Evaluation of herb-drug interaction of ambrisentan with shikonin based on UPLC-MS/MS.

CONTEXT: Ambrisentan is an oral endothelin-receptor antagonist (ERA). However, there is no report on the interaction between ambrisentan and shikonin. OBJECTIVE: To investigate the effect of shikonin on ambrisentan metabolism in vivo and in vitro. MATERIALS AND METHODS: This study developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of ambrisentan and (S)-4-hydroxymethyl ambrisentan in rat plasma. Twelve male Sprague-Dawley (SD) rats were divided into two groups (n = 6): the control group and shikonin (20 mg/kg) group. The pharmacokinetics of ambrisentan (2.5 mg/kg) were investigated after 30 min. Additionally, human and rat liver microsomes were used to investigate the herb-drug interaction. RESULTS: The UPLC-MS/MS method was shown to be accurate, precise and reliable, and was successfully applied to the herb-drug interaction study of ambrisentan with shikonin. When co-administrated with 20 mg/kg shikonin, the Cmax and AUC(0-∞) of ambrisentan were significantly increased by 44.96 and 16.65%, respectively (p < 0.05). In addition, there were modest decreases in (S)-4-hydroxymethyl ambrisentan Cmax and AUC(0-∞) in the presence of shikonin (p < 0.05), which indicated that these results were in accordance with the inhibition of shikonin on ambrisentan metabolism. Moreover, enzyme kinetic study indicated that shikonin had an inhibitory effect on human and rat microsomes where the IC50 values of shikonin were 5.865 and 6.358 µM, respectively. CONCLUSIONS: Our study indicated that shikonin could inhibit ambrisentan metabolism. Further studies need to be carried out to verify whether similar interaction truly apply in humans and whether this interaction has clinical significance.

Daily Brazilian green propolis intake elevates blood artepillin C levels in humans.

BACKGROUND: Propolis is a natural product collected by worker bees from a variety of plant species. As a type of propolis, Brazilian green propolis contains a large amount of artepillin C. Artepillin C is a cinnamic acid derivative and has been shown to have a wide variety of biological functions, including anti-inflammatory, antiviral and antitumor activities, in both cell culture and animal models. However, how propolis is digested and absorbed remains to be elucidated. Moreover, blood artepillin C levels after propolis intake have not been shown in human studies. RESULTS: A randomized, single-blind placebo-controlled study on the effect of Brazilian green propolis on serum artepillin C levels was conducted with healthy volunteers. The participants (n = 133) were randomly allocated in an approximately 2:1 ratio to two groups: propolis (n = 91) and placebo (n = 42). The participants took daily propolis or placebo, and blood tests were performed on day 0 (before propolis intake) and days 1, 3 and 7. Artepillin C was detected in serum in almost all individuals in the propolis groups. No serum artepillin C was detected in the placebo group. Serum artepillin C levels in the female group tended to be higher than those in the male group. In the female group, menstrual status was unrelated to serum artepillin C levels. CONCLUSION: These results suggested that propolis intake might be more effective for females than for males. © 2021 Society of Chemical Industry.

Novel bio-analytical technique for estimation of gamma oryzanol in rat plasma and brain homogenate using HPLC.

Gamma oryzanol, a component of rice bran oil is used for its anticancer and antihyperlipidemic properties. Bioanalytical method for rat plasma and brain homogenate was developed by HPLC system with a PDA detector in which drug elution was performed using C-18 column (4.6mm×150cm, 5µ) with 1% acetic acid in methanol: acetonitrile (65/35, v/v) as mobile phase at 1.2ml/min flow rate and detected at 326nm wavelength. Liquid liquid extraction method was chosen for extraction of oryzanol from plasma as well as brain homogenate as it provided highest recovery (95% in plasma, 85% in brain homogenate). Various extraction solvents for each body fluid were analysed, out of which highest recovery for plasma (95%), in acetone: IPA (1/1, v/v) and for brain homogenate (85%) in isopropyl alcohol (IPA) was observed. Observed linearity was between 500ng/mL-5000ng/mL. The interday and intraday precision (i.e. %RSD) was less than 10% and accuracy was±5%. Selectivity and matrix effect was checked and found as per USFDA criteria. Plasma samples were found to be stable over the analysis period, HQC samples were stable up to third cycle in freeze and thaw stability while LQC samples were stable over fourth cycle. The method proved to be simple, useful and is appropriate, for preclinical and experimental research.

