Developmental Changes in Human Liver CYP2D6...

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Developmental Changes in Human Liver CYP2D6 Expression

Jeffrey C. Stevens, Sandra A. Marsh, Matthew J. Zaya, Karen J. Regina, Min Le, Ronald N. Hines

Pfizer Corporation, Global Research and Development, Chesterfield, MO, USA (J.C.S., S.A.M.,

M.J.Z. and K.J.R.) and, Departments of Pediatrics and Pharmacology/Toxicology and Children’s

Research Institute, Medical College of Wisconsin and Children’s Hospital and Health System,

Milwaukee, WI, USA (M.L. and R.N.H.).

DMD Fast Forward. Published on May 12, 2008 as doi:10.1124/dmd.108.021873

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 12, 2008 as DOI: 10.1124/dmd.108.021873

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Running title: CYP2D6 developmental expression

Corresponding author:

Ronald N. Hines, Ph.D.

Medical College of Wisconsin

TBRC/CRI/CPPT

8701 Watertown Plank Rd.

Milwaukee WI 53226

E-mail: [email protected]

Number of words in Abstract: 224

Number of words in Introduction: 719

Number of words in Discussion: 1,114

Text pages: 25

Tables: 3

Figures: 5

References: 38

Abbreviations: EGA, estimated gestational age; PCR, polymerase chain reaction; PNA, postnatal

age; PMI, postmortem interval


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Abstract

Within the human cytochrome P450 family, specific forms show developmental expression

patterns that can affect drug clearance, efficacy and safety. The objective of this study was to

use dextromethorphan O-demethylase activity and quantitative western blotting to identify

CYP2D6 developmental expression patterns in a large (n=222) and developmentally diverse set

of pediatric liver samples. Immunodetectable levels of CYP2D6 protein determined for selected

samples across all age categories showed a significant correlation with the corresponding

dextromethorphan O-demethylase activity. Of gender, ethnicity, postmortem interval, and

genotype, only increasing gestational age was associated with CYP2D6 activity and protein

content in prenatal samples. In contrast, both age and genotype were associated with CYP2D6

expression in postnatal samples. CYP2D6 expression in liver samples from neonates less than 7

days of age was higher than observed in first and second trimester samples, but not significantly

higher than third trimester fetal samples. In contrast, expression in postnatal samples greater

than 7 days of age was substantially higher than any earlier age category. Higher CYP2D6

expression also was observed in liver samples from Caucasians versus African Americans.

Finally, using phenotype categories inferred from genotype, CYP2D6 activity was higher in

postnatal samples predicted to be extensive or intermediate metabolizers versus poor

metabolizers. These results suggest that age and genetic determinants of CYP2D6 expression

constitute significant determinants of interindividual variability in CYP2D6-dependent

metabolism during ontogeny.


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The cytochromes P450 enzyme superfamily is the predominant system responsible for

xenobiotic metabolic clearance in animals, including humans. These enzymes are characterized

by tremendous diversity in substrate specificity, regulation, and expression that results in

significant interindividual variability in metabolism and clinical pharmacological response

(Guengerich, 1997; Wrighton et al., 1992). In adults, differences in cytochrome P450 expression

and attributable metabolism are determined by genetic variability and, to a lesser extent, by

enzyme induction due to drug therapy, xenobiotic exposure or dietary factors (Guengerich et al.,

1997; Lamba et al., 2002; Ozdemir et al., 2000). Understanding factors influencing metabolism

in a pediatric population presents additional challenges as ontogenesis, or the change in enzyme

expression status as a function of age, is superimposed upon any genetic and/or environmental

factors. Where metabolic clearance is defined largely by a single cytochrome P450, this

phenomenon can result in pediatric patients that present as phenotypically similar but actually

have disparate genotypes that have yet to be realized.

