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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.

The characteristic accumulation of circulating uremic toxins, such as indoxyl sulfate (IS), in chronic kidney disease (CKD) further exacerbates the disease progression. The gut microbiota, particularly gut bacterial-specific enzymes, represents a selective and attractive target for suppressing uremic toxin production and slowing the progression of renal failure. This study investigates the role of 4-phenylbutyrate (PB) and structurally related compounds, which are speculated to possess renoprotective properties in suppressing IS production and slowing or reversing renal failure in CKD. In vitro enzyme kinetic studies showed that 7-phenylheptanoic acid (PH), a PB homologue, suppresses the tryptophan indole lyase (TIL)-catalyzed decomposition of tryptophan to indole, the precursor of IS. A hydroxypropyl β-cyclodextrin (HPβCD) inclusion complex formulation of PH was prepared to enhance its biopharmaceutical properties and to facilitate in vivo evaluation. Prophylactic oral administration of the PH-HPβCD complex formulation reduced circulating IS and attenuated the deterioration of renal function and tubulointerstitial fibrosis in adenine-induced CKD mice. Additionally, treatment of moderately advanced adenine-induced CKD mice with the formulation ameliorated renal failure, although tissue fibrosis was not improved. These findings suggest that PH-HPβCD can slow the progression of renal failure and may have implications for preventing or managing CKD, particularly in early-stage disease.

Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-β, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.

Despite major improvements brought about by the introduction of taste-masked formulations of 4-phenylbutyrate (PB), poor compliance remains a significant drawback to treatment for some pediatric and dysphagic patients with urea cycle disorders (UCDs). This study reports on the development of a cyclodextrin (CD)-based orally disintegrating tablet (ODT) formulation for PB as an alternative to existing formulations. This is based on previous reports of the PB taste-masking potential of CDs and the suitability of ODTs for improving compliance in pediatric and dysphagic populations. In preliminary studies, the interactions of PB with α and βCD in the solid state were characterized using X-ray diffraction, scanning electron microscopy, dissolution, and accelerated stability studies. Based on these studies, lyophilized PB-CD solid systems were formulated into ODTs after wet granulation. Evaluation of the ODTs showed that they had adequate physical characteristics, including hardness and friability and good storage stability. Notably, the developed αCD-based ODT for PB had a disintegration time of 28 s and achieved a slightly acidic and agreeable pH (≈5.5) in solution, which is suitable for effective PB-CD complexation and taste masking. The developed formulation could be helpful as an alternative to existing PB formulations, especially for pediatric and dysphagic UCD patients.

Cisplatin-induced acute kidney injury (AKI) is an important factor that limits the clinical use of this drug for the treatment of malignancies. Oxidative stress and inflammation are considered to be the main causes of not only cisplatin-induced death of cancer cells but also cisplatin-induced AKI. Therefore, developing agents that exert antioxidant and anti-inflammatory effects without weakening the anti-tumor effects of cisplatin is highly desirable. Carbon monoxide (CO) has recently attracted interest due to its antioxidant, anti-inflammatory, and anti-tumor properties. Herein, we report that CO-loaded red blood cell (CO-RBC) exerts renoprotective effects on cisplatin-induced AKI. Cisplatin treatment was found to reduce cell viability in proximal tubular cells via oxidative stress and inflammation. Cisplatin-induced cytotoxicity, however, was suppressed by the CO-RBC treatment. The intraperitoneal administration of cisplatin caused an elevation in the blood urea nitrogen and serum creatinine levels. The administration of CO-RBC significantly suppressed these elevations. Furthermore, the administration of CO-RBC also reduced the deterioration of renal histology and tubular cell injury through its antioxidant and anti-inflammatory effects in cisplatin-induced AKI mice. Thus, our data suggest that CO-RBC has the potential to substantially prevent the onset of cisplatin-induced AKI, which, in turn, may improve the usefulness of cisplatin-based chemotherapy.

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-β1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.

