Design, synthesis and biological evaluation of 2-pyrrolone derivatives as radioprotectors.

It is known that p53 is an important transcription factor and plays a central role in ionizing radiation (IR)-induced DNA damage responses such as cell cycle arrest, DNA repair and apoptosis. We previously reported that regulating p53 protein is an effective strategy for modulating cell fate by reducing the acute side effects of radiation therapy. Herein, we report on the discovery of STK160830 as a new radioprotector from a chemical library at The University of Tokyo and the design, synthesis and biological evaluation of its derivatives. The radioprotective activity of STK160830 itself and its derivatives that were synthesized in this work was evaluated using a leukemia cell line, MOLT-4 cells as a model of normal cells that express the p53 protein in a structure-activity relationships (SAR) study. The experimental results suggest that a direct relationship exists between the inhibitory effect of these STK160830 derivatives on the expression level of p53 and their radioprotective activity and that the suppression of p53 by STK160830 derivatives contribute to protecting MOLT-4 cells from apoptosis that is induced by exposure to radiation.

A Novel RNA Synthesis Inhibitor, STK160830, Has Negligible DNA-Intercalating Activity for Triggering A p53 Response, and Can Inhibit p53-Dependent Apoptosis.

RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.

Evaluation of sodium orthovanadate as a radioprotective agent under total-body irradiation and partial-body irradiation conditions in mice.

PURPOSE: Our previous study indicated that sodium orthovanadate (vanadate), a strong inhibitor of p53, effectively suppressed the lethality from the hematopoietic (HP) and gastrointestinal (GI) syndromes after 12 Gy total-body irradiation (TBI) in mice. This conclusion, however, was inconsistent with the fact that p53 plays a radioprotective role in the intestinal epithelium. The death after TBI of around 12 Gy was attributed to a combined effect of HP and GI syndromes. To verify the effect from prophylactic administration of p53 inhibitor on protection of HP and GI syndromes, in this study, the radioprotective effects from vanadate were investigated in TBI and lower half-body irradiation (partial-body irradiation: PBI) mouse models. METHODS: Female ICR mice were given a single injection of vanadate or vehicle, followed by a lethal dose of TBI or PBI. Radioprotective effects of vanadate against the irradiations were evaluated by analyzing survival rate, body weight, hematopoietic parameters, and histological changes in the bone marrow and intestinal epithelium. RESULTS: TBI-induced HP syndrome was effectively suppressed by vanadate treatment. After TBI, the vanadate-treated mice retained better bone marrow cellularity and showed markedly higher survival rate compared to the vehicle-treated animals. In contrast, vanadate did not relieve loss of intestinal crypts and failed to rescue mice from GI death after PBI. CONCLUSION: Vanadate is a p53 inhibitor that has been shown to be beneficial as a radiation protective agent against HP but was not effective in protecting against acute GI radiation injury.

Protective Effects of p53 Regulatory Agents Against High-LET Radiation-Induced Injury in Mice.

Radiation damage to normal tissues is one of the most serious concerns in radiation therapy, and the tolerance dose of the normal tissues limits the therapeutic dose to the patients. p53 is well known as a transcription factor closely associated with radiation-induced cell death. We recently demonstrated the protective effects of several p53 regulatory agents against low-LET X- or γ-ray-induced damage. Although it was reported that high-LET heavy ion radiation (>85 keV/µm) could cause p53-independent cell death in some cancer cell lines, whether there is any radioprotective effect of the p53 regulatory agents against the high-LET radiation injury in vivo is still unclear. In the present study, we verified the efficacy of these agents on bone marrow and intestinal damages induced by high-LET heavy-ion irradiation in mice. We used a carbon-beam (14 keV/µm) that was shown to induce a p53-dependent effect and an iron-beam (189 keV/µm) that was shown to induce a p53-independent effect in a previous study. Vanadate significantly improved 60-day survival rate in mice treated with total-body carbon-ion (p < 0.0001) or iron-ion (p < 0.05) irradiation, indicating its effective protection of the hematopoietic system from radiation injury after high-LET irradiation over 85 keV/µm. 5CHQ also significantly increased the survival rate after abdominal carbon-ion (p < 0.02), but not iron-ion irradiation, suggesting the moderate relief of the intestinal damage. These results demonstrated the effectiveness of p53 regulators on acute radiation syndrome induced by high-LET radiation.

