Antitumor Activities of IKP-104, a 4(1H)-Pyrizinone Derivative, on Cultured and Implanted Tumors

Antitumor activities of IKP-104, a 4(1 H )-pyrizinone derivative, were investigated with cultured tumor cell lines and implanted tumors in mice. IKP-104 inhibited the growth of cultured murine tumor cell lines (L1210 leukemia, Lewis lung carcinoma and B16 melanoma) and human tumor cell lines (K562 leukemia and HeLa cervical carcinoma). It also had antitumor effects on implanted murine ascitic tumors (L1210 leukemia and sarcoma 180) and a murine solid tumor (Lewis lung carcinoma). IKP-104 could be classified as a phase-dependent cytostatic drug based on the mode of growth inhibition of cultured B16 melanoma cells compared with those of several other antitumor agents. The effect of IKP-104 on the cell cycle traverse of cultured B16 melanoma cells was estimated by morphological and flow cytometric analyses. Cells accumulated in the mitotic phase, and abortive mitosis or polyploidy or multinucleation was induced from 6 h after exposure to IKP-104. Based on these results, IKP-104 is expected to be useful for the treatment of tumors, and its mode of action seemed to be similar to that of metaphase arrestants such as colchicine or vinca alkaloids.     

Effects of the tumor inhibitor IKP-104, a 4(1H)-pyridinone derivative, on cytoskeletal microtubules of cultured tumor cells.

The effects of IKP-104, a 4(1H)-pyridinone derivative, on the mitotic profile and cytoskeletal microtubule dynamics of cultured B16 melanoma cells were examined in order to investigate the mechanism of its antitumor activity. The exposure to IKP-104 caused accumulation of cells in abnormal metaphase with chromosomes scattered within the cytoplasm and induced polyploid and multinucleate cells as detected by differential staining microscopy with brilliant blue R and safranin O. An immunofluorescence study with monoclonal anti-alpha-tubulin antibody revealed that IKP-104 diminished cytoskeletal microtubules of both interphase and mitotic cells, resulting in induction of a few fragments resembling "microtubular bundles" induced by vinblastine (VLB). These results indicated that IKP-104 arrests cells in the mitotic phase by inhibition of polymerization and induction of depolymerization of cytoskeletal microtubules, similarly to VLB.     

Interaction of the tumor inhibitor IKP-104, a 4(1H)-pyridinone derivative, with microtubule proteins.

The effects of a mitotic arrestant, IKP-104, which has an antitumor activity, on the in vitro polymerization and depolymerization of rat brain microtubules were investigated. IKP-104 inhibited microtubule polymerization at concentrations greater than 0.71 x 10(-6) M, and its IC50 value was determined to be 1.31 x 10(-6) M by probit analysis. Fifty-two percent of pre-polymerized microtubules depolymerized at 1.31 x 10(-6) M IKP-104. Electron micrographs of microtubules taken immediately after treatment with 1 x 10(-3) M IKP-104 revealed a fraying of microtubule ends into elongated coil-like filaments, which were composed of 2 or 3 protofilaments. When microtubule protein treated with 1 x 10(-3) M IKP-104 was cleaved by trypsin, fragments of 41, 36, 34, 23, 21, 19 and 16 kilodaltons (kDa) derived from alpha-tubulin were produced. In particular, the 19, 23, and 34 kDa fragments were characteristically observed in the trypsin cleavage of microtubules tested with IKP-104, and these fragments were not observed with untreated microtubules. The effects of IKP-104 on microtubule protein mentioned above were mostly similar to those of vinblastine (VLB) and we suggest that IKP-104 bound to the site or sites near "VLB-binding site or sites" of alpha-tubulin subunit, resulting in induction of conformational changes.     

Interaction of the Tumor Inhibitor IKP-104, a 4(1H)-Pyridinone Derivative, with Microtubule Proteins

