A suitable time point for quantifying the radiochemical purity of 225Ac-labeled radiopharmaceuticals.

BACKGROUND: As 225Ac-labeled radiopharmaceuticals continue to show promise as targeted alpha therapeutics, there is a growing need to standardize quality control (QC) testing procedures. The determination of radiochemical purity (RCP) is an essential QC test. A significant obstacle to RCP testing is the disruption of the secular equilibrium between actinium-225 and its daughter radionuclides during labeling and QC testing. In order to accelerate translation of actinium-225 targeted alpha therapy, we aimed to determine the earliest time point at which the RCP of an 225Ac-labeled radiopharmaceutical can be accurately quantified. RESULTS: Six ligands were conjugated to macrocyclic metal chelators and labeled with actinium-225 under conditions designed to generate diverse incorporation yields. RCP was determined by radio thin layer chromatography (radioTLC) followed by exposure of the TLC plate on a phosphor screen either 0.5, 2, 3.5, 5, 6.5, or 26 h after the plate was developed. The dataset was used to create models for predicting the true RCP for any pre-equilibrium measurement taken at an early time point. The 585 TLC measurements span RCP values of 1.8-99.5%. The statistical model created from these data predicted an independent data set with high accuracy. Predictions made at 0.5 h are more uncertain than predictions made at later time points. This is primarily due to the decay of bismuth-213. A measurement of RCP > 90% at 2 h predicts a true RCP > 97% and guarantees that RCP will exceed 90% after secular equilibrium is reached. These findings were independently validated using NaI(Tl) scintillation counting and high resolution gamma spectroscopy on a smaller set of samples with 10% ≤ RCP ≤ 100%. CONCLUSIONS: RCP of 225Ac-labeled radiopharmaceuticals can be quantified with acceptable accuracy at least 2 h after radioTLC using various methods of quantifying particle emissions. This time point best balances the need to accurately quantify RCP with the need to safely release the batch as quickly as possible.

Production and Supply of α-Particle-Emitting Radionuclides for Targeted α-Therapy.

Encouraging results from targeted α-therapy have received significant attention from academia and industry. However, the limited availability of suitable radionuclides has hampered widespread translation and application. In the present review, we discuss the most promising candidates for clinical application and the state of the art of their production and supply. In this review, along with 2 forthcoming reviews on chelation and clinical application of α-emitting radionuclides, The Journal of Nuclear Medicine will provide a comprehensive assessment of the field.

Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

Construction of a thorium/actinium generator at the Canadian Nuclear Laboratories.

Targeted Alpha Therapy (TAT) has demonstrated considerable promise in the treatment of a range of cancers in both preclinical and, more recently clinical research. In particular, work with the alpha-emitting radionuclide 225Ac has been effectively employed due to the relatively rapid decay cascade that leads to 4 alpha and 2 beta emissions. One limitation for TAT has been caused by access to the vital radionuclide. Traditionally, 225Ac has been sourced from thorium/actinium generators based on the alpha decay of stockpiles of 229Th. 229Th is itself the alpha-decay product from 233U. Due to proliferation issues associated with 233U, only three thorium/actinium generators have been reported in the literature, capable of supporting clinical research. This paper describes the construction and operation of a thorium/actinium radionuclide generator at the Canadian Nuclear Laboratories, capable of supporting preclinical and limited clinical research in the area of TAT. Thorium was recovered and purified by a combination of anion exchange and extraction chromatography from aged 233U stockpiles. A separation scheme for 225Ra and 225Ac has been developed, based upon the chemical composition of the thorium material to allow for regular, routine milkings capable of supplying up to 3.7 GBq (100 mCi) of radiochemically pure 225Ac annually. This routine separation is accomplished using a combination of anion exchange chromatography to separate Ac and Ra isotopes from Th and extraction chromatography employing TEVA and DGA-N resins to separate actinium from radium and breakthrough thorium.

232Th-Spallation-Produced 225Ac with Reduced 227Ac Content.

