Binding of 16α-gitoxin and its 16-acetate to human serum albumin

Summary The binding of semi-synthetic (16-gitoxin and its 16-acetate) and naturally occurring cardioactive glycosides (digitoxin, ouabain) to human serum albumin was studied using equilibrium dialysis, ultracentrifugation and gel filtration. 16-gitoxin was 75% bound and its 16-acetate 95%. The percentage binding of digitoxin and ouabain was in good agreement with values reported in literature.     

How do fatty acids cause allosteric binding of drugs to human serum albumin?

Purpose. This study was undertaken to investigate how fatty acids cause the allosteric binding of drugs to human serum albumin (HSA). The influence of fatty acids on the binding of ketoprofen (KP), an NSAID, to HSA was examined by using a photoaffinity labeling technique. Methods. Ultrafiltration was performed to quantitate the concentration of free KP. HSA, photolabeled with KP in the presence of myristate (MYR), octanoate, and diazepam, was cleaved with cyanogen bromide, separated by Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently analyzed by autoradiography. Results. The addition of MYR at molar ratios from 4 to 5, but not from 1 to 2, causes substantial increases in unbound KP for KP:HSA ratios of 0.5 and 1. The addition of two or more moles of MYR, octanoate, and diazepam per mole of HSA caused a pronounced decrease in the labeling of the 11.6- and 13.5-kDa peptides. However, only MYR showed an increase in labeling of the 20 kDa and, especially, the 9.4-kDa peptides. At MYR:HSA ratios in excess of 3, a decrease in the extent of labeling of the 9.4-kDa peptide was observed. Conclusion. Long-chain fatty acids regulate the binding properties of HSA in a complex manner, in which a simultaneous competitive and allosteric mechanism operates and which mainly involves domain I.     

Binding of aprindine and moxaprindine to human serum, α1-acid glycoprotein and serum of healthy and diseased humans

Summary A comparison was made between the binding of the anti-arrhythmic agents aprindine and moxaprindine to human serum, to human serum albumin (HSA), to ₁-acid glycoprotein (₁-AGP) and to a mixture of HSA and ₁-AGP. In serum from healthy volunteers (n=4) the binding of aprindine-HCl 5 µg/ml (13.8 µM) was 93.8% (SD±1.0), and that of moxaprindine-HCl 5 µg/ml (12.8 µM) was 94.1% (SD±1.1). Their binding to the mixture of ₁-AGP and albumin approximated their binding to serum. For ₁-AGP, the binding was similar for both compounds, whereas for HSA the binding of aprindine was more pronounced than that of moxaprindine: for both products the affinity coefficient for binding to ₁-AGP was about 100 times greater than that for binding to albumin. In serum from rheumatoid patients and from patients with renal failure a small but significant increase in binding of aprindine and moxaprindine was observed, approximately 1%. Increased and decreased binding was seen in serum from cirrhotic patients; for example, for aprindine the range in cirrhosis was 96.7%–79.8%, and the range in controls was 95.0%–92.4%. Free drug fraction and ₁-AGP concentration were inversely correlated. The results show that ₁-AGP plays an important role in the binding of aprindine and moxaprindine, and that alteration in the binding of the two compounds in disease states to a large extent can be explained by changes in serum ₁-AGP concentration.     

Binding of diuretic antihypertensive bendroflumethiazide to human serum albumin studied by 1?F nuclear magnetic resonance method

Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by 1?F nuclear magnetic resonance (NMR) method. The 1?F NMR spectrum of BFZ (200 μM) in a buffer solution showed a sharp signal of its CF? group at 17.8 ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF(3) signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4 ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00 × 10? (M?1) (n=1.1), respectively. The presence of 0.10 M Cl? in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Cl? ions bind again nonspecifically.     

