A Phase 3, Randomized, Controlled Trial of Resmetirom in NASH with Liver Fibrosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive liver disease with no approved treatment. Resmetirom is an oral, liver-directed, thyroid hormone receptor beta-selective agonist in development for the treatment of NASH with liver fibrosis. METHODS: We are conducting an ongoing phase 3 trial involving adults with biopsy-confirmed NASH and a fibrosis stage of F1B, F2, or F3 (stages range from F0 [no fibrosis] to F4 [cirrhosis]). Patients were randomly assigned in a 1:1:1 ratio to receive once-daily resmetirom at a dose of 80 mg or 100 mg or placebo. The two primary end points at week 52 were NASH resolution (including a reduction in the nonalcoholic fatty liver disease [NAFLD] activity score by ≥2 points; scores range from 0 to 8, with higher scores indicating more severe disease) with no worsening of fibrosis, and an improvement (reduction) in fibrosis by at least one stage with no worsening of the NAFLD activity score. RESULTS: Overall, 966 patients formed the primary analysis population (322 in the 80-mg resmetirom group, 323 in the 100-mg resmetirom group, and 321 in the placebo group). NASH resolution with no worsening of fibrosis was achieved in 25.9% of the patients in the 80-mg resmetirom group and 29.9% of those in the 100-mg resmetirom group, as compared with 9.7% of those in the placebo group (P<0.001 for both comparisons with placebo). Fibrosis improvement by at least one stage with no worsening of the NAFLD activity score was achieved in 24.2% of the patients in the 80-mg resmetirom group and 25.9% of those in the 100-mg resmetirom group, as compared with 14.2% of those in the placebo group (P<0.001 for both comparisons with placebo). The change in low-density lipoprotein cholesterol levels from baseline to week 24 was -13.6% in the 80-mg resmetirom group and -16.3% in the 100-mg resmetirom group, as compared with 0.1% in the placebo group (P<0.001 for both comparisons with placebo). Diarrhea and nausea were more frequent with resmetirom than with placebo. The incidence of serious adverse events was similar across trial groups: 10.9% in the 80-mg resmetirom group, 12.7% in the 100-mg resmetirom group, and 11.5% in the placebo group. CONCLUSIONS: Both the 80-mg dose and the 100-mg dose of resmetirom were superior to placebo with respect to NASH resolution and improvement in liver fibrosis by at least one stage. (Funded by Madrigal Pharmaceuticals; MAESTRO-NASH ClinicalTrials.gov number, NCT03900429.).

Discovery of a novel, liver-targeted thyroid hormone receptor-ß agonist, CS271011, in the treatment of lipid metabolism disorders.

Introduction: Thyroid hormone receptor ß (THR-ß) plays a critical role in metabolism regulation and has become an attractive target for treating lipid metabolism disorders in recent years. Thus, in this study, we discovered CS271011, a novel THR-ß agonist, and assessed the safety and efficiency of CS271011 compared to MGL-3196 in vitro and in vivo. Methods: We conducted luciferase reporter gene assays to assess the activation of THR-ß and α in vitro. C57BL/6J mice were fed a high-fat diet for 12 weeks, CS271011 was administered by gavage at the dose of 1 mg/kg and 3 mg/kg, and MGL-3196 was administered at the dose of 3 mg/kg for 10 weeks. Body weight, food intake, serum and hepatic parameters, histological analysis, pharmacokinetic studies, RNA sequencing of the liver and heart, and expression of hepatic lipid-metabolic genes were determined to evaluate the safety and efficiency of CS271011. Results: Compared with MGL-3196, CS271011 showed higher THR-ß activation in vitro. In the diet-induced obesity mice model, CS271011 demonstrated favourable pharmacokinetic properties in mice and was enriched in the liver. Finally, CS271011 improved dyslipidaemia and reduced liver steatosis in the diet-induced obesity murine model. Mechanistically, CS271011 and MGL-3196 showed potent regulation of lipid metabolism-related genes. Conclusions: CS271011 is a potent and liver-targeted THR-ß agonist for treating lipid metabolism disorders.

