Plasma stability-dependent circulation of acyl glucuronide metabolites in humans: How circulating metabolite profiles of muraglitazar and peliglitazar can lead to misleading risk assessment

Article abstract of DOI:10.1124/dmd.110.035048

Muraglitazar and peliglitazar, two structural analogs differing by a methyl group, are dual peroxisome proliferator-activated receptor- α/γ activators. Both compounds were extensively metabolized in humans through acyl glucuronidation to form 1-O-β-acyl g

Full text of DOI:10.1124/dmd.110.035048

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2010/09/28/dmd.110.035048.DC1.html
0090-9556/11/3901-123–131$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:123–131, 2011
Vol. 39, No. 1
35048/3648322
Printed in U.S.A.
Plasma Stability-Dependent Circulation of Acyl Glucuronide
Metabolites in Humans: How Circulating Metabolite Profiles
of Muraglitazar and Peliglitazar Can Lead to Misleading
□
S
Risk Assessment
Donglu Zhang, Nirmala Raghavan, Lifei Wang, Yongjun Xue, Mary Obermeier, Stephanie Chen,
Shiwei Tao, Hao Zhang, Peter T. Cheng, Wenying Li, Ragu Ramanathan, Zheng Yang,
and W. Griffith Humphreys
Pharmaceutical Candidate Optimization (D.Z., N.R., L.W., Y.X., M.O., W.L., R.R., Z.Y., W.G.H.) and Discovery Chemistry
(S.C., S.T., H.Z., P.T.C.), Bristol-Myers Squibb Research and Development, Princeton, New Jersey
Received June 19, 2010; accepted September 28, 2010
ABSTRACT:
Muraglitazar and peliglitazar, two structural analogs differing by a mal incubations. Peliglitazar AG had a greater stability than mura-
methyl group, are dual peroxisome proliferator-activated receptor- glitazar AG in incubations in buffer, rat, or human plasma (pH 7.4).
␣/␥ activators. Both compounds were extensively metabolized in Incubations of muraglitazar AG or peliglitazar AG in plasma pro-
humans through acyl glucuronidation to form 1-O-␤-acyl glucuro- duced more aglycon than acyl migration products compared with
nide (AG) metabolites as the major drug-related components in incubations in the buffer. These data suggested that the difference
bile, representing at least 15 to 16% of the dose after oral admin- in plasma stability, not differences in intrinsic formation, direct
istration. Peliglitazar AG was the major circulating metabolite, excretion, or further oxidation of muraglitazar AG or peliglitazar
whereas muraglitazar AG was a very minor circulating metabolite AG, contributed to the observed difference in the circulation of
in humans. Peliglitazar AG circulated at lower concentrations in these AG metabolites in humans. The study demonstrated the
animal species than in humans. Both compounds had a similar difficulty in doing risk assessment based on metabolite exposure
glucuronidation rate in UDP-glucuronic acid-fortified human liver in plasma because the more reactive muraglitazar AG would not
microsomal incubations and a similar metabolism rate in human have triggered a threshold of concern based on the recent U.S.
hepatocytes. Muraglitazar AG and peliglitazar AG were chemically Food and Drug Administration guidance on Metabolites in Safety
synthesized and found to be similarly oxidized through hydroxyla-
Testing, whereas the more stable peliglitazar AG would have.
tion and O-demethylation in NADPH-fortified human liver microso-
Introduction
and Dickinson, 2003; Stachulski et al., 2006; Corcoran et al., 2001):
1) direct displacement of the acyl residue with a nucleophile to
produce an aglycon (hydrolysis product) and an acylated nucleophile;
and 2) alternatively, migration of the acyl group around the sugar ring
to yield 2-, 3-, and 4-acyl isomers. These isomers may undergo
transient ring opening with concurrent formation of a reactive alde-
hyde group. Either of these pathways could lead to covalent binding
to cellular proteins (Sallustio and Foster, 1995; Akira et al., 2002;
Bailey and Dickinson, 2003). These pathways are effectively cata-
lyzed at physiological pH (pH 7.4) and occur more rapidly under basic
pH conditions (Xue et al., 2008). Acyl glucuronides are relatively
stable under acidic conditions (pH 4–5). Careful acidification and
cooling are required to stabilize acyl glucuronides in biofluids such as
plasma, urine, and bile (Wang et al., 2006).
Acyl glucuronidation is one of the major metabolic clearance
pathways for many carboxylic acid-containing drugs. 1-O-␤-Acyl
glucuronides, which are formed by UDP-glucuronosyltransferase
(UGT) enzymes as the original isomer, can undergo a number of
reactions including hydrolysis, rearrangement via acyl migration (Sta-
chulski et al., 2006; Zhang et al., 2006; Xue et al., 2008), further
metabolism (Kumar et al., 2002; Kochansky et al., 2005; Ogilvie et
al., 2006), and reaction with nucleophiles (Sallustio and Foster, 1995;
Akira et al., 2002; Bailey and Dickinson, 2003). The chemical reac-
tivity of acyl glucuronides proceeds via two distinct pathways (Bailey
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
Muraglitazar and peliglitazar, oxybenzylglycine analogs (nonthia-
zolidinedione), are novel dual peroxisome proliferator-activated re-
ceptor-␣/␥ activators. The two compounds are structurally very sim-
doi:10.1124/dmd.110.035048.
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; MeOH, methanol; AG, 1-O-␤-acyl-glucuronide; THF, tetrahydrofuran; HPLC, high-per-
formance liquid chromatography; MS, mass spectrometry; LC, liquid chromatography; DMSO, dimethyl sulfoxide; UDPGA, UDP-glucuronic acid;
TFA, trifluoroacetic acid; AUC, area under the time-concentration curve.
123

124
ZHANG ET AL.
tific (Gibbstown, NJ). Pooled human liver microsomes (n ϭ 22) were pur-
chased from BD Biosciences (San Jose, CA). The human hepatocytes were
acquired as freshly prepared cell suspensions from Lonza (Walkersville, MD).
All other chemicals used were of reagent grade or better.
Synthesis of Peliglitazar AG and Muraglitazar AG. Muraglitazar AG and
peliglitazar AG were synthesized basically following the procedure described
previously (Kenny et al., 2004; Perrie et al., 2005).
ilar with the difference being the addition of a methyl group (Fig. 1).
After oral administration to humans, both [14C]muraglitazar and
[14C]peliglitazar underwent extensive conjugation, and the major por-
tion of each radioactive dose was excreted in bile as acyl glucuronide
metabolites in humans (Wang et al., 2006, 2010; Zhang et al., 2007b).
