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Cancer Letters 259 (2008) 1–15

www.elsevier.com/locate/canlet

Mini-review

Cytochrome P450s in the development

of target-based anticancer drugs
a,* b,1 b
Kedar Purnapatre , Sunil K. Khattar , Kulvinder Singh Saini
a
Infectious Diseases, New Drug Discovery Research, Ranbaxy Research Laboratories, Plot No. 20, Sector 18,
Udyog Vihar Industrial Area, Gurgaon 122001, Haryana, India
b
Biotechnology, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon 122001, Haryana, India

Received 27 August 2007; received in revised form 16 October 2007; accepted 17 October 2007

Abstract

Enzymes of the cytochrome P450 (CYP) superfamily are the major determinants of half-life and execute pharmacolog-
ical effects of many therapeutic drugs. In new drug discovery research, recombinant (human) CYPs are also used for iden-
tifying active or inactive metabolites that could lead to increased potency or toxicity of a molecule. In addition, CYP
inhibition by anticancer drugs might lead to adverse drug reactions, multiple-drug resistance, and drug–drug interactions.
During the discovery and pre-clinical evaluation of a New Chemical Entity (NCE), large amounts of purified recombinant
CYPs are required for studying metabolism and pharmacokinetic parameters. Therefore, present research efforts are
focused to over-express these human CYPs in bacteria, yeast, insect and mammalian cells, followed by their purification
on an industrial scale to facilitate identification of novel anticancer drugs. This review summarizes the merits and limita-
tions of these expression systems for an optimized production of individual CYP isoforms, and their usefulness in the dis-
covery and development of target-based, safe and efficacious NCEs for the treatment of cancer.
 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Recombinant P450; CYP; Expression; Pharmacokinetics; Drug discovery

1. Introduction window between the drug safety parameters and its

During the drug discovery research and develop-
ment process, studies on the Metabolism and Phar-
macokinetics (PK) of New Chemical Entity (NCE)
play a vital role in the determination of an optimum
*
Corresponding author. Tel.: +91 124 4194400X5312; fax: +91
124 2343544.
E-mail addresses: kedar.purnapatre@ranbaxy.com (K. Pur-
napatre), skhattar@umd.edu (S.K. Khattar).
1
Present address: University of Maryland, Department of
Veterinary Medicine, College Park, MD 20742, USA.
therapeutic potential. Cytochrome P450 (CYP)
superfamily is the major determinant of half-life of
many therapeutic molecules and exerts their physio-
logical effects through modulation of its enzyme
activity and by guiding some of these molecules
on the proteasomal degradation pathway. There
are 57 functional genes (and 58 pseudo genes) which
code for active CYP enzymes present in humans.
The enzymes of the CYP1, 2, and 3 families are
involved in the biotransformation of xenobiotics
and chemicals, while the other CYP families
(CYP4, 11, 17, 19, and 21) are involved in the

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2007.10.024
2 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
‘‘housekeeping’’ metabolism of endogenous mole-
cules [1,2]. The precise biochemical role of 58
pseudo genes, in the overall metabolism of CYPs
needs to be further evaluated, particularly if any
of these pseudo gene(s) have RNA interference
(RNAi) like regulatory properties.
Specific CYPs (primarily 1A2, 3A4, 2D6, 2C9, &
2C19) catalyze oxidative metabolism of over 90% of
the human drugs and are the major determinants of
elimination or systemic clearance and bioavailabil-
ity of therapeutic compounds. These enzymes share
considerable homology at the nucleotide level [3]
and contribution of the major isoforms to overall
drug metabolism is as follows: 1A2 (4%), 2A6
(2%), 2C9 (10%), 2C19 (2%), 2E1 (2%), 2D6
(30%), and 3A4 (50%). Interaction of CYP with
drugs inside human body could lead to various out-
comes, which are listed in Fig. 1. Therefore, early
determination of CYP inhibition potential and iden-
tification of CYP-mediated metabolism of NCEs is
absolutely necessary for the discovery of safe and
efficacious drugs.
The combinatorial synthetic approach used in
the new drug discovery demands screening of a
large number of compounds at early drug discov-
ery phase. Such evaluation of the in vitro pharma-
cokinetics is performed in a high throughput
screening (HTS) mode [4]. Optimization of HTS
has been exploited well in the pharmaceutical
industry. A comparative study evaluating 10-, 3-
and single point at 3 lM screening assays was per-
formed for 569 data points [5]. Statistical analysis
of these data demonstrated that variation and ana-
lytical precision for the IC50 generated by 10-point
and 3-point methods were comparable to single
point assay. Thus, utilization of a single concen-
Fig. 1. Several outcomes of CYP–drug interaction: CYP metabolites can either have an increased potency (bioactivation) or are
hydrophilic intermediates that are eliminated from the body (elimination) or generate toxic intermediates. A drug can either inhibit or
induce CYP leading to drug–drug interaction during multi-drug therapy.
tration of the inhibitor (3 lM) in a 96-well plate
format offers an economical and rapid screening
procedure for predicting drug–drug interactions
in early phase of drug discovery [5]. Large
amounts of CYP enzymes are required to perform
these HTS assays. Currently, there is considerable
effort by the pharmaceutical industry to over-
express human CYPs in bacteria, yeast, insect
and mammalian cells and each of these systems
has been further exploited for the commercial pro-
duction of recombinant CYPs. However, these
expression systems need to be supplemented with
NADPH P450 oxidoreductase (OR) that provides
reducing equivalents from NADPH for the activa-
tion of CYP. In addition, ancillary protein like
cytochrome b5 may also enhance the activity of
P450s.
Cancer patients, at times, undergo multiple treat-
ment regimes, which increases the risk of drug–drug
interactions followed by adverse drug reactions. In
addition, genetic polymorphism in CYP genes could
further complicate such therapy among various eth-
nic populations. Altered isoforms could either
decrease or increase metabolism of certain antican-
cer drugs leading to cytotoxicity in some patients.
Over and above, CYP isoform(s) present in the
tumor cells can play an important role in tumor
development [6]. Inside cancer cells, CYPs could
either inactivate the anticancer drugs, like 2-meth-
oxy estradiol, or activate the tumor-promoting com-
pound like 4-hydroxylestradiol [7,8]. Therefore,
determination of CYP mediated metabolism
appears to be mandatory for the discovery of novel
and safer anticancer therapies.
This review describes methods of production of
recombinant CYPs and their role in the metabolism
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
of existing anticancer molecules, drug–drug interac-
tion and activation of a pro-drug into an active ther-
apeutic agent. In addition, the exploitation of
single-nucleotide-polymorphism (SNP) of CYPs
towards the discovery and development of target-
based, safe, efficacious and personalized therapies
will be discussed. This review also briefly outlines
the ‘‘targeted regulation’’ of CYP genes by RNAi,
like nucleotides.
2. Expression and production of recombinant CYPs
Large number of pharmaceutical R&D compa-
nies are generating in-house recombinant human
CYPs for the determination of in vitro PK proper-
ties of their NCEs [9,10]. Recombinant CYPs with
the catalytic properties comparable to those of the
human liver microsomes (HLM) have now been
expressed in bacteria, such as, Escherichia coli and
Salmonella typhimurium; baker yeast Saccharomyces
cerevisiae. Insect cells with baculoviral vectors, or
mammalian cells such as lymphoblastoid cells,
COS cells, V79 CHO cells, HepG2, etc., with
vaccinia viral vectors have been extensively used
[11–14]. Table 1 summarizes the strengths and chal-
lenges of various recombinant systems used for the
production of CYP enzymes. CYPs expressed using
all these recombinant systems are available from
different commercial sources and these enzymes
have shown comparable properties vis-a-vis HLM.
2.1. Strategies used to enhance expression of
recombinant CYPs
CYPs are associated with microsomal mem-
branes and require NADPH reductase for their
functional activation [33]. Emerging strategies
employed for the expression of a eukaryotic protein
in a bacterial host have been appropriately
addressed and reviewed in a recent article from
our laboratory [34]. Review of the recent literature
strongly suggests utilization of various modifica-
tions in the recombinant CYPs as well as existing
protocols leading to the optimal expression of
recombinant CYPs in various expression systems.
These important observations are discussed in the
following section.
2.1.1. Modification of N-terminal
Deletion of strong secondary sequences around
the initiation codon of human CYPs are required
for the expression of CYPs in E. coli [27,28]. It
3
has been observed that the expression of human
2C9 and 2C19 can be enhanced in bacteria by intro-
ducing eight silent mutations in the N-terminal
codons which reduced the formation of stable sec-
ondary structures [35]. In addition, when the codon
specifying alanine (GCT) at the second position of
transcripts was substituted for tryptophan (TGG),
the resultant enzymes show catalytic properties sim-
ilar to the native enzymes isolated from various ani-
mal sources [36].
2.1.2. Fusion with leader peptide
Presence of hydrophobic membrane anchoring
sequences at the N-terminal of human CYPs is
known to be deleterious for their expression in bac-
teria [15]. This problem was overcome by construct-
ing an in frame fusion of the CYP cDNA with a 21
amino acid long leader sequence of E. coli outer
membrane protein, ompA, in frame with the CYP
initiation codon. In addition, a spacer sequence
for Ala-Pro was added at the ompA–CYP junction
which, leads to the proteolytic degradation of leader
sequence and thereby restores the production of the
native CYP protein. Similarly, CYP reductase fused
to a pelB leader sequence was found to be optimal
for the expression of OR on bacterial membranes.
However, such N-terminal modifications are not
reported for the expression of CYPs in yeast, bacul-
oviral or mammalian expression systems.
2.1.3. Co-expression of CYP and OR
Functional monooxygenase system required for
CYP activity can be reconstituted by the co-expres-
sion of CYP isozyme and OR. To achieve this objec-
tive, the following protocols have been employed
extensively to optimize the expression of individual
CYPs:
In E. coli, co-expression of OR is achieved by
construction of CYP–OR fusion proteins [37], or
CYP and OR are expressed separately using two
different compatible plasmids like CYP gene in
pCWOri+ vector and the OR gene in pACYC-184
vector [38].
In yeast cells, low levels of endogenous OR
(120–140 nmol/min/mg vs. 220 nmol/min/mg in
mammalian tissue) were found to be insufficient
for the activation of CYP [23]. Catalytic properties
of CYP expressed in yeast were improved by the
co-expression of human OR using high copy num-
ber plasmid-based expression system. Recently, a
yeast strain co-expressing hOR and hCYP3A4 has
 4
Table 1
Comparative analysis of various expression systems for P450 production
Expression Timea Typical Strengths Challenges
system yield
E. coli 3 months 150–500 • Availability of multiple cloning vectors & related recombi- • Often requires modifications of N-terminal sequences [26,27]
(bacteria) nmol/L nant technologies [18,19] • Catalytic activity of P450s requires addition of heme precursor like
[15] • Relatively inexpensive compared to other expression sys- delta amino levulinic acid to the growth media [28]
tems [20] • Temperature optimization is necessary to ensure adequate heme
• Requires short culture time incorporation and proper folding of recombinant P450s.
• Low maintenance & ease of purification [21] • Catalytic activity of bacterially expressed P450s requires reconstitu-
• Amenability to large scale production [22] tion with phospholipid(s) and accessory electron transfer proteins,
such as P450 oxidoreductase (OR) and cytochrome b5 [29]
S. cerevisiae 3 months 1–3 nmol/ • Ease of growth and purification • Yeast system possess low level of endogenous yeast reductase, which

