Search

In 1989, Pacific Northwest National Laboratory developed a program, for the U.S. Department of Energy, to involve local citizens in environmental surveillance at the Hanford Site. The Community-Operated Environmental Surveillance Program was patterned after similar community-involvement efforts at the Nevada Test Site and the Three Mile Island nuclear facility. Its purpose is to increase the flow of information to the public, thereby enhancing the public's awareness and understanding of surveillance activities. The program consists of two components: radiological air monitoring at nine offsite locations and agricultural product sampling at selected locations near the site. At each air-monitoring station, two local school teachers collect air particulate samples and operate equipment to monitor ambient radiation levels. Atmospheric tritium samples (as water vapor) are also collected at some locations. Four of the air-monitoring stations include large, colorful informational displays for public viewing. These displays provide details on station equipment, sample types, and sampling purposes. Instruments in the displays also monitor, record, and show real-time ambient radiation readings (measured with a pressurized ionization chamber) and meteorological conditions. Agricultural products, grown primarily by middle-school-aged students, are obtained from areas downwind of the site. Following analysis of these samples, environmental surveillance staff visit the schools to discuss the results with the students and their teachers. The data collected by these air and agricultural sampling efforts are summarized with other routinely collected sitewide surveillance data and reported annually in the Hanford Site environmental report. [ABSTRACT FROM AUTHOR]

The article presents a study which estimates the elimination in tissues and relative rates of quinoline uptake of the crayfish Pacifasticus leniusculus. It states that the interpolation of relative survival rates among control animals and treatments determine the lethal concentration of 50% of the population. The result of the study shows that crayfish were nearly 10 times more tolerant than rainbow trout.

The article presents a study which evaluates the potential ecological consequences from bioaccumulation of fossil fuel chemicals during complex-mixture and single-compound exposures of daphnia magna. The study were done in adult daphnids and analyzes water-soluble fraction (WSF). Results indicate that mean water concentrations of phenol expose to daphnids had minor declined from 696 µ grams per liter (g/L) at zero hours to 634 µg/L at 24 hours.

Background & Aims: Patients with primary biliary cholangitis (PBC) who have an incomplete response to ursodeoxycholic acid remain at risk of disease progression. We investigated the safety and efficacy of elafibranor, a dual PPARα/δ agonist, in patients with PBC., Methods: This 12-week, double-blind phase II trial enrolled 45 adults with PBC who had incomplete response to ursodeoxycholic acid (alkaline phosphatase levels ≥1.67-fold the upper limit of normal (ULN). Patients were randomly assigned to elafibranor 80 mg, elafibranor 120 mg or placebo. The primary endpoint was the relative change of ALP at 12 weeks (NCT03124108)., Results: At 12 weeks, ALP was reduced by -48.3±14.8% in the elafibranor 80 mg group (p <0.001 vs. placebo) and by -40.6±17.4% in the elafibranor 120 mg group (p <0.001) compared to a +3.2±14.8% increase in the placebo group. The composite endpoint of ALP ≤1.67-fold the ULN, decrease of ALP >15% and total bilirubin below the ULN was achieved in 67% patients in the elafibranor 80 mg group and 79% patients in the elafibranor 120 mg group, vs. 6.7% patients in the placebo group. Levels of gamma-glutamyltransferase decreased by 37.0±25.5% in the elafibranor 80 mg group (p <0.001) and 40.0±24.1% in the elafibranor 120 mg group (p <0.01) compared to no change (+0.2±26.0%) in the placebo group. Levels of disease markers such as IgM, 5'-nucleotidase or high-sensitivity C-reactive protein were likewise reduced by elafibranor. Pruritus was not induced or exacerbated by elafibranor and patients with pruritus at baseline reported less pruritic symptoms at the end of treatment. All possibly drug-related non-serious adverse events were mild to moderate., Conclusion: In this randomized phase II trial, elafibranor was generally safe and well tolerated and significantly reduced levels of ALP, composite endpoints of bilirubin and ALP, as well as other markers of disease activity in patients with PBC and an incomplete response to ursodeoxycholic acid., Lay Summary: Patients with primary biliary cholangitis (a rare chronic liver disease) that do not respond to standard therapy remain at risk of disease progression toward cirrhosis and impaired quality of life. Elafibranor is a nuclear receptor agonist that we tested in a randomized clinical trial over 12 weeks. It successfully decreased levels of disease activity markers, including alkaline phosphatase. Thus, this study is the foundation for a larger prospective study that will determine the efficacy and safety of this drug as a second-line therapy., Clinical Trial Registration Number: Clinical Trials.gov NCT03124108., Competing Interests: Conflict of interest JMS reports consultancy: BMS, Boehringer Ingelheim, Echosens, Galmed, Genfit, Gilead Sciences, Intercept Pharmaceuticals, Madrigal, Nordic Bioscience, Novartis, Pfizer, Roche, Sanofi, Zydus. Research Funding: Gilead Sciences. AP has received grant funding, personal fees, and advisory board fees from Intercept Pharmaceuticals; advisory board fees and fees for teaching from Novartis; and personal fees from CymaBay Therapeutics and Inova Diagnostics. KVK: serves as consultant to or an advisory boards for Conatus, CymaBay, Gilead, Intercept, La Jolla, Merck and Novartis. He receives research support from Genfit, Gilead, High Tide, Intercept, NGM Biopharma and Novartis and serves as a speaker for Abbvie, Gilead Sciences and Intercept. MAH: Consultancy for Roche, Novartis, Falk and Intercept. SC received research support from Genfit, Gilead and Zydus. DP: nothing to disclose. AB: nothing to disclose. GMH has consulted for Intercept, Genfit, Novartis, GSK, Cymabay and Gilead. CL reports research grants: Gilead, Intercept, CymaBay, Genfit, Genkyotex, Enanta, GSK, Novartis, NGM, High Tide, Durect, Alnylam, Zydus, Cara Therapeutics, Target PharmaSolutions; Consulting fees/Advisory boards: CymaBay, GSK, Shire, Pliant, Target PharmaSolutions, Flashlight Therapeutics, Cara Therapeutics; Royalties: Up-to-date; other: Editorial board Liver Transplantation. JV Research Grants: Allergan, Arena, CymaBay, Enanta, Genkyotex, Intercept, Lilly, NGM Pharmaceuticals, Novartis, TaiwanJ, Scientific Advisor: Arena, BioIncept, Blade, CymaBay, Enanta, Genkyotex, Glaxo-Smith-Kline, Intercept, Lilly, Novartis, TaiwanJ, Authorship: Up-to-Date Immunosuppression in Liver Transplantation; AASLD Writing Committee AIH Guidance In Press, 2019. DJ reports consultancy and grant funding from Intercept and Consultancy from Novartis. AT has nothing to disclose. BS is consultant and president of the SAB of Genfit SA. SM was a former Genfit employee and has currently no COI. RH, DM and PB are Genfit employees. VL reports consultancy for Genfit., (Copyright © 2021 European Association for the Study of the Liver. All rights reserved.)

