Eiji Kumazawa

Potent and broad antitumor effects of DX-8951f, a water-soluble camptothecin derivative, against various human tumors xenografted in nude mice

Purpose: We have previously reported that DX-8951f, a water-soluble and nonprodrug camptothecin (CPT) derivative, exhibits both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The purpose of this study was to evaluate the therapeutic efficacy of DX-8951f against human tumor xenografts in nude mice and to compare its activity with those of CPT-11 and other current CPT derivatives. Methods: The antitumor activity of DX-8951f against xenografts of several different types of human tumors was determined in nude mice using a schedule in which DX-8951f was administered intravenously every 4th day for a total of four injections. Results: Against both gastric adenocarcinoma SC-6 and its CPT-11-resistant variant, SC-6/CPT-11, DX-8951f demonstrated superior antitumor activity and antitumor activity over a broader range of doses than did CPT-11, SK&F104864 (hycamtin, topotecan) and GG-211 (GI147211). DX-8951f at 75 mg/kg was effective (growth inhibition rate IR≧58%) against 15 of 16 lines of human cancers examined (6 colon cancers, 5 lung cancers, 2 breast cancers, 1 renal cancer and the above 2 gastric cancers), and exhibited excellent antitumor activity (IR≧80%) against 14 of these lines. CPT-11 exhibited antitumor activity with IR values of 58% and higher against 11 lines and IR values of 80% and higher against only eight of the same 16 human tumors. DX-8951f was effective in inhibiting the growth of an SN-38-resistant tumor and some P-glycoprotein-expressing tumors, but CPT-11 was not. Conclusions: DX-8951f exhibited potent antitumor activity against various types of human tumor xenografts. Its in vivo antitumor effects were superior to those of current camptothecin analogs against certain tumors.

Antitumor Effect of DX‐8951, a Novel Camptothecin Analog, on Human Pancreatic Tumor Cells and Their CPT‐11‐resistant Variants Cultured in vitro and Xenografted into Nude Mice

DX‐8951 is a novel water‐soluble derivative of camptothecin. We evaluated the effects of DX‐8951 on the growth of several pancreatic tumor cell lines in vitro and in vivo. In vitro cytotoxic activity of DX‐8951 against SUIT‐2 and KP‐1N cells, as indicated by IC50 value, was several times more potent than that of SN‐38, an active metabolite of CPT‐11, and dozens of times more potent than that of SK&F104864 (topotecan). DX‐8951 also showed the greatest cytotoxicity against CPT‐11‐resis‐tant variants, SUIT‐2/CPT‐11 and KP‐1N/CPT‐11 cells, and the cross‐resistance of these cells to DX‐8951 was lower than that to SN‐38 and SK&F104864. Topoisomerase 1 inhibitory activity of DX‐8951 was about three‐fold stronger than that of SN‐38, as measured in crude nuclear extract obtained from SUIT‐2 cells. DX‐8951 induced DNA fragmentation, a specific feature of apoptosis, in SUIT‐2 cells more effectively than SN‐38. DX‐8951 exhibited potent antitumor effects against SUIT‐2 in a solid tumor model and in a liver metastasis model, in which tumor cells were xenografted sub‐cutaneously and intrasplenically, respectively, into nude mice. The in vivo effects were closely similar to or somewhat superior to those of CPT‐11, DX‐8951 also showed significant antitumor effects against SUIT‐2/CPT‐11 solid tumors, against which CPT‐11 had no effect. These results suggest that, on the basis of its strong antitumor activity and effectiveness against CPT‐11‐resistant tumors, DX‐8951 may be a useful therapeutic agent in the treatment of human cancer. The potent cytotoxicity of DX‐8951 may result from strong inhibition of topoisomerase I, which may then trigger apoptotic cell death.

