Discovery of polar spirocyclic orally bioavailable urea inhibitors of soluble epoxide hydrolase.

Spirocyclic 1-oxa-9-azaspiro[5.5]undecan-4-amine scaffold was explored as a basis for the design of potential inhibitors of soluble epoxide hydrolase (sEH). Synthesis and testing of the initial SAR-probing library followed by biochemical testing against sEH allowed nominating a racemic lead compound (±)-22. The latter showed remarkable (> 0.5 mM) solubility in aqueous phosphate buffer solution, unusually low (for sEH inhibitors) lipophilicity as confirmed by experimentally determined logD7.4 of 0.99, and an excellent oral bioavailability in mice (as well as other pharmacokinetic characteristics). Individual enantiomer profiling revealed that the inhibitory potency primarily resided with the dextrorotatory eutomer (+)-22 (IC50 4.99 ±â€¯0.18 nM). For the latter, a crystal structure of its complex with a C-terminal domain of sEH was obtained and resolved. These data fully validate (+)-22 as a new non-racemic advanced lead compound for further development as a potential therapeutic agent for use in such areas as cardiovascular disease, inflammation and pain.

Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer's Disease : Theme: Drug Discovery, Development and Delivery in Alzheimer's Disease Guest Editor: Davide Brambilla.

PURPOSE: Alzheimer's disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice. METHODS: Eighteen drug transporters and four tight junction-associated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12-16-month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis. RESULTS: The expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls. CONCLUSIONS: The expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain.

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835.

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly's drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC50=55nM) was reached. Four lead compounds (EC50 range 55-410nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F=10.3%) and plasma levels achieved on oral administration.

Methylphenidate modifies overflow and presynaptic compartmentalization of dopamine via an α-synuclein-dependent mechanism.

Methylphenidate (MPD) modulates dopamine (DA) overflow in part by redistributing vesicle pools, a function shared by the presynaptic protein α-synuclein (α-syn). We suggest that α-syn modifies the effect of MPD on DA neurotransmission. The effect was studied in the dorsal striatum in wild-type mice and two mouse lines lacking α-syn by using in vivo voltammetry and microdialysis. MPD (1 mg/kg) attenuated evoked DA overflow only in mice lacking α-syn but produced a similar increase in the extracellular DA levels in all three lines. A kinetic analysis showed that MPD decreased DA release per stimulus pulse in α-syn-deficient mice but increased in wild-type mice. MPD blocked DA reuptake and produced a similar increase in the apparent affinity (K(m)) for DA reuptake in all three lines. Repeated-burst stimulation redistributes vesicular storage pools and facilitates DA overflow, and this form of facilitation is significantly enhanced in α-syn knockout mice. The DA reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) (10 mg/kg) completely blocked the facilitation of DA overflow in all three lines, whereas MPD (1 mg/kg) selectively decreased it only in mice lacking α-syn. MPD (5 mg/kg) and GBR12909 (10 mg/kg) produced equipotent inhibition of DA reuptake (in terms of K(m)), indicating that reuptake inhibition does not explain the MPD selectivity. Our data indicate that MPD decreases DA release probability in the absence of α-syn and increases it in control animals, whereas the effect of MPD on DA reuptake is independent of α-syn. We suggest that this selectivity is based on α-syn-dependent compartmentalization of presynaptic DA.

Phencyclidine-induced cognitive impairments in repeated touchscreen visual reversal learning tests in rats.

Reversal learning, a component of executive functioning, is commonly impaired among schizophrenia patients and is lacking effective treatment. N-methyl-ᴅ-aspartate (NMDA) receptor antagonists, such as phencyclidine (PCP), impair reversal learning of rodents. Touchscreen-based pairwise visual discrimination and reversal test is a translational tool to assess reversal learning in rodents. However, to fully exploit this task in testing of novel compounds, it is necessary to perform several reversal learning experiments with trained animals. Firstly, we assessed whether PCP-induced deficits in visual reversal learning in rats would be detectable with a short (5 sessions) reversal learning phase, and whether the short reversal phases could be repeated with novel stimulus pairs. Secondly, we assessed whether the PCP-induced deficits in reversal learning could be seen upon repeated PCP challenges with the same animals. Finally, we tested the effect of a novel compound, a selective α2C adrenoceptor antagonist, ORM-13070, to reverse PCP-induced cognitive deficits in this model. A 4-day PCP treatment at a dose of 1.5 mg/kg/day impaired early reversal learning in male Lister Hooded rats without inducing non-specific behavioral effects. We repeated the reversal learning experiment four times using different stimulus pairs with the same animals, and the PCP-induced impairment was evident in every single experiment. The α2C adrenoceptor antagonist ameliorated the PCP-induced cognitive deficits. Our results suggest that repeated PCP challenges in the touchscreen set-up induce schizophrenia-like cognitive deficits in visual reversal learning, improve throughput of the test and provide a protocol for testing novel drugs.

