Triamterene-β-cyclodextrin systems: Preparation,characterization and in vivo evaluation

The purpose of this research was to improve the solubility and therefore dissolution and bioavailability of triamterene, a poorly water soluble diuretic, by complexation with β-cyclodextrin. Triamterene has been reported to show low bioavailability after oral administration, with wide intersubject variation. This study presents the formulation of solid dispersions of triamterene with β-cyclodextrin—by cogrinding, kneading, and coevaporation, using low pH conditions—and their characterizations, evaluation of improvement in dissolution profiles, and in vivo advantage. Phase solubility studies indicated complex with possible stoichiometry of 1∶1 and a stability constant of 167.67M⁻¹. The solid dispersions were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, x-ray diffraction, and differential scanning calorimetry studies. The characterization studies confirmed inclusion of the phenyl ring of triamterene within the nonpolar cavity of β-cyclodextrin in the coevaporate. Remarkable improvement in in vitro drug release profiles in 0.1 N HCl and pH 6.8 phosphate buffer was observed with all dispersions, especially the coevaporate. The coevaporate, when administered orally in rats, also exhibited improved in vivo activity, as measured by net sodium ion excretion, as compared with triamterene powder. Thus, coevaporation of the drug and β-cyclodextrin from acidified alcohol provide the optimum condition for inclusion complexation to give a binary system with remarkable improvement in in vitro drug release profile and in vivo performance.     

Spectroscopic characterization of the inclusion complexes of luteolin with native and derivatized β-cyclodextrin

The inclusion complexes of Luteolin (LU) with cyclodextrins (CDs) including β-cyclodextrin (βCD), hydroxypropyl-β-cyclodextrin (HPβCD) and dimethyl-β-cyclodextrin (DMβCD), Scheme 1, have been investigated using the method of steady-state fluorescence. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was obtained in the case of HPβCD followed by DMβCD and βCD. Moreover, ¹H NMR and 2D NMR were carried out, revealing that LU has different form of inclusion which is in agreement with molecular modeling studies. These models confirm that when LU–βCD and LU–DMβCD complexes are formed, the B-ring is oriented toward the primary rim; however, for LU–HPβCD complex this ring is oriented toward the secondary rim. The ESR results showed that the antioxidant activity of luteolin was the order LU–HPβCD > LU–DMβCD > LU–βCD > LU, hence the LU-complexes behave are better antioxidants than luteolin free.     

Physicochemical characterization,in vitro dissolution behavior,and pharmacodynamic studies of rofecoxib-cyclodextrin inclusion compounds. Preparation and properties of rofecoxib hydroxypropyl β-cyclodextrin inclusion complex: A technical note

Conclusion  An inclusion complex of rofecoxib and HPβ-CD was prepared successfully by the spray-drying method in a molar ratio of 1∶1. The inclusion complex was found to have improved in vitro drug release compared with the pure drug. The solubility profile of complexes of rofecoxib prepared using HPβ-CD as the complexing agent in a molar ratio of 1∶1 by the spray-drying method in pH 1.2 and pH 7.4 indicated that the acid solubility of rofecoxib was enhanced considerably by formation of an inclusion complex with HPβ-CD. The above results also clearly demonstrated a significant decrease in the gastric ulcerogenic activity of rofecoxib through complexation with cyclodextrins. Even though the physical mixture of rofecoxib with cyclodextrins reduced ulcer formation, it was the spray-dried complex formation approach that minimized gastric ulceration. These findings are extremely important from a commercial point of view as the prepared complex removes a major drawback for rofecoxib in therapy. Published: September 20, 2005     

Preparation and immunological characterization of β-lactoglobulin-amylose conjugate

β-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.     

