Imaging of Hydroxyl-Radical Generation Using Dynamic Nuclear Polarization-Magnetic Resonance Imaging and a Spin-Trapping Agent.

Reactive oxygen species (ROS) play an important role in cell metabolism, but they can cause oxidative damage to biomolecules. Among ROS, the hydroxyl radical (·OH) is one of the most reactive molecules in biological systems because of its high reaction rate constant. Therefore, imaging of ·OH could be useful for evaluation of the redox mechanism and diagnosis of oxidative diseases. In vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) is a noninvasive imaging method to obtain spatiotemporal information about free radicals with MRI anatomical resolution. In this study, we investigated the visualization of hydroxyl radicals generated from the Fenton reaction by combining DNP-MRI with a spin-trapping agent (DMPO: 5,5-dimethyl-1-pyrroline N-oxide) for ·OH. Additionally, we demonstrated the radical-scavenging effect using four thiol-related reagents by DNP-MRI. We demonstrated that DNP enhancement could be induced by the DMPO-OH radical using the DNP-MRI/spin-trapping method and visualized ·OH generation for the first time. Maximum DNP enhancement was observed at an electron paramagnetic resonance irradiation frequency of 474.5 MHz. Furthermore, the radical-scavenging effect was simultaneously evaluated by the decrease in the DNP image value of DMPO-OH. An advantage of our methods is that they simultaneously investigate compound activity and the radical-scavenging effect.

Effects of propiverine hydrochloride, an anticholinergic agent, on urethral continence mechanisms and plasma catecholamine concentration in rats.

INTRODUCTION AND HYPOTHESIS: Anticholinergics are used to treat overactive bladder. Anticholinergic agents such as propiverine hydrochloride reportedly increase plasma catecholamine levels in rats. It is also known that active urethral closure mechanisms prevents stress urinary incontinence (SUI), which is enhanced by central and peripheral noradrenergic system activation. Therefore, we examined the influence of propiverine hydrochloride on urethral anti-incontinence function in rats. METHODS: Adult female rats were divided into propiverine and vehicle-treated groups. The propiverine group was given propiverine orally once a day for 2 weeks, after which urethral function and plasma concentrations of catecholamine (dopamine, norepinephrine, epinephrine) were tested. RESULTS: Urethral baseline pressure measured by a microtransducer-tipped urethral catheter and leak-point pressure during passive intravesical pressure elevation were significantly increased in the propiverine group compared with the vehicle group. Plasma norepinephrine and epinephrine levels in the propiverine group were also significantly increased. CONCLUSIONS: Propiverine treatment that increases plasma catecholamine levels could contribute to improvement of SUI conditions by increasing urethral resistance.

Possible involvement of host defense mechanism in the suppression of rat acute reflux esophagitis by the particular histamine H2 receptor antagonist lafutidine.

AIMS: Gastroesophageal reflux disease is considered to be caused primarily by gastric juice refluxed into the esophagus. Here, we investigated the possible involvement of host defense mechanisms in the development of acute reflux esophagitis using lafutidine, a histamine H(2) receptor antagonist (H(2)RA) with proven gastric mucosal protective effects. METHODS AND RESULTS: The ligation of both the pylorus and the forestomach of SD rats under anesthesia caused hemorrhagic lesions in the esophageal mucosa at 6 h. Lesion formation was significantly inhibited by treatment with H(2)RAs, including the conventional H(2)RAs famotidine and cimetidine as well as lafutidine. The maximum suppressive abilities of these agents were similar to that of the proton pump inhibitor lansoprazole. Interestingly, unlike famotidine, lafutidine at low doses significantly suppressed esophagitis without inhibiting gastric acid secretion. Note that neither lafutidine nor famotidine inhibited hexosamine output in gastric juice samples obtained 3 h after ligation. Additionally, the protective effect of lafutidine, but not of famotidine, was partly attenuated by the denervation of capsaicin-sensitive afferent nerves with a large dose of capsaicin. CONCLUSION: The present results indicate that esophageal host-defense via capsaicin-sensitive afferent nerves may contribute to the therapeutic action of lafutidine.

