Quantitative evaluation of hepatic integrin αvß3 expression by positron emission tomography imaging using 18F-FPP-RGD2 in rats with non-alcoholic steatohepatitis.

BACKGROUND: Integrin αvß3, which are expressed by activated hepatic stellate cells in non-alcoholic steatohepatitis (NASH), play an important role in the fibrosis. Recently, we reported that an RGD peptide positron emission tomography (PET) probe is useful as a predictor of hepatic fibrosis. Kinetic analysis of the RGD PET probe has been performed in tumours, but not in hepatic fibrosis. Therefore, we aimed to quantify hepatic integrin αvß3 in a model of NASH by kinetic analysis using 18F-FPP-RGD2, an integrin αvß3 PET probe. METHODS: 18F-FPP-RGD2 PET/CT scans were performed in control and NASH rats. Tissue kinetic analyses were performed using a one-tissue, two-compartment (1T2C) and a two-tissue, three-compartment (2T3C) model using an image-derived input function (IDIF) for the left ventricle. We then conducted correlation analysis between standard uptake values (SUVs) or volume of distribution (VT), evaluated using compartment kinetic analysis and integrin αv or ß3 protein expression. RESULTS: Biochemical and histological evaluation confirmed the development of NASH rats. Integrin αvß3 protein expression and hepatic SUV were higher in NASH- than normal rats. The hepatic activity of 18F-FPP-RGD2 peaked rapidly after administration and then gradually decreased, whereas left ventricular activity rapidly disappeared. The 2T3C model was found to be preferable for 18F-FPP-RGD2 kinetic analysis in the liver. The VT (IDIF) for 18F-FPP-RGD2, calculated using the 2T3C model, was significantly higher in NASH- than normal rats and correlated strongly with hepatic integrin αv and ß3 protein expression. The strengths of these correlations were similar to those between SUV60-90 min and hepatic integrin αv or ß3 protein expression. CONCLUSIONS: We have demonstrated that the VT (IDIF) of 18F-FPP-RGD2, calculated using kinetic modelling, positively correlates with integrin αv and ß3 protein in the liver of NASH rats. These findings suggest that hepatic VT (IDIF) provides a quantitative assessment of integrin αvß3 protein in liver.

Pharmacological MRI responses of raclopride in rats: The relationship with D2 receptor occupancy and cataleptic behavior.

Pharmacological magnetic resonance imaging (phMRI) allows the visualization of brain pharmacological effects of drugs using functional MRI (fMRI). phMRI can help us facilitate central nervous system (CNS) drug development. However, there have been few studies demonstrating the dose relationship of the fMRI response induced by CNS drugs to underlying target engagement or behavioral efficacy. To clarify these relationships, we examined receptor occupancy measurements using positron emission tomography (PET) (n = 3~5), fMRI (n = 5~8) and a cataleptic behavior (n = 6) with raclopride, a dopamine D2 receptor antagonist (8, 20, and 200 µg/kg) on Wistar rats. Dopamine D2 receptor occupancy was increased dose dependently by raclopride (41.8 ± 2.7%, 8 µg/kg; 64.9 ± 2.8%, 20 µg/kg; 83.1 ± 3.0%, 200 µg/kg). phMRI study revealed significant positive responses to raclopride at 200 µg/kg specifically in the striatum and nucleus accumbens, related to dopaminergic system. Slight fMRI responses were observed at 20 µg/kg in some areas corresponding to the striatum and nucleus accumbens. There were no noticeable fMRI responses at 8 µg/kg raclopride administration. Raclopride at 200 µg/kg significantly increased the cataleptic score, although, at 8 and 20 µg/kg, raclopride had no significant effects. These findings showed that raclopride-induced fMRI responses were observed at doses inducing cataleptic behavior and high D2 receptor occupancy, suggesting that phMRI can be useful for dose selection in clinical trial as an evaluation method of brain activity, which reflects behavioral responses induced by target engagements.

Evaluation of hepatic function using dynamic contrast-enhanced magnetic resonance imaging in melanocortin 4 receptor-deficient mice as a model of nonalcoholic steatohepatitis.

