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Recent data demonstrated that activation of the muscarinic M1 receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M2 and M3 activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M1 PAMs that address this hypothesis. The lead molecule from this series, compound 1 (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M1-selective PAM with minimal agonist activity. Compound 1 was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings pr...

To enable the clinical development of our CNS casein kinase 1 delta/epsilon (CK1δ/e) inhibitor project, we investigated the possibility of developing a CNS positron emission tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on the initial CK1δ/e inhibitor HTS hits with the goal of identifying a compound that would fulfill a set of recommended PET ligand criteria. We identified [3H]PF-5236216 (9) as a tool ligand that meets most of the key CNS PET attributes including high CNS MPO PET desirability score and kinase selectivity, CNS penetration, and low nonspecific binding. We further used [3H]-9 to determine the binding affinity for PF-670462, a literature CK1δ/e inhibitor tool compound. Lastly, [3H]-9 was used to measure in vivo target occupancy (TO) of PF-670462 in mouse and correlated TO with CK1δ/e in vivo pharmacology (circadian rhythm modulation).

[Image: see text] It has been hypothesized that selective muscarinic acetylcholine receptor (mAChR) M4 subtype activation could provide therapeutic benefits to a number of neurological disorders while minimizing unwanted cholinergic side effects observed due to nonselective mAChR activation. Given the high sequence and structural homology of the orthosteric binding sites among mAChRs, achieving M4 subtype-selective activation has been challenging. Herein, we describe the discovery of a series of M4 subtype-selective agonists bearing novel carbamate isosteres. Comparison of the isosteres’ electrostatic potential isosurface sheds light on key structural features for M4 subtype-selective activation. The identified key features were further illustrated in a proposed receptor–agonist interaction mode.

The rationale for using M1 selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M1 muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M1 muscarinic acetylcholine receptor activation. Five brain penetrant M1-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol ph...

[11C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. [11C]Lu AE92686 has high affinity for PDE10A (IC 50 = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional [11C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain. A total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). [11C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches. Regional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V T) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V T values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V T values increased by ∼30 % with increasing PET measurement duration from 63 to 123 min, while V T values in target regions remained stable. Both pretreatment drugs significantly decreased [11C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP ND) values, derived with the simplified reference tissue model (SRTM), were 13–17 in putamen and 3–5 in substantia nigra and correlated well to values from the Logan plot analysis. The method proposed for quantification of [11C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that [11C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia nigra.

Selective activation of the M1 muscarinic acetylcholine receptor (mAChR) via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. Herein, we describe the characterization of an M1 PAM radioligand, 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one ([(3)H]PT-1284), as a tool for characterizing the M1 allosteric binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 ( 1: )) was shown to potentiate acetylcholine (ACh) in an M1 fluorometric imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and carbachol in a hippocampal slice electrophysiology assay (EC50, 165 nM). PT-1284 ( 1: ) also reduced the concentration of ACh required to inhibit [(3)H]N-methylscopolamine ([(3)H]NMS) binding to M1, left-shifting the ACh Ki approximately 19-fold at 10 μM. Saturation analysis of a human M1 mAChR stable cell line showed that [(3)H]PT-1284 bound to M1 mAChR in the presence of 1 mM ACh with Kd, 4.23 nM, and saturable binding capacity (Bmax), 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit [(3)H]PT-1284 binding in a concentration-responsive manner, whereas M1 allosteric and orthosteric agonists showed weak affinity (>30 μM). A strong positive correlation (R(2) = 0.86) was found to exist between affinity values generated for nineteen M1 PAMs in the [(3)H]PT-1284 binding assay and the EC50 values of these ligands in a FLIPR functional potentiation assay. These data indicate that there is a strong positive correlation between M1 PAM binding affinity and functional activity, and that [(3)H]PT-1284 can serve as a tool for pharmacological investigation of M1 mAChR PAMs.

Rationale This review attempts to summarize the current status in relation to the use of positron emission tomography (PET) imaging in the assessment of synaptic concentrations of endogenous mediators in the living brain. Objectives Although PET radioligands are now available for more than 40 CNS targets, at the initiation of the Innovative Medicines Initiative (IMI) “Novel Methods leading to New Medications in Depression and Schizophrenia” (NEWMEDS) in 2009, PET radioligands sensitive to an endogenous neurotransmitter were only validated for dopamine. NEWMEDS work-package 5, “Cross-species and neurochemical imaging (PET) methods for drug discovery”, commenced with a focus on developing methods enabling assessment of changes in extracellular concentrations of serotonin and noradrenaline in the brain. Results Sharing the workload across institutions, we utilized in vitro techniques with cells and tissues, in vivo receptor binding and microdialysis techniques in rodents, and in vivo PET imaging in non-human primates and humans. Here, we discuss these efforts and review other recently published reports on the use of radioligands to assess changes in endogenous levels of dopamine, serotonin, noradrenaline, γ-aminobutyric acid, glutamate, acetylcholine, and opioid peptides. The emphasis is on assessment of the availability of appropriate translational tools (PET radioligands, pharmacological challenge agents) and on studies in non-human primates and human subjects, as well as current challenges and future directions. Conclusions PET imaging directed at investigating changes in endogenous neurochemicals, including the work done in NEWMEDS, have highlighted an opportunity to further extend the capability and application of this technology in drug development.

