Physical compatibility of lipid emulsions and intravenous medications used in neonatal intensive care settings.

OBJECTIVE: The purpose of this study was to investigate the physical compatibility of intravenous lipid emulsions with parenteral medications used in neonatal intensive care. METHODS: Lipid emulsion and drug solutions were combined 1:1 in glass vials, inspected for physical incompatibility at 0, 1 and 2 hours, and assessed on the basis of lipid droplet size at 0 and 2 hours after mixing. Intravenous fluid controls (Water for Injection, sodium chloride 0.9% w/v, glucose 5% w/v), positive controls (gentamicin, albumin), negative controls (metronidazole, paracetamol, vancomycin) and 21 previously untested drug combinations were evaluated. RESULTS: No phase separation, change in colour, gas production or other visible anomaly was observed. The between-run mean droplet diameter (MDD) for SMOFlipid20% alone (0.301±0.008 µm) was comparable to the lipid emulsion/intravenous fluid and lipid emulsion/drug solution combinations. In addition to gentamicin and albumin, caffeine citrate (20 mg/mL) was shown to be incompatible with the lipid emulsion. All other lipid:drug combinations were compatible, based on the MDD data. CONCLUSION: Intravenous lipid emulsions were found to be compatible with 20 parenteral medications, including antimicrobial agents, inotropes, anti-inflammatory drugs and caffeine base, in simulated Y-site conditions. The lipid emulsion was incompatible with caffeine citrate injection.

A Maleimide-Terminally Modified PEGylated Liposome Induced the Accelerated Blood Clearance Independent of the Production of Anti-PEG IgM Antibodies.

PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. The ABC phenomenon is generally thought to occur when the anti-polyethylene glycol (PEG) antibody (anti-PEG immunoglobulin M (IgM)) expressed in the spleen B cells triggered by the first dose of PL binds to the second and subsequent doses of PL, leading to activation of the complement system. MAL-PEG-DSPE, a PEG lipid with a maleimide (MAL) group at the PEG terminal, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group (PL-OCH3). In this study, we prepared MAL-PEG-DSPE liposomes (PL-MAL) to evaluate the effect of PL-MAL on the ABC phenomenon induction compared to PL-OCH3. Pharmacokinetic, anti-PEG IgM secretion and complement activation analyses of these liposomes were conducted in mice. Interestingly, despite C3 bound to the surface of the initially administered PL-MAL, the administered PL-MAL showed high blood retention, demonstrating the same results as PL-OCH3. On the other hand, although the secretion of anti-PEG IgM induced by PL-MAL was lower than PL-OCH3, the second dose of PL-MAL rapidly disappeared from the blood. These results suggest that the antibody produced from the first dose of PL-MAL binds to the second dose of PL-MAL, thereby activating C3 to act as an opsonin which promotes phagocytic uptake. In conclusion, PL-MAL induced the ABC phenomenon independent of the production of IgM antibodies against PEG. This study provides valuable findings for further studies using ligand-bound liposomes.

The New Delivery Strategy of Albumin Carrier Utilizing the Interaction with Albumin Receptors.

Albumin, the most abundant protein in human serum, is applied to various diseases as a drug delivery carrier because of its superior blood retention, high biocompatibility, and a wide variety of drug binding abilities. Albumin is known to distribute widely in the blood and various interstitial fluids and organs. Different albumin receptors skillfully regulate the distribution characteristics of albumin in the body. Albumin receptors are a group of diverse proteins, such as FcRn, gp60, gp18, megalin, cubilin, SPARC, and CD36. Their tissue distributions in vivo are unique, with different albumin's recognition sites. Therefore, the distribution of albumin in vivo is ingeniously controlled by these multiple albumin receptors. Reevaluation of these albumin receptors opens up new possibilities for applying albumin as a drug delivery carrier. If the tissue distributions of albumin receptors were known and the albumin recognition site of the receptor was identified, organ-specific active targeting would be possible. In this review, we would like to scrutinize what is currently known and share information to develop next-generation albumin carriers that focus on interactions with albumin receptors.

The Therapeutic Effect of Human Serum Albumin Dimer-Doxorubicin Complex against Human Pancreatic Tumors.

