Study of the Structural Chemistry of the Inclusion Complexation of 4-Phenylbutyrate and Related Compounds with Cyclodextrins in Solution: Differences in Inclusion Mode with Cavity Size Dependency.

4-phenylbutyrate (PB) and structurally related compounds hold promise for treating many diseases, including cancers. However, pharmaceutical limitations, such as an unpleasant taste or poor aqueous solubility, impede their evaluation and clinical use. This study explores cyclodextrin (CD) complexation as a strategy to address these limitations. The structural chemistry of the CD complexes of these compounds was analyzed using phase solubility, nuclear magnetic resonance (NMR) spectroscopic techniques, and molecular modeling to inform the choice of CD for such application. The study revealed that PB and its shorter-chain derivative form 1:1 αCD complexes, while the longer-chain derivatives form 1:2 (guest:host) complexes. αCD includes the alkyl chain of the shorter-chain compounds, depositing the phenyl ring around its secondary rim, whereas two αCD molecules sandwich the phenyl ring in a secondary-to-secondary rim orientation for the longer-chain derivatives. ßCD includes each compound to form 1:1 complexes, with their alkyl chains bent to varying degrees within the CD cavity. γCD includes two molecules of each compound to form 2:1 complexes, with both parallel and antiparallel orientations plausible. The study found that αCD is more suitable for overcoming the pharmaceutical drawbacks of PB and its shorter-chain derivative, while ßCD is better for the longer-chain derivatives.

Supramolecular Assembly of Hybrid Pt(II) Porphyrin/Tomatine Analogues with Different Nanostructures and Cytotoxic Activities.

A simple strategy for synthesizing supramolecular hybrids was developed for the preparation of bioavailable nanohybrid photosensitizers by assembling visible-light-sensitive Pt(II) meso-tetrakis(4-carboxyphenyl)porphyrinporphyrin (PtTCPP)/tomatine analogues. The hybrids were self-assembled into nanofibrous or nanosheet structures approximately 3-5 nm thick and several micrometers wide. α-Tomatine generated a unique fibrous vesicle nanostructure based on intermolecular interactions, while dehydrotomatine generated nanosheet structures. Nanoassembly of these fibrous vesicles and sheets directly affected the properties of the light-responsive photosensitizer for tumor photodynamic therapy (PDT), depending on the nanostructure of the hybrid PtTCPP/tomatine analogues. The cytotoxicity of PtTCPP to cancer cells under photoirradiation was significantly enhanced by a tomatine assembly with a fibrous vesicle nanostructure, attributable to increased incorporation of the drug into cells.

Sacran, a sulfated polysaccharide, suppresses the absorption of lipids and modulates the intestinal flora in non-alcoholic steatohepatitis model rats.

AIMS: The objective of this study was to investigate the effects of administering sacran, a sulfated polysaccharide, on liver biology, gut microbiota, oxidative stress, and inflammation on stroke-prone spontaneously hypertensive (SHRSP5/Dmcr) rats that develop fibrotic steatohepatitis with histological similarities to that of non-alcoholic steatohepatitis (NASH). MAIN METHODS: Four groups of 8-week-old SHRSP5/Dmcr rats were fed a high fat-cholesterol (HFC) diet for 4 and 8 weeks and administered either sacran (80 mg/kg/day) or a non-treatment, respectively. Liver function was evaluated by biochemical and histopathological analyses. Hepatic inflammatory markers were measured using mRNA expression. Fecal microbial profiles were determined via 16S rRNA sequencing. A triglyceride (TG) absorption test was administered to the 8-week-old Sprague-Dawley (SD) rats. KEY FINDING: Sacran administration was observed to decrease the extent of oxidative stress and hepatic biochemical parameters in serum and hepatic injury with the levels of transforming growth factor-beta (TGF-ß1) and tumor necrosis factor-alpha (TNF-α), being increased compared to those of the non-treatment group. At the genus level, sacran administration caused a significant decrease in the harmful Prevotella genus, and a significant increase in the useful Blautia genus was observed. Sacran administration also decreased the serum TG increase that was induced by administering corn oil to the SD rats. SIGNIFICANCE: We conclude that sacran administration has the potential to reduce the absorption of lipids into blood and to improve several gut microbiotas, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress and hepatic markers in the systematic circulation on NASH.

Polypseudorotaxane-based supramolecular hydrogels consisting of cyclodextrins and Pluronics as stabilizing agents for antibody drugs.