Although CYP2D6 accounts for 2% or less of the hepatic cytochrome P450 content

(Shimada et al., 1994), this enzyme is responsible for the oxidative metabolism of about 12% of

clinically relevant drugs (Williams et al., 2004). Furthermore, the CYP2D6 locus exhibits a high

degree of genetic polymorphism that clearly has been linked to the variable pharmacological

response to a variety of analgesic, cardiovascular and anti-depressant drugs (Eichelbaum et al.,

1997; Flores et al., 2001; Heim et al., 1992). For these reasons, this enzyme has been the target

of extensive study. Initial phenotyping studies showed that CYP2D6 activity is bimodally

distributed, with poor metabolizer status attributable to approximately 7% of the Caucasian

population. As the number of identified CYP2D6 allelic variants has increased to over 80 and


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the ethnic diversity of subjects studied has expanded, the difficulty in accurately predicting the

metabolic capacity from genotype has become apparent (Griese et al., 1998; Zanger et al., 2001).

These discrepancies, in combination with the delay in the expression of phenotype during

prenatal and postnatal development, may lead to errors in clinical practice unless the application

of genetic medicine is balanced with a complete understanding of CYP2D6 ontogeny. This

concern is particularly relevant, as pharmaceutical companies have recently increased the scope

of pediatric clinical trials in response to the recognition of the unmet medical need (Milne, 1999)

and the objectives outlined in the Pediatric Exclusivity (Food and Drug Administration, 1998)

and Best Pharmaceuticals for Children (2002) Acts.

Due to ethical and logistical concerns related to conducting in vivo studies in pediatric

patients, relatively few studies have been conducted on changes in CYP2D6 expression during

early development. However, the use of in vitro techniques to define human drug metabolism

enzymology and associated interindividual variability is well established scientifically and

endorsed from a regulatory perspective (Lesko et al., 2000; Peck et al., 1993). Treluyer et al.

(1991) reported on CYP2D6 expression in fetal and postnatal liver samples, but the low number

of samples representing infancy and the lack of ethnic information precluded a more complete

analysis. Other studies have shown evidence of birth-dependent CYP2D6 expression but have

lacked sufficient sample size and/or differentiation of pediatric and adult expression (Shimada et

al., 1996; Tateishi et al., 1997). In addition, the improved limits of analytical detection afforded

by mass spectrometry for enzyme assays and chemiluminescence for cytochrome P450

immunoquantitation should allow for improved definition of the low fetal enzyme activity

compared with historical data (Tateishi et al., 1997; Treluyer et al., 1991). Most recently, Blake

et al. (2007) reported on in vivo CYP2D6 ontogeny as measured by dextromethorphan O-


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demethylation in 193 infants during the first year of life. CYP2D6 activity was detectable in

children by 2 weeks of age and was concordant with genotype. Furthermore, no changes in

CYP2D6 activity were observed over the first year of life. However, the interpretation of these

data is controversial, as Johnson et al. (2007) have argued that modeling these data taking into

account the maturation of renal function during the first year of life would suggest a surge in

CYP2D6 expression over the first year of life.

The objective of this study was to further define CYP2D6 ontogeny by the

characterization of marker dextromethorphan O-demethylase activity, CYP2D6 protein levels,

and CYP2D6 genotype in a large liver tissue set that included both fetal and pediatric liver

samples (Koukouritaki et al., 2002).


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Materials and Methods Materials. Human liver samples were obtained from the Brain and Tissue Bank for

Developmental Disorders, University of Baltimore and University of Miami (National Institute

of Child Health and Human Development, NOI-HD-8-3283 and NOI-HD-8-3284, respectively)

and the Central Laboratory for Embryology Research, University of Washington (National

Institute of Child Health and Human Development, HD-00836) as previously described

(Koukouritaki et al., 2002) and in accordance with ethical guidelines. All samples were stored at

–80°C. A total of 222 liver samples representing ages from 8 weeks gestation to 18 years post-

birth were analyzed. Donor estimated gestational age (EGA), postnatal age (PNA) and cause of

death was available for all samples (Table 1). Samples from individuals succumbing to disease

processes that might have involved liver damage or disease were excluded from the tissue bank.

