VOLUME 68, NUMBER 4, JULY/AUGUST 2007
Effects of (R,R)- and (R,R/S,S)-Formoterol on Airway Relaxation and Contraction in an Experimental Rat Model Maroun J. Mhanna, MD1; Jeffrey F. Koester, BS, MBA2; and Robert C. Cohn, MD 1
7Department of Pediatrics, MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio; and 2Sepracor Inc., Marlborough, Massachusetts
ABSTRACT Background: Racemic (R,R/S,S)-formoterol is a long-acting [3-agonist composed of a 50:50 mixture of (R,R)- and (S,S)-enantiomers. Objective: The aim of this study was to determine whether (R,R)-formoterol and (R,R/S,S)-formoterol have differing effects on airway contraction and relaxation in vitro. Methods: Cylindrical airway segments 3-mm long were isolated from the mid-trachea of healthy Sprague-Dawley rats and placed in a modified KrebsHenseleit solution. Dose-response curves of bethanechol-induced contraction (measured as milligrams of tension) and the concentration of bethanechol that elicited 50% to 75% of maximal contraction (EC50_75) were determined. The airway cylinders were then pr e c ont r act ed with bethanechol at the EC50_75 and exposed to different concentrations of (R,R)-formoterol (0.0001-1.0 1JM) or (R,R/S,S)-formoterol (0.0002-2.0 1JM). Each concentration of the 2 formoterol formulations contained the same amount of (R,R)-enantiomer (eg, [R,R]formoterol 0.0001 1JM and [R,R/S,S]-formoterol 0.0002 1JM contained the same amount of [R,R]-enantiomer). The relaxation percentage in response to formoterol was calculated as a reduction in tension (in milligrams) in relation to baseline tension in the precontracted state, with each tracheal cylinder serving as its own control. To determine the effect of (R,R)-formoterol on airway contraction, tracheal cylinders were incubated with (R,R)- or (R,R/S,S)-formoterol before electrical field stimulation (EFS). Results: Tracheae from 56 three-week-old Sprague-Dawley rats were used in the study. The relaxation percentage of p recont ract ed trachea was significantly greater after exposure to (R,R)-formoterol than to (R,R/S,.C)-formoterol at a 2-fold higher concentration (P = 0.03; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in a post hoc analysis, the mean (SE) relaxation percentage of precontracted trachea was significantly greater only after e x p o s u r e to (R,R)-formoterol 0.01 1JM than to (R,R/S,S)-formoterol 0.02 1JM (15.6% [5.8%] vs 39.0% [5.6%]; P < 0.05, unpaired
Acceptedfor publicationApril 19, 2007. Reproduction in whole or part is not permitted.
Copyright © 2007 Excerpta Medica, Inc.
doi:l 0.1016/j.curtheres.2007.08.005 0011-393X/$32.00
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t test). EFS-induced airway contraction was significantly less in tracheal cylinders incubated in (R,R)-formoterol compared with those incubated in (R,R/S,S)formoterol at a 2-fold higher concentration (P = 0.05; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in the post hoc analysis, mean (SE) EFS-induced tracheal contraction was significantly less only in (R,R)-formoterol 0.01 1JM compared with (R,R/S,S)-formoterol 0.02 1JM at 10 V (1070 [55] m g v s 1225 [28] mg; P < 0.05, unpaired t test). Conclusion: We found that (R,R)-formoterol may induce greater relaxation of precontracted airway smooth muscle cells than (R,R/S,S)-formoterol and that (R,R)-formoterol may have a greater inhibitory effect on the endogenous cholinergic and excitatory nonadrenergic, noncholinergic contractile airway responses than (R,R/S,S)-formoterol. We speculate that the presence of the (S~S~)-enantiomer in (R,R/S,S)-formoterol may impair airway relaxation of precontracted trachea in rats. (Curr Ther Res ClinExp. 2007;68:249-261) Copyright © 2007 Excerpta Medica, Inc. Key words: (R,R)-formoterol, (R,R/S~)-formoterol, electrical field stimulation, airway contraction, airway relaxation.
INTRODUCTION In moderate to severe asthma, levalbuterol ([R]-enantiomer of albuterol) has been found to provide a better therapeutic index than racemic albuterol, which is an equal mixture of (R)- and (S)-enantiomers. 1 In an in vitro study, (S)salbutamol was found to significantly increase the contractile response of isolated human bronchi to histamine and leukotrienes. 2 (S)-Albuterol was also found to increase intracellular free calcium by activating muscarinic receptors in airway smooth muscles. 3 (S)-Enantiomers of albuterol and formoterol have also been implicated in the release of inflammatory mediators and the activation of proinflammatory pathways in human airway smooth muscle cells. 4,5 These results may explain some of the adverse events associated with racemic forms of [3-agonists, which have equal mixtures of (R)- and (S)-enantiomers. Racemic (R,R/S,S)-formoterol is a long-acting [3-agonist composed of an equal mixture of (R,R)- and (S,S)-enantiomers. Few studies have examined whether the (S,S)-enantiomer component of the racemic mixture has any detrimental effects on airways in vitro. [3-Adrenergic receptors are extensively distributed in the large and small airways of rats. 6 In vitro isometric relaxation in response to [32-agonists has been reported in tracheae of Sprague-Dawley rats. 7,8 Our previous study 9 and the report by Szarek et al 1° found that precontracted airways at 50% to 75% of the maximal response to bethanechol provided the optimal condition to study bronchodilatation in vitro in Sprague-Dawley rats. The aim of this study was to determine whether (R,R)-formoterol and (R,R/S,S)-formoterol have differing effects on airway contraction and relaxation in vitro.
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MATERIALS AND METHODS The design and conduct of this s t udy were approved by the institutional review board of the MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio.