Recent data demonstrated that activation of the muscarinic M

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

The Group I metabotropic glutamate receptor subtype 5 (mGluR5) is widely distributed in the brain with dense expression in the cerebral cortex, hippocampus, and basal ganglia. These receptors have been implicated in psychiatric and neurological disorders such as schizophrenia, Fragile X syndrome, addiction, anxiety/depression, Parkinson's disease and neuropathic pain. The present study evaluated the effects of the mGluR5 negative allosteric modulators (NAMs) 4-difluoromethoxy-3-(pyridine-2-ylethynyl)phenyl)5H-pyrrolo[3,4-b]pyridine-6(7H)-yl methanone (GRN-529) and methyl (3aR,4S,7aR)-4-hydroxy-4-[(3-methylphenyl)ethynyl]octahydro-1H-indole-1-carboxylate (AFQ056) on polysomnographic (PSG) and quantitative electroencephalographic (qEEG) measures in freely moving rats. Furthermore, the anxiolytic profile of GRN-529 was characterized in anesthetized rats by measuring stimulation-induced hippocampal theta oscillation. The present findings demonstrate that inhibition of mGluR5 via its allosteric site profoundly modulates high-level neuronal network activities as indicated by changes in sleep-wake activity and power distribution of qEEG. Both GRN-529 and AFQ056 reduced the total time spent in rapid-eye movement with AFQ056 producing a significant increase in wakefulness at the highest dose tested. Additionally, qEEG revealed significant compound-induced increases in delta power concomitant with more subtle decreases in theta and alpha band power. Receptor occupancy (RO) studies revealed that GRN-529 and AFQ056 at all doses resulted in over 45% mGluR5 occupancy. Furthermore, GRN-529 dose-dependently decreased elicited hippocampal theta frequency, consistent with previous findings using clinically active anxiolytic compounds. The described changes in neurophysiological signals identified in freely moving rats may be considered suitable translational biomarkers for the clinical evaluation of mGluR5 NAMs.

The rationale for using M

Purpose [11C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. [11C]Lu AE92686 has high affinity for PDE10A (IC 50 = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional [11C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain. Methods A total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). [11C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches. Results Regional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V T) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V T values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V T values increased by ∼30 % with increasing PET measurement duration from 63 to 123 min, while V T values in target regions remained stable. Both pretreatment drugs significantly decreased [11C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP ND) values, derived with the simplified reference tissue model (SRTM), were 13–17 in putamen and 3–5 in substantia nigra and correlated well to values from the Logan plot analysis. Conclusions The method proposed for quantification of [11C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that [11C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia nigra. Electronic supplementary material The online version of this article (doi:10.1007/s00259-016-3544-9) contains supplementary material, which is available to authorized users.

It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimer's disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M1 with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.

The cognitive impairments observed in Alzheimer’s disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT4) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT4 agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure–response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT4 partial agonist candidates 2d and 3 were c...

Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.

Pharmacokinetic–pharmacodynamic (PK–PD) modeling greatly enables quantitative implementation of the “learn and confirm” paradigm across different stages of drug discovery and development. This work describes the successful prospective application of this concept in the discovery and early development of a novel κ-opioid receptor (KOR) antagonist, PF-04455242, where PK–PD understanding from preclinical biomarker responses enabled successful prediction of the clinical response in a proof of mechanism study. Preclinical data obtained in rats included time course measures of the KOR antagonist (PF-04455242), a KOR agonist (spiradoline), and a KOR-mediated biomarker response (prolactin secretion) in plasma. Clinical data included time course measures of PF-04455242 and prolactin in 24 healthy volunteers following a spiradoline challenge and single oral doses of PF-04455242 (18 and 30 mg). In both species, PF-04455242 successfully reversed spiradoline-induced prolactin response. A competitive antagonism model was developed and implemented within NONMEM to describe the effect of PF-04455242 on spiradoline-induced prolactin elevation in rats and humans. The PK–PD model-based estimate of K i for PF-04455242 in rats was 414 ng/mL. Accounting for species differences in unbound fraction, in vitro K i and brain penetration provided a predicted human K i of 44.4 ng/mL. This prediction was in good agreement with that estimated via the application of the proposed PK–PD model to the clinical data (i.e., 39.2 ng/mL). These results illustrate the utility of the proposed PK–PD model in supporting the quantitative translation of preclinical studies into an accurate clinical expectation. As such, the proposed PK–PD model is useful for supporting the design, selection, and early development of novel KOR antagonists.

