Design, synthesis and biological evaluation of novel pyrazolo-pyrimidin-amines as potent and selective BTK inhibitors.

To discover the best-in-class Bruton's Tyrosine Kinase (BTK) inhibitors, for th treatment of autoimmune disorders like cancer (B-Cell Lymphoma (BCL)) and rheumatoid arthritis (RA), in the present investigation, novel structural optimizations were carried out. Introduction of novel bicyclic amine linkers and aromatic backbone led to series of compounds 9a-h and 14a-u. Compound 14b was found to be potent, orally bioavailable, selective and irreversible BTK inhibitor. In vitro, 14b showed IC50 of 1.0 nM and 0.8 nM, in BTK and TMD8 assays, respectively. In vivo,14b displayed robust efficacy in collagen-induced arthritis (CIA) and TMD8 xenograft models, which could be correlated with its improved oral bioavailability. In the repeated dose acute toxicity study, 14b showed no adverse changes, indicating that the BTK inhibitor 14b could be viable therapeutic option for the treatment of autoimmune disorders.

ZYKR1, a novel, potent, and peripherally selective kappa opioid receptor agonist reduces visceral pain and pruritus in animal models.

Opioid receptor agonists are effective analgesic agents. Central activation of the mu and/or kappa opioid receptors (KOR) is associated with CNS side effects, which limits their effectiveness. Recent studies indicated that peripherally restricted, selective KOR agonists were potent analgesics and devoid of CNS-related side effects. To confirm this hypothesis, we designed a novel, potent, and peripherally restricted KOR-selective agonist, ZYKR1. The analgesic efficacy, brain penetration and safety of ZYKR1 were assessed in pre-clinical models. ZYKR1 showed KOR agonistic activity in the cAMP assay, with an EC50 of 0.061 nM and more than 105-fold selectivity over the mu and delta opioid receptors (EC50 > 10 µM). ZYKR1 was not found to bind mu, delta opioid, and NOP receptors in radioligand binding assays. ZYKR1 produced concentration-dependent inhibition of electrically evoked contractions in isolated mouse vas deferens with an IC50 of 1.6 nM ZYKR1 showed peripheral restriction and potent analgesic efficacy in various in-vivo animal models (acetic acid induced visceral pain mouse model, ED50: 0.025 mg/kg, IV; ovariohysterectomy induced postoperative pain rat model, ED50: 0.023 mg/kg, IV; and C48/80 induced pruritus mouse model, ED50: 0.063 mg/kg, IV). In addition, ZYKR1 was devoid of motor coordination, physical dependence, dysphoria, and respiratory depression at 30, 400, 10 and 10-fold of efficacy dose, respectively. In conclusion, ZYKR1 has potent antinociceptive action in visceral pain and pruritus with limited CNS side effects in preclinical models owing to its peripheral restriction.

ZYBT1, a potent, irreversible Bruton's Tyrosine Kinase (BTK) inhibitor that inhibits the C481S BTK with profound efficacy against arthritis and cancer.

Bruton's tyrosine kinase (BTK) plays a central and pivotal role in controlling the pathways involved in the pathobiology of cancer, rheumatoid arthritis (RA), and other autoimmune disorders. ZYBT1 is a potent, irreversible, specific BTK inhibitor that inhibits the ibrutinib-resistant C481S BTK with nanomolar potency. ZYBT1 is found to be a promising molecule to treat both cancer and RA. In the present report we profiled the molecule for in-vitro, in-vivo activity, and pharmacokinetic properties. ZYBT1 inhibits BTK and C481S BTK with an IC50 of 1 nmol/L and 14 nmol/L, respectively, inhibits the growth of various leukemic cell lines with IC50 of 1 nmol/L to 15 µmol/L, blocks the phosphorylation of BTK and PLCγ2, and inhibits secretion of TNF-α, IL-8 and IL-6. It has favorable pharmacokinetic properties suitable for using as an oral anti-cancer and anti-arthritic drug. In accordance with the in-vitro properties, it demonstrated robust efficacy in murine models of collagen-induced arthritis (CIA) and streptococcal cell wall (SCW) induced arthritis. In both models, ZYBT1 alone could suppress the progression of the diseases. It also reduced the growth of TMD8 xenograft tumor. The results suggested that ZYBT1 has high potential for treating RA, and cancer.

