Synthesis of a doxycycline-[(13) CD3 ] standard.

A stable isotope labelled mass spectrometry internal standard of the antibiotic doxycycline was prepared to assist in pharmacokinetic analyses. Our approach was to first N-demethylate doxycycline using a non-classical Polonovski reaction and then re-methylate using methyl-[(13) CD3 ] iodide, which gave doxycycline-[(13) CD3 ] with an isotopic purity of 99%.

Thermodynamics and docking of agonists to the ß(2)-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand.

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 ß(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-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 ß(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The ß(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 ß(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 ß(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 ß(2)-adrenoceptor.

Synthesis of Tritium Labeled (R,R)-4-Methoxyfenoterol.

The preparation of 2',4',6'-[(3)H(3)]-(R,R)-4-methoxyfenoterol, a tritium-labeled derivative of (R,R)-4-methoxyfenoterol was demonstrated on a 15 mCi scale providing material with a specific activity of 57 Ci/mmol.

[¹²5I]AT-1012, a new high affinity radioligand for the α3ß4 nicotinic acetylcholine receptors.

Recent genetic and pharmacological studies have implicated the α3, ß4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3ß4* nAChR subtype has been shown to co-assemble with the α5 or ß3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3ß4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [(125)I]-radiolabeled analog of a high affinity, selective small-molecule α3ß4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3ß4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat α3ß4 and α4ß2 nAChR transfected into HEK cells, as well as at the human α3ß4α5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat α3ß4 nAChR was 1.4 nM, with a B(max) of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [(3)H]epibatidine. Saturation isotherms suggested that [(125)I]AT-1012 binds to a single site on the α3ß4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human α3ß4α5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from α4ß2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3ß4 over α4ß2 nAChR. K(i) values determined for known nAChR compounds using [(125)I]AT-1012 as radioligand were comparable to those obtained with [(3)H]epibatidine. [(125)I]AT-1012 was also used to label α3ß4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3ß4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the α3ß4 nAChR in the presence of other nAChR subtypes.

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome.

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 sub-anesthetic 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.