Assessing the effectiveness of fluorinated and α-methylated 3-boronophenylalanine for improved tumor-specific boron delivery in boron neutron capture therapy.

A [10B]boron agent and a nuclear imaging probe for pharmacokinetic estimation form the fundamental pair in successful boron neutron capture therapy (BNCT). However, 4-[10B]borono-l-phenylalanine (BPA), used in clinical BNCT, has undesirable water solubility and tumor selectivity. Therefore, we synthesized fluorinated and α-methylated 3-borono-l-phenylalanine (3BPA) derivatives to realize improved water solubility, tumor targetability, and biodistribution. All 3BPA derivatives exhibited over 10 times higher water solubility than BPA. Treatment with α-methylated 3BPA derivatives resulted in decreased cell uptake via l-type amino acid transporter (LAT) 2 while maintaining LAT1 recognition, thereby significantly improving LAT1/LAT2 selectivity. Biodistribution studies showed that fluorinated α-methyl 3BPA derivatives exhibited reduced boron accumulation in nontarget tissues, including muscle, skin, and plasma. Consequently, these derivatives demonstrated significantly improved tumor-to-normal tissue ratios compared to 3BPA and BPA. Overall, fluorinated α-methyl 3BPA derivatives with the corresponding radiofluorinated compounds hold potential as promising agents for future BNCT/PET theranostics.

Development of a 2-(2-Hydroxyphenyl)-1H-benzimidazole-Based Fluorescence Sensor Targeting Boronic Acids for Versatile Application in Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is an attractive approach to treating cancers. Currently, only one 10B-labeled boronoagent (Borofalan, BPA) has been approved for clinical BNCT in Japan, and methods for predicting and measuring BNCT efficacy must be established to support the development of next-generation 10B-boronoagents. Fluorescence sensors targeting boronic acids can achieve this because the amount and localization of 10B in tumor tissues directly determine BNCT efficacy; however, current sensors are nonoptimal given their slow reaction rate and weak fluorescence (quantum yield < 0.1). Herein, we designed and synthesized a novel small molecular-weight fluorescence sensor, BITQ, targeting boronic acids. In vitro qualitative and quantitative properties of BITQ were assessed using a fluorophotometer and a fluorescence microscope together with BPA quantification in blood samples. BITQ exhibited significant quantitative and selective fluorescence after reacting with BPA (post-to-pre-fluorescence ratio = 5.6; quantum yield = 0.53); the fluorescence plateaued within 1 min after BPA mixing, enabling the visualization of intracellular BPA distribution. Furthermore, BITQ quantified the BPA concentration in mouse blood with reliability comparable with that of current methods. This study identifies BITQ as a versatile fluorescence sensor for analyzing boronic acid agents. BITQ will contribute to 10B-boronoagent development and promote research in BNCT.

A Red-Emitting Fluorescence Sensor for Detecting Boronic Acid-Containing Agents in Cells.

The amount and localization of boron-10 atoms delivered into tumor cells determines the therapeutic effect of boron neutron capture therapy (BNCT) and, consequently, efforts have been directed to develop fluorescence sensors to detect intracellular boronic acid compounds. Currently, these sensors are blue-emitting and hence are impracticable for co-staining with nucleus staining reagents, such as DAPI and Hoechst 33342. Here, we designed and synthesized a novel fluorescence boron sensor, BS-631, that emits fluorescence with a maximum emission wavelength of 631 nm after reaction with the clinically available boronic acid agent, 4-borono-l-phenylalanine (BPA). BS-631 quantitatively detected BPA with sufficiently high sensitivity (detection limit = 19.6 µM) for evaluating BNCT agents. Furthermore, BS-631 did not emit fluorescence after incubation with metal cations. Notably, red-emitting BS-631 could easily and clearly visualize the localization of BPA within cells with nuclei co-stained using Hoechst 33342. This study highlights the promising properties of BS-631 as a versatile boron sensor for evaluating and analyzing boronic acid agents in cancer therapy.

Development of a switching-type fluorescence sensor for the detection of boronic acid-containing agents.

