Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase: Human UDP-glucuronosyltransferase enzymes involved in lactonization

Article abstract of DOI:10.1080/0049825021000017957

1. Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase little metabolized in hepatic microsomes. Pitavastatin lactone, which can be converted back to the unchanged form, is the major metabolite of pitavastatin in humans. To clarify the mechanism of the lactonization of pitavastatin and the metabolic properties of the lactone, we performed experiments in vitro. 2. On addition of UDP-glucuronic acid, human hepatic microsomes produced pitavastatin lactone and an unknown metabolite (UM-2). UM-2 was converted to its unchanged form by enzymatic hydrolysis and to a lactone form non-enzymatically. Using several human UGT-expressing microsomes, UGT1A3 and UGT2B7 were principally responsible for glucuronidation of pitavastatin leading to lactonization. 3. No marked difference in intrinsic clearance between pitavastatin and its lactone form was detected in human hepatic microsomes. 4. Pitavastatin lactone showed no inhibitory effects on CYP2C9- and CYP3A4-mediated metabolism of model substrates in contrast to other HMG-CoA reductase inhibitors. 5. The mechanism of pitavastatin lactone formation has been clarified, in that glucuronidation by UGT occurs first followed by lactonization via an elimination reaction. It was also found that pitavastatin lactone demonstrates no drug - drug interactions.

Full text of DOI:10.1080/0049825021000017957

xenobiotica, 2003, vol. 33, no. 1, 27–41
Metabolic fate of pitavastatin, a new inhibitor of
HMG-CoA reductase: human UDP-
glucuronosyltransferase enzymes involved in
lactonization
H. FUJINO*, I. YAMADA, S. SHIMADA, M. YONEDA and
J. KOJIMA
Tokyo New Drug Research Laboratories I, Kowa Company Ltd, 2-17-43 Noguchicho,
Higashimurayama, Tokyo 189-0022, Japan
Received 20 May 2002
1. Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase little
metabolized in hepatic microsomes. Pitavastatin lactone, which can be converted back to
the unchanged form, is the major metabolite of pitavastatin in humans. To clarify the
mechanism of the lactonization of pitavastatin and the metabolic properties of the lactone,
we performed experiments in vitro.
2. On addition of UDP-glucuronic acid, human hepatic microsomes produced
pitavastatin lactone and an unknown metabolite (UM-2). UM-2 was converted to its
unchanged form by enzymatic hydrolysis and to a lactone form non-enzymatically. Using
several human UGT-expressing microsomes, UGT1A3 and UGT2B7 were principally
responsible for glucuronidation of pitavastatin leading to lactonization.
3. No marked difference in intrinsic clearance between pitavastatin and its lactone
form was detected in human hepatic microsomes.
4. Pitavastatin lactone showed no inhibitory effects on CYP2C9- and CYP3A4-
mediated metabolism of model substrates in contrast to other HMG-CoA reductase
inhibitors.
5. The mechanism of pitavastatin lactone formation has been clarified, in that
glucuronidation by UGT occurs first followed by lactonization via an elimination
reaction. It was also found that pitavastatin lactone demonstrates no drug–drug
interactions.
Introduction
In general, elevated levels of low-density lipoprotein (LDL)-cholesterol are
considered the most important risk factor for coronary artery disease (Brown et al.
1991). The most effective method of lowering LDL-cholesterol is the administra-
tion of inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA)
reductase, the rate-limiting enzyme of the cholesterol synthesis pathway (Havel
and Rapaport 1995). Pitavastatin is a highly potent inhibitor of HMG-CoA
reductase and causes a significant reduction in serum total cholesterol, LDL-
cholesterol and triglyceride levels in animals (Aoki et al. 1997, Suzuki et al. 1999,
2001). In humans, the persistent effect on serum lipid and safety of pitavastatin
have been confirmed in clinical practice (Kajinami et al. 2000a), and this drug has
an enhancing effect on patients with non-insulin-dependent diabetes mellitus
* Author for correspondence; e-mail: h-fujino@kowa.co.jp
Xenobiotica ISSN 0049–8254 print/ISSN 1366–5928 online # 2003 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/0049825021000017957

28
H. Fujino et al.
(NIDDM) or familial hypercholesterolemia (Kajinami et al. 2000b, Yamada et al.
2000).
Pitavastatin is minimally metabolized in hepaticmicrosomes and there are no
drug–drug interactions between it and either tolbutamide or taxol in vitro (Fujino
et al. 1999a, 2001). Moreover, no induction of the drug-metabolizing enzymes
(aniline hydroxylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethy-
lase and UDP-gulucuronic acid transferase) was found in rat after multiple
administrations of pitavastatin (Fujino et al. 2002). The favourable metabolic
characteristic of this drug support efficacy in clinical studies. Pitavastatin is
metabolized via biotransformation as follows: lactonization; b-oxidative degrada-
tion of the side-chain; hydroxylation of the quinoline ring; and conjugation with b-
glucuronic acid and taurine (Kojima et al. 1999a). The lactone form of pitavastatin,
which can be reversibly converted to the parent drug, is found as a major
metabolite in plasma following oral administration in animals and human (Fujino
et al. 1999b, Kojima et al. 1999b). This suggests that pitavastatin and pitavastatin
lactone coexist at equilibrium in blood after administration of the drug. To date,
only the metabolism of pitavastatin, not of lactone, has been investigated and no
studies have been published about the mechanism of lactonization. To clarify this
latter mechanism, experiments were performed in vitro in the present study.
