[Changes in Attitudes to Medical Care among Pharmacy Pharmacists before and after the COVID-19 Pandemic: A Questionnaire Survey].

The coronavirus disease 2019 (COVID-19) pandemic has had a major negative effect on the number of patients visiting pharmacies in Japan. The decrease in pharmacy visits during the pandemic compared with the pre-pandemic period may have increased the likelihood of adverse health outcomes; thus, it is important that pharmacy pharmacists take measures to prevent health disadvantages. In this study, we distributed a questionnaire survey to 104 pharmacy pharmacists (mainly in Kagoshima and Kumamoto Prefectures), and investigated changes in the extent of implementation and perceptions of measures considered necessary to protect patients' health between the pre-pandemic and pandemic period. The results showed that the proportions of respondents "sharing patient information between primary care doctors and pharmacy pharmacists" and conducting "follow-up after prescribing medications mainly via telephone" increased between the pre-pandemic period and September 2022. The perceived necessity of the above two measures, as well as "online medication instructions" and "a prescription refill system," increased during the same period. However, the proportion of respondents who perceived "0410 correspondence," which was introduced during the pandemic, as a necessity did not change. Moreover, many pharmacists indicated that, at their own discretion, they continued to correspond with patients in relation to the above, and to respond to specific requests during normal daily practice. Our results could help community-based pharmacists tackle serious public health problems, such as COVID-19.

Evaluation of the Safety and Gastrointestinal Migration of Guanidinylated Chitosan after Oral Administration to Rats.

Arginine-rich membrane-permeable peptides (APPs) can be delivered to cells by forming complexes with various membrane-impermeable bioactive molecules such as proteins. We recently reported on the preparation of guanidinylated chitosan (GCS) that mimics arginine peptides, using chitosan, a naturally occurring cationic polysaccharide, and confirmed that it enhances protein permeability in an in vitro cell system. However, studies on the in vivo safety of GCS are not available. To address this, we evaluated the in vivo safety of GCS and its translocation into the gastrointestinal tract in rats after a single oral administration of an excessive dose (500 mg/kg) and observed changes in body weight, major organ weights, and organ tissue sections for periods of up to 2 weeks. The results indicated that GCS causes no deleterious effects. The results of an oral administration of rhodamine-labeled chitosan and an evaluation of its migration in the gastrointestinal tract suggested that the disappearance of rhodamine-labeled GCS from the body appeared to be slower than that of the non-dose group and pre-guanidinylated chitosan due to its mucoadhesive properties. In the future, we plan to investigate the use of GCS to improve absorption using Class III and IV drugs, which are poorly water-soluble as well as poorly membrane-permeable.

The Effects of Sacran, a Sulfated Polysaccharide, on Gut Microbiota Using Chronic Kidney Disease Model Rats.

The aim of this study was to investigate the beneficial effects of sacran, a sulfated polysaccharide, on renal damage and intestinal microflora, in 5/6 nephrectomy rats as a model for chronic kidney disease (CKD). 5/6 Nephrectomy rats were divided into sacran treated and non-treated groups and examined for lethality after 4 weeks. The 5/6 nephrectomy rats were also divided into three groups: sacran treated, non-treated and AST-120 treated groups, and treated orally in a concentration-dependent manner for 4 weeks. Renal function was estimated by biochemical and histopathological analyses. Metagenomic analysis of feces from each group after 4 weeks was also performed and changes in intestinal microflora were compared. The administration of sacran to CKD rats at ≥19 mg/d increased their survival. In addition, the sacran-treated group improved CKD-related parameters in a concentration-dependent manner, and the inhibitory effect of 40 mg/d of sacran was comparable to that of AST-120. The changes in the intestinal microflora of the sacran treated group were positively correlated with an increase in the number of Lactobacillus species, which are known to be rich in beneficial bacteria, and the increment of this beneficial bacteria was negatively correlated with the concentration of indoxyl sulfate, a uremic toxin, in plasma. These results strongly suggest that the oral administration of sacran could contribute to the stabilization of intestinal microflora in CKD rats and to the reduction of oxidative stress as well as the inhibition of progression of CKD.

Supramolecular Assembly of Hybrid Pt(II) Porphyrin/Tomatine Analogues with Different Nanostructures and Cytotoxic Activities.