Recently, antibody drugs have been used worldwide, and based on worldwide sales, 7 of the top 10 pharmaceutical products in 2019 were antibody-based drugs. However, antibody drugs often form aggregates upon thermal and shaking stresses with few efficient stabilizing agents against both stresses. Herein, we developed polypseudorotaxane (PpRX) hydrogels consisting of cyclodextrins (CyDs) and polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG block copolymers (Pluronics F108, F87, F68, and L44), and evaluated their utility as antibody stabilizing agents. α- and γ-CyDs formed PpRX hydrogels with Pluronics, where CyD/F108 gels showed remarkable stabilizing effects for human immunoglobulin G (IgG) against both thermal and shaking stresses beyond CyD/PEG gels or generic gels. The effects were probably due to the interaction between IgG and the free PPG block of Pluronic F108, resulting in the strong IgG retention in the gels. These findings suggest the great potential of CyD/Pluronic gels as pharmaceutical materials for antibody formulations.

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.

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.

Biomaterials based on freeze dried surface-deacetylated chitin nanofibers reinforced with sulfobutyl ether ß-cyclodextrin gel in wound dressing applications.

A freeze-dried gel composed of surface-deacetylated chitin nanofibers (SDACNFs), reinforced with an anionic cyclodextrin, sulfobutyl ether ß-cyclodextrin (SBE-ß-CD) was evaluated for treating wounds in a rat model, and the results were compared with a SDACNFs gel without SBE-ß-CD. The incorporation of prednisolone (PD), a poorly water-soluble drug, in both types of gels and its release from the gels were also compared. In both cases, wound areas were decreased and their effect was higher than that of commercially available wound dressings. The rate of release of PD from the freeze-dried SDACNFs/SBE-ß-CD was much faster than that form SDACNFs alone without SBE-ß-CD, due to fact that the PD is more soluble in the amorphous SBE-ß-CD complex compared to the other preparations. The findings indicate that the freeze-dried SDACNFs/SBE-ß-CD gel would be beneficial as a new biomaterial for the treatment of wounds and for preparing homogeneous high-content gels that contain poorly water-soluble drugs.

In Vitro and In Vivo Evaluation of Hydrophilic C60(OH)10/2-Hydroxypropyl-ß-cyclodextrin Nanoparticles as an Antioxidant.

The objective of this study was to assess the antioxidant ability of C60(OH)10/2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) nanoparticles, by comparing their scavenging ability for reactive nitrogen species, their cytoprotective effects under conditions of oxidative stress, and their therapeutic effects against diseases that are induced by oxidative stress. The C60(OH)10/HP-ß-CD nanoparticles had a higher scavenging activity against nitric acid and peroxynitrite (ONOO(-)) than the other antioxidants such as ascorbic acid, trolox, and edaravone. The cytoprotective effect of C60(OH)10/HP-ß-CD nanoparticles was examined on HeLa and HepG2 cells by monitoring the percentage of cell death induced by H2O2. Treatment with C60(OH)10/HP-ß-CD nanoparticles resulted in an increase in cell viability, due to the suppression of the oxidative stress. Furthermore, the nanoparticles had a high cytoprotective effect, compared with other polyhydroxylated C60 (C60(OH)24 and C60(OH)40). The C60(OH)10/HP-ß-CD nanoparticles were intravenously administered to mice with a liver injury induced by an over dose of acetaminophen. Levels of alanine transaminase and aspartate transaminase were essentially the same as those of normal mice and the survival rate was also prolonged by the intravenous administration of the C60(OH)10/HP-ß-CD nanoparticles. The results indicate that C60(OH)10/HP-ß-CD nanoparticles are a promising antioxidant for use in the treatment of diseases caused by oxidative stresses.

Thermoresponsive Formation of Dimethyl Cyclodextrin Polypseudorotaxanes and Subsequent One-Pot Synthesis of Polyrotaxanes.

We demonstrated a new strategy for efficient preparation of polypseudorotaxanes (PpRXs) and polyrotaxanes (PRXs) with cyclodextrin derivatives, 2,6-di-O-methyl-cyclodextrins (DM-CyDs), by utilizing the cloud points of DM-CyDs. DM-α-CyD and DM-ß-CyD formed PpRXs with polyethylene glycol (PEG) and polypropylene glycol (PPG) in water at >50 °C and >35 °C, respectively, but did not at room temperature. Meanwhile, randomly methylated ß-CyD (RM-ß-CyD) and 2,3,6-tri-O-methyl-ß-CyD (TM-ß-CyD) did not form PpRX with PPG at higher temperature. The driving force of thermoresponsive formation of DM-CyD PpRXs was derived from hydrophobic interaction of methyl groups and a hydrogen bond of hydroxyl groups formed by adjacent DM-CyD molecules. Furthermore, in one pot, DM-CyD PRXs were synthesized by capping the PpRXs with bulky ends in high yields.

