Radiotheranostics Using a Novel 225Ac-Labeled Radioligand with Improved Pharmacokinetics Targeting Prostate-Specific Membrane Antigen.

225Ac-based radiotheranostics targeting prostate-specific membrane antigen (PSMA) has induced impressive responses in patients with metastatic castration-resistant prostate cancer. To enhance the therapeutic effects of radioligands labeled with 225Ac (half-life: 10 days), a radioligand that shows longer tumor retention would be useful. Here, we designed and synthesized a straight-chain PSMA-targeting radioligand, PSMA-DA1, which includes an (iodophenyl)butyric acid derivative as an albumin binder (ALB). We performed preclinical evaluations of PSMA-DA1 as a tool for PSMA-targeting radiotheranostics using 111In, 90Y, and 225Ac. [111In]In-PSMA-DA1 demonstrated significantly greater tumor uptake and retention than a corresponding non-ALB-conjugated compound. In mice, single-photon emission computed tomography performed with [111In]In-PSMA-DA1 produced clear tumor images, and the administration of [90Y]Y-PSMA-DA1 or [225Ac]Ac-PSMA-DA1 inhibited tumor growth. [225Ac]Ac-PSMA-DA1 had antitumor effects in mice at a lower radioactivity level than [225Ac]Ac-PSMA-617, which has been reported to be clinically useful. These results indicate that PSMA-DA1 may be a useful PSMA-targeting radiotheranostic agent.

Correction to: High­Mobility Group Box­1­Induced Angiogenesis After Indirect Bypass Surgery in a Chronic Cerebral Hypoperfusion Model.

The original version of this article unfortunately contained a mistake. Figure 5a, b were incorrect. The correct figures are given below.

Development and characterization of a 68Ga-labeled A20FMDV2 peptide probe for the PET imaging of αvß6 integrin-positive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known to be one of the most lethal cancers. Since the majority of patients are diagnosed at an advanced stage, development of a detection method for PDAC at an earlier stage of disease progression is strongly desirable. Integrin αVß6 is a promising target for early PDAC detection because its expression increases during precancerous changes. The present study aimed to develop an imaging probe for positron emission tomography (PET) which targets αVß6 integrin-positive PDAC. We selected A20FMDV2 peptide, which binds specifically to αvß6 integrin, as a probe scaffold, and 68Ga as a radioisotope. A20FMDV2 peptide has not been previously labeled with 68Ga. A cysteine residue was introduced to the N-terminus of the probe at a site-specific conjugation of maleimide-NOTA (mal-NOTA) chelate. Different numbers of glycine residues were also introduced between cysteine and the A20FMDV2 sequence as a spacer in order to reduce the steric hindrance of the mal-NOTA on the binding probe to αVß6 integrin. In vitro, the competitive binding assay revealed that probes containing a 6-glycine linker ([natGa]CG6 and [natGa]Ac-CG6) showed high affinity to αVß6 integrin. Both probes could be labeled by 67/68Ga with high radiochemical yield (>50%) and purity (>98%). On biodistribution analysis, [67Ga]Ac-CG6 showed higher tumor accumulation, faster blood clearance, and lower accumulation in the surrounding organs of pancreas than did [67Ga]CG6. The αVß6 integrin-positive xenografts were clearly visualized by PET imaging with [68Ga]Ac-CG6. The intratumoral distribution of [68Ga]Ac-CG6 coincided with the αVß6 integrin-positive regions detected by immunohistochemistry. Thus, [68Ga]Ac-CG6 is a useful peptide probe for the imaging of αVß6 integrin in PDAC.

High-Mobility Group Box-1-Induced Angiogenesis After Indirect Bypass Surgery in a Chronic Cerebral Hypoperfusion Model.

High-mobility group box-1 (HMGB1) is a nuclear protein that promotes inflammation during the acute phase post-stroke, and enhances angiogenesis during the delayed phase. Here, we evaluated whether indirect revascularization surgery with HMGB1 accelerates brain angiogenesis in a chronic cerebral hypoperfusion model. Seven days after hypoperfusion induction, encephalo-myo-synangiosis (EMS) was performed with or without HMGB1 treatment into the temporal muscle. We detected significant increments in cortical vasculature (p < 0.01), vascular endothelial growth factor (VEGF) expression in the temporal muscle (p < 0.05), and ratio of radiation intensity on the operated side compared with the non-operated side after EMS in the HMGB1-treated group than in the control group (p < 0.01). Altogether, HMGB1 with EMS in a chronic hypoperfusion model promoted brain angiogenesis in a VEGF-dependent manner, resulting in cerebral blood flow improvement. This treatment may be an effective therapy for patients with moyamoya disease.

