Mass spectrometric characterization of intact desferal-conjugated monoclonal antibodies for immuno-PET imaging.

RATIONALE: Immuno-PET imaging may prove to be a diagnostic and progression/intervention biomarker for Alzheimer's disease (AD) with improved sensitivity and specificity. Immuno-PET imaging is based on the coupling of an antibody with a chelator that captures a radioisotope thus serving as an in-vivo PET ligand. A robust and quality controlled process for linking the chelator to the-antibody is fundamental for the success of this approach. METHODS: The structural integrities of two monoclonal antibodies (trastuzumab and JRF/AßN/25) and the quantity of desferal-based chelator attached following modification of the antibodies were assessed by online desalting and intact mass analysis. Enzymatic steps for the deglycosylation and removal of C-terminal lysine was performed sequentially and in a single tube to improve intact mass data. RESULTS: Intact mass analysis demonstrated that inclusion of enzymatic processing was critical to correctly derive the quantity of chelator linked to the monoclonal antibodies. For trastuzumab, enzymatic cleaving of the glycans was sufficient, whilst additional removal of the C-terminal lysine was necessary for JRF/AßN/25 to ensure reproducible assessment of the relatively low amount of attached chelator. CONCLUSIONS: An efficient intact mass analysis-based process was developed to reproducibly determine the integrity of monoclonal antibodies and the quantity of attached chelator. This technique could serve as an essential quality control approach for the development and production of immuno-PET tracers.

Efficacy and safety of aticaprant, a kappa receptor antagonist, adjunctive to oral SSRI/SNRI antidepressant in major depressive disorder: results of a phase 2 randomized, double-blind, placebo-controlled study.

This was a double-blind, randomized, phase 2 study of adults (18-64 years) with DSM-5 diagnosis of major depressive disorder (MDD), with moderate-to-severe episode severity (Montgomery-Åsberg Depression Rating Scale [MADRS] ≥25) despite an adequate course with ongoing antidepressant for ≥6 weeks to ≤12 months. Following a double-blind placebo lead-in period (up to 3 weeks), participants were randomized to receive once daily aticaprant 10 mg or continue placebo, added to their ongoing treatment, for 6 weeks. Of 184 participants enrolled, 169 were included in safety analyses (aticaprant n = 85, placebo n = 84) and 166 in full intent-to-treat (fITT) efficacy analyses; 121 placebo lead-in non-responders (<30% reduction in MADRS total score) in fITT were included in enriched ITT (eITT) analyses. Improvement (least squares mean difference [upper limit 1-sided 80% CI] versus placebo) in MADRS total score at week 6 for aticaprant was significant versus placebo (eITT: -2.1 [-1.09], 1-sided p = 0.044; effect size (ES) 0.23; fITT -3.1 [2.21], 1-sided p = 0.002; ES 0.36). The between-group difference was larger among participants with Snaith-Hamilton Pleasure Scale (SHAPS) score greater/equal to versus less than baseline median SHAPS. The most common treatment-emergent adverse events reported for aticaprant (versus placebo) were headache (11.8% versus 7.1%), diarrhea (8.2% versus 2.4%), nasopharyngitis (5.9% versus 2.4%), and pruritus (5.9% versus 0%). One participant (1.2%) in each arm discontinued treatment due to an adverse event. In this study of participants with MDD and inadequate response to SSRI/SNRI, adjunctive treatment with aticaprant significantly reduced depressive symptoms versus placebo, without resulting in significant safety signals, supporting further investigation in larger trials.

Concordance of Alzheimer's Disease-Related Biomarkers Between Intraventricular and Lumbar Cerebrospinal Fluid in Idiopathic Normal Pressure Hydrocephalus.