OBJECTIVES: To assess the ability of a previously developed hybrid physiology-based pharmacokinetic-pharmacodynamic (PBPKPD) model in rats to predict the dopamine D2 receptor occupancy (D2RO) in human striatum following administration of antipsychotic drugs.METHODS: A hybrid PBPKPD model, previously developed using information on plasma concentrations, brain exposure and D2RO in rats, was used as the basis for the prediction of D2RO in human. The rat pharmacokinetic and brain physiology parameters were substituted with human population pharmacokinetic parameters and human physiological information. To predict the passive transport across the human blood-brain barrier, apparent permeability values were scaled based on rat and human brain endothelial surface area. Active efflux clearance in brain was scaled from rat to human using both human brain endothelial surface area and MDR1 expression. Binding constants at the D2 receptor were scaled based on the differences between in vitro and in vivo systems of the same species. The predictive power of this physiology-based approach was determined by comparing the D2RO predictions with the observed human D2RO of six antipsychotics at clinically relevant doses.RESULTS: Predicted human D2RO was in good agreement with clinically observed D2RO for five antipsychotics. Models using in vitro information predicted human D2RO well for most of the compounds evaluated in this analysis. However, human D2RO was under-predicted for haloperidol.CONCLUSIONS: The rat hybrid PBPKPD model structure, integrated with in vitro information and human pharmacokinetic and physiological information, constitutes a scientific basis to predict the time course of D2RO in man.

Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies.

(2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide (compound E) is a gamma-secretase inhibitor capable of reducing amyloid beta-peptide (1-40) and amyloid beta-peptide (1-42) levels. In this study we investigated the effect of in vivo administration of compound E on guinea-pig plasma, CSF and cortical amyloid beta-peptide (1-40) concentration. Using repeated sampling of CSF, compound E (30 mg/kg p.o.) was shown to cause a time-dependent decrease in CSF amyloid beta-peptide (1-40) levels, which was maximal at 3 h (70% inhibition), compared to baseline controls. After 3 h administration, compound E (3, 10 and 30 mg/kg p.o.), reduced plasma, CSF and DEA-extracted cortical amyloid beta-peptide (1-40) levels by 95, 97 and 99%; 26, 48 and 78%; 32, 33, and 47%, respectively, compared to vehicle control values. In the same animals, compound E (3, 10 and 30 mg/kg p.o.) inhibited cortical gamma-secretase activity, determined ex vivo using the recombinant substrate C100Flag, by 40, 71 and 79% of controls, respectively. These data demonstrate the value of determining not only the extent by which systemic administration of a gamma-secretase inhibitor reduces amyloid beta-peptide, but also the inhibition of brain gamma-secretase activity, as a more direct estimate of enzyme occupancy.

To assess the ability of a previously developed hybrid physiology-based pharmacokinetic-pharmacodynamic (PBPKPD) model in rats to predict the dopamine D2 receptor occupancy (D2RO) in human striatum following administration of antipsychotic drugs.A hybrid PBPKPD model, previously developed using information on plasma concentrations, brain exposure and D2RO in rats, was used as the basis for the prediction of D2RO in human. The rat pharmacokinetic and brain physiology parameters were substituted with human population pharmacokinetic parameters and human physiological information. To predict the passive transport across the human blood-brain barrier, apparent permeability values were scaled based on rat and human brain endothelial surface area. Active efflux clearance in brain was scaled from rat to human using both human brain endothelial surface area and MDR1 expression. Binding constants at the D2 receptor were scaled based on the differences between in vitro and in vivo systems of the same species. The predictive power of this physiology-based approach was determined by comparing the D2RO predictions with the observed human D2RO of six antipsychotics at clinically relevant doses.Predicted human D2RO was in good agreement with clinically observed D2RO for five antipsychotics. Models using in vitro information predicted human D2RO well for most of the compounds evaluated in this analysis. However, human D2RO was under-predicted for haloperidol.The rat hybrid PBPKPD model structure, integrated with in vitro information and human pharmacokinetic and physiological information, constitutes a scientific basis to predict the time course of D2RO in man.

