Synthesis of pH-sensitive amphotericin B-poly(ethylene glycol) conjugates and study of their controlled release in vitro

New intravenous conjugates of amphotericin B (AMB) with poly(ethylene glycols) (PEG) (M=5000, 10,000, 20,000) have been synthesized and characterised. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AMB gave conjugates containing a pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. All types of the conjugates are relatively stable in phosphate buffer at physiological conditions of pH 7.4 (37 degrees C), less than 5 mol% AMB being split off from them within 24 h. For a model medium of afflicted tissue was used a phosphate buffer (pH 5.5, 37 degrees C), in which controlled release of AMB from the conjugates takes place. The imine linkage is split to give free AMB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights of the PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4 (37 degrees C) with half-lives of 2-5 h depending on molecular weights of the PEGs used (M = 5000, 10,000, 20,000). At first, the splitting of ester linkage produces the relatively stable pro-drug, that is, 4-carboxybenzylideniminoamphotericin B, which is decomposed to AMB and 4-formylbenzoic acid in a goal-directed manner only at pH 7 (t1/2 = 2 min, pH 5.5, 37 degrees C). A goal-directed release of AMB is only achieved by acid catalysed hydrolysis of imine linkage, either from the polymeric conjugate or from the pro-drug released thereof. The LD50 values determined in vivo (mouse) are 20.7 mg/kg and 40.5 mg/kg for the conjugates with ester linkage (M = 10,000 and 5000, respectively), which means that they are ca. 6-11 times less toxic than free AMB.     

In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (±)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 μM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid β-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (?)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 × 10^?4 and 0.177 × 10^?4 mol/mol protein. Multipie dosing increased irreversible binding 3- to 4-fold. © 1993 Wiley-Liss, Inc.     

The effect of structure and conformation on the relative hydrophobicity of dinucleosidephosphates and their analogs

Partition of a number of dinucleosidephosphates, their (2'-5')-isomers and a series of their analogues, in which the ribose ring was replaced by acyclic hydroxyalkyl substituents, in an aqueous biphasic ficoll--dextran system has been studied. Effect of the ionic composition of the biphasic system on partitioning of the compounds was examined. The relative hydrophobicity of the compounds in the presence of 0.11 M phosphate buffer, pH 7.4, and in the presence of 0.15 M NaCl in 0.01 M buffer, pH 7.4, was estimated. The results obtained are considered in regard to the effect of structure and conformation of a molecule on its affinity for an aqueous environment.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

In vitro degradation behavior of microspheres based on cross-linked dextran

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time. The amount and nature of the formed degradation products were established for microspheres with different cross-link densities by FT-IR (Fourier transformed infrared spectroscopy), NMR, mass spectrometry, SEC analysis, and XPS (X-ray photoelectron microscopy). The dex-HEMA microspheres DS 12 (degree of HEMA substitution; the number of HEMA groups per 100 glucose units) incubated at pH 7.4 and 37 degrees C showed a continuous mass loss, leaving after 6 months a residue of about 10% (w/w) of water-insoluble products. NMR, mass spectrometry, and SEC showed that the water-soluble degradation products consisted of dextran, low molecular weight pHEMA (M(n) approximately 15 kg/mol), and small amounts of unreacted HEMA and HEMA-DMAP (intermediate reaction product of the Baylis-Hillman reaction of HEMA with DMAP (4-dimethyl aminopyridine)). Microscopy revealed that the water-insoluble residue consisted of particles with shape and size similar to that of nondegraded microspheres. However, these particles had lost their mechanical strength as evidenced from micromanipulation experiments. FT-IR and XPS (X-ray photoelectron microscopy) revealed that these particles consisted of pHEMA, of which a small fraction was soluble in methanol (M(n) ranging between 27 and 82 kg/mol). The insoluble material likely consisted of lightly cross-linked pHEMA. In conclusion, in vitro degradation of dex-HEMA microspheres results in the formation of water-soluble degradation products (mainly dextran), leaving a small water-insoluble residue mainly consisting of pHEMA.     

