Spectroscopic and in silico study of binding mechanism of cynidine‐3‐O‐glucoside with human serum albumin and glycated human serum albumin

The drug–serum albumin interaction plays a dominant role in drug efficacy and disposition. The glycation of serum albumin that occurs during diabetes may affect its drug‐binding properties in vivo. In order to evaluate the interactivity characteristics of cyanidin‐3‐O‐glucoside (C3G) with human serum albumin (HSA) and glycated human serum albumin (gHSA), this study was undertaken using multiple spectroscopic techniques and molecular modeling analysis. Time‐resolved fluorescence and the thermodynamic parameters indicated that the quenching mechanism was static quenching, and hydrogen bonding and Van der Waals force were the main forces. The protein fluorescence could be quenched by C3G, whereas the polarity of the fluorophore was not obviously changed. C3G significantly altered the secondary structure of the proteins. Furthermore, the interaction force that existed in the HSA–C3G system was greater than that in the gHSA–C3G system. Fluorescence excitation emission matrix spectra, red edge excitation shift, Fourier transform infrared spectroscopy and circular dichroism spectra provided further evidence that glycation could inhibit the binding between C3G and proteins. In addition, molecular modeling analysis supported the experimental results. The results provided more details for the application of C3G in the treatment of diabetes.     

Enhanced intracellular delivery of methotrexate by a receptor-mediated process

In the present investigation we have described a method of enhancing the uptake of methotrexate by macrophages. This enhanced uptake was mediated by endocytosis through the "scavenger receptor" system which recognized maleylated bovine serum albumin. Experimental evidence showed that macrophages internalized methotrexate coupled to maleylated bovine serum albumin through a saturable process at 37 degrees C leading to an eightfold higher concentration of cell-associated methotrexate compared to the free drug. Following uptake, the drug conjugate was degraded in the lysosomes leading to intracellular release of a pharmacologically active form of methotrexate. When administered to macrophages infected with Leishmania mexicana amazonensis, the drug conjugate could eliminate the intracellular amastigotes more efficiently than the free drug. The leishmanicidal effect of the drug conjugate was inhibited in the presence of excess maleylated bovine serum albumin and lysosomal inhibitors such as chloroquine and monensin. Addition of folinic acid to the medium also prevented the elimination of the amastigotes by the drug conjugate. These results suggested that the scavenger receptor-mediated endocytosis of the drug conjugate led to enhanced transport and intracellular release of a pharmacologically active form of methotrexate resulting in more efficient killing of the amastigotes compared to the free drug. This modality of delivering drugs selectively to macrophages might have utility in the chemotherapy of macrophage-associated disorders in general.     

Characterization of the aflatoxin B1-binding site of rat albumin

A fluorescence-enhancement method was used to investigate the non-covalent interaction between aflatoxin B1 and rat albumin. Solvent-induced shifts in the emission spectrum of aflatoxin B1 provided evidence that the aflatoxin B1-binding site of rat albumin is a highly nonpolar environment. A dissociation constant of 20 microM was determined at 20 degrees C. The possibility that aflatoxin B1 binds one of the three major drug sites of albumin was investigated by ligand-displacement experiments. Mechanisms whereby marker ligands displace aflatoxin B1 were further investigated by comparing the experimental binding parameters with those derived theoretically, assuming competitive binding. The results indicate that: aflatoxin B1 and phenylbutazone compete for a common high-affinity site on rat albumin; high-affinity binding of aflatoxin B1 and site-II marker ligands takes place independently; aflatoxin B1 does not compete with either cholate or warfarin for the same high-affinity site, but the simultaneous binding of warfarin or cholate negatively modulates the binding of aflatoxin B1 to albumin. Fluorescence energy-transfer studies show that the lone tryptophan residue, Trp-214, is not associated with the aflatoxin B1-binding site.     

Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin,alpha-globulins,alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6

The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.     

Molecular heterogeneity of Cowpea (Vigna unguiculata Fabaceae) seed storage proteins

81 wild forms and 110 cultivated cowpea,Vigna unguiculata, accessions from 21 countries of Africa were screened for variability in seed storage proteins. Total seed proteins, albumin and globulin fractions were investigated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of nonreduced and/or reduced samples in one- and two-dimensional procedures. The globulin fraction is heterogeneous in molecular weight and contains both legumin-like components and three to six nondisulfide-linked subunits. Three globulin subunits, with molecular weights 110, 76, and 41 kD were found to be composed of disulfide-linked polypeptides. In the nondisulfide-linked fraction, both cultivated and wild forms exhibited patterns of four types (A–D). This fraction contains polypeptide subunits of molecular weights 62, 56, and 52 kD for A type, 62, 56, 54, and 52 kD for B type, 62, 56, 52, and 50 kD for C type, and at least 62, 56, 54, 52, 50, and 49 kD for D type. These subunits present similar multiple charge forms but C and D types possess more basic specific 50 and 49 kD nondisulfide linked components. Major albumin fraction contains subunits of 94, 86, 32, and 24kD. No infraspecific variation was observed in albumin or legumin-like fractions. The discussion is focussed on the relations between genetic variability assessed by storage protein coding genes and phenotypic variability.     

Stability, pKa and plasma protein binding of roscovitine

In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 degrees C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 degrees C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to alpha1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, alpha1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 degrees C compared to 37 degrees C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.     

S-Nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.     

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (-50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin-propranolol complexes.     

Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

Study of biochemical behavior of some exported and nonexported hepatic proteins during an acute inflammatory reaction in the rat

Haptoglobin, albumin, glucose-6-phosphatase, p-nitrophenol uridine diphosphate (UDP)-glucuronosyltransferase and cytochrome P-450 were measured in liver microsomes from normal rats and from rats undergoing an acute inflammatory reaction (AIR) induced either by subcutaneous administration of turpentine or by intrapleural injection of calcium pyrophosphate. 24 h after the beginning of the AIR induced by subcutaneous administration of turpentine, haptoglobin and albumin, two exported proteins, had risen to a peak (+313%), and dropped considerably (-52%) whereas nonexported protein levels did not change except for cytochrome P-450, which diminished (-38%). In the same way, intrapleural injection of calcium pyrophosphate was followed after 24 h by significant but smaller variations in haptoglobin (+60%) and cytochrome P-450 (-20%) concentrations. Albumin levels, glucose-6-phosphatase and p-nitrophenol UDP-glucuronosyltransferase activities were unchanged in this experimental model. The drop in cytochrome P-450 under all these conditions and also the diminution of albumin in the first model suggest that all the proteins produced by liver cells might not be synthesized in equal amounts. The decrease in cytochrome P-450 could interfere in hepatic drug metabolism during an AIR.     

The efficacy of Liv-52 on liver cirrhotic patients: a randomized, double-blind, placebo-controlled first approach.

Cirrhosis is the irreversible sequel of various disorders that damage liver cells permanently over time. Presently, the use of herbal medicines for prevention and control of chronic liver diseases is in the focus of attention for both the physicians and the patients; the reasons for such shift toward the use of herbals include the expensive cost of conventional drugs, adverse drug reactions, and their inefficacy. In the present study, the efficacy of herbal medicine Liv-52 (consisting of Mandur basma, Tamarix gallica and herbal extracts of Capparis spinosa, Cichorium intybus, Solanum nigrum, Terminalia arjuna and Achillea millefolium) on liver cirrhosis outcomes was compared with the placebo for 6 months in 36 cirrhotic patients referred to Tehran Hepatic Center. The outcome measures included child-pugh score, ascites, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total billirubin, albumin, prothrombin time, platelet and white blood cells counts. The indices were recorded in all patients before and after 6 months of drug or placebo treatment. The results demonstrated that the patients treated with Liv-52 for 6 months had significantly better child-pugh score, decreased ascites, decreased serum ALT and AST. In placebo administered patients all the clinical parameters recorded at beginning of the study were not significantly different than after 6 months. We conclude that Liv-52 possess hepatoprotective effect in cirrhotic patients. This protective effect of Liv-52 can be attributed to the diuretic, anti-inflammatory, anti-oxidative, and immunomodulating properties of the component herbs.     

Structural differences between albumin A and albumin C of the house mouse,Mus musculus

Two albumins, albumin A from C3H mice and albumin C isolated from descendents of the wild mice in which the variant was first uncovered, were found to differ in their electrophoretic properties. Albumin C was shown to bind two more H⁺ ions than albumin A at pH 5.4. Peptide mapping after trypsin digestion revealed that albumin C had three peptides (TP-C1, TP-C2, and TP-C3) which were missing in albumin A. The latter likewise had a peptide (TP-A1) which was not found in albumin C. An amino acid analysis of the variant peptides suggests that TP-A1 had been split into TP-C1 and TP-C2 on digestion with trypsin, because a glutamic acid in TP-A1 was replaced by a lysine. This change would also appropriately alter the electrophoretic properties of albumin C. No obvious counterpart was discovered for TP-C3 of albumin C in albumin A.This work was supported by a grant from the National Research Council of Canada.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography.

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.     

Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum

The antiviral antibiotic brefeldin A (BFA) strongly inhibits the protein secretion in cultured rat hepatocytes (Misumi, Y., Misumi, Y., Miki, K., Takatsuki, A., Tamura, G., and Ikehara, Y. (1986) J. Biol. Chem. 261, 11398-11403). We have further examined the inhibitory effect of the drug on intracellular transport of albumin by an immunocytochemical technique with peroxidase-conjugated Fab fragments of anti-rat albumin IgG. In hepatocytes treated with BFA (2.5 micrograms/ml) for 1 h at 37 degrees C, no characteristic structures of the Golgi complex could be observed, and albumin was diffusely distributed in the endoplasmic reticulum (ER), nuclear envelope, and small vesicles around, in contrast to its condensed localization in the Golgi complex in the control cells. Such an unusual distribution of the secretory protein, however, was rearranged to the normal localization in the Golgi complex after 4 h even in the presence of the drug, possibly due to a metabolism of the drug to an inert form. Exposure of the cells to BFA with constant renewals (2.5 micrograms/ml at 1-h intervals) or at a higher concentration (10 micrograms/ml) caused a prolonged accumulation of albumin in the ER, resulting in its dilation. These results indicate that BFA primarily blocks the protein transport from the ER to the Golgi complex, consistent with the biochemical data previously reported.     

Effect of heating temperature and time on pharmaceutical characteristics of albumin microspheres containing 5-fluorouracil

Conclusion  In this study, we found that both heating temperature and heating time affect mean particle size, particle size distribution, and drug entrapment efficiency of albumin microspheres. The change in heating temperature may affect the particle size of the product, especially when heating is carried out at a lower temperature (90°C–120°C). Hence the temperature should be selected on the basis of desired size range. Given that it is desirable for a maximum amount of the drug used in the preparation to become entrapped in microspheres, heating temperature and heating time for denaturation of albumin should be selected cautiously, as both have a significant effect on drug entrapment efficiency. In the present case, the highest entrapment was found in batches prepared by heating at 90°C for 5 minutes. However, the extent of stabilization at the selected temperature and the time of heating should also be taken into consideration, as they may affect the release of drugs to target tissue.     

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.     

Normal atmospheric oxygen tension and the use of antioxidants improve hepatocyte spheroid viability and function

Hepatocyte spheroids have been proposed for drug metabolism studies and in bioartificial liver devices. However, the optimal conditions required to meet the aerobic demands of mitochondria-rich hepatocyte spheroids is not well studied. We hypothesized that an optimal concentration of oxygen could be identified and that the health of hepatocyte spheroids might be further improved by antioxidant therapy. Rat hepatocyte spheroids were maintained in suspension culture for 7 days under a mixture of 5% CO(2) plus O(2):N(2) to achieve fractional oxygen contents of 6%(C1), 21%(C2), 58%(C3), and 95%(C4). Spheroid health was assessed under each condition by vital staining, TEM, oxygen consumption, and mitochondrial counts. Hepatocyte differentiation was assessed by expression of 10 liver-related genes (HNF4a, HNF6, Cyp1A1, albumin, Nags, Cps1, Otc, Ass, Asl, Arg1). Functional markers (albumin and urea) were measured. The influence of oxygen tension and antioxidant treatment on the production of reactive oxygen species (ROS) was assessed by confocal microscopy. We observed that the hepatocyte spheroids were healthiest under normal atmospheric (C2) conditions with antioxidants ascorbic acid and L-carnitine. Cell death and reduced functionality of hepatocyte spheroids correlated with the formation of ROS. Normal atmospheric conditions provided the optimal oxygen tension for suspension culture of hepatocyte spheroids. The formation and deleterious effects of ROS were further reduced by adding antioxidants to the culture medium. These findings have direct application to development of the spheroid reservoir bioartificial liver and the use of hepatocyte spheroids in drug metabolism studies.     

Interaction of antiviral and antitumor photoactive drug hypocrellin A with human serum albumin.

Absorption, resonance Raman, surface-enhanced Raman spectroscopy and differential scanning microcalorimetry were employed to study the interaction of hypocrellin A with human serum albumin. The identification of the binding place for hypocrellin A as well as the model for the albumin-hypocrellin A complex are proposed. In this model hypocrellin A interacts with albumin through more than one binding site placed on the protein surface. This model of non-specific interaction could explain why the absorption spectrum of hypocrellin A does not change in the presence of albumin and why the presence of the drug does not change significantly the thermodynamic parameters of the protein, while the Raman spectra show evident changes concerning both the protein and the drug structure. Even if hypocrellin A does not interact with an interior binding site, it can affect deeply the general albumin structure.