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Imaging of Myeloperoxidase in Mice by Using Novel Amplifiable Paramagnetic Substrates¹

¹ From the Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, 5404 Building 149, 13th St, Charlestown, MA 02129. Received June 15, 2005; revision requested August 16; revision received September 1; accepted September 22; final version accepted October 16. J.W.C. supported in part by the RSNA Research and Education Foundation. R.W. supported in part by NIH grants P50-CA86355, R24-CA92782, and R01-HL078641. A.B. supported in part by NIH grant R01 EB000858. Address correspondence to J.W.C. (e-mail: chenjo{at}helix.mgh.harvard.edu).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 
Purpose: To evaluate whether contrast agents for molecular magnetic resonance (MR) imaging can demonstrate the in vivo activity of myeloperoxidase, an enzyme that is secreted by stimulated polymorphonuclear leukocytes, monocytes, and macrophages during inflammation.

Materials and Methods: Animal experiments were approved by the animal care committee. Protocols for the procurement and use of human blood were approved by the institutional review board. Informed consent was obtained from each donor, and HIPAA guidelines were followed for humans. Two paramagnetic myeloperoxidase substrates—that is, gadolinium-5-hydroxytryptamide–tetraazacyclododecane tetraacetic acid (Gd-5-HT-DOTA) and Gd-bis-5-HT–diethylenetriaminepentaacetic acid (Gd-bis-5-HT-DTPA)—were synthesized. Indium 111-labeled bis-5-HT-DTPA was used to determine biodistribution and target localization. A total of 22 mice were used in three models. In the first model, human myeloperoxidase was embedded in a basement membrane matrix gel and was injected intramuscularly. In the second model, lipopolysaccharide (LPS) from Escherichia coli was embedded in a basement membrane matrix gel and was injected intramuscularly to induce endogenous myeloperoxidase secretion. In the third model, LPS was injected intramuscularly to induce myositis. Statistical significance was calculated for contrast-to-noise ratio (CNR) curves by using the Kolmogorov-Smirnov test.

Results: After the administration of Gd-bis-5-HT-DTPA, strong MR signal enhancement (up to 2.5-fold increase in CNR, P < .001) was observed in vivo for implants that contained human myeloperoxidase. In the LPS-induced myositis model, a smaller visible difference was seen (1.3-fold increase in CNR, P < .001), which was consistent with the fact that endogenous mouse myeloperoxidase is only about 10%–20% as active as human myeloperoxidase. Prolonged contrast material enhancement was observed in the myeloperoxidase-containing areas that were injected with Gd-5-HT-DOTA or Gd-bis-5-HT-DTPA but was not observed in areas that were injected with Gd-DTPA or Gd-dopamine-DOTA (P < .05). Single photon emission computed tomography combined with computed tomography was used to confirm the increased retention of contrast agents at sites that contained human myeloperoxidase, and the results of biodistribution studies demonstrated a more than fourfold increase radiotracer accumulation at these sites.

Conclusion: Human and mouse myeloperoxidase activity in myeloperoxidase implants and inflamed tissues can be visualized and reported in vivo by using myeloperoxidase-sensitive "smart" molecular imaging probes.

© RSNA, 2006

	INTRODUCTION

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Inflammation (host response that is characterized by macrophage and immune cell recruitment) is a central component of infection, rheumatoid arthritis (1), and multiple sclerosis (2). In addition, the role of the inflammatory cascade is critical in emphysema (3), cardiovascular disease (4), and cancer (5). Therefore, the ability to noninvasively image critical molecules of inflammation may be beneficial in a variety of clinical scenarios.

Myeloperoxidase is the most abundant component of azurophilic granules of neutrophils (6). It is also found in the lysosomes of monocytes, polymorphonuclear leukocytes, and macrophages (7). During stimulation, myeloperoxidase is secreted by these cells as a result of degranulation and interacts with H₂O₂ to generate highly reactive molecular species, such as hypochlorite, tyrosyl radicals, and aldehydes, that can further activate cellular inflammatory signaling cascades (8).