Cationic proteins from human neutrophil granulocytes. Evidence for their chymotrypsin-like properties.

Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose. Together with lysozyme, the three cationic proteins exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH, among the granule constituents that are solubilized in 0.1 M phosphate buffer, pH 7.0, containing 1 M NaCl. The three cationic proteins represent a group of "neutral proteases" distinct from elastase and collagenase. They hydrolyze casein, azocasein and the chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester. Optimal activity is found at pH 7.4-7;5. The enzymes are inhibited by the specific chymotrypsin inhibitor N-tosyl-L-phenylalanylchloromethane and by the naturally occurring inhibitors alpha-antichymotrypsin, alpha-1-antitrypsin, as well as by the trypsin inhibitors from soy beans and limabeans.     

Isolation and properties of rat plasma lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.     

Alkylation of brain corticosteroid acetyltransferase by 17-hydroxyprogesterone 17-(9-oxo-10-chlorodecanoate) and related compounds

The addition of 17-hydroxyprogesterone 17-(9-oxo-10-chlorodecanoate) (1) at 1.5 μM to a partially purified preparation of corticosteroid acetyl-transferase from the primate brain at pH 7.4, results in a 50% inhibition of enzymatic activity after 30 minutes at 37°. At this concentration the analogous 9-oxo-10-diazodecanoate, 8-carbomethyoxyoctanoate or 8-carboxyoctanoate esters show no effect on the activity of this enzyme. 9-Oxo-10-chlorodecanoate and its methyl ester are respectively 0.44 and 0.07-fold as effective inhibitors as 1. The inhibition by 1 has been shown to be non-competitive and irreversible. There is no reaction of 1 with amino acids, glutathione, or human or bovine serum mercaptalbumin in a pH 7.4 phosphate buffer at 37°, demonstrating the partial specificity of this alkylating agent.     

Synthesis and characterisation of a new pH-sensitive amphotericin B--poly(ethylene glycol)-b-poly(L-lysine) conjugate

This paper reports on the synthesis, characterisation, and efficiency of a new intravenous conjugate of amphotericin B (AMB). Twelve molecules of AMB were attached to block copolymer poly(ethylene glycol)-b-poly(L-lysine) via pH-sensitive imine linkages. In vitro drug release studies demonstrated the conjugate (M(w)=26,700) to be relatively stable in human plasma and in phosphate buffer (pH 7.4, 37 degrees C). Controlled release of AMB was observed in acidic phosphate buffer (pH 5.5, 37 degrees C) with the half-life of 2 min. The LD(50) value determined in vivo (mouse) is 45 mg/kg.     

Relative hydrophobicity of amino acid residues in homooligopeptides as measured by aqueous two-phase partitioning.

Partitioning of 17 amino acids and their homooligopeptides of different lengths in an aqueous dextran-PEG two-phase system containing 0.15 m NaCl in 0.01 M sodium phosphate buffer, pH 7.4 and 0.11 m sodium phosphate buffer, pH 7.4 was examined. The relative hydrophobicity of the amino acid residues was estimated and expressed in equivalent numbers of methylene units. Analysis of the data shows that the additivity principle does hold for the hydrophobicity of homooligopeptides. The relative hydrophobicity of essentially all amino acid residues is noticeably affected by the ionic composition of aqueous media.     

Recovery in aqueous two-phase systems of lutein produced by the green microalga Chlorella protothecoides

The objective of this study was to develop a chromatographic method for the analysis of the anti-androgen vinclozolin (V) and its metabolites 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) and 3,5-dichloroaniline (M3) in rat serum. V, M1-M3 were resolved using an HPLC gradient program with a mobile phase consisting of 60-75% methanol:acetonitrile (70:30) and 0.05 M monobasic sodium phosphate buffer pH 3.3 at 1 ml/min, a C18 column, and monitored at 212 nm. Incubates of 0.01 M monobasic potassium phosphate buffer (PB) pH 7.4 and rat serum were spiked with V and its metabolites and processed by diluting samples (1:4) with 0.1M PB pH 3.3, to limit methodological hydrolysis of analytes, followed by addition of acetonitrile. Recoveries of V, M1 and M2 ranged from 85 to 105%, whereas recovery of M3 was <25%. V was hydrolyzed to M1 and M2 after incubation in PB pH 7.4 and rat serum, with M1 the predominant metabolite. This method was successfully applied in the analysis of V and its metabolites in the serum of a male rat after oral administration of V (100 mg/kg).     

