Incorporation of the antibacterial agent, miconazole nitrate into a cellulosic fabric grafted with β-cyclodextrin

The aim of the study was to investigate the incorporation of the antibacterial agent, miconazole nitrate into cyclodextrin cavities covalently bonded onto cloth fibers. The cellulosic fabric was grafted with β-cyclodextrin molecules through reaction with monochlorotriaziny β-cyclodextrin (MCT-β-CD). The suitable bonded reaction conditions were found to be MCT-β-CD 60–100 g/L, catalyst Na₂CO₃ 50–60 g/L, the reaction temperature 150–160 °C and the reaction time 5–8 min.

The modified and unmodified fabrics were characterized by UV spectrophotometry. The level of miconazole nitrate entrapped in the fabrics were determined by HPLC and was founded to be much higher (0.458% w/w) for the textile functionalized with MCT-β-CD compared to the unmodified fabric (0.056% w/w). The antibacterial abilities measured by shaker flask method showed that the antibacterial property was markedly enhanced by impregnation with miconazole nitrate of the MCT-β-CD grafted textile. The finished fabric kept the antibacterial abilities more than 70% even after washing 10 times, while the antibacterial activity of the unmodified textile was almost lost.     

Incorporation of chlorohexidin diacetate into cotton fabrics grafted with glycidyl methacrylate and cyclodextrin

Linear electron beam radiation was used to induce grafting of glycidyl methacrylate/β-cyclodextrin mixture onto cotton fabric. Chlorohexidin diacetate was incorporated to the cavities of cyclodextrin fixed on the cotton fabric to form an inclusion complex having antimicrobial activity. After incorporating chlorohexidin diacetate, the fabric was subjected to several washing cycles to examine the durability of the antimicrobial finishing. Control and grafted cotton fabrics (before and after loading with antimicrobial agent) were characterized for their antimicrobial activity against different kinds of bacteria and fungi.Grafted fabrics loaded with antimicrobial agent were found to show good antimicrobial activity in comparison with control and grafted fabrics which are not loaded with antimicrobial agent. The grafted fabrics loaded with antimicrobial agent were found also to exhibit good antimicrobial activity after five washings and this lasting antimicrobial activity can be attributed to the inclusion complex formed between chlorohexidin diacetate molecules and the cavities of cyclodextrin.     

Synthesis and biological evaluation of 2-amino-2-deoxy- and 6-amino-6-deoxy-cyclomaltoheptaose polysulfates as synergists for angiogenesis inhibition

2-Amino-2-deoxy-cyclomaltoheptaose was prepared from β-cyclodextrin perbenzoate [heptakis(2,3,6-tri-O-benzoyl)cyclomaltoheptaose] by a series of reactions including selective de-O-benzoylation at C-2 of one of the perbenzoylated -glucopyranosyl moieties, oxidation to the 2-ulose derivative, oxime formation, and reduction to the 2-amino-2-deoxy- -glucose moiety. This compound and 6-amino-6-deoxycyclomaltoheptaose accessible from β-cyclodextrin through the known procedure were sulfated to give polysulfated aminocyclomaltoheptaose derivatives (3, 5). Employing β-cyclodextrin polysulfate as a reference compound, the synergistic effects of 3 and 5 for cortexolone on angiogenesis inhibitory activity were examined by rabbit-corneal micropocket assay system. In contrast to the significant anti-angiogenesis activity of the β-cyclodextrin polysulfate-cortexolone pair, neither 3 nor 5 showed any cooperative activity with cortexolone in the inhibition of basic FGF-induced angiogenesis.     

Non-aqueous capillary electrophoresis chiral separations with sulfated β-cyclodextrin

This paper reports the application of an anionic cyclodextrin (CD), sulfated β-cyclodextrin with a degree of substitution of four (β-CD-(SO₄⁻)₄, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between β-CD-(SO₄⁻)₄–analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of β-CD-(SO₄⁻)₄ was needed due to the fact that the electrostatic attraction caused stronger binding between β-CD-(SO₄⁻)₄ and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.     