K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15

(yeast) L [15] • Similar to the mammalian cells in protein synthesis, process- results in low specific activity of recombinant human P450 [30]
ing, and membrane compartmentalization [23] • Endogenous cytochrome b5 in yeast is unable to enhance the activity
• Does not require any modification in the cDNA sequence of human CYPs, and is usually supplemented in the microsomal
coding for the P450 protein preparations [23]
• Endogenous membrane anchoring sequences target it to • Provision of heme is often limiting and endogenous CYPs compli-
endoplasmic reticulum and integrate into the ER cate interpretation of spectra and catalytic data in some strains [30]
• Yeast microsomes prepared from such recombinant strains
offer a rich source of P450s for in vitro studies [23]
Insect cells 2–3 103 ± 29 • Expression levels are high as compared to other systems [16] • Requires co-expression of OR, as insect cells lack endogenous OR,
with months nmol/L • Allows proper targeting of the recombinant protein into the and culture media must be supplemented with hemin [31,24]
baculoviral [16] host’s sub-cellular compartment [13] • Microsomes prepared from the insect cells, even in the presence of
vectors hemin contain significant amount of heme lacking apoprotein. This
apoprotein fraction is catalytically inactive and not spectroscopically
detected by CO difference spectra but could easily be detected by
western blotting [32]
• Cumbersome scale-up issues
Mammalian 6 months 20– • Mammalian cell lines endogenously express small quantities • Lymphoblastoid cells used for stable expression of CYPs contain
cells 100 pmol/ of OR and b5, which seems sufficient to activate expressed negligible amount of constitutive CYPs [24]
mg protein recombinant CYPs [15,24] • The expression level of recombinant P450s in mammalian cells is rel-
[17] • They possess the machinery for sub-cellular localization of atively low compared to other expression systems
P450 & electron transport chains. • Expensive growth culture media
• Capable of carrying out second phase metabolism, after the • Industrial production of CYPs is commercially a poor ‘‘return-on-
P450 reaction(s). investment’’ [17]
• Transient expression systems based on viral expression vec-
tors; e.g., SV40 based vectors in COS cells and vaccinia
virus system in HepG2 are available [25]
• The episomal mammalian stable expression system using
Epstein bar virus in human B lymphoid cells is commer-
cially used for the production of CYPs [15]
a
Timelines are only indicative, and denotes authors’ personal experience. It includes cloning, stable expression, and/or purification of a target P450.
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
been shown to have 30-fold enhanced activity of the
CYP3A4, compared to its parental strain [23].
In insect cells, OR required for the catalytically
active CYP isozymes is absent. This deficiency is
overcome by providing OR to the insect cell line
using the following approaches: Both CYP and
OR can be produced individually and mixed
together to reconstitute active CYP system
[31,39,40], or the desired CYP and reductase partner
were co-expressed using a single virus [41]. In
another approach, insect cells were co-infected with
independent viruses containing individual CYP and
a reductase partner [42].
In mammalian lymphoblast cell lines, certain
CYP isoforms require additional expression of
active OR. Here, the activities of 1A2, 2B6, and
2C19 were supported by the endogenously
expressed OR, whereas 2A6, 2C8, 2C9, 2D6, and
3A4 required cotransfection with the OR cDNA
to bring a significant improvement in the enzymatic
activity of individual CYPs [32].
2.1.4. Addition of delta-amino levulinic acid (d-ALA)
d-ALA is the precursor for the heme-moiety in
CYP and needs to be supplemented in the growth
media for optimum production of CYP. In
E. coli, the yield of CYP1B1 could be enhanced
by 20-fold with the inclusion of 2.5 mM d-ALA
in the growth medium [28]. In yeast, excessive glu-
cose (30%), heat shock, and the addition of the d-
ALA favorably improved the activity of CYP3A4.
Additional glucose helps to increase the growth
rate and protein expression levels of yeast cells,
and d-ALA, helps to improve both the stability
and activity of CYP enzymes [23]. In Insect cells,
Hemin chloride or iron-citrate and d-ALA were
added to the culture media as a source of heme
to compensate low levels of endogenous heme. A
sharp increase in the synthesis of functional CYP
2E1 was seen in the Tni cells supplemented with
of 0.2 lg/ml heme-albumin [43].
2.1.5. Addition of cytochrome b5
Cytochrome b5 is an alternate electron carrier for
the second electron transfer involved in the catalytic
activity of CYP. Co-expression of cytochrome b5
along with CYP3A4 and OR has been shown to
increase the oxidation of nifedipine and testosterone
by CYP 3A4 to 167% in bacterial membrane prepa-
rations [44,45].
Recombinant CYPs are required to be supple-
mented with OR, and membrane phospholipids
5
for catalytic activity. However, the conditions for
reconstitution vary for each CYP, e.g., CYP3A4
requires cytochrome b5 for its complete catalytic
activity. The optimal conditions for reconstitution
of CYP1A2, 2C9, 2E1, and 3A4 can be found in a
recent report by Yamazaki and Shimada [46].
2.2. Purification of recombinant CYPs
Microsomes prepared from yeast, insect or mam-
malian cell lines with CYP coding plasmids provide
rich source of CYP enzymes for in vitro PK studies.
For structural studies, CYP isoform 1A2, 2C9, 2D6,
and 3A4 have been purified from yeast microsomes
[47]. CYP proteins were solubilized from yeast
microsomes in the presence of 0.6% sodium cholate
and subjected to octylamino-sepharose 4B column
chromatography. CYPs eluted with 0.2–0.5% Emul-
gen were subjected to DEAE column chromatogra-
phy. The flowthrough fractions containing CYPs
were subjected to hydroxylapatite column chroma-
tography. CYPs were further eluted by applying
0.01–0.35 M Phosphate gradient. Typical yields
obtained were 17 pmol/mg for CYP2C19 and 9–
14 nmol/mg for CYP1A2, 2C9, 2D6, and 3A4 [47].
For the purification of CYP3A4 from insect cells,
microsomes were solubilized with 0.6% cholate and
subjected to DEAE column chromatography. CYP
from flow through and wash fractions was loaded
on to Hydroxylapatite column to get rid of the
detergent. CYP3A4 was recovered by washing the
column with 300 mM phosphate buffer. Typical
yield obtained was 12.7 nmol CYP/mg protein
[16]. From bacterial cells, CYPs were purified from
its membranes fractions. C-terminal His tag CYP
3A4 was solubilized from bacterial membranes by
non-ionic detergents and purified to homogeneity
using sequential DEAE and metal affinity chroma-
tography [21].
For crystallization studies, CYP3A4 with C-ter-
minal His tag was solubilized with CHAPS and
purified by metal ion affinity chromatography fol-
lowed by CM-Sepharose [48].
2.3. Validation of recombinant CYPs
CYP content from various recombinant expres-
sion systems is quantified by calculating the differ-
ence between Fe2+ vs Fe2+-CO absorption
spectrum at 450 nm [49]. CYP reductase is measured
by cytochrome c reductase assay performed at
550 nm [50]. Recombinant CYPs obtained from het-
6 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
erologous expression systems usually retain catalytic
properties, and substrate specificities like their
native human liver microsomes (HLM) counter-
parts. Recombinant CYPs are normally validated
by comparing the specificity, affinity and capacity
of the enzymes with those from the HLM [51].
The validation of specificity requires screening of
the metabolic activity for each isoform towards a
panel of standard substrates. Affinity can be vali-
dated by studying kinetics of biotransformation of
standard substrate. If Km values for standard sub-
strates are similar then affinity of both enzymes is
considered identical. Kcat or turn over number is
another measure of affinity of enzyme towards the
substrate. The concentration of accessory electron
transfer proteins and the lipid composition of the
membrane could alter the kinetic properties of
recombinant CYPs. Optimal molar ratio of OR to
recombinant CYPs is important for the maximum
activity of individual enzyme. For bacterially
expressed 2C19, the OR/2C19 a ratio of 20:1 was
found to be optimal for diazepam-N methylation
activity [52]. In addition, membrane proteins and
lipids from recombinant system might exhibit non-
specific binding to compounds leading to decreased
amount of ‘‘metabolically free’’ drug. Apparently
only unbound drug interacts freely with these
metabolizing enzymes and such non-specific interac-
tions could further lead to altered kinetic properties
of recombinant enzymes [53].
Appropriate purification steps are required for
the recombinant proteins to obtain results compa-
rable with the CYPs expressed from HLM. IC50s
should be determined against a series of standard
inhibitors for both recombinant and HLM
enzymes. Due to high purity and specific activity
of recombinant proteins total protein concentra-
tions tends to be lower in incubation with recombi-
nant enzymes. In a study of recombinant CYP2D6
inhibition, a 10-fold increase in total protein con-
centration with constant CYP2D6 content led to
two to ninefold higher IC50 values for inhibition
of bufuralol 1 hydroxylation by fluoxetine, ezlopit-
ant and imipramine [53]. The use of RAF (relative
activity factor) is recommended to normalize values
to CYP content. RAF is the ratio of Vmax values
for the metabolism of marker substrates in incuba-
tion with HLM and recombinant enzymes [54].
Also, addition of control protein can be optimized
for IC50 determination using recombinant enzymes.
Taken together, these proposed mechanisms clearly
suggest that the optimization of conditions for the
determination of Km and Ki are essential for the
prediction of in vitro–in vivo extrapolations in new
drug discovery.
3. Role of CYPs in anticancer drug discovery
Recombinant CYPs are used routinely in new
drug discovery to predict in vitro PK properties that
can be extrapolated to in vivo PK of the NCE. Var-
ious functional assays are used to screen NCEs in
new drug discovery and some of key strategies for
the use of CYPs in the discovery and development
of new drug candidates are described below:
3.1. Role of CYPs in metabolism of existing
anticancer drugs
Hepatic CYP-mediated oxidation and conjuga-
tion reactions lead to the elimination of compounds
from human body [55]. Thus, the residence time of a
compound in systemic circulation is directly depen-
dent on the resistance to this first pass metabolism.
Intrinsic clearance (CLintr) is determined to extrap-
olate the in vitro data for prediction of in vivo
metabolism. It is a direct measure of enzyme activity
towards a drug and is not influenced by other deter-
minants like hepatic blood flow or plasma protein
binding.
More than 30 CYP isoforms are known to
metabolize xenobiotics and are used extensively to
study the metabolism of NCEs. Several CYPs, most
notably, CYP 1A2, 1B1, 2A6, 2C9, 2C19, 2D6, 2E1,
and 3A4/5 are responsible for the metabolism of
known anticancer drugs [56]. Specific CYP isoforms
and their targeted anticancer drugs are listed in
Table 2.
Endogenous CYP isoforms expressed in tumor
cells may lead to the metabolism of active drug into
inactive or less potent form, thereby, altering the
half-life and kinetics of the administered therapeutic
molecule. One such example is the hydroxylation of
Paclitaxel into 6-hydroxy metabolite that decreases
the potency of the drug by 30-fold [75].
In a rat oncology model, regulation of CYPs was
reported to occur primarily at post-transcriptional
level. Animals were treated with a single dose of Tri-
acid (a metabolite of dehydrotarplatin), which led to
the down-regulation of androgen-dependent male-
specific CYP2C11 mRNA and its apoprotein [76].
Recently, heavy metal ions; arsenate, cadmium,
and chromium, were shown to regulate CYP1A1
mRNA at post-transcriptional level in Hepa
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15 7

