Biochemical alterations and liver toxicity analysis with pioglitazone in healthy subjects

Abstract

Pioglitazone, a member of the thiazolidinediones, is a potent, highly selective agonist for peroxisome proliferator-activated receptor gamma and is an excellent insulin sensitizer used in treating type 2 diabetes mellitus. The present study investigated the effect of pioglitazone on glucose, total cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol, total proteins, albumin (ALB), alanine transaminase (ALT), and aspartate transaminase (AST) levels in 20 healthy Bengali male volunteers in a randomized, placebo-controlled study. Blood samples were collected before and 0.5-24.0 hours after a single oral dose of a 30 mg pioglitazone tablet. Plasma pioglitazone level was determined using a validated method of reverse-phase binary high-performance liquid chromatography. Blood lipid profile and levels of glucose, ALT, and AST were estimated using enzyme assay kits, plasma protein level was estimated by the biuret method, and plasma ALB level was determined colorimetrically. No significant change in blood glucose, total proteins, total cholesterol, triglyceride, HDL, and LDL levels was observed over the 24-hour assessment period, indicating no plasma biochemical alterations. There were no significant differences between baseline and 24-hour values of ALB, ALT, and AST levels, indicating a lack of liver toxicity. Our results indicate that a single dose of 30 mg of pioglitazone has no hypoglycemic or hypolipidemic effect or liver toxicity within 24 hours of treatment among healthy Bengali males.

Full text

1
Introduction
e prevalence of diabetes, especially type 2 diabetes
mellitus (T2DM), has been increasing rapidly, imposing
one of the most challenging public health problems of
the 21st century to Bangladesh and the world. ere is no
known fail-safe method of preventing T2DM. e treat-
ment goals for T2DM include eectively controlling blood
glucose level and maintaining a healthy blood pressure
and lipid prole to avert the serious complications result-
ing from a sustained tissue exposure to excess glucose.
A number of agents have been used in treating T2DM,
but more eective new drugs are necessary (Ripsin et
al., 2009). Some of the new agents include peroxisome
proliferator-activated receptor gamma PPAR-γ agonists,
such as pioglitazone and rosiglitazone. Comprehensive
information on the mechanism(s) of action, ecacy,
pharmacokinetics, pleiotropic eects, drug interactions,
and adverse eects of such drugs are essential. Benecial
or neutral eects on body weight are some of the attrac-
tive features of the new drugs. However, the higher cost
and lack of adequate long-term safety and clinical out-
come data for the agents remain of concern.
Pioglitazone is a thiazolidinedione (TZD) derivative
and a novel oral hypoglycemic agent for the management
of T2DM (Arono et al., 2000). Insulin resistance and
hyperinsulinemia play important roles on the pathogenesis
of T2DM. Hyperinsulinemia is an independent risk
factor for cardiovascular diseases (Uwaifo and Ratner,
2003; Despres et al., 1996). Pioglitazone activates the
transcription of insulin-responsive genes and thus
increases insulin sensitivity (Gillies and Dunn, 2000). e
drug stimulates the uptake of glucose and fatty acids by
RESEARCH ARTICLE
Biochemical alterations and liver toxicity analysis with
pioglitazone in healthy subjects
Sajal Kumar Saha1, Sreedam Chandra Das1, Abdullah-Al-Emran2, Mithun Sarker1,
Md Aftab Uddin2, A.K. Azad Chowdhury1, and Sitesh Chandra Bachar3
Departments of 1Clinical Pharmacy and Pharmacology,2Genetic Engineering and Biotechnology, and
3Pharmaceutical Technology, University of Dhaka, Dhaka, Bangladesh
Abstract
Pioglitazone, a member of the thiazolidinediones, is a potent, highly selective agonist for peroxisome proliferator-
activated receptor gamma and is an excellent insulin sensitizer used in treating type 2 diabetes mellitus. The present
study investigated the eect of pioglitazone on glucose, total cholesterol, triglyceride, low-density lipoprotein (LDL)
cholesterol and high density lipoprotein (HDL) cholesterol, total proteins, albumin (ALB), alanine transaminase (ALT),
and aspartate transaminase (AST) levels in 20 healthy Bengali male volunteers in a randomized, placebo-controlled
study. Blood samples were collected before and 0.5–24.0 hours after a single oral dose of a 30 mg pioglitazone tablet.