Simultaneous determination of TUG-891 and its metabolites in rat plasma using LC-HRMS with application to preclinical pharmacokinetic study.

In this study, a simple and reliable LC-MS/MS method was first proposed for the simultaneous determination of TUG-891 and its metabolites TUG-891-alcohol, TUG-891-aldehyde, and TUG-891-acid in rat plasma. The analytes and fasiglifam (internal standard) were extracted from plasma samples with acetonitrile and separated using an Acquity BEH C18 column (1.7 µm, 2.1 × 50 mm) with water containing 0.05% ammonium hydroxide and acetonitrile containing 0.05% ammonium hydroxide as the mobile phase. A Q-Exactive Orbitrap mass spectrometer in full-scan mode was used for mass detection, and the data analysis was obtained using a mass extraction window of 5 ppm. The calibration curves exhibited excellent linearity (correlation coefficient > 0.9981) in the concentration range of 0.5-1000 ng/mL. The lower limit of quantification was 0.5 ng/mL for all analytes. The intra- and inter-day precision was less than 11.31%, and the accuracy ranged from -11.50 to 9.50%. The extraction recovery of the analytes from rat plasma was greater than 82.31%, and no obvious matrix effect was found. The established method was further applied to the pharmacokinetic study of TUG-891, TUG-891-alcohol, TUG-891-aldehyde, and TUG-891-acid in rat after a single dose of 5-mg/kg treatment of TUG-891. The results demonstrated that TUG-891 was rapidly metabolized into its metabolites and the systemic exposures of the metabolites were much higher than those of TUG-891.

Ambrisentan use in a HIV-1 infected patient with end-stage renal disease and pulmonary hypertension: minimal removal by hemodialysis - a case report.

BACKGROUND: Ambrisentan is a selective endothelin receptor antagonist used for the treatment of pulmonary arterial hypertension (PAH). Little is known about ambrisentan removal by hemodialysis in patients with end-stage renal disease (ESRD). CASE PRESENTATION: A 53-year-old woman with HIV/hepatitis C virus (HCV) co-infection, PAH and ESRD on regular hemodialyis was admitted in our hospital due to refractory heart failure while on treatment with bosentan (125 mg twice daily) and tadalafil (20 mg once daily) for PAH and antiretroviral treatment (cART) including darunavir/cobicistat (800/150 mg once daily). Excessive exposure to bosentan due to drug interactions between bosentan and darunavir/cobicistat was suspected. Bosentan was replaced by ambrisentan, with progressive improvement in her clinical condition. Pre- and postdialyzer cocentrations of ambrisentan in plasma were determined and hemodialysis extraction ratio for ambrisentan was 2%. CONCLUSIONS: Our results suggest that hemodialysis results in minimal ambrisentan removal, and therefore no specific ambrisentan dosage adjustment seems to be required in ESRD patients undergoing hemodialysis.

Quantitative determination of characteristic components from compound of Lysionotus pauciflorus Maxim. by LC-MS/MS and its application to a pharmacokinetic study.

Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim., which have a significant effect on tuberculosis of cervical lymph nodes. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. A selective and sensitive LC-MS/MS method was established and validated in rat plasma for the first time. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 µm). The mobile phase contained 0.1% formic acid aqueous solution and acetonitrile with a flow rate of 0.8 mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of six components and internal standard (IS) sulfamethoxazole were quantified in multiple reaction monitoring (MRM) using QTRAP-3200 MS/MS. The method fulfilled US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery, dilution integrity, and stability. This proposed method was then successfully applied to a pharmacokinetic study after oral administration of 10 mL/kg compound extracts in rats. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine.