It has been previously demonstrated that paramagnetic substrates can be synthesized (9,10) and used to image peroxidase activity (eg, horseradish peroxidase) through polymerization-induced relaxivity amplification (9,11–14). To develop myeloperoxidase-specific contrast agents, we tested various candidate myeloperoxidase substrates and found that paramagnetic substrates composed of 5-hydroxytryptamide (5-HT) demonstrated sufficiently fast kinetics of relaxivity increase in vitro (9). Importantly, the reducing potential of the 5-HT–based molecules was so high that they could outcompete chloride anions, which are one of the natural substrates for myeloperoxidase (14). However, despite these advances in contrast agent design for magnetic resonance (MR) imaging, it is unclear which contrast agents would be best suited for the imaging of myeloperoxidase in vivo.

We hypothesized that, when injected into an animal, these small substrate molecules would extravasate into the interstitium, including the inflamed tissues that contained substantial amounts of myeloperoxidase. The myeloperoxidase-activated molecules would be expected not only to exhibit signal amplification due to polymerization (9) but also to bind (15) or crosslink with proteins (16) in inflamed areas that would serve as "traps" for enzyme-activated contrast agents. Thus, the purpose of our study was to evaluate whether contrast agents for molecular MR imaging can demonstrate the in vivo activity of myeloperoxidase, an enzyme that is secreted by stimulated polymorphonuclear leukocytes, monocytes, and macrophages during inflammation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 
Protocol for the animal experiments was approved by the animal care committee at our institution. Protocol for the procurement and use of human blood was approved by the institutional review board at our institution, and informed consent was obtained from each donor. Guidelines for the Health Insurance Portability and Accountability Act were followed for humans.

Synthesis of Substrates and Analytic Procedures
Mono(hydroxytryptamide)–tetraazacyclododecane tetraacetic acid, gadolinium salt (gadolinium-5-HT–tetraazacyclododecane tetraacetic acid [Gd-5-HT-DOTA]), and Gd-dopamine-DOTA were synthesized as described by Chen et al (9). We also synthesized bis-substituted paramagnetic substrates to take advantage of the potentially increased efficacy of polymerization by having multiple reducing moieties linked to the same paramagnetic chelate. Gd-bis-tyramide–diethylenetriaminepentaacetic acid (Gd-bis-tyramide-DTPA) and Gd-bis-5-HT-DTPA were synthesized as described by Querol et al (10). Indium 111 (¹¹¹In)-labeled bis-5-HT-DTPA was prepared by using transchelation from ¹¹¹In-oxiquinoline complex (Oxine; Cardinal Health, Cleveland, Ohio). To prepare ¹¹¹In-bis-5-HT-DTPA, 50 µg of bis-5-HT-DTPA (1 mg/mL) was mixed with 37 MBq of ¹¹¹In-oxiquinoline solution in 0.7–1.0 mL of saline and was incubated for 1 hour under argon. Transchelation and purity were controlled by using a high-pressure liquid chromatography system (Discovery C-18 column, 25 x 3 mm; Supelco, Bellefonte, Pa) with a 0%–55% gradient of acetonitrile in water. Human myeloperoxidase (BioDesign, Saco, Me) was obtained, and its activity was determined by using a standard method (17).

To determine if the gadolinium compounds were substrates for human myeloperoxidase, myeloperoxidase-mediated oligomerization was monitored by using size-exclusion high-pressure liquid chromatography and by measuring the change in longitudinal relaxation time (T₁) with a spectrometer (Minispec 120 NMR; Bruker BioSpin, Billerica, Mass) that used a standard inversion-recovery sequence (M.Q.S.). The degree of oligomerization was determined by using matrix-assisted laser desorption—ionization mass spectrometry (M.Q.S.).

