Xie QM et al / Acta Pharmacol Sin 2003 Mar; 24 (3): 277-282

Comparison of bronchodilating and antiinflammatory activities of oral formoterol and its (R,R)-enantiomersl

XIE Qiang-Min2, CHEN Ji-Qiang, SHEN Wen-Hui, YANG Qiu-Huo, BIAN Ru-Lian

Zhejiang Respiratory Drugs Research Laboratory of State Drugs Administration of China, Medical School of Zhejiang University, Hangzhou 310031, China

1 Project supported by the National Natural Science Foundation of China, No 39970857.

2 Correspondence to Dr XIE Qiang-Min. Phn 86-571-8721-7380. Fax 86-571-8721-7380. E-mail xieqm@zju.edu.cn

Received 2002-03-04 Accepted 2002-08-05

KEY WORDS formoterol; bronchodilator agents; inflammation; eosinophils; asthma; guinea pigs; mice

ABSTRACT

AIM: To compare the bronchodilating and antiinflammatory effects of oral racemic formoterol (rac-FMT) and (R,R)-formoterol (R,R-FMT). METHODS: The changes of lung resistance (RL), dynamic lung compliance (Cdyn), and the accumulation of inflammatory cells in bronchoalveolar lavage fluids (BALF) induced by ovalbumin aerosol in sensitized guinea pigs and mice were investigated in vivo. RESULTS: Mean value increase of RL and mean value reduction of Cdyn from 1 to 30 min after antigen challenge were up to 101 %±34 % and 42 %±7 %, respectively. rac-FMT 0.5, 1.0, and 2.0 mg/kg, and R,R-FMT 0.25, 0.5, and 1.0 mg/kg ig, induced dose-related inhibition of the bronchoconstrictive responses to aerosolised ovalbumin. ID50 (95% confidence limits, 95 % CL) value of rac-FMT on RL maximal increase and Cdyn maximal reduction at 5 min were 0.64 (0.54-0.76) and 1.02 (0.88-1.18) mg/kg, respectively. For R,R-FMT they were 0.46 (0.40-0.53) and 0.52 (0.45-0.61) mg/kg, respectively. ID50 (95 % CL) value of rac-FMT on RL mean increase and Cdyn mean reduction from 1 to 30 min were 0.96 (0.86-1.07) and 1.59 (1.32-1.92) mg/kg, respectively. For R,R-FMT they were 0.52 (0.45-0.59) and 0.43 (0.37-0.51) mg/kg, respectively. Ovalbumin-aerosol challenge induced an increase of inflammatory cells in BALF in sensitized mice. rac-FMT and RR-FMT caused a dose-dependent and almost complete inhibition at 2.0 mg/kg. ID50 (95 % CL) of rac-FMT on the number of total inflammatory cells and eosinophil in BALF were 1.48 (1.22-1.81) and 0.80 (0.62-1.04) mg/kg, respectively. ID50 (95 % CL) of RR-FMT were 0.80 (0.57-1.13) and 0.60 (0.43-0.83) mg/kg, respectively. CONCLUSION: R,R-FMT protected lung against increase of RL and reduction of Cdyn induced by bronchial challenge of ovalbumin in the asthma model of guinea pigs, and inhibited airway inflammation in the sensitized mice. Efficacy of R,R-FMT was approximately 2-fold than that of rac-FMT.

INTRODUCTION

Racemic formoterol (rac-FMT) is a long acting 2 adrenoceptor agonist which is used as a bron chodilator agent in asthma and chronic obstructive pulmonary disease[1,2]. The marketed form of formoterol is a racemic mixture composed of a 50:50 mixture of R and S isomers. Rac-FMT activity resides in the (R,R)-enantiomer whilst the (S,S) form is essentially inactive. Accordingly, pure (R,R)-formoterol (R,R-FMT) provides bronchodilation at lower doses than racemate, allowing for fewer 2-adrenergic-mediated side effects. In addition, different metabolism may allow for the progressive accumulation of (S,S)-formoterol S,SFMT)[3]. The therapeutically active, long-acting 2 agonist R,R-FMT is currently being developed according to the logic, and preliminary results suggest rapid improvements in forced expiratory volume in first second (FEV1) with up to 24-h duration of action in the clinical test[4]. Pharmacological activities of rac-FMT and R,R-FMT have been compared; all have been in vitro involving airway tissue from horse[5], guinea-pig, and man[6] except anti-bronchoconstrictive activities of inhaled rac-FMT and R,R-FMT in the rhesus monkey in vivo recently[7]. We have compared the bronchodilating effect of rac-FMT and R,R-FMT on Chinese bronchus in vitro (unpublished data made by our Lab). In the present study, we compared the bronchodilating and antiinflammatory activities of oral rac-FMT and R,R-FMT in asthma model in vivo.