Simultaneous quantification of ambrisentan, macitentan and sitaxentan in human plasma using UPLC-MS/MS.

Endothelin receptor antagonists (ERAs) such as, ambrisentan, macitentan and sitaxentan are primarily used for the treatment of pulmonary arterial hypertension. Considering the rise in endothelin in pre-eclampsia, ERAs may also be useful in its treatment. To evaluate the pharmacokinetics of ERAs, a rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated to determine the concentration of ambrisentan, macitentan and sitaxentan in human plasma. Plasma samples were treated with methanol to induce protein precipitation. A chromatographic separation was performed on a C18 column using a gradient of methanol-water containing 0.1% formic acid and 0.013% ammonium acetate and a flow rate of 0.5 ml/min. Multiple reaction monitoring was used for quantification. This method was validated in a linear range of 20.28-2028 µg/l for ambrisentan, 4.052-405.2 µg/l for macitentan and 205.4-10 270 µg/l for sitaxentan. The method was successfully validated according to US Food and Drug Administration guidelines to determine the concentrations of macitentan, ambrisentan and sitaxentan in human plasma. This method is now being used for study samples and clinical patient samples.

Identification of sulfonyl-loxoprofen as novel phase 2 conjugate in rat.

Loxoprofen is a prodrug that exerts strong analgesic and anti-inflammatory effects through its active trans-alcohol metabolite, which is produced in the liver by carbonyl reductase. Previous metabolic studies have evaluated loxoprofen, but its sulfate and taurine conjugates have not yet been studied. We characterized the metabolomic profile of loxoprofen in rat plasma, urine, and feces using high-resolution mass spectrometry. We identified 17 metabolites of loxoprofen in the three different biological matrices, 13 of which were detected in plasma and feces and 16 in urine. Amongst these metabolites, two novel taurine conjugates (M12 and M13) and two novel acyl glucuronides (M14, M15) were identified for the first time in rats. In addition, we detected three novel sulfate conjugates (M9, M10, and M11) of loxoprofen. Further study of these metabolites of loxoprofen is essential in order to assess their potency and toxicity.

Interference of hydroxyphenylpyruvic acid, hydroxyphenyllactic acid and tyrosine on routine serum and urine clinical chemistry assays; implications for biochemical monitoring of patients with alkaptonuria treated with nitisinone.

OBJECTIVES: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1). MATERIALS AND METHODS: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter. RESULTS: No significant effect (p > 0.1) was observed up to 225 µmol/L of HPPA and HPLA, and up to 5000 µmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked. CONCLUSIONS: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory.

Comparison of Blood Profiles of γ-Oryzanol and Ferulic Acid in Rats after Oral Intake of γ-Oryzanol.

γ-Oryzanol (OZ), a bioactive phytochemical abundant in cereals such as rice, has been reported to be mainly hydrolyzed to ferulic acid (FA) in the body. Meanwhile, in our previous study, we revealed that a part of OZ is absorbed into the body and exists in its intact form. However, the comprehensive absorption profile of OZ and its metabolites (e.g., FA) after OZ intake has not been fully elucidated yet. Therefore, in this study, we measured the concentrations of OZ, FA, and FA conjugates (i.e., FA sulfate and glucuronide) in the blood of rats with the use of HPLC-MS/MS after a single oral administration of 300 µmol/kg body weight of rice bran OZ (RBOZ). As a result, intact OZ along with FA and FA conjugates existed in the blood, which implied that these constituents may all contribute to the physiological effects under OZ intake. Additionally, when an equimolar amount of FA (300 µmol/kg body weight) was administered, it was found that the absorption profile of FA was significantly different from that when RBOZ was administered.