Information on sex and ethnicity were provided for some samples. Adult pooled human liver

microsomes were purchased from Xenotech (Kansas City, KS). Human cDNA-expressed

CYP2D6 supersomes and peptide-specific monoclonal antibody against CYP2D6 were

purchased from BD Gentest Corp. (Woburn MA) while horseradish peroxidase (HRP)-

conjugated sheep anti-mouse IgG, nitrocellulose membrane and enhanced chemiluminescence

western blotting kits were obtained from GE Healthcare (Princeton NJ). Ten percent pre-cast

polyacrylamide gels were from BioRad (Hercules CA). Dextromethorphan and dextrorphan were

purchased from Sigma Chemical RBI (St. Louis, MO). DNAzol reagent and QIAmp DNA Mini

kits were obtained from Invitrogen (Carlsbad, CA) and Qiagen (Valencia, CA), respectively.

Oligonucleotide primers for both DNA amplification and single-base extension were from

Integrated Technologies, Inc. (Coralville, IA). The ExoSAP-IT mix, containing both

exonuclease I and shrimp alkaline phosphatase, and shrimp alkaline phosphatase alone were


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purchased from United States Biochemical Corp (Cleveland, OH). CEQ SNP-Primer Extension

kits were from Beckman Coulter, Inc. (Fullerton, CA). All other reagents and materials were

obtained from common commercial sources.

Preparation of hepatic microsomal suspensions. Microsomal suspensions from individual

liver samples were prepared and protein concentrations determined as previously described

(Koukouritaki et al., 2002).

Dextromethorphan O-demethylase activity determination. CYP2D6-dependent

dextromethorphan O-demethylation was measured using 25 µg of human liver microsomal

protein, 20 µM dextromethorphan (saturating based on reported kinetic values) and 50 mM

phosphate buffer (pH 7.4) in a total volume of 0.2 mL. Incubations were performed in a 96-well

cluster tube format. Chilled samples were preincubated for 3 minutes at 37ºC prior to the

addition of NADPH to a final concentration of 1 mM. Following 30 minutes of incubation,

reactions were terminated with 50 µL cold acetonitrile. All activity assays were performed on

the same day and in duplicate and assays on each sample were performed a minimum of two

times. Each 96-well format also contained samples with varying amounts of the metabolite

standard, dextrorphan, in addition to incubations of dextromethorphan with pooled human liver

microsomal samples to assess variability across the 96-well format.

Dextrorphan was quantitated by high pressure liquid chromatography using Shimadzu

LC-10AD pumps (Shimadzu, Kyoto, Japan), an HTC PAL autosampler (Leap Technologies,

Canboro, NC) equipped with a narrow bore reverse phase column (Thermo Electron Corp.,

Aquasil C-18 50 x 2.1 mm, 34 µm, West Palm Beach, FL.) and using a step gradient (5% B for

0.5 minute, to 95% B in 1.0 minute, held for 24.5 minutes) at a flow rate of 0.5 mL/min. The

mobile phase consisted of solvent A, water with 0.1% (V/V) formic acid and solvent B,


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acetonitrile (10:90) with 0.1% (V/V) formic acid. The effluent from the high pressure liquid

chromatography was introduced into a Sciex API 4000 triple quadrupole mass spectrometer

(Thornhill, Ontario, Canada) via a Turbo Ionspray source. Mass spectrophotometric analysis

was performed in the positive ion mode using multiple reaction monitoring at unit resolution for

the transition 258 → 157 m/z. Instrument parameters were optimized for the product ion at m/z

157. Needle voltage was 4500 and the collision energy was set to 53 eV with 5 mtorr nitrogen as

the collision gas. The dextrorphan limit of detection was 0.007 pmol. Unknown dextrorphan

concentration amounts were read from a linear regression of known standards (0.12 pmol to1.00

nmol) using 1/x weighting. All unknown samples fell within the range of concentrations used to

prepare the standard curve, the lower limit of which gave a signal 10-fold higher than

background. All replicates were averaged and calculations of activity (nmol/min/mg) were

performed in Excel (Microsoft Corp., Seattle, WA).

