Naomi Uchida

Fluorine containing amino acids and their derivatives. 7. Synthesis and antitumor activity of α- and γ-substituted methotrexate analogs

Abstract Three types of reactions of α-substituted malonates, difluoromethylation, alkylation with n,n,n-trifluoroalkyl sulfonates, and Michael addition to 2-substituted-acrylates, conveniently afforded a number of fluorine containing α amino acids such as β-fluorinated-alanines, 2-amino-n,n,n-trifluoroalkanoic acids, and fluorinated glutamic acids as well as other γ-heteroatom substituted glutamic acids. Here, an efficient enzymatic optical resolution using hog kidney acylase was conducted to obtain both optical isomers of 2-amino-n,n,n-trifluoroalkanoic acids. In addition, a novel sulfoxide rearrangement was observed in a base-catalyzed reaction of diethyl α-difluoromethyl-α-sulfoxymalonates. Finally, α- and γ-substituted glutamic acids obtained were used for chemical modification of the antitumor agent methotrexate to reveal remarkable structure-activity relationships. In particular, the significant effects of fluorine substitution on the in vivo antitumor activity were observed.

Sequence-dependent antitumor efficacy of combination chemotherapy with nedaplatin, a newly developed platinum, and paclitaxel.

We evaluated the sequence dependency of antitumor efficacy and toxicity in combination therapy of nedaplatin (NDP) with paclitaxel (TXL) against Lewis lung carcinoma. The sequential administration of NDP prior to TXL (NT therapy) resulted in severe body weight loss followed by frequent toxic death of mice. In contrast, the sequential dosing of TXL prior to NDP (TN therapy) resulted in synergistically enhanced inhibition of tumor growth with less toxicity compared with the NT therapy. Comparing the antitumor activity of NDP plus TXL with that of cisplatin (CDDP) plus TXL or carboplatin (CBDCA) plus TXL, the combination effect of NDP plus TXL was significantly higher than that of CDDP plus TXL or CBDCA plus TXL. These results indicate that the TN therapy may have significant potential in clinical use.

A significant enhancement of therapeutic effect against hepatic metastases of M5076 in mice by a liposomal interleukin-2 (mixture).

Recombinant interleukin-2 (IL-2) was strongly and almost completely adsorbed onto small hydrophobic liposomes under optimal conditions (liposome: DSPC-DSPG; molar ratio, 10:1; 30-50 nm in size, ratio of IL-2 to liposome: 4.0 JRU/nmol lipid). This liposomal IL-2 improved the distribution of IL-2 after intravenous administration as reported, previously. Liposomal IL-2 (300-10000 JRU/mouse per day) was significantly more effective than free IL-2 alone for inhibiting against the experimental metastases of M5076 in mice. The inhibitory effect of liposomal IL-2 was greatest in the liver. The ED(50) of liposomal IL-2 and that of free IL-2 in the liver were 1640 and 12500 JRU/mouse per day, respectively. This simple preparation (mixture) using IL-2 and liposome suspension is expected to have potential for increasing therapeutic efficacy against hepatic metastases.

Urinary retention induced by estrogen injections in mice: an analytical model.

Daily subcutaneous injections of pharmacological doses of 17 beta-estradiol (E2, 0.4 micrograms./gm. body weight) resulted in significant urinary retention in the bladder of castrated mice. Although the urinary retention developed in both male and female mice, the increase in urethral resistance to urinary flow and the dilatation of posterior urethra were observed only in castrated male mice receiving E2. Histological changes common to male and female mice were cornification and stratification of urethral epithelium and fibrosis of connective tissues surrounding the urethra, suggesting that these changes may cause the urinary retention. Although the exact mechanism has not been defined, the urinary retention produced by the present method may be useful as a model of human disease.

Improved in vivo Antitumor Efficacy and Reduced Systemic Toxicity of Carboxymethylpullulan-peptide-doxorubicin Conjugates

The antitumor efficacy of the conjugate of doxorubicin (DXR) and carboxymethylpullulan (CMPul) with Phe‐Gly spacer (CMPul‐FG‐DXR) was evaluated using murine tumor models and compared with that of DXR. The conjugate exhibited higher antitumor efficacy against Lewis lung carcinoma than DXR. Complete tumor regression followed by long‐term tumor‐free survival was frequently observed when CMPul‐FG‐DXR was administered i.v. three times at a dose equivalent to 10 mg/kg of DXR. The superior survival as well as anti‐metastatic effect of CMPul‐FG‐DXR in comparison with DXR was also demonstrated with the M5076 murine reticulosarcoma model. Body weight loss in mice treated with the conjugate was less than that in the DXR‐treated group, indicating lower systemic toxicity of CMPul‐FG‐DXR. Simply mixing CMPul with DXR did not enhance the antitumor activity of DXR, showing that the conjugation of DXR with CMPul is necessary for improved antitumor activity. However, no enhanced antitumor efficacy of the conjugates was observed against a non‐solid tumor model such as P388 leukemia. In summary, improved antitumor efficacy with reduced systemic toxicity of CMPul‐FG‐DXR was demonstrated in the present study. CMPul‐FG‐DXR may be useful as a cancer chemotherapy agent against solid tumors and metastases.