MATERIALS AND METHODS

Animals Hartley guinea pigs of either sex weighing (315±s 22 g) and NIH male mice weighing (22.5± s 1.5 g) were perchased from Laboratory Animal Center of Medical School of Zhejiang University (Grade II, Certificate No 220010014 conferred by Zhejiang Medical Laboratory Animal Administration Committee).

Drugs Rac-formoterol (purity >99.9 %) and R,R-formoterol (enantiomeric purity >99.6 %) were synthesized at Tianjing Drugs Research Institute; egg albumin (Reagent grade II and V, Sigma, USA); pentobarbital sodium (Union, Belgium); heparin sodium (Xuzhou Biochemical Pharmaceutical Factory).

Ovalbumin-induced changes of RL and Cdyn in sensitized guinea pig

Sensitizing procedures Guinea pigs were sensitized by a single intraperitoneal injection (ip) of ovalbumin (grade II)10 mg mixed with 100 mg of aluminum hydroxide in saline 1.0 mL per animal. These animals were used 25 - 35 d later for aerosol challenge with ovalbumin.

Treatment procedures To determine dose-effect, in our preliminary experiments, guinea pigs were administered with rac-FMT and R,R-FMT of 0.125, 0.25, 0.5, 1.0 , 2.0, and 3.0 mg/kg by intragastric gavage (ig). To determine time-effect, we referenced human pharmacokinetic parameter of oral rac-FMT[8] and time-effect relation of animal pharmacodynamic parameter of intravenous injection (iv) rac-FMT[9]. Finally, the dose-ranges were definited with rac-FMT 0.5, 1.0, and 2.0 mg/kg or R,R-FMT 0.25, 0.5, and 1.0 mg/kg at 1.0 h before antigen challenge.

RL and Cdyn measurement The sensitized guinea pigs were anesthetized with pentobarbital sodium (30 mg/kg) and placed in a whole body plethysmograph for the measurement of RL and Cdyn[10]. The sensitized animals were treated with rac-FMT, R,R-FMT, and saline 1.0 h before a 1-min exposure to an aerosol of ovalbumin (grade II) 5.0 g/L in saline which was generated in a jet nebulizer (particle size 1-5 mm; BARI, MASTER, Germany). After antigen challenge RL and Cdyn were monitored for 30 min and maximal changes from baseline for each parameter were recorded by a Medlab (V 5.0, Medease Scientific Technic Co Ltd, Nanjing). The effect of rac-FMT or R,R-FMT was determined by comparing the ovalbumin-induced changes in RL and Cdyn after drug treatment with the mean of antigen responses alone in the same guinea pig on previous and successive control periods.

Ovalbumin-induced airway inflammation in sensitized mice

Sensitizing procedures NIH mice were sensitized by subcutaneous injection (sc) at d 0 with antigen ovalbumin (grade V) 1 mg mixed with aluminium hydroxide adjuvant 50 mg in saline 0.2 mL, at footpad, neck, back, and groin. Control animals received saline at the same points[11].

Treatment procedures One time every day for 7 d and 1 h before antigen challenge, treatment mice were administered with rac-FMT or R,R-FMT 0.5, 1.0, and 2.0 mg/kg at 1.0 h before antigen challenge. Challenged control mice were injected ig with saline.

Antigen challenge After treatment, mice were placed in a 4-L bell cover and challenged by exposure to an aerosol of ovalbumin 10 g/L in saline which was generated in a jet nebulizer for 20 min, and one time every day for 7 d. Control mice were similarly exposed to an aerosol of saline.

