Association between risk of myopathy and cholesterol-lowering effect: A comparison of all statins

Article abstract of DOI:10.1016/j.lfs.2008.02.019

In the present study, we examined the mechanisms underlying the cytotoxicity of pitavastatin, a new statin, and we compared the in vitro potencies of muscle cytotoxicity using a prototypic embryonal rhabdomyosarcoma cell line (RD cells), a typical side effect of statins and compared the cholesterol-lowering effects of statins using Hep G2 hepatoma cells. Pitavastatin reduced the number of viable cells and caused caspase-9 and -3/7 activation in a time- and concentration-dependent manner. The comparison of cytotoxities of statins showed that statins significantly reduced cell viability and markedly enhanced activity of caspase-3/7 in concentration-dependent manner. On the other hand, the effects of hydrophilic statins, pravastatin, rosuvastatin were very weak. The rank order of cytotoxicity was cerivastatin > simvastatin acid> fluvastatin > atorvastatin > lovastatin acid > pitavastatin ? rosuvastatin, pravastatin. Statin-induced cytotoxicity is associated with these partition coefficients. On the other hand, the cholesterol-lowering effect of statins did not correlate with these partition coefficients and cytotoxicity. Thus, it is necessary to consider the association between risk of myopathy and cholesterol-lowering effect of a statin for precise use of statins.

Full text of DOI:10.1016/j.lfs.2008.02.019

Life Sciences 82 (2008) 969–975
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Association between risk of myopathy and cholesterol-lowering effect:
A comparison of all statins
Masaki Kobayashi, Ikumi Chisaki, Katsuya Narumi, Kazuhiro Hidaka, Toshiki Kagawa, Shirou Itagaki,
Takeshi Hirano, Ken Iseki ⁎
Laboratory of Clinical Pharmaceutics and Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku,
Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 February 2008
Accepted 29 February 2008
In the present study, we examined the mechanisms underlying the cytotoxicity of pitavastatin, a new statin,
and we compared the in vitro potencies of muscle cytotoxicity using
a prototypic embryonal
rhabdomyosarcoma cell line (RD cells), a typical side effect of statins and compared the cholesterol-
lowering effects of statins using Hep G2 hepatoma cells. Pitavastatin reduced the number of viable cells and
caused caspase-9 and -3/7 activation in a time- and concentration-dependent manner. The comparison of
cytotoxities of statins showed that statins significantly reduced cell viability and markedly enhanced activity
of caspase-3/7 in concentration-dependent manner. On the other hand, the effects of hydrophilic statins,
pravastatin, rosuvastatin were very weak. The rank order of cytotoxicity was cerivastatin N simvastatin acidN
fluvastatin N atorvastatin N lovastatin acid N pitavastatin ≫ rosuvastatin, pravastatin. Statin-induced
cytotoxicity is associated with these partition coefficients. On the other hand, the cholesterol-lowering effect
of statins did not correlate with these partition coefficients and cytotoxicity. Thus, it is necessary to consider
the association between risk of myopathy and cholesterol-lowering effect of a statin for precise use of statins.
Keywords:
Statins
Myopathy
Rhabdomyolysis
Cholesterol-lowering effect
Drug preference
©
2008 Elsevier Inc. All rights reserved.
Introduction
of pitavastatin-induced cytotoxicity, and the associations between risk
of myopathy and cholesterol-lowering effects of all statins, including
pitavastatin, have not been fully investigated.
The aim of this study was to elucidate the mechanisms of the
cytotoxicity of pitavastatin and to compare the in vitro potencies of
muscle cytotoxicity, a typical side effect of statins and to compare the
cholesterol-lowering effects of all statins.
All statins (cerivastatin, simvastatin, fluvastatin, atorvastatin,
lovastatin, pitavastatin, pravastatin and rosuvastatin) lower choles-
terol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-
CoA) reductase, thus preventing the formation of the major building
block of the cholesterol molecule. However, severe adverse events, i.e.,
skeletal muscle abnormalities, including myopathy and rhabdomyo-
lysis, associated with lipophilic statins sometimes limit the lipid-
lowering therapy with these agents (Hodel, 2002; Thompson et al.,
Materials and methods
Chemicals
2
003). Skeletal muscle abnormalities can range from benign myalgia to
myopathy, which is defined as a ten-fold elevation of the serum
creatine kinase concentration (Pedersen et al., 2004). When statins are
prescribed as monotherapy, the incidence of myopathy is approxi-
mately 0.1–0.5% and is dose-related (Garnett,1995; Maron et al., 2000).
The mechanism by which statins cause rhabdomyolysis is not precisely
known, though possibilities include, (1) depletion of secondary
metabolic intermediates such as coenzyme Q10 (Folkers et al., 1990;
De Pinieuxet al.,1996), (2) induction of apoptotic cell death (Guijarro et
al., 1998; Nakagawa et al., 2001), and (3) participation of chloride ion
channels (Pierno et al.,1992; Bramow et al., 2001). Pitavastatin is a new
chemically synthesized statin. It has strong HMG-CoA reductase
inhibiting activity (Aoki et al.,1997). There has been little investigation
Cerivastatin Na, simvastatin, atorvastatin Ca, lovastatin, pravastatin
Na and rosuvastatin Ca were kindly donated by Sankyo (Tokyo, Japan).
Fluvastatin Na was kindly supplied by Novartis Pharma (Tokyo, Japan).
Pitavastatin Ca was kindly donated by Kowa (Tokyo, Japan). Simvas-
tatin acid and lovastatin acid were prepared from their lactone form
by hydrolysis in a 0.05 N NaOH solution with stirring at 20 °C for
3
0 min. The hydrolyzed solution was adjusted to pH 7.4 with 0.2 N HCl
and then stored at 4 °C until use as described previously (Kobayashi et
al., 2006). All other reagents were of the highest grade available and
used without further purification.
Cell culture
RD cells, a prototypic embryonal rhabdomyosarcoma cell line, are
tumor cells of skeletal muscle origin that affect children and young
⁎
Corresponding author. Tel./fax: +81 11 706 3770.
E-mail address: ken-i@pharm.hokudai.ac.jp (K. Iseki).
0
024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2008.02.019

