Yoshihiro Murakami

Expression of Pigment Epithelium-Derived Factor Decreases Liver Metastasis and Correlates with Favorable Prognosis for Patients with Ductal Pancreatic Adenocarcinoma

Pigment epithelium-derived factor (PEDF) is expressed in several normal organs and identified as an inhibitor of neovascularization. In the present study, we screened the expression of PEDF immunohistochemically and investigated its correlation with clinicopathological features in patients who underwent surgery for ductal pancreatic adenocarcinoma. Of the 80 patients, 22 cases (27.5%) were positive for PEDF. A significant association was found between the PEDF expression and low microvessel density (P = 0.0003). No correlation was found between PEDF expression and age, gender, depth of invasion, tumor diameter, lymphatic invasion, venous, invasion or histopathological grading. The patients in pathological stage II had a significantly higher incidence of PEDF-positive expression than those in pathological stage III or IVA (P = 0.0418). PEDF immunoreactivity was inversely associated with liver metastasis (P = 0.0422). The survival of patients that were PEDF positive was significantly longer than that of those with negative expression (P = 0.0026). Multivariate analysis using the Cox regression model indicated that PEDF-positive expression was an independent favorable prognostic factor (risk ratio, 0.394; P = 0.0016). We conclude that PEDF expression suggests a more favorable prognosis than in patients whose carcinomas lack PEDF expression.

Pigment epithelium-derived factor gene therapy inhibits human pancreatic cancer in mice.

Purpose: Pigment epithelium–derived factor (PEDF), which has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, is also expressed in the pancreas. Previously, we have screened the expression of PEDF by immunohistochemical analysis and showed that low expression of PEDF is associated with increased risk of hepatic metastasis and short survival. The purpose of this study was to investigate whether PEDF gene is a potent tumor suppressor and a potential candidate for cancer gene therapy. Experimental Design: We investigated both in vitro and in vivo growth characteristics of human pancreatic adenocarcinoma cell lines that were stably transfected to overexpress human PEDF and therapeutic effects of lentivirus-based vectors expressing PEDF on tumor growth in murine s.c. tumor model. Results: We discovered that cells secreted PEDF protein in the media and this exhibited strong inhibitory effects on proliferation and migration of human umbilical vein endothelial cells. The size of PEDF-overexpressing pancreatic adenocarcinoma tumors was significantly smaller than that of control tumors in s.c. tumor models. Moreover, the growth of PEDF-overexpressing pancreatic adenocarcinoma cells was significantly suppressed in comparison with control cells in peritoneal metastasis models. In gene transfer models, intratumoral injection of a lentivirus vector encoding PEDF (LV-PEDF) caused significant inhibition of tumor growth. The antitumor effect observed after treatment with LV-PEDF was associated with decreased microvessel density in tumors. Conclusion: Our data suggest that PEDF may exert a biological effect on tumor angiogenesis and PEDF gene therapy may provide a new approach for treatment of pancreatic adenocarcinoma.

Cancer Detection Using a PET Tracer, 11C-Glycylsarcosine, Targeted to H+/Peptide Transporter

H+/peptide transporter, PEPT1, is functionally expressed in some human cancer cell lines and might be a candidate molecular target for detection of cancers in vivo using PET. The aim of the present study was to establish a novel tumor-imaging technology using a PET tracer targeted to H+/peptide transporter(s). We also compared the tracer with 18F-FDG, focusing on the specificity of their accumulation between tumor and inflammatory tissues. Methods: A dipeptide PET tracer, 11C-glycylsarcosine (11C-Gly-Sar), was injected intravenously into athymic mice transplanted with human pancreatic, prostate, and gastric cancer cells. The distribution patterns of 11C-Gly-Sar and 18F-FDG in the tumor-bearing mice, and in mice with inflammatory tissue, were assessed by imaging with a positron planar imaging system (PPIS). Tissue distributions of tracer radioactivity were also measured. The expression levels of PEPT1 and PEPT2 (PEPTs) proteins in tumor xenografts and inflammatory tissue were examined by immunohistochemical analysis. The messenger RNA expression levels of PEPTs in 58 available cancer cell lines were quantified by means of real-time polymerase chain reaction. Results: All 3 tumor xenografts were well visualized with the PPIS after injection of 11C-Gly-Sar. Expression of PEPTs in those xenografts was confirmed by immunohistochemical analysis. Tumor-to-blood concentration ratios of 11C-Gly-Sar increased in a time-dependent manner and were much higher than unity. Most of the radioactivity found in the tumor tissue was recovered as the intact tracer. These results indicated that 11C-Gly-Sar was taken up by the PEPTs in tumor xenografts. It is noteworthy that 11C-Gly-Sar was minimally present in inflammatory tissues that expressed no PEPT1 or PEPT2 protein, whereas 18F-FDG was highly accumulated, with the values of the selectivity index being >25.1 and 0.72 for 11C-Gly-Sar and 18F-FDG, respectively. The mRNAs of PEPT1 and PEPT2 were expressed in 27.6% and 93.1%, respectively, of the cancer cell lines examined in the present study. Conclusion: The present study indicates that 11C-Gly-Sar is a promising tumor-imaging agent and is superior to 18F-FDG for distinguishing between tumors and inflammatory tissue. Because PEPTs were ubiquitously expressed in various types of tumor cells examined, 11C-Gly-Sar could be useful for the detection of many types of cancers.