INTRODUCTION: Melanocortin 4 receptor-deficient (MC4R-KO) mice fed a high-fat diet (HFD) develop liver pathology similar to human nonalcoholic steatohepatitis (NASH). However, although liver histology and blood biochemistry have been reported, hepatic function has not been evaluated. In the present study, we evaluated hepatic function in MC4R-KO mice fed an HFD using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with gadolinium­ethoxybenzyl­diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). MATERIALS AND METHODS: Wild type (WT) mice and MC4R-KO mice were fed a standard diet (SD) or an HFD for 20 weeks. The hepatic signal intensity was obtained from DCE-MRI images, and relative enhancement (RE), the time to maximum RE (Tmax), and the half-life of RE elimination (T1/2) were calculated. Histopathological analysis was then performed. RESULTS: Histological analysis with nonalcoholic fatty liver disease activity score (NAS) revealed that MC4R-KO mice fed an HFD achieved the NAS of 5. There was moderate fibrosis in MC4R-KO mice fed an HFD. DCE-MRI with Gd-EOB-DTPA showed that Tmax and T1/2 were significantly longer in MC4R-KO mice fed an HFD compared with wild type (WT) mice (Tmax, WT, 3.9 ±â€¯0.4 min; MC4R-KO, 7.4 ±â€¯1.5 min; T1/2, WT, 23.7 ±â€¯1.9 min; MC4R-KO, 62.5 ±â€¯18.5 min). Tmax and T1/2 were significantly correlated with histopathologic score (steatosis vs. Tmax, rho = 0.48, P = 0.04; steatosis vs. T1/2, rho = 0.50, P = 0.03; inflammation vs. Tmax, rho = 0.55, P = 0.02; inflammation vs. T1/2, rho = 0.61, P < 0.01; ballooning vs. T1/2, rho = 0.51, P = 0.03;fibrosis vs Tmax, rho = 0.72, P < 0.01; fibrosis vs T1/2, rho = 0.75, P < 0.01). CONCLUSIONS: MC4R-KO mice fed an HFD developed obesity and NASH. The liver kinetics of Gd-EOB-DTPA were significantly different in MC4R-KO mice fed an HFD from WT mice, and correlated with the histopathologic score. These results suggest that MC4R-KO mice fed an HFD mimic the hepatic pathology and liver function of human NASH, and therefore might be useful for the study of hepatic dysfunction during the fibrotic stage of NASH.

Coordination-Mediated Synthesis of 67Ga-Labeled Purification-Free Trivalent Probes for in Vivo Imaging of Saturable Systems.

A large excess of unlabeled ligands over gallium-67 (67Ga) provides 67Ga-labeled probes with high radiochemical yields in a short reaction time. However, the unlabeled ligands hinder target accumulation of radiolabeled probes by competing for target molecules. To circumvent the problem, we investigated the way to prepare 67Ga-labeled multivalent probes from monovalent ligands. The reaction of a bi- or tridentate ligand with [67Ga]Ga-citrate resulted in 67Ga-labeled probes of insufficient stability. However, the reaction of [67Ga]Ga-citrate with a mixture of RGD-conjugated salicylaldehyde and triamine provided a 67Ga-labeled trivalent probe with stability sufficient for in vivo applications. Since the free Schiff base ligand decomposed rapidly upon injection, the 67Ga-labeled trivalent probe visualized the murine tumor without postlabeling purification, which was not achieved with a 67Ga-labeled trivalent probe from a trivalent ligand. These findings indicate the availability of Schiff base ligands to prepare 67Ga-labeled trivalent probes by a simple radiolabeling procedure.

Detection of neuroinflammation before selective neuronal loss appearance after mild focal ischemia using [18F]DPA-714 imaging.