Determination of 2-hydroxypropyl-γ-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography–mass spectrometry

This report describes a specific and highly sensitive gas chromatography–mass spectrometry (GC–MS) assay for the analysis of industrially produced 2-hydroxypropyl-γ-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-γ-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-β-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2′-deuteromethoxypropylglucose (representative for HP-γ-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose (representative for the internal standard), respectively, and analyzed by GC–MS. The limit of quantification of this assay was 20 nmol/l.     

Charged cyclodextrin-mediated sample stacking in micellar capillary electrophoresis: A simple method for enhancing the detection sensitivity of hydrophobic compounds

The development of on-line sample stacking techniques for enhancing limits of detection of neutral analytes in micellar capillary electrophoresis (MCE) has recently gained much attention. Utilizing high-conductivity sample matrices to invoke sample stacking is promising, but requires the limited use of sample solubilizing agents such as alcohols in the sample matrix. In this study, we show how simple replacement of the sample solvent (methanol) with a solution of sulfated β-cyclodextrin (sβ-CD) allows a significant increase in the sensitivity of detection of model hydrophobic analytes. This increase in sensitivity is accompanied by significant peak sharpening. Sulfated CDs in the sample matrix allow for effective solubilization of hydrophobic analytes without the use of organic solvents such as methanol. The testing of various sample matrix sβ-CD concentrations for their effect on peak sharpening identified 3 to 5% as optimal for the estrogen standards. The use of a sβ-CD sample matrix allowed for hydrostatic injections (3.5 kPa) of 297 s, compared with 4 s when the analytes were dissolved in methanol. A mechanism explaining the sβ-CD-induced effect involves an analyte transfer mechanism where the sβ-CDs, despite providing anodic mobility to analytes in the sample zone, are able to transfer analytes to trailing separation buffer micelles for “recycling” back into the sample zone without compromising the stacking process. The overall improvement in sensitivity allows detection of estrogens in the parts-per-billion range and stands to improve the utility of MCE as a bioanalytical technique.     

Artemether/hydroxypropyl-β-cyclodextrin host–guest system: Characterization, phase-solubility and inclusion mode

An inclusion complex of the antimalarial artemether (ATM) in hydroxypropyl-β-cyclodextrin (HPβCD) was prepared and characterized. The phase-solubility diagram for the drug showed an increase in water solubility and gave an apparent binding constant of 220 M⁻¹. According to ¹H NMR and 2D NMR spectroscopy (ROESY), the inclusion mode involves two CH₃ from the drug orientated in the HPβCD cavity. The complex was characterized by Powder X-ray diffraction and thermal analysis. In addition, the complex produces a 1.81-fold enhancement in apparent bioavailability compared to artemether.     

Enhancement of 17alpha-hydroxyprogesterone production from progesterone by biotransformation using hydroxypropyl-beta-cyclodextrin complexation technique

An enhancement of 17α-hydroxyprogesterone (17α-HP) production from progesterone by biotransformation using hydroxypropyl-β-cyclodextrin (HPβCD) complexation together with aeration and sonication technique was demonstrated. The progesterone–hydroxypropyl-β-cyclodextrin complex was prepared by co-evaporation method. The percentage yield of 17α-HP from P of 11.26 ± 0.64% at 24 h was observed in Curvularia lunata ATCC 12017. In the complex form of P, together with sonication at 40 kHz for 5 s and aeration, the yield of 17α-HP was increased to 72.92 ± 4.28% which was about 6.5 and 1.3 times of that from the uncomplexed (P) and the complexed (PC), respectively without sonication and aeration. The increased aqueous solubility of P by complexation with HPβCD was the main factor which increased the yield of 17α-HP, while aeration had more effect on P than PC. Sonication did not significantly increased the yield of the product from both P and PC. When both aeration and sonication were used in the PC system, the product yield was increased significantly more than that from P. The result from this study can be applied for the biotransformation of other poor aqueous soluble precursors.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with β-cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- β-cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 μg 100 ml -1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- β-cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- β-cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 μg 100 ml -1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- β-cyclodextrin alone.     

