Preparation and Physicochemical Characterization of Amoxicillin β-cyclodextrin Complexes

Amoxicillin (AMOX), a penicillin A, belongs to the β-lactam family It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Its β-lactamase degradation might be prevented by using a molecular [AMOX:β-CD] complex. The aim of this work was to prepare complexes using two methods and then characterize interactions between AMOX and the native β-CD. The extent of complexation in solution has been evaluated by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and 2D rotating-frame Overhauser enhancement spectroscopy (2D ROESY). Mass changes (TG), calorimetric effects (DSC), and mass spectrometry (MS) were determined on the same sample under identical conditions using the Skimmer coupling system. Skimmer and infrared spectroscopy (FT-IR) were used to characterize the solid state of the binary system. Complexation of AMOX with β-CD was proven by FT-IR, NMR, DSC, and HPLC. The 2D ROESY spectra did not show any dipolar proton interaction of the AMOX with cyclodextrin. The 1:1 stoichiometry of the complex was obtained by HPLC. The stability constant for AMOX with β-CD was determined to be 1,878 M⁻¹. In the [AMOX:β-CD] complex, the phenyl group is included inside the β-CD, and the ionized carboxyl group on the penam ring forms hydrogen bonds with the secondary hydroxyl groups of another β-CD to keep the complex stable. Preparation methods allowed exactly the same complex.     

Preparation and Solid-State Characterization of Inclusion Complexes Formed Between Miconazole and Methyl-β-Cyclodextrin

The aim of this study is to confirm the formation of inclusion complexes between miconazole (MCZ) and two derivatives of beta-cyclodextrin, methyl-beta-cyclodextrin (MβCD) and 2-hydroxypropyl-beta-cyclodextrin (HPβCD) in aqueous solution by phase solubility studies. Inclusion complexes with MβCD in the solid state were then prepared by different methods, i.e., kneading, coevaporation (COE), spray-drying (SD), and lyophilization (LPh). The physicochemical properties of these complexes were subsequently studied by means of differential scanning calorimetry, Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction techniques. Phase solubility diagrams with MβCD and HPβCD were classified as A_P type, indicating the formation of 1:1 and 1:2 stoichiometric inclusion complexes. The apparent stability constants (K_S) calculated from the phase solubility diagram were 145.69 M⁻¹ (K _1:1) and 11.11 M⁻¹ (K _1:2) for MβCD and 126.94 M⁻¹ (K _1:1) and 2.20 M⁻¹ (K _1:2) for HPβCD. The method of preparation of the inclusion complexes in the solid state was shown to greatly affect the properties of the formed complex. Hence, the LPh, SD, and COE methods produce true inclusion complexes between MCZ and MβCD. In contrast, crystalline drug was still clearly detectable in the kneaded (KN) product.     

Physicochemical Properties and Dissolution Studies of Dexamethasone Acetate-β-Cyclodextrin Inclusion Complexes Produced by Different Methods

Inclusion complexes between dexamethasone acetate (DMA), a poorly water soluble drug, and β-cyclodextrin (βCD) were obtained to improve the solubility and dissolution rate of this drug. Phase-solubility profile indicated that the solubility of DMA was significantly increased in the presence of βCD (33-fold) and was classified as A_L-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by different methods (kneading, coevaporation, freeze drying) and physical mixture were characterized by differential scanning calorimetry, thermogravimetry, infrared absorption and optical microscopy. Preparation methods influenced the physicochemical properties of the products. The dissolution profiles of solid complexes were determined and compared with those DMA alone and their physical mixture, in three different mediums: simulated gastric fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and distilled water. The dissolution studies showed that in all mediums DMA presented an incomplete dissolution even in four hours. In contrast, the complexes formed presented a higher dissolution rate in simulated gastric fluid (SGF pH 1.2), which indicate that these have different ionization characteristics. According to the results, the freeze–dried and kneaded products exhibited higher dissolution rates than the drug alone, in all the mediums.     