Evaluation of the Relationship Between Cognitive Impairment, Glycometabolism, and Nicotinic Acetylcholine Receptor Deficits in a Mouse Model of Alzheimer's Disease.

PURPOSE: In patients with Alzheimer's disease (AD), the loss of cerebral nicotinic acetylcholine receptors (nAChRs) that are implicated in higher brain functions has been reported. However, it is unclear if nAChR deficits occur in association with cognitive impairments. The purpose of this study was to assess the relationship between nAChR deficits and cognitive impairments in a mouse model of AD (APP/PS2 mice). PROCEDURES: The cognitive abilities of APP/PS2 and wild-type mice (aged 2-16 months) were evaluated using the novel object recognition test. Double-tracer autoradiography analyses with 5-[125I]iodo-A-85380 ([125I]5IA: α4ß2 nAChR imaging probe) and 2-deoxy-2-[18F]fluoro-D-glucose were performed in both mice of different ages. [123I]5IA-single-photon emission tomography (SPECT) imaging was also performed in both mice at 12 months of age. Furthermore, each age cohort was investigated for changes in cognitive ability and expression levels of α7 nAChRs and N-methyl-D-aspartate receptors (NMDARs). RESULTS: No significant difference was found between the APP/PS2 and wild-type mice at 2-6 months of age in terms of novel object recognition memory; subsequently, however, APP/PS2 mice showed a clear cognitive deficit at 12 months of age. [125I]5IA accumulation decreased in the brains of 12-month-old APP/PS2 mice, i.e., at the age at which cognitive impairments were first observed; this result was supported by a reduction in the protein levels of α4 nAChRs using Western blotting. nAChR deficits could be noninvasively detected by [123I]5IA-SPECT in vivo. In contrast, no significant changes in glycometabolism, expression levels of α7 nAChRs, or NMDARs were associated with cognitive impairments in APP/PS2 mice. CONCLUSION: A decrease in cerebral α4ß2 nAChR density could act as a biomarker reflecting cognitive impairments associated with AD pathology.

Noninvasive evaluation of nicotinic acetylcholine receptor availability in mouse brain using single-photon emission computed tomography with [(123)I]5IA.

INTRODUCTION: Nicotinic acetylcholine receptors (nAChRs) are of great interest because they are implicated in higher brain functions. Nuclear medical imaging is one of the useful techniques for noninvasive evaluation of physiological and pathological function in living subjects. Recent progress in nuclear medical imaging modalities enables the clear visualization of the organs of small rodents. Thus, translational research using nuclear medical imaging in transgenic mice has become possible and helps to elucidate human disease pathology. However, imaging of α4ß2 nAChRs in the mouse brain has not yet been performed. The purpose of this study was to assess the feasibility of single-photon emission computed tomography (SPECT) with 5-[(123)I]iodo-3-[2(S)-azetidinylmethoxy]pyridine ([(123)I]5IA) for evaluating α4ß2 nAChR availability in the mouse brain. METHODS: A 60-min dynamic SPECT imaging session of α4ß2 nAChRs in the mouse brain was performed. The regional distribution of radioactivity in the SPECT images was compared to the density of α4ß2 nAChRs measured in an identical mouse. Alteration of nAChR density in the brains of Tg2576 mice was also evaluated. RESULTS: The mouse brain was clearly visualized by [(123)I]5IA-SPECT and probe accumulation was significantly inhibited by pretreatment with (-)-nicotine. The regional distribution of radioactivity in SPECT images showed a significant positive correlation with α4ß2 nAChR density measured in an identical mouse brain. Moreover, [(123)I]5IA-SPECT was able to detect the up-regulation of α4ß2 nAChRs in the brains of Tg2576 transgenic mice. CONCLUSIONS: [(123)I]5IA-SPECT imaging would be a promising tool for evaluating α4ß2 nAChR availability in the mouse brain and may be useful in translational research focused on nAChR-related diseases.

