Search

Background: Nacubactam (NAC), a new diazabicyclooctane β-lactamase inhibitor, is being developed for use together with aztreonam (AZT) and cefepime (CFPM). However, the effective clinical dosages of AZT/NAC and CFPM/NAC have not yet been established. We have previously shown that free time above instantaneous MIC (fT > MICi) is a valuable pharmacokinetic (PK)/pharmacodynamic parameter for β-lactam (BL)/NAC in a mouse thigh infection model., Objectives: This study simulated the fT > MICi (%) for AZT/NAC and CFPM/NAC against carbapenemase-producing Enterobacterales (CPE) with different MIC in humans to estimate the clinical efficacy at practically achievable combination doses of AZT/NAC and CFPM/NAC., Methods: Using previously reported PK parameters of each drug in humans and chequerboard MIC data, we calculated the fT > MICi (%) for AZT/NAC and CFPM/NAC in 10 000 simulated patients to predict the percentages of target attainment of bacteriostatic and bactericidal efficacies at various combined doses., Results: The results predicted that both BL/NAC combinations could achieve 100% 2 log10-kill against CPE strains at the lowest combination dose (0.5 g/0.5 g q8h). Additionally, in MIC studies examining BLs/NAC at a 1:1 ratio, the dosage regimen for strains with MICcomb ≤ 1 mg/L was expected to offer 100% bactericidal efficacy (2 log10-kill) at 0.5 g/0.5 g q8h or higher doses. For strains with 1 mg/L < MICcomb ≤ 16 mg/L, BLs/NAC at a 2 g/2 g q8h was predicted to produce bactericidal efficacy (1 log10-kill). At MICcomb = 32 mg/L, some bacteriostatic effect was expected at high BL doses., Conclusions: AZT/NAC and CFPM/NAC are bactericidal against CPE at practically achievable dosages., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive form of nonalcoholic fatty liver disease characterised by fat accumulation, inflammation, oxidative stress, fibrosis, and impaired liver regeneration. In this study, we found that heme oxygenase-1 (HO-1) is induced in both MASH patients and in a MASH mouse model. Further, hepatic carbon monoxide (CO) levels in MASH model mice were >2-fold higher than in healthy mice, suggesting that liver HO-1 is activated as MASH progresses. Based on these findings, we used CO-loaded red blood cells (CO-RBCs) as a CO donor in the liver, and evaluated their therapeutic effect in methionine-choline deficient diet (MCDD)-induced and high-fat-diet (HFD)-induced MASH model mice. Intravenously administered CO-RBCs effectively delivered CO to the MASH liver, where they prevented fat accumulation by promoting fatty acid oxidation via AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor induction. They also markedly suppressed Kupffer cell activation and their corresponding anti-inflammatory and antioxidative stress activities in MASH mice. CO-RBCs also helped to restore liver regeneration in mice with HFD-induced MASH by activating AMPK. We confirmed the underlying mechanisms by performing in vitro experiments in RAW264.7 cells and palmitate-stimulated HepG2 cells. Taken together, CO-RBCs show potential as a promising cellular treatment for MASH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Skeletal muscle atrophy is a known risk factor for immunosuppressive conditions and for a poor prognosis in sepsis. However, its immunopathology has not been experimentally elucidated. This study investigated the effects of skeletal muscle atrophy on the immunopathology of sepsis. Male C57BL/6J mice were subjected to sciatic denervation (DN) and caecal ligation and puncture (CLP) to induce muscle atrophy or sepsis. The macrophages, myeloid-derived suppressor cells (MDSC), and T-cells in peritoneal and spleen were analysed using flow cytometry. DN-induced muscle atrophy did not affect macrophage and MDSC populations. In contrast, the percentage of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 + CD4 + T-cells, programmed death (PD)-1 + CD8 + T-cells, regulatory T-cells, and the CTLA-4 + regulatory T-cells was statistically significantly higher in DN-CLP mice than in sham-CLP mice. Skeletal muscle atrophy before sepsis triggers excessive T cell immunosuppression, which may contribute to the exacerbation of sepsis under skeletal muscle atrophy., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Sarcopenia is characterized by loss of muscle strength and muscle mass with aging. The growing number of sarcopenia patients as a result of the aging population has no viable treatment. Exercise maintains muscle strength and mass by increasing peroxisome growth factor activating receptor γ-conjugating factor-1α (PGC-1α) and Akt signaling in skeletal muscle. The present study focused on the carbon monoxide (CO), endogenous activator of PGC-1α and Akt, and investigated the therapeutic potential of CO-loaded red blood cells (CO-RBCs), which is bioinspired from in vivo CO delivery system, as an exercise mimetic for the treatment of sarcopenia. Treatment of C2C12 myoblasts with the CO-donor increased the protein levels of PGC-1α which enhanced mitochondrial biogenesis and energy production. The CO-donor treatment also activated Akt, indicating that CO promotes muscle synthesis. CO levels were significantly elevated in the skeletal muscle of normal mice after intravenous administration of CO-RBCs. Furthermore, CO-RBCs restored the mRNA expression levels of PGC-1α in the skeletal muscle of two experimental sarcopenia mouse models, denervated (Den) and hindlimb unloading (HU) models. CO-RBCs also restored muscle mass in Den mice by activating Akt signaling and suppressing the muscle atrophy factors myostatin and atrogin-1, and oxidative stress. Treadmill tests further showed that the reduced running distance in HU mice was significantly restored by CO-RBC administration. These findings suggest that CO-RBCs have potential as an exercise mimetic for sarcopenia treatment., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Urticaria is induced by the histamine released from mast cells which develops wheals (edema) as a visual feature. In clinical practice, second-generation histamine H 1 -receptor blockers are routinely used as the first-line symptomatic treatment for urticaria. Nevertheless, not much research has directly examined the second-generation histamine H 1 -receptor blockers' ability to reduce edema. In this study, we directly evaluated the anti-edematous activities of three second-generation histamine H 1 -receptor blockers available in the market (epinastine hydrochloride, cetirizine hydrochloride, and levocetirizine hydrochloride) using a λ-carrageenan-induced footpad edema model. One hour before the induction of edema with 1% λ -carrageenan injection, all second-generation histamine H 1 -receptor blockers (5, 10, 50 and 100 mg/kg) were subcutaneously administered to rats. At 0.5 and 3 hours after λ -carrageenan administration, the edema volume was evaluated using a Plethysmometer. Epinastine hydrochloride significantly suppressed the edema growth in a dose-dependent manner. Cetirizine hydrochloride showed a slight anti-edematous effect, while levocetirizine significantly inhibited the development of edema in a dose-dependent manner. On the other hand, dextrocetirizine did not prevent edema from growing. In summary, second-generation histamine H 1 -receptor blockers, at least those examined in this study, may be able to reduce the clinical symptoms of urticaria associated with edema. Levocetirizine hydrochloride is also anticipated to have stronger anti-edematous effects than cetirizine hydrochloride because levocetirizine is responsible for cetirizine's anti-edematous activity.

