Alex J. Poot

PET imaging with radiolabeled antibodies and tyrosine kinase inhibitors: immuno-PET and TKI-PET.

During the last decade, the discovery of critical tumor targets has boosted the design of targeted therapeutic agents with monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) receiving most of the attention. Immuno-positron emission tomography (immuno-PET) and TKI-PET, the in vivo tracking and quantification of mAbs and TKIs biodistribution with PET, are exciting novel options for better understanding of the in vivo behavior and efficacy of these targeted drugs in individual patients and for more efficient drug development. Very recently, current good manufacturing practice compliant procedures for labeling of mAbs with positron emitters have been described, as well as the preparation of some radiolabeled TKIs, while the first proof of principle studies has been performed in patients. In this review, technical developments in immuno-PET and TKI-PET are described, and their clinical potential is discussed. An overview is provided for the most appealing preclinical immuno-PET and TKI-PET studies, as well as the first clinical achievements with these emerging technologies.

PET imaging with small-molecule tyrosine kinase inhibitors: TKI-PET

The discovery and increased understanding of tumor targets has led to the development and approval of 12 small molecule tyrosine kinase inhibitors (TKIs). Despite tremendous efforts in TKI development, treatment efficacies with these therapeutics are still too low and improvements require a personalized medicine approach. Positron emission tomography (PET) with radiolabeled TKIs (TKI-PET) is a tracking, quantification and imaging method, which provides a unique understanding of the behavior of these drugs in vivo and of the interaction with their target(s). In this article we provide an overview of tracer synthesis and development because each TKI requires a tailor made approach. Moreover, we describe current preclinical work and the first proof-of-principle clinical studies on the application of TKI-PET, illustrating the potential of this approach for improving therapy efficacy and personalized cancer treatment.

Development of [18F]afatinib as new TKI-PET tracer for EGFR positive tumors.

INTRODUCTION Afatinib is an irreversible ErbB family blocker that was approved for the treatment of EGFR mutated non-small cell lung cancer in 2013. Positron emission tomography (PET) with fluorine-18 labeled afatinib provides a means to obtain improved understanding of afatinib tumor disposition in vivo. PET imaging with [(18)F]afatinib may also provide a method to select treatment responsive patients. The aim of this study was to label afatinib with fluorine-18 and evaluate its potential as TKI-PET tracer in tumor bearing mice. METHODS A radiochemically novel coupling, using peptide coupling reagent BOP, was explored and optimized to synthesize [(18)F]afatinib, followed by a metabolite analysis and biodistribution studies in two clinically relevant lung cancer cell lines, xenografted in nude mice. RESULTS A reliable [(18)F]afatinib radiosynthesis was developed and the tracer could be produced in yields of 17.0 ± 2.5% calculated from [(18)F]F(-) and >98% purity. The identity of the product was confirmed by co-injection on HPLC with non-labeled afatinib. Metabolite analysis revealed a moderate rate of metabolism, with >80% intact tracer in plasma at 45 min p.i. Biodistribution studies revealed rapid tumor accumulation and good retention for a period of at least 2 hours, while background tissues showed rapid clearance of the tracer. CONCLUSION We have developed a method to synthesize [(18)F]afatinib and related fluorine-18 labeled 4-anilinoquinazolines. [(18)F]Afatinib showed good stability in vivo, justifying further evaluation as a TKI-PET tracer.

A comparative PET imaging study with the reversible and irreversible EGFR tyrosine kinase inhibitors [(11)C]erlotinib and [(18)F]afatinib in lung cancer-bearing mice.

BackgroundTyrosine kinase inhibitors (TKIs) have experienced a tremendous boost in the last decade, where more than 15 small molecule TKIs have been approved by the FDA. Unfortunately, despite their promising clinical successes, a large portion of patients remain unresponsive to these targeted drugs. For non-small cell lung cancer (NSCLC), the effectiveness of TKIs is dependent on the mutational status of epidermal growth factor receptor (EGFR). The exon 19 deletion as well as the L858R point mutation lead to excellent sensitivity to TKIs such as erlotinib and gefitinib; however, despite initial good response, most patients invariably develop resistance against these first-generation reversible TKIs, e.g., via T790M point mutation. Second-generation TKIs that irreversibly bind to EGFR wild-type and mutant isoforms have therefore been developed and one of these candidates, afatinib, has now reached the market. Whether irreversible TKIs differ from reversible TKIs in their in vivo tumor-targeting properties is, however, not known and is the subject of the present study.MethodsErlotinib was labeled with carbon-11 and afatinib with fluorine-18 without modifying the structure of these compounds. A preclinical positron emission tomography (PET) study was performed in mice bearing NSCLC xenografts with a representative panel of mutations: an EGFR-WT xenograft cell line (A549), an acquired treatment-resistant L858R/T790M mutant (H1975), and a treatment-sensitive exon 19 deleted mutant (HCC827). PET imaging was performed in these xenografts with both tracers. Additionally, the effect of drug efflux transporter permeability glycoprotein (P-gp) on the tumor uptake of tracers was explored by therapeutic blocking with tariquidar.ResultsBoth tracers only demonstrated selective tumor uptake in the HCC827 xenograft line (tumor-to-background ratio, [11C]erlotinib 1.9 ± 0.5 and [18F]afatinib 2.3 ± 0.4), thereby showing the ability to distinguish sensitizing mutations in vivo. No major differences were observed in the kinetics of the reversible and the irreversible tracers in each of the xenograft models. Under P-gp blocking conditions, no significant changes in tumor-to-background ratio were observed; however, [18F]afatinib demonstrated better tumor retention in all xenograft models.ConclusionsTKI-PET provides a method to image sensitizing mutations and can be a valuable tool to compare the distinguished targeting properties of TKIs in vivo.

