Preparation and pharmaceutical properties of Hangeshashinto oral ointment and its safety and efficacy in Syrian hamsters with 5-fluorouracil-induced oral mucositis.

Chemotherapy-induced oral mucositis (COM) is a common adverse effect of cancer chemotherapy. Several clinical studies reported that repetitive use of mouthwashes containing 2.5-6.25% Hangeshashinto (HST), a Kampo formula, relieves COM, but the effect is insufficient. To solve this problem, we produced an oral ointment of 12% HST extract (considered quantitatively equivalent to 20% commercially available HST), which will increase the local concentrations of its active ingredients and prolong the contact time with COM. In this study, we evaluated the pharmaceutical properties (spreadability and stability) of HST oral ointment. In addition, its safety (oral mucosal irritation) and therapeutic effects on 5-fluorouracil-induced oral mucositis were evaluated in male Syrian hamsters. The HST ointment showed good spreadability and stability for more than 8 weeks at 4 °C. In the oral mucosal irritation test, topical application of HST ointment (0.2 g) three times per day for 14 days had no adverse effect on the oral mucosa of hamsters. In hamsters treated with 5-fluorouracil (60 mg/kg) twice, COM was induced by a submucosal injection of 5% acetic acid into the cheek pouch. When HST ointment (50 µg) was topically applied to the mucositis area once per day for 12 days, the area and macroscopic score of mucositis were significantly decreased, and the depth of the wound tended to be reduced compared with the lactose ointment-treated control animals. These findings suggest that HST oral ointment shows good properties in spreadability, stability, and safety, and elicits a therapeutic effect in an animal model of COM.

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice.

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3-/-) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3-/- embryos. Slc52a3-/- mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice.

Disruption of Slc52a3 gene causes neonatal lethality with riboflavin deficiency in mice.

Homeostasis of riboflavin should be maintained by transporters. Previous in vitro studies have elucidated basic information about riboflavin transporter RFVT3 encoded by SLC52A3 gene. However, the contribution of RFVT3 to the maintenance of riboflavin homeostasis and the significance in vivo remain unclear. Here, we investigated the physiological role of RFVT3 using Slc52a3 knockout (Slc52a3-/-) mice. Most Slc52a3-/- mice died with hyperlipidemia and hypoglycemia within 48 hr after birth. The plasma and tissue riboflavin concentrations in Slc52a3-/- mice at postnatal day 0 were dramatically lower than those in wild-type (WT) littermates. Slc52a3-/- fetuses showed a lower capacity of placental riboflavin transport compared with WT fetuses. Riboflavin supplement during pregnancy and after birth reduced neonatal death and metabolic disorders. To our knowledge, this is the first report to indicate that Rfvt3 contributes to placental riboflavin transport, and that disruption of Slc52a3 gene caused neonatal mortality with hyperlipidemia and hypoglycemia owing to riboflavin deficiency.

Early Tumor Shrinkage Under Treatment with First-line Tyrosine Kinase Inhibitors as a Predictor of Overall Survival in Patients with Metastatic Renal Cell Carcinoma: a Retrospective Multi-Institutional Study in Japan.

BACKGROUND: Prognostic significance of early tumor shrinkage following treatment with tyrosine kinase inhibitors (TKIs) in patients with metastatic renal cell carcinoma (mRCC) has not been fully elucidated. OBJECTIVE: The aim of this study was to assess the impact of early tumor shrinkage induced by first-line TKIs on overall survival (OS) in mRCC patients. PATIENTS AND METHODS: This study retrospectively included 185 consecutive Japanese patients with mRCC treated with either sunitinib or sorafenib for at least 3 months as first-line molecular-targeted therapy between April 2011 and December 2014 at Kobe University Hospital and its affiliated institutions. RESULTS: Median OS in the 185 patients was 33.6 months. At 12 weeks after the introduction of TKIs, 9 patients had achieved tumor shrinkage from -100 to -50 %, 43 from -49 to -25 %, 61 from -24 to 0 %, and the remaining 72 patients showed an increase in tumor size. The median OS stratified according to tumor shrinkage as shown above was 59.2, 39.1, 31.4, and 16.1 months, respectively. Univariate analysis identified prior nephrectomy, Memorial Sloan Kettering Cancer Center (MSKCC) risk classification, C-reactive protein (CRP) level, liver metastasis, number of metastatic organs, histological subtype, sarcomatoid feature, and early tumor shrinkage as significant predictors of OS. Of these significant factors, only the MSKCC classification, CRP level, liver metastasis, and early tumor shrinkage were shown to be independently associated with OS on multivariate analysis. CONCLUSIONS: Early tumor shrinkage could be a useful predictor of OS in mRCC patients receiving TKIs as a first-line molecular-targeted agent.

