68Ga-labeled Peptides Research Articles

Biological Effects Induced by 68Ga-Conjugated Peptides in Human and Rodent Tumor Cell Lines

Positron emission tomography (PET) with 68Ga-labeled peptides is a promising option in the imaging of tumors expressing peptide-binding receptors. PET scanning is non-invasive but involves internal exposure to ionizing radiation which could subsequently generate biological effects, particularly on targeted cells. The aim of the present study was to investigate the potential biological effects of 68Ga-labeled peptides on different targeted tumor cells in vitro. Human U87MG and HT-29 cells and murine AR42J and B16F10 cells were incubated with the following 68Ga-labeled peptides: 68Ga-NODAGA-c(RGDfK), 68Ga-DOTA-c(RGDfK), 68Ga-NOTA-TATE, 68Ga-DOTA-NOC, 68Ga-NODAGA-NOC, or 68Ga-NOTA-(Cys-39)-Exendin-4. Cell cycle analysis and annexin V/PI assay for apoptosis were performed by flow cytometry. Oxidative stress was evaluated by spectrophotometric measuring the level of reactive oxygen species (ROS) and total thiols. After incubation with 37 kBq/ml (1 µCi/ml) 68Ga-labeled peptides, generally observed an increase of apoptosis and cell cycle arrest, especially in G2/M. Furthermore, the increased ROS generation was mostly correlated with apoptosis. Our data demonstrated the ability of 68Ga-labeled peptides to exert significant inhibitory effects on tumor cell lines, through the cycle dysregulation and induction of apoptosis.

In Vivo Characterization of 4 68Ga-Labeled Multimeric RGD Peptides to Image αvβ3 Integrin Expression in 2 Human Tumor Xenograft Mouse Models.

αvβ3 integrins play an important role in angiogenesis and cell migration in cancer and are highly expressed on the activated endothelial cells of newly formed blood vessels. Here, we compare the targeting characteristics of 4 68Ga-labeled multimeric cyclic arginine-glycine-aspartate (RGD)-based tracers in an αvβ3 integrin-expressing tumor model and a tumor model in which αvβ3 integrin is expressed solely on the neovasculature. Methods: Female BALB/c nude mice were subcutaneously injected with SK-RC-52 (αvβ3 integrin-positive) or FaDu (αvβ3 integrin-negative) tumor cells. 68Ga-labeled DOTA-(RGD)2, TRAP-(RGD)3, FSC-(RGD)3, or THP-(RGD)3 was intravenously administered to the mice (0.5 nmol per mouse, 10-20 MBq), followed by small-animal PET/CT imaging and ex vivo biodistribution studies 1 h after injection. Nonspecific uptake of the tracers in both models was determined by coinjecting an excess of unlabeled DOTA-(RGD)2 (50 nmol) along with the radiolabeled tracers. Results: Imaging and biodistribution data showed specific uptake in the tumors for each tracer in both models. Tumor uptake of 68Ga-FSC-(RGD)3 was significantly higher than that of 68Ga-DOTA-(RGD)2, 68Ga-TRAP-(RGD)3, or 68Ga-THP-(RGD)3 in the SK-RC-52 model but not in the FaDu model, in which 68Ga-FSC-(RGD)3 showed significantly higher tumor uptake than 68Ga-TRAP-(RGD)3 Most importantly, differences were also observed in normal tissues and in tumor-to-blood ratios. Conclusion: All tracers showed sufficient targeting of αvβ3 integrin expression to allow for tumor detection. Although the highest tumor uptake was found for 68Ga-FSC-(RGD)3 and 68Ga-THP-(RGD)3 in the SK-RC-52 and FaDu models, respectively, selection of the optimal tracer for specific diagnostic applications also depends on tumor-to-blood ratio and uptake in normal tissues; these factors should therefore also be considered.

A Systematic Comparative Evaluation of 68Ga-Labeled RGD Peptides Conjugated with Different Chelators.

