Alejandro Amor-Coarasa

Comprehensive Quality Control of the ITG 68Ge/68Ga Generator and Synthesis of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC for Clinical Imaging

A good-manufacturing-practices (GMP) 68Ge/68Ga generator that uses modified dodecyl-3,4,5-trihydroxybenzoate hydrophobically bound to a octadecyl silica resin (C-18) as an adsorbent has been developed that allows for dilute HCl (0.05N) to efficiently elute metal-impurity-free 68Ga3+ ready for peptide labeling. We characterized the performance of this generator system over a year in conjunction with the production of 68Ga-labeled DOTATOC and Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (PSMA-HBED-CC) intended for clinical studies and established protocols for batch release. Methods: A 2,040-MBq self-shielded 68Ge/68Ga generator provided metal-free 68GaCl3 ready for peptide labeling in the fluidic labeling module after elution with 4 mL of 0.05N HCl. The compact system was readily housed in a laminar flow cabinet allowing an ISO class-5 environment. 68Ga labeling of peptides using GMP kits was performed in 15–20 min, and the total production time was 45–50 min. Batch release quality control specifications were established to meet investigational new drug submission and institutional review board approval standards. Results: Over a period of 12 mo, 68Ga elution yields from the generator averaged 80% (range, 72.0%–95.1%), and 68Ge breakthrough was less than 0.006%, initially decreasing with time to 0.001% (expressed as percentage of 68Ge activity present in the generator at the time of elution), a unique characteristic of this generator. The radiochemical purity of both 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC determined by high-performance liquid chromatography analysis was greater than 98%, with a minimum specific activity of 12.6 and 42 GBq/μmol, respectively. The radionuclidic (68Ge) impurity was 0.00001% or less (under the detection limit). Final sterile, pyrogen-free formulation was provided in physiologic saline with 5%–7% ethanol. Conclusion: The GMP-certified 68Ge/68Ga generator system was studied for a year. The generator system is contained within the fluidic labeling module, and it is compact, self-shielded, and easy to operate using simple manual techniques. The system provides radiolabeled peptides with high (>98%) radiochemical purity and greater than 80% radiochemical yield. The 68Ge levels in the final drug products were under the detection limits at all times. 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC investigational radiopharmaceuticals are currently being studied clinically under investigational new drug (IND) applications submitted to the U.S. Food and Drug Administration.

Dual-Target Binding Ligands with Modulated Pharmacokinetics for Endoradiotherapy of Prostate Cancer

Prostate-specific membrane antigen (PSMA)–targeted radiotherapy of prostate cancer (PCa) has emerged recently as a promising approach to the treatment of disseminated disease. A small number of ligands have been evaluated in patients, and although early tumor response is encouraging, relapse rate is high and these compounds localize to the parotid, salivary, and lacrimal glands as well as to the kidney, leading to dose-limiting toxicities and adverse events affecting quality of life. We envision that dual-target binding ligands displaying high affinity for PSMA and appropriate affinity for human serum albumin (HSA) may demonstrate a higher therapeutic index and be suitable for treatment of PCa by targeted α-therapy. Methods: Six novel urea-based ligands with varying affinities for PSMA and HSA were synthesized, labeled with 131I, and evaluated by in vitro binding and uptake assays in LNCaP cells. Four compounds were advanced for further evaluation in a preclinical model of PCa. The compounds were compared with MIP-1095, a PSMA ligand currently in clinical evaluation. Results: The compounds demonstrated affinity for PSMA on the order of 4–40 nM and affinity for HSA in the range of 1–53 μM. Compounds with relatively high affinity for HSA (≤2 μM) showed high and sustained blood-pool activity and reduced uptake in the kidneys. 131I-RPS-027, with a 50% inhibitory concentration (PSMA) of 15 nM and a dissociation constant (HSA) of 11.2 μM, cleared from the blood over the course of 48 h and showed good tumor uptake (10 percentage injected dose per gram) and retention and a greater than 5-fold decrease in kidney uptake relative to MIP-1095. The tumor-to-kidney ratio of 131I-RPS-027 was greater than 3:1 at 24 h after injection, increasing to 7:1 by 72 h. Conclusion: RPS-027 shows dual targeting to PSMA and albumin, resulting in a high tumor uptake, highly favorable tissue distribution, and promising therapeutic profile in a preclinical model of prostate cancer. In comparison to existing ligands proposed for targeted therapy of prostate cancer, RPS-027 has tumor-to-tissue ratios that predict a significant reduction in side effects during therapy. Using iodine/radioiodine as a surrogate for the radiohalogen 211At, we therefore propose dual-target binding ligands such as RPS-027 as next-generation radiopharmaceuticals for targeted α-therapy using 211At.

