Transdermal Treatment with Tulobuterol Increases Isometric Contractile Properties of Diaphragm Muscle in Mice

Tohoku J. Exp. Med., 2007, 212Effects, 309-317 of Tulobuterol on Diaphragm Muscle 309

1 1 2 3 CHIYOHIKO SHINDOH, RINA TSUSHIMA, YURIKO SHINDOH and GEN TAMURA

1Department of Medical Technology, School of Health Sciences, Faculty of Medicine, Tohoku University, Sendai, Japan 2Department of Respiratory Medicine, Sendai City Medical Center, Sendai, Japan 3Department of Infection and Respiratory Medicine, Tohoku University Hospital, Sendai, Japan

SHINDOH, C., TSUSHIMA, R., SHINDOH, Y. and TAMURA, G. Transdermal Treatment with Tulobuterol Increases Isometric Contractile Properties of Diaphragm Muscle in Mice. Tohoku J. Exp. Med., 2007, 212 (3), 309-317 ── Clinically, patients suffering from bronchial asthma are often treated transdermally with tulobuterol patches to dilate the bronchi.

Tulobuterol, a synthetic β 2 agonist, is also thought to act as a diaphragm muscle contractor,

like other β 2 sympathomimetic drugs. However, it has not been clarified that transdermal treatment with tulobuterol influences diaphragm muscle contractility. We therefore examined its effects on contractile properties of such muscles obtained from BALB/c mice. Two systems, a tulobuterol incubation group (in vitro) and a tulobuterol transdermal treatment group (in vivo), were employed. In both groups, the contractile properties of the dissected diaphragm muscles were measured by field stimulation in an organ bath. In the incubation group, the diaphragm muscle of untreated mice was incubated in an organ buffer at 10-7, 10-6, or 10-5 M tulobuterol for 1 hr and then measured for contractility. Tulob- uterol significantly increased force-frequency curves at a concentration of 10-5 M at 1 (p < 0.01), 30, 50, 70, 100, and 120 Hz (p < 0.05, each) compared with the values at 0 M. In the transdermal treatment group, the diaphragm muscle was dissected from animals at 1, 4, 8, 12, or 24 hrs after treatment and measured for contractility, showing that the force- frequency curves were significantly increased and maintained from 4 to 24 hrs (each p < 0.01 as compared with the sham-treated group). We suggest that transdermal tulobuterol treatment in case of bronchial asthma is useful not only for bronchial dilatation, but also