Recent clinical results have demonstrated remarkable treatment responses of late-stage cancer patients when treated with alpha-emitting radionuclides such as actinium-225 (225Ac). The resulting intense global effort to produce greater quantities of 225Ac has triggered a number of emerging technologies to produce this rare, yet important, radionuclide. Accelerator-based methods for increasing global 225Ac production capacity have focused on the high energy (>100 MeV) proton irradiation of thorium, despite the coproduction of the undesirable 227Ac byproduct at 0.1-0.3% of the 225Ac activity. We at TRIUMF have developed a process for the production of a 225Ra/225Ac generator from irradiated thorium that results in an 225Ac product with reduced 227Ac content. 225Ac was separated from irradiated thorium and coproduced radioactive spallation and fission products using a thorium peroxide precipitation method followed by cation exchange and extraction chromatography. Stable and radioactive tracer studies demonstrated the ability of this method to separate Ac from most other elements, providing a directly produced Ac product with measured 227Ac content of (0.15 ± 0.04)%. A second, indirectly produced Ac product with 227Ac content of <7.5 × 10-5% is obtained by repeating the final extraction chromatography step with the 225Ra-containing fraction. The 225Ra-derived 225Ac showed similar or improved quality compared to the initial, directly produced 225Ac product in terms of chemical purity and radiolabeling capability, the latter of which was comparable with other 225Ac sources reported in the literature.

Synthesis and Evaluation of a Macrocyclic Actinium-225 Chelator, Quality Control and In Vivo Evaluation of 225 Ac-crown-αMSH Peptide.

Targeted alpha-therapy (TAT) has great potential for treating a broad range of late-stage cancers by delivering a focused and lethal radiation dose to tumors. Actinium-225 (225 Ac) is an emerging alpha emitter suitable for TAT; however, the availability of chelators for Ac remains limited to a small number of examples (DOTA and macropa). Herein, we report a new Ac macrocyclic chelator named 'crown', which binds quantitatively and rapidly (<10 min) to Ac at ambient temperature. We synthesized 225 Ac-crown-αMSH, a peptide targeting the melanocortin 1 receptor (MC1R), specifically expressed in primary and metastatic melanoma. Biodistribution of 225 Ac-crown-αMSH showed favorable tumor-to-background ratios at 2 h post injection in a preclinical model. In addition, we demonstrated dramatically different biodistrubution patterns of 225 Ac-crown-αMSH when subjected to different latency times before injection. A combined quality control methodology involving HPLC, gamma spectroscopy and radioTLC is recommended.

Comparison of various radioactive payloads for a human monoclonal antibody to glycoprotein 41 for elimination of HIV-infected cells.

BACKGROUND: cART has significantly improved the life expectancy of people living with HIV (PLWH). However, it fails to eliminate the long-lived reservoir of latent HIV-infected cells. Radioimmunotherapy (RIT) relies on antigen-specific monoclonal antibodies (mAbs) for targeted delivery of lethal doses of ionizing radiation to cells. Previously, we have demonstrated that human mAb 2556 against HIV gp41 conjugated with 213Bismuth radioisotope (t1/2 = 46 min, alpha-emitter) selectively killed HIV-infected cells. 225Actinium (t1/2 = 9.92 d, alpha-emitter) and 177Lutetium (t1/2 = 6.7 d, beta-emitter) are two long-lived clinically proven radioisotopes for cancer treatment which might be more effective in killing infected cells systemically and in CNS. METHODS: In this study we have conjugated 2556 mAb with 213Bi, 225Ac and 177Lu, and compared their ability to kill HIV-infected human peripheral blood mononuclear cells (PBMCs) and monocytes. PBMCs and monocytes from healthy donors were infected with HIVp49.5 and treated in vitro with increasing concentrations of 213Bi (4-20 µCi)-, 225Ac (20-100 nCi)- and 177Lu (4-50 µCi)-2556 mAb. RESULTS: After three days post-treatment of infected PBMCs and monocytes, 213Bi- and 177Lu-conjugated 2556 mAb reduced virus production measured by p24 level in a dose-dependent manner, whereas, 225Ac-2556 showed minimal effect. However, seven days post-treatment all three radioisotopes showed significantly more pronounced reduction of virus replication as compared to control labeled mAb with 225Ac-2556 showing the least non-specific killing. CONCLUSION: These results indicate that RIT holds promise as a novel treatment option for the eradication of HIV-infected cells that merits further study in combination with cART and reactivation drugs.

Low-dose radiobiology program at Canadian nuclear laboratories: past, present, and future.

