Patricia Oliver

Improved radioiodination of biomolecules using exhaustive Chloramine-T oxidation.

To improve standardization in analytical reagents we investigated Chloramine-T radioiodination (125I) of several biomolecules based on the use of a single amount of the oxidizing agent Chloramine-T as the limiting reagent being exhausted during the course of the reaction. Whenever the labeling yield resulted in less than one atom 125I/molecule, a second amount of the oxidizing agent was added. Thereafter, the integrity of the various biomolecules was assessed using radioimmunoassays, radioreceptor binding assays, or radioimmunometric assays. Purification yields were done by gel permeation (56% +/- 19%, n=230) or by precipitation with trichloroacetic acid (59% +/- 19%, n=230). Specific activity (117 +/- 61 MBq/nmol) and the degree of iodine incorporation (1.4 +/- 0.8 atoms of 125I/molecule) were achieved after 300 sec of incubation. A second addition of Chloramine-T resulted in an increased labeling yield of all biomolecules tested by a mean factor of 1.8 +/- 0.9. After the second addition of Chloramine-T, we observed for some biomolecules a significant (p<0.001) decreased effect in biological performance. In conclusion, the use of Chloramine-T as a limiting reagent resulted in molecules with appropriate immunological and biological performance. In general, tracers were minimally damaged and assessment of the shelf life as well as storing conditions showed the usefulness of the standardization of biomolecule labeling.

[177Lu]DOTA-anti-CD20: labeling and pre-clinical studies.

Anti-CD20 (Rituximab®), a specific chimeric monoclonal antibody used in CD20-positive Non-Hodgkins Lymphoma, was conjugated to a bifunctional quelate (DOTA) and radiolabeled with (177)Lu through a simple method. [(177)Lu]-DOTA-anti-CD20 was obtained with a radiochemical purity higher than 97%, and showed good chemical and biological stability, maintaining its biospecificity to CD20 antigens. Monte Carlo simulation showed high doses deposited on a spheroid tumor mass model. This method seems to be an appropriate alternative for the production of [(177)Lu]-DOTA-anti-CD20 as therapeutic radiopharmaceutical.

68Ga-NOTA-UBI 29-41 as a PET tracer for detection of bacterial infection

The cationic peptide 68Ga-NOTA-UBI-29-41 was synthesized and characterized. Biodistribution and PET/CT examinations were performed for evaluation of its biologic behavior. Differentiation of infection from sterile inflammation was investigated using microbiology methods at the sites of bacterial infections. Methods: Labeling of UBI-29-41 conjugated with NOTA with 68Ga was optimized at 20°C–100°C and pH 3.5–5.5. Radiochemical purity, stability up to 260 min, and binding to serum proteins were determined. In vitro binding to Staphylococcus aureus was evaluated from 9.14 × 107 to 1.17 × 1010 cfu/mL. Of 3 groups of Mus musculus Swiss male mice, the first was inoculated intramuscularly with 1.2 × 108 cfu of S. aureus to provoke infection, and the second, with 1.2 × 108 cfu of heat shock–treated S. aureus to generate sterile inflammation. The third mouse was not treated and served as a control. After 24 h, 68Ga-NOTA-UBI-29-41 was administrated intravenously, and biodistribution was performed at 30, 60, and 120 min. PET/CT dynamic studies (120 min) were acquired. Sinograms were reconstructed using 3D maximum-likelihood expectation maximization and analyzed with software. Infected or inflamed muscles were dissected, homogenized, and cultured in tryptic soy agar medium. Recovered S. aureus was calculated as cfu/g. Results: 68Ga-NOTA-UBI-29-41 showed high renal excretion (83.2% ± 7.3%) of injected dose and rapid blood clearance. More than 95% was bound in vitro to 5 × 109 cfu/mL. A significantly higher (P < 0.05) accumulation of 68Ga-NOTA-UBI-29-41 was observed at sites of S. aureus inoculation in infected mice (ratio of target to nontarget, 5.0 at 60 min and 4.1 at 120 min) compared with animals with inflammation (ratio of target to nontarget, 1.6 at 60 min and 1.2 at 120 min). Conclusion: The difference in uptake of 68Ga-NOTA-UBI-29-41 in the infected muscles compared with the inflamed muscles was clearly observed in the PET/CT images and positively correlated with the degree of infection.

3-(Benzyloxy)-1-(5-[18F]fluoropentyl)-5-nitro-1H-indazole: a PET radiotracer to measure acetylcholinesterase in brain

AIM Noninvasive studies of the acetylcholinesterase (AChE) level in Alzheimers disease (AD) patients can contribute to a better understanding of the disease and its therapeutic. We propose 3-(benzyloxy)-1-(5-[18F]fluoropentyl)-5-nitro-1H-indazole, [18F]-IND1, structurally related to the AChE-inhibitor CP126,998, as a new positron emission tomography-radiotracer. EXPERIMENTAL Radiosynthesis, with 18F, stability, lipophilicity and protein binding of [18F]-IND1 were studied. In vivo behavior, in normal mice and on AD mice models, were also analyzed. RESULTS [18F]-IND1 was obtained in good radiochemical yield, was stable for at least 2 h in different conditions, and had adequate lipophilicity for blood-brain barrier penetration. Biodistribution studies, in normal mice, showed that [18F]-IND1 was retained in the brain after 1 h. In vivo tacrine-blocking experiments indicated this uptake could be specifically due to AChE interaction. Studies in transgenic AD mice showed differential, compared with normal mice, binding in many brain regions. CONCLUSION [18F]-IND1 can be used to detect AChE changes in AD patients.

Synthesis of [18F]2B-SRF101: A Sulfonamide Derivative of the Fluorescent Dye Sulforhodamine 101

Background: The red fluorescent dye Sulforhodamine 101 (SR101) has been used in neuroscience research as a useful tool for staining of astrocytes, since it has been reported as a marker of astroglia in the neocortex of rodents in vivo. The aim of this work is to label SR101 with positron emission radionuclides, in order to provide a radiotracer to study its biological behavior. This is the first attempt to label SR101 by [18F], using a chem-ical derivatization via a sulfonamide-linker and a commercially available platform. Methods: The synthesis of SR101 N-(3-Bromopropyl) sulfonamide and SR101 N-(3-Fluoropropyl) sulfonamide (2B-SRF101) was carried out. The radiosynthesis of SR101 N-(3-[18F]Fluoropropyl) sulfonamide ([18F]2B-SRF101) was performed in a TRACERlab® FX-FN. Different labeling conditions were tested. Three pilot batches were produced and quality control was performed. Lipophilicity, plasma protein binding and radiochemical sta-bility of [18F]2B-SRF101 in final formulation and in plasma were determined. Results: SR101 N-(3-Bromopropyl) sulfonamide was synthetized as a precursor for radio-labeling with [18F]. 2B-SRF101 was prepared for analytical purpose. [18F]2B-SRF101 was obtained with radiochemical purity of (97.0 ± 0.6%). The yield of the whole synthesis was (11.9 ± 1.7%), non-decay corrected. [18F]2B-SRF101 was found to be stable in final for-mulation and in plasma. The octanol-water partition coefficient was (Log POCT = 1.88 ± 0.14). The product showed a high percentage of plasma protein binding. Conclusions: The derivatization of SR101 via sulfonamide-linker and the first radiosyn-thesis of [18F]2B-SRF101 were performed. It was obtained in accordance with quality con-trol specifications. In vitro stability studies verified that [18F]2B-SRF101 was suitable for preclinical evaluations.

Automated One-pot Radiosynthesis of [11C]S-adenosyl Methionine

Background: Glycine N-methyltransferase is an enzyme overexpressed in some neo-plastic tissues. It catalyses the methylation of glycine using S-adenosyl methionine (SAM or AdoM-et) as substrate. SAM is involved in a great variety of biochemical processes, including transmeth-ylation reactions. Thus, [11C]SAM could be used to evaluate transmethylation activity in tumours. The only method reported for [11C]SAM synthesis is an enzymatic process with several limitations. We propose a new chemical method to obtain [11C]SAM, through a one-pot synthesis. Method: The optimization of [11C]SAM synthesis was carried out in the automated TRACERlab® FX C Pro module. Different labelling conditions were performed varying methylating agent, precur-sor amount, temperature and reaction time. The compound was purified using a semi-preparative HPLC. Radiochemical stability, lipophilicity and plasma protein binding were evaluated. Results: The optimum labelling conditions were [11C]CH3OTf as the methylating agent, 5 mg of precursor dissolved in formic acid at 60 ºC for 1 minute. [11C]SAM was obtained as a diastereomer-ic mixture. Three batches were produced and quality control was performed according to specifica-tions. [11C]SAM was stable in final formulation and in plasma. Log POCT obtained for [11C]SAM was (-2,01 ± 0,07) (n=4), and its value for plasma protein binding was low. Conclusion: A new chemical method to produce [11C]SAM was optimized. The radiotracer was ob-tained as a diastereomeric mixture with a 53:47 [(R,S)-isomer: (S,S)-isomer] ratio. The compound was within the quality control specifications. In vitro stability was verified. This compound is suita-ble to perform preclinical and clinical evaluations.