CYP2D6 Immunoquantitation. Fetal, pediatric, and adult human liver microsome samples

were analyzed for immunodetectable CYP2D6 essentially as described by Koukouritaki et al.

(2002). Briefly, microsomal protein suspensions (1 µg protein) along with known amounts of

cDNA-expressed CYP2D6 supersomes (2.5 to 40 fmoles CYP2D6 protein) were fractionated by

SDS polyacrylamide gel electrophoresis (Laemmli, 1970) and transferred to nitrocellulose

membranes. After blocking and washing, the membranes were incubated with the primary

MAB-2D6 antibody at 1:5000 dilution and secondary HRP-conjugated sheep anti-mouse IgG at

1:2000 dilution at room temperature for 1 hr. The antibody-bound CYP2D6 protein was detected

by chemiluminescence using ECL kits (GE Healthcare). Following film development, the optical

density of the immunoreactive CYP2D6 bands were visualized using a Kodak DC120 digital

camera and digitized using Digital Science 1D v3 software (New Haven CT). CYP2D6


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concentration in the liver samples was determined by linear regression using the known amounts

of CYP2D6 supersome samples as standards (SigmaPlot V 9.01 and SigmaStat V3.11, SysStat

Software, San Jose, CA). If the amount of CYP2D6 in 1 µg of sample liver microsomes was

outside of the standard curve range, the immunoquantitation was repeated using 10 µg of

microsomal protein.

DNA Isolation and Genotype Analysis. Genomic DNA was isolated from tissue samples either

as described (Koukouritaki et al., 2005) or using the QIAmp DNA Mini kit (Qiagen, Valencia,

CA) per the manufacturer’s instructions. Long range polymerase chain reaction (PCR) DNA

amplification was performed as described by Gaedigk et al. (2002) to ascertain the presence or

absence of CYP2D6 deletion (CYP2D6*5) or duplication alleles. In the absence of a

CYP2D6*5/*5 diplotype, a 5.1 kbp long range PCR amplicon served as the template for

subsequent genotype analysis by multiplexed single-base extension (Koukouritaki et al., 2005;

Lindblad-Toh et al., 2000) to detect the CYP2D6 100C>T, 1023C>T, 1659G>A, 1707delT,

1758G>A, 1846G>A, 2549delA, 2617delAGA, 2850C>T, 3183G>A, and 4180G>C single

nucleotide polymorphisms. To assess the relationship between observed dextromethorphan O-

demethylase activity or CYP2D6 protein content and CYP2D6 genotype, the latter was

determined for 145 of the postnatal liver samples for which whole tissue was available. Based

on the determined diplotypes, individual liver samples were grouped into categories of predicted

extensive, intermediate or poor metabolizer phenotype (Table 2). Although not considered poor

metabolizers by convention, individuals genotyped as having one null and one partially active

allele were included in the poor metabolizer phenotype category.

Statistical analysis of data. Stepwise linear regression was used to evaluate factors contributing

to dextromethorphan O-demethylase activity and CYP2D6 protein content. One-way analysis of


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variance (ANOVA) with the Dunnet’s T3 post hoc test were used to compare the effects of age

and genotype on dextromethorphan O-demethylase activity and/or CYP2D6 protein levels while

comparison of CYP2D6 activities among ethnic groups was performed using the Mann-Whitney

U test. In all instances, a P < 0.05 was accepted as significant. The correlation between

CYP2D6 protein and dextromethorphan O-demethylase activity in the tissue samples was

analyzed by simple regression. All analyses were performed using SPSS version 16.0 (SPSS

Science, Chicago, IL).


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Results

Dextromethorphan O-demethylation has been well documented as a sensitive and

selective CYP2D6 probe (Schmid et al., 1985) and as such, was used along with mass

spectrometry detection to determine CYP2D6 activity in 222 fetal and pediatric liver microsome

samples. As shown in Fig. 1A, activity for first and second trimester fetal samples was either

undetectable (41 of 46 samples) or very low (< 0.01 nmol/min/mg microsomal protein).

