Antitumor effect of liposomal histone deacetylase inhibitor-lipid conjugates in vitro

Article abstract of DOI:10.1248/cpb.59.1386

Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strategy for various cancers. In this study, we

Full text of DOI:10.1248/cpb.59.1386

1386
Regular Article
Chem. Pharm. Bull. 59(11) 1386—1392 (2011)
Vol. 59, No. 11
Antitumor Effect of Liposomal Histone Deacetylase Inhibitor-Lipid
Conjugates in Vitro
Yoshiyuki HATTORI, ,a Yasuo NAGAOKA,b Manami KUBO,a Haruka YAMASAKU,a Yuta ISHII,b
*
a
Hiroko OKITA,b Hiroki NAKANO,b Shinichi UESATO,b and Yoshie MAITANI
a Institute of Medicinal Chemistry, Hoshi University; 2–4–41 Ebara, Shinagawa-ku, Tokyo 142–8501, Japan: and b Faculty
of Chemistry, Materials and Bioengineering, Kansai University; Suita, Osaka 564–8680, Japan.
Received August 2, 2011; accepted August 29, 2011; published online September 7, 2011
Histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), approved by the Food and
Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma, is a promising new treatment strat-
egy for various cancers. In this study, we hypothesized that a liposomal formulation of HDACI might efficiently
deliver HDACI into tumors. To incorporate HDACI efficiently into the liposomal membrane, we synthesized six
HDACI-lipid conjugates, in which polyethylene glycol2000 (PEG2000)-lipid or cholesterol (Chol) was linked with a
potent hydroxamic acid, HDACI, SAHA or K-182, by cleavable linkers, such as ester, carbamide and disulfide
bonds. Liposomal HDACI-lipid conjugates were prepared with distearoylphosphatidylcholine (DSPC) and
HDACI-Chol conjugate or with DSPC, Chol and HDACI-PEG-lipid conjugates, and their cytotoxicities were
evaluated for human cervix tumor HeLa and mouse colon tumor Colon 26 cells. Among the liposomes, liposomal
oleyl-PEG2000-SAHA conjugated with SAHA and oleyl-PEG2000 via a carbamate linker showed higher cytotox-
icity via hyperacetylation of histone H3 and induction of caspase 3/7 activity. These results suggested that lipo-
somal HDACI-lipid conjugates may be a potential tool for cancer therapy.
Key words histone deacetylase inhibitor; suberoylanilide hydroxamic acid; liposome; cytotoxicity
Histone deacetylases (HDAC) are a family of enzymes however, in the encapsulation of hydrophobic SAHA into
that have been identified as a promising target to reverse liposome, SAHA must be incorporated into the lipid mem-
aberrant epigenetic states associated with cancer via the reg- brane, which might be limited in its capacity to entrap
ulation of acetylation levels in histone.1,2) Aberrant histone SAHA. We therefore attempted to synthesize hydroxamate-
acetylation has been observed in the development of numer- type HDACI-lipid conjugates as a component of the lipo-
ous malignancies, and HDAC inhibitors (HDACIs) are a some formulation for efficient incorporation into the liposo-
promising new treatment strategy for malignant disease.3) mal membrane. Previously, we have reported that inclusion
The hydroxamic acid trichostatin A (TSA) have been known of HDACI-lipid conjugates into cationic nanopartilces as a
as a differentiation inducer for tumor cells, but its clinical non-viral vector could increase gene expression via hyper
use has been limited by high reactivity and instability. Fur- acetylation of histone.15) In this study, taking into considera-
thermore, TSA has also been reported to be unstable in vivo tion the diverse derivatization, we selected SAHA and one of
following intravenous injection in mice.4,5)
the K-series compounds, K-182, which has two OH groups
Suberoylanilide hydroxamic acid (SAHA, vorinostat, to tether other groups with a biodegradable ester bond, and
Zolinza), is a potent HDACI that has demonstrated antitumor synthesized cholesteryl HDACI and HDACI-polyethylene
activity in vitro against a variety of cell lines and in vivo glycol (PEG)-lipid conjugates to prepare liposomal HDACI-
against several human tumor xenograft models.6—8) Further- lipid conjugates (Fig. 1). Furthermore, we evaluated their
more, SAHA is currently prescribed to treat cutaneous T-cell cytotoxic effect on tumor cells via the acetylation of histone
lymphoma9) and is also being tested in several phase I to III H3 and apoptotic activity.
clinical trials for the treatment of a variety of other cancers,
Experimental
including breast, lung, and colon.10,11) Although SAHA is
Synthesis of HDACI-Lipid Conjugates IR spectra were recorded on a
orally administrated because of its hydrophobic property, it is
easily metabolized in the liver. Pharmacokinetic studies in
patients after administration of SAHA have identified two in-
active metabolites, SAHA-glucuronide and 4-anilino-4-
oxobutanoic acid, which are pharmacologically inactive, and
Shimadzu FTIR-8400 infrared spectrophotometer. FAB-MS spectra were
1
measured on a JEOL JMS-HX 100 instrument. H- and 13C-NMR spectra
1
were recorded on JEOL EX-400 (399.7 MHz for H-NMR and 100.4 MHz
for 13C-NMR) instruments using tetramethylsilane as an internal standard.
Analytical and preparative TLC were performed using Silica gel 60 F254
(Merck, 0.25, 1 mm, respectively) glass plates. Column chromatography was
performed using Silica Gel 60 (70—230 mesh ASTM). CM-K-182 was pre-
pared according to the method described previously.15)
the mean terminal half-life (t1/2) of SAHA was ca. 2.0 h12,13)
;
therefore, the liposomal SAHA formulation warrants in vivo
experiments because it allows intravenous administration and
prevents metabolism in the liver.