Some macrolide antibiotics, which share a basic lactone ring structure, also exhibit anti-inflammatory actions in addition to their antibacterial activities. However, no study has directly compared anti-inflammatory effects on acute inflammation among macrolide antibiotics with the distinct size of the lactone ring. In this study, we evaluated and compared the anti-inflammatory activities of four 14-membered macrolides (erythromycin, clarithromycin, roxithromycin, oleandomycin), one 15-membered macrolide (azithromycin), and three 16-membered macrolides (midecamycin, josamycin, leucomycin) using a rat carrageenan-induced footpad edema model. All macrolide antibiotics were intraperitoneally administered to rats one hour before the induction of inflammatory edema with 1% λ -carrageenan. The anti-inflammatory effects on acute inflammation were evaluated by changing the edema volume. All 14-membered and 15-membered macrolide antibiotics significantly suppressed the development of edema. Conversely, none of the 16-membered macrolide antibiotics inhibited the growth of edema. In conclusion, compared to 16-membered macrolide antibiotics, 14-membered and 15-membered macrolide antibiotics have stronger anti-inflammatory effects. Further research should be done to determine why different lactone ring sizes should have distinct anti-inflammatory effects.

Objectives: In recent years, Vancomycin (VCM) dosing design using area under the concentration-time curve (AUC) has been recommended as a measure of efficacy and safety, but there are fewer reports on pediatric patients than on adults. In this study, we evaluated the threshold of AUC for AKI occurrence in pediatric patients and investigated the factors that contribute to the occurrence of AKI., Methods: Pediatric patients aged 1-15 years on VCM treatment who underwent TDM at Kagoshima University Hospital from April 2016 to March 2022 were included in the computation of AUC using pediatric population pharmacokinetic parameters., Results: The ROC curve showed that the AUC threshold for the risk of developing AKI was 583.0 μg・h/mL, and the AUC-ROC curve was 0.873 (sensitivity 0.930, specificity 0.750). Univariate analysis showed that factors associated with AKI incidence were the duration of VCM administration, ICU admission, and AUC SS . Concomitant medications identified as risk factors for AKI incidence were tazobactam/piperacillin, liposomal amphotericin B, calcineurin inhibitors, contrast agents, and H 2 -receptor blockers. The multivariate analysis showed that AUC ≧ 583.0 μg・h/mL (odds ratio 20.14, 95% CI 3.52-115.22, p < 0.001) and H 2 -receptor blockers (odds ratio 8.70, 95% confidence interval = 1.38-54.87, p = 0.02) were independent factors for AKI incidence., Conclusions: We showed that in pediatric patients receiving VCM, the risk of AKI increases as AUC increases. The findings imply that concurrent use of VCM and H 2 -receptor blockers may increase the risk of AKI., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kazuaki Matsumoto reports a relationship with Meiji Seika Pharma Co., Ltd that includes: funding grants. Kazuaki Matsumoto reports a relationship with Sumitomo Pharma Co., Ltd that includes: funding grants. Kazuaki Matsumoto reports a relationship with Meiji Seika Pharma Co., Ltd that includes: speaking and lecture fees. co-author is the editor of Journal of Infection and Chemotherapy – Kazuaki Matsumoto., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Tedizolid (TZD) is an oxazolidinone anti-methicillin-resistant Staphylococcus aureus (MRSA) drug. Linezolid (LZD), another oxazolidinone, has been shown to have an anti-inflammatory effect. TZD has been shown to exhibit an anti-inflammatory effect in a murine model of hematogenous pulmonary infection. In this study, we further investigated the anti-inflammatory effect of TZDs using a carrageenan-induced rat footpad edema model. TZD was administered at 0, 10, 20, and 40 mg/kg to the carrageenan-induced rat footpad edema model, and the edema rate was measured over time up to 9 h later. The area under the time curve of the edema rate profile (AUC edema0→9 ) decreased in a TZD dose-dependent manner. In addition, the correlation between AUC edema0→9 and the area under the time curve of free TZD plasma concentration (fAUC blood ) obtained from the pharmacokinetic study of TZD in the carrageenan-induced rat footpad edema model was examined. fAUC blood and AUC edema0→9 showed a good negative correlation. These results indicate that TZD suppresses carrageenan-induced footpad edema and that TZD exerts its anti-inflammatory effects in a plasma concentration-dependent manner., Competing Interests: Declaration of competing interest Kazuaki Matsumoto received grant support funding from Meiji Seika Pharma Co., Ltd., Sumitomo Pharma Co., Ltd., and Shionogi & Co., Ltd., and