Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver

Article abstract of DOI:10.1016/j.bcp.2008.07.016

Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned from equine liver and expressed in a baculovirus expression system. The nucleotide sequence of CYP2D50 was highly homologous to that of human CYP2D6 and therefore the activity of the enzyme was characterized using dextromethorphan and debrisoquine, two isoform selective substrates for the human orthologue. CYP2D50 displayed optimal catalytic activity with dextromethorphan using molar ratios of CYP2D50 to NADPH CYP450 reductase of 1:15. Although CYP2D50 and CYP2D6 shared significant sequence homology, there were striking differences in the catalytic activity between the two enzymes. CYP2D50 dextromethorphan-O-demethylase activity was nearly 180-fold slower than the human counterpart, CYP2D6. Similarly, rates of formation of 4-hydroxydebrisoquine activity were 50-fold slower for CYP2D50 compared to CYP2D6. The results of this study demonstrate substantial interspecies variability in metabolism of substrates by CYP2D orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.

Full text of DOI:10.1016/j.bcp.2008.07.016

b i o c h e m i c a l p h a r m a c o l o g y 7 6 ( 2 0 0 8 ) 9 0 4 – 9 1 1
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biochempharm
Complementary DNA cloning, functional expression and
characterization of a novel cytochrome P450, CYP2D50,
from equine liver
*
H.K. DiMaio Knych , S.D. Stanley
K.L. Maddy Equine Analytical Chemistry Laboratory, California Animal Health and Food Safety Laboratory, School of Veterinary Medicine,
University of California, West Health Science Drive, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Members of the CYP2D family constitute only about 2–4% of total hepatic CYP450s, however,
they are responsible for the metabolism of 20–25% of commonly prescribed therapeutic
compounds. CYP2D enzymes have been identified in a number of different species. How-
ever, vast differences in the metabolic activity of these enzymes have been well documen-
ted. In the horse, the presence of a member of the CYP2D family has been suggested from
studies with equine liver microsomes, however its presence has not been definitively
proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned from
equine liver and expressed in a baculovirus expression system. The nucleotide sequence of
CYP2D50 was highly homologous to that of human CYP2D6 and therefore the activity of the
enzyme was characterized using dextromethorphan and debrisoquine, two isoform selec-
tive substrates for the human orthologue. CYP2D50 displayed optimal catalytic activity with
dextromethorphan using molar ratios of CYP2D50 to NADPH CYP450 reductase of 1:15.
Although CYP2D50 and CYP2D6 shared significant sequence homology, there were striking
differences in the catalytic activity between the two enzymes. CYP2D50 dextromethorphan-
O-demethylase activity was nearly 180-fold slower than the human counterpart, CYP2D6.
Similarly, rates of formation of 4-hydroxydebrisoquine activity were 50-fold slower for
CYP2D50 compared to CYP2D6. The results of this study demonstrate substantial inter-
species variability in metabolism of substrates by CYP2D orthologues in the horse and
human and support the need to fully characterize this enzyme system in equids.
# 2008 Elsevier Inc. All rights reserved.
Received 14 May 2008
Accepted 11 July 2008
Keywords:
CYP450
Horse
CYP2D
Dextromethorphan
Debrisoquine
Phase I metabolism
1.
Introduction
species as well as in humans. In humans, more than 50
individual CYP450s have been identified. Of these 50, only six
Cytochrome P450 monooxygenases (CYP450s) are a super-
family of hemeproteins responsible for metabolizing a vast
array of drugs, environmental pollutants and endogenous
compounds. CYP450 enzymes enable a functional group to be
introduced or unmasked in a compound, generating a more
polar intermediate, which is more readily eliminated. CYP450s
have been well characterized in many laboratory animal
have been shown to play a major role in metabolism and
subsequent clearance of drugs. These include members of the
CYP1, CYP2 and CYP3 families. CYP2D enzymes are particu-
larly important in drug metabolism as they are responsible for
metabolism of 20–25% of commonly prescribed drugs, includ-
ing antiarrythmics, b-adrenoreceptor antagonists, neurolep-
tics and tricyclic antidepressants and because there are
* Corresponding author. Tel.: +1 530 752 8700; fax: +1 530 754 5588.
E-mail address: hkknych@ucdavis.edu (H.K. DiMaio Knych).