Longitudinal PET imaging demonstrates biphasic CAR T cell responses in survivors

Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.

Assessment of PSMA targeting ligands bearing novel chelates with application to theranostics: Stability and complexation kinetics of 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+

INTRODUCTION Recent successes in the treatment of metastatic castration-resistant prostate cancer (mCRPCa) by systemic endoradiotherapy has sparked renewed interest in developing small molecule ligands targeting prostate-specific membrane antigen (PSMA) and chelators capable of stable complexation of metal radionuclides for imaging and therapy. As the size and coordination number of metals for imaging, such as 68Ga3+, and for targeted therapy, such as 177Lu3+ and 225Ac3+, are substantially different, they may show a preference for macrocycles of different denticity. We have prepared three simple conjugates that target PSMA and form radiometal complexes through coordination by either octa-, deca-, or dodecadentate tetraazacyclododecane chelators. The complex formation and metal ion selectivity of these constructs were determined at two relevant temperatures, complex stability was examined in vitro, and tumor targeting was demonstrated in preclinical PCa models with a view towards identifying a candidate with potential value as a theranostic agent for the imaging and therapy of mCRPCa. METHODS Three bifunctional chelates with high denticity, including the octadentate chelate DOTA, the decadentate 3p-C-DEPA and a novel dodecadentate analogue of DEPA, were synthesized and conjugated to a glutamate-urea-lysine (EuK) pharmacophore (EuK-DOTA, EuK-107 and EuK-106, respectively) to enable targeting of PSMA. The metal ion selectivity for each construct was determined by incubation at 25 °C and 95 °C with the trivalent radiometals 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+. PSMA binding affinity was determined by competitive binding using LNCaP cells, while in vivo tumor targeting of the 68Ga-labeled constructs was examined by positron emission tomography (PET) in LNCaP xenograft tumor-bearing mice. RESULTS PMSA affinities (IC50 values) were 13.3 ± 0.9 nM for EuK-DOTA, 18.0 ± 3.7 nM for EuK-107 and 42.6 ± 6.6 nM for EuK-106. EuK-107 and EuK-DOTA proved to rapidly and near quantitatively complex 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+ at 95 °C, with EuK-107 also rapidly complexing 111In3+ and 177Lu3+ at 25 °C. The inability of EuK-106 to chelate 177Lu3+ and 225Ac3+ suggests that size of the cavity of the macrocylic ring may be more critical than the number of donor groups for the chelation of larger radiometals. In vivo, 68Ga-EuK-107 proved to have similar uptake to 68Ga-DKFZ-PSMA-617, a theranostic ligand currently in clinical evaluation, in a PSMA positive xenograft tumor model. CONCLUSIONS The broad metal ion selectivity, good in vitro affinity for PSMA and good in vivo tumor targeting suggest that EuK-107, with the 3p-C-DEPA chelator, merits further evaluation as a theranostics construct in prostate cancer.