Preparation and Physicochemical Properties of Catechin/β-cyclodextrin Inclusion Complex Nanoparticles

Nano-size catechin/β-cyclodextrin inclusion complex (CA/β-CD IC) with 1:1 molar ratio was obtained by cooling precipitation at 4 °C. Physicochemical properties of the CA/β-CD IC nanoparticles were characterized. Results of dynamic light scattering and SEM observation showed that CA/β-CD IC molecules underwent a process of assembling and shaping nano-size particles. In the range of 10–14 mM, the higher the concentration of β-CD aqueous solution, the faster the CA/β-CD IC nanoparticles form and the larger the size of the nanoparticles (195.2–438.6 nm). The total recovery, inclusion ratio and loading capacity of the CA/β-CD IC nanoparticles were determined. Results of FT-IR and DSC indicated that stability of CA was enhanced after it was embedded into β-CD cavity. XRD results showed that the strongest three diffraction peaks (located at 2θ = 10.6°, 12.4° and 19.6°) of the CA/β-CD IC nanoparticles was different from that (located at 2θ = 6.6°, 11.7° and 17.7°) of micro-size CA/β-CD IC and the nanoparticles obtained from higher concentration solution possessed higher crystallinity.

     

Hydroxymethylnitrofurazone:Dimethyl-β-cyclodextrin Inclusion Complex: A Physical–Chemistry Characterization

Hydroxymethylnitrofurazone (NFOH) is active against Trypanosoma cruzi; however, its low solubility and high toxicity precludes its current use in treatment of parasitosis. Cyclodextrin can be used as a drug carrier system, as it is able to form inclusion (host–guest) complexes with a wide variety of organic (guest) molecules. Several reports have shown the interesting use of modified β-cyclodextrins in pharmaceutical formulation, to improve the bioavailability of drugs and to decrease their toxicity. The aim of this work was to characterize inclusion complexes formed between NFOH and dimethyl-β-cyclodextrin (DM-β-CD) by complexation/release kinetics and solubility isotherm experiments using ultraviolet (UV)-visible spectrophotometry and by the measurement of the dynamics information obtained from T ₁ relaxation times and diffusion (DOSY) experiments using nuclear magnetic resonance (NMR) spectroscopy. The complex was prepared at different NFOH and DM-β-CD molar ratios. The UV-visible measurements were recorded in a spectrophotometer, and NMR experiments were recorded at 20°C on a NMR spectrometer (Varian Inova) operating at 500 MHz. Longitudinal relaxation times were obtained by the conventional inversion-recovery method and the DOSY experiments were carried out using the BPPSTE sequence. The kinetics of complexation revealed that 30 h is enough for stabilization of the NFOH absorbance in presence of cyclodextrin. Solubility isotherm studies show a favorable complexation and increase in solubility when NFOH interacts with cyclodextrin. The analysis of the NMR-derived diffusion coefficients and T ₁ relaxation times shows that in the presence of DM-β-CD, NFOH decreases its mobility in solution, indicating that this antichagasic compound interacts with the cyclodextrin cavity. The release kinetics assays showed that NFOH changes its release profile when in the presence of cyclodextrin due to complexation. This study was focused on the physicochemical characterization of drug-delivery formulations that may serve as potentially new therapeutic options for the treatment of Chagas’ disease.     

Characterization of Protein–Protein Interfaces

We analyze the characteristics of protein–protein interfaces using the largest datasets available from the Protein Data Bank (PDB). We start with a comparison of interfaces with protein cores and non-interface surfaces. The results show that interfaces differ from protein cores and non-interface surfaces in residue composition, sequence entropy, and secondary structure. Since interfaces, protein cores, and non-interface surfaces have different solvent accessibilities, it is important to investigate whether the observed differences are due to the differences in solvent accessibility or differences in functionality. We separate out the effect of solvent accessibility by comparing interfaces with a set of residues having the same solvent accessibility as the interfaces. This strategy reveals residue distribution propensities that are not observable by comparing interfaces with protein cores and non-interface surfaces. Our conclusions are that there are larger numbers of hydrophobic residues, particularly aromatic residues, in interfaces, and the interactions apparently favored in interfaces include the opposite charge pairs and hydrophobic pairs. Surprisingly, Pro-Trp pairs are over represented in interfaces, presumably because of favorable geometries. The analysis is repeated using three datasets having different constraints on sequence similarity and structure quality. Consistent results are obtained across these datasets. We have also investigated separately the characteristics of heteromeric interfaces and homomeric interfaces.     