BACKGROUND: Translocator protein (TSPO) imaging can be used to detect neuroinflammation (including microglial activation) after acute cerebral infarction. However, longitudinal changes of TSPO binding after mild ischemia that induces selective neuronal loss (SNL) without acute infarction are not well understood. Here, we performed TSPO imaging with [18F]DPA-714 to determine the time course of neuroinflammation and SNL after mild focal ischemia. RESULTS: Mild focal ischemia was induced by middle cerebral artery occlusion (MCAO) for 20 min. In MCAO rats without acute infarction investigated by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, in vitro ARG revealed a significant increase of [18F]DPA-714 binding in the ipsilateral striatum compared with that in the contralateral side at 1, 2, 3, and 7 days after MCAO. Increased [18F]DPA-714 binding was observed in the cerebral cortex penumbra, reaching maximal values at 7 days after MCAO. Activation of striatal microglia and astrocytes was observed with immunohistochemistry of ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) at 2, 3, and 7 days after MCAO. SNL was investigated with Nissl staining and neuronal nuclei (NeuN) immunostaining and observed in the ischemic core region of the striatum on days 3 and 7 after MCAO. We confirmed that total distribution volume of [18F]DPA-714 in the ipsilateral striatum was significantly increased at 2 and 7 days after MCAO using positron emission tomography (PET). CONCLUSIONS: [18F]DPA-714 binding measured with in vitro ARG was increased before SNL appeared, and this change was detected by in vivo PET. These findings suggest that TSPO PET imaging might be useful for detection of neuroinflammation leading to SNL after focal ischemia.

Evaluation of hepatic integrin αvß3 expression in non-alcoholic steatohepatitis (NASH) model mouse by 18F-FPP-RGD2 PET.

BACKGROUND: Activated hepatic stellate cells (HSCs), which express integrin αvß3, are a major fibrogenic factor in NASH pathophysiology. 18F-labeled cyclic arginine-glycine-aspartic acid penta-peptide (18F-FPP-RGD2) has been used as a PET probe for tumors expressing integrin αvß3. The aim of this study was to assess the potential of PET with 18F-FPP-RGD2 to detect hepatic integrin αvß3 expression in non-alcoholic steatohepatitis (NASH) model mice. RESULTS: Thirty-two male C57BL/6 mice aged 6 weeks were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 3 and 8 weeks. 18F-FPP-RGD2 PET imaging of the liver was performed at 3 and 8 weeks after CDAHFD feeding. After PET scanning, levels of hepatic integrin αvß, 3α-smooth muscle actin (α-SMA), and collagen type 1 alpha 1(col1a1) were measured. Histopathological analysis of hepatic steatosis, inflammation, and fibrosis, as well as blood biochemistry analysis, was also performed. CDAHFD for 3 and 8 weeks produced a moderate-to-severe steatosis and inflammation of the liver in mice. NAFLD activity score (NAS) in mice fed the CDAHFD for 3 and 8 weeks were more than 4 indicating NASH or borderline NASH pathology. Fibrosis was observed only in mice fed the CDAHFD for 8 weeks. PET imaging showed that the hepatic standardized uptake value, SUV80-90 min, was increased with prolonged CDAHFD feeding compared with the respective controls (CDAHFD 3 weeks 0.32 ± 0.06 vs 0.48 ± 0.05, p < 0.01; CDAHFD 8 weeks 0.35 ± 0.04 vs 0.75 ± 0.07, p < 0.01, respectively). Prolonged CDAHFD feeding increased hepatic mRNA and protein levels of integrin αv and ß3 at 3 and 8 weeks. Hepatic 18F-FPP-RGD2 uptake and amount of integrin αv and ß3 protein were well correlated (r = 0.593, p < 0.05 and r = 0.835, p < 0.001, respectively). Hepatic 18F-FPP-RGD2 uptake also showed a positive correlation with Sirius red-positive area. CONCLUSIONS: The hepatic uptake of 18F-FPP-RGD2 correlated well with integrin αv and ß3 expression and histological fibrosis in a mouse model of NASH, suggesting the predictability of fibrosis in NASH pathology.

[18F]-BMS-747158-02PET imaging for evaluating hepatic mitochondrial complex 1dysfunction in a mouse model of non-alcoholic fatty liver disease.

