Hakaru Seo

Cross-linked human serum albumin dimer has the potential for use as a plasma-retaining agent for the fatty acid-conjugated antidiabetic drugs.

The half‐life of fatty acid‐conjugated antidiabetic drugs are prolonged through binding to albumin, but this may not occur in diabetic patients with nephropathy complicated with hypoalbuminemia. We previously showed that human serum albumin (HSA) dimerized at the proteins Cys34 by 1,6‐bis(maleimido)hexane has longer half‐life than the monomer under high permeability conditions. The aim of this study was to investigate the superior ability of this HSA dimer as a plasma‐retaining agent for fatty acid conjugated antidiabetic drugs.

γ-シクロデキストリン複合体化によるジゴキシンのバイオアベイラビリティの向上 : ヒトにおける舌下及び経口投与時の評価

The inclusion complex of digoxin with gamma-cyclodextrin was prepared in a molar ratio of 1:4, and evaluated for sublingual and oral administrations in humans. In the dissolution tests of digoxin tablets, the increase in dissolution rate and decrease in acid hydrolysis were attained by gamma-cyclodextrin complexation. The serum levels of digoxin after sublingual and oral administrations to human healthy volunteers in the form of complex tablets were higher than the digoxin alone, particularly in the case of the sublingual form of gamma-cyclodextrin complex. The present data suggested that the sublingual administration of the rapid dissolving form of gamma-cyclodextrin complex may be useful for improving the bioavailability of digoxin due to the prevention of acid hydrolysis in stomach and the enhancement of drug absorption rate.

Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin

A wide variety of drugs bind to human serum albumin (HSA) at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand) and ibuprofen (site II ligand) at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole) to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a ‘competitive-like’ allosteric interaction in the case of the defatted HSA conformer to a ‘nearly independent’ binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a ‘competitive-like’ allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to ‘nearly independent’ binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

Tyrosine411 and Arginine410 of Human Serum Albumin Play an Important Role in the Binding of Sodium 4-Phenylbutyrate to Site II

Sodium 4-phenylbutyrate (PB) has many pharmacological activities; therefore extending its clinical use to the treatment of a wider variety of diseases would be desirable. However, our knowledge of the binding of PB to plasma proteins is not extensive. To address this issue in more detail, we characterized the protein binding of PB. Binding experiments showed that PB mainly binds to human serum albumin (HSA) in plasma. PB was also found to bind to a single site on HSA, which was identified as site II by fluorescent probe displacement experiment. Furthermore, an appropriate alkyl chain length and a carboxylic group in the PB structure were required for PB binding to HSA, suggesting that hydrophobic (and van der Waals) and electrostatic interactions are involved as binding modes. The contributions of hydrogen bonding and/or van der Waals interactions were also indicated by thermodynamic analyses. Tyrosine411 and arginine410 were identified as being involved in the binding of PB to site II, based on binding experiments using chemically modified- and mutant-HSA preparations. In conclusion, the available evidence indicates that PB binds to site II of HSA with assistance by multiple forces and that tyrosine411 and arginine410 both play important roles in this phenomenon.

Biological Responsiveness and Metabolic Performance of Liposome-Encapsulated Hemoglobin (Hemoglobin-Vesicles) in Apolipoprotein E-Deficient Mice after Massive Intravenous Injection.

The hemoglobin-vesicle (HbV), a vesicle in which a concentrated human hemoglobin solution is encapsulated, was developed as an artificial oxygen carrier. Although HbV has a favorable safety, metabolic, and excretion performance in healthy animals, the effect of a massive amount of HbV, which also contains a large amount of a lipid component including cholesterol, on physiological response and metabolic performance under hyperlipidemic conditions is unclear. The aim of this study was to evaluate whether administration of HbV causes toxicity in apolipoprotein E-deficient mice (hyperlipidemic model mice). Apolipoprotein E-deficient mice were given a single injection of HbV (2000 mg hemoglobin/kg), and physiological responses and metabolic profiles were monitored for 14 d thereafter. All the mice tolerated the massive amount of HbV and survived, and adequate biocompatibility was observed. Serum biochemical parameters indicate that liver and kidney function were not remarkably affected, and morphological changes in the liver and spleen were negligible. Lipid parameters in serum were significantly increased until 3 d after HbV administration, but recovered within 7 d after the administration. In a pharmacokinetic study, HbV was mainly found distributed in the liver and spleen, and disappeared from the body within 14 d. In conclusion, even under conditions of hyperlipidemia, a massive dose of HbV and its components resulted in favorable biological compatibility, metabolic, and excretion profiles. These findings provide further support for the safety of HbV for clinical use.

Potential Use of Biological Proteins for Liver Failure Therapy

Biological proteins have unlimited potential for use as pharmaceutical products due to their various biological activities, which include non-toxicity, biocompatibility, and biodegradability. Recent scientific advances allow for the development of novel innovative protein-based products that draw on the quality of their innate biological activities. Some of them hold promising potential for novel therapeutic agents/devices for addressing hepatic diseases such as hepatitis, fibrosis, and hepatocarcinomas. This review attempts to provide an overview of the development of protein-based products that take advantage of their biological activity for medication, and discusses possibilities for the therapeutic potential of protein-based products produced through different approaches to specifically target the liver (or hepatic cells: hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) in the treatment of hepatic diseases.

