Kappa opioid receptors (KOR) are believed to be involved in the pathophysiology of depression, anxiety disorders, drug abuse and alcoholism. To date, only one tracer, the kappa opioid receptor agonist [11C]GR103545, has been reported to be able to image KOR in primates. The goal of the present study was to synthesize the selective KOR antagonist [11C]LY2795050 and evaluate its potential as a PET tracer to image KOR in vivo. METHODS In vitro binding affinity of LY2795050 was measured in radioligand competition binding assays. Ex vivo experiments were conducted using microdosing of the unlabelled ligand in Sprague-Dawley rats, as well as wild-type and KOR knock-out mice, to assess the ligand’s potential as a tracer candidate. Imaging experiments with [11C]LY2795050 in monkeys were carried out on the Focus-220 PET scanner with arterial blood input function measurement. Binding parameters were determined with kinetic modeling analysis. RESULTS LY2795050 displays full antagonist activity and high binding affinity and selectivity for KOR. Microdosing studies in rodents and ex vivo analysis of tissue concentrations with LC/MS/MS identified LY2795050 as an appropriate tracer candidate able to provide specific binding signals in vivo. [11C]LY2795050 was prepared in an average yield of 12% and >99% radiochemical purity. In rhesus monkeys, [11C]LY2795050 displayed a moderate rate of peripheral metabolism, with ∼40% of parent compound remaining at 30 min postinjection. In the brain, [11C]LY2795050 displayed fast uptake kinetics (regional activity peak times < 20 min) and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, iv) resulted in a uniform distribution of radioactivity. Further, specific binding of [11C]LY2795050 was reduced by the selective KOR antagonist LY2456302 in a dose-dependent manner. CONCLUSION [11C]LY2795050 displayed favorable pharmacokinetic properties and binding profiles in vivo, and therefore is a suitable ligand for imaging the KOR in primates. This newly developed KOR antagonist tracer has since been advanced to PET imaging of KOR in humans and constitutes the first successful KOR antagonist radiotracer.

Interest in the phytochemical study of species of the Sideritis genus (Lamiaceae) has been stimulated by their pharmacological activity and their wide range of medicinal folk uses in Spain such as vulnerary, anti-infectious, anti-inflammatory, anti-rheumatic and anti-ulcerous applications. Various flavonoids and terpenoids have been isolated from this genus. 1,2) We have recently reported the anti-inflammatory properties of a lipid fraction obtained from Sideritis javalambrensis and those of the diterpenoid (ent-13(16),14-labdadiene-6a,8a,18triol-andalusol-) isolated from the acetone extract of Sideritis foetens, an endemic plant of Southern Spain. 3,4,5) Inflammation is normally a localized protective response which serves to destroy, dilute, or wall-off both the injurious agent and the injured tissue. The inflammatory response is coordinated by cells such as macrophages, lymphocytes, leukocytes and mast cells. A large number of mediators produced by these cells play a key role, for example, arachidonic acid metabolites such as prostaglandins and leukotrienes, reactive oxygen species, hydrolytic enzymes, histamine, nitric oxide, cytokines, etc. [6][7][8] Acute inflammation is associated with a rapid, early infiltration of polymorphonuclear leukocytes and increased vascular permeability at the site of injury.In this paper, we have evaluated the in vivo anti-inflammatory properties of a sterol fraction obtained from S. foetens on different inflammatory responses in mice. In addition, the interaction of this fraction with the functional properties of leukocytes and mast cells was analyzed in vitro. Immunomodulating activity through interaction with complement activation was also investigated. MATERIALS AND METHODS Plant MaterialThe aerial parts of Sideritis foetens were collected in Sierra de Gádor, Almería province (Spain). A voucher specimen has been deposited in the Department of Botany, Faculty of Pharmacy, Complutense University, Madrid (Spain). Extraction and IdentificationThe carrageenan-induced mouse paw oedema model was used for a bioassay guidedfractionation based on the anti-inflammatory activity.Dried aerial parts of Sideritis foetens (3 kg) were macerated with 99.5% acetone (12 l) at room temperature. The acetone extract was evaporated in vacuo to yield 84.8 g of residue. This extract (11.8 g) was fractionated by flash column chromatography over a silica gel (450 g, 230-400 mesh) column (6ϫ80 cm) using cyclohexane: acetone 70/30 to yield nine fractions. The most active fraction (subfraction B) (1.5 g) was chromatographed over a silica gel column (80 g, 230-400 mesh) using cyclohexane/ethyl acetate 70/30 as solvent, yielding the sterol fraction as white needles (800 mg).Sterol identification was performed by GC analysis [Perkin Elmer Sigma 3B equipped with a SE-30 Sobrechromosob WHP 3/100 column (lenght 2 m, i.d. 0.32 mm). Experimental conditions were: injector temp., 295°C; oven temp., 270°C (isotherm); detection temp., 285°C)]. The relative retention times were compared to standard samples. Campester...