Discovery of a Highly Selective and H435R-Sensitive Thyroid Hormone Receptor ß Agonist.

The design and development of agonists selectively targeting thyroid hormone receptor ß (TRß) and TRß mutants remain challenging tasks. In this study, we first adopted the strategy of breaking the "His-Phe switch" to solve two problems, simultaneously. A structure-based design approach was successfully utilized to obtain compound 16g, which is a potent TRß agonist (EC50: 21.0 nM, 85.0% of the maximum efficacy of 1) with outstanding selectivity for TRß over TRα and also effectively activates the TRßH435R mutant. Then, we developed a highly efficient synthetic method for 16g. Our serials of cocrystal structures revealed detailed structural mechanisms in overcoming subtype selectivity and rescuing the H435R mutation. 16g also showed excellent lipid metabolism, safety, metabolic stability, and pharmacokinetic properties. Collectively, 16g is a well-characterized selective and mutation-sensitive TRß agonist for further investigating its function in treating dyslipidemia, nonalcoholic steatohepatitis (NASH), and resistance to thyroid hormone (RTH).

TG68, a Novel Thyroid Hormone Receptor-ß Agonist for the Treatment of NAFLD.

Activation of thyroid hormone receptor ß (THRß) has shown beneficial effects on metabolic alterations, including non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effect of TG68, a novel THRß agonist, on fatty liver accumulation and liver injury in mice fed a high-fat diet (HFD). C57BL/6 mice fed HFD for 17 or 18 weeks, a time when all mice developed massive steatohepatitis, were then given TG68 at a dose of 9.35 or 2.8 mg/kg for 2 or 3 weeks, respectively. As a reference compound, the same treatment was adopted using equimolar doses of MGL-3196, a selective THRß agonist currently in clinical phase III. The results showed that treatment with TG68 led to a reduction in liver weight, hepatic steatosis, serum transaminases, and circulating triglycerides. qRT-PCR analyses demonstrated activation of THRß, as confirmed by increased mRNA levels of Deiodinase-1 and Malic enzyme-1, and changes in lipid metabolism, as revealed by increased expression of Acyl-CoA Oxidase-1 and Carnitine palmitoyltransferase-1. The present results showed that this novel THRß agonist exerts an anti-steatogenic effect coupled with amelioration of liver injury in the absence of extra-hepatic side effects, suggesting that TG68 may represent a useful tool for the treatment of NAFLD.

Syringic acid, a novel thyroid hormone receptor-ß agonist, ameliorates propylthiouracil-induced thyroid toxicity in rats.

The aim of this study was to evaluate the potential of syringic acid (SA) against propylthiouracil (PTU)-induced hypothyroidism in rats. SA at a prestandardized dose, 50 mg/kg/day, was orally administered to PTU-induced hypothyroid rats for 30 days, and alterations in the levels of serum triiodothyronine (T3 ), thyroxine (T4 ), thyrotropin (TSH), alanine transaminase (ALT), and aspartate transaminase (AST); tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); total cholesterol (CHOL) and triglycerides (TG); hepatic lipid peroxidation (LPO) and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and glutathione content), as well as histological changes in liver and thyroid were examined. The molecular interactions of the ligand, SA, with thyroid-related protein targets, such as human thyroid hormone receptor ß (hTRß), and thyroid peroxidase (TPO) protein, were studied using molecular docking. Whereas in hypothyroid animals, T4 , T3 , and antioxidants were decreased, there was an increase in TSH, TNF-α, IL-6, ALT, AST, and hepatic LPO; administration of SA in PTU-induced animals reversed all these indices to near normal levels. SA also improved the histological features of liver and thyroid gland. Our study clearly demonstrates SA as a novel thyroid agonist for augmenting the thyroid functions in rats. Molecular docking analysis reveals that SA possesses good binding affinity toward both the targets, hTRß and TPO. Through this approach, for the first time we provide the evidence for SA as a novel thyroid agonist and suggest a receptor-mediated mechanism for its thyroid stimulatory potential.