0006-2952/$ – see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2008.07.016

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several functionally significant polymorphisms in the human
CYP2D6 gene [1].
transfer buffer (20ꢀ) were from Invitrogen (Carlsbad, CA).
BsrGI restriction enzyme was purchased from New England
Biosciences (Ipswich, MA). All sequencing primers were
synthesized by IDT (Coralville, IA). TOPO and pDEST8 cloning
vectors, Cellfectin reagent, Spodoptera frugiperda (Sf9) and High
Five cells and human recombinant CYP2D6 were from
Invitrogen (Carlsbad, CA). Kaleidoscope Prestained Standards
were from Biorad (Hercules, CA). Human CYP2D6 antibody
was purchased from ABCAM (Cambridge, MA) and Alexa Fluor
secondary antibody was purchased from Invitrogen (Carlsbad,
CA). NADPH, CHAPS, cytochrome c, dextromethorphan,
debrisoquine and dextrorphan were obtained from Sigma
(St. Louis, MO). 4-Hydroxydebrisoquine was purchased from
US Biological (Swampscott, MS). Acetonitrile and water were
purchased from Burdick & Jackson (Muskegon, MS). Methanol
was purchased from Thermo Fisher Scientific (Fairlawn, NJ).
All solvents were HPLC grade or better.
The most common variants of human CYP2D6 result in
very high activity (as a result of gene duplication), in altered
affinity for some substrates or in no activity which results
from inactive splice variants or point mutations that result in
an unstable product. Some of the variants have low pene-
trance in the human population (1–2%) while others such as
some of the point mutations are present in more than half of
Asians studied. The functional importance of these variants
combined with the relatively high penetrance in the human
population means that doses of those therapeutic agents with
a narrow therapeutic index must be adjusted to obtain
appropriate therapeutic levels. Polymorphic expression of
CYP2D is of particular importance because of the wide array of
therapeutic compounds metabolized by this enzyme.
A
decrease or complete inability to metabolize CYP2D6 sub-
strates presents the potential for adverse drug reactions as a
result of an inability to metabolize and clear the administered
compound from the body.
2.2.
Animals
Although there is considerable experimental evidence
supporting the functional importance of CYP2D6 and its
variants in both humans and animals used for human drug
development, little is known about this protein in other species.
Therapeutic agents used in equine medicine are those that are
effective in other species and dose regimens are established on
a trial and error basis. The studies reported here are part of a
long term effort to systematically evaluate the metabolic
capabilities of the drug metabolizing enzymes in equids.
Although there have been a limited number of studies using
equine liver microsomes [2–4], these provide little information
about individual equine CYP450s, specifically which enzymes
are responsible for the metabolism of specific drugs. Human
CYP450s have been well characterized and various in-vitro
systems, such as recombinant cytochrome P450 enzymes
expressed in E. coli or insect cells, have been developed to
study the metabolism of therapeutic agents. To date, there are
no reports of equine recombinant CYP450 enzymes.
Liver samples were collected from two horses (one female
Tennessee Walker and one male Paint Horse). Samples were
collected from horses being euthanized for other studies
previously approved by the Institutional Animal Care and Use
Committee of the University of California at Davis. Both horses
were determined to be healthy by physical examination prior
to euthanasia.
2.3.
RNA isolation
Liver samples were collected, cut into approximately 3 mm
cubes and stored at ꢁ20 8C in RNA Later (Ambion, Foster City,
CA) until processed. Sections weighing approximately 100 mg
were cut into small pieces, homogenized directly in Trizol
Reagent (Invitrogen, Carlsbad, CA) and total RNA isolated from
each horse. The quality of total RNA was checked by gel
electrophoresis. Concentrations were measured by absor-
bance at 260 nm.
Studies with equine liver microsomes have suggested the
presence of a member of the CYP2D family in the horse, based
on metabolic activity with the human isoform selective
substrate dextromethorphan [2]. In this study, equine liver
microsomes demonstrated more than 20 times the metabolic
turnover of dextromethorphan as compared to human liver
microsomes. However, the only definitive way to determine
whether CYP2D is responsible for metabolism of dextro-
methorphan in the horse, or if there are multiple members of
this family playing a role in its metabolism, is through the use
of a recombinant enzyme. Accordingly, in this study, a
member of the CYP2D family was cloned, sequenced,
expressed and characterized with respect to human CYP2D6
selective substrates.
