Drug Metab. Pharmacokinet. 21 (2): 83–98 (2006).

Review Drug-Metabolizing Ability of Molybdenum Hydroxylases

Shigeyuki KITAMURA,KazumiSUGIHARA and Shigeru OHTA Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Molybdenum hydroxylases, which include and xanthine , are involved in the metabolism of some medicines in humans. They exhibit oxidase activity towards various heterocyclic compounds and aldehydes. The liver cytosol of various mammals also exhibits a signiˆcant reductase activity toward nitro, sulfoxide, N-oxide and other moieties, catalyzed by aldehyde oxidase. There is considerable variability of aldehyde oxidase activity in liver cytosol of mammals: humans show the highest activity, rats and mice show low activity, and dogs have no detectable activity. On the other hand, xanthine oxidoreductase activity is present widely among species. Interindividual variation of aldehyde oxidase activity is present in humans. Drug-drug interactions associated with aldehyde oxidase and xanthine oxidoreductase are of potential clinical signiˆcance. Drug metabolizing ability of molybdenum hydroxylases and the variation of the activity are described in this review.

Key words: molybdenum hydroxylase; aldehyde oxidase; xanthine oxidoreductase; interindividual variation; drug-drug interaction

disease.10,11) Aldehyde oxidase may also play a major Introduction role in the pathogenesis of alcohol-induced liver injury, Molybdenum hydroxylases, which include aldehyde while xanthine oxidoreductase plays a minor role.9) oxidase (EC 1.2.3.1) and xanthine oxidase (EC Aldehyde oxidase and xanthine oxidoreductase both 1.17.3.2)Wxanthine dehydrogenase (EC 1.17.1.4), play important roles in the metabolism of exogenous commonly contain FAD, molybdenum and an iron- compounds. These ‰avoenzymes generally catalyze sulfur center in the . The properties of these nucleophilic oxidation of N-heterocycles, resulting in are closely related.1–3) In mammals, xanthine diŠerent metabolites from those obtained via elec- oxidoreductase exists in two interconvertible forms, trophilic oxidation by the cytochrome P450 system. xanthine dehydrogenase, which predominates in vivo, However, the substrate speciˆcities of aldehyde oxidase and xanthine oxidase. In this review, xanthine and xanthine oxidoreductase diŠer.12) Aldehyde oxidase oxidoreductase is taken to be the total of xanthine in liver of various species catalyzes the oxidation of a oxidase and xanthine dehydrogenase. Aldehyde oxidase number of aldehydes and nitrogenous heterocyclic and xanthine oxidoreductase commonly exist in xenobiotics, such as methotrexate and cyclophospha- vertebrates.4) These cytosolic enzymes have a very close mide,1,13) and also catalyzes the metabolism of physio- evolutionary relationship (based on the recent cloning logical compounds such as retinaldehyde, pyridoxal, of the ), and they show a high degree of amino acid N1-methylnicotinamide and monoamine neurotransmit- .5–8) The physiological function of ters.14–17) The conventionally accepted role of xanthine aldehyde oxidase is not yet fully understood. The oxidoreductase is purine catabolism, in which it cata- ‰avoenzyme may merely play a supplementary role to lyzes the oxidation of hypoxanthine to xanthine, then to . However, aldehyde oxidase uric acid. The two enzymes, in the presence of an has been suggested to be relevant to the pathophysiolo- adequate electron donor, can also mediate the reduction gy of a number of clinical disorders.9) Its relevance to of various other compounds.18) Recently, aldehyde amyotrophic lateral sclerosis is suggested by the oxidase homologues, which may have diŠerent metabol- hypothesis that defects in radical metabolism by ic roles, were identiˆed in mice,8,19,20) and isoforms of the may be involved in the pathogenesis of this the enzyme were also suggested to be present in

Received; February 13, 2006, Accepted; March 10, 2006 To whom correspondence should be addressed: Dr. Shigeyuki KITAMURA, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. Tel. ＋81-82-257-5328, Fax. ＋81-82-257-5329, E-mail: skitamu＠hiroshima-u.ac.jp Abbreviation used are: N1-methyl-2-pyridone-5-carboxamide (2-PY), N1-methyl-4-pyridone-3-carboxamide (4-PY).