Effects of Resmetirom on Noninvasive Endpoints in a 36-Week Phase 2 Active Treatment Extension Study in Patients With NASH.

Resmetirom (MGL-3196), a selective thyroid hormone receptor-ß agonist, was evaluated in a 36-week paired liver biopsy study (NCT02912260) in adults with biopsy-confirmed nonalcoholic steatohepatitis (NASH). The primary endpoint was relative liver fat reduction as assessed by MRI-proton density fat fraction (MRI-PDFF), and secondary endpoints included histopathology. Subsequently, a 36-week active treatment open-label extension (OLE) study was conducted in 31 consenting patients (including 14 former placebo patients) with persistently mild to markedly elevated liver enzymes at the end of the main study. In patients treated with resmetirom (80 or 100 mg orally per day), MRI-PDFF reduction at OLE week 36 was -11.1% (1.5%) mean reduction (standard error [SE]; P < 0.0001) and -52.3% (4.4%) mean relative reduction, P < 0.0001. Low-density lipoprotein (LDL) cholesterol (-26.1% [4.5%], P < 0.0001), apolipoprotein B (-23.8% [3.0%], P < 0.0001), and triglycerides (-19.6% [5.4%], P = 0.0012; -46.1 [14.5] mg/dL, P = 0.0031) were reduced from baseline. Markers of fibrosis were reduced, including liver stiffness assessed by transient elastography (-2.1 [0.8] mean kilopascals [SE], P = 0.015) and N-terminal type III collagen pro-peptide (PRO-C3) (-9.8 [2.3] ng/mL, P = 0.0004 (baseline ≥ 10 ng/mL). In the main and OLE studies, PRO-C3/C3M (matrix metalloproteinase-degraded C3), a marker of net fibrosis formation, was reduced in resmetirom-treated patients (-0.76 [-1.27, -0.24], P = 0.0044 and -0.68, P < 0.0001, respectively). Resmetirom was well tolerated, with few, nonserious adverse events. Conclusion: The results of this 36-week OLE study support the efficacy and safety of resmetirom at daily doses of 80 mg and 100 mg, used in the ongoing phase 3 NASH study, MAESTRO-NASH (NCT03900429). The OLE study demonstrates a potential for noninvasive assessments to monitor the response to resmetirom from an individual patient with NASH.

Regulation of gene transcription by thyroid hormone receptor ß agonists in clinical development for the treatment of non-alcoholic steatohepatitis (NASH).

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRß agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRß-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRß and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolism in vitro using human hepatic cells and in vivo using a rat model. Treatment with GC-1 upregulated the transcription of CPT1A in the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely, ANGPTL4 and DIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liver Dio1 and Me1 RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRß agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

Selective thyroid hormone receptor ß agonists with oxadiazolone acid isosteres.

Use of the oxadiazolone acid isostere in triiodothyronine analogs yielded potent and selective agonists for the thyroid hormone receptor ß. Selected examples showed good in-vivo efficacy in a rat hypercholesterolemic model. One compound was further profiled in a diet-induced mouse model of nonalcoholic steatohepatitis (NASH) and showed robust target engagement and significant histological improvements in both liver steatosis and fibrosis.

Potential role of two novel agonists of thyroid hormone receptor-ß on liver regeneration.

