Additive anti-inflammatory effects of beta 2 adrenoceptor agonists or glucocorticosteroid with roflumilast in human peripheral blood mononuclear cells

https://doi.org/10.1016/j.pupt.2012.01.003Get rights and content

Abstract

The phosphodiesterase 4 inhibitor (PDE4i) roflumilast has been approved in the US and EU for treatment of GOLD stage 3 and 4 chronic obstructive pulmonary disease (COPD). Inhaled β2 adrenoceptor agonist bronchodilators and anti-inflammatory glucocorticosteroids are also used as standard of care in COPD. We investigated the anti-inflammatory interaction of roflumilast in combination with long-acting β2 agonists (LABA), salmeterol or formoterol, or a glucocorticosteroid, dexamethasone, on cytokine production from LPS-stimulated human primary peripheral blood mononuclear cells (PBMC). Salmeterol or formoterol caused a concentration-dependent inhibition of tumor necrosis factor-α (TNFα) secretion with an IC50 of 0.33 pM (C.I. 0.006–19) and 34 pM (C.I. 13–87), respectively. When roflumilast was evaluated, the addition of salmeterol (1 nM) to roflumilast caused the IC50 for roflumilast to shift from 1.8 nM (C.I. 0.8–4) to 4.1 pM (C.I.0.3–69) (p < 0.01), and maximal inhibition increased from 72.5 ± 3.2% to 90.9 ± 3.1%. Addition of formoterol to roflumilast also produced an increased TNFα inhibition more than either drug alone (p < 0.05). The inhibition of TNFα production with salmeterol was both β2 adrenoceptor- and protein kinase A-dependent. Addition of roflumilast (10 nM) in the presence of dexamethasone increased the inhibition of LPS-induced TNFα and CCL3. Roflumilast in combination with salmeterol, formoterol, or dexamethasone increased the inhibition of LPS-induced TNFα from human PBMC, in an additive manner. Addition of roflumilast to either a β2 adrenoceptor agonist or a glucocorticosteroid may provide superior anti-inflammatory activity and greater efficacy in COPD patients and be dose sparing.

Highlights

► Roflumilast combination with salmeterol or formoterol increased TNFα inhibition. ► Salmeterol effects on TNFα inhibition are both β2 adrenoceptor- and PKA-dependent. ► Addition of roflumilast to dexamethasone increased inhibition of TNFα and CCL3.

Introduction

The novel anti-inflammatory phosphodiesterase-4 (PDE4) inhibitor roflumilast (Daxas®; Daliresp™) is efficacious in chronic obstructive pulmonary disease (COPD) and has recently been approved in the US and EU for GOLD stage 3 and 4 disease. In Europe, Daxas® is indicated for maintenance treatment associated with chronic bronchitis in patients with a history of exacerbations as an add-on to bronchodilator treatment. There is a wealth of evidence related to the anti-inflammatory effects of roflumilast which has been extensively studied in immune and resident lung cells as well as in a variety of pre-clinical respiratory disease models [1]. Six large studies ranging from 24 weeks to one year have demonstrated clinical efficacy for roflumilast on lung function (FEV1) and acute exacerbations in COPD [2]. The therapeutic dose of roflumilast is however limited to 500 mcg daily, due to its side-effect profile [3].

Long-acting β2 agonists (LABA), such as salmeterol, are used for symptom relief in asthma and COPD, and have been used for several decades as bronchodilator agents. There is evidence for β2 agonists to have anti-inflammatory effects in a variety of cell types in preclinical systems [4] and some clinical evidence [5], [6], [7], however, there is little evidence to suggest that they have a clinically meaningful anti-inflammatory effect as a monotherapy. There is strong evidence that β2 agonists can augment the anti-inflammatory effects of glucocorticosteroids in asthma [8], [9]. β2 agonists act on seven-transmembrane receptors which are linked to Gs coupled proteins and mediate signaling through elevation of cAMP [10]. Numerous other resident and immune cells besides airway smooth muscle cells express β2 adrenergic receptors [11]. Regular administration of inhaled β2 agonists has been associated with a degree of tolerance with respect to both bronchodilator and non-bronchodilator effects [12]. Asthmatic patients on maintenance therapy with short-acting β2 agonists have also been shown to be more sensitive to bronchoconstrictor agents with respect to late-phase and inflammatory responses [13], [14]. A number of molecular mechanisms underlying β2 receptor tolerance have been identified with several studies indicating that increased intracellular PDE4 activity in human airway smooth muscle or T-cells can contribute to this phenomenon [15], [16].

