Quantitative Systems Pharmacology Model for Alzheimer Disease Indicates Targeting Sphingolipid Dysregulation as Potential Treatment Option.

Alzheimer disease (AD) is a devastating neurodegenerative disorder with high unmet medical need. Drug development is hampered by limited understanding of the disease and its driving factors. Quantitative Systems Pharmacology (QSP) modeling provides a comprehensive quantitative framework to evaluate the relevance of biological mechanisms in the context of disease and to predict the efficacy of novel treatments. Here, we report a QSP model for AD with a particular focus on investigating the relevance of dysregulation of cholesterol and sphingolipids. We show that our model captures the modulation of several biomarkers in subjects with AD, as well as the response to pharmacological interventions. We evaluate the impact of targeting the sphingosine-1-phosphate 5 receptor (S1PR5) as a potential novel treatment option for AD, and model predictions increase our confidence in this novel disease pathway. Future applications for the QSP model are in validation of further targets and identification of potential treatment response biomarkers.

Discovery of Novel Aminotetralines and Aminochromanes as Selective and Competitive Glycine Transporter 1 (GlyT1) Inhibitors.

The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure-activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7 S,8 R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7 S,8 R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED50 of 0.8 mg/kg. All these results suggest (7 S,8 R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.

De Novo Design, Synthesis, and Biological Evaluation of 3,4-Disubstituted Pyrrolidine Sulfonamides as Potent and Selective Glycine Transporter 1 Competitive Inhibitors.

The development of glycine transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-d-aspartate (NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3,4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug-drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure-activity relationship (SAR) studies possessed high potency in ex vivo binding studies and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics, and manageable DDI and bioactivation liabilities.

Drug Tolerance: A Known Unknown in Translational Neuroscience.

In neuropsychiatric drug development, the rate of successful translation of preclinical to clinical efficacy has been disappointingly low. Tolerance, defined as a loss of efficacy with repeated drug exposure, is rarely addressed as a potential source of clinical failures. In this review, we argue that preclinical methods of tolerance development may have predictive validity and, therefore, inclusion of studies using repeated drug exposure early during the drug discovery and development process should serve to mitigate a proportion of clinical failures. Our analysis indicates that many published preclinical efficacy studies in the neuropsychiatry arena are conducted with acute drug administration only. Furthermore, specifically in the field of schizophrenia, there are several examples where tolerance development may be suspected as a factor contributing to translational failures. These and other examples highlight the need for built-for-purpose tolerance studies to be conducted, regardless of the target interaction mode of the drugs (i.e., agonist or antagonist, allosteric or orthosteric). We suggest that, for compounds that have failed in clinical studies, preclinical efficacy data sets need to be revisited to estimate the potential impact of tolerance development, one of the most significant known unknowns in the preclinical-to-clinical translation.

Effects of a positive allosteric modulator of group II metabotropic glutamate receptors, LY487379, on cognitive flexibility and impulsive-like responding in rats.

Orthosteric group II metabotropic glutamate receptor (mGluR) agonists are regarded as novel, effective medications for all major symptom domains of schizophrenia, including cognitive disturbances. mGluR2s also can be affected in a more subtle way by positive allosteric modulators (PAMs) characterized by a unique degree of subtype selectivity and neuronal frequency-dependent activity. Because currently available treatments for schizophrenia do not improve cognitive dysfunction, the main aim of the present study was to examine the effects of a mGluR2 PAM, N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379), on rat cognitive flexibility and impulsive-like responding, assessed in an attentional set-shifting task (ASST) and a differential reinforcement of low-rate 72 s (DRL72) schedule of food reinforcement. In addition, in vivo microdialysis was used to assess the drug's impact on cortical levels of dopamine, norepinephrine, serotonin, and glutamate. Rats treated with LY487379 (30 mg/kg) required significantly fewer trials to criteria during the extradimensional shift phase of the ASST. Under a DRL72 schedule, LY487379 (30 mg/kg) decreased the response rate and increased the number of reinforcers obtained. These effects were accompanied by the shift of the frequency distribution of responses toward longer inter-response time durations. LY487379 significantly enhanced extracellular norepinephrine and serotonin levels in the medial prefrontal cortex. In summary, the present study demonstrates that a mGluR2 PAM, LY487379, promotes cognitive flexibility and facilitates behavioral inhibition. These procognitive effects may contribute to the therapeutic efficacy of agents stimulating mGluR2 in schizophrenia.