Preparation and Characterization of Inclusion Complexes of a Hemisuccinate Ester Prodrug of Δ<Superscript>9</Superscript>-Tetrahydrocannabinol with Modified Beta-Cyclodextrins

Δ⁹-Tetrahydrocannabinol hemisuccinate (THC-HS), an ester prodrug of Δ⁹-tetrahydrocannabinol (THC) has been investigated for its potential to form inclusion complexes with modified synthetic beta-cyclodextrins (CDs). Phase solubility studies were performed to determine the stoichiometric ratio of complexation of THC-HS with random methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl beta-cyclodextrin (HPBCD). THC-HS/RAMEB and THC-HS/HPBCD solid systems were prepared by lyophilization and the lyophilized complexes were characterized by Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic spectroscopy, and molecular modeling techniques. The formation of inclusion complexes of THC-HS/RAMEB and THC-HS/HPBCD was demonstrated by an A_L type curve with the slopes less than unity by the phase solubility method. The association constants for THC-HS/RAMEB and THC-HS/HPBCD were found to be 562.48 and 238.83 M⁻¹, respectively. The stoichiometry of both of the complexes was found to be 1:1 as determined from the Job's plot. This was confirmed by ¹H NMR and FT-IR techniques. The results obtained from the molecular modeling studies were in accordance with the data obtained from nuclear magnetic resonance and FT-IR. The docking studies revealed the most probable mode of binding of THC-HS with RAMEB in which the alkyl chain was submerged in the hydrophobic pocket of the CD molecule and hydrogen bonding interactions were observed between the hemisuccinate ester side chain of THC-HS and the rim hydroxy groups of RAMEB. The solubility of THC-HS was significantly higher in RAMEB compared to HPBCD. Solid dispersions of THC-HS with CDs will be further utilized to develop oral formulations of THC-HS with enhanced bioavailability.     

Interaction Mode between Inclusion Complex of Vitamin K3 with γ- Cyclodextrin and Herring-Sperm DNA

Methods including spectroscopy, electronic chemistry and thermodynamics were used to study the inclusion effect between γ-cyclodextrin (CD) and vitamin K₃(K₃), as well as the interaction mode between herring-sperm DNA (hsDNA) and γ-CD-K₃ inclusion complex. The results from ultraviolet spectroscopic method indicated that VK₃ and γ-CD formed 1:1 inclusion complex, with the inclusion constant K_f = 1.02 × 10⁴ L/mol, which is based on Benesi–Hildebrand's viewpoint. The outcomes from the probe method and Scatchard methods suggested that the interaction mode between γ-CD-K₃ and DNA was a mixture mode, which included intercalation and electrostatic binding effects. The binding constants were K ^θ_25°C = 2.16 × 10⁴ L/mol, and K^θ_37°C = 1.06 × 10⁴ L/mol. The thermodynamic functions of the interaction between γ-CD-K₃ and DNA were Δ_rH_m^θ = ?2.74 × 10⁴ J/mol, Δ_rS_m^θ = 174.74 J·mol^?1K^?1, therefore, both Δ_rH_m^θ (enthalpy) and Δ_rS_m^θ (entropy) worked as driven forces in this action.     

In Vitro–In Vivo Correlation of Efavirenz Tablets Using GastroPlus®

The aim of the present work was to use GastroPlus™ software for the prediction of pharmacokinetic profiles and in vitro–in vivo correlation (IVIVC) as tools to optimize the development of new generic medications. GastroPlus™ was used to simulate the gastrointestinal compartment and was based on the advanced compartmental absorption and transit model. Powder dissolution and efavirenz tablet dissolution studies were carried out to generate data from which correlation was established. The simulated plasma profile, based on the physicochemical properties of efavirenz, was almost identical to that observed in vivo for biobatches A and B. A level A IVIVC was established for the dissolution method obtained for the generic candidate using the Wagner–Nelson (r² = 0.85) and for Loo–Riegelman models (r² = 0.92). The percentage of fraction absorbed indicated that 0.5% sodium lauryl sulfate may be considered a biorelevant dissolution medium for efavirenz tablets. The simulation of gastrointestinal bioavailability and IVIVC obtained from immediate-release tablet formulations suggests that GastroPlus™ is a valuable in silico method for IVIVC and for studies directed at developing formulations of class II drugs.KEY WORDS: bioavailability, computational simulation, efavirenz, GastroPlus™, in vivo–in vitro correlation     

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant K_d ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar K_d were identified.     

