A metabolic screening study of trichostatin A (TSA) and TSA-like histone deacetylase inhibitors in rat and human primary hepatocyte cultures

Article abstract of DOI:10.1124/jpet.106.116202

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, Cα-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amide-linked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance. Copyright

Full text of DOI:10.1124/jpet.106.116202

0022-3565/07/3211-400–408$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 321:400–408, 2007
Vol. 321, No. 1
116202/3189465
Printed in U.S.A.
A Metabolic Screening Study of Trichostatin A (TSA) and TSA-
Like Histone Deacetylase Inhibitors in Rat and Human Primary
Hepatocyte Cultures
G. Elaut, G. Laus, E. Alexandre, L. Richert, P. Bachellier, D. Tourwe´ , V. Rogiers, and
T. Vanhaecke
Departments of Toxicology (G.E., V.R., T.V.) and Organic Chemistry (G.L., D.T.), Vrije Universiteit Brussel, Brussels, Belgium;
Laboratoire de Chirurgie Expe´ rimentale, Fondation Transplantation, Strasbourg, France (E.A., L.R.); and Centre de Chirurgie
Visce´ rale et de Transplantation, Hoˆ pital de Hautepierre, Strasbourg, France (P.B.)
Received October 26, 2006; accepted January 10, 2007
ABSTRACT
Hydroxamic acid (HA)-based histone deacetylase (HDAC) in- inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly im-
hibitors, with trichostatin A (TSA) as the reference compound, proves both potency and metabolic stability, favoring HA re-
are potential antitumoral drugs and show promise in the cre- duction to hydrolysis. On the other hand, C␣-unsaturation and
ation of long-term primary cell cultures. However, their meta- spacer methylation not only reduce HDAC inhibition but also
bolic properties have barely been investigated. TSA is rapidly increase the rate of metabolic inactivation approximately
inactivated in rodents both in vitro and in vivo. We previously 2-fold, mainly through HA reduction. However, in rat hepato-
found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hy- cyte monolayer cultures, compound 1 is shown to be exten-
droxyamide or 4-Me2N-BAVAH (compound 1) is metabolically sively metabolized by phase II conjugation. In conclusion, this
more stable upon incubation with rat hepatocyte suspensions. study suggests that simple structural modifications of amide-
In this study, we show that human hepatocytes also metabolize linked TSA analogs can improve their phase I metabolic stability
TSA more rapidly than compound 1 and that similar pathways in both rat and human hepatocyte suspensions. Phase II glu-
are involved. Furthermore, structural analogs of compound 1 curonidation, however, can compensate for their lower phase I
(compounds 2-9) are reported to have the same favorable metabolism in rat hepatocyte monolayers and could play a yet
metabolic properties. Removal of the dimethylamino substitu- unidentified role in the determination of their in vivo clearance.
ent of compound 1 creates a very stable but 50% less potent
Histone deacetylase inhibitors (HDACi) have a high the- positively affect the maturation of expanded stem cells. Ex-
rapeutic potential as antitumoral drugs (Cohen et al., 1999; citing results have been obtained with respect to the long-
Piekarz and Bates, 2004; Vanhaecke et al., 2004b) and are
being explored as new agents to prevent and/or treat several
inflammatory diseases (Niki et al., 1999; Rombouts et al.,
2002; Hockly et al., 2003; Rahmani et al., 2005; Blanchard et
al., 2005a). In addition, they are components of a new strat-
egy to prevent the spontaneous dedifferentiation of primary
cells when isolated from their originating tissues and to
term cultivation of primary rat hepatocytes and the creation
of hepatocyte-like cells from adult rat and human bone mar-
row (Papeleu et al., 2003; Rogiers et al., 2004; Vanhaecke et
al., 2004a; Snykers et al., 2006; Vinken et al., 2006).
Several structurally divergent classes of HDACi have now
been synthesized or discovered. They are characterized by
the size of their cap group and the nature of their functional
group (Piekarz and Bates, 2004; Vanhaecke et al., 2004b).
Hydroxamic acid (HA)-based small molecule HDACi, struc-
turally derived from the naturally occurring compound (R)-
(ϩ)-trichostatin A (TSA, Table 1), constitute one of the most
important classes because they potently inhibit HDAC in a
reversible way (Finnin et al., 1999). One of these TSA ana-
logs, SAHA (suberoylanilide hydroxamic acid, vorinostat), is
This work was supported by grants from the Fund for Scientific Research-
Flanders (FWO) and the Research Council (OZR) of the Vrije Universiteit
Brussel (Brussels, Belgium). This work is also part of the European Specific
Targeted Research Project-Project Predictomics 504761.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.116202.
ABBREVIATIONS: FBS, fetal bovine serum; HA, hydroxamic acid; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor(s); HPLC,
high-performance (pressure) liquid chromatography; LDH, lactate dehydrogenase; 4-Me2N-BAVAH, 5-(4-dimethylaminobenzoyl)aminovaleric acid
hydroxyamide; QSAR, quantitative structure-activity relationship; SAHA, suberoylanilide hydroxamic acid; TSA, R-(ϩ)-trichostatin A or 7-[4-
(dimethylamino)phenyl]-4,6-dimethyl-7-oxo-hepta-2,4-dienoic acid hydroxamide; DMSO, dimethyl sulfoxide.
