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.

SPIRIT Women, evaluation of the safety and efficacy of the XIENCE V everolimus-eluting stent system in female patients: referral time for coronary intervention and 2-year clinical outcomes.

AIMS: SPIRIT Women is the first interventional trial dedicated exclusively to women, focusing on symptoms at presentation, referral time to coronary intervention and the safety and performance of the XIENCE V stent. METHODS AND RESULTS: SPIRIT Women is a prospective, open-label, multicentre study in which 1,573 women were enrolled at 73 sites outside the United States. The primary endpoint is the composite of all death, Academic Research Consortium (ARC) defined myocardial infarction (MI) and target vessel revascularisation (TVR) at one year. Data collected included symptoms at presentation and referral to coronary intervention. To allow comparison by gender, the latter were compared to data from male patients from the SPIRIT V study. The one- and two-year composite of all death, MI and TVR was 12% and 15%, respectively. Target lesion revascularisation (TLR) and stent thrombosis (definite and probable) rates were 2.4% and 0.59%, respectively, at one year and 3.6% and 0.73%, at two years. The total referral time for coronary intervention in women was four days longer than for men in the SPIRIT V study. CONCLUSIONS: The XIENCE V stent is safe and effective with low TLR and stent thrombosis rates. More efforts remain to be made to increase the awareness of women and physicians of the risk for coronary artery disease (CAD).

The SPIRIT V diabetic study: a randomized clinical evaluation of the XIENCE V everolimus-eluting stent vs the TAXUS Liberté paclitaxel-eluting stent in diabetic patients with de novo coronary artery lesions.

BACKGROUND: Diabetic patients respond less favorably to revascularization and have poorer long-term outcomes. Our main aim was to evaluate the angiographic efficacy of XIENCE V (everolimus-eluting stent, or EES) in diabetic patients compared with TAXUS Liberté (paclitaxel-eluting stent, or PES). METHODS: The SPIRIT V Diabetic Study was a prospective, single-blind, randomized study that enrolled 324 diabetic (insulin and non-insulin dependent) patients at 28 sites in Europe and Asia Pacific. Randomization was 2:1 between EES (n = 218) and PES (n = 106). The primary end point was sequential noninferiority and superiority of EES for in-stent late loss at 9 months. Secondary clinical end points included stent thrombosis, death, myocardial infarction, and revascularization rates up to 1 year. RESULTS: Everolimus-eluting stent was superior to PES for in-stent late loss at 9 months (0.19 mm vs 0.39 mm, respectively; P(superiority) = .0001). The composite rate of death, myocardial infarction, and target vessel revascularization was the same in the 2 groups at 1 year (16.3% vs 16.4%). No stent thromboses (Academic Research Consortium definite and probable) were seen through 1 year with EES compared with 2 of 104 (2%) with PES (P = .11). CONCLUSION: In this prospective, randomized trial in a high-risk group of diabetic patients, implantation of EES compared with PES resulted in significantly better inhibition of intimal hyperplasia with a comparable safety outcome.

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.

Drug metabolism and pharmacokinetics.

In this article, aspects of absorption, distribution, metabolism, and excretion have been described bearing in mind the pathogenesis of allergic diseases and their possible therapeutic opportunities. The importance of the routes of administration of the different therapeutic groups has been emphasized. The classical aspects of drug metabolism and disposition related to oral administration have been reviewed, but special emphasis has been given to intranasal, cutaneous, transdermal, and ocular administration as well as to the absorption and the subsequent bioavailability of drugs. Drug-metabolizing enzymes and transporters present in extrahepatic tissues, such as nasal mucosa and the respiratory tract, have been particularly discussed. As marketed antiallergic drugs include both racemates and enantiomers, aspects of stereoselective absorption, distribution, metabolism, and excretion have been discussed. Finally, a new and promising methodology, microdosing, has been presented, although it has not yet been applied to drugs used in the treatment of allergic diseases.

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.

Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.

Primary hepatocytes and their cultures are a simple but versatile, well-controlled, and relatively easy to handle in vitro system that is well-accepted for investigating xenobiotic biotransformation, enzyme induction and inhibition, and (biotransformation-mediated) hepatotoxicity. In addition, hepatocyte cultures have proven to be valuable tools in the study of liver physiology, viral hepatitis, and liver regeneration and are proposed as an alternative to orthotopic liver transplantation. It has been observed, however, that a number of liver-specific functions are progressively lost with time when hepatocytes are isolated and cultivated. These phenotypic changes are primarily the result of fundamental changes in gene expression concomitant with a diminished transcription of the relevant liver-specific genes, and can be interpreted as a 'dedifferentiation' of the isolated hepatocytes. Ischemia-reperfusion stress induced during the isolation process, disruption of the normal tissue architecture, as well as an adaptation to the in vitro environment are underlying factors and will be extensively discussed. A detailed description of the regulation of the hepatocyte phenotype in vivo in the first section of this review will help to understand the effect of these factors on hepatocyte gene expression. Although different approaches, mainly mimicking the in vivo hepatocyte environment, have been succesfully used to prevent or slow down the dedifferentiation of primary hepatocytes in monolayer culture, the ideal hepatocyte-based culture model, characterized by a long-term expression of hepatocyte-specific functions comparable to the in vivo level, does not exist at the moment. Consequently, alternative strategies should focus on the isolation procedure, during which dedifferentiation is already initiated. In addition, identification of the conditions needed for the full in vitro maturation of hepatic progenitor cells to quiescent, functional hepatocyte-like cells opens promising perspectives.

