Diminished responses to monoaminergic antidepressants but not ketamine in a mouse model for neuropsychiatric lupus.

BACKGROUND: A significant proportion of patients suffering from major depression fail to remit following treatment and develop treatment-resistant depression. Developing novel treatments requires animal models with good predictive validity. MRL/lpr mice, an established model of systemic lupus erythematosus, show depression-like behavior. AIMS: We evaluated responses to classical antidepressants, and associated immunological and biochemical changes in MRL/lpr mice. METHODS AND RESULTS: MRL/lpr mice showed increased immobility in the forced swim test, decreased wheel running and sucrose preference when compared with the controls, MRL/MpJ mice. In MRL/lpr mice, acute fluoxetine (30 mg/kg, intraperitoneally (i.p.)), imipramine (10 mg/kg, i.p.) or duloxetine (10 mg/kg, i.p.) did not decrease the immobility time in the Forced Swim Test. Interestingly, acute administration of combinations of olanzapine (0.03 mg/kg, subcutaneously)+fluoxetine (30 mg/kg, i.p.) or bupropion (10 mg/kg, i.p.)+fluoxetine (30 mg/kg, i.p.) retained efficacy. A single dose of ketamine but not three weeks of imipramine (10 mg/kg, i.p.) or escitalopram (5 mg/kg, i.p.) treatment in MRL/lpr mice restored sucrose preference. Further, we evaluated inflammatory, immune-mediated and neuronal mechanisms. In MRL/lpr mice, there was an increase in autoantibodies' titers, [3H]PK11195 binding and immune complex deposition. There was a significant infiltration of the brain by macrophages, neutrophils and T-lymphocytes. p11 mRNA expression was decreased in the prefrontal cortex. Further, there was an increase in the 5-HT2aR expression, plasma corticosterone and indoleamine 2,3-dioxygenase activity. CONCLUSION: In summary, the MRL/lpr mice could be a useful model for Treatment Resistant Depression associated with immune dysfunction with potential to expedite antidepressant drug discovery.

Preclinical Characterization of (R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169), a Novel, Intravenous, Glutamate N-Methyl-d-Aspartate 2B Receptor Negative Allosteric Modulator with Potential in Major Depressive Disorder.

(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169) and the phosphate prodrug 4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl dihydrogen phosphate (BMS-986163) were identified from a drug discovery effort focused on the development of novel, intravenous glutamate N-methyl-d-aspartate 2B receptor (GluN2B) negative allosteric modulators (NAMs) for treatment-resistant depression (TRD). BMS-986169 showed high binding affinity for the GluN2B subunit allosteric modulatory site (Ki = 4.03-6.3 nM) and selectively inhibited GluN2B receptor function in Xenopus oocytes expressing human N-methyl-d-aspartate receptor subtypes (IC50 = 24.1 nM). BMS-986169 weakly inhibited human ether-a-go-go-related gene channel activity (IC50 = 28.4 µM) and had negligible activity in an assay panel containing 40 additional pharmacological targets. Intravenous administration of BMS-986169 or BMS-986163 dose-dependently increased GluN2B receptor occupancy and inhibited in vivo [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding, confirming target engagement and effective cleavage of the prodrug. BMS-986169 reduced immobility in the mouse forced swim test, an effect similar to intravenous ketamine treatment. Decreased novelty suppressed feeding latency, and increased ex vivo hippocampal long-term potentiation was also seen 24 hours after acute BMS-986163 or BMS-986169 administration. BMS-986169 did not produce ketamine-like hyperlocomotion or abnormal behaviors in mice or cynomolgus monkeys but did produce a transient working memory impairment in monkeys that was closely related to plasma exposure. Finally, BMS-986163 produced robust changes in the quantitative electroencephalogram power band distribution, a translational measure that can be used to assess pharmacodynamic activity in healthy humans. Due to the poor aqueous solubility of BMS-986169, BMS-986163 was selected as the lead GluN2B NAM candidate for further evaluation as a novel intravenous agent for TRD.

Characterization of the adrenocorticotrophic hormone - induced mouse model of resistance to antidepressant drug treatment.

