In Vivo Induction of P-Glycoprotein Function can be Measured with [18F]MC225 and PET

Abstract

P-Glycoprotein (P-gp) is an efflux pump located at the blood-brain barrier (BBB) that contributes to the protection of the central nervous system by transporting neurotoxic compounds out of the brain. A decline in P-gp function has been related to the pathogenesis of neurodegenerative diseases. P-gp inducers can increase the P-gp function and are considered as potential candidates for the treatment of such disorders. The P-gp inducer MC111 increased P-gp expression and function in SW480 human colon adenocarcinoma and colo-320 cells, respectively. Our study aims to evaluate the P-gp inducing effect of MC111 in the whole brain in vivo, using the P-gp tracer [¹⁸F]MC225 and positron emission tomography (PET). Eighteen Wistar rats were treated with either vehicle solution, 4.5 mg/kg of MC111 (low-dose group), or 6 mg/kg of MC111 (high-dose group). Animals underwent a 60 min dynamic PET scan with arterial-blood sampling, 24 h after treatment with the inducer. Data were analyzed using the 1-tissue-compartment model and metabolite-corrected plasma as the input function. Model parameters such as the influx constant (K₁) and volume of distribution (V_T) were calculated, which reflect the in vivo P-gp function. P-gp and pregnane xenobiotic receptor (PXR) expression levels of the whole brain were assessed using western blot. The administration of MC111 decreased K₁ and V_T of [¹⁸F]MC225 in the whole brain and all of the selected brain regions. In the high-dose group, whole-brain K₁ was decreased by 34% (K₁-high-dose = 0.20 ± 0.02 vs K₁-control = 0.30 ± 0.02; p < 0.001) and in the low-dose group by 7% (K₁-low-dose = 0.28 ± 0.02 vs K₁-control = 0.30 ± 0.02; p = 0.42) compared to controls. Whole-brain V_T was decreased by 25% in the high-dose group (V_T-high-dose = 5.92 ± 0.41 vs V_T-control = 7.82 ± 0.38; p < 0.001) and by 6% in the low-dose group (V_T-low-dose = 7.35 ± 0.38 vs V_T-control = 7.82 ± 0.37; p = 0.38) compared to controls. k₂ values did not vary after treatment. The treatment did not affect the metabolism of [¹⁸F]MC225. Western blot studies using the whole-brain tissue did not detect changes in the P-gp expression, however, preliminary results using isolated brain capillaries found an increasing trend up to 37% in treated rats. The decrease in K₁ and V_T values after treatment with the inducer indicates an increase in the P-gp functionality at the BBB of treated rats. Moreover, preliminary results using brain endothelial cells also sustained the increase in the P-gp expression. In conclusion, the results verify that MC111 induces P-gp expression and function at the BBB in rats. An increasing trend regarding the P-gp expression levels is found using western blot and an increased P-gp function is confirmed with [¹⁸F]MC225 and PET.

Keywords: ABC transporters; P-glycoprotein; P-gp inducers; brain Imaging; efflux transporters.

Figure 1

Molecular structures of the P-gp tracer [¹⁸F]MC225 (A) and the P-gp inducer MC111 (B).

Figure 2

Metabolite-corrected SUV–TACs for plasma of the control (blue), low-dose (green), and high-dose groups (red) (A) and the percentage of plasma radioactivity representing [¹⁸F]MC225 as a function of time (B).

Figure 3

SUV–TACs of the whole brain in the control (blue color), low-dose (4.5 mg/kg) (green color), and high-dose groups (6 mg/kg) (red color).

Figure 4

Mean ± SE values of K₁ (A), V_T (B), and k₂ (C) in the control (blue), low-dose (4.5 mg/kg) (green), and high-dose (6 mg/kg) (red) groups.

Figure 5

Parametric images calculated using 1-TCM and 60 min scan duration of three representative subjects of each group: control, low-dose (4.5 mg/kg), and high-dose (6 mg/kg).

Figure 6

P-gp expression levels and western blot bands corresponding to P-gp (band close to 160 kDa predicted molecular weight) and ß-actin as the load control (band close to 42 kDa predicted molecular weight).

Figure 7

PXR expression levels and western blot bands corresponding to PXR (two bands close to 50 kDa predicted molecular weight) and histone H3 as the load control (band close to 17 kDa predicted molecular weight). Notice that in our analysis only the 50 kDa band was quantified.

Figure 8

P-gp expression levels in isolated brain endothelial cells and western blot bands corresponding to P-gp (band close to 160 kDa predicted molecular weight) and ß-actin as the load control (band close to 42 kDa predicted molecular weight).