Selective PPARδ agonist seladelpar suppresses bile acid synthesis by reducing hepatocyte CYP7A1 via the fibroblast growth factor 21 signaling pathway.

Peroxisome proliferator-activated receptor delta (PPARδ) agonists have been shown to exert beneficial effects in liver disease and reduce total bile acid levels. The mechanism(s) whereby PPARδ agonism reduces bile acid levels are, however, unknown, and therefore the aim of the present study was to investigate the molecular pathways responsible for reducing bile acid synthesis in hepatocytes, following treatment with the selective PPARδ agonist, seladelpar. We show that administration of seladelpar to WT mice repressed the liver expression of cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme for bile acid synthesis, and decreased plasma 7α-hydroxy-4-cholesten-3-one (C4), a freely diffusible metabolite downstream of Cyp7a1. In primary mouse hepatocytes, seladelpar significantly reduced the expression of Cyp7a1 independent of the nuclear bile acid receptor, Farnesoid X receptor. In addition, seladelpar upregulated fibroblast growth factor 21 (Fgf21) in mouse liver, serum, and in cultured hepatocytes. We demonstrate that recombinant Fgf21 protein activated the c-Jun N-terminal kinase (JNK) signaling pathway and repressed Cyp7a1 gene expression in primary hepatocytes. The suppressive effect of seladelpar on Cyp7a1 expression was blocked by a JNK inhibitor as well as in the absence of Fgf21, indicating that Fgf21 plays an indispensable role in PPARδ-mediated downregulation of Cyp7a1. Finally, reduction of CYP7A1 expression by seladelpar was confirmed in primary human hepatocytes. In conclusion, we show that seladelpar reduces bile acid synthesis via an FGF21-dependent mechanism that signals at least partially through JNK to repress CYP7A1.

OCE-205 in rats and non-human primates: Pharmacokinetic and pharmacodynamic analysis.

Treatment for complications associated with the hemodynamic consequences of decompensated cirrhosis remains suboptimal. Terlipressin, the latest pharmacological management of hepatorenal syndrome-acute kidney injury (HRS-AKI), targets the vasopressin system but has serious side effects. OCE-205 is a novel peptide designed to target the vasopressin receptor system as a mixed V1a agonist/antagonist, resulting in effective partial agonism without V2 agonism. We examined the in vivo pharmacokinetic/pharmacodynamic properties of OCE-205 in healthy rats and cynomolgus monkeys. OCE-205 was administered by IV or SC bolus injection; arginine vasopressin (AVP) or terlipressin were comparators. After IV OCE-205 administration in rats, mean plasma concentration decreased in a mostly linear manner to 2 mg/mL after 120 min, and for SC administration, slowly decreased to ∼50 ng/mL after 300 min. Compared with pre-test values, arterial blood pressure values significantly increased after all OCE-205 doses tested. For monkeys, the concentration after IV OCE-205 administration was mostly linear to 5 ng/mL after 180 min, and for SC administration, ∼3 ng/mL after 480 min. Subcutaneous OCE-205 administration increased mean arterial pressure (MAP) versus baseline, with ΔMAP in OCE-205-treated animals marked and long-lasting while terlipressin induced an increase from baseline in MAP, with negligible ΔMAP, on average, by 150 min after administration in all groups. AVP, but not OCE-205, significantly increased blood lactate concentrations. OCE-205 was well tolerated in adult male rats and cynomolgus monkeys following single-dose bolus administration. The preclinical results of OCE-205, with its demonstrated V1a selective partial agonist activity and potentially tolerable safety profile, suggest its potential utility for treatment of the cardiovascular complications of cirrhosis. Institutional protocol number: Procedures were approved by the Ferring Research Institute (FRI) Institutional Animal Care and Use Committee (IACUC) on November 27, 2006 under protocol FRI 06-011, and by the Sinclair Research Center IACUC under protocol S11177.

OCE-205, a Selective V1a Partial Agonist, Reduces Portal Pressure in Rat Models of Portal Hypertension.

