Koki Hasegawa

64Cu-DOTA-Trastuzumab PET Imaging in Patients with HER2-Positive Breast Cancer

The purpose of this study was to determine the safety, distribution, internal dosimetry, and initial human epidermal growth factor receptor 2 (HER2)–positive tumor images of 64Cu-DOTA-trastuzumab in humans. Methods: PET was performed on 6 patients with primary or metastatic HER2-positive breast cancer at 1, 24, and 48 h after injection of approximately 130 MBq of the probe 64Cu-DOTA-trastuzumab. Radioactivity data were collected from the blood, urine, and normal-tissue samples of these 6 patients, and the multiorgan biodistribution and internal dosimetry of the probe were evaluated. Safety data were collected for all the patients after the administration of 64Cu-DOTA-trastuzumab and during the 1-wk follow-up period. Results: According to our results, the best timing for the assessment of 64Cu-DOTA-trastuzumab uptake by the tumor was 48 h after injection. Radiation exposure during 64Cu-DOTA-trastuzumab PET was equivalent to that during conventional 18F-FDG PET. The radioactivity in the blood was high, but uptake of 64Cu-DOTA-trastuzumab in normal tissues was low. In 2 patients, 64Cu-DOTA-trastuzumab PET showed brain metastases, indicative of blood–brain barrier disruptions. In 3 patients, 64Cu-DOTA-trastuzumab PET imaging also revealed primary breast tumors at the lesion sites initially identified by CT. Conclusion: The findings of this study indicated that 64Cu-DOTA-trastuzumab PET is feasible for the identification of HER2-positive lesions in patients with primary and metastatic breast cancer. The dosimetry and pharmacologic safety results were acceptable at the dose required for adequate PET imaging.

Molecular imaging analysis of intestinal insulin absorption boosted by cell-penetrating peptides by using positron emission tomography

Molecular imaging technique by use of positron emission tomography (PET) is a noninvasive tool that allows one to quantitatively analyze the function of endogenous molecules and the pharmacokinetics of therapeutic agents in vivo. This technique is expected to be useful for evaluating the effectiveness of diverse drug delivery systems. We demonstrated previously that intestinal insulin absorption is increased significantly by coadministration of cell-penetrating peptides (CPPs), which are taken up effectively by several cells. However, the distribution behavior of insulin whose absorption is increased by CPPs is not clear. We used PET imaging and quantitatively analyzed the intestinal absorption and subsequent distribution of insulin and the effect of CPPs on its absorption and distribution. An unlabeled insulin solution containing tracer insulin, (68)Ga-DOTA-insulin, was administered with or without CPPs into a rat ileal closed loop. PET imaging showed that the CPPs, particularly D-R8 and L-penetratin, significantly increased the (68)Ga-DOTA-insulin level in the liver, kidney, and circulation. After absorption from the intestine, the (68)Ga-DOTA-insulin passed rapidly through the liver and accumulated in the kidney. The increase in the hepatic and renal distribution of (68)Ga-DOTA-insulin by each CPP coadministration was similar manner as that in intestinal absorption, suggesting that the increased accumulation of insulin in the liver and kidney induced by coadministration of CPPs was associated with the increased intestinal absorption of insulin. This is the first study to show that PET imaging enables one to quantitatively analyze the distribution behavior of intestinally absorbed insulin in several organs. This imaging methodology is likely to be useful for developing effective drug delivery systems targeted to specific organs.

Insulinoma-associated protein 1 is a crucial regulator of neuroendocrine differentiation in lung cancer