Tulobuterol, a synthetic β 2-adrenoceptor ago- al. 1975). However, the bronchodilating effect of nist, which is structurally related to isoprenaline, tulobuterol is sustained more than 10 times longer produces a bronchodilating effect 2-10 times than those of isoproterenol and sulbutamol. In stronger than that of clorprenaline and consider- addition, tulobuterol has chronotrophic effects ably weaker than that of isoproterenol (Kubo et through stimulation of cardiac β 2-adrenoceptors, Received February 23, 2007; revision accepted for publication May 24, 2007. Correspondence: Chiyohiko Shindoh, M.D., Prof., Department of Medical Technology, Faculty of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. e-mail: [email protected] 309 310 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 311 resulting in an increase in heart rate, and it also three different concentrations of tulobuterol in increases contractile force and O2 consumption in vitro. We then examined the effects of transder- the heart of dogs (Kubo et al. 1977). These mal tulobuterol treatment on diaphragm contrac- effects, however, are found to be about 1/1,000 tility throughout a 24-hr period. We found that and 1/100 as potent as those of isoproterenol and tulobuterol increased contraction in normal dia- salbutamol, respectively, and as potent as those of phragm muscles (in vitro study) and that a signifi- clorprenaline. cant increase in force-frequency curves continued The β 2-adrenergic receptor agonists (β 2- from 4 to 24 hrs during transdermal treatment (in agonists) relax airway smooth muscle and are vivo study). principal bronchodilator agents used to treat bronchial asthma by metered-dose inhalation (MDI). METHODS Tulobuterol given by inhalation in 100-μg incre- Animal preparation ments up to a cumulative dose of 600 μg has been Experiments were performed on 70 BALB/c mice shown to produce dose-related increases in both weighing 24.9 ± 1.3 g (Charles River Japan, Yokohama), which were divided into 2 groups. (A) In the tulobuterol forced expiratory volume in 1 sec (FEV1) and forced vital capacity, and tulobuterol 200 μg and incubation (in vitro) group, diaphragm muscles extracted from normal animals were incubated in an organ buffer 400 μg aerosol has been reported to inhibit exer- -5 -6 -7 cise-induced asthma following 6-8 min treadmill with concentrations of 0, 10 , 10 , or 10 M of tulobuterol (kindly provided by Abbott Japan Co., Tokyo) (n = exercise, an effect comparable to that of 200 μg 5 each) for 1 hr, and then measured for contractility. (B) of salbutamol aerosol (Patel 1986). On the other In the tulobuterol transdermal treatment (in vivo) group, hand, it has been reported that isoproterenol and a small piece (4 × 4 mm2) of a 0.5 mg tulobuterol patch aminophylline improved contractility of fatigued sheet (16 × 16 mm2) was affixed to the shaved back of canine diaphragm muscle (Howell and Roussos each animal and covered with a small adhesive strip. 1984), that fenoterol improved fatigued canine The diaphragm muscles were dissected and measured for diaphragm muscle (Suzuki et al. 1986), that terbu- contractility at 1, 4, 8, 12, and 24 hrs (n = 5 each) after taline activated contraction in isolated fast- and patch affixation. As a control, the diaphragm muscles of slow-twitch skeletal muscle fibers in rats (Cairns another group of mice with shaved backs to which non- and Dulhunty 1993), and that broxaterol also tulobuterol patches were affixed were dissected and mea- increased force output of fatigued canine dia- sured for contractility at 1, 4, 8, 12, and 24 hrs (n = 5 phragm muscle more than salbutamol (Derom et each) after patch affixation. This study was approved by al. 1997). In addition, long-term clenbuterol the Animal Ethics Committee, Tohoku University, treatment (1 mg/kg subcutaneously twice a day Sendai, Japan. The study was performed from September for 12 week) has been reported to result in an 1, 2005 to January 31, 2006, and supported in part by a increased size of all diaphragm fiber types in nor- grant for physiological research on respiratory muscle. mal and emphysematous hamsters (Heijden et al. Measurement of muscle contraction 1998). These findings suggest thatβ 2-agonists increase contractility in normal and fatigued dia- Muscle strips (3-4 mm wide) were dissected from the right and left hemidiaphragms of each animal under phragm muscles. However, it has not yet been diethyl ether anesthesia and mounted in separate organ clarified whether tulobuterol has inotropic effects baths containing Krebs-Henseleit solution oxygenated on diaphragm muscles nor how long tulobuterol with a 95% O2 - 5% CO2 gas mixture (37.0 ± 0.5°C, pH transdermal treatment affects diaphragm contrac- 7.40 ± 0.05). The composition of the aerated Krebs- tility. We hypothesized that tulobuterol would Henseleit solution in mEq/l was as follows: Na+, 153.8; + 2+ 2+ − − increase diaphragm contractility during transder- K , 5.0; Ca , 5.0; Mg , 2.0; Cl , 145.0; HCO3 , 15.0; 2− 2− mal treatment in an animal model. HPO4 , 1.9; SO4 , 2.0; glucose, 110 mg%; d-tubocura- Accordingly, we first attempted to ascertain rine, 10 μM; and regular crystalline zinc insulin, 50 U/l. the direct effects of tulobuterol on diaphragm Both muscle strips were simultaneously stimulated with muscle by incubating diaphragm muscle strips in supramaximal currents of 200-250 mA (i.e., 1.2 to 1.5 310 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 311