Cell recovery and counts From the airway lumen of mice at 24 h after the last time antigen challenge, mice were anesthetized with urethane 2 g/kg, ip, and the trachea was cannulated. Bronchoalveolar lavage was performed by flushing the airways with saline 1.0 mL per mouse containing 1 % bovine serum albumin and 1 kU/L heparin sodium through the tracheal cannula for three flushing. Counts of total number of inflammatory cells recovered in bronchoalveolar lavage fluids (BALF) were made using a Neubauer chamber. BALF was centrifuged (Eppendorf Centrifuge 5804R, Germany) at 500×g for 10 min at 4 ºC, the cells deposit was resuspended in 0.2 mL saline containing 10 % bovine serum albumin, and smeared on a slide, differential cell analysis was made under light microscopy after Wright-Gieamsa staining.

Statistical analysis Data are expressed as mean±SD. A statistical analysis was performed using a one-way analysis of variance (Student-Newman-Keuls test). ID50 (95% confidence limits) was calculated and compared by weighted probit analysis of Bliss method.

RESULTS

Antigen-induced changes of RL and Cdyn in sensitized guinea pigs Comparision with baseline value before aerosol challenge of antigen, bronchial challenge of ovalbumin-sensitized guinea pigs induced 140 % increase of RL and 49 % reduction of Cdyn with maximal response at 5 min. Mean value increase of RL and mean value reduction of Cdyn from 1 to 30 min after antigen challenge were up to 101 %±34 % and 42 %±7 %, respectively in saline group. rac-FMT 0.5, 1.0, and 2.0 mg/kg, administered by ig, induced dose-related inhibition of the bronchoconstrictive responses to aerosolised ovalbumin. Maximal increase of RL and reduction response of Cdyn at 5 min were 86.0 %, 42.1 %, and 16.9 %, and 36.2 %, 28.7 %, and 9.6 %; ID50 (95 % CL) were 0.64 (0.54-0.76) and 1.02 (0.881.18) mg/kg, respectively. Mean increase of RL and reduction response of Cdyn from 1 to 30 min were 62 %±16 %, 39 %±12 %, and 16 %±10 % and 32 %± 5 % , 19 %±5 %, and 14 %±2 %; ID50 (95 % CL) were 0.96 (0.86-1.07) and 1.59 (1.32-1.92) mg/kg, respectively (Fig 1). R,R-FMT, 0.25, 0.5 and 1.0 mg/kg administered by i g produced dose-related inhibition of the bronchoconstrictive responses to aerosolised ovalbumin. Maximal increase of RL and reduction response of Cdyn at 5 min were 103.6 %, 68.6 %, and 24.7 % and 39.6 %, 21.9 %, and 13.6 %, ID50 (95 % CL) were 0.46 (0.40-0.53) and 0.52 (0.45-0.61) mg/kg, respectively. Mean increase of RL and reduction response of Cdyn from 1 to 30 min were 79 %±24 %, 55 %±20 %, and 22 %±4 % and 29 %±9 % , 19 %± 4 %, and 9 %±3 %; ID50 (95 % CL) were 0.52 (0.450.59) and 0.43 (0.37-0.51) mg/kg, respectively (Fig 2).

Fig 1. Effect of formoterol on lung resistance and dynamic lung compliance challenged with ovalbumin aerosol in sensitized guinea pigs. Saline (), Rac-formoterol 0.5 mg/kg (), Rac-formoterol 1.0 mg/kg (×), Rac-formoterol 2.0 mg/kg (), ig 1 h before exposure to ovalbumin aerosol. n=8 guinea pigs. Mean±SD. bP<0.05, cP<0.01 vs saline.

Fig 2. Effect of R, R-formoterol on lung resistance and dynamic lung compliance challenged with ovalbumin aerosol in sensitized guinea pigs. Saline (), R,R-formoterol 0.25 mg/kg (), R, R-formoterol 0.5 mg/kg (×), R,R-formoterol 1.0 mg/kg (), ig 1 h before exposure to ovalbumin aerosol. n=8 guinea pigs. Mean±SD. bP<0.05, cP<0.01 vs saline.