Combination of Lorcaserin and GLP-1/glucagon Coagonist Improves Metabolic Dysfunction in Diet Induced-obese Mice.

BACKGROUND: Obesity and diabetes are major metabolic disorders that progress to severe morbidity and mortality. Neuroendocrine mechanisms controlling energy balance indicate that combination therapies are needed to sustain weight loss. Lorcaserin was one of the approved therapies for the treatment of obesity, which is recently withdrawn because a safety clinical trial, shows an increased occurrence of cancer. Coagonist of glucagon-like-peptide-1 (GLP-1) and glucagon receptors is a novel investigational therapy demonstrated to have both anti-obesity and anti-diabetic effect. Here, we investigated the effect of combination of lorcaserin and a GLP-1 and glucagon receptors coagonist in diet-induced obese (DIO) mice model. METHODS: The diet-induced obese C57BL/6J mice were used to assess acute and chronic effect of lorcaserin, coagonist of GLP-1and glucagon receptors and their combination on food intake, body weight, and biochemical parameters. RESULTS: In acute study, combination of lorcaserin and coagonist causes synergistic reductions in food intake and body weight. Repeated treatment of combination of lorcaserin and coagonist showed enhanced body weight loss over time, which is due to reduction in fat mass (subcutaneous, retroperitoneal, mesenteric and epididymal fat pad) compared to individual therapy. Also, suppression of locomotor activity seen with lorcaserin was not evident in combination with coagonist. No additive effect was observed in glucose tolerance (intraperitoneal glucose tolerance test or insulin tolerance test), serum lipids, hepatic lipids, and energy expenditure in combination group. CONCLUSION: These data suggest that combination of lorcaserin and coagonist could be a better combination to induce body weight loss.

Discovery of diaminopyrimidine-carboxamide derivatives as JAK3 inhibitors.

Selective inhibition of janus kinase (JAK) has been identified as an important strategy for the treatment of autoimmune disorders. Optimization at the C2 and C4-positions of pyrimidine ring of Cerdulatinib led to the discovery of a potent and orally bioavailable 2,4-diaminopyrimidine-5-carboxamide based JAK3 selective inhibitor (11i). A cellular selectivity study further confirmed that 11i preferentially inhibits JAK3 over JAK1, in JAK/STAT signaling pathway. Compound 11i showed good anti-arthritic activity, which could be correlated with its improved oral bioavailability. In the repeat dose acute toxicity study, 11i showed no adverse changes related to gross pathology and clinical signs, indicating that the new class JAK3 selective inhibitor could be viable therapeutic option for the treatment of rheumatoid arthritis.

Discovery of 1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-ones based novel, potent and PI3Kδ selective inhibitors.

PI3Kδ is implicated in various inflammatory and autoimmune diseases. For the effective treatment of chronic immunological disorders such as rheumatoid arthritis, it is essential to develop isoform selective PI3Kδ inhibitors. Structure guided optimization of an imidazo-quinolinones based pan-PI3K/m-TOR inhibitor (Dactolisib) led to the discovery of a potent and orally bioavailable PI3Kδ isoform selective inhibitor (10h), with an improved efficacy in the animal models.

Activation of GLP-1 and Glucagon Receptors Regulates Bile Homeostasis Independent of Thyroid Hormone.

