Possible Involvement of Protein Binding Inhibition in Changes in Dexmedetomidine Concentration in Extracorporeal Circuits during Midazolam Use.

It was recently reported that the dexmedetomidine concentration within the extracorporeal circuit decreases with co-administration of midazolam. In this study, we investigated whether displacement of dexmedetomidine by midazolam from the binding site of major plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AAG), would increase levels of free dexmedetomidine that could be adsorbed to the circuit. Equilibrium dialysis experiments indicated that dexmedetomidine binds to a single site on both HSA and AAG with four times greater affinity than midazolam. Midazolam-mediated inhibition of the binding of dexmedetomidine to HSA and AAG was also examined. The binding of dexmedetomidine to these proteins decreased in the presence of midazolam. Competitive binding experiments suggested that the inhibition of binding by midazolam was due to competitive displacement at site II of HSA and due to non-competitive displacement at the site of AAG. Thus, our present data indicate that free dexmedetomidine displaced by midazolam from site II of HSA or from AAG is adsorbed onto extracorporeal circuits, resulting in a change in the dexmedetomidine concentration within the circuit.

Qualitative and quantitative status of cytochrome P450s after the administration of a liposomal platelet substitute in rat liver.

In the process of the drug development, studies on the cytochrome P450 (CYP) profiles after its administration provided fundamental information regarding drug interactions with concomitantly administered drugs. Here, we evaluated the influence of the administration of H12-(ADP)-liposomes, a platelet substitute, on the mRNA and protein expression, and metabolic activity of CYPs, with focus on the CYP1A2, CYP2C11 and CYP3A2, in rat liver.At 24 h after administering saline or H12-(ADP)-liposomes (10 mg of lipids/kg), a quantitative RT-PCR and western blot analysis revealed that the mRNA and proteins expression of all of the target hepatic CYP isoforms were not different between the saline and H12-(ADP)-liposome groups. Furthermore, an ex vivo CYP metabolic activity assay showed that hepatic CYP metabolic activities in the H12-(ADP)-liposome group were comparable to the corresponding saline group. On the other hand, the area under the blood concentration-time curve for substitutes for CYP1A2 and CYP2C11 was higher in H12-(ADP)-liposome group than in saline group, but the degree of elevations was negligible levels.At a minimum, based on these results, we conclude that H12-(ADP)-liposomes have no quantitative and qualitative effect on the hepatic CYP isoforms, indicating that the drug interactions of H12-(ADP)-liposomes with CYP-metabolizing drugs would be negligible.

Preparation and in Vitro Characterization of Fatty-Acid Modified Pirarubicin Nanosuspensions Stabilized by Albumin.

Pirarubicin (THP) shows more rapid intracellular uptake, more effective antitumor activity, and less cardiac toxicity, compared to doxorubicin. However, THP is distributed to both tumor and normal tissues indiscriminately. This study aimed to develop a nanosuspension to deliver THP to tumor tissues more efficiently. Fatty-acid-modified THPs (FA-THPs; octanoic acid, dodecanoic acid, palmitic acid-THPs) were synthesized to increase the hydrophobicity of THP. Nanosuspensions of these FA-THPs were then prepared using an antisolvent precipitation technique. Among the FA-THPs, the most efficiently drug-loaded nanosuspension was obtained from palmitic acid-THP (pal-THP) using an aqueous antisolvent containing bovine serum albumin as a stabilizer. The pal-THP nanoparticles in the nanosuspension were confirmed to be of optimal size (100-125 nm) for delivery to tumor tissues using dynamic light scattering and transmission electron microscopy. The pal-THP nanosuspension showed cytotoxicity in colon 26 cells. The nanosuspension was shown to disintegrate in the presence of surfactants such as lecithin, liberating pal-THP, which was converted to free THP in acidic media. It is therefore proposed that pal-THP nanoparticles that reach tumor cells after intravenous administration would exert antitumor effect by liberating pal-THP (i.e., disintegration of nanoparticles by the interaction with cell membrane), followed by the release of free THP in the acidic milieu of tumor cells. These findings indicate that FA-THP nanosuspensions, particularly pal-THP nanosuspension, hold promise as a candidate for cancer treatment. However, further in vivo studies are necessary.

Study of the Structural Chemistry of the Inclusion Complexation of 4-Phenylbutyrate and Related Compounds with Cyclodextrins in Solution: Differences in Inclusion Mode with Cavity Size Dependency.