Implication of the descending dynorphinergic neuron projecting to the spinal cord in the (+)-matrine- and (+)-allomatrine-induced antinociceptive effects.

We previously reported that either (+)-matrine (matridin-15-one) or (+)-allomatrine (the C-6 epimer of matrine)-induced antinociceptive effect was attenuated by s.c. pretreatment with a kappa-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI), indicating the critical role of KORs in antinociceptive effects induced by these alkaloids. In the present study, we found that i.c.v. administration of either (+)-matrine- or (+)-allomatrine induced antinociceptive effects in the mouse tail-flick and warm-plate test, whereas these alkaloids when given spinally failed to induce antinociception. In the guanosine-5'-O-(3-[(35)S]thio)trisphosphate ([(35)S]GTPgammaS) binding assay, we demonstrated that neither (+)-matrine nor (+)-allomatrine produced the stimulation of [(35)S]GTPgammaS binding in the membranes of the spinal cord, indicating that (+)-matrine- and (+)-allomatrine-induced supraspinal antinociceptive actions was not due to a direct stimulation of KORs by these alkaloids. Therefore, we next investigated the involvement of dynorphin A (1-17) release at the spinal or supraspinal site in (+)-matrine- or (+)-allomatrine-induced antinociception. The i.c.v. pretreatment with an antiserum against dynorphin A (1-17) could not affect the antinociceptive effect induced by s.c. treatment of (+)-matrine. In contrast, the s.c.-administered (+)-matrine- and (+)-allomatrine-induced antinociceptive effect was significantly attenuated by i.t. pretreatment of an antiserum against dynorphin A (1-17). The present data suggest that either (+)-matrine or (+)-allomatrine when given i.c.v. may stimulate the descending dynorphinergic neuron, resulting in the stimulation of KORs in the spinal cord, and this phenomenon in turn produces the antinociception in mice.

Analysis of the involvement of hydroxyanthranilate hydroxycinnamoyltransferase and caffeoyl-CoA 3-O-methyltransferase in phytoalexin biosynthesis in oat.

Two oat genes encoding hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT) and S-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), both of which are possibly involved in the biosynthesis of oat avenanthramide phytoalexins, were cloned and their expression profiles in response to biological stress were studied. Four distinct cDNAs of oat HHT (AsHHT1-4) were isolated with the degenerative polymerase chain reaction method. The enzymatic activity of AsHHT1 expressed in E. coli was found using hydroxyanthranilate and hydroxycinnamoyl-CoAs as cosubstrates. Cloned oat CCoAOMT (AsCCoAOMT) encoded a polypeptide of 130 amino acid residues with 77.7 to 80.8% identities to the CCoAOMT sequences from other plant species. The accumulation of AsHHT1 and AsCCoAOMT transcripts increased concomitantly with phytoalexin accumulation by the treatment of victorin, a specific elicitor in oat lines carrying the Pc-2/Vb gene. Pharmacological approaches indicated the involvement of Ca2+, NO, and protein kinases in the signaling pathways of AsHHT1 and AsCCoAOMT mRNA induction. When oat leaves were inoculated with Puccinia coronata, the mRNA expression of AsHHT1 and AsCCOAOMT increased in both incompatible and compatible interactions but more rapidly in incompatible interaction. Interestingly, however, significant phytoalexin accumulation was observed only in incompatible interaction during the experimental period, suggesting that phytoalexin accumulation may be inhibited in one or more posttranscriptional processes in the compatible interaction.