Transport mechanisms of hepatic uptake and bile excretion in clinical hepatobiliary scintigraphy with 99mTc-N-pyridoxyl-5-methyltryptophan.

INTRODUCTION: In clinical hepatobiliary scintigraphy, (99m)Tc-N-pyridoxyl-5-methyltryptophan ((99m)Tc-PMT) is an effective radiotracer among the (99m)Tc-pyridoxylaminates. However, the mechanisms of human hepatic uptake and bile excretion transport of (99m)Tc-PMT have not been determined. We thus investigated the transport mechanisms of human hepatic uptake and bile excretion in hepatobiliary scintigraphy with (99m)Tc-PMT. METHODS: Four solute carrier (SLC) transporters involved in hepatic uptake were evaluated using human embryonic kidney (HEK) and HeLa cells with high expression of SLC transporters (organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporters (OAT)2 and organic cation transporters (OCT)1) after 5 min of (99m)Tc-PMT incubation. Metabolic analysis of (99m)Tc-PMT was performed using pooled human liver S9. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter vesicles human expressing multiple drug resistance 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein or bile salt export pump. (99m)Tc-PMT was incubated for 1, 3 and 5 min with ATP or adenosine monophosphate and these vesicles. SPECT scans were performed in normal and Eisai hyperbilirubinemic (EHBR) model rats, deficient in Mrp2 transporters, without and with verapamil (rat Mdr1 and human MDR1 inhibitor) after intravenous injection of (99m)Tc-PMT. RESULTS: Uptake of (99m)Tc-PMT in HEK293/OATP1B1 and HeLa/OATP1B3 was significantly higher than that in HEK293- and HeLa-mock cells. (99m)Tc-PMT was not metabolized in the human liver S9. In vesicles with high expression of ABC transporters, uptake of MDR1 or MRP2 was significantly higher at all incubation times. Bile excretion of (99m)Tc-PMT was also identified by comparison between normal and EHBR rats with and without verapamil on in-vivo imaging. CONCLUSIONS: Human hepatic uptake of (99m)Tc-PMT was transferred by OATP1B1 and OATP1B3, and excretion into bile canaliculi via MDR1 and MRP2. (99m)Tc-PMT hepatobiliary scintigraphy may be a useful ligand as a noninvasive method of visualizing and quantifying hepatobiliary transporter functionality, which could predict drug pharmacokinetics.

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.

Chemical design of (99m)Tc-labeled probes for targeting osteogenic bone region.

Radionuclide bone imaging using polynuclear (99m)Tc complexes of bisphosphonates is the most common clinical practice in nuclear medicine. However, the improvement in the contrast between normal and osteogenic bone regions has been required. Herein we reported a new (99m)Tc-labeled compound considering the increased vascular permeability of osteogenic region. We selected penta-d-Asp as both a targeting motif to hydroxyapatite (HA) and a molecular size modifier, and two penta-d-Asp molecules were conjugated with the two carboxylate residues of ethylene dicysteine (EC) selected as the (99m)Tc chelating moiety to prepare EC-[(d-Asp)5]2. The molecular size, HA binding, and pharmacokinetics of (99m)Tc-EC-[(d-Asp)5]2 in normal mice and model rats bearing osteogenic tumor were compared to those of (99m)Tc-MDP and (99m)Tc-EC with one (d-Asp)5 motif, (99m)Tc-EC-(d-Asp)5. The molecular size of (99m)Tc-EC-[(d-Asp)5]2 was higher than that of (99m)Tc-MDP and (99m)Tc-EC-(d-Asp)5 when determined by permeability of the (99m)Tc-compounds through a membrane filter (10 kDa). The HA binding of (99m)Tc-EC-[(d-Asp)5]2 was higher than and similar to that of (99m)Tc-EC-(d-Asp)5 and (99m)Tc-MDP. (99m)Tc-EC-[(d-Asp)5]2 exhibited significantly lower accumulation in normal bone of mice than did (99m)Tc-MDP. In osteogenic tumor bearing model rats, (99m)Tc-EC-[(d-Asp)5]2 accumulated in the osteogenic and normal bone region similar to and lower than (99m)Tc-MDP, respectively. Although further studies including the chain length of d-Asp are required, these findings indicated that the present chemical design of (99m)Tc-labeled probe would be applicable to develop (99m)Tc-labeled probes for selective imaging of osteogenic bone region as well as develop therapeutic agents using therapeutic radionuclides such as (90)Y, (177)Lu, (186)Re, or (188)Re and cytotoxic agents to osteogenic bone tumor region.

