Preclinical evaluation of [68Ga]Ga-DFO for molecular imaging of Vibrio cholerae infection

Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen that causes a variety of life-threatening nosocomial infections. Novel strategies for the development of new antibacterial treatments as well as diagnostic tools are needed. One of the novel diagnostic strategies for the detection of infection could be the utilization of siderophores. Siderophores are low-molecular-weight chelators produced by microbes to scavenge essential iron. Replacing iron in siderophores by suitable radiometals, such as Ga-68 for positron emission tomography (PET) imaging, opens approaches for targeted imaging of infection. Here we report on pyoverdine PAO1 (PVD-PAO1), a siderophore produced by P. aeruginosa, labelled with Ga-68 for specific imaging of Pseudomonas infections. PVD-PAO1 was labelled with Ga-68 with high radiochemical purity. The resulting complex showed hydrophilic properties, low protein binding and high stability in human serum. In vitro uptake of 68Ga-PVD-PAO1 was highly dependent on the type of microbial culture. In normal mice 68Ga-PVD-PAO1 showed rapid pharmacokinetics with urinary excretion. PET imaging in infected animals displayed specific accumulation of 68Ga-PVD-PAO1 in infected tissues and better distribution than clinically used 18F-fluorodeoxyglucose (18F-FDG) and 68Ga-citrate. Ga-68 labelled pyoverdine PAO1 seems to be a promising agent for imaging of P. aeruginosa infections by means of PET.

Radiosynthesis and initial preclinical evaluation of [11C]AZD1283 as a potential P2Y12R PET radiotracer

Small-animal positron emission tomography as a tool for neuropharmacology

Noninvasive imaging techniques for the early detection of infections are in high demand. In this study, we present the development of an infection imaging agent consisting of the antimicrobial peptide fragment UBI (31-38) conjugated to the chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which allows for labeling with the positron emitter Ga-68. The preclinical evaluation of [68 Ga]Ga-NODAGA-UBI (31-38) was conducted to investigate its potential for imaging bacterial infections caused by Staphylococcus aureus. The octapeptide derived from ubiquicidin, UBI (31-38), was synthesized and conjugated with the chelator NODAGA. The conjugate was then radiolabeled with Ga-68. The radiolabeling process and the stability of the radio formulation were confirmed through chromatography. The study included both in vitro evaluations using S. aureus and in vivo evaluations in an animal model of infection and inflammation. Positron emission tomography (PET) and Cherenkov luminescence imaging (CLI) were performed to visualize the targeted localization of the radio formulation at the site of infection. Ex vivo biodistribution studies were carried out to quantify the uptake of the radio formulation in different organs and tissues. Additionally, the uptake of [18 F]Fluorodeoxyglucose ([18 F] FDG) in the animal model was also studied for comparison. The [68 Ga]Ga-NODAGA-UBI (31-38) complex consistently exhibited high radiochemical purity (>90%) after formulation. The complex demonstrated stability in saline, phosphate-buffered saline, and human serum for up to 3 h. Notably, the complex displayed significantly higher uptake in S. aureus, which was inhibited in the presence of unconjugated UBI (29-41) peptide, confirming the specificity of the formulation for bacterial membranes. Bacterial imaging capability was also observed in PET and CLI images. Biodistribution results indicated a substantial target-to-nontarget ratio of approximately 4 at 1 h postinjection of the radio formulation. Conversely, the uptake of [18 F]FDG in the animal model did not allow for the discrimination of infected and inflamed sites. Our studies have demonstrated that [68 Ga]Ga-NODAGA-UBI (31-38) holds promise as a radiotracer for imaging bacterial infections caused by S. aureus.

