Yoji Kitamura

Anxiety- and depression-like behavior in mice lacking the CD157/BST1 gene, a risk factor for Parkinson's disease

CD157, known as bone marrow stromal cell antigen-1, is a glycosylphosphatidylinositol-anchored ADP-ribosyl cyclase that supports the survival and function of B-lymphocytes and hematopoietic or intestinal stem cells. Although CD157/Bst1 is a risk locus in Parkinsons disease (PD), little is known about the function of CD157 in the nervous system and contribution to PD progression. Here, we show that no apparent motor dysfunction was observed in young knockout (CD157−/−) male mice under less aging-related effects on behaviors. CD157−/− mice exhibited anxiety-related and depression-like behaviors compared with wild-type mice. These behaviors were rescued through treatment with anti-psychiatric drugs and oxytocin. CD157 was weakly expressed in the amygdala and c-Fos immunoreactivity in the amygdala was less evident in CD157−/− mice than in wild-type mice. These results demonstrate for the first time that CD157 plays a role as a neuro-regulator and suggest a potential role in pre-motor symptoms in PD.

Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers

68Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

Development and Evaluation of a Novel 99mTc-Labeled Annexin A5 for Early Detection of Response to Chemotherapy

99mTc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, 99mTc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel 99mTc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. 99mTc labeling was performed by a ligand exchange reaction with 99mTc-glucoheptonate. Biodistribution experiments for both 99mTc-C3(BHam)2-annexin A5 and 99mTc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of 99mTc-C3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. 99mTc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, 99mTc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than 99mTc-HYNIC-annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of 99mTc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of 99mTc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of 99mTc-C3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that 99mTc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy.

Syntheses and in vitro evaluation of decalinvesamicol analogues as potential imaging probes for vesicular acetylcholine transporter (VAChT)

A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and σ receptors (σ-1, σ-2) were evaluated by in vitro binding assays using rat cerebral or liver membranes. OIDV and OBDV showed greater binding affinity to VAChT (K(i) = 20.5 ± 5.6 and 13.8 ± 1.2 nM, respectively) than did vesamicol (K(i) = 33.9 ± 18.1 nM) with low affinity to σ receptors. A saturation binding assay in rat cerebral membranes revealed that [(125)I]OIDV had a single high affinity binding site with a K(d) value of 1.73 nM and a B(max) value of 164.4 fmol/mg protein. [(125)I]OIDV revealed little competition with inhibitors, which possessed specific affinity to each σ (σ-1 and σ-2), serotonin (5-HT(1A) and 5-HT(2A)), noradrenaline, and muscarinic acetylcholine receptors. In addition, BBB penetration of [(125)I]OIDV was verified in in vivo. The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging probes with high affinity and selectivity.

Development and evaluation of a radiobromine-labeled sigma ligand for tumor imaging

INTRODUCTION Sigma receptors are appropriate targets for tumor imaging because they are highly expressed in a variety of human tumors. Previously, we synthesized a vesamicol analog, (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol ((+)-pIV), with high affinity for sigma receptors, and prepared radioiodinated (+)-pIV. In this study, to develop a radiobromine-labeled vesamicol analog as a sigma receptor imaging agent for PET, nonradioactive and radiobromine-labeled (+)-2-[4-(4-bromophenyl)piperidino]cyclohexanol ((+)-pBrV) was prepared and evaluated in vitro and in vivo. In these initial studies, (77)Br was used because of its longer half-life. METHODS (+)-[(77)Br]pBrV was prepared by a bromodestannylation reaction with radiochemical purity of 98.8% after HPLC purification. The partition coefficient of (+)-[(77)Br]pBrV was measured. In vitro binding characteristics of (+)-pBrV to sigma receptors were assayed. Biodistribution experiments were performed by intravenous administration of a mixed solution of (+)-[(77)Br]pBrV and (+)-[(125)I]pIV into DU-145 tumor-bearing mice. RESULTS The lipophilicity of (+)-[(77)Br]pBrV was lower than that of (+)-[(125)I]pIV. As a result of in vitro binding assay to sigma receptors, the affinities of (+)-pBrV to sigma receptors were competitive to those of (+)-pIV. In biodistribution experiments, (+)-[(77)Br]pBrV and (+)-[(125)I]pIV showed high uptake in tumor via sigma receptors. The biodistributions of both radiotracers showed similar patterns. However, the accumulation of radioactivity in liver after injection of (+)-[(77)Br]pBrV was significantly lower compared to that of (+)-[(125)I]pIV. CONCLUSION These results indicate that radiobromine-labeled pBrV possesses great potential as a sigma receptor imaging agent for PET.