Approximately 30-60% of patients treated with existing antidepressants fail to achieve remission of depressive symptoms leading to Treatment Resistant Depression (TRD). There is an urgent need to develop novel medications, which is highly limited by the non-availability of relevant animal models with good predictive validity. ACTH administration has been shown to result in the resistance to acute and chronic effects of imipramine. However, the pharmacology of the model and the mechanisms contributing to the resistance are not completely understood. Furthermore, it is not known whether the ACTH administered animals show signs of depression-like behavior. Accordingly, we characterized the behavioral profile and sensitivity to antidepressants in BALB/c mice treated with ACTH and to evaluate some of the mechanisms responsible for the behavioral effects. Daily treatment with ACTH for 14, 21 or 28days failed to produce a depression-like phenotype in the sucrose preference test, voluntary wheel running or FST. In contrast, the acute antidepressant response in the FST was no longer observed in ACTH mice treated with fluoxetine, imipramine, duloxetine or bupropion. Interestingly, the combination of fluoxetine and a low dose of olanzapine, or the combination of fluoxetine and bupropion was efficacious in ACTH treated mice. Further, the sensitivity to a GluN2B receptor antagonist, radiprodil was retained in the ACTH model. To understand the mechanism responsible for the diminished response in these mice, we evaluated p11 (S100A10) mRNA expression and 5-HT2A protein expression. p11 expression was decreased and 5-HT2A protein content increased in ACTH treated mice. In summary, this model may have utility for the identification of novel treatments for TRD.

[3H]BMT-046091 a potent and selective radioligand to determine AAK1 distribution and target engagement.

Adaptor-associated kinase 1 (AAK1), a member of the Ark1/Prk1 family of serine/threonine kinases, plays a role in modulating clatherin coated endocytosis of specific surface receptors. We have demonstrated that AAK1 inhibitors are efficacious in rodent models of neuropathic pain (Kostich et al., 2016). Here we have characterized the binding properties and distribution pattern of the tritiated AAK1 radioligand, [3H]BMT-046091, in rodents and cynomolgus monkeys, and used the radioligand to measure the brain target occupancy following systemic administration of AAK1 inhibitors. We have found that [3H]BMT-046091 is potent and selective AAK1 inhibitor. It inhibits AAK1 phosphorylation of a peptide derived from a physiologic substrate, the µ2 subunit of the adaptor protein complex, with an IC50 value of 2.8 nM, and is inactive at >5 µM in a panel of functional or binding assays for receptors, transporters and enzymes. [3H]BMT-046091 binding in the brain is absent in the AAK1 knockout mouse, and is displaceable with a high concentration of AAK1 inhibitors in wild type mice. Specific [3H]BMT-046091 binding is widespread in the brain and spinal cord with the highest density in the cortex, hippocampus, amygdala, striatum and thalamus. In the spinal cord, [3H]BMT-046091 binding appears enriched in the dorsal horn superficial layers. Oral administration of LP-935509, an AAK1 inhibitor, results in a dose-dependent occupation of AAK1 binding sites in the brain and spinal cord. The increase in AAK1 binding site occupancy by LP-935509 correlates with the decrease in antinociceptive responses in the rat chronic constriction injury model of neuropathic pain.

Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Role of hepatic blood flow and metabolism in the pharmacokinetics of ten drugs in lean, aged and obese rats.

1. The effect of age and obesity on the pharmacokinetics (PK), hepatic blood flow (HBF) and liver metabolism of 10 compounds was determined in rats. The animals fed a high-fat diet were defined as the diet-induced obese (DIO) group, while the animals that were aged similar to the DIO rats but not fed with high-fat diet were called the age-matched (AM) group. 2. The clearance (CL) values of high CL compounds (CL > 50 mL/min/kg, namely propranolol, diazepam, phenytoin, ethinylestradiol, lorcaserin and fenfluramine) decreased significantly (1.5- to 6-fold) in DIO and AM rats as compared to lean rats, while there was no clear trend for change in CL for the low-to-moderate CL compounds (CL < 50 mL/min/kg, namely atenolol, chlorzoxazone, vancomycin and sibutramine). Hepatocytes incubations revealed a change in half life (t1/2) only for phenytoin. The body weight normalized liver weights and HBF of AM and DIO rats were found to be 2- to 3-fold lower than in lean rats. 3. Our findings suggest that age, and diet to a lesser extent, can reduce HBF and body normalized liver weights and, hence, also reduce CL values for high CL compounds in rats.

Effects of lipoprotein lipase and statins on cholesterol uptake into heart and skeletal muscle.