A simple strategy for synthesizing supramolecular hybrids was developed for the preparation of bioavailable nanohybrid photosensitizers by assembling visible-light-sensitive Pt(II) meso-tetrakis(4-carboxyphenyl)porphyrinporphyrin (PtTCPP)/tomatine analogues. The hybrids were self-assembled into nanofibrous or nanosheet structures approximately 3-5 nm thick and several micrometers wide. α-Tomatine generated a unique fibrous vesicle nanostructure based on intermolecular interactions, while dehydrotomatine generated nanosheet structures. Nanoassembly of these fibrous vesicles and sheets directly affected the properties of the light-responsive photosensitizer for tumor photodynamic therapy (PDT), depending on the nanostructure of the hybrid PtTCPP/tomatine analogues. The cytotoxicity of PtTCPP to cancer cells under photoirradiation was significantly enhanced by a tomatine assembly with a fibrous vesicle nanostructure, attributable to increased incorporation of the drug into cells.

Sacran, a sulfated polysaccharide, suppresses the absorption of lipids and modulates the intestinal flora in non-alcoholic steatohepatitis model rats.

AIMS: The objective of this study was to investigate the effects of administering sacran, a sulfated polysaccharide, on liver biology, gut microbiota, oxidative stress, and inflammation on stroke-prone spontaneously hypertensive (SHRSP5/Dmcr) rats that develop fibrotic steatohepatitis with histological similarities to that of non-alcoholic steatohepatitis (NASH). MAIN METHODS: Four groups of 8-week-old SHRSP5/Dmcr rats were fed a high fat-cholesterol (HFC) diet for 4 and 8 weeks and administered either sacran (80 mg/kg/day) or a non-treatment, respectively. Liver function was evaluated by biochemical and histopathological analyses. Hepatic inflammatory markers were measured using mRNA expression. Fecal microbial profiles were determined via 16S rRNA sequencing. A triglyceride (TG) absorption test was administered to the 8-week-old Sprague-Dawley (SD) rats. KEY FINDING: Sacran administration was observed to decrease the extent of oxidative stress and hepatic biochemical parameters in serum and hepatic injury with the levels of transforming growth factor-beta (TGF-ß1) and tumor necrosis factor-alpha (TNF-α), being increased compared to those of the non-treatment group. At the genus level, sacran administration caused a significant decrease in the harmful Prevotella genus, and a significant increase in the useful Blautia genus was observed. Sacran administration also decreased the serum TG increase that was induced by administering corn oil to the SD rats. SIGNIFICANCE: We conclude that sacran administration has the potential to reduce the absorption of lipids into blood and to improve several gut microbiotas, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress and hepatic markers in the systematic circulation on NASH.

Glycosylation decreases aggregation and immunogenicity of adalimumab Fab secreted from Pichia pastoris.

Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H chain constant region of adalimumab Fab through site-directed mutagenesis (H:L178N Fab), and the H:L178N Fab was produced in Pichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behaviour of the Fab, L-glyco Fab and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-life in vivo.

The preparation and validation of chitosan tablets that rapidly disperse and disintegrate as an oral adsorbent in the treatment of lifestyle-related diseases.

A carrier and an oral absorbent for the treatment of chronic diseases in the form of a tablet was prepared from granulated chitosan (G-CS) particles. The resulting tablet was highly dispersible and disintegrated rapidly (< 30 s) in aqueous media. The non-granulated chitosan (N-CS) powder partially crystallized (2θ = 12-15° and 20°) during wet granulation to give G-CS crystalline particles. The rate of penetration of water into G-CS aggregates was markedly faster than that for N-CS aggregates, as evidenced by the ease of disintegration of the tablets. The rapid disintegration and dispersion of the tablets in vivo was confirmed by MRI measurements after the oral administration of the both tablets to rats. Some ureic toxins were adsorbed more strongly to G-CS tablets than on N-CS tablets. The results suggest that G-CS tablets have great potential for use as a fast disintegrating carrier and as an oral adsorbent in lifestyle-related diseases.

A Comparison of the Frequency of Prescription and Pharmacy Revisits between Baloxavir Marboxil and Neuraminidase Inhibitors in Influenza-Infected Pediatric Patients during the 2019-2020 Influenza Season.