400

Biotransformation of TSA-Like HDACi
401
TABLE 1
Chemical structures, in vitro rat hepatocyte HDAC inhibition data, and major phase I biotransformation pathways of TSA and the synthesized
amide-linked analogs in rat hepatocyte suspensions
Benzamides 1 to 7 were prepared as described previously by Jung et al. (1997, 1999); compounds 8 and 9 were synthesized as described by Van Ommeslaeghe et al. (2003).
HDAC inhibition determinations and biotransformation experiments were performed as described under Materials and Methods.
IC50 Rat Liver
HDAC
Major Phase I Biotransformation
Pathway
Major Phase I Metabolite(s)
after 3 h of Incubation
Compound
Chemical Structures
nM
TSA
11 Ϯ 2
N-Demethylation, HA reduction
N-Didemethylated amide
1
1920 Ϯ 405
HA hydrolysis, HA reduction
HA hydrolysis
Carboxylic acid, amide
Carboxylic acid
2
3
4
4218 Ϯ 681
Ͼ106
3109 Ϯ 294
HA hydrolysis
HA reduction
Carboxylic acid
Amide
5
6
7
3978 Ϯ 614
Ͼ106
152 Ϯ 15
HA reduction
Amide
Amide
8
9
12,187 Ϯ 1384
359 Ϯ 92
HA reduction
currently in phase I and II clinical trials for the treatment of This could imply that simple structural modifications of the
hematologic and solid tumors (Kelly and Marks, 2005; Krug TSA aliphatic spacer already can generate significantly more
et al., 2006).
stable inhibitors.
Xenobiotic biotransformation is generally recognized as a
We now report data on the biotransformation of eight other
key determinant of the efficacy and toxicity of a potential new amide-linked TSA analogs (compounds 2-9) (Jung et al.,
drug (Li, 2001; Elaut et al., 2002). However, little informa- 1997, 1999; Van Ommeslaeghe et al., 2003) (Table 1) in rat
tion on the biotransformation of HA-based HDACi is avail- hepatocyte suspensions. Furthermore, because no human
able in the current literature, and attempts to optimize their data are available yet in the current literature, the metabolic
metabolic properties have been scarce. We previously showed properties of TSA, compound 1 and the most stable and
that 50 ␮M TSA is completely inactivated within 40 min active analog 7, are studied in human hepatocyte suspen-
upon incubation with rat hepatocyte suspensions (Elaut et sions. Finally, because of the potential use of TSA-like
al., 2002). Another group (Sanderson et al., 2004) subse- HDACi to optimize primary hepatocyte cultures, a series of
quently reported a half-life of 6.3 min upon peritoneal ad- experiments is performed with rat hepatocyte monolayers.
ministration of TSA (0.5 mg ⅐ kg) to mice. Likewise, SAHA This also gives us the opportunity to identify potential phase
displays a low half-life (2–3 min) in rat (Cohen et al., 1999). II biotransformation pathways and elucidate the metabolic
In the search for metabolically more stable TSA deriva- patterns after long-term exposure.
tives, we recently found the amide-linked analog 5-(4-di-
methylaminobenzoyl)aminovaleric acid hydroxyamide or
4-Me2N-BAVAH [further referred to as compound 1 (Table
Materials and Methods
1)] to be considerably more resistant to metabolic inactiva-
tion by rat hepatocytes in suspension (Elaut et al., 2004). ronidase from bovine liver B-3, ␤-glucuronidase-sulfatase mollusk
Chemicals and Reagents. Acetonitrile (HPLC grade), ␤-glucu-

402
Elaut et al.
H-1, bovine insulin, bovine serum albumin fraction, crude collage- cultured on 35-mm (ဧ) Petri dishes at a density of 1.12 ϫ 105
nase type I, HEPES, kanamycin monosulfate, L-glutamine, MgCl2, cells/cm2 at 37°C in an atmosphere of 5% CO2 and 95% air and 100%
TSA or 7-[4-(dimethylamino)phenyl]-4,6-dimethyl-7-oxo-hepta-2,4- relative humidity. After plating in Williams’ E medium supple-
dienoic acid hydroxamide (purity Ն98%), trypan blue, sodium ampi- mented with 10% v/v FBS, 2 mM L-glutamine and antibiotics (7.3
cillin, streptomycin sulfate, and sucrose were purchased from Sigma- IU/ml benzylpenicillin, 50 ␮g/ml streptomycin sulfate, 50 ␮g/ml
Aldrich (Bornem, Belgium). Hydrocortisone sodium hemisuccinate kanamycin monosulfate, and 10 ␮g/ml sodium ampicillin), the cells
came from Pharmacia (Brussels, Belgium), Williams’ E medium was were allowed to attach to the plastic substrate for 4 h. Serum-
from Invitrogen (Brussels, Belgium), and fetal bovine serum (FBS) containing medium then was removed, and fresh serum-free culture
was from Invitrogen. Heparin and glucagon were obtained from Novo
medium supplemented with hydrocortisone sodium hemisuccinate
Nordisk (Copenhagen, Denmark). Trifluoroacetic acid of analytical (0.5 ␮g/ml), glucagon (0.007 ␮g/ml), and bovine insulin (5 ␮g/ml) was
reagent grade, sodium acetate, Tris⅐HCl, KCl, and ethyl acetate were added. One-day-old cultures were exposed to TSA (25 ␮M), com-
from VWR International (Leuven, Belgium). Insta-gel plus scintilla- pound 1 (50 ␮M), or the solvent control (0.05% v/v DMSO) during
tion cocktail came from Packard Bioscience B.V. (Groningen, The 24 h. Medium samples for biotransformation analysis were taken by
Netherlands).
