Direct Multi-Deuterium Labelling of Pirtobrutinib.

Herein, we demonstrate an efficient method for multi-deuterium labelling of pirtobrutinib-a Bruton's tyrosine kinase inhibitor recently approved by the FDA-using a straightforward hydrogen isotope exchange (HIE) reaction. A remarkably high level of deuterium incorporation was achieved using an excess of a Kerr-type iridium catalyst. The key factor in the significant deuterium labelling was the decision to employ a deuterium uniformly labelled solvent, chlorobenzene-d5, at an elevated temperature. Virtually, no d0-d3 species were detected, with only traces of d4-d5 isotopomers (< 5%) observable in the mass spectrum of pirtobrutinib-d8, fulfilling requirements for stable isotope-labelled internal standard. The labelled compound-mainly consisting of isotopomers d6-d9 at 82.4% of the total abundance-was isolated in a high yield (73%) and purity (99%). Noteworthy, fluorine group acting as a directing group was observed for the first time. Significant incorporation of deuterium in ortho-positions, exceeding 87%, was observed. Interestingly, chlorinated solvent used in the HIE reactions was non-specifically deuterated yielding up to 0.42 deuterium per chlorobenzene molecule even at an exceptionally low iridium catalyst loading of 4.17 × 10-2 mol%.

Tacrine and its 7-methoxy derivate; time-change concentration in plasma and brain tissue and basic toxicological profile in rats.

The search for tacrine derivatives, as potential Alzheimer´s disease treatment, is still being at the forefront of scientific efforts. 7-MEOTA was found to be a potent, centrally active acetylcholinesterase inhibitor free of the serious side effects observed for tacrine. Unfortunately, a relevant argumentation about pharmacokinetics and potential toxicity is incomplete; information about tacrine derivatives absorption and especially CNS penetration are still rare as well as detailed toxicological profile in vivo. Although the structural changes between these compounds are not so distinctive, differences in plasma profile and CNS targeting were found. The maximum plasma concentration were attained at 18th min (tacrine; 38.20 ± 3.91 ng/ml and 7-MEOTA; 88.22 ± 15.19 ng/ml) after i.m. application in rats. Although the brain profiles seem to be similar; tacrine achieved 19.34 ± 0.71 ng/ml in 27 min and 7-MEOTA 15.80 ± 1.13 ng/ml in 22 min; the tacrine Kp (AUCbrain/AUCplasma) fit 1.20 and was significantly higher than 7-MEOTA Kp 0.10. Administration of tacrine and 7-MEOTA showed only mild elevation of some biochemical markers following single p.o. application in 24 hours and 7 days. Also histopathology revealed only mild-to-moderate changes following repeated p.o. administration for 14 days. It seems that small change in tacrine molecule leads to lower ability to penetrate through the biological barriers. The explanation that lower p.o. acute toxicity of 7-MEOTA depends only on differences in metabolic pathways may be now revised to newly described differences in pharmacokinetic and toxicological profiles.

Synthesis of [13 C6 ]-ibrutinib.

Convenient and straightforward synthesis of ibrutinib labeled by carbon-13 isotope is reported. Isotopically labeled building block is introduced in the last step of reaction sequence affording sufficient isolated yield (7%) of [13 C6 ]-ibrutinib calculated towards starting commercially available [13 C6 ]-bromobenzene.

Atorvastatin Modulates Bile Acid Homeostasis in Mice with Diet-Induced Nonalcoholic Steatohepatitis.

Bile acids (BA) play a significant role in the pathophysiology of nonalcoholic steatohepatitis (NASH). The present study evaluates the modulation of bile acid metabolomics by atorvastatin, a cholesterol-lowering agent commonly used to treat cardiovascular complications accompanying NASH. NASH was induced in mice by 24 weeks of consuming a high-saturated fat, high-fructose, and high-cholesterol diet (F), with atorvastatin administered orally (20 mg/kg/day) during the last three weeks. Biochemical and histological analyses confirmed the effectiveness of the F diet in inducing NASH. Untreated NASH animals had significantly reduced biliary secretion of BA and increased fecal excretion of BA via decreased apical sodium-dependent bile salt transporter (Asbt)-mediated reabsorption. Atorvastatin decreased liver steatosis and inflammation in NASH animals consistently with a reduction in crucial lipogenic enzyme stearoyl-coenzyme A (CoA) desaturase-1 and nuclear factor kappa light chain enhancer of activated B-cell pro-inflammatory signaling, respectively. In this group, atorvastatin also uniformly enhanced plasma concentration, biliary secretion and fecal excretion of the secondary BA, deoxycholic acid (DCA). However, in the chow diet-fed animals, atorvastatin decreased plasma concentrations of BA, and reduced BA biliary secretions. These changes stemmed primarily from the increased fecal excretion of BA resulting from the reduced Asbt-mediated BA reabsorption in the ileum and suppression of synthesis in the liver. In conclusion, our results reveal that atorvastatin significantly modulates BA metabolomics by altering their intestinal processing and liver synthesis in control and NASH mice.

