Koichiro Ootsu

Ansamitocin, a group of novel maytansinoid antibiotics with antitumour properties from Nocardia

WE have isolated a new group of ansamycin antibiotics with potent antitumour activity, from a fermentation broth of Nocardia sp. No. C-15003 (N-l) and have named it ansamitocin. Structures of ansamitocin were found to be similar to maytansine and related maytansinoids obtained from plant sources by Kupchan et al.1–4 and Wani et al.5. These comounds have strong antitumour activities, but development of production would be difficult, because plants containing maytansinoids are only harvested in tropical areas and their content in the plants is extremely low. Some attempts have been made to find a maytansinoid-producing microorganism6, but no success has been reported. We report here the microbial production, isolation and structural elucidation of these antibiotics and their antitumour activities against several experimental tumours in mice.

Enhancement of anti-tumor activity of recombinant interleukin-2 (rIL-2) by immunocomplexing with a monoclonal antibody against rIL-2.

We have investigated biological properties of an immune complex of recombinant interleukin-2 (rIL-2) and a monoclonal antibody against rIL-2 in mice for induction of killer cells and for anti-tumor activity. We have also examined the clearance of subcutaneously-injected immune complex in mice and compared it with that of rIL-2 alone. Plasma rIL-2 levels were sustained longer in mice given the immune complex than in mice given rIL-2 alone at a dose of 10μg/mouse, and they were detectable even at 24 hours after the administration of the immune complex, while they fell to undetectable levels by 6 hours after the administration of rIL-2 alone. A more significant portion of rIL-2 was detected in lymph nodes after subcutaneous injection of the immune complex than that of rIL-2 alone. Splenic lymphocytes from mice given the immune complex demonstrated a higher killer cell activity against YAC-1 cells than those from mice given rIL-2 alone. The immune complex also exerted more significant anti-tumor effect in a dose-dependent manner in Meth-A fibrosarcoma-bearing mice than rIL-2 alone. Our results indicate that immunocomplexing of rIL-2 with an antibody against rIL-2 provides a useful tool as the drug delivery system for cancer therapy using rIL-2.

Therapeutic efficacy of human recombinant interleukin-2 (TGP-3) alone or in combination with cyclophosphamide and immunocompetent cells in allogeneic, semi-syngeneic, and syngeneic murine tumors

SummaryThe potential for a recombinant human interleukin-2 (rIL-2, TGP-3) alone, in combination with cyclophosphamide, and in combination with cyclophosphamide and normal immunocompetent cells to manifest biological activity in vivo was tested using allogeneic, semi-syngeneic, and syngeneic tumor-host systems in mice. The biological activity of rIL-2 was evaluated by the inhibition of the growth of tumors and the inhibition of metastases in short-term assays and, in long-term assays, the prolongation of the survival time of mice bearing subcutaneously (s.c.) or intradermally transplanted tumors. rIL-2 was injected s. c. daily continuously for up to 40 days or intermittently two to four times into mice bearing established tumors. In the short-term assays, the dose and schedule dependence of activity of rIL-2 alone was significantly manifested against sarcoma 180 in ICR mice (allogeneic) by the regression of the tumor, and was confirmed against Meth-A fibrosarcoma in BALB/c mice (syngeneic) by retarding the growth of the tumor. When assessed using these tumors, it was found that the antitumor activity of rIL-2 was scheduledependent: the growth of tumors was more significantly suppressed when rIL-2 was injected every day for 10 days, starting on the 7th day after tumor transplantation, than when rIL-2 was injected five times every other day or twice every 5th day, even if the total amounts of rIL-2 injected were same. The continuous injection for 10 days was considered to be a standard regimen and the daily effective doses of rIL-2 were 5, 10, and 25 µg/mouse. Using the standard regimen and the effective doses, the activity of rIL-2 alone was also observed against two other syngeneic tumors: Colon carcinoma 26 in BALB/c mice, by retarding the growth of the tumor, and Lewis lung carcinoma in C57BL/6 mice by reducing the formation of lung metastases. When assessed using M5076 reticulum cell sarcoma, in a long-term assay, the activity of rIL-2 alone was not manifested in C57BL/6 mice (syngeneic) even when rIL-2 was injected for a long period (20 days) but it was observed in BDF1 (semi-syngeneic) mice. On the other hand, it was found that rIL-2 was effective in combination with cyclophosphamide in prolonging the survival time of C57BL/6 mice bearing the tumor. After cyclophosphamide (2.0 mg) had been administered orally to mice on the 6th day after tumor transplantation, the tumor regressed temporarily but regrew; however, when rIL-2 at a dose of 10 µg was also injected daily for a long period (40 days), the regrowth was retarded and the survival time of the mice was significantly prolonged. Moreover, when normal immunocompetent cells were transferred at the tumor sites, the regrowth of the tumors was retarded more significantly even at a daily dose of 1 µg or 3 µg rIL-2, and mice were observed to be cured by daily doses over 3 µg. The results obtained in the syngeneic tumor-host systems indicate that the continuous injection of rIL-2 is necessary and important for its activity to be manifest in vivo, and that, when combined with cytotoxic drugs and/or with immunocompetent cells, the potential efficacy of rIL-2 is valuable in cancer therapy.

