Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell

ABSTRACT We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo [3,4-d]pyrimidine-4-one , the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC 50 ϭ 55 nM) and murine (IC 50 ϭ 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca 2ϩ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity

Modulation of hippocampal acetylcholine release after fimbria-fornix lesions and septal transplantation in rats

Female Long-Evans rats sustained electrolytic lesions of the fimbria and the dorsal fornix causing a partial lesion of the septohippocampal pathway. Two weeks later, the rats received intra-hippocampal grafts of fetal septal cell suspensions. Nine to twelve months later, the release of acetylcholine (ACh) in the hippocampus of sham-operated, lesion-only and grafted rats was measured by microdialysis. The extent of cholinergic (re)innervation was determined by acetylcholinesterase (AChE) staining and densitometry. In both lesion-only and grafted rats, the ratio of ACh release to AChE staining intensity was increased as compared to sham-operated rats, indicating a loss of endogenous inhibitory mechanisms. Scopolamine (0.5 mg/kg i.p.), a muscarinic antagonist, increased ACh release in all treatment groups. 8-OH-DPAT (0.5 mg/kg s.c.), an agonist at serotonergic 5HT1A-receptors, induced an increase of hippocampal ACh release in sham-operated rats. This effect was lost in lesion-only rats, but was fully restored by neuronal grafting. As 8-OH-DPAT influences hippocampal ACh release by a postsynaptic action, this finding indicates that the host brain exerts a serotonergic influence on the grafted cholinergic neurons

Oocyte and ovarian tissue cryopreservation in European countries : statutory background, practice, storage and use

STUDY QUESTION: What is known in Europe about the practice of oocyte cryopreservation (OoC), in terms of current statutory background, funding conditions, indications (medical and ‘non-medical’) and specific number of cycles? SUMMARY ANSWER: Laws and conditions for OoC vary in Europe, with just over half the responding countries providing this for medical reasons with state funding, and none providing funding for ‘non-medical’ OoC. WHAT IS ALREADY KNOWN: The practice of OoC is a well-established and increasing practice in some European countries, but data gathering on storage is not homogeneous, and still sparse for use. Ovarian tissue cryopreservation (OtC) is only practiced and registered in a few countries. STUDY DESIGN, SIZE, AND DURATION: A transversal collaborative survey on OoC and OtC, was designed, based on a country questionnaire containing information on statutory or professional background and practice, as well as available data on ovarian cell and tissue collection, storage and use. It was performed between January and September 2015. PARTICIPANTS/MATERIALS, SETTING AND METHODS: All ESHRE European IVF Monitoring (EIM) consortium national coordinators were contacted, as well as members of the ESHRE committee of national representatives, and sent a questionnaire. The form included national policy and practice details, whether through current existing law or code of practice, criteria for freezing (age, health status), availability of funding and the presence of a specific register. The questionnaire also included data on both the number of OoC cycles and cryopreserved oocytes per year between 2010 and 2014, specifically for egg donation, fertility preservation for medical disease, ‘other medical’ reasons as part of an ART cycle, as well as for ‘non-medical reasons’ or age-related fertility decline. Another question concerning data on freezing and use of ovarian tissue over 5 years was added and sent after receiving the initial questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: Out of 34 EIM members, we received answers regarding OoC regulations and funding conditions from 27, whilst 17 countries had recorded data for OoC, and 12 for OtC. The specific statutory framework for OoC and OtC varies from absent to a strict frame. A total of 34 705 OoC cycles were reported during the 5-year-period, with a continuous increase. However, the accurate description of numbers was concentrated on the year 2013 because it was the most complete. In 2013, a total of 9126 aspirations involving OoC were reported from 16 countries. Among the 8885 oocyte aspirations with fully available data, the majority or 5323 cycles (59.9%) was performed for egg donation, resulting in the highest yield per cycle, with an average of 10.4 oocytes frozen per cycle. OoC indication was ‘serious disease’ such as cancer in 10.9% of cycles, other medical indications as ‘part of an ART cycle’ in 16.1%, and a non-medical reason in 13.1%. With regard to the use of OoC, the number of specifically recorded frozen oocyte replacement (FOR) cycles performed in 2013 for all medical reasons was 14 times higher than the FOR for non-medical reasons, using, respectively, 8.0 and 8.4 oocytes per cycle. Finally, 12 countries recorded storage following OtC and only 7 recorded the number of grafted frozen/thawed tissues. LIMITATIONS, REASONS FOR CAUTION: Not all countries have data regarding OoC collection, and some data came from voluntary collaborating centres, rather than a national authority or register. Furthermore, the data related to use of OoC were not included for two major players in the field, Italy and Spain, where numbers were conflated for medical and non-medical reasons. Finally, the number of cycles started with no retrieval is not available. Data are even sparser for OtC. WIDER IMPLICATIONS OF THE FINDINGS: There is a need for ART authorities and professional bodies to record precise data for practice and use of OoC (and OtC), according to indications and usage, in order to reliably inform all stakeholders including women about the efficiency of both methods. Furthermore, professional societies should establish professional standards for access to and use of OoC and OtC, and give appropriate guidance to all involved. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by ESHRE. There are no conflicts of interest.peer-reviewe

Assigning moral roles within the Second World War in Europe: National similarities, differences, and implications for group-level moral representations

The moral roles assigned to nations that took part in the Second World War cast a shadow over contemporary international politics. To understand contemporary moral beliefs about the war, we took 11 mostly student samples from 9 nations that took part in the European theater of war (total N = 1,427). We asked respondents, in free and scaled listings, to identify the war’s heroes, villains, victims, and recipients of help. Nations and individuals seen as heroes, victims and villains could be readily identified by most samples and showed both continuity and difference across nations. Most nations preferentially assigned themselves hero and victim roles, and the two were correlated positively, showing ingroup favoritism linked to victimhood. These findings show the importance of morality to contemporary views of the war and suggest further directions for studying today’s political climate in Europe and elsewhere