[Evaluation of antitumor activity of etoposide administered orally for 21 consecutive days against human uterine cancer subcutaneous and/or orthotopic xenografts in nude mice].

The antitumor activity of etoposide (ETP) against human uterine cancer cell lines were investigated in vitro and in vivo. The cytotoxic activity of ETP against HeLa S3, a human cervical cancer cell line, depended on exposure time. The survival rate with 24 h prolonged exposure was reduced to about 1/200 that with 6 h exposure. The time dependency of antitumor activity of ETP against HeLa S3 subcutaneously transplanted in nude mice was studied. The effect of 21 or 28 consecutive days oral administration was greater than that of 5 or 14 consecutive days. Furthermore, a longer administration schedule was less toxic. The antitumor activity of ETP administered orally for 21 consecutive days was compared with that of CDDP, CPT-11 and 5'-DFUR using both human uterine cancer cell lines (TCO-1, SIHA, UCC08JCK) transplanted subcutaneously in nude mice and human uterine cancer cell lines (HeLa S3, UCC08JCK) transplanted into the uterus of nude mice. ETP showed the same antitumor activity as CPT-11 and 5'-DFUR against TCO-1 and UCC08JCK, human uterine cancer cell lines transplanted subcutaneously in nude mice. ETP also showed anticancer activity against two cell lines transplanted into the uterus. The growth inhibition caused by ETP administered orally at 50 mg/kg against HeLa S3 transplanted subcutaneously was 36.7% while that against the same cell line transplanted into the uterus was 58.5%. 5'-DFUR also showed the same antitumor activity as ETP. These results suggest that long term oral administration of ETP is clinically useful for cervical cancer patients.

ICAM-1 mediates lung leukocyte recruitment but not pulmonary fibrosis in a murine model of bleomycin-induced lung injury.

Bleomycin-induced lung injury has been extensively used as a model of interstitial pneumonia and pulmonary fibrosis. Intercellular adhesion molecule (ICAM)-1 is a ligand for lymphocyte function-associated antigen (LFA)-1alpha and has been shown to be required for leukocyte migration into inflamed areas. The purpose of this report was to investigate the role of the ICAM-1/LFA-1alpha pathway in a murine model of bleomycin-induced lung injury. Animals received 75 mg x kg(-1) bleomycin (BLM) i.v. followed by treatment with phosphate-buffered saline (BLM group), anti-ICAM-1 and LFA-1alpha monoclonal antibodies (mAb) (BLM+mAb group). Inflammatory cell counts of bronchoalveolar lavage (BAL) fluid, hydroxyproline content and histological findings were compared between these groups. In the BLM group, significant increases in total cell count, macrophage count and neutrophil count of BAL fluid were observed on days 7 and 14. In the BLM+mAb group, bleomycin-induced accumulation of neutrophils was significantly reduced on days 7 and 14 (p<0.01). However, the administration of mAb to ICAM-1 and LFA-1alpha did not decrease the lung hydroxyproline content or the histopathological fibrosis grading score, indicating that the antagonism of ICAM-I and LFA-1alpha did not attenuate bleomycin-induced pulmonary fibrosis. This study suggests that the intercellular adhesion molecule-1/lymphocyte function-associated antigen-1alpha pathway mediates the accumulation of inflammatory cells in the injured lung caused by bleomycin; however, other mechanisms are important for the subsequent development of pulmonary fibrosis.

Malignant transformation by overproduction of translation initiation factor eIF4G.

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.

[Our policy and future tasks related to ICH in anti-cancer drug development--a discussion from the viewpoint of an enterprise].

International harmonization of data to be submitted in order to receive governmental authorization for the release of new medicines (ICH) has been promoted by Japan, the USA and European countries. Because of this trend, the Japanese drug authority, the medical institutions which perform clinical trials of not yet authorized new drugs and the pharmaceutical companies which contract these trials to medical institutions, are now required to change greatly their conventional views concerning drug development. The trend towards ICH has also been having a great impact on the development of anti-cancer agents in Japan. The greatest impact of ICH is that it requires pharmaceutical companies to take responsibility for the planning, pursuit and management of clinical trials of new drugs. To meet this requirement, it is urgent that pharmaceutical companies educate and train staff members involved in drug development, so that they attain high levels of medical proficiency in the field concerned. It is also necessary for these companies to organize in house groups of specialists in making clinical trials, who can evaluate clinical data and make decisions about outcomes by themselves. At the same time, medical institutions are required to establish a system which supports the clinical trials carried out within the hospital, while meeting the appropriate guidelines. Thus, medical institutions are required to make greater efforts to ensure adequate disclosure of diagnosis of cancer to a patient, obtain informed consent from patients and develop a hospital system capable of conducting excellent clinical trials. The governmental authority related to drugs is required to improve drug administration, including streamlining regulations and providing consultation services concerning the appropriate strategy for particular clinical trials. If the relevant governmental authority, medical institutions, pharmaceutical companies and mass media cooperate with the goal of improving the environment and systems related to clinical trials, the current system of clinical trials will be improved significantly, allowing more scientific and ethical clinical trials. This, in turn, will promote the smoother development of anti cancer agents in this country. At present, both the views on and the manner of conducting clinical trials (especially phase I clinical trials) differ in Japan and Western countries. These differences cause differences in the scheduling of preclinical studies, possibly leading to delayed commencement of phase I clinical trials in Japan. Among these issues, the procedures for preclinical studies of safety and pharmacokinetics studies (absorption, distribution, metabolism and elimination of drugs) need to be internationally standardized as soon as possible.

