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Review CYP2D in the Brain

Review CYP2D in the Brain

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Drug Metab. Pharmacokin. 18 (6): 337–349 (2003).

Review CYP2D in the Brain

Yoshihiko FUNAE, Wataru KISHIMOTO,ToshioCHO, Toshiro NIWA, and Toyoko HIROI Department of Chemical Biology, Osaka City University, Medical School, Osaka, Japan

Summary: CYP2D1, 2D2, 2D3, and 2D4 are major CYP2D isoforms expressed in the rat. In humans, only CYP2D6 is expressed. In rat brain, the mRNA for CYP2D4 is most abundant in cerebellum, striatum, pons and medulla oblongata. In , CYP2D6 mRNA expression was detected in all regions with highest levels observed in cerebellum. CYP2D isoforms are involved in the of not only xenobiotics such as , b-adrenergic blockers, antiarrhysthmics, and antihypertensives, but also endogenous compounds such as and . Among 11 isoforms of human recombinant P450s, only CYP2D6 exhibit- ed an ability to e‹ciently convert which exists in the brain, to . CYP2D4 and CYP2D6 which are the predominant CYP2D isoforms in the rat and human brain, respectively, possess 21- activity for both progesterone and allopregnanolone. CYP2D4, not P450c21, works as a steroid 21-hydroxylase in the brain. These results suggested that CYP2D in the brain may be involved in the metabolism of neuronal amines and steroids and in the regulation of the central nervous system.

Key words: CYP2D; CYP2D4; CYP2D6; brain; dopamine formation; steroid 21-hydroxylation

system or on the heart. It is thus of immense interest Introduction whether CYP2D6 is not only localized in the liver,8) but Six , named CYP2D1 through CYP2D5 and also found at high levels in these two target tissues, the CYP2D18,werefoundintherat.1–3) CYP2D1 catalyzes brain9,10) and heart.11) a 4-hydroxylase and exhibits a closest The polymorphic hydroxylation of debrisoquine was enzymatic relationship to human CYP2D6. CYP2D7P ˆrst described in the 1970s.12) Since then, the clinical and CYP2D8P are human . No cDNA has pharmacological as well as molecular biological fea- been cloned that corresponds to these genes, and their tures13) of this have been studied. Several expression has never been demonstrated. groups of important drugs, such as antidepres- The levels and speciˆcity of P450s from sants and neuroleptics,14) are metabolized in the liver by humans were systematically characterized.4,5) Antibod- CYP2D6, and this has important clinical implications ies raised against puriˆed P450 which were expressed for the usage of such drugs. In healthy volunteers, a in Saccharomyces cerevisiae were used to measure the signiˆcant diŠerence in personality between extensive levels of hepatic P450s. The level of CYP3A4 was (EM) and poor metabolizers (PM) of debrisoquine highest in human hepatic microsomes, comprising living in Sweden was reported.15) That study compared 30¿40z of total P450. CYP2C9 comprised 10¿20z PM and EM of debrisoquine using the Karolinska of the total. The level of CYP2D6 was 14z.4) It Scales of Personality inventory.15) This indicated that has been estimated that CYP2D6 is responsible for debrisoquine hydroxylase also metabolizes endogenous 20¿30z of the oxidation of all pharmaceutics substrate(s) important for central nervous system used by humans.6,7) Of particular note are tricyclic function.16) Poor metabolizers were more anxiety- antidepressants, selective reuptake inhibitors, prone and less successfully socialized than extensive 5-hydroxytryptamine receptor antagonists, anti- metabolizers of debrisoquine.17) This and a previous psychotics, , and , together with study among subjects in Sweden suggest that there may b-adrenoreceptor antagonists and antidysrhythmic be a relationship between personality and activity of drugs,7,8) all agents that act either in the central nervous CYP2D6. This polymorphic may have an

Received; September 22, 2003, Accepted; October 24, 2003 To whom correspondence should be addressed: Yoshihiko FUNAE, Department of Chemical Biology, Osaka City University, Medical School, Osaka, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585 Japan. Tel. ＋81-6-6645-3915, Fax. ＋81-6-6645-3917, E-mail: funae＠med.osaka-cu.ac.jp

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Table 1. Sequence identities (z) of rat and human CYP2D Table 2. residues of human and rat CYP2D isoforms29) isoforms. Both the overall sequence identity (upper triangle) and that of the active site residues listed in Table 2 (lower triangle) is given. CYP b Residue SRS a number 2D1 2D2 2D3 2D4 2D5 2D6

