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Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of α1-acid glycoprotein (AGP) for this role because it is a nanosize plasma protein and binds a variety of anticancer agents. Pharmacokinetic analyses indicated that AGP is distributed more extensively in tumor tissue than human serum albumin, which was already established as a cancer DDS carrier. AGP is possibly being incorporated into tumor cells via endocytosis pathways. Moreover, a synthetic AGP-derived peptide which possesses a high ability to form an α-helix, as deduced from the primary structure of AGP, was also taken up by the tumor cells. AGP loaded with anticancer agents, such as paclitaxel or nitric oxide, efficiently induced tumor cell death. These results suggest that AGP has the potential to be a novel DDS carrier for anticancer agents.

Recent research findings indicate that the posttranslational modification of human serum albumin (HSA) such as oxidation, glycation, truncation, dimerization, and carbamylation is related to certain types of diseases. We report herein on a simple and rapid analytical method, using an electrospray ionization time-of-flight mass spectrometry technique, that allows posttranslational modifications of HSA to be quantitatively and qualitatively evaluated with a high degree of sensitivity. In patients with chronic liver disease, chronic renal disease, and diabetes mellitus, an increase in the level of oxidized cysteine-34 (Cys-34) of HSA accompanied by a decrease in the level of reduced Cys-34 was observed. The redox status of Cys-34 was correlated with ligand binding and the antioxidative functions of HSA. Available evidence indicates that monitoring the redox state of Cys-34 not only could be a useful marker for evaluating the progression of disease and its complications but also would permit therapeutic efficacy to be predicted. The redox state of Cys-34 was also used as an index of the quality of HSA preparations. These data suggest that monitoring the posttranslational modifications of HSA can be important, because the function of HSA is related not only to its serum concentration but also to the preservation of its structural integrity under disease conditions.

Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of α

Adenosine diphosphate (ADP)-encapsulated liposomes coated with a fibrinogen γ-chain dodecapeptide [H12 (dodecapeptide ( 400 HHLGGAKQAGDV 411 ))-(ADP)-liposome] is a synthetic platelet substitute, in which the surface is covered with polyethylene glycol (PEG). It has been reported that repeated injections of PEGylated liposomes induce an accelerated blood clearance (ABC) phenomenon, which involves a loss in the long-circulation half-life of the material when administered repeatedly to the same animals. The objective of this study was to determine whether the ABC phenomenon was induced by repeated injections of H12-(ADP)-liposome in healthy and anticancer drug-induced thrombocytopenia model rats. The findings show that the ABC phenomenon was induced by healthy rats that were repeatedly injected with H12-(ADP)-liposomes at the interval of 5days at a dose of 10mg lipids/kg. The ABC phenomenon involves the production of anti-H12-(ADP)-liposome immunoglobulin M (IgM) and complement activation. On the other hand, when thrombocytopenia model rats were repeatedly injected with H12-(ADP)-liposomes under the same conditions, no ABC phenomenon, nor was any suppression of anti-H12-(ADP)-liposome IgM-mediated complement activation observed. We thus conclude that the repeated injection of H12-(ADP)-liposome treatment in rat model with anticancer drug-induced thrombocytopenia did not induce the ABC phenomenon. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3084–3091, 2015

Sodium 4-phenylbutyrate (PB) is clinically used as a drug for treating urea cycle disorders. Recent research has shown that PB also has other pharmacologic activities, suggesting that it has the potential for use as a drug for treating other disorders. In the process of drug development, preclinical testing using experimental animals is necessary to verify the efficacy and safety of PB. Although the binding of PB to human albumin has been studied, our knowledge of its binding to albumin from the other animal species is extremely limited. To address this issue, we characterized the binding of PB to albumin from several species (human, bovine, rabbit, and rat). The results indicated that PB interacts with 1 high-affinity site of albumin from these species, which corresponds to site II of human albumin. The affinities of PB to human and bovine albumins were higher than those to rabbit and rat albumin, and that to rabbit albumin was the lowest. Binding and molecular docking studies using structurally related compounds of PB suggested that species differences in the affinity are attributed to differences in the structural feature of the PB-binding sites on albumins (e.g., charge distribution, hydrophobicity, shape, or size).