The novel anti-influenza virus agent baloxavir marboxil is a selective inhibitor of an influenza cap-dependent endonuclease. Although a single oral dose in tablet form of baloxavir marboxil is expected to improve drug compliance and rapidly reduce viral titers for pediatric patients with influenza, there is a concern that baloxavir marboxil-resistant influenza A variants could be generated. In this study, we investigated the frequency of prescription and pharmacy revisits for baloxavir marboxil at an outpatient clinic compared with that of neuraminidase inhibitors in pediatric patients with influenza. A total of 475 pediatric patients who were infected with the influenza virus visited the pharmacy between December 2019 and March 2020. Baloxavir marboxil (n = 149), oseltamivir (n = 161) and laninamivir (n = 162) were mainly prescribed and only a few patients were treated with peramivir (n = 2) or zanamivir (n = 1). Baloxavir marboxil-, oseltamivir- and laninamivir-treated pediatric patients were enrolled, and a log-rank test showed that the revisits of pediatric patients who were taking baloxavir marboxil was lower than those for oseltamivir (p < 0.001). Moreover, Cox proportional hazards models also revealed that baloxavir marboxil decreased the risk of revisits in comparison to oseltamivir (hazard ratio 0.28, 95% confidence interval 0.11-0.70, p = 0.006), while no difference was found between laninamivir and baloxavir marboxil. Although there is a need to acquire appropriate and relevant information concerning resistant viruses, our results suggest that baloxavir marboxil may be a useful drug for treating pediatric patients with influenza infections.

Antioxidative Potency of Dolphin Serum Albumin Is Stronger Than That of Human Serum Albumin Irrespective of Substitution of 34Cysteine With Serine.

Serum albumin (SA), the most abundant protein in circulation, functions as a carrier protein, osmoregulator, and antioxidant. Generally, SA exerts its antioxidative effects by scavenging reactive oxygen species. Because marine mammals are superior divers, they are intermittently exposed to oxidative stress induced by rapid reperfusion of oxygen to ischemic tissues after the dive. Although several antioxidants in marine mammals have been described, SA activity remains largely uncharacterized. In this study, we investigated the antioxidative activity of SA in marine mammals by comparing features of the primary and steric structures, biochemical properties, and antioxidative activities of common bottlenose dolphin SA (DSA) and human SA (HSA). Our results revealed that DSA lacked free cysteine at position 34 that is important for the antioxidative activity of HSA; however, the antioxidative capacity and thiol activity of DSA were stronger than those of HSA. Circular dichroism spectra showed different patterns in DSA and HSA. Ultraviolet fluorescence intensities of DSA were higher than those of HSA, suggesting lower surface hydrophobicity of DSA. Additionally, DSA showed higher excess heat capacity than HSA. We then compared a homology model of DSA with a 3D model of HSA. Our results indicate that DSA was more unstable than HSA at least in the body-temperature range, probably due to the mode of molecules involved in the disulfide bonds and/or the lower surface hydrophobicity, and it may be related to the equivalent or stronger antioxidant potency of DSA. These data show that DSA is an effective antioxidant in the circulation of the dolphin.

Effects of surface-deacetylated chitin nanofibers on non-alcoholic steatohepatitis model rats and their gut microbiota.

Nonalcoholic steatohepatitis (NASH), a more advanced form of nonalcoholic fatty liver disease (NAFLD), is associated with increased cardiovascular and liver-related mortality. Stroke-prone spontaneously hypertensive rats (SHRSP5/Dmcr) that are fed a high-fat and high-cholesterol diet develop hepatic lesions that are similar to those observed in human NASH pathology. We investigated the hepatic protective and antioxidant effects of surface-deacetylated chitin nanofibers (SDACNFs) that were administered to SHRSP5/Dmcr rats for 8 weeks. The administration of SDACNFs (80 mg/kg/day) resulted in a significant decrease in hepatic injury, oxidative stress, compared with the non-treatment. The SDACNFs also caused a reduction in the population of harmful members of the Morganella and Prevotella genus, and increased the abundance of the Blautia genus, a useful bacterium in gut microbiota. We therefore conclude that SDACNF exerts anti-hepatic and antioxidative effects not only by adsorbing lipid substances but also by reforming the community of intestinal microflora in the intestinal tract.

[Evaluation of the Usefulness of a Completely Separated Infection Waiting Room and Non-infection Waiting Room in a Pediatric Neighborhood Pharmacy].

Because patients and their families are at a high risk of contracting infectious diseases in hospital as well as pharmacy, a pharmacy containing the waiting rooms separately for infectious diseases and non-infectious diseases is expected to lead them to visit with confidence and receive appropriate drug administration guidance without the risk of infection. In this study, we examined the potential usefulness of having separate waiting rooms by conducting a questionnaire survey on the structure of the pharmacy waiting room and investigating the visiting situation for influenza-infected pediatric patients from a record of the medicines they received. As a result of the questionnaire tabulated from guardians of 385 pediatric patients who first visited a pharmacy with separate waiting rooms, 70% of the pediatric patients concluded that having separate waiting rooms would be satisfactory. In addition, the possible risk of horizontal transmission of influenza was supposed to be reduced in the pharmacy with separate waiting room in comparison with the pharmacies without among 211 pediatric patients who could not identify the influenza source within their daily living areas, such as home and school. Based on these findings, we anticipate the growing availability of the pharmacies having separation type waiting rooms.

