99mTc-labeled interleukin 8 for the scintigraphic detection of infection and inflammation: first clinical evaluation.

UNLABELLED: Interleukin 8 (IL-8) is a chemotactic cytokine that binds with a high affinity to receptors expressed on neutrophils. Previous studies with various animal models showed that (99m)Tc-labeled IL-8 accumulates specifically and rapidly in infectious and inflammatory foci. The aims of the present study were to evaluate the safety of IL-8 in humans and to assess the value of (99m)Tc-IL-8 scintigraphy in patients with suspected localized infections. METHODS: (99m)Tc-IL-8 was intravenously injected at 400 MBq into 20 patients with various suspected localized infections. Patients were monitored for IL-8-related side effects for 4 h. Whole-body imaging was performed directly after injection and at 4 h after injection. Imaging after 24 h was performed for the first 7 patients and for subsequent patients when the results of (99m)Tc-IL-8 scintigraphy at 4 h after injection were normal or equivocal. Blood was drawn at several time points to determine the total number of leukocytes and leukocyte differentiation (all patients) and to determine pharmacokinetics (6 patients). RESULTS: (99m)Tc-IL-8 scintigraphy was performed for 20 patients (13 men and 7 women) with a mean age of 60 y (range, 21-76 y). No significant side effects were noted. Patients had suspected joint prosthesis infections (n = 9), osteomyelitis (n = 8), liver abscess (n = 1), and soft-tissue infections (n = 2). (99m)Tc-IL-8 was rapidly cleared from the blood and most other organs. In 10 of 12 patients with infections, (99m)Tc-IL-8 localized the infection at 4 h after injection. In 1 patient with vertebral osteomyelitis and in 1 patient with an infected knee prosthesis, (99m)Tc-IL-8 scintigraphy results were false-negative. In 8 patients with noninfectious disorders, no focal accumulation of (99m)Tc-IL-8 was found. CONCLUSION: Injection of (99m)Tc-IL-8 is well tolerated. (99m)Tc-IL-8 scintigraphy is a promising new tool for the detection of infections in patients as early as 4 h after injection.

PET imaging of infection with a HYNIC-conjugated LTB4 antagonist labeled with F-18 via hydrazone formation.

UNLABELLED: It was previously shown that the (99m)Tc-labeled hydrazinonicotinamide (HYNIC)-conjugated LTB4 antagonist MB81 visualized infectious foci in rabbits adequately and within a few hours after injection. Here, the bivalent HYNIC-conjugated LTB4 antagonist MB67 (analog of MB81) was fluorinated with (18)F via hydrazone formation and tested in vivo. METHODS: MB67 was [(18)F]-fluorinated via reaction of the [(18)F]-fluorinated intermediate p-[(18)F]-fluorobenzaldehyde ([(18)F]FB) and the HYNIC moiety of MB67 via hydrazone formation. For comparison, MB67 was also labeled with (99m)Tc. The biodistribution of (18)F- and (99m)Tc-labeled MB67 was investigated in rabbits with intramuscular infection. RESULTS: [(18)F]-MB67 was obtained at a maximum specific activity of 1200 GBq/mmol and proved to be stable in phosphate buffered saline (PBS) at 37 degrees C for at least 4 h. PET images obtained with [(18)F]-MB67 clearly delineated the abscess at 2 and 4 h pi. Counting of dissected tissues at 4 h pi revealed an abscess uptake of 0.073+/-0.005 %ID/g, as compared to 0.160+/-0.010 %ID/g for the (99m)Tc-labeled analog. Abscess-to-muscle ratios were 23+/-4 for [(18)F]-MB67 and 35+/-9 for [(99m)Tc]-MB67. CONCLUSION: The present study showed the feasibility of a new [(18)F]-labeling methodology and its application in the production of a new PET tracer for imaging of infection, [(18)F]-MB67.

Scintigraphic imaging of infectious foci with an 111In-LTB4 antagonist is based on in vivo labeling of granulocytes.

