Doxofylline, a Novofylline Inhibits Lung Inflammation Induced By
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Pulmonary Pharmacology & Therapeutics 27 (2014) 170e178 Contents lists available at ScienceDirect Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt Doxofylline, a novofylline inhibits lung inflammation induced by lipopolysacharide in the mouse Yanira Riffo-Vasquez*, Francis Man, Clive P. Page Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science, King’s College London, UK article info abstract Article history: Rational: Doxofylline is a xanthine drug that has been used as a treatment for respiratory diseases for Received 20 December 2013 more than 30 years. In addition to doxofylline being a bronchodilator, some studies have indicated that Received in revised form doxofylline also has anti-inflammatory properties, although little is known about the effect of this drug 30 December 2013 on lung inflammation. Accepted 2 January 2014 Objectives: We have investigated the actions of doxofylline against the effects of Escherichia coli LPS in the lungs of BALB/c mice. Keywords: Methods: Animals have been treated with doxofylline (0.1, 0.3 and 1 mg/kg i.p.) 24, -and 1 h before, and Doxofylline m Neutrophils 6 h after intra-nasal instillation of LPS (10 g/mouse). Readouts were performed 24 h later. fi LPS Results: Doxofylline at 1 and 0.3, but not at 0.1 mg/kg, signi cantly inhibit neutrophil recruitment to the Lung lung induced by LPS (LPS: 208.4 Æ 14.5 versus doxofylline: 1 mg/kg: 106.2 Æ 4.8; 0.3 mg/kg: 4 Inflammation 105.3 Æ 10.7  10 cells/ml). Doxofylline significantly inhibited IL-6 and TNF-a release into BAL fluid in Mice comparison to LPS-treated animals (LPS: 1255.6 Æ 143.9 versus doxofylline 1 mg/kg: 527.7 Æ 182.9; 0.3 mg/kg: 823.2 Æ 102.3 pg/ml). Intra-vital microscopy of the tracheal tissue demonstrated that dox- ofylline significantly reduced LPS-mediated leukocyte adhesion to the vessel wall (LPS: 5.9 Æ 2.4 versus doxofylline 0.3 mg/kg: 1.78 Æ 0.87 cells/100 mm/15 s). Similarly, intra-vital microscopy of cremaster muscle demonstrated that doxofylline significantly reduced LPS-mediated leukocyte transmigration across the blood vessel wall (LPS: 9.3 Æ 3.5 versus doxofylline0.3 mg/kg: 3.1 Æ 1.87 cells/100 mm2). Doxofylline (0.1e10 mM) also reduced fMLP-induced leukocyte migration in vitro, achieving a maximum effect at 10 mM (fMLP: 37.8 Æ 0.5 versus doxofylline 10 mM: 15.1 Æ 1.2 cells  104/ml). Conclusion: Doxofylline inhibits LPS-induced inflammation in the lungs of mice. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction due to doxofylline lacking either adenosine receptor antagonism or inhibition of phosphodiesterase enzymes (PDEs), such as PDE3 Xanthines are drugs widely used in the treatment of respiratory expressed in the cardiovascular system [4], mechanisms that are diseases and exhibit both bronchodilator and anti-inflammatory thought to contribute to the side effect profile of theophylline [5]. actions [1,2]. However, older xanthines such as theophylline can Whilst there is a considerable amount of information describing present dose-dependent side effects which has led to the search for the anti-inflammatory effects of theophylline [1,2,6] there are newer drugs with an improved safety profile, including enprofyl- limited data demonstrating the anti-inflammatory actions of dox- line, bamifylline and doxofylline which have been referred to as ofylline [7,8]. Indeed the available data is restricted to a report of “novofyllines” [3]. Clinical data has suggested that doxofylline has a the ability of doxofylline to reduce the pleurisy induced by the wider therapeutic window than theophylline, and exhibits less inflammatory mediator platelet activating factor (PAF) in the rat [7] cardiovascular and gastrointestinal side effects (Reviewed in and ex-vivo effects on the proliferation of inflammatory cells ob- Ref. [3]). Recent experimental work has suggested that this may be tained from patients with chronic bronchitis [8]. Neutrophils are now known to be a significant cell involved in the pathogenesis of both severe asthma [9] and COPD [10], and unlike eosinophil recruitment, neutrophil activation is often thought to be resistant * Corresponding author. Sackler Institute of Pulmonary Pharmacology, Institute to treatment with glucocorticosteroids [10]. This has led to the of Pharmaceutical Science, King’s College London, Room 5.17, 150 Stamford Street, fl London SE1 9NH, UK. Tel.: þ44 2078484819; fax: þ44 2078486097. development of new classes of anti-in ammatory drugs targeting E-mail address: [email protected] (Y. Riffo-Vasquez). the neutrophil as treatments for severe asthma and COPD such as 1094-5539/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2014.01.001 Y. Riffo-Vasquez et al. / Pulmonary Pharmacology & Therapeutics 27 (2014) 170e178 171 the selective PDE4 inhibitor roflumilast which has been