submersion of 1-ml aliquots in liquid nitrogen. For studying the
Synthesis of the Amide-Linked Analogs. Benzamides 1-7 (Ta- intracellular biotransformation profiles, the cell monolayer was
ble 1) were prepared as described previously by Jung et al. (1997, scraped off and washed in ice-cold phosphate-buffered saline. The
1999) by coupling of the benzoic acid to the methyl ester of 5-ami-
collected cell pellets were sonicated (Labsonic U; B. Braun Laboser-
novaleric acid, 6-aminohexanoic acid, and (E)-5-amino-2-pentenoic vice N.V.) in methanol (1 ml) and centrifuged (2000g, 30 min, 4°C),
acid, followed by saponification of the ester, formation of the O- and the supernatants were stored at Ϫ80°C until further analysis.
benzyl hydroxamate, and hydrogenolysis. Compounds 8 and 9 (Table
Incubations with Primary Human Hepatocytes. Adult nor-
1) were synthesized as described by Van Ommeslaeghe et al. (2003). mal liver samples were obtained from 16 patients (male/female, aged
The O-benzyl group of compounds 5 and 8 was removed by treatment 22–68) undergoing partial hepatectomy for primary or secondary
with liquid anhydrous hydrogen fluoride. Stock solutions (200 mM) liver tumors. The hepatocytes were isolated by a two-step collage-
of each compound and of TSA were prepared in DMSO, stored at
Ϫ20°C, and diluted as required for each experiment.
nase perfusion through the existing vasculature or by direct injection
of collagenase into the liver parenchyma (Alexandre et al., 2002). All
Determination of in Vitro HDAC Inhibition Potency. The experimental procedures were done in compliance with French laws
inhibition of HDAC activity in crude rat hepatocyte lysates was and regulations and were approved by the National Ethics Commit-
assessed according to the procedure described by Ko¨lle et al. (1998), tee. After Percoll purification (33% v/v) (Chesne´ et al., 1993), cell
with some modifications. Freshly isolated rat hepatocytes (see next viability was estimated by trypan blue dye exclusion (45–86%). The
paragraph) were homogenized by sonication (Labsonic U; B. Braun freshly isolated cells in suspension were either used directly for
Laboservice N.V., Kontich, Belgium) in a 50 mM Tris⅐HCl buffer, pH incubation (three donors) or cryopreserved for later use by the pro-
7.5, containing 0.25 mM sucrose, 25 mM KCl, and 5 mM MgCl2. gressive freezing procedure described previously by Alexandre et al.
Protein concentrations, after centrifugation (10000g, 5 min, 4°C), (2002) (12 donors). To be able to analyze differences induced by the
were determined according to the Bradford procedure (Bradford, cryopreservation procedure, cells isolated from the 16th patient were
1976) using a Bio-Rad protein assay kit (Bio-Rad, Brussels, Bel- partly used directly and partly cryopreserved for later biotransfor-
gium), with bovine serum albumin as a standard. Enzyme prepara- mation experiments.
tion aliquots of 25 ␮l (protein concentration, 6 mg/ml) were mixed
with 5 ␮l of compound solution and preincubated on ice for 15 min.
Incubations were carried out with either 50 ␮M TSA, 50 ␮M
compound 1, 0.025% v/v DMSO, or only buffer in conditions identical
The reaction (30°C, 20 min) was started by the addition of 10 ␮l of to the ones used for rat (Elaut et al., 2004). Cryopreserved cells
[3H]acetate-prelabeled histones (1 mg/ml) and terminated with 36 ␮l (stored for no longer than 6 years) were thawed by immersion in a
of 1 M HCl/0.4 M sodium acetate and 800 ␮l of ethyl acetate. After 37°C water bath. DMSO was removed by dilution with Leibovitz
centrifugation (10000g, 5 min), [3H]acetate in the upper ethyl acetate L-15 medium containing 10% v/v FBS, and the hepatocytes were
phase was estimated by radioactive counting. For determination of washed and suspended in HEPES buffer (pH 7.65, 4°C). Cell num-
the 50% inhibitory concentrations (IC50), six concentrations of each ber, viability, and yield were assessed by trypan blue dye exclusion.
compound (within the range of 5 nM-100 ␮M) were tested in tripli- Percoll (20% v/v) purification of the thawed hepatocytes was only
cate. Homogenization buffer and 0.05% v/v DMSO were used as
negative controls, whereas incubations with a 5-min boiled cell ex-
tract served as a blank.
performed when cell viability was low (Ͻ 60%).
