Parenteral Delivery of HPβCD: Effects on Drug-HSA Binding

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

Target identification of covalently binding drugs by activity-based protein profiling (ABPP)

The characterization of the target proteins of drug molecules has become an important goal in understanding its mode of action and origin of side effects due to off-target binding. This is especially important for covalently binding drugs usually containing electrophilic moieties, which potentially can react with nucleophilic residues found in many proteins. This review gives a comprehensive overview of the use of activity-based protein profiling (ABPP) as an efficient tool for the target identification of covalently binding drugs.     

Drug–drug plasma protein binding interactions of ivacaftor

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR‐protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co‐administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co‐administered CF drugs for human serum albumin (HSA) and α₁‐acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site‐selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug–drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug–drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug–drug interactions of ivacaftor with co‐administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. Copyright © 2015 John Wiley & Sons, Ltd.     

Systematic interaction of plasma albumin with the efficacy of chemotherapeutic drugs

Albumin, as the most abundant plasma protein, plays an integral role in the transport of a variety of exogenous and endogenous ligands in the bloodstream and extravascular spaces. For exogenous drugs, especially chemotherapeutic drugs, binding to and being delivered by albumin can significantly affect their efficacy. Meanwhile, albumin can also bind to many endogenous ligands, such as fatty acids, with important physiological significance that can affect tumor proliferation and metabolism. In this review, we summarize how albumin with unique properties affects chemotherapeutic drugs efficacy from the aspects of drug outcome in blood, toxicity, tumor accumulation and direct or indirect interactions with fatty acids, plus application of albumin-based carriers for anti-tumor drug delivery.     

Endogenous proteins controlling amyloid beta-peptide polymerization. Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues.

We report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid beta-peptide (Abeta) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Abeta. Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by alpha1-antitrypsin and immunoglobulins A and G. Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Abeta, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Abeta into genuine beta-amyloid in tissue section was studied. The Abeta and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin. Tolbutamide displaces Abeta from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid. These findings may provide one explanation as to why beta-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance beta-amyloid formation and promote the development of Alzheimer's disease.     

Characterization of the binding of angiotensin II receptor blockers to human serum albumin using docking and molecular dynamics simulation

Human serum albumin (HSA), the most abundant protein found in blood plasma, transports many drugs and ligands in the circulatory system. The drug binding ability of HSA strongly influences free drug concentrations in plasma, and is directly related to the effectiveness of clinical therapy. In current work, binding of HSA to angiotensin II receptor blockers (ARBs) are investigated using docking and molecular dynamics (MD) simulations. Docking results demonstrate that the main HSA–ARB binding site is subdomain IIIA of HSA. Simulation results reveal clearly how HSA binds with valsartan and telmisartan. Interestingly, electrostatic interactions appear to be more important than hydrophobic interactions in stabilizing binding of valsartan to HSA, and vice versa for HSA–telmisartan. The molecular distance between HSA Trp214 (donor) and the drug (acceptor) can be measured by fluorescence resonance energy transfer (FRET) in experimental studies. The average distances between Trp-214 and ARBs are estimated here based on our MD simulations, which could be valuable to future FRET studies. This work will be useful in the design of new ARB drugs with desired HSA binding affinity.     

Structural basis of the drug-binding specificity of human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that binds a remarkably wide range of drugs, thereby restricting their free, active concentrations. The problem of overcoming the binding affinity of lead compounds for HSA represents a major challenge in drug development. Crystallographic analysis of 17 different complexes of HSA with a wide variety of drugs and small-molecule toxins reveals the precise architecture of the two primary drug-binding sites on the protein, identifying residues that are key determinants of binding specificity and illuminating the capacity of both pockets for flexible accommodation. Numerous secondary binding sites for drugs distributed across the protein have also been identified. The binding of fatty acids, the primary physiological ligand for the protein, is shown to alter the polarity and increase the volume of drug site 1. These results clarify the interpretation of accumulated drug binding data and provide a valuable template for design efforts to modulate the interaction with HSA.     

Identification of protein targets for mycophenolic acid acyl glucuronide in rat liver and colon tissue

Covalent binding of acyl glucuronides to proteins is considered an initiating event for the organ toxicity of drugs containing a carboxylic acid group. An acyl glucuronide (AcMPAG) of the immunosuppressant mycophenolic acid was described and shown to form covalent adducts with plasma albumin in vivo. The aim of the present investigation was to identify AcMPAG target proteins in the liver and colon of rats treated with mycophenolate mofetil, which may contribute to a better understanding of the mechanisms responsible for the development of side effects during therapy with this drug. Mycophenolate mofetil was administered per os in to Wistar rats (40 mg/kg/day) over 21 days. Proteins in liver and colon homogenates were separated by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. AcMPAG labeled protein spots were detected by Western blotting. After in-gel tryptic digestion of the protein spots from parallel gels (n = 2), peptides were characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. Data base searching identified AcMPAG target proteins. Tryptic peptides with sufficient signal intensities were subjected to post-source decay analysis. Three proteins in the liver (ATPase/ATP synthase (alpha and beta subunits), protein disulfide isomerase A3 and selenium binding protein) and one protein in the colon (selenium binding protein) were identified as targets for AcMPAG. ATPase/ATP synthase and protein disulfide isomerase are essential proteins involved in the control of the energy and redox state of the cells, whereas the physiological role of selenium binding protein is not fully understood. This study shows for the first time the formation of adducts between tissue proteins and AcMPAG. Whether this chemical modification is associated with compromised protein function and drug toxicity remains to be investigated.     

