Localization of opioid receptor antagonist [3H]-LY255582 binding sites in mouse brain: comparison with the distribution of mu, delta and kappa binding sites.

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.

Synthesis and biological activity of some known and putative duloxetine metabolites.

Several putative phase I duloxetine metabolites, 4-hydroxy-, 5-hydroxy-, 6-hydroxy-, 5-hydroxy-6-methoxy-, 6-hydroxy-5-methoxy-, 5,6-dihydroxy-, and 4,6-dihydroxyduloxetine were synthesized, and their phase II metabolite as glucuronide or sulfate conjugates were also synthesized. Their in vitro binding activities were compared to that of parent compound duloxetine.

Preclinical characterization of 2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy]benzoic acid metabolism: in vitro species comparison and in vivo disposition in rats.

Assessment of the pharmacokinetics of [14C]2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy-]benzoic acid ([14C]LY293111), an experimental anti-cancer agent, suggested long-lived circulating metabolites in rats. In vivo metabolites of LY293111 were examined in plasma, bile, urine, and feces of Fischer 344 (F344) rats after oral administration of [14C]LY293111. Metabolites were profiled by high-performance liquid chromatography-radiochromatography, and identified by liquid chromatography (LC)/mass spectrometry and LC/NMR. The major in vivo metabolites of LY293111 identified in rats were phenolic (ether), acyl, and bisglucuronides of LY293111. Measurement of radioactivity in rat plasma confirmed that a fraction of LY293111-derived material was irreversibly bound to plasma protein and that this bound fraction increased over time. This was consistent with the observed disparity in half-lives between LY293111 and total radioactivity in rats and monkeys, and is likely due to covalent modification of proteins by the acyl glucuronide. In vitro metabolism of [14C]LY293111 in liver slices from CD-1 mice, F344 rats, rhesus and cynomolgus monkeys, and humans indicates that glucuronidation was the primary metabolic pathway in all species. The acyl glucuronide was the most prevalent radioactive peak (16% of total 14C) produced by F344 rat slices, whereas the ether glucuronide was the major metabolite in all other species (26-36% of total 14C). Several minor hydroxylated metabolites were detected in F344 rat slice extracts but were not observed in other species. The data presented suggest that covalent modification of proteins by LY293111 acyl glucuronide is possible in multiple species, although the relative reactivity of this metabolite appears to be low compared with those known to cause adverse drug reactions.

[3H]LY341495 binding to group II metabotropic glutamate receptors in rat brain.

[3H]LY341495 is a highly potent and selective antagonist for group II metabotropic glutamate (mGlu) receptors (mGlu2 and mGlu3), which has been used to label these receptors in cells expressing recombinant receptor subtypes. In this study, we characterized the kinetics, pharmacology, and distribution of [3H]LY341495 binding to mGlu receptors in rat brain tissue. Equilibrium experiments in the rat forebrain demonstrated binding to a single site that was saturable, reversible, and of high affinity (Bmax, 3.9 +/- 0.65 pmol/mg of protein, Kd, 0.84 +/- 0.11 nM). The relative order of potencies for displacement of [3H]LY341495 by mGlu receptor ligands was LY341495 >> L-glutamic acid > LY354740 > (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine > 4-(2R,4R)-aminopyrrolidine-2,4-dicarboxylate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (R,S)-alpha-methyl-4-phosphonophenylglycine > (R,S)3,5-dihydroxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. [3H]LY341495 was not displaced by the selective ionotropic glutamate receptor agonists N-methyl-D-aspartic acid, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate at concentrations up to 1 mM. Comparison of [3H]LY341495 binding in rat brain with recombinant mGlu receptor subtypes demonstrated a very high correlation with mGlu3 receptor binding (r2 = 0.957), a significant, but lower, correlation with mGlu2 receptor binding (r2 = 0.869), but no significant correlation to mGlu8 receptor binding (r2 = 0.284). Regional studies using autoradiography showed a similar distribution of [3H]LY341495 binding to that for group II mGlu receptors previously reported by others using immunocytochemical techniques. These studies indicate that [3H]LY341495 selectively labels group II (mGlu2/3) receptors, but under the conditions used, [3H]LY341495 may bind predominately to mGlu3 receptor populations in the rat forebrain.

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.

Binding of [3H](2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine ([3H]LY341495) to cell membranes expressing recombinant human group III metabotropic glutamate receptor subtypes.