recently 2.6. Histological analysis approved for the treatment of severe COPD, although this drug is also limited by very significant gastrointestinal side effects [11]. Formalin fixed tissues were embedded in paraffin and 4 mm Given that doxofylline has been demonstrated to have significant sections were cut and stained with H&E (SigmaeAldrich) for clinical benefit in both adults and children with respiratory diseases examining cell infiltration microscopically using a 40 objective such as asthma or COPD, and to have an improved safety profile (Zeiss Axioscope). A Nikon Digital Camera (DXM1200) and Zeiss compared to theophylline, we thought it of value to better under- Axiovision Image Analyser (Zeiss, version 4.61) were used to obtain stand the anti-inflammatory effects of this drug of relevance to images of the airways. airway diseases. Bacterial LPS is known to be a good stimulus to induce neutro- 2.7. In vitro chemotaxis assay phil recruitment into the lung, both experimentally [12] and clin- ically [13,14] and so we have therefore investigated the anti- Bone marrow cell migration was examined using 96-well inflammatory actions of doxofylline against LPS-induced lung chemotaxis plates (ChemoTx, USA) with 3-mm pore size poly- inflammation in the lungs of mice. carbonate filters. Cells were obtained from naive mice, washed and adjusted to a 5  106 cell/ml suspension in DMEM (10% FBS, 2 mM L-glutamine,100 U/ml penicillin,100 mg/ml streptomycin and 1 mM 2. Methods sodium pyruvate). Fifteen minutes prior to carrying out the chemotaxis assay, cells were incubated with doxofylline (0.001e 2.1. Animals 100 mM). Twenty microlitres of this suspension were added to the top wells of the plate. The bottom wells were filled with 30 mlof Male BALB/c, 6e8 weeks old, were used in this study (Harlan, DMEM containing Formyl-Methionyl-Leucyl-Phenylalanine (fMLP UK). Experiments were approved by the Home Office under The 100 mM; previously determined as the optimal concentration to Animals (Scientific Procedures) Act (1986) and local approval from induce leukocyte chemotaxis). As a negative control, some bottom the Ethics Committee of King’s College London. wells were filled with medium alone. After 2 h incubation at 37 C, the membrane was removed and numbers of cells present in the 2.2. Drug treatment bottom wells counted using a Modified Neubauer chamber. Doxofylline (Eurodrug Laboratories, The Hague, The 2.8. Effect of doxofylline on leukocyte activation Netherlands) was administrated i.p. at 0.1, 0.3 and 1 mg/kg À24, À1 before, and 6 h after intra-nasal instillation of 10 mg of LPS Bone marrow cells were collected from naive BALB/c mice, 6 (Escherichia coli, Sigma, UK). Control mice were treated with saline resuspended to 5  10 /ml in DMEM medium and pre-treated with only. doxofylline (0.1e10 mM) for 30 min. Five or 15 ng/ml of murine TNF- a (eBioscience, UK) was added to the cells for stimulation of the expression of CD11b and CD62L, respectively. After a further 20 min 2.3. Bronchoalveolar lavage incubation, cells were stained with the specific antibodies for the leukocyte activation markers CD11b (BD Pharmingen, UK) and Twenty-four hours after LPS instillation, mice were euthanized CD62L (Invitrogen, UK). Negative control cells were stained with with an overdose of urethane (25% solution i.p.; Sigma Chemical isotypes control antibodies. Thirty minutes after incubation with Co.) and a cannula was inserted into the exposed trachea and three the antibodies, cells were washed and expression of markers ana- 0.5 ml aliquots of saline were injected into the lungs. From the BAL lysed by flow cytometry (Coulthard, UK). fluid, an aliquot (50 ml) was added to 50 ml of haemolysis solution (Turk’s solution, Fluka, UK). The total number of cells in the lavage 2.9. Intravital microscopy of trachea was counted with an improved Neubauer haemocytometer. For differential cell counts, cytospin preparations were prepared from Twenty-four hours after intra-nasal instillation of 10 mg of LPS, aliquots of BAL fluid (100 mL) centrifuged at 1000 rpm for 1 min animals received an injection of rodhamine 6G (Sigma, UK, 0.5% using a Shandon Cytospin 2 (Shandon Southern Instruments, solution, s.c). Thirty minutes later animals were anaesthetized with Sewickley, PA, USA) at room temperature. Cells were stained with urethane (2 mg/kg, i.p.) and the trachea was carefully exposed Diff Quick (DADE Behring, Germany) and a total of 100 cells were through a midline incision of the skin and muscle. A cannula was counted to determine the proportion of neutrophils, eosinophils inserted intra-tracheally and animals were immediately connected and monocytes using standard morphological criteria. to a ventilator. The animals were ventilated throughout the whole experiment at 110 breaths per minute with a tidal volume of 0.15e 0.2 ml and positive end expiratory pressure between 3 and 5 cm 2.4.