Background: Non-invasive tests that can identify patients with non-alcoholic steatohepatitis (NASH) at higher risk of disease progression are lacking. We report the development and validation of a blood-based diagnostic test to non-invasively rule in and rule out at-risk NASH (defined as non-alcoholic fatty liver disease [NAFLD] activity score [NAS] ≥4 and fibrosis stage ≥2)., Methods: In this prospective derivation and global validation study, blood samples, clinical data, and liver biopsy results from three independent cohorts with suspected NAFLD were used to develop and validate a non-invasive blood-based diagnostic test, called NIS4. Derivation was done in the discovery cohort, which comprised 239 prospectively recruited patients with biopsy-confirmed NASH (NAFLD NAS ≥3; fibrosis stage 0-3) from the international GOLDEN-505 phase 2b clinical trial. A complete matrix based on 23 variables selected for univariate association with the presence of at-risk NASH and avoiding high multi-collinearity was used to derive the model in a bootstrap-based process that minimised the Akaike information criterion. The overall diagnostic performance of NIS4 was externally validated in two independent cohorts: RESOLVE-IT diag and Angers. The RESOLVE-IT diag cohort comprised the first 475 patients screened for potential inclusion into the RESOLVE-IT phase 3 clinical trial. Angers was a retrospective cohort of 227 prospectively recruited patients with suspected NAFLD and clinical risk factors for NASH or fibrosis stage 2 or more according to abnormal elastography results or abnormal liver biochemistry. Both external validation cohorts were independently analysed and were combined into a pooled validation cohort (n=702) to assess clinical performance of NIS4 and other non-invasive tests., Findings: The derived NIS4 algorithm comprised four independent NASH-associated biomarkers (miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin; area under the receiver operating characteristics curve [AUROC] 0·80, 95% CI 0·73-0·85), and did not require adjustment for age, sex, body-mass index (BMI), or aminotransferase concentrations. Clinical cutoffs were established within the discovery cohort to optimise both rule out and rule in clinical performance while minimising indeterminate results. NIS4 was validated in the RESOLVE-IT diag cohort (AUROC 0·83, 95% CI 0·79-0·86) and the Angers cohort (0·76, 0·69-0·82). In the pooled validation cohort, patients with a NIS4 value less than 0·36 were classified as not having at-risk NASH (ruled out) with 81·5% (95% CI 76·9-85·3) sensitivity, 63·0% (57·8-68·0) specificity, and a negative predictive value of 77·9% (72·5-82·4), whereas those with a NIS4 value of more than 0·63 were classified as having at-risk NASH (ruled in) with 87·1% (83·1-90·3) specificity, 50·7% (45·3-56·1) sensitivity, and a positive predictive value of 79·2% (73·1-84·2). The diagnostic performance of NIS4 within the external validation cohorts was not influenced by age, sex, BMI, or aminotransferase concentrations., Interpretation: NIS4 is a novel blood-based diagnostic that provides an effective way to non-invasively rule in or rule out at-risk NASH in patients with metabolic risk factors and suspected disease. Use of NIS4 in clinical trials or in the clinic has the potential to greatly reduce unnecessary liver biopsies in patients with lower risk of disease progression., Funding: Genfit., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Bile acids (BAs) are signaling molecules controlling lipid and glucose metabolism. Since BA alterations are associated with obesity and insulin resistance, plasma BAs have been considered candidates to predict type 2 diabetes (T2D) risk. We aimed to determine (1) the association of BAs with glucose homeostasis parameters and (2) their predictive association with the risk of conversion from prediabetes to new-onset diabetes (NOD) in a prospective cohort study., Design: 205 patients with impaired fasting glucose (IFG) were followed each year during 5 years in the IT-DIAB cohort study. Twenty-one BA species and 7α-hydroxy-4-cholesten-3-one (C4), a marker of BA synthesis, were quantified by LC/MS-MS in plasma from fasted patients at baseline. Correlations