Regulation of cholesterol metabolism in cultured cells and in the liver is dependent on actions of the LDL receptor. However, nonhepatic tissues have multiple pathways of cholesterol uptake. One possible pathway is mediated by LPL, an enzyme that primarily hydrolyzes plasma triglyceride into fatty acids. In this study, LDL uptake and tissue cholesterol levels in heart and skeletal muscle of wild-type and transgenic mice with alterations in LPL expression were assessed. Overexpression of a myocyte-anchored form of LPL in heart muscle led to increased uptake of LDL and greater heart cholesterol levels. Loss of LDL receptors did not alter LDL uptake into heart or skeletal muscle. To induce LDL receptors, mice were treated with simvastatin. Statin treatment increased LDL receptor expression and LDL uptake by liver and skeletal muscle but not heart muscle. Plasma creatinine phosphokinase as well as muscle mitochondria, cholesterol, and lipid droplet levels were increased in statin-treated mice overexpressing LPL in skeletal muscle. Thus, pathways affecting cholesterol balance in heart and skeletal muscle differ.

Insulin sensitizing property of Indigofera mysorensis extract.

Indigofera mysorens is a shrub used for its antidiabetic activity in rural India. Here, we elucidate the antidiabetic potential of Indigofera mysorensis extract. Ethanolic extract of the whole shrub of Indigofera (EEI) at 300 mg/kg for 10 days, produced a 63% reduction in plasma glucose, 41% reduction in plasma triglyceride and 77% reduction in plasma insulin levels in insulin resistant db/db mice, which is better than insulin sensitizer, troglitazone (400 mg/kg). EEI unlike sulphonylureas failed to show any acute hypoglycemic effect in normoglycemic Swiss albino mice (SAM). Even in a chronic study (10 days) in SAM, EEI (300 mg/kg) like insulin sensitizers showed no effect on plasma glucose, but an 81% reduction in plasma insulin levels. When challenged with 3 gm/kg sucrose, SAM treated with EEI (300 mg/kg, 7 days) failed to show any effect on the absorption of sugar, whereas standard drug, acarbose (10 mg/kg) showed 52% reduction in the area under the plasma glucose curve. EEI failed to show any significant transactivation of PPARgamma, a proposed target of synthetic insulin sensitizers. Taken together, our data indicate that the antidiabetic effect of the ethanolic extract of Indigofera is due to its insulin sensitizing property and is clearly different from that of sulfonylurea or acarbose.

Human aldose reductase expression accelerates diabetic atherosclerosis in transgenic mice.

Direct evidence that hyperglycemia, rather than concomitant increases in known risk factors, induces atherosclerosis is lacking. Most diabetic mice do not exhibit a higher degree of atherosclerosis unless the development of diabetes is associated with more severe hyperlipidemia. We hypothesized that normal mice were deficient in a gene that accelerated atherosclerosis with diabetes. The gene encoding aldose reductase (AR), an enzyme that mediates the generation of toxic products from glucose, is expressed at low levels in murine compared with human tissues. Mice in which diabetes was induced through streptozotocin (STZ) treatment, but not nondiabetic mice, expressing human AR (hAR) crossed with LDL receptor-deficient (Ldlr-/-) C57BL/6 male mice had increased aortic atherosclerosis. Diabetic hAR-expressing heterozygous LDL receptor-knockout mice (Ldlr+/-) fed a cholesterol/cholic acid-containing diet also had increased aortic lesion size. Lesion area at the aortic root was increased by STZ treatment alone but was further increased by hAR expression. Macrophages from hAR-transgenic mice expressed more scavenger receptors and had greater accumulation of modified lipoproteins than macrophages from nontransgenic mice. Expression of genes that regulate regeneration of glutathione was reduced in the hAR-expressing aortas. Thus, hAR increases atherosclerosis in diabetic mice. Inhibitors of AR or other enzymes that mediate glucose toxicity could be useful in the treatment of diabetic atherosclerosis.

