Effects of New NSAID-CAI Hybrid Compounds in Inflammation and Lung Fibrosis

Abstract

Pulmonary fibrosis is a severe lung disease with progressive worsening of dyspnea, characterized by chronic inflammation and remodeling of lung parenchyma. Carbonic anhydrases are a family of zinc-metallo-enzymes that catalyze the reversible interconversion of carbon-dioxide and water to bicarbonate and protons. Carbonic Anhydrase Inhibitor (CAI) exhibited anti-inflammatory effects in animals with permanent-middle-cerebral artery occlusion, arthritis and neuropathic pain. The pharmacological profile of a new class of hybrid compounds constituted by a CAI connected to a Nonsteroidal-Anti-Inflammatory Drug (NSAID) was studied in the modulation of inflammation and fibrosis. In-vitro tests were performed to assess their effects on cyclo-oxygenase enzyme (COX)-1 and COX-2, namely inhibition of platelet aggregation and thromboxane B2 production in the human-platelet-rich plasma, and reduction of Prostaglandin-E2 production in lipopolysaccharide-treated-RAW-264.7 macrophage cell line. The activity of compound 3, one of the most active, was studied in a model of bleomycin-induced lung fibrosis in C57BL/6 mice. The hybrid compounds showed a higher potency in inhibiting PGE₂ production, but not in modifying the platelet aggregation and the TXB₂ production in comparison to the reference molecules, indicating an increased activity in COX-2 inhibition. In the in-vivo murine model, the compound 3 was more effective in decreasing inflammation, lung stiffness and oxidative stress in comparison to the reference drugs given alone or in association. In conclusion, these CAI-NSAID hybrid compounds are promising new anti-inflammatory drugs for the treatment of lung chronic inflammatory diseases.

Keywords: CAI; COX-1; COX-2; NSAIDs; inflammation; pulmonary fibrosis.

Figure 1

Chemical structures. (A) celecoxib 1 and valdecoxib 2. (B) chemical structures and molecular weights of the studied compounds 3–6 [7].

Figure 2

Effects of different concentrations of (A) CAI-ibuprofen, (B) CAI-naproxen, (C) CAI-ketoprofen and (D) CAI-sulindac on lipopolysaccharide (LPS)-induced prostaglandin E₂ (PGE₂) production in RAW 264.7 cells. The cells were pretreated with the studied compounds (100 nM, 1 µM, 10 µM and 100 µM) for 1 h, followed by LPS (1 μg/mL) stimulation for 18 h. PGE₂ production analysis was carried out by using cell culture supernatants. PGE₂ production of Control (naïve cells not treated with LPS) is 36.279 ± 0.67 pg/mL. Values represent the mean ± SEM of eight independent experiments (^# p < 0.05 between a Nonsteroidal Anti-Inflammatory Drug (NSAID) and a Carbonic Anhydrase Inhibitor connected to a Nonsteroidal Anti-Inflammatory Drug (CAI-NSAID)).

Figure 3

Percentage inhibition of platelet aggregation. Effects of different concentrations of (A) CAI-ibuprofen, (B) CAI-naproxen, (C) CAI-ketoprofen and (D) CAI-sulindac after 5 min of ADP (10 µM) incubation in human platelet-rich plasma (PRP). Platelet aggregation baseline is 79.37%, corresponding to 100% of aggregation. Values represent the mean ± SEM of 8 independent experiments, (ns = non-significant between selected columns).

Figure 4

Thromboxane B₂ production. Effects of different concentrations of (A) CAI-ibuprofen, (B) CAI-naproxen, (C) CAI-ketoprofen and (D) CAI-sulindac after 5 min of ADP (10 µM) incubation in human PRP. Values represent the mean ± SEM of eight independent experiments (* p < 0.05 between selected columns; ns = non-significant).

Figure 5

(A) Functional measurement of bronchoconstriction. Lung resistance to airflow measured through the evaluation of pressure at airway opening (PAO). Spirometric evaluation (mm on charts) of different treatments. (B) Variations of body weight after 3 weeks of treatments; (C) measurement of lung weight after bleomycin treatment in presence or absence of various treatments. Values represent the mean ± SEM of 6 individual mice from each group (^### p < 0.001 vs. Naive; ^# p < 0.05 vs. Naive; * p < 0.05 vs. Vehicle).

Figure 6

(A) Lung histology of collagen deposition. Histopathological evaluation of fibrosis by picro-sirius staining. By computer-aided densitometry analysis, it is possible to obtain a semiquantitative measure of this accumulation. n = 6 animals/group. Data are mean ± SEM, ^# p < 0.05 vs. Naive, * p < 0.05 vs. Vehicle. Scale bar, 100 µm (B) Histopathological evaluation of airway remodeling by hematoxylin-eosin staining. n = 6 animals/group. Data are mean ± SEM. ^# p < 0.05 vs. Naive, * p < 0.05 vs. Vehicle. Scale bar, 50 µm (C) Goblet cell number in PAS-stained lung sections (see the arrows) is evaluated in each experimental group. n = 6 animals/group. Data are mean ± SEM. ^# p < 0.05 vs. Naive, * p < 0.05 vs. Vehicle. Scale bar, 50 µm.

Figure 7

Determination of pro-inflammatory (Interleukin-1β (IL-1 β) and Tumor Necrosis Factor-α (TNFα)), anti-inflammatory (IL-10) and pro-fibrotic (Transforming Growth Factor-β (TGF-β)) cytokines. Analysis of (A) IL-1 β, (B) TNFα, (C) IL-10 and (D) TGF-β content in the supernatant of lung tissue homogenates. Values represent the mean ± SEM of six individual mice from each group performed in duplicate and expressed as pg/μg of total proteins determined over an albumin standard curve. ^# p < 0.05 and ^## p < 0.005 vs. Naive; ^### p < 0.001 vs. Naive; * p < 0.05 vs. Vehicle; ** p < 0.005 vs. Vehicle.

Figure 8

Evaluation of oxidative stress parameter in lung tissue. (A) Levels of myeloperoxidase (MPO), a marker for leukocyte accumulation in inflamed tissues. (B) Levels of 8-OHdG, a marker of free radicals-induced DNA damage; (C) dihydroethidium (DHE), a morphological marker of DNA damage and the densitometer evaluation. n = 6 animals/group. Values are mean ± SEM. ^# p < 0.05 vs. Naive. ^### p < 0.001 vs. Naive. * p < 0.05 vs. Vehicle. *** p < 0.001 vs. Vehicle. Scale bar, 100 µm.