Junji Uchino

Prevention of radiation-induced pneumonitis by recombinant adenovirus-mediated transferring of soluble TGF-beta type II receptor gene.

To investigate whether radiation-induced pneumonitis in the mouse-irradiated lung could be prevented by recombinant adenovirus-mediated soluble transforming growth factor-beta (TGF-β) type II receptor gene therapy. Radiation fibrosis-prone mice (C57BL/6J) were randomly divided into four groups consisting of a (1) control group (sham-irradiated); (2) radiation (RT)-alone group; (3) RT+AdCMVsTβR group and (4) RT+AdCMVluc group. The RT-alone and sham-irradiated mice were killed at several time points after thoracic irradiation with a single dose of 9 Gy, and then the TGF-β1 concentrations in serum and broncho-alveolar lavage fluid (BALF) were quantified by enzyme-linked immunosorbent assay (ELISA). We used an adenoviral vector expressing a soluble TGF-β type II receptor (AdCMVsTβR), which can bind to TGF-β and then block the TGF-β receptor-mediated signal transduction. The C57BL/6J mice were intraperitoneally (i.p.) injected with either 5 × 108 plaque-forming units of AdCMVsTβR or AdCMVluc, a control adenovirus-expressing luciferase, a week preceding and a week following the X-ray thoracic irradiation. Four weeks after irradiation, the mice were killed and the concentration of TGF-β1 in the serum and BALF were then measured using ELISA and the lung tissue specimens were examined histopathologically. Following thoracic irradiation with a single dose of 9 Gy, radiation-induced TGF-β1 release in the serum reached the first peak concentration at 12 h and then declined. It reached a maximal value at 2 weeks after irradiation. In the BALF, the TGF-β1 concentration was appreciable within the first hour and thereafter declined. It reached a maximal value at 3 days after irradiation. A one-time i.p. injection of AdCMVsTβR 1 week before irradiation could not completely suppress the two peaks of the radiation-induced TGF-β1 increase, whereas an injection a week preceding and a week following thoracic irradiation was able to suppress those two peaks thoroughly. The TGF-β1 was completely suppressed in the AdCMVsTβR-treated mouse serum and BALF; however, no statistical difference was observed in the serum and BALF between the AdCMVluc-infected mice and the control mice at 4 weeks after irradiation (P<0.05). A histopathological examination showed only mild radiation pneumonitis in the irradiated lungs of AdCMVsTβR-treated mice in comparison to the AdCMVluc-infected and RT-alone mice. Our results demonstrated that TGF-β1 plays an important role in radiation pneumonitis, thus suggesting that the adenovirus-mediated overexpression in soluble TGF-β type II receptor gene therapy may be a potentially feasible and effective strategy for the prevention of radiation pneumonitis.

Combination with low-dose gemcitabine and hTERT-promoter-dependent conditionally replicative adenovirus enhances cytotoxicity through their crosstalk mechanisms in pancreatic cancer

To overcome the limited clinical efficacy of conditionally replicative adenoviruses (CRAds), we investigated the effects of combination therapy with gemcitabine (GEM) and the hTERT-promoter-dependent CRAd (hTERT-CRAd), Ad5/3hTERTE1. This combination therapy exhibited enhanced cytotoxic effects on pancreatic cancer both in vitro and in vivo. Furthermore, we revealed that this enhancement effect was due to the multiple bidirectional interactions between hTERT-CRAd and GEM. The GEM-sensitizing effect of E1 expression derived from hTERT-CRAd, and the enhancement effect by GEM on hTERT promoter activity which led to the increase of adenovirus E1 and viral infectivity. This combination therapy may be a promising therapeutic approach for pancreatic cancer.

Infectivity enhanced, hTERT promoter-based conditionally replicative adenoviruses are useful for SCLC treatment

Treatment of advanced small-cell lung cancer (SCLC) remains one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and it also has an extremely poor prognosis. As a result, new therapeutic approaches are needed. Telomere maintenance to the regulation of replicative lifespan strongly implies that alterations in telomere biology play an important role during malignant transformation. Cancers that exhibit high levels of telomerase activity, such as all of the SCLC, were examined in a previous study. In this study, we turned the expression of human telomerase reverse transcriptase (hTERT) by tumors to a therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 (early region 1) is controlled by the hTERT promoter. This virus achieved good levels of viral replication in SCLC cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with a tropism modification of the virus to express the knob domain of Ad3 (serotype 3 adenovirus), and this improved infectivity for cancer cells. Conversely, the hTERT promoter has low activity in normal tissues, and the CRAd caused no damage to normal lung fibroblast cells. Since the telomerase activity is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that the use of hTERT promoter-based CRAds may be a potentially effective strategy for cancer treatment.

