Clinical Remission in Severe Asthma: A Pooled Post Hoc Analysis of the Patient Journey with Benralizumab.

INTRODUCTION: Consensus definitions for clinical remission and super-response were recently established for severe asthma. Benralizumab is an interleukin-5 (IL-5) receptor α-directed monoclonal antibody for severe, uncontrolled asthma; efficacy and safety were demonstrated in previous pivotal phase 3 trials (SIROCCO, CALIMA, ZONDA). This analysis applied a composite remission definition to characterize individual responses to benralizumab after 6 and 12 months. METHODS: In previous phase 3 studies, eligible patients were those with severe, uncontrolled asthma receiving medium- or high-dosage inhaled corticosteroids plus long-acting ß2-agonists. This post hoc analysis included patients randomized to the approved benralizumab dose and not receiving oral corticosteroids (OCS) at baseline (SIROCCO/CALIMA) or OCS ≤ 12.5 mg per day (ZONDA). Individual remission components were zero exacerbations; zero OCS use; Asthma Control Questionnaire-6 (ACQ-6) score < 1.5 or ≤ 0.75; and pre-bronchodilator forced expiratory volume in 1 s (FEV1) increase ≥ 100 mL; clinical remission incorporated zero exacerbations, zero OCS use, ACQ-6 score ≤ 0.75, and pre-bronchodilator FEV1 increase ≥ 100 mL after 6 or 12 months. RESULTS: Overall, 609 patients (N = 301 and N = 308) and 586 patients (N = 293 and N = 293) receiving benralizumab in SIROCCO and CALIMA were included at 6 and 12 months, respectively; 40 ZONDA patients were included after 6 months. In SIROCCO/CALIMA, similar to 6-month findings, approx. 83% and approx. 49% receiving benralizumab, and 77% and 37% on placebo achieved ≥ 2 and ≥ 3 remission components after 12 months; 14.5% (85/586) on benralizumab and 7.7% (48/620) on placebo achieved clinical remission at 12 months. Among ZONDA patients, 75% and approx. 48% on benralizumab and 35% and 20% on placebo achieved ≥ 2 and ≥ 3 remission components at 6 months, respectively; 22.5% (9/40) on benralizumab and 7.5% on placebo achieved clinical remission. CONCLUSIONS: This analysis demonstrates clinical remission is achievable by targeting the underlying drivers of inflammation. Precision medicines can help shift treatment paradigms toward treat-to-target, with clinical remission as the ultimate therapeutic goal in severe asthma. CLINICAL TRIAL REGISTRATION: SIROCCO (NCT01928771); CALIMA (NCT01914757); ZONDA (NCT02075255).

Widely accepted definitions for disease remission are already established for the treatment of rheumatoid arthritis, ulcerative colitis, and cancer, among others. Two separate expert groups recently collaborated to discuss clinical remission/super-response to treatment in patients with severe asthma. Both groups developed separate, yet similar ways to determine whether a patient should be considered "in remission." In this study, we used the results from three previous trials (SIROCCO, CALIMA, and ZONDA) that were conducted to assess a therapy called benralizumab in patients with severe asthma to identify patients who met some or all of the criteria for disease remission in severe asthma. These criteria included zero asthma exacerbations; zero oral steroid (OCS) use; asthma control score; and improvement in lung function. Across all three trials, about three quarters of the patients achieved two or more remission components and about half achieved three or more remission components after 6 months of treatment; furthermore, these rates were generally similar to the numbers of patients who achieved two or more components and three or more components of remission after 12 months of treatment. Overall, 15­23% of patients achieved clinical remission in 6 months, and approximately 15% achieved remission within 12 months. The results show that biologic therapies like benralizumab help improve the symptoms of severe asthma and allow patients to achieve disease remission.

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response.

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow.

BACKGROUND: The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. RESULTS: Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3beta, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1alpha, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)alpha, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. CONCLUSION: hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.

Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes.

Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4alpha) and CCAAT/enhancer binding protein alpha (C/EBPalpha) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells.

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3beta decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1alpha were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450-dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development.

Connexins and their channels in cell growth and cell death.