The acyl glucuronide metabolites of muraglitazar and peliglitazar
seemed to be stable in ex vivo plasma, urine, and bile under acidic and
low temperature conditions because no significant amounts of acyl
migration isomers were formed in plasma, urine, and bile of animals
and humans that were acidified and stored at low temperature. In
addition, the parent concentrations were not increased over time
(Wang et al., 2006, 2010). The major circulating drug-related com-
ponent (Ͼ90%) after oral administration in humans was muraglitazar;
however, both peliglitazar and peliglitazar AG were major drug-
related components (approximately equal concentrations) in humans
after oral administration of peliglitazar. There was no protein co-
valently bound radioactivity found in the protein pellet after extraction
with organic solvents from plasma samples of humans with oral
administration of either 14C-labeled compound.
The circulation of a metabolite, in general, would mainly depend on
its formation, further metabolism, and direct clearance into urine and
bile. For reactive metabolites such as acyl glucuronides, there could
also be reactions to form various small metabolites or large molecular
adducts. In this study we investigated the mechanism responsible for
the distributional difference between muraglitazar and peliglitazar,
two structurally similar analogs, by studying formation, stability,
excretion, and further metabolism of their glucuronide metabolites.
Allyl glucuronate. To a solution of D-glucuronic acid (5.0 g, 25.7 mmol) in
dimethyl formamide (50 ml) at 20°C was added 1,8-diazabicyclo[5.4.0]undec-
7-ene (4.3 ml, 28.2 mmol). The mixture was stirred for 15 min, after which
allyl bromide (2.8 ml, 30.8 mmol) was added. The reaction was stirred
overnight, after which volatile compounds were removed in vacuo at 60°C.
The crude residue was then purified by flash chromatography twice on a 120-g
silica gel column with a continuous gradient from 0 to 20% methanol (MeOH)
in CH2Cl2. The isolated material was then dissolved in MeOH (5 ml) and
concentrated in vacuo. CH2Cl2 (40 ml) was added, and the solution was cooled
to 0°C for 0.5 h. The precipitated solid was filtered off and washed with
ice-cold CH2Cl2 and then dried in vacuo to give the allyl glucuronate (3.4 g,
57% yield, ␣/␤-anomer ratio at 2:1).
Peliglitazar ␤-anomer allyl ester. To a Ϫ5°C solution of peliglitazar (5.5 g,
10.4 mmol) and PPh3 (2.72 g, 10.4 mmol) in tetrahydrofuran (THF) (40 ml)
and dimethyl formamide (5 ml) was added diisopropyl azodicarboxylate (2.0
ml, 10.4 mmol). After 10 min, a solution of the allyl glucuronate (1.44 g, 6.15
mmol) in THF (10 ml) and dimethyl formamide (2.5 ml) was added slowly
over 10 min using a syringe pump. After the addition was complete, the
reaction solution had turned dark brown. The reaction was stirred at Ϫ5°C for
2 h, and the reaction was monitored by thin-layer chromatography until
completion. The formation of product(s) was monitored by HPLC-MS analysis
on a Luna C18 50 ϫ 4.6 mm column with a 0 to 100% B linear gradient in 4
min with solvent A (10% MeOH in 10 mM ammonium acetate solution) and
solvent B (90% MeOH in 10 mM ammonium acetate solution at a flow rate of
4 ml/min). Volatile compounds were removed in vacuo, and the crude residue
was purified by flash chromatography twice on a 120-g silica gel column with
a continuous gradient of 0 to 10% ethanol in CH2Cl2. The resulting material
contained the desired ␣- and ␤-anomers as major products. The ␣- and
Materials and Methods
Materials. Muraglitazar, peliglitazar, and their 14C-labeled materials were
synthesized at Bristol-Myers Squibb (Princeton, NJ). Their structures are
shown in Fig. 1. The 14C-labeled materials had a radiochemical purity of
Ͼ99%. Acetonitrile and trifluoroacetic acid were purchased from EM Scien-
500
muraglitazar
Human plasma 1 h
400
300
200
100
O
O
OH
O
N
N
*
O
O
O
muraglitazar
*
Indicates C-14 label
muraglitazar
glucuronide
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
FIG. 1. Metabolite profiles in 1 h human plasma samples after
Time (min)
single oral administration of 14C-labeled muraglitazar or peligli-
tazar. The samples were analyzed by HPLC system II as described
under Materials and Methods.
200
150
100
50
Human plasma 1 h
O
O
OH
O
N
peliglitazar
peliglitazar
glucuronide
N
*
*
O
O
O
Peliglitazar
*
Indicates C-14 label
0
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Time (min)

STABILITY-DEPENDENT CIRCULATION OF AGs IN HUMANS
125
␤-anomers with a 2 to 3:1 ratio had been partially separated by the flash
The following resonance signals corresponded to the minor rotational con-
chromatography procedure described above, and the mixture was further
former: 4.58 ppm (dd, 1 H), 5.38 (J ϭ 8.25 Hz), 7.31 (d, J ϭ 8.25 Hz, 2H),
purified by preparative HPLC. The preparative HPLC conditions used a 7.01 (d, J ϭ 8.80 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) ␦ ppm: 10.95
Synergi Hydro-RP 80A column (250 ϫ 21 mm 4 ␮m; Phenomenex, Torrance, (s, 1C), 26.69 (s, 2C), 49.50 (s, 1C), 51.88 (br s, 1C), 56.47 (s, 1C), 67.29 (s, 1C), 72.98
CA) at room temperature with a flow rate of 25 ml/min and a mobile phase of
0.05% acetic acid (pH 3.3)-acetonitrile (50:50, v/v) of a run time of 48 min at
278 nm. A portion of 35 mg was injected on the column each time. After
careful removal of acetonitrile in vacuo at room temperature, the remaining
aqueous solution was extracted with ethyl acetate (300 ml), and the organic
(s, 1C), 73.52 (br s, 1C), 75.69 (s, 1C), 77.36 (s, 1C), 96.15 (br s, 1C),
115.10–115.47 (m, 2C), 115.65 (br s, 2C), 123.30–124.14 (m, 2C), 126.53 (s,
2C), 128.22 (s, 1C), 130.03 (s, 1C), 130.14 (s, 1C), 130.39 (s, 1C), 131.14 (s,
2C), 133.78 (s, 1C), 145.51 (s, 1C), 146.20 (s, 1C), 155.71 (s, 1C), 157.65 (br
s, 1C), 158.86 (s, 1C), 159.47 (s, 1C), 169.30 (s), 169.62 (s, 1C), 172.61 (br s,
phase was concentrated and dried in vacuo to provide the ␣- and ␤-anomer 1C). The carbonyl signal at 169.30 corresponded to the minor rotational
esters as white powders. The ␤-anomer was recovered in a 10% yield.
Peliglitazar AG. To a 0°C solution of the ␤-anomer allyl ester (370 mg,
conformer.