This research was conducted with the aim of estimating the human absorbed dose of [68Ga]Ga-α-melanocyte-stimulating hormone ([68Ga]Ga-αMSH), a new agent for positron emission tomography (PET) imaging of melanoma cancer. 68Ga was eluted from an in-house developed 68Ge/68Ga generator in the form of [68Ga]GaCl3. αMSH was labeled with 68Ga under optimized conditions by adjusting various parameters that affect the peptide labeling process. The radiochemical purity (RCP) of the radiotracer was assessed using radio thin layer chromatography/high-performance liquid chromatography (RTLC/HPLC). The stability of the complex in human serum and PBS buffer was monitored for up to 2 h post-incubation. The biodistribution of the radiolabeled peptide was studied in tumor-bearing C57 mice. Medical Internal Radiation Dose (MIRD) and mass extrapolation methods were utilized to estimate the absorbed dose of human organs. [68Ga]Ga-αMSH was prepared with RCP>99% and a specific activity> 118 GBq/µmol. The radiolabeled peptide is stable in human serum and PBS buffer. Biodistribution studies in tumor-bearing mice revealed that majority of activity remained in the animal’s body was accumulated at the tumor site. Estimations of absorbed doses in human organs indicated that the kidneys received the highest dose (0.055 mGy/MBq) due to their high uptake and route of activity excretion. The equivalent dose for humans following injection of the [68Ga]Ga-αMSH complex was estimated to be 0.011 mGy/MBq. The results showed that [68Ga]Ga-αMSH has a high potential as a PET imaging agent in melanoma cancer patients. It is considered safe compared to other radiotracers used in clinical studies.

Targeting the programmed death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint blockade therapy plays a critical role in cancer therapy. However, not all patients benefit from this approach, with PD-L1 expression levels being a significant contributing factor. Positron emission tomography (PET) imaging of PD-L1 offers a noninvasive, whole-body, and dynamic assessment of its expression. This study aims to develop a novel peptide-based PD-L1 tracer, [68Ga]HF12, to quantitatively evaluate PD-L1 expression in tumors, thereby offering clinical guidance. HF12 was successfully synthesized and radiolabeled with 68Ga to yield [68Ga]HF12. In vitro binding assays confirmed the specific binding affinity of HF12 for PD-L1 using CHO-hPD-L1 and CHO cell lines. Subsequent in vivo positron emission tomography (PET) imaging and biodistribution studies assessed [68Ga]HF12 for monitoring PD-L1 expression levels in tumor-bearing mice, including those subjected to immunotherapy. Furthermore, PD-L1 expression in tumor tissues was evaluated by using autoradiography, Western blotting, and immunohistochemical (IHC) analysis. The synthesis of [68Ga]HF12 was successfully achieved with a radiochemical purity and yield exceeding 95%. Cellular uptake studies indicated that [68Ga]HF12 demonstrated both high specificity and significant uptake in PD-L1-positive CHO-hPD-L1 cells. Micro-PET imaging and biodistribution studies revealed that [68Ga]HF12 was preferentially accumulated in CHO-hPD-L1 tumors compared to PD-L1-negative CHO tumors. Treatment with Atezolizumab resulted in a significant reduction in [68Ga]HF12 uptake in CHO-hPD-L1 tumors relative to pretreatment levels, whereas no significant changes were observed in the phosphate-buffered saline (PBS) control group. Subsequent biodistribution studies, along with Western blotting and immunohistochemical analyses, confirmed that PD-L1 expression levels in tumors were reduced following immunotherapy, consistent with the results obtained from PET imaging. [68Ga]HF12 was successfully synthesized as a radiotracer for noninvasive quantitative PET imaging of PD-L1 expression levels. This radiotracer exhibited the potential to quantify PD-L1 expression across various tumors, thereby facilitating the prediction of patient response to anti-PD-1 and anti-PD-L1 immunotherapies and monitoring therapeutic efficacy.

Editorial Review of 2014

Positron Emission Tomography—the Promise of Metabolic Imaging

Cyclin-dependent kinase 4 and 6 (CDK4/6) have emerged as interesting therapeutic drug targets with many potential applications in anti-tumors, especially in breast cancer. A novel CDK4/6 kinase-derived positron emission tomography (PET) imaging agent was designed based on palbociclib modified with a chelator DOTA. This new compound with a chelator DOTA-palbociclib was radiolabeled with gallium 68 (68Ga). After labeling, the purity and stability were evaluated, and the blood pharmacokinetics were carried out in normal healthy mice. Human breast cancer MCF-7 (ER+/HER2−) cells were used for in vitro cell uptake tests. PET imaging and ex vivo biodistribution were conducted in MCF-7 tumor-bearing mice. Specific binding of tumors was evaluated by the blocking assay. Furthermore, the uptake of 68Ga-DOTA-palbociclib in tumors was studied by autoradiography of tissue sections followed by immunofluorescence evaluation of CDK4 and CDK6. 68Ga-DOTA-palbociclib was synthesized very simply in a high labeling rate and radiochemical purity in 10 min. The labeling compound showed excellent stability both in vitro and in vivo and exhibited good pharmacokinetics, making it suitable for in vivo imaging. Cell uptake studies display that co-incubation with palbociclib can inhibit cellular uptake of 68Ga-DOTA-palbociclib. In vivo imaging and ex vivo biodistribution in mice bearing MCF-7 tumors both showed obvious radioactive uptake in the tumor and higher tumor-to-muscle ratios, while the tumor radioactivity accumulation was significantly decreased when prior administered with an excess of cold palbociclib, confirming CDK4/6 specific binding of 68Ga-DOTA-palbociclib in vivo. Autoradiography of the avid tumor section showed a high correlation between immunofluorescence with the CDK4/6 positive areas of the tumor, further demonstrating that 68Ga-DOTA-palbociclib specifically targeted CDK4/6 positive tumors. We synthesized 68Ga-DOTA-palbociclib, a new CDK4/6 kinase PET imaging agent, and validated its excellent stability, pharmacokinetics, and specific tumor binding. Based on our primary results, 68Ga-DOTA-palbociclib is a promising imaging agent with the potential to tailor a precise treatment program for CDK4/6 inhibitors.

The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [(99m)Tc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([(99m)Tc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [(68)Ga]GSA for the potential use with positron emission tomography (PET). Labelling of GSA with (68)Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [(68)Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [(68)Ga]GSA distribution in Lewis rats was compared with [(99m)Tc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [(68)Ga]GSA and [(99m)Tc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90). [(68)Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free (68)Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98%). Stability after 120 min incubation at 37 °C was high in PBS (>95% intact tracer) and low in human serum (∼27% intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [(68)Ga]GSA compared to [(99m)Tc]GSA. The [(99m)Tc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [(68)Ga]GSA curves. The mean [(68)Ga]GSA t90 was higher than the mean t90 for [(99m)Tc]GSA. The mean [(68)Ga]GSA t50 was not significantly different from the mean t50 for [(99m)Tc]GSA. This study provides a promising new (68)Ga-labelled compound based on a commercially used kit for imaging the functional hepatocellular mass.

Riboflavin receptor 3 (RFVT3) is a key protein in energetic metabolism reprogramming and is overexpressed in multiple cancers involved in malignant proliferation, angiogenesis, chemotherapy resistance, and immunosuppression. To enable non-invasive real-time quantification of RFVT3 in tumors, we sought to develop a suitable PET probe that would allow specific and selective RFVT3 imaging in vivo. A novel radiofluorinated riboflavin probe (18F-RFTA) based on riboflavin was synthesized and characterized in terms of radiochemical purity, hydrophilicity, binding affinity, and stability. Positron emission tomography (PET) imaging of 18F-RFTA was performed in U87MG tumor-bearing mice. Immunohistochemistry staining was carried out to determine the expression of RFVT3 in U87MG tumors. 18F-RFTA was characterized by high radiochemical purity and RFVT3 binding affinity, and remarkable stability in vitro and in vivo. Small-animal PET imaging with 18F-RFTA revealed significantly higher uptake in RFVT3-expressing U87MG tumors than in muscle. In conclusion, we have developed the first radiofluorinated riboflavin-based PET probe that is suitable for imaging RFVT3-positive tumors. The new target/probe system can be leveraged for extensive use in the diagnosis and treatment of RFVT3 overexpressing diseases, such as oncologic, cardiovascular, and neurodegenerative diseases.

BackgroundHeat shock proteins (HSPs) are present throughout the brain. They function as molecular chaperones, meaning they help with the folding and unfolding of large protein complexes. These chaperones are vital in the development of neuropathological conditions such as Alzheimer’s disease and Lewy body disease, with HSP90, a specific subtype of HSP, playing a key role. Many studies have shown that drugs that inhibit HSP90 activity have beneficial effects in the neurodegenerative diseases. Therefore, HSP90 PET imaging ligand can be used effectively to study HSP90 in neurodegenerative diseases. Among four HSP90 isoforms, two cytosolic isoforms (HSP90α and HSP90β) thought to be involved in the structural homeostasis of the proteins related to the neurodegenerative diseases. Currently, no useful PET imaging ligands selectively targeting the two cytosolic isoforms of HSP90 have been available yet.ResultsIn this study, we developed a novel positron emission tomography (PET) imaging ligand, [11C]BIIB021, by 11C-radiolabeling (a positron emitter with a half-life of 20.4 min) 6-Chloro-9-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-9H-purin-2-amine (BIIB021), an inhibitor with a high affinity for and selectivity to HSP90α and HSP90β. [11C]BIIB021 was synthesized with a high yield, molar activity and radiochemical purity. [11C]BIIB021 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90β proteins. Radioactivity was well detected in the rat brain (SUV 1.4). It showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections. Radiometabolite was detected in the brain, however, total distribution volume was well quantified using dual-input graphical model. Inhibition of p-glycoprotein increased brain radioactivity concentrations. However, total distribution volume values with and without p-glycoprotein inhibition were nearly the same.ConclusionsWe have developed a new PET imaging agent, [11C]BIIB021, specifically targeting HSP90α/β. We have been successful in synthesizing [11C]BIIB021 and in vitro and in vivo imaging HSP90α/β. However, the quantification of HSP90α/β is complicated by the presence of radiometabolites in the brain and the potential to be a substrate for p-glycoprotein. Further efforts are needed to develop radioligand suitable for imaging of HSP90α/β.

[68Ga]Ga-PSMA-11, a urea-based peptidomimetic, is a diagnostic radiopharmaceutical for positron emission tomography (PET) imaging that targets the prostate-specific membrane antigen (PSMA). The recent Food and Drug Administration approval of [68Ga]Ga-PSMA-11 for PET imaging of patients with prostate cancer, expected follow-up approval of companion radiotherapeutics (e.g., [177Lu]Lu-PSMA-617, [225Ac]Ac-PSMA-617) and large prostate cancer patient volumes requiring access are poised to create an unprecedented demand for [68Ga]Ga-PSMA-11 in nuclear medicine clinics around the world. Meeting this global demand is going to require a variety of synthesis methods compatible with 68Ga eluted from a generator or produced on a cyclotron. To address this urgent need in the PET radiochemistry community, herein we report detailed protocols for the synthesis of [68Ga]Ga-PSMA-11, (also known as HBED-CC, Glu-urea-Lys(Ahx)-HBED-CC and PSMA-HBED-CC) using both generator-eluted and cyclotron-produced 68Ga and contrast the pros and cons of each method. The radiosyntheses are automated and have been validated for human use at two sites (University of Michigan (UM), United States; Royal Prince Alfred Hospital (RPA), Australia) and used to produce [68Ga]Ga-PSMA-11 for patient use in good activity yields (single generator, 0.52 GBq (14 mCi); dual generators, 1.04-1.57 GBq (28-42 mCi); cyclotron method (single target), 1.47-1.89 GBq (40-51 mCi); cyclotron method (dual target), 3.63 GBq (98 mCi)) and high radiochemical purity (99%) (UM, n = 645; RPA, n > 600). Both methods are appropriate for clinical production but, in the long term, the method employing cyclotron-produced 68Ga is the most promising for meeting high patient volumes. Quality control testing (visual inspection, pH, radiochemical purity and identity, radionuclidic purity and identity, sterile filter integrity, bacterial endotoxin content, sterility, stability) confirmed doses are suitable for clinical use, and there is no difference in clinical prostate cancer PET imaging using [68Ga]Ga-PSMA-11 prepared using the two production methods.

Stimulator of interferon genes (STING) protein plays a vital role in the immune surveillance of tumor microenvironment. Monitoring STING expression in tumors benefits the relevant STING therapy. This study aimed to develop a novel 18F-labeled agonist, dimeric amidobenzimidazole (diABZI), and firstly evaluate the feasibility of noninvasive positron emission tomography (PET) imaging of STING expression in the tumor microenvironment. An analog of the STING agonist NOTA-DABI was synthesized and labeled with 18F via Al18F-NOTA complexation (denoted as [18F]F-DABI). Physicochemical properties, STING protein-binding affinity, and specificity of [18F]F-DABI were evaluated using cell uptake and docking assays. In vivo small-animal PET imaging and biodistribution studies of [18F]F-DABI in tumor-bearing mice were performed to verify the pharmacokinetics and tumor targeting ability. The correlation between tumor uptake and STING expression was also analyzed. [18F]F-DABI was produced conveniently with high radiochemical yield (44 ± 15%), radiochemical purity (> 97%) and molar activity (15-30GBq/μmol). In vitro binding assays demonstrated that [18F]F-DABI has a favorable affinity and specificity for STING with a KD of 12.98 ± 2.07nM. In vivo studies demonstrated the specificity of [18F]F-DABI for PET imaging of STING expression with B16F10 tumor uptake of 10.93 ± 0.93%ID/g, which was significantly different from that of blocking groups (3.13 ± 0.88%ID/g, ***p < 0.0001). Furthermore, tumor uptake of [18F]F-DABI was well positively correlated with STING expression in different tumor types. Biodistribution results demonstrated that [18F]F-DABI was predominately uptaken in the liver and intestines, indicating its hepatobiliary elimination. This proof-of-concept study demonstrated a STING-binding radioligand for PET imaging, which could be used as a potential companion diagnostic tool for related STING-agonist therapies.

Both NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) derivatives have been used as bifunctional chelating agents (BFCAs) for the preparation of 68Ga-labeled target-specific agents having potential for positron emission tomography (PET) imaging of cancerous lesions. In the present work, the authors have attempted a comparative pharmacokinetic evaluation between 68Ga-labeled porphyrins prepared using NOTA and DOTA derivatives as the BFCAs. A symmetrical porphyrin derivative, 5,10,15,20-tetrakis(p-carboxymethyleneoxyphenyl)porphyrin, was synthesized and coupled with two different BFCAs viz. p-NH2-benzyl-NOTA and p-NH2-benzyl-DOTA. Both the porphyrin-BFCA conjugates were radiolabeled with 68Ga. A comparative bioevaluation involving pharmacokinetics and tumor affinity was performed in a tumor-bearing small animal model. Gallium-68-labeled porphyrin-amido-benzyl-NOTA and porphyrin-amido-benzyl-DOTA complexes were prepared with high radiochemical purity. Both radiolabeled complexes exhibited almost similar stability in human serum and near-identical tumor affinity and pharmacokinetic behavior in animal studies. The present study demonstrates that the pharmacokinetic behavior of 68Ga-labeled porphyrin derivatives, prepared using either NOTA or DOTA derivatives as BFCAs, remains almost identical and hence both NOTA and DOTA derivatives could be considered equivalent for developing 68Ga-based PET agents for imaging of tumorous lesions.