Background: Quercetin-3,5,7,3’,4’-O-pentamethylether (QPME) was reported to the most potently relax histamine-, carbachol- and KCl-induced contractions in isolated guinea pig tracheas. Whether QPME has a potential to reverse airway hyperresponsiveness (AHR) in murine allergic asthma is the aim of this study.

Methods: The Lineweaver-Burk analyses of QPME were performed. The AHR was analyzed by determining airway resistance (RL) and lung dynamic compliance (Cdyn) using the FlexiVent system. Cytokines in the bronchoalveolar lavage fluid (BALF) were determined using mouse T helper (Th)1/Th2 cytokine cytometric bread array (CBA) kits. Total and ovalbumin (OVA)-specific immunoglobulin (Ig)E in the BALF and serum were determined using enzyme-linked immunosorbent assay (ELISA) kits. The number of inflammatory cells in the BALF were counted under microscope. QPME whether reverses xylazine/ketamine-induced anesthesia in mice as a surrogate for determining its emetic side effects.

Results: QPME may be a dual PDE3/4 inhibitor, because of their dissociation constant for inhibitory binding (Ki) values did not significantly differ from each other. QPME (10~100 μmol/kg, intraperitoneally) significantly attenuated RL and enhanced Cdyn values in control mice. It significantly inhibited all inflammatory cells and cytokines, including interleukin (IL)-2, IL-4, IL-5, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the BALF. QPME significantly reduced the total and OVA-specific IgE in the serum and BALF. QPME may inhibit degranulation of mast cells.

Conclusion: Thus, QPME may be useful for treating allergic asthma.

Abbreviations: AHR: Airway Hyperresponsiveness; BALF: Bronchoalveolar Lavage Fluid; BSA: Bovine Serum Albumin; cAMP: adenosine 3',5' cyclic monophosphate; CBA: Cytometric Bread Array; C_dyn: Lung Dynamic Compliance; cGMP: guanosine 3',5' cyclic monophosphate; DMSO: Dimethyl sulfoxide; EDTA: Ethylenediaminetetraacetic Acid; EHNA: erythro-9-(2-hydroxy-3-nonyl)-adenine HCl; ELISA: Enzyme-linked Immunosorbent Assay; IFN-γ: interferon-γ; Ig: Immunoglobulin; IL: Interleukin; Ki: Dissociation constant for inhibitory binding; MCh: Methacholine; OVA: Ovalbumin; PBS: phosphate-buffered saline; PDE: phosphodiesterase; PMSF: phenylmethanesulfonyl fluoride; QPME: quercetin-3,5,7,3’,4’-O-pentamethylether; RL: airway resistance; Ro 20-1724: 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone; Th: T helper; TNF-α: tumor necrosis factor-α.

Phosphodiesterases (PDEs) hydrolyze adenosine 3',5' cyclic monophosphate (cAMP) and/or guanosine 3',5' cyclic monophosphate (cGMP) and have 11 distinct enzyme families nowadays [1]. Rolipram, a prototype PDE4-selective inhibitor, has high (PDE4_H) and low (PDE4_L) affinities for PDE4, respectively. It is believed that PDE4_H and PDE4_L are respectively related to an adverse effect and to inflammatory suppression and bronchodilatory actions. Thus, the PDE4_H/PDE4_L ratio is used as a therapeutic ratio of selective PDE4 inhibitors for treating asthma and chronic obstructive pulmonary disease (COPD) [2,3].

Men with higher quercetin intakes from fruits and vegetables have lower lung cancer and asthma incidences [4]. Among quercetin and quercetin-methylethers, such as quercetin-3-O-methylether (3-MQ), quercetin-4’-O-methylether (tamarixetin), quercetin-3,4’,7-O-trimethylether (ayanin), quercetin-3,3’,4’,7-O-tetramethylether (QTME), and quercetin-3,5,7,3’,4’-O-pentamethylether (QPME), QPME has been reported to the most potently relax histamine-, carbachol- or KCl-induced contractions in isolated guinea pig tracheas. The potency order is QPME>3-MQ>quercetin, ayanin>tamarixetin ≈ QTME [5]. The mechanisms of the relaxant action of QPME were reported to inhibit calcium release from intracellular calcium stores, calcium influx from the extracellular space, and activities of cAMP- and cGMP-PDE, in which the inhibition on cAMP- was significantly greater than that on cGMP-PDE by QPME [6]. Whether QPME has inhibitory actions on airway hyperresponsiveness (AHR) in a mouse model of allergic asthma was the aim of this study.