BACKGROUND: Alzheimer's disease cerebrospinal fluid (CSF) biomarkers amyloid-ß 1-42 (Aß42), total tau (T-tau), and phosphorylated tau 181 (P-tau181) are widely used. However, concentration gradient of these biomarkers between intraventricular (V-CSF) and lumbar CSF (L-CSF) has been demonstrated in idiopathic normal pressure hydrocephalus (iNPH), potentially affecting clinical utility. OBJECTIVE: Here we aim to provide conversion factors for clinical and research use between V-CSF and L-CSF. METHODS: Altogether 138 iNPH patients participated. L-CSF samples were obtained prior to shunt surgery. Intraoperative V-CSF samples were obtained from 97 patients. Post-operative follow-up L- and V-CSF (shunt reservoir) samples of 41 patients were obtained 1-73 months after surgery and then after 3, 6, and 18 months. CSF concentrations of Aß42, T-tau, and P-tau181 were analyzed using commercial ELISA assays. RESULTS: Preoperative L-CSF Aß42, T-tau, and P-tau181 correlated to intraoperative V-CSF (ρ= 0.34-0.55, p < 0.001). Strong correlations were seen between postoperative L- and V-CSF for all biomarkers in every follow-up sampling point (ρs Aß42: 0.77-0.88, T-tau: 0.91-0.94, P-tau181: 0.94-0.96, p < 0.0001). Regression equations were determined for intraoperative V- and preoperative L-CSF (Aß42: V-CSF = 185+0.34*L-CSF, T-tau: Ln(V-CSF) = 3.11+0.49*Ln(L-CSF), P-tau181: V-CSF = 8.2+0.51*L-CSF), and for postoperative V- and L-CSF (Aß42: V-CSF = 86.7+0.75*L-CSF, T-tau: V-CSF = 86.9+0.62*L-CSF, P-tau181: V-CSF = 2.6+0.74*L-CSF). CONCLUSION: Aß42, T-tau, and P-tau181 correlate linearly in-between V- and L-CSF, even stronger after CSF shunt surgery. Equations presented here, provide a novel tool to use V-CSF for diagnostic and prognostic entities relying on the L-CSF concentrations and can be applicable to clinical use when L-CSF samples are not available or less invasively obtained shunt reservoir samples should be interpreted.

Identification of a series of 1,3,4-oxadiazol-2-amines as potent alpha-7 agonists with efficacy in the novel object recognition model of cognition.

A series of α7 nicotinic acetylcholine receptor full-agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Systematic exploration of the structure-activity relationships for both α7 potency and selectivity with respect to interaction with the hERG channel are described. Further profiling led to the identification of compound 22, a potent full agonist showing efficacy in the novel object recognition model of cognition enhancement.

The discovery of 2-fluoro-N-(3-fluoro-4-(5-((4-morpholinobutyl)amino)-1,3,4-oxadiazol-2-yl)phenyl)benzamide, a full agonist of the alpha-7 nicotinic acetylcholine receptor showing efficacy in the novel object recognition model of cognition enhancement.

A series of α7 nicotinic acetylcholine receptor full agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Early lead 1 was found to have a limited therapeutic index with respect to its potential for cardiovascular side effects. Further optimisation of this series led to the identification of 22 a potent full agonist showing efficacy at a dose of 0.1mg/kg in the novel object recognition model of cognition enhancement. Comparison of 1 with 22 demonstrated the latter to have an improved oral pharmacokinetic profile and cardiovascular therapeutic index.

The effects of inhibition of fatty acid amide hydrolase (FAAH) by JNJ-42165279 in social anxiety disorder: a double-blind, randomized, placebo-controlled proof-of-concept study.