The effects of a mitotic arrestant, IKP-104, which has an antitumor activity, on the in vitro. , polymerization and depolymerization of rat brain microtubules were investigated. IKP-104 inhibited microtubule polymerization at concentrations greater than 0.71 × 10^-6 M. ,a nd its IC₅₀ value was determined to be 1.31 × 10^-6 M by probit analysis. Fifty-two percent of pre-polymerized microtubules depolymerized at 1.31 × 10^-6 M IKP-104. Electron micrographs of microtubules taken immediately after treatment with 1 × 10^-3 M IKP-104 revealed a fraying of microtubule ends into elongated coil-like filaments, which were composed of 2 or 3 protofilaments. When microtubule protein treated with 1 × 10^-3 M IKP-104 was cleaved by trypsin, fragments of 41,36, 34, 23,21,19 and 16 kilodaltons (kDa) derived from a-tubulin were produced. In particular, the 19, 23 and 34 kDa fragments were characteristically observed in the trypsin cleavage of microtubules treated with IKP-104, and these fragments were not observed with untreated microtubules. The effects of IKP-104 on microtubule protein mentioned above were mostly similar to those of vinblastine (VLB) and we suggest that IKP-104 bound to the site or sites near "VLB-binding site or sites" of α-tubulin subunit, resulting in induction of conformational changes.     

Effects of the Tumor Inhibitor IKP-104, a 4(lH)-Pyridinone Derivative, on Cytoskeletal Microtuhules of Cultured Tumor Cells

The effects of IKP-104, a 4(l H )-pyridinone derivative, on the mitotic profile and cytoskeletal microtubule dynamics of cultured B16 melanoma cells were examined in order to investigate the mechanism of its antitumor activity. The exposure to IKP-104 caused accumulation of cells in abnormal metaphase with chromosomes scattered within the cytoplasm and induced polyploid and multinucleate cells as detected by differential staining microscopy with brilliant blue R and safranin O. An immunofluorcsceiicc study with monoclonal anti-α-tubulin antibody revealed that IKP-104 diminished cytoskeletal microtubules of both interphase and mitotic cells, resulting in induction of a few fragments resembling "microtubular bundles" induced by vinblastine (VLB). These results indicated that IKP-104 arrests cells in the mitotic phase by inhibition of polymerization and induction of depolymerization of cytoskeletal microtubules, similarly to VLB.     

Antitumor activity of a nitrosourea derivative,CNUA, on murine tumors

Summary The antitumor activity of a new derivative of nitrosourea, 3-[3-(2-chloroethyl)-3-nitrosoureido]-3-deoxy-d-allose (CNUA), against murine tumors was studied. CNUA showed significant antitumor activity against L1210 leukemia, Lewis lung carcinoma, B-16 melanoma and autochthonous lung tumor induced by 1-ethyl-1-nitrosourea. The effect of CNUA, chlorozotocin, and ACNU on the peripheral white blood cell count (WBC) in normal CDF₁ mice was examined. The lowest WBC count occurred 3 days after administration at the therapeutic dose level and the decreased value returned to the normal level 7–14 days following administration of CNUA and chlorozotocin. CNUA also exerted a depressive action on both humoral and cell-mediated immune response to sheep red blood cells determined by the serum hemagglutinin titer, plaque-forming cells in the spleen, and delayed-type hypersensitivity reaction, while the suppression was almost the same or less than that obtained with chlorozotocin when compared at the dose resulting in similar antitumor activity. These findings suggest that the antitumor activity of CNUA was not at all inferior to those of other nitrosoureas. The bone marrow toxicity was moderate and did not last long.     

Antitumor activities of a newly synthesized shikonin derivative, 2-hyim-DMNQ-S-33

2- or 6-(1-hydroxyiminoalkyl)-5,8-dimethoxy-1, 4-naphthoquinone(2- or 6-hyim-DMNQ) derived from the roots of Lithospermum erythrorhizon was synthesized for the evaluation of antitumor activities. Among those derivatives, 2-hyim-DMNQ-S33 was found to be a potent anticancer agent. This compound suppressed the proliferation of Radiation Induced Fibrosarcoma (RIF) cells in a dose-dependent manner. 2-hyim-DMNQ-S33 significantly prolonged the survival time by 239% as compared with Sarcoma 180 tumor-bearing control mice in vivo. We found that the compound significantly suppressed phosphorylation of extracellular signal-regulated kinase (pERK) and activated c-jun-N-terminal kinase (JNK) and protein kinase C (PKC)-alpha following 4 h-treatment. These findings indicate that 2-hyim-DMSQ-S33 exerts antitumor activities by regulating pERK, JNK and PKC-alpha.     

