Parkinson’s disease (PD) affects 0.1-0.2% of the general population with increasing prevalence with age, and up to 1% in individuals above 60 years of age, resulting in an estimated 7 to 10 million people with PD worldwide (Tysnes and Storstein, 2017). The physical burdens for PD patients includes motor symptoms, such as bradykinesia, rigidity, resting tremor, and postural instability, and non-motor symptoms, including autonomic disturbances, depression and cognitive impairment (Boland and Stacy, 2012). PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and by the appearance of Lewy bodies, which are intracellular inclusions of aggregated α-synuclein (α-syn), a protein which plays multiple roles in the pathogenesis of PD and which has therefore become a target for novel therapeutics aimed at reducing its toxicity (Dehay et al., 2015).
A collaborative study involving researchers based in UE, USA and Canada now shows that β2-adrenoceptors (β2-AR) are linked to the transcription of α-syn and increased risk of PD, suggesting that they might represent novel targets for the development of antiparkinson therapeutics (Mittal et al., 2017). Evidence provided in the study has been obtained by means of several different and multidisciplinary approaches. First, a high-throughput gene expression assay was developed in human SK-N-MC neuroblastoma cells for endogenous expression of human SNCA, the gene encoding α-syn. Researchers screened 1126 compounds, including drugs approved by the U.S. Food and Drug Administration (FDA) and a diverse set of natural products, vitamins, health supplements, and alkaloids. Screening was followed by replication and confirmation experiments, eventually leading to the identification of four SNCA expression–lowering compounds, including three β2-AR agonists, namely metaproterenol, salbutamol (also known as albuterol) and clenbuterol. Interestingly, the fourth compound was riluzole, which is currently approved for amyotrophic lateral sclerosis and may attenuate dopaminergic neurodegeneration in rodent models of PD.
In a second experimental approach, it was shown that clenbuterol administered intraperitoneally in mice lowered the expression of endogenous α-syn protein and mRNA levels in the PD-vulnerable substantia nigra. In vitro experiments were thereafter performed in primary neurons from mice carrying a deletion of the β2-AR gene (Adrb2), as well as in human SK-N-MC cells after silencing of the β2-AR gene. In both cell types, SNCA mRNA and α-syn protein levels were increased. Further support to the role of β2-AR in the control of α-syn expression came from experiments in wild-type SK-N-MC cells, where the β-AR antagonist propranolol increased endogenous SNCA mRNA and α-syn protein levels, and genetic silencing of β2-AR or co-treatment with propranolol blocked clenbuterol’s SNCA expression–lowering effects.
Perhaps the most impressive evidence however comes from a pharmacoutilization study performed in a wide Norwegian population, and from evidence of neuroprotective effects in a mouse model of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–induced neurodegeneration, as well as from results in an induced pluripotent stem cell (iPSC)–derived neuronal culture system from a patient with autosomal dominant PD due to a triplication of the SNCA locus. Using the Norwegian Prescription Database (NorPD), the entire living Norwegian population as of January 1, 2004 was included, and after adjustment for sex, age, and level of education, it was shown that use of the β2-AR agonist salbutamol was associated with decreased risk of PD (rate ratio, 0.66 [95% confidence interval (CI), 0.58 to 0.76]), while use of the β-AR antagonist propranolol was associated with a markedly increased risk of PD (rate ratio of 2.20 [95% CI, 1.62 to 3.00]). In MPTP-treated mice, clenbuterol treatment abrogated the MPTP-induced loss of TH+ neurons, and in iPSC-derived neurons, which constitutively overexpress endogenous α-syn, clenbuterol reduced SNCA mRNA expression and α-syn protein levels and protected cells against the toxicity of rotenone, another chemical which -like MPTP – induces dopaminergic neurotoxicity.
Although altogether the evidence provided appears massive and impressive, the study has nonetheless many potential limitations and caveats which must be discussed. First, the concentrations of β2-AR agonists used in in vitro experiments are very high, in the range 5-10 mM, which is much higher than the therapeutic concentrations of e.g. clenbuterol (Cmax = 0.13 mM with 80 mg oral dose) or salbutamol (Css = 0.04 mM with 400 mg intravenous bolus followed by 10 mg/min infusion) (Yamamoto et al., 1985; Morgan et al., 1986), as well as their affinity for β2-AR, (pKi = 7.9 for clenbuterol and 5.8–6.1 for salbutamol) (Isogaya et al., 1999; Baker et al., 2010).