970
M. Kobayashi et al. / Life Sciences 82 (2008) 969–975
adults and express a number of muscle-specific proteins (Knudsen et
al., 1998), and these cells have been used as a model for studying the
myotoxicity effects of statins (Nishimoto et al., 2003). RD cells were
maintained in plastic culture flasks (Corning Incorporated Corning) as
described previously (Kobayashi et al., 2005).
reagent (Promega, Madison, WI). Protein concentration of the cell
lysate was adjusted to 10 µg/mL, and the cell lysate was assayed for
caspase-3/7 and caspase-9 colorimetric protease assays measuring Ac-
DEVD-pNa and Ac-LEHD-pNa cleavage (Promega, Madison, WI) as
described in the manufacturer's protocol (Promega, Madison, WI).
Hep G2 cells were kept in Dulbecco's modified Eagle's medium
(
Sigma) with 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora,
Uptake experiments
2
OH) and 1% penicillin-streptomycin at 37 °C under 5% CO .
The uptake experiments were performed as described previously
(Kobayashi et al., 2007a,b). Since we previously examined the
accumulation of 100 µM cerivastatin Na, pravastatin Na and
rosuvastatin Ca in RD cells, we selected other statin concentrations
at 100 µM in order to compare all statins.
MTT assay
The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bro-
mide (MTT) assay was performed as described previously (Mosmann,
1
983). MTT is normally reduced by dehydrogenases of viable cells and
In the experiments on uptake of cerivastatin Na, simvastatin acid,
fluvastatin Na, atorvastatin Ca, lovastatin acid, pitavastatin Ca,
pravastatin Na and rosuvastatin Ca, after removal of the growth
medium, cells were washed with HEPES buffer (25 mM D-glucose,
transformed to formazan. The assay detects living, but not dead, cells
and the signal generated is dependent on the degree of activation of
cells. For the MTTassay, RD cells were seeded on 96-well plastic plates.
Following cell attachment (24 h), various concentrations of statins
were added for the times indicated. At 4 h before the end of treatment,
137 mM NaCl, 5.37 mM KCl, 0.3 mM Na , 0.44 mM KH
2
HPO
4
2 4
PO ,
4.17 mM NaHCO , 1.26 mM CaCl , 0.8 mM MgSO and 10 mM HEPES,
3
2
4
1
0 µL of PBS-containing MTT solution (0.5%) was added, and the cells
pH 7.4) and preincubated at 37 °C for 10 min with 1 mL of HEPES buffer
(pH 7.4). Uptake was initiated by applying HEPES buffer (pH 7.4)
containing 100 µM statins. The uptake study was performed at 37 °C.
After a predetermined time period, uptake was terminated by
suctioning off the applied solution and immersing the plates in ice-
cold HEPES buffer (pH 7.4) and then suspending in 0.5 mL of ice-cold
water. The cellular protein content was determined by the method of
Lowry et al. (1951) with bovine serum albumin as a standard.
were incubated for a further 4 h. The MTT medium was then replaced
with 0.2 mL dimetylsulfoxide, and absorbance was read at 590 nm.
Absorbance measured in MTT assays was expressed as percent of the
control (defined as 100%).
Caspase assay
The caspase assay was performed as described previously
Kobayashi et al., 2007a). Cells were lysed with a cell culture lysis
All statins were determined using an HPLC system equipped with a
UV detector. Cerivastatin, pravastatin and rosuvastatin concentrations
(
Fig. 1. Effect of pitavastatin on viability of RD cells (a). Cell viability was measured by the MTT assay. RD cells were exposed to various concentrations of pitavastatin for 24–72 h. Each
point represents the mean±S.D. of six–twelve determinations. Effect of pitavastatin on caspase-3/7 activity ratio in RD cells (b). RD cells were exposed to various concentrations of
pitavastatin for 24–72 h, and the cell lysate was used to measure caspase-3/7 activity ratio. Each column represents the mean with S.D. of three-eighteen determinations. ⁎;
significantly different from control (no addition) at pb0.05. Effect of caspase-9 inhibitor on caspase-9 (c) and caspase-3/7 (d) activity ratio in pitavastatin-treated RD cells. RD cells
were exposed to 10 µM pitavastatin in the absence or presence of 10 µM caspase-9 inhibitor for 24–48 h, and the cell lysate was used to measure caspase-9 and -3/7 activity ratio. Each
†
column represents the mean with S.D. of three–eighteen determinations. ⁎; significantly different from control (no addition) at pb0.05, ; significantly different from pitavastatin
alone at pb0.05.