83 84 Shigeyuki KITAMURA et al. monkeys and humans.21,22) Marked inter-species varia- tion of the enzyme activity in oxidative and reductive reactions was reported.23,24) This review focuses on the metabolic ability of molybdenum hydroxylases toward medicines, as well as the role of these enzymes in drug-drug interactions, and interindividual diŠerences in their activities. Characterization of Molybdenum Hydroxylases Xanthine oxidoreductase and aldehyde oxidase are complex ‰avoproteins comprising two identical subunits of molecular weight 145,000. Each subunit contains one molybdenum, one FAD, and two nonidentical iron- sulfur redox centers as an electron reservoir.13) The optimal pH of these enzymes is estimated to be 9–10.5.25,26) Garattini et al.20) demonstrated that subunits of xanthine oxidoreductase have a tripartite structure, consisting of the 2FeW2S N-terminal domain (20 kDa), Fig. 1. Intra-molecular electron transport system of molybdenum ‰avin-containing region (40 kDa), and Mo-containing hydroxylases, aldehyde oxidase and xanthine oxidoreductase. C-terminal domain (substrate ; 85 kDa). This ˆgure is modiˆed from the scheme of Bhushan et al. (2003). Arrows indicate the direction of electron ‰ow. Branzoli and Massey27) showed that de‰avo aldehyde oxidase can reduce cytochrome c, but cannot transfer electrons to oxygen. It is presumed that electron donors Aldehyde oxidase was also puriˆed from ˆsh liver.43) such as xanthine act at the molybdenum site, reducing it This enzyme functions as a reductase toward from Mo(VI) to the Mo(IV) state. The reducing equiva- nitropolycyclic aromatic hydrocarbons and sulfoxide lents transfer directly to the FAD site or via the iron-sul- compounds in ˆsh.43–45) Xanthine oxidoreductase has fur center, and the enzyme is reoxidized by interaction been puriˆed from the livers of various species and from with oxygen.28) Then, an oxygen atom from water binds milk, and its properties were compared with those of with the electron donor which lost electron. In this case, aldehyde oxidase.2,37,46,47) the iron-sulfur center functions not only as the pathway Species and Tissue DiŠerences of Molybdenum of electron ‰ow, but also as an electron sink to provide Hydroxylases electrons to the FAD center.29) The proposed internal electron transport system is summarized in Fig. 1. Molybdenum hydroxylases are found in many spe- Xanthine dehydrogenase is easily converted to cies, including ˆsh and insect.4,43,48,49) However, there xanthine oxidase by proteolysis.30) Bothformsofthe are marked species and strain diŠerences (in rats and enzyme can reduce molecular oxygen, although xan- mice) and sex diŠerence (in mice) of liver aldehyde thine dehydrogenase can reduce NAD, which is its oxidase.4,50–54) Broadly speaking, high activity is ob- preferred electron acceptor. Reduction of oxygen leads served in monkey and humans, followed by hamster, to the formation of superoxide anion and hydrogen rabbit, guinea pig, rat and mouse, and no activity in peroxide. This capacity has attracted attention to the dog.17,55) Aldehyde oxidase is also found in ˆsh and possible role of xanthine oxidoreductase as a source of insects, but not in birds.48) Beedham et al.56) suggested reactive oxygen species, particularly as a pathogenic that the marked species diŠerences of aldehyde oxidase agent in many forms of ischemia-reperfusion injury.31) activity toward 1-substituted phthalazines are due to The role of the enzyme in post-ischemic reperfusion diŠerences in the size of the active site. It was also tissue injury has been widely investigated.32) However, shown that the substrate speciˆcities of the hepatic xanthine oxidoreductase may also play beneˆcial roles. enzyme from guinea pig, rabbit, baboon and human for Tubaro et al.33) suggested that superoxide radical substituted quinazolines and phthalazines are due to the produced by xanthine oxidoreductase acts as an lipophilic substituents, and the species diŠerences are antimicrobial agent. It was also proposed that uric acid due to the size of the binding site.57) Schoˆeld et al.24) produced by xanthine oxidoreductase forms a part of reported that guinea pig and rat aldehyde oxidase the protective system against oxygen radicals.34,35) preparations are not suitable for predicting aldehyde Aldehyde oxidase and xanthine oxidoreductase have oxidase-mediated drug-drug interactions in humans, been puriˆed from many species. Aldehyde oxidase has because of diŠerences in the inhibitory activity of SKF been puriˆed from livers of rat,36,37) mouse,38) guinea 525-A on aldehyde oxidase activity towards N-[(2?- pig,39) rabbit,26,27,40,41) hog,26) monkey22) and hamster.42) dimethylamino)ethyl]acridine-4-carboxamide (an an- Drug Metabolism by Molybdenum Hydroxylases 85 ticancer agent) as a substrate among liver preparations and their analogs. In general, aldehyde oxidase has the from humans, and rats and guinea pigs. ability to oxidize a wide variety of substrates, compared Molybdenum hydroxylases have been shown to be with xanthine oxidoreductase. distributed in many tissues.4) Xanthine oxidoreductase is Some anticancer agents are metabolized by aldehyde detected in liver, small intestine, forestomach, vagina, oxidase, as shown in Fig. 2. Methotrexate is oxidized to penis, oral and nasal cavities, esophagus, tongue and 7-hydroxymethotrexate by aldehyde oxidase in the livers uterus.9) Immunohistochemical analysis also conˆrmed of rats, guinea pigs, rabbits and humans.53,66–68) Conver- that xanthine oxidoreductase is distributed in epidermis, sion of acridine carboxamide, an antitumor agent, to an sebaceous glands and hair follicle epithelium in rats, acridone derivative is mediated by aldehyde oxidase.69) while aldehyde oxidase is also similarly distributed, N-[(2?