A Phase I Study to Show the Relative Bioavailability and Bioequivalence of Fixed-Dose Combinations of Ambrisentan and Tadalafil in Healthy Subjects.

PURPOSE: Pulmonary arterial hypertension (PAH) is a life-threatening disease that typically causes shortness of breath and exercise intolerance. Combination therapy with ambrisentan and tadalafil has proven to be more effective at preventing clinical failure events in patients with PAH than either drug alone. The aim of this study was to evaluate the bioequivalence of an ambrisentan/tadalafil fixed-dose combination (FDC) compared with co-administration of the 2 monotherapies. METHODS: This 3-part, randomized, single-dose, open-label crossover study was conducted in healthy volunteers. The first part of the study consisted of a 5-way crossover that compared the relative bioavailability of 4 FDC formulations (10-mg ambrisentan + 40-mg tadalafil) with co-administered reference monotherapies. One formulation was selected and its relative bioavailability was assessed when produced in 3 different granulation sizes during the second part of the study. In the third part of the study, the bioequivalence of the candidate FDC with the reference monotherapies was evaluated for the 10-mg/40-mg dose strength, in addition to 2 other dose strengths (5 mg/20 mg and 5 mg/40 mg). For all parts of the study, blood samples were taken at regular intervals after each dose, ambrisentan and tadalafil concentrations determined, and pharmacokinetic (PK) parameters (Cmax, AUC0-∞, and AUC0-t) obtained. Test/reference ratios of the geometric means of PK parameters were used to evaluate bioequivalence. Safety and tolerability were assessed by recording adverse events and monitoring vital signs, ECGs, and clinical laboratory data. FINDINGS: Of the 174 subjects screened for eligibility, 112 were allocated to a randomized treatment sequence across all study parts, and 100 completed their full assigned treatments. All 4 FDC formulations tested during part 1 of the study yielded PK parameters similar those of the reference treatments. In part 2, granulation size was found to not affect the relative bioavailability of the selected formulation. In part 3, the selected FDC was found to be bioequivalent to co-administration of the monotherapies in both the fasted and fed states. The FDC was also found to be bioequivalent to the reference treatments at the 2 additional dose strengths. All but one of the adverse events was mild to moderate in intensity, and no serious adverse events were reported. IMPLICATIONS: An ambrisentan/tadalafil FDC was bioequivalent to concurrently administered monotherapies and therefore represents a viable alternative treatment to co-administration. Use of an FDC is likely to be associated with reduced costs and improved patient compliance. ClinicalTrials.gov identifier: NCT02688387.

Plasma alkylresorcinol metabolite, a biomarker of whole-grain wheat and rye intake, and risk of ischemic stroke: a case-control study.

Background: Epidemiologic studies on whole grains and risk of stroke have reported inconsistent results, with some suggesting a protective effect but others showing a null association. Objectives: The aim of this study was to examine whether plasma 3-(3,5-dihydroxyphenyl)-1-propanoic acid (DHPPA), a biomarker of whole-grain wheat and rye intake, is associated with risk of ischemic stroke. Methods: A hospital-based case-control study was conducted between March 2011 and May 2016. Cases (n = 990) with first ischemic stroke were matched to controls (n = 990) by sex and age. Concentrations of plasma DHPPA were determined by high-performance liquid chromatography-tandem mass spectrometry. We calculated ORs for the association of plasma DHPPA concentrations with ischemic stroke risk through the use of logistic regression. Results: Plasma DHPPA was inversely associated with ischemic stroke risk. After adjustment for potential confounding factors, the ORs for ischemic stroke across increasing quartiles of plasma DHPPA concentrations were 1 (referent), 0.76 (95% CI: 0.58, 0.99), 0.71 (95% CI: 0.54, 0.92), and 0.59 (95% CI: 0.45, 0.77), respectively (P-trend = 0.001). The inverse association was also observed in all subgroups of participants according to sex, age, body mass index, smoking status, alcohol consumption, history of hypertension, and history of diabetes. Conclusions: Our study showed that higher plasma DHPPA concentrations were associated with lower risk of ischemic stroke. This finding provides further evidence to support the health benefits of whole-grain consumption.