Determination of Cell Membrane Damage. Hepatocyte mem-
brane damage was evaluated by determination of the LDH index (ϭ
Incubations with Primary Rat Hepatocytes. A two-step col- 100 ϫ LDH activity in the supernatant divided by the sum of LDH
lagenase perfusion technique (Papeleu et al., 2006) was used to activity in the supernatant and in the cells) using a Merckotest
isolate the hepatocytes from the liver of male outbred Sprague-
Dawley rats (200–300 g; Charles River Laboratories, Brussels, Bel-
(VWR International, Leuven, Belgium).
Analysis of Biotransformation. Samples taken for determina-
gium), which were kept under controlled environmental conditions tion of the intracellular amounts of parent compounds and metabo-
(12-h light/dark cycle) and fed a standard diet (Animalabo A 04) with lites were analyzed directly after thawing without further treatment.
water ad libitum. Procedures for the accomodation of the animals Samples for extracellular medium profile determinations were cen-
and for the isolation and cultivation of the rat hepatocytes were trifuged (120g, 2 min, 4°C), and their supernatants were cleaned up
approved by the local ethical committee of the Vrije Universiteit by solid-phase extraction (Waters Oasis HLB Cartridges; Waters
Brussel (Brussels, Belgium).
Corporation, MA) as described previously (Elaut et al., 2002, 2004).
Freshly isolated hepatocyte suspensions (2 ϫ 106 cells/ml, HEPES Information on reversible protein binding of the metabolites was
buffer, pH 7.65, 37°C) were exposed to TSA, the synthesized HA- obtained through treatment of the extracellular medium samples
containing analogs, or the solvent control (0.1% v/v DMSO) for 3 h. with an equal volume of acetonitrile:methanol (1:1), followed by
Samples for biotransformation (extracellular medium) and lactate centrifugation (200g, 30 min, 4°C). After drying (N2, 45°C) and
dehydrogenase (LDH) index analyses were taken (Elaut et al., 2002, dissolving the samples in 60 mM sodium acetate/acetic acid buffer
2005). Nonmetabolic degradation was assessed by incubation with containing 0.11 M NaCl, they were extracted in a way similar to
2-min boiled cells.
nontreated extracellular supernatants.
To investigate long-term biotransformation and in particular po-
Separation of the mother compounds and their metabolites was
tential phase II conjugation of TSA and compound 1, rat hepatocyte performed by reversed-phase HPLC on a Discovery C18 (5 ␮m, 250 ϫ
monolayer cultures were used. Freshly isolated rat hepatocytes were 4.6 mm, Supelco; Sigma-Aldrich) or Alltima HP C18 column (5 ␮m,

Biotransformation of TSA-Like HDACi
403
250 ϫ 4.6 mm; Alttech Associates Inc., Lokeren, Belgium) using a
Gilson chromatographic system (Gilson International B.V. Rijswijk,
The Netherlands), according to the method described previously
(Elaut et al., 2002, 2004), albeit with small variations of the mobile
phase gradient. UV detection was performed at 266 (TSA) and 255
nm (amide-linked structural analogs). For identification of the me-
tabolites, the eluate was split (Acurate; LC Packings, Amsterdam,
The Netherlands) to direct 10% to a VG Quattro II triple mass
spectrometer with electrospray ionization interface applied in the
positive mode with a mass range from m/z 110 to 850 (Micromass,
Manchester, UK). Further structural identification was performed
by tandem mass spectrometry fragmentation, with detection over a
mass range of m/z 75 to 350 (Elaut et al., 2002).
To determine free and conjugated parent compounds and phase I
metabolites, 500 ␮l of supernatant fractions were incubated (37°C,
12 h) with 700 ␮l of 60 mM acetate/0.11 M NaCl buffer supplemented
with 10 mM saccharonolactone, 540 IU/ml ␤-glucuronidase-sulfatase
H1, or 580 IU/ml ␤-glucuronidase B3. The relative abundances of the
glucuronide and sulfate conjugates was determined semiquantitatively
by comparing HPLC-UV peak areas before and after hydrolysis.
Statistical Analysis. Unless specified, the results are expressed
as the mean (Ϯ S.D.) of at least three independent experiments. The
group means were compared by a paired Student’s t test (metabolic
degradation, LDH index in rat hepatocyte cultures), an unpaired
Student’s t test (metabolic degradation rat/human, humans interin-
dividually), or a two-way analysis of variance followed by a Student-
Newman-Keuls test (metabolite formation). A p Ͻ 0.05 was consi-
dered to be statistically significant.
