Glutamate is the major excitatory transmitter in the brain. Recent developments in the molecular biology and pharmacology of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype of glutamate receptors have led to the discovery of selective, potent and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and play important roles in plasticity and cognitive processes. In the present studies we characterized a novel AMPA receptor potentiator, LY503430, on recombinant human GLU(A1-4) and native preparations in vitro, and then evaluated the potential neuroprotective effects of the molecule in rodent models of Parkinson's disease. Results indicated that at submicromolar concentrations LY503430 selectively enhanced glutamate-induced calcium influx into HEK293 cells transfected with human GLU(A1), GLU(A2), GLU(A3), or GLU(A4) AMPA receptors. The molecule also potentiated AMPA-mediated responses in native cortical, hippocampal and substantia nigra neurones. LY503430 had good oral bioavailability in both rats and dogs. We also report here that LY503430 provided dose-dependent functional and histological protection in animal models of Parkinson's disease. The neurotoxicity following unilateral infusion of 6-hyrdoxydopamine (6-OHDA) into either the substantia nigra or the striatum of rats and that following systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice were reduced. Interestingly, LY503430 also had neurotrophic actions on functional and histological outcomes when treatment was delayed until well after (6 or 14 days) the lesion was established. LY503430 also produced some increase in brain derived neurotrophic factor (BDNF) in the substantia nigra and a dose-dependent increase in growth associated protein-43 (GAP-43) expression in the striatum. Therefore, we propose that AMPA receptor potentiators such as LY503430 offer the potential of a new disease modifying therapy for Parkinson's disease.

(+)-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (1), also known as LY354740, is a highly potent and selective agonist for group II metabotropic glutamate receptors (mGlu receptors 2 and 3) tested in clinical trials. It has been shown to block anxiety in the fear-potentiated startle model. Its relatively low bioavailability in different animal species drove the need for an effective prodrug form that would produce a therapeutic response at lower doses for the treatment of anxiety disorders. We have investigated the increase of intestinal absorption of this compound by targeting the human peptide transporter hPepT1 for active transport of di- and tripeptides derived from 1. We have found that oral administration of an N dipeptide derivative of 1 (12a) in rats shows up to an 8-fold increase in drug absorption and a 300-fold increase in potency in the fear-potentiated startle model in rats when compared with the parent drug 1.

2-(2-Amino-2-methyl-propionylamino)-5-phenyl-pentanoic acid [1-[1-(4-methoxy-phenyl)-1-methyl-2-oxo-2-pyrrolidin-1-yl-ethyl]-1H-imidazol-4-yl]-amide (LY444711, 6) is an orally active ghrelin agonist that binds with high affinity to and is a potent activator of the growth hormone secretagogue receptor 1a (GHS-R1a) receptor. In rat models of feeding behavior and pharmacology, 6 creates a positive energy balance and induces adiposity by stimulating food consumption and sparing fat utilization. As an orally active ghrelin agonist, 6 represents a new pharmacological tool to investigate the orexigenic role of ghrelin in regulating energy homeostasis.

The melanocortin receptors have been implicated as potential targets for a number of important therapeutic indications, including inflammation, sexual dysfunction, and obesity. We identified compound 1, an arylpiperazine attached to the dipeptide H-d-Tic-d-p-Cl-Phe-OH, as a novel melanocortin subtype-4 receptor (MC4R) agonist through iterative directed screening of nonpeptidyl G-protein-coupled receptor biased libraries. Structure-activity relationship (SAR) studies demonstrated that substitutions at the ortho position of the aryl ring improved binding and functional potency. For example, the o-isopropyl-substituted compound 29 (K(i) = 720 nM) possessed 9-fold better binding affinity compared to the unsubstituted aryl ring (K(i) = 6600 nM). Sulfonamide 39 (K(i) = 220 nM) fills this space with a polar substituent, resulting in a further 2-fold improvement in binding affinity. The most potent compounds such as the diethylamine 44 (K(i) = 60 nM) contain a basic group at this position. Basic heterocycles such as the imidazole 50 (K(i) = 110 nM) were similarly effective. We also demonstrated good oral bioavailability for sulfonamide 39.

Glycogen synthase kinase-3 (GSK3) is involved in signaling from the insulin receptor. Inhibitors of GSK3 are expected to effect lowering of plasma glucose similar to insulin, making GSK3 an attractive target for the treatment of type 2 diabetes. Herein we report the discovery of a series of potent and selective GSK3 inhibitors. Compounds 7-12 show oral activity in an in vivo model of type II diabetes, and 9 and 12 have desirable PK properties.

Incubation of (R)-tazofelone and (S)-tazofelone in rat, dog, and human liver microsomes demonstrated that the (R)-tazofelone enantiomer was more rapidly metabolized, with two diastereomeric sulfoxides as the major metabolites formed in all three species. The two diasteresomers epimerized at physiological pH, therefore total sulfoxide formation rates were measured. The formation of the total sulfoxide metabolites followed Michaelis-Menten kinetics. The K(m), Vmax, and intrinsic formation clearance (Vmax/K(m)) values were determined in rat, dog, and human liver microsomes. The intrinsic formation clearance of sulfoxide from (R)-tazofelone exceeded that of (S)-tazofelone in all three species. In vivo studies in rats and dogs dosed orally and intravenously confirmed the stereoselective metabolism of tazofelone observed in vitro. Plasma concentrations of (S)-tazofelone exceeded (R)-tazofelone in rats and dogs by a factor of 3 to 4. In rat portal plasma, both enantiomers were of approximately equal concentration after oral dosing, indicating similar absorption. The half-lives of tazofelone and total sulfoxides in rats were 3.5 and 2.8 h, respectively. In dogs, the half-lives of tazofelone and total sulfoxides were 2.2 and 5.5 h, respectively. Plasma clearance was 2.3 l/h in rats and 1.4 l/h in dogs, and the volumes of distribution were 12 and 4.5 l, respectively, in rats and dogs. Both enantiomers were highly bound to plasma proteins to a similar extent in both species.

