Binding of sulphonylureas to plasma proteins - A KATP channel perspective

Abstract

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug's therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion.

Fig 1. Effect of BSA on gliclazide block of the β-cell K_ATP current.

(A,B) Representative whole-cell K_ATP currents recorded from mouse pancreatic β-cells in response to alternating ±20mV steps from a holding potential of -70mV. Gliclazide (1μmol/l) was added (as indicated by the bars) in the absence (A) or presence (B) of 0.9mmol/l BSA. The dotted line indicates the zero current level. (C) Concentration-response relationships for gliclazide inhibition of whole-cell K_ATP currents in mouse β-cells in the absence (○, n = 6) or presence (●, n = 5) of 0.9mmol/l BSA. Current is expressed relative to that in the absence of gliclazide. The solid lines are the best fit of Eq 2 to the mean data: (○) IC₅₀ = 190nmol/l, h = 0.97, a = 0.05; (●) IC₅₀ = 320nmol/l, h = 0.90, a = 0.03. This shift predicts 60% of drug is bound and a dissociation constant (K_d) for drug binding to BSA of ~1.3mmol/l (Eq 3). The dotted line is the estimated gliclazide block in the presence of human plasma (HP) assuming gliclazide binds to plasma proteins with a K_d of 155μmol/l. The width of the grey bar indicates the mean C_SS±SEM (steady-state total plasma concentrations of sulphonylurea drugs) of the total gliclazide concentration in the plasma estimated from a daily dose of 80mg and an AUC (the area under the plasma concentration against time curve) of 44μg.h/ml [26].

Fig 2. Effect of BSA on glibenclamide and gliclazide block of recombinant β-cell K_ATP channels.

(A,B) Representative Kir6.2/SUR1 currents recorded at -60mV from cell-attached patches on Xenopus oocytes. Currents were recorded in the presence of 3mmol/l Na-azide (top trace), 3mmol/l Na-azide plus 1μmol/l glibenclamide (A, middle) or 3mmol/l Na-azide plus 1μmol/l gliclazide (B, middle), and 3mmol/l Na-azide, 1μmol/l glibenclamide and 0.9mmol/l BSA (A, bottom) or 3mmol/l Na-azide, 1μmol/l gliclazide and 0.9mmol/l BSA (C, bottom). The dotted line indicates the zero current level. (C) Concentration-response relationships for glibenclamide inhibition of Kir6.2/SUR1 currents in the absence (○, n = 10) and presence (●, n = 10) of 0.9mmol/l BSA. Open probability (P_O) was recorded in the cell-attached configuration and is expressed relative to that in the absence of glibenclamide. The lines are the best fit of Eq 2 to the mean data: (○) IC₅₀ = 1.2nmol/l, h = 1.1; (●) IC₅₀ = 1.6μmol/l, h = 0.95. a was set at 0 in both cases. The dotted line is the estimated glibenclamide block in the presence of human plasma (HP) assuming the drug binds to plasma proteins with a K_d of 0.44μmol/l. The width of the grey bar indicates the mean C_SS±SEM of the total glibenclamide concentration in the plasma of patients with type 2 diabetes [28]. (D) Concentration-response relationships for gliclazide inhibition of Kir6.2/SUR1 currents in the absence (○, n = 10) and presence (●, n = 10) of 0.9mmol/l BSA. Open probability (P_O) was recorded in the cell-attached configuration and is expressed relative to that in the absence of gliclazide. The lines are the best fit of Eq 2 to the mean data: IC₅₀ = 128nmol/l, h = 1.3 (○); IC₅₀ = 217nmol/l, h = 1.3 (●). a was set at 0. The IC₅₀ obtained in the absence of BSA (128nmol/l) is similar to that previously reported for whole-cell Kir6.2/SUR1 currents in oocytes (108nmol/l; [27]). The width of the grey bar indicates the mean C_SS±SEM of the total gliclazide concentration in the plasma estimated from a daily dose of 80mg and an AUC of 44μg.h/ml [26].

Fig 3. Effect of BSA on glibenclamide stimulated insulin release.

Insulin secretion was evaluated in islets from 8–10 week-old male mice in response to 60–120 minute stimulation with various glucose and glibenclamide concentrations, as indicated, in the presence of (A) 15μmol/l BSA or (B) 0.9mmol/l BSA (n = 3 animals, 3 technical replicates per condition). Insulin secretion is expressed as a percentage of the insulin content. Insulin secretion in the presence of glucose and glibenclamide was significantly affected by the BSA concentration (F(5,12) = 10.77; p = 0.004). For both incubation times and for all drug concentrations tested, insulin secretion was significantly lower in the presence of 0.9mmol/l BSA than 15μM BSA (p<0.05).

Fig 4. Glucose and glibenclamide block of the K_ATP channel.

(A) Concentration-response relationships for glucose inhibition of K_ATP conductance in pancreatic β-cells from wild-type (○, n = 5) and βV59M (●, n = 8) mice, measured using the perforated patch configuration. Data are the same as those in [16] but are replotted as nS/pF. (B) Concentration-response relationships for glibenclamide inhibition of K_ATP conductance in pancreatic β-cells from wild-type (○, n = 6) and βV59M (●, n = 6) mice, measured using the perforated patch configuration. Data are the same as those in [18] but are replotted as nS/pF. Note the ‘pedestal’ (arrowed) in the dose-response curve for glibenclamide inhibition. This results because glibenclamide binds to SUR1 with high affinity and acts a partial antagonist of the K_ATP channel, with a maximal block of ~60–80% [14]. It also produces a low affinity block at Kir6.2. However, like other sulphonylureas [39], glibenclamide also displaces MgADP from NBD2 of SUR1, which prevents MgADP activation and thereby reveals the full extent of ATP block at Kir6.2. Thus, in the presence of intracellular nucleotides, inhibition is the sum of the high-affinity glibenclamide block at SUR1, ATP block at Kir6.2, and a low-affinity block by glibenclamide at Kir6.2.