Synthesis of Cholest-5-en-3-yl (2R)-2-{[(9H-Fluoren-9-ylmethoxy)car-
bonyl]amino}-3-{[(4-methoxyphenyl)(diphenyl)methyl]sulfanyl}-
propanoate (2) A solution of Fmoc-Cys (Mmt) –OH (1) (500 mg, 0.81
mmol), cholesterol (314 mg, 0.81 mmol), dimethylaminopyridine (DMAP)
(99 mg, 0.81 mmol) and dicyclohexylcarbodiimide (DCC) (178 mg,
0.81 mmol) in CH2Cl2 (3 ml) was stirred at room temperature for 3 h (Chart
1). After the removal of DCU by filtration, the solution was diluted with
CHCl3, washed with 0.1 M HCl, 5% NaHCO3 and saturated NaCl and
then dried over Na2SO4. Concentration and column chromatography
(EtOAc/hexaneꢀ1/5) gave 2 (581 mg, 72.9%) as a colorless oil. IR (neat)
Liposomes have the advantage of an enhanced permeabil-
ity and retention effect (EPR), increasing their tumor accu-
mulation when they are intravenously injected.14) Such a type
of nanocarrier is supposed to improve the pharmacokinetic
and pharmacodistribution of the encapsulated drug. Liposo-
mal LAQ824, hydrophilic hydroxamic acid HDACI, was
shown to be both long-circulating and highly stable in vivo14); cmꢁ1: 3428, 3028, 2951, 2855, 1728, 1669, 700. 1H-NMR (399.65 MHz,
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yhattori@hoshi.ac.jp

November 2011
1387
Chart 1. Synthesis Scheme for CCS-K-182
CDCl3) d: 0.68—2.04 (44H, m, chol), 2.30 (2H, m, S–CH2–CH), 2.64 (1H, d: 11.84, 18.70, 18.81, 19.28, 21.01, 22.54, 22.80, 23.81, 24.25, 25.04,
m, COO–CH), 3.76 (3H, s, O–CH3), 4.24 (1H, m, CH2–CH–(Ar)2), 4.35 27.67, 27.99, 28.16, 28.20, 29.68, 31.82, 31.87, 35.76, 36.17, 36.55, 36.89.
(2H, m, O–CH2–CH), 4.63 (1H, m, NH2–CH–COO), 5.32 (1H, m, 37.99, 39.50, 39.70, 42.29, 49.98, 51.89, 53.82, 56.12, 56.66, 58.35, 58.95,
CH2–CH–C), 6.80—6.78 (2H, m, O–Ar–H), 7.18—7.77 (20H, m, Ar–H). 52.82, 65.61, 65.88, 75.28, 102.80, 122.96, 125.66, 126.02, 126.90, 127.25,
13C-NMR (100.40 MHz, CDCl3) d: 11.85, 14.10, 18.70, 19.31, 21.02, 22.55,
22.64, 22.80, 23.82, 24.27, 27.64, 28.00, 28.21, 31.84, 31.89, 34.32, 35.78,
36.17, 36.55, 36.87, 37.84, 39.51, 39.71, 42.31, 47.09, 49.97, 53.05, 55.051,
56.13, 56.67, 66.44, 67.17, 75.66, 113.25, 119.95, 122.98, 125.15, 126.79,
127.36, 127.66, 128.08, 128.87, 133.29, 136.34, 143.79, 154.90. HR-FAB-
MS m/z: 1026.5697 (Calcd for C59H84N3O8S2 [MꢂHꢂ], 1026.5700).
Synthesis of Cholest-5-en-3-yl (13R)-13-Amino-2-{4-[(hydroxyamino)-
carbonyl]benzyl}-1-(2-naphthyl)-6-oxo-5,7-dioxa-10,11-dithia-2-aza-
127.08, 127.70, 127.97, 129.39, 129.42, 130.73, 136.40, 139.31, 141.26, tetradecan-14-oate (CCS-K182) A mixture of 5 (20.0 mg, 0.0195 mmol)
143.78, 143.91, 144.60, 144.64, 155.57, 158.21, 169.89. High resolution in CH3CO2H (0.8 ml), tetrahydrofuran (THF) (0.8 ml) and H2O (0.2 ml) was
(HR)-FAB-MS m/z:1006.5414 (Calcd for C65H77NNaO5S [MꢂNaꢂ], stirred at 60 °C for 2.5 h (Chart 1). Concentration of the reaction mixture,
1006.5420).
followed by purification of the residue with preparative SiO2 TLC (EtOAc)
Synthesis of Cholest-5-en-3-yl (2R)-2-Amino-3-{[(4-methoxyphenyl)-
(diphenyl)methyl]sulfanyl}propanoate (3) A solution of 2 (330 mg,
afforded CCS-K-182 (16 mg, 87% yield) as a colorless oil. IR (neat) cmꢁ1
:
1
3400, 3019, 2930, 2855, 1740, 1650, 706. H-NMR (399.65 MHz, CDCl3)
0.34 mmol) in 20% piperidine/CH2Cl2 (4 ml) was stirred at room tempera- d: 0.67—2.01 (47H, m), 2.31 (2H, m, SS–CH2–CH), 2.77 (2H, m, N–CH2–
ture for 20 min (Chart 1). The solution was diluted with CHCl3, washed suc- CH2), 2.94 (2H, m, CH2–CH2–SS), 3.13 (1H, m, NH2–CH–COO), 3.64 (2H,
cessively with water and saturated NaCl, and then dried over Na2SO4. Con- s, N–CH2–Ar), 3.80 (2H, s, Ar–CH2–N), 4.23 (2H, m, CH2–CH2–OCOO),
centration and SiO2 column chromatography (EtOAc/hexaneꢀ1/5) gave 3
4.33 (2H, m, OCOO–CH2–CH2), 4.66 (1H, m, O–CH), 5.34 (1H, m,
(101 mg, 39% yield) as a colorless oil. IR (neat) cmꢁ1: 3400, 3050, 2951, CH2–CHꢀC), 7.44—7.82 (11H, m, Ar–H). 13C-NMR (100.40 MHz CDCl3)
2855, 1718, 1650, 701; 1H-NMR (399.65 MHz, CDCl3) d: 0.68—1.99 (43H, d: 11.86, 14.13, 18.73, 19.30, 21.03, 22.57, 22.70, 22.82, 23.85, 24.28,
m, chol), 2.25 (2H, m, CH-CH2-CꢀCH), 2.49 (2H, m, S–CH2–CH), 3.20
27.67, 28.02 28.22, 29.37, 29.70, 31.83, 31.89, 35.80, 36.20, 36.55, 36.89,
(1H, t, Jꢀ8.6 Hz, NH2–CH–COO), 3.78 (3H, s, O–CH3), 4.52—4.60 (1H, 36.98, 37.99, 39.53, 39.72, 42.32, 49.98, 52.20, 56.15, 56.67, 58.34, 59.26,
m, O–CH), 5.36—5.36 (1H, m, CH2–CHꢀC), 6.80—6.82 (2H, m, 65.65, 65.76, 70.53, 75.58, 123.02, 125.71, 126.07, 126.95, 127.43, 127.68,
O–Ar–H), 7.20—7.43 (12H, m, Ar–H); 13C-NMR (100.40 MHz CDCl3) d:
11.85, 14.19, 18.71, 19.31, 21.03, 22.54, 22.80, 23.83, 24.27, 27.65, 28.00,
28.21, 31.86, 31.89, 35.78, 36.18, 36.56, 36.91, 37.20, 37.93, 39.52, 39.72,
42.32, 50.01, 54.12, 55.20, 56.15, 56.68, 60.35, 66.37, 74.79, 113.18,
128.15, 128.92, 132.86, 133.31, 136.35, 139.24, 144.03, 154.91. HR-FAB-
MS m/z: 942.5115 (Calcd for C54H76N3O7S2 [MꢂHꢂ], 942.5125).