JNJ-42165279 is a selective inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of fatty acid amides (FAA) including anandamide (AEA), palmitoylethanolamide (PEA), and N-oleoylethanolamide (OEA). We assessed the efficacy, safety, tolerability, pharmacokinetics, and pharmacodynamics of treatment with JNJ-42165279 in subjects with social anxiety disorder (SAD). This was a multicenter, double-blind, placebo-controlled study randomizing subjects to 12 weeks of treatment with either JNJ-42165279 (25 mg daily) or placebo (PBO). The primary endpoint was the change in the Liebowitz Social Anxiety Scale (LSAS) total score from baseline to end of study. Secondary endpoints included the Hamilton Anxiety Scale (HAM-A), Hamilton Depression Rating Scale (HDRS17), and the Clinical Global Impression-Improvement (CGI-I). Samples were collected for plasma concentration of AEA, PEA, OEA, and JNJ-42165279. A total of 149 subjects were enrolled with a mean baseline LSAS total score of 102.6 (SD 16.84). The mean change from baseline (SD) in LSAS total score at week 12 was numerically greater for JNJ-42165279: -29.4 (27.47) compared to PBO: -22.4 (23.57) but not significant. The percentage of subjects with ≥30% improvement from baseline in the LSAS total score was significantly higher for JNJ-42165279 (42.4%) compared to PBO (23.6%) (p value = 0.04). The percentage of subjects with a CGI-I score of much or very much improved was also significantly higher for JNJ-42165279 (44.1%) than for PBO (23.6%) (p value = 0.02). The drug was well tolerated. JNJ-42165279 appears to elicit an anxiolytic effect in subjects with SAD although trough concentrations with 25 mg once daily appeared to be insufficient to completely inhibit FAAH activity which may have led to suboptimal efficacy. ClinicalTrials.gov Identifier: NCT02432703.

Time Trends of Cerebrospinal Fluid Biomarkers of Neurodegeneration in Idiopathic Normal Pressure Hydrocephalus.

BACKGROUND: Longitudinal changes in cerebrospinal fluid (CSF) biomarkers are seldom studied. Furthermore, data on biomarker gradient between lumbar (L-) and ventricular (V-) compartments seems to be discordant. OBJECTIVE: To examine alteration of CSF biomarkers reflecting Alzheimer's disease (AD)-related amyloid-ß (Aß) aggregation, tau pathology, neurodegeneration, and early synaptic degeneration by CSF shunt surgery in idiopathic normal pressure hydrocephalus (iNPH) in relation to AD-related changes in brain biopsy. In addition, biomarker levels in L- and V-CSF were compared. METHODS: L-CSF was collected prior to shunt placement and, together with V-CSF, 3-73 months after surgery. Thereafter, additional CSF sampling took place at 3, 6, and 18 months after the baseline sample from 26 iNPH patients with confirmed Aß plaques in frontal cortical brain biopsy and 13 iNPH patients without Aß pathology. CSF Amyloid-ß42 (Aß42), total tau (T-tau), phosphorylated tau (P-tau181), neurofilament light (NFL), and neurogranin (NRGN) were analyzed with customized ELISAs. RESULTS: All biomarkers but Aß42 increased notably by 140-810% in L-CSF after CSF diversion and then stabilized. Aß42 instead showed divergent longitudinal decrease between Aß-positive and -negative patients in L-CSF, and thereafter increase in Aß-negative iNPH patients in both L- and V-CSF. All five biomarkers correlated highly between V-CSF and L-CSF (Aß42 R = 0.87, T-tau R = 0.83, P-tau R = 0.92, NFL R = 0.94, NRGN R = 0.9; all p < 0.0001) but were systematically lower in V-CSF (Aß42 14 %, T-tau 22%, P-tau 20%, NFL 32%, NRGN 19%). With APOE genotype-grouping, only Aß42 showed higher concentration in non-carriers of allele ɛ4. CONCLUSION: Longitudinal follow up shows that after an initial post-surgery increase, T-tau, P-tau, and NRGN are stable in iNPH patients regardless of brain biopsy Aß pathology, while NFL normalized toward its pre-shunt levels. Aß42 as biomarker seems to be the least affected by the surgical procedure or shunt and may be the best predictor of AD risk in iNPH patients. All biomarker concentrations were lower in V- than L-CSF yet showing strong correlations.

Rasagiline improves learning and memory in young healthy rats.

The effect of rasagiline on learning and memory in Lister-Hooded rats was investigated in this study. Two cognitive tests were used: a 24-h temporal deficit novel object recognition test and a modified water maze task. Rasagiline (0.3 and 1 mg/kg) was administered subcutaneously 15 min before the cognitive tests. In a novel object recognition test, rasagiline treatment enhanced object recognition memory. A small effect was observed with 0.3 mg/kg rasagiline; at 1 mg/kg, rasagiline-treated animals spent twice as much time exploring the novel object. On the water maze test, the use of an on-demand platform allowed adjustment of the difficulty of this spatial learning task. This enabled the detection of a small positive effect of rasagiline (1 mg/kg) on spatial learning, which was not observed in earlier reports. For the first time, our study has showed the procognitive effect of rasagiline in young healthy rats. On the basis of these findings, a monoamine oxidase-B inhibitor would seem to be a potential symptomatic treatment for cognitive impairments affecting patients with neurodegenerative disorders.

