LEGACY: Phase 2a Trial to Evaluate the Safety, Pharmacokinetics, and Pharmacodynamic Effects of the Anti-EL (Endothelial Lipase) Antibody MEDI5884 in Patients With Stable Coronary Artery Disease.

OBJECTIVE: Functional HDL (high-density lipoprotein) particles that facilitate cholesterol efflux may be cardioprotective. EL (endothelial lipase) hydrolyzes phospholipids promoting catabolism of HDL and subsequent renal excretion. MEDI5884 is a selective, humanized, monoclonal, EL-neutralizing antibody. We sought to determine the safety, pharmacokinetics, and pharmacodynamic effects of multiple doses of MEDI5884 in patients with stable coronary artery disease. Approach and Results: LEGACY was a phase 2a, double-blind, placebo-controlled, parallel-design trial that randomized 132 patients with stable coronary artery disease receiving high-intensity statin therapy to 3 monthly doses of 1 of 5 dose levels of MEDI5884 (50, 100, 200, 350, or 500 mg SC) or matching placebo. The primary end point was the safety and tolerability of MEDI5884 through the end of the study (day 151). Additional end points included change in HDL cholesterol and cholesterol efflux from baseline to day 91, hepatic uptake of cholesterol at day 91, changes in various other lipid parameters. The incidence of adverse events was similar between the placebo and MEDI5884 groups. In a dose-dependent manner, MEDI5884 increased HDL cholesterol up to 51.4% (P<0.0001) and global cholesterol efflux up to 26.2% ([95% CI, 14.3-38.0] P<0.0001). MEDI5884 increased HDL particle number up to 14.4%. At the highest dose tested, an increase in LDL (low-density lipoprotein) cholesterol up to 28.7% (P<0.0001) and apoB (apolipoprotein B) up to 13.1% (P=0.04) was observed with MEDI5884. However, at the potential target doses for future studies, there was no meaningful increase in LDL cholesterol or apoB. CONCLUSIONS: Inhibition of EL by MEDI5884 increases the quantity and quality of functional HDL in patients with stable coronary artery disease on high-intensity statin therapy without an adverse safety signal at the likely dose to be used. These data support further clinical investigation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03351738.

Blocking endothelial lipase with monoclonal antibody MEDI5884 durably increases high density lipoprotein in nonhuman primates and in a phase 1 trial.

Cardiovascular disease (CVD) is the leading global cause of death, and treatments that further reduce CV risk remain an unmet medical need. Epidemiological studies have consistently identified low high-density lipoprotein cholesterol (HDL-C) as an independent risk factor for CVD, making HDL elevation a potential clinical target for improved CVD resolution. Endothelial lipase (EL) is a circulating enzyme that regulates HDL turnover by hydrolyzing HDL phospholipids and driving HDL particle clearance. Using MEDI5884, a first-in-class, EL-neutralizing, monoclonal antibody, we tested the hypothesis that pharmacological inhibition of EL would increase HDL-C by enhancing HDL stability. In nonhuman primates, MEDI5884 treatment resulted in lasting, dose-dependent elevations in HDL-C and circulating phospholipids, confirming the mechanism of EL action. We then showed that a favorable lipoprotein profile of elevated HDL-C and reduced low-density lipoprotein cholesterol (LDL-C) could be achieved by combining MEDI5884 with a PCSK9 inhibitor. Last, when tested in healthy human volunteers, MEDI5884 not only raised HDL-C but also increased HDL particle numbers and average HDL size while enhancing HDL functionality, reinforcing EL neutralization as a viable clinical approach aimed at reducing CV risk.

Mode of action framework analysis for receptor-mediated toxicity: The peroxisome proliferator-activated receptor alpha (PPARα) as a case study.

Several therapeutic agents and industrial chemicals induce liver tumors in rodents through the activation of the peroxisome proliferator-activated receptor alpha (PPARα). The cellular and molecular events by which PPARα activators induce rodent hepatocarcinogenesis has been extensively studied and elucidated. This review summarizes the weight of evidence relevant to the hypothesized mode of action (MOA) for PPARα activator-induced rodent hepatocarcinogenesis and identifies gaps in our knowledge of this MOA. Chemical-specific and mechanistic data support concordance of temporal and dose-response relationships for the key events associated with many PPARα activators including a phthalate ester plasticizer di(2-ethylhexyl) phthalate (DEHP) and the drug gemfibrozil. While biologically plausible in humans, the hypothesized key events in the rodent MOA, for PPARα activators, are unlikely to induce liver tumors in humans because of toxicodynamic and biological differences in responses. This conclusion is based on minimal or no effects observed on growth pathways, hepatocellular proliferation and liver tumors in humans and/or species (including hamsters, guinea pigs and cynomolgous monkeys) that are more appropriate human surrogates than mice and rats at overlapping dose levels. Overall, the panel concluded that significant quantitative differences in PPARα activator-induced effects related to liver cancer formation exist between rodents and humans. On the basis of these quantitative differences, most of the workgroup felt that the rodent MOA is "not relevant to humans" with the remaining members concluding that the MOA is "unlikely to be relevant to humans". The two groups differed in their level of confidence based on perceived limitations of the quantitative and mechanistic knowledge of the species differences, which for some panel members strongly supports but cannot preclude the absence of effects under unlikely exposure scenarios.

