Cholesteryl-Conjugated Ribonuclease A Exhibits Enzyme Activity in Aqueous Solution and Resistance to Dimethyl Sulfoxide.

Using bovine pancreatic ribonuclease A (RNase A) and cholesterol, we synthesized cholesteryl-conjugated ribonuclease A (CHRNase A) to evaluate the influence of a conjugated hydrophobic moiety on protein function. Nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight spectrometry suggested that one cholesteryl group was conjugated to RNase A. Differential scanning calorimetry indicated that CHRNase A was denatured in the solid state but was folded in phosphate buffer (0.05 mol/L, pH 6.5). CHRNase A resembled RNase A in its secondary structure, but circular dichroism (CD) spectra revealed that the helical content of CHRNase A was decreased and the tertiary structure of CHRNase A differed from that of RNase A. Furthermore, fluorescence measurements, CD spectra, an 8-anilino-1-naphthalenesulfonic acid ammonium salt-based assay, and surface tension measurements suggested that cholesterol was conjugated to a tyrosine residue on the protein surface. The relative activity of CHRNase A to RNase A was 79 ± 7%, and the enzyme activity of CHRNase A by adding ß-cyclodextrin (ß-CyD) increased to 129 ± 7%. Therefore, we considered that the cholesteryl group interacted with substrate (cytidine 2'3'-cyclic monophosphate monosodium salt) to inhibit the enzyme reaction. Finally, the environment around tyrosine residues in CHRNase A in dimethyl sulfoxide was similar to that of native RNase A in phosphate buffer (0.05 mol/L, pH 6.5). These results suggest that cholesterol conjugation to RNase A altered RNase A functionality, including improvement of RNase A resistance to dimethyl sulfoxide and modulation of the ability of ß-CyD to control RNase A enzymatic activity.

Synthesis and Characterization of Cholesteryl Conjugated Lysozyme (CHLysozyme).

Hydrophobic interaction is important for protein conformation. Conjugation of a hydrophobic group can introduce intermolecular hydrophobic contacts that can be contained within the molecule. It is possible that a strongly folded state can be formed in solution compared with the native state. In this study, we synthesized cholesteryl conjugated lysozyme (CHLysozyme) using lysozyme and cholesterol as the model protein and hydrophobic group, respectively. Cholesteryl conjugation to lysozyme was confirmed by nuclear-magnetic resonance. Differential-scanning calorimetry suggested that CHLysozyme was folded in solution. CHLysozyme secondary structure was similar to lysozyme, although circular dichroism spectra indicated differences to the tertiary structure. Fluorescence measurements revealed a significant increase in the hydrophobic surface of CHLysozyme compared with that of lysozyme; CHLysozyme self-associated by hydrophobic interaction of the conjugated cholesterol but the hydrophobic surface of CHLysozyme decreased with time. The results suggested that hydrophobic interaction changed from intramolecular interaction to an intermolecular interaction. Furthermore, the relative activity of CHLysozyme to lysozyme increased with time. Therefore, CHLysozyme likely forms a folded state with an extended durability of activity. Moreover, lysozyme was denatured in 100% DMSO but the local environment of tryptophan in CHLysozyme was similar to that of a native lysozyme. Thus, this study suggests that protein solution stability and resistance to organic solvents may be improved by conjugation of a hydrophobic group.

Crystallographic evaluation of the conformation of quetiapine included in ß-cyclodextrin.

Single-crystal X-ray diffraction and theoretical calculations were conducted for insights into the ß-cyclodextrin (ß-CD)-quetiapine inclusion complex structure. ß-CD and quetiapine form a host-guest inclusion complex at a ratio of 2:1 in which the ß-CD molecules form head-to-head dimers with their secondary hydroxyl groups linked by multiple hydrogen bonds. Quetiapine is totally contained within the ß-CD cavity and exhibits two kinds of disorder (parts 1 and 2) in opposite directions in the ß-CD complex. To clarify the mobility of the guest molecule in the ß-CD cavity, theoretical molecular conformational calculations, crystal optimization and crystal energy calculations were conducted using CONFLEX software. The results of theoretical molecular conformation calculations showed that the mobility of quetiapine is restricted because its tricyclic structure is covered by ß-CD. The results of crystal energy calculations indicated that the conformation of disorder part 1, which has high occupancy, was more stable.

Improving the Solid-State Photostability of Furosemide by Its Cocrystal Formation.