-Dimethylamino)ethyl]acridine-4-carboxamide is except in epidermis.9,58) Moreover, in mice, mRNA of also metabolized to the 9(10H)-acridone by aldehyde aldehyde oxidase was detected in esophagus, lung, liver oxidase in rats, mice, guinea pigs and humans.24,54) and testis, but not skin.59) Tereo et al.7) cloned cDNA for An anticancer drug, XK469 (2(R)-[4-(7-chloro-2-quino- AOH1 and AOH2, novel aldehyde oxidase analogues, xalinyl)oxyphenoxy]propionic acid, is metabolized to and demonstrated that mRNA of AOH2 is present in the oxo derivative by aldehyde oxidase.70) the basal layer of the epidermis and hair follicles in Cinchona antimalarial alkaloids, quinine, quinidine, mice. cinchonine, cinchonidine, epiquinidine and epiquinine, Recently, we detected aldehyde oxidase and xanthine are oxidized by aldehyde oxidase, as shown in Fig. 2.71) oxidoreductase activities in skin of various mammals, Itoh et al.72,73) also reported the aldehyde oxidase- and found clear species diŠerences in the levels of their catalyzed stereospeciˆc oxidation of a quinine-related activities. The aldehyde oxidase and xanthine ox- compound, RS-8359, which is a potent inhibitor of idoreductase activities in skin of various species ap- MAO-A. Brimonidine, an ocular hypotensive agent, is peared to be complementary, i.e., when one was high, activated by aldehyde oxidase.74) Kawashima et al.75) the other was low. In addition, the pattern of species reported aldehyde oxidase-dependent metabolism of a diŠerence of these enzymes in skin cytosol was similar to sedative-hypnotic, zaleplon, and noted a marked species that in liver.60,61) However, the physiological roles of diŠerence between monkey and rats. Dibenz[b,f]- these enzymes in skin are still unknown. Possible roles 1,4-oxazepine (a potent sensory irritant), hydralazine include homeostatic control of vitamins, such as (an antihypertensive agent), and metyrapone are also retinoids, and other biologically active molecules, and oxidized to the lactam metabolites by aldehyde oxi- elimination of biogenic wastes or breakdown products dase.13,76) Acyclovir, which is highly active toward generated by ultraviolet radiation and other environ- herpes virus, is detoxiˆed to 8-hydroxyacyclovir by mental stresses.20,62) Aldehyde oxidase in bovine eye aldehyde oxidase.13,77) Purines are also oxidized by ciliary body was puriˆed and characterized.63) However, aldehyde oxidase, and in this case, hydrophobicity and the physiological role of the enzyme in ciliary body is an electron-withdrawing 6-substituent are important also not known. As regards subcellular distribution, factors determining the e‹cency of the reaction.65) xanthine oxidoreductase and aldehyde oxidase are Some prodrugs for anticancer agents are also activat- mainly distributed in the cytosol fraction of tissues. ed by aldehyde oxidase, as shown in Fig. 2.5-Fluoro-2- Critchley et al.64) reported that xanthine oxidoreductase pyrimidinone and 5-ethynyl-2(1H)-pyrimidinone are and aldehyde oxidase are localized in mitochondrial activated by aldehyde oxidase to 5-‰uorouracil and 5- fraction of guinea pig liver. However, we could not ˆnd ethynyluracil, respectively.78,79) 5-Ethynyluracil activates the activity of either of these ‰avoenzymes in liver the pharmacological action of 5-‰uorouracil by inac- mitochondrial fraction of guinea pigs. tivating dihydropyrimidine dehydrogenase, a detoxiˆca- tion enzyme of 5-‰uorouracil.78) Harrell et al.80) showed Oxidative Drug-Metabolism by Molybdenum that BRL 55792, BRL 55791 and BRL 55039, deoxygua- Hydroxylases nine analogues that are prodrugs of an antiviral agent, Aldehyde oxidase and xanthine oxidoreductase are 9-(3-hydroxypropoxy)guanine, were activated by xan- known to promote nucleophilic attack at an electron- thine oxidoreductase in rats and by aldehyde oxidase in deˆcient carbon atom adjacent to a ring nitrogen atom, humans. Thioguanine is activated to 8-hydroxythiogua- e.g., in pyridine, pyrazine, purine, pyrimidine, quino- nine by human aldehyde oxidase.81) Oxidation of fam- line and pteridine, although the attack does not neces- ciclovir and 6-deoxypenciclovir to penciclovir is mediat- sarily occur at a single site in N-containing heterocy- ed by aldehyde oxidase.52) Clarke et al.82) reported a role cles.12) Hall and Krenitsky65) demonstrated that the of aldehyde oxidase in the in vitro conversion of fam- electron-withdrawing character of 6-substituents of ciclovir to the active antiviral penciclovir in human purine and their hydrophobicity are important factors liver. determining aldehyde oxidase activity toward purines Nicotine is metabolized to nicotine D1?(5?) iminium ion 86 Shigeyuki KITAMURA et al.

