• By their nature, radioactive isotopes are unstable and can change to more stable forms by radioactive decay and emission of different types of radiation.  This emitted radiation is useful for cancer treatment which includes placement of “sealed” radioactive sources physically close to the disease site (brachytherapy), or by focusing the radiation emitted by an external source to the disease site (radiotherapy). These are practiced by radiation oncologists.  In another clinical specialty of nuclear medicine, radioisotopes are used in a different manner for both diagnostic and therapeutic procedures by intravenous injection of “unsealed” radiopharmaceutical agents. Nuclear medicine allows visualization of the cancer sites by an imaging technique after specifically targeting diagnostic radiopharmaceuticals and subsequently treating them with therapeutic radioactive molecules capable of killing the cancer cells. Combination of such therapy based on evidence derived diagnosis is called theranostics. Lutetium-177 is an important radioisotope used for targeted therapy.

    Fig. 1. Single Photon Emission Tomography (SPECT) images of two patients treated with 177Lu agents for cancer therapy. The small dark circular areas represent tumor sites. Images were obtained following the injection and illustrate targeting of tumors for therapy. 177Lu-DOTATATE tumors expressing somatostatin receptors (A, Left) and 177Lu-PMSA agent illustrating well defined prostate metastases (B, Right) (Images courtesy: Ajit Joy, Arun Sasikumar, Raviteja Nanabala, KIMS-DDNMRC, Thiruvanthapuram, Kerala, India).

    The strategy for targeted therapy involves stable chemical attachment of 177Lu to molecules which target the disease sites after injection.  Radioactive decay of 177Lu emits low energy gamma photons which traverse through tissue and are used for targeting visualization by external imaging.  In addition, beta particles are also emitted from 177Lu decay but only travel short distances in tissue, the energy of which is absorbed by the disease target sites killing the cancer cells. The therapy is performed in many parts of the world, as the radioactive levels of 177Lu decrease by only 50% every 6.64 days (half-life) permitting its shipment from the reactor production facility to the clinical sites.

    Major clinical applications using 177Lu include treatment of neuroendocrine tumors (NETs) where cellular receptors over-expressed on the surface of the cancer cells are targets for the 177Lu-“DOTATATE” agent, which is an analogue of the hormone somatostatin. The clinical use of this agent is expanding in several countries. Other key examples of over-expressed cancer cell target receptors, many of which are being evaluated for therapeutic targeting with the 177Lu, include bombesin/GRP, CCK/gastrin, endothelin, extendin, integrin, neurotensin, oxytocin and substance P. Another therapeutic example is radioimmunotherpay with 177Lu-labeled rituximab®, a monoclonal antibody useful for treating Non-Hodgkin’s lymphoma.  In addition, use of the 177Lu-“EDTMP” agent is progressing in clinical trials for the reduction of metastatic bone pain originating from the spread of cancers from the prostate, ovaries and lungs.

    Because of the very high incidence of metastatic prostate cancer throughout the world, a very exciting recent advance uses a 177Lu-based agent for therapy of prostate cancer metastases. The prostate specific membrane antigen (PSMA) protein  is expressed in very high levels on prostate parenchyma but in low levels on most other normal cells and is the target for this radiopharmaceutical. PSMA is significantly over-expressed in prostate cancer cells and some other solid tumours. PSMA is an enzyme which hydrolyses the dipeptide, n-acetylaspartylglutamate (NAAG) molecule.

    177Lu-NAAG (commonly called 177Lu-PSMA) is emerging as a successful therapeutic agent for metastatic prostate cancer. 177Lu-NAAG binds to PMSA molecules found on the cell surfaces and the complex is then internalized into the target cells providing very high intracellular levels of 177Lu emitting beta radiation for killing the cancer cells. Clinical trials with 177Lu-PSMA are in progress in many countries and have demonstrated effective therapy with very few side effects. The above examples reflect the widespread interest in the clinical community for therapeutic applications of 177Lu-  radiopharmaceuticals which are expected to result in regulatory approval and introduction of some of these agents for routine applications.

    FF (Russ) Knapp1 and MRA Pillai2
    1Emeritus, Medical Radioisotopes Program, Oak Ridge National Laboratory (ORNL), Oak Ridge, TN, USA
    2Molecular Group of Companies, Puthuvype, Ernakulam, Kerala, India

    Publication
    Evolving Important Role of Lutetium-177 for Therapeutic Nuclear Medicine.
    Pillai AM, Knapp FF Jr.
    Curr Radiopharm. 2015

    The information comes from:
    https://atlasofscience.org/lutetium-177-radioisotope-targeted-therapy-for-treatment-of-cancer-and-other-diseases/

  • BACKGROUND:
    A remarkable therapeutic efficacy has been demonstrated with 225Ac-prostate-specific membrane antigen (PSMA)-617 in heavily pre-treated metastatic castration-resistant prostate cancer (mCRPC) patients. We report our experience with 225Ac-PSMA-617 therapy in chemotherapy-naïve patients with advanced metastatic prostate carcinoma.

  • Iodine-125 seed implantation for synchronous pancreatic metastases from hepatocellular carcinoma: A case report and literature review.

    Rationale The image-guided iodine-125 seed implantation has been widely used for a variety of tumors, including prostatic cancer, pulmonary cancer, hepatocellular carcinoma and pancreatic cancer. However, the clinical value of iodine-125 seed implantation for the treatment of pancreatic metastasis from hepatocellular carcinoma has not been reported. We presented the first case with ultrasound-guided iodine-125 seed implantation for this disease. Patient concerns We presented the case of a 48-year-old man patient with primary hepatocellular carcinoma and pancreatic metastasis who was managed with ultrasound-guided iodine-125 seeds implantation. Diagnoses She was diagnosed with synchronous pancreatic metastases from hepatocellular carcinoma. Interventions Puncture biopsy and ultrasound-guided iodine-125 seeds implantation. Outcomes The hepatic and pancreatic tumors were obviously reduced after 15 months. Moreover, the liver function test was mildly abnormal in glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase. Lessons The image-guided iodine-125 seeds implantation was an important therapeutic approache to unresectable hepatocellular carcinoma with pancreatic metastasis. However, more related cases should be reported for further evaluating the value of the way.

    The information comes from:
    https://www.researchgate.net/publication/321066757_Iodine-125_seed_implantation_for_synchronous_pancreatic_metastases_from_hepatocellular_carcinoma_A_case_report_and_literature_review

  • Objective:

    Brachytherapy employing iodine-125 seeds is an established treatment for low-risk prostate cancers. Post-implant dosimetry (PID) is an important tool for identifying suboptimal implants. The aim of this work was to improve suboptimal implants by a subsequent iodine-125 seed top-up (reimplantation), based on the PID results.

    Methods:

    Of 255 patients treated between 2009 and 2012, 6 were identified as having received suboptimal implants and were scheduled for seed top-up. Needle configurations and the number of top-up seeds were determined based on post-implant CT images as well as a reimplantation treatment plan. An average of 14 seeds per patient were implanted during each top-up. Dosimetric outcome was assessed via target parameters and doses received by organs at risk.

    Results:

    All six patients had a successful top-up, with a 67% increase in the mean dose delivered to 90% of the prostate volume and a 40% increase in the volume that receives 100% of the prescribed dose. However, the final dosimetric assessment was based on the same seed activity, as the planning system does not account for the decay of the initially implanted seeds. Although physical dosimetry is not influenced by different seed activities (doses are calculated to infinity), the radiobiological implications might be slightly different from the situation when optimal implantation is achieved with one treatment only.

    Conclusion:

    Seed reimplantation in suboptimal prostate implants is feasible and leads to successful clinical outcomes.

    Advances in knowledge:

    Suboptimal prostate implants can occur for various reasons. This work shows that seed reimplantation as salvage therapy can lead to an optimal dosimetric outcome with manageable normal tissue effects.

    Low dose rate (LDR) brachytherapy employing radioactive seeds is a well-established treatment for low-risk prostate cancers. In our centre, implants are conducted with iodine-125 seeds (Oncura RAPID Strand, model 6711; Oncura Inc., Arlington Heights, IL) with an average seed activity of 0.395 mCi to deliver a prescribed dose of 145 Gy (to >98% of the prostate). Treatment planning and post-implant dosimetry (PID) are completed using SPOT-PRO™ v. 3.1 (Nucletron, Utrecht, Netherlands) software based on transrectal ultrasound images (for treatment planning) and CT images (for PID).

    The most commonly reported parameters that are indicative of the dosimetric quality of the implant are D90 (the dose delivered to 90% of the prostate volume) and V100 (the volume that receives 100% of the prescribed dose). Several studies showed a link between the quality of implants and the biochemical outcome []. Therefore, to minimise the risk of recurrence, it is recommended to achieve a post-implant D90>140 Gy and V100>90% [].

    PID is an important quantitative tool for the assessment of LDR implants; therefore, it is recommended as a routine procedure by several professional organisations []. Besides evaluating the overall quality of the implant, PID can assist in the dosimetric assessment of the organs at risk (OARs). Although the dosimetry of OARs cannot be adjusted if overdosed, the radiation oncologist can have a closer follow-up of those patients at risk of developing normal tissue sequelae.

    Another role of PID is to identify suboptimal implants that can arise owing to organ movement during the procedure, geographical misses of seeds or technical equipment errors. Despite all the efforts and experience of the brachytherapy team, suboptimal implants do occur and they have to be dealt with. Although several centres encounter such events, there is a lack of guidelines or even indications as to how to proceed to improve the final outcome. The major challenge is perhaps the planning, which cannot be done in a conventional way, i.e. based on the ultrasound study of the transrectal volume, owing to lack of previously implanted seed visibility []. Therefore, post-implant CT images are the most convenient to use for this task, as the original seeds can be seen and extra seeds can be added to cover the underdosed areas of the prostate.

    The aim of this work was to present our experience with iodine-125 seed reimplantation (top-up) in a cohort of six patients whose initial implant was suboptimal as identified by PID. The technical and dosimetric challenges of seed top-up implants are investigated.

    MATERIALS AND METHODS

    Of 255 patients treated with iodine-125 permanent seed brachytherapy between January 2009 and July 2012 at the Royal Adelaide Hospital, SA, 6 were identified as having received suboptimal implants and were scheduled for seed top-up. The suboptimal implants were attributed to equipment/technical problems (two cases), patient movement during implantation (one case) and uncommonly large oedema (one case); however, for two patients, the causes of underdosage were unidentified. Therefore, the cold spots were random from patient to patient, although underdosage of the base was more common than the involvement of other prostate regions.

    The decision to undergo reimplantation was based on the PID results using ultrasound–CT image coregistration, which our centre’s standard technique (the methods are described in detail in a previous report []). However, in all situations, the main dosimetric parameters were independently confirmed by a second medical physicist, and in some cases, when the result was uncertain, an alternative PID technique was also used, such as CT-delineation-based PID or MR–CT image coregistration-based PID. As with all our PID data, the results were reviewed by the patient’s radiation oncologist, who, after following the PID prostate D90 criteria given below, decided whether the patient should be scheduled for a seed top-up or not.

    At our centre, we categorise the quality of the implants according to the following PID D90 values:

    • D90>145 Gy—good implant [as recommended by Groupe Européen de Curiethérapie–European Society for Radiotherapy & Oncology (GEC-ESTRO) guidelines] []

    • 130 Gy<D90<145 Gy—adequate by local definition (within 10% of the prescribed dose).

    • 110 Gy<D90<130 Gy—potentially acceptable, subject to re-assessment of PID with another PID technique and evaluation of cold spots. If cold spots are identified in areas that were not positive on transrectal ultrasound biopsy, we consider the implant acceptable. If the implant is subsequently deemed inadequate, the patient is scheduled for a seed top-up procedure.

    • D90<110 Gy—the implant is inadequate [,]. The patient is scheduled for a seed top-up procedure.

    For those patients identified as having an inadequate implant, a top-up treatment plan is generated. This is designed using the initial PID ultrasound–CT images as the reference to determine the location and the number of seeds required to improve the dosimetry.

    The prostate target (hereafter referred to as the target) is defined as the clinical target volume, and acceptable target volumes for initial implants are between 15 and 55 cm3. A planning target volume (PTV) is also defined during preplanning but is not considered in this report, as its dosimetry trends reflect those of the target values.

    The information comes from:
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664978/

  • Abstract

    OBJECTIVE:
    (99m)Tc-MDP (technetium-99(m)-labeled methylene diphosphonate) has been widely used as a radiopharmaceutical for bone scintigraphy in cases of metastatic bone disease. (177)Lu is presently considered as an excellent radionuclide for developing bone pain palliation agents. No study on preparing a complex of (177)Lu with MDP has been reported yet. Based on these facts, it was hypothesized that a bone-seeking (177)Lu-MDP (lutetium-177-labeled MDP) radiopharmaceutical could be developed as an agent for palliative radiotherapy of bone pain due to skeletal metastases. Biodistribution studies after intravenous injection of (177)Lu-MDP complex in rats may yield important information to assess its potential for clinical use as a bone pain palliation agent for the treatment of bone metastases.

    METHODS:
    (177)Lu was produced by irradiating natural Lu(2)O(3) (10 mg) target at a thermal flux ∼ 8.0 × 10(13) n/cm(2) per second for 12 h in the swimming pool-type reactor.(177)Lu was labeled with MDP by adding nearly 37 MBq (1.0 mCi) of (177)LuCl(3) to a vial containing 10 mg MDP. The radiochemical purity and labeling efficiencies were determined by thin layer chromatography. Labeling of (177)Lu with MDP was optimized, and one sample was subjected to high-performance liquid chromatography (HPLC) analysis. Twelve Sprague-Dawley rats were injected with 18.5 MBq (0.5 mCi). (177)Lu-MDP in a volume of 0.1 ml was injected intravenously and then sacrificed at 2 min, 1 h, 2 h and 22 h (three rats at each time point) after injection. Samples of various organs were separated, weighed and measured for radioactivity and expressed as percent uptake of injected dose per gram. Bioevaluation studies with rats under gamma-camera were also performed to verify the results.

    RESULTS:
    The quality control using thin layer chromatography has shown >99% radiochemical purity of (177)Lu-MDP complex. Chromatography with Whatman 3MM paper showed maximum labeling at pH = 6, incubation time = 30 min, and ligand/metal ratio = 60:1. HPLC analysis showed 1.35 ± 0.05 min retention time of (177)Lu-MDP complex. No decrease in labeling was observed at higher temperatures, and the stability of the complex was found adequate. Biodistribution studies of (177)Lu-MDP revealed high skeletal uptake, i.e., 31.29 ± 1.27% of the injected dose at 22 h post injection. Gamma-camera images of (177)Lu-MDP in Sprague-Dawley rats also showed high skeletal uptake and verified the results.


    CONCLUSION:
    The study demonstrated that MDP could be labeled with (177)Lu with high radiochemical yields (>99%). The in vitro stability of the complex was found adequate. Biodistribution studies in Sprague-Dawley rats indicated selective bone accumulation, relatively low uptake in soft tissue (except liver) and higher skeletal uptake, suggesting that it may be useful as a bone pain palliation agent for the treatment of bone metastases.

    Copyright © 2011 Elsevier Inc. All rights reserved.

    The information comes from:
    https://www.ncbi.nlm.nih.gov/pubmed/21492790

  • Abstract

    Theranostics, a modern approach combining therapeutics and diagnostics, is among the most promising concepts in nuclear medicine for optimizing and individualizing treatments for many cancer entities. Theranostics has been used in clinical routines in nuclear medicine for more than 60 y—as 131I for diagnostic and therapeutic purposes in thyroid diseases. In this minireview, we provide a survey of the use of 2 different radioiodine isotopes for targeting the sodium–iodine symporter in thyroid cancer and nonthyroidal neoplasms as well as a brief summary of theranostics for neuroendocrine neoplasms and metastatic castration-refractory prostate cancer. In particular, we discuss the role of 124I-based dosimetry in targeting of the sodium–iodine symporter and describe the clinical application of 124I dosimetry in a patient who had radioiodine-refractory thyroid cancer and who underwent a redifferentiation treatment with the mitogen-activated extracellular signal–related kinase kinase inhibitor trametinib.

    Theranostics is a diagnostic approach coupled with treatment modalities in a personalized fashion to improve therapeutic effects and reduce treatment toxicities (1). This term was initially coined by John Funkhouser in a 1998 press release in connection with personalized treatment. Translating theranostics to the field of nuclear medicine means labeling of a compound with different radionuclides for both diagnostic and therapeutic purposes for a specific target. Ideally, the radionuclides used for both diagnostic and therapeutic purposes are derived from the same element, but for chemical and physical reasons this is often not practicable. In this mini review, we provide a brief survey of 2 theranostic approaches for neuroendocrine tumors and prostate cancer, with a focus on targeting of the sodium–iodine symporter (NIS) in thyroidal disorders, and we discuss issues regarding 124I and 131I targeting of the NIS in extrathyroidal disorders.

    CLINICAL USE OF THERANOSTIC AGENTS OTHER THAN IODINE

    Neuroendocrine neoplasms and prostate cancer are currently the most prominent targets of nonthyroidal theranostic agents (24). Because of a lack of efficient treatment options, metastatic, well-differentiated neuroendocrine neoplasms are challenging tumors. These tumors express somatostatin receptors, which can be imaged by PET with, for instance, 68Ga-labeled DOTATATE, DOTATOC, or DOTANOC (5). PET enables in vivo quantification of the tumoral expression level and of the background uptake level in the surrounding tissues or organs at risk. For treatment, the same compound can be labeled with a β-particle emitter (177Lu or 90Y) to target metastatic sites. The higher the level of expression in the tumor (and, thus, the higher the tumor-to-background ratio), the lower the radiation-related toxicity effects in the surrounding tissues.

    Regarding toxicity, protection of the kidneys is important, because a large portion of the injected amount of a radiolabeled compound is eliminated via renal excretion. To address this issue, patients receive an infusion of an amino acid cocktail during treatment to saturate renal reabsorption mechanisms (6). The efficacy of this approach was recently shown in a prospective multicenter trial (2).

    Another promising theranostic approach is being used for metastatic castration-resistant prostate cancer. The target in this challenging disease is the prostate-specific membrane antigen (PSMA), which is expressed at high levels, particularly in recurrent prostate cancer; additionally, expression is not lost with dedifferentiation, making it an ideal target. PSMA-targeting molecules can be labeled with different positron emitters, such as 124I, 18F, 64Cu, or 68Ga (3,7). At present, PSMA ligands are more frequently labeled with 68Ga for PET imaging. For therapeutic purposes, they are usually labeled with the β-particle emitter 177Lu. However, there has even been a report on the labeling of a PSMA ligand with the α-particle emitter 225Ac for the treatment of patients whose disease progressed after 177Lu-PSMA ligand treatment (8).

    THERANOSTICS WITH IODINE

    Radioiodine treatment (using 131I) has been the main pillar of nuclear medicine for more than 60 y. 131I not only is a β-particle emitter but also has penetrating γ-radiation, which makes this tracer trackable in vivo through imaging. However, 131I is not the ideal tracer for quantitative imaging purposes because of poor spatial resolution and quantification capacity using SPECT (Table 1). With the increasing availability of PET scanners during the last 15 y, 124I became the tracer of first choice for the imaging of thyroid disorders, mainly in patients with high-risk and recurrent thyroid cancer (9,10). The properties of 124I and 131I are juxtaposed in Table 1, demonstrating the superiority of 124I for imaging. Moreover, 124I allows more reliable dosimetry, the 2 main pillars of which are shown in Figure 1.

    TABLE 1

    Physical Half-Lives and Qualitative Comparison of Common Radioiodine Isotopes for Imaging in Theranostics

    FIGURE 1.
    FIGURE 1.

    Simplified illustration of 2 main pillars of 124I dosimetry concept. LDpA = lesion-absorbed dose per administered activity; MTA = maximum tolerable activity.

    Most patients typically undergo several radioiodine treatments during their disease history, and each additional radioiodine treatment increases the risk of radiation-associated detrimental effects. To reduce or at least estimate the risks, as well as to increase the efficacy of radioiodine treatment, an individual assessment of absorbed radiation doses to the tumors and the organs at risk is crucial. According to Maxon et al., an absorbed dose of 85 Gy or higher is associated with an 80%–90% likelihood of a therapy response in lymph node metastases (Table 2) (11). The absorbed dose thresholds for other metastatic tissues (and thyroid remnants), derived from current 124I PET dosimetry studies, are also shown in Table 2 (12,13). Of note, a response to radioiodine is already expected for absorbed doses exceeding 20 Gy (11,12). Moreover, the bone marrow is often the dose-limiting organ in the application of high therapy activities.

    TABLE 2

    Relationship Between Absorbed Dose Thresholds and Associated Complete Response Rates for Metastases and Thyroid Remnants

    Pretherapy blood dosimetry has been developed to estimate the toxicity of radioiodine with the aim of avoiding possible life-threatening, radiation-induced bone marrow suppression (Fig. 1). In this organ-at-risk dosimetry approach, the maximum tolerable 131I activity that can be safely administered without producing toxic effects is calculated. Through collection of blood samples and determination of external whole-body counts over a period of 4 d or longer, the maximum tolerable 131I activity can be estimated using an absorbed dose limit of 2 Gy to blood (as a surrogate for bone marrow toxicity) (1416). The key quantities for estimating the absorbed dose to the tumor are mass (or volume), the initial uptake value (for instance, at 24 h), and the effective 131I half-life. The mass can be estimated from CT or 124I PET using sophisticated threshold-based segmentation algorithms. Determination of the 24-h uptake—mainly mediated through the NIS—and the predicted effective 131I half-life requires serial 124I PET/CT scans (17). Pretherapy dosimetry results enable the selection of an optimized therapeutic activity—that is, the activity achieving a high tumor dose (such as 85 Gy for lymph node metastases) while maintaining a dose less than the 2-Gy limit to blood (Fig. 1). Thus, 124I dosimetry is suitable for individual therapy assessment.

    IODINE METABOLISM

    A better understanding of 124I PET dosimetry can be gained through an examination of iodine metabolism (Fig. 2). In brief, iodide is transported actively into the cell via the NIS, which is located in the basolateral membrane of thyrocytes. Next, this iodide enters the colloid on the apical membrane, located on the opposite side, via pendrin or other unspecified channels (18). In the colloid, iodide is oxidized, is bound to thyroglobulin (via the enzyme thyroid peroxidase), and either remains in the colloid or exits the cell as the end product—the thyroid hormone triiodothyronine or tetraiodothyronine. The active transport of iodide into the cell via the NIS is correlated with the level of expression of this symporter and can be estimated in vivo through 124I PET imaging. The effective half-life—that is, the decrease in accumulated radioiodine uptake over time—cannot be quantified on the basis of a single PET scan. Therefore, serial PET scans over time are needed to estimate the kinetics of radioiodine. Even though the precision of the quantification of radioiodine accumulation over time increases with the number of PET scans, these scans are limited in clinical settings for time and economic reasons. Therefore, a 2-time-point model that reasonably balances precision and effort or cost in clinical settings has been developed (19).

    FIGURE 2.
    FIGURE 2.

    Thyroid follicle showing iodine uptake and residence. Expression of NIS is essential for iodide uptake. Iodide is transported via pendrin to colloid in which iodide is bound to thyroglobulin. The latter is crucial to increasing average time on site of radioiodide in the follicle, which is associated with an increased absorbed radiation dose. Nonthyroidal cells expressing NIS lack this storing mechanism. DIT = diiodotyrosine; MIT = monoiodotyrosine; TSH = thyroid-stimulating hormone; TSH-R = TSH receptor. The function of TSH is to stimulate NIS expression.

    An example is provided in Figure 3, which shows 124I PET/CT images of a thyroid cancer patient and describes the lesions along with their predicted absorbed doses. 124I PET was capable of quantifying the increase in NIS expression in this patient with radioiodine-refractory thyroid cancer after redifferentiation treatment with trametinib, a mitogen-activated extracellular signal–related kinase kinase inhibitor. This is the first report of trametinib treatment of a patient with radioiodine-refractory thyroid cancer. Basically, as shown by the 124I PET results, the effective half-lives of the lesions remained similar (Fig. 4). Because of previous experience with mitogen-activated extracellular signal–related kinase kinase inhibition in this setting, we expected an increase in NIS expression without a significant increase in the effective half-lives, as shown in Figure 4 (17). The effects in our patient with radioiodine-refractory thyroid cancer were in line with this expectation. The 124I PET results revealed a 10-fold increase in iodide uptake with a nonsignificant change in the effective half-life.

    FIGURE 3.
    FIGURE 3.

    PET/CT images of 69-y-old patient with follicular thyroid carcinoma diagnosed in 1999. Patient underwent surgery and many treatments with radioiodine and experienced disease progression involving bone and lymph node metastases. Patient had undergone tyrosine kinase inhibitor treatment with sorafenib and lenvatinib but discontinued treatment because of disease progression. Patient was introduced to our hospital for redifferentiation therapy. After confirmation of BRAF-WT mutation status using archival tumor tissue, patient underwent pretreatment lesion dosimetry under thyrotropin stimulation with recombined human thyrotropin (A and C). Target lesions showed absorbed doses of 1–10 Gy/GBq. After 4 wk of trametinib treatment, 124I PET lesion dosimetry revealed absorbed doses of 10–322 Gy/GBq for most metastases (B and D). Blood dosimetry estimated maximum tolerable activity of 7 GBq. Patient was treated with 6 GBq of 131I. This example demonstrates importance of in vivo dosimetry in estimating redifferentiation effects and evaluating radioiodine treatment of nonthyroidal tumors.

    FIGURE 4.
    FIGURE 4.

    Predicted 131I uptake curve derived from 124I PET–based lesion dosimetry before (A) and after (B) redifferentiation. Lines were calculated using 2-point approach (19), and symbols represent measured PET-derived uptake values. Uptake values before differentiation were lower by factor of 10, demonstrating similar effective half-lives but 10-fold-lower 24-h uptake per gram.

    These findings were most likely due to the fact that in such tumors, mitogen-activated extracellular signal–related kinase kinase inhibition alone is not sufficient to reestablish the polarity of the cells and, thus, reshape a functioning colloidal structure. The latter is crucial for proper binding of iodide to thyroglobulin, resulting in an increased effective half-life. Nevertheless, the increased level of NIS expression alone was sufficient to increase the estimated absorbed dose significantly. The dosimetry results showed that the patient should have been treated with 17 GBq of 131I. However, the decision about a treatment must also be based on the blood dosimetry results, which limited the amount of treatment activity to 7 GBq of 131I. Even though there is no single clinical study analyzing the predictive value of pretherapeutic 124I PET dosimetry, the common consensus is that 124I PET dosimetry contributes significantly to pretherapeutic absorbed dose estimations. The example provided here not only illustrates the potential of 124I PET dosimetry but also underlines the importance of applying this dosimetry approach to redifferentiation treatments in clinical routines (20).

    THERANOSTICS WITH IODINE IN NONTHYROIDAL CANCER

    The simplicity and efficacy of radioiodine for the imaging and treatment of thyroid cancer patients attracted many research groups to investigate NIS expression in nonthyroidal tumor entities with the aim of treating these entities with radioiodine as well (21,22). In this context, some research groups investigated the efficacy of transfection of the NIS gene to tumor cells to make them targets for radioiodine. Table 3 shows the results of a study in which NIS expression in nonthyroidal tumors was investigated. Investigating radioiodine accumulation in nonthyroidal tissue requires an appreciation of the lack of a colloidal structure in the tumor cells and, thus, the absence of an ability to metabolize iodine and the consequent negative impact on the effective half-life of iodine. Given these circumstances, the level of expression of the NIS in nonthyroidal tumor cells should compensate for these shortcomings to achieve a significant absorbed dose. This goal is challenging and is probably the main reason why no clinical data showing the efficacy of radioiodine in nonthyroidal tumors have yet been published.

    TABLE 3

    Extrathyroidal Tissues Expressing NIS

    Another issue is the presence of thyroid in patients with nonthyroidal cancer. Because these patients have a functioning thyroid, the applied radioiodine will be actively transported into thyroid cells; therefore, the amount of radioiodine delivered to the targeted tumor cells will be reduced. More important than this reduced efficacy is unintended radiation damage to thyroid cells. However, thyroid uptake can be reduced through the coapplication of triiodothyronine (or tetraiodothyronine) and methimazole. Triiodothyronine downregulates the thyrotropin level (through a feedback loop), resulting in a reduction in NIS expression and, consecutively, a reduction in iodine uptake. Methimazole inhibits the enzyme thyroperoxidase, which catalyzes the binding of iodine to thyroglobulin and, thus, reduces the effective half-life of iodine (23).

    IODINE AND BREAST CANCER

    Breast cancer was one of the first nonthyroidal tumor entities in which NIS expression was convincingly shown (by messenger RNA levels and immunohistochemical staining). Therefore, many groups proposed radioiodine treatment for patients with breast cancer expressing the NIS (24,25). However, the in vivo imaging of NIS expression (131I or 99mTc scans) in the tumor cells did not correlate with NIS expression shown by messenger RNA levels and immunohistochemical staining. This apparently contradictory result is more likely to be due to the fact that the increased expression of the NIS on messenger RNA and protein levels is not translated into a functioning NIS located on the basolateral membrane. The latter is crucial for proper functioning of this symporter.