Table 2
Current anticancer drugs that are substrate for CYP 450 enzymes
Cancer Drugs CYP isoforms involved in References
Metabolism Activation
Breast Docetaxel CYP3A, CYP1B1 [57,58]
Ellipticine CYP3A4, CYP1A1, CYP1A2, [59]
CYP1B1, CYP2C9
Exemestane, Fulvestrant, CYP3A [57,60,61]
Vinblastine, Vinorelbine
Letrozole CYP3A, CYP2A6 [60]
Mitoxantrone CYP3A, CYP1B1 [58,62]
Paclitaxel CYP3A, CYP2C8 [58,63]
Tamoxifen CYP3A, CYP2D6, CYP2C9, [58,62–64,67,65]
CYP1B1, CYP2C19
Toremifene CYP3A, CYP1A2 [60]
Tegafur CYP2A6, CYP2C8, [66–68]
CYP1A2
Thiotepa CYP3A, CYP2B6 [69]
NSCLC Vinorelbine, Erlotinib, CYP3A4 [57,60,61]
Vindesine
Docetaxel CYP3A, CYP1B1 [57,58]
Gefitinib CYP3A, CYP2D6 [70]
Paclitaxel CYP3A, CYP2C8 [58,63,71]
SCLC Etoposide CYP3A4, CYP2E1, CYP1A2 [57,72,73]
Topotecan CYP3A [74]
Leukemias Idarubicin CYP2D6, CYP2C9 [60]
Imatinib, Teniposide, CYP3A [73]
Vindesine
Mitoxantrone CYP3A, CYP1B1 [58,62]
Cyclophosphamide CYP2B6, CYP2C19, [57,66]
CYP3A4
Sarcoma Paclitaxel CYP3A, CYP2C8 [63,71]
Vinblastine, Vincristine CYP3A [60,57,61]
Ifosfamide CYP3A, CYP2B6 [57,66]
Ovarian Paclitaxel CYP3A, CYP2C8 [58,63]
Topotecan CYP3A [74]
Thiotepa CYP3A, CYP2B6 [60]
Prostate Docetaxel CYP3A, CYP1B1 [60]
Flutamide CYP1A2 [60]
Colon Irinotecan CYP3A [66]
Tegafur CYP2A6, CYP2C8, [66–68]
CYP1A2
Lymphomas Vinblastine, Vincristine CYP3A [60,57,61]
Cyclophosphamide CYP2B6, CYP2C19, [57,66]
CYP3A4