BACKGROUND: Balanced coagonists of glucagon-like peptide-1 (GLP-1) and glucagon receptors are emerging therapies for the treatment of obesity and diabetes. Such coagonists also regulate lipid metabolism, independent of their body weight lowering effects. Many actions of the coagonists are partly mediated by fibroblast growth factor 21 (FGF21) signaling, with the major exception of bile homeostasis. Since thyroid hormone is an important regulator of bile homeostasis, we studied the involvement of thyroid hormone in coagonist-induced changes in lipid and bile metabolism. METHODS: We evaluated the effect of a single dose of coagonist Aib2 C24 chimera2 at 150 to 10000 µg/kg on tetraiodothyronine (T4) and triiodothyronine (T3) in high-fat diet-induced obese (DIO) mice and chow-fed mice. Repeated dose treatment of coagonist (150 µg/kg, subcutaneously) was assessed in four mice models namely, on lipid and bile homeostasis in DIO mice, propylthiouracil (PTU)-treated DIO mice, methimazole (MTM)-treated DIO mice and choline-deficient, L-amino acid-defined, highfat diet (CDAHFD)-induced nonalcoholic steatohepatitis (NASH). RESULTS: Single dose treatment of coagonist did not alter serum T3 and T4 in chow-fed mice and DIO mice. Coagonist treatment improved lipid metabolism and biliary cholesterol excretion. Chronic treatment of GLP-1 and glucagon coagonist did not alter serum T3 in hypothyroid DIO mice and CDAHFDinduced NASH. Coagonist increased serum T4 in DIO mice after 4 and 40 weeks of treatment, though no change in T4 levels was observed in hypothyroid mice or mice with NASH. CONCLUSION: Our data demonstrate that coagonist of GLP-1 and glucagon receptors does not modulate bile homeostasis via thyroid signaling.

Coagonist of glucagon-like peptide-1 and glucagon receptors ameliorates kidney injury in murine models of obesity and diabetes mellitus.

AIM: To investigate the role of glucagon-like peptide-1 (GLP-1)/glucagon receptors coagonist on renal dysfunction associated with diabetes and obesity. METHODS: Chronic high-fat diet fed C57BL/6J mice, streptozotocin-treated high-fat diet fed C57BL/6J mice and diabetic C57BLKS/J db/db mice were used as models of diabetes-induced renal dysfunction. The streptozotocin-treated high-fat diet fed mice and db/db mice were treated with the GLP-1 and glucagon receptors coagonist (Aib2 C24 Chimera2, 150 µg/kg, sc) for twelve weeks, while in chronic high-fat diet fed mice, coagonist (Aib2 C24 Chimera2, 150 µg/kg, sc) treatment was continued for forty weeks. Kidney function, histology, fibrosis, inflammation, and plasma biochemistry were assessed at the end of the treatment. RESULTS: Coagonist treatment decreased body weight, plasma lipids, insulin resistance, creatinine, blood urea nitrogen, urinary albumin excretion rate and renal lipids. In kidney, expression of lipogenic genes (SREBP-1C, FAS, and SCD-1) was decreased, and expression of genes involved in ß-oxidation (CPT-1 and PPAR-α) was increased due to coagonist treatment. In plasma, coagonist treatment increased adiponectin and FGF21 and decreased IL-6 and TNF-α. Coagonist treatment reduced expression of inflammatory (TNF-α, MCP-1, and MMP-9) and pro-fibrotic (TGF-ß, COL1A1, and α-SMA) genes and also improved histological derangement in renal tissue. CONCLUSION: Coagonist of GLP-1 and glucagon receptors alleviated diabetes and obesity-induced renal dysfunction by reducing glucose intolerance, obesity, and hyperlipidemia.

Coagonist of glucagon-like peptide-1 and glucagon receptors ameliorates nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. Coagonists of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) are under clinical investigation for the treatment of obesity and type 2 diabetes. In this study, we have demonstrated the effect of a balanced coagonist in the treatment of NAFLD using mouse models. GLP-1R agonist exendin-4, glucagon, and coagonist (Aib2 C24 chimera2) were administered to C57BL6/J mice, in which NAFLD was induced by carbon tetrachloride (CCl4) treatment after high-fat diet (HFD) feeding, and choline-deficient, L-amino-acid-defined HFD (CDAHFD) feeding. Repeated dose administration of coagonist significantly attenuated liver inflammation and steatosis induced by acute and long-term treatment with CCl4 in HFD-fed mice. Coagonist markedly attenuated the CDAHFD-induced expression of TIMP-1, MMP-9, TNF-α, MCP-1, COL1A1, and α-SMA. It also inhibited progression of hepatic steatosis and fibrosis in mice. Exendin-4 was better than glucagon, but coagonist was most effective in reduction of hepatic inflammation as well as steatosis. Coagonist of GLP-1R and GCGR improved NAFLD in C57BL6/J mice. This effect is mediated by reduction in lipotoxicity and inflammation in liver.

