Pharmacological effects of mTORC1/C2 inhibitor in a preclinical model of NASH progression.

Knowledge of the benefits of mTOR inhibition concerning adipogenesis and inflammation has recently encouraged the investigation of a new generation of mTOR inhibitors for non-alcoholic steatohepatitis (NASH). We investigated whether treatment with a specific mTORC1/C2 inhibitor (Ku-0063794; KU) exerted any beneficial impacts on experimentally-induced NASH in vitro and in vivo. The results indicated that KU decreases palmitic acid-induced lipotoxicity in cultivated primary hepatocytes, thus emerging as a successful candidate for testing in an in vivo NASH dietary model, which adopted the intraperitoneal KU dosing route rather than oral application due to its significantly greater bioavailability in mice. The pharmacodynamics experiments commenced with the feeding of male C57BL/6 mice with a high-fat atherogenic western-type diet (WD) for differing intervals over several weeks aimed at inducing various phases of NASH. In addition to the WD, the mice were treated with KU for 3 weeks or 4 months. Acute and chronic KU treatments were observed to be safe at the given concentrations with no toxicity indications in the mice. KU was found to alleviate NASH-related hepatotoxicity, mitochondrial and oxidative stress, and decrease the liver triglyceride content and TNF-α mRNA in at least one set of in vivo experiments. The KU modulated liver expression of selected metabolic and oxidative stress-related genes depended upon the length and severity of the disease. Although KU failed to completely reverse the histological progression of NASH in the mice, we demonstrated the complexity of mTORC1/C2 signaling regulation and suggest a stratified therapeutic management approach throughout the disease course.

Differentiated modulation of signaling molecules AMPK and SIRT1 in experimentally drug-induced hepatocyte injury.

AIM: Currently available medicines have little to offer in terms of supporting the regeneration of injured hepatic cells. Previous experimental studies have shown that resveratrol and metformin, less specific activators of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), can effectively attenuate acute liver injury. The aim of this experimental study was to elucidate whether modulation of AMPK and SIRT1 activity can modify drug/paracetamol (APAP)-induced hepatocyte damage in vitro. METHODS: Primary rat hepatocytes were pretreated with mutual combinations of specific synthetic activators and inhibitors of SIRT1 and AMPK and followed by a toxic dose of APAP. At the end of cultivation, medium samples were collected for biochemical analysis of alanine-aminotransferase and nitrite levels. Hepatocyte viability, thiobarbituric reactive substances, SIRT1 and AMPK activity and protein expression were also assessed. RESULTS: The harmful effect of APAP was associated with decreased AMPK and SIRT1 activity and protein expression alongside enhanced oxidative stress in hepatocytes. The addition of AMPK activator (AICAR) or SIRT1 activator (CAY10591) significantly attenuated the deleterious effects of AMPK inhibitor (Compound C) on the hepatotoxicity of APAP. Furthermore, CAY10591 but not AICAR markedly decreased the deleterious effect of APAP in combination with SIRT1 inhibitor (EX-527). CONCLUSION: Our findings demonstrate that decreased AMPK activity is associated with the hepatotoxic effect of APAP which can be significantly attenuated by the administration of a SIRT1 activator. These findings suggest that differentiated modulation of AMPK and SIRT1 activity could therefore provide an interesting and novel therapeutic opportunity in the future to combat hepatocyte injury.

Detection of galanin receptors in the spinal cord in experimental autoimmune encephalomyelitis.

AIMS: The neuropeptide galanin is a widely distributed neurotransmitter/neuromodulator that regulates a variety of physiological processes and also participates in the regulation of stress responses. The aims of the present study were to investigate the expression of galanin receptors (GalR1, GalR2, GalR3) in the spinal cords in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) using qPCR analysis and to determine GalR1 cellular localization (oligodendrocytes, microglia, astrocytes, ependymal cells, and endothelial cells in the capillaries) by immunohistochemistry. METHODS: Twelve samples from the EAE group and 14 samples from the control group were analyzed. Spinal cords samples were obtained at the peak of the EAE disease. RESULTS: The GalR1 mRNA level was significantly decreased in the EAE mice compared with the controls (P=0.016), whereas the mRNA levels of GalR2 and GalR3 were not significantly different for the EAE and the control mice. No significant correlations were found between the severity of the EAE disease and the mRNA levels of GalR1, GalR2 and GalR3. Immunochemical detection of the GalR1 revealed its expression in the ependymal and endothelial cells. Additionally, a weak GalR1 immunoreactivity was occasionally detected in the oligodendrocytes. CONCLUSION: This study provides additional evidence of galanin involvement in EAE pathophysiology, but this has to be further investigated.

