Determination of chiglitazar, a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist, in human plasma by liquid chromatography-tandem mass spectrometry

Chiglitazar is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist. A LC-MS/MS method for the determination of chiglitazar was developed and validated. The assay used 0.2 mL of plasma. 90% acetonitrile containing internal standard was used for protein precipitation. The mobile phase contained 70/30 (v/v) of methanol and water at a flow rate of 0.25 mL/min. Detection was by negative ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 2 to 1500 ng/mL, was fitted to a 1/x weighted quadratic regression model. The validation results demonstrated that the method was sensitive, rapid, selective and robust and provided satisfactory precision and accuracy. The method has been successfully used for the analysis of clinical samples in pharmacokinetic studies of chiglitazar.     

Design and synthesis of subtype- and species-selective peroxisome proliferator-activated receptor (PPAR) alpha ligands

The molecular pharmacological discovery of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha) as the master regulator of lipid and lipoprotein homeostasis, and the rapid development of a parallel screening approach to evaluate activity towards other PPAR subtypes (PPAR delta, and PPAR gamma) have provided an opportunity to develop novel PPAR alpha-selective, PPAR alpha/gamma dual, and PPAR pan agonists. This review focuses on the molecular pharmacology of PPAR alpha, and summarizes our current design, synthesis, and evaluation of subtype-selective PPAR alpha agonists. The species selectivity of several classes of PPAR alpha selective agonists in response to in vitro PPAR alpha transactivation activity is also reported. These studies should help us to understand the structure-activity relationships and the mode of interaction between ligands and PPAR alpha, and also help to create novel therapeutic choices for the treatment of metabolic disorders.     

Biochemical and morphological effects of K-111, a peroxisome proliferator-activated receptor (PPAR)alpha activator, in non-human primates

K-111 has been characterized as a potent peroxisome proliferator-activated receptor (PPAR)alpha activator. Antidiabetic potency and amelioration of disturbed lipid metabolism were demonstrated in rodents, which were accompanied by elevations of peroxisomal enzymes and liver weight. To examine the possible therapeutic application of K-111 we have now assessed its efficacy in non-human primates with high transferability to humans. For this purpose obese, hypertriglyceridaemic, hyperinsulinaemic prediabetic rhesus monkeys were dosed sequentially with 0, 1, 3 and 10mg/kg per day orally over a period of 4 weeks each. In addition, the effect of K-111 on the peroxisome compartment was analyzed in cynomolgus monkeys using liver samples obtained following a 13-week oral toxicity study. In prediabetic monkeys, the reduction of hyperinsulinaemia and improvement of insulin-stimulated glucose uptake rate indicated amelioration of insulin resistance. These effects were nearly maximal at a dose of 3mg/kg per day, while triglycerides and body weight were lowered significantly in a dose-dependent manner. This reduction of body weight contrasts sharply with the adipogenic response observed with thiazolidinediones, another family of insulin-sensitizing agents. In young cynomolgus monkeys at a dosage of 5mg/kg per day and more, K-111 induced an up to three-fold increase in lipid beta-oxidation enzymes with an 1.5- to 2-fold increase in peroxisome volume density. This moderate increase in peroxisomal activity by K-111 in monkeys is consistent with its role as an PPARalpha activator and corresponds to the observations with fibrates in other low responder mammalian species. The increase in beta-oxidation may explain, at least in part, the lipid modulating effect as well as the antidiabetic potency of K-111. This pharmacological profile makes K-111 a highly promising drug candidate for clinical applications in the treatment of type 2 diabetes, dyslipidaemia, obesity and the metabolic syndrome.     

