CNS Drugs 16: 1-8, No. 1, 2002

LEADING ARTICLE

CNS Drugs 2002; 16 (1): 1-8 1172-7047/02/0001-0001/$22.00/0 © Adis International Limited. All rights reserved.

Potential of β2-Adrenoceptor Agonists as Add-On Therapy for Multiple Sclerosis Focus on Salbutamol (Albuterol) Karim Makhlouf, Howard L. Weiner and Samia J. Khoury Center for Neurologic Diseases, Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

Abstract

The β2-adrenergic receptor agonist salbutamol (albuterol) has been used for many years in the treatment of bronchospasm in patients with asthma. In this patient group, salbutamol is a relatively safe and inexpensive drug, and is easy to administer. Within the last few years, there has been increasing evidence that salbutamol might have immunomodulatory properties both in vitro and in vivo, in different animal models as well as in humans. This has led researchers to consider salbutamol as a potential therapy for several autoimmune diseases, including multiple sclerosis (MS). In this article, we review the literature presenting such evidence, and discuss the possible mechanisms by which salbutamol influences the immune system. We conclude that salbutamol might be an interesting add-on therapy in patients with MS and that further research is warranted.

Multiple sclerosis (MS) is one of the most common neurological diseases in young adults. Although its precise aetiology is still unknown, it is generally viewed as an immune-mediated disease. It is characterised by a chronic inflammation in the CNS, leading to demyelination and axonal loss, and ultimately to functional disability. There is no cure for the disease, but tremendous research efforts and a better understanding of the immunological mechanisms involved have led to the development of disease-modifying drugs, such as β-interferons and glatiramer acetate, which are able to delay symptom progression by interfering with the immune system. Salbutamol (albuterol) has recently been shown to have immunomodulatory proper-

ties, in addition to its well known bronchodilating effect, and this has stimulated research into its possible use in a number of autoimmune disorders, including MS. 1. Pharmacodynamic and Pharmacokinetic Properties of Salbutamol: A Brief Reminder Introduced in the early 1970s, salbutamol is a β2adrenergic receptor agonist (figure 1). The principal effect of the drug is to induce bronchodilatation as the result of immediate relaxation of tracheal and bronchial smooth muscles, mediated through binding of the drug to β2-adrenergic receptors. The same receptors also mediate uterine relaxation and

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skeletal muscle increased contractility (responsible for the most common adverse effect of the drug, tremor), glycogenolysis and neoglycogenesis in the liver, and arteriole dilatation. Because it is a selective β2-agonist, salbutamol has reduced β1-mediated cardiac adverse effects compared with nonselective β-agonists. It can be administered by several routes: orally (as syrup or tablets), by inhalation (through metered-dose inhalers or non-metered devices like nebulisers), or parenterally. The following provides a brief review of the metabolism of salbutamol; more details are beyond the scope of this paper and are available elsewhere.[2,3] Although the main metabolic pathway for salbutamol in animals is coupling to glucuronic acid, sulphate conjugates are the main metabolites in humans.[2] The degree of metabolite formation, and hence the percentage of available active drug and the rate of adverse effects, all depend on the route of administration. After intravenous injection, most of the dose is secreted into the urine as intact, nonmetabolised drug and only 20% is conjugated. Biliary excretion is negligible. The elimination halflife of salbutamol after parenteral administration is about 3 to 4 hours in healthy adult volunteers, with a plasma clearance of approximately 500 ml/min and a renal clearance of 300 ml/min.[4] It is well known that intravenous administration yields the most adverse effects. Salbutamol is most often administered as an inhalation aerosol, or orally. Less than 20% of a single salbutamol dose is absorbed after administration via a nebuliser, the remaining being recovered from the apparatus or in the expired air.[5] Most of the absorbed dose is recovered in the urine within 24 hours. However, the onset of the bronchodilating effect is observed within seconds. Salbutamol is rapidly and well absorbed after oral administration, although 50% of the given dose is recovered in the urine as a sulphate conjugate, due to an extensive first pass effect, related to both strong hepatic metabolism and presystemic metabolism in the intestinal mucosa.[2] The peak plasma salbutamol concentration is observed 2 hours after oral administration, and the elimination half-life is 5 to 6 hours. The  Adis International Limited. All rights reserved.

Makhlouf et al.