Prostate cancer at the peripheral end of a prostate biopsy specimen as assessed by a novel marking technique may indicate increased risk of locally advanced disease.

The objective of this study was to test a novel technique of processing a prostate biopsy (PB) specimen by marking its peripheral end (PE) as a predictive tool for locally advanced prostate cancer (PC) or margin-positive resection (R1) after radical prostatectomy (RP). Prospective evaluation of a consecutive cohort of men who underwent PB and subsequent RP was carried out. Transrectal ultrasound-guided 10-20 core PB was performed according to a standardized protocol. Each biopsy core was inked at the PE and classified as PE positive or negative. The study cohort comprised 100 men with a mean age of 62.3 years (41-75 years). Overall, PE-positive cores were found in 71 men, postoperative tumour (pT)3/pT4 stages were diagnosed in 33 men and R1 status in 45 men after RP. In univariate analysis, the presence of at least one PE-positive core was correlated to an increased risk for pT3/pT4 stage (relative risk (RR): 3.15; 95% confidence interval (95% CI): 1.1-9.9; P = 0.03) and R1 status (RR: 2.9; 95% CI: 1.1-7.5; P = 0.03). In multivariate analysis including Gleason score, total number of positive cores, PE positivity and PSA, PE positivity was correlated to pT3/pT4 stage (P = 0.04). In conclusion, PC at the PE of a PB specimen predicts non-organ-confined tumour stage in subsequent prostatectomy. This simple, new technique may contribute to increasing the accuracy of risk stratification for curative treatment of PC.

Camptothecin induces the ubiquitin-like protein, ISG15, and enhances ISG15 conjugation in response to interferon.

Interferon (IFN)-stimulated gene (15 kDa) (ISG15) is a ubiquitin-like protein that forms covalent conjugates with cellular proteins. ISG15 is induced by IFN, microbial challenge, and p53, suggesting that it represents a genetic response that is shared among diverse stress stimuli. To investigate the regulation of this posttranslational modification pathway by a genotoxic chemotherapeutic agent, we examined ISG15 induction and conjugation in cells treated with the topoisomerase I (topoI) poison, camptothecin (CPT). CPT induced ISG15mRNA, and induction required protein synthesis and a functional p53 protein. However, IFN and the Jak-Stat components of the IFN signaling pathway were dispensable for CPT induction of ISG15. CPT induced free ISG15 and conjugates in a dose-dependent and time-dependent manner. A single 55-kDa protein was the prominent CPT-induced ISG15 conjugate and localized to the nuclear compartment. CPT-induced ISG15 conjugates were distinct from those induced by IFN; however, CPT treatment dramatically enhanced ISG15 conjugation in response to IFN. These findings provide the first evidence of a stimulus-specific induction of discrete ISG15 conjugate species and demonstrate that treatment with a combination of cancer therapeutic agents can cooperate to enhance ISG15 conjugation. Identification of the specific ISG15 conjugates induced by chemotherapeutic agents may reveal novel molecular targets.

Chromosome 18 suppresses prostate cancer metastases.

Loss of heterozygosity and allelic imbalance data has shown that there are two distinct regions of loss on chromosome 18q associated with the progression of prostate cancer (CaP). To investigate the functional significance of chromosome 18q loci in CaP, we utilized the technique of microcell-mediated chromosome transfer to introduce an intact chromosome 18 into the human prostate cancer cell line, PC-3. Three of the resulting hybrid lines were compared to the PC-3 cells in vitro and in vivo. The hybrid cell lines, containing an intact copy of the introduced chromosome 18, exhibited a substantial reduction in anchorage-dependent and independent growth in vitro. These hybrid cell lines also made smaller tumors in nude mice following subcutaneous injection compared to PC-3 cells. Because tumor growth was not completely eliminated by introduction of chromosome 18, we assessed the ability of the hybrids to metastasize to bone after intra-cardiac inoculation in a nude mouse model. Mice inoculated with PC-3 hybrids containing intact copies of chromosome 18 had significantly fewer bone metastases and dramatically improved survival compared to PC-3 cells. In addition, the introduction of chromosome 18 significantly reduced tumor burden in extraskeletal sites. This was not because of differences in growth rates because mice bearing hybrids were monitored for metastases over twice as long as mice bearing PC-3 cells. Taken together, these data suggest that chromosome 18 has a functional role in CaP to suppress growth and metastases. Identification of the responsible gene(s) may lead to molecular targets for drug discovery.