LY341495 is a highly potent and selective antagonist for group II mGlu receptors (mGlu2 and mGlu3). High affinity binding of [3H]LY341495 to recombinant human group II mGlu receptors (mGlu2 and mGlu3), and in rat brain homogenates (Kd approximately 1 nM), has been previously described. Although LY341495 is a very selective nM-potent antagonist for group II mGlu receptors, it is also a relatively potent antagonist for group III mGlu receptors at high nanomolar to low micromolar concentrations. In this study we examined and characterized the binding of [3H]LY341495 to membranes of cells expressing recombinant human group III mGlu receptors. Using up to 100 nM of [3H]LY341495, the level of specific binding in human mGlu4a receptor-expressing cell membranes was not appreciable and binding to this site was not examined further. In contrast, we demonstrated sufficient specific binding of [3H]LY341495 to human mGlu6, mGlu7a and mGlu8a receptor-expressing cell membranes to allow for further characterizations. [3H]LY341495 binding was saturable and rapidly reversible. [3H]LY341495 bound to a single site in each cell line, with Kd and Bmax values of 31.6+/-6.8 nM and 3.3+/-0.7 pmol/mg protein (mGlu6), 72.7+/-22.0 nM and 3.7+/-0.4 pmol/mg protein (mGlu7a), and 14.0+/-1.1 nM and 3.0+/-0.2 pmol/mg protein (mGlu8a). [3H]LY341495 binding to mGlu6, 7a and 8a was displaceable by compounds which interact functionally with group III mGlu receptors. For example, L-AP4 displaced [3H]LY341495 with Ki values of 6.8+/-3.1 microM (mGlu6), 211+/-43 microM (mGlu7a) and 1.6+/-0.3 microM (mGlu8a). With L-glutamate, we obtained Ki values of 12.3+/-3.5, 869+/-154 and 4.5+/-0.83 microM, for mGlu6, mGlu7a and mGlu8a, respectively. Ki values for unlabelled LY341495 were 0.058+/-0.008, 0.22+/-0.05 and 0.029+/-0.008 microM, respectively. These studies demonstrated that [3H]LY341495 is a useful radioligand for studying the pharmacology and expression of recombinant mGlu6, 7a and 8a receptors in cell lines.

[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.

[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.

Biotransformation of the antiviral agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and characteristics of a mesoionic ribose metabolite.

The biotransformation of the antiinfluenza agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896, I) was studied. In addition to a urea metabolite (II) formed by transformation of the cyanamide functionality, another highly polar metabolite was found in mouse urine and in BSC-1, MDCK, and other cell culture incubations of [14C]LY217896. Using 13C-labeled LY217896 together with NMR and MS techniques, this highly polar metabolite was identified as a ribose derivative (III), which apparently exists in a mesoionic form (i.e. positive and negative charges within the same ring system). It was also found that this ribose is formed from LY217896 and ribose-1-phosphate in a reaction catalyzed by the enzyme purine nucleoside phosphorylase, but that the reverse reaction (cleavage of the ribose) is not observed under the conditions used. When tested in vitro using the same assay as that used to measure the antiviral activity of LY217896, this ribose and the urea metabolite exhibit essentially no activity. The presence of a ribose has been implicated in the activity of antiviral compounds such as ribavirin and anticancer agents like 2-aminothiadiazole and tiazofurin, which are structurally similar to LY217896. These activities have been postulated to involve either mono- or triphosphorylated forms, or NAD-type analogs. Possible implications of the formation of this mesoionic ribose metabolite for the mechanism of antiviral activity of LY217896 are discussed.

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.

Mitotic inhibition and chromosome displacement induced by estradiol in Chinese hamster cells.

