Search

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing., Recently, the first orally-administered ultra-long acting insulin was shown to have clinical efficacy. Here, the authors report the molecular engineering, as well as the biological and pharmacological properties of these insulin analogues.

The use of translationally relevant animal models is essential, also within the field of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Compared to frequently used mouse and rat models, the hamster may provide a higher degree of physiological similarity to humans in terms of lipid profile and lipoprotein metabolism. However, the effects in hamsters after long-term exposure to a NASH diet are not known. Male Syrian hamsters were fed either a high-fat, high-fructose, high-cholesterol diet (NASH diet) or control diets for up to 12 months. Plasma parameters were assessed at two weeks, one, four, eight and 12 months and liver histopathology and biochemistry was characterized after four, eight and 12 months on the experimental diets. After two weeks, hamsters on NASH diet had developed marked dyslipidemia, which persisted for the remainder of the study. Hepatic steatosis was present in NASH-fed hamsters after four months, and hepatic stellate cell activation and fibrosis was observed within four to eight months, respectively, in agreement with progression towards NASH. In summary, we demonstrate that hamsters rapidly develop dyslipidemia when fed a high-fat, high-fructose, high-cholesterol diet. Moreover, within four to eight months, the NASH-diet induced hepatic changes with resemblance to human NAFLD.

Background Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent comorbidities in patients with Type 2 diabetes. While many of these patients eventually will need treatment with insulin, little is known about the effects of insulin treatment on histopathological parameters and hepatic gene expression in diabetic patients with co-existing NAFLD and NASH. To investigate this further, we evaluated the effects of insulin treatment in NASH diet-fed hamsters with streptozotocin (STZ) -induced hyperglycemia. Methods Forty male Syrian hamsters were randomized into four groups (n = 10/group) receiving either a NASH-inducing (high fat, fructose and cholesterol) or control diet (CTRL) for four weeks, after which they were treated with STZ or sham-injected and from week five treated with either vehicle (CTRL, NASH, NASH-STZ) or human insulin (NASH-STZ-HI) for four weeks by continuous s.c. infusion via osmotic minipumps. Results NASH-STZ hamsters displayed pronounced hyperglycemia, dyslipidemia and more severe liver pathology compared to both CTRL and NASH groups. Insulin treatment attenuated dyslipidemia in NASH-STZ-HI hamsters and liver pathology was considerably improved compared to the NASH-STZ group, with prevention/reversal of hepatic steatosis, hepatic inflammation and stellate cell activation. In addition, expression of inflammatory and fibrotic genes was decreased compared to the NASH-STZ group. Conclusions These results suggest that hyperglycemia is important for development of inflammation and profibrotic processes in the liver, and that insulin administration has beneficial effects on liver pathology and expression of genes related to inflammation and fibrosis in a hyperglycemic, dyslipidemic hamster model of NAFLD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Diabetic human patients have increased risk of heart failure compared to healthy subjects. The underlying mechanisms for this are not fully understood, and to help develop improved treatment strategies, well-characterized animal models are essential. To investigate cardiac dysfunction in diabetes, this study evaluated myocardial changes in 10 aging rhesus monkeys with and without diabetes. Based on evaluation of plasma glycosylated hemoglobin and glucose, 7 of 10 rhesus macaques had diabetes for a minimum of 11 months, while 3 of 10 were categorized as nondiabetic. A detailed histological examination of formalin-fixed left ventricular myocardial samples was followed by a semiquantitative evaluation of myocardial fibrosis and fat infiltration; digital quantifications of myocardial collagen, lipofuscin, and nuclear area fractions; and measurements of cardiomyocyte diameter. Histological myocardial evaluation revealed the presence of lipofuscin; large nuclei; interstitial, replacement, and vascular fibrosis; adipocyte infiltration; and vacuolar degeneration with atrophy of cardiomyocytes and fibrosis. However, there were no differences between groups for semiquantitative fat infiltration, fibrosis, cardiomyocyte size, collagen, or nuclear and lipofuscin area fraction. Lipofuscin area fraction correlated with plasma insulin, triglyceride, total cholesterol, and high-density lipoprotein cholesterol concentrations. In conclusion, myocardial pathological changes were found in left ventricular myocardium in aged rhesus macaques, independent of the stage of diabetes. The duration of diabetes might have been too short to cause differences between groups.