Plasma pioglitazone level was determined using a validated method of reverse-phase binary high-performance liquid
chromatography. Blood lipid prole and levels of glucose, ALT, and AST were estimated using enzyme assay kits,
plasma protein level was estimated by the biuret method, and plasma ALB level was determined colorimetrically. No
signicant change in blood glucose, total proteins, total cholesterol, triglyceride, HDL, and LDL levels was observed
over the 24-hour assessment period, indicating no plasma biochemical alterations. There were no signicant
dierences between baseline and 24-hour values of ALB, ALT, and AST levels, indicating a lack of liver toxicity. Our
results indicate that a single dose of 30 mg of pioglitazone has no hypoglycemic or hypolipidemic eect or liver
toxicity within 24 hours of treatment among healthy Bengali males.
Keywords: Biochemical alterations, Bengali population, liver toxicity, pioglitazone, PPAR-γ
Address for Correspondence: Sajal Kumar Saha, Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Curzon Hall,
Dhaka 1000, Bangladesh; Fax: +880 28615583; E-mail: sajal331@yahoo.com
(Received 20 November 2011; revised 15 January 2012; accepted 16 January 2012)
Drug and Chemical Toxicology, 2012; Early Online: 1–6
© 2012 Informa Healthcare USA, Inc.
ISSN 0148-0545 print/ISSN 1525-6014 online
DOI: 10.3109/01480545.2012.658920
Drug and Chemical Toxicology
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00
1
6
20November2011
15January2012
16January2012
0148-0545
1525-6014
© 2012 Informa Healthcare USA, Inc.
10.3109/01480545.2012.658920
2012
Pioglitazone in healthy subjects
S. K. Saha et al.
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2 S. K. Saha et al.
Drug and Chemical Toxicology
promoting the expression of cellular glucose and fatty acid
transporters (Gillies and Dunn, 2000). Similar to other
TZDs, pioglitazone ameliorates insulin resistance without
stimulating insulin release by the pancreatic β cells, thus
lowering the risk of hypoglycemia (Madan, 2005). It has
been demonstrated that pioglitazone improves glycemic
control and glycated hemoglobin, fasting glucose and
high-density lipoprotein (HDL) levels, and signicantly
decreases triglyceride (Tg) level without eecting total
cholesterol and low-density lipoprotein (LDL) levels
(Dormandy et al., 2005).
A measurable level of pioglitazone appears in the
plasma of fasting subjects within 30 minutes of oral
administration and peak plasma concentration and is
attained within 2 hours (Eckland and Danhof, 2000;
Baba, 2001). e absolute bioavailability of the drug
ranges between 70 and 96%, with a mean value of 83%
(Hanefeld, 2001). Over 99% of plasma pioglitazone is
found to be associated with plasma proteins, mainly to
plasma albumin (ALB) (Li, 2006). After a single-dose
administration, the mean apparent volume of distribu-
tion of pioglitazone was observed at 0.63 ± 0.41 L/kg of
body weight and the drug was excreted primarily in the
form of metabolites and their conjugates (Waugh et
al., 2006). Identication of these metabolites and their
routes of excretion will be helpful in deciphering the
mechanism of pioglitazone clearance (Torii et al., 1984).
e above summary indicates that much is known on the
pharmacokinetics of the drug.
Bangladesh has over 3.2 million diabetic patients
(Wild et al., 2004) and pioglitazone has been prescribed
alone or with other drugs in Bangladesh since 1999, but
there are no data on the clinical eect of the drug on
Bengali populations. e present work was designed to
study the eect of pioglitazone on hypoglycemic activity,
hypolipidemic activity, and liver toxicity among healthy
Bangladeshi subjects.