The photostability of three types of furosemide (FUR) cocrystal (FUR-caffeine, FUR-urea, and FUR-nicotinamide cocrystals) was studied under irradiation with a D65 fluorescent lamp. The coloration of the FUR-urea pellets was significantly faster than that of the intact FUR, whereas the coloration of FUR-nicotinamide was suppressed compared with that of intact FUR and the other cocrystals. In the case of FUR-urea, the chemical degradation of FUR increased by approximately 6.6% after irradiation for 90 d. On the other hand, FUR-nicotinamide showed better chemical stability, with only 1.3% of FUR degraded, which was significantly lower than the other cocrystals. The FUR-urea pellets showed a UV-Visible absorption spectrum similar to that of intact FUR, while the absorption range of FUR-nicotinamide shifted to a shorter wavelength. The light sensitivity of FUR-nicotinamide was improved because of the much lower emission of the D65 fluorescent lamp in the absorption range of the cocrystal.

Effect of sulfobutyl ether-ß-cyclodextrin and propylene glycol alginate on the solubility of clozapine.

Clozapine (CLZ) is an atypical antipsychotic medication used in the treatment of schizophrenia and is poorly soluble in water (0.05 mM). In this study, we have investigated the effect of ß-cyclodextrin (CD) and its derivatives on the solubility of CLZ. The solubility of the CLZ was measured to generate a phase solubility diagram, and the interaction between CLZ and sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) in aqueous solution was observed by 1H- and 2D rotating-frame Overhauser enhancement spectroscopy (ROESY)-NMR methods. Moreover, the synergistic effect of SBE-ß-CD and water-soluble polymers, including polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose sodium salt, polyvinyl alcohol, sodium alginate, and propylene glycol alginate (PGA), on the solubility of CLZ was investigated. The results show that the solubility of CLZ with 1 w/v% PGA was 7.6 mM, which was almost four times greater than that of CLZ without PGA in a 15 mM SBE-ß-CD solution. In contrast, the solubility of CLZ with 1 w/v % PGA in an aqueous solution decreased by one-third relative to that of CLZ in a 15 mM SBE-ß-CD solution. 2D ROESY-NMR indicated that a CLZ/SBE-ß-CD/PGA ternary complex formed. It was found that the combination of PGA and SBE-ß-CD enhanced the solubility of CLZ.

Crystallographic and theoretical studies of an inclusion complex of ß-cyclodextrin with fentanyl.

The crystal structure of an inclusion complex of ß-cyclodextrin (ß-CD) with fentanyl was determined by single crystal X-ray diffraction analysis. The crystal belongs to the triclinic space group P1 and the complex comprises one fentanyl, two ß-CD, and several water molecules. ß-CD and fentanyl form a host-guest inclusion complex at a ratio of 2:1 and the asymmetric unit of the complex contains two host molecules (ß-CDs) in a head-to-head arrangement that form dimers through hydrogen bonds between the secondary hydroxyl groups of ß-CD and one guest molecule. Fentanyl is totally contained within the ß-CD cavity and the structure of the phenylethyl part of fentanyl inside the dimeric cavity of the complex is disordered. Furthermore, theoretical molecular conformational calculations were conducted to clarify the mobility of the guest molecule in the ß-CD cavity using CONFLEX software. Crystal optimization and crystal energy calculations were also conducted. The results of the theoretical calculations confirmed that the conformation of disorder part 1, which was high in occupancy by crystal structure analysis, was more stable. The phenylethyl part of fentanyl existed in several stable conformations.

Enhanced dissolution and skin permeation profiles of epalrestat with ß-cyclodextrin derivatives using a cogrinding method.

Epalrestat (EPL) is a water-insoluble drug (14µM) that inhibits aldose reductase. This study investigated the interactions between ß-cyclodextrin (CD) derivatives and EPL to determine the solubilizing effect on EPL from phase solubility diagrams. We improved the solubility of EPL in water by adding ß-CD derivatives. Moreover, the solubility of EPL mixed with ß-CD derivatives by cogrinding in a ball mill method was about 2-3 times higher than those of EPL with the same CD concentration (5mM) calculated from phase solubility diagrams. In addition, we investigated the effect of ß-CD derivatives on in vitro percutaneous absorption of EPL through hairless mouse skin. Among the coground mixtures of EPL and ß-CD derivatives, the mixture containing methyl (ME)-ß-CD showed the strongest enhancement of EPL skin permeation. Furthermore, adding 10wt% urea as a skin permeation enhancer after cogrinding with ME-ß-CD improved the flux of EPL 300 times compared to the flux of EPL alone. This result indicates the ME-ß-CD ground mixture system with urea has potential as a new transdermal drug delivery system of EPL for diabetic neuropathy.

