An investigation of the in vitro reversibility of MAO inhibition by lazabemide
4.1. Introduction
The monoamine oxidase (MAO) enzymes exist as two isoforms, MAO-A and MAO-B, and are key enzymes for the metabolism of monoamine neurotransmitters in the peripheral and central tissues. Serotonin is a specific substrate of MAO-A while the false neurotransmitter, β- phenylethylamine, is a MAO-B specific substrate. The catecholamines, dopamine (DA), noradrenaline and adrenaline, as well as the dietary amines, tryptamine and tyramine, are substrates for both MAO isoforms (Youdim et al., 2006). Inhibitors of MAO have been used as antidepressant drugs for over 50 years and act by raising neurotransmitter levels in the brain (Ramsay et al., 2016). In this respect, MAO-A specific inhibitors are used for major depressive disorders and therapy-resistant depression (Lum & Stahl, 2012). Specific inhibitors of MAO-B are used in Parkinson’s disease (PD) and act by preventing the MAO-B-catalysed depletion of central DA (Youdim et al., 2006; Youdin & Bakhle, 2006). In PD, MAO-B inhibitors are frequently combined with L-dopa in an effort to bolster the enhancement of DA levels in the brain. Currently two MAO-B inhibitors are registered for the treatment of PD, selegiline and rasagiline (Fig. 4.1). These are irreversible mechanism-based inhibitors of the propargylamine class. A reversible inhibitor, safinamide, was recently approved in Europe for the treatment of PD (Müller, 2016).
Figure 4.1: The structures of (R)-deprenyl (selegiline), rasagiline and safinamide.
MAO inhibitors may also find future applications in other disease states. For example, MAO- A levels are elevated in certain types of cancer tissue such as prostate cancer, and MAO-A inhibition may, in synergism with surviving suppressants, inhibit cancer cell growth, migration and invasion (Xu et al., 2015; Wu et al., 2014). MAO-B inhibitors, in turn, are under investigation for the treatment of Alzheimer’s disease, possibly acting by reducing hydrogen peroxide and aldehyde intermediates formed by the MAO catalytic cycle (Sturm et al., 2016).
These are potentially injurious to neuronal cells and may contribute to disease pathogenesis.
By similarly decreasing the MAO-B-catalysed generation of these injurious species in the Parkinsonian brain, MAO-B inhibitors have been proposed to be neuroprotective in PD (Youdim & Bakhle, 2006; Edmondson, 2014). For neurodegenerative disorders such as Alzheimer’s disease and PD, the MAO-B isoform seems to be the more relevant target for neuroprotection since MAO-B activity in the brain increases with age while MAO-A activity remains largely unchanged (Fowler et al., 1997). Conversely, hydrogen peroxide formed by MAO-A in the heart has been linked to age-related cardiac cellular degeneration in rats (Maurel et al., 2003), thus providing a possible role for MAO-A inhibitors in the therapy for certain cardiomyopathies. Interestingly, MAO-B inhibitors have also been advocated as an aid to smoking cessation (Berlin et al., 2002).
Due to the role of MAO in neurotransmitter metabolism, and the potential applications of MAO inhibitors in various disease states, the discovery of novel compounds that potently inhibit the MAOs has been pursued for many decades (Ramsay, 2013). In this effort, compounds that exhibit potentially useful mechanisms of inhibitory action are of particular interest. One such compound is lazabemide [Ro 19-6327; N-(2-aminoethyl)-5-chloro-2-pyridinecarboxamide], a MAO-B specific inhibitor discovered in the 1980s (Fig.4.2) (Cesura et al., 1990; Cesura et al., 1999). Lazabemide and related N-(2-aminoethyl)carboxamides (e.g. Ro 41-1049, Ro 16- 6491) have the distinction of acting as mechanism-based inhibitors with a reversible mode of action. These inhibitors exhibit an initial competitive mode of binding, but are subsequently activated by MAO to form reversible adducts with the enzyme. The result is rapid and complete MAO-B inhibition with enzyme activity returning to baseline values by 36 h after drug discontinuation (Dingemanse et al., 1997; Fowler et al., 1993). Following inhibition with irreversible MAO-B inhibitors, the recovery period can be 40 days (Fowler et al., 2005; Fowler et al., 2015). Furthermore, for a pharmacological effect >90% of MAO-B should be inhibited (Ramsay et al., 2016; Fowler et al., 2005). A dose of at least 0.4 mg/kg lazabemide given every 12 h provides >90% inhibition of brain MAO B in patients with early PD (Fowler et al., 1993). Unfortunately the development of lazabemide has been discontinued due to liver toxicity (Berlin et al., 2002).
Figure 4.2: The structures of lazabemide, Ro 41-1049 and Ro 16-6491.
The mechanism by which lazabemide inhibits MAO-B is not completely understood. Structural evidence with N-(2-aminoethyl)-p-chlorobenzamide (Ro 16-6491) shows that an adduct forms at the N(5) position of the flavin, which is support for a mechanism-based mode of inhibition (Fig. 4.3) (Binda et al., 2003; Edmondson et al., 2004). The N(5) position also is the site of covalent attachment of virtually all irreversible MAO inhibitors (including propargylamines).
The only exceptions are cyclopropylamine inhibitors which form a flavin C(4a) adduct (Edmondson et al., 2009). Evidence for the reversibility of MAO-B inhibition by lazabemide is provided by the relatively short enzyme recovery period after drug discontinuation in clinical studies (Dingemanse et al., 1997; Fowler et al., 1993). Furthermore, after denaturation of the enzyme, no covalent adducts with the flavin are found. A covalent adduct is only detected in peptide fragments after borohydride reduction (Cesura et al., 1989). With MAO-B in brain and platelet membranes, the binding of radiolabeled Ro 16-6491 is fully reversible. However, irreversible attachment to the membranes occurs by treatment with borohydride (Cesura et al., 1988).
Figure 4.3: The adduct that forms with the inhibition of human MAO-B by N-(2-aminoethyl)-p- chlorobenzamide (Ro 16-6491) (Edmondson et al., 2004).
Although the development of lazabemide has been discontinued, this [and other N-(2- aminoethyl)carboxamides] are still being used as reference MAO-B inhibitors in the in vitro screening of experimental MAO inhibitors (Petzer et al., 2013). For comparison with experimental inhibitors, the behaviour of lazabemide in in vitro MAO inhibition studies should thus be defined, particularly with respect to reversibility of inhibition on the time scale (10–60 min) of a typical in vitro experiment. An important consideration when comparing in vitro IC50
values of experimental and reference inhibitors is reversibility of inhibition. The IC50 value recorded for an irreversible inhibitor will vary with different exposure times of the enzyme to the inhibitor prior to the addition of the substrate. Typically the longer the enzyme and inhibitor are preincubated, the higher the degree of inhibition in as much that enzyme activity may be supressed to a level much lower than expected from a literature IC50 value. With this in mind, the present study aimed to characterise the in vitro MAO inhibition properties of lazabemide with respect to potency, isoform selectivity and reversibility.