Methods
Study subjects
Twenty healthy Bengali male adult volunteers (mean
age, 23.93 ± 2.73 year; range, 20–30; mean body weight,
61.40 ± 7.98 kg; range, 58–70; mean height, 164.93 ± 4.87
cm; range, 166–185; mean body mass index, 22.57 ± 1.47
kg/m2) were enrolled in this study. Before enrollment,
each subject was screened for good health through a
routine physical checkup and laboratory tests through
qualied healthcare providers. None of the volunteers
used any medications, including the test drug, within 2
weeks before and throughout the study. Exclusion cri-
teria included any history of signicant gastrointestinal
conditions that could potentially impair the absorption
or disposition of the drug, previous history of allergy to
any medications, donation of blood or plasma within 30
days preceding the study, and the use of the investiga-
tional agent within 30 days of the study. Volunteers were
asked to abstain from smoking and from taking alcohol
or caeine for at least 48 hours before and throughout
the study. Volunteers were informed of the risks, ben-
ets, procedures, and aims of the study as well as their
rights as research subjects. Informed signed consent was
obtained from each of the volunteers, and the study pro-
tocol was approved by the Institutional Review Board of
the Department of Clinical Pharmacy and Pharmacology
at the University of Dhaka (Dhaka, Bangladesh).
Reagent and chemicals
Pioglitazone hydrochloride (99.98% purity), for use as a
reference standard, and rosiglitazone (99.96% purity),
for use as an internal standard, were purchased from Dr.
Reddy’s Laboratories Ltd. (Hyderabad, India). Sodium
dihydrogen phosphate, disodium hydrogen phosphate,
and glacial acetic acid were of analytical reagent grade.
All solvents were of high-performance liquid chromatog-
raphy (HPLC) grade and were obtained from Scharlau
(Sentmenat, Spain). Reagent-grade water was obtained
from the Center of Excellence at the University of Dhaka
(Dhaka, Bangladesh).
Chromatography
e HPLC system consisted of a Shimadzu prominence
module with ultraviolet-visible (UV/vis) (SPD20AVP;
Shimadzu) detector (Shimadzu, Kyoto, Japan).
Chromatographic separation was carried out on a Luna
C18 (250 × 4.6 mm) column (Phenomenex, Torrence,
California, USA). e mobile phase was comprised of
acetonitrile and 20 mM of ammonium acetate buer (pH
4.5 ± 0.2; 60:40, v/v). All separations were performed at a
ow rate of 1.0 mL/min. Injection volume was 20 μL, and
the column was maintained at an ambient temperature.
Peaks were determined using a UV detector set at a wave-
length of 269 nm.
Blood collection
Venous blood samples (6.0 mL) were collected in hepa-
rinized tubes immediately before and 0.5, 1.0, 1.5, 2.0,
2.5, 3.0, 5.0, 8.0, 12.0, and 24.0 hours after drug adminis-
tration. Plasma was separated by centrifuging the tubes
at 1,000 relative centrifugal force (RCF) for 20 minutes.
Plasma samples were stored at –20°C until analysis.
Detection and quantification of analytes
e level of the drug in the plasma samples was deter-
mined using HPLC. Concentrations of the other com-
ponents of plasma samples were determined using the
following methods: total protein by the biuret method,
ALB by the colorimetric method, glucose by the glu-
cose-oxidase method, and alanine transaminase (ALT),
aspartate transaminase (AST), and lipid prole (total
cholesterol, Tg, and HDL and LDL) using appropriate
enzymatic assay kits and a biochemical analyzer.