Development and evaluation of a novel radioiodinated vesamicol analog as a sigma receptor imaging agent

BackgroundSigma receptors are highly expressed in human tumors and should be appropriate targets for developing tumor imaging agents. Previously, we synthesized a vesamicol analog, (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol ((+)-p IV), with a high affinity for sigma receptors and prepared radioiodinated (+)-p IV. As a result, (+)-[125I]p IV showed high tumor uptake in biodistribution experiments. However, the accumulation of radioactivity in normal tissues, such as the liver, was high. We supposed that some parts of the accumulation of (+)-p IV in the liver should be because of its high lipophilicity, and prepared and evaluated a more hydrophilic radiolabeled vesamicol analog, (+)-4-[1-(2-hydroxycyclohexyl)piperidine-4-yl]-2-iodophenol ((+)-IV-OH).Methods(+)-[125I]IV-OH was prepared by the chloramine T method from the precursor. The partition coefficient of (+)-[125I]IV-OH was measured. Biodistribution experiments were performed by intravenous administration of a mixed solution of (+)-[125I]IV-OH and (+)-[131I]p IV into DU-145 tumor-bearing mice. Blocking studies were performed by intravenous injection of (+)-[125I]IV-OH mixed with an excess amount of ligand into DU-145 tumor-bearing mice.ResultsThe hydrophilicity of (+)-[125I]IV-OH was much higher than that of (+)-[125I]p IV. In biodistribution experiments, (+)-[125I]IV-OH and (+)-[131I]p IV showed high uptake in tumor tissues at 10-min post-injection. Although (+)-[131I]p IV tended to be retained in most tissues, (+)-[125I]IV-OH was cleared from most tissues. In the liver, the radioactivity level of (+)-[125I]IV-OH was significantly lower at all time points compared to those of (+)-[131I]p IV. In the blocking studies, co-injection of an excess amount of sigma ligands resulted in significant decreases of tumor/blood uptake ratios after injection of (+)-[125I]IV-OH.ConclusionsThe results indicate that radioiodinated (+)-IV-OH holds a potential as a sigma receptor imaging agent.

Evaluation of Chlorella as a Decorporation Agent to Enhance the Elimination of Radioactive Strontium from Body

Background Release of radionuclides, such as 137Cs and 90Sr, into the atmosphere and the ocean presents an important problem because internal exposure to 137Cs and 90Sr could be very harmful to humans. Chlorella has been reported to be effective in enhancing the excretion of heavy metals; thus, we hypothesized that Chlorella could also enhance the elimination of 137Cs or 90Sr from the body. We evaluated the potential of Chlorella as a decorporation agent in vitro and in vivo, using 85Sr instead of 90Sr. Methods In vitro experiments of adsorption of 137Cs and 85Sr to Chlorella were performed under wide pH conditions. The maximum sorption capacity of Chlorella to strontium was estimated using the Langmuir model. A 85Sr solution was orally administrated to mice pretreated with Chlorella. At 48 h after 85Sr administration, the biodistribution of radioactivity was determined. Results In the in vitro experiments, although 85Sr barely adsorbed to Chlorella at low pH, the 85Sr adsorption ratio to Chlorella increased with increasing pH. The maximum sorption capacity of Chlorella to strontium was 9.06 mg / g. 137Cs barely adsorbed to Chlorella under any pH conditions. In the biodistribution experiments, bone accumulation of radioactivity after 85Sr administration was significantly decreased in the Chlorella pretreatment group compared with the non-treatment control group. Conclusions In conclusion, these results indicated that Chlorella could inhibit the absorption of 90Sr into the blood and enhance the elimination of 90Sr from the body through adsorption in intestine. Further studies are required to elucidate the mechanism and the components of Chlorella needed for adsorption to strontium and could promote the development of more effective decorporation agents.