Antitumor activity of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothec in, a novel water-soluble derivative of camptothecin, against murine tumors

The search for new water-soluble analogues of camptothecin (CPT) with higher activity and less toxicity has led to the development of a novel compound, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (CPT-11), which showed significant antitumor activity against a broad spectrum of experimental tumor models by i.p., i.v., or oral administration. When its activity against L1210 was compared with that of CPT and known derivatives, CPT-11 was most effective, giving the highest maximum increase in life span (ILS) and showing good activity over a wide dose range. The antitumor activity of CPT-11 was shown against tumors not only in the ascites form but also in the solid form. Included among the more susceptible murine tumors are S180, Meth A fibrosarcoma, Lewis lung carcinoma, Ehrlich carcinoma, MH134 hepatoma, mammary carcinoma of C3H/HeN mice, L1210, and P388 leukemia. Probable cures of these tumors were induced frequently by CPT-11. The antitumor activity of CPT-11 against i.p.-implanted L1210 was superior to that of Adriamycin in maximum ILS, the number of cured mice, and the therapeutic ratio. CPT-11 at a dose of 100 mg/kg produced an ILS in excess of 300% with five of six mice surviving tumor free, and effected 100% tumor regression at 200 mg/kg, whereas the optimum dose of Adriamycin, 12.5-25 mg/kg, brought about 114-129% ILS with one of six mice surviving. The acute toxicity of CPT-11 was extremely low, particularly in the case of oral administration. CPT-11 is expected to be clinically useful.     

Antitumor activity on murine tumors of a novel antitumor benzoylphenylurea derivative,HO-221

Summary A novel antitumor compound,N-[4-(5-bromo-2-pyrimidinyloxy)-3-chlorophenyl]-N-(2-nitrobenzoyl) urea (HO-221) was evaluated for its antitumor activity in experimental tumor models. HO-221 preparation was given orally to tumor-bearing animals. The compound exhibited significant effects against various tumors such as P388 and L1210 leukemias; M5076 reticulum-cell sarcoma; colon 38 carcinoma; human xenografts MX-1, LX-1, GA-1, and Co-1; Lewis lung carcinoma; sarcoma 180; and Walker 256 carcinosarcoma and was especially effective against solid tumors. However, its effect on murine B16 melanoma was moderate. Intermittent administration of HO-221 produced better results. The effects of HO-221 on human tumor xenografts were compared with those of other antitumor agents. HO-221 showed activity against LX-1 lung and Co-1 gastrointestinal tumor and was also effective against advanced-stage L1210 leukemia and Lewis lung carcinoma. Furthermore, the effect of HO-221 on drug-resistant tumors was examined using murine leukemias L1210 and P388. It showed no cross-resistance with the known antitumor agents Adriamycin (ADM), daunomycin (DM), vincristine (VCR), mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5-FU), cytosine arabinoside (Ara-C), methotrexate (MTX), cyclophosphamide (CPA), or carboquone (CQ), and collateral sensitivity to HO-221 was found in MMC-, CDDP-, and CPA-resistant sublines. HO-221 exhibits significant reproducible, broadspectrum antitumor activity against experimental tumors as well as human neoplasms.     

Antitumor activities and schedule dependence of orally administered MST-16, a novel derivative of bis(2,6-dioxopiperazine)

Summary We studied bioavailability, treatment schedule dependence, and therapeutic efficacy of orally administered MST-16, a novel derivative of bis(2,6-dioxopiperazine), against murine tumors and human tumor xenografts. The rate of its intestinal absorption was about 50%, and it was immediately metabolized to its parent compound, ICRF-154. Therapeutic efficacy of MST-16 was heavily dependent on the treatment schedule: 9 daily oral administrations and treatment every 4 h on day 1 only were much more effective against s.c.-implanted L1210 leukemia than a single dose or five daily administrations giving the same total dose. Orally administered MST-16 showed potent lifeprolonging effects (196%, 219% and 148%) in mice inoculated i.p. with P388, L1210 leukemia, and C-26 colon adenocarcinoma, respectively, but had no effect on B16 melanoma inoculated in the same way. MST-16 inhibited more than 80% growth of Lewis lung carcinoma, B16 melanoma, and C-38 colon adenocarcinoma implanted s.c., but had only a minor effect on M5076 fibrosarcoma. Lung metastasis of Lewis lung carcinoma was also effectively suppressed. Furthermore, MST-16 significantly inhibited growth of human colon, lung and breast cancers implanted s.c. in nude mice. We also made a kinetic analysis of the in vitro cell-killing effect by ICRF-154, the active form of MST-16 in vivo. It demonstrated a cell cycle phase-specific and time-dependent action, providing a reasonable explanation for the schedule-dependent therapeutic effect of MST-16.     

