A phase 2 randomized, double-blind study of AMG 108, a fully human monoclonal antibody to IL-1R, in patients with rheumatoid arthritis

Introduction

Preclinical work has suggested that IL-1 plays a critical role in the pathogenesis of rheumatoid arthritis (RA). The objective of the present study was to determine the effect of a long-acting IL-1 receptor inhibitor, AMG 108, in a double-blind, placebo-controlled, parallel-dosing study in patients with active RA who were receiving stable methotrexate (15 to 25 mg/week).

Methods

Patients were randomized equally to receive placebo or 50, 125, or 250 mg AMG 108 subcutaneously every 4 weeks for 6 months. The primary efficacy endpoint was a 20% improvement in the American College of Rheumatology response (ACR20) at week 24; other efficacy endpoints included the ACR50, the ACR70, and the RA disease activity score (28-joint count Disease Activity Score) responses, patient-reported outcomes, and pharmacokinetic parameters. Safety endpoints included treatment-emergent adverse events (AEs), infectious AEs, serious AEs, serious infections, injection site reactions, laboratory abnormalities, and antibodies to AMG 108.

Results

Of 813 patients enrolled in the study, 204 patients were randomized to the 50 mg group, 203 to the 125 mg group, 203 to the 250 mg group, and 203 to placebo. At week 24, 40.4% of the 250 mg group, 36% of the 125 mg group, 30.9% of the 50 mg group, and 29.1% of the placebo group achieved an ACR20 (P = 0.022, 250 mg vs. placebo). Of the individual ACR components, numerical dose-dependent improvements were only seen in tender joint counts, pain (visual analog scale), and the acute phase reactants, erythrocyte sedimentation rate and C-reactive protein. No dose-related increase was observed in the incidence of treatment-emergent AEs. No deaths were reported, and the incidence of AEs and infections, serious AEs and infections, and withdrawals from study for safety were similar in the AMG 108 and placebo groups.

Conclusions

This large double-blind randomized trial with a long-acting IL-1 receptor blocker, AMG 108, is consistent with the experience of other IL-1 blockers, represents a definitive experiment showing that IL-1 inhibition provides only moderate symptomatic amelioration of arthritis activity in the majority of RA patients, and provides an answer to a question that has been discussed for many years in the rheumatologic community.

Trial Registration

ClinicalTrials.gov NCT00293826     

Identification of a patient with intellectual disability and de novo 3.7 Mb deletion supports the existence of a novel microdeletion syndrome in 2p14–p15

Microdeletions spanning 2p14–p15 have recently been described in two patients with developmental and speech delay and intellectual disability but no congenital malformations or severe facial dysmorphism. We report a 4-year-old boy with a de novo 3.7 Mb long deletion encompassing the region deleted in the previous cases. The patient had clinical features partly consistent with the published cases including intellectual disability, absent speech, microcephaly, long face, bulbous nasal tip and thin upper lip, but his overall clinical picture was more severe compared to the published patients. The identification of this additional patient and a detailed analysis of deletions identified in various patient cohorts and in normal individuals support the existence of a new rare microdeletion syndrome in 2p14–p15. Its critical region is in the vicinity of but clearly separate from the minimal region deleted in the well established 2p15–p16.1 microdeletion syndrome. A thorough comparison of the deletions and phenotypes indicates that multiple genes located in this region may be involved in intellectual functioning, and that some patients may show composite and more complex phenotypes due to deletions spanning both critical regions.     

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.     

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus

Bestrophin-1 modulates currents through voltage-dependent L-type Ca(2+) channels by physically interacting with the β-subunits of Ca(2+) channels. The main function of β-subunits is to regulate the number of pore-forming Ca(V)-subunits in the cell membrane and modulate Ca(2+) channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca(2+) channel subunits, together with modulation of Ca(V)1.3 Ca(2+) channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and Ca(V)1.3 subunits partly lost membrane localization. Currents from Ca(V)1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca(2+) channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming Ca(V) Ca(2+)-channel subunits.     

Photoprotective effects of glucomannan isolated from Candida utilis

Glucomannans belong to yeast and fungal cell wall polysaccharides with known immunostimulatory and radioprotective effects. However, glucomannan protective effects against pathological consequences of skin exposure to short wavelength solar light, ultraviolet (UV) radiation, are unclear. Herein, a highly branched glucomannan (GM) isolated from the cell wall of Candida utilis, a member of the alpha-(1-->6)-D-mannan group, was tested for its photoprotective effects in an in vitro model of UVB-irradiated human keratinocytes and an in vivo model of UV-induced erythema formation in human volunteers. GM suppressed the UVB-induced decrease of keratinocyte viability, which was connected with the suppression of UVB-induced keratinocyte apoptosis. GM reduced UVB-mediated caspase activation together with suppression of DNA fragment release into the cytoplasm. Furthermore, GM suppressed UVB-induced gene expression of pro-inflammatory markers including nuclear factor kappa B, inducible nitric oxide synthase, interleukins 8 and 1, together with suppression of prostaglandin E2 and interleukin 1alpha protein release. In vivo, GM decreased UV-induced skin erythema formation, which was correlated with a decrease of phosholipase A(2) activity within the stratum corneum. It could be concluded that GM isolated from C. utilis possesses significant photoprotective effects on human keratinocytes in vitro as well as in vivo.     