    There are few reports on the imaging of increased NIS expression in breast cancer xenografts and in brain metastases of breast cancer in mice. Kelkar et al. discussed a potential treatment for patients (25). However, no data analyzing this hypothesis in clinical settings have yet been published. The main shortcoming of the published in vivo data is that the role of tumor dosimetry—that is, an estimation of the absorbed doses delivered to tumors—was not analyzed. As described earlier, quantifying the uptake, effective half-life, and tumor mass is crucial for calculating the doses delivered to tumors; these data, in turn, predict the response to radioiodine.

    Figure 5 shows an estimation of the model-based absorbed doses for a spheric tumor as a function of 24-h 131I uptake per gram and at various effective half-lives. As shown in Figure 4, the 24-h 131I uptake per gram was approximately 0.16%/g after redifferentiation, and the effective half-life was estimated to be 1 d. As shown in Figure 5, the estimated absorbed dose was about 10 Gy/GBq, a value that was similar to the calculated one. This approach may suggest the extent to which the iodide uptake must be increased to achieve a tumoricidal absorbed dose.

    FIGURE 5.
    FIGURE 5.

    Model-based relationship between absorbed dose and actual 24-h 131I uptake per gram of tissue for 1-mL spheric lesion at different effective half-lives (in days) (shown close to straight lines); values within parentheses are estimated slopes (in Gy/GBq per unit percentage uptake per gram) for assessing absorbed doses beyond axis scale limit. Uptake curves decreased monoexponentially using the respective effective half-lives. For volumes ranging from 0.1 to 5 mL, absolute percentage absorbed dose deviations from 1-mL volume were less than or equal to 5%. Nonlinear relationship between slope and half-life resulted from extrapolation from 24-h uptake value to zero time point.

    CONCLUSION

    Theranostics with the matched pair 124I/131I in high-risk or progressive thyroid cancer enables an individualized dosimetry approach to delivering high absorbed doses to tumors and reducing radiation-related toxicity primarily to bone marrow. The target dose delivered through 131I depends on iodine uptake and effective half-life. In this context, NIS expression is critical for iodine uptake and a colloidal configuration with polarized thyrocytes for the effective half-life of radioiodine. Therefore, applying radioiodine isotopes to nonthyroidal tumor cells remains challenging, but individualized dosimetry at least facilitates proper analysis of the expected effectiveness.

    DISCLOSURE

    No potential conflict of interest relevant to this article was reported.

    The information comes from:
    http://jnm.snmjournals.org/content/58/Supplement_2/34S.long

  • Abstract

    CD147 is highly expressed in hepatocellular carcinoma (HCC) and associated with the invasion and metastasis of HCC. The efficacy of I131‐metuximab (I131‐mab), a newly developed agent that targets CD147, as a radio‐immunotherapy for local HCC, has been validated in clinical practice. However, the synergistic anticancer activity and molecular mechanism of different conjugated components within I131‐mab remain unclear. In this study, the cytological experiments proved that I131‐mab inhibited the proliferation and invasion of HCC cells. Mechanically, this inhibition effect was mainly mediated by the antibody component part of I131‐mab, which could reverse the epithelial–mesenchymal transition of HCC cells partially by suppressing the phosphorylation of VEGFR‐2. The inhibitory effect of I131 on HCC cell proliferation and invasion is limited, whereas, when combined with metuximab, I131 significantly enhanced the sensitivity of HCC cells to CD147‐mab and consequently reinforced the anticancer effects of CD147‐mab, suggesting that the two components of I131‐mab exerted synergistic anti‐HCC capability. Furthermore, the experiments using SMMC‐7721 human HCC xenografts in athymic nude mice showed that I131‐mab and CD147‐mab significantly inhibited the growth of xenograft tumors and that I131‐mab was more effective than CD147‐mab. In conclusion, our results elucidated the mechanism underlying the anti‐HCC effects of I131‐mab and provided a theoretical foundation for the clinical application of I131‐mab.

    1 INTRODUCTION

    Hepatocellular carcinoma (HCC) is a common malignant tumor that is commonly treated with surgery, minimally invasive treatment and transcatheter arterial chemoembolization (TACE) (Li & Yeo, 2017; Ramaswami et al., 2016). However, postoperative recurrence and metastasis remain important factors that affect the survival of patients with HCC (Grandhi et al., 2016; Yagci, Cetin, & Ercin, 2017). In particular, in HCC treated with chemotherapy or TACE, the residual cancer cells are found to be more invasive and metastatic (Su, 2016; Wan et al., 2016; Yu, Park, Park, & Yoon, 2016). Thus, further studying the genetic and biological properties of HCC is important to identify specific molecular markers of HCC recurrence and metastasis, develop new anti‐HCC agents and ultimately improve the quality of life of HCC patients.

    Immunotherapy is an important approach of comprehensive cancer therapy. Specifically, HCC cells strongly express CD147 antigen, which is related to the invasion and metastasis of HCC (Chen et al., 2016). Thus, CD147, a highly glycosylated transmembrane protein and a member of the immunoglobulin superfamily, is an effective target for HCC immunotherapy. CD147 expression is up‐regulated in many tumors and is especially high in HCC tissue and HCC cell lines, where it is expressed in up to 75%. Conversely, it is not expressed in normal hepatic tissue and normal hepatic cell lines (Dai et al., 2009; Gou et al., 2009). Moreover, the over‐expression of CD147 can induce the expression and secretion of matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9), which degrade extracellular matrix (ECM) and promote tumor invasion and metastasis by interfering with mesenchymal cells (Cui et al., 2012; Wang et al., 2010). To target CD147, a highly specific monoclonal antibody, HAB18 or metuximab, was developed and conjugated to the radioisotope I131. The resultant I131‐metuximab (I131‐mab) is a radio‐immunotherapy injection that was officially approved by the CFDA in 2007, being under the trade name Licartin for the treatment of local HCC (Ma & Wang, 2015; Wu, Shen, Xia, & Yang, 2016). I131‐mab exhibits several advantages, such as its specificity, rapid response and lethality to cancer cells. The preliminary results of a phase IV clinical trial of TACE combined with I131‐mab for the treatment of advanced HCC at eight Chinese institutions showed that I131‐mab is a safe and effective agent for the treatment of advanced HCC (Xu et al., 2007) and can effectively delay HCC relapse after hepatic transplantation (Wu et al., 2010, 2012). Moreover, the combination of TACE and I131‐mab can significantly prolong progression‐free survival and the overall survival of patients with advanced HCC according to the Barcelona clinical staging (BCLC) (Li et al., 2009). Among the 167 patients with HCC in this study, the 1‐year survival rate was significantly higher for patients receiving the combination treatment than that of patients only receiving the TACE treatment. The extrahepatic metastasis incidence in the combined treatment group was 2.94%, which was significantly lower than the 8.57% rate in the TACE group. This difference suggests that I131‐mab can reduce the extrahepatic metastasis incidence in patients after TACE treatment.

    The molecular mechanisms underlying the acceleration effect of CD147 on HCC invasion and metastasis and the synergistic inhibition effect of I131‐mab on HCC progression have been reported (Bian et al., 2014; Gou et al., 2016). Specifically, CD147 can activate the transforming growth factor‐β (TGF‐β) signaling pathway to promote the expression and secretion of MMPs in tumor cells and mesenchymal cells, which induces the epithelial–mesenchymal transition (EMT) in cancer cells and contributes to tumor invasion and metastasis (Ru, Wu, Chen, & Bian, 2015; Xu et al., 2007). Growing evidence shows that EMT plays an important role in the molecular mechanisms underlying HCC occurrence and development. During the process of EMT, cells of epithelial origin transform into mesenchymal cells under specific physiological and pathological conditions, and this transition is accompanied by a series of phenotypic and behavioral changes within cells (Jayachandran, Dhungel, & Steel, 2016). EMT is not only involved in embryonic development but also plays an important role in the pathology of many diseases, such as fibrosis and cancer (Nieto, Huang, Jackson, & Thiery, 2016). In cancer cells, EMT can decrease cell adhesion and increase cell motility, and these changes promote invasion, metastasis and the formation of a new tumor (Giannelli, Koudelkova, Dituri, & Mikulits, 2016; Polyak & Weinberg, 2009). I131‐mab specifically binds to CD147, which is located on the surface of HCC cells, to inhibit TGF‐β signaling and MMP secretion. This inhibition consequently interrupts EMT process and suppresses the invasion and metastasis of cancer cells.

    The efficacy of TACE in combination with I131‐mab has been confirmed, and the mechanisms underlying this effect have also been partially elucidated. However, multiple factors are involved in these mechanisms. Do the mechanisms interact? Is the effect synergistic? In addition to the I131‐mab‐mediated inhibition of TGF‐β signaling to interrupt EMT, are other signaling pathways involved in this process? Furthermore, studies are needed to answer these questions. The work described herein shows that I131‐mab can reverse the EMT of HCC cells, and metuximab, a component of I131‐mab, plays an important role in the reversion effect. I131 can increase sensitivity of HCC cells to metuximab to synergistically enhance the efficacy of treatment. Moreover, the mechanism by which I131‐mab reverses the EMT of HCC cells is related to VEGFR‐2 signaling. Overall, this work showed that I131‐mab may interrupt EMT of HCC by inhibiting VEGFR‐2 signaling to prevent HCC metastasis.

    2 RESULTS

    2.1 Inhibition of HCC cell proliferation by I131‐mab

    Western blotting was used to measure the expression levels of CD147 in a variety of cell lines, and CD147 was expressed in HepG2, Hep3B, SMMC‐7721, MHCC97H and MHCC97L cells, with HepG2, Hep3B and MHCC97H cells expressing higher levels of CD147. MHCC97L cells expressed the lowest level of CD147, and WRL‐68 cells were negative for the expression of CD147 (Figure 1a). Based on these results, we selected CD147‐negative WRL‐68 cells to investigate the cytotoxicity of I131‐mab and CD147 antibody (CD147‐mab). CD147‐mab did not affect cell viability, whereas I131‐mab exerted a dose‐dependent effect on cell viability. The IC20 and IC50 values of I131‐mab on WRL‐68 cells were 8.09 μCi/100 μl and 12.69 μCi/100 μl, respectively (Figure 1b). Because WRL‐68 cells did not express CD147 and were not inhibited by CD147‐mab, the cytotoxicity of I131‐mab was attributed to the conjugated I131. Because the unit of measurement of I131‐mab and I131 was μCi and the unit of measurement of CD147‐mab is μM, therefore, to establish the concentration equivalence relation between I131‐mab and CD147‐mab, we regarded that the concentrations of I131‐mab corresponding to IC20 and IC50 could be served as the reference concentrations of I131 and consequently calculated the molar concentrations of CD147‐mab to be 5.62 μM/100 μl and 8.82 μM/100 μl, respectively.

    image
    Inhibition of HCC cell proliferation by I131‐mab. (a) Experimental cell lines were seeded in a 24‐well plate (1 × 105 cells per well) and cultured for 48 hr. Western blotting was used to measure CD147 expression in the harvested cells. GAPDH was used as the internal control. (b) WRL‐68 cells were seeded in 96‐well plate (1 × 104 cells/100 μl per well) and cultured for 24 hr. I131‐mab and CD147‐mab were diluted in serum‐free medium. The cells were treated with 0, 1.0, 5.0, 10.0 and 15.0 μCi (per 100 μl) I131‐mab and 0, 2.5, 5.0, 7.5 and 10.0 μM (per 100 μl) CD147‐mab. After 2 hr of incubation, the medium was replaced with medium supplemented with 10% FBS (100 μl/well). After 48 hr of incubation, MTT reagent was added, and the cells were incubated overnight. The absorbance was measured at 490 nm, and cell viability curves were plotted. (c) HCC cells were seeded in a 96‐well plate (1 × 104 cells/100 μl per well) and cultured for 24 hr. I131‐mab (8.09 μCi/100 μl), I131 (12.69 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl or 8.82 μM/100 μl) were used to treat cells to examine the cell visibility as described above. Compared with the parental cell group (upper panel) or I131 group (lower panel): *< .05, **< .01, and ***< .001

    The tested cells were treated with I131‐mab, I131 (both agents administered at doses of 8.09 μCi/100 μl or 12.69 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl and 8.82 μM/100 μl corresponding to the concentration of I131‐mab) to examine their effects on the proliferation of HCC cells. The results showed that I131‐mab was significantly more cytotoxic to all HCC cell lines than CD147‐mab or I131 alone, and specifically, viability was lowest in HepG2, Hep3B and MHCC97H cells, and doses of 8.09 μCi/100 μl and 12.69 μCi/100 μl resulted in viabilities lower than 30% and 10%, respectively. In contrast, I131‐mab was weakly cytotoxic to MHCC97L, resulting in viabilities exceeding 50% at doses of 8.09 μCi/100 μl and 12.69 μCi/100 μl. Moreover, CD147‐mab was significantly more cytotoxic to HepG2, Hep3B, SMMC‐7721 and MHCC97H cells than I131, whereas the effects of I131 and CD147‐mab on MHCC97L cells did not significantly differ (Figure 1c).

    2.2 Inhibition of HCC cell invasion by I131‐mab

    To verify the effect of I131‐mab on HCC cell invasion, a Transwell device was used to assess cell invasion. Both I131‐mab and CD147‐mab significantly inhibited the invasion of the HCC cell lines. Specifically, I131‐mab inhibited HepG2, Hep3B, SMMC‐7721, MHCC97H and MHCC97L cell invasion more effectively than CD147‐mab, whereas the effects of CD147‐mab on the invasion of Hep3B and MHCC97L did not significantly differ from the control group. These results suggested that inhibition of HCC cells by I131‐mab or CD147‐mab was closely associated with CD147 expression. I131 alone exerted a limited effect on HCC cell invasion, and this effect only differed between the treated and control groups for MHCC97L cells (Figure 2).

    image
    Inhibition of HCC invasion by I131‐mab. The upper Transwell chambers were coated with 50 μl of 1:6 diluted Matrigel (BD Biosciences, San Jose, USA), and the lower chambers were filled with 500 μl of 10% fetal bovine serum, and the Transwell chambers were placed in a well of a 24‐well plate. HCC cells were seeded into the upper chamber of the Transwell (1 × 105 cells/well) and cultured for 24 hr; the cells were treated with I131‐mab, I131 (8.09 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl) were used to treat cells for 48 hr. The Transwell chambers were collected, and the cells were stained with 0.1% crystal violet for 20 min. Positive cells were counted. Compared with the control group: *< .05, **< .01 and ***< .001

    2.3 I131‐mab reversed HCC cell EMT via VEGFR‐2 signaling

    To elucidate the mechanism by which I131‐mab inhibits HCC cells, we compared some markers related to EMT in HCC cells treated with I131‐mab, I131 and CD147‐mab. E‐cadherin expression was significantly increased, whereas the expression levels of N‐cadherin and vimentin were decreased in all HCC cells treated with I131‐mab and CD147‐mab. Conversely, I131 did not affect the expression levels of these proteins (Figure 3a). These results suggested that I131‐mab reversed HCC cell EMT via the CD147 antibody in the conjugated molecules but not via I131.

    image
    Impact of I131‐mab on EMT and VEGFR‐2 expression in HCC cells. (a) HCC cells were seeded in 24‐well plates (1 × 105 cells/100 μl/well) and cultured for 24 hr. I131‐mab and CD147‐mab were diluted in serum‐free medium. The cells were then treated with I131‐mab, I131 (8.09 μCi/10 0 μl) or CD147‐mab (5.62 μM/100 μl) for 48 hr. The cells were harvested, total protein was extracted, and the protein concentration was measured. Western blotting was used to measure the expression of E‐cadherin, N‐cadherin and vimentin. GAPDH was used as a loading control. Compared with the parental cell group: *< .05, **< .01 and ***< .001. (b) Cells were cultured and treated as described above. Western blotting was used to measure the expression of VEGFR‐1, p‐VEGFR‐1, VEGFR‐2 and p‐VEGFR‐2. GAPDH was used as a loading control. Compared with the parental cell group: **< .01 and ***< .001

    VEGFR expression was further studied in HCC cells treated with I131‐mab, CD147‐mab and I131. The results showed that VEGFR‐2 phosphorylation (p‐VEGFR‐2) significantly decreased in HCC cells treated with I131‐mab and CD147‐mab, whereas the levels of VEGFR‐1, p‐VEGFR‐1 and VEGFR‐2 did not change. I131 alone did not affect the expression levels of these proteins in HCC cells (Figure 3b). These results suggest that I131‐mab reversed EMT by inhibiting the phosphorylation of VEGFR‐2 in HCC cells.

    2.4 I131 increases the sensitivity of HCC cells to the cytotoxicity of CD147‐mab

    By MTT assay, we found that I131 did not inhibit the proliferation of all HCC cells at doses <8.09 μCi/100 μl, whereas the cytotoxicity of 5.62 μM/100 μl CD147‐mab (concentration corresponding to the I131 dose) varied among HCC cell lines. We cotreated cells with I131 (8.09 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl) to compared the effect of this treatment to that of CD147‐mab alone. We found that the viability of all HCC cell lines significantly decreased in response to the combined treatment (Figure 4a). The results of cell invasion capability were coincident with the MTT assay (Figure 4b,c). The results suggested that I131 increases the sensitivity of HCC cells to CD147‐mab and consequently enhances the cytotoxicity of this agent.

    image
    I131 enhanced the inhibition of CD147‐mab on HCC cells. (a) HCC cells were seeded in 96‐well plates (1 × 104 cells/100 μl/well) and cultured for 24 hr. I131 (8.09 μCi/100 μl) and CD147‐mab (5.62 μM/100 μl) were combined to treat cells. The control group was treated with the same dose of I131 alone or CD147‐mab alone. The cell viabilities were carried out as described in Figure 1b. Compared with the parental cell group: #< .05 and ##< .01; compared with the parental cell group, the I131 group or the CD147‐mab group: ***< .001. (b) The transwell assay was carried out as described in Figure 2 to compare the capability of HCC cell invasion after treated with I131, CD147‐mab or combination of them. (c) The invasive cells were counted within five high‐magnification fields by three persons. Compared with the parental cell group: **< .01 and ***< .001

    2.5 Inhibition of SMMC‐7721 human hepatoma xenografts by I131‐mab

    The inhibition of HCC by I131‐mab and the molecular mechanisms underlying this inhibition were verified in nude mice harboring SMMC‐7721 human hepatoma xenografts. Treating these xenografts with I131‐mab injection markedly inhibited tumor growth speed; specifically, the tumor volume was significantly smaller in the I131‐mab treatment group than that in the control group 7 days after the first injection, and this difference increased over time. The efficacy of CD147‐mab group was evident 14 days after the start of treatment, whereas no noticeable effects were observed in the I131 treatment group, even at the end of the observation period (Figure 5a). After 21 days of treatment, the tumor volume in the control group exceeded the standard limit, and the observation was ended. The tumors were dissected and weighed, and the tumor weight was lowest in the I131‐mab group, followed by the CD147‐mab group. There is no difference in tumor weight between the I131 group and the control group (Figure 5b). Tissue sections were obtained from the hepatoma xenografts, and immunohistochemistry staining was used to measure E‐cadherin and p‐VEGFR‐2 expression. The results showed that E‐cadherin expression was up‐regulated, whereas p‐VEGFR‐2 expression was down‐regulated in the I131‐mab and CD147‐mab groups (Figure 5c).

    image
    Inhibition of SMMC‐7721 human hepatoma xenografts by I131‐mab. (a) SMMC‐7721 cell suspension (5 × 105 cells/100 μl per mouse) was injected subcutaneously into the right flanks of the nude mice. Tumors formed 12 days after inoculation. The mice were then randomly divided into four groups of five mice each (the I131‐mab group, CD147‐mab group, I131 group and control group). The injections were carried out every other day for 5 times. Each mouse in the I131 and I131‐mab groups received 12.69 μCi/100 μl I131 or I131‐mab, respectively, whereas each mouse in the CD147‐mab group received 8.82 μM/100 μl CD147‐mab. The control group mice were injected with saline (100 μl per mouse per injection). After treatment, tumor size was measured weekly, and the tumor volume was calculated with the following formula: “maximum diameter × minimum diameter2 × 0.5.” Compared with the control group: *< .05, **< .01 and ***< .001. (b) At the end of observation, the mice were killed, and the tumor specimens were harvested and weighed. Compared with the control group: **< .01 and ***< .001; (c) sections of hepatoma xenografts were obtained. The expression levels of E‐cadherin and p‐VEGFR‐2 were measured by immunohistochemical staining. Stained cells were counted in five high‐magnification fields, and the percentages of positive cells were determined. Compared with the control group: *< .05 and ***< .001

    3 DISCUSSION

    EMT has been observed in many human tumors, and extracellular signaling molecules that induce EMT in tumor cells include MMP‐2, MMP‐3, MMP‐9, type I or III collagen, hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF‐β and tumor necrosis factor‐α (TNF‐α). EMT also modulates the activity of multiple signaling pathways, such as TGF‐β, Wnt, PDGF, Notch, Hedgehog, Akt, PI3K, NF‐κB and Ras, via the Snail family of zinc finger transcription factors (Snail1, Snail2 and Snail3) and the helix‐loop‐helix structure of transcription factors (Twist, ZEB 1 and ZEB2/SIP1) to down‐regulate the epithelial markers E‐cadherin and keratin or up‐regulate the mesenchymal markers N‐cadherin and vimentin (Hanna & Shevde, 2016; Lee & Kong, 2016; Moustakas & Heldin, 2016; Xu, Yang, & Lu, 2015; Zhang, Tian, & Xing, 2016). EMT increases the malignancy of cancer cells because cells lose apical–basal polarity and cell junctions and acquire a migratory mesenchymal phenotype. These cells then invade the lymphatic or vascular system and spread to different sites or organs to grow and form metastatic tumors (Jayachandran et al., 2016; Kölbl, Jeschke, & Andergassen, 2016).

    TACE is an important treatment for HCC but has been clinically associated with increases in distant metastasis due to residual cancer cells in many patients with HCC (Xue et al., 2016). This phenomenon has greatly compromised the long‐term efficacy of TACE. Moreover, TACE can induce tissue hypoxia, which down‐regulates E‐cadherin and up‐regulates of N‐cadherin in residual cancer cells. Thus, hypoxia may mediate the negative effects of TACE by inducing EMT in cancer cells (Fransvea, Angelotti, Antonaci, & Giannelli, 2008; Xue et al., 2015). EMT enhances the invasion and metastasis of cancer cells, reduces the sensitivity or increases the resistance of HCC to chemotherapeutic agents (Bae et al., 2014; Nishida, Kitano, Sakurai, & Kudo, 2015) and contributes to the immune escape of HCC cells (Chockley & Keshamouni, 2016; Ye et al., 2016).

    The studies of the relationship between EMT and the metastasis of HCC have provided an effective target for HCC treatment. LY2109761, a TGF‐β 1 receptor kinase inhibitor, inhibits EMT to reduce vascular invasion and metastasis in HCC cells (Baldassarre et al., 2012). Blocking the effects of upstream loop regulatory factors of EMT, such as inhibition of NF‐κB/miR‐448, can improve the response of cancer cells to chemotherapeutic agents (Li et al., 2011). CD147, under the regulation of slug at transcriptional level, can promote EMT process in HCC via TGF‐β signaling (Wu et al., 2011). Furthermore, hypoxia caused by TACE can up‐regulate CD147 expression in HCC cells (Gou et al., 2016), and the over‐expression of CD147 enhances EMT in cancer cells by activating TGF‐β signaling. Thus, I131‐mab, which targets CD147, may specifically inhibit EMT to attenuate tumor development and metastasis. In nude mouse HCC models, I131‐mab effectively inhibited the growth and metastasis of HCC and inhibited MMPs and VEGF expression in the para‐tumor microenvironment (Wu et al., 2011). We conducted a prospective controlled clinical study in which the combination of TACE and I131‐mab was used to treat intermediate‐stage HCC (BCLC staging). Specifically, 68 patients with intermediate‐stage HCC were included in the combined treatment group, and 70 patients were included in the TACE alone group. The median survival time was 26.7 months in the combined treatment group but only 20.6 months in the control group, and the overall survival in the combined treatment group was significantly better than that in the control group (= .038). The median time to progression was 18.6 months in the combined treatment group and superior to only 12.5 months in the control group (= .046). In particular, the extrahepatic metastasis rate was 2.94% in the combined treatment group and 8.57% in the TACE group. These results suggested that I131‐mab reduces the risk of extrahepatic metastasis after TACE treatment (Wu et al., 2012).

    In this work, the cytological experiments proved that I131‐mab inhibits the proliferation and invasion of HCC cells, and this inhibition is closely related to the level of CD147 expression. HCC cells expressing high levels of CD147 are more sensitive to I131‐mab. The inhibition effect of I131‐mab on HCC cells was mostly attributed to the CD147 antibody within the conjugated molecule. Although the inhibition of HCC cell proliferation and invasion by I131 alone is limited, when combined with CD147‐mab, I131 significantly enhanced the sensitivity of cancer cells to CD147‐mab and consequently enhanced the cytotoxicity of anticancer antibodies. This finding suggested that the two components of I131‐mab synergistically inhibited HCC. Both I131‐mab and CD147‐mab can reverse the EMT of HCC cells partially by inhibiting the phosphorylation of VEGFR‐2 and then reduce the capabilities of proliferation and metastasis of HCC cells. The experiments using SMMC‐7721 human hepatoma xenografts in athymic nude mice also proved that I131‐mab and CD147‐mab significantly inhibited xenograft tumors, and I131‐mab was more effective than CD147‐mab in this inhibition. The synergistic effect of conjugated I131‐mab was attributed to I131 because I131 alone did not significantly inhibit the proliferation and invasion of HCC. Our results elucidated the mechanism underlying the antiproliferative and antimetastatic effects of I131‐mab and provided a theoretical foundation for the clinical application of I131‐mab.

    4 EXPERIMENTAL PROCEDURES

    4.1 Cells and reagents

    Human hepatoma cell lines (HepG2, Hep3B, SMMC‐7721, MHCC97H and MHCC97L) and normal hepatic cells (WRL‐68) were provided by Cell Bank, Institute of Cell Biochemistry, Chinese Academy of Sciences Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured according to the manufacturer's protocol. I131‐mab was developed by the Chengdu Huasun Group Inc., Ltd. (Chengdu, China). CD147‐mab and Sodium Iodide [131] Capsules (I131) were purchased from Abcam Trading Company Ltd. (Shanghai, China) and Atom‐Hitech Co., Ltd. (Beijing, China), respectively.

    4.2 Cell proliferation assay

    A tetrazolium colorimetric assay (MTT assay) was used to assess the effects of I131‐mab, CD147‐mab and I131 on the proliferation of HCC and normal cells. Briefly, cells were harvested during the logarithmic growth phase and seeded in 96‐well plates at 1 × 104 cells/100 μl/well, and then cultured for 24 hr; serum‐free culture medium was used to dilute I131‐mab, and cells were treated with various concentrations of this agent, with eight duplicates per concentration. After incubated for 2 hr, the medium was then replaced with medium containing 10% serum (100 μl/well). After 48 hr of culture, the medium was replaced with 0.1 M PBS solution (100 μl/well), and MTT labeling reagent (10 μl/well for a final concentration of 0.5 mg/ml; Roche Diagnostics GmbH, Shanghai, China) was then added. After 4 hr of incubation, the solubilization solution (10% SDS in 0.01 mol/L HCl, 100 μl/well) was added for overnight, the absorbance was measured at 490 nm, and the values were plotted to assess cell viability and calculate the IC20 and IC50 of each concentration of I131‐mab and its controls.

    4.3 Cell invasion assay

    The upper Transwell chambers (8 μm pore size, Corning, Tewksbury, USA) were coated with 50 μl of 1:6 diluted Matrigel (BD Biosciences, San Jose, USA), and the lower chambers were filled with 500 μl of 10% fetal bovine serum. The Transwell chambers were placed in a well of a 24‐well plate. Harvested cells were seeded into the upper Transwell chamber at 1 × 105 cells/well and cultured for 24 hr; I131‐mab was diluted in serum‐free culture medium and added to the wells for a variety of final concentrations. The cells were incubated for 2 hr, and the medium was replaced with medium containing 10% serum (100 μl/well) after 48 hr of incubation. The Transwell inserts were removed, and the cells were stained with 0.1% crystal violet for 20 min before being counted under a microscope; three random fields were photographed (200× magnification).

    4.4 Immunoblotting

    The above‐studied cells were seeded in a 24‐well plate at 1 × 105 cells/well and cultured for 24 hr. I131‐mab was diluted in serum‐free culture medium and added to the culture wells at various concentrations (based on the protocol). These cells were cultured for 2 hr. Medium containing 10% serum (100 μl/well) was used to replace the previous medium. After 48 hr of incubation, the cells were harvested. Western blotting was used to measure the expression of the target proteins. The antibodies used in the experiments are listed below: rabbit anti‐CD147 (Abcam Trading Company Ltd., Shanghai, China); mouse anti‐E‐cadherin, mouse anti‐N‐cadherin, mouse antivimentin, rabbit anti‐VEGF‐C antibodies (Cell Signaling Technology, Danvers, MA, USA); and mouse anti‐VEGFR‐1, mouse anti‐VEGFR‐2, rabbit anti‐phospho‐VEGFR‐1 and rabbit anti‐p‐VEGFR‐2 (Cell Applications Inc., CA, USA).