Fig. 2. Aldehyde oxidase-catalyzed oxidation of various drugs (I). Arrowsindicatethesiteoxidizedbyaldehydeoxidase. by the cytochrome P450 system, and to cotinine by cynomolgus monkeys and humans.85) Furthermore, aldehyde oxidase.83) Prolintane, an sympathomimetic Taylor et al.86) reported that quinolines are oxidized to amine which is used to stimulate appetite, is also 2- and 4-quinolones by aldehyde oxidase, but quinolini- oxidized to oxoprolintane by aldehyde oxidase, via an um cations after N-alkylation are more oxidizable than iminium ion intermediate formed by the cytochrome quinolines (Fig. 3). P450 system.84) Azapetine, an antihypertensive agent, is The oxidase activity of aldehyde oxidase towards also converted to a lactam metabolite by aldehyde aldehyde compounds is generally lower than toward oxidase after biotransformation to the iminium ion by nitrogen-containing heterocyclic compounds. The cytochrome P450.13) In contrast, nicotinamide and anticancer drug cyclophosphamide and its isomeric ethionamide are not oxidized by aldehyde oxidase. analogue, ifosphamide, are activated to 4-hydroxyl However, ethionamide, a tuberculostatic agent, is ˆrst metabolites, in equilibrium with the ring-opened form methylated, and then the N-methylated ethionamide aldophosphamides, by CYP2B6 and CYP3A4, respec- cation, which undergoes nucleophilic attack more tively.87) Aldophosphamides undergo chemical decom- readily than ethionamide, is oxidized to the 2-pyridone position to yield mustard-type alkylating agents by aldehyde oxidase.13) Nicotinamide is also oxidized to (phosphoramide mustard and ifosphoramide mustard). 2- and 4-pyridones after N-methylation as described These aldophosphamides are metabolized to the inactive later (see: Individual Variation of Drug-Metabolizing carboxyphosphamides by aldehyde oxidase.88) Pyridoxal Ability of Molybdenum Hydroxylases). ABT-418, a and vitamin A aldehyde are also suggested to be selective cholinergic channel activator, is similarly oxidized by aldehyde oxidase.14,89,90) Tolbutamide oxidized by aldehyde oxidase in livers of rats, dogs, aldehyde, which is formed from tolbutamide by the Drug Metabolism by Molybdenum Hydroxylases 87