Animal Preparation Healthy weanling Sprague-Dawley rats were used in the study. The t racheae of 1 group of rats were used to determine the concentration of bethanechol that elicited 50% to 75% of maximal contraction (EC50_75) of the trachea. Another group was used to determine the relaxation percentage with (R,R)- or (R,R/S,S)-formoterol of t r a c he a e p r e c o n t r a c t e d with bethanechol. To st udy the endogenous cholinergic and excitatory nonadrenergic, noncholinergic (NANC) contractile airway responses to electrical field stimulation (EFS), a third group was used to s t udy EFS-induced tracheal contraction in the presence of (R,R)- or (R,R/S,S)-formoterol. The fourth group was used to study bethanecholinduced tracheal c o n t r a c t i o n in t r a c h e a e incubated with (R,R)- or (R,R/S,S)formoterol. The rats were euthanized and the trachea of each was removed. Under the dissecting microscope, each trachea was freed of adventitia and fat tissue, as described in previous studies. 9,1° One cylindrical airway segment 3 mm in length was isolated from the mid-trachea of each animal and placed in a modified Krebs-Henseleit solution with the following composition (mM): sodium chloride, 118.2; sodium bicarbonate, 25; potassium chloride, 4.6; potassium phosphate, 1.2; magnesium sulfate, 1.2; calcium chloride, 2.5; and 10% dextrose; with the pH adjusted to 7.4. The solution was continuously aerated with 5% carbon dioxide balanced with oxygen. The tracheal cylinders were then s us p ended between a sturdy glass rod and a force displacement transducer (FT03, Grass Instruments, Quincy, Massachusetts) connected to an amplifier. Generated force was continuously monitored and recorded on a rectilinear chart recorder (LDS Test and Measurement, Middleton, Wisconsin). The cylinders were allowed to equilibrate in the organ bath for 40 to 45 minutes before being challenged. The optimal length at which maximal isometric force developed was obtained for each cylinder by 0.1-g increments of load until EFS (5-V alternating current applied through platinum electrodes, 250 mA/cm 2) applied for 10 seconds at 4-minute intervals gave a reproducible maximal response. 9 A cumulative concentration-response curve to bethanechol and the resultant concentration of bethanechol that elicited EC50_75 were obtained. 9 Relaxation Studies The tracheal cylinders were washed, equilibrated, and precont ract ed with bethanechol to their EC50_75, after which t hey were exposed to different concentrations of (R,R)-formoterol (0.0001-1.0 1JM) or (R,R/S,S)-formoterol (0.00022.0 1JM). Each concentration of the 2 formoterol formulations contained the
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s a m e a m o u n t of (R,R)-enantiomer (eg, [R,R]-formoterol 0.0001 1JM and [R,R/S,S]f o r m o t e r o l 0.0002 1JM c o n t a i n e d the s a m e a m o u n t of [R,R]-enantiomer). The relaxation p e r c e n t a g e in r e s p o n s e to f o r m o t e r o l was calculated from the reduction in tension (in mg) in relation to baseline tension in the p r e c o n t r a c t e d state, with each t r a c h e a l s e g m e n t serving as its own control (before and after drug exposure). When airway cylinders were p r e c o n t r a c t e d to EC50_75, baseline tension was o b s e r v e d to d e c r e a s e over time. After the application of (R,R)or (R,R/S,S)-formoterol, t h e r e was a relaxation of the airway cylinders followed b y a r e c o v e r y and a return to a new baseline tension that was lower than the original baseline tension. The new higher c o n c e n t r a t i o n of the drug was not applied to the t r a c h e a l cylinders until the cylinders had r e a c h e d the new baseline tension.
Contraction Studies To s t u d y airway c o n t r a c t i o n in r e s p o n s e to EFS in the p r e s e n c e of (R,R)- or (R,R/S,S)-formoterol, tracheal cylinders from a s e p a r a t e group of rats were isolated, and a d o s e - r e s p o n s e c u r v e to EFS-induced tracheal c o n t r a c t i o n was obtained in the p r e s e n c e of (R,R)- or (R,R/S,S)-formoterol. Tracheal cylinders were i n c u b a t e d with (R,R)-formoterol 0.01 1JM and 1.0 1JM or (R,R/S,S)-formoterol 0.02 1JM and 2.0 1JM for 20 minutes before e x p o s u r e to EFS at different voltages. EFS was applied for 10 s e c o n d s to each t r a c h e a at 2, 4, 6, 8, and 10 V s e p a r a t e d b y 4-minute intervals. To s t u d y airway c o n t r a c t i o n in r e s p o n s e to b e t h a n e c h o l , t r a c h e a e were r e m o v e d from a n o t h e r group of rats, and a c o n c e n t r a t i o n - r e s p o n s e c u r v e to b e t h a n e c h o l was o b t a i n e d in the p r e s e n c e of (R,R)- or (R,R/S~S~)-formoterol. Tracheal cylinders were i n c u b a t e d with (R,R)-formoterol 0.01 1JM or 1.0 1JM or with (R,R/S,S)-formoterol 0.02 1JM or 2.0 1JM for 20 minutes before e x p o s u r e to different c o n c e n t r a t i o n s of b e t h a n e c h o l (0.3-1000.0 1JM).
Statistical Analysis Comparisons among the various experimental groups were made using a general linear model with repeated measures. The dependent variable was the relaxation percentage of precontracted tracheae over time. The independent variable was (R,R)-formoterol versus (R,R/S,S)-formoterol, with the repeated measure being increasing drug concentrations on the tracheal sections. A similar analysis was used to assess both EFS-induced airway contraction and bethanecol-induced airway contraction of tracheae preincubated with (R,R)- or (R,R/S,S)-formoterol over time. The assumption of homogeneity of variances was met in all the analyses (Levine's test of equality-of-error variances). If the between-subject effects were statistically significant, post hoc, pooled-variance, unpaired t tests were used for each of the time periods (different drug concentrations) to determine the differences between the 2 study drugs. P ~ 0.05 was considered statistically significant. Data were analyzed using SPSS version 14.0 (SPSS Inc., Chicago, Illinois). All data are e x p r e s s e d as mean (SE).
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RESULTS Fifty-six 3-week-old Sprague-Dawley rats were divided into the 4 study groups, as mentioned previously. The concentration-response curve to bethanechol was obtained (n = 8 rats). Bethanechol 10 pM was found to be the optimal concentration necessary to elicit EC50_75. Bethanechol-induced airway contraction increased from a mean (SE) tension of 40 (18) mg at 0.3 pM to 2594 (156) mg at 1000 pM (Figure 1). To determine the effect of (R,R)-formoterol on airway relaxation of precontracted trachea, tracheal cylinders (n = 16) incubated in bethanechol 10 pM (EC50_75) were exposed to different concentrations of (R,R)-formoterol or (R,R/S,S)-formoterol. The relaxation percentage of precontracted trachea in response to (R,R)-formoterol was significantly greater than the relaxation percentage in response to (R,R/S,S)-formoterol at equivalent concentrations (P = 0.03, general linear model with repeated measures analysis comparing the 2 groups of animals) (Figure 2). However, in a post hoc analysis, the mean (SE) relaxation percentage of precontracted trachea was significantly greater only after exposure to (R,R)-formoterol 0.01 pM than to (R,R/S,S)-formoterol 0.02 pM (15.6% [5.8%] vs 39.0% [5.6%]; P < 0.05, unpaired t test). The relaxation percentage varied from 0% to 46.7% (4.2%) with (R,R)-formoterol 0.0001 pM and 1 pM, respectively, and from 0% to 33.8% (5.9%) with (R,R/S,S)-formoterol 0.0002 pM and 2 pM, respectively. In a post hoc analysis, there was only a statistically sig-
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(R,R)- or (R,R/S,5)-Formoterol Concentrations (IJM) Figure 2. The relaxation percentage of tracheae from Sprague-Dawley rats precontracted w i t h bethanechol in response to (R,R)- and (R,R/S,S)-formoterol at concentrations ranging from 0.0001 to 1.0 IJM and 0.0002 to 2.0 IJM, respectively (n = 16, 8 animals in each group; P = 0.03, general linear model w i t h repeated measures analysis comparing the 2 groups of animals). *P < 0.05, by unpaired t test.