However, 10 of 14 samples (71%) from the third trimester fetal group showed measurable

dextromethorphan O-demethylase activity. Following birth, a substantial increase in enzyme

activity was observed compared with the prenatal age group (Fig. 1B) that did not appear to

change further as a function of postnatal age (Fig. 1C).

Given potential issues with donor sample quality due to the degradation of enzymes as a

function of postmortem interval (PMI), immunodetectable levels of CYP2D6 protein were

determined to assess whether measurements of enzyme activity reflected hepatic CYP2D6

protein levels. No correlation was observed between either dextromethorphan O-demethylase

activity or CYP2D6 protein content and the PMI (data not shown), consistent with other studies

using these same tissue samples [e.g., (Koukouritaki et al., 2004; Stevens et al., 2003)]. In

contrast, there was a good correlation between dextromethorphan O-demethylase activity and

CYP2D6 protein in individual tissue samples (Fig. 2) (rs = 0.686, P < 0.01).

In addition to PMI, the relative contribution of age, gender, ethnicity, and genotype on

dextromethorphan O-demethylase activity and CYP2D6 protein levels initially was evaluated.

For the fetal samples, increasing gestational age was the only factor significantly associated with

increasing dextromethorphan O-demethylase activity and CYP2D6 protein (P<0.001 and P =


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0.008, respectively) (Table 3). Among those tissue samples from individuals born prematurely,

increasing postnatal age was associated with increasing CYP2D6 protein content (P = 0.005) but

only marginally associated with increasing dextremethorphan O-demethylase activity (P =

0.086). In contrast, for the postnatal samples, both age and genotype were significantly

associated with increasing dextromethorphan O-demethylase activity (P = 0.018 and P = 0.003,

respectively) while age (P<0.001), genotype (P = 0.009) and ethnicity (P = 0.007) were

associated with increasing CYP2D6 protein (Table 3).

Based on this preliminary analysis and visual inspection of the scatter plots (Fig. 1), both

dextromethorphan O-demethylase activity and CYP2D6 protein content were grouped into one

of four age groups: gestational age <26 weeks (first and second trimester), gestational age 26 to

40 weeks (third trimester), postnatal ages 0 to 7 days, and postnatal ages >7 days to 18 years

(Fig. 3A and B). Because genotype was an important covariate in determining expression in the

postnatal sample set, predicted poor metabolizers (Table 2) were excluded from the analysis of

postnatal samples. Attempts were made to subdivide the >7 days to 18 years group into more

defined age brackets, e.g., >7 days to 28 days, >28 days to 6 months, and > 6 months to 18 years.

However, no statistical differences were observed among these latter categories (data not

shown). Although there is a trend for increased activity and CYP2D6 protein content between

the grouped first and second trimester and third trimester liver samples, there was no statistical

difference between these age groups. There also was no statistical difference in CYP2D6 protein

content or dextromethorphan O-demethylase activity between third trimester liver samples and

tissues from neonates less than 7 days of age. The difference between dextromethorphan O-

demethylate activity and CYP2D6 protein in neonates less than 7 days of age and the activity

observed in first and second trimester fetal samples (10 to 26 weeks gestational age) was


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significant, but not compared to third trimester fetal samples. In contrast, there was a highly

significant difference between both CYP2D6 activity and protein in the postnatal samples >7

days to 18 years of age when compared to either fetal age group or the neonatal 0 to 7 day age

group. The role of ethnicity in expression of hepatic CYP2D6 also was examined for the

postnatal age group. Because of the relatively small number of Hispanic samples in the dataset,

the analysis of the postnatal samples was limited to Caucasians and African Americans. Among

these two postnatal groups, the observed activity in African Americans was less than that

observed in Caucasians (Fig. 4). Among all postnatal pediatric samples, the observed median

activity of 0.032 nmol/min/mg microsomal protein was considerably less than that determined in

a pooled sample of adult liver microsomes, 0.168 nmol/min/mg microsomal protein.