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

It is important to calculate the drug removal by hemodialysis (HD) for drug dosing regimens in HD patients. However, there are limited and inconsistent information about the dialyzability of drugs by HD. Therefore, the aim of our study is to evaluate drug removal by utilizing a rat model of HD (HD rat) and to extrapolate this result to the drug removal rate in HD patients. HD rats received bilateral nephrectomy and HD for 2 h. The dialysis removal of 6 drugs was evaluated in HD rats. Dialysis efficiency, plasma protein binding rate (PBR) and distribution volume (Vd) of drugs were also measured. Furthermore, we examined the correlation between the dialyzability of drug in HD rats and humans and constructed the prediction formula of the drug dialyzability in HD patients. The clearance of urea and creatinine and normalized dialysis dose in HD rats were 0.83 ± 0.07 mL/min, 0.70 ± 0.08 mL/min, and 0.13 ± 0.06, respectively. The drug dialyzability in HD rats was similar to reported clinical data except for doripenem. A higher correlation was observed between drug dialyzability in reported clinical data and HD rats which were adjusted for PBR (r2 = 0.936; p < 0.001) compared to unadjusted (r2 = 0.812; p = 0.009). Therefore, we constructed the prediction formula of the drug dialyzability in HD patients by utilizing the HD rat model and PBR. This study is useful for evaluating the dialyzability of high-risk drugs in a clinical setting and might provide appropriate preclinical dialyzability data for new drug.

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

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. Keywords: Ultraviolet irradiation, Human serum albumin, Reactive sulfur species, Whitening agent, Oxidative stress

Sodium 4-phenylbutyrate (PB) is an orphan drug for the treatment of urea cycle disorders. It also inhibits the development of endoplasmic reticulum stress, the action of histone deacetylases and as a regulator of the hepatocanalicular transporter. PB is generally considered to have the potential for use in the treatment of the diseases such as cancer, neurodegenerative diseases and metabolic diseases. In a previous study, we reported that PB is primarily bound to human serum albumin (HSA) in plasma and its binding site is drug site 2. However, details of the binding mode of PB to HSA remain unknown. To address this issue, we examined the crystal structure of HSA with PB bound to it. The structure of the HSA–PB complex indicates that the binding mode of PB to HSA is quite similar to that for octanoate or drugs that bind to drug site 2, as opposed to that for other medium-chain length of fatty acids. These findings provide useful basic information related to drug–HSA interactions. Moreover, the information presented herein is valuable in terms of providing safe and efficient treatment and diagnosis in clinical settings. Keywords: Human serum albumin, X-ray crystallography, Sodium 4-phenylbutyrate, Drug interaction, Drug site 2

Macrophages play a central role in various inflammatory disorders and are broadly divided into two subpopulations, M1 and M2 macrophage. In the healing process in acute inflammatory disorders, shifting the production of M1 macrophages to M2 macrophages is desirable, because M1 macrophages secrete pro-inflammatory cytokines, whilst the M2 variety secrete anti-inflammatory cytokines. Previous findings indicate that when macrophages are treated with carbon monoxide (CO), the secretion of anti-inflammatory cytokine is increased and the expression of pro-inflammatory cytokines is inhibited, indicating that CO may have a potential to modulate the production of macrophages toward the M2-like phenotype. In this study, we examined the issue of whether CO targeting macrophages using a nanotechnology-based CO donor, namely CO-bound hemoglobin vesicles (CO-HbV), modulates their polarization and show therapeutic effects against inflammatory disorders. The results showed that the CO-HbV treatment polarized a macrophage cell line toward an M2-like phenotype. Furthermore, in an in vivo study using acute pancreatitis model mice as a model of an inflammatory disease, a CO-HbV treatment also tended to polarize macrophages toward an M2-like phenotype and inhibited neutrophil infiltration in the pancreas, resulting in a significant inflammation. In addition to the suppression of acute pancreatitis, CO-HbV diminished a subsequent pancreatitis-associated acute lung injury. This could be due to the inhibition of the systemic inflammation, neutrophil infiltration in the lungs and the production of HMGB-1. These findings suggest that CO-HbV exerts superior anti-inflammatory effects against inflammatory disorders via the regulation of macrophage and neutrophil activity.

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.

