The influence of pH on the interaction of lipophilic anthracyclines with bovine serum albumin. Quantitative characterization by measurement of fluorescence quenching.

We have investigated the interaction of the lipophilic anthracyclines 4'-iodo-4'-deoxydoxorubicin (IDX) and 4-demethoxy-daunorubicin (DDN) with bovine serum albumin by the quantitation of fluorescence quenching. The protein binding of IDX was extremely sensitive to the pH of the solution in which the complex was formed and paralleled the effect of pH on dimerization of the drug. The effect of pH on the protein binding and self-association of DDN was less extensive. Both compounds exhibited curvilinear Scatchard plots indicating apparent cooperativity in the binding process. Because of the self-association of the drugs in aqueous solution, we attempted to resolve this cooperativity in terms of the preferential binding of the dimer to the acceptor. However, we found that similar Scatchard plots could be simulated by using slightly erroneous estimates of the fluorescence yield of the complex, rendering any such analysis inconclusive. Consequently, the relationship between acceptor concentration and the fraction of ligand bound was considered to be fitted adequately in terms of a single acceptor site per albumin molecule. The pH dependence of the association constants for bovine serum albumin was described best by the hydrophobic interaction of neutral drug monomer with a binding site with titratable affinity. We postulate that the pH-dependent binding of some anthracyclines with albumin may lead to their enhanced uptake, relative to that of non-target organs, into tumours with an acidotic extracellular milieu.     

Albumin-Encapsulated Liposomes: A Novel Drug Delivery Carrier With Hydrophobic Drugs Encapsulated in the Inner Aqueous Core

Liposomes are clinically used in drug delivery, but loading hydrophobic substances is limited to the hydrophobic space of a lipid membrane, despite the fact that it is favorable to encapsulate substances into the inner aqueous core of liposome, from a drug stability of view. We report herein on the preparation of a liposome with bovine serum albumin encapsulated (BSA-liposome). Using this system, it is possible to encapsulate hydrophobic drugs in the inner aqueous core of the liposome based on the hypothesis that the water solubility of hydrophobic drugs is increased when bound to albumin. The physicochemical properties of the prepared BSA-liposomes could be easily regulated and the loading of hydrophobic drugs in the inner aqueous core of the liposome was dramatically improved by virtue of the drug-binding properties of albumin. An in vivo safety and pharmacokinetic study showed that BSA-liposomes possess favorable properties as a drug carrier, including biocompatibility and a stealth effect. This new type of hydrophobic drug carrier, an albumin-liposome, has the potential for use in delivering numerous hydrophobic drugs that typically bind to albumin.     

Biodegradable cross-linked starch/protein microcapsules containing proteinase inhibitor for oral protein administration.

The objective of this study is to demonstrate the feasibility of microcapsules containing a protein and a proteinase inhibitor in order to allow the oral administration of proteic or peptidic drug. Starch/bovine serum albumin mixed-walled microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride. The microcapsules were loaded with native or amino-protected aprotinin by incorporating protease inhibitors in the aqueous phase during the cross-linking process. Microcapsules can be degraded in the presence of alpha-amylase. The influence of the formulation parameters on the in vitro release of the inhibitor activity and the protein was studied. The protective effect of microcapsules with aprotinin for bovine serum albumin was revealed in vitro. The presence of the native bovine serum albumin was demonstrated after incubation of the microcapsules with aprotinin in a mixture of alpha-amylase (5.4 U/ml) and trypsin (900 spectrophotometric BAEE units/ml) for 3 h at 37 degrees C, whereas the protein was completely degraded in the release medium of the microcapsules without aprotinin.     

灯盏乙素与牛血清白蛋白相互作用的研究

目的采用荧光、紫外光谱法研究在模拟人体生理条件下灯盏乙素与牛血清白蛋白(BSA)之间的相互作用。方法将灯盏乙素对牛血清白蛋白进行荧光淬灭滴定,利用Scatchard模型和F"rster非辐射能量转移理论得出灯盏乙素和牛血清白蛋白的结合参数。结果灯盏乙素与BSA的结合常数K=1.240×106,结合距离r=2.94nm,相互作用力主要为疏水作用力。结论阐明了灯盏乙素和牛血清白蛋白相互作用的机制,建立了灯盏乙素和牛血清白蛋白的结合模型。     

Carrier proteins determine local pharmacokinetics and arterial distribution of paclitaxel.

