Radiosynthesis and initial preclinical evaluation of [11C]AZD1283 as a potential P2Y12R PET radiotracer

Abstract

Intro: 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.

Methods: 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.

Results: 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)).

Conclusion: 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.

Keywords: Carbon-11; Cyanation; Microglia; Neuroinflammation; P2Y12R; PET.

Fig. 1.

P2Y12R Antagonists (A) AZD1283 with potential radio-labelling site highlighted in red, (B) [¹¹C]5, and (C), (D) new thienopyridine based radiotracers with labelling site highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

(A) Radiosynthetic scheme for [¹¹C]AZD1283. (B) HPLC chromatograms confirming identity of [¹¹C]AZD1283 (gamma, red) co-injected with cold AZD1283 standard (UV 214 nm, blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

HPLC analysis of human plasma samples after 0 (red), 5 (blue), 15 (green), 30 (pink), and 60 (brown) minutes incubation showed [¹¹C]AZD1283 to be metabolically stable in vitro, with no loss of parent tracer or appearance of radio-metabolites. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Summed representative PET/CT images of a naïve wild-type female mouse at (A) 0–45 s, (B) 10–20 min, (C) 30–40 min and (D) 50–60 min after intravenous injection of [¹¹C]AZD1283. H = heart, Liv = liver, and black arrows point to carotid arteries (i.e., tracer signal in blood pool).

Fig. 5.

(A) Time-activity curves (TACs) showing PET signal in the whole brain of 3 naïve wild-type mice and 1 P2Y12 KO mouse over the duration of a 60 min dynamic scan. (B) TAC of PET signal in the liver of 3 naïve wild-type mice throughout 60 min dynamic scan. (C) Graph of radioactive signal in tissues from naïve wild-type mice (n = 4) using ex vivo gamma counting. (D) HPLC chromatograms of processed supernatant extracted from liver and blood samples from mice perfused 30 min after injection of [¹¹C]AZD1283.

Fig. 6.

Sagittal PET image of rhesus macaque summed 0–60 min showed negligible CNS uptake/binding, reflected by (A) Sagittal microPET brain images (n = 2), and (B) low peak brain SUV (peak SUV = 0.37).