Birtamimab plus standard of care in light-chain amyloidosis: the phase 3 randomized placebo-controlled VITAL trial.

Amyloid light-chain (AL) amyloidosis is a rare, typically fatal disease characterized by the accumulation of misfolded immunoglobulin light chains (LCs). Birtamimab is an investigational humanized monoclonal antibody designed to neutralize toxic LC aggregates and deplete insoluble organ-deposited amyloid via macrophage-induced phagocytosis. VITAL was a phase 3 randomized, double-blind, placebo-controlled clinical trial assessing the efficacy and safety of birtamimab + standard of care (SOC) in 260 newly diagnosed, treatment-naive patients with AL amyloidosis. Patients received 24 mg/kg IV birtamimab + SOC or placebo + SOC every 28 days. The primary composite end point was the time to all-cause mortality (ACM) or centrally adjudicated cardiac hospitalization ≥91 days after the first study drug infusion. The trial was terminated early after an interim futility analysis; there was no significant difference in the primary composite end point (hazard ratio [HR], 0.826; 95% confidence interval [CI], 0.574-1.189; log-rank P = .303). A post hoc analysis of patients with Mayo stage IV AL amyloidosis, those at the highest risk of early mortality, showed significant improvement in the time to ACM with birtamimab at month 9 (HR, 0.413; 95% CI, 0.191-0.895; log-rank P = .021). At month 9, 74% of patients with Mayo stage IV AL amyloidosis treated with birtamimab and 49% of those given placebo survived. Overall, the rates of treatment-emergent adverse events (TEAEs) and serious TEAEs were generally similar between treatment arms. A confirmatory phase 3 randomized, double-blind, placebo-controlled clinical trial of birtamimab in patients with Mayo stage IV AL amyloidosis (AFFIRM-AL; NCT04973137) is currently enrolling. The VITAL trial was registered at www.clinicaltrials.gov as #NCT02312206.

First-in-human assessment of PRX002, an anti-α-synuclein monoclonal antibody, in healthy volunteers.

BACKGROUND: α-Synuclein is a major component of pathologic inclusions that characterize Parkinson's disease. PRX002 is an antibody that targets α-synuclein, and its murine parent antibody 9E4 has been shown in preclinical studies to reduce α-synuclein pathology and to protect against cognitive and motor deteriorations and progressive neurodegeneration in human α-synuclein transgenic mice. METHODS: This first-in-human, randomized, double-blind, placebo-controlled, phase 1 study assessed the impact of PRX002 administered to 40 healthy participants in 5 ascending-dose cohorts (n = 8/cohort) in which participants were randomly assigned to receive a single intravenous infusion of study drug (0.3, 1, 3, 10, or 30 mg/kg; n = 6/cohort) or placebo (n = 2/cohort). RESULTS: PRX002 demonstrated favorable safety, tolerability, and pharmacokinetic profiles at all doses tested, with no immunogenicity. No serious adverse events, discontinuations as a result of adverse events, or dose-limiting toxicities were reported. Serum PRX002 exposure was dose proportional; the average terminal half-life across all doses was 18.2 days. A significant dose-dependent reduction in free serum α-synuclein (unbound to PRX002) was apparent within 1 hour after PRX002 administration, whereas total α-synuclein (free plus bound) increased dose-dependently, presumably because of the expected change in kinetics following antibody binding. CONCLUSIONS: This study demonstrates that serum α-synuclein can be safely modulated in a dose-dependent manner after single intravenous infusions of an anti-α-synuclein antibody. These findings support continued development of PRX002, including further characterization of its safety, tolerability, pharmacokinetics, and pharmacodynamic effects in the central nervous system in patients with Parkinson's disease. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

The mechanism of action, pharmacological characteristics, and clinical utility of the amyloid depleter birtamimab for the potential treatment of AL amyloidosis.