Peroxisome proliferator-activated receptor agonists modulate heart function in transgenic mice with lipotoxic cardiomyopathy.

hLpL(GPI) transgenic mice that overexpress human lipoprotein lipase (hLpL) with a glycosylphosphatidylinositol anchor on cardiomyocytes develop lipotoxic cardiomyopathy associated with increased cardiac uptake of plasma lipids. We hypothesized that peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, or a PPARalpha/gamma agonist would alter cardiac function by modulating lipid uptake by the heart. hLpL(GPI) mice were administered rosiglitazone (10 mg/kg/day), fenofibrate (100 mg/kg/day), or DRF2655, an alkoxy propanoic acid analog (10 mg/kg/day), for 16 days. Rosiglitazone reduced plasma triglyceride (TG) from 107.63 +/- 6.98 to 77.61 +/- 3.98 mg/dl, whereas fenofibrate had no effect. DRF2655 reduced TG to 33.17 +/- 4.12 mg/dl. Rosiglitazone and DRF2655 decreased heart TG and total cholesterol; fenofibrate had no effect. Molecular markers for cardiac dysfunction, atrial natriuretic factor, brain natriuretic peptide, and tumor necrosis factor-alpha were decreased with rosiglitazone and increased with fenofibrate. Echocardiographic measurements showed reduced fractional shortening and increased left ventricular systolic dimension with fenofibrate. No changes in these parameters were observed with rosiglitazone or DRF2655 treatment. Muscle-specific carnitine palmitoyltransferase-1 and fatty acid transporter protein-1 gene expression were increased with fenofibrate and DRF2655 treatment; no change in expression of these genes was noted with rosiglitazone treatment. Rosiglitazone and DRF2655 reduced TG uptake by the heart, and fenofibrate treatment increased fatty acid uptake. Thus, in a lipotoxic cardiomyopathy mouse model, a PPARgamma agonist reduced cardiac lipid and markers of cardiomyopathy, whereas an agonist of PPARalpha did not improve cardiac lipids and worsened heart function. These changes were paralleled by alterations in heart lipid uptake. Overall, PPAR activators exhibit differential effects in this model of lipotoxic dilated cardiomyopathy.

Atherosclerosis in perlecan heterozygous mice.

The hypothesis that lipoprotein association with perlecan is atherogenic was tested by studying atherosclerosis in mice that had a heterozygous deletion of perlecan, the primary extracellular heparan sulfate proteoglycan in arteries. We first studied the expression of perlecan in mouse lesions and noted that this proteoglycan in aorta was found in the subendothelial matrix. Perlecan was also a major component of the lesional extracellular matrix. Mice with a heterozygous deletion had a reduction in arterial wall perlecan expression. Atherosclerosis in these mice was studied after crossing the defect into the apolipoprotein E (apoE) and LDL receptor knockout backgrounds. At 12 weeks, chow-fed apoE null mice with a heterozygous deletion had less atherosclerosis. However, at 24 weeks and in the LDL receptor heterozygous background, the presence of a perlecan knockout allele did not significantly alter lesion size. Thus, it appears that loss of perlecan leads to less atherosclerosis in early lesions. Although this might be attributable to a decrease in lipoprotein retention, it should be noted that perlecan might mediate multiple other processes that could, in sum, accelerate atherosclerosis.

Antidiabetic and hypolipidemic potential of DRF 2519--a dual activator of PPAR-alpha and PPAR-gamma.

We investigated the biological activity of Dr. Reddy's Research Foundation (DRF) 2519, a benzoxazinone analogue of the thiazolidinedione class of compounds. In the in vitro transactivation assay, DRF 2519 showed interesting dual activation of Peroxisome Proliferator Activated Receptor (PPAR) alpha and gamma. In insulin-resistant ob/ob mouse model, DRF 2519 showed significant alleviation of insulin resistance and dyslipidemia, which is better than rosiglitazone. Fatty Zucker rats treated with DRF 2519 showed better reduction of plasma insulin, triglyceride and free fatty acid levels than those treated with rosiglitazone. In addition, these rats were able to clear plasma lipids better when challenged with exogenous lipid (i.v.). DRF 2519 treatment resulted in improved plasma lipid profiles in high-fat-fed Sprague-Dawley rats. Treated rats showed better plasma lipid clearance and hepatic triglyceride secretion. When compared to DRF 2519, fenofibrate was comparatively less efficacious while rosigltiazone showed no activity in these models. In ex vivo studies, DRF 2519 showed induction of liver acyl CoA oxidase mRNA and increase in lipoprotein lipase (LPL) protein expression and activity in adipose tissue. In the in vitro studies, DRF 2519 inhibited the lipid biosynthesis and secretion of apolipoprotein B from human hepatoma (Hep)G2 cells. It also enhanced insulin-induced relaxation of rat aortic smooth muscle. These results indicate that DRF 2519, a dual activator of PPAR-alpha and gamma, could be an interesting development candidate in the management of metabolic disorders and associated complications.

Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression.

Fatty acids are the primary energy source for the heart. The heart acquires fatty acids associated with albumin or derived from lipoprotein lipase (LpL)-mediated hydrolysis of lipoprotein triglyceride (TG). We generated heart-specific LpL knock-out mice (hLpL0) to determine whether cardiac LpL modulates the actions of peroxisome proliferator-activated receptors and affects whole body lipid metabolism. Male hLpL0 mice had significantly elevated plasma TG levels and decreased clearance of postprandial lipids despite normal postheparin plasma LpL activity. Very large density lipoprotein-TG uptake was decreased by 72% in hLpL0 hearts. However, heart uptake of albumin-bound free fatty acids was not altered. Northern blot analysis revealed a decrease in the expression of peroxisome proliferator-activated receptor alpha-response genes involved in fatty acid beta-oxidation. Surprisingly, the expression of glucose transporters 1 and 4 and insulin receptor substrate 2 was increased and that of pyruvate dehydrogenase kinase 4 and insulin receptor substrate 1 was reduced. Basal glucose uptake was increased markedly in hLpL0 hearts. Thus, the loss of LpL in the heart leads to defective plasma metabolism of TG. Moreover, fatty acids derived from lipoprotein TG and not just albumin-associated fatty acids are important for cardiac lipid metabolism and gene regulation.

Ragaglitazar: a novel PPAR alpha PPAR gamma agonist with potent lipid-lowering and insulin-sensitizing efficacy in animal models.

Ragaglitazar [(-) DRF 2725; NNC 61-0029] is a coligand of PPARalpha and PPARgamma. In ob/ob mice, ragaglitazar showed significant reduction in plasma glucose, triglyceride and insulin (ED50 values <0.03, 6.1 and <0.1 mg kg-1). These effects are three-fold better than rosiglitazone and KRP-297. In Zucker fa/fa rats, ragaglitazar showed dose-dependent reduction in triglyceride and insulin, hepatic triglyceride secretion and triglyceride clearance kinetics (maximum of 74, 53, 32 and 50% at 3 mg kg-1), which are better than rosiglitazone and KRP-297. In a high-fat-fed hyperlipidaemic rat model, the compound showed an ED50 of 3.95, 3.78 mg kg-1 for triglyceride and cholesterol lowering, and 0.29 mg kg-1 for HDL-C increase. It also showed improvement in clearance of plasma triglyceride and hepatic triglyceride secretion rate. All these effects are 3-10-fold better than fenofibrate and KRP-297. Ragaglitazar treatment showed significant reduction in plasma Apo B and Apo CIII levels, and increase in liver CPT1 and CAT activity and ACO mRNA. Significant increase of both liver and fat LPL activity and fat aP2 mRNA was also observed. In a high-fat-fed hamster model, ragaglitazar at 1 mg kg-1 showed 83 and 61% reduction in triglyceride and total cholesterol, and also 17% reduction in fat feed-induced body weight increase. In these hyperlipidaemic animal models, PPARgamma ligands failed to show any significant efficacy. Taken together, ragaglitazar shows better insulin-sensitizing and lipid-lowering potential, as compared to the standard compounds.

PAT5A: a partial agonist of peroxisome proliferator-activated receptor gamma is a potent antidiabetic thiazolidinedione yet weakly adipogenic.

PAT5A [5-[4-[N-(2-pyridyl)-(2S)-pyrrolidine-2-methoxyl]phenylmethylene[thiazolidine-2,4-dione, malic acid salt]], a chemically distinct unsaturated thiazolidinedione, activates peroxisome proliferator-activated receptor gamma (PPARgamma) submaximally in vitro with the binding affinity approximately 10 times less than that of rosiglitazone, a highly potent thiazolidinedione. PAT5A reduces plasma glucose level and improves insulin sensitivity in insulin resistant db/db mice, similar to that of rosiglitazone, while exerting a relatively weak adipogenic effect. In contrast to rosiglitazone, PAT5A inhibits cholesterol and fatty acid biosynthesis suggesting that PAT5A possesses a unique receptor-independent non-PPAR related property. PAT5A induces qualitatively similar but quantitatively different protease digestion patterns and interacts with PPARgamma differently than rosiglitazone. PAT5A shows differential cofactor recruitment and gene activation than that of rosiglitazone. Thus, the partial agonism of PAT5A to PPARgamma together with its receptor independent effects may contribute to its antidiabetic potency similar to rosiglitazone in vivo despite reduced affinity for PPARgamma. These biological effects suggest that PAT5A is a PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

DRF 2655: a unique molecule that reduces body weight and ameliorates metabolic abnormalities.