Amphetamine decreases behavioral inhibition by stimulation of dopamine D2, but not D3, receptors.

Behavioral disinhibition is a manifestation of impulsive behavior that is prominent in the psychopathology of various psychiatric disorders such as addiction, attention-deficit hyperactivity disorder, mania, and personality disorders. Impulsivity may be studied by measuring anticipatory responses made before the presentation of a food-predictive, brief light stimulus in a two-choice serial reaction time task. In such serial reaction time tasks, amphetamine has been shown to produce dose-dependent increases in premature responding in a manner dependent on dopamine D(2)-like receptor stimulation. So far, it is unknown whether it is the D(2) or D(3) receptor that is involved in this form of impulsivity. In this study, rats were trained in a two-choice serial reaction time task until baseline performance was stable. Next, effects of the dopamine D(2) preferring antagonist L-741,626 and selective D(3) antagonist SB-277011 were assessed alone and in the presence of amphetamine. Neither L-741,626 nor SB-277011 affected behavioral inhibition, although the latter significantly increased reaction time at 10 mg/kg. Amphetamine dose-dependently increased impulsivity. The effect of amphetamine was attenuated by L-741,626 (3 mg/kg), whereas SB-277011 (3 mg/kg) had no effect. Therefore, amphetamine-induced behavioral disinhibition depends on D(2), but not D(3), receptor stimulation.

Habituation deficits induced by metabotropic glutamate receptors 2/3 receptor blockade in mice: reversal by antipsychotic drugs.

Cortical metabotropic glutamate receptors (mGluRs) seem to be involved in habituation of simple stimulus-bound behaviors (e.g., habituation to acoustic startle or odor-elicited orienting response). Habituation deficits may contribute to the cognitive symptoms of schizophrenia. In the present study, male NMRI mice were injected with mGluR2/3 antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY-341495) 30 min before being placed into novel arenas for automatic motor activity recording (2-h sessions). Administration of LY-341495 (1-10 mg/kg s.c.) dose-dependently prevented the habituation of the locomotor activity. Effects of LY-341495 (10 mg/kg) were fully and dose-dependently reversed by i.p. administration of haloperidol (0.03-0.3 mg/kg), clozapine (1-10 mg/kg), risperidone (0.01-0.1 mg/kg), olanzapine (0.3-3 mg/kg), aripiprazole (1-10 mg/kg), and sulpiride (3-30 mg/kg), each of which was given 15 min before the test. Effects of antipsychotic drugs were observed at the dose levels that did not affect spontaneous motor activity. LY-341495-induced delayed hyperactivity was also partially attenuated by lithium (50-200 mg/kg), amisulpride (1-10 mg/kg), and the selective dopamine D3 antagonist trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (SB-277011A; 3-30 mg/kg). Application of diazepam, imipramine, or several agonists and/or antagonists acting at various receptors that are thought to be relevant for antipsychotic treatment [e.g., 5-hydroxytryptamine (5-HT)(2A), 5-HT(3), and 5-HT(6) antagonists; 5-HT(1A) agonist; D4 antagonist; CB1 antagonist; ampakines; and glycine transporter inhibitor) had no appreciable effects. Thus, behavioral deficits induced by mGluR2/3 blockade (such as delayed motor hyperactivity) are selectively reversed by clinically used antipsychotic drugs.

Effect of dopamine D3 antagonists on PPI in DBA/2J mice or PPI deficit induced by neonatal ventral hippocampal lesions in rats.