nificant difference between tracheae exposed to (R,R)-formoterol 0.01 tiM and (R,R/S,S)-formoterol 0.02 tiM (Figure 2) (39.0% [5.6%] vs 15.6% [5.8%]; P < 0.05, unpaired t test). To determine the effect of (R,R)-formoterol on airway contraction, tracheal cylinders (n = 16) were incubated with (R,R)- or (R,R/S,S)-formoterol before EFS. EFS-induced tracheal contraction was significantly lower in the presence of (R,R)-formoterol 0.01 tiM versus (R,R/S,~C)-formoterol 0.02 tiM (Figure 3A) (P = 0.05, general linear model with repeated measures analysis comparing the 2 groups of animals). EFS-induced tracheal contraction increased from 0 mg to 1070 (55) mg at 0 V and 10 V, respectively, with (R,R)-formoterol 0.01 tiM (n = 8) and from 0 mg to 1225 (28) mg at 0 V and 10 V, respectively, with (R,R/S,S)formoterol 0.02 tiM (n = 8) (Figure 3A). However, in a post hoc analysis, EFSinduced tracheal contraction was significantly less only in (R,R)-formoterol 0.01 tiM compared with (R,R/S,S)-formoterol 0.02 tiM at 10 V (1070 [55] mg vs 1225 [28] mg; P< 0.05, unpaired t test). No significant differences were seen with (R,R)-formoterol 1.0 tiM compared with (R,R/S,~C)-formoterol 2.0 tiM (n = 8) (Figure 3B). There was no significant difference in bethanechol-induced airway
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contraction in the presence of (R,R)-formoterol or ferent concentrations (n = 16) (Figure 4).
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DISCUSSION We found that the relaxation of bethanechol-precontracted trachea with (R,R)formoterol was greater than that induced by equivalent concentrations of (R,R/S,S)-formoterol in this animal model. We also found that that EFS-induced airway contraction was greater with (R,R/S,S)-formoterol than with (R,R)formoterol in vitro, while no differences were found in bethanechol-induced contractions. Our findings suggest that (S,S)-formoterol impairs the ability of (R,R)-formoterol to relax the airway and to protect against contraction induced by EFS but not by bethanechol in vitro. In a study in ovalbumin-sensitized and -nonsensitized guinea pigs, Handley et a111 used isolated strips of guinea pig tracheae that had been precontracted with histamine. The addition of (R,R)-formoterol produced greater dose-related airway relaxation than (R,R/S,S)-formoterol. These data are consistent with our findings. In our study using tracheal preparations from nonsensitized SpragueDawley rats, (R,R)-formoterol produced concentration-related airway relaxation of p r eco n tr act ed tracheal cylinders that was greater than that seen with (R,R/S,S)-formoterol. However, Schmidt et a112did not find a difference in relaxation of human and guinea pig bronchi pr econt ract ed with carbachol or histamine when treated with (R,R)- or (R,R/S,S)-formoterol. Their results might have differed from ours because the airway cylinders t hey used were precontracted to a greater extent (EC90) and with different spasmogens (carbachol and histamine). The tracheal cylinders used in our s tudy may have also behaved differently from the bronchiolar cylinders used in their study. EFS induces the release of acetylcholine and excitatory NANC substances from airway s e n s o r y nerves. 13,14 Verleden et a113 found that formoterol produced concentration-dependent inhibition of the NANC contraction of guinea pig bronchi in vitro. To determine if (R,R)-formoterol inhibited the NANC contraction induced by EFS more than (R,R/S,S)-formoterol, we incubated tracheal cylinders in (R,R)- or (R,R/S~S~)-formoterol prior to EFS exposure at different voltages. (R,R)-Formoterol had a greater inhibitory effect on EFS-induced airway contraction than (R,R/S,S)-formoterol. Our findings were consistent with previous studies that indicated that [32-agonists inhibit NANC neural bronchoconstrictor responses in vitro 13 and that [3-agonists inhibit cholinergic nerve-induced contractions. 15 However, we were the first to report that (R,R)formoterol induced greater inhibition of EFS-induced airway contraction compared with (R,R/S,S)-formoterol. One potential explanation might be exaggerated release of endogenous acetylcholine after EFS in the presence of the (S~S~)-enantiomer, as previously found by Zhang et al. 16 These authors reported that (S,S)-formoterol significantly increased acetylcholine release with EFS in equine tracheal smooth muscles. Increased intracellular calcium concentration
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Bethanechol (pM) Figure 4. Bethanechol=induced tracheal contraction in the presence of (A) (R,R)= and (R,R/S,S)=formoterol at 0.01 pM (n = 8) and 0.02 pM (n = 8), respectively, and (B) (R,R)= and (R,R/S,S)=formoterol at 1 pM (n = 8) and 2 pM (n = 8), respectively.