Investigation of Discoloration of Furosemide Tablets in a Light-Shielded Environment.

We observed that uncoated furosemide tablets turned yellow in a light-shielded automatic packaging machine and discoloration of the furosemide tablets was heterogeneity and occurred on the surface of the tablets only. The machine was equipped with an internal blower to maintain a constant temperature. Therefore, we investigated the effect of air flow on the discoloration of the furosemide tablets using a blower in a dark environment. The color difference (ΔE) of the furosemide tablets increased linearly as the blowing time increased. We performed structural analysis of the yellow compound in the furosemide tablets by LC-MS and identified the compound as a hydrolysate of furosemide. This suggested that furosemide hydrolysis was accelerated by the air flow. The furosemide tablets were prepared with the most stable furosemide polymorph, form I. X-Ray powder diffractometry and IR spectroscopy showed that during tablet preparation, no crystal transition occurred to an unstable furosemide polymorph. Furthermore, IR spectroscopy showed that the crystal form of furosemide in the yellow portion of the tablets was form I. To elucidate the factors producing the discoloration, we investigated the effect of humidity and atmosphere (air, oxygen, and nitrogen) on the discoloration of the furosemide tablets. The results suggested that the discoloration of the furosemide tablets was accelerated by oxidation, although humidity did not affect the hydrolysis. Therefore, we concluded that the discoloration of the furosemide tablets in the automatic packing machine was caused by acceleration of oxidative degradation by air flow.

Interaction of fentanyl with various cyclodextrins in aqueous solutions.

OBJECTIVES: Water-soluble fentanyl citrate salt has been used in sublingual or buccal formulations for the breakthrough pain treatment. However, fentanyl absorption through the lipid mucosal membrane may be improved by enhancing the non-ionic lipophilic fentanyl base solubility. Therefore, the interaction between cyclodextrins (CDs) and fentanyl base has been evaluated to obtain basic information for its application. METHODS: Parent CDs (α-, ß- and γ-CD) as well as α- and ß-CD derivatives were used for solubility studies with fentanyl base. Nuclear magnetic resonance (NMR) studies were applied in a system including ß-CD or glucosyl-ß-CD (G1-ß-CD) with fentanyl base or fentanyl citrate. (1) H- and (13) C-NMR studies and a two-dimensional rotating frame Overhauser effect spectroscopy (ROESY) study were conducted to confirm inclusion complexes formation. KEY FINDINGS: Parent CDs displayed BS type phase solubility diagrams; ß-CD exhibited a strong interaction with fentanyl base. Hydrophilic ß-CD derivatives, such as G1-ß-CD, displayed AL type phase diagrams and higher solubilizing effects compared with parent CDs. ROESY study suggested that fentanyl phenyl groups were included in ß-CD cavity. CONCLUSIONS: This study revealed that hydrophilic ß-CD derivatives, such as G1-ß-CD, could be useful pharmaceutical additives for oral mucosal formulations because of the improved fentanyl base solubility via inclusion complexation.

Identification of Furosemide Photodegradation Products in Water-Acetonitrile Mixture.

The aim of this study was to identify the chemical structure of the photodegradation products of furosemide in a water-acetonitrile mixture (1 : 1). Furosemide solution was irradiated with a D65 fluorescent lamp and the products were isolated by preparative HPLC. The fractions were evaporated to dryness in vacuo. The purity of the photodegradation products was measured by HPLC. The purity of products 1, 3, and 4 was greater than 90%, whereas that of product 2 was 13%, therefore, photodegradation product 2 was unstable. We identified photodegradation products 1 and 3 as 4-chloro-5-sulfamoylanthranilic acid and 4-hydroxy-N-furfuryl-5-sulfamoylanthranilic acid, respectively, by LC/MS and NMR. Additionally, we assumed that photodegradation product 4 was methyl 2-((furan-2-ylmethyl)amino)-4-hydroxy-3-(methyleneamino)-5-sulfamoylbenzoate by LC/MS and NMR. This showed that furosemide underwent hydrolysis and substitution, and reacted with the acetonitrile under the light of a D65 fluorescent lamp. We were furthermore able to determine the elution times of the photodegradation products of furosemide by applying the Japanese Pharmacopoeia chromatographic method for related substances to the isolated products.