Results
Biotransformation of TSA and Compound
1 in
Freshly Isolated and Cryopreserved Human Hepa-
tocyte Suspensions. Suspensions of freshly isolated and
thawed cryopreserved human hepatocytes were exposed to
TSA and compound 1 under conditions similar to those de-
scribed previously for rat hepatocytes (Elaut et al., 2004). In
preliminary experiments, the LDH indices of the hepatocytes
exposed to 50 ␮M of each compound within 3 h were evalu-
ated. No increases in LDH index were observed in thawed
cryopreserved or freshly isolated cells compared with control
suspensions in buffer (results not shown).
The metabolic degradation of TSA by a suspension of
thawed cryopreserved human hepatocytes pooled from five
different donors (Fig. 1A) was slower compared with the rat
hepatocyte suspensions. The latter fully degraded TSA
within the 1st hour of incubation (Elaut et al., 2002). This
was not due to the cryopreservation procedure, because no
differences in the time-dependent breakdown of TSA be- trations in the extracellular medium were followed by HPLC-UV (at 266
tween freshly isolated and thawed cryopreserved human
hepatocytes, prepared from a same donor, were observed
(Fig. 1B). Similar to rat hepatocytes (Elaut et al., 2002),
Fig. 1. A and B, metabolic degradation of 50 ␮M TSA and compound 1 in
a pool of five cryopreserved hepatocyte suspensions (2 ϫ 106/ml; viability
61.8%) as a function of the incubation time (A) and comparison of the
metabolic degradation of 50 ␮M TSA in fresh and cryopreserved human
hepatocytes obtained from the same donor (B). Parent compound concen-
and 255 nm for TSA and compound 1, respectively). Results are expressed
as percentage of unmetabolized compound (percentage of the peak area in
samples taken immediately after addition of the compound stock solu-
tions) (n ϭ 3).
human cells preferentially reduced and N-dealkylated TSA,
resulting in the formation of TSA amide, and N-mono- and went hydrolysis to produce the carboxylic acid (Table 2).
N-didemethylated TSA or TSA amide metabolites. There N-Monodemethyl and carboxylic acid metabolites constituted
were no large differences in the nature of the major phase I approximately half of the total amount of metabolites pro-
metabolites produced in suspensions of thawed cryopre- duced after 2 h of incubation of compound 1 with cryopre-
served and freshly isolated human hepatocytes after 2 h of served human hepatocytes. A slightly more extensive N-
incubation (Table 2).
demethylation of compound 1 was observed in freshly
As observed in rat hepatocyte suspensions, the metabolic isolated cells, which resulted in the additional formation of
stability of compound 1 in a pool of thawed cryopreserved small amounts of N-didemethylated 1 (Table 2).
human hepatocytes was significantly higher than observed
For both TSA and compound 1, no significant amounts of
for TSA (Fig. 1A). Although the corresponding amide was a phase II metabolites could be detected upon treatment of the
major metabolite of TSA, compound 1 preferentially under- samples with ␤-glucuronidase H3/B1. Dinor dihydro acid me-

404
Elaut et al.
TABLE 2
Comparison of the relative abundances of the major phase I metabolites of TSA and compound 1 upon their incubation with suspension and
cryopreserved and freshly isolated human hepatocytes within 2 h
The experiments were performed as described under Materials and Methods. For TSA, hepatocytes from a single donor were used. In the case of compound 1, the indicated
values are the average of results obtained with cryopreserved cells from 10 donors and with three different freshly isolated human hepatocyte incubations. For both inhibitors,
a 50 ␮ M starting concentration was used. Peak areas obtained after HPLC-UV analysis of the samples were used to determine the relative abundances, e.g., the amount
of each metabolite relative to the total amount of metabolite ϩ parent compound (%).
Cryopreserved
Hepatocytes
Freshly Isolated
Hepatocytes
Metabolite
%
TSA
TSA amide
20
3
22
2
Trichostatic acid
N-Monodemethylated TSA
N-Monodemethylated TSA amide
N-Didemethylated TSA
4
5
2
0
10
4
6
N-Didemethylated TSA amide
3
Compound 1
1-Amide
2
7
6
0
4
6
9
2
2
9
1-Acid
N-Monomethylated 1
N-Didemethylated 1
N-Monodemethylated 1-acid
tabolites could only be identified by tandem mass spectrom- compounds 2 and 5, respectively (Elaut et al., 2004). There-
etry upon manifold concentration of the samples (under N2, fore, we screened their necrotic effects in freshly isolated rat
45°C).
hepatocyte suspensions and used their major metabolites to
Effect of Structural Modifications of Compound 1 on identify secondary metabolites of their HA-containing parent
HDAC Inhibition Potency and Biotransformation Prop- compounds.
erties in Freshly Isolated Rat Hepatocyte Suspensions.
