RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Synthesis, Cytotoxicity, and Phase-Solubility Study of Cyclodextrin Click Clusters HOA THI LE,1 HYUN MI JEON,1 CHOON WOO LIM,2 TAE WOO KIM1 1 2
Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea Department of Chemistry, College of Life Science and Nano-Technology, Hannam University, Daejeon 305-811, Republic of Korea
Received 24 February 2014; revised 28 June 2014; accepted 8 July 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24107 ABSTRACT: To explore the possibility of cyclodextrin click clusters (CCCs) as a new cyclodextrin-based excipient, we prepared three different CCCs; heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}--cyclodextrin (HT--CD), heptakis{6-(4-hydroxymethyl1H-[1,2,3]triazol-1-yl)-6-deoxy}{2,3-di-O-methyl}--cyclodextrin (HT--CD(OMe)2 ), and heptakis{6-(4-sulfonylmethyl-1H-[1,2,3]triazol1-yl)-6-deoxy}--cyclodextrin (ST--CD). The CCCs were prepared using copper(I)-catalyzed azide-alkyne cycloaddition from 6-azido6-deoxy--CD and their water solubility, cytotoxicity, and drug-solubilizing effect were investigated. Water turbidity testing of the CCCs showed that the minimum water solubility of the CCCs is at least 20 times higher than that of -CD. An MTT cell viability assay performed on HeLa cells demonstrated a low cytotoxicity of the CCCs compared with 2,6-dimethyl--cyclodextrin. HT--CD(OMe)2 and ST--CD did not demonstrate any cytotoxicity within the experimental concentration (∼5 mM) like 2-hydroxypropyl--CD. A phase-solubility study of prednisolone with the CCCs suggested that CCCs showed increased solubility of prednisolone in the presence of increasing concentrations of the CCCs. The comparison between the conventional CD derivatives and CCCs on solubility, cytotoxicity, and binding property implies C 2014 that CCCs are alternative cyclodextrin derivatives useful for overcoming the restrictions of conventional cyclodextrin chemistry. Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: cyclodextrins; complexation; excipients; solubility; encapsulation; copper(I)-catalyzed azide-alkyne cycloaddition; cyclodextrin click cluster; cytotoxicity
INTRODUCTION Cyclodextrin click clusters (CCCs) are a kind of chemically modified cyclodextrin (CD) derivative, in which all the primary alcohols of CDs are transformed to triazole units by copper(I)catalyzed azide-alkyne cycloaddition (CuAAc) (Scheme 1a). The second face of CDs can be also modified through CuAAc, but the cases of secondary face modification are relatively rare.1 The CuAAC reaction is modular, specific, wide in scope, and provides high yields. The two reaction counterparts, azide and alkyne, are inert to most biological and organic conditions, including highly functionalized biological molecules, water, and the majority of common reaction conditions in organic synthesis.2 Thus, the CuAAC reaction was adapted by many fields of organic chemistry.3 In particular, the CuAAC reaction was quickly introduced to the chemistry of CDs, and since 2007, the amount of work published dealing with CDs and CuAAC has increased greatly.4 In addition to the synthetic advantages, CCCs have unique, structural characteristics including well-defined structures, precise molecular weights, and multivalent functionalization sites. CCCs have been extensively exploited in polymers,5,6 nucleic acid carriers,7–9 sensors,10 glycoconjugates,11–13 multivalent scaffolds,14–16 and magnetic resonance imaging probes.17,18
Correspondence to: Choon W. Lim (Telephone: 82-42-6298812; Fax: 8242-6298811; E-mail: [email protected]); Tae W. Kim (Telephone: +82-31-2012189; Fax: +82-31-6229639; E-mail: tw1275@ khu.ac.kr) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
Several CDs are used industrially in pharmaceutical and allied applications.19 The ability to utilize the hydrophobic cavity of CD to encapsulate bioactive molecules in water has drawn tremendous interest from the pharmaceutical industry because encapsulation improves the stability and bioavailability of cargo compounds.20 Natural "-, $-, (-CD, which consist of six, seven, or eight glucose units, were widely used, but the chemically modified CD derivatives, like hydroxypropyl-$-CD, hydroxypropyl-(-CD, sulfobutyl ether-$-CD, methyl $-CD (Scheme 1b) were already marketed as excipients in the pharmaceutical formulation.21 Chemically modified CD derivatives have merits beyond natural CDs in some applications. For example, sulfobutyl ether-$CD can solve the nephrotoxicity of $-CD observed in parenteral injection. $-CD itself is the most commonly used CD, although it is the least soluble (solubility in water at 20◦ C; 1.85 g/100 mL). When parenterally administered, $-CD is not metabolized but accumulates in the kidneys as insoluble cholesterol complexes, resulting in severe nephrotoxicity.22 Sulfobutyl ether-$-CD has been developed as a means for solving the solubility problem observed with parenteral $-CD administration.23 However, the conventional modification methods of CDs have a few drawbacks. The common products of conventional synthesis are not homogeneous; they are mixtures of compounds with different degrees of substitution.24 This heterogeneity of modified $-CD derivatives makes it difficult for researchers to precisely investigate the inclusion phenomena at a molecular level. Usually, the reagents and conditions of conventional CD modification are limited to either propylene oxide ring opening for 2-hydroxypropyl CD or 1,4-butane sultone ring opening for 4-sulfobutyl ether CD. The reactions offer low efficiency and
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on a Bruker Advance III 600 NMR spectrometer (Seoul, South Korea) equipped with a PABBO BB-1H Z GRD probe head. The other NMR spectra were measured on a JNM-AL300 (JEOL) spectrometer (Seoul, South Korea). Chemical shifts were reported as * in units of parts per million (ppm), and J-values were noted in Hz. MALDI–MS (matrix-assisted laser desorption/ionization mass spectrometry) spectra were measured on a Voyager-DETM STR Biospectrometry Workstation (Applied Biosystems supplied in Seoul, South Korea). A CEM DiscoverTM microwave reactor was applied for the microwave-assisted organic synthesis. Heptakis-(6-chloro-6-deoxy)-$-CD (Cl-$-CD), heptakis(6-azido-6-deoxy)-$-CD (azido-$-CD), heptakis{6-(4hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-$-CD (HT -$-CD), heptakis{6-(4-sulfonylmethyl-1H-[1,2,3]triazol-1yl)-6-deoxy}-$-CD (ST-$-CD), heptakis(2,3-di-O-methyl)tris(3(6-azido-6-deoxy)-$-CD (azido-$-CD(OMe)2 ), hydroxypropyltriazolylmethyl)amine (THPTA), and sodium propargyl sulfonate were prepared via literature procedures, and the 1 H and 13 C NMR spectra of the compounds were consistent with the literatures (Supporting Information S1). The synthesis and NMR characterization of heptakis{6(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}{2,3-di-Omethyl}-$-CD (HT-$-CD(OMe)2 ) were reported in Supporting Information S1 and S2. Turbidity Measurement To each CD, 150 :L of deionized distilled water was added and the mixtures were sonicated (Hwa Shin Power Sonic 410) for 30 min. The mixture was vigorously vortexed and 120 :L of the mixture was quickly transferred into a 96-microwell plate. The visible absorption at 595 nm was measured by microplate reader (PerkinElmer VICTORTM 3, photometry mode, supplied in Seoul, South Korea). R
Scheme 1. (a) General molecular structure of cyclodextrin click clusters. (b) Molecular structures of $-CD and two reference cyclodextrin derivatives.