Fig. 4. Metabolism of aldehyde compounds catalyzed by aldehyde oxidase (AO) and xanthine oxidoreductase (XO).

mide, is converted to the 5-oxo metabolite by xanthine Fig. 3. Aldehyde oxidase-catalyzed oxidation of various drugs (II). oxidoreductase and aldehyde oxidase.96,98) 5-Fluoroura- Oxidations in this ˆgure involve iminium ion or nitrogen cation cil, an antineoplastic agent, is also oxidized by these two formation in the reaction sequences. enzymes13) (Fig. 5). Reductive Drug-Metabolism by Molybdenum cytochrome P450 system and alcohol dehydrogenase, is Hydroxylases oxidized to carboxytolbutamide, a major urinary metabolite of tolbutamide,91) as shown in Fig. 4. Aldehyde oxidase and xanthine oxidoreductase, in the Xanthine oxidoreductase participates in metabolism presence of an adequate electron donor, can mediate the of purines, such as caŠeine, hypoxanthine and xan- reduction of aromatic nitro compounds, such as thine.92) 6-Deoxyacyclovir, a highly absorbed prodrug nitrobenzoic acid, 1-nitropyrene, 2-nitro‰uorene, 4- of acyclovir, is activated by xanthine oxidoreductase to nitroquinoline N-oxide and nitrofurazone.99–102) In nitro acyclovir.77,93) 6-Mercaptopurine is oxidized by liver reduction with xanthine oxidoreductase, the enzyme aldehyde oxidase of rabbits and humans and bovine exhibited uniformly high activity toward nitrofuran milk xanthine oxidoreductase to 6-mercaptopurin-8-one derivatives. However, in the case of nitrobenzene and 6-thiouric acid, respectively.12,94) Famciclovirisalso derivatives, high reducing ability was observed when an metabolized by human liver xanthine oxidoreductase.95) electron-withdrawing group was substituted at the p- Allopurinol, an antihyperuricemic agent, is oxidized to position of the nitrobenzene. On the contrary, when an the active metabolite, oxypurinol, by xanthine ox- electron-donating group was present at the p-position, idoreductase and aldehyde oxidase.96) Azathioprine is the activity was low compared with that towards oxidized to 8-hydroxyazathioprine by aldehyde oxidase, nitrobenzene.103) These enzymes also show reductase and further metabolized to thiouric acid by xanthine activity toward some aminoazo compounds and oxidoreductase.97) An antituberculous agent, pyrazina- nicotinamide N-oxide.104–107) 88 Shigeyuki KITAMURA et al.

