Automated GMP compliant production of [18F]AlF-NOTA-octreotide.
ABSTRACT: BACKGROUND:Gallium-68 labeled synthetic somatostatin analogs for PET/CT imaging are the current gold standard for somatostatin receptor imaging in neuroendocrine tumor patients. Despite good imaging properties, their use in clinical practice is hampered by the low production levels of 68Ga eluted from a 68Ge/68Ga generator. In contrast, 18F-tracers can be produced in large quantities allowing centralized production and distribution to distant PET centers. [18F]AlF-NOTA-octreotide is a promising tracer that combines a straightforward Al18F-based production procedure with excellent in vivo pharmacokinetics and specific tumor uptake, demonstrated in SSTR2 positive tumor mice. However, advancing towards clinical studies with [18F]AlF-NOTA-octreotide requires the development of an efficient automated GMP production process and additional preclinical studies are necessary to further evaluate the in vivo properties of [18F]AlF-NOTA-octreotide. In this study, we present the automated GMP production of [18F]AlF-NOTA-octreotide on the Trasis AllinOne® radio-synthesizer platform and quality control of the drug product in accordance with GMP. Further, radiometabolite studies were performed and the pharmacokinetics and biodistribution of [18F]AlF-NOTA-octreotide were assessed in healthy rats using ?PET/MR. RESULTS:The production process of [18F]AlF-NOTA-octreotide has been validated by three validation production runs and the tracer was obtained with a final batch activity of 10.8?±?1.3?GBq at end of synthesis with a radiochemical yield of 26.1?±?3.6% (dc), high radiochemical purity and stability (96.3?±?0.2% up to 6?h post synthesis) and an apparent molar activity of 160.5?±?75.3?GBq/?mol. The total synthesis time was 40?±?3?min. Further, the quality control was successfully implemented using validated analytical procedures. Finally, [18F]AlF-NOTA-octreotide showed high in vivo stability and favorable pharmacokinetics with high and specific accumulation in SSTR2-expressing organs in rats. CONCLUSION:This robust and automated production process provides high batch activity of [18F]AlF-NOTA-octreotide allowing centralized production and shipment of the compound to remote PET centers. Further, the production process and quality control developed for [18F]AlF-NOTA-octreotide is easily implementable in a clinical setting and the tracer is a potential clinical alternative for somatostatin directed 68Ga labeled peptides obviating the need for a 68Ge/68Ga-generator. Finally, the favorable in vivo properties of [18F]AlF-NOTA-octreotide in rats, with high and specific accumulation in SSTR2 expressing organs, supports clinical translation.
Project description: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.
Project description:Dosimetry models using preclinical positron emission tomography (PET) data are commonly employed to predict the clinical radiological safety of novel radiotracers. However, unbiased clinical safety profiling remains difficult during the translational exercise from preclinical research to first-in-human studies for novel PET radiotracers. In this study, we assessed PET dosimetry data of six 18F-labelled radiotracers using preclinical dosimetry models, different reconstruction methods and quantified the biases of these predictions relative to measured clinical doses to ease translation of new PET radiotracers to first-in-human studies. Whole-body PET images were taken from rats over 240 min after intravenous radiotracer bolus injection. Four existing and two novel PET radiotracers were investigated: [18F]FDG, [18F]AlF-NOTA-RGDfK, [18F]AlF-NOTA-octreotide ([18F]AlF-NOTA-OC), [18F]AlF-NOTA-NOC, [18F]ENC2015 and [18F]ENC2018. Filtered-back projection (FBP) and iterative methods were used for reconstruction of PET data. Predicted and true clinical absorbed doses for [18F]FDG and [18F]AlF-NOTA-OC were then used to quantify bias of preclinical model predictions versus clinical measurements. Our results show that most dosimetry models were biased in their predicted clinical dosimetry compared to empirical values. Therefore, normalization of rat:human organ sizes and correction for reconstruction method biases are required to achieve higher precision of dosimetry estimates.
Project description:BACKGROUND:68Ga-labelled peptides targeting somatostatin receptor 2 (SSTR2) have demonstrated encouraging results in managing patients with neuroendocrine tumours (NETs). In addition to metal chelation, bifunctional chelators have also been found to impact imaging outcomes due to their differences in stability, charge, hydrophilicity, etc. In the present work, a comparative pharmacokinetic evaluation and imaging characteristics were performed between 68Ga-labelled somatostatin analogues (TATE) using NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as bifunctional chelating agents (BFCAs). RESULTS:Both 68Ga-NOTA-TATE and 68Ga-DOTA-TATE were obtained with high radiochemical purity. 68Ga-NOTA-TATE demonstrated higher in vitro stability (? 99%) than 68Ga-DOTA-TATE (? 95%) after 3?h of incubation. The water solubilities (partition coefficients, - 1.76 ± 0.06 vs. - 2.72 ± 0.16) and plasma protein binding rates (12.12% vs. 30.6%) were lower for 68Ga-NOTA-TATE than for 68Ga-DOTA-TATE. Differential pharmacokinetics and comparable tumour affinities (within 1?h) were observed in AR42J tumour-bearing mice. Healthy volunteer imaging studies showed comparable distribution patterns of these two imaging agents. However, the maximum standardized uptake values (SUVmax) of the two tracers varied in each organ. The two PET agents demonstrated almost identical SUVmax values in the kidneys. 68Ga-NOTA-TATE did have a lower SUVmax in most other organs compared with 68Ga-DOTA-TATE, including the liver (4.2 vs. 10.1), potentially due to the lower protein binding rate. CONCLUSION:68Ga-NOTA-TATE and 68Ga-DOTA-TATE demonstrated comparable tumour uptake in an AR42J mouse model. An initial clinical study revealed that 68Ga-NOTA-TATE may have reduced background uptake in the major organs such as the liver. Although the subject numbers were limited, further investigation of 68Ga-NOTA-TATE is warranted for detecting SSTR2-positive neuroendocrine tumours.
