Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE Advance Publication DOI:10.1248/cpb.c17-00824 December 22, 2017
Chemical and Pharmaceutical Bulletin
Regular Article
Supramolecular Complex of Methyl-β-cyclodextrin with Adamantane- grafted Hyaluronic Acid as a Novel Antitumor Agent
Khaled M. Elamina, Yuki Yamashitaa, Taishi Higashia, Keiichi Motoyamaa,
Hidetoshi Arimaa,b,*
a Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences,
Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
b Program for Leading Graduate Schools “HIGO (Health life science: Interdisciplinary and
Glocal Oriented) Program”, Kumamoto University
*Corresponding author: Hidetoshi Arima
E-mail address: [email protected]
Ⓒ 2017 The Pharmaceutical Society of Japan
ABSTRACT
Methyl-β-cyclodextrin (M-β-CyD) exhibits cytotoxic activity, and has the potentials as an
antitumor agent. However, a tumor-selectivity of M-β-CyD is low, leading to low antitumor
activity and the adverse effects. Meanwhile, hyaluronic acid (HA) is known as a promising tumor targeting ligand, because various cancer cells overexpress CD44, a HA-binding glycoprotein. In the present study, to develop a tumor-selective delivery system for M-β-
CyD, we designed a supramolecular complex of M-β-CyD with adamantane-grafted HA (Ad-
HA/M-β-CyD) and evaluated it as a tumor-selective antitumor agent. M-β-CyD formed a
4 -1 stable complex with Ad-HA (Kc > 10 M ). In addition, Ad-HA/M-β-CyD formed slightly a
negative-charged nanoparticle with ca. 140 nm of a particle size, indicating the favorable physicochemical properties for antitumor agents. Ad-HA/M-β-CyD showed the superior
cytotoxic activity via CD44-mediated endosomal pathways in HCT116 cells (CD44 (+)), a
human colon cancer cell line. In addition, cytotoxic activity of Ad-HA/M-β-CyD was induced by apoptosis. These results suggest that Ad-HA/M-β-CyD has the potentials as a
tumor-selective supramolecular antitumor agent.
Keywords:
Methyl-β-cyclodextrin; Hyaluronic acid; Adamantine; Supramolecular complex; Antitumor
agent
Chemical and Pharmaceutical Bulletin Advance Publication
Introduction
In recent years, various antitumor agents, such as small molecules, proteins, antibodies, genes
and small interfering RNA (siRNA), have been developed.1, 2) In addition, drug delivery
systems (DDS), such as an active targeting based on ligands modification and passive
targeting based on the enhanced permeability and retention (EPR) effect, are also utilized to
improve the antitumor effects and to reduce adverse effects of antitumor agents.3, 4) However,
in spite of evolution of cancer chemotherapy, low therapeutic effects, serious side effects,
drug resistance, and high cost are important issues in the clinical field.5) Therefore,
development of new kinds of antitumor agents is strongly required.
Recently, cyclodextrins (CyDs), typical pharmaceutical excipients, have attracted
considerable attention because they show therapeutic effects by themselves against Niemann-
Pick disease type C (NPC),6) familial amyloid polyneuropathy (FAP),7) Alzheimer's disease,8) atherosclerosis,9) septic shock,10, 11) and so on. Notably, CyDs are also acknowledged to
show antitumor activity. For instance, Grosse et al. reported that methyl-β-CyD (M-β-CyD)
shows antitumor activity in human tumor xenografted athymic nude mice after intraperitoneal
administration.12) In addition, we previously reported that M-β-CyD induces apoptosis in KB
cells, a human oral squamous carcinoma cell line, Ihara cells, a highly pigmented human
melanoma cell line, and M213 cells, a human cholangiocarcinoma cell line, through
cholesterol depletion in plasma membranes, and drastically inhibits the tumor growth after
intratumoral administration to Colon-26 cells-bearing mice.13) We also demonstrated that 2-
hydroxypropyl-β-CyD (HP-β-CyD) inhibits leukemic cell proliferation at physiologically
available doses without significant adverse effects.14) More recently, we prepared tumor
targeting ligand-appended M-β-CyD, namely folate-appended M-β-CyD (FA-M-β-CyD), to
achieve tumor-selective antitumor activity.15) FA-M-β-CyD induced strong antitumor
Chemical and Pharmaceutical Bulletin Advance Publication
activity beyond M-β-CyD and doxorubicin without significant adverse effects.16)
Importantly, FA-M-β-CyD was selectively recognized by folate receptor-α overexpressing cancer cells, and induced mitophagy-mediated cell death, not apoptosis.17)
Thus, FA-M-β-CyD has the great potentials as novel antitumor agents. However, few reports are available on tumor targeting ligand-appended M-β-CyD except for FA-M-β-
CyD.