Liver Microsomal and Hepatocyte Incubations. Incubations (250 ␮l)
0.44 mmol) in THF (2.0 ml) was added (PPh3)4Pd (59.2 mg, 0.051 mmol) contained muraglitazar or peliglitazar (10 ␮M, 2.5 mM stock solution in 50:50,
followed by pyrrolidine (35.2 ␮l, 0.44 mmol). The mixture was stirred at 0°C v/v, acetonitrile-Tris buffer), human liver microsomes (1 mg/ml protein),
for 30 min, after which volatile compounds were immediately removed in
vacuo to give the crude carboxylic acid. LC-MS using the previously described
Luna C18 column method indicated the formation of desired product. The
crude product was purified by previously described preparative HPLC on a
Synergi Hydro-RP column. After removal of acetonitrile, the remaining solu-
UDPGA (2 mM), magnesium chloride (10 mM), and Tris buffer (100 mM, pH
7.4). Incubations were initiated by addition of a substrate at 37°C and
quenched after 15 min by addition of 1 volume of acetonitrile to the incubation
mixture. After centrifugation to remove the precipitated microsomal proteins,
the clear supernatant (100 ␮l) was analyzed by LC-MS. HPLC system I
tion was lyophilized to give the desired acyl glucuronide ␤-anomer as a white consisted of a Waters 600 pump, a 717 autosampler, and a 996 photodiode
powder (98% pure by HPLC, 300 mg, at 75% yield). 1H NMR (500 MHz, array detector. The column used was a C18 YMC ODS-AQ reverse-phase
DMSO-d6) ␦ ppm: 1.50 (d, J ϭ 7.15 Hz, 3H), 1.57 (d, J ϭ 6.60 Hz, 2H), 2.36 column (4.6 ϫ 150 mm, 3 ␮m) maintained at room temperature with a flow
(s, 3H) 2.93 (t, J ϭ 6.60 Hz, 2H), 3.07–3.12 (m, 1H), 3.15 (s, 1H), 3.23 (s, 1H), rate of 0.7 ml/min. The mobile phase A consisted of 5% acetonitrile in water
3.35 (d, J ϭ 9.90 Hz, 1H), 3.38 (d, J ϭ 9.90 Hz, 1H), 3.73 (s, 3H), 3.84 (d, containing 0.1% TFA and the mobile phase B consisted of 95% acetonitrile
J ϭ 18.70 Hz, 1H), 3.90 (d, J ϭ 18.15 Hz, 1H), 4.07 (d, J ϭ 18.50 Hz, 1H), containing 0.1% TFA. A linear gradient was 10 to 100% B in 20 min and then
4.11 (d, J ϭ 18.15 Hz, 1H), 4.22 (t, J ϭ 6.60 Hz, 2H), 5.30 (q, J ϭ 7.70 Hz,
held at 100% B for 10 min. The retention times were 12.1, 7.3, 15.4, and 8.6
1H), 5.35 (q, J ϭ 7.70 Hz, 1H), 5.39 (d, J ϭ 8.25 Hz, 1H), 6.89 (d, J ϭ 8.80 min for muraglitazar, muraglitazar AG, peliglitazar, and peliglitazar AG,
Hz, 2H), 6.94 (d, J ϭ 8.80 Hz, 2H), 6.98 (d, J ϭ 8.80 Hz, 1H), 7.29 (d, J ϭ respectively. The HPLC was interfaced to a LCQ mass spectrometer (Thermo
8.25 Hz, 2H), 7.34 (d, J ϭ 8.25 Hz, 2H), 7.49 (t, J ϭ 7.88 Hz, 2H), 7.49 (s, Fisher Scientific, Waltham, MA) operated in the positive ionization mode to
1H), 7.92 (dd, J ϭ 7.70, 1.10 Hz, 2H). The anomeric proton of the ␤-anomer acquire full-scan LC-MS data with a mass scan range of 200 to 1000 Da. The
was at ␦5.39, with J ϭ 8.25 Hz, which is typical for the ␤ configuration. The percent metabolism was calculated on the basis of the ratio of peak areas of the
following resonance signals corresponded to the minor rotational conformer: metabolite versus metabolite plus the parent (metabolite/metabolite ϩ parent)
␦1.57 (proton 18); ␦3.90 (15); ␦5.30 (17); ␦5.35 (2); ␦6.98 (24, 26); ␦7.34 (23, from the UV chromatogram (at 278 nm).
27). 13C NMR (126 MHz, DMSO-d6) ␦ ppm: 10.44 (s, 1C), 17.42 (s, 1C),
26.16 (s, 1C), 39.61 (s, 1C), 45.31 (s, 1C), 54.11 (s, 1C), 55.90 (s, 1C), 66.74
Hepatocyte incubations were performed with cells in suspension in 24-well
tissue culture plates shaken at 90 rpm on an orbital shaker. The incubations
(s, 1C), 72.47 (s, 1C), 73.03 (s, 1C), 75.08 (s, 1C), 77.00 (s, 1C), 95.58 (s, 1C), were in Krebs-Henseleit buffer in a 5% CO2/95% air atmosphere at 37°C with
114.69 (s, 2C), 115.02 (s, 2C), 123.00 (s, 1C), 123.55 (s, 2C), 126.00 (s, 2C),
127.69 (s, 1C), 128.91 (s, 1C), 129.62 (s, 2C), 130.61 (s, 3C), 133.11 (s, 1C),
133.26 (s, 1C), 145.01 (s, 1C), 145.67 (s, 1C), 154.69 (s, 1C), 157.04 (s, 1C), 158.21
(s, 1C), 158.94 (s, 1C).
muraglitazar or peliglitazar (5 ␮M, 0.5 mM stock in 50:50 acetonitrile-
potassium phosphate, v/v) and 1 ϫ 106 hepatocytes/ml. Incubation time was
1 h. The samples were quenched by addition of an equal volume of acetonitrile.
The quenched samples were treated and analyzed by LC-MS with HPLC
1-O-Acyl-␣-glucuronide of peliglitazar. The isolated acyl glucuronide system I as described for the microsomal incubations.
