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 2.5 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 2.0 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 (302.5 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 20C. 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. wise stated. For the determination of the SSAO activity in the micro- The oxidation of methylamine by SSAO was determined by coupling the somal preparation it was preincubated at 37C for 30 min with clorgyline formation of formaldehyde to the reduction of NADþ in the presence of plus deprenyl (1 mM each), to inhibit MAO. It was found that MAO-B formaldehyde dehydrogenase (Lizcano et al., 2000). The ability of hydro- contributed approximately 22% of the total activity of the microsomal xyzine to act as a substrate for the amine oxidase was assessed by the preparation in the absence of these inhibitors. Mitochondrial samples determination of hydrogen peroxide formation. Both the fluorimetric were incubated with 1 mM semicarbazide, to inhibit SSAO, before assay method of Tipton (1969) and by the spectrophotometric procedure of Holt of MAO activity. et al. (1997) were used. The oxidation of 5-hydroxytrypamine, 2-phenethylamine (substrates for In all cases, assay conditions used were those determined from prelimin- MAO A and MAO B, respectfully) was determined according to the method ary studies to correspond to the linear, initial velocity, period of product of Tipton et al. (2000). SSAO activity was assayed with benzylamine as the formation and a linear dependence of this velocity on the concentration of

Fig. 2. The kinetics of the inhibition of bovine plasma SSAO by hydroxyzine. All data were obtained using the enzyme from Sigma. Data points are mean valuess.e.m. from triplicate determinations. A The enzyme was preincubated with hydroxyzine for 0 () and 60 (œ)minat37Cbeforethe reaction was started by the addition of benzylamine. The curves are those obtained by fitting to the inhibition equation described in the text by non- linear regression. B Michaelis-Menten curves for the oxidation of benzylamine in the presence of following fixed concentrations of hydroxyzine 0 (), 2.5 (4), 7.5 (hi), 10 () and 15 () mM. The curves are those obtained by non-linear regression. C Double-reciprocal plots of the data in B, presented to illustrate the type of inhibition. D. Plot of the apparent values of Km=Vmax values, determined from B, as a function of the hydroxyzine concentration 108 J. O’Sullivan et al. the tissue sample. Samples were assayed in triplicate. Protein concentrations Table 1. The inhibition of amine oxidases by hydroxyzine were determined according to the method of Markwell et al. (1978). For kinetic studies, initial rates were determined by varying the substrate con- Enzyme IC50 (mM) IC50 (mM) Inhibition Ki (mM) centration at a constant specific radioactivity and kinetic parameters were preparation 0 min 60 min determined by non-linear regression, using the programme MacCurveFit, Bovine plasma 17 0.83 20 0.85 Competitive 1.8 0.39 Version 1.5 (Kevin Raner Software, Australia). IC values (the inhibitor 50 SSAO (Sigma) concentration that gives 50% inhibition of the reaction) were also deter- Bovine plasma 23 0.5 20 0.42 Competitive 1.47 0.79 mined by using the same software to fit data to the equation. SSAO (Purified) 100ð10ðxþaÞÞ Bovine lung 1050 43 870 28 n.d. n.d. FðxÞ¼ 10ðxþaÞ þ 1 microsomal SSAO Reversibility of inhibition was assessed by dilution experiments. The Rat liver MAO-A 631 13 676 16 n.d. n.d. enzyme (bovine plasma amine oxidase) from Sigma or partially purified Rat liver MAO-B 40 1.5 38 0.9 Competitive 19 1.3 enzyme, at a concentration of 1.2 mg protein ml1, was incubated with in- hibitor until inhibition was essentially complete, using previous time-course experiments as a guide. 20 ml samples were then removed and diluted into the standard benzylamine assay mixture, to give a final protein concentra- and Table 1). The Ki value for the inhibition of benzyla- 1 tion of 0.024 mg ml , for activity determinations. SSAO activity was then mine oxidation by bovine plasma SSAO was determined, assayed as described above. A control sample was also taken through the same procedure where inhibitor was added to the desired final concentration by nonlinear regression of the curves shown in Fig. 2B, to after dilution. be 3.8 0.39 mM. As illustrated by the double-reciprocal Reversibility of MAO inhibition was assessed by centrifugation and re- and secondary plots in Fig. 2C and D, the inhibition was suspension. 1 ml of a 10 mg=ml suspension of rat liver mitochondria was incubated for 1 hour with sufficient inhibitor to give essentially complete simple competitive in nature. inhibition. The inhibitor was then depleted by centrifugation at 14000 rpm The microsomal membrane-bound SSAO was found to for 10 min. The supernatant was discarded and the mitochondrial pellet was be considerably less sensitive to inhibition by hydroxyzine. resuspended in a fresh phosphate buffer (100 mM, pH 7.5) to give a con- centration of approximately 10 mg=ml. The MAO activity was then assayed There was no time-dependence, with IC50 values deter- as described previously. mined as 1.0 0.04 and 0.870 0.03 mM after enzyme- inhibitor preincubation for 0 min and 60 min respectively (Fig. 3). The high IC value made determination of the K Results 50 i impractical. The inhibition of bovine plasma SSAO (from Sigma) by Since the SSAO preparation obtained from Sigma was hydroxyzine was found to be time-independent, with no found, by polyacrylamide-gel electrophoresis in the pre- significant change in the extent of inhibition occurring over sence of sodium dodecyl sulphate (SDS-PAGE), to contain a 60 min period of enzyme-inhibitor preincubation (Fig. 2A multiple impurities (data not shown), the possibility was