2.4.
Cloning of equine CYP2D50
The cDNA for equine CYP2D50 was obtained by amplification
of total RNA prepared from liver samples from two individual
horses. The cDNA transcripts were generated by reverse
transcription using the Superscript III One Step RT-PCR kit
with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA)
with the forward (50-CACCATGGGGCTGCT GACCTGG-30) and
reverse (50-AGGACCTGATCCTGAGGAGGCTGCT-30) primers
designed against predicted CYP2D sequences from the equine
genome project (GI:149743367). The PCR products were cloned
into pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) and the
two DNA sequences confirmed using an automated sequencer
(ABI 3730, Applied Biosystems, Foster City, CA). The deduced
sequence was submitted to the P450 nomenclature committee
for name designation and then to GenBank (EU190996).
2.
Materials and methods
2.1.
Chemicals
2.5.
Expression of CYP2D50 in insect cells
Superscript III One Step RT-PCR Kit, Novex mini-gel system,
10% Bis-Tris precast gels, NuPage loading dye and NuPage
The cDNA insert encoding equine CYP2D50 was directionally
cloned into the multiple cloning site of pDEST8 (Invitrogen,

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Carlsbad, CA). The integrity and orientation of the pDEST8/
2D50 clone was verified by forward sequencing. The cDNA
encoding CYP2D50 from pDEST8/2D50 was site specifically
transposed into the baculovirus genome as described in the
manufacturer’s protocol (Bac-to-Bac Expression System; Invi-
trogen, Carlsbad, CA). The high molecular weight genomic
DNA was purified from recombinant clones and used to
transfect Sf9 cells for the production of baculovirus stocks.
Baculovirus stocks were amplified in Sf9 cells and the viral
titer was determined using the BacPAK Baculovirus Rapid
Titer Kit (BD Biosciences Clontech; Palo, Alto, CA). High Five
cells (Invitrogen, Carlsbad, CA), grown as monolayers in
serum-free medium supplemented with L-glutamine were
used to optimize expression and produce active enzyme.
Multiplicity of infection (MOI) and time to collection of cells
were determined by infecting at an MOI of 0.1, 1, 5 and 10 pfu/
cell and using post infection harvest times of 24, 48 and 72 h.
The product was verified by Western blot analysis. Production
of functional protein was performed using the optimal MOI in
the presence of 300 mM 5-aminolevulinic acid (ALA) and
200 mM ferric citrate (FC). Cell lysates were prepared by
pelleting cells at 1000 ꢀ g for 5 min at 4 8C followed by
resuspension in 0.1 M phosphate buffer, pH 7.4, with 0.25 M
sucrose, 1 mM EDTA and 0.5 mM PMSF. The resuspended
pellet was then centrifuged at 100,000 ꢀ g for 1 h at 4 8C. The
supernatant was discarded and the pellet resuspended in
phosphate storage buffer (0.1 M KPO4, pH 7.4 containing 20%
glycerol) and stored at ꢁ80 8C. P450 levels were determined by
obtaining the difference spectra of sodium dithionate-
reduced vs CO-bubbled samples at 500–400 nm according to
the methods of Omura and Sato [5].
kinetic studies were conducted within the linear portion of the
rate curve.
2.8.
Enzyme activity and kinetics
Dextromethorphan metabolism by equine recombinant
CYP2D50 and equine liver microsomes was carried out in
250 ml reaction volumes. Recombinant assays included
2.5 pmol of recombinant CYP2D50 or CYP2D6, 37.5 pmol
NADPH CYP450 reductase (rCYP2D50), 1 mM CHAPS, 100 mM
potassium phosphate buffer (pH 7.4) and varying substrate
concentrations. Microsomal incubations consisted of 1 mg/ml
protein in 100 mM potassium phosphate buffer (pH 7.4). All
reactions were incubated at 37 8C for 2 min prior to initiation of
the reaction by the addition of 1 mM NADPH. Reactions were
allowed to proceed for either 15 min (rCYP2D50 and rCYP2D6)
or 5 min (liver microsomes). Reactions were terminated by the
addition of 250 ml of acetonitrile containing levallorphan as
the internal standard. Reaction rates were measured under
linear conditions to obtain values for Km or Vmax using a
varying number of substrate concentrations ranging from 0 to
400 mM (rCYP2D50 and rCYP2D6) or
0 to 1000 mM (liver
microsomes). The data was evaluated by plotting velocity
(V) vs V/[Substrate] (Eadie Hofstee plot) and the intercepts
calculated by linear regression analysis. Debrisoquine hyrox-
ylase activity measurements were carried out for 10 min with
saturating substrate concentrations (1000 mM) and activity
calculated as pmol product/min/pmol CYP450.
