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. 2023 Oct 25;6(11):1734–1744. doi: 10.1021/acsptsci.3c00175

Molecular Imaging of Monoamine Oxidase A Expression in Highly Aggressive Prostate Cancer: Synthesis and Preclinical Evaluation of Positron Emission Tomography Tracers

Kevin Zirbesegger †,‡, Laura Reyes †, Andrea Paolino †, Rosina Dapueto †, Florencia Arredondo †, Juan P Gambini †, Eduardo Savio †,*, Williams Porcal §,*
PMCID: PMC10653014  PMID: 37982127

Abstract

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The role of monoamine oxidase A (MAO-A) in the aggressiveness of prostate cancer (PCa) has been established in recent years. The molecular imaging of MAO-A expression could offer a noninvasive tool for the visualization and quantification of highly aggressive PCa. This study reports the synthesis and preclinical evaluation of 11C- and 18F-labeled MAO-A inhibitors as positron emission tomography (PET) tracers for proof-of-concept studies in animal models of PCa. Good manufacturing practice production and quality control of these radiotracers using an automated platform was achieved. PET imaging was performed in an LNCaP tumor model with high MAO-A expression. The tumor-to-muscle (T/M) uptake ratio of [11C]harmine (4.5 ± 0.5) was significantly higher than that for 2-[18F]fluoroethyl-harmol (2.3 ± 0.7) and [11C]clorgyline (2.0 ± 0.1). A comparable ex vivo biodistribution pattern in all radiotracers was observed. Furthermore, the tumor uptake of [11C]harmine showed a dramatic reduction (T/M = 1) in a PC3 tumor model with limited MAO-A expression, and radioactivity uptake in LNCaP tumors was blocked in the presence of nonradioactive harmine. Our findings suggest that [11C]harmine may serve as an attractive PET probe for the visualization of MAO-A expression in highly aggressive PCa. These radiotracers have the potential for clinical translation and may aid in the development of personalized therapeutic strategies for PCa patients.

Keywords: molecular imaging, positron emission tomography, carbon-11, fluorine-18, prostate cancer, MAO-A inhibitors

1.

Prostate cancer (PCa) is a significant public health issue as it is the second most frequently diagnosed cancer in men and the eighth largest cause of cancer-related deaths.1 Approximately one million cases of PCa were diagnosed worldwide in 2018, resulting in an estimated 359,000 deaths, representing 7.1% of the total male cancer mortality. Accurate diagnosis, grading, and staging of PCa are crucial for identifying the most appropriate treatments for each patient. However, the heterogeneity of this disease from both biological and clinical perspectives presents a significant challenge for evaluations involving imaging.2−4 Molecular imaging can provide a valuable tool that can be used to elucidate the molecular mechanisms involved in the development and progression of PCa.5−7 The development of new radiopharmaceuticals is key in the search for molecules that can specifically be directed toward molecular targets, allowing the characterization of pathological processes using noninvasive techniques, such as positron emission tomography (PET). PET radiotracers that can be applied in oncology are among the most active areas of radiopharmaceutical research. Two of the most commonly used positron-emitting radionuclides are carbon-11 and fluorine-18. 11C and 18F-labeled molecules, such as drugs, amino acids, peptides, and small biomolecules, have gained much attention for PET imaging in various disease conditions.8 Carbon-11 allows isotopic labeling, and its radiochemistry has been well studied in recent decades, but its short half-life (20.4 min) limits its use. In this context, fluorine-18 is more commonly used in radiolabeling due to its longer half-life (110 min), which provides advantages in radiosynthesis steps and for transporting it to other institutes.8 Numerous studies have demonstrated that malignant tumors can be detected with high sensitivity and specificity using PET imaging.9−11 PET imaging of PCa was initially demonstrated with various small-molecule radiopharmaceuticals as [11C]-choline, [11C]acetate and [18F]fluciclovine. Both [11C]choline and [18F]fluciclovine PET have been shown to detect nodal and osseous disease at higher rates compared to FDG-PET but offer no additional benefit in detecting prostate disease, especially in primary staging.12 Recently, studies with these tracers have shown lack of significant correlation for tumor uptake with tumor aggressiveness and limited accuracy in the diagnosis in PCa.12 The expression of prostate-specific membrane antigen (PSMA) has shown a positive correlation with tumor aggressiveness, metastatic disease, and castration resistance. This led to the development of various PSMA inhibitors labeled with carbon-11, fluorine-18, and gallium-68, showing remarkable results in both preclinical and clinical studies.13 However, PSMA is expressed in nonprostatic tissues as well as in other pathologic conditions. PSMA uptake is not prostate-specific and can be taken up physiologically and pathologically in nonprostatic tissue. It is important for reporting physicians to recognize these pitfalls.14 Enzymes are a desirable target for PET imaging because radiolabeled inhibitors can measure enzyme expression. Their associated changes can play a significant role in pathology.15,16 Furthermore, recent studies have established a direct relationship between the high degree of aggressiveness of PCa and the increased expression of the enzyme monoamine oxidase A (MAO-A).17,18 MAO-A is a mitochondria-bound enzyme that catalyzes the degradation of monoamine neurotransmitters and dietary amines via oxidative deamination. Several works have highlighted the need to validate MAO-A as a potential therapeutic target in the treatment of PCa.19−21 Different studies have emphasized the implications of MAO-A as a mediator of tumorigenesis and in the metastasis of PCa.21−23 The pharmacological inhibition of MAO-A activity and MAO-A knockdown has been shown to reduce or even eliminate prostate tumor growth and metastasis in PCa xenograft mouse models. High MAO-A expression has also been correlated with worse clinical outcomes in PCa patients and higher levels of serum prostate-specific antigen in patients at the diagnosis stage.24,25 Androgen deprivation therapy is the standard treatment for metastatic PCa, but most available antiandrogens eventually fail, including the recently developed potent antiandrogen enzalutamide.26,27 Therefore, there is a need for alternative treatment options. Recent studies have shown that MAO-A inhibitors, such as clorgyline and phenelzine (an FDA-approved nonselective MAO-A inhibitor for depression), could reverse enzalutamide resistance in castration-resistant prostate cancer patients.28 These results highlight the potential of MAO-A as a molecular target of already known antidepressants as a new therapy, either as a single drug or in combination with current standard antiandrogens. Currently, phenelzine is under phase II clinical trials for nonmetastatic recurrent PCa (ClinicalTrials.gov Identifier: NCT02217709).29 Given this context, MAO-A is a viable molecular imaging target in high-grade aggressive PCa.24,30 The ability of PET techniques to noninvasively molecular image biochemical processes has numerous implications for clinical as well as basic scientific research.31 For example, the translation of molecular imaging to clinical applications to achieve personalized treatment will require specific tracer approaches.32,33 Using such a method, valuable information regarding the stage and degree of malignancy and metastasis in PCa could be provided as well as the possibility to propose specific therapies for patients with tumors that overexpress MAO-A. Thus, MAO-A would have a dual function in both diagnosis and therapy. These characteristics reinforce the need to study MAO-A as a target of molecular imaging for its applicability in the evaluation and diagnosis of highly aggressive PCa. Such an understanding is key for targeted imaging and therapy and for predicting and evaluating treatment responses and prognosis. In this work, we describe the synthesis and evaluation of 11C- and 18F-labeled MAO-A inhibitor molecules to enable in vivo proof-of-concept studies in animal models of PCa. This goal was achieved through in vivo PET imaging studies and ex vivo biodistribution assays in LNCaP (MAO-A positive cell line) and PC3 (MAO-A negative cell line) tumor-bearing nude mice. The tumor uptake of the irreversible MAO-A inhibitor [11C]clorgyline and reversible MAO-A inhibitors [11C]harmine and 2-[18F]fluoroethyl-harmol was evaluated (Figure 1).