Species differences in the intra-brain distribution of an L-type amino acid transporter 1 (LAT1) -utilizing compound between mice and rats.

The prodrug approach targeting influx transporters has been extensively studied as a means of central nervous system drug delivery. Transporter and enzyme expression, localization and activity may contribute to significant species differences in preclinical studies. However, data about the possible species differences in the intra-brain distribution of transporter utilizing compounds is scarce. Here, we investigated the species differences in the intra-brain distribution of an L-type amino acid transporter 1 (LAT1)-utilizing L-lysine analogue of ketoprofen (KPF) (compound 1) and KPF itself by the whole tissue and brain microdialysis methods in mice, and compared the results to those previously reported in rats. Their pharmacodynamic responses in both species were assessed by measuring the brain prostaglandin E2 (PGE2) levels by LC-MS/MS. The intracellular delivery of compound 1 was much lower in mice than in rats. Higher target site concentrations of compound 1 and released KPF were reflected on a more pronounced effect on PGE2 levels in the rat brain. In conclusion, these results highlight the need for cross-species characterization of prodrug pharmacokinetics and pharmacodynamics in preclinical studies.

Alcohol Co-Administration Changes Mephedrone-Induced Alterations of Neuronal Activity.

Mephedrone (4-MMC), despite its illegal status, is still a widely used psychoactive substance. Its effects closely mimic those of the classical stimulant drug methamphetamine (METH). Recent research suggests that unlike METH, 4-MMC is not neurotoxic on its own. However, the neurotoxic effects of 4-MMC may be precipitated under certain circumstances, such as administration at high ambient temperatures. Common use of 4-MMC in conjunction with alcohol raises the question whether this co-consumption could also precipitate neurotoxicity. A total of six groups of adolescent rats were treated twice daily for four consecutive days with vehicle, METH (5 mg/kg) or 4-MMC (30 mg/kg), with or without ethanol (1.5 g/kg). To investigate persistent delayed effects of the administrations at two weeks after the final treatments, manganese-enhanced magnetic resonance imaging brain scans were performed. Following the scans, brains were collected for Golgi staining and spine analysis. 4-MMC alone had only subtle effects on neuronal activity. When administered with ethanol, it produced a widespread pattern of deactivation, similar to what was seen with METH-treated rats. These effects were most profound in brain regions which are known to have high dopamine and serotonin activities including hippocampus, nucleus accumbens and caudate-putamen. In the regions showing the strongest activation changes, no morphological changes were observed in spine analysis. By itself 4-MMC showed few long-term effects. However, when co-administered with ethanol, the apparent functional adaptations were profound and comparable to those of neurotoxic METH.

Selective adrenergic alpha2C receptor antagonist ameliorates acute phencyclidine-induced schizophrenia-like social interaction deficits in rats.

RATIONALE: Social withdrawal is a core feature of the negative symptoms of schizophrenia. Currently available pharmacotherapies have only limited efficacy towards the negative symptoms, i.e., there is a significant unmet medical need in the treatment of these symptoms. OBJECTIVE: We wanted to confirm whether selective adrenergic α2C receptor (AR) antagonist therapy could ameliorate acute phencyclidine (PCP)-induced schizophrenia-like social interaction deficits in rats, and to compare the effects of an α2C AR antagonist to another putative therapeutic alternative, an α7 nicotinic acetylcholine receptor (nAChR) partial agonist, as well against three commonly used atypical antipsychotics. METHODS: Here, we used acute PCP administration and modified a protocol for testing social interaction deficits in male Wistar rats and then used this model to compare the effects of an α2C AR antagonist (ORM-13070 0.3 and 1.0 mg/kg s.c.) with an α7 nAChR partial agonist (EVP-6124 0.3 mg/kg s.c.) and three atypical antipsychotics (clozapine 2.5 mg/kg i.p., risperidone 0.04 and 0.08 mg/kg s.c., olanzapine 0.125 and 0.5 mg/kg s.c.) on social interaction behavior. RESULTS: Acute PCP (1.5 mg/kg s.c.) produced robust and reproducible deficits in social interaction behavior without affecting locomotor activity. The selective α2C AR antagonist significantly ameliorated PCP-induced social interaction deficits. In contrast, neither the partial α7 nAChR agonist nor any of the three atypical antipsychotics were able to reverse the behavioral deficits at the selected doses. CONCLUSION: Our findings confirm that α2C AR antagonism is a potential mechanism for the treatment of the negative symptoms of schizophrenia.