The growing use of local drug delivery to vascular tissues has increased interest in hydrophobic compounds. The binding of these drugs to serum proteins raises their levels in solution, but hinders their distribution through tissues. Inside the arterial interstitium, viscous and steric forces and binding interactions impede drug motion. As such, this might be the ideal scenario for increasing the amount of drug delivered to, and residence time within, arterial tissues. We quantified carrier-mediated transport for paclitaxel, a model hydrophobic agent with potential use in proliferative vascular diseases, by determining, in the presence or absence of carrier proteins, the maximum concentration of drug in aqueous solution, the diffusivity in free solution, and the diffusivity in arterial tissues. Whereas solubility of paclitaxel was raised 8.1-, 21-, and 57-fold by physiologic levels of alpha(1)-acid glycoproteins, bovine serum albumin, and calf serum over that in protein-free solution, diffusivity of paclitaxel in free solution was reduced by 41, 49, and 74%, respectively. When paclitaxel mixed in these solutions was applied to arteries both in vitro and in vivo, drug was more abundant at the tissue interface, but protein carriers tended to retain drug in the lumen. Once within the tissue, these proteins did not affect the rate at which drug traverses the tissue because this hydrophobic drug interacted with the abundant fixed proteins and binding sites. The protein binding properties of hydrophobic compounds allow for beneficial effects on transvascular transport, deposition, and distribution, and may enable prolonged effect and rationally guide local and systemic strategies for their administration.     

盐酸西布曲明与牛血清白蛋白结合作用的光谱学研究

目的运用光谱学方法研究在生理pH值条件下盐酸西布曲明与牛血清白蛋白之间的结合作用。方法 通过荧光法和吸收光谱法确定了盐酸西布曲明对牛血清白蛋白的荧光猝灭机制。依据Scatchard方程测定了不同温度下该结合反应的结合常数和结合位点数。根据热力学方程讨论了两者间的主要作用力类型。结合同步荧光分析了盐酸西布曲明对牛血清白蛋白构象的影响。结果盐酸西布曲明对牛血清白蛋白的荧光猝灭机制为静态猝灭。在8，25和37 ℃时盐酸西布曲明与牛血清白蛋白的结合常数分别为1.21×10⁵，8.31×10⁴和6.97×10⁴ L·mol^-1，结合位点数均为1。结合反应的热力学参数为ΔH=-9.70 kJ·mol^-1，ΔS=56.41 J·mol^-1·K^-1。结论 两者结合的主要作用力类型是静电作用。盐酸西布曲明在体内能够被血清蛋白存储和转运且结合时对蛋白构象无影响。     

Spray coated pellets as carrier system for mucoadhesive drug nanocrystals.

High pressure homogenization can be employed to produce drug nanocrystals with a number of advantages, like improved solubility behaviors, better drug targeting or even increased mucoadhesiveness. To obtain a controlled drug delivery system it is necessary to transform the resulting nanosuspension into a solid dosage form. The present study shows the feasibility to use a mucoadhesive nanosuspension of poorly soluble hydrocortisone acetate produced by high pressure homogenization as layering dispersion in a fluidized bed process, followed by the application of an enteric coating to achieve a controlled drug release. To point out the advantages of drug nanocrystals the new fomulation was compared with a formulation containing micronized drug. Both formulations were characterized with regard to their particle size and crystallinity by using laser diffractometry, photon correlation spectroscopy and X-ray diffraction. The pellet morphology was characterized by using the environmental scanning electron microscopy (ESEM). In the in vitro dissolution tests an accelerated dissolution velocity and an increased drug release could be shown for the pellets containing drug nanocrystals.     

ZnO-ZnS QDs interfacial heterostructure for drug and food delivery application: enhancement of the binding affinities of flavonoid aglycones to bovine serum albumin

Zero-dimensional nanostructures are green nanomaterials that have recently attracted increasing attention. However, very little information is available on whether or not these heterostructures affect drug transport in blood. In current work, flavonoid aglycones were studied for their affinities for bovine serum albumin (BSA) in the presence and absence of zinc oxide-zinc sulfide quantum dots (ZnO-ZnS QDs) in vitro. The fluorescence intensity of BSA decreased remarkably with increasing concentration of ZnO-ZnS QDs, resulting in an obvious red-shift of the maximum emission of BSA from 340 to 348 nm. The magnitudes of binding constants in the presence of QDs ranged from 10(4) to 10(6) L/mol, and the number of binding sites per BSA molecule (n) was determined as 1.12 ± 0.17. Although ZnO-ZnS QDs significantly increased the affinities for BSA of myricetin, luteolin, gallocatechin gallate, tectorigenin, and formononetin, they barely affected the binding affinities of flavone, (-)-epicatechin gallate, and quercetin. FROM THE CLINICAL EDITOR: Serum albumins are major transport proteins in blood that reversibly bind fatty acids, amino acids, drugs, and inorganic ions, which interactions have important effects on the distribution, free concentration, and metabolism of drugs in blood. In this research nine flavonoid aglycones were studied for their affinities for bovine serum albumin (BSA). Interestingly it was found that presence of ZnO-ZnS QDs significantly increased the affinities of BSA for several of these aglycones.     