Amyloid imaging in aged and young macaques with [11C]PIB and [18F]FDDNP

[11C]PIB and [18F]FDDNP were examined on five aged and five young adult male rhesus macaques using positron emission tomography. Both tracers showed increased accumulation in the striatum, thalamus, cingulate and pons in the aged group. Compared to [11C]PIB, [18F]FDDNP showed higher accumulation in the cortical regions of aged animals as well as young animals. Although [18F]FDDNP may have possible usefulness for imaging, including other proteins, [11C]PIB may be better for amyloid imaging owing to lower non‐specific binding. Synapse 62:472–475, 2008.

Brain penetration of telmisartan, a unique centrally acting angiotensin II type 1 receptor blocker, studied by PET in conscious rhesus macaques☆

INTRODUCTION Telmisartan is a widely used, long-acting antihypertensive agent. Known to be a selective angiotensin II type 1 (AT(1)) receptor (AT(1)R) blocker (ARB), telmisartan acts as a partial agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and inhibits centrally mediated effects of angiotensin II in rats following peripheral administration, although the brain penetration of telmisartan remains unclear. We investigated the brain concentration and localization of telmisartan using (11)C-labeled telmisartan and positron emission tomography (PET) in conscious rhesus macaques. METHODS Three male rhesus macaques were bolus intravenously administered [(11)C]telmisartan either alone or as a mixture with unlabeled telmisartan (1mg/kg). Dynamic PET images were acquired for 95min following administration. Blood samples were collected for the analysis of plasma concentration and metabolites, and brain and plasma concentrations were calculated from detected radioactivity using the specific activity of the administered drug preparation, in which whole blood radioactivity was used for the correction of intravascular blood radioactivity in brain. RESULTS Telmisartan penetrated into the brain little but enough to block AT(1)R and showed a consistently increasing brain/plasma ratio within the PET scanning period, suggesting slow clearance of the compound from the brain compared to the plasma clearance. Brain/plasma ratios at 30, 60, and 90min were 0.06, 0.13, and 0.18, respectively. No marked localization according to the AT(1)R distribution was noted over the entire brain, even on tracer alone dosing. CONCLUSIONS Telmisartan penetrated into the brain enough to block AT(1)R and showed a slow clearance from the brain in conscious rhesus macaques, supporting the long-acting and central responses of telmisartan as a unique property among ARBs.

Brain Adenosine A2A Receptor Occupancy by a Novel A1/A2A Receptor Antagonist, ASP5854, in Rhesus Monkeys: Relationship to Anticataleptic Effect

The purpose of the present study was to measure adenosine A2A receptor (A2AR) occupancy in the brain by a novel adenosine A1/A2A antagonist, 5-[5-amino-3-(4fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), and to determine the degree of receptor occupancy necessary to inhibit haloperidol-induced catalepsy in rhesus monkeys. Methods: A2AR occupancy by ASP5854 (0.001–0.1 mg/kg) was examined in the striatum using an A2AR-specific radiotracer, 11C-SCH442416, and PET in conscious rhesus monkeys. A2AR occupancy was monitored after a single intravenous administration of ASP5854 in 3 animals, and a dynamic PET scan was performed at 1, 4, and 8 h after an intravenous bolus injection of the tracer for approximately 740 MBq. Catalepsy was induced by haloperidol (0.03 mg/kg, intramuscularly) and examined for incidence and duration. Results: ASP5854 dose-dependently increased A2AR occupancy in the striatum and showed long-lasting occupancy even after the reduction of plasma concentration. Haloperidol induced severe catalepsy at 40 min after intramuscular injection. The incidence and duration of cataleptic posture were dose-dependently reduced by ASP5854 at 1 h after oral administration, and the minimum ED50 value was 0.1 mg/kg. Administration of a dose of 0.1 mg/kg yielded a plasma concentration of 97 ± 16.3 ng/mL, which corresponded to 85%–90% of A2AR occupancy. Conclusion: These results showed that ASP5854 antagonized A2AR in the striatum, and the dissociation from A2AR was relatively slow. In addition, more than 85% A2AR occupancy by ASP5854 resulted in an inhibition of haloperidol-induced catalepsy. Thus, such a pharmacodynamic study directly demonstrates both the kinetics of a drug in the brain and the relationship between dose-dependent receptor occupancy and plasma level.

