2022 Volume 45 Issue 6 Pages 803-805
Nateglinide (NAT) is used to treat diabetes, stimulating pancreatic islet β-cells with residual insulin secretory capacity to increase insulin secretion. NAT has been reported to bind to human serum albumin (HSA), but the detail is still unclear. In the current study, we investigated the location and the affinity for the binding of NAT to HSA. Quantitative analysis data from the ultrafiltration experiment indicated that NAT binds strongly to a primary site on HSA with a high affinity. The presence of diazepam (DZP) or ibuprofen (IB), the specific site II ligands of HSA, decreased the binding constants of NAT respectively, without the significant changes in the number of binding sites. Whereas warfarin (WF), a site I specific ligand, did not affect the binding of NAT. Fluorescent replacement experiment showed that NAT replaced dansylsarcosine (DNSS), a site II probe of HSA, but not WF. An increasing level of myristate and uremic toxins, indoxyl sulphate (IS), indoxyl acetate (IA) and p-cresyl sulphate (PCS), during renal disease significantly increased the concentration of unbound NAT. These findings suggest that NAT specifically binds to site II of HSA and the binding capacity and pharmacokinetics of NAT change in renal diseases.
Nateglinide (NAT) is a phenylalanine derivative that blocks the K+ channels of pancreatic β-cells and promotes insulin secretion.1) NAT lowers blood glucose levels by stimulating β-cell insulin secretion relative to blood glucose levels. NAT is metabolized mainly by CYP2C9 in the liver. The metabolites have also been investigated in detail.2,3) NAT has been reported to bind to human serum albumin (HSA) with a high affinity in serum,4) but the details of its binding property to HSA have not been clarified.
HSA is the most abundant protein in serum and binds endogenous substances such as fatty acids and many drugs.5) HSA has two primary drug binding sites, site I and site II. NAT has been reported to bind to site II of HSA, but no detailed experimental data are available. In diabetic disease, a high concentration of glucose in serum caused glycation of HSA, resulting in a decrease in the binding affinity of NAT.6,7) Renal failure disease such as diabetic nephropathy causes an increase in various endogenous substances in serum. Among them, uremic toxins such as indoxyl sulphate (IS), indole acetate (IA) and p-cresyl sulphate (PCS) specifically bind to site II of HSA,8,9) causing the displacement of drugs bound to HSA. In addition, fatty acids such as myristate, which has several binding sites on HSA, increased free drug fractions in serum.10,11)
In this study, we investigated the detailed drug-binding properties of NAT to HSA and the effects of increased concentration of uremic toxins and fatty acids in serum on the binding of NAT to HSA.
HSA, essentially fatty acid-free, was purchased from Sigma-Aldrich (Darmstadt, Germany). NAT was purchased from Combi-Blocks (CA, U.S.A.). PCS was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). IS and IA were purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals and solvents were purchased from commercial sources and were of analytical grade.
HPLC ConditionsThe HPLC system used in this study consisted of a Hitachi model D-2000 Elite HPLC system (Hitachi, Ltd., Tokyo, Japan). YMC Triart C18 column (YMC Co., Ltd., Kyoto, Japan) as the stationary phase at 40 °C and acetonitrile and water as the mobile phase were used. The linear gradient of the mobile phase solution was set to be 40% of acetonitrile to 80% over 10 min. The flow rate of the mobile phase was set constant at 1.0 mL/min. The detection wavelength was fixed at 210 nm.
Binding ParametersTo obtain the binding parameters of NAT to HSA, we used the ultrafiltration method. Ultrafiltration was performed using Amicon Ultra-0.5 mL Centrifugal Filters (10 kDa cutoff, 500 µL) from Merck Millipore (Darmstadt, Germany). After centrifugation at 5000 × g for 30 min at 25 °C, the concentrations of NAT in the 50 µL of filtrate, the free concentration, were measured by HPLC. Free fractions were calculated using the values of [Df] and [Dt] as follows:
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where [Df] is the concentration of the unbound NAT, [Dt] is the total NAT concentration. Binding parameters were obtained from the Scatchard plot using the following equation.
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where K, n, and r are the binding affinity, the number of binding sites of NAT and the number of moles of NAT bound per mole of HSA, respectively. Inhibition mode of warfarin (WF), diazepam (DZP) and ibuprofen (IB) to the binding of NAT to HSA was analyzed by Klotz plots using the following equation.
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WF and dansylsarcosine (DNSS) were used as site I and site II fluorescent probes on HSA, respectively. Fluorescence spectra of probes were measured using a Hitachi F-2500 fluorescence spectrophotometer (Hitachi) at 25 °C. The excitation wavelengths for WF and DNSS were 320 and 350 nm, respectively. The degree of displacement of these probes was calculated using the following equation,
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where F1 and F2 represent the maximum fluorescent intensity of the probe in HSA solution without and with displacers, respectively.
