0090-9556/00/2807-0772–780$03.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
DMD 28:772–780, 2000 /1754/835200
Vol. 28, No. 7
Printed in U.S.A.
ABSORPTION, DISPOSITION, AND METABOLISM OF ROSIGLITAZONE, A POTENT
THIAZOLIDINEDIONE INSULIN SENSITIZER, IN HUMANS
PETER J. COX, DAVID A. RYAN, FRANK J. HOLLIS, ANN-MARIE HARRIS, ANN K. MILLER,
MARIKA VOUSDEN, AND HUGH COWLEY
Drug Metabolism and Pharmacokinetics Department (P.J.C., D.A.R., F.J.H., A.-M.H., A.K.M.) and Clinical Pharmacology Unit (M.V., H.C.),
SmithKline Beecham Pharmaceuticals, Welwyn, United Kingdom
(Received July 27, 1999; accepted April 10, 2000)
This paper is available online at http://www.dmd.org
ABSTRACT:
of radioactivity and the elimination half-life for two metabolites in
plasma were significantly longer than for rosiglitazone itself (4–6 h
versus 0.5–1 h, and ca. 5 days versus 3–7 h). Radioactivity was
excreted primarily via the urine (⬃65%) and was excreted similarly
after oral and i.v. dosing. The major routes of metabolism were
N-demethylation and hydroxylation with subsequent conjugation,
of which neither was affected by the route of drug administration.
The major metabolites, those of intermediate importance, and
nearly all of the trace metabolites in humans have been identified
previously in preclinical studies. Rosiglitazone was well tolerated
in all formulations.
Rosiglitazone [AVANDIA, BRL-49653C, (⫾)-5-[[4-[2-methyl-2(pyridinylamino)ethoxy]phenyl]methyl]methyl]-2,4-thiazolidinedione (Z)-2-butenedioate (1:1)] is a potent antihyperglycemic agent
(Patel et al., 1998; Young et al., 1998) that reduces insulin resistance
in patients with type 2 diabetes (Matthews et al., 1999). Rosiglitazone
is a member of the thiazolidinedione class, a group of oral antidiabetic
agents that exert their glucose-lowering effect by binding to peroxisome proliferator-activated receptors gamma (PPAR␥) (Lehmann et
al., 1995; Berger et al., 1996; Willson et al., 1996; Young et al., 1998),
which preferentially bind to DNA as heterodimers and activate transcription of a wide variety of metabolic regulators (Forman et al.,
1996). These regulators are associated with the differentiation of stem
cells into adipocytes (Lehmann et al., 1995; Gimble et al., 1996; Tai
et al., 1996; Adams et al., 1997) and increased expression of a number
of genes involved in the regulation of glucose and lipid metabolism
(Tontonoz et al., 1995; Pearson et al., 1996; Schoonjans et al., 1996;
Lefebvre et al., 1997; Martin et al., 1997; Motojima et al., 1998). Like
all drugs of this class, which includes pioglitazone, englitazone, and
troglitazone (Perry and Petrie, 1998), rosiglitazone contains a thiazolidinedione core, but differs from other thiazolidinediones in the
presence of an aminopyridyl side chain (Henry, 1997; Young et al.,
1998). Such substitutions among side chains are believed to be responsible for differences in disposition, metabolism, and antidiabetic
efficacy among thiazolidinediones (Lehmann et al., 1995; Berger et
al., 1996).
The objectives of this study were 3-fold: 1) to determine the
disposition and routes of elimination of rosiglitazone in humans after
oral and i.v. dosing of solution; 2) to quantify and characterize the
major compound-related components of rosiglitazone in human
plasma and excreta; and 3) to obtain estimates of the absolute bioavailability of rosiglitazone from both the radiolabeled solution dose
and the nonradiolabeled tablet dose.
1
Abbreviations used are: LC-MS, liquid chromatography-mass spectrometry;
LSC, liquid scintillation counting; SPE, solid-phase extraction; Cmax, maximum
observed concentration; Tmax, the time to reach Cmax; AUC0-inf, area under the
plasma concentration-time curve from time zero to infinity.
2
Nomenclature of metabolites. During preclinical metabolism studies (Bolton
et al., 1996), metabolite structure names were not based on the IUPAC numbering
system. Thus, metabolites previously designated as 3-hydroxy and 5-hydroxy
should more correctly, under the IUPAC system, be designated 5-hydroxy and
3-hydroxy, respectively, a source of potential confusion. In this study, metabolites
containing the IUPAC substructure 2-aminopyridinyl-5-hydroxy are referred to as
para-hydroxy metabolites, reflecting hydroxylation at a position para to the amino
side chain. Similarly, metabolites containing the pyridinyl-3-hydroxy substructure
are referred to as ortho-hydroxy, reflecting hydroxylation at a position ortho to the
amino side chain.
Materials and Methods
Rosiglitazone (BRL-49653C). All dose amounts and concentrations are
expressed in terms of pure base. Ampoules of nonradiolabeled rosiglitazone
injection solution (0.098 mg/ml) and tablets (4 mg) were supplied by Pharmaceutical Technologies, SmithKline Beecham (Upper Merion, PA) (See
Scheme 1.).
