Preparation of 99mTc-PQQ and preliminary biological evaluation for the NMDA receptor

Abstract

Pyrroloquinoline quinone (PQQ), an essential nutrient, antioxidant, redox modulator and nerve growth factor found in a class of enzymes called quinoproteins, was labeled with ^99mTc by using stannous fluoride (SnF₂) method. Radiolabeling qualification, quality control and characterization of ^99mTc-PQQ and its biodistribution studies in mice were performed and discussed. Effects of pH values, temperature, time and reducing agents concentration on the radiolabeling yield were investigated. The quality control procedure of ^99mTc-PQQ was determined by thin layer chromatography (TLC), radio high-performance liquid chromatography (RHPLC) and paper electrophoresis methods. The average radiolabeling yield was 94 ± 1% under optimum conditions of 0.99 mg of PQQ, 30 μg of SnF₂, 0.5 mg of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) and 18.5 MBq of Na^99mTcO₄ at pH 6 and 25 °C with a response volume of 1 ± 0.1 mL. ^99mTc-PQQ was stable and anionic. Lipid-water partition coefficient of ^99mTc-PQQ was -1.49 ± 0.16. The pharmacokinetics parameters of ^99mTc-PQQ were t_1/2α = 18.16 min, t_1/2β = 100.45 min, K₁₂ = 0.013 min^-1, K₂₁ = 0.017 min^-1, K_e = 0.016 min^-1, AUC (area under the curve) = 1040.78 ID% g^-1 min and CL (plasma clearance) = 0.096 mL min^-1. The dual-exponential equation was Y = 10.88e^-0.038t + 5.21e^-0.0069t . The biodistribution of ^99mTc-PQQ was studied in ICR (Institute for Cancer Research 7701 Burhelme Are., Fox Chase, Philadelphia, PA 1911 USA) mice. In vitro autoradiographic studies clearly showed that the ^99mTc-PQQ radioactivity accumulated predominantly in the hippocampus and cortex, which had a high density of N-methyl-d-aspartate Receptor (NMDAR). The enrichment can be blocked by NMDAR redox modulatory site antagonists-ebselen (EB) and ^99mTc-PQQ is therefore a promising candidate for the molecular imaging of NMDAR. To date, however, there have been no studies characterizing ^99mTc-PQQ.

Keywords: 99mTc; 99mTc-PQQ; NMDAR; Pyrroloquinoline quinone.

Fig. 1

The chemical structure of PQQ, shown here as 4,5-dioxo-H-pyrrolo(2,3-f) quinoline-2,7,9-tricarboxylic acid

Fig. 2

HPLC chromatogram of PQQ

Fig. 3

HPLC chromatogram (dash line) and RHPLC chromatograms (solid line) (PQQ rt = 10.8 min at UV detector λ = 249 nm) (^99mTc-PQQ rt = 5.2 min, R-^99mTc rt = 2.2 min and ^99mTcO₄ ⁻ rt = 2.5 min at Cd (Te) detector)

Fig. 4

Stability of ^99mTc-PQQ at room temperature

Fig. 5

Time course of ^99mTc-PQQ (closed square) in blood after intravenous administration of ^99mTc-PQQ (9.9 mg kg⁻¹) to ICR mice (n = 6, mean %ID g⁻¹ ±SD)

Fig. 6

Autoradiography of NMDAR in vitro by storage phosphor imaging in vitro autoradiography of sagittal brain sections of rat shown the distribution of ^99mTc-PQQ. (A, A′: brain section without EB pretreated. B, B′: control section of EB-pretreated, which displays a relatively low uptake of ^99mTc-PQQ in the hippocampus.)