[A Case of Venous Malformation that Occurred in the Retroperitoneum].

A 70-year-old man complaining of pain in his right leg presented to the Department of Orthopedics in our hospital. X-ray findings revealed calcifications around the left kidney. He was referred to our department for further examination. Computed tomography revealed a tumor 3 cm in diameter with calcifications and an obscure border that was located on the caudal side of the pancreas, anterior to the left iliopsoas muscle and at the left side of the aorta. Magnetic resonance imaging showed that the tumor had comparatively low intensity in diffusion-weighted images and the cell density was not high. The contrast of the tumor by enhanced computed tomography was weak, and we had difficulty judging whether the tumor was benign or malignant. Each tumor marker, immunity factor, and hormone-like catecholamine were within the normal range. We considered the retroperitoneal tumor with calcifications as Castleman disease or tumor of nerve origin. It is believed that most retroperitoneal tumors are malignant. We performed laparoscopic surgery to resect the retroperitoneal tumor. Histopathological diagnosis was a primary retroperitoneal venous malformation. Vascular malformation derived from the retroperitoneum is rare. Furthermore, very few cases of venous malformation in the retroperitoneum have been reported.

Effective syntheses of 2',4'-BNANC monomers bearing adenine, guanine, thymine, and 5-methylcytosine, and the properties of oligonucleotides fully modified with 2',4'-BNANC.

We efficiently synthesized 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNANC) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2'-O-substituted 5-methyluridines to evaluate the effects of 2'-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2',4'-BNANC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2',4'-BNANC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2',4'-BNANC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2',4'-BNANC monomers in both or either strands were found to possess excellent thermal stabilities.

Quantitative evaluation of the improvement in the pharmacokinetics of a nucleic acid drug delivery system by dynamic PET imaging with (18)F-incorporated oligodeoxynucleotides.

Recently, we demonstrated the utility of positron emission tomography (PET) imaging-based pharmacokinetic evaluation studies for preclinical experiments and microdose clinical trials, mainly focused on low molecular weight compounds. In order to investigate the pharmacokinetics of nucleic acid drugs and their drug delivery systems (DDSs) in vivo by using PET imaging, we developed a novel and efficient method for radiolabeling oligodeoxynucleotides with the positron-emitting radionuclide (18)F (stoichiometry-focused Huisgen-type (18)F labeling). By using this method, we succeeded in synthesizing a variety of (18)F-labeled oligodeoxynucleotides with not only phosphodiesters (PO) in natural forms, but also phosphorothioate (PS) and bridged nucleic acid (BNA) in artificial forms, and then performed PET studies and radioactive metabolite analyses of these (18)F-labeled oligodeoxynucleotides. The tissue-distribution and dynamic changes in radioactivity showed significantly different profiles between these antisense oligodeoxynucleotides. The radioactivity of (18)F-labeled PO-DNA and PO-BNA rapidly accumulated in the kidneys and liver and then moved to the renal medulla, ureter, bladder, and intestine. However, the radioactivity of (18)F-labeled PS-DNA and PS-BNA, possessing PS backbone structures, was retained in the blood for relatively long periods and then gradually accumulated in the liver and kidneys. The metabolite analysis showed that (18)F-labeled PO-DNA rapidly degraded by 5min and (18)F-labeled PO-BNA gradually degraded over time by 60min. Conversely, (18)F-labeled PS-DNA and PS-BNA were shown to be much more stable. To demonstrate the usefulness of the PET imaging technique for evaluating the improved targeting potential of the DDS, we designed and synthesized a cholesterol-modified oligodeoxynucleotide, that we developed as an antisense nucleic acid drug against proprotein convertase subtilisin/kexin type 9 (PCSK9) for hypercholesterolemia therapy, and evaluated its pharmacokinetics using PET imaging. As expected, the (18)F-labeled cholesterol-modified PS-BNA-type oligodeoxynucleotide showed much higher and more rapid accumulation in the delivery target organ, that is, the liver, which encourages us to develop this drug. These results suggest that dynamic PET studies using (18)F-incorporated oligodeoxynucleotide synthesized by stoichiometry-focused Huisgen-type labeling is useful for quantitative pharmacokinetic evaluation of nucleic acid drugs and their delivery systems.

