Evaluation of [(11)C]MRB for assessment of occupancy of norepinephrine transporters: Studies with atomoxetine in non-human primates

Abstract

[(11)C]MRB is one of the most promising radioligands used to measure brain norepinephrine transporters (NET) with positron emission tomography (PET). The objective of this study was to evaluate the suitability of [(11)C]MRB for drug occupancy studies of NET using atomoxetine (ATX), a NET uptake inhibitor used in the treatment of depression and attention-deficit hyperactivity disorder (ADHD). A second goal of the study was identification of a suitable reference region. Ten PET studies were performed in three anesthetized rhesus monkeys following an infusion of ATX or placebo. [(11)C]MRB arterial input functions and ATX plasma levels were also measured. A dose-dependent reduction of [(11)C]MRB volume of distribution was observed after correction for [(11)C]MRB plasma free fraction. ATX IC(50) was estimated to be 31 ± 10ng/mL plasma. This corresponds to an effective dose (ED(50)) of 0.13mg/kg, which is much lower than the therapeutic dose of ATX in ADHD (1.0-1.5mg/kg). [(11)C]MRB binding potential BP(ND) in the thalamus was estimated to be 1.8 ± 0.3. Defining a reference region for a NET radiotracer is challenging due to the widespread and relatively uniform distribution of NET in the brain. Three regions were evaluated for use as reference region: caudate, putamen and occipital cortex. Caudate was found to be the most suitable for preclinical drug occupancy studies in rhesus monkeys. The IC(50) estimate obtained using MRTM2 BP(ND) without arterial blood sampling was 21 ± 3ng/mL (using caudate as the reference region). This study demonstrated that [(11)C]MRB is suitable for drug occupancy studies of NET.

Fig. 1

Study timelines for the two-day, four-dose studies (Day 1: baseline and medium-dose studies; Day 2: low- and high-dose studies). The solid line represents the ATX infusion rates (i.e., loading rate followed by maintenance rate for each study), as used for monkey #3 (i.e., the maintenance rates were 0.01 mg/kg/h (low dose), 0.03 mg/kg/h (medium dose) and 0.09 mg/kg/h (high dose)). The grey areas highlight the PET scanning times. The black triangles represent the PK sampling times.

Fig. 2

(A, B, C) ATX plasma concentration measured for monkeys 1, 2, and 3, respectively, for the low (circle), medium (square) and high (triangle) dose scans. (D) Correlation between the ATX plasma levels and the final infusion rates (infusion II) for monkey 1 (+), 2 (×) and 3 (*), respectively. The regression line was obtained by pooling data from all monkeys (slope=1.04 kg×h/L, r²=0.902, n=7).

Fig. 3

(A) Unchanged fraction of [¹¹C]MRB in plasma (average±SD, n=10). The solid line represents the average of the fitted f_H curves used for metabolite correction. (B) [¹¹C]MRB plasma free fraction measured by ultrafiltration for the baseline (black), low (dark grey), medium (light grey) and high (white) ATX dose scans. Errors bars represent the standard errors estimated from the triplicate measurements of plasma and ultrafiltrate activity.

Fig. 4

Example of regional time activity curves obtained in one monkey (#2) at baseline in the caudate nucleus (open squares), putamen (open circles), occipital cortex (open triangles), anterior cingulate cortex (solid triangles) and thalamus (solid circles). Solid lines correspond to the MA1 fits.

Fig. 5

Dose-dependent occupancy of ATX in NET-rich regions in three monkeys. (A to C) Normalized volume of distribution (V_T/f_P) computed using MA1 at baseline (black bars) and at the low (dark grey bars), medium (light grey bars) and high (white bars) doses of ATX in the monkeys 1 to 3. The brain regions are: Ca = caudate, Pu = putamen, Oc = occipital cortex, AC = anterior cingulate cortex, Th = thalamus, Br = brainstem, Mi = midbrain. Error bars represent the estimated standard errors. (D) From top to bottom: Anatomic MR image, and parametric images of the normalized volume of distribution (V_T/f_P) at baseline and after blockade with 0.03 mg/kg/h, 0.06 mg/kg/h and 0.12 mg/kg/h of ATX. Images are from monkey #1 and were computed using MA1.

Fig. 6

The relationship between the normalized distribution volumes (V_T/f_P) and the plasma levels of ATX three NET-rich regions: thalamus (A), brainstem (B) and anterior cingulate cortex (C); and in three candidate reference regions: caudate (D), putamen (E) and occipital cortex (F). Data obtained from monkey 1, 2 and 3 and represented with circles, diamonds and squares, respectively. In subfigures A to C, the solid line represents the fit obtained using Eq. (5) to estimate the ATX IC₅₀. The parameters IC₅₀ and V_ND/f_P were shared across all NET-rich regions. In subfigures D to F, the two lines represent the fits obtained using Eq. (5), with the IC₅₀ set to the value estimated in NET-rich regions (32 ng/mL), and B_max/K_d set to zero (dashed line) or fitted (solid line). According to the F test, B_max/K_d was significantly different from zero in the occipital only.

Fig. 7

Lassen plots obtained for the low (open circles), medium (solid circles) and high (open diamonds) doses for monkeys 1 to 3 are displayed in subfigures A to C. The solid lines represent the regression lines for each set of data. In subfigure D, occupancies estimated as the slope of these regression lines are plotted versus the ATX plasma concentration (monkey 1: open circles; monkey 2: open diamonds, monkey 3: solid circles). Solid line represent the line of best fit obtained with Eq. (8) (pooling data from all monkeys), and corresponding to an IC₅₀ estimate of 28±7 ng/mL.