Modification of Drug Crystallization by Cyclodextrins in Pre-formulation Study.

Controlling drug crystallization is one of the important issues in pre-formulation study. In recent years, advanced approaches including the use of tailor-made additives have gathered considerable attention to control crystallization behavior of drugs. This review focuses on the use of hydrophilic cyclodextrins (CDs) as additives for controlling drug crystallization. CDs affect the crystallization of drugs in solution and in solid state based on a host-guest interaction. For example, 2,6-di-O-methyl-ß-CD and 2-hydroxybutyl-ß-CD suppressed solution-mediated transition of drugs during crystallization by the host-guest interaction; as a result, metastable forms selectively precipitated in solution. The use of CDs in crystal engineering provided an opportunity for the detection of a new polymorph by changing the crystallization pathway. It was also possible to modify crystal morphology (i.e., crystal habit) by selective suppression of crystal growth on a certain direction based on the host-gust interaction. For solid formulation, stable amorphous drug/CDs complex under humid conditions was prepared using two different CDs. An overview of some recent progress in the use of CDs in crystal engineering and in amorphous formulation is described in this review.

The Use of Enteric Capsules for Releasing a Fragrance over an Extended Period of Time.

A system for releasing a fragrance, citral (CR) over an extended period of time using three types of enteric capsules is reported. The L- and M-type capsules released CR into media with a pH above 6, while the H-type capsule released CR at a pH above 7. The pH of the releasing medium was controlled by sodium borate (SB), i.e., by adding SB-methylcellulose (MC) prepared in different weight ratios (SB-MC 1 : 2, 1 : 1 and 2 : 1) to tablets and by compressing them at different pressures. The tablet containing a large amount of SB and that was pressed at higher pressures permitted the pH of the releasing medium to be changed from 5 to 9, at 4-5 h after the addition of SB to the tablets, while negligible changes were observed for tablets containing low amounts of SB and which were compressed at lower pressures. Reflecting these pH changes, CR was released after different periods of time when SB-MC tablets and capsules containing CR were simultaneously added to the releasing medium. When enteric capsules containing CR and the pH adjusting tablets were simultaneously added to a benzyl acetate (BA) solution, BA was released at a constant rate, while CR was released for different periods of time depending on the type of capsule used. The results suggest that fragrances could be released over different time frames by using enteric capsules and pH adjusting agents, for example, the release of fragrances with sedative effects at night time and with stimulating effects in the morning.

Glucose Responsive Rheological Change and Drug Release from a Novel Worm-like Micelle Gel Formed in Cetyltrimethylammonium Bromide/Phenylboronic Acid/Water System.

A novel glucose (Glc)-responsive gel formed by worm-like micelles (WLMs) has the potential to provide a self-regulating insulin delivery system. We have prepared a WLM gel system using 75 mM cetyltrimethylammonium bromide, 75 mM phenylboronic acid, and water. At pH 9.4, this gel-like system was highly viscous and supported its own weight, and dynamic viscoelasticity measurement indicated that it contained long and entangled WLMs. The visual observation of gels prepared to include >6 mM Glc revealed that these adopted a sol-like appearance, whereas those prepared to include a control compound (2-10 mM diethylene glycol) retained their gel-like appearance. The storage modulus ( G') of this system decreased as the Glc concentration increased (2-10 mM), indicating a gradual shortening of the WLMs. In vitro release was evaluated using a test compound (fluorescein isothiocyanate dextran) in a microsized flow system. By 120 min, the release of this compound from the WLM gel was around 27-fold greater in the presence of 100 mM Glc than without Glc or with 100 mM diethylene glycol. This demonstrated the successful preparation of a WLM gel that showed an altered drug release rate, depending on Glc concentration.

Hydrophobically Modified Polymer/α-Cyclodextrin Thermoresponsive Hydrogels for Use in Ocular Drug Delivery.

