Neil Stahl

Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration.

OBJECTIVE Two similarly designed, phase-3 studies (VEGF Trap-Eye: Investigation of Efficacy and Safety in Wet AMD [VIEW 1, VIEW 2]) of neovascular age-related macular degeneration (AMD) compared monthly and every-2-month dosing of intravitreal aflibercept injection (VEGF Trap-Eye; Regeneron, Tarrytown, NY, and Bayer HealthCare, Berlin, Germany) with monthly ranibizumab. DESIGN Double-masked, multicenter, parallel-group, active-controlled, randomized trials. PARTICIPANTS Patients (n = 2419) with active, subfoveal, choroidal neovascularization (CNV) lesions (or juxtafoveal lesions with leakage affecting the fovea) secondary to AMD. INTERVENTION Patients were randomized to intravitreal aflibercept 0.5 mg monthly (0.5q4), 2 mg monthly (2q4), 2 mg every 2 months after 3 initial monthly doses (2q8), or ranibizumab 0.5 mg monthly (Rq4). MAIN OUTCOME MEASURES The primary end point was noninferiority (margin of 10%) of the aflibercept regimens to ranibizumab in the proportion of patients maintaining vision at week 52 (losing <15 letters on Early Treatment Diabetic Retinopathy Study [ETDRS] chart). Other key end points included change in best-corrected visual acuity (BCVA) and anatomic measures. RESULTS All aflibercept groups were noninferior and clinically equivalent to monthly ranibizumab for the primary end point (the 2q4, 0.5q4, and 2q8 regimens were 95.1%, 95.9%, and 95.1%, respectively, for VIEW 1, and 95.6%, 96.3%, and 95.6%, respectively, for VIEW 2, whereas monthly ranibizumab was 94.4% in both studies). In a prespecified integrated analysis of the 2 studies, all aflibercept regimens were within 0.5 letters of the reference ranibizumab for mean change in BCVA; all aflibercept regimens also produced similar improvements in anatomic measures. Ocular and systemic adverse events were similar across treatment groups. CONCLUSIONS Intravitreal aflibercept dosed monthly or every 2 months after 3 initial monthly doses produced similar efficacy and safety outcomes as monthly ranibizumab. These studies demonstrate that aflibercept is an effective treatment for AMD, with the every-2-month regimen offering the potential to reduce the risk from monthly intravitreal injections and the burden of monthly monitoring. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Scrapie prion protein contains a phosphatidylinositol glycolipid

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.

Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.

Many members of the cytokine receptor superfamily initiate intracellular signaling by activating members of the Jak family of tyrosine kinases. Activation of the same Jaks by multiple cytokines raises the question of how these cytokines activate distinct intracellular signaling pathways. Selection of particular substrates--the transcriptional activator Stat3 and protein tyrosine phosphatase PTP1D--that characterize responses to the ciliary neurotrophic factor-interleukin-6 cytokine family depended not on which Jak was activated, but was instead determined by specific tyrosine-based motifs in the receptor components--gp130 and LIFR--shared by these cytokines. Further, these tyrosine-based motifs were modular, because addition of a Stat3-specifying motif to another cytokine receptor, that for erythropoietin, caused it to activate Stat3 in a ligand-dependent fashion.