Substrate Binding to Plasma Components
The following experiments were performed to determine the effect of myeloperoxidase-activated substrate oligomerization with plasma proteins to form larger aggregates. Human plasma was extracted from two healthy male donors (ages 36 and 45 years) by using a sterile endotoxin-tested density gradient solution (Polymorphprep; Accurate Chemicals, Westbury, NY), and the samples were centrifuged according to the manufacturer's instructions. Aliquots of a 3 mmol/L solution of Gd-bis-5-HT-DTPA were mixed with aliquots of ¹¹¹In-bis-5-HT-DTPA, which served as a tracer. Aliquots were mixed with or without human plasma and with or without myeloperoxidase and H₂O₂. These solutions were loaded on minicolumns (Biospin P6; Bio-Rad Laboratories, Hercules, Calif), and the plasma was separated according to the method suggested by the manufacturer. Retained and eluted radioactivity were counted separately in a gamma counter (1480 Wizard; Perkin-Elmer, Wellesley, Mass); the fraction of radioactive tracer that was bound to the proteins was then calculated in three independent experiments. Results were evaluated by all authors in consensus.

MR Imaging
MR imaging was performed by using a 1.5-T MR imager (Excite; GE Healthcare, Waukesha, Wis) and a 4.7-T MR imager (Pharmascan; Bruker BioSpin MRI). For all experiments, precontrast T2-weighted fast spin-echo MR imaging at both 1.5 T (2000/100 [repetition time msec/echo time msec], echo train length of eight, four signals acquired, and acquisition time of 4 minutes 57 seconds) and 4.7 T (2650/56.7, echo train length of eight, six signals acquired, acquisition time of 6 minutes 36 seconds) and T1-weighted spin-echo MR imaging at both 1.5 T (500/11, four signals acquired, and acquisition time of 5 minutes 59 seconds) and 4.7 T (565/8.33, four signals acquired, and acquisition time of 7 minutes 17 seconds) were initially performed with fat saturation to locate the gels or area of inflammation. Parameters for 1.5-T MR imaging included a matrix size of 256 x 128, field of view of 10 x 7.5 cm, section thickness of 1 mm, and 22 sections acquired. Parameters for 4.7-T MR imaging included a matrix size of 192 x 192, field of view of 4 x 4 cm, section thickness of 1 mm, and 22 sections acquired. All MR images were obtained in the coronal plane except for those that were acquired during the lipopolysaccharide (LPS)-induced myositis experiments; in these experiments, MR images were obtained in the sagittal plane.

Gadolinium substrates were injected into the mouse via the tail vein; 0.3 mmol/kg, which is triple the normal clinical dose of 0.1 mmol/kg, was selected to increase the absolute difference in signal intensity between activated contrast agents and nonactivated contrast agents. For a typical mouse weighing 30 g, this resulted in 0.009 mmol of gadolinium substrate in an average volume of 250 µL. The mouse was imaged immediately after the injection of the contrast agent by using multiple T1-weighted MR imaging sequences with fat saturation for at least 3 hours.

Mouse Models
Ten-week-old male C57BL6 mice were obtained from Jackson Laboratories (Bar Harbor, Me). A total of 22 mice were used for the study. In the following experiments, all injections were performed by one author (J.W.C.), and the results were evaluated by all authors in consensus.

Myeloperoxidase and basement membrane matrix gel experiments.—A Ewing sarcoma basement membrane matrix gel (Matrigel; Beckton-Dickinson, San Jose, Calif) was used to immobilize human myeloperoxidase and glucose oxidase (Calbiochem, San Diego, Calif), which supplies the H₂O₂ that is required for detecting the oxidative activity of myeloperoxidase. This system was chosen to reflect the deposits of human myeloperoxidase in inflamed tissue in a mouse model. A 400-µL mixture of basement membrane matrix gel and MEM medium (Cambrex, East Rutherford, NJ) was injected slowly into the thighs of 15 mice 1 hour prior to MR imaging. The mixture that was injected into the right thigh contained 15 U of myeloperoxidase and 4 U of glucose oxidase, while the mixture that was injected into the left thigh contained no enzyme and served as an internal control. A total of 15 mice (three each for Gd-5-HT-DOTA, Gd-bis-5-HT-DTPA, ¹¹¹In-bis-5-HT-DTPA, Gd-bis-tyramide-DTPA, and Gd-DTPA) were used.

