Synthesis and in Vitro and in Vivo Evaluation of ¹⁸F-Labeled Positron Emission Tomography (PET) Ligands for Imaging the Vesicular Acetylcholine Transporter

A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the benzovesamicol structure was synthesized, and analogues were evaluated in vitro. (±)-trans-2-Hydroxy-3-(4-(4-[18F]fluorobenzoyl)piperidino)tetralin (9e) has Ki values of 2.70 nM for VAChT, 191 nM for σ1, and 251 nM for σ2. The racemic precursor (9d) was resolved via chiral HPLC, and (±)-[18F]9e, (−)-[18F]9e, and (+)-[18F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with [18F]/F− and Kryptofix/K2CO3 in DMSO with radiochemical yields of ∼50−60% and specific activities of >2000 mCi/μmol. (−)-[18F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT with an initial uptake of 0.911 %ID/g in rat striatum and a striatum/cerebellum ratio of 1.88 at 30 min postinjection (p.i.). MicroPET imaging of macaques demonstrated a 2.1 ratio of (−)-[18F]9e in putamen versus cerebellum at 2 h p.i. (−)-[18F]9e has potential to be a PET tracer for clinical imaging of the VAChT

Fluorine-18-Labeled Benzamide Analogues for Imaging the σ₂ Receptor Status of Solid Tumors with Positron Emission Tomography

A series of fluorine-containing benzamide analogs was synthesized and evaluated as candidate ligands for positron emission tomography (PET) imaging of the sigma-2 (σ2) receptor status of solid tumors. Four compounds having a moderate to high affinity for σ2 receptors and a moderate to low affinity for sigma-1 (σ1) receptors were radiolabeled with fluorine-18 via displacement of the corresponding mesylate precursor with [18F]fluoride. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated that all four F-18-labeled compounds had a high tumor uptake (2.5−3.7% ID/g) and acceptable tumor/normal tissue ratios at 1 and 2 h post-i.v. injection. An analysis of the chemistry and biodistribution data suggested that N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-methylbenzamide ([18F]3c) and N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-iodo-3-methoxybenzamide ([18F]3f) are acceptable compounds for imaging the σ2 receptor status of solid tumors

Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS

Correlation of PET measures of tumor proliferation to subsequent changes in tumor volume.

<p>(<b>A</b>) Correlation between normalized FDG total uptake and percent change in volume between consecutive imaging sessions in untreated MNU rats. (<b>B</b>) Correlation between normalized [¹⁸F]ISO-1 total uptake and percent change in volume between consecutive imaging sessions in untreated MNU rats. The correlation coefficient is denoted by R = 0.68 significant at P<0.003. MRI and PET images were typically acquired within a ±1 day, which could explain some of the variability along the fitted line in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074188#pone-0074188-g007" target="_blank">Figure 7B</a>.</p

MR Tumor volume measured at baseline and at 2-week intervals.

<p>(A) MR-derived tumor volume (B) Percent change in tumor volume between consecutive imaging sessions for a select group of tumors. In analyzing the data, we exploited on the lack of consistency in tumor increase and correlated changes in tumor volume to baseline measures of normalized [¹⁸F]ISO-1 uptake with the underlying notion that change in tumor volume is predicated on the proliferative status of the tumor. (C) Representative time course imaging of MNU-induced tumors at baseline, 2 weeks, 4 weeks, 6 weeks, and 8 weeks with MRI, FDG and [¹⁸F]ISO-1. Note: time-course images are on differing color scale for each time point.</p

Time-course of FDG and [¹⁸F]ISO-1 mean SUV normalized to baseline for bexarotene- and Vorozile-treated MNU rats.

<p>Top row: (<b>A</b>) FDG mean SUV and (<b>B</b>) [¹⁸F]ISO-1 mean SUV for bexarotene-treated rats. Bottom row: (<b>C</b>) FDG mean SUV and (<b>D</b>) [¹⁸F]ISO-1 mean SUV for Vorozole-treated rats. Treatment was provided for 8 weeks. Following the imaging session at week 8, treatment was withdrawn. (<b>E</b>) Time course of treatment is segmented into the short term efficacy (week 0–2) and tumor response to treatment withdrawal (weeks 8–10).</p

Normalized tumor volume in bexarotene- treated MNU rats (A) and Vorozole (B).

<p>The legend in each plot denotes tumor ID which matches tumor IDs of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074188#pone-0074188-g006" target="_blank">Figure 6</a>, where applicable. Treatment was provided as described in the methods section for 8 weeks. Following the imaging session at week 8, treatment was withdrawn. (<b>C</b>) Time course of treatment is segmented into the short term efficacy (week 0–2) and tumor response to treatment withdrawal (weeks 8–10).</p

Characterization of the pharmacokinetics of [¹⁸F]ISO-1 and in-vitro determination of Sigma-2 Receptor Density.

<p>(<b>A</b>) A 2-hour sum image depicting two MNU-induced tumors and the submandibular (S/M). The liver is evident in the coronal slices. (<b>B</b>) Time activity curves of the two tumors, muscle, and the left-ventricular blood pool. Inset figure depicts kinetics at initial 5 min. (<b>C</b>) Representative saturation binding experiments which show the total bound, non-specific bound and specific bound. (<b>D</b>) Representative Scatchard plots which were used to determine <i>K_d</i>, <i>B_max</i> and <i>n</i>_H values.</p

SUV 10-minute sum images at 60-minute post-injection of FDG (top row) and [¹⁸F]ISO-1 (middle row).

<p>Bottom row depicts the corresponding MR image at each time point (week 8 image is missing). Panel depicts treatment of tumor C1-1_L3 with bexarotene. Note that following the imaging session at week 8, treatment was withdrawn.</p

Validation of in-vivo measures of tumor proliferation using mammary 66 tumors.

<p>(<b>A</b>) Sum microPET images of sigma-2 ligand [¹⁸F]ISO-1 (top panel) and [¹⁸F]FDG (bottom panel) in 66 mammary tumors with varying P:Q ratios. Correlation between [¹⁸F]ISO-1<b>,</b> [¹⁸F]FDG PET measures and P:Q ratio. Radiotracer tracer uptake tumor to background ratio was plotted against the P:Q ratio and linear regression analysis were performed. (<b>B</b>) [¹⁸F]ISO-1 tumor to background ratio linearly correlates with P:Q ratio with a correlation coefficient R = 0.92 and steep slope of 0.47; (<b>C</b>) [¹⁸F]FDG tumor to background ratio slightly correlates with P:Q ratio, R = 0.37, with a slope of 0.16.</p