Amyloid light chain (AL) amyloidosis is a progressive plasma cell disorder caused by amyloid deposition resulting in organ damage and failure. Current standard-of-care treatments target clonal plasma cells, the source of misfolded light chains (amyloid precursors), yet only half of patients with advanced disease survive ≥6 months. The amyloid depleter birtamimab is an investigational humanized monoclonal antibody that binds misfolded κ and λ light chains with high specificity and was designed to neutralize soluble toxic light chain aggregates and promote phagocytic clearance of deposited amyloid. Post hoc analyses from the Phase 3 VITAL trial suggested birtamimab plus standard of care confers a survival benefit in patients with advanced (Mayo Stage IV) AL amyloidosis. AFFIRM-AL (NCT04973137), a Phase 3 confirmatory trial of birtamimab plus standard of care in patients with Mayo Stage IV AL amyloidosis, is ongoing. This review summarizes birtamimab's mechanism of action, attributes, and potential clinical utility.

Neutralization of soluble, synaptotoxic amyloid ß species by antibodies is epitope specific.

Several anti-amyloid ß (Aß) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aß antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aß plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aß plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aß species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aß(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aß moieties. Importantly C-terminal anti-Aß antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aß antibodies effectively interact with both soluble and insoluble forms of Aß and therefore appear particularly well suited for testing the Aß hypothesis of AD.

Vascular alterations in PDAPP mice after anti-Aß immunotherapy: Implications for amyloid-related imaging abnormalities.

BACKGROUND: Clinical studies of ß-amyloid (Aß) immunotherapy in Alzheimer's disease (AD) patients have demonstrated reduction of central Aß plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Aß pathology and after anti-Aß immunotherapy. METHODS: We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Aß pathology and after anti-Aß immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed. RESULTS: The central vasculature displayed morphological abnormalities associated with vascular Aß deposits. Treatment with 3D6 antibody induced clearance of vascular Aß that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Aß deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Aß accumulation. CONCLUSIONS: These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Aß. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies.

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.

The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.

Inhibition of calcineurin-mediated endocytosis and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors prevents amyloid beta oligomer-induced synaptic disruption.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (Abeta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Abeta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.

A Phase II Study to Evaluate the Safety and Efficacy of Prasinezumab in Early Parkinson's Disease (PASADENA): Rationale, Design, and Baseline Data.

Background: Currently available treatments for Parkinson's disease (PD) do not slow clinical progression nor target alpha-synuclein, a key protein associated with the disease. Objective: The study objective was to evaluate the efficacy and safety of prasinezumab, a humanized monoclonal antibody that binds aggregated alpha-synuclein, in individuals with early PD. Methods: The PASADENA study is a multicenter, randomized, double-blind, placebo-controlled treatment study. Individuals with early PD, recruited across the US and Europe, received monthly intravenous doses of prasinezumab (1,500 or 4,500 mg) or placebo for a 52-week period (Part 1), followed by a 52-week extension (Part 2) in which all participants received active treatment. Key inclusion criteria were: aged 40-80 years; Hoehn & Yahr (H&Y) Stage I or II; time from diagnosis ≤2 years; having bradykinesia plus one other cardinal sign of PD (e.g., resting tremor, rigidity); DAT-SPECT imaging consistent with PD; and either treatment naïve or on a stable monoamine oxidase B (MAO-B) inhibitor dose. Study design assumptions for sample size and study duration were built using a patient cohort from the Parkinson's Progression Marker Initiative (PPMI). In this report, baseline characteristics are compared between the treatment-naïve and MAO-B inhibitor-treated PASADENA cohorts and between the PASADENA and PPMI populations. Results: Of the 443 patients screened, 316 were enrolled into the PASADENA study between June 2017 and November 2018, with an average age of 59.9 years and 67.4% being male. Mean time from diagnosis at baseline was 10.11 months, with 75.3% in H&Y Stage II. Baseline motor and non-motor symptoms (assessed using Movement Disorder Society-Unified Parkinson's Disease Rating Scale [MDS-UPDRS]) were similar in severity between the MAO-B inhibitor-treated and treatment-naïve PASADENA cohorts (MDS-UPDRS sum of Parts I + II + III [standard deviation (SD)]; 30.21 [11.96], 32.10 [13.20], respectively). The overall PASADENA population (63.6% treatment naïve and 36.4% on MAO-B inhibitor) showed a similar severity in MDS-UPDRS scores (e.g., MDS-UPDRS sum of Parts I + II + III [SD]; 31.41 [12.78], 32.63 [13.04], respectively) to the PPMI cohort (all treatment naïve). Conclusions: The PASADENA study population is suitable to investigate the potential of prasinezumab to slow disease progression in individuals with early PD. Trial Registration: NCT03100149.