Previous studies in rats and humans demonstrated poor oral bioavailability of potent in vitro 2-aminobenzimidazole inhibitors of rhinovirus replication due to significant first-pass elimination and possibly also to poor aqueous solubility. Estimations of aqueous solubility, as well as measurements of caco-2 permeability and NADPH dependent compound loss in rat liver microsomal incubations were employed alongside traditional in vivo experiments in rats to guide subsequent chemistry efforts. Retention of activity upon replacement of the metabolically labile vinyl oxime in the lead molecule with a vinyl carboxamide was a major breakthrough; however, oral bioavailability among the latter compounds was variable. Based on the ability to independently measure solubility, permeability, and metabolic stability of new compounds, variable solubility across the series (ranging from approximately 1 to 10 microg/mL) was identified as the cause of the inconsistent performance. Subsequent efforts to improve solubility led to the discovery of highly soluble (>10 mg/mL) and potent dessulfonyl vinyl carboxamide benzimidazoles. Determination of the metabolic stability of these compounds as a surrogate of the extent of their first-pass elimination supported a prediction of excellent oral bioavailability. In comparison to the sulfonyl-containing vinyl carboxamides, caco-2 permeabilities were reduced 5 to 10-fold; however, these were considered to be in the range of well-absorbed compounds based on comparison to a series of reference compounds of known percentage absorption in humans. Subsequent experiments in the rat verified the oral bioavailability of these N-alkyl compounds, with one compound (368177) having an absolute oral bioavailability of 89.4%. The application of solubility and caco-2 permeability as surrogates for oral absorption potential, in conjunction with the use of microsomal incubations as a surrogate for first-pass metabolism, was shown to augment a rational chemistry approach to discover orally bioavailable inhibitors of rhinovirus replication. Future expanded use of these surrogates is planned.

Tazofelone is a new inflammatory bowel disease agent. The biotransformation of tazofelone in human livers and the cytochrome P450 responsible for the biotransformation has been studied. Two metabolites of tazofelone were formed in vitro. A sulfoxide metabolite was identified by cochromatography with authentic standards, and a quinol metabolite of tazofelone was identified by mass spectrometry and proton NMR. Sulfoxidation was catalyzed by a single enzyme system while formation of the quinol metabolite was catalyzed by a two enzyme system. The Km and Vmax values for sulfoxidation were 12.4 microM and 0.27 nmol/min/mg protein, respectively. The high affinity Km and Vmax values for the formation of the quinol metabolite were 7.5 microM and 0.17 nmol/min/mg protein, respectively. Tazofelone was incubated at 20 microM concentration with human microsomes to determine which of the cytochrome P450 isozyme(s) is involved in the oxidation of tazofelone. A strong correlation was found between the immunoquantified concentrations of CYP3A and the rates of formation of the sulfoxide and quinol metabolites of tazofelone. Similarly, significant correlations were observed between the formation of midazolam 1'-hydroxylation and the rates of formation of both metabolites of tazofelone. Inhibition studies have indicated that triacetyloleandomycin, a CYP3A specific inhibitor, almost completely inhibited the formation of both of these tazofelone metabolites. Incubations with specific cDNA expressed microsomes indicated that the formation of both the sulfoxide and quinol metabolites was highest with CYP3A4 containing microsomes. The correlation data was confirmed by inhibition studies and cDNA expressed cytochrome P450 systems demonstrating that the biotransformation of tazofelone to its metabolites is primarily mediated by CYP3A.

The purpose of this study was to determine whether the selective type IV cAMP-phosphodiesterase inhibitor rolipram could reduce the reperfusion injury that occurs during myocardial infarction in the anesthetized dog. This question was tested in pentobarbital-anesthetized dogs subject to 90 min of regional myocardial ischemia and 5 h of reperfusion. Dogs were treated with 1 mg/kg of rolipram (i.v., 15 min before reperfusion) followed by a 1 mg/kg/h infusion over the duration of the 5 h of reperfusion. Rolipram was tested in vitro for efficacy in inhibition of isolated human neutrophil superoxide generation. Rolipram produced significant inhibition of superoxide production over the concentration range of 0.1-100 microM rolipram when neutrophils were stimulated with a 10(-7) M concentration of the chemotactic peptide f-Met-Leu-Phe. Rolipram significantly inhibited superoxide generation from human and canine granulocytes in whole blood stimulated by zymosan. Therapeutic concentrations of rolipram in the blood of dogs were achieved during the course of the experiments with a plasma concentration of 0.761 +/- 0.095 micrograms/ml (2.76 +/- 0.34 microM) at the time of reperfusion, and 0.574 +/- 0.098 micrograms/ml (2.08 +/- 0.36 microM) at the end of the reperfusion period. The relative severity of myocardial ischemia between the two treatment groups was similar as assessed with radiolabeled microsphere measurement of myocardial blood flow. Transmural myocardial blood flows were not significantly different between the two groups after coronary occlusion (control, 0.05 +/- 0.01 ml/min/g, n = 6, vs. rolipram, 0.18 +/- 0.07 ml/min/g, n = 6; p = 0.48).(ABSTRACT TRUNCATED AT 250 WORDS)