Drug Binding Capacity Research Articles

Effect of chain length on binding of fatty acids to Pluronics in microemulsions

We investigated the effect of fatty acid chain length on the binding capacity of drug and fatty acid to Pluronic F127-based microemulsions. This was accomplished by using turbidity experiments. Pluronic-based oil-in-water microemulsions of various compositions were synthesized and titrated to turbidity with concentrated Amitriptyline, an antidepressant drug. Sodium salts of C 8, C 10, or C 12 fatty acid were used in preparation of the microemulsion and the corresponding binding capacities were observed. It has been previously determined that, for microemulsions prepared with sodium caprylate (C 8 fatty acid soap), a maximum of 11 fatty acid molecules bind to the microemulsion per 1 molecule of Pluronic F127 and a maximum of 12 molecules of Amitriptyline bind per molecule of F127. We have found that with increasing the chain length of the fatty acid salt component of the microemulsion, the binding capacity of both the fatty acid and the Amitriptyline to the microemulsion decreases. For sodium salts of C 8, C 10 and C 12 fatty acids, respectively, a maximum of approximately 11, 8.4 and 8.3 molecules of fatty acid molecules bind to 1 Pluronic F127 molecule. We propose that this is due to the decreasing number of free monomers with increasing chain length. As chain length increases, the critical micelle concentration (cmc) decreases, thus leading to fewer monomers. Pluronics are symmetric tri-block copolymers consisting of propylene oxide (PO) and ethylene oxide (EO). The polypropylene oxide block, PPO is sandwiched between two polyethylene oxide (PEO) blocks. The PEO blocks are hydrophilic while PPO is hydrophobic portion in the Pluronic molecule. Due to this structure, we propose that the fatty acid molecules that are in monomeric form most effectively diffuse between the PEO “tails” and bind to the hydrophobic PPO groups.

Determination of Drug and Fatty Acid Binding Capacity to Pluronic F127 in Microemulsions

We propose that one can deduce very insightful information regarding the drug and fatty acid binding capacity of microemulsions through simple turbidity experiments. Pluronic F127-based oil-in-water microemulsions of various compositions were synthesized and titrated to turbidity with concentrated amitriptyline, an antidepressant drug. We observed that, above certain Pluronic F127 concentrations, turbidity was never observed, irrespective of how much amitriptyline was added to the microemulsion. We also observed that whenever sodium caprylate fatty acid was not included in the microemulsion formulation, turbidity never occurred. On the basis of these findings, we were able to determine the point at which all sodium caprylate present in the microemulsion formulation was bound to the F127 in the microemulsion (i.e., no fatty acid was free in the bulk in monomer form). By the same logic we were also able to determine how much amitriptyline was binding to the microemulsions. We also measured the dynamic surface tension, foamability, and fabric wetting time of the microemulsion formulations to further prove the hypothesis that all fatty acid is bound to the F127 in the microemulsion above a critical Pluronic F127 concentration. On the basis of this research, we have concluded that there are approximately 11 molecules of sodium caprylate fatty acid bound per molecule of Pluronic F127 and approximately 12 molecules of amitriptyline bound per molecule of Pluronic F127 in the optimal microemulsion formulation. These findings give us valuable information about the charge density at the oil/water interface and about the mechanism of binding of the drug to the microemulsion.

Binding of Anti‐HIV Drugs to Human Serum Albumin

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (i.e., drug) binding capacity. Here, values of the dissociation equilibrium constant (Kd)for the binding of HIV protease and reverse transcriptase inhibitors to HSA are reported. The binding of abacavir, atazanavir,didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir,nevirapine, ritonavir, saquinavir, stavudine, zalcitabine, and zidovudine to the Sudlow site I (i.e., the warfarin cleft) located in the subdomain IIA involves the alteration of the HSA structure around Trp214 and induces intrinsic tryptophan fluorescence quenching. Accordingly, ibuprofen that primarily binds to the Sudlow site II located in the subdomain IIIA does not affect the HSA intrinsic tryptophan fluorescence and the binding of anti-HIV drugs to the Sudlow site 1. Accounting for the physiological concentration of HSA (= 7.0 x 10(-4) M), the average anti-HIV drug concentration in plasma (= 1.0 x 10(-4) M), and Kd values for the binding of anti-HIV drugs to HSA (ranging between 4.4 x 10(-5)M and 3.8 x 10(-4) M), it appears that the fraction of HIV protease and reverse transcriptase inhibitors bound to HSA ranges between 63% and 91%. This represents a significant drawback in the anti-HIV therapy and management, the anti-HIV drug concentration required to achieve 90% protease and reverse transcriptase inhibition in the presence of plasma proteins appears to be at least one order of magnitude higher than that required in their absence.

