Radiation dosimetry and efficacy of an 89Zr/225Ac-labeled humanized anti-MUC5AC antibody.

INTRODUCTION: Theranostic applications are currently difficult to achieve owing to the limited evaluation of suitable chelators for therapeutic nuclides, such as 225Ac and 227Th. With a focus on targeted α therapy and theranostics using human IgG as a drug-delivery system (i.e., combining highly cytotoxic α-particle emitter radiation with efficient tumor targeting), we developed a recombinant humanized Nd2 (hNd2) as an anti-MUC5AC antibody since MUC5AC is highly expressed in patients with pancreatic cancer. Therefore, we aimed to evaluate the performance of 89Zr- (for diagnosis) and 225Ac- (for therapy) labeling of these antibodies using well-controlled radioisotope (RI)-labeling technology in pancreatic cancer mouse models. METHODS: 89Zr-labeled hNd2 (NMK89) and 225Ac-labeled hNd2 (NMT25) were manufactured by chemical conjugation using affinity peptides. A binding assay and the evaluation of plasma stability were performed in vitro to confirm the properties of NMK89 and NMT25. In vivo, we evaluated biodistribution, positron emission tomography (PET)/computed tomography (CT) imaging, antitumor effects, and toxicity. Moreover, the exposure dose in humans was estimated based on the biodistribution evaluation in normal mice. RESULTS: NMK89 and NMT25 showed binding specificity to MUC5AC and stability with radiochemical purity ≥90% in mice and human plasma following incubation for 168 h. NMK89 showed high accumulation in tumors and low non-specific accumulation in normal tissues. The antitumor effect of NMT25 was dose-dependent and significantly suppressed tumor growth in the NMT25 treatment groups compared with the control group (p < 0.05). NMK89 and NMT25 showed similar pharmacokinetics and biodistribution characteristics. Additionally, the human estimated exposure dose of NMK89 and NMT25 was confirmed, and the effective dose of NMK89 and NMT25 was 0.33 mSv/MBq and 177.5 mSv/MBq, respectively. CONCLUSION: NMK89 showed specific accumulation in the MUC5AC-expressing tumors, while NMT25 showed strong antitumor effects. These results suggest NMK89 and NMT25 as promising theranostic agents for pancreatic cancer.

Comparative evaluation of [18F]DiFA and its analogs as novel hypoxia positron emission tomography and [18F]FMISO as the standard.

INTRODUCTION: Hypoxia, a common feature of most solid tumors, is an important predictor of tumor progression and resistance to radiotherapy. We developed a novel hypoxia imaging probe with optimal biological characteristics for use in clinical settings. METHODS: We designed and synthesized several new hypoxia probes with additional hydrophilic characteristics compared to [18F]fluoromisonidazole ([18F]FMISO). These were 1-(2,2-Dihydroxy-methyl-3-[18F]-Fluoropropyl) azomycin ([18F]DiFA, formerly [18F]HIC101) and its analogs ([18F]F1 and [18F]F2). Biodistribution studies with EMT6 mammary carcinoma cell-bearing mice were performed 1 and 2 h after injection of each probe. Small-animal positron emission tomography (PET) imaging studies were conducted using [18F]DiFA and [18F]FMISO in the same mice. Tumoral hypoxia was confirmed via pimonidazole staining. Ex vivo digital autoradiographs were obtained for confirming the co-localization of [18F]DiFA and pimonidazole in the tumor tissues. RESULTS: The EMT6 tumors used had pimonidazole-positive regions. In biodistribution studies, the tumor-to-blood ratio and tumor-to-muscle ratio of [18F]DiFA was significantly higher than the respective [18F]FMISO ratios 1 h after injection. Hence, we selected [18F]DiFA as the best hypoxia probe among those tested. Small-animal PET imaging studies showed time-dependent increases in the tumor-to-normal tissue ratio of [18F]DiFA uptake. Rapid clearance from the rest of the body was observed primarily via the renal system. Ex vivo autoradiography showed a positive correlation between [18F]DiFA uptake and the regions of pimonidazole distribution, indicating that [18F]DiFA selectively accumulated in the tumor tissue's hypoxic region. CONCLUSIONS: A better contrast image and a shorter waiting time may be obtained with [18F]DiFA than with [18F]FMISO. ADVANCES IN KNOWLEDGE: By optimizing LogP based on the [18F]FMISO structure, we demonstrated that [18F]DiFA could detect tumor hypoxia regions at an early time point. IMPLICATIONS FOR PATIENT CARE: [18F]DiFA imaging facilitates the evaluation of various cancer hypoxic states due to the lower uptake of normal tissues and could contribute to novel treatment development.