Inhibition of oligopeptide transporter suppress growth of human pancreatic cancer cells

Oligopeptide transporters are abundantly expressed in various types of cancer cells. We here synthesized two novel dipeptides, l-phenylalanyl sarcosine (Phe-Sar) and 4-(4-methoxyphenyl)-l-phenylalanyl sarcosine (Bip(OMe)-Sar), and examined their effect on the growth of human pancreatic cancer AsPC-1 cells, which are known to highly express oligopeptide transporter PEPT1/SLC15A1. Growth of AsPC-1 cells was inhibited by these two peptides and a typical PEPT1/SLC15A1 substrate Gly-Sar. Growth inhibition by Gly-Sar, Phe-Sar and Bip(OMe)-Sar was concentration-dependent with half-maximal inhibitory concentration of 50, 0.91 and 0.55mM, respectively. These peptides also inhibited PEPT1-mediated [(3)H]Gly-Sar uptake with half-maximal inhibitory concentration of 2.6, 0.81 and 0.27mM, respectively. Thus, the rank order of the tumor cell growth inhibition by these three peptides was the same as that of PEPT1-inhibitory activity. Growth of AsPC-1 cells was also inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), which is a typical inhibitor of amino acid transporter system L. The growth inhibition by BCH and Gly-Sar was additive, suggesting that these compounds act at distinct loci. Oligopeptide transporters thus appear to be a promising target for inhibition of pancreatic cancer progression. These results also proposed the idea that oligopeptide transporter is required for growth of AsPC-1 cells.

A PET study following treatment with a pharmacological stressor, FG7142, in conscious rhesus monkeys.

FG7142 is a benzodiazepine partial inverse agonist, which is known as a pharmacological stressor. Several reports demonstrated that FG7142 produced anxiety in humans, non-human primates, and rodents, and impaired working memory in non-human primates and rodents. In this study, we examined the effect of FG7142 on cerebral blood flow and glucose metabolism using positron emission tomography (PET) in conscious rhesus monkeys. Male rhesus monkeys were intramuscularly treated with FG7142 (0.2 or 1.0 mg/kg, n=5, respectively), and regional cerebral blood flow (rCBF) and regional cerebral metabolic rate of glucose (rCMRglc) were measured by PET 20 min and 40 min after treatment, respectively. During PET measurement, physiological parameters and plasma cortisol levels were monitored. FG7142 significantly decreased rCBF in the thalamus and rCMRglc in all brain regions examined in a dose-dependent manner without changes in physiological parameters. FG7142 also significantly increased plasma cortisol levels. The present study may provide an important insight into the understanding of the pathophysiology of anxiety and stress-related disorders in humans, and strongly suggesting that prevention of anxiety or stress is important when measuring conscious brain function.

Total synthesis of bicyclomycin

Abstract Total synthesis of (±)bicyclomycin was achieved in 19 steps starting from diketopyperazine.

Radiosynthesis, biodistribution and imaging of [11C]YM155, a novel survivin suppressant, in a human prostate tumor-xenograft mouse model

INTRODUCTION Sepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [(11)C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts. METHODS Methods utilizing [(11)C]acetyl chloride and [(11)C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [(11)C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [(11)C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS). RESULTS Sufficient quantities of radiopharmaceutical grade [(11)C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16-22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29-60 GBq/μmol (EOS). High uptake levels of radioactivity (%ID/g, mean±SE) were observed in tumor (0.0613±0.0056), kidneys (0.0513±0.0092), liver (0.0368±0.0043) and cecum (0.0623±0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [(11)C]YM155, at 40 min after injection, were 26.5 (±2.9) and 25.6 (±3.6), respectively. CONCLUSION A rapid method for producing a radiopharmaceutical grade [(11)C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [(11)C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.