Cisplatin (CDDP) is an essential anti-tumor agent for chemotherapeutic regimens against various types of cancer. However, the progression of nephrotoxicity, which is the main adverse effect of CDDP, leads to discontinuation of CDDP chemotherapy. Therefore, development of a renoprotectant against CDDP-induced nephrotoxicity is crucial. Here, the potential of a carbon monoxide (CO)-loaded hemoglobin-vesicle (CO-HbV) as a renoprotectant for CDDP-induced nephrotoxicity was evaluated for its renoprotective effects against CDDP-induced nephrotoxicity, inhibitory effects on the anti-tumor activity of CDDP, and anti-tumor activity. In healthy mice, after pretreatment with either saline, HbV, or CO-HbV prior to CDDP administration, only the CO-HbV pretreatment group ameliorated the progression of CDDP-induced nephrotoxicity by suppressing apoptosis via caspase-3. In experiments using B16-F10 melanoma cells, the half-maximal inhibitory concentration of CDDP decreased with co-incubation with CO-HbV, owing to the anti-tumor activity of CO. CO-HbV pretreatment had no impact on the anti-tumor activity of CDDP in B16-F10 melanoma cell-bearing mice, which was consistent with the results of the cell experiment. Furthermore, CO-HbV pretreatment improved body growth and survival rates. In conclusion, CO-HbV pretreatment is a potent renoprotectant for CDDP-induced nephrotoxicity, allowing treatment with CDDP to be conducted without failure of cancer treatment.

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.

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.

In a previous study, we reported on the development of a synthetic polymer conjugate of pirarubicin (THP) that was formed via an acid-labile hydrazone bond between the polymer and the THP. However, the synthetic polymer itself was non-biodegradable, which could lead to unexpected adverse effects. Human serum albumin (HSA), which has a high biocompatibility and good biodegradability, is also a potent carrier for delivering antitumor drugs. The objective of this study was to develop pH-sensitive HSA conjugates of THP (HSA-THP), and investigate the release of THP and the cytotoxicity under acidic conditions in vitro for further clinical development. HSA-THP was synthesized by conjugating maleimide hydrazone derivatives of THP with poly-thiolated HSA using 2-iminothiolane, via a thiol-maleimide coupling reaction. We synthesized two types of HSA-THP that contained different amounts of THP (HSA-THP2 and HSA-THP4). Free THP was released from both of the HSA conjugates more rapidly at an acidic pH, and the rates of release for HSA-THP2 and HSA-THP4 were similar. Moreover, both HSA-THPs exhibited a higher cytotoxicity at acidic pH than at neutral pH, which is consistent with the effective liberation of free THP under acidic conditions. These findings suggest that these types of HSA-THPs are promising candidates for further development.

4'-cyano-2'-deoxyinosine (SK14-061a), a novel nucleoside analog based on inosine, has antiviral activity against the human immunodeficiency virus type 1 that has the ability to acquire resistance against many types of reverse transcriptase inhibitors based on nucleosides. The aim of this study was to investigate the pharmacokinetics studies after its oral administration to rats. For this purpose, we first developed and validated an analytical method for quantitatively determining SK14-061a levels in biological samples by a UPLC system interfaced with a TOF-MS system. A rapid, simple and selective method for the quantification of SK14-061a in biological samples was established using liquid chromatography mass spectrometry (LC-MS) with solid phase extraction. The pharmacokinetic properties of SK14-061a in rats after oral administration were then evaluated using this LC-MS method. SK14-061a was found to be relatively highly bioavailable, is rapidly absorbed from the intestinal tract, and is then mainly distributed to the liver and then ultimately excreted via the urine in an unchanged form. Furthermore, the simultaneous administration of SK14-061a with the nucleoside analog, entecavir, led to a significant alteration in the pharmacokinetics of SK14-061a. These results suggest that the SK14-061a has favorable pharmacokinetic properties with a high bioavailability with the potential for use in oral pharmaceutical formulations, but drug-drug interactions should also be considered.

