CGRP blockade by galcanezumab was not associated with reductions in signs and symptoms of knee osteoarthritis in a randomized clinical trial.

OBJECTIVE: This study tested whether galcanezumab, a humanized monoclonal antibody with efficacy against migraine, was superior to placebo for the treatment of mild or moderate osteoarthritis (OA) knee pain. METHOD: In a multicenter, double-blind, placebo- and celecoxib-controlled trial, patients with moderate to severe OA pain were randomized to placebo; celecoxib 200 mg daily for 16 weeks; or galcanezumab 5, 50, 120, and 300 mg subcutaneously every 4 weeks, twice. The primary outcome was change from baseline at Week 8 in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain subscore measured by 100 mm visual analog scale (VAS). The trial was considered positive if ≥1 dose of galcanezumab demonstrated ≥95% Bayesian posterior probability of superiority to placebo and ≥50% posterior probability of superiority to placebo by ≥9 mm. A planned interim analysis allowed termination of the study if posterior probability of superiority to placebo by ≥9 mm was ≤5%. Secondary endpoints included WOMAC function subscore and Patient Global Assessment (PGA) of OA. Safety and tolerability were also assessed. RESULTS: The study was terminated after interim analysis suggested inadequate efficacy. Celecoxib significantly reduced WOMAC pain subscore compared with placebo [-12.0 mm; 95% confidence interval (CI) -23 to -2 mm]. None of the galcanezumab arms demonstrated clinically meaningful improvement (range: 1.5 to -5.0 mm) or met the prespecified success criteria. No improvement in any secondary objective was observed. Galcanezumab was well tolerated by OA patients. CONCLUSIONS: This study failed to demonstrate sufficient statistical evidence that galcanezumab was efficacious for treating OA knee pain. STUDY IDENTIFICATION: NCT02192190.

Evaluation of immunogenicity of LY2963016 insulin glargine compared with Lantus® insulin glargine in patients with type 1 or type 2 diabetes mellitus.

AIMS: To compare the immunogenicity profiles and the potential effects on clinical outcomes of LY2963016 insulin glargine (LY IGlar) and Lantus® insulin glargine (IGlar), products with identical primary amino acid sequences, in patients with type 1 or type 2 diabetes mellitus (T1DM or T2DM). METHODS: To assess immunogenicity, anti-insulin glargine antibodies (measured as percent binding) were compared between treatments in 52-week (open-label) and 24-week (double-blind) randomized studies in total study populations of patients with T1DM (N = 535) and T2DM (N = 756), respectively, and two subgroups of patients with T2DM: insulin-naïve patients and those reporting prestudy IGlar treatment (prior IGlar). Relationships between insulin antibody levels and clinical outcomes were assessed using analysis of covariance and partial correlations. Insulin antibody levels were assessed using Wilcoxon rank sum. Treatment comparisons for treatment-emergent antibody response (TEAR) and incidence of detectable antibodies were analysed using Fisher's exact test. RESULTS: No significant treatment differences were observed for insulin antibody levels, incidence of detectable anti-insulin glargine antibodies, or incidence of TEAR [overall and endpoint, by last-observation-carried-forward (LOCF)] in patients with T1DM or patients with T2DM, including the insulin-naïve subgroup. A statistically significant difference was noted in the overall incidence of detectable antibodies but not at endpoint (LOCF) nor in TEAR for the prior IGlar subgroup of patients with T2DM. Insulin antibody levels were low (<5%) in both treatment groups. Insulin antibody levels or developing TEAR was not associated with clinical outcomes. CONCLUSIONS: LY IGlar and IGlar have similar immunogenicity profiles; anti-insulin glargine antibody levels were low for both treatments, with no observed effect on efficacy and safety outcomes.

Short-term administration of the glucagon receptor antagonist LY2409021 lowers blood glucose in healthy people and in those with type 2 diabetes.