Bioactive gaseous molecules, such as oxygen (O2) and carbon monoxide (CO), are essential elements for most living organisms to maintain their homeostasis and biological activities. An accumulating body of evidence suggests that such molecules can be used in clinics as a medical gas in the treatment of various intractable disorders. Recent developments in hemoglobin-encapsulated liposomes, namely hemoglobin vesicles (HbV), possess great potential for retaining O2 and CO and could lead to strategies for the development of novel pharmacological agents as medical gas donors. HbV with either O2 or CO bound to it has been demonstrated to have therapeutic potential for treating certain intractable disorders and has the possibility to serve as diagnostic and augmenting product by virtue of unique physicochemical characteristics of HbV. The present review provides an overview of the present status of the use of O2- or CO-binding HbV in experimental animal models of intractable disorders and discusses prospective clinical applications of HbV as a medical gas donor.

Red blood cell (RBC) transfusions are the gold standard in cases of massive hemorrhage, but induce hepatic ischemia-reperfusion injury, a serious complication associated with hemorrhage and RBC resuscitation. Thus, the development of a novel resuscitable fluid that is not associated with hepatic ischemia-reperfusion injury would be desirable. It was reported that exogenous carbon monoxide (CO) treatment ameliorated hepatic ischemia-reperfusion injury accompanying liver transplantation. This suggests that transfusions with CO-bound RBC (CO-RBC) might protect against hepatic ischemia-reperfusion injury following massive hemorrhage and resuscitation compared with RBC resuscitation. To investigate this, we created a hemorrhagic shock model rat, followed by resuscitation with RBC and CO-RBC. Hepatic ischemia-reperfusion injury and the destruction of hepatic cytochrome P450 (CYP) were significantly ameliorated in the CO-RBC resuscitation group compared with the RBC resuscitation group. The free heme derived from the destruction of hepatic CYP was correlated with hepatic oxidation and injury, suggesting that CO-RBC was a major factor in the amelioration of hepatic ischemia-reperfusion injury induced by hemorrhage and resuscitation via hepatic CYP protection. These results indicate that CO-RBC has potential for use as a resuscitative fluid in blood transfusion and does not suffer from the limitations associated with the RBC transfusions that are currently in use.

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

Human α(1) -acid glycoprotein (AGP), a serum glycoprotein, is known to have anti-inflammatory activity. We recently reported that AGP was mainly incorporated into the liver in mice via a receptor-mediated pathway, although the mechanism for this was largely unknown. The objective of this study was to identify the specific cellular surface protein that recognizes the peptide moiety of AGP. Pharmacokinetic studies of (111) In-AGP and (111) In -recombinant glycan-deficient AGP (rAGP) in mice demonstrated that both AGPs are mainly distributed to the liver and kidney, but hepatic and renal uptake clearance of rAGP was higher than that for AGP. Hepatic uptake of rAGP was inhibited in the presence of 100-fold excess of unlabeled AGP, indicating that the hepatic uptake of rAGP shared a common route with that of AGP and that it recognized the peptide moiety of AGPs. In ligand blotting analyses using crude cellular membrane fraction of mice liver, a band corresponding to a 16 kDa protein was observed to bind to both AGPs. Interestingly, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MALDI-TOF-MS and western blotting analyses indicated that this 16 kDa protein is the hemoglobin β-chain (HBB). It, therefore, appears that HBB is associated with the hepatic uptake of AGP via a direct interaction with its peptide moiety.

Human α(1) -acid glycoprotein (AGP), a lipocalin family member, serves as a carrier for basic drugs and endogenous hormones. It is mainly distributed in the liver and also has anti-inflammatory effects. We previously discovered a protein in liver parenchymal cells that interacts with AGP and it was identified as hemoglobin β-chain (HBB). The purpose of this study was to clarify the role of HBB in the hepatic cellular uptake of AGP. Ligand blotting experiments showed that the interaction of (125) I-AGP with hemoglobin was saturable and was significantly suppressed in the presence of excess unlabeled AGP. In addition, the cellular uptake of fluorescein isothiocianate-AGP by HepG2 cells was saturable and temperature dependent. This uptake was inhibited by fillipin and methyl-β-cyclodextrin, but not chlorpromazine, suggesting that AGP is taken up via caveolae/lipid rafts endocytic pathway. Immunostaining showed that HBB and caveolin-1, exclusively expressed in caveolae, were partially colocalized on the plasma membranes of HepG2 cells. HBB knockdown with siRNA decreased the uptake of AGP by HepG2 cells by 40%, and exogenous hemoglobin inhibited the uptake by 40%-50%. These findings indicate that HBB is located on the liver plasma membrane and that it contributes to the intracellular uptake of AGP.