Solubility enhancement of mefenamic acid by inclusion complex with β-cyclodextrin: in silico modelling,formulation, characterisation,and in vitro studies

The aim of this study was to prepare and characterise inclusion complexes of a low water-soluble drug, mefenamic acid (MA), with β-cyclodextrin (β-CD). First, the phase solubility diagram of MA in β-CD was drawn from 0 to 21 × 10⁻³ M of β-CD concentration. A job’s plot experiment was used to determine the stoichiometry of the MA:β-CD complex (2:1). The stability of this complex was confirmed by molecular modelling simulation. Three methods, namely solvent co-evaporation (CE), kneading (KN), and physical mixture (PM), were used to prepare the (2:1) MA:β-CD complexes. All complexes were fully characterised. The drug dissolution tests were established in simulated liquid gastric and the MA water solubility at pH 1.2 from complexes was significantly improved. The mechanism of MA released from the β-CD complexes was illustrated through a mathematical treatment. Finally, two in vitro experiments confirmed the interest to use a (2:1) MA:β-CD complex.     

TGF-β: from latent to active

The transforming growth factor-betas (TGF-βs) are synthesized as precursor proteins that are modified intracellularly prior to secretion. One of the most relevant intracellular modifications is the cleavage of the C-terminal pro-region from the N-terminal portion of the protein. The C-terminal pro-region is referred to as the latency-associated peptide (LAP) while the N-terminal region is called the mature TGF-β or active TGF-β. However, with some exceptions the LAP noncovalently associates with the mature TGF-β prior to secretion. When the mature TGF-β is associated with the LAP it is called L-TGF-β and cannot interact with its receptor and has no biological effect. The TGF-βs and their receptors are very ubiquitously expressed, suggesting that the regulation of TGF-β activity is likely to be complex and multifactorial. However, one of the most important means of controlling the biological effects of TGF-β is the regulation of converting L-TGF-β to active TGF-β. The current literature supports two major mechanisms of activation of L-TGF-β and suggests that the mechanism of activation of L-TGF-β may be varied and context-dependent. For TGF-β to become biologically active the LAP has to be either released from its associations with L-TGF-β or undergo conformational change such that the LAP is not released from the L-TGF-β complex but exposes the TGF-β receptor binding site. Since TGF-β has been associated with the pathogenesis of numerous diseases, the various mechanisms of activation of L-TGF-β in context offer the possibility of controlling TGF-β activity localized to the organ of involvement and to a more specific disease process.     

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

The interaction of estrogen receptor α and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor α in cardiomyocytes, we investigate whether the membrane estrogen receptor α associates with caveolin-3 and whether this association is linked to the 17β-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor α co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor α was not seen following treatment with the cholesterol-depleting agent methyl-β-cyclodextrin (5 mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor α. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor α, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17β-estradiol (200 nM). The effect of 17β-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor α docking at caveolae, which provide a unique compartment for conveying 17β-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Comparison of capillary electrophoresis and liquid chromatography for the enantiomeric separation of α-phosphonosulfonic acids

The enantiomers of α-phosphonosulfonic acids were completely resolved by capillary electrophoresis using β-cyclodextrin as a chiral selector in a borate electrolyte and HPLC using a chiral AGP column. The methods were used to quantitate the R-enantiomer present as an impurity in the S-enantiomer, a potential cardiovascular drug candiate. Chirality 9:104–108, 1997. © 1997 Wiley-Liss, Inc.     

Catalytic function and affinity purification of site-directed mutant β-cyclodextrin glucanotransferase from alkalophilic Bacillus firmus var. alkalophilus

Subsites −3 and −7 in the active site of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilic Bacillus firmus var. alkalophilus were modified through site-directed mutagenesis to obtain novel mutant CGTases. Four mutant CGTases, H59Q, Y96M, 90-PPI-92, and Δ(154–160) were constructed and produced using a recombinant E. coli with a secretive expression system extracellularly. The secreted mutant β-CGTases were purified by one-step affinity adsorption chromatography using a β-cyclodextrin (CD) polymer as an adsorbent to nearly homogeneous purity. The catalytic activities were modified significantly compared to the wild-type. In particular, the Y96M and Δ(154–160) mutants increased cyclization activity around 1.5 times without any significant reduction of coupling and hydrolyzing activities. Meanwhile, the Y96M and Δ(154–160) mutants exhibited a much higher conversion yield into CDs from 28.6 to 39% without any recognizable change in the CD ratio. The conversion yield into linear maltooligosaccharides was also significantly reduced. The catalytic functions of subsites −3 and −7 in the active site of β-CGTase would appear to be related to the overall productivity of CDs rather than the product specificity.