Introduction In animal models of non-alcoholic fatty liver disease (NAFLD), assessment of disease severity and treatment effects of drugs rely on histopathological scoring of liver biopsies. However, little is known about the sampling variation in liver samples from animal models of NAFLD, even though several histopathological hallmarks of the disease are known to be affected by sampling variation in patients. The aim of this study was to assess the sampling variation in multiple paired liver biopsies from three commonly used diet-induced rodent models of NAFLD. Methods Eight male C57BL/6 mice, 8 male Sprague Dawley rats and 16 female Hartley guinea pigs were fed a NAFLD-inducing high-fat diet for 16 weeks (mice and rats), 20 or 24 weeks (guinea pigs). After the initial diet period, liver sections were sampled and subsequently assessed by histopathological scoring and biochemical analyses. Results Fibrosis was heterogeneously distributed throughout the liver in mice, manifesting as both intra- and interlobular statistically significant differences. Hepatic triglyceride content showed interlobular differences in mice, and both intra- and interlobular differences in guinea pigs (24-week time point) all of which were statistically significant. Also, hepatic cholesterol content was subject to significant intra-lobular sampling variation in mice, and hepatic glycogen content differed significantly between lobes in mice and guinea pigs. Discussion Dependent on animal model, both histopathological and biochemical end-points differed between sampling sites in the liver. Based on these findings, we recommend that sample site location is highly standardized and properly reported in order to minimize potential sampling variation and to optimize reproducibility and meaningful comparisons of preclinical studies of NAFLD.

Background: In humans and animal models, excessive intake of dietary fat, fructose and cholesterol has been linked to the development of non-alcoholic fatty liver disease (NAFLD). However, the individual roles of the dietary components remain unclear. To investigate this further, we compared the effects of a high-fat diet, a high-fructose diet and a combination diet with added cholesterol on the development of NAFLD in rats. Methods: Forty male Sprague-Dawley rats were randomized into four groups receiving either a control-diet (Control: 10% fat); a high-fat diet (HFD: 60% fat, 20% carbohydrate), a high-fructose diet [HFr: 10% fat, 70% carbohydrate (mainly fructose)] or a high-fat/high-fructose/high-cholesterol-diet (NASH: 40% fat, 40% carbohydrate (mainly fructose), 2% cholesterol) for 16 weeks. Results: After 16 weeks, liver histology revealed extensive steatosis and inflammation in both NASH- and HFD-fed rats, while hepatic changes in HFr-rats were much more subtle. These findings were corroborated by significantly elevated hepatic triglyceride content in both NASH- (p < 0.01) and HFD-fed rats (p < 0.0001), elevated hepatic cholesterol levels in NASH-fed rats (p < 0.0001), but no changes in HFr-fed rats, compared to Control. On the contrary, only HFr-fed rats developed dyslipidemia as characterized by higher levels of plasma triglycerides compared to all other groups (p < 0.0001). Hepatic dysfunction and inflammation was confirmed in HFD-fed rats by elevated levels of hepatic MCP-1 (p < 0.0001), TNF-alpha (p < 0.001) and plasma β-hydroxybutyrate (p < 0.0001), and in NASH-fed rats by elevated levels of hepatic MCP-1 (p < 0.01), increased hepatic macrophage infiltration (p < 0.001), and higher plasma levels of alanine aminotransferase (p < 0.0001) aspartate aminotransferase (p < 0.05), haptoglobin (p < 0.001) and TIMP-1 (p < 0.01) compared to Control. Conclusion: These findings show that dietary fat and cholesterol are the primary drivers of NAFLD development and progression in rats, while fructose mostly exerts its effect on the circulating lipid pool.

