Simultaneous Inhibition of Angiopoietin-2 and Vascular Endothelial Growth Factor-A with Faricimab in Diabetic Macular Edema: BOULEVARD Phase 2 Randomized Trial.

PURPOSE: The phase 2 BOULEVARD trial compared safety and efficacy of faricimab, a novel bispecific antibody targeting angiopoietin-2 and vascular endothelial growth factor-A (VEGF-A), with ranibizumab in patients with diabetic macular edema (DME). DESIGN: The BOULEVARD trial (ClinicalTrials.gov identifier, NCT02699450) was a prospective, randomized, active comparator-controlled, double-masked, multicenter, phase 2 study conducted at 59 sites in the United States. PARTICIPANTS: The trial enrolled patients 18 years of age or older with center-involving DME, best-corrected visual acuity (BCVA) of 73 to 24 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, and central subfield thickness (CST) of 325 µm or more. METHODS: Anti-VEGF treatment-naïve patients were randomized 1:1:1 to intravitreal 6.0 mg faricimab, 1.5 mg faricimab, or 0.3 mg ranibizumab, and patients previously treated with anti-VEGF were randomized 1:1 to 6.0 mg faricimab or 0.3 mg ranibizumab. Patients were dosed monthly for 20 weeks, followed by an observation period up to week 36 to assess durability. MAIN OUTCOME MEASURES: The prespecified primary outcome measure was mean change in BCVA from baseline at week 24 for faricimab versus ranibizumab in treatment-naïve patients. Key secondary and exploratory outcome measures included CST, Diabetic Retinopathy Severity Scale (DRSS) score, and durability as assessed by time to re-treatment. RESULTS: The trial enrolled 229 patients (168 treatment-naïve and 61 previously treated with anti-VEGF). In treatment-naïve patients, 6.0 mg faricimab, 1.5 mg faricimab, and 0.3 mg ranibizumab resulted in mean improvements of 13.9, 11.7, and 10.3 ETDRS letters from baseline, respectively. The 6.0-mg faricimab dose demonstrated a statistically significant gain of 3.6 letters over ranibizumab (P = 0.03). In both patient populations, faricimab resulted in dose-dependent reductions in CST, improvements in DRSS score, and longer time to re-treatment during the observation period compared with ranibizumab. Faricimab showed no new or unexpected safety signals. CONCLUSIONS: The BOULEVARD trial met its primary end point; faricimab demonstrated statistically superior visual acuity gains versus ranibizumab at week 24 in treatment-naïve patients. Central subfield thickness reduction, DRSS score improvement, and extended durability outcomes support the primary outcome. These findings suggest the benefit of simultaneous inhibition of angiopoietin-2 and VEGF-A with faricimab for patients with DME.

A novel tumor necrosis factor-mediated mechanism of direct epithelial sodium channel activation.

RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

The effect of cholesteryl ester transfer protein inhibition on lipids, lipoproteins, and markers of HDL function after an acute coronary syndrome: the dal-ACUTE randomized trial.

AIMS: The effects of cholesteryl ester transfer protein (CETP) inhibition on lipids, inflammation, and markers of high-density lipoprotein (HDL) function, following an acute coronary syndrome (ACS), are unknown. METHODS AND RESULTS: The dal-ACUTE study randomized 300 patients (1 : 1) to dalcetrapib 600 mg/day or placebo within 1 week of an ACS. The primary endpoint was per cent change in HDL-cholesterol (HDL-C) after 4 weeks. Secondary endpoints included apolipoprotein levels, markers of HDL function, and inflammation. Dalcetrapib treatment increased HDL-C and apolipoprotein A1 by 33.7 and 11.8%, respectively (both P < 0.001) and total cholesterol efflux by 9.5% (P = 0.003) after 4 weeks, principally via an increase in non-ATP-binding cassette transporter (ABC) A1-mediated efflux, without statistically significant changes in pre-ß1-HDL levels. The increase in total efflux with dalcetrapib correlated most strongly with increases in apolipoprotein A1 and HDL-C (r = 0.46 and 0.43, respectively) rather than the increase in pre-ß1-HDL (r = 0.32). Baseline and on-treatment ABCA1-mediated efflux correlated most strongly with pre-ß1-HDL levels; in contrast, non-ABCA1-mediated efflux correlated better with apolipoprotein A1 and HDL-C levels. CONCLUSIONS: High-density lipoprotein raised through CETP inhibition with dalcetrapib improves cholesterol efflux, principally via a non-ABCA1-mediated pathway. While HDL-C was increased by one-third, apolipoprotein A1 and total efflux were increased only by one-tenth, supporting the concept of dissociation between improvements in HDL function and HDL-C levels, which may be of relevance to ongoing trials and the development of therapeutic interventions targeting HDL.