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might be another potential mechanism responsible for the exaggerated airway contraction in the presence of the (S,S)-enantiomer. Mitra et al 3 reported that another ineffective but not inert isomer of albuterol, (S)-albuterol, increased intracellular calcium concentration, whereas the effective isomer, (R)-albuterol, decreased intracellular calcium concentration in bovine tracheal smooth muscle cells. In our study, the difference in EFS-induced contraction between the (R,R)- and (R,R/S,S)-exposed tracheae might have been the result of increased intracellular calcium concentration in the presence of the (S,S)-enantiomer in
(R,R/S,S)-formoterol. The (S,S)-enantiomer of (R,R/S,S)-formoterol may be responsible for the exaggerated NANC contractile responses seen in our experimental model. Our findings are consistent with those of other investigators. Keir et a117 found that treating guinea pigs with racemic or (S)-albuterol increased airway responsiveness to bradykinin and capsaicin, indicating the role of the s e n s o r y nerves in bronchial hyperresponsiveness seen with the chronic use of racemic albuterol. Mazzoni et a118 reported that IV infusion of (S)-salbutamol induced airway hyperresponsiveness to histamine in guinea pigs and that hyperresponsiveness was abolished by bilateral vagal nerve section. We did not find any difference in bethanechol-induced airway contraction between tracheal cylinders preincubated with (R,R)- or (R,R/S~S~)-formoterol. One explanation for our findings might be associated with the greater inhibitory effect of [3-agonists on EFS-induced contraction than on exogenous cholinergicinduced contraction. 15,19Rhoden el a115 found that [3-agonists inhibit cholinergic n e r v e - i n d u c e d contractions of human bronchi more potently than contractions induced by exogenous acetylcholine. Bergendal et a119 also found that (R,R/S,S)formoterol p r o duc e d a greater inhibitory effect in preparations contracted by EFS than in preparations contracted by exogenously added acetylcholine. It is possible that in our study the effect of (R,R)- versus (R,R/S~S~)-formoterol might not be detected because of the small changes in inhibition of airway contractions induced by bethanecol (an exogenous cholinergic substance).
Study Limitations The results of this study might have differed if it had been conduct ed in vivo. Some clinical studies have s u p p o r t e d 2°-24 and others have not s u p p o r t e d 25-28 the use of [3-agonists without the (S,S)-enantiomers. In addition, we studied tracheal cylinders. Large airways might respond differently to [3-agonists and EFS than smaller airways and bronchi. The number of [32-receptors is higher in the smooth muscle of the small airways than the large airways in humans and rats, 29 and [3-adrenergic responses are weaker in the large peripheral airways than in the small peripheral airways. 3° Our results might have differed if bronchial cylinders had been studied instead of tracheal cylinders. When airway cylinders are precontracted to 50% to 75% of their maximal contraction for a prolonged time, there will be a time-dependent loss of their baseline tension, a factor that might have affected the accuracy of our relaxation percentage findings.
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In vivo s t u d i e s are n e e d e d to validate the significance of our in vitro findings. Future s t u d i e s are n e e d e d to d e t e r m i n e the clinical i m p o r t a n c e of the differe n c e s s e e n in vitro of t r a c h e a l relaxation and c o n t r a c t i o n in r e s p o n s e to (R,R)and (R,R/S,S)-formoterol.
CONCLUSION (R,R)-Formoterol m a y i n d u c e g r e a t e r r e l a x a t i o n of p r e c o n t r a c t e d a i r w a y s m o o t h m u s c l e cells t h a n (R,R/S,~C)-formoterol, and (R,R)-formoterol m a y h a v e a g r e a t e r i n h i b i t o r y effect on the e n d o g e n o u s cholinergic and e x c i t a t o r y NANC c o n t r a c t i l e a i r w a y r e s p o n s e s t h a n (R,R/S,S)-formoterol.
ACKNOWLEDGMENTS This w o r k w a s s u p p o r t e d b y a grant from S e p r a c o r Inc. ( M a r l b o r o u g h , Massachusetts). Dr. M h a n n a h a s r e c e i v e d r e s e a r c h funds from S e p r a c o r Inc. Mr. K o e s t e r is a s t o c k h o l d e r of S e p r a c o r Inc. Dr. Cohn has b e e n on the s p e a k e r ' s b u r e a u for and h a s r e c e i v e d grant funding from S e p r a c o r Inc.; A s t r a Z e n e c a , Wilmington, Delaware; GlaxoSmithKline, R e s e a r c h Triangle Park, N o r t h Carolina; G e n e n t e c h Inc., South San Francisco, California; Novartis P h a r m a c e u t i c a l s Corp., East Hanover, New Jersey; and Merck & Co. Inc., Rahway, New Jersey.
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8. Fayon M, Dumas De La Roque E, Berger P, et al. Increased relaxation of immature airways to [32-adrenoceptor agonists is related to a t t e n u a t e d expression of postjunctional s m o o t h muscle muscarinic M2 receptors. JAppl Physiol. 2005;98:1526-1533. 9. Mhanna MJ, Dreshaj IA, Haxhiu MA, Martin RJ. Mechanism for s u b s t a n c e P-induced relaxation of p r e c o n t r a c t e d airway s m o o t h muscle during development. Am J Physiol. 1999;276:L51-L56. 10. Szarek JL, Stewart NL, Spurlock B, Schneider C. S e n s o r y nerve- and neuropeptidem e d i a t e d relaxation r e s p o n s e s in airways of Sprague-Dawley rats. J Appl Physiol. 1995;78:1679-1687. 11. Handley DA, Senanayake CH, Dutczak W, et al. Biological actions of formoterol isomers. Pulm Pharmacol Ther. 2002;15:135-145. 12. Schmidt D, Kallstrom BL, Waldeck B, et al. The effect of the enantiomers of formoterol on inherent and i n d u c e d tone in guinea-pig t r a c h e a and h u m a n bronchus. Naunyn Schmiedebergs Arch Pharmacol. 2000;361:405--409. 13. Verleden GM, Belvisi MG, Rabe KF, et al. [32-Adrenoceptor agonists inhibit NANC neural b r o n c h o c o n s t r i c t o r r e s p o n s e s in vitro. JAppl Physiol. 1993;74:1195-1199. 14. Barnes PJ. Modulation of neurotransmission in airways. Physiol Rev. 1992;72:699729. 15. Rhoden KJ, Meldrum LA, Barnes PJ. Inhibition of cholinergic n e u r o t r a n s m i s s i o n in h u m a n airways b y [32-adrenoceptors. J Appl Physiol. 1988;65: 700-705. 16. Zhang XY, Zhu FX, Olszewski MA, Robinson NE. Effects of enantiomers of [32-agonists on ACh release and s m o o t h muscle contraction in the trachea. Am J Physiol. 1998;274:L32-L38. 17. Keir S, Page C, Spina D. Bronchial h y p e r r e s p o n s i v e n e s s induced b y chronic treatment with albuterol: Role of s e n s o r y nerves. JAllergy Clin Immunol. 2002;110:388-394. 18. Mazzoni L, Naef R, Chapman ID, Morley J. H y p e r r e s p o n s i v e n e s s of the airways following e x p o s u r e of guinea-pigs to racemic mixtures and d i s t o m e r s of [32-selective s y m p a t h o m i m e t i c s . Pulm Pharmacol. 1994;7:367-376. 19. Bergendal A, Linden A, Lotvall J, et al. Different effects of salmeterol, formoterol and salbutamol on cholinergic r e s p o n s e s in the ferret trachea. Br J Pharmacol. 1995;114: 1478-1482. 20. Berger WE, Ames DE, Harrison D. A patient satisfaction s u r v e y comparing levalbuterol with racemic albuterol in children. Allergy Asthma Proc. 2004;25:437-444. 21. Carl JC, Myers TR, Kirchner HL, Kercsmar CM. Comparison of racemic albuterol and levalbuterol for treatment of acute asthma. J Pediatr. 2003; 143:731-736. 22. Milgrom H, Skoner DP, Bensch G, et al, for the Levalbuterol Pediatric Study Group. Low-dose levalbuterol in children with asthma: Safety and efficacy in c o m p a r i s o n with p l a c e b o and racemic albuterol. JAllergy Clin Immunol. 2001;108:938-945. 23. Nowak R, Emerman C, Hanrahan JP, et al, for the XOPENEX Acute Severe Asthma Study Group. A c o m p a r i s o n of levalbuterol with racemic albuterol in the treatment of acute severe a s t h m a e x a c e r b a t i o n s in adults. Am JEmerg Med. 2006;24:259-267. 24. Schreck DM, Babin S. Comparison of racemic albuterol and levalbuterol in the treatment of acute a s t h m a in the ED. Am J Emerg Med. 2005;23:842--847.