MTT Assay are difficult to precisely control. In addition, they would not be suitable for the incorporation of further functional groups on CDs. The bioorthogonality of the CuAAC reaction gives a distinct advantage over traditional CD modification to CCCs.25 In order to apply CCCs to the field of drug, food, and cosmetic formulation, three important considerations should be taken into account; CCC synthesis, cellular safety, and cargo encapsulation ability. Unfortunately, CCC studies in the pharmaceutical field are rare. To answer these questions, we prepared three CCC derivatives and investigated water solubility, cytotoxicity, and the drug inclusion properties of each derivative, which were compared with the properties of $-CD and two conventional CD derivatives.
EXPERIMENTAL
HeLa (human cervical adenocarcinoma cell line) cells were seeded in 96-well culture plate at 2 × 104 cells/well and incubated in DMEM (Gibco/BRL, Dulbecco’s Modified Eagle’s Medium) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotics (Gibco/BRL) for 24 h. The stock solutions (10 mM) of CDs were prepared in DMEM medium. HeLa cells on a 96-well plate were treated with a series of CD solutions (0, 0.01, 0.1, 1, 2, and 5 mM) and the plate was incubated in a CO2 incubator at 37◦ C for 1 day. To each well, 100 :L of MTT was added [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL Stock in PBS, Sigma M5655]. After a 2-h incubation at 37◦ C, the medium was removed. To dissolve the formazan crystals, 100 :L of dimethyl sulfoxide (Sigma–Aldrich 472301) was added and mixed well via pipette. Then, the absorbance values at 570 nm were measured by microplate reader (PerkinElmer VICTORTM 3, photometry mode).
Materials $-Cyclodextrin and prednisolone were purchased from TCI PN C0777and P0637 (Seoul, South Korea). 2-Hydroxypropyl$-CD (HP-$-CD) and heptakis(2,6-di-O-methyl)-$-CD (DM-$CD) were purchased from Aldrich PN 389145, H0513 (Yongin, South Korea). All purchased reagents were used without purifications. 1 H, 13 C NMR (nuclear magnetic resonance), HH COSY (homonuclear correlation spectroscopy), and CH HSQC (heteronuclear single quantum coherence) spectra were measured Le et al., JOURNAL OF PHARMACEUTICAL SCIENCES
Phase Solubility Studies HPLC was performed on an Agilent 1100 series liquid chromatography system with a diode array detector interfaced with an Agilent Chem Station for data analysis. The column was a 4.6 mm ID × 150 mm Extend-C18 column (5 :m pore size; Hewlett Packard, CA, Seoul, South Korea) with a Security GuardTM guard cartridge (3.0 × 4.0 mm, Phenomenex, CA, Seoul, South Korea). The mobile phase consisted of DOI 10.1002/jps.24107
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Scheme 2. Reactions and reagents. (a) Methanesulfonyl chloride, DMF, 65◦ C; (b) NaN3 , DMSO, MW; (c) propargyl alcohol, CuSO4 /THPTA, sodium ascorbate, THF/phosphate buffer, MW; (d) sodium propargylsulfonate; (e) MeI, NaH, DMF; (f) propargyl alcohol, CuSO4 /THPTA, sodium ascorbate, THF/phosphate buffer/ EtOH, 80◦ C, 20 h. THPTA = tris(3-hydroxypropyltriazolylmethyl)amine, MW = microwave irradiation.
acetonitrile/water (30/70, v/v). Excess prednisolone was added to aqueous 0–0.1 M CD solutions (0–0.014 M in the case of $-CD). The suspensions were equilibrated in an Eppendorf Thermomixer Comfort (25◦ C, 1000 rpm) for 48 h. The prepared samples were filtered through a 0.2 :m RC Phenomenex membrane, and the filtrates were centrifuged at 10,000g (5 min; Hanil Science Industrial Company Micro 17TR). After proper dilution with HPLC mobile phase solution (acetonitrile/water, 30/70, v/v), the amount of prednisolone in the supernatant of samples was quantified by absorbance at 254 nm. R
R
RESULTS AND DISCUSSION
efficiency in aqueous medium. THPTA developed by the Finn group has proven to be invaluable for bioconjugation in water.28 Complete triazole formation of HT-$-CD(OMe)2 was evident by the presence of axial symmetry in the NMR spectra. Only a single doublet for all H1 protons appeared in the 1 H NMR spectra [* = 5.40 ppm, JH1-H2 = 3.4 Hz for HT-$-CD(OMe)2 , vs. * = 5.12 ppm, JH1-H2 = 3.2 Hz for HT-$-CD and * = 5.15 ppm, JH1-H2 = 3.4 Hz for ST-$-CD], and the corresponding C1 atoms also appeared as one singlet [* = 97.2 ppm for HT-$-CD(OMe)2 vs. * = 103.2 ppm for HT-$-CD and * = 102.1 ppm for ST-$CD] in the H1 decoupled 13 C NMR spectrum. All protons and carbons of HT-$-CD(OMe)2 were fully assigned by HH COSY and CH HSQC (Supporting Information S2).
Synthesis of 6-Triazolyl-6-Deoxy--CD Derivatives
Water Solubility of CCCs
The core starting material, 6-azido-6-deoxy-$-CD was prepared from $-CD by a two-step reaction: (1) chlorination of the primary alcohol and (2) azide substitution at a gram scale without column chromatography. Three different CCCs were designed and synthesized through CuAAC (Scheme 2). HT-$-CD and ST-$-CD were prepared by following our group’s report.26 In order to control the hydrophobicity of the CD cavity, the secondary alcohols of 6-azido-6-deoxy-$-CD were fully methylated by Williamson ether synthesis. The resulting Azido-$CD(OMe)2 was further click-functionalized with propargyl alcohol. DiaionTM HP-20 column chromatography afforded the desired product HT-$-CD(OMe)2 at a good yield (74%). Copper(I)-catalyzed azide-alkyne cycloaddition reactions are reliable and efficient, but copper ligands are often employed both to enhance the rate of reaction and to protect Cu(I) from oxidation in the presence of adventurous oxygen.27 Thus, we also used a water-soluble ligand, THPTA to maximize our CuAAC
The water solubility of CDs was compared by turbidity measurement.29 As compared with the relatively low water solubility of $-CD, the solutions containing HT-$-CD, HT-$CD(OMe)2 , and ST-$-CD looked clean at the full experimental concentration (Fig. 1 and Supporting Information S3). These results suggest that the minimum water solubility of HT-$CD/HT-$-CD(OMe)2 /ST-$-CD is higher than 50 mg/150 :L; ca. 20 times higher than that of $-CD (solubility in water at 20◦ C, 1.85 mg/100 :L). Intramolecular hydrogen bonding of $CD can result in relatively unfavorable enthalpies of solution and low aqueous solubility. Substitution of any of the hydrogen bond forming hydroxyl groups, even by hydrophobic moieties such as methoxy groups, will result in a dramatic increase in water solubility (the water-solubility of DM-$-CD at 20◦ C, 60 mg/100 :L).30 Actually, HT-$-CD, HT-$-CD(OMe)2 , and ST$-CD are freely miscible in excess of 100 mg/100 :L and result in viscous media, which is approximately a 50-fold
DOI 10.1002/jps.24107
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Table 1. Binding Constants for Inclusion Complexation of Prednisolone with Various $-CDs
Figure 1. The turbidity changes of $-CD, two $-CD derivatives (HP$-CD, DM-$-CD), and three CCCs (HT-$-CD, HT-$-CD(OMe)2, ST$-CD). Absorption at 595 nm was measured by microplate reader (PerkinElmer VICTORTM 3, photometry mode).
