Kazuaki Taguchi

An Oral Adsorbent, AST-120 Protects Against the Progression of Oxidative Stress by Reducing the Accumulation of Indoxyl Sulfate in the Systemic Circulation in Renal Failure

PurposeThe effect of AST-120, an oral adsorbent, on oxidative stress in the systemic circulation in chronic renal failure (CRF) was examined and the potential role of indoxyl sulfate (IS), an uremic toxin adsorbed by AST-120, in inducing the formation of reactive oxygen species (ROS) in the vascular system was studied, in vitro and in vivo.Materials and methodsThe level of oxidized albumin, a marker for oxidative stress in the systemic circulation was determined by HPLC, as previously reported. The mRNA levels of TGF-β1 and Oat1 were measured by quantitative RT-PCR. The IS induced ROS generation in cultured human umbilical vein endothelial cells (HUVECs) was estimated using a fluorescence microplate reader.ResultsAn increase in the ratio of oxidized to unoxidized albumin was determined using 5/6 nephrectomized rats (CRF rats) compared to a control group. The ratio was significantly reduced in the group that received AST-120 of 4xa0weeks, suggesting that AST-120 inhibits oxidative stress in CRF. An anti-oxidative effect of AST-120 was also observed in CRF rats with a similar renal function. The ratio of oxidized albumin was correlated with serum IS levels in vivo. The same relationship was also observed in CRF rats with the continued administration of IS. In addition, IS dramatically increased the generation of ROS in both a dose- and time- dependent manner in HUVEC, suggesting that accumulated IS may play an important role in enhancing intravascular oxidative stress.ConclusionWe propose that AST-120 reduces IS concentrations in the blood that induces ROS production in endothelial cells, thereby inhibiting the subsequent occurrence of oxidative stress in the systemic circulation in renal failure.

Hemoglobin vesicles, polyethylene glycol (PEG)ylated liposomes developed as a red blood cell substitute, do not induce the accelerated blood clearance phenomenon in mice.

The hemoglobin vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a liposome of which the surface is covered with polyethylene glycol (PEG). It was recently reported that repeated injections of PEGylated liposomes induce the accelerated blood clearance (ABC) phenomenon, in which serum anti-PEG IgM plays an essential role. To examine this issue, we investigated whether HbV induces the ABC phenomenon in mice at a dose of 0.1 mg Hb/kg, a dose that is generally known to induce the ABC phenomenon, or at 1400 mg Hb/kg, which is proposed for clinical use. At 7 days after the first injection of nonlabeled HbV (0.1 mg Hb/kg), the mice received HbV in which the Hb had been labeled with 125I. After a second injection, HbV was rapidly cleared from the circulation, and uptake clearances in liver and spleen were significantly increased. In contrast, at a dose of 1400 mg Hb/kg, the pharmacokinetics of HbV was negligibly affected by repeated injection. It is interesting to note that IgM against HbV was produced 7 days postinjection at both of the above doses, and their recognition site was determined to be 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG in HbV. These results suggest that a clinical dose of HbV does not induce the ABC phenomenon, and that suppression of ABC phenomenon is caused by the saturation of phagocytic processing by the mononuclear phagocyte system. Thus, we conclude that induction of the ABC phenomenon would not be an issue in the dose regimen used in clinical settings.

Evaluation of carboxymethyl-β-cyclodextrin with acid function: Improvement of chemical stability, oral bioavailability and bitter taste of famotidine

The objective of the present study was to evaluate the potential influence of carboxymethyl-beta-cyclodextrin (CM-beta-CyD) on the aqueous solubility, chemical stability and oral bioavailability of famotidine (FMT) as well as on its bitter taste. We examined the effect of the CM-beta-CyD on the acidic degradation of FMT compared with that for sulfobutyl-ether-beta-cyclodextrin (SBE-beta-CyD). The potential use of CM-beta-CyD for orally disintegrating tablets (ODTs) was evaluated in vitro and in vivo. A taste perception study was also carried out. A strong stabilizing influence of CM-beta-CyD was observed against the acidic degradation, in sharp contrast to SBE-beta-CyD which induced a weird destabilizing effect on FMT. (13)C NMR was used to investigate the interaction mode between FMT and the 2 CyDs. In vivo study of ODTs indicated a significant increase in C(max), AUC and oral bioavailability in the case of FMT-CM-beta-CyD tablets, compared with plain drug tablets. However, no significant difference in T(max) and t(1/2) was observed. CM-beta-CyD complexation appears to be an acceptable strategy for enhancing the oral bioavailability of FMT owing to its dramatic effect on the aqueous solubility and chemical stability of the drug. In addition, it has a pronounced effect on masking the bitter taste of FMT.

