Polymorphic cytochrome P450 2D6: humanized mouse model and endogenous substrates.

Cytochrome P450 2D6 (CYP2D6) is the first well‐characterized polymorphic phase I drug‐metabolizing enzyme, and more than 80 allelic variants have been identified for the CYP2D6 gene, located on human chromosome 22q13.1. Human debrisoquine and sparteine metabolism is subdivided into two principal phenotypes—extensive metabolizer and poor metabolizer—that arise from variant CYP2D6 genotypes. It has been estimated that CYP2D6 is involved in the metabolism and disposition of more than 20% of prescribed drugs, and most of them act in the central nervous system or on the heart. These drug substrates are characterized as organic bases containing one nitrogen atom with a distance about 5, 7, or 10 Å from the oxidation site. Aspartic acid 301 and glutamic acid 216 were determined as the key acidic residues for substrate‐enzyme binding through electrostatic interactions. CYP2D6 transgenic mice, generated using a lambda phage clone containing the complete wild‐type CYP2D6 gene, exhibits enhanced metabolism and disposition of debrisoquine. This transgenic mouse line and its wild‐type control are models for human extensive metabolizers and poor metabolizers, respectively, and would have broad application in the study of CYP2D6 polymorphism in drug discovery and development, and in clinical practice toward individualized drug therapy. Endogenous 5‐methoxyindolethylamines derived from 5‐hydroxytryptamine were identified as high‐affinity substrates of CYP2D6 that catalyzes their O‐demethylations with high enzymatic capacity and specificity. Thus, polymorphic CYP2D6 may play an important role in the interconversions of these psychoactive tryptamines, including a crucial step in a serotonin‐melatonin cycle.

Poor metabolizer (PM) and extensive metabolizer (EM) are generally recognized as the two major CYP2D6 phenotypes (Eichelbaum, 1982;Evans et al., 1980;Schmid et al., 1985). As new information became available, the ultrarapid metabolizer (UM) and intermediate metabolizer (IM) subgroups were classified to yield a range of phenotypes with modestly decreased and increased activity, respectively (Bathum et al., 1998;Dahl et al., 1995;Daly, 1995;Raimundo et al., 2000). The incidence of CYP2D6 PM was investigated extensively in different ethnic populations containing small to large numbers of subjects. One study (Bertilsson et al., 1992) examined 1011 Swedish Caucasians and 695 Chinese and found that debrisoquine PMs occur among 6.28% of the Swedish Caucasian population and only 1.01% of the Chinese (Fig. 1). This finding is similar to results reported for European and American Caucasians (Alvan et al., 1990;Droll et al., 1998;Llerena et al., 1993;Marez et al., 1997;Nakamura et al., 1985;Sachse et al., 1997), and Japanese and Korean Orientals (Horai et al., 1989;Nakamura et al., 1985;Sohn et al., 1991) performed before and after that study. Moreover, debrisoquine hydroxylation in Asian EMs is slower than Caucasian EMs, as judged by the population mean of the metabolic ratio (MR; % dose excreted as debrisoquine/% dose excreted as 4-hydroxydebrisoquine). Most Caucasian EMs have an MR less than 1.0, whereas most Chinese EMs have an MR value of more than 1.0. As Figure 1. Shown is the distribution of urinary debrisoquine/4-hydroxydebrisoquine metabolic ratio (MR) in 695 Chinese and 1011 Swedish healthy subjects. The arrows indicate MR value of 12.6, an antimode between extensive metabolizers (EMs) and poor metabolizers. A line is drawn at MR = 1.0. Most Chinese EMs have MR > 1.0, whereas Caucasian EMs have MR < 1.0. This figure was reprinted from Clinical Pharmacology & Therapeutics, 51(4), 1992. Pronounced differences between native Chinese and Swedish populations in the polymorphic hydroxylations of debrisoquin and S-mephenytoin, 388 -397, (1992) with permission from Elsevier. ORDER REPRINTS shown in Fig. 1, urinary debrisoquine MR distribution is shifted to the right in Chinese EMs compared with Caucasian EMs.