WY-14 643, a selective PPAR{alpha} agonist, induces proinflammatory and proangiogenic responses in human ocular cells

Peroxisome proliferator-activated receptor α (PPARα) agonism in ocular inflammation has not been thoroughly investigated. The objective of this investigation was to determine the effect of WY-14 643, a selective PPARα agonist, on inflammatory cytokine release in human ocular cells. Stimulation of primary human corneal epithelial cells, keratocytes, and retinal endothelial cells with 1 to 10 ng/mL interleukin 1β (IL-1β) resulted in a significant increase in numerous inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α (TNF-α); and dexamethasone was able to significantly inhibit these effects. However, WY-14 643 did not effectively block IL-1β-induced cytokine release in ocular cells; rather, significant increases in IL-1β-induced inflammatory cytokines were observed in these cells but not in aortic smooth muscle cells. WY-14 643 also significantly upregulated vascular endothelial growth factor (VEGF) expression in corneal epithelial cells and keratocytes. These studies demonstrate for the first time that PPARα agonism may be proinflammatory and proangiogenic in a variety of ocular cells and suggest that therapeutic applications of such agents in ophthalmology may be limited.     

Highly sensitive upregulation of apolipoprotein A-IV by peroxisome proliferator-activated receptor α (PPARα) agonist in human hepatoma cells

Peroxisome proliferator-activated receptor α (PPARα) is a key regulator in hepatic lipid metabolism and a potential therapeutic target for dyslipidemia. However, in humans hepatic PPARα-regulated genes remain unclear. To investigate the effect of PPARα agonism on mRNA expressions of lipid metabolism-related genes in human livers, a potent PPARα agonist, KRP-101 (KRP), was used to treat the human hepatoma cell line, HepaRG cells. KRP did not affect AOX or L-PBE, which are involved in peroxisomal β-oxidation. KRP increased L-FABP, CPT1A, VLCAD, and PDK4, which are involved in lipid transport or oxidation. However, the EC₅₀ values (114–2500 nM) were >10-fold weaker than the EC₅₀ value (10.9 nM) for human PPARα in a transactivation assay. To search for more sensitive genes, we determined the mRNA levels of apolipoproteins, apoA-I, apoA-II, apoA-IV, apoA-V, and apoC-III. KRP had no or little effect on apoA-I, apoC-III, and apoA-II. Interestingly, KRP increased apoA-IV (EC₅₀, 0.99 nM) and apoA-V (EC₅₀, 0.29 nM) with high sensitivity. We identified apoA-IV as a PPARα-upregulated gene in a study using PPARα siRNA. Moreover, when administered orally to dogs, KRP decreased the serum triglyceride level and increased the serum apoA-IV level in a dose-dependent manner. These findings suggest that apoA-IV, newly identified as a highly sensitive PPARα-regulated gene in human livers, may be one of the mechanisms underlying PPARα agonist-induced triglyceride decrease and HDL elevation.     

Peroxisome proliferator-activated receptor delta agonists attenuated the C-reactive protein-induced pro-inflammation in cardiomyocytes and H9c2 cardiomyoblasts

C-reactive protein (CRP) has emerged as a new marker for cardiovascular diseases. Activation of peroxisome proliferator-activated receptor δ (PPARδ) plays beneficial roles in cardiac disorders. However, the relationship between CRP and PPARδ in cardiac cells remains unclear. This study focused on the underlying molecular mechanisms of CRP and PPARδagonists. Cardiomyocytes and cardiomyoblast cell line (H9c2) were used in different groups: Untreated; 15 μg/ml CRP with or without 1 μM PPARδ agonists (L-165041). CRP increased PPARδ and interleukin-6 expression in cardiomyocytes and H9c2 cardiomyoblasts. NF-κB inducing kinase (NIK) and NF-κB pathway also activated by CRP stimulation. These changes could be inhibited by L-165041 through p38MAPK and c-JNK pathways. However, transfection with siRNA of CD32 CRP receptor did not decrease CRP signaling or reverse the effects of L-165041 in CRP-treated cardiomyocytes and H9c2. Pretreatment with L-165041 attenuated apoptosis induced by hypoxia with or without CRP in H9c2 cardiomyoblasts. CRP up-regulated PPARδ expression in cardiomyocytes and H9c2. L-165041 attenuated CRP-induced pro-inflammatory signaling through p38MAPK and c-JNK in H9c2 cardiomyoblasts. However, PPARδ activation attenuated CRP-induced NF-κB pathway may be independent of CD32. These results may provide new evidence of PPARδ beneficial effects for inflammatory cardiomyopathy.     