It is well established that the NMDA receptor antagonists block hippocampal long-term potentiation and impair acquisition in the Morris watermaze task, although the role of individual NMDA receptor subtypes is largely unknown. In the present study, we compared the effects of (±)-CP-101,606, an antagonist selective for NMDA receptor NR1/NR2B subunit-containing receptors and the nonselective NMDA receptor antagonist MK-801, on acquisition in the Morris watermaze. Male hooded Lister rats were given 4 trials/day to find a fixed hidden platform submerged beneath the opaque water of the Morris watermaze. Twenty-four hours after the last acquisition trial, a ‘probe trial’ was conducted to assess the rat's spatial memory for the location of the hidden platform. Those rats treated with MK-801 (0.1 mg/kg, i.p.) 60 min prior to the acquisition and probe trials took significantly longer to find the hidden platform during training and spent significantly less time searching the platform's location during the probe trial than vehicle-treated rats. In contrast, 60-min pretreatment with (±)-CP-101,606 (60 mg/kg, p.o.), a dose that fully occupied hippocampal NR1/NR2B subunit-containing receptors, as determined by ex vivo NMDA receptor-specific [3H]ifenprodil binding immediately following watermaze experiments, had no effect on acquisition or the probe trial. These results suggest that antagonists selective for NR1/NR2B subunit-containing receptors may not impair spatial memory in rats in the Morris watermaze.

A 45 Ca 2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45 Ca 2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45 Ca 2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45 Ca 2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45 Ca 2+ influx into cells expressing NR1a/NR2A receptors (IC 50 = 408 μM) and NR1a/NR2B receptors (EC 50 = 37.3 μM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC 50 = 0.099 μM) compared with NR1a/NR2A receptors (IC 50 = 164 μM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg 2+ and Zn 2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45 Ca 2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.

Transfection of mouse L(tk-) cells with human N-methyl-D-aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist D, L-(epsilon)-2-[3H]amino-4-propyl-5-phosphono-3-pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6-5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.

Homoquinolinate, a derivative of the endogenous NMDA agonist, quinolinate, has been shown to display higher affinity for Xenopus oocytes expressing NR2A- and NR2B-containing receptors, compared to NR2C- and NR2D-containing receptors, whilst autoradiographical experiments subsequently showed that [3H]homoquinolinate labelled a subpopulation of NMDA receptors in rat brain sections, with a similar distribution to NR2B-containing receptors. In this study, we have shown that NMDA-specific [3H]homoquinolinate binding to rat brain membranes comprised 44% of total binding with a Bmax value of 5.73 pmol/mg protein, which was inhibited by NMDA with Ki=0.867 µm. However, NMDA-specific [3H]homoquinolinate binding was not observed for a number of human recombinant NMDA receptors investigated, suggesting that there are subtle differences between the binding sites of recombinant and native receptors. Electrophysiological experiments revealed that homoquinolinate activated human recombinant NR1a/NR2A, NR1a/NR2B and NR1a/NR2A/NR2B receptors with EC50 values of 25.2, 13.8 and 9.04 µm, respectively, with intrinsic activities of 148, 93.3 and 125%, respectively, compared to glutamate (=100%). In contrast to an autoradiographical study, these radioligand binding and electrophysiological experiments suggest that homoquinolinate is not highly selective for NR2B-containing receptors.

Objectives Dopamine D-2 receptor occupancy (D2RO) is the major determinant of efficacy and safety in schizophrenia drug therapy. Excessive D2RO (>80%) is known to cause catalepsy (CAT) in rats and extrapyramidal side effects (EPS) in human. The objective of this study was to use pharmacokinetic and pharmacodynamic modeling tools to relate CAT with D2RO in rats and to compare that with the relationship between D2RO and EPS in humans.Methods Severity of CAT was assessed in rats at hourly intervals over a period of 8 h after antipsychotic drug treatment. An indirect response model with and without Markov elements was used to explain the relationship of D2RO and CAT.Results Both models explained the CAT data well for olanzapine, paliperidone and risperidone. However, only the model with the Markov elements predicted the CAT severity well for clozapine and haloperidol. The relationship between CAT scores in rat and EPS scores in humans was implemented in a quantitative manner. Risk of EPS not exceeding 10% over placebo correlates with less than 86% D2RO and less than 30% probability of CAT events in rats.Conclusion A quantitative relationship between rat CAT and human EPS was elucidated and may be used in drug discovery to predict the risk of EPS in humans from D2RO and CAT scores measured in rats.

Herein we report the identification of (+)- N -(2-((1 H -pyrazol-1-yl)methyl)-3-((1 R ,3 r ,5 S )-6′-fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1′-isochroman]-8-yl)propyl)- N -[ 3 H ]-methylacetamide {[ 3 H]PF-7191 [(+)- 11 ]} as a promising radiotracer for the nociceptin opioid peptide (NOP) receptor. (+)- 11 demonstrated high NOP binding affinity ( K i = 0.1 nM), excellent selectivity over other opioid receptors (>1000×) and good brain permeability in rats ( C b,u / C p,u = 0.29). Subsequent characterization of [ 3 H](+)- 11 showed a high level of specific binding and a brain bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in vivo brain binding of [ 3 H](+)- 11 in rats was inhibited by a selective NOP receptor antagonist in a dose–responsive manner. This overall favorable profile indicated that [ 3 H](+)- 11 is a robust radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher species.