1C1C7 cells, where chromium increased the half-life
of CYP1A1 [77]. Relatively little is known with
respect to the stability of various CYP mRNAs
and their proteins after treatment with different che-
motherapeutic agents.
3.2. CYP metabolism and the discovery of new
anticancer molecules
A significant mechanistic insight into the role of
CYPs in the metabolism of anticancer drugs has
now been elucidated. A clear understanding of these
metabolic pathways involved will allow us to predict
with certainty, the efficacy and safety of novel anti-
cancer molecules under development [78]. Antican-
cer agents are incubated with HLM or
recombinant cytochrome CYPs and their metabo-
lites are identified by Mass Spectrometry. The data
generated from such studies will help us in:
(1) Designing new chemotherapeutic agent which
retain anticancer properties but show reduced
CYP inhibitory activity or CYP induction
potential. Such drugs will also help in reduc-
8 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
tion of drug–drug interactions in patients
undergoing multiple therapies. A SAR of
ADS102550, thienyl based hydroxamic acids
series of HDAC inhibitors has lead to identifi-
cation of a compound with reduced CYP inhi-
bition potential, retaining the HDAC
inhibitory activity [79].
(2) Further identification of active metabolites
can be useful in understanding mechanism of
action of new chemotherapeutics. Novel anti-
neoplastic agent, ellipticine, was shown to
act by intercalation into DNA and inhibition
of DNA topoisomerase II. Recently, a novel
mode of action mediated by CYP3A was iden-
tified. 9-hydroxy and 7-hydroxyellipticine
metabolites of ellipticine by CYP3A and per-
oxidases lead to formation of DNA adducts
increasing the susceptibility to ellipticine [80].
(3) Inhibitors or substrates of CYP responsible
for the metabolism of anticancer agents can
be co-administered to increase the half-life of
drug and thus reduce dosing in patients. Co-
administration of cyclosporine (substrate for
CYP3A4) increased oral bioavailability of
docetaxol in cancer patients [81].
(4) CYP metabolism of inactive pro-drug into
cytotoxic drug can be explored in designing
safer potent anticancer agents. Several new
agents now entering clinical trials (e.g., Phor-
tress and AQ4N) are specifically designed to
exploit tumor cytochrome CYP, resulting in
local bioactivation of the cytotoxicity within
the tumor cells [82].
3.3. Polymorphism in CYPs and anticancer therapy
Reaction phenotyping is employed to identify the
isoform(s) that catalyze the metabolism of a particu-
lar drug. Several polymorphs of human CYPs are
reported in the literature and have been extensively
categorized. These reactions are used for predicting
the consequences of drug administration to a partic-
ular patient population, which are carrier of a partic-
ular allele. Enzyme deficiency due to CYP2C19
alleles, 2C19*2 and 2C19*3, are predominant
amongst Asian populations, and should be included
in reaction phenotyping, while conducting clinical tri-
als [83]. Several recombinant human isoforms are
available commercially for reaction phenotyping.
Recently CYPs have been classified into two
groups based on the functional characterization of
its polymorphs [60]. Class I enzymes are well con-
served and are active in the metabolism of drugs
and pre-carcinogens & includes CYP1A1, 1A2,
2E1, and 3A4, whereas Class II category of enzymes
have important functional polymorphs and are
involved in the metabolism of drugs. This latter
class includes CYP 2B6, 2C9, 2A6, and 2D6.
Identification of CYP SNPs is done using poly-
merase chain reaction-restriction fragment polymor-
phism genotyping. Genotyping of 500 Caucasian
patients from Europe have identified the
CYP3A5*11 allele. Functional characterization of
recombinant CYP3A5*11 purified from E. coli dem-
onstrated lower 3A5 activity [84]. Another SNP
CYP 2A6*7 represents gene deletion mostly in Asian
population, at a frequency of 7–22% as compared to
0.5–1% in Caucasians. In a recent study, patients
with poor metabolizing activity of tegafur were
found to be heterozygous for 2A6*4 and 2A6*11
[85]. These observations clearly make a point that
before including patients in clinical trials, they
should be screened for appropriate CYP poly-
morph(s) to avoid adverse drug reactions. Important
CYP polymorphs metabolizing existing anticancer
drugs are listed in Table 3. A Database of ethnic fre-
quency of clinically relevant CYP polymorphs and
adverse drug reaction of commonly used medicines
has been used in creating a pharmocogenetic drug
monitoring (PDM) system in Korea [86]. This
PDM demonstrated that CYP 2C9, 19, and 2D6
are major CYP enzymes of clinical importance for
pharmacological effects and safety in the Korean
population [86]. Therefore, it appears highly likely
that recombinant polymorphic CYPs will be useful
in predicting certain pharmacological effects of exist-
ing chemotherapeutics in a particular population.
3.4. Role of CYPs in activation of anticancer pro-
drugs
Idiosyncratic effects of a drug can occur by pro-
longed exposure in humans. It has been suggested
that bioactivation caused by CYPs could lead to
formation of reactive metabolites that are electro-
philic in nature and are capable of causing covalent
modification of protein or nucleic acids [87]. Some
of these reactions are performed by pre-incubating
high concentrations of compounds with an excess
of enzyme in the presence of glutathione or cyanide.
Resulting metabolites are purified by chromatogra-
phy and further detected by mass spectroscopy [88].
Recombinant enzymes are preferred in these reac-
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15 9

Table 3
Polymorphic P450 enzymes metabolizing anticancer drugs*
CYP isozymes No. of variants Important isoform Functional alteration Altered amino acid
1A1 15
1A2 36
2A6 52 2A6*4 Gene deletion (inactive)
2B6 53
2C8 12 2C8*3 Altered substrate specificity R139K,
K399R
2C8*4 I264M
2C9 37 2C9*2 Decreased enzyme activity Cys144 (R144C)
2C9*3 Decreased enzyme activity Leu359 (I359L)
2C19 25 2C19*2 Premature stop codon (inactive) splicing defect; I331V
2C19*3 W212X, I331V
2C19*17 Mutation in promoter. Increased transcription I331V
2D6 106 2D6*1
2D6*10 P34S, S486T
2E1 13 2E*3 Active V389I
2E*2 30% active R76H
3A4 40 3A4*1B Gene alteration in promoter
3A 4*2 Decreased enzyme activity S222P
1B1 23 1B1*7 Decreased Estradiol metabolism R48G, A119S, L432V, A443G
*Compiled from CYP allele homepage (http://www.imm.ki.se/cypalleles), [56,60].