Formation of Liquid-Crystalline Dispersions of Double-Stranded DNA–Chitosan Complexes

Right-handed helical double-stranded DNA molecules were shown to interact with chitosans to form under certain conditions (chitosan molecular weight, content of amino groups, distance between amino groups, ionic strength and pH of solution) cholesteric liquid-crystalline dispersions characterized by abnormal positive band in CD spectrum in the absorption region of DNA nitrogen bases. Conditions were found for the appearance of intense negative band in CD spectrum upon dispersion formation. In some cases, no intense band appeared in CD spectrum in spite of dispersion formation. These results indicate not only the multiple forms of liquid-crystalline dispersions of DNA–chitosan complexes but also a possibility to control the spatial properties of these complexes. The multiplicity of liquid-crystalline forms of DNA–chitosan complexes was attempted to explain by the effect of character of dipoles distribution over the surface of DNA molecules on the sense of spatial twist of cholesteric liquid crystals resulting from molecules of the complexes.     

Formulation and In Vivo Evaluation of Orally Disintegrating Tablets of Clozapine/Hydroxypropyl-β-cyclodextrin Inclusion Complexes

The aim of this study was to improve the solubility and oral bioavailability of clozapine (CLZ), a poorly water-soluble drug subjected to substantial first-pass metabolism, employing cyclodextrin complexation technique. The inclusion complexes were prepared by an evaporation method. Phase solubility studies, differential scanning calorimetry, X-ray powder diffraction, and Fourier transform infrared spectroscopy were used to evaluate the complexation of CLZ with hydroxypropyl-β-cyclodextrin (HP-β-CD) and the formation of true inclusion complexes. Characterization and dissolution studies were carried out to evaluate the orally disintegrating tablets (ODTs) containing CLZ/HP-β-CD complexes prepared by direct compression. Finally, the bioavailability studies of the prepared ODTs were performed by oral administration to rabbits. The ODTs showed a higher in vitro dissolution rate and bioavailability compared with the commercial tablets. It is evident from the results herein that the developed ODTs provide a promising drug delivery system in drug development, owing to their excellent performance of a rapid onset of action, improved bioavailability, and good patient compliance.     

Physicochemical Aspects of Reaction of Ozone with Galactolipid and Galactolipid–Tocopherol Layers

The impact of reaction of galactolipids with ozone on the physicochemical properties of their monolayers was examined. In Megli and Russo (Biochim Biophys Acta, 1778:143–152, 2008), Cwiklik and Jungwirth (Chem Phys Lett, 486:99–103, 2010), Jurkiewicz et al. (Biochim Biophys Acta, 1818:2388–2402, 2012), Khabiri et al. (Chem Phys Lett, 519:93–99, 2012), and Conte et al. (Biochim Biophys Acta, 1828:510–517, 2013), the properties of layers formed from model mixtures composed of chosen lipids and selected oxidation products were studied, whereas in this work, question was raised as to how the oxidation reactions taking place in situ affect the physical properties of the galactolipid layers. So, set experiment should take into account the effect of all reaction products. The mechanical characteristics of monolayers of monogalactosyldiacyl-glycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were determined by Langmuir trough technique, and the electrical properties of liposomes formed from these lipids by measuring their electrophoretic mobility. Considerable loss of galactolipid molecules forming monolayers was found at ozone concentrations (in aqueous medium) higher than 0.1 ppm with a stronger effect measured for MGDG. That goes along with the greater amounts of MDA found in the extracts of oxidized MGDG films compared with DGDG. Based on this, it was concluded that an additional galactose group present in DGDG molecules acts protectively under oxidative conditions. The surface tension of the solutions (of small volume) contacting the oxidized galactolipids films was significantly reduced, indicating the presence of soluble in polar media, surface active reaction products. The presence of α-tocopherol in mixtures with tested galactolipids at a molar ratio of lipid to tocopherol equal to 1.7:1 caused some inhibition of lipid oxidation, reducing the decrease of amount of lipid particles forming the monolayer. Here, also protective effect of α-tocopherol was greater for the MGDG compared to DGDG.     