Purpose: Management of decompensated cirrhosis may include the use of vasoconstrictors that can lead to serious adverse events. OCE-205 was designed as a highly selective V1a receptor partial agonist, intended to have a wider therapeutic window than full vasopressin agonists. Methods: We aimed to characterize the activity of OCE-205 treatment in two rat models of portal hypertension (PHT). For both models, OCE-205 was administered as a subcutaneous bolus injection. Thirty male Wistar rats were fed a methionine/choline-deficient (MCD) diet to model PHT. Animals received OCE-205 (10, 25, 100, or 500 µg/kg) or intra-arterial terlipressin (100 µg/kg). In a more severe model of PHT, 11 male Sprague Dawley rats had the common bile duct surgically ligated (BDL) and received OCE-205. Portal pressure (PP) and mean arterial pressure (MAP) were measured. Results: For PP in the MCD model, MAP increased while PP decreased in rats treated with OCE-205 or terlipressin; the peak changes to MAP were 14.7 and 33.5 mmHg, respectively. Changes in MAP began to plateau after 10 min in the OCE-205 groups, whereas in the terlipressin group, MAP rapidly increased and peaked after 20 min. Across all treatment groups in the BDL model, a dose-related decrease from baseline in PP was observed following OCE-205, plateauing as the dose increased. In all treatment groups, PP change remained negative throughout the 30-min testing period. In both PHT rat models, a reduction in PP was coupled to an increase in MAP, with both plateauing in dose-response curves. Conclusion: Data support OCE-205 as a promising candidate for further development. Institutional Protocol Number: Procedures were approved by the Ferring Research Institute (FRI) Institutional Animal Care and Use Committee on July 13, 2011, under protocol FRI-07-0002.

The selective PPAR-delta agonist seladelpar reduces ethanol-induced liver disease by restoring gut barrier function and bile acid homeostasis in mice.

Alcohol-associated liver disease is accompanied by dysregulation of bile acid metabolism and gut barrier dysfunction. Peroxisome proliferator-activated receptor-delta (PPARδ) agonists are key metabolic regulators and have anti-inflammatory properties. Here, we evaluated the effect of the selective PPAR-delta agonist seladelpar (MBX-8025) on gut barrier function and bile acid metabolism in a mouse model of ethanol-induced liver disease. Wild type C57BL/6 mice were fed LieberDeCarli diet containing 0%-36% ethanol (caloric) for 8 weeks followed by a single binge of ethanol (5 g/kg). Pair fed mice received an isocaloric liquid diet as control. MBX-8025 (10 mg/kg/d) or vehicle were added to the liquid diet during the entire feeding period (prevention), or during the last 4 weeks of Lieber DeCarli diet feeding (intervention). In both prevention and intervention trials, MBX-8025 protected mice from ethanol-induced liver disease, characterized by lower serum alanine aminotransferase (ALT) levels, hepatic triglycerides, and inflammation. Chronic ethanol intake disrupted bile acid metabolism by increasing the total bile acid pool and serum bile acids. MBX-8025 reduced serum total and secondary bile acids, and the total bile acid pool as compared with vehicle treatment in both prevention and intervention trials. MBX-8025 restored ethanol-induced gut dysbiosis and gut barrier dysfunction. Data from this study demonstrates that seladelpar prevents and treats ethanol-induced liver damage in mice by direct PPARδ agonism in both the liver and the intestine.

Reduction of hepatic steatosis in rats and mice after treatment with a liver-targeted thyroid hormone receptor agonist.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver disease, with a prevalence ranging from 10% to 30%. The use of thyroid hormone receptor (TR) agonists for the treatment of NAFLD has not been considered viable because thyroid hormones increase free fatty acid (FFA) flux from the periphery to the liver, induce hepatic lipogenesis, and therefore could potentially cause steatosis. MB07811 is an orally active HepDirect prodrug of MB07344, a liver-targeted TR-beta agonist. The purpose of these studies was to assess the effects of MB07811 on whole body and liver lipid metabolism of normal rodents and rodent models of hepatic steatosis. In the current studies, MB07811 markedly reduced hepatic steatosis as well as reduced plasma FFA and triglycerides. In contrast to MB07811, T(3) induced adipocyte lipolysis in vitro and in vivo and had a diminished ability to decrease hepatic steatosis. This suggests the influx of FFA from the periphery to the liver may partially counteract the antisteatotic activity of T(3). Clearance of liver lipids by MB07811 results from accelerated hepatic fatty acid oxidation, a known consequence of hepatic TR activation, as reflected by increased hepatic mitochondrial respiration rates, changes in hepatic gene expression, and increased plasma acyl-carnitine levels. Transaminase levels remained unchanged, or were reduced, and no evidence for liver fibrosis or other histological liver damage was observed after treatment with MB07811 for up to 10 weeks. Additionally, MB07811, unlike T(3), did not increase heart weight or decrease pituitary thyroid-stimulating hormone beta (TSHbeta) expression. CONCLUSION: MB07811 represents a novel class of liver-targeted TR agonists with beneficial low-density lipoprotein cholesterol-lowering properties that may provide additional therapeutic benefit to hyperlipidemic patients with concomitant NAFLD.