Heterogeneity of platelet monoamine oxidase in man

Multiplicity of platelet MAO was studied. Multiple forms of enzyme was separated with the use of 1.5% Tritone x-100 and, 1.3 M urea at pH 7.4 in, 0.01 M K-Na phosphate buffer. Three forms of MAO--MAO-I, MAO-II, and MAO-III were obtained under separation of solubilized proteins by affinity chromatography on AN-Sepharose 4B. Deprenyl in 10(-5) M inhibited all forms of the activity completely. Relation between multiple forms of the brain and platelet was discussed.     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.     

Use of Horseradish-Peroxidase Labelled Antibodies in ELISA for Plant Virus Detection

Detection of plant viruses by ELISA using horseradish peroxidase for antibody labelling (ELISA-peroxidase) has been standardized by evaluating variants of the procedure, regarding composition and concentration of buffers and additives. Immunoglobulins (IgG) are isolated from antisera by precipitation with ammonium-sulphate and by purification with DEAE-52 (Whatman) cellulose. IgG are conjugated with horseradish peroxidase by a modified oxidation-periodate method. In ELISA-peroxidase 0.05 M carbonate-bicarbonate coating buffer pH 9.6 has been substituted by 0.01 M carbonate buffer pH 9.2. Extraction buffer is used with 0.5% bovine serum albumin (BSA), without polyvinylpyrrolidone (PVP). Samples are diluted in, phosphate buffered saline (PBS) pH 7.2 with 0.05% Tween 20 and 0.5% BSA. IgG are conjugated with horseradish peroxidase, diluted in 0.1 M Tris-HCl, pH 7.4 with 0.05% Tween 20 and 1% BSA. The substrate is incubated in the darkness for 20 min at room temperature. ELISA-peroxidase proved to be equivalent in sensitivity and specificity with ELISA using alkaline phosphatase for antibody labelling. Its advantage is a lower cost of chemicals used in the test.     

Fine tuning of the pH-dependent drug release rate from polyHPMA-ellipticinium conjugates

Polymer conjugates of anticancer drugs have shown high potential for assisting in cancer treatments. The pH-labile spacers allow site-specific triggered release of the drugs. We synthesized and characterized model drug conjugates with hydrazide bond-containing poly[N-(2-hydroxypropyl)methacrylamide] differing in the chemical surrounding of the hydrazone bond-containing spacer to find structure–drug release rate relationships. The conjugate selected for further studies shows negligible drug release in a pH 7.4 buffer but released 50% of the ellipticinium drug within 24 h in a pH 5.0 phosphate saline buffer. The ellipticinium drug retained the antiproliferative activity of the ellipticine.     

Simple and sensitive determination of five quinolones in food by liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of five different quinolones: enrofloxacin, ciprofloxacin, sarafloxacin, oxolinic acid and flumequine in pork and salmon muscle. The method includes one extraction and clean-up step for the five quinolones together which are detected in two separated HPLC runs by means of their fluorescence. The proposed analytical method involves homogenizing of the tissue sample with 0.05 M phosphate buffer, pH 7.4 and clean-up by Discovery DS-18 cartridges. For chromatographic separation a Symmetry C(18) column is used in two different runs: (1) ciprofloxacin, enrofloxacin and sarafloxacin with acetonitrile-0.02 M phosphate buffer pH 3.0 (18:82) as mobile phase and the detector at excitation wavelength: 280 nm and emission wavelength 450 nm; and (2) oxolinic acid and flumequine with acetonitrile-0.02 M phosphate buffer pH 3.0 (34:66) as mobile phase and excitation wavelength: 312 nm and emission wavelength: 366 nm. Detection limit was as low as 5 ng g(-1), except for sarafloxacin which had a limit of 10 ng g(-1). Standard curves using blank muscle tissues spiked at different levels showed a good linear correlation coefficient, r(2) higher than 0.999 for all quinolones.     

