Dual 5-HT2A and 5-HT2C Receptor Inverse Agonist That Affords In Vivo Antipsychotic Efficacy with Minimal hERG Inhibition for the Treatment of Dementia-Related Psychosis.

Psychosis is a distressing symptom commonly occurring in people with dementia. To treat Parkinson's disease psychosis, pimavanserin (1), a 5-HT2A receptor inverse agonist having minimal 5-HT2C receptor affinity and no dopamine D2 receptor affinity, was approved in the United States, but not for dementia-related psychosis due to limited efficacy issues. Herein, we report on the identification of a potent and dual 5-HT2A and 5-HT2C receptor inverse agonist 8 having minimal hERG inhibition, after having demonstrated the involvement of both 5-HT2A and 5-HT2C receptors to deliver antipsychotic efficacy in an MK-801-induced locomotor model and having conducted 5-HT2A and 5-HT2C occupancy studies including a surrogate method. The introduction of a spirocyclopropyl group boosting 5-HT2C affinity in 1 followed by further optimization to control lipophilicity resulted in balanced dual potency and metabolic stability, and mitigating hERG inhibition led to 8 that showed significant antipsychotic efficacy due to the involvement of both receptors.

The nociceptin/orphanin FQ receptor partial agonist sunobinop promotes non-REM sleep in rodents and patients with insomnia.

The potent and partial agonist sunobinop activates the nociceptin/orphanin-FQ peptide receptor and promotes non-REM sleep in rodents and patients with insomnia.

Relationship between receptor occupancy and the antinociceptive effect of mu opioid receptor agonists in male rats.

The analgesic mechanisms of mu opioid receptor (MOR) agonists, including receptor occupancy at the site of action, are not completely understood. The aims of the present study were to evaluate: (i) receptor occupancy in the rat brain after administration of MOR agonists; (ii) the relationship between occupancy and the antinociceptive effect. Morphine (2 or 4 mg/kg) or oxycodone (1 or 3 mg/kg) was subcutaneously administered to rats. The antinociceptive effect of these drugs was measured by the hot-plate test. MOR occupancy in the thalamus was assessed by conducting an ex vivo receptor binding assay using [3H] [D-Ala2, N-MePhe4, Gly-ol]-enkephalin, followed by autoradiographic analysis. Both drugs produced antinociception in a dose-dependent manner, and these effects disappeared after the time point at which the maximal effect was elicited. Thalamic MOR occupancy was observed in a dose-dependent manner at the time point at which maximal antinociception was elicited, and relatively low occupancy was observed when the antinociceptive effect was decreasing. Good correlation between thalamic MOR occupancy and the antinociceptive effect was observed. These findings provide direct evidence for the receptor occupancy of MOR agonists at the site of action and its relationship with the analgesic effect.

Imaging of drug and metabolite distribution by MS: case studies.

Analysis of drug and metabolite distribution is essential for understanding of the mechanisms underlying the pharmacological or toxicological effects. MS imaging (MSI) can visualize the distribution of drugs or biological molecules in tissue sections without radiolabeling, and distinguish between the distribution of a drug and that of its metabolites in tissue sections. Therefore, it is expected to be a potent imaging technique for drug distribution studies. This article includes cases in which MSI was used to analyze drug and metabolite distribution, and discusses the impact of data obtained by MSI in drug discovery and development.

Imaging of reactive oxygen species in focal ischemic mouse brain using a radical trapping tracer [(3)H]hydromethidine.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in the pathophysiology of the brain after ischemic stroke. In this study, we investigate the generation of brain ROS after transient focal ischemia in mice using a radical trapping radiotracer, [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]hydromethidine), which we recently reported as a ROS imaging probe. We also examined the effect of dimethylthiourea (DMTU), a hydroxyl radical scavenger, on brain ROS generation and infarct volume after transient focal ischemia in mice. METHODS: [(3)H]Hydromethidine was intravenously injected into mice at 1, 2, 5, and 7 h after transient middle cerebral artery occlusion (tMCAO), and then, the brain autoradiogram was acquired at 60 min after tracer injection. Brain infarct volumes at 24 h after tMCAO were assessed by 2,3,5-triphenyltetrazolium chloride staining. RESULTS: Accumulation of radioactivity was observed in the ipsilateral striatum and cortex at 1 h after tMCAO. The increase of radioactivity was attenuated at 2 h after tMCAO and then became maximized at 5 h. The high accumulation of radioactivity remained until 7 h after tMCAO. DMTU treatment significantly attenuated the accumulation of radioactivity in the ipsilateral hemisphere at 1, 5, and 7 h after tMCAO. Brain infarct volumes were also significantly reduced in DMTU-treated mice at 24 h after tMCAO. CONCLUSIONS: These results indicated that [(3)H]hydromethidine is a useful radiotracer for detecting in vivo brain ROS generation such as hydroxyl radical after ischemic injury.