Probing the mechanism of drug/lipid membrane interactions using Biacore

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug’s dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome’s drug-binding capacity and affinity depended on both the lipid headgroup and the drug’s structure. Although a drug’s charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.

Pharmaceutical-grade albumin: impaired drug-binding capacity in vitro

BackgroundAlbumin is the most abundant protein in blood plasma, and due to its ligand binding properties, serves as a circulating depot for endogenous and exogenous (e.g. drugs) compounds. Hence, the unbound drug is the pharmacologically active drug. Commercial human albumin preparations are frequently used during surgery and in critically ill patients. Recent studies have indicated that the use of pharmaceutical-grade albumin is controversial in critically ill patients. In this in vitro study we investigated the drug binding properties of pharmaceutical-grade albumins (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn), native human serum, and commercially available human serum albumin from Sigma Chemical Company.MethodsThe binding properties of the various albumin solutions were tested in vitro by means of ultrafiltration. Naproxen, warfarin, and digitoxin were used as ligands. HPLC was used to quantitate the total and free drug concentrations. The data were fitted to a model of two classes of binding sites for naproxen and warfarin and one class for digitoxin, using Microsoft Excel and Graphpad Prism.ResultsThe drugs were highly bound to albumin (95–99.5%). The highest affinity (lowest K1) was found with naproxen. Pharmaceutical-grade albumin solutions displayed significantly lower drug-binding capacity compared to native human serum and Sigma albumin. Thus, the free fraction was considerably higher, approximately 40 times for naproxen and 5 and 2 times for warfarin and digitoxin, respectively. The stabilisers caprylic acid and N-acetyl-DL-tryptophan used in the manufacturing procedure seem to be of importance. Adding the stabilisers to human serum and Sigma albumin reduced the binding affinity whereas charcoal treatment of the pharmaceutical-grade albumin from Octapharma almost restored the specific binding capacity.ConclusionThis in vitro study demonstrates that the specific binding for warfarin and digitoxin is significantly reduced and for naproxen no longer detectable in pharmaceutical-grade albumin. It further shows that the addition of stabilisers may be of major importance for this effect.

Modulation of drug sensitivity in yeast cells by the ATP-binding domain of human DNA topoisomerase IIalpha.

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIalpha (htopoIIalpha), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIalpha N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.

Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump.

Multidrug efflux pumps cause serious problems in cancer chemotherapy and treatment of bacterial infections. Yet high-resolution structures of ligand transporter complexes have previously been unavailable. We obtained x-ray crystallographic structures of the trimeric AcrB pump from Escherichia coli with four structurally diverse ligands. The structures show that three molecules of ligands bind simultaneously to the extremely large central cavity of 5000 cubic angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding.

SPR Sensor as a Tool to Evaluate Binding Capacity of Gastric Ulcer Drugs with Mucous Membrane

Covalent immobilization of mucin molecules was carried out on a gold surface of surface plasmon resonance sensor. Interaction of three types of gastric ulcer drug with mucin was detected by measuring the shift of resonance angle. The two gastric proton pump inhibitors have shown drastic change, while antagonist of histamine H2 receptor, showed insignificant change. The result suggests that SPR-based measurement is capable of evaluating binding capacity between an intestinal membrane and corresponding biosubstance, such as a gastric ulcer agent and mucin.

Antibody C219 recognizes an alpha-helical epitope on P-glycoprotein.

The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 A of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic alpha-helical conformation, a secondary structure not previously observed in structures of antibody-peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin--implications for further purging strategies.