LPS and basement membrane matrix gel experiments.—This model was developed to induce endogeneous mouse myeloperoxidase secretion and activation. A total of 200 µL of basement membrane matrix gel that contained 10% heparin was injected slowly into the thighs of three mice 4 days prior to MR imaging. The mixture that was injected into the right thigh contained 10 µg/mL of Escherichia coli LPS (Escherichia coli O55:B5; Sigma, St Louis, Mo), while the mixture that was injected into the left thigh contained no LPS and served as an internal control. One mouse was sacrificed for immunohistochemical analysis.

LPS-induced myositis experiments.—A total of 100 µg/mL of LPS in phosphate-buffered saline was injected intramuscularly into the right flank of four mice to produce myositis. Mice underwent MR imaging 24 hours after injection.

Biodistribution
A total of 2.96–3.70 MBq of ¹¹¹In-bis-5-HT-DTPA in 200 µL of saline was injected intravenously into three mice, which were prepared in the same manner as those that underwent implantation with basement membrane matrix gel and myeloperoxidase. Fused single photon emission computed tomographic (SPECT) and computed tomographic (CT) images of the animals were obtained 3 hours after injection by using a dual modality system (X-SPECT/CT; Gamma Medica, Northridge, Calif). On average, the acquisition time was approximately 2 hours. Subsequent to SPECT/CT imaging (about 6 hours after injection), the mice were sacrificed and biodistribution was determined in major organs. The percentage of radiotracer dose accumulation per gram of major organ tissue was determined by using a gamma counter (1480 Wizard; Perkin-Elmer).

Immunohistochemical Analysis
The tissues were stained for the presence of myeloperoxidase, and the resultant slides were examined by the authors (J.W.C., A.B., and R.W., with at least 10 years of experience in slide interpretation). The tissues were fixed in paraformaldehyde, immersed in 30% sucrose, and used for paraffin embedding within several days. A diluted solution of rabbit polyclonal anti-myeloperoxidase antibody (1:10 dilution in 10% horse serum and phosphate-buffered saline; AbCam, Cambridge, Mass) was used as the primary antibody, which was followed by anti-rabbit peroxidase conjugate and diaminobenzidine staining. The antibodies had cross-reactivity with mouse myeloperoxidase and were suitable for mouse tissues.

Statistical Analysis
For normalized signal intensity measured on T1-weighted MR images, the mean ± standard deviation was obtained in regions of interest. One region of interest per time point per animal was measured. Contrast-to-noise ratios (CNRs) were computed for each region of interest according to the formula CNR = (ROI_site – ROI_muscle)/SD_noise, where ROI_site is the region of interest at a location of possible myeloperoxidase activity, ROI_muscle is the region of interest of muscle, SD_noise is the standard deviation of noise.

Within each animal, CNR was normalized to the maximum value to compute relative CNR. The statistical significance of the relative CNR curves that were induced by the injection of the contrast agent was calculated by using the Kolmogorov-Smirnov test.

For the experiments that were performed with the basement membrane matrix gel, we compared the relative CNR curve for the test side (ie, the side that contained either myeloperoxidase or LPS) with the relative CNR curve for the control side within the same animal for each contrast agent. For these experiments, the Kolmogorov-Smirnov test was modified to account for within-animal correlation by computing the test statistic for 10000 random permutations of the paired data (ie, by switching the paired values of the test site and control site at each time point) to determine the number of permutations that generated a test statistic that was at least as extreme as the unpaired statistic.

