Optimal treatment schedule and antitumor spectrum of 4-carbamoylimidazolium 5-olate (SM-108) in murine tumors.

By designing optimal administration schedules, it was found that 4-carbamoylimidazolium 5-olate (SM-108) showed an excellent antitumor potency against a number of murine tumors. The optimal administration schedule of SM-108 was an intermittent multiple administration, in which the drug was multiply administered to mice at definite intervals of less than 3 hr for about 1 day on Days 1, 5, and 9 following tumor implantation. Although usual daily administration of SM-108 exhibited poor efficacy, the intermittent multiple administration of SM-108 exhibited potent antitumor activity against a wide variety of tumors, such as Ehrlich carcinoma, P388, 6-mercaptopurine-sensitive and -resistant L1210, Lewis lung carcinoma, Colon 26 adenocarcinoma, and Sarcoma 180. Among them, Ehrlich carcinoma showed the most prominent susceptibility to SM-108. With the intermittent multiple administration of SM-108, complete suppression was obtained in both the ascitic and solid forms of this tumor over a wide dose range. The schedule dependency of the antitumor effect of SM-108 described above was reasonably explained by its in vitro growth-inhibitory effects and pharmacokinetics in the mice.

Improved survival with an innovative approach to the treatment of severely burned patients: development of a burn treatment manual.

The management of severely burned patients remains a major issue worldwide as indicated by the high incidence of permanent debilitating complications and poor survival rates. In April 2012, the Advanced Emergency & Critical Care Medical Center of the Okayama University Hospital began implementing guidelines for severely burned patients, distributed as a standard burn treatment manual. The protocol, developed in-house, was validated by comparing the outcomes of patients with severe extensive burns (SEB) treated before and after implementation of these new guidelines at this institution. The patients included in this study had a burn index (BI) ≥30 or a prognostic burn index (PBI = BI + patient's age) ≥100. The survival rate of the patients with BI ≥30 was 65.2% with the traditional treatment and 100% with the new guidelines. Likewise, the survival rate of the patients with PBI ≥100 was 61.1% with the traditional treatment compared to 100% with the new guidelines. Together, these data demonstrate that the new treatment guidelines dramatically improved the treatment outcome and survival of SEB patients.

La prise en charge des patients gravement brûlés est toujours un problème majeur dans le monde, avec une mortalité élevée et de lourdes séquelles chez les survivants. En Avril 2012, le Centre de l'Hôpital de l'Université d'Okayama a commencé à distribuer un manuel pour le traitement des patients gravement brûlés. Notre protocole a été validé en comparant les résultats des patients souffrant de brûlures étendues traités avant et après la mise en oeuvre de ces nouvelles lignes directrices. Les patients inclus dans cette étude avaient une surface brûlée (SB) ≥30% ou un index de Baux (IBx= SB + âge du patient) ≥100. Le taux de survie chez les patients atteints sur ≥30% SB était de 65.2% avant et 100% après. Le taux de survie chez les patients avec un IBx ≥100 était de 61.1% avant et 100% après. Ces données démontrent que les nouvelles lignes directrices de traitement ont amélioré considérablement la survie chez ces patients.

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60.

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.

Immunomagnetic separation of scum-forming bacteria using polyclonal antibody that recognizes mycolic acids.

Mycolic acid-containing bacteria (mycolata) are thought to be involved in scum formation in aeration basins of activated sludge plants due to their ability to produce biosurfactants and their cell surface hydrophobicity. To isolate these bacteria, immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody was investigated. IMS that targeted Gordonia amarae SC1 exhibited a 100% recovery at 5x10(3) CFU ml(-1). At cell concentration of 7.8x10(6) CFU ml(-1), the recovery was lowered, but 80% of cells were still captured. Effect of bead concentrations on the recovery of SC1 at 10(6) CFU ml(-1) was examined. The results showed that addition of more than 6-7x10(6) beads for 1x10(6) CFU reached a maximum recovery (83%). Furthermore, the IMS procedure optimized with SC1 cells was tested with another mycolata. The results suggested that variation of the recovery for each mycolata is dependent on the specificity of the polyclonal antibody and that mycolata which are recognized by the antibody can be recovered by this procedure.

Rapid detection of Nocardia amarae in the activated sludge process using enzyme-linked immunosorbent assay (ELISA).

Nocardia amarae, a mycolic acid-containing bacterium, has often been reported to cause foaming of activated sludge in wastewater treatment plants. In this study, the number of N. amarae cells in the activated sludge process was estimated by enzyme-linked immunosorbent assay (ELISA) with anti-N. amarae polyclonal antibody. Use of the antibody enabled N. amarae to be detected at levels of 10(4) to 10(7) colony forming units. On the other hand, the antibody reacted with only a small portion of activated sludge, in which no N. amarae cells were detected by the plate count method. Competitive ELISA was employed to estimate the N. amarae cells in samples taken from a municipal wastewater treatment plant, including raw wastewater and activated sludge foam. The cell numbers estimated by competitive ELISA corresponded well with those obtained by plate counts. Hence, the antibody produced in this study was shown to be effective for the rapid monitoring of N. amarae in the activated sludge process.