2.9.
Sample analysis
The liquid chromatography–mass spectrometry (LC–MS) sys-
tem consisted of an Aquity UPLC system (Waters, Milford, MA),
an LTQ Orbitrap XL mass spectrometer (Thermo Fisher
Scientific, Bremen, Germany) and an HPLC column (HSS T3
2.1 mm ꢀ 50 mm, 1.8 mm, Waters, Milford, MA). UPLC was
performed using a gradient of 3% B to 90% B over 6.0 min
(solvent A, H2O with 0.2% formic acid; solvent B, acetonitrile
with 0.2% formic acid), followed by an immediate return to
initial conditions that were maintained for 3.0 min with a flow
rate of 400 ml/min. The UPLC gradient system started with 10%
B and linearly increased to 80% B in 15 min, followed by an
increase to 100% B in 3 min prior to column re-equilibration.
All experimental data were acquired using external calibration
5 days prior to data acquisition, and all collected mass scan
data were recorded in the centroid mode in order to minimize
data file sizes.
2.6.
Reductase purification
NADPH cytochrome P450 reductase was purified from equine
liver microsomes as described by Yasukochi and Masters [6].
Specific activity was determined by the cytochrome
c
reductase assay with one unit being defined as 1 mmol of
cytochrome c reduced per minute.
2.7.
Optimization of CYP2D50 metabolism
Earlier work has demonstrated the importance of optimizing
conditions of incubation for studies examining the kinetics of
substrate metabolism catalyzed by recombinant proteins [7].
Accordingly, prior to conducting enzyme kinetic studies, the
ratio of recombinant CYP2D50 to equine NADPH cytochrome
P450 oxidoreductase was determined by adding increasing
quantities of equine NADPH cytochrome P450 oxidoreductase
to the incubation. Incubations contained 5 pmol of recombi-
nant CYP2D50, 1 mM CHAPS, 100 mM potassium phosphate
buffer, pH 7.4 and 1 mM dextromethorphan in a total volume
of 250 ml. Incubation vessels were incubated for 2 min in a
37 8C shaking water bath prior to the addition of 1 mM NADPH.
Following an additional 15 min incubation, all reactions were
terminated by the addition of ice cold acetonitrile. Reaction
conditions for recombinant CYP2D6 were according to the
manufacturer’s protocol with the addition of 1 mM CHAPS.
Linearity was established with protein and with time for
equine and human recombinant enzymes and all subsequent
2.9.1. MS of dextromethorphan and analogues – quadrupole-
linear ion trap analyzer
High-resolution/accurate mass measurement of fragment
ions of dextrophan in MS2 and MS3 experiments was
accomplished using a solution at 10 mg/ml in acetonitrile:-
water (1:1, v/v) with 0.2% formic acid. The protonated
precursor ion at m/z 258 was isolated at a width of 1.5 u and
dissociated at a normalized collision energy value of 35%. In
the case of MS3, the second precursor at m/z 201 was
dissociated at 35% of the normalized collision energy. Thirty
product ion mass spectra were recorded with the Orbitrap
from 50 to 265 m/z at a resolution of 60,000.

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2.9.2. MS of debrisoquine and analogues – quadrupole-linear
ion trap analyzer
obtained by processing the full-scan MS data set using
potential dextromethorphan and debrisoquine metabolite
ions with 5 ppm mass tolerance. The potential dextromethor-
phan and debrisoquine metabolite ions were calculated from a
default metabolite list in MetWorks 1.1 (Thermo Fisher
Scientific).
