Preparation Of R-and S-terbutaline And Comparison Of Their Effects On Airways In OVA-sensitized Asthmatic Mice

Terbutaline,as one of the most common short actingβ₂‐agonists in clinic,remains the first-line medication in alleviating asthma symptoms due to their ability to promote bronchodilation.Terbutaline is a chiral drug,and consists of equimolar amount of R-enantiomer and S-enantiomer.Commercial products of terbutaline are all in racemic mixture sulfate.However,it has been reported that R-enantiomer ofβ₂-agonist generates the therapeutic effect,while S-enantiomer is inert and always relates to the side effects of racemate.Thus,the aim of our investigation is to synthesize single enantiomer of terbutaline through chiral resolution method,and then investigate their differential effects involved in airway inflammation,AHR and airway remodeling and the related molecular mechanisms based on animal model and in vitro cell line studies.Moreover,we also explore the differential effects of terbutaline enantiomers on cardiac function using Langendorff isolated heart perfusion system.The detailed researches and results are as follows:1.In the first part,R-and S-terbutaline with high chemical purity and optical purity were synthesized through chiral resolution from racemic terbutaline sulfate.DTTA was used as the chiral resolving agent.The overall yield is 53.6%（calculated by half quantity of racemate）.The chemical purity of R-terbutaline is 99.72%and the optical purity is 99.9%by HPLC analysis.While 99.69%and 99.8%are for that of S-terbutaline.Then DSC and Polarimeter were used to analyze the physical and chemical properties of R-terbutaline.The optical rotation([α]_D²⁰)of R-terbutaline is-38.5.The melting point is 236℃,and the DSC curve of R-terbutaline reveals that it is in a mono-morphology.There exists one impurity in R,S-terbutaline hydrochloride.The impurity 3 has been predicated as the 7-OH esterified product,3,2-（tert-butylamino）-1-（3,5-dihydroxyphenyl）ethyl acetate by LC-MS and timsTOF analysis.Impurity 3 shows no carcinogenic and mutagenic toxicity on rodents predicated by DS TOPKAT software.2.Then we utilized a mouse model of allergic asthma induced by OVA to study the differential effects of R-and S-terbutaline,as well as racemic mixture,via.intranasal inhalation for consecutive seven days,and explore the related molecular mechanisms involved in airway inflammation and airway remodeling.Airway responsiveness to methacholine was measured by the plethysmography in conscious mice.Eosinophils counts in blood and bronchoalveolar fluid（BALF）were determined.The OVA-sIgE in plasma and inflammatory cytokines and mediators in BALF or lung tissue were analyzed by ELISA,qRT-PCR or western blotting.Airway inflammation and remodeling were evaluated with hematoxylin and eosin（HE）,periodic acid-Schiff（PAS）,and Masson staining.Drug distribution and deposition after inhalation were determined by LC-MS.After inhalation,most of active drugs can deposit in the lung.R-terbutaline preferentially retains in the system,whereas S-terbutaline exhibits a greater elimination behavior than its counterpart R-form.However,the concentration of S-terbutaline in heart was with no significant difference compared with that of R-terbutaline and racemic.Our data shows that R-terbutaline efficiently ameliorates asthma responses,including airway hyperresponsiveness,eosinophils influx and IL-5 in BALF,plasma OVA-sIgE and significantly reduced pulmonary inflammation,peribronchial smooth muscle layer thickness,goblet cell hyperplasia,and deposition of collagen fibers,as well as down regulation of p38 MAPK phosphorylation and NF-κB expression.Racemic mixture exhibits diminished effects while S-terbutaline enhances airway responsiveness to methacholine and elevates eosinophilia in blood,and significantly increases IL-4 and IL-13 levels in BALF,and promotes smooth muscle thickness and mucus hypersecretion,possibly by activating NF-κB activity and downregulating AMPKαexpression and its phosphorylation.3.We examined and compared the differential effects of R,S-terbutaline on cell phenotype differentiation in normal human lung fibroblasts（HLF）and TGF-β1 induced HLF cell model.RT-PCR and western blot were performed to detect the mRNA levels ofα-SMA and fibronectin and the expression of proliferative protein AMPKα.The result reveals that 0.1-10μM S-terbutaline can stimulate fibroblast differentiation to myofibroblast and the effect is dose-dependent.1μM S-terbutaline shows an enhanced tendency to promote fibroblast differentiation to myofibroblast in TGF-β1 induced HLF cells,including increased mRNA levels ofα-SMA and fibronectin.The active effects of S-terbutaline on fibroblast differentiation to myofibroblast might be through downregulation of AMPKαexpression and its phosphorylation.In contrast with S-terbutaline,R-terbutaline has no effect on normal HLF.Moreover,1μM R-terbutaline significantly reduced the increased mRNA levels ofα-SMA and fibronectin in TGF-β1 induced HLF cells.When compared with 1μM R-terbutaline,2μM rac-terbutaline exhibits a diminished effect.4.We assessed the differential effects of terbutaline enantiomers on cardiac inotropy and heart rate using Langendorff isolated heart perfusion system.It shows that R-terbutaline has a positive inotropic effect on isolated rat heart.100μM R-terbutaline can markedly increase LVP and HR.This effect of 100μM R-terbutaline could mostly be blocked byβ₂ antagonist ICI 118,551,and completely blocked byβ₁ antagonist metoprolol.While S-terbutaline exhibits a weak inhibitory effect on cardiac function of isolated rat heart,including decreased LVP and depressed heart rate.When compared with R-terbutaline,S-terbutaline could significantly increase the times of cardiac arrhythmia on isolated rat heart.Taken together,our results demonstrate that“Chiral switch”product R-terbutaline has a superiority over racemate in reducing side effects and systemic metabolic burden due to its lower dose,suggesting that inhaled low dose of R-terbutaline is an efficient therapeutic candidate against asthma.