Oral fluoropyrimidine may augment the efficacy of aromatase inhibitor via the down-regulation of estrogen receptor in estrogen-responsive breast cancer xenografts.

The present preclinical study was designed to evaluate a new combination therapy comprised of the aromatase inhibitor anastrozole (ANA) and the oral fluoropyrimidines, UFT and S-1 against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7/Arom 14, which was stably transfected with the cDNA of human aromatase. MCF-7/Arom 14 cells showed a high aromatase activity and notably were able to grow in the presence of testosterone and estradiol (E(2)) in vitro. ANA and 5-fluorouracil (5-FU) inhibited cell growth at concentrations of 0.005-10 and 0.2-5 µM, respectively, and the combination of both drugs additively inhibited cell growth. The growth of MCF-7/Arom 14 tumors was significantly inhibited by ANA and S-1 or UFT in vivo. The combination of ANA with S-1 or UFT administered using a 21-day consecutive, metronomic-like regimen significantly enhanced the antitumor efficacy, suppressing tumor growth for 2-4 times longer than monotherapy. To investigate the mechanisms by which S-1 enhances the antitumor activity of ANA, the protein and mRNA expression levels of ER-α in tumor tissue after treatment with S-1, ANA, and the typical chemotherapeutic agents doxorubicin (ADM) or paclitaxel (TXL) were analyzed. The protein and mRNA expression levels of ER-α in the tumor tissue were markedly decreased after treatment with S-1 or S-1 + ANA, but not after treatment with either ADM or TXL. The reduced ER-α level after S-1 treatment might contribute to the increased antitumor activity of ANA by reducing ER-α-induced growth signaling in addition to the decrease in estrogen production induced by ANA. Based on these results, the combination of ANA and S-1 might yield a greater benefit than other chemotherapeutic agents in postmenopausal women with ER-positive breast cancer.

Up-regulation of α1a and α1d-adrenoceptors in the prostate by administration of subtype selective α1-adrenoceptor antagonist tamsulosin in patients with benign prostatic hyperplasia.

PURPOSE: We examined the change in α(1)-adrenoceptor subtype expression in the prostate due to chronic tamsulosin administration in a benign prostatic hyperplasia rat model and in patients. MATERIALS AND METHODS: We measured α(1)-adrenoceptor subtype expression after tamsulosin administration in the prostate of the benign prostatic hyperplasia rat model using TaqMan® reverse transcriptase-polymerase chain reaction. We also measured expression before and after 12-week tamsulosin treatment in the prostate of patients with benign prostatic hyperplasia. We examined the correlation between the change in α(1)-adrenoceptor expression due to tamsulosin treatment and acute urinary retention during long-term followup. RESULTS: The expression of α(1a) and α(1d)-adrenoceptors was significantly increased in dose dependent fashion by tamsulosin in the benign prostatic hyperplasia rat model. Median mRNA expression of subtypes α(1a) and α(1d)-adrenoceptors was 1.4 (IQR 0.6, 3.0) and 1.7 × 1,000 copies per 1 ng ß-actin (IQR 0.9, 2.4) before treatment, and 6.0 (IQR 2.0, 8.0) and 2.2 × 1,000 copies per 1 ng ß-actin (IQR 1.7, 3.6), respectively, after treatment. The expression of α(1a) and α(1d)-adrenoceptors significantly increased after tamsulosin treatment (p <0.01 and <0.05, respectively). This increase was observed in 10 patients in whom acute urinary retention did not develop during long-term followup but not in 4 in whom acute urinary tract retention developed. CONCLUSIONS: Tamsulosin up-regulated α(1a) and α(1d)-adrenoceptors, suggesting that it has clinical selectivity for α(1a) and α(1d)-adrenoceptors. Up-regulation of α(1)-adrenoceptors subtype expression is considered an adaptive response to chronic tamsulosin administration. The difference in the response to α(1)-adrenoceptors antagonists among patients may contribute to the diversity in the long-term efficiency of α(1)-adrenoceptor antagonists.