␣-anomer allyl ester obtained by preparative HPLC was deprotected by a
procedure identical to that described for the synthesis of the ␤-anomer acid as
Muraglitazar AG or peliglitazar AG at 20 ␮M was separately incubated for
15 min with human liver microsomes (2 mg/ml protein) in 1 ml of 50 mM
a white powder (98% purity by HPLC) after lyophilization. 1H NMR (500 sodium phosphate buffer with and without 1 mM NADPH. The samples were
MHz, DMSO-d6) ␦ ppm: 1.47 (d, J ϭ 6.60 Hz, 3H), 1.55 (d, J ϭ 6.60 Hz, 3H), separated by HPLC system II using a Shimadzu LC-10AT system with the
2.37 (s, 3H), 2.94 (t, J ϭ 6.32 Hz, 2H), 3.15 (d, J ϭ 8.80 Hz, 1H), 3.73 (s, 3H),
analytical column used for the microsomal incubation samples. The mobile
4.22 (t, J ϭ 6.32 Hz, 2H), 4.94 (d, J ϭ 3.85 Hz, 1H), 5.06 (d, J ϭ 6.60 Hz, phase consisted of two solvents: solvent A (0.06% TFA in water) and solvent
1H), 5.34 (d, J ϭ 7.15 Hz, 2H), 5.39 (d, J ϭ 6.60 Hz, 2H), 5.97 (d, J ϭ 2.61 B (0.06% TFA in acetonitrile). The gradient consisted of the following steps:
Hz, 1H), 6.02 (d, J ϭ 3.30 Hz, 1H), 6.89 (d, J ϭ 6.86 Hz, 2H), 6.91 (d, J ϭ solvent B started at 5%, then increased linearly to 25% at 5 min, to 40% at 20
6.89 Hz, 2H), 6.95 (d, J ϭ 8.25 Hz, 2H), 7.02 (d, J ϭ 8.80 Hz, 2H), 7.28 (d,
J ϭ 8.25 Hz, 2H), 7.33 (d, J ϭ 8.80 Hz, 2H), 7.49 (s, 1H), 7.50 (t, J ϭ 7.15
Hz, 2H), 7.92 (d, J ϭ 7.70 Hz, 2H). Some of the signals corresponded to the
minor rotational conformer, including the ␦1.55 doublet (for proton 18) and
␦5.97 doublet (for the anomeric proton 2). Because the amount of material was
limited for carbon-proton NMR heterocorrelations, the chemical shift (at 6
min, to 53% at 60 min, to 60% at 63 min, and to 90% at 65 min, held at 90%
for 7 min, and then decreased to 5% at 75 min. The HPLC effluent was 1
ml/min. The quantities of muraglitazar and peliglitazar AGs were estimated by
HPLC separation with UV detection at 278 nm. The metabolites were identi-
fied by LC-MS as described in the supplemental data.
Study Subjects, Dosing, and Sample Collection. All animal housing and
ppm) and coupling (Ͻ4 Hz) of the anomeric proton were used to determine the care conformed to the standards recommended by the Guide for the Care and
molecule to be in an ␣ conformation. Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
Muraglitazar AG. This compound was synthesized with procedures sim- Animal rooms were maintained on a 12-h light/dark cycle. The human study
ilar to those for peliglitazar AG at an overall 3.3% yield and the purity of 98%. was performed in accordance with the principles of the Declaration of Helsinki
1H NMR (500 MHz, DMSO-d6) ␦ ppm: 2.36 (s, 3H), 2.93 (t, J ϭ 6.60 Hz, 2H), and its amendments, and the study protocol was approved by the Institutional
3.07–3.16 (m, 2H), 3.20–3.27 (m, 1H), 3.35 (t, J ϭ 9.62 Hz, 1H), 3.73 (s, 3H), Review Board and Radiation Safety Committee at the investigational site. All
4.09–4.25 (m, 4H), 4.45 (dd, 2H), 4.58 (dd, 1H), 5.38 (d, J ϭ 8.25 Hz, 0H), subjects were in good health and gave written, informed consent to participate
5.42 (d, J ϭ 7.70 Hz, 1H), 6.94 (dd, 4H), 7.01 (d, J ϭ 8.80 Hz, 1H), 7.08 (d, in the study.
J ϭ 8.80 Hz, 1H), 7.26 (d, J ϭ 8.25 Hz, 1H), 7.31 (d, J ϭ 8.25 Hz, 1H),
7.43–7.55 (m, 3H), 7.91 (d, J ϭ 7.15 Hz, 2H). The anomeric proton of the
Male CD-1 mice (n ϭ 5, 25–35 g), male Sprague-Dawley rats (n ϭ 3,
250–280 g), and male cynomolgus monkeys (n ϭ 3, 3–5 kg) were fasted for
␤-anomer was at 5.38 ppm (J ϭ 8.25 Hz) for the minor rotational conformer approximately 8 h before dosing. Each animal received an oral gavage dose of
and at 5.42 ppm (J ϭ 7.70 Hz) for the major conformer. The downfield 30 mg/kg (300 ␮Ci/kg, 5 ml/kg) for mouse, 15 mg/kg (150 ␮Ci/kg, 5 ml/kg)
chemical shift and large coupling observed are typical for the ␤ configuration. for rat, and 3 mg/kg (30 ␮Ci/kg, 1 ml/kg) for monkey of [14C]peliglitazar

126
ZHANG ET AL.
dissolved in PEG-400. Human studies were conducted as described previously time point) after the same quenching procedure. The samples were mixed well
(Wang et al., 2006). Four healthy male subjects, aged 18 to 45 years, each
and immediately frozen at Ϫ80°C until analysis. The entire experiment was
received a single dose of [14C]muraglitazar (20 mg) or [14C]peliglitazar (10 done on ice-water bath. Before analysis, samples were taken out and thawed in
mg) containing approximately 100 ␮Ci of radioactivity as an oral solution in
PEG-400 after at least an 8-h overnight fast.
an ice-water bath. Likewise, for incubations in phosphate buffer, 0.75 ml of 1
M phosphate buffer at pH 7.4 and 14.1 ml of water were mixed with 0.15 ml
of 5 mg/ml 1-O-␤-acyl glucuronide stock solution. The remaining steps were
the same as those for the incubations for buffered plasma.
Blood samples (terminal for mouse and rat and via the cephalic vein for
monkey and direct venipuncture using Vacutainers for human) were collected
at 1, 4, 12, and 24 h using K2EDTA as the anticoagulant. Plasma was prepared
by centrifugation at approximately 1000g for 15 min at 4°C. Acetic acid was
added to plasma to a final concentration of 5% (v/v, 0.83 M) immediately after
processing. All plasma samples were frozen and stored at Ϫ20°C.
1-O-␤-Acyl glucuronides and their isomers as well as aglycons were sep-
arated isocratically by HPLC system III using a mobile phase composed of
65:35 (v/v) acetonitrile-water containing 0.05% formic acid at a flow rate of
0.3 ml/min. The separation was performed using a Luna C18(2) analytical
column (3 ϫ 150 mm, 3 ␮m; Phenomenex, Torrance, CA) operated at 25°C.