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25. Hardasmalani MD, DeBari V, Bithoney WG, Gold N. Levalbuterol versus racemic albuterol in the treatment of acute exacerbation of asthma in children. Pediatr Emerg Care. 2005;21:415-419. 26. Ralston ME, Euwema MS, Knecht KR, et al. Comparison of levalbuterol and racemic albuterol combined with ipratropium bromide in acute pediatric asthma: A randomized controlled trial. J Emerg Med. 2005;29:29-35. 27. Qureshi F, Zaritsky A, Welch C, et al. Clinical efficacy of racemic albuterol versus levalbuterol for the treatment of acute pediatric asthma. Ann Emerg Med. 2005;46:29-36. 28. Thompson M, Wise S, Rodenberg H. A preliminary comparison of levalbuterol and albuterol in prehospital care. JEmerg Med. 2004;26:271-277. 29. Hamid QA, Mak JC, Sheppard MN, et al. Localization of [32-adrenoceptor messenger RNA in human and rat lung using in situ hybridization: Correlation with receptor autoradiography. Eur J Pharmacol. 1991;206:133-138. 30. Vornanen M. Adrenergic responses in different sections of rat airways. Acta Physiol Scand. 1982;114:587-591.
Address correspondence to: M a r o u n J. M h a n n a , MD, M e t r o H e a l t h M e d i c a l Center, 2500 M e t r o H e a l t h Drive, C l e v e l a n d , OH 44109. E-mail: m m h a n n a @ metrohealth.org.
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Effects of (R,R)- and (R,R/S,S)-Formoterol on Airway Relaxation and Contraction in an Experimental Rat Model Maroun J. Mhanna, MD1; Jeffrey F. Koester, BS, MBA2; and Robert C. Cohn, MD 1
7Department of Pediatrics, MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio; and 2Sepracor Inc., Marlborough, Massachusetts
ABSTRACT Background: Racemic (R,R/S,S)-formoterol is a long-acting [3-agonist composed of a 50:50 mixture of (R,R)- and (S,S)-enantiomers. Objective: The aim of this study was to determine whether (R,R)-formoterol and (R,R/S,S)-formoterol have differing effects on airway contraction and relaxation in vitro. Methods: Cylindrical airway segments 3-mm long were isolated from the mid-trachea of healthy Sprague-Dawley rats and placed in a modified KrebsHenseleit solution. Dose-response curves of bethanechol-induced contraction (measured as milligrams of tension) and the concentration of bethanechol that elicited 50% to 75% of maximal contraction (EC50_75) were determined. The airway cylinders were then pr e c ont r act ed with bethanechol at the EC50_75 and exposed to different concentrations of (R,R)-formoterol (0.0001-1.0 1JM) or (R,R/S,S)-formoterol (0.0002-2.0 1JM). Each concentration of the 2 formoterol formulations contained the same amount of (R,R)-enantiomer (eg, [R,R]formoterol 0.0001 1JM and [R,R/S,S]-formoterol 0.0002 1JM contained the same amount of [R,R]-enantiomer). The relaxation percentage in response to formoterol was calculated as a reduction in tension (in milligrams) in relation to baseline tension in the precontracted state, with each tracheal cylinder serving as its own control. To determine the effect of (R,R)-formoterol on airway contraction, tracheal cylinders were incubated with (R,R)- or (R,R/S,S)-formoterol before electrical field stimulation (EFS). Results: Tracheae from 56 three-week-old Sprague-Dawley rats were used in the study. The relaxation percentage of p recont ract ed trachea was significantly greater after exposure to (R,R)-formoterol than to (R,R/S,.C)-formoterol at a 2-fold higher concentration (P = 0.03; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in a post hoc analysis, the mean (SE) relaxation percentage of precontracted trachea was significantly greater only after e x p o s u r e to (R,R)-formoterol 0.01 1JM than to (R,R/S,S)-formoterol 0.02 1JM (15.6% [5.8%] vs 39.0% [5.6%]; P < 0.05, unpaired
Acceptedfor publicationApril 19, 2007. Reproduction in whole or part is not permitted.
Copyright © 2007 Excerpta Medica, Inc.
doi:l 0.1016/j.curtheres.2007.08.005 0011-393X/$32.00
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t test). EFS-induced airway contraction was significantly less in tracheal cylinders incubated in (R,R)-formoterol compared with those incubated in (R,R/S,S)formoterol at a 2-fold higher concentration (P = 0.05; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in the post hoc analysis, mean (SE) EFS-induced tracheal contraction was significantly less only in (R,R)-formoterol 0.01 1JM compared with (R,R/S,S)-formoterol 0.02 1JM at 10 V (1070 [55] m g v s 1225 [28] mg; P < 0.05, unpaired t test). Conclusion: We found that (R,R)-formoterol may induce greater relaxation of precontracted airway smooth muscle cells than (R,R/S,S)-formoterol and that (R,R)-formoterol may have a greater inhibitory effect on the endogenous cholinergic and excitatory nonadrenergic, noncholinergic contractile airway responses than (R,R/S,S)-formoterol. We speculate that the presence of the (S~S~)-enantiomer in (R,R/S,S)-formoterol may impair airway relaxation of precontracted trachea in rats. (Curr Ther Res ClinExp. 2007;68:249-261) Copyright © 2007 Excerpta Medica, Inc. Key words: (R,R)-formoterol, (R,R/S~)-formoterol, electrical field stimulation, airway contraction, airway relaxation.