4-phenylbutyrate (PB) and structurally related compounds hold promise for treating many diseases, including cancers. However, pharmaceutical limitations, such as an unpleasant taste or poor aqueous solubility, impede their evaluation and clinical use. This study explores cyclodextrin (CD) complexation as a strategy to address these limitations. The structural chemistry of the CD complexes of these compounds was analyzed using phase solubility, nuclear magnetic resonance (NMR) spectroscopic techniques, and molecular modeling to inform the choice of CD for such application. The study revealed that PB and its shorter-chain derivative form 1:1 αCD complexes, while the longer-chain derivatives form 1:2 (guest:host) complexes. αCD includes the alkyl chain of the shorter-chain compounds, depositing the phenyl ring around its secondary rim, whereas two αCD molecules sandwich the phenyl ring in a secondary-to-secondary rim orientation for the longer-chain derivatives. ßCD includes each compound to form 1:1 complexes, with their alkyl chains bent to varying degrees within the CD cavity. γCD includes two molecules of each compound to form 2:1 complexes, with both parallel and antiparallel orientations plausible. The study found that αCD is more suitable for overcoming the pharmaceutical drawbacks of PB and its shorter-chain derivative, while ßCD is better for the longer-chain derivatives.

Effect of Fatty Acids and Uremic Toxins on the Binding of Nateglinide, an Insulin Secretagogue, to Site II on Human Serum Albumin.

Nateglinide (NAT) is used to treat diabetes, stimulating pancreatic islet ß-cells with residual insulin secretory capacity to increase insulin secretion. NAT has been reported to bind to human serum albumin (HSA), but the detail is still unclear. In the current study, we investigated the location and the affinity for the binding of NAT to HSA. Quantitative analysis data from the ultrafiltration experiment indicated that NAT binds strongly to a primary site on HSA with a high affinity. The presence of diazepam (DZP) or ibuprofen (IB), the specific site II ligands of HSA, decreased the binding constants of NAT respectively, without the significant changes in the number of binding sites. Whereas warfarin (WF), a site I specific ligand, did not affect the binding of NAT. Fluorescent replacement experiment showed that NAT replaced dansylsarcosine (DNSS), a site II probe of HSA, but not WF. An increasing level of myristate and uremic toxins, indoxyl sulphate (IS), indoxyl acetate (IA) and p-cresyl sulphate (PCS), during renal disease significantly increased the concentration of unbound NAT. These findings suggest that NAT specifically binds to site II of HSA and the binding capacity and pharmacokinetics of NAT change in renal diseases.

Interaction of Benzbromarone with Subdomains IIIA and IB/IIA on Human Serum Albumin as the Primary and Secondary Binding Regions.

Benzbromarone has been used for the treatment of gout for more than 30 years. Although it shows a high level of binding to plasma proteins (>99%), our knowledge of this binding is not sufficiently extensive to permit us to understand its pharmacokinetics and pharmacodynamics. To address this issue in more detail, we characterized the binding of benzbromarone to human serum albumin (HSA), the most abundant protein in plasma. Equilibrium dialysis and circular dichroism findings indicated that benzbromarone binds strongly to one primary as well as to multiple secondary sites on HSA and that the bromine atoms of benzbromarone play important roles in this high affinity binding. An X-ray crystallographic study revealed that benzbromarone molecules bind to hydrophobic pockets within subdomains IB, IIA, and IIIA. Inhibition experiments using site specific ligands (subdomain IB; fusidic acid, IIA; warfarin, IIIA; diazepam) indicated that the primary and secondary binding sites that benzbromarone binds to are within subdomains IIIA and IB/IIA, respectively. Lastly, a study of the effect of fatty acids on the benzbromarone-HSA interaction suggested that benzbromarone, when displaced from subdomain IIIA by sodium oleate, could transfer to subdomains IB or IIA. Thus, these data will permit more relevant assessments of the displacement interactions of benzbromarone especially in cases of co-administered drugs or endogenous compounds that also bind to subdomain IIIA. In addition, the findings presented herein will also be useful for designing drug combination therapy in which pharmacokinetic and pharmacodynamic performance need to be controlled.

Possible Role of Electrolytes on the Formation of Precipitates during the Infusion of Nafamostat Mesilate in Hemodialysis.