mTOR as an eligible molecular target for possible pharmacological treatment of nonalcoholic steatohepatitis.

Currently, non-alcoholic fatty liver disease (NAFLD) progressing into chronic non-alcoholic steatohepatitis (NASH), liver cirrhosis, and eventually hepatocellular cancer has emerged as an epidemiological concern due to lack of proven treatment. Our review briefly comprises of the mechanism of pathogenesis and inflammation corresponding to the disease, and all the offered insights of mechanistic pathways that could be targeted in the progression of NASH. The review principally focuses on mTOR (mammalian target of rapamycin) as a promising target highlighting its immense role in lipogenesis and alleviating inflammation and fibrosis. A detailed description of signaling pathways of mTORC1 and mTORC2 that are inhibited by rapamycin and other mTOR inhibitor analogues is accentuated. The exploration of mTOR inhibitors clearly explains the exigent molecular aspects of mTOR in regulating adipocyte and lipogenic marker genes (e.g. those encoding PPARγ, SREBP1c). The literature on available mTOR inhibitors and their classification so far could be extremely useful in highlighting mTOR as a favorable drug target in the indication of NASH in the near future.

Validity of cycloheximide chylomicron flow blocking method for the evaluation of lymphatic transport of drugs.

BACKGROUND AND PURPOSE: Lymphatic transport of drugs after oral administration is an important mechanism for absorption of highly lipophilic compounds. Direct measurement in lymph duct cannulated animals is the gold standard method, but non-invasive cycloheximide chylomicron flow blocking method has gained popularity recently. However, concerns about its reliability have been raised. The aim of this work was to investigate the validity of cycloheximide chylomicron flow blocking method for the evaluation of lymphatic transport using model compounds with high to very high lipophilicity, that is, abiraterone and cinacalcet. EXPERIMENTAL APPROACH: Series of pharmacokinetic studies were conducted with abiraterone acetate and cinacalcet hydrochloride after enteral/intravenous administration to intact, lymph duct cannulated and/or cycloheximide pre-treated rats. KEY RESULTS: Mean total absolute oral bioavailability of abiraterone and cinacalcet was 7.0% and 28.7%, respectively. There was a large and significant overestimation of the lymphatic transport extent by the cycloheximide method. Mean relative lymphatic bioavailability of abiraterone and cinacalcet in cycloheximide method was 28-fold and 3-fold higher than in cannulation method, respectively. CONCLUSION AND IMPLICATIONS: Cycloheximide chylomicron flow blocking method did not provide reliable results on lymphatic absorption and substantially overestimated lymphatic transport for both molecules, that is, abiraterone and cinacalcet. This non-invasive method should not be used for the assessment of lymphatic transport and previously obtained data should be critically revised.

Preclinical evaluation of new formulation concepts for abiraterone acetate bioavailability enhancement based on the inhibition of pH-induced precipitation.

Abiraterone acetate is a potent drug used for the treatment of metastatic castration resistant prostate cancer. However, currently marketed product containing crystalline abiraterone acetate exhibits strong positive food effect which results in strict dosing regimen. In the present work, a rational approach towards design of novel abiraterone acetate formulations that would allow increased bioavailability on a fasting stomach and thus decreased food effect is presented. Precipitation experiments in biorelevant media were designed to assess pH induced precipitation of the drug and a pool of polymeric excipients was then screened for their potential to inhibit precipitation. The best performing polymeric excipients were subsequently used as carriers for the preparation of amorphous solid dispersions. Two main approaches were followed in order to formulate the drug. The first approach relies on the suppression of precipitation from a supersaturated solution whereas the second one is based on the hypothesis that when the release of the drug is tuned, optimal uptake of the drug can be reached. Optimized formulation prototypes were tested in a rat animal model in an incomplete block, randomized bioequivalence study to assess their relative bioavailability under fasting conditions. We show that both formulation approaches lead to increased bioavailability of abiraterone acetate on a fasting stomach with bioavailability in rats being enhanced up to 250% compared to the original drug product containing crystalline drug.