Prednisolone Molar Intrinsic Solubility = 2.8 × 10−4 mol/L (Sparingly Soluble)a log P = 1.635 ± 0.526a Solubility (mg/mL) in 50 mM CD Literatureb
Cyclodextrin
K1:1 (M−1 ) ± SD
Type
$-CD HP-$-CD DM-$-CD HT-$-CD HT-$CD(OMe)2 d ST-$-CD
2744 ± 39 1907 ± 39 2279 ± 158 2253 ± 14 1243 ± 42
AL AL AL AL AL
NDc 5.1 11.6 7.2 6.3
3000, 3479 1319, 2428 No report – –
704 ± 17
AL
4.2
–
The binding constant, K1:1 , was averaged from three independent measurements. a Molecular structure of prednisolone, and its water solubility and partition coefficient (log P = log ([Solute]octanol /[Solute]water ). The molar intrinsic solubility and log P of prednisolone were calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02. b Cited from Ref. 34 and the references in there. c The prednisolone solubility in 50 mM of $-CD is not determined because of the low solubility of $-CD (solubility = 1.85 mg/100 :L; saturation concentration = 16 mM). d The K1:1 was determined using 2% ethanolic water (98:2, water–ethanol, v/v).
Figure 2. MTT of CD derivatives on HeLa cell viability. After 24 h of CD treatment, the visible absorption of formazan crystals was measured at 570 nm and the cell viability was normalized by the control value ([CD] = 0). Results represent the mean of five independent experiments.
improvement compared with $-CD’s solubility (Supporting Information Fig. S3). From the turbidity testing of various CD derivatives, we suggest that the triazolyl modification of CCCs dramatically improves water solubility. Cytotoxicity of CCCs The MTT assay provides a read-out for cytotoxicity.31 The cytotoxicity of new chemically modified $-CD derivatives is critical for future drug delivery and food/cosmetic formulation applications. We measured the effect of CCCs on cell viability using the MTT assay, performed on HeLa cells (Fig. 2). As reference CDs, we selected 2-hydroxypropyl-$-CD (HP-$-CD, average Mw ∼ 1540) and heptakis(2,6-di-O-methyl)-$-CD (DM$-CD). HP-$-CD is commercially available and considered a safe CD derivative (an active deodorant ingredient of Febreze ; Procter & Gamble). Salem et al.32 also reported on the cytotoxicity of methylated-$-CD derivatives. As expected, HP-$-CD showed no cytotoxicity over the entire range of experimental concentrations and DM-$-CD reduced cellular viability dose-dependently over 0.1 mM. When compared with the cellular viability of HP-$-CD and DM-$CD, HT-$-CD(OMe)2 and ST-$-CD did not adversely influence HeLa cell viability at experimental concentrations (∼5 mM). However, HT-$-CD showed a slight dose-independent reducR
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tion in cellular viability (∼80%). It is remarkable that the two methylated CDs, DM-$-CD and HT-$-CD(OMe)2 , showed very different cytotoxicity profiles. DM-$-CD showed toxicity at concentrations over 0.1 mM, but HT-$-CD(OMe)2 is not toxic until around 5 mM. This result implies that the CCC approach is an alternative solution to reduce cytotoxicity of conventional DM-$-CD. Prednisolone Phase Solubility Studies with CCCs Phase solubility analysis provides valuable data on the effect of various CDs on the solubility of drugs with poor or limited aqueous solubility.33 A phase-solubility study on CCC will provides evidence for application of CCC as a drug solubilizer. Prednisolone (a poorly water-soluble glucocorticoid) was selected as a model compound for the phase-solubility study (Table 1).34 CDs, especially $- and (-CD, have been shown to form complexes with many steroids, leading to improved solubility and dissolution rates compared with the pure steroids.35 A phase-solubility profile of prednisolone with various CDs at 25◦ C was obtained according to the methods of Hirayama and Uekama.36 Establishment of equilibrium was verified by the comparison of HP-$-CD phase solubility after 24, 48, and 72 h. The results showed no significant differences, and the equilibration time was set to 48 h (Supporting Information S4). A linear standard curve was obtained in the concentration range from 2.4 × 10−4 to 1.5 × 10−2 M for prednisolone. The standard curve showed good linearity in the concentration range (Supporting Information S5). Phase solubility diagrams show that all CDs exhibited AL type diagrams in the chosen concentration range (Fig. 3). AL type phase-solubility profile is obtained when the solubility of prednisolone increases with CD concentration. In general, DOI 10.1002/jps.24107
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 3. Phase solubility diagrams of prednisolone–CD systems in a series from the first independent set (25◦ C in water). Upper box: expansion of the low concentration region for clarity of the $-CD phase solubility diagram.
water-soluble CD derivatives form A-type phase solubility profiles. In the AL case and assuming that the complex exhibits 1:1 binding, the stability constant of the complex can be calculated from the slope of the isotherm: K 1:1 =
Slope S0 (1 − slope)
Three independent measurements were performed for each CD case (Fig. 4 and Supporting Information S6). The intrinsic solubility in water of prednisolone (S0 ) was determined from the intercept. The binding constant of the prednisolone complex with CD derivatives is summarized in Table 1. Our phase-solubility study demonstrates the effect of CCCs on CD-drug encapsulation ability. The binding constants of $CD derivative decrease in the order of $-CD (2744) > DM-$-
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CD (2279) ≈ HT-$-CD (2253) > HP-$-CD (1907) >> ST-$-CD (704). The solubility of the prednisolone complex with native $-CD is relatively limited because of the limited solubility of native $-CD. In contrast, a large linear increase in prednisolone solubility was observed with HT-$-CD. However, the binding constant of HT-$-CD-(OMe)2 cannot be directly compared with those of the other CD derivatives because of the difference in the binding solvent. Interestingly, we observed that the phase solubility diagram of HT-$-CD(OMe)2 in pure water was abnormal [regression coefficients (R2 ) = 0.95–0.97, high SD; Supporting Information S7]. This phenomenon may be due to the low water solubility of the HT$-CD(OMe)2 –prednisolone complex. Thus, 2% ethanolic water (98:2, water:ethanol, v/v) was used for the phase solubility test of HT-$-CD(OMe)2 instead of pure water. The binding constant of CD-guest complexation mainly depends on the hydrophobicity of the inner cavity and the solvophobicity. Increasing the number of methyl groups in the $-CD molecule will increase the hydrophobicity of the inner cavity. Kiss et al.37 reported that the cholesterol-solubilizing effect of various methylated-CD derivatives was proportional to the methyl group number of CD at a certain range (number of methyl group, 0–15). However, the binding constant is not a simple function of the methyl group number of CD. Wenz38 reported that stabilizing intramolecular hydrogen bonds between the glucose units were made responsible for the high binding potentials of those $-CD derivatives that possess secondary hydroxy groups. Schneider et al.39 reported that all log Ka values with "-CD and p-nitrphenolate correlate linearly with solvophobicity parameters of the corresponding medium for a series of aqueous methanol mixtures; adding methanol to water reduces the binding constant of the CD-guest complexation. The lower Ka of HT-$-CD(OMe)2 (1243) than that of HT$-CD (2253) can be explained by the above two parameters; destabilization of intramolecular hydrogen bond and solvophobicity.