Coagonist of GLP-1 and glucagon decreases liver inflammation and atherosclerosis in dyslipidemic condition.

Dyslipidemia enhances progression of atherosclerosis. Coagonist of GLP-1 and glucagon are under clinical investigation for the treatment of obesity and diabetes. Earlier, we have observed that coagonist reduced circulating and hepatic lipids, independent of its anorexic effects. Here, we investigated the role of coagonist of GLP-1 and glucagon receptors in complications of diet-induced dyslipidemia in hamsters and humanized double transgenic mice. Hamsters fed on high fat high cholesterol diet were treated for 8 weeks with coagonist of GLP-1 and glucagon receptors (75 and 150 µg/kg). Pair-fed control was maintained. Cholesterol fed transgenic mice overexpressing hApoB100 and hCETP with coagonist (300 µg/kg) for 4 weeks. After the completion of treatment, biochemical estimations were done. Coagonist treatment reduced triglycerides in plasma, liver and aorta, plasma cholesterol and hepatic triglyceride secretion rate. Expressions of HMG-CoA reductase and SBREBP-1C were reduced and expressions of LDLR, CYP7A1, ABCA1 and ABCB11 were increased in liver, due to coagonist treatment. Coagonist treatment increased bile flow rate and biliary cholesterol excretion. IL-6 and TNF-α were reduced in plasma and expression of TNF-α, MCP-1, MMP-9 and TIMP-1 decreased in liver. Treatment with coagonist reduced oxidative stress in liver and aorta. Energy expenditure was increased and respiratory quotient was reduced by coagonist treatment. These changes were correlated with reduced hepatic inflammation and lipids in liver and aorta in coagonist treated hamsters. Coagonist treatment also reduced lipids in cholesterol-fed transgenic mice. These changes were independent of glycaemia and anorexia observed after coagonist treatment. Long term treatment with coagonist of GLP-1 and glucagon receptor ameliorated diet-induced dyslipidemia and atherosclerosis by regulating bile homeostasis, liver inflammation and energy expenditure.

Coagonist of GLP-1 and Glucagon Receptor Ameliorates Development of Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Obesity, diabetes and dyslipidemica are the key pathogenic stimulus that enhances progression of Non-Alcoholic Fatty Liver Disease (NAFLD). Coagonist of Glucagon Like- Peptide-1 (GLP-1) Receptor (GLP-1R) and Glucagon Receptor (GCGR) are being evaluated for obesity and diabetes. GLP-1 analogs have shown to reverse diabetes and obesity. Glucagon treatment reduces lipids after acute and chronic treatment. OBJECTIVE: In this study, we have investigated the effect of co-agonist on the prevention of NAFLD induced by long-term feeding of High Fat Diet (HFD). METHOD: We have used HFD to induce NAFLD after chronic feeding in mice. Co-agonist treatment (150 µg.kg-1, s.c.) was initiated with induction of HFD, which was continued for 40 weeks. Body weight, food intake, glucose homeostasis, lipid profile, inflammatory and fibrotic markers were assessed at the end of treatment. RESULTS: Co-agonist treatment prevented body weight gain, glucose intolerance and insulin resistance. Treatment with co-agonist reduced NEFA, increased FGF21 and adiponectin levels. Co-agonist increased glycerol release and energy expenditure, while decreased respiratory quotient. Co-agonist reduced lipids in circulation and liver. Expression of SREBP-1C, SCD-1, ACC and FAS were decreased, while ACOX1 and CPT1 were increased after co-agonist treatment. Inflammatory cytokine TNF-α and IL-6 in plasma and expression of MCP-1, TGF-ß, MMP-9, TNF-α, TIMP-1, α-SMA, and COL1A1 were decreased after co-agonist treatment. Plasma transaminases, hepatic TBARS, hepatic hydroxyproline and relative liver weight were suppressed after co-agonist treatment. Fat accumulation, inflammation and fibrosis were reduced in histological assessment of liver in co-agonist treated animals. CONCLUSION: Co-agonist prevented development of HFD-induced NAFLD by ameliorating obesity, diabetes, inflammation and fibrosis.