Based on the stepwise linear regression, genotype also contributes significantly to the

differences in dextromethorphan O-demethylase activity in the postnatal age groups. When the

postnatal samples were grouped based on predicted phenotype (Table 2), the observed

dextromethorphan O-demethylase activity of the poor metabolizer phenotype group was

significantly less than that for either the intermediate or extensive metabolizer phenotype groups

(Fig. 5). However, the difference between the intermediate and extensive metabolizer phenotype

groups was not statistically significant.


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Discussion

Low levels of CYP2D6 protein and activity were observed in a small percentage of first

or second trimester fetal liver samples. Although the percentage of samples exhibiting detectable

protein and activity increased in third trimester fetal liver samples, the values remained no

greater than 3 to 5% of the levels observed in adult liver. CYP2D6 protein and activity remained

similar to third trimester fetal levels during the first week of life, but then increased significantly

thereafter. Furthermore, there were no significant differences in either protein or activity with

increasing age after one week postnatal age. These data are consistent with the in vivo

longitudinal data reported by Blake et al. (2007) who suggested that phenotype reflects CYP2D6

genotype by 2 weeks of age. The low fetal levels of CYP2D6-dependent activity and protein

also are consistent with the results of Treluyer et al. (1991), as was the lack of difference

between CYP2D6 activity and protein in the first week of life versus the third trimester. Because

CYP2D6 protein was independent of gestational age in their sample set, Treluyer et al. (1991)

concluded an important contribution from a birth-dependent control process. Similar to the

conclusion of these authors, of the factors examined (estimated gestational age, postnatal age,

postmortem interval, gender and ethnicity), only postnatal age was significantly associated with

increasing CYP2D6 activity, consistent with a major contribution from an unidentified birth-

dependent process.

Johnson et al. (2007) have argued that adjusting the in vivo data reported by Blake et al.

(2007) for maturation of renal function would suggest a gradual increase of hepatic CYP2D6

activity such that adult enzyme levels would not be achieved until approximately 6 months of

age. However, the initial assessment of the data reported herein would be more consistent with a


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rapid rise in hepatic CYP2D6 activity because there was no discernable increase in activity in

liver samples beyond one week after birth. Yet, the median activity for the >7 days to 18 year

old age group of 0.033 nmol/min/mg protein is at the low range of reported values for adult

human liver microsomes (Gorski et al., 1999; Pearce et al., 1996), and considerably lower than

the activity determined for a pooled adult human liver microsomal sample (0.168 nmol/min/mg).

This observation is likely due to the purposeful weighting of this sample set with earlier ages.

The median age of all postnatal samples was only 3.7 months (Table 1) and the >7 days to 18

year old age group only 5.4 months. Thus, these data also would be consistent with a longer

period of maturation as suggested by Johnson et al. (2007), but have insufficient power to detect

an intermediate level of CYP2D6 activity in tissue samples from older neonates and infants less

than six months of age. Clearly, the ultimate goal of being able to reliably predict the disposition

of and response to the administration of compounds to children of different ages that depend on

CYP2D6 for elimination remains to be achieved.

Another possible explanation for the observed low activity in the >7 days to 18 year old

age group would be sample quality. However, the lack of correlation between either CYP2D6

activity or protein levels and PMI argues against this explanation. Furthermore, for enzymes that

show little change with ontogeny, reported protein and activity levels with these same tissue

samples was consistent with adult values (Koukouritaki et al., 2004; Stevens et al., 2003), again

arguing against sample quality being a significant issue.

