Radiosynthesis and in Vivo and ex Vivo Evaluation of Isomeric [11C]methoxy Analogs of Nimesulide as Brain Cyclooxygenase-2-Targeted Imaging Agents.

Our previous studies identified that nimesulide analogs which bear a methoxy substituent at the para-position of the phenyl ring could be potential radiotracer candidates for detecting disorders related to cyclooxygenase-2 (COX-2) expression and activity in vivo using positron emission tomography (PET) in the brain. The present study was conducted to evaluate the in vivo characteristics of 11C-labeled para-methoxy nimesulide ([11C]1d) as a brain COX-2-targeted imaging agent compared to other isomeric methoxy analogs of nimesulide ([11C]1b and [11C]1c). [11C]1b-d were synthesized with reasonable yield and purity by the methylation of the O-desmethyl precursor with [11C]methyl triflate in the presence of NaOH at room temperature. We performed in vivo biodistribution analysis, brain PET imaging, ex vivo autoradiography, and metabolite analysis in mice. The uptake of [11C]1b-d was lower in the brain than in other tissues, including in the blood, and both [11C]1c and [11C]1d were rapidly metabolized. However, [11C]1d showed a small, but significant, specific signal and heterogeneous distribution in the brain. In vivo evaluation suggested that [11C]1d might correlate with COX-2 expression in the brain. Given its instability in vivo, [11C]1d seems unsuitable as a brain-COX-2 radioimaging agent. Further structural refinement of these radiotracers is necessary to enhance their uptake in the brain and to achieve sufficient metabolic stability.

Involvement of APOBEC3B in mutation induction by irradiation.

To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

Synthesis and characterization of 11 C-labeled benzyl amidine derivatives as PET radioligands for GluN2B subunit of the NMDA receptors.

GluN2B-containing NMDA receptors (NMDARs) play fundamental roles in learning and memory, although they are also associated with various brain disorders. In this study, we synthesized and evaluated three 11 C-labeled N-benzyl amidine derivatives 2-[11 C]methoxybenzyl) cinnamamidine ([11 C]CBA), N-(2-[11 C]methoxybenzyl)-2-naphthamidine ([11 C]NBA), and N-(2-[11 C]methoxybenzyl)quinoline-3-carboxamidine ([11 C]QBA) as PET radioligands for these receptors. The 11 C-benzyl amidines were synthesized via conventional methylation of corresponding des-methyl precursors with [11 C]CH3 I. In vitro binding characteristics were examined in brain sagittal sections using various GluN2B modulators and off-target ligands. Further, in vivo brain distribution studies were performed in normal mice. The 11 C-labeled benzyl amidines showed high-specific binding to the GluN2B subunit at in vitro. In particular, the quinoline derivative [11 C]QBA had the best binding properties in terms of high-brain localization to GluN2B-rich regions and specificity to the GluN2B subunit. Conversely, these 11 C-radioligands showed the brain distributions were inconsistent with GluN2B expression in biodistribution experiments. The majority of the radiolabeled compounds were identified as metabolized forms of which amido derivatives seemed to be the major species. Although these 11 C-ligands had high-specific binding to the GluN2B subunit, significant improvement in metabolic stability is necessary for successful positron emission tomography (PET) imaging of the GluN2B subunit of NMDARs.

ddY Mice Fed 10% Fat Diet Exhibit High p27KIP Expression and Delayed Hepatocyte DNA Synthesis During Liver Regeneration.

BACKGROUND: Excessive intake of a high-calorie diet has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Several studies have investigated the effect of NAFLD on liver regeneration, but the effects of simple steatosis have been found to be inconsistent. We aimed to assess whether the initial phase of diet-induced lipid accumulation, induced by a diet containing moderate levels of fat, impairs liver regeneration after partial hepatectomy (PHx) of mice. METHODS: Male ddY mice are prone to obesity, even when fed a relatively low-fat diet (FD). A model of early simple steatosis was created by feeding a 10% FD for 6 weeks to male ddY mice. Liver regeneration rate, DNA synthesis in hepatocytes, and DNA damage were then assessed. RESULTS: FD-fed mice had a slightly higher body mass (44.5 ± 2.6 grams vs. 48.1 ± 3.6 grams, P < 0.05), but not liver mass (2.55 ± 0.37 grams vs. 2.69 ± 0.26 grams). Lipid droplets appeared in FD-fed mouse hepatocytes and Oil Red O staining was five times as intense as in control mice. In FD-fed mice, liver regeneration rate after two-thirds PHx was lower and impaired DNA replication was also observed. FD induced the expression of the cyclin-dependent kinase inhibitor p27KIP, but not p21CIP. Moreover, greater histone 2A.X phosphorylation was observed, indicating that FD caused DNA damage. CONCLUSIONS: Even short-term feeding of a moderately high FD to male ddY mice results in lipid accumulation in hepatocytes, DNA damage, and greater expression of p27KIP, implying lower DNA synthesis.