Dupilumab in Persistent Asthma with Elevated Eosinophil Levels

BACKGROUND Moderate-to-severe asthma remains poorly treated. We evaluated the efficacy and safety of dupilumab (SAR231893/REGN668), a fully human monoclonal antibody to the alpha subunit of the interleukin-4 receptor, in patients with persistent, moderate-to-severe asthma and elevated eosinophil levels. METHODS We enrolled patients with persistent, moderate-to-severe asthma and a blood eosinophil count of at least 300 cells per microliter or a sputum eosinophil level of at least 3% who used medium-dose to high-dose inhaled glucocorticoids plus long-acting beta-agonists (LABAs). We administered dupilumab (300 mg) or placebo subcutaneously once weekly. Patients were instructed to discontinue LABAs at week 4 and to taper and discontinue inhaled glucocorticoids during weeks 6 through 9. Patients received the study drug for 12 weeks or until a protocol-defined asthma exacerbation occurred. The primary end point was the occurrence of an asthma exacerbation; secondary end points included a range of measures of asthma control. Effects on various type 2 helper T-cell (Th2)-associated biomarkers and safety and tolerability were also evaluated. RESULTS A total of 52 patients were assigned to the dupilumab group, and 52 patients were assigned to the placebo group. Baseline characteristics were similar in the two groups. Three patients had an asthma exacerbation with dupilumab (6%) versus 23 with placebo (44%), corresponding to an 87% reduction with dupilumab (odds ratio, 0.08; 95% confidence interval, 0.02 to 0.28; P<0.001). Significant improvements were observed for most measures of lung function and asthma control. Dupilumab reduced biomarkers associated with Th2-driven inflammation. Injection-site reactions, nasopharyngitis, nausea, and headache occurred more frequently with dupilumab than with placebo. CONCLUSIONS In patients with persistent, moderate-to-severe asthma and elevated eosinophil levels who used inhaled glucocorticoids and LABAs, dupilumab therapy, as compared with placebo, was associated with fewer asthma exacerbations when LABAs and inhaled glucocorticoids were withdrawn, with improved lung function and reduced levels of Th2-associated inflammatory markers. (Funded by Sanofi and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT01312961.).

CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130

Ciliary neurotrophic factor (CNTF) has a variety of actions within the nervous system. While some of the actions of leukemia inhibitory factor (LIF) on neurons resemble those of CNTF, LIF also has broad actions outside of the nervous system that in many cases mimic those of interleukin-6 (IL-6). Comparison of the tyrosine phosphorylations and gene activations induced by CNTF and LIF in neuron cell lines reveals that they are indistinguishable and also very similar to signaling events that characterize LIF and IL-6 responses in hematopoietic cells. We provide a basis for the overlapping actions of these three factors by demonstrating that the shared CNTF and LIF signaling pathways involve the IL-6 signal transducing receptor component gp130. Thus, the receptor system for CNTF is surprisingly unlike those used by the nerve growth factor family of neurotrophic factors, but is instead related to those used by a subclass of hematopoietic cytokines.

Effect of a monoclonal antibody to PCSK9 on LDL cholesterol.

BACKGROUND Proprotein convertase subtilisin/kexin 9 (PCSK9), one of the serine proteases, binds to low-density lipoprotein (LDL) receptors, leading to their accelerated degradation and to increased LDL cholesterol levels. We report three phase 1 studies of a monoclonal antibody to PCSK9 designated as REGN727/SAR236553 (REGN727). METHODS In healthy volunteers, we performed two randomized, single ascending-dose studies of REGN727 administered either intravenously (40 subjects) or subcutaneously (32 subjects), as compared with placebo. These studies were followed by a randomized, placebo-controlled, multiple-dose trial in adults with heterozygous familial hypercholesterolemia who were receiving atorvastatin (21 subjects) and those with nonfamilial hypercholesterolemia who were receiving treatment with atorvastatin (30 subjects) (baseline LDL cholesterol, >100 mg per deciliter [2.6 mmol per liter]) or a modified diet alone (10 subjects) (baseline LDL cholesterol, >130 mg per deciliter [3.4 mmol per liter]). REGN727 doses of 50, 100, or 150 mg were administered subcutaneously on days 1, 29, and 43. The primary outcome for all studies was the occurrence of adverse events. The principal secondary outcome was the effect of REGN727 on the lipid profile. RESULTS Among subjects receiving REGN727, there were no discontinuations because of adverse events. REGN727 significantly lowered LDL cholesterol levels in all the studies. In the multiple-dose study, REGN727 doses of 50, 100, and 150 mg reduced measured LDL cholesterol levels in the combined atorvastatin-treated populations to 77.5 mg per deciliter (2.00 mmol per liter), 61.3 mg per deciliter (1.59 mmol per liter), and 53.8 mg per deciliter (1.39 mmol per liter), for a difference in the change from baseline of -39.2, -53.7, and -61.0 percentage points, respectively, as compared with placebo (P<0.001 for all comparisons). CONCLUSIONS In three phase 1 trials, a monoclonal antibody to PCSK9 significantly reduced LDL cholesterol levels in healthy volunteers and in subjects with familial or nonfamilial hypercholesterolemia. (Funded by Regeneron Pharmaceuticals and Sanofi; ClinicalTrials.gov numbers, NCT01026597, NCT01074372, and NCT01161082.).