Parenteral Delivery of HPβCD: Effects on Drug-HSA Binding

It is thought that cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin (HPβCD), will at high concentration affect pharmacokinetics of drugs through competitive binding with plasma proteins. Albumin is the major component of plasma proteins responsible for plasma protein binding. The purpose of this study was to evaluate in vitro the competitive binding of drugs between human serum albumin (HSA) and HPβCD in isotonic pH 7.4 phosphate buffer saline solution (PBS) at ambient temperature. Eight model drugs were selected based on their physicochemical properties and ability to form complexes with HSA and HPβCD. The drug/HPβCD stability constants (K _1:1) were determined by the phase-solubility method and HSA/HPβCD competitive binding determined by an equilibrium dialysis method. Protein binding of drugs that are both strongly protein bound and have high affinity to HPβCD (i.e., have high K _1:1 value) is most likely to be affected by parenterally administered HPβCD. However, this in vitro study indicates that even for those drugs single parenteral dose of HPβCD has to be as high as 70 g to have detectable effect on their protein binding. Weakly protein bound drugs and drugs with low affinity towards HPβCD are insensitive to the cyclodextrin presence regardless their lipophilic properties.     

Synthesis and characterization of η-cyclopentadienyl-silylallyl niobium and tantalum complexes

Reaction of the disilylcyclopentadiene 1,1-[SiMe₂(CH₂CHCH₂)]₂C₅H₄ with NbCl₅ gave the new allylsilyl-substituted monocyclopentadienyl niobium complex [Nb{η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}Cl₄]. This compound was reacted with LiNHtBu or NH₂tBu to give the imido derivative [Nb{η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}(NtBu)Cl₂], which was further alkylated to the imido alkyl complexes [Nb{η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}(NtBu)R₂] (R = Me, CH₂Ph) and [Nb{η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}(NtBu)Cl (CH₂Ph)]. Reaction of the imido complexes with the corresponding lithium cyclopentadienides gave the dicyclopentadienyl-imido complexes [M(η⁵-C₅R₅){η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}(NtBu)Cl] (M = Nb, Ta; R = H, Me). Metallocene dichlorides [M(η⁵-C₅R₅){η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}Cl₂] (M = Nb, Ta; R = H, Me) were easily prepared by reduction with Na/Hg and simultaneous transmetallation of [Ta(η⁵-C₅R₅)Cl₄] with Li[C₅H₄SiMe₂(CH₂CHCH₂)] and of [Nb{η⁵-C₅H₄SiMe₂(CH₂CHCH₂)}Cl₄] with Li(C₅R₅). All of the new compounds have been characterized by elemental analysis, and IR and NMR spectroscopy.     

Binary and ternary complexes of FLNa-Ig21 with cytosolic tails of αMß2 integrin reveal dual role of filamin mediated regulation

BackgroundCytoskeletal protein filamin A is critical for the outside-in signaling of integrins. Although molecular mechanisms of filamin-integrin interactions are not fully understood. Mostly, the membrane distal (MD) part of the cytosolic tail (CT) of β subunit of integrin is known to interact with filamin A domain 21 (FLNa-Ig2). However, binary and ternary complexes of full-length CTs of leucocyte specific ß2 integrins with FLNa-Ig21 are yet to be elucidated.MethodsBinding interactions of the CTs of integrin αMß2 with FLNa-Ig21 are extensively investigated by NMR, ITC, cell-based functional assays and computational docking.ResultsThe αM CT demonstrates interactions with FLNa-Ig21 forming a binary complex. Filamin/αM interface is mediated by sidechain-sidechain interactions among non-polar and aromatic residues involving MP helix of αM and the canonical CD face of FLNa-Ig21. Functional assays delineated an interfacial residue Y1137 of αM CT is critical for in-cell binding to FLNa-Ig2. In addition, full-length ß2 CT occupies two distinct binding sites in complex with FLNa-Ig21. A ternary complex of FLNa-Ig21 with CTs has been characterized. In the ternary complex, αM CT moves away to a distal site of FLNa-Ig21 with fewer interactions.ConclusionOur findings demonstrate a plausible dual role of filamin in integrin regulation. The molecular interactions of the ternary complex are critical for the resting state of integrins whereas stable FLNa-Ig21/αM CT binary complex perhaps be required for the activated state.General significanceFilamin binding to both α and β CTs of other integrins could be essential in regulating bidirectional signaling mechanisms.     