More recently, the molecular basis of the CYP2D6 polymorphism has been intensively studied. The CYP2D6 gene exhibits more than 80 allelic variations among different ethnic populations (http://www.imm.ki.se/CYPalleles/cyp2d6.htm). The recessive PM phenotype occurs among individuals carrying two null CYP2D6 alleles, arising from a broad range of DNA sequence variations, from single nucleotide substitution to deletion of the complete gene. This may result in a CYP2D6 protein that is unable to bind the substrate; a truncated protein unable to bind heme and, therefore, unable to produce recognizable P450 enzymatic activity; or simply no CYP2D6 protein at all (Haining and Yu, 2003). Other CYP2D6 alleles contain point mutations resulting in one or more amino acid changes in the proteins compared with wild-type CYP2D6.1, and may lead to slightly decreased or increased activity . Generally, the CYP2D6 polymorphism stratifies the population, depending on the copy number of wild-type alleles: PM, zero; IM, one; EM, two; and UM, multiple copies (Corchero et al., 2001;Gonzalez, 1996).

PHENOTYPE AND GENOTYPE
PMs lacking CYP2D6 activity are believed to be physiologically normal, although no comprehensive investigation has ever been carried out. However, the CYP2D6 polymorphism is expected to influence the therapeutic efficacy and adverse drug reactions of common drugs such as b-blockers, selective serotonin reuptake inhibitors (SSRIs), and tricyclic antidepressants during clinical practice (Bertilsson et al., 2002;Gonzalez and Idle, 1994;Ingelman-Sundberg et al., 1999;Kroemer and Eichelbaum, 1995;Wolf and Smith, 1999;Wolf et al., 2000). For drug substrates with narrow therapeutic windows, serious consequences may result. Indeed, with fluoxetine (Prozac), a known substrate and inhibitor of CYP2D6, several phenotype-related fatality cases have been documented (Kincaid et al., 1990;Sallee et al., 2000). Nevertheless, it is not known whether these toxic events were related to drug metabolism. With the benefits of well-established phenotyping and rapidly developing genotyping methodologies, polymorphism information can be obtained and included in the patient's medical records. Here, it could be used to perform individualized drug therapy by adjusting the dose or selecting an alternative drug, which might reduce the incidence of similar adverse events (Bertilsson et al., 2002;Idle and Smith, 1995;Ingelman-Sundberg et al., 1999).
The UM phenotype, defined as subjects with debrisoquine MR less than 0.20 (Dahl et al., 1995) or sparteine MR less than 0.15 (Bathum et al., 1998), is reported to be present at relatively high frequency among Saudi Arabians (20%) (McLellan et al., 1997) and Ethiopians (29%) (Aklillu et al., 1996). This group of CYP2D6 phenotype can be explained by the occurrence of multiple copies of active CYP2D6 alleles, and enhanced expression of stable and active protein among these populations (Aklillu et al., 1996;Dahl et al., 1995;Johansson et al., 1993). More recently, CYP2D6*35 (Lovlie et al., 2001) was identified in Caucasian UMs without a CYP2D6 gene duplication (duplication negative) at significantly higher frequency than control EMs. However, in vitro functional analysis revealed that the enzymatic activity of its resulting allelic isoform CYP2D6.35 is comparable with the wild-type CYP2D6.1 isoform Table 1. Drug substrates and their metabolic pathways catalyzed by CYP2D6 and selected CYP2D6 inhibitors.
Various drugs of abuse are known as substrates (e.g., codeine, dextromethorphan, hydrocodone) or inhibitors [e.g., (-)-cocaine, pentazocine] of CYP2D6. Recreational drugs such as 3,4-methylenedioxymethamphetamine (''Ecstasy''), amphetamine, and methamphetamine are also oxidized by polymorphic CYP2D6. The metabolism and disposition, pharmacokinetics, and pharmacodynamics for some of these substrate drugs of abuse would be expected to vary among people due to CYP2D6 polymorphism. For other drugs, CYP2D6 may not contribute significantly to their overall disposition, but may catalyze the formation of highly active metabolites, such as codeine to morphine, hydrocodone to hydromorphone, and oxycodone to oxymorphone, and thus impact largely on their efficacy. In drug abuse, the CYP2D6 polymorphism is believed to play an important protective role as well as being a risk factor (Sellers and Tyndale, 2000;Sellers et al., 1997).