Combination Chemotherapy with Nedaplatin and Cyclophosphamide in Human Ovarian Cancer Model

The antitumor efficacy of the combination of nedaplatin (NDP) with cyclophosphamide (CPM) was evaluated using human ovarian cancer models. Since NDP has been found to have greater anti‐tumor activity and lower nephrotoxicity than cisplatin (CDDP), we also compared the antitumor activity of NDP plus CPM with that of CDDP plus CPM. Increasing doses of NDP (16.5, 33 and 66 mg/kg as a total dose) and a fixed amount of CPM (174 or 348 mg/kg as a total dose) were injected three times at intervals of 7 days via the tail vein into mice implanted with RMUG‐S, OC9‐JCK or KF‐28 human ovarian cancer. Simultaneous administration of NDP with CPM resulted in markedly enhanced inhibition of tumor growth for all cancers tested. The growth inhibition and survival effect of the combination therapy of NDP with CPM against KF‐28 and OC9‐JCK were as potent as those of CDDP plus CPM. Neither increased hematotoxicity nor a significant difference in maximum concentration, half time or area under the curve of platinum or CPM in plasma between the single and combined treatment was found. These results suggest that the combination of NDP with CPM may be clinically effective.

DEVELOPMENT OF SPINDLE-SHAPED CELLS AND CHONDROID CELLS FROM ANDROGEN-DEPENDENT SHIONOGI CARCINOMA 115: A Light and Electron Microscopic Study

Androgen‐dependent Shionogi carcinoma 115 (SC115) is an undifferentiated medullary carcinoma consisting of compact round cells. However, when host male DS mice were castrated 2 weeks after tumor transplantation, tumors composed of compact round cells, spindle‐shaped cells and chondroid cells grew 4 weeks after castration. Compact round cells with desmosomes were arranged in solid nests and exhibited immunoreactivity for keratin protein. Spindle‐shaped cells had prominent rough endoplasmic reticulum, and appeared to secrete collagen. Chondroid cells had the characteristics of chon‐drocytes. The light and electron microscopic features were highly suggestive of a transition from compact round cells to spindle‐shaped cells, and from spindle‐shaped cells to chondroid cells. The histology of this tumor thus suggests that SC115 cells are able to change into chondroid cells via spindle‐shaped cells. ACTA PATHOL JPN 38: 1405–1416, 1988.

Adjuvant chemo-endocrine therapy for androgen-dependent mammary tumor in mice

SummaryAbout 500 male DS mice grafted with androgen-dependent Shionogi carcinoma 115 (SC115) were used. When the tumor diameter reached about 20 mm (approximately 25 days after transplantation), excision of the tumor and/or castration were carried out. The injection of cyclophosphamide (80 mg/kg body weight × 3 at 7-day intervals) was started from the day after excision.In mice with excised tumor, adjuvant chemo-endocrine therapy was the most effective treatment examined; cumulative 120-day mortalities after transplantation of tumors in non-treated, adjuvant endocrine therapy, adjuvant chemotherapy and adjuvant chemo-endocrine therapy groups were 91%, 29%, 21% and 0%, respectively. Castration induced development of clusters of androgen-independent cancer cells in androgendependent SC115 tumor. In mice without tumor excision, the chemo-endocrine therapy was again the most effective treatment, though 86% of mice died by the 120th day after tumor transplantation. These findings suggest the usefulness of adjuvant chemo-endocrine therapy for achieving complete remission in hormonedependent tumors.

Maintenance of androgen-, glucocorticoid- or estrogen-responsive growth in shionogi carcinoma 115 subline sustained in castrated mice with high dose of estrogen for 30 generations (3 years).

Shionogi carcinoma 115 (SC115), an androgen‐dependent mouse mammary tumor, rapidly loses its androgen responsiveness after androgen withdrawal. The growth of this tumor can also be stimulated by high doses of estrogen or glucocorticoid. In the present study, the maintenance of hormone‐responsive growth of SC115 tumors with a high dose of estrogen was examined in castrated male mice using an SCI 15 subline obtained by serial transplantations of SCI 15 tumors in estrogen‐treated castrated mice for 3 years (30 generations) (subline E2). Seed tumors from both SC115 and subline E2 could rapidly grow in castrated mice given daily injections of testosterone propionate (TP), 17β‐estradiol (E2), or dexamethasone (Dex) (100 μg/mouse/day) but not in those given vehicle alone. Although SCI 15 and subline‐E2 tumors grown with TP or Dex showed temporary regression after steroid withdrawal, the tumors grown with E2 did not show such temporary regression. The TP‐, E2‐, or Dex‐induced growth of subline‐E2 tumors was almost the same as that of the original SCI 15 tumors. However, responsiveness to androgen, estrogen or glucocorticoid of both tumors disappeared within one passage in steroid‐depleted castrated mice. The present findings demonstrate that the loss of responsiveness to androgen as well as to high doses of estrogen or glucocorticoid of SCI 15 tumors can be prevented in castrated mice not only with androgen but also with high doses of estrogen.

Increase in collagen production with loss of androgen responsiveness in cultured androgen-responsive Shionogi carcinoma 115 cells

The collagen production of androgen-responsive and -unresponsive Shionogi carcinoma 115 cells was investigated by culturing them in a medium with or without testosterone. Androgen-unresponsive cells were obtained by culturing a cloned androgen-responsive cell in a testosterone-free medium for 12 weeks. The collagen production of androgen-responsive cells slightly increased in the absence of testosterone, whereas testosterone did not affect the collagen production of androgen-unresponsive cells. Androgen-unresponsive cells produced 3-4 times more collagen than androgen-responsive cells. The major collagen produced by both androgen-responsive and - unresponsive cells migrated to the same position in sodium dodecylsulfate:polyacylamide gel electrophoresis. The present results indicate that the collagen production of androgen-responsive Shionogi carcinoma 115 cells increases with the loss of androgen responsiveness in culture.