Statistical AnalysisThe overall differences between groups were determined with a one-way ANOVA.
To investigate the binding capacity of NAT to HSA, we examined the binding affinity constant and the number of binding sites of NAT on the HSA molecule (Table 1). NAT is strongly bound to a single binding site on HSA, because the binding affinity of DZP and IB are 1.3 × 105 (M−1) and 3.5 × 106 (M−1) respectively.12) HSA is well known to have two drug binding sites, site I and site II. Therefore, we investigated which sites of HSA NAT binds to using WF (a site I-binding drug) and DZP and IB (site II-binding drugs) (Fig. 1). In the presence of WF, a slight decrease of the binding constant of NAT was observed, whereas, in the presence of DZP or IB, the binding constants of NAT were drastically decreased. The number of binding sites in the presence of any inhibitors was around 1.00. To confirm the binding of NAT to site II of HSA, we investigated whether NAT displaces the fluorescent probe, DNSS, bound to site II (Fig. 2). NAT significantly replaced DNSS, but not WF. These results suggest that NAT primarily binds to site II of HSA.
| K (×106) M−1 a) | na) | |
|---|---|---|
| NAT | 7.99 ± 1.57 | 0.89 ± 0.03 |
| NAT + WF | 3.08 ± 2.87 | 1.15 ± 0.09 |
| NAT + DZP | 0.69 ± 0.28 | 0.72 ± 0.12 |
| NAT + IB | 0.31 ± 0.10 | 1.24 ± 0.16 |
a) K and n represent the binding constant and the number of binding sites, respectively.
For the measurement, 50 µM of NAT and 50 µM of HSA and each concentration (0–250 µM) of WF, DZP and IB were used, respectively. Solid, dotted, grey and dashed lines represent NAT only, NAT in the presence of WF, DZP or IB, respectively. Each plot was obtained from independent three experiments.
Fluorescent intensities of 10 µM concentrations of WF and DNSS were measured in the presence of 10 µM of HSA and each concentration (0–30 µM) of NAT. The values are represented as the mean ± standard deviation (S.D.) of three independent experiments. *Symbol represent significantly different vs. values in the absence of NAT, p < 0.05.
We investigated the effect of uremic toxins or myristate on the binding of NAT. As a result, all uremic toxins significantly increased the free fraction of NAT. Myristate has multiple binding sites in HSA molecules, including site II, which is not the primary site for myristate. As shown in Fig. 3, a high concentration of myristate inhibited NAT binding.
The free fractions of NAT were measured in the presence of 100 µM of NAT and 50 µM of HSA, and each concentration (0–250 µM) of uremic toxins or myristate respectively. The values are represented as the mean ± S.D. of three independent experiments. *Symbol represent significantly different vs. values in the absence of uremic toxins or myristate, p < 0.05.
Our study reveals that NAT specifically binds to site II of albumin. From the Klotz plot analysis results, WF did not significantly affect the binding of HSA in NAT, while DZP and IB reduced the binding constant without affecting the number of binding sites in NAT. This meant that DZP and IB competitively inhibited NAT binding. In the fluorescent probe displacement experiment, only DNSS, a site II probe, was significantly replaced by NAT. This result supports our conclusion that NAT binds to site II.
High serum glucose levels observed during diabetes cause glycation of HSA. Much research has been conducted on HSA glycation, and it is known that glycation of basic amino acid residues around site II reduces the binding of site II drugs.13,14) It is known that the binding property of NAT to glycated HSA is diminished.6,7) Thus, changes in drug–HSA interactions can affect the pharmacokinetics of binding drugs during pathological conditions. It is known that the concentration of uremic toxins such as IS, IA and PCS and fatty acids such as myristate increases during renal diseases, including diabetic nephropathy.8,11) Watanabe et al. report that these uremic toxins bind to site II of HSA and affect the pharmacokinetics of site II drugs. Myristate is known to increase in concentration up to 5 times that of HSA during renal disease.11) Therefore, to compare and examine the effects of uremic toxins and myristate on the NAT binding to HSA equivalently, the change of free fraction of NAT in the presence of uremic toxins or myristate at up to 5.0 times to HSA were measured. All uremic toxins significantly inhibited NAT binding to HSA. The order of the binding constant magnitudes of these uremic toxins is IS> PCS> IA.8,9) This is consistent with the degree of binding inhibition observed in our results. In the case of myristate, inhibition of NAT binding was observed only in the presence of high concentrations of myristate. This is because myristate has about seven binding sites on HSA, and site II is not the primary binding site.15,16)
Our current data indicate that NAT binds to site II of HSA with high affinity and specificity. During renal disease, increased endogenous substances such as uremic toxins and fatty acids affect the pharmacokinetics of NAT through the change of interaction between NAT and HSA.
The authors declare no conflict of interest.