These known CYP2D6 drug substrates and inhibitors are characterized as organic bases containing at least one nitrogen atom serving as an electron donor. The oxidation site, about 5 or 7 Å from the basic nitrogen, possesses a flat hydrophobic area close to it (de Groot et al., 1997;Koymans et al., 1992;Strobl et al., 1993). However, the distance between the basic nitrogen and reaction site is around 10 Å in a few of the substrates (de Groot et al., 1999a,b). Site-directed mutagenesis and molecular modeling revealed that the basic nitrogen atoms in the substrates can interact with the negatively charged carboxyl group of aspartic acid 301 and glutamic acid (de Groot et al., 1999a,b;Ellis et al., 1995;Guengerich et al., 2003;Paine et al., 2003). Thus, it is likely that both of these acidic amino acids are key residues for CYP2D6-substrate binding through electrostatic interactions. Besides, CYP2D6 may provide more than one binding orientation or site of metabolism for the same substrate (Yu et al., 2001. Like other P450-catalyzed oxidations, most of the reactions mediated by CYP2D6 are aliphatic/aromatic hydroxylations and O-demethylation (Table 1). However, some drug (and other chemical) substrates are N-demethylated by CYP2D6, which was initially seen as an atypical and rare metabolic pathway, and is now a generally accepted pathway (Coutts et al., 1994;de Groot et al., 1999a) as more and more chemicals have been shown to undergo N-demethylation. Dextromethorphan, the widely used probe drug both in vitro and in vivo, was both O-and N-demethylated by highly purified and well-characterized CYP2D6 isoforms (Ramamoorthy et al., 2002;Yu and Haining, 2001a,b;Yu et al., 2001). A combined protein and pharmacophore model has also been generated for CYP2D6 in order to elucidate all these reactions including N-demethylation (de Groot et al., 1999a,b), which would provide helpful information for the research on drug metabolism and drug -drug interactions.

SUSCEPTIBILITY TO DISEASE
It is reasoned that the mutations and polymorphism of P450 genes might lead to altered individual risk of disease because these enzymes are responsible for the biosynthesis and biodegradation of physiological compounds, as well as the metabolism and disposition of environmental chemicals (Gonzalez and Idle, 1994;Guengerich, 2003;Huber et al., 2002;Ingelman-Sundberg, 2001). It is also known that few common diseases are monogenetic in origin; many diseases are caused by multiple factors such as multiple genes, diet, exposure to environmental factors, or a combination of these. Therefore, caution must be exercised before drawing a conclusion about the genetic determination of a certain disease.
More and more evidence has accumulated during the past decades, in support of the association of P450 genes with diseases (Guengerich, 2003;Huber et al., 2002;Ingelman-Sundberg, 2001). For examples, CYP1B1 has been identified as a major genetic determinant of primary congenital glaucoma, besides the risk for developing prostate, ovarian, lung, and breast cancer. This has been confirmed by analysis of the CYP1B1-null mouse model (Libby et al., 2003). CYP19, also named aromatase, which produces estrogen from androgen, is associated with the risk of breast cancer ORDER REPRINTS (Huber et al., 2002). Deficiency of CYP27, which encodes the mitochondrial sterol 27hydroxylase playing a key role in bile acid biosynthesis, causes cerebrotendinous xanthomatosis, an autosomal recessive sterol storage disease characterized by the accumulation of a bile alcohol in diverse tissues. Almost all these associations can be bridged through a defect in biotransformation of endogenous compounds or activation of exogenous chemicals.