times the current required to elicit maximal twitch ten- by dividing the muscle weight by the product of the strip sion, with a pulse duration of 0.2 msec) by a constant- muscle length and muscle density (1.06 g/cm3) (Close current stimulus isolation unit (SS-302J, Nihon Kohden, 1972), and tension was calculated as force per unit area Tokyo) driven by a stimulator (SEN-3201, Nihon (N/cm2). Data were averaged by the number of muscle Kohden, Tokyo). The tensions elicited were measured strips for force-frequency curves, twitch kinetics and by a force transducer (UL-100GR, Minebea Co., fatigue. Parameters were compared using the unpaired Fujisawa). The length of each muscle strip was changed Student’s t-test and two-way repeated-measures ANOVA. by moving the position of the force transducer with a All data are expressed as the means ± S.E.M. (Standard micrometer-controlled rack-and-pinion gear (accuracy of error of the mean). P < 0.05 was considered to be statis- displacement, 0.05 mm; Mitsutoyo Co., Kawasaki) and tically significant. measured with a micrometer in close proximity to the muscle. The optimal length of the muscle (Lo) was RESULTS defined as the muscle length at which twitch tension Changes in contractility in the tulobuterol development was maximal, and this Lo was maintained in subsequent measurements. incubation group The diaphragm force-frequency relationship was The force-frequency curves for the tulob- assessed by sequentially stimulating muscles at 1, 10, 20, uterol incubation (in vitro) group are summarized 30, 50, 70, 100, and 120 Hz. Each stimulus train was in Fig. 1. The peak tensions of the force-frequency curves at 10-5 M were 0.178 ± 0.010 N/cm2, applied for approximately 1 sec, and adjacent trains were -6 applied at approximately 10-sec intervals. The tensions and mostly increased, while those at 10 M were 2 -7 of both muscle strips were recorded by a hot-pen record- 0.164 ± 0.006 N/cm and those of 10 M were 2 er (RECTI-HORIZ-8K, San-ei, Tokyo). The force- 0.156 ± 0.008 N/cm , with a dose-dependent frequency curves obtained from the groups studied were increase compared with 0 M (0.147 ± 0.006 2 displayed as elicited tensions (N/cm2) on the Y-axis and N/cm ). As shown in Fig. 1, there were signifi- stimulating frequencies on the X-axis. cant increases at 1 (p < 0.01), 30, 50, 70, 100, and Twitch contraction was elicited by single-pulse 120 Hz for 10-5 M (p < 0.05, each) compared with stimulation (with 0.2-msec pulse duration), and the trace those for 0 M. of the twitch contraction was recorded at high speed (10 Table 1 summarizes the twitch contraction cm/sec). The twitch kinetics were assessed based on kinetics in the tulobuterol incubation group. The 2 twitch tension (TT, N/cm ), contraction time (CT, the twitch tension at 10-5 M (0.054 ± 0.005 N/cm2, time required to develop peak tension, milliseconds), and p < 0.01) was significantly higher than that at 0 M, half-relaxation time (HRT, the time required for peak and contraction time at 10-6 M (0.029 ± 0.001 sec, tension to fall by 50%, msec) during a single muscle p < 0.05) was significantly higher than that at 0 M. contraction. For analysis of rate of twitch contractions, The TT/CT at 10-5 M (1.98 ± 0.15 N/cm2/sec, p < TT/CT (slope during contraction time) and (TT/2)/HRT 0.01) was significantly higher than that at 0 M, (slope during half-relaxation time) were calculated from -5 2 and the (TT/2)/HRT at 10 M (0.72 ± 0.04 N/cm / the curve of the twitch contraction trace. sec, p < 0.001) was also significantly higher than Muscle fatigue was then assessed by examining the that at 0 M. There were no significant changes in rate of the decrease of tension over a 5-min period of rhythmic contraction, which was induced by applying half-relaxation time or fatigue. These results trains of 20 Hz stimuli (train duration, 0.3 sec; rest dura- show that diaphragm muscle tension increased and contraction became faster after 1 hr incubation, 0.7 sec) at a rate of 60 trains/min. Muscle fatigue -5 was expressed as a percentage of the final remaining ten- tion with 10 M tulobuterol in vitro compared sion (%) in relation to initial tension. After completion with the case of 0 M. of this protocol, the muscle strip was removed from the organ bath and weighed. Changes in contractility in the tulobuterol transdermal treatment group Data analysis The force-frequency curves with or without The cross-sectional area of the strip was calculated in vivo tulobuterol transdermal treatment are sum- 312 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 313

Fig. 1. Changes in force-frequency curves in the tulobuterol incubation group at 0 M (closed circles), 10-7 M (open circles), 10-6 M (open triangles), and 10-5 M (open squares). *p < 0.05, **p < 0.01, compared with each frequency of control.