Among postnatal liver samples, median dextromethorphan O-demethylase activity was

higher in Caucasians (0.043 nmol/min/mg protein) than African Americans (0.029 nmol/min/mg

protein). This observation is consistent with the report of a lower CYP2D6 functional capacity

in black Americans, as measured by the urinary dextromethorphan/dextrorphan metabolic ratio


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(Gaedigk et al., 2002). Several investigators have reported a population shift toward lower

CYP2D6 activity for populations of African descent, likely due to a higher incidence of the

reduced function CYP2D6*17 and CYP2D6*29 alleles when compared to the Caucasian

population. Both the CYP2D6*17 and CYP2D6*29 alleles are prevalent in African Americans,

with frequencies of 21% and 7%, respectively (Bradford, 2002) with even higher frequencies in

African populations in which there is less admixture with other ethnic groups (Bradford et al.,

1998; Masimirembwa et al., 1996). These genetic differences likely explain the differences

observed in the current study in that the allelic frequencies of the CYP2D6*17 and *29 alleles

were 20.9% and 7.2%, respectively, in the liver samples from African American donors in

contrast to 1.3% and 0%, respectively, in the liver samples from Caucasian donors. However,

extrapolation of these genotypes to phenotypes is complicated by evidence of altered substrate

specificity for CYP2D6*17 and *29, such that the concordance between phenotype is dependent

upon the probe substrate used and the genotypic composition of the ethnic population under

investigation (Wennerholm et al., 1999; Wennerholm et al., 2001). CYP2D6 probe-dependent

analysis was not feasible for the current study, due to constraints of tissue sample amounts.

CYP2D6 is highly polymorphic with more than 80 known alleles, several of which have a

significant functional impact. Although a large number of alleles have been identified, many of

these are relatively rare. Using the long-range PCR DNA amplification approach described by

Gaedigk et al. (2002) to detect gene duplication and deletion together with multiplexed single-

base extension to detect the presence or absence of 11 single nucleotide polymorphisms, it was

possible to identify 14 CYP2D6 alleles that account for greater than 90% of the variability

observed in the population groups included in this study (Bradford, 2002; Gaedigk et al., 2002;

Gaedigk et al., 2007). Consistent with the concept that during early life stages, individuals must


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mature into a phenotype consistent with genotype, genetic variability was not associated with

CYP2D6 protein or dextromethorphan O-demethylase activity in fetal samples. However, both

age and genotype were strongly associated with CYP2D6 protein content and activity in the

postnatal liver samples with significantly higher activity observed in samples predicted to be

extensive or intermediate metabolizers versus those predicted to be poor metabolizers. Also

consistent with this concept, the percentage of samples with non-detectable dextromethorphan O-

demethylase activity decreased from 87% in first or second trimester samples, to 29 to 38% in

third trimester or one week postnatal samples, respectively, to only 13% in tissue samples from

individuals older than 7 days of age. The latter percentage is similar to the expected percentage

of poor metabolizers in the study population. Combined with the inability to discern significant

increases in either CYP2D6 protein or activity in postnatal samples from individuals over 7 days

of age, these data would suggest that CYP2D6 variability in individuals older than one to two

weeks of age largely is due to genetic variability.


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References

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Bradford LD (2002) CYP2D6 allele frequency in European Caucasians, Asians, Africans and

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Bradford LD, Gaedigk A, and Leeder JS (1998) High frequency of CYP2D6 poor and

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Products; Final Rule. Federal Registry 63:66631-66672.

Gaedigk A, Bradford LD, Marcucci KA, and Leeder JS (2002) Unique CYP2D6 activity

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Figure Legends

Fig. 1. CYP2D6 developmental expression pattern. (A) Scatter plot analysis of

dextromethorphan O-demethylase activity observed in individual fetal liver samples as a

function of gestational age. (B) Scatter plot analysis of dextromethorphan O-demethylase activity

observed in individual postnatal liver samples as a function of postnatal age up to 1 year. (C)

Scatter plot analysis of dextromethorphan O-demethylase activity observed in individual

postnatal liver samples as a function of postnatal age.

Fig. 2. Correlation of immunodetectable CYP2D6 protein levels and dextromethorphan O-

demethylase activities in selected fetal and postnatal pediatric liver microsome samples. Liver

microsomal samples were included in the analysis in which both dextromethorphan O-

demethylase activity and immunodectable CYP2D6 protein content were available.