DE‐310, a novel macromolecular carrier system for the camptothecin analog DX‐8951f: Potent antitumor activities in various murine tumor models

DE‐310 is a novel macromolecular conjugate composed of DX‐8951f, a camptothecin analog, and a carboxymethyldextran polyalcohol carrier, which are covalently linked via a peptidyl spacer. In a murine Meth A (fibrosarcoma) solid tumor model, once daily×5 treatments (qd×5) with DX‐8951f at the maximum tolerated dose (MTD) were required to shrink the tumor, and DX‐8951f (qd×5) at 1/4 MTD was required to inhibit tumor growth. A single treatment (qd×1) with DE‐310 at the MTD or 1/4 MTD shrank the tumor, with no body weight loss occurring at 1/4 MTD. Even at 1/16 MTD, DE‐310 inhibited tumor growth. In a long‐term assay, Meth A solid tumors disappeared in mice treated with DE‐310 (qd×1) at the MTD and 1/2 MTD, and all 6 mice remained tumor‐free on the 60th day after administration. Repeated injection (4 times) on schedules of every 3 days, 7 days or 14 days demonstrated that multiple treatment with DE‐310 produced greater tumor growth delay than a single treatment with DE‐310. Against 5 human tumor (colon and lung cancer) xenografts in mice, DE‐310 (qd×1) was as effective as DX‐8951f administered once every 4 days, 4 times. The life‐prolonging activity of DE‐310 was assessed in lung (3LL, Lewis lung carcinoma) and liver (M5076, histiocytoma) metastasis models. Against 3LL, DE‐310 (qdx1) at the MTD to 1/3 MTD significantly prolonged survival, with an increase in life span (ILS) of 4.8‐ to 1.6‐fold, respectively, over that in untreated control mice. Also, DE‐310 (qd×1) significantly prolonged survival in the liver metastasis model of M5076. These results demonstrate that DE‐310 is a promising agent for the treatment of cancer.

Antitumour activity of DX-8951f: a new camptothecin derivative.

DX-8951f is a water-soluble camptothecin analogue with a unique hexacyclic structure. Compared to other current camptothecin derivatives, DX-8951f is the most effective topoisomerase I (topo I) inhibitor and has the most potent cytotoxic activity against various tumour cell lines in vitro. Of particular interest is DX-8951fs significant effect on certain tumour cell lines resistant to other camptothecin derivatives, as well as on multi-drug resistant variants that overexpress P-glycoprotein. In addition, in in vivo xenograft systems using nude mice, DX-8951f strongly inhibits the growth of human solid tumours, including resistant tumours. Its antitumour effects and resulting life prolongation in tumour-bearing mice have also been confirmed in several metastasis models. DX-8951f provides greater therapeutic efficacy and broader effective dose ranges using multiple injections than with a bolus injection and simple intermittent applications. The in vivo effects of the compound are superior to those of CPT-11 and SK&F104864, suggesting that DX-8951f is a promising therapeutic agent for the treatment of cancer patients. Phase I clinical trials are ongoing in Europe, the USA and Japan.

Excretion Into Gastrointestinal Tract of Irinotecan Lactone and Carboxylate Forms and Their Pharmacodynamics in Rodents

AbstractPurpose. To investigate the excretion of irinotecan hydrochloride (CPT-11) and its active metabolite, SN-38, into the gastrointestinal lumen via the biliary and/or intestinal membrane route after dosing with lactone and carboxylate forms of CPT-11, and to evaluate the toxic and antitumor effects of the two forms. Methods. The excretions of CPT-11 and SN-38 were investigated by the in situ perfusion technique using rats. The incidence of delayed diarrhea was evaluated after i.v. dosing (60 mg/kg) with CPT-11 lactone and carboxylate forms for 4 days. Antitumor activity and changes in body weight were investigated in mice with Meth A tumors. Results. The excretion of CPT-11 into bile was greater in dosing with CPT-11 carboxylate than that with its lactone form, whereas the exsorption across intestinal membrane was greater in dosing with CPT-11 lactone than that with its carboxylate form. Dosing with CPT-11 lactone dose-dependently inhibited the increase in tumor weights in Meth A tumor mice, whereas the dosing with its carboxylate form reduced the antitumor effect. Conclusions. The decreased antitumor effect caused by dosing with the CPT-11 carboxylate form could be due to less accumulation in the tissue including tumor cells resulting from the rapid elimination of the form in the body.

Significant antitumor effect of a synthetic lipid A analogue, DT-5461, on murine syngeneic tumor models

SummaryThe antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.

