCT: 01713036) investigated the absolute bioavailability, mass balance and metabolite profile of pimasertib in a new design combining these investigations in a single group of patients. METHODS: Six male patients with pathologically confirmed, locally advanced or metastatic solid tumours were enrolled. Exclusion criteria included Eastern Cooperative Oncology Group performance status >1. In Part A of the trial, patients received a 60 mg oral dose of unlabelled pimasertib followed by an intravenous (i.v.) tracer dose of [14 C]pimasertib 2 µg (equalling 9 kBq) as a bolus injection, one hour after the oral dose, on Day 1. On Day 8, all patients received 60 mg pimasertib capsules spiked with 2.6 MBq of [14 C]pimasertib. Patients received 60 mg oral unlabelled pimasertib twice daily from Day 3 to Day 21 of Part A and in subsequent 21-day cycles in Part B. RESULTS: Following i.v. administration, [14 C]pimasertib exhibited a geometric mean total body clearance of 45.7 l h-1 (geometric coefficient of variation [geometric CV]: 47.2%) and a volume of distribution of 229 l (geometric CV: 42.0%). Absolute bioavailability was 73%. The majority of the oral [14 C] dose (85.1%) was recovered in excreta. Total radioactivity was mainly excreted into urine (52.8%) and faeces (30.7%) with 78.9% of the [14 C] dose recovered as metabolites. Two major circulating metabolites were identified in plasma: a carboxylic acid (M445) and a phosphoethanolamine conjugate (M554). The safety profile was in line with the published pimasertib trials. CONCLUSION: Pimasertib showed a favourable pharmacokinetic profile with high absolute bioavailability and a unique metabolic pathway (conjugation with phosphoethanolamine).

Plasma concentrations of amino acid and nicotinamide metabolites in rheumatoid arthritis--potential biomarkers of disease activity and drug treatment.

This study aimed to evaluate changes in plasma amino acid and nicotinamide metabolites concentrations in rheumatoid arthritis (RA) in a search for potential biomarkers of the disease activity and the effect treatment. Analysis of plasma metabolite patterns with liquid chromatography/mass spectrometry revealed specific changes in RA as well as correlations with clinical parameters. Combined concentration parameter calculated as [aspartic acid] + [threonine] + [tryptophan] - [histidine] - [phenylalanine] offered the strongest correlation (p < 0.001) with pain joint count, swollen joint count and DAS 28. Such analysis of amino acid and related metabolite pattern offers potential for diagnosis as well as for monitoring disease progression and therapy in RA.