Morphometric Analysis of Paramylon Particles Produced by Euglena gracilis EOD-1 Using FIB/SEM Tomography.

Euglena gracilis EOD-1, a microalgal strain, produces large quantities of paramylon, a class of polymers known as ß-1,3-glucans and has been reported to function as a dietary fiber and to improve the metabolic syndrome including obesity. However, despite its importance, the morphometric analysis of paramylon has not been conducted so far. In this study, we attempted to observe the detailed three-dimensional structure of paramylon by focused ion beam/scanning electron microscopy (FIB/SEM). Paramylon samples were fixed and three-dimensional image reconstruction and segmentation of the image stack were created using computer software (Amira v6.0.1, FEI). The results indicated that the inside of paramylon particles (diameter: 5 µm, thickness: 3 µm) was comprised of a dense structure with no evidence of the presence of large pores and gaps, although a small 100 nm crack was observed. The specific surface area of paramylon particles measured by the Brunauer-Emmet-Teller (BET) method, was not as large as activated charcoal, but similar to those of plant starches, indicating that the cholesterol-lowering effect of paramylon cannot be simply attributed to its adsorption ability. The FIB/SEM method was found to be useful for elucidating the internal structure of small solid particles.

A thermoresponsive hydrophobically modified hydroxypropylmethylcellulose/cyclodextrin injectable hydrogel for the sustained release of drugs.

The objective of this study was to develop a thermoresponsive injectable hydrogel for the sustained release of drugs by taking advantage of host-guest interactions between a hydrophobically modified hydroxypropylmethyl cellulose (HM-HPMC) and cyclodextrin (CD). A thermoresponsive injectable hydrogel was prepared by simply adding CDs to HM-HPMC hydrogel. The HM-HPMC hydrogel was converted into a sol with a low viscosity through host-guest interactions with CDs. The HM-HPMC/ß-CD hydrogel became a gel near body temperature where the host dissociated from the hydrophobic moieties of the polymer in response to the temperature. The yield stress of the HM-HPMC became progressively lower on the addition of ß-CD which was desirable in the case of developing an injectable formulation. When the HM-HPMC/ß-CD hydrogel containing indocyanine green (ICG) was subcutaneously administered to mice, the fluorescence of the ICG remained relatively constant for 24 h after the administration, which was substantially longer than that for ICG alone or an HPMC formulation. The plasma insulin level was maintained for a longer period of time when the HM-HPMC/ß-CD containing insulin was administered and the MRT value was increased by 1.6 times compared to a solution of insulin alone. In addition, the HM-HPMC/ß-CD hydrogel formulation showed a prolonged hypoglycemic effect in response to the insulin which was slowly released from the hydrogel. A thermoresponsive injectable hydrogel was successfully constructed from the highly viscous HM-HPMC and ß-CD, and the resulting formulation functioned as a sustained release carrier for drugs.

C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond.

Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1 : C177-CL : C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.

Modification of Drug Crystallization by Cyclodextrins in Pre-formulation Study.

Controlling drug crystallization is one of the important issues in pre-formulation study. In recent years, advanced approaches including the use of tailor-made additives have gathered considerable attention to control crystallization behavior of drugs. This review focuses on the use of hydrophilic cyclodextrins (CDs) as additives for controlling drug crystallization. CDs affect the crystallization of drugs in solution and in solid state based on a host-guest interaction. For example, 2,6-di-O-methyl-ß-CD and 2-hydroxybutyl-ß-CD suppressed solution-mediated transition of drugs during crystallization by the host-guest interaction; as a result, metastable forms selectively precipitated in solution. The use of CDs in crystal engineering provided an opportunity for the detection of a new polymorph by changing the crystallization pathway. It was also possible to modify crystal morphology (i.e., crystal habit) by selective suppression of crystal growth on a certain direction based on the host-gust interaction. For solid formulation, stable amorphous drug/CDs complex under humid conditions was prepared using two different CDs. An overview of some recent progress in the use of CDs in crystal engineering and in amorphous formulation is described in this review.

The Use of Enteric Capsules for Releasing a Fragrance over an Extended Period of Time.