CONVERGENT APPROACH TO WATER SOLUBLE CAMPTOTHECIN DERIVATIVES

Abstract New water soluble camptothecin derivatives with excellent antitumor activity have been designed and synthesized. The synthetic approach includes an efficient route to 7-aminoethyl and 7-aminomethyl camptothecins using either N-Boc protected aminopropion(or aceto)aldehyde or N-methylacetamide as alkylating agents.

Combination Therapy of Colon Carcinoma 26 in Mice with Recombinant Human Interleukin-2 and Interferon-α A/D: Occurrence of Large Granular Cells in the Tumor

The antitumor effects of recombinant human interleukin‐2 (rIL‐2), in combination with recombinant human interferon‐α A/D hybrid (rIFN‐α A/D) on colon carcinoma 26 (colon 26) in mice were examined histologically. Colon 26 was transplanted subcutaneously into female BALB/c mice on day 0. The mice bearing the tumor received intramuscular injection of rIL‐2, rIFN‐α A/D or the combination of rIL‐2 and rIFN‐α A/D for 2–10 consecutive days starting on day 7. Mice were killed on days 9, 13, 17 and 21. After day 13, growth of the tumor was significantly suppressed in the mice treated with rIL‐2 or rIFN‐α A/D alone and was stopped in the mice treated with rIL‐2 in combination with rIFN‐α A/D. Histologically, tumor necrosis developed in all treated groups, though the degree was the most severe in the group receiving combination treatment. Many large cells (about 15 30 μm in diameter) infiltrated into the tumor, and they had Thy‐1 surface antigen and many periodic acid‐Schiff‐positive round grannies in the cytoplasm. The incidence of these large granular cells was correlated well with the reduction in tumor weight. The ultrastructural features of the large granular cells were very similar to those of murine large granular lymphocyte‐like cells maintained in vitro in an IL‐2‐containing medium. The present large granular cells appear to be a kind of activated lymphoid cells.

Antitumor Effect of Recombinant Human Interleukin‐2 on the Growth of Murine Hemangioendothelioma D14 in Nude Mice: Occurrence of Large Granular Cells in the Tumor

The antitumor effect of recombinant human interleukin‐2 (rIL‐2) on murine hemangioendothelioma D14 (D14) in female BALB/c‐nu/nu mice was examined histologically. D14 cells which had been maintained in vitro were transplanted subcutaneously into nude mice on day 0 (1 × 107 cells/mouse). The mice with established tumor on day 28 received rIL‐2 subcutaneously at a dose of 20 μg/mouse/ day for 35 days. On day 63, the mice were killed, and the tumor, spleen and bone marrow were examined histologically. In the mice that had received rIL‐2, tumor growth was significantly suppressed. Histologically, there was marked infiltration of large granular cells (about 15‐30 μ in diameter) in the tumors. In the adjacent areas, there was a significant increase in the number of tumor cells showing karyorrhexis. The large granular cells (LGC) contained periodic acid Schiff‐positive round granules in the cytoplasm and were stained positively for Thy‐1.2 surface antigen. The LGC were also positive for asialo GM1 surface antigen but not for Lyt‐1, Lyt‐2 or IgG surface antigens. This evidence suggests that the LGC are lymphokine‐activated killer‐like cells which were derived from a natural killer cell lineage. The concomitant increases in the number of LGC and the number of cells showing karyorrhexis in the tumors of the mice treated with rIL‐2 suggest that LGC play an important role in the destruction of tumor cells.

An electrophoretic polymorphism in the serum transferrins of Praomys (Mastomys) natalensis

Three serum transferrin phenotypes of Praomys (Mastomys) natalensis, an African rodent having a high incidence of many varieties of spontaneous neoplasms, were detected by electrophoresis and autoradiography. On electrophoresis, transferrin of Trf-K was found to be a protein that migrates slowly toward the cathode, whereas that of Trf-M migrates rapidly. Trf-KM is a heterozygous phenotype of the above two. Progeny testing demonstrated that these three serum transferrin phenotypes in Mastomys are probably controlled by two codominant allelic genes, Trfkand Trfm, at a single autosomal locus. The genotypes of these phenotypes may be Trfk/Trfk, Trfm/Trfm, and Trfk/Trfm.