Materials and Methods

Reagents and animals

QPME (mol. wt., 372) was synthesized by ourselves according to the previous method.7 Bovine serum albumin (BSA), chloralose, dimethyl sulfoxide (DMSO), Erythro-9-(2-hydroxy-3-nonyl)-adenine HCl (EHNA), ketamine, methacholine (MCh), milrinone, ovalbumin (OVA), Ro 20-1724, rolipram, urethane, vinpocetin, xylazine and zaprinast were purchased from Sigma Chemical (St. Louis, MO, USA). Freund’s adjuvant (Mycobacterium butyricum) was purchased from Pierce Biotechnology (Rockford, IL, USA). Mouse T helper (Th)1/Th2 cytokine CBA kits were purchased from Pharmingen (San Diego, CA, USA). Polyethyleneglycol (PEG) 400 and ethyl alcohol were purchased from Merck (Darmstadt, Germany). QPME was dissolved in a mixture of ethyl alcohol and DMSO (1: 1). Other drugs were dissolved in distilled water. The final concentration of ethyl alcohol or DMSO was ≤ 0.1%, and did not significantly affect isolated guinea pig tracheal contractions and behavior of normal mice.

Female BALB/c mice at 8~12 weeks of age and male Hartley guinea pigs (500~600 g) were purchased from the Animal Center of Ministry of Science and Technology, Taipei, Taiwan. Under a protocol approved by the Animal Care and Use Committee of Taipei Medical University, the following in vivo and in vitro experiments were performed.

Inhibition of PDE3 and PDE4 activities by QPME

According to the previous method, [7,8] the Lineweaver-Burk analyses for QPME and selective PDE1-5 inhibitors, such as vinpocetine [9], EHNA [10], milrinone [11], rolipram [12], and zaprinast [13], as reference drugs at various concentrations including its vehicle (0 μM, control) were performed. The total protein was assayed according to the previous method [14]. PDE activities are reported as nmol/mg/min.

AHR in vivo

In accordance with the previous method and schedule [15], ten mice in each group were sensitized by an intraperitoneal (i.p.) injection of 20 μg of OVA emulsified in 2.25 mg aluminum hydroxide gel in a total volume of 100 μl on days 0 and 14. On days 28, 29, and 30, these mice were challenged via the airway with 1% OVA in saline for 30 min by ultrasonic nebulization. On day 32, the AHR of female BABL/c mice was assessed by determining airway resistance (R_L, cmH₂O/ml/sec) and lung dynamic compliance (C_dyn, ml/cmH₂O) before and after challenge with aerosolized methacholine (MCh, 0.78~25 mg/ml) using the FlexiVent system (SCIREQ, Montreal, Quebec, Canada). Under anesthesia (urethane 600 mg/kg and chloralose 120 mg/kg, i.p.), the tracheostomized (stainless-steel cannula, 18 G) mice were ventilated mechanically at 150 breaths/min, with a tidal volume of 10 ml/kg, positive end-expiratory pressure of 3 cmH2O.

Unrestrained mice, sensitized and challenged as same as the above described method, were placed into the main chamber of a whole-body plethysmograph, and nebulized with phosphate-buffered saline (PBS) on day 32 in each group. Subsequently nebulized with MCh 6.25~50 mg/ml for 3 min at each increasing concentration. Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and blood of mice were collected under anesthesia by pentobarbital 50 mg/kg (i.p.). Cytokines in the BALF were determined using mouse T helper (Th)1/Th2 cytokine CBA kits (Pharmingen, San Diego, CA, USA) [15]. Total and OVA-specific immunoglobulin (Ig)E in the BALF and serum were determined using ELISA kits (Pharmingen, San Diego, CA, USA) [15,16]. The number of inflammatory cells in the BALF and differentiation were performed as the previous method [15]. All undetectable data (< 1 pg/ml) of cytokines were taken as 0.5 pg/ml.