Synthesis of 3-(2-Pyridinyldisulfanyl)propanoic Acid (7) 3-(2-
Pyridinyldisulfanyl)propanoic acid (7) was synthesized according to the pro-
122.81, 126.66, 127.90, 129.51, 130.80, 136.66, 139.45, 144.91, 144.94, cedure described previously.16) 1H-NMR (399.65 MHz, CDCl3) d: 2.80 (2H,
158.16, 173.23; HR-FAB-MS m/z: 784.4734 (Calcd for C50H67NNaO3S,
784.4739).
Synthesis of Cholest-5-en-3-yl (13R)-13-amino-1-(2-naphthyl)-6-oxo-
2-(4-{[(tetrahydro-2H-pyran-2-yloxy)amino]carbonyl}benzyl)-5,7-dioxa-
t, Jꢀ6.8 Hz), 3.07 (2H, t, Jꢀ6.8 Hz), 7.16 (1H, ddd, Jꢀ7.8, 5.2, 1.6 Hz),
7.64 (1H, dd, Jꢀ7.8, 1.6 Hz), 7.67 (1H, dt, Jꢀ7.8, 1.6 Hz), 8.48 (1H, dd,
Jꢀ5.2, 1.6 Hz).
Synthesis of Distearoylphosphatidylethanolamine (DSPE)-PEG-3-(2-
pyridyldithio)propanoate (8) To a solution of 7 (43 mg, 0.2 mmol) in
CH2Cl2 (1 ml) was added N-hydroxysuccineimide (HOSu, 23 mg, 0.2 mmol)
and (DCC, 41 mg, 0.2 mmol) at 0 °C and the mixture was stirred at room
temperature for 6 h (Chart 2). After the removal of precipitates by filtration,
DSPG-PEG-NH2 (50 mg, ca. 17.4 mmol) was added to the solution and
stirred for another 18 h under the same conditions. The reaction mixture was
10,11-dithia-2-azatetradecan-14-oate (5)
A solution of 3 (35.1 mg,
0.0462 mmol), trifluoroacetic acid (TFA) (16 ml), and triethylsilane (20 ml) in
CH2Cl2 (2.0 ml) was stirred at 0 °C for 4 h under Ar atomosphere (Chart 1).
After the solution was concentrated in vacuo, the residue was dissolved in
dimethyl sulfoxide (DMSO) and to this was added 415) (30 mg, 0.069 mmol).
After stirring for another 2 h at room temperature, the solution was diluted
with CHCl3, washed with water and saturated NaCl, dried over Na2SO4 and concentrated, applied to Sephadex LH-20 gel filtration column chromatogra-
concentrated. Purification with preparative SiO2 thin layer chromatography phy (1.5 cm i.d.ꢃ45 cm) and eluted with methanol. The fractions of the elu-
(EtOAc/hexaneꢀ3/2) gave 5 (30 mg, 71% yield) as a colorless oil. IR (neat) ate from 27 to 32 ml were collected and evaporated in vacuo to gave 8
cmꢁ1: 3400, 3030, 2935, 2855, 1734, 1684, 699. 1H-NMR (399.65 MHz,
CDCl3) d: 0.67—2.04 (53H, m), 2.31—2.33 (2H, d, Jꢀ7.6 Hz, SS–CH2–
CH), 2.81 (2H, t, Jꢀ8.4 Hz, N–CH2–CH2), 2.96 (2H, t, Jꢀ10.2 Hz, CH2–
(29 mg, ca. 9.4 mmol) as a colorless oil. Molecular weights of the compo-
nents were calculated from electrospray ionization (ESI)-MS (ꢂ) multiply
charged ion peaks for 2806 (m/z 936 (Mꢂ3Hꢂ), 562 (Mꢂ5Hꢂ)), 2850 (m/z
CH2–SS), 3.09—3.14 (1H, m, NH2–CH–COO), 3.64 (2H, s, N–CH2–Ar), 951 (Mꢂ3Hꢂ), 713 (Mꢂ4Hꢂ)), 2894 (m/z 966 (Mꢂ3Hꢂ), 724 (Mꢂ4Hꢂ),
3.72 (2H, m, O–CH2–CH2), 3.80 (2H, s, Ar–CH2–N), 4.25 (2H, t, Jꢀ8.4 Hz,
CH2–CH2–OCOO), 4.36 (2H, t, Jꢀ9.6 Hz, OCOO–CH2–CH2), 4.64—4.67
579 (Mꢂ5Hꢂ)), 3026 (m/z 1009 (Mꢂ3Hꢂ), 757 (Mꢂ4Hꢂ), 606 (Mꢂ5Hꢂ)),
3114 (m/z 1039 (Mꢂ3Hꢂ), 757 (Mꢂ4Hꢂ), 624 (Mꢂ5Hꢂ)), 3158 (m/z 1054
(1H, m, O–CH), 5.07 (1H, m, O–CH–O), 5.30—5.36 (1H, d, Jꢀ23.6 Hz, (Mꢂ3Hꢂ), 790 (Mꢂ4Hꢂ)), 3202 (m/z 1068 (Mꢂ3Hꢂ), 641 (Mꢂ5Hꢂ)) and
CH2–CHꢀC), 7.43—7.81 (11H, m, Ar–H); 13C-NMR (100.40 MHz, CDCl3)
3246 (m/z 1083 (Mꢂ3Hꢂ), 650 (Mꢂ5Hꢂ)), These values corresponded to a

1388
Vol. 59, No. 11
Chart 2. Synthesis Scheme for DSPE-PEG-K-182
mass number of 8, whose polymerization degrees of PEG were from 40 to
applied to preparative SiO2 thin layer chromatography (EtOAc/hexaneꢀ1/1)
for separation to give 12 (80.6 mg, 50%) as a brownish oil. IR (neat) cmꢁ1
50.