The injection volume was 5 ␮l, and the run time was 12.0 min. Under these
conditions, the retention time was 6.17, 7.04, 7.55, 8.13, 8.35, 8.82, and 15.08
min, respectively, for isomer 1, isomer 2, muraglitazar AG, isomer 3, isomer
4, isomer 5, and muraglitazar. The retention time was 7.51, 8.62, 9.55, 10.25,
11.05, and 14.80 min, respectively, for isomer 1, isomer 2, peliglitazar AG,
isomer 3, isomer 4, and peliglitazar. The HPLC effluent was monitored at 278
nm and analyzed by a LTQ mass spectrometer (Thermo Fisher Scientific).
Sample preparation and analysis. Pooled plasma samples were prepared
separately by mixing an equal volume (0.2–0.5 ml) of plasma sample from
each subject. Portions (0.5–1 ml) of the pooled plasma samples were extracted
by addition of a mixture of 1 volume of methanol and 3 volumes of acetonitrile
and mixed on a Vortex mixer. The mixtures were centrifuged at 2000g at 10°C
for 30 min, and then the supernatants were transferred into a polypropylene
centrifuge tube. The extraction was repeated two more times, and all super-
natants were combined. The recovery of radioactivity in the supernatant after
extraction was quantitative. The plasma protein pellets were digested in 1 M
NaOH for 12 h before being neutralized with 1 M HCl and counted for
radioactivity in 15 ml of Ecolite scintillation fluid. The combined supernatants
were concentrated under a stream of nitrogen, the residues were then recon-
stituted in 0.2 to 0.5 ml of a solution of 70% of HPLC mobile phase A (0.06%
TFA in water) and 30% mobile phase B (0.06% TFA in acetonitrile), vortexed, and
centrifuged at 2000g for 10 min, and 100 ␮l of the supernatant was used for the
HPLC analysis. Metabolites in plasma were analyzed by HPLC system II using a
Shimadzu LC-10AT system as described in the preceding section. HPLC effluent
(1 ml/min) was collected in a Deepwell LumaPlate-96 (PerkinElmer Life and
Analytical Sciences, Waltham, MA) at 0.26 min. The plates were dried with a
SpeedVac (Thermo Fisher Scientific) and counted for 10 min/well with a Top-
Count analyzer (PerkinElmer Life and Analytical Sciences) to quantify radioac-
tivity. Biotransformation profiles were prepared by plotting the resulting net counts
per minute values versus time after injection. Radiochromatograms were recon-
structed from the TopCount data using Microsoft Excel software.
Results
Table 1 and Fig. 2 show the exposures of peliglitazar and peligli-
tazar AG in the plasma of mouse, rat, monkey, and human. The
exposure multiples of the maximum concentration (Cmax) and AUC
values for the parent compound in mouse, rat, and monkey were Ͼ21.
The exposure multiples of Cmax and AUC values for the major
glucuronide metabolite, peliglitazar AG, were 3.8 to 6.4 in the mouse,
1 to 2 in monkey, and Ͻ0.5 in rat from the ADME studies. The
projected exposure multiples of Cmax and AUC values for the metab-
olite were higher (all were Ͼ1) in mouse, rat, and even monkey with
linearly scaled doses for the term toxicological studies. However, the
exposure multiples of Cmax and AUC values for the metabolite were
still Ͻ1 with the projected carcinogenicity testing doses. Overall,
peliglitazar AG exposure multiples of Cmax and AUC in toxicological
species were marginal even at the projected high toxicological doses.
Figure 1 shows that peliglitazar AG circulated in humans as a major
metabolite, whereas muraglitazar AG had little circulation in humans in the
1-h plasma samples. Radioactivity profiles of human plasma at 4, 12, and
24 h also showed that peliglitazar AG but not muraglitazar AG was a
significant circulating metabolite in humans. There were minimal to no acyl
Stability studies in incubations in buffer and plasma. A portion of 0.75 ml
of 1 M phosphate buffer at pH 7.4 and 14.1 ml of human or rat plasma were
mixed, and 0.15 ml of 5 mg/ml 1-O-␤-acyl glucuronide stock solution in
acetonitrile and water (50:50, v/v) was spiked to that solution to give a final
concentration of 50 ␮g/ml 1-O-␤-acyl glucuronide. Each spiked plasma solu-
tion was aliquoted into a 0.5-ml portion of 27 tubes. Twenty-four of them were
placed in the water bath at 37°C, and three of them were quenched with 3
volumes of 1.35% acetic acid in acetonitrile/methanol (1:1, v/v). The remain-
ing samples were quenched at 0.25, 0.5, 1, 2, 4, 6, 24, and 48 h (three tubes per isomers detected in the plasma samples from subjects after administration of
TABLE 1
Estimated exposures of peliglitazar and its acyl glucuronide in mice, rats, monkeys, and humans after a single oral dose of peliglitazar
Study
Analyte
Species
Doses
Conc.c at 1 h
Conc. Exposure Multiplesa
AUC0–t
AUC Exposure Multiplesa
mg/kg
␮M
␮M ꢀ h
Radiolabeled ADME
Peliglitazar
Mouse
Rat
Monkey
Humanb
Mouse
Rat
Monkey
Humanb
Mouse
Rat
Monkey
Mouse
Rat
30
15
3
10
30
15
3
10
250
300
2.5
10
30
59.58
4.51
3.64
0.15
1.18
0.07
0.34
0.18
9.8
397
30
24
1
6.4
0.4
1.8
1
54
7.8
1.6
2.2
0.7
247.4
17.6
15.5
0.74
4.72
0.21
1.45
1.23
39.3
4.2
333
24
21
1
Radiolabeled ADME
Peliglitazar AG
3.84
0.17
1.18
1
31.9
3.41
1.0
Long-term toxicity studiesd,e
Carcinogen studiese,f
Peliglitazar AG
Peliglitazar AG
1.4
0.28
0.39
0.14
1.21
1.57
0.42
1.28
0.34
ADME, absorption, distribution, metabolism, and excretion.
a Exposure multiples were calculated by dividing Cmax or AUC values in animals by that in humans.
b Data are milligrams per subject.
c Concentration is estimated from the relative distribution of the parent or acyl glucuronide metabolites in the plasma, the total concentration of radioactivity, and the specific activity
of the administered drug; AUC0–t is estimated from the concentration in plasma at limited time points (1, 4, and 12 h) using the trapezoidal rule.
d Doses are the highest dose from the 6-month rat, 3-month mouse, and 1-year monkey toxicology studies.
e Concentration and AUC values are scaled linearly with regard to dose values in ADME studies.
f Highest projected doses for the carcinogenicity studies.