1 103ProLeuGluProProPro 105 Pro Pro Pro His Pro Pro 106 Ile Ile Ile Phe Ile Ile CYP2D1 CYP2D2 CYP2D3 CYP2D4 CYP2D5 CYP2D18 CYP2D6 112 Val Tyr Tyr Phe Val Phe CYP2D1  73 79 72 95 72 71 120 Ile Val Val Val Val Phe CYP2D2 45  78 73 73 73 71 121 Leu Leu Leu Leu Phe Leu CYP2D3 59 59  75 79 75 72 2 213 Leu Phe Met Leu Leu Leu CYP2D4 50 41 45  73 99 77 216 Val Asp Gln Glu Val Glu CYP2D582415550 73 71 217 Ser Thr Thr Ser Ser Ser CYP2D18 50 41 45 100 50  77 3 243 Gly Lys Gly Gly Gly Phe CYP2D6594155595059 244 Gln Leu Gln Lys Gln Gln 4 301AspAspAspAspAspAsp 304 Thr Met Gly Met Thr Ser 305AlaAlaAlaAlaAlaAla endogenous neuroactive substrate or product, such as a 308 Val Val Val Val Val Val biogenic amine. There is now substan- 309ThrThrThrThrThrThr 5 369IleIleIleIleIleIle tial evidence that CYP2D6 is present and functions also 370 Ala Val Val Leu Ala Val 18–20) in the human brain, although its activity is lower 374 Leu Ile Leu Val Leu Val than in the liver. 375ProProProProProThr In this review, we describe the genetic and catalytic 6 483 Phe Leu Phe Ala Ile Phe properties of rat and human CYP2D isoforms. 483 Pro Pro Leu Leu Ser Leu CYP2D4 and 2D6, which are expressed in the brain, a Residue numbering is that of CYP2D6. catalyze endogenous substrates such as amines and b SRS: substrate recognition sites. steroids. The pharmacological and physiological sig- niˆcance of the presence of CYP2D in the brain is described. CYP2D6 and rat CYP2D1-4, as deduced from rigid docking studies are shown in Table 2. The analogous Isoforms of the CYP2D family in rat and human residues in CYP2D5 and 2D18 have also been included. Six homologues of CYP2D6 have been isolated CYP2D4 and 2D18 were found to diŠer in the identity from the rat,1,2,21–23) namely, CYP2D1, 2D2, 2D3, 2D4, of only four residues, none of which were located in the 2D5, and 2D18. Human CYP2D6 is one of the most CYP2D active site. When the active site residues of the important phase I involved in the metabolism human and rat CYP2D isoforms listed in Table 2 are of therapeutic drugs. Another feature that has sig- examined, it is striking how few residues in the models niˆcantly contributed to the attention CYP2D6 has are completely conserved. Only ˆve of the 22 residues obtained concerns its polymorphic nature.24,25) To date are invariant in all human and rat CYP2D isozymes, of more than 70 diŠerent have been identiˆed.26) which four are located in the I helix (SRS 4). The eŠects of these polymorphic genes range from a Correspondingly, sequence identities between CYP2D complete loss of functional protein to an increase in isoforms were much lower for the active site residues enzyme activity. DiŠerences in substrate speciˆcity may than for the overall sequences (Table 1) except between also arise. As a result, drug treatment in polymorphic CYP2D4 and 2D18. The most notable diŠerences in individuals may cause adverse eŠects or a lack of drug active site residues of the homology models between e‹cacy.27) human and rat isoforms involved residues 120 and 216. Immunoblotting studies have demonstrated hepatic As described above, residue 120 constitutes a phenylala- expression of CYP2D1, 2D2, 2D4, and 2D5, but not nine in CYP2D6 and provides p-stacking interactions CYP2D3, in various rat strains.28) CYP2D18 is believed with ligands. In all rat isoforms, residue 120 is nonaro- to be the rat brain variant of CYP2D4.23) The rat and matic and therefore only capable of contributing to human CYP2D isoforms share high sequence identity ligand binding a‹nities by van der Waals interactions. (À70z)29) (Table 1). Nevertheless, signiˆcant diŠer- Rigid docking studies with CYP2D6 identiˆed Glu216 ences in binding and regioselectivity of metabolism have as a key ligand-binding residue forming hydrogen bonds been observed between these CYP2D isoforms.28–32) For with several substrates. In the cases of CYP2D1W5and example, R- was N-oxidated by only CYP2D1 2D3, a valine and glutamine, respectively, are located at while 8-hydroxylation was oxidated by all isoforms this position. These isoforms are unable to form a salt investigated.30) The active site residues of human bridge with the basic nitrogen of ligands. p337 p.3 [100%]