The efficacy of mafosfamide purging depends on factors like incubation time, drug and erythrocyte concentration. To determine the influence of protein-bound SH groups in the incubation medium, the cytotoxicity of mafosfamide on G-CSF mobilized CD34+/- cells was evaluated by short-term culture assays and drug concentration measurements. 100 micromol/ml mafosfamide was incubated for 30 min in five buffers (PBS, PBS with 1%, 5% and 10% BSA and plasma). The mean calculated areas under the concentration-time curves (AUC) were 2489 +/- 198, 1561 +/- 286, 976 +/- 201, 585 +/- 62 and 605 +/- 196 micromol/l/min. The mean reductions of CFU-GM growth were 79.4%, 73.0%, 62.5%, 30.3%, 6.2% respectively. Similar results were obtained for BFU-E. Regression analysis showed a good correlation between cytotoxicity and AUCs (CFU-GM: r = 0.8195; BFU-E: r = 0.8207). This effect is well explained by the different concentrations of SH moieties in the incubation medium resulting in a higher drug binding capacity. The profound difference between AUCs and CFU-GMs in plasma and 10% BSA cannot be explained by the quantity of SH-groups. It is probably due to an additional enzymatic drug degeneration by the 3'-5'exonuclease subsite of plasma DNA polymerase. In conclusion, the concentration of albumin-associated SH groups strongly influences the cytotoxicity of mafosfamide. It has to be considered as a new and important aspect in ex vivo bone marrow purging.

Preparation and characterization of Ca-alginate microspheres by a new emulsification method

The preparation and characterization of Ca-alginate microspheres obtained by a new procedure from alginic acid, treated with an excess of NaOH and subsequently with a very concentrated solution of CaCl 2 are described. The produced particles have a spherical shape with an average diameter of 350 μm. The particles show a relative dense and homogeneous internal structure in comparison with previously reported alginate microcapsules (the bulk density being 0.47 g/cm 3), consequently the particle matrix is characterized by a high density of charged carboxylic groups. Particles produced with a contact time of 1 h showed a degree of crosslinking of 54%. Microspheres have a good stability, and despite of very low degree of swelling, they have a good solvents regain, and an excellent ionic binding capacity of cationic drugs. After preparation the beads were loaded by ionic complexation with an antitumor aromatic tetramidine. In addition, the in vitro release of drug has been studied.

Influence of glycosylation on the drug binding of human serum albumin.

The influence of glycosylation on the drug binding of human serum albumin (HSA) was studied using HSA containing different amounts and degrees of glycosylated HSA. The drugs used were furosemide, naproxen, procaine, phenylbutazone, salicylic acid, sulphamethoxazole, tolbutamide and warfarin. The drug-HSA parameters (lognK) were measured by the ultrafiltration method, frontal analysis and a modified Hummel-Dreyer method. The modified Hummel-Dreyer method was the simplest method with high precision and required the smallest amounts of proteins. The lognK values were well correlated with the octanol-water partition coefficients; the correlation coefficients were over 0.95. The results suggested that hydrophobic interaction is the predominant force for the drug binding. The early stage of glycosylation of HSA did not significantly affect the drug-binding capacity. Generally, the binding affinity of HSA decreased, perhaps due to a conformational change or steric hindrance (except naproxen) when further glycosylation occurred.

Binding of dihydroartemisinin to hemoglobin H: role in drug accumulation and host-induced antimalarial ineffectiveness of alpha-thalassemic erythrocytes.

Dihydroartemisinin and other artemisinin derivatives are relatively ineffective against Plasmodium falciparum infecting alpha-thalassemic erythrocytes, namely hemoglobin (Hb) H or HbH/Hb Constant Spring erythrocytes, as compared with those infecting genetically normal erythrocytes. The variant erythrocytes accumulate radiolabeled dihydroartemisinin to a much higher extent than the normal ones, and the accumulated drug was retained after extensive washing, in contrast to the drug in normal erythrocytes which was mostly removed. At initial drug concentration of 1 mM, most (82-88%) of the drug was found in the cytosol fraction of both variant and normal erythrocytes. Binding of the drug to hemoglobins accounted for 40-70% of the total uptake. Hb H accounted for 10.9 +/- 2.7% and 12.4 +/- 6.2% of total protein in HbH and HbH/Hb Constant Spring erythrocytes. HbH bound with 28.7 +/- 6.7% of the drug, whereas HbH/Hb Constant Spring erythrocytes bound with 21.8 +/- 8.3% of the drug. Binding experiments showed that Hb H had 5-7 times the drug-binding capacity of Hb A. For Hb H, the maximum binding capacity (Bmax) = 1.67 +/- 0.17 mol/mol Hb, and the dissociation constant (Kd) = 66 +/- 17 microM, and for Hb A, Bmax = 0.74 +/- 0.18 mol/mol Hb and Kd = 224 +/- 15 microM. It is concluded that preferential binding of dihydroartemisinin to Hb H over Hb A accounts partly for the higher accumulation capacity of the alpha-thalassemic erythrocytes, which leads to its antimalarial ineffectiveness.

