[Quantitative Determination Method of Aconitum Monoester Alkaloids Using Relative Molar Sensitivity (RMS) for the Assay in the Japanese Pharmacopoeia].

Recently, a novel quantitative method using relative molar sensitivity (RMS) was applied to quantify the ingredients of drugs and foods. An important development in this regard can be observed in the Japanese Pharmacopoeia (JP) 18, where the quantification of perillaldehyde, an unstable compound, in crude drug "Perilla Herb," was revised to incorporate the RMS method. In this study, the primary objective was to improve the tester safety and reduce the amount of reagents used in the JP test. To achieve this, the quantification of three toxic Aconitum monoester alkaloids (AMAs) was explored using the RMS method, employing a single reference compound for all three targets. These AMAs, namely benzoylmesaconine hydrochloride, benzoylhypaconine hydrochloride, and 14-anisoylaconine hydrochloride, which are the quantitative compounds of Kampo extracts containing Aconite Root (AR), were quantified using the reference compound benzoic acid (BA). Reliable RMS values were obtained using both 1H-quantitative NMR and HPLC/UV. Using the RMS of three AMAs relative to the BA, the AMA content (%) in commercial AMAs quantitative reagents were determined without analytical standards. Moreover, the quantitative values of AMAs using the RMS method and the calibration curve method using the three analytical standards were similar. Additionally, similar values were achieved for the three AMAs in the Kampo extracts containing AR using the RMS and the modified JP18 calibration curve methods. These results suggest that the RMS method is suitable for quantitative assays of the Kampo extracts containing AR and can serve as an alternative to the current method specified in the JP18.

Celecoxib exerts antitumor effects in canine mammary tumor cells via COX­2­independent mechanisms.

Celecoxib plays antitumor roles via multiple mechanisms in a variety of human cancers. The aim of this study was to clarify the mechanism of action of celecoxib in canine mammary tumors. We examined the antitumor effects of celecoxib in AZACB canine mammary tumor cells expressing low levels of cyclooxygenase­2 (COX­2) to minimize the effect of COX­2 on its activity. Our data revealed that celecoxib inhibited cell proliferation mainly via COX­2­independent mechanisms. Specifically, celecoxib decreased the proportion of cells in S phase and increased G2/M arrest, which was associated with increased expression of the cyclin­dependent kinase inhibitors (CDKIs) p21 and p27. In addition, treatment with celecoxib downregulated COX­2 expression, and induced apoptosis via both the intrinsic and extrinsic pathways. These findings suggest that celecoxib might be a useful agent for the treatment of canine mammary tumors, regardless of COX­2 expression. In the future, it might be possible to use a combination of celecoxib and other antitumor agents to treat canine mammary tumors.

Profiles of serum amino acids to screen for catabolic and inflammation status in calves with Mycoplasma bronchopneumonia.

The aim of the present study was to investigate the relationships between serum amino acid profiles in normal and calves with Mycoplasma bronchopneumonia. Serum free amino acid concentrations in serum obtained from 34 calves with or without Mycoplasma bronchopneumonia were determined by high-performance liquid chromatography. The calves with Mycoplasma were characterized by significantly lower total amino acid and total essential amino acid concentrations and molar ratios of branched-chain amino acid (BCAA) to aromatic amino acid (BCAA/AAA) and BCAA to tyrosine (BTR), and by a significantly higher molar ratio of serine phosphorylation (SPR). The proposed diagnostic cutoffs for BCAA/AAA, BTR and SPR in serum based on ROC analysis for detection of catabolic states associated with Mycoplasma bronchopneumonia were set at <1.75, <2.86 and >0.85, respectively. Our results suggest that determining the profiles of amino acids, especially BTR and SPR, could provide useful diagnostic information in terms of predicting protein catabolism in Mycoplasma bronchopneumonia.

Photodynamic therapy using a novel irradiation source, LED lamp, is similarly effective to photodynamic therapy using diode laser or metal-halide lamp on DMBA- and TPA-induced mouse skin papillomas.