Evaluation of trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid accumulation in low-grade glioma in chemically induced rat models: PET and autoradiography compared with morphological images and histopathological findings.

INTRODUCTION: Magnetic resonance imaging (MRI) can have a problem to delineate diffuse gliomas with an intact blood-brain barrier (BBB) especially when a marked peritumoral edema is present. We evaluated the potential of trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) positron emission tomography (PET) to delineate the extent of diffuse gliomas by comparing PET findings with autoradiography, in vivo and ex vivo MRI, and histopathology findings. METHODS: Dynamic PET was performed in rats with N-ethyl-N-nitrosourea-induced glioma for 60 min after anti-(18)F-FACBC injection. Contrast-enhanced MRI was performed before or after PET. The PET images were fused with in vivo and ex vivo MR images, and histopathological images for direct comparisons. Autoradiograms were compared with the results of Evans Blue (EB) extravasation (to assess BBB integrity) and hematoxylin-eosin staining. RESULTS: Histopathological examination, including EB extravasation assessment, and enhanced T1-weighted MRI identified several diffuse gliomas with slight BBB disruption, similar to low-grade human gliomas. Anti-(18)F-FACBC uptake was specific and high in the gliomas, irrespective of BBB integrity. Higher anti-(18)F-FACBC uptake corresponded to areas of T2 hyperintensity, independent of gadolinium enhancement. Ex vivo autoradiography also showed high anti-(18)F-FACBC accumulation in tumors lacking EB extravasation and a correlation between anti-(18)F-FACBC accumulation and tumor cell density, but not EB extravasation. CONCLUSIONS: Anti-(18)F-FACBC-PET allowed visualization of gliomas irrespective of BBB integrity. The tumor-to-normal uptake ratio of anti-(18)F-FACBC generally correlated with the relative cell density. Anti-(18)F-FACBC PET combined with MRI shows promise for preoperative glioma delineation. ADVANCES IN KNOWLEDGE: Radiopharmaceuticals that cross the BBB, such as anti-(18)F-FACBC, are taken up by low-grade gliomas with equivocal MRI findings due to an intact BBB. IMPLICATIONS FOR PATIENT CARE: Surgery is the first-line therapy for low-grade gliomas; therefore, delineation of their extent in the presence of an intact BBB is essential to planning surgery that removes the entire neoplasm, which will positively affect long-term survival.

Comparison of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid (anti-[18F]FACBC) accumulation in lymph node prostate cancer metastasis and lymphadenitis in rats.