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Although it is known that rat CYP2D2, similar to human CYP2D6, is capable of metabolizing debriso- quine, bufuralol, and other substrates,1,33,34) the catalytic characteristics of most rat CYP2D enzymes remain to be elucidated. Therefore, we expressed four rat P450 isoforms (CYP2D1, 2D2, 2D3, and 2D4) placed in the CYP2D subfamily in yeast and compared their biochemical properties and substrate speciˆcity simul- taneously.32) We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simultane- ously. Three are believed to be alleles of CYP2D1, 2D2, and2D3,respectively,basedonhighnucleotide sequence similarity, while CYP2D4 had sequences of both CYP2D4 and CYP2D18. Expression plasmids carrying CYP2D cDNAs were transformed into Saccharomyces cerevisiae. Typical P450 CO-diŠerence Fig. 1. mRNA expression of rat CYP2D isoforms in tissues. spectra with an absorbance maximum at 448 nm were recorded with microsomal preparations from the yeast cells expressing the four CYP2D forms. A catalytic study of these CYP2D forms was done with debriso- and small intestine mucosa, which were exposed to quine, bufuralol, and . CYP2D2 had the xenobiotics such as drugs, food components and highest debrisoquine 4-hydroxylation activity (2.2 nmol environmental contaminations. mRNA of CYP2D4 was WminWnmol P450), similar to that (2.2 nmolWminWnmol expressed in liver, kidney, small intestine mucosa and P450) of human CYP2D6 expressed in yeast cells. brain. CYP2D3 had high lidocaine N-deethylation activity Although the amount of P450 in the brain is a small (43 nmolWminWnmol P450), and both CYP2D3 and fraction of that in liver, the presence and activity of 2D2 exhibited high lidocaine 3-hydroxylation activity many diŠerent isoforms have been demonstrated in (2.4 and 1.6 nmolWminWnmol P450, respectively). rat,37) dog,19) monkey38) and human39) brain tissue. The Bufuralol 1?-hydroxylation catalytic capabilities were overall metabolic capacities of these enzymes are low comparable among the four isoforms. The activity of in the whole brain, but recent immunocytochemical CYP2D1 was relatively low toward the three substrates investigations show that P450s are unevenly distributed (debrisoquine, 0.091; bufuralol, 1.5; lidocaine 3- across brain regions. In rat brain, the mRNA for hydroxylation, 0.019; lidocaine N-deethylation, 2.8 CYP2D436) and for CYP2D140) have been isolated, with nmolWminWnmol P450). These ˆndings indicate that that for CYP2D4 being the most abundant.41) The RT- debrisoquine, a typical substrate for CYP2D forms, was PCR product from CYP2D4, the predominant CYP2D mainly metabolized by CYP2D2 but not CYP2D1 in rat isoform in rat brain, was more abundant in cerebellum, liver and that the CYP2D forms have diŠerent substrate striatum, pons and medulla oblongata. Similar to the speciˆcity. distribution of CYP2D4 mRNA, all CYP2D mRNAs showed a consistent distribution pattern, indicating that Localization the majority of CYP2D4 expression occurs in rat brain. In mammals, the liver is generally accepted to be the In contrast to rats, humans possess only one functional major organ involved in CYP-mediated metabolism, CYP2D6 and two putative pseudogenes, CYP2D7 and but there is increasing evidence that these enzymes are CYP2D8.42) In human brain, CYP2D6 mRNA expres- present in extrahepatic tissues and that they may sion was detected in all regions and localized to speciˆc contribute to the local in situ metabolism of drugs and regions with highest levels observed in cerebellum.9) of carcinogens and toxins.35) In rats, the Western blot analysis is more useful for estimating the tissue distributions of four isoforms (CYP2D1, 2D2, amount of CYP2Ds than RT-PCR analysis because 2D3 and 2D4) in the CYP2D subfamily were investigat- some discrepancy was observed between the mRNA and ed in twelve kinds of tissue (liver, kidney, brain, lung, protein level. However, it is di‹cult to prepare speciˆc heart, spleen, adrenal gland, small intestine mucosa, antibodies to distinguish individual rat CYP2Ds due to bladder, testis, ovary and gonecystis) by RT-PCR36) high homology (77z to 83z). Using CYP2D4 speciˆc (Fig. 1). mRNA of CYP2D1 was expressed in all tissues antibodies, CYP2D4 protein in rat brain was revealed to used in this study except brain. mRNAs of CYP2D2 be expressed in regions containing CYP2D4 mRNA. In and CYP2D3 were mainly expressed in liver, kidney addition, a strong relationship between CYP2D4 p337 p.4 [100%]

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protein levels and CYP2D4 speciˆc catalytic activities was observed. It is intriguing that CYP2D isoforms are expressed in brain because many centrally acting drugs and toxins such as tricyclic antidepressants, selective serotonin reuptake inhibitors, monoamine inhibitors, amphetamines, codeines and neuroleptics are metabolized by CYP2Ds,43) although the physiological and pharmacological signiˆcance of CYP2D isoforms in brain is not fully clariˆed. CYP2D6, an important isoform of , mediates the metabolism of several psychoactive drugs in liver. Quantitatively, liver is the major drug metabolizing organ, however metabolism of drugs in brain could modulate pharmacological and phar- macodynamic eŠects of psychoactive drugs at their site of action and explain some of the variation typically seen in patient populations. Chinta et al. has measured P450 content and examined the constitutive expression of CYP2D mRNA and protein in human brain by RT- PCR, Northern blotting and immunoblotting and local- ized the expression by in situ hybridization and im- munohistochemistry.44) CYP2D mRNA was expressed constitutively in neurons of the cerebral cortex, Pur- kinje and granule cell layers of the cerebellum, reticular neurons of the midbrain and pyramidal neurons of subˆelds CA1, CA2 and CA3 of the hippocampus. Immunoblot studies demonstrated the presence of Fig. 2. Metabolic pathway of mianserin by CYP2D isoforms. cytochrome P4502D protein in cortex, cerebellum, mid- brain, striatum and thalamus of human brain. Immuno- histochemical localization showed the predominant presence of CYP2D6 not only in neuronal soma but also exogenous substrates of each CYP2D isoform in rats in dendrites of Purkinje and cortical neurons. These stu- and humans, we isolated full-length cDNAs of dies demonstrate a constitutive expression of CYP2D6 CYP2D1, CYP2D2, CYP2D3, CYP2D4 and CYP2D6, in the neuronal cell population in human brain, indicat- and expressed each enzymatic protein in yeast cells.4,32) ing a possible role in the metabolism of psychoactive Each of the recombinant enzymes was used as an drugs directly at or near their site of action, in neurons, isolated enzyme. in human brain. Mianserin metabolism by CYP2D Exogenous substrate of CYP2D Mianserin, a , is mainly The CYP2D isoforms are involved in the metabolism metabolized by CYP2D6 and CYP3A4 in humans and of xenobiotics including drugs such as antidepressants its major metabolites are 8-hydroxymianserin, N- (e.g. , mianserin, ‰uoxetine),30,45–48) b- demethylmianserin, mianserin N-oxide and 8-hydroxy- adrenergic blockers (e.g. , bufuralol, N-demethylmianserin (Fig. 2).30,46,47) Catalytic activities bunitolol),31,49–53) antiarrhythmics (e.g. , of CYP2D isoforms toward mianserin were measured at ),54,55) antihypertensives (e.g. debrisoquine, the substrate concentration of 100 mM(Table 3). The guanoxan),12,28,56) (e.g. , CYP2D isoforms (CYP2D1, 2D2, 2D3, 2D4 and 2D6) , )37,56–58) and neuroleptics (e.g. showedsimilarlevelsof8-hydroxylationactivity , ).59,60) In humans, only ranging from 1.15 to 2.53 nmolWminWnmol P450. By one isoform named CYP2D6 is expressed in various contrast, N- activity varied considerably tissues, whereas six isoforms (CYP2D1, CYP2D2, among the CYP2D isoforms; with that of CYP2D3 CYP2D3, CYP2D4, CYP2D5 and CYP2D18) have been and 2D4 several fold that of CYP2D1 and 2D6. The identiˆed in rats by genomic analysis. The physiological catalytic activity of CYP2D2 in this reaction was roles of the multiple rat CYP2D isoforms are not negligible. The order of N-demethylation activity was known, nor is the isoform that corresponds to human CYP2D3À2D4:2D1À2D6:2D2. Mianserin N-oxida- CYP2D6. To examine the catalytic speciˆcity toward tion activity was speciˆc to CYP2D1 though its level was p337 p.5 [100%]