For the basement membrane matrix gel experiments, a comparison of the relative ratios between different contrast agents in different animals was also performed. These comparisons were made by using the equation rCNR_site/rCNR_control, where rCNR_site is the relative CNR of the myeloperoxidase-sensitive contrast agents (Gd-5-HT-DOTA and Gd-bis-5-HT-DTPA) and rCNR_control is the relative CNR of the contrast agents that were used as a control (Gd-DTPA and Gd-bis-tyramide-DTPA).

For the LPS-induced myositis experiments, the Kolmogorov-Smirnov test was used to evaluate the relative CNR curves for different contrast agents in different animals. One data point per animal per time was used. A P value of less than .05 was considered to indicate a statistically significant difference. Statistical computations were performed by using a statistical software package (R, version 2.1.1; R Foundation for Statistical Computing, Vienna, Austria).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 
Figure 1 shows the structure of the myeloperoxidase substrates—that is, Gd-5-HT-DOTA (9) and Gd-bis-5-HT-DTPA (10)—and demonstrates the changes that occurred in the presence of myeloperoxidase and proteins. When activated by myeloperoxidase and H₂O₂, these substrates are oligomerized up to five monomeric substrate units in length, as is shown on the images obtained during mass spectroscopy for Gd-bis-5-HT-DTPA (Fig 1). Furthermore, in the presence of plasma proteins and after activation by myeloperoxidase and H₂O₂, there is a marked increase in the elution of ¹¹¹In radioactivity (Fig 1, C), which demonstrates the association between myeloperoxidase-activated products and plasma proteins. The monomers did not show substantial plasma protein binding.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 1: A, Chemical structures (left) and mechanisms of action (middle) for myeloperoxidase (MPO)-sensitive contrast agents. After activation by myeloperoxidase, contrast agents are radicalized to form oligomers. Blue spheres indicate the formation of myeloperoxidase-activated complexes. B, Mass spectrum demonstrates that, with each successive group of peaks, there is an increasing number of units (up to five) from myeloperoxidase oligomerization. Slightly different peaks are due to associations with other solutes in test solution. C, Graph demonstrates that both radicals and oligomers can bind to proteins, as was shown in the radiolabeled elution experiment in which only the molecules that were bound to large proteins were eluted. Subsequent increases in relaxivity (r1) are noted in A.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 2a: Coronal 1.5-T fat-saturated MR images (450/11, 10 x 7.5-cm field of view, 256 x 128 matrix, 22 sections acquired, 1-mm section thickness, four signals acquired, 90° flip angle) and corresponding CNR curves in four mice injected with (a) Gd-DTPA, (b) Gd-bis-tyramide-DTPA, (c) Gd-5-HT-DOTA, and (d) bis-5-HT-DTPA. On each MR image, the right side (R) contains human myeloperoxidase embedded in a basement membrane matrix gel and the left side (L) contains only the basement membrane matrix gel. Note that there is a substantial increase in contrast material enhancement on the right side of the mice in c and d that is not seen in a or b. Red outlines indicate the regions of the implants.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 2b: Coronal 1.5-T fat-saturated MR images (450/11, 10 x 7.5-cm field of view, 256 x 128 matrix, 22 sections acquired, 1-mm section thickness, four signals acquired, 90° flip angle) and corresponding CNR curves in four mice injected with (a) Gd-DTPA, (b) Gd-bis-tyramide-DTPA, (c) Gd-5-HT-DOTA, and (d) bis-5-HT-DTPA. On each MR image, the right side (R) contains human myeloperoxidase embedded in a basement membrane matrix gel and the left side (L) contains only the basement membrane matrix gel. Note that there is a substantial increase in contrast material enhancement on the right side of the mice in c and d that is not seen in a or b. Red outlines indicate the regions of the implants.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 2c: Coronal 1.5-T fat-saturated MR images (450/11, 10 x 7.5-cm field of view, 256 x 128 matrix, 22 sections acquired, 1-mm section thickness, four signals acquired, 90° flip angle) and corresponding CNR curves in four mice injected with (a) Gd-DTPA, (b) Gd-bis-tyramide-DTPA, (c) Gd-5-HT-DOTA, and (d) bis-5-HT-DTPA. On each MR image, the right side (R) contains human myeloperoxidase embedded in a basement membrane matrix gel and the left side (L) contains only the basement membrane matrix gel. Note that there is a substantial increase in contrast material enhancement on the right side of the mice in c and d that is not seen in a or b. Red outlines indicate the regions of the implants.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 2d: Coronal 1.5-T fat-saturated MR images (450/11, 10 x 7.5-cm field of view, 256 x 128 matrix, 22 sections acquired, 1-mm section thickness, four signals acquired, 90° flip angle) and corresponding CNR curves in four mice injected with (a) Gd-DTPA, (b) Gd-bis-tyramide-DTPA, (c) Gd-5-HT-DOTA, and (d) bis-5-HT-DTPA. On each MR image, the right side (R) contains human myeloperoxidase embedded in a basement membrane matrix gel and the left side (L) contains only the basement membrane matrix gel. Note that there is a substantial increase in contrast material enhancement on the right side of the mice in c and d that is not seen in a or b. Red outlines indicate the regions of the implants.