Synthesis of 2-{[(4-Methoxyphenyl)(diphenyl)methyl]sulfanyl}ethanol 3400, 3030, 2837, 1741, 1660; 1H-NMR (399.65 MHz, CDCl3) d: 2.50 (2H,
(10) To a solution of 4-methoxy triphenyl methyl chloride (2.00 g,
6.4 mmol) in N,N-dimethylformamide (DMF) (12 ml) was added 2-mercap-
toethanol (0.45 ml, 6.4 mmol) and triethylamine (0.90 ml, 6.4 mmol) and the
solution was stirred at room temperature for 3 h (Chart 2). After the addition
of CHCl3 (30 ml), the organic layer was washed with water (30 ml) and then
dried over Na2SO4. Concentration and silica gel column chromatography
m, CH2–CH2–STmt), 2.74 (2H, m, N–CH2–CH2), 3.51—4.00 (9H, m,
Ar–CH2–N, N–CH2–Ar, COO–CH2–CH2, O–CH3), 4.12 (2H, t, Jꢀ6.0 Hz,
CH2–CH2–OCOO), 6.80—7.77 (15H, m, Ar–H); 13C-NMR (100.40 MHz
CDCl3) d: 29.65, 30.67, 51.75, 55.22, 58.23, 58.80, 65.67, 66.15, 66.45,
113.22, 125.61, 125.99, 126.73, 126.86, 127.31, 127.62, 126.86, 127.31,
127.62, 127.94, 129.40, 130.74, 132.76, 133.23, 136.52, 144.79, 154.72,
(EtOAc/tolueneꢀ5/1) gave 10 (2.30 g, 70%) as yellow crystals. IR (neat) 158.11, 171.20; HR-FAB-MS m/z: 727.2855 (Calcd for C44H43N2O6S
cmꢁ1 3590, 1722, 1602, 1506, 1250; 1H-NMR (399.65 MHz, CDCl3) d: 2.40 [MꢂHꢂ], 727.2842).
(2H, t, Jꢀ6.0 Hz), 3.31 (2H, t, Jꢀ6.0 Hz), 3.74 (3H, s), 6.73 (2H, d,
Synthesis of DSPE-PEG-K-182 To a solution of 12 (30 mg, 0.039
Jꢀ8.4 Hz), 7.1—7.5 (12H, m); 13C-NMR (100.40 MHz CDCl3) d: 35.2, mmol) in CH2Cl2 (2.0 ml) was added CF3CO2H (16 ml) and Et3SiH (20 ml)
55.2, 60.8, 66.1, 113.1, 126.7, 127.9, 129.4, 130.7, 136.8, 145.0, 158.1; HR- and the solution was stirred at 0 °C for 4 h (Chart 2). After confirmation of
FAB-MS m/z: 351.1416 (Calcd for C22H23O2S [MꢂHꢂ], 351.1419).
the complete deprotection of the p-methoxytrityl group by TLC, the solution
Synthesis of 2-{[(4-Methoxyphenyl)(diphenyl)methyl]sulfanyl}ethyl 2- was evaporated to remove the solvent and regents. The residual oily mixture
[(2-Naphthylmethyl)(4-{[(tetrahydro-2H-pyran-2-yloxy)amino]carbon-
benzyl)amino]ethyl Carbonate (11) To a solution of 915) (100 mg,
0.23 mmol) and DMAP (169 mg, 1.38 mmol) in CH2Cl2 (2.00 ml) was added
triphosgene (24 mg, 0.081 mmol) (Chart 2). After stirring at room tempera-
ture for 15 min, 10 (80.6 mg, 0.23 mmol) was added and the solution was
stirred for another 7 h under the same conditions. The reaction mixture was
was applied to 8 (29 mg, ca. 9.4 mml) in DMF (0.5 ml) and the solution was
stirred for another 20 h at room temperature. The resulting mixture was ap-
plied to Sephadex LH-20 column chromatography (1.5 cm i.d.ꢃ45 cm),
eluted with methanol, and the eluate was between 20 and 26 ml, in which
compounds with an average molecular weight were collected. Evaporation
of the solvent gave DSPE-PEG-K-182 (25 mg, ca. 7.8 mmol) as a brownish
diluted with CHCl3, washed with water, and then dried over Na2SO4. oil. Molecular weight of the components was calculated from ESI-MS (ꢂ)
Concentration and preparative thin layer silica gel chromatography
(EtOAc/hexaneꢀ3/2) gave 11 (102 mg, 55%) as a brownish oil. IR (neat)
cmꢁ1 3406, 3026, 2837, 1732, 1685; 1H-NMR (399.65 MHz, CDCl3) d:
1.60—1.87 (6H, m, 3CH2 of THP), 2.51 (2H, t, Jꢀ6.0 Hz, CH2–CH2–S),
2.77 (2H, m, N–CH2–CH2), 3.65 (1H, m, O–CHa–CH2 of THP), 3.70 (2H, s,
multiply charged ion peaks for 3192 (m/z 1065 (Mꢂ3Hꢂ), 799 (Mꢂ4Hꢂ),
640 (Mꢂ5Hꢂ)), 3237 (m/z 1080 (Mꢂ3Hꢂ), 810 (Mꢂ4Hꢂ), 648 (Mꢂ5Hꢂ)),
3325 (m/z 1109 (Mꢂ3Hꢂ), 832 (Mꢂ4Hꢂ), 666 (Mꢂ5Hꢂ)), 3369 (m/z 1124
(Mꢂ3Hꢂ), 843 (Mꢂ4Hꢂ), 675 (Mꢂ5Hꢂ)), 3545 (m/z 1183 (Mꢂ3Hꢂ), 887
(Mꢂ4Hꢂ), 710 (Mꢂ5Hꢂ)) and 3589 (m/z 1197 (Mꢂ3Hꢂ), 898 (Mꢂ4Hꢂ),
N–CH2–Ar), 3.76 (3H, s, O–CH3), 3.76 (2H, s, Ar–CH2–N), 3.86 (2H, t, 719 (Mꢂ5Hꢂ)). These values corresponded to the mass number of DSPE-
Jꢀ6.0 Hz, COO–CH2–CH2), 3.98 (1H, m, O–CHb–CH2 of THP), 4.19 (2H,
t, Jꢀ5.2 Hz, CH2–CH2–OCOO), 5.06 (1H, m, O–CH–O), 6.79 (2H, d,
Jꢀ8.0 Hz, Ar–H), 6.18—7.78 (13H, m, Ar–H); 13C-NMR (100.40 MHz
CDCl3) d: 18.64, 25.01, 28.05, 29.80, 30.63, 51.75, 55.20, 58.31, 58.81,
62.66, 65.71, 66.11, 66.45, 102.69, 113.20, 125.61, 125.99, 126.73, 126.86,
127.21, 127.33, 127.65, 127.94, 128.06, 128.90, 129.42, 130.76, 132.78,
133.25, 136.36, 136.54, 143.87, 144.81, 154.73, 158.13, 166.05; HR-FAB-
MS m/z: 811.3425 (Calcd for C49H51N2O7S [MꢂHꢂ], 811.3417).