We report herein on the preparation of thermoresponsive hydrogels by taking advantage of the interaction of cyclodextrins (CDs) and a hydrophobically modified polymer. A hydrophobically modified hydroxypropyl methylcellulose (HM-HPMC) gel formed thermoresponsive hydrogels when small amounts of α-CD were added to the solution. The HM-HPMC/α-CD showed reversible sol-gel transition in the physiological temperature range that was completely opposite to the temperature dependency shown by the original HM-HPMC. The thermoresponsive gelation was attributed to the temperature dependency of the interaction between α-CD and the hydrophobic moiety of HM-HPMC. The potency of the HM-HPMC/α-CD sol-gel transition system in ophthalmic formulation was tested on the eyes of a rabbit. The use of HM-HPMC/α-CD significantly improved the ocular absorption of a drug, diclofenac sodium, by virtue of the rapid formation of a gel on the ocular surface. That is, the HM-HPMC/α-CD was in a low viscous sol state at room temperature, which made administration easy, but it rapidly formed a viscous hydrogel on the ocular surface at physiological temperature. The thermoresponsive hydrogel based on the hydrophobically modified polymer and CD promises to have widespread applications in drug delivery.

Formation of the ternary inclusion complex of limaprost with α- and ß-cyclodextrins in aqueous solution.

The inclusion mode of Limaprost in the presence of α- and ß-cyclodextrins (CDs) was investigated to gain insight into the stabilization mechanism of Limaprost-alfadex upon the addition of ß-CD in the solid state. The inclusion sites of α- and ß-CDs were studied by NMR spectroscopic and kinetic methods. With the addition of α- and ß-CDs, displacements in (13)C chemical shifts of prostaglandin F2α (PGF2α) were observed in the ω-chain and the five-membered ring, respectively, of the drug. Similar shift changes were observed with the addition of both α- and ß-CDs. In two-dimensional (2D) (1)H-NMR spectra, intermolecular correlation peaks were observed between protons of PGF2α and protons of both α- and ß-CDs, suggesting that PGF2α interacts with α- and ß-CDs to form a ternary complex by including the ω-chain with the former CD and the five-membered ring with the latter. In kinetic studies in aqueous solution, Limaprost was degraded to 17S,20-dimethyl-trans-Δ(2)-PGA1 (11-deoxy-Δ(10)) and 17S,20-dimethyl-trans-Δ(2)-8-iso-PGE1 (8-iso). The addition of α-CD promoted the dehydration to 11-deoxy-Δ(10), while ß-CD promoted the isomerization to 8-iso, under these conditions. In the presence of both α- and ß-CDs, dehydration and isomerization were also accelerated, supporting the formation of the ternary Limaprost/α-CD/ß-CD complex.

Stabilizing effect of ß-cyclodextrin on Limaprost, a PGE1 derivative, in Limaprost alfadex tablets (Opalmon) in highly humid conditions.

Stabilization against humidity of Limaprost (a prostaglandin E1 derivative), which is currently marketed as Opalmon, was undertaken using ß-cyclodextrin (ß-CD). Aqueous solutions of Limaprost alfadex/dextran 40 were lyophilized with and without ß-CD. Limaprost alfadex lyophilized with ß-CD was more chemically stable in humid conditions than that without ß-CD. Moreover, the addition of ß-CD as an excipient to tablets of these lyophilized composites remarkably improved the stability of Limaprost, and Limaprost in this moisture-resistant formulation was chemically stable for 19 weeks at 30°C, 75% relative humidity (R.H.). Chemical analysis of Limaprost and its degradation products indicated that degradation proceeded in the inclusion form (i.e., within the CD cavity). Solid (2)H-NMR spectroscopic studies showed that ß-CD constrained the molecular mobility of water in the solid state. These results suggested that the stabilization of Limaprost by ß-CD was at least partly due to the restricted molecular mobility of water, which acted as a catalytic species for the degradation, and also to the protection of the five-membered ring of Limaprost from water catalytic dehydration through inclusion complex formation with ß-CD.

Formation of stable hydrophilic C60 nanoparticles by 2-hydroxypropyl-ß-cyclodextrin.