Optimizing Behavioral Paradigms to Facilitate Development of New Treatments for Anhedonia and Reward Processing Deficits in Schizophrenia and Major Depressive Disorder: Study Protocol.

Background: Behavioral tasks focusing on different subdomains of reward processing may provide more objective and quantifiable measures of anhedonia and impaired motivation compared with clinical scales. Typically, single tasks are used in relatively small studies to compare cases and controls in one indication, but they are rarely included in larger multisite trials. This is due to limited systematic standardization as well as the challenges of deployment in international studies and stringent adherence to the high regulatory requirements for data integrity. The Reward Task Optimization Consortium (RTOC) was formed to facilitate operational implementation of reward processing tasks, making them suitable for use in future large-scale, international, multisite drug development studies across multiple indications. The RTOC clinical study aims to conduct initial optimization of a set of tasks in patients with major depressive disorder (MDD) or schizophrenia (SZ). Methods: We will conduct a multicenter study across four EU countries. Participants (MDD = 37, SZ = 37, with ≤80 age- and gender-matched healthy volunteers) will attend a study visit comprising screening, self-report and clinically rated assessments of anhedonia and symptom severity, and three reward processing tasks; specifically, the Grip Strength Effort task, the Doors task, and the Reinforcement Learning Working Memory task. The Grip Strength Effort and Doors tasks include simultaneous electroencephalography/event-related potential recordings. Outcomes will be compared using a two-way group design of MDD and SZ with matched controls, respectively. Further analyses will include anhedonia assessment scores as covariates. Planned analyses will assess whether our findings replicate previously published data, and multisite deployment will be evaluated through assessments of quality and conduct. A subset of participants will complete a second visit, to assess test-retest reliability of the task battery. Discussion: This study will evaluate the operational deployment of three reward processing tasks to the regulatory standards required for use in drug development trials. We will explore the potential of these tasks to differentiate patients from controls and to provide a quantitative marker of anhedonia and/or impaired motivation, establishing their usefulness as endpoints in multisite clinical trials. This study should demonstrate where multifaceted reward deficits are similar or divergent across patient populations. Registration: ClinicalTrials.gov (NCT04024371).

Effect of donepezil on reversal learning in a touch screen-based operant task.

Impairments in reversal learning, which are commonly observed in patients with psychiatric disorders, remain difficult to treat. There is still a debate over the beneficial effects of cholinergic enhancers on improving behavioural flexibility. The objective of this study was to investigate the effect of an acetylcholinesterase inhibitor, donepezil, on the performance of a rodent Probabilistic Reversal Learning task. Lister-Hooded rats were trained to retrieve food rewards by discriminating two computer-generated stimuli in an automated touch screen-based operant task. When a steady performance was achieved, the stimulus-reward rule was reversed. Each rat was given a 30-min training session daily for 24 days and donepezil was administered 30 min before each training session. Systemic treatment with donepezil had no effect on trial accuracy of the two-stimulus discrimination training. However, the donepezil group showed enhanced performance in the reversal learning. Our result showed that treatment with donepezil significantly enhanced Probabilistic Reversal Learning performance in healthy animals. On the basis of this finding, the inhibition of the central acetylcholinesterase would seem to be a potential therapeutic approach to treat behavioural inflexibility.

Relating constructs of attention and working memory to social withdrawal in Alzheimer's disease and schizophrenia: issues regarding paradigm selection.

