In vivo evaluation of limiting brain penetration of probes for α(2C)-adrenoceptor using small-animal positron emission tomography

Abstract

To evaluate in vivo brain penetration of α(2C)-adrenoceptor (α(2C)-AR) antagonists as a therapeutic agent, we synthesized two new (11)C-labeled selective α(2C)-AR antagonists 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methyl-2-aryl-7-methoxybenzofuran ([(11)C]MBF) and acridin-9-yl-[4-(4-methylpiperazin-1-yl)phenyl]amine ([(11)C]JP-1302) as α(2C)-AR-selective positron emission tomography (PET) probes. The radiochemical yield, specific activity, and radiochemical purity of these probes was appropriate for injection. To evaluate whether the brain penetration of these probes is related to the function of two major drug efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), we performed PET studies using wild-type and P-gp/Bcrp knockout mice. In wild-type mice, the radioactivity level after injection with [(11)C]MBF initially increased and effluxed immediately from the brain, whereas that with [(11)C]JP-1302 was distributed throughout the brain. However, the regional distribution of radioactivity after injection with [(11)C]JP-1302 in the brain was different from that of α(2C)-ARs. In P-gp/Bcrp knockout mice, uptake of [(11)C]MBF was approximately 3.7-fold higher and that of [(11)C]JP-1302 was approximately 1.6-fold higher than those in wild-type mice. These results indicate that brain penetration of the two PET probes was affected by modulation of P-gp and Bcrp functions.

Keywords: 11C; 4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)methyl-2-aryl-7-methoxybenzofuran (MBF); P-glycoprotein (P-gp); acridin-9-yl-[4-(4-methylpiperazin-1-yl)phenyl]amine (JP-1302); breast cancer resistance protein (Bcrp); α2C-Adrenoceptor.

Figure 1

Synthesis of precursor 6 (A) and [¹¹C]MBF (B). Reagents and conditions: (i) boron tribromide, CH₂Cl₂, 0 °C, 2 h; (ii) chloromethyl methyl ether, N,N′-diisopropylethylamine, CH₂Cl₂, room temperature (rt), 4 h; (iii) normal butyl lithium, THF then DMF, −78 °C− to rt, 1 h; (iv) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, sodium tri(acetoxy)borohydride, CH₂Cl₂, rt, 16.5 h; (v) p-toluenesulfonic acid, methanol, rt, 7 h, 55.3% yield from 1, 98.7% chemical purity; (vi) [¹¹C]CH₃I, tetrabutylammonium hydroxide, DMF, 80 °C, 5 min, 27% radiochemical yield (RCY) from [¹¹C]CO₂.

Figure 2

Synthesis of precursor 11 (A) and [¹¹C]JP-1302 (B). Reagents and conditions: (i) K₂CO₃, methyl chloroformate, CH₂Cl₂, rt, 0.5 h; (ii) 10% Pd/C, H₂, ethanol, rt, 2 h; (iii) 9-chloroacridine, ethylene glycol, 150 °C, 1 h; (iv) KOH, water, ethylene glycol, rt, 150 °C, 4 h, 18.8% yield from 7, 95.2% chemical purity; (v) [¹¹C]methyl triflate, acetone, rt, 26% RCY from [¹¹C]CO₂.

Figure 3

In vivo distribution of radioactivity in the brain and blood after injection of [¹¹C]MBF (A) and [¹¹C]JP-1302 (B) into mice (n = 3 per group). Values are means ± standard deviation (SD). Injected dose of ¹¹C ligands was 7.4−12 MBq/0.07−0.12 nmol. SUV, standardized uptake value.

Figure 4

Effects of treatment with α_2C-adrenoceptor (α_2C-AR) ligands on the brain-to-blood ratio at 15 min after the injection of [¹¹C]MBF or at 30 min after the injection of [¹¹C]JP-1302 into mice (n = 3−7 per group). Values are means ± SD. Injected dose of ¹¹C-labeled ligands was 5.9−11 MBq/0.13−0.25 nmol. One of the α_2C-AR ligands (MBF, JP-1302, MK912; 1.0 mg/kg) was intravenously co-injected with [¹¹C]MBF or injected 15 min before administration of [¹¹C]JP-1302. The ∗ indicates P < 0.05 compared with control (one-way ANOVA and Dunnett’s post hoc tests).

Figure 5

Effects of the inhibition of P-gp and Bcrp functions on the brain-to-blood ratio at 30 min after the injection of [¹¹C]MBF (A) and [¹¹C]JP-1302 (B). The ¹¹C-labeled ligands (11−12 MBq/0.12−0.17 nmol), P-gp/BCRP inhibitor GF120918 (5.0 mg/kg), or cold ligand (MBF or JP-1302; 1.0 mg/kg) were intravenously co-injected into mice (n = 4 per group). Values are means ± SD; ∗ indicates P < 0.05 (one-way ANOVA and Bonferroni’s multiple comparison tests).

Figure 6

Typical transaxial positron emission tomography (PET) images showing [¹¹C]MBF (A, C) and [¹¹C]JP-1302 (B, D) in the brain of a wild-type mouse (A, B) and a P-gp/Bcrp knockout mouse (C, D). Injected dose was 3.7−4.7 MBq/0.071−0.56 nmol. PET images were acquired from 5 to 60 min after the injection. Mice were anesthetized with isoflurane and fixed in a prone position on the bed of the scanner.

Figure 7

Time−activity curves of the brain after injection of [¹¹C]MBF (A) and [¹¹C]JP-1302 (B) in wild-type mice and P-gp/Bcrp knockout mice (n = 3 per group). Values are means ± SD. The injected dose was 3.6−8.1 MBq/0.033−1.22 nmol. SUV, standardized uptake value.