Antitumor activity of ME2303, a fluorine-containing anthracycline, against human tumors implanted in nude mice

A fluorine-containing anthracycline, ME2303, given intravenously once a week for 4 weeks at the maximum tolerated doses showed better therapeutic effects against 2 gastric, 3 lung and 2 human breast tumor xenografts than did adriamycin (ADM) at the maximum tolerated dose. Among the tumors, ME2303 showed a better effect against St-40, a well-differentiated human gastric adenocarcinoma, against which ADM showed only a marginal effect. Likewise, ME2303 was more effective against Lu-24 human small cell carcinoma and MX-1 human medullary tubular adenocarcinoma than ADM. Notably, the Lu-24 tumor, developed in nude mice, disappeared after the treatment in 3 out of 6 mice. ME2303 would be an interesting compound for phase I and II clinical studies in the future.     

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.     

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.     

Antitumor activity of a new fluoropyrimidine derivative, 5'-deoxy-5-fluorouridine, against murine and human experimental tumors

5'-Deoxy-5-fluorouridine (5'-DFUR) was evaluated for antitumor activity against four murine tumors (L1210 leukemia, P388 leukemia, Lewis lung carcinoma, and B16 melanoma) and a human mammary carcinoma (MX-1) xenografted in athymic mice. Intraperitoneal administration of 5'-DFUR was ineffective against B16 melanoma implanted intraperitoneally and showed less marked antitumor activity against P388 and L1210 leukemias implanted intraperitoneally or intravenously as compared with that of 5-fluorouracil (5-FU) or 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207), while oral administration of 5'-DFUR showed a similar or superior antitumor activity to that of 5-FU or FT-207 against L1210 leukemia implanted subcutaneously. 5'-DFUR showed a marked antitumor activity against MX-1 implanted subcutaneously and also showed slight antitumor activity against Lewis lung carcinoma implanted subcutaneously, while 5-FU and FT-207 did not show any significant antitumor activity against these tumors. These results suggest that 5'-DFUR may be worthy of clinical trial against solid tumors, especially cancers of the breast.     

Antitumor activities of newly synthesized N4-acyl-1-beta-D-arabinofuranosylcytosine.

New derivatives of 1-beta-D-arabinofuranosylcytosine were synthesized and their antitumor activities were tested against mouse leukemia L1210. Among the 50 compounds investigated, a series of N4-acyl derivatives with long-chain saturated fatty acids were found to be highly active. The most active derivatives were N4-stearoly-1-beta-D-arabinofuranosylcytosine, which was administered in the form of suspension, and N4-behenoyl-1-beta-D-arabinofuranosylcytosine given in the form of solution. They were superior to the parent compound, 1-beta-D-arabinofuranosylcytosine, in that smaller dosages exhibited strong activities regardless of the treatment schedule, and they were also resistant to cytidine deaminase.     

Effects of a new amsacrine derivative, N-5-dimethyl-9-(2-methoxy-4-methylsulfonylamino)phenylamino-4- acridinecarboxamide, on cultured mammalian cells

The effects of N-5-dimethyl-9-(2-methoxy-4-methylsulfonylamino)-phenylamino-4- acridinecarboxamide (CI-921; NSC 343499), a lipophilic and water-soluble derivative of amsacrine (NSC 249992), on cell viability, growth, clonogenicity, and progression through the cell cycle were investigated in suspension cultures of Friend erythroleukemic cells and in in suspension cultures of Friend erythroleukemic cells and in adherent cultures of Chinese hamster ovary cells. CI-921 was less toxic toward stationary than toward exponentially growing Chinese hamster ovary cells; colony formation was inhibited by 50% following a 1-h pulse of 190 versus 80 nM CI-921, respectively. Cell viability was unaffected in Friend erythroleukemic cell cultures at concentrations up to 50 nM, although growth was inhibited by 50% following 24 h of continuous exposure to 9.5 nM or a 1 h pulse of 67.5 nM CI-921. Constant exposure of Friend erythroleukemic cells to 10 nM CI-921 slowed proliferation and resulted in prolongation of cell transit through late S and G2 phases. Higher drug concentrations (50 nM) caused a complete cessation of growth marked by greatly suppressed cell transit through S phase and an irreversible block in G2 phase, about 30 min prior to division. In such cases, unbalanced growth was observed with total RNA and protein content of drug-treated cells increasing by 74 and 34%, respectively. Pulse exposure of cells to CI-921 resulted in transient accumulations of cells in S and/or G2 phase depending upon dose. The cell cycle distribution of stationary cultures treated for 1 h with drug and replated at a low cell density were identical to that of controls. Binding of the drug affected the sensitivity of DNA in situ to acid denaturing conditions which provides additional evidence that CI-921 binds to DNA by intercalation.     