The present paper reports a systematic study on the effect of bifunctional chelators (BFC) namely, NOTA, DOTA, and DTPA, on the radiochemical formulation, in vitro stability, and in vivo biological properties of 68Ga-labeled RGD peptide derivatives. The three RGD conjugates namely, NOTA-Bn-E-[c(RGDfk)]2, DOTA-Bn-E-[c(RGDfk)]2, and DTPA-Bn-E-[c(RGDfk)]2 were radiolabeled with 68Ga and the radiolabeling was optimized with respect to the ligand amount, radiolabeling time, and temperature. Further, the 68Ga complexes were assessed for their in vitro and in vivo stabilities. The biodistribution studies of the three radiolabeled conjugates were carried out in C57BL/6 mice bearing melanoma tumor at 30min and 1h post-adimistration. NOTA-Bn-E-[c(RGDfk)]2 could be radiolabeled with 68Ga at room temperature while DOTA-Bn-E-[c(RGDfk)]2 and DTPA-Bn-E-[c(RGDfk)]2 were radiolabeled at high temperature. 68Ga-NOTA-Bn-E-[c(RGDfk)]2 was found to be the most kinetically rigid in in vitro stability assay. The uptake of the three radiolabeled peptide conjugates in melanoma tumor was comparable at 1h post-administration (NOTA; DOTA; DTPA (% I.D./g):: 2.78±0.38; 3.08±1.1; 3.36±0.49). However, the tumor/background ratio of 68Ga-NOTA-Bn-E-[c(RGDfk)]2 was the best amongst the three radiotracers. 68Ga-complexes of NOTA-Bn-E-[c(RGDfk)]2 and DOTA-Bn-E-[c(RGDfk)]2 showed excellent in vivo stability while 68Ga-DTPA-Bn-E-[c(RGDfk)]2 showed significant metabolic degradation. These studies show that 68Ga-NOTA-Bn-E-[c(RGDfk)]2 would be the most appropriate 68Ga-labeled radiotracer and the most amenable for kit formulation.

Challenges for chemistry in molecular imaging.

Healthcare practice, both in the UK and internationally is in a state of flux. Change is arising both from economic stringencies and new opportunities, underpinned by emerging scientific and technologically driven developments. Arguably, some of the most significant developments involve the recent rapid advances in medical imaging. The technological pull is most acute in the management of chronic disease, where the current population-based treatment strategies ignore many of the opportunities for personalization of care. These opportunities could be seized through early diagnosis and more effective treatments for the individual patient. Advances in imaging are revolutionizing the way to treat diseases, whether it be accurately locating the position of a tumour or providing a method that allows early diagnosis of a disease. In the clinic, around 45 million magnetic resonance imaging (MRI) scans are taken every year, of which 40% use a contrast agent to enhance signal intensity selectively. Radioisotope tracers are equally widely used, e.g. for blood perfusion imaging using positron emission tomography (PET, e.g. 18F-labelled probes such as fluorodeoxyglucose (FDG) or 68Ga-labelled peptides and proteins) or gamma-imaging (SPECT, e.g. with 99mTc, 111In). However, the basic approach in such studies has not changed through the course of this period, and most imaging studies rely on relatively simple …

Evaluation of agonist and antagonist radioligands for somatostatin receptor imaging of breast cancer using positron emission tomography