UNLABELLED: Radiolabeled leukotriene B4 (LTB4) antagonist DPC11870 is able to reveal infectious and inflammatory foci in distinct animal models. Because previous studies showed that accumulation of (111)In-DPC11870 in the abscess continued although the tracer had cleared from the circulation, we decided to investigate the pharmacodynamics of (111)In-DPC11870 and determine the mechanism of accumulation of the radiolabeled LTB4 antagonist in the abscess. METHODS: (111)In-DPC11870 was intravenously injected in healthy New Zealand White rabbits and rabbits with intramuscular Escherichia coli infection. Pharmacodynamics were studied by serial imaging and by ex vivo counting of dissected tissues. The mechanism of visualization of the abscess was investigated in rabbits with intramuscular infection that was induced 16 h after intravenous administration of (111)In-DPC11870. In addition, heterologous leukocytes and bone marrow cells of a donor rabbit were labeled with (111)In-DPC11870 in vitro and the biodistribution of these in vitro radiolabeled cells was compared with that of (111)In-DPC11870 in rabbits with an infection. RESULTS: The LTB4 antagonist (111)In-DPC11870 revealed the intramuscular abscess in rabbits only a few hours after injection. Quantitative analysis of the images confirmed accumulation of (111)In-DPC11870 in the abscess although the compound had cleared almost completely from the circulation. Radioactivity concentration in the bone marrow decreased more rapidly in infected animals than in healthy animals. Therefore, we hypothesized that (111)In-DPC11870 associates with receptor-positive (bone marrow) cells and accumulated in the abscess because of subsequent migration from the bone marrow to the abscess. Accumulation of radioactivity in the abscess induced 16 h after (111)In-DPC11870 injection was similar to that in animals intravenously injected with the tracer 24 h after induction of the abscess (0.37 +/- 0.16 percentage injected dose [%ID]/g). Moreover, differences in radioactivity concentration in the bone marrow of healthy and infected animals (0.67 +/- 0.29 %ID/g and 0.15 +/- 0.03 %ID/g at 24 h, respectively, after injection) supported our hypothesis. Additional studies with peripheral blood leukocytes and bone marrow cells that were labeled ex vivo with (111)In-DPC11870 showed the ability of these cells to migrate to the abscess (0.40 %ID/g and 0.52 %ID/g for (111)In-DPC11870 bone marrow cells and (111)In-DPC11870 peripheral blood leukocytes, respectively, 24 h after injection). CONCLUSION: The (111)In-labeled LTB4 antagonist DPC11870 accumulates in infectious and inflammatory foci because of binding to LTB4 receptors expressed on activated hematopoietic cells that subsequently migrate to the site of infection, which leads to visualization of the infectious lesions.

Kinetics of 99mTc-labeled interleukin-8 in experimental inflammation and infection.