Numerous studies have been reported with the intention to link specific disease to polymorphic CYP2D6, for which exist large numbers of allelic variants with high frequencies, significant interethnic differences, and multiple drugs and chemical neurotoxin substrates. Those examined have included PD, Alzheimer's disease, and various types of cancer (Gonzalez and Idle, 1994). However, the results obtained from these association studies have been inconsistent, even with the determination of specific null alleles by genotyping. For the susceptibility to PD, CYP2D6 has been the most extensively examined candidate gene, probably evoked by its metabolism of MPTP that causes immediate dopaminergic neuronal damage and irreversible Parkinsonism. MPTP is activated to neurotoxic MPP + by monoamine oxidase B, whereas it is detoxicated by N-demethylation, largely by CYP2D6. Thus, there have been many commentaries predicting a protective role for polymorphic CYP2D6 in MPTP-induced PD. The variable results of these studies on the association between CYP2D6 genotype and PD may be attributed to many of the studies employing only small numbers of patients. Thus, a metaanalysis of 11 studies was carried out and showed a small, yet significant (P = 0.01) odds ratio (1.47) for the association between the poor metabolizer genotypes and PD (McCann et al., 1997). However, a study (Payami et al., 2001) containing 566 PD patients and 247 control subjects, using standard diagnostic and genotyping techniques, revealed that the CYP2D6*4 allele, which is the most common variant among CYP2D6 PMs, is not associated with earlier PD onset. On the contrary, apolipoprotein E has been consistently identified to be associated with onset age of PD (Kruger et al., 1999;Maraganore et al., 2000;Zareparsi et al., 1997) and is so far the only recognized susceptibility gene. Although the causes of the common forms of PD are still unknown, it would be helpful to examine the major risk factors together, including candidate genes, age, family history, and environmental exposure markers. Chemicals such as b-carboline alkaloids contained in the diet or formed from its components are known for their neurotoxicity and induction of PD similarly to MPTP. CYP2D6 has been shown to be involved in their metabolism as well as CYP1A2 (Yu et al., 2003d).

HUMANIZED MOUSE MODEL FOR CYP2D6 POLYMORPHISM
Clinical studies are fundamental to the identification of human pharmacogenetic polymorphisms, and for the establishment of pharmacokinetic profiles and drug -drug interaction effects. However, to determine how a drug is metabolized, what toxic effects it might produce, or how pathophysiological conditions affect drug metabolism at early stages of drug development, animal models or in vitro systems must be developed. Due to marked differences between humans and experimental animals, the results from animal studies can be misleading and need to be interpreted cautiously. The CYP2D family in humans has a single active member CYP2D6 that is highly polymorphic, whereas rats and mice have at least five genes (Gonzalez and Nebert, 1990;Nelson et al., 1996). Debrisoquine is hydroxylated to 4-hydroxydebrisoquine by humans and by Sprague-Dawley rats. However, female Dark Agouti (DA) rats have been found to possess a low capacity to metabolize debrisoquine (Al-Dabbagh et al., 1981). Similarly, no significant formation of 4-hydroxydebrisoquine was detected by liver microsomes from three strains of mice and by purified CYP2D9-11 (Masubuchi et al., 1997). Although the female DA rat was proposed early on as a model for the human PM phenotype, in which to evaluate the role of the debrisoquine 4hydroxylation polymorphism in drug and chemical toxicity (Al-Dabbagh et al., 1981). Employing two inbred strains of rat as models for two human phenotypes was soon recognized as having practical limitations. For example, using DA (PM) and Lewis (EM) female rats, it was proposed that the reduced hepatotoxicity of aflatoxin B 1 (AFB1) in the DA rat was due to its relative inability to activate metabolically AFB1 (Hietanen et al., 1986;Ritchie and Idle, 1982). Subsequently, it emerged that DA rats Hybridization signals were present only in Tg-CYP2D6 mice, and their sizes were as expected from the CYP2D6 sequence. C, PCR genotyping of wild-type and Tg-CYP2D6 mice. Tail DNA was amplified with mEH [internal polymerase chain reaction (PCR) control] and CYP2D6 gene-specific primers. The PCR products (341 bp for mEH, 241 bp for CYP2D6) were separated on a 1.5% agarose gel. D, Western blot analysis of CYP2D6 protein expression in wild-type and Tg-CYP2D6 mice. Liver (L), intestine (I), and kidney (K) microsomal proteins (40 mg) were separated by sodium dodecyl sulfate polyacrylamide gel electophoresis (SDSPAGE) and transferred to a nitrocellulose membrane. A CYP2D6-specific monoclonal antibody (Krausz et al., 1997) was used to assess CYP2D6 protein expression. The antibody only reacted against CYP2D6-expressed protein, but did not recognize any of the mouse CYP2D proteins. Human liver microsomes (HLM) was used as a control. had the highest microsomal epoxide hydrolase activity of 22 rat strains tested (Oesch et al., 1983), and this would appear to be the best explanation of the observed interstrain difference in AFB1 activation and hepatotoxicity, rapid metabolic clearance of the procarcinogenic AFB1 exo-8,9-epoxide. Thus, inbred strains, with their manifold genetic and biochemical differences, are imperfect models for the investigation of the biological consequences of human single polymorphisms.