OBJECTIVES: Although the hepatomitogenic activity of triiodothyronine (T3) is well established, the wide range of harmful effects exerted by this hormone precludes its use in liver regenerative therapy. Selective agonists of the beta isoform of thyroid hormone receptor (TRß) do not exhibit T3-induced cardiotoxicity and show a good safety profile in patients with NASH. The aim of this study was to investigate whether two novel TRß agonists, the prodrug TG68 and the active compound IS25 could stimulate hepatocyte proliferation without T3/TRα-dependent side effects. METHODS: Rats were treated with three different doses (12.5, 25 and 50 µg/100 g body weight) for one week. Hepatocyte proliferation, liver injury and serum biochemical parameters were measured by immunohistochemistry, qRT-PCR and Western blot. RESULTS: Both drugs increased hepatocyte proliferation as assessed by bromodeoxyuridine incorporation (from 14% to 28% vs 5% of controls) and mitotic activity. Enhanced proliferation occurred in the absence of significant signs of liver injury as shown by lack of increased serum transaminase levels or of apoptosis. No cardiac or renal hypertrophy typically associated with treatment with T3 was observed. Importantly, no proliferation of pancreatic acinar cells, such as that seen after administration of T3 or the TRß agonist GC1 was detected following either TG68 or IS25, demonstrating the hepato-specificity of these novel TRß agonists. CONCLUSIONS: The present study shows that TG68 and IS25 induce massive hepatocyte proliferation without overt toxicity. Hence, these agents may have a significant clinical application for regenerative therapies in liver transplantation or other surgical settings.

Design, synthesis and biological evaluation of novel TRß selective agonists sustained by ADME-toxicity analysis.

Although triiodothyronine (T3) induces several beneficial effects on lipid metabolism, its use is hampered by toxic side-effects, such as tachycardia, arrhythmia, heart failure, bone and muscle catabolism and mood disturbances. Since the α isoform of thyroid hormone receptors (TRs) is the main cause of T3-related harmful effects, several efforts have been made to develop selective agonists of the ß isoform that could induce some beneficial effects (i.e. lowering triglyceride and cholesterol levels reducing obesity and improving metabolic syndrome), while overcoming most of the adverse T3-dependent side effects. Herein, we describe the drug discovery process sustained by ADME-Toxicity analysis that led us to identify novel agonists with selectivity for the isoform TRß and an acceptable off-target and absorption, distribution metabolism, excretion and toxicity (ADME-Tox) profile. Within the small series of compounds synthesized, derivatives 1 and 3, emerge from this analysis as "potentially safe" to be engaged in preclinical studies. In in vitro investigation proved that both compounds were able to reduce lipid accumulation in HepG2 and promote lipolysis with comparable effects to those elicited by T3, used as reference drug. Moreover, a preliminary in vivo study confirmed the apparent lack of toxicity, thus suggesting compounds 1 and 3 as new potential TRß-selective thyromimetics.

Evaluation of a panel of in vitro methods for assessing thyroid receptor ß and transthyretin transporter disrupting activities.

We developed a thyroid testing panel to assess endocrine disrupting chemicals (EDCs) capacities to bind either the thyroid receptor ß (TRß) or the thyroid hormones transporter transthyretin (TTR). We first stably transfected a human U2OS cell line with TRß and a luciferase reporter construct to develop the TRß CALUX® reporter gene assay to assess chemicals' potential to interact with TRß. Secondly, we combined a TTR-binding assay with the TRß CALUX (TTR-TRß CALUX) and optimized the system to evaluate the competitive properties of EDCs towards T4 for TTR binding. Both systems were evaluated with a range of known thyroid-disrupting compounds. The agonistic/antagonistic TRß CALUX successfully predicted 9/9 and 9/12 test compounds, respectively. The TTR-TRß CALUX predicted 9/9 compounds and demonstrated competitive activities when analyzing waste water samples. We concluded that the proposed test battery is a promising screening method able to efficiently generate data on thyroid hormone interferences by chemicals.

Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis, inflammation, hepatocellular injury, and progressive liver fibrosis. Resmetirom (MGL-3196) is a liver-directed, orally active, selective thyroid hormone receptor-ß agonist designed to improve NASH by increasing hepatic fat metabolism and reducing lipotoxicity. We aimed to assess the safety and efficacy of resmetirom in patients with NASH. METHODS: MGL-3196-05 was a 36-week randomised, double-blind, placebo-controlled study at 25 centres in the USA. Adults with biopsy confirmed NASH (fibrosis stages 1-3) and hepatic fat fraction of at least 10% at baseline when assessed by MRI-proton density fat fraction (MRI-PDFF) were eligible. Patients were randomly assigned 2:1 by a computer-based system to receive resmetirom 80 mg or matching placebo, orally once a day. Serial hepatic fat measurements were obtained at weeks 12 and 36, and a second liver biopsy was obtained at week 36. The primary endpoint was relative change in MRI-PDFF assessed hepatic fat compared with placebo at week 12 in patients who had both a baseline and week 12 MRI-PDFF. This trial is registered with ClinicalTrials.gov, number NCT02912260. FINDINGS: 348 patients were screened and 84 were randomly assigned to resmetirom and 41 to placebo at 18 sites in the USA. Resmetirom-treated patients (n=78) showed a relative reduction of hepatic fat compared with placebo (n=38) at week 12 (-32·9% resmetirom vs -10·4% placebo; least squares mean difference -22·5%, 95% CI -32·9 to -12·2; p<0·0001) and week 36 (-37·3% resmetirom [n=74] vs -8·5 placebo [n=34]; -28·8%, -42·0 to -15·7; p<0·0001). Adverse events were mostly mild or moderate and were balanced between groups, except for a higher incidence of transient mild diarrhoea and nausea with resmetirom. INTERPRETATION: Resmetirom treatment resulted in significant reduction in hepatic fat after 12 weeks and 36 weeks of treatment in patients with NASH. Further studies of resmetirom will allow assessment of safety and effectiveness of resmetirom in a larger number of patients with NASH with the possibility of documenting associations between histological effects and changes in non-invasive markers and imaging. FUNDING: Madrigal Pharmaceuticals.

A Liver-Specific Thyromimetic, VK2809, Decreases Hepatosteatosis in Glycogen Storage Disease Type Ia.

Background: Glycogen storage disease type Ia (GSD Ia), also known as von Gierke disease, is the most common glycogen storage disorder. It is caused by the deficiency of glucose-6-phosphatase, the enzyme that catalyzes the final step of gluconeogenesis and glycogenolysis. The accumulation of glucose-6-phosphate leads to increased glycogen and triglyceride levels in the liver. Patients with GSD Ia can develop steatohepatitis, cirrhosis, and increased risk for hepatocellular adenomas and carcinomas. We previously showed that animal models of GSD Ia had defective autophagy and dysfunctional mitochondria. In this study, we examined the effect of VK2809, a liver-specific thyroid hormone receptor ß agonist, on hepatic steatosis, autophagy, and mitochondrial biogenesis in a mouse model of GSD Ia. Methods:G6pc-/--deficient (GSD Ia) mice were treated with VK2809 or vehicle control by daily intraperitoneal injection for four days. The hepatic triglyceride and glycogen were determined by biochemical assays. Autophagy and mitochondrial biogenesis were measured by Western blotting for key autophagy and mitochondrial markers. Results: VK2809 treatment decreased hepatic mass and triglyceride content in GSD Ia mice. VK2809 stimulated hepatic autophagic flux as evidenced by increased microtubule-associated protein light chain 3-II (LC3B-II), decreased p62 protein levels, activation of AMP-activated protein kinase (AMPK), inhibition of the mammalian target of rapamycin (mTOR) signaling, enhancement of protein levels of ATG5-ATG12, and increased lysosomal protein expression. VK2809 also increased the expression of carnitine palmitoyltransferase 1a (CPT1α) and fibroblast growth factor 21 (FGF21), as well as mitochondrial biogenesis to promote mitochondrial ß-oxidation. Conclusions: In summary, VK2809 treatment decreased hepatic triglyceride levels in GSD Ia mice through its simultaneous restoration of autophagy, mitochondrial biogenesis, and ß-oxidation of fatty acids. Liver-specific thyromimetics represent a potential therapy for hepatosteatosis in GSD Ia as well as nonalcoholic fatty liver disease.

Potential Role of Thyroid Receptor ß Agonists in the Treatment of Hyperlipidemia.