A number of papers have reported that the large cavity of γ-CyD is favorable for inclusion of C(60) and forms a 1:2 (C(60):γ-CyD) complex, whereas it is thought to be difficult for ß-CyD to form a complex at the molecular level. This is because the cavity size of ß-CyD (0.78 nm) is smaller than the van der Waals diameter of C(60) (1.0 nm). In this paper, we will report on the formation of the stable C(60) nanoparticles by the hydrophilic 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) layer through weak interaction on the surface of the nanoparticles. C(60) was coground with ß-CyD, γ-CyD or HP-ß-CyD mainly in a 1:2 molar ratio by an automatic magnetic agitating mortar, the coground powders were dispersed in water, and the resulting solutions were filtered through a pore size of 0.8 µm filter. The γ-CyD and HP-ß-CyD systems gave transparent colloidal solutions consisting of C(60)/CyD nanoparticles with the size lower than 100 nm, with high yields (about 100%). The C(60)/HP-ß-CyD nanoparticles are physically stable, keeping a small size for more than 28 days, whereas the γ-CyD nanoparticles are readily aggregated to form large particles (>800 nm). Solid and liquid NMR spectroscopic studies including measurements of spin-lattice relaxation times indicated that C(60) interacted with γ-CyD and HP-ß-CyD in the solid and colloidal solutions. When compared with the γ-CyD nanoparticles, adsorption studies of a hydrophobic dye on the surface of C(60)/CyD nanoparticles indicated that the surface of the HP-ß-CyD nanoparticles is largely covered by HP-ß-CyD molecules forming hydrophilic hydration layers. The present results suggest that HP-ß-CyD is useful for the preparation of C(60) nanoparticles and medical applications such as photodynamic therapy, in spite of having a cavity size smaller than that of γ-CyD.

A thermoresponsive hydrophobically modified hydroxypropylmethylcellulose/cyclodextrin injectable hydrogel for the sustained release of drugs.

The objective of this study was to develop a thermoresponsive injectable hydrogel for the sustained release of drugs by taking advantage of host-guest interactions between a hydrophobically modified hydroxypropylmethyl cellulose (HM-HPMC) and cyclodextrin (CD). A thermoresponsive injectable hydrogel was prepared by simply adding CDs to HM-HPMC hydrogel. The HM-HPMC hydrogel was converted into a sol with a low viscosity through host-guest interactions with CDs. The HM-HPMC/ß-CD hydrogel became a gel near body temperature where the host dissociated from the hydrophobic moieties of the polymer in response to the temperature. The yield stress of the HM-HPMC became progressively lower on the addition of ß-CD which was desirable in the case of developing an injectable formulation. When the HM-HPMC/ß-CD hydrogel containing indocyanine green (ICG) was subcutaneously administered to mice, the fluorescence of the ICG remained relatively constant for 24 h after the administration, which was substantially longer than that for ICG alone or an HPMC formulation. The plasma insulin level was maintained for a longer period of time when the HM-HPMC/ß-CD containing insulin was administered and the MRT value was increased by 1.6 times compared to a solution of insulin alone. In addition, the HM-HPMC/ß-CD hydrogel formulation showed a prolonged hypoglycemic effect in response to the insulin which was slowly released from the hydrogel. A thermoresponsive injectable hydrogel was successfully constructed from the highly viscous HM-HPMC and ß-CD, and the resulting formulation functioned as a sustained release carrier for drugs.

Potential use of 2-hydroxypropyl-beta-cyclodextrin for preparation of orally disintegrating tablets containing dl-alpha-tocopheryl acetate, an oily drug.

To expand the application of a drug in orally disintegrating tablets, the potential use of beta-cyclodextrin (beta-CyD) and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) as excipients for the tablets containing dl-alpha-tocopheryl acetate (VE), an oily drug, was evaluated. HP-beta-CyD, not beta-CyD, solubilized VE in water through the formation of higher order of complexes at the molar ratio of 1 : 2 (VE : HP-beta-CyD). When prepared under the optimal preparation conditions, the VE tablets containing lactose and 5% (w/w) of HP-beta-CyD, not beta-CyD, had high hardness more than 4 kg and rapid disintegration within 100 s both in vitro and in vivo. In addition, VE tablets containing lactose and 5% (w/w) of HP-beta-CyD, not beta-CyD, maintained the high hardness and rapid disintegration under the accelerated stability test using different conditions for 4 weeks. Therefore, these results suggest the potential use of HP-beta-CyD, not beta-CyD, as an excipient for orally disintegrating tablets containing VE, an oily drug, in the molding method.

Preparation of four types of coenzyme Q10/gamma-cyclodextrin supramolecular complexes and comparison of their pharmaceutical properties.