Are antimuscarinic drugs effective against urinary frequency mediated by atropine-resistant contractions?

In the disease states of urinary frequency and urgency, atropine-resistant contractions are known to be involved, in addition to contractions mediated by cholinergic nerves. This study was undertaken to investigate the mechanism underlying the development of atropine-resistant contractions using the representative antimuscarinic drugs solifenacin and tolterodine and also propiverine that has Ca(2+) channel-antagonizing activity in addition to antimuscarinic activity. Rat models of urinary frequency were established by intravesical infusion of acetylcholine (ACh) (cholinergic nerve-mediated urinary frequency model), acetic acid (AcOH) [non-adrenergic non-cholinergic nerve (NANC)-mediated urinary frequency model], or CaCl(2) (atropine-resistant contractions-mediated urinary frequency model). Cystometrograms were obtained to measure the micturition parameters following oral administration of the aforementioned drugs. Propiverine increased the micturition weight in all the urinary frequency models. Solifenacin and tolterodine increased the micturition weight in the ACh-induced urinary frequency model but neither had any effect in the AcOH- or CaCl(2)-induced urinary frequency models. While antimuscarinic drugs are, in general, effective for the control of urinary frequency and incontinence, use of drugs possessing inhibitory effects on contractions mediated by cholinergic as well as NANC nerve transmission or Ca(2+) influx into smooth muscles is recommended for management of the symptoms in disease states in which atropine-resistant contractions, such as Ca(2+)- and capsaicin-sensitive sensory nerves, are involved.

Prostate growth inhibition by subtype-selective alpha(1)-adrenoceptor antagonist naftopidil in benign prostatic hyperplasia.

BACKGROUND: Recently, alpha(1)-adrenoceptors (alpha(1)-ARs) have been reported to play a prominent role in the growth of a variety of cells; however, little is known about prostate growth and subtype-specific effects on cell proliferation. We examined the role of alpha(1d)-AR in prostate growth and the effect of subtype-selective alpha(1)-AR antagonist, naftopidil, which has relatively higher affinity for alpha(1d)-AR, on prostate growth in vitro and in vivo. METHODS: First, we examined the effect of naftopidil on the cell proliferation of PrEC, PrSC, and PrSMC using WST-1 assay. Second, we performed real-time RT-PCR to quantify each alpha(1)-AR subtype mRNA expression level in a benign prostate hyperplasia (BPH) model rat, which was recently established to pathologically resemble human BPH patients. In addition, naftopidil was given to this model orally for 21 days and the proliferative and apoptotic indexes measured. Third, 18 BPH patients were administered naftopidil for 12 weeks and the proliferative and apoptotic indexes were compared before and after naftopidil administration. RESULTS: Naftopidil significantly inhibited cell proliferation dose-dependently in all cell lines that expressed alpha(1d)-AR mRNA. The expression level of alpha(1d)-AR during the growth process of the prostate in the BPH model rat was significantly higher than that in the normal prostate (P < 0.001). Naftopidil administration inhibited cell proliferation without apoptosis in the BPH model rat and BPH patients. CONCLUSIONS: alpha(1d)-AR may play an important role in the regulation of cellular proliferation in the prostate, and alpha(1d)-AR blockage by naftopidil may not only improve lower urinary tract symptoms but also inhibit prostate growth in BPH patients.

New histopathological experimental model for benign prostatic hyperplasia: stromal hyperplasia in rats.