Vascular endothelial growth factor promoter-based conditionally replicative adenoviruses for pan-carcinoma application

Treatment of advanced lung cancer is one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis. Thus, new therapeutic approaches are needed. Lung tumor formation depends on angiogenesis in which the vascular endothelial growth factor (VEGF) produced by cancer cells plays a pivotal role. Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth; however, the anticancer effect with this therapy is weakened after the intratumoral vascular network is completed. In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter. This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells. These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer. Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage to normal bronchial epithelial cells. Since tumor-associated angiogenesis via VEGF signalling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment.

Tumor targeting carboxylesterase fused with anti-CEA scFv improve the anticancer effect with a less toxic dose of irinotecan

Irinotecan (CPT-11) is a key drug for the treatment of various cancers. CPT-11 can be considered to be a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. However, CPT-11 may induce severe diarrhea and bone marrow suppression as adverse effects, thus leading to treatment interruption. The tumor-specific activation of CPT-11 is a possible strategy to avoid the severe toxicities by reducing the serum concentration of CPT-11. In this study, we constructed human liver carboxylesterase-2 fused with anticarcinoembryonic antigen (CEA) scFv as a targeting molecule. The recombinant enzyme anchors onto the tumor cell surface CEA, and thus metabolize CPT-11 extracellularly. In addition a secreted tumor-targeted form of carboxylesterase should help prevent the leakage of the enzyme from the site of the tumor into the circulation. This fusion protein showed CPT-11 activation to SN-38 and specific binding to CEA-expressing cells. In combination with CPT-11, the recombinant carboxylesterase protein exerted antiproliferative effects on human cancer cells. This recombinant enzyme is, therefore, a promising new tool in enzyme prodrug therapy for the treatment of carcinoma with CPT-11.

First reported case of hemoglobin lansing in Asia detected by false low oxygen saturation on pulse oximetry

In recent years, the pulse oximeter has become a widely used medical instrument, which facilitates the estimation of arterial oxygen saturation, and it is not invasive compared with arterial blood oxygen saturation measurement by arterial puncture (SaO2). Here, we report the first case in an Asian setting of hemoglobin (Hb) lansing, a type of Hb variant, in a patient who underwent thorough laboratory testing for persistent asymptomatic low pulse oximeter’s estimate of oxygen saturation (SpO2). A 52-year-old woman was referred to our hospital due to a low SpO2 detected by her primary care physician. She had been followed up for cough variant asthma for 5 years, which was characterized by a chronic dry cough and low SpO2. Her nephew had also suffered from asthma, with a history of hospitalization caused by status asthmaticus complicated by low SpO2, but his SpO2 remained low (less than 90 %) even after he recovered from symptoms of asthma, resulting in a prolonged hospital stay. Her physical examination was unremarkable, but SpO2 was 83–86 % using different oximeters and different fingers. Complete blood counts showed: Hb, 13.3 g/dl; hematocrit, 40.2 %; mean corpuscular volume, 87.0 fl; and reticulocyte count, 70,000/lL. There was no evidence of hemolysis with total bilirubin, 0.7 mg/dl and haptoglobin, 34 mg/dl. Arterial blood gas in room air showed partial pressure of oxygen of 97.5 mmHg and SaO2 of 96.1 %. The level of metHb in arterial blood was 0.1 %. The presence of unstable Hbs was suspected, as obvious precipitation was observed on an isopropanol test (Fig. 1a). Hb electrophoresis at an alkaline pH (pH 8.6) on cellulose acetate revealed that the percentage of HbA2 and HbF was 2.3 % (normal 2.0–3.1 %) and 0.5 % (normal 0–1.0 %), respectively, and the presence of an abnormal band in the position of approximate HbF (Fig. 1b). Finally, direct DNA sequence analysis of aand b-globin genes revealed a CAC to CAG mutation at codon 87 of the a2 gene, resulting in histidine to glutamine substitution (Fig. 1c). The same Hb variant first observed in a Hispanic man and his daughter was reported in 2009, and was termed ‘‘Hb lansing’’ [1]. Six patients from four different families observed in African-American, Caucasian, and Mexican have been listed in HbVar, a database of Human Hemoglobin Variants and Thalassemias (http:// globin.bx.psu.edu/hbvar/menu.html), therefore, our current patient represents the first reported case in Asian. The patient’s nephew may have the same Hb variant, and we proposed further analysis of her family’s Hb, but this was not accepted. More than 1,000 variant Hbs have been identified, and a small proportion of them are associated with abnormal SpO2 readings [2]. OxyHb and deoxyHb show unique absorbance at 660 and 940 nm, respectively, and the pulse oximeter determines SpO2 by measuring a ratio of pulsatile light transmission through a cutaneous vascular bed at the two wavelengths [3]. Since the pulse oximeter uses only two wavelengths for measuring oxyHb and deoxyHb, it is impossible to determine arterial oxygen saturation K. Ishitsuka (&) K. Tamura Division of Medical Oncology, Hematology and Infectious Disease, Fukuoka University School of Medicine, 7-45-1 Nanakuma Jonan, Fukuoka 814-0180, Japan e-mail: kenjiishitsuka@fukuoka-u.ac.jp