表面活性剂对高水溶性药物阿魏酸钠白蛋白纳米粒的载药特性影响

目的:制备高水溶性药物白蛋白纳米粒,考察表面活性剂对高水溶性药物的包封作用。方法:以牛血清白蛋白为载体材料,阿魏酸钠为高水溶性药物模型,采用去溶剂化法制备阿魏酸钠白蛋白纳米粒。用低温超速离心法、层析-离心法、层析-酶解法对纳米粒包封率和载药量进行测定评价,并考察表面活性剂对纳米粒包封率、载药量及得率的影响。结果和结论:层析-离心法测定结果可靠。亲水性表面活性剂0.3%洛泊沙姆和亲脂性表面活性剂0·48%卵磷脂联合使用,有利于高水溶性药物的包封,包封率为28.42%,载药量为10.5%。     

5-羟甲基糠醛与不同血清白蛋白的结合反应机制研究

利用光谱实验结合计算机模拟技术研究中药活性成分5-羟甲基糠醛 (5-HMF) 与人血清白蛋白 (HSA) 及牛血清白蛋白 (BSA) 的结合反应机制。结果表明, 5-HMF与HSA及BSA均结合生成静态复合物, 但结合强度存在一定的差异, 5-HMF与HSA及BSA分子的结合距离不同, 且r值均很小, 说明发生了能量转移现象。药物分子与血清白蛋白 (SA) 相互作用过程中, 药物分子对HSA及BSA构象均产生影响, 使结合位域的疏水性发生改变, 但对不同血清白蛋白影响程度迥异。应用荧光相图法解析出5-HMF与BSA及HSA反应构象形态的变迁均为“二态”模型。金属离子Co (Ⅱ) 介导药物与SA相互作用的程度随SA种类不同而存在差别。依据计算机模拟建立的分子对接结果显示, 5-HMF与血清白蛋白的相互作用主要为疏水作用和氢键, 分子模拟结果与光谱实验结果一致, 此为5-HMF的药理作用机制研究提供一定理论参考。     

Albumin inhibition of the antileukemic activity of hydroxylated phenothiazines

Trifluoperazine (TFP) shows cytotoxic activity against human acute lymphatic leukemia (ALL) in vitro. This activity is inhibited by increasing serum concentration and by albumin. Despite its in vitro activity, the drug is inactive in vivo. To determine if increased phenothiazine hydrophilicity could protect against albumin inhibition of antileukemic activity, we compared ALL cytotoxic median effective dose concentrations of a series of hydroxylated phenothiazines in 5% fetal bovine serum (FBS) and in 5% FBS supplemented with albumin. Albumin inhibits the activity of all drugs. A representative derivative 7,8-dihydroxychlorpromazine, although active in vitro, is inactive against L1210 and P388 murine leukemias in vivo.     

Mechanism of interaction of the non-steroidal antiinflammatory drugs meloxicam and nimesulide with serum albumin

The mechanism of interaction of the non-steroidal antiinflammatory drugs meloxicam and nimesulide with human and bovine serum albumin has been studied using fluorescence spectroscopy. There was only one high affinity site on serum albumin for both the drugs with association constants of the order of 10(5). Negative enthalpy (DeltaH(0)) and positive entropy (DeltaS(0)) values in the case of both meloxicam and nimesulide showed that both hydrogen bonding and hydrophobic interactions play a role in the binding of these drugs. Binding studies in the presence of the hydrophobic probe 1-anilinonaphthalene-8-sulfonate (ANS) showed that the binding of meloxicam and nimesulide to serum albumin involves predominantly hydrophobic interactions. Stern-Volmer analysis of the quenching data showed that quenching is highly efficient and that the tryptophan residues in hydrophobic regions of the proteins are fully exposed to the drugs. Thus these drugs are bound to albumin by hydrophobic interactions as well as hydrogen bonding at a site, which is close to the tryptophan residues. An increase of the pH and ionic strength caused an increase in the concentration of free drug, although the effect was not very significant.     