Imaging of reactive oxygen species using [(3)H]hydromethidine in mice with cisplatin-induced nephrotoxicity.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in cisplatin-induced nephrotoxicity. The aim of this study was to investigate the potential of using [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]hydromethidine) for ex vivo imaging of regional ROS overproduction in mouse kidney induced by cisplatin. METHODS: Male C57BL/6 J mice were intraperitoneally administered with a single dose of cisplatin (30 mg/kg). Renal function was assessed by measuring serum creatinine and blood urea nitrogen (BUN) levels and morphology by histological examination. Renal malondialdehyde levels were measured as a lipid peroxidation marker. Autoradiographic studies were performed with kidney sections from mice at 60 min after [(3)H]hydromethidine injection. RESULTS: Radioactivity accumulation after [(3)H]hydromethidine injection was observed in the renal corticomedullary area of cisplatin-treated mice and was attenuated by pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger. Cisplatin administration significantly elevated serum creatinine and BUN levels, caused renal tissue damage, and promoted renal lipid peroxidation. These changes were significantly suppressed by DMTU pretreatment. CONCLUSIONS: The present study showed that [(3)H]hydromethidine was rapidly distributed to the kidney after its injection and trapped there in the presence of ROS such as hydroxyl radicals, suggesting that [(3)H]hydromethidine is useful for assessment of the renal ROS amount in cisplatin-induced nephrotoxicity.

Using improved serial blood sampling method of mice to study pharmacokinetics and drug-drug interaction.

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8 ± 4.0% and 64.4%, fexofenadine: 6.5 ± 0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies.

Using Improved Serial Blood Sampling Method of Mice to Study Pharmacokinetics and Drug-Drug Interaction.

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8±4.0% and 64.4%, fexofenadine: 6.5±0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies.

In vivo imaging of reactive oxygen species in mouse brain by using [3H]hydromethidine as a potential radical trapping radiotracer.

To assess reactive oxygen species (ROS) production by detecting the fluorescent oxidation product, hydroethidine has been used extensively. The present study was undertaken to evaluate the potential of the hydroethidine derivative as a radiotracer to measure in vivo brain ROS production. [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]Hydromethidine) was synthesized, and evaluated using in vitro radical-induced oxidization and in vivo brain ROS production model. In vitro studies have indicated that [(3)H]Hydromethidine is converted to oxidized products by a superoxide radical (O(2)(•)-) and a hydroxyl radical (OH(•)-) but not hydrogen peroxide (H(2)O(2)). In vivo whole-body distribution study showed that [(3)H]Hydromethidine rapidly penetrated the brain and then was washed out in normal mice. Microinjection of sodium nitroprusside (SNP) into the brain was performed to produce ROS such as OH(•)- via Fenton reaction. A significant accumulation of radioactivity immediately after [(3)H]Hydromethidine injection was seen in the side of the brain treated with SNP (5 and 20 nmol) compared with that in the contralateral side. These results indicated that [(3)H]Hydromethidine freely penetrated into the brain where it was rapidly converted to oxidized forms, which were trapped there in response to the production of ROS. Thus, [(3)H]Hydromethidine should be useful as a radical trapping radiotracer in the brain.

Quantification of small molecule drugs in biological tissue sections by imaging mass spectrometry using surrogate tissue-based calibration standards.

