interleukin-8 and hydrazino-nicotinamide

Radiolabeled autologous leukocytes (WBCs) are the gold standard for imaging inflammatory bowel disease (IBD). For the rapid and adequate management of patients with IBD, there is need for a new agent at least as good as radiolabeled WBCs, but easier to prepare and without its inherent risks. In this study, the potential of interleukin-8 (IL-8) labeled with (99m)Tc using hydrazinonicotinamide (HYNIC) to image IBD was investigated in a rabbit model of acute colitis and compared with that of (99m)Tc-HMPAO-labeled granulocytes.. In rabbits with chemically induced acute colitis, inflammatory lesions were scintigraphically visualized after injection of either IL-8 or purified granulocytes, both labeled with (99m)Tc. Gamma camera images were acquired at 2 min and at 1, 2, and 4 h after injection. Four hours after injection, the rabbits were killed, and the uptake of the radiolabel in the dissected tissues was determined. The dissected colon was imaged and the inflammatory lesions were scored macroscopically. For each affected colon segment, the colitis index (affected colon-to-normal colon uptake ratio, CI) was calculated and correlated with the macroscopically scored severity of inflammation.. Both agents visualized the colitis within 1 h after injection. (99m)Tc-HYNIC-IL-8 images of the colonic abnormalities were more accurate and the intensity of uptake in the affected colon continuously increased until 4 h after injection, whereas no further increase 1 h after injection was noticed scintigraphically for (99m)Tc-HMPAO-granulocytes. The absolute uptake in the affected colon was much higher for IL-8 than for the radiolabeled granulocytes with the percentage injected dose per gram (%ID/g) 0.41 +/- 0.04 %ID/g and 0.09 +/- 0.05 4 %ID/g h after injection, respectively. With increasing severity, the CI at 4 h after injection for (99m)Tc-HYNIC-IL-8 was 4.4 +/- 0.6, 13.5 +/- 0.5, and 25.8 +/- 1.0; for granulocytes, the CI at 4 h after injection was 1.5 +/- 0.1, 3.4 +/- 0.2, and 6.4 +/- 0.5, respectively. The CI correlated with the severity of the inflammation (r = 0.95, P < 0.0001 for IL-8; r = 0.95, P < 0.0001 for granulocytes).. Within 1 h after injection, visualization of the extent of colonic inflammation in vivo was possible with (99m)Tc-HYNIC-IL-8 and (99m)Tc-HMPAO-granulocytes. Within 2 h after injection, (99m)Tc-IL-8 allowed a good evaluation, and within 4 h after injection, a meticulous evaluation of the severity of IBD. Although (99m)Tc-HMPAO-granulocytes were able to delineate the extent of IBD within 2 h after injection, an accurate estimation of severity of inflammation was not possible. (99m)Tc-HYNIC-IL-8 is an inflammation-imaging agent that showed promising results in this study. (99m)Tc-IL-8 can be prepared off-the-shelf and yields excellent imaging with high target-to-background ratios.

Topics: Acute Disease; Animals; Colitis; Colon; Female; Gamma Cameras; Granulocytes; Hydrazines; Interleukin-8; Niacinamide; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Technetium; Technetium Tc 99m Exametazime; Trinitrobenzenesulfonic Acid

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.

Topics: Aprotinin; Biological Assay; Chromatography, High Pressure Liquid; Cytochrome c Group; Humans; Hydrazines; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-8; Lactalbumin; Leukocytes; Muramidase; Niacinamide; Proteins; Receptors, Immunologic; Sialoglycoproteins; Technetium