N-isopropyl-p-iodoamphetamine hydrochloride is predominantly metabolized by CYP2C19

Article abstract of DOI:10.1124/dmd.111.043893

[¹²³I]N-Isopropyl-p-iodoamphetamine hydrochloride ([ ¹²³I]IMP) is clinically used to evaluate blood flow in the brain on single photon emission-computed tomography. This is a rare radiopharmaceutical that undergoes metabolism. The first step is reported to be [ ¹²³I]p-iodoamphetamine formation. The drug-metabolizing enzyme(s) involved remain(s) unclear. This study examined the roles of human cytochrome P450 (P450) in the metabolism of nonradiolabeled IMP with the use of human liver microsomes (HLM) and recombinant human CYP1A1, -1A2, -1B1, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -3A4, and -3A5. Disappearance of IMP was examined because p-iodoamphetamine was not available. IMP (0.5 μM) time-dependently disappeared when HLM and NADPH-generating system were added to the reaction mixture. (S)-Mephenytoin (1 mM) inhibited the IMP disappearance by approximately 90%. The disappearance of IMP was predominantly catalyzed by recombinant CYP2C19, with K_m and V_max of 8.6 μM and 9.7 nmol · min^-1 · nmol P450^-1, respectively. IMP disappearance in CYP2C19-deficient HLM (CYP2C19*2/*2) was approximately 30% of that in the presence of HLM harboring wild-type CYP2C19, indicating that IMP is polymorphically metabolized by CYP2C19. High-performance liquid chromatography of the incubation mixture of IMP and CYP2C19 revealed an unidentified peak. As the area of the IMP peak decreased, the area of this unidentified peak increased in a time-dependent fashion. The peak was also detectable on incubation of IMP with HLM. Mass spectrometry revealed that the molecular weight of a compound in this unidentified peak was the same as that of p-iodoamphetamine. Thus, we demonstrated that IMP was predominantly metabolized by CYP2C19 to form p-iodoamphetamine. Copyright