Phase I Biotransformation in Freshly Isolated Rat
As shown in Table 1, the synthesized analogs are structurally Hepatocyte Suspensions. No acute necrotic effects (i.e.,
characterized by the linkage of an aromatic benzoyl nucleus to increases in LDH index) were observed during a 3-h exposure
an aliphatic side chain through an amide bond. Whereas the of freshly isolated rat hepatocytes to a 100 ␮M concentration
p-substituent on the benzoyl fragment differs for compounds 1, of HA-containing analogs 2, 4, 5, 7, 8 and 9. Likewise, no
2, and 4 (-N(CH3)2, -H, and -OCH3, respectively), analog 5 has cytotoxicity was observed upon exposure of the hepatocytes
an unsaturated (C␣) aliphatic chain. Compounds 1 to 6 share to 25 ␮M of the acid compound 3. However, 25 ␮M of the acid
an aliphatic chain of four methylene groups, whereas com- metabolite 6 of compound 5 significantly decreased hepato-
pounds 7 to 9 possess a 5-carbon chain. With the exception of cyte viability (results not shown).
carboxylic acids 3 and 6, which are potential phase I metabo-
The metabolic breakdown of HA-containing amide-linked
lites of compounds 2 and 5, respectively (Elaut et al., 2002, compounds 1, 2, 4, 5, 7, and 9 (50 ␮M) in isolated rat hepa-
2004), the compounds contain a HA functional group. Because tocyte suspensions is depicted in Fig. 2. Their major phase I
of the presence of a methyl substituent adjacent to the benzoyl biotransformation pathways identified are summarized in
fragment on their 5-carbon side chains, compounds 8 and 9 are Table 1. p-Methoxy substitution of the benzoyl fragment
structurally more similar to TSA.
(compound 4) accelerated metabolic degradation compared
Inhibition of Rat Hepatocyte HDAC. Table 1 shows
that the synthesized amide-linked compounds are 10- to
1000-fold less potent in inhibiting rat HDAC than the refer-
ence compound TSA (IC50 ϭ 11 nM). Similar to the results
obtained by Jung et al. (1999) in maize HD-2, compound 7
(152 nM) is one order of magnitude more potent than 1 (1920
nM), demonstrating the importance of the spacer length (5-
carbon versus 4-carbon spacer, respectively). The newly syn-
thesized compound 1 derivatives, characterized by a removed
p-dimethylamino substituent (compound 2) and unsaturated
aliphatic chain (compound 5), display a 50% reduced enzyme
inhibitory activity. Although derivatives 8 and 9 bear greater
structural resemblance to TSA due to the presence of an
aliphatic methyl substituent, their inhibiting powers are
lower than compound 7. The HDAC inhibitory activity drops
with two orders of magnitude in the case of the diene 8.
Because of its low activity, this compound was not considered
for biotransformation studies. As expected from other struc-
ture-activity data (Massa et al., 2001; Mai et al., 2003) and
the HDAC inhibition mechanism (Finnin et al., 1999), the
acid metabolites 3 and 6 do not significantly inhibit rat
hepatocyte HDAC in the concentration range tested. How-
Fig. 2. Metabolic stabilities of the HA-containing TSA analogs 1, 2, 4, 5,
7, and 9 in suspensions of freshly isolated rat hepatocytes. Two million
hepatocytes per milliliter were exposed to 50 ␮M TSA or HA-based analog
within 3 h. Parent compound concentrations in the extracellular medium
were followed by HPLC-UV (at 266 and 255 nm for TSA and the analogs,
respectively). Results are expressed as percentage of unmetabolized com-
pound (percentage of the peak area in samples taken immediately after
addition of the compound stock solutions) (n ϭ 3) as a function of the
ever, they may constitute important phase I metabolites of incubation time.

Biotransformation of TSA-Like HDACi
405
with p-dimethylamino derivative 1. This was not due to a
lower inherent stability of the methoxy substituent, because
only minor amounts of O-demethylated metabolites were
found after 3 h of incubation. Unsubstituted compound 2 was
more stable (37% compound 2 still present after 3 h).
Whereas unsaturation of the aliphatic linker of compound 1
resulted in a more rapid degradation, lengthening to five
methylene groups increased metabolic stability (compound
7). This effect was abolished by methylation (compound 9) of
the aliphatic chain. The HA-moieties of compounds with a
5-C spacer were preferentially reduced, although the analogs
with a shorter spacer were more readily hydrolyzed to car-
boxylic acids. The introduction of a double bond at the C␣
position of compound 5 seemed to favor HA reduction.
Biotransformation of Compound 7 in Human Hepa-
tocyte Suspensions. Because of its potent inhibitory capac-
ity toward rat HDAC and its good metabolic stability in the
presence of freshly isolated rat hepatocytes in suspension,
compound 7 was selected for further incubations with a pool
of five thawed cryopreserved human hepatocyte suspensions.
The measured LDH indices showed that 50 ␮M was a suit-
able testing concentration (results not shown). As also ob-
served in rat hepatocyte suspensions, compound 7 was found
to be slightly more stable than compound 1. It preferably
underwent HA reduction and oxidative N-dealkylation
(Fig. 3).
Biotransformation of TSA and Compound 1 in Mono-
layer Cultures of Freshly Isolated Rat Hepatocytes. As
50 ␮M TSA induced an increase in LDH index after a 24-h
incubation with rat hepatocyte monolayer cultures, its con-
centration was reduced to 25 ␮M. No significant differences
were observed when the monolayers were exposed for 24 h to
50 ␮M compound 1 or the control solvent (0.05 v/v% DMSO).