Fig. 3. The inhibition of bovine lung membrane-bound SSAO by hydroxyzine. The enzyme was preincubated with hydro- xyzine for ¼ 0 min and œ ¼ 60 min at 37C before the reaction was started by the addition of benzylamine. The curves are those obtained by fitting to the inhibition equation described in the text by non-linear regression. Data points are mean values s.e.m. from triplicate determinations Amine oxidase inhibition by hydroxyzine 109 considered that degradation during the purification proce- dure used might have contributed to the higher sensitivity of the plasma enzyme. The bovine plasma SSAO, purified as described above, which showed only minor impurities on SDS-PAGE, was studied for comparison. The purified and Sigma SSAO preparations showed similar Km values towards benzylamine, 1088 73 mM and 997 59 mM, respectively, and methylamine, 317 64 mM and 305

32 mM, respectively. For comparison, the Km for the micro- somal membrane bound SSAO towards benzylamine was determined as 76 29 mM. The two preparations of the plasma enzyme also behaved similarly in their sensitivities to inhibition by hydroxyzine, as shown in Table 1. Incuba- tion of the microsomal fraction in the presence of endogly- cosidase-H (1 unit) for 20 h at 37C before assay decreased the sensitivity to inhibition by hydroxyzine; an IC50 value of 2.9 0.79 mM being obtained. Incubation of the puri- fied plasma SSAO with endoglycosidase-H under similar conditions did not significantly affect the activity towards benzylamine or the sensitivity towards inhibition by 20 mM Fig. 4. The effects of pretreatment with endoglycosidase-H (Endo) on the hydroxyzine, as compared to a control incubated under the inhibition of purified beef plasma SSAO by hydroxyzine. Experimental details are described in the text. Values are means s.e.m. from triplicate same conditions in the absence of the endoglycosidase determinations (Fig. 4). Rat liver MAO-A, determined towards 5-HT, was rela- times less potent an inhibitor of MAO-B, as compared to tively insensitive to inhibition by hydroxyzine, which was the plasma SSAO (Table 1). not time dependent (Fig. 5). The high IC50 value, shown in The inhibition of the amine oxidase preparations, used in Table 1, made determination of the Ki impractical. In con- this study, by hydroxyzine was found to be freely reversible trast, hydroxyzine was a much more potent inhibitor of with essentially complete recoveries of activity after MAO-B (Fig. 6) The inhibition, which showed no time removal of the inhibitor, by dilution in the case of SSAO dependence, was found to be competitive, with respect to and by sedimentation for MAO. Hydroxyzine was found 2-phenylethylamine, as shown, for illustrative purposes, in not to be a substrate for any of the enzymes studied, as

Fig. 6B and C. The Ki value indicated it to be about 10 determined by H2O2 release.