Production of anti-Gordonia amarae mycolic acid polyclonal antibody for detection of mycolic acid-containing bacteria in activated sludge foam.

Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti-Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 microg. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti-G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge.

[Evaluation of amrubicin with a 5 day administration schedule in a mouse model].

It was reported that amrubicin hydrochloride (9-aminoanthracycline SM-5887), showed a higher therapeutic activity than doxorubicin against human tumor xenografts implanted into nude mice with a single treatment schedule. In order to find a more effective treatment schedule, the efficacy, toxicity and pharmacokinetic properties with a 5 consecutive day treatment schedule were investigated. The total amount of the maximum tolerated dose and tumor growth inhibiting activity with a 5 day schedule was found to be higher than with a single administration. High levels of amrubicinol, the active metabolite of amrubicin, was detected in the tumor tissue. It was thus assumed that the improved efficacy with the 5-day schedule resulted from the high accumulation of amrubicinol. Bone marrow suppression at the MTD with the 5 day schedule was severer than with a single dose, but recovery was rapid, similar to that following a single dose. In conclusion, it was demonstrated that a 5 day treatment schedule was more effective than a single administration.

[Antitumor activities of orally administered 4-carbamoylimidazolium-5-olate (SM-108)].

We investigated the antitumor effect of orally administered SM-108. The drug showed strong antitumor activities against Ehrlich carcinoma, sarcoma 180, P388 leukemia, L1210 leukemia, colon 26 adenocarcinoma, colon 38 adenocarcinoma, and Lewis lung carcinoma. The antitumor activity of SM-108 against Ehrlich carcinoma was so remarkable, in fact, that all mice in a group survived for a long time. The antitumor effect of SM-108 depends on its administration schedule. Treatment involving a schedule of one dose every 6 h for 24 h at intervals of 3 days brought about a much stronger effect than daily single treatment. The maintenance of a high serum level of SM-108 for 24 h by the former treatment is responsible for the strong therapeutic effects, because the action of SM-108 is time-dependent. The antitumor activities of SM-108 administered orally are excellent enough to be comparable with those obtained by intraperitoneal administration as previously reported.

Toxicological aspects of a novel 9-aminoanthracycline, SM-5887.

The toxicological characteristics of SM-5887 were evaluated in mice after a bolus intravenous injection, and compared with those of adriamycin (ADR). The acute toxic signs observed after SM-5887 administration were body weight decrease, ataxia, hair loss, and myelosuppression. They were qualitatively comparable to those induced by ADR. The 50% lethal dose values determined by 14-day observation after drug administration were in the range of 32 to 50 mg/kg for SM-5887 and 16 to more than 20 mg/kg for ADR in four strains of mice. The maximum tolerated doses (MTD) were estimated to be 25 mg/kg for SM-5887 and 12.5 mg/kg for ADR (no death or body weight loss of more than 3 g occurred). When 14-day survivors were further observed until 90 days after drug administration, ADR frequently and dose-independently showed delayed-type lethal toxicity at doses of more than 10 mg/kg, whereas SM-5887 did not. The myelosuppression of SM-5887 was more severe even at a half of the MTD than that of ADR at the MTD, but its recovery was more rapid than that after ADR. In addition, when the drugs were injected into the subplantar region of mouse hind paws, ADR induced a severe inflammatory reaction, whereas SM-5887 yielded only a slight one. The data suggest that toxic effects of SM-5887 are more reversible and more controllable than those of ADR.

In vivo efficacy and tumor-selective metabolism of amrubicin to its active metabolite.

The tissue distribution of a novel antitumor anthracycline antibiotic, amrubicin, was studied using seven human tumor xenografts implanted into nude mice, in order to identify the principal factors determining its therapeutic efficacy. We found a good correlation between the level of the metabolite amrubicinol in the tumor and the in vivo efficacy. High metabolic activity of amrubicin to amrubicinol was detected in tumor tissue homogenates, especially in cell lines highly sensitive to amrubicin in vivo. In contrast to amrubicin, the administration of amrubicinol showed less tumor-selective toxicity in these human tumor xenograft models. These data indicate that the tumor-selective metabolism of amrubicin to amrubicinol resulted in a tumor-selective disposition of amrubicinol, leading to good efficacy in in vivo experimental therapeutic models.

Tumor-selective distribution of an active metabolite of the 9-aminoanthracycline amrubicin.

It has been reported that the 9-aminoanthracycline amrubicin shows good efficacy in human tumor xenograft models. We studied the disposition and metabolism of amrubicin in mice, in comparison with those of doxorubicin. Amrubicinol, a 13-hydroxy metabolite of amrubicin, which is 10 to 100 times more cytotoxic than amrubicin, was detected as a major metabolite in blood and tissues, and aglycones of amrubicin were also detected. A pharmacokinetic study revealed that amrubicin had a smaller distribution volume and a shorter half-life than doxorubicin. In several normal tissues, the levels of amrubicin and amrubicinol were lower than those of doxorubicin. In contrast, the tumor levels of amrubicinol in the mice treated with amrubicin were higher than those of doxorubicin in the mice treated with that drug, in tumors that are sensitive to amrubicin. These results suggest that the potent therapeutic activity of amrubicin is caused by the selective distribution of its highly active metabolite, amrubicinol, in tumors.