Project description:BACKGROUND:Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([18F]FDG PET), [18F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover. OBJECTIVES:This study tested the efficacy of gallium-68-labeled DOTATATE (68Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation. METHODS:We confirmed 68Ga-DOTATATE binding in macrophages and excised carotid plaques. 68Ga-DOTATATE PET imaging was compared to [18F]FDG PET imaging in 42 patients with atherosclerosis. RESULTS:Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). 68Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [18F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [18F]FDG mTBRmax differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [18F]FDG spillover rendered coronary [18F]FDG scans uninterpretable in 27 patients (64%). Coronary 68Ga-DOTATATE PET scans were readable in all patients. CONCLUSIONS:We validated 68Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that 68Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [18F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188).
Project description:C-X-C motif chemokine receptor 4 (CXCR4) and somatostatin receptors (SSTR) are overexpressed in gastro-entero-pancreatic neuroendocrine tumors (GEP-NET). In this study, we aimed to elucidate the feasibility of non-invasive CXCR4 positron emission tomography/computed tomography (PET/CT) imaging in GEP-NET patients using [68Ga]Pentixafor in comparison to 68Ga-DOTA-D-Phe-Tyr3-octreotide ([68Ga]DOTATOC) and 18F-fluorodeoxyglucose ([18F]FDG). Twelve patients with histologically proven GEP-NET (3xG1, 4xG2, 5xG3) underwent [68Ga]DOTATOC, [18F]FDG, and [68Ga]Pentixafor PET/CT for staging and planning of the therapeutic management. Scans were analyzed on a patient as well as on a lesion basis and compared to immunohistochemical staining patterns of CXCR4 and somatostatin receptors SSTR2a and SSTR5. [68Ga]Pentixafor visualized tumor lesions in 6/12 subjects, whereas [18F]FDG revealed sites of disease in 10/12 and [68Ga]DOTATOC in 11/12 patients, respectively. Regarding sensitivity, SSTR-directed PET was the superior imaging modality in all G1 and G2 NET. CXCR4-directed PET was negative in all G1 NET. In contrast, 50% of G2 and 80% of G3 patients exhibited [68Ga]Pentixafor-positive tumor lesions. Whereas CXCR4 seems to play only a limited role in detecting well-differentiated NET, increasing receptor expression could be non-invasively observed with increasing tumor grade. Thus, [68Ga]Pentixafor PET/CT might serve as non-invasive read-out for evaluating the possibility of CXCR4-directed endoradiotherapy in advanced dedifferentiated SSTR-negative tumors.
Project description:The trend to inform personalized molecular radiotherapy with molecular imaging diagnostics, a concept referred to as theranostics, has transformed the field of nuclear medicine in recent years. The development of theranostic pairs comprising somatostatin receptor (SSTR)-targeting nuclear imaging probes and therapeutic agents for the treatment of patients with neuroendocrine tumors (NETs) has been a driving force behind this development. With the Neuroendocrine Tumor Therapy (NETTER-1) phase 3 trial reporting encouraging results in the treatment of well-differentiated, metastatic midgut NETs, peptide radioligand therapy (RLT) with the 177Lu-labeled somatostatin analog (SSA) [177Lu]Lu-DOTA-TATE is now anticipated to become the standard of care. On the diagnostics side, the field is currently dominated by 68Ga-labeled SSAs for the molecular imaging of NETs with positron emission tomography-computed tomography (PET/CT). PET/CT imaging with SSAs such as [68Ga]Ga-DOTA-TATE, [68Ga]Ga-DOTA-TOC, and [68Ga]Ga-DOTA-NOC allows for NET staging with high accuracy and is used to qualify patients for RLT. Driven by the demand for PET/CT imaging of NETs, a commercial kit for the production of [68Ga]Ga-DOTA-TATE (NETSPOT) was approved by the U.S. Food and Drug Administration (FDA). The synthesis of 68Ga-labeled SSAs from a 68Ge/68Ga-generator is straightforward and allows for a decentralized production, but there are economic and logistic difficulties associated with these approaches that warrant the search for a viable, generator-independent alternative. The clinical introduction of an 18F-labeled SSTR-imaging probe can help mitigate the shortcomings of the generator-based synthesis approach, but despite extensive research efforts, none of the proposed 18F-labeled SSAs has been translated past prospective first-in-humans studies so far. Here, we review the current state of probe-development from a translational viewpoint and make a case for a clinically viable, 18F-labeled alternative to the current standard [68Ga]Ga-DOTA-TATE.
Project description:The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [18F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH2 (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many in vivo tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (18F) is the optimal PET radioisotope, and the creation of a [18F]AlF-peptide complex is a simple procedure.C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [18F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [18F]AlF-NOTA-C6 were tested in vitro and in vivo.The non-decay corrected yield of [18F]AlF-NOTA-C6 was 46.2-64.2%, and the radiochemical purity exceeded 95%. [18F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [18F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.[18F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.
Project description:Single domain antibody fragments (sdAbs) labeled with 18F have shown promise for assessing the status of oncological targets such as the human epidermal growth factor receptor 2 (HER2) by positron emission tomography (PET). Earlier, we evaluated two residualizing prosthetic agents for 18F-labeling of anti-HER2 sdAbs; however, these methods resulted in poor labeling yields and high uptake of 18F activity in the kidneys. To potentially mitigate these limitations, we have now developed an 18F labeling method that utilizes the trans-cyclooctene (TCO)-tetrazine (Tz)-based inverse-electron demand Diels-Alder reaction (IEDDAR) in tandem with a renal brush border enzyme-cleavable glycine-lysine (GK) linker in the prosthetic moiety. The HER2-targeted sdAb 2Rs15d was derivatized with TCO-GK-PEG4-NHS or TCO-PEG4-NHS, which lacks the cleavable linker. As an additional control, the non HER2-specific sdAb R3B23 was derivatized with TCO-GK-PEG4-NHS. The resultant sdAb conjugates were labeled with 18F by IEDDAR using [18F]AlF-NOTA-PEG4-methyltetrazine. As a positive control, the 2Rs15d sdAb was radioiodinated using the well-characterized residualizing prosthetic agent, N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB). Synthesis of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d was achieved with an overall radiochemical yield (RCY) of 17.8 ± 1.5% ( n = 5) in 90 min, a significant improvement over prior methods (3-4% in 2-3 h). In vitro assays indicated that [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d bound with high affinity and immunoreactivity to HER2. In normal mice, when normalized to coinjected [125I]SGMIB-2Rs15d, the kidney uptake of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d was 15- and 28-fold lower ( P < 0.001) than that seen for the noncleavable control ([18F]AlF-NOTA-Tz-TCO-2Rs15d) at 1 and 3 h, respectively. Uptake of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d in HER2-expressing SKOV-3 ovarian carcinoma xenografts implanted in athymic mice was about 80% of that seen for coinjected [125I]SGMIB-2Rs15d. On the other hand, kidney uptake was 5-6-fold lower, and as a result, tumor-to-kidney ratios were 4-fold higher for [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d than those for [125I]SGMIB-2Rs15d. SKOV-3 xenografts were clearly delineated even at 1 h after administration of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d by Micro-PET/CT imaging with even higher contrast observed thereafter. In conclusion, this strategy warrants further evaluation for labeling small proteins such as sdAbs because it offers the benefits of good radiochemical yields and enhanced tumor-to-normal tissue ratios, particularly in the kidney.
Project description:The 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.Peptides 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).The 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.
Project description:Phosphaturic mesenchymal tumors (PMTs) are small, typically difficult to localize, and express somatostatin receptors. Recent work suggests imaging studies using 68Gallium (68Ga)-conjugated somatostatin peptide analogues, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)TATE, which enables somatostatin receptor imaging with positron emission tomography (PET), may be useful at identifying these tumors.Our objective was to evaluate the use of 68Ga-DOTATATE PET/computed tomography (CT) for tumor localization in tumor-induced osteomalacia (TIO).This was a single-center prospective study of patients with TIO.The study was conducted at the National Institutes of Health Clinical Center between February 2014 and February 2015.Eleven subjects (six females, five males) with TIO were included.Subjects underwent 68Ga-DOTATATE PET/CT in addition to 111In-pentetreotide single-photon emission CT (Octreoscan- SPECT/CT) and fluorodeoxyglucose-PET/CT (18F FDG-PET/CT) scan.Localization of PMTs on the previously described imaging modalities were determined.The tumor was successfully localized in 6/11 (54.5%) subjects (one was metastatic). The tumor was identified by 68Ga-DOTATATE in all six cases. Both Octreoscan-SPECT/CT and 18F FDG-PET each identified the tumor in 4/6. In no cases was 68Ga-DOTATATE the only imaging study to identify the tumor.In this first prospective study comparing 68Ga-DOTATATE PET/CT to Octreoscan-SPECT/CT and 18F FDG-PET in TIO localization, 68Ga-DOTATATE PET/CT demonstrated the greatest sensitivity and specificity, suggesting that it may be the best single study for localization of PMTs in TIO.