Development and validation of liquidchromatographic method for quantitative determination of Loxoprofen in mobilephase and in human plasma.

A Simple, sensitive and accurate high-performance liquid chromatographic (HPLC) method for effective and specific analysis of Loxoprofen (LXP) in the mobilephase and human plasma was developed. Effective chromatographic separation was attained on a Mediterranean Sea C18 column (250×4.6mm, 5um) with mobilephase containing acetonitrile and 0.01 M NaH2PO4 buffer (55:45) by adjusting pH 6.5 with sodium dihydrogen phosphate buffer at a flow rate of 1ml/min. Calibration ranges from 0.1ppm to 10 ppm with a coefficient of relation value (R2=0.999) by using a linear regression method and lower limit of quantification was 0.1ppm. The current method showed inter-day and intra-day accuracy and precision within the range of ±10%. % RSD was found to be less than 5 %. Analytical recovery was more than 90% which confirmed the reliability of current method. The proposed method was found appropriate for assessment of LXP in pharmacokinetic and bioequivalence study.

A sensitive LC-MS/MS-based bioanalytical method for quantification of salviaflaside and rosmarinic acid in rat plasma and its application in a pharmacokinetic study.

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of salviaflaside and rosmarinic acid in rat plasma. Sample preparation was carried out through liquid-liquid extraction with ethyl acetate using curculigoside as internal standard (IS). The analytes were determined by selected reaction monitoring operated in the positive ESI mode. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column (100 × 4.6 mm, 1.8 µm) with a mobile phase consisting of methanol-water-formic acid (50:50:0.1, v/v/v) at a flow rate of 0.3 mL/min. The run time was 1.9 min per sample and the injection volume was 5 µL. The method had an LLOQ of 1.6 ng/mL for salviaflaside and 0.94 ng/mL for rosmarinic acid in plasma. The linear calibration curves were fitted over the range of 1.6-320 ng/mL for salviaflaside and 0.94-188 ng/mL for rosmarinic acid in plasma with correlation coefficients (r2 ) >0.99. Intra- and inter-day precisions (relative standard deviation) were < 13.5%, and accuracies (relative error) were between -8.6% and 14.5% for all quality control samples. The method was validated and applied to the pharmacokinetics of salviaflaside and rosmarinic acid in plasma after oral administration of Prunella vulgaris extract to rats.

Metabolite characterization of ambrisentan, in in vitro and in vivo matrices by UHPLC/QTOF/MS/MS: Detection of glutathione conjugate of epoxide metabolite evidenced by in vitro GSH trapping assay.

The focus of the present study is on in vitro and in vivo metabolite identification of ambrisentan (AMBR) a selective endothelin type - A (ETA) receptor antagonist using quadruple time-of-flight mass spectrometry (QTOF/MS). in vitro metabolism study was conducted by incubating AMBR in rat liver microsomes (RLM), rat and human liver S9 fractions. In vivo study was carried out through the collection of urine, faeces and plasma samples at various time points after oral administration of AMBR in suspension form at a dose of 25 mg/kg to six male Sprague - Dawley (SD) rats. The samples were prepared using an optimized sample preparation techniques involving protein precipitation (PP), freeze liquid extraction (FLE) and solid phase extraction (SPE). The extracted samples were further concentrated and analyzed by developing a sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method. A total of seventeen metabolites were identified in in vivo samples which includes hydroxyl, demethylated, demethoxylated, hydrolytic, decarboxylated, epoxide and glucuronide metabolites. Most of the metabolites were observed in faeces and urine matrices and few were observed in the plasma matrix. Only ten metabolites were identified in in vitro study which was commonly observed in in vivo study. The detailed structural elucidation of all the metabolites was done using UHPLC/QTOF/MS/MS in combination with accurate mass measurements. The toxicity profile of AMBR and its metabolites were predicted using TOPKAT software. In addition, a mass spectrometric method was developed for the detection and characterization of GSH-trapped reactive epoxide metabolitein human liver S9 fraction supplemented with glutathione (GSH) as trapping agent.