TABLE 1. Changes of twitch kinetics and fatigue in the incubation groups. 0 M 10-7 M 10-6 M 10-5 M TT (N/cm2) 0.037 ± 0.002 0.043 ± 0.003 0.043 ± 0.003 0.054 ± 0.005** CT (sec) 0.025 ± 0.001 0.028 ± 0.001 0.029 ± 0.001* 0.027 ± 0.001 HRT (sec) 0.043 ± 0.005 0.040 ± 0.003 0.041 ± 0.003 0.038 ± 0.004 TT/CT (N/cm2/sec) 1.45 ± 0.08 1.53 ± 0.12 1.48 ± 0.07 1.98 ± 0.15** (TT/2)/HRT (N/cm2/sec) 0.45 ± 0.05 0.54 ± 0.04 0.54 ± 0.04 0.72 ± 0.04*** Fatigue (%) 34.4 ± 2.5 36.5 ± 1.5 34.6 ± 1.3 34.1 ± 0.9 TT: Twitch tension, CT: Contraction time, HRT: Half relaxation time. Significant difference compared with 0 M:* p < 0.05; **p < 0.01; ***p < 0.001. marized in Fig. 2. In the non-tulobuterol trans- tension in the force-frequency curve at 1 hr (0.148 dermal treatment group, there were no significant ± 0.006 N/cm2), although, there were significant changes in peak tensions in the force-frequency changes at 4 hrs (0.170 ± 0.004 N/cm2, p < 0.01), curves at 1 hr (0.145 ± 0.008 N/cm2), 4 hrs (0.137 8 hrs (0.174 ± 0.009 N/cm2, p < 0.05), 12 hrs ± 0.007 N/cm2), 8 hrs (0.136 ± 0.010 N/cm2), 12 (0.172 ± 0.005 N/cm2, p < 0.01), and 24 hrs (0.168 hrs (0.136 ± 0.011 N/cm2), or 24 hrs (0.131 ± 0.011 ± 0.013 N/cm2, p < 0.05) compared with the N/cm2). In the tulobuterol transdermal treatment curves without transdermal treatment. The incre- group, there was no significant increase in peak ment in diaphragm muscle contraction in the 312 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 313

Fig. 2. Changes in force-frequency curves at 1, 4, 8, 12, and 24 hrs with (open circles) and without tulobuterol transdermal treatment (closed circles). *p < 0.05, **p < 0.01, compared with each frequency with and without treatment. ##p < 0.01, compared between groups with and without treatment by ANOVA.

tulobuterol transdermal treatment group thus Fig. 4 shows the contractile rates in the commenced at 4 hrs and was maintained up to 24 tulobuterol transdermal treatment group. Both hrs (each p < 0.01 as compared with the non- TT/CT and (TT/2)/HRT with tulobuterol transder- treatment groups). mal treatment were increased at 8 hrs, and then Fig. 3 shows the twitch contraction kinetics decreased at 24 hrs. The TT/CT at 8 hrs (p < in the tulobuterol transdermal treatment group. 0.05), and (TT/2)/HRT at 8 and 12 hrs (p < 0.01 The twitch tensions with the tulobuterol treatment each) were significantly increased in the tulob- gradually increased at 4 and 8 hrs, while those at uterol transdermal treatment group. These results 12 hrs gradually decreased in a fashion similar to indicate that both contraction and relaxation that of the force-frequency curves, however, there speeds of diaphragm muscle increases with tulob- were not significant changes. Half-relaxation uterol transdermal treatment. times at 1 hr (0.049 ± 0.004 sec, p < 0.01) with the tulobuterol transdermal treatment were sig- DISCUSSION nificantly increased compared with those for mice In the present study, tulobuterol incubation without tulobuterol transdermal treatment. At no shifted the force-frequency curves upward dose- time point were the contraction times or fatigue dependently and increased the rates of contraction -5 with transdermal treatment significantly different (TT/CT) and relaxation ([TT/2]/HRT) at 10 M. from those for mice without transdermal treat- In the tulobuterol transdermal treatment group, ment. force-frequency curves were significantly 314 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 315

Fig. 3. Changes in twitch kinetics and fatigue with (white columns) and without tulobuterol transdermal treatment (black columns). A, twitch tension; B, contraction time; C, half-relaxation time; D, fatigue. #p < 0.05, compared with at 1 hr in the half-relaxation time of groups with and without treatment.

Fig. 4. Mean changes in slopes of twitch contraction traces at 1, 4, 8, 12, and 24 hrs with (white columns) and without tulobuterol transdermal treatment (black columns). A, TT/CT; B, (TT/2)/HRT. *p < 0.05, **p < 0.01, compared within the group with treatment. 314 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 315