INTRODUCTION: Trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) is a positron emission tomography (PET) tracer used to visualize prostate cancer (PCa). In this study, we investigated the differences in anti-[(18)F]FACBC accumulation between metastatic and inflamed lymph node (LN) lesions. METHODS: A PCa LN metastasis (PLM) model was developed by inoculating a rat PCa cell line, MAT-Ly-Lu-B2, into popliteal LNs of Copenhagen rats. Acute lymphadenitis (AL) was induced by injecting concanavalin A (Con A) into the hind footpad, and chronic lymphadenitis (CL) was induced by daily injection of Con A into the tissues surrounding the popliteal LNs for 2weeks. Main lesions of all animal models were established in lumbar and/or inguinal LNs. Biodistribution and dynamic PET imaging data were acquired after tracer injection. T2-weighted magnetic resonance (MR) images were registered with PET images. RESULTS: In the biodistribution study, the uptake ratios of PLM-to-lymphadenitis in lesional lumbar and inguinal LNs were 0.97-1.57 and 1.47-2.08 at 15 and 60min post-anti-[(18)F]FACBC injection respectively. In PET imaging, the lesional lumbar LNs of CL and PLM, but not of AL, were visualized on anti-[(18)F]FACBC-PET/MR fusion images without disturbance from radioactivity from urine, and　the rank order of anti-[(18)F]FACBC accumulation at 50-60 post-injection in lesional lumbar LNs was PLM>CL>AL. CONCLUSIONS: Anti-[(18)F]FACBC accumulation in LNs with PLM was higher than that in inflamed LNs. ADVANCES IN KNOWLEDGE: The study showed that although low but significant levels of anti-[(18)F]FACBC uptake by chronic inflamed lesions might cause false-positives in anti-[(18)F]FACBC-PET in some PCa patients, uptake of the tracer at acutely inflamed sites was minimal. IMPLICATIONS FOR PATIENT CARE: The findings of this study suggest the potential of Anti-[(18)F]FACBC for distinguishing between tumors and acute inflammation in clinical practice.

Highly mobile gapless excitations in a two-dimensional candidate quantum spin liquid.

The nature of quantum spin liquids, a novel state of matter where strong quantum fluctuations destroy the long-range magnetic order even at zero temperature, is a long-standing issue in physics. We measured the low-temperature thermal conductivity of the recently discovered quantum spin liquid candidate, the organic insulator EtMe3Sb[Pd(dmit)2]2. A sizable linear temperature dependence term is clearly resolved in the zero-temperature limit, indicating the presence of gapless excitations with an extremely long mean free path, analogous to excitations near the Fermi surface in pure metals. Its magnetic field dependence suggests a concomitant appearance of spin-gap-like excitations at low temperatures. These findings expose a highly unusual dichotomy that characterizes the low-energy physics of this quantum system.

Inflammatory cytokines and hypoxia contribute to 18F-FDG uptake by cells involved in pannus formation in rheumatoid arthritis.

UNLABELLED: Assessment of the activity of rheumatoid arthritis (RA) is important for the prediction of future articular destruction. (18)F-FDG PET is known to represent the metabolic activity of inflammatory disease, which correlates with the pannus volume measured by MRI or ultrasonography. To evaluate the correlation between (18)F-FDG accumulation and RA pathology, we assessed (18)F-FDG accumulation in vivo using collagen-induced arthritis (CIA) animal models and (3)H-FDG uptake in vitro using various cells involved in arthritis. METHODS: (18)F-FDG PET images of rats with CIA were acquired on days 10, 14, and 17 after arthritis induction. The specimens were subsequently subjected to macroautoradiography, and the (18)F-FDG accumulation was compared with the histologic findings. (3)H-FDG uptake in vitro in inflammatory cells (neutrophils, macrophages, T cells, and fibroblasts) was measured to evaluate the contributions of these cells to (18)F-FDG accumulation. In addition, the influence on (3)H-FDG uptake of inflammatory factors, such as cytokines (tumor necrosis factor alpha [TNFalpha], interleukin 1 [IL-1], and IL-6), and hypoxia was examined. RESULTS: (18)F-FDG PET depicted swollen joints, and (18)F-FDG accumulation increased with the progression of arthritis. Histologically, a higher level of (18)F-FDG accumulation correlated with the pannus rather than the infiltration of inflammatory cells around the joints. In the in vitro (3)H-FDG uptake assay, fibroblasts showed the highest (3)H-FDG uptake, followed by neutrophils. Although only a small amount of (3)H-FDG was incorporated by resting macrophages, a dramatic increase in (3)H-FDG uptake in both fibroblasts and macrophages was observed when these cells were exposed to inflammatory cytokines, such as TNFalpha and IL-1, and hypoxia. Although neutrophils showed relatively high (3)H-FDG uptake without activation, no increase in (3)H-FDG uptake was observed in response to inflammatory cytokines. (3)H-FDG uptake by T cells was much lower than that by other cells. Thus, fibroblasts and activated macrophages contribute to a high level of (18)F-FDG accumulation in the pannus, and hypoxia as well as cytokine stimulation significantly increases (18)F-FDG uptake by these cells. CONCLUSION: (18)F-FDG accumulation in RA reflects proliferating pannus and inflammatory activity enhanced by inflammatory cytokines and hypoxia. (18)F-FDG PET should be effective for quantifying the inflammatory activity of RA.

Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus.

Nobiletin isolated from citrus peels prevents bulbectomy- and amyloid-beta protein-induced memory impairment in rodents. In the present study, using combined methods of biochemistry and electrophysiology, we examined the effects of nobiletin on phosphorylation of GluR1 receptor, the subunit of alpha-amino-3-hydroxy-5-methyl-D-aspartate (AMPA) receptors, and the receptor-mediated synaptic transmission in the hippocampus, a region implicated in memory formation, in culture and/or in slices. Western blot analysis showed that nobiletin-stimulated phosphorylation of multiple protein kinase A (PKA) substrates at 10 min following the treatment in cultured hippocampal neurons. In the cultured neurons, this natural compound also increased not only PKA activity, but also phosphorylation of GluR1 receptor at a PKA phosphorylation site, Ser 845, which has been demonstrated to be critical for synaptic plasticity, including enhancement of postsynaptic glutamate response, and important for spatial memory in vivo. The increased phosphorylation of GluR1 receptor at Ser 845 was abolished by H89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride), the PKA inhibitor, but not U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, in the cultured neurons. An increment of the phosphorylation of GluR1 receptor at Ser 845 was induced by nobiletin in the hippocampal slices as well. Furthermore, our electrophysiological analysis showed that nobiletin potentiated the AMPA receptor-mediated synaptic transmission at Schaffer collateral-CA1 pyramidal cell synapses in the hippocampal slices. This potentiation induced by the natural compound was not accompanied by the changes in paired-pulse ratio, and partially occluded the long-term potentiation, indicating the possible involvement of the postsynaptic mechanism. These findings suggest that nobiletin probably up-regulates synaptic transmission via the postsynaptic AMPA receptors at least partially by stimulation of PKA-mediated phosphorylation of GluR1 receptor in the hippocampus.

Nobiletin, a citrus flavonoid, reverses learning impairment associated with N-methyl-D-aspartate receptor antagonism by activation of extracellular signal-regulated kinase signaling.

Recent studies have indicated that learning-induced activation of extracellular signal-regulated kinase (ERK) signaling via N-methyl-D-aspartate (NMDA) receptors is required for consolidation of the resultant learning. These findings raise an idea that control of ERK signaling may be a potential target for treatment of cognitive dysfunction. Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from Citrus depressa, enhances cAMP/protein kinase A/ERK signaling in cultured rat hippocampal neurons and PC12D cells. Here, we, for the first time, present the evidence that this natural compound reverses learning impairment associated with NMDA receptor antagonism by activation of ERK in the hippocampus. Treatment with 50 mg/kg nobiletin reversed the NMDA receptor antagonist MK-801 (dizocilpine maleate)-induced learning impairment in mice. Western blot analysis also showed that nobiletin reversed MK-801-induced inhibition of learning-associated ERK activation in the hippocampus of the animals. Furthermore, consistent with these results, in cultured rat hippocampal neurons, nobiletin restored MK-801-induced impairment of NMDA-stimulated phosphorylation of ERK in a concentration-dependent manner. Taken together, the present study suggests that compounds that activate ERK signaling improve cognitive deficits associated with NMDA receptor hypofunction and that nobiletin may give us a new insight into therapeutic drug development for neurological disorders exhibiting cognitive impairment accompanied by a hypofunction of NMDA receptor-ERK signaling.