Preclinical pharmacokinetics of a HepDirect prodrug of a novel phosphonate-containing thyroid hormone receptor agonist.

The prodrug [(2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811)] of a novel phosphonate-containing thyroid hormone receptor agonist [3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxylmethylphosphonic acid (MB07344)] is the first application of the HepDirect liver-targeting approach to a non-nucleotide agent. The disposition of MB07811 was characterized in rat, dog, and monkey to assess its liver specificity, which is essential in limiting the extrahepatic side effects associated with this class of lipid-lowering agents. MB07811 was converted to MB07344 in liver microsomes from all species tested (CL(int) 1.23-145.4 microl/min/mg). The plasma clearance and volume of distribution of MB07811 matched or exceeded 1 l/h/kg and 3 l/kg, respectively. Although absorption of prodrug was good, its absolute oral bioavailability as measured systemically was low (3-10%), an indication of an extensive hepatic first-pass effect. This effect was confirmed by comparison of systemic exposure levels of MB07811 after portal and jugular vein administration to rats, which demonstrated a hepatic extraction ratio of >0.6 with liver CYP3A-mediated conversion to MB07344 being a major component. The main route of elimination of MB07811 and MB07344 was biliary, with no evidence for enterohepatic recirculation of MB07344. Similar metabolic profiles of MB07811 were obtained in liver microsomes across the species tested. Tissue distribution and whole body autoradiography confirmed that the liver is the major target organ of MB07811 and that conversion to MB07344 was high in the liver relative to that in other tissues. Hepatic first-pass extraction and metabolism of MB07811, coupled with possible selective distribution of MB07811-derived MB07344, led to a high degree of liver targeting of MB07344.

Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid.

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.

The lipophilic iron compound TMH-ferrocene [(3,5,5-trimethylhexanoyl)ferrocene] increases iron concentrations, neuronal L-ferritin, and heme oxygenase in brains of BALB/c mice.

Mismanagement of intracellular iron is a key pathological feature of many neurodegenerative diseases. Our long-term goal is to use animal models to investigate the mechanisms of iron neurotoxicity and its relationship to neurodegenerative pathologies. The immediate aim of this experiment was to determine regional distribution of iron and cellular distribution of iron storage proteins (L- and H-ferritin) and an oxidative stress marker (heme oxygenase-1) in brains of mice fed the lipophilic iron compound (3,5,5-trimethylhexanoyl) (TMH)-ferrocene. We fed male and female weanling BALB/cj mice diets either deficient in iron (0 mg Fe/kg diet), adequate in iron (35 mg Fe/kg diet; control mice), or adequate in iron and supplemented with 0.1 or 1.0 g TMH-ferrocene/kg diet for 8 wk. Iron concentrations in cerebrum were higher in mice fed 1.0 g TMH-ferrocene/kg diet than in control mice (p < 0.05). Liver iron concentrations were eightfold higher in mice fed 1.0 g TMH-ferrocene/kg diet than in control mice (p < 0.0001). L-Ferritin and heme oxygenase-1 expression were elevated in striatum in mice fed 1.0 g TMH-ferrocene/kg diet. We conclude that administration of the lipophilic iron compound TMH-ferrocene leads to subtle perturbations of cellular iron within the brain, potentially representing a model of iron accumulation similar to that seen in various neuropathological conditions.

A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis.

AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that regulates cellular energy metabolism by affecting energy-consuming pathways such as de novo lipid biosynthesis and glucose production as well as energy-producing pathways such as lipid oxidation and glucose uptake. Accordingly, compounds that activate AMPK represent potential drug candidates for the treatment of hyperlipidemia and type 2 diabetes. Screening of a proprietary library of AMP mimetics identified the phosphonic acid 2 that bears little structural resemblance to AMP but is capable of activating AMPK with high potency (EC50 = 6 nM vs AMP EC50 = 6 µM) and specificity. Phosphonate prodrugs of 2 inhibited de novo lipogenesis in cellular and animal models of hyperlipidemia.

Synthesis and biological evaluation of a series of liver-selective phosphonic acid thyroid hormone receptor agonists and their prodrugs.