between plasma BA species and metabolic parameters at baseline were assessed by Spearman's coefficients and the association between BAs and NOD was determined using Cox proportional-hazards models., Results: Among the analyzed BA species, total hyocholic acid (HCA) and the total HCA/total chenodeoxycholic acid (CDCA) ratio, reflecting hepatic BA 6α-hydroxylation activity, negatively correlated with BMI and HOMA-IR. The total HCA/total CDCA ratio also correlated negatively with HbA 1C . Conversion from IFG to NOD occurred in 33.7% of the participants during the follow-up. Plasma BA species were not independently associated with the conversion to NOD after adjustment with classical T2D risk factors., Conclusions: Fasting plasma BAs are not useful clinical biomarkers for predicting NOD in patients with IFG. However, an unexpected association between 6α-hydroxylated BAs and glucose parameters was found, suggesting a role for this specific BA pathway in metabolic homeostasis. IT-DIAB study registry number: NCT01218061., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background & Aims: Elafibranor is an agonist of the peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-δ. Elafibranor improves insulin sensitivity, glucose homeostasis, and lipid metabolism and reduces inflammation. We assessed the safety and efficacy of elafibranor in an international, randomized, double-blind placebo-controlled trial of patients with nonalcoholic steatohepatitis (NASH)., Methods: Patients with NASH without cirrhosis were randomly assigned to groups given elafibranor 80 mg (n = 93), elafibranor 120 mg (n = 91), or placebo (n = 92) each day for 52 weeks at sites in Europe and the United States. Clinical and laboratory evaluations were performed every 2 months during this 1-year period. Liver biopsies were then collected and patients were assessed 3 months later. The primary outcome was resolution of NASH without fibrosis worsening, using protocol-defined and modified definitions. Data from the groups given the different doses of elafibranor were compared with those from the placebo group using step-down logistic regression, adjusting for baseline nonalcoholic fatty liver disease activity score., Results: In intention-to-treat analysis, there was no significant difference between the elafibranor and placebo groups in the protocol-defined primary outcome. However, NASH resolved without fibrosis worsening in a higher proportion of patients in the 120-mg elafibranor group vs the placebo group (19% vs 12%; odds ratio = 2.31; 95% confidence interval: 1.02-5.24; P = .045), based on a post-hoc analysis for the modified definition. In post-hoc analyses of patients with nonalcoholic fatty liver disease activity score ≥4 (n = 234), elafibranor 120 mg resolved NASH in larger proportions of patients than placebo based on the protocol definition (20% vs 11%; odds ratio = 3.16; 95% confidence interval: 1.22-8.13; P = .018) and the modified definitions (19% vs 9%; odds ratio = 3.52; 95% confidence interval: 1.32-9.40; P = .013). Patients with NASH resolution after receiving elafibranor 120 mg had reduced liver fibrosis stages compared with those without NASH resolution (mean reduction of 0.65 ± 0.61 in responders for the primary outcome vs an increase of 0.10 ± 0.98 in nonresponders; P < .001). Liver enzymes, lipids, glucose profiles, and markers of systemic inflammation were significantly reduced in the elafibranor 120-mg group vs the placebo group. Elafibranor was well tolerated and did not cause weight gain or cardiac events, but did produce a mild, reversible increase in serum creatinine (effect size vs placebo: increase of 4.31 ± 1.19 μmol/L; P < .001)., Conclusions: A post-hoc analysis of data from trial of patients with NASH showed that elafibranor (120 mg/d for 1 year) resolved NASH without fibrosis worsening, based on a modified definition, in the intention-to-treat analysis and in patients with moderate or severe NASH. However, the predefined end point was not met in the intention to treat population. Elafibranor was well tolerated and improved patients' cardiometabolic risk profile. ClinicalTrials.gov number: NCT01694849., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