    4.5 Human hepatoma xenografts in nude mice

    A total of 25 healthy, purebred BALB/C male mice aged 4 weeks were purchased from the SLAC Experimental Animal Centre of the Chinese Academy of Sciences (Shanghai, China). SMMC‐7721 cells were harvested during the logarithmic growth phase, and cell suspension was prepared. The cells (5 × 105 cells in 100 μl per mouse) were subcutaneously injected into the right flanks of nude mice, and tumors formed 12 days after inoculation. The three mice with the largest tumors and the two mice with the smallest tumors were excluded. The remaining 20 mice were randomly divided into four groups (the I131‐mab group, the CD147‐mab group, the I131 group and the control group). Animals in the I131 and I131‐mab groups received injections of I131 or I131‐mab at multiple sites (dose: 12.69 μCi/100 μl per mouse); animals in the CD147‐mab group received CD147‐mab at a dose of 8.82 μM/100 μl per mouse. The animals were injected every other day for a total of five injections. The control mice were injected with saline (100 μl per mouse per injection). After treatment, tumor size was measured weekly, and the following formula was used to calculate tumor volume: “maximum diameter × minimum diameter2 × 0.5.” The experiment was terminated immediately when the mean tumor volume exceed 2,000 mm3 in any group, as defined by the Animal Ethics Committee of the Second Military Medical University. At the end of observation, the mice were killed with over‐doses anesthesia. The tumor specimens were harvested and weighed, and the tumor tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and immunohistochemically stained.

    4.6 Immunohistochemistry

    The sections of paraffin‐embedded SMMC‐7721 hepatoma xenografts were subjected to streptavidin–peroxidase immunohistochemistry to detect the expression of E‐cadherin and p‐VEGFR‐2. The antibodies used for staining were the same as those used for Western blotting. The percentages of positive cells in all sections were determined by counting the cells in five high‐magnification fields.

    4.7 Statistical analysis

    The cytological experiments were repeated three times independently, and a total of five animals were included in each group. The data are expressed as the mean ± SD. An analysis of variance (ANOVA) and a t test were used for the statistical analysis with the PASW Statistics 18 software. p value less than .05 was considered significant.

    ACKNOWLEDGMENTS

    This work was supported by the National Natural Science Foundation of China (81301302; 81773251; 81702735), and the Science and Technology Commission of Shanghai Municipality (15411966100).

      AUTHOR CONTRIBUTIONS

      L.W., B.S., X.L. and C.L. conceived the experiments. H.Q., L.C. and C.S. conducted the experiments. L.W., B.S., X.L., Y.Y. and F.S. analyzed the results. All authors reviewed the manuscript.

      The information comes from:
      https://onlinelibrary.wiley.com/doi/full/10.1111/gtc.12545

    1. Background: Rapid I-131 turnover Graves’ disease patients have low cure rate. We aimed to compare cure percentage at 12 months among 3 treatment doses of I-131 with or without lithium carbonate (LiCO3) in rapid turnover Graves’ disease patients.

      Methods: Sixty Graves’ disease patients referred for radioactive iodine treatment were randomised into three arms of treatment: Group A, 3.7 MBq I-131/g thyroid plus 600 mg/day LiCO3, Group B, 5.55 MBq I-131/g plus 600 mg/day LiCO3, and Group C, 7.4 MBq I-131/g without LiCO3. Data were collected at baseline, 3, 6, 9, and 12 months. The primary endpoint were cure rates (percentage of euthyroid or hypothyroid) at 12 months. Pairwise comparisons were made across 3 groups using an equality of proportions test. The secondary endpoint, the odds of cure over the total follow-up for group B and C versus group A, was analyzed using generalized estimating equation (GEE). Side effects of I-131 and LiCO3 treatment were evaluated at 1 to 2 weeks after treatment.

      Results: The cure rate at 12 months was 45% (9/20) for group A, 60% (12/20) for group B and 80% (16/20) for group C. The mean difference in proportion cured at 12 months between group C and group A was 35 (7.0 to 66.8)%; P-value = .02. There was a statistically significant difference between cure rates over all follow-up of group C and A after adjustment for sex (adjusted OR = 3.09; 95%CI = 1.32–7.20; P-value = .009), but no significant difference was found between group B and A or C and B in the primary and/or secondary efficacy endpoints. Side effects from the treatment were found in 12% (7/60); 2 in group A, 4 in group B, and 1 in group C. Four of these were likely due to LiCO3 side effects.

      Conclusions: Treatment of rapid turnover Graves’ disease patients with high dose I-131 (7.4 MBq/g) provides significantly higher cure rates at 12 months, and 3 times odds of cure than 3.7 MBq/g I-131 plus LiCO3 with lesser side effects. We thus recommend 7.4 MBq I-131/g for treatment in these patients.

      1 Introduction

      Graves’ disease in the most common cause of hyperthyroidism.[1] Among patients with Graves’ disease, about 15% have a rapid I-131 turnover (4/24 hours uptake ratio > 1),[2] which is associated with I-131 treatment failure of approximately 50% using a standard I-131 treatment dose of 3.70–5.55 MBq/g (100–150 microCi/g) of thyroid tissue.[3] Studies have shown that lithium carbonate (LiCO3) increases intrathyroidal iodine content by inhibiting the discharge of I-131 from the thyroid gland.[4] This results in increased I-131 resident time in the thyroid and thus may lead to increased cure rates when LiCO3 is used together with I-131. However, the role of using LiCO3 together with I-131 to enhance the cure rate in hyperthyroidism is still controversial and studies have shown conflicting results.[5–8] Although LiCO3 decreases rapid turnover in Graves’ disease patients,[9,10] the cure rate after a single dose of I-131 treatment with this regimen has not been extensively studied. Until now, there is no consensus for the optimal regimen to treat Graves’ disease with rapid turnover.

      The purpose of this study was to compare the effectiveness of treatment among standard dose 3.7 MBq/g (100 microCi/g) I-131 plus LiCO3, high dose 5.55 MBq/g (150 microCi/g) I-131 plus LiCO3 and high dose 7.4 MBq/g (200 microCi/g) I-131 in rapid turnover Graves’ disease patients.

      2 Material and methods

      2.1 Study design

      This was a parallel, 3-arm randomized trial, which the Graves’ disease patients with rapid I-131 turnover (4 hours uptake/24 hours uptake > 1) were randomised into three treatment groups by block of 3 with allocation concealment using envelopes in a box. After written informed consent was obtained, a nurse not involved in the study was assigned to blindly pick one envelope containing the sequence of treatment assignment, for example, A–B–C or A–C–B, etc. After knowing the sequence of treatment group, the nurse then informed the physician who treated the patient of the sequence of treatment for every 3 patients. Treatment in each group was assigned as follows: Group A, 3.7 MBq/g thyroid (100 microCi/g) I-131 plus 600 mg/day LiCO3 for 7 days after I-131 treatment; group B, 5.55 MBq/g (150 microCi/g) I-131 plus 600 mg/day LiCO3 for 7 days after I-131 treatment; and group C, high dose 7.4 MBq/g (200 microCi/g) without LiCO3.

      This study was approved by the institutional review board of the Faculty of Medicine, Chulalongkorn University (COA No. 253/2015). The study was registered in the Thai Clinical Trials Registry website before enrollment of the first patient at www.clinicaltrials.in.th (TCTR20151023001).

      2.2 Participants

      Sixty Graves’ disease patients with rapid I-131 turnover who were referred to the Nuclear Medicine division, Department of Radiology, King Chulalongkorn Memorial Hospital for radioactive iodine treatment between April 2015 and June 2016 were enrolled. The inclusion criteria were thyroid gland weight >50 grams and age >18 years. The exclusion criteria were previous treatment with I-131 or thyroid surgery, pregnancy or lactation.

      2.3 Procedure

      Thyroid uptake was performed 4 and 24 hours after 25 μCi I-131 oral administration using an Atomlab 950 thyroid uptake probe (Biodex, NY) counting at the thyroid for 60 seconds with photopeak of 364 keV ± 15% window. Thyroid scan was performed for 15–30 minutes, following intravenous injection of 74 to 185 MBq (2–5 mCi) of Tc-99m pertechnetate after finishing the thyroid uptake procedure on the second day. The image was acquired in the anterior view with a 256x256 matrix, LEHR collimator, photopeak 140 keV ± 20% window using an e.cam SPECT camera (Siemens, Erlangen, Germany). Thyroid gland size in grams was calculated using the Allen and Goodwin formula.[11] Actual dosage of I-131 treatment for each study group as stated above was then calculated using the formula: [12,13]

      2.4 Outcome measures and statistical analysis

      The cure rate (percentage of patients with euthyroid or hypothyroid status) in each group was evaluated at 3, 6, 9, and 12 months by monitoring thyroid gland size and thyroid function tests (FT3, FT4 and TSH). A repeat dose of I-131 treatment was allowed during the follow up period if the attending physician thought that there was no, or minimal response to I-131 treatment. In these patients, we defined their status as “not cured” throughout the remaining follow-up.

      Side effects of I-131 and LiCO3 treatment were evaluated by telephone interview, 1–2 weeks after treatment. Any new symptoms occurring after I-131 and/or LiCO3 treatment were recorded and defined as side effects. Other factors including age, sex, family history of hyperthyroidism, the duration of disease prior to radioactive iodine treatment, type and duration of treatment by antithyroid drug (ATD), thyroid gland size by thyroid scan were recorded in the study database.

      Statistical analyses were performed using Stata 15.1 (Statacorp, College Station, TX).[14] Statistically significance was set at P-values <.05. Sample size calculations were based on an improvement in the cure rates at 12 months in groups B or C versus group A. We assumed the cure rate in group A would be 45%. Enrolling 20 patients per group would allow us to assess a doubling of the cure rate to 90% in groups B or C at 80% power and a 2-sided significance level of 5%. Baseline categorical characteristics were reported as N (%) and continuous covariates were described as median (interquartile range, IQR). The primary endpoint was the proportion of patients cured at 12 months; pairwise comparisons of proportions cured were made across the 3 groups using an equality of proportions test, to calculate the difference in the cured proportions (95%CI). The primary analysis was intention to treat (ITT), where patients were analyzed according to their randomised arm, and missing data were imputed as failure (i.e., not cured). A secondary analysis excluded missing data.

      We also compared the odds of cure over the total follow-up as a secondary endpoint for group B and C versus group A as a reference group, using a generalized estimating equation (GEE) with a logit link and an exchangeable correlation matrix, to give population averaged estimates. Finally, as there was a sex imbalance among groups, we conducted a sensitivity analysis adjusting for sex.

      3 Results

      A total of 60 patients (12 males and 48 females) were recruited into this study and randomly assigned into 3 treatment groups. Baseline characteristics of each treatment group are shown in Table 1. The age ranged from 18 to 84 years (mean 34.4 ± 13.2 years). Most had medication failure (methimazole in 52 patients and propylthiouracil in 7 patients); one patient had never taken antithyroid drugs due to a drug allergy. The average duration of hyperthyroid therapy before I-131 ablation was 12 months. Thyroid gland size ranged from 50.1 to 288.2 grams (mean 96.2 ± 42.3 grams). The median of 4 to 24 hours thyroid uptake ratio was 1.07.

      Table 1

      Table 1

      The median I-131 treatment dose in group A was 351.5 MBq or 9.5 mCi (range 203.5–1,924 MBq or 5.5–52.0 mCi), group B was 610.5 MBq or 16.5 mCi (range 370–1,202.5 MBq or 10.0–32.5 mCi) and group C was 925 MBq or 25.0 mCi (range 444–1,813 MBq or 12.0–49.0 mCi). According to Regulatory Guide 8.39 of the Nuclear Regulatory Commission (NRC) of the United States, an assigned dose of I-131 as outpatient treatment should be limited to 1,221 MBq (33 mCi). Therefore, 4 patients where the calculated dose of I-131 exceeded this level (1 patient with 1,924 MBq (52 mCi) in group A and 3 patients with 1,369 MBq (37.0 mCi), 1,535,5 MBq (41.5 mCi) and 1,813 MBq (49.0 mCi) in group C) were treated by splitting the dose over 2 consecutive days (The first day treated with 1,110 MBq (30.0 mCi) and the remaining dose given on the second day).

      During follow-up, 6 patients received a second dose of I-131 due to no or minimal response to the first dose (1 in group A, 3 in group B and 2 in group C). Of these, 3 patients (1 in group A and 2 in group B) were cured after a second dose of treatment, but the remaining still had hyperthyroidism. One patient in Group C received a third dose of I-131 treatment and still had hyperthyroidism until 12 months. All of these patients were classified as cure failures over all follow-up in both ITT and available data analyses.

      One patient in group A did not attend a 9-month follow-up visit; In group C, there were 1 patient, 3 patients, 2 patients and 1 patient who failed to attend their follow-up visits at 3, 6, 9, and 12 months, respectively. Tables 2 and 3 show the number of patients (%) cured in each group at each study week in the ITT and available data population. Figure 1 shows cure percentage at each follow up visit, by treatment group, in the ITT population. A higher proportion of patients were cured in group B and C than group A at all time points.

      Table 2

      Table 2

      Table 3

      Table 3

      Figure 1

      Figure 1

      The primary endpoint analysis, which was the difference in cure percentage at 12 months, is shown in Table 4. A higher percentage of patients in group C were cured at 12 months compared to group A. The difference in percentage was 35 (95%CI 7.0–66.8)%; P = .02. The result was comparable in magnitude and statistical significance in the available data analysis. There were no differences in the cure proportions at 12 months in group B versus group A or group B versus group C in ITT or available data analyses.

      Table 4

      Table 4

      As a secondary endpoint, we analysed the odds of cure in groups B and C versus group A as a reference (Table 5). The data were presented in both unadjusted and adjusted for gender difference due to the imbalance in the number of males assigned to each treatment group.

      Table 5

      Table 5

      Patients in group C had 3.09 times higher odds of being cured at all points over follow-up compared to group A after adjusting for sex. Patients in group B also had higher odds of 2.18 of cure over all follow-up visits compared to group A, although the difference was not significant. Sex was not significantly associated with the outcome, and adjustment for sex had only a small impact on the odds ratios.

      There were 7 patients who had side effects after the treatments as shown in Table 6: 2 in group A, 4 in group B and 1 in group C.

      Table 6

      Table 6

      4 Discussion

      An additive effect of lithium on radioactive iodine for the treatment of hyperthyroid patients has shown conflicting results for cure in a meta-analysis.[15] In observational trials, the cure rate was significantly higher in the lithium with I-131 treatment groups. However, interventional trials have not shown any significant additive effect. This might be due to smaller amounts of I-131 given, and shorter duration of antithyroid drug cessation, in a large randomized control trial compared with smaller trials, thereby affecting the pooled effect size in the interventional trials analysis. Another possible explanation is that selection and other biases inherent in observational studies, have influenced the findings. Using lithium carbonate with 3.7 mBq/g thyroid of I-131 for the treatment of Graves’ disease patients with rapid turnover uptake is a standard practice at our institution. This is because LiCO3 is known to improve I-131 retention in the thyroid gland, and is thus suitable for hyperthyroid patients with rapid turnover uptake.[9] However, it was observed that even with this regimen, the cure rate was still unimpressive. This might be due to similarly low amount of I-131 to the large randomized controlled trial in the meta-analysis mentioned above.[6] Such amount of I-131 given to the rapid turnover Graves’ disease patients may not be enough to render cure even though LiCO3 is given. Thus, the role of LiCO3 is unclear for this amount level of I-131. Factors leading to treatment failure are thyroid volume greater than 50 mL and 4/24-hour uptake ratio 0.8 or greater.[16] All of the patients in our study had thyroid gland size ≥50 grams, and all had a 4/24-hour uptake ratio of ≥1, factors known to be associated with high failure rates. The usual regimen for LiCO3 treatment in our institution is prescribing LiCO3 after I-131 treatment. I-131 is given in the late morning, then the patient is instructed to take first pill of LiCO3 after lunch on the day of treatment. We chose this regimen because many of our patients are sent from other hospitals and it is not always convenient to start LiCO3 before I-131 treatment. Furthermore, evidence suggests that prescribing LiCO3 after I-131 treatment produces similarly good outcomes to the pretreatment regimen.[8,17] This may be due to the fact that the peak serum concentration of LiCO3 is reached 0.5–2 hours after ingestion.

      The design of this study was different from previous studies in that we compared standard I-131 dose (3.7 MBq/g thyroid) plus LiCO3 with the other two groups that we designed to give higher I-131 doses. One group with slightly higher dose plus LiCO3 and another with even higher dose without LiCO3, hypothesizing we would see higher cure rates in the latter two groups. The cure rate of patients at 12 months in group C who received the highest dose of I-131 of 7.4 MBq/g thyroid without LiCO3, was significantly higher than those receiving 3.7 MBq/g plus LiCO3 (group A). This was in line with the study by de Jong et al[16] who reported more cures in those receiving 7.4 MBq/ml when compared with those receiving 3.7 MBq/ml, but without prescribing LiCO3. We found that although we added LiCO3 to the 3.7 MBq/g group, there was still a significantly higher percentage of cures in the 7.4 MBq/mL group. The rate of cure in group B at 3 to 6 months after I-131 treatment was equal to group C, but the rate decreased at later time points (Fig. 1). This resulted in the non-significant difference of cure rate in group B when compared to group A. Therefore the dose of 1.5 times standard dose of I-131 plus LiCO3 (group B) may not be adequate to render enough cure even though Pusuwan et al[18] showed treatment with 5.5 MBq/g led to more cures than 3.7 MBq/g (70% vs 52%). The cure rate of group B in our study, which was 60% at 1 year, is less than that of Pusuwan's study. This may be because all patients in our study were Graves’ disease patients with rapid turnover, which is known to have a high treatment failure rate.[3] The decrease in the group B cure rate at 9 months and 12 months is likely due to transient hypothyroid at 3 to 6 months post I-131 treatment.[19,20] The mechanism underlying transient hypothyrodism is still unclear. A number of possibilities have been suggested, including transient hypofunction of hypothalamic-pituitary axis, depletion of thyroidal iodine storage by pretreatment antithyroid drugs, immunogenic (a shift between TSH receptor blocking and stimulating antibodies), or transient hibernation.[20] This effect was also seen in group A with an almost parallel curve to group B, though the cure rate was higher in group B (Fig. 1). The effect of transient hypothyroid was less in group C, probably due to the more permanent damage to the thyroid cells when compared to the other 2 groups.

      Although group C did not show a statistically significant difference in cures compared to group B, the high rate of side effects of LiCO3 in this study found in groups A and B (Table 6) cannot be ignored. The reported side effect and toxicities of lithium [21] encompass tremor, nausea, vomiting, and diarrhea which are common in early treatment phases. Thirst, excessive urination, and weight gain are, in contrast, commonly found after long-term treatment. Cognitive impairment, dermatological effects (acne and psoriasis) seem to be dose related. There are long term effects on renal, thyroid and parathyroid function. Other than these side effects, fatigue, irritability and palpitation were also reported.[22] Mild lithium toxicity includes weakness, tremor, mild ataxia, poor concentration and diarrhea. The more toxic effect results in vomiting, gross tremor, slurred speech, confusion and lethargy. Some of these toxicities overlap with lithium side effects. A large interventional trial showed that approximately 10% of patients receiving lithium experienced mild to moderate side effects.[6] In our study, thirst was likely caused by LiCO3. Nausea and fatigue could be caused by either I-131 or LiCO3, while rash was thought to be associated with LiCO3.[23] Sialadenitis that occurred in a patient who received 29.5 mCi of I-131 was obviously due to I-131 treatment.[24] Irritability, palpitations, sweating and auditory hallucinations experienced by a patient in group B was more likely to be caused by I-131 than LiCO3. This is because the half-life of LiCO3 is 18–24 hours, thus any symptoms occurred after 4 to 6 half-lives is less likely to be caused by lithium. Symptoms in this patient which occurred after 14 days of I-131 treatment, may be due to more thyroid hormone release into the blood stream after I-131 treatment. The symptoms of so-called thyrotoxic psychosis, as previously reported.[25]

      From Table 1, all baseline characteristics are balanced due to the randomised nature of this study, except for a higher proportion of females in groups A and B versus group C. In a sensitivity analysis, we performed an adjusted analysis of our secondary endpoint corrected for sex, but there was no important impact on the result (Table 5). In an ITT population, group C had 2.85 times of odds of cure versus group A in nongender adjusted model and 3.09 times in sex-adjusted model. When analyzed using available data only, there was 3.22 and 3.62 times of odds of cure comparing group C and A in the nonadjusted and gender-adjusted model, respectively.

      There were 4 cases (group A and group C) that the treatment was divided into two consecutive days due to the calculated I-131 dose of more than 1,221 MBq (33.0 mCi). Previous studies showed that the thyroid gland can still take up I-131 without a stunning effect, when the first and second doses of I-131 are given up to 48 hours apart. If the second dose I-131 is given after 72 hours, the ability to capture I-131 drops dramatically (a phenomenon known as thyroid stunning).[26,27] Based on these studies, 2 patients in group C who received 1,535.5 MBq (41.5 mCi) and 1,813 MBq (49.0 mCi) were found to have cure at 12 months. Another 2 patients, 1 in group A (1,924 MBq or 52.0 mCi) and 1 in group C (1,369 MBq or 37.0 mCi), were not cured.

      There are a number of limitations of this study. First, our study was small with limited power to discern if there was a difference in efficacy between groups B and C, and therefore we recommend additional larger studies examine this issue. Second, some side effects of I-131 or LiCO3 can also be caused by other health conditions, for example nausea/vomiting can be due to gastritis or other underlying gastrointestinal conditions, and auditory hallucination can be caused by psychosis. To mitigate the risk of wrongly attributing these adverse events to a study drug, we checked that the symptoms were new and only occurred after study drug administration.

      In conclusion, treatment of rapid turnover Graves’ disease patients with high dose (7.4 MBq/g or 200 microCi/g) I-131 has high cure rate at 12 months (80%) with lower side effects when compared to other groups. It provides approximately 3 times higher odds of cure over follow-up than those using 3.7 MBq/g or 100 microCi/g I-131 plus LiCO3. We thus recommend this treatment regimen in Graves’ disease patients with rapid turnover uptake.

      The information comes from:
      https://journals.lww.com/md-journal/Fulltext/2019/05100/Finding_the_best_effective_way_of_treatment_for.83.aspx

    2. Background

      Although the use of Sr-89 chloride in the treatment of patients with prostate and breast cancer has been widely reported, little information is available about its use for other malignancies. Here, we retrospectively analyzed the clinical profile of Sr-89 chloride in various patients with painful bone metastases.

      Methods

      Entry criteria were a pathologically proven malignancy, clinically diagnosed multiple bone metastases, and adequate organ function. Sr-89 chloride (Metastron) was given by single intravenous infusion at 2 MBq/kg over 2 min. Self-reported outcome measures were used as a response index, including pain diary data on a 0–10 numeric rating scale (NRS).

      Results

      Fifty-four consecutive patients with painful bone metastases were treated with Sr-89 chloride at the National Cancer Center Hospital East between March 2009 and July 2011, consisting of 26 with breast/prostate cancer and 28 with other malignancies (lung 8, head and neck 6, colorectal 6, others 8). Thirteen (24 %) patients experienced a transient increase in pain, which was categorized as a flare-up response. Grade 3–4 anemia was observed in 6 patients, 3 of whom required blood transfusion. Regarding efficacy, response rates and complete response rates were 71.2 % and 34.6 %, respectively, and time to response from the initiation of treatment was 36 days (range, 13–217). No significant difference in response rates was seen between patients with breast/prostate cancer and other cancers (breast/prostate 69.2 %, other 73.1 %; p = 0.76).

      The information comes from:
      https://link.springer.com/article/10.1007%2Fs10147-013-0597-7

    3. Abstract


      Objective: The aim of this study was to comparatively evaluate the efficacy of strontium-89 chloride (89 SrCl2) in treating bone metastasis-associated pain in patients with lung, breast, or prostate cancer.
      Materials and Methods: The 126 patients with lung cancer included 88, 16, 15, 4, and 3 patients with adenocarcinoma, squamous cell carcinoma, nonsmall cell carcinoma, mixed carcinoma, and small cell carcinoma, respectively, and the control group consisted of patients with breast (71 patients) or prostate cancer (49 patients) who underwent 89 SrCl2 treatment during the same period. The treatment dose of 89 SrCl2 was 2.22 MBq/kg.
      Results: The efficacy rate of treatment in the lung cancer group was 75.4%, compared to 95.0% in the control group. Approximately 67% of patients with lung cancer and bone metastases and 47% of control patients exhibited mild-to-moderate reductions of leukocyte and platelet counts 4 weeks after 89 SrCl2 treatment.
      Conclusions: 89 SrCl2 can safely and effectively relieve bone pain caused by bone metastasis from lung cancer. However, its efficacy was lower in patients with lung cancer with bone metastasis than in those with breast or prostate cancer with bone metastasis, and its effects on the peripheral hemogram were also significantly stronger in the lung cancer group.

      Keywords: Bone metastasis, lung cancer, strontium-89 chloride

      How to cite this article:
      Ye X, Sun D, Lou C. Comparison of the efficacy of strontium-89 chloride in treating bone metastasis of lung, breast, and prostate cancers. J Can Res Ther 2018;14, Suppl S1:36-40

      How to cite this URL:
      Ye X, Sun D, Lou C. Comparison of the efficacy of strontium-89 chloride in treating bone metastasis of lung, breast, and prostate cancers. J Can Res Ther [serial online] 2018 [cited 2019 May 17];14:36-40. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/36/181172


      Introduction


      More than 50% of patients with cancer experience bone pain, and breast-, lung-, and prostate cancer-associated metastatic bone diseases account for 80% of cases of bone pain among cancer patients.[1] Bone metastasis can cause unbearable intractable pain, leading to reductions in quality of life. Strontium-89 chloride (89 SrCl2) is a radiopharmaceutical of palliative care used to treat metastatic bone cancer-associated pain.[2],[3] The agent can effectively kill tumor cells, induce tumor cell apoptosis,[4],[5] and restore patients' immune functions.[6]89 SrCl2 is the palliative care mainly used to treat pain caused by the bone metastasis of prostate and breast cancers.[7],[8] The treatment is considered optimal for this indication,[9],[10] and in some cases, metastases might subside.[11] Lung cancer is currently the malignant tumor with the highest incidence and mortality in China,[12] and as its incidence continues to increase, research on the application of 89 SrCl2 to treat the bone metastasis of lung cancer is also increasing, although studies have reported varying effects. To further investigate the clinical application value and characteristics of 89 SrCl2 in treating pain caused by the bone metastasis of lung cancer, we followed up patients with lung cancer and bone metastasis who underwent 89 SrCl2 treatment and compared the treatment outcomes with those of patients with prostate or breast cancer and bone metastasis. Meanwhile, the related literature was reviewed to analyze the possible factors that might affect treatment efficacy.


      Materials and methods



      Patient information

      Patients with lung cancer, prostatic cancer, and breast cancer, who were treated by 89 SrCl2 in Shao Yifu Affiliated Hospital and the Second Affiliated Hospital of School of Medicine in Zhejiang University were collected from 2009 to 2013. Patients were excluded if they had not bone X-ray films, computed tomography/magnetic resonance imaging (CT/MRI), or pathological examination had diagnosed with extensive metastatic bone tumors, or survival time was <3 months after treated by 89 SrCl2. A total of 246 patients were obtained, and all of these patients had been signed informed consents before treatment. Among the 126 patients with lung cancer, 88 were male, and 38 were female. The patients, including 88, 16, 15, 4, and 3 patients with adenocarcinoma, squamous cell carcinoma, nonsmall cell carcinoma, mixed carcinoma, and small cell carcinoma, respectively, were 40–81 years old. The control group included 120 patients who underwent 89 SrCl2 treatment during the same period, including 71 patients with breast cancer and 49 patients with prostate cancer. The age of the control group ranged from 36 to 79 years, and the duration of primary diseases varied from newly diagnosed to 5 years. All patients were confirmed to have an extensive metastatic bone tumor by bone scintigraphy, radiography, CT/MRI, or pathological examination. The bone metastatic lesions were observed as abnormal radioactive concentrated shadows (osteoblastic lesions) by bone scintigraphy. The patients had various degrees of bone pain, with or without activity limitation, and some patients had experienced treatment failure after radiotherapy and/or chemotherapy at least 2 months before 89 SrCl2 treatment. The examination results for hepatonephric function were normal for all patients, and there were no cases of spinal pathological fracture or spinal cord compression. Each patient had a leukocyte count of >3.5 × 109/L and a platelet count of >80 × 109/L.

      Treatment

      The treatment medicine (89 SrCl2) was provided by Shanghai Kexing Co., China. The therapy was intravenously administrated at a dosage of 1.48–2.22 MBq (40–60 μCi)/kg body weight. The detailed information regarding the patients' body weights, the drug dosages administrated, the batch numbers used, and possible side effects or discomforts experienced during drug administration was recorded.