AIM: To describe the clinical effects of single and multiple doses of a potent, selective, orally administered, small-molecule antagonist of the human glucagon receptor, LY2409021, in healthy subjects and in patients with type 2 diabetes. METHODS: LY2409021 was administered in dose-escalation studies to healthy subjects (n = 23) and patients with type 2 diabetes (n = 9) as single doses (Study 1) and daily to patients with type 2 diabetes (n = 47) for 28 days (Study 2). Safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) assessments were made after single doses and in patients receiving once-daily doses of LY2409021 (5, 30, 60 or 90 mg) for 28 days. RESULTS: LY2409021 was well tolerated at all dose levels in both studies. Fasting and postprandial glucose were reduced and glucagon levels increased after single and multiple dosing, with reductions in fasting serum glucose of up to ∼1.25 mmol/l on day 28. Serum aminotransferases increased in a dose-dependent manner with multiple dosing and reversed after cessation of dosing. Significant glucose-lowering was observed with LY2409021 at dose levels associated with only minor aminotransferase increases. CONCLUSION: Blockade of glucagon signalling in patients with type 2 diabetes is well tolerated and results in substantial reduction of fasting and postprandial glucose with minimal hypoglycaemia, but with reversible increases in aminotransferases. Inhibition of glucagon signalling by LY2409021 is a promising potential treatment for patients with type 2 diabetes and should be evaluated in longer clinical trials to better evaluate benefits and risks.

[Health-economic assessment of combination therapy for rheumatoid arthritis with methotrexat and etanercept based on the TEMPO Study]. / Gesundheitsökonomische Bewertung der Kombinationstherapie der rheumatoiden Arthritis mit Methotrexat und Etanercept auf der Basis der TEMPO-Studie.

BACKGROUND: The TEMPO study has shown that the combination of etanercept and methotrexat (MTX) in the treatment of rheumatoid arthritis (RA) is superior to monotherapy. It further suggested that remission of RA is a realistic treatment objective. A health-economic assessment of the combination needs to demonstrate the suitability of the combination for daily clinical practice taking economic aspects into consideration. PATIENTS AND METHODS: For the 686 patients in the TEMPO study, a re-analysis was carried out in the form of a Monte-Carlo-Markov-Chain simulation. Study types were cost-effectiveness analysis and cost-utility analysis. Comparators were combined etanercept and MTX vs. MTX alone; the perspective was that of society as a whole. RESULTS: The incremental cost-effectiveness ratio of the combination is 21,300 per life year in remission as compared with MTX alone. The incremental cost-utility ratio of the combination is 38,700 per quality-adjusted life year. CONCLUSION: Both health-economic parameters suggest to adopt the combination therapy into daily clinical practice of RA patients.

A comparison of cardiovascular disease risk factor biomarkers in African Americans and Yoruba Nigerians.

OBJECTIVE: Classical risk factors for coronary artery disease are changing in the developing world while rates of cardiovascular disease are increasing in these populations. Newer risk factors have been identified for cardiovascular disease, but these have been rarely examined in elderly populations and not those of developing countries. METHODS: This study was a cross-sectional comparison from a longitudinal, observational, epidemiologic study in which participants are interviewed at three-year intervals. The sample included 1510 African Americans from Indianapolis, Indiana, and 1254 Yoruba from Ibadan, Nigeria. We compared anthropomorphic measurements; biomarkers of endothelial dysfunction (plasminogen activator inhibitor type 1 [PAI-1 and E-selectin), inflammation (C-reactive protein), and lipid oxidation (8-isoprostane); and levels of lipids, homocysteine, folate, and vitamin B12. RESULTS: Cholesterol, triglycerides, and low-density lipoprotein cholesterol levels were higher in African Americans. For markers of endothelial dysfunction, E-selectin and homocysteine differed between men, and PAI-1 was higher in the Yoruba. C-reactive protein differed only in women, but 8-isoprostane was higher in the Yoruba. CONCLUSION: Higher lipid levels in African Americans are consistent with their Western diet and lifestyle. Oxidative stress appears to be higher in the Yoruba than in African Americans, which may be secondary to dietary differences. Whether these differences in classical and emerging risk factors account for the different rates of cardiovascular disease, dementia, or other morbidities in these two populations remains to be determined.