Studies on the kinetic stabilities of the Gd(3+) complexes formed with the N-mono(methylamide), N'-mono(methylamide) and N,N"-bis(methylamide) derivatives of diethylenetriamine-N,N,N',N",N"-pentaacetic acid

High kinetic stability is an important requirement for the Gd(3+) complexes used as contrast enhancement agents in magnetic resonance imaging. The kinetic stabilities of the Gd(3+) complexes formed with DTPA-N-mono(methylamide) (L(3)), DTPA-N'-mono(methylamide) (L(2)) and DTPA-bis(methylamide) (L(1)) are characterized by the rates of the exchange reactions with Eu(3+) and the endogenous Cu(2+) and Zn(2+). The exchange reactions occur via the proton-assisted dissociation of the complexes and direct attack of the exchanging metal ions on the complex. On the basis of the line-shape analysis of the 1H NMR spectra of the LaL(2), obtained in the pH range 2.5-3.5, we assume that for the proton-assisted dissociation of the complexes the formation of an intermediate containing a free iminodiacetate group must be followed with the rupture of the metal-central nitrogen bond. At about pH > or = 5, the reactions between GdL(2) or GdL(3) and Cu(2+) or Zn(2+) proceed predominantly by direct reaction of the reactants, through the formation of dinuclear intermediates. The contribution of the proton-assisted dissociation is highly important for GdL(1), but its reaction with Zn(2+) is significantly slower than the reactions of GdL(2) and GdL(3). The overall rates of dissociation of GdL(1), GdL(2), GdL(3) and Gd(DTPA)(2-) through H(+) (pH 7.4), Cu(2+) (1 x 10(-6) M) and Zn(2+) (1 x 10(-5) M)-assisted reactions are surprisingly very similar. Replacement of one or two carboxylates with amide groups results in significantly decreased stability constants, but has practically no effect on the kinetic stability of the Gd(3+) complexes, indicating the lower reactivity of the amide groups with Cu(2+) and Zn(2+).     

Kinetic studies on elemental sulfur oxidation by Acidithiobacillus ferrooxidans: sulfur limitation and activity of free and adsorbed bacteria

The kinetics of sulfur oxidation by Acidithiobacillus ferrooxidans in shaking flasks and a 10-L reactor was studied. The observed linearity of growth and sulfur oxidation was explained by sulfur limitation. Total cell yield was not significantly different for exponential growth as compared to growth during the sulfur-limiting phase. Kinetic studies of sulfur oxidation by growing and nongrowing bacteria indicated that both free and adsorbed bacteria oxidize sulfur. Changes in the number of free bacteria rather than cells adsorbed on sulfur were better predictors of the kinetics of sulfur oxidation, indicating that the free bacteria were performing sulfur oxidation. The active growth phase always followed adsorption of bacteria on sulfur; however, the special metabolic role of adsorbed bacteria was unclear. Their activity in sulfur solubilization was considered.     

Identification and characterization of phosphorylated proteins in the human pituitary

Post-translational modifications of proteins from the human pituitary gland play an important role in the regulation of different body functions. We report on the application of a liquid chromatography-tandem mass spectrometry (MS/MS) based approach to detect and characterize phosphorylated proteins in a whole human pituitary digest. By combining an immobilized metal affinity column-based enrichment method with MS/MS conditions that favor the neutral loss of phosphoric acid from a phosphorylated precursor ion, we identified several previously undescribed phosphorylated peptides. The identified peptides were matched to the sequences of six pituitary proteins: the human growth hormone, chromogranin A, secretogranin I, 60S ribosomal protein P1 and/or P2, DnaJ homolog subfamily C member 5, and galanin. The phosphorylation sites of these important regulatory proteins were determined by MS/MS and MS(3) analysis.     

Basic residues in the Mason-Pfizer monkey virus gag matrix domain regulate intracellular trafficking and capsid-membrane interactions

Mason-Pfizer monkey virus (M-PMV) capsids that have assembled in the cytoplasm must be transported to and associate with the plasma membrane prior to being enveloped by a lipid bilayer during viral release. Structural studies have identified a positive-charge density on the membrane-proximal surface of the matrix (MA) protein component of the Gag polyprotein. To investigate if basic amino acids in MA play a role in intracellular transport and capsid-membrane interactions, mutants were constructed in which lysine and arginine residues (R10, K16, K20, R22, K25, K27, K33, and K39) potentially exposed on the capsid surface were replaced singly and in pairs by alanine. A majority of the charge substitution mutants were released less efficiently than the wild type. Electron microscopy of mutant Gag-expressing cells revealed four distinct phenotypes: K16A and K20A immature capsids accumulated on and budded into intracellular vesicles; R10A, K27A, and R22A capsid transport was arrested at the cellular cortical actin network, while K25A immature capsids were dispersed throughout the cytoplasm and appeared to be defective at an earlier stage of intracellular transport; and the remaining mutant (K33A and K39A) capsids accumulated at the inner surface of the plasma membrane. All mutants that released virions exhibited near-wild-type infectivity in a single-round assay. Thus, basic amino acids in the M-PMV MA define both cellular location and efficiency of virus release.     

Highly hydrophilic cationic gold nanorods stabilized by novel quaternary ammonium surfactant with negligible cytotoxicity

The photothermal cancer therapy using cationic gold nanorods (GNRs) stabilized by quaternary ammonium salts (QAS) have a great potential to enhance conventional cancer treatment as it promises the effective eradication of cancer cells including cells resistant to radio‐ and chemo‐therapy and the stimulation of anti‐tumor immune response. However, as the cytotoxicity of the conventional alkanethiol‐QAS compounds limits their utility in medicine, here we developed GNRs modified by novel highly hydrophilic cationic surfactant composed of the quaternary ammonium group and ethylene glycol chain N,N,N‐trimethyl‐3,6,9,12,15‐pentaoxaheptadecyl‐17‐sulfanyl‐1‐ammonium bromide (POSAB) showing insignificant cytotoxicity in the free state. Surface modification of GNRs by POSAB allowed to prepare nanoparticles with good stability in water, high cellular uptake and localization in lysosomes that are a promising alternative to alkanethiol‐stabilized GNRs especially for biomedical applications.