Introduction Kappa opioid receptors (KOR) are implicated in several brain disorders. In this report, a first-in-human positron emission tomography (PET) study was conducted with the potent and selective KOR agonist tracer, [11C]GR103545, to determine an appropriate kinetic model for analysis of PET imaging data and assess the test–retest reproducibility of model-derived binding parameters. The non-displaceable distribution volume (VND) was estimated from a blocking study with naltrexone. In addition, KOR occupancy of PF-04455242, a selective KOR antagonist that is active in preclinical models of depression, was also investigated. Methods For determination of a kinetic model and evaluation of test–retest reproducibility, 11 subjects were scanned twice with [11C]GR103545. Seven subjects were scanned before and 75 min after oral administration of naltrexone (150 mg). For the KOR occupancy study, six subjects were scanned at baseline and 1.5 h and 8 h after an oral dose of PF-04455242 (15 mg, n = 1 and 30 mg, n = 5). Metabolite-corrected arterial input functions were measured and all scans were 150 min in duration. Regional time-activity curves (TACs) were analyzed with 1- and 2-tissue compartment models (1TC and 2TC) and the multilinear analysis (MA1) method to derive regional volume of distribution (VT). Relative test–retest variability (TRV), absolute test–retest variability (aTRV) and intra-class coefficient (ICC) were calculated to assess test–retest reproducibility of regional VT. Occupancy plots were computed for blocking studies to estimate occupancy and VND. The half maximal inhibitory concentration (IC50) of PF-04455242 was determined from occupancies and drug concentrations in plasma. [11C]GR103545 in vivo KD was also estimated. Results Regional TACs were well described by the 2TC model and MA1. However, 2TC VT was sometimes estimated with high standard error. Thus MA1 was the model of choice. Test–retest variability was ~ 15%, depending on the outcome measure. The blocking studies with naltrexone and PF-04455242 showed that VT was reduced in all regions; thus no suitable reference region is available for the radiotracer. VND was estimated reliably from the occupancy plot of naltrexone blocking (VND = 3.4 ± 0.9 mL/cm3). The IC50 of PF-04455242 was calculated as 55 ng/mL. [11C]GR103545 in vivo KD value was estimated as 0.069 nmol/L. Conclusions [11C]GR103545 PET can be used to image and quantify KOR in humans, although it has slow kinetics and variability of model-derived kinetic parameters is higher than desirable. This tracer should be suitable for use in receptor occupancy studies, particularly those that target high occupancy.

In this study, we have further delineated the domains of the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine co-agonist binding site. Taking an iterative approach, we have constructed truncation mutants of the NR1 subunit, transiently expressed them in HEK-293 cells, and determined the binding of the glycine site antagonist [3H]L-689,560. Amino acids 380-811 were sufficient to form a glycine binding site with affinities for [3H]L-689,560 and glycine that were not significantly different from wild-type NR1. More extensive deletions, from either the amino- or the carboxy-terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380-843 of NR1, replacing the transmembrane (TMI-TMIII) domain with intervening linker sequences while retaining the TMIV domain so as to anchor the polypeptide to the membrane. Although robust amounts of polypeptides were synthesised by transfected cells, only low levels of [3H]L-689,560 binding sites could be detected. This suggests that only a small proportion of the synthesised polypeptide folds in the appropriate manner so as to form a ligand binding site. These data indicate that although it is possible to reduce the glycine binding site to minimal so-called S1 and S2 domains, efficient folding of the polypeptide so as to form a ligand binding site may require sequences within the TMI-TMIII domain.

4-Substituted-3-phenylquinolin-2(1H)-ones have been synthesized and evaluated in vitro for antagonist activity at the glycine site on the NMDA (N-methyl-D-aspartate) receptor and in vivo for anticonvulsant activity in the DBA/2 strain of mouse in an audiogenic seizure model. 4-Amino-3-phenylquinolin-2(1H)-one (3) is 40-fold lower in binding affinity but only 4-fold weaker as an anticonvulsant than the acidic 4-hydroxy compound 1. Methylsulfonylation at the 4-position of 3 gives an acidic compound (6, pKa = 6.0) where affinity is fully restored but in vivo potency is significantly reduced (Table 1). Methylation at the 4-position of 1 to give 18 results in the abolition of measurable affinity, but the attachment of neutral hydrogen bond-accepting groups to the methyl group of 18 produces compounds with comparable in vitro and in vivo activity to 1 (e.g., 23 and 28, Table 2). Replacement of the 4-hydroxy group of 1 with an ethyl group abolishes activity (42), but again, incorporation of neutral hydrogen bond acceptors to the terminal carbon atom restores affinity (e.g., 36, 39, and 40, Table 3). Replacement of the 4-hydroxy group of the high-affinity compound 2 with an amino group produces a compound with 200-fold reduced affinity (43; IC50 = 0.42 microM, Table 4) which is nevertheless still 10-fold higher in affinity than 3. The results in this paper indicate that anionic functionality is not an absolute requirement for good affinity at the glycine/NMDA site and provide compelling evidence for the existence of a ligand/receptor hydrogen bond interaction between an acceptor attached to the 4-position of the ligand and a hydrogen bond donor attached to the receptor.