Immunoquantitation and enzyme activities were determined as described in Materials and

Methods. Spearman’s rank correlation coefficient (rs) was 0.686 (P<0.001).

Fig. 3. CYP2D6 interindividual variability and ontogeny. A box-plot of (A) CYP2D6 protein

content and (B) dextromethorphan O-demethylase activities in different age groups is depicted in

which median (bar), interquartile (box) and 10th to 90th percentile (whiskers) values are shown.

Outliers (•) are plotted that were 1.5-fold outside the interquartile values. Based on genotype-

predicted phenotype (Table 2), poor metabolizers were excluded from the postnatal dataset for

this analysis. Sample sizes for each age group also are shown (*, P<0.05; ***, P<0.001).


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Fig. 4. Ethnic differences in CYP2D6 expression. Dextromethorphan O-demethylase activity in

the postnatal liver samples from Caucasians and African Americans were compared. The data

are depicted as a box plot in which median (bar), interquartile (box) and 10th to 90th percentile

(whiskers) values are shown. Outliers (•) are plotted that were 1.5-fold outside the interquartile

values (*, P<0.05).

Fig. 5. Effect of CYP2D6 genotype on CYP2D6 activity. Based on genotype, tissue samples

were grouped into predicted extensive (EM), intermediate (IM), or poor (PM) metabolizer

phenotypes (Table 2) and the dextromethorphan O-demethylase activities in each group

compared. The data are depicted as a box plot in which median (bar), interquartile (box) and 10th

to 90th percentile (whiskers) values are shown. Outliers (•) are plotted that were 1.5-fold outside

the interquartile values (**, P<0.01; ***, P<0.001).


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Table 1

Liver donor demographics

Variable Median (Range)

Age at death Fetal (n = 62, weeks gestational age) 20.0 (11.7-41)

Postnatal (n = 160, months) 3.7 (0-215)

Time between death and tissue processing (PMI) (hrs) 17.0 (1-41)

N (%)

Sex Male 133 (60)

Female 78 (35)

Unknown 11 (5)

Ethnicity N. European 93 (42)

African 83 (37)

Hispanic 19 (9)

Other 4 (2)

Unknown 23 (10)


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Table 2

Predicted phenotypes from CYP2D6 genotype and genotype frequencies

EMa % Frequency IMb % Frequency PMc % Frequency

*1/*1 13.1 *1/*3 0.7 *4/*4 1.4

*1/*2 22.1 *1/*4 15.6 *4/*5 1.4

*1XN/*2 2.8 *1/*5 1.4 *4/*9 0.7

*2/*2 8.3 *1/*10 4.1 *4/*17 1.4

*2XN/*1 0.7 *1/*17 3.4 *5/*10 0.7

*1/*29 3.4 *5/*17 0.7

*2/*4 4.8 *5/*29 0.7

*2/*5 1.4

*2/*6 0.7

*2/*9 0.7

*2/*10 2.1

*2/*17 1.4

*10/*10 1.4

*10/*17 0.7

*17/*17 2.1

*17/*29 1.4

a Extensive metabolizer phenotype (n = 68)

b Intermediate metabolizer phenotype (n = 67)

c Poor metabolizer phenotype (n = 10)


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Table 3

Factors associated with increasing CYP2D6 activity or protein in fetal and postnatal human liver microsomal samplesa

Age Group Dependent

Variable

Factor Standardized β t P-Value

Fetal Activity Increasing gestational age 0.601 4.452 <0.001

Protein Increasing gestational age 0.640 3.116 0.008

Premature Activity Increasing postnatal age 0.365 1.799 0.086

Protein Increasing postnatal age 0.688 3.422 0.005

Postnatal Activity Genotype 0.245 3.025 0.003

Increasing postnatal age 0.194 2.393 0.018

Protein Increasing postnatal age 0.462 4.812 <0.001

Ethnicity -0.268 -2.756 0.007

Genotype 0.257 2.677 0.009

a Other variables tested included gender, ethnicity, and post-mortem interval.

This article has not been copyedited and form

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