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We recently reported that aripiprazole binds strongly to human albumin. In continuing our investigations, we investigated the mechanism responsible for the binding and the related interactions of aripiprazole with α1-acid glycoprotein (AGP). The extrinsic Cotton effects for the binding of aripiprazole and its derivatives to AGP were generated, but the magnitudes of the induced circular dichroism intensities did not correlate with those for the binding affinities. It therefore appears that the binding mode of aripiprazole with AGP is somewhat complicated, compared with that of albumin. Isothermal titration calorimetry data obtained for the binding of aripiprazole with AGP were different from that for albumin systems in that the 3 driving reactions, entropy-driven, enthalpy-driven, and the entropy-enthalpy mixed type, were all found for the AGP system, but not albumin. Moreover, the weak binding mode of aripiprazole with the 2 proteins were supported by a molecular docking model analysis. The concentration of albumin in plasma is about 50 times higher than those of AGP, but AGP levels in plasma are increased by about 10 times under inflammatory disease. Therefore, the involvement of these 2 plasma proteins should be considered in more depth for understanding the pharmacokinetics of aripiprazole.

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.

We recently reported that aripiprazole binds strongly to human albumin. In continuing our investigations, we investigated the mechanism responsible for the binding and the related interactions of aripiprazole with α1-acid glycoprotein (AGP). The extrinsic Cotton effects for the binding of aripiprazole and its derivatives to AGP were generated, but the magnitudes of the induced circular dichroism intensities did not correlate with those for the binding affinities. It therefore appears that the binding mode of aripiprazole with AGP is somewhat complicated, compared with that of albumin. Isothermal titration calorimetry data obtained for the binding of aripiprazole with AGP were different from that for albumin systems in that the 3 driving reactions, entropy-driven, enthalpy-driven, and the entropy-enthalpy mixed type, were all found for the AGP system, but not albumin. Moreover, the weak binding mode of aripiprazole with the 2 proteins were supported by a molecular docking model analysis. The concentration of albumin in plasma is about 50 times higher than those of AGP, but AGP levels in plasma are increased by about 10 times under inflammatory disease. Therefore, the involvement of these 2 plasma proteins should be considered in more depth for understanding the pharmacokinetics of aripiprazole.

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

Previously, we reported on the high-affinity binding of aripiprazole (ARP), an antipsychotic drug, to human albumin and the role of the chlorine atom of ARP on this binding. In this study, we investigated the binding mode of ARP to human albumin in detail using ARP derivatives and several animal-derived albumins. ARP bound strongly to human and dog albumin. The circular dichroism (CD) spectra of ARP bound to human and dog albumin were also similar. Deschloro-ARP bound less strongly to all of the albumin species compared to ARP, and the shapes of CD spectra were similar for all albumin species. CD spectra of dimethyl-ARP, for which chlorine atoms were substituted methyl groups, were quite similar to that of deschloro-ARP. In displacement experiments, competitive binding was observed between ARP and deschloro-ARP. These results suggest that the chlorine atoms in ARP are involved in the binding modes of ARP for human and dog albumins, whereas ARP and deschloro-ARP appear to share the same binding region in site II. The aforementioned results imply that compounds having a chlorine atom bind more strongly to plasma proteins, resulting in a long blood retention time. Therefore, findings reported here may provide the basically useful data for drug design.

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.

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.