Phosphodiesterase (PDE) enzymes consist of a family of 11 different isozymes, which have different tissue distribution and properties. The PDE4 isozymes modulate the breakdown of intracellular cAMP in both inflammatory cells and structural cells in the lung [17], [18]. The combination of a β2 agonist with a PDE4 inhibitor therefore provides the potential to elevate and maintain cellular cyclic AMP levels, which may offer a potential molecular mechanism for additive anti-inflammatory interactions between these two drugs. This provides an attractive therapeutic proposition as it could provide maximal anti-inflammatory activities at lower PDE4 inhibitor doses.

PDE4 inhibitors have been extensively evaluated for their ability to inhibit lipopolysaccharide (LPS)-induced cytokine production from both human peripheral blood mononuclear cells (PBMC) and monocytes [19], [20], [21], [22]. In the present studies we have combined the PDE4 inhibitor roflumilast with the long acting β2 receptor agonists salmeterol or formoterol to evaluate their anti-inflammatory activity on TNFα release following LPS stimulation of PBMC. Additionally, we have looked at the effects of addition of a glucocorticosteroid (dexamethasone) to roflumilast on cytokine release from these cells. Our data indicates that roflumilast in combination with a β2 agonist or glucocorticosteroid has superior anti-inflammatory effects compared to roflumilast alone. These data may support a role for roflumilast in combination with either agent or as a triple therapy.

Section snippets

Reagents

Cell culture reagents RPMI, fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from Gibco (Invitrogen, Carlsbad, CA). Dimethyl sulfoxide (DMSO), LPS, H89, ICI 118551 and dexamethasone were purchased from Sigma–Aldrich (St. Louis, MO). Roflumilast, formoterol and salmeterol were synthesized in the laboratories of Gilead Sciences.

PBMC cell isolation and culture

All donors gave informed consent and were healthy, non-smoking male donors. Blood was drawn into K2EDTA vacutainers, and PBMC were isolated by

Addition of salmeterol to roflumilast increases inhibition of TNFα production by LPS-stimulated PBMC

PBMC were stimulated with LPS, and the mean TNFα levels post-LPS stimulation were 385 ± 111 pg/mL (Fig. 1A) and 481 ± 70 pg/mL (Fig. 1B and C). Evaluation of the LABA salmeterol showed a very shallow concentration-response curve for TNFα inhibition, with the maximal inhibition of LPS-stimulated TNFα of 52 ± 4% (0.1 μM) and IC50 of 0.33 pM (C.I. 0.006–19) (Fig. 1A). PBMC pretreatment with roflumilast resulted in a concentration-dependent inhibition of LPS-induced TNFα secretion with an IC50 of

Discussion

A recent advance in COPD treatment was the US and European approval of roflumilast, which can be used as an add-on therapy to LABA treatment. The mechanism of action of both salmeterol and roflumilast suggests that the combined use of these drugs may be complementary, providing both bronchodilatory and anti-inflammatory effects, and potentially providing a more efficacious alternative treatment. Recent clinical trial results showed that when roflumilast treatment is given in combination with

Conflict of interest statement

The authors of this manuscript are all employees of Gilead Sciences Incorporated.

Acknowledgments

The authors would like to thank Prof. Mark Giembycz and Prof. Robert Newton from the University of Calgary for their helpful discussions during the course of these studies, and Lixin Shao from the Gilead department of Clinical Data Management for assistance with statistical analysis.

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