Endogenous Mouse Myeloperoxidase
We found that, at the site where the basement membrane matrix gel was embedded with LPS, there was recruitment of a large number of cells that stained positive for myeloperoxidase (Fig 3, D). The site without LPS had almost no cells, and the few cells that were present stained negative for myeloperoxidase. We additionally found that, in the presence of endogenous mouse myeloperoxidase, there was a 1.3-fold increase in contrast material enhancement on the side that contained myeloperoxidase (P < .001) when Gd-bis-5-HT-DTPA was administered. No significant difference in contrast material enhancement was seen after Gd-DTPA administration (P = .88). It is not surprising that the increase for mouse myeloperoxidase is less than that for human myeloperoxidase, because mouse myeloperoxidase is only about 10%–20% as active as human myeloperoxidase (18).

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   Figure 3: Sagittal 4.7-T fat-saturated MR images (565/8.33, 4 x 4-cm field of view, 192 x 192 matrix, 16 sections acquired, 1-mm section thickness, four signals acquired, 90° flip angle) in mice with LPS-induced myositis. Mice were injected with, A, Gd-DTPA, which was used as a control, and, B, Gd-5-HT-DOTA. A washout of contrast material enhancement can be seen in A at 50 minutes, which is reinforced by, C, the relative CNR time curve. Gd-dopamine-DOTA and Gd-DTPA behaved similarly, as is shown in C. A noticeable increase in contrast material enhancement could still be seen at 50 minutes in B and C. D, Immunohistochemical slide of the site with embedded LPS demonstrates a large number of cells that stained positive for myeloperoxidase (brown areas), while immunohistochemical slide of the control site, which did not contain LPS, demonstrates very few cells and did not stain positive for myeloperoxidase. (Original magnification, x40.)

Scintigraphic Confirmation of Target Accumulation
We intravenously injected ¹¹¹In-bis-5-HT-DTPA into three mice that underwent basement membrane matrix gel and myeloperoxidase implantation, as described for the MR imaging experiments. SPECT/CT was performed 3 hours after injection because the data from the CNR time curves suggested that washout was slightly increased for contrast agents without myeloperoxidase activation 3 hours after Gd-bis-5-HT-DTPA injection (Fig 2d). Fused SPECT/CT images demonstrated high radiotracer retention in the region that contained human myeloperoxidase (Fig 4a). Conversely, the left basement membrane matrix gel implant, which did not contain myeloperoxidase, showed less activity. There was a 2.3-fold increase (standard deviation, 0.3) in radioactivity in the right leg, which contained myeloperoxidase, compare with the left leg, which did not contain myeloperoxidase.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 4a: (a) 111In scintigraphic image of fused SPECT/CT image demonstrates a large amount of radiotracer retention on the right side (R), which contained myeloperoxidase, 3 hours after the injection of 111In-bis-5-HT-DTPA. The left side (L) was used as a control. Colors indicate the intensity of radioactivity, with red indicating the highest level of radioactivity and yellow indicating the second highest level of radioactivity. (b) Biodistribution results indicate that most of the contrast agents were distributed in the spleen, kidneys, bowel, and liver. Error bars = standard deviation.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 4b: (a) 111In scintigraphic image of fused SPECT/CT image demonstrates a large amount of radiotracer retention on the right side (R), which contained myeloperoxidase, 3 hours after the injection of 111In-bis-5-HT-DTPA. The left side (L) was used as a control. Colors indicate the intensity of radioactivity, with red indicating the highest level of radioactivity and yellow indicating the second highest level of radioactivity. (b) Biodistribution results indicate that most of the contrast agents were distributed in the spleen, kidneys, bowel, and liver. Error bars = standard deviation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

