Design, synthesis and structure activity relationships of indazole and indole derivatives as potent glucagon receptor antagonists.

A novel series of indazole/indole derivatives were discovered as glucagon receptor (GCGR) antagonists through scaffold hopping based on two literature leads: MK-0893 and LY-2409021. Further structure-activity relationship (SAR) exploration and optimization led to the discovery of multiple potent GCGR antagonists with excellent pharmacokinetic properties in mice and rats, including low systemic clearance, long elimination half-life, and good oral bioavailability. These potent GCGR antagonists could be used for potential treatment of type II diabetes.

Design, synthesis and biological evaluation of (2S,3R,4R,5S,6R)-5-fluoro-6-(hydroxymethyl)-2-aryltetrahydro-2H-pyran-3,4-diols as potent and orally active SGLT dual inhibitors.

A new series of (2S,3R,4R,5S,6R)-5-fluoro-6-(hydroxymethyl)-2-aryltetrahydro-2H-pyran-3,4-diols as dual inhibitors of sodium glucose co-transporter proteins (SGLTs) were disclosed. Two methods were developed to efficiently synthesize C-fluoro-lactones 3 and 4, which are key intermediates to the C-fluoro-hexose based C-aryl glucosides. Compound 2b demonstrated potent hSGLT1 and hSGLT2 inhibition (IC = 43 nM for SGLT1 and IC = 9 nM for SGLT2).

Discovery of N-arylpyrroles as agonists of GPR120 for the treatment of type II diabetes.

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.

FcRn binding is not sufficient for achieving systemic therapeutic levels of immunoglobulin G after oral delivery of enteric-coated capsules in cynomolgus macaques.

Although much speculation has surrounded intestinally expressed FcRn as a means for systemic uptake of orally administered immunoglobulin G (IgG), this has not been validated in translational models beyond neonates or in FcRn-expressing cells in vitro. Recently, IgG1 intestinal infusion acutely in anesthetized cynomolgus resulted in detectable serum monoclonal antibody (mAb) levels. In this study, we show that IgG2 has greater protease resistance to intestinal enzymes in vitro and mice in vivo, due to protease resistance in the hinge region.

Characterization of the ZDSD Rat: A Translational Model for the Study of Metabolic Syndrome and Type 2 Diabetes.

Metabolic syndrome and T2D produce significant health and economic issues. Many available animal models have monogenic leptin pathway mutations that are absent in the human population. Development of the ZDSD rat model was undertaken to produce a model that expresses polygenic obesity and diabetes with an intact leptin pathway.

Design and synthesis of polyethylene glycol-modified biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s.

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.

Improved pharmacokinetic and bioavailability support of drug discovery using serial blood sampling in mice.

Pharmacokinetic studies in mice traditionally require one animal per time point, resulting in dosing and euthanizing a large number of animals and producing suboptimal quality of pharmacokinetic data due to inter-animal variability and dosing error. These studies are time-consuming and labor-intensive. To improve the throughput and quality of pharmacokinetic evaluation in mice, we have developed a serial blood sampling methodology using the lateral saphenous vein puncture technique.

Isobaric metabolite interferences and the requirement for close examination of raw data in addition to stringent chromatographic separations in liquid chromatography/tandem mass spectrometric analysis of drugs in biological matrix.

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS.

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif.

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.

Pharmacokinetics of TDP223206 following intravenous and oral administration to intact rats and intravenous administration to bile duct-cannulated rats.

The pharmacokinetics of TDP223206 was studied following single intravenous and oral administrations in rats. A mixture of TDP223206 and (14)C-TDP223206 were administered to intact and bile duct-cannulated rats. Following intravenous administration, plasma concentrations declined biphasically.

Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s.

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.

Pharmacokinetic properties of TDP4815 after single intravenous and oral administrations to rat, rabbit, monkey, dog and in vitro drug metabolism.

The pharmacokinetics of TDP4815 was evaluated in rats, rabbits, dogs and monkeys. After intravenous administration, TDP4815 achieved C(O) of 3255 ng/ml in rats at 5 mg/kg, 9066 ng/ml in rabbits and 7858 ng/ml in monkeys at 6 mg/kg, and 4457 ng/ml in dogs at 3 mg/kg. The clearance (C(L)) was 3105, 1692, 835 and 640 ml/h/kg in rats, rabbits, monkeys and dogs, respectively.

Studies with an orally bioavailable alpha V integrin antagonist in animal models of ocular vasculopathy: retinal neovascularization in mice and retinal vascular permeability in diabetic rats.

The alpha(V) integrins are key receptors involved in mediating cell migration and angiogenesis. In age-related macular degeneration (AMD) and diabetic retinopathy, angiogenesis plays a critical role in the loss of vision. These ocular vasculopathies might be treatable with a suitable alpha(V) antagonist, and an oral drug would offer a distinct advantage over current therapies.

Use of a trapping agent for simultaneous capturing and high-throughput screening of both "soft" and "hard" reactive metabolites.