Furthermore, in view of the widespread clinical use of pitavastatin and the
importance of drug–drug interactions, the inhibitory effect of pitavastatin lactone
on cytochrome P450 enzymes was also investigated.
Materials and methods
Chemicals and reagents
Pitavastatin, monocalcium bis[(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]3,5-di-
hydroxy-6-hepteonate] and its lactone form were synthesized by Nissan Chemical Industries (Chiba,
Japan). Fluvastatin and atorvastatin lactone were synthesized in our laboratory. The chemical purities
of all synthesized compounds were >98.3%. 4-Hydroxytolbutamide and 6b-hydroxytestosterone were
purchased from Ultrafine Chemicals (Manchester, UK). Ketoprofen and p-nitrophenol as chemical
inhibitors of UGT were purchased from Wako Pure Chemicals (Osaka, Japan). The NADPH-
regenerating system (b-NADPþ, glucose 6-phosphate and glucose 6-phosphate dehydrogenase) and
UDP-glucronic acid were obtained commercially. All other chemicals and reagents used were
commercially available and of guaranteed purity.
Radioisotope-labelled chemicals
(Fluorobenzene-U-14C( pitavastatin was synthesized by Amersham Co. (Little Chalfort, UK). The
specific radioactivity of the labelled compound was 2.2 MBq mgÀ1, and the radiochemical purity was
>99.2%. [Ring-U-14C] tolbutamide (Amersham, 2.22 GBq mmolÀ1) and [4-14C] testosterone (Amer-
sham, 2.11 GBq mmolÀ1) were purchased commercially and the radiochemical purities were >98.4%.
Microsomes
Human hepaticmicrosomes (H23, H30, H42, H66, H70, H93, H112, H161, HK27 and HK31) and
recombinant microsomes derived from baculovirus-expressing human UGT (control, UGT1A1,
UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B7 and UGT2B15) and
human cytochrome P450s (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were
purchased from Gentest Co. (Woburn, MA, USA). Human pooled renal microsomes (HRM-005),
intestinal microsomes (HMPM-INT) and pooled monkey hepatic microsomes were purchased from the
International Institute for Advancement of Medicine (Exton, PA, USA) and In Vitro Technologies
(Baltimore, MD, USA), respectively.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
29
In vitro metabolism of pitavastatin
In Phase 1 reaction experiments, the incubation mixture was prepared as follows: a 0.5 ml reaction
mixture containing 1 mg protein mlÀ1 microsome, 1.3 mm NADPþ, 3.3 mm glucose 6-phosphate, 0.4
U mlÀ1 glucose 6-phosphate dehydrogenase, 3.3 mm magnesium chloride and 2.5 mm 14C-pitavastatin in
100 mm sodium phosphate buffer (pH 7.4) was incubated for 2 h. In Phase 2 reaction experiments, a
0.5 ml reaction mixture containing 1 mg protein mlÀ1 microsomes, 1 mm uridine diphosphate glucuronic
acid (UDPGA), 10 mm magnesium chloride and 2.5 mm 14C-pitavastatin in 50 mm Tris (pH 7.5) was
incubated for 2 h. After incubation at 378C, the reaction was quenched by adding an equal volume of
acetonitrile. The reaction-terminated samples were centrifuged at 6000g and the supernatant analysed.
Isolation of 14C-pitavastatin lactone
14C-pitavastatin lactone was isolated according to our previous method (Yamada et al. 2001). After
incubation of 14C-pitavastatin (50 mm) with monkey microsomes in the Phase 2 reaction experiment for
2 h, pitavastatin lactone was fractionated from the HPLC elutes while monitoring UV absorption
(254 nm) at a retention time of about 20 min. The fractionated pitavastatin lactone solution was purified
by a solid-phase extraction method as follows: the solution was diluted with 6 vols water and absorbed
on a solid-phase extraction column, Bond Elut C18 (300 mg, Varian Sample Preparation Products,
Habor, CA, USA). After being washed with 2 ml water, the residual pitavastatin lactone was eluted with
3 ml ethanol. The radiochemical purity of the isolated 14C-pitavastatin lactone was >99%. All
operations were carried out in the dark to avoid decomposition.
Isolation of unknown metabolites (UM-2)
In the Phase 2 reaction, UM-2 was fractionated from the HPLC elutes while monitoring UV
absorption (254 nm) at a retention time of about 7 min. Fractionated UM-2 was evaporated to dryness
and subjected to enzymatic hydrolysis with b-glucuronidase/arylsulfatase (5000 Fishman units and 80
units, respectively) at 378C for 2 h or alkaline treatment with 0.1 N NaOH at room temperature for
10 min.