Fig. 5. Functions of aldehyde oxidase (AO) and xanthine oxidoreductase (XO) in drug-oxidation. Oxidations catalyzed by aldehyde oxidase andWor xanthine oxidoreductase are shown in this ˆgure.

Moreover, aldehyde oxidase can mediate the reduc- epoxide reductase activity toward benzo[a]pyrene 4,5- tion of various compounds other than those mentioned oxide and naphthalene 1,2-oxide.116) The azide group of above, as shown in Fig. 6. Sulfoxide compounds such as 3?-azido-3?-deoxythymidine (AZT), which is used for sulindac (an anti-in‰ammatory agent), phenothiazine the treatment of the acquired immune deˆciency syn- sulfoxide and diphenyl sulfoxide are reduced to the cor- drome (AIDS), is reduced to an amino group by alde- responding sulˆde compounds by aldehyde oxidase.108) hyde oxidase, a reaction which is similar to that with the N-Oxide reductase activity of aldehyde oxidase toward cytochrome P450 system.117–119) imipramine N-oxide, cyclobenzaprine N-oxide, nicotine The 1,2-benzisoxazole ring of zonisamide, an an- N-oxide and brucine N-oxide has been reported.109–111) ticonvulsant agent, is reductively cleaved to the ketimine N-Nitrosodiphenylamine is also reduced to 1,1- derivative, and the unstable intermediate is chemically diphenylhydrazine by aldehyde oxidase.112) Hydroxamic hydrolyzed to an oxo derivative.23) Reductive meta- acids, such as benzohydroxamic acid, salicylhydroxamic bolism of ziprasidone, an antipsychotic agent, to acid, a tuberculostatic agent, anthranylhydroxamic dihydroziprasidone by aldehyde oxidase was report- acid, nicotinohydroxamic acid and N-hydroxy-2- ed.120,121) In this case, the benzisothiazole ring is reduc- acetylamino‰uorene are reduced to the corresponding tively cleaved via the 2-hydroxythiazole intermediate to amides by aldehyde oxidase.113,114) Aldehyde oxidase an amidine-type metabolite.121,122) Aldehyde oxidase also exhibits reductase activity toward azo compounds, showed oxime-reductase activity toward camphor such as p-dimethylaminoazobenzene, amaranth, methyl oxime, acetophenone oxime, salicylaldoxime and ben- red and methyl orange, which are not reduced by xan- zamidoxime, aŠording the corresponding ketimines, thine oxidoreductase.106,115) The ‰avo enzyme also shows and other oxime compounds (except benzamidoxime) Drug Metabolism by Molybdenum Hydroxylases 89

Fig. 6. Reduction of various drugs by aldehyde oxidase.

are chemically hydrolyzed to oxo compounds.123) Alde- acetylcholine receptors in insects.124,125) hyde oxidase also functions as a nitroreductase of Both of the molybdenum hydroxylases contribute to neonicotinoids, nitroguanidine-type insecticides, such the nitroreduction of nitropolycyclic aromatic as imidacloprid and thiamethozan. These insecticides hydrocarbons in skin, which is one of target organs for are reduced by aldehyde oxidase via two- and six- environmental contaminants. The skin cytosols of electron reduction to nitrosoguanidine and amino- rabbits, guinea pigs, mice and rats all exhibit signiˆcant guanidine-type metabolites, respectively, and this reduc- nitroreductase activity. Aldehyde oxidase participates tive metabolism alters the agonistic potency at nicotinic predominantly in the reduction in skins of hamsters 90 Shigeyuki KITAMURA et al.

