Evaluation of [18F]Fluoromethyl PBR28 as a Translocator Protein (18 kDa) PET Ligand for Imaging Neuroinflammation

Abstract

목적: 중추 신경계 질환에서 미세아교세포의 translocator protein (18 kDa)(TSPO)의 발현은 신경 염증 과정 중의 세포 활성화를 평가하는 생체 내 바이오 마커로 활용할 수 있다. 본 연구는 새로운 TSPO PET 방사성추적자로 18F 표지 fluoromethyl moiety를 가진 PBR28 유도체([18F]FM-PBR28)의 합성 및 신경 염증 질환에서 [18F]FM-PBR28의 유용성을 평가하고자 한다. 방법: [18F]FM-PBR28은 PBR28-OH에 triazolium triflate를 도입한 화합물을 전 구체로 사용하고 일 단계 과정으로 18F를 치환하는 새로운 방법으로 합성하였다. [11C]PBR28은 기존의 알려진 방법에 따라 phenolic PBR28 전 구체에 [11C]MeOTf를 이용, FX C pro module에서 합성하였다. FM-PBR28과 PBR28의 TSPO에 대한 체외 affinity(IC50)는 사람 백혈구 막에서 [3H]PK11195와의 경쟁적 결합으로 그리고 lipophilicity는 octanol-buffer 분배 계수 (partition coefficient)로 측정하였다. [18F]FM-PBR28 및 [11C]PBR28 PET study는 lipopolysaccharide (LPS)를 편측 선조체에 주입 후 4일 된 신경 염증 쥐 모델에서 시행하였다 (each group, n=5). 또한, [18F]FM-PBR28의 특이도(specificity)와 선택성(selectivity)을 평가하고자 PBR28 (5 mg/kg)를 사용하여 displacement PET study (n=4)를 하였고, FM-PBR28 (5 mg/kg) 그리고 flumazenil (5 mg/kg)를 사용하여 co-injection PET study (each group, n=3)를 시행하였다. 결과: [18F]FM-PBR28의 방사화학적 수율은 25.8 ± 3.2% 이었으며, 방사화학적 순도는 99% 이상이었다. [18F]FM-PBR28은 reverse-phase HPLC system으로 분리/정제하였으며, tC18 Sep-Pak cartridge를 이용, 5% EtOH/saline으로 제조하였다. [11C]CO2를 이용한 [11C]PBR28의 방사화학적 수율은 27.1 ± 4.9 % 이었다. FM-PBR28과 PBR28의 체외 affinity (IC50)와 log D는 서로 유사한 함을 확인하였다(IC50: 8.28 versus 8.07 nM

Log D: 2.85 versus 3.01). 신경염증 모델 쥐에서 촬영한 [18F]FM-PBR28 PET study에서 LPS를 주사한 편측 선조체는 반대측 선조체에 비하여 3.4배 이상의 높은 섭취를 보였으며(p = 0.008), [11C]PBR28영상과 비교하였을 때 조기에 높은 좌우 선조체 비를 보였다(3.9 at 35 분 versus 4.3 at 115 분). Displacement PET study에서 [18F]FM-PBR28 주사 30분 후에 PBR28의 주입은 편측 선조체의 급격한 섭취감소를 보임으로써 [18F]FM-PBR28의 특이적인 TSPO binding을 확인할 수 있었다 (p = 0.029). Co-injection PET study에서 [18F]FM-PBR28과 동시에 FM-PBR28를 주입하였을 때, 편측 선조체의 섭취가 효과적으로 저해됨을 확인하였다 (p = 0.036). 그러나 central benzodiazepine receptor에 특이적으로 결합하는 flumazenil의 동시주입은 편측 선조체의 섭취에 영향을 주지 않았으며 (p = 0.250), 이는 [18F]FM-PBR28이 TSPO에 선택적으로 결합 함을 알 수 있었다. 결론: [18F]FM-PBR28는 반감기가 긴 18F 표지 fluoromethyl moiety를 가지고 합성된 TSPO PET 방사성 추적자로서 신경염증 쥐 모델의 좌우 선조체에서 유의한 섭취 차이를 보였고, 더하여 [11C]PBR28보다 초기에 높은 좌우 선조체 대조도를 가졌다. 그러므로 [18F]FM-PBR28는 중추 신경계 염증 질환의 평가에 유용하게 활용될 수 있을 것으로 기대된다.