Antigen-induced changes of airway inflammatory cells in BALF In sensitized mice, after 7× ovalbumin-aerosol challenge, antigen induced a significant increase of inflammatory cells in BALF. The number of inflammatory cells in BALF in antigen challenged group was significantly higher than that in control group (P<0.01). Rac-FMT and R,R-FMT caused a dosedependent and almost complete inhibition at 2.0 mg/kg (Tab 1). ID50 (95 % CL) of rac-FMT on the number of total inflammatory cells, eosinophil, and lymphocyte and macrophage in BALF were 1.48 (1.22-1.81) , 0.80 (0.62-1.04), and 1.73 (1.45-2.05) mg/kg, respectively. ID50 (95 % CL) of R, R-FMT were 0.80 (0.57-1.13), 0.60 (0.43-0.83), and 0.79 (0.52-1.21) mg/kg, respectively (Tab 1).

Tab 1. Inhibition of antigen-induced lung inflammatory cells in bronchoalveolar lavage fluid by rac-formoterol and R,R-formoterol. Mean±SD. bP<0.05, cP<0.01 vs saline. eP<0.05 vs rac-FMT 1.0 mg/kg.

DISCUSSION

Rac-FMT contains two chiral carbons and, therefore, the synthetic molecular product contains two enantiomers (RR and SS) and two diastereomers (RS and SR)[4]. The marketed product contains only the (RR)- and (SS)-non-superimposable enantiomers. Since rac-FMT is a 50:50 mixture of the two enantiomers, the distomer (S,S-FMT) is inactive at the same dose of the eutomer which showed activity and the eudismic ratio is close to 1000[6], the result is exactly to be expected. In our results, rac-FMT and R,R-FMT present dose-related inhibition of the bronchoconstrictive responses and airway inflammation to aerosolised ovalbumin in the asthma models of guinea pig and mouse. ID50 value of rac-FMT is approximately two-fold than that of R,R-FMT, which shows the bronchodilating and antiinflammatory effects of the eutomer, R,R-FMT, are qualitatively similar to those of racemic rac-FMT, that at a half dose of the racemate the eutomer has a similar effect. Thus, the bronchodilating and antiinflammatory effects of the racemate can be attributed entirely to the R,R-enantiomers. Waldeck found that the (R,R)- enantiomer was by far the most potent. However, the relative potencies obtained for the (R,S)-, (S,R)-, and (S,S)-isomers were critically dependent on the degree of enantiomeric purity[12]. The results suggest the relative potency as that of receptor binding affinities. Handley and his colleagues described the binding affinities (kd) of R,R-FMT and S,S-FMT for the 2 receptor were 2.9 nmol and 3100 nmol, respectively, whereas the kd for rac-FMT was 5.2 nmol. Therefore, R,R-FMT is 1000-fold than that of S,S-FMT and 2-fold than that of receptor-binding activity of racFMT[4]. However, the presence of the distomer in the racemate does not influence the bronchodilating and antiinflammatory activity of the eutomer. Similar findings were reported from in vivo anti-bronchoconstrictive activity in monkey[7] and, from in vitro studies with animal tissues[5,6] and, more recently, from an analysis of the relaxant activities of the enantiomers of rac-FMT against resting tone and high tone induced by histamine and carbamylcholine in isolated Eurasian[6] and Chinese bronchi (unpublished data made by our Lab). Collectively, the results indicate that R,R-FMT would offer no benefit over the racemic mixture as far as the bronchodilating and antiinflammatory activity is concerned except that the dose could be halved. However it should be emphasized, the possibility of disadvantages or adverse effects of the distomer cannot be ruled out here.

In summary R,R-FMT protects lung against increase of RL and reduction of Cdyn induced by bronchial challenge of ovalbumin in the asthma model of guinea pig, and inhibits airway inflammation in the sensitized mouse. Efficacy of R,R-FMT is approximately two-fold than that of rac-FMT. The bronchodilating and antiinflammatory activity of rac-FMT resides in the R,R-enantiomers and S,S-enantiomers does not interfere with the activity when present in the racemic form.

REFERENCES