[(76) Br]BMK-152, a nonpeptide analogue, with high affinity and low nonspecific binding for the corticotropin-releasing factor type 1 receptor

Abstract

Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF(1) or CRF(2) . A PET ligand able to monitor changes in CRF(1) receptor occupancy in vivo would aid in understanding the pathophysiology of stress-related diseases as well as in the clinical development of nonpeptide antagonists with therapeutic value. We have radiolabeled the CRF(1) receptor ligand, [8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine (BMK-152) (ClogP = 2.6), at both the 3 and 4 position with [(76) Br]. Using in vitro autoradiography saturation studies the 4-[(76) Br]BMK-152 exhibited high affinity binding to both rat (K(d) = 0.23 ± 0.07 nM; n = 3) and monkey frontal cortex (K(d) = 0.31 ± 0.08 nM; n = 3) consistent with CRF(1) receptor regional distribution whereas with the 3-[(76) Br]BMK-152, the K(d) s could not be determined due to high nonspecific binding. In vitro autoradiography competition studies using [(125) I]Tyr(0) -o-CRF confirmed that 3-Br-BMK-152 (K(i) = 24.4 ± 4.9 nM; n = 3) had lower affinity (70-fold) than 4-Br-BMK-152 (K(i) = 0.35 ± 0.07 nM; n = 3) in monkey frontal cortex and similiar studies using [(125) I]Sauvagine confirmed CRF(1) receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wild-type littermates (WT) showed that the brain uptake of 3-[(76) Br]BMK/4-[(76) Br]BMK was increased less than twofold in KO versus WT indicating that 3-[(76) Br]BMK-152/4-[(76) Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[(76) Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF(1) receptor distribution and potential as a PET ligand for in vivo imaging of CRF(1) receptors.

Fig. 1

Structures of compounds 1 (CP-154,526), 2 (antalarmin) and 3 (MJL-1-109-2).

Fig. 2

Structures of compounds 4 (4-Br-BMK-152) and 5 (3-Br-BMK-152).

Fig. 3

Representative autoradiograms comparing 3-[⁷⁶Br]BMK-152 and 4-[⁷⁶Br]BMK-152 regional distribution to [¹²⁵I]oCRH, a known CRF₁R ligand in: A) rhesus monkey prefrontal cortex; B) rat brain (sagittal slice ~3.8 mm from midline).

Fig. 4

Quantitative regional distribution of 4-[⁷⁶Br]BMK in rat brain determined by in vitro autoradiography which corresponds to CRF₁ receptor binding. The quanitative data was obtained by drawing ROI’s expressed as photostimulated luminescence units per mm² (PSL/mm²). Non-specific binding determined using 4-BMK (10⁻⁵M) and quantitated in the same manner (Each bar represents the mean ± SD; n=3).

Fig. 5

Regional Localization of 4-[⁷⁶Br]BMK and non-specific binding (10⁻⁵M 4-BMK) determined from in vitro autoradiography: A) rhesus monkey prefrontal cortex; B) Rat brain (sagittal section ~3.6 mm from midline).

Fig. 6

Uptake of parent 3- and 4-[⁷⁶Br]BMK (corrected for metabolites) in brain and blood of PgP knockout mice and wildtypes from 15 min to 120 min. Mice were injected IV (via tail vein with mixture of 3- and 4-[⁷⁶Br]BMK) awake under temporary restraint and then allowed to free roam for the appropriate uptake period. Each time point represents DUR ± SD of parent 3- and 4-[⁷⁶Br]BMK [n= 3 (15 min groups only) or 4 (all other time points)].