PEG-K-182, whose polymerization degrees of PEG were from 40 to 50.
Synthesis of DSPE-PEG-SAHA To a solution of SAHA17) (26 mg,
98 mmol) in DMF (0.3 ml) was added 3-(N-succinimidyloxyglutaryl)amino-
propyl, polyethyleneglycol-carbamyldistearoylphosphatidyl-ethanolamine
(DSPE-PEG-NHS) (30 mg, ca. 9.8 mmol) and the solution was stirred at
room temperature for 12 h. After concentration of the reaction mixture, the
residue was applied to Sephadex LH-20 gel filtration column chromatogra-
phy (1.5 cm i.d.ꢃ45 cm) and eluted with methanol. The fractions of the elu-
ate from 20 to 28 ml were collected and evaporated in vacuo to gave DSPE-
Synthesis of 2-[{4-[(Hydroxyamino)carbonyl]benzyl}(2-naphthyl-
methyl)amino]ethyl 2-{[(4-Methoxyphenyl)(diphenyl)methyl]sulfanyl}- PEG-SAHA (21 mg, ca. 9.4 mmol) as a colorless oil. Molecular weights of
ethyl Carbonate (12) A mixture of 11 (90 mg, 0.11 mmol) in CH3CO2H
the components were calculated from ESI-MS (ꢂ) multiply charged ion
(0.8 ml), THF (0.4 ml) and H2O (0.2 ml) was stirred at 60 °C for 6 h (Chart
peaks for 2806 (m/z 936 (Mꢂ3Hꢂ), 562 (Mꢂ5Hꢂ)), 2850 (m/z 951
2). After concentration of the reaction mixture, the resulting crude oil was (Mꢂ3Hꢂ), 713 (Mꢂ4Hꢂ)), 2894 (m/z 966 (Mꢂ3Hꢂ), 724 (Mꢂ4Hꢂ), 579

November 2011
1389
(Mꢂ5Hꢂ)), 3026 (m/z 1009 (Mꢂ3Hꢂ), 757 (Mꢂ4Hꢂ), 606 (Mꢂ5Hꢂ)),
3114 (m/z 1039 (Mꢂ3Hꢂ), 757 (Mꢂ4Hꢂ), 624 (Mꢂ5Hꢂ)), 3158 (m/z 1054
(Mꢂ3Hꢂ), 790 (Mꢂ4Hꢂ)), 3202 (m/z 1068 (Mꢂ3Hꢂ), 641 (Mꢂ5Hꢂ)) and
3246 (m/z 1083 (Mꢂ3Hꢂ), 650 (Mꢂ5Hꢂ)), These values corresponded to a
molecular weight of 13, whose polymerization degrees of PEG were from 40
to 50.
quadrupole mass spectrometer (API-3000; Applied Biosystems). The instru-
ment was operated with a turbo ion spray interface in positive ion mode. An-
alyst 1.4.1 software (Applied Biosystems) was used to control equipment,
data acquisition, and the MRM experiment. The parameters in the method
are as follows: dwell time, 1000 ms; MRM transition, m/z 265 in Q1 and m/z
232 in Q3; declustering potential, 45 V; collision energy, 25 V; source tem-
perature, 350 °C and ion spray voltage, 5000 V. The amount of SAHA in the
mixture was determined from the calibration curves constructed from four
standards ranging from 0.05 to 1.00 mg/ml of SAHA.
Cell Culture Human cervix epithelial adenocarcinoma HeLa cells were
obtained from the European Collection of Cell Culture (Wiltshire, U.K.).
Murine colon carcinoma Colon 26 cells were obtained from the Cell Re-
source Center for Biomedical Research, Tohoku University (Miyagi, Japan).
Colon 26 cells were grown in RPMI-1640 medium (Invitrogen, Carlsbad,
CA, U.S.A.) and HeLa cells in Eagle’s Minimum Essential Medium (Invitro-
gen), supplemented with 10% heat-inactivated fetal bovine serum (FBS, In-
vitrogen) and kanamycin (100 mg/ml) at 37 °C in a 5% CO2 humidified
atmosphere.
Antiproliferative Activity The cells were seeded in 96-well plates 24 h
prior to transfection. Cells at 30% confluences in the well were treated with
various concentrations of HDACI-lipid conjugates or liposomal HDACI-
lipid conjugates for 48 h. Cell viability (%) was measured by WST-8 assay
(Dojindo Laboratories, Kumamoto, Japan) as previously reported.18)
Western Blot Analysis Western blot analysis for acetylated histone H3
and b-actin protein was performed as previously reported.19) Briefly, Colon
26 cells were seeded in a 35-mm culture dish and incubated overnight. The
cells at 30% confluency were treated with liposomal HDACI-lipid conju-
gates (10 mM as a concentration of HDACI-lipid) or liposomes (L or L-
DSPE-PEG) and then incubated for 24 h. The cells were suspended in lysis
buffer (1% Triton-X 100 in phosphate-buffered saline pH 7.4 (PBS)), and
then Western blotting was performed as previously reported.19) Acetylated
histone H3 was identified using rabbit anti-acetyl histone H3 antibody
(Sigma Chemical Co., St. Louis, MO, U.S.A.) and goat anti-rabbit im-
munoglobulin G (IgG) peroxidase conjugate (Santa Cruz Biotechnology,
Santa Cruz, CA, U.S.A.) as the secondary antibody. The blots of b-actin
protein were probed with a mouse anti b-actin IgG peroxidase conjugate
(b-actin (C4) HRP; Santa Cruz Biotechnology).
Synthesis of Oleyl-PEG-SAHA To a solution of SAHA17) (63 mg,
0.24 mmol) in DMF (3 ml) was added oleyl-PEG-NHS (100 mg, ca.