Insulinoma-associated protein 1 (INSM1) is expressed exclusively in embryonic developing neuroendocrine (NE) tissues. INSM1 gene expression is specific for small-cell lung cancer (SCLC), along with achaete-scute homolog-like 1 (ASCL1) and several NE molecules, such as chromogranin A, synaptophysin, and neural cell adhesion molecule 1. However, the underlying biological role of INSM1 in lung cancer remains largely unknown. We first showed that surgically resected SCLC samples specifically expressed INSM1. Forced expression of the INSM1 gene in adenocarcinoma cell lines (H358 and H1975) induced the expression of ASCL1, brain-2 (BRN2), chromogranin A, synaptophysin, and neural cell adhesion molecule 1; in contrast, knockdown of the INSM1 gene by siRNA in SCLC (H69 and H889) decreased their expression. However, forced/knockdown expression of ASCL1 and BRN2 did not affect INSM1 expression. A chromatin immunoprecipitation study revealed that INSM1 bound to the promoter region of the ASCL1 gene. A xenotransplantation assay using tet-on INSM1 gene-transfected adenocarcinoma cell lines demonstrated that INSM1 induced NE differentiation and growth inhibition. Furthermore, we found that INSM1 was not expressed in non-small-cell lung cancer and some SCLC cell lines expressing Notch1-Hes1. By forced/knockdown expression of Notch1 or Hes1 genes, we revealed that Notch1-Hes1 signaling suppressed INSM1, as well as ASCL1 and BRN2. INSM1, expressed exclusively in SCLC, is a crucial regulator of NE differentiation in SCLCs, and is regulated by the Notch1-Hes1 signaling pathway.

Notch1 controls cell invasion and metastasis in small cell lung carcinoma cell lines

INTRODUCTION Notch signaling plays a key role in a wide variety of human neoplasms, and it can be either oncogenic or anti-proliferative. Moreover, Notch function in regulating cancer is unpredictable, and its outcome is strictly context-dependent. AIM To study the role of Notch1 signaling in human small cell lung carcinoma (SCLC) and its effect on cell invasion and metastasis. MATERIALS AND METHODS We used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 SCLC cells. On the other hand, we up-regulated Notch1 in H69 and H1688 SCLC cells through transfection with venus Notch1 intracellular domain (v.NICD) plasmid. In addition, H69 cells with v.NICD were xenotransplanted into immune-compromised Rag2(-/-) Jak3(-/-) mice, for analysis of ex vivo tumor epithelial mesenchymal transition (EMT) phenotype and for detection of metastatic cancer cells in the lung tissues. Moreover, we examined the metastatic ability for H69AR and SBC3 cells transfected with siRNA against Notch1, compared to their subsequent controls, by use of tail vein xenograft mouse models. RESULTS Notch1 controls cell adhesion and EMT. Overexpression of Notch1 in SCLC switched off EMT, cell motility and cell metastatic potential. CONCLUSION Our results demonstrate that activation of Notch1 signaling pathway may represent a new strategy for treating human SCLC.

Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

BACKGROUND Sensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte-endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. METHODS AND RESULTS A (64)Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody ((64)Cu-DOTA-anti-P-selectin mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by (64)Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3MBq of (64)Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180min PET scan, autoradiography and biodistribution of (64)Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. (64)Cu-DOTA-anti-P-selectin mAb accumulated selectively in aortic atherosclerotic plaques and was detectable by PET and CT fusion imaging in Ldlr-/- mice. CONCLUSIONS P-selectin is a candidate target molecule for early-phase detection by PET and CT fusion imaging of atherosclerotic plaques.

Notch1 controls cell chemoresistance in small cell lung carcinoma cells

Small cell lung carcinoma (SCLC) is characterized by a high rate of relapse and failure of chemotherapy because of the emergence of drug resistant cells. Notch signaling controls carcinogenesis in several human malignancies and could be involved in the resistance of cells to several chemotherapeutic agents. Herein, we analyzed the role of Notch1 signaling in the resistance of human SCLC cells to doxorubicin.

Positron emission tomography study on pancreatic somatostatin receptors in normal and diabetic rats with 68Ga-DOTA-octreotide: A potential PET tracer for beta cell mass measurement

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and the loss or dysfunction of pancreatic beta cells has been reported before the appearance of clinical symptoms and hyperglycemia. To evaluate beta cell mass (BCM) for improving the detection and treatment of DM at earlier stages, we focused on somatostatin receptors that are highly expressed in the pancreatic beta cells, and developed a positron emission tomography (PET) probe derived from octreotide, a metabolically stable somatostatin analog. Octreotide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating agent, and labeled with (68)Gallium ((68)Ga). After intravenous injection of (68)Ga-DOTA-octreotide, a 90-min emission scan of the abdomen was performed in normal and DM model rats. The PET studies showed that (68)Ga-DOTA-octreotide radioactivity was highly accumulated in the pancreas of normal rats and that the pancreatic accumulation was significantly reduced in the rats administered with an excess amount of unlabeled octreotide or after treatment with streptozotocin, which was used for the chemical induction of DM in rats. These results were in good agreement with the ex vivo biodistribution data. These results indicated that the pancreatic accumulation of (68)Ga-DOTA-octreotide represented specific binding to the somatostatin receptors and reflected BCM. Therefore, PET imaging with (68)Ga-DOTA-octreotide could be a potential tool for evaluating BCM.