Effects of loxoprofen on the apical root resorption during orthodontic tooth movement in rats.

Studies have revealed that severe apical root resorption during tooth movement is caused by the noninfective inflammatory reaction of apical root tissues. We hypothesized that loxoprofen can suppress apical root resorption during tooth movement. Cyclic tensile force (CTF) of 10 kPa was applied to the human pulp cells for 48 hours by the Flexcell Strain Unit. Loxoprofen (10 and 100 µM) was added to the culture cells, and expression of cyclooxygenase (COX)-1, COX-2, interleukin (IL)-1ß, receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF) were examined. To determine the effects of loxoprofen sodium on apical root reabsorption during tooth movement, the upper first molars of 7-week-old rats were subjected to mesial movement by 10g force for 30 days with or without the oral administration of loxoprofen. Gene expression and protein concentration of COX-1, COX-2, IL-1ß, TNF-α, RANKL and M-CSF were significantly higher in the CTF group than in the control group. However, these levels were decreased by loxoprofen administration. After orthodontic tooth movement, the expression of IL-1ß, TNF-α, RANKL and M-CSF decreased in the loxoprofen group than in the control group by immunohistochemical staining. In comparison to control group, less number of odontoclasts and a decrease in the amount of apical root resorption was observed in the loxoprofen group. Many osteoclasts became visible on the pressure side of the alveolar bone in the both groups, and the amount of tooth movement did not show a significant difference. These findings demonstrate that severe apical root resorption may be suppressed by loxoprofen administration, without a disturbance of tooth movement.

Acidosis and acute kidney injury in severe malaria.

BACKGROUND: In severe falciparum malaria metabolic acidosis and acute kidney injury (AKI) are independent predictors of a fatal outcome in all age groups. The relationship between plasma acids, urine acids and renal function was investigated in adult patients with acute falciparum malaria. METHODS: Plasma and urinary acids which previously showed increased concentrations in proportion to disease severity in patients with severe falciparum malaria were quantified. Patients with uncomplicated malaria, sepsis and healthy volunteers served as comparator groups. Multiple regression and multivariate analysis were used to assess the relationship between organic acid concentrations and clinical syndromes, in particular AKI. RESULTS: Patients with severe malaria (n = 90), uncomplicated malaria (n = 94), non-malaria sepsis (n = 19), and healthy volunteers (n = 61) were included. Univariate analysis showed that both plasma and creatinine-adjusted urine concentrations of p-hydroxyphenyllactic acid (pHPLA) were higher in severe malaria patients with AKI (p < 0.001). Multiple regression analysis, including plasma or creatinine-adjusted urinary acids, and PfHRP2 as parasite biomass marker as independent variables, showed that pHPLA was independently associated with plasma creatinine (ß = 0.827) and urine creatinine (ß = 0.226). Principal component analysis, including four plasma acids and seven urinary acids separated a group of patients with AKI, which was mainly driven by pHPLA concentrations. CONCLUSIONS: Both plasma and urine concentrations of pHPLA closely correlate with AKI in patients with severe falciparum malaria. Further studies will need to assess the potential nephrotoxic properties of pHPLA.

Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD.

