Kit-like 18F-labeling of RGD-19F-Arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging
Introduction
Positron Emission Tomography (PET) Imaging is a powerful technique for visualizing and quantifying the dynamic distribution of target-specific ligands, and is particularly useful for imaging solid cancers in regards to specific cellular targets [1], [2], [3], [4], [5], [6]. Peptides as potential imaging agents are relatively large water-soluble ligands that exhibit high target selectivity yet clear rapidly so as to ensure high tumor-to-blood and tumor-to-muscle (T:NT) ratios. Once a peptide sequence has been identified and optimized for target binding and specificity, usually a site (e.g. N-terminus, C-terminus, pendant lysine) can also be identified for appending a suitable linker arm without reducing affinity or target specificity. This linker arm is then conjugated to a suitable radio-prosthetic that, with a robust labeling strategy, provides high specific activities. Of several available β+-isotopes, 18F-fluorine is often the isotope of choice owing to its excellent nuclear properties and on-demand production at Curie levels in hospital cyclotrons [7].
Although anionic 18F-fluoride is routinely produced at high specific activity, its lack of reactivity in water [8], along with a relatively short half-life (109.8 min), makes single-step 18F-labeling of peptides challenging. Generally, 18F-labeling of peptides usually proceeds via the radiosynthesis of an 18F-labeled prosthetic that is conjugated to the peptide in a second step, typically via acylation, oxime ligation, thiol-alkylation [9], or various bio-orthogonal click chemistries, which have been touted in terms of a general ease of labeling [10], [11], [12], [13], [14]. Nevertheless, most two-step methods still suffer from relatively long synthesis times (100–180 min) [15], [16], [17] that further erode specific activities to ~ 1 Ci/μmol [9], [10], [11], [15], [16], [18]. In contrast, methods for direct labeling on carbon [19], silicon [14], [20], [21], [22], [23], [24], [25], [26], [27], [28], boron [29], [30], [31], and aluminum chelates [32], [33], [34], [35], [36], [37], which have been recently reviewed [25], [27], [38], [39], are now receiving increasing attention owing to the inherent radiosynthetic simplicity of a single step. Such reactivity would enable the use of radiolabeling kits containing aliquots of precursors for on-demand labeling that ideally would obviate the need for very dry 18F-fluoride while requiring little in the way of radiosynthetic skill.
Irrespective of isotope, labeling procedure, or prosthetic composition, the specific activity of a radiotracer, defined as Ci/μmol radiotracer, represents an impartial measure of radiotracer quality with important repercussions for imaging low-abundance targets as well as meeting regulations for microdosing (vide infra) [40], [41]. While the specific activity of carrier-free 18F-fluorine is 1720 Ci/μmol, the specific activity of no-carrier added (NCA) 18F-fluoride ion obtained directly following bombardment falls in the range of 15–30 Ci/μmol [40], [42], [43]. Anion exchange trapping, which is often used to concentrate the fluoride, and which may be required to remove contaminating radioactive metals that cannot be injected into patients, further erodes the specific activity to approximately < 10 Ci/μmol. Hence, most small molecule tracers as well as most radio-prosthetics are labeled at specific activities of 8 Ci/μmol, or less [13], [44], [45], [46], [47], [48]. For 18F-labeling of peptides, which often requires two steps, there are few examples where specific activities exceed 2 Ci/μmol [19], [47], [49], [50], [51] and to the best of our knowledge there is no example of routine peptide labeling at > 10 Ci/μmol. Corroboration of this assertion is found in a literature survey, where to date, a value of 1 Ci/μmol is often described as “high” [9], [48], [52] in contrast to more standard values of 0.5 Ci/μmol [53], [54], [55], [56] and even much lower values of < 0.25 Ci/μmol, which nevertheless have been sufficient for the publication of animal and human PET images [57], [58], [59].
Over the past few years, we have sought to exploit the well-known fluorophilicity of boron to capture aqueous 18F-fluoride in one step to provide an in vivo stable 18F-ArBF3− conjugate. This method was used to directly label biotin [30], Lymphoseek [60], Marimastat [31], and RGD [61], while a one-pot-two-step copper click labeling was used to label and image bombesin [62]. In these examples, specific activities were calculated to be 0.16–0.5 Ci/μmol yet were never measured. Both the relatively low specific activities that were calculated along with the lack of concrete measurement thereof raised concerns over the utility, if not the validity, of this approach. In light of these concerns, we improved this method to demonstrate extraordinarily high specific activities (~ 15 Ci/μmol) in good-to-excellent radiochemical yields at record synthetic times of 15 min [63], [64]. In order to directly measure such values, we converted a boronate ester/borimidine to the corresponding 18F-ArBF3− using ~ 10 mCi NCA 18F-fluoride (specific activity of which was measured independently at 5 Ci/μmol) and then click-conjugated it to a fluorophore, which provided an unambiguous and direct measurement of 3-fold higher specific activity [64]. Notably, calculated values approximated measured values, within experimental error. In order to label with production levels of fluoride e.g. 400–1000 mCi, we featured 19F-18F isotope exchange (IEX) [63], which was first elegantly disclosed by Schirrmacher et al. [24], for use with silylfluorides and later by Li et al. [65], who provided an 18F-ArBF3−, albeit at very low specific activities in the absence of any peptide labeling or tumor targeting. Likewise, the fluorophore provided unambiguous proof that the specific activities were as high as 15 Ci/μmol when IEX was performed.