Purpose: The neurodegenerative condition with neuronal loss was well correlated with increased upregulation of the translocator protein (18 kDa) (TSPO), which was formerly known as the peripheral benzodiazepine receptor by activated microglia. Here, we detail the synthesis of a newly developed [18F]fluoromethyl-PBR28 (N-acetyl-N-(2-[18F]fluoromethoxybenzyl)-2-phenoxy-5-pyridinamine) as a derivative of PBR28 ([18F]FM-PBR28) and evaluate the suitability of [18F]FM-PBR28 as a biomarker of neuroinflammatory disease. Materials and Methods: [18F]FM-PBR28 was prepared from nucleophilic aliphatic substitution on the triazolium triflate precursor of the PBR28 precursor with fluorine-18 in a single-step radiolabeling procedure. [11C]PBR28 was produced in FX C pro module using [11C]MeOTf from the phenolic PBR28 precursor according to the literature. The TSPO binding affinity and lipophilicity were measured by competition with [3H]PK11195 in the membrane of a human leukocyte and the octanol-buffer partition coefficient, respectively (n=4). Four days after injection of lipopolysaccharide (LPS) in the ipsilateral striatum, neuroinflammatory positron emission tomography (PET) studies using [18F]FM-PBR28 and [11C]PBR28 were performed (n=5). Additional neuroinflammatory PET studies involving the displacement with unlabeled PBR28 (5 mg/kg, n=4), and the co-injection with unlabeled FM-PBR28 (5 mg/kg, n=3), and flumazenil (5 mg/kg, n=3) were performed to evaluate the specificity and selectivity of [18F]FM-PBR28. Results: [18F]FM-PBR28 has been efficiently synthesized in 25.8 ± 3.2% radiochemical yield. The final pure [18F]FM-PBR28 was purified by a reverse-phase HPLC system, and the collected solution was reformulated with 5% EtOH/saline using a tC18 Sep-Pak cartridge with a radiochemical purity of over 99%. [11C]PBR28 was produced in 27.1 ± 4.9% of radiochemical yield based on [11C]CO2. FM-PBR28 and PBR28 exhibited similar in vitro binding affinity (IC50) and log D values (8.28 versus 8.07 nM and 2.85 versus 3.01, respectively). In a neuroinflammatory rat model, an over 3.4 higher level of uptake of [18F]FM-PBR28 than that of contralateral striatum (p = 0.008) was observed in the ipsilateral striatum. [18F]FM-PBR28 PET studies exhibited an early peak uptake ratio of the ipsilateral and contralateral striatum than that of [11C]PBR28 (3.9 at 35 min versus 4.3 at 115 min). The displacement PET study using unlabeled PBR28 30 min after the injection of [18F]FM-PBR28 resulted in PET ligand washout, indicating the specific TSPO binding of [18F]FM-PBR28 (p = 0.029). Co-injection with unlabeled FM-PBR28 effectively inhibited the uptake of [18F]FM-PBR28 in the ipsilateral striatum (p = 0.036), but not with unlabeled flumazenil (p = 0.250), which is known to bind to the central benzodiazepine receptor, demonstrating [18F]FM-PBR28 selectivity and specificity to the TSPO. Conclusion: This study demonstrated that [18F]FM-PBR28 is a promising TSPO PET ligand, using a novel direct radiofluorination method, for the introduction of the [18F]fluoromethyl moiety. In addition, [18F]FM-PBR28 exhibited an excellent TSPO specific ratio at early times compared with that of [11C]PBR28. Thus, [18F]FM-PBR28 is a specific TSPO PET ligand with a long half-life that provides an appropriate tool for the evaluation of neuroinflammatory disease.