Since the therapeutic effect of boron neutron capture therapy is influenced by the intracellular distribution profile of boronoagents containing 10B atoms, it is necessary to establish a method that can determine the intracellular distribution profile of boronoagents. We aimed to develop a small molecule-based fluorescence sensor that changes its fluorescence properties upon complexation with the boronic acid moiety of a boronoagent. Thus, we designed a 2-(2-pyridyl)phenol derivative PPN-1 by introducing a N,O ligand substructure into a zinc sensor probe with excellent fluorescence properties. To investigate the effectiveness of PPN-1, we synthesized PPN-1 and evaluated its fluorescence properties compared to DAHMI, a current available boronic acid sensor. Consequently, PPN-1 showed favorable off/on fluorescence switching ability with a large Stokes shift after the addition of p-boronophenylalanine (BPA). Notably, after adding BPA, PPN-1 exhibited a rapid increase and reached a fluorescence plateau within 5 min, which is much shorter than the 2 h needed for DAHMI. Further, PPN-1 has excellent selectivity and detection and quantification limits similar to those of ICP-OES. These results demonstrated that PPN-1 is a practical scaffold for the detection and quantification of boronic acids and will provide essential insights for the development of boronic acid-targeted fluorescent sensors in the future.

Radiobrominated probe targeting activated p38α in inflammatory diseases.

OBJECTIVE: p38α, a member of the mitogen-activated protein kinase superfamily, is activated by external stimuli, followed by nuclear translocation for the regulation of inflammatory responses at the transcriptional and translational levels in inflammatory diseases. Thus, activated p38α would be an appropriate target molecule for in vivo noninvasive imaging and targeted radionuclide therapy. For this purpose, we designed a radiobrominated compound, 6-(4-[77Br]bromo-2-fluorophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-ylamino)-pyrido[2,3-d]pyrimidin-7(8H)-one ([77Br]4-BR), based on a potent p38α selective inhibitor, R1487, for use with single-photon emission computed tomography. We synthesized [77Br]4-BR and evaluated its effectiveness as an activated p38α imaging probe compared with our previous radioiodinated probe (6-(2-fluoro-4-[125I]iodophenoxy)-8-methyl-2-(tetrahydro-2H-pyran-4-ylamino)-pyrido[2,3-d]pyrimidin-7(8H)-one ([125I]4-IR)) in a mouse inflammatory model. METHODS: We designed [77Br]4-BR by replacing the radioiodine of [125I]4-IR or the fluorine of R1487 with radiobromine at the 4-position of the phenoxy ring. We synthesized 4-BR via a four-step process. The inhibitory potency of 4-BR was measured using an ADP-Glo™ kinase assay system. Radiosynthesis of [77Br]4-BR was performed via an organotin-radiobromine exchange reaction using the corresponding tributyltin precursor. Radioactivity biodistribution was evaluated in normal ddY mice and turpentine oil-induced inflammation model mice for 120 min after intravenous administration of [77Br]4-BR. The temporal changes in radioactivity in blood fractions were compared between [77Br]4-BR and [125I]4-IR. RESULTS: 4-BR was synthesized at a total yield of 9.1% and showed a p38α inhibitory potency similar to that of 4-IR. [77Br]4-BR was successfully obtained from a tributyltin precursor with high radiochemical yield (89.9%), purity (95.9%), and molar activity (2.0 TBq/µmol). [77Br]4-BR showed accumulation of high radioactivity in the inflamed tissue (3.4% ± 0.9% ID/g, peaking at 15 min), rapid delivery throughout the body, and rapid blood clearance with approximately half of the blood radioactivity existing as an intact form at 60 min. Although the maximum radioactivity accumulation in inflamed tissue after [77Br]4-BR administration was approximately half that of [125I]4-IR because of its faster blood clearance and lower free fraction in the input function, the inflamed tissue-to-blood ratio was comparable between [77Br]4-BR and [125I]4-IR. CONCLUSIONS: [77Br]4-BR would be a promising imaging agent for detecting activated p38α in inflammatory diseases.

Evaluation of 3-Borono-l-Phenylalanine as a Water-Soluble Boron Neutron Capture Therapy Agent.