Direct communication between cells, mediated by gap junctions, is nowadays considered as an indispensable mechanism in the maintenance of cellular homeostasis. In fact, gap junctional intercellular communication is actively involved in virtually all aspects of the cellular life cycle, ranging from cell growth to cell death. For a long time, it was believed that this was merely a result of the capacity of gap junctions to control the direct intercellular exchange of essential cellular messengers. However, recent data show that the picture is more complicated than initially thought, as structural precursors of gap junctions, connexins and gap junction hemichannels, can affect the cellular homeostatic balance independently of gap junctional intercellular communication. In this paper, we summarize the current knowledge concerning the roles of connexins and their channels in the control of cellular homeostasis, with the emphasis on cell growth and cell death. We also briefly discuss the role of gap junctional intercellular communication in carcinogenesis and the potential use of connexins as tools for cancer therapy.

Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF-kappaB is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF-kappaB also suppresses TNF-mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF-kappaB inhibitors in the liver. This dual function of NF-kappaB emphasizes the need for therapeutics that can inhibit both TNF-induced NF-kappaB activation and cell death. Here we describe that adenoviral expression of the NF-kappaB inhibitory protein ABIN-1, but not an IkappaBalpha superrepressor (IkappaBalpha(s)), completely prevents lethality in the TNF/D-(+)-galactosamine-induced model of liver failure. Protection was associated with a significant decrease in TNF-induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN-1 and IkappaBalpha(s) suggest a role for an NF-kappaB independent function of ABIN-1. Indeed, ABIN-1 was found to prevent not only NF-kappaB activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF-induced liver failure. In conclusion, ABIN-1 has a dual NF-kappaB inhibitory and anti-apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases.

Differential effects of histone deacetylase inhibitors in tumor and normal cells-what is the toxicological relevance?

Histone deacetylase (HDAC) inhibitors target key steps of tumor development: They inhibit proliferation, induce differentiation and/or apoptosis, and exhibit potent antimetastatic and antiangiogenic properties in transformed cells in vitro and in vivo. Preliminary studies in animal models have revealed a relatively high tumor selectivity of HDAC inhibitors, strenghtening their promising potential in cancer chemotherapy. Until now, preclinical in vitro research has almost exclusively been performed in cancer cell lines and oncogene-transformed cells. However, as cell proliferation and apoptosis are essential for normal tissue and organ homeostasis, it is important to investigate how HDAC inhibitors influence the regulation of and interplay between proliferation, differentiation, and apoptosis in primary cells as well. This review highlights the discrepancies in molecular events triggered by trichostatin A, the reference compound of hydroxamic acid-containing HDAC inhibitors, in hepatoma cells and primary hepatocytes (which are key targets for drug-induced toxicity). The implications of these differential outcomes in both cell types are discussed with respect to both toxicology and drug development. In view of the future use of HDAC inhibitors as cytostatic drugs, it is highly recommended to include both tumor cells and their healthy counterparts in preclinical developmental studies. Screening the toxicological properties of compounds early in their development process, using a battery of different cell types, will enable researchers to discard those compounds bearing undesirable adverse activity before entering into expensive clinical trials. This will not only reduce the risk for harmful exposure of patients but also save time and money.

Trichostatin A-like hydroxamate histone deacetylase inhibitors as therapeutic agents: toxicological point of view.

Modulation of chromatin structure through histone acetylation/deacetylation is known to be one of the major mechanisms involved in the regulation of gene expression. Two opposing enzyme activities determine the acetylation state of histones: histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively acetylating or deacetylating the epsilon-amino groups of lysine residues located in the amino-terminal tails of the histones. In general, transcriptionally active chromatin is associated with hyperacetylated histones, whilst silenced chromatin is linked to hypoacetylated histones. A number of structurally divergent classes of HDAC inhibitors have been identified. They have been shown to induce cell cycle arrest, terminal differentiation and/or apoptosis in various cancer cell lines and inhibit tumor growth in animals. In particular, the reversible HDAC inhibitor Trichostatin A (TSA) and its hydroxamate analogues can effectively and selectively induce tumor growth arrest at very low concentrations (nano- to micromolar range). They form a group of so-called promising antitumor agents of which some are currently under clinical trial. Since the selection of a molecule for further drug development requires a balance of biological potency, safety and pharmacokinetics, it is of paramount importance to elucidate the pharmacokinetic and toxicological properties of these HDAC inhibitors before they can be considered as potential new drugs. Primary hepatocytes and their cultures are well-differentiated in vitro models and can be used to study simultaneously the biological effects of HDAC inhibitors and their biotransformation. The present review provides a state-of-the-art of our current knowledge of the pharmacological and toxicological effects on proliferating cells of TSA and its hydroxamate-based structural analogues. Besides a theoretical basis, an overview of the experimental results, obtained by the authors using primary rat hepatocytes as an in vitro model, is given.

