Interactions of some modified mono- and bis-beta-cyclodextrins with bovine serum albumin

Two mono-substituted beta-cyclodextrins and two bridged bis-beta-cyclodextrins, that is, mono(6-(2-aminoethylamino)-6-deoxy)-beta-cyclodextrin (1), mono(6-(2-(2-aminoethylamino)ethylamino)-6-deoxy)-beta-cyclodextrin (2), ethylene-1,2-diamino bis-6-(6-deoxy-beta-cyclodextrin) (3), and iminodiethylene-2,2'-diamino bis-6-(6-deoxy-beta-cyclodextrin) (4), were prepared from beta-cyclodextrin. Their binding ability with bovine serum albumin as a model protein was investigated through proton magnetic resonance (1H NMR), ultraviolet visible spectroscopy (UV-vis), circular dichroism (CD), and fluorescence spectroscopy. In the 1H NMR spectra of the modified cyclodextrins, the resolution of proton signals decreases after the addition of BSA. From the UV and CD spectra, it is found that both the UV absorption and the alpha-helix content of BSA increase with the concentration of the modified cyclodextrins. The protein-ligand interactions cause a fluorescence quenching. The quenching constants are determined using the Stern-Volmer equation to provide an observation of the binding affinity between modified cyclodextrins and BSA. All these results indicate that the modified cyclodextrins can interact with BSA and the bridged bis(beta-cyclodextrin)s (3 and 4) have much stronger interactions than the mono-substituted beta-cyclodextrins (1 and 2). The strong binding stability of bis-cyclodextrins should be attributed to the cooperative effect of two adjacent cyclodextrin moieties. Job's plot shows that the complex stoichiometries of BSA to the modified cyclodextrins were 1:4 for 1 and 2, as well as 1:3 for 3 and 4, respectively.     

Physico-Chemical Characterization and In Vitro Dissolution Assessment of Clonazepam—Cyclodextrins Inclusion Compounds

The objectives of this research were to prepare and characterize inclusion complexes of clonazepam with β-cyclodextrin and hydroxypropyl-β-cyclodextrin and to study the effect of complexation on the dissolution rate of clonazepam, a water-insoluble lipid-lowering drug. The phase-solubility profiles with both cyclodextrins were classified as A_P-type, indicating the formation of 2:1 stoichiometric inclusion complexes. Gibbs free energy ( DG_tr^o ) \left( {\Delta {G_{tr}}^o} \right) values were all negative, indicating the spontaneous nature of clonazepam solubilization, and they decreased with increase in the cyclodextrins concentration, demonstrating that the reaction conditions became more favorable as the concentration of cyclodextrins increased. Complexes of clonazepam were prepared with cyclodextrins by various methods such as kneading, coevaporation, and physical mixing. The complexes were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry studies. These studies indicated that complex prepared kneading and coevaporation methods showed successful inclusion of the clonazepam molecule into the cyclodextrins cavity. The complexation resulted in a marked improvement in the solubility and wettability of clonazepam. Among all the samples, complex prepared with hydroxypropyl-β-cyclodextrin by kneading method showed highest improvement in in vitro dissolution rate of clonazepam. Mean dissolution time of clonazepam decreased significantly after preparation of complexes and physical mixture of clonazepam with cyclodextrins. Similarity factor indicated significant difference between the release profiles of clonazepam from complexes and physical mixture and from plain clonazepam. Tablets containing complexes prepared with cyclodextrins showed significant improvement in the release profile of clonazepam as compared to tablet containing clonazepam without cyclodextrins.     

Retinoic acid metabolism inhibition by 3-azolylmethyl-1H-indoles and 2, 3 or 5-(alpha-azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or alpha-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl) (1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 microM) ketoconazole exerted similar inhibitory effect (70% inhibition).     