Solid-state characterization of sertraline base-ß-cyclodextrin inclusion complex.

Sertraline is one of the serotonin-specific reuptake inhibitors that is effective in treating several disorders such as major depression, obsessive-compulsive disorder, panic disorder, and social phobia. It is marketed in the form of its hydrochloride salt, which exhibits better solubility in water than its free base form. However, the absorption of sertraline through biological membranes could be improved by enhancing the solubility of its base because it is more hydrophobic than sertraline hydrochloride. To clarify the mechanism for the interaction of sertraline base with ß-CD, it is important to study the basic interaction between the ß-CD ring and sertraline base. Therefore, in this study, the currently used hydrochloride salt form was converted into the free base and ß-CD was used as a model for ß-CD derivatives to evaluate the interaction between ß-CD and the sertraline base. The solid-state physicochemical characteristics of the sertraline-ß-CD complex were investigated by the phase solubility method, differential scanning calorimetry, Fourier transform IR spectroscopy, FT-Raman spectroscopy, powder X-ray diffraction, and (13)C cross-polarization magic-angle spinning NMR measurements. The results showed that sertraline base and ß-CD form an inclusion complex, and the stoichiometric ratio of the solid-state sertraline base-ß-CD complex is 1:1, which was estimated by the (1)H NMR measurements of the complex dissolved in DMSO-d6.

Effect of terpenes on the skin permeation of lomerizine dihydrochloride.

PURPOSE: Lomerizine dihydrochloride (LOM) is a Ca2+ channel blocker used as an antimigraine drug, which is currently administered orally in Japan. We therefore investigated the effect of terpenes in propylene glycol (PG) solvent on the percutaneous absorption of LOM by hairless mouse skin. METHODS: Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), confocal laser scanning microscopy (CLSM), and small angle X-ray scattering (SAXS) were carried out to analyze the effects of terpene enhancers on the biophysical properties of the stratum corneum (SC) of the skin. RESULTS: Of the terpenes tested, the highest permeation rate of LOM (28.8 mg/cm2/h) was observed with 1,8-cineole, while nerolidol conferred the lowest enhancement of LOM flux (14.2 mg/cm2/h). ATR-FTIR studies revealed that terpenes/PG induced higher CH2 stretching frequencies of SC lipids than PG alone. The extent of penetration of the lipophilic fluorescence probes Nile Red and DiI was measured by CLSM in in vitro skin permeation studies, using either PG or terpenes/PG as skin permeation enhancers. With PG alone, both fluorescence dyes were undetectable in the skin. In contrast, when co-administered with terpenes/PG, both probes were distributed into the intercellular space between corneocytes and detected in the deeper layers of the skin. SAXS measurements showed that in SC treated with a combination of 1,8-cineole and PG, the scattering peak of the SC was broad and very weak in intensity compared to untreated SC, whereas pretreatment with PG alone did not alter the peak profile. CONCLUSION: A combination of terpenes and PG enhance the skin permeation of LOM. Our findings suggest that the mechanism for this effect involves the ability of terpenes to increase the fluidity of SC lipids, thus enhancing the distribution of LOM into the intercellular region of the SC. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Quetiapine free base complexed with cyclodextrins to improve solubility for parenteral use.

Quetiapine, an antipsychotic drug used for schizophrenia treatment, is poorly water soluble, and therefore, administration of the more water-soluble quetiapine fumarate is preferred. Absorption of quetiapine through biological membranes may be improved by enhancing the solubility of the quetiapine base, the non-ionic form. In this study, the currently used salt form was converted into the free base (oily material). We employed cyclodextrins (CDs) as pharmaceutical additives to improve the solubility of the quetiapine base. The formation of quetiapine-ß-cyclodextrin (ß-CD) complexes was studied by phase solubility studies, continuous variation method, NMR spectroscopy, and powder X-ray diffraction. The formation of a poorly water-soluble complex was confirmed by the phase solubility study, and the interaction between quetiapine and ß-CD in water was confirmed by NMR spectroscopy. In addition, the effects of ß-CD derivatives (glucosyl-ß-CD, maltosyl-ß-CD, 2-hydroxypropyl-ß-CD, dimethyl-ß-CD, and trimethyl-ß-CD) on the solubility of the quetiapine base were studied. The findings indicated that the aforementioned hydrophilic ß-CD derivatives could be used as pharmaceutical additives of quetiapine for parenteral formulations as a result of the improved solubility of the quetiapine base because of inclusion complexation. Therefore, converting the currently used salt form into the free base, investigating the free base as a candidate for CD inclusion, and converting the oily material such as the free base into a powder by forming an inclusion complex that is easy to deal with is considered a worthwhile approach that may lead to novel formulations of the drug in question.