We report here the efficacy and safety of GFT505, a novel liver-targeted peroxisome proliferator-activated receptor alpha/delta (PPARα/δ) agonist, in the db/db mouse model of diabetes. Mice were treated with vehicle, GFT505, PPARγ agonist rosiglitazone or dual-PPARα/γ agonist aleglitazar for up to 8 weeks. All compounds comparably reduced fasting glycaemia and HbA1c and improved insulin sensitivity. The glucose-lowering effect of GFT505 was associated with decreased hepatic gluconeogenesis, correlating with reduced expression of gluconeogenic genes. In contrast with the PPARγ-activating drugs, treatment with GFT505 did not affect heart weight and did not increase plasma adiponectin concentrations. This absence of cardiac effects of GFT505 was confirmed after 12 months of administration in cynomolgus monkeys, by the absence of echocardiographic and histological findings. Moreover, long-term GFT505 administration to monkeys induced no change in haematological parameters or in bone marrow differential cell counts. Compared to PPARγ-activating drugs, the dual-PPARα/δ agonist GFT505 therefore shows an improved benefit/risk ratio, treating multiple features of type 2 diabetes without inducing the cardiac side-effects associated with PPARγ activation., (© The Author(s) 2014.)

There is now substantial data in the literature showing that statins can protect against cerebral ischemia. This neuroprotective potency is related to their pleiotropic effects that modulate various pathways implicated in the pathophysiology of stroke. It has been demonstrated that statins exert anti-inflammatory and vasculoprotective effects, thus contributing to a reduction in infarct size. The underlying mechanisms are still incompletely known. As a cross-talk between statins and the nuclear receptor PPARα has been described, we hypothesized that this cross-talk is necessary to neuroprotection in stroke. We studied the effects of a 14-day preventive atorvastatin treatment (10 mg/kg/day) on C57Bl6 wild-type and PPARα-KO mice submitted to experimental stroke. PPARα was involved in the atorvastatin-induced neuroprotective effect, as confirmed by the measurement of infarct volumes. We also evidenced that the anti-inflammatory action of atorvastatin is mediated, at least partly, by PPARα. The decrease in IL-6 plasmatic levels was PPARα dependent. The cerebral expression of the adhesion molecules ICAM-1 and vascular cell adhesion molecule was reduced by the atorvastatin treatment, and this effect was PPARα dependent in the cortex but not in the striatum of treated animals. Atorvastatin also diminished the cerebral expression of iNOS in the cortex, but had no effect in the striatum of treated mice, whatever the PPARα status. At the vascular level, we found that the atorvastatin-related endothelial nitric oxide synthase upregulation was regulated by PPARα in the aorta, while there was no effect in the brain. We demonstrate here that PPARα is a key mediator of the multitargeted neuroprotective effects of statins in stroke., (© 2013 The Authors Fundamental and Clinical Pharmacology © 2013 Société Française de Pharmacologie et de Thérapeutique.)

Unlabelled: Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. To date, no pharmacological treatment is approved for NAFLD/NASH. Here, we report on preclinical and clinical data with GFT505, a novel dual peroxisome proliferator-activated receptor alpha/delta (PPAR-α/δ) agonist. In the rat, GFT505 concentrated in the liver with limited extrahepatic exposure and underwent extensive enterohepatic cycling. The efficacy of GFT505 was assessed in animal models of NAFLD/NASH and liver fibrosis (Western diet [WD]-fed human apolipoprotein E2 [hApoE2] transgenic mice, methionine- and choline-deficient diet-fed db/db mice, and CCl4 -induced fibrosis in rats). GFT505 demonstrated liver-protective effects on steatosis, inflammation, and fibrosis. In addition, GFT505 improved liver dysfunction markers, decreased hepatic lipid accumulation, and inhibited proinflammatory (interleukin-1 beta, tumor necrosis factor alpha, and F4/80) and profibrotic (transforming growth factor beta, tissue inhibitor of metalloproteinase 2, collagen type I, alpha 1, and collagen type I, alpha 2) gene expression. To determine the role of PPAR-α-independent mechanisms, the effect of GFT505 was assessed in hApoE2 knock-in/PPAR-α knockout mice. In these mice, GFT505 also prevented WD-induced liver steatosis and inflammation, indicating a contribution of PPAR-α-independent mechanisms. Finally, the effect of GFT505 on liver dysfunction markers was assessed in a combined analysis of four phase II clinical studies in metabolic syndrome patients. GFT505 treatment decreased plasma concentrations of alanine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase., Conclusion: The dual PPAR-α/δ agonist, GFT505, is a promising liver-targeted drug for treatment of NAFLD/NASH. In animals, its protective effects are mediated by both PPAR-α-dependent and -independent mechanisms., (© 2013 by the American Association for the Study of Liver Diseases.)