Soluble Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Participating in Aggravation of NASH-Related Changes in Mouse Liver.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis with inflammation and fibrosis. Membrane endoglin (Eng) expression is shown to participate in fibrosis, and plasma concentrations of soluble endoglin (sEng) are increased in patients with hypercholesterolemia and type 2 diabetes mellitus. We hypothesize that NASH increases both hepatic Eng expression and sEng in blood and that high levels of sEng modulate cholesterol and bile acid (BA) metabolism and affect NASH progression. Three-month-old transgenic male mice overexpressing human sEng and their wild type littermates are fed for six months with either a high-saturated fat, high-fructose high-cholesterol (FFC) diet or a chow diet. Evaluation of NASH, Liquid chromatography-mass spectrometry (LC/MS) analysis of BA, hepatic expression of Eng, inflammation, fibrosis markers, enzymes and transporters involved in hepatic cholesterol and BA metabolism are assessed using Real-Time Quantitative Reverse Transcription Polymerase Chain reaction (qRT-PCR) and Western blot. The FFC diet significantly increases mouse sEng levels and increases hepatic expression of Eng. High levels of human sEng results in increased hepatic deposition of cholesterol due to reduced conversion into BA, as well as redirects the metabolism of triglycerides (TAG) to its accumulation in the liver, via reduced TAG elimination by ß-oxidation combined with reduced hepatic efflux. We propose that sEng might be a biomarker of NASH development, and the presence of high levels of sEng might support NASH aggravation by impairing the essential defensive mechanism protecting NASH liver against excessive TAG and cholesterol accumulation, suggesting the importance of high sEng levels in patients prone to develop NASH.

Iron depletion induces hepatic secretion of biliary lipids and glutathione in rats.

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

Cardioprotective effects of inorganic nitrate/nitrite in chronic anthracycline cardiotoxicity: Comparison with dexrazoxane.

Dexrazoxane (DEX) is a clinically available cardioprotectant that reduces the toxicity induced by anthracycline (ANT) anticancer drugs; however, DEX is seldom used and its action is poorly understood. Inorganic nitrate/nitrite has shown promising results in myocardial ischemia-reperfusion injury and recently in acute high-dose ANT cardiotoxicity. However, the utility of this approach for overcoming clinically more relevant chronic forms of cardiotoxicity remains elusive. Hence, in this study, the protective potential of inorganic nitrate and nitrite against chronic ANT cardiotoxicity was investigated, and the results were compared to those using DEX. Chronic cardiotoxicity was induced in rabbits with daunorubicin (DAU). Sodium nitrate (1g/L) was administered daily in drinking water, while sodium nitrite (0.15 or 5mg/kg) or DEX (60mg/kg) was administered parenterally before each DAU dose. Although oral nitrate induced a marked increase in plasma NOx, it showed no improvement in DAU-induced mortality, myocardial damage or heart failure. Instead, the higher nitrite dose reduced the incidence of end-stage cardiotoxicity, prevented related premature deaths and significantly ameliorated several molecular and cellular perturbations induced by DAU, particularly those concerning mitochondria. The latter result was also confirmed in vitro. Nevertheless, inorganic nitrite failed to prevent DAU-induced cardiac dysfunction and molecular remodeling in vivo and failed to overcome the cytotoxicity of DAU to cardiomyocytes in vitro. In contrast, DEX completely prevented all of the investigated molecular, cellular and functional perturbations that were induced by DAU. Our data suggest that the difference in cardioprotective efficacy between DEX and inorganic nitrite may be related to their different abilities to address a recently proposed upstream target for ANT in the heart - topoisomerase IIß.

Boldine Inhibits Mouse Mammary Carcinoma In Vivo and Human MCF-7 Breast Cancer Cells In Vitro.