Figure 4. An example of a phase-solubility study of the prednisolone–ST-$-CD system. The binding constant, K1:1 was averaged from three independent measurements at 25◦ C after 24 h equilibration. The error bars in the charts resulted from three repeated injections per an independent sample. S0 (intrinsic solubility) was experimentally determined by the intercept. DOI 10.1002/jps.24107
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The introduction of an anion charge to CDs is an important strategy to solve the parenteral delivery problem of native $CD. Stella and coworkers40,41 reported the drug inclusion ability of sulfoalkyl ether CD derivatives. For instance, sulfobutyl ether-$-CD (SBE-$-CD), sold under the name CAPTISOL (degree of substitution ∼7), has been components of six United States Food and Drug Administration-approved parenteral products and is a component of a number of other parenteral products in late stage development.23 As a CCC analogue of SBE-$-CD, ST-$-CD, a sulfonated CCC was tested. The phase solubility diagram showed that ST-$-CD has a lower binding constant than HT-$-CD and HT-$-CD(OMe)2 . The aqueous solvation shell associated with sulfonate substituents may diminish the size of the cavity opening, resulting in a reduced accessibility of guest molecules.42 R
CONCLUSIONS In order to maximize the synthetic advantages of CCCs beyond the conventional CD modification chemistry and to confirm CCC application, three CCCs were prepared through CuAAc. HT-$-CD, HT-$-CD(OMe)2 , and ST-$-CD are freely miscible in an excess of 100 mg/100 :L water. This result suggested that the triazolyl modification of CCCs dramatically improves water solubility. MTT cell viability assay on HeLa cells demonstrated the low cytotoxicity of the CCCs. HT-$-CD(OMe)2 and ST-$-CD do not have any cytotoxicity within the experimental concentration (∼5 mM) but DM-$-CD was cytotoxic over 0.1 mM. This result implies that a CCC approach will be an alternative solution to reduce cytotoxicity of conventional DM$-CD. The binding constants of $-CD derivatives against prednisolone decrease in the order of $-CD > DM-$-CD ≈ HT-$-CD > HP-$-CD >> ST-$-CD in pure water, and the binding constant of HT-$-CD(OMe)2 is 1243 in 2% ethanolic water. Our experiments are the first systematic trial to explore the possibility of CCCs as a new CD-based excipient. The comparison between the conventional CD derivatives and the CCCs on solubility, cytotoxicity, and binding properties implies that CCCs are alternative CD derivatives useful for overcoming the restriction of conventional CD chemistry.
ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2065643). This research was Research Foundation of Korea (supported by the Basic Science Research Program through the National NRF) funded by the Ministry of Education, Science and Technology (2011–0024970).
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4. Faugeras PA, Bo¨ens B, Elchinger PH, Brouillette F, Montplaisir D, Zerrouki R, Lucas R. 2012. When cyclodextrins meet click chemistry. Eur J Org Chem 4087–4105. 5. Hoogenboom R, Moore BC, Schubert US. 2006. Synthesis of starshaped poly(epsilon-caprolactone) via’click’ chemistry and ‘supramolecular click’ chemistry. Chem Commun 14:4010–4012. 6. Xu J, Liu S. 2009. Synthesis of well-defined 7-arm and 21-arm poly(N-isopropylacrylamide) star polymers with $-cyclodextrin cores via click chemistry and their thermal phase transition behavior in aqueous solution. J Polym Sci A 47:404–419. 7. Srinivasachari S, Fichter KM, Reineke TM. 2008. Polycationic $cyclodextrin “click clusters”: Monodisperse and versatile scaffolds for nucleic acid delivery. J Am Chem Soc 130:4618–4627. 8. M´endez-Ardoy A, Guilloteau N, Di Giorgio C, Vierling P, Santoyo´ ´ Gonzalez F, Ortiz Mellet C, Garc´ıa Fernandez JM. 2011. $Cyclodextrin-based polycationic amphiphilic “click” clusters: Effect of structural modifications in their DNA complexing and delivery properties. J Org Chem 76:5882–5894. ´ Bienvenu C, Jim´enez Blanco JL, Vierling P, Mel9. Mart´ınez A, ´ let CO, Garc´ıa Fernandez JM, Di Giorgio C. 2013. Amphiphilic oligoethyleneimine-$-cyclodextrin “click” clusters for enhanced DNA delivery. J Org Chem 78:8143–8148. 10. David O, Maisonneuve S, Xie J. 2007. Generation of new fluorophore by Click chemistry: Synthesis and properties of $-cyclodextrin substituted by 2-pyridyl triazole. Tetrahedron Lett 48:6527–6530. ˜ 11. P´erez-Balderas F, Ortega-Munoz M, Morales-Sanfrutos J, ´ Hernandez-Mateo F, Calvo-Flores FG, Calvo-As´ın JA, Isac-Garc´ıa J, ´ Santoyo-Gonzalez F. 2003. Multivalent neoglycoconjugates by regiospecific cycloaddition of alkynes and azides using organic-soluble copper catalysts. Org Lett 5:1951–1954. ˜ 12. Ortega-Munoz M, Morales-Sanfrutos J, Perez-Balderas F, Hernandez-Mateo F, Giron-Gonzalez MD, Sevillano-Tripero N, SaltoGonzalez R, Santoyo-Gonzalez F. 2007. Click multivalent neoglycoconjugates as synthetic activators in cell adhesion and stimulation of monocyte/machrophage cell lines. Org Biomol Chem 5:2291–2301. ´ T, Ortiz Mellet 13. Decroocq C, Joosten A, Sergent R, Mena Barragan C, Compain P. 2013. The multivalent effect in glycosidase inhibition: Probing the influence of valency, peripheral ligand structure, and topology with cyclodextrin-based iminosugar click clusters. Chembiochem 14:2038–2049. 14. Kim HY, Sohn J, Wijewickrama GT, Edirisinghe P, Gherezghiher T, Hemachandra M, Lu PY, Chandrasena RE, Molloy ME, Tonetti DA, Thatcher GR. 2010. Click synthesis of estradiol-cyclodextrin conjugates as cell compartment selective estrogens. Bioorg Med Chem 18:809– 821. 15. D´ıaz-Moscoso A, M´endez-Ardoy A, Ortega-Caballero F, Benito JM, Ortiz Mellet C, Defaye J, Robinson TM, Yohannes A, Karginov VA, ´ Garc´ıa Fernandez JM. 2011. Symmetry complementarity-guided design of anthrax toxin inhibitors based on $-cyclodextrin: Synthesis and relative activities of face-selective functionalized polycationic clusters. ChemMedChem 6:181–192. 16. Joshi A, Kate S, Poon V, Mondal D, Boggara MB, Saraph A, Martin JT, McAlpine R, Day R, Garcia AE, Mogridge J, Kane RS. 2011. Structure-based design of a heptavalent anthrax toxin inhibitor. Biomacromolecules 12:791–796. 17. Bryson JM, Chu WJ, Lee JH, Reineke TM. 2008. A $-cyclodextrin “click cluster” decorated with seven paramagnetic chelates containing two water exchange sites. Bioconjug Chem 19:1505–1509. 18. Song Y, Kohlmeir EK, Meade TJ. 2008. Synthesis of multimeric MR contrast agents for cellular imaging. J Am Chem Soc 130:6662– 6663. 19. Davis ME, Brewster ME. 2004. Cyclodextrin-based pharmaceutics: Past, present and future. Nat Rev Drug Discov 3:1023–1035. 20. Laza-Knoerr AL, Gref R, Couvreur P. 2010. Cyclodextrins for drug delivery. J Drug Target 18:645–656. 21. Bilensoy E, editor. 2011. Cyclodextrin in pharmaceutics, cosmetics, and biomedicine; current and future industrial applications. Hoboken, New Jersey: John Wiley & Sons Inc., pp 91–122. DOI 10.1002/jps.24107