Nafamostat mesilate (NFM) is used as an anticoagulant during hemodialysis in patients who have had complications due to hemorrhages. The formation of precipitates, which could lead to the interruption of hemodialysis has been reported when NFM is infused into blood during hemodialysis. We report herein on an examination of possible factors that could cause this. The effects of electrolytes such as phosphates, citrates or succinates on the formation of precipitates were examined by mixing NFM with aqueous solutions or plasma that contained these electrolytes. The formation of precipitates was observed in all electrolyte solutions when higher concentrations of NFM were mixed at around physiological pH. In the case of plasma, precipitates were observed when solutions containing higher concentrations of NFM were mixed with plasma that contained phosphate and citrate. In addition, the formation of precipitates under dynamic conditions where NFM was infused into flowing electrolyte solutions was also evaluated. The data suggested that such precipitates might be formed and disrupt the blood flow and/or an NFM infusion when NFM is infused into blood flowing in the hemodialysis circuit. The findings presented herein suggest the serum levels of anionic electrolytes (e.g., phosphate), the type of excipients present in pharmaceutical products (e.g., succinic acid or citric acid), the concentration of NFM used for the infusion or the rates of NFM infusion and blood flow are all factors that could affect precipitate formation during NFM infusions for hemodialysis.

Effects of Uremic Toxins on the Binding of Aripiprazole to Human Serum Albumin.

We recently reported that aripiprazole (ARP), an antipsychotic drug, binds strongly to human serum albumin (HSA), the major drug binding protein in serum. It is known that uremic toxins that accumulate during renal disease affect the interaction between HSA and drug binding. In this study, the issue of how uremic toxins (indoxyl sulfate, indole acetic acid and p-cresyl sulfate) affect the binding of ARP to HSA was investigated. Equilibrium dialysis experiments revealed that all uremic toxins inhibited the binding of ARP to HSA although the inhibitory effects differed, depending on the specific uremic toxin. The potency of inhibition can be partially explained by the affinities of uremic toxins to HSA. Fluorescence displacement experiments suggested that ARP as well as all uremic toxins bind to site II of HSA. The inhibitory effects of the toxins on the binding of ARP for the drugs binding to the diazepam subsite are significantly larger, comparing with those for binding to arylpropionic acids subsite. Interestingly, induced circular dichroism (CD) spectra indicated that the spatial orientation of p-cresyl sulfate in the binding pocket is different from that for indoxyl sulfate and indole acetic acid. The limited findings obtained herein are important data in considering the effects of uremic toxins on the pharmacokinetics of ARP and the drugs that bind to site II on HSA, particularly drugs binding to diazepam binding site in site II.

Pharmacokinetics of 4'-cyano-2'-deoxyguanosine, a novel nucleoside analog inhibitor of the resistant hepatitis B virus, in a rat model of chronic kidney disease.

INTRODUCTION: The novel nucleoside analog, 4'-cyano-2'-deoxyguanosine (CdG), possesses inhibitory activity against both the wild-type and resistant hepatitis B virus. Since the dosage of the currently available nucleoside analog preparations needs to be adjusted, depending on renal function, we investigated the effect of renal dysfunction on the pharmacokinetics of CdG in a rat model of chronic kidney disease (CKD). METHODS: CKD model rats were either intravenously or orally administered CdG at a dose of 1 mg/kg. The concentration of CdG in plasma, organs (liver and kidney) and urine samples were determined by means of a UPLC system interfaced with a TOF-MS system. RESULTS: Following intravenous administration, the plasma retention of CdG was prolonged in CKD model rats compared to healthy rats. In addition, the clearance of CdG was well correlated with plasma creatinine levels in CKD model rats. Similar to the results for intravenous administration, the plasma concentration profiles of CdG after oral administration were also found to be much higher in CKD model rats than in healthy rats. However, the results for the organ distribution and urinary excretion of CdG, the profiles of which were similar to that of healthy rats, indicated that CdG did not accumulate to a significant extent in the body. CONCLUSION: The extent of renal dysfunction has a direct influence on the pharmacokinetics (plasma retention) of CdG without a significant accumulation, indicating that the dosage of CdG will be dependent on the extent of renal function. .

Serum Albumin, Lipid and Drug Binding.

Albumin is widely conserved from vertebrates to invertebrates, and nature of mammalian albumins permit them to bind various endogenous ligands and drugs in the blood. It is known that at least two major ligand binding sites are present on the albumin molecule, which are referred to as Site I and Site II. These binding sites are thought to be almost completely conserved among mammals, even though the degree of binding to these sites are different depending on the physical and chemical properties of drugs and differences in the microenvironment in the binding pockets. In addition, the binding sites for medium and long-chain fatty acids are also well conserved among mammals, and it is considered that there are at least seven binding sites, including Site I and Site II. These bindings properties of albumin in the blood are also widely known to be important for transporting drugs and fatty acids to various tissues. It can therefore be concluded that albumin is one of the most important serum proteins for various ligands, and information on human albumin can be very useful in predicting the ligand binding properties of the albumin of other vertebrates.