Inhibition of lactonization by chemical UGT inhibitors
The experiments with known chemical inhibitors of UGT were performed at a substrate concen-
tration of 2.5 mm and inhibitor concentrations of 150 and 750 mm for ketoprophen and 200 and 1000 mm
for p-nitrophenol using human hepatic microsomes. Pitavastatin was co-incubated with these UGT
inhibitors for 2 h. The inhibitory effect was estimated from the amount of pitavastatin lactone formed in
the presence and absence of inhibitor.
Possible role for UGT enzymes in pitavastatin metabolism
Using nine recombinant human UGT-expressing microsomes, the human UGT enzymes partici-
pating in the lactonization of pitavastatin to pitavastatin lactone were investigated. At pitavastatin
concentrations of 2.5, 10 and 50 mm, the reaction mixtures were incubated for 30 min with UDPGA. In
addition, incubation with control microsomes (native insect cells, BTI-TN-5B1-4) was performed in
parallel. The incubation conditions were the same as for the Phase 2 reaction.
Kinetic analysis of lactonization
The Michaelis–Menten parameters of the apparent Km and Vmax for pitavastatin lactonization were
estimated by the analysis of Lineweaver–Burk reciprocal plots. The pitavastatin concentration was 1–
80 mm and incubation was carried out for 30 min in human hepatic and renal microsomes, respectively.
Metabolic stability of pitavastatin lactone
Pitavastatin and its lactone form at a concentration of 2.5 mm were incubated with human hepatic
microsomes and an NADPH-regenerating system and then the metabolic stability was evaluated. The
remaining pitavastatin or lactone form were measured at 0, 15, 30, 60 and 120 min after the addition of
microsomes. In the determination of in vitro clearance (Clint), the slope of the linear regression from the
log concentration of actual data versus incubation time relationships ðÀkÞ was used in the calculation as
follows:
Clint ¼ k  (ml incubation)/(mg microsome).
In the evaluation of lactone clearance, the unchanged form which was converted from the lactone form
non-enzymatically was calculated as the amount of pitavastatin lactone remaining.

30
H. Fujino et al.
Role of CYP enzymes in pitavastatin lactone metabolism
Using six recombinant CYP-expressing microsomes, the human CYP enzymes participating in the
metabolism of pitavastatin lactone were investigated. Pitavastatin lactone (2.5 mm) and the reaction
mixtures were incubated for 30 min with the NADPH-regenerating systems. The incubation conditions
were as for the Phase 1 reaction.
Correlation of pitavastatin lactone metabolism with CYP activities
Using 10 individual human hepatic microsomes, the correlation between the metabolism of
pitavastatin lactone and activity of CYP markers was studied for CYP1A2 (phenacetin O-deethylase),
CYP2A6 (coumarin 7-hydroxylase), CYP2C9 (diclofenac 40-hydroxylase), CYP2C19 (mephenytoin 40-
hydroxylase), CYP2D6 (bufuralol 10-hydroxylase), CYP2E1 (chlorzoxazone 6-hydroxylase), CYP3A4
(testosterone 6b-hydroxylase) and CYP contents. Pitavastatin lactone at a concentration of 2.5 mm was
incubated for 2 h with several human hepatic microsomes and total metabolism was estimated from the
pitavastatin and lactone remaining.
Effect of pitavastatin lactone on the metabolism of several CYP markers
The inhibitory effect of pitavastatin lactone on CYP-mediated metabolism was examined using 14C-
tolbutamide and 14C-testosterone in the concentration range 40–800 mm. In the inhibition experiment
for CYP2C9, pitavastatin lactone (1–10 mm) was co-incubated with tolbutamide. In the presence or
absence of pitavastatin lactone, the CYP2C9-mediated production of 4-hydroxytolbutamide was
evaluated. In the inhibition experiment for CYP3A4, pitavastatin lactone (0.5–5 mm) was co-incubated
with testosterone. Fluvastatin and atorvastatin lactone were used as inhibitors of CYP2C9- and
CYP3A4-mediated metabolism, respectively. The inhibition experiments for these inhibitors were
carried out in the same manner as for pitavastatin. The inhibition constant (Ki) was estimated by Dixon
plot analysis.
Analytical methods
Total radioactivity was measured with a liquid scintillation counter (Tri-Carb 1500) for 10 min after
addition of a scintillation cocktail (Hionic-Fluor, all Packard Instrument Co., Meriden, CT, USA).
