Slow receptor dissociation kinetics differentiate macitentan from other endothelin receptor antagonists in pulmonary arterial smooth muscle cells

Abstract

Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP(1)) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K(b) values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt(1/2)) compared to bosentan and ambrisentan (ROt(1/2):17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP(1) assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt(1/2) rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies.

Figure 1. Contribution of endothelin receptor subtypes to calcium signaling in human primary PASMC.

PASMC or CHO-ET_A or CHO-ET_B cells were pre-incubated with reference ERA dilution series for 120 min and then stimulated in the FLIPR with ET-1 (EC₅₀–EC₇₀). IC₅₀ values were calculated from fluorescence peak responses and transformed into K_b values as described in Methods. Correlation scatter plot of K_b values obtained in PASMC versus CHO-ET_A cells (A) or versus CHO-ET_B cells (B). The correlation line and R² value was generated by linear regression of the double-logarithmic data. Geometric means of n = 3–8 determinations are shown.

Figure 2. Antagonistic potency and apparent association kinetics of different ERAs using calcium release assays in PASMC.

Cells were pre-incubated with antagonist dilution series for 10 min or 120 min and then stimulated in the FLIPR with ET-1 (EC₅₀–EC₇₀). IC₅₀ values were calculated from fluorescence peak responses and transformed into K_b values as described in Methods. Correlation scatter plot of the K_b values generated for 10 min versus 120 min antagonist pre-incubation times (geometric means of n = 6 determinations).

Figure 3. Dissociation kinetics of macitentan, ambrisentan and bosentan determined by calcium release assays in PASMC.

Cells were pre-incubated for 120 min with antagonist dilution series and then either directly stimulated with ET-1 (EC₅₀–EC₇₀) or subjected to a compound washout procedure followed by stimulation with ET-1 at the indicated time points after washout. The peak calcium responses were used to calculate IC₅₀ values which were transformed into K_b values as described in Methods. Shown are the time-dependent changes in K_b after washout. Geometric means are displayed +/− SEM. K_b values after washout significantly different (p<0.05) from the K_b at 0 min are indicated with an asterisk. Test: one-way ANOVA, Dunnett’s post test.

Figure 4. Effect of bosentan, ambrisentan and macitentan on ET-1-induced IP₁ accumulation.

Human PASMCs were pre-incubated with dilution series of antagonists for 120 min followed by the addition of a dilution series of ET-1. After 20 min of stimulation, cells were lysed and the IP₁ content was determined. Raw data from 4 independent experiments were normalized, and average values +/− SEM are shown.

Figure 5. Time dependence of macitentan insurmountability determined in IP₁ accumulation assays.

Human PASMCs were pre-incubated with dilution series of antagonists for 120 min followed by the addition of a dilution series of ET-1. After 20 min (A) or 90 min (B) of stimulation, cells were lysed and the IP₁ content was determined. Shown are the raw data +/− SD of duplicates of one representative experiment of a total of n = 3.

Figure 6. Characterization of the biphasic intracellular calcium response to ET-1 in PASMC.

Cells were stimulated with a dilution series of ET-1 and the biphasic calcium response was recorded for 25 minutes. Shown are the raw FLIPR fluorescence traces for 200 nM ET-1 and vehicle treatment covering the first transient response phase and the second sustained response phase (A), the concentration-response-curves of ET-1 obtained for the first phase using peak fluorescence values +/− SD of duplicates (B) and the concentration-response-curves of ET-1 obtained for the second phase using area under the curve values between 3 min and 23 min after stimulation +/− SD of duplicates (C).

Figure 7. Effect of bosentan, ambrisentan and macitentan on both phases of the ET-1-induced calcium response.

Human PASMCs were pre-incubated with dilution series of antagonists for 120 min, stimulated with a dilution series of ET-1 and the biphasic calcium response was recorded. Shown is the effect of antagonists on the ET-1 concentration-response-curves of the first response phase (A, using peak fluorescence values +/− SD of duplicates) and the second sustained response phase (C, using area under the curve values +/− SD of duplicates). Furthermore, the concentration-response-curves of the three antagonists for antagonizing peak responses induced by 1 µM ET-1 are shown +/− SD of duplicates (B) with the approximate localization of intermediate plateaus of antagonist efficacy. Shown are the results of one representative experiment out of n = 3 experiments.