James E. Bupp

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.

Tricyclic analogues of artemisinin: synthesis and antimalarial activity of (+)-4,5-secoartemisinin and (–)-5-nor-4,5-secoartemisinin

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

The distribution and clearance of (2S,6S)-hydroxynorketamine, an active ketamine metabolite, in Wistar rats

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.

Effect that Various Electron Donating Functional Groups have Regarding Nitromethane's Inability to Quench Fluorescence Emission of Nonalternant Fluoranthenoid Polycyclic Aromatic Hydrocarbons

Abstract Nitromethane selectively quenches fluorescence emission of alternant polycyclic aromatic hydrocarbons (PAHs) via an electron transfer mechanism. Emission intensities of nonalternant PAHs, for the most part, are unaffected. To better understand the quenching mechanism, the fluorescence behavoir of 1-methylfluoranthene, 2-methylfluoranthene, 3-methylfluoranthene, 7-methylfluoranthene, 8-methylfluoranthene, t-butyldibenzo[ghi, mno]fluoranthene, 10-methylbenzo[b]fluoranthene, 12-methoxybenzo[b]fluoranthene, 10-methoxybenzo[b]fluoranthene, 10-hydroxybenzo[b]fluoranthene, 10-hydroxybenzo[j]fluoranthene, 10-methoxybenzo[j]fluoranthene, 3-metnoxybenzo[k]fluoranthene, 3-hydroxy-benzo[k]fluoranthene, 9-methoxybenzo[k]fluoranthene and 9-hydroxybenzo[k]fluoranthene dissolved in neat acetonitrile, toluene-acetonitrile and ethyl acetate-acetonitrile solvents was measured at different nitromethane concentrations. Experimental results show that electron donating methyl-, t-butyl, methoxy- and hydroxy-groups do n...

Thermodynamics and Docking of Agonists to the β2-Adrenoceptor Determined Using [3H](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 β2-adrenergic receptor, we developed a new agonist radioligand for binding assays. [3H](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 β2-adrenoceptor (AR) population as determined with the antagonist [3H]CGP-12177. The β2-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and Ki values determined for fenoterol analogs model much better the cAMP activity of the β2-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 [3H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β2-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β2-adrenoceptor.

[125I]AT-1012, a new high affinity radioligand for the α3β4 nicotinic acetylcholine receptors

Recent genetic and pharmacological studies have implicated the α3, β4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 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 α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat α3β4 and α4β2 nAChR transfected into HEK cells, as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat α3β4 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 α3β4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human α3β4α5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3β4 over α4β2 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 α3β4 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 α3β4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the α3β4 nAChR in the presence of other nAChR subtypes.

Radiolabeled antimalarials: Synthesis of 14C-artemisinin

The stereoselective total synthesis of the antimalarial agent 14 C-(+)-artemisinin (1), incorporating 14 C at C-16 (C-9 methyl) is reported, in 18% radiochemical yield from acid 3.

Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein

Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]-benzonic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)be nzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED500.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID50 0.9 microM and 0.85 microM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at -78 degrees C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p-chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.

Combined chromatographic—isotopic dilution analysis of fecapentaenes in human feces

Fecapentaene-12 (FP-12) and fecapentaene-14 (FP-14) are genotoxic unsaturated ether lipids produced by colonic bacteria in man. We have developed and applied to feces collections from normal volunteers direct isotopic dilution procedures using tritium-labeled (at C5) FP-12 and FP-14 for measuring these compounds. FPs were recovered from feces by solvent extraction, silica cartridge clean-up, and analytical liquid chromatography. Low levels of FP-12 and FP-14 (less than 0.1 to 2.4 micrograms/g of freeze-dried feces) were observed. Identity of chromatographic peaks was established by co-elution and by ultraviolet absorption spectra obtained via photodiode array scanning. Two unknown peaks were tentatively identified from absorption spectra as closely related compounds with increased (hexane?) or decreased (tetraene?) number of double bonds. Levels of FPs increased after incubation of feces at 37 degrees C for 96 h under anaerobic conditions and pre-FP-12 and pre-FP-14 peaks were observed, which showed identical spectra with authentic FPs. These were interpreted to be isomeric forms of the all-trans-[3H]FPs used for the isotopic dilution analysis. Total FPs (including pre-FP) yielded a range of 0.3-80 micrograms FP-12 and 2.8-44 micrograms FP-14 per g of freeze-dried feces from the study group.

Synthesis of a doxycycline-[13CD3] 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%.