Antitumor effect of DT-5461a, a synthetic low-toxicity lipid a analog, involves endogenous tumor necrosis factor induction subsequent to macrophage activation

We previously showed that the synthetic lipid A derivative DT-5461a exhibited significant antitumor effects against various murine solid tumors, probably via activation of host immune systems. To clarify the participation of the macrophage-stimulating effect of DT-5461a in the antitumor mechanisms, we studied the ability of this compound to induce cytostatic macrophages and TNF production in murine systems. Cytostatic macrophages were induced by treatment with DT-5461a either in vitro or in vivo. DT-5461a also induced TNF production by resident peritoneal macrophages or spleen cells obtained from untreated mice. When spleen cells prepared from DT-5461a-treated mice were re-stimulated in vitro with DT-5461a, no TNF was produced by cells obtained at 1 day after the treatment. This may be due to transient refractoriness of macrophages to the compound, since the response to re-stimulation with DT-5461a recovered in cells obtained at 3 or 5 days after treatment. Moreover, while the serum TNF production and antitumor effects by DT-5461a decreased on daily administration, they were elicited by intermittent administration at intervals of 3 days or more. This suggests that the antitumor effects of DT-5461a depend on the TNF-producing activity of macrophages. These results indicate that DT-5461a possesses significant macrophage-stimulating activity, and that macrophages so activated mediate the DT-5461a-induced augmentation of host response against solid tumors.

Intratumoral production of tumor necrosis factor augmented by endogenous interferons results in potent antitumor effects of DT-5461, a synthetic lipid A analog

Summary: We previously reported that DT-5461 exhibits potent antitumor effects on various murine syngeneic tumors, probably via activation of host immune systems. Of the various systemic administration routes, intravenous (i.v.) administration gave the best antitumor effects. When the total dose was fixed, multiple and intermittent applications resulted in greater therapeutic efficacy than single and daily applications, respectively. The therapeutically effective applications of DT-5461 induced endogenous tumor necrosis factor (TNF) activity in serum and tumor tissue. The TNF activity peaked at 1–2 h after the administration. Although TNF activity in the serum declined to an undetectable level by 4 h, intratumoral TNF activity persisted even at 16 h. TNF-α messenger RNA (mRNA) was clearly expressed in the tumor tissues as early as 0.5 h after the DT-5461 administration. DT-5461 also caused increases in interferon activity in tumor-bearing mice. In vivo treatment with anti-interferon-α/β serum or anti-interferon-γ serum, as well as with anti-TNF-α serum, significantly reduced the antitumor effect of DT-5461. DT-5461-induced endogenous TNF production was also inhibited by treatment with either of these anti-interferon antisera alone. These results suggest that intermittent i.v. administration is optimal for cancer treatment with DT-5461, and that the optimal application of DT-5461 causes a long-lasting production of intratumoral TNF-α that may play a crucial role in the antitumor mechanisms of this compound. Furthermore, endogenous interferons induced by DT-5461 are involved in the antitumor mechanisms of this compound, probably by regulating the intratumoral TNF induction.

Antitumor Effect of DT-5461, a Lipid a Derivative, Against Human Tumor Xenografts Is Mediated by Intratumoral Production of Tumor Necrosis Factor and Affected by Host Immunosuppressive Factors in Nude Mice

We previously reported that DT-5461, a synthetic low-toxic lipid A analog, inhibits growth of various murine tumors through activation of host immune systems. In the present study, DT-5461 also exhibited significant antitumor effects against 5 out of 6 human tumor xenografts in nude mice. The antitumor activity was similar to or greater than those of chemotherapeutics. Antitumor effects of DT-5461 significantly correlated with intratumoral levels of tumor necrosis factor (TNF) induced by the compound (r = 0.701, p < 0.05). In vitro TNF production by DT-5461-stimulated macrophages was augmented by tumor cells, and the augmentative effect correlated with TNF activity detected in these tumor tissues. Meanwhile, a weaker therapeutic efficacy of DT-5461 was observed against certain tumors that caused a significant increase in the level of immunosuppressive factors in host blood. These findings support the idea that intratumoral TNF plays a crucial role in the antitumor mechanisms of DT-5461 and suggest that its antitumor action is influenced by an augmentative effect of tumor cells on TNF production and by blood levels of immunosuppressive factors.