ω-Imidazolyl-alkyl derivatives as new preclinical drug candidates for treating non-alcoholic steatohepatitis.

Cytochrome P450 2E1 (CYP2E1)-associated reactive oxygen species production plays an important role in the development and progression of inflammatory liver diseases such as alcoholic steatohepatitis. We developed two new inhibitors for this isoenzyme, namely 12-imidazolyl-1-dodecanol (I-ol) and 1-imidazolyldodecane (I-an), and aimed to test their effects on non-alcoholic steatohepatitis (NASH). The fat-rich and CYP2E1 inducing Lieber-DeCarli diet was administered over 16 weeks of the experimental period to induce the disease in a rat model, and the experimental substances were administered simultaneously over the last four weeks. The high-fat diet (HFD) pathologically altered the balance of reactive oxygen species and raised the activities of the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP) and γ-glutamyl-transferase (γ-GT); lowered the level of adiponectine and raised the one of tumor necrosis factor (TNF)-α; increased the hepatic triglyceride and phospholipid content and diminished the serum HDL cholesterol concentration. Together with the histological findings, we concluded that the diet led to the development of NASH. I-ol and, to a lesser extent, I-an shifted the pathological values toward the normal range, despite the continued administration of the noxious agent (HFD). The hepatoprotective drug ursodeoxycholic acid (UDCA), which is used off-label in clinical practice, showed a lower effectiveness overall. I-ol, in particular, showed extremely good tolerability during the acute toxicity study in rats. Therefore, cytochrome P450 2E1 may be considered a suitable drug target, with I-ol and I-an being promising drug candidates for the treatment of NASH.

Drug targeting CYP2E1 for the treatment of early-stage alcoholic steatohepatitis.

BACKGROUND AND AIMS: Alcoholic steatohepatitis (ASH)-the inflammation of fatty liver-is caused by chronic alcohol consumption and represents one of the leading chronic liver diseases in Western Countries. ASH can lead to organ dysfunction or progress to hepatocellular carcinoma (HCC). Long-term alcohol abstinence reduces this probability and is the prerequisite for liver transplantation-the only effective therapy option at present. Elevated enzymatic activity of cytochrome P450 2E1 (CYP2E1) is known to be critically responsible for the development of ASH due to excessively high levels of reactive oxygen species (ROS) during metabolization of ethanol. Up to now, no rational drug discovery process was successfully initiated to target CYP2E1 for the treatment of ASH. METHODS: In this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies. RESULTS: A new class of drug candidates was generated successfully. Two of the most promising small compounds named 12-Imidazolyl-1-dodecanol (abbr.: I-ol) and 1-Imidazolyldodecane (abbr.: I-an) were selected at the end of this process of drug discovery and developability. These new ω-imidazolyl-alkyl derivatives act as strong chimeric CYP2E1 inhibitors at a nanomolar range. They restore redox balance, reduce inflammation process as well as the fat content in the liver and rescue the physiological liver architecture of rats consuming continuously a high amount of alcohol. CONCLUSIONS: Due to its oral application and therapeutic superiority over an off-label use of the hepatoprotector ursodeoxycholic acid (UDCA), this new class of inhibitors marks the first rational, pharmaceutical concept in long-term treatment of ASH.

Antidiabetic effects and erythrocyte stabilization by red cabbage extract in streptozotocin-treated rats.

The protective effect of red cabbage extract (RCE) was evaluated in rats with streptozotocin-induced diabetes, assessing a probable role of this extract in the prevention of erythrocyte impairments associated with a high risk of vascular complications in diabetes. RCE was analyzed by ultrahigh performance liquid chromatography and mass spectrometry, and 11 anthocyanins, 3 hydroxybenzoic acids and 9 hydroxycinnamic acids were identified. Type 1 diabetes was induced by streptozotocin (60 mg kg-1) in Wistar male rats (n = 8 per group). After 7 days of acclimatization, streptozotocin-treated rats were given RCE (800 mg kg-1) or vehicle by intragastric administration for 4 weeks. The RCE treatment lowered blood glucose, and glycated and fetal hemoglobin concentrations and improved glucose tolerance as well as considerably raised serum insulin, proinsulin and C-peptide levels in streptozotocin-treated rats. Simultaneously, RCE improved pancreatic islet morphology, increasing the amount of pancreatic ß-cells in diabetic animals. The RCE administration prevented anemia in rats with streptozotocin-induced diabetes, enhanced erythrocyte resistance to acid hemolysis, and normalized reticulocyte production as well as sialic acid content in erythrocyte membranes. The enhanced lectin-induced erythrocyte aggregation in diabetic rats was significantly lowered after the RCE treatment. RCE demonstrated a significant antioxidant effect, decreasing MDA and protein carbonyl contents and increasing catalase and glutathione peroxidase activities in erythrocytes. These results indicate that RCE can be considered as a promising candidate for use as a drug or a food supplement to alleviate diabetes and its vascular complications.