Image-guided Spine Stabilization for Traumatic Or Osteoporotic Spine Injury: Radiological Accuracy and Neurological Outcome.

Significant progress has been made in image-guided surgery (IGS) over the last few decades. IGS can be effectively applied to spinal instrumentation surgery. In the present study, we focused our attention on the feasibility and safety of image-guided spine stabilization for traumatic or osteoporotic spine injury. The IGS spine fixation with or without minimally invasive surgery (MIS) techniques such as percutaneous screw placement, balloon kyphoplasty (BKP), or vertebroplasty (VP) were accomplished in 80 patients with traumatic or osteoprotic spine injury between 2007 and 2015. The injured vertebral levels included the following: cervical spine, 41; thoracic spine, 22; and lumbar spine, 17. Neurological condition before and after surgery was assessed using the American Spinal Injury Association Impairment Scale (AIS). A total of 419 pedicle, lateral mass, or laminar screws were placed, and 399 screws (95.2%) were found to be placed correctly based on postoperative computed tomography scan. Although 20 screws (4.8%) were found to be unexpectedly placed incorrectly, no neural or vascular complications closely associated with screw placement were encountered. Neurological outcomes appeared to be acceptable or successful based on AIS. The IGS is a promising technique that can improve the accuracy of screw placement and reduce potential injury to critical neurovascular structures. The integration of MIS and IGS has proved feasible and safe in the treatment of traumatic or osteoporotic spine injury, although a thorough knowledge of surgical anatomy, spine biomechanics, and basic technique remain the most essential aspects for a successful surgery.

Hepatitis C virus infection as a risk factor for increased aortic stiffness and cardiovascular events in dialysis patients.

BACKGROUND: Although links have been found between microorganisms and cardiovascular diseases, the role of hepatitis C virus (HCV) infection in the pathogenesis of arteriosclerosis and cardiovascular events is unclear. The primary objective of this research was to examine whether HCV infection is associated with increased aortic stiffness and cardiovascular events in chronic hemodialysis patients. SUBJECTS AND METHODS: A prospective cohort study was conducted in 94 dialysis outpatients from October 2002 to October 2004. Measurements included carotid-femoral pulse wave velocity (PWV), echocardiographic parameters, serum HCV-RNA (positive in 17 patients), and several items of biochemical data. Multiple logistic regression and the Cox proportional hazard model were used to assess independent determinants of high aortic PWV (> or =10.0 m/sec, mean) and cardiovascular events (including cerebral and peripheral vascular events), adjusting for several risk factors and duration of dialysis. RESULTS: The HCV-positive group had higher aortic PWV and lower serum cholesterol levels. Multivariate analysis indicated mean blood pressure, hemoglobin A1c and HCV viremia to be independent determinants of high PWV. During the follow-up period, 13 patients suffered from cardiovascular events. Prevalence of the diseases at baseline, pulse pressure, left ventricular mass index, HCV viremia and aortic PWV were associated with cardiovascular events. The Kaplan-Meier analysis indicated a significant difference in event-free rates between HCV-positive and HCV-negative patients. CONCLUSION: HCV infection is closely associated with increased aortic stiffness and cardiovascular event in dialysis patients.

A proposed mechanism for amitriptyline neurotoxicity based on its detergent nature.

Although amitriptyline has gained attention as a potent local anesthetic, recent animal studies showed that it can cause irreversible neural impairment. We hypothesized that nerve membrane disruption caused by solubilization, a common detergent property, accounted for amitriptyline neurotoxicity. We used a two-phase approach to test our hypothesis. Firstly, we determined (1) the molecular aggregation concentration of amitriptyline, (2) the concentration of amitriptyline that disrupts artificial lipid membranes and (3) the concentration of amitriptyline that causes hemolysis. Secondly, we compared these levels with neurotoxic concentrations determined from assessment in a rat model of spinal anesthesia using changes in cutaneous stimulus threshold (CST). Amitriptyline concentrations that caused molecular aggregation, model membrane disruption and hemolysis were 0.46%, 0.35% and 0.3%, respectively. Animal study showed a significant increase in CST at >or=0.3% of amitriptyline, indicating neurological impairment. Since amitriptyline caused model membrane disruption and hemolysis at the molecular aggregation concentration, solubilization plays a role in the destruction of artificial membranes and erythrocytes. Furthermore, these concentrations are also in good agreement with the minimum concentration causing neurological injury. Therefore, while additional studies, including histopathology, are necessary to clarify this observation, amitriptyline neurotoxicity appears to be associated with its detergent nature.