Central administration of coagonist of GLP-1 and glucagon receptors improves dyslipidemia.

Coagonists of Glucagon-like peptide-1 (GLP-1) and glucagon receptors are under clinical investigation for treatment of obesity associated with diabetes. In addition to their role in glucose homeostasis, GLP-1 and glucagon modulate lipid metabolism. In this study, we have investigated the role of central GLP-1 receptor (GLP-1R) and glucagon receptor (GCGR) activation in regulation of lipid metabolism in cholesterol-fed hamsters. Hamsters were treated with coagonist alone (0.3 µg) or in combination with either GLP-1R antagonist (0.15 µg) or GCGR antagonist (0.3 µg) for 4 weeks by intracerebroventricular route (icv). A pair-fed control to coagonist was included in the experiment. In a separate experiment, vagotomized hamsters were treated with coagonist (0.3 µg) for four weeks. At the end of the treatment, plasma and hepatic lipids, bile homeostasis, and hepatic gene expression were determined. Coagonist treatment caused a reduction in plasma and liver lipids, and reduced triglyceride absorption from intestine. Also, hepatic triglyceride secretion, bile flow, and biliary cholesterol excretion were increased by the coagonist treatment. Coagonist treatment exhibited increased energy expenditure and reduced the expression of SREBP-1C, HMG-CoA reductase, SCD-1, FAS and ACC in liver. Increase in the expression of LDLR, ACOX1, CPT-1, PPAR-α, CYP7A1, ABCA1 and ABCB11 was also observed in liver. The effect of coagonist on lipids was partially blocked by either GLP-1R or GCGR antagonist. Coadministration of GLP-1R antagonist blocked the effect of coagonist on bile flow, while effect of coagonist on biliary cholesterol was blocked by co-administration of GCGR antagonist. Coagonist did not affect lipid metabolism in vagotomized hamsters. It appears that central administration of coagonist reduces dyslipidemia by activation of GLP-1R and GCGR, independent of its anorectic effect.

Balanced Coagonist of GLP-1 and Glucagon Receptors Corrects Dyslipidemia by Improving FGF21 Sensitivity in Hamster Model.

Hyperlipidemia is often associated with obesity and diabetes, and can lead to serious complications like atherosclerosis and fatty liver disease. Coagonist of GLP-1 and glucagon receptors is a therapy under clinical investigation for treatment of obesity and diabetes. In this study, we have characterized the mechanism of hypolipidemic effect of a balanced coagonist using high cholesterol-fed hamsters. Tyloxapol-induced hypertriglyceridemia, lipolysis in adipose tissue, and bile homeostasis were assessed after repeated dose treatment of the coagonist of GLP-1 and glucagon receptors (Aib2 C24 chimera 2, SC). Antagonists of GLP-1, glucagon, and FGF21 receptors were coadministered, and FGF21 sensitivity was determined in liver and adipose tissue. Repeated dose treatment of coagonist reduced cholesterol and increased FGF21 in blood and liver. Coagonist treatment reduced hepatic triglyceride secretion, increased lipolysis and reduced body weight. Antagonism of GLP-1 and glucagon receptors partially blocked the effect of the coagonist on lipid metabolism in circulation and liver, while FGF21 receptor antagonist completely abolished it. Glucagon and GLP-1 receptors antagonists blocked the action of coagonist on cholesterol excretion and bile flow in liver, but FGF21 antagonist was not effective. Treatment with the coagonist increased expression of FGF21, FGF21R and cofactor ßKlotho in liver and adipose. In conclusion, coagonist of GLP-1 and glucagon receptors improved lipid metabolism in liver of dyslipidemic hamsters. This effect is partially mediated by GLP-1 and glucagon receptors, and the improved FGF21 sensitivity could be the mechanism of hypolipidemic action of the coagonist of GLP-1/glucagon receptors.