      Follow-up and therapeutic evaluation

      The follow-up period after 89 SrCl2 treatment in the two groups ranged from 3 months to 4 years. In addition to regular hemogram analysis, the patients were mainly observed for improvements in pain, sleep, and activities to determine the efficacy of treatment. According to World Health Organization (WHO), the grades of pain includes O grade, no pain; I grade, mild pain, is intermittent pain, and drug may be not used; II grade, moderate pain, is continuous pain and interfere with good rest, and paregoric is needed; III grade, severe pain, is continuous pain and pain is not relieved if drug is not used; IV grade, very severe pain, is continuous sharp pain with changes of blood pressure and pulse. The evaluation of therapeutic efficiency of 89 SrCl2 treatment includes (1) Complete remission (CR), entirely painless after treatment; (2) partial remission (PR), pain is obviously relieved, and patient can live a normal life and sleep is not basically interfered; (3) mild remission (MR), pain is relieved but patient still feels pain, and sleep is interfered; (4) invalid, pain is not relieved than before treatment. Of these, CR + PR + MR is believe as effective.

      Hematologic toxic reactions

      The patients' hematologic toxic reactions were judged with reference to the indexing criteria (adult) of WHO hematologic acute and subacute toxic reactions [Table 1]. Before treatment, some patients' leukocyte counts were 3.5 × 109–3.9 × 109/L, and their platelet counts were 80 × 109–99 × 109/L. If the leukocyte count remained at >3.5 × 109/L, and the platelet count remained at >80 × 109/L within 3 months after 89 SrCl2 treatment, a grade of 0, indicating no change, was assigned. Meanwhile, if the leukocyte count declined to (3 × 109–3.4 × 109/L and the platelet count fell to (75 × 109–79 × 109/L, a grade of I was given.

      Table 1: Indexing criteria of hematologic toxic reactions and follow up results after the 89Sr treatment

      Click here to view



      Statistical analysis

      The Chi-square test was used to compare treatment efficacy between the two groups using SPSS 19.0 software, IBM company (Armonk, New york, USA). P < 0.05 was considered to indicate a statistically significant difference.

      Results



      Efficacy analysis

      Among the 126 patients in the lung cancer group, the efficacy of treatment was graded as aggravated, invalid, effective, and significantly effective in 0, 31 (24.6%), 79 (63.0%), and 16 patients (12.7%), respectively, resulting in an efficacy rate of 75.4%. According to the type of cancer, treatment was effective in 60 patients with adenocarcinoma (68.2%), 8 patients with squamous cell carcinoma (50.0%), 9 patients with nonsmall cell carcinoma (60.0%), and 2 patients with mixed cancer. Meanwhile, treatment was significantly effective in 13 patients with adenocarcinoma (14.8%), 2 patients with squamous cell carcinoma (12.5%), and 1 patient with mixed cancer. Comparatively, among the 120 patients with breast or prostate cancer, the efficacy of treatment was graded as invalid, effective, and significantly effective in 6 (5.0%), 66 (55.0%), and 48 patients (40.0%), respectively, resulting in an efficacy rate of 95.0%. The differences in efficacy between the two groups were statistically significant (P < 0.05).

      The analgesic effects (efficacy) experienced by most patients with lung cancer and bone metastasis occurred 1–2 weeks after 89 SrCl2 treatment. Meanwhile, the earliest effects were observed 24 h after the injection, and the latest effect occurred 46 days after treatment. The duration of efficacy of a single injection varied from 56 days to 9 months (most commonly 3–6 months). Treatment was repeated a maximum of 3 times, and the longest duration of efficacy was more than 3 years. Efficacy was observed between 1 and 33 days in the control group (most commonly 3–10 days), and the duration of efficacy ranged from 3 to 10 months (most commonly 4–10 months). Treatment was repeated a maximum of 6 times, and the longest duration of efficacy following a single injection was 4.5 years.

      Toxic reactions

      Approximately 67% of patients with lung cancer and bone metastasis exhibited mild-to-moderate reductions of their leukocyte and platelet counts 3–4 weeks after 89 SrCl2 treatment, although leukocyte and platelet counts returned to normal or pretreatment levels within 3–12 months. The remaining patients in the lung cancer group exhibited no significant changes, or a slight decline of the peripheral hemogram leukocyte count following treatment, with the value remaining in the normal range. Concerning the control group, approximately 45% of patients exhibited mild-to-moderate reductions of their leukocyte and platelet counts 4 weeks after 89 SrCl2 treatment, and their leukocyte and platelet counts returned to normal or pretreatment levels within 3–9 months [Table 2].

      Table 2: Follow up results of hematologic toxic reactions and follow up results after the 89Sr treatment

      Click here to view



      Neither group exhibited allergic reactions and side effects such as rash, proteinuria, and hematuria during 89 SrCl2 treatment, whereas approximately 2% of patients experienced a mild fever or gastrointestinal reactions after 89 SrCl2 treatment.


      Discussion



      89 SrCl2 had a relatively long half-life (50.6 days), and 90 days after the injection, the amount of the compound retained inside the bone metastatic lesion ranged from 20% to 88%. Consequently, the efficacy of therapy could be maintained, thus providing more extensive and long-lasting pain relief, improving patients' quality of life,[13] and increasing their survival time.[14]

      89 SrCl2 was an effective treatment for the bone pain of patients with prostate or breast cancer and bone metastasis.[15] In this study, among the 120 patients in the control group, 114 experienced significant analgesic effects after 89 SrCl2 treatment, and the total efficacy rate was 95.0%.89 SrCl2 could safely and effectively relieve the bone pain caused by the bone metastasis of lung cancer, prolong patients' survival time, and reduce the annual incident rate of skeletal-related events.[16] However, the efficacies reported by most domestic and foreign studies varied from 46.2% to 96.8%,[17] and the differences were relatively large, which might be related to the differences in the pathological compositions and efficacy criteria. Among the patients with lung cancer, adenocarcinoma had the highest incidence of bone metastasis, followed by squamous cell carcinoma and small cell lung cancer, which was consistent with the finding in this study that adenocarcinoma most commonly resulted in bone metastasis. The efficacy of 89 SrCl2 against the bone metastatic pain of lung adenocarcinoma was superior to that against metastatic squamous cell carcinoma because the blood supply inside adenocarcinoma is richer than that inside squamous carcinoma; thus, the blood supply inside the metastatic lesion might also be abundant. Meanwhile, the affinity of 89 SrCl2 for bone resulted in its substantial accumulation inside the blood supply-rich bone metastatic lesions of adenocarcinoma and stronger cytotoxic effects.

      The efficacy rate of 89 SrCl2 treatment in the lung cancer group was 75.4%, lower than that of the control group, and the effects of the therapy on the peripheral hemogram in the lung cancer group were also significantly greater than those observed in the control group. A number of factors may have affected the efficacy and hematologic toxicities of 89 SrCl2 in treating bone metastasis from lung cancer. First, the mechanism of relieved pain using 89 SrCl2 in patients with osseous metastasis remains unclear. Autoradiography of bone slices of patients following 89 SrCl2 treatment confirmed that the compound was obviously deposited and retained in the sites with active osteoblastic cells near the metastatic lesions.[18],[19] Therefore,89 SrCl2 may have exhibited better efficacy against osteoblastic metastatic lesions.[3],[20] Bone metastasis from lung cancer was mainly osteolytic, whereas that from prostate cancer was mainly osteoblastic. The characteristics of bone metastasis from breast cancer were intermediate between those of bone metastasis from lung cancer and prostate cancer. Bone metastasis from lung cancer arose primarily via bone resorption caused by osteoclasts, mostly resulting in osteolytic lesions. After lung cancer cells were transferred to the bones, they released soluble mediators and activated osteoclasts and osteoblasts. The osteoclast-released cytokines further promoted the tumor cells to secrete the bone dissolution medium, thus forming a vicious cycle.89 SrCl2 could play a therapeutic role against osteoblastic metastasis, but it effects against osteolytic metastasis were limited. Second, the primary treatment of breast cancer is surgical removal, whereas prostate cancer is generally treated via castration therapy. Systematic chemotherapy and radiotherapy are rarely applied in the treatment of either malignancy. However, the primary treatment strategy for lung cancer is surgical resection and postoperative chemotherapy and/or radiotherapy. At the time of diagnosis, many patients were no longer eligible for surgery, and they could only undergo chemotherapy. Therefore, most patients with lung cancer and bone metastasis underwent multiple rounds of chemotherapy and/or radiotherapy prior to 89 SrCl2 treatment. The efficacy of chemotherapy against lung cancer was not ideal, and the side effects were significant, which could damage the peripheral hemogram, significantly reduce systemic immune functions, and increase disease resistance. The application of 89 SrCl2 treatment at this time point might both affect the efficacy of treatment and more strongly suppress bone marrow function. Based on our experience, medications could be administered first for treatment and conditioning, after which the patients' condition could be permitted to stabilize for 2–3 months prior to 89 SrCl2 treatment. Caution should be exercised for patients whose hemogram was extremely suppressed by recently administered chemotherapy, or 89 SrCl2 treatment should be delayed. If 89 SrCl2 therapy was necessary, then supportive therapy to maintain bone marrow functioning might be considered at the same time to avoid or mitigate hematologic toxic reactions. Third, the severity of disease among patients with lung cancer was generally greater than that among patients with prostate or breast cancer, and the possibility of an early diagnosis was lower. The data illustrated that 80–85% of patients with lung cancer patients had advanced disease during the first treatment, including distant metastasis, which was normally accompanied by systematic dysfunction. The survival period of the lung cancer group was shorter, the 5-year survival rate was low, and the sensitivity and tolerance toward 89 SrCl2 were poor, thus limiting the therapeutic effects of 89 SrCl2 treatment. Among the patients who underwent 89 SrCl2 treatment but did not survive for more than 3 months, more than two-thirds of these patients had bone metastasis from lung cancer, whereas all treated patients with breast or prostate cancer survived for more than 3 months. As the efficacy of 89 SrCl2 treatment is limited among patients with advanced cancer, Yamaguchi emphasized that the indication of 89 SrCl2 should be changed from advanced disease to early-stage disease.[21] Fourth, patients with lung cancer and bone metastasis may also experience metastasis to other organs, and thus, the pain they experienced might not be simply caused by bone metastasis. In addition,89 SrCl2 was ineffective against pain caused by potential ectosteal factors. The pain of some patients might be caused by other benign bone lesions (such as fractures, gout, and hypertrophic pulmonary osteoarthropathy), and the efficacy of 89 SrCl2 against pain caused by these lesions has not been clarified. Finally, some patients with lung cancer and bone metastasis in this study received long-term treatment with morphine analgesia before 89 SrCl2 treatment and exhibited addiction that could not be alleviated after 89 SrCl2 treatment. This finding may also explain the “unsatisfactory efficacy” experienced by some patients.


      Conclusions


      Although the efficacy of 89 SrCl2 treatment against bone pain caused by the bone metastasis of lung cancer was significantly lower than the that observed for breast and prostate cancer, and the effects of treatment on the peripheral hemogram were significantly better in the lung cancer group, we believe that if patients can be carefully chosen according to their pathological types and treatment situations,89 SrCl2 could have greater safety and efficacy in relieving bone pain caused by bone metastasis.

      The information comes from:
      http://www.cancerjournal.net/article.asp?issn=0973-1482;year=2018;volume=14;issue=8;spage=36;epage=40;aulast=Ye

    4. Abstract

      The inverse electron-demand Diels-Alder reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooct-2-ene (TCO) has gained increasing attraction among extensive studies on click chemistry due to its exceptionally fast reaction kinetics and high selectivity for in vivo pretargeting applications including PET imaging. The facile two-step approach utilizing TCO-modified antibodies as targeting structures has not made it into clinics yet. An increase in the blood volume of humans in comparison to mice seems to be the major limitation. This study aims to show if the design of multimeric Tz-ligands by chelator scaffolding can improve the binding capacity and may lead to enhanced PET imaging with gallium-68. We utilized for this purpose the macrocyclic siderophore Fusarinine C (FSC) which allows conjugation of up to three Tz-residues due to three primary amines available for site specific modification. The resulting mono- di- and trimeric conjugates were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, stability, binding towards TCO modified rituximab (RTX)) and in vivo (biodistribution- and imaging studies in normal BALB/c mice using a simplified RTX-TCO tumour surrogate). The 68Ga-labelled FSC-based Tz-ligands showed suitable hydrophilicity, high stability and high targeting specificity. The binding capacity to RTX-TCO was increased according to the grade of multimerization. Corresponding in vivo studies showed a multimerization typical profile but generally suitable pharmacokinetics with low accumulation in non-targeted tissue. Imaging studies in RTX-TCO tumour surrogate bearing BALB/c mice confirmed this trend and revealed improved targeting by multimerization as increased accumulation in RTX-TCO positive tissue was observed.

      Keywords:pretargeting, Fusarinine C, rituximab, click chemistry, multimerization, PET, gallium-68

      1. Introduction

      Immunoglobulins, in particular monoclonal antibodies (mAbs) are highly attractive targeting structures due to their extraordinary specificity and selectivity and are well established for therapeutic applications, particularly in the field of oncology [,]. Because of their favorable targeting abilities and therefore an ever increasing clinical importance, mAbs have regained interest also as imaging agents, in particular for positron emission tomography (PET) applications [,,]. In principle mAbs can be directly radiolabelled either by direct incorporation of radiohalogens or by attaching a chelator to the protein in case of radiometals. Prolonged circulation time and slow distribution within the organism, however, restricts its use to long-lived radionuclides e.g., zirconium-89 (3.26 d) and iodine-124 (4.18 d). This multiday circulation paired with slow radioactive decay provides unfavorable radiation to healthy tissue and adds significantly to the overall radiation burden of patients.

      In order to overcome this problem, various pretargeting methodologies have been reported and reviewed recently [,,], enabling a straight forward two-step approach. Thereby the modified antibody is administered, allowed to accumulate at the target site and be eliminated from the bloodstream followed by injection of the radioactive payload to form the radioimmunoconjugate in vivo. This provides certain advantages as it facilitates the use of short-lived radioisotopes for PET applications e.g., gallium-68 (1.13 h), fluorine-18 (1.83 h) and copper-64 (12.7 h) and significantly reduces the radiation dose to healthy tissue, since the radioligand either finds its binding partner to form stable conjugates or is rapidly eliminated due to its small size. Furthermore, it allows to apply radiolabelling at high temperatures using high concentrations of organic solvents if necessary—harsh conditions, where the structural integrity of antibodies would be severely in danger when direct labelling was performed.

      Among these pretargeting strategies the inverse electron-demand Diels-Alder reaction (IEDDA) between 1,2,4,5-tetrazines (Tz) and trans-cyclooct-2-enes (TCO) has gained enormous attention mainly due to the exceptionally fast reaction kinetics and high selectivity between the reaction partners even in complex biological systems as encountered in vivo [,]. Various preclinical studies demonstrated the feasibility of this approach for molecular imaging using PET-radioisotopes with promising results [,,,]. The application on humans, however, remains unsuccessful due to the increased blood volume in humans and may therefore lead to insufficient accumulation due to accelerated elimination of the small-sized radioligand.

      Related to this, recent study investigated whether an increase of Tz motifs by chelator scaffolding, i.e., multimerization, can improve the binding efficiency and thereby improve imaging contrast. This could contribute in particular to the application of gallium-68 for pretargeted immuno-PET imaging. For this purpose we utilized the macrocyclic chelator Fusarinine C for the design of mono- and multimeric Tz-conjugates as presented in Scheme 1 for a proof-of-concept study, on potentially improved pretargeting for imaging with gallium-68 by applying multimerization. We chose non-internalizing anti-CD20 antibody rituximab (RTX) modified with TCO as targeting vector. Radiolabelling was conducted at room temperature within minutes and the 68Ga-labelled conjugates showed reasonable hydrophilicity and excellent stability in human serum. Protein binding, however, remained comparable within the conjugates but was generally high. The 68Ga-labelled multimeric conjugates showed a higher binding capacity towards TCO-motif bearing RTX. Furthermore, cell-binding studies revealed highly specific targeting properties and the binding of [68Ga]Ga-FSC-Tz multimers to CD20-expressing Raji cells increased with the number of Tz-motifs attached to the chelator. Imaging studies in a simplified pretargeting mouse model proved the trend for improved targeting. We therefore conclude, that multimerization bears a great potential to improve IEDDA related pretargeting when short-lived PET-radioisotopes, particularly gallium-68, are used. Further investigations in established tumor models are warranted to confirm these promising findings.

      2. Results

      2.1. (Radio) Chemistry

      FSC-based mono- and multimeric Tz-conjugates were accessible in a three-step synthesis to give the corresponding conjugates in good yields and high chemical purity (>95%; analytical RP-HPLC, UV absorption at λ = 220 nm). The results from mass analysis were in good agreement with the calculated values. The structure of the Tz-conjugates was further confirmed by 1H-NMR spectroscopy. The singlets at 6.30 and 1.86 ppm which can be assigned to the -C=O-CH=C(CH3)C-substructure were used as the marker signals of the FSC subunit. The singlet at 1.83 ppm corresponds to the methyl protons of the acetyl group(s). The singlet at 10.56 ppm is highly characteristic for the tetrazine moiety. The doublets at 8.44 and 7.53 ppm with coupling constants of 8.4 Hz are characteristic for the para-substituted phenyl ring, and the triplet at 8.50 ppm as well as the doublet at 4.40 ppm with a coupling constant of 6.0 Hz can be assigned to the -NH-CH2 group of the PEG5-Tz subunit(s). The ratio of the integrals of these marker signals is summarized in Table 1 and corresponding 1H-NMR spectra are presented in Figures S1–S3. Radiolabelling with gallium-68 was quantitative within minutes at RT, thus exhibiting fast labelling kinetics. Corresponding (radio-)RP-HPLC chromatograms are presented in Figure S4.

      Table 1

      1H-NMR data (chemical shifts and integrals) of characteristic signals of FSC-based Tz-conjugates and N,N′,N′′-triacetylfusarinine (TAFC) as a reference.

      FSC Subunit Acetyl PEG5-Tz Subunit
      3× CH 3× CH3 CH3 Tetrazine p-Phenylen NH-CH2 NH-CH2
      6.3 ppm 1.86 ppm 1.83 ppm 10.56 ppm 8.44 ppm 7.53 ppm 8.50 ppm 4.40 ppm
      Tz-monomer 3 H 9 H 6 H 1 H 2 H 2 H 1 H 2 H
      Tz-dimer 3 H 9 H 3 H 2 H 4 H 4 H 2 H 4 H
      Tz-trimer 3 H 9 H none 3 H 6 H 6 H 3 H 6 H
      TAFC 3 H 9 H 9 H none none none none none

      2.2. In Vitro Evaluation

      Stability studies of 68Ga-labelled conjugates in fresh human serum and PBS as control revealed high stability as no major degradation was observed over a period of 4 h. Corresponding radio-RP-HPLC chromatograms are presented in Figure S5. The results of logD studies and the ability to bind to serum proteins of 68Ga-labelled conjugates are summarized in Table 2. They revealed suitable hydrophilicity with minor decrease when increasing the number of Tz residues. All conjugates showed very high protein binding with minor differences between mono- and multimeric [68Ga]Ga-Tz-ligands.

      Table 2

      Distribution coefficient (logD) and protein binding of 68Ga-labelled FSC-based Tz-conjugates.

      68Ga-Labelled
      Compound
      LogD
      (pH 7.4)
      Protein Binding (%)
      1 h 2 h 4 h
      Tz-monomer −1.64 ± 0.02 61.8 ± 0.2 63.8 ± 2.1 64.0 ± 1.4
      Tz-dimer −1.35 ± 0.01 67.0 ± 2.4 65.9 ± 1.3 68.4 ± 0.3
      Tz-trimer −1.00 ± 0.06 70.5 ± 0.7 69.5 ± 0.4 67.8 ± 0.4

      Data are presented as mean ± SD (n = 3)

      The non-internalizing anti-CD20 monoclonal antibody rituximab was modified with the TCO motif similar to a previously published procedure [] and corresponding FACS analysis of CD20-expressing Raji cells incubated with both, TCO-modified and non-modified RTX showed high target specificity (Figure S6), thus demonstrating that the binding ability was not altered by the TCO modification.

      The binding capacity of 68Ga-labelled mono- and multimeric Tz-ligands was assessed via competitive binding on immobilized RTX-TCO using the non-labelled conjugates as competitor and is presented in Figure 1. The binding of the [68Ga]Ga-Tz-monomer was reduced by 50% at a competitor concentration of 486 ± 52 nM when challenged with the non-labelled monomer, whereas the non-labelled dimer (112 ± 6 nM) and trimer (100 ± 10 nM) reduced the binding at significantly lower concentrations. The binding of the [68Ga]Ga-Tz-dimer was reduced by half at 95 ± 25 nM in competition with its non-labelled counterpart and at a comparable concentration with the non-labelled trimer (92 ± 15 nM), whereas a decrease to 50% was only achieved at a much higher concentration in competition with non-labelled monomer (865 ± 263 nM). Binding studies of the [68Ga]Ga-Tz-trimer showed a comparable trend as the non-labelled trimer reduced the binding by 50% at 147 ± 49 nM and the dimer at 258 ± 60 nM; whereby a significantly higher amount of the monomer (2987 ± 1664 nM) was needed for a 50% binding reduction. Overall in all assays improved binding of di- and trimer over monomer was observed.

      An external file that holds a picture, illustration, etc.
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      An external file that holds a picture, illustration, etc.
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      Competitive binding studies of [68Ga]Ga-Tz-monomer (A), [68Ga]Ga-Tz-dimer (B) and [68Ga]Ga-Tz-trimer (C) on immobilized RTX-TCO using the non-labelled counterparts as competitor.

      The results of cell-binding studies on CD20-expressing Raji cells pre-treated with RTX or RTX-TCO prior to incubation with 68Ga-labelled Tz-ligands are presented in Figure 2. All 68Ga-labelled conjugates showed highly specific targeting properties as the amount of unspecific bound radioligand to RTX pre-treated Raji cells was negligible low (<1%). The binding of 68Ga-labelled Tz-ligands on RTX-TCO bound Raji cells increased with the grade of multimerization and was 4.01 ± 0.24% for [68Ga]Ga-Tz-monomer, 7.35 ± 0.77% for [68Ga]Ga-Tz-dimer and 15.93 ± 0.88 for [68Ga]Ga-Tz-trimer, respectively.

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      Cell-binding studies of 68Ga-labelled FSC-based Tz-ligands on CD20-expressing Raji cells pre-treated with anti-CD20 antibody RTX (negative control, black bars) and its TCO modified counterpart (white bars).

      2.3. In Vivo Evaluation

      Biodistribution studies in non-tumour xenografted BALB/c mice 1 h after administration of the 68Ga-labelled Tz-ligands are shown in Figure 3. In general, accumulation in non-targeted tissue was significantly lower for the [68Ga]Ga-Tz-monomer compared to the [68Ga]Ga-Tz-trimer, whereby the difference between the multimers was less pronounced. In particular, the multimeric Tz-ligands showed slower blood clearance and higher accumulation in renal tissue compared to the monomeric conjugate. The conjugates generally showed low accumulation in non-targeted tissue and low retention in critical organs (e.g., muscle, bone).

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      Biodistribution studies of 68Ga-labelled mono- and multimeric FSC-based pretargeting agents in normal BALB/c mice 1 h p.i. presented as percentage of total injected activity per gram tissue (n = 3).

      Imaging studies using BALB/c mice i.m. injected with RTX and RTX-TCO as tumour surrogate 5 h prior to r.o. administration of the radioligand and imaging performed 90 min p.i. are presented in Figure 4. The results mainly confirmed the suitable in vivo distribution profile of FSC-based Tz-ligands radiolabelled with gallium-68 with main activity in kidneys and bladder and some blood pool activity for the 68Ga-labelled multimers. Moreover, the accumulation in RTX-TCO positive muscle tissue increased with ascending numbers of Tz motifs and was ~1% for the [68Ga]Ga-Tz-monomer, ~2% for the [68Ga]Ga-Tz-dimer and ~3% for the [68Ga]Ga-Tz-trimer. Surprisingly, the accumulation in TCO negative tissue was also approximately 1% showing negligible differences between the different radioligands.

      An external file that holds a picture, illustration, etc.
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      Static µPET/CT image of i.m. RTX(-TCO) pre-treated normal BALB/c mice 5 h after treatment and 90 min p.i. of the 68Ga-labelled Tz-monomer (a), Tz-dimer (b) and Tz-trimer (c). (1 = coronal slice; 2 = 3D volume rendered projections; both prone position).

      3. Discussion

      IEDDA-based pretargeting has become increasingly popular for molecular imaging as well as radioimmunotherapy over the past few years [,,,,,]. Despite recent advancements towards structural improvement of cyclen- and TACN-based Tz-probes [,] or the synthesis of TCO-bearing dendrimers [], the design of targeting probes bearing multiple Tz-motifs for potentially improved pretargeting has scarcely been investigated. Devaraj and co-workers reported on polymer modified tetrazines (PMT) radiolabelled with fluorine-18 [] and gallium-68 [] for PET applications while Zlatni et al. recently demonstrated the targeting applicability for microbubbles bearing multiple Tz-motifs towards prostate specific membrane antigen for ultrasound related diagnostic purposes []. However, investigations on the use of small-sized multimeric Tz-ligands have not been reported yet.

      FSC is a suitable chelating scaffold for PET radiometals, particularly gallium-68 and zirconium-89 [,]. Its unique structural properties enable straight forward mono- and multimeric tracer design [,,,]. This novel chelator, therefore, represented a very suitable scaffold for the synthesis of mono- and multimeric small-sized Tz-bearing probes for radiolabelling with gallium-68 to evaluate potential benefits for IEDDA-based pretargeting by increasing the number of Tz-residues. The resulting conjugates showed reasonable hydrophilicity and increasing the number of Tz-residues from one to three did not alter the water solubility too much, although being somewhat lower in comparison to linker modified monomeric conjugates []. Unexpectedly, all FSC-based Tz-conjugates showed high protein binding with negligible differences between the compounds. This can be related to the introduction of the pegylated Tz residue, since non-modified 68Ga-labelled FSC-based precursors showed a protein binding of <3% (Kaepookum et al. unpublished results). This phenomenon has not been reported in the case of monomeric DOTA- and TACN-based derivatives [,] and might be disadvantageous by slowing down the distribution of the tracer to the TCO-interaction site. In vivo results, however, did not really reveal a major problem with too slow pharmacokinetics, since the washout from non-targeted tissue was still sufficiently rapid. Competitive binding assessments showed significantly reduced binding of [68Ga]Ga-Tz-monomer by a factor of five when challenged with the multimeric conjugates in comparison with the non-labelled monomer. This corresponded with the significantly higher amounts of Tz-monomer needed, 8 to 20 fold respectively, to reduce the binding of the 68Ga-labelled multimers by 50%, thus indicating that the binding capacity increased with the number of Tz residues. This was substantiated by the results of the cell binding studies, since the binding to CD20-expressing Raji cells increased by a factor of 1.8 for the [68Ga]Ga-Tz-dimer and 3.9 for the [68Ga]Ga-Tz-trimer in comparison to the [68Ga]Ga-Tz-monomer. Biodistribution studies in non-tumour xenografted healthy BALB/c mice indicated suitable pharmacokinetics and showed a multimerization typical profile exhibiting slower blood clearance and increased kidney retention, similar to prior findings with the FSC-scaffold [,]. Imaging studies using a tumour surrogate model confirmed this trend and demonstrated increased binding of the 68Ga-labelled multimeric Tz-conjugates. The accumulation in RTX-TCO pretreated muscle tissue doubled for the [68Ga]Ga-Tz-dimer and was 3-fold higher for the [68Ga]Ga-Tz-trimer compared to the [68Ga]Ga-Tz-monomer.

      In regard to imaging in living mice there was a clear improvement of target specific accumulation when switching from mono- to multimers. The use of our simplified tumour surrogate animal model, however, requires further investigations in established animal models to address the limitations of this study:

      (1) The accumulation in non-targeted tissue, i.e., i.m. injection of RTX resulted to be ~1%. This effect was even more pronounced when choosing a shorter time interval of 2 h between i.m. administration of the mAb and radioligand injection (data not shown) and was only seen at the injection site but generally not in other tissue. We speculate that this is related to the injection of the antibody leading to tissue damage with increased tissue permeability and unspecific accumulation of the radioligand.

      (2) Intravenously injected mAb stimulates accumulation at the target interaction site, but a non-negligible amount remains in circulation, thereby increasing the background signal when administering the radioligand. In order to reduce this amount, high molecular weight TCO-scavenger molecules exhibiting low vascular permeability (clearing agents) have been established with great success, significantly improving target-to-background (TTB) ratios [,]. The need for clearing agents was completely neglected in this study and we are fully aware that our animal model does not reflect the real situation regarding TTB ratios.

      In summary, we have been able to show that FSC is a suitable scaffold for the design of multimeric Tz-conjugates for radiolabelling with gallium-68. Although multimeric Tz-ligands exhibited significant improvements towards IEDDA-based pretargeting additional studies in tumour models are warranted to explore the full potential of this promising concept. Furthermore, a highly interesting therapeutic approach is the release of drugs directly at the interaction site from antibody-drug conjugates (ADCs) mediated via IEDDA reaction between Tz and TCO [,,]. It might be of interest for future perspectives if multimeric Tz-conjugates can boost the release of drugs improving this highly promising “click-to-release” strategy.