[14C]Rosiglitazone injection solution (chemical purity 99.4%, radiochemiSend reprint requests to: Dr. Peter J. Cox, SmithKline Beecham Pharmaceutical purity 99.5%, 0.100 mg/ml, 2.56 ␮Ci/ml) was formulated for human use by
cals, The Frythe, Welwyn, Herts. AL6 9AR, UK. E-mail: [email protected]
Pharmaceutical Technologies, SmithKline Beecham, as follows. Appropriate
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Rosiglitazone is a potent peroxisome proliferator-activated receptor gamma agonist that decreases hyperglycemia by reducing
insulin resistance in patients with type 2 diabetes mellitus. The
disposition of 14C-labeled rosiglitazone was determined after oral
and i.v. dosing of rosiglitazone solution, and the disposition of
nonradiolabeled rosiglitazone was determined after oral dosing of
tablets in this open-label, three-part, semirandomized, crossover
study. The absorption of rosiglitazone was rapid and essentially
complete, with absolute bioavailability estimated to be ⬃99% after
oral tablet dosing and ⬃95% after oral solution dosing, and clearance was primarily metabolic. The time to maximal concentration
HUMAN METABOLISM OF ROSIGLITAZONE, A NEW INSULIN SENSITIZER
SCHEME 1.
UK). All samples were assayed for radioactivity by liquid scintillation counting (LSC) using Tri-Carb liquid scintillation spectrometers (2200CA, 2500TR,
and 2700TR). Quench correction was achieved using an automatic external
standard ratio method. Unless stated otherwise, 10 ml of Ultima Gold scintillant was added to each sample before LSC for 10 min, with automatic
background subtraction. When possible, radio-HPLC data were captured online by homogeneous detection. Alternatively, where levels of radioactivity
were too low for on-line radiodetection, HPLC-radio data were captured by
off-line fraction collection into minivials followed by direct LSC of the
individual fractions. Fractions collected during off-line radio-HPLC analysis
were counted for 2 min (without background subtraction) after the addition of
4 ml of Ultima Gold MV scintillant. UV data for these analyses were captured
by Laura v1.2 (Lablogic, Sheffield, UK).
Radiochemical Purity Determinations. Aliquots (100 or 200 ␮l) of each
dose were analyzed by reverse-phase HPLC with on-line radiodetection (Canberra Packard A-525AX radiodetector, with a 500-␮l homogeneous flow cell).
They were applied to a Kromasil C18 column (5 ␮m, 150 ⫻ 4.6 mm), which
was operated at 40°C. The UV absorbance was monitored at 247 nm. Solvent
A of the mobile phase was 50 mM sodium acetate (pH 5.0), and solvent B of
the mobile phase was acetonitrile. A gradient was used, starting at 100%
solvent A at 0 min, changing to 50% solvent B over 15 min, and held isocratic
for an additional 25 min. The flow rates were 1.0 ml/min for the mobile phase
and 3.0 ml/min for the scintillant. The HPLC column recoveries were calculated from the total dpm recovered in the eluate during the entire gradient run
as a percentage of the total dpm injected.
Radioassay of Samples. Two or three weighed samples of each dose
solution (approximately 0.2 g) and dose residue were diluted by weight and
radioassayed in triplicate. Plasma and urine samples were radioassayed using
three and four aliquots, respectively. Each fecal homogenate was assayed using
six aliquots. During sample extractions, recovery of radioactivity was determined at each procedural step using two or three aliquots.
Plasma Analysis of Rosiglitazone. Plasma concentrations of rosiglitazone
were quantified using a validated automated Automated Sequential Trace
Enrichment of Dialysates/HPLC assay using fluorescence detection. The lower
limit of quantification for rosiglitazone was 3.00 ng/ml using a 200-␮l sample
volume. Quality control samples were assayed with each batch of samples
against calibration standards to assess the reproducibility of the assay.
Sample Preparation for HPLC Radiometabolite Profiling. Urine. For
each subject, a pooled sample representative of the urinary excretion over the
first 8 days was prepared by mixing an equal percentage (by weight) of the
sample collection of each day. Urine samples (12–24 h and day 8) were also
analyzed. On-line radiodetection was used to produce radiochromatograms
from unextracted urine samples (0 – 8 day pooled and 12–24 h) and from
solid-phase extracts (day 8 urine and urine hydrolysates). All urine samples
and urine extracts were centrifuged (ca. 11,000gav, 5 min) before HPLC
analysis.
Enzymic hydrolysis of urine samples. Selected urine samples were subjected
to enzymic hydrolysis to aid metabolite structural identification. Three enzyme
preparations were used in these analyses: 1) an enzyme solution containing
␤-glucuronidase (EC 3.2.1.21, Sigma type B-1, bovine liver, 1000 U/ml) in 0.2
M sodium acetate buffer pH 5.0; 2) a solution containing ␤-glucuronidase and
the ␤-glucuronidase inhibitor, D-saccharic acid-1,4-lactone (Sigma, 7.5 mg/
ml); and 3) a solution containing sulfatase (EC 3.1.61, Sigma Type H-2, Helix
pomatia, 115 U/ml). Incubations were prepared by diluting the enzyme and
enzyme plus inhibitor 1:1 with buffer/sample mixtures. Identical control incubations were prepared in each case, replacing enzyme with buffer. Samples
were incubated overnight at 37°C in a shaking water bath.