Balaglitazone is a novel thiazolidinedione in clinical development for the treatment of type 2 diabetes. Common side effects associated with PPARgamma receptor agonists are weight gain, oedema and adipogenesis. Balaglitazone is a selective partial PPARgamma agonist and it has been speculated that such compounds have a more favourable safety margin than full agonists. We have compared impact of equi-efficacious antihyperglycaemic doses of balaglitazone with full PPARgamma agonist rosiglitazone on body fluid accumulation, cardiac enlargement, and adipogenesis. Equi-efficacious antihyperglycaemic doses (ED(90)) of balaglitazone (3 mg/kg/day) and rosiglitazone (6 mg/kg/day) were determined in male diabetic db/db mice. In adult male rats treated for up to 42 days, feeding, drinking, anthropometry, and plasma volumes were measured. Total plasma volume was measured with dye dilution technique. Compared to vehicle, rosiglitazone consistently increased food intake throughout the 42 day treatment period. In contrast, balaglitazone increased food intake in the last week of the experiment. However, both rosiglitazone and balaglitazone increased water intake. After 42 days, rosiglitazone treated rats displayed significantly elevated adiposity. Rosiglitazone increased total blood and plasma volumes throughout the treatment. Twenty-one days of balaglitazone treatment had no significant impact on blood or plasma volumes, whilst 42 days of balaglitazone increased plasma volume but to a significantly lesser extent than seen for rosiglitazone (vehicle: 46.1+/-1.5; balaglitazone: 50.8+/-1.21; rosiglitazone: 54.6+/-1.6 ml/kg). Heart weight was significantly elevated only in rosiglitazone treated animals. At doses inducing comparable antihyperglycaemic control, the full PPARgamma agonist, rosiglitazone, induces more pronounced body fluid retention and heart enlargement than seen for the partial PPARgamma agonist, balaglitazone. Thus, partial agonists may pose safer alternative to current anti-diabetic therapy with full PPARgamma agonist.

Computational analysis of the ligand binding pocket of the three PPAR receptor subtypes was utilized in the design of potent PPARα agonists. Optimum PPARα potency and selectivity were obtained with substituents having van der Waals volume around 260. Compound 6 had a PPARα potency of 0.002 μM and a selectivity ratio to PPARγ and PPARδ of 410 and 2000, respectively.

The aim was to identify a novel selective PPARdelta agonist with full efficacy on free fatty acid (FFA) oxidation in vitro and plasma lipid correction in vivo. Using the triple PPARalpha,gamma,delta agonist 1 as the structural starting point, we wanted to investigate the possibility of obtaining selective PPARdelta agonists by modifying only the acidic part of 1, while holding the lipophilic half of the molecule constant. The structure-activity relationship was guided by in vitro transactivation data using the human PPAR receptors, FFA oxidation efficacy performed in the rat muscle L6 cell line, and in vivo rat pharmacokinetic properties. Compound 7 ([4-[3,3-bis-(4-bromo-phenyl)-allylthio]-2-chloro-phenoxy]-acetic acid) was identified as a selective, partial agonist with good oral pharmacokinetic properties in rat. Chronic treatment of high fat fed ApoB100/CETP-Tgn mice with 7 corrected the plasma lipid parameters and improved insulin sensitivity. These data suggest that selective PPARdelta agonists have the potential to become a novel treatment of dyslipidemia.

Using a known dual PPARα/γ activator (5) as a structural template, SAR evaluations led to the identification of triple PPARα/γ/δ activators (18–20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARα/γ/δ activation.

