New lyophilized kit for rapid radiofluorination of peptides.

Radiolabeling compounds with positron-emitting radionuclides often involves a time-consuming, customized process. Herein, we report a simple lyophilized kit formulation for labeling peptides with (18)F, based on the aluminum-fluoride procedure. The prototype kit contains IMP485, a NODA (1,4,7-triazacyclononane-1,4-diacetate)-MPAA (methyl phenylacetic acid)-di-HSG (histamine-succinyl-glycine) hapten-peptide, [NODA-MPAA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH(2)], used for pretargeting, but we also examined a similar kit formulation for a somatostatin-binding peptide [IMP466, NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl] bearing a NOTA ligand to determine if the benefits of using a kit can be extended to other AlF-binding peptides. The NODA-MPAA ligand forms a single stable complex with (AlF)(2+) in high yields. In order to establish suitable conditions for a facile kit, the formulation was optimized for pH, peptide to Al(3+) ratio, bulking agent, radioprotectant, and the buffer. For optimal labeling, the kit was reconstituted with an aqueous solution of (18)F(-) and ethanol (1:1), heated at 100-110 °C for 15 min, and then simply and rapidly purified using one of two equally effective solid-phase extraction (SPE) methods. Al(18)F-IMP485 was isolated as a single isomer complex, in high yield (45-97%) and high specific activity (up to 223 GBq/µmol), within 20 min. The labeled product was stable in human serum at 37 °C for 4 h and in vivo, urine samples showed the intact product was eliminated. Tumor targeting of the Al(18)F-IMP485 in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody showed very low uptake (0.06% ± 0.02 ID/g) in bone, further illustrating its stability. At 1 h, pretargeted animals had high Al(18)F-IMP485 tumor uptake (28.1% ± 4.5 ID/g), with ratios of 9 ± 4, 123 ± 38, 110 ± 43, and 120 ± 108 for kidney, liver, blood and bone, respectively. Tumor uptake remained high at 3 h postinjection, with increased tumor/nontumor ratios. The NOTA-somatostatin-binding peptide also was fluorinated with good yield and high specific activity in the same kit formulation. However, yields were somewhat lower than those achieved with IMP485 containing the NODA-MPAA ligand, likely reflecting this ligand's superior binding properties over the simple NOTA. These studies indicate that (18)F-labeled peptides can be reproducibly prepared as stable Al-F complexes with good radiochemical yield and high specific activity using a simple, one-step, lyophilized kit followed by a rapid purification by SPE that provides the (18)F-peptide ready for patient injection within 30 min.

Optimized labeling of NOTA-conjugated octreotide with F-18.

We recently reported a facile method based on the chelation of [(18)F]aluminum fluoride (Al(18)F) by NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Here, we present a further optimization of the (18)F labeling of NOTA-octreotide (IMP466). Octreotide was conjugated with the NOTA chelate and was labeled with (18)F in a two-step, one-pot method. The labeling procedure was optimized with regard to the labeling buffer, ionic strength, peptide concentration, and temperature. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice. In addition, microPET/CT images were acquired. IMP466 was labeled with Al(18)F in a single step with 97% yield in the presence of 80% (v/v) acetonitrile or ethanol. The labeled product was purified by HPLC to remove unlabeled peptide and unbound Al(18)F. The radiolabeling, including purification, was performed for 45 min. Specific activities of 48,000 GBq/mmol could be obtained. (18)F-IMP466 showed a high tumor uptake and excellent tumor-to-blood ratios at 2 h post-injection. In addition, the low bone uptake indicated that the Al(18)F-NOTA complex was stable in vivo. PET/CT scans revealed excellent tumor delineation and specific accumulation in the tumor. Uptake in receptor-negative organs was low. NOTA-octreotide could be labeled with (18)F in quantitative yields using a rapid two-step, one-pot, method. The compound was stable in vivo and showed rapid accretion in SSTR(2)-receptor-expressing AR42J tumors in nude mice. This method can be used to label other NOTA-conjugated compounds such as RGD peptides, GRPR-binding peptides, and Affibody molecules with (18)F.

High-yielding aqueous 18F-labeling of peptides via Al18F chelation.

The coordination chemistry of a new pentadentate bifunctional chelator (BFC), NODA-MPAA 1, containing the 1,4,7-triazacyclononane-1,4-diacetate (NODA) motif with a methylphenylacetic acid (MPAA) backbone, and its ability to form stable Al(18)F chelates were investigated. The organofluoroaluminates were easily accessible from the reaction of 1 and AlF(3). X-ray diffraction studies revealed aluminum at the center of a slightly distorted octahedron, with fluorine occupying one of the axial positions. The tert-butyl protected prochelator 7, which can be synthesized in one step, is useful for coupling to biomolecules on solid phase or in solution. High yield (55-89%) aqueous (18)F-labeling was achieved in 10-15 min with a tumor-targeting peptide 4 covalently linked to 1. Defluorination was not observed for at least 4 h in human serum at 37 °C. These results demonstrate the facile application of Al(18)F chelation using BFC 1 as a versatile labeling method for radiofluorinating other heat-stable peptides for positron emission imaging.

Improved 18F labeling of peptides with a fluoride-aluminum-chelate complex.

We reported previously the feasibility to radiolabel peptides with fluorine-18 ((18)F) using a rapid one-pot method that first mixes (18)F(-) with Al(3+) and then binds the (Al(18)F)(2+) complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al(18)F NOTA complexes were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100 degrees C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide could be labeled up to 115 GBq/micromol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.

A novel method of 18F radiolabeling for PET.