Glutathione (GSH) has been widely used for in vitro trapping and subsequently detecting reactive metabolites using liquid chromatography-mass spectrometry. A major drawback of GSH is its low trapping efficiency for "hard" reactive metabolites such as reactive aldehydes. In the present study, a bifunctional trapping agent (gamma GSK, gamma-glutamylcysteinlysine) is investigated as an alternative of GSH for simultaneous trapping both "hard" and "soft" reactive metabolites.

Metabolism and bioactivation of 3-methylindole by human liver microsomes.

Metabolism and bioactivation of 3-methylindole (3MI) were investigated in human liver microsomes. The metabolism of two deuterium-labeled analogues of 3MI permitted a relatively unambiguous identification of multiple metabolites and glutathione (GSH) adducts of reactive intermediates. A total of eight oxidized metabolites were detected, five of which were assigned as previously identified 3-methyloxindole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole, 5-hydroxy-3-methylindole, and 6-hydroxy-3-methylindole.

Human oral drugs absorption is correlated to their in vitro uptake by brush border membrane vesicles.

Brush border membrane vesicles (BBMV) were prepared from the rabbit small intestine for testing drug absorption potency through the enterocyte's apical membrane, which is an important compartment for drug oral absorption. Some modifications have been made to the traditional vesicle assay for adapting it to the 96-well plate format. The accumulation of 23 reference drugs was measured, and the data showed a good correlation with human oral absorption with a correlation coefficient R=0.

Discovery, SAR, and X-ray structure of novel biaryl-based dipeptidyl peptidase IV inhibitors.

The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.

Rapid detection and characterization of minor reactive metabolites using stable-isotope trapping in combination with tandem mass spectrometry.

Stable-isotope trapping combined with mass spectrometry (MS) neutral loss scanning has recently been developed as a high-throughput method for the in vitro screening of major reactive metabolites. In fact, detection and identification of minor reactive metabolites are equally important since the minor metabolites, even though at low levels, may be highly reactive and also play an important role in drug-induced adverse reactions. In this study, 2-acetylthiophene, clozapine, troglitazone and 7-methylindole were selected as model compounds to further validate the advantages of this method for rapid detection and structural characterization of minor glutathione (GSH) adducts derived from reactive metabolites.

Bioactivation of 4-methylphenol (p-cresol) via cytochrome P450-mediated aromatic oxidation in human liver microsomes.

It has previously been proposed that 4-methylphenol (p-cresol) is metabolically activated by oxidation of the methyl group to form a reactive quinone methide. In the present study a new metabolism pathway is elucidated in human liver microsomes. Oxidation of the aromatic ring leads to formation of 4-methyl-ortho-hydroquinone, which is further oxidized to a reactive intermediate, 4-methyl-ortho-benzoquinone.

Structure-based design, synthesis, and biological evaluation of novel 1,4-diazepines as HDM2 antagonists.

Crystallographic analysis of ligands bound to HDM2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the alpha-helix of p53 peptide and may represent a promising scaffold to develop HDM2-p53 antagonists. To verify this hypothesis, we synthesized and biologically evaluated 5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1,4-diazepin-1-yl]valeric acid (10) and 5-[(3S)-7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl]valeric acid (11). Preliminary in vitro testing shows that 10 and 11 substantially antagonize the binding between HDM2 and p53 with an IC(50) of 13 and 3.

Detection of a novel reactive metabolite of diclofenac: evidence for CYP2C9-mediated bioactivation via arene oxides.

A new glutathione adduct (M4) was tentatively identified, likely as 2'-hydroxy-3'-(glutathione-S-yl)-monoclofenac, using liquid chromatography-tandem mass spectrometry analysis of incubations of diclofenac with human liver microsomes. The same conjugate was not detected in incubations with either rat or monkey liver microsomes. Formation of M4 was mediated specifically by CYP2C9 in human liver microsomes, as evidenced by the following observations: 1) cDNA-expressed CYP2C9-catalyzing formation of M4; 2) inhibition of M4 formation by sulfaphenazole, a CYP2C9-selective inhibitor; and 3) strong correlation between the production of M4 and CYP2C9-mediated tolbutamide 4-hydroxylase activities in a panel of human liver microsome samples.

Using in vitro human tissues to predict pharmacokinetic properties.

Pressure on drug discovery teams to predict the success of drug candidates earlier in the drug discovery process has led to the development of in vitro assays using human tissues, cells or fluids that aid chemists and biologists in their decision-making process. The use of human material is often related to the species-specific nature of the enzymatic biotransformations or transport processes that are involved in drug bioavailability. In vitro assays have been developed to indicate liabilities in scaffolds relating to the absorption, distribution or metabolism of new chemical entities.

A method using a liquid chromatographic-electrospray-mass spectrometric assay for the determination of antimigraine compounds: preliminary pharmacokinetics of MDL 74,721, sumatriptan and naratriptan, in rabbit.

MDL 74,721 (I), sumatriptan(II) and naratriptan(III) are new 5-HT1-like agonists that have potential as a novel treatment for migraine. Liquid chromatographic-electrospray-mass spectrometric (LC-ESI-MS) assay have been developed to compare the pharmacokinetics of these three antimigraine compounds. The concentration of each parent drug was determined using a solid-phase extraction method and LC-ESI-MS analysis demonstrating the high sensitivity and specificity of the methods down to subnanogram levels in rabbit plasma samples.