Molecular modelling of the peroxisome proliferator-activated receptor alpha (PPAR alpha) from human, rat and mouse, based on homology with the human PPAR gamma crystal structure.

The generation of homology models of human, rat and mouse peroxisome proliferator-activated receptor alpha (PPAR alpha) are reported, based on the recently published crystal structure of the human PPAR gamma ligand-binding domain (LBD) with bound ligand, rosiglitazone. It is found that a template of peroxisome proliferating fibrate drugs and related compounds can fit within the putative ligand-binding site of rat PPAR alpha, via contacts with amino acid residues which are consistent with their biological potency for peroxisome proliferation, site-directed mutagenesis experiments and with quantitative structure-activity relationship (QSAR) analysis studies. The experimental binding affinity of leukotriene B(4) (LTB(4)) for the mouse PPAR alpha agrees closely with the calculated value based on the modelled interactions, whereas selective PPAR alpha ligands such as clofibric acid are able to fit the human PPAR alpha binding site in agreement with reported site-directed mutagenesis information.     

Antidiabetic and hypolipidemic effects of a novel dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, E3030, in db/db mice and beagle dogs

We investigated the antidiabetic effects of E3030, which is a potent dual activator of peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma, in an animal model of diabetes, C57BL/KsJ-db/db mice (db/db mice), and the lipidemic effects of E3030 in beagle dogs, whose PPARalpha and PPARgamma transactivation responses to E3030 were similar to those of humans. E3030 activated human PPARalpha, mouse PPARalpha, dog PPARalpha, human PPARgamma, mouse PPARgamma, and dog PPARgamma with EC(50) values of 65, 920, 87, 34, 73, and 34 nM, respectively, in the chimeric GAL4-PPAR receptor transactivation reporter assay. In db/db mice orally administered E3030 decreased blood glucose, triglyceride (TG), non-esterified fatty acids (NEFA), and insulin levels and increased blood adiponectin levels during a 14-day experimental period. Significant effects on blood glucose and adiponectin levels were observed at a dose of 3 mg/kg or greater. Furthermore, significant effects on blood TG, NEFA, and insulin levels were observed at doses of 1 mg/kg or more. An oral glucose tolerance test (OGTT) performed on Day 15 showed that E3030 at 3 mg/kg improved glucose tolerance in this model. Fourteen days of oral treatment with E3030 at a dose of 0.03 mg/kg or greater showed remarkable TG- and non high-density lipoprotein (non-HDL) cholesterol-lowering effects in beagle dogs. These results were similar to those observed for the PPARalpha agonist fenofibrate. E3030 also reduced apo C-III levels on Days 7 and 14, and elevated lipoprotein lipase (LPL) levels on Day 15. These results indicate that the TG- and non-HDL cholesterol-lowering actions of E3030 involve combined effects on reduction of apo C-III and elevation of LPL, resulting in increased lipolysis. The experimental results in animals suggest that E3030 has potential for use in the treatment of various aspects of metabolic dysfunction in type 2 diabetes, including dyslipidemia, hyperglycemia, hyperinsulinemia, and impaired glucose disposal.     

Review of the expression of peroxisome proliferator-activated receptors alpha (PPAR alpha), beta (PPAR beta), and gamma (PPAR gamma) in rodent and human development

The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary subtypes, PPARalpha, beta, and gamma. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis. These nuclear receptors have important roles in reproduction and development and their expression may influence the responses of an embryo exposed to PPAR agonists. PPARs are relevant to the study of the biological effects of the perfluorinated alkyl acids as these compounds, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), activate PPARalpha. Exposure of the rodent to PFOA or PFOS during gestation results in neonatal deaths, developmental delay and growth deficits. Studies in PPARalpha knockout mice demonstrate that the developmental effects of PFOA, but not PFOS, depend on expression of PPARalpha. This review provides an overview of PPARalpha, beta, and gamma protein and mRNA expression during mouse, rat, and human development. The review presents the results from many published studies and the information is organized by organ system and collated to show patterns of expression at comparable developmental stages for human, mouse, and rat. The features of the PPAR nuclear receptor family are introduced and what is known or inferred about their roles in development is discussed relative to insights from genetically modified mice and studies in the adult.     