Single-step isolation method for six glycoforms of human α 1-acid glycoprotein by hydroxylapatite chromatography and study of their binding capacities for disopyramide

A single-step isolation method for the glycoforms of human serum α 1-acid glycoprotein (AAG) using a hydroxylapatite column under a gradient elution program was developed. The concentrations of N-acetylneuraminic acid and monosaccharides (fucose, N-acetylglucosamine, galactose and mannose) of six AAG glycoforms were determined by the pulsed-amperometric detection method. For each AAG glycoform, significant sex-related differences in carbohydrate content have been observed only for AAG glycoforms two and six, and not for each AAG glycoform. The relationship between the extent of the branch in the glycan chain and the binding capacity to disopyramide were examined. Female AAG contained highly sialylated AAG glycoforms compared to male glycoforms. Conversely, male AAG was rich in the lower sialylated AAG glycoform. Furthermore, it was found that the drug binding capacity decreases with increasing branching of the glycan chain. This suggests that the binding sites of AAG are hindered by a relatively large carbohydrate moiety, such as tetraantennary structures.

Low Density Lipoproteins as Drug Carriers in the Therapy of Macrophage-Associated Diseases

Low density liporoteins (LDL) are internalised by the LDL receptor, which is expressed on the surface of eucaryotic cells. Metabolisation and chemical modification of LDLs lead to a shift in receptor affinity: modified LDLs are internalised through scavenger receptors, which are expressed on cells of the monocyte/macrophage lineage. Coupling of substances with pharmacological activity, such as azidothymidine, to LDL results in a cell specific uptake of these drugs into macrophages by the scavenger receptor. Thus, drug–LDL derivatives might work as tools for macrophage specific drug targeting. In this context, it is essential to know the drug binding capacity, the optimal derivatization conditions, and the amount of drug molecules covalently bound to the LDL particle. In this study, we compared methods for optimal derivatisation and estimation of coupling efficiency, such as semiquantitative lipoprotein gel electrophoresis, ultraviolet (UV) spectrophotometry, radiometric quantification, and specific protein hydrolysis.

Adsorption Characteristics of Norfloxacin to Pharmaceutical Additives

AbstractNorfloxacin is a very potent drug, adsorption of even a few milligrams of this drug may account for a significant fraction of total dose, hence, adsorption effect might be of great significance. The results of in vitro adsorption studies of norfloxacin to different pharmaceutical additives are plotted on the basis of Langmuir Equation. The linear nature of the adsorption isotherms shows that the adsorption process is due to monomolecular layer. Adsorption constants were calculated to determine the binding capacity of this drug to various additives. The adsorbents with their decreasing binding capacity are as follows:Activated charcoal (33.0 × 10−3) > Bentonite (17.0 × 10−3) > Kaolin (9.5 × 10−3) > Methyl cellulose (8.6 × 10−3) > Potato starch (7.4 × 10−3) > Talc (6.0 × 10−3)The mechanism of adsorption has been discussed in light of physical characteristics of the adsorbents.

The pharmacokinetics of antiarrhythmic agents in pregnancy and lactation.