Discovery of N-{[1-(propylsulfonyl)-4-pyridin-2-ylpiperidin-4-yl]methyl}benzamides as novel, selective and potent GlyT1 inhibitors.

Employing an iterative analogue library approach, novel potent and selective glycine transporter 1 (GlyT1) inhibitors containing a 4-pyridin-2-ylpiperidine sulfonamide have been discovered. These inhibitors are devoid of time-dependent CYP inhibition activity and exhibit improved aqueous solubility versus the corresponding 4-phenylpiperidine analogues.

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties.

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.

Safety and Tolerability of Multiple Ascending Doses of PRX002/RG7935, an Anti-α-Synuclein Monoclonal Antibody, in Patients With Parkinson Disease: A Randomized Clinical Trial.

Importance: Aggregated α-synuclein is believed to be central to the pathogenesis of Parkinson disease (PD). PRX002/RG7935 (PRX002) is a humanized monoclonal antibody designed to target aggregated forms of α-synuclein, thereby inhibiting neuron-to-neuron transfer of presumed pathogenic forms of α-synuclein, potentially resulting in neuronal protection and slowing disease progression. Objective: To evaluate the safety and tolerability of multiple intravenous infusions of PRX002 in patients with idiopathic PD. Design, Setting, and Participants: Multicenter, randomized, double-blind, placebo-controlled, multiple ascending-dose trial at 8 US study centers from July 2014 to September 2016. Eligible participants were aged 40 to 80 years with mild to moderate idiopathic PD (Hoehn and Yahr stages 1-3). Interventions: Participants were enrolled into 6 ascending-dose cohorts and randomly assigned to receive PRX002 (0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg, or 60 mg/kg) or placebo. Participants received 3 intravenous infusions every 4 weeks of PRX002 or placebo and were monitored during a 24-week observational period. Main Outcomes and Measures: Safety and tolerability assessments included physical and neurological examinations, laboratory tests, vital signs, and adverse events. Pharmacokinetic parameters included maximum PRX002 concentration, area under the curve, and half-life. Results: Of the 80 participants, most were white (97.5%; n = 78) and male (80%; n = 64); median (SD) age was 58 (8.4) years. PRX002 was generally safe and well tolerated; no serious or severe PRX002-related treatment-emergent adverse events (TEAEs) were reported. The TEAEs experienced by at least 5% of patients receiving PRX002, irrespective of relatedness to study drug, were constipation (9.1%; n = 5), infusion reaction (7.3%; n = 4), diarrhea (5.5%; n = 3), headache (5.5%; n = 3), peripheral edema (5.5%; n = 3), post-lumbar puncture syndrome (5.5%; n = 3), and upper respiratory tract infection (5.5%; n = 3). No antidrug antibodies were detected. Serum PRX002 levels increased in an approximately dose-proportional manner; mean terminal elimination half-life was similar across all doses (10.2 days). Rapid dose- and time-dependent mean reductions from baseline vs placebo in free serum α-synuclein levels of up to 97% were seen after a single infusion at the highest dose (F78,284 = 1.66; P = .002), with similar reductions after 2 additional infusions. Mean cerebrospinal fluid PRX002 concentration increased with PRX002 dose and was approximately 0.3% relative to serum across all dose cohorts. Conclusions and Relevance: Single and multiple doses of PRX002 were generally safe and well tolerated and resulted in robust binding of peripheral α-synuclein and dose-dependent increases of PRX002 in cerebrospinal fluid, reaching cerebrospinal fluid concentrations that may be expected to engage extracellular aggregated α-synuclein in the brain. Findings support the design of an ongoing phase 2 clinical study (NCT03100149). Trial Registration: ClinicalTrials.gov Identifier: NCT02157714.