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Table 3. Catalytic activities of expressed CYP2D isoforms toward mianserin

Activity (nmolWminWnmol P450)

Reaction Rat Human

2D12D22D32D42D6

8-Hydroxylation 1.15±0.22 2.08±0.35 1.37±0.20 2.53±0.27 2.01±0.18 N-Demethylation 2.20±0.15 0.42±0.08 10.2±1.1 8.22±0.66 1.05±0.18 N-Oxidation 0.56±0.06 N.D. N.D. N.D. N.D. 8-Hydroxy-N-demethylation N.D. N.D. N.D. 0.78±0.07 0.33±0.04

Each value represents the mean±S.D. of three determinations. Substrate: Mianserin (100 mM). N.D.: Not detected.

Table 4. Kinetic parameters for 8-hydroxylation and N-demethyla- serin, also have antidepressant eŠects.61) The rat tion of mianserin in expressed CYP2D isoforms CYP2D4, like human CYP2D6, catalyzed the forma- Rat Human tion of three metabolites, 8-hydroxymianserin, N- Reaction demethylmianserin and 8-hydroxy-N-demethylmianse- 2D1 2D2 2D3 2D4 2D6 rin. It is therefore possible that CYP2D4 is involved in

Km 36 5.9 69 130 3.7 the metabolism of mianserin in the brain, and CYP2D1, 8-Hydroxylation Vmax 1.8 2.3 2.7 9.3 2.9 2D2 and 2D3 catalyze its metabolism in the liver. Vmax WKm 0.050 0.39 0.039 0.072 0.78 Bufuralol metabolism by CYP2D Km 160 N.C. 85 400 5.1 N-Demethylation Vmax 4.4 N.C. 15 65 1.2 Bufuralol is a typical substrate for CYP2D isoforms. W Vmax Km 0.028 N.C. 0.18 0.16 0.24 Though 1?-hydroxybufuralol was a main metabolite of Each value represents the mean of three determinations. bufuralol, 1?2?-ethenylbufuralol was also formed by 51) Km: mM, Vmax:nmolWminWnmol P450, Km WVmax:mLWminWnmol P450. CYP2D isoforms (Fig. 3). Catalytic activities of N.C.: Not calculated. CYP2D isoforms toward bufuralol were measured at the substrate concentrations of 20 and 100 mM (Table 5). All human and rat CYP2D isoforms exam- low. The formation of 8-hydroxy-N-demethyl mianse- ined had 1?-hydroxylation activity, a potential probe of rin was speciˆc to CYP2D4 and CYP2D6. CYP2D activity.33) The 1?-hydroxylation activity is The kinetic parameters for 8-hydroxylation and N- considered to be common to CYP2D isoforms. We have

demethylation of mianserin in the expressed CYP2D demonstrated that CYP2D2 had an extremely low Km isoforms are listed in Table 4.TheKm value of 8- value (0.044 mM) for bufuralol 1?-hydroxylation activity hydroxylation for CYP2D2 (5.9 mM) was much smaller compared to other CYP2D isoforms.31) Therefore, in than those for other rat CYP2D isoforms and was close rats, CYP2D2 is likely to predominantly catalyze the 1?-

to that for human CYP2D6 (3.7 mM). The Vmax WKm hydroxylation of bufuralol, though all CYP2D isoforms value for CYP2D2 was also comparable to that for have this activity. CYP2D4 and CYP2D6 had strong