tions as they exhibit higher specific activity com-
pared to HLM.
Various CYP isoforms are quite often over-
expressed during the development phase of a cancer
cell from a benign form to a highly metastatic malig-
nant phenotype. Two recent examples include the
over-expression of 1A1 & 1B1 isotypes in various
cancers [89,90]. This ability of using these over-
expressed CYP isoforms for the bioactivation of
inactive pro-drugs into an active cytotoxic molecule
in tumor tissues is being exploited for designing che-
motherapeutic agents. CYP1B1 has been shown to
convert inactive pro-drug, reseveratrol, into active
anticancer agent piceatannol in cancer cells [91]
and was also reported to convert DMU 135 to
DMU 117. DMU 117 is a non-selective tyrosine
kinase (and COX) inhibitor that is being explored
as a therapeutic molecule against gastrointestinal
cancers [92]. In solid tumors, such bio-activation
of a pro-drug by CYPs, will aid in decreasing the
cytotoxicity to the adjacent normal cells, and will
also go a long way in targeting tumor-specific acti-
vation of chemotherapeutic agents.
3.5. CYP inhibition potential of anticancer drugs and
its implications for drug–drug interactions
Inhibition of CYP could affect accumulation or
elimination of drugs administered simultaneously,
and might cause irreversible tissue damage to
patients undergoing multiple therapies [93]. Com-
mercially available recombinant CYP inhibition kits
offer HTS mode of predicting inhibition potential of
a large number of compounds under investigation.
Two different approaches are normally followed to
streamline the screening of the compounds. (1) If
a particular class of drugs has liability with respect
to a single CYP, screening can be done initially
for that isoform to narrow down the number of
compounds to be screened with other isoforms.
While working with macrolides, we screen initially
for liability against CYP3A4. Compounds showing
reduced liabilities can be taken further to determine
liability for other major hepatic CYP isoforms. (2)
In another parallel approach, liabilities of major
CYPs can be checked at a single concentration of
compounds, usually at 25 lM. Exact IC50s could
be determined further for selected compounds that
show greater inhibition at this single concentration.
IC50s of compounds for all five major CYPs (1A2,
2D6, 3A4, 2C9, & 2C19) play important role in
deciding the molecules to be taken forward for
developmental studies.
Anticancer agents, like other therapeutics, are
known to have its own pharmacological profile and
PK properties, which may influence the PK parame-
ters of another drug administered simultaneously
[93]. This will require careful monitoring, as patients
with advanced cancer quite often require other med-
ications as well. Such drug–drug interaction could
10 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
alter the efficacy of both the drugs and might lead to
adverse reactions in some patients. E7070 was dis-
covered as novel sulfonamide anticancer agent that
arrests cancer cells at the G1/S boundary of the cell
cycle. In vitro studies showed that E7070 has the
potential to inhibit several CYP enzymes, including
CYP2C9, CYP2C19, CYP2D6, CYP2E1, and
CYP3A4. The oral anticoagulant, Acenocoumaro,
is metabolized in humans by CYP2C9. Upon co-
administration, E7070 decreased metabolism of Ace-
nocoumarol even at a very low concentration
(2.1 lM), thereby reducing the systemic clearance
of Acenocoumarol [94]. In the absence of careful
analysis of individual patients, this drug–drug inter-
action may result in hypoprothrombinemia and a
hemorrhagic tendency [94].
Changes in PK properties of co-administered
drugs are also regulated by drug efflux pump; P-gly-
coprotein (Pgp, product of MDR1) [93]. Most com-
pounds, which are substrates for Pgp are also
substrates for CYP3A4 and can act synergistically
in vivo. Gleevac (imatinib mesylate) is administered
orally for the treatment of chronic myeloid leuke-
mia (CML). It is a substrate for 3A4 but simulta-
neously inhibits CYP3A4, 2C9, 2D6. Therefore,
agents like Erythromycin or ketokonazole (3A4
inhibitors) and dexamethhazone or carbamazepine
(3A4 inducers) will change concentrations and bio-
availability of Gleevac in a multi-drug administra-
tion scenario, and might lead to adverse events in
patients [93,95].
Determination of the in vitro CYP inhibition
potential of novel anticancer drugs is a tool, which
could help us in predicting possible drug–drug inter-
actions. It may also be useful in designing a treat-
ment regime to be followed in patients undergoing
chemotherapy, where some of these CYPs could
be monitored as prognostic bio-markers.
3.6. CYPs as prognostics/diagnostic markers for the
detection of tumors
Certain CYP isoforms like 1B1 and 1A2 are
induced in response to environmental mutagens like
UV [96]. In addition, altered gene regulation in
tumor cells could lead to the activation of endoge-
nously expressed CYPs. Over-expression of
CYP1B1, CYP2J2, and CYP2W1 has been reported
in various cancer tissues [90,97,98]. Furthermore,
several carcinogens that are metabolized by CYP
enzymes binds to AhR, thereby inducing the expres-
sion of CYP1B1 in prostate, kidney, ovarian, breast,
and colon cancers. CYP2J2 was found to be over-
expressed in esophageal, adenocarcinoma, pulmon-
ary squamous carcinoma, and breast carcinoma
tissues [98]. Thus careful determination of expres-
sion levels of these CYPs could also serve as a good
bio-marker for early detection of cancer in tumor
biopsies. Indeed, CYP1B1 is already being used as
a prognostic marker for cancer [Patent No:
WO0135810]. Depending upon the secretion &
detection of some of these CYPs into blood, urine,
sputum & stool, non-invasive diagnostic and/or
prognostic test could also be developed for various
cancers.
3.7. CYPs inhibitors as anticancer drugs
Are tumor cells capable of efflux of chemothera-
peutic drugs out of their cellular compartments with
help from the CYP enzymes? Inhibition of these
endogenous CYPs in cancerous cells might thus offer
a novel target for designing new anticancer agents,
whereas simultaneous activation of drug-specific
CYPs inside the normal cells (adjacent to the tumors)
might help in reducing adverse reactions. Extra-
hepatic CYPs, that are involved in the metabolism
of hormones, and are not responsible for xenobiotic
metabolism, are also being targeted using this
approach. For example, inhibitors of CYPs responsi-
ble for the metabolism of hormones or vitamins, are
being used as anticancer drugs for hormone depen-
dent cancers. CYP 19, an aromatase, catalyses a
rate-limiting step in the conversion of androgen to
estrogen. Aromatase inhibitors, like exemestane, or
non-steroidal trazoles, letrazole(femera), and anas-
trozole are used successfully to maintain low levels
of estrogen in post-menopausal women patients with
hormone sensitive breast cancer [99].
Vitamin D3 metabolite, 1a,25(OH)2D3 (calci-
trol), inhibits proliferation, promotes differentiation
and induces apoptosis in colon and prostrate cancer
cells [100]. However, deactivation of 1a,25(OH)2D3
occurs due to hydroxylation at C4 via CYP 24
expressed in kidney cells. QW-1642, a synthetic ana-
logue of 1a,25(OH)2D3 was identified, which inhib-
its the expression of CYP24, and further inhibited
cell proliferation. This synthetic analogue has
shown good efficacy in pre-clinical studies and could
be developed as a chemotherapeutic agent for the
prostate cancer [101].
Various strategies to inhibit or exploit CYP
enzymes for the treatment of cancer have been
recently reviewed by Bruno and Njar [99].
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
3.8. SiRNA and CYP metabolism
RNAi offers a powerful tool to achieve target
selective knockdown of specific genes. Recently
hPXR-siRNA was used for down-regulating the
expression of CYP genes controlled by the PXR ele-
ment in primary human hepatocytes [102]. These
siRNAs not only suppressed 3A4 transcript levels
to about 18% in primary hepatocytes but it also sup-
pressed the induction of 3A4 by inducers like Phe-
nobarbital (1 mM) and Clotrimazole (10 lM).
Besides CYP3A4, levels of 2A6, 2C8, and 3A5 were
also suppressed by these siRNAs. In another study,
3A4III siRNAs (specific for suppression of
CYP3A4) significantly diminished cytotoxicity of
two CYP3A4 substrate drugs, cyclophosphamide
and ifosamide in CHL-3A4 cells [103]. It is clear
that the siRNA technology could be explored fur-
ther to suppress organ specific toxicity caused by
induction or suppression of different CYP isoforms