Figure 1.

Figure 1

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Chemical structures of selected 11C- and 18F-labeled MAO-A tracers.

2. Results and Discussion

The selection of the PET radiotracers labeled with carbon-11 was based on their wide use in the study of the enzymatic properties of MAO-A in the central nervous system, both in animal models and in humans.30,34−36 In this context, Långström’s group reported the development and application of PET tracers, such as [11C]clorgyline and [11C]harmine.37 Their studies have also shown the high expression of MAO-A in urinary bladder cancer, neuroendocrine tumors, and pancreas cancer, initially demonstrated in vitro and in vivo studies and then in patients using [11C]harmine.38−40 These promising results suggested the possibility of performing in vivo characterization of MAO-A using [11C]harmine PET for diagnostic purposes or for treatment monitoring. More recently, other groups have shown that a derivative of harmine labeled with fluorine-18 (2-[18F]fluoroethyl-harmol) exhibited excellent affinity for MAO-A both in vitro and in vivo, proving again that this radiotracer is a promising candidate for the visualization of MAO-A by PET.41,42 In this context, our first step was to obtain the corresponding labeling precursors and the analytical standards for the selected 11C- and 18F-labeled MAO-A tracers.

2.1. Chemistry

2.1.1. Chemical Synthesis

The precursor nor-clorgyline 4 and nonradioactive standard clorgyline 5 were synthesized according to the routes shown in Scheme 1 using 2,4-dichlorophenol as the starting material. The first reaction of the synthetic pathway was adapted from Ohmomo et al. and involved O-alkylation on 2,4-dichlorophenol using 3-bromo-1-propanol as an alkylating agent to obtain an excellent yield of alcohol 1.43 Next, the activation of alcohol 1 by tosylation was performed according to Berardi et al., with slight modifications to yield compound 2.44 To obtain the analytical standard of [11C]clorgyline, compound 5, the next step was performed according to the reaction between N-methylpropargylamine and compound 2, obtaining a 66% yield (condition e, Scheme 1).45 To synthesize the labeling precursor nor-clorgyline 4, we explored a methodology that would minimize competitive reactions of polyalkylation on derivative 2. Using the solid-phase Staudinger’s methodology from azide derivatives and triphenylphosphine resin, followed by N-alkylation and subsequent cleavage, efficiently yielded secondary amines.46 Based on this, first, we carried out the previous transformation of compound 2 in azide 3. Next, the reaction between azide 3 and polymer-bound triphenylphosphine generated the corresponding supported iminophosphorane (Aza–Wittig reaction), which via reaction with propargyl bromide and 2% KOH in MeOH yielded desired nor-clorgyline 4 (condition d, Scheme 1).

Scheme 1. Synthesis Route of the Labelling Precursor and the Analytical Standard for Clorgyline.

Scheme 1

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Reagents and conditions: (a) 3-bromo-1-propanol, NaOH (aq), EtOH, reflux, 4 h, 96%; (b) p-TsCl, Et3N, CH2Cl2, rt, 25 h, 67%; (c) NaN3, DMF, 65 °C, 2 h, 88%; (d) (i) triphenylphosphine resin, THF, 100 °C, 1 h; (ii) propargyl bromide, 85 °C, 22 h; (iii) MeOH, KOH 2% (in MeOH), reflux 2 h, 30%; and (e) N-methylpropargylamine, Et3N, DMF, rt, 7 d, 66%.

Harmol 7 was used both as the labeling precursor of [11C]harmine and in the synthesis of the labeling precursor and analytical standard of 2-[18F]fluoroethyl-harmol, derivatives 8 and 9, respectively (Scheme 2). Harmol was obtained from the demethylation of commercial harmine performed in typical conditions, as described by Schieferstein et al.42 Precursor 8 was obtained by monosubstitution of ethylene glycol ditosylate 10 with harmol using the slightly modified protocol of Blom et al. (Scheme 2).47 The reference compound 2-fluoroethyl-harmol 9 was synthesized via the O-alkylation of phenol 7 with 2-fluoroethyltosylate 11 under refluxing in ethanol with a good yield. Previously, derivative 11 was obtained via the reaction between potassium fluoride/Kryptofix 222 and ditosylate 10 in refluxing acetonitrile. The structure of compounds was confirmed using analytical and spectroscopic techniques, such as 1H NMR mono and bidimensional, 13C NMR, heteronuclear single quantum coherence spectroscopy, and heteronucear multiple bond correlation experiments. The signals were coincident with those previously reported.