Slow-release of famotidine from tablets consisting of chitosan/sulfobutyl ether ß-cyclodextrin composites.

An intermolecular complex formed from a 1:1 weight ratio of chitosan (CS, molecular weight 30 kDa) and sulfobutyl ether ß-cyclodextrin (SBE-ß-CyD, degree of substitution 7) was less soluble than either of the original components. The release of famotidine from tablets composed of a simple mixture of CS and SBE-ß-CyD is slower in media at pH 1.2 than at 6.8. Macroscopic observation of tablets and a kinetic analysis of release profiles suggested that, at pH 1.2, the drug was slowly released from the less-soluble CS/SBE-ß-CyD complex formed on the surface of the tablet immediately after exposure to water, accompanied by the dissolution of the interpolymer complex and, ultimately, the erosion and disintegration of the tablet. In the case of the medium at pH 6.8, the formation of a gel by CS was the cause of the slow release, especially for CS/SBE-ß-CyD tablets which were significantly gelated and both the diameter and thickness of the tablet had expanded. The in vitro slow releasing characteristic of the CS/SBE-ß-CyD tablet was reflected in the in vivo absorption of the drug after oral administration to rats. These results suggest that a simple mixing of CS and SBE-ß-CyD is potentially useful for the controlled release of a drug.

Preparation of hydrophilic C60(OH)10/2-hydroxypropyl-ß-cyclodextrin nanoparticles for the treatment of a liver injury induced by an overdose of acetaminophen.

Stable hydrophilic C60(OH)10 nanoparticles were prepared from 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and applied to the treatment of an acetaminophen overdose induced liver Injury. C60(OH)10 nanoparticles were produced by cogrinding α-CD, ß-CD, γ-CD and HP-ß-CD and characterized in terms of solubility, mean particle diameter, ζ-potential and long term dispersibility in water. Hydrophilic C60(OH)10 nanoparticles with particle sizes less than 50 nm were effectively produced by cogrinding HP-ß-CD with C60(OH)10 at a molar ratio of 1:3 (C60(OH)10:CD). The resulting C60(OH)10/HP-ß-CD nanoparticles were stable in water and showed no aggregation over a 1 month period. The C60(OH)10/CDs nanoparticles scavenged not only free radicals (DPPH and ABTS radicals) but also reactive oxygen species (O2(•-) and •OH). When C60(OH)10/HP-ß-CD nanoparticles were intraperitoneally administered to mice with a liver injury induced by an overdose of acetaminophen (APAP), the ALT and AST levels were markedly reduced to almost the same level as that for normal mice. Furthermore, the administration of the nanoparticles prolonged the survival rate of liver injured mice, while all of the mice that were treated with APAP died within 40 h. To reveal the mechanism responsible for liver protection by C60(OH)10 nanoparticles, GSH level, CYP2E1 expression and peroxynitrite formation in the liver were assessed. C60(OH)10/HP-ß-CD nanoparticles had no effect on CYP2E1 expression and GSH depletion, but suppressed the generation of peroxynitrite in the liver. The findings indicate that the protective effect of C60(OH)10/HP-ß-CD nanoparticles was due to the suppression of oxidative stress in mitochondria, as the result of scavenging ROS such as O2(•-), NO and peroxynitrite, which act as critical mediators in the liver injuries.

Possible enhancing mechanisms for gene transfer activity of glucuronylglucosyl-ß-cyclodextrin/dendrimer conjugate.