OVA-induced contractions in vitro

Guinea pigs were sensitized by intramuscular injections of 0.7 ml of 5% (w/v) OVA in saline on days 1, 4, and 43, and of adjuvant on days 25 and 39 into each thigh, respectively, according to the previous method [17]. Three days after the last injection, under anesthesia by pentobarbital 50 mg/kg (i.p.), the sensitized guinea pigs were sacrificed by cervical dislocation, and their tracheas were dissected. Each trachea was cut into six segments consisting of three cartilage rings for each. All segments were cut open opposite the trachealis. The segments were placed in 5 ml of normal Krebs solution containing indomethacin (3 μM) and gassed with a mixture of 95% O₂ plus 5% CO₂ at 37 °C for the isometric recording of tension changes via force displacement transducers (Grass FT03, Quincy, MA, USA) on a polygraph (Gould RS3200, Cleveland, OH, USA). The composition of the normal Krebs solution was (mM): NaCl 118, KCl 4.7, MgSO₄ 1.2, KH2PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25, and dextrose 10.1. The tissues were equilibrated under an initial tension of 1.5 g and washing at 15-min intervals for at least 1 h. After pre-contracted with KCl (60 mM) and washed with normal Krebs solution, OVA (0.1~100 μg/ml) was cumulatively added, and contractions were allowed to reach a steady state at each concentration. To evaluate the suppressive effect of QPME on OVA-induced contractions, each tissue was pre-incubated with each concentration (3~100 μM) of QPME or its vehicle for 15 min and then challenged with cumulative OVA again. Therefore, the log concentration-response curves of OVA were constructed in the absence and presence of QPME. The tension of the pre-contraction induced by KCl was set to 100%.

Xylazine/ketamine-induced anesthesia

According to the previous methods [15,18], the ability of reversing xylazine/ketamine-induced anesthesia, a surrogate for determining gastrointestinal (GI) side effects, such as nausea, vomiting and gastric hypersecretion, by QPME, Ro 20-1724 or their vehicle was determined in normal female BALB/c mice, respectively.

Statistical analysis

For comparison between two groups, Student’s t-test was applied. When more than two groups were compared, one-way analysis of variance (ANOVA) was used followed by Dunnett’s test. The results are presented as the means ± standard error of the mean (SEM). A P value < 0.05 was considered statistically signi?cant.

Results

Competitive inhibition of PDE1-5 activities by QPME

QPME (3~100 μM) competitively inhibited PDE1-5 activities (Figures 1A-1E upper panel), as 1/V_max values were not significantly influenced by various concentrations in the Lineweaver-Burk analysis. So did the reference drugs, selective PDE1-5 inhibitors used (Figures 1A-1E lower panel). The dissociation constant for inhibitory binding (K_i) values of QPME were respectively calculated to be 16.2 ± 0.2 (n=3), 20.5 ± 0.3 (n=3), 5.3, ± 0.2 (n=3), 6.9, ± 0.1 (n=3) and 27.3 ± 5.6 (n=4) μM (Figures 1A-1E upper inset). The K_i values for PDE3 and PDE4 did not significantly differ from each other, but were significantly less than those for PDE1, 2 and 5 (one-way ANOVA and then Dunnett’s test), suggesting that the affinities of QPME for PDE3 and PDE4 were similar, but greater than those for PDE1, 2 and 5. Thus QPME may be a relatively dual PDE3/4 inhibitor.

Suppression of AHR in vivo

Methacholine (MCh, 0.78~25 mg/ml) concentration-dependently and significantly enhanced airway resistance (R_L) values (Figure 2A), and reduced lung dynamic compliance (C_dyn) values (Figure 2B) in the control sensitized and challenged mice compared to the non-challenged mice. QPME at 10 and 30 μmol/kg (i.p.) significantly reversed these two changes at 25 mg/ml of MCh. The change of RL even at 12.5 mg/ml of MCh was significantly reversed by QPME 30 μmol/kg. QPME at 100 μmol/kg (i.p.) significantly reversed these two changes at 3.125~25 mg/ml of MCh.  The change of RL even at 1.56 mg/ml of MCh was significantly reversed by QPME at the largest dose (Figures 2A and 2B).

Inhibition of inflammatory cells in BALF

Total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils from the BALF of control mice significantly increased when compared to non-challenged mice (Figure 3A). QPME (10~100 μmol/kg, i.p.) significantly inhibited the increase in the all inflammatory cells detected with the exception of macrophages at a dose of 10 μmol/kg (Figure 3A). Interestingly, lymphocytes, neutrophils, and eosinophils were almost completely abolished at each dose used (Figure 3A).