HOH2C CH3 HO

CH− CH2 NH

C

OH

CH3

CH3

Fig. 1. Chemical structure of the β 2-adrenoceptor agonist salbutamol (albuterol) [α 1-[(tert-butylamino)methyl]-4-hydroxy-mxylene-α ,α ’ - d i o l ] . S a l b u t a m o l e x i s t s i n t w o d i ff e r e n t stereoisomeric forms and is administered clinically as a racemic mixture, but only one of the forms (the eutomer) is active; the other (distomer) has been implicated in toxicity.[1]

mean steady-state peak and trough plasma concentrations vary between 6.7 and 14.8 µg/L, and 3.8 and 8.6 µg/L, respectively, with habitual therapeutic dosages.[5]Plasma protein binding is rather low, at around 7%.[4] Finally, it is important to mention that salbutamol does not cross the blood-brain barrier (BBB), at least in animals.[5] 2. Basic Immunological Concepts in Multiple Sclerosis (MS) Although MS is clinically and pathologically heterogenous, demyelination is a key feature of the disease. Very early in the course of the disease, axonal damage also occurs[6] and leads to the irreversible clinical symptoms of the chronic phase of MS. MS is considered an immune-mediated disease, but genetic and environmental factors (such as viral infections) may also play a pathogenic role.[7] Experimental autoimmune encephalomyelitis (EAE) is the most widely used animal model of MS. The demyelinating process in MS involves immunoglobulins, complement molecules and, more importantly, T cells and monocytes/macrophages, which can all cross the BBB and into the brain. At this site, they release cytokines, chemokines, adhesion molecules, metalloproteinases, nitric oxide and oxygen metabolites, all molecules that participate in the effector stages of the disease. MS is considered to be a Th1 cell-mediated autoimmune disease,[8] characterised by chronic inflammation in the CNS, with perivascular infiltration of mononuclear cells. Th1 cells are helper T cells that CNS Drugs 2002; 16 (1)

Salbutamol in Multiple Sclerosis

produce Th1 cytokines, also called pro-inflammatory cytokines, such as interleukin (IL) 2 (IL-2), tumour necrosis factor-α (TNF-α) and interferon (IFN)-γ, mainly under the influence of IL-12. Th2 cells produce Th2 cytokines, such as IL-4, IL-5, IL-10 and IL-13. Th1 and Th2 cytokines tend to suppress each other’s effect. The now classical polarised Th1/Th2 paradigm may not be able to explain all the experimental observations in the study of chronic inflammatory and/or autoimmune diseases,[9] but it still is a useful model to explain the pathogenic mechanisms of several immunologic diseases. IL-12 is one of the most potent inducers of IFNγ and the Th1 response.[10] It is a heterodimeric cytokine made of two disulphide-linked chains, p35 and p40, and produced by B cells, monocytes/macrophages and dendritic cells. As IL-12 is considered a key cytokine in the pathogenesis of MS,[11,12] any drug that decreases its production is of potential therapeutic interest. Several treatments used in MS, like IFN-β, do in fact decrease IL-12 production[12] and we have previously shown that IL-12 production is increased in patients with progressive MS,[13,14] and that expression of this cytokine is positively correlated with disease activity, and is normalised by pulse cyclophosphamide therapy.[14] 3. Evidence of Immunomodulatory Effects of β2-Agonists in Animal Models and Humans 3.1 In Animal Models

Although no work has been published with salbutamol specifically, several papers from the same group showed that both the nonspecific β-adrenergic agonist, isoprenaline (isoproterenol), and the specific β2-adrenergic agonist, terbutaline, were able to suppress the first acute attack and the number of further relapses in a chronic/relapsing EAE model of MS in the Lewis rat, even when the treatment with either β-agonist was started after the onset of the disease.[15-17] Salbutamol is reported to suppress collagen-induced arthritis, a murine model for the human autoimmune disease rheumatoid arthritis.[18]  Adis International Limited. All rights reserved.

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3.2 In Humans

Knowing that IL-12 production in human macrophages can be inhibited by prostaglandin E2, through increased intracellular cyclic adenosine monophosphate (cAMP),[19] and since β2-agonists also elevate intracellular cAMP levels,[20,21] PaninaBordignon et al.[22] assessed the effect of β2-agonists such as salbutamol on IL-12. They reported that these agents selectively inhibited the production of IL-12 by human monocytes in vitro and in vivo in healthy subjects, through increased intracellular cAMP level. Interestingly, β2-adrenergic receptor expression is increased on peripheral blood mononuclear cells in patients with MS,[23] and this is positively correlated with clinical and magnetic resonance imaging (MRI) disease activity.[24] In an open pilot study involving 21 untreated patients who had secondary progressive MS, using flow cytometry, we measured the percentage of IL-12– producing monocytes in the peripheral blood before and after treatment with salbutamol.[25] The treatment was given orally for 2 weeks, using the protocol shown in figure 2. We first confirmed that baseline IL-12 production was higher in patients with MS than in healthy controls. Most importantly, we showed that oral salbutamol treatment induced a significant decrease in the percentage of IL-12– producing monocytes and dendritic cells. This effect could already be detected 2 hours after oral intake of the drug (figures 3a, b and c). Moreover, the IL-12 level was still decreased for at least 1 week after the end of therapy (figure 3d). Although in three patients the level of IL-12 at week 3 was clearly higher than at baseline (figure 3d), this longterm effect of salbutamol treatment lasted far beyond the half-life of the drug for the majority of the tested patients. In a subset of five patients, we tested the effect of salbutamol on other cytokines and found that treatment with oral salbutamol induced a significant increase in the percentage of IL-10–, IL-4– and IL-5–producing T cells, and a decrease in IFN-γ– producing T cells. These results suggest that salbutamol not only decreases IL-12 production, but seems CNS Drugs 2002; 16 (1)

