DMD#34876 Rapid Communication ASSESSMENT OF A...
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Rapid Communication
ASSESSMENT OF A MICROPATTERNED HEPATOCYTE CO-CULTURE SYSTEM
TO GENERATE MAJOR HUMAN EXCRETORY AND CIRCULATING DRUG
METABOLITES
Wendy WeiWei Wang, Salman R. Khetani, Stacy Krzyzewski, David B. Duignan, and R.
Scott Obach
Pfizer Global Research and Development
Groton, CT 06340 (WWW, DBD, and RSO)
and
Hepregen Corporation
Medford, MA 02155 (SRK and SK)
DMD Fast Forward. Published on July 1, 2010 as doi:10.1124/dmd.110.034876
Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: Metabolite Generation in Micropatterned Hepatocyte Co-Culture
Address for Correspondence:
R.S. Obach
Pfizer, Inc.
Eastern Point Rd.
Groton, CT 06340
Number of:
Words in Abstract: 255
Words in Introduction: 749
Words in Discussion: 908
References: 12
Tables: 3
Figures: 2
Abbreviations: ADME: absorption, distribution, metabolism, and excretion; HPLC: high
pressure liquid chromatography
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ABSTRACT
Metabolism is one of the important determinants of the overall disposition of drugs and
the profile of m etabolites can have an impact on effi cacy and safety. Pred icting which
drug metabolites will be quantitatively predominant in humans has become increasingly
important in the r esearch and de velopment o f new dr ugs. In t his s tudy, a novel
micropatterned hepatocyte co -culture s ystem w as e valuated f or i ts ability to g enerate
human in vivo metabolites. Twenty seven compounds of diverse chemical structure and
subject to a ra nge of d rug bi otransformation re actions w ere a ssessed for me tabolite
profiles i n th e m icropatterned c o-culture s ystem u sing p ooled c ryopreserved hu man
hepatocytes. The ability of this system to generate metabolites that are >10% of dose in
excreta or >10% of total drug-related material in circulation was assessed and compared
to previously reported data obtained in human hepatocyte suspensions, liver S-9 fraction,
and li ver mi crosomes. T he mi cropatterned co-culture sy stem was i ncubated for up to
seven days without a change in medium which offered an ability to generate metabolites
for s lowly m etabolized co mpounds. T he micropatterned co -culture s ystem g enerated
82% of the exc retory m etabolites tha t exc eed 10% of dose and 75% of the circulating
metabolites that exceed 10% of total circulating drug-related material. This exceeds the
performance of hepatocyte s uspension incubations and oth er in vit ro s ystems. P hase 1
and phase 2 metabolites were generated, as well as metabolites that arise via two or more
sequential r eactions. T hese r esults s uggest th at this i n v itro s ystem o ffers the h ighest
performance a mong in vi tro m etabolism s ystems t o p redict m ajor hum an in vi vo
metabolites.
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INTRODUCTION
Data on the b iotransformation of a n ew drug represents a c ritical pi ece of
information used to understand its overall disposition in humans and laboratory animals.
Knowledge o f the m ain m etabolism r outes is im portant in u nderstanding the m ain
clearance mechanisms, potential pharmacologically active metabolites, potential for inter-
patient differences in exposure, and drug-drug interactions. It is also critically important
to c ompare the m ain m etabolism pa thways of a n ew d rug can didate in humans vs .
animals, since laboratory animal species are used in safety evaluations. It is expected that
major human metabolites are represented in animal safety s tudies; i.e. that each human
metabolite that is present in circulation at 10% or more of the total drug-related material
will be present in at least one animal species that is used in safety evaluations at equal or
greater exp osure le vels (A trakchi, 200 9; R obison and J acobs, 2009; I nternational
Conference on Ha rmonization, 2009). S uch exp ectations ha ve b een d escribed in
regulatory guidance, and are laid out to ensure that the human metabolites of new drugs
have be en ade quately te sted f or s afety. How ever, in the ty pical dr ug de velopment
process, quantitative information on the circulating and excretory metabolites in humans
is on ly a vailable la ter s ince th e s tudies n eeded t o ga ther s uch in formation a re r esource
intensive. Many research organizations wait until after a new compound shows clinical
promise in a t argeted in dication be fore inv esting in the s tudies to g ain q uantitative
metabolite profiles (i.e. radiolabel ADME studies).