Radiogallium Complex-Conjugated Bifunctional Peptides for Detecting Primary Cancer and Bone Metastases Simultaneously

(68)Ga (T(1/2) = 68 min, a generator-produced nuclide) is an interesting radionuclide for clinical positron emission tomography (PET). Recently, it was reported that radiogallium-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (Asp)n peptide [Ga-DOTA-(Asp)n] has great potential for bone metastases imaging. In the current study, a compound containing an aspartic acid peptide linker (D11) as a carrier to bone metastases, an RGD peptide [c(RGDfK) peptide] as a carrier to the primary cancer, and Ga-DOTA as a stable radiometal complex for imaging in one molecule, Ga-DOTA-D11-c(RGDfK), was designed, prepared, and evaluated to detect both the primary cancer and bone metastases simultaneously using (67)Ga, which is easy to handle. After DOTA-D11-c(RGDfK) was synthesized using Fmoc-based solid-phase methodology, (67)Ga-DOTA-D11-c(RGDfK) was prepared by complexing DOTA-D11-c(RGDfK) with (67)Ga. Hydroxyapatite binding assays, integrin binding assays, biodistribution experiments, and single photon emission tomography (SPECT) imaging using tumor-bearing mice were performed using (67)Ga-DOTA-D11-c(RGDfK). (67)Ga-DOTA-D11-c(RGDfK) was prepared with a radiochemical purity of >97%. In vitro, (67)Ga-DOTA-D11-c(RGDfK) had a high affinity for hydroxyapatite and αvβ3 integrin. In vivo, (67)Ga-DOTA-D11-c(RGDfK) exhibited high uptake in bone and tumor. The accumulation of (67)Ga-DOTA-D11-c(RGDfK) in tumor decreased when it was co-injected with c(RGDfK) peptide. (68)Ga-DOTA-D11-c(RGDfK) has great potential as a PET tracer for the diagnosis of both the primary cancer and bone metastases simultaneously.

Regional brain imaging of vesicular acetylcholine transporter using o-[125I]iodo-trans-decalinvesamicol as a new potential imaging probe

In this study, the regional rat brain distribution of radioiodinated o‐iodo‐trans‐decalinvesamicol ([125I]OIDV) was determined in vivo to evaluate its potential as a single‐photon emission computed tomography (SPECT) imaging probe for vesicular acetylcholine transporter (VAChT). Following intravenous injection, [125I]OIDV passed freely across the blood–brain barrier and accumulated in rat brain. The accumulation of [125I]OIDV in rat brain was significantly reduced by coadministration of (+/−)‐vesamicol (0.125 µmol). In contrast, the coadministration of σ‐receptor ligands, such as (+)‐pentazocine (0.125 µmol) as a σ‐1 receptor ligand and (+)−3‐(3‐hydroxyphenyl)‐N‐propylpiperidine (0.125 µmol) as a σ‐1 and σ‐2 receptor ligands, barely affected the accumulation of [125I]OIDV in rat brain. These findings in vivo were corroborated by autoradiographic analysis ex vivo. The authors found that the tracer binds with pharmacological selectivity to VAChT in rat brain and predicted that it may likewise serve in translational SPECT imaging studies of this marker in the integrity of cholinergic innervations. Synapse 68:107–113, 2014.

Design, synthesis and biological evaluation of negatively charged 111In-DTPA-octreotide derivatives

Our previous studies indicated that (111)In-diethylenetriaminepentaacetic acid ((111)In-DTPA)-octreotide derivatives with an additional negative charge by replacing N-terminal d-phenylalanine (d-Phe) with an acidic amino acid such as l-aspartic acid (Asp) or its derivative exhibited low renal radioactivity levels when compared with (111)In-DTPA-D-Phe(1)-octreotide. On the basis of the findings, we designed, synthesized and evaluated two Asp-modified (111)In-DTPA-conjugated octreotide derivatives, (111)In-DTPA-Asp(1)-octreotide and (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide. While (111)In-DTPA-Asp(1)-octreotide showed negligible AR42J cell uptake, (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide exhibited AR42J cell uptake similar to that of (111)In-DTPA-D-Phe(1)-octreotide. When administered to AR42J tumor-bearing mice, (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide exhibited renal radioactivity levels significantly lower than did (111)In-DTPA-D-Phe(1)-octreotide at 1 and 3 h post-injection. No significant differences were observed in tumor accumulation between (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide and (111)In-DTPA-D-Phe(1)-octreotide after 1 and 3h injection. The findings in this study suggested that an interposition of an Asp at an appropriate position in (111)In-DTPA-D-Phe(1)-octreotide would constitute a useful strategy to develop (111)In-DTPA-D-Phe(1)-octreotide derivatives of low renal radioactivity levels while preserving tumor accumulation.