Sembragiline in Moderate Alzheimer's Disease: Results of a Randomized, Double-Blind, Placebo-Controlled Phase II Trial (MAyflOwer RoAD).

BACKGROUND: Sembragiline is a potent, selective, long-acting, and reversible MAO-B inhibitor developed as a potential treatment for Alzheimer's disease (AD). OBJECTIVE: To evaluate the safety, tolerability, and efficacy of sembragiline in patients with moderate AD. METHODS: In this Phase II study (NCT01677754), 542 patients with moderate dementia (MMSE 13-20) on background acetylcholinesterase inhibitors with/without memantine were randomized (1:1:1) to sembragiline 1 mg, 5 mg, or placebo once daily orally for 52 weeks. RESULTS: No differences between treated groups and placebo in adverse events or in study completion. The primary endpoint, change from baseline in ADAS-Cog11, was not met. At Week 52, the difference between sembragiline and placebo in ADAS-Cog11 change from baseline was - 0.15 (p = 0.865) and 0.90 (p = 0.312) for 1 and 5 mg groups, respectively. Relative to placebo at Week 52 (but not at prior assessment times), the 1 mg and 5 mg sembragiline groups showed differences in ADCS-ADL of 2.64 (p = 0.051) and 1.89 (p = 0.160), respectively. A treatment effect in neuropsychiatric symptoms (as assessed by the difference between sembragiline and placebo on BEHAVE-AD-FW) was also seen at Week 52 only: - 2.80 (p = 0.014; 1 mg) and - 2.64 (p = 0.019; 5 mg), respectively. A post hoc subgroup analysis revealed greater treatment effects on behavior and functioning in patients with more severe baseline behavioral symptoms (above the median). CONCLUSIONS: This study showed that sembragiline was well-tolerated in patients with moderate AD. The study missed its primary and secondary endpoints. Post hoc analyses suggested potential effect on neuropsychiatric symptoms and functioning in more behaviorally impaired study population at baseline.

Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity.

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

Enhancement of the NMDA receptor function by reduction of glycine transporter-1 expression.

The occupation of the glycine binding-site is a prerequisite for NMDA receptor activation by glutamate. To analyze the regulation of NMDA receptor function by the glycine transporter 1 (GlyT1), we generated heterozygous constitutive GlyT1 knockout mice (GlyT1tm1.1(+/-)). These animals were fully viable. Using a newly generated antibody, the pattern of GlyT1 expression in brain was found to be unaltered in the mutants while the level of expression was strongly reduced in all brain regions, as shown immunohistochemically. In hippocampal slices the ratio of the peak amplitude of NMDA and AMPA receptor evoked excitatory postsynaptic currents (EPSCs), recorded in CA1 pyramidal cells, was significantly enhanced by 36% in Glyt1tm1.1(+/-) compared to wild-type slices. The frequency and amplitude of AMPA miniature events in Glyt1tm1.1(+/-) mice were indistinguishable from those recorded in wild type. These results provide proof that the NMDA receptor function is enhanced by a reduction of GlyT1 expression. Thus, GlyT1 function is a controlling factor for an enhancement of the NMDA receptor response. These findings are of relevance for the development of GlyT1 inhibitory drugs.

Glycine transporter 1 as a potential therapeutic target for schizophrenia-related symptoms: evidence from genetically modified mouse models and pharmacological inhibition.

