Tomoyuki Ohya

In Vivo Measurement of the Affinity and Density of Metabotropic Glutamate Receptor Subtype 1 in Rat Brain Using 18F-FITM in Small-Animal PET

Metabotropic glutamate receptor subtype 1 (mGluR1) is a crucial molecular target in the central nervous system disorders. 4-18F-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide (18F-FITM) has been recently developed as a useful PET ligand for mGluR1 imaging in our laboratory. In this study, we aimed to measure the affinity and density of mGluR1 using PET with 18F-FITM in rat brain under the in vivo conditions. Methods: Binding potentials (BPND) and amounts of specific binding (bound ligand concentration) at equilibrium state in brain regions were noninvasively estimated using the equilibrium analysis combined with the receptor-blocked approach (EA RBA) for kinetic analysis of 18F-FITM PET results in place of reference tissue methods. Using BPND and specific binding values of rats treated with multidose ligand, we performed Scatchard analyses for in vivo measurements of mGluR1 density (maximum number of binding sites, or Bmax) and ligand affinity (dissociation constant, or Kd) in brain regions, respectively. Results: The pretreatment of rats with unlabeled FITM (1 mg/kg) occupied an mGluR1 binding site of 18F-FITM by more than 99% and did not affect the input function. Hence, we used the tissue time–activity curve for receptor-blocked rats as representative of the nondisplaceable (free and nonspecific binding of radioligand) compartment. The BPND based on EA RBA showed a high correlation with the BPND based on invasive Logan plot graphical analysis in the thalamus, hippocampus, striatum, and cingulate cortex. The Kd (nM) and Bmax (pmol/mL) obtained by the Scatchard analyses with the multidose ligand assays were 2.1 and 36.3, respectively, for the thalamus; 2.1 and 27.5, respectively, for the hippocampus; 1.5 and 22.2, respectively, for the striatum; and 1.5 and 20.5, respectively, for the cingulate cortex with a high confidence. Conclusion: Our study is the first to our knowledge to measure the in vivo affinity (Kd and binding potential) of 18F-FITM and mGluR1 density (Bmax) with a high correlation to in vitro values in rat brain regions. This measurement using PET with 18F-FITM would be a useful index for research about mGluR1 functions in central nervous system disorders and development of new pharmaceuticals.

An Analysis of Long-Distance Water Transport in the Soybean Stem Using H215O

The lateral water movement in the intact stem of a transpiring soybean plant was analyzed quantitatively by a real-time measurement system utilizing labeled water, H(2)(15)O and gamma ray detectors. A large volume of water escaping from xylem vessels during its transport was detected. The escape of water was not influenced by evaporation from the stem surface or mass flow in the sieve tubes. It was assumed that the total amount of water transported through xylem vessels was kept almost completely constant along the internode. As a result, most of the escaped water was found to re-enter the xylem vessels, i.e. water exchange occurred. The analysis of radiographs of tritiated water suggested that the self-diffusion effect of water was strong for lateral water movement, although another driving force besides thermal motion was included in the process, and that the process was also affected by the water permeability of the plasma membrane. An analysis based on a mathematical model showed that the net volume of water which escaped from xylem vessels was not dependent on the transpiration rate of the plant.

Efficient preparation of high-quality 64Cu for routine use

INTRODUCTION Copper-64 is an attractive radionuclide for positron emission tomography and is emerging as a radiotherapeutic agent. The demand of 64Cu with low metallic impurities has increased because of its wide applications when incorporated with antibodies, peptides, and proteins. In this study, we propose a new separation method to produce high-quality 64Cu using a cation exchange column, as well as an automated separation system suitable for large-scale production. METHODS 64Cu was produced from an electrodeposited 64Ni target via the 64Ni(p,n)-reaction with a 24MeV HH+ beam at 10eμA (electrical microampere) conducted for 1-3h. The irradiated target was transported to a hot cell and disassembled remotely. 64Cu was separated by a solvent mixture of HCl and acetone on a cation-exchange resin, AG50W-X8. The chemical purity of 64Cu final product was evaluated using ion-chromatography coupled with a UV detector and inductively coupled plasma mass spectroscopy for quality as well as metallic impurities. RESULTS We obtained 64Cu in dried form at a yield of 5.2-13GBq at the end of separation, or 521±12MBq/eμAh as the final product within 2.5h of processing time. The metallic impurities were a satisfactory low level in the order of ppb. Major contaminants of Co and Ni were lower than those samples obtained by a widely accepted separation using an anion-exchange resin. CONCLUSION Using a cation-exchange resin and a systematic operation, we successfully reduced the contamination level of the 64Cu product. As a straightforward separation method, which shortened the entire processing time, we obtained a satisfactory amount of high-quality 64Cu available for routine use.