STABILITY-DEPENDENT CIRCULATION OF AGs IN HUMANS
127
5,000
4,000
Peliglitazar
Mouse 1 h plasma
3,000
2,000
1,000
0
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
4,000
Rat 1 h plasma
3,000
2,000
1,000
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
9,000
7,000
5,000
3,000
Monkey 1 h plasma
1,000
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
0
120
80
Peliglitazar
Human 1 h plasma
Peliglitazar glucuronide
40
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
Time (minutes)
FIG. 2. Radioactivity profiles in 1 h plasma of mice (30 mg/kg), rats (15 mg/kg), monkeys (3 mg/kg), and humans (10 mg/subject) following single oral doses of
14C]peliglitazar. Arrows point to the position of peliglitazar AG elution.
[
[14C]peliglitazar or [14C]muraglitazar. In addition, the plasma protein pellets binding suggest ex vivo stabilization of the plasma samples by addition of
after extraction with the organic solvent contained no detectable levels of acetic acid and low levels of total plasma radioactivity. Overall, muraglitazar
radioactivity. Together, the low levels of isomerization and protein covalent AG represented approximately 4% of the muraglitazar AUC0–24 h, whereas

128
ZHANG ET AL.
TABLE 2
Glucuronidation of muraglitazar and peliglitazar in human liver microsomes and hepatocytes and excretion of acyl glucuronide metabolites in humans after oral
administration of 14C-labeled compounds
In Vitro Glucuronidation Ratesa
Glucuronide Excretionb
HLM
Hepatocytesc
Urine (0–168 h)
Bile (3–8 h)
pmol/(min ꢀ mg protein)
pmol/(min ꢀ 106 cells)
%dose
Muraglitazar
Peliglitazar
33
32
60
49
0.81
0.24
15
16
a The substrate concentration was 5 and 10 ␮M, respectively, for hepatocyte and microsomal incubations.
b Excretion of muraglitazar AG and peliglitazar AG in humans after oral administration of 14C-labeled compounds (20 mg/subject, 100 ␮Ci). Urinary elimination represented 2.13 and 1.24%
of dose and biliary elimination represented 39.9 and 24.43% of dose, respectively, for muraglitazar and peliglitazar.
TABLE 3
peliglitazar AG represented 240% of the peliglitazar AUC0–24 h. With con-
centrations of peliglitazar AG of 102, 48.8, and 21.3 ng/ml at 1, 4, and 12 h
postdose, the estimated half-life of the peliglitazar AG was approximately 3 h
based on the limited data points (Wang et al., 2010). Estimation of half-life
for muraglitazar AG was not possible. Although both muraglitazar and
peliglitazar underwent extensive conjugation metabolism to form AGs as the
major metabolite, only peliglitazar AG was the major circulating component
after oral administration of the 14C-labeled drug. A similar distributional
difference was observed in monkeys as well as in rats after oral administra-
tion of 14C-labeled muraglitazar or peliglitazar (data not shown).
Stability of acyl glucuronides of muraglitazar and peliglitazar in buffer
and human plasma incubations at 50 ␮g/ml and 37°C
The buffer was 50 mM sodium phosphate, pH 7.4. Plasma samples were buffered with 50
mM phosphate, pH 7.4.
Compounds
Products
Buffer
Human Plasma
Disappearance of glucuronide (t1/2, h)a
Muraglitazar AG
N.A.
N.A.
1.23
10.1
0.33
2.3
Peliglitazar AG
b
Aglycon formation rates ͓␮g/(ml ꢀ h)͔
Muraglitazar AG
Peliglitazar AG
Muraglitazar
Peliglitazar
3.5
0.5
16.7
5.9
Table 2 shows the glucuronidation rates of muraglitazar and pe-
liglitazar in human liver microsomes and hepatocytes and biliary/
urinary excretion of acylglucuronide metabolites in humans after oral
administration of 14C-labeled compounds. At clinically relevant con-
centrations, 5 ␮M in hepatocytes and 10 ␮M in human liver micro-
somes (Zhang et al., 2006; Wang et al., 2010), muraglitazar and
peliglitazar showed similar glucuronidation rates in incubations with
both UDPGA-fortified human liver microsomes and human hepato-
cytes. In humans, muraglitazar and peliglitazar AGs showed similar
elimination profiles with at least 15 to 16% of dose excreted in the bile
over the 3- to 8-h postdose collections and 0.2 to 0.8% of the dose in
the urine over 0- to 168-h collections after oral administration of
14C-labeled compounds. These data suggest that muraglitazar AG was
formed in the liver and excreted into the urine and bile in a manner
similar to that of peliglitazar AG.
N.A., not applicable.
a t1/2 was calculated from initial rates of glucuronide disappearance (t1/2 ϭ 0.693/slope).
b The value was the average of formation rates within the linear ranges (Ͻ3 h).
NADPH. Multiple hydroxylated metabolites and the O-demethylated
metabolite of muraglitazar AG were observed in the incubation start-
ing with muraglitazar and peliglitazar AGs, and metabolite identifi-
cation is shown in the supplemental data. The glucuronide oxidation
rates were estimated to be 22 and 24 pmol/(min ꢀ mg), respectively,
for muraglitazar AG and peliglitazar AG.
Discussion
Fig. 6 illustrates the potential factors that would be expected to
affect the circulating levels of an acyl glucuronide metabolite include
Table 3 shows the stability of muraglitazar AG and peliglitazar AG
in incubation in buffer or human plasma. Figure 3 shows distinct
concentration-time profiles of muraglitazar AG and peliglitazar AG
degradation in the buffer and rat or human plasma samples. Muragli-
tazar AG degraded in a phosphate buffer or human plasma at approx-
imately 7 to 8 times faster than peliglitazar AG by hydrolysis to
aglycons and to a lesser extent by acyl migration to positional isomers.
Muraglitazar and peliglitazar AGs degraded approximately 3 to 5
times faster in human plasma than in the buffer (Table 3). Both
glucuronides had degradation that was faster in rat plasma than in
buffer but was different from that in human plasma (Fig. 3). Good
separation of the positional isomers of muraglitazar AG and peligli-
tazar AG and their degradation products was achieved by simple
reverse-phase HPLC using an isocratic elution as described by Xue et al.
(2008). The hydrolysis rates for formation of muraglitazar and peligli-
tazar were 5 to 10 times faster in plasma than in buffer (Figs. 4 and 5).
In the incubation with muraglitazar AG in human plasma, the acyl
migration isomers 3 and 4 were formed and then quickly hydrolyzed
to form the aglycon (Fig. 4B); however, both the isomers and the
aglycon were formed slowly in the buffer. In addition, the acyl
isomers degraded to aglycon at a slower rate than that in plasma (Fig.
4A). Similar degradation profiles were observed with peliglitazar AG
but at a much slower rate than with muraglitazar AG (Fig. 5).