by anticancer drugs [103]. Furthermore, RNAi tech-
nology may be able to target multiple genes in met-
abolically diverse pathways, and eventually help us
override drug-resistance issues normally encoun-
tered with single target-based drugs. Co-administra-
tion of siRNAs along with a therapeutic drug will
not only reduce drug–drug interaction but can also
offer altered metabolism leading ultimately to an
increased specificity and reduced toxicity. Most
importantly, unlike the small molecules that inhibit
various CYP polymorphs differentially, siRNAs will
not discriminate amongst these isoforms and can
regulate all the polymorphs uniformly.
A detailed analysis of this very powerful technol-
ogy in cancer is beyond the scope of this review,
although two recent reviews outlining RNAi-medi-
ated anti-tumorigenesis for drug-target validation
have appeared in the literature [104,105]. Effective
applications of such siRNAs further warrant the
development of more efficient in vivo siRNA deliv-
ery systems and then analyzing their therapeutic
potential in animal models of cancer.
4. Conclusion and future directions
In this review, we have summarized methods of
production of recombinant CYPs in various expres-
sion systems. E. coli and yeast offer simple & eco-
nomical modes of production, while insect cells
with baculovirus expression system have shown
highest yields. Human lymphoblast cells are also
11
used commercially as they possess most of the ancil-
lary proteins required for the activation of CYPs.
Recombinant CYPs play a major role in the dis-
covery of novel anticancer compounds. Individual
CYP enzymes are used to study the metabolism
and inhibition of a potential new molecule. Inside
the tumor cell, metabolism by the endogenous
CYPs is being exploited for the activation of a
non-toxic pro-drug into a cytotoxic drug. Simulta-
neously, such studies could give insights into possi-
ble drug–drug interactions and they may also help
in determining the safety and efficacy of these
NCEs.
Polymorphism in CYPs could lead to the identi-
fication of the genetic parameters which may cause
varied therapeutic response in different ethnic
groups. Recombinant allelic CYPs are being used
to predict the efficacy of new molecules in geneti-
cally diverse populations, and some of these individ-
ual CYP SNPs data has been compiled onto
microarray slides, which are now available from
commercial vendors. Inhibitors of endogenously
expressed CYPs, like 1B1, are being explored by
many pharmaceutical companies as possible thera-
peutic targets. Over-expressed CYPs, with an
altered substrate/inhibitor specificity, offer great
advantage for the designing of safe anticancer mol-
ecules, as levels of these CYPs are either undetect-
able or miniscule in normal cells. Recombinant
CYPs will enable us to screen compounds that mod-
ulate CYP levels inside the tumor cells. Co-adminis-
tration of anticancer drugs with CYP activity
modulators can activate/inactivate pro-drugs, or
could reduce the formation of cytotoxic metabo-
lite(s), thereby leading to the discovery and develop-
ment of safe and efficacious NCEs. It remains to be
seen that at what stage of cancer development such
a combination strategy will be most effective and
will be close to the tolerated doses. Further, with
RNAi technology, we may be able to dissect not
only the mechanism of action of NCEs under devel-
opment, but also design compounds which will tar-
get multiple ‘‘culprit genes’’ in the signal
transduction pathway of cancer.
Acknowledgements
This work was funded by Ranbaxy Research
Laboratories, India. We thankfully acknowledge
our recombinant P450 group: Dr. Deepika Singh,
Ms. Alka Khanna, Ms. Pratibha Sharma and Mr.
Hemant Kumar Chavan for their assistance during
12 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
the compilation of this review and for generating the
unpublished data.
References
[1] F.J. Gonzalez, The molecular biology of cytochrome P450s,
Pharmacol. Rev. 40 (1988) 243–288.
[2] B.S. Kalra, Cytochrome P450 enzyme isoforms and their
therapeutic implications: an update, Indian J. Med. Sci. 61
(2007) 102–116.
[3] A. Juneja, K.S. Saini, R. Soni, Discerning relationships
among human cytochrome P450s by computational anal-
yses, Bioinf. Trends 1 (2006) 1–12.
[4] G. Zlokarnik, P.D. Grootenhuis, J.B. Watson, High
throughput P450 inhibition screens in early drug discovery,
Drug Discov. Today 10 (2005) 1443–1450.
[5] F. Gao, D.L. Johnson, S. Ekins, Optimizing higher
throughput methods to assess drug–drug interactions for
CYP1A2, CYP2C9, CYP2C19, CYP2D6, rCYP2D6, and
CYP3A4 in vitro using a single point IC(5), J. Biomol.
Screen. 7 (2002) 373–382.
[6] M.C. McFadyen, W.T. Melvin, G.I. Murray, Cytochrome
P450 enzymes: novel options for cancer therapeutics, Mol.
Cancer Ther. 3 (2004) 363–371.
[7] N.J. Lakhani, M.A. Sarkar, J. Venitz, W.D. Figg, 2-
Methoxyestradiol, a promising anticancer agent, Pharma-
cotherapy 23 (2003) 165–172.
[8] S. Jennewein, C.D. Rithner, R.M. Williams, R.B. Croteau,
Taxol biosynthesis: taxane 13 alpha-hydroxylase is a
cytochrome P450-dependent monooxygenase, Proc. Natl.
Acad. Sci. USA 98 (2001) 13595–13600.
[9] C.M. Masimirembwa, C. Otter, M. Berg, M. Jönsson, B.
Leidvik, E. Jonsson, T. Johansson, A. Bäckman, A.
Edlund, T.B. Andersson, Heterologous expression and
kinetic characterization of human cytochromes P-450:
validation of a pharmaceutical tool for drug metabolism
research, Drug Metab. Dispos. 27 (1999) 1117–1122.
[10] R.B. Vail, M.J. Homann, I. Hanna, A. Zaks, Preparative
synthesis of drug metabolites using human cytochrome
P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase
expressed in Escherichia coli, J. Ind. Microbiol. Biotechnol.
32 (2005) 67–74.
[11] F.P. Guengerich, A. Parikh, Expression of drug-metabo-
lizing enzymes, Curr. Opin. Biotechnol. 8 (1997) 623–628.
[12] P. Urban, C. Mignotte, M. Kazmaier, F. Delorme, D.
Pompon, Cloning, yeast expression, and characterization of
the coupling of two distantly related Arabidopsis thaliana
NADPH-cytochrome P450 reductases with P450
CYP73A5, J. Biol. Chem. 272 (1997) 19176–19186.
[13] C.A. Lee, T.A. Kost, C.J. Serabjit-Singh, Recombinant
baculovirus strategy for coexpression of functional human
cytochrome P450 and P450 reductase, Methods Enzymol.
272 (1996) 86–95.
[14] C.L. Crespi, V.P. Miller, The use of heterologously
expressed drug metabolizing enzymes-state of the art and
prospects for the future, Pharmacol. Ther. 84 (1999) 121–
131.
[15] T. Friedberg, M.P. Pritchard, M. Bbandera, S.P. Hanlon,
D. Yao, L.A. McLaughlin, S. Ding, B. Burchell, C.R. Wolf,
Merits and limitations of recombinant models for the study
of human P450-mediated drug metabolism and toxicity: an
intralaboratory comparison, Drug Metabol. Rev. 31 (1999)
523–544.
[16] J.T. Buters, K.R. Korzekwa, K.L. Kunze, Y. Omata, J.P.
Hardwick, F.J. Gonzalez, cDNA-directed expression of
human cytochrome P450 CYP3A4 using baculovirus, Drug
Metab. Dispos. 22 (1994) 688–692.
[17] F.J. Gonzale, K.R. Korzekawa, Cytochrome P450 expres-
sion systems, Annu. Rev. Pharmacol. Toxicol. 35 (1995)
369–390.
[18] F.P. Guengerich, A. Parikh, E.F. Johnson, T.H. Richard-
son, von C. Wachenfeldt, J. Cosme, F. Jung, C.P. Strass-
burg, M.P. Manns, R.H. Tukey, M. Pritchard, S. Fournel-
Gigleux, B. Burchell, Heterologous expression of human
drug-metabolizing enzymes, Drug Metab. Dispos. 25 (1997)
1234–1241.
[19] T. Uno, A. Nakao, S. Masuda, Y. Taniguchi, K. Kana-
maru, H. Yamagata, M. Nakamura, H. Imaishi, K. Oono,
Modification of small molecules by using cytochrome P450
expressed in Escherichia coli, J. Ind. Microbiol. Biotechnol.
33 (2006) 1043–1050.
[20] M.P. Pritchard, L. McLaughlin, T. Friedberg, Establish-
ment of functional human cytochrome P450 monooxygen-
ase systems in Escherichia coli, Methods Mol. Biol. 320
(2006) 19–29.
[21] F.P. Guengerich, M.V. Martin, Purification of cytochromes
P450: products of bacterial recombinant expression sys-
tems, Methods Mol. Biol. 320 (2006) 31–37.
[22] Y. Kanamori, K. Fujita, K. Nakayana, H. Kawai, T.
Kamataki, Large-scale production of genetically engineered
CYP3A4 in E. coli: application of a Jarfermenter, Drug
Metab. Pharmacokin. 18 (2003) 42–47.
[23] J. Cheng, D.F. Wan, J.R. Gu, Y. Gong, S.L. Yang,
D.C. Hao, L. Yang, Establishment of a yeast system
that stably expresses human cytochrome P450 reductase:
application for the study of drug metabolism of
cytochrome P450s in vitro, Protein Expr. Purif. 47
(2006) 467–476.
[24] C.L. Crespi, B.W. Penman, Use of cDNA-expressed human