BACKGROUND: Mitochondrial dysfunction is one of the main causes of non-alcohol fatty liver disease (NAFLD). [18F]-BMS-747158-02 (18F-BMS) which was originally developed as a myocardial perfusion imaging agent was reported to bind mitochondrial complex-1 (MC-1). The aim of this study was to investigate the potential use of 18F-BMS for evaluating hepatic MC-1 activity in mice fed a methionine- and choline-deficient (MCD) diet. Male C57BL/6J mice were fed a MCD diet for up to 2 weeks. PET scans with 18F-BMS were performed after 1 and 2 weeks of the MCD diet. 18F-BMS was intravenously injected into mice, and the uptake (standardized uptake value (SUV)) in the liver was determined. The binding specificity for MC-1 was assessed by pre-administration of rotenone, a specific MC-1 inhibitor. Hepatic MC-1 activity was measured using liver homogenates generated after each positron emission tomography (PET) scan. Blood biochemistry and histopathology were also assessed. RESULTS: In control mice, hepatic 18F-BMS uptake was significantly inhibited by the pre-injection of rotenone. The uptake of 18F-BMS was significantly decreased after 2 weeks of the MCD diet. The SUV at 30-60 min was well correlated with hepatic MC-1 activity (r = 0.73, p < 0.05). Increases in plasma ALT and AST levels were also noted at 1 and 2 weeks. Mild hepatic steatosis with or without minimal inflammation was histopathologically observed at 1 and 2 weeks in mice liver on the MCD diet. However, inflammation was observed only at 2 weeks in mice on the MCD diet. CONCLUSIONS: The present study demonstrated that 18F-BMS is a potential PET probe for quantitative imaging of hepatic MC-1 activity and its mitochondrial dysfunction induced by steatosis and inflammation, such as in NAFLD.

Evaluation of a filament perforation model for mouse subarachnoid hemorrhage using 7.0 Tesla MRI.

The filament perforation model (FPM) in mice is becoming increasingly popular to elucidate the molecular pathogenesis of neuronal injury after subarachnoid hemorrhage (SAH). We evaluated brain MRI in a mouse FPM. A total of 28 male C57Bl/6J mice were used. Seventeen animals underwent SAH induction by FPM. In two animals, transient middle cerebral artery occlusion (MCAo) was induced. Nine mice served as controls. T1-weighted images (T1WI), T2-weighted images (T2WI), T2(∗)-weighted images (T2*WI) and apparent diffusion coefficient maps were acquired at day 0 and at various time points following SAH (range: day 1-6 after SAH). Cerebral blood flow (CBF) analysis by (14)C-iodoamphetamine ((14)C-IMP) autoradiography was conducted in nine animals. Hemorrhage could be best confirmed using T2*WI. The degree of hemorrhage varied. All animals evaluated for ⩾2days were hydrocephalic, which was best seen on T2WI. T2-hyperintensity of the corpus callosum and external capsule, indicating white matter (WM) injury, was present after SAH. Ventricle and WM injury volumes were statistically significantly higher at day 3 compared to day 0. Territorial ischemia was detectable in MCAo but not in SAH. Markedly hypointense cortical veins were visible in the hyperacute and delayed phase after SAH on T2*WI. The (14)C-IMP analysis indicated decreased CBF after SAH. MRI is feasible and useful in evaluating pathophysiological changes over time. T2*WI seems best for SAH detection and grading. The chronological change of hydrocephalus and WM injury could be analyzed. T2*WI illustrated specific signal changes of cortical veins, possibly caused by increased oxygen extraction fraction due to decreased CBF.

Gd-EOB-DTPA-enhanced-MR imaging in the inflammation stage of nonalcoholic steatohepatitis (NASH) in mice.