To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).

A series of analogues of the novel diiodotyrosine derived NMDA glycine-site ligand (R)-4 was prepared in which the aryl substitution, chain length and amino acid groups were varied. The key structural features for binding are the α-amino acid function, having the (R) absolute stereochemistry, the 3,5-diiodo substituted aromatic ring and a lipophilic group attached at the phenolic oxygen of the tyrosine moiety.

Tetramic acids ( 9 ) have been designed as novel glycine site N-methyl-D-aspartate receptor antagonists.

The effect of reducing the benzo ring of a series of 4-hydroxyquinolone Glycine/NMDA antagonists is described. It is important that the ring be present, but not that it is aromatic. Provided that the correct lipophilic interactions are present, high affinity is found in 4-hydroxy-5,6,7,8-tetrahydroquinolones.

Almost all of the existing known antagonists at the glycine site of the N-methyl-D-aspartate (NMDA) receptor have a low propensity for crossing the blood-brain barrier. It has been suggested that in many cases this may be due to the presence of a carboxylic acid which is a common feature of most of the potent full antagonists at this receptor. In this study, 2-aryl-1H-pyrazolo[3,4-c]quinoline-1,4(2H)-diones were found to have high-affinity binding at the glycine receptor. In particular, structure-activity studies identified 7-chloro-3,5-dihydro-2-(4-methoxyphenyl)-1H-pyrazolo[3,4-c]quinoline- 1,4(2H)-dione as the most potent of a series of analogues with an IC50 of 3.3 nM. The measured pKa values in this class of compounds (typically 4.0) indicate they are of equivalent acidity to carboxylic acids. Functional antagonism was demonstrated by inhibition of NMDA-evoked responses in rat cortical slices. Anticonvulsant activity in DBA/2 mice was achieved after dosing by direct injection into the cerebral ventricles, but no activity was seen after systemic administration, suggesting low brain penetration with this class of antagonists.

The cognitive impairments observed in Alzheimer's disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT(4)) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT(4) agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure-response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT(4) partial agonist candidates 2d and 3 were chosen for clinical development.

5-Hydroxytryptamine (5-HT)(4) receptor agonists reportedly stimulate brain acetylcholine (ACh) release, a property that might provide a new pharmacological approach for treating cognitive deficits associated with Alzheimer's disease. The purpose of this study was to compare the binding affinities, functional activities, and effects on neuropharmacological responses associated with cognition of two highly selective 5-HT(4) receptor agonists, prucalopride and 6,7-dihydro-4-hydroxy-7-isopropyl-6-oxo-N-[3-(piperidin-1-yl)propyl]thieno[2,3-b]pyridine-5-carboxamide (PRX-03140). In vitro, prucalopride and PRX-03140 bound to native rat brain 5-HT(4) receptors with K(i) values of 30 nM and 110 nM, respectively, and increased cAMP production in human embryonic kidney-293 cells expressing recombinant rat 5-HT(4) receptors. In vivo receptor occupancy studies established that prucalopride and PRX-03140 were able to penetrate the brain and bound to 5-HT(4) receptors in rat brain, achieving 50% receptor occupancy at free brain exposures of 330 nM and 130 nM, respectively. Rat microdialysis studies revealed that prucalopride maximally increased ACh and histamine levels in the prefrontal cortex at 5 and 10 mg/kg, whereas PRX-03140 significantly increased cortical histamine levels at 50 mg/kg, failing to affect ACh release at doses lower than 150 mg/kg. In combination studies, donepezil-induced increases in cortical ACh levels were potentiated by prucalopride and PRX-03140. Electrophysiological studies in rats demonstrated that both compounds increased the power of brainstem-stimulated hippocampal θ oscillations at 5.6 mg/kg. These findings show for the first time that the 5-HT(4) receptor agonists prucalopride and PRX-03140 can increase cortical ACh and histamine levels, augment donepezil-induced ACh increases, and increase stimulated-hippocampal θ power, all neuropharmacological parameters consistent with potential positive effects on cognitive processes.