Figure 4, A and B, shows the time-dependent breakdown of
TSA and compound 1 and formation of their major phase I
and phase II metabolites, respectively, in the extracellular
medium of 1-day-old adult rat hepatocyte monolayer cul-
tures. Consistent with the results obtained in rat and human
hepatocyte suspensions, TSA was readily N-demethylated.
As a result, N-didemethylated TSA was the major metabolite
in the extracellular medium during the first 2 h of incuba-
tion. Furthermore, because of the onset of a rapid HA-reduc-
Fig. 4. A and B, phase I and phase II biotransformation of TSA (A) and
compound 1 (B) in rat hepatocyte monolayer cultures. One-day-old hepa-
tocytes were exposed to 25 ␮M TSA and 50 ␮M compound 1, and samples
were taken immediately after the addition of the medium containing the
inhibitors (0 min) and after 1, 2, 3, and 6 h of exposure. Parent compound
and metabolite concentrations in the extracellular medium were followed
by HPLC-UV (TSA, 266 nm; compound 1, 255 nm). Results (n ϭ 3) are
expressed as percentage of the total peak area in the samples taken at the
indicated time points; means Ϯ S.D. were omitted for clarity reasons.
Because the extinction coefficients of the metabolites at the detection
wavelength are not known, the results are semiquantitative. The
amounts of glucuronide metabolites were derived from the differences in
peak areas between nonhydrolyzed and ␤-glucuronidase B3-treated sam-
ples as described under Materials and Methods.
Fig. 3. Kinetics of compound 7 consumption and metabolite production in
suspensions of cryopreserved human hepatocytes (pool of cells obtained
from five patients). Two million hepatocytes per milliliter were exposed to
50 ␮M compound 7 for 3 h. The peak areas detected at 255 nm are shown
as a function of the incubation time (n ϭ 2).
tion process, N-didemethylated TSA, TSA amide, and N-
didemethylated TSA amide were among the major phase I
metabolites detected in 6-h samples. Monolayer cultures of

406
Elaut et al.
rat hepatocytes also formed relatively large amounts of N- production of N-monodemethylated 1-acid, N-monode-
monodemethylated dinor dihydro trichostatic acid (approxi- methylated 1-acid glucuronide, and N-monodemethylated
mately 25% of the total amount of metabolites starting from 1 as major metabolites after 24 h. In addition, considerable
1 h of incubation), a metabolite produced in small amounts amounts of the reduced metabolite, 1-amide, were formed
upon in vivo administration of TSA to mice (Sanderson et al., (Table 3). Protein precipitation of the 24-h samples re-
2004) but not detectable in hepatocyte suspensions. Tricho- vealed that the sulfate conjugates of compound 1, 1-amide,
static acid, N-monodemethylated trichostatic acid, and dihy- and N-monodemethylated 1-acid had a high protein affin-
dro trichostatic acid were minor metabolites. Whereas no ity (Table 3).
phase II conjugations of TSA nor those of its phase I meta-
bolites could be observed previously in hepatocyte suspen-
sions (Elaut et al., 2002), a gradual glucuronidation of TSA,
Discussion
its N-demethylated amide metabolites, and trichostatic acid
Among the different classes of HDACi now available, TSA
was seen in the monolayer cultures. TSA glucuronide, N- and its structural analogs remain among the most interest-
monodemethylated dinor dihydro trichostatic acid, and N- ing because of their potent and reversible inhibitory proper-
didemethylated TSA amide were among the major meta- ties, as well as their relatively easy synthesis (Vanhaecke et
bolites after 24 h of exposure (Table 3). The site of al., 2004b; Elaut et al., 2006). However, they are metaboli-
glucuronidation is currently unknown as a comparison of cally unstable (Cohen et al., 1999; Elaut et al., 2004; Sand-
peak areas before and after hydrolysis was used here to erson et al., 2004). Thus, it seems interesting to look for
determine the amount of phase II conjugates. No intracel- possibilities to improve their biotransformation properties.
lular accumulation of the parent compound or its metabo- This will aid in the identification of the most appropriate
lites could be detected. However, upon protein precipita- compounds for further drug development and/or for the pre-
tion before clean-up by solid-phase extraction, a 1.5-fold vention of dedifferentiation of primary cells in culture.
increase in total peak area was obtained. Comparison of
In this study, we compared the rat hepatocyte HDAC in-
the individual metabolite peak areas before and after pro- hibition potencies and in vitro biotransformation properties
tein precipitation revealed that N-didemethylated TSA of nine structural amide-linked analogs of TSA (Table 1) in
amide glucuronide, trichostatic acid glucuronide, and di- rat and man. We found that, although they are less potent
hydro dinor trichostatic acid were extensively bound to inhibitors than TSA, all analogs are more resistant to phase
cellular and/or excreted proteins (Table 3).