UNLABELLED: Small biomolecules are typically radiolabeled with (18)F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein (18)F is first attached to aluminum as Al(18)F, which is then bound to a chelate attached to a peptide, forming a stable Al(18)F-chelate-peptide complex in an efficient 1-pot process. METHODS: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl(3).6H(2)O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of (18)F to form the Al(18)F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH(2)) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100 degrees C for 15 min. In vitro and in vivo stability and targeting ability of the Al(18)F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). RESULTS: The radiolabeled peptide was produced in 5%-20% yield with an estimated specific activity of 18,500-48,100 GBq (500-1,300 Ci)/mmol. The Al(18)F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al(18)F had higher tissue uptake, particularly in the bones, than the chelated Al(18)F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al(18)F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. CONCLUSION: The ability to bind highly stable Al(18)F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.

The radiolabeling of proteins by the [18F]AlF method.

A new ([(18)F]AlF)(2+)-binding ligand that contains 1,4,7-triazacyclononane-1,4-diacetate (NODA) attached to a methyl phenylacetic acid group (MPA) was conjugated to N-(2-aminoethyl)maleimide (EM) to form NODA-MPAEM. The NODA-MPAEM was labeled with ([(18)F]AlF)(2+) at 105°C in 49-82% yield and conjugated at room temperature to an antibody Fab' fragment in 69-80% yield (total time ∼50min) and with retention of immunoreactivity. These data indicate that the rapid and simple [(18)F]AlF-labeling method can be easily adapted for preparing heat-sensitive compounds with (18)F quickly and in high yields.

PET of tumors expressing gastrin-releasing peptide receptor with an 18F-labeled bombesin analog.

UNLABELLED: The gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer. Bombesin (BBN) is a neurotransmitter of 14 amino acids and binds with selectivity and with high affinity to GRPRs. We have synthesized a NOTA-conjugated bombesin derivative, NOTA-8-Aoc-BBN(7-14)NH(2), to label this analog with (18)F using the new Al(18)F method. In this study, the GRPR-targeting potential of (18)F-labeled NOTA-8-Aoc-BBN(7-14)NH(2) was studied using (68)Ga-NOTA-8-Aoc-BBN(7-14)NH(2) as a reference. METHODS: The NOTA-conjugated bombesin analog was synthesized and radiolabeled with (68)Ga or (18)F. For (18)F labeling, we used our new 1-pot, 1-step method. The labeled product was purified by reversed-phase high-performance liquid chromatography. The log P values of the radiotracers were determined. The tumor-targeting characteristics of the compounds were assessed in mice with subcutaneously growing PC-3 xenografts. GRPR-binding specificity was studied by coinjection of an excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2). Small-animal PET/CT images were acquired. RESULTS: NOTA-8-Aoc-BBN(7-14)NH(2) could be efficiently labeled with (18)F or with (68)Ga. NOTA-8-Aoc-BBN(7-14)NH(2) was labeled with (18)F in a single step, with 50%-90% yield. Radiolabeling, including purification, was performed in 45 min and resulted in a specific activity of greater than 10 GBq/µmol. The log P values of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) were -1.47 ± 0.05 and -1.98 ± 0.03, respectively. In mice, both radiolabeled compounds cleared rapidly from the blood (<0.07 percentage injected dose per gram at 1 h after injection), mainly via the kidneys. At 1 h after injection, the uptake of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) in the PC-3 tumors was 2.15 ± 0.55 and 1.24 ± 0.26 percentage injected dose per gram, respectively. GRPR-binding specificity was demonstrated by reduced tumor uptake of radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) after coinjection of a 100-fold excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2) peptide. The accumulation of (18)F-NOTA-8-Aoc-BBN(7-14)NH(2) in the subcutaneous PC-3 tumors could be visualized via small-animal PET. CONCLUSION: NOTA-8-Aoc-BBN(7-14)NH(2) could be labeled rapidly and efficiently with (18)F using a 1-pot, 1-step method. Radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) specifically accumulated in the GRPR-expressing PC-3 tumors and should be evaluated clinically.

Antibody conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) for targeted cancer chemotherapy.

CPT-11 is a clinically used cancer drug, and it is a prodrug of the potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin). To bypass the need for the in vivo conversion of CPT-11 and increase the therapeutic index, bifunctional derivatives of SN-38 were prepared for use in antibody-based targeted therapy of cancer. The general synthetic scheme incorporated an acetylene-azide click cycloaddition step in the design, a short polyethylene glycol spacer for aqueous solubility, and a maleimide group for conjugation. Conjugates of a humanized anti-CEACAM5 monoclonal antibody, hMN-14, prepared using these SN-38 derivatives were evaluated in vitro for stability in buffer and human serum and for antigen-binding and cytotoxicity in a human colon adenocarcinoma cell line. Conjugates of hMN-14 and SN-38 derivatives 16 and 17 were found promising for further development.

Transition-metal-promoted hydroboration of alkenes: a unique reversal of regioselectivity.

When hydroboration of 1-octene is carried out in the presence of catalytic amounts of rhodium trichloride followed by the usual oxidation (hydrogen peroxide in aqueous alkali), only minor proportions of 1-octanol (2.4%) are formed accompanied by very significant amounts of 2- (17.4%), 3- (36.9%), and 4-octanol (43.3%). These product compositions are obtained in good overall yield when the borane-THF complex is slowly added to a stirred solution of 1-octene in THF solvent containing the rhodium trichloride. Isomerization of 1-octene to 2-, 3-, and 4-octene in the presence of rhodium trichloride alone is far too slow to account for the foregoing results. The mechanism likely involves multiple and reversible addition/elimination of a Rh-activated B-H species across the double bonds.