Schizophrenia is characterized by positive symptoms such as hallucinations, negative symptoms such as blunted affect, and symptoms of cognitive deficiency such as deficits in working memory and selective attention. N-methyl-d-aspartate receptor (NMDAR) hypofunction has been implicated in all three pathophysiological aspects of the disease. Due to the severe side effects of direct NMDAR agonists, targeting the modulatory co-agonist glycine-B site of the NMDAR is considered to be a promising strategy to ameliorate NMDAR hypofunction. To assess the antipsychotic and pro-cognitive potential of this approach, we examine the strategies designed to enhance glycine-B site occupancy through glycine transporter 1 (GlyT1) blockade. Among the existing transgenic mouse models with GlyT1 deficits, the one specifically targeting forebrain neuronal GlyT1 has yielded the most promising data on cognitive enhancement. Parallel advances in the pharmacology of GlyT1 inhibition point not only to an enhancement of attention, learning and memory but also include suggestions of mood enhancing effects that might be valuable for treating negative symptoms. Thus, interventions at GlyT1 are highly effective in modifying multiple brain functions, and dissection of their respective mechanisms is expected to further maximize their therapeutic potential for human mental diseases.

Pharmacological characterization of senktide-induced tail whips.

The tachykinin NK(3) receptor shows promise as a novel target for antipsychotics, but knowledge of downstream activity following tachykinin NK(3) receptor activation is lacking. To determine the practical utility of senktide-induced tail whips in mice as a tool for determining and characterizing downstream activity following tachykinin NK(3) receptor activation, mice were injected with 0.05 nmol of senktide i.c.v. and the number of tail whip bouts was counted for 20 min. Strain differences were observed, with NMRI mice showing a stronger tail whip response than C57Bl/6J mice. Tachykinin NK(3) receptor specificity was confirmed by the absence of the senktide-induced tail whip response in tachykinin NK(3) receptor knockout mice. Effects of tachykinin receptor pharmacological agents were tested by pretreatment with tachykinin NK(3) receptor antagonists (SB222200, talnetant and osanetant), which attenuated senktide-induced tail whips, and the tachykinin NK(1) receptor antagonist MK869, which had no effect on senktide-induced tail whips. Pharmacological interactions with other neurotransmitter systems were determined by pretreatment with dopamine D(1), D(2), and D(3) receptor antagonists, atypical antipsychotics, serotonin 5HT(1a) receptor antagonists, serotonin 5HT(2a/c) receptor antagonists, benzodiazepine and putative anxiolytics, antidepressants, and an anticholinergic. Senktide-induced tail whips were attenuated by dopamine D(2) receptor antagonists, atypical antipsychotics, serotonin 5HT(2a/c) antagonists, and benzodiazepine anxiolytics, but unaffected by drugs from other classes. Thus, the senktide-induced tail whip response is easily quantifiable, specific to the tachykinin NK(3) receptor, and provides valuable information on the downstream pharmacology of tachykinin NK(3) receptor activation.

Characterization of behavioral response to amphetamine, tyrosine hydroxylase levels, and dopamine receptor levels in neurokinin 3 receptor knockout mice.

The neurokinin 3 (NK3) receptor is a novel target under investigation for improvement of symptoms of schizophrenia, because of its ability to modulate dopaminergic signaling. To further understanding of the function of this receptor, sensitivity to dopaminergic stimuli and levels of dopaminergic receptors and tyrosine hydroxylase in NK3 receptor knockout mice were studied. Knockout of the receptor was confirmed by lack of NK3 protein and lack of electrophysiological responsivity of presumed dopaminergic neurons to senktide. NK3 receptor knockout mice showed mild hyperlocomotion and deficits on the rotarod. NK3 receptor knockout mice did not show significant differences in sensitivity to locomotor effects of acute amphetamine (0.3, 1, and 3 mg/kg subcutaneously) or significant alterations in sensitization to locomotor effects of amphetamine, but did show nonsignificant hyperreactivity to 1 mg/kg amphetamine and a nonsignificantly increased propensity to develop sensitization. A small decrease in D1 receptor binding was seen in the dorsal striatum and olfactory tubercle, and a small decrease of in tyrosine hydroxylase in the olfactory tubercle, but no change was seen in D2 receptor binding. Together, these results support a role for the NK3 receptor in reactivity to dopaminergic stimuli, but the lack of robust changes indicates that the sensitivity to dopamine may be activity-dependent or benign in nature.