Antitumor and antivascular effects of AC-7700, a combretastatin A-4 derivative,against rat liver cancer

Background. Unlike the many chemotherapeutic agents that do not effectively stop blood flow or induce necrosis in hepatocellular carcinoma, AC-7700 has been shown to inhibit tubulin polymerization and selectively stop tumor blood flow. The aim of this study was to elucidate the antivascular and antitumor effects of AC-7700 on rat hepatoma. Methods. AH-130 cells, a rat hepatoma cell line, were solidified and implanted into the liver of Donryu rats. Vascularity of the liver tumor was directly identified by in-vivo fluorescence microscopy from 0 to 60 min after the injection of 10 mg/kg AC-7700. To observe the antivascular effect of AC-7700, the vascular density of the tumor was measured and assessed as the ratio of preinjection to postinjection values. The antitumor effects were evaluated with histopathologic findings and analysis of animal survival. Results. In-vivo microscopic observation showed that tumor perfusion diminished within 30 min after AC-7700 administration. Vascular density in the AC-7700 group was significantly less than that in the control group at 60 min (AC-7700, 26.3 ± 16.4%; control, 88.5 ± 9.2%; P < 0.001). After AC-7700 injection, marked necrosis of tumor cells was observed histologically, and tumor area was decreased significantly (AC-7700, 11.5 ± 15.4 mm²; control, 43.5 ± 18.3 mm²; P < 0.05). The survival rate (50%) of the AC-7700 group animals was better than that of the control group (0%; P < 0.01). Conclusion. Markedly decreased tumor perfusion was induced by AC-7700 within 30 min, and this decrease may have contributed to the tumor necrosis and favorable outcome in the treatment group. AC-7700 appears to be a promising agent for the treatment of hepatocellular carcinoma. Received: September 14, 2001 / Accepted: February 21, 2002     

Antitumor activity of a human cytotoxic T-cell line (TALL-104) in brain tumor xenografts

Malignant glioma in adults and primitive neuroectodermal tumors/medulloblastomas in children are the most common malignant primary brain tumors that either respond poorly to current treatment or tend to recur. Adoptive therapy with TALL-104 cells-an IL-2-dependent, major histocompatibility complex nonrestricted, cytotoxic T-cell line-has demonstrated significant antitumor activity against a broad range of implanted or spontaneously arising tumors. This study investigates distribution of systemically and locally administered TALL-104 cells and their efficacy in effecting survival of a rat model of human brain tumor. In vitro, TALL-104 cells showed significant cytotoxic activity when added to human glioblastoma cell lines U-87 MG, U-251 MG, and A1690; the medulloblastoma cell lines DAOY, D283 Med, and D341 Med; and the epidermoid cancer cell line A431. In brain tumor-bearing rats, the amount of fluorescent dye-labeled TALL-104 cells in brain increased after they were given by intracarotid injection as compared with i.v. cell administration. However, TALL-104 cells rapidly decreased to low levels within 1 h after intracarotid injection. This finding suggests that TALL-104 cells given systemically may not invade brain or tumor tissues, but rather may remain in the vascular system, making this approach less efficient for brain tumor treatment. In a model of athymic rats engrafted with human A431 carcinoma brain tumor, repetitive local administration of TALL-104 cells directly into the tumor bed resulted in a significant increase in survival time compared with control animals. Therefore, local therapy with TALL-104 cells may be a novel and highly effective treatment approach for malignant brain tumors.     

Antitumor effect of CPT-11, a new derivative of camptothecin,against pleiotropic drug-resistant tumors in vitro and in vivo

Summary CPT-11, a new derivative of camptothecin, was effective against tumor cells, especially vincristine (VCR)-and adriamycin (ADM)-resistant P388 leukemia, compared to either VCR or ADM. The drug showed superior chemotherapeutic effects over VCR and ADM in sensitive P388 leukemia-bearing mice, and was also effective in VCR-and ADM-resistant P388 leukemia-bearing mice. These latter survival advantages with CPT-11 were almost equal to those obtained by CPT-11 against sensitive P388 leukemia. CPT-11 was found to be effective against human tumor cells, especially various pleiotropically drug-resistant human tumor lines, compared to VCR and ADM. CPT-11 should be considered for further development as a new chemotherapeutic agent potentially effective against pleiotropically drug-resistant tumors.