The measurement of pitavastatin and its metabolite was performed according to an HPLC-
radioluminography method (Baba et al. 1994, Kimata et al. 1998) as follows: the HPLC system (L-
6000 series, Hitachi, Tokyo, Japan) equipped with a UV detector, a reversed-phase column (Inertsil
ODS-3V, 4.6 Â 150 mm, GL Science, Inc., Tokyo, Japan) and auto-injector (AS-4000; Hitachi) was
operated with two mobile phases. The separation was achieved with the following program: the initial
solvent (A), 0.1 m acetate buffer (pH 3.5)/acetonitrile (55:45 v/v), was delivered for 7.5 min, then the
mobile phase was changed with a linear gradient over a period of 25 min to solvent (B), 0.1 m acetate
buffer (pH 4.5)/acetonitrile (30:70 v/v), which was delivered for 5 min. The flow rate of the mobile phase
was 1 ml minÀ1. The HPLC elute was fractionated into the wells of polystyrene flat-bottomed
microplates (RLG plate Beta48, Toyobo, Inc., Osaka, Japan) at 15-s intervals using a fraction collector
(Liquid Handler 222XL, Gilson, Middlenton, WI, USA). The plates were allowed to stand for 12 h at
room temperature for evaporation of the solvent.
The measurement of 4-hydroxytolbutamide was performed by TLC (Ludwig et al. 1998). The
analysis of 6b-hydroxytestosterone was carried out as follows: the incubates were applied to TLC plates
(Silicagel 60F254, Merck, Darmstadt, Germany) and developed with dichloromethane–acetone (4:1 v/v)
to 16 cm in a horizontal TLC chamber. The amount of unchanged drug and its metabolites were
determined using a BAS-2500 (Fuji Photo Film Co., Tokyo, Japan). The radioactive metabolites were
positively identified by a comparison of retention times or R f using authenticunlabelled standard.
Results
Characteristics of pitavastatin in human microsomes
Figure 1 shows typical chromatograms derived from a reaction mixture using
human hepatic, renal and intestinal microsomes. In the Phase 1 reaction, 14C-
pitavastatin was metabolized only marginally to M-13 with hepaticmicrosomes
but no metabolite was detected with other microsomes. The amount of fractional
radioactivity of M-13 in hepatic microsomes was 16.3%. On addition of UDPGA,
microsomes produced pitavastatin lactone and an unknown metabolite (UM-2) at
retention times of 20 and 7 min in the Phase 2 reaction. The fractional radioactivity

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
31
Figure 1. Representative HPLC patterns of radioactivity in human microsomes. PV, pitavastatin;
M-13, 8-OH pitavastatin; UM-2, unknown metabolite; lactone, pitavastatin lactone.
Table 1. Composition of pitavastatin and its metabolites after incubation
with human tissue microsomes.
Percentage of composition
Reaction
Phase 1
Liver
Kidney
Intestine
Pitavastatin
M-13
unknown
91.5 ꢀ 0.8
6.7 ꢀ 0.7
1.8 ꢀ 0.2
99.7 ꢀ 0.2
0.0
0.3 ꢀ 0.2
99.7 ꢀ 0.1
0.0
0.3 ꢀ 0.2
Phase 2
Pitavastatin
Lactone
unknown
88.6 ꢀ 1.9
9.6 ꢀ 1.5
1.8 ꢀ 0.4
72.8 ꢀ 0.5
22.1 ꢀ 0.4
10.1 ꢀ 0.4
96.0*
2.6*
1.5*
Data are the mean ꢀ SE of three samples.
* n ¼ 2
of pitavastatin lactone in hepatic, renal and intestinal microsomes was 9.6, 22.1 and
2.6%, respectively (table 1). These latter peaks disappeared on incubation without
UDPGA (data not shown.). In addition, among the UGT chemical inhibitors
tested, ketoprofen and p-nitrophenol had extensive inhibitory effects on pitavas-
tatin lactone formation (figure 2).
Identification of UM-2 in human microsomes
UM-2 was fractionated from the HPLC elutes by monitoring of UV absorption
(254 nm), and the metabolicproperties were analysed as follows. Injection of UM-
2 led to the formation of pitavastatin lactone. UM-2 was degraded to its unchanged

32
H. Fujino et al.
Figure 2. Inhibition of lactonization of pitavastatin in human hepatic microsomes by several UGT
inhibitors. The formation of pitavastatin lactone in control samples was 9.6%. Data are the
average of duplicate determinations.
or lactone form by enzymatic hydrolysis with b-glucronidase and also converted
completely to the unchanged form by treatment with 0.1 N NaOH (figure 3).
Intrinsic metabolic clearance of pitavastatin lactonization
Lactonization of pitavastatin in human hepatic and renal microsomes with
UDPGA followed Michaelis–Menten kinetics as demonstrated by Lineweaver–
Burk plots and the curves were all linear (data not shown). The apparent Km for
pitavastatin lactone in hepatic and renal microsomes were 49.4 and 48.8 mm,
respectively, and the Vmax were 124.6 and 115.6 pmol minÀ1 mgÀ1 protein,
respectively. The intrinsic clearances (Vmax/Km) were 2.5 and 2.4 ml minÀ1 mgÀ1
protein, respectively (table 2).