Analyses of dose-response in radiotherapy for patients with mature T/NK-cell lymphomas according to the WHO classification.

BACKGROUND AND PURPOSE: This study was conducted to analyze the influence of radiotherapy doses and chemotherapy doses and clinical parameters on in-field disease control in order to assess the optimal radiation doses for treatment of mature T/NK-cell lymphomas according to the newly proposed WHO classification. PATIENTS AND METHODS: Subjects consisted of 62 patients with mature T/NK-cell lymphomas treated with radiotherapy at four Japanese institutions between 1983 and 2002. We reevaluated all histopathological specimens of non-Hodgkin's lymphomas (NHL), using the WHO classification. Radiation therapy was usually delivered to the involved field. The majority of patients also received adriamycin-based chemotherapy such as CHOP, modified CHOP, or more intensive chemotherapy. RESULTS: There were no significant differences in radiosensitivity among subtypes of mature T/NK-cell lymphomas, at least between extranodal NK/T-cell lymphomas, nasal type and peripheral T-cell lymphomas, unspecified. There was a radiation dose-response in non-bulky mature T/NK-cell lymphomas, indicating that radiation doses of more than 52 Gy may be required to obtain in-field control. However, it was difficult to obtain local control of bulky T-cell lymphomas, even with high doses of irradiation. CONCLUSIONS: Mature T/NK-cell lymphomas were more radioresistant than B-cell lymphomas such as diffuse large B-cell lymphomas (DLBCL). The chemotherapy including adriamycin did not improve the in-field control of mature T/NK-cell lymphomas. These results were obtained by using non-randomized data and the significance of these results is limited by bias in data. However, our results suggest that the treatment strategy which is usually used for DLBCL, that is, a combined modality of CHOP and around 40 Gy of radiotherapy, may not be sufficiently effective for mature T/NK-cell lymphomas.

Quadruple immunosuppression with basiliximab, tacrolimus, mycophenolate mofetil and prednisone is safe and effective for renal transplantation.

INTRODUCTION: Recent immunosuppression with tacrolimus and mycophenolate mofetil has improved the results of renal transplantation. In this study, we analyzed the effect and safety of basiliximab as an induction therapy. MATERIAL AND METHODS: Forty-nine kidney recipients were given tacrolimus, mycophenolate mofetil and prednisone (non-Bas group), and 31 recipients were given basiliximab as an induction therapy in addition to the triple immunosuppressants (Bas group). Graft function, incidence of acute rejection (AR), findings of protocol graft biopsy and adverse effects were compared. RESULTS: Serum creatinine within 1 yr post-transplant was comparable between the two groups. Incidence of biopsy-proven AR within 6 months post-transplant was less in the Bas group than in the non-Bas group. Borderline change at 3 months post-transplant was less in the Bas group when compared to the non-Bas group. The frequency and severity of tubulitis were higher in the non-Bas group than in the Bas group. The addition of basiliximab did not increase opportunistic infection, but reduced tacrolimus nephrotoxicity. CONCLUSION: The addition of basiliximab to the tacrolimus-based triple immunosuppressive regimen enabled us to reduce the doses of immunosuppressants and tacrolimus nephrotoxicity without increasing early rejection or infection. This regimen is safe and effective for application during the early period after renal transplantation.