Photodynamic therapy (PDT) is useful for superficial skin tumors such as actinic keratosis and Bowen disease. Although PDT is non-surgical and easily-performed treatment modality, irradiation apparatus is large and expensive. Using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-ο-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin papilloma model, we compared the efficacy of TONS501- and ALA-PDT with a LED lamp, a diode laser lamp or a metal-halide lamp on the skin tumor regression. TONS501-PDT using 660 nm LED lamp showed anti-tumor effect at 1 day following the irradiation and the maximal anti-tumor effect was observed at 3 days following the irradiation. There was no significant difference in the anti-tumor effects among TONS501-PDT using LED, TONS501-PDT using diode laser, and 5-aminolevulinic acid hydrochloride (ALA)-PDT using metal-halide lamp. Potent anti-tumor effect on DMBA- and TPA-induced mouse skin papilloma was observed by TONS501-PDT using 660 nm LED, which might be more useful for clinical applications.

Synthesis and biological evaluation of new BSH-conjugated chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

New disodium mercaptoundecahydro-closo-dodecaborate (BSH)-conjugated chlorin derivatives 11, 12, 16 and 20 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized. The in vivo biodistribution and clearance of 11, 12, 16 and 20 were investigated in tumor-bearing mice. Compounds 12 and 16 showed good tumor-selective accumulation among the four derivatives. The time to maximum accumulation of compound 16 in tumor tissue was one-fourth of that of compound 12, and clearance from normal tissues of compound 16 was similar to that of compound 12. The in vivo therapeutic efficacy of PDT using 16, which has twice as many boron atoms as 12, was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 6h after injection of 16. Tumor growth was significantly inhibited by PDT using 16. These results suggested that 16 is a good candidate for both PDT and BNCT of cancer.

Usefulness of selective COX-2 inhibitors as therapeutic agents against canine mammary tumors.

Cyclooxygenase-2 (COX-2) is a key enzyme for converting arachidonic acids to prostanoids, which are known to be induced during inflammation and cancer initiation. Previously, it has been reported that COX inhibitors, such as aspirin, reduce the incidence of human colorectal cancer; therefore, it is widely believed that COX-2 is a potential therapeutic and chemoprevention target for several types of human cancer. However, whether selective COX-2 inhibitors have antitumor effects against canine mammary tumor cells remains unclear. In the present study, to elucidate the antitumor effect of selective COX-2 inhibitors against canine mammary tumors, we investigated the antitumor effects of meloxicam, etodolac and celecoxib using COX-2-expressing canine mammary tumor (CF33) cells. We analyzed the effects of selective COX-2 inhibitors on COX-2 protein expression levels in CF33 cells. Celecoxib (100 µM) was found to induce downregulation of COX-2 protein expression. We examined the effect of selective COX-2 inhibitors on CF33 cell proliferation. All the selective COX-2 inhibitors suppressed CF33 cell growth. Specifically, etodolac and celecoxib inhibited cell proliferation via a decrease in S-phase cells and an increase in G0/G1 arrest. We examined the apoptotic effect of selective COX-2 inhibitors on CF33 cells. Our data suggested that etodolac and celecoxib induced apoptosis in CF33 cells. In particular, celecoxib led to apoptosis mediated by the activation of the mitochondrial apoptosis pathway, including the upregulation of BAX expression, downregulation of Bcl-2 expression and activation of caspase-3/7. Furthermore, celecoxib increased the percentages of cells in both early apoptosis and late apoptosis. Our results revealed that celecoxib induced apoptosis and cell cycle arrest in CF33 cells. The data suggested that celecoxib is the most viable candidate as a therapeutic agent for the treatment of canine mammary tumors. Furthermore, our findings provide the first indication that COX-2 inhibition can provide a new therapeutic strategy for treating canine mammary tumors.

Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and L-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer.

Bronchoalveolar lavage fluid and serum canine surfactant protein A concentrations in dogs with chronic cough by bronchial and interstitial lung diseases.

We measured bronchoalveolar lavage fluid (BALF) and serum canine surfactant protein (cSP)-A concentrations in dogs with chronic cough. There were no significant differences between bronchial and interstitial lung diseases in BALF cSP-A concentrations. However, serum cSP-A concentrations in dogs with the interstitial lung disease as diffuse panbronchiolitis and idiopathic pulmonary fibrosis were significantly higher than those in dogs with the bronchial disease as chronic bronchitis. These results suggest that serum cSP-A concentrations may be a useful and noninvasive biomarker to understand the existence of interstitial lung damage in dogs with chronic cough.

Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy.

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3h after intravenous injection and rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.

The hyaluronan synthesis inhibitor 4-methylumbelliferone exhibits antitumor effects against mesenchymal-like canine mammary tumor cells.

Hyaluronan (HA), a principal constituent of the extracellular matrix (ECM), mediates growth and metastasis of tumor cells. The role of HA in the epithelial-mesenchymal transition (EMT) is well known, and increased ECM remodeling is observed in mesenchymal-like cells. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) is anti-tumorigenic for various malignant tumors. However, the antitumor effect of 4-MU against canine mammary tumor cells that possess a mesenchymal-like phenotype is unclear. We examined the antitumor effect of 4-MU on CF41.Mg mesenchymal-like canine mammary tumor cells. We investigated the influence of 4-MU on the expression of HA synthase (HAS) 1-3 mRNA and observed dose-dependent downregulation of HAS2 mRNA at 24-72 h; in contrast, HAS3 expression was elevated at 24 h. Thus, 4-MU inhibited HA synthesis via HAS2 repression. 4-MU also inhibited cell proliferation and induced apoptosis in the CF41.Mg cells. Our experiments showed that 4-MU-induced apoptosis in CF41.Mg cells involved induction of the pro-apoptotic gene BAX. We also assessed motility and found that 4-MU reduced chemokinesis and chemotaxis in CF41.Mg cells. Our data suggest that 4-MU may serve as a candidate therapeutic agent for the treatment of canine mammary tumors. Since 4-MU exhibits antitumor activity in mesenchymal-like cells, it may also be a useful inhibitor of canine mammary tumor invasion and metastasis.

4-methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells.

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is synthesized by HA synthase (HAS) 1, HAS2 and HAS3 and is intricately involved in cell growth and metastasis. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has been reported to exhibit anticancer properties in various types of malignant tumors. However, the underlying mechanisms at the molecular and cellular levels remain unclear. In this study, to establish an animal model for studying the function of HA in human breast cancer, we investigated the antitumor effects of 4-MU using canine mammary tumor (CF33) cells. First, we investigated the effects of 4-MU on HA production in CF33 cells. Quantitative analysis of HA in culture media showed that 4-MU inhibited HA synthesis, accompanied by downregulation of HAS2 mRNA levels, in a dose-dependent manner at 24-72 h. Additionally, we observed a 4-MU-mediated decrease in the extent of the cell-associated HA matrix. We examined the effect of 4-MU on cell growth and apoptosis in CF33 cells. 4-MU markedly inhibited cell proliferation and induced apoptosis in CF33 cells. In particular, our experiments showed that the mechanism of 4-MU-induced apoptosis in CF33 cells involved increased levels of expression of pro-apoptotic BAX mRNA and protein molecules. These data suggest that 4-MU may be a candidate therapeutic agent for the treatment of canine mammary tumors. Furthermore, this study provides the first indication that the canine mammary tumor may be a suitable model for comparative study of the function of HA in human breast cancer.

Photodynamic therapy using a novel photosensitizer, TONS501, is similarly effective to ALA and EC036 photodynamic therapy on DMBA-and TPA-induced mouse skin papilloma.

BACKGROUND: Although topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is applied for skin tumors including actinic keratosis, Bowen disease, and squamous cell carcinoma, there are no approved photosensitizers in dermatological field in Japan. TONS501 and TONS504 are novel hydrophobic photosensitizers with anionic and cationic chemical characteristics, respectively. OBJECTIVE: Using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-ο-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin papilloma model, we compared the efficacy of ALA-, TONS501-, and TONS504-PDT on the skin tumor regression. METHODS: Following application of ALA, TONS501, TONS504 ointment or TONS501 lotion on DMBA- and TPA-induced mouse papillomas, 670 nm laser irradiation by LD670-05 diode laser was performed. Then tumor regression rate was calculated at the indicated time. RESULTS: The anti-tumor effect of ALA, TONS501, and TONS504 ointment was detected at 24 h and the maximal response was observed at 3 day following the PDT treatment. The maximal response was observed at 150 J/cm(2) irradiation in all 3 photosensitizers. Although both ALA, TONS501 (ointment)-PDT showed more potent anti-tumor effect compared with that of TONS504 ointment or TONS501 lotion, no significant difference was detected between ALA ointment and TONS501 ointment. CONCLUSION: A novel TONS501ointment-PDT shows potent anti-tumor effect on DMBA- and TPA-induced mouse skin papilloma and might be more useful for the clinical applications.