ZY15557, a novel, long acting inhibitor of dipeptidyl peptidase-4, for the treatment of Type 2 diabetes mellitus.

BACKGROUND AND PURPOSE: Dipeptidyl peptidase (DPP)-4 inhibitors increase levels of glucagon-like peptide-1 (GLP-1) and provide clinical benefit in the treatment of type 2 diabetes mellitus. As longer acting inhibitors have therapeutic advantages, we developed a novel DPP-4 inhibitor, ZY15557, that has a sustained action and long half-life. EXPERIMENTAL APPROACH: We studied the potency, selectivity, efficacy and duration of action of ZY15557, in vitro, with assays of DPP-4 activity. In vivo, the pharmacodymamics and pharmacokinetics of ZY15557 were studied, using db/db mice and Zucker fatty rats, along with normal mice, rats, dogs and non-human primates. KEY RESULTS: ZY15557 is a potent, competitive and long acting inhibitor of DPP-4 (Ki 5.53 nM; Koff 3.2 × 10-4 ·s-1 , half-life 35.8 min). ZY15557 treatment inhibited DPP-4 activity, and enhanced active GLP-1 and insulin in mice and rats, providing dose-dependent anti-hyperglycaemic effects. Anti-hyperglycaemic effects were also observed in db/db mice and Zucker fatty rats. Following oral dosing, ZY15557 significantly inhibited plasma DPP-4 activity, determined ex vivo, in mice and rats for more than 48 h, and for up to 168 h in dogs and non-human primates. Allometric scaling predicts a half-life for ZY15557 in humans of up to 60 h. CONCLUSIONS AND IMPLICATIONS: ZY15557 is a potent, competitive and long acting DPP-4 inhibitor. ZY15557 showed similar DPP-4 inhibition across different species. ZY15557 showed excellent oral bioavailability in preclinical species. It showed a low plasma clearance (CL) and large volume of distribution (Vss ) across species, resulting in an extended half-life.

Sensitive and specific LC-ESI-MS/MS method for determination of ZYDPLA1, a novel long-acting dipeptidyl peptidase 4 inhibitor in rat plasma: An application for toxicokinetic study in rats.

A rapid and highly specific assay was developed and validated for the estimation of ZYDPLA1 in rat plasma using liquid chromatography coupled to tandem mass spectrometry with positive electrospray ionization. Method validation comprised of parameters such as specificity, matrix effect, precision, accuracy, recovery, stability, etc. The assay procedure involved a simple protein precipitation of ZYDPLA1 and alprazolam (internal standard) from rat plasma using acetonitrile. Chromatographic separation was achieved with a gradient mobile phase comprising: (A) 0.2% ammonia in purified water; (B) 0.1% formic acid in isopropyl alcohol/methanol (1: 1 v/v); and (C) acetonitrile at a flow rate of 1 mL/min on an ACE-5, C18 (4.6 × 50 mm) column with a run time of 5.5 min. The quantitation of ZYDPLA1 was achieved by the summation of four multiple reaction mode transitions (m/z 399.7 → 383.0, 399.7 → 276.10, 399.7 → 153.20 and 399.7 → 127.20), while that of the internal standard was by a single multiple reaction mode transition (m/z 309.10 → 281.00). The lower limit of quantitation achieved was 0.01 µg/mL and the method showed linearity from 0.01 to 25 µg/mL. The intra- and inter-day precision (%CV) of the quality control samples was within 8.81% and accuracy was ±10% of nominal values. This novel method was applied for evaluation of toxicokinetics of ZYDLA1 in rats.

Central and Peripheral Glucagon Reduces Hyperlipidemia in Rats and Hamsters.