Central nervous system diseases are not currently diagnosed based on knowledge of biological mechanisms underlying their symptoms. Greater understanding may be offered through an agnostic approach to traditional disease categories, where learning more about shared biological mechanisms across conditions could potentially reclassify sub-groups of patients to allow realisation of more effective treatments. This review represents the output of the collaborative group "PRISM", tasked with considering assay choices for assessment of attention and working memory in a transdiagnostic cohort of Alzheimer's disease and schizophrenia patients exhibiting symptomatic spectra of social withdrawal. A multidimensional analysis of this nature has not been previously attempted. Nominated assays (continuous performance test III, attention network test, digit symbol substitution, N-back, complex span, spatial navigation in a virtual environment) reflected a necessary compromise between the need for broad assessment of the neuropsychological constructs in question with several pragmatic criteria: patient burden, compatibility with neurophysiologic measures and availability of preclinical homologues.

In vitro and in vivo characterization of the non-peptide NK3 receptor antagonist SB-223412 (talnetant): potential therapeutic utility in the treatment of schizophrenia.

Neurokinin-3 (NK3) receptors are concentrated in forebrain and basal ganglia structures within the mammalian CNS. This distribution, together with the modulatory influence of NK3 receptors on monoaminergic neurotransmission, has led to the hypothesis that NK3 receptor antagonists may have therapeutic efficacy in the treatment of psychiatric disorders. Here we describe the in vitro and in vivo characterization of the highly selective NK3 receptor antagonist talnetant (SB-223412). Talnetant has high affinity for recombinant human NK3 receptors (pKi 8.7) and demonstrates selectivity over other neurokinin receptors (pKi NK2 = 6.6 and NK1<4). In native tissue-binding studies, talnetant displayed high affinity for the guinea pig NK3 receptor (pKi 8.5). Functionally, talnetant competitively antagonized neurokinin B (NKB)-induced responses at the human recombinant receptor in both calcium and phosphoinositol second messenger assay systems (pA2 of 8.1 and 7.7, respectively). In guinea pig brain slices, talnetant antagonized NKB-induced increases in neuronal firing in the medial habenula (pKB = 7.9) and senktide-induced increases in neuronal firing in the substantia nigra pars compacta (pKB = 7.7) with no diminution of maximal agonist efficacy, suggesting competitive antagonism at native NK3 receptors. Talnetant (3-30 mg/kg i.p.) significantly attenuated senktide-induced 'wet dog shake' behaviors in the guinea pig in a dose-dependent manner. Microdialysis studies demonstrated that acute administration of talnetant (30 mg/kg i.p.) produced significant increases in extracellular dopamine and norepinephrine in the medial prefrontal cortex and attenuated haloperidol-induced increases in nucleus accumbens dopamine levels in the freely moving guinea pigs. Taken together, these data demonstrate that talnetant is a selective, competitive, brain-penetrant NK3 receptor antagonist with the ability to modulate mesolimbic and mesocortical dopaminergic neurotransmission and hence support its potential therapeutic utility in the treatment of schizophrenia.

Fatty Acid Amide Hydrolase Inhibition by JNJ-42165279: A Multiple-Ascending Dose and a Positron Emission Tomography Study in Healthy Volunteers.

Inhibition of fatty acid amide hydrolase (FAAH) potentiates endocannabinoid activity and is hypothesized to have therapeutic potential for mood and anxiety disorders and pain. The clinical profile of JNJ-42165279, an oral selective FAAH inhibitor, was assessed by investigating the pharmacokinetics, pharmacodynamics, safety, and binding to FAAH in the brain of healthy human volunteers. Concentrations of JNJ-42165279 (plasma, cerebrospinal fluid (CSF), urine) and fatty acid amides (FAA; plasma, CSF), and FAAH activity in leukocytes was determined in a phase I multiple ascending dose study. A positron emission tomography study with the FAAH tracer [11 C]MK3168 was conducted to determine brain FAAH occupancy after single and multiple doses of JNJ-42165279. JNJ-42165279 administration resulted in an increase in plasma and CSF FAA. Significant blocking of brain FAAH binding of [11 C]MK3168 was observed after pretreatment with JNJ-42165279. JNJ-42165279 produces potent central and peripheral FAAH inhibition. Saturation of brain FAAH occupancy occurred with doses ≥10 mg of JNJ-42165279. No safety concerns were identified.