Isomeric iodinated analogs of nimesulide: Synthesis, physicochemical characterization, cyclooxygenase-2 inhibitory activity, and transport across Caco-2 cells.

Isomeric iodinated derivatives of nimesulide, with an iodine substituent on the phenoxy ring, were prepared with the aim of identifying potential candidate compounds for the development of imaging agents targeting cyclooxygenase-2 (COX-2) in the brain. Both the experimental logP7.4 and pKa values for these iodinated analogs were in the acceptable range for passive brain penetration. The para-iodo-substituted analog was a more potent and selective COX-2 inhibitor than nimesulide, with a potency that was comparable to the reference drug, celecoxib. Iodination at the ortho- or meta-position of the phenoxy ring was associated with a substantial loss of COX-2 inhibitory activity. Transport studies across Caco-2 cell monolayers in the presence and absence of a P-glycoprotein (P-gp) inhibitor, verapamil, indicated that the para-iodo-substituted analog was not a P-gp transport substrate; this feature is a prerequisite for potential in vivo brain imaging compounds. The para-iodo-substituted analog of nimesulide appears to be an attractive candidate for the development of radioiodine-labeled tracers for in vivo brain imaging of COX-2 levels.

Isomeric methoxy analogs of nimesulide for development of brain cyclooxygense-2 (COX-2)-targeted imaging agents: Synthesis, in vitro COX-2-inhibitory potency, and cellular transport properties.

Nimesulide analogs bearing a methoxy substituent either at the ortho-, meta- or para-position on the phenyl ring, were designed, synthesized, and evaluated for potential as radioligands for brain cyclooxygenase-2 (COX-2) imaging. The synthesis of nimesulide and regioisomeric methoxy analogs was based on the copper-mediated arylation of phenolic derivatives for the construction of diaryl ethers. These isomeric methoxy analogs displayed lipophilicity similar to that of nimesulide itself, as evidenced by their HPLC logP7.4 values. In vitro inhibition studies using a colorimetric COX (ovine) inhibitor-screening assay demonstrated that the para-methoxy substituted analog retains the inhibition ability and selectivity observed for parent nimesulide toward COX-2 enzyme, whereas the meta- and ortho-methoxy substituents detrimentally affected COX-2-inhibition activity, which was further supported by molecular docking studies. Bidirectional transport cellular studies using Caco-2 cell culture model in the presence of the P-glycoprotein (P-gp) inhibitor, verapamil, showed that P-gp did not have a significant effect on the efflux of the para-methoxy substituted analog. Further investigations using the radiolabeled form of the para-methoxy substituted analog is warranted for in vivo characterization.

Guided acoustic-wave Brillouin scattering characteristics of few-mode fiber.

We experimentally investigate the modal dependence of the guided-acoustic wave Brillouin scattering (GAWBS) characteristics in a few-mode fiber (FMF). We clarify that launching higher-order modes into FMF induces different peak frequencies from those of single-mode fiber (SMF) in the GAWBS spectrum and that the GAWBS spectrum strongly depends on the mode excitation power ratio between the LP(01) and LP(11) modes. We evaluate the mode excitation power ratio at a splice between an FMF and an SMF by utilizing the GAWBS spectrum, which we realize by detecting only the fundamental mode at the output of the FMF and without the use of a mode demultiplexer.

Loss reduction in few-mode photonic crystal fiber by reducing inner surface imperfections in air holes.