speaker honoraria from Meiji Seika Pharma Co., Ltd., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Competing interests: None declared., (© European Association of Hospital Pharmacists 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Nacubactam (NAC) is a novel diazabicyclooctane β-lactamase inhibitor used in combination with cefepime (CFPM). In this study, we aimed to determine the target pharmacokinetics (PK) and pharmacodynamics (PD) values of CFPM/NAC in mice infected with β-lactamase-producing Enterobacterales, such as the carbapenemase-producing Enterobacterales., Methods: Three strains of β-lactamase-producing Enterobacterales, Klebsiella pneumoniae MSC 21444, Escherichia coli MSC 20662, and K. pneumoniae ATCC BAA-1898, were used for checkerboard assays and fractionation studies and dose-range studies. A PK study was performed in neutropenic mice. Additionally, PK/PD analysis was performed based on the instantaneous minimum inhibitory concentration (MIC i ) concept., Results: Checkerboard measurements revealed that higher NAC concentrations decreased the CFPM MIC in a concentration-dependent manner. In all tested strains, fT > MIC i calculated from the PK experiments showed a high correlation with the mean change in the bacterial count of thigh-infected mice in the in vivo PD study, suggesting that fT > MIC i is an optimal PK/PD parameter for monitoring the CFPM/NAC combination. The target fT > MIC i values for CFPM/NAC to achieve a bacteriostatic effect, 1-log 10 -kill, and 2-log 10 -kill values were 30, 49, and 94%, respectively., Conclusions: Our results indicate that fT > MIC i is a PK/PD parameter is suitable for monitoring the CFPM/NAC combination. The minimum target value for achieving a static effect against β-lactamase-producing Enterobacterales is 30%., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Nacubactam, a new β-lactamase inhibitor with antibacterial activity, is being developed as a single drug to be co-administered with cefepime or aztreonam. However, determining pharmacokinetics/pharmacodynamics (PK/PD) parameters in β-lactam/β-lactamase inhibitor combinations remains challenging. We aimed to establish a practical PK/PD analysis method for aztreonam/nacubactam that incorporates instantaneous MIC (MICi)., Methods: Based on chequerboard MIC measurements, MICi of aztreonam against carbapenemase-producing Klebsiella pneumoniae in the presence of nacubactam was simulated., Results: The mean change in the bacterial count of thigh-infected mice in an in vivo PD study was plotted based on %fT>MICi and analysed using the inhibitory effect sigmoid Imax model. fT>MICi calculated from the PK experiments showed a high correlation with the in vivo bactericidal effect, suggesting that fT>MICi is the optimal PK/PD parameter for aztreonam/nacubactam. The target values of fT>MICi achieving growth inhibition, 1 log10 kill and 2 log10 kill, were 22, 38% and 75%, respectively., Conclusions: The PK/PD analysis method proposed in this study is promising for determining practical PK/PD parameters in combination therapy. In addition, this is the first report of aztreonam/nacubactam showing a potent in vivo therapeutic effect against NDM-producing K. pneumoniae., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Biologic medications have dramatically improved the treatment outcomes of immunological inflammatory diseases, but their immunosuppressive effects put patients at risk for tuberculosis (TB). We investigated the risk factors for developing TB in patients treated for latent tuberculosis infection (LTBI) who also had experience of using biologic medications. At Keio University Hospital, we retrospectively investigated patients treated with anti-mycobacterial drugs before or concurrently with biologic medications from January 2012 to August 2020. Patients in the 'follow-on cases group' who had a positive TB screening test after initiating biologic medications and subsequently started LTBI treatment were excluded. We researched and compared the patient characteristics for TB and non-TB patient groups. Of the 146 eligible patients, 5 (3.4%) developed TB. The incidence rate was 600/100000 person-years. There were no significant differences between TB and non-TB patient groups in the history of TB, interferon-gamma release assay (IGRA), duration of biologic medication therapy, LTBI treatment periods, concomitant use of calcineurin inhibitors or anti-rheumatic drugs. The percentage of patients who received prednisolone at a dose of ≥15 mg for more than 1 month was higher in those who developed TB than in those who did not (40.0 vs. 7.1%, p = 0.054); however, this difference was not statistically significant. Regular monitoring of TB is necessary for long-term concomitant use of high prednisolone doses during and after the administration of biologic medications.