Cytoprotection of pancreatic ß-cells and hypoglycemic effect of 2-hydroxypropyl-ß-cyclodextrin: sertraline complex in alloxan-induced diabetic rats.

Sertraline, a selective inhibitor of serotonin reuptake, is widely used as antidepressant in diabetic patients for improvement of depression and glycemic control. Sertraline is poorly soluble in water, which limits its oral applicability. In this work we tried to improve the pharmaceutical properties of sertraline by complexation with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and evaluated the efficacy of the HPßCD:sertraline complex in prevention of alloxan-induced lesions in rats. The solubility of sertraline increased in the presence of HPßCD and the association constant for sertraline and HPßCD was equal to 4000 ± 1000 M(-1). Two-week treatment of diabetic animals with the HPßCD:sertraline complex improved pancreatic islet morphology and ß-cell survival, which, in turn, reduced the severity of diabetes, as evidenced by lowering of blood glucose and glycated hemoglobin contents as well as normalization of serum insulin level and insulin sensitivity (HOMA-IR). The effect of the HPßCD:sertraline complex was strongly expressed in comparison with the antidiabetic effect of both the monopreparations, HPßCD and sertraline. It is suggested that the cyclodextrin derivative increased the pharmacological effect of sertraline, probably due to enhanced drug bioavailability.

Pituitary tumor transforming gene as a novel regulatory factor of liver fibrosis.

AIMS: Pituitary tumor-transforming gene (PTTG) is involved in multiple cellular pathways. We studied the development of liver fibrosis induced by thioacetamide (TAA) in knockout (PTTG-/-) and wildtype (PTTG+/+) mice. MAIN METHODS: Liver fibrosis in PTTG+/+ and PTTG-/- mice was induced by escalating dose TAA treatment (50-400mg/kg, i.p.) for 12 weeks and assessed by histochemistry, immunohistochemistry, liver hydroxyproline, serum fibrosis markers and fibrosis-related mRNA expression by real-time PCR determination. KEY FINDINGS: Both PTTG+/+ and PTTG-/- mice treated with TAA developed signs of fibrosis and inflammatory cell infiltration. However, histological signs of bridging fibrosis and connective tissue square morphometry were significantly attenuated in mice lacking PTTG. α-SMA immunohistochemistry revealed that hepatic stellate cell activation was markedly reduced in PTTG-/- mice compared to wildtype controls. Hepatic hydroxyproline levels were significantly lower in fibrotic PTTG-/- group. The serum TNFα and hepatic TNFα mRNA expression were significantly lower in fibrotic PTTG-/- animals, as well as hepatic TGFß and VEGF mRNA levels compared to TAA-treated wildtype controls. Serum hyaluronate and TGFß levels were markedly elevated in fibrotic mice of both genotypes, but were not altered by the absence of PTTG. SIGNIFICANCE: TAA-induced fibrosis development is significantly ameliorated in PTTG-/- mice. These animals demonstrated diminished stellate cell activation, suppressed circulating serum markers of inflammation, fibrogenesis and angiogenesis. The presented findings suggest that PTTG is functionally required for hepatic fibrosis progression in an animal model of chronic liver injury. PTTG can be considered as a new important target for prevention and treatment of liver fibrosis/cirrhosis.

Protective Effects of Norursodeoxycholic Acid Versus Ursodeoxycholic Acid on Thioacetamide-induced Rat Liver Fibrosis.