The described experiments were performed to address two major questions: (a) What is the most probable mechanism of contrast material enhancement in myeloperoxidase-containing tissues in the presence of gadolinium- and indium-labeled reducing substrates, and (b) would the above substrates be useful for imaging myeloperoxidase activity in vivo? The performed experiments allowed us to pinpoint two factors that contributed to an increase in signal intensity; both of these factors were the direct result of myeloperoxidase-mediated oxidation of the substrates. The first factor is the myeloperoxidase-mediated oligomerization of the substrates (imaging probes), which results in increased molecular mass and, consequently, allows for (a) increased MR signal intensity and (b) delayed clearance from tissue. The second factor is that the myeloperoxidase-mediated oxidation products bind to and/or crosslink with plasma proteins, thereby causing more local accumulation. The association with proteins then further contributes to an increase in MR signal intensity due to increased relaxivity.

We tested several peroxidase contrast agents for MR imaging. In the human myeloperoxidase implant experiments (which simulate myeloperoxidase-rich tissues), Gd-bis-tyramide-DTPA did not produce increased contrast material enhancement in the presence of myeloperoxidase. This was perhaps not too surprising because of the slow kinetics that Gd-tyramide-DOTA exhibited in the presence of myeloperoxidase (9). However, we found that both of the 5-HT–based contrast agents were highly sensitive to myeloperoxidase, with Gd-bis-5-HT-DTPA resulting in the highest CNR and Gd-5-HT-DOTA resulting in a slightly lower CNR. At 4.7 T, a similar increase was found for Gd-bis-5-HT-DTPA. It is important that a substantial relative increase in contrast material enhancement is still obtained at high magnetic field strengths because imaging at higher magnetic field strengths, such as 3 T, is being increasingly used in the clinical setting because of increased resolution, better signal-to-noise ratio, and shorter imaging times.

In addition to increased CNR, there is prolonged contrast material enhancement for the myeloperoxidase substrates; this contrast material enhancement persists at a very high level for nearly an hour before decreasing gradually and was demonstrated in the LPS-induced myositis experiments. Therefore, Gd-5-HT-DOTA and Gd-bis-5-HT-DTPA have substantially different pharmacokinetics than Gd-dopamine-DOTA and Gd-DTPA, which served as controls. These findings are consistent with our hypothesis that, because of their increased size, myeloperoxidase-converted oligomers cannot quickly diffuse out of the area and thus remain at the area of inflammation longer than the unconverted smaller substrates.

This hypothesis was also supported by the results of the radiolabeled elution experiments. In these experiments, the columns only eluted molecules that were larger than 6000 Da. Therefore, only molecules that were bound to plasma proteins would have been eluted. After activation with myeloperoxidase in the presence of plasma, a much larger fraction of radioactivity was eluted, which is consistent with increased binding to plasma proteins. This is also consistent with the findings of Heuther et al (15), who found that the compounds resulting from peroxidase oxidation of 5-HT, but not 5-HT itself, demonstrated substantial binding to albumin, plasma proteins, and tissues. Together with the larger size of the myeloperoxidase-activated products, this increased binding affinity of the activated product serves to prolong the pharmacokinetics of these contrast agents in the presence of myeloperoxidase.