Improvement of the sensitivity of EPR spin trapping in biological systems by cyclodextrins: A model study with thylakoids and photosystem II particles

Electron paramagnetic resonance (EPR) spin trapping spectroscopy is an important method used in free radical research; however, its application in biological systems is hindered by EPR silencing of spin adducts. Previous studies in superoxide-generating chemical systems have shown that spin adducts can be partially stabilized by cyclodextrins. In this work, for the first time, this proposed protective effect of cyclodextrins is investigated in a real biological sample—in isolated thylakoid membranes and photosystem II (PSII) particles with EMPO as a spin trap. It is shown that (i) randomly methylated β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin form inclusion complexes with EMPO–superoxide adducts (EMPO-OOH), (ii) both cyclodextrins increase the intensity of the EMPO-OOH EPR signal in PSII particles up to five times, (iii) higher EMPO-OOH EPR signal intensity is a result of increased stability of EMPO-OOH, and (iv) the extent of the protection of EMPO-OOH adduct provided by cyclodextrins is different in thylakoids and PSII particles. Along with the spin trapping data, the toxicity of cyclodextrins is also discussed with particular focus on photosynthetic preparations. The presented data show that both tested cyclodextrins can be used as valuable tools to improve the sensitivity of spin trapping in biological samples.     

Synthesis and characterisation of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins: application to the complexation of acyclovir

The synthesis of sulfated amphiphilic alpha-, beta- and gamma-cyclodextrins was achieved according to the standard protection-deprotection procedure. The formation of inclusion complexes between the amphiphilic alpha-, beta- and gamma-cyclodextrins and an antiviral molecule, acyclovir (ACV) was investigated by UV-visible spectroscopy (UV-Vis) and electrospray ionisation mass spectrometry (ESIMS). UV-Vis spectroscopy allowed determination of the stoichiometry and stability constants of complexes, whereas ESIMS, a soft ionisation technique, allowed the detection of the inclusion complexes. The results showed that the non-sulfated amphiphilic cyclodextrins exhibit a 1:2 stoichiometry with acyclovir, while sulfated amphiphilic cyclodextrins, except gamma-cyclodextrin, exhibit a 1:1 stoichiometry indicating the loss of one interaction site. Non-covalent interactions between acyclovir and non-sulfated amphiphilic cyclodextrins appear to take place both in the cavity of the cyclodextrin and inside the hydrophobic zone generated by alkanoyl chains. In contrast, in the case of sulfated amphiphilic cyclodextrins, the interactions appear to involve only the hydrophobic region of the alkanoyl chains.     

Study of isopropyl myristate microemulsion systems containing cyclodextrins to improve the solubility of 2 model hydrophobic drugs

The objectives of this project were to evaluate the effect of alkanols and cyclodextrins on the phase behavior of an isopropyl myristate microemulsion system and to examine the solubility of model drugs. Triangular phase diagrams were developed for the microemulsion systems using the water titration method, and the solubility values of progesterone and indomethacin were determined using a conventional shake-flask method. The water assimilation capacities were determined to evaluate the effective microemulsion formation in different systems. The alkanols showed higher microemulsion formation rates at higher concentrations. A correlation between the carbon numbers of the alkanol and water assimilation capacity in the microemulsions studied was observed; isobutanol and isopentanol produced the best results. The addition of cyclodextrins showed no effect or had a negative effect on the microemulsion formation based on the type of cyclodextrin used. Isopropyl myristate-based microemulsion systems alone could increase the solubility values of progesterone and indomethacin up to 3300-fold and 500-fold, respectively, compared to those in water. However, the addition of cyclodextrins to the microemulsion systems did not show a synergistic effect in increasing the solubility values of the model drugs. In conclusion, microemulsion systems improve the solubility of progesterone and indomethacin. But the two types of cyclodextrins studied affected isopropyl myristatebased microemulsion systems negatively and did not improve the solubilization of 2 model drugs.     