Increased lipid levels in blood contribute to increasing the risk of diabetic complications. Glucagon exerts lipid lowering effects in diabetic state. However, the mechanism behind the lipid reduction by glucagon independent of glucose homeostasis is not well understood. We assessed the actions of glucagon on lipid modulation in blood and markers in liver in hyperlipidemic hamsters and rats. Male Sprague Dawley rats and Golden Syrian hamsters on a hyperlipidemic diet for 2 weeks were administered a single dose of glucagon by subcutaneous (SC, 150 and 300 µg/kg) or intracerebroventricular (ICV, 15 and 30 µg/animal) route. Effect of acute treatment was observed on tyloxapol-induced hypertriglyceridemia, corn oil-induced post-prandial lipemia, and bile flow. A repeated dose treatment by subcutaneous (300 µg/kg) or intracerebroventricular (30 µg/animal) route was done for 2 weeks, following which circulating and hepatic lipids, hepatic markers of lipid metabolism and bile flow were assessed. Acute administration of glucagon (SC and ICV) decreased triglyceride absorption, hepatic triglyceride secretion rate and increased excretion of cholesterol in bile fluid in dose related manner. Repeated dose treatment reduced circulating and hepatic lipids and mainly LDL, and enhanced cholesterol excretion in bile. In liver, expression of HMG-CoA reductase was reduced while that of ABCA1 was increased after repeated treatment, whereas pair fed group did not show significant changes when compared to the control group. These findings demonstrate that central as well as peripheral glucagon effectively reduces hyperlipidemia in rat and hamster model, by modulating hepatic lipid metabolism.

Is the Inhibition of Dipeptidyl Peptidase-4 (DDP-4) Enzyme Route Dependent and/or Driven by High Peak Concentration?- Seeking Answers with ZYDPLA1, a Novel Long Acting DPP-4 Inhibitor, in a Rodent Model.

ZYDPLA1 is a long acting enzyme dipeptidyl peptidase-4 (DPP-4) inhibitor. The comparative effect of DPP-4 inhibition after intravenous (IV) and oral administration of ZYDPLA1 in a rat model was evaluated to answer the question of route dependency and/or the need of high plasma levels of ZYDPLA1. The study was conducted using parallel design in male Wistar rats for IV/oral route (n=9 and 6, for IV and oral respectively). A single 30 mg/kg dose of ZYDPLA1 was administered. Plasma samples were analysed for ZYDPLA1 concentration and DPP-4 inhibition. Pharmacokinetic analysis was carried out to assess peak concentration, area under the concentration-time curve, total body clearance, elimination half-life, and mean residence time. The PK/PD correlation was performed using standard sigmoidal Emax modelling to derive; maximum effect (Emax) and concentration to exert 50% Emax effect (EC50). ZYDPLA1 showed rapid absorption, high volume of distribution, low clearance, and complete oral bioavailability. The Emax derived after both routes and corresponding PK/PD profile showed comparable DDP-4 inhibition. The EC50 for IV (0.021 µg/mL) was comparable to the oral route (0.019 µg/mL). ZYDPLA1 showed full DPP-4 inhibition without regard to the route of administration. Higher systemic peak levels showed no bearing on the DDP-4 inhibition.

Central GLP-1 receptor activation improves cholesterol metabolism partially independent of its effect on food intake.

Glucagon-like peptide-1 (GLP-1) receptor agonists modulate lipid metabolism, apart from controlling glucose homeostasis. We investigated the role of central GLP-1 receptor (GLP-1R) agonism in regulation of hepatic lipid metabolism in cholesterol-fed hamsters. Cholesterol-fed hamsters were treated by intracerebroventricular (i.c.v.) route with exendin-4, as acute or repeated dose regimen and compared with hamsters pair-fed to the exendin-treated hamsters and with hamsters co-treated with GLP-1 antagonist exendin-9. Effect of acute treatment was observed on food intake, tyloxapol-induced hypertriglyceridemia, and corn oil induced post prandial lipemia. Plasma and hepatic lipids and changes in the expression of hepatic genes involved in lipid metabolism were assessed after chronic administration. Acute, as well as repeated dose, treatment of exendin-4 showed significant changes in hepatic lipids, circulating fatty acids, triglycerides, LDL, and cholesterol. Expression of SREBP-1c was reduced while that of LDLR and CYP7A1 was increased after the repeated dose treatment, and there was no change in HMG CoA reductase. These changes were blocked by co-treatment of exendin-9, and not replicated by pair feeding to the significant extent. Central GLP-1 receptor activation showed profound effects on peripheral lipid metabolism, which were partially independent of its effect on food intake.