Preparation of stock and standard solutions
Base stock solutions (200 µg/mL) of pioglitazone and the
internal standard were prepared separately in dimethyl
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Pioglitazone in healthy subjects 3
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sulfoxide (DMSO). e stock solution was diluted as
necessary using the mobile phase as the diluent. Working
standard solutions of pioglitazone were prepared by
mixing the secondary standard solutions with drug-free
human plasma to a nal pioglitazone concentration of
0, 0.05, 0.10, 0.25, 0.50, 1.0, and 2.0 μg/mL. e working
internal standard solution with a concentration of 0.05
μg/mL was prepared by diluting the stock solution with
the mobile phase.
Preparation of samples
Plasma (1.0 mL) was mixed with 0.25 mL of 0.1 mol/L of
K2HPO4 in a 10 mL glass tube, and the tube was applied
on a vortex mixer for 30 seconds. An aliquot of 5.0 mL of
ethyl acetate was added, and the tube was applied again
to a vortex mixer for 3 minutes. e tube was centrifuged
at 1,300 RCF for 6 minutes. A sample of the supernatant
(4.0 mL, exclusively the organic phase) was removed to
a separate tube, and the uid phase was evaporated by
placing the tube in a 45°C water bath under a stream of
nitrogen gas. e dried residue was completely dissolved
in 0.1 mL of DMSO. A 20 μL volume of the solution was
injected onto the HPLC column.
Bioanalytical method validation
Linearity
e calibration curve was linear over the range of 0.05–
2.0 μg/mL in human plasma. e linear equation was
typically Y = 64,510 X –1,128.6, r2 = 0.9995 [Y = peak area
and X = concentration (μg/mL)]. e extracted recovery
was >80%, and relative standard deviation for intra- and
interday assay was less than 10%. Limit of quantication
was 0.05 μg/mL.
Accuracy and precision
Accuracy and precision (i.e., intra- and interday) was
ascertained on the basis of quality-control samples (QCs)
analysis. A result obtained from six replicate injections of
the QCs in plasma is summarized in Table 1.
Recovery
Recovery was examined from QCs for low-, medium-,
and high-concentration ranges in plasma samples.
Recovery was expressed as the percentage of analytes
recovered by the assay. In plasma, average recovery was
>80%. e high recovery conrmed the suitability of the
method for analysis of pioglitazone in the given samples.
Statistical analyses
e values for the control and test subjects were compared
using analysis of variance (paired t-test), followed by least
signicant dierence analysis using the SPSS software
bundle (Dublin, Ireland). Results were expressed as mean
± standard deviation, where P ≤ 0.05 was considered sig-
nicant and P ≤ 0.01 was considered highly signicant.
Results
e plasma concentration of pioglitazone rose rapidly and
peaked at 2.514 ± 0.735 hours (tmax) post-administration. e
peak plasma concentration (Cmax) was 1.117 ± 0.315 μg/mL
(Figure 1). e plasma drug concentration declined rapidly
for aproximately 2 hours after the Cmax and then slowly for
the next 9 hours to 0.109 ± 0.059 μg/mL at 24 hours past the
time of administration of the single dose (Figure 1A).
To investigate whether the single dose of the drug
aected the metabolic homeostasis of the subjects,
Table 1. Accuracy and precision in determining plasma pioglitazone concentrations using HPLC based on six replicate injections
reecting intra- and interday variations.
Intraday Interday
Cadded (µg/mL) Cobs (µg/mL) RSD Recovery (%) Cobs (µg/mL) RSD Recovery (%)
0.05 0.043 ± 0.01 1.98 80.75 0.039 ± 0.01 2.56 79.28
0.10 0.031 ± 0.01 1.76 82.10 0.080 ± 0.01 1.74 80.36
0.50 0.400 ± 0.02 2.20 80.64 0.390 ± 0.01 1.62 79.53
Figure 1. Pattern of changes of plasma concentrations of pioglitazone, glucose, and total proteins over a 24-hour assessment period. (A)
Change of plasma pioglitazone and glucose concentration. (B) Change of plasma pioglitazone and total protein concentration. Values at
the 0 time point indicate the concentrations of pioglitazone, glucose, and total proteins before drug administration.
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4 S. K. Saha et al.
