Phase 1 Evaluation of Elezanumab (Anti-Repulsive Guidance Molecule A Monoclonal Antibody) in Healthy and Multiple Sclerosis Participants.

OBJECTIVE: This study was undertaken to describe the safety, tolerability, pharmacokinetics, and immunogenicity of elezanumab (ABT-555), a fully human monoclonal antibody (mAb) directed against repulsive guidance molecule A (RGMa), in healthy and multiple sclerosis (MS) study participants. METHODS: The single-center, first-in-human, single ascending dose (SAD) study evaluated elezanumab (50-1,600mg intravenous [IV] and 150mg subcutaneous) in 47 healthy men and women. The multicenter multiple ascending dose (MAD; NCT02601885) study evaluated elezanumab (150mg, 600mg, and 1,800mg) in 20 adult men and women with MS, receiving either maintenance or no immunomodulatory treatment. RESULTS: No pattern of study drug-related adverse events was identified for either the SAD or MAD elezanumab regimens. Across both studies, the Tmax occurred within 4 hours of elezanumab IV infusion, and the harmonic mean of t1/2 ranged between 18.6 and 67.7 days. Following multiple dosing, elezanumab Cmax , area under the curve, and Ctrough increased dose-proportionally and resulted in dose-dependent increases in elezanumab cerebrospinal fluid (CSF) concentrations. Elezanumab CSF penetration was 0.1% to 0.4% across both studies, with CSF levels of free RGMa decreased by >40%. Changes in CSF interleukin-10 (IL-10) and free RGMa demonstrated dose/exposure-dependence. INTERPRETATION: The elezanumab pharmacokinetic profile supports monthly, or bimonthly, administration of up to 1,800mg with the option of a loading dose of 3,600mg. Elezanumab partitioning into CSF is within the range expected for mAbs. Reduced CSF levels of free RGMa demonstrate central nervous system target binding of elezanumab with an apparent maximal effect at 1,800mg IV. Exposure-associated increases in CSF IL-10, an anti-inflammatory cytokine with neuroprotective/neurorestorative properties, support potential pathway modulation in MS participants. ANN NEUROL 2023;93:285-296.

Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity.

Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.

Protein geranylgeranylation controls collagenase expression in osteoarthritic cartilage.

OBJECTIVE: Statins possess anti-inflammatory properties. This study was undertaken to characterize the mechanism of action of statin drugs on collagenase expression in primary human osteoarthritic cartilage tissue. METHOD: Human articular chondrocytes and cartilage explants from osteoarthritic donors were exposed to simvastatin in the presence or absence of interleukin-1 beta (IL-1beta). Collagenase expression was determined by quantifying levels of matrix metalloproteinase 13 (MMP-13) and MMP-1 mRNA and MMP-13 protein. The mechanism of statin action was tested by addition of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) or by using inhibitors of farnesyl transferase (FT) and geranylgeranyl transferase (GGT-1). RESULTS: Treatment of osteoarthritic chondrocytes with simvastatin decreased mRNA levels of MMP-13 and MMP-1 whether under basal conditions or during stimulation with IL-1beta. MMP-13 protein secreted into the culture media was also decreased. Genes involved in cartilage synthesis (type II collagen and aggrecan) were not down-regulated by simvastatin. Exogenous addition of GGPP completely reversed the statin-mediated decrease in MMP-13 mRNA and protein levels whereas FPP partially reversed the statin-mediated effect. An inhibitor of GGT-1 mimicked the simvastatin-mediated reduction in MMP-13 expression by chondrocytes. Finally, consistent with impacts on MMP-13 and MMP-1 expression, simvastatin as well as the GGT-1 inhibitor both blocked type II collagen degradation in primary human articular cartilage explants. CONCLUSION: These results suggest that statins modulate chondrocyte metabolism by reducing prenylation of key signaling molecules that control the expression of collagen-degrading enzymes. Our results strongly support the hypothesis that protein prenyltransferases including geranylgeranyl transferase regulate chondrocyte collagenase expression in osteoarthritis.

Plasma 3-nitrotyrosine is a biomarker in animal models of arthritis: Pharmacological dissection of iNOS' role in disease.

The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA). Quantification of nitrotyrosine was performed using immuno-affinity 2-D LC-MS/MS assay. This assay is a very specific and reproducible and is amenable to a number of biological fluids. Plasma levels of 3-NT were significantly elevated in an acute model of inflammation (rat LPS) and in models of rheumatoid arthritis (adjuvant- and collagen-induced arthritis), and osteoarthritis (monoiodoacetate-induced arthritis). Plasma 3-NT correlated with the severity of the inflammatory response; thus, a 20-fold increase was observed in the rat LPS model, a 10-fold increase in AIA, and only a 2.5-fold elevation in CIA. Pharmacological intervention with iNOS inhibitors decreased 3-NT levels and associated pathology. 3-NT determination allowed for better elucidation of the role of iNOS in RA and OA disease pathology and provided proof of pharmacology for NOS inhibitors in animal models of RA and OA.

Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids: development of a clinically translatable biomarker.

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.

Multiplexed immunoassay panel identifies novel CSF biomarkers for Alzheimer's disease diagnosis and prognosis.

BACKGROUND: Clinicopathological studies suggest that Alzheimer's disease (AD) pathology begins ∼10-15 years before the resulting cognitive impairment draws medical attention. Biomarkers that can detect AD pathology in its early stages and predict dementia onset would, therefore, be invaluable for patient care and efficient clinical trial design. We utilized a targeted proteomics approach to discover novel cerebrospinal fluid (CSF) biomarkers that can augment the diagnostic and prognostic accuracy of current leading CSF biomarkers (Aß42, tau, p-tau181). METHODS AND FINDINGS: Using a multiplexed Luminex platform, 190 analytes were measured in 333 CSF samples from cognitively normal (Clinical Dementia Rating [CDR] 0), very mildly demented (CDR 0.5), and mildly demented (CDR 1) individuals. Mean levels of 37 analytes (12 after Bonferroni correction) were found to differ between CDR 0 and CDR>0 groups. Receiver-operating characteristic curve analyses revealed that small combinations of a subset of these markers (cystatin C, VEGF, TRAIL-R3, PAI-1, PP, NT-proBNP, MMP-10, MIF, GRO-α, fibrinogen, FAS, eotaxin-3) enhanced the ability of the best-performing established CSF biomarker, the tau/Aß42 ratio, to discriminate CDR>0 from CDR 0 individuals. Multiple machine learning algorithms likewise showed that the novel biomarker panels improved the diagnostic performance of the current leading biomarkers. Importantly, most of the markers that best discriminated CDR 0 from CDR>0 individuals in the more targeted ROC analyses were also identified as top predictors in the machine learning models, reconfirming their potential as biomarkers for early-stage AD. Cox proportional hazards models demonstrated that an optimal panel of markers for predicting risk of developing cognitive impairment (CDR 0 to CDR>0 conversion) consisted of calbindin, Aß42, and age. CONCLUSIONS/SIGNIFICANCE: Using a targeted proteomic screen, we identified novel candidate biomarkers that complement the best current CSF biomarkers for distinguishing very mildly/mildly demented from cognitively normal individuals. Additionally, we identified a novel biomarker (calbindin) with significant prognostic potential.

Optical tomographic imaging discriminates between disease-modifying anti-rheumatic drug (DMARD) and non-DMARD efficacy in collagen antibody-induced arthritis.

INTRODUCTION: Standard measurements used to assess murine models of rheumatoid arthritis, notably paw thickness and clinical score, do not align well with certain aspects of disease severity as assessed by histopathology. We tested the hypothesis that non-invasive optical tomographic imaging of molecular biomarkers of inflammation and bone turnover would provide a superior quantitative readout and would discriminate between a disease-modifying anti-rheumatic drug (DMARD) and a non-DMARD treatment. METHODS: Using two protease-activated near-infrared fluorescence imaging agents to detect inflammation-associated cathepsin and matrix metalloprotease activity, and a third agent to detect bone turnover, we quantified fluorescence in paws of mice with collagen antibody-induced arthritis. Fluorescence molecular tomographic (FMT) imaging results, which provided deep tissue detection and quantitative readouts in absolute picomoles of agent fluorescence per paw, were compared with paw swelling, clinical scores, a panel of plasma biomarkers, and histopathology to discriminate between steroid (prednisolone), DMARD (p38 mitogen-activated protein kinase (MAPK) inhibitor) and non-DMARD (celecoxib, cyclooxygenase-2 (COX-2) inhibitor) treatments. RESULTS: Paw thickness, clinical score, and plasma biomarkers failed to discriminate well between a p38 MAPK inhibitor and a COX-2 inhibitor. In contrast, FMT quantification using near-infrared agents to detect protease activity or bone resorption yielded a clear discrimination between the different classes of therapeutics. FMT results agreed well with inflammation scores, and both imaging and histopathology provided clearer discrimination between treatments as compared with paw swelling, clinical score, and serum biomarker readouts. CONCLUSIONS: Non-invasive optical tomographic imaging offers a unique approach to monitoring disease pathogenesis and correlates with histopathology assessment of joint inflammation and bone resorption. The specific use of optical tomography allowed accurate three-dimensional imaging, quantitation in picomoles rather than intensity or relative fluorescence, and, for the first time, showed that non-invasive imaging assessment can predict the pathologist's histology inflammation scoring and discriminate DMARD from non-DMARD activity.