Hesperetin-5,7,3’-O-triacetate suppresses airway hyperresponsiveness in ovalbumin-sensitized and challenged mice without reversing xylazine/ketamine-induced anesthesia in normal mice

We recently reported that hesperetin-5,7,3’-O-triacetate (HTA) dually inhibited phosphodiesterase (PDE)3/4 with a therapeutic ratio of 20.8. The application and development of PDE4 inhibitors for treating asthma or COPD are limited by their side effects, such as nausea, vomiting and gastric hypersecretion. PDE4 inhibitors were reported to reverse xylazine/ketamine-induced anesthesia in rats and triggered vomiting in ferrets. Thus the reversing effect of HTA on xylazine/ketamine-induced anesthesia in mice was studied to assess emetic effect of HTA. The aim of this study was to prove the therapeutic effect of HTA without vomiting effect at an effective dose for treating COPD. Ten female BALB/c mice in each group were sensitized by ovalbumin (OVA) on days 0 and 14. On day 21, these mice were emphasized the sensitization by Freund’s complete adjuvant. Mice were challenged by 1% OVA nebulization on days 28, 29, and 30. Airway hyperresponsiveness (AHR) was assessed on day 32 in each group, using the FlexiVent system to determine airway resistance (RL) and lung dynamic compliance (Cdyn) in anesthetized ovalbumin (OVA)-sensitized and challenged mice. Each group was orally administered HTA (10 ~ 100 μmol/kg), roflumilast (1 and 5 mg/kg) or vehicles (controls) 2 h before and 6 and 24 h after OVA provocation. For comparison, sham-treated mice were challenged with saline instead of 1% OVA. The ability to reverse xylazine/ketamine-induced anesthesia by HTA or roflumilast for 3 h was determined in normal mice. We used roflumilast, a selective PDE4 inhibitor and bronchodilator for severe COPD approved by the US Food and Drug Administration, as a reference drug. In the results, HTA (100 μmol/kg, p.o.) or roflumilast (5 mg/kg, p.o.) significantly suppressed all RL values of MCh at 0.78 ~ 25 mg/mL and enhanced Cdyn values of MCh at 3.125 ~ 25 mg/mL compared to OVA-sensitized and -challenged control mice. Orally administered 1, 3 or 10 mg/kg roflumilast, but not 30 or 100 μmol/kg HTA, significantly reversed xylazine/ketamine-induced anesthesia. In contrast to roflumilast, HTA may ameliorate COPD but induce few side effects of nausea, vomiting and gastric hypersecretion at an effective dose for treating COPD, because HTA did not reverse xylazine/ketamine-induced anesthesia in mice.