M. Kobayashi et al. / Life Sciences 82 (2008) 969–975
971
were determined using an HPLC system as described previously
Results
(
(
Kobayashi et al., 2007a,b). The columns were Mightysil RP-8 GP
5 µm, 250 mm×4.6 mm i.d.). The mobile phase of cerivastatin Na and
Mechanism underlying the cytotoxicity of pitavastatin in RD cells
simvastatin acid consisted of 2.5 mM CH
that of fluvastatin Na, atorvastatin Ca and lovastatin acid consisted of
.5 mM CH COONH :CH CN (1:2, v/v), and that of pitavastatin Ca,
pravastatin Na and rosuvastatin Ca consisted of 2.5 mM CH COONH
CH CN (1:1, v/v).
3 4 3
COONH :CH CN (1:4, v/v),
To clarify the mechanism of the cytotoxicity of pitavastatin, we
performed an MTT assay and examined the effects of pitavastatin, a
new statin, on RD cell viability. As shown in Fig. 1(a), pitavastatin
reduced the number of viable cells in a time- and concentration-
dependent manner. To clarify whether pitavastatin-induced cytotoxi-
city is associated with apoptosis, we examined the activation of
caspases. The effector caspase-3/7 plays a central role in apoptosis
since it translocates from the cytosol into the nucleus upon activation
(Ferri and Kroemer, 2001). As shown in Fig.1(b), pitavastatin markedly
enhanced the activity of caspase-3/7 in a time- and concentration-
dependent manner. Moreover, the activation of caspase-9 and that of
caspase-3/7 were blocked by the caspase-9 inhibitor zLEHD-fmk (Fig.
1(c,d)). Accordingly, pitavastatin mainly induced apoptosis via a
mitochondrial stress-induced cascade in RD cells. These results are
consistent with the results for another statin, cerivastatin (Kobayashi
et al., 2007a).
2
3
4
3
3
4
:
3
Measurement of cellular cholesterol contents
5
HepG2 cells were seeded at a density of 5×10 cells/mL on 12-well
plastic plates. Following cell attachment (24 h), 1 µM statins were
added for 24 h or 48 h. After the medium had been removed, the cells
were washed two timees with PBS and cellular cholesterol were
extracted with 1.5 mL of n-hexane:isopropanol (3:2, v/v) for 30 min.
The lipid phase was evaporated to dryness and resuspended in 300 µL
of hexane. Cellular total cholesterol was measured by using a
Cholesterol assay kit (Cayman Chemical).
Statistical analyses
Statin-induced skeletal muscle toxicity: a comparison of all statins
Student's t-test was used to determine the significance of
differences between two group means. Statistical significance
among means of more than two groups was determined by one-way
analysis of variance (ANOVA).
To compare the cytotoxicities of statins, we examined the effects of
all statins on RD cell viability. As shown in Fig. 2(a), lipophilic statins,
cerivastatin, simvastatin acid, fluvastatin, atorvastatin, lovastatin acid
and pitavastatin, significantly reduced cell viability in a concentration-
dependent manner. On the other hand, the effects of hydrophilic
statins, pravastatin and rosuvastatin were very weak (Table 1). As
Statistical significance was defined as pb0.05. The IC50 value for
each drug was determined from the dose–response curve generated
®
by using Origin (version 6.1J).
Fig. 2. Effects of all statins on viability of RD cells (a). Cell viability was measured by the MTT assay. RD cells were exposed to various concentrations of all statins for 48 h. Each point
represents the mean±S.D. of three–eighteen determinations. Effect of cerivastatin on the viability of RD cells was investigated in our previous studies (Kobayashi et al., 2007a).
Correlation between the cytotoxicity and partition coefficients of statins (b). Effects of all statins on caspase-3/7 activity ratio in RD cells (c). RD cells were exposed to various
concentrations of statins for 48 h, and the cell lysate was used to measure caspase-3/7 activity ratio. Each column represents the mean with S.D. of three-eighteen determinations. ⁎;
significantly different from control (no addition) at pb0.05. Effects of cerivastatin and rosuvastatin on caspase-3/7 activity ratio were investigated in our previous studies (Kobayashi
et al., 2007a,b).