Metabolism of pitavastatin by UGT enzymes
Nine microsomal samples expressing human UGTs were used to examine
pitavastatin lactonization. The catalytic properties of each human UGT enzyme
for the formation of pitavastatin lactone are shown in figure 4. Among the
recombinant UGTs examined, UGT1A3 showed the highest activity at all
concentrations. UGT2B7 was also capable of catalysing pitavastatin lactonization,
although other UGT1A enzymes including UGT1A1, 1A4 and 1A6 were involved
as well. In contrast, little metabolite was formed in the presence of the other UGT
enzymes up to a concentration of 50 mm.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
33
Figure 3. Radiochromatograms of pitavastatin and its metabolities after isolation of the unknown
metabolite (UM-2) by several treatments. (1) Direct injection, (2) enzymatic hydrolysis and (3)
alkaline treatment. PV, pitavastatin; UM-2, unknown metabolite; lactone, pitavastatin lactone.
Table 2. Kinetic constants of the apparent metabolic reaction of pitavastatin after incubation with
microsomes
Km
(mm)
Vmax
Vmax=Km
Tissue
Metabolites
(pmol minÀ1 mgÀ1 protein)
(ml minÀ1 mgÀ1 protein)
tonHeepaticlac49.4
Microsomes
Renal
124.6
2.52
77.4
115.6
–
M-13*
lactone
M-13
45.1
48.8
–
1.79
2.37
–
Microsomes
* Quoted from Xenobiotic Metabololism and Disposition (1999), 14, 415–424.
–, Unable to be calculated
Metabolic stability of pitavastatin lactone
Figure 5 shows the metabolicstability of pitavastatin and its lactone in human
hepatic microsomes. The disappearance curves for both compounds were linear up
to 120 min. The intrinsic clearance of pitavastatin and its lactone in human hepatic
microsomes was 3.4 and 4.6 ml minÀ1 mgÀ1 protein, respectively.
Metabolism of pitavastatin lactone by various CYP enzymes
Among the six recombinant CYPs examined (figure 6), CYP3A4 catalysed the
metabolism of pitavastatin lactone and the formation of M-3 was predominant.
CYP2D6 was also capable of catalysing the formation of M-13. In contrast,
metabolite formation was not observed in the presence of the other CYP enzymes
at a concentration of 2.5 mm.

34
H. Fujino et al.
Figure 4. Formation of pitavastatin lactone in microsomes by human UDP-glucuronosyl transferase-
expressed enzymes.
Figure 5. Comparison of intrinsic clearance of pitavastatin and its lactone form after incubation with
human hepaticmicrosomes (1 mg protein ml À1).
Correlation of pitavastatin lactone metabolism with CYP activities
Figure 7 shows the correlation between the metabolism of pitavastatin latone
and several CYP marker substrates in ten human hepaticmircosome samples.
A good correlation was observed for testosterone 6b-hydroxylase (CYP3A4,

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
35
Figure 6. Formation of metabolite in microsomes by CYP-expressed enzymes after incubation of
pitavastatin lactone.
R2 ¼ 0:911) and total CYP content (R2 ¼ 0:634), whereas a lower correlation
(R2 ¼ 0:036) was observed for lactone metabolism and CYP2C9-mediated dicro-
fenac 4-hydroxylase activity. Only low correlations (R2 < 0:06) were observed for
lactone metabolism and markers of enzymic activities catalysed by CYP1A2,
CYP2A6, CYP2C19, CYP2D6 or CYP2E1 isoforms.
Effect of pitavastatin on the metabolism of CYP marker substrates
Figure 8 shows the Dixon plots of tolbutamide 4-hydroxylation and testoster-
one 6b-hydroxylation, respectively. In the presence of pitavastatin and its lactone,
parallel lines without a focal point were obtained, demonstrating that there was no
inhibition of CYP2C9- and CYP3A4-mediated metabolism by pitavastatin or
pitavastatin lactone. In the presence of fluvastatin or atorvastatin lactone, the
metabolicinhibition of tolbutamide hydroxylation and testosterone 6 ꢀ-hydroxyla-
tion were observed and the Ki were 0.5 and 0.6 mm, respectively.
Discussion
Statins can be classified into the following two groups: those administered as a
prodrug, i.e. the lactone form including simvastatin and lovastatin, and those
administered in the active form, i.e. the acid form such as pravastatin, fluvastatin,
cerivastatin and atorvastatin (Alberts 1990, Lennernas and Fager 1997, Christians
et al. 1998). The absolute mechanism of lactonization for each statin has not been
reported, although the lactone form has been reported to be a metabolite of the
acid form (Komai et al. 1992, Dain et al. 1993, Kearney et al. 1993, Boberg et al.
1998). The lactone form of pitavastatin was detected although at significantly
lower levels than the parent drug in plasma of rabbits, dogs and monkeys (Fujino

36
H. Fujino et al.
Figure 7. Correlation between total metabolism of lactone and CYP activities in human hepatic
microsomes.
et al. 1999b). In humans, the area under the curve for pitavastatin lactone was
almost the same as for the parent drug (Kojima et al. 1999b), suggesting that
lactonization is the major pathway in the metabolism of pitavastatin. To clarify the
mechanism of lactonization and the metabolic properties of pitavastatin lactone,
experiments were performed in vitro in the current study.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
37
Figure 8. Inhibition of tolbutamide 4-hydroxylation and testosterone 6b-hydroxylation by HMG-
CoA reductase inhibitors in human hepatic microsomes.