Although 4-borono-l-phenylalanine (4-BPA) is currently the only marketed agent available for boron neutron capture therapy (BNCT), its low water solubility raises concerns. In this study, we synthesized 3-borono-l-phenylalanine (3-BPA), a positional isomer of 4-BPA, with improved water solubility. We further evaluated its physicochemical properties, tumor accumulation, and biodistribution. The water solubility of 3-BPA was 125 g/L, which is more than 100 times higher than that of 4-BPA. Due to the high water solubility, we prepared the administration solution of 3-BPA without a solubilizer sugar, which is inevitably added to 4-BPA preparation and has adverse effects. In in vitro and in vivo experiments, boron accumulation in cancers after administration was statistically equivalent in both sugar-complexed 3-BPA and 4-BPA. Furthermore, the biodistribution of 3-BPA was comparable with that of sugar-complexed 3-BPA. Since 3-BPA has high water solubility and tumor targetability equivalent to 4-BPA, 3-BPA can replace 4-BPA in future BNCT.

Development of radioiodinated pyrimidinopyridone derivatives as targeted imaging probes of activated p38α for single photon emission computed tomography.

OBJECTIVE: p38α, a member of the mitogen-activated protein kinase superfamily, is ubiquitously expressed in a variety of mammalian cells. Activated p38α induces inflammatory responses to external stimuli, suggesting that non-invasive detection of activated p38α would be valuable for diagnosing inflammatory diseases. For this purpose, we designed radiolabeled compounds [123I]2-IR and [123I]4-IR based on a potent p38α selective inhibitor R1487 for use with single photon emission computed tomography (SPECT). In this study, we used 125I instead of 123I due to its more usable radiochemical properties, synthesized [125I]2-IR and [125I]4-IR, and evaluated their effectiveness as activated p38α imaging probes. METHODS: [123I]2-IR and [123I]4-IR were designed by introduction of a 123I atom at the 2- or 4-ositions of the phenoxy ring, preserving the pyrimidinopyridone structure of R1487. We synthesized 2-IR and 4-IR via a 7-step process. The inhibitory potencies of 2-IR, 4-IR, and p38α inhibitors were measured using an ADP-Glo™ kinase assay system. Radioiodination of 2-IR and 4-IR was performed via an organotin-radioiodine exchange reaction using the corresponding tributyltin precursors. Biodistributions were evaluated by determining radioactivity in tissues of interest after intravenous administration of [125I]2-IR and [125I]4-IR in normal ddY mice and turpentine oil-induced inflammation model mice. In vivo inhibition study was also performed in inflammation model mice after intravenous administration of [125I]4-IR with pretreatment of p38α inhibitors. RESULTS: We synthesized 2-IR and 4-IR at total yields of 17.5% and 19.2%, respectively. 4-IR had higher p38α inhibitory potency than 2-IR; both compounds were significantly less potent than R1487. [125I]2-IR and [125I]4-IR were successfully obtained from tributyltin precursors with high radiochemical yield (> 65%), purity (> 97%), and molar activity (~ 81 GBq/µmol). [125I]4-IR showed high radioactivity accumulation in the inflamed tissue (7.0 ± 1.2%D/g), rapid delivery throughout the body, and rapid blood clearance, resulting in a high inflammation-to-blood ratio (6.2 ± 0.4) and a high inflammation-to-muscle ratio (5.2 ± 1.3) at 30 min, while [125I]2-IR showed low radioactivity accumulation in inflamed tissue over the experimental period. Further, radioactivity accumulation in inflamed tissue after [125I]4-IR administration was significantly decreased by pretreatment with selective inhibitors. CONCLUSIONS: [123I]4-IR would be a promising imaging agent for detection of activated p38α.

Diagnostic performance of 68Ga-DOTATOC PET/CT in tumor-induced osteomalacia.

OBJECTIVE: Tumor-induced osteomalacia (TIO) is caused by typically small tumors that secrete fibroblast growth factor 23 (FGF23). As tumor resection is the only effective treatment for TIO, it is important to detect the culprit tumor. We aimed to assess the utility of 68Gallium-DOTA-D-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) PET/CT in TIO and the correlation between biochemical parameters and the PET/CT results. METHODS: Thirty-five patients with clinically suspected TIO who had undergone 68Ga-DOTATOC PET/CT were retrospectively analyzed. 68Ga-DOTATOC PET/CT results were compared with biochemical parameters and the final diagnosis, including histopathology. RESULTS: 68Ga-DOTATOC PET/CT detected focal uptake consistent with TIO in 21/35 patients, one of which was considered false positive. In 16 patients, the cause of osteomalacia was confirmed histologically as phosphaturic mesenchymal tumor (n = 15) or fibrous dysplasia (n = 1). The other four patients were judged clinically as true positive by subsequent MRI and the clinical course. Overall, the detection rate of 68Ga-DOTATOC PET/CT was 57% (20/35). Median tumor maximum standardized uptake value (SUVmax) was 6.9 (range 1.5-37.7). There was no significant difference in serum intact FGF23 level between DOTATOC-positive and DOTATOC-negative cases, and no significant correlation was observed between intact FGF23 level and tumor SUVmax. CONCLUSIONS: 68Ga-DOTATOC PET/CT was clinically useful in detecting culprit tumors and subsequent patient management in TIO.