In addition, no pharmacological experiments include concentration-response curves, and most of them are just performed with only agonists or antagonists, therefore not well controlled by showing that the effects of agonists are selectively counteracted in the presence of antagonists. Finally, it is hard to understand why in in vivo experiments the authors used clenbuterol rather than salbutamol. Actually, they write that clenbuterol “can be efficiently administered intraperitoneally”, however this applies also to salbutamol (see e.g. Choucair-Jaafar et al., 2009), which rapidly penetrates the ‘blood-brain barrier’ and reaches brain concentrations amounting to about 5% of the plasma concentrations (Caccia and Fong, 1984). In addition, clenbuterol is not approved for human use in many countries including Norway, the USA and Canada, and it has anabolic effects on skeletal muscle, including the activation of PI3K/Akt signaling (Lynch and Ryall, 2008), which led the World Anti-Doping Agency to prohibit its use in IOC-controlled athletes.
Despite these limitations, the study by Mittal et al. (2017) has the merit to attract novel attention on the possibility to repurpose as antiparkinson drugs the β2-AR agonists, which are well established as bronchodilators in asthma, where they are used with favorable safety profiles and high therapeutic indexes.
Actually, β2-AR agonists are not an absolute novelty as therapeutics for PD patients. Indeed, the notion that β2-AR regulate the transport of large neutral amino acids across the ‘blood-brain barrier’ dates back almost 30 years ago (Edwards et al., 1989). In particular, the transport of levodopa into the brain is reduced in monkeys with severe PD and it could be increased by prior administration of the β2-AR agonists isoproterenol (Alexander et al., 1994). The effect is apparently mediated peripherally, does not involve changes in the cerebral blood flow, and is due to activation of β2-AR. In rodents, it is exerted by both isoproterenol and clenbuterol, which respectively don’t cross and cross the ‘blood-brain barrier’, it occurs in the absence of changes in the regional blood flow, and it is blocked by administration of the peripheral β-AR antagonist nadolol (Uc et al., 2002). Clinical experience with β2-AR agonists is based on such evidence and consists so far of three studies.
In the first one (Hishida et al., 1992), nine PD patients with wearing-off (mean age: 62 years; mean duration of disease: 11 years; all 3 on the Hoehn and Yahr scale and all taking levodopa, on average 456 mg daily) were observed for two weeks and then given salbutamol 6 mg daily in three divided doses for an additional 2 weeks. Co-administration of salbutamol increased the mean daily total ‘on’ phase and shortened latency. One patient complained of postural tremor after salbutamol.
In a second study, the effects of salbutamol were studied in an open trial on eight male PD patients (mean age: 70.9 years; mean duration of disease: 11.9 years; all 3 or 4 on the Hoehn and Yahr scale and all taking levodopa, on average 800 mg daily) (Alexander et al., 1994). The study was performed over five days, and salbutamol 2 mg was administered to six of the eight patients on the third and fourth day, 1 hour prior to their morning and first afternoon levodopa doses. The other two patients received a placebo. Patients treated with salbutamol had reduced parkinsonian symptoms, while those on placebo showed no changes. Three patients discharged on salbutamol (2 mg tid), together with antiparkinson drugs, maintained the improvement within the first week following discharge, and two of them reduced daily levodopa by 29-38%. Adverse events attributable to salbutamol were nervousness and tremor in one patient, mild tachycardia without blood pressure increase in three patients, and an increase in the peak-dose dyskinesias in two patients, which led to the above mentioned levodopa dose reduction.
The third study was an open-label study in eight PD patients on levodopa with response fluctuations but no disabling dyskinesia (mean age: 59.8 years), all taking levodopa, on average 481 mg daily (Uc et al., 2003). In this case, treatment with salbutamol (4 mg daily) lasted 14 weeks and before and after treatment a whole array of evaluations were performed, including Unified Parkinson’s Disease Rating Scale (UPDRS) motor, tapping, stand–walk–sit tests every 30 minutes between 8 AM and 5 PM, body composition analyses, muscle strength tests, and asked about the quality of their life using the the Parkinson’s Disease Questionnaire (PDQ-39). Seven patients completed the study, and one withdrew after 2 weeks due to headache, jitteriness, and anxiety. In the seven patients who completed the study, there was an improvement of the UPDRS motor scores, and an increase of the thigh muscle cross-sectional area and of fat-free mass. No changes were observed in the stand-walk-sit test, muscle strength tests, other UPDRS sections, daily OFF time, or PDQ-39. At the end of the study, three patients chose to continue salbutamol.