Trace and major elements status in bronchoalveolar lavage fluid in dogs with or without bronchopneumonia.

The aim of this study was to investigate the relationships between the bronchopneumonia and mean concentrations of those trace elements in bronchoalveolar lavage fluid (BALF). Twenty-nine dogs were included this study (17 healthy dogs and 12 dogs with respiratory disease). Each BALF sample had been obtained during bronchoscope examination by use of a standardized method. The concentrations of Al, Br, Ca, Cu, Fe, K, Ni, P, Si, Sr and Zn in BALF were measured by the particle-induced X-ray emission method. We found no relationship between the bronchopneumonia and the levels of elements in the BALF, except Ca, P and Zn. The dogs with respiratory disease were found to have a large amount of Ca and Zn, and a high Ca/P and Zn/Cu ratios in BALF compared to those without respiratory disease.

The effect of 7.2% hypertonic saline solution with 6% dextran 70 on cardiac contractility as observed by an echocardiography in normovolemic and anesthetized dogs.

We studied the effect of a small volume of 7.2% hypertonic saline solution (HSS) or HSS with 6% dextran 70 (HSD) on hemodynamic status, especially on cardiac contractility, in anesthetized dogs using the left ventricular end-systolic volume index (ESVI) and ejection fraction (EF), which can be obtained in noninvasive echocardiography. In the present study, the mean values of ESVI were unaffected by HSS and HSD infusion, whereas the left ventricular end-diastolic volume index (EDVI) was markedly and significant increased. As a result of the changes in EDVI but not in ESVI, EF increased transiently and significantly in the HSS and HSD group, whereas no such significant change was observed in the dogs that received isotonic saline solution. In addition, as a result of the increases in cardiac index but not arterial pressure, system vascular resistances (SVR) decreased transiently and significantly in the HSS and HSD groups, whereas no such significant change was observed in the ISS group. Therefore, the positive inotropic effects of HSS and HSD may be attributable to the increase in left ventricular preload and decreases in SVR rather than direct changes in myocardial contractility.

Connexin 43-dependent tumor-suppressing effect of the Bowman-Birk protease inhibitor on M5076 ovarian sarcoma-bearing mice.

The present study was designed to confirm whether the Bowman-Birk inhibitor (BBI) induces an increase in p27 accumulation without S phase kinase-associated protein 2 (skp2) degradation by means of the expression of connexin (Cx) 43 as a gap junctional intercellular communication (GJIC)-dependent pathway in mice with M5076 ovarian sarcoma. M5076 ovarian sarcomas (1x105 cells/animal) were subcutaneously transplanted onto the backs of BDF1 mice receiving 10, 20 or 40 mg/kg of purified BBI intraperitoneally. Relative tumor weight (p<0.01, r=0.503) was negatively correlated with the dose of BBI. In contrast, the relative density of Cx43 mRNA (p<0.01, r=0.570) and Cx43 (p<0.01, r=0.718) was positively correlated with the dose of BBI, as were p21 (p<0.01, r=0.633), p27 (p<0.01, r=0.561) and skp2 (p<0.01, r=0.733). We therefore suggest that the anti-carcinogenic effects of BBI induce negative growth control by means of an increase in p27 accumulation caused by the expression of Cx43 as a GJIC pathway.

Effects of a single-dose administration of Bowman-Birk inhibitor concentrate on anti-proliferation and inhabitation of metastasis in M5076 ovarian sarcoma-bearing mice.

