Anti-inflammatory effect of a selective IκB kinase-beta inhibitor in rat lung in response to LPS and cigarette smoke

Abstract

Rationale

IκB kinase (IKK) activates NF-κB which plays a pivotal role in pro-inflammatory response in the lung. NF-κB has been shown to be activated in alveolar macrophages and peripheral lungs of smokers and patients with chronic obstructive pulmonary disease. We investigated the anti-inflammatory effect of a highly selective and novel IKKβ/IKK2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[γ]indazole-3-carboxamide], in lungs of rat in vivo.

Methods

Adult Sprague-Dawley rats were administered orally with PHA-408 (15 and 45 mg/kg) daily for 3 days and exposed to LPS aerosol (once on day 3, 2 h post-last PHA-408 administration) or cigarette smoke (CS; 2 h after PHA-408 administration for 3 days). Animals were sacrificed at 1, 4 and 24 h after the last exposure, and lung inflammatory response and NF-κB activation were measured.

Results

Oral administration of IKKβ/IKK2 inhibitor PHA-408 significantly inhibited LPS- and CS-mediated neutrophil influx in bronchoalveolar lavage (BAL) fluid of rats. The levels of pro-inflammatory mediators in BAL fluid (CINC-1) and lungs (IL-6, TNF-α, IL-1β and GM-CSF) were also reduced by PHA-408 administration in response to LPS or CS exposures. The reduced pro-inflammatory response in PHA-408-administered rats was associated with decreased nuclear translocation and DNA binding activity of NF-κB in response to LPS or CS.

Conclusion

These results suggest that IKKβ/IKK2 inhibitor PHA-408 is a powerful anti-inflammatory agent against LPS- and CS-mediated lung inflammation.

Introduction

Cigarette smoking is the most important risk factor for the development of chronic obstructive pulmonary disease (COPD), which is characterized by chronic inflammatory response in the lungs with a progressive and irreversible airflow limitation [1], [2]. COPD is the sixth leading cause of death in the world and is predicted to become the third most common cause of death by 2020 [3], [4]. Unfortunately, none of the currently available drugs are effective in slowing/controlling the progression of COPD or suppressing the pro-inflammatory response in the lung [5]. In recent years, an increasing number of studies have been conducted to investigate the therapeutic potential of small molecule inhibitors of signal transduction proteins such as IκB kinase (IKK), phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase to inhibit various pro-inflammatory pathways [5], [6], [7], [8], [9], [10], [11], [12]. We are interested in nuclear factor-kappaB (NF-κB) pathway as a target for therapeutic intervention since NF-κB is activated in lungs of patients with COPD and plays a pivotal role in chronic pro-inflammatory response seen in COPD [13], [14], [15], [16], [17], [18]. NF-κB is an inducible pleiotropic transcription factor that plays a key role in the expression of multiple genes, leading to the synthesis of pro-inflammatory mediators such as cytokines and chemokines [12], [19], [20], [21]. Therefore, inhibition of cigarette smoke (CS)-induced in lung pro-inflammatory response using small molecule inhibitors for NF-κB activation is an area of research interest for therapeutic approaches and strategies to treat patients with COPD.

In resting cells, the majority of NF-κB RelA/p65-p50 is bound to IκBα protein that holds the complex inactive in the cytoplasm. Upon stimulation/pro-inflammatory stimuli, IKK is activated, which leads to phosphorylation (at Ser32 and Ser36 residues) and subsequent proteasomal degradation of IκB protein. Upon degradation of IκBα protein, NF-κB is translocated into the nucleus and binds to the consensus sequences on DNA, which can lead to pro-inflammatory gene transcription. Thus, degradation of IκBα plays a crucial role in activation of NF-κB and pro-inflammatory gene transcription. It has been shown that IKKβ/IKK2-mediated degradation of IκBα and NF-κB activation is induced by a number of pro-inflammatory stimuli including CS, lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α and interleukin (IL)-1β [22], [23], [24], [25]. Therefore, modulation of NF-κB activity by IKK inhibitors could be useful in controlling the lung inflammation in patients with COPD.

IKK complex consists of IKKα/IKK1, IKKβ/IKK2 and the regulatory subunit IKKγ/NEMO. IKK2 is the primary kinase that phophorylates IκBα [10], [22] and is required for the cytokine-mediated activation of NF-κB [26]. Due to the critical role of IKK2 in inflammation, a number of small molecule IKK2 inhibitors are under development or in preclinical/clinical trials to prevent inflammation against various pro-inflammatory stimuli in vitro or in vivo[7], [27], [28].

In the light of activation of NF-κB in COPD, we hypothesized that inhibition of endogenous IKK2 using a selective small molecule inhibitor would reduce NF-κB activation and the ensuing lung inflammatory response to pro-inflammatory stimuli. To test this hypothesis, we used acute LPS- and CS-exposure as a model of lung inflammation in rat lungs in vivo to determine the anti-inflammatory effect of a selective IKK2 inhibitor PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[γ]indazole-3-carboxamide] [29], [30], [31]. PHA-408 is a novel, highly selective IKK2 inhibitor, which has greater selectivity for IKK2 over IKK1 and number of other kinases with the pharmacokinetics of IC₅₀ (40 ± 2 nM) in vitro, EC₅₀ (27–29 mg/kg) [29], [30] with a half-life of 3.4 h and good oral bioavailability (50–60%) in rats [31]. It is distinct from other IKK2 inhibitors because it binds IKK2 tightly with slow off-rate kinetics/clearance (11.5 ± 2.1 mL/min/kg), and inhibits IKK2 in cell-free systems and living cells with equal potency [29]. Hence, we determined whether targeted inhibition of IKK2 by PHA-408 decreases the LPS- or CS-mediated inflammation in vivo in rat lung in a dose- and time-dependent manner.