Glycosylphosphatidylinositol-specific phospholipase D is expressed by macrophages in human atherosclerosis and colocalizes with oxidation epitopes.

BACKGROUND: Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) may play an important role in inflammation, because it can hydrolyze the GPI anchors of several inflammatory membrane proteins (eg, CD106, CD55, and CD59) and its hydrolytic products upregulate macrophage cytokine expression (eg, interleukin-1 and tumor necrosis factor-alpha). Because of its potential regulatory role in inflammatory reactions, we hypothesized that GPI-PLD might be expressed in atherosclerosis. METHODS AND RESULTS: Immunohistochemistry using human GPI-PLD-specific rabbit polyclonal antiserum was performed on a total of 83 nonatherosclerotic and atherosclerotic human coronary arteries from 23 patients. Macrophages, smooth muscle cells, apoA-I, and oxidation epitopes also were identified immunohistochemically. Cell-associated GPI-PLD was detected in 95% of atherosclerotic segments, primarily on a subset of macrophages. Extracellular GPI-PLD was present in only 30% of atherosclerotic segments and localized to regions with extracellular apoA-I. In contrast, GPI-PLD was not detected in nonatherosclerotic segments. Expression of GPI-PLD mRNA by human macrophages was confirmed in vitro by reverse transcription/polymerase chain reaction. Further studies demonstrated that GPI-PLD-positive plaque macrophages contained oxidation epitopes, suggesting a link between oxidant stress and GPI-PLD expression. This possibility was supported by studies in which exposure of a macrophage cell line to H2O2 led to a 50+/-3% increase in steady-state GPI-PLD mRNA levels. CONCLUSIONS: Collectively, these results suggest that oxidative processes may regulate GPI-PLD expression and suggest a role for GPI-PLD in inflammation and in the pathogenesis of atherosclerosis.

Inositol glycan phosphate derived from human erythrocyte acetylcholinesterase glycolipid anchor and inositol cyclic 1,2-phosphate antagonize glucagon activation of glycogen phosphorylase.

In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of glycosylphosphatidylinositol-specific phospholipase D secretion from beta TC3 cells.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian serum, but the source of the circulating enzyme is unknown. Pancreatic islets have been reported to contain and secrete GPI-PLD. In this report we examined the regulation of GPI-PLD secretion from beta TC3 cells, a mouse insulinoma cell line. In the absence of glucose, phorbol myristic acid (0.1 microM) stimulated insulin secretion by 2.5-fold and GPI-PLD secretion by 2-fold. Carbachol (5 microM), glucagon-like peptide I-(7-36) amide (0.1 microM), and isobutylmethylxanthine (0.1 mM) had no significant effect on insulin or GPI-PLD secretion in the absence of glucose. Glucose (16.7 mM) stimulated both GPI-PLD and insulin secretion from beta TC3 cells by 55% and 235%, respectively. In addition, glucose potentiated the secretagogue effect of isobutylmethylxanthine, phorbol myristic acid, and glucagon-like peptide I on both insulin and GPI-PLD secretion. By immunohistochemistry and confocal microscopy, beta TC3 cells contain both insulin and GPI-PLD, which generally colocalized intracellularly. However, GPI-PLD secretion differed from insulin secretion by a higher rate of basal release (2.8% vs. 0.23%/h), a lower magnitude of response to secretagogues, and a more prolonged period of increased secretion. These results demonstrate that beta TC3 cells secrete GPI-PLD in response to insulin secretagogues and suggest that GPI-PLD may be secreted via the regulated pathway in these cells.

Glycosylphosphatidylinositol-phospholipase D: a tool for glycosylphosphatidylinositol structural analysis.