Expression and localization of tumour necrosis factor-alpha and its converting enzyme in human abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by chronic aortic wall inflammation and loss of matrix components. Proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) are thought to be involved in this inflammatory process and, therefore, to play an important role in the pathogenesis of human AAA. TNF-alpha-converting enzyme (TACE) has recently been purified and cloned as a disintegrin and metalloproteinase that converts TNF-alpha precursor into its mature form. The aim of the present study was to determine whether TNF-alpha and TACE were expressed and localized in aortic tissues in human AAA. Infrarenal aortic tissues were obtained from AAA patients (n=19) undergoing elective aneurysm reconstruction and from autopsy cases without cardiovascular disorders as normal controls (n=5). Internal thoracic artery samples were also obtained from patients with coronary artery disease undergoing coronary artery bypass grafting to represent biopsied conduit vessels (n=5). The AAA specimens were taken from the mid-portion of the aneurysm and from the longitudinal transition zone between the non-dilated aorta and the proximal aspect of the aneurysm. TNF-alpha and TACE mRNA levels were determined by real-time quantitative reverse transcriptase-PCR. Expression levels of both TNF-alpha mRNA and TACE mRNA were significantly greater in the transition zone than in the mid-portion (both P<0.05). Expression levels of both forms of mRNA were significantly higher in AAA samples than in control aortas or atherosclerotic arteries. There was a significant correlation between the expression of TNF-alpha mRNA with that of TACE mRNA in AAA (r=0.54, P<0.005). Immunostaining was positive for both TNF-alpha and TACE in CD68-positive macrophages in the media and adventitia obtained from the transition zone in AAA, whereas neither TNF-alpha nor TACE was expressed in control vessels. In conclusion, the concomitant activation and localization of TNF-alpha and TACE in the media and adventitia of the transition zone in human AAA underlines the importance of this system in the pathogenesis of this disorder.

Aldosterone synthase (CYP11B2) expression and myocardial fibrosis in the failing human heart.

The pathway of tissue aldosterone production may exist in the heart, and may be an important contributory factor to myocardial fibrosis and cardiac remodelling in the failing heart. CYP11B2 (aldosterone synthase) catalyses the final step of aldosterone production. The aim of the present study was to determine whether CYP11B2 and CYP11B1 (11beta-hydroxylase) are expressed in myocardial tissues, and whether these enzymes contribute to collagen accumulation and myocardial dysfunction in the failing human heart. Endomyocardial tissues were obtained from 23 patients with chronic heart failure (CHF) and 10 controls. CYP11B2 and CYP11B1 mRNA levels were measured by real-time quantitative reverse transcriptase-PCR. The myocardial collagen volume fraction (CVF) was determined by digital planimetry. CYP11B2 mRNA expression was greater in the CHF group than in the controls (P<0.05), while CYP11B1 mRNA was barely expressed in either group. There was a positive correlation between CYP11B2 mRNA levels and CVF (r=0.64, P=0.001). CYP11B2 mRNA was particularly highly expressed in subgroups of CHF patients with a large left ventricular end-systolic diameter (>55 mm) or a low left ventricular ejection fraction (<30%). CYP11B2 mRNA expression and CVF were lower in a CHF subgroup treated with a combination of spironolactone and angiotensin-converting enzyme inhibitors (ACEIs) than in a subgroup not treated with these drugs. In conclusion, this study has shown that increased myocardial expression of CYP11B2 mRNA is associated with increased myocardial fibrosis and with the severity of left ventricular dysfunction in human CHF. In addition, CYP11B2 expression and cardiac fibrosis are found to be decreased in CHF patients on drug therapy comprising spironolactone combined with ACEIs.

Reversible restrictive cardiomyopathy due to light-chain deposition disease.

Systemic light-chain deposition due to plasma cell dyscrasias manifests as a form of restrictive cardiomyopathy with diastolic ventricular dysfunction. Although these manifestations are likely to be cardiac amyloidosis, whether these pathological conditions are reversible after treatment of the underlying plasma cell disorders is unknown. To our knowledge, we describe the first patient with cardiac light-chain deposition due to multiple myeloma in whom echocardiographic and biochemical factors of cardiac function were ameliorated dramatically after remission of this disorder. We emphasize that restrictive cardiomyopathy due to light-chain deposition may be reversible and have a relatively better prognosis after remission of plasma cell dyscrasias.