BACKGROUND/OBJECTIVES: Effects of norursodeoxycholic acid (norUDCA) and ursodeoxycholic acid (UDCA) on liver fibrosis progression and liver fibrosis reversal in thioacetamide (TAA)-treated rats were studied. METHODS: Advanced liver fibrosis was induced by TAA treatment (200 mg/kg, i.p.) for 12 weeks. In the second experiment resolution of liver fibrosis was assessed after 8 weeks of TAA withdrawal. During 8 last weeks of each trial, fibrotic rats were daily administered with UDCA (80 mg/kg) and norUDCA (equimolar to 80 mg/kg of UDCA) by oral gavage. Liver fibrosis was assessed by Sirius red staining, liver hydroxyproline and serum fibrosis markers determination. RESULTS: The TAA treatment resulted in advanced fibrosis and increase in liver hydroxyproline content and serum fibrosis markers. These signs of fibrosis were less pronounced in rats after TAA withdrawal. Treatment with of norUDCA significantly decreased the total and relative liver hydroxyproline contents in rats with fibrosis reversal, whereas UDCA did not change these parameters. Both compounds decreased serum TGFß and type IV collagen contents, whereas other serum markers did not differ from the placebo group. In the fibrosis progression model the square of connective tissue was decreased by norUDCA. Serum type IV collagen and procollagen III-NT contents in these experiments were lowered by both UDCA and norUDCA, whereas rest of serum fibrosis markers were diminished only by norUDCA. CONCLUSIONS: Both norUDCA and UDCA showed therapeutic and prophylactic antifibrotic effect in rats with TAA-induced liver fibrosis. For most of tested parameters norUDCA was more effective than UDCA, especially in the experiment with liver fibrosis regression.

Ursodeoxycholic acid dose-dependently improves liver injury in rats fed a methionine- and choline-deficient diet.

AIM: The data on the beneficial effect of ursodeoxycholic acid (UDCA) in non-alcoholic steatohepatitis (NASH) are controversial. The difference of opinion is connected with UDCA dosage to be used. Therefore, we evaluated the dose-dependent efficacy of UDCA in experimental NASH. METHODS: Male Wistar rats were fed the methionine- and choline-deficient (MCD) diet for 10 weeks. Rats were administrated UDCA (10, 20, 40 and 80 mg/kg bodyweight intragastrically) after 6 weeks of the MCD diet. RESULTS: Animals fed the MCD diet developed severe steatohepatitis. Treatment with UDCA dose-dependently decreased liver damage, but only high-dose UDCA (80 mg/kg) significantly diminished ultrastructural changes in addition to preventing steatosis, ballooning and inflammatory changes in the liver. The activities of serum marker enzymes and the content of liver triglyceride and blood glucose were increased in MCD diet-fed rats, but decreased in all the UDCA-treated groups. Serum insulin concentration was decreased whereas the quantitative insulin sensitivity check index did not changed in MCD diet-fed groups. Serum tumor necrosis factor-α content was strongly increased after MCD diet and normalized in the UDCA-treated rats, with the most pronounced effect in the highest dose groups, 40 and 80 mg/kg. The contents of endogenous ethanol in blood and intestinal mucus were increased in MCD diet-fed rats which were significantly lowered by UDCA (40 and 80 mg/kg per day). CONCLUSION: The present data demonstrate a beneficial effect of UDCA that manifested by the decrease of liver steatosis, inflammatory signs and serum tumor necrosis factor-α content especially of the highest 40 and 80 mg/kg day doses.

The protective effect of ursodeoxycholic acid in alloxan-induced diabetes.

The purpose of this work was to study the effect of ursodeoxycholic acid (UDCA) on the morphological and functional alterations in pancreatic islet beta-cells in rats with diabetes induced by alloxan (150 mg kg(-1), i.p.). UDCA (40 mg kg(-1), i.g.) was administered daily from the fifth to the 35th day after the alloxan treatment. The treatment of diabetic rats with UDCA improved the pancreatic morphology disturbed by the alloxan treatment: UDCA increased the number of pancreatic islets and beta-cells, the beta-/alpha-cell ratio and decreased the number of alpha-cells. As the morphometric data suggest, the treatment of diabetic animals with UDCA significantly increased the area of beta-cell cytoplasmatic granules stained by paraldehyde-fuchsin. The concentration of blood glucose in diabetic rats was gradually decreased after the UDCA treatment, and at the end of the experiment reached the control value. The treatment with UDCA raised the serum insulin level in diabetic rats about 2.5-fold, but this concentration was significantly lower as compared to the control group. The content of lipid peroxidation end-products, hydroxyalkenals and malondialdehyde, was significantly elevated in the alloxan-treated rats, whereas the treatment with UDCA normalized these parameters. The present data indicate that UDCA acts as an effective antidiabetic agent in alloxan-induced diabetes and its beneficial effects in diabetic rats can be related to the antioxidant properties of UDCA.