Cleavage by the GPI-PLD provides definitive evidence of a minimal GPI structure: glucosamine-phosphatidylinositol. Unlike the case for PI-PLC, cleavage by the GPI-PLD is unaffected by acylation of the inositol ring. Thus the GPI-PLD provides an excellent simple enzymatic tool for analyzing the basic core structure of GPI anchors.

Palmitoyl derivatives of GpMBP epitopes: T-cell response and peptidases susceptibility.

We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a palmitoyl moiety at the N(alpha)-terminal of Gln(74) or at the epsilon-NH(2) of Lys(75) of GpMBP(74-85) induced increased T-cell responsiveness compared to the native nonlipidated peptide. On the other hand, lipoderivatives of GpMBP(82-98) did not increase the T-cell response, demonstrating that the superagonist inducing effect of lipoconjugation is epitope-specific. Digestion of the two native peptides with cathepsin D and L, both implicated in antigen processing, and with a complete lysosomal fraction of a EBV-transformed B cell line shows that GpMBP(74-85) is resistant to cellular proteases, while GpMBP(82-98) is easily digested by these enzymes. These results suggest that the first prerequisite for increasing the T-cell response by lipoconjugation is the stability of the native peptide to peptidases, providing an important insight into the understanding of the immunoadjuvant effect of lipoderivative antigens.

Glucose and insulin regulate glycosylphosphatidylinositol-specific phospholipase D expression in islet beta cells.

Insulin resistance is associated with a compensatory islet hyperactivity to sustain adequate insulin biosynthesis and secretion to maintain near euglycemia. Both glucose and insulin are involved in regulating proteins required for insulin synthesis and secretion within the islet and islet hypertrophy. We have determined that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is present within the secretory granules of islet beta cells. To determine if GPI-PLD is regulated in islet beta cells, we examined the effect of glucose and insulin on GPI-PLD expression in rat islets and murine insulinoma cell lines. Glucose (16.7 mmol/L) increased cellular GPI-PLD activity and mRNA levels 2- to 7-fold in isolated rat islets and betaTC3 and betaTC6-F7 cells. Insulin (10(-7) mol/L) also increased GPI-PLD mRNA levels in rat islets and betaTC6-F7 cells 2- to 4-fold commensurate with an increase in GPI-PLD biosynthesis. To determine if islet GPI-PLD expression is increased in vivo under conditions of islet hyperactivity, we compared GPI-PLD mRNA levels in islets and liver from ob/ob mice and their lean littermates. Islet GPI-PLD mRNA was increased 5-fold while liver mRNA and serum GPI-PLD levels were reduced 30% in ob/ob mice compared with lean littermate controls. These results suggest that glucose and insulin regulate GPI-PLD mRNA levels in isolated islets and beta-cell lines. These regulators may also account for the increased expression of GPI-PLD mRNA in islets from ob/ob mice, a model of insulin resistance and islet hyperactivity.

Effect of dihydralazine on the renal kallikrein-kinin system of the rat.

Dihydralazine (0.1 mg/kg), injected intravenously into male Sprague-Dawley rats, caused a decrease in mean arterial blood pressure and an increase in renal plasma flow, while urine volume remained unchanged. Dihydralazine had no effect on kallikrein excretion in the urine and on kallikrein activity in the renal cortex. No correlation was found between renal kallikrein and either renal plasma flow or mean arterial blood pressure. The excretion of kinins in the urine rose markedly after the administration of dihydralazine; no correlation between urinary kinins and urinary or renal kallikrein was observed. Dihydralazine had no influence on the kininogen content of blood-free renal cortex. The enzymatic activity of kininase II in renal cortex was not impaired by dihydralazine. It is suggested that the increased formation of kinins within the kidney could be involved in the vasodilating and blood pressure lowering effect of dihydralazine.

Chemical modifications of bafilomycin-type 16-membered dienlactone macrolides.

