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1.
J Biochem Mol Toxicol ; 33(8): e22345, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31066974

ABSTRACT

For fasiglifam (TAK875) and its metabolites the substance-specific mechanisms of liver toxicity were studied. Metabolism studies were run to identify a putatively reactive acyl glucuronide metabolite. In vitro cytotoxicity and caspase 3/7 activation were assessed in primary human and dog hepatocytes in 2D and 3D cell culture. Involvement of glutathione (GSH) detoxication system in mediating cytotoxicity was determined by assessing potentiation of cytotoxicity in a GSH depleted in vitro system. In addition, potential mitochondrial liabilities of the compounds were assessed in a whole-cell mitochondrial functional assay. Fasiglifam showed moderate cytotoxicity in human primary hepatocytes in the classical 2D cytotoxicity assays and also in the complex 3D human liver microtissue (hLiMT) after short-term treatment (24 hours or 48 hours) with TC50 values of 56 to 68 µM (adenosine triphosphate endpoint). The long-term treatment for 14 days in the hLiMT resulted in a slight TC50 shift over time of 2.7/3.6 fold lower vs 24-hour treatment indicating possibly a higher risk for cytotoxicity during long-term treatment. Cellular GSH depletion and impairment of mitochondrial function by TAK875 and its metabolites evaluated by Seahorse assay could not be found being involved in DILI reported for TAK875. The acyl glucuronide metabolites of TAK875 have been finally identified to be the dominant reason for liver toxicity.


Subject(s)
Benzofurans/toxicity , Fatty Acids, Nonesterified/metabolism , Liver/drug effects , Receptors, G-Protein-Coupled/agonists , Sulfones/toxicity , Animals , Benzofurans/metabolism , Cells, Cultured , Dogs , Glutathione/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Rats , Receptors, G-Protein-Coupled/metabolism , Sulfones/metabolism
2.
Endocrinology ; 159(8): 3105-3119, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29992313

ABSTRACT

We assessed the therapeutic contribution of the individual components of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonists alone and in combination upon energy homeostasis and glycemic control in diet-induced obese, diabetic nonhuman primates. The pharmacological active dose ranges of selective agonists were established through a dose-finding study, followed by a 6-week chronic study. Repeated subcutaneous administration of a selective GCGR agonist (30 µg/kg once daily) did not affect food intake or body weight, whereas the selective GLP-1R agonist (3 µg/kg once daily) alone decreased energy intake by 18% and body weight by 3.8% ± 0.9%. Combination of both agonists reduced significantly cumulative food intake by 27% and body weight by 6.6% ± 0.9%. Fasting plasma glucose (FPG) was improved by GLP-1R agonist (baseline vs end of study, 176.7 ± 34.0 vs 115.9 ± 16.1 mg/dL). In contrast, groups exposed to GCGR agonist experienced nonsignificant elevations of FPG. More accurate assessment of therapeutic interventions on glucose homeostasis was tested by an IV glucose tolerance test. Glucose excursion was significantly elevated by chronic GCGR agonist administration, whereas it was significantly decreased in GLP-1R agonist-treated monkeys. In the combination group, a nonsignificant increase of glucose excursion was seen, concomitantly with significantly increased insulin secretion. We conclude that chronic glucagon agonism does not affect energy homeostasis in nonhuman primates. In combination with GLP-1R agonism, glucagon agonism synergistically enhances negative energy balance with resulting larger body weight loss. However, adding GCGR to GLP-1R agonism diminishes glycemic control in diabetic monkeys. Therefore, long-term therapeutic implications of using GLP-1R/GCGR coagonists for weight management in diabetes warrants further scrutiny.