Pharmacokinetic Properties of Orally Administered 4'-Cyano-2'-deoxyguanosine, a Novel Nucleoside Analog Inhibitor of the Hepatitis B Virus, in Viral Liver Injury Model Rats.

A nucleoside analog, 4'-cyano-2'-deoxyguanosine (CdG), which was developed as an inhibitor of the chronic hepatitis B virus (HBV), exhibited a superior antiviral activity against both wild-type and drugs-resistant HBV to marketed nucleoside analogs. In addition to previous pharmacokinetic studies of CdG in healthy rats, this study reports on an evaluation of the pharmacokinetic characteristics of CdG in a rat model of viral liver injury (VLI) induced by treatment with concanavalin A. Following an intravenous administration of CdG at a dose of 1 mg/kg, the plasma concentration profile of CdG in VLI model rats was found to be similar to that of healthy rats with no significant difference in kinetic parameters. However, when CdG was orally administered at a dose of 1 mg/kg, the maximum blood concentration was much lower in VLI model rats than in healthy rats. Interestingly, the amount of residual food in the stomachs in VLI model rats was significantly larger than that in healthy rats, indicating that the adsorption of CdG in the gastrointestinal tract was inhibited in the presence of food as well as other marketed nucleoside analogs. As observed in healthy rats, CdG was largely distributed to the liver compared to the kidney in the VLI model. These results suggest that liver pathology has only a minor effect on the pharmacokinetic properties of CdG, but the influence of food on CdG absorption needs to be considered.

Effects of Oxidation of Human Serum Albumin on the Binding of Aripiprazole.

Aripiprazole (ARP) is one of antipsychotics and binds to human serum albumin (HSA) with a high affinity. In this study, we investigated the binding characteristics of ARP to oxidized HSA as observed in chronic disease conditions. Oxidized HSAs were prepared using chloramine-T (CT-HSA) or metal-catalyzed oxidation system (MCO-HSA) in vitro, respectively. An increase in the carbonyl content was confirmed in oxidized HSAs. From the results of circular dichroism (CD) and tryptophan fluorescence spectra, no significant structural change of oxidized HSAs was observed. These results indicate that prepared HSAs are mildly oxidized and well reflects the status of HSA during chronic diseases. However, oxidized HSAs were observed to have a significant decrease in binding to ARP. The results of the induced CD spectrum suggested that ARP bound to oxidized HSAs with a similar orientation. These results suggest that oxidation of HSA during chronic disease state significantly affected the microenvironment of the binding site for ARP and binding capacity of HSA to ARP.

Characterization of Bovine Lactoferrin Nanoparticle Prepared by Desolvation Technique.

Lactoferrin (Lf) nanoparticles have been developed as a carrier of drugs and gene. Two main methods, desolvation technique and emulsification method, for preparation of protein nanoparticles have been reported so far, but most of the previous reports of Lf nanoparticles preparation are limited to emulsification method. In this study, we investigated the optimal conditions by desolvation technique for the preparation of glutaraldehyde-crosslinked bovine Lf (bLf) nanoparticles within the size range of 100-200 nm, and evaluated their properties as a carrier for oral and intravenous drug delivery. The experimental results of dynamic light scattering and Transmission Electron Microscope suggested that glutaraldehyde-crosslinked bLf nanoparticles with 150 nm in size could be produced by addition of 2-propanol as the desolvating solvent into the bLf solution adjusted to pH 6, followed by crosslinking with glutaraldehyde. These cross-linked bLf nanoparticles were found to be compatible to blood components and resistant against rapid degradation by pepsin. Thus, cross-linked bLf nanoparticles prepared by desolvation technique can be applied as a drug carrier for intravenous administration and oral delivery.

Preparation and in Vitro Analysis of Human Serum Albumin Nanoparticles Loaded with Anthracycline Derivatives.

Nanoparticles prepared using human serum albumin (HSA) have emerged as versatile carriers for improving the pharmacokinetic profile of drugs. The desolvation of HSA using ethanol followed by stabilization through crosslinking with glutaraldehyde is a common technique for preparing HSA nanoparticles, but our knowledge concerning the characteristics (or functions) of HSA nanoparticles and their efficiency when loaded with drugs is limited. To address this issue in more detail, we prepared anthracycline-loaded HSA nanoparticles. Doxorubicin-loaded HSA nanoparticles with a size similar to doxorubicin-unloaded particles could be prepared by desolvating at a higher pH (8-9), and the size (100-150 nm) was optimum for delivery to tumor tissues. Using this procedure, HSA nanoparticles were loaded with other anthracycline derivatives, and all showed cytotoxicity in cancer cells. However, the efficiency of drug loading and dissolution rate were different among them possibly due to the differences in the type of association of the drugs on nanoparticles (doxorubicin and daunorubicin; covalently bound to nanoparticles, pirarubicin; both covalently bound to and adsorbed on nanoparticles, aclarubicin; adsorbed on nanoparticles). Since the formulation of such drug-loaded HSA nanoparticles should be modified for efficient delivery to tumors, the findings reported herein provide the useful information for optimizing the formulation and the production process for the HSA nanoparticles using a desolvation technique.