Visualization and Quantitative Assessment of the Brain Distribution of Insulin through Nose-to-Brain Delivery Based on the Cell-Penetrating Peptide Noncovalent Strategy

Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive (64)Cu-NODAGA-insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of (64)Cu-NODAGA-insulin with l- or d-penetratin. The brain accumulation of (64)Cu-NODAGA-insulin with penetratin was confirmed by ELISA using unlabeled insulin in which intact insulin was delivered to the brain after intranasal coadministration with l- or d-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with l-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure.

Identification of nuclear phosphoproteins as novel tobacco markers in mouse lung tissue following short-term exposure to tobacco smoke.

Smoking is a risk factor for lung diseases, including chronic obstructive pulmonary disease and lung cancer. However, the molecular mechanisms mediating the progression of these diseases remain unclear. Therefore, we sought to identify signaling pathways activated by tobacco‐smoke exposure, by analyzing nuclear phosphoprotein expression using phosphoproteomic analysis of lung tissue from mice exposed to tobacco smoke. Sixteen mice were exposed to tobacco smoke for 1 or 7 days, and the expression of phosphorylated peptides was analyzed by mass spectrometry. A total of 253 phosphoproteins were identified, including FACT complex subunit SPT16 in the 1‐day exposure group, keratin type 1 cytoskeletal 18 (K18), and adipocyte fatty acid‐binding protein, in the 7‐day exposure group, and peroxiredoxin‐1 (OSF3) and spectrin β chain brain 1 (SPTBN1), in both groups. Semi‐quantitative analysis of the identified phosphoproteins revealed that 33 proteins were significantly differentially expressed between the control and exposed groups. The identified phosphoproteins were classified according to their biological functions. We found that the identified proteins were related to inflammation, regeneration, repair, proliferation, differentiation, morphogenesis, and response to stress and nicotine. In conclusion, we identified proteins, including OSF3 and SPTBN1, as candidate tobacco smoke‐exposure markers; our results provide insights into the mechanisms of tobacco smoke‐induced diseases.

Comparison of 64Cu and 68Ga for Molecular Imaging of Atherosclerosisusing the Apolipoprotein A-I Mimetic Peptide FAMP

Background: Molecular imaging for detection of the atherosclerotic plaque burden has been highlighted as a modality for the diagnosis of atherosclerosis. We recently developed a novel and noninvasive positron emission tomography (PET) that was functionalized with an apolipoprotein (Apo) A-I mimetic peptide [known as Fukuoka University Apo A-I mimetic peptide (FAMP)] radiolabeled with gallium-68 (68Ga) - 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) to specifically image the status of atherosclerotic plaque in myocardial infarctionprone Watanabe heritable hyperlipidemic rabbits (WHHL-MI). Methods and Results: To achieve more sensitive molecular imaging, FAMP was modified with 4, 11 - bis (carboxymethyl) - 1, 4, 8, 11 - tetraazabicyclo (6.6.2) hexadecane (CB-TE2A) and radiolabeled with copper-64 (64Cu) for PET, and the ability of 64Cu-TE2A-FAMP to image plaque was compared with that of 68Ga-DOTA-FAMP. Japanese white normal (JW) and WHHL-MI rabbits were intravenously injected with 64Cu-CB-TE2A-FAMP or 68Ga- DOTA-FAMP, and subjected to continuous PET (25-30 MBq). Interestingly, 64Cu-CB-TE2A-FAMP was not taken up by atherosclerotic lesions in the aorta of WHHL-MI, whereas 68Ga-DOTA-FAMP was dramatically illuminated in the aorta of WHHL-MI. Moreover, 64Cu-CB-TE2A-FAMP was rapidly decomposed and 64Cu was excreted to the intestine, liver or urinary bladder in both JW and WHHL-MI rabbits. Conclusions: These results demonstrated that FAMP may be a target molecule for atherosclerotic molecular imaging with 68Ga-DOTA, but not with 64Cu-CB-TE2A. The selection of a suitable radio-nuclide and chelator might be important for HDL functioning imaging.