Comparison Of SPFF, A Novel β₂-adrenoceptor Agonist, And Its Enantiomers In Antiasthmatic Activity

Comparison of SPFF,a novelβ₂-adrenoceptor agonist,and its enantiomers in antiasthmatic activityβ_-adrenoceptor agonists,the most effective bronchodilator,has long been widely used in symptomatic therapy of asthma.A variety ofβ₂-adrenoreceptor agonists are currently available,such as salbutamol,terbutaline,salmeterol,formoterol and procaterol. SPFF[2-(4-amino-3-chloro-5-trifluomethyl-phenyl)-2-tert-butylamino-ethanol hydrochloride] is synthesized and exploited by drug synthesis laboratory of Shenyang Pharmaceutical University as a novelβ₂-adrenoreceptor agonist.Leling Gan et al has validated that it was a potent,long-acting bronchodilator with relative higherβ₂-adrenoceptor selectivity.There is a chiral center(asymmetric carbon atom)in its structures,therefore SPFF exists as a pair of enantiomers.Based on the conformational stereochemistry,enantiomers vary greatly in their pharmacokinetic parameters and pharmacological effects on target tissue(s).In the present study we compared the pharmacological properties of racemic SPFF with its individual enantiomers on antiasthmatic action and potency in experiments in vivo and in vitro.We studied the bronchodilative effects of SPFF and its enantimers on isolated guinea pig trachea strips with or without the precontraction of bronchocontractors(His and Ach)taking isoprenaline(ISO)as referenced drug.(-)-SPFF,(±)-SPFF,(+)-SPFF and ISO concentration-dependently decreased the resting tension of isolated guinea pig trachea strips with pD₂(95%confidence limit)values of 7.77(7.68～7.86),77.01(6.97～7.05),6.73 (6.33～7.14)and 8.51(8.06～8.95)respectively,which indicated that(-)-SPFF was 5.8 times more potent than(±)-SPFF and 10.9 times more potent than(+)-SPFF.Whereas the maximal relaxations produced by(-)-,(±)- and(+)-SPFF were almost equal to that of isoprenaline, indicating that both(-)- and(+)-isomers posses similar intrinsic activity and behave as aβ₂-adrenoreceptor full agonist like racemic SPFF and isoprenaline.In addition,(-)-,(±)-and (+)-SPFF exhibited a concentration -dependent relaxation of trachea strips contracted by acetylcholine and histamine,the potency was in the rank order of(-)-SPFF＞(±)-SPFF＞(+)-SPFF in term of EC₅₀.Besides,we confirmed that the bronchodilator effect of(-)- and (+)-enantiomers was due to activation ofβ₂-adrenoceptor because this effect was antagonized by a specificβ₂-adrenorecepter antagonist,ICI-118551,with the similar pA₂ values to that of (+)-SPFF.Furthermore,(-)-SPFF potently inhibited the bronchoconstrictive effect of histamine in isolated lung perfusion model in guinea pigs,it was 23.2 times more potent than (+)-SPFF in term of ID₅₀.We have found that(+)-SPFF has no interaction with(-)-SPFF on the relaxation of normal tone of isolated guinea pigs trachea strips.(±)-SPFF and its enantiomers exerted bronchodilative effect on guinea pigs trachea smooth muscle in vivo.(-)-SPFF displayed almost the same protective effect against bronchospasm induced by histamine- acetylcholine aerosol in conscious guinea pigs as (±)-SPFF did.In the Konzett and Rossler experiment,the inhibition of(-)-SPFF in the increase of airway resistance induced by histamine in anesthetized guinea pigs was 1.6 and 6.3 times more potent than(±)-SPFF and(+)-SPFF respectively.Subsequently,in the inhibition of histamine-induced increase of pulmonary resistance(R_L)in anesthetized guinea pigs,(-)-SPFF was 1.3 and 3.5 times more potent than(±)- and(+)-SPFF,meanwhile in inhibiting the decrease of pulmonary compliance(C_L),the potency of(-)- and (+)-enantiomers were approximately equivalent to that of(±)-SPFF.Furthermore,the study on the long-lasting action of the test drugs had shown that the effects of(-)-SPFF(30μg·kg^-1), (±)-SPFF(30μg·kg^-1)and(+)-SPFF(100μg·kg^-1)in inhibiting the increase of R_L all lasted for 4hr.Nevertheless,the effects of(-)- and(+)-enantiomers degraded slightly in 4hr after intraduodenal administration in inhibiting the decrease of C_L.Moreover,(±)-SPFF and its enantiomers(1×10^-8～1×10^-6mol·L^-1)inhibited the release of histamine in ovalbumin-allergic rats lung fragments.In the investigation of(±)-SPFF and its enantiomers on mucociliary activity,it was shown that(-)-SPFF(0.04,0.10mg·kg^-1)and(±)-SPFF(0.10,0.25 mg·kg^-1) improved markedly the cilium transportation function on the trachea of conscious pigeon while had no effect on the air way mucus excretion in mice。Correspondingly,(+)-SPFF neither enhanced cilium transportation in pigeon nor promoted mucus excretion in mice. These results implied that,by prompting the clearance of airway sputum,(-)-SPFF and (±)-SPFF could have an additional superiority in the alleviation of asthma symptom than (+)-SPFF.In the following study,the receptor-binding technique was used to determine the affinity and selectivity of(±)-SPFF and its isomers forβ₁- andβ₂-adrenoceptor,using(-)[¹²⁵I]pindolol as radioligand.The result showed that(-)-SPFF's binding affinity toβ₂-adrenoceptor was about 6 and 164 times greater than that of(±)- and(+)-SPFF,which may account predominantly for its more potent efficacy in experiments in vitro and in vivo.Furthermore, the overall selectivity ratio estimated from K₁(lung)/K₁(atria)in radioligand binding experiment suggested that(-)-SPFF appears to possess a higher selectivity forβ₂-AR(lung tissue containing about 80%β₂-AR)relative toβ₁-AR(atria tissue containing about 80%β₁-AR)compared with(±)- and(+)-SPFF,which will contribute to its lack of cardiovascular side effects when used as a bronchodilator,especially when it is administered orally,which provides a large therapeutic width.Finally,in an attempt to clarify the intracellular effect of (±)-SPFF and its enantiomers,we determined intracellular free Ca²⁺concentration([Ca²⁺]_i)using fluorescent Calcium indicator Fura-2/AM via primary culture of guinea pig trachea smooth muscle cells.The results indicated that(±)-SPFF,(+)-SPFF and(-)-SPFF can significantly decreased the Ca²⁺concentration in cultured trachea smooth muscle cells, particularly,the effect of(-)-SPFF(6×10^-10mol·L^-1)on decrease[Ca²⁺]_i was more significantly (P＜0.05)than that of(+)-SPFF(6×10^-10mol·L^-1).On the basis of the present study,it can be concluded that(-)-SPFF has various advantages over(±)- and(+)-SPFF including greater efficacy and high affinity and selectivity toβ₂-adrenoreceptor and may be a preferred substitute to racemic SPFF in the treatment of asthma.