We previously reported that glucuronylglucosyl-ß-cyclodextrin (GUG-ß-CyD) conjugate with polyamidoamine starburst dendrimer (GUG-ß-CDE conjugate) with the average degree of substitution (DS) of cyclodextrin (CyD) of 1.8 (GUG-ß-CDE conjugate (DS 1.8)), showed remarkably higher gene transfer activity than α-CyD/dendrimer conjugate (α-CDE conjugate (DS 1.2)) and ß-CyD/dendrimer conjugate (ß-CDE conjugate (DS 1.3)) in vitro and in vivo. In this study, to clarify the enhancing mechanism for high gene transfer activity of GUG-ß-CDE conjugate (DS 1.8), we investigated the physicochemical properties, cellular uptake, endosomal escape and nuclear translocation of the plasmid DNA (pDNA) complexes as well as pDNA release from the complexes. The particle size, ζ-potential and cellular uptake of GUG-ß-CDE conjugate (DS 1.8)/pDNA complex were mostly comparable to those of α-CDE conjugate (DS 1.2) and ß-CDE conjugate (DS 1.3). Meanwhile, GUG-ß-CDE conjugate (DS 1.8)/pDNA complex was likely to have high endosomal escaping ability and nuclear localization ability in A549 and RAW264.7 cells. In addition, the pDNA condensation and decondensation abilities of GUG-ß-CDE conjugate (DS 1.8) were lower and higher than that of α-CDE conjugate (DS 1.2) or ß-CDE conjugate (DS 1.3), respectively. These results suggest that high gene transfer activity of GUG-ß-CDE conjugate (DS 1.8) could be, at least in part, attributed to high endosomal escaping ability, nuclear localization ability and suitable pDNA release from its complex.

Evaluation of photodynamic activity of C60/2-hydroxypropyl-ß-cyclodextrin nanoparticles.

The objective of this study is to evaluate the ability of C(60)/2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) naonparticles to generate reactive oxygen species (ROS) and to induce cell toxicity by the photoirradiation. C(60) nanoparticles were prepared by cogrinding with HP-ß-CyD for 3 h at 4°C under reduced pressure. The photodynamic activity of C(60)/HP-ß-CyD nanoparticles was evaluated by spectroscopic methods, including the electron spin resonance spin-trapping method, and by the cell viability test using Hela cells. C(60)/HP-ß-CyD nanoparticles efficiently generated not only superoxide anion radical (O(2)(·-)) and hydroxyl radical (·OH), but also singlet oxygen ((1)O(2)) through photoirradiation. The ROS generation was enhanced by decreasing the mean particle diameter of C(60) nanoparticles, and the particle size smaller than 90 nm showed a high generation of ·OH and (1)O(2). In addition, HP-ß-CyD enhanced the generation of (1)O(2), compared with polyvinylpyrrolidone (an effective solubillizer for C(60)), due to partial disposition of C(60) in the hydrophobic CyD cavity. Furthermore, C(60) /HP-ß-CyD nanoparticles showed cell toxicity after the light irradiation, but no toxicity was observed without the light irradiation. Therefore, HP-ß-CyD is useful for the preparation of stable C(60) nanoparticles with high ROS generation ability, and C(60)/HP-ß-CyD nanoparticles are a promising photosensitizer for photodynamic therapy.

Potential use of glucuronylglucosyl-ß-cyclodextrin/dendrimer conjugate (G2) as a DNA carrier in vitro and in vivo.

In this study, we evaluated the polyamidoamine starburst dendrimer (dendrimer, generation 2: G2) conjugate with 6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-ß-cyclodextrin (GUG-ß-CDE (G2)) as a gene transfer carrier. The in vitro gene transfer activity of GUG-ß-CDE (G2, degree of substitution (DS) of cyclodextrin (CyD) 1.8) was remarkably higher than that of dendrimer (G2) conjugate with α-CyD (α-CDE (G2, DS 1.2)) and that with ß-CyD(ß-CDE (G2, DS 1.3)) in A549 and RAW264.7 cells. The particle size, ζ-potential, DNase I-catalyzed degradation, and cellular association of plasmid DNA (pDNA) complex with GUG-ß-CDE (G2, DS 1.8) were almost the same as those of the other CDEs. Fluorescent-labeled GUG-ß-CDE (G2, DS 1.8) localized in the nucleus 6 h after transfection of its pDNA complex in A549 cells, suggesting that nuclear localization of pDNA complex with GUG-ß-CDE (G2, DS 1.8), at least in part, contributes to its high gene transfer activity. GUG-ß-CDE (G2, DS 1.8) provided higher gene transfer activity than α-CDE (G2, DS 1.2) and ß-CDE (G2, DS 1.3) in kidney with negligible changes in blood chemistry values 12 h after intravenous injection of pDNA complexes with GUG-ß-CDE (G2, DS 1.8) in mice. In conclusion, the present findings suggest that GUG-ß-CDE (G2, DS 1.8) has the potential for a novel polymeric pDNA carrier in vitro and in vivo.

Inhibitory effect of siRNA complexes with polyamidoamine dendrimer/α-cyclodextrin conjugate (generation 3, G3) on endogenous gene expression.