We studied both theoretically and experimentally the additional loss in photonic crystal fiber (PCF) that results from inner surface imperfections such as contamination and the surface roughness of air holes. We estimated the modal loss dependence of these imperfections using a model with a "defective layer" for the first time. The theoretical studies suggest that higher order modes have a larger loss due to imperfections in the air holes. By minimizing the inner surface imperfections of the six innermost air holes, we can theoretically expect any additional loss to be reduced to a negligible level. Moreover, we examined our theoretical prediction experimentally. We fabricated few-mode PCFs by employing a suitable inner surface treatment for just the six innermost holes. As expected theoretically, the transmission loss was greatly reduced by employing these processes. The lowest transmission losses in the 1550 nm band were 0.31 dB/km for the LP01 mode and 0.43 dB/km for the LP11 mode. Our theoretical model will be useful with a view to realizing few-mode PCF with a loss comparable to that of conventional fibers.

Loss of EGF-dependent cell proliferation ability on radioresistant cell HepG2-8960-R.

Acquired radioresistance of cancer cells interferes with radiotherapy and increases the probability of cancer recurrence. HepG2-8960-R, which is one of several clinically relevant radioresistant (CRR) cell lines, has a high tolerance to the repeated clinically relevant doses of X-ray radiation. In this study, HepG2-8960-R had slightly lower cell proliferation ability than HepG2 in the presence of FBS. In particular, epidermal growth factor (EGF) hardly enhanced cell proliferation and DNA synthesis in HepG2-8960-R. Additionally, EGF could not induce the activation of Erk1/2, because the expression of EGF receptor (EGFR) protein decreased in HepG2-8960-R in accordance with the methylation of the EGFR promoter region. Therefore, cetuximab did not inhibit HepG2-8960-R cell proliferation. Our study showed that HepG2-8960-R had radioresistant and cetuximab-resistant abilities.

PLC-based LP11 mode rotator for mode-division multiplexing transmission.

A PLC-based LP11 mode rotator is proposed. The proposed mode rotator is composed of a waveguide with a trench that provides asymmetry of the waveguide. Numerical simulations show that converting LP11a (LP11b) mode to LP11b (LP11a) mode can be achieved with high conversion efficiency (more than 90%) and little polarization dependence over a wide wavelength range from 1450 nm to 1650 nm. In addition, we fabricate the proposed LP11 mode rotator using silica-based PLC. It is confirmed that the fabricated mode rotator can convert LP11a mode to LP11b mode over a wide wavelength range.

Mode multi/demultiplexing with parallel waveguide for mode division multiplexed transmission.

We propose a planar lightwave circuit (PLC)-based mode multi/demultiplexer (MUX/DEMUX) with an asymmetric parallel waveguide for mode division multiplexed (MDM) transmission. The PLCb-ased mode MUX/DEMUX has advantage of selectively exciting higher-order mode. We realize three-mode (LP(01), LP(11)a, and LP(21)a) multiplexing by using an asymmetric parallel waveguide. We then design and fabricate a PLC-based mode MUX/DEMUX on one chip by using our proposed LP(11) mode rotator to allow us to utilize the LP(11)b mode. We successfully multiplex the LP(01), LP(11)a, and LP(11)b modes and achieve a relatively low insertion loss over the C-band using our fabricated mode MUX/DEMUX. Our results indicate that the PLC-based mode MUX/DEMUX with a uniform height has the potential to increase the mode number by using an LP(11)b mode.

Experimental and numerical evaluation of inter-core differential mode delay characteristic of weakly-coupled multi-core fiber.

We investigated the inter-core differential mode delay (DMD) characteristic of a weakly-coupled homogeneous multi-core fiber with a view to utilizing inter-core crosstalk compensation with MIMO processing. We confirmed experimentally that the bend induced inter-core DMD is lower than the simulated results, which we expected owing to the twist of the fiber. We also revealed numerically that the refractive index profile variation of each core greatly increases inter-core DMD. Finally, we conducted a 4 × 4 MIMO transmission experiment using a weakly-coupled 4-core fiber and successfully compensated for the inter-core crosstalk.

Two-mode PLC-based mode multi/demultiplexer for mode and wavelength division multiplexed transmission.