Fig. 7. Comparison of the RP values (the relative amounts of N1-methylnicotinamide and its metabolites in urine) for several strains of rats (A) and correlation between in vivo RP values and in vitro aldehyde oxidase activity in six strains of rats (B). The RP values were calculated from the amounts of N1-methylnicotinamide, 2-PY and 4-PY excreted in the urine of rats according to the following formula. RP (z)＝[(2PY＋4PY)W(N1-methylnicotinamide＋2PY＋4PY)]×100. The aldehyde oxidase activities toward N1-methylnicotinamide in rat livers were measured according to the method of Sugihara et al. (2006). Aldehyde oxidase activity is positively correlated with in vivo RPvaluesinsixstrainsofrats(Spearman'srankcorrelationcoe‹cientrs＝1, P＝0.001). Each value represents the mean±SD of four rats. and rabbits, whereas xanthine oxidoreductase partici- WKAWSea rats showed the lowest value. The value of pates predominantly in mice and rats, and both ‰avoen- Sea:SD rats was about 146-fold higher than that of zymes participate in guinea pigs.60,61) Aldehyde oxidase WKAWSea rats.128) We have also found a marked strain in bovine ciliary body also exhibits drug-reductase diŠerence of liver aldehyde oxidase activity in twelve activity toward nicotinamide N-oxide, nicotinohydrox- strains of rats in an assay using benzaldehyde or amic acid, sulindac, 1-nitropyrene and chloram- methotrexate as a substrate. Among the strains tested, phenicol.126,127) the highest activity was observed with Sea:SD rats and In ˆsh, liver aldehyde oxidase also catalyzes the the lowest with WKA WSea rats. The diŠerences between reductive metabolism of 2-nitro‰uorene (sea bream), the activities toward benzaldehyde and methotrexate in 1-nitropyrene (carp and sea bream) and fenthion sul- the two strains of rats were 63.5- and 104-fold, respec- foxide (goldˆsh and sea bream).43–45) This cytosolic tively.50,53) We suggested that quantitative, but not enzyme seems to play an important role in drug qualitative, diŠerences of aldehyde oxidase exist among metabolism in ˆsh. diŠerent strains of rats, since there was little variation of Km values of the enzyme among the strains of rats Individual Variation of Drug-Metabolizing Ability of examined, and Western blot analysis showed that the Molybdenum Hydroxylases strains possessed diŠerent quantities of the enzyme. N1-Methylnicotinamide, which is formed from Furthermore, we showed that the strain diŠerences of nicotinamide by nicotinamide methyltransferase, is aldehyde oxidase in rat liver with N1-methy- widely distributed in animals, like nicotinamide. lnicotinamide as a substrate were well correlated with N1-Methylnicotinamide is neurotoxic, and is detoxiˆed the activity in vivo128) (Fig. 7). by oxidation to N1-methyl-2-pyridone-5-carboxamide Aldehyde oxidase activity is regulated by androgen in (2-PY) and N1-methyl-4-pyridone-3-carboxamide mice, which contain four isozymes of the oxidase (4-PY). The conversion of N1-methylnicotinamide to (AOX1, AOH1, AOH2 and AOH3).8) The aldehyde 2-PY and 4-PY has been reported to be catalyzed by oxidase cluster in rat 9 is similar to aldehyde oxidase.14,15) The N1-methylnicotinamide that in mice, and contains Aox1, Aoh1, Aoh2 and oxidase activity shows marked variations among various Aoh3. AOX1 and AOH1 are active aldehyde oxidase strains of rats. The activity was highest in Sea:SD, species in mouse liver, and they have similar substrate followed by Jcl:SD, and Slc:WistarWST rats, while speciˆcities, while AOH2 and AOH3 are observed at Drug Metabolism by Molybdenum Hydroxylases 91 low levels in skin and olfactory mucosa, respectively.8) oxidoreductase activity was not.137) Inhibitory eŠects of A similar gene cluster exists in humans.20) some medicinal drugs on human liver aldehyde oxidase Individual variation of xanthine oxidoreductase activity have been reported.138,139) For example, aldehyde activity was examined by measurement of the metabo- oxidase activity is suppressed by raloxifene in human lites of caŠeine after a single dose. The amounts of liver.138) Raloxifene inhibited oxidations of phthalazine, 7-demethylated metabolite formed by CYP1A2 diŠered vanilin and nicotine iminium ion mediated by aldehyde depending upon race, sex and age, but no diŠerence of oxidase. However, raloxifene had only a small eŠect the amounts of 8-hydroxylated metabolite generated by on xanthine oxidoreductase.138) An inhibititory eŠect of xanthine oxidase was observed.92) Patients with xanthine cimetidine on zaleplon metabolism in human liver oxidoreductase deˆciency (xanthinuria) have been in vitro was also reported.140) Bioavailability of zebula- classiˆed into two groups; one that lacks xanthine rine, which is metabolized by aldehyde oxidase, was oxidoreductase, and has oxidation ability for al- enhanced by a potent inhibitor, raloxifene, but the eŠect lopurinol and pyrazinamide, and another that lacks varied markedly in hepatic cytosols from humans, these activities. As aldehyde oxidase oxidizes these monkeys, dogs, rats, and mice due to species diŠer- substrates, a combined deˆciency of xanthine ox- ence.141) In contrast, medicinal drugs that inhibit idoreductase and aldehyde oxidase may exist in the oxidation reactions catalyzed by aldehyde oxidase do latter group.129,130) Johnson et al.131) also reported not inhibit the reductive metabolism of ziprasidone to combined deˆciency of xanthine oxidoreductase and its major metabolite, S-methyldihydroziprasidone, sulˆte oxidase in xanthinuric patients. in vitro.142) Variation of aldehyde oxidase activity in humans has As regards inducing eŠects on both enzyme activities, been discussed in several papers.21,24,132,133) Marked Dietrich143) demonstrated that xanthine oxidoreductase interindividual variation in methotrexate hydroxylase activity is increased about twice by xanthine i.p. injec- activity was found in human liver.134) Zaleplon, a tion for 6–12 days at 120 mgWkgWday. Johnson et al.144) sedative-hypnotic, was reported to be metabolized by showed that phthalazine (50 mgWkg) and its hydroxylat- aldehyde oxidase and the cytochrome P450 system, and ed metabolite, 1-hydroxyphthalazine (10 mgWkg), in- the metabolic pattern was in‰uenced by the level of duce both aldehyde oxidase and xanthine oxidoreduc- aldehyde oxidase.75) We recently examined developmen- tase activities in rabbit liver. Recently, we showed that tal changes of aldehyde oxidase activity in rats. Little or 2,3,7,8-tetrachlorodibenzo-p-dioxin induces xanthine no activity was observed in fetal or newborn rats, but oxidoreductase activity mediated by aryl hydrocarbon the activity markedly increased after birth. At 2–3 weeks receptor.145) In this case, aldehyde oxidase activity was of age, the activity was about the same as in adult rats induced only 1.5-fold by the treatment. Ethacrynic acid, (Tayama et al., unpublished data). As is the case in carbon tetrachloride and buthionine sulfoxime also humans, rat studies involving treatment with drugs slightly induce xanthine oxidoreductase activity by that are metabolized by aldehyde oxidase require about 1.5-fold.146) The enzyme is also induced by caution. Interindividual variations in aldehyde oxidase bacterial lipopolysaccaride and hypoxia.147,148) However, activity are of considerable importance for the clinical we found that inducers of the cytochrome P450 system, application of any drug metabolized by the enzyme, such as phenobarbital, 3-methylcholanthrene and including methotrexate, cyclophosphamide, zaleplon dexamethasone, did not induce these molybdenum and allopurinol. hydroxylases in vivo in rats. Xanthine oxidoreductase in the presence of a suitable Drug-Drug Interaction via Aldehyde Oxidase electron donor yields superoxide anion, which is associ- Various medicinal drugs have inducing or inhibitory ated with edema, ischemia and sepsis syndrome.31,149) eŠects on aldehyde oxidase and xanthine oxidoreduc- To prevent these injurious eŠects, attempts have been tase. In particular, many chemicals inhibit aldehyde made to ˆnd natural products with an inhibitory eŠect oxidase.135) Aldehyde oxidase-linked activities are on xanthine oxidoreductase. Chang et al.150) reported markedly inhibited by menadione, isovanillin, chlor- inhibitory eŠects of some ‰avonoids; baicalein was the promazine and estradiol, and xanthine oxidoreductase most potent. Lin et al.151) also found inhibitory eŠects of is inhibited by oxypurinol (allopurinol). SKF 525-A and ‰avonoids, and noted that the most potent inhibitor is a narcotic analgesic, methadone, show a potent inhibi- apigenin. In contrast, Rivera et al.152) reported that tory eŠect on aldehyde oxidase, and the antihistamines aldehyde oxidase and its homologue (AOH1) in mice are diphenhydramine, pheniramine, doxylamine, orphena- induced 2- to 3-fold at the protein levels by dioxin. drine, methapyrilene and pyrilamine also inhibit with A similar eŠect was seen in Hepa-1 cells, but the mRNA IC50 values of 100–500 mM.136) Aldehyde oxidase level in the cells, which lack aryl hydrocarbon receptor activity was inhibited after administration of hydrala- or aryl hydrocarbon receptor nuclear translocator, was zine (10 mgWkgWday for 7 days) to rabbits, but xanthine not enhanced. 92 Shigeyuki KITAMURA et al.