We tested diethylstilbestrol (DES) and 17 beta-estradiol as mitotic arrestants to determine their effects on chromosome distribution, spindle microtubules, and the cytoplasmic microtubule complex (CMTC) in the Chinese hamster strain Don. Cytological experiments assessed micronuclei induction, chromosome displacement, and anaphase recovery. Indirect immunofluorescence microscopy with antibody to tubulin and electron microscopy were used to illustrate effects on microtubules. Both DES and estradiol were potent inhibitors of mitosis when applied to cells in vitro. Estradiol induced micronuclei at a greater frequency than did DES. Estradiol-arrested metaphases often contained misaligned chromosomes despite the presence of a bipolar spindle and an equatorial plate. Equatorial plates were not observed in DES-arrested cells. Cells recovered quickly from estradiol exposure upon removal of the steroid. The frequency of abnormal metaphases and abnormal anaphases declined as the recovery period increased. Microtubule experiments showed that DES inhibited spindle assembly and disassembled the CMTC, whereas estradiol, at similar concentrations, arrested mitosis in a manner that allowed spindle assembly. A definite effect on the CMTC by estradiol could not be determined. However, changes in cell morphology were observed. In the presence of estradiol, centrosomes organized microtubules that joined with kinetochores of chromosomes at the equatorial plate as well as with those of misaligned chromosomes. Misaligned chromosomes appeared predominantly at polar regions of mitotic cells. Following drug removal, the pole-oriented chromosomes reoriented at the equatorial plate. The unique arresting properties of estradiol may prove useful in studies of chromosome migration and segregation during mitosis.

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro.

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.

The synthesis and biological evaluation of 7 beta-[2-(2-aminooxazol-4-yl)-2-Z-methoximinoacetamido]-cep halosporin antibiotics.

7 beta-[2-(2-Aminooxazol-4-yl)-2-Z-methoximinoacetamido]-3-cep hem -4-carboxylic acids 12 and 13 were synthesized. The microbiological activity of 12 and 13 as well as the beta-lactamase stability of 12 were discussed. Both 12 and 13 were quite active against a wide variety of microorganisms although usually less active than cefotaxime.

The synthesis and biological evaluation of 7 beta-[2-(5-amino-[1,2,4] oxadiazol-3-yl)-2-Z-methoximinoacetamido]-cephalosporin derivatives.

A series of 7 beta-[2-(5-aminooxadiazol-3-yl)-2-Z-methoximinoacetamido] -3-cephem-4-carboxylic acids (7a-g) were synthesized and evaluated microbiologically Although somewhat less active than cefotaxime 7a-g showed good antimicrobial activity against a wide variety of Gram-positive and Gram-negative bacteria. The beta-lactamase stability of 7a and 7f was also discussed.

Metaphase arrest, anaphase recovery and aneuploidy induction in cultured Chinese hamster cells following exposure to mitotic arrestants.

Metaphases of Chinese hamster cells in culture, when blocked by mitotic arrestants, regardless of their mechanisms, would recover when a proper concentration was used. Arrested metaphases would quickly enter anaphase and karyokinesis ensued. However, the frequency of abnormal anaphases would increase when the period of arrest was prolonged, and the frequency of aneuploidy in the recovering cell generations would likewise increase. It appears that in the future, assays for aneuploidy induction by mitotic arrestants can be simplified by determining their potency of mitotic block without the tedious chromosome counts.

Inhibition of estrogen-induced renal carcinoma in Syrian hamsters by vitamin C.

The ability of vitamin C to inhibit induction of renal carcinoma by estrogens was tested in male Syrian hamsters in vivo. The animals received estrogen (estradiol or diethylstilbestrol) implants s.c. Hamsters which were continuously given vitamin C, administered in the drinking water for estradiol-treated or in the food for diethylstilbestrol-treated animals, were observed to develop renal carcinoma with a significantly lower incidence (10 of 33 animals with estradiol implants; 14 of 29 animals with diethylstilbestrol implants) than animals which did not receive vitamin C supplementation (16 of 23 animals with estradiol implants; 11 of 13 animals with diethystilbestrol implants). Administration of vitamin C to estradiol-treated hamsters for only the first 3 months of the carcinogenesis experiment had no effect on tumor incidence, but vitamin C in drinking water for the last 3 months also lowered incidence. Vitamin C supplementation did not significantly alter the absorption of estrogen from the implant; it did not change the estrogenic effect on the hamsters nor did it significantly influence estrogen-dependent H-301 tumor cell growth. The results were taken as evidence for a mechanism of tumor induction via oxidation of estrogens to reactive metabolites capable of inducing kidney tumors.

Bis-trimethylsilylcefamandole: an unusually stable trimethylsilyl-intermediate.

The preparation of crystalline bis-trimethylsilylcefamandole (7) and its utility in the preparation and purification of cefamandole are described. Although stable to solvolysis in isopropyl alcohol, 7 underwent smooth conversion to cefamandole in the presence of water, methanol, or ethanol.