Indirectly radioiodinated exendin-4 as an analytical tool for in vivo detection of glucagon-like peptide-1 receptor in a disease setting.

OBJECTIVE: Glucagon-like peptide-1 receptor agonist (GLP-1RA) has been reported to have therapeutic effects on diabetes and various diseases. Precise detection of GLP-1 receptor (GLP-1R) can be useful to diagnose and elucidate the mechanism of such diseases. Here we aimed to develop an imaging probe based on GLP-1RA that has high molar activity and sensitivity for detection of low-level GLP-1R expression in non-pancreatic diseases. METHODS: We selected the agonist exenatide (Ex4) as the parent peptide of a GLP-1R targeting probe and prepared Cys-Ex4 by addition of an N-terminal Cys residue and labeling with the prosthetic agent N-(3-[125I]iodophenyl)maleimide ([125I]IPM) to generate [125I]Ex4ipm. We evaluated the affinity of [125I]Ex4ipm for GLP-1R, as well as cellular binding profiles in insulinoma and prostate cancer cell lines, and in vivo biodistributions in normal and tumor-bearing mice to assess GLP-1R-dependent accumulation of radioactivity in tissues. RESULTS: [125I]Ex4ipm was easily synthesized with high radiochemical yield (73%), radiochemical purity (> 99%), and molar activity (81 GBq/µmol) via a thiol/maleimide reaction. Following administration to mice, [125I]Ex4ipm accumulated to high levels in the pancreas (23.3% ID/g), with radioactivity co-localizing in areas having insulin-positive ß cells. High amounts of radioactivity also accumulated in insulinomas that overexpressed GLP-1R (27.5% ID/g). In contrast, low amounts of [125I]Ex4ipm accumulation, corresponding to low expression levels of GLP-1R, were observed in prostate cancer cells and xenografts used as a model of non-pancreatic applications. CONCLUSION: Our results suggested that [123I]Ex4ipm could be valuable for GLP-1R imaging in diabetes, insulinomas, and various diseases related to GLP-1R.

Intravenously delivered multilineage-differentiating stress enduring cells dampen excessive glutamate metabolism and microglial activation in experimental perinatal hypoxic ischemic encephalopathy.

Perinatal hypoxic ischemic encephalopathy (HIE) results in serious neurological dysfunction and mortality. Clinical trials of multilineage-differentiating stress enduring cells (Muse cells) have commenced in stroke using intravenous delivery of donor-derived Muse cells. Here, we investigated the therapeutic effects of human Muse cells in an HIE model. Seven-day-old rats underwent ligation of the left carotid artery then were exposed to 8% oxygen for 60 min, and 72 hours later intravenously transplanted with 1 × 104 of human-Muse and -non-Muse cells, collected from bone marrow-mesenchymal stem cells as stage-specific embryonic antigen-3 (SSEA-3)+ and -, respectively, or saline (vehicle) without immunosuppression. Human-specific probe revealed Muse cells distributed mainly to the injured brain at 2 and 4 weeks, and expressed neuronal and glial markers until 6 months. In contrast, non-Muse cells lodged in the lung at 2 weeks, but undetectable by 4 weeks. Magnetic resonance spectroscopy and positron emission tomography demonstrated that Muse cells dampened excitotoxic brain glutamatergic metabolites and suppressed microglial activation. Muse cell-treated group exhibited significant improvements in motor and cognitive functions at 4 weeks and 5 months. Intravenously transplanted Muse cells afforded functional benefits in experimental HIE possibly via regulation of glutamate metabolism and reduction of microglial activation.

Synthesis and evaluation of novel radioiodinated anthranilate derivatives for in vivo imaging of vascular endothelial growth factor receptor with single-photon emission computed tomography.