Quantitative analysis of administered drugs in biological tissues is essential for understanding the mechanisms underlying their efficacy or toxicity. Imaging mass spectrometry (IMS) may allow the quantification of targeted drugs in tissue sections along with the visualization of their spatial distribution. In this study, surrogate tissue-based calibration standards were prepared to quantify a small molecule drug (S-777469 or raclopride) in tissue sections of mice administered with the drug, followed by analysis with a linear ion trap mass spectrometer equipped with a matrix-assisted laser desorption/ionization (MALDI) source. The distribution of the drugs in the dissected organs was clearly visualized by MALDI-IMS. The drug concentration determined using the calibration standards prepared for MALDI-IMS analysis was highly consistent with that determined by liquid chromatography-tandem mass spectrometry, and the quantification in multiple organs was enabled. The results of this study show that MALDI-IMS can be used to quantify small molecule drugs in biological tissue sections using surrogate tissue-based calibration standards.

Quantitative imaging of a therapeutic peptide in biological tissue sections by MALDI MS.

BACKGROUND: Therapeutic peptides and proteins are being increasingly explored as potential therapeutic agents for molecular-targeted therapy, and the requirement for quantitative bioanalytical tools for such molecules has been discussed. RESULTS: The distribution of octreotide, a synthetic octapeptide analog of somatostatin, in the liver and kidney of mice administered with the analog was clearly visualized by MALDI-Imaging MS (IMS). The developed MALDI-IMS analytical method successfully quantified the amount of octreotide on tissue sections (accuracy was 76-127%) and the 2,5-dihydroxybenzoic acid-based normalization method was effective. CONCLUSION: The results of this study suggest that MALDI-IMS enables the quantification of an administered therapeutic peptide on biological tissue sections, as well as visualization of the in vivo distribution of the peptide.

Quantitative analysis of pharmaceutical drug distribution in multiple organs by imaging mass spectrometry.

RATIONALE: Recently, the requirement for a quantitative research method using imaging mass spectrometry (IMS) to be developed has been discussed. Specifically, the simultaneous quantification of a drug in multiple organs by using whole-body sections could be insightful for the pharmaceutical industry in the study of drug distribution. METHODS: Frozen whole-body sections were obtained from mice injected with raclopride, a dopamine D2 receptor selective antagonist, and coated with a matrix-assisted laser desorption/ionization (MALDI) matrix compound. The whole-body sections were then analyzed using a linear ion trap mass spectrometer equipped with a MALDI source. The concentration of raclopride in each tissue was determined using liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: The IMS-based signal intensity of raclopride strongly correlated with the concentration of the drug in the tissue samples (R=0.94; p <0.001) of six different organs. Furthermore, the spatial information obtained by IMS was very similar to that obtained by autoradiography, which is a traditional technique used for the study of drug distribution. CONCLUSIONS: This study suggests that IMS enables the quantitative analysis of drug distribution in multiple organs simultaneously. In addition, it enhances ideal drug candidate selection in terms of efficient evaluations.

Tissue factor detection for selectively discriminating unstable plaques in an atherosclerotic rabbit model.

UNLABELLED: Tissue factor (TF), a transmembrane glycoprotein that acts as an essential cofactor to factor VII/VIIa, initiates the exogenous blood coagulation cascade leading to thrombin generation and subsequent thrombus formation in vivo. TF expression is closely related to plaque vulnerability, and high TF expression is shown in macrophage-rich atheromatous lesions, making TF a potential target for detecting atheromatous lesions in vivo. Thus, we prepared (99m)Tc-labeled anti-TF-monoclonal antibody (TF-mAb) IgG as a molecular probe and evaluated its usefulness to achieve TF-specific imaging using myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. METHODS: Anti-TF-mAb was created using a standard hybridoma technique and was labeled by (99m)Tc with 6-hydrazinonicotinic acid (HYNIC) as a chelating agent to obtain (99m)Tc-TF-mAb. The immunoreactivity of HYNIC-TF-mAb was estimated by flow cytometry. WHHLMI and control rabbits were injected intravenously with (99m)Tc-TF-mAb. Twenty-four hours after the injection, the aorta was removed and radioactivity was measured. Autoradiography and histologic studies were performed using serial aorta sections. Subclass matched antibody (IgG(1)) was used as a negative control. RESULTS: HYNIC-TF-mAb showed 93% immunoreactivity of the anti-TF-mAb. The radioactivity accumulation in WHHLMI aortas was 6.1-fold higher than that of control rabbits. Autoradiograms showed a heterogeneous distribution of radioactivity in the intima of WHHLMI aortas. Regional radioactivity accumulation was positively correlated with TF expression density (R = 0.64, P < 0.0001). The highest radioactivity accumulation in percentage injected dose × body weight/mm(2) × 10(2) was found in atheromatous lesions (5.2 ± 1.9) followed by fibroatheromatous (2.1 ± 0.7), collagen-rich (1.8 ± 0.7), and neointimal lesions (1.8 ± 0.6). In contrast, (99m)Tc-IgG(1) showed low radioactivity accumulation in WHHLMI aortas that was independent of the histologic grade of lesions. CONCLUSION: The TF-detecting ability and preferential accumulation in atheromatous lesions of (99m)Tc-TF-mAb were demonstrated, indicating its potential for selective imaging of macrophage-rich atheromatous lesions in vivo.

Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques.

PURPOSE: Membrane type 1 matrix metalloproteinase (MT1-MMP) activates pro-MMP-2 and pro-MMP-13 to their active forms and plays important roles in the destabilization of atherosclerotic plaques. This study sought to determine the usefulness of (99m)Tc-labelled monoclonal antibody (mAb), recognizing MT1-MMP, for imaging atherosclerosis in a rabbit model (WHHLMI rabbits). METHODS: Anti-MT1-MMP monoclonal IgG(3) and negative control IgG(3) were radiolabelled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid (HYNIC) to yield (99m)Tc-MT1-MMP mAb and (99m)Tc-IgG(3), respectively. WHHLMI and control rabbits were injected with these radio-probes. The aorta was removed and radioactivity was measured at 24 h after the injection. Autoradiography and histological studies were performed. RESULTS: (99m)Tc-MT1-MMP mAb accumulation in WHHLMI rabbit aortas was 5.4-fold higher than that of control rabbits. Regional (99m)Tc-MT1-MMP mAb accumulation was positively correlated with MT1-MMP expression (r = 0.59, p < 0.0001), while (99m)Tc-IgG(3) accumulation was independent of MT1-MMP expression (r = 0.03, p = NS). The highest (99m)Tc-MT1-MMP mAb accumulation was found in atheromatous lesions (4.8 ± 1.9, %ID×BW/mm(2) × 10(2)), followed in decreasing order by fibroatheromatous (1.8 ± 1.3), collagen-rich (1.6 ± 1.0) and neointimal lesions (1.5 ± 1.5). In contrast, (99m)Tc-IgG(3) accumulation was almost independent of the histological grade of lesions. CONCLUSION: Higher (99m)Tc-MT1-MMP mAb accumulation in grade IV atheroma was shown in comparison with neointimal lesions or other more stable lesions. Nuclear imaging with (99m)Tc-MT1-MMP mAb, in combination with CT and MRI, could provide new diagnostic imaging capabilities for detecting vulnerable plaques, although further investigations to improve target to blood ratios are strongly required.

Development of a radiolabeled probe for detecting membrane type-1 matrix metalloproteinase on malignant tumors.

Membrane type-1 matrix metalloproteinase (MT1-MMP) expressed on the tumor cell surface activates pro-MMP-2 and pro-MMP-13 to exacerbate the malignancy, suggesting its suitability as a target molecule for diagnosis by in vivo molecular imaging. Thus, we prepared radiolabeled anti-MT1-MMP monoclonal antibody (mAb) as a novel radiolabeled probe for detecting MT1-MMP in vivo and evaluated its usefulness in breast tumor-bearing rodents. (99m)Tc-anti-MT1-MMP mAb was prepared using HYNIC as a bifunctional chelating agent and immunoreactivity was evaluated by flow cytometry. MT1-MMP expression in breast carcinoma cells (rat: Walker-256 and MRMT-1, mouse: FM3A) was measured by Western blotting. In vivo biodistribution was examined for 48 h using tumor-implanted rodents followed by estimation of radiation absorbed by a standard quantitation platform Organ Level Internal Dose Assessment (OLINDA). (99m)Tc-anti-MT1-MMP mAb was obtained with 84% immunoreactivity to MT1-MMP and more than 92% radiochemical purity. MT1-MMP was highly expressed in all malignant cells. Tumor radioactivity increased with time after administration and reached 3 to 5 times higher values at 24 h post-injection than those at 1 h. Other organs, including the stomach, showed decreasing values over time. Tumor to blood ratios increased with time and reached more than 1.3 at 48 h. The effective dose was <5.0 muSv/MBq. The results suggest that (99m)Tc-anti-MT1-MMP mAb is a promising probe for future diagnosis of breast tumors by in vivo nuclear medical imaging.