Involvement of cell junctions in hepatocyte culture functionality.

In liver, like in other multicellular systems, the establishment of cellular contacts is a prerequisite for normal functioning. In particular, well-defined cell junctions between hepatocytes, including adherens junctions, desmosomes, tight junctions, and gap junctions, are known to play key roles in the performance of liver-specific functionality. In a first part of this review article, we summarize the current knowledge concerning cell junctions and their roles in hepatic (patho)physiology. In a second part, we discuss their relevance in liver-based in vitro modeling, thereby highlighting the use of primary hepatocyte cultures as suitable in vitro models for preclinical pharmaco-toxicological testing. We further describe the actual strategies to regain and maintain cell junctions in these in vitro systems over the long-term.

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.

Isolation of rat hepatocytes.

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined. Although applicable to the liver of various species, including human, the practical aspects of the method are explained here for rat liver. Critical parameters for the successful isolation of primary rat hepatocytes are highlighted and a troubleshooting guide is provided. In addition, a new development based on the inhibition of histone deacetylase activity is presented. This approach allows inhibition of cell-cycle reentry during hepatocyte isolation, a process known to underlie the dedifferentiation process of cultured hepatocytes.

Rat hepatocyte cultures: conventional monolayer and cocultures with rat liver epithelial cells.

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended lifespan and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions. In this chapter, techniques for setting up conventional monolayer cultures and cocultures of hepatocytes with RLECs (including isolation, culture, and cryopreservation of RLECs) are described in detail. In addition, comments derived from our own experience are given for successfully culturing primary hepatocytes.

Rat hepatocyte cultures: collagen gel sandwich and immobilization cultures.

Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g., biotransformation capacity) for more than 6 wk. We describe how to set up both types of organotypical hepatocyte culture systems. Besides a detailed protocol, we give some practical tips, taken from our own experience with long-term hepatocyte culture.

Hepatocytes in suspension.

Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the "gold standard" for investigating xenobiotic biotransformation and metabolic bioactivation. When used in suspension, they provide an easy-to-handle and relatively cheap in vitro system that can be used for up to 4 h. The use of animal- and human-derived hepatocytes allows interspecies comparisons of metabolic properties. In contrast with microsomes, which are easily prepared from human liver tissue and can be stored in liquid nitrogen with minimal loss of functionality, cryopreservation of isolated human hepatocytes has been shown to be more difficult: after thawing losses of cell viability and biotransformation capacity occur. We provide general recommendations for the appropriate use of hepatocytes in suspension for pharmaco-toxicological studies. We also provide protocols for the cryopreservation of freshly isolated hepatocytes and their handling on thawing.

Trichostatin a enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.

The effects of histone deacetylase inhibitor Trichostatin A (TSA) on connexin (Cx) expression and gap junctional intercellular communication (GJIC) were investigated in primary cultures of adult rat hepatocytes. GJIC was monitored by using the scrape-loading/dye transfer method. Immunoblotting and immunocytochemistry were used to investigate Cx protein levels and localization. Cx gene expression was studied by means of quantitative reverse transcriptase-polymerase chain reaction. TSA increased Cx32 protein levels and affected negatively the Cx26 protein levels. The latter was preferentially located in the cytosol of cultured cells. TSA also promoted the appearance of Cx43 in the nuclear compartment of primary cultured hepatocytes. Overall, this resulted in enhanced GJIC activity. It is important to note that the time of onset of TSA treatment was crucial for the extent of its outcome and that the effects of TSA on Cx protein levels occurred independently of transcriptional changes. TSA differentially affects Cx proteins in primary rat hepatocyte cultures, suggesting distinct regulation and/or distinct roles of the different Cx species in the control of hepatic homeostasis. TSA enhances GJIC between primary cultured rat hepatocytes, an interesting finding supporting its use to further optimize liver-based in vitro models for pharmacotoxicological purposes.

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.

Rat Hepatocyte Cultures : Collagen Gel Sandwich and Immobilization Cultures.

Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g., biotransformation capacity) for more than 6 wk. We describe how to set up both types of organotypical hepatocyte culture systems. Besides a detailed protocol, we give some practical tips, taken from our own experience with long-term hepatocyte culture.

Rat Hepatocyte Cultures : Conventional Monolayer and Cocultures With Rat Liver Epithelial Cells.

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended life-span and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions. In this chapter, techniques for setting up conventional monolayer cultures and cocultures of hepatocytes with RLECs (including isolation, culture, and cryopreservation of RLECs) are described in detail. In addition, comments derived from our own experience are given for successfully culturing primary hepatocytes.

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.