Validation of a Gyrolab™ assay for quantification of rituximab in human serum

IntroductionGyrolab? technology presents a technology breakthrough for large molecule bioanalysis to support biologic drug development. The advantages of this innovative platform include fully automated nanoscale immunoassay capability, better assay reproducibility and data quality, small reagent and sample volumes, and rapid assay development and validation as a result of reduced run time. Although Gyrolab has been increasingly used in method development in discovery environment, few fully validated Gyrolab assays have been reported. Here we report a method validation of a Gyrolab assay to determine rituximab levels in human serum.MethodsRituximab is captured on a Bioaffy? CD by a biotinylated rat anti-idiotypic monoclonal antibody against rituximab and detected by an Alexa Fluor®-labeled anti-human IgG antibody. Assay conditions were optimized to give required sensitivity and dynamic range. The assay validation was conducted according to the current industry standards for GLP-regulated immunoassays.ResultsThe intrabatch precision and accuracy for the assay were determined using spiked human serum samples and shown to have a coefficient of variation (CV) of < 11% with a mean bias < 20%. The interbatch precision (CV) and absolute mean bias were both < 12% with the total error < 25%. Adequate spike recovery was demonstrated in serum samples of healthy individuals and solid tumor patients. The dilutional linearity test showed that the determined concentrations adjusted with various dilution factors had a linear relationship with the expected concentrations and that there was no hook effect. The method has been validated for the quantification of rituximab in human serum from 90 to 60,000 ng/mL with a minimum required dilution of 30.DiscussionThe Gyrolab assay was proved to be accurate, precise and selective, with a comparable sensitivity to the ELISA method, but provided an automated nanoscale assay with a significantly wider assay dynamic range for the determination of rituximab in human serum during pharmacokinetics/toxicokinetics studies.     

Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Göttingen minipigs exposed to sarin vapor

Human serum butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a prophylactic countermeasure against organophosphorus nerve agents. This study was designed to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to a lethal dose of sarin (GB) vapor. Male Göttingen minipigs were subjected to: air exposure, GB vapor exposure, or pretreatment with Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection 24 h prior to exposure to 4.1 mg/m³ of GB vapor for 60 min. Electrocardiograms (ECG), electroencephalograms (EEG), and pupil size were recorded throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB present. Untreated animals exposed to GB vapor exhibited cardiac abnormalities and generalized seizures, ultimately succumbing to respiratory failure. Pretreatment with 3.0 or 6.5 mg/kg of Hu BChE delayed blood gas and acid–base disturbances and the onset of cardiac and neural toxic signs, but failed to increase survivability. Pretreatment with 7.5 mg/kg of Hu BChE, however, completely prevented toxic signs, with blood chemistry and ECG and EEG parameters indistinguishable from control during and after GB exposure. GB bound in plasma was 200-fold higher than plasma from pigs that did not receive Hu BChE, suggesting that Hu BChE scavenged GB in blood and prevented it from reaching other tissues. Thus, prophylaxis with Hu BChE alone not only increased survivability, but also prevented cardiac abnormalities and neural toxicity in minipigs exposed to a lethal dose of GB vapor.     

Characterization of the mangiferin–human serum albumin complex by spectroscopic and molecular modeling approaches

The interactions between mangiferin and human serum albumin (HSA) were investigated by spectroscopy and molecular modeling. The results proved the formation of complex between mangiferin and HSA. Hydrophobic interaction dominated in the association reaction. Mangiferin statically quenched the fluorescence of HSA in a concentration dependent manner positively deviating from the linear Scatchard equation. The binding of mangiferin to HSA lead to changes in the conformation of HSA according to synchronous fluorescence spectra, FT-IR, UV–vis and CD data. The presence of amino acids and metal ion affected the binding constant of mangiferin–HSA complex. Computational mapping of the possible binding sites of mangiferin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

Does Haber's law apply to human sensory irritation?