Objective: The development of new insulin sensitizers is an unmet need for the treatment of type 2 diabetes. We investigated the effect of GFT505, a dual peroxisome proliferator-activated receptor (PPAR)-α/δ agonist, on peripheral and hepatic insulin sensitivity., Research Design and Methods: Twenty-two abdominally obese insulin-resistant males (homeostasis model assessment of insulin resistance>3) were randomly assigned in a randomized crossover study to subsequent 8-week treatment periods with GFT505 (80 mg/day) or placebo, followed by a two-step hyperinsulinemic-euglycemic insulin clamp with a glucose tracer to calculate endogenous glucose production (EGP). The primary end point was the improvement in glucose infusion rate (GIR). Gene expression analysis was performed on skeletal muscle biopsy specimens., Results: GFT505 improved peripheral insulin sensitivity, with a 21% (P=0.048) increase of the GIR at the second insulin infusion period. GFT505 also enhanced hepatic insulin sensitivity, with a 44% (P=0.006) increase of insulin suppression of EGP at the first insulin infusion period. Insulin-suppressed plasma free fatty acid concentrations were significantly reduced on GFT505 treatment (0.21±0.07 vs. 0.27±0.11 mmol/L; P=0.006). Neither PPARα nor PPARδ target genes were induced in skeletal muscle, suggesting a liver-targeted action of GFT505. GFT505 significantly reduced fasting plasma triglycerides (-21%; P=0.003) and LDL cholesterol (-13%; P=0.0006), as well as liver enzyme concentrations (γ-glutamyltranspeptidase: -30.4%, P=0.003; alanine aminotransferase: -20.5%, P=0.004). There was no safety concern or any indication of PPARγ activation with GFT505., Conclusions: The dual PPARα/δ agonist GFT505 is a liver-targeted insulin-sensitizer that is a promising drug candidate for the treatment of type 2 diabetes and nonalcoholic fatty liver disease.

Aims: Peroxisome proliferator-activated receptor (PPAR)-α is a transcription factor controlling lipid metabolism in liver, heart, muscle, and macrophages. Peroxisome proliferator-activated receptor-α activation increases plasma HDL cholesterol and exerts hypotriglyceridaemic actions via the liver. However, the intestine expresses PPAR-α, produces HDL and chylomicrons, and is exposed to diet-derived PPAR-α ligands. Therefore, we examined the effects of PPAR-α activation on intestinal lipid and lipoprotein metabolism., Methods and Results: The impact of PPAR-α activation was evaluated in term of HDL-related gene expression in mice, ex vivo in human jejunal biopsies and in Caco-2/TC7 cells. Apolipoprotein-AI/HDL secretion, cholesterol esterification, and trafficking were also studied in vitro. In parallel to improving plasma lipid profiles and increasing liver and intestinal expression of fatty acid oxidation genes, treatment with the dual PPAR-α/δ ligand GFT505 resulted in a more pronounced increase in plasma HDL compared with fenofibrate in mice. GFT505, but not fenofibrate, increased the expression of HDL production genes such as apolipoprotein-AI and ATP-binding cassette A1 transporter in murine intestines. A similar increase was observed upon PPAR-α activation of human biopsies and Caco-2/TC7 cells. Additionally, HDL secretion by Caco-2/TC7 cells increased. Moreover, PPAR-α activation decreased the cholesterol esterification capacity of Caco-2/TC7 cells, modified cholesterol trafficking, and reduced apolipoprotein-B secretion., Conclusion: Peroxisome proliferator-activated receptor-α activation reduces cholesterol esterification, suppresses chylomicron, and increases HDL secretion by enterocytes. These results identify the intestine as a target organ of PPAR-α ligands with entero-hepatic tropism to reduce atherogenic dyslipidaemia.

The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. POR's unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump-probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP(+) (PChlide/NADP(+)/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S(1) excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP(+)/POR complex, whereas the nonradiative rates are decreased by ∼30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (S(ICT)) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the S(ICT) state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.