67Cu-Radiolabeling of a multimeric RGD peptide for α v β 3 integrin-targeted radionuclide therapy: Stability, therapeutic efficacy, and safety studies in mice

Objective Copper-67 (67Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of 67Cu. Encouraged by these, we performed 67Cu-radiolabeling of a tetrameric cyclic Arg–Gly–Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets &agr;V&bgr;3 integrin (&agr;V&bgr;3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. Materials and methods 67Cu, produced through the 64Ni(&agr;, p)67Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous &agr;V&bgr;3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: 67Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. Results 67Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of 67Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in 67Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. Conclusion 67Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for &agr;V&bgr;3 integrin-targeted internal radiotherapy.

Effect of radiolabeled metabolite elimination from the brain on the accuracy of cerebral enzyme activity estimation using positron emission tomography with substrate tracers.

Cerebral enzyme activity can be quantified using positron emission tomography (PET) in conjunction with a radiolabeled enzyme substrate. We investigated the relationship between the elimination rate (k(el)) of tracer metabolites from the brain and the precision of target enzyme activity estimation (k(3)). An initial simulation study indicated that the precision of k(3) estimates was highly dependent on k(el), and was characterized by several kinetic parameters including the ratio of k(el) and the efflux rate (k(2)) of authentic tracer (β≡k(el)/k(2)). The optimal tracer condition for high sensitivity was found to be β<0.1. To verify the simulation results, we performed a PET study with a single monkey using two PET tracers, N-[(18)F]fluoroethylpiperidin-4-ylmethyl acetate ([(18)F]FEP-4MA) and N-[(11)C]methylpiperidin-4-yl acetate ([(11)C]MP4A). Both of these substrate type tracers were developed for measuring cerebral acetylcholinesterase activity. There was good retention of the radioactive metabolite of [(11)C]MP4A in the brain (k(el)=0.0036±0.0013 min(-1), β=0.028), whereas that of [(18)F]FEP-4MA was eliminated from the brain (k(el)=0.012±0.0010 min(-1), β=0.085). A non-linear least square analysis for simultaneous estimation of all parameters showed that the precision of the k(3) estimate for [(18)F]FEP-4MA was as high (7.4%) as that for [(11)C]MP4A (10%). These results indicate that tracers with metabolites that are eliminated from the brain at a slow rate (β<0.1) may be useful for the quantitative measurement of target enzyme activity.

Improved Visualization and Specific Binding for Metabotropic Glutamate Receptor Subtype 1 (mGluR1) Using [11C]ITMM with Ultra-High Specific Activity in Small-Animal PET.

Metabotropic glutamate receptor subtype 1 (mGluR1) is a crucial target in the development of new medications to treat central nervous system (CNS) disorders. Recently, we developed N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[11C]methoxy-N-methyl-benzamide ([11C]ITMM) as a useful positron emission tomography (PET) probe for mGluR1 in clinical studies. Here, we aimed to improve visualization and threshold of specific binding for mGluR1 using [11C]ITMM with ultra-high specific activity (SA) of > 3,500 GBq/μmol in rat brains. A two-tissue compartment model indicated large differences between the two SAs in the constants k3 and k4, representing binding ability for mGluR1, while constants K 1 and k2 showed no differences. The total distribution volume (VT) values of conventional and ultra-high SA were 9.1 and 11.2 in the thalamus, 7.7 and 9.7 in the striatum, and 6.4 and 8.5 mL/cm3 in the substantia nigra, respectively. The specific binding of [11C]ITMM with ultra-high SA was significantly higher than the conventional SA, especially in the basal ganglia. Parametric PET images scaled with VT of the ultra-high SA clearly identified regional differences in the rat brain. In conclusion, PET studies using [11C]ITMM with ultra-high SA could sufficiently improve visualization and specific binding for mGluR1, which could help further understanding for mGluR1 functions in CNS disorders.