Scheme 2. Synthesis Routes of the Labelling Precursors and Analytical Standards for Harmine Derivatives.

Scheme 2

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Reagents and conditions: (a) HBr, AcOH, reflux, 24 h, 90%; (b) Cs2CO3, DMF, 10, rt, 26 h, 46%; (c) 11, dry EtOH, reflux, 6 h, 89%; (d) p-TsCl, Et3N, CH2Cl2, rt, 7 d, 70%; and (e) KF, K2.2.2, MeCN, reflux, 4 h, 16%.

2.1.2. Radiochemistry

Radiosynthesis of [11C]clorgyline ([11C]5) was initially reported by the N-[11C]methylation of the desmethyl precursor 4 using [11C]MeI as the methylating agent.48 Here, we performed the radiolabeling using the more reactive [11C]methyl trifluoromethanesulfonate ([11C]MeOTf) (Scheme 3) and a purification method based on our previous reports (see the Experimental Section).45,49 The fully automated synthesis of [11C]5 was performed using the commercial platform GE TRACERlab FX C Pro (Figure S6). This methodology afforded us the potential of [11C]MeOTf in the improvement and reproducibility of radiochemical yields. [11C]MeOTf was prepared by passing [11C]CH3I through a heated column filled with silver triflate and a GraphPAC-GC 80/100. The resulting [11C]MeOTf was subsequently trapped in a solution of 4 in anhydrous methyl ethyl ketone (MEK) at −15 °C and then heated at 80 °C for 1 min. The labeled product [11C]clorgyline was obtained with a 44 ± 12% (end of synthesis, decay-corrected from[11C]MeI) radiochemical yield from [11C]MeI after 38 ± 3 min synthesis time, with molar activities of 416 ± 62 GBq/μmol and a radiochemical purity over 97 ± 1% (including HPLC purification). We also adapted a previously reported methodology in our platform to obtain [11C]clorgyline using [11C]MeI as the 11C-methylating agent.50 However, the results were not as encouraging as the results obtained using our developed methodology (Table S1). Harmine was labeled with carbon-11 via the O-[11C]methylation of harmol using [11C]MeI as the methylating agent (Scheme 3). The fully automated preparation of [11C]harmine ([11C]6) was performed as reported previously by Philippe et al. with slight modifications, such as a different semipreparative RP-HPLC column and a C18 SPE cartridge with less charge (SEP-PAK C18 light).51 Furthermore, formulation was done with 10 mL of physiological saline (0.9%) as performed for [11C]clorgyline, with a final total volume of 11 mL (8% ethanol). The labeled product [11C]harmine was obtained with a 45 ± 6% radiochemical yield (end of synthesis, decay-corrected from [11C]MeI), molar activity of 513 ± 155 GBq/μmol, and radiochemical purity over 99.9 ± 0.1% (including HPLC purification). It should be noted that we initially tried to reproduce the labeling conditions described by Murthy et al. for [11C]harmine.52 Using harmol as the precursor and [11C]MeOTf as the methylating agent for 5 min of labeling at room temperature in anhydrous ketone, the radiotracer [11C]6 was not obtained. We observed that harmol has poor solubility in the anhydrous ketone. Furthermore, our attempts to perform the same labeling conditions in anhydrous DMSO were unsuccessful in obtaining [11C]6. The radiosynthesis of 2-[18F]fluoroethyl-harmol ([18F]9) was achieved via direct 18F-fluorination of tosylated precursor 8 (Scheme 3). The fully automated radiosynthesis of [18F]9 was performed in the Synthra RN Research Plus platform (Figure S11). The radiolabeling conditions of derivative 8 to obtain [18F]9 were adapted from Cumming et al. (Scheme 3).53 The purification by semipreparative HPLC using the process described in the literature was unsuccessfully performed in our platform. Thus, we set up the conditions for purification by semipreparative HPLC by adapting the analytical HPLC method (see the Experimental Section). The labeled 2-[18F]fluoroethyl-harmol was obtained with a 19.5 ± 4% radiochemical yield (end of synthesis, decay-corrected) and a radiochemical purity over 99.6 ± 0.1% (including HPLC purification) after a total synthesis time of 58–62 min (molar activity of 900 ± 300 GBq/μmol).

Scheme 3. Radiosynthesis of [11C]Clorgyline, [11C]Harmine, and 2-[18F]Fluoroethyl-harmol.

Scheme 3

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Reagents and conditions: (a) [11C]MeOTf, MEK, 80 °C, 1 min; (b) [11C]MeI, NaOH (5 M), DMSO, 80 °C, 2 min; and (c) K18F, K2.2.2, DMSO, 120 °C, 5 min.