All reference standards were dissolved in methanol at
concentrations of 1 mg/ml and analyzed by electrospray
ionization (ESI) and collision-induced dissociation (CID) on a
LTQ Orbitrap XL at room temperature. High-resolution/
accurate mass measurement of fragment ions of 4-hydro-
xydebrisoquine in MS2 and MS3 experiments was accom-
plished using a solution at 10 mg/ml in acetonitrile:water (1:1,
v/v) with 0.2% formic acid. The protonated precursor ion at m/z
192 was isolated at a width of 1.5 u and dissociated at a
normalized collision energy value of 37%. In the case of MS3,
the second precursor at m/z 174 was dissociated at 35% of the
normalized collision energy. Thirty product ion mass spectra
were recorded with the Orbitrap from 50 to 200 m/z at a
resolution of 60,000.
3.
Results
3.1.
Cloning and sequencing of equine CYP2D50
RT-PCR products from two different horses were generated.
The consensus sequence was 1503 base pairs. The sequence
was submitted to the CYP450 Nomenclature Committee and
was given the novel designation of CYP2D50. The deduced
amino acid sequence of CYP2D50 (accession no. EU0190996)
showed a homology of 80% to cattle CYP2D14 and CYP2D43.
The amino acid sequence alignment of CYP2D50 with that of
human CYP2D6 (Fig. 1) revealed that CYP2D50 is 77%
homologous to CYP2D6.
The calculation of chemical formulae from the accurate
measurement of m/z values and data processing of EIC were
carried out using the Qual Browser of Xcaliber 2.0.7 (Thermo
Fisher Scientific, San Jose, CA). High-resolution EICs of
dextromethorphan and debrisoquine metabolites were
Fig. 1 – Amino acid alignment of CYP2D50 from equine and CYP2D6 from human liver. Key: (*) denotes conserved amino acid
residues among the CYP enzymes.

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3.2.
Expression of CYP2C92 in insect cells
3.4.
Kinetic properties
An entry vector containing CYP2D50 from one of the horses
was used in the Bac-to-Bac system to generate a recombinant
bacmid construct. The bacmid DNA was then used to
transfect Sf9 cells to generate a recombinant baculovirus,
which was further amplified by an additional two rounds to
make a high titer baculovirus stock for expression studies.
The expression of full-length recombinant CYP2D50 by Sf9
cells was assessed using Western blot analysis. Protein
expression was apparent at 72 h post infection in Sf9 cells
from the third round of amplification. Western blot analysis
was also used to optimize MOI and harvest times after
infection in High Five cells that were infected with amplified
baculovirus stock. Expression levels were determined to be
the greatest at an MOI of 10 and accordingly a MOI of 10 pfu/
cell was chosen for subsequent expression studies. There was
a noticeable difference in expression levels between a harvest
time of 24, 48 and 72 h post infection with maximum
expression being apparent at 72 h post infection. Easily
detectable quantities of CYP with a soret maximum at
452 nm were observed following the addition of ferric citrate
and ALA to the medium.
Kinetic parameters (Km and Vmax) for the metabolism of
dextromethorphan by recombinant CYP2D50 were estab-
lished with the optimized system described previously. For
comparison, incubations of dextromethorphan with human
CYP2D6 recombinant enzyme were performed according to
the manufacturer’s protocol with the addition of 1 mM
CHAPS. Incubations for dextromethrophan metabolism
were performed for 5 min (liver microsomes) or 15 min
(CYP2D50 or CYP2D6) using substrate concentrations ran-
ging from 0 to 1000 mM (liver microsomes) or 0 to 400 mM
(CYP2D50 and CYP2D6). The data was evaluated by plotting
velocity (V) vs V/[Substrate] (Eadie Hofstee plot) and the
intercepts calculated by linear regression analysis. The Km
value for CYP2D6 is nearly five times lower when compared
with the equine orthologue, CYP2D50 (Fig. 3A and B).
Similarly the maximal rates of substrate metabolism in
the incubations containing the human recombinant CYP2D6
were more than 180-fold higher (Table 1) than CYP2D50. The
calculated Vmax value for dextromethorphan-O-demethy-
lase activity in equine liver microsomes was 27.6 pmol/min/
pmol CYP450. In addition to dextrorphan, a hydroxylated
metabolite was detected in incubations with CYP2D50 and
CYP2D6. Dextromethorphan-O-demethylation was the pre-
dominant reaction in liver microsome incubations, however
low concentrations of additional metabolites were detected
including a hydroxylated, a di-demethylated, and a methy-
lated product.