972
M. Kobayashi et al. / Life Sciences 82 (2008) 969–975
Fig. 2 (continued).
shown in Fig. 2 (b), statins induced the reduction of cell viability
correlated with these partition coefficients. Next, we compared the
caspase-3/7 activation potencies of all statins. As shown in Fig. 2(c),
lipophilic statins significantly enhanced the activity of caspase-3/7 in a
concentration-dependent manner. On the other hand, the effects of
hydrophilic statins were very weak. These results suggest that statin-
induced apoptosis is associated with these partition coefficients.
greater than that of hydrophilic statins. Moreover, accumulation of
statins correlated with cytotoxicity of all statins. These results
suggest that statin-induced cytotoxicity is associated with intracel-
lular accumulation of statins.
Association between risk of myopathy and cholesterol-lowering effect: a
comparison of all statins
Accumulation of statins in skeletal muscle cells: a comparison of all
statins
The association between risk of myopathy and cholesterol-
lowering effect must be clarified for more precise use of statins. We
examined the cholesterol-lowering effects of all statins using an in
vitro hepatocyte model, HepG2 cells. All statins significantly lowered
intracellular cholesterol amounts after 24 or 48 h of incubation
(Tables 2 and 3). As shown in Fig. 4, cholesterol-lowering effect of
statins did not correlate with cytotoxicity.
To clarify the difference in the cytotoxicities of statins, we
examined the accumulation of statins in RD cells. As shown in Fig. 3,
the time courses of accumulation of all statins reached an
equilibrium in 30 min, and accumulation of lipophilic statins was
Discussion
Table 1
IC50 values of statins on the growth of RD cells
HMG-CoA reductase inhibitors such as statins are the most widely
used cholesterol-lowering agents for prevention of cardiovascular
disease (Havel and Rapaport, 1995; Jukema et al., 1995; Downs et al.,
1998). However, severe adverse events, including myopathy and
rhabdomyolysis, associated with lipophilic statins sometimes limit the
lipid-lowering therapy with these agents (Hodel, 2002; Thompson et
al., 2003). There have been many studies on statin-induced myopathy
in animals and humans (Smith et al., 1991; Baker and Tarnopolsky,
Statins IC50
Values (μM)
Cerivastatin
Simvastatin
Fluvastatin
Atorvastatin
Lovastatin
Pitavastatin
Pravastatin
Rosuvastatin
1.77
3.99
8.34
9.26
52.2
117
4890
N100
2
2
001; Evans and Rees, 2002; Thompson et al., 2003; Schaefer et al.,
004), but the precise mechanism has not been elucidated. Pitavas-
Data were taken from Fig. 2(a).
tatin is a new chemically synthesized statin. There has been little