Gastric pH in Rats: Key Determinant for Preclinical Evaluation of pH-dependent Oral Drug Absorption.

Data on gastric pH in rats to be used in preclinical models for pH-dependent drug absorption are still limited or contradictory. The aim of this study was to describe gastric pH in rats at fasted state and to evaluate its changes induced by pentagastrin or omeprazole in order to mimic gastric pH at fasted and fed human subjects. Twenty Wistar rats, fasting for 12 h, were randomly assigned into four treatment groups (n=5): control, pre-treated with omeprazole 2 h before pH measurement, pre-treated with omeprazole 12 h before pH measurement, and pre-treated with pentagastrin 20 min before pH measurement. An incision on the stomach wall was made in anesthetized animals, and pH of gastric juice was measured. The observed pH values were significantly different among groups (p=0.0341), with the median (IQR) values of gastric pH of 3.5 (2.7-4.2), 6.7 (4.7-7.0), 5.6 (3.5-6.4) and 2.2 (1.6-3.1) in control, omeprazole 2 h, omeprazole 12 h and pentagastin group, respectively. We recommend using short interval pentagastrin and 2 h omeprazole pre-treatment in fasting animals to model similar gastric pH as is expected in human fasted and fed state pharmacokinetic studies, respectively.

Hyperbilirubinemia in Gunn Rats is Associated with Decreased Inflammatory Response in LPS-Mediated Systemic Inflammation.

Decreased inflammatory status has been reported in subjects with mild unconjugated hyperbilirubinemia. However, mechanisms of the anti-inflammatory actions of bilirubin (BR) are not fully understood. The aim of this study is to assess the role of BR in systemic inflammation using hyperbilirubinemic Gunn rats as well as their normobilirubinemic littermates and further in primary hepatocytes. The rats were treated with lipopolysaccharide (LPS, 6 mg/kg intraperitoneally) for 12 h, their blood and liver were collected for analyses of inflammatory and hepatic injury markers. Primary hepatocytes were treated with BR and TNF-α. LPS-treated Gunn rats had a significantly decreased inflammatory response, as evidenced by the anti-inflammatory profile of white blood cell subsets, and lower hepatic and systemic expressions of IL-6, TNF-α, IL-1ß, and IL-10. Hepatic mRNA expression of LPS-binding protein was upregulated in Gunn rats before and after LPS treatment. In addition, liver injury markers were lower in Gunn rats as compared to in LPS-treated controls. The exposure of primary hepatocytes to TNF-α with BR led to a milder decrease in phosphorylation of the NF-κB p65 subunit compared to in cells without BR. In conclusion, hyperbilirubinemia in Gunn rats is associated with an attenuated systemic inflammatory response and decreased liver damage upon exposure to LPS.

The involvement of sirtuin 1 and heme oxygenase 1 in the hepatoprotective effects of quercetin against carbon tetrachloride-induced sub-chronic liver toxicity in rats.