Targeting of lectinlike oxidized low-density lipoprotein receptor 1 (LOX-1) with 99mTc-labeled anti-LOX-1 antibody: potential agent for imaging of vulnerable plaque.

UNLABELLED: Lectinlike oxidized low-density lipoprotein (LDL) receptor 1 (LOX-1), a cell surface receptor for oxidized LDL, has been implicated in vascular cell dysfunction related to plaque instability, which could be a potential target for an atherosclerosis imaging tracer. In this study, we designed and prepared (99m)Tc-labeled anti-LOX-1 monoclonal IgG and investigated its usefulness as an atherosclerosis imaging agent. METHODS: Anti-LOX-1 monoclonal IgG and control mouse IgG2a were labeled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid to yield (99m)Tc-LOX-1-mAb and (99m)Tc-IgG2a, respectively. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits (atherosclerosis model) and control rabbits were injected intravenously with these probes, and in vivo planar imaging was performed. At 24 h after the injection, the aortas were removed, and radioactivity was measured. Autoradiography and histologic studies were performed with serial aortic sections. RESULTS: The level of (99m)Tc-LOX-1-mAb accumulation was 2.0-fold higher than the level of (99m)Tc-IgG2a accumulation in WHHLMI rabbit aortas, and the level of (99m)Tc-LOX-1-mAb accumulation in WHHLMI rabbit aortas was 10.0-fold higher than the level of (99m)Tc-LOX-1-mAb accumulation in control rabbit aortas. In vivo imaging clearly visualized the atherosclerotic aortas of WHHLMI rabbits. Autoradiography and histologic studies revealed that regional (99m)Tc-IgG2a accumulation was independent of the histologic grade of the lesions; however, regional (99m)Tc-LOX-1-mAb accumulation was significantly correlated with LOX-1 expression density and the vulnerability index. The highest level of (99m)Tc-LOX-1-mAb accumulation, expressed as {radioactivity in region of interest (Bq/mm(2))/[injected radioactivity (Bq)/animal body weight (g)]} x 10(2), was found in atheromatous lesions (3.8 +/- 1.1 [mean +/- SD]), followed in decreasing order by fibroatheromatous lesions (2.0 +/- 1.0), collagen-rich lesions (1.6 +/- 0.8), and neointimal lesions (1.4 +/- 0.7). CONCLUSION: The level of (99m)Tc-LOX-1-mAb accumulation in grade IV atheroma was higher than that in neointimal lesions or other, more stable lesions. Nuclear imaging of LOX-1 expression with (99m)Tc-LOX-1-mAb may be a useful means for predicting atheroma at high risk for rupture.

Prominent lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1) expression in atherosclerotic lesions is associated with tissue factor expression and apoptosis in hypercholesterolemic rabbits.

BACKGROUND: Despite increasing in vitro evidence that lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL, is implicated in the atherogenesis and thrombus formation, its in vivo participation to the atherosclerotic plaque destabilization, rupture and thrombus formation remains unclear. Here, we compared the in vivo expression of LOX-1, with tissue factor (TF) expression and cell apoptosis, in atherosclerotic lesions of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. METHODS AND RESULTS: We prepared sixty series of cross sections in the aortic arch and the thoracic aorta from four WHHLMI rabbits. LOX-1 and TF expression, as well as apoptotic events were determined by immunohistochemical staining and TUNEL methods, respectively. LOX-1 expression was mainly observed in the macrophage-rich lipid areas of vulnerable plaque-like atheromatous lesions where TF expression and apoptotic events were prominent. LOX-1 expression was positively correlated with TF expression (r=0.53, p<0.0001), apoptotic events (r=0.52, p<0.0001) and morphological vulnerability (r=0.63, p<0.0001). CONCLUSIONS: LOX-1 expression appears to be closely associated with TF expression, apoptotic events and the morphological vulnerability, suggesting the in vivo involvement of LOX-1 in the destabilization and rupture of atherosclerotic lesions and the subsequent thrombus formation. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis.