Solid-phase extraction (SPE) of urine samples. Solid-phase extracts were
prepared for radiochromatography of day 8 urine and for those urine samples
to be analyzed by liquid chromatography-mass spectrometry (LC-MS). SPE
cartridges (Varian Mega Bond Elut C18, 60 cc) were preconditioned with
methanol and distilled water. Samples of urine (approximately 10 –70 ml) were
run through the column. The column was washed with distilled water (approximately 25 ml) and then eluted with methanol (approximately 25 ml). The
eluent was dried under N2 at room temperature before being resuspended in
deionized water (0.5–7 ml) and the resulting solution was centrifuged before
HPLC analysis. On average, recovery of radioactivity after SPE was 98%
(range, 87–113%) and at subsequent reconstitution was 87% (range, 60 –
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amounts of sodium chloride and citric acid were dissolved sequentially in
Water for Injection (Fresenius, Runcorn, UK). [14C]Rosiglitazone [specific
activity 19.2 ␮Ci/mg (712 kBq/mg), synthesized and supplied by the Synthetic
Isotope Chemistry Unit, SmithKline Beecham, Harlow, UK] was then dissolved in this solution. The weight of the final solution was made up as
required with Water for Injection and filtered (0.2-␮m polyvinylidene difluoride, Minisart NML; Sartorius, Epsom, UK). The filtrate was finally refiltered
into washed and sterilized 5-ml ampoules, which were then sealed and stored
protected from light.
For i.v. dosing, this solution was used directly. For oral administration,
individual radiolabeled doses were prepared on the day of dosing by mixing
[14C]rosiglitazone injection solution (28.7 ml) and nonradiolabeled rosiglitazone injection solution (53.3 ml) in prewashed and sterilized bottles (100 ml).
The solutions were protected from light until administration. Immediately
before dosing, each bottle of dose solution was weighed. Approximately 2 g of
each solution was removed for the assessment of radiochemical purity and
concentration, and each bottle was reweighed.
Study Subjects. Four healthy male volunteers, aged between 40 and 65,
with normal medical history, vital signs (blood pressure and heart rate),
12-lead ECG, clinical chemistry, hematology, and urinalysis participated in the
study. Exclusion criteria included: 1) receipt of a total body radiation dose
greater than 5.0 mSv or participation in a study with radiolabeled medication
in the 12 months before the study start; 2) use of any prescribed medication
within 7 days of the study start; 3) a family history of diabetes in first degree
relatives; and 4) a history of any condition known to interfere with the
absorption, distribution, metabolism, and excretion of drugs. The study was
conducted in accordance with Good Clinical Practice guidelines and the
Declaration of Helsinki. The protocol was approved by the Independent Ethics
Committee in Harlow, UK, before the start of the study, and all subjects
provided their written informed consent.
Treatment Protocol. On the first study day, subjects received [14C]rosiglitazone either as an i.v. infusion (2 mg, 20 ml, 50 ␮Ci) over 60 min or as an oral
solution (8 mg, 80 ml, 70 ␮Ci, followed by two 50-ml water washings of the
solution container). Subjects crossed over to the other regimen on the second
study day, after a 50-day washout period. On the third study day, 42 days later,
subjects received nonradiolabeled rosiglitazone tablets (2 ⫻ 4 mg), administered with 180 ml of water. Subjects fasted for at least 7.5 h before administration of all study drugs and for an additional 4 h after dosing.
Subjects receiving oral formulations remained ambulatory throughout the
procedure; those receiving i.v. rosiglitazone solution were dosed in bed and
remained there for 3 h after dosing. Adverse events, vital signs, ECG, clinical
chemistry, hematology, urinalysis, and physical examinations were recorded
throughout the study. Safety assessments were also made at a follow-up visit
7 to 14 days after the last study dose. Spontaneously reported adverse experiences were also recorded. Subjects remained in the Clinical Pharmacology
Unit for 24 h after dosing.
Collection of Biological Specimens. Blood, urine, and feces were collected
at appropriate times after each dose from all subjects. Blood samples were
collected into potassium EDTA tubes, mixed gently, and centrifuged (ca.
1500gav) at 4°C without delay. During and after i.v. dosing, blood samples
were obtained from the contralateral arm. Blood samples were collected
predose (baseline sample), at a dozen time points within the first 24 h, and 2,
3, 4, 7, 14, 21, and 42 days after dosing. The sampling schedule had to be
extended from that initially protocoled, due to the slower than anticipated
clearance of radioactivity from the plasma. Based on the results from the first
period, the sampling schedule was modified so that samples were collected
predose and at 14 or 15 time points up to 21 days postdose. In the third study
period, blood samples were collected at 10 time points up to 48 h after dosing.
Radiodetection and Quantification. All materials and instruments used in
the radioassay of samples were obtained from Canberra-Packard (Pangbourne,
773
774
COX ET AL.
Data shown are for a representative subject (1).
100%). Column recoveries for radioactivity were on average 103% (range,
100 –106%). Therefore, no adjustments for this have been made in the calculations.
