Systemic Administration of Β2-Adrenoceptor Agonists
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British Journal of Pharmacology (2006) 147, 587–595 & 2006 Nature Publishing Group All rights reserved 0007–1188/06 $30.00 www.nature.com/bjp Systemic administration of b2-adrenoceptor agonists, formoterol and salmeterol, elicit skeletal muscle hypertrophy in rats at micromolar doses 1James G. Ryall, 2Martin N. Sillence & *,1Gordon S. Lynch 1Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Grattan Street, Victoria 3010, Australia and 2School of Agricultural and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2678, Australia 1 b2-Adrenoceptor agonists provide a potential therapy for muscle wasting and weakness, but their use may be limited by adverse effects on the heart, mediated in part, by b1-adrenoceptor activation. 2 Two b2-agonists, formoterol and salmeterol, are approved for treating asthma and have an extended duration of action and increased safety, associated with greater b2-adrenoceptor selectivity. 3 The pharmacological profiles of formoterol and salmeterol and their effects on skeletal and cardiac muscle mass were investigated in 12-week-old, male F344 rats. Formoterol and salmeterol were each administered via daily i.p. injection at one of seven doses (ranging from 1 to 2000 mgkgÀ1 dayÀ1), for 4 weeks. Rats were anaesthetised and the EDL and soleus muscles and the heart were excised and weighed. Dose–response curves were constructed based on skeletal and cardiac muscle hypertrophy. 4 Formoterol was more potent than salmeterol, with a significantly lower ED50 in EDL muscles (1 and 130 mgkgÀ1 dayÀ1, P o0.05), whereas salmeterol had greater intrinsic activity than formoterol in both EDL and soleus muscles (12% greater hypertrophy than formoterol). The drugs had similar potency and intrinsic activity in the heart, with a smaller leftward shift for formoterol than seen in skeletal muscle. A dose of 25 mgkgÀ1 dayÀ1 of formoterol elicited greater EDL and soleus hypertrophy than salmeterol, but resulted in similar b-adrenoceptor downregulation. 5 These results show that doses as low as 1 mg kgÀ1 dayÀ1 of formoterol can elicit significant muscle hypertrophy with minimal cardiac hypertrophy and provide important information regarding the potential therapeutic use of formoterol and salmeterol for muscle wasting. British Journal of Pharmacology (2006) 147, 587–595. doi:10.1038/sj.bjp.0706669; published online 23 January 2006 Keywords: Adrenergic; heart; cardiac hypertrophy; receptors Abbreviations: BM, body mass; CI, confidence intervals; ED50, drug dose at half of the maximum effect; EDL, extensor digitorum longus; Emax, maximum hypertrophic effect; i.p., intraperitoneal; MW, molecular weight Introduction A severe loss of muscle mass and strength is associated with (Emery et al., 1984). Since then, b2-agonists such as clenbuterol numerous conditions and disease states, including: dystrophy, and fenoterol have been examined in numerous animal models cancer cachexia, chemotherapy, sepsis, acquired immune of muscle wasting (Maltin et al., 1987; Chen & Alway, 2000; deficiency syndrome, burn injury, and sarcopenia (Barton & Sneddon et al., 2000; Zeman et al., 2000; Beitzel et al., 2004; Morris, 2003; Jackman & Kandarian, 2004). The continual Ryall et al., 2004). Some agonists, such as albuterol, have been loss of muscle protein can lead to an increase in morbidity, and used in clinical trials for the treatment of neuromuscular in extreme cases, an increased mortality rate. Thus, therapies disorders (Martineau et al., 1992; Kissel et al., 1998; 2001; that aim to alleviate the symptoms of muscle wasting are Fowler et al., 2004). The first clinical trials using albuterol to directed towards preserving existing muscle fibres, enhancing treat young boys with facioscapulohumeral dystrophy, found muscle fibre regeneration, and promoting muscle fibre growth. that year-long administration at doses of 16 and 32 mg dayÀ1 Agents that stimulate an increase in muscle size (hypertrophy), had only limited beneficial effects on strength, and was by either increasing protein synthesis or decreasing protein associated with some adverse cardiac-related events (Kissel degradation, or both, have the potential to be applied clinically et al., 1998). In a more recent study, Fowler et al. (2004) to combat muscle wasting conditions (Lynch, 2004). administered a lower dose of 8 mg dayÀ1 of albuterol for 28 Synthetic b2-adrenoceptor agonists (b2-agonists) were initi- weeks to boys with Duchenne and Becker muscular dystrophy, ally developed to facilitate bronchodilation to relieve asthma and found modest increases in strength with no reported side (Solis-Cohen, 1900). However, it became apparent that at effects. These results suggested that low doses