The role of amyloid-beta derived diffusible ligands (ADDLs) in Alzheimer's disease.

The amyloid-beta (Abeta) cascade hypothesis of Alzheimer's disease (AD) has dominated research and subsequent therapeutic drug development for over two decades. Central to this hypothesis is the observation that Abeta is elevated in AD patients and that the disease is ultimately characterized by the central deposition of insoluble senile plaques. More recent evidence, however, suggests that the presence or absence of plaque is insufficient to fully account for the deleterious role of elevated Abeta in AD. Such studies support the basis for an alternate interpretation of the Abeta cascade hypothesis. Namely, that soluble oligomers of Abeta (i.e., ADDLs) accumulate and cause functional deficits prior to overt neuronal cell death or plaque deposition. Accordingly, the following review focuses on research describing the preparation and functional activity of ADDLs in vitro and in vivo. These studies provide the basis for an alternate, ADDL-based, view of the Abeta cascade hypothesis and accounts for the disconnect between plaque burden and cognitive deficits. Possible therapeutic approaches aimed at lowering ADDLs in AD patients are also considered.

Progress towards validating the NMDA receptor hypofunction hypothesis of schizophrenia.

This article describes recent progress towards validation of the N-methyl-D-aspartate (NMDA) receptor hypofunction hypothesis of schizophrenia in preclinical models. Schizophrenia, a complex disease characterized by positive, negative and cognitive symptoms, affects 1% of the world population and requires lifelong, daily maintenance therapy. For the last several decades, thinking in this field has been dominated by the hypothesis that hyperfunction of dopamine pathways played a key role in schizophrenia. However, the therapeutic agents developed from this hypothesis have a slow onset of action and tend to improve only the positive symptoms of the disease. The NMDA receptor antagonist PCP has been shown to induce the positive, negative and cognitive symptoms of schizophrenia in healthy patients and cause a resurgence of symptoms in stable patients. These observations led to the NMDA receptor hypofunction hypothesis as an alternative theory for the underlying cause of schizophrenia. According to this hypothesis, any agent that can potentiate NMDA receptor currents has the potential to ameliorate the symptoms of schizophrenia. To date, NMDA receptor currents can be modulated by either direct action on modulatory sites on the NMDA receptor (i.e., the glycine co-agonist binding site) or indirectly by activation of G-protein coupled receptors (GPCRs) known to potentiate NMDA receptor function (i.e., mGluR5). This review will discuss the NMDA receptor hypofunction hypothesis, the NMDA receptor as an emerging target for the development of novel antipsychotic agents and progress towards in vivo target validation with GlyT1 inhibitors and mGluR5 positive allosteric modulators. Other potential targets for modulating NMDA receptor currents (polyamine sites, muscarinic receptors, etc...) will also be addressed briefly.

Amphetamine-induced locomotor activity is reduced in mice following MPTP treatment but not following selegiline/MPTP treatment.

MPTP treatment has been used in mice to cause dopaminergic neuronal cell loss and subsequent behavioral abnormalities. As such, this animal model is often used as a method for the characterization of putative novel therapeutics for disease states characterized by dopamine loss, such as Parkinson's disease. Previous reports of behavioral abnormalities in mice following MPTP intoxication, however, have been conflicting. For example, open field spontaneous activity has been reported to increase, decrease or not change in MPTP treated mice. Accordingly, a more robust and direct functional measure of MPTP-induced central dopamine depletion is needed. In the present manuscript, we report on the characterization of amphetamine-induced locomotor activity as a sensitive functional endpoint for dopamine loss following MPTP treatment. We found that the amphetamine-induced locomotor activity of C57BL/6 mice was reduced in a dose-dependent manner following treatment with MPTP. This reduction of activity was associated with decreases in central dopamine levels. Further, the potential for use of this endpoint to evaluate putative therapeutics is exemplified by the amelioration of these effects following pre-treatment with the MAO-B inhibitor selegiline.