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22. Kibbe AH, editor. 2000. Handbook of pharmaceutical excipients, 3rd ed. Washington, DC: American Pharmaceutical Association and Pharmaceutical Press, pp 165–168. 23. Antle VD, Pipkin JD. 2012. Solubility-considering modified cyclodextrins as a tool for solving parenteral solubility challenges. Pharmaceutical Formulation & Quality. 24. For instance, the average degree of substitution of HP-$-CD used in this experiment (Aldrich, PN 389145) is 0.6–0.8 units of 2hydroxypropyl per glucose unit. 25. Sletten EM, Bertozzi CR. 2011. From mechanism to mouse: A tale of two bioorthogonal reactions. Acc Chem Res 44:666–676. 26. Le HT, Jeon HM, Lim CW, Kim TW. 2014. 6-Triazolyl-6-deoxy$-cyclodextrin derivatives: Synthesis, cellular toxicity, and phasesolubility study. Carbohydr Res 391:22–28. 27. Presolski SI, Hong V, Cho SH, Finn MG. 2010. Tailored ligand acceleration of the Cu-catalyzed azide-alkyne cycloaddition reaction: Practical and mechanistic implications. J Am Chem Soc 132:14570– 14576. 28. Hong V, Presolski SI, Ma C, Finn MG. 2009. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew Chem Int Ed Engl 48:9879–9883. 29. Harada A, Kamachi M. 1990. Complex formation between poly(ethylene glycol) and "-cyclodextrin. Macromolecules 23:2821– 2823. 30. Loftsson T, Brewster ME. 1996. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J Pharm Sci 85:1017–1025. 31. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. 32. Salem LB, Bosquillon C, Dailey LA, Delattre L, Martin GP, Evrard B, Forbes B. 2009. Sparing methylation of $-cyclodextrin mitigates cytotoxicity and permeability induction in respiratory epithelial cell layers in vitro. J Control Release 136:110–116.
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33. Chaudhary VB, Patel JK. 2013. Cyclodextrin inclusion complex to enhance solubility of poorly water soluble drugs: A review. Int J Pharm Sci Res 4:68–76. 34. Larsen KL, Aachmann FL, Wimmer R, Stella VJ, Kjolner UM. 2005. Phase solubility and structure of the inclusion complexes of prednisolone and 6 "-methyl prednisolone with various cyclodextrins. J Pharm Sci 94:507–515. 35. Mikhail VR, Yoshihisa I. 1998. Complexation thermodynamics of cyclodextrins. Chem Rev 98:1875–1918. 36. Hirayama F, Uekama K. 1987. Methods of investigating and preparing inclusion compounds. In Duchˆene D, editor. Cyclodextrins ´ and their industrial uses. Paris: Editions de Sant´e, pp 131–172. ´ ´ ´ 37. Kiss T, Fenyvesi F, Bacskay I, Varadi J, Fenyvesi E, Ivanyi R, Szente L, T´osaki A, Vecserny´es M. 2010. Evaluation of the cytotoxicity of $-cyclodextrin derivatives: Evidence for the role of cholesterol extraction. Eur J Pharm Sci 40:376–380. 38. Wenz G. 2012. Influence of intramolecular hydrogen bonds on the binding potential of methylated $ -cyclodextrin derivatives. Beilstein J Org Chem 8:1890–1895. 39. Schneider HJ, Kramer R, Simova S, Schneider U. 1988. Solvent and salt effects on binding constants of organic substrates in macrocyclic host compounds. A general equation measuring hydrophobic binding contributions. J Am Chem Soc 110:6442–6448. 40. Tongiani S, Velde DV, Ozeki T, Stella VJ. 2005. Sulfoalkyl etheralkyl ether cyclodextrin derivatives, their synthesis, NMR characterization and binding of 6 alfa-methylpredonisolone. J Pharm Sci 94:2380–2392. 41. Tongiani S, Ozeki T, Stella VJ. 2009. Sulfobutyl ether-alkyl ether mixed cyclodextrin derivatives with enhanced inclusion ability. J Pharm Sci 98:4769–4980. 42. Kim DH, Jang JG, Le HT, Kim JY, Lim CW, Kim TW. 2012. 6Hydroxymethyltriazolyl-6-deoxy-$-cyclodextrin: A highly water soluble and structurally well-defined $-cyclodextrin click cluster. Tetrahedron Lett 53:5791–5795.
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Synthesis, Cytotoxicity, and Phase-Solubility Study of Cyclodextrin Click Clusters HOA THI LE,1 HYUN MI JEON,1 CHOON WOO LIM,2 TAE WOO KIM1 1 2
Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea Department of Chemistry, College of Life Science and Nano-Technology, Hannam University, Daejeon 305-811, Republic of Korea
Received 24 February 2014; revised 28 June 2014; accepted 8 July 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24107 ABSTRACT: To explore the possibility of cyclodextrin click clusters (CCCs) as a new cyclodextrin-based excipient, we prepared three different CCCs; heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}--cyclodextrin (HT--CD), heptakis{6-(4-hydroxymethyl1H-[1,2,3]triazol-1-yl)-6-deoxy}{2,3-di-O-methyl}--cyclodextrin (HT--CD(OMe)2 ), and heptakis{6-(4-sulfonylmethyl-1H-[1,2,3]triazol1-yl)-6-deoxy}--cyclodextrin (ST--CD). The CCCs were prepared using copper(I)-catalyzed azide-alkyne cycloaddition from 6-azido6-deoxy--CD and their water solubility, cytotoxicity, and drug-solubilizing effect were investigated. Water turbidity testing of the CCCs showed that the minimum water solubility of the CCCs is at least 20 times higher than that of -CD. An MTT cell viability assay performed on HeLa cells demonstrated a low cytotoxicity of the CCCs compared with 2,6-dimethyl--cyclodextrin. HT--CD(OMe)2 and ST--CD did not demonstrate any cytotoxicity within the experimental concentration (∼5 mM) like 2-hydroxypropyl--CD. A phase-solubility study of prednisolone with the CCCs suggested that CCCs showed increased solubility of prednisolone in the presence of increasing concentrations of the CCCs. The comparison between the conventional CD derivatives and CCCs on solubility, cytotoxicity, and binding property implies C 2014 that CCCs are alternative cyclodextrin derivatives useful for overcoming the restrictions of conventional cyclodextrin chemistry. Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: cyclodextrins; complexation; excipients; solubility; encapsulation; copper(I)-catalyzed azide-alkyne cycloaddition; cyclodextrin click cluster; cytotoxicity