Evaluation of the interaction between ß-cyclodextrin and psychotropic drugs by surface plasmon resonance assay with a Biacore ® system.

Phase-solubility studies have been used to evaluate the solubilizing effects of cyclodextrins (CDs) on lipophilic, water-insoluble drugs. However, large amounts of CDs and drugs are required to measure solubility by phase-solubility studies. Thus, more efficient approaches to evaluate the interaction of CDs with drugs are needed. Herein we introduce a method that evaluates the interaction between immobilized ß-cyclodextrin and psychotropic drugs by surface plasmon resonance assay with a Biacore(®) system. Association constants and stoichiometries observed were generally consistent with values calculated by traditional methods, such as phase-solubility and continuous variation methods. Results showed that the analytical method using Biacore(®) was suitable to evaluate CD-drug interactions.

yCD/HPyCD mixtures as solubilizer: solid-state characterization and sample dexamethasone eye drop suspension.

PURPOSE: Study the complexation of dexamethasone in combinations of γ-cyclodextrin (γCD) and 2-hydroxypropyl-γ-cyclodextrin (HPγCD) with emphasis on solid characterization and development of aqueous dexamethasone eye drop suspension for drug delivery through sclera. METHODS: Dexamethasone/cyclodextrin (dexamethasone/CD) solid complex systems were prepared and characterized by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and by in vitro drug dissolution testing. Sample eye drop suspensions were prepared applying solubilizer/suspender consisting of γCD/HPγCD mixtures, poloxamer 407 (P407) and polyvinylpyrrolidone. The eye drop suspension was characterized by its physicochemical properties. RESULTS: The solid characterization techniques applied suggested that solid complexes were being formed. The results indicated that dexamethasone formed non-inclusion or micelle-like aggregates with HPγCD and the γCD/HPγCD mixture. The dissolution and dexamethasone release from the solid dexamethasone/γCD/HPγCD complexes was much faster than from the solid dexamethasone/γCD and dexamethasone/HPγCD complexes. The diameter of the solid particles in the dexamethasone eye drop suspension formulations were in all cases less than 10 µm with a mean diameter from 2.5 to 5.8 µm. The particle size decreased with increasing amount of P407. Permeation studies through semipermeable membrane and porcine sclera showed that increasing the amount HPγCD could enhance drug transport through the membrane barriers and this was related to enhanced drug solubility. The permeation rates were, however, decreased compared to formulation containing γCD alone due to larger hydrodynamic diameter of dexamethasone/γCD/HPγCD complex aggregates. All formulations were both chemically stable for at least 8 months at 25°C and 40°C. CONCLUSIONS: Combination of γCD and HPγCD, i.e., formation of dexamethasone/γCD/HPγCD complexes, resulted in synergistic effect. That is the mixture had greater solubilizing effect than the individual CD, resulted in enhanced dissolution and drug delivery through membranes. Furthermore, it is possible to control the drug release rate by adjusting the γCD:HPγCD ratio in the solid dexamethasone/γCD/HPγCD complexes.

Effects of cogrinding with ß-cyclodextrin on the solid state fentanyl.

Fentanyl base and ß-cyclodextrin (ß-CD) were coground at 1:1 and 1:2 molar ratios (fentanyl: ß-CD) and the physicochemical characteristics of the mixtures were studied using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), solid state (13)C nuclear magnetic resonance (NMR) spectroscopy and powder X-ray diffraction (PXRD) measurement. Additionally, portions of the coground samples were exposed to high relative humidity to investigate fentanyl and ß-CD interactions. The results of DSC and PXRD analyses indicate that the ground mixtures are in an amorphous state, and the FTIR measurements show hydrogen bonding interactions between fentanyl and ß-CD. Solid state (13)C NMR indicates that a fentanyl/ß-CD inclusion compound is formed in the humidified mixture. Furthermore, PXRD data from the humidified mixtures are similar to the PXRD patterns from the inclusion complex.