Background
It is known that phosphodiesterases (PDEs) comprise at least 11 distinct enzyme families that hydrolyze adenosine 3′,5′ cyclic monophosphate (cAMP) and/or guanosine 3′,5′ cyclic monophosphate (cGMP) [1]. PDE3 and PDE4 families are cGMP-inhibited and cAMP-specific, respectively. PDE4 may have high (PDE4 H ) and low (PDE4 L ) affinities for rolipram. In general, it is believed that inhibition of PDE4 H is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, while inhibition of PDE4 L is associated with anti-inflammatory and bronchodilating effects. Therefore, the therapeutic ratio of selective PDE4 inhibitors for treating asthma and chronic obstructive pulmonary disease (COPD) is defined as the PDE4 H /PDE4 L ratio [2].
Hesperetin (5,7,3'-trihydroxy-4'-methoxyflavanone) was reported to selectively inhibit PDE4 activity [3], and is used as a lead compound to synthesize hesperetin-5,7,3'-O-triacetate (HTA), a more-liposoluble derivative of hesperetin. HTA was reported to dually inhibit PDE3/4 with a therapeutic (PDE4 H /PDE4 L ) ratio of 20.8 [4], which is greater than that of roflumilast [5], a selective PDE4 inhibitor. Roflumilast was approved by the European Commission [6], and the US Food and Drug Administration (FDA) [4] as an adjunct to bronchodilator therapy for severe COPD associated with chronic bronchitis in adults with a history of frequent exacerbations. However, dual PDE3/4 inhibitors are reported to have additive or synergistic antiinflammatory and bronchodilator effects compared to PDE3 or PDE4 inhibitors alone [7]. In other words, the real therapeutic ratio of dual PDE3/4 inhibitors should be greater than that reported [4]. Therefore, we were interested in investigating the suppressive effects of HTA on ovalbumin (OVA)-induced airway hyperresponsiveness (AHR), and clarifying its potential for treating atypical asthma and COPD [8]. In this animal model, the number of neutrophils in the bronchoalveolar lavage fluid of control sensitized and challenged mice was significantly greater than that of eosinophils [8]. AHR was previously assessed by barometric plethysmography [9] using a whole-body plethysmograph in unrestrained animals. However, the determination of enhanced pause does likely not reflect lung mechanics [10,11]. Thus AHR in the present study was assessed using the FlexiVent system to determine the airway resistance (R L ) and lung dynamic compliance (C dyn ) in anesthetized ventilated mice. The application and development of PDE4 inhibitors for treating asthma and COPD are limited by their side effects, such as nausea, vomiting and gastric hypersecretion [2]. PDE4 inhibitors were reported to reverse xylazine/ketamine-induced anesthesia in rats [12] and triggered vomiting in ferrets [13]. Thus the reversing effect of HTA on xylazine/ketamine-induced anesthesia in mice was used to assess emetic effect of HTA. The aim of this study was to prove the therapeutic effect of HTA without vomiting effect at effective dose for treating COPD. To compare the therapeutic and gastrointestinal (GI) side effects of HTA, roflumilast was used as a reference drug.
Female BABL/c mice at 8~12 weeks old were purchased from the Animal Center of the Ministry of Science and Technology (Taipei, Taiwan), housed in ordinary cages at 22 ± 1°C with a humidity of 50%~60% under a constant 12/12-h light/dark cycle and provided with OVA-free food and water ad libitum [8]. Under a protocol approved (LAC-100-0152) on May 4, 2012 by

AHR in vivo
In accordance with a previously published protocol [8], ten female BALB/c mice in each group were sensitized by an intraperitoneal (i.p.) injection of 20 μg of OVA emulsified in 2.25 mg of an aluminum hydroxide gel, prepared from aluminum sulfate hexadecahydrate, in a total volume of 100 μL on days 0 and 14. On day 21, these mice were (i.p.) injected with 100 μL of a mixture of 1% OVA and Freund's complete adjuvant (1:1). Mice were challenged via the airway using 1% OVA in saline for 30 min on days 28, 29, and 30 by ultrasonic nebulization. After the last OVA challenge [15], AHR was assessed on day 32 (48 h after 1% OVA provocation) in each group. Each group of mice was orally (p.o.) administered HTA (10~100 μmol/ kg), roflumilast (1 and 5 mg/kg) or vehicles (controls) 2 h before and 6 and 24 h after OVA provocation. For comparison, sham-treated mice were challenged with saline instead of 1% OVA (non-challenged). A mixture of DMSO: ethyl alcohol: PEG 400: saline (0.5: 0.5: 1: 8, v/v) or PBS was the vehicle for the control of HTA or roflumilast, respectively. The vehicles were administered (p.o.) at a volume of 0.01 mL/g of body weight. Mice showed no abnormal behavior after oral administration of the vehicle.
In accordance with a previously described method [8], anesthetized (urethane 600 mg/kg and chloralose 120 mg/kg, i.p.), tracheostomized (stainless-steel cannula, 18 G) mice were mechanically ventilated (at 150 breaths/min, with a tidal volume of 10 mL/kg and a positive end-expiratory pressure of 3 cmH 2 O). Prior to PBS nebulization for 10 s the baseline R L and C dyn were determined. Then the AHR of mice was assessed by measuring changes in the R L and C dyn after being challenged with aerosolized MCh (0.78, 1.563, 3.125, 6.25, 12.5, and 25 mg/mL) for 10 s using the FlexiVent system (SCIREQ, Montreal, Quebec, Canada), in which these data were automatically saved for 3 min after 10 s of nebulization.