      4. Materials and Methods

      4.1. Instrumentation

      4.1.1. Analytical [radio]-RP-HPLC

      Reversed-phase high-performance liquid chromatography analysis was performed with the following instrumentation: UltiMate 3000 RS UHPLC pump, UltiMate 3000 autosampler, UltiMate 3000 column compartment (25 °C oven temperature), UltiMate 3000 Variable Wavelength Detector (Dionex, Germering, Germany; UV detection at λ = 220 nm) a radio detector (GabiStar, Raytest; Straubenhardt, Germany), Jupiter 5 μm C18 300 Å 150 × 4.6 mm (Phenomenex Ltd. Aschaffenburg, Germany) column with acetonitrile (ACN)/H2O/0.1% trifluoroacetic acid (TFA) as mobile phase; flow rate of 1 mL/min; gradient: 0.0–1.0 min 10% ACN, 1.0–12.0 min 10–60% ACN, 13.0–15.0 min 60–80% ACN, 15.0–16.0 min 80–10% ACN, 16.0–20.0 min 10% ACN.

      4.1.2. Preparative RP-HPLC

      Sample purification via RP-HPLC was carried out as follows: Gilson 322 Pump with a Gilson UV/VIS-155 detector (UV detection at λ = 220 nm) using a PrepFC™ automatic fraction collector (Gilson, Middleton, WI, USA), Eurosil Bioselect Vertex Plus 30 × 8 mm 5 μm C18A 300 Å pre-column and Eurosil Bioselect Vertex Plus 300 × 8 mm 5 μm C18A 300 Å column (Knauer, Berlin, Germany) and following ACN/H2O/0.1% TFA gradients with a flow rate of 2 mL/min: gradient A: 0.0–5.0 min 0% ACN, 5.0–35.0 min 0–50% ACN, 35.0–38.0 min 50% ACN, 38.0–40.0 min 50–0% ACN. Gradient B: 0.0–5.0 min 10% ACN, 5.0–40.0 min 10–60% ACN, 41.0–45.0 min 60% ACN, 46.0–50.0 min. 60–80% ACN, 51.0–55.0 min 80–10% ACN.

      4.1.3. MALDI-TOF MS

      Mass spectrometry was conducted on a Bruker microflexTM bench-top MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) with a 20 Hz laser source. Sample preparation was performed according to dried-droplet method on a micro scout target (MSP96 target ground steel BC, Bruker Daltonics) using α-cyano-4-hydroxycinnamic acid (HCCA, Sigma-Aldrich, Handels GmbH, Vienna, Austria) as matrix. Flex Analysis 2.4 software was used for processing of the recorded data.

      4.1.4. 1H-NMR Spectroscopy

      1H-NMR spectra of the FSC-based Tz-conjugates were recorded on a “Saturn” 600 MHz Avance II+ spectrometer and the NMR of the reference compound (TAFC) was recorded on a “Mars” 400 MHz Avance 4 Neo spectrometer, both from Bruker Corporation (Billerica, MA, USA). The centre of the solvent multiplet (DMSO-d6) was used as internal standard (chemical shifts in δ ppm), which was related to TMS with δ 2.49 ppm. TopSpin 3.5 pl7 software (Bruker) was used for data processing.

      4.2. Synthesis

      4.2.1. General Information

      All chemicals and solvents were purchased as reagent grade from commercial sources unless otherwise stated. trans-Cyclooctene-NHS ester and tetrazine-PEG5-NHS ester were bought from Click Chemistry Tools (Scottsdale, AZ, USA). Rituximab (MabThera®, Roche Pharma AG, Grenzach-Wyhlen, Germany) was of pharmaceutical grade and was a kind gift from the University Hospital of Innsbruck.

      4.2.2. [Fe]Fusarinine C ([Fe]FSC)

      The cyclic siderophore Fusarinine C (FSC) was obtained from iron deficient fungal culture and the extraction of FSC was conducted with a slightly modified method as described before []. Briefly, 1 L of iron saturated culture media was flushed through a C18-Reveleris flash cartridge (40 μm, 12 g; Grace, MD, USA) by using a REGLO tubing pump (Type ISM795, Ismatec SA, Glattbrugg-Zurich, Switzerland) with a flow rate of 10 mL/min. [Fe]FSC fixed on the cartridge was washed with 50 mL of water and eluted afterwards with 50 mL H2O/ACN (20/80% v/v). After evaporation to dryness ~300 mg [Fe]FSC were obtained as red−brown coloured solid in high purity (>90%). Analytical data: RP-HPLC tR = 6.95 min; MALDI TOF-MS: m/z [M + H]+ = 779.93 [C33H51FeN6O12; exact mass: 779.63 (calculated)].

      4.2.3. Acetylation of [Fe]FSC

      [Fe]FSC was dissolved in methanol to a final concentration of 30 mg/mL (38.5 mM). An aliquot of 300 µL was reacted with 10 µL of acetic anhydride for 5 min at room temperature (RT) under vigorous shaking followed by subsequent purification of the resulting mixture of mono-, di- and triacetylfusarinine C via preparative RP-HPLC using gradient A to obtain N-monoacetylfusarinine C ([Fe]MAFC, tR = 20.1 min) and N,N’-diacetylfusarinine C ([Fe]DAFC, tR = 24.5 min). Analytical data: [Fe]MAFC: RP-HPLC tR = 7.67 min; MALDI TOF-MS: m/z [M + H]+ = 822.04 [C35H53FeN6O13; exact mass: 821.67 (calculated)]. [Fe]DAFC: RP-HPLC tR = 8.49 min; MALDI TOF-MS: m/z [M + H]+ = 864.02 [C37H55FeN6O14; exact mass: 863.71 (calculated)].

      4.2.4. Conjugation of Tetrazine-PEG5 Motif

      Iron protected FSC (1.0 mg, 1.28 µmol), MAFC (2.0 mg, 2.43 µmol) or DAFC (2.0 mg, 2.32 µmol) were dissolved in 500 µL anhydrous DMF and after addition of Tetrazine-PEG5-NHS ester, 1.5 equivalents (2.10 mg, 3.48 µmol) in case of DAFC, 2.5 equivalents (3.67 mg, 6.08 µmol) in case of MAFC and 3.5 equivalents (2.71 mg, 4.48 µmol) in case of FSC, pH was adjusted to 9.0 using DIPEA and the reaction mixtures were maintained for 4 h at RT. Finally the organic solvent was evaporated and the crude mixture was used without further purification.

      4.2.5. Demetallation

      For the purpose of iron removal, corresponding conjugates were dissolved in 1 mL H2O/ACN solvent 50% (v/v) and 1 mL of aqueous Na2EDTA solution (200 mM) was added. The resulting mixtures were stirred for 4 h at ambient temperature followed by preparative RP-HPLC purification to give slightly to intensively pink coloured, iron free FSC-based Tz-conjugates after lyophilisation.

      • DAFC-PEG5-Tz (=Tz-monomer): 2.35 mg [1.81 µmol, 78%], gradient B (tR = 31.1 min); Analytical data: RP-HPLC tR = 11.5 min; MALDI TOF-MS: m/z [M + H]+ = 1303.95 [C60H89N11O21; exact mass: 1300.41 (calculated)]

      • MAFC-(PEG5-Tz)2 (=Tz-dimer): 3.65 mg [2.09 µmol, 86%], gradient B (tR = 35.3 min); Analytical data: RP-HPLC tR = 12.5 min; MALDI TOF-MS: m/z [M + H]+ = 1748.25 [C81H118N16O27; exact mass: 1747.89 (calculated)]

      • FSC-(PEG5-Tz)3 (=Tz-trimer): 1.68 mg [0.77 µmol, 60%], gradient B (tR = 37.9 min); Analytical data: RP-HPLC tR = 13.2 min; MALDI TOF-MS: m/z [M + H]+ = 2200.20 [C102H147N21O33; exact mass: 2195.38 (calculated)]

      4.2.6. Modification of Rituximab (RTX)

      Rituximab was obtained in solution (Mabthera®, 10 mg/mL, Roche Pharma AG, Grenzach-Wyhlen, Germany) and a PD-10 (GE Healthcare, Vienna, Austria) size exclusion column was used for buffer exchange according to manufacturer’s protocol to give RTX in 0.1 M NaHCO3 solution (7 mg/mL). For the conjugation of trans-cyclooctene (TCO), 2 mL of the RTX solution were mixed with 20 molar equivalent of TCO-NHS ester dissolved in DMSO and the reaction was stirred for 30 min at ambient temperature followed by incubation overnight at 4 °C under light exclusion. Subsequently, the modified antibody (RTX-TCO) was purified using size exclusion chromatography (PD-10) to give 10 mg of RTX-TCO dissolved in PBS. The antibody was treated following the same procedure as described above without adding TCO-NHS ester, in order to obtain a non-modified RTX counterpart as negative control.

      4.3. Fluorescence Activated Cell-Sorting (FACS)

      The humanoid lymphoblast-like CD20-expressing B-lymphocyte cells (Raji cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Flow cytometric analysis of CD20 expression was evaluated on (Raji cells) adjusted to 5 × 105 cells/sample in DMEM (including 10% FCS, 1% Pen/Strep). Cells were incubated with FC-Block (containing CD16/CD32 (1:200), e-Bioscience, Thermo Fisher Scientific, Vienna, Austria) for 15 min at 4 °C in order to avoid signals from non-specific binding. The following antibodies were used: RTX-TCO and RTX, both at 20 µM (f. c.). As secondary antibody a APC-labelled anti-human IgG Fc (clone: HP6017; BioLedgend, San Diego, CA, USA) at a dilution of 1:100 was used. Antibodies were incubated for at least 15 min at 4 °C. FACS analysis was performed on BD LSRFortessa™ (Cell Analyzer, BD Bioscience, San Jose, CA, USA). As internal control samples unstained or stained with either primary or secondary antibodies were analyzed. FACS-data was analyzed by FlowJo v10 software.

      4.4. Radiolabelling of FSC-Based Tz-Conjugates with Gallium-68

      Gallium-68 was obtained as [68Ga]GaCl3 (gallium chloride) by fractioned elution of a 68Ge/68Ga-generator (IGG100, nominal activity 1100 MBq, Eckert & Ziegler, Berlin, Germany) with 0.1 M hydrochloric acid (HCl, Rotem Industries Ltd., Beer-Sheva, Israel). Hereafter, 500 µL of eluate (100 MBq) were mixed with 100 µL sodium acetate solution (1.14 M) to give a pH of 4.5 followed by addition of 10 µg (4.55–7.68 nmol) of corresponding FSC-Tz conjugate. After 5 min incubation at RT the radiolabelling solution was analyzed using radio-RP-HPLC.

      4.5. In Vitro Characterization.

      4.5.1. Distribution Coefficient (LogD)

      To determine the distribution of the 68Ga-labelled conjugates between an organic (octanol) and aqueous (PBS) layer, aliquots (50 µL) of the tracers (~5 µM) were diluted in 1 mL of octanol/PBS (1:1, v/v). The mixture was vortexed at 1400 rpm (MS 3 basic vortexer, IKA, Staufen, Germany) for 15 min at RT followed by centrifugation for 2 min at 4500 rpm. Subsequently, aliquots (50 µL) of both layers were collected and measured in the gamma counter (Wizard2 3”, Perkin Elmer, Waltham, MA, USA) followed by logD calculation (n = 3, six replicates).

      4.5.2. Protein Binding

      Serum protein binding was determined using Sephadex G-50 (GE Healthcare Vienna, Austria) size exclusion chromatography []. Aliquots (50 µL, n = 3) of the radioligand solution (~10 µM) were incubated in 450 µL freshly prepared human serum or 450 µL PBS (controls) and were kept at 37 °C. After 1, 2, and 4 h aliquots (25 µL) were directly transferred to the column (MicroSpin G-50, GE Healthcare) and after centrifugation (2 min, 2000 rcf) the column containing the free conjugate and the eluate containing the protein-bound conjugate were measured in the gamma counter. The percent of activity in both fractions was calculated thereafter.

      4.5.3. Stability Studies in Human Serum

      Stability of the radioligands was evaluated in human serum as described in []. Briefly, 50 µL of the radioligand solution (~10 µM, n = 2) were mixed with 950 µL freshly prepared serum or 950 µL PBS (controls). The mixtures were then maintained at 37 °C. At designated time points, 1, 2 and 4 h respectively, aliquots (100 µL) were mixed with 0.1% TFA/ACN, centrifuged for 2 min at 14 × 103 rcf. The supernatant was diluted with H2O (1:1, v/v) and analyzed by analytical RP-HPLC for decomposition without filtration prior to injection.

      4.5.4. Competitive Binding Assay

      Binding on immobilized RTX-TCO was conducted using high protein-binding capacity Nunc MaxiSorp™ 96-well plates (Thermo Fisher Scientific, Vienna, Austria). Coating was performed by adding 20 µg of antibody (RTX or RTX-TCO) dissolved in 100 µL coating buffer (0.1 M NaHCO3, pH 8.5) to each well and after 2 h incubation at RT the plate was left at 4 °C overnight both under the exclusion of light. After removal of the coating solution 200 µL of blocking buffer (1% BSA in PBS) was added, left for 1 h at room temperature and subsequently each well was washed twice with 200 µL binding buffer (0.1% BSA in PBS). Hereafter, the radioligand was mixed with increasing concentrations of the competitor (=non-labelled conjugate), diluted in binding buffer and 100 µL of the mixture was added to each well. After 30 min at RT the supernatant was removed, each well was washed three times with 150 µL binding buffer and the coating film was finally detached with 2 × 150 µL of hot (80 °C) 2 N sodium hydroxide (NaOH). The NaOH fraction was taken for gamma counter measurement to determine the percentage of binding in contrast to the standard followed by non-liner curve fitting using Origin 6.1 software (Origin Inc., Northampton, MA, USA) to calculate the apparent half maximum inhibitory concentration of the competitor (n = 3, 4 replicates).

      4.5.5. Cell Binding

      Raji-cells were seeded in tissue culture flasks (Cellstar; Greiner Bio-One, Kremsmuenster, Austria) using RPMI-1640 medium supplemented with fetal bovine serum (FBS) to a final concentration of 10% (v/v). In order to do studies on cell binding 10 × 106 cells were washed twice with fresh media, diluted with PBS to a final concentration of 1 × 106 cells per mL and 500 µL of cell suspension was transferred to Eppendorf tubes. Hereafter, 50 µL of RTX-TCO or non-modified RTX as negative control (both 0.5 µM) were added and the cell suspension was maintained at 37 °C under gentle shaking. After 1 h the suspension was centrifuged (2 min, 11 × 103 rcf), the supernatant was discarded, the cells were washed twice and finally resuspended with 450 µL PBS. Subsequently, 50 µL of the radioligand solution (22 nM in PBS) was added and the suspension was incubated for 30 min at 37 °C. After centrifugation and two washing steps with 600 µL PBS, the cells were resuspended in 500 µL PBS and transferred to polypropylene vials for gamma counter measurement followed by calculation from cell-associated activity in comparison to the standard (n = 3, six replicates).

      4.6. In Vivo Characterization

      4.6.1. Ethics Statement

      All animal experiments were performed in accordance with regulations and guidelines of the Austrian animal protection laws and the Czech Animal Protection Act (No. 246/1992), with approval of the Austrian Ministry of Science (BMWF-66.011/0161-WF/V/3b/2016), the Czech Ministry of Education, Youth, and Sports (MSMT-18724/2016-2), and the institutional Animal Welfare Committee of the Faculty of Medicine and Dentistry of Palacky University in Olomouc.

      4.6.2. Biodistribution Studies

      Biodistribution of 68Ga-labelled conjugates was conducted in healthy 5-week-old female BALB/c mice (Charles River Laboratories, Sulzfeld, Germany). Animals (n = 3) were injected via lateral tail vain with 1 nmol of conjugate and a total activity of approximately 6 MBq. Mice were sacrificed by cervical dislocation 1 h p.i. followed by collection of the main organs and tissue, subsequent gamma counter measurement and calculation of the percentage of injected activity per gram tissue (% IA/g).

      4.6.3. Imaging Studies

      MicroPET/CT images were acquired with an Albira PET/SPECT/CT small animal imaging system (Bruker Biospin Corporation, Woodbridge, CT, USA). Mice were pre-treated by intramuscular (i.m.) injection of 50 µL of RTX-TCO to the left hind muscle and 50 µL of RTX to the right hind muscle. Pre-treated mice were retro-orbitally (r.o.) injected with radiolabelled tracer in a dose of 5–10 MBq corresponding to 1–2 μg of conjugate per animal 5 h after the pre-treatment. Anaesthetized (2% isoflurane (FORANE, Abbott Laboratories, Abbott Park, IL, USA)) animals were placed in a prone position in the Albira system before the start of imaging. Static PET/CT images were acquired over 30 min starting 90 min p.i. A 10-min PET scan (axial FOV 148 mm) was performed, followed by a double CT scan (axial FOV 65 mm, 45 kVp, 400 μA, at 400 projections). Scans were reconstructed with the Albira software (Bruker Biospin Corporation) using the maximum likelihood expectation maximization (MLEM) and filtered backprojection (FBP) algorithms. After reconstruction, acquired data was viewed and analyzed with PMOD software (PMOD Technologies Ltd., Zurich, Switzerland). 3D volume rendered images were obtained using VolView software (Kitware, Clifton Park, NY, USA).

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      Synthetic strategy for FSC-based tetrazine (Tz) conjugates [a: methanol and acetic anhydride (MetOH/Ac2O); b: Tz-PEG5-NHS/DMF/DIPEA; c: EDTA] radiolabelled with gallium-68.

      Acknowledgments

      We gratefully acknowledge Christoph Kreutz from the Institute of Organic Chemistry, (Leopold-Franzens University, Innsbruck, Austria) for performing NMR analysis, the staff from the hospital pharmacy for supplying us with Rituximab, the Austrian Science Foundation (FWF) and the Czech Ministry of Education Youth and Sports for funding.

      Abbreviations

      ACN acetonitrile
      BSA bovine serum albumin
      CD cluster of differentiation
      DAFC N,N′-diacetylfusarinine C
      DIPEA N,N-diisopropylamine
      DMF N,N-dimethylformamide
      EDTA ethylenediaminetetraacetic acid
      FSC fusarinine C
      IEDDA inverse electron-demand Diels-Alder
      i.m. intramuscular
      mAb monoclonal antibody
      MAFC N-monoacetylfusarinine C
      NHS N-hydroxysuccinimide
      p.i. post injection
      PBS phosphate buffered saline
      PET/CT positron emission computed tomography
      r.o. retro-orbitally
      RP-HPLC reversed phase high performance liquid chromatography
      RT room temperature
      RTX rituximab
      TCO trans-cyclooctene
      TFA trifluoracetic acid
      Tz tetrazine

      Supplementary Materials

      The following are available online at http://www.mdpi.com/1424-8247/11/4/102/s1, Figure S1: 1H-NMR spectrum of DAFC-PEG5-Tz (Tz-monomer), Figure S2: 1H-NMR spectrum of of MAFC-(PEG5-Tz)2 (Tz-dimer), Figure S3: 1H-NMR spectrum of FSC-(PEG5-Tz)3 (Tz-trimer), Figure S4: Representative RP-HPLC chromatograms of mono- and multimeric FSC-Tz conjugates (A, UV/vis chromatograms) and their 68Ga-labelled counterparts (B, radio-chromatograms), Figure S5: Representative radio-RP-HPLC chromatograms of the stability assessment of 68Ga-labelled mono- and multimeric FSC-Tz conjugates incubated with fresh human serum (A) and PBS (B), Figure S6: Fluorescence-activated cell sorting of unstained Raji cells, RTX, RTX-TCO, secondary antibody (APC), RTX + APC and RTX-TCO + APC (order from left to right).

      Author Contributions

      L.V. carried out the synthesis of FSC-Tz conjugates under guidance of B.M. and both provided the corresponding part of the manuscript; B.M. evaluated the NMR data; D.S. evaluated the protocol for radiolabelling with gallium-68, reviewed the literature, summarized the results and has written the major part of the manuscript; C.R. performed cell culture, animal housing and animal care; S.M. modified the antibody and evaluated the radiolabelled conjugates in vitro as well as in vivo. K.S. provided the Raji cells and carried out FACS analysis of the antibodies. M.P. evaluated the tumour-surrogate model and performed in vivo imaging studies. C.D. was responsible for conceptualization, funding acquisition, project administration and supervision.

      Funding

      This research was funded by the Austrian Science Foundation (FWF) grant P 25899-B23 and by the Czech Ministry of Education Youth and Sports grant LO1304.

      Conflicts of Interest

      The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

      The information comes from:
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316846/

    5. Abstract

      Colorectal cancer is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women worldwide. We have recently reported that curcuminoid complexes labelled with gallium-68 have demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compared to normal human lymphocytes. In the present study, we report a new gallium-68-labelled curcumin derivative (68Ga-DOTA-C21) and its initial validation as marker for early detection of colorectal cancer. The precursor and non-radioactive complexes were synthesized and deeply characterized by analytical methods then the curcuminoid was radiolabelled with gallium-68. The in vitro stability, cell uptake, internalization and efflux properties of the probe were studied in HT29 cells, and the in vivo targeting ability and biodistribution were investigated in mice bearing HT29 subcutaneous tumour model. 68Ga-DOTA-C21 exhibits decent stability (57 ± 3% after 120 min of incubation) in physiological media and a curcumin-mediated cellular accumulation in colorectal cancer cell line (121 ± 4 KBq of radiotracer per mg of protein within 60 min of incubation). In HT29 tumour-bearing mice, the tumour uptake of 68Ga-DOTA-C21 is 3.57 ± 0.3% of the injected dose per gram of tissue after 90 min post injection with a tumour to muscle ratio of 2.2 ± 0.2. High amount of activity (12.73 ± 1.9% ID/g) is recorded in blood and significant uptake of the radiotracer occurs in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). Further studies are needed to understand the mechanism of accumulation and clearance; however, 68Ga-DOTA-C21 provides a productive base-structure to develop further radiotracers for imaging of colorectal cancer.

      1. Introduction

      Colorectal cancer (CRC) is the third most commonly occurring cancer in men and the second in women worldwide, with more than 1.4 million new cancer cases every year []. India has a relatively low prevalence of CRC in comparison to other countries, mainly attributed to differences in dietary patterns and lifestyles []. It is widely known that curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a phyto-polyphenol pigment isolated from the dried rhizomes of Curcuma longa L., has been used for centuries in Asian countries as a dietary spice and traditional drug to treat many different diseases []. Besides having antioxidant, anti-inflammatory, anti-bacterial and anti-amyloid properties, curcumin has been shown also to possess anti-cancer activities [,]. In particular, the ability to inhibit proliferation of a wide variety of tumour types, including colorectal cancer, has been highlighted by recent studies in vitro and in vivo [,,]. Several studies suggest that curcumin changes the gene expression profiles and signalling pathways such as those involving COX-2 enzymes as well as NF-κB, cyclin D1 and p53 proteins [,,]. Although the mechanism of curcumin’s high accumulation and cytotoxic activity against HeLa, MCF-7, HT29, and HCT-116 cancer cell lines are still unclear [,], curcumin itself is a promising tumour-targeting moiety for the development of early diagnosis and therapeutic agents. Nuclear medicine imaging provides information about biological processes occurring at a molecular level in vivo; this occurs by following the fate of radiolabelled compounds. Such radiotracers can be substrates for metabolic pathways overexploited in tumour cells or have particular affinity for receptors overexpressed in tumours. Hence, curcumin derivatives labelled with proper β+ emitter radionuclides or curcumin-based radio-metal complexes have the potential to be diagnostic tools for positron emission tomography (PET). Currently, radiolabelled curcuminoids have been mostly investigated for imaging of the central nervous system (CNS) where several curcumin-based probes labelled with fluorine-18 have demonstrated promise in the early detection of Alzheimer’s disease [,]. In oncology, curcuminoids as imaging agents are underinvestigated. We have recently reported that curcuminoid complexes labelled with gallium-68 demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compare to normal human lymphocytes. In that study, the core radio-metal was directly linked to two curcuminoid molecules through the keto-enol moiety [,]. The curcuminoid complexes showed good stability in PBS and human serum but were rapidly degraded in whole human blood. These findings, in addition to the low stability and solubility of curcumin structures in physiological media [], limited the possibility to further study these kind of compounds in vivo. In the present study, we report a new gallium-68-labelled curcumin derivative with curcumin linked to an efficient gallium-68 chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and its initial validation as a marker for early detection of CRC. The synthetic pathway, chemical characterization, labelling methods, stability, cell uptake, internalization and efflux studies on HT29 cells and in vivo micro-PET imaging and biodistribution in colorectal tumour bearing mice of this radiotracer are herein reported.

      2. Results

      2.1. Synthesis and Chemical Characterization

      Synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic(1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (DOTA-C21) was carried out in two steps as shown in Figure 1. The curcumin-based conjugate was identified and completely characterized by ESI-LC/MS (Figure S1) and 1H-,13C-NMR spectroscopy (Figures S2, S3). ESI-LC/MS spectrum exhibits two isomeric peaks (m/z = 798.3; [M + H]+) that were attributed to contemporary presence of two main isomeric forms (diketo and keto-enol) as previously reported for curcumin and its derivatives [,]. Gallium complexes were obtained by reacting a DOTA-C21 solution with a Ga(NO3)3 solution at 95 °C for 30 min in a 1.5:1 metal to ligand molar ratio using a 0.4 M ammonium acetate solution as buffer. Complexes formation was identified by ESI-LC/MS analysis thanks to the typical isotopic pattern of gallium ion (Figures S4, S5). Similar to the free ligand analysis, the spectrum exhibits two compounds with the same m/z (retention times: 2.8 and 3.3 min, respectively) both corresponding to Ga-DOTA-C21 complexes (m/z = 864.2 [M69Ga + H]+ – 866.2 [M71Ga + H]+). To elucidate chelation mode and binding sites, 1H-NMR titration of DOTA-C21 with Ga3 + was also performed (Figure S6) and the gallium(III)-complex was characterized by NMR (Figure 2, Figure S7) as well as LC-MS/MS fragmentation experiments (Figure 3, Figure S8).

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      Synthetic pathway for the preparation of DOTA-C21 and its complexation with Ga(III) by reacting DOTA-C21 with Ga(NO3)3 at 1.5:1 metal-to-ligand molar ratio in 0.4 M ammonium acetate solution for 30 min at 95 °C.

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      Aliphatic region overlap of 1H-, 13C-HSQC-NMR spectra of DOTA-C21 (red (CH2) and purple (CH/CH3)) and Ga-DOTA-C21 (green (CH2) and blue (CH/CH3)). All the spectra were acquired in MeOD-d4 at 25 °C ([DOTA-C21] = 0.63 mM). Atom numbering refers to Figures 11 and 12.

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      LC-MS/MS fragmentation experiments on Ga-DOTA-C21 complexes with m/z 864.5 [M + H]+. HPLC-MS chromatogram (A). Fragmentation pathway of the m/z 864.5 [M + H]+ ion corresponding to the peak with retention time 5.9 min (B). Fragmentation pathway of the m/z 864.5 [M + H]+ ion corresponding to the peak with retention time 6.5 min (C).

      1H- and 13C-NMR spectra displayed a strong shift of both protons and carbon with respect to the free ligand when gallium(III) is added. In particular, methylene protons of the chelator ring, briefly r1 and r2, are no longer chemical shift equivalent since the interconversion between pseudo-axial (Ha) and pseudo-equatorial (He) conformation is prevented by metal complexation. Geminal protons Ha(r1) and He(r1) appear as broad triplets at 4.00 ppm and 3.38, respectively, due to similar values of geminal (2JHH) and vicinal (3JHH) coupling constants (~14Hz). The same situation occurs for Ha(r2) and He(r2) that fall at 3.47 ppm and 3.41 ppm, respectively. Assignments of ring protons was confirmed by 1H,13C-HSQC spectrum, Ha/He(r1) show cross-peaks with carbon at 54.65 ppm (C(r1)) and Ha/He(r2) with a carbon at 57.11 ppm (C(r2)). The methylene groups of acetate arms are not chemical shift equivalent in gallium complex and appear as broad singlets in proton spectrum. The following reasonable assignments can be proposed (for atom numbering refers to Section 4.1.1 and Section 4.1.2): CH2 in α to amide and nitrogen (atom numbering 15) 1H- 3.79 ppm and 13C- 60.64 ppm; CH2 in α to carboxylic group involved in gallium coordination (atom numbering 24 and 26) 1H- 3.83 ppm and 13C- 59.41 ppm; CH2 in α to free carboxylic group (atom numbering 25) 1H- 3.74 ppm, 13C- 61.68 ppm.

      Moreover, UV absorption and fluorescent emission spectra upon increasing addition of Ga3+ from free ligand were acquired (Figures S9, S10). These analyses showed that DOTA-C21 displays an absorption maximum at 410 nm, typical of curcumin structure in the keto-enol form. Upon addition of gallium (III) solution, an increase in absorbance is observed at 290 nm, while no changes are observed for the absorption maximum. Fluorescence emission spectra of DOTA-C21 (λex 410 nm) do not show any significant change after gallium(III) addition.