The present study was designed to evaluate the therapeutic potential of quercetin in a sub-chronic model of hepatotoxicity. The roles of putative antioxidant enzymes, sirtuin 1 (SIRT1) and heme oxygenase 1 (HO-1), in hepatoprotection were also addressed. Sub-chronic liver injury was induced in rats by intraperitoneal administration of 0.5 ml/kg carbon tetrachloride (CTC), once every 3 days, for 2 weeks. Some CTC rats were concurrently treated with 100 mg/kg quercetin, intragastrically, once every day, for 2 weeks. The effects of these drugs in the liver were evaluated by biochemical, histological, immunohistochemical and molecular biological studies. CTC triggered oxidative damage to the liver as unanimously shown by altered biochemical parameters and liver morphology. Furthermore, CTC highly upregulated HO-1 and SIRT1 expression levels. Concomitant treatment of rats with quercetin downregulated SIRT1 expression and ameliorated the hepatotoxic effects of CTC. However, quercetin did not have any significant effect on HO-1 expression and bilirubin levels. Collectively, these results suggest that the antioxidant and cytoprotective effects of quercetin in CTC treated rats were SIRT1 mediated and less dependent on HO-1. Thus, pharmacologic modulation of SIRT1 could provide a logic therapeutic approach in sub-chronic hepatotoxicity.

[Potential interactions between drugs and dietary supplements]. / Potenciální interakce mezi lécivy a doplnky stravy rostlinného puvodu*

Purified active plant constituents were isolated and assessed for their pharmacological activities that constitute a basis of modern drug development. The situation with herbal supplements is different because the extract or dried herb or mixture of herbs contains several substances beside the beneficial one(s) that might produce drug interaction with the conventional medicine(s). Most patients are misinformed and believe that anything "natural" must be safe. This article is focusing on plant-based substances referred as dietary supplements (DS). Examples of reported drug interactions and contraindications associated with DS with two case studies are presented. As supplements are typically not prescribed, many doctors seem to have no interest in drug-DS interactions since a typical medical history of the patients does not include any questions about self-prescribed remedies of this nature. Rather, patients are left alone when they are tempted to try this or that DS and tend to rely on advice from friends, or on material they read on internet. A better quality control, compliance, public awareness and healthcare professionals vigilance for potential interactions are needed. It is of utmost importance to appreciate the impact of supplements on different stages of pharmacokinetics, especially on drug absorption and metabolism.

[Perspectives on pharmacological and clinical benefits from sirtuin 1 activators in oxidative damage]. / Perspektivy farmakologického a klinického prínosu aktivátoru sirtuinu 1 pri oxidativním poskození.

Sirtuins, named after their homology to the Saccharomyces cerevisiae Silent Information Regulator Two, constitute a family of highly conserved nicotinamide adenine dinucleotide-dependent enzymes that deacetylate histones and residues of acetylated lysine. The main aim of this article is to put forward the pharmacological importance of major sirtuin 1 activators of natural or synthetic origin tested in last years in cases of oxidative tissue damage. The related bioactivity of these activators as "leading" compounds in the search for new drugs and remedies is also described. With the recent development of our knowledge on the cross talks between sirtuin 1 and its modulators (e.g. resveratrol), pharmacological and clinical research on this topic is getting a new horizon.

Cryopreservation of precision cut tissue slices (PCTS): investigation of morphology and reactivity.

Precision cut tissue slices (PCTS) represent a suitable and convenient tool for pharmacological, toxicological and morphological studies. Cryopreservation would enable to overcome the shortage of liver tissue, in particular in settings using human liver tissue. We investigated the potential of cryopreservation of porcine PCTS as a morphological tool by rapid freezing with 10% and 30% dimethyl sulfoxide as cryopreservation agents and with or without medium using a Brendel/Vitron tissue slicer. Incubation after thawing was done in a static incubation system. Slices were cultured for 3 h, 6 h, 24 h and 48 h and assessed histologically and immunohistologically for proliferation (Ki67) and spontaneous as well as induced apoptotic activity (M30Cytodeath). Vitality was tested using the Tox-8 test. After cryopreservation, morphology of PCTS was well preserved up to 24 h. A reduction of vitality rate took place. Compared to non-frozen PCTS, the rate of spontaneous proliferation of Kupffer cells and apoptosis of hepatocytes were significantly reduced independent of the freezing conditions. The reactivity of PCTS to apoptotic stimuli was significantly reduced in tissue slices after cryopreservation. Apoptotic stimuli could not induce the same amount of cell deaths compared to non-frozen sections. Thus, cryopreservation of PCTS does interfere with pathomechanisms of apoptosis in PCTS.