Protein-bound uremic toxins, such as indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, p-cresyl sulfate, hippuric acid, and indoleacetic acid, have been the subjects of extensive investigations. In this review, we summarized the recent works providing the new insight on the pharmacokinetics and redox properties of these uremic toxins. They have a common characteristic of being difficult to remove by conventional dialysis because they all bind tightly to serum albumin. They are transported via organic anion transporters to various tissues, and accumulate not only in the kidney but also in other tissues including vascular endothelial cells, smooth muscle cells, osteoblasts, and the central nervous system. Accumulated uremic toxins alter nonrenal drug clearance. Intracellular accumulated uremic toxins have been linked to the induction of oxidative stress and the stimulation of proinflammatory cytokines through the production of reactive oxygen species, which play a role in the progression of chronic kidney disease and the development of complications. Unfortunately, despite the massive amount of information on the undesirable effects of uremic toxins, methods for improving the detoxification of these toxins appear to be lacking. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3682–3695, 2011

Human α1-acid glycoprotein (AGP), a major carrier of many basic drugs in circulation, consists of at least two genetic variants, namely A and F1*S variant. Interestingly, the variants of AGP have different drug-binding properties. The purpose of this study was to identify the amino acid residues that are responsible for the selectivity of drug binding to genetic variants of AGP using site-directed mutagenesis. First, we screened amino acid residues in the region proximal to position 100 that are involved in binding of warfarin and dipyridamole, which are F1*S-specific ligands, and of propafenone, which is an A-specific ligand, using ultrafiltration. In the F1*S variant, His97, His100, and Trp122 were involved in either warfarin- or dipyridamole-binding, while Glu92, His100, and Trp122 participated in the binding of propafenone in the A variant. Exchange of the residue at position 92 between AGP variants reversed the relative strength of propafenone binding to the two variants, but had a markedly different effect on binding of warfarin and dipyridamole. These findings indicate that the amino acid residue at position 92 plays a significant role in drug-binding selectivity in AGP variants, especially for drugs that preferentially bind to the A variant. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4316–4326, 2009

In the process of drug development, preclinical testing using experimental animals is an important aspect, for verification of the efficacy and safety of a drug. Serum albumin is a major binding protein for endogenous and exogenous ligands and regulates their distribution in various tissues. In this study, the structural and drug-binding properties of albumins on a biomembrane surface were investigated using reverse micelles as a model membrane. In reverse micelles, the secondary structures of all albumins were found, to varying degrees, to be intermediate between the native and denatured states. The tertiary structures of human and bovine albumin were similar to those of the native and intermediate states, respectively, whereas those of the dog, rabbit, and rat were in a denatured state. Thus, bovine albumin is an appropriate model for studying structural changes in human albumin in a membrane-water phase. Binding studies also showed the presence of species difference in the change in binding capacity of albumins during their interaction with reverse micelles. Among the albumins, rat albumin appears to be a good model for the protein-mediated drug uptake of human albumin in a biomembrane environment. These findings are significant in terms of the appropriate extrapolation of pharmacokinetics and pharmacodynamics data in various animals to humans.

A preclinical study of dodecapeptide ((400)HHLGGAKQAGDV(411)) (H12)-(adenosine diphosphate, ADP)-liposomes for use as a synthetic platelet (PLT) substitute under conditions of red blood cell (RBC) transfusion-induced dilutional thrombocytopenia is limited to pharmacological effect. In this study, the pharmacokinetics of H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were evaluated. As evidenced by the use of (14) C, (3) H double-radiolabeled H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane were labeled with (14) C and (3) H, respectively, the H12-(ADP)-liposomes remained intact in the blood circulation for up to 3 h after injection, and were mainly distributed to the liver and spleen. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane was mainly eliminated in the feces. These successive pharmacokinetic properties of the H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were similar to those in healthy rats, except for the shorter retention time in the circulation. When H12-(ADP)-liposomes were repeatedly injected into RBC transfusion-induced dilutional thrombocytopenic rats at intervals of 5 days at a dose of 10 mg lipids/kg, the second dose of injected H12-(ADP)-liposomes were rapidly cleared from the circulation, namely, via the accelerated blood clearance phenomenon. These novel pharmacokinetic findings provide useful information for the further development of H12-(ADP)-liposomes as a PLT substitute.

