A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)-and (S)-Ket, in Complex Regional Pain Syndrome (CRPS) patients receiving a 5-day continuous infusion of a subanesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)-and (S)-norKet and dehydronorketamine, (R)-and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R, 6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C 18 stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)-and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)-and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary, metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients.

The distribution, clearance, and bioavailability of (2S,6S)-hydroxynorketamine has been studied in the Wistar rat. The plasma and brain tissue concentrations over time of (2S,6S)-hydroxynorketamine were determined after intravenous (20 mg/kg) and oral (20 mg/kg) administration of (2S,6S)-hydroxynorketamine (n = 3). After intravenous administration, the pharmacokinetic parameters were estimated using noncompartmental analysis and the half-life of drug elimination during the terminal phase (t1/2) was 8.0 ± 4.0 h and the apparent volume of distribution (Vd) was 7352 ± 736 mL/kg, clearance (Cl) was 704 ± 139 mL/h per kg, and the bioavailability was 46.3%. Significant concentrations of (2S,6S)-hydroxynorketamine were measured in brain tissues at 10 min after intravenous administration, ∼30 μg/mL per g tissue which decreased to 6 μg/mL per g tissue at 60 min. The plasma and brain concentrations of (2S,6S)-hydroxynorketamine were also determined after the intravenous administration of (S)-ketamine, where significant plasma and brain tissue concentrations of (2S,6S)-hydroxynorketamine were observed 10 min after administration. The (S)-ketamine metabolites (S)-norketamine, (S)-dehydronorketamine, (2S,6R)-hydroxynorketamine, (2S,5S)-hydroxynorketamine and (2S,4S)-hydroxynorketamine were also detected in both plasma and brain tissue. The enantioselectivity of the conversion of (S)-ketamine and (R)-ketamine to the respective (2,6)-hydroxynorketamine metabolites was also investigated over the first 60 min after intravenous administration. (S)-Ketamine produced significantly greater plasma and brain tissue concentrations of (2S,6S)-hydroxynorketamine relative to the (2R,6R)-hydroxynorketamine observed after the administration of (R)-ketamine. However, the relative brain tissue: plasma concentrations of the enantiomeric (2,6)-hydroxynorketamine metabolites were not significantly different indicating that the penetration of the metabolite is not enantioselective.

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the ␤ 2 -adrenergic receptor, we developed a new agonist radioligand for binding assays. [3 H](R,RЈ)-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total ␤ 2 -adrenoceptor (AR) population as determined with the antagonist [ 3 H]CGP-12177. The ␤ 2 -AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K i values determined for fenoterol analogs model much better the cAMP activity of the ␤ 2 -AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [ 3 H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel ␤ 2 -AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the ␤ 2 -adrenoceptor.

Two ring-A cleaved analogues of the natural product artemisinin have been synthesized and examined for in vitro anti ma la rial activity.