cytochrome P450 enzymes to study potential drug–drug
interactions, Adv. Pharmacol. 43 (1997) 171–188.
[25] T. Aoyama, S. Yamano, P.S. Guzelian, H.V. Gelboin, F.J.
Gonzalez, Five of 12 forms of vaccinia virus-expressed
human hepatic cytochrome P450 metabolically activate
aflatoxin B1, Proc. Natl. Acad. Sci. USA 87 (1990) 4790–
4793.
[26] M.P. Pritchard, R. Ossetian, D.N. Li, C.J. Henderson, B.
Burchell, C.R. Wolf, T. Friedberg, A general strategy for
the expression of recombinant human cytochrome P450s in
Escherichia coli using bacterial signal peptides: expression
of CYP3A4, CYP2A6, and CYP2E1, Arch. Biochem.
Biophys. 345 (1997) 342–354.
[27] T.D. Porter, S. Chang, Strategies to enhance the coexpres-
sion of cytochrome P450 2E1 and reductase in bacteria,
Drug Metab. Rev. 31 (1999) 159–174.
[28] I. Jansson, I. Stoilov, M. Sarfarazi, J.B. Schenkman,
Enhanced expression of CYP1B1 in Escherichia coli,
Toxicology 144 (2000) 211–219.
[29] P.M. Shaw, N.A. Hosea, D.V. Thompson, J.M. Lenius,
F.P. Guengerich, Reconstitution premixes for assays using
purified recombinant human cytochrome P450, NADPH-
cytochrome P450 reductase, and cytochrome b5, Arch.
Biochem. Biophys. 348 (1997) 107–115.
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
[30] P. Urban, C. Cullin, D. Pompon, Maximizing the expres-
sion of mammalian cytochrome P-450 monooxygenase
activities in yeast cells, Biochimie 72 (1990) 463–472.
[31] S. Tamura, K.R. Korzekwa, S. Kimura, H.V. Gelboin, F.J.
Gonzalez, Baculovirus-mediated expression and functional
characterization of human NADPH-P450 oxidoreductase,
Arch. Biochem. Biophys. 293 (1992) 219–223.
[32] K. Venkatakrishnan, L.L. VonMoltke, D.J. Greenblatt,
Human drug metabolism and the cytochromes P450:
application and relevance of in vitro models, J. Clin.
Pharmacol. 41 (2001) 1149–1179.
[33] I.F. Sevrioukova, J.A. Peterson, NADPH-P-450 reductase:
structural and functional comparisons of the eukaryotic
and prokaryotic isoforms, Biochimie 77 (1995) 562–572.
[34] S. Sahdev, S.K. Khattar, K.S. Saini, Production of active
eukaryotic proteins through bacterial expression systems: a
review of the existing biotechnology strategies, Mol. Cell.
Biochem., in press.
[35] T.H. Richardson, F. Jung, K.J. Griffin, M. Wester, J.L.
Raucy, B. Kemper, L.M. Bornheim, C. Hassett, C.J.
Omiecinski, E.F. Johnson, A universal approach to the
expression of human and rabbit cytochrome P450s of the
2C subfamily in Escherichia coli, Arch. Biochem. Biophys.
323 (1995) 87–96.
[36] K. Fujita, T. Kamataki, Genetically engineered bacterial
cells co-expressing human cytochrome P450 with NADPH-
cytochrome P450 reductase: prediction of metabolism and
toxicity of drugs in humans, Drug Metab. Pharmacokinet.
17 (2002) 1–22.
[37] A. Parikh, E.M. Gillam, F.P. Guengerich, Drug metabo-
lism by Escherichia coli expressing human cytochromes
P450, Nat. Biotechnol. 15 (1997) 784–788.
[38] F.P. Guengerich, Cytochrome P450 proteins and potential
utilization in biodegradation, Environ. Health Perspect. 103
(1995) 25–28.
[39] J.T.M. Buters, M. Shou, J.P. Hardwick, K.R. Korzekwa,
F.J. Gonzalez, cDNA-directed expression of human cyto-
chrome P450 CYP1A1 using Baculovirus, Drug Metab.
Dispos. 23 (1995) 696–701.
[40] C.A. Lee, S.H. Kandwell, T.A. Kost, C.J. Serabjit-Singh,
CYP3A4 expressed by insect cells infected with a recombi-
nant baculovirus containing both CYP3A4 and human
NADPH-cytochrome P450 reductase is catalytically similar
to human liver microsomal CYP3A4, Arch. Biochem.
Biophys. 319 (1995) 157–167.
[41] A. Yu, H. Dong, D. Lang, R.L. Haining, Characterization
of dextromethorphan O- and N-demethylation catalyzed by
highly purified recombinant human CYP2D6, Drug Metab.
Dispos. 29 (2001) 1362–1365.
[42] L. Chen, J.T. Buters, J.P. Hardwick, S. Tamura, B.W.
Penman, F.J. Gonzalez, C.L. Crespi, Coexpression of
cytochrome P4502A6 and human NADPH-P450 oxidore-
ductase in the baculovirus system, Drug Metab. Dispos. 25
(1997) 399–405.
[43] J. Grogan, M. Shou, E.A. Andrusiak, S. Tamuro, J.T.M.
Buters, F.J. Gonzalez, K.R. Korzekwa, Cytochrome P450
2A1, 2E1, and 2C9 cDNA-expression by insect cells and
partial purification using hydrophobic chromatography,
Biochem. Pharmacol. 50 (1995) 1509–1515.
[44] H. Yamazaki, M. Nakajima, M. Nakamura, S. Asahi, N.
Shimada, E.M. Gillam, F.P. Guengerich, T. Shimada, T.
Yokoi, Enhancement of cytochrome P-450 3A4 catalytic
13
activities by cytochrome b (5) in bacterial membranes, Drug
Metab. Dispos. 27 (1999) 999–1004.
[45] M.W. Voice, Y. Zhang, C.R. Wolf, B. Burchell, T.
Friedberg, Effects of human cytochrome b5 on CYP3A4
activity and stability in vivo, Arch. Biochem. Biophys. 366
(1999) 116–124.
[46] H. Yamazaki, T. Shimada, Cytochrome P450 reconstitu-
tion systems, Methods Mol. Biol. 320 (2006) 61–71.
[47] S. Imaoka, T. Yamada, T. Hiroi, K. Hayashi, T. Sakaki, Y.
Yabusaki, Y. Funae, Multiple forms of human P450
expressed in Saccharomyces cerevisiae. Systematic charac-
terization and comparison with those of the rat, Biochem.
Pharmacol. 51 (1996) 1041–1050.
[48] J.K. Yano, M.R. Wester, G.A. Schoch, K.J. Griffin, C.D.
Stout, E.F. Johnson, The structure of human microsomal
cytochrome P450 3A4 determined by X-ray crystallography
to 2.05-A resolution, J. Biol. Chem. 279 (2004) 38091–
38094.
[49] T. Omura, R. Sato, The carbon monoxide-binding pigment
of liver microsomes. I. Evidence for its hemoprotein nature,
J. Biol. Chem. 239 (1964) 2370–2378.
[50] I.R. Phillips, E.A. Shephard, Cytochrome P450 protocols,
Methods Mol. Biol. 320 (2006) 1–10.
[51] K. Venkatakrishnan, L.L. von Moltke, D.J. Greenblatt,
Application of the relative activity factor approach in
scaling from heterologously expressed cytochromes P450 to
human liver microsomes: studies on amitriptyline as a
model substrate, J. Pharmacol. Exp. Ther. 297 (2001) 326–
337.
[52] D.F. McGinnity, R.J. Riley, Predicting drug pharmacoki-
netics in humans from in vitro metabolism studies,
Biochem. Soc. Trans. 29 (2001) 135–139.
[53] J.M. Margolis, R.S. Obach, Impact of nonspecific binding
to microsomes and phospholipid on the inhibition of
cytochrome P4502D6: implications for relating in vitro
inhibition data to in vivo drug interactions, Drug Metab.
Dispos. 31 (2003) 606–611.
[54] W. Tang, R.W. Wang, A.Y. Lu, Utility of recombinant
cytochrome P450 enzymes: a drug metabolism perspective,
Curr. Drug Metab. 6 (2005) 503–517.
[55] S.A. Wrighton, J.C. Stevens, The human hepatic cyto-
chromes P450 involved in drug metabolism, Crit. Rev.
Toxicol. 22 (1992) 1–21.
[56] I. Sekine, N. Saijo, Polymorphisms of metabolizing
enzymes and transporter proteins involved in the clearance
of anticancer agents, Ann. Oncol. 12 (2001) 1515–1525.
[57] K.T. Kivisto, H.K. Kroemer, M. Eichelbaum, The role of
human cytochrome P450 enzymes in the metabolism of
anti-cancer agents: implications for drug interactions, Br. J.
Clin. Pharmacol. 40 (1995) 523–530.
[58] K.M.L. Crommentuyn, J.H.M. Schellens, J.D. vander
Berg, J.H. Beijnen, In vitro metabolism of anti-cancer
drugs, methods and applications: paclitaxel, docetaxel,
tamoxifen and ifosfamide, Cancer Treat. Rev. 24 (1998)
345–366.
[59] M. Stiborova, C.A. Bieler, M. Wiessler, E. Frei, The
anticancer agent ellipticine on activation by cytochrome
P450 forms covalent DNA adducts, Biochem. Pharmacol.
62 (2001) 1675–1684.
[60] C. Rodriguez-Antona, M. Ingelman-Sundberg, Cyto-
chrome P450 pharmacogenetics and cancer, Oncogene 25
(2006) 1679–1691.
14 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
[61] D. Yao, S. Ding, B. Burchell, C.R. Wolf, T. Friedberg,
Detoxication of Vinca alkaloids by human P450 CYP3A4-
mediated metabolism: implications for the development of
drug resistance, J. Pharmacol. Exp. Ther. 294 (2000) 387–
395.
[62] B. Rochat, J.M. Morsman, G.I. Murray, W.D. Figg, H.L.
McLeod, Human CYP1B1 and anticancer agent metabo-
lism: mechanism for tumor-specific drug inactivation?, J
Pharmacol. Exp. Ther. 296 (2001) 537–541.
[63] S.S. Dehal, D. Kupfer, CYP2D6 catalyzes tamoxifen 4-
hydroxylation in human liver, Cancer Res. 57 (1997) 3402–
3406.
[64] H.K. Crewe, L.M. Notley, R.M. Wunsch, M.S. Lennard,
E.M. Gillam, Metabolism of tamoxifen by recombinant
human cytochrome P450 enzymes: formation of the 4-
hydroxy, 4 0 -hydroxy and N-desmethyl metabolites and
isomerization of trans-4-hydroxytamoxifen, Drug Metab.
Dispos. 30 (2002) 869–874.
[65] H.K. Crewe, S.W. Ellis, M.S. Lennard, G.T. Tucker,
Variable contribution of cytochromes P450 2D6, 2C9
and 3A4 to the 4-hydroxylation of tamoxifen by human
liver microsomes, Biochem. Pharmacol. 53 (1997) 171–
178.