INTRODUCTION Cyclodextrin click clusters (CCCs) are a kind of chemically modified cyclodextrin (CD) derivative, in which all the primary alcohols of CDs are transformed to triazole units by copper(I)catalyzed azide-alkyne cycloaddition (CuAAc) (Scheme 1a). The second face of CDs can be also modified through CuAAc, but the cases of secondary face modification are relatively rare.1 The CuAAC reaction is modular, specific, wide in scope, and provides high yields. The two reaction counterparts, azide and alkyne, are inert to most biological and organic conditions, including highly functionalized biological molecules, water, and the majority of common reaction conditions in organic synthesis.2 Thus, the CuAAC reaction was adapted by many fields of organic chemistry.3 In particular, the CuAAC reaction was quickly introduced to the chemistry of CDs, and since 2007, the amount of work published dealing with CDs and CuAAC has increased greatly.4 In addition to the synthetic advantages, CCCs have unique, structural characteristics including well-defined structures, precise molecular weights, and multivalent functionalization sites. CCCs have been extensively exploited in polymers,5,6 nucleic acid carriers,7–9 sensors,10 glycoconjugates,11–13 multivalent scaffolds,14–16 and magnetic resonance imaging probes.17,18
Correspondence to: Choon W. Lim (Telephone: 82-42-6298812; Fax: 8242-6298811; E-mail: [email protected]); Tae W. Kim (Telephone: +82-31-2012189; Fax: +82-31-6229639; E-mail: tw1275@ khu.ac.kr) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
Several CDs are used industrially in pharmaceutical and allied applications.19 The ability to utilize the hydrophobic cavity of CD to encapsulate bioactive molecules in water has drawn tremendous interest from the pharmaceutical industry because encapsulation improves the stability and bioavailability of cargo compounds.20 Natural "-, $-, (-CD, which consist of six, seven, or eight glucose units, were widely used, but the chemically modified CD derivatives, like hydroxypropyl-$-CD, hydroxypropyl-(-CD, sulfobutyl ether-$-CD, methyl $-CD (Scheme 1b) were already marketed as excipients in the pharmaceutical formulation.21 Chemically modified CD derivatives have merits beyond natural CDs in some applications. For example, sulfobutyl ether-$CD can solve the nephrotoxicity of $-CD observed in parenteral injection. $-CD itself is the most commonly used CD, although it is the least soluble (solubility in water at 20◦ C; 1.85 g/100 mL). When parenterally administered, $-CD is not metabolized but accumulates in the kidneys as insoluble cholesterol complexes, resulting in severe nephrotoxicity.22 Sulfobutyl ether-$-CD has been developed as a means for solving the solubility problem observed with parenteral $-CD administration.23 However, the conventional modification methods of CDs have a few drawbacks. The common products of conventional synthesis are not homogeneous; they are mixtures of compounds with different degrees of substitution.24 This heterogeneity of modified $-CD derivatives makes it difficult for researchers to precisely investigate the inclusion phenomena at a molecular level. Usually, the reagents and conditions of conventional CD modification are limited to either propylene oxide ring opening for 2-hydroxypropyl CD or 1,4-butane sultone ring opening for 4-sulfobutyl ether CD. The reactions offer low efficiency and
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on a Bruker Advance III 600 NMR spectrometer (Seoul, South Korea) equipped with a PABBO BB-1H Z GRD probe head. The other NMR spectra were measured on a JNM-AL300 (JEOL) spectrometer (Seoul, South Korea). Chemical shifts were reported as * in units of parts per million (ppm), and J-values were noted in Hz. MALDI–MS (matrix-assisted laser desorption/ionization mass spectrometry) spectra were measured on a Voyager-DETM STR Biospectrometry Workstation (Applied Biosystems supplied in Seoul, South Korea). A CEM DiscoverTM microwave reactor was applied for the microwave-assisted organic synthesis. Heptakis-(6-chloro-6-deoxy)-$-CD (Cl-$-CD), heptakis(6-azido-6-deoxy)-$-CD (azido-$-CD), heptakis{6-(4hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-$-CD (HT -$-CD), heptakis{6-(4-sulfonylmethyl-1H-[1,2,3]triazol-1yl)-6-deoxy}-$-CD (ST-$-CD), heptakis(2,3-di-O-methyl)tris(3(6-azido-6-deoxy)-$-CD (azido-$-CD(OMe)2 ), hydroxypropyltriazolylmethyl)amine (THPTA), and sodium propargyl sulfonate were prepared via literature procedures, and the 1 H and 13 C NMR spectra of the compounds were consistent with the literatures (Supporting Information S1). The synthesis and NMR characterization of heptakis{6(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}{2,3-di-Omethyl}-$-CD (HT-$-CD(OMe)2 ) were reported in Supporting Information S1 and S2. Turbidity Measurement To each CD, 150 :L of deionized distilled water was added and the mixtures were sonicated (Hwa Shin Power Sonic 410) for 30 min. The mixture was vigorously vortexed and 120 :L of the mixture was quickly transferred into a 96-microwell plate. The visible absorption at 595 nm was measured by microplate reader (PerkinElmer VICTORTM 3, photometry mode, supplied in Seoul, South Korea). R
Scheme 1. (a) General molecular structure of cyclodextrin click clusters. (b) Molecular structures of $-CD and two reference cyclodextrin derivatives.
MTT Assay are difficult to precisely control. In addition, they would not be suitable for the incorporation of further functional groups on CDs. The bioorthogonality of the CuAAC reaction gives a distinct advantage over traditional CD modification to CCCs.25 In order to apply CCCs to the field of drug, food, and cosmetic formulation, three important considerations should be taken into account; CCC synthesis, cellular safety, and cargo encapsulation ability. Unfortunately, CCC studies in the pharmaceutical field are rare. To answer these questions, we prepared three CCC derivatives and investigated water solubility, cytotoxicity, and the drug inclusion properties of each derivative, which were compared with the properties of $-CD and two conventional CD derivatives.
EXPERIMENTAL
HeLa (human cervical adenocarcinoma cell line) cells were seeded in 96-well culture plate at 2 × 104 cells/well and incubated in DMEM (Gibco/BRL, Dulbecco’s Modified Eagle’s Medium) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotics (Gibco/BRL) for 24 h. The stock solutions (10 mM) of CDs were prepared in DMEM medium. HeLa cells on a 96-well plate were treated with a series of CD solutions (0, 0.01, 0.1, 1, 2, and 5 mM) and the plate was incubated in a CO2 incubator at 37◦ C for 1 day. To each well, 100 :L of MTT was added [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL Stock in PBS, Sigma M5655]. After a 2-h incubation at 37◦ C, the medium was removed. To dissolve the formazan crystals, 100 :L of dimethyl sulfoxide (Sigma–Aldrich 472301) was added and mixed well via pipette. Then, the absorbance values at 570 nm were measured by microplate reader (PerkinElmer VICTORTM 3, photometry mode).