Hyaluronic acid (HA) is one of the promising tumor targeting ligands, because it is recognized by CD44, a cell surface HA-binding glycoprotein that is overexpressed on various cancer cells, such as pancreatic cancer cells, lung cancer cells, breast cancer cells, and tumor initiating cancer stem like cells. 18, 19) HA shows high binding affinity with
-12 CD44 (Kd ≈ 10 M) and high safety, thus various HA-based drug carriers for antitumor
agents have been developed.20) The combination systems of HA and CyDs are also
developed as advanced drug carriers for antitumor agents. For instance, Yang et al. developed a supramolecular nanoparticle consisting of adamantane (Ad)-grafted HA (Ad-
HA) and camptothecin/β-CyD conjugate.21) Moreover, Badwaik et al. reported a plasmid
DNA carrier consisting of a supramolecular complex of Ad-HA with cationic β-CyD.22)
Yin et al. prepared HA conjugate with oligoethylenimine-grafted β-CyD as a tumor- selective gene carrier.23) In these cases, CyDs are used as the building block of drug carriers; however, little is known about the use of HA for delivery of CyDs to tumor cells, namely the use of combination systems of HA and CyDs as antitumor agents.
Based on these backgrounds, we herein prepared a supramolecular complex of M-β-
CyD with Ad-HA (Ad-HA/M-β-CyD), and evaluated its in vitro antitumor activity.
Firstly, Ad-HA was synthesized, and then Ad-HA/M-β-CyD was prepared by mixing the both components. Next, physicochemical properties of Ad-HA/M-β-CyD were examined
Chemical and Pharmaceutical Bulletin Advance Publication
by 1H-NMR and dynamic light scattering. In addition, cytotoxic activity, cellular association, and mechanism for cytotoxic activity of Ad-HA/M-β-CyD were also examined in HCT116 cells (CD44 (+)), a human colon cancer cell line, and NIH3T3 cells (CD44 (-)), a mouse fibroblast cell line.
Experimental
Materials
HA (M.W. 50 kDa) was supplied by Kewpie Corporation (Tokyo, Japan). 1-Adamantane methyl amine was purchased from Wako Pure Chemical Industries (Osaka, Japan). 1-Ethyl-
3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole
(HoBt), and Annexin V-FITC Apoptosis Detection Kit were purchased from Nacalai Tesque
(Kyoto, Japan). M-β-CyD (degree of substitution (DS) of methyl group of 12.2) was obtained from Tokyo Kasei (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Nissui Pharmaceuticals (Tokyo, Japan) and Nichirei (Tokyo, Japan), respectively. CD44 siRNA was purchased from Hokkaido
System Science, Co., Ltd. (Hokkaido, Japan). LipofectamineTM2000, cytochrome c monoclonal antibody-FITC conjugate, and 5-(4,6-dichlorotriazinyl)aminofluorescein (5-
DTAF) were obtained from Thermo Fisher Scientific Inc (Tokyo, Japan). Sephadex® G-15 was obtained from GE Healthcare UK Ltd. (Buckinghamshire, UK). All other chemicals and solvents were of analytical reagent grade, and deionized double-distilled water was used throughout the study.
Synthesis of Ad-HA
HA (200 mg) was dissolved in 50 mL of water by agitation for 3 h. Then, EDC (122 mg),
HOBt (4 mg) and 1-adamantne methyl amine (18 mg) were dissolved in 50 mL of DMSO,
Chemical and Pharmaceutical Bulletin Advance Publication
and mixed with the HA solution. After agitation for 24 h at 45°C, 500 mL of acetone was
added to yield the white precipitates, and the precipitates were collected by centrifugation
(12,000 rpm, 10 min). After washing 3 times with n-hexane (50 mL), the product was dried overnight under the reduced pressure. The resulting Ad-HA was dissolved in deuterium
1 oxide (D2O), and characterized by H-NMR (JEOL JNM-R 500 instrument, Tokyo, Japan),
operating at 500 MHz for protons at 25°C.
Stability Constant
The stability constant (Kc) of Ad-HA/M-β-CyD was determined by the analysis of peak
1 shifting in H-NMR at different concentration of M-β-CyD (0-5 mM) in D2O. The Kc value
was obtained from the following Benesi-Hildebrand Equation, assuming the 1:1 guest/host
interaction (Ad moiety of Ad-HA/M-β-CyD).