Suppression of cytokines in BALF

The levels of cytokines in the BALF of control mice significantly increased when compared to those of non-challenged mice (Figure 3B). QPME (10~100 μmol/kg, i.p.) also significantly suppressed the increases in levels of these cytokines, with the exceptions of IL-5 and IFN-γ at a dose of 10 μmol/kg (Figure 3B). Noticeably, the levels of TNF-α in control mice were abolished at each dose used (Figure 3B).

Suppression of total and OVA-specific IgE levels in serum and BALF

Total and OVA-specific IgE levels in the serum and BALF of control mice significantly increased when compared to non-challenged mice (Figure 4). QPME (10~100 μmol/kg, i.p.) concentration-dependently and significantly suppressed the increases in total (Figures 4A and 4C) and OVA-specific IgE (Figures 4B and 4D) in the serum (Figures 4A and 4B) and BALF (Figures 4C and 4D) of control mice with the exception of total IgE in serum at a dose of 10 μmol/kg (Figure 4A).

Suppression of OVA-induced contractions in vitro

KCl (60 mM) evoked contractions of isolated sensitized guinea pig trachea and increased tension to 0.63 ± 0.06 g (n=7), which was set as 100%. QPME (10, 30, and 100 μM) concentration-dependently and significantly relaxed the trachealis by 53.2 ± 6.8% (n=5), 59.3 ± 9.7% (n=5), and 276.7 ± 31.8% (n=5) of the baseline tension, respectively, when compared to its control (vehicle, Figure 5A). OVA (0.01~100 μg/ml) alone concentration-dependently enhanced tension from the baseline to 111.7% ± 13.9% (n=6) of the KCl-induced contraction (Figure 5B). The log concentration-response curve of OVA was uninfluenced by 1 μM nifedipine, a selective voltage-dependent calcium channel blocker.19 QPME (10~100 μM) concentration-dependently and significantly inhibited OVA (10~100 μg/ml)-induced contractions, when compared to the vehicle (Figure 5B).

Xylazine/ketamine-induced anesthesia

The durations of xylazine/ketamine-induced anesthesia in vehicle (control) of QPME- and Ro 20-1724-treated mice were 25.4 ± 3.3 and 20.4 ± 2.4 min, respectively, which did not significantly differ from each other. QPME (10~100 μmol/kg, s.c.) did not influence the duration (Figure 6A), but Ro 20-1724 at 0.1 and 1 μmol/kg (s.c.) significantly reversed the duration (Figure 6B).

Allergic asthma is a chronic respiratory disease characterized by AHR, mucus hypersecretion, bronchial inflammation, and elevated IgE levels. Th2 cells, together with other inflammatory cells such as eosinophils, B cells, and mast cells, have been proposed to play critical roles in the initiation, development, and chronicity of this disease [20]. One hypothesis emphasizes an imbalance in the Th cell population favoring expression of Th2 over Th1 cells. Cytokines released from Th2 cells are IL-4, -5, -6, -9, -13, and TNF-α, and those from Th1 cells are IL-2, -12, and IFN-γ [21]. In the present results, QPME suppressed levels of IL-2, -4, -5, TNF-α, and IFN-γ, suggesting that QPME selectively suppresses neither Th1 nor Th2 cells. The Th1 and Th2 cells are respectively implicated in autoimmune and atopic diseases [22]. QPME also suppressed total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils in the BALF of mice.