To circumvent all these problems, a transgenic mouse line expressing CYP2D6 would offer a unique approach to answering fundamental questions about the specific role of CYP2D6 in drug metabolism and drug interactions. Such experiments would be performed in the context of the entire animal, and overcome many limitations inherent in in vitro experiments. To this end, the complete wild-type allele of the human CYP2D6 gene (Fig. 2), including its regulatory sequence, was microinjected into a fertilized FVB/N mouse egg, and a CYP2D6 transgenic (Tg-CYP2D6) mouse line has been produced (Corchero et al., 2001). Tg-CYP2D6 mouse carries 5 ± 1 copies of CYP2D6 transgene per haploid genome. Active CYP2D6 enzyme is expressed in liver, intestine, and kidney of Tg-CYP2D6 mice (Fig. 2), which was confirmed with a specific monoclonal antibody (Krausz et al., 1997). Metabolism and disposition of debrisoquine in Tg-CYP2D6 mice is enhanced compared with control wild-type mice. After a single oral dose of debrisoquine (2.5 mg/kg), both Tg-CYP2D6 heterozygous and homozygous mice had debrisoquine serum levels significantly lower than in wild-type (Fig. 3A). Consistently, 4hydroxydebrisoquine levels are highest in Tg-CYP2D6 homozygous, intermediate in Tg-CYP2D6 heterozygous, and lowest in the wild-type (Fig. 3B). Pharmacokinetic analysis showed that the debrisoquine AUC is about three-fold and six-fold higher in wild-type mice than in heterozygous, and homozygous Tg-CYP2D6 mice, respectively (Table 1). This is illustrated by differences in the elimination half-life of debrisoquine, which is 2.1 and 1.4 times shorter in the heterozygous and homozygous Tg-CYP2D6 mice than in wild-type mice. Accordingly, Tg-CYP2D6 mice showed a clearance about six-and three-fold higher than wild-type mice (Corchero et al., 2001).
CYP2D6 integration in the mouse genome does not affect any other physiological parameters such as renal function. Twenty-four hours after a single oral dose of debrisoquine, Tg-CYP2D6 mice excreted significantly higher amounts of 4-hydroxydebrisoquine (28.9 ± 12.5% of dose) and lower amounts of debrisoquine (14.6 ± 6.4%) than the wild-type mice (6.2 ± 3.1% and 61.0 ± 9.0%, respectively). Urinary MR of debrisoquine for the wild-type mice was 12.1 ± 7.3%, which was decreased to 0.5 ± 0% with expression of the human transgene. Total recoveries of debrisoquine plus 4-hydroxydebrisoquine were 67.2 ± 10.7% and 43.5 ± 18.9% for the wild-type and Tg-CYP2D6 mice, respectively (Corchero et al., 2001). This latter finding perhaps indicates that the human CYP2D6 gene may provoke the metabolism of debrisoquine to other metabolites ( Table 2).

CYP2D6 Transgenic Mice and Endogenous Substrates
ORDER REPRINTS debrisoquine 4-hydroxylation activity is significantly decreased more than 50%. With the Tg-CYP2D6 mouse model, it is the first time that CYP2D6 gene has been demonstrated to be regulated by HNF4a in vivo (Corchero et al., 2001). The Tg-CYP2D6 mouse model solves the problems of species differences, and offers a unique in vivo system to study drug metabolism and disposition, pharmacokinetics, and drug -drug interactions for the prediction of the effects of drugs, drug candidates, and environmental chemicals in humans. Moreover, this mouse line can serve as a whole intact animal model for exploring endogenous substrates for CYP2D6, investigating their biotransformations, and elucidating physiological significance and its polymorphism.