In the present study, we prepared the small interfering RNA (siRNA) complexes with polyamidoamine (PAMAM) dendrimer (G3) conjugate with α-cyclodextrin (α-CDE (G3)), and examined the physicochemical properties, serum resistance, in vitro RNAi effects on endogenous gene expression, cytotoxicity, interferon response, hemolytic activity, cellular association and intracellular distribution. In addition, these results were compared to the siRNA complexes with the commercial transfection reagents such as linear polyethyleneimine (PEI), Lipofectamine™2000 (L2) and RNAiFect™ (RF). α-CDE (G3) interacted with siRNA, and suppressed siRNA degradation by serum. The siRNA complex with α-CDE (G3) showed the potent RNAi effects against Lamin A/C and Fas expression with negligible cytotoxicity and hemolytic activity, compared to those of the transfection reagents in Colon-26-luc cells and NIH3T3-luc cells. Cell-death patterns induced by siRNA polyplexes with α-CDE (G3) and PEI were different from siRNA lipoplexes with L2 and RF. α-CDE (G3) delivered fluorescent-labeled siRNA to cytoplasm, not nucleus, after transfection in NIH3T3-luc cells. Taken together, α-CDE (G3) could be potentially used as a siRNA carrier to provide the RNAi effect on endogenous gene expression with negligible cytotoxicity.

Effect of sulfobutyl ether-ß-cyclodextrin on bioavailability of insulin glargine and blood glucose level after subcutaneous injection to rats.

Insulin glargine is the first long-acting basal insulin analogue used for subcutaneous administration once daily in patients with type 1 or type 2 diabetes mellitus. To obtain the further bioavailability and the sustained glucose lowering effect of insulin glargine, in the present study, we investigated the effect of sulfobutyl ether-ß-cyclodextrin (SBE4-ß-CyD), with the degree of substitution of sulfobutyl ether group of 3.9, on pharmaceutical properties of insulin glargine and the release of insulin glargine after subcutaneous injection to rats. SBE4-ß-CyD increased the solubility and suppressed aggregation of insulin glargine in phosphate buffer at pH 9.5, probably due to the interaction of SBE4-ß-CyD with aromatic amino acid residues such as tyrosine of insulin glargine. In addition, SBE4-ß-CyD accelerated the dissolution rate of insulin glargine from its precipitates, compared to that of insulin glargine alone. Furthermore, we revealed that subcutaneous administration of an insulin glargine solution with SBE4-ß-CyD to rats enhanced the bioavailability of insulin glargine and sustained the glucose lowering effect, possibly due to the inhibitory effects of SBE4-ß-CyD on the enzymatic degradation at the injection site. These results suggest that SBE4-ß-CyD can be a useful excipient for sustained release of insulin glargine.

Enhancement of the aqueous solubility and masking the bitter taste of famotidine using drug/SBE-beta-CyD/povidone K30 complexation approach.

The objective of the present study was to evaluate the potential of ternary system (comprised of famotidine, beta-cyclodextrin (beta-CyD) or its derivatives and a hydrophilic polymer) as an approach for enhancing the aqueous solubility and masking the bitter taste of famotidine. The aqueous solubility of famotidine increased in the presence of beta-CyDs, particularly sulfobutyl ether beta-CyD (SBE-beta-CyD), and it was further enhanced by the combination of SBE-beta-CyD and polyvinyl pyrrolidone (Povidone) K30. The solid binary (drug-beta-CyDs) and ternary (drug-beta-CyDs-Povidone K30) systems were prepared by the kneading and freeze-drying methods. The dissolution rates of these solid systems were much faster than that of the drug alone. A taste perception study was carried out, initially using a taste sensory machine and subsequently on human volunteers to evaluate the taste masking ability of the ternary complexation. Our results indicated that the combination of SBE-beta-CyD and Povidone K30 is effective not only in the enhancement of the solubility and dissolution rate of famotidine, but also in masking of the bitter taste of the drug. This technique may be of value for the pharmaceutical industries, especially in preparation of rapidly disintegrating tablets dealing with bitter drugs to improve patient compliance and thus effective pharmacotherapy.

5-Fluorouracil acetic acid/beta-cyclodextrin conjugates: drug release behavior in enzymatic and rat cecal media.