Samples of 0 to 8 day pooled and day 8 urine intended for enzymic
hydrolysis were prepared as described above. These samples were re-extracted
after incubation by SPE before LC-MS analysis. SPE cartridges (Varian C18,
100 mg, 1 cc) were preconditioned with methanol and distilled water. Urine
enzymic hydrolysates (approximately 1–2 ml) were then run through the
column. The column was washed with distilled water and then eluted with
methanol. The eluent was dried under N2 at room temperature before reconstitution in deionized water (0.5–1.5 ml). On average, recovery of radioactivity
at SPE elution was 106% (range, 99 –120%) and at subsequent reconstitution
was 78% (range, 72– 82%).
Feces. For each subject, fecal subsamples from days 0 to 7 (0 – 6 day,
subject 4, i.v.; and 0 –9 day, subject 3, i.v.) were pooled by total homogenate
wet weight ratio and were analyzed by HPLC. To each fecal subsample,
one-quarter volume of 0.1 M citric acid was added to improve recovery of
radioactivity and the suspension was mixed by vortexing. An equal volume of
acetonitrile to fecal homogenate was added, the suspension was mixed and
centrifuged (ca. 1300gav, 5 min), the supernatant was removed and retained,
and the pellet was extracted twice more. The three supernatants for each
sample were combined and recovery of radioactivity was determined. The total
volume of the extract was reduced under N2 at room temperature and the
samples were frozen with ethanol/dry ice and freeze-dried. The dry extracts
TABLE 1
Mean (S.D., n ⫽ 4) pharmacokinetic parameter values for rosiglitazone and total radioactivity after single oral or i.v. doses of rosiglitazone
Rosiglitazone
Total Radioactivity
Dose
Tmaxa
Cmax
AUC0-inf
t1/2
CL
VSS
l/h
l
F
Tmaxa
Cmax
h
ng/ml
ng 䡠 h/ml
h
%
h
ng eq./ml
0.5
564
2,900
4.65
95
5.0
885
(0.5–1.0)
(19)
(548)
(1.51)
(88–101)
(4.0–6.0)
(173)
Oral dose of rosiglitazone (tablet), 8-mg
0.75
(0.75–1.0)
603
(332)
2,930
(473)
4.06
(1.23)
99
(86–106)
Dose of [14C]rosiglitazone, 2-mg i.v.
1.0
(0.75–1.0)
146
(40)
744
(142)
4.16
(1.64)
6.0
(6.0–6.7)
226
(43)
Oral dose of [14C]rosiglitazone (solution),
8-mg
a
Values expressed as median (range).
2.78
(0.63)
15.1
(4.1)
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FIG. 1. Plasma concentrations of rosiglitazone (nanograms per milliliter) and of
radioactivity (nanograms rosiglitazone equivalents per milliliter) after i.v. (2 mg,
open symbols) and oral (8 mg, filled symbols) administration of
[14C]rosiglitazone.
were suspended in approximately 15 ml of methanol. Particulates in the
suspension were removed by centrifugation, and radioactivity in the supernatant was determined before reducing the preparation to near dryness under N2
at room temperature. The resulting residue was resuspended in 1 to 2 ml of
methanol, and the recovery of radioactivity was determined. Before analysis by
HPLC, particulates in each extract were removed by centrifugation (ca.
11,000gav, 5 min) and the recovery of radioactivity was again determined.
Radioactivity was extracted for LC radioprofiling with an average efficiency of 84% (range, 77– 89%). Recovery of radioactivity after reconstitution
for HPLC was on average 86% after the five extraction steps (range, 75–
100%). Column recoveries for radioactivity were, on average, 91.5% (range,
90 –93%); no corrections to the data have been made to account for this.
Fecal extracts were prepared for LC-MS by omitting the addition of 0.1 M
citric acid in the first step and mixing the final methanol solutions with equal
volumes of distilled water before centrifugation. In the absence of citric acid,
the overall recovery of radioactivity was lower (44 – 61%), but unchanged in
composition.
Plasma. HPLC analysis was performed on pooled plasma samples from day
1 and on individual plasma samples from day 2. An equal volume of 0.1 M
citric acid was added to each plasma sample (2 or 4 ml), and the suspension
was mixed by vortexing. An equal volume of acetonitrile was then added and
the suspension was thoroughly mixed before being centrifuged (ca. 11,000gav).
The supernatant was removed and retained, and the pellet was extracted twice
more. The three supernatants for each sample were combined and recovery of
radioactivity was determined (99%, range 80 –133%). The extract was reduced
to dryness under N2 at room temperature, and the resulting residue was
resuspended in distilled water. Before HPLC analysis, particulates were removed by centrifugation and recovery of radioactivity was again determined
(86%, range 49 –112%). Plasma extracts obtained on study day 1 were combined according to dose route and time point for radioprofiling and LC-MS.
Plasma extracts obtained on study day 2 were analyzed separately for each
subject and time point. An average radioactive concentration value was used
for samples from the first dose administration that had been combined. Column
recoveries for radioactivity were 92.5% (range, 90 –95%); no corrections to the
data have been made to account for this.