Efficacy of aprepitant in patients with advanced or recurrent lung cancer receiving moderately emetogenic chemotherapy

AIMS AND BACKGROUND To evaluate the efficacy of a combination of aprepitant and conventional antiemetic therapy in patients with advanced or recurrent lung cancer receiving moderately emetogenic chemotherapy (MEC). METHODS Patients with advanced or recurrent lung cancer who were treated with MEC regimens at the Department of Respiratory Medicine, Fukuoka University Hospital, were included and classified into the following groups: control group (treatment: 5-HT3 receptor antagonists + dexamethasone) and aprepitant group (treatment: 5-HT3 receptor antagonists + dexamethasone + aprepitant). The presence or absence of chemotherapy-induced nausea and vomiting (CINV) was evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.0; patients with grade 1 or above were considered positive for CINV. Food intake per day, completion of planned chemotherapy, and progression-free survival (PFS) achieved by chemotherapy were investigated. RESULTS The complete suppression rate of nausea in the aprepitant group was significantly higher than that in the control group (p = 0.0043). Throughout the study, the food intake in the aprepitant group was greater than that in the control group, with the rate being significantly higher, in particular, on day 5 (p = 0.003). The completion rate of planned chemotherapy was also higher in the aprepitant group (p = 0.042). PFS did not differ significantly, but tended to be improved in the aprepitant group. CONCLUSIONS The aprepitant group showed significantly higher complete suppression of nausea, food intake on day 5, and completion of planned chemotherapy than the control group.

Anamorelin (ONO-7643) for the treatment of patients with non–small cell lung cancer and cachexia: Results from a randomized, double-blind, placebo-controlled, multicenter study of Japanese patients (ONO-7643-04)

Cachexia, described as weight loss (mainly in lean body mass [LBM]) and anorexia, is common in patients with advanced cancer. This study examined the efficacy and safety of anamorelin (ONO‐7643), a novel selective ghrelin receptor agonist, in Japanese cancer patients with cachexia.

Critical role of tumor necrosis factor receptor 1 in the pathogenesis of pulmonary emphysema in mice

COPD is a major cause of chronic morbidity and mortality throughout the world. Although tumor necrosis factor-α (TNF-α) has a critical role in the development of COPD, the role of different TNF receptors (TNFRs) in pulmonary emphysema has not been resolved. We aimed to clarify the role of TNFRs in the development of pulmonary emphysema. TNF-α transgenic mice, a murine model of COPD in which the mice spontaneously develop emphysema with a large increase in lung volume and pulmonary hypertension, were crossed with either TNFR1-deficient mice or TNFR2-deficient mice. After 6 months, the gross appearance of the lung, lung histology, and pulmonary and cardiac physiology were determined. In addition, the relationship between apoptosis and emphysema was investigated. Pulmonary emphysema-like changes disappeared with deletion of TNFR1. However, slight improvements were attained with deletion of TNFR2. Apoptotic cells in the interstitium of the lung were observed in TNF-α transgenic mice. The apoptotic signals through TNFR1 appear critical for the pathogenesis of pulmonary emphysema. In contrast, the inflammatory process has a less important role for the development of emphysema.

Chronic Pseudomonas aeruginosa infection-induced chronic bronchitis and emphysematous changes in CCSP-deficient mice.

The club cell secretory protein (CCSP) is a regulator of lung inflammation following acute respiratory infection or lung injury. Recently, the relationship between CCSP and COPD has been reported. Since COPD results from an abnormal inflammatory response, we hypothesized that CCSP could have a protective role against chronic inflammation-induced lung damage. To address this issue, the pathophysiology of chronic lung inflammation induced by Pseudomonas aeruginosa in CCSP-deficient mice was determined. A tube of 5 mm in length was soaked in a fluid containing P. aeruginosa (PAO01 strain) for 1 week and inserted into the trachea of CCSP-deficient mice. One week later, P. aeruginosa was administered into the trachea. Five weeks after insertion of tube, the mice were sacrificed. Bronchoalveolar lavage fluids were collected to determine the bacterial growth, and the lung histology and physiology were also examined. P. aeruginosa was continuously detected in bronchoalveolar lavage fluids during the study. Neutrophils were increased in the bronchoalveolar lavage fluids from the CCSP-deficient mice in comparison to wild-type mice. A histological study demonstrated chronic inflammation around bronchus, serious bronchial stenosis, and alveolar enlargement in the CCSP-deficient mice. The lung physiology study demonstrated an increase in the lung compliance of the CCSP-deficient mice. Chronic P. aeruginosa inflammation resulted in chronic bronchitis and emphysematous changes in the CCSP-deficient mice. CCSP could play an important role in protecting the host from the chronic inflammation-induced lung damage.