Site I of human serum albumin is an important and complex region for high‐affinity binding of drugs. Equilibrium dialysis showed independent binding of dansyl‐ L ‐asparagine (DNSA) and n ‐alkyl p ‐aminobenzoates ( p ‐ABEs) to regions Ib and Ic, respectively, in the pH range 6.0–9.0. However, individual binding of DNSA increased with pH in the same range. Binding of the four n ‐alkyl p ‐ABEs strongly perturbed the circular dichroism spectrum of bound DNSA, and the effect increased with concentration and the number of carbon atoms in the alkyl moiety. A similar effect was observed by increasing pH from 6.0 to 9.0, a pH range in which human serum albumin is known to undergo the neutral‐to‐base transition. The spectral changes propose spatial orientation changes of DNSA at region Ib. This proposal was supported by increased fluorescence anisotropy values: n ‐alkyl p ‐ABEs binding and the pH‐dependent conformational change each restricted the mobility of the naphthalene ring of bound DNSA. Despite the similar effects on the spatial orientation of DNSA, clear differences were observed between the effects of n ‐alkyl p ‐ABEs and neutral‐to‐base transition. The former hardly changed the affinity and maximum fluorescence emission wavelength of bound DNSA; in contrast, the latter significantly affected them. The results give new information about site I and, according to our knowledge, represent a new type of ligand interaction, because the binding site of DNSA could be changed by simultaneous binding of the n ‐alkyl p ‐ABEs without affecting the binding constant. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:3004–3012, 2004

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. We have previously demonstrated that poly-nitric oxide (NO) conjugated human serum albumin (Poly-SNO-HSA) has the potential to be a DDS carrier capable of accumulating NO in tumors. However, the stability of Poly-SNO-HSA in the circulation has to be improved, and its optimal molecular size for using the EPR effects has to be evaluated. In the present study, we performed two tuning methods for refining Poly-SNO-HSA, namely, pegylation and dimerization. We observed that pegylation enhanced the stability of Poly-SNO-HSA both in vitro and in vivo, and that dimerization of Poly-SNO-HSA enhanced the antitumor activity via more efficient delivery of NO in Colon 26 tumor-bearing mice. Intriguingly, dimerization resulted in a 10 times higher antitumor activity. These data suggest that pegylation and dimerization of Poly-SNO-HSA are very important tuners to optimize NO stability and accumulation, and thereby effect, in tumors. Thus, polyethylene glycol-Poly-SNO-HSA dimer seems to be a very appealing and safe NO carrier and thereby a strong candidate as an antitumor drug in future development of cancer therapeutics.

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitric oxide metabolite and an important second messenger. 8-Nitro-cGMP reacts with sulfhydryl groups forming a novel posttranslational modification, namely, S -guanylation. In this work, we found, by using a quantitative competition enzyme-linked immunosorbent assay procedure, that S -guanylated human serum albumin ( S -cGMP-HSA) is a component of normal plasma, and that hemodialysis patients decrease its concentration, on an average, from 68 to 34nM. End-stage renal disease is often accompanied by septicemia, and we found that S -cGMP-HSA possesses an in vitro antibacterial effect with half maximal inhibitory concentration of approximately 2μM against Escherichia coli American Type Culture Collection. Our findings indicate that S -cGMP-HSA can be regarded as an endogenous antibacterial agent in healthy conditions and as a useful new class of antibacterial agents with a circulation time sufficient for in vivo biological activity. The clinical development of S -cGMP-HSA as a safe and strong antibacterial agent arisen from endogenous posttranslational modification would be expected. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association.

The purpose of this study was to quantitatively evaluate the extent of contribution of cysteine-34 (34Cys) on the antioxidant effect of human serum albumin (HSA) and to elucidate the physiological implication in hemodialysis (HD) patients. The ratio of oxidized albumin correlated directly with the thiol content in plasma of the HD patients who received intravenous iron. Moreover, the degree of oxidation of 34Cys in HSA purified from the HD patients' plasma correlated with the thiol content in plasma. The radical scavenging activity of purified HSA was dependent on the degree of 34Cys oxidation. A recombinant mutant, C34S, was produced to confirm the role of 34Cys. The activity of C34S was about half of that of wild-type HSA (WT-HSA). Consistent with this observation, the protective effects of C34S were significantly lower than those of WT-HSA in suppressing cytotoxicity and vascular endothelial growth factor production induced by fenton reaction in a human vascular endothelial cell model. These results indicate that the 34Cys residue of HSA may account for more than 40% of the antioxidant effect of HSA in vivo, and thus may exert a protective effect on vascular endothelium function via an antioxidative mechanism in chronic renal disease. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3968–3976, 2011