Peroxisome proliferator-activated receptor gamma (PPARgamma) activation and its consequences in humans

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the superfamily of nuclear receptors. It binds and is activated by natural polyunsaturated fatty acids, eicosanoids, synthetic thiazolidinediones and related analogues. Biological effects exerted by PPARγ ligands are mostly concerned with differentiation processes, sensitization to insulin and atherogenesis, and are paradigmatically ascribed to PPARγ transactivation of PPARγ-responsive genes. The PPARγ paradigm and its consequences in humans are analyzed here in terms of the tissue specificity of PPARγ, loss and gain of function mutants of PPARγ, PPARγ-responsive genes and clinical effects of PPARγ ligands. Differentiation, as well as some of the atherogenic effects induced by PPARγ ligands, does conform to the PPARγ paradigm. However, sensitization to insulin as well as some of the antiatherogenic effects of PPARγ ligands is not accounted for by PPARγ activation, thus calling for an alternative target for insulin sensitizers.     

Peroxisome proliferator-activated receptor alpha is involved in the regulation of lipid metabolism by ginseng

1. Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the expression of the key genes involved in lipid metabolism following activation of this receptor by various ligands. Ginseng, a highly valuable medicine in oriental societies, is also reported to modulate lipid metabolism, although the mechanism of its action remains unknown. In order to test our hypothesis that ginseng exerts its effects by altering PPARalpha-mediated pathways, the effects of Korean red ginseng on PPARalpha function and serum lipid profiles were investigated using in vivo and in vitro approaches. 2. In vivo administration of ginseng extract (GE) and ginsenosides (GS) not only inhibited mRNA levels of acyl-CoA oxidase, a rate-limiting enzyme for PPARalpha-mediated peroxisomal fatty acid beta-oxidation, induced by the potent PPARalpha ligand Wy14,643 in a dose- and time-dependent manner, but also inhibited the induction of PPARalpha target genes expected following treatment with Wy14,643. 3. Consistent with the in vivo data, both GE and GS caused dose-dependent decreases in the endogenous expression of a luciferase reporter gene containing the PPAR responsive element (PPRE), while GS significantly decreased the magnitude of reporter gene activation in the presence of Wy14,643. 4. Serological studies demonstrated that, compared with vehicle-treated mice, treatment with GS significantly increased serum concentrations of total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol. Compared to groups treated with Wy14,643 alone, which significantly decreased serum triglyceride and HDL cholesterol levels versus controls, coadministration of either GE or GS with Wy14,643 modestly increased serum triglycerides and HDL cholesterol. 5. These results indicate that the effects of ginseng on serum lipid profiles may be mediated by changes in the expression of PPARalpha target genes, providing the first evidence that in vivo and in vitro treatments of ginseng modulate PPARalpha action. In addition, these data suggest that ginseng can act as an inhibitor of PPARalpha function, which may have therapeutic implications.     

