Radiosynthesis and Preclinical Evaluation of [⁶⁸Ga]Ga-NOTA-Folate for PET Imaging of Folate Receptor β Positive Macrophages

ABSTRACT

Folate receptor β (FR-β) is one of the markers expressed on macrophages and a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [⁶⁸Ga]Ga-NOTA-folate (⁶⁸Ga-FOL). First, we determined the affinity of ⁶⁸Ga-FOL using human FR-β expressing cells. Then, we studied atherosclerotic mice with ⁶⁸Ga-FOL and ¹⁸F-FDG PET/CT. After sacrifice, the tissues excised were measured with a γ-counter for ex vivo biodistribution. Further, the tracer distribution and co-localization with macrophages in aorta cryosections were studied using autoradiography, hematoxylin-eosin staining and immunostaining with anti-Mac-3 antibody. Specificity of ⁶⁸Ga-FOL was assessed in a blocking study with excess of folate glucosamine. As a last step, human radiation doses were extrapolated from rat PET data. We were able to produce ⁶⁸Ga-FOL at high radioactivity concentration, with high molar activity and radiochemical purity. The cell binding studies showed high (5.1 ± 1.1 nM) affinity of ⁶⁸Ga-FOL to FR-β. The myocardial uptake of ⁶⁸Ga-FOL (SUV 0.43 ± 0.06) was 20-folds lower compared to ¹⁸F-FDG (SUV 10.6 ± 1.8, P = 0.001). The autoradiography and immunohistochemistry of aorta revealed that ⁶⁸Ga-FOL radioactivity co-localized with Mac-3-positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of ⁶⁸Ga-FOL (2.44 ± 0.15) was significantly higher than that of ¹⁸F-FDG (1.93 ± 0.22, P = 0.005). Blocking studies verified ⁶⁸Ga-FOL specificity to FR. As estimated from rat data the human effective dose was 0.0105 mSv/MBq. The organ with highest absorbed dose was kidney (0.1420 mSv/MBq). In conclusion, ⁶⁸Ga-FOL is a promising new FR-β-targeted tracer for imaging macrophage-associated inflammation.

INTRODUCTION

Folate receptor (FR) over-expression on cancer cells and during inflammation has been frequently used as a diagnostic and therapeutic tool to allow targeted delivery to tumors and inflammation¹. The beta isoform of the folate receptor (FR-β), distinctly expressed on activated macrophages, has been recognized as a promising imaging marker for inflammatory conditions such as rheumatoid arthritis². The imaging of inflammation by positron emission tomography/computed tomography (PET/CT) is currently done mainly with a glucose analog 2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG), which reflects high consumption of glucose by macrophages and other inflammatory cell types. However, due to the non-specific nature of ¹⁸F-FDG, detection of inflammation adjacent to metabolically active tissues, such as the heart is difficult using ¹⁸F-FDG. Therefore, the development of PET tracers targeting exclusive markers are vital for the specific detection of inflammation. FR-β-targeted PET tracers investigated for this purpose so far include [¹⁸F]AlF-NOTA-folate (¹⁸F-FOL)^3–5, [¹⁸F]fluoro-PEG-folate⁶ and 3’-Aza-2’-[¹⁸F]-fluoro-folic acid (¹⁸F-AzaFol)^7,8, with the latter two already reaching initial clinical phase^9,10. Other recently developed FR-targeted tracers include reduced ¹⁸F-folate conjugates¹¹, ⁵⁵Co-labeled albumin-binding folate derivatives¹² and [⁶⁸Ga]NOTA-folate¹³, which have been investigated for imaging FR-overexpressing tumors in preclinical studies. In a previous study, we showed that ¹⁸F-FOL PET successfully visualized FR-β-positive macrophages in mouse and rabbit models of atherosclerosis³. Atherosclerotic lesions display chronic inflammation associated with accumulation of macrophages in the affected area, providing a basis for investigating macrophage-targeted tracers.