Materials $-Cyclodextrin and prednisolone were purchased from TCI PN C0777and P0637 (Seoul, South Korea). 2-Hydroxypropyl$-CD (HP-$-CD) and heptakis(2,6-di-O-methyl)-$-CD (DM-$CD) were purchased from Aldrich PN 389145, H0513 (Yongin, South Korea). All purchased reagents were used without purifications. 1 H, 13 C NMR (nuclear magnetic resonance), HH COSY (homonuclear correlation spectroscopy), and CH HSQC (heteronuclear single quantum coherence) spectra were measured Le et al., JOURNAL OF PHARMACEUTICAL SCIENCES
Phase Solubility Studies HPLC was performed on an Agilent 1100 series liquid chromatography system with a diode array detector interfaced with an Agilent Chem Station for data analysis. The column was a 4.6 mm ID × 150 mm Extend-C18 column (5 :m pore size; Hewlett Packard, CA, Seoul, South Korea) with a Security GuardTM guard cartridge (3.0 × 4.0 mm, Phenomenex, CA, Seoul, South Korea). The mobile phase consisted of DOI 10.1002/jps.24107
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Scheme 2. Reactions and reagents. (a) Methanesulfonyl chloride, DMF, 65◦ C; (b) NaN3 , DMSO, MW; (c) propargyl alcohol, CuSO4 /THPTA, sodium ascorbate, THF/phosphate buffer, MW; (d) sodium propargylsulfonate; (e) MeI, NaH, DMF; (f) propargyl alcohol, CuSO4 /THPTA, sodium ascorbate, THF/phosphate buffer/ EtOH, 80◦ C, 20 h. THPTA = tris(3-hydroxypropyltriazolylmethyl)amine, MW = microwave irradiation.
acetonitrile/water (30/70, v/v). Excess prednisolone was added to aqueous 0–0.1 M CD solutions (0–0.014 M in the case of $-CD). The suspensions were equilibrated in an Eppendorf Thermomixer Comfort (25◦ C, 1000 rpm) for 48 h. The prepared samples were filtered through a 0.2 :m RC Phenomenex membrane, and the filtrates were centrifuged at 10,000g (5 min; Hanil Science Industrial Company Micro 17TR). After proper dilution with HPLC mobile phase solution (acetonitrile/water, 30/70, v/v), the amount of prednisolone in the supernatant of samples was quantified by absorbance at 254 nm. R
R
RESULTS AND DISCUSSION
efficiency in aqueous medium. THPTA developed by the Finn group has proven to be invaluable for bioconjugation in water.28 Complete triazole formation of HT-$-CD(OMe)2 was evident by the presence of axial symmetry in the NMR spectra. Only a single doublet for all H1 protons appeared in the 1 H NMR spectra [* = 5.40 ppm, JH1-H2 = 3.4 Hz for HT-$-CD(OMe)2 , vs. * = 5.12 ppm, JH1-H2 = 3.2 Hz for HT-$-CD and * = 5.15 ppm, JH1-H2 = 3.4 Hz for ST-$-CD], and the corresponding C1 atoms also appeared as one singlet [* = 97.2 ppm for HT-$-CD(OMe)2 vs. * = 103.2 ppm for HT-$-CD and * = 102.1 ppm for ST-$CD] in the H1 decoupled 13 C NMR spectrum. All protons and carbons of HT-$-CD(OMe)2 were fully assigned by HH COSY and CH HSQC (Supporting Information S2).
Synthesis of 6-Triazolyl-6-Deoxy--CD Derivatives
Water Solubility of CCCs
The core starting material, 6-azido-6-deoxy-$-CD was prepared from $-CD by a two-step reaction: (1) chlorination of the primary alcohol and (2) azide substitution at a gram scale without column chromatography. Three different CCCs were designed and synthesized through CuAAC (Scheme 2). HT-$-CD and ST-$-CD were prepared by following our group’s report.26 In order to control the hydrophobicity of the CD cavity, the secondary alcohols of 6-azido-6-deoxy-$-CD were fully methylated by Williamson ether synthesis. The resulting Azido-$CD(OMe)2 was further click-functionalized with propargyl alcohol. DiaionTM HP-20 column chromatography afforded the desired product HT-$-CD(OMe)2 at a good yield (74%). Copper(I)-catalyzed azide-alkyne cycloaddition reactions are reliable and efficient, but copper ligands are often employed both to enhance the rate of reaction and to protect Cu(I) from oxidation in the presence of adventurous oxygen.27 Thus, we also used a water-soluble ligand, THPTA to maximize our CuAAC
The water solubility of CDs was compared by turbidity measurement.29 As compared with the relatively low water solubility of $-CD, the solutions containing HT-$-CD, HT-$CD(OMe)2 , and ST-$-CD looked clean at the full experimental concentration (Fig. 1 and Supporting Information S3). These results suggest that the minimum water solubility of HT-$CD/HT-$-CD(OMe)2 /ST-$-CD is higher than 50 mg/150 :L; ca. 20 times higher than that of $-CD (solubility in water at 20◦ C, 1.85 mg/100 :L). Intramolecular hydrogen bonding of $CD can result in relatively unfavorable enthalpies of solution and low aqueous solubility. Substitution of any of the hydrogen bond forming hydroxyl groups, even by hydrophobic moieties such as methoxy groups, will result in a dramatic increase in water solubility (the water-solubility of DM-$-CD at 20◦ C, 60 mg/100 :L).30 Actually, HT-$-CD, HT-$-CD(OMe)2 , and ST$-CD are freely miscible in excess of 100 mg/100 :L and result in viscous media, which is approximately a 50-fold
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Table 1. Binding Constants for Inclusion Complexation of Prednisolone with Various $-CDs
Figure 1. The turbidity changes of $-CD, two $-CD derivatives (HP$-CD, DM-$-CD), and three CCCs (HT-$-CD, HT-$-CD(OMe)2, ST$-CD). Absorption at 595 nm was measured by microplate reader (PerkinElmer VICTORTM 3, photometry mode).
Prednisolone Molar Intrinsic Solubility = 2.8 × 10−4 mol/L (Sparingly Soluble)a log P = 1.635 ± 0.526a Solubility (mg/mL) in 50 mM CD Literatureb
Cyclodextrin
K1:1 (M−1 ) ± SD
Type
$-CD HP-$-CD DM-$-CD HT-$-CD HT-$CD(OMe)2 d ST-$-CD
2744 ± 39 1907 ± 39 2279 ± 158 2253 ± 14 1243 ± 42
AL AL AL AL AL
NDc 5.1 11.6 7.2 6.3
3000, 3479 1319, 2428 No report – –
704 ± 17
AL
4.2
–
The binding constant, K1:1 , was averaged from three independent measurements. a Molecular structure of prednisolone, and its water solubility and partition coefficient (log P = log ([Solute]octanol /[Solute]water ). The molar intrinsic solubility and log P of prednisolone were calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02. b Cited from Ref. 34 and the references in there. c The prednisolone solubility in 50 mM of $-CD is not determined because of the low solubility of $-CD (solubility = 1.85 mg/100 :L; saturation concentration = 16 mM). d The K1:1 was determined using 2% ethanolic water (98:2, water–ethanol, v/v).
Figure 2. MTT of CD derivatives on HeLa cell viability. After 24 h of CD treatment, the visible absorption of formazan crystals was measured at 570 nm and the cell viability was normalized by the control value ([CD] = 0). Results represent the mean of five independent experiments.