Evaluation of drug-human serum albumin binding interactions with support vector machine aided online automated docking

MOTIVATION: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform (http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated.     

Role of binding in distribution of furosemide: where is nonrenal clearance?

Furosemide is an acidic drug that binds tightly to anionic binding sites on albumin, but has negligible binding to tissue proteins. As a consequence, it has a low volume of distribution of total drug at steady state (Vdss total), which is less than extracellular fluid volume. With decreases in serum albumin concentration, plasma protein binding decreases, total Vdss increases, and Vdss of free drug decreases in a manner that is quantitatively consistent with no change in tissue binding. The elimination of furosemide is partly by renal clearance of unchanged drug (predominantly because of renal tubule secretion) and partly by nonrenal routes. Glucuronidation appears to occur in extrahepatic sites in dogs and, possibly, humans. Residual nonrenal elimination is not influenced by liver disease but is reduced by probenecid pretreatment and uremia. Furthermore, there is appreciable recovery of 35S-labeled furosemide in feces after i.v. administration without the appearance of 35S in the bile. These observations are consistent with the hypothesis of active secretion of furosemide into the gut. The independent variables of distribution and elimination contribute to determine intersubject variability of plasma concentration-time profiles in health and disease and, therefore, are important in determining the extent and duration of diuretic response.     

Molecular dynamics simulation as a tool for assessment of drug binding property of human serum albumin

Human serum albumin (HSA) is a major plasma protein and binding of drugs with this plasma protein has a great importance. It possess esterase activity which can cleave the drugs containing ester bond and thus, can regulate the effect of drugs. Till date no systematic study has been done to analyse binding of such drugs and to compare the results with the drugs which do not have ester bond. Therefore, in the present study two different categories—ester and non-ester drugs have been considered to analyse their interaction with HSA at two principle drug binding sites using molecular modelling tools. It is observed that the drugs irrespective of ester or non-ester nature prefer either Sudlow site I or II by hydrogen bond and hydrophobic interactions. The information obtained from the study can assist to study pharmacokinetics of the drugs and that in turn will help in noval drug discoveries.     

Pharmaceutical-grade albumin: impaired drug-binding capacity <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Albumin 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.     

Competitive binding of bismuth to transferrin and albumin in aqueous solution and in blood plasma

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi(3+) to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of (1)H and (13)C NMR resonances of isotopically labeled [epsilon-(13)C]Met transferrin. We show that Met(132) in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi(3+) binds to the specific Fe(3+) sites of transferrin and the observed shifts of Met resonances suggest that Bi(3+) induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi(3+) binding to albumin is nonspecific and Cys(34) is not a major binding site, which is surprising because Bi(3+) has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi(3+)) in proteins depend not only on their presence but also on their accessibility. Bi(3+) binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.     

The extraordinary ligand binding properties of human serum albumin

Human serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites. HSA provides a depot for many compounds, affects pharmacokinetics of many drugs, holds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and acts as a NO-carrier. The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins. Here, structural, functional, biotechnological, and biomedical aspects of ligand binding to HSA are summarized.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

蛋白多肽类药物长效化技术研究策略

多肽/蛋白质类药物普遍存在体内半衰期短的问题,使其应用受到了限制。近年来,蛋白质半衰期的研究取得了很大的进展,许多技术已经被提出并测试延长治疗性蛋白的作用时间,如与其他蛋白基因融合(免疫球蛋白域或血清蛋白,如白蛋白)、与聚合物结合(PEG化修饰、聚唾液酸化、HEP化等)。这些技术主要基于本身较长的半衰期及循环机制调控以延长多肽/蛋白质类药物的半衰期。针对上述的多种长效化方式及相关产品进行了综述与讨论,以期为此类药物的长效化研究提供思路。     

Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302)

In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.     

Measurement of drug-protein binding by immobilized human serum albumin-HPLC and comparison with ultrafiltration

An HPLC method employing CHIRAL-I (150 mm x 3 mm), 5 microm column from Chrom. Tech., immobilized with human serum albumin (HSA), was used to determine in vitro protein binding of several compounds. Experimentally obtained plasma protein data exhibited good correlation with the reported values. The method was compared with the conventional ultra filtration technique and both yielded similar results. Proprietary compounds that could not be analyzed by ultra filtration due to high non-specific binding to filter membrane were successfully analyzed by HSA-HPLC method. On the other hand, two proprietary compounds did not elute from HSA column due to strong binding, but were successfully analyzed by ultra filtration. This proves that both the techniques have their own merits and demerits and should be exploited judiciously as per the requirement. The plasma protein binding studies conducted on four gyrase inhibitors in rat and human plasma exhibited no interspecies difference via ultra filtration method. Further, it was also observed that the protein binding obtained for the four gyrase inhibitors by HSA-HPLC method was not only similar to that obtained by ultra filtration in human plasma but was also in accordance with ex vivo and in vitro protein binding obtained for rat plasma after ultra filtration because these compounds predominantly bind to HSA The binding of several compounds to alpha1-acid glycoprotein (AGP), another important plasma protein, was also examined using AGP immobilized column. However, the data could not be relied upon since some anti-bacterials and non-steroidal anti-inflammatory drugs (NSAIDS), which are known to predominantly bind to HSA, were also found to bind to AGP.     

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.