Quantitative HPLC Radiometabolite Profiling. HPLC analysis of radiometabolites in plasma, urine, hydrolyzed urine, and feces was performed as
follows. Authentic rosiglitazone and synthetic metabolites (SB-243914, SB244675, and SB-237216) were obtained from Chemical Development, SmithKline Beecham (Harlow, UK) and were used as chromatographic and MS
standards. A Canberra Packard A-525AX radiodetector with a 500-␮l homogeneous flow cell was used. The UV absorbance was monitored at 247 nm. For
analysis of all samples, a Hypersil BDS C18 column (5 ␮m, 150 ⫻ 4.6 mm)
was used. The column was operated at 40°C. Solvent A of the mobile phase
was 50 mM ammonium acetate (pH 8.0). Solvent B of the mobile phase was
acetonitrile/methanol (1:1). A gradient was used, starting at 100% solvent A
held for 5 min, changing to 50% solvent B at a rate of 1% per min for 50 min,
finally increasing to 95% solvent B over 5 min and held for an additional 10
min. The flow rates were 1.0 ml/min for the mobile phase and 3.0 ml/min for
the scintillant. All samples were coinjected with rosiglitazone, SB 243914, SB
244675, and SB 237216 to confirm retention times.
HUMAN METABOLISM OF ROSIGLITAZONE, A NEW INSULIN SENSITIZER
775
TABLE 2
Excretion of radioactivity (percentage of dose) over 21 days after a single
administration of [14C]rosiglitazone
Sample
Subject 1
Subject 2
Subject 3
Subject 4
Mean
(S.D.)
Excluding
Subject 2
Oral dose (8-mg)
Urine
67.6
53.9
58.0
69.6
Feces
28.1
12.2
22.2
23.9
Total
95.6
66.1
80.2*
93.5
62.3
(7.5)
21.6
(6.7)
83.8
(13.7)
65.0
(6.2)
24.7
(3.0)
89.8
(8.4)
66.5
57.4
67.9
73.4
Feces
28.3
26.5
27.6
18.3
Total
94.8
83.9
95.5
91.6**
66.3
(6.6)
25.2
(4.6)
91.4
(5.3)
69.2
(3.6)
24.7
(5.6)
94.0
(2.1)
i.v. dose (2-mg)
Urine
* Estimated 8% not collected.
** Estimated 3% not collected.
been shown over the relevant dose range and plasma concentrations (D. A.
Boyle, personal communication). Thus the use of dose-normalized AUC
values for the i.v. dose (2 mg) and the oral dose (8 mg) was valid in the
calculation of absolute bioavailability. Descriptive statistics (mean, S.D., median, minimum, and maximum) were determined to summarize the data for
each pharmacokinetic parameter.
Results and Discussion
Tolerability. Four healthy male Caucasian subjects aged 52 ⫾ 6
years, with a mean weight of 80 ⫾ 16 kg and a mean height of 174 ⫾
8 cm, enrolled into and completed the study. Rosiglitazone was
generally well tolerated during administration. A total of six adverse
events were reported during the study, all of which occurred only
once, and none were considered likely to be drug-related. There were
no clinically important changes in ECG, laboratory parameters, blood
pressure, or heart rate. One subject did not complete the study due to
a falling hemoglobin concentration, which was most likely due to the
intensive blood sampling schedule.
Plasma Concentration-Time Profiles. Rosiglitazone was rapidly
cleared from plasma in all subjects, being quantifiable only up to 24 h
after dosing. Plasma concentration-time data obtained after both oral
and i.v. administration displayed an overall monoexponential decline
from peak values (Fig. 1). Pharmacokinetic parameter values are
shown in Table 1. On the other hand, plasma concentrations of
radioactivity changed little between Cmax (ca. 6 h) and 24 h after
dosing, and then displayed an overall biexponential decline (Fig. 1),
being measurable for the entire 21-day sampling period. Thus, systemic exposure to rosiglitazone, after both oral and i.v. administration,
was a small percentage (⬍5%) of the exposure to all drug-related
materials (total radioactivity).
Mass Balance. The overall excretion of radioactivity in urine and
feces is summarized in Table 2, and mean cumulative excretion data
are shown in Fig. 2. Elimination of radioactivity was similar after
either route of administration, and was predominantly into the urine.
There were two known instances of significant noncompliance with
urine and fecal collections. Samples for days 9 to 17 were not
collected for subject 3 who, after oral dosing, unexpectedly traveled
abroad for 1 week. After i.v. administration, subject 4 mistakenly
thought he had completed the study and the day 7 samples were not
collected. Estimation of the losses, by comparison with the excretion
patterns of the other volunteers over the relevant periods, suggested
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LC-MS. The HPLC system, column, and conditions were essentially identical with those described under Quantitative HPLC Radiometabolite Profiling
except that detection was by MS and the column eluent was split in an
approximate ratio of 3:1, to give an approximate flow rate of 250 ␮l/min into
the MS interface. For MS, a VG Quattro mass spectrometer with a PC running
Mass Lynx v2.10 data system was used. The ionization mode for MS was
electrospray, positive and negative ion, and selected ion recording (SIR) was
used. For tandem mass spectrometry, the ionization mode was electrospray,
positive ion, the collision energy was 25 eV, and the mass range was 50 to 300
Da (daughters of 281). All samples analyzed by MS were compared with
control (predose) samples, prepared, and analyzed under identical conditions.
Data Analysis. Excretion. The total radioactivity in each collection of
excreta has been expressed as a percentage of the total administered radioactive dose. The total radioactivity in plasma has been expressed as nanograms
rosiglitazone equivalents per milliliter.