CYP2D6. The Km values for N-demethylation by bufuralol 1?2?-ethenylayion activity. CYP2D1 did not CYP2D1, 2D3 and 2D4 were much larger than that of have this activity. CYP2D2 and CYP2D3 showed this CYP2D6, and that for CYP2D2 could not be calculated activity but at low levels: less than 15z of CYP2D4 because of low activity. 8-Hydroxylation activities of activity. At a low concentration of bufuralol such as CYP2D isoforms toward mianserin were similar, but 20 mM, 1?2?-ethenylation activity was speciˆc to rat N-demethylation activity varied considerably among CYP2D4 and human CYP2D6. As CYP2D4 is CYP2D isoforms and N-oxidation activity was speciˆc expressed in rat brain, the catalytic reactions speciˆc to to CYP2D1 (Fig. 2). The formation of 8-hydroxy-N- CYP2D4 are useful for investigating the physiological demethyl mianserin was speciˆc to rat CYP2D4 and roles of CYP2D4 in the brain. human CYP2D6 because they have high levels of Endogenous substrate for CYP2D4 and 2D6 catalytic activity for both N-demethylation of mianserin and 8-hydroxylation of N-demethylmianserin compared CYP2D isoforms are expressed not only in the liver with other CYP2D isoforms. Rat CYP2D isoforms, but also in the brain,62,63) although their physiological CYP2D4 and 2D18, have been reported to be expressed and pharmacological functions in the brain are in the brain.23,36,41) Mianserin is an antidepressant and its unknown. Because the CYP2D subfamily is responsible metabolites, 8-hydroxymianserin and N-demethylmian- for the metabolism of many drugs acting on the central p337 p.6 [100%]

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Fig. 3. Metabolic pathway of bufuralol by CYP2D isoforms.

Table 5. Catalytic activities of expressed CYP2D isoforms toward bufuralol

Activity (nmolWminWnmol P450) Substrate (Concentration) Metabolite Rat Human 2D1 2D2 2D3 2D4 2D6

Bufuralol (100 mM) 1?-Hydroxylation 1.31±0.08 7.66±0.30 2.41±0.25 5.96±1.47 22.38±5.61 1?2?-Ethenylation N.D. 0.25±0.13 0.05±0.01 1.65±0.37 1.34±0.30 Bufuralol (20 mM) 1?-Hydroxylation 0.44±0.15 6.29±1.35 0.24±0.02 2.14±0.45 20.48±7.54 1?2?-Ethenylation N.D. N.D. N.D. 0.32±0.06 0.98±0.49

Each Value represents the mean±S.D. of three determination. N.D.: Not detected.

nervous system (CNS) and CYP2D6 polymorphism cytochrome P450 expressed in yeast, only CYP2D6 seems to have some relationship with personality and exhibited an ability to e‹ciently convert p-tyramine and Parkinson's disease,16,17,64) this subfamily is of interest in m-tyramine to dopamine. In studies with human hepatic brain pharmacology and toxicology. In addition, many microsomes, the hydroxylation of tyramine to dopa- investigators have shown the interaction of CNS-active mine was inhibited by bufuralol, a typical substrate for drugs with the CYP2D subfamily. In addition to the CYP2D isoforms, and anti-CYP2D1 antiserum. This is function of CYP2Ds in brain the local in situ mediators the ˆrst report that CYP2D is capable of converting 65) for drug metabolism, CYP2D was revealed to metabo- tyramine to dopamine. The Km values of CYP2D6, lize endogenous substances (Table 6). expressed in yeast, for p-tyramine and m-tyramine were 190.1±19.5 mMand58.2±13.8 mM, respectively. Amine Tyramine is an endogenous compound which exists in Dopamine is formed from L- by tyrosine the brain as a trace amine but is also an exogenous hydroxylase and aromatic L- decarboxylase. compound found in foods such as cheese and wine. In addition to this pathway, however, the formation of Our results suggest that dopamine is formed from , including dopamine, from trace amines endogenous andWor exogenous tyramine by this CYP2D such as tyramine by hepatic microsomes has been isoform. We have demonstrated that only CYP2D6 has demonstrated.65) Tyramine, an endogenous compound strong activity for the formation of dopamine from which exists in the brain as a trace amine, is a neuro- p-tyramine, and that the formation of transmitter and a precursor of norepinephrine and and epinephrine from p- and p-, epinephrine (Fig. 4). We investigated the formation of respectively, is catalyzed by CYP2D6 (Fig. 4).65) dopamine from trace amines, using human hepatic The fact that CYP2D6 is expressed not only in liver microsomes and human cytochrome P450 isoforms but also in brain and the clinical association of this expressed in yeast. Among the 11 isoforms of human cytochrome with Parkinson's disease suggests the p337 p.7 [100%]

CYP2D in the Brain 343

Table 6. Summary for endogenous substances metabolized by CYP2Ds

Category Substance Reaction Reference

Amine Neurotransmitter Tryptophol formation (66) Neurotransmitter Tyramine Dopamine formation (65) Steroid Sex hormone 17a-Estradiol C2-hydroxylation (87) Sex hormone 18a-Estradiol C4-hydroxylation (87) Sex hormone Pregnenolone C21-hydroxylation (80) Sex hormone Progesterone C6-hydroxylation (79), (80) Sex hormone Progesterone C16-hydroxylation (79), (80) Sex hormone Progesterone C6-hydroxylation (80) Sex hormone Progesterone C2-hydroxylation (79) Sex hormone Progesterone C21-hydroxylation (79) Sex hormone 17b-Hydroxyprogesterone C21-hydroxylation Sex hormone Allopregnanolone C21-hydroxylation Sex hormone 5-Dihydroprogesterone C21-hydroxylation

