Kenneth E. Carlson

A selective small molecule agonist of the melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice

It is well established that melanocortins are peptides that have potent anti‐inflammatory activity. Recent research has focused on understanding which of the known melanocortin receptors mediates the anti‐inflammatory actions of the melanocortins. The aim of this study was to assess the anti‐inflammatory activity of a synthetic MC‐1R agonist. BMS‐470539 is a potent, selective, full agonist of human and murine MC‐1R with EC50 values in a cAMP accumulation assay of 16.8 and 11.6 nM, respectively. BMS‐470539 dose‐dependently inhibited TNF‐α‐induced activation of a NF‐κB transcriptional reporter in human melanoma cells, which endogenously express MC‐1R. In vivo studies with BMS‐470539 demonstrated that subcutaneous administration of BMS‐470539 resulted in a dose‐dependent inhibition of LPS‐induced TNF‐α production in BALB/c mice. In this model, the compound had an ED50 of approximately 10 μmol/kg and a pharmacodynamic half‐life of ∼8 h. Pharmacokinetic analysis of the compound indicated that the compound had a t1/2 of 1.7 h. In a model of lung inflammation, administration of 15 μmol/kg BMS‐470539 resulted in a 45% reduction in LPS‐induced leukocyte infiltration (an infiltrate comprised primarily of neutrophils). The compound was also effective in a model of delayed‐type hypersensitivity, reducing paw swelling by 59%, comparable with that seen with 5 mg/kg dexamethasone. These studies demonstrate that a selective small molecule agonist of the melanocortin‐1 receptor is a potent anti‐inflammatory agent in vivo and provides compelling evidence for the involvement of this receptor in the modulation of inflammation.

Immobilized α-melanocyte stimulating hormone 10–13 (GKPV) inhibits tumor necrosis factor-α stimulated NF-κB activity

Abstract α-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of α-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-α (TNF-α) stimulated activation of NF-κB in HBL cells stably transfected with an NF-κB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (α-MSH or GKPV) or non-functionalized beads to inhibit TNF-α stimulated activation of NF-κB. GKPV peptide functionalized beads significantly inhibited NF-κB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble α-MSH and GKPV peptides were also confirmed to inhibit NF-κB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties.

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

CANNABINOID RECEPTOR ANTAGONIST-INDUCED STRIATED MUSCLE TOXICITY AND ETHYLMALONIC-ADIPIC ACIDURIA IN BEAGLE DOGS

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.

Solid phase synthesis of 1,5-diarylpyrazole-4-carboxamides: discovery of antagonists of the CB-1 receptor.

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor.

Cannabinoid CB1 receptor ligand binding and function examined through mutagenesis studies of F200 and S383

The cannabinoid CB(1) G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptors three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB(1) receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB(1) receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB(1) receptor ligands.

Characterization of a novel and selective CB1 antagonist as a radioligand for receptor occupancy studies

Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease. CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affinity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radioligand was used to conduct ex vivo receptor occupancy studies.