Interaction of Benzbromarone with Subdomains IIIA and IB/IIA on Human Serum Albumin as the Primary and Secondary Binding Regions.

Benzbromarone has been used for the treatment of gout for more than 30 years. Although it shows a high level of binding to plasma proteins (>99%), our knowledge of this binding is not sufficiently extensive to permit us to understand its pharmacokinetics and pharmacodynamics. To address this issue in more detail, we characterized the binding of benzbromarone to human serum albumin (HSA), the most abundant protein in plasma. Equilibrium dialysis and circular dichroism findings indicated that benzbromarone binds strongly to one primary as well as to multiple secondary sites on HSA and that the bromine atoms of benzbromarone play important roles in this high affinity binding. An X-ray crystallographic study revealed that benzbromarone molecules bind to hydrophobic pockets within subdomains IB, IIA, and IIIA. Inhibition experiments using site specific ligands (subdomain IB; fusidic acid, IIA; warfarin, IIIA; diazepam) indicated that the primary and secondary binding sites that benzbromarone binds to are within subdomains IIIA and IB/IIA, respectively. Lastly, a study of the effect of fatty acids on the benzbromarone-HSA interaction suggested that benzbromarone, when displaced from subdomain IIIA by sodium oleate, could transfer to subdomains IB or IIA. Thus, these data will permit more relevant assessments of the displacement interactions of benzbromarone especially in cases of co-administered drugs or endogenous compounds that also bind to subdomain IIIA. In addition, the findings presented herein will also be useful for designing drug combination therapy in which pharmacokinetic and pharmacodynamic performance need to be controlled.

Effects of Uremic Toxins on the Binding of Aripiprazole to Human Serum Albumin.

We recently reported that aripiprazole (ARP), an antipsychotic drug, binds strongly to human serum albumin (HSA), the major drug binding protein in serum. It is known that uremic toxins that accumulate during renal disease affect the interaction between HSA and drug binding. In this study, the issue of how uremic toxins (indoxyl sulfate, indole acetic acid and p-cresyl sulfate) affect the binding of ARP to HSA was investigated. Equilibrium dialysis experiments revealed that all uremic toxins inhibited the binding of ARP to HSA although the inhibitory effects differed, depending on the specific uremic toxin. The potency of inhibition can be partially explained by the affinities of uremic toxins to HSA. Fluorescence displacement experiments suggested that ARP as well as all uremic toxins bind to site II of HSA. The inhibitory effects of the toxins on the binding of ARP for the drugs binding to the diazepam subsite are significantly larger, comparing with those for binding to arylpropionic acids subsite. Interestingly, induced circular dichroism (CD) spectra indicated that the spatial orientation of p-cresyl sulfate in the binding pocket is different from that for indoxyl sulfate and indole acetic acid. The limited findings obtained herein are important data in considering the effects of uremic toxins on the pharmacokinetics of ARP and the drugs that bind to site II on HSA, particularly drugs binding to diazepam binding site in site II.

Effects of Oxidation of Human Serum Albumin on the Binding of Aripiprazole.

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.

Rhabdomyolysis caused by interaction between rosuvastatin and vadadustat: a case control study.

BACKGROUND: Rhabdomyolysis is a potentially life-threatening disease caused by melting or necrosis of skeletal muscle cells and leakage of muscle components into the bloodstream. It has been reported that the interaction of the HMG-CoA reductase inhibitor rosuvastatin with the renal anemia drug vadadustat increases the blood concentration of rosuvastatin in vitro. In this study, we report a case of suspected rhabdomyolysis caused by the drug interaction of rosuvastatin and vadadustat in clinical practice. CASE PRESENTATION: A 62-year-old male with medical records of hypertension, myocardial infarction, chronic renal failure, renal anemia, dyslipidemia, and alcoholic liver disease. The patient had been diagnosed with chronic kidney disease (CKD) at the Department of Nephrology, and treated by outpatient care with renal support therapy for the past two years. On X-63 day, his prescription was rosuvastatin (10 mg/day) and a continuous erythrocyte-stimulating agent, epoetin beta pegol (genetical recombination, 100 µg). X-Day 0, blood tests revealed creatine phosphokinase (CPK) 298 U/L, serum creatinine (SCr) 5.26 mg/dL, and hemoglobin (Hb) 9.5 g/dL; thus, the prescription was changed from epoetin beta pegol 100 µg to vadadustat 300 mg/day. On X + day 80, a prescription for a diuretic (azosemide 15 mg/day) was added for swelling of the lower extremities. On X + day 105, we found CPK 16,509 U/L, SCr 6.51 mg/dL, and Hb 9.5 g/dL. The patient was diagnosed as rhabdomyolysis and hospitalized. After hospitalization, rosuvastatin and vadadustat were discontinued and we administered intravenous fluids. Thereafter, CPK and SCr values of the patient improved. On X + day 122, CPK improved to 29 U/L, SCr to 2.6 mg/dL, and Hb to 9.6 g/dL, and he was discharged on X + day 124. At discharge, rosuvastatin 2.5 mg/day was resumed. A blood test on X + day 133 showed CPK 144 U/L and SCr 4.2 mg/dL. CONCLUSION: We experienced a case of rhabdomyolysis caused by drug interactions between rosuvastatin and vadadustat.