Boldine is an aporphine alkaloid widely consumed in the folk medicine of some regions. Its anticancer potential has been shown but not yet elucidated. We compared the antitumor effect of orally and parenterally applied boldine in mice bearing solid Ehrlich tumor. We also explored the effects of boldine on breast adenocarcinoma MCF-7 cells in vitro. Repeated i. p. injections of 30, 60, or 90 mg boldine/kg, either alone or combined with doxorubicin, slowed tumor growth in vivo. The latter two doses also prolonged the post-therapeutic survival of the mice. When fed food supplemented with boldine at a dose of 90 mg/kg, the tumor-bearing mice survived significantly longer, but there was no effect on tumor size. Interestingly, continuous p. o. administration did not produce detectable levels of boldine in plasma or tissue samples, in contrast to high but short-lived concentrations after i. p. injections. There was neither antagonism nor synergism between boldine and doxorubicin, except a possible synergism of i. p. boldine 90 mg/kg combined with doxorubicin when compared with doxorubicin alone.Boldine was cytotoxic to MCF-7 cells and reduced their viability and proliferation in vitro. Exposure to boldine decreased bromodeoxyuridine incorporation and histone H3 phosphorylation but did not induce apoptosis. Boldine treatment resulted in p38, ERK, and JNK activation in the mitogen-activated protein kinase pathway in a dose-dependent manner. Since bioavailability in mice seems to be different from that reported in rats, pharmacokinetic studies in humans are needed to evaluate the role of boldine in the beneficial effects of Boldo infusions.

LC-MS/MS method for the determination of haemanthamine in rat plasma, bile and urine and its application to a pilot pharmacokinetic study.

Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 µmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.

IL-1 receptor blockade alleviates endotoxin-mediated impairment of renal drug excretory functions in rats.

The aim of our study was to investigate whether two potent anti-inflammatory agents, dexamethasone and anakinra, an IL-1 receptor antagonist, may influence acute kidney injury (AKI) and associated drug excretory functions during endotoxemia (LPS) in rats. Ten hours after LPS administration, untreated endotoxemic rats developed typical symptoms of AKI, with reduced GFR, impaired tubular excretion of urea and sodium, and decreased urinary excretion of azithromycin, an anionic substrate for multidrug resistance-transporting proteins. Administration of both immunosuppressants attenuated the inflammatory response, liver damage, AKI, and increased renal clearance of azithromycin mainly by restoration of GFR, without significant influence on its tubular secretion. The lack of such an effect was related to the differential effect of both agents on the renal expression of individual drug transporters. Only dexamethasone increased the urinary clearance of bile acids, in accordance with the reduction of the apical transporter (Asbt) for their tubular reabsorption. In summary, our data demonstrated the potency of both agents used for the prevention of AKI, imposed by endotoxins, and for the restoration of renal drug elimination, mainly by the improvement of GFR. The influence of both drugs on altered tubular functions and the expression of drug transporters was differential, emphasizing the necessity of knowledge of transporting pathways for individual drugs applied during sepsis. The effect of anakinra suggests a significant contribution of IL-1 signaling to the pathogenesis of LPS-induced AKI.

Boldine enhances bile production in rats via osmotic and farnesoid X receptor dependent mechanisms.

Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 µM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine.

Single- and multiple-dose pharmacokinetics of arginase inhibitor Nω-hydroxy-nor-L-arginine, and its effect on plasma amino acids concentrations in Wistar rats.

Arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) augments synthesis of nitric oxide (NO) exerting therapeutic effects in rodent models for cardiovascular and airway diseases. This study examined single- and multiple-dose pharmacokinetics and effects of nor-NOHA on plasma amino acids in Wistar rats. Animals were administered 30 mg/kg nor-NOHA in a single bolus intravenous (i.v.) or intraperitoneal (i.p.) injection, or five once-daily i.p. injections at the same dose, or vehicle. Nor-NOHA and amino acids were assayed in blood plasma by high-performance liquid chromatography. After a bolus i.v. injection, the elimination of nor-NOHA was rapid (the mean residence time was 12.5 min). The area under the concentration-time curve and maximum concentration were higher by 17% and 31%, respectively, after the fifth as compared to the first i.p. injection. A shift in arginine utilization towards the synthesis of NO was indicated by elevated citrulline-to-ornithine and citrulline-to-arginine ratios. No changes in plasma arginine were observed. Increased glutamine concentrations might indicate an alternative detoxification pathway for ammonia due to inhibition of hepatic arginase. In conclusion, pharmacokinetic data of the present study can guide rational dosing of nor-NOHA in future studies. Limitations of the strategy of NO modulation via arginase inhibition should be further explored.