Aripiprazole (ARP), an antipsychotic drug, binds more strongly to human serum albumin (HSA) than the other ARP derivatives. In addition, the signs for the extrinsic Cotton effects for HSA complexed with ARP or deschloro-ARP are reversed. In this study, we report on a structural-chemical approach using circular dichroism (CD) spectroscopic analysis, X-ray crystallographic analysis, and molecular dynamics simulations. The objective was to examine the relationship between the induced CD spectra and the structural features of the HSA complexes with ARP or deschloro-ARP. The intensity of the induced CD spectra of the HSA complexes with ARP or deschloro-ARP was reduced with increasing temperature. We determined the crystal structure of the HSA complexed with deschloro-ARP in this study and compared it to HSA complexed with ARP that we reported previously. The comparison of these structures revealed that both ARP and deschloro-ARP were bound at the site II pocket in HSA and that the orientation of the molecules was nearly identical. Molecular dynamics simulations indicated that the molecular motions of ARP and deschloro-ARP within the site II pocket were different from one another and the proportion of stacking interaction formations of Tyr411 with the dihydroquinoline rings of ARP and deschloro-ARP was also different. These findings indicate that the induced CD spectra are related to the molecular motions and dynamic interactions of ARP and deschloro-ARP in HSA and may help to understand the molecular recognition and motion that occurs within the binding site for the other HSA ligands more clearly., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The effects of myristate on the induced circular dichroism spectra of aripiprazole (ARP) bound to human serum albumin (HSA) were investigated. High concentrations of myristate reversed the Cotton effects induced in the ARP-HSA system. The observed ellipticities increased with increasing drug concentration up to an ARP-to-HSA molar ratio of 1:1 and then decreased, indicating that the extrinsic Cotton effects were generated by the binding of ARP molecules to the high- and low-affinity sites in HSA. The data for the concentration of free ARP show that myristate displaces ARP molecules from HSA. Moreover, the free fractions of ARP in the ARP-HSA-myristate system increased significantly when adding fusidic acid, a subdomain IB ligand. In the crystal structure of the ARP-HSA-myristate ternary complex, one ARP molecule is bound to subdomain IB, and the interaction between the carbonyl group of ARP and the aromatic ring of Tyr138 in subdomain IB is essential for binding to occur. Meanwhile, the ARP molecule in the ARP-HSA binary complex structure is bound only to subdomain IIIA. Consequently, the inversion in the extrinsic Cotton effects in the ARP-HSA system can be attributed to the modification of the geometry within the binding pocket, in addition to the transfer of ARP from subdomain IIIA to subdomain IB through the displacement as a result of the binding of myristate to subdomain IIIA., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