Drug and Chemical Toxicology
plasma glucose and total protein levels were determined
concomitantly. Plasma glucose concentration increased
slightly and peaked at 8 hours post–drug administration
(most likely because of dietary reasons) before returning
to the base level (Figure 1A). Likewise, plasma protein
concentration increased slightly after drug administra-
tion and peaked at 3 hours and then decreased to baseline
(Figure 1B). e slight wavy nature of the plasma protein
concentration curve, both in test and control subjects
(Figures 2B), was most likely a result of the limitations of
the biuret method of protein assay. Overall, there was no
signicant dierence in plasma glucose and total protein
concentrations of the test subjects and the control sub-
jects (Figure 2).
To investigate whether administration of pioglitazone
caused any alterations in lipid prole, the prole at
baseline and 24 hours after administration of the drug
were examined. Table 2 shows that there was no sig-
nicant dierence in total cholesterol (P < 0.472), Tg
(P < 0.814), HDL (P < 0.600), and LDL (P < 0.098) levels in
the pre- and post-treatment samples. Finally, to investi-
gate whether administration of the drug caused any hep-
atotoxicity, levels of two important indicator enzymes in
the pretreatment and 24-hour post-treatment samples
were determined. As shown in Table 2, there was no sig-
nicant change in the levels of ALT (P < 0.485) and AST
(P < 0.053) in the two types of samples. Notably, ALB
binds the majority of plasma pioglitazone, but there was
no signicant change in plasma ALB levels (P < 0.650) in
the pretreatment and 24-hour post-treatment samples
(Table 2).
Discussion and conclusion
Success of a drug and a drug delivery system depend
on the fate of the drug in the human body (Tsuchida
and Abe, 1982). A large body of literature has identied
inappropriate pharmacokinetics as the major cause
for the failure of drug development projects (Prentis
et al., 1988). Appropriate consideration regarding
safety and ecacy of a drug requires an analysis of
the links between systemic exposure and eects of the
drug (Walker, 2004). Hereditary dierences aect drug
metabolism and thus play a role as a major determinant
of variable drug exposure and response (Flockhart and
Desta, 2009). Yet, many drugs are developed, tested, and
formulated in one country and then exported and used
in another. Besides genetic dierences, environmental
dierences may also play important roles in drug
metabolism, exposure, and response (Belle and Singh,
2008). erefore, there is a need for studying the
biodisposition of drugs in target populations residing in
diverse environments.
Here, we studied the biodisposition of pioglitazone,
an imported drug in Bangladesh, using healthy Bengali
volunteers as the test subjects. In this population, the
Cmax of the drug was observed at 1.117 ± 0.315 μg/mL
and the tmax was 2.54 ± 0.735 hours postadministration
of a single 30-mg tablet (Figure 1). Plasma drug con-
centration was negligible (0.109 ± 0.059 μg/mL) at 24
hours after administration. Wittayalertpanya et al. (2006)
examined the similar parameters on a ai population
and observed a Cmax of 1.14 ± 0.29 μg/mL at the tmax of 2.00
± 1.61 hours. Budde et al. (2003) used a single dose of
45 mg of pioglitazone on a sample of a German popula-
tion and observed a Cmax 1.329 ± 0.667 µg/ mL at the tmax of
2.0 hours (range, 1.0–4.0). ese values are comparable
to the values we observed for the Bengali population.
However, Zhang et al. (2004) observed a Cmax of 1.85 µg/
Table 2. Plasma concentrations of selected metabolites and
enzymes before and 24 hours after a single 30-mg dose of
pioglitazone administration among healthy Bengali subjects.