HPLC. On-line radio-HPLC data were processed and calculated using the
software Radiomatic Flo-One, version 3.52 (Packard Instrument Company,
Meriden, CT). The LSC data obtained after off-line radiodetection were
converted into a format compatible with Labchrom version 2.10 (Lablogic,
Sheffield, UK) using its LSC import function to reconstruct radio-HPLC
chromatograms. Radioactive peaks were marked using an on-screen cursor.
The software integrated the area under the radiochromatogram and also expressed each region of interest as a fraction of the total number of counts
detected. The metabolites in urine and feces were quantified in terms of
percentage of the radioactive dose administered, and those in plasma as a
percentage of the total radioactivity in the sample.
Mass spectral characterization of metabolites. Peak assignments were made
by comparison of chromatographic retention times with the corresponding
radiotraces. Numbers were assigned to each individual metabolite identified
together with descriptive names.2 Structural assignments were made from
molecular ion and retention time data by reference to the fuller mass spectral
and NMR data obtained for these metabolites in the preclinical species (Bolton
et al., 1996).
Pharmacokinetic analysis. Plasma concentration-time data for each subject
in each regimen were analyzed by noncompartmental methods using a validated in-house computer software program, #PROTOCOL (version 1.2;
SmithKline Beecham). All calculations were based on actual sampling times.
Pharmacokinetic parameter values determined included maximum observed
concentration (Cmax), the time to reach Cmax (Tmax), elimination phase half-life
(t1/2), and the area under the plasma concentration-time curve from time zero
to infinity (AUC0-inf). AUC0-inf was calculated using the linear trapezoidal rule
for each incremental trapezoid and the log trapezoidal rule for each decremental trapezoid (Chiou, 1978). In addition, plasma clearance and the volume of
distribution at steady state were calculated for rosiglitazone using the plasma
concentration data obtained after i.v. administration of [14C]rosiglitazone. The
observed absolute bioavailability was calculated as dose-normalized AUC0-inf
oral/dose-normalized AUC0-inf i.v. Pharmacokinetic linearity previously had
FIG. 2. Mean recovery (n ⫽ 4) of radioactivity after i.v. (2 mg, open symbols)
and oral (8 mg, filled symbols) administration of [14C]rosiglitazone.
776
COX ET AL.
TABLE 3
Retention time (RT), MS data, and structures for the human metabolites of rosiglitazone
Metabolite
No.
RT
Metabolite
Molecular
Iona
Fragment
Ion
Anticipated
Deconjugation
Product
Molecular Structure
12
site of conjugation not determined
site of conjugation not determined
min
17.4
phenoxyacetic acid derivative
280 (⫺ve)b
15
27.8
N-desmethyl glucuronide
520 (⫹ve)
16
30.8
N-despyridinyl
281 (⫹ve)
2
35.1
N-desmethyl glucuronide
520 (⫹ve)b
12
3
35.9
ortho-O-glucuronide
550 (⫹ve)b
11
4
36.9
N-desmethyl-para-O-sulfate
438 (⫺ve)
360 (⫹ve)
7
5
38.4
para-O-glucuronide
550 (⫹ve)
374 (⫹ve)
13
6
38.9
N-desmethyl-ortho-O-sulfate
438 (⫺ve)
360 (⫹ve)
9
9
41.3
N-desmethyl-ortho-hydroxy (SB 243914)
360 (⫹ve)
137 (⫹ve)
7c
41.7
N-desmethyl-para-hydroxy
360 (⫹ve)
8
41.9
ortho-O-sulfate
452 (⫺ve)
17
43.6
10
44.3
374 (⫹ve)
11
360 (⫹ve)b
para-O-sulfate
452 (⫺ve)
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1
Unknown
374 (⫹ve)
13
HUMAN METABOLISM OF ROSIGLITAZONE, A NEW INSULIN SENSITIZER
777
TABLE 3
(Continued)
Metabolite
No.
RT
Metabolite
Molecular
Iona
Fragment
Ion
Anticipated
Deconjugation
Product
Molecular Structure
min
49.9
ortho-hydroxy (SB 244675)
374 (⫹ve)
151 (⫹ve)
12
51.7
N-desmethyl (SB 237216)
344 (⫹ve)
121 (⫹ve)
13
53.7
para-hydroxy
374 (⫹ve)
14
59.3
rosiglitazone
358 (⫹ve)
135 (⫹ve)
Molecular Ion ⫽ Pseudo-molecular ion: [M⫹H]⫹ in (⫹) ion mode, [M⫺H]⫺ in (⫺) ion mode.
Not observed during LC-MS (selected ion recording) of urine in this study.
c
Retention time of M7 obtained from sulfatase-deconjugated urine.
a
b
FIG. 3. Representative radiochromatogram from the analysis of 12–24 h urine (subject 1) after oral administration (8 mg) of [14C]rosiglitazone.
that overall recovery for subject 4 (i.v.) should have been ca. 3%
higher and for subject 3 (oral) ca. 8% higher, which would have given
an acceptable mass balance. There was, however, a very poor mass
balance for subject 2 after both doses (10% lower than the mean for
the other three subjects after i.v. dosing, 24% lower after oral administration). No errors could be detected in either the dose estimation or
the radioassay results for urine and feces, thus giving no reason for the
poor recoveries other than the possibility of consistent noncompliance
in sample collection.