Derivatives of the 16-membered dienlactone bafilomycins were prepared in order to study the structure-activity relationship of these compounds. Some derivatives formed by hydrolysis, O-alkylation, O-acylation, trans-esterification and epoxidation were isolated and their structures determined by two-dimensional (2D) NMR studies and mass spectroscopy.

GPI-specific phospholipase D as an apolipoprotein.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) has recently been shown to be associated with high-density lipoproteins (HDL) in bovine serum. To determine the distribution of GPI-PLD among lipoproteins and characterize the GPI-PLD-containing lipoproteins in human plasma, we used dextran sulfate and immunoaffinity chromatography to isolate apolipoprotein-specific lipoproteins. This procedure allowed fractionation of lipoprotein particles into those containing apolipoprotein B (Lp B), apolipoproteins AI and AII (Lp AI/AII), or apolipoprotein AI only (Lp AI). In five plasma samples with HDL cholesterol ranging from 40 to 129 mg/dl, 75 +/- 12% (mean +/- SD) of the GPI-PLD activity was associated with Lp AI, 11 +/- 13% with Lp AI/AII, while only 13 +/- 9% was present in plasma devoid of these lipoproteins, suggesting that most of the GPI-PLD in human plasma is associated with apolipoprotein AI. No GPI-PLD activity was detected in Lp B. Further characterization of the GPI-PLD-containing lipoproteins by gel filtration chromatography, nondenaturing poly-acrylamide and agarose gel electrophoresis revealed that GPI-PLD was restricted to an apolipoprotein AI-containing particle or complex that was small (apparent mean Mw of 140 kDa) and distinct from the bulk of HDL. Thus, the majority of plasma GPI-PLD appears to be specifically associated with a small, minor fraction of apolipoprotein AI.

Interrelation between renal kallikrein and diuresis in rats.

In rats diuresis was produced by 1. furosemide (5 mg/kg/s.c.), 2. triamterene (50 mg/kg/p.o.), 3. mannitol (250 microliters, 30%) and 4. change (+ 35 mm Hg) in perfusion pressure in the isolated perfused rat kidney. In all experiments a prolonged increase in diuresis and natriuresis was effected. Urinary potassium excretion was markedly enhanced by furosemide and change in perfusion pressure, moderately increased by mannitol, and significantly depressed by triamterene. Urinary kallikrein excretion, however, showed in all of the experiments a biphasic course with an initial increase and secondary decrease. The increase of kallikrein excretion was observed only in the periods when urine flow started to increase. Thus, no correlation was found between kallikrein excretion and either urine volume or urinary sodium excretion. Kallikrein excretion correlated with potassium excretion in urine after furosemide, triamterene, and mannitol, however, it did not after increase in perfusion pressure. In renal cortical tissue kallikrein activity was reduced after all experiments, independently of the mechanisms, diuresis was induced.

Resistance to dermcidin-derived peptides is independent of bacterial protease activity.

Dermcidin (DCD) is an antimicrobial peptide constitutively expressed in eccrine sweat glands in human skin. By post-secretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides that are able to kill several Gram-positive and Gram-negative bacteria but are only weakly active against Pseudomonas aeruginosa. Here, we questioned whether bacterial resistance to DCD peptides is mediated by proteolytic degradation. It was shown that DCD-derived peptides are degraded by purified bacterial proteases and by extracellular proteases secreted by P. aeruginosa in a concentration-dependent manner. However, protease-deficient mutants of P. aeruginosa PAO1 lacking either lasA, lasB (elastase) or both showed a similar sensitivity towards DCD-derived peptides as the wild-type strain. Finally, inhibition of total protease activity indicated that proteases secreted by P. aeruginosa are not responsible for the poor activity of DCD-derived peptides against P. aeruginosa. These data suggest that the decreased sensitivity of P. aeruginosa to DCD-derived peptides is not mediated by proteolytic degradation under physiological conditions.