I-dependent metabolic inactivation in both rat and human
Whereas TSA was more susceptible to phase I biotransfor- hepatocyte suspensions. Compounds 1, 2, and 7 were found
mation than glucuronidation, a large amount (80%) of com- to display the best balance between inhibition potency and
pound 1 was glucuronidated immediately upon exposure to metabolic stability. Importantly, structural modifications of
the hepatocyte monolayers (Fig. 4B). As observed in suspen- the aliphatic spacer seem to affect both the rate and pathway
sions, the monolayers initially did not N-demethylate com- of metabolic inactivation of the functional group.
pound 1 but preferentially formed the corresponding acid
Some observations recently made by our research team in
metabolite. Glucuronidated 1 and 1-acid glucuronide were primary hepatocyte cultures support the significance of a
the major metabolites during the first 6 h of exposure (Fig. higher metabolic stability with respect to cellular efficacy.
4B). During the following hours, N-demethylation of both the Although the IC50 values of TSA and compound 1 in rat
parent compound and the 1-acid metabolite resulted in the hepatocyte lysates differ almost 200-fold (Table 1), only 50-
TABLE 3
Relative abundances of the phase I and II metabolites of TSA and compound 1 after 24 h of incubation with 1-day-old rat hepatocyte monolayer
cultures and the influence of protein binding
The experiments were performed as described under Materials and Methods. Starting concentrations of TSA and compound 1 were 25 and 50 ␮ M, respectively. Peak areas
obtained after HPLC-UV analysis of the samples were used to determine the relative abundances, e.g., the amount of each metabolite relative to the total amount of
metabolite ϩ parent compound (%).
Metabolite
Free
Protein Bound ϩ Free
%
TSA
Trichostatic acid
9
26
0
25
27
0
7
20
6
19
20
3
N-Didemethylated TSA amide
Dihydro dinor trichostatic acid
N-Monodemethylated dihydro dinor trichostatic acid
TSA glucuronide
Trichostatic acid glucuronide
N-Monodemethylated TSA amide glucuronide
N-Didemethylated TSA amide glucuronide
13
0
9
16
Compound 1
1-Amide
17
3
12
1
1-Acid
N-Monomethylated 1
N-Monomethylated 1-Acid
1-Sulfate
1-Glucuronide
1-Acid sulfate
1-Acid glucuronide
1-Amide sulfate
N-Monodemethylated 1-acid sulfate
N-Monodemethylated 1-acid glucuronide
20
18
1
8
13
29
1
1
1
10
14
10
2
1
2
6
0
30

Biotransformation of TSA-Like HDACi
407
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It is not yet clear whether the inhibitors are more rapidly
degraded by rat than human hepatocytes. Freshly isolated
and thawed cryopreserved human hepatocytes lost approxi-
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of incubation, after which viabilities remained stable, which
is in line with the observations made by Blanchard et al.
(2005b) and Richert et al. (2006). In contrast, the viabilities
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during the entire 3-h incubation period (Elaut et al., 2005).
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metabolic pathways are involved. Analogous results were
obtained in cryopreserved and freshly isolated human hepa-
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the major phase I biotransformation pathways of TSA (i.e.,
HA reduction) is catalyzed by nonmicrosomal enzymes (Elaut
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and not human liver microsomes might be an appropriate
tool for the future phase I biotransformation screening of a
larger number of structurally related, potential drug candi-
dates.
Experiments in monolayer cultures of primary adult rat
hepatocytes showed that glucuronidation can be a major
elimination pathway of TSA and, in particular, of compound
1. However, the importance of this phase II detoxification
pathway in vivo is as yet unknown. When TSA was admin-
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in plasma (Sanderson et al., 2004). However, rodent hepato-
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molecular mass higher than 250 Da into bile (Parkinson,
1996). Therefore, enterohepatic recirculation might play a
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Acknowledgments
Papeleu P, Loyer P, Vanhaecke T, Elaut G, Geerts A, Guguen-Guillouzo C, and
Rogiers V (2003) Trichostatin A induces differential cell cycle arrests but does not
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J Hepatol 39:374–382.
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We are grateful to Prof. Daniel Jaeck and team from the Centre de
Chirurgie Visce´rale et de Transplantation, Hoˆpital de Hautepierre
(Strasbourg, France) for providing the human hepatocytes. We also
thank S. Coppens, B. Degreef, E. Desmedt, G. De Pauw, and H.
Mertens (Department of Toxicology, Vrije Universiteit Brussel,
Brussels, Belgium) for excellent technical assistance. We sincerely
appreciate the contribution of Dr. P. Papeleu (Department of Toxi-
cology, Vrije Universiteit Brussel, Brussels, Belgium) in optimizing
the HDAC inhibition assay and express our thanks to Dr. G. Brosch
(Department of Microbiology, University of Innsbruck, Medical
School, Innsbruck, Austria) for providing the [3H]acetate-prelabeled
chicken reticulocyte core histones.
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Address correspondence to: Prof. Dr. Tamara Vanhaecke, Department of
Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels,
Belgium. E-mail: tamara.vanhaecke@vub.ac.be