The oxidative metabolism of muraglitazar AG and peliglitazar AG
60.0
Buffer muraglitazar-AG
50.0
Human plasma muraglitazar-AG
Rat plasma muraglitazar-AG
40.0
Buffer peligltazar-AG
Human plasma peliglitazar-AG
30.0
Rat plasma peliglitazar-AG
20.0
10.0
0.0
0
10
20
30
40
50
60
Incubation Time (h)
FIG. 3. Concentration-time degradation profiles of muraglitazar AG and peligli-
tazar AG at 50 ␮g/ml in incubations at 37°C in the buffer, rat and human plasma at
pH 7.4. The samples were analyzed by HPLC system III as described under
was investigated in human liver microsomes in the presence of Materials and Methods.

STABILITY-DEPENDENT CIRCULATION OF AGs IN HUMANS
129
100
90
80
70
60
50
40
30
20
10
0
120
A
Isomer 1
isomer 2
Muraglitazar-AG
Isomer 3+4
isomer 5
Isomer 1
Isomer 2
Peliglitazar-AG
Isomer 3
Isomer 4
A
100
80
60
40
20
0
Muraglitazar
Peliglitazar
0
10
20
30
40
50
60
0
10
20
30
Time (h)
40
50
60
Time (h)
90
80
70
60
50
40
30
20
10
0
Isomer 1
Isomer 2
Peliglitazar-AG
Isomer 3
Isomer 4
Peliglitazar
90
80
70
60
50
40
30
20
10
0
B
B
Isomer 1
Isomer 2
Muraglitazar-AG
Isomer 3+4
Muraglitazar
0
10
20
30
40
50
60
0
10
20
30
Time (h)
40
50
60
Time (h)
FIG. 5. Hydrolysis and isomerization profiles of peliglitazar AG (50 ␮g/ml) in
incubations in a buffer or buffered human plasma (pH 7.4) at 37°C. A, buffer;
B, human plasma. The samples were analyzed by HPLC system III as described
under Materials and Methods.
FIG. 4. Hydrolysis and isomerization profiles of muraglitazar AG (50 ␮g/ml) in
incubations in a buffer or buffered human plasma (pH 7.4) at 37°C. A, buffer;
B, human plasma. The samples were analyzed by HPLC system III as described
under Materials and Methods.
ence of NADPH produced hydroxylated and O-demethylated gluc-
uronide metabolites. However, the oxidation of both muraglitazar AG
and peliglitazar AG was similarly slow and cannot be used to explain
their differences in the circulation.
In incubations in pH 7.4 buffer at 37°C, muraglitazar AG degraded
Ͼ7 to 8 times faster than peliglitazar AG (Fig. 3; Table 3). In
incubations of muraglitazar and peliglitazar AGs in human plasma,
formation rate, biliary and renal excretion, further metabolism, tissue
distribution, and reactions with small and large molecules. The dis-
proportional exposures to peliglitazar AG across species and the
striking differences in exposures to AGs of peliglitazar and muragli-
tazar in humans lead us to evaluate factors that may influence AG
exposures for these two compounds. Intrinsic formation in vitro and
direct excretion for acyl glucuronide metabolites in vivo were found
to be similar for muraglitazar and peliglitazar. Incubations of mura-
glitazar and peliglitazar in UDPGA-fortified human liver microsomes
or in hepatocytes generated the acyl glucuronides as the major me-
tabolites at similar formation rates. Examination of the acyl glucuro-
nide excretion profile revealed that the acyl glucuronide was excreted
as the major metabolite in the bile of humans and to the same extent.
Urinary excretion was a minor clearance pathway for both com-
pounds. There was no tissue accumulation or other major differences
in tissue distribution patterns in rats after oral administration of
[14C]muraglitazar or [14C]peliglitazar (data not shown). These obser-
vations do not explain the in vivo data, which showed that muragli-
tazar AG had a much lower exposure than peliglitazar AG. Thus, the
difference in chemical stability or further metabolism of their gluc-
uronide metabolites may have contributed to their very different
circulation profiles in humans. Further metabolism of glucuronide
metabolites was demonstrated in the in vitro incubations using
NADPH-fortified human liver microsomes. The human liver micro-
some incubations of muraglitazar AG or peliglitazar AG in the pres-
Bile
Biliary Excretion
Formation
Further metabolism
Drug
Acyl-gluc
Metabolite(s)
Liver
Distribution
Chemical degradation
Acyl-gluc
Drug
Blood
Urinary excretion
Distribution
Urine
Other tissues
/organs
FIG. 6. Factors affecting circulation of an acyl glucuronide metabolite.

130
ZHANG ET AL.
migration isomers were formed to lesser amounts, and these incuba- cder/guidance) defines a rigorous set of experiments to ensure that
tions generated aglycons as the dominant products (Figs. 4B and 5B).
In addition, the acyl migration isomers quickly degraded to the agly-
con in the plasma incubations. For comparison with incubations in
buffer (Figs. 4A and 5A), the aglycon was formed 3 to 5 times more
slowly than in human plasma. In addition, more acyl isomers were
formed and then slowly degraded to the aglycon in the buffer. The
greater formation of aglycon in plasma may be due to hydrolysis
catalyzed by ␤-glucuronidases and esterases or an unknown hydro-
lase. The differences in the plasma stability of muraglitazar and
peliglitazar AGs could be governed by the steric hindrance of nearby
groups to the sugar aglycon C–O–CϭO bond for hydrolysis or mi-
gration. The additional methyl group that is near the sugar aglycon
bond in peliglitazar compared with muraglitazar might provide the
steric hindrance to prevent hydrolysis of peliglitazar AG. This hy-
pothesis of a steric effect on hydrolysis may be tested with model
compounds in the future. Differences in the chemical stability of
structurally related acyl glucuronides have been well documented
(Spahn-Langguth and Benet, 1992; Bailey and Dickinson, 2003).