of b2-agonists doses higher than used therapeutically, b2-agonists were were well tolerated, but elicited only a modest improvement in capable of eliciting significant skeletal muscle hypertrophy skeletal muscle mass and strength. In the past 15–20 years, much research on asthma has focused on extending the bronchodilating actions of b2- *Author for correspondence; E-mail: [email protected] agonists, while maintaining or improving their safety profile 588 J.G. Ryall et al Low dose b-agonists elicit muscle growth (Lo¨ fdahl and Svedmyr, 1989; Ball et al., 1991; Guhan et al., free access to drinking water and food. Rats were housed 2000). To this end, two b2-agonists, formoterol fumarate under an artificial light : dark cycle with light between 0600 and salmeterol xinafoate, have been recently approved in the and 1800 h. U.S. for the treatment of asthma. These b2-agonists have an extended duration of action in relaxing smooth muscle b2-Agonist administration and tissue collection compared with traditional asthma medications, such as albuterol and terbutaline (Lo¨ fdahl & Svedmyr, 1989; Roux Male 3-month-old Fischer 344 rats (F344, n ¼ 128 rats, body et al., 1996; Waldeck, 1996). In addition to an extended mass (BM) B270 g) obtained from the Animal Resource duration of action, the safety profiles of formoterol and Centre (Canning Vale, Western Australia) were allocated into salmeterol, defined as the frequency of adverse events, have either control or one of two b2-agonist-treated groups. Treated been improved significantly, because of an increased selectivity rats (n ¼ 112, 8 rats doseÀ1) received 1,10, 25, 250, 500, 1000, or for the b2-adrenoceptor (Guhan et al., 2000; Pearlman et al., 2000 mgkgÀ1 of formoterol in saline, while salmeterol was 2002). Since the skeletal muscle adrenoceptor population administered in a 1 : 10 cremphor : saline vehicle due to its consists predominantly of b2-adrenoceptors, and cardiac highly lipophilic nature. b2-Agonist treatment consisted of muscle consists primarily of b1-adrenoceptors (Ryall et al., once daily intraperitoneal (i.p.) injections for a period of 4 2002; 2004; Gregorevic et al., 2005), a more selective b2-agonist weeks. Control rats received an identical volume of saline only is likely to lessen any adverse effects on cardiac performance. (n ¼ 8), or a cremphor : saline mix (for salmeterol control, While cardiac muscle consists primarily of b1-adrenoceptors, n ¼ 8). All animals were weighed daily throughout the study. there is a significant proportion of b2-adrenoceptors, generally Following 4 weeks of treatment, rats were anaesthetised in a ratio of 2 : 1 (Brodde, 1991; Ryall et al., 2002; Gregorevic with sodium pentobarbitone (Nembutal, Rhone Merieux, et al., 2005). The cardiac b2-adrenoceptor is known to have Pinkenba, QLD, Australia: 60 mg kgÀ1, i.p.), with supplemen- dual coupling properties (Xiao et al., 1999; Kilts et al., 2000), tal doses administered to maintain an adequate depth of whereby stimulation activates not only the traditional adre- anaesthesia, such that there was no response to tactile noceptor–Gs–cAMP–PKA pathway, but also a Gi-mediated stimulation. The EDL (fast-twitch) and the soleus (slow- signal, believed to be involved in cell survival (Foerster et al., twitch) muscles were surgically excised from both hindlimbs, 2003; Po¨ nicke et al., 2003; Bernstein et al., 2005). Formoterol blotted on filter paper, trimmed of their tendons, and weighed is reported to have a b2 : b1 adrenoceptor selectivity ratio of on an analytical balance. The rats were killed by opening the between 200 and 400, while salmeterol has a selectivity ratio of thoracic cavity and immediate cardiac excision. The heart was 410,000 (Anderson, 1993; Johnson et al., 1993). blotted on filter paper, trimmed of large vessels, and weighed. The exact mechanism for the extended duration of action of All tissues were frozen immediately in thawing isopentane and both formoterol and salmeterol is still unclear, although two then stored at À801C for later biochemical analyses. hypotheses have been proposed. The first, and most popular explanation, is known as the ‘diffusion microkinetic hypo- Muscle protein concentration thesis’, and it relates to the greater lipophilic nature of formoterol and salmeterol (compared to other b-agonists, such To determine the effect of daily injection of cremphor vehicle, as fenoterol and clenbuterol; Waldeck, 1996), a property skeletal muscle protein concentration was determined in saline conferred by a long carbon side-chain (Anderson, 1993). The control- and cremphor vehicle-treated animals using a second explanation relates to the presence of a secondary Bradford protein assay (Bio-Rad, Hercules, CA, U.S.A.), with binding site, or ‘exo-site’ (Johnson et al., 1993). bovine