Comparative pharmacokinetics of N(ω)-hydroxy-nor-L-arginine, an arginase inhibitor, after single-dose intravenous, intraperitoneal and intratracheal administration to brown Norway rats.

1. Rodent studies have documented that N(ω)-hydroxy-nor-L-arginine (nor-NOHA), an arginase inhibitor, has therapeutic potential in the treatment of cardiovascular and obstructive airway diseases. However, its bioavailability and pharmacokinetics have not been described so far. 2. Anesthetized brown Norway rats were administered single doses of nor-NOHA (10, 30 or 90 mg/kg) intravenously (i.v.), intraperitonealy (i.p.) or via intratracheal (i.t.) instillation of aerosol. Plasma nor-NOHA was assayed using a validated HPLC method. 3. Upon i.v. administration, the mean concentration showed a biphasic decline and its value dropped below 10% of the maximum after 20 min. The pharmacokinetics were linear with the total and inter-compartmental clearances of 33 and 17 mL/min/kg, central and peripheral volumes of distribution of 0.19 and 0.43 L/kg and terminal half-life of 30 min. 4. The average absolute bioavailability of nor-NOHA after i.p. and i.t. delivery was 98% and 53%, respectively. The absorption from the airways was rate-limiting and its extent decreased with the dose. 5. In conclusion, nor-NOHA is rapidly cleared from the plasma in concordance with the short time window of its in vivo inhibitory activity reported in the literature. I.t. instillation of aerosol for topical effects of nor-NOHA in the airways is characterized with significant systemic availability.

Pharmacokinetic study of two acetylcholinesterase reactivators, trimedoxime and newly synthesized oxime K027, in rat plasma.

K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-propane dibromide] is a promising new reactivator of organophosphate- or organophosphonate-inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 ± 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg(-1)) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed-phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R(2) > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1-100 µg ml(-1). Near-identical plasma profiles were obtained for both compounds. No differences were found in the mean ± SD values of C(max) (18.6 ± 2.5 vs 20.0 ± 6.3 µg ml(-1), P = 0.72) and AUC(0-180min) (2290 ± 304 vs 2269 ± 197 min µg ml(-1), P = 0.84). However, the percentage coefficient of variation of the first-order rate constant of absorption (k(a)) was 3-fold higher (P < 0.01) providing evidence for more erratic absorption of intramuscular trimedoxime as compared with K027. In conclusion, oxime K027 might have superior pK properties that may be translated in its faster absorption and subsequent tissue distribution.

An alternative approach to validation of liquid chromatography-mass spectrometry methods for the quantification of endogenous compounds.

Despite the progress in the quantification of xenobiotics, the development and validation of methods designed for endogenous substances still remain challenging due to the natural presence of the analytes in a biological matrix, leading to the inability to obtain a blank sample. Several generally recognized procedures are described to solve this issue, like using surrogate or analyte-depleted matrices or surrogate analytes. However, the workflows used do not always meet the requirements for developing a reliable analytical method or are cost-intensive. This study aimed to design an alternative approach for preparing validation reference samples using authentic analytical standards while preserving the nature of the biological matrix and solving the problem with the inherent presence of analyzed compounds in a studied matrix. The methodology used is based on the standard-addition type procedure. However, unlike the original method, the addition is modified according to a previously measured basal concentration of monitored substances in the pooled biological sample to obtain a predefined concentration in reference samples according to the European Medicines Agency (EMA) validation guideline. The study shows the advantages of described approach on an example of LC-MS/MS analysis of 15 bile acids in human plasma and compares it with other methods commonly used in this field. The method was successfully validated according to the EMA guideline with lower limit of quantification of 5 nmol/L and linearity in the range of 5 - 2000 nmol/L. Finally, the method was used in a metabolomic study on a cohort of pregnant women (n = 28) to confirm intrahepatic cholestasis, the major liver disease observed in pregnancy.

Labetalol and soluble endoglin aggravate bile acid retention in mice with ethinylestradiol-induced cholestasis.