2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †.

Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency-approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T-positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

First-in-Human Phase I/II Study of NEOD001 in Patients With Light Chain Amyloidosis and Persistent Organ Dysfunction.

PURPOSE: Light chain (AL) amyloidosis is caused by the accumulation of misfolded proteins, which induces the dysfunction of vital organs. NEOD001 is a monoclonal antibody targeting these misfolded proteins. We report interim data from a phase I/II dose-escalation/expansion study of NEOD001 in patients with AL amyloidosis and persistent organ dysfunction (NCT01707264). PATIENTS AND METHODS: Patients who had completed at least one previous anti-plasma cell-directed therapy, had partial hematologic response or better, and had persistent organ dysfunction received NEOD001 intravenously every 28 days. Dose levels of 0.5, 1, 2, 4, 8, 16, and 24 mg/kg were evaluated (3 + 3 study design). Primary objectives were to determine the maximum tolerated dose and the recommended dose for future studies and to evaluate safety/tolerability. Secondary and exploratory objectives included pharmacokinetics, immunogenicity, and organ responses on the basis of published consensus criteria. RESULTS: Twenty-seven patients were enrolled in seven cohorts (dose-escalation component). No drug-related serious adverse events (AEs), discontinuations because of drug-related AEs, dose-limiting toxicities, or antidrug antibodies were reported. The most frequent AEs were fatigue, upper respiratory tract infection, cough, and dyspnea. Recommended dosing was 24 mg/kg. Pharmacokinetics support intravenous dosing every 28 days. Of 14 cardiac-evaluable patients, eight (57%) met the criteria for cardiac response and six (43%) had stable disease. Of 15 renal-evaluable patients, nine (60%) met the criteria for renal response and six (40%) had stable disease. CONCLUSION: Monthly infusions of NEOD001 were safe and well tolerated. Recommended future dosing was 24 mg/kg. Organ response rates compared favorably with those reported previously for chemotherapy. A phase II expansion is ongoing. A global phase III study (NCT02312206) has been initiated. Antibody therapy targeting misfolded proteins is a potential new therapy for the management of AL amyloidosis.

Novel conformation-specific monoclonal antibodies against amyloidogenic forms of transthyretin.

INTRODUCTION: Transthyretin amyloidosis (ATTR amyloidosis) is caused by the misfolding and deposition of the transthyretin (TTR) protein and results in progressive multi-organ dysfunction. TTR epitopes exposed by dissociation and misfolding are targets for immunotherapeutic antibodies. We developed and characterized antibodies that selectively bound to misfolded, non-native conformations of TTR. METHODS: Antibody clones were generated by immunizing mice with an antigenic peptide comprising a cryptotope within the TTR sequence and screened for specific binding to non-native TTR conformations, suppression of in vitro TTR fibrillogenesis, promotion of antibody-dependent phagocytic uptake of mis-folded TTR and specific immunolabeling of ATTR amyloidosis patient-derived tissue. RESULTS: Four identified monoclonal antibodies were characterized. These antibodies selectively bound the target epitope on monomeric and non-native misfolded forms of TTR and strongly suppressed TTR fibril formation in vitro. These antibodies bound fluorescently tagged aggregated TTR, targeting it for phagocytic uptake by macrophage THP-1 cells, and amyloid-positive TTR deposits in heart tissue from patients with ATTR amyloidosis, but did not bind to other types of amyloid deposits or normal tissue. CONCLUSIONS: Conformation-specific anti-TTR antibodies selectively bind amyloidogenic but not native TTR. These novel antibodies may be therapeutically useful in preventing deposition and promoting clearance of TTR amyloid and in diagnosing TTR amyloidosis.

