Anticonvulsant evaluation of novel non-imidazole histamine H3R antagonists in different convulsion models in rats.

Novel non-imidazole histamine H3 receptor (H3R) antagonists (2-8) were developed and assessed for in-vitro antagonist binding affinities at the human histamine H1-H4R. These novel H3R antagonists (2-8) were examined in-vivo for anticonvulsant effects in three different convulsion models in male adult rats. Compound 6 significantly and dose-dependently exhibited decreased duration of tonic hind limb extension (THLE) in the maximal electroshock (MES)- and fully protected animals against pentylenetetrazole (PTZ)-induced convulsion, following acute systemic administration (5, 10, and 20 mg/kg, i.p.). Contrary, all compounds 2-8 showed moderate protection in the strychnine (STR)-induced convulsion model following acute pretreatment (10 mg/kg, i.p.). Moreover, the acute systemic administration of H3R antagonist 6 (10 mg/kg, i.p.) significantly prolonged latency time for MES convulsions. Furthermore, the anticonvulsant effect observed with compound 6 in MES-model was entirely abrogated when rats were co-injected with the brain penetrant H1R antagonist pyrilamine (PYR) but not the brain penetrant H2R antagonist zolantidine (ZOL). However, PYR and ZOL failed to abolish the full protection provided by the H3R antagonist 6 in PTZ- and STR-models. No mutagenic or antiproliferative effects or potential metabolic interactions were shown for compound 6 when assessing its antiproliferative activities and metabolic profiling applying in-vitro methods. These findings demonstrate the potential of non-imidazole H3R antagonists as novel antiepileptic drugs (AEDs) either for single use or in addition to currently available epilepsy medications.

Novel naphthyloxy derivatives - Potent histamine H3 receptor ligands. Synthesis and pharmacological evaluation.

A series of 1- and 2-naphthyloxy derivatives were synthesized and evaluated for histamine H3 receptor affinity. Most compounds showed high affinities with Ki values below 100 nM. The most potent ligand, 1-(5-(naphthalen-1-yloxy)pentyl)azepane (11) displayed high affinity for the histamine H3 receptor with a Ki value of 21.9 nM. The antagonist behaviour of 11 was confirmed both in vitro in the cAMP assay (IC50 = 312 nM) and in vivo in the rat dipsogenia model (ED50 = 3.68 nM). Moreover, compound 11 showed positive effects on scopolamine induced-memory deficits in mice (at doses of 10 and 15 mg/kg) and an analgesic effect in the formalin test in mice with ED50 = 30.6 mg/kg (early phase) and ED50 = 20.8 mg/kg (late phase). Another interesting compound, 1-(5-(Naphthalen-1-yloxy)pentyl)piperidine (13; H3R Ki = 53.9 nM), was accepted for Anticonvulsant Screening Program at the National Institute of Neurological Disorders and Stroke/National Institute of Health (Rockville, USA). The screening was performed in the maximal electroshock seizure (MES), the subcutaneous pentylenetetrazole (scPTZ) and the 6-Hz psychomotor animal models of epilepsy. Neurologic deficit was evaluated by the rotarod test. Compound 13 inhibited convulsions induced by the MES with ED50 of 19.2 mg/kg (mice, i.p.), 17.8 (rats, i.p.), and 78.1 (rats, p.o.). Moreover, 13 displayed protection against the 6-Hz psychomotor seizures (32 mA) in mice (i.p.) with ED50 of 33.1 mg/kg and (44 mA) ED50 of 57.2 mg/kg. Furthermore, compounds 11 and 13 showed in vitro weak influence on viability of tested cell lines (normal HEK293, neuroblastoma IMR-32, hepatoma HEPG2), weak inhibition of CYP3A4 activity, and no mutagenicity. Thus, these compounds may be used as leads in a further search for histamine H3 receptor ligands with promising in vitro and in vivo activity.

Biphenyloxy-alkyl-piperidine and azepane derivatives as histamine H3 receptor ligands.

Novel biphenyloxy-alkyl derivatives of piperidine and azepane were synthesized and evaluated for their binding properties at the human histamine H3 receptor. Two series of compounds were obtained with a meta- and a para-biphenyl moiety. The alkyl chain spacer contained five and six carbon atoms. The highest affinity among all compounds was shown by 1-(6-(3-phenylphenoxy)hexyl)azepane (13) with a Ki value of 18nM. Two para-biphenyl derivatives, 1-(5-(4-phenylphenoxy)pentyl)piperidine (14; Ki=25nM) and 1-(5-(4-phenylphenoxy)pentyl)azepane (16; Ki=34nM), classified as antagonists in a cAMP accumulation assay (IC50=4 and 9nM, respectively), were studied in detail. Compounds 14 and 16 blocked RAMH-induced dipsogenia in rats (ED50 of 2.72mg/kg and 1.75mg/kg respectively), and showed high selectivity (hH4R vs hH3R>600-fold) and low toxicity (hERG inhibition: IC50>1.70µM; hepatotoxicity IC50>12.5µM; non-mutagenic up to 10µM). Furthermore, the metabolic stability was evaluated in vitro on human liver microsomes (HLMs) and/or rat liver microsomes (RLMs). Metabolites produced were analyzed and tentatively identified by UPLC-MS techniques. The results demonstrated easy hydroxylation of the biphenyl ring.