We then reasoned that, if our hypothesis regarding the accumulation of the activated products in myeloperoxidase-containing sites is correct, we should also be able to perform scintigraphic imaging of myeloperoxidase activity. The results of ¹¹¹In-bis-5-HT-DTPA scintigraphic imaging confirm that there is large focal accumulation of myeloperoxidase-converted products. This approach has several potential advantages compared with the existing methods for imaging inflammation. Unlike white blood cell–labeled indium, the proposed scintigraphic contrast agent does not require the extraction and manipulation of the patient's blood but still retains the advantage of ¹¹¹In use. It is potentially more specific for inflammation because it shows only the sites where myeloperoxidase is active (ie, where there is active inflammation that causes damage). Gadolinium-labeled contrast agents give the same functionality to MR imaging but with the added benefit of the much higher resolution that is achievable with MR imaging. Because of the signal amplification that is activated by myeloperoxidase, there would be dosimetry advantages as well.

A potential limitation of our study is the fact that the newly developed myeloperoxidase contrast agents were tested in mouse models only. Mice have different pharmacokinetics compared with other species, and other studies will ultimately have to be performed prior to clinical use. However, it is important to note that both the laboratory scale synthesis and high-pressure liquid chromatographic purification of the myeloperoxidase substrates are labor intensive, which is part of the reason why we used mouse models. Another reason for using mouse models is the fact that they now represent universally accepted models in biomedical research; many immunologic reagents and knock-out models of myeloperoxidase are available (19) and will be tested in the future.

Another limitation of our study was that no internal control was included in the LPS-induced myositis experiments. An internal control in which saline was injected without LPS was not feasible because the saline would have been absorbed. Therefore, only the differences in pharmacokinetics could be directly observed but not the increase in signal intensity from myeloperoxidase activation. Nonetheless, a 1.3-fold increase in signal intensity for endogenous mouse myeloperoxidase may be inferred from the experiments in which LPS was embedded in the basement membrane matrix gel.

The power of this class of contrast agents lies in the fact that they can be used to demonstrate sites of active inflammation not only for infection or injury but also for the wide variety of pathologic processes that myeloperoxidase is implicated in, such as Alzheimer disease (20), multiple sclerosis (2), lung cancer (21), and leukemia (22). In addition, there is an accumulating body of evidence that myeloperoxidase plays an important role in atherosclerosis (23–30). There is the potential that, in the future, radiologists may be able use this class of contrast agents to pinpoint specific vulnerable plaques before the plaques become culprit lesions and to facilitate focused treatment. Moreover, because there is an increased realization that patients often have more than one vulnerable plaque in different vascular territories, scintigraphic and/or MR imaging with these contrast agents may play an important role in the future screening of patients for systemic anti-inflammatory treatment (31,32).

	ADVANCES IN KNOWLEDGE

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 

• In vivo MR imaging of myeloperoxidase activity is possible through enzymatic modification of gadolinium-based contrast agents.
  
• Improved MR contrast agent relaxivity can be achieved through enzymatic amplification.
  
• Improved in vivo contrast agent retention can be attained through protein binding by means of enzymatic activation.
  

	FOOTNOTES

 

Abbreviations: CNR = contrast-to-noise ratio • DOTA = tetraazacyclododecane tetraacetic acid • DTPA = diethylenetriaminepentaacetic acid • 5-HT = 5-hydroxytryptamide • LPS = lipopolysaccharide

² Current address: Department of Radiology, University of Massachusetts Medical School, Worcester, Mass.

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, J.W.C., A.B., R.W.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, J.W.C.; experimental studies, all authors; statistical analysis, J.W.C.; and manuscript editing, J.W.C., R.W.

	References

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ADVANCES IN KNOWLEDGE References

 

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