Expression of Smad4 in the FaDu cell line partially restores TGF-beta growth inhibition but is not sufficient to regulate fibronectin expression or suppress tumorigenicity.

Mutations of the Smad4 gene, a member of a group of TGF-beta signal transduction components, occur in several types of cancer suggesting that its inactivation significantly affects TGF-beta responsiveness in these tumors. To further investigate the role of Smad4 with respect to TGF-beta signaling and carcinogenesis, we re-expressed the Smad4 gene in the Smad4-deficient cancer cell line FaDu by microcell-mediated chromosome transfer (MMCT) and retroviral infection to closely approximate physiological protein levels. The Smad4-expressing FaDu clones were then evaluated for TGF-beta responsiveness to assess the role of Smad4 in TGF-beta-induced growth inhibition and target gene regulation. We found that the re-expression of the Smad4 gene by either method partially restored TGF-beta responsiveness in FaDu cells with respect to both growth inhibition and expression of p21WAF1/CIP1 and p15INK4B. However, only the microcell hybrids showed growth retardation in organotypic raft culture and an enhanced ability to upregulate fibronectin. In contrast, the re-expression of Smad4 by either method failed to suppress tumorigenicity. These results suggest that in addition to a homozygous deletion of Smad4, FaDu cells contain additional defects within the TGF-beta signaling pathway, thereby limiting the extent of TGF-beta responsiveness upon Smad4 re-expression and perhaps accounting for the inability to induce p15INK4B to a high level. They also demonstrate the advantages of providing a physiological extracellular environment, when assessing TGFbeta responsiveness.

Role for p53 in gene induction by double-stranded RNA.

Cross talk between p53 and interferon-regulated pathways is implicated in the induction of gene expression by biologic and genotoxic stresses. We demonstrate that the interferon-stimulated gene ISG15 is induced by p53 and that p53 is required for optimal gene induction by double-stranded RNA (dsRNA), but not interferon. Interestingly, virus induces ISG15 in the absence of p53, suggesting that virus and dsRNA employ distinct signaling pathways.

[Elastase-induced hyperfunction of submucous glands develops independent of elastase-induced emphysema]. / Die durch Elastase induzierte Uberfunktion submuköser Drüsen entwickelt sich unabhängig vom elastaseinduzierten Emphysem.

We instilled 2 mg of pancreatic elastase into the lungs, and insufflated a further 2 mg into the trachea of 6 male ferrets. Six control animals were given 0.9% NaCl (lungs) and glucose powder (trachea). This combined treatment was administered on days 1, 8 and 15. On these days, and again on days 29 and 39, we obtained chest x-rays. On the 39th day, we removed the lungs and measured the mean linear intercept. In tracheal sections, we measured goblet cell density, glandular structure and function by quantitative histology and autoradiography. Although, up to day 39, no emphysema developed, both structural and functional changes occurred in the glands: the volume density of the serous glands (S) decreased, that of the mucous glands (M) increased, the ratio S/M decreased from 2.0 to 1.5 (p = 0.05). The turnover of radioactive sulphur (indication of mucus synthesis) was significantly increased in animals treated with elastase (p = 0.001). Goblet cells increased from 0.9 +/- 0.4 to 3.4 +/- 1.8 cells per mm epithelium (p = 0.05). The results show that elastase induces structural and functional changes in submucosal glands and goblet cell hyperplasia, independently of emphysema.

[Effect of repeated nicotine administration on mucus secretion, bronchodilation and circulatory variables: inhibitory effect of autonomic antagonists]. / Wirkung wiederholter Nikotinapplikation auf Schleimsekretion, Bronchialweite und Kreislaufvariable: Hemmwirkung autonomer Antagonisten.

To investigate the mechanisms of action of nicotine we studied the effect of muscarinic and adrenergic receptor antagonists on nicotine-induced changes in the lungs and circulation. Nicotine increased mucus secretion from tracheal submucosal glands and caused bronchocon-striction. Cardiovascular effects were a bradycardia followed by tachycardia, hypertension and a biphasic increase in cardiac output. All of these effects were completely blocked by prior administration of hexamethonium. Propranolol and phentolamine prevented the increase in heart rate, and reduced the increase in blood pressure. Only atropine inhibited nicotine-induced hypersecretion and bronchocon-striction.