OBJECTIVE: Angiogenesis facilitates tumor survival and promotes malignancy. The vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) tyrosine kinase (TK) signaling pathway is a key factor mediating angiogenesis, suggesting that this pathway may be a target for diagnosis and therapy. In this study, we aimed to develop small molecule radioiodinated probes applicable for in vivo VEGFR imaging considering the versatility and usefulness of single-photon emission computed tomography (SPECT). METHODS: We designed and synthesized four radioiodinated anthranilate compounds (6a-d) based on the structure of an anticancer drug targeting VEGFR-TK. The inhibitory potencies of corresponding cold compounds 4a-d and in vitro stability of compounds 6a-d were assessed by cellular proliferation inhibition assays and radio thin-layer chromatography after incubation in neutral solution. In vivo biodistributions were evaluated by determining radioactivity in tissues of interest after intravenous injection of test compounds in tumor-bearing mice. In vitro and in vivo blocking experiments using a selective VEGFR-TK inhibitor and SPECT/computed tomography (CT) imaging were performed in tumor-bearing mice. RESULTS: The radioiodinated compounds 6a-d were obtained with more than 68.0% radiochemical yield and more than 95% radiochemical purity. Because compounds 4a-d showed high inhibitory potencies and compounds 6c and 6d showed high in vitro stability, 6c ([125I]m-NPAM) and 6d ([125I]p-NPAM) were further evaluated. Analysis of the in vivo biodistribution revealed a tumor to blood radioactivity ratio of greater than 4 at 24 h after [125I]p-NPAM administration. Accumulation of radioactivity in cultured tumor cells and tumor xenografts after [125I]p-NPAM administration was significantly blocked by inhibitor pretreatment. Tumors were clearly imaged at 24 h after [125I]p-NPAM injection with SPECT/CT in comparison to that in inhibitor-pretreated tumor-bearing mice. CONCLUSION: [125I]p-NPAM may have potential applications as a lead compound for future development of a clinically usable VEGFR imaging probe for SPECT.

Radioiodinated bicyclic RGD peptide for imaging integrin αvß3 in cancers.

Integrin αvß3 is an effective marker of angiogenesis in cancer, and αvß3-specific imaging can yield important details about this complex physiological process. We utilized the recently reported and highly αvß3-specific peptide, bicyclic RGD (bcRGD), as the basic structure of an in vivo αvß3 imaging probe, and synthesized a radioiodinated form of bcRGD, namely [125I]bcRGD, with high radiochemical purity (>99%) and high molar activity (81 GBq/µmol). As expected, [125I]bcRGD exhibited high selectivity for αvß3 compared with αvß5 and α5ß1in vitro. [125I]bcRGD showed significantly higher accumulation in U-87MG cells (1.6% dose/mg) with high expression of αvß3 compared to A549 cells (0.3% dose/mg) with only moderate expression. Furthermore, 30 min after administration to tumor-bearing mice, [125I]bcRGD showed significantly higher accumulation in U-87MG tumors (3.8% ID/g) than in A549 tumors (2.1% ID/g), and the radioactivity accumulation ratios of U-87MG tumor/blood and U-87MG tumor/muscle were 4.0 and 6.0, respectively. These results highlight the promising properties of [123/125I]bcRGD for use as an in vivo αvß3 imaging probe, as well as the utility of bcRGD as a basic structure of molecular probes for both imaging and therapeutic applications. bcRGD may exhibit broad use in future theranostics applications targeting integrin αvß3-related diseases.

Synthesis and characterization of novel radiofluorinated probes for positron emission tomography imaging of monoamine oxidase B.

Monoamine oxidase B (MAO-B), predominantly expressed in glial cells, plays an important role in neurotransmitter regulation, and MAO-B activity relates to several neuronal diseases. Here, we aimed to develop a radiofluorinated MAO-B imaging probe based on the structure of a selective MAO-B inhibitor, MD-230254. We synthesized and evaluated a series of compounds in vitro and in vivo. A series of fluorinated analogs of MD-230254 were synthesized and evaluated for inhibitory potency and selectivity toward MAO-B. 5-[4-(2-[18 F]Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (2-[18 F]FBPO) was synthesized from a corresponding tributylstannyl precursor and [18 F]CH3 COOF. Biodistribution after intravenous injection of 2-[18 F]FBPO was evaluated in male ddY mice with or without pretreatment by inhibitors. Among the compounds synthesized and evaluated, 2-FBPO showed high inhibitory potency and selectivity toward MAO-B comparable with MD-230254. 2-[18 F]FBPO was successfully synthesized by an electrophilic reaction with a high radiochemical purity of more than 99%. 2-[18 F]FBPO was efficiently taken up by the brain and showed rapid blood clearance, which provided a brain/blood radioactivity ratio of 3.7 at 90 minutes postinjection. The brain radioactivity was significantly decreased by pretreatment with an MAO-B selective inhibitor. The great potential of 2-[18 F]FBPO as an MAO-B imaging probe, applicable to a variety of diseases, is indicated.