UNLABELLED: The cytokine interleukin-8 (IL-8) binds with high affinity to the CXCR1 and CXCR2 receptors on neutrophils. In previous studies, we showed that (99m)Tc-IL-8 could rapidly and effectively delineate foci of infection and inflammation in rabbit models of intramuscular infection, colitis, and osteomyelitis. Here, the in vivo kinetics and pharmacodynamics of (99m)Tc-IL-8 are studied in detail. A derivative of hydrazinonicotinamide (HYNIC) was used as a bifunctional coupling agent to label the protein with (99m)Tc. METHODS: To address specificity of uptake of (99m)Tc-IL-8 in the abscess, uptake in turpentine-induced abscesses in neutropenic rabbits was compared with uptake in turpentine-induced abscesses in normal rabbits. The pharmacokinetics of (99m)Tc-IL-8 were studied in neutropenic rabbits and compared with those in normal rabbits. To investigate the interaction of (99m)Tc-IL-8 with blood cells in circulation in normal rabbits, the distribution of the radiolabel over circulating white and red blood cells and plasma was determined. The in vivo kinetics of (99m)Tc-IL-8 were studied by quantitative analysis of whole-body images acquired between 0 and 6 h after injection. The results of this analysis (in vivo biodistribution) were validated by ex vivo counting of radioactivity in dissected tissues. RESULTS: The abscess uptake (percentage of injected dose per gram of tissue [%ID/g] +/- SEM) in immunocompetent rabbits (0.41 +/- 0.05) was 10 times higher than that in neutropenic rabbits (0.038 +/- 0.014), demonstrating specificity of the target uptake of (99m)Tc-IL-8. Abscess-to-muscle ratios +/- SEM were also 10 times higher (110 +/- 10 vs. 10 +/- 5). Lung and spleen uptake in normal rabbits was 3 times higher than that in neutropenic rabbits. The blood clearance of the radiolabel in neutropenic rabbits was similar to that in normal rabbits. In circulation, most of (99m)Tc-IL-8 (70%) was found in the plasma fraction. Less than one third was associated with red blood cells, and only a very low percentage (<2.5%) was associated with white blood cells. Image analysis revealed a gradually increasing abscess uptake over time up to >15%ID, which was confirmed by ex vivo gamma-counting of the infected muscle. The highest increase in uptake in the abscess was observed after 2 h following injection, when most of (99m)Tc-IL-8 was cleared from the blood, suggesting specific neutrophil-mediated accumulation of (99m)Tc-IL-8 in the abscess. Furthermore, region-of-interest analysis revealed that gradual accumulation of (99m)Tc-IL-8 in the abscess was accompanied by a simultaneous clearance of activity from the lungs, suggesting that neutrophil-associated (99m)Tc-IL-8 that was initially trapped in the lungs migrates to the abscess at later time points, favoring neutrophil-bound transportation from the lungs to the abscess. CONCLUSION: Substantial support is given for the hypothesis that (99m)Tc-IL-8 localizes in the abscess, mainly bound to peripheral neutrophils. Accumulation in the abscess is a highly specific, neutrophil-driven process. As assessed by in vivo and ex vivo analysis, the total fraction that accumulates in the inflamed tissue is extremely high (up to >15 %ID) compared with that of other agents used for imaging infection and inflammation.

Relationship between neutrophil-binding affinity and suitability for infection imaging: comparison of (99m)Tc-labeled NAP-2 (CXCL-7) and 3 C-terminally truncated isoforms.

UNLABELLED: The CXC chemokines are a family of closely related chemoattractant cytokines that bind to, attract, and activate neutrophils to variable degrees. In this study, the relationship between neutrophil-binding affinity and suitability for infection imaging was investigated in a selected group of CXC chemokines. Neutrophil-activating peptide-2 (NAP-2, 70 residues; also called CXCL7) binds with high affinity to the CXCR2 receptor on neutrophils. Recently, C-terminally truncated NAP-2-variants have been described that have enhanced neutrophil-binding affinity and neutrophil-stimulating capacity. Here, NAP-2 and its C-terminal shortened variants NAP-2(1-68), NAP-2(1-66), and NAP-2(1-63) were labeled with (99m)Tc via the hydrazinonicotinamide (HYNIC) chelator and their potential for imaging of infection was investigated in a rabbit model of infection. The CXC chemokine interleukin-8 (IL-8) was used for comparison. In addition, a series of (99m)Tc-labeled CXC chemokines were screened for their potential to image infection, including CTAP-III, GCP-2, ENA-78, PF-4, and IP-10. METHODS: The receptor-binding affinity of HYNIC-conjugated NAP-2 and its analogs was compared in competitive binding assays on Jurkat cells transfected with the CXCR2 receptor gene. Biodistribution of labeled NAP-2 (analogs) and other CXC chemokines in rabbits with intramuscular Escherichia coli infections was determined both by gamma-camera imaging and by counting dissected tissues at 6 h after injection. RESULTS: The CXCR2-binding affinity of the HYNIC-conjugated NAP-2 analogs relative to NAP-2 was as follows: NAP-2(1-68), 2.5-fold; NAP-2(1-66), 10-fold; and NAP-2(1-63), 3-fold. In the rabbit model, uptake in the abscess (in percentage injected dose per gram +/- SEM) was 0.084 +/- 0.015 for NAP-2, 0.098 +/- 0.010 for NAP-2(1-68), 0.189 +/- 0.044 for NAP-2(1-66), and 0.114 +/- 0.017 for NAP-2(1-63) at 6 h after injection. In comparison, higher uptake in the abscess was found for labeled IL-8, a modest uptake was found for GCP-2 and ENA-78, and a low uptake was found for CTAP-III, PF-4, and IP-10. CONCLUSION: This study showed a clear relationship between affinity to receptors on neutrophils and suitability for infection imaging. Of the NAP-2 variants, NAP-2(1-66) combined highest affinity to CXCR2 with the best characteristics for imaging. IL-8 binds to both CXCR1 and CXCR2 with high affinity and showed a superior imaging quality. The other CXC chemokines tested bind to neutrophils with lower affinity and were shown to be less suitable for infection imaging in this study.

