J Neural Transm (2006) [Suppl] 71: 105–112 # Springer-Verlag 2006 Inhibition of amine oxidases by the histamine-1 receptor antagonist hydroxyzine J. O’Sullivan1;2, M. I. O’Sullivan2, K. F. Tipton1, G. Davey1 1 Department of Biochemistry, and 2 Dublin Dental School and Hospital, Trinity College, Dublin, Ireland Summary The effects of the drug hydroxyzine on the activities of the rat amine oxidases, as they are not inhibited by the mono- liver monoamine oxidases (EC 1.4.3.6; MAO) and the membrane-bound amine oxidase (MAO; EC 1.4.3.6) inhibitor clorgyline. and soluble forms of bovine semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) were studied. Hydroxyzine was found to be a competitive They are a ubiquitous group of enzymes found in plants, inhibitorofMAO-B(Ki 38 mM), whereas it had a low potency towards animals and microbes (Lewinsohn, 1984; Callingham et al., > MAO-A (IC50 630 mM). Although it was a relatively potent competitive 1995; Lyles, 1996; Houen, 1999). Most mammalian spe- inhibitor of bovine plasma SSAO (K 1.5 mM), it was a weak inhibitor of i cies contain a soluble form of SSAO in the blood plasma the membrane-bound form of the enzyme from bovine lung (IC50 1mM). These findings extend our knowledge of the drug binding capabilities of the plus a membrane bound form of the enzyme that has a amine oxidases and suggest that these interactions may contribute to the relatively high activity in cardiovascular tissue, lung and complex actions of this drug. adipocytes (Boomsma et al., 2000; Lyles, 1995, 1996). Abbreviations: MAO monoamine oxidase, SSAO semicarbazide-sensitive Although the physiological functions of SSAO are by amine oxidase. no means fully understood, a primary role of SSAO is believed to be the scavenging of primary amines, either Introduction endogenous or xenobiotic (Tipton and Strolin Benedetti, 2001). The active site of the membrane bound form of The semicarbazide sensitive amine oxidases (SSAO) be- the enzyme appears to be externally facing, thus allowing long to the copper-containing group of amine oxidases the metabolism of extracellular amines. Methylamine and (EC 1.4.3.6; amine: oxygen oxidoreductase (deaminating) aminoacetone are specific substrates for SSAO, in that (copper containing)). They catalyse the oxidative de- MAO does not oxidise them (Lyles and Chalmers, 1992; amination of primary amines according to the overall reaction: Lizcano et al., 1994). SSAO and MAO, which is an in- tracellular enzyme, have been shown to work in concert RCH2NH2 þ O2 þ H2O ! RCHO þ NH3 þ H2O2: to facilitate the removal of tyramine in the isolated per- fused mesenteric arterial bed of the rat (Elliott et al., Their sensitivity to inhibition by semicarbazide and other 1989). The metabolism of dopamine may also involve carbonyl reagents results from the presence of a 3,4,6,tri- both SSAO and MAO, as shown in rat vas deferens (Lizcano hydroxyphenylalanine (TOPA) residue that serves as a co- et al., 1991). factor in the reaction. Several other functions of SSAO have been identified They have been known by several different names, (O’Sullivan et al., 2004; Tipton et al., 2003, for reviews). including benzylamine oxidase, owing to their preference At least in some tissues, it functions as a vascular-adhesion for benzylamine as a substrate, and clorgyline-resistant protein (vascular-adhesion protein-1; VAP-1) (Jalkanen and Salmi, 2001). It may also modulate cellular glucose trans- port by stimulating membrane glucose transporter re- Correspondence: Dr. J. O’Sullivan, Department of Biochemistry, Trinity College, Dublin 2, Ireland cruitment (Enrique-Tarancon et al., 1998, 2000). Roles e-mail: [email protected] for SSAO=VAP-1 have also been identified in adipocyte 106 J. O’Sullivan et al. Fig. 1. Chemical structure of hydroxyzine (2-[2-[4-[(4-chloro- phenyl)phenylmethyl]-1-piperazinyl]ethoxy]ethanol) development (Mercier et al., 2001) and in extracellular selectivity as an antagonist of the histamine H-1 receptor matrix formation (Langford et al., 2002; Gokturk et al., resulting in a reduction in sedation and brain penetration 2003). It appears that SSAO, may also function as a (Gillard et al., 2001). drug-binding protein. Imidazolines, such as amiloride, Since, histamine is a substrate for semicarbazide-sensi- and guanidine compounds, which are used clinically, as tive amine oxidase whereas MAO-B oxidizes its metabolite diuretic and hypertensive agents have been shown to bind NT-methylhistamine, we have investigated the possibility to MAO, SSAO and other amine oxidases (Mu et al., 1994; that hydroxyzine, might interact with these amine oxidases. Remaury et al., 2000; Holt et al., 2004). However the phy- siological significance of these drug-binding sites remains obscure (e.g., Eglen et al., 1988). Materials and methods The heterocyclic piperazine derivative hydroxyzine 