Thyroid hormones have important effects on cellular development, growth, and metabolism and are necessary for the healthy function of almost all tissues. Hyperthyroid patients with excess thyroid hormone levels experience tachycardia, fatigue, muscle wasting, and osteoporosis. However, although high thyroid hormone levels have adverse effects, efforts have been made to harness the beneficial effects, such as reduced serum low-density lipoprotein (LDL) cholesterol levels, elevated basal metabolic rate, and weight loss. Thyroid hormones interact with nuclear thyroid hormone receptors (TRs), and cholesterol levels are reduced through TRß, whereas extrahepatic adverse actions are primarily connected to TRα. Thus, to develop a useful compound for clinical use, efforts have been focusing on developing compounds with isomer-specific functions based on the structure of thyroid hormones, i.e., thyromimetics that are liver and/or TRß specific. In this short review, we discuss the development of the early thyromimetics that enabled, through modern molecular techniques, the progress towards improved design of TRß-selective thyromimetics. We also address the early promise shown in human clinical trials and the current status of these drugs and other emerging compounds.

GC-1: A Thyromimetic With Multiple Therapeutic Applications in Liver Disease.

Thyroid hormones (THs), namely, 3,5,3'-triiodo-l-thyronine (T3) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine or T4), influence a variety of physiological processes that have important implications in fetal development, metabolism, cell growth, and proliferation. While THs elicit several beneficial effects on lipid metabolism and improve myocardial contractility, these therapeutically desirable effects are associated to a thyrotoxic state that severely limits the possible use of THs as therapeutic agents. Therefore, several efforts have been made to develop T3 analogs that could retain the beneficial actions (triglyceride, cholesterol, obesity, and body mass lowering) without the adverse TH-dependent side effects. This goal was achieved by the synthesis of TRß-selective agonists. In this review, we summarize the current knowledge on the effects of one of the best characterized TH analogs, the TRß1-selective thyromimetic, GC-1. In particular, we review some of the effects of GC-1 on different liver disorders, with reference to its possible clinical application. A brief comment on the possible therapeutic use of GC-1 in extrahepatic disorders is also included.

The identification of the metabolites of chlorothalonil in zebrafish (Danio rerio) and their embryo toxicity and endocrine effects at environmentally relevant levels.

Chlorothalonil is a broad spectrum fungicide with high annual production and environmental contamination. Despite its high consumption, studies regarding the potential ecological risks of chlorothalonil, especially its metabolites, to aquatic organisms are still limited. In this study, we selected the zebrafish (Danio rerio) as the in vivo model and first identified the metabolite (4-hydroxychlorothalonil) of chlorothalonil in zebrafish by tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF). Then, the in vivo and in vitro models were applied to comprehensively estimate the embryo toxicity and potential endocrine effect of chlorothalonil and 4-hydroxychlorothalonil. The data from zebrafish embryo toxicity showed that the lowest observed effect concentrations of both chlorothalonil and 4-hydroxychlorothalonil were 50 µg/L after 96 h of exposure. The mortality rate of the 4-hydroxychlorothalonil was 2.6-fold higher than that of the parent compound at the concentration of 50 µg/L. Dual-luciferase reporter gene assays indicated that both chlorothalonil and 4-hydroxychlorothalonil exerted estrogen receptor α (ERα) agonist activity with REC20 values of 2.4 × 10-8 M and 3.6 × 10-8 M, respectively. However, only 4-hydroxychlorothalonil exhibited both thyroid receptor ß (TRß) agonistic and antagonistic activities. Lastly, we employed molecular docking to predict the binding affinity of chlorothalonil and 4-hydroxychlorothalonil with ERα or TRß. The results revealed that the potential endocrine effect of chlorothalonil and 4-hydroxychlorothaloni might be attributed to the different binding affinities with the receptors. In conclusion, our studies revealed that 4-hydroxychlorothalonil exhibited potent endocrine-disrupting effects compared to its parent compound, chlorothalonil. The results provided here remind us that the assessment of the potential ecological and health risks of the metabolites of fungicides in addition to their parent compounds should arouse great concerns.

Sustained zero-order delivery of GC-1 from a nanochannel membrane device alleviates metabolic syndrome.