99mTc-HMPAO-labeled autologous versus heterologous leukocytes for imaging infection.

UNLABELLED: Radiolabeled autologous leukocytes are the gold standard for imaging infectious foci in patients. Good results have also been reported for radiolabeled heterologous leukocytes from noninfected donors. Until now, the 2 methods have not been directly compared. In this study, we compared the infection-imaging potential of 99mTc-hexamethylpropyleneamine oxime (HMPAO)-labeled autologous granulocytes with that of 99mTc-HMPAO-labeled granulocytes from either infected or noninfected donors in rabbits with Escherichia coli infection. METHODS: The radiolabeled granulocyte preparations were studied in rabbits with an E. coli infection in the left calf muscle. The soft-tissue infections were scintigraphically visualized after injection of 18 MBq of either 99mTc-HMPAO purified autologous granulocytes or radiolabeled purified heterologous granulocytes from infected or noninfected donor rabbits. Gamma camera images were acquired at 2 min and at 1, 2, and 4 h after injection. After the last image, the rabbits were killed and uptake of the radiolabel in the dissected tissues was determined. RESULTS: The 99mTc-HMPAO autologous granulocytes and heterologous granulocytes from infected donors accurately revealed the infectious focus in the calf muscle at 2 h after injection. At 4 h after injection, a significantly better (P < 0.05) delineation of the infection was established with the 99mTc-HMPAO autologous granulocytes and 99mTc-HMPAO heterologous granulocytes from the infected rabbits than with the heterologous granulocytes from noninfected donors. With both cell preparations, the intensity of uptake in the infected calf muscle continuously increased until 4 h after injection. The 99mTc-HMPAO heterologous granulocytes from noninfected donors showed no significant increase in contrast after 2 h after injection. Absolute uptake in the infected calf muscle was much higher for 99mTc-HMPAO autologous granulocytes (7.81 +/- 1.21 percentage injected dose [%ID]) and 99mTc-HMPAO heterologous infected granulocytes (8.91 +/- 1.92 %ID) than for the radiolabeled heterologous noninfected granulocytes (2.32 +/- 0.75 %ID) (P < 0.04) at 4 h after injection. The ratio of infected muscle to noninfected contralateral muscle was significantly higher for 99mTc-HMPAO autologous granulocytes and 99mTc-HMPAO heterologous granulocytes from infected donors than for 99mTc-HMPAO heterologous granulocytes from noninfected donors (5.53 +/- 1.09, 3.86 +/- 0.75, and 1.86 +/- 0.31, respectively; P < 0.05). CONCLUSION: For nuclear medicine imaging of infection, purified granulocytes derived from infected rabbits are superior to purified granulocytes derived from noninfected donor rabbits. In addition, autologous granulocytes gave similar results to heterologous granulocytes from infected donor rabbits, suggesting the need for intrinsic cell activation for specific granulocyte migration.

Scintigraphic detection of pulmonary aspergillosis in rabbits with a radiolabeled leukotriene b4 antagonist.