Target gene knockdown by 2',4'-BNA/LNA antisense oligonucleotides in zebrafish.

Gene knockdowns using oligonucleotide-based approaches are useful for studying gene function in both in vitro cell culture systems and in vivo animal models. We evaluated the efficacy of 2',4'-bridged nucleic acids (BNA)-modified antisense oligonucleotides (AONs) for gene knockdown in zebrafish. We used the tcf7l1a gene as a model for testing the knockdown efficacy of 2',4'-BNA AONs and examined how the target sites/affinity and RNase H induction activity of 2',4'-BNA AONs affect knockdown efficacy. We found that tcf7l1a gene function was knocked down by 2',4'-BNA AONs that target the start codon and induce RNase H activity. Although nonspecific p53-mediated developmental defects were observed at higher doses, the effective dose of the 2',4'-BNA AONs for tcf7l1a is much lower than that of morpholino oligonucleotides. Our data thus show a potential application for 2',4'-BNA AONs in the downregulation of specific genes in zebrafish.

[Long-term survival of metastatic clear cell adenocarcinoma of the female urethra by multidisciplinary treatment: a case report].

We report a case of clear cell adenocarcinoma of the female urethra. A 57-year-old woman presented with complaint of gross hematuria. Abdominal ultrasonography, cystourethroscopy, computed tomography (CT) and magnetic resonance imaging (MRI) revealed the urethral tumor was invasive to bladder neck. Clinical stage was determined as cT3N1M0, then anterior pelvic exenteration and ileal conduit formation were performed. The pathological diagnosis was clear cell adenocarcinoma of urethra and the stage was pT3N1. The patient received TS-1 and cisplatin for postoperative recurrence, but she died from multiple lung metastasis 54 months after the operation. Clear cell adenocarcinoma of the female urethra is rare case in the Japanese literatures. Pathogenesis and management of this rare condition are discussed.

Superior Silencing by 2',4'-BNA(NC)-Based Short Antisense Oligonucleotides Compared to 2',4'-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors.

The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2',4'-BNA(NC) antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2',4'-BNA(NC) was directly compared to its conventional bridged (or locked) nucleic acid (2',4'-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between 2',4'-BNA(NC)-based antisense oligonucleotides and the target mRNA surpassed those of 2',4'-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2',4'-BNA/LNA-based counterpart, the corresponding 2',4'-BNA(NC)-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2',4'-BNA(NC)-based 20-mer AON exhibited the highest affinity but the worst IC(50) value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2',4'-BNA(NC) may be a better alternative to conventional 2',4'-BNA/LNA, even for "short" antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.

Design and concise synthesis of a novel type of green fluorescent protein chromophore analogue.

A small molecular model compound for the green fluorescent protein chromophore was readily synthesized by a novel condensation reaction of (thio)imidate with imino-ester via an aziridine intermediate. This compound showed fluorescence in the solid and frozen solution states but not in the solution state. Its fluorescent property was successfully applied in the detection of dsDNA.

Synthesis and evaluation of novel caged DNA alkylating agents bearing 3,4-epoxypiperidine structure.

Previously, we reported that the 3,4-epoxypiperidine structure, whose design was based on the active site of DNA alkylating antitumor antibiotics, azinomycins A and B, possesses prominent DNA cleavage activity. In this report, novel caged DNA alkylating agents, which were designed to be activated by UV irradiation, were synthesized by the introduction of four photo-labile protecting groups to a 3,4-epoxypiperidine derivative. The DNA cleavage activity and cytotoxicity of the caged DNA alkylating agents were examined under UV irradiation. Four caged DNA alkylating agents showed various degrees of bioactivity depending on the photosensitivity of the protecting groups.