A convenient, general, and high yielding Pd-catalyzed cross-coupling of methanesulfonamide with aryl bromides and chlorides is reported. The use of this method eliminates concern over genotoxic impurities that can arise when an aniline is reacted with methanesulfonyl chloride. The application of this method to the synthesis of dofetilide is also reported.

Evaluation of the sequence selectivity, noncovalent association, and orientation of the DNA cross-linking agent azinomycin B on its duplex DNA receptor is described. A strong correlation between sequence nucleophilicity and cross-linking yield was observed, and steric effects due to the thymine C5-methyl group were identified. Detailed studies on the role of the azinomycin naphthoate using viscometry, fluorescence contact energy transfer, and DNA unwinding assays point to a nonintercalative binding mode for this group. A kinetic assay for agent regioselectivity was used to determine the orientation of binding and covalent cross-link formation.

The κ-opioid receptor (KOR) has been implicated in depression, addictions, and other central nervous system disorders and, thus, is an important target for drug development. We previously developed several C-labeled PET radiotracers for KOR imaging in humans. Here we report the synthesis and evaluation ofF-LY2459989 as the first F-labeled KOR antagonist radiotracer in nonhuman primates and its comparison withC-LY2459989. The novel radioligandF-LY2459989 was synthesized by F displacement of a nitro group or an iodonium ylide. PET scans in rhesus monkeys were obtained on a small-animal scanner to assess the pharmacokinetic and in vivo binding properties of the ligand. Metabolite-corrected arterial activity curves were measured and used as input functions in the analysis of brain time-activity curves and the calculation of binding parameters. With the iodonium ylide precursor, F-LY2459989 was prepared at high radiochemical yield (36% ± 7% [mean ± SD]), radiochemical purity (>99%), and mean molar activity (1,175 GBq/μmol; = 6). In monkeys,F-LY2459989 was metabolized at a moderate rate, with a parent fraction of approximately 35% at 30 min after injection. Fast and reversible kinetics were observed, with a regional peak uptake time of less than 20 min. Pretreatment with the selective KOR antagonist LY2456302 (0.1 mg/kg) decreased the activity level in regions with high levels of binding to that in the cerebellum, thus demonstrating the binding specificity and selectivity of F-LY2459989 in vivo. Regional time-activity curves were well fitted by the multilinear analysis 1 kinetic model to derive reliable estimates of regional distribution volumes. With the cerebellum as the reference region, regional binding potentials were calculated and ranked as follows: cingulate cortex> insula > caudate/putamen > frontal cortex > temporal cortex > thalamus, consistent with the reported KOR distribution in the monkey brain. The evaluation ofF-LY2459989 in nonhuman primates demonstrated many attractive imaging properties: fast tissue kinetics, specific and selective binding to the KOR, and high specific binding signals. A side-by-side comparison of F-LY2459989 andC-LY2459989 indicated similar kinetic and binding profiles for the 2 radiotracers. Taken together, the results indicated that F-LY2459989 appears to be an excellent PET radiotracer for the imaging and quantification of the KOR in vivo.