increased at 4 hrs after treatment, and this over 25°C. On the other hand, the cross-sectional increase was maintained up to 24 hrs after treat- area of the diaphragm muscle in our experiment ment compared with the control group. The rates was square with a thickness of about 0.25 mm. of contraction (TT/CT) and relaxation ([TT/2]/ Thus, we could neglect such a possibility in the HRT) at 8, and 8 and 12 hrs, respectively, after tu- present study. lobuterol transdermal treatment were increased Thirdly, it has also been reported that hyper- significantly. These increases in contractile rates oxia (tissue bath Po2 83.3 ± 0.9 kPa) or severe appear comparable with those observed in the in hypoxia (Po2 7.1 ± 0.3 kPa) was observed and that vitro study. this resulted in a severe decrease in diaphragm There are several concerns regarding the twitch force in rat (Heijden et al. 1999). The Po2 method of the present study. Firstly, we used a in the baths was 65.3 kPa during the present 2 1/16 (4 × 4 mm ) portion of a commercially avail- study, suggesting that the value of Po2 is suffi- able 0.5 mg tulobuterol patch (16 × 16 mm2, equal ciently hyperoxic to avoid hypoxia. to 0.2 mg/cm2) in these experiments. The dosage Subsequent to the report that adrenaline and per mouse of tulobuterol with a mean weight of other sympathomimetic amines increase the maxi- 24.9 ± 1.3 g was about 33 times that for a 60-kg mal twitch tension of unfatigued skeletal muscles human subject with a 0.5 mg patch, even though (Bowman et al. 1958), the pharmacokinetic the animal was shaved prior to transdermal treat- actions of β-adrenergic agonists have been exten- ment. This size of patch was necessary to main- sively studied. Furthermore, it appears that these tain attachment to the skin of an animal for 24 effects on muscle contractility may arise through hrs. It has been reported that finger tremor, an activation of the adenyl cyclase/cyclic 3′, 5′- inevitable concomitant of bronchodilatory therapy adenosine monophosphate (AMP) system, which with 2 mg tulobuterol oral administration, in turn leads to changes in the rates of uptake of becomes tolerable, at least after a certain period Ca2+ by the sarcoplasmic reticulum (Bowman et of habituation (Schaffler and Reeh 1987). al. 1969). Radioligand-binding studies have Uematsu et al. (1993) reported peak serum con- shown that up to 40% of β 2-adrenoceptors in the centration to be about 4 ng/ml with a 0.2 mg/cm2 ventricle and up to 55% of such receptors in the of transdermal tulobuterol patch affixed to the atrium are of β 2-subtype (Bristow et al. 1986). chest skin. If the drug absorption rate from a Myocardial β 1-adrenoceptors are functionally patch is similar to 0.2 mg/cm2 of a transdermal active and evoke chronotropic effects, which in tulobuterol patch, the serum concentration can be turn increase the heart rate and at least partially expected to be about 4 ng/ml in an animal model. mediate inotropic effects (Lipworth et al. 1991). We suggest that this serum concentration is too At concentrations which produce effects on mus- low to induce tremor based on the fact that we did cle contractility, salbutamol has been found to not observe such tremor in any animal in the pres- significantly elevate cyclic AMP (cAMP) concen- ent experiment. trations in both types of muscle (Al-Jeboory and Secondly, it has been reported that a progres- Marshall 1978). Thus, it is currently considered sively larger hypoxic core was observed at incu- that β -adrenergic agonists stimulate adenylyl bation temperatures above 25°C (Segal and cyclase, elevating cAMP, which activates cAMP- Faulkner 1985). However, we did not experience dependent protein kinase (PKA) and which phos- such a phenomenon, when we tested twitch stimu- phorylates several key proteins, including 1) lation every 10 min for 4 hrs in our experimental troponin I (reducing Ca affinity of TnC), 2) sarco- setup. We suggest that there are histological dif- lemmal Ca channels (increasing Ica), 3) phos- ferences, that is, the cross-sectional area of soleus pholamban (increasing SR Ca pump rate) and 4) (SOL) and extensor digitorum longus (EDL) SR Ca release channels (modifying RyR gating) muscles in that paper was round and had a central (Bers 2001). We suggest that the augmentation of core which induced hypoxia during incubation contractile and relaxation rates by tulobuterol 316 C. Shindoh et al. Effects of Tulobuterol on Diaphragm Muscle 317 observed in the present study is an overall effect In conclusion, tulobuterol has direct inotro- of Ca changes in these rates in the diaphragm pic and chronotropic effects on the diaphragm muscle. muscle, and these effects on diaphragm muscle Although we measured isometric tension in with transdermal treatment are maintained for the present study, diaphragm flattening is some- about 24 hrs. We suggest that these effects of times observed in patients with chronic obstruc- tulobuterol, which increase diaphragm contractili- tive pulmonary disease (COPD). A previous ty with bronchial dilatation, may complement study has shown that the β 2-adrenergic agent sal- each other and may relieve dyspnea. butamol has inotropic effects on rat diaphragm contractility and that these effects are potentiated References by foreshortening (Heijden et al. 1997). In addi- Al-Jeboory, A.A. & Marshall, R.J. 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