UNLABELLED: Radiolabeled chemotactic peptides have been studied for their applicability to the visualization of infectious and inflammatory foci. Because a radiolabeled leukotriene B4 (LTB4) antagonist allowed visualization of intramuscular E. coli abscesses in rabbits within a few hours after injection, we decided to test the imaging characteristics of this agent in a more clinically relevant model of pulmonary aspergillosis. The pharmacokinetics and imaging characteristics of the 111In-labeled LTB4 antagonist DPC11870 were studied in New Zealand White rabbits with experimental pulmonary aspergillosis infection. The imaging characteristics of 111In-DPC11870 were compared with those of 67Ga-citrate, a radiopharmaceutical commonly used to detect pulmonary infections in patients. METHODS: Pulmonary aspergillosis was induced in the left lung of rabbits by intratracheal inoculation of 1 x 10(8) conidia of Aspergillus fumigatus. Three days after the inoculation, the rabbits received 111In-DPC11870 or 67Ga-citrate intravenously. Images were acquired at several time points up to 24 h after injection. RESULTS: Pulmonary aspergillosis was visualized with both agents. Images acquired after injection of 111In-DPC11870 showed uptake in the pulmonary lesions from 6 h after injection. Because of accumulation at the site of infection and clearance from the background, the images improved with time. Region-of-interest analysis at 24 h after injection revealed infected lung-to-normal lung ratios of 5.0 +/- 1.5 for 111In-DPC11870 and 2.9 +/- 0.6 for 67Ga-citrate. CONCLUSION: The radiolabeled LTB4 antagonist DPC11870 clearly delineated experimentally induced pulmonary aspergillosis in rabbits. Images acquired at 24 h after injection of 111In-DPC11870 were superior to those obtained after injection of 67Ga-citrate.

A bivalent leukotriene B(4) antagonist for scintigraphic imaging of infectious foci.

UNLABELLED: Several radiolabeled chemotactic peptides have been tested for their suitability to show infection and inflammation. Leukotriene B(4) (LTB(4)) receptor-binding ligands could be useful agents for revealing neutrophilic infiltrations because the LTB(4) receptor is abundantly expressed on neutrophils after an inflammatory stimulus. In this study, we investigated the in vivo and in vitro characteristics of a new hydrophilic (111)In-labeled LTB(4) antagonist. METHODS: The LTB(4) antagonist DPC11870-11 was labeled with (111)In and intravenously injected into New Zealand White rabbits with Escherichia coli infection in the left thigh muscle. The pharmacokinetics and biodistribution were studied by serial scintigraphic imaging (0-24 h after injection) and by ex vivo counting of dissected tissues (6 and 24 h after injection). The receptor-mediated in vivo localization of the compound was investigated in 3 rabbits that received an excess of nonradioactive indium-labeled agent 2 min before the administration of the (111)In-labeled LTB(4) antagonist. RESULTS: In rabbits with intramuscular E. coli infection, the abscess was visualized as early as 2 h after injection. Accumulation in the abscess increased with time, resulting in excellent images at 6 h after injection. Blood clearance was rapid in the first hours after injection (alpha-half-life = 30 +/- 6 min, 85%; beta-half-life = 25.7 +/- 0.8 h, 15%). Abscess-to-background ratios, as derived from the region-of-interest analysis, increased to 34 +/- 7 at 24 h after injection. The images of both groups showed moderate uptake in the liver, spleen, kidneys, and bone marrow. No activity was seen in the bladder, indicating almost complete retention in the kidneys. The uptake in the abscess could be blocked completely by injection of an excess of nonradioactive agent, indicating a specific receptor-ligand interaction of the radiolabeled agent in the infected tissue. Biodistribution data showed that after saturation of the LTB(4) receptor, the abscess uptake, in percentage injected dose per gram, was significantly reduced (0.03 +/- 0.02 vs. 0.24 +/- 0.06, P = 0.008). CONCLUSION: The modified LTB(4) antagonist showed infectious foci rapidly after injection because of specific receptor-ligand interaction. Because of the high abscess-to-background ratios that were obtained and the fact that no accumulation of radioactivity was observed in the gastrointestinal tract, this compound has excellent characteristics for revealing infectious and inflammatory foci.

99mTc-labeled interleukin-8 for scintigraphic detection of pulmonary infections.