A system for releasing a fragrance, citral (CR) over an extended period of time using three types of enteric capsules is reported. The L- and M-type capsules released CR into media with a pH above 6, while the H-type capsule released CR at a pH above 7. The pH of the releasing medium was controlled by sodium borate (SB), i.e., by adding SB-methylcellulose (MC) prepared in different weight ratios (SB-MC 1 : 2, 1 : 1 and 2 : 1) to tablets and by compressing them at different pressures. The tablet containing a large amount of SB and that was pressed at higher pressures permitted the pH of the releasing medium to be changed from 5 to 9, at 4-5 h after the addition of SB to the tablets, while negligible changes were observed for tablets containing low amounts of SB and which were compressed at lower pressures. Reflecting these pH changes, CR was released after different periods of time when SB-MC tablets and capsules containing CR were simultaneously added to the releasing medium. When enteric capsules containing CR and the pH adjusting tablets were simultaneously added to a benzyl acetate (BA) solution, BA was released at a constant rate, while CR was released for different periods of time depending on the type of capsule used. The results suggest that fragrances could be released over different time frames by using enteric capsules and pH adjusting agents, for example, the release of fragrances with sedative effects at night time and with stimulating effects in the morning.

Design and Evaluation of An Extended-Release Olmesartan Tablet Using Chitosan/Cyclodextrin Composites.

Sustained-release olmesartan tablets (OLM) were prepared by the simple, direct compression of composites of anionic sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) and cationic spray-dried chitosan (SD-CS), and were evaluated for use as a sustained release preparation for the treatment of hypertension. An investigation of the interaction between OLM and SBE-ß-CD by the solubility method indicated that the phase diagram of the OLM/SBE-ß-CD system was the AL type, indicating the formation of a 1:1 inclusion complex. The release of OLM from tablets composed of the SD-CS/SBE-ß-CD composite was slow in media at both pH 1.2 and at 6.8. The in vitro slow release characteristics of the SD-CS/SBE-ß-CD composite were reflected in the in vivo absorption of the drug after normal rats were given an oral administration of the preparation. Furthermore, the SD-CS/SBE-ß-CD composite continuously increased the antihypertensive effect of OLM in hypertensive rats, compared with that of the drug itself. These results suggest that a simple mixing of SD-CS and SBE-ß-CD can be potentially useful for the controlled release of a drug for the continuous treatments of hypertension.

Preparation and evaluation of freeze dried surface-deacetylated chitin nanofiber/sacran pellets for use as an extended-release excipient.

Pelleted preparations were formulated from sacran (Sac), an anionic, sulfated, carboxyl-containing polysaccharide, which is extracted from the Japanese indigenous cyanobacterium Aphanothece sacrum, and surface-deacetylated chitin nanofibers (SDACNF). The use of this material as an extended-release excipient for tetrahydrocurcumin (THC), a model drug that is used to treat wounds via its radical scavenging ability was examined. The THC used in the study was complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), which increases its water solubility. The radical scavenging activity of the THC/HP-ß-CD complex (molar ratio of 1:1) was significantly higher than the values for SDACNF or Sac alone. The rate of release of THC from the Sac/SDACNF pellets containing the THC/HP-ß-CD complex decreased with increasing Sac content in the pellet, suggesting that Sac/SDACNF (1:1) and Sac alone pellets function as extended-release excipients for THC. The findings reported here indicate that this can be attributed to the ability of the Sac component to retain fluids, thus extending the effects of the drug. In view of the above experimental outcomes, i.e. wound healing efficacy, fluid absorption, retention and the extended drug release of the system indicates that this preparation, in the appropriate ratios, has the potential for use as a controlled-release drug in wound healing.

Antioxidant activities of chitosans and its derivatives in in vitro and in vivo studies.

This review focuses on the in vitro and in vivo antioxidant activities of various chitosan preparations, including those with different molecular weights and degrees of acetylation and the nanofibers produced from them. In in vitro studies, low molecular weight (LMW) chitosan with high degrees of deacetylation has more potent antioxidant properties than those of high molecular weight (HMW) chitosan. On the other hand, HMW chitosan has higher adsorption properties than those of LMW chitosan. On the basis of the in vitro results obtained, the ingestion of chitosan and nanofiber derived from it, with moderate MW and degrees of acetylation results in a significant reduction in oxidative stress in several chronic oxidative stress related diseases such as the metabolic syndrome and renal failure. In the future, chitosan and related nanofibers with presumed antioxidant properties may be used as a new source of antioxidant, as a possible food supplement, as an ingredient or in the pharmaceutical industry.

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites.

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.