OBJECTIVE: Preclinical evaluation of DRF 2655, a peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma agonist, as a body-weight lowering, hypolipidemic and euglycemic agent. RESEARCH METHODS AND PROCEDURES: DRF 2655 was studied in different genetic, normal, and hyperlipidemic animal models. HEK 293 cells were used to conduct the reporter-based transactivation of PPARalpha and PPARgamma. To understand the biochemical mechanism of lipid-, body-weight-, and glucose-lowering effects, activities of key beta-oxidation and lipid catabolism enzymes and gluconeogenic enzymes were studied in db/db mice treated with DRF 2655. 3T3L1 cells were used for adipogenesis study, and HepG2 cells were used to study the effect of DRF 2655 on total cholesterol and triglyceride synthesis using [(14)C]acetate and [(3)H]glycerol. RESULTS: DRF 2655 showed concentration-dependent transactivation of PPARalpha and PPARgamma. In the 3T3L1 cell-differentiation study, DRF 2655 and rosiglitazone showed 369% and 471% increases, respectively, in triglyceride accumulation. DRF 2655 showed body-weight lowering and euglycemic and hypolipidemic effects in various animal models. db/db mice treated with DRF 2655 showed 5- and 3.6-fold inhibition in phosphoenolpyruvate carboxykinase and glucose 6-phosphatase activity and 651% and 77% increases in the beta-oxidation enzymes carnitine palmitoyltransferase and carnitine acetyltransferase, respectively. HepG2 cells treated with DRF 2655 showed significant reduction in lipid synthesis. DISCUSSION: DRF 2655 showed excellent euglycemic and hypolipidemic activities in different animal models. An exciting finding is its body-weight lowering effect in these models, which might be mediated by the induction of target enzymes involved in hepatic lipid catabolism through PPARalpha activation.

Antidiabetic and hypolipidemic activity of Helicteres isora in animal models.

Helicteres isora (Sterculiaceae) root juice has been used in the treatment of diabetes by several ethnic groups in different parts of India. A program was initiated to elucidate the scientific basis for the antidiabetic activity of H. isora. Ethanolic extract of H. isora root caused significant reduction in plasma glucose, triglyceride and insulin levels at 300 mg/kg dose after 9 days of administration to insulin resistant and diabetic C57BL/KsJdb/db mice. In normoglycemic and mildly hypertriglyceridemic Swiss albino mice, the extract also showed significant reduction in plasma triglyceride and insulin levels, without affecting plasma glucose level. An ethanolic extract showed activity distinctly different from glybenclamide and acarbose but similar to troglitazone in these models. In high fat fed hamster model, the extract showed significant reduction in plasma lipid levels. In order to identify the active pharmacophore, the ethanolic extract was further subjected to sequential partitioning with low, medium and high polarity solvents, which yielded a semipurified fraction having both euglycemic and lipid-lowering activity. Our study suggests that the extract of H. isora has insulin-sensitizing and hypolipidemic activity and has the potential for use in the treatment of type-2 diabetes.

Synthesis and biological activity of novel pyrimidinone containing thiazolidinedione derivatives.

A series of pyrimidinone derivatives of thiazolidinediones were synthesized. Their biological activity were evaluated in insulin resistant, hyperglycemic and obese db/db mice. In vitro PPARgamma transactivation assay was performed in HEK 293T cells. PMT13 showed the best biological activity in this series. PMT13 (5-[4-[2-[2-ethyl-4-methyl-6-oxo-1,6-dihydro-1-pyrimidinyl]ethoxy]phenylmethyl]thiazolidine-2,4-dione) showed better plasma glucose, triglyceride and insulin-lowering activity in db/db mice than rosiglitazone and pioglitazone. PMT13 showed better PPARgamma transactivation than the standard compounds. Pharmacokinetic study in Wistar rats showed good systemic exposure of PMT13. Twenty-eight day oral toxicity study in Wistar rats did not show any treatment-related adverse effects.