Diffusion kurtosis imaging allows the early detection and longitudinal follow-up of amyloid-ß-induced pathology.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in the elderly population. In this study, we used the APP/PS1 transgenic mouse model to explore the feasibility of using diffusion kurtosis imaging (DKI) as a tool for the early detection of microstructural changes in the brain due to amyloid-ß (Aß) plaque deposition. METHODS: We longitudinally acquired DKI data of wild-type (WT) and APP/PS1 mice at 2, 4, 6 and 8 months of age, after which these mice were sacrificed for histological examination. Three additional cohorts of mice were also included at 2, 4 and 6 months of age to allow voxel-based co-registration between diffusion tensor and diffusion kurtosis  metrics and immunohistochemistry. RESULTS: Changes were observed in diffusion tensor (DT) and diffusion kurtosis (DK) metrics in many of the 23 regions of interest that were analysed. Mean and axial kurtosis were greatly increased owing to Aß-induced pathological changes in the motor cortex of APP/PS1 mice at 4, 6 and 8 months of age. Additionally, fractional anisotropy (FA) was decreased in APP/PS1 mice at these respective ages. Linear discriminant analysis of the motor cortex data indicated that combining diffusion tensor and diffusion kurtosis metrics permits improved separation of WT from APP/PS1 mice compared with either diffusion tensor or diffusion kurtosis metrics alone. We observed that mean kurtosis and FA are the critical metrics for a correct genotype classification. Furthermore, using a newly developed platform to co-register the in vivo diffusion-weighted magnetic resonance imaging with multiple 3D histological stacks, we found high correlations between DK metrics and anti-Aß (clone 4G8) antibody, glial fibrillary acidic protein, ionised calcium-binding adapter molecule 1 and myelin basic protein immunohistochemistry. Finally, we observed reduced FA in the septal nuclei of APP/PS1 mice at all ages investigated. The latter was at least partially also observed by voxel-based statistical parametric mapping, which showed significantly reduced FA in the septal nuclei, as well as in the corpus callosum, of 8-month-old APP/PS1 mice compared with WT mice. CONCLUSIONS: Our results indicate that DKI metrics hold tremendous potential for the early detection and longitudinal follow-up of Aß-induced pathology.

S-[18F]THK-5117-PET and [11C]PIB-PET Imaging in Idiopathic Normal Pressure Hydrocephalus in Relation to Confirmed Amyloid-ß Plaques and Tau in Brain Biopsies.

BACKGROUND: Detection of pathological tau aggregates could facilitate clinical diagnosis of Alzheimer's disease (AD) and monitor drug effects in clinical trials. S-[18F]THK-5117 could be a potential tracer to detect pathological tau deposits in brain. However, no previous study have correlated S-[18F]THK-5117 uptake in PET with brain biopsy verified tau pathology in vivo. OBJECTIVE: Here we aim to evaluate the association between cerebrospinal fluid (CSF) AD biomarkers, S-[18F]THK-5117, and [11C]PIB PET against tau and amyloid lesions in brain biopsy. METHODS: Fourteen patients with idiopathic normal pressure hydrocephalus (iNPH) with previous shunt surgery including right frontal cortical brain biopsy and CSF Aß1 - 42, total tau, and P-tau181 measures, underwent brain MRI, [11C]PIB PET, and S-[18F]THK-5117 PET imaging. RESULTS: Seven patients had amyloid-ß (Aß, 4G8) plaques, two both Aß and phosphorylated tau (Pτ, AT8) and one only Pτ in biopsy. As expected, increased brain biopsy Aß was well associated with higher [11C]PIB uptake in PET. However, S-[18F]THK-5117 uptake did not show any statistically significant correlation with either brain biopsy Pτ or CSF P-tau181 or total tau. CONCLUSIONS: S-[18F]THK-5117 lacked clear association with neuropathologically verified tau pathology in brain biopsy probably, at least partially, due to off-target binding. Further studies with larger samples of patients with different tau tracers are urgently needed. The detection of simultaneous Aß and tau pathology in iNPH is important since that may indicate poorer and especially shorter response for CSF shunt surgery compared with no pathology.

In Vivo Amyloid-ß Imaging in the APPPS1-21 Transgenic Mouse Model with a (89)Zr-Labeled Monoclonal Antibody.