Purification and characterization of HIV–human protein complexes

To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.     

Preparation,characterization and in vitro activity of a docetaxel–albumin conjugate

Docetaxel is one of the most effective anticancer drugs. However, the current formulation of docetaxel contains Tween 80 and ethanol as the solvent, which can cause severe side effects. Consequently, the development of new type of formulation of docetaxel with high efficiency and low side effects is a very important issue. In this study, we explored the covalent linking of docetaxel and albumin via one organic linker. 6-Maleimidocaproic acid was applied to link the C2′ hydroxyl group of docetaxel with the cysteine-34 of albumin to obtain 1:1 docetaxel–albumin conjugate. The synthesized conjugate can control the release of docetaxel in the bovine serum. Furthermore, in vitro cell cytotoxicity experiments indicated that the docetaxel–albumin conjugate have high activities for human prostate cancer cell line PC3 and human breast cancer cell line MCF-7. The present study provides a valuable strategy for further development of a new type of docetaxel–albumin prodrug.     

The fate of DDT in soils treated with hydroxypropyl-β-cyclodextrin (HPβCD)

Point Pelee National Park (PPNP) is highly contaminated with dichlorodiphenyltrichloroethane (DDT) due to the historical use of this persistent organochlorine pesticide. Hydroxypropyl-β-cyclodextrin (HPβCD) has previously been investigated for its role in the remediation of polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). In the present study, HPβCD's ability to promote DDT microbial degradation, enhance DDT phytoextraction by two native grasses (Schizachyrium scoparium and Panicum virgatum), and increase DDT bioavailability to redworms (Eisenia fetida) was investigated. Using a range of HPβCD concentrations (2.5% to 10%), it was determined that it did not promote DDT microbial degradation in PPNP soils, however, it was able enhance the DDT phytoextraction ability of S. scoparium plants due to the increased water solubility of DDT. Although HPβCD application to PPNP soil did not increase DDT bioavailability to redworms, its enhanced solubility allowed it to move through the soil column, and hence groundwater contamination is a possibility. Due to this important issue, in situ use of HPβCD to remediate DDT contamination is not recommended unless measures are in place to mitigate movement into groundwater.     

Preparation and characterization of gas-filled liposomes: can they improve oil recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (T(c)), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their T(c) was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 microm, after 7 days storage at 25 degrees C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 +/- 0.3 mum and 12.3 +/- 1.0 microm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12-13 microm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the zeta potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1-8 microm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.     

Preparation and characterization of a novel polymorph of indiplon, Form α

A new polymorph α of indiplon was discovered, initially prepared by two methods, and further characterized by various means including single-crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), variable temperature powder X-ray diffraction (VT-PXRD), differential scanning calorimetry (DSC), thermogravimetry analysis (TGA), Fourier transform Raman (FT-Raman) spectroscopy and solubility determination. The crystal structure of Form α as analyzed by SCXRD differ from the three previously reported polymorphs, Form I, II, and III. In addition, PXRD and solubility measurements could clearly distinguish between Form α and Form I. Slight differences between the two forms were also detected by FT-Raman. No differences between Form α and I were observed by DSC, which was explained by VT-PXRD results showing a solid-solid phase change from Form α to Form I during the heating process. Solubility measurements at various temperatures showed that the two polymorphs were mutually monotropic and that Form I was the relatively thermodynamically stable crystal form.     