Schizophrenic patients typically exhibit impairment of sensorimotor gating, which can be modeled in animal models such as the test of prepulse inhibition of startle response (PPI) in rodents. It has been found that antipsychotics enhanced PPI in DBA mice and reversed the PPI deficit induced by neonatal ventral hippocampal (NVH) lesions in rats. However, the relative involvement of D(3) and D(2) receptors in these effects is unknown since all antipsychotics are D(2)/D(3) antagonists with limited binding preference at D(2) receptors. Therefore, in the current study, we investigated the influence of several dopamine antagonists with higher selectivity at D(3) vs D(2) receptors on PPI in DBA/2J mice and in NVH-lesioned rats. The PPI in DBA/2J mice was enhanced by the nonselective D(2)/D(3) antagonists, haloperidol at 0.3-3 mg/kg, or risperidone at 0.3-1 mg/kg, while PPI-enhancing effects were observed after the administration of higher doses of the preferential D(3)/D(2) antagonist, BP 897 at 8 mg/kg, and the selective D(3) antagonists, SB 277011 at 30 mg/kg and A-437203 at 30 mg/kg. No effect was observed following the treatment with the selective D(3) antagonist, AVE 5997 up to 30 mg/kg. The PPI deficits induced by NVH lesions were reversed by haloperidol but not by the more selective D(3) antagonists, A-437203 and AVE 5997. BP 897 enhanced PPI nonselectivity, that is, in both lesioned and nonlesioned rats. In summary, the present study indicates that PPI-enhancing effects induced by antipsychotics in DBA/2J mice and in NVH-lesioned rats are unlikely to be mediated by D(3) receptors.

The use of stereological counting methods to assess immediate early gene immunoreactivity.

The issue of whether profile and stereological counting methods are interchangeably accurate when assessing immediate early gene expression still needs to be resolved. To compare these two counting techniques, we quantified the expression of c-fos in the nucleus accumbens core and shell, and in the lateral septum as a control structure, of rats treated with neuroleptics. With the profile counting method, which relies on selective placement of a counting grid within a structure, we evaluated the density of c-fos labeled cells within a box of fixed dimension. With stereology, which applies random and systematic sampling methods, we used the optical fractionator method and counted the absolute number of c-fos labeled cells within the contours of each structure examined. Our results showed that the substantial increase in c-fos expression in the shell and core induced by haloperidol treatment was detected by both stereological and profile counting methods; in contrast, the weaker effect of clozapine on c-fos expression was detected differentially by the two methods. Whereas the profile counting method reported a reduction of c-fos in the core by clozapine, and an increase in c-fos in the lateral septum, these effects were not replicated using stereology. These findings suggest that stereological and profile counting methods do not always produce equivalent results. This may be particularly relevant when a measured effect is relatively small, and it is not distributed homogeneously within a structure. In this respect, the random and systematic sampling methods of stereology may yield more accurate and unbiased results than the profile counting method, and therefore may be preferred for a more accurate and thorough investigation of a treatment effect on immediate early gene expression in a specific brain region.

A differential involvement of the shell and core subterritories of the nucleus accumbens of rats in memory processes.

The role of the core and the shell subterritories of the nucleus accumbens in conditioned freezing and spatial learning was investigated by means of selective N-methyl-D-aspartate lesions. Shell-lesioned rats showed reduced conditioned freezing to context and a tendency toward reduced freezing to the discrete stimulus compared with controls. However, lesions of the core did not modify the freezing response either to the context or to the discrete stimuli. Although spatial memory, as assessed by a water-maze paradigm, was not disrupted by the lesions, in a 4-arm baited, 4-arm unbaited radial-arm maze paradigm, the shell-lesioned rats showed selective deficits in working memory, but not in reference memory. In contrast, core-lesioned rats showed no memory deficits.

Sensitized Fos expression in subterritories of the rat medial prefrontal cortex and nucleus accumbens following amphetamine sensitization as revealed by stereology.

Behavioral sensitization to the locomotor activating effects of amphetamine refers to the progressive, long lasting increase in locomotor activity that occurs with repeated injections. This phenomenon is thought to result from neuroadaptations occurring in the projection fields of mesocorticolimbic dopaminergic neurons. In the present study, we investigated the effects of amphetamine sensitization on Fos immunoreactivity (Fos-IR) in subterritories of the nucleus accumbens (core and shell) and medial prefrontal cortex (mPFC; dorsal and ventral) using stereology. Rats received five daily injections of amphetamine (1.5 mg/kg, i.p.) or saline. Behavioral sensitization was measured 48 h following the last injection, in response to a challenge injection of 1.5 mg/kg amphetamine. Sensitized rats showed a greater enhancement of locomotor activity upon drug challenge compared with their saline counterparts. Densities of Fos-positive nuclei were enhanced more in the dorsal than the ventral mPFC subterritory, whereas in the nucleus accumbens, densities of Fos-positive nuclei were increased more in the core than the shell of amphetamine-sensitized rats compared to controls. These results represent, to our knowledge, the first published report using stereological methods to quantify Fos-IR in the brain and suggest functional specialization of cortical and limbic regions in the expression of behavioral sensitization to amphetamine.