PURPOSE: Histological observations of clinical benign prostatic hyperplasia specimens show that benign prostatic hyperplasia tissue is mainly composed of stromal components, smooth muscle and fibrous tissue, so-called stromal hyperplasia. However, little is understood regarding the pathogenesis of this stromal hyperplasia due to no suitable stromal hyperplasia model to elucidate the pathology of benign prostatic hyperplasia. We created a novel model of benign prostatic hyperplasia accompanied by clinically relevant stromal hyperplasia. MATERIALS AND METHODS: The urogenital sinus isolated from male rat 20-day embryos was implanted into pubertal male rat ventral prostates. Two to 8 weeks after the operation the implanted urogenital sinus was isolated, weighed and subjected to histochemical analysis. To distinguish between and characterize the epithelial and stromal components we stained for collagen, smooth muscle components, growth factors and proliferating cell nuclear antigen. In addition, to determine whether the implanted urogenital sinus had differentiated into functional prostate we stained for androgen receptor and dorsolateral prostatic secretory protein. RESULTS: Urogenital sinuses removed from male rat 20-day embryos initially weighed approximately 1 mg. After implantation into host rat ventral prostates they grew in time dependent fashion with no apparent change in the original ventral prostate weight in the host rat. Implanted urogenital sinus weight was more than 100 mg 3 weeks after implantation. Histological observation demonstrated that the ratio of stromal to total area was approximately 70%, which was much higher than that in age matched rat ventral prostates and in a testosterone induced epithelial hyperplasia model (approximately 20% and 15%, respectively). This predominantly stromal tissue composition was maintained up to 8 weeks after implantation. Proliferating cell nuclear antigen staining revealed that the ratio of proliferating cells in stroma was equal to or greater than that in epithelium. In this model the antiandrogen agent chlormadinone acetate (Wako Pure Chemicals Industries, Osaka, Japan) at a dose of 10 mg/kg prevented the increase in implanted urogenital sinus weight (19.1%) but its potency was less than that seen in the testosterone induced epithelial hyperplasia model, that is 93.4% at the 10 mg/kg dose. CONCLUSIONS: We have established a new experimental stromal hyperplasia model corresponding to clinical benign prostatic hyperplasia in terms of the composition of stromal components and functional differentiation of the prostate. Furthermore, the localization and time course of growth factor expression were also similar to those in men with benign prostatic hyperplasia.

Monitoring trifluridine incorporation in the peripheral blood mononuclear cells of colorectal cancer patients under trifluridine/tipiracil medication.

Trifluridine/tipiracil (TFTD, TAS-102) is an orally administrated anti-cancer drug with efficacy validated for patients with metastatic colorectal cancer (mCRC). Trifluridine (FTD) is an active cytotoxic component of TFTD and mediates the anticancer effect via its incorporation into DNA. However, it has not been examined whether FTD is incorporated into the tissues of patients who received TFTD medication. By detecting FTD incorporation into DNA by a specific antibody, we successfully detected FTD in the bone marrow and spleen cells isolated from FTD-challenged mice as well as human peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-P and exposed to FTD in vitro. FTD was also detected in PBMCs isolated from mCRC patients who had administrated TFTD medication. Intriguingly, weekly evaluation of PBMCs from mCRC patients revealed the percentage of FTD-positive PBMCs increased and decreased in parallel with the administration and cessation of TFTD medication, respectively. To our knowledge, this is the first report to detect an active cytotoxic component of a chemotherapeutic drug in clinical specimens using a specific antibody. This technique may enable us to predict the clinical benefits or the adverse effects of TFTD in mCRC patients.

The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2'-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells.

Angiogenesis is a complex process involving an ECM and vascular endothelial cells (EC), and is regulated by various angiogenic factors including VEGF. The ability to form a capillary/tube-like network is a specialized function of EC. Therefore, in vitro angiogenesis was assessed by a capillary/tube-like network formation assay. There are three angiogenic parameters: capillary length, number of capillaries, and relative capillary area per field. We evaluated capillary length per field in the assay. VEGF promoted capillary/tube-like network formation of EC in a type I collagen gel matrix in vitro. Moreover, we demonstrated the involvement of ILK in a VEGF signaling pathway mediating capillary/tube-like network formation of EC using dominant-negative, kinase deficient ILK. This is a straightforward assay to monitor responses of human vascular endothelial cells.

Redox imaging of skeletal muscle using in vivo DNP-MRI and its application to an animal model of local inflammation.