The present study was designed to investigate the effects of Bowman-Birk inhibitor concentrate (BBIC) on anti-proliferation, up-regulation of Connexin 43 (Cx43) expression and inhabitation of hepatic metastasis in mice with M5076 ovarian sarcoma. M5076 ovarian sarcomas (1x106 cells/animal) were subcutaneously transplanted into the back of BDF1 mice. The 'pre-treated' (n=10) and 'post-treated' (n=10) groups were fed a standard diet (CE-2) compounded with 0.5% BBI from 3 weeks before and from the day of tumor inoculation, respectively, until 4 weeks after tumor inoculation. The 'control group' (n=10) was fed CE-2 alone. Relative tumor weights in the pre- (0.013±0.010) and post- (0.012±0.013) treated groups were significantly reduced by 30.0 and 32.5%, respectively, as compared to the control group (0.040±0.022, p<0.01). The relative densities of Cx43 mRNA and Cx43 protein were significantly higher in the BBIC-treated groups than in the control group. The median numbers of macroscopic spontaneous metastases were significantly lower in the pre- (1.0±2.3) and post- (1.9±3.6) treated groups than in the control group (71.4±97.3). These results suggest that BBIC reduces proliferation as a result of increased expression of Cx43 genes in mice with M5076 ovarian sarcoma. In addition, BBIC inhibits hepatic metastasis in M5076-bearing mice.

Relationship between hair elements and severity of atrioventricular block in horses.

The aim of this study was to investigate the relationships between the mean concentrations of trace elements and the severity of the second-degree atrioventricular (AV) block in the mane hair of horses. Electrocardiographs of horses were continually recorded for 6 h using a holter cardiac monitor to determine dropped ventricular beats (DVBs) which can be used as an indicator of the severity of the AV block. Mane hair Ca, Cu, Mg, and Zn concentrations were measured by the particle-induced X-ray emission method. The Zn/Cu ratio and Ca concentration in mane hair were significantly and positively correlated with the hourly DVBs in horse with a second-degree AV block (p<0.01, r2=0.485; p<0.05, r2=0.351, respectively). Proposed diagnostic cutoff points for hair Ca concentration and Zn/Cu ratio based on receiver operating characteristics curves analysis in detecting second-degree AV block were set at 1536 microg/g and 26.0, respectively. Those results with horse hair suggest that the evaluation of the Ca, Cu, Mg, and Zn status in mane hair by this method is strongly related to the severity of second-degree AV block and might predict the susceptibility of an individual much before the development of the symptom.

Sonodynamic therapy of cancer using a novel porphyrin derivative, DCPH-P-Na(I), which is devoid of photosensitivity.

To improve the efficacy of sonodynamic therapy of cancer using photosensitizers, we developed a novel porphyrin derivative designated DCPH-P-Na(I) and investigated its photochemical characteristics and sonotoxicity on tumor cells. DCPH-P-Na(I) exhibited a minimum fluorescent emission by excitation with light, compared with a strong emission from ATX-70, which is known to reveal both photo- and sonotoxicity. According to this observation, when human tumor cells were exposed to light in the presence of DCPH-P-Na(I) in vitro, the least phototoxicity was observed, in contrast to the strong phototoxicity of ATX-70. However, DCPH-P-Na(I) exhibited a potent sonotoxicity on tumor cells by irradiation with ultrasound in vitro. This sonotoxicity was reduced by the addition of L-histidine, but not D-mannitol, thus suggesting that singlet oxygen may be responsible for the sonotoxicity of DCPH-P-Na(I). DCPH-P-Na(I) demonstrated significant sonotoxicity against a variety of cancer cell lines derived from different tissues. In addition, in a mouse xenograft model, a potent growth inhibition of the tumor was observed using sonication after the administration of DCPH-P-Na(I) to the mouse. These results suggest that sonodynamic therapy with DCPH-P-Na(I) may therefore be a useful clinical treatment for cancers located deep in the human body without inducing skin sensitivity, which tends to be a major side-effect of photosensitizers.

2-O-alpha-D-glucopyranosyl-L-ascorbic acid scavenges 1,1-diphenyl-2-picrylhydrazyl radicals via a covalent adduct formation.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.