Preclinical in vitro and in vivo evaluation of [11C]ORM-13070 as PET ligand for alpha-2C adrenergic receptor occupancy using PET imaging in non-human primates

Abstract

This paper describes the preclinical validation of the radioligand [¹¹C]ORM-13070 and its tritiated analog for addressing selectivity and occupancy of the selective alpha-2C adrenergic receptor (α_2CR) antagonist BAY 292 in the cynomolgus brain. BAY 292 is a novel drug candidate being developed for the treatment of obstructive sleep apnea (OSA) via binding to central α_2CR. In vitro autoradiography studies with sections from non-diseased post-mortem human caudate revealed an excellent specific binding window (>80%) using [³H]ORM-13070. BAY 292 bound to the same binding site as [³H]ORM-13070 and generated a good specific binding signal, with greater selectivity for α_2CR. In non-human primates in vivo, [¹¹C]ORM-13070 demonstrated a reversible behavior, with uptake at baseline highest in striatum (putamen, caudate, ventral striatum, and pallidum) and low in the cerebellar cortex, consistent with the known distribution of the α_2CR. A dose dependent increase in receptor occupancy after BAY 292 administration was observed, confirming BBB penetration and target engagement. The estimated EC₅₀ for BAY 292 is 33.39 ± 11.91 ng/mL. This study aimed to demonstrate the suitability of [¹¹C]ORM-13070 as a PET-radioligand for the study of α_2CR in the non-human primate brain, and to pave the way for future clinical PET tracer studies with BAY 292.

Keywords: ORM-13070; alpha-2C adrenergic receptor; non-human primate; positron emission tomography; receptor occupancy.

Figure 1.

Study Design of PET imaging study in cynomolgus monkeys. CRI: constant rate infusion; IV: intravenous

Figure 2.

In vitro autoradiography of [³H]ORM-13070 binding in human caudate. (a) Representative autoradiograms of [³H]ORM-13070 binding in human caudate tissue under TOP: total binding conditions, MIDDLE: non-specific binding as determined with 10 µM unlabeled ORM-13070, BOTTOM: non-specific binding as determined with 10 µM BAY 292. Specific binding of [³H]ORM-13070 in control human caudate (n = 3, mean ± SD) expressed as (b) fmol/mg tissue and (c) percentage of total binding. Normal distribution was confirmed by the Shapiro-Wilk test.

Figure 3.

[¹¹C]ORM-13070 images of the two monkey subjects (a) M1, and (b) M2 acquired at baseline and competition with BAY 292 administered as IV infusion at total doses of 0.3, 0.35, 0.37, 0.6, 0.75 (n = 2), 2.25, and 6.45 mg/kg. PET images (SUV) were averaged from 20 to 60 min post tracer injection. All images were normalized to a common cynomolgus MRI brain template. Each monkey’s normalized MRI is shown in the top row.

Figure 4.

(a, b) Examples of regional time activity curves from one monkey (M2) during the PET scan, (c) BAY 292 plasma concentration profiles, and (d) α_2CR occupancy vs. BAY 292 plasma concentration. (a) baseline and (b) competition with BAY 292 0.75 mg/kg in the caudate nucleus (solid triangles), putamen (solid circles), ventral striatum (open triangles), pallidum (solid squares) and cerebellar cortex (open circles). Solid lines correspond to the MA1 fits.The arrow in subplot C indicates start of PET tracer injection.

Figure 5.

Occupancy plots of the two monkey subjects M1 (a, c), and M2 (b, d) at eight doses of BAY 292 using only V_T data from α_2C-rich regions (a, b), or using V_T data from α_2C-rich regions + cerebellar cortex (c, d). Fractional occupancy values (slope), and V_ND (x-int) are displayed in the inset tables.