Huub J.J.M. Rennen

Labeling proteins with Tc-99m via hydrazinonicotinamide (HYNIC): optimization of the conjugation reaction

At present there is considerable interest in labeling peptides with Tc-99m for the development of target specific radiopharmaceuticals for imaging purposes. In the present study the conjugation of the bifunctional coupling agent succinimidyl-hydrazinonicotinamide (S-HYNIC) was studied and optimized in a series of peptides [molecular weight (MW) 6.5-14.3 kDa]. Aprotinin (MW 6.5 kDa), cytochrome C (MW 12.4 kDa), alpha-lactalbumin (MW 14.2 kDa), and lysozyme (MW 14.3 kDa) were conjugated with S-HYNIC via the epsilon amino groups of their lysine residues. The effects of molar conjugation ratio, reaction temperature, pH, and protein concentration were studied. Reaction products were analyzed both with respect to the HYNIC-substitution ratio (spectrophotometrically) as well as to the labeling efficiency silica gel-instant thin layer chromatography (SG-ITLC) and molecular size fast performance liquid chromatography (FPLC). The effects of conjugation on biological activity were studied in three proteins binding to receptors on leukocytes: interleukin-8 (MW 8.5 kDa), interleukin-1alpha (MW 17 kDa), and interleukin-1 receptor antagonist (MW 17 kDa). The labeling efficiency of aprotinin, cytochrome c, alpha-lactalbumin, and lysozyme conjugated under optimal conjugation conditions exceeded 90%. Specific activities obtained were up to 7.5 MBq/microg. Conjugation was most efficient at 0 degrees C (as compared to 20 and 40 degrees C), at pH 8.2 (as compared to 6.0, 7.2, and 9.5), and at protein concentrations > or = 2. 5 mg/mL. In general, efficiency increased with increasing molar conjugation ratio (protein-HYNIC-ratio 1:3 < 1:6 < 1:15<1:30). For the receptor binding proteins, biological activity was preserved only under the mildest conjugation conditions. For each of these proteins an inverse relation between labeling efficiency and receptor binding capacity was found. Labeling proteins with (99m)Tc using S-HYNIC is easy, rapid, and efficient, and preparations with high specific activity can be obtained. However, biological activity of proteins may be lost at high HYNIC-substitution ratios. With the proteins tested here a careful balancing of reaction conditions resulted in acceptable, although suboptimal, labeling efficiencies and preservation of biological activity.

The effect of molecular weight on nonspecific accumulation of 99mT-labeled proteins in inflammatory foci

UNLABELLED Although several proteins have been proposed and tested for scintigraphic detection of infection, the most optimal characteristics of a protein for this application have not yet been determined. Molecular weight (MW) of the protein, its charge, shape, carbohydrate content, characteristics of the radionuclide and receptor interactions are factors that could affect the in vivo behavior of the infection imaging agent. The effect of molecular weight on nonspecific accumulation of (99m)Tc-labeled proteins in inflammatory foci was studied in a rat model. METHODS Eleven proteins whose MWs ranged from 2.5 kDa up to 800 kDa were labeled with (99m)Tc using the hydrazinonicotinamide (HYNIC) chelator. Rats with S. aureus infection were injected i.v. with 15 MBq (99m)Tc-labeled protein. Gamma camera images were acquired and biodistribution of the radiolabel was determined ex vivo. RESULTS From biodistribution data no significant correlation was found between abscess uptake and molecular size of the (99m)Tc-labeled proteins that were studied. Fast blood clearance with predominant uptake in liver and spleen was found for the largest proteins (MW 669 kDa-800 kDA). For proteins of intermediate size (MW 66 kDa -206 kDa) we found relatively slow blood clearance with relatively moderate uptake in liver and spleen. For smaller proteins (MW 2.5 kDa -29 kDa) rapid blood clearance with predominant kidney uptake was observed. The abscess uptake of the (99m)Tc-labeled proteins (%ID/g, 24 h p.i.) was highest for serum proteins IgG and BSA. Abscess uptake correlated well with blood levels: r = 0.95 and 0.84 at 4 and 24 h respectively (P < 0.005). The abscess-to-muscle ratios varied from 2.1 to 17.8 at 24 h p.i. with highest values for alpha-2 macroglobulin (MW 725 kDa) and the intermediate sized proteins (MW 66-206 kDa). Gamma camera imaging showed localization of all radiotracers at the site of infection with abscess-to-background ratios (A/B) ranging from 1.4 to 7.0 (IgG) at 20 h p.i. The serum proteins IgG and BSA showed highest blood levels and best infection imaging characteristics. CONCLUSION Not molecular weight but blood residence time is the principal factor that determines localization of a nonspecific tracer protein in infectious foci. The ideal nonspecific infection imaging agent is a protein with a long circulatory half-life. From the proteins tested here IgG and albumin showed the best characteristics for an infection imaging agent.

Tc-99m-labeled C5a and C5a des Arg74 for infection imaging

UNLABELLED The complement anaphylatoxin C5a and its natural metabolite C5a des Arg(74) (C5adR) are involved in several stages of the inflammatory process. Both act on a common receptor expressed on different cell types, including neutrophils and monocytes. The receptor binding affinity of C5a is in the nanomolar range and exceeds that of C5adR by 1-2 orders of magnitude. The biologic potency of C5a is considerably higher than that of C5adR. Here we tested both proteins labeled with (99m)Tc for imaging of infection. METHODS The proteins were labeled with (99m)Tc via the hydrazinonicotinamide (HYNIC) chelator. The preparations were tested for imaging of infection in a rabbit model of intramuscular infection. Biodistribution of the radiolabel was determined by gamma-camera imaging and by counting dissected tissues at 5 h p.i. RESULTS C5a and C5adR showed in vivo abscess uptakes of 0.12 and 0.025%ID/g, abscess/muscle ratios of 76 and 14, abscess/blood ratios of 9.1 and 2.6, and ROI derived target-to-background ratios of 5.9 and 2.1, respectively at 5 h p.i. CONCLUSION For infection imaging (99m)Tc-labeled C5a showed excellent in vivo characteristics. However, C5a is a very bioactive protein, impeding its clinical use as an infection imaging agent. The naturally occurring partial agonist C5adR has less biological effect but showed suboptimal imaging characteristics. The present study showed that for adequate localization of a receptor binding ligand affinities for the receptor in the nanomolar range are required.

Imaging Infection and Inflammation

H. J. J. M. Rennen, PhD Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands C. P. Bleeker-Rovers, MD Department of Nuclear Medicine and Department of Internal Medicine, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands W. J. G. Oyen, MD Professor, Department of Nuclear Medicine (565), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands 7.

Molecular Imaging of Infection and Inflammation

In response to tissue damage, powerful defense mechanisms are activated, consisting of cells (leukocytes) and plasma proteins (antibodies, complement). Besides, a complex variety of mediators, both vasoactive and chemotactic, is involved in the process. These mediators are generated in the focus of inflammation and amplify the local response by the recruitment of cells and plasma components from the blood. Vasodilatation and increased endothelial permeability are induced to facilitate the extravasation of proteins and cells. Tracers in nuclear medicine such as 67Ga-citrate and radiolabeled polyclonal immunoglobulins, liposomes, and avidin-biotin accumulate in the inflammatory lesion based on a nonspecific process of leakage out of dilated capillaries.

Specific and rapid scintigraphic detection of infection with Tc-99m-labeled interleukin-8

UNLABELLED Interleukin-8 (IL-8) is a chemotactic cytokine involved in activation and recruitment of neutrophils to areas of infection. In our previous studies in rabbits we tested 123I-labeled IL-8 for its potential to image infections and showed that IL-8 rapidly and efficiently accumulated in infectious foci. However, labeling of IL-8 with 123I is costly and laborious and the specific activity of the preparation was low. In this study IL-8 was labeled with 99mTc through the hydrazinonicotinamide (HYNIC) chelator. METHODS The leukocyte receptor-binding capacity of the preparation was determined in vitro. Rabbits with Escherichia coli abscesses were injected intravenously with 7 MBq 99mTc-HYNIC-IL-8. Biodistribution of the radiolabel was determined by gamma camera imaging and tissue counting at 8 h after injection. 99mTc-HYNIC-lysozyme was used as a size-matched control. RESULTS The leukocyte receptor-binding capacity of the 99mTc-HYNIC-IL-8 preparation was preserved as determined in vitro, but labeling efficiency was modest with a specific activity of 3 MBq/microg. 99mTc-HYNIC-IL-8 accumulated rapidly in the abscess up to 0.33 +/- 0.06 percentage injected dose per gram (%ID/g) at 8 h after injection (vs. 0.025 +/- 0.003 %lD/g for 99mTc-HYNIC-lysozyme). Total uptake in the abscess was 4.9 +/- 0.7 %ID (vs. 0.44 +/- 0.05 %ID for 99mTc-HYNIC-lysozyme). Abscess-to-contralateral muscle ratios increased up to 127 +/- 23 (compared with 6.7 +/- 1.1 for 99mTc-HYNIC-lysozyme) and abscess-to-blood ratios increased to 11.9 +/- 2.2 (0.24 +/- 0.03 for 99mTc-HYNIC-lysozyme). The radiolabel was excreted renally, with a retention in the kidneys of 28 %ID. Gamma camera imaging rapidly visualized the abscess from 1 h after injection onward, with abscess-to-background ratios improving with time up to 22 at 8 h after injection (vs. 2.7 for 99mTc-HYNIC-lysozyme), as determined by quantitative analysis of the images. Most important, only a transient (30 min) moderate drop of leukocyte counts and no leukocytosis were observed after injection of an imaging dose of 99mTc-HYNIC-IL-8. CONCLUSION IL-8 can be labeled with 99mTc using HYNIC as a chelator. By this method the leukocyte receptor-binding capacity is preserved. The preparation allows rapid visualization of infection in a rabbit model with high target-to-background ratios. The mild transient drop of leukocyte counts and the absence of leukocytosis suggest that 99mTc-HYNIC-IL-8 may be used as an imaging agent with only mild and transient side effects.