Labetalol is used for the therapy of hypertension in preeclampsia. Preeclampsia is characterized by high soluble endoglin (sEng) concentration in plasma and coincides with intrahepatic cholestasis during pregnancy (ICP), which threatens the fetus with the toxicity of cumulating bile acids (BA). Therefore, we hypothesized that both labetalol and increased sEng levels worsen BA cumulation in estrogen-induced cholestasis. C57BL/6J, transgenic mice overexpressing human sEng, and their wild-type littermates were administrated with ethinylestradiol (EE, 10 mg/kg s.c., the mice model of ICP) and labetalol (10 mg/kg s.c.) for 5 days with sample collection and analysis. Plasma was also taken from healthy pregnant women and patients with ICP. Administration of labetalol to mice with EE cholestasis aggravated the increase in BA plasma concentrations by induction of hepatic Mrp4 efflux transporter. Labetalol potentiated the increment of sEng plasma levels induced by estrogen. Increased plasma levels of sEng were also observed in patients with ICP. Moreover, increased plasma levels of human sEng in transgenic mice aggravated estrogen-induced cholestasis in labetalol-treated mice and increased BA concentration in plasma via enhanced reabsorption of BAs in the ileum due to the upregulation of the Asbt transporter. In conclusion, we demonstrated that labetalol increases plasma concentrations of BAs in estrogen-induced cholestasis, and sEng aggravates this retention. Importantly, increased sEng levels in experimental and clinical forms of ICPs might present a novel mechanism explaining the coincidence of ICP with preeclampsia. Our data encourage BA monitoring in the plasma of pregnant women with preeclampsia and labetalol therapy.

Carvedilol impairs bile acid homeostasis in mice: implication for nonalcoholic steatohepatitis.

Carvedilol is a widely used beta-adrenoreceptor antagonist for multiple cardiovascular indications; however, it may induce cholestasis in patients, but the mechanism for this effect is unclear. Carvedilol also prevents the development of various forms of experimental liver injury, but its effect on nonalcoholic steatohepatitis (NASH) is largely unknown. In this study, we determined the effect of carvedilol (10 mg/kg/day p.o.) on bile formation and bile acid (BA) turnover in male C57BL/6 mice consuming either a chow diet or a western-type NASH-inducing diet. BAs were profiled by liquid chromatography-mass spectrometry and BA-related enzymes, transporters, and regulators were evaluated by western blot analysis and qRT-PCR. In chow diet-fed mice, carvedilol increased plasma concentrations of BAs resulting from reduced BA uptake to hepatocytes via Ntcp transporter downregulation. Inhibition of the ß-adrenoreceptor-cAMP-Epac1-Ntcp pathway by carvedilol may be the post-transcriptional mechanism underlying this effect. In contrast, carvedilol did not worsen the deterioration of BA homeostasis accompanying NASH; however, it shifted the spectra of BAs toward more hydrophilic and less toxic α-muricholic and hyocholic acids. This positive effect of carvedilol was associated with a significant attenuation of liver steatosis, inflammation, and fibrosis in NASH mice. In conclusion, our results indicate that carvedilol may increase BAs in plasma by modifying their liver transport. In addition, carvedilol provided significant hepatoprotection in a NASH murine model without worsening BA accumulation. These data suggest beneficial effects of carvedilol in patients at high risk for developing NASH.

Chronic anthracycline cardiotoxicity: molecular and functional analysis with focus on nuclear factor erythroid 2-related factor 2 and mitochondrial biogenesis pathways.

Anthracycline anticancer drugs (e.g., doxorubicin or daunorubicin) can induce chronic cardiotoxicity and heart failure (HF), both of which are believed to be based on oxidative injury and mitochondrial damage. In this study, molecular and functional changes induced by chronic anthracycline treatment with progression into HF in post-treatment follow-up were analyzed with special emphasis on nuclear factor erythroid 2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) pathways. Chronic cardiotoxicity was induced in rabbits with daunorubicin (3 mg/kg, weekly for 10 weeks), and the animals were followed for another 10 weeks. Echocardiography revealed a significant drop in left ventricular (LV) systolic function during the treatment with marked progression to LV dilation and congestive HF in the follow-up. Although daunorubicin-induced LV lipoperoxidation was found, it was only loosely associated with cardiac performance. Furthermore, although LV oxidized glutathione content was increased, the oxidized-to-reduced glutathione ratio itself remained unchanged. Neither Nrf2, the master regulator of antioxidant response, nor the majority of its target genes showed up-regulation in the study. However, down-regulation of manganese superoxide dismutase and NAD(P)H dehydrogenase [quinone] 1 were observed together with heme oxygenase 1 up-regulation. Although marked perturbations in mitochondrial functions were found, no induction of PGC1α-controlled mitochondrial biogenesis pathway was revealed. Instead, especially in the post-treatment period, an impaired regulation of this pathway was observed along with down-regulation of the expression of mitochondrial genes. These results imply that global oxidative stress need not be a factor responsible for the development of anthracycline-induced HF, whereas suppression of mitochondrial biogenesis might be involved.