Health risks associated with the exposure of humans to low-dose ionizing radiation are currently estimated using the Linear-No-Threshold model. Over the last few decades, however, this model has been widely criticized for inconsistency with a large body of experimental evidence. Substantial efforts have been made to delineate biological mechanisms and health-related outcomes of low-dose radiation. These include a large DOE-funded Low Dose program operated in the 2000s, as well as the EU funded programs, previously NOTE and DOREMI and currently MELODI. Although not as widely known, the Atomic Energy of Canada Limited (AECL) in Chalk River, operated a low-dose radiobiology program since as early as 1948. The Canadian Nuclear Laboratories (CNL), the successor to AECL since 2015, has expanded this program into new areas making it the world's most robust, centrally coordinated and long-lived research efforts to delineate the biological effects of low-dose radiation. The purpose of this review is to provide a high-level overview of the low-dose radiobiology program maintained at CNL while capturing the historical perspectives. Past studies carried out at CNL have substantially influenced the area of low-dose radiobiology, exemplified by highly cited papers showing delays in spontaneous tumorigenesis in low-dose irradiated mice. The current low-dose research program at CNL is not only addressing a wide range of mechanistic questions about the biological effects of low doses - from genetic to epigenetic to immunological questions - but also moving toward novel areas, such as the dosimetry and health consequences of space radiation and the use of low-dose radiation in cancer therapy and regenerative medicine.

Self-radiolysis of tritiated water. 4. The scavenging effect of azide ions (N3 -) on the molecular hydrogen yield in the radiolysis of water by 60Co γ-rays and tritium ß-particles at room temperature.

The effect of the azide ion N3 - on the yield of molecular hydrogen in water irradiated with 60Co γ-rays (∼1 MeV Compton electrons) and tritium ß-electrons (mean electron energy of ∼7.8 keV) at 25 °C is investigated using Monte Carlo track chemistry simulations in conjunction with available experimental data. N3 - is shown to interfere with the formation of H2 through its high reactivity towards hydrogen atoms and, but to a lesser extent, hydrated electrons, the two major radiolytic precursors of the H2 yield in the diffusing radiation tracks. Chemical changes are observed in the H2 scavengeability depending on the particular type of radiation considered. These changes can readily be explained on the basis of differences in the initial spatial distribution of primary radiolytic species (i.e., the structure of the electron tracks). In the "short-track" geometry of the higher "linear energy transfer" (LET) tritium ß-electrons (mean LET ∼5.9 eV nm-1), radicals are formed locally in much higher initial concentration than in the isolated "spurs" of the energetic Compton electrons (LET ∼0.3 eV nm-1) generated by the cobalt-60 γ-rays. As a result, the short-track geometry favors radical-radical reactions involving hydrated electrons and hydrogen atoms, leading to a clear increase in the yield of H2 for tritium ß-electrons compared to 60Co γ-rays. These changes in the scavengeability of H2 in passing from tritium ß-radiolysis to γ-radiolysis are in good agreement with experimental data, lending strong support to the picture of tritium ß-radiolysis mainly driven by the chemical action of short tracks of high local LET. At high N3 - concentrations (>1 M), our H2 yield results for 60Co γ-radiolysis are also consistent with previous Monte Carlo simulations that suggested the necessity of including the capture of the precursors to the hydrated electrons (i.e., the short-lived "dry" electrons prior to hydration) by N3 -. These processes tend to reduce significantly the yields of H2, as is observed experimentally. However, this dry electron scavenging at high azide concentrations is not seen in the higher-LET 3H ß-radiolysis, leading us to conclude that the increased amount of intra-track chemistry intervening at early time under these conditions favors the recombination of these electrons with their parent water cations at the expense of their scavenging by N3 -.

The synthesis, magnetic purification and evaluation of 99mTc-labeled microbubbles.