Development of a p38α-selective radioactive probe for qualitative diagnosis of cancer using SPECT.

OBJECTIVE: p38 mitogen-activated protein (MAP) kinase (p38α) has drawn attention as a new target molecule for the treatment and diagnosis of cancer, and its overexpression and activation have been reported in various types of cancer. In this study, a single photon emission computed tomography (SPECT) imaging probe of p38α was developed to noninvasively image p38α activity for effective qualitative diagnosis of cancer. METHODS: Pyrrolepyridine derivatives, m-YTM and p-YTM, were designed and synthesized based on the structure of the p38α-selective inhibitor. Radioactive iodine-labeled m-YTM, [125I]m-YTM, was synthesized because m-YTM greatly inhibited the phosphorylation of p38α upon examining the inhibitory effects of the compounds. After investigating the binding affinity of [125I]m-YTM to the recombinant p38α, a saturation binding experiment using activated p38α and inactive p38α was performed to determine the binding site. Uptake of [125I]m-YTM into various cancer cell lines was investigated, and the pharmacokinetics was evaluated using tumor-bearing mice. RESULTS: The inhibitory activity of m-YTM was approximately 13 times higher than that of SB203580, a p38α-selective inhibitor. The binding site of [125I]m-YTM was estimated to be the p38α activating site, similar to that of SB203580, because the [125I]m-YTM bound strongly to both activated p38α and inactive p38α. Various different cancer cells incorporated [125I]m-YTM; however, its accumulation was significantly reduced by treatment with SB203580. Pharmacokinetics study of [125I]m-YTM in B-16 tumor-bearing mice was examined which revealed high accumulation of radioactivity in tumor tissues. The ratios of radioactivity in the B-16 tumor to that in blood were 3.1 and 50 after 1 and 24 h, respectively. The ratio of radioactivity in the tumor to that in blood in the tumor-bearing mice generated using other cancer cell lines was also ≥ 1 at 1 h after the administration of the probe. CONCLUSIONS: This study suggests that [123I]m-YTM has potential as a p38α imaging probe effective for various cancer types.

One-pot enzymatic synthesis of l-[3-11C]lactate for pharmacokinetic analysis of lactate metabolism in rat brain.

INTRODUCTION: Lactate could serve as an energy source and signaling molecule in the brain, although there is insufficient in vivo evidence to support this possibility. Here we aimed to use a one-pot enzymatic synthetic procedure to synthesize l-[3-11C]lactate that can be used to evaluate chemical forms in the blood after intravenous administration, and as a probe for pharmacokinetic analysis of lactate metabolism in in vivo positron emission tomography (PET) scans with normal and fasted rats. METHODS: Racemic [3-11C]alanine obtained from 11C-methylation of a precursor and deprotection was reacted with an enzyme mixture consisting of alanine racemase, d-amino acid oxidase, catalase, and lactate dehydrogenase to yield l-[3-11C]lactate via [3-11C]pyruvate. The optical purity was measured by HPLC. Radioactive chemical forms in the arterial blood of Sprague Dawley rats with or without insulin pretreatment were evaluated by HPLC 10 min after bolus intravenous injection of l-[3-11C]lactate. PET scans were performed on normal and fasted rats administered with l-[3-11C]lactate. RESULTS: l-[3-11C]Lactate was synthesized within 50 min and had decay corrected radiochemical yield, radiochemical purity, and optical purity of 13.4%, >95%, and >99%, respectively. The blood radioactivity peaked immediately after l-[3-11C]lactate injection, rapidly decreased to the minimum value within 90 s, and slowly cleared thereafter. HPLC analysis of blood samples revealed the presence of [11C]glucose (78.9%) and l-[3-11C]lactate (12.1%) 10 min after administration of l-[3-11C]lactate. Insulin pretreatment partly inhibited glyconeogenesis conversion leading to 55.4% as [11C]glucose and 38.9% as l-[3-11C]lactate simultaneously. PET analysis showed a higher SUV in the brain tissue of fasted rats relative to non-fasted rats. CONCLUSIONS: We successfully synthesized l-[3-11C]lactate in a one-pot enzymatic synthetic procedure and showed rapid metabolic conversion of l-[3-11C]lactate to [11C]glucose in the blood. PET analysis of l-[3-11C]lactate indicated the possible presence of active lactate usage in rat brains in vivo.