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.

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-OCH 3 ). 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-OCH 3 . 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-OCH 3 . On the other hand, although the secretion of anti-PEG IgM induced by PL-MAL was lower than PL-OCH 3 , 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 binding of drugs to plasma protein is frequently altered in certain types of renal diseases. We recently reported on the effects of oxidation and uremic toxins on the binding of aripiprazole (ARP) to human serum albumin. In our continuing investigations, we examined the binding of ARP to plasma pooled from patients with chronic renal dysfunction. We examined the issue of the molecular basis for which factors affect the changes in drug binding that accompany renal failure. The study was based on the statistical relationships between ARP albumin binding and biochemical parameters such as the concentrations of oxidized albumin and uremic toxins. The binding of ARP to plasma from chronic renal patients was significantly lower than healthy volunteers. A rational relationship between the ARP binding rate and the concentration of toxins, including indoxyl sulphate (IS) and p-cresyl sulphate (PCS), was found, particularly for IS. Moreover, multiple regression analyses that involved taking other parameters such as PCS or oxidized albumin ratio to IS into account supports the above hypothesis. In conclusion, the limited data reported in this present study indicates that monitoring IS in the blood is a very important determinant in the dosage plan for the administration of site II drugs such as ARP, if the efficacy of the drug in renal disease is to be considered.

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.

Many macromolecular antitumor drugs were developed based on the enhanced permeability and retention (EPR) effect, for example, albumin-bound paclitaxel nanoparticles (nab-PTX and Abraxane) and pegylated liposomal doxorubicin (Doxil). However, these EPR effect-based therapeutic systems are less effective in malignant tumors with low vascular permeability, such as pancreatic tumors. Because the EPR effect depends on nanoparticles' size, we first determined nanoparticles' size associated with a high tumor-targeting rate in a human pancreatic tumor xenograft model with low vascular permeability. Abraxane appears to behave as an albumin monomer (7 nm) in the blood circulation following intravenous injection. The in vitro and in vivo tumor-targeted delivery and antitumor activity of PTX-loaded albumin nanoparticles were significantly improved by optimizing the mean nanoparticle diameter to 30 nm. Furthermore, nitric oxide was added to 30 nm PTX-loaded albumin nanoparticles to examine the feasibility of albumin nanoparticles as a platform for multiple drug delivery. Their antitumor effect was evaluated in an orthotopic transplantation mouse model of a human pancreatic tumor. The nitric oxide PTX-loaded 30 nm albumin nanoparticle treatment on model mice achieved a significantly higher survival rate than Abraxane treatment. These findings suggest that 30 nm albumin nanoparticles have a high therapeutic effect as a useful platform for multiple drugs against human pancreatic tumors.