3.3.
Optimization of dextromethorphan metabolism
Prior to conducting kinetic studies, optimal incubation
conditions for dextromethorphan metabolism by recombi-
nant CYP2D50 were established. The optimal ratio of CYP2D50
to NADPH cytochrome P450 reductase was determined in
incubations containing 5 pmol of CYP2D50 and increasing
amounts of reductase (1:1–1:20). The rate of dextromethor-
phan metabolism increased up to a ratio of 1:15 (Fig. 2). A
decrease in substrate turnover was seen after 1:15 and
Debrisoquine is another isoform selective substrate for
human CYP2D6, and was tested as a possible substrate for
recombinant CYP2D50. For this experiment, saturating con-
centrations of debrisoquine (1000 mM) were incubated with
both CYP2D50 and CYP2D6 and samples removed following a
10 min incubation. CYP2D50 activity was minimal (nearly 50-
fold less) as compared to CYP2D6. Liver microsomes exhibited
minimal debrisoquine 4-hydroxylase activity with a calcu-
lated Vmax of 1.41 pmol/min/pmol CYP450. In addition to 4-
hydroxydebrisoquine, a ketone metabolite was detected in
incubations with CYP2D50. Products detected in equine liver
microsomal incubations included 4-hydroxydebrisoquine, a
dehydrogenated product, a reduced hydroxy and a di-hydroxy
metabolite.
therefore
a ratio of 1:15 was used for all subsequent
metabolism studies. The optimal CHAPS concentration of
1 mM was determined in a previous study.
3.5.
Sample analysis
For incubations with dextromethorphan, mass accuracy of
less than 2 ppm was obtained with external calibration for all
selected fragment ions allowing for the assignment of a single
elemental composition given candidate compositions encom-
passing C17H24O1N1 at nominal precursor m/z of 258.1848
corresponding to the O-desmethyl product. The full-scan MS
data set contained rich information on protonated molecules
of dextromethorphan metabolites with high mass accuracy.
For debrisoquine samples, mass accuracy of less than 2 ppm
was obtained with external calibration for all selected
Fig. 2 – Effect of increasing amounts of NADPH CYP450
reductase on the rate of dextromethorphan metabolism by
recombinant CYP2D50. Incubations containing 5 pmol of
recombinant CYP2D50, 1 mM CHAPS, 100 mM potassium
phosphate buffer, pH 7.4, 1 mM dextromethorphan and
varying amounts of equine liver reductase in a total
volume of 250 ml were incubated at 37 8C for 20 min.
Values are the mean of two separate incubations.
fragment ions allowing for the assignment of
a single
elemental composition given candidate compositions encom-
passing C10H14O1N3 at nominal precursor m/z of 192.1133
corresponding to the hydroxylated product. The full-scan MS

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is important to note that interspecies differences in in-vitro
metabolism as well as substrate specificity have been well
documented. In this study, we have cloned and identified a
member of the CYP2D family in the horse, given the novel
designation CYP2D50. In humans, CYP2D6 constitutes only
about 2–4% of total CYP450s, however, it is responsible for the
metabolism of
a broad range of commonly prescribed
therapeutic compounds. Through studies utilizing equine
liver microsomes and isoform selective substrates for CYP2D6,
it has been suggested that a member of the CYP2D family is
present in the horse liver that may be capable of metabolizing
similar substrates to that of CYP2D6 [2]. However, the only
definitive way of determining precisely which enzyme is
responsible for the metabolism of a given drug and which
metabolites a specific enzyme produces, is by constructing a
recombinant enzyme. To that end, in the present study,
CYP2D50 cDNA was cloned and expressed in insect cells in
order to characterize and compare enzymatic activity to the
human orthologue, CYP2D6.
The amino acid sequence of equine CYP2D50 is 77%
homologous to that of human CYP2D6. However when the
catalytic properties of the two enzymes were compared with
respect to O-demethylation of dextromethrophan and debri-
soquine 4-hydroxylation, two markers of CYP2D6 activity,
some distinct differences in activity were observed. Dextro-
methorphan-O-demethylase activity levels for CYP2D6 were
nearly 180 times higher than those observed for CYP2D50.