In this study, we prepared four kinds of complexes of Coenzyme Q10 (CoQ10) with gamma-cyclodextrin (gamma-CyD) by the kneading methods and the solubility methods with or without heating, and compared their pharmaceutical properties. Differential scanning calorimetrical curves and powder X-ray diffraction patterns showed that the complexes formed pseudorotaxane-like supramolecular structure, although included free gamma-CyD and CoQ10, when prepared by the kneading method and solubility method without heating. At the preparation process, a heating improved the complexation of CoQ10 with gamma-CyD in the both methods. The dispersion rate of CoQ10 in water increased in the order of CoQ10 alone approximately physical mixture with gamma-CyD<

Design and evaluation of polypseudorotaxanes of pegylated insulin with cyclodextrins as sustained release system.

Supramolecular assemblies have attracted a great attention, due to their intriguing topologies and their application in various fields such as nanodevices, sensors, molecular switches, and drug delivery systems. In this study, we prepared the monosubstituted insulin with poly(ethylene glycol) (PEG, MW about 2200) and its cyclodextrin (CyD) polypseudorotaxanes. The pegylated insulin formed polypseudorotaxanes with alpha- and gamma-CyDs, by inserting one PEG chain in the alpha-CyD cavity and two PEG chains in the gamma-CyD cavity. The pegylated insulin/alpha- and gamma-CyD polypseudorotaxanes were less soluble in water and the release rate of the drug decreased in the order of drug alone>the gamma-CyD polypseudorotaxane>the alpha-CyD polypseudorotaxane. The plasma levels of the pegylated insulin after subcutaneous administration of the gamma-CyD polypseudorotaxane to rats were significantly prolonged, accompanying an increase in the area under plasma concentration-time curve, which was clearly reflected in the prolonged hypoglycemic effect. The results indicated that the pegylated insulin/CyD polypseudorotaxanes can work as a sustained drug release system, and the polypseudorotaxane formation with CyDs may be useful as a sustained drug delivery technique for other pegylated proteins and peptides.

Preparation of amorphous indomethacin from aqueous 2,6-di-O-methyl-beta-cyclodextrin solution.

Indomethacin precipitated exclusively in an amorphous form from aqueous 2,6-di-O-methyl-beta-cyclodextrin solutions, whereas it precipitated in Form V polymorph from the solutions of the drug alone, parent cyclodextrins and 2-hydroxypropyl-cyclodextrins. The polymorphic transition of the amorphous form to Form V crystals in aqueous solution was markedly inhibited by the addition of 2,6-di-O-methyl-beta-cyclodextrin, keeping the amorphous state for at least 5 days at 4 degrees C, whereas it quickly transformed to Form V crystals in the absence of 2,6-di-O-methyl-beta-cyclodextrin. 2,6-Di-O-methyl-beta-cyclodextrin suppressed the solution-mediated polymorphic transition of amorphous form of indomethacin to Form V crystals in aqueous solution. The current results suggested that 2,6-di-O-methyl-beta-cyclodextrin is useful for isolation of amorphous indomethacin that occurs at an early stage of crystallization according to "Ostwald's Rule of Stages".

Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies.

This review focuses on the in vitro and in vivo antioxidant activities of various chitosan preparations, including those with different molecular weights and degrees of acetylation and the nanofibers produced from them. In in vitro studies, low molecular weight (LMW) chitosan with high degrees of deacetylation has more potent antioxidant properties than those of high molecular weight (HMW) chitosan. On the other hand, HMW chitosan has higher adsorption properties than those of LMW chitosan. On the basis of the in vitro results obtained, the ingestion of chitosan and nanofiber derived from it, with moderate MW and degrees of acetylation results in a significant reduction in oxidative stress in several chronic oxidative stress related diseases such as the metabolic syndrome and renal failure. In the future, chitosan and related nanofibers with presumed antioxidant properties may be used as a new source of antioxidant, as a possible food supplement, as an ingredient or in the pharmaceutical industry.

An oral absorbent, surface-deacetylated chitin nano-fiber ameliorates renal injury and oxidative stress in 5/6 nephrectomized rats.