The glycine transporter type 1 inhibitor N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine potentiates NMDA receptor-mediated responses in vivo and produces an antipsychotic profile in rodent behavior.

Glycine acts as a necessary coagonist for glutamate at the NMDA receptor (NMDAR) complex by binding to the strychnine-insensitive glycine-B binding site on the NR1 subunit. The fact that glycine is normally found in the brain and spinal cord at concentrations that exceed those required to saturate this site has led to the speculation that glycine normally saturates NMDAR-containing synapses in vivo. However, additional lines of evidence suggest that synaptic glycine may be efficiently regulated in synaptic areas by the glycine transporter type 1 (GlyT1). The recent description of a potent and selective GlyT1 inhibitor (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine [NFPS]) provides a tool for evaluation of the hypothesis that inhibition of GlyT1 may increase synaptic glycine and thereby potentiate NMDAR function in vivo. In the present study, we found that (+)-NFPS demonstrated >10-fold greater activity in an in vitro functional glycine reuptake assay relative to the racemic compound. In vivo, (+/-)-NFPS significantly enhanced long-term potentiation in the hippocampal dentate gyrus induced by high-frequency electrical stimulation of the afferent perforant pathway. Furthermore, (+)-NFPS induced a pattern of c-Fos immunoreactivity comparable with the atypical antipsychotic clozapine and enhanced prepulse inhibition of the acoustic startle response in DBA/2J mice, a strain with low basal levels of prepulse inhibition. Collectively, these data suggest that selective inhibition of GlyT1 can enhance NMDAR-sensitive activity in vivo and also support the idea that GlyT1 may represent a novel target for developing therapeutics to treat disorders associated with NMDAR hypofunction.

Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse.

The globus pallidus (GP) is a key GABAergic nucleus in the basal ganglia (BG). The predominant input to the GP is an inhibitory striatal projection that forms the first synapse in the indirect pathway. The GP GABAergic neurons project to the subthalamic nucleus, providing an inhibitory control of these glutamatergic cells. Given its place within the BG circuit, it is not surprising that alterations in GP firing pattern are postulated to play a role in both normal and pathological motor behavior. Because the inhibitory striatal input to the GP may play an important role in shaping these firing patterns, we set out to determine the role that the group III metabotropic glutamate receptors (GluRs) play in modulating transmission at the striatopallidal synapse. In rat midbrain slices, electrical stimulation of the striatum evoked GABA(A)-mediated IPSCs recorded in all three types of GP neurons. The group III mGluR-selective agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) inhibited these IPSCs through a presynaptic mechanism of action. L-AP4 exhibited high potency and a pharmacological profile consistent with mediation by mGluR4. Furthermore, the effect of L-AP4 on striatopallidal transmission was absent in mGluR4 knock-out mice, providing convincing evidence that mGluR4 mediates this effect. The finding that mGluR4 may selectively modulate striatopallidal transmission raises the interesting possibility that activation of mGluR4 could decrease the excessive inhibition of the GP that has been postulated to occur in Parkinson's disease. Consistent with this, we find that intracerebroventricular injections of L-AP4 produce therapeutic benefit in both acute and chronic rodent models of Parkinson's disease.

Discovery of positive allosteric modulators for the metabotropic glutamate receptor subtype 5 from a series of N-(1,3-diphenyl-1H- pyrazol-5-yl)benzamides that potentiate receptor function in vivo.

This report describes the discovery of the first centrally active allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGluR5). Appropriately substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides (e.g., 8) have been identified as a novel class of potent positive allosteric modulators of mGluR5 that potentiate the response to glutamate. An iterative analogue library synthesis approach provided potentiators with excellent potency and selectivity for mGluR5 (vs mGluRs 1-4, 7, 8). Compound 8q demonstrated in vivo proof of concept in an animal behavior model where known antipsychotics are active, supporting the development of new antipsychotics based on the NMDA hypofunction model for schizophrenia.