[ 18 F ]RPS-544: A PET tracer for imaging the chemokine receptor CXCR4

INTRODUCTION CXCR4 specific [18F]-labeled positron emission tomography (PET) imaging agents are needed which would enable general distribution of the radiotracer for clinical investigation. We sought to synthesize, radiolabel and evaluate [18F]RPS-544, a novel non-peptide CXCR4 antagonist as a CXCR4 specific probe. We compared [18F]RPS-544 with the previously published [18F]-3 ([18F]RPS-510 in this paper) in a bi-lateral tumor model of differential CXCR4 expression for its ability to selectively target CXCR4 expression. METHODS Radiolabeling of [18F]RPS-544 and [18F]RPS-510 was performed by aromatic substitution on a 6-nitropyridyl group using no-carrier-added [18F]fluoride under basic conditions. 18F incorporation was determined by radioHPLC. Semi-preparative HPLC was used to purify the final product prior to reformulation. Imaging and biodistribution was performed in nude mice with bilateral PC3 (CXCR4+ and WT) xenograft tumors at 1, 2 and 4 h post injection. RESULTS RPS-544 bound CXCR4 with an IC50 of 4.9 ± 0.3 nM. [18F]RPS-544 showed preferential uptake in CXCR4+ tumors, with a CXCR4/WT ratio of 3.3 ± 1.3 at 1 h p.i. and 2.3 ± 0.5 at 2 h p.i. Maximum uptake in the CXCR4+ tumors was 3.4 ± 1.2%ID/g at 1 h p.i., significantly greater (p = 0.003) than the uptake in the WT tumor. Tumor/blood ratios were 2.5 ± 0.4 and 3.6 ± 0.3 at 1 and 2 h p.i. Tumor/muscle ratios were >4 at all time-points. Tumor/lung ratios were >2 at 1 h and 2 h p.i. Substantial uptake was observed in the liver (15-25%ID/g), kidneys (25-35%ID/g), the small intestine (1-7%ID/g) and the large intestine (1-12%ID/g). Blood concentrations varied over time (0.5-2%ID/g). All other organs showed uptake of <1%ID/g at all time points studied with clearance profiles similar to blood clearance. CONCLUSIONS Here we present, to the best of our knowledge, the first high affinity [18F]-labeled tracer, suitable for in vivo PET imaging of CXCR4. [18F]RPS-544 displayed high affinity for CXCR4 and good tumor uptake with a maximum uptake at 1 h p.i.. CXCR4 dependent uptake was demonstrated using bilateral tumors with differential CXCR4 expression as well as pharmacological blockade using the known CXCR4 antagonist, AMD-3100. Tissue contrast as judged by tumor to normal tissue ratios was positive in several key tissues. The structural and pharmacological similarities between [18F]RPS-544 and the approved drug AMD-3465, combined with the ease of synthesis and high molar activity (>185 GBq/μmol) achieved during radiosynthesis could lead to accelerated translation into the clinic.

66Ga: A Novelty or a Valuable Preclinical Screening Tool for the Design of Targeted Radiopharmaceuticals?

Emerging interest in extending the plasma half-life of small molecule radioligands warrants a consideration of the appropriate radionuclide for PET imaging at longer time points (>8 h). Among candidate positron-emitting radionuclides, 66Ga (t1/2 = 9.5 h, β+ = 57%) has suitable nuclear and chemical properties for the labeling and PET imaging of radioligands of this profile. We investigated the value of 66Ga to preclinical screening and the evaluation of albumin-binding PSMA-targeting small molecules. 66Ga was produced by irradiation of a natZn target. 66Ga3+ ions were separated from Zn2+ ions by an optimized UTEVA anion exchange column that retained 99.99987% of Zn2+ ions and allowed 90.2 ± 2.8% recovery of 66Ga3+. Three ligands were radiolabeled in 46.4 ± 20.5%; radiochemical yield and >90% radiochemical purity. Molar activity was 632 ± 380 MBq/µmol. Uptake in the tumor and kidneys at 1, 3, 6, and 24 h p.i. was determined by µPET/CT imaging and more completely predicted the distribution kinetics than uptake of the [68Ga]Ga-labeled ligands did. Although there are multiple challenges to the use of 66Ga for clinical PET imaging, it can be a valuable research tool for ligand screening and preclinical imaging beyond 24 h.

Continuation of comprehensive quality control of the itG 68Ge/68Ga generator and production of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC for clinical research studies

Performance of a second itG 68Ge/68Ga generator system and production of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC were tested over one year as an accompaniment to a previously published study (J Nucl Med. 2016;57:1402-1405). METHODS Performance of a 1951MBq 68Ge/68Ga generator was characterized and the eluate used for preparation of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC. Weekly elution profiles of 68Ga elution yield and 68Ge breakthrough were determined. RESULTS 68Ga elution yields averaged 82% (61.8-98.4%) and 68Ge breakthrough averaged 0.002% (0.0007% to 0.004%). The radiochemical purities of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC were determined by HPLC analysis to be >98% and specific activity was 12.6 and 42GBq/μmol, respectively. 68Ge contamination in the product was under the detection limit (0.00001%). Final sterile, pyrogen-free formulation of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC in physiologic saline with 5%-7% ethanol was achieved. CONCLUSION Performance of a 68Ge/68Ga generator was studied over one year with satisfactory results. The generator eluate was used to synthesize 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC on a routine basis in high purity.