Disorders of skeletal muscle are often associated with inflammation and alterations in redox status. A non-invasive technique that could localize and evaluate the severity of skeletal muscle inflammation based on its redox environment would be useful for disease identification and monitoring, and for the development of treatments; however, no such technique currently exists. We describe a method for redox imaging of skeletal muscle using dynamic nuclear polarization magnetic resonance imaging (DNP-MRI), and apply this method to an animal model of local inflammation. Female C57/BL6 mice received injections of 0.5% bupivacaine into their gastrocnemius muscles. Plasma biomarkers, myeloperoxidase activity, and histological sections were assessed at 4 and 24h after bupivacaine injection to measure the inflammatory response. In vivo DNP-MRI was performed with the nitroxyl radicals carbamoyl-PROXYL (cell permeable) and carboxy-PROXYL (cell impermeable) as molecular imaging probes at 4 and 24h after bupivacaine administration. The images obtained after carbamoyl-PROXYL administration were confirmed with the results of L-band EPR spectroscopy. The plasma biomarkers, myeloperoxidase activity, and histological findings indicated that bupivacaine injection caused acute muscle damage and inflammation. DNP-MRI images of mice treated with carbamoyl-PROXYL or carboxy-PROXYL at 4 and 24h after bupivacaine injection showed similar increases in image intensity and decay rate was significantly increased at 24h. In addition, reduction rates in individual mice at 4h and 24h showed faster trends with bupivacaine injection than in their contralateral sides by image-based analysis. These findings indicate that in vivo DNP-MRI with nitroxyl radicals can non-invasively detect changes in the focal redox status of muscle resulting from locally-induced inflammation.

Trifluridine Induces p53-Dependent Sustained G2 Phase Arrest with Its Massive Misincorporation into DNA and Few DNA Strand Breaks.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2'-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102.

Peroral TAS-202 reduced vessel density in rats with adjuvant-induced arthritis.

The present study was designed to investigate blood vessel density interpreted as an indirect measurement of angiogenesis following 4-(3,4,5-trimethoxyphenyl)-6-(2,4,5-trimethoxyphenyl)-2-diethylamino-pyrimidine (TAS-202) treatment in a rat model of arthritis. Male Lewis rats were inoculated intradermally with Mycobacterium butyricum into the hind paw and the arthritic responses were evaluated by measuring the changes in paw volume. Both peroral TAS-202 (10 or 30 mg/kg/day) and indomethacin (1 mg/kg/day) inhibited the autoimmune phase of the arthritic response. However, while the increase in blood vessel density in the synovial tissue was significantly inhibited by TAS-202 (10 and 30 mg/kg/day), indomethacin did not exert this effect (1 mg/kg/day). These results, together with the observation that TAS-202 in combination with indomethacin or prednisolone maintained its ability to exert an antiangiogenic effect, indicate that TAS-202 may offer promise as an oral pro-drug for the treatment of rheumatoid arthritis, through its inhibitory effect on angiogenesis at the inflammation site.

Anti-hyperlipidemic action of a newly synthesized benzoic acid derivative, S-2E.

A newly synthesized benzoic acid derivative, (+)-(S)-p-[1-(p-tert-butylphenyl)-2-oxo-4-pyrrolidinyl]methoxybenzoic acid (S-2E), has the capacity to inhibit the biosynthesis of both sterol and fatty acids. Here, we report the mechanism by which S-2E lowers blood cholesterol and triglyceride levels. In the liver, S-2E was converted into its active metabolite, S-2E-CoA. S-2E-CoA noncompetitively inhibited the enzymatic activities of both 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase and acetyl-CoA carboxylase at K(i)=18.11 microM and K(i)=69.2 microM, respectively. Interestingly, pharmacokinetic experiments in rats showed that the concentration of S-2E-CoA in the liver was sufficient to inhibit the activities of HMG-CoA reductase and acetyl-CoA carboxylase, for example, when orally given to rats at 10 mg/kg. Indeed, S-2E (3-30 mg/kg) given orally suppressed the secretion rate of very-low-density lipoprotein (VLDL)-cholesterol and triglyceride in Triton WR-1339-injected rats. Furthermore, S-2E lowered the blood total cholesterol and triglyceride levels simultaneously in Zucker fatty rats. Collectively, S-2E may be useful in the treatment of familial hypercholesterolemia and mixed hyperlipidemia.