5-Fluorouracil-1-acetic acid (5-FUA) was prepared and covalently conjugated to beta-cyclodextrin (beta-CyD) through ester or amide linkage, and the drug release behavior of the conjugates in enzymatic solutions and rat cecal contents were investigated. The 5-FUA/beta-CyD ester conjugate was slowly hydrolyzed to 5-FUA in aqueous solutions (half lives (t(1/2))=38 and 17h at pH 6.8 and 7.4, respectively, at 37 degrees C), whereas the amide conjugate was hardly hydrolyzed at these physiological conditions, but hydrolyzed only in strong alkaline solutions (>0.1M NaOH) at 60 degrees C. Both ester and amide conjugates were degraded in solutions of a sugar-degrading enzyme, alpha-amylase, to 5-FUA/maltose and triose conjugates, but the release of 5-FUA was only slight in alpha-amylase solutions. In solutions of an ester-hydrolyzing enzyme, carboxylic esterase, the ester conjugate was hydrolyzed to 5-FUA at the same rate as that in the absence of the enzyme, whereas the amide conjugate was not hydrolyzed by the enzyme. On the other hand, 5-FUA was rapidly released when the ester conjugate was firstly hydrolyzed by alpha-amylase, followed secondly by carboxylic esterase. The results indicated that the ester conjugate was hydrolyzed to 5-FUA in a consecutive manner, i.e. it was firstly hydrolyzed to the small saccharide conjugates, such as the maltose conjugate, by alpha-amylase, and the resulting small saccharide conjugates having less steric hindrance was susceptible to the action of carboxylic esterase, giving 5-FUA. The in vitro release behavior of the ester conjugate was clearly reflected in the hydrolysis in rat cecal contents and in the in vivo release after oral administration to rats.

Involvement of PI3K-Akt-Bad pathway in apoptosis induced by 2,6-di-O-methyl-beta-cyclodextrin, not 2,6-di-O-methyl-alpha-cyclodextrin, through cholesterol depletion from lipid rafts on plasma membranes in cells.

Cyclodextrins (CyDs), which are widely used to increase the solubility of drug in pharmaceutical fields, are known to induce hemolysis and cytotoxicity at high concentrations. However, it is still not unclear whether cell death induced by CyDs is apoptosis or not. Therefore, in the present study, we investigated the effects of various kinds of CyDs on apoptosis in the cells such as NR8383 cells, A549 cells and Jurkat cells. Of various CyDs, methylated CyDs inducted cell death under the present experimental conditions, but hydroxypropylated CyDs or sulfobutyl ether-beta-CyD (SBE7-beta-CyD) did not. Of methylated CyDs, 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TM-beta-CyD) markedly caused apoptosis in NR8383 cells, A549 cells and Jurkat cells, through cholesterol depletion in cell membranes. In sharp contrast, 2,6-di-O-methyl-alpha-cyclodextrin (DM-alpha-CyD) and methyl-beta-cyclodextrin (M-beta-CyD) induced cell death in an anti-apoptotic mechanism. DM-beta-CyD induced apoptosis through the inhibition of the activation of PI3K-Akt-Bad pathway. Neither p38 MAP kinase nor p53 was contributed to the induction of apoptosis by DM-beta-CyD. Additionally, DM-beta-CyD significantly decreased mitochondrial transmembrane potential, and then caused the release of cytochrome c from mitochondria to cytosol in NR8383 cells. Furthermore, we confirmed that down-regulation of pro-caspase-3 and activation of caspase-3 after incubation with DM-beta-CyD. These results suggest that of methylated CyDs, DM-beta-CyD, not DM-alpha-CyD, induces apoptosis through the PI3K-Akt-Bad pathway, resulting from cholesterol depletion in lipid rafts of cell membranes.

Polypseudorotaxane formation of randomly-pegylated insulin with cyclodextrins: slow release and resistance to enzymatic degradation.

Pegylation technology has been widely used to improve therapeutic efficacies of protein drugs and a number of selective- or randomly-substituted pegylated proteins are on the market. In this study, we prepared a insulin derivative substituted randomly with poly(ethylene glycol) (PEG, MW about 2200) and its polypseudorotaxanes with cyclodextrins (CyDs). 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/CyD polypseudorotaxanes were less soluble in water. The release rate of the pegylated protein from its polypseudorotaxanes decreased in the order of drug alone>the gamma-CyD polypseudorotaxane>the alpha-CyD polypseudorotaxane. The pegylated insulin/gamma-CyD polypseudorotaxane displayed a significantly higher resistance to proteolysis. The results indicated that the CyD polypseudorotaxanes could be formed with randomly-pegylated insulin and work not only as a sustained release system, but also as a stabilizing agent to enzymatic degradations of pegylated insulin.