Subject(s)
Blood Glucose/drug effects , Body Weight/drug effects , Diabetes Mellitus, Type 2/metabolism , Eating/drug effects , Glucagon-Like Peptide-1 Receptor/agonists , Obesity/metabolism , Receptors, Glucagon/agonists , Animals , Bariatric Surgery , Blood Glucose/metabolism , Diabetes Mellitus/metabolism , Diabetes Mellitus, Type 2/surgery , Drug Therapy, Combination , Energy Metabolism/drug effects , Macaca fascicularis , Mice , Obesity/surgery
3.
Diabetes Obes Metab ; 20(8): 1836-1851, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29938884

ABSTRACT

AIM: We performed acute and chronic studies in healthy and diet-induced obese animals using mouse-specific or monkey-specific dual GLP-1R/GCGR agonists to investigate their effects on food intake, body weight, blood glucose control and insulin secretion. The selective GLP-1R agonist liraglutide was used as comparator. METHODS: The mouse-specific dual agonist and liraglutide were tested in lean wild type, GLP-1R knockout and diet-induced obese mice at different doses. A chronic study was performed in DIO mice to investigate the effect on body weight, food consumption and total energy expenditure (TEE) in obese and diabetic monkeys with a focus on body weight and energy intake. RESULTS: The mouse-specific dual agonist and liraglutide similarly affected glycaemic control. A higher loss in body weight was measured in dual agonist-treated obese mice. The dual agonist significantly enhanced plasma glucose excursion in overnight fed GLP-1R-/- mice, probably reflecting a potent GCGR agonist activity. It increased TEE and enhanced fat and carbohydrate oxidation, while liraglutide produced no effect on TEE. In obese and diabetic monkeys, treatment with the monkey-specific dual agonist reduced total energy intake to 60%-70% of baseline TEI during chronic treatment. A decrease in body weight and significant improvement in glucose tolerance was observed. CONCLUSIONS: In DIO mice and non-human primates, dual agonists elicited robust glycaemic control, similar to the marketed GLP-1R agonist, while eliciting greater effects on body weight. Results from DIO mice suggest that the increase in TEE is caused not only by increased fat oxidation but also by an increase in carbohydrate oxidation.


Subject(s)
Appetite Depressants/therapeutic use , Diabetes Mellitus, Type 2/drug therapy , Glucagon-Like Peptide-1 Receptor/agonists , Hyperglycemia/prevention & control , Hypoglycemic Agents/therapeutic use , Obesity/drug therapy , Receptors, Glucagon/agonists , Animals , Animals, Outbred Strains , Appetite Depressants/administration & dosage , Appetite Depressants/adverse effects , Body Weight/drug effects , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Diet, High-Fat/adverse effects , Dose-Response Relationship, Drug , Drug Therapy, Combination/adverse effects , Energy Intake/drug effects , Energy Metabolism/drug effects , Female , Glucagon-Like Peptide-1 Receptor/genetics , Glucagon-Like Peptide-1 Receptor/metabolism , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/adverse effects , Insulin Secretion/drug effects , Macaca fascicularis , Male , Mice, Inbred C57BL , Mice, Knockout , Obesity/blood , Obesity/etiology , Obesity/metabolism , Random Allocation , Receptors, Glucagon/metabolism
5.
J Med Chem ; 60(10): 4293-4303, 2017 05 25.
Article in English | MEDLINE | ID: mdl-28448133

ABSTRACT

Dual activation of the glucagon-like peptide 1 (GLP-1) and glucagon receptor has the potential to lead to a novel therapy principle for the treatment of diabesity. Here, we report a series of novel peptides with dual activity on these receptors that were discovered by rational design. On the basis of sequence analysis and structure-based design, structural elements of glucagon were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in hybrid peptides with potent dual GLP-1/glucagon receptor activity. Detailed structure-activity relationship data are shown. Further modifications with unnatural and modified amino acids resulted in novel metabolically stable peptides that demonstrated a significant dose-dependent decrease in blood glucose in chronic studies in diabetic db/db mice and reduced body weight in diet-induced obese (DIO) mice. Structural analysis by NMR spectroscopy confirmed that the peptides maintain an exendin-4-like structure with its characteristic tryptophan-cage fold motif that is responsible for favorable chemical and physical stability.