123I-Labeled oxLDL Is Widely Distributed Throughout the Whole Body in Mice.

PURPOSE: Oxidized low-density lipoprotein (oxLDL) plays a key role in endothelial dysfunction, vascular inflammation, and atherogenesis. The aim of this study was to assess blood clearance and in vivo kinetics of radiolabeled oxLDL in mice. METHODS: We synthesized 123I-oxLDL by the iodine monochloride method, and performed an uptake study in CHO cells transfected with lectin-like oxLDL receptor-1 (LOX-1). In addition, we evaluated the consistency between the 123I-oxLDL autoradiogram and the fluorescence image of DiI-oxLDL after intravenous injection for both spleen and liver. Whole-body dynamic planar images were acquired 10 min post injection of 123I-oxLDL to generate regional time-activity curves (TACs) of the liver, heart, lungs, kidney, head, and abdomen. Regional radioactivity for those excised tissues as well as the bladder, stomach, gut, and thyroid were assessed using a gamma counter, yielding percent injected dose (%ID) and dose uptake ratio (DUR). The presence of 123I-oxLDL in serum was assessed by radio-HPLC. RESULTS: The cellular uptakes of 123I-oxLDL were identical to those of DiI-oxLDL, and autoradiograms and fluorescence images also exhibited consistent distributions. TACs after injection of 123I-oxLDL demonstrated extremely fast kinetics. The radioactivity uptake at 10 min post-injection was highest in the liver (40.8 ± 2.4% ID). Notably, radioactivity uptake was equivalent throughout the rest of the body (39.4 ± 2.7% ID). HPLC analysis revealed no remaining 123I-oxLDL or its metabolites in the blood. CONCLUSION: 123I-OxLDL was widely distributed not only in the liver, but also throughout the whole body, providing insight into the pathophysiological effects of oxLDL.

Comparison of 125I- and 111In-labeled peptide probes for in vivo detection of oxidized low-density lipoprotein in atherosclerotic plaques.

OBJECTIVE: Oxidized low-density lipoprotein (OxLDL) plays a pivotal role in atherosclerotic plaque destabilization, which suggests its potential as a nuclear medical imaging target. We previously developed radioiodinated 125I-AHP7, a peptide probe carrying a 7-residue sequence from the OxLDL-binding protein Asp-hemolysin, for specific OxLDL imaging. Although 125I-AHP7 recognized OxLDL, it had low stability. Thus, to improve stability, we designed radiolabeled 22-residue peptide probes, 125I-AHP22 and 111In-AHP22, which include the entire AHP7 sequence, and evaluated the stability, activity, and applications of these probes in vitro and in vivo. METHODS: Probes consisting of a 21-residue peptide derived from the Asp-hemolysin sequence and an N-terminal Cys or aminohexanoic acid for labeling with 125I-N-(3-iodophenyl)maleimide or 111In diethylene triamine pentaacetic acid were termed 125I-AHP22 and 111In-AHP22. An in vitro-binding inhibition assay with OxLDL was performed using 125I-AHP7 as a radiotracer. Radioactivity accumulation in the atherosclerotic aorta and plasma intact fraction was evaluated 30 min after intravenous administration of probes in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. RESULTS: 125I-AHP22 and 111In-AHP22 were synthesized in ~ 360 and 60 min, respectively, with > 98% radiochemical purities after RP-HPLC purification. An in vitro-binding assay revealed similar or greater inhibition of OxLDL binding by both In-AHP22 and I-AHP22 compared to I-AHP7. The fraction of intact 125I-AHP22 and 111In-AHP22 in plasma was estimated to be approximately tenfold higher than that of 125I-AHP7. Both probes were rapidly cleared from the blood. 111In-AHP22 had a 2.3-fold higher accumulation in WHHLMI rabbit aortas compared to control rabbits, which was similar to 125I-AHP7. However, 125I-AHP22 accumulated to similar levels in aortas of WHHLMI and control rabbits due to high nonspecific accumulation in normal aortas that could be due to high lipophilicity. CONCLUSIONS: 111In-AHP22, easily prepared within 1 h, showed moderate affinity for OxLDL, high stability in vivo, and high accumulation in atherosclerotic aortas. 111In-AHP22 could be a potential lead compound to develop future effective OxLDL imaging probes.