Distribution profiles of membrane Type-1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) in rabbit atherosclerosis: comparison with plaque instability analysis.

BACKGROUND: Despite increasing evidence that membrane type 1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2), and cyclooxygenase-2 (COX-2) are involved in the pathogenesis of atherosclerosis, the possible links among these enzymes remain unclear. Accordingly, we investigated the distribution of MT1-MMP, MMP-2, and COX-2 immunohistologically in the atherosclerotic lesions of hypercholesterolemic (WHHLMI) rabbits. METHODS AND RESULTS: Distribution of MT1-MMP, MMP-2, and COX-2 was examined by immunohistochemical staining using sixty cross sections of the ascending-arch and thoracic aortas prepared from 4 WHHLMI rabbits. MT1-MMP and MMP-2 staining was prominently observed in the macrophage-rich regions of the atheromatous lesions, and was positively correlated with morphological vulnerability (r=0.63 for MT1-MMP; r=0.60 for MMP-2; p<0.0001). MT1-MMP staining was positively correlated with MMP-2 staining (r=0.61, p<0.0001). COX-2 staining was also the highest in the macrophage-rich regions of the atheromatous lesions, with relatively high staining levels in other more stable lesions. CONCLUSIONS: Co-distribution of MT1-MMP, MMP-2, and COX-2 was demonstrated in grade IV atheroma, indicating a possible link among these enzymes in the destabilization of atherosclerotic plaques. The relatively high COX-2 distribution in other more stable lesions may indicate its additional roles in the stabilization of atherosclerotic lesions. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis and provide useful information for the development of new therapeutic and diagnostic (imaging) agents that target MMPs and COX-2 in atherosclerosis.

99mTc-Annexin A5 for noninvasive characterization of atherosclerotic lesions: imaging and histological studies in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits.

PURPOSE: Apoptosis is commonly observed in advanced atherosclerotic lesions. 99mTc-annexin A5 (99mTc-annexin V) has been proposed as a potential tracer for imaging apoptosis in atherosclerotic plaques. Accordingly, we determined the usefulness of 99mTc-annexin A5 as an atherosclerosis imaging tracer in a rabbit model (myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits; WHHLMI rabbits) of spontaneous atherosclerosis. METHODS: The WHHLMI and control rabbits were injected intravenously with 99mTc-annexin A5. After in vivo planar imaging, the radioactivity in the aorta was measured. Autoradiography, TUNEL staining, Azan-Mallory staining and immunohistological studies were performed serially throughout the aorta. RESULTS: 99mTc-Annexin A5 accumulation in the aorta of the WHHLMI rabbits was 5.6-fold higher than in that of control rabbits. Autoradiography showed heterogeneous multifocal accumulation of 99mTc-annexin A5 in WHHLMI rabbits. 99mTc-Annexin A5 accumulation was highest in the atheromatous lesions (6.2+/-2.5, %IDxBW/mm2x10(3)), followed in decreasing order by neointimal (4.9+/-1.3), fibroatheromatous (4.5+/-1.9), and collagen-rich lesions (3.3+/-1.4). The regional 99mTc-annexin A5 accumulation was significantly correlated with the TUNEL-positive cell density, macrophage density and "vulnerability index," an index of the morphological destabilized characteristics. The in vivo imaging clearly visualized the atherosclerotic lesions in WHHLMI rabbits. CONCLUSION: The present study in WHHLMI rabbits showed higher 99mTc-annexin A5 accumulation in grade IV atheroma than in other more stable lesions. 99mTc-Annexin A5 may be useful in identifying atheroma that is at higher risk for rupture and possibly in assessing the response to anti-atherosclerotic therapy.