Appropriate collimators in a small animal SPECT scanner with CZT detector.

OBJECTIVE: Almost all small animal SPECT is performed with pinhole collimators (PH), including single-PH (SPH) and multi-PH (MPH). In the clinical study, not only PH but also parallel-hole collimator (PAH) is often used in planar and SPECT imaging. However, there have been no comparative studies on image quality with various collimators on the small animal imaging. This study compared the basic characteristics of PH and PAH in small animal imaging. METHODS: Performance of planar and SPECT images was evaluated using (99m)TcO4(-) and SPH, MPH and PAH with low energy and high resolution on the SPECT/CT scanner FX3200. We measured sensitivity, resolution, concentration linearity and uniformity. Planar imaging of mice with (99m)Tc-labeled mercaptoacetyltriglycine ((99m)Tc-MAG3) was performed using SPH and PAH. SPECT imaging with (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) was performed using all collimators. RESULTS: With SPH, MPH and PAH, sensitivity was 43.5, 211.2 and 926.5 cps/MBq, respectively, and spatial resolution was 0.60/0.56, non/0.96, 5.20/5.34 mm full-width half maximum (planar/SPECT), respectively. There were marked correlations between the radioactivity counts on images and radioactivity with all collimators. Values of % standard deviation on planar imaging showed small differences between the SPH and PAH, while the values were the smallest on SPECT imaging with MPH. On imaging of mice, SPH yielded high-quality (99m)Tc-MAG3-planar images when compared with PAH. MPH yielded sharper (99m)Tc-MDP-SPECT images than SPH and PAH. CONCLUSIONS: The characteristics of PH and PAH differed on small animal imaging. Although sensitivity was higher with PAH, PH showed higher resolution. Among the PH collimators, SPH was more appropriate for planar imaging, and MPH was more suitable for SPECT imaging in a small animal imaging scanner with CZT detector.

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.

Appropriate parameters of the ordered-subset expectation maximization algorithm on measurement of myocardial blood flow and oxygen consumption with 11C-acetate PET.

OBJECTIVE: The purpose of this study was to evaluate the appropriate parameters of a filter and of subsets (S) and iterations (I) of the ordered-subset expectation maximization (OSEM) algorithm in 11C-acetate PET. METHODS: A Hanning filter (HF) and a Gaussian filter (GF) were selected for filtered back-projection (FBP) and the OSEM algorithm, respectively. After evaluation of the optimal HF size, the GF size was optimized using healthy volunteers (HV). Myocardial blood flow (MBF) and oxygen consumption (k(mono)) values were calculated by combining 4S, 16S, or 28S with 2I, 4I, 6I, or 8I of the OSEM (MBF(OSEM) and k(monoOSEM), respectively) in eight HV and eight coronary artery disease (CAD) patients. These MBF(OSEM) and k(monoOSEM) values were compared with those obtained using FBP (MBF(FBP) and k(monoFBP), respectively). RESULTS: Optimal HF and GF (10.0GF) sizes for the FBP and OSEM algorithms, respectively, were 10.0 mm full-width resolution at half-maximum. MBF(OSEM) was changed by modifying the parameters of the OSEM algorithm. The best correlations were between MBF(FBP) and MBF(OSEM), with 28S6I and 10.0GF for HV patients and 28S8I for CAD patients. However, the MBF(OSEM) with 28S8I was significantly different from MBF(FBP) at the global myocardium in HV. The k(monoOSEM) with 28S6I was not significantly different from k(monoFBP) in HV or CAD patients. CONCLUSION: Appropriate parameters are 28S6I with a 10.0GF on the MBF(OSEM) and k(monoOSEM) measurement using 11C-acetate. Diagnostic performance will improve using noiseless, artifact-reduction images, and accurate quantitative values that are provided by the OSEM algorithm with the appropriate parameters.