William J. Wheeler

[3H]-LY341495 as a novel antagonist radioligand for group II metabotropic glutamate (mGlu) receptors: characterization of binding to membranes of mGlu receptor subtype expressing cells

Metabotropic glutamate (mGlu) receptors are a family of eight known subtypes termed mGlu1-8. Currently, few ligands are available to study the pharmacology of mGlu receptor subtypes. In functional assays, we previously described LY341495 as a highly potent and selective mGlu2 and mGlu3 receptor antagonist. In this study, radiolabeled [3H]-LY341495 was used to investigate the characteristics of receptor binding to membranes from cells expressing human mGlu receptor subtypes. Using membranes from cells expressing human mGlu2 and mGlu3 receptors, [3H]-LY341495 (1 nM) specific binding was > 90% of total binding. At an approximate K(D) concentration for [3H]-LY341495 binding to human mGlu2 and mGlu3 receptors (1 nM), no appreciable specific binding of [3H-]LY341495 was found in membranes of cells expressing human mGlu1a, mGlu5a, mGlu4a, mGlu6, or mGlu7a receptors. However, modest (approximately 20% of mGlu2/3) specific [3H]-LY341495 (1 nM) binding was observed in human mGlu8 expressing cells. [3H]-LY341495 bound to membranes expressing human mGlu2 and mGlu3 receptors in a reversible and saturable manner with relatively high affinities (Bmax 20.5 +/- 5.4 and 32.0 +/- 7.0 pmol/mg protein; and K(D) = 1.67 +/- 0.20 and 0.75 +/- 0.43 nM, respectively). The pharmacology of [3H]-LY341495 binding in mGlu2 and mGlu3 expressing cells was consistent with that previously described for LY341495 in functional assays. [3H]-LY341495 binding provides a useful way to further investigate regulation of receptor expression and pharmacological properties of mGlu2 and mGlu3 receptor subtypes in recombinant systems.

Novel Pharmacologic Approaches to the Management of Sepsis: Targeting the Host Inflammatory Response

Sepsis is currently the 10(th) leading cause of death overall and accounts for significant healthcare expenditures in the developed world. There are now more deaths attributable to sepsis than coronary artery disease, stroke, or cancer, and it is widely believed that the incidence of sepsis and sepsis-related mortality will continue to rise. Based on these sobering statistics, there is great interest in identifying novel treatments for managing critically ill children and adults with sepsis. Unfortunately, to date, there have been very few successful therapeutic agents employed in the clinical setting. Despite these disappointing results, new therapeutic agents continue to be identified, and there is reason for optimism and hope for the future. Herein, we will briefly review several novel therapeutic adjuncts for the management of critically ill patients with sepsis. We will largely focus on those therapies that directly target the host inflammatory response, specifically those that result in activation of the transcription factor, nuclear factor (NF)-kappaB. We will also reference some of the patents recently filed that pertain to the host innate immune response and sepsis.

[3H]LY341495, a highly potent, selective and novel radioligand for labeling group II metabotropic glutamate receptors

We report herein the synthesis and pharmacological characterization of a tritiated version of the potent and selective cyclopropyl amino acid LY341495 as a radioligand to label group II metabotropic glutamate receptors in rat brain homogenates.

Synthesis of [11C]dapoxetine · HCl, a serotonin re-uptake inhibitor: Biodistribution in rat and preliminary PET imaging in the monkey

[11C]Dapoxetine.HCl, S-(+)-N,N-dimethyl-a-[2-(naphthalenyloxy)ethyl] benzenemethanamine hydrochloride, a potent serotonin re-uptake inhibitor was prepared from its mono-methyl precursor, S-(+)-N-methyl-a-[2-(naphthalenyloxy)ethyl]benzene methanamine hydrochloride. Biodistribution was determined in rats at 5, 30 and 60 min after injection and preliminary PET studies were performed in a Rhesus monkey. 11CH3I was bubbled into a solution of S-(+)-N-methyl-alpha-[2-(naphthalenyloxy)ethyl]benzene methanamine hydrochloride (3.0 mg in DMSO) and the mixture was heated at 110 degrees C for 8 min. [11C]Dapoxetine.HCl was purified by HPLC on a C18 cartridge eluted with MeOH:phosphate buffer, pH 7,2 (75:25) with a 10% yield (end of synthesis). The time required for the synthesis was 40 min, from the end of bombardment. Radiochemical purity of the final product was > 99% and specific activity was routinely > 400 mCi/mumol [EOS]. In the biodistribution studies the highest concentration (%ID/g +/- SEM) of dapoxetine.HCl was detected in lung: 4.56 +/- 0.27 (5 min), 1.28 +/- 0.18 (30 min) and 0.67 +/- 0.04 (60 min). Brain accumulation was 0.76 +/- 0.02 (5 min), 0.46 +/- 0.04 (30 min) and 0.27 +/- 0.01 (60 min). Preliminary PET studies demonstrated significant displaceable binding in the cerebral cortex and subcortical grey matter. These results demonstrate that [11C]dapoxetine.HCl can be prepared in high purity and may be useful for the in vivo evaluation of serotonin re-uptake mechanisms.