0.041 mmol), which was stirred at room temperature for 20 h. After concen-
tration of the reaction mixture, the residue was applied to Sephadex LH-20
gel filtration column chromatography (1.5 cm i.d.ꢃ45 cm) and eluted with
methanol. The fractions of the eluate from 25 to 30 ml were collected and
evaporated in vacuo to give oleyl-PEG-SAHA (OL-PEG-SAHA, 107 mg,
ca. 0.040 mmol) as a colorless oil. Molecular weights of the components
were calculated from ESI-MS (ꢂ) multiply charged ion peaks for 2022 (m/z
1012 (Mꢂ2Hꢂ), 675 (Mꢂ3Hꢂ)), 2066 (m/z 1034 (Mꢂ2Hꢂ), 690
(Mꢂ3Hꢂ)), 2110 (m/z 1056 (Mꢂ2Hꢂ), 704 (Mꢂ3Hꢂ)), 2154 (m/z 1078
(Mꢂ2Hꢂ), 719 (Mꢂ3Hꢂ)), 2198 (m/z 1100 (Mꢂ2Hꢂ), 733 (Mꢂ3Hꢂ)),
2242 (m/z 1122 (Mꢂ2Hꢂ), 748 (Mꢂ3Hꢂ)), 2286 (m/z 1144 (Mꢂ2Hꢂ), 763
(Mꢂ3Hꢂ)), 2330 (m/z 1166 (Mꢂ2Hꢂ), 778 (Mꢂ3Hꢂ), 583 (Mꢂ4Hꢂ)),
2374 (m/z 1188 (Mꢂ2Hꢂ), 792 (Mꢂ3Hꢂ), 594 (Mꢂ4Hꢂ)), 2418 (m/z 1210
(Mꢂ2Hꢂ), 807 (Mꢂ3Hꢂ), 605 (Mꢂ4Hꢂ)), 2462 (m/z 1232 (Mꢂ2Hꢂ), 822
(Mꢂ3Hꢂ), 616 (Mꢂ4Hꢂ)) and 2506 (m/z 1254 (Mꢂ2Hꢂ), 866 (Mꢂ3Hꢂ),
649 (Mꢂ4Hꢂ)). These values corresponded to molecular weight of OL-
PEG-SAHA, whose polymerization degrees of PEG were from 32 to 45.
Preparation of Liposomal HDACI-Lipid Conjugate Liposomes (L)
were prepared from distearoylphosphatidylcholine (DSPC)/cholesterol
(Chol) (55/45, molar ratio) by a modified ethanol injection method, as re-
ported previously.15) For preparation of liposomal cholesteryl HDACI, Chol
of liposome (L) formulation was substituted with CCS-K-182, CM-K-182 or
CM-SAHA (L-CCS-K-182, L-CM-K-182 and L-CM-SAHA, respectively)
(Table 1). For liposomal HDACI-PEG-lipid conjugates, 5 mol% of OL-
PEG2000-SAHA, DSPE-PEG2000-SAHA and DSPE-PEG2000-K-182 were
added to the formulation of L (L-OL-PEG-SAHA, L-DSPE-PEG-SAHA
and L-DSPE-PEG-K-182, respectively) (Table 1). As a control of liposomal
HDACI-PEG-lipid conjugates, 5 mol% PEG2000-DSPE was added to the for-
mulation of L (L-DSPE-PEG). The particle size distributions and the z-po-
tentials were measured by the dynamic light scattering method and the elec-
trophoresis light scattering method, respectively (ELS-Z2; Otsuka Electron-
ics Co., Ltd., Osaka, Japan), at 25 °C after dispersion and dilution to an ap-
propriate volume with water.
Entrapment Efficiency of HDACI-Lipid Conjugate into Liposome
Unencapsulated SAHA-lipid conjugates were removed using a Sephadex G-
50 column eluted with saline adjusted to pH 7.4. After purification, the con-
centration of phospholipid (DSPC) in liposome suspension was measured
with the Phospholipids C-test Wako (Wako Pure Chemical Industries, Ltd.).
The amount of SAHA-lipid conjugate in liposomes was quantified as its acid
hydrolysate, intact SAHA. Quantification of SAHA in the sample and stan-
dard mixture was performed with multiple reaction monitoring (MRM)
analysis using MS/MS without chromatographic purification. To each of the
standard SAHAs or SAHA-lipid conjugates in liposomal suspension
(0.65 ml) was added ethanol (0.25 ml) and 12 M HCl (0.15 ml). After incu-
bating for one hour at 50 °C, the mixture was neutralized to pH 7 with 6 M
NaOH at 0 °C and extracted twice with ethyl acetate. The organic layer was
collected, washed with brine and concentrated. The resulting extract was dis-
solved in methanol (0.20 ml) to afford a standard or sample solution of
SAHA.
Caspase 3/7 Activities Colon 26 cells were seeded in a 35-mm culture
dish and incubated overnight. The cells at 30% confluences in the well were
treated with liposomal HDACI-lipid conjugates for 24 h. To measure caspase
3/7 activity, a homogenous assay (Caspase-GloTM 3/7 assay; Promega, Madi-
son, WI, U.S.A.) was performed as previously reported.18)
Statistical Analysis Significant differences in the mean values were
evaluated by Student’s unpaired t-test. A p value of 0.05 or less was consid-
ered significant.
Results and Discussion
SAHA is orally administrated because of its hydrophobic
property, and is easily metabolized in the liver12,13); therefore,
we decided to incorporate a potent hydroxamic acid, HDACI,
SAHA or K-182,15) into the liposomal membrane to allow
their intravenous administration and increase their accultura-
tion in the tumor by the EPR effect. Liposomal HDACI for-
mulation warrants in vivo experiment; however, the amount
of hydrophobic drugs incorporated into liposomal membrane
was limited. Therefore, we decided to synthesize HDACI-
For MS/MS analysis, SAHA was analyzed by ESI-LC-MS/MS on a triple-
Table 1. Formulation, Particle Size and z-Potential of Liposomal HDACI-Lipid Conjugates
Liposome Formulation (mol% ratio)
DSPC/Chol (55 : 45)
z-Potential
Size (nm)
Polydispersity index
(mV)
L
138.9
981.5
311.1
153.8
86.6
110.5
131.2
90.2
0.28
0.35
0.16
0.20
0.26
0.13
0.16
0.23
1.9ꢄ1.1
18.5ꢄ1.2
26.4ꢄ1.6
L-CM-SAHA
DSPC/CM-SAHA (55 : 45)
L-CM-K-182
DSPC/CM-K-182 (55 : 45)
L-CCS-K-182
DSPC/CCS-K-182 (55 : 45)
36.3ꢄ0.5
L-DSPE-PEG
DSPC/Chol/DSPE-PEG2000 (52 : 43 : 5)
DSPC/Chol/DSPE-PEG2000-SAHA (52 : 43 : 5)
DSPC/Chol/OL-PEG2000-SAHA (52 : 43 : 5)
DSPC/Chol/DSPE-PEG2000-K-182 (52 : 43 : 5)
ꢁ63.4ꢄ2.9
ꢁ43.6ꢄ1.6
ꢁ31.6ꢄ2.9
ꢁ29.8ꢄ0.4
L-DSPE-PEG-SAHA
L-OL-PEG-SAHA
L-DSPE-PEG-K-182
L: Liposome.