Fig. 5. The inhibition of rat liver mitochondrial MAO-A by hydroxyzine. The enzyme was preincubated with hydroxy- zine for ¼ 0 min and œ ¼ 60 min at 37C before the reac- tion was started by the addition of 5-hydroxytryptamine. The curves are those obtained by fitting to the inhibition equation described in the text by non-linear regression. Data points are mean values s.e.m. from triplicate determinations 110 J. O’Sullivan et al.

Fig. 6. The inhibition of rat liver mitochondrial MAO-B by hydroxyzine. A. The enzyme was preincubated with hydroxyzine for ¼ 0 min and œ ¼ 60 min at 37C before the reaction was started by the addition of 2-phenylethylamine. The curves are those obtained by fitting to the in- hibition equation described in the text by non-linear regression. Data points are mean values s.e.m. from triplicate determinations. B. Dou- ble-reciprocal plots of the data of the initial rates of 2-phenylethylamine oxidation at the following fixed concentrations of hydroxyzine 0 (), 10 (4), 100 (hi), 175 () and 250 () mM, presented to illustrate the type of

inhibition. Plot of the apparent values of Km=Vmax values, determined from non-linear regression fitting of the initial-rate data, as a function of the hydroxyzine concentration

Discussion involved. However, treatment of the microsomal pre- paration with endoglycosidase-H resulted in a decreased The reversible inhibition of bovine plasma SSAO and sensitivity to inhibition by hydroxyzine. Thus, although MAO-B by hydroxyzine adds a further potential complex- the glycosylation of SSAO may affect the accessibility ity to the mechanism of action of this drug. In contrast, the of hydroxyzine it would appear to enhance, rather than low inhibitory potency towards MAO-A and the mem- impede, access. brane-bound SSAO suggests that the interactions with these The membrane location may play a role in this differ- enzymes are unlikely to be important. The difference ence. Lizcano et al. (1998) have reported inhibitor-sensi- between the behaviour of the plasma and membrane-bound tivity differences between bovine membrane-bound and SSAO is likely to be a result of some processing event, plasma SSAO. For example, amiloride to be ineffective since it appears that they are both encoded by the same as an inhibitor of membrane bound SSAO (IC > 50 mM), gene (Zhang and McIntire, 1996). It does not appear that 50 mM), although the inhibitor sensitivity increased somewhat artefactual degradation during purification of the plasma after extraction and purification (K ¼ 750 100 mM). Pur- enzyme may have resulted in a greater sensitivity, since i ification also resulted in a 50-fold increase in the sensitivity two different preparations gave similar results. Since SSAO of the bovine lung enzyme to inhibition by phenylhydra- is a heavily glycosylated protein, differences in glyco- zine (Lizcano et al., 1998). sylation between membrane-bound SSAO and the plasma The competitive nature of the inhibition of plasma SSAO form, which are believed to be associated with cleavage and mitochondrial MAO-A might suggest that hydroxyzine of the membrane form (Kurkij€aarvi et al., 1998), might be binds to the active sites of these amine oxidases, although it Amine oxidase inhibition by hydroxyzine 111 showed no detectable activity as a substrate. These results rally occurring amines and their structural analogues. Biochem Pharmacol 38: 1507–1515 extend our knowledge of the drug-binding capabilities of Enrique-Tarancon G, Marti L, Morin N, Lizcano JM, Unzeta M, Sevilla L, these enzymes, which are already known to