Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

Abstract

Background and purpose: A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF(1)) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan.

Experimental approach: A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF(1) antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic-pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF(1) receptor binding of novel compounds can be predicted on the basis of in vitro assays.

Key results: The non-competitive antagonist behaviour appeared to be correlated to the CRF(1) receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations.

Conclusions and implications: This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF(1) receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF(1) receptor antagonists in an efficient manner in a drug discovery setting.

Figure 1

Inhibition of specific [³H]-SN003 binding to rCRF₁ receptor by CRF₁ non-peptide antagonists. Data points represent mean of n = 4 ± SEM.

Figure 2

(A) Effect of a single concentration of antagonist on oCRF-induced cAMP accumulation in CHO-pro5 cells expressing rCRF₁ receptor. (B) Percentage suppression of 20 µM oCRF response caused by 20 µM of each of the CRF₁ antagonists expressed as a ratio of its K_i derived from competition binding studies. Data points represent mean of n = 4 ± SEM.

Figure 3

Effects of multiple concentrations of CRF₁ antagonists (A, PF-4325743; B, DMP904; C, R121919; D, PF-4734666; E, PF-4659901; and F, SN003) on oCRF-induced cAMP accumulation in CHO-pro5 cells expressing recombinant rCRF₁ receptor. Assays were incubated for 90 min. Representative data from one experiment.

Figure 4

Effect of single and combined concentrations of DMP904 (0.06 µM) and SN003 (2 µM) on oCRF-induced cAMP accumulation in CHO-pro5 cells expressing recombinant rCRF₁ receptor. Assays were incubated for either (A) 5 h or (B) 20 h. Data points represent n = 4 ± SEM.

Figure 5

Effect of incubation time on (A) stability of oCRF response and the surmountability of either (B) 6 µM or (C) 20 µM DMP904 by oCRF-induced cAMP accumulation in CHO-pro5 cells expressing recombinant rCRF₁ receptor.

Figure 6

Effects of the seven CRF₁ antagonists on the receptor–tracer concentration in the in vitro non-equilibrium binding kinetic assay. Representative data from one experiment; n = 3–5 experiments performed in duplicate. Data fitted to Equations 5–7.

Figure 7

Relationship between unbound plasma concentration of DMP904 at the time of brain removal and ex vivo CRF₁ receptor occupancy. Data points represent mean of n = 4 ± SEM.

Figure 8

In vivo receptor occupancy time-course profiles of a single oral dose of four CRF₁ antagonists determined using the ex vivo[³H]-SN003 binding competition assay. (A) Observed receptor occupancy () and model fit (solid black line), and plasma concentration from receptor occupancy study (), or satellite oral pharmacokinetic study () and model fit (solid orange line) versus time relationships. Since DMP904 did not display a significant decay in receptor occupancy during the time course of the experiment, no attempt was made to fit a PKPD model to the data for this ligand. (B) In vivo concentration versus receptor occupancy relationship: () = observed data, solid red line = model fit. Since DMP904 did not display a significant decay in receptor occupancy during the time course of the experiment, no attempt was made to fit a PKPD model to the data for this ligand.

Figure 8

In vivo receptor occupancy time-course profiles of a single oral dose of four CRF₁ antagonists determined using the ex vivo[³H]-SN003 binding competition assay. (A) Observed receptor occupancy () and model fit (solid black line), and plasma concentration from receptor occupancy study (), or satellite oral pharmacokinetic study () and model fit (solid orange line) versus time relationships. Since DMP904 did not display a significant decay in receptor occupancy during the time course of the experiment, no attempt was made to fit a PKPD model to the data for this ligand. (B) In vivo concentration versus receptor occupancy relationship: () = observed data, solid red line = model fit. Since DMP904 did not display a significant decay in receptor occupancy during the time course of the experiment, no attempt was made to fit a PKPD model to the data for this ligand.

Figure 9

Simulations of receptor occupancy versus time profiles using the in vitro non-equilibrium binding kinetic k_on and k_off values (solid line), or k_on optimized fit (dashed line), superimposed on observed receptor occupancy data (). (A) DMP904, (B) R121919, (C) PF-4734666, (D) PF-4850890.

Figure 10

Correlation between the off rate (min⁻¹) of each antagonist from the rat CRF₁ receptor and percentage suppression of a 20 µM oCRF response by 20 µM of each of the CRF₁ antagonists in the cAMP functional assay.

Figure 11

Simulation of R121919 receptor occupancy versus concentration profile using rat pharmacokinetic parameters and k_on and k_off rates determined from in vitro non-equilibrium binding kinetic experiments. Arrows indicate the time order of concentration data, the light blue line represent concentration data not captured by in vivo receptor occupancy study and the red line represent concentration range covered with the in vivo study.

Figure 12

Simulations of an 80 mg clinical dose of the CRF₁ antagonist, R121919 (see text for details). Solid line represents a simulation using the in vitro derived parameters, whereas the dotted and dashed lines represent a fivefold increase and decrease of k_on respectively.