When methotrexate is used clinically, the main- measured the ratio of the amount of pyridones to the tenance of an eŠective level is critical. 7-Hydrox- total amount of N1-methylnicotinamide and pyridones ymethotrexate is less active, but cytotoxic, and also has (RP value) in the urine of rats, marked intraspecies other pharmacological eŠects. Clinically, combined variations were observed. usage of methotrexate with cyclosporin resulted in a RP (z)＝[(2-PY＋4-PY)W(N1-methylnicotinamide decrease in the level of 7-hydroxymethotrexate, in blood of patients, and thereby potentiated methotrexate e‹ca- ＋2-PY＋4-PY)]×100 cy.153) Therefore, external inhibition of methotrexate The variation in RP value among strains was closely hydroxylation by aldehyde oxidase could be important related to the diŠerences of liver aldehyde oxidase in the clinical application of methotrexate. Possible activity measured with N1-methylnicotinamide as a drug-drug interaction through modulation of drug- substrate. RP values after administration of N1-methy- metabolizing activity based on aldehyde oxidase should lnicotinamide to diŠerent strains of rats conˆrmed the always be taken into consideration in the clinical setting. existence of strain diŠerences of aldehyde oxidase The purine analogue, allopurinol, has been in clinical activity in vivo. We also conˆrmed the strain diŠerences use for more than 30 years as an inhibitor of xanthine of aldehyde oxidase in rat liver with N1-methy- oxidoreductase in the treatment of hyperuricemia and lnicotinamide as a substrate, and showed that the gout. However, folate compounds and ‰avanoids also oxidase activity correlated with the activity in vivo inhibit xanthine oxidoreductase.154) Drug-drug interac- (Fig. 7). Thus, the quantitative variation of the enzyme tion based on inhibition of the enzyme is involved in the in liver appears to be the cause of strain diŠerences of interaction of 6-mercaptopurine with allopurinol.155) N1-methylnicotinamide metabolism in rats in vivo. It is recommended that the dose of 6-mercaptopurine is Therefore, we had demonstrated that measurement of reduced by about half when it is coadministered with RP values in urine is a useful method to predict the allopurinol. Pea156) also reported that when allopurinol in vivo level of aldehyde oxidase in animals and is coadministered with azathiopurine and 6-mer- humans.128) Reiter et al.129) suggested a deˆciency of caputopurine, their toxic eŠects are potentiated. In this aldehyde oxidase in humans by using a method similar case, it was suggested that azathiopurine exhibits toxici- to ours, that involved measuring the amounts of ty after conversion to 6-mercaptopurine by reaction pyridones of N1-methylnicotinamide in human urine. with glutathione.157) Interaction of cyclophosphamide However, whether there is a genetic deˆciency of and allopurinol has also been reported. Although aldehyde oxidase in humans is unclear. Furthermore, it allopurinol pretreatment resulted in a longer half-life was shown by us that the degree of 7-hydroxylation of of cyclophosphamide, urinary metabolites and cyclo- methotrexate in rats in vivo closely re‰ects strain phosphamide excretion were unchanged.158) However, diŠerences of aldehyde oxidase-based methotrexate 7- Witten et al.159) reported that although the half-life of hydroxylase activity in the liver (Moriyasu et al., unpub- cyclophosphamide is not signiˆcantly in‰uenced by lished data). allopurinol treatment, that of its metabolite was Recently, chimeric mice, which contain human liver increased. In contrast, it was demonstrated that cells, have been produced for use in preclinical studies. allopurinol protects rat hepatocytes against the reactive We attempted to estimate in vivo aldehyde oxidase oxygen-based toxicity of azathiopurine and 6-mercap- activity in these chimeric mice. It was established that topurine.97) human-type metabolism of N1-methylnicotinamide by aldehyde oxidase occurred in these mice in vivo,and Estimation of Aldehyde Oxidase Levels In Vivo that the aldehyde oxidase in these mice is a typical The pharmacological action of a drug is closely relat- human-type aldehyde oxidase, but not mouse-type ed to its metabolism. Therefore, variations in aldehyde aldehyde oxidase.160) oxidase activity are of considerable importance for the Conclusion clinical application of drugs. Variations of aldehyde oxidase in Caucasians and Japanese have been report- Recently, increased attention has been paid to drug ed.21,132) However, no report was available on the predic- metabolism mediated by aldehyde oxidase and xanthine tion of aldehyde oxidase levels in vivo. A method for oxidoreductase, and to the possibility of drug-drug estimation of the enzyme level in vivo is therefore interaction associated with interindividual variation of important to prevent drug overdose. these enzyme activities. It appears that variation of We examined the in vivo conversion ratio of N1- these enzyme activities can markedly in‰uence the methylnicotinamide to 2-PY and 4-PY as a parameter disposition of medicines that are metabolized by them, for the estimation of aldehyde oxidase level in rats. N1- and can be clinically signiˆcant. Furthermore, as the Methylnicotinamide and its pyridones (2-PY and 4-PY) activity of aldehyde oxidase in humans is very high are usually detected in the urine of rats. When we compared with those in experimental animals, the Drug Metabolism by Molybdenum Hydroxylases 93 results of drug disposition studies in rats and mice can Hitchings, G. H.: A comparison of the speciˆcities of not necessarily be extrapolated to humans. 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