2.2. Biological Assays

2.2.1. Small Animal PET Imaging

PET studies combined with computed tomography (PET/CT) were carried out with [11C]clorgyline, 2-[18F]fluoroethyl-harmol, and [11C]harmine to evaluate the tumor uptake of the tracers in a tumor-bearing nude mice model. To determine the MAO-A expression in the LNCaP and PC3 cell lines, immunoblotting was performed. The cell lines showed high (LNCaP) or reduced (PC3) basal levels of MAO-A expression (Figure S14), which was consistent with previous studies.21 The histological grade of tumors extracted from the LNCaP xenograft mice model confirmed highly aggressive PCa (Gleason Score 5, Figure S15).18 At the beginning of our experiments, PET studies with [11C]clorgyline were carried out in animals inoculated with the LNCaP cell line using a high-resolution PET scanner. Imaging data were processed and analyzed using PMODE software to establish a volume of interest (VOI) in the region where the tumor was located and another VOI in the contralateral side muscle. PET images showed marked accumulation of [11C]clorgyline in the tumor and liver at 20 min p.i. (Figure 2 shows a representative PET-CT image). The highest tumor/muscle (T/M) uptake value (2.0 ± 0.1) was achieved in frame 1 (5–20 min, see Figure 2). Furthermore, in frame 2 (20–40 min) T/M uptake value decreased to 1.5 ± 0.1. Next, PET-CT studies were carried out with 2-[18F]fluoroethyl-harmol in four groups of animals inoculated with LNCaP. Images were preprocessed to obtain three frames of 15 min for each group of animals. The average T/M uptake ratio increased significantly from 30 to 90 min p.i. (frames 2–5), with the highest T/M uptake ratio value 2.3 ± 0.7 (n = 5) obtained in frame 5 (75–90 min) (Figure 3). The tumor was clearly visible with high contrast to the contralateral background (Figure 3). High brain uptake was observed due to basal MAO-A expression in this organ. No significant increase in the T/M uptake ratio of 2-[18F]fluoroethyl-harmol after 90 min was observed (Figure 3).

Figure 2.

Figure 2

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PET-CT imaging of [11C]clorgyline in a LNCaP tumor-bearing mouse. Representative images of [11C]clorgyline distribution in Frame 1 (5–20 min p.i.). Left: coronal plane. The purple circle corresponds to the VOI of the tumor. Within the green circle, a region on the contralateral side was delimited, which represents healthy muscle tissue. Right: sagittal plane.

Figure 3.

Figure 3

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Left: PET-CT imaging of 2-[18F]fluoroethyl-harmol in a LNCaP tumor-bearing mouse. Representative image (coronal and sagittal planes) of 2-[18F]fluoroethyl-harmol distribution in Frame 5 (75–90 min p.i.). The purple circle corresponds to the VOI of the tumor. Within the green circle, a region on the contralateral side was delimited, which represents healthy muscle tissue. Right: Time–T/M graph after IV injection of 2-[18F]fluoroethyl-harmol from 15 to 225 min (n = 5).

Finally, [11C]harmine was administered intravenously following the same [11C]clorgyline acquisition protocol. Initially, studies were performed with a group of 5 animals 15 min after intravenous administration and up to 60 min. Since the T/M uptake ratio of [11C]harmine showed an increase over time (Figure 4), a second group of 7 animals was studied from 60 to 90 min p.i. PET images in the LNCaP tumor-bearing mice model showed marked accumulation of [11C]harmine in tumors up to 90 min p.i. (Figure 4). The highest T/M uptake ratio value of 4.5 ± 0.5 was obtained between 75 and 90 min p.i. (Figure 4), making [11C]harmine the most effective radiolabeled MAO-A inhibitor assayed. A similar tumor uptake was previously observed for PSMA radiotracers, [18F]AlF-PSMA and [68Ga]-PSMA when PET studies were carried out in animals inoculated with the LNCaP cell line.54,55 These results also highlight the potential of MAO tracers as promising imaging biomarkers for highly aggressive PCa. PET-CT studies were also carried out in PC3 tumor-bearing nude mice, which showed limited expression of MAO-A, to confirm the specificity of [11C]harmine for the molecular target. Studies were performed from 45 min after intravenous administration up to 90 min based on the best results previously obtained in the LNCaP model (Figure 4). Images were preprocessed to obtain three frames of 15 min as previously performed to enable the comparison of the T/M uptake value with the LNCaP model (high MAO-A expression). As expected, significantly reduced uptake of [11C]harmine was observed in the PC3 tumor-bearing mice model, with the highest T/M uptake ratio value being 1.2 ± 0.2 obtained at 75–90 min (Figure 4). Brain uptake of [11C]harmine was high in both the LNCaP and the PC3 models. These findings validate our proof of concept, confirming the specificity of [11C]harmine for the molecular target.21

Figure 4.

Figure 4

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Above: PET-CT imaging of [11C]harmine in a LNCaP (left) and PC3 (right) tumor-bearing mouse. Representative images (coronal and sagittal planes) of [11C]harmine distribution in Frame 3 (75–90 min p.i.). The purple circle corresponds to the VOI of the tumor. Within the green circle, a region on the contralateral side was delimited, which represents healthy muscle tissue. Below: Time–T/M graph after IV injection of [11C]harmine from 45 to 90 min (n = 12).

2.2.2. Ex Vivo Biodistribution

To compare the in vivo distribution determined by analyzing PET-CT images with that obtained from the measurement of the activity in each organ/tissue, ex vivo biodistribution assays of [11C]clorgyline, 2-[18F]fluoroethyl-harmol, and [11C]harmine were performed in tumor-bearing nude mice. The biodistribution time was determined in agreement with the highest T/M uptake value obtained in PET-CT imaging studies.

Table 1 shows the results expressed as a percentage of injected dose per organ (%ID) in the most significant organs. First, [11C]clorgyline showed a T/M uptake value similar to that in the PET imaging results in LNCaP tumor-bearing mice at 20 min p.i. (T/M = 1.8 ± 0.1). However, T/M ratio uptake of 2-[18F]fluoroethyl-harmol at 90 min p.i. (T/M = 1.3 ± 0.1, Table S2) was lower than that in the PET imaging results (T/M = 2.3 ± 0.7, frame: 75–90 min, Figure 3). Nevertheless, the biodistribution of 2-[18F]fluoroethyl-harmol showed retention in the tumor over time. In fact, the highest T/M ratio uptake of 2-[18F]fluoroethyl-harmol was obtained at 225 min p.i. (Table 1), in agreement with the PET imaging results (2.3 ± 0.7, 75–90 min, Figure 3). Moreover, the biodistribution results of [11C]harmine in both LNCaP and PC3 tumor-bearing nude mice showed T/M uptake values that agree with the PET-CT imaging results, with T/M = 3.6 ± 0.5 vs 4.5 ± 0.5 in LNCaP and T/M = 1.6 ± 0.3 vs 1.2 ± 0.2 in PC3 (Table 1 and Figure 4, Table S3). Specificity of the radiotracer [11C]harmine for MAO-A was demonstrated through blocking experiments. Ex vivo biodistribution confirmed substantial decrease of radioactivity uptake (about 70% after 60 min) in the absence and presence of nonradioactive harmine in the LNCaP tumor (7.8% Act/g vs 2.6% Act/g, respectively, Supporting Information). Uptake in the brain was also reduced by about 82%. Taken together, these results confirm the previous findings obtained by PET imaging.

Table 1. Tissue Distribution of Radioactivity in Mice after Intravenous Injection of [11C]Clorgyline, 2-[18F]Fluoroethyl-harmol, and [11C]Harminea.
tissue % ID/g 20 min [11C]clorgyline % ID/g 225 min 2-[18F]fluoroethyl-harmol % ID/g 90 min [11C]harmine
  LNCaP (n = 4) LNCaP (n = 3) LNCaP (n = 4) PC3 (n = 3)
blood 1.8 ± 0.4 1.5 ± 0.3 1.3 ± 0.3 1.5 ± 0.5
liver 5.5 ± 0.6 1.3 ± 0.01 4.8 ± 1.0 4.4 ± 1.0
heart 2.2 ± 0.5 1.9 ± 0.04 1.8 ± 0.4 1.2 ± 0.1
lung 8.2 ± 2.8 7.2 ± 0.3 3.7 ± 0.5 2.8 ± 0.3
kidney 7.2 ± 1.5 4.0 ± 0.7 6.1 ± 1.0 3.9 ± 0.5
muscle 1.5 ± 0.2 1.6 ± 0.1 1.0 ± 0.1 0.9 ± 0.2
tumor 2.7 ± 0.5 3.2 ± 0.8 3.7 ± 0.5 1.3 ± 0.2
brain 3.4 ± 1.3 5.9 ± 0.2 4.7 ± 0.8 3.1 ± 0.6
T/M 1.8 ± 0.2 2.0 ± 0.4 3.6 ± 0.7 1.6 ± 0.3
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a

Mean % injected dose ± SD per gram of tissue of n animals. T/M: tumor/muscle uptake.

3. Conclusions

We evaluated the feasibility of using three selective MAO-A inhibitors labeled with carbon-11 or fluorine-18 to visualize in vivo MAO-A expression through PET images using a PCa xenograft mice model. All analytical standards and labeling precursors for [11C]clorgyline, [11C]harmine, and 2-[18F]fluoroethyl-harmol were efficiently synthesized, and labeling conditions were screened and optimized. We developed a fully automated radiosynthesis protocol to obtain [11C]clorgyline with [11C]MeOTf as the methylating agent in a TRACERLAB FX C Pro module as well as 2-[18F]fluoroethyl-harmol using a Synthra RN Research Plus module. To the best of our knowledge, this is the first study to describe a fully automated radiosynthesis protocol to obtain [11C]clorgyline and 2-[18F]fluoroethyl-harmol. PET tracers were evaluated in a tumor model with high MAO-A expression (using the LNCaP cell line) through small animal PET imaging studies and ex vivo biodistribution assays. We also assessed the most promising tracer, [11C]harmine, in a tumor-bearing mice model with limited MAO-A expression (using the PC3 cell line) as a negative control. We confirmed the specificity of [11C]harmine for the molecular target through both PET imaging and ex vivo biodistribution assays. Our proof-of-concept results suggest that 11C- and 18F-labeled MAO-A inhibitors could serve as promising imaging biomarkers for investigations into the involvement of MAO-A expression in preclinical models of highly aggressive PCa. These PET tracers for molecular imaging of MAO-A expression could help visualize and quantify highly aggressive PCa and could also drive new treatment opportunities for MAO-A-overexpressed tumors. Taken together, our results and previous animal and human data support the feasibility of [11C]harmine as a potential imaging tool for clinical translation, diagnosis, and therapeutic purposes in the treatment of PCa.

4. Experimental Section

4.1. Radiosynthesis

All PET tracers are >95% pure by HPLC analysis.

4.2. Materials

All chemicals and solvents were purchased from commercial sources (Merck, Sigma-Aldrich, and Carlo Erba). They were of analytical grade and used without further purification. The Sep-Pak C18 light and QMA plus light cartridges and 0.22 μm sterilizing filters were purchased from Waters. The semipreparative HPLC columns were a VP 250/10 mm Nucleosil 100–5 C18 Nautilus (Macherey-Nagel) used in the purification of 11C radiotracers and a VP 250/10 mm Nucleosil 100–5 C18 (Phenomenex) for the purification of 2-[18F]Fluoroethyl-harmol. The analytical HPLC column was an EC 250/4.6 mm Nucleodur 100–5 C18ec (Macherey-Nagel). The analytical gas chromatography (GC) column was a DB-WAX 30 m long, 0.53 mm in diameter, and with 1.00 mm film thickness (Agilent).

4.3 Instruments

[11C]CO2 and [18F]Fluoride were produced in a PET Trace 16.5 MeV cyclotron (GE Healthcare). Radiosynthesis of 11C and 18F radiotracers was carried out using either a TRACERlab FX C PRO module (GE Healthcare) and a Synthra RN Plus Research module (Synthra GmbH), respectively. Activity measurements were performed with a dose calibrator Capintec CRC 25R, CRC 25 PET or a 3” × 3” well type NaI (Tl) solid scintillation detector coupled to a multichannel analyzer ORTEC. HPLC analyses were performed with a Shimadzu UFLC instrument equipped with diode array and gamma detectors. The GC analyses of ethanol and residual solvents were done with a Shimadzu Nexis GC-2030 Plus equipped with an FID detector and an automatic injector Shimadzu AOC-20i Plus.

4.3.1. General Methodology of Radiosynthesis Procedures

Before the three validation production runs of all radiotracers were performed, the corresponding standard operating procedure protocols and documentation were created. All productions for in vivo assays were performed in clean areas in agreement with good manufacturing practice procedures. The conditioning of the TRACERlab FX C PRO module (GE Healthcare), preliminary steps, and the production of secondary precursors as [11C]MeI and [11C]MeOTf were performed as previously described by Buccino et al.49 The radiochemical purity was calculated considering the portion of carbon-11 or fluorine-18 of the radiolabeled product in relation to the total radioactivity. The specific activity was determined considering the total radiopharmaceutical activity at the end of synthesis and the amount of the unlabeled product. The residual solvents (such as acetone, MEK, DMSO, and acetonitrile) and the ethanol were analyzed by GC in accordance with USP general chapter ⟨467⟩. The appearance of the solution was checked by visual inspection, and pH was determined using a calibrated pH-meter. Radionuclidic purity was assessed by recording the corresponding gamma spectrum and the radionuclidic identity by measuring the physical half-life. Pyrogen was tested in a Endosafe PTS Cartridge (Charles River). The sterility test was performed according to chapter ⟨71⟩ of the USP [The United States Pharmacopeia (USP-40) and the National Formulary (NF-25)2017].

4.4. Radiosynthesis and Quality Control of [11C]Clorgyline

The automated radiosynthesis of [11C]clorgyline was performed on an TRACERlab FX C pro with the configuration as shown in Figure S6.

4.4.1. Radiolabeling Step

The radiolabeling process was performed in the reactor of the automated module. During test runs, the precursor solution contained 1.0 mg of 4 free base in 0.35 mL of different solvents: DMF/DMSO or methylethylketone (butanone, MEK). The mixture was loaded into the sealed reactor through a septum. The reaction mixture was heated at 80 °C for 1 min; after that, all the incoming activity in the form of the aforementioned radioactive precursors had been trapped. After labeling time, the crude reaction mixture was cooled to 40 °C.

4.4.2. Purification and Formulation

After labeling time and cooling, the crude reaction mixture was diluted by adding 1 mL of semipreparative HPLC eluent from vial 3 to the reactor. The reactor content was then transferred to the 5 mL injection loop of the HPLC system. The purification of the desired 11C-labeled compounds was performed by injection of the diluted crude reaction mixture controlled by an automated fluid detector. Semipreparative HPLC was performed using MeCN/NH4OAc 0.1 M (70:30) as a mobile phase with an isocratic flow of 8.0 mL/min. Chromatograms were registered using a UV detector (210 nm) and a gamma detector in series. The fraction corresponding to [11C]5 (eluting between 6 and 7 min) was collected at and diluted with 50 mL of water for injection into a collection flask (CF). The diluted solution was passed through a C18-SPE cartridge (Sep-Pak C18 light; preactivated with 5 mL of absolute ethanol followed by 10 mL of water for injection), previously installed in the synthesis module between V11 and V12, and then washed with 10 mL of water for injection (from Vial 6). The trapped product was eluted with 1 mL of absolute ethanol (from Vial 5) and collected in a two-neck vial (TNV) containing 5 mL of 0.9% NaCl solution. Finally, formulation was performed passing 5 mL of 0.9% NaCl solution through the cartridge (from Vial 4) and collecting it in the TNV. The final solution was transferred to a sterile vial through a 0.22 μm sterilizing filter. The total radiotracer solution volume was 11 mL.

4.4.3. Quality Control

Chemical and radiochemical impurities were detected and quantified by using radio-HPLC: H2O (TFA 0.1%):MeCN (62.5:37.5, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min on a C18 column. The whole HPLC analysis was completed within 10 min. The retention times of the precursor (4) and the product ([11C]5) were 6.6 and 8.0 min, respectively. The chemical identity of [11C]5 was determined by comparing the retention time of the unlabeled reference compound (5). Chromatograms were registered using a UV detector (λ: 210 nm) and a gamma detector in series.

4.5. adiosynthesis and QC of [11C]Harmine

The procedure was adapted from ref (51), with slight modifications according to the following stepwise procedure:

4.5.1. Formulation of [11C]Harmine

The [11C]6 fraction was collected into the CF (containing 50 mL of water for injection) through V14 (Supporting Information [SI], Figure S6). The resulting solution was then pushed through a C18 SPE light cartridge. After washing with 10 mL of water (Vial 6), the purified product was completely eluted with 1.0 mL of ethanol (Vial 5) and 5 mL of 0.9% saline (Vial 4). Formulation was done with a further 5 mL of physiological saline (0.9%) in the product collection vial. The final solution was transferred through a sterile filter (0.22 μm) into a sterile vial. The final total volume was 11.0 mL (containing 9.0% ethanol).

4.5.2. Quality Control

Chemical and radiochemical impurities were detected and quantified using radio-HPLC: ammonium formiate 0.1 M (pH = 4) and acetonitrile (75/25, v/v) were used as the mobile phase at a flow rate of 1.5 mL/min on a C18 column. The whole HPLC analysis was completed within 10 min. The retention time of the precursor (7) and the product ([11C]6) were 2.7 and 6.0 min, respectively. Chromatograms were registered using a UV detector (λ: 330 nm) and a gamma detector in series.

4.6. Radiosynthesis and QC of 2-[18F]Fluoroethyl-harmol

4.6.1. Conditioning of the Synthetic Module and Preliminary Steps

The reactor was washed with water for injection and analytical grade acetone (from A1, A3 and A6) and then dried in helium. Vials containing purification and formulation solutions (C1, C2, and C3) and other reservoirs such as the vial SPE 2 and the Product vial (where the radiopharmaceutical is ultimately formulated) were rinsed with water for injection and with absolute ethanol and then dried thoroughly in a helium stream.

4.6.2. Production and Azeotropic Drying of [18F]Fluoride

The synthetic process was performed in the automated synthetic platform Synthra RN Plus Research with the configuration shown in Figure S11. [18F]fluoride (solubilized in [18O]H2O) was produced in the cyclotron via the nuclear reaction 18O(p,n)18F using 2.5 mL of enriched water in a Niobium target. To remove the enriched water, the aqueous [18F]fluoride was trapped on an anion exchange cartridge (SEP-PAK QMA, preactivated with 5 mL of K2CO3 0.5 M, followed by 10 mL of water for injection). The fluoride anion was then eluted into Reactor 1 using a solution of aqueous potassium carbonate (3.5 mg in 100 μL of water) mixed with a solution of Kryptofix 2.2.2 (15 mg in 900 μL of acetonitrile). The solution was concentrated to dryness to remove both the acetonitrile and the water. The drying was carried out at 65 °C for 5 min, followed by 95 °C for 3 min under reduced pressure and a stream of helium. After removal of the solvents, vacuum was applied for 30 s at 60 °C, and helium was injected at atmospheric pressure.

4.6.3. Radiolabeling Step

A solution of the precursor 8 (2.5 mg) in anhydrous DMSO (0.4 mL) was added to the Reactor 1 over the activated [18F]fluoride from vial A3. The reaction was heated at 120 °C for 5 min. After the labeling time, the crude reaction mixture was cooled to 60 °C.

4.6.4. Purification and Formulation

The crude solution was diluted with a mixture of H2O/MeCN (2 mL, 1:1, from vial A6) and transferred to an automatic dispenser. The mixture was then transferred to the injection loop and purified by semipreparative HPLC using NaH2PO4 (aq, 25 mM):MeCN (70:30 v/v) as the mobile phase with an isocratic flow of 4.0 mL/min. Chromatograms were registered using an UV detector (λ: 330 nm) and a gamma detector in series. The fraction corresponding to [18F]9 (eluting between 8 and 10 min) was collected and diluted with 50 mL of water for injection into the Vial SPE 2. The diluted solution was passed through a C18 light-SPE cartridge (preactivated with 5 mL of absolute ethanol followed by 10 mL of water for injection). The cartridge was washed with 10 mL of water for injection (from vial C3). The trapped product was eluted with 1 mL of absolute ethanol (from vial C2) and collected in the Product vial containing 5 mL of 0.9% NaCl solution. Finally, formulation was done passing 5 mL of 0.9% NaCl solution through the cartridge (from vial C1) and collected it into the Product vial. The final formulation then passed from the Product vial through a 0.22 μm sterilizing filter to a sterile vial. The total radiotracer solution volume was 11 mL.

4.6.5. Quality Control

Chemical and radiochemical impurities were detected and quantified by using radio-HPLC on a C18 column. The mobile phase was Na2HPO4 25 mM (A) and MeCN (B); gradient: 0–6 min: 30% B, 6–8 min: from 30% to 80% B, and 8–15 min: 80% B; flow rate: 1.0 mL/min; UV detection at 330 nm. The whole HPLC analysis was completed within 15 min. The retention times of the precursor and the product were 6.5 and 10.1 min, respectively. The concentration of Kryptofix 2.2.2 in the final product was assessed with an iodine spot test.

4.7. Cell Line and Animal Models

4.7.1. Cell Cultures

LNCaP (ATCC CRL-1740) and PC3 (ATCC CRL-1435) cell lines were obtained from ATCC. LNCaP cells were grown and maintained in a growth medium consisting of RPMI 1640 with stable glutamine (Capricorn Scientific) supplemented with100 μg/mL streptomycin, 100 U/l penicillin G (Sigma, St Louis, MO), and 10% fetal bovine serum (FBS) (Gibco) in T25 cm3 culture flasks (Greiner). Cultures were fed twice a week with 90% complete growth medium and 10% of their own conditioned medium. When they reached a near-confluent state 1 week later, subculturing was carried out using 0.05% trypsin and 0.02% EDTA (Gibco) in sterile PBS. PC3 cells were grown and maintained in a growth medium consisting of DMEM high glucose 4.5 g/L, with stable glutamine and sodium pyruvate (Capricorn Scientific) supplemented with 100 μg/mL streptomycin, 100 U/l penicillin G (Sigma, St Louis, MO), and 10% FBS (Gibco) in T75 cm3 culture flasks (Greiner). Cultures were fed twice a week with complete growth medium. When they reached near-confluent state 1 week later, subculturing was carried out using 0.05% trypsin, 0.02% EDTA (Gibco) in sterile PBS. Both cells lines were maintained in a 37 °C incubator containing a water-saturated atmosphere with 5% CO2.

4.7.2. Statement of Ethics

All protocols for animal experimentation were performed in accordance with institutional, national, and international guidelines for the use of animals, with the approval of the CUDIM Bioethics Committee’s requirements (protocol number: 19012401) and under the current ethical regulations of the national law on animal experimentation no. 18.611 (Commission of ethics for animal studies; National commission of experimentation with animals).

4.7.3. Cell Transplantation

Seven- to ten-week-old male athymic nude mice (J:Nu-Foxn1nu, homozygous) were subcutaneously implanted in the left flank with 5 × 106 cells (150 μL, 1:1, v/v, Cells in RPMI 1640:Geltrex) for the LNCaP cell line or 3 × 106 cells (150 μL, 1:1, v/v, Cells in DMEM: Geltrex) in the case of the PC3 cell line. A small portion of testosterone gel (Androgel 50 mg/5 g) was applied daily to the skin of the mice inoculated with LNCaP until the appearance of the tumors as a small dark-blue dot. Tumor growth was monitored by external caliper measurements [(short axis)2 × (long axis) × π]/6. Animals were housed in racks with filtered air under controlled conditions: temperature (24 ± 1) °C and relative humidity (40–60%). They were maintained on a 14:10 h light/dark cycle 7 in the CUDIM animal facility with food and water ad libitum. Imaging studies were performed once tumors reached a volume of 100–300 mm3, approximately 4 weeks post inoculation.