Three different in vitro approaches using human microsomes, isolated UGT
enzymes and UGT chemical inhibitors demonstrated that UGTs play an import-
ant role in the lactonization of pitavastatin as follows. The first step in the
lactonization of pitavastatin is glucuronidation. UGT involves the formation of
UM-2 as an intermediate to the lactone form and catalyses the transfer of
glucuronic acid to the carboxyl of the side chain. The glucuronic acid moiety is
subsequently converted non-enzymatically to its lactone form. After conversion,
some of the resulting pitavastatin lactone is converted to the open acid form. We
also found that a portion of pitavastatin lactone is metabolized in human hepatic
microsomes. CYP3A4-mediated M-3 formation and CYP2D6-mediated M-13
formation were observed for the metabolism of pitavastatin lactone in the current
study. Interestingly, CYP2C9 was the major enzyme responsible for the formation
of M-13 from pitavastatin (Fujino et al. 1999b), but not in the metabolism of
pitavastatin lactone. A good correlation with pitavastatin lactone metabolism was
obtained for CYP3A4-mediated testosterone 6b-hydroxylase activity and for total
CYP content. This may be attributable to the fact that the CYP3A4 enzymes are
relatively abundant in human hepaticmircosomes. On the other hand, no
correlation was found for CYP2D6, indicating that the CYP2D6-mediated meta-
bolism of pitavastatin lactone was negligible. We propose the metabolic pathway
shown in figure 9, where UGT and multiple P450 enzymes are involved in the
metabolism of pitavastatin in humans.
With regard to the metabolism in extrahepatictissues by UGT enzymes, it has
been demonstrated that, as in liver, pitavastatin undergoes Phase 2 metabolism in
the kidney and intestine, resulting in the formation of pitavastatin lactone. The
results of our in vitro investigation revealed that human renal and intestinal
microsomes catalyse lactone formation. Kinetic analysis of the lactonization

38
H. Fujino et al.
Figure 9. Postulated metabolicpathways of CYP and UGT-mediated pitavastatin metabolism. The
unidentified metabolite of pitavastatin is shown in brackets. *Quoted from Xenobiotic Metabolism
and Disposition (1999), 14, 415–424.
revealed relatively low Vmax/Km in hepaticand renal microsomes. However, the
Vmax/Km of lactonization in both types of microsomes was about twofold higher
than that of M-13 formation (Fujino et al. 1999a), suggesting that lactonization is
the major metabolic pathway in man. This knowledge could be retrospectively
applied to provide a pharmacokinetic rationale for our in vivo findings (Fujino et al.
1999b).
The formation of acyl glucuronides is a major metabolic pathway for many
compounds containing a carboxylic acid (Faed 1984). These acyl glucuronides can
undergo hydrolysis and are capable of reacting with protein nucleophiles to form
covalent adducts (Ding et al. 1995). On the other hand, the characteristic
migration of the acyl group, which occurs in ester-type glucuronides, was not
observed in the metabolism of pitavastatin (Kojima et al. 1999a). These findings
implied that the elimination of pitavastatin acyl glucuronides to its lactone form
was rapid, indicating the covalent adducts might be negligible in pitavastatin
metabolism.
To date, at least 70 cDNA-encoding animal and human UGTs have been
isolated, composing a superfamily of proteins that have been identified as UGT1
and UGT2 families based upon sequence similarities in humans (Mackenzie et al.
1997). The substrate specificity of human UGTs is generally broad, and several
genetic polymorphisms have been recently identified in these families (Burchell
et al. 1995, Tukey and Strassburg 2000). In the present study, mainly UGT1A3
and UGT2B7 catalysed the glucuronidation of pitavastatin, although other UGT
enzymes such as UGT1A1, 1A4 and 1A6 are involved. Therefore, we consider that
at least five UGT enzymes contribute to the lactonization of pitavastatin in human
microsomes, indicating that drug–drug interaction was absent in the competitive
inhibition of UGT.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
39
The intrinsic clearance of atorvastatin lactone, which was mediated by
CYP3A4-dependent metabolism, was 83-fold greater than that of its acid form
(Jacobsen et al. 2000). No marked difference was noted in the intrinsic clearance of
pitavastatin and its lactone in the current study. Furthermore, we performed an
inhibition analysis using several CYP substrates to examine drug–drug inter-
actions. Simvastatin, lovastatin, atorvastatin, cerivastatin and the corresponding
lactone forms, all reported to be substrates for CYP3A4, are known to inhibit the
metabolism of mexazolam and a strong inhibitory effect on CYP has been reported
for the lactone form compared with the acid form of all statins (Ishigami et al.