[11C]Sorafenib: Radiosynthesis and preclinical evaluation in tumor-bearing mice of a new TKI-PET tracer

INTRODUCTION Tyrosine kinase inhibitors (TKIs) like sorafenib are important anticancer therapeutics with thus far limited treatment response rates in cancer patients. Positron emission tomography (PET) could provide the means for selection of patients who might benefit from TKI treatment, if suitable PET tracers would be available. The aim of this study was to radiolabel sorafenib (1) with carbon-11 and to evaluate its potential as TKI-PET tracer in vivo. METHODS Synthetic methods were developed in which sorafenib was labeled at two different positions, followed by a metabolite analysis in rats and a PET imaging study in tumor-bearing mice. RESULTS [methyl-(11)C]-1 and [urea-(11)C]-1 were synthesized in yields of 59% and 53%, respectively, with a purity of >99%. The identity of the products was confirmed by coinjection on HPLC with reference sorafenib. In an in vivo metabolite analysis [(11)C]sorafenib proved to be stable. The percentage of intact product in blood-plasma after 45 min was 90% for [methyl-(11)C]-1 and 96% for [urea-(11)C]-1, respectively. Due to the more reliable synthesis, further research regarding PET imaging was performed with [methyl-(11)C]-1 in nude mice bearing FaDu (head and neck cancer), MDA-MB-231 (breast cancer) or RXF393 (renal cancer) xenografts. Highest tracer accumulation at a level of 2.52 ± 0.33%ID/g was observed in RXF393, a xenograft line extensively expressing the sorafenib target antigen Raf-1 as assessed by immunohistochemistry. CONCLUSION In conclusion, we have synthesized [(11)C]sorafenib as PET tracer, which is stable in vivo and has the capability to be used as PET tracer for imaging in tumor-bearing mice.

Imaging of TKI–Target Interactions for Personalized Cancer Therapy

The discovery of critical tumor targets during the past decade has boosted the design of targeted drugs such as tyrosine kinase inhibitors (TKIs). Positron emission tomography with radiolabeled TKIs (TKI‐PET) allows in vivo analysis of these agents to better understand the therapeutic potential of TKIs in individual patients. The highlights of this new field of imaging, including the first clinical studies with great potential for personalized medicine, are discussed.

Improved synthesis and application of [C-11]benzyl iodide in positron emission tomography radiotracer production

Positron emission tomography has increased the demand for new carbon-11 radiolabeled tracers and building blocks. A promising radiolabeling synthon is [(11) C]benzyl iodide ([(11) C]BnI), because the benzyl group is a widely present functionality in biologically active compounds. Unfortunately, synthesis of [(11) C]BnI has received little attention, resulting in limited application. Therefore, we investigated the synthesis in order to significantly improve, automate, and apply it for labeling of the dopamine D2 antagonist [(11) C]clebopride as a proof of concept. [(11) C]BnI was synthesized from [(11) C]CO2 via a Grignard reaction and purified prior the reaction with desbenzyl clebopride. According to a one-pot procedure, [(11) C]BnI was synthesized in 11 min from [(11) C]CO2 with high yield, purity, and specific activity, 52 ± 3% (end of the cyclotron bombardment), 95 ± 3%, and 123 ± 17 GBq/µmol (end of the synthesis), respectively. Changes in the [(11) C]BnI synthesis are reduced amounts of reagents, a lower temperature in the Grignard reaction, and the introduction of a solid-phase intermediate purification. [(11) C]Clebopride was synthesized within 28 min from [(11) C]CO2 in an isolated decay-corrected yield of 11 ± 3% (end of the cyclotron bombardment) with a purity of >98% and specific activity (SA) of 54 ± 4 GBq/µmol (n = 3) at the end of the synthesis. Conversion of [(11) C]BnI to product was 82 ± 11%. The reliable synthesis of [(11) C]BnI allows the broad application of this synthon in positron emission tomography radiopharmaceutical development.