Rat hepatocyte suspensions as a suitable in vitro model for studying the biotransformation of histone deacetylase inhibitors.

This paper focuses on the use of liver-derived in vitro systems for biotransformation studies during early drug development, as exemplified by the two molecules recently studied in our laboratory: Trichostatin A (TSA) and its structural analogue 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me2N-BAVAH). Phase I biotransformation of TSA, a histone deacetylase inhibitor with promising antifibrotic and antitumoural properties, was investigated in liver microsomal (rat and human) and in hepatocyte (rat) suspensions. Within 40 minutes, 50 microM of TSA was completely metabolised by 2 x 10(6) hepatocytes/ml. Reduction of the hydroxamic acid function to its corresponding amide and N-demethylation were the two major phase I biotransformation pathways, while hydrolysis products of TSA were minor metabolites. Lower concentrations of TSA (5 microM and 25 microM) were N-demethylated faster. Liver microsomes, however, metabolised TSA incompletely with the formation of two major metabolites, N-mono- and N-didemethylated TSA. Unlike TSA, 4-Me2N-BAVAH (50 microM) could still be detected after 3 hours of incubation with 2 x 10(6) rat hepatocytes/ml suspension. Hydrolysis and reduction of the hydroxamic acid function to its corresponding acid and amide, respectively, were shown to be the major phase I biotransformation pathways. Lower concentrations of 4-Me2N-BAVAH were hydrolysed more readily. 4-Me2N-BAVAH and its metabolites were less subjected to N-demethylation than TSA.

Trichostatin A induces differential cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes.

BACKGROUND/AIMS: The effects of Trichostatin A (TSA), a drug candidate for cancer therapy, on proliferation and survival of primary hepatocytes, the major site of xenobiotic biotransformation and primary target of drug-induced toxicity, were investigated. METHODS: DNA replication was measured using [methyl-3H]-thymidine incorporation. Cell cycle markers were analyzed by Western and Northern blottings. Necrosis and apoptosis were monitored by LDH release, caspase-3-activation, respectively. RESULTS: We identified two distinct cell cycle arrests, prior DNA replication, in two experimental conditions. First, perfusion of the liver in presence of TSA, prevented c-jun and cyclin D1 induction, characteristic for G1 entry and progression through late G1, respectively. Secondly, TSA treatment of isolated hepatocytes, located in early G1, led to an early S-phase arrest evidenced by the absence of the S/G2/M marker, CDK1. TSA upregulated the expression of the anti-apoptotic protein Bcl(xL) and did not increase caspase-3-activity and LDH release. CONCLUSIONS: TSA inhibits hepatocyte proliferation at different steps of the cell cycle. Our data suggest that this inhibition may involve downregulation of distinct subsets of genes. TSA does not induce apoptosis in primary hepatocytes, in contrast to what has been observed in hepatoma cells. This finding supports its use in the treatment of proliferative disorders.

Structure and function of the N-cadherin/catenin complex in retinoblastoma.

PURPOSE: To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion. METHODS: The cadherin/catenin complex was characterized in surgical retinoblastoma specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/catenin complex in aggregation and collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay. RESULTS: In retinoblastoma and normal retina N-cadherin associated with alpha-catenin and beta-catenin but not E- or P-cadherin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The N-cadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/catenin complex indicated that its linkage with the cytoskeleton were weak or absent. CONCLUSIONS: Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter.