HIGH SPECIFIC ACTIVITY PREPARATION OF F-18 TETRAFLUOROBORATE

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priorit to U.S, Provisional Application No. 62/327, 13 entitled "High Specific Activity Preparation of F-18 Tetrafluoroborate" filed on April 25, 2016 which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not Applicable.

BACKGROUND

[0003] The sodium/iodide symporter (NIS) is an intrinsic membrane glycoprotein, which mediates the uptake of iodide in the thyroid gland and other NIS expressing cells or tissues [1-3]. The active ttansport of iodide is the basis for the diagnosis and therapeutic treatment of thyroid disease and thyroid cancer. The clinical application of radioiodine also builds the foundation of modem nuclea medicine [4], The identification and characterization o human NIS (hMS) in 1996 [5, 6] created new opportunities for the use of hN IS as a reporter gene in viral therapy investigations and imaging of cell migration and differentiation. Despite considerable success in single photon imaging of the thyroid and thyroid cancers with ¹²³ i, ¹³¹ L or [ ^9¾n Tc]pertechnetate [2, 3], there remain obvious limitations for use of these radioisotopes for diagnostic imaging. Both ^l23 I (Ti/2-13.13 h) and ^{1 ι} ί d) are true iodine imaging radiotracers, but have longer half lives than required for a diagnostic study, which result in unnecessarily high dose of irradiation to patients and staff. [ ^99m l c]perteehnetate (Tio- 6 h) has found use as a radioiodine analo in thyroid diseases [7, 8], and has suitable properties for single photon emissio computed tomography (SPECT). Nevertheless, SPECT has the limited resolutio and sensitivity, especially in detection of small metastases and low volume diseases. Positron emission tomography (PET) has significantly better sensitivity and quantitative accuracy tha SPECT, particularly for accumulations in small regions. The positron emitter ^!2 I .£T|¾= 4.2 d) has been used in NIS imaging ^], £0©04] However, the unnecessarily long half-life of ^m l and its complex emission properties that include high energy gamma photons are drawbacks for diagnostic imaging applications. Also, the production of ¹²⁴ I requires specialized solid target systems which are not available in most cyclotron facilities [10], Positron emitting ¹⁸ F-fti¾aride ([ ¹⁸ F], Ti/ ₂ = 109.7 min) is the most commonly employed radioisotope for PET imaging. It has favorable physical decay properties of 97% positron emission and low positron energy (β+max ^a 0.635 M©V) and is produce by all PET cyclotrons. The development o ¹⁸ F- fluoride based PET tracers for NIS: imaging would be encouraging. Since various anions (e.g., 1 ^" , SeCN ^" , SCN ^" , Te04 ^" , NOf) are transported by NIS [6], the critical physicoehemieal features of these well transported substrates is anionic monovalency with similar size and space-filling properties as the iodide ion. Before the advent of clinical PET, in 1950s and early 1960s, Anbar et al, [1 1, 12] reported that the tetraftuoroborate (TFB, ΒΡ ₄ ^' ) anion was effective to inhibit thyroid uptake of iodide ion. These researchers also showed that [ ^{l 8} F] labeled TFB, synthesized from reactor produced ^{, 8} F-fluoride, specifically accumulated in rat thyroid. The initial labeling of TFB was accomplished by an ion exchange reaction of KBF ₄ in acid at room temperature or heating to make ^F-TFB, and the potassium salt was purified by recrystallization after neutralizatio [13], The isotopic exchange labelin approach inherently results in low specific activity ^t8 F-TFB. Recently, Jauregui-Osoro et al. j 14] updated the labeling of ⁱ⁸ F-TFB using current ^!S F-fluoride production and purification methods. With the same mechanism, ^I8 F-TFB was synthesized in mixture of 1 mg NaBF ₄ and 1 , 5 HCl at 120 ^° C, After filtration through a Dionex AG cartridge (a cation exchange cartridge loaded with silver ions) and alumina cartridge, ¹⁸ F-TFB of >96% radiochemical purity was obtained in approximately 10% yield. The specific activity was ~1 q^mol with starting activities of 12- 18 GBq of ¹⁸ F-fluoride [14], PET/CT imaging in normal mice with transgenic thyroid tumors showed ^t8 F-TFB to delineate uptake in normal tissues expressin NIS (thyroid, stomach, salivary glands) and enhanced uptake in thyroid tumor. Researchers from the same institution also showed that ¹⁸ F-TFB was effective as a NIS probe in the NlS ransfected colon carcinoma cell line, HCTi 16 [15], However, as pointed A by Youn et al [16], specific activities achieved by the reported method are substantially lower than those typically required for receptor-mediated radiopharmaceuticals (~30 GBq&tmol). Therefore, there is a need for synthesis of ⁱ⁸ F-TFB with higher specific activity,

SUMMARY OF THE INVENTIO [0005] The present disclosure describes in one aspect a method of radiolabeling tetrafluoroborate (T B with ^lS F-fluQride _s the method comprising direct radiofluormation n boron trifiuoride (BFs) by reactin the BF ₃ directly with ^!¾ F»fluoride.

[0006] I another aspect th present disclosure provides, a method of radiolabelin rafluoroborate with '^-fluoride, the method comprising; (a) reactin ^ls F-fliiofide with BF ₃ and (b) isolating the ¹⁸ F-TFB f om unreacted ^{l s} F-fiuoride and BF ₃ , In some aspects, the ^{i S} F- fluoride is trapped on an anion exchange cartridge and the reacting of step (a) of ^!¾ F-ftuoride with BF ₃ i performed on the anio exchange cartridge.

[00071 & anothe aspect, the method of radiolabeling tetrafluoroborate with [ ⁱ⁸ F]fluoride, the method consisting essentially of: (a) trapping ^ls F-fiu0ride on an anion exchange column; (b} reacting ^l8 F]fiuoride with BF3 and (e) isolatin the ^l8 F~TFB from urireaeted ^ts F-fluoride and BF ₃ from the anion exchange column, wherein the resultant ¹⁸ F-TF has a specific activity of at least 5 GB ^ ol.

[0008] In another aspect, a high specific activity preparation of ¹⁸ F-tetrafluoroborate is provided. The high specific activity preparation is prepared by the methods disclosed herein. In some aspects, the preparation has at least 95% radiochemical purity. In some aspects, the specific activity is at least 8 GB<q imoI,

[0009] In yet another aspect, methods of use of the high specific activity preparation of ^¾ F~ tetrafluoroborate are provided. Suitable uses include, for example, imaging the thyroid, breast, stomach, salivary glands or kidneys of a subject. Other suitable uses include imaging of th roid or breast cancer in subject. Another suitable use is for monitoring gene therapies that employ the hNlS reporte gene,

[0010| These and other features, aspects, and advantages of the present invention will become better understood upon consideration of the following detailed description, drawings, and appended claims.

DESCRIPTION OF THE DRAWINGS

[001 1 ] Fig. Ϊ A is a schematic showing the preparation of BF3 ^■ THF FE complex solution.

[0012] Fig, IB is schematic of an alternative simplified method of preparing BF ₃ THF/PE complex solution. O 13] Fig, 2 a depiction of the in-house-made Le watit ^® MP-64 cartridge.

[00143 Fig. 3 is a schematic of automated module for the pfeparaiio f ¹⁸ F-TFB, Valves V i-YS are composed of a single-use cass ette that i s mounted to the front of the module, All o ther valves are non-disposable Teflon diaphragm solenoid valves.

[0015] Wig, 4 depicts the relationshi of radiochemical yields (uncorrected) to the amount of unlabeled BF#- produced as found using different amounts of MP-64 resin to pre-Slter the BI¾'THF PB eagent (data from Table 1).

fOO 16] Fig.. 5 is a graph showing radio-TLC of purified ^l8 F-TFB (Rf =* 0,8 - 0 JS) with the silica gel stationary phase and methanol mobile phase. If present, unreacted ¹⁸ F-fluoride would remain at the origin,

[0017] Fig. 6 A depicts HPLC anal sis of the ¹⁸ F-TFB product in saline, conductivity data for 10 pg/mL.NaF standard,

[0018] Fig. 6B depicts HPLC analysis of the ⁱ⁸ F-TFB product in saline, conductivit data fo 10 ag mL NaBF standard,

[0019] Fig, depicts HPLC analysis of the ^!8 F~TP product in saline, radioactivity data fo 1.1 MBq ^1¾ F-fluoride standard,

[0O2Q] Fig. £B depicts HPLC anafysis of the ¹⁸ F-TFB product in saline, conductivity data for purified ^{, 8} F-TFB in saline,

[0021 ] Fig.€E depicts HPLC analysis of the ^l8 F-TFB product in saline, radioactivity data for purified ¹⁸ F*TP in saline,

[0022] Fig. ? shows GC analysis of residual organic solvents in the final ¹⁸ F-TpB product. N obvious petroleum ether peaks were observed,

[0023] Fig. 8 is a schematic of the putative reaction scheme for formation of * ^¾ F/ ¹⁸ F-TFB from BFs of the preparation of BFrTHF/PE complex solution,

[0024] Fig, 9 is a graph depicting time dependence of ¹⁸ F-TFB uptake (SUV) in different organs in representative hNlS-expressing C6 glioma xenografted mice at high specific activit of ^iS F-TFB (13 M q/pmol or 0.37 mg/kg mouse weight). [0025] Fig _* 9B is a graph depictin time dependence of ^{] 8} F« FB uptake (SUV) in. different organs in representative h S-expressin C6 glioma xenografted miee at low specific activity of ¹⁸ F-TFB (3 MBq/uinoI or 1 ,6 mg/k mouse weight).

[0026] fig 10 depicts PET images o ¹⁸ F-TFB distribution in control mouse (left) and a mouse bearing hNIS-positive and hNlS-negative C6 glioma xenografts (right). The overlaid reference bone atlas is computer generated,

[0027] Fig, 11A depicts dependence of tumor of ^iS F-TFB uptake on administered mass of TFB to hNIS-expressing C6 glioma xenografted mice.

[0028] Fig, 11B depicts dependence of stomach of ^! %-TFB uptake on administered mass of TFB to M EIS-expressing C6 glioma xenografted mice.

[0029] Fig, llC depicts dependence of tumor/stomach ratio of ¹⁸ F-TFB uptake on administered mass of TFB to hNIS -expressing C6 glioma xenografted mice. The tumor stomaeh ratio data for administered mass >0,5 mg/kg was fit to a bi-exponential clearance model using non-linear least-squares regression, Microsoft Excel Solver was used to regress the tumor stomach ratios using a nonlinear least-squares regression algorithm.

[0030] Fig, 12 are pictures representing immunostaining o hMS-expressing C6 glioma xenografts (10X magnification) showing iritra-tumoral and inter-turnoral variability of expression of hNIS. (Green: Alexa Fluor 488 antibod bound hNIS and Blue; DAM stained nuclei)

[0031 Fig. 13 is a schematic representatiOn of the imaging protocol. Patient were screened on day 1 and imaged by ^{1 S} F-TFB PET/CT on day 2.

[0032] Fig, 14 depicts graph showing time-activity curves of ^is F-TFB in healthy mail participant, LVBP= left-ventricular blood pool.

[0033] Fig, ISA are corona] PET/CT images of ^!S F-TFB in healthy male participants at 2 hour post-injection. Physiologic distribution of ¹⁸ F-TFB is seen in thyroid, salivary glands, stomach and intestines. Prominent excretion of radioactivity is seen in urinary bladder.

[0034] Fig, 15B are coronal PET/C Images of ^l8 F-TFB in healthy female participants at 2 hour post-injection. Physiologic distribution of ¹⁸ F-TF is seen in thyroid, salivary glands, stomach and intestines. Prominent excreti on of radioactivity is seen in urinary bladder. DETAILED DESCRIPTION

[0035] Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited i its application to the details of construction and the arrangement of components set forth in the following description or illustrate in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways, Also _* it is to be understood that the phraseolog and terminology used herein is for the purpose of descri tioii and should not be regarded as limiting. The use of "including/' "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as ell as additional items,

[0< ¾1 he following discussion is presented to enable a person s lled in the art to make and use embodiments of the invention. Various modifications to the illustrated embodiments will be readily appa ent to those skilled in the art, and the generic principle herei can be applied to Mother embodiments and applications without departing from embodiments of the in ention:, Thus, embodiments of the invention are not intended to be limited to embodiments shown, but are to be accorded the widest scope consistent with the principles and features disclosed herein, The following detailed description is to be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended t limit the scope o embodiments of the invention. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the invention,

[0(137] A method of radiolabeling tetrafiuoroborate (TFB) with ^l8 F-fiuoride is provided. The method comprises the direct radiofluorination on boron triSuoride (BF ₃ by reacting the BF ₃ directly with ^t8 F-fluoride. This method provides ^I8 F-TFB with an enhanced labeling yield and higher specific activity.

£003.8] In some embodiments, the method com (a) reacting ^l8 F-fluoride with Bf¾ and (b) isolating the ^l8 F-TFB from unreaeted ^ls F-fiuoride and BFs. In some embodiments, the ¹⁸ F-fluoride is trapped on an anion exchange cartridge or column and the reactin of ste (a) of ^iS F-fluoride with BF ₃ is performed on the anion exchange cartridge o column, In this method, the reacted ^{! 8} F-TFB remains on the anion exchange cartridge or column to be separated from the unreaeted '^-fluoride and BF ₃ . [0039J Suitable anion exchang columns or cartridges are known in the art- For example, in some embodiments, the anion exchange cartridge is a quaternary methyl ammonium anion exchange (QMA) cartridge. Suitable anion exchange resins will have a sufficient affinity for ¹⁸ F-TFB s to be able to separate the ^,:¾ F-TFB from the unreacted ^l8 F-fiUQrIde and BF ₃ .

[0040]; In some embodiments, the anion exchange cartridge or column containing the ^iS F- TFB is washed at least once to remove the unreacted BF ₃ and residual solvent from the cartridge. The anion exchange cartridge or column may be washed with a suitable wash solution. Suitable wash solutions include, but not limited to _* for example, THF and/or water to remove the unreacted BF ₃ and residual solvent In some embodiments, the wash solution comprises ether. In some embodiments, the cartridge or eolurnn is washed at least one, at least twice, at least three times. In some embodiments, the column is first washed with THF and then washed ith distilled water. In another embodiment, the column is washed sequentiall with THF and water. In some embodiments, th column is washed with about 10 hiL of THF followed by 10 m L of water,

[0041] Th ^{I 8} F-TFB is eluted from the anion exchange column. In some embodiments, the ¹⁸ F-TF8 is eluted using saline. In other embodiments, the ^{) S} F-TFB is eluted with solution selected fro the group consisting of phosphate buffer saline (PBS), sodium citrate, sodium chloride and sodium bicarbonate. Suitable concentrations of saline for removal o the ⁱ⁸ F-TF are known in the art and include., but are not limited to, about 0.9% Nad,

[0042] The eluted crude ¹⁸ F-TFB may be further purified by passing through a second column or cartridge comprising alumina wit a neutral surface chemistry. Suitable columns or cartridges include, but are not limited to. for example, aiumina-N SepPak® Plu cartridge (!fe Pak®). The second column or cartridge is used to remove unreacted ⁱ⁸ F-fluoride from the solution. The el ted crude ^l8 F-TFB may be passed over at least one second column or cartridge, in som embodiments, the eluted crude ^{i 8} F-TFB may be passed over at least two second column or cartridges. In some embodiments, the eluted crude ¹⁸ F-TFB may be passed over at least three second column or cartridges, in some embodiments, the eluted ¹⁸ F-TFB is passage through a sterilizing filter. In some embodiments, the sterilizing filter is 0.2 μΜ.

[0043] For the methods provided herewith,; BF ₃ may be provided in a solution of BF ₃ THF comple in petroleum ether, BFrTHF may be produced by methods known in the art, for example fro BF ₃ gas dissolved in THF using petroleum ether. In some instances, the BF THF complex was filtered to remove BF ₄ - before reactin with ¹⁸ F-fiuoride. In some embodiments, the BF THF comple was passed over a anion exchang resin, for example, an anion exchange resin that contains crosslinfced polystyrene matrix with tertiary amine and quaternary ammonium functional groups (e.g. Lewatit® MP-64).

|0044] In other embodiments, the BFs may be provided as, for e ample; BF3 -pyridine^ BFrEtsN, BFrMeOH, BFs'EtOH, BF ₃ -gt ₂ 0 and BFs'THF, among: others. fThe ^IS F-fluoride may be prepared by known methods in the art, for example, by irradiation of ^O-water, Suitable methods of irradiating ^{i 8} Q-water are known in the art, and include, using 50-70: uA (e.g. 65 uA) for about 10-30 (e. g. 15) minutes in cyclotron _*

[0045] In some embodiments, the ^l8 F-fluoride used has radioactivities of about 10 to about 50 Bq. In some embodiments, the ¹⁸ F-8uoride used has r dioactivities of about 15 to about 37 MBq,

:[004β] The disclosed methods provide purified ^{i 8} F-TFB with of specific activity of at least 5 In some instances, the methods provide ¹⁸ F-TFB with a specific activit of at least 8 GBq i oh In one embodiment, the purified ¹⁸ F-TFB has a specific activity of at least 8 J GBq/jamoL

[0047] The disclosed methods synthesize ^{1 &} F-TFB with a radiochemical yield of at least 15% with greater than about 95% purity. In some embodiments, methods provide ¹⁸ F-TFB with a radiochemical yield of up to 40% with greater than about 95% purity. In some instances, the radiochemical yield Is at least about 10%, at least about 20%, at least about 30%, at least about 40%,

[0048] The disclosure provides ¹⁸ F-TFB made by the methods provided. The ^t8 F-TFB is at least about 95% pure, in some embodiments, at least 98 pure. In some embodiments, the methods synthesize ¹⁸ F-TF! with at least about 95 purity. I some embodiments, the methods synthesize ^!8 F-TFB with about 98 purity. The ¹⁸ F-TFB provide has a specific activity of at least 3 GBq/ noL. alternatively at least 5 GBq pmol, alternatively at least 8 GBq/uirnol.

[0049] I n some embodiments, a method of radlolabeilng tetraffuoroborate with ¹⁸ F-fluoride consists essentiall ofi (a) trapping ¹⁸ F-fluoride on an anion exchange column; (b) reacting ¹⁸ F- fluoride with BF ₃ ; and (c) isolating the -F-TFB from unreacted ¹⁸ F~iluorid and BF ₃ from the anion exchange column, wherein the resultant ¹⁸ F-TFB has specific activity of at least 5 GBq/pmol. In this method, the %-TFB remains attached to th column or cartridge until e!uted using an isotonic saline solution. The column or cartridge containing ¹⁸ F-TFB may be washed with at least one wash solution before eluting the ¹⁸ F-TFB. The method may further include purifying the ⁱ⁸ F~TFB by runnin ove a column or cartridge comprising alumina ith a neutral surface chemistry. Suitable columns or cartridges include, but are not limited to, for example, alumina-N SepPak® Plus cartridge (SepPal®), I some embodiments, the eluted ^!8 F*TFB is passage through a sterilizing filter,

[0050] This disclosure provides methods of synthesizing ^{1 S} F-TFB in less than 30 minutes, alternativel less than 20 minutes, alternatively less than 10 minutes, In one embodiment, the method of synthesizing ¹⁸ F-TP is performed in about 10 minutes, in some instances in about 9 to 15 minutes. The following Examples are offered for illustrative purposes only, and .are not intended to limit the scope of the present invention in any way . Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoin description and the following examples and fall within the scope of the appended claims.

EXAMPLES

Example T Synthesis of ^{i 8} F-TetrafIuoroborate ( ^{i 8} F-TFB) via Pvadiofluorinatio of Boron

Trifluoride and Evaluation in Murine C6-GHoma Tumor Model

Materials and methods

[0051] Lewatit ^® MP-64 chloride form resin was procured from Sigma Aldrieh (St. Louis, Mo) and preconditioned with 1 M 2CO3 solution (1 equivalent), water { 100 equivalent, w/w) and tetra ydrofuran (THF) (100 equivalent, w/w) in colum _s and then dried u der nitrogen overnight. Acetone, THF and a BF gas cylinder were purchased from Sigma Aldrieh. Petroleum ether was procured from Fisher Scientific. Sep-Pak Aecell Plus QM A Carbonate Plus Light (46 mg sorbeht per cartridge, 40 ίη particle size), Alumma-M SepPak Plus cartridges and Alumina- N SepPak Light cartridges were obtained from Waters Corporation (Waltbam, MA). A Mini- scan radio-TLC scanner from Bioscan, Inc was used to monitor the radiochemicai purity. Anion chromatography HPLC (Bionex IC-210 , AS 1 analytical eolumn 4,7 x 150 mm, eluent 35 mM ROB, sample volume 25 uL. flow rate 1 mL/rntri) was calibrated to measure ^1:& F-TFB and ^{j 8} F- TFB concentrations with conductivity and radioactivity detectors i series. The method was validated by separation of a series of anions (F ^" , CI ^" , Bf , Γ, NC ^" , and BF ₄ ^* ) and OH ^" was negated by the system. Residual organic solvents were analyzed by GC (helium carrier gas flow at 10 ec/min through a MXTWAX column (Restek, Bellefonte, PA, 0.53 mm ID, 30 m length)). The temperature program was 4 min at 35 'C, followed by a temperature ramp of 4 ^° C per min to a maximum of 150 X.

Preparation of BPs-THF complex solutio

A solution o BFj-TFlF complex in petroleum ether was prepared freshly within 30 min of the radiosynthesis of ^{, a} F-TFB (Fig. lA). The luer opening of a 20 mL polypropylene syringe was melted closed. A silicone stopper was placed in the opposite opening. A 20 mL sterile vial with PEEK tubing was connected to the 20 mL syringe as shown in Figure 1. The syring body was completely filled with petroleum ether and about half of the sterile vial was also fi lled with petroleum ether (PE), BF3 gas ·-· 5 mL) was flowed into the syringe from a W cylinder through a PEEK, tube, thereby displacing the corresponding petroleum ether to the sterile vial. After removal of the BF3 addition tube, 0.5 mL TffF was injected through the silicone stopper. The BF3 gas was disSOlved in the added THF quickly, and petroleum ether was sucked back into the syringe from the auxiliary vial without entry of atmospheric air. The tube was removed and the mixture became a homogenous solution with gentle shaking. The concentration of the BF3 in the solution was ~ 1 ,8 ^moI/mL. (Fig, I A) Fig, IB shows an alternative method usin a simple mani fold/syringe system. As shown in Fig. 1 _t Step 1 yo fill the syringe A with 5 ml BP? and

Syringe B with PE/THF mixture; step 2 you switch the valve to the right and step 3 you push PE/THF mixture from Syringe B to the syringe A, Preparation of in-house-made Lewatit ^® MP-

64 cartridge

[0052] A 1 mL syringe body was filled with 300 mg Lewatit® MP-64 (carbonate form) and stoppered with some degreasing cotton at both sides. The syringe was cut off and jointed with another ImL syringe body using a sheath of silicone tubing (Fig, 2),

[ ¹⁸ F]TFB synthesis method [0053 An automated synthesis of ^1S F-TFB was developed for the preparatio of ¹⁸ F«TFB (Fig, 3), ^{l 8} F-fiuaride (37.6-40,5 GBq was made hy irradiation of 2.5 mL [ ^!8 0] wate with 65 uA for 15 mill in OE PETtrace cyclotron, and then delivered to the hot cell and trapped on a QMA (46 mg, carbonate form) cartridge. ¹⁸ G-enriched water was collected using valve V13. The QMA cartridge was rinsed with 1 thL anhydrous acetone, and flushed with nitroge for 100 seconds. The freshly prepared BFrTHF complex solution (5 mL)- was filtered by a In-house-made Lewatit® MP-64 cartridge (200-400 mg), and then passed through the: QMA cartridge within 1 s to react with the trapped ^{) 8} F-fluoride t form ^t8 F-TFB> The QMA cartridge was rinsed with a solution of 10 mL HF, flushed fo 100 s with trogeii, and rinsed with 13 mL water to furt er remove: impurities. The QMA was then treated with 5 mL sterile 0,9% NaCl, DSP solution (saline) to elute into a product vial pre-loaded with an additio l 5 mL saline.

[0054]: The crude ¹⁸ F-TFB product solution was further purified fr m unreaeted ^!8 F-fmoride by passin three alumina-N SepPafc Light ca tridges before passage thr ugh a 0,2 u sterilizing filter with final collection of the ^{i a} F~TFB in a product vial, (Fig, 3)

Quality control

[0055] The produet ¹⁸ F-TFB was analyzed for radiochemical purity by both radio-TLC (MeOti, f ~ 0.8-0.85) and anion chromatograph HPLC with radioactivity detector (Dionex iC-2100, AS 19 analytical column: 4.7 x 150 mm; eluetit: 3 M KOH; sample volume: 25 X; flow rate: 1 mL/min retention times: 7.6 min for ^!8 F-TFB, 7.8 min for ¹⁸ F-TFB), Chemical purity and specific activity were analyzed b anion chromatography HPLC (retention times: 3,5 min for fluoride, 4,3 min for chloride, and 7,6 mi for BP4-), Residual organic solvents were analyzed by OC (helium carrier gas flow at 10 o mrn through a MXT X column (Restek, Bellefonte, PA, 0.53 mm ID, 30 m length)).

In Viv Imaging

[0056] Studies with mice were performed under approval of the Mayo Clinic Institutional Animal Care and Use Committee. Dynamic PIT imaging was performed in hNIS-expressisg 06 glioma xenografted athymie mice following retro-orbital injection ~1.1 MBq Na ^{l 8} F-TFB at different specific activities ( 10 -0.001 mg TFB/Kg mouse) to assess MS activity with ¹⁸ F-TFB. In this xenograft model, one flank has hNlS-negative C gliom tumor xenograft and the other Sank had hNrS-expressing C6 glioma xenograft, [0057] PET scans were acquired for 60 min followed by an X-ray scan using the GEM1SYS4 P T imaging system (Sofie Biosciences, CA). the images from 40 hlNtlS-expressing C6 gliom xenografted mice were analyze for Standardized Uptake Valu (SUV) in tumor, stomach, and thyroid using AMIDE image processing software (2§)< The SUV in tumor was normalized with SUV in stomac to account for difference in ^1S F-TFB bioavailability- in different animals due to competing uptake in normal organs like thyroid, salivary glands and stomach,

im anohistoehemistry

[0058] Tumors from 5 age-matehed hNIS-expressing 06 glioma xenografted mice were harvested and formalin-fixed. The tumors were then equilibrated in 15% and 30% sucrose with phosphate buffer for 4 days, and frozen for cryo-sectioning. A series of adjacent sections were cut o a cryostat Each section was 10 m thick, and mounted onto charged slide Superfrost Plus slides,. Pisherhrand , After drying, the sections were blocked with 1 % goat serum for 4h at room temperature, followed by overnight incubation wit 1 : 4000 dilution of rabbit Anti-huma HIS antibody SJl (Imanis Life Sciences, Rochester, MM) in PBS with 10% goat serum at 4 °C, The non-specific anti-human HIS antibody SJl in sections was 3X washed in PB -Tween 20 (0.05%) for 15 mi each at room temperature. The sections were incubated with secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (i l + L) antibody (Life Technologies, CA) at a dilution of 1 :4000 in PBS fo 45 min at room temperature. The non-specific secondary antibody wa thfiee washed in PBS- een 20 (0.05%) for 1 min each at room temperature:, Following washing, the sections were counter-stained with nuclear OAPI stain. The sections were then cover-slipped with mounting medium and imaged using Nikon Eclipse Ti inverted microscope at 10X magn¾cation.

Data Analysis and Statistics

[0059] Data Is expressed as mean ± SD« Microsoft Excel Solver was used to regress the tumor/stomach ratios of ' ^& F-TFB uptake using a nonlinear least-squares regression algorithm.

Results

Radiosyn hesls and quality control of ^t8 F-TFB

[0060] After irradiation, ^l8 F-ftuoride in ^l8 0-enrIched water was delivered to the hot eel and quantitatively trapped on the QMA cartridge. The QMA cartridge Was rinsed with acetone (10 mL) and flushed with nitrogen for J 00 s. Tie fteshiy prepared BFs'THF complex solution (5 niL) was passed through an inhouse-rnade Lewatit® MP-6 cartridge and the QMA cartridge as a single bulk passage of solvent lasting ~10 s. ^I8 F-TFB largely remained on the cartridge while 20 -40% of the ^! ¾¾luoride was released from the QMA cartridge, possibly by formation of ^iS F- HF under the acidic conditions caused by BF ₃ , The QMA cartridge was rinsed with a solutio ©f 10 mL THF and 13 mL water to remove the impurities from the QMA cartridge, To decrease the residual acetone and THF in the final product, 100 s of nitrogen flush was applied after THF rinsing, ^l8 F-TPB was eluted from the QMA cartridge wit 5 mL sterile saline to the product vial, in which 5 mL sterile saline was added in advance for further dilution of the product. The crude product solution was purified by trapping the unreached ^{l s} F-fiuorkle on three alamina-N SepPak Light cartridges. Using two alun ina-N SepPak Light cartridges, the radiochemical purities were 93-96%. Radiochemical purity was increased to >98% by use of an additional alumina-M SepPak Light cartridge. The amount of starting BF ₃ for the reaction was found to be critical to determine the radiochemical yields and specific activities. Firstly, 5 mL BF ₃ 'THF/PE solution (~ 43 urno!) was used in the reaction and 8,33-0.6$ y ol unlabeled TFB with 35, + 3.6% yields were obtained. To decrease the amount of BF ₃ . fa the reaction, the Bl¾ TFIF/PE solution as passed through Lewatit® MP-64 resin immediatel before passage through the QMA cartridge, Lewatit® MP-6 is an anion exchange resiii, which contains erosslinked polystyrene matrix with tertiary amine and quaternar ammonium mnctional groups. Separate analysis of the post- Lewatit® MP-64 filtrate showed that 70%„ 80%. and 90% of the BF ₃ in the original S mL BF ₃ 'TFIF/PE solution was retained on 20¾, 300 and 400 mg Lewatit® MP-64 resin, respectively, Thus, the influence of the Lewatit® MP-64 was no only to retain unlabeled TFB, but also to reduce the amount BF ₃ reaetant. Evidently, the BFa tertiary amine complex formed on the Lewatit® MP-6 was stronger than the BFj'THF complex in the solution, which result in retention of BFj on the resin,

[0061] Investigations with 20 to 40 mg Lewatit® MP-64 resin (Table 1) resulted in the production of unlabeled TFB and product ^!8 F~TFB in proportional amounts (Fig. 4). More Lewatit® MP-64 resin gave less BF ₃ in the reaction,, resulting in lower radiochemical yield with proportionately less unlabeled TFB (Table I ). Th _^ the specific activity of the '%-TFB product improved as the amount of Lewatit® MP-64 resin is increased, however, at the cost of decreased radiochemical yield, Table 1 : Dependence of overall radiochemical yield and specifie activity (N ^« *3) of ⁱ⁸ F-TFB pfodtiet on the amount of Lewatifw P-64 resin. Reactions were performed with starting ^t8 F- fluoride radioactivities of 15-37 MB .

[0062]: A 300 mg Lewaiit ^® MP-6 resin was used for a high radioactivity level synthesis (40-44 GBq) as a compromise between radiochemical yield and specifie activity. The radioehemieal yield of ' ⁸ P~TFB was 20.0 ± 0.7% (n— 3) uncoreeeted in a synthesis time of 10 min. Radiochemical purity was >98% as show on silica gel TLC (Fig. 5) and anion chromatography HPLC (Fig, 6). Specific activities of 8.8 ± 0.56 G ^mol (n ~ 3) were achieved fro starting ^{! 8} F-flnoride activities of 40-44 GBq. In the HPLC analysis of the final product, a carbonate peak at 3.6 min and an unknown impurity peak at 3.8 min were initiall obs rved _^ Investigatio revealed these to be contaminants present on the stock QMA and alumina cartridges. By pretreating the cartridges with 20 mL 0.9% saline and 20 mL water,; the peaks were reduced to a trace amount.

[0063] Residual acetone and f. 1 11· ^' were obtained in concentrations of 6 - 135 pj?r and 24 - 27 ppm,. respectively, whic were well under the allowe solvent concentration limits (acetone 5000 ppm, HF 720 ppm) set by the International Conference on Harmonization (ICH) for technical requirements for registration of pharmaceuticals for human use (Fig, 7).

Stability of ⁱ⁸ F-TFB

[0064] After the synthesis, a sample was diluted with wate for the analysis. With the eluent of methanol on silica gel plate, purities of 98% were obtained on the radio-TLC scanner. After 20 h, r¾dlo--tLC gave a 96%. radiochemical purity and HPLC analysis of the product sample showed the ¹⁸ F-TFB peak was chemically stable. A slow hydration of ^r8 F- FB in water may resulted in the slow decrement of the radiochemical purity, HPLC analysis of a NaBF ₄ stock solution (stored fo 7 month at room temperature) showed 50% of TFB was decayed and gave a peak of fluoride at 34 min.

In Vivo Imaging Studies

[0065] Eobust uptake of '%-TFB was observed i thyroid _* stomach, bladder and hNlS- expressing tumors of the C6-glioma xenograft mouse model (Fig 9 and 10). No uptake of ^{, 8} F- TFB was observed in hNIS-negative tumors, confirmin specificity of uptake of ^{l g} F-TFB to hNIS-expressing tumors. ¹⁸ F-TFB uptake in thyroid and h IS-expressin tumor showed a bi- phasic kinetic: rapid uptak over the first 10 min was followed by slower uptake until 30 min with little subsequen change. The stomach showed linear increase in ¹⁸ F-TFB uptake over 60 min. Stomach uptake was independent of specific activity of ¹⁸ F-TFB while uptake in hNIS- expressing tumor and thyroid was dependent on specific activity of ¹⁸ F-TFB. The uptake of ^,8 F- TFB by hNIS-expressing tumor was higher at hig specific aetivity f>13 ΜΒ /μηιοΙ) as compared to lower specific aetivity (3 MB /μκωΙ). This trend was also seen for thyroid,

[0066] For a more detailed analysis of the effect o specifie activity of ¹⁸ F-TFB on uptake in hNIS-expressing tumors, a 60 mitt time-point was chosen and a range of specifie activities (10- 0,001 mg TFB injected/kg mouse weight) was tested. The uptake in tumor was normalized to stomach uptake to account fo differences in bioavailability of ¹⁸ F-TFB across animals, The uptake of ¹⁸ F-TFB in hNIS-expressing C6 tumor showed dependence on specific activity at 10- 0,5 mg TFB/Kg mouse weight or 0- 1 ΜΒα ιηοΙ considering ~U MBq radioactivity injected in a ~25 g mice. But this trend was not observed for higher specific activitie .(> 10 MBq/μηιοΙ TFB or < 0,5 mg TFB/Kg mouse weight) (Fig, 1 1). At these higher specific activities the uptake of ¹⁸ F-TFB was no longer dependent on specifie activity but exhibited high variability. Immunohistochemistr analysis for hNIS in tumor sections fma 5 m ce showed significant intra- tumoral and inter-tumoral variability of expression of hNIS (Fig- 12) that may play a role in the variability of ¹⁸ F-TFB uptake observed in tumors.

Discussion [0067] Βϊ¾ is a versatile Lewis acid and widely used in the chemistry. It is known that BR will form TFB in the presence of water via a fluoride exchange reaction, which was well documented: [17, 18]. Therefore, the present disclosure aimed to synthesize high specific activit ^{! 8} F-TFB with t e reaction of BF¾ and ⁱ⁸ F-fluoride. The; ⁱ⁸ F*fIuoride was conveniently trapped on a QMA cartridge and the reaction was found to proceed, by addition of a BF ₃ complex in solution, A variety of BF ₃ complexes (e.g. BFj'pyridine, BFrltsN, BFrMeO L B a-EtOH, BFs^EtaO and BF ₃ -THF) were tested in the reaction. With respect to yield and specific activity, BFj'THF complex gave the best results. Therefore, BFa-THF complex was used for the further optimization. In initial exploration, only 5 - 10% radiochemical yields of ^1¾ F-TFB were obtained with commercialiy available BIVTHF solutions. To improve radiochemical yields of ¹⁸ -TFB, a BFrTHF FE solution was freshly prepared with BF ₃ gas in a simple apparatus described above (Fig. I A r IB). To improve specific activity, the freshly prepared s- TFIF/PE solution was passed through Lewatit® MP-64 resin immediately before passage through the QMA cartridge. As shown ¼ Table 1 , depending on the a o t of Lewatit® resin used in the rocess^ a trade-off was found to exist between radiochemical yield and specific activity of the ^[ F-TFB product.

[006f| Using 300 mg Lewatit® MP- 6 resin was selected for a high radioactivity level synthesis (40-44 GBq) as a compromise between radiochemical yiel and specific activity. Three runs in this conditio achieved specific activities of 8,84 + Q56 GBq/. ol and uncorrected radiochemical yields of 20.0 ± 0,7%, In some embodiments, to optimize specific activity at the sacrifice of radiochemical yi ld, the use of 400 mg Lewatit® MP-64 resin and 8 GBq starting activity would result in %-TFB specific activity of approximately 30 ΟΒς/μηιοί with an: overall uncorrected radiochemical yield near about 12%, The high specific activities obtained by our method ate unlikely to be obtained with the previously reported isotope exchange method [1 ],

|006 ] The methods provided improve upon the isotope exchange method by providing si nficantly enhanced radiochemical yields (-31 with use of 200 mg Lewatit® MP-64 resin versus -1 for isotopic exchange [M at moderate specific a tivity ( - 5 GBq^mol (estimated) versus -4 B ^rnol [14]), A putative chemical reaction mechanism for synthesis of ^1S F-TFB via BF3 is shown in scheme ί , BFa hy drates with water quickly to ^' form HBF3OH [ 17] .. Although the BF TH complex solution is prepared in near anhydrous conditions, the QMA cartridge is likely containing trace amounts of water after trapping of the ^{i a} F-fluoride and treatment with acetone and nitrogen. Indeed, the ^{, 8} F-fluoride itself may be in a hydrated form [19} on the QMA cartridge. Although it is possible that BFi reacts directly with ¹⁸ F-fiuoride ion, not to be bound by any theory, it is believed thai HBF3OH is the more likely intermediate t react with ¹⁸ F- fluoride to make ¹⁸ F-TFB. In the presence of water, MBF3OH may react further to form a series of intermediates, which ends with the release of boric acid and HF [17], The release of ^l9 F- fluoride from B ₃ may represent a source of unlabeled fluoride that can decrease the specific activity of the product. (Scheme 1 )

[00701 Jauregui-Osoro et al. (14) estimated that th administration of -400 MBq ^ls F-TFB synthesized by the conventional method to a huma subject would result in a plasma concentration of -0,1 M TFB. Considering the ICso of TFB to be 0,1-1 μ for inhibition of iodine uptake by NIS in thyroid (21 it is desirable to increase ^l8 F~TFB specific activity above -5 GBq/umol to avoid a pharmacological effect (14). The presently reported synthesis method for ¹⁸ F-TFB achieves this goal. At a specific activity of 8 GBq/pmol, a -400 MBq administered dose of ^!8 F-TFB would give an estimated in vivo concentration of -0.02 μΜ TFB which should not exhibit a pharmacologic effect.

[0071] To evaluate the influence of ¹⁸ F-TFB specific activity on in vivo uptake by MIS- expressing tissues, we employed a hNIS-expressin 05 tumor xenograft mouse model. As expected, ¹⁸ F-TFB was taken up by selected organs expressing NIS in th xenograft mouse model. Amon the select organs, thyroid and hMlS-expressing tumor were sensitive to specific activity of ^l8 F-TFB as ^,8 F-TFB was being transported using NIS in these organs. On the other hand, in stomach, which also possesses NIS (22-24), the uptake of ¹⁸ F-TFB was found to be independent of its specific activity, The insensitiviry of ^l8 F-TFB uptake to speeifie activity in stomach was not clarified, but ma point to different kinetic properties o the NIS protei in gastric mucosal cells as compared to thyroid and hNIS-expressing tumor 23,24), Anothe possible explanation is that ⁱ⁸ F-TFB entering th gastric epithelial cells is immediately eHluxed into the stomach lumen such that the intracellular concentration (e.g. in gastric parietal cells) is never in equilibrium with the interstitial fluid so unidirectional transport continues unabated, A biphasic response was observed in hNIS-expressing tumor uptake of ^l8 F-TFB versus administered dose of unlabeled TFB. The ^,8 F-TFB uptake in tumor decreased with increasing amount of administered TFB over the range of 0.5*10 mg TFB/kg mouse weight, but this trend was not observed for higher specific activities (< 0,5 mg TFB/kg mouse weight). Rather, the tumor uptake was constant but highly variable for administered TFB doses < 0,5 mg kg. The reason for ( s trend is not clear but it is possible that at high specific activity, factors other than specific activity contribute to the variability, such as variability the inter- and intra-tumoral expression and/or activity of hNIS, number of tumor cells expressing hNIS, heterogeneity of tumor perfusion and/or oxygenation, variability of tumor size o the indirect influence of differences in the phy siological distribution of radiotracer to other areas of the body . The large variation seen in hNIS expression levels in the 06 glioma xenografts may reflect the fact that the C6-hNIS transduced cell line was not a clonal population but included high, low and negative expressin cells. The ability of the ^t? F-TFB-PET method to reliably report on viral infection depends on hNIS expression within infected cells an possibly p st-translational events that influence hNIS transporte activity (25). These considerations must also be kept in mind for future studies in monitoring hNI transduction in human studies that may also entail significant het^ogeneity of hNIS expression followin viral therapies. Nonetheless, it was encouragin to observe a broad range of specific activities of ^l8 F~TFB over which tumor u take was robust.

Conclusion

0:?2] A solid-phase supported synthesis of ^I8 F-TFB was developed via radiofiuorinatioo of BF ₃ . With the optimized condition, the radiochemical yield of ^K F-TFB was 20 ± 0.7% ( - 3) uncorrected i a synthesis time of 10 min. Specific activities of 8.84 + 0.56 GBq^mol (n = 3) were achieved with startin ^! *fiuorlde radioactivities f 40 - 44 GBq. This method offers a convenient synthesis of high specific activity ¹⁸ F TFB. A positive correlation was observed between specific activity of ^l8 F-TFB and hNIS-e pressing C glioma xenografts for lower specific activities resul ting in administration of TFB exceedin 0.5 mg/kg in mice. The increased specific activit of ^1S F-TFB ma allow for enhanced PET imaging of hNIS reporte in future human studies.

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2Qi%37:21Q8-16.

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[18] Wamser CA, Hydrolysis of Fluoboric Acid in Aqueous Solution, J. Am. Chem, Soc 1948;70: 1209 15.

[19] Cai L, Lu S, and Pike VW, Chemistry with F-18 fluoride ion, Eur. J. Org. Chem 2008:2853 73.

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[21]. Lecat«Guiilet N, Ambroise Y. Discovery of aryltrifluoroborates as potent sodium/iodide symporter (NIS) inhibitors, CheraMedChem. 2008;3:1207-1209.

[22]i. Portulano C, Paroder-Belenitsky M, Carrasco N, The i¾+/I- symporter (NIS): mechanism and medical impact. Endocr v. 2014;35: 106-149.

[23.] Wolosin JM. Ion transport studies with H+-K+-ATPase~rieh vesicles: implications for HCI secretion and parietal cell physiology. Am J Physiol. 1985;248;G595-607. [24]. R, McG, Harden, , D. Alexander, J. Sliimmins, D, Chishoim, A comparison between the gastric and salivary concentration of iodide, pertechnetate, and bromide in man. Gut

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The citation of an do cument i s not to be construed as an admission that it is prior art with respect to the present invention.

[0073] Example 2; Bidistribution and Radiation Dosimetry of ^I8 F-Tetrafluoroborate ( ¹⁸ F- TFB) in Healthy Human Subjects

10074] ¹⁸ P -tetrafluoroborate ( ⁱ⁸ F-TFB); has bee identified as a promising iodide analog for imaging of thyroid cancer and monitoring of NIS expression as a reporter probe in viral therapy applications, This Example demonstrates the pharmacokinetics, biodistribution and radiation dosimetry of high- specific activity ¹⁸ F-TFB in healthy human subjectSv ethods:; ^t8 F-TFB was synthesized via ¹⁸ F-fiuorinafion of BF3 with specific activity of 2,2 ± 0 J GBej/^mol. Dynamic FET/CT imaging over 4 h was performed after intravenous administration of ⁱ⁸ F-TFB 333-407 MBq) in 4 female and 4 male health volunteers (35 ± 10 year old). Initial scanning over the heart allowed definition of the biood pharmacokinetics, while whole body scans at 2 and 3.5 h provided data for biodistribution and radiation dosimetry. Samples of venous blood and urine were collected over the imaging period and analyzed by ion-ehromatography HPLC to determine tracer stability. Vital signs and clinieal laboratory safety assays were measured to determine the safety of ¹⁸ F-TFB administration. Results; ^{i 8} F-TFB administration was well tolerated with no significant findings on vital sighs or clinical laboratory assays. Left-ventricular blood pool time- activity curves showed a multi-phasic blood clearance of radioactivity with the two rapid clearance phases over the first 30-45 min, followed by a slowe clearance phase, HPLC analysis showed insignificant ^l8 F-labele<i metabolites in the blood and urine over the length of the study (4 h). At 2 h p.i., high uptakes were seen in thyroid, stomach, salivary glands, and kidney. Urinary clearance of ^{i S} F-TFB was prominent. Minor changes were seen in ^t8 F-TFB biodistribution f om 2-4 h p.i. A low level of metabolic defluorination was evidenced by tow accumulatio of ^t8 F-radioactivity in bone (SUV ~ 1. ±0.5 in males, SUV = L3±Q.9 in females at 3,5 h), CoBefesioii; This initial study in healthy human subjects showed ^{i 8} F-TFB to be safe, metabolically stable, and distribute in the human bod similar to other iodide analogs with prominent physiologic distribution to thyroid, stomach, salivary glands and bladder, with thyroid as the dose-critical organ. ¹⁸ F-TPB ca be used as a hNlS gene reporter and imaging biomarke for thyroid cancer and other disease processes that import iodide,

[0875] Example 1 demonstrates high specific radioactivity syntheses of ¹⁸ F-TF via the reaction of boron trifluoride (BF3) and ^l8 F-fluoride, We demonstrated the relationship of ^lS F- TFB specific radioactivity and FE -delineated radiotracer uptake in MS-trarisificted C6-gMo a xenograft bearing mice, confirming the desirability of high specific radioactivity to avoid saturation effects at the MS transporter. In this study, we report PET CT imaging data with ^l8 F- TFB in healthy male and female participants to describe physiologic distribution of the radiotracer and allow calculation of radiation dosimetry estimates.

Materials and ethods:

[0076]; Radiotracer synthesis

[0077] ¹⁸ F-TFB was prepared and formulated in sterile 0.9% NaQ under cGMP conditions as previously described (12). Beeay-corrected radiochemical yields of 32 ± 2% and radiochemical purities ( CPs >98% were obtained. Specifie radioactivity of 2. ± 0.9 GBq/μηιοΙ was acMeved from starting ^l8 F-fiu«ide radioactivit of 20-31 GBq, in vitro RCP remained >96% up to 8 h at room temperature.

[007S] Fluman participants

[0079]; Approval of the study was obtained from the Mayo Clinic institutional Review Board and all participants provided informed consent. Four male (36 ± 14 y) and four female (35 ^■ ± 8 y) health volunteers were enrolled in th study. Participants were excluded that had previous diagnosis of cancer or clinically sig ficant cardiovascular., renal, pulmonary, metabolic^ and endocrin (thyroid) diseases. Participants were not required to fast prior to the imaging study but instructed, to remain well-hydrated.

[0080] PET/CT imaging protocol

[0081] The imaging protocol is illustrated in Figure 13. After voiding of the bladder, participants were positioned in a GE 690XT PET C scanner (GE BealthCare, Waukesha, WI) in a supine position with the heart in the center of the axial ield of view. Intravenous catheters were pla¾ed in both arms for radiotracer injectio and blood sampling. Computed tomography scans of the thorax were initially acquired in the preparation for a dynamic PET data acquisition over the heart for the first 45 min following commencement of radiotracer administration. ¹⁸ F- TFB (333-407 MBq) was administered over I minute into one o the ca heters. The frame sequence for initial dynamic PET acquisition was 15 x 4, S x 15, 4 x 30. and 8 x 300 seconds. Following the dynamic heart scan, the participants were allowed a break from the scanner, during which time voided their bladders. o additional PET/CT scans, from the vertex of the skull to mid-thigh, were performed at 2 hours and 3,5 hours respectively. The last PET/CT scan was completed ~4 h post-injection,

[0082 Measurements ⁱⁿ venous blood and urine samples

[0083] Urine was collected after each PET/CT scan (approximately 55, 160, 250 rain), and venous blood samples were collected at 40, 1 5 and 235 min, post-injection, as shown in figure 13. Blood samples (3-4 ml) were collected in heparinized tubes and placed on ice. The blood samples were centrifuged at 3000 g for 5 min to obtain plasma. Urine was measured for urine volume and radioactivity concentration as measured in a calibrated gamm counter. Analysis for metabolites in plasma and urine was performed by ion-chromatography HPLC, allowing estimation of the fraction of radioactivity as lionmetabolized ¹⁸ F-TFB .

[0084] Safet measurements for ⁱ⁸ F-TFB administration

[0085] Vital signs (heart rate, systolic and diastolic blood pressures, respiratory rate, and temperature) were measured prior to ^1S F-TFB administration and at 45 and 240 mi post- injection. Venous blood samples were taken prior to ⁱ⁸ F-TF administration and at -240 min post-injection for a panel of clinical laboratory tests to evaluate the safety of the radiotracer administration,

[0086] PET/CT image analysis

[0087] All PET scans images were reconstructed using 3D OSEM and time-of-flight reconstruction. Volume of interest (VOFs) of organs were drawn using PMC© software (Ver. 3.5). Standardized Uptake Values (SUVs) normalized to body weight were then calculated for each VOL Time-activity curves (TACs) were evaluated from the initial dynamic sca over the heart for left-ventricular blood pool, lung and liver regions..

[0088] Radiation dosimetry estimation

[0089] Radiation dosimetry estimates: were calculated from organ residence times using OTINBA software (Ver. 1.1), assuming a bladder voiding interval of 3.5 h ^, Gender-speeif c organ masses for the conversion of SUV to disu tegration per organ per unit radioactivity administered (Bq-hr/Bq) were taken from Sehfck et af (15). QMj Statistics

[0091 ] Data are expressed as mean ± SB, Statistical significance of differences i SUV values between: PBT/CT scans were determined by ANO VA.

Results

[0092] Pharmacokirteties of ^{I S} F-TFB

[0093] The initial 45-rnin dynamic PET CT scan over the heart: allowed evaluation of the early kinetics of ^1;8 F-TFB in the lefr-ventricu!ar blood pool (LVBP) as in indication of the: pharmacokinetics of this radiotracer. Figure 14 shows the time-activity curves in LVBP _S lung, and live in a representative participant. Peak values of radioactivity concentratio were seen at ~4 min for LVBP and lung regions, whereas the peak in liver wa at ~2 min post administratio of ^1S F-TF . Blood clearance was multiphasic, with clearance proceeding to the end of the 4-h measurement period. Continued radiotracer washout was also see in lun and liver.

[0094] Metabolite analysis

[0095] HPLC metabolite analysis of plasma samples at 40 min p.i, showed >97% of radioactivit to be in the form of etabolcally intact ⁱ⁸ F-TFB: (Table 2). Similarly, all urine samples taken from urine collections at approximately 50, 160, 250 min showed >97% of radioactivity in the farm of ⁱ⁸ F-TFB, The accumulated percentage of administered ⁱ⁸ F-TFB in the urine at the end of study (~250 min) was 40 ± 4% for males and 47 ± 7% for females. Thus, renal clearance of aonmetabolized ⁱ⁸ F-TFB is major excretion pathwa for ^{¾ 8} F-TFB, 40 min ¹ ' 145 min ^a 235 min ^a Accumulative

Males Females Males Females Males Females Males Fema]

Whole blood (SUV) 3.0 ± 0,2 3 A ± 0.9 2,1 ± 0.2 L7 ± 1.7 ± 0,2 1.4 ± 0.4 - 0.4

Plasma (SUV) 3.8 ± 0.2 3.8 ± 1 ,0 2.8 ± 0.3 2.1 ± 2.0 ± 0.1 1.7 ± 0.5 - 0.5

Urine (% ose) 15 ± 2 ^b 16 ± 3 13 ± 3 18 ± 3 10 ± 2 ^fe 12 ± 2 40 ± 4 ^b 47 ± %lntaet Plasma 97 ± 2 97 ± 3 ^C

l ⁸ F-TFB Urine 96 ± 2 98 ± 1 97 ± 2 98 ± 1 98 ± 1 97 ± 2

Values are mean ± SE (/? = = 4) aTime-points are shown for blood samples; urine samples were colleeted --10 min later,

¾te urine in third collection in one of the male subjects,

cOne plasma sample was not used because precipitate was observed in the BPLC analysis.

dMet¾bolite data were not obtained in second and third plasma samples analysis because the low radioactivity levels were below detection limit,

[0097] Biodistribution of ^ia F-TFB

{©098} Representative images from the whole-body PET/CT scan at 2 h post-injection are shown in Figure 15 and the biodistribution data are summarized in Table 3, Robust uptake of 1 ^S F-TFB was observed in thyroid, stomach, salivary glands, and kidney. Prominent clearance of tracer through kidney to bladder was also observed. Minor differences were observed in the SUV values between the first ( h) and second (3. h) whole body PET/CT scans, showin the tracer distribution to be stable at least after 2 h. Bone uptake was moderately increased from 2 to 3,5 h in males, indicating a low level of defluorination of ^l F-TFB, but the increase was not statistically significant in females.

[00991 Tafcie 3L PETyCT-derived distribution of ^SS F-TFB i healthy participants

2 h postinfectio (StJV) 3 ,5 h post-injection (SUV)

Organ Males Females Males Females

Bone OJO ± 0,33 0,74 ±0.20 1.4 ±0.5* 1.3 ± 0.9

Brain GJ9±0,14 0.42 ±0,15 0.74 ± 0.26 0,61 ± 0.34

Breast - 2.8 ± 0.4 3.9 ± 1.4

Gallbladder 2.7 ± 1,5 3.7 ± 1.0 4.2 ± 1.7 5,0 ±1,6

Intestines 2.9 ±1,0 5,0 ±2,7 4.4 ± 1.0 6,0 ±3.1

Kidney 7.0 ±2. 5.6 ±0.7 11 ±3 6.3 ± 2,

Liver 2.3 ±1.0 2.7 ± 0, 3.6 ±1.2 3.3 ± 1.6

Lang 1.0 ±0,4 1.2 ±0.4 1.7 ±0, 1.5 ±0,8

Muscle 0,71 ±0.31 0,65 ± 0,35 0.96 ±0.31 0,78 ± 0.54

Myocardiu 3.2 ±0.9 2.8 ±0,5 5,0 ± Q J 3J± 1.8

Pancreas 3,4 ±0.7 3,1 ± 1.7 6.2 ±2.1 3.0 ±1.2

Parotid U±9 20± 11 16 ±13 25 ±15

Spleen 4.1 ±1.2 4.3 vL 1.0 6,3 ± 1.1 5L5 ±2.8

Stomach 33 ± 15 72 ± 1 70 ±3 51 ±18

Thyroid 55 ± 1. 50 ± 11 82 ±42* 58 ± 12

Values are mean ± SBM (/? = 4) *p iQ.05 ersus:

{00100] Radiation dosimetry estimates

[00101] Table 4 shows: organ residence times derived from the biodistributio data. Bladder residence time was estimated by the bladder emptying model within the 0LIMDA software, assuming a 3,5 h bladder voiding period. Estimated organ absorbed doses are shown in Table 5. The dose-critical organ is thyroid, with dose estimates of 0.26 and 0.36 mSv/MBq: in males and females, respectively. The prominent excretion through the bladder resulted in moderately high doses to the bladder wall, with doses depending on voiding frequency. Effective doses are show in Table 6. Effective dose was higher in fe ales (0,065 mSv/MBq) relative to males (0.03 mSv/MBq).

Breast - 2 J ± 0.9

Gallfeiadder 0.06 ± o:.03 0.09 ± 0.04

Intestines. 5, ± ! J 13 ±7

Kidney 4.2 ± I, .2 3 ,9 ± 0,8:

Liver ±.3 ,9 10 ± 2

Ltmg 2.1 ± 0,7 2,4 ± 0.2

Mnsete 36 ± 14 26 ± 8

Myocardium 2.1 ± 0.6 1.8 ± 0□

Pancreas: 0.7 ± 0.3 0.58 ± 0.08

Parotid 1.7 ± U 3.3. ± 1.2

Spleen 1 ,5 ± 0,4 1 ,6 ± 0.2

Stomach 1 1 ± 5 :24 ± I2

Thyroid 2,0 ± 0.7 2:3 ± 0,:9

Bladder 17 ± 2 19 ± 4

Values are mean ± SEM (n =4)

[00103] Table 5 Estimated absorbed radiation dose for ^l8 F-TFB (mSyr Bq)

Organ Pose (mSv/MBq

Males Females

Adrenals 0.00:8 0.012

Brain 0.004 0:.0O

Breasts 0.002 0,028

Gallbladder Wall 0,012 0,022

Lower Large Intestine Wall 0.00 OJiS

Smalt Intestine Wall 0,027 0,066:

Stomac Wail 0.076 0,184

Upper Large Intestine Wall 0.04 0.109

Heart a 0.024 0,030

Kidneys 0.049 Θ.052

Liver 021 0.033:

Langs 0,009 0,014

Muscle 0.008 0.01 1

Ovaries _* 0.020

Pancreas 0.034 0.043

Red Marrow 0.005 0,008

Osteogenic Cells 0.0Θ8 0,009

Skin 0.OQ3 0.004

Spleen 0.033 0.047

Testes 0,005 -

Thymus 0,004 0,005

Thyroid 0:,26 0,36

Urinary Bladder Wall 0,14 0.22 tltertis - sm

Total Body 0.008: 0.01 1: [00104] Table 6. Effective dose for ^{1 S} F-TFB in healthy participants

Mates Females

Effective Dose (mSv/MBq) 0.036 0.065

[00105] ¹⁸ F-TFB Safety data

[00106] Vital signs (heart rate, diastolic and systolic blood pressures, and respiratory rate) were monitored before ^{i 8} F-TFB administration and throughout the PET/CT imaging period. No significant changes in the vital signs were found after ¹⁸ F-TFB administration (data not shown). Likewise, a panel of clinical laboratory blood tests was measured to assess for effects of ^{f 8} F-TPB administration on blood chemistries, including electrolytes, and liver and kidney funetional tests (Supplemental data no shown). No significant changes were observed in the clinical laboratory test values for the samples acquired after ¹⁸ F-TFB administration relativ to baseline.

Discussion

[00107] This Example evaluated the pharmacokinetics, biodistribution and radiation dosimetry of high specific radioactivity ¹⁸ F-TFB in eight healthy human participants. The tracer was well-tolerated and no adverse effects were noted. hMIS is known to be highly expressed in certain tissues (thyroid, breast, stomach and salivary glands) as well as hNIS-transfected tissues. Thus, non-invasive PET imaging of hNIS activity could be used to f cilitate the treatment of thyroid and breast cancer and gene therapies that employ hNIS as a reporter gene, ¾ principle, the ^1S F-TFB PET method may also enable quantitative estimation of hNIS activity in tissues. Therefore, it may be useful to monitor changes over time for understanding the progression of disease and serial assessments of therapy response .

[00108] Since only high specific radioactivity ^iS F-TFB was administered in this study we did not explore the effects of specific radioactivity over a broader range. However, in our previous preclinical study with NlS-transfected C6 glioma xenografts in mice we showed mat as specific radioactivity was decreased such that TFB administration levels exceeded -0,5 mg/kg, there was decreases seen in both thyroid and tumor uptake, Since thyroid uptake levels in the healthy participants in this study were very high, it is inferred that the specific radioactivity levels were sufficient to avoid any saturation effects do to administered TF mass.

[00109] The regional distribution of ¹⁸ F-TFB in healthy participants: was found to be consistent with known hNI expression levels throughout the body tissues. The slow accumulation of ^I8 F-radioactivity seen in SUV values between the 2 and 3,5 h imaging time points could be evidence of a minor degree of radiotracer defluorination, but since routin ¹⁸ F- TFB PE images will likely be acquired in the 1 -2 h post-injection period, the impact of this accumulation is of minor significance, indeed, bone uptake was not qualitatively remarkable in either the 2 or 3.5 fa images. Overall, the biodistribution data confirm ¹⁸ F-TFB to be an excellent iodide analog radiotracer with excellent in vivo stability.

[00110] The estimated radiatio doses were higher in thyroid, urinary bladder wall, lower large Intestine wall, small intestine wall, upper large intestine wall, heart wall, kidneys, liver, pancreas, and spleen, but on par with other ¹⁸ F-labeled radiopharmaceuticals and appropriate for clinical use. Further decrease in adde wall doses can be realized with good hydration and more frequent voiding of the bladder. Estimated effective doses were 0.036 mSv/MBq in males and

0.06 mSv/MBq in females, Our data on the biodistribution a d dosimetry estimates for ¹⁸ F* TFB are in general agreement with the results very recently published O'Boherty et al. (11) in five patients with thyroid cancer. In that study, 2 male and 3 female subjects were studied and the results from both genders were pooled. The specific radioactivity of their ¹ F-TFB preparations (24±13 MBq/jAg) was similar to the specific radioactivity obtained in this study. Conclusion

[001 1 1] The pharmacokinetics and biodistribution of high-specific activity ^{! 8} F-TFB in healthy human participants support its use as an iodide analog radiotracer for evaluation o thyroid and breast cancers and monitoring of gene therapies that employ the tiNlS reporter gene. The radiation dosimetry estimates are on par with other ^{i 8} F-Iabeled radiopharmaceuticals with prominent renal excretion (e.g. ¹⁸ F-FDG) and are acceptable for clinical imaging purposes.

[001 12] References from Example 2

1. Chung JK. Sodium iodide symporter: Its role in nuclear medicine. J Nucl Med. 20Q2;43;1188- 1200.

2. Penheiter AR, Russell SI, and Carison SK. The sodium iodide symporter (NIS) as an imaging reporter for gene, viral, and cell-based therapies. Cmr Ge e Ther. 2 12; 12:33-47.

3. Arm B-C. Sodium Iodide Symporter for Nuclear Molecular Imaging and Gene Therapy: From Bedside to Bench and Back, Themmstics, 2012;2:392-402.

4. Dai G, Levy O, and Carrasco N, Cloning and characterization of the thyroid iodide transporter. Nature. 1996;379:458-460. 5. Eskandari S, Loo DBF, Dai G, Levy O, Wright EM, and Carrasco N. Thyroid Na+Z Sjroporter: Mechanism, Stoichiometry, and Specificity. J Biol Chem. 1 97;272:27230-27238,

6. Miler A and Russell S J. The use o f the NIS reporter gene for optimizing oncolytic; virotherapy. Expert Opin on Biol Th&r. 2016;16: 5-32.

7. Jauregui-Osoro M, Sunassee Κ,. Weeks AJ, Berry DJ _> Paul RL,. Cleij M, et al Synthesis and biological evaluation of F-18 tetrafluoroborate: a PET imaging agent for thyroid disease and reporter gene imaging of the sodium/iodide symporter. Eur J N t Me Mgl Imaging.. 2010;37:2108-21 16.

8. Weeks A.!, Jauregui-Osoro M, Ckij M, Blower JE, Ballinger JR, and Blower PJ. Evaluation of F-18 etxaf oroborate as a potential PET imaging agent for the human sodium/iodide symporter in a new colon carcinoma cell line. HCT116, expressing hNlS, Nuel Med Comm. 201 1 32:98- 105,

9. Marti-Climent JM, Collantes M, Jauregui-Osoro M, Quineoces G, Prieto E, Bilbao I, et al. Radiation dosimetry and biodistribution in non-human primates of the sodium/iodide PE ligand F-18-ietrafmoroborate. EJNMMI Research 20I 5;5:70.

10. Yo n Ff, Jeong JM _S and Chung J-K, A new PET probe, F-18 _' tetrafluoroborate. for the sodium/iodide symporter: possible impacts on nuclear medicine. Eur J Nucl Med Mot imaging. 2010;37:2105-2107.

1 1. 0' Doherty I, Jauregui-Osoro M ₅ Brothwood T, Szyszko T, Marsden P, O' Doherty M,€¾ok G, Blower P, Lewingto V. ^l8 F-tetrafluorQborate ( ^l8 F-TFBX a PET probe for imaging sodium- iodide symporter expression: Whole-body biodistribution, safety and radiation dosimetry in thyroid cancer patients. J Nucl Med. 2017 pii: jnumed. l 17.192252. doi: 10.2967 jnumed.117.1 2252. [Epub ahead of printj

12. Jiang HL, Bansal A, Pandey MK, Peng KW, Suksanpaisan L, Russell SJ, et al. Synthesis of F 8-Tetrafiuoroborate via Radiofiuorination of Boron Trifluoride and Evaluation in a Murine C6-GMo a Tumor Model. J Nmi Med 2016;57:1454-1459.

1 . Khoshnevis n A, Jauregui-Osoro M, Shaw K, Torres JB _? Young JD, Ramakrishnan NK, et al. F-18 tetrafluoroborate as a PET tracer for the sodium/iodide symporter: the importance of specific activity. EJNMMI Research. 2Q16;6:34. [QQl 13] It will be appreciated b those skilled in the art that while the invention has been described above in connection with particular embodirnents and examples, the inventio is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures feom the embodiments, examples and uses are intended to be encompassed b the claims attached hereto.