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Vol. 59, No. 11
lipid conjugates to increase the amount of HDACI incorpo- SAHA and L-DSPE-PEG-K-182, respectively) (Table 1).
rated into the liposomal membrane. The liposomal HDACI-PEG-lipid conjugates were about
Accordingly, we designed lipid conjugates of SAHA and 90—130 nm with narrow size dispersions and negative z-po-
K-182, in which their hydroxyl (OH) group is tethered to an tentials, which might be influenced by PEG coating (Table
aliphatic fatty acid or a cholesteryl group via linkers con- 1). The liposomal HDACI-Chol conjugates, L-CM-K-182
nected with biodegradable ester or disulfide carbonate bonds and L-CCS-K-182 were about 150—300 nm, but L-CM-
(Fig. 1). The disulfide carbonate linker in a cholesteryl K- SAHA was large (about 1 mm) (Table 1). Among liposomal
182, CCS-K-182, and a PEG2000-lipid conjugate of K-182, HDACI-Chol conjugates, L-CCS-K-182 and L-CM-K-182
DSPE-PEG-K-182, is designed to release unmodified K-182 had positive charges in z-potential, which might be affected
in the reductive cytosolic condition.15) We synthesized these by a ternary amine of K-182. When size and z-potential
compounds using the procedures shown in Charts 1 and 2. measurements were conducted over a period of one month at
The cholesteryl group of CM-K-182 and CM-SAHA is 4 °C, no physicochemical changes were observed. After gel
linked with succinic diester bonds, formed by coupling cho- filtration, we measured the entrapment efficiency of HDACI-
lesteryl succinate to the original HDACIs, as described previ- lipid conjugate into the liposome. Entrapment efficiency of
ously.15) The PEG2000-lipid conjugates of SAHA, DSPE- HADCI-Chol conjugates into liposomes was almost 100%,
PEG-SAHA and OL-PEG-SAHA, were furnished with a re- but that of HDACI-PEG-lipid conjugates was about 60—
action between SAHA and commercially available N-hydrox- 70% (data not shown), suggesting that the lipid anchor of
ysuccinimide (NHS) ester of DSPE-PEG2000 or oleyl-PEG2000 HDACI-lipid conjugate might affect modification of the sur-
followed by purification with LH-20 gel filtration column face of liposomes by HDAC-lipid; however, in subsequent
chromatography.
For preparation of liposomal HDACI-lipid conjugates, the concentration among liposomal HDACI-lipids.
formulation of DSPC/Chol (L) or DSPC/Chol/DSPE- To investigate whether liposomal HDACI-lipid conjugates
experiments, we used unpurified liposomes for uniform lipid
PEG2000 (L-DSPE-PEG), which was often used for in vivo had antitumor effects on tumor cells, we treated HDACI-lipid
experiments,20—22) was modified by substitution of a compo- conjugates or liposomal HDACI-lipid conjugates with HeLa
nent (Chol in L or DSPE-PEG2000 in L-DSPE-PEG) with and Colon 26 cells for 48 h (Table 2). In HDACI-lipid conju-
HDACI-lipid conjugate. For preparation of liposomal gates, CCS-K-182, DSPE-PEG-K-182, DSPE-PEG-SAHA
HDACI-Chol conjugate, Chol of liposome (L) formulation and OL-PEG-SAHA showed high cytotoxicity for both cell
was substituted with CCS-K182, CM-K182 and CM-SAHA lines, but CM-K-182 and CM-SAHA only weakly. In liposo-
(L-CCS-K-182, L-CM-K-182 and L-CM-SAHA, respec- mal HDACI-lipid conjugates, L-OL-PEG-SAHA strongly ex-
tively) (Table 1). For liposomal HDACI-PEG-lipid conju- hibited cytotoxicity for Colon 26 and HeLa cells (IC50ꢀ1.4,
gates, DSPE-PEG2000 of L-DSPE-PEG formulation was sub- 4.0 mM, respectively), L-DSPE-PEG-SAHA, L-DSPE-PEG-
stituted with OL-PEG-SAHA, DSPE-PEG-SAHA and K-182 and L-CCS-K-182 moderately for HeLa cells
DSPE-PEG-K-182 (L-OL-PEG-SAHA, L-DSPE-PEG- (IC50ꢀ13.3, 9.9, 3.5 mM, respectively), but L-CM-SAHA and
Fig. 1. Chemical Structures of K-182, SAHA, K-182 and SAHA Derivatives

November 2011
1391
Table 2. IC50 Values for HeLa and Colon 26 Cells by HDACI-Lipid Con-
jugates and Liposomal HDACI-Lipid Conjugates
IC50 of HDACI or HDACI-
IC50 of liposomal HDACI-
Drugs
lipid conjugates (mM)
lipid conjugates (mM)
HeLa
Colon 26
HeLa
Colon 26
SAHA
K-182
1.9
1.0
9.5
2.3
—
—
—
—
CM-SAHA
19.0
27.4
4.6
ꢆ40.0
ꢆ40.0
17.1
10.7
2.4
18.6
ꢆ40.0
3.5
ꢆ40.0
ꢆ40.0
19.4
CM-K-182
CCS-K-182
DSPE-PEG-SAHA
OL-PEG-SAHA
DSPE-PEG-K-182
7.6
13.3
1.4
ꢆ20.0
4.0
1.8
5.1
7.8
9.9
ꢆ20.0
IC50 was calculated as a concentration of HDACI or HDACI-lipid conjugate.
L-CM-K-182 did not (Table 2). In particulars, L-OL-PEG-
SAHA showed higher cytotoxicity than free SAHA for both
cell lines (IC50 of free SAHAꢀ1.9, 9.5 mM for HeLa and
Colon 26 cells, respectively), suggesting that hydroxamic
acid of SAHA might be protected from hydrolysis in the
medium by conjugation with lipid. When L and L-DSPE-
PEG were treated at the same lipid concentrations with lipo-
somal HDACI-lipid conjugates, they did not show any cyto-
toxic effects (data not shown). Generally, positively charged
liposomes can be easily taken up by the cells via electrostatic
interaction compared with negatively charged ones, however,
cytotoxicties by liposomal HDACI-lipid conjugates were not
affected by the physicochemical property and were depen-
dent on structure of HDACI-lipid conjugates.