Human serum albumin (HSA) is a versatile drug carrier with active tumor targeting capacity for an antitumor drug delivery system. Nanoparticle albumin-bound (nab)-technology, such as nab-paclitaxel (Abraxane®), has attracted significant interest in drug delivery research. Recently, we demonstrated that HSA dimer (HSA-d) possesses a higher tumor distribution than HSA monomer (HSA-m). Therefore, HSA-d is more suitable as a drug carrier for antitumor therapy and can improve nab technology. This study investigated the efficacy of HSA-d-doxorubicin (HSA-d-DOX) as next-generation nab technology for tumor treatment. DOX conjugated to HSA-d via a tunable pH-sensitive linker for the controlled release of DOX. Lyophilization did not affect the particle size of HSA-d-DOX or the release of DOX. HSA-d-DOX showed significantly higher cytotoxicity than HSA-m-DOX in vitro. In the SUIzo Tumor-2 (SUIT2) human pancreatic tumor subcutaneous inoculation model, HSA-d-DOX could significantly inhibit tumor growth without causing serious side effects, as compared to the HSA binding DOX prodrug, which utilized endogenous HSA as a nano-drug delivery system (DDS) carrier. These results indicate that HSA-d could function as a natural solubilizer of insoluble drugs and an active targeting carrier in intractable tumors with low vascular permeability, such as pancreatic tumors. In conclusion, HSA-d can be an effective drug carrier for the antitumor drug delivery system against human pancreatic tumors.

Quality of benzathine penicillin G: A multinational cross-sectional study.

Benzathine penicillin G (BPG) is used as first-line treatment for most forms of syphilis and as secondary prophylaxis against rheumatic heart disease (RHD). Perceptions that poor quality of BPG is linked to reported adverse effects and therapeutic failure may impact syphilis and RHD control programs. Clinical networks and web-based advertising were used to obtain vials of BPG from a wide range of countries. The quality of BPG was assessed using a high performance liquid chromatography assay capable of detecting relevant impurities and degradation products. Tests for water content, presence of heavy metals and physical characteristics of BPG, including particle size analysis and optical microscopy, also were conducted. Thirty-five batches of BPG were sourced from 16 countries across 4 WHO regions. All batches passed the US Pharmacopeia requirements for BPG injection (content), with no evidence of breakdown products or other detected contaminants. Water content and heavy metal analysis (n = 11) indicated adherence to regulatory standards and Good Manufacturing Practice. Particle size analysis (n = 20) found two batches with aggregated particles (>400 µm) that were dispersed following sonication. Current batches of BPG were of satisfactory pharmaceutical quality but aggregated particles were found in a modest proportion of samples. Future studies should focus on the physical characteristics of BPG which may contribute to variations in plasma penicillin concentrations an observed needle blockages in clinical practice. Pharmacopeial monographs could be revised to include standards on particle size and crystal morphology of BPG.

Ergometrine stability in postpartum haemorrhage kits: Does temperature and light matter?

BACKGROUND: Postpartum haemorrhage (PPH) kits containing uterotonics are used on obstetric units for the timely management of PPH. Visible discolouration of ergometrine and ergometrine-oxytocin injections was observed in PPH kits stored in medical refrigerators on the obstetric unit at our hospital. AIM: To investigate the stability of ergometrine and ergometrine-oxytocin injections in PPH kits under simulated clinical storage conditions and to determine the potency of ampoules quarantined from PPH kits on our obstetric unit. MATERIAL AND METHODS: Ergometrine and ergometrine-oxytocin injection ampoules were stored exposed to and protected from light at 4°C and room temperature (25°C) for up to three months, and assayed by high-performance liquid chromatography. Stability was based on the time for the ergometrine or oxytocin concentration to fall to 90% of the original concentration (t90 ). The potency of quarantined discoloured ampoules also was determined. RESULTS: Ergometrine was stable at both temperatures for >6 months, when stored protected from light in simulated clinical conditions. When exposed to light, ergometrine was stable for approximately 4 days at 25°C and 10 days at 4°C. Discoloured ergometrine and ergometrine-oxytocin injection ampoules were found to be <90% of the nominal concentration. CONCLUSION: Stability of ergometrine in PPH kits is largely unaffected by temperature fluctuations (at 4°C and 25°C) over 6 months when protected from light. Ergometrine and ergometrine-oxytocin ampoules should be inspected prior to use and any discoloured ampoules discarded.

Distribution of Polysulfide in Human Biological Fluids and Their Association with Amylase and Sperm Activities.

Intracellular polysulfide could regulate the redox balance via its anti-oxidant activity. However, the existence of polysulfide in biological fluids still remains unknown. Recently, we developed a quantitative analytical method for polysulfide and discovered that polysulfide exists in plasma and responds to oxidative stress. In this study, we confirmed the presence of polysulfide in other biological fluids, such as semen and nasal discharge. The levels of polysulfide in these biological fluids from healthy volunteers (n = 9) with identical characteristics were compared. Additionally, the circadian rhythm of plasma polysulfide was also investigated. The polysulfide levels detected from nasal discharge and seminal fluid were approximately 400 and 600 µM, respectively. No correlation could be found between plasma polysulfide and the polysulfide levels of tear, saliva, and nasal discharge. On the other hand, seminal polysulfide was positively correlated with plasma polysulfide, and almost all polysulfide contained in semen was found in seminal fluid. Intriguingly, saliva and seminal polysulfide strongly correlated with salivary amylase and sperm activities, respectively. These results provide a foundation for scientific breakthroughs in various research areas like infertility and the digestive system process.