      2.2. Radiolabelling of the Chelator-Curcuminoid Derivatives with Gallium-68

      Radiolabelling was performed using two methods. In the first, postprocessing of the generator elution was performed by cation exchange purification [] and kinetics of the labelling were studied between 1 and 20 min by TLC. Kinetics were fast and the incorporation yield was 97 ± 2% after 5 min at 95 °C (n = 3). Postprocessing of the generator elution minimizes the metal cation impurities allowing a complete radiolabelling of DOTA-C21 by using only 10 nmol of the precursor. Representative TLC analyses are shown in Figure S11. In the second method, labelling without processing of the elution was attempted with increasing amount of precursor (20, 40, 60, 80 nmol). In this case, a reproducible radiolabelling could only be achieved with 80 nmol of DOTA-C21 after 10 min at 95 °C, with an incorporation yield ranging from 60 to 80%. The crude mixture was further purified by SPE to eliminate unreacted gallium-68 and hydrolysed products. Radiochemical purity was assessed by UHPLC (RCP > 95%, RCY = 55 ± 5%, n = 10). Representative UHPLC chromatograms (radiochemical detector) of a 68Ga-DOTA-C21 preparation before and after SPE purification are shown in Figure 4. Similarly, to the complexation reaction with natural gallium, two main radioactive species were obtained with a retention time of 4.7 and 5.1 min, respectively. Based on the data gathered for the non-radioactive preparation, the peaks were assigned to two 68Ga-DOTA-C21 isomeric complexes. A further peak at 5.3 min was recorded (ca. 4% of total radioactivity) and was likely due to a gallium-68 complexes with a degraded form of the precursor at labelling conditions. In both methods, the pH was kept constant around 4.

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      Representative UHPLC chromatograms (radiochemical detector) of a 68Ga-DOTA-C21 preparation before (A) and after (B) SPE purification. (1) 68Ga-free, (2) 68Ga-hydrolyzed products, (3) 68Ga-labelled byproducts, (4) and (5) 68Ga-DOTA-C21 isomers, (6) 68Ga-labelled degradation product.

      2.3. In Vitro Stability Studies

      In vitro stability of 68Ga-DOTA-C21 complexes in PBS, human serum (HS) and whole human blood (HB) were determined according to peak integration of analytical UHPLC (Figure 5). Stability of 68Ga-DOTA-C21 in PBS was more than 88 ± 3% after 120 min at 37 °C. In HS, the radiotracer was also stable with 85 ± 3% of intact compound after 120 min of incubation. On the contrary, in whole blood, 68Ga-DOTA-C21 was partially degraded to more polar metabolites already after 10 min of incubation. The amount of intact compound ranged from 74 ± 2% after 10 min to 57 ± 3% after 120 min of incubation.

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      Stability of 68Ga-DOTA-C21 complex incubated over time (10, 40, 70, 120 min.) in different media (n = 3, mean ± SD).

      2.4. Uptake, Internalization and Efflux in Colorectal Cancer Cell Line

      In vitro 68Ga-DOTA-C21 uptake studies demonstrated time-dependent cellular accumulation in HT29 colorectal cancer cell line. Total uptake increased continuously in the first 60 min of incubation up to 121 ± 4 KBq of radiotracer per mg of protein (n = 3). At 60 min post incubation, 83% of total radioactivity was internalized in the cells. Conversely, 68GaCl3, used as a negative control, demonstrated significantly lower in vitro cell uptake (20 ± 4 KBq/mg of protein after 60 min of incubation, n = 3) confirming the curcuminoid-mediated uptake by HT29 cells (Figure 6A,B). The cellular uptake of 68Ga-DOTA-C21 is comparable to that of the compound obtained by direct labelling of curcumin with gallium-68 (i.e., 68Ga(CUR)2 complexes []), however the internalized fraction was more than doubled. To test for receptor-mediated uptake, HT29 cells were preincubated with a 200-fold excess of calcitriol, the natural ligand for vitamin D receptor (VDR), and then 68Ga-DOTA-C21 was added. The experiments were performed to assess whether 68Ga-DOTA-C21 uptake was mediated by VDR since it has been reported that curcumin has a structure suitable to bind nuclear vitamin D receptor. The difference between uptake with or without calcitriol was not statistically significant (n = 3, Figure 6C) suggesting that the VDR receptor was not involved in the uptake or that the receptor cannot be saturated in these conditions.

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      Uptake and internalization at different time points of 68Ga-DOTA-C21 in HT-29 colorectal cancer cell line (n = 3, mean ± SD) (A). Comparison between the uptake of 68Ga-DOTA-C21 and 68GaCl3 in HT-29 cells indicating curcumin structure dependent accumulation (n = 3, mean ± SD) (B). Comparison between the uptake of 68Ga-DOTA-C21 and 68Ga-DOTA-C21 + calcitriol in HT-29 cells (n = 3, mean ± SD) (C).

      For efflux studies, cells were incubated with 68Ga-DOTA-C21 and the uptake was stopped after 60 min by removing the supernatant. Then, cells were washed, fresh medium was added and they were incubated again for different periods of time. Media were collected and radioactivity released from the cells was measured.68Ga-DOTA-C21 showed a slow externalization pattern with around 75% (n = 3) of the radiotracer remaining within the cell after 60 min of incubation (Figure 7).

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      Externalization of 68Ga-DOTA-C21 in HT-29 colorectal cancer cell line at different time points (n = 3, mean ± SD).

      2.5. PET Imaging and Biodistribution

      HT29 tumour-bearing mice were injected i.p. with 68Ga-DOTA-C21 and PET/CT imaging was taken 1 h and 2 h post injection (Figure 8). The following day, the same cohort of mice was injected i.v. with 68Ga-DOTA-C21 and dynamic PET/CT imaging was registered for the first 10 min to assess initial distribution of the tracer. The 1 h post injection time point was retaken to evaluate potential differences in the biodistribution pathway related to the type of injection. Mice were sacrificed at 1.5 h post injection, and ex-vivo biodistribution performed. As per PET/CT imaging analysis, upon i.v. injection, 68Ga-DOTA-C21 was taken up rapidly in the heart and liver, and began accumulating into the tumour within the first minute of circulation. The tumour was immediately visible over background (muscle). At 1 h post injection, tumour accumulation of the tracer was 2.27 ± 0.85% ID/cc (n = 3) with a tumour to muscle ratio (T/M) of 1.91 ± 0.47 (n = 3). 68Ga-DOTA-C21 appeared to have both renal and hepatic clearance. Radioactivity in the heart, which reflected the blood pool, was surprisingly high at 1 h post injection (5.8 ± 0.7% ID/cc, n = 3). Similar biodistribution was observed when the tracer was delivered i.p. At 1 h post injection, the tumour had an uptake of 1.89 ± 0.45% ID/cc (n = 5) and a T/M of 3.59 ± 2.76 (n = 5). At 2 h post injection, tumour uptake increased to 2.60 ± 0.27% ID/cc (n = 5) which was reflected by an increase in the T/M to 5.41 ± 3.62 (n = 5) (Figure 9 and Figure S12A–C). It is worth noting that, when analysing tumour uptake in each single animal, there was no appreciable variation between 1 h and 2 h post injection in animals with an initial low tumour uptake. On the contrary, animals with a tumour uptake greater than 4% ID/cc after one hour, tended to accumulate more radiotracer (Figure S12D). No correlation was found between T/M ratio and tumour size (Figure S12E). Ex-vivo biodistribution confirmed the described accumulation (Figure 9). Tumour uptake was 3.08 ± 0.53% ID/cc (n = 5) with a T/M of 2.29 ± 0.62 (n = 5). As already observed by PET, clearance occurred via both renal and hepatobiliary excretion, bone uptake was barely detectable over background and no significant radioactivity was seen in the intracranial region, suggesting that 68Ga-DOTA-C21 is unlikely to cross an intact blood–brain barrier. Blood radioactivity was high, suggesting that a high amount of the radiotracer was in circulation or bound to blood constituents such as serum albumin. Further studies evaluating the clearance mechanism of 68Ga-DOTA-C21 are underway to understand the high blood circulation, however one explanation may be that 68Ga-DOTA-C21 pharmacokinetics follow a two-compartment model [,].

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      Representative microcoronal-PET/CT scans of HT29 colorectal tumour bearing mouse at 60 min (A) and 120 min (B) post i.p. injection of ca. 37 MBq of 68Ga-DOTA-C21.

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      Biodistribution in multiple organs of 68Ga-DOTA-C21 in HT-29 colorectal cancer bearing nude mice (n = 5, mean ± SD) at 90 min post i.v. injection. (A). Ratios of the tumour uptake to major organs (n = 5, mean ± SD) for 68Ga-DOTA-C21 at 90 min post i.v. injection (B).

      2.6. Metabolism

      Mouse blood was drawn 1.25–1.5 h post injection, processed and analysed by radio-HPLC. Out of the two isomers present in the parent compound, one disappears in the blood and only the more polar one (Rt = 7.7 min) persists in all the analysed mice (n = 5) (Figure S13). It is likely that physiological media influence the keto-enol tautomerism of 68Ga-DOTA-C21, stabilizing the diketo form [].

      3. Discussion

      The use of curcumin and its derivatives as potential therapeutics in many medical applications has been largely attested by a long list of scientific literature [,,]. We previously demonstrated that 68Ga-labelled curcumin complexes had preferential accumulation in HT29 colorectal cancer cells compared to other cancer cell lines and lymphocyte cultures []. However, the stability of these complexes in blood was low as the curcuminoid backbone was directly linked to the radio-metal through the keto-enol moiety, resulting in positively charged 2:1 ligand to metal ratio complexes. The present study is focused on evaluating whether a suitable curcumin derivative that is more stably labeled with gallium-68 provides a radiotracer that preferentially accumulates in CRC lesions, and could be a useful early detection agent which enables nuclear medicine imaging techniques. CRC is globally the third most common type of cancer and is usually diagnosed by invasive techniques like sigmoidoscopy or colonoscopy, during which a colon specimen is collected. These procedures are then followed by medical imaging (usually CT scan) to determine if the disease has spread. While small polyps may be removed during colonoscopy, the cancerous state of large polyps may be assessed by a biopsy []. The introduction of a PET radiotracer that specifically accumulates in CRC might be useful for verifying the nature of these lesions without resort to invasive methods. Moreover, treatments used for colorectal cancer may include some combination of surgery, radiation therapy, chemotherapy and targeted therapy and nuclear medicine imaging may be a suitable technique to stage and assess follow-up of the patients.

      In the present study, the structure of the first generation of gallium-68 curcumin complexes has been improved by adding DOTA, a strong gallium-68 chelator. DOTA was linked through an (amino)ethyl spacer to one of the phenol groups of the curcumin backbone. With the addition of DOTA, the new bio-conjugate (namely DOTA-C21) has an enhanced water solubility and a higher stability of the complex with gallium-68 is achieved in physiological conditions. Despite its solubility in water, 1H-NMR spectrum of DOTA-C21 in D2O (Figure S2A) exhibited extremely broad signals in the aromatic region, suggesting that the hydrophobic backbone of curcumin interacted strongly by intramolecular π−π stacking. The signals could be sharpened by reducing the solvent polarity, for example, switching to methanol-d4, which reduced aggregation. The 1H-NMR spectrum in methanol-d4 (Figure S2B) provided signals in the aromatic region attributable to the asymmetric curcumin moiety of DOTA-C21, while methylene signals of the ethyl spacer and DOTA were found in the aliphatic region (3–4.5 ppm). The structure of curcumin in DOTA-C21 is asymmetric due to the alkylation of one phenol group with the (amino)ethyl spacer, as clearly shown by two slightly shifted spin systems with equivalent spectral pattern (Figures S2, S3). Complete assignment and chemical structure with atom numbering is reported in Section 4.1.1.

      As shown in the ESI-LC-MS analysis (Figure S1), DOTA-C21 was present in two isomeric forms that could be assigned to the keto-enol and di-keto tautomer, respectively [,]. As the equilibrium was influenced by solvent polarity and pH, the ratio between the two forms could vary over the different analysis conditions. Due to the rapid interconversion between the two isomeric forms also in physiological conditions, it was not expected that the two isomers exhibited different in vivo properties but this assumption should be verified with further studies.

      When Ga-DOTA-C21 complexes were synthesized mimicking the radiolabelling experimental conditions (95 °C, 30 min, L:M 1.5:1, 0.4 M NH4Ac), complex formation was confirmed by mass spectrometry and two compounds with the same m/z, corresponding to gallium-DOTA-C21 complexes at 1:1 metal to ligand molar ratio, were identified by ESI-LC/MS (Figure S4). The isotopic pattern (dashed square, Figures S4C and S5) highlighted the presence of gallium with its characteristic isotopic distribution in both compounds. Further LC/MS fragmentation experiments were carried out in order to exclude the involvement of the keto-enol moiety in the coordination sphere. As reported in Figure 3, the fragmentation pattern of the two isobaric peaks (panels B and C) were completely equivalent and could be attributed to cleavage of the curcumin structure, while gallium was always strongly bound by DOTA (Figure 10). This finding supported the hypothesis that the two isobaric peaks were due to the keto-enol and di-keto tautomers and not to different coordination modes.

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      Molecular fragments of Ga-DOTA-C21 attributed to the correspondent m/z signals detected in panel B and C of Figure 3.

      To elucidate chelation mode and binding sites in depth, 1H-NMR titration of DOTA-C21 with Ga3+ was performed in methanol-d4 at 25 °C (Figure S6). The signals at 3.2 ppm (methylene groups of tetraazacyclododecane) and at 3.87 ppm (methylene groups in α to carboxylic groups) in the free ligand broadened and shifted at the first Ga3 + addition, suggesting the involvement of the chelator in gallium coordination. A complete overview of 1:1 metal to ligand molar ratio complex formation for both proton and carbon resonances was shown by the overlap of 1H-,13C-HSQC-NMR spectra of the free ligand (DOTA-C21) and its gallium complex (Ga-DOTA-C21) (Figure 2). The metal coordination induces a de-shielding effect on all methylene groups of the chelator ring resulting in an increase of both proton and carbon chemical shifts. Particularly, metal complexation removes chemical shift equivalence of ring methylene protons, as clearly showed by 1H-,1H-COSY and 1H-,13C-HSQC-NMR spectra (Figure S7 and Figure 4). Methylene group r1 shifts from 1H/13C δ (ppm) 3.35/49.77 to 4.00/54.65 and 3.38/54.65, respectively. A lower shift is observed for methylene group r2 from 1H/13C δ (ppm) 3.26/49.64 to 3.47/57.11 and 3.41/57.11, respectively. These outcomes suggest the assignment of type r1 to those methylene groups in α position to carboxylic moiety directly involved in metal coordination (atom numbering 17, 18, 21 and 22). All atom numbering herein reported refers to Figure 11 and Figure 12), while r2 type can be attributed to those methylene groups in α position to non-coordinating carboxylic (amidic) moiety (atom numbering 16, 19, 20 and 23). Methylene groups of the carboxylic arms in α to both nitrogen and carboxylic (amidic) moiety undergo the effect of metal coordination in a different way if the carboxylic group is involved in coordination or not. As shown in Figure 4, methylenes of the carboxylic arms shift from 1H/13C δ (ppm) 3.75–3.87/54.20 to 3.83/59.41, 3.79/60.64 and 3.74/61.68, respectively. Conversely, the chemical shifts of the ethyl spacer (H12 and H13) are unaffected by metal coordination.

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      Chemical structure of DOTA-C21 with atom numbering.

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      Chemical structure of Ga-DOTA-C21 with atom numbering.

      Curcumin and curcuminoids act as fluorophores in the visible spectrum with strong solvent dependence, and consequently may be employed as fluorescent probes for distribution studies in cells and tissues. The typical main absorbance, due to the keto-enol group, was around 420 nm and the emission was around 500 nm, when excited at 420 nm wavelength []. In particular, the absorption of DOTA-C21 showed a maximum at 410 nm in water solution. When a titration with Ga3 + was performed, no variations were observed at λmax suggesting that the keto-enol moiety was not involved in gallium complexation. On the other hand, an increase in absorbance around 300 nm upon metal addition was detected, supporting that DOTA was the main moiety involved in metal chelation (Figure S9). The free ligand showed weak fluorescence emission upon excitation at λmax (410 nm) with a maximum at 525 nm (Figure S10). These results differ from previously reported curcumin studies. In fact, while fluorescence quenching occurred due to gallium chelation by the keto-enol moiety [], herein, emission spectra of DOTA-C21 were only slightly affected by gallium addition. This opposite behaviour furthermore confirmed the involvement of DOTA in gallium chelation rather than the keto-enol group, confirming what was seen by MS and NMR analyses.

      Initially, DOTA-C21 was labelled with gallium-68 by preprocessing the generator elution by cation exchange purification []. This resulted in efficient labelling with high RCY and RCP after 5 min of incubation of just 10 nmol of precursor. In order to simplify the procedure, labelling was also attempted without preprocessing of the generator elutions. In these conditions, labelling was less effective, further purification of the final product was necessary due to the presence of high amounts of hydrolysed products, and 80 nmol of precursor was needed to drive the reaction to completion. This behavior could be ascribed to the presence of high levels of metal impurities that come from the generator and potentially compete with gallium-68 in the coordination with DOTA []. It is worth noting, that DOTA is known to have slow kinetics of complexation and requires harsh conditions for labelling. For these reasons, use of DOTA as a chelator for gallium-68 may enhance hydrolysis of the product and decomposition of the precursor. Use of chelators with smaller cavities such as NOTA and derivatives, or some acyclic chelators recently reported in literature such as HBED-CC or THP, would likely resolve these problems [].

      Similar to other gallium-68 labelled curcuminoid derivatives reported in literature [] but differently from 68GaCl3, 68Ga-DOTA-C21 exhibited a time-dependent uptake in HT29 cells suggesting a relationship between the curcumin-like structure and the ability of the cells to bind and internalize the radiotracers. In particular, the addition of a specific chelator connected to a phenolic moiety appears to be advantageous for the internalization of the compound when compared to the our previously reported tracers where the keto-enol group was exploited to form gallium-68 complexes []. Once internalized or bound to the cell membrane, 68Ga-DOTA-C21 showed a slow efflux rate and 75% of the compound was still retained by the cells after 60 min of incubation. The mechanism by which curcumin or its derivatives are taken up in colorectal tumours has not been elucidated so far. It has been reported that curcumin has a structure suitable to bind nuclear vitamin D receptor and plays a role in colon cancer chemoprevention thanks to this property [,] but no specific experiments have been performed by the authors to confirm this finding. To test the hypothesis of VDR-mediated curcumin uptake, we analysed the VDR expression in HT29 cells and we found that this expression was higher than in other tumour cell lines and human lymphocytes. Then, to assess VDR-specificity, calcitriol, the hormonally active metabolite of vitamin D and VDR natural ligand, was used as a blocking agent. This would clarify the potential involvement of these receptors in 68Ga-DOTA-C21 uptake. Unfortunately, uptake of 68Ga-DOTA-C21 was not influenced by the presence of the excess of calcitriol (Figure 6C) therefore providing evidence that curcumin and calcitrol are not competitive substrates for the same binding site on VDR. In conclusion, seeing as this is a topic of great interest, further studies are necessary to evaluate the cellular uptake mechanism of curcumin and its radiolabelled derivatives. When the mechanism of accumulation will be elucidated, considerations about the structure-affinity of 68Ga-DOTA-C21 in comparison to other compounds will be possible.

      The stability tests in whole blood (Figure 7) attested to a certain lability of the curcumin backbone that was probably partially metabolized into more polar products, which then underwent renal excretion in physiological conditions, as proved during the biodistribution study (kidney uptake 11.86 ± 1.1% ID/g at 1.5 h post injection). However, it was shown that 57 ± 3% of 68Ga-DOTA-C21 complexes remained intact after 120 min of incubation in human blood, comparing favourably with our other previously reported gallium-68 complexes [], where the metal, coordinated by the keto-enol moiety, was rapidly released. Stability of 68Ga-DOTA-C21 in mouse blood appears comparable, if not superior, to previously reported F-18 labelled curcumin derivatives where the amount of unchanged radiotracer in rat plasma after 10 min post injection comprised only 45% of the total injected radioactivity []. 68Ga-DOTA-C21 exhibited a slow absorption rate into the main organs and a high amount of activity (12.73 ± 1.9% ID/g) was recorded in blood after 1.5 h post injection suggesting a strong binding interaction with serum albumin as reported for curcumin and their derivatives in general [,]. Significant uptake of 68Ga-DOTA-C21 occurred in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). 68Ga-DOTA-C21 rapidly accumulated in colorectal xenograft tumours with an uptake of 3.57 ± 0.3% ID/g after 1.5 h post injection. Compared to previously reported 18F-labelled curcumin derivatives, which exhibit fast clearance from the blood to the liver and fast hepatobiliary excretion to the intestine, 68Ga-DOTA-C21 blood clearance was slow and it showed both renal and hepatobiliary excretion. Generally, higher kidney and lower intestine uptake with respect to the fluorinated compounds were found. However, the amount of radiotracer accumulated in the intestine might still be a concern since it could mask the colorectal cancer uptake in a potential human examination. Liver uptake was comparable, indicating that 68Ga-DOTA-C21 remained partially metabolised, likely via hexahydrocurcumin-glucuroniside conjugation route. Similar to 18F-radiolabelled compounds, brain uptake was almost negligible [,].

      4. Materials and Methods

      4.1. General Procedures and Chemicals

      All chemicals were reagent grade and used without further purification unless otherwise specified.

      Complete information related to general laboratory procedures and instrumental analyses are reported in the Supplementary Information file.

      4.1.1. Synthesis of 1,4,7,10-tetraazacyclododecane -1,4,7,10-tetraacetic (1,7-bis(4-hydroxy-3-methoxyphenyl)- 1,6-heptadiene-3,5-dione (DOTA-C21)

      An anhydrous DMF (4 mL) solution of curcumin (100 mg, 0.27 mmol) and potassium carbonate (45 mg, 0.33 mmol) was added to 2-(Boc-amino)ethyl bromide (61 mg, 0.27 mmol). The mixture was stirred for 48 h at room temperature and then concentrated under reduced pressure. The residue was purified by flash silica-gel column chromatography (2% methanol/dichloromethane) to obtain compound 1 (16 mg, 12%) as a red-solid. 1H-NMR (600 MHz, CDCl3) δ 1.45 (s, 9H), 3.54–3.62 (m, 2H), 3.90 (s, 3H), 3.95 (s, 3H), 4.08–4.12 (m, 2H), 5.16–5.18 (m, 1H), 5.79 (s, 1H), 5.85 (s, 1H), 6.49 (dd, 2H, J = 6.0, 16.00 Hz), 6.92 (dd, 2H, J = 8.0, 15.6 Hz), 7.10 (ddd, 4H, J = 2.0, 5.6, 22.4 Hz), 7. 60 (d, 2H, J = 16.0 Hz).

      An anhydrous dichloromethane (2.4 mL) solution of 1 (10.6 mg, 0.02 mmol) was added to TFA (0.6 mL). The mixture was sonicated for 20 min and then concentrated under reduced pressure. The residue was dissolved in DMF (3 mL) and diisopropylethylamine (21 μL, 0.12 mmol) and DOTA-NHS ester (19 mg, 0.025 mmol) were added. The mixture was stirred overnight at room temperature, and then concentrated under reduced pressure. The residue was purified by flash silica-gel column chromatography (20–40% acetonitrile/water containing 0.1% TFA) and freeze-dried to obtain 2 (10 mg, 38.5%) as a yellow-red colour solid. MS (ESI) m/z 798.4 [M + H]+.

      1H-NMR (600 MHz, MeOD-d4) δ: 3.2–3.3 (H16→H23, m broad, 16H), 3.87 (H15/H24/H25/H26, m broad, 8H), 3.65 (H13, t, 2H), 3.93 (H11′, s, 3H), 3.94 (H11, s, 3H), 4.17 (H12, t, 2H), 6.00 (H1, s, 1H), 6.66 (H4′, d, 1H), 6.70 (H4, d, 1H), 6.85 (H9′, d, 1H), 7.05 (H9, d, 1H), 7.14 (H10′, dd, 1H), 7.22 (H10, dd, 1H), 7.24 (H6′, d, 1H), 7.29 (H6, d, 1H), 7.61 (H3′, d, 1H), 7.62 (H3, d, 1H). 13C-NMR (150.9 MHz, MeOD-d4) δ: 184.0 (C2′), 182.7 (C2), 150.1 (C8), 149.6 (C7), 149.2 (C8′), 148.0 (C7′), 141.1 (C3), 140.0 (C3′), 128.8 (C5), 127.1 (C5′), 122.8 (C10′), 122.5 (C10), 122.0 (C4), 120.8 (C4′), 115.2 (C9′), 113.0 (C9), 110.4 (C6/C6′), 100.7 (C1), 67.0 (C12), 55.2 (C11′), 55.1 (C11), 54.4 (C15), 54.1 (C24/C25/C26), 50.0 (C16→C23), 38.6 (C13). Atom numbering refers to Figure 11.

      4.1.2. Preparation of natGa-DOTA-C21 Complexes

      Compound 2 was dissolved in 0.4 M ammonium acetate (450 μg of ligand in 600 μL of buffer, pH 4.5) and stirred at 95 °C for 5 min. Then, 85 µL of 10 mM Ga(NO3)3·9H2O solution were added to the mixture in order to have a 1.5:1 metal to ligand molar ratio. The advancement of the complexation reaction was monitored by ESI-LC-MS. Reaction was completed after 30 min of heating (m/z 864.2–866.2). Complete data on chemical characterization of DOTA-C21 and its gallium complexes are reported in the supplementary material (Figures S1–S10).

      4.2. Radiolabelling of DOTA-C21 with Gallium-68

      Postprocessing based labelling protocol: 68Ge/68Ga generator (EZAG, Berlin, Germany) was eluted with 5 mL 0.1 M HCl and gallium-68 was passed and fixed on a AG50W-X4, 200-400mesh hydrogen form, cartridge (Bio-Rad, Milan, Italy). Gallium-68 was eluted with 0.4 mL of a 97.56% acetone, 0.05 M HCl solution and an aliquot (80 MBq) was added to a vial containing 10 nmol of DOTA-C21 in a 0.2 M ammonium acetate solution (pH 4). The mixtures were incubated at 95 °C and kinetic studies were performed at 1, 3, 5, 10 and 20 min by TLC analysis.

      Direct labelling protocol: 68Ge/68Ga generators were manually eluted with 4 mL of HCl solution (0.1 N or 0.05 N depending whether EZAG or ITG generator was used, respectively). 700 μL of eluates, containing around 200 MBq of gallium-68, were collected in a disposable vial containing 80 nmol of precursors and 65 μL of a 1.5 M sodium acetate in order to maintain the pH of the reaction around 4. The mixture was heated at 95 °C up to 10 min and consequently passed through a light C18 cartridge (Waters, Milan, Italy) to eliminate unlabelled gallium-68 and polar by-products. The cartridge was washed with 3 mL of saline and 1 mL of 10% ethanol solution and then eluted with 1 mL of 95% ethanol solution followed by 2 mL water. Aliquots of the crude mixture and of the final product after purification were collected to assess the radiochemical purity of the product (RCP) by UHPLC analysis. Every preparation was performed at least in triplicate and all the incorporation yields were computed by considering the radiochemical purity (RCP) obtained from the UHPLC or TLC analyses.

      4.3. In Vitro Stability Studies

      For assessing the stability, aliquots of 68Ga-DOTA-C21 solution (1 mL, approx. 5 nmol, 37 MBq) were incubated with 1 mL of i) PBS (0.2M pH = 7.2), ii) human serum (HS), iii) human whole blood (HB) at 37 °C for different time points (10, 40, 70, 120 min). Samples incubated with HB were centrifuged at 3000 rpm for 10 min to precipitate the blood cells and a solution (200 μL) of ACN/H2O/TFA 50/45/5 v/v/v was added to 400 μL of the supernatant. Samples incubated with HS were treated only with ACN/H2O/TFA 50/45/5 v/v/v solution. After another centrifugation under the same conditions, the supernatant was injected into an UPLC for assessing the stability of the preparation.

      4.4. Cell Culture and Animal Models

      HT29 (colorectal adenocarcinoma) cells were kindly provided by Dr Alessandro Zerbini from the Unit of Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria di Parma (Parma, Italy). The cells were grown in DMEM + 10% FBS supplemented with penicillin and streptomycin at 37 °C in a 5% CO2 incubator. To determine protein yield, cells were lysed with a radio immune-precipitation assay (RIPA) buffer (Santa Cruz Biotechnology) and protein concentration was determined with the detergent compatible (DC) protein assay (Bio-Rad) following the manufacturer’s instructions using bovine serum albumin as protein standard.

      All animal experiments were performed in compliance with the guidelines for the care and use of research animals established by The University of Texas MD Anderson Cancer Center (ethic approval number 1179). Mice were maintained in sterile conditions and could eat and drink ad libitum. Mice were housed in a 12 h light-dark cycle. Adult female mice (athymic nude, Taconic Biosciences, 7–8 weeks old) were injected subcutaneously with 9 × 106 HT29 cells in 50% matrigel on the right flank.