Binding of an AMPA receptor potentiator ([3H]LY395153) to native and recombinant AMPA receptors

LY395153 is a member of a newly described class of arylpropylsulfonamide AMPA receptor potentiators. Here, we characterize and compare [(3)H]LY395153 binding to native AMPA receptors from rat cerebral cortex and recombinant human GluR4(flip) receptors expressed in HEK293 cells. L-Glutamate and AMPA increased [(3)H]LY395153 binding to both native and recombinant AMPA receptors in a concentration dependent and stereoselective manner; this effect of AMPA receptor agonists reflects an apparent increase in ligand affinity. In the presence of L-glutamate (500 microM), [(3)H]LY395153 binding is saturable; the affinity of this radioligand is slightly, albeit statistically significantly higher at human GluR4(flip) (K(d)=55.6+/-5.3nM) than rat cortical receptors (K(d)=110+/-15.1nM). NBQX competitively inhibited L-glutamate-induced increases in [(3)H]LY395153 binding in both native and recombinant receptors, whilst LY303070 (the active isomer of GYKI53655) noncompetitively inhibited this effect in native, but not recombinant receptors. The prototypic AMPA receptor potentiator cyclothiazide competitively inhibited [(3)H]LY395153 binding with a potency (K(i) approximately 7 microM) comparable to EC(50) values reported in electrophysiological studies. In contrast, the structurally unrelated AMPA receptor potentiator CX 516 did not inhibit [(3)H]LY395153 binding at concentrations of up to 600 microM. Further, at concentrations reported to facilitate AMPA receptor desensitization, thiocyanate acts as a competitive inhibitor of [(3)H]LY395153 binding. [(3)H]LY395153 binding was unaffected by a variety of structurally (and mechanistically) diverse compounds tested at a concentration of 10 microM. These data indicate [(3)H]LY395153 is a useful probe for labeling a unique modulatory site on both native and recombinant AMPA receptors.

Wittig methylenation of 9,10-didehydro-6-methylergolin-8-one, a novel synthesis of lysergene and its subsequent conversion to agroclavine

Abstract N-p-Ts-lysergene ( 6 ) was synthesized by the reaction of N-p-Ts-9,10-didehydro-6-methylergolin-8-one ( 5 ) with methylene triphenylphosphorane. Subsequent lithium/ammonia reduction of 6 yielded agroclavine ( 3 ).

Evaluation of [3H]LY341495 for labeling group II metabotropic glutamate receptors in vivo

New glutamatergic drugs are being developed as potential therapies for neurodegenerative disorders, anxiety disorders, and psychoses. The development of effective mGluR radiotracers would provide essential tools with which to probe these sites in living humans, providing critical information about certain disease processes involving the glutamaterigic system and its regulation in humans. As a first step towards this goal, the tritiated form of the high affinity group II metabotropic glutamate receptor (mGluR) antagonist LY341495 [K(D) (mGluR(2)) = 1.67 +/- 0.20 nM, K(D) (mGluR(3)) = 0.75 +/- 0.43 nM] was evaluated to determine its potential to label mGluRs in vivo. Dissection analysis of the regional brain distribution over time of [(3)H]LY341495 in male rats revealed low brain uptake and no significant demonstrable saturable binding of this tracer. A group II mGluR tracer possessing higher affinity than [(3)H]LY341495 and an absence of carboxylic acid groups is likely required for in vivo PET imaging purposes.

Catalytic hydrogenolysis of poly-iodinated recombinant human insulin-like growth factor II (IGF-II) : a potentially useful method for the tritiation of IGF-II

A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Mono- and di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the iodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.

Metabolism of LY654322, a Growth Hormone Secretagogue, to an Unusual Diimidazopyridine Metabolite

2-Methylalanyl-N-{1-[(1R)-1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidin-1-ylethyl]-1H-imidazol-4-yl}-5-phenyl-d-norvalinamide (LY654322) was rapidly cleared in rats and dogs by renal excretion of parent and metabolism (oxidative and hydrolytic). Among the metabolites identified in the urine of rats and dogs was M25, which was structurally unusual. Indeed, the characterization of M25 and investigation into its disposition relied on the convergence of diverse analytical methodologies. M25 eluted after the parent on reverse-phase chromatography with an MH+ at m/z 598 (parent + 35 Da). Given its increased lipophilicity and its mass difference compared with the parent, it was evident that M25 was not a phase 2 conjugate. Subsequent liquid chromatography with multiple-stage tandem mass spectrometry and accurate mass experiments identified the structure of M25 as having two replicates of the 1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidinyl substructure flanking a central aromatic core of composition C7H3N5 that was refractory to fragmentation. Compared with the UV spectrum of the parent (λmax = 213 nm), M25 displayed a bathochromic shift (λmax = 311 nm), which substantiated extensive conjugation within the central core. Subsequent NMR analysis of M25 isolated from dog urine coupled with molecular modeling revealed the structure to be consistent with a diimidazopyridine core with two symmetrically substituted 1-(4-fluorophenyl)-1-methyl-2-oxo-2-pyrrolidinyl moieties. Using a structural analog with a chromophore similar to M25, LC-UV was used to quantitate M25 and determine its urinary disposition. The formation of M25 appears consistent with hydrolysis of LY654322 to an aminoimidazole, dimerization of the latter with the loss of NH3, C-formylation, and subsequent ring closure and aromatization with loss of H2O.

Corrigendum to “Synthesis and biological activity of some known and putative duloxetine metabolites”[Bioorg. Med. Chem. Lett. 14 (2004) 3481]

Compound 2 had moderate to weak activity in all of the membranes; however, this compound is unstable so the values may not be accurate. Although compounds 2, 3, 4, and 7 showed some degree of in vitro affinity, these compounds do not appear to contribute to the in vivo activity of duloxetine, since they circulate in human plasma at such low concentrations. The circulating metabolites are in the conjugated forms and do not appear to be active. The conjugated metabolites tested (10, 11, 12, 13, 15) were devoid of any significant binding to any of the three transporters.