Methods for in vitro percutaneous absorption studies III: hydrophobic compounds

The absorption of two hydrophobic compounds through rat skin was measured by in vivo and in vitro techniques. The permeation of the fragrance ingredients 3-phenyl-2-propenyl 2-aminobenzoate (I) and 1-(3-ethyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)+ ethanone (II) was measured from a petrolatum and an acetone vehicle. Increases in permeation of 8-fold (I) and 95-fold (II) were observed when the compounds were tested in vivo under conditions similar to in vitro procedures. The apparent inability of the compounds to freely enter the diffusion cell receptor fluid was partially reversed by replacing normal saline with other fluids: rabbit serum, 3% bovine serum albumin, organic solvents, and dilutions of four nonionic surfactants. The effect of the receptor fluids on the integrity of the skin barrier was assessed by measuring the permeability of control compounds (cortisone, urea, and water). A 6% solution of polyethylene glycol 20 oleyl ether was the receptor fluid of choice. Without apparent damage to the skin, 61% (petrolatum vehicle) or 73% (acetone vehicle) of the in vivo absorption of I was obtained. With II, only 32% of the in vivo absorption was achieved (petrolatum vehicle). Even when the surfactant solution is used, significant differences may still remain between in vivo and in vitro results.     

Risperidone interacts with serum albumin forming complex

The aim of the work is to study the mechanisms of the interaction of risperidone with human and bovine serum albumins using the fluorescence quenching technique. Risperidone is an atypical antipsychotic drug used to treat many psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with a 290 nm wavelength light, and observed quenching by titrating human and bovine serum albumin solutions with risperidone. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that the drug quenches the fluorescence of the human serum albumin by the formation of a complex risperidone-albumin. Association constants calculated from Stern-Volmer equation for low concentrations (lower than 1:10 ratio risperidone/albumin) were of 2.56 × 10(5)M(-1), at 25 °C, and 1.43 × 10(5)M(-1), at 37 °C. As the quenching intensity of bovine serum albumin, which contains two tryptophan residues, was found to be higher than that of human serum albumin, which contains only one tryptophan residue. Hence, we suggest that the primary binding site for risperidone in albumin should be located in sub domain IB.     

Some properties of the interaction between 2,2'-diselenadibenzoic acid and serum albumins

The binding of 2,2'-diselenadibenzoic acid to bovine serum albumin (BSA) and human serum albumin (HSA) was studied by using fluorescence spectroscopy. The measurement was performed in Tris-HCl buffer aqueous medium at pH = 7.40. The quenching constant at 303 K was (3.277 +/- 0.046) x 10(13) L mol(-1) s(-1) for BSA, and (3.946 +/- 0.002) x 10(12) L mol(-1) s(-1) for HSA. Decreased quenching was observed in association with increased temperature. Our findings show that the observed binding constant is dependent on the ionic strength of the medium. It is said that electrostatic interactions play a role in the binding of 2,2'-diselenadibenzoic acid to serum albumin, in addition to the hydrophobic association. The decrease of the linearity of S-V plot demonstrates reduced binding of ligand to the protein in the presence of anionic surfactants such as sodium dodecyl sulfate (SDS), which indicates that 2,2'-diselenadibenzoic acid most likely binds to the hydrophobic pockets within sub-domain IIA of serum albumin, the same site as SDS.     

Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells.

The effects of various experimental conditions on in vitro drug permeability to Caco-2 monolayers were investigated to determine the optimized conditions for the prediction of intestinal drug absorption. Concerning the pH of the transport medium in the Caco-2 study, two different pH values, 6.0 and 7.4, were tested for the apical medium with the pH of the basolateral medium fixed to 7.4. The change in the apical pH showed pronounced effects on the permeability of both passively and actively transported drugs. It was found that the transport study under the condition of an apical pH value of 6.0 showed a better prediction of in vivo drug absorption in human. The appropriate conditions for determining the permeability of poorly soluble drugs were also examined. First, the effects of bile acids, surfactant and some agents used for solubilizing drugs on the permeability and transepithelial electrical resistance (TEER) of Caco-2 monolayers were investigated. Taurocholic and cholic acid showed no effects on the permeability of 3H-Dexamethasone (DEX) and TEER at 10 mM concentration, suggesting the possibility of use in the Caco-2 study. Polyethyleneglycol-400 and dimethylsulfoxide reduced the permeability of DEX concentration dependently, whereas ethanol induced no significant changes in the permeability. Furthermore, it was demonstrated that the addition of plasma protein (bovine serum albumin) to the basolateral medium apparently facilitated the transport of poorly soluble drugs with high lipophilicity across Caco-2 monolayers. These findings clearly suggest the importance of considering the physiological conditions of in vivo drug absorption in optimizing the in vitro experimental conditions for transport study using Caco-2 cells, in order to obtain a satisfactory in vitro-in vivo correlation.     

Importance of the test medium for the release kinetics of a somatostatin analogue from poly(D,L-lactide-co-glycolide) microspheres.