ENDOGENOUS SUBSTRATES FOR CYP2D6
Since the discovery of the CYP2D6 polymorphism, there has been speculation about potential physiologically important substrates for CYP2D6 in humans (Kroemer and Eichelbaum, 1995;Llerena et al., 1989Llerena et al., , 1993Nadir et al., 1982). Could the PM have an advantage in development, reproduction, or behavior? The difference in personality between EM and PM individuals reported by Llerena and colleagues (Llerena et al., 1989(Llerena et al., , 1993 suggests that CYP2D6 may be involved in the metabolism of one or more endogenous neuroactive substances. This hypothesis is strongly supported by the expression of CYP2D6 in neurons of the human CNS, which has been demonstrated using a variety of techniques, including immunoblotting (Fonne-Pfister et al., 1987;Miksys et al., 2002;Siegle et al., 2001), in situ hybridization (Gilham et al., 1997;Siegle et al., 2001), reverse transcription-polymerase chain reaction (RT-PCR) (McFayden et al., 1998), and metabolism of the CYP2D6 probe drug dextromethorphan (Voirol et al., 2000) by microsomes prepared from brain tissues. One report localized the expression of CYP2D6 to the pigmented cells of the substantia nigra (Gilham et al., 1997), whereas another detected CYP2D6 mRNA in the neocortex, caudate nucleus, putamen, globus pallidus, hippocampus, thalamus, substantia nigra, and cerebellum (Siegle et al., 2001). CYP2D6 protein, however, was only detected in the large principal neurons in the cortex, hippocampus, and cerebellum (Siegle et al., 2001). If CYP2D6 was associated with the endothelial cells lining the 650 km of blood capillary found in the human brain, then a case could be made that it functioned as part of the blood -brain barrier and its role was as a ''last line of defense,'' preventing toxins from entering the brain, but this does not appear to be the case. Many toxic alkaloids, including MPTP-like b-carbolines, are CYP2D6 substrates (Yu et al., 2003d). However, all studies would appear to show that CYP2D6 within the CNS is neuronal in origin (Gilham et al., 1997;McFayden et al., 1998;Siegle et al., 2001), and this brings into question the function of this enzyme in the CNS. The possibility that CYP2D6 may have endogenous psychoactive substrates in the human brain would link all these evidence together and provide reasonable explanation for these phenomena.
Tryptamine, one of the trace amines found at very low concentrations in the mammalian CNS, but localized in neurons with a very high turnover and short half-life (Jones, 1982), exhibits high affinity to a new family of 15 G protein-coupled receptors recently identified (Borowsky et al., 2001). These receptors, called trace amine (TA) receptors, are distinct from the classical biogenic amine receptors, those for 5-HT, Table 3. 12.1 ± 1.3 (Granvil et al., 2002) p-Tyramine

CONCLUSION
Since the discovery of debrisoquine/sparteine polymorphism in the late 1970s, significant interethnic difference in phenotype frequencies have been reported. Comprehensive studies on CYP2D6 genotypes provided satisfactory molecular explanation for the distribution of phenotypes. Due to the lack of a robust animal model for the study of the CYP2D6 polymorphism, CYP2D6 humanized mice have been generated and validated by molecular methods and debrisoquine phenotyping. This mouse model has been applied to the search for endogenous substrates for CYP2D6, which catalyzes the O-demethylation of a number of psychotropic methoxyindolethylamines. This mouse model could have broad applications for predicting the variation of metabolism and disposition of drugs or drug candidates, in vivo drug -drug interactions, and pharmacokinetics and pharmacodynamics for individualized drug therapy in the human population. This humanized mouse will also permit investigation into the physiological significance of these endogenous substrates of CYP2D6 and its polymorphism.

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