Altogether, available evidence so far obtained in open-label studies performed in small groups of PD patients suggests that salbutamol improves the therapeutic response to levodopa, and possibly increases muscle mass and improves motor performance, usually with minor adverse effects. Unfortunately, no double-blind, placebo-controlled studies have been ever performed to better establish the efficacy and safety of salbutamol and/or of other β2-AR agonists in PD.
Support to the opportunity to test β2-AR agonists in PD actually comes also from a completely different line of evidence. Indeed, an emerging issue in PD regards the role of peripheral adaptive immunity, possibly triggering neuroinflammation ultimately leading to neurodegeneration. T lymphocytes have been shown to occur in the substantia nigra of parkinsonian brains and in the MPTP mouse model of PD, where CD4+ T cells likely determine T cell-mediated dopaminergic cell death (McGeer et al., 1989; Brochard et al., 2009). Moreover, at least in vitro, α-syn has been reported to increase CD4+ T memory cells in PD patients and in healthy subjects (Kustrimovic et al., 2016), and T cells from PD patients recognize α-syn peptides (Sulzer et al., 2017). CD4+ T cells may acquire proinflammatory phenotypes, such as T helper (Th) 1 and Th17, as well as antiinflammatory phenotypes, such as Th2 and the T regulatory (Treg) one, and evidence from animal models of PD suggests that Th1 and Th17 may be detrimental while Th2 and Treg may be protective (González et al., 2015; Mosley and Gendelman, 2017).
In this regard, it is well established that β2-AR are the main interface between sympathoadrenergic nerves and immune cells (Elenkov et al., 2000; Marino and Cosentino, 2013; Scanzano and Cosentino, 2015), and that stimulation of β2-AR on CD4+ T lymphocytes inhibits Th1 and stimulates Th2 cytokines (Elenkov et al., 2000; Marino and Cosentino, 2013), and may even enhance Treg suppressive functions (Guereschi et al., 2013).
Repurposing β2-AR agonists as antiinflammatory agents has been proposed in multiple sclerosis (Cosentino and Marino, 2013), and salbutamol was tested in multiple sclerosis patients in a prospective open-label study showing its ability to reduce monocyte IL-12 production (Makhlouf et al., 2001), and thereafter in double-blind clinical trial as add-on to conventional immunomodulation with glatiramer, resulting well tolerated and improving clinical outcomes (Khoury et al., 2010). β2-AR agonists might therefore benefit PD patients also by acting on peripheral adaptive immunity, cutting the proinflammatory profile of CD4+ T cells as well as of other immune cells, and favoring an antiinflammatory profile.
In summary, evidence supporting β2-AR agonists as novel antiparkinson drugs so far consists of short term open label studies showing the ability of salbutamol to enhance the therapeutic response to levodopa as well as to improve motor performance, possibly through an anabolic effect on skeletal muscle mass. Emerging evidence however points to β2-AR agonists as drugs able to also target neuroinflammation and neurodegeneration, as these drugs can act directly on CD4+ T cells to inhibit Th1 responses, likely involved in neuroinflammation, and to enhance Th2 and Treg, which may be neuroprotective. The recently described ability of β2-AR agonists to inhibit α-syn expression in the brain and possibly to protect mice from neurotoxin induced neurodegeneration might unveil an additional beneficial mechanism of these drugs in PD. It would be interesting to study whether such effect occurs also in peripheral tissues, as clinical and pathological evidence supports the possibility that PD starts in periphery, and specifically in the gut, where α-syn-related neurodegeneration of the enteric nerves is an early manifestation of PD (Klingelhoefer and Reichmann, 2015).
For all these reasons, β2-AR agonists might be ideal candidates to be tested as novel nonconventional antiparkinson drugs in well-designed double-blind, placebo-controlled studies aimed at assessing their potential neuroprotective effects. Indeed, if it were possible to repurpose β2-AR agonists for PD then patients could benefit from drugs with established favorable therapeutic index and low price, which is in the interest of both patients as well as the healthcare systems.
Marco Cosentino, Natasa Kustrimovic, Franca Marino – Center of Research in Medical Pharmacology, University of Insubria, Via Ottorino Rossi n. 9, 21100 Varese, VA, Italy. Phone: +39 0332 217410, Fax: +39 0332 217409; email: firstname.lastname@example.org
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