Because of their wide range of actions on glucose homeostasis, lipid metabolism and vascular inflammation, peroxisome proliferator-activated receptors (PPARs) are promising targets for the development of new drugs for the treatment of metabolic disorders such as diabetes, dyslipidemia and atherosclerosis. In clinical practice, PPARalpha agonists, such as the already available fibrates, improve dyslipidemia, while PPARgamma agonists, such as thiazolidinediones, improve insulin resistance and diabetes. The complementary action of simultaneous activation of each PPAR in patients suffering from metabolic syndrome and type 2 diabetes has led to new pharmacological strategies focused on the development of agonists targeting more than one receptor such as the dual PPARalpha/gamma agonists. However, despite the proven benefits of targeting PPARs, safety concerns have recently led to late stage development failures of various PPAR agonists including novel specific PPARgamma agonists and dual PPARalpha/gamma agonists. These safety concerns include potential carcinogenicity in rodents, signs of myopathy and rhabdomyolysis, increase in plasma creatinine and homocysteine, weight gain, fluid retention, peripheral edema and potential increased risk of cardiac failure. Although the discontinued compounds shared common side effects, the reason for discontinuation was always compound specific and the toxicological or adverse effects which have motivated the discontinuation could be either due to the activation of PPARgamma, PPARalpha or both (class effect) or due to a PPAR unrelated effect. Thus, the risk evaluation of each adverse effect should be viewed on a case by case basis considering both the PPAR profile of the drug, its absorption/distribution profile, the nature of the side effect and the putative PPAR-related mechanism of action. This review mainly focuses on the preclinical and clinical adverse events of PPAR agonists that could be of concern when considering the development of new PPAR agonists. The selective modulation of PPAR activities is a promising approach to develop new drugs with preserved efficacy but diminished adverse effects.

1. We investigated the role of arachidonic acid metabolism and assessed the participation of mast cells and leukocytes in neurogenic inflammation in rat paw skin. We compared the effect of lipoxygenase (LOX) and cyclo-oxygenase (COX) inhibitors on oedema induced by saphenous nerve stimulation, substance P (SP), and compound 48/80. 2. Intravenous (i.v.) pre-treatment with a dual COX/LOX inhibitor (RWJ 63556), a dual LOX inhibitor/cysteinyl-leukotriene (CysLt) receptor antagonist (Rev 5901), a LOX inhibitor (AA 861), a five-lipoxygenase activating factor (FLAP) inhibitor (MK 886), or a glutathione S-transferase inhibitor (ethacrynic acid) significantly inhibited (40 to 60%) the development of neurogenic oedema, but did not affect cutaneous blood flow. Intradermal (i.d.) injection of LOX inhibitors reduced SP-induced oedema (up to 50% for RWJ 63556 and MK 886), whereas ethacrynic acid had a potentiating effect. 3. Indomethacin and rofecoxib, a highly selective COX-2 inhibitor, did not affect neurogenic and SP-induced oedema. Surprisingly, the structurally related COX-2 inhibitors, NS 398 and nimesulide, significantly reduced both neurogenic and SP-induced oedema (70% and 42% for neurogenic oedema, respectively; 49% and 46% for SP-induced oedema, respectively). 4. COX-2 mRNA was undetectable in saphenous nerves and paw skin biopsy samples, before and after saphenous nerve stimulation. 5. A mast cell stabilizer, cromolyn, and a H(1) receptor antagonist, mepyramine, significantly inhibited neurogenic (51% and 43%, respectively) and SP-induced oedema (67% and 63%, respectively). 6. The co-injection of LOX inhibitors and compound 48/80 did not alter the effects of compound 48/80. Conversely, ethacrynic acid had a significant potentiating effect. The pharmacological profile of the effect of COX inhibitors on compound 48/80-induced oedema was similar to that of neurogenic and SP-induced oedema. 7. The polysaccharide, fucoidan (an inhibitor of leukocyte rolling) did not affect neurogenic or SP-induced oedema. 8. Thus, (i) SP-induced leukotriene synthesis is involved in the development of neurogenic oedema in rat paw skin; (ii) this leukotriene-mediated plasma extravasation might be independent of mast cell activation and/or of the adhesion of leukocytes to the endothelium; (iii) COX did not appear to play a significant role in this process.

1. Intravital microscopy technique was used to determine the distribution of a fluorescent plasma marker (fluorescein-isothiocyanate-dextran, 150 kD; FD-150) into venular and interstitial compartments of dorsal skin fold preparations in conscious hamsters. 2. One mg kg(-1) histamine (i.v.) caused a biphasic decrease in venular fluorescence due to FD-150 extravasation in all organs (general extravasation). Immediately after injection, the venular fluorescence decreased and plateaued in 60 min. Ninety minutes after histamine injection, venular fluorescence further decreased until 180 min. Prior treatment with indomethacin (0.1 mg kg(-1), i.v.) did not modify the time-course of general extravasation but prevented histamine-induced venule dilatation. 3. Prior treatment with the 5-lipoxygenase activating protein (FLAP) inhibitor, 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-d imethyl-propanoic acid sodium (MK-886)(10 microg kg(-1), i.v.), the leukotriene receptor antagonist, benzenemethanol a-pentyl-3-(2-quinolinylmethoxy) (REV-5901)(1 mg kg(-1), i.v.), or the glutathione-S-transferase inhibitor, ethacrynic acid (1 mg kg(-1), i.v.), delayed by 60 min the onset of general extravasation caused by 1 mg kg(-1) histamine. 4. Prior treatment with lipoxygenase pathway inhibitors and N(G)-nitro-L-arginine-methylester (L-NAME)(100 mg kg(-1), i.v.) abolished the general extravasation and venule dilatation induced by 1 mg kg(-1) histamine. 5. Injection of 1 microg kg(-1) (i.v.), of leukotriene-C4 (LTC4) or -D4 (LTD4) induced immediate and sustained general extravasation and reduction in venule diameter, these effects being blocked by REV-5901. 6. Histamine (1 mg kg(-1), i.v.) induced biphasic decline in mean arterial blood pressure (MAP). An initial phase (from 0 to 60 min) was followed by a late phase beginning 90 min after histamine injection. L-NAME (100 mg kg(-1), i.v.) and aminoguanidine (1 mg kg(-1), i.v.) prevented the late phase of histamine-induced hypotension. 7. Thus, plasma histamine can trigger both an immediate cysteinyl-leukotriene (Cys-LT)-dependent and a late nitric oxide (NO)-mediated inflammatory cascade. Although the cyclo-oxygenase (COX) pathway might account for histamine-induced venule dilatation, it would not influence histamine-induced extravasation.