Abraxane, an albumin-bound paclitaxel nanoparticle formulation, is superior to conventional paclitaxel preparations because it has better efficacy against unresectable pancreatic cancer. Previous reports suggest that this better efficacy of Abraxane than conventional paclitaxel preparation is probably due to its transport through Gp60, an albumin receptor on the surface of vascular endothelial cells. The increased tumor accumulation of Abraxane is also caused by the secreted protein acid and rich in cysteine in the tumor stroma. However, the uptake mechanism of Abraxane remains poorly understood. In this study, we demonstrated that the delivery of Abraxane occurred via different receptor pathways from that of endogenous albumin. Our results showed that the uptake of endogenous albumin was inhibited by a Gp60 pathway inhibitor in the process of endocytosis through endothelial cells or tumor cells. In contrast, the uptake of Abraxane-derived HSA was less affected by the Gp60 pathway inhibitor but significantly reduced by denatured albumin receptor inhibitors. In conclusion, these data indicate that Abraxane-derived HSA was taken up into endothelial cells or tumor cells by a mechanism different from normal endogenous albumin. These new data on distinct cellular transport pathways of denatured albumin via gp family proteins different from those of innate albumin shed light on the mechanisms of tumor delivery and antitumor activity of Abraxane and provide new scientific rationale for the development of a novel albumin drug delivery strategy via a denatured albumin receptor.

Albumin, the most abundant protein in plasma, possesses some inherent beneficial structural and physiological characteristics that make it suitable for use as a drug delivery agent, such as an extraordinary drug-binding capacity and long blood retention, with a high biocompatibility. The use of these characteristics as a nanoparticle drug delivery system (DDS) offers several advantages, including a longer circulation time, lower toxicity, and more significant drug loading. To date, many innovative liposome preparations have been developed in which albumin is involved as a DDS. These novel albumin-containing liposome preparations show superior deliverability for genes, hydrophilic/hydrophobic substances and proteins/peptides to the targeting area compared to original liposomes by virtue of their high biocompatibility, stability, effective loading content, and the capacity for targeting. This review summarizes the current status of albumin applications in liposome-based DDS, focusing on albumin-coated liposomes and albumin-encapsulated liposomes as a DDS carrier for potential medical applications.

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., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

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 (t 90 ). 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., (© 2019 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

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.

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.

We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg) 12 . While it is possible that the palmitoyl-cyclic-(D-Arg) 12 /HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg) 12 /HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg) 12 /HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg) 12 /HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg) 12 /HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg) 12 /HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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 (IC 50  = 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., Competing Interests: Authors report no disclosures., (© 2018 The Authors.)

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.

Oxidative stress induced by hyperuricemia is closely associated with the renin-angiotensin system, as well as the onset and progression of cardiovascular disease (CVD) and chronic kidney disease (CKD). It is therefore important to reduce oxidative stress to treat hyperuricemia. We previously found that benzbromarone, a uricosuric agent, has a direct free radical scavenging effect in vitro . The antioxidant effects of benzbromarone were evaluated in vivo via oral administration of benzbromarone for 4 weeks to model rats with angiotensin II- and salt-induced hypertension. Benzbromarone did not alter plasma uric acid levels or blood pressure but significantly reduced the levels of advanced oxidation protein products, which are oxidative stress markers. Furthermore, dihydroethidium staining of the kidney revealed a reduction in oxidative stress after benzbromarone administration. These results suggest that benzbromarone has a direct antioxidant effect in vivo and great potential to prevent CVD and CKD.

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg) 12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg) 12 /HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications., (Copyright © 2018. Published by Elsevier B.V.)

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 (Na 2 S n ) react readily with HSA. An assay for estimating the elimination of sulfide from polysulfide showed that almost all of the sulfur released from Na 2 S n bound to HSA. The Na 2 S n -treated HSA was found to efficiently scavenge ROS and NO produced from chemical reagents. The Na 2 S n -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 Na 2 S n -treated HSA also inhibited the levels of ROS and NO induced by UV radiation. Finally, the Na 2 S n -treated HSA inhibited melanin synthesis from L-DOPA and mushroom tyrosinase and suppressed the extent of aggregation of melanin pigments. These data suggest that Na 2 S n -treated HSA inhibits tyrosinase activity for melanin synthesis via two pathways; by directly inhibiting ROS signaling and by scavenging NO. These findings indicate that Na 2 S n -treated HSA has potential to be an attractive and effective candidate for use as a skin whitening agent., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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 Cu 2+ /Zn 2+ -induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu 2+ /Zn 2+ -induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu 2+ /Zn 2+ -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 Cu 2+ and Zn 2+ after Cu 2+ /Zn 2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu 2+ /Zn 2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu 2+ /Zn 2+ -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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 LD 50 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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

Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') 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, α 1 -anti-trypsin, α 1 -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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 Na 2 S. 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., (Copyright © 2016 Elsevier Inc. All rights reserved.)