Characterization of the Y-Chromosome Sardinian–Corsican Haplotype

The Y chromosome of 523 Italian male subjects was examined for the 49a,f TaqI haplotype XII and for two microsatellites, YCAIIa and YCAIIb. Results were then compared to other populations living in the western Mediterranean basin whom we had previously studied: 419 French (including 328 Corsicans), 46 Italians from Milan, and 73 Tunisians. Haplotype XII is present in 127 out of the 1061 examined samples (11.9%), and most of the haplotype XII subjects are of the compound haplotype YCAIIa-21 and YCAIIb-11. Two peaks of haplotype XII frequencies occur in the north of Sardinia (35%) and in the central area of Corsica (17.4%).     

Characterization of Wistar–Kyoto rats showing hyperadiponectinemia

AimsWistar–Kyoto rats (HA-WKY) kept in the author's laboratory showed higher levels of serum adiponectin (approximately 4-fold) compared with Wistar–Kyoto/Izm rats (WKY/Izm), a WKY standard strain, at 6 weeks old. In a preliminary study, HA-WKY but not WKY/Izm showed hyperinsulinemia and severe hypercholesterolemia when fed a high-fat diet. This study analyzed the differences between HA-WKY and WKY/Izm to investigate the causes of hyperadiponectinemia.Main methodsSix-week-old male HA-WKY and WKY/Izm were used for an analysis of adiponectin-related factors.Key findingsThe main intermediates in the adiponectin signaling pathway, AMP-activated protein kinase and peroxisome proliferator-activated receptor α, were activated at similar levels in liver and skeletal muscle between HA-WKY and WKY/Izm, although HA-WKY had not only higher adiponectin concentrations but also extremely high levels of high-molecular weight (HMW, polymer) adiponectin, which is the active form. The main difference between HA-WKY and WKY/Izm was the existence of adiponectin, mainly middle-molecular weight (MMW, hexamer) and HMW adiponectin multimers, in skeletal muscle extracts from WKY/Izm but not HA-WKY. Skeletal muscle in WKY/Izm expressed much higher amounts of T-cadherin, a receptor for MMW and HMW adiponectin multimers, than that in HA-WKY. In contrast, there was no significant difference in the expression level of adiponectin receptor 2 for trimer, MMW, and HMW adiponectin multimers.SignificanceThe results suggested that the existence of adiponectin in WKY/Izm skeletal muscle was due to the binding of serum adiponectin. The difference in serum adiponectin concentrations between HA-WKY and WKY/Izm could come from the difference in adiponectin binding to skeletal muscle.     

Studies on the Effect of Water-Soluble Polymers on Drug–Cyclodextrin Complex Solubility

The effect of complexation of irbesartan (IRB), a practically water-insoluble drug, with cyclodextrins in presence of different concentrations of water-soluble polymers (PEG 4000 and PVP K-90) on the dissolution rate of the drug has been investigated. Phase solubility studies were carried out to evaluate the solubilizing power of βCD in association with water-soluble polymers towards IRB and to determine the apparent stability constant (K _S) of the complexes. Improvement in K _S value for ternary complexes (IRB–βCD–polymers) clearly proved the benefit on the addition of water-soluble polymer to increase complexation efficiency. The dissolution rate of the drug from ternary systems containing PEG 4000 and PVP K-90 was higher as compared to the binary system. An optimum increase in the dissolution rate of the drug was observed at a polymer concentration of 5% w/w for PVP K-90 and 10% w/w for PEG 4000. DSC, FTIR, SEM, and XRD studies were carried out to characterize the complexes.     

Screening and Characterization of Fructosyl-Valine–Utilizing Marine Microorganisms

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val–oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H₂O₂ with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast. Received October 21, 1999; accepted September 12, 2000     

Preparation and Characterization of Collagen–Chitosan–Chondroitin Sulfate Composite Membranes

Collagen (Col)–chitosan (Chi) membrane was modified by a hot dehydrogenation cross-linking method. Carbodiimide was added for further crossing modification. Chondroitin sulfate (CS) was added so that Col–Chi sulfate composite membranes were prepared. The structure of the composite membranes was characterized by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, and its mechanical properties, degradation, and cytotoxicity were characterized. The composite membrane was applied to a full-thickness skin injury in animal experiments performed in rabbits. Strong interactions and good compatibility among Col, Chi, and CS in the composite membrane were present. The good mechanical properties, biocompatibility, digestion resistance, and wound healing promotion of the composite membrane make it a potential wound dressing or skin scaffold for tissue engineering.