IL-4 and IL-13 have been shown to induce AHR in mouse asthmatic models [23,24]. IL-4 has three primary effects. First, IL-4 promotes B cell differentiation to plasma cells that secrete antigen-specific IgE antibodies. Second, promotes mast cell proliferation. Third, upregulates endothelial cell expression of adhesion molecules for eosinophils [25]. IL-13 was reported to be a central mediator of AHR [26,27]. In addition, IL-5 mobilizes and activates eosinophils, leading to the release of a major basic proteins, cystenyl-leukotrienes, and eosinophil peroxidase that contribute to the tissue damage and AHR [24,28]. Phosphoinositide 3-kinase d (p110d) has been shown to play a crucial role in the development, differentiation, and antigen receptor-induced proliferation of mature B cells [29,30] and inhibition of p110d attenuates allergic airway inflammation and AHR in a mouse asthmatic model [30,31]. IL-4 and IL-13 are also important in directing B cell growth and differentiation, and secretion of IgE [32]. The activities of IgE are mediated through the high-affinity IgE receptor (FceRI) on mast cells and basophils. Cross-linking of the FceRI initiates signaling cascades leading to cellular degranulation and activation [33,34]. The activity of p110d was reported to be critical for allergen-IgE-induced mast cell degranulation and release of cytokines [35]. Inhibition of p110d therefore attenuates the production of IgE as well as allergen-IgE-induced mast cell activation during allergic inflammation. The calcium channels in mast cell membranes have been proposed to differ from those in cardiovascular and other smooth muscle tissues [36], which are sensitive to nifedipine. In the present in vitro results, QPME concentration-dependently inhibited the cumulative OVA-induced contractions of isolated sensitized guinea pig trachealis, which were uninfluenced by 1 μM nifedipine, suggesting that QPME inhibits calcium channels and degranulation of mast cells. Therefore, this inhibition by QPME may be associated to its relatively selective inhibition on PDE3/4.

Cyclic AMP has important regulatory roles in inflammation. Selective PDE4 inhibitors specifically prevent the hydrolysis of cAMP, and therefore have broad anti-inflammatory effects such as inhibition of cell trafficking, and cytokine and chemokine release from inflammatory cells. These PDE4 inhibitors may be limited by their clinical potency when using doses that have minimal nausea and vomiting. The PDE4_H/PDE4_L ratios of cilomilast and roflumilast have respectively been reported to be 1 [37] and 3 [38], respectively. Owing to its low PDE4_H/PDE4_L ratio, cilomilast was discontinued for use against asthma after phase II clinical trials in 2003 [39]. Roflumilast was approved by the European Commission [40] and the US Food and Drug Administration [2] as a bronchodilator therapy for severe COPD, and recently reported to reverse xylazine/ketamine-induced anesthesia in mice suggesting that roflumilast may have emetic side effects [41]. Thus, selective PDE4 inhibitors were limited their development [42]. The PDE4_H/PDE4_L ratio of AWD 12-281, another selective PDE4 inhibitor, has been calculated to be approximately 11 [43]. AWD 12-281 has been undergoing clinical development phase IIa trials for COPD, and has been reported to be a unique potential drug for the topical treatment of asthma and COPD [44]. Recently, AWD 12-281 was reported to be a very promising drug candidate not only for treating lung inflammation by inhalation but also for treating atopic dermatitis [45].

Similar to AWD 12-281, the PDE4_H/PDE4_L ratio of QPME was also reported to be 11 [46]. QPME dually and selectively inhibits PDE3/4 in the present results. Owing to dual PDE3/4 inhibitors are reported to have additive or synergistic anti-inflammatory and bronchodilator effects compared to PDE3 or PDE4 inhibitors alone [42]. Thus, the real therapeutic ratio of dual PDE3/4 inhibitors should be greater than that reported. In the present results, QPME (10-100 µmol/kg, s.c.) did not influence xylazine/ketamine-induced anesthesia suggesting that QPME may have few or no GI adverse effects. In contrast, Ro 20-1724, a selective PDE4 inhibitor, reversed the anesthesia. The reversing effect has been suggested to occur through presynaptic α2-adrenoceptor inhibition [47], because MK-912, an α2-adrenoceptor antagonist, was reported to reverse xylazine/ketamine-induced anesthesia in rats [48] and trigger vomiting in ferrets [47]. In contrast, clonidine, an α2-adrenoceptor agonist, prevented emesis induced by PDE4 inhibitors in ferrets [47].

QPME, a quercetin-methylether, may have few GI side effects. Therefore, QPME has the potential for treating allergic asthma.

Authors contributions: WCK conceived and designed the study. SMT performed the experiments and analyzed the data. CMC synthesized and identified the compound QPME. WCK and SMT wrote the manuscript. All the authors read and approved the final manuscript.

Conflict of interests: The authors declare that there is no con?ict of interest regarding the publication of this paper.

Acknowledgements: We gratefully acknowledge that this work was supported by a grant (96TMU-WFH-07) from Taipei Municipal Wan-Fang Hospital, Taipei, Taiwan.

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