A New Radioligand Binding Assay to Measure the Concentration of Drugs in Rodent Brain Ex Vivo

ABSTRACT We have developed a new radioligand binding assay method to measure the concentration of nonradiolabeled drugs in the brain ex vivo. This new method fuses the concepts of standard competition and saturation binding assays and uses a transformed version of the Cheng-Prusoff equation (Biochem Pharmacol 22: 3099 -3108, 1973) to calculate the drug concentration. After testing the validity of this method, we demonstrated its utility by measuring the brain concentration of sazetidine-A, a newly developed nicotinic receptor ligand, and its elimination rate after a single subcutaneous administration. Our results indicate that sazetidine-A reaches brain concentrations that are known to occupy and desensitize the majority of neuronal nicotinic acetylcholine receptor binding sites. Furthermore, using this method, we estimated the half-life of sazetidine-A in the rat brain to be ϳ65 min. It is important to note that the method described here to measure sazetidine-A in brain should be generalizable to other drugs acting at any receptor that can be reliably measured with a radiolabeled ligand

Chronic Nicotine Differentially Regulates ␣6-and ␤3- Containing Nicotinic Cholinergic Receptors in Rat Brain

ABSTRACT We investigated the effects of chronic nicotine on ␣6-and ␤3-containing nicotinic acetylcholine receptors (nAChRs) in two rat brain regions using three methodological approaches: radioligand binding, immunoprecipitation, and nicotine-stimulated synaptosomal release of dopamine. Nicotine was administered by osmotic minipumps for 2 weeks. Quantitative autoradiography with [ 125 I]␣-conotoxin MII to selectively label ␣6* nAChRs showed a 28% decrease in binding in the striatum but no change in the superior colliculus. Immunoprecipitation of nAChRs labeled by [ 3 H]epibatidine in these two regions showed that chronic nicotine increased ␣4-and ␤2-containing nAChRs by 39 to 67%. In contrast, chronic nicotine caused a 39% decrease in ␣6-containing nAChRs in striatum but no change in superior colliculus. No changes in ␤3-containing nAChRs were seen in either region after chronic nicotine. The decreased expression of ␣6-containing nAChRs persisted for at least 3 days, recovering to baseline by 7 days after removal of the pumps. There was a small but significant decrease in total nicotine-stimulated dopamine release in striatal synaptosomes after nicotine exposure. However, the component of dopamine release that was resistant to ␣-conotoxin MII blockade was unaffected, whereas dopamine release that was sensitive to blockade by ␣-conotoxin MII was decreased by 56%. These findings indicate that the ␣6* nAChR is regulated differently from other nAChR subtypes, and they suggest that the inclusion of a ␤3 subunit with ␣6 may serve to inhibit nicotineinduced down-regulation of these receptors

Identification of six new susceptibility loci for invasive epithelial ovarian cancer.

Genome-wide association studies (GWAS) have identified 12 epithelial ovarian cancer (EOC) susceptibility alleles. The pattern of association at these loci is consistent in BRCA1 and BRCA2 mutation carriers who are at high risk of EOC. After imputation to 1000 Genomes Project data, we assessed associations of 11 million genetic variants with EOC risk from 15,437 cases unselected for family history and 30,845 controls and from 15,252 BRCA1 mutation carriers and 8,211 BRCA2 mutation carriers (3,096 with ovarian cancer), and we combined the results in a meta-analysis. This new study design yielded increased statistical power, leading to the discovery of six new EOC susceptibility loci. Variants at 1p36 (nearest gene, WNT4), 4q26 (SYNPO2), 9q34.2 (ABO) and 17q11.2 (ATAD5) were associated with EOC risk, and at 1p34.3 (RSPO1) and 6p22.1 (GPX6) variants were specifically associated with the serous EOC subtype, all with P < 5 × 10(-8). Incorporating these variants into risk assessment tools will improve clinical risk predictions for BRCA1 and BRCA2 mutation carriers.COGS project is funded through a European Commission's Seventh Framework Programme grant (agreement number 223175 ] HEALTH ]F2 ]2009 ]223175). The CIMBA data management and data analysis were supported by Cancer Research.UK grants 12292/A11174 and C1287/A10118. The Ovarian Cancer Association Consortium is supported by a grant from the Ovarian Cancer Research Fund thanks to donations by the family and friends of Kathryn Sladek Smith (PPD/RPCI.07). The scientific development and funding for this project were in part supported by the US National Cancer Institute GAME ]ON Post ]GWAS Initiative (U19 ]CA148112). This study made use of data generated by the Wellcome Trust Case Control consortium. Funding for the project was provided by the Wellcome Trust under award 076113. The results published here are in part based upon data generated by The Cancer Genome Atlas Pilot Project established by the National Cancer Institute and National Human Genome Research Institute (dbGap accession number phs000178.v8.p7). The cBio portal is developed and maintained by the Computational Biology Center at Memorial Sloan ] Kettering Cancer Center. SH is supported by an NHMRC Program Grant to GCT. Details of the funding of individual investigators and studies are provided in the Supplementary Note. This study made use of data generated by the Wellcome Trust Case Control consortium, funding for which was provided by the Wellcome Trust under award 076113. The results published here are, in part, based upon data generated by The Cancer Genome Atlas Pilot Project established by the National Cancerhttp://dx.doi.org/10.1038/ng.3185This is the Author Accepted Manuscript of 'Identification of six new susceptibility loci for invasive epithelial ovarian cancer' which was published in Nature Genetics 47, 164–171 (2015) © Nature Publishing Group - content may only be used for academic research