Development of matrix metalloproteinase-targeted probes for lung inflammation detection with positron emission tomography.

As matrix metalloproteinases (MMPs), especially MMP-9 and MMP-12 are involved in the pathological processes associated with chronic obstructive pulmonary disease (COPD), we developed a novel radiofluorinated probe, 18F-IPFP, for MMPs-targeted positron emission tomography (PET). 18F-IPFP was designed by iodination of MMP inhibitor to enhance the affinity, and labelled with a compact prosthetic agent, 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP). As a result, IPFP demonstrated the highest affinity toward MMP-12 (IC50 = 1.5 nM) among existing PET probes. A COPD model was employed by exposing mice to cigarette smoke and the expression levels of MMP-9 and MMP-12 were significantly increased in the lungs. Radioactivity accumulation in the lungs 90 min after administration of 18F-IPFP was 4× higher in COPD mice than normal mice, and 10× higher than in the heart, muscle, and blood. Ex vivo PET confirmed the radioactivity distribution in the tissues and autoradiography analysis demonstrated that accumulation differences in the lungs of COPD mice were 2× higher than those of normal mice. These results suggest that 18F-IPFP is a promising probe for pulmonary imaging and expected to be applied to various MMP-related diseases for early diagnosis, tracking of therapeutic effects, and new drug development in both preclinical and clinical applications.

Synthesis of radioiodinated probes targeted toward matrix metalloproteinase-12.

Matrix metalloproteinase-12 (MMP-12, macrophage elastase) is a member of the MMP family that is responsible for the degradation of extracellular matrix, and is associated with the inflammatory process of chronic obstructive pulmonary disease (COPD). COPD, characterized by progressive and irreversible airflow obstruction, is recently a major cause of mortality and morbidity worldwide. Herein, to develop radioiodinated probes for the early diagnosis of COPD, we designed and synthesized novel MMP-12-targeted dibenzofuran compounds (1-3) with a variety of linker structures (carbamate, amide, and sulfonamide). In competitive enzyme activity assays, it was revealed that the linker structures significantly affected the inhibitory activity against and selectivity for MMP-12. Compound 1, with carbamate linker, demonstrated potent MMP-12 inhibitory activity (IC50 = 8.5 nM) compared to compound 2, with amide linker, and compound 3, with sulfonamide linker. Using bromo-substituted carbamate 13 as a radioiodination precursor, [125I]1 was successfully prepared to high radiochemical purity (over 98%) and good specific radioactivity (4.1 GBq/µmol). These results suggest that radioiodinated compound 1 is potent as a novel MMP-12-targeted probe.

Sequential PET estimation of cerebral oxygen metabolism with spontaneous respiration of 15O-gas in mice with bilateral common carotid artery stenosis.

Positron emission tomography with 15O-labeled gases (15O-PET) is important for in vivo measurement of cerebral oxygen metabolism both in clinical and basic settings. However, there are currently no reports concerning 15O-PET in mice. Here, we developed an 15O-PET method applicable to mice with spontaneous respiration of 15O-gas without a tracheotomy catheter. Sequential 15O-PET was also performed in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) induced by placement of microcoils. 15O-gas with isoflurane was supplied to the nose of mouse with evacuation of excess 15O-gas surrounding the body. 15O-PET was performed on days 3, 7, 14, 21, and 28 after surgery. Cerebral blood flow (CBF), cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2) were calculated in whole brains. A significant decrease in CBF and compensatory increase in OEF in the BCAS group produced CMRO2 values comparable to that of the sham group at three days post-operation. Although CBF and OEF in the BCAS group gradually recovered over the first 28 days, the CMRO2 showed a gradual decrease to 68% of sham values at 28 days post-operation. In conclusion, we successfully developed a noninvasive 15O-PET method for mice.