BACKGROUND: Interleukin (IL)-8 is a chemotactic cytokine that binds with high affinity to receptors on neutrophils. Previously we showed that (99m)Tc-labeled IL-8 is highly suitable for scintigraphic imaging in rabbit models of IM infection and of colitis. STUDY DESIGN: (99m)Tc-labeled IL-8 was tested for its potential to image pulmonary infection in three experimental rabbit models: aspergillosis in immunocompromised rabbits, pneumococcal (Gram-positive) pneumonia, and Escherichia coli-induced (Gram-negative) pneumonia in immunocompetent rabbits (four rabbits in each group). A derivative of hydrazinonicotinamide was used as bifunctional coupling agent to label IL-8 with (99m)Tc. Biodistribution of (99m)Tc IL-8 was determined both by gamma-camera imaging and by counting dissected tissues at 6 h after injection. RESULTS: (99m)Tc IL-8 enabled early (within 2 h after injection) and excellent visualization of localization and extent of pulmonary infection in each of the three experimental models of pulmonary infection. Uptake of (99m)Tc IL-8 in the infected lung and the contralateral lung was (in percentage of the injected dose per gram of tissue +/- SEM) at 6 h after injection 0.63 +/- 0.12 and 0.12 +/- 0.02 (aspergillosis), 0.89 +/- 0.04 and 0.44 +/- 0.04 (pneumococcal pneumonia), and 1.53 +/- 0.12 and 0.36 +/- 0.06 (E coli pneumonia), respectively. In the E coli model, uptake of (99m)Tc IL-8 in the focus of infection even exceeded uptake in the kidneys, the main clearing organs. CONCLUSION: (99m)Tc IL-8 offers many advantages over the conventionally used radiopharmaceuticals to image pulmonary infection, (67)Ga citrate and radiolabeled leukocytes, ie, rapid and easy preparation, short time span between injection and imaging, low radiation burden and, most importantly, clear delineation of the infectious foci.

Effects of coligand variation on the in vivo characteristics of Tc-99m-labeled interleukin-8 in detection of infection.

In our previous studies, interleukin-8 (IL-8) was labeled with (99m)Tc using hydrazinonicotinamide (HYNIC) as bifunctional coupling agent and tricine as coligand. This preparation showed excellent characteristics for imaging of infection in a rabbit model of soft-tissue infection. In the present study, the propylaldehyde hydrazone formulation of HYNIC was introduced to stabilize HYNIC-IL-8. (99m)Tc-HYNIC-IL-8 was prepared using 5 different coligand formulations. The effect of these coligand formulations on the in vitro characteristics and in vivo behavior of (99m)Tc-HYNIC-IL-8 was investigated. HYNIC-conjugated IL-8 was labeled with (99m)Tc in the presence of either (A) tricine, (B) ethylenediaminediacetic acid (EDDA), (C) tricine and trisodium triphenylphosphinetrisulfonate (TPPTS), (D) tricine and nicotinic acid (NIC), or (E) tricine and isonicotinic acid (ISONIC). These preparations were characterized in vitro by RP-HPLC, determination of the octanol/water partition coefficient, stability studies, and receptor binding assays. The in vivo biodistribution of the radiolabel in rabbits with E. coli-induced soft-tissue infection was determined both by gamma-camera imaging as well as by tissue counting at 6 h pi. Specific activity (MBq/microg) was highest for (ISO)NIC (up to 80) > TPPTS (40) > tricine (15) > EDDA (7). RP-HPLC and octanol/water partition coefficients showed a shift toward higher lipophilicity for the TPPTS preparation. The leukocyte receptor binding fractions were around 40-55% for all preparations except for TPPTS, which showed predominantly nonspecific binding. All preparations were stabilized in serum, but the stability in PBS was highest for NIC and TPPTS > EDDA > ISONIC > tricine. The in vivo biodistribution showed highest abscess/muscle for NIC and ISONIC (>200) > EDDA and tricine (approximately 100) > TPPTS (<40). Gamma camera imaging rapidly visualized the abscess from 2 h pi onward for all formulations. The abscess/background (A/B) at 6 h pi for ISONIC was significantly higher (P < 0.05) than that of tricine and the A/B of TPPTS was significantly lower (P < 0.05). IL-8 can be rapidly and easily labeled with (99m)Tc using HYNIC as a chelator in combination with various coligands. The most optimal infection imaging characteristics were found for formulations using nicotinic acid/tricine as coligand system combining a high specific activity and high in vitro stability with high abscess/muscle ratios (>200) and high abscess/background ratios (>20). Protein doses to be administered were as low as 70 ng/kg bodyweight. At these low protein doses, side effects are not to be expected in the human system. This paves the way for infection imaging studies in patients.