INTRODUCTION: Ultrasound (US) contrast agents based on microbubbles (MBs) are being investigated as platforms for drug and gene delivery. A methodology for determining the distribution and fate of modified MBs quantitatively in vivo can be achieved by tagging MBs directly with (99m)Tc. This creates the opportunity to employ dual-modality imaging using both US and small animal SPECT along with quantitative ex vivo tissue counting to evaluate novel MB constructs. METHODS: A (99m)Tc-labeled biotin derivative ((99m)TcL1) was prepared and incubated with streptavidin-coated MBs. The (99m)Tc-labeled bubbles were isolated using a streptavidin-coated magnetic-bead purification strategy that did not disrupt the MBs. A small animal scintigraphic/CT imaging study as well as a quantitative biodistribution study was completed using (99m)TcL1 and (99m)Tc-labeled bubbles in healthy C57Bl-6 mice. RESULTS: The imaging and biodistribution data showed rapid accumulation and retention of (99m)Tc-MBs in the liver (68.2±6.6 %ID/g at 4 min; 93.3±3.2 %ID/g at 60 min) and spleen (214.2±19.7 %ID/g at 4 min; 213.4±19.7 %ID/g at 60 min). In contrast, (99m)TcL1 accumulated in multiple organs including the small intestine (22.5±3.6 %ID/g at 4 min; 83.4±5.9 %ID/g at 60 min) and bladder (184.0±88.1 %ID/g at 4 min; 24.2±17.7 %ID/g at 60 min). CONCLUSION: A convenient means to radiolabel and purify MBs was developed and the distribution of the labeled products determined. The result is a platform which can be used to assess the pharmacokinetics and fate of novel MB constructs both regionally using US and throughout the entire subject in a quantitative manner by employing small animal SPECT and tissue counting.

A robust strategy for the preparation of libraries of metallopeptides. A new paradigm for the discovery of targeted molecular imaging and therapy agents.

A robust method for synthesizing structurally diverse metallopeptide libraries using a Re(i) complex of a non-natural amino acid was developed as a way to accelerate the discovery of novel molecular imaging probes.

Expedient multi-step synthesis of organometallic complexes of Tc and Re in high effective specific activity. A new platform for the production of molecular imaging and therapy agents.

For over thirty years, instant labeling kits which involve no purification steps have been the only method used to prepare (99m)Tc radiopharmaceuticals for clinical studies. To address the limitations imposed by instant kits, which is hindering the development of molecularly targeted Tc- and Re-based imaging and therapy agents, a new strategy for the rapid multistep synthesis and purification of organometallic technetium-based molecular probes and corresponding rhenium-based therapeutic analogues was developed. Beginning with MO4(-) (M = (99m)Tc, (186/188)Re), the carbonyl precursor [M(CO)3(H2O)3](+) was synthesized in 3 min in quantitative yield in a microwave reactor. A dipicolyl ligand was added and the chelate complex was formed in high yield in 2 min using microwave heating at 150 degrees C. This was followed by a new purification strategy to remove unlabeled ligand which entailed using a copper resin/C18 solid phase extraction protocol giving the desired product in greater than 78% decay corrected yield (dcy). Conversion to the corresponding succinimidyl active ester was achieved following a 5 min microwave irradiation at 120 degrees C and C18 solid phase extraction purification in 60% dcy. A series of amides were prepared subsequently by microwave heating at 120 degrees C for 5 min producing the desired targets in greater than 86% dcy. The reported method represents a move away from traditional instant kits toward more versatile platform synthesis and purification technologies that are better suited for producing modern molecular imaging and therapy agents.

Synthesis and screening of mono- and di-aryl technetium and rhenium metallocarboranes. A new class of probes for the estrogen receptor.

A series of mono and diaryl rhenium(I)-carborane derivatives were prepared using microwave heating and screened for their affinity for two isoforms of the estrogen receptor (ER). The rhenacarborane derivative [(RR'C 2B9H9)Re(CO)3](-) (R = p-PhOH, R' = H), which was generated by taking advantage of a recently discovered cage isomerization process, and the neutral nitrosated analogue [(RR'C2B9H9)Re(CO)2(NO)] (R = p-PhOH, R' = H) showed the highest affinities of the compounds screened. As a result, the (99m)Tc analogue of one of the leads was produced in high yield (84%) and specific activity in a manner that is suitable for routine production in support of future preclinical and molecular imaging studies.

Microwave-assisted synthesis of 3,1,2- and 2,1,8-Re(I) and 99mTc(I)-metallocarborane complexes.

Microwave heating was used to prepare eta5-rhenium carborane complexes in aqueous reaction media. For carboranes bearing sterically demanding substituents, isomerization of the cage from 3,1,2 to 2,1,8 derivatives occurred concomitantly with complexation. Microwave heating was equally effective at the tracer level using technetium-99m, affording access to a new class of synthons for designing novel molecular imaging agents.