Cytotoxicity of amrubicin, a novel 9-aminoanthracycline, and its active metabolite amrubicinol on human tumor cells.

Amrubicin, a completely synthetic 9-aminoanthracycline derivative, was previously shown to have potent antitumor activities against various human tumor xenografts. In this study, the in vitro activities of amrubicin and its major metabolite, amrubicinol, were examined using 17 human tumor cell lines. Amrubicinol was 5 to 54 times more potent than amrubicin, and as potent as doxorubicin, in inhibiting the growth of the cells following 3-day continuous drug exposure. Amrubicinol closely resembled doxorubicin in its profile of activities on the 17 human tumor cell lines. Cells were incubated with the drugs for 1 h, and the intracellular drug concentration and cell growth inhibition after 3 days were determined. Amrubicinol attained similar intracellular concentrations at lower medium concentrations compared to amrubicin, and the intracellular concentration of amrubicinol necessary to produce 50% cell growth inhibition was 3 to 8 times lower than that of amrubicin in 4 cell lines tested. Amrubicinol has a higher activity level inside the cells than does amrubicin. When cells were incubated with amrubicin for 5 h, a substantial amount of amrubicinol, more than 9% of that of amrubicin, was found in cells in 4 of the 8 cell lines tested. Amrubicinol may contribute to the in vitro growth-inhibitory effect of amrubicin on these cells. The results suggest that amrubicinol plays an important role in the in vivo antitumor effect of amrubicin as an active metabolite.

Antitumor activities of a novel 9-aminoanthracycline (SM-5887) against mouse experimental tumors and human tumor xenografts.

The antitumor effects of SM-5887, a totally synthetic 9-aminoanthracycline derivative, were evaluated in six murine experimental tumor systems (P388, Ehrlich carcinoma, sarcoma 180, Lewis lung carcinoma, B16 melanoma and colon 38) and nine human tumor-nude mouse systems (one breast cancer, two lung cancers and six gastric cancers). Characteristically SM-5887 showed excellent antitumor activities, superior to adriamycin (ADR), against human tumor xenografts, although its activities against murine experimental tumors were almost equal to those of ADR. When the human tumors were implanted sc in female athymic mice (BALB/c, nu/nu) and their volume reached 100-300 mm3, SM-5887 and ADR were injected iv. All nine human tumors tested showed statistically significant responses to SM-5887, and 7 of them were strongly suppressed in their growth by SM-5887 so that minimum T/C values were less than 30% at the maximum tolerated dose (MTD, 25 mg/kg) with a single iv injection. Compared with ADR, SM-5887 was statistically more effective in five tumors (one breast, one lung and three gastric), equal in two tumors (two gastric), and less potent in two tumors (one lung and one gastric). In addition, the 10-day-interval repeated iv treatments with SM-5887 at the MTD (25 mg/kg) resulted in remarkably potent antitumor effects (including complete regression) against human gastric cancer, 4-1ST, implanted in nude mice without enhancement of toxic effects. SM-5887 was also effective against ip-inoculated P388 by oral administration as well as iv injection.

Uptake and intracellular distribution of amrubicin, a novel 9-amino-anthracycline, and its active metabolite amrubicinol in P388 murine leukemia cells.

Amrubicin, a 9-aminoanthracycline anti-cancer drug, and its C-13 hydroxyl metabolite amrubicinol, were examined for growth-inhibitory activity as well as cellular uptake and distribution in P388 murine leukemia cells and doxorubicin-resistant P388 cells. Also discussed are the differences in the mechanisms of action among amrubicin, amrubicinol and doxorubicin in terms of their cellular pharmacokinetic character. In P388 cells, amrubicinol was about 80 times as potent as amrubicin, and about 2 times more potent than doxorubicin in a 1-h drug exposure growth-inhibition test. A clear cross-resistance was observed to both amrubicin and amrubicinol in doxorubicin-resistant P388 cells, though the resistance index was lower for amrubicin. The intracellular concentration of amrubicinol was about 6 times and 2 times higher than those of amrubicin and doxorubicin, respectively. Compared to doxorubicin, amrubicin and amrubicinol were released rapidly after removal of the drugs from the medium. A clear correlation was found between the growth-inhibiting activity and the cellular level of amrubicin and amrubicinol in P388 cells. About 10 to 20% of amrubicin or amrubicinol taken up by the cells was detected in the cell nuclear fraction, whereas 70 to 80% of doxorubicin was localized in this fraction. These results suggest that amrubicin and amrubicinol exert cytotoxic activity via a different mechanism from that of doxorubicin.