Full text of DOI:10.1124/dmd.111.043893

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2012/01/31/dmd.111.043893.DC1.html
1521-009X/12/4005-843–846$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD 40:843–846, 2012
Vol. 40, No. 5
43893/3761827
Short Communication
N-Isopropyl-p-iodoamphetamine Hydrochloride Is Predominantly
□
S
Metabolized by CYP2C19
Received November 20, 2011; accepted January 31, 2012
ABSTRACT:
123I]N-Isopropyl-p-iodoamphetamine hydrochloride ([123I]IMP) of IMP was predominantly catalyzed by recombinant CYP2C19,
is clinically used to evaluate blood flow in the brain on single with Km and Vmax of 8.6 ␮M and 9.7 nmol ꢀ min؊1 ꢀ nmol P450؊1
[
,
photon emission-computed tomography. This is a rare radio- respectively. IMP disappearance in CYP2C19-deficient HLM
pharmaceutical that undergoes metabolism. The first step is (CYP2C19*2/*2) was approximately 30% of that in the presence
reported to be [123I]p-iodoamphetamine formation. The drug- of HLM harboring wild-type CYP2C19, indicating that IMP is
metabolizing enzyme(s) involved remain(s) unclear. This study polymorphically metabolized by CYP2C19. High-performance
examined the roles of human cytochrome P450 (P450) in the liquid chromatography of the incubation mixture of IMP and
metabolism of nonradiolabeled IMP with the use of human liver CYP2C19 revealed an unidentified peak. As the area of the IMP
microsomes (HLM) and recombinant human CYP1A1, -1A2, peak decreased, the area of this unidentified peak increased in
-1B1, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -3A4, and -3A5. a time-dependent fashion. The peak was also detectable on
Disappearance of IMP was examined because p-iodoamphet- incubation of IMP with HLM. Mass spectrometry revealed that
amine was not available. IMP (0.5 ␮M) time-dependently disap- the molecular weight of a compound in this unidentified peak was
peared when HLM and NADPH-generating system were added the same as that of p-iodoamphetamine. Thus, we demonstrated
to the reaction mixture. (S)-Mephenytoin (1 mM) inhibited the that IMP was predominantly metabolized by CYP2C19 to form
IMP disappearance by approximately 90%. The disappearance p-iodoamphetamine.
Introduction
by sequential generation of [123I]p-iodobenzoic acid and [123I]p-
iodohippuric acid (Baldwin and Wu, 1988). The biotransformation
of [123I]IMP to metabolite(s) with lower lipid solubility could
practically impair penetration of 123I to the brain. Therefore, to
accurately evaluate blood flow in the brain, radioactivity derived
from the water-soluble metabolite(s), which cannot reach the brain,
has to be subtracted from the total radioactivity of all components
with 123I. To date, drug-metabolizing enzyme(s) involved in the
metabolism of nonradiolabeled IMP remain(s) unclear, although
one study has demonstrated binding of IMP and its initial metab-
olite to rat cytochrome P450 (P450), suggesting that P450 plays a
[
123I]N-Isopropyl-p-iodoamphetamine hydrochloride ([123I]IMP) is
a radiopharmaceutical used clinically to evaluate blood flow in the
brain on single photon emission-computed tomography (see chem-
ical structure in Baldwin and Wu, 1988). This radiopharmaceutical
has been amply demonstrated to be distributed in brain tissue in a
clinically useful fashion. With the application of nuclear medicine
techniques in the 1980s, pharmacokinetic analysis revealed uptake
of [123I]IMP by the liver after a high uptake by lung during the first
pass (Moretti et al., 1983). Subsequently, [123I]IMP was found to
undergo biotransformation in the body (Baldwin and Wu, 1988),
although few imaging diagnostics were thought to be metabolized. role in the metabolism of IMP (Joulin et al., 1992).
Analyses of human or rat plasma and urine samples obtained after
This study first examined whether or not nonradiolabeled IMP was
metabolized by drug-metabolizing enzyme(s) in human liver micro-
somes (HLM) and then attempted to elucidate human P450 isoform(s)
responsible for the biotransformation of IMP. Because the first me-
tabolite p-iodoamphetamine was not available, IMP disappearance
was examined.
[
123I]IMP administration have revealed that the first step in me-
tabolism was the formation of [123I]p-iodoamphetamine followed
This study was supported in part by a Grant-in-Aid for Cancer Research from the
Ministry of Health, Labor and Welfare of Japan [21S-8-1]; a Grant-in-Aid for Scientific
Research (C) [23590198] from Japan Society for the Promotion of Science; and a
Grant-in-Aid for “Support Project of Strategic Research Center in Private Universi-
ties” from the Ministry of Education, Culture, Sports, Science and Technology (to
Saitama Medical University Research Center for Genomic Medicine).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
Materials and Methods
Human Liver Microsomes and Recombinant Human P450 Isoforms.
Pooled HLM (wild-type CYP2C19) and HLM harboring CYP2C19*2/*2
(CYP2C19*2/*2 HLM) were obtained from BD Biosciences (Woburn, MA).
Twelve recombinant P450 isoforms (i.e., CYP1A1, -1A2, -1B1, -2A6, -2B6,
-2C8, -2C9.1, -2C19, -2D6.1, -2E1, -3A4, and -3A5) were purchased from BD
Biosciences.
http://dx.doi.org/10.1124/dmd.111.043893.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: [123I]IMP, [123I]N-isopropyl-p-iodoamphetamine hydrochloride; P450, cytochrome P450; HLM, human liver microsomes; HPLC,
high-performance liquid chromatography.
843