In this study, we report that surface-deacetylated chitin nano-fibers (SDACNFs) are more effective in decreasing renal injury and oxidative stress than deacetylated chitin powder (DAC) in 5/6 nephrectomized rats. An oral administration of low doses of SDACNFs (40mg/kg/day) over a 4 week period resulted in a significant decrease in serum indoxyl sulfate, creatinine and urea nitrogen levels, compared with a similar treatment with DAC or AST-120. The SDACNFs treatment also resulted in an increase in antioxidant potential, compared with that for DAC or AST-120. Immunohistochemical analyses also demonstrated that SDACNFs treated CRF rats showed a decrease in the amount of accumulated 8-OHdG compared with the CRF group. These results suggest that the ingestion of SDCH-NF results in a significant reduction in the levels of pro-oxidants, such as uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation.

Surface-deacetylated chitin nanofibers reinforced with a sulfobutyl ether ß-cyclodextrin gel loaded with prednisolone as potential therapy for inflammatory bowel disease.

Surface-deacetylated chitin nanofibers (SDACNFs) reinforced with a sulfobutyl ether ß-cyclodextrin (SBE-ß-CD) (NFs-CDs) gel were developed to obtain a controlled release carrier of prednisolone (PD) for the treatment of colitis. PD was released slowly from the gel at both pH 1.2 and 6.8. The in vitro slow release of PD from the NFs-CDs gel was reflected in the in vivo absorption of the drug after oral administration to rats. These results suggest that a simple gel composed of a mixture of SDACNFs and SBE-ß-CD has the potential for use in the controlled release of PD. We also evaluated the therapeutic effects of the NFs-CDs gel containing PD on dextran sulfate sodium (DSS)-induced colitis model mice. The administration of the NFs-CDs gel at intervals of 3days from the beginning of the DSS treatment resulted in a significant improvement, not only in colitis symptoms but also histopathological changes in colon tissue. In addition, the therapeutic effects of the NFs-CDs gel on colitis can be attributed to decreased levels of neutrophil infiltration and the development of oxidative stress. These efficacy profiles of the NFs-CDs gel containing PD suggest that it has the potential for use in the treatment of, not only colitis, but also a variety of other disorders associated with inflammation and oxidative injuries.

Enhancement of gene transfer activity mediated by mannosylated dendrimer/alpha-cyclodextrin conjugate (generation 3, G3).

To enhance gene transfer activity of dendrimers, we prepared its conjugate (generation 3, G3) with alpha-cyclodextrin bearing mannose (Man-alpha-CDE conjugates) with various degrees of substitution of the mannose moiety (DSM5, 10, 13, 20) and compared their cytotoxicity and gene transfer activity, and elucidated the enhancing mechanism for the activity. Of the various carriers used here, Man-alpha-CDE conjugate (G3, DSM10) provided the highest gene transfer activity in NR8383, A549, NIH3T3 and HepG2 cells, being independent of the expression of mannose receptors. Gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) was not decreased by the addition of 10% serum in A549 cells. Cytotoxicity of the polyplex with Man-alpha-CDE conjugates (G3, DSM10) was not observed in A549 and NIH3T3 cells up to the charge ratio of 200/1 (carrier/pDNA). The gel mobility and particle size of polyplex with Man-alpha-CDE conjugate (G3, DSM10) were relevant to those with alpha-CDE conjugate (G3), but zeta-potential, DNase I stability, pDNA condensation of the former polyplex were somewhat different from those of the latter one. Cellular association of polyplex with Man-alpha-CDE conjugate (G3, DSM10) was almost comparable to that with dendrimer (G3) complex and alpha-CDE conjugate (G3). The addition of mannan and mannose attenuated gene transfer activity of Man-alpha-CDE conjugate (G3, DSM10) in A549 cells. Alexa-pDNA complex with TRITC-Man-alpha-CDE conjugate (G3, DSM10), but not the complex with TRITC-alpha-CDE conjugate (G3), was found to translocate to nucleus at 24 h after incubation in A549 cells. HVJ-E vector including mannan, but neither the vector alone nor the vector including dextran, suppressed the nuclear localization of TRITC-Man-alpha-CDE conjugate (G3, DSM10) to a striking degree after 24 h incubation in A549 cells. These results suggest that Man-alpha-CDE conjugate (G3, DSM10) has less cytotoxicity and prominent gene transfer activity through not only its serum resistant and endosome-escaping abilities but also nuclear localization ability.

Cyclodextrin-based isolation of Ostwald's metastable polymorphs occurring during crystallization.