The Role of <Emphasis Type="SmallCaps">l</Emphasis>-arginine in Inclusion Complexes of Omeprazole with Cyclodextrins

In this study, we investigate how the effect of l-arginine (ARG) and cyclodextrins upon omeprazole (OME) stability and solubility. The effect of the presence of ARG on the apparent stability constants (K_1:1) of the inclusion complexes formed between OME and each cyclodextrin, β-cyclodextrin (βCD), and methyl-β-cyclodextrin (MβCD) is studied by phase solubility diagrams and nuclear magnetic resonance (NMR) spectroscopy. The interaction of OME with those cyclodextrins, in the presence of ARG, is characterized using NMR spectroscopy and molecular dynamics simulations. ARG significantly increases the drug solubility and complex stability, in comparison to inclusion complexes formed in its absence. The effect is more pronounced for the OME:βCD complex. ARG also contributes to a larger stability of OME when free in aqueous solution. The combination of ARG with cyclodextrins can represent an important tool to develop stable drug formulations.     

The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 implications for product inhibition and product specificity.

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active-site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products. Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins. Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47-->Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47-->Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity.     

Enhancement of phytosterols, taraxasterol and induction of extracellular pathogenesis-related proteins in cell cultures of Solanum lycopersicum cv Micro-Tom elicited with cyclodextrins and methyl jasmonate

Suspension-cultured cells of Solanum lycopersicum cv Micro-Tom were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses. An extracellular accumulation of two sterols (isofucosterol and β-sitosterol) and taraxasterol, a common tomato fruit cuticular triterpene, were observed. Their levels were higher in Micro-Tom tomato suspension cultured cells elicited with cyclodextrins than in control and methyl jasmonate-treated cells. Also, their accumulation profiles during the cell growth phase were markedly different. The most striking feature in response to cyclodextrin treatments was the observed enhancement of taraxasterol accumulation. Likewise, the exogenous application of methyl jasmonate and cyclodextrins induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a cationic peroxidase and a biotic cell death-associated protein, which suggests that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in S. lycopersicum cv Micro-Tom.     

Influence of cyclodextrin ring substituents on folding-related aggregation of bovine carbonic anhydrase.

A common obstacle to proper renaturation of an unfolded protein is aggregation, an intermolecular side reaction of immense importance in biotechnology and in the pathogenesis of several neurodegenerative diseases. Cyclic sugars known as cyclodextrins have been used as protein-folding aids. The effect of cyclodextrin chemistry on aggregation and refolding of carbonic anhydrase was evaluated in this study. Size-exclusion HPLC showed that cyclodextrins inhibit aggregate formation without interfering with the correct renaturation of carbonic anhydrase. PAGE of refolded enzyme provides further evidence of inhibition of folding-related aggregation by natural and chemically modified cyclodextrins. Although the amount of aggregate formed and recovery of active enzyme was dependent on cavity size, the nature of the chemical substituents found on the rims of the sugar molecule seems to play a more important role in cyclodextrin-assisted refolding of carbonic anhydrase. In general, neutral or cationic cyclodextrins with small cavities were found to be better folding aids than anionic cyclodextrins with larger holes. Although the exact prediction of the effect of a cyclodextrin substitution on protein refolding is not possible at present, these results clearly show that modified cyclodextrins can be designed that effectively inhibit protein aggregation.     

New aromatic esters of progesterone as antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17alpha-(pchlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1-4 have a weak inhibitory activity on 5alpha-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114 microM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds. The binding efficiency of the synthesized steroids 1-4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1-4.     

Hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins.

The oxidation of xenobiotics by the hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins was studied. These produced an inhibitory effect on xenobiotics oxidation, based on their degree of hydrophobicity and the charge (isoproterenol < 4-methyl-catechol (4MC) < 4-tert-butylcatechol (TBC) < 4-tert-octylcatechol (TOC)). This inhibitory effect was due to the complexation of xenobiotics in the hydrophobic cavity of cyclodextrins. The complexation constant Kc was calculated by nonlinear regression of the inhibition curves obtained in the presence of cyclodextrins, and the values obtained were 400, 16,250, and 35,127 M-1 for 4MC, TBC, and TOC, respectively. The validity of these values was checked at different points of the Michaelis-Menten saturation curve, and a sigmoidal inhibition curve was obtained at the saturating concentration of the o-diphenol, TBC, with no change in the Kc value. This demonstrates the validity of the equations used to calculate Kc for the complete range of the Michaelis-Menten equation.     