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FUJITA ET AL.
IMP Metabolism by Pooled Human Liver Microsomes. NADPH-
CYP2C19 or CYP1A1 are shown in Supplemental Fig. 1. The appar-
ent Km and Vmax were calculated to be 8.6 Ϯ 0.8 ␮M and 9.7 Ϯ 0.4
nmol ꢀ minϪ1 ꢀ nmol P450Ϫ1 for CYP2C19 and 18 Ϯ 5.0 ␮M and
7.3 Ϯ 1.0 nmol ꢀ minϪ1 ꢀ nmol P450Ϫ1 for CYP1A1. The intrinsic
metabolic clearance calculated with the Km and Vmax values obtained
for CYP2C19 was approximately 2.5-fold higher than that for
CYP1A1. We could not determine these apparent enzyme kinetic
parameters for CYP1A2 because of a slight decrease in IMP at
concentrations higher than 8.0 ␮M where IMP disappearance was not
saturated. (S)-Mephenytoin, a specific CYP2C19 substrate (Wrighton
et al., 1993), inhibited IMP disappearance catalyzed by pooled HLM
in a concentration-dependent manner (Fig. 1C). IMP disappearance
was inhibited by 1 mM (S)-mephenytoin by approximately 90%. IMP
disappearance activities seen in CYP2C19*2/*2 HLM for respective
concentrations of microsomal protein were approximately 30% com-
pared with those seen in the pooled HLM (wild-type CYP2C19) (Fig.
1D). These results indicate that IMP is predominantly metabolized
polymorphically by CYP2C19.
dependent IMP metabolism by pooled HLM was examined in the presence
or absence of an NADPH-generating system. The mixture without the
addition of the pooled HLM and NADPH-generating system served as the
control. IMP metabolism was monitored based on the disappearance of
IMP. When the inhibition of IMP disappearance by (S)-mephenytoin was
examined, the inhibitor was added 5 min before the initiation of metabo-
lism. IMP was analyzed with a high-performance liquid chromatography
(HPLC) system.
IMP Metabolism by P450 Isoforms. IMP metabolism by 12 recombinant
P450 isoforms was examined based on the disappearance of IMP. IMP disap-
pearance in the absence of the NADPH-generating system for each P450
served as the control.
Enzyme Kinetics of IMP Metabolism. Substrate disappearance velocity
(v) was calculated as [(Cs,initial Ϫ Cs,t)/incubation time/P450 concentration],
where Cs,initial was the substrate concentration at time 0 and Cs,t was the
substrate concentration after t min incubation with each P450 (Li et al., 2007).
Kinetic constants (Km and Vmax) are reported as the means Ϯ S.E.
IMP Metabolism by Human Liver Microsomes Harboring
CYP2C19*2/*2. IMP metabolism was monitored based on the disappear-
ance of IMP. The incubation period of 0.5 ␮M IMP was 60 min. IMP
disappearance velocity (v) was calculated as [(Cs,initial Ϫ Cs,60) ϫ volume
of reaction mixture/60 min].
During the HPLC analyses of the IMP disappearance by
CYP2C19, an unidentified peak was found on HPLC chromato-
grams (Fig. 2, A–C). As the area of the IMP peak decreased, the
area of this unidentified peak increased in a time-dependent fash-
Analysis of IMP Metabolite by Liquid Chromatography-Electrospray
Ionization Mass Spectrometry. Liquid chromatography-electrospray ion-
ization mass spectrometry was performed with a micrOTOF-Q II mass ion (Fig. 2D). The unidentified peak was also detectable when IMP
spectrometer (Bruker Daltonics, Billerica, MA) to identify the IMP metab-
olite produced by CYP2C19. Detailed descriptions of the methods are
given in the supplemental data.
was incubated with pooled HLM (wild-type CYP2C19) (Supple-
mental Fig. 2A). Ultraviolet spectra of both peaks seen with pooled
HLM and recombinant CYP2C19 were nearly the same (Supple-
mental Fig. 2, B and C). The unidentified peak was fractionated
and analyzed with liquid chromatography-electrospray ionization
mass spectrometry (Fig. 2E and Supplemental Fig. 3). A major
peak at 232 nm was detected at 11.2 min (Supplemental Fig. 3),
and a parent mass ([M ϩ H]ϩ) was observed at m/z 262.0085 (Fig.
2E). High-resolution mass spectrometry (electrospray ionization)
calculated for C9H12IN [M ϩ H]ϩ:(262.0087) was found at
Results and Discussion
IMP time-dependently disappeared when pooled HLM and an
NADPH-generating system were contained in the reaction mixture
(Fig. 1A). Among 12 recombinant P450 forms, CYP2C19 was dem-
onstrated to predominantly catalyze the reaction (Fig. 1B). The effects
of substrate concentrations on IMP disappearance in the presence of
FIG. 1. Disappearance of IMP in the presence
of both pooled HLM and NADPH-generating
system (A), catalyzed predominantly by recom-
binant CYP2C19 (B), showing inhibition of
HLM-catalyzed IMP disappearance by (S)-me-
phenytoin (C), and demonstrating polymorphic
metabolism of IMP by CYP2C19 (D). The IMP
concentration used was 0.5 ␮M. A, open bars,
the presence of pooled HLM and absence of
NADPH-generating system; closed bars, pres-
ence of both pooled HLM and NADPH-gener-
ating system; D, open bars, CYP2C19*2/*2
HLM; closed bars, pooled HLM (wild-type
CYP2C19). Each point shows the mean of three
independent analyses with S.D. bars.