OBJECTIVE: The purpose of this study is to investigate the correlation between the liver kinetics of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) and liver histopathology in a mouse model of NASH by using dynamic contrast-enhanced MRI. MATERIALS AND METHODS: Twenty male C57/BL6 mice aged 8weeks were fed a methionine-choline-deficient (MCD) diet for 2, 4 and 6weeks (MCD groups: MCD 2w, 4w, or 6w). Gd-EOB-DTPA-enhanced MR imaging of the liver was performed at 2, 4 and 6weeks after the MCD feeding. The signal intensity of the liver was obtained from dynamic MR images and relative enhancement (RE), and the time to maximum RE (Tmax) and half-life of elimination RE (T1/2) were calculated. After MRI scan, histopathological scores of hepatic steatosis and inflammation and blood biochemistry data, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, were obtained. RESULTS: Plasma AST and ALT levels were significantly increased in mice fed MCD. Histopathological scores indicated that steatohepatitis progressed with the MCD feeding period from 2 to 6weeks, but significant fibrosis was observed only in mice fed MCD for 6weeks. Gd-EOB-DTPA-enhanced MRI showed that Tmax was significantly prolonged in the livers of the 6-week group compared to the control group (control, 4.0±0.7min; MCD 6w, 12.1±1.6min), although there was no alteration in the 2- and 4-week groups. T1/2 was significantly prolonged in mice fed MCD for 4 and 6weeks compared to the control group (control, 19.9±2.0min; MCD 4w, 46.7±8.7min; MCD 6w, 65.4±8.8min). The parameters of Gd-EOB-DTPA kinetics (Tmax and T1/2) in the liver were positively correlated with the liver histopathological score (steatosis vs Tmax, rho=0.69, P=0.0007; inflammation vs Tmax, rho=0.66, P=0.00155; steatosis vs T1/2, rho=0.77, P<0.0001; inflammation vs T1/2, rho=0.73, P=0.0003). CONCLUSIONS: The liver kinetics of Gd-EOB-DTPA correlated well with the inflammation score in the mouse model of NASH, suggesting the possibility of detecting the steatohepatitis stage without fibrosis by Gd-EOB-DTPA-enhanced MR imaging.

Time Course of Diffusion Kurtosis in Cerebral Infarctions of Transient Middle Cerebral Artery Occlusion Rat Model.

OBJECTIVE: To evaluate the relationship between fiber bundle direction and changes in diffusion kurtosis, we evaluated the apparent diffusion kurtosis coefficients (AKCs) that were perpendicular to and parallel to the principal diffusion tensor eigenvector. MATERIALS AND METHODS: Adult male Wistar rats were subjected to 30 or 60 minutes of middle cerebral artery occlusion and imaged with a 7T Magnetic Resonance Imager System (Varian MRI System 7T/210: Agilent Technologies, CA). Diffusion kurtosis images were obtained before middle cerebral artery (MCA) reperfusion and 3, 6, and 24 hours after reperfusion to generate the apparent diffusion coefficient (ADC), fractional anisotropy (FA), mean apparent diffusion kurtosis coefficient (mAKC), AKC axial to the eigenvector (axAKC), and AKC radial to the eigenvector (radAKC) images. The time course of the region/normal ratio was evaluated for the above parameters in the caudoputamen and white matter. RESULTS: Relative FA and relative ADC values decreased 3 hours after MCA reperfusion and remained decreased until 24 hours. Relative mAKC, axAKC, and radAKC values were increased 3 hours after MCA reperfusion, peaked after 6 hours, and slightly decreased after 24 hours. In the white matter, axAKC showed larger changes than radAKC. CONCLUSION: The time course of the diffusion kurtosis value showed earlier pseudonormalization than the ADC value of the lesions. For white matter lesions, the increase in axAKC was larger than that in radAKC, suggesting that the tissue changes after infarction mainly produce reduced diffusivity along the fibers and lead to increased inhomogeneity of the diffusion.

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.

Pharmacological MRI response to a selective dopamine transporter inhibitor, GBR12909, in awake and anesthetized rats.

Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, d-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies.

(67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of (67)Ga-labeled Fabs for low renal radioactivity levels.

Potential of (99m)Tc-MIBI SPECT imaging for evaluating non-alcoholic steatohepatitis induced by methionine-choline-deficient diet in mice.