Phosphonic acid (PA) thyroid hormone receptor (TR) agonists were synthesized to exploit the poor distribution of PA-based drugs to extrahepatic tissues and thereby to improve the therapeutic index. Nine PAs showed excellent TR binding affinities (TRbeta(1), K(i) < 10 nM), and most of them demonstrated significant cholesterol lowering effects in a cholesterol-fed rat (CFR) model. Unlike the corresponding carboxylic acid analogue and T(3), PA 22c demonstrated liver-selective effects by inducing maximal mitochondrial glycerol-3-phosphate dehydrogenase activity in rat liver while having no effect in the heart. Because of the low oral bioavailability of PA 22c, a series of prodrugs was synthesized and screened for oral efficacy in the CFR assay. The liver-activated cyclic 1-(3-chlorophenyl)-1,3-propanyl prodrug (MB07811) showed potent lipid lowering activity in the CFR (ED(50) 0.4 mg/kg, po) and good oral bioavailability (40%, rat) and was selected for development for the treatment of hypercholesterolemia.

Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index.

Despite efforts spanning four decades, the therapeutic potential of thyroid hormone receptor (TR) agonists as lipid-lowering and anti-obesity agents remains largely unexplored in humans because of dose-limiting cardiac effects and effects on the thyroid hormone axis (THA), muscle metabolism, and bone turnover. TR agonists selective for the TRbeta isoform exhibit modest cardiac sparing in rodents and primates but are unable to lower lipids without inducing TRbeta-mediated suppression of the THA. Herein, we describe a cytochrome P450-activated prodrug of a phosphonate-containing TR agonist that exhibits increased TR activation in the liver relative to extrahepatic tissues and an improved therapeutic index. Pharmacokinetic studies in rats demonstrated that the prodrug (2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811) undergoes first-pass hepatic extraction and that cleavage of the prodrug generates the negatively charged TR agonist (3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methylphosphonic acid (MB07344), which distributes poorly into most tissues and is rapidly eliminated in the bile. Enhanced liver targeting was further demonstrated by comparing the effects of MB07811 with 3,5,3'-triiodo-l-thyronine (T(3)) and a non-liver-targeted TR agonist, 3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid (KB-141) on the expression of TR agonist-responsive genes in the liver and six extrahepatic tissues. The pharmacologic effects of liver targeting were evident in the normal rat, where MB07811 exhibited increased cardiac sparing, and in the diet-induced obese mouse, where, unlike KB-141, MB07811 reduced cholesterol and both serum and hepatic triglycerides at doses devoid of effects on body weight, glycemia, and the THA. These results indicate that targeting TR agonists to the liver has the potential to lower both cholesterol and triglyceride levels with an acceptable safety profile.

Expression of E-cadherin and other paracellular junction genes is decreased in iron-loaded hepatocytes.

Iron overload in the liver may occur in the clinical conditions hemochromatosis and transfusion-dependent thalassemia or by long-term consumption of large amounts of dietary iron. As iron concentrations increase in the liver, cirrhosis develops, and subsequently the normal architecture of the liver deteriorates. The underlying mechanisms whereby iron loading of hepatocytes leads to the pathology of the liver are not understood. Similarly, a direct relationship between the expression levels of paracellular junction genes and altered hepatocellular physiology has been reported; however, no relationship has been identified between iron loading and the expression of paracellular junction genes. Here, we report that the expression of numerous paracellular junction genes was decreased in iron-loaded hepatocytes, leading to increased cellular permeability, increased baculovirus-mediated gene transfer, and decreased gap junction communication. Iron loading of hepatocytes resulted in decreased E-cadherin promoter activity and subsequently decreased E-cadherin mRNA and protein expression. The data presented in this study describe a clear relationship between iron overload and decreased expression of paracellular junction genes in hepatic cells of rat and human origin.

Comparison of anti-hepatitis B virus activities of lamivudine and clevudine by a quantitative assay.

In this study, we used a quantitative assay to measure the concentration-dependent effects of antivirals on extracellular hepatitis B virus (HBV) DNA as well as on different cytoplasmic and nuclear forms of HBV DNA that participate in HBV replication. HBV recombinant baculovirus, which efficiently delivers the HBV genome to HepG2 cells, was used for this study because (i) antivirals can be administered prior to initiation of HBV infection or after HBV infection and (ii) sufficiently high HBV replication levels are achieved that HBV covalently closed circular (CCC) DNA can be easily detected and individual HBV DNA species can be quantitatively analyzed separately from total HBV DNA. The results showed that the levels of HBV replicative intermediate and extracellular DNA decreased in a concentration-dependent fashion following antiviral treatment. The 50% effective concentration (EC(50)) and EC(90) values and the Hill slopes differed for the different HBV DNA species analyzed. The data clearly indicated that (i) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (ii) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form. This report presents the first in vitro comparison of the effects of two antivirals administered prior to initiation of HBV infection and the first thorough in vitro quantitative study of concentration-dependent antiviral effects on HBV CCC DNA.