The most notable feature of the excretion of radioactivity in humans
was the prolonged period over which it occurred, in comparison to the
rate of excretion in the rat and dog. In the preclinical species, approximately 90% of the excreted radioactivity was recovered within 48 h
of dosing (Bolton et al., 1996). In humans, however, only about 35%
of the administered radioactivity was recovered in 48 h and it took
over a week to recover 90% of the excreted radioactivity. This slow
excretion of radioactivity mirrored the slow elimination from the
systemic circulation.
This prolonged elimination of radioactivity could be a consequence of
the extremely high plasma protein binding of M10 (SB-332650), the
principal drug-related component in the circulation after 24 h, and the
dominant component in urine on day 8 (see below). Binding in human
plasma was measured using ultrafiltration and an LC-tandem mass spectrometric assay (detection limit of 0.5 ng/ml) and found to be in excess of
99.99%, compared with 99.72 ⫾ 0.04% for rosiglitazone (A-M. Harris,
S. E. Fowles, and P. B. East, unpublished data). Other mechanisms, e.g.,
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
11
778
COX ET AL.
TABLE 4
Mean percentage of dose associated with each identified metabolite in 0 – 8 day pooled urine and 0 –7 day pooled fecal extracts after administration of
[14C]rosiglitazone to four volunteers
Mean Percentage of Administered [14C]Rosiglitazone Dose
Metabolite
No.
Metabolite
8-mg oral dose
Urine
a
phenoxyacetic acid derivative
N-desmethyl glucuronide
N-despyridinyl
N-desmethyl glucuronide
ortho-O-glucuronide
N-desmethyl-para-O-sulfate
para-O-glucuronide
N-desmethyl-ortho-O-sulfate
N-desmethyl-ortho-hydroxy (SB 243914)
N-desmethyl-para-hydroxy
ortho-O-sulfate
unknown
para-O-sulfate
ortho-hydroxy (SB 244675)
N-desmethyl (SB 237216)
para-hydroxy
rosiglitazone
Total assigned 0–8 day (urine) or 0–7 day (fecal extracts)b
0–8 day urinary or 0–7 day fecal excretion
Percentage of 0–8 day urinary or 0–7 day fecal excretion assigned
Total urinary or fecal excretion 0–21 day
Percentage of 0–21 day urinary or fecal excretion assigned
Urine
Feces
3.7
1.6
1.2
1.8
0.3
17.7
3.8
0.7
1.1
nd
3.5
0.3
15.9
0.1
0.5
0.1
0.1
nd
nd
nd
nd
nd
nd
nd
nd
0.7
6.1
nd
nd
nd
0
nd
9.0
nd
3.6
1.5
1.0
2.1
0.2
18.4
3.4
1.0
1.0
nd
3.3
0.3
17.5
0.2
0.2
0.1
0.0
nd
nd
nd
nd
nd
nd
nd
nd
0.2
6.9
nd
nd
nd
0.2
nd
10.8
nd
52.3
(46.1)
55.3
94.6
(83.4)b
62.3
84.2
(74.0)b
15.8
53.7
(47.5)
56.9
94.5
(83.5)b
66.3
81.2
(71.6)b
18.0
17.7
88.9
21.6
73.1
20.5
87.5
25.2
71.3
a
Assigned by retention time only—no MS data.
Values in parentheses for metabolites unambiguously assigned (MS plus retention time data).
nd, Not detected.
b
TABLE 5
Radiometabolite quantification in plasma from human subjects after administration of [14C]rosiglitazone to four volunteers (mean)
Percentage of Plasma Radioactivity
Metabolite No.
Metabolite
1h
4h
8h
24 h
Day 4
N-desmethyl-para-O-sulfate
ortho-O-sulfate
para-O-sulfate
N-desmethyl (SB 237216)
rosiglitazone
0
0
18
11
71
100
2
2
46
15
34
99
3
2
60
6
19
90
10
1
65
17
2
95
11
2
80
4
0
97
N-desmethyl-para-O-sulfate
ortho-O-sulfate
para-O-sulfate
N-desmethyl (SB 237216)
rosiglitazone
0
0
9
5
86
101
3
1
44
16
34
98
5
2
60
17
15
99
10
1
65
19
3
98
12
0
86
2
0
100
8-mg oral dose [14C]rosiglitazone
4
8
10
12
14
Total identified
2-mg i.v. dose [14C]rosiglitazone
4
8
10
12
14
Total identified
enterohepatic recycling, may play contributory roles. In the rat, biliary
secretion of the para-O-sulfates M4 and M10 was substantial, suggesting
the possibility of enterohepatic recycling, but excretion of radioactivity
was essentially complete within 48 h in both the intact dog (Bolton et al.,
1996) and intact rat (S. M. Wheeler, A. W. Harrell, P. J. Cox, R. J.
Chenery, and J. P. Keogh, unpublished data). This excretion pattern was
consistent with the rapid clearance of radioactivity from the plasma of the
preclinical species. Thus it cannot be assumed that biliary recycling is
responsible for the prolonged elimination of rosiglitazone metabolites in
humans.
Characterization of Human Urinary, Fecal, and Plasma Radio-
metabolites. Molecular ion and retention time data for metabolites
and authentic reference compounds (rosiglitazone, SB-243914, SB244675, and SB-237216) are given in Table 3.