INTRODUCTION: The accumulation of amyloid-ß is a pathological hallmark of Alzheimer's disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-ß antibody (JRF/AßN/25) to non-invasively assess amyloid-ß burden in aged transgenic mice (APPPS1-21) with µPET imaging. METHODS: We investigated the antibody JRF/AßN/25 that binds to full-length Aß. JRF/AßN/25 was radiolabeled with a [(89)Zr]-desferal chelate and intravenously injected into 12-13 month aged APPPS1-21 mice and their wild-type (WT) controls. Mice underwent in vivo µPET imaging at 2, 4, and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution, and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [(89)Zr]-labeled antibody (trastuzumab) as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/AßN/25 and finally we assessed the possible confounding effects of blood retention. RESULTS: Voxel-wise analysis of µPET data demonstrated significant [(89)Zr]-Df-Bz-JRF/AßN/25 retention in APPPS1-21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [(89)Zr]-Df-Bz-JRF/AßN/25 was found at 4 and 7 days pi in APPPS1-21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/AßN/25 only partially blocked [(89)Zr]-Df-Bz-JRF/AßN/25 uptake indicative of a high contribution of non-specific binding. CONCLUSION: Amyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibody in addition to non-specific binding prevented an accurate estimation of plaque burden. However, it should be noted that [(89)Zr]-Df-Bz-JRF/AßN/25 nevertheless demonstrated in vivo binding and strategies to increase brain penetrance would likely achieve better results.

Synthesis and Evaluation of a Zr-89-Labeled Monoclonal Antibody for Immuno-PET Imaging of Amyloid-ß Deposition in the Brain.

PURPOSE: The aim of this study was to evaluate the in vitro and in vivo characteristics of [(89)Zr]JRF/AßN/25, a radiolabeled monoclonal antibody directed against amyloid-ß (Aß). PROCEDURES: JRF/AßN/25 was labeled with (89)Zr following modification with desferal. The affinity of the tracer for Aß1-40 was determined in a saturation binding assay. In vitro stability was evaluated, and in vivo plasma stability and biodistribution of [(89)Zr]Df-Bz-JRF/AßN/25 were determined in wild-type mice. To evaluate whether the antibody can cross the blood-brain barrier, brain uptake in wild-type mice was additionally assessed by ex vivo autoradiography. RESULTS: [(89)Zr]Df-Bz-JRF/AßN/25 was obtained in an average radiochemical yield of 50 % and a radiochemical purity of >97 %. A saturation binding assay demonstrated specific binding of [(89)Zr]Df-Bz-JRF/AßN/25 to Aß1-40 with nanomolar affinity. The tracer was stable in buffer and proved to be stable in vivo with >92 % intact monoclonal antibody (mAb) remaining in the plasma at 48 h post injection. A biodistribution study showed a slow blood clearance with no significant accumulation of activity in any of the organs. Furthermore, [(89)Zr]Df-Bz-JRF/AßN/25 demonstrated modest brain penetration, which slowly decreased in time. This cerebral uptake was confirmed by ex vivo autoradiography. CONCLUSIONS: [(89)Zr]Df-Bz-JRF/AßN/25 binds with high affinity to Aß1-40. The tracer displays an acceptable in vivo stability and is able to cross the blood-brain barrier. [(89)Zr]Df-Bz-JRF/AßN/25 might therefore be a potential candidate for in vivo imaging of Aß deposition in the brain.

Synaptic transmission changes in fear memory circuits underlie key features of an animal model of schizophrenia.