The Binding of Silibinin, the Main Constituent of Silymarin, to Site I on Human Serum Albumin

Silibinin is the main constituent of silymarin, an extract from the seeds of milk thistle (Silybum marianum). Because silibinin has many pharmacological activities, extending its clinical use in the treatment of a wider variety of diseases would be desirable. In this study, we report on the binding of silibinin to plasma proteins, an issue that has not previously been extensively studied. The findings indicated that silibinin mainly binds to human serum albumin (HSA). Mutual displacement experiments using ligands that primarily bind to sites I and II clearly revealed that silibinin binds tightly and selectively to site I (subsites Ia and/or Ic) of HSA, which is located in subdomain IIA. Thermodynamic analyses suggested that hydrogen bonding and van der Waals interactions are major contributors to silibinin-HSA interactions. Furthermore, the binding of silibinin to HSA was found to be decreased with increasing ionic strength and detergent concentration of the media, suggesting that electrostatic and hydrophobic interactions are involved in the binding. Trp214 and Arg218 were identified as being involved in the binding of silibinin to site I, based on binding experiments using chemically modified- and mutant-HSAs. In conclusion, the available evidence indicates that silibinin binds to the region close to Trp214 and Arg218 in site I of HSA with assistance by multiple forces and can displace site I drugs (e.g., warfarin or iodipamide), but not site II drugs (e.g., ibuprofen).

Establishment of a model of acetaminophen-induced hepatotoxicity in different weekly-aged ICR mice

Acetaminophen (APAP), a widely used analgesic and antipyretic drug, has the potential to cause lethal hepatotoxicity. Mice are widely used for developing murine models of APAP-induced hepatotoxicity, and many researchers have used these models for APAP-related studies including the fields of biology, pharmacology and toxicology. Although drug-induced hepatotoxicity is dependent on a number of factors (species, gender and age), very few studies have investigated the effect of aging on APAP hepatotoxicity. In this study, we evaluated the effect of age on APAP-induced hepatotoxicity in different weekly-aged mice to establish a model of APAP-induced hepatotoxicity that is an accurate reflection of general experimental conditions. Male ICR mice 4, 6, 8, 10 and 12 weeks old were given APAP intraperitoneally, and mortality, hepatic damage and the plasma concentration of APAP metabolites were evaluated. It was found that younger male ICR mice were relatively resistant to hepatotoxicity induced by intraperitoneal APAP administration. In addition, the APAP-glucuronide concentration in plasma remained essentially the same among the differently-aged mice, while APAP-sulfate levels were dramatically decreased in an age-dependent manner. Thus, it is recommended that mice of the same ages be used in studies related to APAP-induced hepatotoxixity. These results provide evidence in support of not only the age-related changes in susceptibility to APAP-derived hepatotoxicity in mice but also in developing mouse models for APAP-related studies.

An evaluation of novel biological activity in a crude extract from Hemerocallis fulva L. var. sempervirens M. Hotta.

Hemerocallis fulva L. var. sempervirens M. Hotta (kwanso) represents an exceptional resource for identifying and developing new phytomedicines for the treatment and prevention of disease. The aim of this study was to conduct a detailed investigation of the biological activities of kwanso. Our study resulted in four major findings. First, kwanso scavenges hydroxyl radicals generated by H2O2/UV light system in vitro in a dose-dependent manner. Second, hepatic glutathione levels were significantly increased when kwanso was orally administered to mice. Third, the oral administration of kwanso to mice showed a tendency to suppress hepatic injury induced by acetaminophen. Finally, kwanso slightly inhibited cytochrome P450 3A activity. These results provide useful evidence in support of the development of kwanso as a candidate raw material for the treatment and prevention of disease.

Species Differences in the Binding of Sodium 4-Phenylbutyrate to Serum Albumin

Sodium 4-phenylbutyrate (PB) is clinically used as a drug for treating urea cycle disorders. Recent research has shown that PB also has other pharmacologic activities, suggesting that it has the potential for use as a drug for treating other disorders. In the process of drug development, preclinical testing using experimental animals is necessary to verify the efficacy and safety of PB. Although the binding of PB to human albumin has been studied, our knowledge of its binding to albumin from the other animal species is extremely limited. To address this issue, we characterized the binding of PB to albumin from several species (human, bovine, rabbit, and rat). The results indicated that PB interacts with 1 high-affinity site of albumin from these species, which corresponds to site II of human albumin. The affinities of PB to human and bovine albumins were higher than those to rabbit and rat albumin, and that to rabbit albumin was the lowest. Binding and molecular docking studies using structurally related compounds of PB suggested that species differences in the affinity are attributed to differences in the structural feature of the PB-binding sites on albumins (e.g., charge distribution, hydrophobicity, shape, or size).