Parameters Baseline After 24 hours P–value
Glucose (mg/dL) 81.095 ± 5.542 81.513 ± 3.999 0.424
Total cholesterol
(mg/dL)
130.957 ± 21.998 114.042 ± 20.098 0.472
Tg (mg/dL) 100.721 ± 21.819 111.78 ± 31.285 0.814
HDL (mg/dL) 7.691 ± 1.879 7.447 ± 2.316 0.600
LDL(mg/dL) 103.049 ± 23.381 84.238 ± 24.925 0.098
Total proteins
(mg/mL)
67.71 ± 3.330 67.70 ± 3.230 0.440
ALB (g/dL) 5.045 ± 0.030 5.034 ± 0.030 0.650
ALT (U/L) 7.027 ± 1.608 7.550 ± 1.607 0.485
AST (U/L) 6.250 ± 1.927 7.602 ± 1.633 0.053
e high P-values indicate a lack of signicant dierence in each
biochemical parameter.
Figure 2. Change of plasma glucose and total protein concentrations between test and control subjects before and up to 24 hours after
administration of pioglitazone. (A) Comparison of glucose concentration changes between control and test group. (B) Comparison of
protein concentration changes between control and test group.
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Pioglitazone in healthy subjects 5
© 2012 Informa Healthcare USA, Inc.
mL at the tmax of 1.80 ± 0.6 hours using the same dose in
Chinese population. e observation reects that there
is indeed a dierence in pioglitazone metabolism among
dierent ethnic groups.
e safety of the oral administration of pioglitazone in
humans has been examined in acute settings previously,
and no signicant adverse eect has been observed
(Erdmann et al., 2007; Hanefeld et al., 2007); however,
a drop in plasma glucose level was observed by some
groups (Miyazaki et al., 2001; Bajaj et al., 2003). In the
present study, we observed no signicant change in
plasma glucose level, irrespective of the plasma drug con-
centration. Plasma glucose level changed slightly during
the 24-hour observation period, and maximum (129.471
± 7.414 mg/dL) and minimum (81.513 ± 3.99 mg/dL)
plasma glucose concentrations were recorded at 8 and
24 hours post-administration, respectively (Figure 1A).
e above values were not signicantly dierent from the
values for the control subjects (Figure 2A). e slight dif-
ference in plasma glucose level, both in control and test
groups, was most likely a result of dietary reasons. us,
our results indicate that a single dose of pioglitazone has
no signicant eect on plasma glucose concentration.
A previous report indicated that pioglitazone does not
cause hypoglycemia in nondiabetic animals (Stevenson
et al., 1991).
Because pioglitazone interacts with plasma proteins,
the drug may aect plasma protein levels (Li, 2006).
However, we observed no signicant change in total
plasma protein in the 24-hour observation period after
the administration of the drug in test and control subjects
(Figure 2B). Maximum (68.02 ± 3.33 mg/mL) and
minimum (67.39 ± 3.69 mg/mL) plasma protein levels
were observed at 3 and 5 hours post–drug administration,
respectively (Figure 1B). Drugs used in treating T2DM
may aect lipid metabolism (Reed et al., 1999). However,
we observed no signicant dierences between baseline
values and values 24 hours after administration for
total cholesterol, Tg, and HDL and LDL levels (Table 2).
Many drugs are hepatotoxic, and the toxicity is reected
by the activation of certain liver enzymes. e present
study indicated no signicant changes in the level of
two important indicator enzymes (i.e., ALT and AST)
within the 24-hour study period after administration of
pioglitazone. Although our study detected no eect of
pioglitazone in glucose and lipid metabolism and no
hepatotoxic eect among healthy Bengali males within
24 hours of administration of 30 mg of pioglitazone, it
remains to be observed whether chronic administration
of the drug may aect the above parameters of healthy
subjects and the subjects with T2DM.
Acknowledgments
e authors are grateful to Professor Ruhul H. Kudddus
of Utah Valley University (Orem, Utah, USA), for critically
reviewing the article, and the volunteers who partici-
pated in this study.
Declaration of interest
is work was supported solely through internal fund-
ing of the Departments of Clinical Pharmacy and
Pharmacology, Genetic Engineering and Biotechnology
and Pharmaceutical Technology, University of Dhaka,
(Dhaka, Bangladesh).
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