S-Nitrosated alpha-1-acid glycoprotein exhibits antibacterial activity against multidrug-resistant bacteria strains and synergistically enhances the effect of antibiotics.

Alpha-1-acid glycoprotein (AGP) is a major acute-phase protein. Biosynthesis of AGP increases markedly during inflammation and infection, similar to nitric oxide (NO) biosynthesis. AGP variant A (AGP) contains a reduced cysteine (Cys149). Previously, we reported that S-nitrosated AGP (SNO-AGP) synthesized by reaction with a NO donor, possessed very strong broad-spectrum antimicrobial activity (IC50 = 10-9-10-6 M). In this study, using a cecal ligation and puncture animal model, we confirmed that AGP can be endogenously S-nitrosated during infection. Furthermore, we examined the antibacterial property of SNO-AGP against multidrug-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa to investigate the involvement of SNO-AGP in the host defense system. Our results showed that SNO-AGP could inhibit multidrug efflux pump, AcrAB-TolC, a major contributor to bacterial multidrug resistance. In addition, SNO-AGP decreased biofilm formation and ATP level in bacteria, indicating that SNO-AGP can revert drug resistance. It was also noteworthy that SNO-AGP showed synergistic effects with the existing antibiotics (oxacillin, imipenem, norfloxacin, erythromycin, and tetracycline). In conclusion, SNO-AGP participated in the host defense system and has potential as a novel agent for single or combination antimicrobial therapy.

Thioredoxin-albumin fusion protein prevents copper enhanced zinc-induced neurotoxicity via its antioxidative activity.

Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer's disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu2+/Zn2+-induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu2+/Zn2+-induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu2+/Zn2+-induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu2+ and Zn2+ after Cu2+/Zn2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu2+/Zn2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu2+/Zn2+-induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases.

A novel S-sulfhydrated human serum albumin preparation suppresses melanin synthesis.

Products of ultraviolet (UV) irradiation such as reactive oxygen species (ROS) and nitric oxide (NO) stimulate melanin synthesis. Reactive sulfur species (RSS) have been shown to have strong ROS and NO scavenging effects. However, the instability and low retention of RSS limit their use as inhibitors of melanin synthesis. The free thiol at Cys34 on human serum albumin (HSA) is highly stable, has a long retention and possess a high reactivity for RSS. We report herein on the development of an HSA based RSS delivery system. Sulfane sulfur derivatives released from sodium polysulfides (Na2Sn) react readily with HSA. An assay for estimating the elimination of sulfide from polysulfide showed that almost all of the sulfur released from Na2Sn bound to HSA. The Na2Sn-treated HSA was found to efficiently scavenge ROS and NO produced from chemical reagents. The Na2Sn-treated HSA was also found to inhibit melanin synthesis in B16 melanoma cells and this inhibition was independent of the number of added sulfur atoms. In B16 melanoma cells, the Na2Sn-treated HSA also inhibited the levels of ROS and NO induced by UV radiation. Finally, the Na2Sn-treated HSA inhibited melanin synthesis from L-DOPA and mushroom tyrosinase and suppressed the extent of aggregation of melanin pigments. These data suggest that Na2Sn-treated HSA inhibits tyrosinase activity for melanin synthesis via two pathways; by directly inhibiting ROS signaling and by scavenging NO. These findings indicate that Na2Sn-treated HSA has potential to be an attractive and effective candidate for use as a skin whitening agent.

Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albumin-protein interactions using S-nitrosated human serum albumin dimer.

In the latest trend of anticancer chemotherapy research, there were many macromolecular anticancer drugs developed based on enhanced permeability and retention (EPR) effect, such as albumin bound paclitaxel nanoparticle (nab- PTX, also called Abraxane®). However, cancers with low vascular permeability posed a challenge for these EPR based therapeutic systems. Augmenting the intrinsic EPR effect with an intrinsic vascular modulator such as nitric oxide (NO) could be a promising strategy. S-nitrosated human serum albumin dimer (SNO-HSA Dimer) shown promising activity previously was evaluated for the synergistic effect when used as a pretreatment agent in nab-PTX therapy against various tumor models. In the high vascular permeability C26 murine colon cancer subcutaneous inoculation model, SNO-HSA Dimer enhanced tumor selectivity of nab-PTX, and attenuated myelosuppression. SNO-HSA Dimer also augmented the tumor growth inhibition of nab-PTX in low vascular permeability B16 murine melanoma subcutaneous inoculation model. Furthermore, nab-PTX therapy combined with SNO-HSA Dimer showed higher antitumor activity and improved survival rate of SUIT2 human pancreatic cancer orthotopic model. In conclusion, SNO-HSA Dimer could enhance the therapeutic effect of nab-PTX even in low vascular permeability or intractable pancreatic cancers. The possible underlying mechanisms of action of SNO-HSA Dimer were discussed.