Small-scale production of 67Cu for a preclinical study via the 64Ni(α, p)67Cu channel

INTRODUCTION Copper-67 is an attractive beta emitter for targeted radionuclide therapy. However, the availability of 67Cu limits its potential use in a wide range of applications. In this study, we propose an easy small-scale production of 67Cu using 64Ni target for a preclinical study. METHODS 67Cu was produced from an electrodeposited 64Ni target via the 64Ni(α, p)67Cu-reaction with a 36 MeV alpha beam at 15 eμA (electrical microampere) conducted for 7 h. The chemical separation process of 67Cu from the 64Ni target was performed following by our routine procedure of 64Cu production using cation exchange resin, AG50W-X8, with minor modification. The target and its holder were redesigned in the preparation. RESULTS The 67Cu product was obtained with a yield of 55 ± 10 MBq at the end of bombardment (EOB), and the yield was 527 ± 96 kBq/μAh at the EOB. The copper impurity in the product was low (0.71 ± 0.21 μg) and the product was suitable for a preclinical study. CONCLUSIONS We produced 67Cu with sufficient activity and quality for a preclinical study using a 64Ni-target. This production method also showed advantages as a routine method, i.e., shorten the processing time, reducing the radiation exposure and ready target recycling, when compared with that of a conventional Zn-target used for 67Cu production.

First demonstration of in vivo mapping for regional brain monoacylglycerol lipase using PET with [11C]SAR127303

ABSTRACT Monoacylglycerol lipase (MAGL) is a main regulator of the endocannabinoid system within the central nervous system (CNS). Recently, [11C]SAR127303 was developed as a promising radioligand for MAGL imaging. In this study, we aimed to quantify regional MAGL concentrations in the rat brain using positron emission tomography (PET) with [11C]SAR127303. An irreversible two‐tissue compartment model (2‐TCMi, k4=0) analysis was conducted to estimate quantitative parameters (k3, Ki2‐TCMi, and &lgr;k3). These parameters were successfully obtained with high identifiability (<10 %COV) for the following regions ranked in order from highest to lowest: cingulate cortex>striatum>hippocampus>thalamus>cerebellum>hypothalamus≈pons. In vitro autoradiographs using [11C]SAR127303 showed a heterogeneous distribution of radioactivity, as seen in the PET images. The Ki2‐TCMi and &lgr;k3 values correlated relatively highly with in vitro binding (r>0.4, P<0.005). The Ki2‐TCMi values showed high correlation and low underestimation (<10%) compared with the slope of a Patlak plot analysis with linear regression (KiPatlak). In conclusion, we successfully estimated regional net uptake value of [11C]SAR127303 reflecting MAGL concentrations in rat brain regions for the first time. HIGHLIGHTSCompartment model analysis for PET with [11C]SAR127303 was successfully conducted.Net uptake value of [11C]SAR127303 highly correlated with MAGL concentrations.In vivo mapping for brain MAGL concentration was demonstrated for the first time.

67Cu-Radiolabeling of a multimeric RGD peptide for αVβ3 integrin-targeted radionuclide therapy

Objective Copper-67 (67Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of 67Cu. Encouraged by these, we performed 67Cu-radiolabeling of a tetrameric cyclic Arg–Gly–Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets &agr;V&bgr;3 integrin (&agr;V&bgr;3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. Materials and methods 67Cu, produced through the 64Ni(&agr;, p)67Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous &agr;V&bgr;3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: 67Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. Results 67Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of 67Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in 67Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. Conclusion 67Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for &agr;V&bgr;3 integrin-targeted internal radiotherapy.

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Objective Copper-67 (67Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of 67Cu. Encouraged by these, we performed 67Cu-radiolabeling of a tetrameric cyclic Arg–Gly–Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets &agr;V&bgr;3 integrin (&agr;V&bgr;3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. Materials and methods 67Cu, produced through the 64Ni(&agr;, p)67Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous &agr;V&bgr;3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: 67Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. Results 67Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of 67Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in 67Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. Conclusion 67Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for &agr;V&bgr;3 integrin-targeted internal radiotherapy.