The N- methyl- d -aspartate (NMDA) receptor complex possesses distinct recognition sites for glutamate, glycine and polyamines, which appear to be allosterically linked. We have investigated the effects of polyamines on the binding of the glycine site antagonist [ 3 H ](±)-4-(trans)-2- carboxy -5,7- dichloro -4- phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline ([3H]L-689,560), using rat cortex/hippocampus P2 membranes. Spermine and spermidine partially inhibited [3H]L-689-560 binding under non-equilibrium conditions, with IC50 values of 25.9 and 106 μM, respectively. The putative polyamine site antagonists arcaine, 1,10-diaminodecane, diethylenetriamine and putrescine had no effect on [3H]L-689,560 binding per se at 1 mM. The inhibition of [3H]L-689,560 binding by spermine was antagonised by arcaine in a competitive manner, but not by 1,10-diaminodecane, diethylenetriamine or putrescine. Kinetic analysis revealed that spermine (100 μM) decreased the association and dissociation rates of [3H]L-689,560 binding. In saturation experiments 100 μM spermine increased the KD for [3H]L-689,560 binding from 1.99 nM to 4.03 nM, with no effect on the number of binding sites. Spermine increased the affinity of glycine site agonists in displacing [3H]L-689,560 binding, with no effect on inhibition by partial agonists or antagonists, suggesting that spermine promotes an ‘agonist-preferring’ state. Modulation of [3H]L-689,560 binding by agonists for the polyamine and glutamate sites on the NMDA receptor did not appear to be additive in nature.

The κ-opioid receptor is a widely expressed G-protein-coupled receptor that has been implicated in biological responses to pain, stress, anxiety, and depression, and its potential as a therapeutic target in these syndromes is becoming increasingly apparent. However, the prototypical selective κ-opioid antagonists have very long durations of action that have been attributed to c-Jun N-terminal kinase (JNK) 1 activation in vivo. To test generality of this proposed noncompetitive mechanism, we used C57BL/6 wild type mice to determine the durations of antagonist action of novel κ-opioid receptor ligands and examined their efficacies for JNK1 activation compared with conventional competitive antagonists. Of the 12 compounds tested, 5 had long durations of action that positively correlated with JNK activation: RTI-5989-97 [(3S)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylpropyl]-2-methyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide], RTI-5989-194 [(3R)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylbutyl]-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide], RTI-5989-241 [(3R)-7-hydroxy-N-[(1S)-1-{[(3R,4R)-4-(3-methoxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide)], nor-binaltorphimine (nor-BNI); and (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). Seven had short durations of action and did not increase phospho-JNK-ir: RTI-5989-212[(3R)-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-(2-methylpropyl]-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide], RTI-5989-240 [(3R)-7-hydroxy-N-[(1S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-(2-methylpropyl]-3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide], JSPA0658 [(S)-3-fluoro-4-(4-((2-(3,5-dimethylphenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide], JSPA071B [(S)-3-fluoro-4-(4-((2-(3,5-bis(trifluoromethyl)phenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide]. PF-4455242 [2-methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine], PF-4455242 [2-methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine], FP3FBZ [(S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide], and naloxone. After long-acting antagonist treatment, pJNK-ir did not increase in mice lacking the κ-opioid receptor; increased pJNK-ir returned to baseline by 48 h after treatment; and a second challenge with nor-BNI 72 h after the first did not increase pJNK-ir. Long-lasting antagonism and increased phospho-JNK-ir were not seen in animals lacking the JNK1 isoform. These results support the hypothesis that the duration of action of small molecule κ-opioid receptor antagonists in vivo is determined by their efficacy in activating JNK1 and that persistent inactivation of the κ-receptor does not require sustained JNK activation.

2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242) is a novel κ-opioid receptor (KOR) antagonist with high affinity for human (3 nM), rat (21 nM), and mouse (22 nM) KOR, a ∼ 20-fold reduced affinity for human μ-opioid receptors (MORs; K(i) = 64 nM), and negligible affinity for δ-opioid receptors (K(i) > 4 μM). PF-04455242 also showed selectivity for KORs in vivo. In rats, PF-04455242 blocked KOR and MOR agonist-induced analgesia with ID(50) values of 1.5 and 9.8 mg/kg, respectively, and inhibited ex vivo [(3)H](2-(benzofuran-4-yl)-N-methyl-N-((5S,7R,8R)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-yl)acetamide ([(3)H]CI977) and [(3)H](2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl) propanoyl]amino]propanoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide ([(3)H]DAMGO) binding to KOR and MOR receptors with ID(50) values of 2.0 and 8.6 mg/kg, respectively. An in vivo binding assay was developed using (-)-4-[(3)H]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(3)H]PF-04767135), a tritiated version of the KOR positron emission tomography ligand (-)-4-[(11)C]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(11)C]GR103545) in which PF-04455242 had an ID(50) of 5.2 mg/kg. PF-04455242 demonstrated antidepressant-like efficacy (mouse forced-swim test), attenuated the behavioral effects of stress (mouse social defeat stress assay), and showed therapeutic potential in treating reinstatement of extinguished cocaine-seeking behavior (mouse conditioned place preference). KOR agonist-induced plasma prolactin was investigated as a translatable mechanism biomarker. Spiradoline (0.32 mg/kg) significantly increased rat plasma prolactin levels from 1.9 ± 0.4 to 41.9 ± 4.9 ng/ml. PF-04455242 dose-dependently reduced the elevation of spiradoline-induced plasma prolactin with an ID(50) of 2.3 ± 0.1 mg/kg, which aligned well with the ED(50) values obtained from the rat in vivo binding and efficacy assays. These data provide further evidence that KOR antagonists have potential for the treatment of depression and addiction disorders.