The hemoglobin-vesicle (HbV) is a cellular, hemoglobin-based oxygen carrier. Our previous pharmacokinetic studies demonstrated that the liver is strongly associated with the metabolism and excretion of HbV. The aim of this study was to evaluate the pharmacokinetics of HbV in a chronic cirrhosis rat (CCR) model induced by carbon tetrachloride (CCl(4) ) and explore whether liver functional parameters and Kupffer cell (KC) phagocyte activity are related to the pharmacokinetics of HbV. The CCRs were induced three times weekly by intraperitoneal administration of CCl(4) for 8 weeks and categorized as Child-Pugh grade B. To analyze the pharmacokinetics, the CCRs were given a single intravenous injection of (3) H-HbV (1400 mg of Hb/kg). The total clearance and hepatic distribution of HbV were negatively correlated with plasma aspartate aminotransferase (AST) levels (p = 0.007). In addition, the phagocyte index was negatively correlated with plasma AST levels (p = 0.047). The excretion of lipid components in feces was also negatively correlated with plasma AST levels (p = 0.049). In conclusion, alteration in the pharmacokinetics of HbV in CCRs can be attributed to a decrease in KC phagocyte activity and the extent of damage to parenchymal cells. This represents the first demonstration of the pharmacokinetics of a liposome preparation in chronic liverimpairment.

The binding of nine diazepines to human serum albumin (HSA) and to alpha 1-acid glycoprotein (AGP) was investigated by means of fluorescence and circular dichroism (CD) spectroscopies. The binding parameters of diazepam obtained from fluorescence agreed with those obtained from CD measurements. Diazepines have one tight binding site on both HSA and AGP. The binding parameters (nK) of the diazepine: HSA systems are slightly higher than those of diazepine:AGP systems. The relationship between the binding parameters for these two serum proteins and the physicochemical parameters of diazepines was studied by multiple regression analysis to elucidate the binding mode. Moreover, the effects of long-chain fatty acids and cesium chloride on the binding of diazepines to HSA and AGP were also studied. The driving force for the binding of diazepines to both proteins appears to be hydrophobic interaction. In addition, steric effects and electrostatic interactions may also contribute to the binding of diazepines to HSA and AGP, respectively.

Advanced glycation end products (AGEs) are generated not only from glucose but also from several aldehydes such as methylglyoxal, glyoxal, and glycolaldehyde. The aim of the present study was to investigate the effect of several aldehydes on human serum albumin (HSA) in terms of the physicochemical properties and formation of AGE structures. HSA modified with methylglyoxal, generated by the glycolysis pathway and degradation of the Schiff base, showed the highest increase in the molecular weight and net negative charge, whereas glucose modification caused a small increase in the molecular weight even incubation for after 4 weeks. N(epsilon)-(carboxymethyl)lysine (CML), N(epsilon)-(carboxyethyl)lysine (CEL), and imidazolone increased in modified HSA in correlation with their lysine and arginine modification, whereas high amounts of GA-pyridine was detected in HSA modified with glycolaldehyde. Furthermore, the binding ability of HSA to warfarin and ketoprofen was more effectively decreased by methylglyoxal modification than the other aldehydes. These results indicated that changes in the physicochemical properties and the formation of AGE structures are highly dependent on the aldehydes.

The fluorescence of phenprocoumon is greatly enhanced on binding to a single site on alpha 1-acid glycoprotein (alpha 1-AGP). Advantage is taken of this phenomenon to estimate a binding constant for the binding of phenprocoumon to alpha 1-AGP. The fluorescence intensity and binding constant of the phenprocoumon: alpha 1-AGP complex decreased with pH from 6.5 to 8.5, suggesting that phenprocoumon binding to alpha 1-AGP is significantly affected by microenvironmental change in alpha 1-AGP. A variety of drugs, including chlorpromazine and dicumarol, significantly inhibited phenprocoumon binding to alpha 1-AGP. Fatty acids seem to displace phenprocoumon from its binding site on alpha 1-AGP, whereas the addition of neutral salts and sialic acid did not cause the displacement of phenprocoumon. It is concluded that the phenprocoumon binding site is located in the hydrophobic protein structure of alpha 1-AGP.