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.

Apoptosis induction of poly-S-nitrosated human serum albumin in resistant solid tumor under hypoxia can be restored by phosphodiesterase 5 inhibition.

Poly-S-nitrosated human serum albumin (Poly-SNO-HSA) delivered and accumulated nitric oxide (NO) in tumors and induces apoptosis. Tumor hypoxia is strongly associated with malignant progression and tumor resistance to therapy. In this study, we examined the cytotoxic effect of Poly-SNO-HSA under hypoxia on the murine colon 26 adenocarcinoma (C26) cells in vitro and in vivo. Under hypoxia, at about 4 times LD50 dose of Poly-SNO-HSA in vitro, the reactive oxygen species production was hindered but apoptotic cells were induced via cGMP pathway as the effect was suppressed by a soluble guanylate cyclase inhibitor, NS2028. The apoptosis induction effect of low dose Poly-SNO-HSA on C26 cells in vitro under hypoxia can be restored by a phosphodiesterase 5 (PDE5) inhibitor, vardenafil. In C26-bearing mice, Poly-SNO-HSA/vardenafil combination treatment significantly suppressed the tumor volume compared with Poly-SNO-HSA or vardenafil treatment alone. Furthermore, the core tumor tissues showed increased expression of caspase-3 than the non-core tissue. The expression of caspase-3 appeared to overlap with the hypoxic zone of tumor tissues. Similar results were also obtained when the experiments were repeated using Epimedium extract, a natural herbal supplement with PDE5 inhibition activity. In conclusion, Poly-SNO-HSA/PDE5 inhibitors combination therapy is a promising approach for enhancing the anticancer therapeutic effects of Poly-SNO-HSA against not only anti-cancer drug resistance but also hypoxic stress related solid tumor resistance.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 µM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease.

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.

S-Nitrosated human serum albumin dimer as novel nano-EPR enhancer applied to macromolecular anti-tumor drugs such as micelles and liposomes.

The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3-4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.

Pleiotropic Effects of Levofloxacin, Fluoroquinolone Antibiotics, against Influenza Virus-Induced Lung Injury.

Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-γ in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs.

Therapeutic impact of human serum albumin-thioredoxin fusion protein on influenza virus-induced lung injury mice.

Reactive oxygen species (ROS) are the primary pathogenic molecules produced in viral lung infections. We previously reported on the use of a recombinant human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) for extending the half-life Trx, an endogenous protein with anti-oxidant properties. As a result, it was possible to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx. We hypothesized that HSA-Trx would attenuate the enhanced ROS production of species such as hydroxyl radicals by neutrophils during an influenza viral infection. The levels of 8-hydroxy-2'-deoxyguanosine and 3-nitrotyrosine were used as indices of the anti-oxidant activity of HSA-Trx. In addition, the cytoprotective effects of HSA-Trx were examined in PR8 (H1N1) influenza virus-induced lung injured mice. The findings show that HSA-Trx reduced the number of total cells, neutrophils, and total protein in BALF of influenza virus-induced lung injured mice. The HSA-Trx treatment significantly decreased the level of 8-hydroxy-2'-deoxyguanosine and 3-nitrotyrosine, but failed to inhibit inducible nitric oxide synthase expression, in the lungs of the virus-infected mice. On the other hand, Tamiflu(®) treatment also significantly suppressed the production of inflammatory cells and neutrophil infiltration, as well as the protein level in BALF and lung histopathological alterations caused by the influenza virus. The suppressive effect of Tamiflu(®) was slightly stronger than that of HSA-Trx. Interestingly, Tamiflu(®) significantly decreased virus proliferation, while HSA-Trx had no effect. These results indicate that HSA-Trx may be of therapeutic value for the treatment of various acute inflammatory disorders such as influenza-virus-induced pneumonia, by inhibiting inflammatory-cell responses and suppressing the overproduction of NO in the lung.

A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy.

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin-Trx fusion protein (HSA-Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA-Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N-acetyl-ß-D-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA-Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA-Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA-Trx has the potential to effectively prevent CIN.

Albumin domain II mutant with high bilirubin binding affinity has a great potential as serum bilirubin excretion enhancer for hyperbilirubinemia treatment.

BACKGROUND: 4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function. METHODS: Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice. RESULTS: After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment. CONCLUSIONS: These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia. GENERAL SIGNIFICANCE: This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.