We proposed a PLC-based mode multi/demultiplexer (MUX/DEMUX) with an asymmetric parallel waveguide for mode division multiplexed (MDM) transmission. The mode MUX/DEMUX including a mode conversion function with an asymmetric parallel waveguide can be realized by matching the effective indices of the LP(01) and LP(11) modes of two waveguides. We report the design of a mode MUX/DEMUX that can support C-band WDM-MDM transmission. The fabricated mode MUX/DEMUX realized a low insertion loss of less than 1.3 dB and high a mode extinction ratio that exceeded 15 dB. We used the fabricated mode MUX/DEMUX to achieve a successful 2 mode x 4 wavelength x 10 Gbps transmission over a 9 km two-mode fiber with a penalty of less than 1 dB.

Radiosynthesis and initial evaluation of (18)F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide.

INTRODUCTION: With the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named "Lactosome", which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the (18)F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out. METHODS: (18)F-labeled Lactosomes were prepared by a film hydration method under sonication in water at 50°C from a mixture of 4-[(18)F]fluoro-benzoyl poly-L-lactic acid ((18)F-BzPLLA30) and the amphiphilic polydepsipeptide. For biodistribution studies, BALB/cA Jcl-nu/nu mice bearing HeLa cells in the femur region were used. We took both PET and near-infrared fluorescence (NIRF) images of tumor bearing mice after co-injection of (18)F-labeled Lactosome and NIRF-labeled Lactosome. RESULTS: (18)F-labeled Lactosomes were prepared at good yields (222-420MBq) and more than 99% of (18)F-BzPLLA30 was incorporated into (18)F-labeled Lactosome. The radioactivity of (18)F-labeled Lactosome was found to be stable and maintained at high level for up to 6h after injection into the blood stream. Tumor uptake increased gradually after the injection. The uptake ratio of tumor/muscle was 2.7 at 6h from the time of injection. Tumor PET imaging with (18)F-labeled Lactosome was as capable as tumor NIRF imaging with NIRF-labeled Lactosome. CONCLUSION: Tumor PET imaging using Lactosome as a nanocarrier may be therefore a potential candidate for a facile and general solid tumor imaging technique.

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes.

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

Pharmacokinetic change of nanoparticulate formulation "Lactosome" on multiple administrations.

Lactosome, which is a polymer micelle composed of poly(lactic acid)-b-poly(sarcosine), was applied successfully for solid tumor imaging. Lactosome is considered to escape from the reticuloendothelial system recognition, and shows prolonged in vivo blood clearance time. In vivo disposition of Lactosome, however, changed upon multiple dosages. Lactosome at the 2nd dosage was cleared from the blood stream by trapping at liver. This accelerated blood clearance (ABC) phenomenon is explained by production of anti-Lactosome IgM and IgG(3) through the immune response related with B-lymphocyte cells. The memory effect of B-lymphocyte cells lasted nearly for six months in mouse. The epitope moiety of Lactosome is concluded to be poly(sarcosine) based on the competitive inhibition assay. Since the ABC phenomenon was also reported with PEGylated liposome, nanoparticles in general may be potential in triggering the immune system.

Control of in vivo blood clearance time of polymeric micelle by stereochemistry of amphiphilic polydepsipeptides.

Polymeric micelle, "Lactosome", is composed of amphiphilic polydepsipeptide with a hydrophobic block of helical poly(L-lactic acid) (PLLA) and a hydrophilic block of poly(sarcosine). Lactosome was labeled by incorporation of poly(lactic acid) having a near-infrared fluorescence (NIRF) chromophore, and studied on blood clearance and tumor imaging. In vivo blood clearance time of Lactosome was prolonged with incorporation of poly(D-lactic acid) (PDLA), but decreased with poly(D,L-lactic acid) (PDLLA). NIRF imaging with applying these Lactosomes to tumor-bearing mice revealed that the tumor/background intensity ratio increased with incorporation of PDLLA. Stereochemistry in the hydrophobic core of self-assemblies is thus an important factor for determining physical stability in the blood stream and consequently contrast in imaging.

5-O-(4-[125 I]Iodobenzyl)-L-ascorbic acid: electrophilic radioiodination and biodistribution in mice.