[Rupture of renal artery aneurysm into the renal pelvis].

We report a case of ruptured renal artery aneurysm into the renal pelvis. A 48-year-old woman presented with complaints of gross hematuria and right back pain. Abdominal ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI) revealed the aneurysm, which was 5 x 5 cm in diameter. Enhansed CT revealed blood flow from the renal artery aneurysm into the renal pelvis. Radical nephrectomy was performed. Rupture of renal artery aneurysm into the renal pelvis is the 3rd case in the Japanese literatures. Pathogenesis and management of this rare condition are discussed.

Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice.

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2',4'-BNA (also called as locked nucleic acid (LNA)) and 2',4'-BNA(NC) chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2',4'-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2',4'-BNA-AON, 2',4'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

Cleavage of oligonucleotides containing a P3'→N5' phosphoramidate linkage mediated by single-stranded oligonucleotide templates.

Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3'→N5' phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.

Characteristics of aggressive variants in T1a renal cell carcinoma.

PURPOSE: To explore factors associated with metastasis and prognosis in T1a renal cell carcinoma (RCC). METHODS: We retrospectively reviewed 451 cases of sporadic T1aRCC among 1,060 patients admitted to the Department of Urology at Hamamatsu University Hospital and affiliated hospitals between 1978 and 2007. Clinicopathological factors were analyzed for metastatic and prognostic risks. RESULTS: We identified 32 RCC patients with metastatic disease, 22 with synchronous and 10 with metachronous metastatic RCC. Patients with metastatic disease had a significantly higher incidence of symptomatic cancer, as well as greater tumor size, C-reactive protein (CRP) level, sarcomatoid component ratio, histological grade 3 and microvascular invasion than those without metastasis. Among the 32 patients with metastasis, there is no significant difference in clinicopathological factors. The most common site of metastasis was bone. Among patients with metastatic T1aRCC, findings at diagnosis of a symptomatic cancer, CRP level of 0.4 mg/dL or more, tumor size of 3.0 cm or greater, histological grade 3, a sarcomatoid component and microvascular invasion appeared to be significant and independent risk factors. Significant independent risk factors with metachronous metastatic RCC were a symptomatic cancer and a sarcomatoid component at diagnosis. A CRP level of 0.4 mg/dL or more was also an independent prognostic factor for overall survival. CONCLUSION: RCC patients with findings at diagnosis of a symptomatic cancer, a sarcomatoid component and CRP level of 0.4 mg/dL or more require intensive follow-up.

2'-O,4'-C-methyleneoxymethylene bridged nucleic acids (2',4'-BNA(COC)).

The synthesis of 2'-O,4'-C-methyleneoxymethylene bridged nucleoside (2',4'-BNA(COC)) phosphoramidites and oligonucleotides containing 2',4'-BNA(COC) are described. 2',4'-BNA(COC) phosphoramidites bearing natural nucleobases, such as thymine, cytosine, 5-methylcytosine, adenine, and guanine were synthesized. Moreover, fully or partially 2',4'-BNA(COC)-modified oligonucleotides can be prepared by using a standard protocol except for a prolonged coupling time on an automated DNA synthesizer.

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH.

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition.

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.

Stoichiometry-focused (18)F-labeling of alkyne-substituted oligodeoxynucleotides using azido([(18)F]fluoromethyl)benzenes by Cu-catalyzed Huisgen reaction.

A novel method for (18)F-radiolabeling of oligodeoxynucleotides (ODNs) by a Cu-catalyzed Huisgen reaction has been developed by using the lowest possible amount of the precursor biomolecule for the realization of stoichiometry-oriented PET (positron emission tomography) chemistry. Under the optimized cyclization conditions of p- or m-azido([(18)F]fluoromethyl)benzene and alkyne-substituted ODN (20nmol) at 40°C for 15min in the presence of CuSO(4), TBTA [tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine], and sodium ascorbate (2:1:2), the synthesis of (18)F-labeled ODNs with sufficiently high radioactivities of 2.1-2.5GBq and specific radioactivities of 1800-2400GBq/µmol have been accomplished for use in animal and human PET studies.