improvement compared with $-CD’s solubility (Supporting Information Fig. S3). From the turbidity testing of various CD derivatives, we suggest that the triazolyl modification of CCCs dramatically improves water solubility. Cytotoxicity of CCCs The MTT assay provides a read-out for cytotoxicity.31 The cytotoxicity of new chemically modified $-CD derivatives is critical for future drug delivery and food/cosmetic formulation applications. We measured the effect of CCCs on cell viability using the MTT assay, performed on HeLa cells (Fig. 2). As reference CDs, we selected 2-hydroxypropyl-$-CD (HP-$-CD, average Mw ∼ 1540) and heptakis(2,6-di-O-methyl)-$-CD (DM$-CD). HP-$-CD is commercially available and considered a safe CD derivative (an active deodorant ingredient of Febreze ; Procter & Gamble). Salem et al.32 also reported on the cytotoxicity of methylated-$-CD derivatives. As expected, HP-$-CD showed no cytotoxicity over the entire range of experimental concentrations and DM-$-CD reduced cellular viability dose-dependently over 0.1 mM. When compared with the cellular viability of HP-$-CD and DM-$CD, HT-$-CD(OMe)2 and ST-$-CD did not adversely influence HeLa cell viability at experimental concentrations (∼5 mM). However, HT-$-CD showed a slight dose-independent reducR
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tion in cellular viability (∼80%). It is remarkable that the two methylated CDs, DM-$-CD and HT-$-CD(OMe)2 , showed very different cytotoxicity profiles. DM-$-CD showed toxicity at concentrations over 0.1 mM, but HT-$-CD(OMe)2 is not toxic until around 5 mM. This result implies that the CCC approach is an alternative solution to reduce cytotoxicity of conventional DM-$-CD. Prednisolone Phase Solubility Studies with CCCs Phase solubility analysis provides valuable data on the effect of various CDs on the solubility of drugs with poor or limited aqueous solubility.33 A phase-solubility study on CCC will provides evidence for application of CCC as a drug solubilizer. Prednisolone (a poorly water-soluble glucocorticoid) was selected as a model compound for the phase-solubility study (Table 1).34 CDs, especially $- and (-CD, have been shown to form complexes with many steroids, leading to improved solubility and dissolution rates compared with the pure steroids.35 A phase-solubility profile of prednisolone with various CDs at 25◦ C was obtained according to the methods of Hirayama and Uekama.36 Establishment of equilibrium was verified by the comparison of HP-$-CD phase solubility after 24, 48, and 72 h. The results showed no significant differences, and the equilibration time was set to 48 h (Supporting Information S4). A linear standard curve was obtained in the concentration range from 2.4 × 10−4 to 1.5 × 10−2 M for prednisolone. The standard curve showed good linearity in the concentration range (Supporting Information S5). Phase solubility diagrams show that all CDs exhibited AL type diagrams in the chosen concentration range (Fig. 3). AL type phase-solubility profile is obtained when the solubility of prednisolone increases with CD concentration. In general, DOI 10.1002/jps.24107
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Figure 3. Phase solubility diagrams of prednisolone–CD systems in a series from the first independent set (25◦ C in water). Upper box: expansion of the low concentration region for clarity of the $-CD phase solubility diagram.
water-soluble CD derivatives form A-type phase solubility profiles. In the AL case and assuming that the complex exhibits 1:1 binding, the stability constant of the complex can be calculated from the slope of the isotherm: K 1:1 =
Slope S0 (1 − slope)
Three independent measurements were performed for each CD case (Fig. 4 and Supporting Information S6). The intrinsic solubility in water of prednisolone (S0 ) was determined from the intercept. The binding constant of the prednisolone complex with CD derivatives is summarized in Table 1. Our phase-solubility study demonstrates the effect of CCCs on CD-drug encapsulation ability. The binding constants of $CD derivative decrease in the order of $-CD (2744) > DM-$-
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CD (2279) ≈ HT-$-CD (2253) > HP-$-CD (1907) >> ST-$-CD (704). The solubility of the prednisolone complex with native $-CD is relatively limited because of the limited solubility of native $-CD. In contrast, a large linear increase in prednisolone solubility was observed with HT-$-CD. However, the binding constant of HT-$-CD-(OMe)2 cannot be directly compared with those of the other CD derivatives because of the difference in the binding solvent. Interestingly, we observed that the phase solubility diagram of HT-$-CD(OMe)2 in pure water was abnormal [regression coefficients (R2 ) = 0.95–0.97, high SD; Supporting Information S7]. This phenomenon may be due to the low water solubility of the HT$-CD(OMe)2 –prednisolone complex. Thus, 2% ethanolic water (98:2, water:ethanol, v/v) was used for the phase solubility test of HT-$-CD(OMe)2 instead of pure water. The binding constant of CD-guest complexation mainly depends on the hydrophobicity of the inner cavity and the solvophobicity. Increasing the number of methyl groups in the $-CD molecule will increase the hydrophobicity of the inner cavity. Kiss et al.37 reported that the cholesterol-solubilizing effect of various methylated-CD derivatives was proportional to the methyl group number of CD at a certain range (number of methyl group, 0–15). However, the binding constant is not a simple function of the methyl group number of CD. Wenz38 reported that stabilizing intramolecular hydrogen bonds between the glucose units were made responsible for the high binding potentials of those $-CD derivatives that possess secondary hydroxy groups. Schneider et al.39 reported that all log Ka values with "-CD and p-nitrphenolate correlate linearly with solvophobicity parameters of the corresponding medium for a series of aqueous methanol mixtures; adding methanol to water reduces the binding constant of the CD-guest complexation. The lower Ka of HT-$-CD(OMe)2 (1243) than that of HT$-CD (2253) can be explained by the above two parameters; destabilization of intramolecular hydrogen bond and solvophobicity.
Figure 4. An example of a phase-solubility study of the prednisolone–ST-$-CD system. The binding constant, K1:1 was averaged from three independent measurements at 25◦ C after 24 h equilibration. The error bars in the charts resulted from three repeated injections per an independent sample. S0 (intrinsic solubility) was experimentally determined by the intercept. DOI 10.1002/jps.24107
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
The introduction of an anion charge to CDs is an important strategy to solve the parenteral delivery problem of native $CD. Stella and coworkers40,41 reported the drug inclusion ability of sulfoalkyl ether CD derivatives. For instance, sulfobutyl ether-$-CD (SBE-$-CD), sold under the name CAPTISOL (degree of substitution ∼7), has been components of six United States Food and Drug Administration-approved parenteral products and is a component of a number of other parenteral products in late stage development.23 As a CCC analogue of SBE-$-CD, ST-$-CD, a sulfonated CCC was tested. The phase solubility diagram showed that ST-$-CD has a lower binding constant than HT-$-CD and HT-$-CD(OMe)2 . The aqueous solvation shell associated with sulfonate substituents may diminish the size of the cavity opening, resulting in a reduced accessibility of guest molecules.42 R
CONCLUSIONS In order to maximize the synthetic advantages of CCCs beyond the conventional CD modification chemistry and to confirm CCC application, three CCCs were prepared through CuAAc. HT-$-CD, HT-$-CD(OMe)2 , and ST-$-CD are freely miscible in an excess of 100 mg/100 :L water. This result suggested that the triazolyl modification of CCCs dramatically improves water solubility. MTT cell viability assay on HeLa cells demonstrated the low cytotoxicity of the CCCs. HT-$-CD(OMe)2 and ST-$-CD do not have any cytotoxicity within the experimental concentration (∼5 mM) but DM-$-CD was cytotoxic over 0.1 mM. This result implies that a CCC approach will be an alternative solution to reduce cytotoxicity of conventional DM$-CD. The binding constants of $-CD derivatives against prednisolone decrease in the order of $-CD > DM-$-CD ≈ HT-$-CD > HP-$-CD >> ST-$-CD in pure water, and the binding constant of HT-$-CD(OMe)2 is 1243 in 2% ethanolic water. Our experiments are the first systematic trial to explore the possibility of CCCs as a new CD-based excipient. The comparison between the conventional CD derivatives and the CCCs on solubility, cytotoxicity, and binding properties implies that CCCs are alternative CD derivatives useful for overcoming the restriction of conventional CD chemistry.
ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2065643). This research was Research Foundation of Korea (supported by the Basic Science Research Program through the National NRF) funded by the Ministry of Education, Science and Technology (2011–0024970).
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