Relationship between peroxisome proliferator-activated receptors (PPAR alpha and PPAR gamma) and endothelium-dependent relaxation in streptozotocin-induced diabetic rats

(1) The aim of the present study was to investigate the causal relationship between peroxisome proliferator-activated receptor (PPAR) and endothelium-dependent relaxation in streptozotocin (STZ)-induced diabetic rats. (2) Acetylcholine (ACh)-induced endothelium-dependent relaxation was significantly weaker in diabetic rats than in age-matched controls. The decreased relaxation in diabetes was improved by the chronic administration of bezafibrate (30 mg kg-1, p.o., 4 weeks). (3) The expressions of the mRNAs for PPARalpha and PPARgamma were significantly decreased in STZ-induced diabetic rats (compared with the controls) and this decrease was restored partially, but not completely, by the chronic administration of bezafibrate. (4) Superoxide dismutase activity in the aorta was not significantly different between diabetic rats and bezafibrate-treated diabetic rats. (5) The expression of the mRNA for the p22phox subunit of NAD(P)H oxidase was significantly higher in diabetics than in controls, but it was lower in bezafibrate-treated diabetic rats than in nontreated diabetic rats. Although the expression of the mRNA for prepro ET-1 (ppET-1) was markedly increased in diabetic rats (compared with controls), this increase was prevented to a significant extent by the chronic administration of bezafibrate. (6) These results suggest that downregulations of PPARalpha and PPARgamma may lead to an increased expression of ppET-1 mRNA in diabetic states and this increment may trigger endothelial dysfunction.     

Design, synthesis, and structure-activity relationship study of peroxisome proliferator-activated receptor (PPAR) delta-selective ligands

Improvements in our understanding of the functions of peroxisome proliferator-activated receptor (PPAR) subtypes as master regulators of many biological functions have made it possible to develop novel PPAR ligands with characteristic subtype selectivity as biochemical tools and/or candidate drugs for the treatment of PPAR-dependent diseases such as metabolic syndrome, which includes type II diabetes, dyslipidemia, obesity, hypertension, and inflammation. Based on the findings that the glitazone-class antidiabetic agents, and fibrate-class antidyslipidemic agents are ligands of PPARgamma and PPARalpha respectively, much research interest has been focused on these two subtypes as therapeutic targets for the treatment of type II diabetes and dyslipidemia. In contrast, research interest in PPARdelta has been limited. However, since 2001, the availability of PPARdelta knockout animals and selective ligands has led to the uncovering of possible roles of PPARdelta in fatty acid metabolism, insulin resistance, reverse cholesterol transport, inflammation, and so on. It has become clear that ligands able to modulate PPARdelta-mediated pathways are candidates for the treatment of altered metabolic function. This review focuses on recent medicinal chemical studies to identify PPARdelta-selective agonists.     

Highly sensitive upregulation of apolipoprotein A-IV by peroxisome proliferator-activated receptor alpha (PPARalpha) agonist in human hepatoma cells

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator in hepatic lipid metabolism and a potential therapeutic target for dyslipidemia. However, in humans hepatic PPARalpha-regulated genes remain unclear. To investigate the effect of PPARalpha agonism on mRNA expressions of lipid metabolism-related genes in human livers, a potent PPARalpha agonist, KRP-101 (KRP), was used to treat the human hepatoma cell line, HepaRG cells. KRP did not affect AOX or L-PBE, which are involved in peroxisomal beta-oxidation. KRP increased L-FABP, CPT1A, VLCAD, and PDK4, which are involved in lipid transport or oxidation. However, the EC(50) values (114-2500 nM) were >10-fold weaker than the EC(50) value (10.9 nM) for human PPARalpha in a transactivation assay. To search for more sensitive genes, we determined the mRNA levels of apolipoproteins, apoA-I, apoA-II, apoA-IV, apoA-V, and apoC-III. KRP had no or little effect on apoA-I, apoC-III, and apoA-II. Interestingly, KRP increased apoA-IV (EC(50), 0.99 nM) and apoA-V (EC(50), 0.29 nM) with high sensitivity. We identified apoA-IV as a PPARalpha-upregulated gene in a study using PPARalpha siRNA. Moreover, when administered orally to dogs, KRP decreased the serum triglyceride level and increased the serum apoA-IV level in a dose-dependent manner. These findings suggest that apoA-IV, newly identified as a highly sensitive PPARalpha-regulated gene in human livers, may be one of the mechanisms underlying PPARalpha agonist-induced triglyceride decrease and HDL elevation.