[Stimulation of mucous glands of the respiratory tract by neuropeptides: comparison of exogenous administration and endogenous liberation]. / Stimulierung von Schleimdrüsen der Atemwege durch Neuropeptide: Vergleich exogener Zufuhr und endogener Freisetzung.

Exogenous substance P is a powerful stimulant of tracheal gland secretion but the contribution of endogenous neuropeptides to neurogenic gland secretion is unknown. Using the Ussing chamber technique, we measured the secretion of radiolabeled macromolecules from submucosal glands in the ferret. Neurokinins caused gland secretion through a neurokinin-1 (NK-1) receptor. Gland secretion caused by electric field stimulation of postganglionic nerve endings was not inhibited by a substance P antagonist and was not augmented by agents known to prevent neurokinin degradation in the tissue. We conclude that the release of endogenous neurokinins plays no rôle in neurally evoked gland secretion. This is despite the fact that endogenous neurokinins have been shown to be involved in vagally induced airway smooth muscle contraction and increased capillary permeability in the same species.

Stimulation of submucosal glands by nicotine applied locally to the airway mucosa.

Chronic bronchitis is characterized by hypersecretion of mucus and is caused by cigarette smoking. We investigated the effect of nicotine on mucus secretion from tracheal submucosal glands, applying nicotine to the airway mucosa in vitro. We anesthetized 50 ferrets with pentobarbital (66 mg/kg), excised their tracheae and mounted tracheal segments in Ussing chambers. We added 50 microCi Na(2)35SO4 (35S) to the submucosal side and determined nondialyzable 35S in medium collected from the luminal side at 15 min intervals. Nicotine was a powerful stimulant of mucus secretion with threshold effects at 10(-5) M and peak effects at 3 x 10(-4) M. Percentage increases were the same for males and females, but absolute increases in mucus secretion were significantly larger in males than in females. Luminal nicotine was more effective than submucosal nicotine, especially when nicotine bitartrate was used (increase above baseline, 150 +/- 54 vs 22 +/- 12 cpm/15 min, 10(-4) M, n = 4, bitartrate). Effects of luminal nicotine sulfate were larger than those of luminal nicotine bitartrate (303 +/- 88 vs 120 +/- 38 cpm/15 min, P less than 0.05, n = 6, 10(-4) M). Two applications of nicotine 1.5 h apart had similar effects up to 3 x 10(-5) M. At higher concentrations the second response was significantly weaker than the first (tachyphylaxis). Secretory effects of nicotine were prevented completely by atropine and were reduced significantly by hexamethonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of systemic nicotine on mucus secretion from tracheal submucosal glands and on cardiovascular, pulmonary, and hematologic variables.

To investigate effects of systemic nicotine on mucus secretion from tracheal submucosal glands we anesthetized 13 ferrets (5 male, 8 female; average weight 1138 +/- 469 g, mean +/- SD) with pentobarbital (initial dose, 62 +/- 26, total dose, 90 +/- 26 mg/kg), cannulated the jugular vein for i.v. application of infusions and drugs and cannulated the femoral artery for determination of blood pressure, heart rate, blood cells, and blood gases. We opened the thorax, cannulated the trachea 1 cm above the carina and ventilated the lungs through the lower airways with a Harvard respirator. We placed an electromagnetic flow probe around the ascending aorta for measurement of cardiac output. We measured transpulmonary pressure as tracheal pressure with a strain gauge transducer. We created an isolated segment of trachea between the larynx and the tracheal cannula. We perfused the segment with medium M-199 containing 30 microCi/ml Na(2)35SO4 (35S) and we injected 100 microCi 35S intravenously. After 90 min we drained the radioactive solution from the luminal side and replaced it with nonradioactive medium which we collected at 5-min intervals for determination of nondialyzable radioactivity. At 25 min we injected nicotine sulfate (5 x 10(-7)-10(-5) M/kg) and continued to collect the perfusate every 5 min. Systemic nicotine had profound effects on circulatory and ventilatory variables and on gland secretion. Initial hypertension was followed by bradycardia and a fall in blood pressure and cardiac output.(ABSTRACT TRUNCATED AT 250 WORDS)

Photooxidation of DDT and DDE.

The pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane] and its metabolite DDE [ 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene] can be photoOxidized in methanol. Photolytic generation of free radicals that may abstract hydrogen from solvent, react with oxygen, or abstract hydrogen from unreacted substrate occurs. Further decomposition of short-lived intermediates yields many compounds. Oxidation products include benzoic acids, aromatic ketones, and chlorinated phenols. The DDE also undergoes photocyclization to give dichlorofluorene derivatives.