The pharmacokinetics of various drugs may be profoundly altered during different stages of pregnancy, parturition, and lactation. Gastrointestinal absorption or bioavailability of drugs may vary due to changes in gastric secretion and motility. Various haemodynamic changes such as an increase in cardiac output, blood volume, and renal plasma flow may affect drug disposition and elimination. The increase in blood volume and total body water which occurs during pregnancy can alter the volume of distribution for various drugs. Although exact quantifications are not easy, these changes in pharmacokinetic parameters should be considered when dosing antiarrhythmic agents in pregnant women. Plasma protein concentrations and drug binding capacity are altered in the mother and fetus as pregnancy advances. With highly protein bound drugs, these changes may be clinically significant, as the pharmacological efficacy and toxicity are presumed to be related to the concentration of free drug in both the mother and fetus. In some instances, the fetus may be susceptible to greater drug toxicity as free drug concentrations may be underestimated by measurement of total drug concentrations. Changes in maternal drug metabolism and metabolism by the fetoplacental unit also contribute to alterations in the pharmacokinetics of drugs. As the placenta contains many metabolising enzymes, biotransformation of drugs at this site could potentially convert a drug into an active metabolite, or prevent fetal exposure to a toxic drug. Placental transfer of drugs, leading to toxicity in the fetus, is a major concern in the pharmacological management of the pregnant patient. The passage of individual drugs will vary depending on their apparent volumes of distribution, degree of protein binding the rates of metabolic conversion and excretion within the placenta and fetus, the pH difference between the maternal and fetal fluids, and maternal haemodynamic changes. Drug properties such as lipid solubility, protein binding characteristics, and ionisation constant (pKa) also influence the placental passage of drugs. For weakly basic antiarrhythmic agents, the fetal drug concentration may potentially exceed the maternal plasma concentration when the fetal pH is lowered as in the case of fetal acidosis; this is due to 'ion trapping'. Additionally, higher free drug concentrations of these basic drugs may exist, due to decreased alpha 1-acid glycoprotein concentration and binding affinity in the fetus. Lignocaine (lidocaine) has been shown to enter fetal plasma rapidly with fetal-maternal concentration ratios in the range of 0.52 to 0.66.(ABSTRACT TRUNCATED AT 400 WORDS)

Nonenzymatic glucosylation of human serum albumin and its influence on binding capacity of sulfonylureas

To estimate the functional change occurring in human serum albumin by nonenzymatic glucosylation, glucosylated human serum albumin was prepared by in vitro incubation with glucose. The rate of glucosylation proceeded as a first-order reaction. The binding of sulfonylureas to serum albumin was determined by equilibrium gel filtration. Through this method, it was possible to estimate the binding capacity of a low water solubility drug with a high affinity to protein. The amounts of the sulfonylureas bound to glucosylated HSA decreased by 44% with tolazamide and acetohexamide, 50% with glibenclamide, and 52% with tolbutamide, compared to human serum albumin (HSA). This suggests that a high concentration of glucosylated HSA in diabetic patients may possibly cause an increase in free drug concentration exceeding normal levels. This study shows that the decrease in the binding capacity of sulfonylureas with protein is due to the modification of albumin molecules by the covalent binding of glucose.

ESR study of intercalation: Quantitative evaluation of drug-DNA binding through competition with a spin-labeled 9-aminoacridine

Interaction of a spin-labeled 9-aminoacridine with DNA was studied by electron spin resonance spectroscopy. Accurate determination of the binding parameters, equilibrium dissociation constant ( K D), and total number of ligand-binding sites was obtained using Scatchard and Lineweaver-Burk plots. The competition between 9-aminoacridine and its spin-labeled derivative was examined by a similar analysis of the spin-label signals. The practical interest of this method lies in the fact that the precision and the simplicity of the measurements allow the quantitative determination of the binding capacity of any intercalative drug which interacts specifically with adenine/thymine bases of DNA.

猫心筋のSarcoplasmic reticulumおよび細胞膜の強心配糖体結合

The binding capacity of ouabain or digitoxin by sarcoplasmic reticulum (SRF) isolated from cat heart muscle was found to be about 7-9 times as much as that by cell membrane from cat heart muscle. Acetone treatment of SRF caused a marked decrease in the binding capacity of ouabain and digitoxin. SRF pretreated with phospholipase C was decreased in content of phospholipid, in the ATPase activity, in the Ca-binding capacity and in the binding capacity of ouabain or digitoxin, while SRF pretreated with digitonin was decreased in content of cholesterol, but not in phospholipid content, in ATPase activity, in the Ca-binding capacity and in the binding capacity of ouabain or digitoxin. From these findings, it might be concluded that SRF was the major binding site for cardiac glycosides and that the content of phospholipid of SRF plays an important role in the drug-binding capacity.