Thus, in vitro approaches and systems from which reliable predictions of in vivo
human m etabolite pr ofiles ca n be m ade ar e h ighly de sired. Co mparisons o f i n v itro
metabolism profiles across species can provide an early warning as to whether humans
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could have a different major pathway of metabolism than animals, and appropriate action
can be taken earlier so as to not delay the clinical development process. However in vitro
systems can possess so me sh ortcomings w hen u sed t o predict t he total i n v ivo
metabolism profiles in humans. Some systems, such as subcellular fractions, are limited
by the complement o f dr ug-metabolizing e nzymes pr esent, a nd t hus do not pr ovide a
complete picture of the metabolism. In other cases, some drugs are metabolized through
multiple sequential reactions in vivo before the drug-related material is excreted, while
the in vitro systems will only carry out one or two sequential reactions. Finally, when
attempting to pr edict w hether a metabolite w ill be pr esent i n circulation, o ther
dispositional a nd distributional properties o f the m etabolite w ill hav e a n impact, a nd
presently this is difficult to predict. In a previous investigation, human liver microsomes,
human liver S-9 fraction, and human hepatocyte suspensions were tested for their success
rates in the generation of major human metabolites that had been observed in previously
run r adiolabeled hu man A DME s tudies (Da lvie, et a l., 2009). W hile i t w as rea dily
demonstrated that major metabolites of many compounds could be generated by in v itro
systems, rat es o f su ccess g enerally were i n t he ran ge o f 5 0%. S imilar fi ndings w ere
made by Anderson, et al., (2009). Thus, there is considerable room for improvement for
in vitro systems as applied to the generation of relevant human metabolite profiles.
Hepatocytes presently offer the most complete complement of drug-metabolizing
enzymes for the generation of metabolite profiles. The typical use of human hepatocytes
in m etabolite gen eration in volves i ncubations of new c ompounds w ith s uspensions of
primary cells (fresh or cryopreserved). However these studies are limited by the duration
over which incubations can be run, since metabolic capacity declines after a few hours,
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and i n m any cas es m etabolite pr ofiles o f co mpounds that ar e e ither s lowly and/o r
extensively metabolized are inadequate. H epatocyte culture systems in w hich the cells
are l onger-lived have generally no t been u sed f or the generation o f m etabolite profiles
because it is well-known that the expression levels of various drug-metabolizing enzymes
change (Hewett, et al., 2007; Guillouzo and Guguen-Guillouzo, 2008). Thus, unrealistic
metabolite profiles w ould b e obt ained. R ecently, a n ovel m icropatterned c o-culture
system has be en de veloped an d i t h as been de monstrated that im portant drug-
metabolizing enzyme expression levels are maintained over extended time periods (24-42
days) (Khetani and Bhatia, 2008). Such a system offers the potential to generate superior
human m etabolite p rofiles, s ince en zyme l evels a re m aintained an d the pot ential exi sts
for le ngthy inc ubation t imes ( i.e. m ultiple day s) to be tter han dle s lowly m etabolized
drugs. T he o bjective o f t his s tudy w as to de termine t he s uccess r ate o f this
micropatterned co -culture s ystem to g enerate m ajor h uman m etabolites. A s et o f 27
compounds (Figure 1) for w hich metabolite profile data from human radiolabel ADME
studies were available had been previously used to assess the ability of human in vitro
systems to g enerate m ajor human m etabolites. T his s ame s et w as us ed in t he present
study and the metabolite profile data from the micropatterned co-culture were compared
to the previous data.
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METHODS
Preparation of Micropatterned Hepatocyte Co-Culture. Plateable cryopreserved primary
human hepatocyte v ials were purchased f rom Celsis In Vitro Technologies (Baltimore,
MD) (lots RCP and BOB), and BD Biosciences (Woburn, MA) (lot 109). Cryopreserved
hepatocyte vials were thawed at 370C for 90 -120 seconds followed by d ilution with 50
mL of warm Hepregen-customized and proprietary hepatocyte culture medium (HCM).
The cell suspension was spun at 50xg for 5 minutes. The supernatant was discarded, cells
were r esuspended i n H CM, a nd v iability w as as sessed using T rypan blue e xclusion
(typically 70–90%). Liver-derived nonparenchymal cells, as judged by their size (~10 um
in diameter) and morphology (nonpolygonal), were consistently found to be less than 1%
in these preparations.