1. Arachidonic acid (0.01-1 microM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1 microM. Concentrations higher than 1 microM were required to induce dose-dependent contraction of vena cava and thoracic aorta from the same animals. 2. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3 microM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. 3. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration-relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid-induced relaxations were prevented by indomethacin (10 microM) pretreatment. 4. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration-relaxation curves of this tissue to arachidonic acid in a dose-dependent manner. 5. In the presence of 1 microM arachidonic acid, venous rings produced 8-10 fold more PGE2 than did aorta whereas 6keto-PGF1alpha and TXB2 productions remained comparable. 6. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L-NAME (100 microM) or indomethacin (10 microM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L-NAME. 7. We conclude that stimulation of the cyclo-oxygenase pathway by arachidonic acid induced endothelium-dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187-induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.

We investigated the effects of L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) on macromolecule extravasation in the microcirculation of awake hamsters by computer-assisted image analysis of the distribution of FITC (fluorescein isothiocyanate)-dextran fluorescence in dorsal fold skin preparations. This analysis made it possible to simultaneously study the time course of local (skin) and general (all irrigated organs) extravasation in 180-min experiments. Bolus injection of 30 or 150 mg/kg (i.v.) L-arginine induced immediate local and general macromolecule leakage and delayed venule dilation beginning 1 h later. Injection of 20 or 100 mg/kg (i.v.) L-NAME caused rapid venule constriction followed by local and general extravasation beginning 45-60 min later. These effects of L-arginine and L-NAME were not mimicked by their biologically inactive isomers, D-arginine and D-NAME. Simultaneous bolus injection of 20 mg/kg L-NAME and 150 mg/kg L-arginine caused no significant change in fluorescence distribution or venule diameter. L-arginine effects on macromolecule extravasation were mimicked by sodium nitroprusside (10 microg/kg, i.v.) and by 8-bromo-cGMP (1 mg/kg, i.v.). Sodium nitroprusside was ineffective on venule diameter. The effects of both L-arginine and sodium nitroprusside on FITC-dextran extravasation were prevented by simultaneous injection (10 microg/kg, i.v.) of the specific inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). This dose of ODQ mimicked the effects of L-NAME on macromolecule extravasation and venule diameter. Taken together, these results suggest that activation or inhibition of basal NO synthesis might induce macromolecule leakage in the microcirculation of awake hamsters via temporally distinct cGMP-dependent mechanisms.

1. Late effects (up to 3 h) of intravenously-injected histamine on FITC-dextran extravasation were investigated in the conscious hamster, by use of computer-assisted image analysis of fluorescence distribution in a microscopic window of dorsal skin fold preparations. This analysis allowed measurement of local (skin) and general (all organs) extravasations caused by a bolus injection of histamine (1 mg kg(-1), i.v.) 2. Histamine doses higher than 0.01 mg kg(-1) caused biphasic local and general extravasations. Initial phases developed fully within 15 min (for local) and 60 min (for general) and were followed by late phases beginning 90 min after histamine injection. Although the initial and late phases of histamine-induced extravasations had differential apparent reactivities to the autacoid, all the effects of histamine on the microcirculation (1 mg kg[-1]) were inhibited by pyrilamine (1 mg kg(-1), i.v.) but not by cimetidine (1 mg kg(-1), i.v.). 3. Pretreatment with N(G)-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1), i.v.) or N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg kg(-1), i.v.) did not affect the initial phases but did prevent the late phases of local and general extravasations triggered by 1 mg kg(-1) histamine. The inhibitory effects of L-NAME were reversed by L-arginine (30 mg kg[-1]) but not by D-arginine (30 mg kg[-1]) according to the enantioselectivity of nitric oxide synthase (NOS). A late NO-mediated venular dilatation occurred in response to plasma histamine. 4. A low dose of aminoguanidine (1 mg kg(-1), i.v.), a selective inhibitor of the inducible isoform of NOS (iNOS), mimicked the inhibitory effects of L-NAME on the late phases of histamine-induced macromolecular extravasations and venular dilatation. 5. Pretreatment with dexamethasone (1 mg kg(-1), i.v.) prevented both the initial and late phases of histamine-induced extravasations. Fucoidan (1 or 25 mg kg(-1), i.v.) prevented the late phases without affecting initial phases, consistent with a role for leukocytes adhesion in the development of the late NO-mediated effects of histamine. 6. We conclude that intravenous injection of histamine triggers a biphasic inflammatory cascade via initial activation of H1 receptors which induces a late NO-mediated PMN-dependent extravasation process.