BACKGROUND: Hepatic mitochondrial dysfunction has been implicated in pathological conditions leading to non-alcoholic steatohepatitis (NASH). Technetium-99 m-2-methoxyisobutyl-isonitrile ((99m)Tc-MIBI), a lipophilic cationic myocardial perfusion agent, is retained in the mitochondria depending on membrane potential. The aim of this study was to investigate the feasibility of (99m)Tc-MIBI for evaluating the hepatic mitochondrial dysfunction induced by methionine-choline-deficient (MCD) diet in mice. METHODS: Male C57Black6J/jcl mice were fed a MCD diet for up to 4 weeks. SPECT scan (N =6) with (99m)Tc-MIBI was performed at 2 and 4 weeks after MCD diet. Mice were imaged with small-animal SPECT/CT under isoflurane anesthesia. Radioactivity concentrations of the liver were measured, and the time of maximum (T max) and the elimination half-life (T 1/2) were evaluated. After SPECT scan, liver histopathology was analyzed to evaluate steatosis and inflammation. Non-alcoholic fatty liver disease (NAFLD) activity score was obtained from the histological score of hepatic steatosis and inflammation. Blood biochemistry and hepatic ATP content were also measured (N =5 to 6). RESULTS: Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly elevated at 2 and 4 weeks after MCD diet. A decrease in hepatic ATP content was also observed in MCD-fed mice. (99m)Tc-MIBI SPECT imaging clearly showed the decrease of hepatic (99m)Tc-MIBI retention in MCD-fed mice compared to control mice. T 1/2 after (99m)Tc-MIBI injection was significantly decreased in the liver of MCD-fed mice (control, MCD 2 weeks, and MCD 4 weeks, T 1/2 = 57.6, 37.6, and 19.8 min, respectively), although no change in T max was observed in MCD-fed mice. SPECT data and histological score showed that the negative correlation (r = -0.74, p <0.05) between T 1/2 and NAFLD activity score was significant. CONCLUSIONS: Hepatic (99m)Tc-MIBI elimination was increased with increase in NAFLD activity score (NAS) in mice fed MCD diet for 2 and 4 weeks. These results suggest that (99m)Tc-MIBI SPECT imaging might be useful for detecting hepatic mitochondrial dysfunction induced by steatosis and inflammation such as NAFLD or NASH.

Synthesis and evaluation of diastereoisomers of 1,4,7-triazacyclononane-1,4,7-tris-(glutaric acid) (NOTGA) for multimeric radiopharmaceuticals of gallium.

In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7-triazacyclononane (NOTGA), four isomeric species are usually generated by the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To estimate their biological efficacies as well as their stability and radiochemistry, the RRR/SSS and RRS/SSR NOTGA-(t)Bu prochelators were isolated and the corresponding cyclic RGDfK (RGD) conjugates with triethylene glycol linkages were prepared. The RRR/SSS and RRS/SSR diastereomers were obtained in 69% and 17% yields, respectively. In the complexation reaction with (67)GaCl(3), both diastereomers provided >98% radiochemical yields at pH 5 within 10 min when the reaction was conducted at room temperature. However, the RRR/SSS diastereomer exhibited more pH-sensitive radiochemical yields between pH 3.5 to 4.5. Despite their diasteromeric nature, both (67)Ga-labeled RGD-NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin α(v)ß(3) and biodistribution with predominant renal excretion. Similar tumor uptake was also observed in mice bearing U87MG tumor xenograft, which resulted in impressively high contrast SPECT/CT images. These findings indicate that the RGD-NOTGA conjugates of both diastereomers presented here possess equivalent biological efficacies and their combined usage would be feasible. It is worth noting that specific properties of a given biomolecule, cell expression levels of the corresponding target molecule, and presence or absence of a pharmacokinetic modifier would affect the structural differences between diastereomers on the ligand-receptor interactions and pharmacokinetics. Thus, the preparation of corresponding conjugates and evaluation of their chemical and biological performances still remains important for applying NOTGA to other biomolecules of interest using the diastereomerically pure NOTGA-(t)Bu prochelator.