Irritation of the eyes, nose, and throat by airborne chemicals--also referred to as "sensory irritation"--is an important endpoint in both occupational and environmental toxicology. Modeling of human sensory irritation relies on knowledge of the physical chemistry of the compound(s) involved, as well as the exposure parameters (concentration and duration). A reciprocal relationship between these two exposure variables is postulated under Haber's law, implying that protracted, low-level exposures may be toxicologically equivalent to brief, high-level exposures. Although time is recognized as having an influence on sensory irritation, the quantitative predictions of Haber's Law have been addressed for only a handful of compounds in human experimental studies. We have conducted a systematic literature review that includes a semiquantitative comparison of psychophysical data extracted from controlled human exposure studies versus. the predictions of Haber's law. Studies containing relevant data involved exposures to ammonia (2), chlorine (2), formaldehyde (1), inorganic dusts such as calcium oxide (1), and the volatile organic compound 1-octene (1). With the exception of dust exposure, varying exposure concentration has a proportionally greater effect on sensory irritation than does changing exposure duration. For selected time windows, a more generalized power law model (c(n) x t = k) rather than Haber's law per se (c x t = k) yields reasonably robust predictions. Complicating this picture, however, is the frequent observation of intensity-time "plateauing," with time effects disappearing, or even reversing, after a relatively short period, depending on the test compound. The implications of these complex temporal dynamics for risk assessment and standard setting have been incompletely explored to date.     

Characterization of thymoquinone binding to human α₁-acid glycoprotein

Thymoquinone (TQ) is the main bioactive component isolated from Nigella sativa essential oil and seeds and has been used for the treatment of inflammations, liver disorders, arthritis, and is of great importance as a promising therapeutic drug for different diseases including cancer. This paper reports the first experimental evidence on binding of TQ to human α(1)-acid glycoprotein (AGP), an important drug-binding glycoprotein in human plasma, which affects pharmacokinetic properties of various therapeutic agents. The interaction of TQ with AGP has been characterized by Fourier transform infrared (FTIR) and fluorescence spectroscopy, as well as by molecular docking experiments. FTIR spectroscopy showed that the binding of TQ to AGP slightly increases its thermal stability and shifts the existence of a molten globule-like state observed in a previous study to higher temperature. The binding constants K(a); the number of binding sites n; and the corresponding thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated through fluorescence spectroscopy. Fluorescence quenching experiments indicated that TQ binding involves hydrophobic interactions and to a lower extent hydrogen bonds, in agreement with molecular docking experiments. The data on binding ability of TQ to AGP represent basic information for the TQ pharmacokinetics such as drug metabolism and distribution in the body.     

LC-MS/MS assays to determine olmesartan concentration in human plasma

LC-MS/MS方法测定人血浆中奥美沙坦@陈英$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @谭志荣$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @周宏灏$Institute of Clinic     

Recombinant human tumor necrosis factor-α covalently conjugated to long-circulating liposomes

Recombinant tumor necrosis factor-α (rHuTNF) was covalently conjugated to a phospholipid, N-glutaryl phosphatidylethanolamine (NGPE). The resultant rHuTNF-NGPE conjugates were incorporated into liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) with or without polyethyleneglycol conjugated to phosphatidylethanolamine (PEG3000-PE). Efficient incorporation (35–50%) of rHuTNF-NGPE conjugates into liposomes was obtained for both PC/Chol and PC/Chol/PEG3000-PE liposomes. An in vitro cytotoxicity assay showed that rHuTNF-NGPE conjugates incorporated into liposomes exhibit a reduced biological activity as compared to the free rHuTNF. Biodistribution studies using ¹²⁵I-labeled rHuTNF showed a significant increase in the circulation time of rHuTNF by incorporation into PC/Chol/PEG3000-PE liposomes, but not conventional PC/Chol liposomes. However, studies using a radioactive lipid as a liposome marker showed that incorporation of rHuTNF-NGPE conjugates resulted in increased clearance from the blood and accumulation in the spleen and liver of both liposomal formulations. The liposome clearance from the blood depends on the protein/lipid ratio of liposomes. The higher the protein/lipid ratio, the higher the liposome clearance from the blood and accumulation in the spleen and liver, suggesting that accumulation of rHuTNF-bound liposomes in the spleen and liver involves interactions with TNF-receptors in these organs.     