Fig. 4. synthesis pathways. TyrOH, ; PheOH, hydroxylase; AADC, aromatic L-amino acid decarbosylase; DBH, dopamine b-hydroxylase; PNMT, N-.

existence of some endogenous substrate, and among the endogenous 2-phenylethylamine, octopamine, syn- which one or more could be metabo- ephrine, 3-methoxy-p-tyramine, 4-methoxy-m-tyra- lized by CYP2D6. Martinez et al.66) explored such a mine, metanephrine, and normetanephrine, nor the possibility by studying the modulation of CYP2D6 indolethylamines tryptamine, serotonin, 6-methoxy- activity by several neurotransinitters. Their ˆndings tryptamine, and melatonin, nor the b-carbolines conˆrmed a competitive inhibition of dextromethor- harman, norharman and tryptoline. However, the phan O-demethylation in the presence of tryptamine, indolethylamines 5-methoxy-N,N-dimethyltryptamine

with a Ki value of 44.6 mM. Tryptamine was metabo- (5-MDMT) and (6-methoxy-1,2,3,4-tetra- lized in human liver microsomes by an enzyme activity hydro-b-carboline) showed relatively high a‹nity for

with a Km of 3.6±0.9 mM. Such activity is NADPH- CYP2D6 in a spectral binding assay and were O- dependent and is inhibited by and CYP2D6- demethylated only by CYP2D6 in a panel of 15 recom- speciˆc substrates. The product of the reaction is binant common human P450s. Pinoline and 5-MDMT tryptophol. These results suggest that tryptamine may O- activities were 35- and 11-fold greater in be an endogenous substrate of CYP2D6. liver microsomes from CYP2D6-humanized mice, Endogenous substrates for CYP2D6 were screened respectively, than liver microsomes from control mice. by Yu et al.67) CYP2D6 does not signiˆcantly metabolize Moreover, the increased activities were completely p337 p.8 [100%]

344 Yoshihiko FUNAE, et al.

inhibited by an anti-CYP2D6 monoclonal antibody. Table 7. Progesterone hydroxylation activities of CYP2D isoforms These two substrates can be added to 5-methoxytrypta- Rat mine, a metabolite and precursor of melatonin, which Metabolites Human: 2D6 was reported to be a speciˆc and high-turnover en- 2D1 2D2 2D3 2D4 dogenous CYP2D6 substrate. CYP2D6 is therefore a 2b-OH 0.03±0.01 ND ND ND 1.59±0.49 relatively highly speciˆc, high-a‹nity, high-capacity 6b-OH 0.29±0.16 0.16±0.08 ND ND ND 5-methoxyindolethylamine O-demethylase. The poly- 16a-OH 0.08±0.04 0.04±0.02 ND ND ND morphic cytochrome CYP2D6 may therefore exert 21-OH 0.18±0.09 ND ND ND 1.06±0.17 an in‰uence on mood and behavior through the Substrate progesterone (25 mM). Activities are expressed as nanomoles O-demethylation of these 5-methoxyindolethylamines per minWnmol P450. Values are expressed as the mean±SD of experi- found in the brain and pineal gland. These processes ments performed in triplicate. ND, Not detected. may also impact on mental and neurological health. The ˆndings may open new vistas for the determination of CYP2D6 . speciˆcally to CYP2D1 and CYP2D6 among eight forms of rat CYPs and ten forms of human CYPs.74) EŠects of antagonist of dopamine and serotonin Additionally, H receptor antagonists, includ- transporter on CYP2D6 1 ing , and mequitazine, The dopamine uptake sites in the brain have been inhibit CYP2D6-mediated-bufuralol 1?-hydroxylation studied in binding experiments with several tritiated in human liver microsomes, and CYP2D6 is the prin- ligands, including 3H- and 3H-madindol,68,69) and cipal CYP responsible for the metabolism of prometha- competition binding experiments with the dopamine zine.75) It has been shown that a partial amino acid transporter, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl- sequence of binding protein, which is quite