Effect of beta-cyclodextrin on the degradation rate of amoxicillin in acidic solution.

The formation of an inclusion complex of amoxicillin (AMX) with beta-cyclodextrin (beta-CD) in aqueous solution was confirmed by a solubility method and proton nuclear magnetic resonance (1H-NMR) spectroscopy. The apparent stability constant for the inclusion complex was 10.72 M(-1) in water at 25 degrees C. The effect of alpha-CD, beta-CD, and gamma-CD on the degradation of AMX in a pH 1.2 solution at 37 degrees C was investigated. beta-CD and gamma-CD reduced the rate of degradation. alpha-CD had no effect. These results were consistent with those of 1H-NMR spectroscopy. The effect of beta-CD on the degradation rate was studied in more detail. The apparent first order rate constant for the degradation of AMX in the pH 1.2 solution at 37 degrees C was 0.1121 h(-1) (t(1/2)=6.18 h), which decreased with the addition of beta-CD. The rate constants and t(1/2) values for the concentrations of beta-CD added, corresponding to molar ratios of AMX to beta-CD of 1:0.5, 1:1, 1:2, 1:5, and 1:10, were 0.1051 h(-1) and 6.59 h, 0.0992 h(-1) and 6.98 h, 0.0893 h(-1) and 7.76 h, 0.0697 h(-1) and 9.95 h, and 0.0509 h(-1) and 13.61 h, respectively. The activation energy for the degradation of AMX in the pH 1.2 solution was increased from 6.9 x 10(4) J/mol (AMX alone) to 8.0 x 10(4) J/mol (AMX:beta-CD=1:10).

Carbon-13 NMR relaxation study of the internal dynamics in cyclodextrins in isotropic solution.

(13)C nuclear spin relaxation processes in seven cyclodextrins (from six-membered alpha to twelve-membered eta) were investigated in (2)H(2)O solution at multiple magnetic fields. Detailed analysis of (13)C longitudinal relaxation in laboratory and rotating frames and (13)C{(1)H} nuclear Overhauser enhancement in these molecules yielded their rotational diffusion tensors and a semiquantitative picture of their internal dynamics.

Study on preparation and formation mechanism of n-alkanol/water emulsion using alpha-cyclodextrin.

Surfactants are usually used for the preparation of emulsions; however, some have an adverse effect on the human body such as skin irritation, hemolysis, and protein denaturation, etc. In this study, we examined the preparation and formation mechanism of n-alkanol/water emulsions using alpha-cyclodextrin (alpha-CD) as an emulsifier. Emulsions were prepared by mixing oil and water phases for 4 min at 2500 rpm using a vortex mixer. The mechanism of emulsification was investigated with some physico-chemical techniques. From phase diagrams of n-alkanol/alpha-CD/water systems, the emulsion phase extended as the chain length of n-alkanols and the amount of alpha-CD added increased. Furthermore, the emulsion was not formed in the region where the n-alkanol/alpha-CD complex didn't precipitate; however, the emulsion was formed in the region where the complex precipitated. In addition, it was clear that the emulsions have a yield stress value and correspond to the Maxwell model from rheological measurement. Our experiments clearly showed that the stable emulsions are formed because the precipitated complexes form a dense film at the oil-water interface and prevent aggregation among dispersed phases. Furthermore, it is suggested that the creation of a three-dimensional network structure formed by precipitated complexes in the continuous phase contributes to the stabilization of the emulsion. Thus, we concluded that the n-alkanol/water emulsions using alpha-cyclodextrin were a kind of the Pickering emulsion.

Effects of various cyclodextrins on the stability of freeze-dried lactate dehydrogenase.

The effects of eight cyclodextrins (CDs) on the stability of freeze-dried lactate dehydrogenase (LDH) were investigated. Five low-molecular weight saccharides were used as a reference. In the samples without saccharides, LDH activity was decreased by freeze-drying. The LDH activities of the samples with 2-hydroxypropylated CDs (HP-CDs) such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) showed only a small decrease and were more effective protein stabilizers than low-molecular weight saccharides. HP-beta-CD more effectively stabilized LDH than trehalose, at a sugar concentration of less than 1.0 w/w% and furthermore, a close relationship existed between the highly stabilizing effect and the degree of substitution of polar substituents.