1. A fast perfusion system was used to analyse the kinetics of the response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.

The ability of acetylcholine (ACh) to inhibit beta-agonist stimulated calcium current was compared to its ability to activate the inwardly rectifying potassium current IK(ACh) in frog atrial myocytes. As suggested by previous studies, ACh inhibited the calcium current at concentrations (EC50 = 8 nM) significantly lower than those required for the activation of IK(ACh) (EC50 = 101 nM). The pharmacological profiles of the two responses suggest that despite the differences in agonist sensitivity, both are mediated by the same (m2) type of muscarinic receptors. Intracellular application of GDP beta S, an inhibitor of G protein function, completely abolished both responses, implying that both actions of ACh are coupled to effectors by G proteins. In contrast, intracellular application of pertussis toxin (PTX) shifted to higher concentrations (EC50 = 170 nM) but did not abolish inhibition of the calcium current by ACh even though the block of the IK(ACh) response was complete. Increasingly large PTX concentrations and/or prolonged PTX treatments revealed a limiting, PTX-resistant inhibitory component that appears to be mediated by a PTX-insensitive G protein distinct from that mediating IK(ACh). For the PTX-sensitive components, the different agonist dependencies of IK(ACh) activation and calcium current inhibition may imply that different G proteins mediate each response although alternate possibilities involving the same G protein either functionally sequestered and/or differentially affected by interactions with effectors, can not be ruled out.

1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose-dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (-)-Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF-DX 116 >> pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.

The diterpene forskolin is widely known for its ability to directly activate adenylyl cyclase and consequently increase intracellular cAMP. In cardiac cells, one result is a cAMP-mediated increase in the L-type Ca2(+)-channel current (ICa). However, forskolin was also shown recently to affect a number of ionic channels in noncardiac cells by mechanisms that do not involve activation of adenylyl cyclase. The present study reveals such an effect of forskolin on cardiac Ca2+ channels. Indeed, under appropriate conditions, forskolin was found to cause an inhibition of ICa. Although the stimulation of adenylyl cyclase and ICa requires micromolar concentrations of forskolin, the inhibitory effect of forskolin was observed in the nanomolar range of concentrations, i.e., 2-3 orders of magnitude lower. This high affinity forskolin inhibition of ICa was observed when ICa was previously enhanced via a cAMP-dependent pathway, but not when ICa was at its basal level or when the current was elevated by the dihydropyridine Bay K 8644. The inhibitory effect occurred at a site of action remote from adenylyl cyclase, because forskolin similarly inhibited ICa that had been previously elevated by isoprenaline (a beta-adrenergic agonist) or directly by intracellular perfusion with cAMP. Under these conditions, forskolin was inhibitory when applied to either side of the cell membrane, but only in its lipid-soluble form. The inhibitory effect of forskolin appeared to be independent of membrane potential and was not accompanied by a change in the time constants of ICa activation and inactivation. This may indicate that forskolin mainly reduces the number of functional Ca2+ channels without changing the gating of individual channels. However, the reduction in ICa amplitude was not equally distributed among the different exponential components that constitute ICa, which suggests that forskolin also modifies the resting state of the channels. This novel high affinity forskolin inhibition of ICa may take place at some step in the pathway between cAMP and Ca2+ channel phosphorylation and/or at Ca2+ channels only after they have been phosphorylated.

The sodium-calcium exchange activity has been studied in sarcolemmal vesicles isolated from rat ventricles hypertrophied by pressure overload. 4 weeks after aortic stenosis the degree of hypertrophy varied from 30 to 70%. The Na+-dependent 45Ca2+ influx and efflux were up to 50% decreased and the sensitivity to Ca2+ was 13-fold lower in vesicles from hypertrophied heart as compared to those from normal heart. However, the Na+,K+-ATPase activity, the orientation of the vesicles and the passive Ca2+ permeability were found to be similar in the two heart groups. These results indicate that the sarcolemmal Na+/Ca2+ exchange activity could be qualitatively and/or quantitatively changed in hypertrophied rat heart.