M. Kobayashi et al. / Life Sciences 82 (2008) 969–975
973
Fig. 3. Accumulation of all statins in RD cells. The accumulation of 100 µM statins was measured at pH 7.4. Each column represents the mean with S.D. of three–eight determinations.
Insert: correlation between a 60-min uptake and the cytotoxicity of statins. The accumulation of cerivastatin, pravastatin and rosuvastatin was investigated in our previous studies
(
Kobayashi et al., 2007a,b).
investigation of pitavastatin-induced cytotoxicity. Therefore, we
examined the mechanism of the cytotoxicity of pitavastatin. Pitavas-
tatin reduced the number of viable cells and elevated caspase-3/7
activity in a time- and concentration-dependent manner (Fig. 1(a,b)).
Caspases, a family of cysteine proteases, is one of the best-studied
apoptotic pathways. Caspases are synthesized in the cytosol as
inactive proenzymes and, in response to severe stress, become
activated in a particular sequential cascade. Once activated, execu-
tioner caspases contribute to dismantle the cell through direct
proteolysis of cell structures and repair enzymes and regulatory
proteins through initiator caspases (i.e., caspase-2, -8, -9, or -12) or
effector (downstream) caspases (i.e., caspase-3, -6, or -7). Activation of
caspase-3 represents a commitment for cell disassembly and is a
hallmark for apoptosis. Three different pathways are recognized as the
initiator caspases involved in activating apoptosis (Loeffler and
Kroemer, 2000; Walczak and Krammer, 2000). The receptor-mediated
pathway includes the Fas/Fas-ligand cell-surface receptors, which are
activated and subsequently lead to caspase-8 activation (Chang et al.,
ingly, we speculate that pitavastatin mainly induced apoptosis via a
mitochondrial stress-induced cascade in RD cells. Next, to compare
the cytotoxicities of statins, we examined the effects of all statins on
RD cell viability. Lipophilic statins, cerivastatin, simvastatin acid,
fluvastatin, atorvastatin, lovastatin acid and pitavastatin, significantly
reduced cell viability in a concentration-dependent manner. On the
other hand, the effects of hydrophilic statins, pravastatin and
rosuvastatin, were very weak (Fig. 2(a), Table 1). The rank order of
the cytotoxicity was cerivastatin N simvastatin acidN fluvastatin N
atorvastatin N lovastatin acid N pitavastatin ≫ rosuvastatin, pravas-
tatin and cytotoxicity of statins correlated with these partition
coefficients (Fig. 2(b)). We also examined whether all cytotoxicity of
statins was associated with apoptosis. Lipophilic statins significantly
enhanced the activity of caspase-3/7 in a concentration-dependent
manner (Fig. 2(c)). The rank order of caspase-3/7 activation was
cerivastatin N simvastatin acid N fluvastatin N atorvastatin N lovastatin
acid N pitavastatin ≫ rosuvastatin, pravastatin. These results suggest
that statin-induced apoptosis is associated with these partition
coefficients. To clarify the difference in the cytotoxicities of statins,
we examined the accumulation of all statins in RD cells. Accumulation
of statins correlated with the cytotoxicity of all statins (Fig. 3). These
results suggest that statin-induced skeletal muscle cytotoxicity is
associated with intracellular accumulation of statins.
1995). Alternatively, the mitochondrial pathway includes the activa-
tion of caspase-9, is associated with mitochondrial stress, and leads to
cytochrome c release (Cecconi et al., 1998). The newly recognized
caspase-12 is activated by endoplasmic reticulum stress and induces
the cleavage of caspase-3 in a cytochrome c-independent manner
(
Nakagawa and Yuan, 2000). The activation of caspase-9 and -3/7 was
In terms of the more precise use of statins, we examined the
association between risk of myopathy and cholesterol-lowering
blocked by the caspase-9 inhibitor zLEHD-fmk (Fig. 1(c,d)). Accord-
Table 2
Table 3
Cholesterol-lowering effect of statins in Hep G2 cells
Cholesterol-lowering effect of statins in Hep G2 cells
Control
Cholesterol (% of control)
00.0±25.8
Control
Cholesterol (% of control)
100.0±23.8
1
Cerivastatin
Simvastatin
Fluvastatin
Atorvastatin
Lovastatin
Pitavastatin
Pravastatin
Rosuvastatin
72.6±7.0
83.0±10.0
68.3±11.1
59.6±7.0
71.1±16.5
59.3±10.1
62.9±9.7
62.1±2.3
Cerivastatin
Simvastatin
Fluvastatin
Atorvastatin
Lovastatin
Pitavastatin
Pravastatin
Rosuvastatin
93.0±29.5
62.7±16.4
91.1±26.5
93.7±36.5
55.1±30.7
63.1±18.7
97.1±44.9
92.1±41.5
Hep G2 cells were exposed to 1 µM statins for 24 h. Each value represents the mean±S.
Hep G2 cells were exposed to 1 µM statins for 48 h. Each value represents the mean±S.D.
D. of 4–16 measurements.
of 13–31 measurements.

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