Xylazine/Ketamine-induced anesthesia
According to previously reported methods [8,16], after loss of the righting reflex (i.e., when a mouse remains on its back and no longer spontaneously rights itself to a prone position), the duration of anesthesia was measured until its return as the endpoint. The ability to reverse xylazine/ketamine-induced anesthesia by oral administration of HTA, roflumilast or their vehicles for 3 h was determined in female BALB/c mice.

Statistical analysis
Differences among values given as the mean ± standard error of the mean (SEM) were calculated by a one-way analysis of variance (ANOVA), and then determined by Dunnett's test. The difference between two values, however, was determined by Student's t-test. Significance was accepted when p < 0.05.

Discussion
HTA dually inhibits PDE3/4, whereas roflumilast selectively inhibits PDE4 activity. Thus degradation of cAMP, an important secondary messenger, is prevented by them and the intracellular cAMP content indirectly increases [15,[17][18][19]. Increased cAMP activates cAMP-dependent protein kinase, inhibits myosin light-chain kinase, and results in bronchodilation. Thus the R L decreased and the C dyn was enhanced. These results suggest that HTA would have benefits in treating COPD, although no evidence was found to support it having benefits for treating atypical asthma.
The application and development of PDE4 inhibitors in treating asthma and COPD are limited by their side effects, such as nausea, vomiting and gastric hypersecretion [2]. Rolipram, a first generation PDE4 inhibitor, has a therapeutic ratio of 0.002 [20] and has many side effects. Cilomilast and roflumilast have therapeutic ratios of 1 and 3, respectively [5,21]. Recently, roflumilast was approved by the European Commission [6] and the US FDA [4] as an add-on to bronchodilator therapy for maintenance treatment of severe COPD associated with chronic bronchitis in adults with a history of frequent exacerbations.
Robichaud et al. reported that MK-912, an α 2 -adrenoceptor antagonist, reversed xylazine/ketamine-induced anesthesia in rats [12] and triggered vomiting in ferrets [13]. In contrast, clonidine, an α 2 -adrenoceptor agonist, prevented emesis in ferrets [13]. Thus they suggested that the reversing effect occurred through presynaptic Fig. 3 Effects of orally administered HTA (a) and roflumilast (b) on the duration of xylazine (10 mg/kg, i.p.)/ketamine (70 mg/kg, i.p.)-induced anesthesia in mice. * p < 0.05, *** p < 0.001, compared to the control. Each value represents the mean ± SEM. The number of mice in each group was 5~8 α 2 -adrenoceptor inhibition [13]. They also found that PDE4 inhibitors reversed xylazine/ketamine-induced anesthesia in rats and ferrets [12,13]. Thus the reversing effect of PDE4 inhibitors on xylazine/ketamine-induced anesthesia in rats or mice is convenient and could be a surrogate for assessing the emetic effects of these drugs, as rodents have no emetic reflex and we cannot observe emesis. In the present results, orally administered HTA at 300 μmol/kg (approximately 128.5 mg/kg) and roflumilast at 1~10 mg/kg significantly reversed xylazine/ketamine-induced anesthesia in mice, whereas orally administered HTA at 100 μmol/kg or roflumilast at 5 mg/kg significantly reduced the R L and enhanced the C dyn . HTA even at 30 μmol/kg also reduced the R L , although did not enhance the C dyn .

Conclusions
In contrast to roflumilast, HTA may ameliorate COPD but induce few side effects of nausea, vomiting and gastric hypersecretion at a dose effective for treating COPD, because HTA did not reverse xylazine/ketamineinduced anesthesia in mice.