Title Page Title: A New Radioligand Binding Assay to Measure the Concentration of Drugs in Rodent Brain Ex Vivo Running Title Page Running Title: A New Competitive-Saturation Binding Assay

Abstract We have developed a new radioligand binding assay method to measure the concentration of non-radiolabeled drugs in the brain ex vivo. This new method fuses the concepts of standard competition and saturation binding assays, and utilizes a transformed version of the Cheng-Prusoff equation to calculate the drug concentration. After testing the validity of this method, we demonstrated its utility by measuring the brain concentration of sazetidine-A, a newly developed nicotinic receptor ligand, and its elimination rate after a single subcutaneous administration. Our results indicate that sazetidine-A reaches brain concentrations that are known to occupy and desensitize the majority of nAChR binding sites. Furthermore, using this method, we estimated the half-life of sazetidine-A in the rat brain to be ~65 min. It is important to note that the method described here to measure sazetidine-A in brain should be generalizable to other drugs acting at any receptor that can be reliably measured with a radiolabeled ligand

Effects of chronic phencyclidine on fixed-ratio responding: No relation to neurotransmitter receptor binding in rat cerebral cortex

The effects of chronic phencyclidine (3.2 mg/kg for 25 days) on responding maintained under a fixed-ratio 30 schedule of food presentation were studied in rats. Initially phencyclidine produced large decreases in the overall rate of responding. This decrease was due primarily to long pauses in responding and secondarily to a decrease in local rates of responding. Although tolerance developed to the rate-decreasing effects of phencyclidine in each subject, the extent and pattern of its development differed among the subjects. After the chronic drug regimen, the rats were sacrificed. Ligand binding to muscarinic cholinergic, opiate, adrenergic, and serotonergic receptors in cortex was then compared to that in rats which received saline with operant training, phencyclidine alone, or saline alone. Neither operant behavior alone, phencyclidine alone, nor the interaction of phencyclidine and operant behavior was found to alter binding to these receptors. The results indicate that behavioral tolerance develops to phencyclidine, but it is not accompanied by changes in binding to the receptors studied. © 1982

Electroconvulsive shocks increase the concentration of neocortical and hippocampal neuropeptide Y (NPY)-like immunoreactivity in the rat

Studies on humans and rats have suggested that neuropeptide Y (NPY) is involved in major depression and anxiety. Therefore, we conducted the present study in order to elucidate the effect of repeated (13 or 14 days) treatment of rats with electroconvulsive shocks (ECS) on the concentration of NPY-like immunoreactivity (-LI) in various brain regions, adrenals and plasma. In addition, the effect of ECS on 125I-NPY binding was studied in 3 brain regions. The effects of ECS were compared to effects of 3 control treatments: one group not being handled at all during the time period, one group handled like the ECS-group but not receiving shocks, and one group receiving shocks below the threshold for induction of convulsions. The latter group developed behavioural signs reminiscent of the inescapable shock-induced ‘learned helplessness’ syndrome (a proposed animal model of depression). We found that the concentration of NPY-LI in the frontal and parietal cortex and in the hippocampus were approximately doubled in the ECS-group as compared to the 3 control groups. No changes in NPY-LI were detected in the striatum, hypothalamus, pons, olfactory bulbs or cerebellum, nor in plasma or adrenals. In spite of the marked changes NPY-LI concentration, the binding characteristics of 125I-NPY in the frontal and parietal cortex and in the hippocampus were similar in all 4 groups of rats. Finally, we confirmed the previous observationnthat ECS increase [ 3H]prazosin binding in cortex. In conclusion, ECS treatment increases neocortical and hippocampal NPY-LI concentrations, while leaving 125I-NPY binding unaffected. Subconvulsive shocks were without effect