Possibility of the long-term fixation of the cells of a HeLa culture without disturbing their ultrastructure

It is shown that the composition of fixatives (2.5% glutaraldehyde in 0.1 M phosphate buffer with pH 7.2-7.4, and the mixture of 2.5% glutaraldehyde with 2% paraformaldehyde in 0.1 M phosphate buffer with pH 7.2-74) and duration of fixation (30 minutes, 24 hours, 7 days, and 30 days) under the room temperature exerts no influence on preservation of HeLa cultured cells. The ultrastructure of all the organelles of these cells is similar in any cases examined. All the membrane structures are well preserved; no condensation of chromatin is observed; the widths of the canals of endoplasmic reticulum, and of the intracristal and lateral spaces of mitochondria are invariable. Polysomes are present in the cytoplasm throughout the period of fixation.     

Solubility of palmitoyl-coenzyme A in acyltransferase assay buffers containing magnesium ions

The solubility of palmitoyl-CoA is strongly affected by Mg2+ concentrations commonly used in acyltransferase reactions. In 0.10 M Tris-HCl buffer at pH 7.4 or 8.5, all of the palmitoyl-CoA in 10 microM solutions and 90% of the palmitoyl-CoA in 100 microM solutions are precipitated by 1 mM Mg2+. In 0.05 M phosphate at pH 7.4, and in 0.10 M Tris-HCl containing 0.4 M KCl, the substrate remains soluble at Mg2+ concentrations below 4-5 mM. Above 5 mM Mg2+, palmitoyl-CoA is insoluble in all of these buffers. Substrate solubility could therefore be a limiting factor when free Mg2+ and fatty acyl-CoAs are present together during acyltransferase assays.     

Mechanism of Cu2+-induced elevation of [3H]cimetidine binding to membranes in rat brain

The mechanism of increasing effect of CuCl₂ on specific [³H]cimetidine binding was examined in brain membranes of rats. CuCl₂-Induced elevation of [³H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl₂ (5–50 mM) inhibited effect of CuCl₂, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl₂ (5–50 mM) did not. CuCl₂ (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl₂ in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl₂ mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu²⁺ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl₂ also caused a marked elevation in [³H]cimetidine binding, seeming to be due to formation of cimetidine-Pd²⁺ chelate complex. There were two types of [³H]cimetidine binding in the presence of 20 nM PdCl₂: high affinity binding with K_d = 0.7 ± 0.1 nM and low affinity binding with K_d = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu²⁺ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone.     

The Effect of Polysorbate 20 on Solubility and Stability of Candesartan Cilexetil in Dissolution Media

The addition of polysorbate 20 (T20) is required to achieve “sink” conditions during a dissolution test for tablets with candesartan cilexetil (CC). Polysorbate 20 (0.35%–0.7% w/w) added to 0.05 mol/L of phosphate buffer pH 6.5 dramatically increased the apparent solubility of the drug from 0.8 μg/ml even to 353 μg/ml, while its effect in lower pH or in water was much smaller (20 μg/ml in pH 4.5). The increased concentration of phosphate salts (0.2 mol/l) at pH 6.5 in the presence of 0.7% of polysorbate 20, resulted in further increase of candesartan cilexetil solubility to 620 μg/ml. The change of pH from 1.2 to 7.4 resulted in a 1.5-fold increase of the activation energy and, depending on temperature, 8–14-fold decrease of the degradation rate. When polysorbate 20 increased the activation energy 2-fold, independent of pH, it protected candesartan cilexetil from degradation; however, this effect was temperature dependent and was very small at 310 K—the degradation rate in pH 6.5 decreased by 13% only. It was calculated that in the phosphate buffer pH 6.5 with polysorbate, one can expect during 24 h the degradation at the level of 9.3%, thus a flow-through dissolution apparatus was recommended for testing prolonged release dosage forms.