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.

Efficacy and safety of rilonacept (interleukin-1 trap) in patients with cryopyrin-associated periodic syndromes: Results from two sequential placebo-controlled studies†

OBJECTIVE To assess the efficacy and safety of rilonacept (Interleukin-1 [IL-1] Trap), a long-acting and potent inhibitor of IL-1, in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS). METHODS Forty-seven adult patients with CAPS, as defined by mutations in the causative NLRP3 (CIAS1) gene and pathognomonic symptoms, were enrolled in 2 consecutive phase III studies. Study 1 involved a 6-week randomized double-blind comparison of weekly subcutaneous injections of rilonacept (160 mg) versus placebo. Study 2 consisted of 9 weeks of single-blind treatment with rilonacept (part A), followed by a 9-week, randomized, double-blind, placebo-controlled withdrawal procedure (part B). Primary efficacy was evaluated using a validated composite key symptom score. RESULTS Forty-four patients completed both studies. In study 1, rilonacept therapy reduced the group mean composite symptom score by 84%, compared with 13% with placebo therapy (primary end point; P < 0.0001 versus placebo). Rilonacept also significantly improved all other efficacy end points in study 1 (numbers of multisymptom and single-symptom disease flare days, single-symptom scores, physicians and patients global assessments of disease activity, limitations in daily activities, and C-reactive protein and serum amyloid A [SAA] levels). In study 2 part B, rilonacept was superior to placebo for maintaining the improvements seen with rilonacept therapy, as shown by all efficacy parameters (primary end point; P < 0.0001 versus placebo). Rilonacept was generally well tolerated; the most common adverse events were injection site reactions. CONCLUSION Treatment with weekly rilonacept provided marked and lasting improvement in the clinical signs and symptoms of CAPS, and normalized the levels of SAA from those associated with risk of developing amyloidosis. Rilonacept exhibited a generally favorable safety and tolerability profile.

Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: antagonism by noggin.

Bone morphogenetic proteins (BMPs) have been heretofore implicated in the induction of osteoblast differentiation from uncommitted progenitors during embryonic skeletogenesis and fracture healing. We have tested the hypothesis that BMPs are also involved in the osteoblastogenesis that takes place in the bone marrow in postnatal life. To do this, we took advantage of the properties of noggin, a recently discovered protein that binds BMP‐2 and −4 and blocks their action. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibited both osteoblast and osteoclast formation; these effects were reversed by exogenous BMP‐2. Consistent with these findings, BMP‐2 and −4 and BMP‐2/4 receptor transcripts and proteins were detected in these primary cultures, in a bone marrow–derived stromal/osteoblastic cell line, as well as in murine adult whole bone; noggin expression was also documented in all these preparations. Moreover, addition of antinoggin antibody caused an increase in osteoblast progenitor formation. These findings suggest that BMP‐2 and −4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts and osteoclasts; and that, in fact, BMP‐2/4‐induced commitment to the osteoblastic lineage is a prerequisite for osteoclast development. Hence, BMPs, perhaps in balance with noggin and possibly other antagonists, may provide the tonic baseline control of the rate of bone remodeling on which other inputs (e.g., hormonal, biomechanical, etc.) operate.