We describe a novel approach for the selective isolation of Ostwald's intermediate metastable polymorphs occurring during an early stage of crystallization, by utilizing the inclusion complex formed with a cyclic oligosaccharide derivative, 2,6-di-O-methyl-beta-cyclodextrin.

Enhancement of antitumor effect of doxorubicin by its complexation with gamma-cyclodextrin in pegylated liposomes.

In the present study, we examined tissue distribution and the antitumor effect of doxorubicin (DOX) after intravenous injection of the pegylated liposomes entrapping the DOX complex with gamma-cyclodextrin (gamma-CyD) (complex-in-liposome) in BALB/c mice bearing colon-26 tumor cells, compared with those of DOX solution, pegylated liposomes entrapping DOX alone (DOX-in-liposome), pegylated liposomes entrapping gamma-CyD (CyD-in-liposome) and the binary system of DOX-in-liposome and CyD-in-liposome. When injected to the mice, complex-in-liposome provided the high DOX levels in plasma and solid tumors, compared with the other preparations. Reflecting the result, complex-in-liposome elicited the retardation of tumor growth and the improvement of survival rate without suppression of increase in the body weight of mice. These results suggest the potential use of pegylated liposomes entrapping the DOX complex with gamma-CyD for a promising carrier for improvement of antitumor effects of DOX.

Enhanced bioavailability of a new thiazolidine derivative FPFS-410, an antidiabetic and lipid-lowering drug, after oral administration of its hydroxypropyl-beta-cyclodextrin complex to bile duct-cannulated rats.

The effect of bile acids on bioavailability of FPFS-410 (2-(N-Cyanoimino)-5-{(E)-4-styrylbenzylidene}-4-oxothiazolidine) after oral administration of the drug and its 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) complex was investigated. The complexation with HP-beta-CyD increased the oral bioavailability of FPFS-410 in normal rats in a HP-beta-CyD concentration-dependent manner, compared with that of drug alone. In bile duct-cannulated rats, bile acid concentrations in pylic serum and biliary were decreased to 18% and 14% of sham-operated rats, respectively. After oral administration of the HP-beta-CyD complex, the plasma levels of FPFS-410 were lower in bile duct-cannulated rats than in sham-operated rats up to 1 h, however, this order reversed from 2 to 12 h. The plasma levels of M1, a dominant metabolite of FPFS-410 in rats, significantly decreased until 2 h after administration of the complex in bile duct-cannulated rats, compared with in sham-operated rats. Bioconversion of FPFS-410 to M1 and CYP3A2 expression in the liver was markedly lowered by bile duct-cannulation. Bile duct-cannulation did not, however, affect the serum levels of estradiol. These results suggest that bile acids have a pivotal role for bioavailability of FPFS-410 after oral administration of the FPFS-410 complex with HP-beta-CyD through CYP3A2 activity in liver of rats.

Effects of cyclodextrins on chemically and thermally induced unfolding and aggregation of lysozyme and basic fibroblast growth factor.

Effects of cyclodextrin (CyDs) on unfolding and aggregation of lysozyme and basic fibroblast growth factor (bFGF) were investigated. CyDs inhibited the chemically induced aggregation and its inhibition was generally in the order of gamma-CyDs < alpha-CyDs < beta-CyDs. Among these CyDs, branched beta-CyDs and dimethyl-beta-CyD (DM-beta-CyD) significantly reduced the aggregation of lysozyme. Hydrophilic CyDs reduced the thermally induced unfolding of lysozyme as shown by a decrease in the thermal unfolding temperature (T(m)) value of lysozyme, suggesting that CyDs destabilize native lysozyme or stabilize the unfolded state of lysozyme. In the case of bFGF, branched beta-CyDs showed greater effects on inhibition of the chemically and thermally induced denaturation. Interestingly, sulfobutyl ether beta-CyD (SBE-beta-CyD), which was not effective in case of lysozyme, provided the inhibitory effect for bFGF on the chemically, thermally and acid-induced denaturation, suggesting that both the inclusion and electrostatic interaction may be operative in the inhibition of aggregation of the positively charged protein. The results indicated that the use of CyDs for protein stabilization is dependent not only on the structure and property of CyDs but also on the nature of the denaturing stimuli, and the most appropriate CyD should be used for the stabilization of each protein.