As a part of our efforts to develop potential imaging agents for ascorbate bioactivity, 5-O-(4-[(125)I]iodobenzyl)-L-ascorbic acid ([(125)I]1) was prepared through a two-step sequence which involved radioiodo-destannylation of a protected tributylstannyl precursor 6, followed by hydrolysis in acidic methanol of the protecting groups in 61% overall radiochemical yield, with a radiochemical purity of over 98% and a specific activity of more than 15.4 GBq/µmol. Tissue distribution of [(125)I]1 in tumor-bearing mice showed signs of distribution profiles similar to the reported results for 6-deoxy-6-[(18)F]fluoro-L-ascorbic (6-(18)FAsA) acid and 6-deoxy-6-[(131)I]iodo-L-ascorbic acid (6-(131)IAsA) but with notable differences in the adrenal glands, in which considerably lower uptake of radioactivity and rapid clearance with time were observed. Pretreatment of mice with a known inhibitor of ascorbate transport, sulfinpyrazone, did not produce any significant change in the adrenal uptake of radioactivity after injection of [(125)I]1 compared to the control, suggesting that uptake in the adrenal glands is independent of the sodium-dependent vitamin C transporter 2 transport mechanism. Introduction of a bulky substituent at C-5 on AsA, such as an iodobenzyloxy group, may not be suitable for the design of analogs that may still be able to maintain characteristic distribution properties in vivo seen with AsA itself.

¹¹C-labeled analogs of indomethacin esters and amides for brain cyclooxygenase-2 imaging: radiosynthesis, in vitro evaluation and in vivo characteristics in mice.

There is great potential in the use of positron emission tomography (PET) and suitable radiotracers for the study of cyclooxygenase type 2 (COX-2) enzyme in living subjects. In the present study, we prepared and evaluated five ¹¹C-labeled ester and amide analogs derived from indomethacin as potential PET imaging agents for the in vivo visualization of the brain COX-2 enzyme. Five ¹¹C-labeled COX-2 inhibitors, with different lipophilicities and moderate COX-2 inhibitory activity, were prepared by treatment of the corresponding O-desmethyl precursors with [¹¹C]methyl triflate and purified by HPLC (radiochemical yields of 55-71%, radiochemical purity of >93%, and the specific activities of 22-331 GBq/µmol). In mice, radioactivity in the brain for all radiotracers was low, with very low brain-to-blood ratios. A clear inverse relationship was observed between brain uptake at 1 min postinjection and the lipophilicity (experimental log P7.4) of the studied ¹¹C-radiotracers. Pretreatment of mice with cyclosporine A to block P-glycoproteins caused a significant increase in brain uptake of radioactivity following injection of the ¹¹C-radiotracer compared to control. HPLC analysis showed that each radiotracer was rapidly metabolized, and a few metabolites, which were more polar than the original radiotracers, were found in both plasma and brain. No specific binding of the tracers towards the COX-2 enzyme in the brain was clearly revealed by in vivo blocking study. Further structural refinement of the tracer agent is necessary for better enhancement of brain uptake and for sufficient metabolic stability.

1-(3'-[125I]Iodophenyl)-3-methy-2-pyrazolin-5-one: preparation, solution stability, and biodistribution in normal mice.

3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), known as a potent free radical scavenger, has been developed as a medical drug for the treatment of acute cerebral infarction. With the aim of developing radiotracers for imaging free radicals in vivo, 1-(3'-[(125)I]iodophenyl)-3-methy-2-pyrazolin-5-one ((125)I-2) was synthesized by two methods, via isotopic exchange and interhalogen exchange under solvent-free conditions, in which iodo- and bromo-derivatives were used as labeling precursors, respectively. After HPLC purification, (125)I-2 was obtained in modest isolated radiochemical yields (ca. 20%) with high radiochemical purities by both methods. The former gave specific activities of 0.2-0.6 kBq/micromol, whereas the latter approach achieved specific activities of more than 0.14 GBq/micromol. On attempting to prepare an injectable formulation for (125)I-2 with high specific activity, its radiochemical purities dropped to about 60-70%. Unlabeled analog 2 was found to have lipophilic and antioxidant properties similar to edaravone. Intravenous injection of (125)I-2 with low specific radioactivity into normal mice showed signs of distribution profiles similar to reported results for (14)C-labeled edaravone in normal rats.