[66] L.H. Patterson, G.I. Murray, Tumor cytochrome P450 and
drug activation, Curr. Pharm. Des. 8 (2002) 1335–1447.
[67] J. Kajita, E. Fuse, T. Kuwabara, H. Kobayashi, The
contribution of cytochrome P450 to the metabolism of
tegafur in human liver, Drug Metab. Pharmacokinet. 18
(2003) 303–309.
[68] K. Ikeda, K. Yoshisue, E. Matsushima, S. Nagayama, K.
Kobayashi, C.A. Tyson, K. Chiba, Y. Kawaguchi, Bioac-
tivation of tegafur to 5-fluorouracil is catalyzed by cyto-
chrome P-450 2A6 in human liver microsomes in vitro,
Clin. Cancer Res. 6 (2000) 4409–4415.
[69] P.A. Jacobson, K. Green, A. Birnbaum, R.P. Remmel,
Cytochrome P450 isozymes 3A4 and 2B6 are involved in
the in vitro human metabolism of thiotepa to TEPA,
Cancer Chemother. Pharmacol. 49 (2002) 461–467.
[70] D. McKillop, A.D. McCormick, A. Millar, G.S. Miles, P.J.
Phillips, M. Hutchison, Cytochrome P450-dependent
metabolism of gefitinib, Xenobiotica 35 (2005) 39–50.
[71] T. Cresteil, B. Monsarrat, J. Dubois, M. Sonnier, P.
Alvinerie, F. Gueritte, Regioselective metabolism of taxoids
by human CYP3A4 and 2C8: structure–activity relation-
ship, Drug Metab. Dispos. 30 (2002) 438–445.
[72] T. Kawashiro, K. Yamashita, X.J. Zhao, E. Koyama, M.
Tani, K. Chiba, T. Ishizaki, A study on the metabolism of
etoposide and possible interactions with antitumor or
supporting agents by human liver microsomes, J. Pharma-
col. Exp. Ther. 286 (1998) 1294–1300.
[73] M.V. Relling, J. Nemec, E.G. Schuetz, J.D. Schuetz, F.J.
Gonzalez, K.R. Korzekwa, O-demethylation of epipodo-
phyllotoxins is catalyzed by human cytochrome P450 3A4,
Mol. Pharmacol. 45 (1994) 352–358.
[74] G. Vassal, C. Pondarre, I. Boland, C. Cappelli, A. Santos,
C. Thomas, E. Lucchi, K. Imadalou, F. Pein, J. Morizet, A.
Gouyette, Preclinical development of camptothecin deriv-
atives and clinical trials in pediatric oncology, Biochimie 80
(1998) 271–280.
[75] R. Taniguchi, T. Kumai, N. Matsumoto, M. Watanabe, K.
Kamio, S. Suzuki, S. Kobayashi, Utilization of human liver
microsomes to explain individual differences in paclitaxel
metabolism by CYP2C8 and CYP3A4, J. Pharmacol. Sci.
97 (2005) 83–90.
[76] A. Nannelli, A. Messina, S. Marini, S. Trasciatti, V. Longo,
P.G. Gervasi, Effects of the anticancer dehydrotarplatin on
cytochrome P450 and antioxidant enzymes in male rat
tissues, Arch. Toxicol. 81 (2007) 479–487.
[77] R.H. Elbekia, A.O. El-Kadi, Transcriptional activation and
post-transcriptional modification of CYPlal by arsenite,
cadmium, and chromium, Toxicol. Lett. 172 (2007) 106–
119.
[78] C.D. Scripture, A. Sparreboom, W.D. Figg, Modulation of
cytochrome P450 activity: implications for cancer therapy,
Lancet Oncol. 6 (2005) 780–789.
[79] S. Price, W. Bordogna, R. Braganza, R.J. Bull, H.J. Dyke,
S. Gardan, M. Gill, N.V. Harris, R.A. Heald, M. van den
Heuvel, P.M. Lockey, J. Lloyd, A.G. Molina, A.G. Roach,
F. Roussel, J.M. Sutton, A.B. White, Identification and
optimisation of a series of substituted 5-pyridin-2-yl-thio-
phene-2-hydroxamic acids as potent histone deacetylase
(HDAC) inhibitors, Bioorgan. Med. Chem. Lett. 17 (2007)
363–369.
[80] M. Stiborova, M. Rupertova, H.H. Schmeiser, E. Frei,
Molecular mechanisms of antineoplastic action of an
anticancer drug elipticine, Biomed. Pap. Med. Fac. Univ.
Palacky Olomouc Czech Repub. 150 (2006) 13–23.
[81] M.M. Malingre, D.J. Richel, J.H. Beijnen, H. Rosing, F.J.
Koopman, W.W. Tem Bokkel Huinink, M.E. Schot, J.H.
Schellens, Coadministration of cyclosporine strongly
enhances the oral bioavailability of docetaxel, J. Clin.
Oncol. 19 (2001) 1160–1166.
[82] P.H. Rooney, C. Telfer, M.C. McFadyen, W.T. Melvin,
G.I. Murray, The role of cytochrome P450 in cytotoxic
bioactivation: future therapeutic directions, Curr. Cancer
Drug Targets 4 (2004) 257–265.
[83] Z. Desta, X. Zhao, J.G. Shin, D.A. Flockhart, Clinical
significance of the cytochrome P450 2C19 genetic polymor-
phism, Clin. Pharmacokinet. 41 (2002) 913–958.
[84] S. Lee, I.P. van der Heiden, J.A. Goldstein, R.H.N. van
Schaik, A new CYP3A5 variant, CYP3A5*11, is shown to
be defective in Nifedipine metabolism in a recombinant
cDNA expression system, Drug Metab. Dispos. 35 (2007)
67–71.
[85] S. Daigo, Y. Takahashi, M. Fujieda, N. Ariyoshi, H.
Yamazaki, W. Koizumi, S. Tanabe, K. Saigenji, S. Nagay-
ama, K. Ikeda, Y. Nishioka, T. Kamataki, A novel mutant
allele of the CYP2A6 gene (CYP2A6*11) found in a cancer
patient who showed poor metabolic phenotype towards
tegafur, Pharmacogenetics 12 (2002) 299–306.
[86] Y.M. Kim, S.H. Yoo, R.Y. Kang, M.J. Kim, Y.Y. Bae,
Y.K. Lee, S.J. Jeon, K.J. Chon, S.M. Shin, S.G. Kim, K.H.
Park, I.J. Son, Identifying drugs needing pharmacogenetic
monitoring in a Korean hospital, Am. J. Health Syst.
Pharm. 64 (2007) 166–175.
[87] B.K. Park, M. Pirmohamed, N.R. Kitteringham, The role
of cytochrome P450 enzymes in hepatic and extrahepatic
human drug toxicity, Pharmacol. Ther. 68 (1995) 385–424.
[88] W. Tang, Miller, in: Z. Yan Caldwell (Ed.), Methods in
Pharmacology and Toxicology: Optimization in Drug
Discovery: In vitro Methods, Humana Press, Totowa, NJ,
2004, pp. 369–383.
[89] S. Wenzlaff, M.L. Cote, C.H. Bock, S.J. Land, S.K. Santer,
D.R. Schwartz, A.G. Schwartz, CYP1A1 and CYP1B1
 K. Purnapatre et al. / Cancer Letters 259 (2008) 1–15
polymorphisms and risk of lung cancer among never
smokers: a population-based study, Carcinogenesis 26
(2005) 2207–2212.
[90] T.M. Sissung, D.K. Price, A. Sparreboom, W.D. Figg,
Pharmacogenetics and regulation of human cytochrome
P450 1B1: implications in hormone-mediated tumor metab-
olism and a novel target for therapeutic intervention, Mol.
Cancer Res. 4 (2006) 135–150.
[91] G.A. Potter, L.H. Patterson, E. Wanogho, P.J. Perry, P.C.
Butler, T. Ijaz, K.C. Ruparelia, J.H. Lamb, P.B. Farmer,
L.A. Stanley, M.D. Burke, The cancer preventative agent
resveratrol is converted to the anticancer agent piceatannol
by the cytochrome P450 enzyme CYP1B1, Br. J. Cancer 86
(2002) 774–778.
[92] S. Sale, R.G. Tunstall, K.C. Ruparelia, P.C. Butler, G.A.
Potter, W.P. Steward, A.J. Gescher, Effects of the potential
chemopreventive agent DMU-135 on adenoma develop-
ment in the ApcMin+ mouse, Invest. New Drugs. 24 (2006)
459–464.
[93] D. Pal, A.K. Mitra, CYP3A4 and MDR mediated interac-
tions in drug therapy, Clin. Res. Regul. Affairs 23 (2006)
125–163.
[94] H.J.G.D. van den Bongard, R.W. Sparidans, D.J.P.
Critchley, J.H. Beijnen, J.H.M. Schellens, Pharmacokinetic
drug–drug interaction of the novel anticancer agent E7070
and acenocoumarol, Invest. New Drugs 22 (2004) 151–158.
[95] Y. Sai, R. Dai, T.J. Yang, K.W. Krausz, F.J. Gonzalez,
H.V. Gelboin, M. Shou, Assessment of specificity of eight
chemical inhibitors using cDNA-expressed cytochromes
P450, Xenobiotica 30 (2000) 327–343.
[96] N. Ahmad, H. Mukhtar, Cytochrome p450: a target for
drug development for skin diseases, J. Invest. Dermatol.
123 (2004) 417–425.
15
[97] M. Karlgren, M. Ingelman-Sundberg, Tumour-specific
expression of CYP2W1: its potential as a drug target in
cancer therapy, Expert Opin Ther. Targets 11 (2007) 61–67.
[98] J.G. Jiang, C.L. Chen, J.W. Card, S. Yang, J.X. Chen, X.N.
Fu, Y.G. Ning, X. Xiao, D.C. Zeldin, D.W. Wang,
Cytochrome P450 2J2 promotes the neoplastic phenotype
of carcinoma cells and is up-regulated in human tumors,
Cancer Res. 65 (2005) 4707–4715.
[99] R.D. Bruno, V.C. Njar, Targeting cytochrome P450
enzymes: a new approach in anti-cancer drug development,
Bioorg. Med. Chem. 15 (2007) 5047–5060.
[100] K.K. Deeb, D.L. Trump, C.S. Johnson, Vitamin D
signaling pathways in cancer: potential for anticancer
therapeutics, Nat. Rev. Cancer 7 (2007) 684–700.
[101] S. Sundaram, M.J. Beckman, A. Bajwa, J. Wei, K.M.
Smith, G.H. Posner, D.A. Gewirtz, QW-1624F2-2, a
synthetic analogue of 1,25-dihydroxyvitamin D3, enhances
the response to other deltanoids and suppresses the
invasiveness of human metastatic breast tumor cells, Mol.
Cancer Ther. 5 (2006) 2806–2814.
[102] K. Kojima, K. Nagata, T. Matsubara, Y. Yamazoe, Broad
but distinct role of pregnane X receptor on the expression
of individual cytochrome P450s in human hepatocytes,
Drug Metab. Pharmacokinet. 22 (2007) 276–286.
[103] J. Chen, X.X. Yang, M. Huang, Z.P. Hu, M. He, W. Duan,
E. Chan, F.S. Sheu, X. Chen, S.F. Zhou, Small interfering
RNA-mediated silencing of cytochrome P450 3A4 gene,
Drug Metab. Dispos. 34 (2006) 1650–1657.
[104] M. Izquierdo, Short interfering RNAs as a tool for cancer
gene therapy, Cancer Gene. Ther. 12 (2005) 217–227.
[105] S.l. Pai, Y.Y. Lin, B. Macaes, A. Meneshian, C.F. Hung,
T.C. Wu, Prospects of RNA interference therapy for
cancer, Gene Ther. 13 (2006) 464–477.