Non-competitive antagonists of the N-methyl-d-aspartate receptor (NMDA) such as phencyclidine (PCP) elicit schizophrenia-like symptoms in healthy individuals. Similarly, PCP dosing in rats produces typical behavioral phenotypes that mimic human schizophrenia symptoms. Although schizophrenic behavioral phenotypes of the PCP model have been extensively studied, the underlying alterations of intrinsic neuronal properties and synaptic transmission in relevant limbic brain microcircuits remain elusive. Acute brain slice electrophysiology and immunostaining of inhibitory neurons were used to identify neuronal circuit alterations of the amygdala and hippocampus associated with changes in extinction of fear learning in rats following PCP treatment. Subchronic PCP application led to impaired long-term potentiation (LTP) and marked increases in the ratio of NMDA to 2-amino-3(5-methyl-3-oxo-1,2-oxazol-4-yl)propionic acid (AMPA) receptor-mediated currents at lateral amygdala (LA) principal neurons without alterations in parvalbumin (PV) as well as non-PV, glutamic acid decarboxylase 67 (GAD 67) immunopositive neurons. In addition, LTP was impaired at the Schaffer collateral to CA1 hippocampal pathway coincident with a reduction in colocalized PV and GAD67 immunopositive neurons in the CA3 hippocampal area. These effects occurred without changes in spontaneous events or intrinsic membrane properties of principal cells in the LA. The impairment of LTP at both amygdalar and hippocampal microcircuits, which play a key role in processing relevant survival information such as fear and extinction memory concurred with a disruption of extinction learning of fear conditioned responses. Our results show that subchronic PCP administration in rats impairs synaptic functioning in the amygdala and hippocampus as well as processing of fear-related memories.

PNU-120596, a positive allosteric modulator of α7 nicotinic acetylcholine receptors, reverses a sub-chronic phencyclidine-induced cognitive deficit in the attentional set-shifting task in female rats.

The α7 nicotinic acetylcholine receptors (nAChRs) have been highlighted as a target for cognitive enhancement in schizophrenia. Adult female hooded Lister rats received sub-chronic phencyclidine (PCP) (2 mg/kg) or vehicle i.p. twice daily for 7 days, followed by 7 days' washout. PCP-treated rats then received PNU-120596 (10 mg/kg; s.c.) or saline and were tested in the attentional set-shifting task. Sub-chronic PCP produced a significant cognitive deficit in the extra-dimensional shift (EDS) phase of the task (p < 0.001, compared with vehicle). PNU-120596 significantly improved performance of PCP-treated rats in the EDS phase of the attentional set-shifting task (p < 0.001). In conclusion, these data demonstrate that PNU-120596 improves cognitive dysfunction in our animal model of cognitive dysfunction in schizophrenia, most likely via modulation of α7 nACh receptors.

Memantine-induced brain activation as a model for the rapid screening of potential novel antipsychotic compounds: exemplified by activity of an mGlu2/3 receptor agonist.

RATIONALE: Schizophrenia is a severe, disabling chronic disorder affecting approximately 1% of the population. Improvements and development of more robust and hopefully predictive screening assays for this disease should enhance the identification and development of novel treatments. The present study describes a rapid and robust method for the testing of potential novel antipsychotics by utilising a simplified [(14)C]2-deoxyglucose (2-DG) autoradiography method following memantine-induced brain activation. METHODS: Male C57BL/6JCRL mice were given vehicle, ketamine or memantine (10, 20 and 30 mg/kg, subcutaneously (s.c.)) and sacrificed 45 min post-[(14 C)]2-DG administration. In subsequent reversal studies, the memantine challenge was further validated with haloperidol (0.32 mg/kg, s.c.) and clozapine (2.5 and 10 mg/kg, s.c.) in parallel with the ketamine model (Duncan et al. 1998a). Lastly, the effects of an mGlu2/3 receptor agonist, LY404039 (10 mg/kg, s.c.), on both ketamine and memantine-induced brain activation was determined. RESULTS: Both N-methyl-d-aspartate (NMDA) antagonists dose-dependently induced significant region-specific increases in 2-DG uptake. Interestingly, memantine elicited a considerably greater brain activation signature with a larger dynamic window than ketamine. The "atypical" antipsychotic clozapine significantly reversed memantine-induced 2-DG uptake whilst the "typical" antipsychotic haloperidol was inactive. Pre-treatment with LY404039 fully reversed both the ketamine- and memantine-induced increase in 2-DG uptake without effects on basal 2-DG uptake. CONCLUSION: This novel pre-clinical imaging methodology displays potential for the screening of compounds targeting the NMDA receptor hypofunction hypothesis of schizophrenia and should assist in developing compounds from the bench to clinic.