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to induce a shift towards a Th2 (anti-inflammatory) cytokine production pattern. Our study was not designed to detect any clinical changes due to salbutamol. No serious adverse reactions occurred during the study, and no patient interrupted the treatment. 4. Mechanisms of Action Salbutamol mediates its effects via an agonist effect at the β2-adrenergic receptor. This receptor is a transmembrane molecule coupled to a G-protein (figure 4). The binding of salbutamol to its receptor stimulates the G-protein, which activates an enzyme, adenylate cyclase, which in turn catalyses the degradation of adenosine triphosphate (ATP) into cAMP. cAMP activates another enzyme, a protein kinase A (PKA), which is the immediate effector of elevated cytosolic cAMP levels, as it activates the transcription of specific genes. PKA enters the nucleus and phosphorylates the cAMP-responsive element binding protein (CREB), which binds to a short spe-

Week 1

D1

Week 2

W2

W1

4mg SB orally × 1/day

Week 3

4mg SB orally × 3/day

W3

No drug

Patient visit Blood sampling

Fig. 2. Schedule for blood sampling and drug intake in a study

of the effect of salbutamol (albuterol) on interleukin 12 (IL-12) production in 21 otherwise untreated patients with secondary progressive multiple sclerosis. All patients received on the first day (D1) a single dose of salbutamol (SB) 4mg orally. Blood was obtained immediately before the intake of the drug (pre-D1), and after 2 hours, corresponding to the peak serum concentration of salbutamol (post-D1). Patients received salbutamol 4 mg/day in the morning for one week, then 4mg three times a day for the next week. No medication was given during the third and last week of the study. Blood for assessment of IL-12 expression was also obtained at the end of weeks 1 (W1) and 2 (W2), again just before and 2 hours after the intake of the drug (pre- and post-W1, and pre- and post-W2, respectively). Finally, a blood sample was obtained at the end of the third week (W3).

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cific DNA sequence called cAMP-responsive element (CRE) at the regulatory region of genes that are regulated by cAMP.[26] cAMP-induced PKA activation can also lead to the suppression of the nuclear factor κB (NFκB), as a potent PKA inhibitor, H89, has been shown to abrogate the suppression of NFκB induced by elevated cAMP.[27] NFκB is a highly regulated dimeric transcription factor, that usually remains inactivated in the cytoplasm, due to association with inhibitory IκB proteins (α and β).[28] Several extracellular stimuli (figure 4) can induce rapid degradation of IκB by phosphorylation. This leads to the release of p50/p65 NFκB proteins, which translocate to the nucleus, bind the NFκB sequence on the DNA and induce transcriptional activation of specific genes. One of the mechanisms by which elevated cAMP decreases NFκB activity is by stabilisation of IκB proteins. Thus, any cAMP-increasing drug may, through PKA activation, induce a suppression of NFκB. As this transcription factor is implicated in the synthesis of several cytokines such as IL-12p40,[29] TNFα and others,[30] it becomes easy to understand how salbutamol or other cAMP-increasing drugs may decrease the expression of these cytokines. However, it is probably less straightforward in reality: for example, PKA activation may not always lead to suppression of NFκB, but to its activation, an effect independent of cAMP.[31] Also, the catalytic subunit of PKA (PKAc) is maintained in an inactive state through association with IκB-α or IκBβ in an NFκB-IκB-PKAc complex.[32] To further complicate the picture, there are data suggesting that activation of the PKA pathway inhibits NFκB transcription by phosphorylating CREB.[33] cAMP-responsive elements are also involved in the transcriptional activation of the human IL-10 gene.[34] There is also evidence that the nonspecific β-agonist isoprenaline increases IL-10 production.[35] This is in accordance with our data on five patients with secondary progressive MS, in whom oral salbutamol increased the percentage of IL-10–producing T cells. CNS Drugs 2002; 16 (1)