IMP IS PREDOMINANTLY METABOLIZED BY CYP2C19
845
FIG. 2. HPLC chromatograms of IMP and un-
identified peak obtained by the incubation of
IMP with CYP2C19 for reaction times of 0 (A),
10 (B), and 20 min (C), showing a reaction-
time-dependent decrease in IMP (᭜) and an
increase in the unidentified peak (F) (D) and
demonstrating total mass spectra between 11.1
and 11.5 min and assignment of fragmentations
(E). D, ratio of peak area was calculated by
dividing the peak area of IMP or unidentified
peak by that of internal standard. Each data
point in D represents the mean of triplicate
determinations and is shown with S.D. bars.
262.0085. The fragments at m/z 244.99 and 216.95 can be assigned genotyping should be examined. In conclusion, our results showed
as successive neutral losses of NH3 (⌬m ϭ 17.0265) and C2H2
(⌬m ϭ 28.0313), both determined with mass accuracies below 3
mDa (Fig. 2E). These results suggest that the unknown metabolite
is p-iodoamphetamine [1-(4-iodophenyl)-propan-2-amine].
that IMP metabolism was mainly catalyzed polymorphically by
CYP2C19.
In clinical practice, the metabolism of lipid soluble [123I]IMP to
water-soluble metabolites, most likely [123I]p-iodobenzoic acid
and [123I]p-iodohippuric acid, can impair the penetration of 123I to
the brain. Therefore, to accurately evaluate blood flow in the brain,
radioactivity derived from water-soluble metabolites must be sub-
tracted from the total radioactivity derived from all components
labeled with 123I. The water-soluble metabolites are usually sepa-
rated by octanol/water extraction of blood samples collected after
Authorship Contributions
Participated in research design: Fujita, Kunishima, and Kawai.
Conducted experiments: Sugiyama, Akiyama, Hioki, and Nishi.
Performed data analysis: Sugiyama, Fujita, Hioki, and Kunishima.
Wrote or contributed to the writing of the manuscript: Fujita, Kunishima,
Kobayashi, Kawai, and Sasaki.
Other: Fujita and Sasaki acquired funding for the research.
[
123I]IMP injection, and the radioactivity in the water phase is Department of Medical Oncology,
KEN-ICHI FUJITA
MINAKO SUGIYAMA
YUKO AKIYAMA
subsequently quantified. If the formation of water-soluble metab-
olites in vivo is predominantly determined by CYP2C19 activity, Center-Comprehensive
International Medical
radioactivity in the water phase might parallel CYP2C19 activity.
Cancer Center (K.F., M.S.,
Y.A., Y.S.) and
KAZUHITO HIOKI
MUNETAKA KUNISHIMA
KODAI NISHI
MASATO KOBAYASHI
KEIICHI KAWAI
YASUTSUNA SASAKI
The quantification of water-soluble metabolites of [123I]IMP might
be applied to CYP2C19 phenotyping to identify poor metabolizers. Project Research Laboratory,
Our CYP2C19-phenotyping method might facilitate patient selec- Research Center for Genomic
tion or dose adjustment for medicines that are substrates of poly- Medicine (K.F., M.S., Y.A., Y.S.),
morphic CYP2C19 (http://www.cypalleles.ki.se/cyp2c19.htm). Saitama Medical University,
However, future studies are necessary to put our idea into clinical Hidaka, Japan; Faculty of
practice. First, the role(s) of CYP2C19 in the subsequent steps of Pharmaceutical Sciences,
IMP metabolism should be comprehensively clarified. We are Kobe Gakuin University, Minatojima,
synthesizing p-iodoamphetamine to examine secondary metabo- Chuo-ku, Kobe, Japan (K.H.);
lism in more detail. Second, whether [123I]IMP is predominantly Faculty of Pharmaceutical
metabolized by CYP2C19 should be confirmed with a variety of Sciences, Institute of Medical,
recombinant human P450s and pooled HLM at concentrations Pharmaceutical and Health Sciences,
similar to those in human plasma, which are much lower than the Kanazawa University,
concentrations used in this study. Our preliminary study using Kakuma-machi, Kanazawa, Japan
radiolabeled IMP and HPLC equipped with a radioactivity detector (M.Ku.); and Faculty of Health
indicated that recombinant CYP2C19 and pooled HLM-mediated Sciences, Institute of Medical, Pharmaceutical
metabolism of labeled IMP. Third, after comprehensive in vitro
studies of metabolism, in vivo correlations between CYP2C19
phenotyping as measured by the proposed methods and CYP2C19
and Health Sciences,
Kanazawa University, Kodatsuno,
Kanazawa, Japan (K.N., M.Ko., K.K.)

846
FUJITA ET AL.
Wrighton SA, Stevens JC, Becker GW, and VandenBranden M (1993) Isolation and character-
ization of human liver cytochrome P450 2C19: correlation between 2C19 and S-mephenytoin
4Ј-hydroxylation. Arch Biochem Biophys 306:240–245.
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Address correspondence to: Dr. Ken-ichi Fujita, Department of Medical
Oncology, International Medical Center-Comprehensive Cancer Center, Saitama
Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298, Japan. E-mail:
fujitak@saitama-med.ac.jp