Fig. 2. Acetylation of Core Histone H3 Protein (A) and Induction of Cas-
pase 3/7 Activity (B) by Liposomal HDACI-Lipid Conjugates in Colon 26
Cells
The cells were treated with a medium containing liposomal HDACI-lipid conjugates
or free HDACIs. L-DSPE-PEG and L were treated at the same lipid concentration of
liposomal HDACI-lipid conjugations. In (A), after 24 h incubation in the medium, cel-
lular proteins were isolated from the cells and resolved on 12.5% SDS-PAGE (10 mg
protein in each well), followed by Western blot analysis for acetylated histone H3
protein and b-actin. In (B), after 48 h incubation in the medium, caspase 3/7 activity in
Colon 26 cells was measured. Each column shows the meanꢄS.D. (nꢀ3). ∗∗ pꢅ0.01,
compared with untreated cells.
Histone H3 is one of the core histone proteins in the chro-
matin of eukaryotic cells. Hyperacetylation of lysine
residues, i.e. lysine 9, in the N-terminal tails of histone H3
loosens the histone-DNA binding and activates gene tran-
scription. On the other hand, deacetylation of acetylated ly- ciently release SAHA in the cells and inhibit HDAC in the
sine residues leads to tight histone-DNA binding, which re- nucleus, resulting in the hyperacetylation of histone H3.
stricts the access of transcriptional factors. Histone acetyl-
In antitumor activity, L-CCS-K-182 had antitumor activity
transferases (HATs) and HDACs play a crucial role in this re- via acetylation of histone H3, but L-CM-K-182 and L-CM-
versible acetylation and deacetylation of histones regulating SAHA did not, suggesting that the disulfide carbonate bond
gene expression. Inhibition of HDACs therefore induces his- between HDACI and Chol was easily cleavable in cytoplasm,
tone hyperacetylation and activates gene transcription.23) but the ester bond was not. In contrast, L-DSPE-PEG-
SAHA selectively induces the expression of specific genes SAHA, L-DSPE-PEG-K-182 and L-OL-PEG-SAHA had
such as p21WAF1/CIP1 cyclin-dependent kinase inhibitor to ef- tumor suppressive effects on HeLa cells, indicating that K-
fect cell-cycle arrest, resulting in the induction of apopto- 182 or SAHA on the PEG-terminal of PEG-lipid via a car-
sis.24)
bamide bond was cleavable in the cells. Among the liposo-
We evaluated the acetylation of histone H3 and induction mal HDACI-lipid conjugates, L-OL-PEG-SAHA showed the
of apoptosis by liposomal HDACI-lipid conjugates (Fig. 2). highest antitumor effect on both HeLa and Colon 26 cells.
When liposomal HDACI-lipid conjugates were treated with DSPE-PEG-SAHA was anchored in liposomes via two alkyl
Colon 26 cells for 24 h, L-CCS-K-182, L-DSPE-PEG-SAHA chains, which could be easily retained liposome compared to
and L-OL-PEG-SAHA strongly induced the acetylation of OL-PEG-SAHA, which was anchored via one alkyl chain.
histone H3 (Fig. 2A). Furthermore, to examine the effect of Previously, we synthesized K-182-lipid conjugate, which
histone hyperacetylation on the activity of apoptosis-associ- bound to lipid via a carbamide or disulfide linker, into
ated enzymes, we measured caspase 3/7 activity 24 h after cationic nanoparticles and confirmed that the conjugates
treatment with liposomal HDACI-lipid conjugates at 10 mM were activated by cleavage between K-182 and lipid.15)
as a concentration of K-182 or SAHA (Fig. 2B). Among the Therefore, OL-PEG-SAHA might be efficiently released
liposomal HDACI-lipid conjugates, L-OL-PEG-SAHA from L-OL-PEG-SAHA in the cells, and converted to SAHA
strongly increased caspase 3/7 activities about 3-fold higher by the cleavage of carbamide bond. However, it was not clear
than untreated cells. In L-CCS-K-182, the caspase 3/7 activ- why L-OL-PEG-SAHA could induce higher cytotoxicity
ity was not increased because CCS-K-182 concentration in than free SAHA, but OL-PEG-SAHA might be stabilized hy-
the medium was under the IC50 for Colon 26 cells (19.4 mM). droxamic acid by conjugation with PEG-lipid. We confirmed
This observation indicated that L-OL-PEG-SAHA could effi- that oleyl-PEG2000 did not show cytotoxicity at the concentra-

1392
Vol. 59, No. 11
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tions used in this study (data not shown).
7) Butler L. M., Agus D. B., Scher H. I., Higgins B., Rose A., Cordon-
Cardo C., Thaler H. T., Rifkind R. A., Marks P. A., Richon V. M., Can-
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Liposomes take advantage of the EPR effect for increasing
their tumor accumulation when they are intravenously in-
jected.14) Liposomal HDACI-lipid conjugates might improve
the pharmacokinetic and pharmacodistribution of the encap-
sulated drug. Liposomal LAQ824, hydrophilic hydroxamic
acid HDAC inhibitor, was shown to be both long-circulating
and highly stable in vivo.14) In further study, we need to eval-
uate whether liposomal HDACI-lipid conjugation has antitu-
mor effects by intravenous injection. Liposomal HDACI-
lipid conjugates might have potential as an effective vector in
cancer therapy.
In this study, we synthesized HDACI-lipid conjugates and
applied liposomal formulations. Liposomal HDACI-lipid
conjugates could induce antitumor activity via acetylation of
histone H3 and activation of caspase 3/7 in HeLa and Colon
26 tumor cells. These findings indicate that liposomal
HDACI-lipid conjugates have potential as an effective vector
in cancer therapy.
13) Kelly W. K., Richon V. M., O’Connor O., Curley T., MacGregor-
Curtelli B., Tong W., Klang M., Schwartz L., Richardson S., Rosa E.,
Drobnjak M., Cordon-Cordo C., Chiao J. H., Rifkind R., Marks P. A.,
Scher H., Clin. Cancer Res., 9, 3578—3588 (2003).
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15) Ishii Y., Hattori Y., Yamada T., Uesato S., Maitani Y., Nagaoka Y., Eur.
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Acknowledgements This project was supported in part by a Grant-in-
Aid for Scientific Research (C) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, KAKENHI (21590131), and by
the Open Research Center Project.
16) Xie H., Braha O., Gu L. Q., Cheley S., Bayley H., Chem. Biol., 12,
109—120 (2005).
17) Gediya L. K., Chopra P., Purushottamachar P., Maheshwari N., Njar V.
C., J
. Med. Chem., 48, 5047—5051 (2005).
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1039—1046 (2010).
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