Similar to the findings for CYP2D50 and CYP2D6 reported here,
there are a number of other reports of interspecies differences
in dextromethorphan-O-demethylase activity for members of
the CYP2D family. The rate of dextromethorphan-O-demethy-
lation by human CYP2D6 has been shown to be 10- and 12-fold
higher in comparison to CYP2D17 (cynomologous monkey) [8]
and CYP2D15 (dog) [9], respectively. LC–MS analysis of
CYP2D50 and CYP2D6 incubations, performed in full-scan
mode, revealed that in addition to dextrorphan, both species
produce a hydroxylated product, albeit at low concentrations
relative to dextrorphan. This suggests that the substrate,
dextromethorphan, orients in the same manner in both the
human and equine recombinant enzyme and that the other
metabolites of dextromethorphan observed in equine liver
microsomal incubations likely arise as a result of metabolism
by enzymes other than CYP2D50.
Fig. 3 – (A) Eadie Hofstee plot for the determination of the
apparent Kmax and Vmax values for dextromethorphan
metabolism by equine recombinant CYP2D50. Incubations
contained 2.5 pmol of CYP2D50, 37.5 pmol of CYP
reductase, NADPH and dextromethorphan (0, 2.5, 5, 10, 25,
50, 200 and 400 mM) and were incubated for 15 min at
37 8C. Values are the mean from data obtained from two
separate incubations at each substrate concentration. (B)
Double reciprocal plot for the determination of the
apparent Kmax and Vmax values for dextromethorphan
metabolism by human recombinant CYP2D6. Incubations
contained 2.5 pmol of CYP2C9, 2.5 pmol of CYP reductase,
NADPH and dextromethorphan (0, 2.5, 5, 10, 25, 50, 200
and 400 mM) and were incubated for 15 min at 37 8C.
Values are the mean from data obtained from two
separate incubations at each substrate concentration.
4-Hydroxydebrisoquine activity proved to be very different
between the human and horse orthologues as well. Full
enzyme kinetic studies were not performed with debrisoquine
as very little metabolite was detected in incubations with
CYP2D50. Following a 10 min incubation at saturating sub-
strate concentrations, CYP2D6 demonstrated a nearly 50-fold
higher rate of production of the 4-hydroxydebrisoquine
metabolite as compared to CYP2D50. Furthermore, liver
microsomes proved to have very low debrisoquine 4-hydro-
xylase activity with a Vmax of 1.4 pmol/min/pmol CYP450. In
addition, the Km value was very high and the Vmax/Km was
calculated at 0.03, indicating highly inefficient debrisoquine 4-
hydroxylase activity. The results from equine liver microsome
incubations suggests that the horse CYP450s are either very
inefficient at metabolizing debrisoquine or that the horse may
produce metabolites other than the 4-hydoxy product. LC–MS
analysis revealed three additional metabolites in liver micro-
data set contained rich information on protonated molecules
of debrisoquine metabolites with high mass accuracy.
4.
Discussion
Due to their large size, the expense associated with main-
tenance and drug administration as well as limited access to
research animals, performing drug metabolism studies in
the horse, metabolism data from other species must fre-
quently be extrapolated for equine veterinary use. However, it

910
b i o c h e m i c a l p h a r m a c o l o g y 7 6 ( 2 0 0 8 ) 9 0 4 – 9 1 1
Table 1 – Catalytic activities of recombinant CYP2D50, CYP2D6 and equine liver microsomes with two CYP2D substrates
d
Substrate
Apparent Km (mM)
Vmax (pmol/min/pmol CYP450)
Vmax/Km
Dextromethorphan
rCYP2D50a
rCYP2D6a
5.60
1.30
0.24
0.15
25.6
27.5
0.03
19.7
114.5
ELMb
Debrisoquine
rCYP2D50a
rCYP2D6a
ELMb
NDc
NDc
48.0
0.11
5.4
NDc
NDc
0.03
1.4
a
rCYP2D50, rCYP2D6: average (n = 2 incubations).
ELM: equine liver microsomes, average (n = 3, duplicate determinations).