Pharmacokinetic study of enclosed hemoglobin and outer lipid component after the administration of hemoglobin vesicles as an artificial oxygen carrier.

The hemoglobin vesicle (HbV) is an artificial oxygen carrier that encapsulates a concentrated Hb solution in lipid vesicles (liposomes). The pharmacokinetic properties of HbV were investigated in mice and rats. With use of HbV in which the internal Hb was labeled with 125I (125I-HbV) and cell-free 125I-Hb, it was found that encapsulation of Hb increased the half-life by 30 times, accompanied by decreased distribution in both the liver and kidney. The half-life of HbV was increased, and the uptake clearance for the liver and spleen were decreased with increasing doses of HbV. In an in vitro study, the specific uptake and degradation of HbV in RAW 264.7 cells were found, but this was not the case for parenchymal and endothelial cells. The pharmacokinetics of HbV components (internal Hb and liposomal lipid) were also investigated using 125I-HbV and 3H-HbV (liposomal cholesterol was radiolabeled with tritium-3). The time courses for the plasma concentration curves of 125I-HbV, 3H-HbV, and iron derived from HbV suggest that HbV maintain an intact structure in the blood circulation up to 24 h after injection. 125I-HbV and 3H-HbV were distributed mainly to the liver and spleen. Internal Hb disappeared from both the liver and spleen 5 days after injection, and the liposomal cholesterol disappeared at approximately 14 days. Internal Hb was excreted into the urine and cholesterol into feces via biliary excretion. These results suggest that the HbV has a reasonable blood retention and metabolic and excretion performance and could be used as an oxygen carrier.

Pharmacokinetics of single and repeated injection of hemoglobin-vesicles in hemorrhagic shock rat model

Hemoglobin-vesicles (HbV) are liposomal artificial oxygen carriers that may be useful as a resuscitation fluid during hemorrhagic shock (HS). It is well-known that the pharmacokinetic properties of liposome change in response to both pathological conditions and repeated administration. Therefore, we compared the pharmacokinetics of single versus repeated administration of HbV during HS. HS was induced by withdrawal of 40% of total blood volume. The normal (non-HS) and HS1 group was received an injection of 125I-labeled HbV (125I-HbV). The HS2 group was resuscitated with non-labeled HbV, and 1 h later, it received an injection of 125I-HbV. The half-life was shorter in HS1 rats, but it returned to non-HS levels after the second HbV injection. During 12 h after administration of HbV, tissue distribution of HbV was greatest in the HS1 group; however, the HS2 group had the greatest tissue distribution at subsequent time points. Excretion into urine, major elimination pathway, did not differ between non-HS and HS1 rats, but was significantly reduced in the HS2 group. Furthermore, the half-life of HbV in humans was estimated to be approximately 3-4 days using an allometric equation. This suggests that HbV may be a useful artificial oxygen carrier in HS based on HbV pharmacokinetics.

Pharmaceutical aspects of the recombinant human serum albumin dimer: structural characteristics, biological properties, and medical applications.

Human serum albumin is the most abundant protein in the blood. It is clinically used in the treatment of severe hypoalbuminemia and as a plasma expander. The use of albumins as a carrier for drugs is currently being developed, and some are now in the preclinical and clinical trial stages. The main technologies for utilizing an albumin as a drug carrier are protein fusion, polymerization and surface modification, and so on. Among these technologies, albumin dimerization has wide clinical applications as a plasma expander as well as a drug carrier. Despite the fact that many reports have appeared on drugs using an albumin dimer as a carrier, our knowledge of the characteristics of the albumin dimer itself is incomplete. In this review, we summarize the structural characteristics of recombinant albumin dimers produced by two methods, namely, chemical linkage with 1,6-bis(maleimido)hexane and genetically linked with an amino acid linker, and the physicochemical characteristics and biological properties of these preparations. Finally, the potential for pharmaceutical applications of albumin dimers in clinical situations is discussed.

Human serum albumin-thioredoxin fusion protein with long blood retention property is effective in suppressing lung injury

Thioredoxin (Trx) is a redox-active protein with anti-inflammatory effects but with a short half life of 1 h. Genetic fusion of Trx to human serum albumin (HSA) extended its half life without causing significant loss of its biological activities. HSA-Trx caused a decrease in the number of cells in brochoalveolar lavage fluid, the wet/dry ratio and the inflammation at the respiratory tract of the ovalbumin (OVA) induced lung injury model mouse. Three intraperitoneal doses of Trx alone produced the same extent of suppression of those three detrimental effects of OVA as one intravenous dose of HSA-Trx. Inhibition experiments confirmed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) involved in the progression of the injury. HSA-Trx inhibited the production of ROS as confirmed in the EPR experiment, but lung tissue staining suggested that induced nitrogen oxide synthase (iNOS) was not suppressed by the fusion protein. Instead, the production of nitrotyrosine, 8-nitro-cGMP, and 8-hydroxy-2-deoxyguanosine downstream to the iNOS has been inhibited. This suggested that HSA-Trx produced lung protection effect via different mechanisms from Trx alone. HSA-Trx retains the biological properties of Trx thus has great potential in treating oxidative stress related diseases.

Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXα revealed by phage display.

4Z,15Z‐bilirubin‐IXα (4Z,15Z‐BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domainu2003II was constructed, after three rounds of panning against immobilized 4Z,15Z‐BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positionsu2003195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand‐alone domainu2003II were expressed in Pichiau2003pastoris for ligand‐binding studies. The binding of 4Z,15Z‐BR to the K195A and K199A mutants was decreased in both whole HSA and the domainu2003II proteins. The P‐helicity conformer (P‐form) of 4Z,15Z‐BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M‐form bound to the K199A mutants. Photoconversion experiments showed that the P‐form of 4Z,15Z‐BR was transformed into highly water‐soluble isomers at a much faster rate than the M‐form. In addition, the M‐form of 4Z,15Z‐BR showed higher affinity for domainu2003I than for domainu2003II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomainu2003IIA are important for the high‐affinity binding of 4Z,15Z‐BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z‐BR.

Hepatically-metabolized and -excreted artificial oxygen carrier, hemoglobin vesicles, can be safely used under conditions of hepatic impairment

The hemoglobin vesicle (HbV) is an artificial oxygen carrier in which a concentrated Hb solution is encapsulated in lipid vesicles. Our previous studies demonstrated that HbV is metabolized by the mononuclear phagocyte system, and the lipid components are excreted from the liver. It is well-known that many hepatically-metabolized and -excreted drugs show altered pharmaceutics under conditions of liver impairment, which results in adverse effects. The aim of this study was to determine whether the administration of HbV causes toxicity in rats with carbon tetrachloride induced liver cirrhosis. Changes in plasma biochemical parameters, histological staining and the pharmacokinetic distribution of HbV were evaluated after an HbV injection of the above model rats at a putative clinical dose (1400 mgHb/kg). Plasma biochemical parameters were not significantly affected, except for a transient elevation of lipase, lipid components and bilirubin, which recovered within 14 days after an HbV infusion. Negligible morphological changes were observed in the kidney, liver, spleen, lung and heart. Hemosiderin, a marker of iron accumulation in organs, was observed in the liver and spleen up to 14 days after HbV treatment, but no evidence of oxidative stress in the plasma and liver were observed. HbV is mainly distributed in the liver and spleen, and the lipid components are excreted into feces within 7 days. In conclusion, even under conditions of hepatic cirrhosis, HbV and its components exhibit the favorable metabolic and excretion profile at the putative clinical dose. These findings provide further support for the safety and effectiveness of HbV in clinical settings.

Liposome-encapsulated hemoglobin (hemoglobin-vesicle) is not transferred from mother to fetus at the late stage of pregnancy in the rat model.

AIMSnLiposome-encapsulated hemoglobin (hemoglobin vesicles: HbV; diameter 250 nm) is reconstructed from human hemoglobin and developed as an artificial oxygen carrier for use as a transfusion alternative. Previous studies using rodent models closely investigated the safety of daily repeated infusions (DRI) of HbV and reported that the reticuloendothelial system was physiologically capable of degrading HbV to maintain plasma clinical chemistry within normal ranges. The present study examined the effect of DRI of HbV on the pregnant rat mother and fetal development, focusing on placental transfer of HbV in pregnancy.nnnMAIN METHODSnPregnant rats intravenously received HbV bolus injections at 2 ml/kg/day for the last 7 consecutive days till term. The cumulative infusion volume (14 ml/kg) was equal to 25% of the whole blood volume (56 ml/kg).nnnKEY FINDINGSnMaternal DRI of HbV had no obvious side effects on the pregnant mother or on fetal development. Maternal vital signs, plasma clinical chemistry, and blood gas parameters were overall normal after DRI of HbV. In addition, maternal/fetal transfer of HbV was limited to the placenta and HbV did not reach the fetus. Histopathological examination with human hemoglobin antibody detected HbV accumulation in the maternal spleen, liver, kidney, and placenta, but not in the fetuses. These results were also confirmed by a pharmacokinetic study using (125)I-labeled HbV.nnnSIGNIFICANCEnThis safety study of HbV use in the pregnant mother and fetus will contribute to a possible application of HbV as a potential treatment for fetal hypoxia by supplying oxygen through the placenta.