High-throughput salting-out assisted liquid-liquid extraction using a 3D printed device and its application in the quantification of ibrutinib and its metabolite PCI-45227 in human serum.

Liquid-liquid extraction methods are widely used for sample treatment in bioanalysis, although their implementation poses a common speed-limiting step in the analytical process when fast separation and detection methods such as UHPLC-MS are used. This study aimed to develop high-throughput salting-out assisted liquid-liquid extraction on a 96-well plate in combination with fast LC-MS analysis of ibrutinib and its active metabolite PCI-45227 (dihydrodiol ibrutinib) in human serum. A specially designed 3D printed extraction device developed in our laboratory allowed for the precise and rapid collection of the organic phase from the 96-well plate using a multichannel pipette, without the risk of aspiration of the bottom aqueous layer. The application of this device significantly accelerated sample preparation and allowed the processing of up to 96 samples in 1 h. The method was successfully validated according to EMA guidelines in the concentration range of 0.1-200 ng/mL for both analytes, providing lower limit of quantification at 100 pg/mL. The intra-day accuracy for IBT was in the range of - 1.67-5.67 %, while the inter-day accuracy was in the range of 0.20-6.90 %. The intra-day precision for IBT was in the range of 3.20-4.37 % and 3.13-4.73 % for the inter-day measurement. PCI-45227 showed intra-day accuracy in the range of - 11.2-3.71 % and the inter-day accuracy in the range of - 5.76-0.92 %. The intra-day precision for PCI was in the range of 3.49-7.64 % and 3.63-8.61 % for the measurement between days. In addition to increasing the speed of sample preparation, this method also offers low consumption of the sample and extraction solvent and can be utilized for other similar small-volume in-well plate extractions where organic solvents of lower density than water are used. The method was successfully applied to the analysis of serum samples (n = 5) of patients with chronic lymphoblastic leukaemia at the trough level (ibrutinib concentration range: 1.63-3.78 ng/mL, PCI-45227 concentration range: 1.84-14.02 ng/mL) and 2 h postdose (ibrutinib concentration range: 7.34-89.0 ng/mL, PCI-45227 concentration range: 5.64-124 ng/mL).

Multidrug Resistance-Associated Protein 2 Deficiency Aggravates Estrogen-Induced Impairment of Bile Acid Metabolomics in Rats.

Multidrug resistance-associated protein 2 (Mrp2) mediates biliary secretion of anionic endobiotics and xenobiotics. Genetic alteration of Mrp2 leads to conjugated hyperbilirubinemia and predisposes to the development of intrahepatic cholestasis of pregnancy (ICP), characterized by increased plasma bile acids (BAs) due to mechanisms that are incompletely understood. Therefore, this study aimed to characterize BA metabolomics during experimental Mrp2 deficiency and ICP. ICP was modeled by ethinylestradiol (EE) administration to Mrp2-deficient (TR) rats and their wild-type (WT) controls. Spectra of BAs were analyzed in plasma, bile, and stool using an advanced liquid chromatography-mass spectrometry (LC-MS) method. Changes in BA-related genes and proteins were analyzed in the liver and intestine. Vehicle-administered TR rats demonstrated higher plasma BA concentrations consistent with reduced BA biliary secretion and increased BA efflux from hepatocytes to blood via upregulated multidrug resistance-associated protein 3 (Mrp3) and multidrug resistance-associated protein 4 (Mrp4) transporters. TR rats also showed a decrease in intestinal BA reabsorption due to reduced ileal sodium/bile acid cotransporter (Asbt) expression. Analysis of regulatory mechanisms indicated that activation of the hepatic constitutive androstane receptor (CAR)-Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by accumulating bilirubin may be responsible for changes in BA metabolomics in TR rats. Ethinylestradiol administration to TR rats further increased plasma BA concentrations as a result of reduced BA uptake and increased efflux via reduced Slco1a1 and upregulated Mrp4 transporters. These results demonstrate that Mrp2-deficient organism is more sensitive to estrogen-induced cholestasis. Inherited deficiency in Mrp2 is associated with activation of Mrp3 and Mrp4 proteins, which is further accentuated by increased estrogen. Bile acid monitoring is therefore highly desirable in pregnant women with conjugated hyperbilirubinemia for early detection of intrahepatic cholestasis.