INTRODUCTION In moderate to severe asthma, levalbuterol ([R]-enantiomer of albuterol) has been found to provide a better therapeutic index than racemic albuterol, which is an equal mixture of (R)- and (S)-enantiomers. 1 In an in vitro study, (S)salbutamol was found to significantly increase the contractile response of isolated human bronchi to histamine and leukotrienes. 2 (S)-Albuterol was also found to increase intracellular free calcium by activating muscarinic receptors in airway smooth muscles. 3 (S)-Enantiomers of albuterol and formoterol have also been implicated in the release of inflammatory mediators and the activation of proinflammatory pathways in human airway smooth muscle cells. 4,5 These results may explain some of the adverse events associated with racemic forms of [3-agonists, which have equal mixtures of (R)- and (S)-enantiomers. Racemic (R,R/S,S)-formoterol is a long-acting [3-agonist composed of an equal mixture of (R,R)- and (S,S)-enantiomers. Few studies have examined whether the (S,S)-enantiomer component of the racemic mixture has any detrimental effects on airways in vitro. [3-Adrenergic receptors are extensively distributed in the large and small airways of rats. 6 In vitro isometric relaxation in response to [32-agonists has been reported in tracheae of Sprague-Dawley rats. 7,8 Our previous study 9 and the report by Szarek et al 1° found that precontracted airways at 50% to 75% of the maximal response to bethanechol provided the optimal condition to study bronchodilatation in vitro in Sprague-Dawley rats. The aim of this study was to determine whether (R,R)-formoterol and (R,R/S,S)-formoterol have differing effects on airway contraction and relaxation in vitro.
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MATERIALS AND METHODS The design and conduct of this s t udy were approved by the institutional review board of the MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio.
Animal Preparation Healthy weanling Sprague-Dawley rats were used in the study. The t racheae of 1 group of rats were used to determine the concentration of bethanechol that elicited 50% to 75% of maximal contraction (EC50_75) of the trachea. Another group was used to determine the relaxation percentage with (R,R)- or (R,R/S,S)-formoterol of t r a c he a e p r e c o n t r a c t e d with bethanechol. To st udy the endogenous cholinergic and excitatory nonadrenergic, noncholinergic (NANC) contractile airway responses to electrical field stimulation (EFS), a third group was used to s t udy EFS-induced tracheal contraction in the presence of (R,R)- or (R,R/S,S)-formoterol. The fourth group was used to study bethanecholinduced tracheal c o n t r a c t i o n in t r a c h e a e incubated with (R,R)- or (R,R/S,S)formoterol. The rats were euthanized and the trachea of each was removed. Under the dissecting microscope, each trachea was freed of adventitia and fat tissue, as described in previous studies. 9,1° One cylindrical airway segment 3 mm in length was isolated from the mid-trachea of each animal and placed in a modified Krebs-Henseleit solution with the following composition (mM): sodium chloride, 118.2; sodium bicarbonate, 25; potassium chloride, 4.6; potassium phosphate, 1.2; magnesium sulfate, 1.2; calcium chloride, 2.5; and 10% dextrose; with the pH adjusted to 7.4. The solution was continuously aerated with 5% carbon dioxide balanced with oxygen. The tracheal cylinders were then s us p ended between a sturdy glass rod and a force displacement transducer (FT03, Grass Instruments, Quincy, Massachusetts) connected to an amplifier. Generated force was continuously monitored and recorded on a rectilinear chart recorder (LDS Test and Measurement, Middleton, Wisconsin). The cylinders were allowed to equilibrate in the organ bath for 40 to 45 minutes before being challenged. The optimal length at which maximal isometric force developed was obtained for each cylinder by 0.1-g increments of load until EFS (5-V alternating current applied through platinum electrodes, 250 mA/cm 2) applied for 10 seconds at 4-minute intervals gave a reproducible maximal response. 9 A cumulative concentration-response curve to bethanechol and the resultant concentration of bethanechol that elicited EC50_75 were obtained. 9 Relaxation Studies The tracheal cylinders were washed, equilibrated, and precont ract ed with bethanechol to their EC50_75, after which t hey were exposed to different concentrations of (R,R)-formoterol (0.0001-1.0 1JM) or (R,R/S,S)-formoterol (0.00022.0 1JM). Each concentration of the 2 formoterol formulations contained the
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s a m e a m o u n t of (R,R)-enantiomer (eg, [R,R]-formoterol 0.0001 1JM and [R,R/S,S]f o r m o t e r o l 0.0002 1JM c o n t a i n e d the s a m e a m o u n t of [R,R]-enantiomer). The relaxation p e r c e n t a g e in r e s p o n s e to f o r m o t e r o l was calculated from the reduction in tension (in mg) in relation to baseline tension in the p r e c o n t r a c t e d state, with each t r a c h e a l s e g m e n t serving as its own control (before and after drug exposure). When airway cylinders were p r e c o n t r a c t e d to EC50_75, baseline tension was o b s e r v e d to d e c r e a s e over time. After the application of (R,R)or (R,R/S,S)-formoterol, t h e r e was a relaxation of the airway cylinders followed b y a r e c o v e r y and a return to a new baseline tension that was lower than the original baseline tension. The new higher c o n c e n t r a t i o n of the drug was not applied to the t r a c h e a l cylinders until the cylinders had r e a c h e d the new baseline tension.
Contraction Studies To s t u d y airway c o n t r a c t i o n in r e s p o n s e to EFS in the p r e s e n c e of (R,R)- or (R,R/S,S)-formoterol, tracheal cylinders from a s e p a r a t e group of rats were isolated, and a d o s e - r e s p o n s e c u r v e to EFS-induced tracheal c o n t r a c t i o n was obtained in the p r e s e n c e of (R,R)- or (R,R/S,S)-formoterol. Tracheal cylinders were i n c u b a t e d with (R,R)-formoterol 0.01 1JM and 1.0 1JM or (R,R/S,S)-formoterol 0.02 1JM and 2.0 1JM for 20 minutes before e x p o s u r e to EFS at different voltages. EFS was applied for 10 s e c o n d s to each t r a c h e a at 2, 4, 6, 8, and 10 V s e p a r a t e d b y 4-minute intervals. To s t u d y airway c o n t r a c t i o n in r e s p o n s e to b e t h a n e c h o l , t r a c h e a e were r e m o v e d from a n o t h e r group of rats, and a c o n c e n t r a t i o n - r e s p o n s e c u r v e to b e t h a n e c h o l was o b t a i n e d in the p r e s e n c e of (R,R)- or (R,R/S~S~)-formoterol. Tracheal cylinders were i n c u b a t e d with (R,R)-formoterol 0.01 1JM or 1.0 1JM or with (R,R/S,S)-formoterol 0.02 1JM or 2.0 1JM for 20 minutes before e x p o s u r e to different c o n c e n t r a t i o n s of b e t h a n e c h o l (0.3-1000.0 1JM).