2001). A large difference was noted in the inhibitory effect of the lactone form
between pitavastatin and atorvastatin on CYP3A4-mediated metabolism in the
present study. CYP2C9 plays an important role in the metabolism of fluvastatin
(Fischer et al. 1999). CYP2C9-mediated metabolism is inhibited in the presence of
fluvastatin but not with pitavastatin or its lactone. Therefore, no inhibitory effect
was found on CYP2C9- and CYP3A4-mediated metabolism by either pitavastatin
or its lactone form, the main metabolite in humans, indicating that the likelihood
of metabolic drug–drug interactions is minimal compared with other HMG-CoA
reductase inhibitors.
Acknowledgements
The authors are grateful to Ms Keiko Maekawa for technical assistance.
References
Alberts, A. W., 1990, Lovastatin and simvastatin inhibitor of HMG-CoA reductase and cholesterol
biosynthesis. Cardiology, 77, 14–21.
Aoki, T., Nishimura, H., Nakagawa, S., Kojima, J., Suzuki, H., Tamaki, T., Wada, Y., Yokoo, N.,
Sato, F., Kimata, H., Kitahara, M., Toyoda, K., Sakashita, M. and Saito, Y., 1997,
Pharmacological profile of novel synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzme A
reductase. Arzneim Forsch/Drug Research, 47, 904–909.
Baba, S., Terasawa, Y., Kimata, H., Shinohara, Y., Akira, K. and Hasegawa, H., 1994, Application of
radioluminography to off-line counting of radioactivity in high-performance liquid
chromatographic elutes. Journal of Chromatography (A), 663, 35–41.
Boberg, M., Angerbauer, R., Kanhai, W. K., Karl, W., Kern, A., Radtke, M. and Steinke, W., 1998,
Biotransformation of cerivastatin in mice, rats, and dogs in vivo. Drug Metabolism and
Disposition, 26, 640–652.
Brown, B. G., Zhao, X. Q., Sacco, D. E. and Albers, J. J., 1993, Lipid lowering and plaque regression:
new insights into prevention of plaque disruption and clinical events in coronary disease.
Circulation, 87, 1781–1791.
Burchell, B., Brierley, C. H. and Rance, D., 1995, Specificity of human UDP-glucuronosyl-
transferases and xenobioticglucuronidation. Life Sciences, 57, 1819–1831.
Christians, U., Jacobsen, W. and Floren, L., 1998, Metabolism and drug interactions 3-hydroxy-3-
methylglutaryl coenzyme A reductase inhibitor in transplant patients: are the statins
mechanistically similar? Pharmacology and Therapy, 80, 1–34.
Dain, J. G., Fu, E., Gorski, J., Nicoletti, J. and Scallen, T. J., 1993, Biotransformation of fluvastatin
sodium in humans. Drug Metabolism and Disposition, 21, 567–572.
Ding, A., Zia-Amirhosseini, P., McDonagh, A. F., Burlingame, A. L. and Benet, L. Z., 1995,
Reactivity of tolmetin glucuronide with human serum albumin: identification of binding sites
and mechanism of reaction by tandem mass spectrometry. Drug Metabolism and Disposition, 23,
369–376.
Faed, E. M., 1984, Properties of acyl glucuronides: implications for studies of the pharmacokinetics and
metabolism of acidic drugs. Drug Metabolism Reviews, 15, 1213–1249.

40
H. Fujino et al.
Fischer, V., Johanson, L., Heitz, F., Tullman, R., Graham, E., Baldeck, J. P. and Robinson, W. T.,
1999, The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin: effect on
human cytochrome P-450 and implications for metabolic drug interactions. Drug Metabolism and
Disposition, 27, 410–416.
Fujino, H., Kojima, J., Yamada, Y., Kanda, H. and Kimata, H., 1999a, Studies on the metabolicfate of
NK-104, a new inhibitor of HMG-CoA reductase (4):Interspecies variation in laboratory
animals and humans. Xenobiotics and Metabolic Disposition, 14, 79–91.
Fujino, H., Yamada, I., Kojima, J., Hirano, M., Matsumoto, H. and Yoneda, M., 1999b, Studies on
the metabolicfate of NK-104, a new inhibitor HMG-CoA reductase (5): in vitro metabolism and
plasma protein binding in animals and human. Xenobiotics and Metabolic Disposition, 14, 415–
424.
Fujino, H., Yamada, I., Shimada, S. and Yoneda, M., 2001, Simultaneous determination of taxol and its
metabolites in microsomal samples by a simple thin layer chromatography assay-inhibitory effect
of NK-104, a new inhibitor of HMG-CoA reductase. Journal of Chromatography (B), 757, 143–
150.
Fujino, H., Yamada, I., Shimada, S. and Yoneda, M., 2002, The metabolicfate of pitavastatin (NK-
104), a new inhibitor of HMG-CoA reductase: Effects on drug-metabolizing systems in rats and
humans. Arzneim Forsch/Drug Research (in press).
Havel, R. J. and Rapaport, E., 1995, Management of primary hyperlipidemia. New England Journal of
Medicine, 332, 1301–1307.