a

5

b

c

d

% IL-12–producing monocytes

80

60

40

20

0 Pre -D1

Post -D1

Pre -W1

Post -W1

Pre -W2

Post -W2

Pre -D1

W3

Fig. 3. Percentage of interleukin 12 (IL-12)–producing monocytes in the blood of patients with secondary progressive multiple sclerosis who were given salbutamol (albuterol) [according to the scheduled outlined in figure 2]. (a) baseline and 2 hours after oral intake of salbutamol 4mg [p = 0.0001]; (b) before and 2 hours after the last dose of the first week [p = 0.0004]; (c) before and 2 hours after the last morning dose of the second week [p < 0.0001]; and (d) baseline and 3 weeks later (2 weeks with treatment, 1 week

without) [p = 0.0256]. All timepoint comparisons were done using the Wilcoxon paired, two-tailed test. The Kruskal-Wallis test showed a strongly significant effect of oral salbutamol on IL-12 production in monocytes (p < 0.0001). See legend to figure 2 for definitions of D1, W1, W2 and W3.

Finally, salbutamol has been shown to decrease the expression of CD80 on the surface of monocytes in vitro.[25] CD80 (or B7-1) is an important molecule involved in co-stimulation of T cells. 5. Conclusion – Potential Place of Salbutamol in the Treatment of MS The existing approved therapies for MS are only partially effective, so there is a need for new treatments that may be more effective or that can be used as add-on therapies. Salbutamol may theoretically have a potential therapeutic role in MS because of its ability to regulate the expression of several cytokines, mainly through an increase in intracytoplasmic cAMP level. To our knowledge, there have been no published reports on salbutamol in MS therapy. While our study[25] provided evidence of the immunological effects of salbutamol  Adis International Limited. All rights reserved.

in MS, it did not assess the effect of the drug on symptoms of the disease. However, we are currently starting a clinical trial to test the efficacy of salbutamol as an add-on therapy to glatiramer acetate in patients with MS. Encouragingly, salbutamol has a known safety profile, is well tolerated, inexpensive, and easy to administer. Another class of drugs, the phosphodiesterase (PDE) inhibitors (PDEIs), also target cAMP and increase intracytoplasmic levels by inhibiting degradation by PDE (figure 4). Rolipram, a type IV PDEI, is the most extensively studied of this class of drugs and it has been shown to suppress IL-12 production in mice,[36] and to prevent EAE in rats[37] and in nonhuman primates.[38] An additional protective mechanism for rolipram in EAE in mice is its ability to reduce BBB permeability.[39] Although mostly known as an antidepressant in humans, rolipram has, like salbutamol, a therapeutic potential in Th1-mediated CNS Drugs 2002; 16 (1)

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G-protein–coupled receptor G-protein

γ β

Adenylate cyclase

Extracell Cell membrane

α

Intracell

Gs Gi ATP

cAMP

PDE

5′AMP

Nucleus membrane Inactive PKA

Inactive CREB

Activated PKA

Transcription effects

Metabolic effects Activation of protein phosphatases

DNA

Action on NFκB production, inhibitory or stimulatory

Activated CREB CRE Gene activation (physiological effects)

p50

p65

κB binding site near gene of interest

IκB-α p50

p65

NFκB

Extracell. stimuli: • LPS • Th1cytokines • viral infection • agonists for G-protein–couped receptor

IκB-α

Fig. 4. Cyclic adenosine monophosphate (cAMP) and nuclear factor κ B (NFκ B) pathways. 5′AMP = 5′-adenosine monophosphate; ATP = adenosine triphosphate; CRE = cAMP-responsive element; CREB = cAMP-responsive element binding protein; Gi = inhibitory subunit of G-protein; Gs = stimulatory subunit of G-protein; LPS = lipopolysaccharide; PDE = phosphodiesterase; PKA = protein

kinase A.

autoimmune diseases,[40] and is now being tested in a clinical trial in patients with MS. Acknowledgements The authors have been supported by NIH grants N01A105411 (SJ Khoury), IP01NS38037 (HL Weiner), the National Multiple Sclerosis Society pilot grant PPD643 (SJ Khoury) and the Nancy Davis Centre Without Walls. The  Adis International Limited. All rights reserved.

authors have no potential conflicts of interest that are directly relevant to the contents of this article.

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Correspondence and offprints: Dr Samia J. Khoury, Center for Neurologic Diseases, Brigham & Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. E-mail: [email protected]

CNS Drugs 2002; 16 (1)