For radiosynthesis of ⁶⁸Ga-radiopharmaceuticals, the use of ⁶⁸Ge/⁶⁸Ga-generators is a common method to obtain ⁶⁸Ga-radionuclide and can be conveniently implemented in a lab. In ⁶⁸Ge/⁶⁸Ga-generators, a certain amount of ⁶⁸Ge is immobilized on a stationary phase, where the mother radionuclide decays into ⁶⁸Ga. However, one drawback for the usage of ⁶⁸Ge/⁶⁸Ga-generators is that the elution capacity of ⁶⁸Ga-radioactivity decreases as the ⁶⁸Ge decays (physical half-life of 271 days). The reduced capacity may become an issue not only for the batch size of each production but also for the radioactivity concentration of the end product. To extent the usable lifespan of ⁶⁸Ge/⁶⁸Ga-generators, we have set up a remotely operated system to concentrate the ⁶⁸Ga-eluate into small volumes from two parallel generators, which would otherwise have low individual ⁶⁸Ga yields.

In this study, we have evaluated [⁶⁸Ga]Ga-NOTA-folate (⁶⁸Ga-FOL), which shares the same precursor structure as our previously studied ¹⁸F-FOL³, for imaging of inflammation. While Al¹⁸F labelling of NOTA-conjugates requires cyclotron facilities for the production of [¹⁸F]fluorine, the generator-produced ⁶⁸Ga offers a convenient and cost-effective option for radiolabeling. First, we determined the binding affinity of ⁶⁸Ga-FOL to human FR-β using transfected cells. Next, we investigated the uptake and specificity of intravenously (i.v) administered ⁶⁸Ga-FOL for the detection of inflamed atherosclerotic lesions in mice and compared the tracer with ¹⁸F-FDG. In addition, we determined the whole-body distribution kinetics with and without blocking agent folate glucosamine in healthy rats, and estimated the human radiation dose of ⁶⁸Ga-FOL.

EXPERIMENTAL SECTION

General Materials and Equipment

NOTA-folate precursor was synthesized as previously described¹⁴. ⁶⁸GaCl₃ was obtained from ⁶⁸Ge/⁶⁸Ga IGG-100 generators (Eckert & Ziegler, Valencia, CA, USA) via elution with 0.1 M hydrochloric acid (HCl) in water. TraceSELECT^™ water (Honeywell, Morristown, NJ, USA) was used for radiosynthesis. Other chemicals were purchased from commercial suppliers. Chinese hamster ovary cells stably transfected with human FR-β (CHO-hFRb; CHO-FR-β⁺) were a generous gift from Philip S. Low, Purdue University, USA. FR-β negative CHO cells (CHO-FR-β⁻ control) were a generous gift from Sirpa Jalkanen, MediCity Research Laboratory, University of Turku, Finland. A dedicated small animal PET/CT (Inveon Multimodality, Siemens Medical Solutions, Knoxville, TN, USA) was used for PET/CT imaging and a gamma counter (1480 Wizard 3”, PerkinElmer/Wallac, Turku, Finland or Triathler 3”, Hidex, Turku, Finland) for radioactivity measurement of ex vivo tissues, blood and plasma samples. Tracer quality control and plasma metabolite analysis were performed with a LaChrom high-performance liquid chromatography (HPLC) system (Hitachi; Merck, Darmstadt, Germany) equipped with a Radiomatic 150TR flow-through radioisotope detector (Packard, Meriden, CT, USA) (radio-HPLC). Photomicroscopy images were taken with a digital slide scanner (Pannoramic 250 Flash or Pannoramic P1000, 3DHistec Ltd., Budapest, Hungary).

⁶⁸Ga-FOL Radiosynthesis

Method 1

A fraction of ⁶⁸Ga-eluate (0.5-1.0 mL) was mixed with an aqueous solution of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, 50–100 μL at a concentration of 1.2 g/mL). Then, NOTA-folate precursor (10-20 nmol in 20-40 μL water) was added, vortexed and incubated for 10 minutes at 80 °C. The mixture was then cooled down and brought to a pH of ~ 6.5 by adding 55 μL of 1 M sodium hydroxide (NaOH). The product was used without further purification. Radiochemical purity was analyzed with HPLC. The radio-HPLC conditions were as follows: 150 × 4.6 mm Jupiter 5μ C18 300 Å column (Phenomenex, Torrance, CA, USA); flow rate =□1 mL/min; wavelength λ = 220 nm; solvent A =□0.1% trifluoroacetic acid (TFA) in water; solvent B□= 0.1% TFA in acetonitrile; gradient: during 0–14 min 3% B to 25% B; 14–15 min from 25% B to 3% B.

Method 2

In a typical synthesis, one or two IGG-100 ⁶⁸Ge/⁶⁸Ga-generators were eluted with 0.1 M HCl (7.0 mL/generator) through a Strata SCX cartridge into a waste container. The cartridge was then eluted with 600 μL of 1.0 M sodium chloride/0.1 M HCl solution, from which 550 μL was transferred to the reaction vial preloaded with a mixture of HEPES (66 mg) and NOTA-folate (5–20 nmol) in 50 μL of water. Gentisic acid (10 μL, 0.1 M in water) was added to avoid radiolysis. The reaction mixture was then incubated at 40 °C for 10 minutes. The mixture was cooled down after incubation and brought to a pH of 5.5–6.5 by an addition of 30 μL of 2 M NaOH. Radiochemical purity was analyzed with HPLC similarly as described in Method 1, as well as with instant thin-layer chromatography (iTLC) (Figure S1). A 1.0 μL sample of the end product or reaction mixture was applied to a silica gel-based iTLC strip (iTLC-SG; Agilent, Santa Clara, CA, USA) and developed with 50 mM citric acid. Unbound ⁶⁸Ga migrated up with the mobile phase with a retention factor (R_f) of 0.8–1.0, while ⁶⁸Ga-FOL remained at the application point (R_f = 0). To measure the unbound and tracer bound ⁶⁸Ga fractions, the strip was cut into two pieces from the middle line between the baseline and the solvent front, and each piece was measured separately in a gamma counter.

Lipophilicity of ⁶⁸Ga-FOL (distribution coefficient LogD) was determined as previously described¹⁵. To evaluate ⁶⁸Ga-FOL stability in the injectable formulation, we kept the end product at room temperature (RT) and took samples for radio-HPLC analysis at time intervals of up to 3 hours.

Quantification ⁶⁸Ga-FOL Binding Affinity to FR-β

Binding specificity of ⁶⁸Ga-FOL to FR-β was evaluated using CHO-FR-β⁺ and CHO-FR-β⁻ cells (control). The cells were cultured in growth medium - Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-Thermofisher Scientifc, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) at 37□°C in a CO₂ incubator.

To verify FR-β expression, the cultured cells were harvested and incubated with either fluorescein isothiocyanate (FITC)-conjugated anti-human FR-β antibody (m909¹⁶, a gift from Philip S. Low) or allophycocyanin (APC)-conjugated anti-human FR-β antibody (mouse IgG2a, BioLegend, San Diego, CA, USA), and isotype controls (mouse IgG-FITC, mouse IgG2a-APC; BioLegend). Then, the cells were fixed using paraformaldehyde and analyzed using a fluorescence-activated cell sorting device (FACS, Fortessa flow cytometer; BD Biosciences, Franklin Lakes, NJ, USA) and Flowing software (Cell Imaging and Cytometry Core, Turku Bioscience, Turku, Finland).

After verifying the presence of FR-β on the cells, either CHO-FR-β⁺ or CHO-FR-β⁻ cells were cultured on one side of a 92-mm petri dish in a tilted position (manufacturer’s protocol, Ridgeview for ligand binding studies) in growth medium at 37□°C in a CO₂ incubator. The other side of the petri dish with no cells was used as background control for non-specific binding of ⁶⁸Ga-FOL. Once the cells attained a confluent monolayer, the growth medium from the petri dishes was removed and phosphate-buffered saline (PBS) containing calcium and magnesium with 10% FBS (binding medium) was added and the cells were incubated at 37□°C in a CO₂ incubator for 30 minutes, to starve the cells of folate. After incubation, the cells were rinsed with binding medium (2 × 2 mL). A LigandTracer Yellow instrument (Ridgeview Instruments AB, Uppsala, Sweden) was then used to measure the dissociation constant (K_D) for ⁶⁸Ga-FOL. The assay protocol with LigandTracer Yellow contains consecutive radioactivity measurements of the target (cell region) and of the opposite background (no cell region on the petri dish).

Radioactivity was measured in each region for 30 seconds as raw counts per second (cps) with a delay of 5 seconds over the time course of the experiment. The target regions (cps) were corrected for background signal and for radioactive decay. To detect the background radioactivity or noise picked by the instrument, 5 mL of binding medium was added to the cells on the petri dish. After 15 minutes, ⁶⁸Ga-FOL was added stepwise to achieve concentration range of 1 nM to 80 nM, followed by replacement with fresh binding medium to measure the dissociation. The ratio of bound ⁶⁸Ga-FOL (to the cells) to background (petri dish) and K_D were calculated with TraceDrawer software (Ridgeview Instruments AB).

Animal Experiments

Low-density lipoprotein receptor deficient mice expressing only apolipoprotein B100 (LDLR^−/−ApoB^100/100, strain #003000, The Jackson Laboratory, Bar Harbor, ME, USA) were used to induce atherosclerosis. The mice were fed with high-fat diet (HFD; 0.2% total cholesterol, TD 88137, Envigo, Madison, WI, USA) starting at the age of 2 months and maintained for 3-5 months. C57BL/6JRj mice (Central Animal Laboratory of the University of Turku) fed with a regular chow diet were used as healthy controls. In total, 17 LDLR^−/− ApoB^100/100 (34.7 ± 5.5 g) and 6 healthy control mice (29.65 ± 1.9 g) were studied. In addition, 6 Sprague-Dawley rats (135.9 ± 17.1 g) from the Central Animal Laboratory of the University of Turku were studied.

All animals were housed at the Central Animal Laboratory of the University of Turku and had ad libitum access to water and food throughout the study. All animal experiments were approved by the national Animal Experiment Board in Finland (license number ESAVI/4567/2018) and were carried out in compliance with European Union directive (2010/63/EU).

Mouse Studies

PET/CT Imaging

The mice were fasted for 4 hours prior to imaging, anesthetized with isoflurane (4–5% induction, 1–2% maintenance), and placed on a heating pad. Then, mice were i.v. administered with ¹⁸F-FDG (14.4 ± 0.2 MBq) via a tail vein cannula and again on the following day with ⁶⁸Ga-FOL (20.1 ±1.0 MBq). Immediately after PET, an iodinated contrast agent (100 μL of eXIATM160XL, Binitio Biomedical Inc., Ottawa, ON, Canada) was i.v. injected and a high-resolution CT was performed for anatomical reference. To analyze PET/CT images, we used the free Carimas 2.10 software (Turku PET Centre, Turku, Finland, www.turkupetcentre.fi/carimas/). We defined regions of interest (ROIs) for the myocardium in coronal PET/CT images, using the contrast-enhanced CT as an anatomical reference as previously described². The results were expressed as normalized for the injected radioactivity dose and animal body weight, i.e. as standardized uptake values (SUVs).

Ex Vivo Biodistribution

To study the specificity of ⁶⁸Ga-FOL uptake, an in vivo blocking study was performed with another group of HFD-fed LDLR^−/−ApoB^100/100 mice i.v. administered with ⁶⁸Ga-FOL alone or with 100-fold molar excess of folate glucosamine together with ⁶⁸Ga-FOL. Mice were i.v. injected with ⁶⁸Ga-FOL (11.3 ± 0.8 MBq) and euthanized after 60 minutes. Various tissues were excised and weighed, and their radioactivity was measured with a γ-counter (Triathler 3”, Hidex, Turku, Finland). After compensating for radioactivity remaining in the tail and cannula, the ex vivo biodistribution of ⁶⁸Ga-radioactivity results were expressed SUV, and blocking vs. non-blocking results were compared.

Autoradiography, Histology and Immunostainings

Following PET/CT imaging, the dissected aortic arch was prepared into 20 μm and 8 μm cryosections. The 20 μm cryosections were used for digital autoradiography analysis as previously described³. Briefly, the sections were apposed on an Imaging Plate BAS-TR2025 (Fuji, Tokyo, Japan), and the plates were subsequently scanned on Fuji Analyzer BAS-5000 (Fuji, Tokyo, Japan) after an exposure time of 3 hours for ⁶⁸Ga-FOL and at least 4 hours for ¹⁸F-FDG. After the scanning, sections were stored at −70°C until staining with hematoxylin and eosin (H&E) and scanned with a Pannoramic digital slide scanner. Autoradiographs were analyzed using Tina 2.1 software (Raytest Isotopemessgeräte, GmbH, Straubenhardt, Germany) and the uptake of ⁶⁸Ga-FOL and ¹⁸F-FDG was corrected for injected radioactivity dose per unit body mass and radioactive decay during exposure and expressed as photostimulated luminescence per square millimeter (PSL/mm²). For immunohistochemistry, adjacent 8 μm sections were used to investigate co-localization of ⁶⁸Ga-FOL with Mac-3-positive macrophages. The sections were incubated with anti-mouse Mac-3 antibody (1:1000, BD Biosciences, Franklin Lakes, NJ, USA) and a color reaction was subsequently developed using 3.3’-diaminobenzidine (Bright-DAB, BS04-110).

In Vivo Stability

To determine the in vivo stability of ⁶⁸Ga-FOL, plasma samples collected from atherosclerotic mice (n = 3) at 60 minutes post-injection were analyzed using radio-HPLC. Blood samples were collected in heparinized tubes and centrifuged in 4 ^oC for 5 minutes at 2,118 × g. Plasma proteins were precipitated with 10% sulfosalicylic acid (1:1 v/v) followed by centrifugation for two minutes at 14,000 × g at RT. The supernatant was analyzed with radio-HPLC. Standard samples were prepared by adding ⁶⁸Ga-FOL tracer to 500 μL of plasma supernatant collected from mice without the administration of tracer. Both standard and metabolite samples applied to radio-HPLC analysis were normalized to a final volume of 1 mL by dilution with radio-HPLC solvent A if necessary. The radio-HPLC conditions were as follows: 250 × 10 mm Jupiter Proteo 5μ C18 90 Å column (Phenomenex, Torrance, CA, USA); flow rate = □5 mL/min; solvent A = □ 0.1% TFA in water; solvent B = 0.1% TFA in acetonitrile; gradient was during 0-11 min from 3% B to 25% B, during 11–12 min from 25% B to 100% B, during 12–14 min 100% B.

Rat Studies

In order to determine distribution kinetics and estimate human radiation dose, a dynamic whole-body ⁶⁸Ga-FOL PET/CT was performed in six healthy rats. In addition, three of the rats were also subjected to a blocking experiment with a co-injection of a 100-fold excess of folate glucosamine. The rats were injected with 10.3 ± 0.4 MBq of ⁶⁸Ga-FOL and PET imaged for 60 minutes. After the imaging, the rats were euthanized, various tissues excised, weighed and measured for radioactivity and plasma samples were analyzed using radio-HPLC as described above. Using CT as an anatomical reference, quantitative PET image analysis was performed by defining ROIs on the main organs and time-activity curves were extracted with Carimas software. Human radiation dosimetry was estimated from the rat data using the OLINDA/EXM 2.2 software¹⁷.

Statistical Analysis

Results are presented as mean ± SD. Differences between groups were analyzed by the unpaired Student t-test using Microsoft Excel. P values of less than 0.05 were considered statistically significant.

RESULTS

Radiosynthesis

Method 1

⁶⁸Ga-FOL was produced with 345.8 ±118.9 MBq/mL (n = 13) radioactivity concentration and high radiochemical purity (96.6 ± 2.7%). The molar activity was 21.8 ± 6.9 GBq/μmol at the end of radiosynthesis. Distribution coefficient LogD of ⁶⁸Ga-FOL was −3.28 ± 0.33 (n = 3), indicating high hydrophilicity.

Method 2

We set up a straightforward system using commonplace hospital pressure IV-tubing (Argon, Frisco, TX, USA) and one-way check valves (B. Braun, Melsungen, Germany) and a 3-way valve (Bürkert, Huntersville, USA) to allow the elution of two generators through an SCX cartridge in a remotely operated manner. The system is outlined in Figure 1A. When two parallel ⁶⁸Ge/⁶⁸Ga-generators (aged 16 and 20 months with nominal radioactivities of 1.85 GBq and 2.20 GBq at the time of manufacture, respectively) we were able to produce 492.9 ± 24.2 MBq (n = 5) of ⁶⁸Ga-FOL at high radioactivity concentration (782.5 ± 38.4 MBq/mL) and radiochemical purity (99.3 ± 0.2%). The molar activity was 49.3 ± 2.42 GBq/μmol.

Figure 1.

(A) The remotely operated system for the concentration of ⁶⁸GaCl₃ eluted from two parallel generators and a flow chart for ⁶⁸GaCl₃ elution and concentration. The molecular weight of ⁶⁸Ga-FOL is 908.82 Da. (B) The effect of gentisic acid on the stability of the end product as determined with radio-HPLC (n = 3, reaction conditions: 40 °C, 15 nmol of precursor). (C) ⁶⁸Ga-FOL fraction of total radioactivity during incubation, measured with iTLC-SG (n = 3, reaction conditions: 15 nmol of precursor, 10 μL of 0.1 M gentisic acid). (D) Effect of precursor amount on radiochemical purity at the end of synthesis (EOS) as measured with iTLC-SG (n = 3, reaction conditions: 40 °C, 10 μL 0.1 M gentisic acid).

In Vitro Quantification of ⁶⁸Ga-FOL Binding Affinity to FR-β

FACS analyses verified that FR-β was clearly expressed on CHO-FR-β⁺ cells but not on CHO-FR-β⁻ cells (Figure 2A-C). In binding assays, we found that with a stepwise increase in concentration of ⁶⁸Ga-FOL from 1 nM to 80 nM, the binding of ⁶⁸Ga-FOL to CHO-FR-β⁺ cells was gradually increased, and showed a KD of 5.1 ± 1.1 nM (n = 3). Whereas there was no clear accumulation of ⁶⁸Ga-FOL to CHO-FR-β⁻ cells even at concentrations up to 40 nM (Figure 2D).

Figure 2.

(A) Representative figures of flow cytometric analyses showing FR-β expression on human FR-β-negative and FR-β-positive CHO cells stained with FITC-conjugated anti-FR-β antibody (red) or APC-conjugated anti-FR-β antibody (blue) and isotope controls (black). Quantification of FR-β expression presented as (B) percentage and (C) mean fluorescence intensity. (D) Representative real time binding affinity of ⁶⁸Ga-FOL measurement with LigandTracer. The graph generated with TracerDrawer, shows raw counts per second (cps) after corrected for background signal and for radioactive decay.

⁶⁸Ga-FOL Detects Macrophage-rich Lesions in Atherosclerotic Mice

We evaluated the biodistribution of i.v. administered ⁶⁸Ga-FOL in mice using in vivo PET/CT, ex vivo gamma counting of excised tissues and ex vivo autoradiography of aorta cryosections. To study the specificity of ⁶⁸Ga-FOL to FR-β, blocking with co-inj ection of a molar excess of folate glucosamine was performed. In addition, ⁶⁸Ga-FOL was compared with ¹⁸F-FDG in a head-to-head PET/CT imaging setting and by ex vivo autoradiography.

The in vivo stability of ⁶⁸Ga-FOL was good; 60 minutes after i.v. injection, the amount of intact tracer was 63.3 ± 1.2% of the total plasma radioactivity in LDLR^−/−ApoB^100/100 mice (n = 3, Figure S2).

Our ex vivo results revealed that the aortic uptake of ⁶⁸Ga-FOL was higher in atherosclerotic mice (SUV 0.75 ± 0.12) than in healthy controls (SUV 0.41 ± 0.10, P = 0.004) or atherosclerotic mice from the blocking study (SUV 0.09 ± 0.03, P = 0.001). Further, the atherosclerotic aorta radioactivity concentration was 3-fold greater than that of blood (SUV 0.23 ± 0.09). The highest radioactivity uptake was seen in FR-positive kidneys¹⁸ in both atherosclerotic and control mice (SUV 22.30 ± 3.28 and 20.27 ± 5.48, respectively, P = 0.49) and it was significantly reduced in the blocking study of atherosclerotic mice (SUV 2.65 ± 1.80, P = 0.0002). The radioactivity of other tissues was much lower than that of the kidneys. Besides kidneys, the folate glucosamine blocking in atherosclerotic mice reduced the radioactivity concentration in many other tissues too, importantly in the aorta by 88% (Supporting Information Table S1).

A comparison of the two tracers by in vivo PET/CT revealed that ⁶⁸Ga-FOL uptake in myocardium (SUV 0.43 ± 0.06) was significantly lower than that of ¹⁸F-FDG (SUV 10.6 ± 1.88, P = 0.001, Figure 3A,B).

Figure 3.

(A) Coronal PET/CT and CT images of atherosclerotic mouse administered with ⁶⁸Ga-FOL or ¹⁸F-FDG. White arrows show aortic arch and red arrows show myocardium region, respectively. (B) Quantification of myocardial PET data showing significant difference between the tracers. (C) Hematoxylin-eosin (H&E) staining and autoradiography images from representative aorta cryosections, and Mac-3 macrophage marker staining on consecutive aorta cryoscetions, Black rectangle indicates the plaque region, which is zoomed. (D) Quantification of autoradiography ⁶⁸Ga-FOL data showing significant difference between the groups. (E) Quantification of autoradiography data showing significant difference between the tracers. Scale bar = 0.5 mm. A = arch; AA = ascending aorta; B = brachiocephalic artery; D = descending thoracic aorta; L = lesion; LC = left common carotid artery; LS = left subclavian artery; W = wall.

In order to further elucidate ⁶⁸Ga-FOL and ¹⁸F-FDG uptake in the aortas of atherosclerotic mice in more detail, we analyzed radioactivity using autoradiography and H&E staining of aortic cryosections followed by macrophage-detecting immunohistochemistry on adjacent tissue cryosections. The results revealed that ⁶⁸Ga-FOL and ¹⁸F-FDG radioactivity co-localized with Mac-3-positive macrophage-rich plaques (Figure 3C). The plaque-to-healthy vessel wall ratio of ⁶⁸Ga-FOL (2.44 ± 0.15) was significantly higher than that of ¹⁸F-FDG (1.93 ± 0.22, P = 0.005, Figure 3D).

Distribution Kinetics in Rats and Estimate Human Radiation Dose of ⁶⁸Ga-FOL

As in mice, ⁶⁸Ga-FOL showed fast renal excretion and the highest uptakes in kidneys, urine, salivary glands, liver and spleen (Figure 4). Co-injection of ⁶⁸Ga-FOL and molar excess of folate glucosamine, clearly decreased tracer uptake in several organs but increased urinary excretion.

Figure 4.

Time-activity curves of healthy rat tissues after i.v. injection of ⁶⁸Ga-FOL or co-injection of ⁶⁸Ga-FOL and molar excess of folate glucosamine (blocking experiment).

Radio-HPLC analysis of plasma samples revealed good in vivo stability of ⁶⁸Ga-FOL (Figure S2); at 60 minutes post-injection 71.8 ± 1.5% of the total radioactivity was still counting from the intact tracer in healthy rats (n = 3) without blocking and 88.0 ± 0.7% (n = 3, P = 0.0002) when blocked with folate glucosamine. Extrapolating from the rat PET data, the estimated human effective dose for a 73 kg man was 0.0105 mSv/MBq. The most critical organ was the kidney (0.1420 mSv/MBq) (Supporting Information Table S2).

DISCUSSION

In this study, we developed a new method for effective synthesis of ⁶⁸Ga-FOL, which could be applicable to generate other ⁶⁸Ga-tracers. We report that ⁶⁸Ga-FOL binds to FR-β with high affinity, and when i.v. administered accumulates in atherosclerotic lesions in mice. Importantly, ⁶⁸Ga-FOL showed lower myocardial uptake and higher plaque-to-healthy vessel wall ratio compared to ¹⁸F-FDG. Human radiation dose of ⁶⁸Ga-FOL was low as estimated from the rat data.

To produce ⁶⁸Ga-FOL, we have used a fractionation method to obtain ⁶⁸GaCl₃ from a ⁶⁸Ge/⁶⁸Ga-generator for a chelation reaction with the precursor compound NOTA-folate (Radiosynthesis, Method 1). This is a well-established method that we have used previously and total radiosynthesis takes less than 20 minutes. The radioactivity concentrations are sufficiently high for applications such as cell binding experiments. However, for in vivo studies in mice, it is increasingly difficult to produce sufficiently high radioactivity concentration by using fractionation-based synthesis as the generator’s ⁶⁸Ga yield decreases over time. This has motivated us to explore alternative methods to utilize the generators in a more effective way. In our lab, we indeed have fully automated ⁶⁸Ga-radiosynthesis devices for ⁶⁸Ga-eluate concentration in the production of ⁶⁸Ga-radiopharmaceuticals compliant with Good Manufacturing Practice (GMP), but it is more cost-effective to build up a simpler system for the synthesis of preclinical non-GMP tracers. Similar approaches have been reported from other labs as well¹⁹. Accordingly, we have used a 3-way tubing system to connect two parallel generators to a SCX-cartridge abstracting ⁶⁸Ga-nuclide from generator eluates, and a remotely controlled 3-way valve to direct the liquids either to a waste vial or a vial for receiving concentrated ⁶⁸Ga-radioactivity (Figure 1A). It is optional to elute ⁶⁸Ga from both generators or from either of them, which increases flexibility. With this system we have managed to concentrate > 90% radioactivity from the generators into a volume of 0.6 mL at radioactivity concentrations up to 1 GBq/mL approximately, when using two generators at ages 16 and 20 months, respectively. This is a large improvement for radiolabeling applications but initially lead to other issues. When using concentrated ⁶⁸Ga-eluate, we have concurrently observed a significant amount of radioactive side product as detected by radio-HPLC quality analysis of ⁶⁸Ga-FOL. Based on our experiences in the radiosynthesis of the corresponding ¹⁸F-FOL³, we have presumed that the side product is caused by radiolysis at high radioactivity concentrations. By adding gentisic acid as a radical scavenger, the formation of the side product has been effectively prevented, the ⁶⁸Ga-FOL being stable for at least 3 hours (Figure 1B, stability for longer time has not been tested). Furthermore, we have observed that the chelation reaction with this type of post-processed ⁶⁸Ganuclide is rather efficient. Even at RT, the reaction can reach near completion in 10 minutes (Figure 1C) and the total radiosynthesis time is only 5 minutes longer than the synthesis with the fractionation method. In typical cases, 10 nmol of precursor is sufficient for each synthesis (Figure 1D), affording the formation of ⁶⁸Ga-FOL with high molar activities (49.3 ± 2.4 GBq/μmol). Thus, this indicates the approach is effective for the synthesis of ⁶⁸Ga-FOL.

Previously, ^99mTc-EC20 and ¹¹¹In-EC0800, two folate-based imaging agents for singlephoton emission computed tomography (SPECT) have been shown to detect atherosclerotic lesions in mice^20,21. Additionally, PET tracer 3′-aza-2′-¹⁸F-fluorofolic acid has been shown to detect FR-β-positive macrophages in human atherosclerotic plaques in vitro⁸. In our previous studies, we have reported ¹⁸F-FOL specificity to FR-β-positive macrophages and detection of inflamed atherosclerotic plaques in mice and rabbits as well as in human tissue sections³. However, ⁶⁸Ga-FOL binding affinity to human FR-β and ability to detect atherosclerotic lesions in comparison to ¹⁸F-FDG has not been evaluated before. Our in vitro binding assay of ⁶⁸Ga-FOL with CHO-FR-β⁺ and CHO-FR-β⁻ cells showed good specificity and high affinity to FR-β (5.1 ± 1.1 nM), which falls close to the binding affinity of ¹⁸F-FOL (1.0 nM) to FR–positive tumor xenografts reported earlier¹⁴. Our blocking studies in mice and rats further supported tracer’s specificity to FR. Mouse studies confirmed the ability to detect macrophage-rich inflammatory lesions. The observed low myocardial uptake is beneficial for detection of atherosclerotic lesions in coronary arteries in prospective PET/CT studies of patients with coronary heart disease. When compared to our earlier ¹⁸F-FOL study, ⁶⁸Ga-FOL showed similar plaque-to-healthy vessel wall ratios (2.44 ± 0.15 for ⁶⁸Ga-FOL and 2.60□±□0.58 for ¹⁸F-FOL). However, the in vivo stability of ⁶⁸Ga-FOL in mice (63 ± 1 % intact tracer at 60 minutes post-injection) was slightly lower compared to our previous studies with ¹⁸F-FOL (85 ± 6% at 60 min post-injection)³. The human effective dose of ⁶⁸Ga-FOL extrapolated from rat data (0.0105 mSv/MBq) is low and within the same range as other ⁶⁸Ga-tracers^22-24.

In conclusion, we demonstrated the feasibility to produce ⁶⁸Ga-FOL at high radioactivity concentration, with high molar activity and radiochemical purity by using a straightforward system for extracting ⁶⁸Ga-nuclide from aged ⁶⁸Ge/⁶⁸Ga-generators. Similar method may be applicable for producing other ⁶⁸Ga-tracers. The preclinical results of ⁶⁸Ga-FOL are in the line with our previous studies using ¹⁸F-FOL and corroborate FR-β as an imaging target for detection of inflamed atherosclerotic lesions.

SUPPORTING INFORMATION

Figure S1: Representative iTLC-SG autoradiographs and chromatographs; Figure S2: Representative radio-HPLC chromatograms; Table S1: Ex vivo biodistribution in mice; Table S2: Human radiation doses extrapolated from the rat data.

ACKNOWLEDGMENTS

The authors thank Aake Honkaniemi and Timo Kattelus for technical assistance. This work was financially supported by the Academy of Finland (grant numbers 314553, 314554, 314556), Sigrid Jusélius Foundation and Jane and Aatos Erkko Foundation.

Footnotes

• Authors' ORCID IDs were added