Ishigami, M., Honda, T., Takasaki, W., Ikeda, T., Komai, T., Ito, K. and Sugiyama, Y., 2001, A
comparison of the effects of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase
inhibitors on the CYP3A4-dependent oxidation of mexazolam in vitro. Drug Metabolism and
Disposition, 29, 282–288.
Jacobsen, W., Kuhn, B., Soldner, A., Kirchner, G., Sewing, K. F., Kollman, P. A., Benet, L. Z. and
Christians, U., 2000, Lactonization is the critical first step in the disposition of the 3-hydroxy-3-
methylglutaryl-CoA reductase inhibitor atorvastatin. Drug Metabolism and Disposition, 28, 1369–
1378.
Kajinami, K., Koizumi, J., Ueda, K., Miyamoto, S., Takegoshi, T. and Mabuchi, H., 2000b, Effect of
NK-104, a new hydroxymethylglutaryl-coenzyme reductase inhibitor, on low density lipoprotein
cholesterol in heterozygous familial hypercholesterolemia. Hokuriku NK-104 study group.
American Journal of Cardiology, 15, 178–183.
Kajinami, K., Mabuchi, H. and Saito, Y., 2000a, NK-104, a novel syntheticHMG-CoA reductase
inhibitor. Expert Opinions and Investigations in Drugs, 9, 2653–2661.
Kearney, A. S., Crawford, L. F., Mehta, S. C. and Radebaugh, G. W., 1993, The interconversion
kinetics, equilibrium, and solubilities of the lactone and hydroxyacid forms of the HMG-CoA
reductase inhibitor, CI-981. Pharmaceutical Research, 10, 1461–1465.
Kimata, H., Fujino, H., Koide, T., Yamada, Y., Tsunenari, Y., Yonemitsu, M. and Yanagawa, Y.,
1998, Studies on the metabolicfate of NK-104, a new inhibitor of HMG-CoA reductase (1):
absorption, distribution, metabolism and excretion in rats. Xenobiotics and Metabolic Disposition,
13, 484–498.
Kojima, J., Fujino, H., Abe, H., Yoshimura, M., Kanda, H. and Kimata, H., 1999a, Identification of
metabolites of NK-104, an HMG-CoA reductase inhibitor, in rat, rabbit and dog bile. Biological
and Pharmacological Bulletin, 22, 142–150.
Kojima, J., Fujino, H., Yoshimura, M., Morikawa, H. and Kimata, H., 1999b, Simultaneous
determination of NK-104 and its lactone in biological samples by column-switching high-
performance liquid chromatography with ultraviolet detection. Journal of Chromatography (B),
724, 173–180.
Komai, T., Kawai, K., Tokui, T., Tokui, Y., Kuroiwa, C., Shigehara, E. and Tanaka, M., 1992,
Disposition and metabolism of pravastatin sodium in rats, dogs and monkeys. European Journal
of Drug Metabolism Pharmacokinetics, 17, 103–111.
Lennernas, H. and Fager, G., 1997, Pharmacodynamics and pharmacokinetics of the HMG-CoA
reductase inhibitors. Clinical Pharmacokinetics, 32, 403–425.
Ludwig, E., WolFInger, H. and Ebner, T., 1998, Assessment of microsomal tolbutamide hydroxylation
by a simple thin-layer chromatography radioactivity assay. Journal of Chromatography (B), 707,
347–350.
Mackenzie, P. I., Owens, I. S., Burchell, B., Bock, K. W., Bairoch, A., Belaner, A., Fournel-
Gigleux, S., Green, M., Hum, D. W., Iyanagi, T., Lancet, D., Louisot, P., Magdalou, J.,
Chowdhury, J. R., Ritter, J. K., Schachter, H., Tephly, T. R., Tipton, K. F. and
Nebert, D. W., 1997, The UDP glycosyltransferase gene superfamily: recommended
nomenclature update based on evolutionary divergence. Pharmacogenetics, 7, 255–269.
Suzuki, H., Aoki, T. and Tamaki, T., 1999, Hypolipidemiceffect of NK-104, a potent HMG-CoA
reductase inhibitor, in guinea pigs. Atherosclerosis, 146, 259–270.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase
41
Suzuki, H., Yamazaki, H., Aoki, T. and Tamaki, T., 2001, Hypolipidemiceffect of NK-104 and other
3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors in guinea pigs. Arzneim Forsch/
Drug Research, 51, 38–45.
Tukey, R. H. and Strassburg, C. P., 2000, Human UDP-glucuronosyltransferases: metabolism,
expression, and disease. Annual Reviews in Pharmacology and Toxicology, 40, 581–616.
Yamada, I., Fujino H., Shimada, S. and Yoneda, M., 2001, Studies on the metabolicfate of pitavastatin
(NK-104), a new inhibitor of HMG-CoA reductase — Is the metabolism of pitavastatin in
monkey similar to that in human? Xenobiotic Metabolism and Disposition, 16 (suppl.), 291.
Yamada, N., Saito, Y. and Akanuma, Y., 2000, Effect of itavastatin on lipid metabolism in NIDDM
patients with hyperlipidemia. Atherosclerosis, 151, 48.