BackgroundThe somatostatin receptor subtype 2 (sstr2) is expressed on a majority of luminal breast cancers, however SPECT and scintigraphy imaging with agonistic sstr2 probes has been sub-optimal. High affinity antagonists can access more binding sites on the cell surface, resulting in higher tumor uptake and improved sensitivity. We compared the tumor uptake and biodistribution of the antagonist 68Ga-NODAGA-JR11 with two agonists 68Ga-DOTA-Tyr3-octreotide (68Ga-DOTATOC) and 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE), in the human, sstr2-positive, luminal breast cancer model: ZR-75-1.ResultsPeptides were assayed for binding affinity using a filtration-based competitive assay to sstr2. natGa-DOTATOC and natGa-DOTATATE had excellent affinity (inhibition constant Ki: 0.9 ± 0.1 nM and 1.4 ± 0.3 nM respectively) compared to natGa-NODAGA-JR11 (25.9 ± 0.2 nM). The number of binding sites on ZR-75-1 cells was determined in vitro by saturation assays. Agonist 67/natGa-DOTATOC bound to 6.64 ± 0.39 × 104 sites/cells, which was 1.5-fold higher than 67/natGa-NODAGA-JR11 and 2.3-fold higher than 67/natGa-DOTATATE. All three 68Ga-labeled peptides were obtained in good decay-corrected radiochemical yield (61-68%) and were purified by high performance liquid chromatography to ensure high specific activity (137 – 281 MBq/nmol at the end of synthesis). NOD scid gamma mice bearing ZR-75-1 tumors were injected intravenously with the labeled peptides and used for PET/CT imaging and biodistribution at 1 h post-injection. We found that 68Ga-DOTATOC had the highest tumor uptake (18.4 ± 2.9%ID/g), followed by 68Ga-DOTATATE (15.2 ± 2.2%ID/g) and 68Ga-NODAGA-JR11 (12.2 ± 0.8%ID/g). Tumor-to-blood and tumor-to-muscle ratios were also higher for the agonists (>40 and >150 respectively), compared to the antagonist (15.6 ± 2.2 and 45.2 ± 11.6 respectively).ConclusionsThe antagonist 68Ga-NODAGA-JR11 had the lowest tumor uptake and contrast compared to agonists 68Ga-DOTATOC and 68Ga-DOTATATE in ZR-75-1 xenografts.The main contributing factor to this result could be the use of an endogenously expressing cell line, which may differ from previously published transfected models in the number of low-affinity, antagonist-specific binding sites. The relative merit of agonists versus antagonists for sstr2 breast cancer imaging warrants further investigation, first in preclinical models with other sstr2-positive breast cancer xenografts, and ultimately in luminal breast cancer patients.

Practıcal Aspects of 68Ga-Labelled Dota-Peptides for Peptide-Receptor Radionuclide Therapy

68Gallium-labeled receptor ligands, such as 68Gallium DOTATOC, 68Gallium-DOTANOC, and 68Gallium-DOTATATE which are used in PET are becoming gold standard of 68Ga-labeled peptides. The 68Ga-labeled peptides show a rapid renal clearance and are rapidly accumulated in the tumors (80% within 30 minutes); concentration in tissues without expression of somatostatin receptors is low, providing higher contrast imaging. Here, this paper presents the practical aspects and the results of preparation and radiolabelling DOTA-peptides with TiO2-based commercially available 68Ge/68Ga generator by using fully automated radiopharmaceutical synthesis device. The radiochemical purity (RCP) of 68Ga-labeled DOTA-peptides has been investigated using high performance liquid chromatography (HPLC) and instant thin layer chromatography (ITLC). The labeling procedure resulted in high radiochemical yield. The system allows the fully automated, efficient and rapid preparation of 68Ga-DOTA-derivatized peptides.

Targeting Gastrointestinal Stromal Tumor with 68Ga-Labeled Peptides: An In Vitro Study on Gastrointestinal Stromal Tumor-Cell Lines.

The gastrointestinal stromal tumor (GIST) is a rare disease with limited therapeutic options when resistance to tyrosine kinase inhibitor (TKI) treatment occurs. The authors investigated binding of various 68Ga-labeled peptides, targeting receptors reported to be overexpressed in GIST, in different cell lines. For this purpose, three GIST cell lines were tested: GIST-T1, GIST882 (Imatinib sensitive), and GIST430 (Imatinib resistant). DOTA-NT 8-13 (targeting NTR1), DOTA-TATE (targeting SSTR2), CP04 (a minigastrin derivative targeting CCK2-R), VIP-DOTA (targeting VPAC2-R), and 2 DOTA-bombesin derivatives [targeting gastrin releasing peptide receptors (GRPR)] were radiolabeled with 68Ga and incubated with the respective tumor cell and control cell lines. Membrane-bound and internalized activity was measured. Very low or no specific binding to GIST cells was found for all 68Ga-labeled DOTA peptides except for bombesin derivatives indicating no or very low expression of respective receptors. Related to GRPR a pronounced specific binding to all GIST cell lines with no preference related to TKI resistance status was found, both for an agonist (AMBA) with high internalization and for an antagonist (NeoBOMB1) with mainly membrane-bound activity (with up to >80% bound/mg protein). GRPR expression was confirmed by immunohistochemistry. The results show that radiolabeled bombesin analogues, especially antagonists are very promising candidates for targeting GIST.