The determination of in vitro release kinetics of peptides from poly(d,l-lactide-co-glycolide) (PLGA) microspheres generally requires optimization of the test conditions for a given formulation. This is particularly important when in vitro/in vivo correlation should be determined. Here, the somatostatin analogue vapreotide pamoate, an octapeptide, was microencapsulated into PLGA 50:50 by spray-drying. The solubility of this peptide and its in vitro release kinetics from the microspheres were studied in various test media. The solubility of vapreotide pamoate was approximately 20-40 microg/ml in 67 mM phosphate buffer saline (PBS) at pH 7.4, but increased to approximately 500-1000 microg/ml at a pH of 3.5. At low pH, the solubility increased with the buffer concentration (1-66 mM). Very importantly, proteins (aqueous bovine serum albumin (BSA) solution or human serum) appeared to solubilize the peptide pamoate, resulting in solubilities ranging from 900 to 6100 microg/ml. The release rate was also greatly affected by the medium composition. Typically, in PBS of pH 7.4, only 33+/-1% of the peptide were released within 4 days, whereas 53+/-2 and 61+/-0.9% were released in 1% BSA solution and serum, respectively. The type of medium was found critical for the estimation of the in vivo release. The in vivo release kinetics of vapreotide pamoate from PLGA microspheres following administration to rats were qualitatively in good agreement with those obtained in vitro using serum as release medium. Finally, sterilization by gamma-irradiation had only a minor effect on the in vivo pharmacokinetics. Copyright     

Hollow poly(MPC-g-PEG-b-PLA) graft copolymer microcapsule as a potential drug carrier

In this article, an amphiphilic graft copolymer composed of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as the hydrophilic backbone, poly(L-lactic acid) (PLA) as the hydrophobic side-chains and polyethylene glycol (PEG) as the spacer was synthesized. Transmission electron microscopy revealed that the graft copolymer could self-assemble into hollow microcapsules when dialyzed in aqueous solution and particle sizes ranged from 200 to 300 nm, while the graft copolymer formed core-shell microspheres with the absence of PEG spacer. X-ray photoelectron microscope showed that MPC polymers were located at the surface of the microcapsules. The amounts of adsorbed bovine serum albumin and Fg on the microcapsules were significantly decreased than that on the conventional PLA particles (74% and 60%, respectively), well indicating the anti-adhesive property of the microcapsules. Paclitaxel was chosen as a prototype anticancer drug for the encapsulation and release studies, the results showed that the drug encapsulation efficiency was 89.3?±?1.2% and the microcapsules exhibited controlled release behaviour.     

Mechanisms of corneal drug penetration. II: Ultrastructural analysis of potential pathways for drug movement

Ultrastructure analysis was conducted in an effort to augment the results of classical kinetic studies. Scanning electron microscopy (SEM) allowed visual inspection of cellular junctions on corneal epithelium and endothelium. The addition of calcium-chelating agents to in vivo and in vitro mounted corneas demonstrated a concentration-dependent progressive expansion of the intercellular spaces of epithelium and endothelium, as seen by SEM. The expansion of these cellular junctions correlates with increases in permeability of the cornea to glycerol under similar conditions. The size of the intercellular space was estimated by transmission electron microscopy. Use of lanthanum as a marker of aqueous diffusional pathways demonstrated that the epithelial surface is not a totally occlusive barrier to transport of small hydrophilic compounds. Development of a method whereby an administered drug could be visualized in its actual pathway of movement through the cornea was undertaken, involving precipitation of specific compounds in the tissue with osmium tetroxide vapor. Results suggest that separate pathways of drug movement exist in the cornea for hydrophilic and hydrophobic compounds. Hydrophilic compounds were preferentially located in intercellular spaces, whereas hydrophobic compounds were associated with the lipid structures of the tissue. The results of these studies are consistent with a currently proposed 'pore' model for the penetration of drugs through the cornea.     

Hydrolysis and protein binding of melphalan

Melphalan (30 microgram/ml) is completely hydrolyzed in water at 37 degrees after 8 hr. At lower temperatures, hydrolysis proceeds at slower rates. The presence of bovine serum albumin retards hydrolysis of melphalan (30 microgram/ml) in water. The melphalan hydrolysis rate is directely releated to the bovine serum albumin concentration. At 37 degrees, 8 g of bovine serum albumin/100 ml of water gives a recovery rate of melphalan similar to that of human plasma. In vitro alkylation of melphalan at 37 degrees with human plasma containing 30 microgram/ml, calculated by equilibrium dialysis, methanol extraction, and high-pressure liquid chromatographic analysis, is 30% after 8 hr.