Studies on the hydrolyzing mechanism for cyclodextrins of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structure of the mutant E354A complexed with beta-cyclodextrin, and kinetic analyses on cyclodextrins

Crystals of the mutant E354A of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) complexed with beta-cyclodextrin were prepared by a soaking method, and the diffraction data were collected at 100 K, using Synchrotron radiation (SPring-8). The crystals belong to an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions a = 111.1 A, b = 117.7 A, c = 113.3 A, which is almost isomorphous with crystals of the wild-type TVAII, and the structure was refined to an R-factor = 0.208 (R(free) = 0.252) using 3.0 A resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of beta-cyclodextrin at the hydrolyzing site are important for TVAII to recognize cyclodextrins as substrates. This observation from the X-ray structure was supported by kinetic analyses of cyclodextrins using the wild-type TVAII, the mutant F286A and F286L. These studies also suggested that the TVAII-hydrolyzing mechanism for cyclodextrins is slightly different from that for starch.     

Synthesis and anti-virus activity of some nucleosides analogues

New 3'-, 5'-, 5-bromo-2'-deoxyuridine (3a-g) and 3'-, 5'-thymidine (4a-i) analogues with amino acid and peptide residues were synthesized and evaluated for antiviral activity. The influence of long peptide chains, essential amino acids and the effect of this structural modification on the antiviral activity has been also reported. Three 5-bromo-2'-deoxyuridine derivatives containing glycyl-, glycyl-glycyl- and glycyl-glycyl-glycyl- residues (3a, 3b, 3c) showed a strong activity against the herpes virus PsRV and a moderate one vs. HSV-1. The corresponding thymidine analogues were considerably less effective, and only compounds 4d and 4h showed a borderline effect against PsRV.     

Use of cyclodextrins in capillary zone electrophoresis for the separation of optical isomers: Resolution of racemic tryptophan derivatives

In this study capillary zone electrophoresis has been used for the separation of racemic tryptophan derivatives in their enantiomers. The effect of cyclodextrins with different shape, added to the background electrolyte, on the migration time of 10 compounds, including methyl tryptophan, hydroxy tryptophan, and tryptophan ester derivatives, has been studied. Furthermore, the effect of cyclodextrins with different shape and that of the composition of the background electrolyte on the enantiomer resolution are discussed. Among different cyclodextrins used α-cyclodextrin and heptakis(2,6-di-O-methyl)-β-cyclodextrin were found to possess the best complexing capacity and thus the resolution power toward analysed compounds.     

Stimulation of root development with cyclodextrins on jojoba shoots in vitro

Summary We studied the effect of α- and β-cyclodextrins on in vitro rooting of jojoba (Simmondsia chinensis (Link) Schn.) shoots. When 0.03–0.5 mM of cyclodextrins were added to modified Murashige and Skoog culture medium containing 0.015 mM of indolebutyric acid as 1-wk pulse treatment, rooting percentage increased by approximately 100% with respect to controls. Addition of cyclodextrins induced earlier rooting. An increase in rooting following treatment with cyclodextrins was also observed in the absence of indolebutyric acid, indicating that both compounds promote rooting per se.     

Structural background of cyclodextrin-protein interactions

Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological applications. We have studied the interaction between beta-cyclodextrin and four non-carbohydrate-binding model proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6 and insulin SerB9Asp by NMR spectroscopy at varying structural detail. We demonstrate that the interaction of beta-cyclodextrin and our model proteins takes place at specific sites on the protein surface, and that solvent accessibility of those sites is a necessary but not compelling condition for the occurrence of an interaction. If this behaviour can be generalized, it might explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically 'masked' by cyclodextrin), alteration of function (if residues involved in function are 'masked' by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be related to the particular structure of this protein.     