Pharmacological characterization of ZYDPLA1, a novel long-acting dipeptidyl peptidase-4 inhibitor.

OBJECTIVE: Dipeptidyl peptidase-4 (DPP-4) is responsible for degradation of glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), the endogenous incretins that stimulate glucose-dependent insulin secretion. The objective was to evaluate preclinical profile of a novel DPP-4 inhibitor ZYDPLA1. METHODS: In vitro inhibition potency and selectivity were assessed using recombinant enzymes and/or plasma. In vivo efficacy was determined in oral glucose tolerance test or mixed meal tolerance test in C57BL/6J mice, db/db mice and Zucker fatty rats. Pharmacokinetics/pharmacodynamics was studied in mice, rats, dogs, and non-human primates. RESULTS: ZYDPLA1 is a potent, competitive and long acting inhibitor of DPP-4 (Ki 0.0027 µM; Koff 2.3 × 10(-4 ) s(-1) ). ZYDPLA1 was more than 7000-fold selective for recombinant DPP-4 relative to DPP-8 and DPP-9, and more than 60 000-fold selective relative to fibroblast activation protein (FAP) in vitro. DPP-4 inhibition was comparable across species. In vivo, oral ZYDPLA1 elevated circulating GLP-1 and insulin levels in mice and rats and showed dose-dependent anti-hyperglycemic effect. Anti-hyperglycemic effect was also observed in db/db mice and Zucker fatty rats. ZYDPLA1 showed low clearance, large volume of distribution, and a long half-life with excellent oral bioavailability in all species. It significantly inhibited plasma DPP-4 activity in mice and rats for more than 48 h, and for up to 168 h in dogs and non-human primates. Allometric scaling predicted a half-life in humans of 53 to 166 h. CONCLUSION: ZYDPLA1 is a potent, selective, long-acting oral DPP-4 inhibitor with potential to become once-a-week therapy for treatment of type 2 diabetes mellitus.

Cannabinoid receptor 1 antagonist treatment induces glucagon release and shows an additive therapeutic effect with GLP-1 agonist in diet-induced obese mice.

Cannabinoid 1 (CB1) receptor antagonists reduce body weight and improve insulin sensitivity. Preclinical data indicates that an acute dose of CB1 antagonist rimonabant causes an increase in blood glucose. A stable analog of glucagon-like peptide 1 (GLP-1), exendin-4 improves glucose-stimulated insulin secretion in pancreas, and reduces appetite through activation of GLP-1 receptors in the central nervous system and liver. We hypothesized that the insulin secretagogue effect of GLP-1 agonist exendin-4 may synergize with the insulin-sensitizing action of rimonabant. Intraperitoneal as well as intracerebroventricular administration of rimonabant increased serum glucose upon glucose challenge in overnight fasted, diet-induced obese C57 mice, with concomitant rise in serum glucagon levels. Exendin-4 reversed the acute hyperglycemia induced by rimonabant. The combination of exendin-4 and rimonabant showed an additive effect in the food intake, and sustained body weight reduction upon repeated dosing. The acute efficacy of both the compounds was additive for inducing nausea-like symptoms in conditioned aversion test in mice, whereas exendin-4 treatment antagonized the effect of rimonabant on forced swim test upon chronic dosing. Thus, the addition of exendin-4 to rimonabant produces greater reduction in food intake owing to increased aversion, but reduces the other central nervous system side effects of rimonabant. The hyperglucagonemia induced by rimonabant is partially responsible for enhancing the antiobesity effect of exendin-4.