To test whether this IEX method could be extended to peptides of clinical relevance, here we focus our efforts on RGD, which was chosen on two accounts: i) its clinical relevance [66] to human and animal images of the αvβ3 integrin receptor, a well-defined prognostic indicator for several different types of cancers [10], [19], [67], [68], [69]; and ii) the identical RGD-18F-ArBF3− bioconjugate (Fig. 1, below) had been previously imaged at low specific activity (0.06–0.16 Ci/μmol) following both one-step and one-pot-two-step click labeling to provide apparent tumor uptake values of ~ 2% ID in the raw image [61]. Here, using IEX on the same RGD-tracer, we demonstrate radiochemical yields in excess of 50% at specific activities that are 100 fold higher than we previously reported and 14 fold higher than values normally described as “high”. This method affords easy operation in fully shielded hot cells with up to 1 Ci 18F-activity, which should be of immediate interest for use in production labs. Moreover this work demonstrates that IEX labeling can easily be extended to peptides of clinical relevance while preliminary in vivo data with blocking controls show statistically significant specific tumor uptake with good tumor:blood ratios. The potential advantages of routine labeling at 14 Ci/μmol are discussed.
Section snippets
General information
Amino acids and resin for the solid-phase synthesis of RGD were obtained from Novabiochem, KHF2 was obtained from Acros, Tetraphenylpinacol, piperazine, and succinic anhydride were obtained from Alfa-Aesar, Butyl-lithium, 4 M HCl in dioxane, trimethoxyborane, and HFIP were obtained from Sigma-Aldrich. Trifluorobenzene was obtained from Oakwood Products Inc. 18F Trap & Release Columns were purchased from ORTG Inc. (Oakdale, TN) and C18 Sep-Pak cartridge (Vac 1 cc, 50 mg) was obtained from Waters.
HPLC methods
Unless otherwise stated, all samples were resolved on a Phenomenex Jupiter 10 μ C18 300 Å 4.6 × 250 mm analytical column. Gradients for purification are listed below: Gradient A: Solvent A: 0.1% TFA water; solvent B: 0.05% TFA MeCN; 0 to 6 min: 10% to 10% B, 6 to 10 min, 10% to 15% B, 10 to 13 min: 15% to 100% B, 13 to 15 min: 100% to 10% B, 15 to 16 min: 10% to 10% B. Flow rate: 1 mL/min, column temperature: 19 to 21 °C; Gradient B: Solvent A: 0.04 M ammonium formate pH 6.8; solvent B: MeCN; 0 to 5 min: 0%
Results
For the radiolabeling featured in Fig. 2, 927 mCi NCA 18F-fluoride in a polyester tube was concentrated at 110 °C for 8–10 min. Decay after 10 min left > 850 mCi. A single radiosynthesis kit containing 50 nmol of lyophilized RGD-19F-ArBF3− was resuspended in buffered DMF-water and combined with the NCA 18F-fluoride. After 15 min the contents of the vial were resuspended in 2 mL of quench solution at pH 7.5. As with all high level radiosyntheses, only a small portion (~ 50 μL, containing approximately 15–20
Discussion
Here we used 800–1000 mCi NCA 18F-Fluoride ion to label ~ 50 μg quantities of lyophilized precursor with 50% radiochemical yields within 15 min in one-step. HPLC purification, which required another 15 min, yielded a radiochemically pure tracer at specific activities of up to 14 Ci/μmol, a value that is up to 10 fold higher than most other radiosyntheses. While IEX provides a rapid and robust means of labeling, the mechanism of IEX is unclear. Indeed, the appearance of free arylboronic acid suggests
Conclusion
Here we have shown that isotope exchange on microgram quantities of RGD-19F-ArBF3− precursor in the presence of production level quantities of 18F-activity provides excellent radiochemical yields in near record reaction times of 15 min. HPLC purification provided radiochemically pure tracer at specific activities of 14 Ci/μmol within 15 min (total time ~ 30–35 min). These values are consistent with previous values obtained with a fluorescent-18F-ArBF3− and demonstrate the reliability and generality
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Current address: Department of Chemistry, School of Environmental Science & Engineering, Nanjing University of Information Science & Technology, Nanjing 210044, P.R. China.