Statistical Analysis Comparisons among the various experimental groups were made using a general linear model with repeated measures. The dependent variable was the relaxation percentage of precontracted tracheae over time. The independent variable was (R,R)-formoterol versus (R,R/S,S)-formoterol, with the repeated measure being increasing drug concentrations on the tracheal sections. A similar analysis was used to assess both EFS-induced airway contraction and bethanecol-induced airway contraction of tracheae preincubated with (R,R)- or (R,R/S,S)-formoterol over time. The assumption of homogeneity of variances was met in all the analyses (Levine's test of equality-of-error variances). If the between-subject effects were statistically significant, post hoc, pooled-variance, unpaired t tests were used for each of the time periods (different drug concentrations) to determine the differences between the 2 study drugs. P ~ 0.05 was considered statistically significant. Data were analyzed using SPSS version 14.0 (SPSS Inc., Chicago, Illinois). All data are e x p r e s s e d as mean (SE).
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RESULTS Fifty-six 3-week-old Sprague-Dawley rats were divided into the 4 study groups, as mentioned previously. The concentration-response curve to bethanechol was obtained (n = 8 rats). Bethanechol 10 pM was found to be the optimal concentration necessary to elicit EC50_75. Bethanechol-induced airway contraction increased from a mean (SE) tension of 40 (18) mg at 0.3 pM to 2594 (156) mg at 1000 pM (Figure 1). To determine the effect of (R,R)-formoterol on airway relaxation of precontracted trachea, tracheal cylinders (n = 16) incubated in bethanechol 10 pM (EC50_75) were exposed to different concentrations of (R,R)-formoterol or (R,R/S,S)-formoterol. The relaxation percentage of precontracted trachea in response to (R,R)-formoterol was significantly greater than the relaxation percentage in response to (R,R/S,S)-formoterol at equivalent concentrations (P = 0.03, general linear model with repeated measures analysis comparing the 2 groups of animals) (Figure 2). However, in a post hoc analysis, the mean (SE) relaxation percentage of precontracted trachea was significantly greater only after exposure to (R,R)-formoterol 0.01 pM than to (R,R/S,S)-formoterol 0.02 pM (15.6% [5.8%] vs 39.0% [5.6%]; P < 0.05, unpaired t test). The relaxation percentage varied from 0% to 46.7% (4.2%) with (R,R)-formoterol 0.0001 pM and 1 pM, respectively, and from 0% to 33.8% (5.9%) with (R,R/S,S)-formoterol 0.0002 pM and 2 pM, respectively. In a post hoc analysis, there was only a statistically sig-
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(R,R)- or (R,R/S,5)-Formoterol Concentrations (IJM) Figure 2. The relaxation percentage of tracheae from Sprague-Dawley rats precontracted w i t h bethanechol in response to (R,R)- and (R,R/S,S)-formoterol at concentrations ranging from 0.0001 to 1.0 IJM and 0.0002 to 2.0 IJM, respectively (n = 16, 8 animals in each group; P = 0.03, general linear model w i t h repeated measures analysis comparing the 2 groups of animals). *P < 0.05, by unpaired t test.
nificant difference between tracheae exposed to (R,R)-formoterol 0.01 tiM and (R,R/S,S)-formoterol 0.02 tiM (Figure 2) (39.0% [5.6%] vs 15.6% [5.8%]; P < 0.05, unpaired t test). To determine the effect of (R,R)-formoterol on airway contraction, tracheal cylinders (n = 16) were incubated with (R,R)- or (R,R/S,S)-formoterol before EFS. EFS-induced tracheal contraction was significantly lower in the presence of (R,R)-formoterol 0.01 tiM versus (R,R/S,~C)-formoterol 0.02 tiM (Figure 3A) (P = 0.05, general linear model with repeated measures analysis comparing the 2 groups of animals). EFS-induced tracheal contraction increased from 0 mg to 1070 (55) mg at 0 V and 10 V, respectively, with (R,R)-formoterol 0.01 tiM (n = 8) and from 0 mg to 1225 (28) mg at 0 V and 10 V, respectively, with (R,R/S,S)formoterol 0.02 tiM (n = 8) (Figure 3A). However, in a post hoc analysis, EFSinduced tracheal contraction was significantly less only in (R,R)-formoterol 0.01 tiM compared with (R,R/S,S)-formoterol 0.02 tiM at 10 V (1070 [55] mg vs 1225 [28] mg; P< 0.05, unpaired t test). No significant differences were seen with (R,R)-formoterol 1.0 tiM compared with (R,R/S,~C)-formoterol 2.0 tiM (n = 8) (Figure 3B). There was no significant difference in bethanechol-induced airway
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contraction in the presence of (R,R)-formoterol or ferent concentrations (n = 16) (Figure 4).