By use of parallel chemistry coupled with physicochemical property design, a series of selective κ opioid antagonists have been discovered. The parallel chemistry strategy utilized key monomer building blocks to rapidly expand the desired SAR space. The potency and selectivity of the in vitro κ antagonism were confirmed in the tail-flick analgesia model. This model was used to build an exposure-response relationship between the κ K(i) and the free brain drug levels. This strategy identified 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242, which entered phase 1 clinical testing and has demonstrated target engagement in healthy volunteers.

associated to aging and AD. (Kalpouzos 2009, Mosconi, 2007). We focused on the small primate (Microcebus murinus (M.m.), during aging some animals develop cognitive impairments (Picq, 2007), amyloid plaque depositions, and altered Tau protein accumulation associated with cerebral atrophy (Kraska, 2009). Brain atrophy is predictive of cognitive alterations (Picq, 2010). The aim of the current study was to evaluate age-associated evolution of glucose uptake in M.m. by using non-invasive FDG PET. Methods: SeventeenM.m. (1.5 to 9 years) were evaluated by PET with MicroPET Focus220 system (FDG: 900mCurie/100g, i.v.) and 3D-MR images were recorded (PharmaScan Bruker 7T). M.m were anesthetized by isoflurane duringimages acquisition. MR and PET images were co-registered by rigid transformations to define volumes of interest (VOI) in brain and appraise Standard UptakeValues (SUV) as well as relative values as compared to whole brain. Results: MR images didn’t reveal any severe atrophy this study is focused on normal aging. PET study revealed a relationship between age and SUVs in the frontal cortex, the cerebellum, and in the whole brain. However, relative values were not correlated with age. The cohort was split by theM.m.midlife (4.5 years) in two groups, young (1.960.2 years) and old animals (6.460.5 years).This second analyse showed a difference between young and old animals in thewhole brain, frontal cortex and cerebellum (t-test).Conclusions:Anage-associated evolution of glucose uptake as measured by SUVs could bedetected in non-severely atrophied M.m. and resembles evolution of the regionalcerebral metabolic rates for glucose (rCMRGlc) observed in healthy aged humans.This study is a first step toward the characterization of age-associated changesof brain glucose uptake inM.m. and will be useful to understand brain aging inM.m.

Purpose A mechanism-based PK-PD model was developed to predict the time course of dopamine D2 receptor occupancy (D2RO) in rat striatum following administration of olanzapine, an atypical antipsychotic drug. Methods A population approach was utilized to quantify both the pharmacokinetics and pharmacodynamics of olanzapine in rats using the exposure (plasma and brain concentration) and D2RO profile obtained experimentally at various doses (0.01–40 mg/kg) administered by different routes. A two-compartment pharmacokinetic model was used to describe the plasma pharmacokinetic profile. A hybrid physiology- and mechanism-based model was developed to characterize the D2 receptor binding in the striatum and was fitted sequentially to the data. The parameters were estimated using nonlinear mixed-effects modeling . Results Plasma, brain concentration profiles and time course of D2RO were well described by the model; validity of the proposed model is supported by good agreement between estimated association and dissociation rate constants and in vitro values from literature. Conclusion This model includes both receptor binding kinetics and pharmacokinetics as the basis for the prediction of the D2RO in rats. Moreover, this modeling framework can be applied to scale the in vitro and preclinical information to clinical receptor occupancy.

Anticonvulsant activity has been optimized in a series of glycine-site NMDA antagonists based on 2-carboxy tetrahydroquinoline, leading to the benzylamine 7 (L-690,590), its methyl ester prodrug 13 (L-691,470) and the phenylalanine 8 (L-696,833) which have ED 50 values of 39, 31.5 and 29 mg/kg (i.p.) respectively in the DBA/2 mouse audiogenic seizure model. Correlations between in vivo and in vitro activities suggest that systemic anticonvulsant action of glycine antagonists depends on both brain penetration as well as ‘access’ to receptors within the brain.