Effects of Myristate on the Induced Circular Dichroism Spectra of Aripiprazole Bound to Human Serum Albumin: A Structural-Chemical Investigation.

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.

An in-vitro comparative study of the binding of caspofungin and micafungin to plasma proteins.

OBJECTIVES: Echinocandins are widely used for the treatment of invasive fungal diseases. While they bind strongly to plasma proteins, our knowledge of this process is not sufficient to permit their pharmacokinetics and pharmacodynamics targets to be discussed. In this study, we characterized the binding of two echinocandins, caspofungin and micafungin, to plasma proteins, human serum albumin (HSA) and human α 1-acid glycoprotein (AAG). METHODS: The binding parameters, number of binding sites (n) and association constant (K) for caspofungin and micafungin to HSA and AAG were determined by equilibrium dialysis. The binding site on HSA for these echinocandins was identified by conducting inhibition experiments. KEY FINDINGS: Caspofungin was found to bind strongly to a single site on HSA (n = 1.26, K = 0.45 × 106 M-1) and AAG (n = 0.99, K = 0.29 × 106 M-1). Micafungin was found to bind more strongly to HSA (n = 1.35, K = 1.44 × 106 M-1) and AAG (n = 1.32, K = 1.16 × 106 M-1). The binding site for these drugs on HSA appears to be within subdomain IA. CONCLUSIONS: Free fraction of caspofungin and micafungin in patients may not be substantially affected due to the contribution of AAG to the overall protein binding and the binding to subdomain IA on HSA, which is different from the major drug-binding sites within subdomains IB, IIA and IIIA.

The Binding of Aripiprazole to Plasma Proteins in Chronic Renal Failure Patients.

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.

Characterization of the Interaction of Daptomycin With Site II on Human Serum Albumin.

Daptomycin, a cyclic lipopeptide antibiotic, is clinically used for the treatment of infections caused by Gram-positive bacteria, including the methicillin-resistant Staphylococcus aureus and the vancomycin-resistant Enterococci. While daptomycin shows high plasma protein binding (90-93%), our knowledge of the binding process is not extensive. To address this issue in more detail, we characterized the binding of daptomycin to plasma proteins and the findings indicate that the association constant for the binding of daptomycin to human serum albumin (HSA) is much higher than that for α1-acid glycoprotein, another plasma protein. Daptomycin was also found to bind to a single site on HSA, which was identified as site II. The findings also suggest that the n-decanoyl moiety of daptomycin penetrates into the hydrophobic pocket of site II and that this acyl moiety interacts with Tyr411 at the entrance to site II. Due to this selective interaction with site II, daptomycin binding was significantly inhibited by drugs (ibuprofen or diazepam) and endogenous compounds (uremic toxins or fatty acids) which also strongly bind to site II. In diseased states, such an inhibition in the binding could result in the pharmacokinetics and therapeutic action of daptomycin being substantially altered.

Interaction of Aripiprazole With Human α1-Acid Glycoprotein.

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.

Further Evidence Regarding the Important Role of Chlorine Atoms of Aripiprazole on Binding to the Site II Area of Human Albumin.

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.

Analysis of the Binding of Aripiprazole to Human Serum Albumin: The Importance of a Chloro-Group in the Chemical Structure.

Aripiprazole (ARP), a quinolinone derivative, is an atypical antipsychotic drug that is used in the treatment of schizophrenia. ARP has an extensive distribution and more than 99% of the ARP and dehydro-ARP, the main active metabolite, is bound to plasma proteins. However, information regarding the protein binding of ARP is limited. In this study, we report on a systematic study of the protein binding of ARP. The interaction of ARP and structurally related compounds with human serum albumin (HSA) was examined using equilibrium dialysis, circular dichroism (CD) spectroscopy, fluorescent probe displacement, and an X-ray crystallographic analysis. The binding affinities (nK) for ARP and its main metabolite, dehydro-ARP with HSA were found to be significantly higher than other structurally related compounds. The results of equilibrium dialysis experiments and CD spectral data indicated that the chloro-group linked to the phenylpiperazine ring in the ARP molecule plays a major role in the binding of these ligands to HSA. Furthermore, fluorescent probe displacement results indicated that ARP appears to bind at the site II pocket in subdomain III. A detailed CD spectral analysis suggests that the chloro-group linked to the phenylpiperazine ring may control the geometry of the ARP molecule when binding in the site II binding pocket. X-ray crystallographic analysis of the ARP-HSA complex revealed that the distance between the chlorine atom at the 3-positon of dichlorophenyl-piperazine on ARP and the sulfur atom of Cys392 in HSA was 3.4-3.6 Å. A similar halogen bond interaction has also been observed in the HSA structure complexed with diazepam, which also contains a chloro-group. Thus, the mechanism responsible for the binding of ARP to a protein elucidated here should be relevant for assessing the pharmacokinetics and pharmacodynamics of ARP in various clinical situations and for designing new drugs.