ABSTRACT
Glucagon-like peptide 1 (GLP-1) is a 30 or 31 amino acid peptide hormone that contributes to the physiological regulation of glucose homeostasis and food intake. Herein, we report the discovery of a novel class of 11 amino acid GLP-1 receptor agonists. These peptides consist of a structurally optimized 9-mer, which is closely related to the N-terminal 9 amino acids of GLP-1, linked to a substituted C-terminal biphenylalanine (BIP) dipeptide. SAR studies resulted in 11-mer GLP-1R agonists with similar in vitro potency to the native 30-mer. Peptides 21 and 22 acutely reduced plasma glucose excursions and increased plasma insulin concentrations in a mouse model of diabetes. These peptides also showed sustained exposures over several hours in mouse and dog models. The described 11-mer GLP-1 receptor agonists represent a new tool in further understanding GLP-1 receptor pharmacology that may lead to novel antidiabetic agents.
Subject(s)
Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Receptors, Glucagon/agonists , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Dose-Response Relationship, Drug , Glucagon-Like Peptide-1 Receptor , Humans , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/pharmacokinetics , Male , Mice , Models, Molecular , Molecular Sequence Data , Oligopeptides/chemical synthesis , Oligopeptides/pharmacokinetics , Protein ConformationABSTRACT
In order to explore the relationship between the anorectic effect of 3-carboxy-4-octyl-2-methylenebutyrolactone (C75) and its pharmacokinetic properties, studies of in vivo and in vitro pharmacological characterization of C75 were performed in Fischer rats. In a quantitative measurement of food intake, we determined that appetite suppression by C75 takes place within 4 h. The C(max) for C75 of 2.6+/-1.5 microM was reached within 1-4 h after intraperitoneal administration at 30 mg/kg, a drug level that causes complete blockade of food intake. However, this concentration is substantially lower than the effective concentration used to inhibit rat fatty acid synthase enzyme activity in vitro (IC50: approximately 200 microM) and hypothalamic enzyme activity was found not to be inhibited by intraperitoneal administration of C75 at 30 mg/kg. Instead, a dramatic induction of c-Fos expression was found in area postrema. Collectively, these data indicate that the anorectic effect of C75 is independent of its inhibition of fatty acid synthase in the hypothalamus.
Subject(s)
4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Fatty Acid Synthases/antagonists & inhibitors , Hypothalamus/drug effects , 4-Butyrolactone/blood , 4-Butyrolactone/pharmacokinetics , Animals , Appetite Depressants/pharmacokinetics , Appetite Depressants/pharmacology , Behavior, Animal/drug effects , Blotting, Northern , Cyclobutanes/pharmacology , Dose-Response Relationship, Drug , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Fenfluramine/pharmacology , Food Deprivation , Hypothalamus/enzymology , Immunoblotting , Injections, Intraperitoneal , Injections, Intraventricular , Kinetics , Male , Phentermine/pharmacology , RNA/genetics , RNA/metabolism , Random Allocation , Rats , Rats, Inbred F344 , Time FactorsABSTRACT
In this work, a high-throughput and high-performance bioanalytical system is described that is capable of extracting and analyzing 1152 plasma samples within 10 hours. A Zymark track robot system interfaced with a Tecan Genesis liquid handler was used for simultaneous solid-phase extraction of four 96-well plates in a fully automated fashion. The extracted plasma samples were injected onto four parallel monolithic columns for separation via a four-injector autosampler. The use of monolithic columns allowed for fast and well-resolved separations at a considerably higher flow rate without generating significant column backpressure. This resulted in a total chromatographic run cycle time of 2 min on each 4.6 x 100 mm column using gradient elution. The effluent from the four columns was directed to a triple quadrupole mass spectrometer equipped with an indexed four-probe electrospray ionization source (Micromass MUX interface). Hence, sample extraction, separation, and detection were all performed in a four-channel parallel format that resulted in an overall throughput of about 30 s per sample from plasma. The performance of this system was evaluated by extracting and by analyzing twelve 96-well plates (1152) of human plasma samples spiked with oxazepam at different concentrations. The relative standard deviation (RSD) of analyte sensitivity (slope of calibration curve) across the four channels and across the 12 plates was 5.2 and 6.8%, respectively. An average extraction recovery of 77.6% with a RSD of 7.7% and an average matrix effect of 0.95 with a RSD of 5.2% were achieved using these generic extraction and separation conditions. The good separation efficiency provided by this system allowed for rapid method development of an assay quantifying the drug candidate and its close structural analog metabolite. The method was cross-validated with a conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay.