Evaluation of neuroendocrine tumors with 99mTc-EDDA/HYNIC TOC.

This paper is the short review of our preliminary results obtained with 99mTc-EDDA/HYNIC-TOC. The total of 495 patients with different neuroendocrine tumors were investigated during last few years. There have been 334 true positive (TP), 73 true negative (TN), 6 false positive (FP) and 82 false negative findings (FN). Diagnosis was made according to SPECT findings in 122 patients (25%). The mean T/NT ratio for TP cases was significantly higher (p < 0.01) on SPECT (3.12 ± 1.13) than on whole body scan (2.2 ± 0.75). According to our results, overall sensitivity of the method is 80%, specificity 92%, positive predictive value 98%, negative predictive value 47% and accuracy 82%. Fifteen TP patients underwent therapy with 90Y-DOTATATE. Scintigraphy of neuroendocrine tumors with 99mTc-Tektrotyd is a useful method for diagnosis, staging and follow up of the patients suspected to have neuroendocrine tumors. SPECT had important role in diagnosis. It is also helpful in the appropriate choice of the therapy, including the peptide receptor radionuclide therapy. In the absence of 68Ga-labeled peptides and PET/CT, the special emphasize should be given to application of SPECT/CT as well as to the radioguided surgery.

(18)F- and (68)Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1.

The neurotensin (NT) receptor-1 (NTS1) is overexpressed in a variety of carcinomas and is therefore an interesting target for imaging with positron emission tomography (PET). The aim of this study was the development of new NT derivatives based on the metabolically stable peptide sequence NLys-Lys-Pro-Tyr-Tle-Leu suitable for PET imaging. The NT peptides were synthesized by solid-phase supported peptide synthesis and elongated with respective chelators (NODA-GA, DOTA) for (68)Ga-labeling or propargylglycine for (18)F-labeling via copper-catalyzed azide-alkyne cycloaddition. Receptor affinities of the peptides for NTS1 were in the range of 19-110 nM. Biodistribution studies using HT29 tumor-bearing mice showed highest tumor uptake for [(68)Ga]6 and [(68)Ga]8 and specific binding in small-animal PET studies. The tumor uptake of (68)Ga-labeled peptides in vivo significantly correlated with the in vitro Ki values for NTS1. [(68)Ga]8 displayed an excellent tumor-to-background ratio and could therefore be considered as an appropriate molecular probe for NTS1 imaging by PET.

Radiolabeling of DOTA-like conjugated peptides with generator-produced (68)Ga and using NaCl-based cationic elution method.

Gallium-68 (68Ga) is a generator-produced radionuclide with a short half-life (t½ = 68 min) that is particularly well suited for molecular imaging by positron emission tomography (PET). Methods have been developed to synthesize 68Ga-labeled imaging agents possessing certain drawbacks, such as longer synthesis time because of a required final purification step, the use of organic solvents or concentrated hydrochloric acid (HCl). In our manuscript, we provide a detailed protocol for the use of an advantageous sodium chloride (NaCl)-based method for radiolabeling of chelator-modified peptides for molecular imaging. By working in a lead-shielded hot-cell system, 68Ga3+ of the generator eluate is trapped on a cation exchanger cartridge (100 mg, ∼8 mm long and 5 mm diameter) and then eluted with acidified 5 M NaCl solution directly into a sodium acetate-buffered solution containing a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or DOTA-like chelator-modified peptide. The main advantages of this procedure are the high efficiency and the absence of organic solvents. It can be applied to a variety of peptides, which are stable in 1 M NaCl solution at a pH value of 3–4 during reaction. After labeling, neutralization, sterile filtration and quality control (instant thin-layer chromatography (iTLC), HPLC and pH), the radiopharmaceutical can be directly administered to patients, without determination of organic solvents, which reduces the overall synthesis-to-release time. This procedure has been adapted easily to automated synthesis modules, which leads to a rapid preparation of 68Ga radiopharmaceuticals (12–16 min).

44Sc for labeling of DOTA- and NODAGA-functionalized peptides: preclinical in vitro and in vivo investigations

BackgroundRecently, 44Sc (T1/2 = 3.97 h, Eβ+av = 632 keV, I = 94.3 %) has emerged as an attractive radiometal candidate for PET imaging using DOTA-functionalized biomolecules. The aim of this study was to investigate the potential of using NODAGA for the coordination of 44Sc. Two pairs of DOTA/NODAGA-derivatized peptides were investigated in vitro and in vivo and the results obtained with 44Sc compared with its 68Ga-labeled counterparts.DOTA-RGD and NODAGA-RGD, as well as DOTA-NOC and NODAGA-NOC, were labeled with 44Sc and 68Ga, respectively. The radiopeptides were investigated with regard to their stability in buffer solution and under metal challenge conditions using Fe3+ and Cu2+. Time-dependent biodistribution studies and PET/CT imaging were performed in U87MG and AR42J tumor-bearing mice.ResultsBoth RGD- and NOC-based peptides with a DOTA chelator were readily labeled with 44Sc and 68Ga, respectively, and remained stable over at least 4 half-lives of the corresponding radionuclide. In contrast, the labeling of NODAGA-functionalized peptides with 44Sc was more challenging and the resulting radiopeptides were clearly less stable than the DOTA-derivatized matches. 44Sc-NODAGA peptides were clearly more susceptible to metal challenge than 44Sc-DOTA peptides under the same conditions. Instability of 68Ga-labeled peptides was only observed if they were coordinated with a DOTA in the presence of excess Cu2+. Biodistribution data of the 44Sc-labeled peptides were largely comparable with the data obtained with the 68Ga-labeled counterparts. It was only in the liver tissue that the uptake of 68Ga-labeled DOTA compounds was markedly higher than for the 44Sc-labeled version and this was also visible on PET/CT images. The 44Sc-labeled NODAGA-peptides showed a similar tissue distribution to those of the DOTA peptides without any obvious signs of in vivo instability.ConclusionsAlthough DOTA revealed to be the preferred chelator for stable coordination of 44Sc, the data presented in this work indicate the possibility of using NODAGA in combination with 44Sc. In view of a clinical study, thorough investigations will be necessary regarding the labeling conditions and storage solutions in order to guarantee sufficient stability of 44Sc-labeled NODAGA compounds.

Recommended administered activities for (68)Ga-labelled peptides in paediatric nuclear medicine.

The aim of this study was to establish a method for determining administered activities for (68)Ga-labelled peptides. Dose calculations were based on the weight-independent effective dose model proposed by the EANM paediatric dosage card for use in paediatric nuclear medicine. Previously published time-integrated activity coefficients for (68)Ga-DOTATATE, (68)Ga-DOTATOC and (68)Ga-pentixafor were used to calculate age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card to determine the radiopharmaceutical class of (68)Ga-labelled peptides ("multiples") and to calculate the baseline activities based on an upper limit for administered activity (185MBq) in an adult. All calculated normalization factors suggest that the (68)Ga-labelled peptides are class "B" radiopharmaceuticals. The baseline activity for all compounds is 12.8MBq. In analogy to (18)F-fluoride, we recommend a minimum activity of 14MBq. For paediatric nuclear medicine applications involving (68)Ga-labelled peptides, we suggest determining administered activities based on the formalism proposed in this work. The corresponding effective doses from these procedures will remain age-independent.

68Ga somatostatin analog radiolabelling: The radiopharmacist's point of view

68Ga-labelled peptides imaging has emerged during the last decade. This was possible thanks to innovative development in 68Ge/68Ga generator or bifunctional chelates (BFC) able to complex 68Ga and to be linked to various peptides and thus optimize radiolabelling procedures. This present review focuses on radiolabelling of somatostatine (SMS) analogs with 68Ga for clinical applications. In that sense, is discussed the key parameters influencing the radiolabelling, from the elution of 68Ge/68Ga generator to purification of final product, and through the quality control of 68Ga-SMS analog radiopharmaceuticals, as well as the full automation of the radiolabelling process.

Formulation and radiochemical evaluation of a freeze-dried mixed peptide kit for the preparation of 68Ga-labeled peptides for PET imaging of somatostatin receptor positive neuroendocrine cancers

68Ga-labeled DOTA-coupled somatostatin analogue peptides are regularly being used for the PET imaging of patients suspected to be suffering from various types of neuroendocrine cancers over-expressing somatostatin receptors. The article describes the formulation of a freeze-dried mixed peptide kit containing equal amounts of DOTA-TATE and DOTA-NOC and the radiochemical evaluation of the kit for the easy and convenient preparation of 68Ga-labeled mixed radiopeptides. The simultaneous use of two different peptides in the kit is expected to perceive more cancerous lesions and may have a wider applicability in the diagnosis of neuroendocrine cancers.

PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

A comparison of three (67/68)Ga-labelled exendin-4 derivatives for β-cell imaging on the GLP-1 receptor: the influence of the conjugation site of NODAGA as chelator.

BackgroundVarious diseases derive from pathologically altered β-cells. Their function can be increased, leading to hyperinsulinism, or decreased, resulting in diabetes. Non-invasive imaging of the β-cell-specific glucagon-like peptide receptor-1 (GLP-1R) would allow the assessment of both β-cell mass and derived tumours, potentially improving the diagnosis of various conditions. We tested three new 67/68Ga-labelled derivatives of exendin-4, an agonist of GLP-1R, in vitro and in vivo. We determined the influence of the chelator NODAGA conjugated to resident lysines either at positions 12 and 27 or the C-terminally attached lysine at position 40 on the binding and kinetics of the peptide.MethodsBinding and internalisation of 67Ga-labelled Ex4NOD12, Ex4NOD27 and Ex4NOD40 were tested on Chinese hamster lung (CHL) cells stably transfected to express the GLP-1 receptor (GLP-1R). In vivo biodistribution of 68Ga-labelled peptides was investigated in CD1 nu/nu mice with subcutaneous CHL-GLP-1R positive tumours; the specificity of the binding to GLP-1R was determined by pre-injecting excess peptide.ResultsAll peptides showed good in vitro binding affinities to GLP-1R in the range of 29 to 54 nM. 67/68Ga-Ex4NOD40 and 67/68Ga-Ex4NOD12 show excellent internalisation (>30%) and high specific uptake in GLP-1R positive tissue, but high activity was also found in the kidneys.ConclusionsWe show that of the three peptides, Ga-Ex4NOD40 and Ga-Ex4NOD12 demonstrate the most favourable in vitro properties and in vivo binding to GLP-1R positive tissue. Therefore, we conclude that the lysines at positions 12 and 40 might preferentially be utilised for modifying exendin-4.

Development of Single Vial Kits for Preparation of 68Ga-Labelled Peptides for PET Imaging of Neuroendocrine Tumours

The present work was aimed at the formulation and evaluation of freeze-dried kits of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptides for the preparation of (68)Ga-labelled peptides for PET imaging of neuroendocrine tumours. The (68)GaCl3 was obtained from the locally produced nanoceria-PAN, composite-sorbent-based (68)Ge/(68)Ga generator. Single vial kits of somatostatin analogues DOTA-[Tyr(3)]-octreotide (DOTA-TOC), DOTA-[NaI(3)]-octreotide (DOTA-NOC) and DOTA-Tyr(3)-Thre(8)-octreotide (DOTA-TATE) were formulated. Optimization of radiolabelling with (68)Ga from the in-house generator, characterization, long term evaluation of stability of kits and bioevaluation studies in animals was carried out. DOTA-TOC, DOTA-NOC and DOTA-TATE kits could be successfully formulated. Consistently high radiochemical yields (>95 %) were obtained on radiolabelling with (68)Ga. The radiolabelled peptides exhibited excellent in vitro stability. Biodistribution studies in normal non-tumour bearing Swiss mice revealed fast clearance of activity via renal route as reported for the respective peptides. Availability of ready to use DOTA-peptide kits in conjunction with (68)Ge/(68)Ga generators would pave way for the establishment of (68)Ga radiopharmacy, a long-felt need of the nuclear medicine community.

Cationic eluate pretreatment for automated synthesis of [68Ga]CPCR4.2

Fostered by the clinical success of sst-ligands, the development and evaluation of (68)Ga-labeled peptides have become a very active field in radiopharmaceutical chemistry. Consequently, various new peptide tracers have been developed, e.g. [(68)Ga]CPCR4.2 for in vivo imaging of solid and haematological tumors or [(68)Ga]TRAP(RGD)₃ for imaging of α(v)β₃ integrin expression. As a consequence of different matrices (TiO₂, SnO₂, polymers) exploited in commercial (68)Ge/(68)Ga-generators, HCl of different concentrations (0.05...1.0 M) is used to obtain (68)Ga as starting material for automated syntheses. We have developed a purification method which reduces the eluate volume and adjusts the HCl concentration. The method may potentially allow standardization of the eluate composition of different commercial generators prior to labeling. Recently, a cationic purification process has been reported which allows the pre-fixation of (68)Ga on a Varian SCX cation exchange cartridge and subsequent elution of (68)Ga with acidified NaCl solutions. As part of the development of ready-to-use cassettes for the automated production of (68)Ga-CPCR4.2 using a SCINTOMICS GRP module and an iThemba Labs generator that is eluted with 0.6...1.0 M HCl, we tested and compared the (68)Ga-trapping efficiency of various commercial available cation exchange cartridges, the efficiency of subsequent (68)Ga-elution from these cartridges by means of various protocols and the influence of these variations on the labeling efficiency of [(68)Ga]CPCR4.2, [(68)Ga]TRAP(RGD)₃ and [(68)Ga]DOTATATE/[(68)Ga]DOTANOC. Finally, we transferred the optimized method to the automated, cassette based synthesis of [(68)Ga]CPCR4.2 and the aforementioned peptides. From seven tested cation exchange cartridges, Chromafix PS-H(+) gave the best extraction results (>95%). Moreover, we observed that acidified solutions of 5 M NaCl or 2.5 M CaCl₂ can be used for efficient cartridge elution. Using a disposable cGMP-compliant cassette system, we obtained [(68)Ga]CPCR4.2 in 80% decay-corrected yield and >99% purity. These data were confirmed by the production of [(68)Ga]DOTATATE, [(68)Ga]DOTANOC and [(68)Ga]TRAP(RGD)3 on the otherwise identical cassette system.

18F-Fluorodihydroxyphenylalanine vs other radiopharmaceuticals for imaging neuroendocrine tumours according to their type

6-Fluoro-(18F)-L-3,4-dihydroxyphenylalanine (FDOPA) is an amino acid analogue for positron emission tomography (PET) imaging which has been registered since 2006 in several European Union (EU) countries and by several pharmaceutical firms. Neuroendocrine tumour (NET) imaging is part of its registered indications. NET functional imaging is a very competitive niche, competitors of FDOPA being two well-established radiopharmaceuticals for scintigraphy, 123I-metaiodobenzylguanidine (MIBG) and 111In-pentetreotide, and even more radiopharmaceuticals for PET, including fluorodeoxyglucose (FDG) and somatostatin analogues. Nevertheless, there is no universal single photon emission computed tomography (SPECT) or PET tracer for NET imaging, at least for the moment. FDOPA, as the other PET tracers, is superior in diagnostic performance in a limited number of precise NET types which are currently medullary thyroid cancer, catecholamine-producing tumours with a low aggressiveness and well-differentiated carcinoid tumours of the midgut, and in cases of congenital hyperinsulinism. This article reports on diagnostic performance and impact on management of FDOPA according to the NET type, emphasising the results of comparative studies with other radiopharmaceuticals. By pooling the results of the published studies with a defined standard of truth, patient-based sensitivity to detect recurrent medullary thyroid cancer was 70 % [95 % confidence interval (CI) 62.1–77.6] for FDOPA vs 44 % (95 % CI 35–53.4) for FDG; patient-based sensitivity to detect phaeochromocytoma/paraganglioma was 94 % (95 % CI 91.4–97.1) for FDOPA vs 69 % (95 % CI 60.2–77.1) for 123I-MIBG; and patient-based sensitivity to detect midgut NET was 89 % (95 % CI 80.3–95.3) for FDOPA vs 80 % (95 % CI 69.2–88.4) for somatostatin receptor scintigraphy with a larger gap in lesion-based sensitivity (97 vs 49 %). Previously unpublished FDOPA results from our team are reported in some rare NET, such as small cell prostate cancer, or in emerging indications, such as metastatic NET of unknown primary (CUP-NET) or adrenocorticotropic hormone (ACTH) ectopic production. An evidence-based strategy in NET functional imaging is as yet affected by a low number of comparative studies. Then the suggested diagnostic trees, being a consequence of the analysis of present data, could be modified, for some indications, by a wider experience mainly involving face-to-face studies comparing FDOPA and 68Ga-labelled peptides.

Comparison of cyclic RGD peptides for αvβ3 integrin detection in a rat model of myocardial infarction

BackgroundExpression of αvβ3 integrin is increased after myocardial infarction as part of the repair process. Increased expression of αvβ3 has been shown by molecular imaging with 18F-galacto-RGD in a rat model. The 68Ga-labelled RGD compounds 68Ga-NODAGA-RGD and 68Ga-TRAP(RGD)3 have high specificity and affinity, and may therefore serve as alternatives of 18F-galacto-RGD for integrin imaging.MethodsLeft coronary artery ligation was performed in rats. After 1 week, rats were imaged with [13N]NH3, followed by 18F-galacto-RGD, 68Ga-NODAGA-RGD or 68Ga-TRAP(RGD)3 using a dedicated animal PET/CT device. Rats were killed, and the activity in tissues was measured by gamma counting. The heart was sectioned for autoradiography and histology. Immunohistochemistry was performed on consecutive sections using CD31 for the endothelial cells and CD61 for β3 expression (as part of the αvβ3 receptor).ResultsIn vivo imaging showed focal RGD uptake in the hypoperfused area of infarcted myocardium as defined with [13N]NH3 scan. In autoradiography images, augmented uptake of all RGD tracers was observed within the infarct area as verified by the HE staining. The tracer uptake ratios (infarct vs. remote) were 4.7 ± 0.8 for 18F-galacto-RGD, 5.2 ± 0.8 for 68Ga-NODAGA-RGD, and 4.1 ± 0.7 for 68Ga-TRAP(RGD)3. The 68Ga-NODAGA-RGD ratio was higher compared to 68Ga-TRAP(RGD)3 (p = 0.04), but neither of the 68Ga tracers differed from 18F-galacto-RGD (p > 0.05). The area of augmented 68Ga-RGD uptake was associated with β3 integrin expression (CD61).Conclusion68Ga-NODAGA-RGD and 68Ga-TRAP(RGD)3 uptake was equally increased in the infarct area at 1 week post infarction as 18F-galacto-RGD. These results show the potential of 68Ga-labelled RGD peptides to monitor integrin expression as a part of myocardial repair and angiogenesis after ischaemic injury in vivo.