Arteriosclerosis related factors had no clinical significant correlation with resistive index in symptomatic benign prostatic hyperplasia.

OBJECTIVE: To investigate whether the resistive index (RI) in symptomatic benign prostatic hyperplasia (BPH) could be used as a surrogate index of the severity of lower urinary tract symptoms (LUTS) due to BPH, and whether arteriosclerosis-related factors were associated with the RI in LUTS due to BPH. METHODS: From January 2005 to April 2008, a total of 625 men with LUTS due to BPH were prospectively enrolled. Patients with heart failure, liver cirrhosis, prostatic cancer, neurogenic bladder, acute prostatitis, acute urinary retention, urethral stenosis, history of transurethral resection or any drug treatment for BPH, or currently under drug treatment for type 2 diabetes mellitus or dyslipidemia were excluded. Variables analyzed included estimated smoking status, blood pressure, body mass index (BMI), serum fasting glucose (FBS), lipid profile (low-density lipoprotein-cholesterol, high density lipoprotein-cholesterol and triglyceride), serum prostate-specific antigen, International Prostatic Symptom Score (IPSS), quality of life score, maximum urinary flow rate (Q(max.)), and postvoid residual urine volume (PVR). We also measured total prostate volume, transition zone (TZ) index, and RI using transrectal ultrasonography. Correlations among parameters were statistically examined. RESULTS: RI was significantly correlated with IPSS, Q(max.), and PVR, but not with blood pressure, BMI, or FBS. On multiple regression analysis, RI was a significant independent variable of IPSS, TZ index, and PVR. CONCLUSIONS: These findings suggest that RI might represent a surrogate index of the severity of LUTS due to BPH, and that RI might have no clinically significant relationship with arteriosclerosis-related factors.

A novel bridged nucleoside bearing a conformationally switchable sugar moiety in response to redox changes.

A novel nucleoside analog with a disulfide bridge structure at the sugar moiety, which shows redox-responsive reversibility of the sugar conformation due to formation and scission of the disulfide bond, was designed and synthesized.

Resistive index as risk factor for acute urinary retention in patients with benign prostatic hyperplasia.

OBJECTIVES: To examine the usefulness of several parameters obtained by transrectal ultrasonography in predicting acute urinary retention (AUR). METHODS: The present study consecutively enrolled 1962 men with a complaint of lower urinary tract symptoms. Of these men, 245 were found to have AUR on examination at our clinic. We assessed the International Prostate Symptom Score (IPSS), maximal urinary flow rate, and postvoid residual urine volume and measured the total prostate volume, transition zone index (TZI), and resistive index (RI) using transrectal ultrasonography. To compare the usefulness of these indexes for predicting AUR, we calculated the area under the receiver operating characteristic curve for each index and for age. RESULTS: In patients without AUR, age, prostate-specific antigen level, IPSS, maximal urinary flow rate, and postvoid residual urine volume were significantly correlated with both the TZI and the RI (P < .001). Multiple regression analysis demonstrated that age, maximal urinary flow rate, postvoid residual urine volume, and TZI were significant independent determinants of the RI (P < .001). Patients with AUR were, on average, older and had an elevated prostate-specific antigen level, increased IPSS, and greater TZI and RI than patients without AUR (P < .001). The area under the receiver operating characteristic curve was 0.640 (95% confidence interval [CI] 0.618-0.662) for age, 0.674 (95% CI 0.653-0.695) for prostate-specific antigen level, 0.787 (95% CI 0.768-0.805) for total prostate volume, 0.821 (95% CI 0.803-0.838) for IPSS, 0.860 (95% CI 0.844-0.875) for TZI, and 0.867 (95% CI 0.851-0.882) for RI. CONCLUSIONS: The RI and TZI obtained using transrectal ultrasonography correlated with the incidence of AUR and are useful predictors of AUR in patients with benign prostatic hyperplasia.