2-propyl) (GBR-12935), reveal the presence diŠerent from the , agrees with of multiple binding protein populations in the brain. that of rat CYP2D isoforms, though the molecular One of these sites displays high a‹nity for dopamine weight of mepyramine binding protein diŠered.76) uptake inhibitors such as and cocaine, and Therefore, we have investigated the immunochemical corresponds to the . The other site homology between CYP2D1 and mepyramine binding displays rather high a‹nity for piperazine derivatives, protein by Western blotting, using speciˆc antibodies and has been termed the piperazine acceptor site.70) An against CYP2D1. The speciˆc antibodies against inhibition study with CYP2D inhibitors and Western CYP2D6 fully recognize mepyramine binding protein, blotting and immunoprecipitation analysis with suggesting similarity between CYP2D isoforms and CYP2D1 antibody suggested that the piperazine accep- mepyramine binding protein.74) torsiteisCYP2D.19) Recently, we have reported that Steroid GBR-12935 has the highest binding activity to CYP2D6 among ten forms of human CYPs, and that the Steroid compounds have been reported to be biosyn- GBR-12935 binding is reduced by CYP2D inhibitors, thesized in the central nervous system, independently of such as and quinidine, and typical substrates for their biosynthesis in peripheral steroidogenic tissues. CYP2D, including propranolol, bufuralol, imipramine, Steroid compounds that are biosynthesized in the cen- and , whereas the binding sites are insensi- tral nervous system and have neural eŠects are referred tive to dopamine.71) Thus, we demonstrate that the to as neurosteroids. Progesterone is a with CYP2D isoform is one of the GBR-12935 binding sites the ability to increase myelin-speciˆc protein levels77) that is insensitive to dopamine. and to enhance g-aminobutyric acid-induced chloride current.78) Progesterone was metabolized by CYP2D Inhibition of CYP2D by CNS-active drugs isoforms in an isoform-dependent manner (Table 7). Selective serotonin re-uptake inhibitors (SSRI), CYP2D isoforms could catalyze hydroxylation of including ‰uoxetine and , inhibit CYP2D6 progesterone at position C2b,C6b,C16a and C21. activity in human liver microsomes.48) In addition, Among rat CYP2D isoforms, CYP2D1 and CYP2D4 is a moderate inhibitor.72) In particular, showed a pole apart substrate recognition, that is, is reported to be a mechanism-based CYP2D1 possessed C6b-andC16a-hydroxylation 73) inhibitor of CYP2D6. Histamine H1 receptor an- activities whereas CYP2D4 possessed C2b-andC21- tagonists, such as mepyramine, , hydroxylation activities. Rat CYP2D2 and CYP2D3 chlorpheniramine, and , competitively showed no catalytic activity toward progesterone. As inhibit CYP2D-mediated lidocaine 3-hydroxylation in for human CYP2D6, it exhibited all four hydroxylation rat liver microsomes and debrisoquine 4-hydroxylation activities. Among these activities, CYP2D4-mediated in human liver microsomes, and mepyramine binds progesterone C2b- and C21-hydroxylation activities p337 p.9 [100%]

CYP2D in the Brain 345

Fig. 5. Synthetic pathways of neurosteroids.

80) were the strongest, and the Km values of CYP2D4 for lone, 5a-dihydroprogesterone and allopregnanolone. these were considered to be su‹cient for Allopregnanolone promptly decreased brain excitabil-

in vivo reactions (the Km values of CYP2D4 for C2b- ity, acting as a potent allosteric modulator of g- and C21-hydroxylation were 17.7 mMand16.6mM, aminobutyric acid action.81) Consequently, it has poten- respectively.)79) These results support the view that tial value in the treatment of various neuropsychiatric CYP2D isoforms in the brain positively regulate the disorders.82) Several recent studies have pointed to levels of neurosteroids such as progesterone. commonly used SSRIs as potential modulators of Furthermore, the fact that rat CYP2D4 and human allopregnanolone synthesis in the brain. , a CYP2D6, not rat CYP2D1, 2D2 or 2D3, possess representative SSRI, increased the level of allopreg- progesterone 21-hydroxylation activity leads us to nanolone in the brain of rats83) and humans.84) However, speculate that these brain-predominant CYP2D the mechanism of its eŠect is poorly understood, isoforms function as steroid 21-hydroxylases in the although ‰uoxetine is a potent CYP2D inhibitor.85) It is brain. In general, steroid 21-hydroxylation is mediated explicable that brain CYP2D-mediated regulation of the by P450 21-hydroxylase (P450c21) and is an obligatory allopregnanolone level in the brain may be interfered conversion for the biosynthesis of the main adrenal with by ‰uoxetine, resulting in an increase of the steroids, aldosterone, cortisol and corticosterone. In allopregnanolone level in the brain. adrenal gland, P450c21 converts progesterone and Consequently, rat CYP2D4 and human CYP2D6 are 17a-hydroxyprogesterone to 11-deoxycorticosterone considered to be involved in the regulation of neu- and 11-deoxycortisol, respectively. However, there is no rosteroids such as progesterone and allopregnanolone published evidence concerning de novo synthesis of (Fig. 5). Furthermore, the interactions of xenobiotics adrenal steroids by P450c21 in the brain. Indeed, with CYP2D-mediated regulation of endogenous CYP2D4 and CYP2D6 in the brain showed 21-hydroxy- substances may produce central eŠects since many lation not only for progesterone79) but also for 17a- central acting drugs are metabolized by CYP2Ds. hydroxyprogesterone, indicating that the brain CYP2D Induction of CYP2D by CNS-active drugs isoforms work as steroid 21-hydroxylases, instead of P450c21, although the physiological relevance of the A neuroleptic drug , as well as toluene, potential for 21-hydroxylated adrenal steroid produc- induces CYP2D4 in rat brain, whereas no induction of

tion in the brain is as yet unknown. In addition to CYP2D4 is observed with the dopamine D2 receptor that of progesterone and 17a-hydroxyprogesterone, blockers, , , and sulpiride or CYP2D4 could catalyze 21-hydroxylation of pregneno- with the serotonin receptor blocker mianserin.86) p337 p.10 [100%]