BACKGROUND/OBJECTIVES: Our objective was to assess the sustained, low-dose and constant administration of the thyroid receptor-ß (TRß)-selective agonist GC-1 (sobetirome) from a novel nanochannel membrane device (NMD) for drug delivery. As it known to speed up metabolism, accomplish weight loss, improve cholesterol levels and possess anti-diabetic effects, GC-1 was steadily administered by our NMD, consisting of an implantable nanochannel membrane, as an alternative to conventional daily administration, which is subject to compliance issues in clinical settings. SUBJECTS/METHODS: Diet-induced obese C57BL/J6 male mice were fed a very high-fat diet (VHFD) and received NMD implants subcutaneously. Ten mice per group received capsules containing GC-1 or phosphate-buffered saline (control). Weight, lean and fat mass, as well as cholesterol, triglycerides, insulin and glucose, were monitored for 24 days. After treatment, plasma levels of thyroid-stimulating hormone (TSH) and thyroxine were compared. mRNA levels of a panel of thermogenic markers were examined using real-time PCR in white adipose tissue (WAT) and brown adipose tissue (BAT). Adipose tissue, liver and local inflammatory response to the implant were examined histologically. Pancreatic islet number and ß-cell area were assessed. RESULTS: GC-1 released from the NMD reversed VHFD-induced obesity and normalized serum cholesterol and glycemia. Significant reductions in body weight and fat mass were observed within 10 days, whereas reductions in serum cholesterol and glucose levels were seen within 7 days. The significant decrease in TSH was consistent with TRß selectivity for GC-1. Levels of transcript for Ucp1 and thermogenic genes PGC1a, Cidea, Dio2 and Cox5a showed significant upregulation in WAT in NMD-GC-1-treated mice, but decreased in BAT. Although mice treated by NMD-GC-1 showed a similar number of pancreatic islets, they exhibited significant increase in ß-cell area. CONCLUSIONS: Our data demonstrate that the NMD implant achieves steady administration of GC-1, offering an effective and tightly controlled molecular delivery system for treatment of obesity and metabolic disease, thereby addressing compliance.

Thyroid Hormone Receptor ß Agonist Induces ß-Catenin-Dependent Hepatocyte Proliferation in Mice: Implications in Hepatic Regeneration.

Triiodothyronine (T3) induces hepatocyte proliferation in rodents. Recent work has shown molecular mechanism for T3's mitogenic effect to be through activation of ß-catenin signaling. Since systemic side effects of T3 may preclude its clinical use, and hepatocytes mostly express T3 hormone receptor ß (TRß), we investigated if selective TRß agonists like GC-1 may also have ß-catenin-dependent hepatocyte mitogenic effects. Here we studied the effect of GC-1 and T3 in conditional knockouts of various Wnt pathway components. We also assessed any regenerative advantage of T3 or GC-1 when given prior to partial hepatectomy in mice. Mice administered GC-1 showed increased pSer675-ß-catenin, cyclin D1, BrdU incorporation, and PCNA. No abnormalities in liver function tests were noted. GC-1-injected liver-specific ß-catenin knockouts (ß-catenin LKO) showed decreased proliferation when compared to wild-type littermates. To address if Wnt signaling was required for T3- or GC-1-mediated hepatocyte proliferation, we used LRP5-6-LKO, which lacks the two redundant Wnt coreceptors. Surprisingly, decreased hepatocyte proliferation was also evident in LRP5-6-LKO in response to T3 and GC-1, despite increased pSer675-ß-catenin. Further, increased levels of active ß-catenin (hypophosphorylated at Ser33, Ser37, and Thr41) were evident after T3 and GC-1 treatment. Finally, mice pretreated with T3 or GC-1 for 7 days followed by partial hepatectomy showed a significant increase in hepatocyte proliferation both at the time (T0) and 24 h after surgery. In conclusion, like T3, TRß-selective agonists induce hepatocyte proliferation through ß-catenin activation via both PKA- and Wnt-dependent mechanisms and confer a regenerative advantage following surgical resection. Hence, these agents may be useful regenerative therapies in liver transplantation or other surgical settings.