The Development and Validation of Radiopharmaceuticals Targeting Bacterial Infection.

The International Atomic Energy Agency organized a technical meeting at its headquarters in Vienna, Austria, in 2022 that included 17 experts representing 12 countries, whose research spanned the development and use of radiolabeled agents for imaging infection. The meeting focused largely on bacterial pathogens. The group discussed and evaluated the advantages and disadvantages of several radiopharmaceuticals, as well as the science driving various imaging approaches. The main objective was to understand why few infection-targeted radiotracers are used in clinical practice despite the urgent need to better characterize bacterial infections. This article summarizes the resulting consensus, at least among the included scientists and countries, on the current status of radiopharmaceutical development for infection imaging. Also included are opinions and recommendations regarding current research standards in this area. This and future International Atomic Energy Agency-sponsored collaborations will advance the goal of providing the medical community with innovative, practical tools for the specific image-based diagnosis of infection.

Bulk Scale Formulation of Therapeutic Doses of Clinical Grade Ready-to-Use 177Lu-DOTA-TATE: The Intricate Radiochemistry Aspects.

INTRODUCTION: 177Lu-DOTA-TATE is a clinically useful and promising therapeutic radiopharmaceutical for peptide receptor radionuclide therapy of neuroendocrine tumors (NETs) overexpressing somatostatin receptors. Currently, the radiopharmaceutical is prepared in-house at nuclear medicine centers, thereby restricting its use to limited centers only. In this article, the authors describe systematic studies toward bulk scale formulation of "ready-to-use" 177Lu-DOTA-TATE using medium specific activity 177Lu (740-1110 GBq/mg) at a centralized radiopharmacy facility. METHODS: In an optimized protocol, 177Lu-DOTA-TATE synthesis was carried out by direct heating of 177LuCl3 (Sp. act. 740-1110 GBq/mg) with DOTA-TATE peptide (1.5-3.0 equivalents) in ammonium acetate buffer (0.2 M) containing 2,5-dihydroxy benzoic acid (gentisic acid). Thereafter, the crude labeled product was purified using a Sep-Pak® C18 column and diluted with acetate buffer-gentisic acid (1.5% w/v) solution to final radioactive concentration of 740 MBq/mL. This was further sterilized and dispensed as 7.4 GBq patient dose/vial with 2 days postformulation calibration. RESULTS: A peptide/metal ratio of 1.5-3.0 is essential for complexation wherein radiolabeling yields >90% are obtained minimizing free 177Lu waste. For formulation of 7.4 GBq patient dose (2 days postproduction), even specific activity of about 555 GBq/mg was found to be adequate for the radiometal. The ready-to-use 740 MBq/mL 177Lu-DOTA-TATE formulation with gentisic acid (1.5% w/v) is observed to be safe for human use for more than 1 week (radiochemical purity >98%) from the day of production when stored at -70°C. However, the target specificity may get affected beyond 2 days as the total peptide content for 7.4 GBq dose may exceed the critical peptide limit of 300 µg. Patient treatment carried with several batches of present formulation in diseased NET patients exhibited desired distribution at the tumor and its metastatic site. CONCLUSIONS: A ready-to-use formulation of 177Lu-DOTA-TATE was successfully prepared and optimized for regular bulk scale production and supply to distant nuclear medicine centers.

Clinical utility of 99mTc-ubiquicidin (29-41) as an adjunct to bone scan in differentiating infected versus noninfected loosening of prosthesis before revision surgery.

PURPOSE: The aim of this study was to evaluate the utility of Tc-ubiquicidin (Tc-UBI) (29-41) as an adjunct to an methylene diphosphonate (MDP) bone scan in differentiating septic versus aseptic loosening in patients with painful prosthesis posted for revision surgery. PATIENTS AND METHODS: A two-vial cold kit of UBI (29-41) was prepared and utilized for the preparation of patient dose of Tc-UBI (29-41). Twenty two patients with painful hip or knee prosthesis and scheduled for revision surgery were included in the study. Overall, 370-555 MBq of Tc-UBI (29-41) was injected intravenously in all the patients. A blood pool image at 20 min after injection was followed by spot views of the suspected region of infection (target) and a corresponding normal area (nontarget) at 60 min. All patients underwent a routine Tc-MDP three-phase whole-body bone scan, followed by single-photon emission computed tomography/computed tomography of the prosthesis within a week of the Tc-UBI (29-41) study. For Tc-UBI scans, a visual score (0-3) was used to categorize studies as positive or negative, with scores of 0 (minimal or no uptake; less than soft tissue or contralateral extremity) and 1 (mild; equivalent to soft tissue or contralateral extremity) being considered negative and scores of 2 (moderate; uptake greater than soft tissue or contralateral extremity, but less than the liver) or 3 (intense; uptake greater than soft tissue or contralateral extremity and equivalent to the liver) being considered positive. The final correlation was on the basis of bacterial culture as the major criterion and the results of clinical tests, radiography, fluorine-18-fluorodeoxyglucose PET-CT, and three-phase bone scanning as the minor criteria. RESULTS: In all, 22 studies were carried out with Tc-UBI (29-41). Of these, 16 scans were considered positive and six were negative for infection foci. All negative scans were subsequently confirmed to be true negative. Adverse reactions were not observed during image acquisition and within 5 days after the study. The overall sensitivity, specificity, and positive and negative predictive values were 100, 85.7, 93.75, and 100%, respectively. A combination of an MDP bone scan and UBI scans was considered to yield maximum confidence toward reporting for the presence of infection. CONCLUSION: Patient dose of Tc-UBI (29-41) was prepared successfully and a simple quality control method to check radiolabeling yield was used at the hospital radiopharmacy. Tc-UBI (29-41) showed promise in localizing foci of infection, with optimal visualization at 20-60 min, for the evaluation of prosthesis loosening.

Studies on batch formulation of a kit for the preparation of the 99mTc-Ubiquicidin (29-41): An infection imaging agent.

The aim of this study was to formulate an indigenous cold kit of Ubiquicidin, UBI (29-41), for easy preparation of (99m)Tc-UBI (29-41) to be used as an infection imaging agent. A two component kit with the peptide and SnCl2 as vial 1 and optimum amount of NaOH as vial 2 was successfully formulated as seen from the consistent radiochemical and pharmaceutical purity of the product over six consecutive batches of kits. The utility of the kit could be demonstrated through in-vitro and in vivo specificity of (99m)Tc-UBI (29-41).

(68) Ga-labeled Ciprofloxacin Conjugates as Radiotracers for Targeting Bacterial Infection.

With an aim of developing a bacteria-specific molecular imaging agent, ciprofloxacin has been modified with a propylamine spacer and linked to two common bifunctional chelators, p-SCN-Bz-DOTA and p-SCN-Bz-NOTA. The two ciprofloxacin conjugates, CP-PA-SCN-Bz-DOTA (1) and CP-PA-SCN-Bz-NOTA (2), were radiolabeled with (68)Ga in >90% radiochemical yield and were moderately stable in vitro for 4 h. The efficacy of (68)Ga-1 and (68)Ga-2 has been investigated in vitro in Staphylococcus aureus cells where bacterial binding of the radiotracers (0.9-1.0% for (68)Ga-1 and 1.6-2.3% for (68)Ga-2) could not be blocked in the presence of excess amount of unlabeled ciprofloxacin. However, uptake of radiotracers in live bacterial cells was significantly higher (p < 0.01) than that in non-viable bacterial cells. Bacterial infection targeting efficacy of (68)Ga-1 and (68)Ga-2 was tested in vivo in rats where the infected muscle-to-inflamed muscle ((68)Ga-1: 2 ± 0.2, (68)Ga-2: 3 ± 0.5) and infected muscle-to-normal muscle ratios ((68)Ga-1: 3 ± 0.4, (68)Ga-2: 6.6 ± 0.8) were found to improve at 120 min p.i. Fast blood clearance and renal excretion was observed for both the radiotracers. The two (68)Ga-labeled infection targeting radiotracers could discriminate between bacterial infection and inflammation in vivo and are worthy of further detailed investigation as infection imaging agents at the clinical level.

Limulus amebocyte lysate testing: adapting it for determination of bacterial endotoxin in 99mTc-labeled radiopharmaceuticals at a hospital radiopharmacy.

UNLABELLED: A bacterial endotoxin test (BET) is required to detect or quantify bacterial endotoxin that may be present in radiopharmaceutical preparations. The test uses Limulus amebocyte lysate, which, in the presence of bacterial endotoxin and divalent calcium ions, causes the formation of a coagulin gel. (99m)Tc-labeled radiopharmaceuticals have chelating ligands such as diethylene triamine pentaacetic acid (DTPA), ethylene dicysteine (EC), L,L-ethyl cysteinate dimer (ECD), N-[2,4,6-trimethyl-3 bromoacetanilid] iminodiacetic acid (mebrofenin), dimercapto succinic acid-III (DMSA-III), dimercapto succinic acid-V (DMSA-V), and several others, which form a coordination complex with Na-(99m)Tc-O4 in the presence of reducing agents. During BET by the gel-clot method, the free sulfhydryl (-SH) and carboxyl (-COOH) in some of the chelating agents in the final (99m)Tc-labeled radiopharmaceuticals decrease the free divalent calcium ion concentration, which in turn inhibits coagulin gel formation. This study was designed using the premise that addition of calcium chloride solution to the reaction mixture would nullify this effect. METHODS: We present here the data obtained from BET assay analysis of (99m)Tc-labeled radiopharmaceuticals and the cold kits from which they are made (EC, ECD, methoxyisobutylisonitrile, DTPA, mebrofenin, methylene diphosphonic acid [MDP], DMSA-III, and DMSA-V) using 2 different dilutions, maximum valid dilution (MVD) and half maximum valid dilution (MVD/2), with and without the addition of calcium chloride at a final concentration of 300 µM. RESULTS: It was observed that at MVD and MVD/2 all of the (99m)Tc-labeled kits exhibited interference in coagulin gel formation with the exception of (99m)Tc-methoxyisobutylisonitrile, (99m)Tc-MDP, (99m)Tc-mebrofenin, and (99m)Tc-ECD. However, only the cold kits of methoxyisobutylisonitrile and MDP did not show inhibition. An addition of calcium chloride solution nullified this interference at both MVD and MVD/2 in all of the (99m)Tc-labeled radiopharmaceuticals in which interference was observed. CONCLUSION: In practice, Limulus amoebocyte lysate testing is not a method of choice for (99m)Tc-labeled radiopharmaceuticals because these radiopharmaceuticals exhibit interference. However, our study proves the hypothesis that the addition of calcium chloride can circumvent this problem. The addition of calcium chloride provides an enhanced biologic quality control testing option for the final formulation of (99m)Tc-labeled radiopharmaceuticals at the hospital radiopharmacy end.