Crystallographic analysis of the ternary complex of octanoate and N-acetyl-l-methionine with human serum albumin reveals the mode of their stabilizing interactions.

During pasteurization and storage of albumin products, Sodium octanoate (Oct) and N-acethyl-l-tryptophan (N-AcTrp) are used as the thermal stabilizer and the antioxidant for human serum albumin (HSA), respectively. We recently reported that N-acethyl-l-methionine (N-AcMet) is an antioxidant for HSA, which is superior to N-AcTrp when it is especially exposed to light during storage. The objective of the present study is to clarify the molecular mechanism responsible for the HSA protective effect of Oct and N-AcMet based on their ternary complex structure. Crystal structure of the HSA-Oct-N-AcMet complex showed that one N-AcMet molecule is bound to the entrance of drug site 1 of HSA, and its side chain, which is susceptible to the oxidation, is exposed to the solvent. At the same time, two Oct binding sites are observed in drug sites 1 and 2 of HSA, respectively, and each Oct molecule occupies the hydrophobic cavity in them. These results indicate the molecular mechanism responsible for the HSA stabilization by these small molecules as follows. N-AcMet seals the entrance of drug site 1 while it acts as an antioxidant for HSA. Oct is chiefly bound to drug site 2 of HSA and it increases the thermal stability of HSA because of the occupying the largest intra-cavity of sub-domain IIIA in HSA. These findings suggest that N-AcMet acts positively as useful stabilizer for albumin formulated products such as functionalized HSA and HSA fusion proteins.

Recombinant Human Serum Albumin Containing 3 Copies of Domain I, Has Significant in Vitro Antioxidative Capacity Compared to the Wild-Type.

Human serum albumin (HSA), the most abundant protein in serum, functions as carrier of drugs and contributes to maintaining serum colloid osmotic pressure. We report herein on the preparation of a genetic recombinant HSA, in which domains II and III were changed to domain I (triple domain I; TDI). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the purity of the TDI was equivalent to that of the wild type (WT). Both far- and near-UV circular dichroism (CD) spectra of the TDI showed that its structural characteristics were similar to the WT. Ligand binding capacity was examined by an ultrafiltration method using 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and ketoprofen as markers for site I and site II, respectively. The binding capacity of TDI for both ligands was lower than that for the wild type. TDI significantly suppressed the oxidation of dihydrorhodamine 123 (DRD) by H2O2 compared to the WT. Our current results suggest that TDI has great potential for further development as HSA a product having antioxidative functions.

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).

Effect of a Ferric Citrate Formulation, a Phosphate Binder, on Oxidative Stress in Chronic Kidney Diseases-Mineral and Bone Disorder Patients Receiving Hemodialysis: A Pilot Study.

A ferric citrate formulation for treating hyperphosphatemia is a new therapeutic that not only suppresses the accumulation of phosphorus in patients with chronic kidney disease-mineral bone disorders (CKD-MBD), but also ameliorates anemia caused by iron deficiency. In contrast, it has been demonstrated that intravenous iron injection markedly increases oxidative stress. This study was designed to investigate the effect of a ferric citrate formulation on oxidative stress in CKD-MBD patients receiving hemodialysis therapy. Fifteen CKD-MBD patients undergoing dialysis were enrolled in this study. The patients were orally administered a ferric citrate formulation for 6 months. Their plasma phosphorus concentrations remained unchanged with the switch from other phosphorus adsorbents to the ferric citrate formulation. In addition, the ferric citrate formulation generally allowed for dose reduction of an erythropoiesis stimulating agent with an increased hematopoietic effect. The average values of plasma ferritin level increased after the introduction of a ferric citrate formulation, but did not exceed 100 (ng/mL). Interestingly, oxidative stress markers did not increase significantly, and anti-oxidative capacity was not significantly decreased at 6 months after the drug administration. Similarly, no change was observed in any inflammation markers. The ferric citrate formulation induces negligible oxidative stress in CKD-MBD patients receiving dialysis under the present clinical condition.

N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-L-tryptophan.

BACKGROUND: Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products. METHODS: Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration. RESULTS: N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present. CONCLUSION: The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations. GENERAL SIGNIFICANCE: N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.

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.