Species-specific differences in the glucocorticoid receptor transactivation function upon binding with betamethasone-esters.

Glucocorticoids (GCs) are the most effective drugs for anti-inflammatory diseases. A number of adverse side effects, however, limit chronic treatment with GCs. To improve their therapeutic usefulness, attempts have been made to dissociate the two main actions of the glucocorticoid receptor (GR), transactivation and transrepression, which are believed to be responsible for the side effects and anti-inflammatory effects, respectively. We report here species-specific differences in the transactivation response mediated by GR. Dexamethasone (DEX), betamethasone (BM), and their esterified-derivatives had full transrepression agonistic activity in a reporter assay using CV-1 cells transfected with either human or rat GR. These GCs also had full transactivation agonistic activity in CV-1 cells transfected with human GR. The esterified-BM, however, had only partial transactivation agonistic activity in cells transfected with rat GR, whereas BM and esterified-DEX had full transactivation agonistic activity. Moreover, in rat hepatoma H4-II-E cells, the esterified-BM failed to induce tyrosine aminotransferase, which is regulated by GR-mediated transactivation activity. There were no significant differences between the binding affinity of these GCs to human and rat GR. Consistent with the weak transactivation activity of esterified-BM mediated by rat GR, there were few side effects, evaluated by thymus involution and body weight loss, in an antigen-induced asthmatic model in rats. These results suggest that the potency of esterified-BM to induce transactivation activity is different between species and that this difference is not due to differences in receptor binding.

Modulation of Bladder Afferent Activity by Propiverine and its Active Metabolites (M-1 and M-2) in Rats.

OBJECTIVES: To evaluate the effects of propiverine and its active metabolites (M-1 and M-2) on bladder function through modulation of afferent activity in rats. METHODS: Cystometry was performed in urethane anesthetized female rats. We examined the effects of intravesical administration of propiverine, M-1 and M-2 on bladder overactivity induced by oxotremorine-M (Oxo-M; non-selective mAChR agonist). RESULTS: Intravesical administration of Oxo-M (200 µM) elicited bladder overactivity as evidenced by decreased intercontraction interval (ICI) and pressure threshold (PT) without changing maximum voiding pressure or baseline pressure. These effects were blocked by intravesical administration of propiverine (30 µM) or M-2 (300 µM). Intravesical administration of M-1 (30 µM) alone increased ICI and PT, but did not prevent Oxo-M-induced decreases in ICI and PT. CONCLUSION: These results suggest that propiverine and M-2 have anticholinergic effects on bladder afferent activity and that M-1 has an inhibitory effect through the mechanism other than muscarinic receptor modulation. Thus, clinical benefits of propiverine in patients with overactive bladder could be mediated by multiple actions of propiverine and its active metabolites.

Combination therapy using oral S-1 and targeted agents against human tumor xenografts in nude mice.

In this study, combination therapies using the oral fluoropyrimidine tegafur-gimeracil-oteracil (S-1) with several targeted agents or antibodies, were evaluated. First, the effects of tyrosine kinase inhibitors (erlotinib hydrochloride, sorafenib tosilate and sunitinib malate) against human non-small cell lung cancer (NSCLC), breast cancer and colorectal cancer were evaluated in vivo. The effects of the combination of S-1 and targeted antibodies (bevacizumab and cetuximab) against human colorectal cancers was also evaluated in vivo. S-1 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, showed a significant inhibition of growth in human NSCLC (Lu-99 and PC-9 cell lines). The antitumor activity of the combination of S-1 and erlotinib against Lu-99 and PC-9 cancer cell lines was significantly superior to either monotherapy (P<0.05). Combination therapy using the multi-tyrosine kinase inhibitors, sorafenib or sunitinib, with S-1 against breast cancer (MX-1 cell line) and NSCLC (NCI-H460 cell line) was significantly superior to either monotherapy (P<0.01). The combination of the anti-vascular endothelial growth factor antibody bevacizumab or the anti-EGFR antibody, cetuximab, with S-1 against human colorectal cancer [Col-1, KM20C (bevacizumab) and DLD-1 (cetuximab) cell lines] and a 5-fluorouracil (5-FU)-resistant cell line (KM12C/5-FU) was significantly superior to either monotherapy (p<0.01). In particular, the growth of the Col-1 cells was completely inhibited by the combination of S-1 and bevacizumab. No toxic mortalities and no significant difference in the body weight changes of the animals treated with S-1 combined with the targeted agents or with the mono-therapies were observed; therefore, the treatments appeared to be well-tolerated. Our preclinical findings indicate that the combination therapies of S-1 and targeted agents are promising treatment options.