Previous studies have shown that gut-microbiome is associated with nonalcoholic fatty liver disease (NAFLD). We aimed to examine if serum metabolites, especially those derived from the gut-microbiome, have a shared gene-effect with hepatic steatosis and fibrosis. This is a cross-sectional analysis of a prospective discovery cohort including 156 well-characterized twins and families with untargeted metabolome profiling assessment. Hepatic steatosis was assessed using magnetic-resonance-imaging proton-density-fat-fraction (MRI-PDFF) and fibrosis using MR-elastography (MRE). A twin additive genetics and unique environment effects (AE) model was used to estimate the shared gene-effect between metabolites and hepatic steatosis and fibrosis. The findings were validated in an independent prospective validation cohort of 156 participants with biopsy-proven NAFLD including shotgun metagenomics sequencing assessment in a subgroup of the cohort. In the discovery cohort, 56 metabolites including 6 microbial metabolites had a significant shared gene-effect with both hepatic steatosis and fibrosis after adjustment for age, sex and ethnicity. In the validation cohort, 6 metabolites were associated with advanced fibrosis. Among them, only one microbial metabolite, 3-(4-hydroxyphenyl)lactate, remained consistent and statistically significantly associated with liver fibrosis in the discovery and validation cohort (fold-change of higher-MRE versus lower-MRE: 1.78, P < 0.001 and of advanced versus no advanced fibrosis: 1.26, P = 0.037, respectively). The share genetic determination of 3-(4-hydroxyphenyl)lactate with hepatic steatosis was RG :0.57,95%CI:0.27-0.80, P < 0.001 and with fibrosis was RG :0.54,95%CI:0.036-1, P = 0.036. Pathway reconstruction linked 3-(4-hydroxyphenyl)lactate to several human gut-microbiome species. In the validation cohort, 3-(4-hydroxyphenyl)lactate was significantly correlated with the abundance of several gut-microbiome species, belonging only to Firmicutes, Bacteroidetes and Proteobacteria phyla, previously reported as associated with advanced fibrosis. Conclusion: This proof of concept study provides evidence of a link between the gut-microbiome and 3-(4-hydroxyphenyl)lactate that shares gene-effect with hepatic steatosis and fibrosis. (Hepatology 2018).

Novel polymorph of ambrisentan: Characterization and stability.

The present work highlights a novel polymorph (form II) of ambrisentan (AMT), a selective endothelin type A (ETA) receptor antagonist used in the treatment of pulmonary arterial hypertension (PAH). Form II was isolated by solution crystallization and characterised by differential scanning calorimetry, powder X-ray diffraction, solution calorimetry and aqueous solubility. The single crystal X-ray diffraction shows that it crystallizes in monoclinic system with space group P21/c different from the form I (commercial form). Form II was found to be enantiotropically related to form I. Apparent solubility of form II was performed in 0.1 N HCl (pH 1.2) was found to be higher (1.5 fold) than of form I. Solution mediated and stress-induced phase transformation studies revealed conversion of form II to form I. Accelerated stability studies (40 °C & 75% RH) also reveal that form II converted to form I after one month. However, this does not belittle the improved solubility of a new solid form.

Electrodialytic extraction of anionic pharmaceutical compounds from a single drop of whole blood using a supported liquid membrane.

A method to introduce target analytes to a chromatograph from a single drop of whole blood was investigated for minimally invasive monitoring of anionic pharmaceuticals. In this work, salicylate and loxoprofen were examined as organic anions. A micro ion extractor (MIE) has been developed for extraction of inorganic trace anions from whole blood, but this device is not suitable for extraction of pharmaceuticals. In the present study, we improved and optimized the MIE device for organic anion extraction. Various supported liquid membranes were evaluated for use as the ion transfer membrane, with each membrane placed between a droplet sample (donor) and an acceptor solution. A supported liquid membrane of porous polypropylene impregnated with 1-butanol was selected. In addition, the methods for electric field creation and electrode contact were examined to improve the characteristics of the MIE device. The current and extraction time were also optimized. With the optimized method, salicylate and loxoprofen were successfully extracted from a single drop of whole blood. Changes in the concentrations of these pharmaceuticals in blood over time were monitored after administration. As only 25µL of whole blood was required for analysis, repeat measurements could be conducted to monitor changes in the concentrations. This MIE will be useful for monitoring pharmaceutical concentrations in blood.