include the Camps M, Palacin M, Testar X, Carpene C, Zorzano A (1998) Role imidazolines. b-Carbolines and harmala alkaloids bind to of semicarbazide-sensitive amine oxidase on glucose transport and MAO (Miralles et al., 2005; Herraiz and Chaparro, 2006) GLUT4 recruitment to the cell surface in adipose cells. J Biol Chem 273: 8025–8032 and a number of transport-inhibiting antidepressants, such Enrique-Tarancon G, Castan I, Morin N, Marti L, Abella A, Camps M, as imipramine, maprotiline, nomifensine and zimeldine, Casamitjana R, Palacin M, Testar X, Degerman E, Carpene C, bind as inhibitors both to MAO (Egashira et al., 1999) Zorzano A (2000) Substrates of semicarbazide-sensitive amine oxi- dase co-operate with vanadate to stimulate tyrosine phosphorylation and SSAO (Obata and Yamanaka, 2000), although the nat- of insulin-receptor-substrate proteins, phosphoinositide 3-kinase ure of these interactions appears to show considerable spe- activity and GLUT4 translocation in adipose cells. Biochem J cies differences. In none of these cases is it known whether 350: 171–180 Ferreri M, Hantouche EG (1988) Recent clinical trials of hydroxyzine or not this binding significantly affects the bioavailability in generalized anxiety disorder. Acta Psychiatr Scand [Suppl 393]: of these drugs or might give rise to undesirable drug-drug 102–108 interactions. Fowler CJ, Tipton KF (1981) Concentration dependence of the oxidation of Histamine, which may play important role in cell adhe- tyramine by the two forms of rat liver mitochondrial monoamine oxidase. Biochem Pharmacol 30: 3329–3332 sion (Andriopoulou et al., 1999; Winter et al., 1999), is a Gillard M, van der Perren C, Moguilevsky N, Massingham R, Chatelain P relatively poor substrate for semicarbazide-sensitive amine (2002) Binding characteristics of cetirizine and levocetirizine to oxidase (Raimondi et al., 1997; Lizcano et al., 1998) and is human H1 histamine receptors: contribution of Lys191 and Thr194. Mol Pharmacol 61: 392–399 not oxidized by MAO, although its metabolite, NT-methyl- Gitomer WL, Tipton KF (1983) Determination of Km and V of an enzyme histamine, is a substrate for MAO-B (Waldmeier et al., for an unstable substrate and its application to the oxidation of T 1997) and SSAO (Gitomer and Tipton, 1983). Inhibition N -methylimidazol-3-ylacetaldehyde by aldehyde dehydrogenase. J Biochem 211: 277–280 of SSAO might affect its other diverse functions and a Giustizieri ML, Albanesi C, Fluhr J, Gisondi P, Norgauer J, Girolomoni G major consequence of MAO-B inhibition is the elevation (2004) H1 histamine receptor mediates inflammatory responses in of 2-phenylethylamine. Further work would be necessary human keratinocytes. J Allergy Clin Immunol 114: 1176–1182 to show whether the amine oxidase inhibition contributes Gokturk C, Nilsson J, Nordquist J, Kristensson M, Svensson K, Soderberg C, Israelson M, Garpenstrand H, Sjoquist M, Oreland L, significantly to the diverse actions of hydroxyzine. Forsberg-Nilsson K (2003) Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal structure of the aortic elastic laminas. Am J Pathol 163: 1921–1928 Acknowledgement Herraiz T, Chaparro C (2006) Human monoamine oxidase enzyme inhibi- tion by coffee and beta-carbolines norharman and harman isolated Financial assistance from the Health Research Board (Ireland) is gratefully from coffee. Life Sci 178: 795–802 acknowledged. Holt A, Wieland B, Baker GB (2004) Allosteric modulation of semicarba- zide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands. 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