346 Yoshihiko FUNAE, et al.

Twenty-four hours after a single dose of the neuroleptic 3) Sakamoto, K., Kirita, S., Baba, T., Nakamura, Y., drug clozapine, CYP2D4 immunoreactivity, which was Yamazoe, Y., Kato, R., Takanaka, A. and Matsubara, barely detectable in the brains of untreated rats, was T.: A new cytochrome P450 form belonging to the clearly evident in neurons of the pars CYP2D in dog liver microsomes: puriˆcation, cDNA compacta, ventral tegmental area, granular neurons of cloning, and enzyme characterization. Arch. Biochem. Biophys., 319: 372–382 (1995). the olfactory bulb, and Purkinje and granular neurons 4)Imaoka,S.,Yamada,T.,Hiroi,T.,Hayashi,K., of the cerebellum. Induction was maintained with daily Sakaki, T., Yabusaki, Y. and Funae, Y.: Multiple forms administration for 3 weeks. The mRNA for CYP2D4 of human P450 expressed in Saccharomyces cerevisiae. was detected by Northern blotting and localized by Systematic characterization and comparison with those in situ hybridization in neurons throughout the brain of the rat. Biochem. Pharmacol., 51: 1041–1050 (1996). and in the Bergman glia in the cerebellum. There were 5) Shimada, T., Yamazaki, H., Mimura, M., Inui, Y. and no detectable changes in the distribution or quantity of Guengerich, F. P.: Interindividual variations in human CYP2D4 mRNA after treatment with clozapine. The liver cytochrome P-450 enzymes involved in the oxida- overall P450 content of the brain increased with daily tion of drugs, carcinogens and toxic chemicals: studies administration to 7-fold after 3 weeks of clozapine with liver microsomes of 30 Japanese and 30 Caucasians. treatment. A clozapine-like induction of CYP2D4 was J. Pharmacol. Exp. Ther., 270: 414–423 (1994). obtained on administration of toluene to rats. The 6) Guengerich,F.P.,Miller,G.P.,Hanna,I.H.,Sato,H. and Martin, M. V.: Oxidation of methoxyphenethyla- speciˆcity of the induction of CYP2D4 in the brain with mines by cytochrome P450 2D6. Analysis of rate-limit- respect to both the drugs that induce it and the cells in ing steps. J. Biol. Chem., 277: 33711–33719 (2002). which it is induced suggests that the induction of this en- 7) Evans, W. E. and Relling, M. V.: : zyme could be involved in the therapeutic action of translating functional genomics into rational therapeu- clozapine. The similarity of induction of CYP2D4 elicit- tics. Science, 286: 487–491 (1999). ed by clozapine and by the neuro-toxine toluene sug- 8) Zanger, U. M., Fischer, J., Raimundo, S., Stuven, T., gests that more information is needed before a beneˆcial Evert, B. O., Schwab, M. and Eichelbaum, M.: Compre- or toxicological role can be assigned to this isozyme. hensive analysis of the genetic factors determining expression and function of hepatic CYP2D6. Phar- Conclusion macogenetics, 11: 573–585 (2001). 9) Miksys,S.,Rao,Y.,HoŠmann,E.,Mash,D.C.and Four diŠerent CYP2D isoforms are present in the Tyndale, R. F.: Regional and cellular expression of rats. CYP2D2 eŠectively metabolizes drugs in the liver CYP2D6 in human brain: higher levels in alcoholics. J. and CYP2D4 is eŠective in the brain. Catalytic proper- Neurochem., 82: 1376–1387 (2002). ties of CYP2D2 are a great deal diŠerent from those of 10) Siegle,I.,Fritz,P.,Eckhardt,K.,Zanger,U.M.and CYP2D4;CYP2D4 eŠectively catalyzes neurosteroids Eichelbaum, M.: Cellular localization and regional such as progesterone, and so on, whereas CYP2D2 distribution of CYP2D6 mRNA and protein expression doesn't catalyze them. In the brain, the constitutive in human brain. Pharmacogenetics, 11: 237–245 (2001). function of CYP2D4 is considered to oxidize physiolog- 11) Thum, T. and Borlak, J.: in distinct ically important compounds, such as neurosteroids and regions of the heart. Lancet, 355: 979–983 (2000). amines. It is very interesting that human CYP2D6 12) Mahgoub,A.,Idle,J.R.,Dring,L.G.,Lancaster,R. possesses all catalytic properties that CYP2D1, 2D2, and Smith, R. L.: Polymorphic hydroxylation of Debrisoquine in man. Lancet, 2: 584–586 (1977). 2D3, and 2D4 have. Functions of CYP2D4 in the brain 13) Heim, M. and Meyer, U. A.: Genotyping of poor should be elucidated more for the study of the metabolisers of debrisoquine by -speciˆc PCR pathophysiology of brains. ampliˆcation. Lancet, 336: 529–532 (1990). References 14) Bertilsson, L., Dahl, M. L., Ekqvist, B., and Llerena, A.: Genetic regulation of the disposition of psychotropic 1) Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. drugs. In: Meltzer, H. Y., Nerozzi, D., ed. Current prac- A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, tices and future developements in the pharmacotherapy J.,Gelboin,H.V.andHardwick,J.P.:Debrisoquine of mental disorders. 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