Lafutidine, a unique histamine H2-receptor antagonist, inhibits distention-induced gastric acid secretion through an H2 receptor-independent mechanism.

Gastric acid secretion during the daytime has been implicated in the pathogenesis of acid-related diseases. Although daytime acid secretion is mainly governed by the parasympathetic vagal nerve, clinical observations have been accumulated that the H(2)-receptor antagonist lafutidine may have a strong effect. Here, we examined the actions of H(2)-receptor antagonists in a rat model of gastric acid secretion induced by stomach distention, a major post-meal stimulus. Indeed, the acid output during a 3h period after the instillation of saline into pylorus-ligated SD rats under urethane anesthesia was dependent on the instilled volume and was strongly suppressed by a vagotomy or the intraduodenal administration of atropine. Interestingly, lafutidine, but not famotidine or cimetidine, administered at a sufficient dose to block histamine-dependent acid secretion was capable of inhibiting distention-induced acid secretion. Moreover, gastric acid secretion induced by the intravenous perfusion of carbachol into SD rats was strongly inhibited by lafutidine but only partially inhibited by famotidine. The antisecretory action of lafutidine under these conditions was partly reversed by the co-administration of the nitric oxide synthase inhibitor L-NMMA, but was hardly affected by denervation with capsaicin or by the administration of the transient receptor potential channel V1 (TRPV1) antagonist capsazepine. Together with the observation that lafutidine increased the amount of intragastric nitric oxide, the present results suggest that lafutidine inhibits daytime gastric acid secretion not only by blocking H(2) receptors, but also through nitric oxide-mediated and histamine-independent indirect actions.

In vivo evidence for a significant role of folylpolyglutamate synthase in combined chemotherapy with oral fluoropyrimidine, UFT or S-1, and leucovorin.

Combined chemotherapy with 5-fluorouracil and leucovorin (LV) has been widely used for the treatment of patients with colorectal cancer. Given that LV effects are attributable to increased levels of reduced folate in cancer cells, we attempted here to show the in vivo role of folylpolyglutamate synthetase (FPGS), which stabilizes intracellular reduced folate, in the anticancer activities of oral fluoropyrimidines, UFT or S-1, combined with LV. To this end, HCT-15 human colon cancer cells were knocked down for FPGS expression by RNA interference. The cell line stably expressing FPGS shRNA (FPGS shRNA HCT-15) was cloned and transferred subcutaneously into nude mice fed a low-folate diet. FPGS shRNA HCT-15 tumors expressed a significantly lower level of FPGS at protein and mRNA levels than parental HCT-15 cells, and the levels of reduced folate in FPGS shRNA HCT-15 tumors became 57% of those in parent after a single administration of 10 mg/kg of LV. Notably, FPGS downregulation did not affect the tumor growth or sensitivity to fluoropyrimidine. Importantly, we observed that LV given for 14 days failed to enhance the anticancer effects of UFT and S-1 in FPGS shRNA HCT-15. This was in keeping with the results that LV did not increase the ternary complex of TS, FdUMP and reduced folate. In conclusion, the present results provide in vivo evidence that intratumor FPGS plays an important role in the efficacy of oral fluoropyrimidine plus LV therapy for colorectal cancer.