Urine. Analysis of 12–24 h, 0 – 8 day pooled, and day 8 urine after
either the oral or i.v. dose showed that at least 15 radiometabolites
were detected by radio-HPLC analysis (Fig. 3). The 0 – 8 day pooled
urine radiochromatograms showed very little variation between dose
route or subject. Table 4 shows that metabolites M10 and M4, both
para-hydroxylated sulfate conjugates, together accounted for approximately 35% of the dose excreted over 8 days. All other metabolites
individually accounted for ⬍4% of the dose (Table 4).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
1
15
16
2a
3a
4
5
6
9
7
8
17a
10
11
12
13
14a
2-mg i.v. dose
Feces
HUMAN METABOLISM OF ROSIGLITAZONE, A NEW INSULIN SENSITIZER
779
Plasma. Analysis of 1, 4, 8, and 24 h as well as day 4 plasma
extracts, after either the oral or i.v. dose, showed that at least four
metabolites and parent were present. Rosiglitazone was the predominant plasma component at 1 h postdose. Peaks for M10 and M12
could also be observed at this time point (Table 5). Plasma from i.v.
dosed subjects in comparison to orally dosed subjects showed lower
proportions of metabolites compared with rosiglitazone at 1 h after
dosing, as expected. From 4 h onwards, oral and i.v. plasma extract
radiochromatograms were very similar, with M10 as the predominant
component and M4 and M12 also at significant levels. From 24 h
onwards, plasma radioactivity was due almost exclusively to M4 and
M10, and therefore the decline in radioactivity concentrations may be
used to calculate an approximate half-life for these para-hydroxy
sulfate metabolites of about 5 days. The persistence in the systemic
circulation of M10, M4, and, for 24 h, of the N-desmethyl metabolite
M12, suggests that these slowly cleared metabolites are likely to
accumulate on repeated daily dosing of rosiglitazone to humans.
The potential contribution of some of these metabolites to the
pharmacodynamic activity of rosiglitazone has been studied by comparing their abilities to activate proliferated-activated receptors
gamma (PPAR␥) in a cell-based gene transcription assay (Lehmann et
al., 1995). The phase I metabolites, N-desmethyl-rosiglitazone (M12,
SB-237216) and unconjugated para-hydroxyrosiglitazone (M13, SB275286, not detected in plasma) were 20-fold less potent than and
equipotent with rosiglitazone, respectively. However, the conjugated
and highly protein-bound plasma metabolite, rosiglitazone-para-Osulfate (M10, SB-332650), was 55-fold less potent than rosiglitazone
in this assay (S.A. Smith, personal communication). Thus, although
theoretically possible, it seems unlikely that the slowly cleared plasma
metabolites contribute significantly to the pharmacodynamic activity
of rosiglitazone.
Feces. Analysis of extracts of feces collected during days 1 or 2 and
6 or 7, together with pooled day 0 to 7 feces extracts, after either the
oral or i.v. dose, gave similar profiles and showed that at least four
radiometabolites were present. Metabolites M7 and M13, both parahydroxylated metabolites, were predominant (Table 4).
Total Percentage of Dose to Which Structures Were Assigned.
Approximately 62 and 65% of the administered dose were unambiguously assigned structures after oral administration of 8 mg of
[14C]rosiglitazone and i.v. administration of 2 mg of [14C]rosiglitazone, respectively. Tentative assignments were made for another 6%
by both dosing routes. Urine and feces samples collected from about
day 8 on contained insufficient radioactivity to allow radiochromatographic analysis. These samples accounted for 11 and 14% of the dose
(oral and i.v., respectively). On the assumption that the metabolic
profiles in urine and feces collected from about day 8 onward were
similar to the profiles obtained for day 7 or 8 postdose, the overall
percentage of the dose to which structures could be assigned is 79 and
86% for oral and i.v. doses, respectively. The remaining radioactivity
was present as components that were too small to be distinguished
above background radioactivity.
Metabolic Scheme. A proposed scheme for the metabolism of
rosiglitazone in humans, based on the results of this study, is presented in Fig. 4 and is closely similar to that proposed for rat and dog
(Bolton et al., 1996). The major routes of metabolism in humans were
N-demethylation, hydroxylation, and subsequent conjugation, and
these were unaffected by the route of dose administration. Approximately 44% of the metabolites were N-demethylated, and about 76%
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 14, 2017
FIG. 4. Proposed metabolic scheme for rosiglitazone (M14) in humans.
780
COX ET AL.
had been hydroxylated on the pyridinyl ring, with or without Ndemethylation. The ratio of ortho- to para-hydroxylation was approximately 1 to 9. The hydroxylated products were extensively conjugated, accounting for about two-thirds of the total metabolites. The
ratio of sulfation to glucuronidation was approximately 6 to 1. It
should be noted that only unconjugated metabolites were detected in
the feces. It is likely that these were first secreted into the bile as
conjugates and then hydrolyzed by intestinal microflora. Thus it can
be estimated that 90% of the phase I metabolites of rosiglitazone are
initially conjugated. Unlike the preclinical species, cleavage of the
molecule to give a phenoxyacetic acid metabolite (M1) was a minor
route of elimination in humans, accounting for less than 4% of the
dose.
Although there were differences between species in the persistence
of the circulating metabolites of rosiglitazone (measured as total
radioactivity), its principal metabolites were accurately predicted from
preclinical studies.
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