Michaelis–Menten kinetic analysis of drugs of abuse to estimate their affinity to human P-glycoprotein

The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug–drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug–drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis–Menten kinetics and assessing their K_m and V_max values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics could be modeled for the entactogen 3,4-methylenedioxy-α-ethylphenethylamine (3,4-BDB), the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), the abused alkaloid glaucine, the opioid-like drugs N-iso-propyl-1,2-diphenylethylamine (NPDPA), and N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), with K_m and V_max values within the same range as for verapamil or sertraline. As a consequence interactions with other drugs being P-gp substrates might be considered to be very likely and further studies should be encouraged.     

Generation of an esterolytic and kinin-forming kallikrein-α2-macroglobulin complex in human serum by treatment with acetone

Summary Kinin-forming and esterolytic activity in human citrate plasma has been activated by treatment of the plasma with acetone. By far most of the esterolytic activity if not all of it was recovered in an 2-macroglobulin (2M) kallikrein complex (SI) which was characterized and purified by chromatography. Little if any esterolytic activity was present which could be ascribed to free plasma kallikrein. The 2M-kallikrein complex had kinin-releasing activity though much less than free plasma kallikrein, relative to esterolytic potency. This explains that a considerable fraction of the kinin-forming potential of acetone-activated plasma resides in free plasma kallikrein although it represents only a very small portion of the total kallikrein store. Like free plasma kallikrein the 2M complex releases kinin from LMW-kininogen less efficiently than from HMW, in systems of purified components. In whole plasma, the efficiencies change: whereas plasma kallikrein is rapidly inactivated by endogenous inhibitors, the 2M complex is protected from further inactivation and capable of releasing kinin continuously if slowly, attacking also LMW-kininogen after HMW-kininogen has been consumed by free kallikrein. While the 2M-complex in this respect differs functionally from free plasma kallikrein and explains earlier observations suggesting the presence of two kininogenases, it seems doubtful now that two truly different kininogenases exist in human plasma.The results suggest that acetone predominantly inactivates full inhibitors of kallikrein such as C1INH whereas 2M is somewhat more resistant and (pre-)-kallikrein even more. Depending on the time and temperature of acetone treatment one obtains more or less total kallikrein and varying proportions of free to bound enzyme. It is likely that acetone does not truly trigger an activation of prekallikrein but supports spontaneous activation by slowing down the control of the feedback reinforcement of this activation, by damaging inhibitors.Supported by Deutsche Forschungsgemeinschaft.Some of the results have been presented at the Conference on Chemistry and Biology of the Kallikrein-Kinin System, Reston, Virginia, 1974, and at the Symposium on Vasopeptides, Florence 1975.     

Xenobiotic metabolism gene expression in the EpiDerm™ in vitro 3D human epidermis model compared to human skin

There is an urgent need to validate in vitro human skin models for use in safety testing. An important component of validation is characterizing the metabolizing capacity of these models. We report comparison of the expression of 139 genes encoding xenobiotic metabolizing enzymes in the EpiDerm™ model and human skin. In microarray analysis, the expression of 87% of the genes was consistent between the EpiDerm™ model and human skin indicating the presence of similar metabolic pathways suggesting commonality in function. Analysis of EpiDerm™ models constructed from four donors showed highly comparable expression of xenobiotic metabolizing genes demonstrating reproducibility of the model. Overall, the expression of Phase II enzymes appeared to be more pronounced in human skin and the EpiDerm™ model than that of Phase I enzymes, consistent with the role of skin in detoxification of xenobiotics. Though the basal expression of CYPs in particular was low in EpiDerm™, significant induction of CYP1A1/1B1 activity was observed following treatment with 3-methylcholanthrene. These results indicate that the xenobiotic metabolizing capacity of the EpiDerm™ model appears to be representative of human skin. Models such as EpiDerm™ provide a valuable in vitro approach for evaluation of metabolism and toxicity of cutaneous exposures to xenobiotics.