4.8. Small Animal PET-CT Imaging

4.8.1. Image Acquisition

Small animal PET-CT imaging was performed in a nanoScan PET/CT Mediso Preclinical Imaging based on LYSO cintillators. The spatial resolution of the scanner was 0.9 mm, and the transaxial field of view was 8.0 cm. The data were acquired in list mode in a 212 × 212 × 235 matrix with a pixel size of 0.4 × 0.4 × 0.4 mm and a coincidence window width of 1.0 ns. The animals were anesthetized with 2% isofluorane in an oxygen flow of 2 L/min, placed in the prone position on the scanner bed, and injected i.v. via the caudal tail vein with 100–200 μL of [11C]clorgyline, [11C]harmine or 2-[18F]fluoroethyl-harmol (25 MBq for all tracers). “Cold” mass of the radioligands administered were 19.7 nmol (clorgyline), 17.5 nmol (harmine), and 11.9 nmol (2-fluoroethyl-harmol). For [11C]clorgyline PET images (static studies), acquisition started 5 min after radiotracer administration and performed over 40 min. For [11C]harmine PET images (static studies), acquisition started 15 min after radiotracer administration and performed over 45 min in a first group of animals and 45 min after radiotracer administration and performed over 90 min in a second group of animals. For 2-[18F]fluoroethyl-harmol, PET images (static studies) were obtained in four groups of animals. In the first group, acquisition started 15 min after radiotracer administration over 60 min, 60–90 min in the second group, 120–165 in the third group, and 180–225 min in the last group. Sinograms were reconstructed using 3D maximum likelihood expectation maximization with 4 iterations and 6 subsets.

4.8.2. Image Analysis

Semiquantitative analysis was done using PMOD software, version 3.8 (PMOD Technologies, Ltd., Zurich, Switzerland). PET studies were coregistered with the corresponding CT scan studies for anatomical localization. The images were displayed as coronal, sagittal, and axial slices. VOIs were drawn manually over the tumor and contralateral muscle in order to generate time–activity curves and calculate T/M ratios. The activity concentration within each VOI was expressed as the hot spot average over the whole VOI (kBq/cc).

4.9. Ex Vivo Biodistribution Studies

Tumor-bearing mice of 24–28 g were injected intravenously through the caudal tail vein with [11C]clorgyline, [11C]harmine or 2-[18F]fluoroethyl-harmol (13–18 MBq). At the desired time interval after administration, the animals were sacrificed by cervical dislocation. Organs of interest were excised and weighed; samples of urine, blood, muscle, and bone were also collected and weighed. The amount of radioactivity was determined in a gamma counter (a 3 in. × 3 in. well–type NaI [Tl] solid scintillation detector coupled to a multichannel analyzer, ORTEC. The results were expressed in terms of the percentage of the injected dose per gram of tissue sample. Corrections by different sample geometry were applied when necessary. For blocking studies, harmine (2 mg) was dissolved in a solution of NaCl 0.9% (0.940 mL), HCl 1 M (0.01 mL), and DMSO (0.05 mL). A dose of 10 mg/kg of harmine was injected i.p. 90 min prior to the injection of [11C]harmine. Biodistribution was performed as previously described 60 min after i.v. administration of 15 ± 3 MBq of [11C]harmine.

Acknowledgments

We gratefully acknowledge the Centro Uruguayo de Imagenologia Molecular (CUDIM) for funding this work, with special emphasis on its previous director; MD. Henry Engler, as well as its current director MD. Pablo Duarte. The research was supported by CSIC initiation to research grant (UdelaR) and PEDECIBA-Química, Uruguay. We thank Agencia Nacional de Investigación e Innovación (ANII) for the scholarship to K.Z. We thank Dr. Dardo Centurión (Cátedra de Anatomía Patológica, Hospital de Clínicas, Universidad de la República) for pathological studies.

Glossary

Abbreviations

MAO-A

monoamine oxidase A

ESI

electrospray ionization

FID

flame ionization detector

GC

gas chromatography

GMP

good manufacturing practice

PCa

prostate cancer

PET

positron emission tomography

p-TsCl

para-toluenesulfonyl chloride

DMF

dimethylformamide

DMSO

dimethyl sulfoxide

Kryptofix 222

4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane

MeCN

acetonitrile

MeOTf

methyl trifluoromethanesulfonate

MEK

methyl ethyl ketone

HRMS

high resolution mass spectrometry

MS

mass spectrometry

NMR

nuclear magnetic resonance

COSY

correlation spectroscopy

HSQC

heteronuclear single quantum coherence spectroscopy

HMBC

heteronucear multiple bond correlation

Ph Eur

European pharmacopoeia

QC

quality control

RCP

radiochemical purity

RCY

radiochemical yield

rt

room temperature

RSD

relative standard deviation

SUV

standard uptake value

TLC

thin layer chromatography

RP-HPLC

reversed phase high-performance liquid chromatography

SPE

solid phase extraction

USP

United States pharmacopoeia

VOI

volume of interest

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00175.

  • Chemical synthesis and additional figures and data sets complementing 1H NMR spectra of compounds 4, 5, 7, 8, and 9; and description of tubings’ and valves’ configuration in synthesis modules used for the automated synthesis, chromatogram and radiochromatogram of the purifications, Western blotting, pathological studies, whole-body microPET images for each tracer and biodistribution studies (PDF)

  • Crystallographic data of the compounds (XLSX)

Author Contributions

All authors have given approval to the final version of the manuscript. W.P. and E.S. conceived the study. K.Z. performed the organic synthesis and radiosynthesis. W.P., E.S., and K.Z. designed the preclinical protocol. K.Z., L.R., and A.P. performed the preclinical experiments (cell transplantation, PET images, and ex vivo biodistribution). R.D. and F.A. performed the cell studies. K.Z., L.R, and R.D. analyzed the data. W.P., K.Z., E.S., and J.P.G. interpreted the data and wrote the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt3c00175_si_001.pdf (1.3MB, pdf)
pt3c00175_si_002.xlsx (8.2KB, xlsx)

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