Biological proteins have unlimited potential for use as pharmaceutical products due to their various biological activities, which include non-toxicity, biocompatibility, and biodegradability. Recent scientific advances allow for the development of novel innovative protein-based products that draw on the quality of their innate biological activities. Some of them hold promising potential for novel therapeutic agents/devices for addressing hepatic diseases such as hepatitis, fibrosis, and hepatocarcinomas. This review attempts to provide an overview of the development of protein-based products that take advantage of their biological activity for medication, and discusses possibilities for the therapeutic potential of protein-based products produced through different approaches to specifically target the liver (or hepatic cells: hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) in the treatment of hepatic diseases.

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.

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.

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

Photoaffinity labeling is a powerful technique for identifying a target protein. A high degree of labeling specificity can be achieved with this method in comparison to chemical labeling. Human serum albumin (HSA) and α1-acid glycoprotein (AGP) are two plasma proteins that bind a variety of endogenous and exogenous substances. The ligand binding mechanism of these two proteins is complex. Fatty acids, which are known to be transported in plasma by HSA, cause conformational changes and participate in allosteric ligand binding to HSA. HSA undergoes an N-B transition, a conformational change at alkaline pH, that has been reported to result in increased ligand binding. Attempts have been made to investigate the impact of fatty acids and the N-B transition on ligand binding in HSA using ketoprofen and flunitrazepam as photolabeling agents. Meanwhile, plasma AGP is a mixture of genetic variants of the protein. The photolabeling of AGP with flunitrazepam has been utilized to shed light on the topology of the protein ligand binding site. Furthermore, a review of photoaffinity labeling performed on other major plasma proteins will also be discussed. Using a photoreactive natural ligand as a photolabeling agent to identify target protein in the plasma would reduce non-specific labeling.

Hemoglobin-vesicles (HbV), encapsulating highly concentrated human hemoglobin in liposomes, were developed as a substitute for red blood cells (RBC) and their safety and efficacy in transfusion therapy has been confirmed in previous studies. Although HbV suspensions are structurally and physicochemically stabile for least 1-year at room temperature, based on in vitro experiments, the issue of whether the use of long-term stored HbV after a massive hemorrhage can be effective in resuscitations without adverse, post-transfusion effects remains to be clarified. We report herein on a comparison of the systemic response and the induction of organ injuries in hemorrhagic shock model rats resuscitated using 1-year-stored HbV, freshly packed RBC (PRBC-0) and by 28-day-stored packed RBC (PRBC-28). The six-hour mortality after resuscitation was not significantly different among the groups. Arterial blood pressure and blood gas parameters revealed that, using HbV, recovery from the shock state was comparable to that when PRBC-0 was used. Although no significant change was observed in serum parameters reflecting liver and kidney injuries at 6 hours after resuscitation among the three resuscitation groups, results based on Evans Blue and protein leakage in bronchoalveolar lavage fluid, the lung wet/dry weight ratio and histopathological findings indicated that HbV as well as PRBC-0 was less predisposed to result in a post-transfusion lung injury than PRBC-28, as evidenced by low levels of myeloperoxidase accumulation and subsequent oxidative damage in the lung. The findings reported herein indicate that 1-year-stored HbV can effectively function as a resuscitative fluid without the induction of post-transfused lung injury and that it is comparable to fresh PRBC, suggesting that HbV is a promising RBC substitute with a long shelf-life.

A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

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.

Hemoglobin (Hb) is an ideal material for use in the development of an oxygen carrier in view of its innate biological properties. However, the vascular retention of free Hb is too short to permit a full therapeutic effect because Hb is rapidly cleared from the kidney via glomerular filtration or from the liver via the haptogloblin-CD 163 pathway when free Hb is administered in the blood circulation. Attempts have been made to develop alternate acellular and cellular types of Hb based oxygen carriers (HBOCs), in which Hb is processed via various routes in order to regulate its pharmacokinetic properties. These HBOCs have been demonstrated to have superior pharmacokinetic properties including a longer half-life than the Hb molecule in preclinical and clinical trials. The present review summarizes and compares the pharmacokinetic properties of acellular and cellular type HBOCs that have been developed through different approaches, such as polymerization, PEGylation, cross-linking, and encapsulation.

Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds.

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.