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DISCUSSION We found that the relaxation of bethanechol-precontracted trachea with (R,R)formoterol was greater than that induced by equivalent concentrations of (R,R/S,S)-formoterol in this animal model. We also found that that EFS-induced airway contraction was greater with (R,R/S,S)-formoterol than with (R,R)formoterol in vitro, while no differences were found in bethanechol-induced contractions. Our findings suggest that (S,S)-formoterol impairs the ability of (R,R)-formoterol to relax the airway and to protect against contraction induced by EFS but not by bethanechol in vitro. In a study in ovalbumin-sensitized and -nonsensitized guinea pigs, Handley et a111 used isolated strips of guinea pig tracheae that had been precontracted with histamine. The addition of (R,R)-formoterol produced greater dose-related airway relaxation than (R,R/S,S)-formoterol. These data are consistent with our findings. In our study using tracheal preparations from nonsensitized SpragueDawley rats, (R,R)-formoterol produced concentration-related airway relaxation of p r eco n tr act ed tracheal cylinders that was greater than that seen with (R,R/S,S)-formoterol. However, Schmidt et a112did not find a difference in relaxation of human and guinea pig bronchi pr econt ract ed with carbachol or histamine when treated with (R,R)- or (R,R/S,S)-formoterol. Their results might have differed from ours because the airway cylinders t hey used were precontracted to a greater extent (EC90) and with different spasmogens (carbachol and histamine). The tracheal cylinders used in our s tudy may have also behaved differently from the bronchiolar cylinders used in their study. EFS induces the release of acetylcholine and excitatory NANC substances from airway s e n s o r y nerves. 13,14 Verleden et a113 found that formoterol produced concentration-dependent inhibition of the NANC contraction of guinea pig bronchi in vitro. To determine if (R,R)-formoterol inhibited the NANC contraction induced by EFS more than (R,R/S,S)-formoterol, we incubated tracheal cylinders in (R,R)- or (R,R/S~S~)-formoterol prior to EFS exposure at different voltages. (R,R)-Formoterol had a greater inhibitory effect on EFS-induced airway contraction than (R,R/S,S)-formoterol. Our findings were consistent with previous studies that indicated that [32-agonists inhibit NANC neural bronchoconstrictor responses in vitro 13 and that [3-agonists inhibit cholinergic nerve-induced contractions. 15 However, we were the first to report that (R,R)formoterol induced greater inhibition of EFS-induced airway contraction compared with (R,R/S,S)-formoterol. One potential explanation might be exaggerated release of endogenous acetylcholine after EFS in the presence of the (S~S~)-enantiomer, as previously found by Zhang et al. 16 These authors reported that (S,S)-formoterol significantly increased acetylcholine release with EFS in equine tracheal smooth muscles. Increased intracellular calcium concentration
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might be another potential mechanism responsible for the exaggerated airway contraction in the presence of the (S,S)-enantiomer. Mitra et al 3 reported that another ineffective but not inert isomer of albuterol, (S)-albuterol, increased intracellular calcium concentration, whereas the effective isomer, (R)-albuterol, decreased intracellular calcium concentration in bovine tracheal smooth muscle cells. In our study, the difference in EFS-induced contraction between the (R,R)- and (R,R/S,S)-exposed tracheae might have been the result of increased intracellular calcium concentration in the presence of the (S,S)-enantiomer in
(R,R/S,S)-formoterol. The (S,S)-enantiomer of (R,R/S,S)-formoterol may be responsible for the exaggerated NANC contractile responses seen in our experimental model. Our findings are consistent with those of other investigators. Keir et a117 found that treating guinea pigs with racemic or (S)-albuterol increased airway responsiveness to bradykinin and capsaicin, indicating the role of the s e n s o r y nerves in bronchial hyperresponsiveness seen with the chronic use of racemic albuterol. Mazzoni et a118 reported that IV infusion of (S)-salbutamol induced airway hyperresponsiveness to histamine in guinea pigs and that hyperresponsiveness was abolished by bilateral vagal nerve section. We did not find any difference in bethanechol-induced airway contraction between tracheal cylinders preincubated with (R,R)- or (R,R/S~S~)-formoterol. One explanation for our findings might be associated with the greater inhibitory effect of [3-agonists on EFS-induced contraction than on exogenous cholinergicinduced contraction. 15,19Rhoden el a115 found that [3-agonists inhibit cholinergic n e r v e - i n d u c e d contractions of human bronchi more potently than contractions induced by exogenous acetylcholine. Bergendal et a119 also found that (R,R/S,S)formoterol p r o duc e d a greater inhibitory effect in preparations contracted by EFS than in preparations contracted by exogenously added acetylcholine. It is possible that in our study the effect of (R,R)- versus (R,R/S~S~)-formoterol might not be detected because of the small changes in inhibition of airway contractions induced by bethanecol (an exogenous cholinergic substance).
Study Limitations The results of this study might have differed if it had been conduct ed in vivo. Some clinical studies have s u p p o r t e d 2°-24 and others have not s u p p o r t e d 25-28 the use of [3-agonists without the (S,S)-enantiomers. In addition, we studied tracheal cylinders. Large airways might respond differently to [3-agonists and EFS than smaller airways and bronchi. The number of [32-receptors is higher in the smooth muscle of the small airways than the large airways in humans and rats, 29 and [3-adrenergic responses are weaker in the large peripheral airways than in the small peripheral airways. 3° Our results might have differed if bronchial cylinders had been studied instead of tracheal cylinders. When airway cylinders are precontracted to 50% to 75% of their maximal contraction for a prolonged time, there will be a time-dependent loss of their baseline tension, a factor that might have affected the accuracy of our relaxation percentage findings.
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In vivo s t u d i e s are n e e d e d to validate the significance of our in vitro findings. Future s t u d i e s are n e e d e d to d e t e r m i n e the clinical i m p o r t a n c e of the differe n c e s s e e n in vitro of t r a c h e a l relaxation and c o n t r a c t i o n in r e s p o n s e to (R,R)and (R,R/S,S)-formoterol.
CONCLUSION (R,R)-Formoterol m a y i n d u c e g r e a t e r r e l a x a t i o n of p r e c o n t r a c t e d a i r w a y s m o o t h m u s c l e cells t h a n (R,R/S,~C)-formoterol, and (R,R)-formoterol m a y h a v e a g r e a t e r i n h i b i t o r y effect on the e n d o g e n o u s cholinergic and e x c i t a t o r y NANC c o n t r a c t i l e a i r w a y r e s p o n s e s t h a n (R,R/S,S)-formoterol.
ACKNOWLEDGMENTS This w o r k w a s s u p p o r t e d b y a grant from S e p r a c o r Inc. ( M a r l b o r o u g h , Massachusetts). Dr. M h a n n a h a s r e c e i v e d r e s e a r c h funds from S e p r a c o r Inc. Mr. K o e s t e r is a s t o c k h o l d e r of S e p r a c o r Inc. Dr. Cohn has b e e n on the s p e a k e r ' s b u r e a u for and h a s r e c e i v e d grant funding from S e p r a c o r Inc.; A s t r a Z e n e c a , Wilmington, Delaware; GlaxoSmithKline, R e s e a r c h Triangle Park, N o r t h Carolina; G e n e n t e c h Inc., South San Francisco, California; Novartis P h a r m a c e u t i c a l s Corp., East Hanover, New Jersey; and Merck & Co. Inc., Rahway, New Jersey.
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Address correspondence to: M a r o u n J. M h a n n a , MD, M e t r o H e a l t h M e d i c a l Center, 2500 M e t r o H e a l t h Drive, C l e v e l a n d , OH 44109. E-mail: m m h a n n a @ metrohealth.org.
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