背景:慢性神经炎症和小胶质细胞功能障碍是许多神经系统疾病的关键特征,包括阿尔茨海默病和多发性硬化症。虽然不幸的是缺乏用于体内研究小胶质细胞的高选择性分子成像生物标志物/探针,P2Y12R已成为有吸引力的候选PET生物标志物,正在为此目的进行探索。重要的是,P2Y12R在CNS的小胶质细胞上选择性表达,并根据炎症背景经历表达的动态变化(例如,毒性与有益/愈合状态),从而有可能揭示活体受试者中有关小胶质细胞的功能信息。在这里,我们发现了一种高亲和力,小分子P2Y12R拮抗剂(AZD1283)的放射性标记,并通过野生型和完全敲除小鼠和非人灵长类动物的体外测定和体内正电子发射断层扫描(PET)成像评估为候选放射性示踪剂。
方法:首先,我们在体外评估了非放射性AZD1283的代谢稳定性和被动通透性。接下来,我们用放射性前体[11C]NH4CN放射性标记[11C]AZD1283,并确定了在制剂和人血浆中的稳定性。最后,我们通过原始野生型小鼠的动态PET成像研究了[11C]AZD1283的体内稳定性和动力学,P2Y12R基因敲除小鼠,和一只恒河猴.
结果:我们确定AZD1283在小鼠和人肝微粒体中的半衰期为37和>160分钟,分别,并预测中枢神经系统的被动摄取与少量的主动外排,使用Caco-2测定。我们的放射性标记工作提供了[11C]AZD1283,其活性为12.69±10.64mCi,具有高化学和放射化学纯度(>99%),摩尔活性为1142.84±504.73mCi/μmol(n=3的平均值)。值得注意的是,我们发现[11C]AZD1283在体外是高度稳定的,在制剂中孵育90分钟后和在人血清中孵育60分钟后存在>99%完整示踪剂。PET成像在健康野生型小鼠(n=3)和P2Y12敲除小鼠(注射后5分钟时0.55±0.37%ID/g)中显示可忽略的脑信号。引人注目的是,在给药的前20分钟内,在所有小鼠的肝脏中检测到高信号(注射后5分钟,峰值摄取=58.28±18.75%ID/g),并在扫描的剩余持续时间内持续存在.注射后60分钟小鼠组织的离体γ计数反映了体内数据,血液中的平均%ID/g为0.9%±0.40、0.02%±0.01和106±29.70%,大脑,还有肝脏,分别(n=4)。鼠类血液和肝脏代谢物样品的高效液相色谱(HPLC)分析显示单个放射性峰(峰下相对面积:100%),代表完整的示踪剂。最后,恒河猴的PET成像还显示在猴脑中可忽略的CNS摄取/结合(峰值摄取=0.37标准摄取值(SUV))。
结论:尽管我们最初令人鼓舞的肝微粒体和Caco-2单层数据,除了观察到[11C]AZD1283在制剂和人血清中的高稳定性外,体内脑摄取可忽略不计,并且在幼稚野生型和P2Y12R敲除小鼠的肝脏中观察到快速积累。肝脏信号似乎独立于代谢和P2Y12R表达,这是由于对于野生型和P2Y12R敲除小鼠都确认了该组织中的完整示踪剂。在恒河猴,[11C]AZD1283脑的可忽略的摄取表明缺乏体内翻译或其进一步研究的潜力。P2Y12R是一个非常有前途的潜在的PET生物标志物,这里提供的数据表明,这种支架具有令人鼓舞的代谢稳定性;然而,在进一步开发类似物之前,应阐明小鼠肝脏摄取的机制。
Chronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer\'s Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly,
P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule
P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate.
First, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [11C]AZD1283 with radioactive precursor [11C]NH4CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [11C]AZD1283 via dynamic PET imaging of naïve wild-type mice,
P2Y12R knockout mouse, and a rhesus macaque.
We determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and > 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [11C]AZD1283 in an activity of 12.69 ± 10.64 mCi with high chemical and radiochemical purity (>99%) and molar activity of 1142.84 ± 504.73 mCi/μmol (average of n = 3). Of note, we found [11C]AZD1283 to be highly stable in vitro, with >99% intact tracer present after 90 min of incubation in formulation and 60 min of incubation in human serum. PET imaging revealed negligible brain signal in healthy wild-type mice (n = 3) and a P2Y12 knockout mouse (0.55 ± 0.37%ID/g at 5 min post injection). Strikingly, high signal was detected in the liver of all mice within the first 20 min of administration (peak uptake = 58.28 ± 18.75%ID/g at 5 min post injection) and persisted for the remaining duration of the scan. Ex vivo gamma counting of mouse tissues at 60 min post-injection mirrored in vivo data with a mean %ID/g of 0.9% ± 0.40, 0.02% ± 0.01, and 106 ± 29.70% in the blood, brain, and liver, respectively (n = 4). High performance liquid chromatography (HPLC) analysis of murine blood and liver metabolite samples revealed a single radioactive peak (relative area under peak: 100%), representing intact tracer. Finally, PET imaging of a rhesus macaque also revealed negligible CNS uptake/binding in monkey brain (peak uptake = 0.37 Standard Uptake Values (SUV)).
Despite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [11C]AZD1283 in formulation and human serum, in vivo brain uptake was negligible and rapid accumulation was observed in the liver of both naïve wildtype and P2Y12R knockout mice. Liver signal appeared to be independent of both metabolism and
P2Y12R expression due to the confirmation of intact tracer in this tissue for both wildtype and
P2Y12R knockout mice. In Rhesus Macaque, negligible uptake of [11C]AZD1283 brain indicates a lack of potential for translation or its further investigation in vivo. P2Y12R is an extremely promising potential PET biomarker, and the data presented here suggests encouraging metabolic stability for this scaffold; however, the mechanism of liver uptake in mice should be elucidated prior to further analogue development.