<Emphasis Type="Italic">In Vitro</Emphasis> antiviral activity of red alga, <Emphasis Type="Italic">Polysiphonia morrowii</Emphasis> extract and its bromophenols against fish pathogenic infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus

Our previous investigation revealed that 80% methanolic extract of the red alga Polysiphonia morrowii has significant antiviral activities against fish pathogenic viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). The present study was conducted to identify compounds attributed for its antiviral activities and investigate their antiviral activities against IHNV and IPNV. Activity-guided fractionation for 80% methanolic extract of Polysiphonia morrowii using a cell-based assay measuring virus-induced cytopathic effect (CPE) on cells yielded a 90% methanolic fraction, which showed the highest antiviral activity against both viruses among fractions yielded from the extract. From the fraction, two bromophenols were isolated and identified as 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) based on spectroscopic analyses. For both compounds, the concentrations to inhibit 50% of flounder spleen cell (FSP cell) proliferation (CC₅₀) and each viral replication (EC₅₀) were measured. In the pretreatment test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihy-droxybenzaldehyde (2) exhibited significant antiviral activities showing selective index values (SI = CC₅₀/EC₅₀) of 20 to 42 against both IHNV and IPNV. In direct virucidal test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) showed significant antiviral activités against both viruses while 3-bromo-4,5-dihydroxybenzaldehyde (2) was significantly effective against only IHNV. Although antiviral efficacies of both compounds against IHNV and IPNV were lower than those of ribavirin used as a positive control, our findings suggested that the red alga Polysiphonia morrowii and isolated two bromophenols may have potential as a therapeutic agent against fish viral diseases.     

Synthesis and characterization of water-soluble hydroxybutenyl cyclomaltooligosaccharides (cyclodextrins)

We have examined the synthesis of hydroxybutenyl cyclomaltooligosaccharides (cyclodextrins) and the ability of these cyclodextrin ethers to form guest-host complexes with guest molecules. The hydroxybutenyl cyclodextrin ethers were prepared by a base-catalyzed reaction of 3,4-epoxy-1-butene with the parent cyclodextrins in an aqueous medium. Reaction byproducts were removed by nanofiltration before the hydroxybutenyl cyclodextrins were isolated by co-evaporation of water-EtOH. Hydroxybutenyl cyclodextrins containing no unsubstituted parent cyclodextrin typically have a degree of substitution of 2-4 and a molar substitution of 4-7. These hydroxybutenyl cyclodextrins are randomly substituted, amorphous solids. The hydroxybutenyl cyclodextrin ethers were found to be highly water soluble. Complexes of HBen-beta-CD with glibenclamide and ibuprofen were prepared and isolated. In both cases, the guest content of the complexes was large, and a significant increase in the solubility of the free drug was observed. Dissolution of the complexes in pH 1.4 water was very rapid, and significant increases in the solubility of the free drugs were observed. Significantly, after reaching equilibrium concentration, a decrease in the drug concentration over time was not observed.     

Stereoselective phosphorylation of branched cyclodextrins with inorganic cyclo-triphosphate

The phosphorylation by inorganic sodium cyclo-triphosphate (P(3m)) having a six-membered ring was examined for cyclomaltohexaose (alpha-cyclodextrin) and branched cyclodextrins (mono-6-O-alpha-D-glucopyranosylcyclomaltohexaose, mono-6-O-alpha-D-maltosylcyclomaltohexaose, mono-6-O-alpha-D-glucopyranosylcyclomaltoheptaose, and mono-6-O-alpha-D-maltosylcyclomaltoheptaose) in aqueous solution. For all cyclomaltooligosaccharides (cyclodextrins) studied, the 2-OH group was stereoselectively phosphorylated. In the reaction of branched cyclodextrins and P(3m), only the 2-OH on the alpha-D-glucopyranosyl group of the cyclodextrin rings was phosphorylated with maximum yields of more than 27%. The phosphorylation mechanism of branched cyclodextrins with P(3m) is also discussed.