Synthesis and structure-activity relationships of tricyclic alpha-ethoxy-phenylpropionic acid derivatives guided by in vitro PPARalpha and PPARgamma transactivation data and computer modeling led to the identification of the novel carbazole analogue, 3q, with dual PPARalpha (EC(50) = 0.36 microM) and PPARgamma (EC(50) = 0.17 microM) activity in vitro. Ten days treatment of db/db mice with 3q improved the insulin sensitivity, as measured by OGTT, better than that seen with both pioglitazone and rosiglitazone treatment, suggesting in vivo PPARgamma activity. Likewise, 3q lowered plasma triglycerides and cholesterol in high cholesterol fed rats after 4 days treatment, indicating in vivo PPARalpha activity. Investigations of the pharmacokinetics of selected compounds suggested that extended drug exposure improved the in vivo activity of in vitro active compounds.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Several reports have shown that PPARdelta is involved in lipid metabolism, increasing fat oxidation and depleting lipid accumulation. Whether PPARdelta is involved in the regulation of glucose metabolism is not completely understood. In this study, we examined effects of long-term PPARdelta activation on glycemic control, islet function and insulin sensitivity in diabetic db/db mice. Male db/db mice were administered orally once daily with a selective and partial PPARdelta agonist (NNC 61-5920, 30mg/kg) for eight weeks; control mice received vehicle. Fasting and non-fasting plasma glucose were reduced, reflected in reduced hemoglobinA(1c) (3.6+/-1.6% vs. 5.4+/-1.8 in db/db controls, P

Objective Severe insulin resistance and impaired pancreatic beta-cell function are pathophysiological contributors to type 2 diabetes, and ideally, antihyperglycaemic strategies should address both. Research design and methods Therapeutic benefits of combining the long-acting human glucagon-like peptide-1 (GLP-1) analog, liraglutide (0.4 mg/kg/day), with insulin sensitizer, pioglitazone (10 mg/kg/day), were assessed in severely diabetic Zucker diabetic fatty rats for 42 days. Impact on glycaemic control was assessed by glycated haemoglobin (HbA(1C)) at day 28 and by oral glucose tolerance test at day 42. Results Liraglutide and pioglitazone synergistically improved glycaemic control as reflected by a marked decrease in HbA(1C) (liraglutide + pioglitazone: 4.8 +/- 0.3%; liraglutide: 8.8 +/- 0.6%; pioglitazone: 7.9 +/- 0.4%; vehicle: 9.7 +/- 0.3%) and improved oral glucose tolerance at day 42 (area under the curve; liraglutide + pioglitazone: 4244 +/- 445 mmol/l x min; liraglutide: 7164 +/- 187 mmol/l x min; pioglitazone: 7430 +/- 446 mmol/l x min; vehicle: 8093 +/- 139 mmol/l x min). A 24-h plasma glucose profile at day 38 was significantly decreased only in the liraglutide + pioglitazone group. In addition, 24-h insulin profile was significantly elevated only in the liraglutide + pioglitazone group. Liraglutide significantly decreased food intake alone and in combination with pioglitazone, while pioglitazone alone increased cumulated food intake. As a result, rats on liraglutide alone gained significantly less weight than vehicle-treated rats, whereas rats on pioglitazone alone gained significantly more body weight than vehicle-treated rats. However, combination therapy with liraglutide and pioglitazone caused the largest weight gain, probably reflecting marked improvement of energy balance because of reduction of glucosuria. Conclusions Combination therapy with insulinotropic GLP-1 agonist liraglutide and insulin sensitizer, pioglitazone, improves glycaemic control above and beyond what would be expected from additive effects of the two antidiabetic agents.

Computational analysis of the ligand binding pocket of the three PPAR receptor subtypes was utilized in the design of potent PPARalpha agonists. Optimum PPARalpha potency and selectivity were obtained with substituents having van der Waals volume around 260. Compound 6 had a PPARalpha potency of 0.002 microM and a selectivity ratio to PPARgamma and PPARdelta of 410 and 2000, respectively.

Type 2 diabetes is a metabolic disease characterized by increased plasma glucose and insulin as well as dyslipidemia. If left untreated, chronic diseases will develop that are associated with neuropathic damage and higher mortality risk. Using a rational drug design, novel compounds have been developed that selectively activate the human PPAR receptors, leading to lessening of hyperglycemia and hyperinsulinemia as well as reduction of lipid levels in conjunction with an increase of the beneficial HDL-cholesterol. These PPAR agonists showed increased potency and efficacy compared to previously marketed insulin sensitizers. Lead compounds with desirable pharmacokinetic properties were chosen for further testing in several animal models. The in vivo activity of some synthetic ligands, capable of activating two or all three members of peroxisome proliferator-activated receptors (PPAR) family of receptors, suggested that they may have improved efficacy in type 2 diabetes therapy. Here, we briefly summarize the development of some novel PPAR agonists identified by our group in recent years.