Two anti-angiogenic TKI-PET tracers, [11C]axitinib and [11C]nintedanib: Radiosynthesis, in vivo metabolism and initial biodistribution studies in rodents

INTRODUCTION Tyrosine kinase inhibitors (TKIs) are very attractive targeted drugs, although a large portion of patients remains unresponsive. PET imaging with EGFR targeting TKIs ([(11)C]erlotinib and [(18)F]afatinib) showed promise in identifying treatment sensitive tumors. The aim of this study was to synthesize two anti-angiogenic TKI tracers, [(11)C]axitinib and [(11)C]nintedanib, and to evaluate their potential for PET. METHODS Following successful tracer synthesis, biodistribution studies in VU-SCC-OE and FaDu xenograft bearing mice were performed. Furthermore, tracer stability studies in mice were performed employing (radio-)HPLC and LC-MS/MS techniques. For [(11)C]nintedanib an LC-MS/MS method was developed to detect the primary carboxylic acid metabolite, resulting from methylester cleavage, in plasma and tumors, because this metabolite is postulated to be important for nintedanib efficacy. LC-MS/MS was also explored to assess the metabolic fate of [(11)C]axitinib in vivo, since axitinib has an isomerizable double bond. RESULTS [(11)C]axitinib and [(11)C]nintedanib were successfully synthesized with 10.5±2.6% and 25.6±3.3% radiochemical yield (corrected for decay), respectively. Biodistribution studies only demonstrated tumor uptake of [(11)C]nintedanib in FaDu xenografts of 1.66±0.02% ID/g at 60min p.i. In vivo stability analysis of [(11)C]axitinib at 45min p.i. revealed the formation of predominantly non-polar metabolites (36.6±6.8% vs 47.1±8.4% of parent tracer and 16.3±2.1% of polar metabolites), while for [(11)C]nintedanib mostly polar metabolites were found (70.9±4.1 vs 26.7±3.9% of parent tracer and only 2.4±1.6 of a non-polar metabolites). No isomerization of [(11)C]axtinib was observed in vivo; however, a sulfoxide metabolite could be detected using LC-MS/MS. For [(11)C]nintedanib, LC-MS/MS revealed formation of the reported primary carboxylic acid metabolite when in vitro plasma incubations were performed, with large differences in plasmas from different species. In vivo metabolite analysis, however, did not demonstrate the presence of the carboxylic acid in plasma or tumor tissue. CONCLUSIONS Reliable syntheses of [(11)C]axitinib and [(11)C]nintedanib were successfully developed. Tumor uptake was observed for [(11)C]nintedanib, albeit modest. The metabolic profiles of the tracers suggest that rapid metabolism is partly responsible for the modest tumor targeting observed.

From Carbon-11-Labeled Amino Acids to Peptides in Positron Emission Tomography: the Synthesis and Clinical Application

Radiolabeled amino acids, their derivatives and peptides have a broad scope of application and can be used as receptor ligands, as well as enzyme substrates for many different diseases as radiopharmaceutical tracers. Over the past few decades, the application of molecular imaging techniques such as positron emission tomography (PET) has gained considerable importance and significance in diagnosis in today’s advanced health care. Next to that, the availability of cyclotrons and state-of-the-art radiochemistry facilities has progressed the production of imaging agents enabling the preparation of many versatile PET radiotracers. Due to many favorable characteristics of radiolabeled amino acids and peptides, they can be used for tumor staging and monitoring the progress of therapy success, while aromatic amino acids can be employed as PET tracer to study neurological disorders. This review provides a comprehensive overview of radiosynthetic and enzymatic approaches towards carbon-11 amino acids, their analogues and peptides, with focus on stereoselective reactions, and reflects upon their clinical application.

Synthesis and Preclinical Evaluation of the First Carbon-11 Labeled PET Tracers Targeting Substance P1-7

Two potent SP1–7 peptidomimetics have been successfully radiolabeled via [11C]CO2-fixation with excellent yields, purity, and molar activity. l-[11C]SP1–7-peptidomimetic exhibited promising ex vivo biodistribution profile. Metabolite analysis showed that l-[11C]SP1–7-peptidomimetic is stable in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic stability can be significantly improved by substituting l-Phe for d-Phe, preserving 70% more of intact tracer and resulting in better brain and spinal cord tracer retention. Positron emission tomography (PET) scanning confirmed moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for l- and d-[11C]SP1–7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment with natural peptide SP1–7 in spinal cord for l-[11C]SP1–7-peptidomimetic. On the contrary, blocking using cold analogues of l- and d-[11C]tracers did not reduce the tracers’ brain and spinal cord exposure. In summary, PET scanning of l- and d-[11C]SP1–7-peptidomimetics confirms rapid blood–brain barrier and blood–spinal-cord barrier penetration. Therefore, further validation of these two tracers targeting SP1–7 is needed in order to define a new PET imaging target and select its most appropriate radiopharmaceutical.