Even stereo isomers (R- versus S-) of acyl glucuronides have been
shown to have a 2-fold difference in reactivity (Fenselau, 1994; Akira
et al., 2000; Hasegawa et al., 2001; Mortensen et al., 2001). Compared
with literature first-order degradation half-lives in a pH 7.4 aqueous
buffer of AGs of tolmetin (0.26 h), probenecid (0.4 h), diclofenac acid
(0.51 h), R-naproxen (0.92 h), salicylic acid (1.3 h), S-naproxen (1.8
h), ibuprofen (3.3 h), bilirubin (4.4 h), mefenamic acid (16.5 h),
gemfibrozil (44 h), and valproic acid (79 h) (Ebner et al., 1999;
Shipkova et al., 2003), and dabigatran (1 h) (Ebner et al., 2010),
muraglitazar AG (1.23 h) and peliglitazar AG (10.1 h) showed mod-
erate aqueous buffer stability. However, reactivity evaluation in buffer
disproportionally exposed human metabolites are adequately evalu-
ated in toxicology animal species (Baillie et al., 2002; Humphreys and
Unger, 2006; Zhu et al., 2009). The exposure multiples found for
peliglitazar AG (AUC) were marginal with the doses used in long-
term toxicology studies compared with those seen in humans after a
dose thought to be in the appropriate range. The marginal multiples
did not precipitate any toxicology studies with peliglitazar AG as the
administered agent, but they did prompt significant analysis for
plasma AG levels (Xue et al., 2008). It is possible that the company
(Bristol-Myers Squibb) could have been asked to consider such a
toxicology study by a regulatory agency. In either case, further ex-
amination of the situation with these two compounds leads to the
conclusion that plasma monitoring of reactive metabolites may pro-
vide misleading information. Peliglitazar AG is obviously less reac-
tive, which leads in part to greater circulating concentrations in
humans and could have triggered additional toxicology studies. On the
other hand, muraglitazar AG is more reactive, which leads to lower
plasma concentrations and thus no further consideration of follow-up
toxicology studies. This type of scenario could lead to a sponsor
de-risking a relatively stable AG with a set of studies and not follow-
ing up on the more reactive AG product. Overall, this does not seem
to provide the best development path for either compound.
LC-MS-based quantitation methods generally apply to chemically
stable metabolites and the exposure to a reactive metabolite could
only be assessed when the reactive species reacts with an endogenous
component to produce a stable form such as an albumin adduct
(Zhang et al., 2005, 2007a). Chemical instability adds complications
for quantification of those unstable metabolites such as acyl glucu-
was not sufficient because both muraglitazar and peliglitazar AGs ronides (Xue et al., 2008). Although the ex vivo chemical stability of
showed approximately 4-fold additional instability in plasma com-
pared with buffer. This additional degradation of muraglitazar AG and
peliglitazar AG is consistent with a catalytic hydrolysis by an un-
known hydrolase for acyl glucuronides in plasma samples, which
could be the reason for the inconsistent correlation between buffer
degradation stability and plasma exposures of various acyl glucu-
ronides (Ebner et al., 2010). Although the reactivity in plasma may not
completely correlate with chemical stability either, as demonstrated in
this study, the real exposure to an acyl glucuronide metabolite in
animals or humans is a dynamic process and difficult to assess,
especially when one is dealing with a metabolite that had a different
degradation rate in plasma samples of different species. This study
provides an excellent example to show that muraglitazar glucuronida-
tion as a major metabolic pathway resulted in a minor circulating
metabolite due to chemical degradation in humans.
Another potential difference in disposition of AGs that could lead
to different circulating levels is further metabolism. Multiple oxida-
tion metabolites of muraglitazar and peliglitazar AGs were identified
(as described in the supplemental data). The extent of oxidative
metabolism was similar for both muraglitazar AG and peliglitazar
AG. Therefore, the elimination of muraglitazar and peliglitazar AGs
may partially depend on oxidation of the AGs themselves, but that
does not appear to explain the differential exposure seen in humans.
Therefore, although other factors such as differential transport of AGs
out of the hepatocytes into blood may also play a role, the chemical
stability differences certainly would be expected to contribute to the
observed exposure differences between muraglitazar AG and peligli-
tazar AG.
an acyl glucuronide can be improved through sample acidification and
storage at low temperature, the chemical instability in vivo is, in
general, difficult to measure directly. In this study, the chemical
stability of muraglitazar AG or peliglitazar AG was assessed by in
vitro incubations under a neutral condition followed by HPLC sepa-
ration and quantification of degradation components in plasma or
buffer fortified with a synthetic glucuronide. In vivo, the hydrolysis of
muraglitazar AG or peliglitazar AG catalyzed by an unknown hydro-
lase formed the parent compounds, which also complicated risk as-
sessment of parents, especially when these acyl glucuronides showed
species-dependent degradations (Fig. 3). In addition, conversion of
glucuronide metabolites to parent compounds should lead to a higher
apparent bioavailability for muraglitazar than for peliglitazar, assum-
ing that all other dispositional properties are similar between two
compounds. Unfortunately, bioavailability was not determined for
either compound to make that comparison. Furthermore, there were
limited data points for peliglitazar AG and no data points for mura-
glitazar AG to perform any detailed pharmacokinetic analysis for
these metabolites.
In summary, different circulating levels of AGs of muraglitazar and
peliglitazar were shown to result from the difference in their chemical
stability in human plasma, not their production, direct excretion, or
further metabolism. The focus on the disproportionate amount of
peliglitazar AG in human plasma would meet the requirement set out
in the Metabolites in Safety Testing guidance; however, it is ques-
tionable whether studies of this AG would add anything for the risk
assessment of the compound. The more meaningful risk assessment in
this case should not be studies with the more stable peliglitazar AG
observed in plasma, but rather studies with the more reactive mura-
The recent U.S. Food and Drug Administration guidance for the
pharmaceutical industry on metabolites in safety testing (Guidance for
Industry: Metabolites in Safety Testing, 2008, http://www.fda.gov/ glitazar AG not observed in plasma because of inherent reactivity.

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Zhang D, Raghavan N, Chando T, Gambardella J, Fu Y, Zhang D, Unger SE, and Humphreys
WG (2007a) LC-MS/MS-based approach for obtaining exposure estimates of metabolites in
early clinical trials using radioactive metabolites as reference standards. Drug Metab Lett
1:293–298.
Zhang D, Wang L, Raghavan N, Zhang H, Li W, Cheng PT, Yao M, Zhang L, Zhu M, Bonacorsi
S, et al. (2007b) Comparative metabolism of radiolabeled muraglitazar in animals and humans
by quantitative and qualitative metabolite profiling. Drug Metab Dispos 35:150–167.
Zhu M, Zhang D, Zhang H, and Shyu WC (2009) Integrated strategies for assessment of
metabolite exposure in humans during drug development: analytical challenges and clinical
development considerations. Biopharm Drug Dispos 30:163–184.
Authorship Contributions
Participated in research design: D. Zhang, Raghavan, Wang, Xue, Li,
Humphreys, and Cheng.
Conducted experiments: D. Zhang, Raghavan, Wang, and Obermeier.
Contributed new reagents or analytic tools: D. Zhang, Xue, Cheng, Chen,
H. Zhang, and Tao.
Performed data analysis: D. Zhang, Raghavan, Wang, Obermeier, Ra-
manathan, Li, Yang, and Humphreys.
Wrote or contributed to the writing of the manuscript: D. Zhang, Ra-
manathan, Yang, and Humphreys.
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Address correspondence to: W. Griffith Humphreys, Pharmaceutical Candi-
date Optimization Bristol-Myers Squibb Research and Development, Princeton,
NJ 08543. E-mail: william.humphreys@bms.com