A series of dimeric PPAR agonists were designed and tested for PPAR activity in vitro. The SAR showed that dimeric ligands with a common group or full dimeric ligands had retained or even increased PPARγ potency. The dimeric agonist concept can be used to fine tune the subtype selectivity of PPAR agonists. The PPARγ potency could, at least partly, be explained using molecular modeling.

Peroxisome proliferator-activated receptor (PPAR)-alpha controls the transcription of a variety of genes involved in lipid metabolism and is the target receptor for the hypolipidemic drug class of fibrates. In the present study, the molecular and physiological effects of seven different PPAR-activating drugs have been examined in a rodent model of dyslipidemia. The drugs examined were selected to display varying potencies and efficacies toward PPAR-alpha. To help elucidate the link between the gene regulation elicited by PPAR-alpha ligands and the concomitant physiological changes, we have used cDNA microarray analysis to identify smaller gene sets that are predictive of the function of these ligands. A number of genes showed strong correlations to the relative PPAR-alpha efficacy of the drugs. Furthermore, using multivariate analysis, a strong relationship between the drug-induced triglyceride lowering and the transcriptional profiles of the different drugs could be found.

Two potent nonselective, but PPARalpha-preferring, PPAR agonists 5 and 6 were designed and synthesized in high yields. The concept of dimeric ligands in transcription factors was investigated by synthesizing and testing the corresponding dimers 7, 8a, and 8b in PPAR transactivation assays. The three dimeric ligands all showed agonist activity on all three PPAR receptor subtypes, but with different profiles compared to the monomers 5 and 6. Despite breaking all the "rule of five" criteria, the dimers had excellent oral bioavailability and pharmacokinetic properties, resulting in good in vivo efficacy in db/db mice. X-ray crystal structure and modeling experiments suggested that the dimers interacted with the AF-2 helix as well as with amino acid residues in the lipophilic pocket close to the receptor surface.

Chronic treatment with compounds activating peroxisome proliferator-activated receptor (PPAR)gamma and -alpha influences body energy stores, but the underlying mechanisms are only partially known. In a chronic-dosing study, equiefficacious antihyperglycemic doses of the PPAR gamma agonist pioglitazone and PPAR alpha/gamma dual activator ragaglitazar were administered to obesity-prone male rats. The PPAR alpha agonist fenofibrate had no effect on insulin sensitivity. Pioglitazone transiently increased and fenofibrate transiently decreased food intake, whereas ragaglitazar had no impact on feeding. As a result, body adiposity increased in pioglitazone-treated rats and decreased in fenofibrate-treated rats. PPAR gamma compounds markedly increased feed efficiency, whereas PPAR alpha agonist treatment decreased feed efficiency. In fenofibrate-treated rats, plasma acetoacetate was significantly elevated. Plasma levels of this potentially anorectic ketone body were unaffected in pioglitazone- and ragaglitazar-treated rats. High-fat feeding markedly increased visceral fat pads, and this was prevented by pioglitazone and ragaglitazar treatment. Pioglitazone treatment enlarged subcutaneous adiposity in high-fat-fed rats. In conclusion, PPAR gamma activation increases both food intake and feed efficiency, resulting in net accumulation of subcutaneous body fat. The impact of PPAR gamma activation on feeding and feed efficiency appears to be partially independent because the PPAR alpha component of ragaglitazar completely counteracts the orexigenic actions of PPAR gamma activation without marked impact on feed efficiency.

Using a known dual PPARalpha/gamma activator (5) as a structural template, SAR evaluations led to the identification of triple PPARalpha/gamma/delta activators (18-20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARalpha/gamma/delta activation.