The role of PPARγ in TBBPA-mediated endocrine disrupting effects in human choriocarcinoma JEG-3 cells.

The goal of the present study was to investigate the action of TBBPA on PPARγ protein expression in vitro in human choriocarcinoma-derived placental JEG-3 cells. We also analyzed TBBPA for its action on placental secretion of progesterone and ß-hCG, cell viability, and apoptosis. Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level. This alteration in PPARγ protein expression was accompanied by a decreased ß-hCG level. Interestingly, co-treatment with the PPARγ antagonist GW9662 reversed the TBBPA-mediated changes in PPARγ protein expression but, according to ß-hCG secretion, potentiated an inhibitory effect of TBBPA. Additionally, in our study, we assessed the ability of TBBPA to increase progesterone levels in JEG-3 cells compared with those of controls. Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control. The apoptotic action of TBBPA was also confirmed by Hoechst 33342 staining. These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells. Although co-treatment with antagonist of PPARγ reversed the TBBPA-mediated increase in this protein expression and restored it to the control level, it did not reverse the effect on ß-hCG secretion. This indicated that the mechanism of TBBPA-induced changes in ß-hCG secretion is PPARγ-independent.

Modulation of estradiol synthesis and aromatase activity in human choriocarcinoma JEG-3 cells exposed to tetrabromobisphenol A.

The goal of the present study was to investigate the impact of tetrabromobisphenol A (TBBPA) on human choriocarcinoma-derived placental JEG-3 cells in vitro. We determined the effect of this compound on estradiol secretion, aromatase protein expression and activity in vitro in the JEG-3 cell line. We assessed the ability of TBBPA to increase intracellular levels of cAMP as well as its effect on cell viability and proliferation. Our results indicated that TBBPA, at a wide range of concentrations (1×10(-8)-5×10(-5)M), significantly induced estradiol secretion by JEG-3 cells compared to that of controls after 24, 48 or 72 h of exposure. This effect was accompanied by an increase in the aromatase protein expression in JEG-3 cells treated with 100 nM and 10 µM of TBBPA for 24 h. Additionally, in our study, we confirmed that TBBPA-induced changes in aromatase protein expression were associated w ith the up-regulation of aromatase activity and cAMP levels. No tested doses of TBBPA inhibited JEG-3 cell proliferation, except for the highest dose of 100 µM, which had a toxic effect on cell viability at all time points. The present study clearly indicates that TBBPA alters JEG-3 cells estrogen synthesis due to its action on CYP19 protein expression and thus this compound may interfere with normal placental development during early pregnancy.

The effect of triclosan on hormone secretion and viability of human choriocarcinoma JEG-3 cells.

Triclosan is an antimicrobial agent frequently used in pharmaceuticals and personal care products. We analyzed triclosan for its action on placental secretion of progesterone, estradiol and human chorionic gonadotropin in vitro in the JEG-3 cells. We also investigated its action on cell viability, proliferation and apoptosis. The JEG-3 cells were cultured with increasing doses of triclosan (1×10(-9)-1×10(-4) M) for 24, 48 and 72 h. Triclosan was found to increase estradiol and progesterone secretion after short- and long-term exposure. The stimulatory effect was observed up to 10 µM after short- and long-term exposure to triclosan. In addition, triclosan caused an adverse effect on ß-hCG secretion. The highest doses of triclosan (50 and 100 µM) showed a strong cytotoxic effect. Anti proliferative and pro-apoptotic effects were also observed. Overall, this study demonstrates that triclosan may indirectly disrupt steroidogenesis which may, in turn, affect placental development and consequently fetal growth.

Effects of two isomers of DDT and their metabolite DDE on CYP1A1 and AhR function in human placental cells.

The aim of this study was to investigate the actions of two isomers of DDT (p,p'-DDT, o,p'-DDT) and DDE (p,p'-DDE, o,p'-DDE) on the human placenta. We studied the effects of DDT and its metabolite DDE on CYP1A1 activity and on CYP1A1 and aryl hydrocarbon receptor (AhR) protein expression in placental cells. We used explants from third-trimester human placental tissue and JEG-3 cells, which are first-trimester human placenta cells. The main finding of this study was that the activity of CYP1A1 in the human placenta, measured in terms of ethoxyresorufin-O-deethylase (EROD) activity, was suppressed by treatment of 1, 10, and 100 ng/ml p,p'-DDT, o,p'-DDT, p,p'-DDE and o,p'-DDE. Immunoblot analyses indicated that both isomers of DDT and DDE inhibited the expression of CYP1A1 most effectively at 48 h and/or 72 h after the treatment. Because CYP1A1 activity is mediated by AhR, we evaluated the expression of AhR in placental tissue exposed to DDT and DDE for 1 h to 72 h. Our data showed that DDT and DDE gradually decreased the level of AhR protein, starting at 3 h or 24 h after the start of the experiment. Our results strongly support the involvement of the AhR/CYP1A1 signaling pathway in the mechanism of action of DDT and DDE in the human placenta.