Subject(s)
Drug Design , Glucagon-Like Peptide 1/agonists , Glucagon-Like Peptide-1 Receptor/agonists , Peptides/chemistry , Peptides/pharmacology , Venoms/chemistry , Venoms/pharmacology , Amino Acid Sequence , Animals , Blood Glucose/analysis , Blood Glucose/metabolism , Body Weight/drug effects , Exenatide , Female , Glucagon/metabolism , Glucagon-Like Peptide 1/chemistry , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/chemistry , Glucagon-Like Peptide-1 Receptor/metabolism , Hypoglycemic Agents/blood , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Mice, Inbred C57BL , Mice, Obese , Molecular Docking Simulation , Obesity/drug therapy , Obesity/metabolism , Peptides/blood , Structure-Activity Relationship , Swine , Venoms/blood
6.
Chembiochem ; 17(9): 861-5, 2016 05 03.
Article in English | MEDLINE | ID: mdl-26812365

ABSTRACT

Mast cells and microglia play a critical role in innate immunity and inflammation and can be activated by a wide range of endogenous and exogenous stimuli. Lysophosphatidic acid (LPA) has recently been reported to activate mast cells and microglia. Using the human mast cell line HMC-1 and the mouse microglia cell line BV-2, we show that LPA-mediated activation can be prevented by blockade of the LPA receptor 5 (LPA5) in both cell lines. The identification of new LPA5-specific antagonists as tool compounds to probe and modulate the LPA5/LPA axis in relevant in vitro and in vivo assays should contribute to better understanding of the underlying role of LPAs in the development and progression of (neuro-) inflammatory diseases.


Subject(s)
Gene Expression/drug effects , Lysophospholipids/pharmacology , Receptors, Lysophosphatidic Acid/metabolism , Administration, Oral , Animals , Cell Line , Cell Membrane Permeability/drug effects , Chemokine CCL2/metabolism , Half-Life , Humans , Kinetics , Lysophospholipids/chemistry , Lysophospholipids/pharmacokinetics , Male , Mast Cells/cytology , Mast Cells/drug effects , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Microglia/cytology , Microglia/drug effects , Microglia/metabolism , Microsomes, Liver/metabolism , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysophosphatidic Acid/genetics
7.
Diabetologia ; 58(3): 549-57, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25417214

ABSTRACT

AIMS/HYPOTHESIS: Islet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage. METHODS: Mouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1ß, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-S-hydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice. RESULTS: Inflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines. CONCLUSIONS/INTERPRETATION: Selective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.


Subject(s)
Arachidonate 12-Lipoxygenase/metabolism , Enzyme Inhibitors/pharmacology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/metabolism , Animals , Apoptosis/drug effects , Cell Line , Cell Survival/drug effects , Enzyme-Linked Immunosorbent Assay , Humans , In Vitro Techniques , Interleukin-1beta/metabolism , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/metabolism
8.
Bioorg Med Chem Lett ; 14(16): 4191-5, 2004 Aug 16.
Article in English | MEDLINE | ID: mdl-15261268

ABSTRACT

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for neutral ligands, which bind in the S1 pocket of factor Xa were investigated with the 2-carboxyindole scaffold. This privileged fragment assembly approach yielded a set of equipotent, selective inhibitors with structurally diverse neutral P1 substituents.


Subject(s)
Factor Xa Inhibitors , Indoles/chemistry , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Humans , Molecular Conformation
9.
Bioorg Med Chem Lett ; 14(11): 2801-5, 2004 Jun 07.
Article in English | MEDLINE | ID: mdl-15125936

ABSTRACT

A series of novel, highly potent, achiral factor Xa inhibitors based on a benzoic acid scaffold and containing a chlorophenethyl moiety directed towards the protease S1 pocket is described. A number of structural features, such as the requirements of the P1, P4 and ester-binding pocket ligands were explored with respect to inhibition of factor Xa. Compound 46 was found to be the most potent compound in a series of antithrombotic secondary assays.


Subject(s)
Benzoates/pharmacology , Factor Xa Inhibitors , Fibrinolytic Agents/chemical synthesis , Benzoates/chemical synthesis , Blood Coagulation Tests , Drug Stability , Fibrinolytic Agents/pharmacology , Humans , Ligands , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Protein Binding , Structure-Activity Relationship
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