ND = not determined.
b
c
d
Expressed as minꢁ1 mmolꢁ1
.
some incubations and one additional product in the CYP2D50
incubations. Based on signals at m/z of 174.013, 194.1288 and
208.1081 for the dehydrogenated, the reduced hydroxyl and
the di-hydroxy metabolite, respectively and assuming that the
ionization efficiencies were similar to 4-hydroxydebrisoquine,
we found that the formation of these additional products
occurred at far lower rates as compared to the rate of
formation of the primary product, 4-hydroxydebrisoquine.
These findings suggest that 4-hydroxydebrisoquine is the
major product of debrisoquine metabolism in the equine
species, however, metabolism of this compound by equine
CYP450s appears to be very slow.
length variants demonstrated enzymatic activity with the
isoform selective substrate, but showed very different rates of
catalytic activity. It is possible that one or more transcript
variants may exist in the horse and that this may explain why
the CYP2D50 enzyme described here does not metabolize
dextromethorphan very efficiently as compared to the human
enzyme. Equine liver microsomes used in this study demon-
strated significant dextromethorphan-O-demethylase activity
indicating that the horse is capable of metabolizing dextro-
methorphan at high rates. Additional studies are required to
determine whether another enzyme is capable producing the
dextrorphan metabolite or whether the activity demonstrated
by the liver microsomes can be attributed to CYP2D50 variants.
The recently released Equine Genome Project has identified
four additional CYP2D sequences, which appear to be highly
homologous. Based on a nucleotide sequence homology of 98%
with CYP2D50, it is highly likely that these sequences are
transcript variants of CYP2D50 and that one of the variants
may have higher catalytic activities with dextromethorphan
and/or debrisoquine.
Two members of the CYP2D family have been identified in
the marmoset, CYP2D19 and CYP2D30 [10]. Even though both
enzymes are members of the CYP2D family, they demon-
strated very different catalytic capabilities. In incubations
with debrisoquine, CYP2D30 exhibited a debrisoquine 4-
hydroxylase activity of 10.8 pmol/min/pmol CYP450. However
CYP2D19 yielded non-detectable levels of the 4-hydroxydeb-
risoquine metabolite. It is possible that similar to the
marmoset, there are other CYP2D variants in the horse
capable of higher rates of dextromethorphan-O-demethyla-
tion and debrisoquine 4-hydroxylation. In the current study
two horses were evaluated and only one member of the CYP2D
family was cloned. This however, does not rule out the
existence of additional members of this family in the horse. It
is possible that the discovery of only one member of the CYP2D
family in the horse can be attributed to the methodology used
in this study. RT-PCR was employed to amplify the CYP2D gene
and primers were designed based on the Equine Genome
Project predicted sequence for the equine orthologue of
CYP2D6. This methodology would not allow for amplification
of sequences that are not highly homologous in the region in
which the primers anneal. Further work screening for CYP2D
variants in a cDNA library from equine liver is underway.
In humans, CYP2D6 is highly polymorphic, with nearly 50
allelic variants identified thus far [1]. Individuals carrying this
polymorphic gene, can exhibit either low or very high hepatic
clearance of drugs that are substrates for CYP2D6 which can
result in therapeutic failure or adverse reactions. CYP2D
transcript variants have also been identified in other species.
In the dog, investigators have isolated two full-length
In this study, we have established the presence of a
member of the CYP2D family in the horse that is capable of
metabolizing isoform selective substrates for CYP2D6, albeit at
dramatically different rates from the human orthologue.
Further investigation, including characterizing the substrate
specificities of other equine CYP450 isoforms, is required
before dextromethorphan-O-demethylase and debrisoquine
4-hydroxylase activities can be assigned to CYP2D50. Further-
more, additional studies are necessary to determine the role of
CYP2D50 in the metabolism of other therapeutic agents
commonly administered to the horse. CYP2D50’s role in the
metabolism of endogenous compounds also warrants further
investigation. In the pig, CYP2D25 is involved in the metabo-
lism of vitamin D3 [12] and has the ability to reduce N-
hydroxylated compounds [13]. The current study is only the
first step in characterizing the metabolism of compounds by
members of the CYP2D family in the horse.
Acknowledgements
The authors would like to thank Dr. Alan R. Buckpitt, Dr. Alan J.
Conley, Jo Corbin and Dexter Morin for their helpful sugges-
tions and Dan McKemie for technical assistance.
transcript variants of CYP2D15 as well as
a third non-
functional splice variant devoid of exon 3 [11]. Both full-

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