2- [7-14C] Benzylamine hydrochloride (Specific activity 59 mCi=mmol), 5- [2-[4-[(4-chlorophenyl)phenyl-methyl]-1-piperazinyl]eth- Hydoxy [side-chain 2-14C]tryptamine creatine sulphate (Specific activity oxy]ethanol) (Fig. 1) is a histamine-1 receptor antagonist, 57 mCi=mmol) and 2-Phenylethylamine[ethyl-1-14C]hydrochloride (Spe- = with anticholinergic properties, which has a wide variety of cific activity 57 mCi mmol) were from Amersham. l-Deprenyl HCl was from Fluka and unlabelled substrates and other chemicals were from Sigma. therapeutic applications, including the treatment of allergic Rat liver mitochondria, prepared as previously described (Fowler and skin disorders and the control the nausea and vomiting Tipton, 1981) were used as the source of MAO. Bovine lung microsomes caused by various conditions, including motion sickness. were prepared as a source of membrane-bound SSAO (Lizcano et al., 1998) and bovine plasma SSAO was from Sigma. It is also used as a tranquilliser for the symptomatic man- More highly purified bovine plasma SSAO was prepared by a modifica- agement of conditions, such as generalised anxiety disorder tion of the procedure of Wang et al. (1994). 10 L of bovine blood were (GAD) and the tension associated with psychoneuroses mixed with 1 L of 2.5% sodium citrate solution and centrifuged at 3000 g for 20 min. Ammonium sulphate was added, with continuous stirring, to 4 L of (Lader et al., 1998; Ferreri and Hantouche, 1988), as well the supernatant to give 35% saturation. Stirring was continued for 2 h as in the treatment of the symptoms of alcohol withdrawal. before centrifugation at 10,000 g for 20 min. The supernatant was retained It is also used in the management of pruritis associated with and ammonium sulphate was added, with continuous stirring, to give 55% allergic conditions, such as chronic urticaria and histamine- saturation. Stirring was continued for 30 min before centrifugation at 10,000 g for 20 min. The precipitate taken up in 200 ml of 100 mM potas- mediated pruritus. Hydroxyzine has been shown to inhibit sium phosphate buffer, pH 7.2 and applied, at a flow rate of 150 ml=h, to a neurogenic mast-cell activation by a mechanism that Q-Sepharose column (30 Â2.5 cm), which had been equilibrated with is unrelated to its H-1 receptor antagonistic properties 10 mM potassium phosphate buffer, pH 7.5. After washing with 500 ml of the 10 mM phosphate buffer, SSAO was eluted, at a flow rate of 150 ml=h, (Minogiannis et al., 1998). It has also been shown to inhibit with a linear concentration gradient from 0.05 to 0.35 M NaCl in the experimental allergic encephalomyelitis and the associated same buffer. Fractions with a specific activity equal to or higher than brain mast cell activation and may also be effective in 0.4 mmol=min=mg protein were pooled, mixed with an equal volume = of 100 mM potassium phosphate buffer, pH 7.2, and applied to a Con the prevention of relapsing remitting multiple sclerosis A-Sepharose column (30 Â2.0 cm) that had been equilibrated with the same (Dimitriadou et al., 2000). buffer. The column was washed with 500 ml of 100 mM phosphate buffer Cetirizine, the carboxylic acid metabolite of hydroxy- and then with 300 ml of 20 mM methyl-a-D-glucopyranoside in that buffer zine, may also suppress inflammatory responses, as it pos- before the SSAO was eluted with 200 ml of 0.5 M methyl-a-D-mannopyr- anoside in the same potassium buffer. Fractions (6 ml) with specific activ- sesses an ability to inhibit both macrophage inhibitory ities equal or higher than 0.95 mmol=min=mg were pooled and applied to a factor (MIF) and IL-8 production (Shimizu et al., 2004; Q-Sepharose (30Â2.5 cm) column, previously equilibrated with 20 mM Giustizieri et al., 2004). It has been also been suggested to potassium phosphate buffer (pH 6.8). The column was then washed with 300 ml of the buffer and eluted with a continuous gradient from 0.05 to be effective against autoimmune diseases (Namazi, 2004). 0.35 M NaCl in that. The fractions with SSAO specific activity higher than The carboxyl group results in this derivative having high equal or higher than 15 mmol=min=mg protein were then pooled, dialysed Amine oxidase inhibition by hydroxyzine 107 against 20 mM potassium phosphate buffer, pH 6.8, for 6 h and stored in substrate by the radiochemical method of Tipton et al. (2000) or by a mod- aliquots of 300 ml in 20% glycerol at À20C. ification of the method of Tabor et al. (1954). The standard reaction mixture contained 50 mM potassium phosphate buffer, pH 7.2, and 0.024 mg=ml SSAO in a final volume of 1 ml. The reaction was initiated by the addition Assay of SSAO and MAO activities of the substrate benzylamine and the rate of change of absorbance at 250 nm All enzyme assays were performed at 37C and at pH 7.2, unless other- was monitored using a Cary 300-Biospectrophotometer.