Synthesis,characterization and biodistribution of tris(β-diketonato)technetium(III) complexes

New tris(β-diketonato) complexes of trivalent ⁹⁹Tc/^99mTc with the ligands hexane-2,4-dione, heptane-2,4-dione, heptane-3,5-dione, and octane-3,5-dione were synthesized by reduction of pertechnetate with dithionite in the presence of excess β-diketone. The complexes were purified by HPLC, identified by elemental analysis and FAB mass spectrometry, and characterized by vis./u.v./i.r. spectrophotometry. The hexane-2,4-dionato complex crystallizes in the monoclinic space group P2₁/c, isostructurally with pentane-2,4-dionatotechnetium(III). Biodistribution measurements in mice showed the neutral and lipophilic ^99mTc-diketonato complexes to penetrate the blood-brain barrier. However, increasing lipophilicity decreased the brain uptake except for the heptane-2,4-dionato complex, which displayed the highest uptake of 0.82% injected dose/g.     

Binary complexes of Mg(II) and Ca(II) in water–surfactant mixtures

Abstract

Chemical speciation of Mg(II) and Ca(II) complexes of L-histidine in the presence of water–surfactant mixtures in the concentration range 0.0–2.5% w/v CTAB and SDS, 0.0–5.0% v/v TX-100 maintaining an ionic strength of 0.16 mol dm^?3 at 303 K has been studied pH metrically. The active forms of the ligand are LH₃²⁺, LH₂⁺, LH and L^?. The models containing different numbers of species were refined by using the computer program, MINIQUAD75. The predominant species detected were ML₂H₄⁴⁺, ML₂H₃³⁺, ML₂H₂²⁺, and ML₂. The best fit chemical models were arrived at based on statistical parameters. The trend in variation of complex stability constants with change in the composition of the medium is explained on the basis of electrostatic and non-electrostatic forces. The effect of errors in the stability constants was also studied. Chemical speciation was also discussed based on the distribution diagrams.     

Preparation and characterization of 3H-labeled α-bungarotoxin

α-Bungarotoxin has been labeled with [³H]pyridoxamine phosphate, by reaction with pyridoxal phosphate followed by reduction with sodium boro[³H]hydride. Specific activities of up to 27 Ci/mmol have been obtained. Mono- and dilabeled toxins bind irreversibly to the acetylcholine receptor from Torpedo electroplax, despite a change in isoelectric point from 9.2 for native toxin to 6.2 for dilabeled toxin. The ³H-labeled α-bungarotoxin is usable for over a year.     

Preparation of highly pure daidzin on oligo-β-cyclodextrin-Sepharose HP and investigation of chromatographic behavior of isoflavones by molecular docking

A novel method using column chromatography on oligo-β-cyclodextrin-Sepharose HP for the preparation of high purity daidzin from crude soybean samples was proposed in this work. The isoflavone of daidzin in sample A and B was purified under the optimum mobile phase composed of methanol/acetic acid/water=20.0/8.0/72.0 (v/v/v) at a flow-rate of 1.0 mL/min in one-step operation with a purity of 97.2% and 98.1%, a recovery of 95.3% and 96.3% respectively. The target products in isolated fraction were detected and characterized by HPLC analysis and ESI-MS spectrum. Preparative separation with sample-load of up to 2.42 mg/mL medium gave satisfactory results for daidzin with the purities over 97% and recoveries approximately 90%. Molecular docking simulations were utilized to help demonstrate the inclusion complexation between β-cyclodextrin and the isoflavones in samples through inclusion geometries and calculations of the binding energies. The prediction of the elution orders with AUTODOCK and SURFLEX-DOCK were validated by the chromatographic results.     