To c reate m icropatterned co-cultures in 24-well p lates, w e fi rst p roduced a
hepatocyte pa ttern by s eeding hepatocytes ( pooled s uspension f rom the three s eparate
hepatocyte donors) on collagen- patterned substrates that mediate selective cell adhesion.
The cells were washed with medium 4-6 hours later to remove unattached cells (leaving
~30,000 a ttached hepatocytes on 91 collagen-coated i slands) a nd in cubated in HCM.
Stromal cells were seeded 12–24 h later t o create co-cultures (Khetani and Bhatia, 2008).
Culture medium was replaced every 2 days (400 uL per well) pr ior to incubation with
compounds.
Metabolism Incubations. T wo s eparate s tudies ut ilizing a ll of t he 2 7 c ompounds were
conducted. F or e ach st udy, m icropatterned co -cultures co ntaining po oled he patocytes
from 3 se parate donors w ere a llowed 7 days t o fu lly st abilize w ith re spect t o li ver-
specific functions. Cultures were washed to remove serum and compounds in serum-free
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HCM (400 uL per well) were added to the cells. At 2 and 7 days the culture medium was
removed f rom 2 r epresentative w ells and immediately f rozen on dr y ice . T ime po ints
selected w ere bas ed o n a n initial a ssessment o f metabolite pr ofiles o bserved a t time
points ranging from 4 h r to 10 days for six of the compounds. Samples were kept a t -
800C until further analysis.
HPLC-MS/MS Analysis. A nalyses of i ncubation s amples for 2 7 c ompounds w ere
performed on a Thermo LTQ system. The system consisted of an Agilent HPLC injector,
a HP-11 00 qu aternary gra dient pump, an d a HP-11 00 diode a rray d etector (A gilent
Technologies, Palo Alto, CA) in line with a LTQ mass spectrometer (Thermo, Waltham,
MA). The chr omatography was pe rformed us ing a P olaris C18 (4.6 × 250 m m; 5 μm;
Varian, Lake Forest, CA) column. The mobile phase consisted of 0.1% formic acid (A)
and ac etonitrile (B ), and w as delivered a t a flow ra te of 0 .8 m L/min. T he gra dient
consisted of 5% B for 5 min followed by a linear gradient to 80% B at 50 min. This was
followed by a 10 m in re-equilibration of the column at 95% A. The effluent was passed
through the diode array detector operated in the wavelength range of 200 to 400 nm. This
was followed by introduction, at a split, of approximately 20 to 1, into the source of the
mass spectrometer. The mass spectrometer was operated in a p ositive ion mode and was
equipped with an electrospray ionization source. The source parameters for the LTQ were
source potential, 4.5 kV; capillary potential, 2 V; source temperature, 3500C. The mass
spectrometer was op erated in a data dependent scanning mode t o MS3. Th e n ormalized
collision energy for the data dependent scanning was 30-40%.
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RESULTS AND DISCUSSION
In a p reviously reported s tudy, 27 compounds for w hich quantitative metabolite
profiles in cir culation and e xcreta w ere av ailable f rom hum an r adiolabel s tudies w ere
examined in three in v itro systems to determine how well these in v itro systems could
generate m etabolites th at r epresent at l east 1 0% o f do se o r 10% o f c irculating dr ug-
related m aterial (Da lvie, et a l, 2009). T his s ame s et of c ompounds w as u sed in th e
present an alysis o f the m icropatterned co -culture sy stem so that a c omparison o f
performance could be made. The 27 compounds represent a range of structure types and
a variety of drug biotransformation reactions (Table 1). Also, some of the metabolites are
the product of a s ingle bi otransformation reac tion w hile ot hers a rise via t wo or m ore
sequential reactions. For these 27 compounds, a total of 56 metabolites were observed in
humans at 10% or more of ei ther c irculating d rug-related material or exc reted dose (or
both). In the previous work, liver microsomes, liver S-9 fraction, and human hepatocyte
suspension produced 22(39%), 26(46%), and 31(55%) of these metabolites, respectively
(Dalvie, e t al ., 2009). T he m icropatterned co-culture sy stem can be i ncubated m uch
longer than hepatocyte suspensions, and 2 and 7 day incubations of this system yielded
38 ( 68%) a nd 43 ( 73%) o f the se m etabolites, r espectively. A list o f the m etabolites
detected in each of the in vitro systems is shown in Table 2. The in vivo metabolites that
were not generated were similar across al l four systems; there was only one metabolite
that w as not o bserved i n the c o-culture s ystem t hat had been previously ob served i n
hepatocyte suspension.
The data are broken down by categories of metabolites in Table 3. In a ll cases,
the c o-culture s ystem ou tperformed a ll ot her i n vi tro s ystems. F or t he 39 exc retory
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metabolites, a seven day incubation of the micropatterned co-culture system yielded 82%
of t hese, a s c ompared t o hepatocyte s uspension w hich y ielded 6 4%. E xcretory
metabolites t hat were one s tep away f rom the par ent drug were generated in 15 o f 16
cases in the 7-day incubation, while those requiring two or more steps were generated at a
lower f requency, al beit at a notable im provement o ver o ther in v itro s ystems.
Metabolites ar ising v ia functionalization ( phase 1) an d conjugation (phase 2) r eactions
were generated at equal frequencies.
For some of the compounds, a direct comparison of the micropatterned co-culture
system and suspension incubations were made using the same pool of hepatocytes. This
was do ne to e nsure th at the co -culture s ystem was no t o utperforming the pr evious
suspension incubations by virtue of different metabolic capacities of different hepatocyte
pools. Example HPLC-UV chromatograms for linezolid and ziprasidone incubations are
shown in Figure 2. Ziprasidone has four important human metabolites which arise via N-
dealkylation, sulfoxidation, reduction, and methylation reactions (Prakash, et al., 1997).
The m icropatterned c o-culture s ystem gen erated th ree of f our of t hese m etabolites in
good quantity while the suspension incubation yielded just two and in low quantities. For
linezolid, w hich is a r elatively s lowly m etabolized co mpound ( Slatter, e t al ., 2 001;
Wienkers, 2000), the major oxidative metabolites were not identified in suspension, but
both were shown in the micropatterned co-culture system.
The generation of relevant human metabolites using in vitro systems represents an
important need i n t he d iscovery and de velopment o f ne w dr ugs. T here has be en a n
increasing e mphasis o n e nsuring th at m ajor h uman metabolites ar e ade quately
represented in animal toxicology studies of the parent molecule (Atrakchi, 2009; Robison
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and Jacobs, 2010; Smith and Obach, 2009). Identifying a major human metabolite for the
first time in a clinical study can cause delays in the development of a new drug, since that
metabolite will req uire a ttention t o assure th at it s r isk has been adequately q ualified in
preclinical s afety s tudies b efore s tudies c an c ontinue. T hus ga ining in sight int o which
metabolites may be quantitatively important in humans in v ivo prior to clinical s tudies
would be adv antageous, s ince t hen it can be determined w hether t hese m ajor h uman
metabolites w ere pr esent i n l aboratory ani mal s pecies us ed to test f or t oxicity o f the
parent co mpound. I n v itro m etabolism e xperiments using w idely av ailable hum an-
derived r eagents ( e.g. l iver m icrosomes, S -9 f raction, he patocytes) hav e bee n us ed
routinely to better understand the metabolism of new compounds. However, while these
systems ar e us eful, t hey do not generate al l im portant cir culating and e xcretory
metabolites. One of the reasons for this may be that the incubations do not remain active
for e xtended incubation pe riods. A lso, t he determinants o f w hich m etabolites w ill be
major in humans in vivo is driven not only by the extent of their generation, but also their
individual dispositional properties (e.g. rate of subsequent metabolism, rate and extent of
active s ecretion in to exc retory b iofluids, and di stribution into ti ssues). T he
micropatterned co -culture s ystem o ffers an ab ility to car ry out m etabolism incubations
for periods of up to 7 days without changing the medium. T hus, a drug added to this
system c an be s ubjected to m ultiple s equential m etabolic rea ctions. A lso, m etabolites
generated from drugs that are very slowly but extensively metabolized can be observed in
a 7-day incubation.
In co nclusion, t he m icropatterned human hepatocyte co -culture s ystem o ffers a
superior in vitro approach to generate major human metabolites. S uch a system can be
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used for generating human metabolite profiles that are more realistic to the in vivo profile.
Furthermore, the potential exists for applying this system to other types of in v itro drug
metabolism e xperiments t hat are us ed to pr edict a nd/or understand the human
dispositional pr ofile o f dr ugs, s uch as p harmacokinetics a nd dr ug-drug inte ractions.
Investigations into these areas are ongoing.
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REFERENCES
Anderson S, L uffer-Atlas D, a nd K nadler M P ( 2009) Pre dicting C irculating H uman
Metabolites: How Good Are We? Chem. Res. Toxicol. 22: 243–256.
Atrakchi A H ( 2009) I nterpretation an d Considerations o n the S afety Evaluation o f
Human Drug Metabolites. Chem. Res. Toxicol. 22: 1217-1220.
Dalvie D, Ob ach RS, Kang P, Pra kash C, Loi C M, Hu rst S, Ne dderman A , Gou let L,
Smith E, Bu HZ, and Smith DA. (2009) Assessment of Three Human in Vitro Systems
in the Generation of Major Human Excretory and Circulating Metabolites. C hem. Res.
Toxicol. 22: 357-368.
Guillouzo A and Guguen-Guillouzo C. (2008) Evolving concepts in liver tissue modeling
and implications for in vitro toxicology. Expert Opin. Drug Metab. Toxicol. 4: 1279-1294.
Hewitt N J, L echon MJ G, H ouston J B, e t al . ( 2007) Pri mary h epatocytes: c urrent
understanding of t he regu lation of m etabolic en zymes a nd t ransporter p roteins, an d
pharmaceutical practice f or the us e of he patocytes in m etabolism, e nzyme in duction,
transporter, clearance, and hepatotoxicity studies. Drug Metab. Rev. 39: 159-234.
International Conference on Ha rmonization (200 9) G uidance o n nonclinical s afety
studies f or t he co nduct o f human cl inical tr ials and m arketing a uthorization f or
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pharmaceuticals M 3(R2). http://www.ich.org/cache/compo/276-254-1.html ( accessed,
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development. Nat. Biotechnol. 26: 120-126.
Prakash C, Kamel A, Gummerus J, and Wilner K. (1997) Metabolism and excretion of a
new antipsychotic drug, ziprasidone, in humans. Drug Metab. Dispos. 25: 863-872.
Robison TW and Jacobs A ( 2009) Metabolites in safety testing. Bioanalysis 1: 1193-
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Slatter JG, S talker DJ, F eenstra KL, Welshman IR, Bruss JB, Sams JP , Johnson MG,
Sanders PE, H auer MJ, F agerness PE, St ryd R P, Pe ng G W, a nd Sh obe EM (200 1)
Pharmacokinetics, m etabolism, a nd excretion o f l inezolid f ollowing an o ral do se o f
[14C]linezolid to healthy human subjects. Drug Metab. Dispos. 29: 1136-1145.
Smith DA and Obach RS (2009) Metabolites in Safety Testing (MIST): Considerations of
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Toxicol 22: 267-279.
Wienkers LC. (2000) Oxidation of the novel oxazolidinone antibiotic linezolid in human
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FIGURE LEGENDS
FIGURE 1. Chemical structures of the 27 drugs used in this analysis.
FIGURE 2 . HPL C-UV c hromatograms of lin ezolid (left ) a nd zi prasidone (ri ght) in
micropatterned human hepatocyte co-cultures (0 hr, 4 hr , 48 hr , 7 days) and suspended
human he patocytes ( 4 hr-s). L = l inezolid, a a nd b = morpholine r ing o pened ac id
metabolites, Z = ziprasidone, 1 = N-dealkylziprasidone S-oxide, 2 = ziprasidone S-oxide,
3 = S-methyldihydroziprasidone.
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TABLE 1. Metabolites for the 27 compounds utilized in this analysis and their amount in human circulation and excreta. The amount in circulation or excreta are included only if values exceed 10%.
compound name in vivo human metabolites
Ph 1 or
Ph 2
primary or
secondary
metabolite
% in
excreta
% of
circulating
radioactivity
gemcabene gem cabene glucuronide 2 1 44
avasimibe de hydrogenated avasimibe 1 1 16
h ydroxyavasimibe 1 1 10
pagoclone h ydroxypagoclone 1 1 65
axitinib hy droxyaxitinib 1 1 11
a xitinib sulfoxide 1 1 16
a xitinib glucuronide 2 1 50
capravirine hy droxycapravirine sulfoxide 1 2 11
hy droxycapraivirinesulfone N-oxide 1 2 10
CJ-13610 C J-13610 sulfoxide 1 1 36 17
C J-13610 sulfone 1 2 21 10
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traxoprodil traxoprodil methoxy sulfate 2 2 40 28
traxoprodil methoxy glucuronide 2 2 10 18
CP-122721 desmethyl CP-122721 glucuronide 2 2 27 14
N-dealkylated CP-122721 glucuronide 2 2 11
de smethylhydroxyCP-122721 glucuronide 2 2 25
5 -trifluoromethoxysalicylic acid 1 2 56
tofimulast di hydroxytofimulast 1 2 23 14
r ing-opened tofimulast 1 2 33
d esthiophene tofimulast 1 2 13
lasofoxifene la sofoxifene glucuronide 2 1 22
capromorelin carboxylic acid of capromorelin 1 2 11 14
O -debenzyl hydroxycapromorelin 1 2 12
carboxylic acid of N-desmethyl-O-
debenzylcapromorelin 1 2 12
N -desmethyl-O-debenzyl hydroxycapromorelin 1 2 15
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torcetrapib bistrifluoromethyl benzoic acid 1 2 50 50
7- trifluoromethylquinaldic acid 1 2 29 63
CP-533536 h ydroxy CP-533536 1 1 70
C P-533536 sulfate 2 1 11
CP-547632 N-d ealkyl CP-547632 1 1 15
zoniporide hy droxyzoniporide 1 1 52 64
carboxylic acid of zoniporide 1 2 17
celecoxib carboxylic acid of celecoxib 1 2 73 20
c elecoxib glucuronide 2 2 15
CP-690550 h ydroxy CP-690550 1 1 20 12
d ihydroxy CP-690550 1 2 12 12
dihydroxy CP-690550 glucuronide 2 2 11 13
ziprasidone zi prasidone sulfoxide 1 1 18 69
S -methyldihydroziprasidone 1 2 18 69
N -dealkylziprasidone S-oxide 1 2 11 21
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N -dealkylziprasidone sulfone 1 2 11 21
sunepitron hy droxysunepitron 1 1 17 61
trovafloxacin t rovafloxacin glucuronide 2 1 13 22
linezolid r ing-opened linezolid 1 2 45
r ing-opened linezolid 1 2 10
sunitinib N-d ealkyl sunitinib 1 1 32 21
irinotecan ring opened carboxylic acid 1 2 11
delavirdine N -dealkyldelavirdine 1 1 46 25
de pyridinyl delavirdine 1 1 38
valdecoxib h ydroxyvaldecoxib glucuronide 2 2 23
va ldecoxib N-glucuronide 2 1 20
eplerenone 6β-hydroxyeplerenone 1 1 32 16
6β,21-dihydroxeplerenone 1 2 21
maraviroc h ydroxymethyl maraviroc 1 1 13
N-d esalkyl maraviroc 1 2 22
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N-desalkyl hydroxymethyl maraviroc 1 2 11
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TABLE 2. Human metabolites detected in incubations with micropatterned human hepatocyte co-culture as compared with other in
vitro human liver systems. There was 100% concordance in metabolite formation between the two separate micropatterned co-culture
studies. Data for hepatocytes, S9 and microsomes are taken from Dalvie et al, 2009.
Metabolites Detected in:
Micropatterned Co-
Culture Incubated for:
Compound In Vivo Human Metabolites Hepatocytes S9 Microsomes 48 hr 7 day
gemcabene gem cabene glucuronide no no no yes yes
avasimibe de hydrogenated avasimibe no no no yes yes
hy droxyavasimibe no no no no no
pagoclone hydroxypagoclone yes yes n o yes yes
axitinib hy droxyaxitinib yes yes y es yes yes
a xitinib sulfoxide yes yes yes yes yes
axitinib glucuronide yes n o n o yes yes
capravirine h ydroxycapravirine sulfoxide yes yes yes yes yes
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hydroxycapraivirinesulfone N-oxide yes yes n o yes yes
CJ-13610 C J-13610 sulfoxide yes yes yes yes yes
C J-13610 sulfone yes yes yes yes yes
traxoprodil traxoprodil methoxy sulfate yes no no yes yes
traxoprodil methoxy glucuronide yes no no yes yes
CP-122721 desmethyl CP-122721 glucuronide yes yes yes yes yes
N-dealkylated CP-122721 glucuronide no no no no no
desmethylhydroxy CP-122721 glucuronide no no no no no
5- trifluoromethoxysalicylic acid no no no no no
tofimulast d ihydroxytofimulast no yes yes yes yes
ring-opened tofimulast yes y es no yes yes
d esthiophene tofimulast no no no yes yes
lasofoxifene la sofoxifene glucuronide yes yes yes yes yes
capromorelin carboxylic acid of capromorelin no no no no no
O -debenzyl hydroxycapromorelin no no yes yes yes
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carboxylic acid of N-desmethyl-O-
debenzylcapromorelin no no no no no
N-desmethyl-O-debenzyl hydroxycapromorelin no no no yes y es
torcetrapib bistrifluoromethyl benzoic acid no no no no no
7 -trifluoromethylquinaldic acid no yes yes yes yes
CP-533536 h ydroxy CP-533536 yes yes yes yes yes
C P-533536 sulfate no no no yes yes
CP-547632 N -dealkyl CP-547632 no no no no no
zoniporide h ydroxyzoniporide yes yes yes yes yes
zoniporide carboxylic acid yes no no no yes
celecoxib carboxylic acid of celecoxib yes yes no yes yes
celecoxib glucuronide yes n o n o yes yes
CP-690550 h ydroxy CP-690550 no no yes no yes
d ihydroxy CP-690550 yes yes yes yes yes
dihydroxy CP-690550 glucuronide no no no no no
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ziprasidone zi prasidone sulfoxide yes yes yes yes yes
S-methyldihydroziprasidone yes y es no yes yes
N -dealkylziprasidone S-oxide no no no yes yes
N -dealkylziprasidone sulfone no no no no no
sunepitron h ydroxysunepitron yes yes yes yes yes
trovafloxacin t rovafloxacin glucuronide yes yes yes no yes
linezolid r ing-opened linezolid no no no no yes
r ing-opened linezolid no no no no yes
sunitinib N-d ealkyl sunitinib yes yes yes yes yes
irinotecan ring opened carboxylic acid yes yes yes yes yes
delavirdine N-d ealkyldelavirdine yes yes yes yes yes
de pyridinyl delavirdine no no no no no
valdecoxib hydroxyvaldecoxib glucuronide h metabolite yes yes no yes yes
valdecoxib N-glucuronide yes n o n o yes yes
eplerenone 6β-hydroxyeplerenone yes yes yes yes yes
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6β,21-dihydroxeplerenone yes no no no no
maraviroc h ydroxymethyl maraviroc yes yes yes yes yes
N -desalkyl maraviroc no no no no no
N-desalkyl hydroxymethyl maraviroc no no no yes yes
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TABLE 3. Success of the Micropatterned Hepatocyte Co-Culture System at Generation of Major Human Metabolites for 27 Compounds As Compared to Other In Vitro Systemsb.
Micropatterned Co-
Culture
In Vivo Microsomesa S-9a Hepatocyte
Suspensiona 48 hr 7 days
Excretory Metabolites >10% of Dose:
all excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)
metabolites arising by phase 1 reactions only 29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)
metabolites arising by a phase 2 reaction 10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)
metabolites that are one reaction from parent (primary) 16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)
metabolites that are two or more reactions from parent (secondary) 23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)
Circulatory Metabolites >10% of Total Drug-Related Material:
all circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)
metabolites arising by phase 1 reactions only 31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)
metabolites arising by a phase 2 reaction 9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)
metabolites that are one reaction from parent (primary) 16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)
metabolites that are two or more reactions from parent (secondary) 24 6 (25) 8 (33) 9 (38) 16 (67) 16 (67) aData from Dalvie, et al., 2009; bValues in parentheses represent success rates expressed as percentages.
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N
N
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NN
NN NCl N
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capravirine
avasimibe
axitinibpagoclone
CP-122721traxoprodilCJ-13610
lasofoxifene capromorelin torcetrapib
CP-533536
zoniporidecelecoxib
CP-547632
ziprasidone sunipetron
trovafloxacin
linezolid
sunitinibirinotecan
delavirdinevaldecoxib eplerenone
maraviroc
CP-690550
gemcabene
tofimilast
Figure 1
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Figure 2
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