Dose-response effect of acute phencyclidine on functional connectivity and dopamine levels, and their association with schizophrenia-like symptom classes in rat.

Current drug treatments for schizophrenia (SCZ) can alleviate positive symptoms, but have little effect on the negative symptoms and cognitive deficits that are difficult to translate into preclinical models for drug development. Therefore, we aimed to determine the dose-response effects of acute phencyclidine (PCP, 1.0-5.0 mg/kg) on rat brain connectivity and detect markers for different SCZ-like symptoms. Pharmacological functional magnetic resonance imaging (phMRI) and microdialysis were used to investigate PCP-induced effects on functional connectivity (FC) and dopamine levels, respectively. Next, we evaluated the association between PCP-induced changes in imaging parameters and behavior. PCP at doses of 3.0-5.0 mg/kg induced fMRI signal changes in several brain regions associated with SCZ. Additionally, the FC was globally disturbed, dopamine levels increased, and locomotor activity increased, reflecting the manifestation of SCZ-like positive symptoms. A distinct pattern in the measures was observed at lower PCP doses (1.0-2.0 mg/kg); PCP induced fMRI signal changes in the fronto-cortical regions, and increased dopamine levels in the medial prefrontal cortex. In addition to the dysconnectivity of these regions, the hippocampal FC was disrupted. These observations are consistent with the induction of SCZ-like cognitive deficits and negative symptoms, which were observed as impaired novel object recognition and decreased social interaction. No indicators for positive symptoms were observed at lower PCP doses. We conclude that acute PCP induces SCZ-like symptom classes in a dose-dependent manner; PCP doses of 1.0-2.0 mg/kg are more suitable for modeling SCZ-like negative symptoms and cognitive deficits, while SCZ-like positive symptoms dominate at doses of 3.0-5.0 mg/kg.

Comparison of phencyclidine-induced spatial learning and memory deficits and reversal by sertindole and risperidone between Lister Hooded and Wistar rats.

Visual learning and memory are one of the key cognitive domains disturbed in schizophrenia. Glutamate NMDA receptors play a crucial role in spatial learning and memory and NMDA receptor antagonists, such as phencyclidine (PCP), impair spatial learning and memory. Pigmented rat strains have superior vision than albino rat strains and are therefore commonly used in visually-demanding cognitive tests. However, all previous water maze experiments using acutely administered PCP to induce schizophrenia-like cognitive deficits have been conducted with albino Wistar rats. This study was designed to assess whether pigmented Lister Hooded (LH) rats would be more suitable in modeling acute PCP-induced deficits in Morris water maze (MWM) task than Wistar rats. We also evaluated whether the efficacy of atypical antipsychotics in reversing PCP-induced spatial navigation deficits was dependent on the rat strain. First, we compared the PCP dose-response in the range of 1.3-2.0mg/kg (s.c.) at causing deficits in MWM performance. Then, the efficacies of sertindole 1.6mg/kg (s.c.) and risperidone 0.04mg/kg (s.c.) in reversing PCP-induced spatial navigation deficits were investigated. Drug-naïve LH rats showed a better spatial memory than Wistar rats. Furthermore, PCP induced deficits in spatial navigation at lower doses in LH than in Wistar rats. In addition, PCP-induced deficits were partly reversed by sertindole in LH but not in Wistar rats. Our results suggest that the deficits in spatial learning and memory that resemble memory deficits found in schizophrenia patients are better modeled by PCP in LH rats than Wistar rats.