Synthesis and characterization of the inclusion compound of a ferrocenyldiimine dioxomolybdenum complex with heptakis-2,3,6-tri-O-methyl-β-cyclodextrin

A dioxomolybdenum(VI) complex bearing the diimine ligand N,N′-bis(ferrocenylmethylene)ethylenediamine (FcNN) has been prepared in good yield by the reaction of FcNN with MoO₂Cl₂(THF)₂. One isomeric form was identified by ¹H NMR (including NOE experiments), corresponding to the cis,cis geometry with respect to the CN bonds of the free ligand. The polynuclear complex was immobilized in permethylated β-cyclodextrin (TRIMEB) by addition of the guest to a solution of TRIMEB in a mixture of dichloromethane and nitromethane. Removal of the solvent led to the isolation of an inclusion compound with a 2:1 host:guest stoichiometry. For comparison, an inclusion compound containing just the ligand FcNN and TRIMEB was prepared using a similar method. The products were characterized in the solid state by powder X-ray diffraction (XRD), thermogravimetric analysis (TGA), FT-IR and ¹³C CP MAS NMR spectroscopy. UV-Vis measurements were also carried out in solution. Both the complex MoO₂Cl₂(FcNN) and its inclusion compound with TRIMEB catalyze with high selectivity the liquid phase epoxidation of cyclooctene using tert-butyl hydroperoxide (TBHP) as the oxidant. In general, the catalytic behavior of the Mo^VI complex was not markedly affected by encapsulation in TRIMEB, although observed activities were slightly lower.     

Preparation and characterization of biotinylated probes for the β-interferon receptor

• 1.1. Recently we described the isolation of the β-interferon receptor [Zhang et al. (1986) J. biol. Chem. 261, 8017–8021]. A highly purified product was obtained but in low quantities.
• 2.2. The use ofbiotinylated β-interferon as a ligand represents an alternate approach to receptor isolation.
• 3.3. We have prepared and characterized the derivatives N-(biotinyl)- and N-(biotinyl-ϵ-aminocaproyl)-recombinant human [Ser¹⁷-interferon β (B- and BC-recHulFNβ).
• 4.4. Biotin incorporation does not result in any loss of antiviral activity, demonstrating the recognition of the derivative by the cell receptor.
• 5.5. The biotinylated recHuIFNβ binds specifically and reversibly to succinoylavidin or guanidine thiocyanate-stripped succinoylavidin linked to a Sepharose matrix.
• 6.6. Comparison of the competition curves obtained with [¹⁴C]biotin and [³H]biotinyl recHuIFN, in the presence of increasing concentrations of biotin suggests that the IFN moiety of the derivative has little effect on the affinity of biotin for avidin.
• 7.7. Biotinylated recHuIFNβ derivatives represent useful probes for the β-IFN receptor.

     

Ion-exchange and potentiometric characterization of Al–cystine and Al–cysteine complexes

The interaction between aluminium and cysteine and cystine was evaluated by means of ion-exchange experiments and potentiometry. Ion-exchange experiments included other ligands with affinity for aluminium and two kinds of resins, either a Na⁺-form or an Al³⁺-form exchanger. The ability of the ligands to keep aluminium in solution in the presence of the Na⁺ exchanger or to withdraw it from the Al³⁺-form resin was evaluated. Aluminium quantification was carried out by either graphite-furnace or flame atomic absorption spectrometry. Aluminium extraction isotherms were linearised using the Scatchard plot, and stability constants were obtained from the curves’ slopes. The experiments showed that the ability of the ligands to withdraw aluminium from the Al³⁺-form resin increased following the order cysteine < oxalate < citrate = cystine < nitrilotriacetic acid < ethylenediaminetetraacetic acid. Potentiometric titrations, carried out in aqueous solution with constant ionic strength and temperature, showed that the predominant species in solution have a metal–ligand proportion of 1:1 for both amino acids. The main species are Al(OH)₃L, with log K of 6.2 for cysteine, and AlL and Al(OH)L, with log K of 10.3 and 1.7, respectively, for cystine. Stability constants obtained from the Scatchard plots showed a linear correlation with the stability constants obtained by potentiometry for cystine and cysteine in this work and those collected from the literature for the other ligands. These results show that cysteine and cystine extract and maintain aluminium in solution, which may explain elevated concentrations of aluminium in parenteral nutrition solutions containing these amino acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .