Radiolabeled compounds in diagnosis of infectious and inflammatory disease.

Nuclear medicine offers powerful noninvasive techniques for visualization of infectious and inflammatory disorders using whole body imaging enabling the determination of both localization and number of inflammatory foci. A wide variety of approaches depicting the different stages of the inflammatory response have been developed. Non-specific radiolabeled compounds, such as 67Ga-citrate and radiolabeled polyclonal human immunoglobulin accumulate in inflammatory foci due to enhanced vascular permeability. Specific accumulation of radiolabeled compounds in inflammatory lesions results from binding to activated endothelium (e.g. radiolabeled anti-E-selectin), the enhanced influx of leukocytes (e.g. radiolabeled autologous leukocytes, anti-granulocyte antibodies or cytokines), the enhanced glucose-uptake by activated leukocytes (18F-fluorodeoxyglucose) or direct binding to micro-organisms (e.g. radiolabeled ciprofloxacin or antimicrobial peptides). Scintigraphy using autologous leukocytes, labeled with 111In or 99mTc, is still considered the "gold standard" nuclear medicine technique for the imaging of infection and inflammation, but the range of radiolabeled compounds available for this indication is still expanding. Recently, positron emission tomography with 18F-fluorodeoxyglucose has been shown to delineate various infectious and inflammatory disorders with high sensitivity. New developments in peptide chemistry and in radiochemistry will result in specific agents with high specific activity. A gradual shift from non-specific, cumbersome or even hazardous approaches to more sophisticated, specific approaches is ongoing. In this review, the different approaches to scintigraphic imaging of infection and inflammation, already in use or under investigation, are discussed.

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.

(99m)Tc-interleukin-8 for imaging acute osteomyelitis.

UNLABELLED: Early and accurate diagnosis of osteomyelitis remains a clinical problem. Acute osteomyelitis often occurs in infants and most often is located in the long bones. Radiologic images show changes only in advanced stages of disease. Scintigraphic imaging with (99m)Tc-methylene diphosphonate (MDP), or bone scanning, is much more sensitive in detecting acute osteomyelitis but lacks specificity. We evaluated the performance of (99m)Tc-interleukin-8 (IL-8) in an experimental model of acute osteomyelitis. METHODS: Acute pyogenic osteomyelitis was induced in 10 rabbits by inserting sodium morrhuate and Staphylococcus aureus into the medullary cavity of the right femur. The cavity was closed with liquid cement. A sham operation was performed on the left femur. Routine radiographs were obtained just before scintigraphy. Ten days after surgery, the rabbits were divided into 2 groups of 5 animals, received an injection of either 18.5 MBq (111)In-granulocytes or 18.5 MBq (67)Ga-citrate, and were imaged both 24 h after injection and 48 h after injection. On day 12, the rabbits received either 18.5 MBq (99m)Tc-MDP or 18.5 MBq (99m)Tc-IL-8, and serial images were acquired at 0, 1, 2, 4, 8, 12, and 24 h after injection. Uptake in the infected femur was determined by drawing regions of interest. Ratios of infected femur (target) to sham-operated femur (background) (T/Bs) were calculated. After the final images were obtained, the rabbits were killed and the right femur was dissected and analyzed for microbiologic and histopathologic evidence of osteomyelitis. RESULTS: Acute osteomyelitis developed in 8 of 10 rabbits. All imaging agents correctly detected the acute osteomyelitis in these animals. The extent of infection was optimally visualized with (67)Ga-citrate and delayed bone scanning, whereas diaphyseal photopenia was noted with both (99m)Tc-IL-8 and (111)In-granulocytes. In 1 rabbit with osteomyelitis, imaging results were falsely negative with (111)In-granulocytes and falsely positive with (99m)Tc-MDP. Quantitative analysis of the images revealed that the uptake in the infected region was highest with (67)Ga-citrate (4.9 +/- 0.8 percentage injected dose [%ID]) and (99m)Tc-MDP (4.7 +/- 0.7 %ID), whereas the uptake in the infected area was significantly lower with (99m)Tc-IL-8 (2.2 +/- 0.2 %ID) and (111)In-granulocytes (0.8 +/- 0.2 %ID) (P < 0.0042). In contrast, the T/Bs were significantly higher for (99m)Tc-IL-8 (T/B, 6.2 +/- 0.3 at 4 h after injection) than for (67)Ga-citrate, (99m)Tc-MDP, and (111)In-granulocytes, which had ratios of 1.5 +/- 0.4, 1.9 +/- 0.2, and 1.4 +/- 0.1, respectively (P < 0.0001). Radiography correctly revealed acute osteomyelitis in only 2 of 8 rabbits. CONCLUSION: In this rabbit model of osteomyelitis, (99m)Tc-IL-8 clearly revealed the osteomyelitic lesion. Although the absolute uptake in the osteomyelitic area was significantly lower than that obtained with (99m)Tc-MDP and (67)Ga-citrate, the T/Bs were significantly higher for (99m)Tc-IL-8 because of fast background clearance. The ease of preparation, good image quality, and lower radiation burden suggest that (99m)Tc-IL-8 may be a suitable imaging agent for the scintigraphic evaluation of acute osteomyelitis.

New concepts in infection/inflammation imaging.

Although autologous leukocytes, labelled with 111In or 99mTc, is still considered the "gold standard" nuclear medicine technique to image infection and inflammation, there is a great need for a less cumbersome and less hazardous approach. Over the last few decades the range of radiopharmaceuticals to investigate infectious and non-microbial inflammatory disorders is vastly expanding. Radiolabelled monoclonal antibodies and antibody-fragments, radiolabelled chemotactic peptides and cytokines, and radiolabelled antibiotics are promising new approaches in the field of nuclear medicine. Recently, positron emission tomography (PET) with 18FJDG has been introduced and has been shown to delineate infectious and inflammatory foci with high sensitivity. Here, a survey is presented of the different approaches in use or under investigation.

The effect of molecular weight on nonspecific accumulation of (99m)T-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.

Rapid imaging of experimental colitis with (99m)Tc-interleukin-8 in rabbits.

UNLABELLED: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Imaging infection/inflammation in the new millennium.

In the closing half of the past century a wide variety of approaches were developed to visualise infection and inflammation by gamma scintigraphy. Use of autologous leucocytes, labelled with indium-111 or technetium-99m, is still considered the "gold standard" nuclear medicine technique for the imaging of infection and inflammation. However, the range of radiopharmaceuticals used to investigate infectious and non-microbial inflammatory disorders is expanding rapidly. Developments in protein/peptide chemistry and in radiochemistry should lead to agents with very high specific activities. Recently, positron emission tomography with fluorine-18 fluorodeoxyglucose has been shown to delineate infectious and inflammatory foci with high sensitivity. The third millennium will witness a gradual shift from basic (non-specific) or cumbersome, even hazardous techniques (radiolabelled leucocytes) to more sophisticated approaches. Here a survey is presented of the different approaches in use or under investigation.

Specific and rapid scintigraphic detection of infection with 99mTc-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.

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.

Lanthionine, a protein cross-link in cataractous human lenses.

Oxidative processes are suggested to be a main cause of the covalent cross-linking of lenticular proteins. For a major part these cross-links are disulfides (cystine). With progression of nuclear cataract, cystine may be oxidized further to cysteic acid. Another oxidative degradation product of cystine is the symmetric thioether lanthionine. In this study, we clearly demonstrate that lanthionine is a protein cross-link of cataractous human lenses. Possible mechanisms of its formation are discussed.