      4.5. In Vitro Uptake, Internalization and Efflux in Colorectal Cancer Cell Line

      Cellular uptake, internalization, blocking and efflux of 68Ga-DOTA-C21 were studied in HT29 colon carcinoma cells. All experiments were performed in triplicate (unless otherwise stated). For uptake studies, HT29 cells were incubated at 37 °C with 20 µL (ca. 2 MBq, 0.2 nmol, 0.1 µM final concentration) of 68Ga-DOTA-C21 and 68GaCl3 as a negative control. For blocking experiments HT29 cells were preincubated for 1 h at 37 °C with 100 μL of 1 mg/mL calcitriol solution and then with 20 µL (ca. 2 MBq, 0.2 nmol, 0.1 µM final concentration) of 68Ga-DOTA-C21. Uptake was stopped after 5, 10, 30 and 60 min by removing the medium and the cells were washed twice with ice-cold PBS or 0.1 M (pH 2.9) ice-cold glycine solution for discriminating between the total bound activity and the internalized activity, respectively. Finally, the cells were detached with 2 mL of trypsin/EDTA 0.25% solution at 37 °C and centrifuged in order to separate the supernatant from the cells pellet. The radioactivity associated to the pellets was measured in the dose calibrator and normalized for the protein contents. For efflux studies, cells were incubated with 20 µL (ca. 2 MBq, 0.2 nmol, 0.1 µM final concentration) of 68Ga-DOTA-C21 for 1 h at 37 °C. The uptake was stopped by removing the supernatant and cells were washed twice with ice-cold PBS. Then 1 mL of fresh prewarmed (37 °C) medium was added and cells were newly incubated at 37 °C for 5, 10, 30, 60 min. After these time points, the media were collected and the radioactivity released from the cells was measured in the dose calibrator.

      4.6. Biodistribution of 68Ga-DOTA-C21

      68Ga-DOTA-C21 biodistribution was determined in mice bearing HT29 subcutaneous tumour model (n = 5) at 1.5 h post tail vein injection of 3.7 MBq (1 mL, 0.2 µM solution) of radiotracer. Blood samples were drawn from the right leg using a femoral vein puncture as well as an intra-cardiac puncture to draw blood from the heart to assess the in vivo stability of the radiotracer. The animals were euthanized using 2% isoflurane and exsanguinated, and the thoracic cavity opened. Organs were excised, washed with saline, dried with absorbent tissue paper, counted on a gamma-counter (Packard BioScience Cobra II Auto-Gamma, Meriden, USA) and weighed. Organs of interest included: tumour, heart, spleen, lungs, liver, kidneys, stomach, small intestine, large intestine, muscle, bone and brain. The uptake in each organ was calculated as a percentage of the injected dose per gram of tissue (% ID/g). Blood samples were immediately centrifuged to precipitate the blood cells and a solution (200 μL) of ACN/H2O/TFA 50/45/5 v/v/v was added to 400 μL of the supernatant. After a further centrifugation, the extracted solution was injected into an HPLC.

      4.7. PET Imaging of Tumour Bearing Mice and Analysis

      Mice were briefly anaesthetized (<5 min) using 1% to 2% isoflurane with O2 as a carrier. Mice were injected i.v. or i.p. with 68Ga-DOTA-C21 in sterile phosphate-buffered saline (PBS) (10% EtOH or 50% EtOH respectively) with a target of 3.7 MBq (1 mL, 0.2 µM solution) per mouse. Actual injected dose was calculated based on measuring the pre and postinjection activity in the syringe with a dose calibrator (Capintec, Florham Park, NJ, USA). Mice were then returned to their cages, quickly became ambulatory and could move freely, eat and drink ad libitum for ~45 min. Mice were then anaesthetized using 1% and 3% isoflurane, transferred to a preclinical PET/SPECT/CT system (Albira PET/SPECT/CT, Bruker, Ettlingen, Germany) and maintained at 0.5% to 2% isoflurane with continuous monitoring of respiration during the acquisition. PET images were acquired for 10 min using a 15 cm FOV centred on the tumour; CT images were acquired for fusion using a 7 cm FOV also centred on the tumour. The same procedure was repeated for the 1h and 2 h PET/CT scan. The 10 min PET/CT dynamic scan was recorded immediately after injection of the tracer, and then mice were allowed to awake and freely move around their cages until the 1 h time point. Images were reconstructed using MLEM and FBP for PET and CT, respectively, and automatically fused by the software. Image data were decay corrected to injection time (Albira, Bruker, Ettlingen, Germany) and expressed as % ID/cc (PMOD, PMOD Technologies). Tumour-to-muscle ratios (T/M) were calculated by dividing the activity present in the tumour by the activity present in the muscle.

      4.8. Statistical Analysis

      Student’s t-test was used to determine whether there were any statistically significant differences between the means of two independent (unrelated) groups. The threshold for statistical significance was set at p < 0.05.

      5. Conclusions

      The new DOTA derivative of curcumin (DOTA-C21) displayed coordinating property typical of DOTA structure, with the involvement of nitrogen atoms of the rings and two carboxylic arms, as demonstrated by NMR and MS analysis. Gallium-68 labelling conditions are harsh (95 °C, 5′), however they provide good RCY and RCP in spite of the methods used for the reaction (prepurification of the eluate or direct labelling and postpurification). Despite high instability of curcumin in physiological conditions, stability of 68Ga-DOTA-C21 in mouse blood appears comparable, if not superior, to previously reported 18F-labelled curcumin derivatives, and these data are compatible with diagnostic applications. 68Ga-DOTA-C21 rapidly accumulated in colorectal xenograft tumours but after 1.5 h post injection, exhibits a slow absorption rate into the main organs and a high amount of activity in blood. Unfortunately, the high uptake in blood, liver and intestines prevents the direct use of this derivatives as an imaging agent for colorectal cancer. However, further studies are needed to understand the mechanism of accumulation of 68Ga-DOTA-C21 and to design strategies to increase its selectivity for colorectal tumour since the molecular structure of this radiotracer is an interesting foundation to develop further compounds with improved stability and pharmacokinetics.

      Acknowledgments

      The authors want to thank the University of Modena and Reggio Emilia for financial support (FONDO DI ATENEO PER LA RICERCA ANNO 2015-FAR2015). The authors thank Jean-Philippe Sinnes for the laboratory assistance and Chiara Coruzzi for the bibliographic research as well. The Small Animal Imaging Facility at UT MD Anderson Cancer Center is also acknowledged for assistance with injections and imaging and was supported by NIH P30CA016672.

      Abbreviations

      PBS phosphate buffer saline
      i.p. intra-peritoneal injection
      i.v. intra-venous injection
      DOTA-NHS tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester
      ESI-LC/MS liquid chromatography/mass spectrometry electrospray ionisation
      NH4Ac ammonium acetate
      NMR nuclear magnetic resonance
      HBED-CC: N,N’-Bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N’-diacetic acid
      THP tris(hydroxypyridinone)
      TLC thin layer chromatography; SPE: solid phase extraction
      UHPLC ultra-high performance liquid chromatography
      RCP radiochemical purity
      RCY radiochemical yield
      DMF: N,N-dimethylformamide
      ACN Acetonitrile
      TFA trifluoroacetic acid
      DMEM Dulbecco modified eagle medium
      FBS fetal bovine serum
      EDTA ethylenediaminetetraacetic acid
      PET/SPECT/CT positron emission tomography/single photon emission computed tomography/computed tomography
      FOV field of view; MLEM: maximum likelihood expectation maximization
      FBP filtered back projection

      Supplementary Materials

      The following supplementary materials are available online http://www.mdpi.com/1420-3049/24/3/644/s1: Chemical characterization of ligand and complexes, Quality controls on radioactive preparations, Figure S1: ESI-LC-MS analysis of DOTA-C21,Figure S2: 1H-NMR spectra of DOTA-C21 in D2O and in MeOD-d4 at 25 °C, Figure S3: 13C-NMR spectra of DOTA-C21 in MeOD-d4 at 25 °C, Figure S4: ESI-LC/MS analysis of Ga-DOTA-C21, Figure S5: Detailed ESI-LC/MS of one Ga-DOTA-C21 isomer, Figure S6: 1H-NMR titration of DOTA-C21 with Ga3+, Figure S7: Aliphatic region of 1H-,1H-COSY-NMR spectrum of Ga-DOTA-C21 in MeOD-d4 at 25 °C ([DOTA-C21] = 0.63 mM), Figure S8: LC/MS fragmentation experiments on curcumin, Figure S9: UV–vis absorption spectra of the system Ga3+/DOTA-C21, Figure S10: Fluorescence emission spectra of the system Ga3 + /DOTA-C21,Figure S11: TLC analyses of 68Ga-DOTA-C21 prepared by using the post processing method, Figure S12: Average biodistribution of 68Ga-DOTA-C21 extracted from micro-PET data at various time points post i.p. and i.v. injection. Correlation between T/M ratio and tumour size, Figure S13: In vivo stability of 68Ga-DOTA-C21 in murine blood samples.

      The information comes from:
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384893/

    6. Abstract

      Somatostatin receptors (SSTRs) are variably expressed by a variety of malignancies. Using radiolabeled somatostatin analogs (SSAs), the presence of SSTRs on tumor cells may be exploited for molecular imaging and for peptide receptor radionuclide therapy. 111In-DTPA-octreotide has long been the standard in SSTR scintigraphy. A major leap forward was the introduction of gallium-68 labeled SSAs for positron emission tomography (PET) offering improved sensitivity. Tracers currently in clinical use are 68Ga-DOTA-Tyr3-octreotide (68Ga-DOTATOC), 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE) and 68Ga-DOTA-1-NaI3-octreotide (68Ga-DOTANOC), collectively referred to as 68Ga-DOTA-peptides. 68Ga-DOTA-peptide PET has superseded 111In-DTPA-octreotide scintigraphy as the modality of choice for SSTR imaging. However, implementation of 68Ga-DOTA-peptides in routine clinical practice is often limited by practical, economical and regulatory factors related to the use of the current generation of 68Ge/68Ga generators. Centralized production and distribution is challenging due to the low production yield and relatively short half-life of gallium-68. Furthermore, gallium-68 has a relatively long positron range, compromising spatial resolution on modern PET cameras. Therefore, possibilities of using other PET radionuclides are being explored. On the other hand, new developments in SSTR PET ligands are strongly driven by the need for improved lesion targeting, especially for tumors with low SSTR expression. This may be achieved by using peptide vectors having a higher affinity for the SSTR or a broader affinity profile for the different receptor subtypes or by using compounds recognizing more binding sites, such as SSTR antagonists. This review gives an overview of recent developments leading to the next generation of clinical PET tracers for SSTR imaging.

      Keywords:Somatostatin receptor, PET, SPECT, agonist, antagonist, DOTATATE, DOTATOC, DOTANOC, radionuclide

      Introduction and background

      Somatostatin receptors (SSTRs) are G-protein coupled membrane receptors that were first described in rat pituitary tumor cells by Schonbrunn and Tashjian in 1978 []. Five different human subtypes have been identified, named SSTR1 to 5 []. While the genes for SSTR1, 3, 4 and 5 are intronless, the SSTR2 gene produces two splice variants, SSTR2A and B, differing only in the length of their cytoplasmic tail [,]. SSTRs are expressed by a wide variety of normal human tissues, both in various regions of the brain and peripheral organs, such as the spleen, adrenals, pituitary gland, pancreas, liver, gastro-intestinal tract, kidneys and lungs, each exhibiting a characteristic expression pattern of the different SSTR subtypes [-]. SSTRs have also been identified in several human tumor types. Neuroendocrine tumors (NETs) represent one of the groups with the highest incidence of SSTR expression []. For instance in gastroenteropancreatic (GEP) NETs, SSTRs are present in 80 to 100% of cases, except for insulinomas, which have a lower incidence of 50 to 70% []. Other NETs expressing SSTRs include pituitary adenomas, pheochromocytomas, paragangliomas, lung carcinoids, small-cell lung cancers, Merkel cell carcinomas, medullary thyroid carcinomas and neuroblastomas []. A wide variability in receptor density and subtype expression has been observed across the different NET types, but also within individual tumor types [,]. In the majority of cases, SSTR2 is most abundant, even when other subtypes are present [,]. A large variety of other solid and hematological malignancies may also variably express SSTRs. These include meningiomas, gliomas, lymphomas, and breast, lung, renal cell, pancreato-biliary tract, liver cell, colorectal, ovarian and prostatic carcinomas [,,].

      The presence of SSTRs on tumor cells may open up an important window of opportunity for the clinical management of those tumors in terms of imaging and therapeutic options. Especially in NETs this opportunity has already been extensively exploited. SSTR overexpression is the foundation on which the use of somatostatin analogs (SSAs) such as octreotide in the pharmacological treatment of NETs is based []. According to the most recent European Neuroendocrine Tumor Society (ENETS) consensus guidelines, SSAs are indicated for symptom relief in case of functioning tumors that cause hormone production, as well as for tumor growth inhibition [].

      Furthermore, SSAs can be labeled with radionuclides. These radionuclides either have a heavy nucleus (Z > 83) or possess an imbalance in proton/neutron ratio, or are in a metastable energy state and will undergo radioactive decay. The excess of energy in the nucleus of the unstable element can result in emission of either particles (α, β+/-) and/or electromagnetic radiation (gamma ray photons (γ)) and as a secondary effect X-rays, conversion electrons and Auger electrons. The specific decay characteristics of the radionuclide attached to the vector molecule determine if the radiopharmaceutical can be used for diagnostic (molecular imaging) or therapeutic (targeted radionuclide therapy) purposes. As such, SSTR imaging now occupies a key position in the clinical management of NETs [-]. Moreover, radiolabeling of SSTR targeting agents with therapeutic radionuclides may allow for vectorized radionuclide therapy, also called peptide receptor radionuclide therapy (PRRT). Currently, PRRT by means of radiolabeled SSAs represents an established, evidence-based treatment modality in case of inoperable/metastatic well-differentiated NETs [] and its role has been enforced by the excellent results obtained in the randomized, controlled NETTER-1 trial [].

      In this review, we will focus on molecular imaging of the SSTR and more specifically on the developments leading to the next generation of clinical PET tracers targeting the SSTR. Peptides can be radiolabeled with radiohalogens (e.g. iodine isotopes and fluorine-18) by standard carbon-halogen bond formation or with radiometals (e.g. indium-111 and gallium-68) using suitable bifunctional chelators that are covalently linked to the biologically active peptide []. Therefore, peptide-based radiopharmaceuticals typically consist of the vector moiety (the biologically active peptide) which is linked by means of a chelator, and possibly through an additional linker, to the radionuclide []. Figure 1 shows the chemical structure of all relevant chelators discussed in this review with their matching radionuclides, in combination with the different SSAs that have been applied in clinical practice or clinical studies. Crucial for an effective SSTR tracer is its potential to bind the relevant SSTR []. It is important to realize that even small changes in the amino acid sequence of the peptide or a different choice of chelator or radionuclide might result in a different affinity profile [,] (see also Tables 1 and and2).2). Recent advances in molecular imaging of the SSTR, relating to the choice of radionuclide and vectors molecules, will be discussed. Figure 2 gives an overview of the likely future directions in the field of clinical SSTR PET imaging.

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      Chemical structures of the relevant SSTR tracers that have been applied in clinical practice or clinical studies: radionuclides with their matching chelators or fluorine-18 labeled constructs plus somatostatin receptor agonists or antagonists. (BASS: pNO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-TyrNH2; JR11: Cpa-c[D-Cys-Aph(Hor)D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2).

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      Overview of the likely future directions in the field of clinical SSTR PET imaging.

      Table 1

      In vitro affinity profile (50% inhibitory concentration (IC50) in nM ± standard error of the mean) for the human somatostatin receptor of several somatostatin analogs

      Somatostatin analog SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 Data from
      Octreotide > 10,000 2.0 ± 0.7 187 ± 55 > 1,000 22 ± 6 []
      In-DTPA-octreotide > 10,000 22 ± 3.6 182 ± 13 > 1,000 237 ± 52 []
      Ga-DOTATATE > 10,000 0.2 ± 0.04 > 1,000 300 ± 140 377 ± 18 []
      Ga-DOTATOC > 10,000 2.5 ± 0.5 613 ± 140 > 1,000 73 ± 12 []
      Ga-DOTANOC > 10,000 1.9 ± 0.4 40 ± 5.8 260 ± 74 7.2 ± 1.6 []
      Gluc-Lys-FP-TOCA > 10,000 2.8 ± 0.4 > 1,000 437 ± 84 123 ± 8.8 []
      F-FET-βAG-TOCA NA 4.7 NA 8,600 NA []
      DOTA-lanreotide > 10,000 26 ± 3.4 771 ± 229 > 10,000 73 ± 12 []
      Y-DOTA-lanreotide > 10,000 23 ± 5 290 ± 105 > 10,000 16 ± 3.4 []
      Ga-KE88 9.5 ± 4.3 4.1 ± 1.4 2.7 ± 1.0 4.9 ± 1.4 2.25 ± 0.5 []
      AM3 119 ± 6 2.3 ± 0.2 4.0 ± 0.03 97 ± 21 27 ± 1 []
      DOTA-LTT-SS28 9.8 ± 0.2 2.5 ± 0.3 2.2 ± 0.5 4.8 ± 1.1 2.8 ± 0.3 []
      In-DOTA-LTT-SS28 14 ± 1.2 1.8 ± 0.2 4.0 ± 0.2 5.4 ± 0.3 1.4 ± 0.2 []
      DOTA-BASS > 1,000 1.5 ± 0.4 > 1,000 287 ± 27 > 1,000 []
      In-DOTA-BASS > 1,000 9.4 ± 0.4 > 1,000 380 ± 57 > 1,000 []
      NODAGA-JR11 > 1,000 4.1 ± 0.2 > 1,000 > 1,000 > 1,000 []
      Ga-NODAGA-JR11 > 1,000 1.2 ± 0.2 > 1,000 > 1,000 > 1,000 []
      DOTA-JR11 > 1,000 0.72 ± 0.12 > 1,000 > 1,000 > 1,000 []
      Ga-DOTA-JR11 > 1,000 29 ± 2.7 > 1,000 > 1,000 > 1,000 []
      Lu-DOTA-JR11 > 1,000 0.73 ± 0.15 > 1,000 > 1,000 > 1,000 []

      Structures are explained in Figure 1, except for KE88 (DOTA-D-Dab-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe), AM3 (DOTA-Tyr-cyclo(DAB-Arg-cyclo(Cys-Phe-D-Trp-Lys-Thr-Cys))) and SS28 (somatostatin-28).

      Table 2

      IC50 values (in nM ± standard error of the mean (SEM)) from competitive binding assays in the rat pancreatic cancer cell line AR42J with high SSTR2 expression for several somatostatin analogs

      Somatostatin analog IC50 ± SEM Data from
      In-DTPA-octreotide 6.3 ± 0.9 []
      Ga-DOTATATE 0.20 ± 0.18 []
      Ga-NOTA-octreotide 13 ± 3 []
      AlF-NOTA-octreotide 3.6 ± 0.6 []
      Sc-DOTATATE 0.70 ± 0.20 []

      From SPECT to PET. SSTR imaging: current status

      SSTR imaging was first performed in humans in the late 1980s using 123I-Tyr3-octreotide []. However, due to several disadvantages such as a cumbersome radiolabeling procedure, high cost, limited availability of Na123I, and considerable amount of intestinal accumulation of activity complicating interpretation of images, iodine-123 was soon replaced by indium-111, bound to the peptide by means of the chelator diethylenetriaminepentaacetic acid (DTPA) [,]. 111In-DTPA-octreotide (or 111In-pentetreotide) has long been the standard in SSTR imaging [,]. Indium-111 is a γ-emitting radionuclide, thus imaging is performed by means of planar scintigraphy or single photon emission computed tomography (SPECT), with or without computed tomography (CT). However, there are some drawbacks to the use of indium-111, such as unfavorable nuclear physical characteristics resulting in suboptimal image quality and relatively high effective doses, limited availability and high costs [,]. Successful efforts have been made to label SSAs with technetium-99m instead [-]. One of these compounds is 99mTc-ethylenediamine-N,N’-diacetic acid/hydrazinonicotinamide-Tyr3-octreotide (99mTc-EDDA/HYNIC-TOC) (see Figure 1), which is registered in Poland (99mTc-Tektrotyd; Polatom) and used in several mainly Eastern European countries.

      A major leap forward was the introduction of SSAs labeled with the positron-emitting radionuclide gallium-68 for positron emission tomography (PET) applications. This was made possible by the development of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a macrocyclic chelator capable of forming stable complexes with a multitude of 2+ and 3+ charged radiometals [,] that can be coupled to SSAs (see Figure 1). PET offers several advantages over SPECT, such as a higher sensitivity and spatial resolution and the possibility for straightforward image quantification []. The first clinical publication in this field reported on the use of 68Ga-DOTA-TOC (68Ga-DOTATOC) in patients with meningiomas [], closely followed by a publication on the application of 68Ga-DOTATOC in NET patients [], both in 2001. Since then, 68Ga-labeled SSA PET has rapidly emerged as an established technique for SSTR imaging, especially in NETs where it represents the molecular imaging modality of choice [,]. Tracers currently in clinical use are 68Ga-DOTATOC, 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE) and 68Ga-DOTA-1-NaI3-octreotide (68Ga-DOTANOC), collectively referred to as 68Ga-DOTA-peptides (see Figure 1).

      Table 1 shows the affinity profiles, determined by means of in vitro binding studies, of most of the SSTR PET ligands discussed in this review as compared to 111In-DTPA-octreotide and octreotide. All listed 68Ga-DOTA-peptides show higher affinity for SSTR2 than 111In-DTPA-octreotide [,]. Therefore, in tumors where SSTR2 is the most overexpressed subtype, such as NETs, this offers an additional benefit on top of the physical advantages associated with PET. As such, smaller lesions as well as lesions with low-to-moderate SSTR expression can be detected using 68Ga-DOTA-peptide PET imaging versus 111In-DTPA-octreotide SPECT []. Indeed, several studies comparing both techniques in a head-to-head manner consistently reported a superior performance of 68Ga-DOTA-peptide PET in NET patients [-] (see Table 3). An example is shown in Figure 3. An additional advantage is the fact that 68Ga-DOTATATE and 68Ga-DOTATOC are the theranostic twins of 177Lu-DOTATATE and 90Y-DOTATOC, currently the most frequently used radiopharmaceuticals for PRRT, and are as such ideally suited to identify eligible patients [].

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      Head-to-head comparison of 111In-DTPA-octreotide scintigraphy (A: planar anterior, B: planar posterior, C: transversal SPECT image) and 68Ga-DOTATOC PET (D: maximal-intensity projection, E: transversal slice) of a patient with ileal NET and liver, lymph node and peritoneal metastases (patient data from: []). More lesions can be visualized on the 68Ga-DOTATOC PET images. Dashed lines in (A, B and D) denote the level of transversal slices in (C and E). Scale bar applies to PET images. (SUV = standardized uptake value).

      Table 3

      Comparison between the sensitivity of 111In-DTPA-octreotide planar and/or SPECT and 68Ga-DOTA-peptide PET as reported in several publications

      Study n Gallium-68 peptide Analysis level Sensitivity 111In-DTPA-octreotide Sensitivity 68Ga-DOTA-peptide Δ
      Gabriel et al. [] 84 -TOC Patient 52% 97% 45%
      Buchmann et al. [] 27 -TOC Region 65.1% 97.6% 32.5%
      Van Binnebeek et al. [] 53 -TOC Lesion 60.1% 99.9% 39.8%
      Morgat et al. [] 19 -TOC Lesion 20% 76% 56%
      Srirajaskanthan et al. [] 51 -TATE Lesion 11.9% 74.3% 62.4%
      Deppen et al. [] 78 -TATE Patient 72% 96% 24%
      Sadowski et al. [] 131 -TATE Lesion 30.9% 95.1% 64.2%

      (n = number of patients; Δ = difference between the sensitivity of 111In-DTPA-octreotide and 68Ga-DOTA-peptide imaging).

      The affinity profiles of the 68Ga-DOTA-peptides show some differences (see Table 1), with the most prominent being the fact that 68Ga-DOTANOC also has high affinity for SSTR5 and to a lesser extent for SSTR3, while 68Ga-DOTATOC shows some affinity towards SSTR5 and 68Ga-DOTATATE only binds to SSTR2 [,]. On the other hand, the affinity of 68Ga-DOTATATE for SSTR2 is an order of magnitude higher than that of the other 68Ga-DOTA-peptides. Therefore, some differences in lesion detection rate might be expected. A meta-analysis regarding the diagnostic role of 68Ga-DOTATOC and 68Ga-DOTATATE reported a high sensitivity (93% and 96%, respectively) and specificity (85% and 100%, respectively) for both tracers []. A head-to-head comparison of 68Ga-DOTATOC and 68Ga-DOTATATE in 40 NET patients showed a comparable diagnostic accuracy, although significantly fewer lesions were detected with the latter (262 vs. 254) []. In a similar head-to-head comparison of 68Ga-DOTATATE and 68Ga-DOTANOC in 20 NET patients, a comparable diagnostic accuracy was found as well with both tracers, with a slight, but not statistically significant difference in the number of detected lesions (130 vs. 116) []. Conversely, in another comparison study in 18 GEP NET patients, 68Ga-DOTANOC PET detected significantly more lesions than 68Ga-DOTATATE (238 vs. 212 out of 248 lesions), but the authors state that the clinical relevance of this observation has to be confirmed in larger trials []. Currently, there is no recommendation on which type of 68Ga-DOTA-peptide is preferred [,,] and logistic reasons such as availability of the precursor peptide will guide the choice in clinical practice. Moreover, there are some practical obstacles to the implementation of 68Ga-DOTA-peptide PET that may differ from country to country. Examples are lack of 68Ge/68Ga generators registered or cleared for human use, no availability of precursor peptide for human use, no reimbursement or the implementation is economically not viable.

      Radionuclide

      Gallium-68 has the theoretical advantage that it is available from 68Ge/68Ga generators and as such cyclotron independent. However, despite the excellent results achieved with 68Ga-DOTA-peptides, their use in routine clinical practice is often limited to large nuclear medicine departments. Unlike the 99Mo/99mTc generator, the current generation of 68Ge/68Ga generators requires a dedicated radiopharmacy staff. Moreover, production and quality control of gallium-68 radiopharmaceuticals is subject to strict regulations imposed by pharmaceutical legislation [,]. Nevertheless, due to the significant boost to clinical PET by the introduction of 68Ga-PSMA-HBED-CC [], more and more nuclear medicine departments installed these 68Ge/68Ga generators and production facilities. Furthermore, the aforementioned issues may be largely solved with the next generation 68Ge/68Ga generators that have received regulatory approval (e.g. IRE ELiT, Fleurus, Belgium) and the use of kit-based labeling approaches, such as SomaKit TOCTM (Advanced Accelerator Applications S.A.) and NETSPOT® (Advanced Accelerator Applications USA), that have received approval by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA), respectively. As the overall activity yield per production batch is low (capacity of two to four patients per production) and half-life of gallium-68 is relatively short (68 minutes), there is only limited potential for centralized production and distribution. However, some possibilities may be opened up in this field by advances in the cyclotron production of gallium-68 [].

      Another disadvantage of gallium-68 is its relatively high positron energy (Emean = 0.83 MeV) and thus relatively long positron range (Rmean = 3.5 mm), which may compromise spatial resolution []. Therefore, the possibilities of using other PET radionuclides for SSTR imaging are currently being explored. The physical characteristics of all radionuclides relevant for PET imaging discussed below are summarized in Table 4.

      Table 4

      Physical characteristics relevant for PET imaging of the discussed radionuclides, with Emean and Emax the mean and maximum positron energy, respectively, and Rmean and Rmax the mean and maximum positron range calculated in water, respectively

      Isotope Half-life Positron branching ratio (%) Emean (MeV) Emax (MeV) Rmean (mm) Rmax (mm) Gamma branching ratio (%) Eγ (MeV)
      Fluorine-18 109.8 min 96.9 0.250 0.634 0.6 2.4 - -
      Scandium-44 3.97 h 94.3 0.632 1.474 2.4 6.9 99.9 1.157
      Copper-64 12.7 h 17.5 0.278 0.653 0.8 2.5 0.47 1.346
      Gallium-68 67.8 min 87.7 0.836 1.899 3.5 9.2 3.2 1.077
      1.2 0.353 0.822 1.1 3.4
      88.9 0.829 3.5
      Terbium-152 17.5 h 8.0 1.337 2.97 6.2 15.0 63.5 0.344
      5.9 1.186 2.62 5.4 13.1 9.5 0.271
      20.3 1.14 5.1

      For gallium-68 and terbium-152 only the positrons from the two highest positron branching ratios are listed in italics. The total positron branching ratio, Emean and Rmean are listed in bold. Furthermore, the most relevant prompt gammas are given. Data from the Laboratoire National Henri Becquerel (http://www.nucleide.org/Laraweb), Brookhaven National Laboratory (http://www.nndc.bnl.gov/nudat2), and National Institute of Standards and Technology (https://www.nist.gov/pml/radiation-dosimetry-data).

      Copper-64

      A frequently used non-standard PET radionuclide is copper-64 [,]. Its half-life (12.7 hours) allows for centralized production, while its low positron energy (Emean = 0.28 MeV) corresponding to a short positron range (Rmean = 0.8 mm) allows for high spatial resolution PET imaging [,]. Copper-64 has a relatively low positron branching ratio of 17.5% and its decay is accompanied by emission of β- particles and Auger electrons adding up to its radiation burden. Therefore, it could be used for therapeutic purposes as well, making it suited for theranostic applications [], taking into account extensive shielding needed in practice due to the simultaneous high energy gamma and positron emission. Copper-64 can either be produced in a reactor or with a cyclotron [,].

      As early as 2001, Anderson et al. reported a first clinical evaluation of the dosimetry and pharmacokinetics of 64Cu-TETA-octreotide - copper-64 bound to octreotide through the chelator 1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA) (see Figure 1) - in eight NET patients and its diagnostic properties were compared to 111In-DTPA-octreotide []. In two patients, 64Cu-TETA-octreotide detected clearly more lesions than 111In-DTPA-octreotide. In one patient mild uptake in a lung lesion was observed with 111In-DTPA-octreotide, but not picked up by 64Cu-TETA-octreotide. However, delayed images which might have shown the lesion were not available for this patient. Pharmacokinetic assessment revealed fast blood clearance with partial urinary excretion. On the other hand, the percentage injected activity in the liver increased with time due to dissociation of the copper-64 isotope, indicating poor in vivo stability [,].

      The next clinical studies on copper-64 labeled SSAs date from more than 10 years later. Pfeifer et al. prospectively evaluated 64Cu-DOTATATE in a first-in-human study in 14 NET patients []. PET images with high spatial resolution were obtained. High and stable tumor-to-background ratios were observed on both the early (one hour post injection (p.i.)) and late (three hours p.i.) PET scans, indicating a high tracer internalization rate. Although some dissociation of copper-64 was suggested by increasing hepatic activity, in vivo stability of the tracer was sufficient for imaging purposes. All patients underwent conventional 111In-DTPA-octreotide SPECT/CT as well. Additional lesions were detected on 64Cu-DOTATATE PET in six patients (43%) and in five of these patients lesions were identified in organs not previously known as disease-involved. No lesions were observed with 111In-DTPA-octreotide SPECT that were not revealed by 64Cu-DOTATATE PET.

      In a subsequent larger prospective study, Pfeifer et al. confirmed the diagnostic superiority of 64Cu-DOTATATE PET over 111In-DTPA-octreotide SPECT by means of a head-to-head comparison in 112 NET patients []. PET images were acquired one hour after injection. The sensitivity and diagnostic accuracy of 64Cu-DOTATATE PET (both 97%) were significantly higher than those of conventional 111In-DTPA-octreotide SPECT (87% and 88%, respectively). Twice as many lesions were detected using 64Cu-DOTATATE PET, and more importantly, in 40 patients (36%) lesions were identified in organs not previously known as disease-involved. In 35 of these 40 patients the true-positive nature of these supplemental involved organs was confirmed by long-term follow-up of 42-60 months.

      Of special interest is a recent study published by Johnbeck et al. comparing 64Cu-DOTATATE and 68Ga-DOTATOC PET in 59 NET patients on a head-to-head basis []. On a patient level, 64Cu-DOTATATE and 68Ga-DOTATOC performed equally well. However, 64Cu-DOTATATE detected significantly more additional true-positive lesions, confirmed by at least 30 months of follow-up, than 68Ga-DOTATOC (33 versus 7). The authors attributed this difference in lesion detection rate to the shorter positron range of copper-64, with consequent higher spatial resolution and less partial volume effect, rather than to the use of a different peptide. Figure 4 shows an example of a patient where more lesions are seen in the intestinal region with 64Cu-DOTATATE than with 68Ga-DOTATOC. Tumor-to-background ratios as a measure for image contrast were not significantly different between the two tracers. Although the radiation burden of 64Cu-DOTATATE is higher than that of 68Ga-DOTATOC (5.7-8.9 mSv vs. 2.8-4.6 mSv), the use of 64Cu-DOTATATE offers several advantages for use in routine clinical practices associated to the half-life of copper-64, such as supply to peripheral sites from a central production unit and a more flexible scanning window ranging from one hour to at least three hours after injection. A preclinical evaluation of 64Cu-DOTATOC has been published [], but to our knowledge no subsequent clinical studies have been performed.

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      PET/CT (left) and PET (right) scans with 68Ga-DOTATOC and 64Cu-DOTATATE of a patient with intestinal NET and multiple metastases. Additional lesions are seen in the intestinal region with 64Cu-DOTATATE. This research was originally published in JNM. Johnbeck CB, Knigge U, Loft A, Berthelsen AK, Mortensen J, Oturai P, Langer SW, Elema DR and Kjaer A. Head-to-head comparison of 64Cu-DOTATATE and 68Ga-DOTATOC PET/CT: a prospective study of 59 patients with neuroendocrine tumors. J Nucl Med. 2017;58:451-457. © SNMMI.

      Copper-64 has also been successfully coupled to TATE by means of a new bifunctional chelator, 5-(8-methyl3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), forming 64Cu-SARTATE []. Initial preclinical results are promising showing a high uptake in SSTR2-positive tumors []. Further preclinical and clinical studies on this new radiopharmaceutical are ongoing (Australian New Zealand Clinical Trials Registry (ANZCTR) identifier: ACTRN12615000727549) [].

      Fluorine-18

      Among β+-emitting radioisotopes, fluorine-18 is the most commonly used PET radionuclide in clinical practice and offers several logistic and physical advantages over gallium-68. Large amounts of fluorine-18 activity (> 370 GBq) can be produced with a cyclotron and the half-life (109.8 minutes) is long enough to allow transport to remote hospitals without an on-site cyclotron and it is short enough to avoid extended irradiation of patients. Furthermore, it predominantly decays by positron emission (96.9%) with a low positron energy (Emean = 0.25 MeV) leading to a short positron range (Rmean = 0.6 mm) [].

      Meisetschläger et al. evaluated the fluorine-18 labeled SSA, Gluc-Lys-(18F-fluoropropionyl)-Lys-Tyr3-octreotate (Gluc-Lys-[18F]FP-TOCA) (see Figure 1), in 25 patients with SSTR-positive tumors seen on 111In-DTPA-octreotide scan and performed a direct comparison in 16 of these patients []. Gluc-Lys-[18F]FP-TOCA showed a fast and high tumor uptake and was rapidly cleared from the blood, mainly through the kidneys. Tumor uptake reached a plateau at about 40 minutes after injection. In contrast to SSAs labeled with radiometals whose fragments remain trapped after cellular internalization, no such trapping has been observed for SSAs-based radiopharmaceuticals labeled with fluorine-18 such as Gluc-Lys-[18F]FP-TOCA [,]. Nevertheless, more than twice as many lesions were observed with Gluc-Lys-[18F]FP-TOCA than with 111In-DTPA-octreotide. However, a serious impediment to the implementation of Gluc-Lys-[18F]FP-TOCA in routine clinical practice is its time-consuming multistep synthesis with limited radiochemical yield (20%-30%) []. Gluc-Lys-[18F]FP-TOCA has been applied in a few small clinical studies [-], but to our knowledge no further large clinical trials have been performed.

      18F-fluoroethyl-triazole-Tyr3-octreotate (18F-FET-βAG-TOCA) (see Figure 1) represents an alternative 18F-octreotate radioligand with a more practical and shorter synthesis route and reasonable radiochemical yield []. Following the promising results in preclinical models [], Dubash et al. carried out a first-in-human study in nine NET patients evaluating the biodistribution and dosimetry of 18F-FET-βAG-TOCA []. The tracer showed a rapid blood clearance with both renal and biliary elimination, whereas 68Ga-DOTA-peptides are mainly eliminated through the kidneys. As such, the highest absorbed dose was received by the gallbladder. Overall, the dosimetry of 18F-FET-βAG-TOCA was similar to other fluorine-18 labeled tracers. Tumor-to-background ratios were high and comparable to values reported for 68Ga-DOTA-peptide PET, resulting in images with excellent contrast. Larger clinical trials for this promising tracer, including a direct comparison with 68Ga-DOTATATE PET/CT are currently ongoing [].

      Another fluorine-18 based SSA that is subject of recently initiated clinical trials in NET patients is Al18F-1,4,7-triazacyclononane-1,4,7-triacetate-octreotide (Al18F-NOTA-octreotide) (see Figure 1) (clinicaltrials.gov identifier: NCT03511768). The Al18F-labeling method developed by McBride et al. combines the advantages of a chelator-based radiolabeling method with the unique properties of the radionuclide of choice, fluorine-18 []. In this method, fluorine is firmly bound to Al3+ forming [18F]AlF which is then complexed by a suitable chelator, conjugated to a vector molecule of interest []. Al18F-NOTA-octreotide was developed by Laverman et al. and has been compared to 111In-DTPA-octreotide and 68Ga-NOTA-octreotide in preclinical models [,]. Al18F-NOTA-octreotide proved to have the highest in vitro binding affinity for the SSTR (see Table 2), while a biodistribution in AR42J tumor-bearing mice showed that both tumor uptake and pharmacokinetics were similar with an excellent in vitro and in vivo stability.

      Other promising fluorine-18 based tracers for SSTR imaging identified in preclinical studies include 18F-silicon-fluoride-acceptor (SiFA) and 18F-SiFAlin octreotate derivatives [-], 18F-trifluoroborate octreotate (18F-AMBF3-TATE) [] and 18F-fluoroglycosylated octreotate (18F-FGlc-TATE) [].

      Scandium-44

      Scandium-44 has more recently emerged as a promising radionuclide for PET imaging. There are several methods to produce the radionuclide, for instance by means of a 44Ti/44Sc generator, or using a cyclotron allowing to produce higher quantities [,,]. Scandium-44 mainly decays through positron emission (94.3%) with a somewhat lower positron energy than gallium-68 (Emean = 0.63 MeV) and accordingly lower positron range (Rmean = 2.4 mm). Its half-life of 3.97 hours is convenient for centralized production and distribution []. Even more promising is its use in theranostic applications. Although very similar to gallium(III)-68, the chemical behavior of scandium(III)-44 even more closely resembles that of the therapeutic radiometals, such as lutetium-177 and yttrium-90 []. Therefore, scandium-44 may represent an attractive alternative to gallium-68 for imaging and dosimetry prior to lutetium-177 based therapy []. However, the need for pre-therapy dosimetry has diminished due to the favorable results of the NETTER-1 trial. Moreover, since the half-life of scandium-44 is still limited compared to lutetium-177 (3.97 hours vs. 6.65 days) evaluation at time points later than three days is not possible. With the isotope scandium-47 (100% β- emission), scandium-44 potentially also possesses a true therapeutic match, although research in this area is still in its infancy [,].

      Several preclinical studies have been published on various scandium labeled SSAs, such as 44Sc-DOTATOC [,], natSc-DOTATATE [], 44Sc-DOTANOC [,] and 44Sc-1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid(NODAGA)-NOC [].

      Rösch et al. performed a clinical proof-of-principle study using generator-produced 44Sc-DOTATOC [,]. High quality PET images of a patient with SSTR-positive liver metastases were acquired at early time points and up to 18 hours after injection. Singh et al. published a proof-of-concept study using cyclotron-produced 44Sc-DOTATOC in two patients with metastatic NET []. Interestingly, scandium-44 was produced at the cyclotron facility at the Paul Sherrer Institut (PSI) in Switzerland and subsequently shipped over 600 km to Zentralklinik Bad Berka (ZBB) in Germany, requiring two and a half half-lives. Eight PET/CT scans were performed at various time points up to 23.5 hours after injection. Excellent tumor uptake was observed with increasing tumor-to-background values over the first four hours after injection. Further clinical studies in larger patient cohorts are scheduled.

      Terbium-152

      In light of theranostic applications, terbium gained the interest of researchers due to its four medically relevant radioisotopes: terbium-152 and terbium-155 for PET and SPECT imaging, respectively, and terbium-161 and terbium-149 for β- and α-therapy, respectively [,]. The latter also offers the possibility of PET imaging, as demonstrated in a preclinical study evaluating 149Tb-DOTANOC in AR42J tumor-bearing mice []. Terbium-152, suited for diagnostic PET imaging, has a half-life of 17.5 hours and a positron branching ratio of 20.3% with a relatively high positron energy (Emean = 1.14 MeV) and thus higher positron range (Rmean = 5.1) than gallium-68. Terbium-152 - as well as terbium-149 and -155 - can be produced by high-energy proton-induced spallation in tantalum foil targets []. Just like lutetium-177, it belongs to the group of radiolanthanides. Therefore, it can be stably coupled to the chelator DOTA and used for radiolabeling of SSAs []. In a preclinical study in AR42J tumor-bearing mice, the biodistribution of 152Tb-DOTANOC was found to be in good agreement with that of 177Lu-DOTANOC []. These findings suggest that terbium-152 could serve as a theranostic agent for lutetium-177 based therapy. A clinical proof-of-concept was published by Baum et al. in 2017 using 152Tb-DOTATOC in a patient with well-differentiated metastatic NET of the terminal ileum []. Terbium-152 was produced at the Isotope mass Separator On-Line (ISOLDE) facility in CERN in Switzerland, shipped to PSI for separation from the collection matrix and quality control and finally transported to ZBB in Germany for radiolabeling. PET/CT images were acquired at various time points up to 24 hours after injection. All known tumor lesions, visualized on a previous 68Ga-DOTATOC PET scan, were clearly identified. Images were noisier, compared to this previous gallium-68 based PET. This was attributed to the lack of prompt γ-correction by the PET software. Even at 24 hours after injection, increased uptake was observed in several metastases. The authors concluded that the longer half-life of terbium-152, as compared to gallium-68, enabling imaging at later time-points, makes terbium-152 particularly valuable for dosimetry prior to radionuclide therapy []. However, the production of terbium-152 is challenging and currently imposes an important constraint on its implementation in routine clinical practice.

      Vector

      Developments in SSTR PET radiopharmaceuticals do not only focus on the choice of radionuclide but also on the characteristics of the vector molecule. The radiopharmaceuticals described above contain the somatostatin receptor agonist octreotide, or an analog of octreotide, as vector molecule, with a predominance of TOC and TATE. Improved tumor targeting may be achieved for instance by using vector molecules having a higher binding affinity for the SSTR or a broader affinity profile for the different receptor subtypes or by using compounds recognizing a higher number of binding sites, such as the SSTR antagonists.

      Somatostatin receptor agonists

      Many tumor types that show SSTR expression, predominantly express SSTR2 [], which makes it the main target for the development of SSTR ligands for imaging and therapy. However, as mentioned above, a wide variability in subtype expression has been observed across and within different tumor types []. Therefore, there is great interest in SSTR ligands with a broader affinity profile to increase tumor uptake and to expand the number of tumors eligible for SSTR imaging []. Of the 68Ga-DOTA-peptides currently used in clinical practice, 68Ga-DOTANOC has the widest affinity profile with high affinities for SSTR2, SSTR5 and to a lesser extent SSTR3 []. However, as discussed above, there is no conclusive evidence to prefer this ligand in clinical practice and large trials are warranted [], but properly powered prospective trials testing this hypothesis will probably not be available for a long time. Another SSA that has been evaluated early on in clinical trials is the long-acting SSA lanreotide (see Figure 1). Coupled to the chelator DOTA, DOTA-lanreotide (DOTALAN) and the radiolabeled compounds 111In/90Y-DOTALAN showed a high affinity for SSTR subtype 2-5 []. However, this was only confirmed for SSTR2 and 5 by Reubi et al. []. Following promising clinical results with 111In-DOTALAN [,], DOTALAN was labeled with gallium-68 to allow PET imaging. After an initial positive evaluation of 68Ga-DOTALAN in 11 patients with lung cancer (three small cell and three non-small cell lung cancer) or thyroid cancer (two medullary and three radioiodine negative thyroid cancer) [], the tracer was used to identify patients who might benefit from PRRT with 90Y-DOTALAN in a group of NET patients not qualified for PRRT with 68Ga-DOTATOC despite progressive disease []. Tumor-to-background ratios were significantly higher for 68Ga-DOTATOC and more tumor sites (106 vs. 53) were detected with 68Ga-DOTATOC than with 68Ga-DOTALAN. Demirci et al. compared 68Ga-DOTALAN with 68Ga-DOTATATE in a group of 11 NET patients and one patient with meningioma. 68Ga-DOTATATE performed better than 68Ga-DOTALAN with a significantly higher lesion uptake and higher lesion detection rate (63 vs. 23 out of a total of 67 tumor lesions detected with both tracers) []. Traub-Weidinger et al. evaluated the SSTR status in a heterogeneous group of thyroid cancer patients with progressive disease using two tracers with a distinct SSTR subtype affinity profile, 68Ga-DOTALAN and 68Ga-DOTATOC, in 28 patients []. On a patient basis, 12 patients were negative with both SSTR tracers, while mixed results were observed in three patients (two patients negative with 68Ga-DOTALAN, but positive with 68Ga-DOTATOC and one patient vice versa). On a region-based analysis half of the 38 regions positive on SSTR imaging (out of a total of 196 regions) showed mixed results. The authors concluded that, due to the heterogeneous SSTR profile of thyroid cancer lesions, patients with progressive disease may benefit from imaging with different SSTR PET tracers for individualized targeted therapy stratification.

      In a first attempt to develop an actual pansomatostatin radiopharmaceutical with high affinity for all SSTR subtypes, the cyclooctapeptide KE108 with pansomatostatin characteristics was modified to couple the chelator DOTA (DOTA-D-Dab-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe or KE88) and allow subsequent radiolabeling with indium-111 and gallium-68 []. Although the resulting tracers were able to bind all SSTR subtypes with high affinity, in vitro internalization of the ligand-receptor complex for the SSTR2 was low compared to the SSTR3 and this was reflected by a low in vivo uptake observed in SSTR2-expressing tumors and fast wash out []. In another preclinical study, Fani et al. synthesized and evaluated several bicyclic somatostatin-based analogs of which DOTA-Tyr-cyclo(DAB-Arg-cyclo(Cys-Phe-D-Trp-Lys-Thr-Cys)) or AM3, showing a high affinity for SSTR2, SSTR3 and SSTR5, was the most promising []. Efficient background clearance and high tumor uptake with 68Ga-AM3 were observed in SSTR2 tumor-bearing mice. Tatsi et al. used the native peptide hormone somatostatin-14 (SS14) as a basis to develop two SS14-derived analogs with a high affinity for all SSTR subtypes and label them with indium-111 []. However, both tracers showed poor in vivo stability. Similarly, Maina et al. used the more stable native peptide somatostatin-28 (SS28) as a basis for the development of DOTA-Ser,Leu,D-Trp,Tyr-SS28 (DOTA-LTT-SS28) and the radioligand 111In-DOTA-LTT-SS28 []. The compounds displayed a high affinity for all SSTR subtypes and triggered internalization of SSTR2, SSTR3 and SSTR5. 111In-DOTA-LTT-SS28 showed a high and specific uptake in SSTR2-, SSTR3- and SSTR5-expressing xenografts in mice and a much higher in vivo stability than the SS14-derived tracers developed by Tatsi et al. The authors concluded that 111In-DOTA-LTT-SS28 is the first true (pan)somatostatin radioligand and may serve as a model for the further development of pansomatostatin radioligands []. Very recently, Liu et al. reported the development of gallium-68 labeled pasireotide (SOM230 or PA1), a longacting synthetic SSA with a high affinity for SSTR1, SSTR2, SSTR3 and SSTR5 [,]. The resulting radiotracer 68Ga-DOTA-PA1 displayed a significantly higher in vitro uptake than 68Ga-DOTATATE in three human lung cancer cell lines: lung adenocarcinoma (A549), lung squamous carcinoma (H520) and pulmonary giant cell carcinoma (PG). PET images of A549 tumor-bearing mice showed a high tumor uptake and better signal-to-noise ratio with 68Ga-DOTA-PA1 than with 68Ga-DOTATATE and the PET signal correlated with the total expression of SSTRs and not only SSTR2, as determined by Western blotting. The authors concluded that 68Ga-DOTA-PA1 and its analogs may hold potential for SSTR imaging in clinical practice, especially lung tumors [].

      Therapeutic purposes have also driven some new developments in the vector part of SSTR ligands aiming for higher tumor retention to improve therapeutic efficiency and preferably lower kidney dose to reduce PRRT toxicity. A recent example is given by Tian et al. who conjugated an Evans blue (EB) analog onto octreotate which allows reversible binding of EB-TATE to albumin to prolong half-life in blood, resulting in a SSA with long circulation time []. EB-TATE was then labeled with yttrium-90 through the chelator DOTA and injected in AR42J tumor bearing mice. 90Y-DOTA-EB-TATE showed high tumor uptake resulting in a complete regression of the tumors []. These promising results were quickly translated into a first-in-human clinical trial by Zhang et al. evaluating the safety, pharmacokinetics and dosimetry of 177Lu-DOTA-EB-TATE in five NET patients as compared to 177Lu-DOTATATE in three other NET patients []. The new compound was well tolerated without adverse symptoms. Tumor dose was 7.9-fold higher with 177Lu-DOTA-EB-TATE, while the effective dose was not significantly different between 177Lu-DOTA-EB-TATE and 177Lu-DOTATATE. However, kidney and bone marrow dose increased 3.2- and 18.2-fold, respectively [], so this radiopharmaceutical does not offer an improved therapeutic ratio compared to the current radiopharmaceutical of choice, 177Lu-DOTATATE.

      Somatostatin receptor antagonists

      All SSTR-targeting radiopharmaceuticals described above are somatostatin agonists. After binding to the SSTR, the ligand-receptor complex is usually internalized, allowing tracer metabolites to accumulate in the target cells []. For a long time, it was believed that this process of internalization and subsequent accumulation of the radioligands was essential for high-contrast imaging of SSTR-positive lesions []. However, in 2006, Ginj et al. observed in cell cultures expressing human SSTR2 and SSTR3, that antagonists labeled considerably more receptor sites than agonists []. This was reflected by the significantly higher tumor uptake seen in mice bearing SSTR2 and SSTR3-expressing tumors after injection with the corresponding antagonist as compared to those injected with the agonist []. Especially striking was the fact that counterintuitive to this observation the SSTR2 antagonist, 111In-DOTA-pNO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)D-TyrNH2 (111In-DOTA-BASS) (see Figure 1), showed a more than sevenfold lower affinity for the SSTR2 than the SSTR2 agonist 111In-DTPA-TATE in this study (50% inhibitory concentration (IC50) of 9.4 ± 0.4 nM vs. 1.3 ± 0.2 nM) []. In 2011, Wild et al. published a pilot study in five patients, one metastatic follicular thyroid carcinoma and four NETs, evaluating the biodistribution, tumor uptake and detection of tumor lesions with the SSTR antagonist 111In-DOTA-BASS in a head-to-head comparison with 111In-DTPA-octreotide []. Tumor uptake was up to four times higher with the antagonist, while renal, liver and spleen uptake were lower. 111In-DOTA-BASS detected more lesions than 111In-DTPA-octreotide (25 vs. 17 out of 28). The three missed bone lesions were negative on the 111In-DTPA-octreotide scan as well.

      Further advances in the preclinical setting in search of more potent SSTR2 antagonists labeled with PET radioisotopes led to the identification of Cpa-c[D-Cys-Aph(Hor)D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH2 (JR11) (see Figure 1) as a promising compound for implementation in the clinical field []. Based on the results of previous affinity studies and an in vivo biodistribution study [], 68Ga-NODAGA-JR11 (68Ga-OPS202) was selected for a first clinical evaluation by Nicolas et al. [,]. 12 patients with GEP NET and a positive 68Ga-DOTATOC PET/CT scan in the previous six months were administered two microdoses of 68Ga-NODAGA-JR11 with ascending peptide masses at different study visits and underwent subsequent PET/CT scans. 68Ga-NODAGA-JR11 showed a fast blood clearance and favorable biodistribution with both peptide doses as compared to 68Ga-DOTATOC (lower hepatic, pancreatic, gastro-intestinal and splenic uptake with 68Ga-NODAGA-JR11) [,]. This was reflected by higher tumor-to-background ratios observed with 68Ga-NODAGA-JR11 and further translates to a significantly higher number of detected tumor lesions and higher lesion-based overall sensitivity (94% and 88% for 50 µg and 15 µg 68Ga-NODAGA-JR11, respectively, vs. 59% for 68Ga-DOTATOC) []. An example is shown in Figure 5. The effective dose is in line with 68Ga-DOTA-peptides (24 ± 2 µSv/MBq for 68Ga-NODAGA-JR11 vs. 21 ± 3 µSv/MBq for 68Ga-DOTATATE and 68Ga-DOTATOC []), although some differences in organ doses are observed due to a slightly different biodistribution []. Overall, 68Ga-NODAGA-JR11 was well tolerated []. However, SSTR antagonists could possibly counteract the effects of SSAs, which may be important in patients with functioning NETs []. Therefore, caution is required in anticipation of more safety data []. Currently a multicenter clinical trial evaluating the optimal dose and safety of 68Ga-NODAGA-JR11 for PET imaging is ongoing (clinicaltrials.gov identifier: NCT03220217).

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      Maximal-intensity projections (A and C) and PET/CT (B and D) scans with 68Ga-NODAGA-JR11 (A and B) and 68Ga-DOTATOC of a patient with ileal NET and bilobar liver metastases. Liver magnetic resonance imaging (MRI) was performed four months later, with delayed post-contrast acquisitions (E) and diffusion-weighted images (F), confirming the additional metastases missed or questionable (arrow with question mark) with 68Ga-DOTATOC. Note the lower background activity in the liver, intestine and thyroid with 68Ga-NODAGA-JR11. Dashed lines in (A and C) denote the level of transversal slices in (B, D, E and F). This research was originally published in JNM. Nicolas GP, Schreiter N, Kaul F, Uiters J, Bouterfa H, Kaufmann J, Erlanger TE, Cathomas R, Christ E, Fani M and Wild D. Sensitivity comparison of 68Ga-OPS202 and 68Ga-DOTATOC PET/CT in patients with gastroenteropancreatic neuroendocrine tumors: a prospective phase II imaging study. J Nucl Med. 2018;59:915-921. © SNMMI.

      The higher tumor uptake achieved with SSTR antagonists may also prove useful for therapeutic purposes. Preclinical studies comparing 177Lu-DOTA-JR11 (177Lu-OPS201) with 177Lu-DOTATATE observed higher tumor uptake and longer residence times, resulting in higher tumor doses delivered by the antagonist as compared to the agonist [,]. In a clinical pilot study by Wild et al. in four patients with progressive NETs, more than threefold higher tumor doses and twofold higher tumor-to-kidney and tumor-to-bone marrow dose ratios were observed using a test dose of 177Lu-DOTA-JR11 as compared to 177Lu-DOTATATE []. All patients were subsequently treated with 177Lu-DOTA-JR11, resulting in partial remission in two patients, mixed response in one patient and stable disease in the last patient, and as such proving the clinical feasibility of PRRT using radiolabeled SSTR antagonists. Currently, a multicenter clinical trial evaluating the safety and efficacy of 177Lu-DOTA-JR11 (clinicaltrials.gov identifier: NCT02592707) as well as a study evaluating the theranostic couple 68Ga-DOTA-JR11 and 177Lu-DOTA-JR11 (clinicaltrials.gov identifier: NCT02609737) are ongoing.

      Radiolabeled SSTR antagonists might also prove to be especially useful for imaging and therapy of cancer types with a typically lower SSTR expression such as breast cancer []. Several preclinical studies observed enhanced tumor targeting in various human SSTR2-expressing tumor samples, including breast carcinoma, by means of in vitro autoradiography using an SSTR2 antagonist in comparison to the SSTR2 agonist [-]. This finding was not confirmed in a recent preclinical study by Dude et al. using the human luminal breast cancer model, ZR-75-1, with endogenous SSTR2 expression and negligible expression of other SSTR subtypes, where 68Ga-NODAGA-JR11 had a lower tumor uptake than 68Ga-DOTATOC and 68Ga-DOTATATE []. This was tentatively explained by the authors by the fact that they used an endogenously expressing cell line, which may have a lower amount of low-affinity, antagonist-specific binding sites. Interestingly, although 68Ga-DOTATATE has a higher affinity for the SSTR2 than 68Ga-DOTATOC, the latter was found to have the highest tumor uptake. Additional studies are warranted to further investigate the role of SSTR2 antagonists in breast cancer imaging [].

      Conclusion

      Advances in SSTR PET ligands occur on two major fronts: the radionuclide and the peptide vector. Other radionuclides could offer a solution to practical, economical and regulatory barriers to the adoption of 68Ga-DOTA-peptide PET, with additional physical advantages such as lower positron range and longer half-life. Developments concerning peptide vectors are mainly driven by the need for improved lesion targeting, especially for tumors with low SSTR expression. Therefore, advances on both fronts are largely complementary. Several promising new PET ligands for clinical SSTR imaging are currently in the pipeline and good results have been demonstrated in phase II trials. Clinical adoption in the near future is a realistic scenario.

      Acknowledgements

      This research was funded by the project from “Kom op tegen Kanker”: “PET/MR imaging of the norepinephrine transporter and somatostatin receptor in neural crest and neuroendocrine tumors for better radionuclide therapy selection” and received support from Research Foundation-Flanders (FWO) (G0D8817N). Frederik Cleeren is a Postdoctoral Fellow of FWO (12R3119N). Christophe M. Deroose is a Senior Clinical Investigator at the FWO.

      Disclosure of conflict of interest

      Christophe M. Deroose received grants and personal fees from Novartis, Terumo, AAA, Ipsen, Sirtex, Bayer outside the submitted work.

      The information comes from:
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261874/