Triple paraneoplastic syndrome of hypercalcemia, leukocytosis and cachexia in two human tumor xenografts in nude mice.

Nude mice bearing the human oral cavity carcinoma cell line OCC-1, and the lung cancer cell line LC-1, developed a triple paraneoplastic syndrome consisting of hypercalcemia, cachexia and leukocytosis. All of these abnormalities disappeared rapidly after surgical resection of the tumors, suggesting their ectopic humoral nature. Search for the factors responsible for the respective abnormalities revealed that the production of parathyroid hormone-related protein and colony-stimulating factors (CSFs), mainly granulocyte-CSF, by the tumors could explain the hypercalcemia and leukocytosis, respectively. With regard to the severe cachexia, the production of two cachexia-associated cytokines, interleukin-6 and leukemia inhibitory factor, was able to explain the syndrome in OCC-1 bearing nude mice; however, the factor responsible in LC-1 bearing nude mice could not be identified. The triple paraneoplastic syndrome that developed in these two animal models could be explained partly by concomitant production of the peptide hormone and cytokines by cancer cells. These animal models may be very useful for the evaluation of diagnostic and therapeutic modalities for humoral abnormalities.

Inhibition of the growth of various human and mouse tumor cells by 1,15-bis(ethylamino)-4,8,12-triazapentadecane.

Effects of 1,15-bis(ethylamino)-4,8,12-triazapentadecane (BE3333), the least toxic bis(ethyl)pentaamine, on the growth of tumor cells were studied in in vitro systems and with tumor xenografts in mice. BE3333 suppressed ornithine decarboxylase and S-adenosylmethionine decarboxylase, induced spermidine/spermine N1-acetyltransferase, and thus decreased the amount of polyamines. BE3333 accumulated in cells at a concentration 3-5-fold that of spermine in control cells through the polyamine transport system. The accumulated BE3333 inhibited protein synthesis, especially mitochondrial protein synthesis, and decreased the amount of ATP. The inhibition of protein synthesis was correlated with the subsequent inhibition of cell growth. BE3333 showed inhibitory effects in in vitro systems against the growth of mouse FM3A mammary carcinoma cells, human SW480 and SW620 colon tumor cells, Lu-65A and A549 lung tumor cells, MCF-7 breast tumor cells, and MALME-3M and A375 melanoma cells at a range of 0.5-10 microM. Intravenous (30 mg/kg) or i.p. (50 mg/kg) daily injections of BE3333 for 5 or 7 days greatly suppressed the growth of human colon tumor SW620 xenotransplanted into nude mice. Similar antitumor activity was obtained with continuous infusion of BE3333 into the peritoneal cavity (80 mg/kg), but not with p.o. administration (200 mg/kg). BE3333 also showed inhibitory effects against the growth of lung tumors (Lu-65, Lx-1, Lc-1, and Lu-61), stomach tumors (Sc-6 and St-15), and melanoma (SEKI) xenotransplanted into nude mice. The results indicate that BE3333 is effective against both rapid- and slow-growing tumors, with reasonable short-term host toxicity.

Antitumoral efficacy and pharmacokinetic properties of pirarubicin upon hepatic intra-arterial injection in the rabbit V x 2 tumour model.

To improve the efficiency of hepatic intra-arterial (h.i.a.) chemotherapy, we selected pirarubicin (THP) because it shows good properties for h.i.a. chemotherapy, such as fast and efficient cellular uptake, and used it for h.i.a. chemotherapy in rabbits with V x 2 tumour implanted in the liver. The anti-tumour effect of THP upon h.i.a. administration was compared with that upon intravenous (i.v.) injection and also with the anti-tumour activity of epirubicin (EPI) upon h.i.a. injection using optimal and maximal tolerated doses of each drug. When tumour growth rates and morphometric examinations were evaluated, it was found that THP and EPI were effective against V x 2 tumour when injected via the h.i.a. route. The activity of THP was stronger than that of EPI. As regards h.i.a. injection-related complication, plasma transaminase levels were temporarily elevated. To demonstrate higher anti-tumour activity and other advantages of h.i.a. injection of THP, plasma and tumour drug concentrations were determined by high-performance liquid chromatography after THP or EPI was administered at an equal dose to the rabbit V x 2 model. Hepatic intra-arterial injection of THP accomplished a selective and higher uptake into the tumour and lower effusion into the plasma than i.v. injection of THP or h.i.a. injection of EPL. Our findings indicate that THP is the better candidate of the two drugs tested for the h.i.a. chemotherapy because of its greater anti-tumour activity and the lower systemic drug exposure achieved upon h.i.a. injection.

Characteristic antitumor activity of cytarabine ocfosfate against human colorectal adenocarcinoma xenografts in nude mice.

The antitumor activity of cytarabine ocfosfate (SPAC) was tested against human colorectal, gastric and lung adenocarcinoma xenografts in nude mice in comparison with the activities of various antitumor drugs used clinically. SPAC showed higher therapeutic efficacy against human colorectal adenocarcinoma xenografts than against human gastric and lung adenocarcinoma xenografts. SPAC was effective against three of four human colorectal adenocarcinoma xenografts, with efficacy higher than that of 1-beta-D-arabinofuranosylcytosine, fluorouracil, cisplatin, doxorubicin, pirarubicin and vindesine sulfate, but lower than that of mitomycin C and cyclophosphamide. These results indicate that SPAC may be useful for induction and/or postoperative chemotherapy against colorectal adenocarcinomas.

Phenotypic reversion induced by anthracyclines in ras oncogene-expressed cells; structure-activity relationships.

Several antitumor anthracyclines, including those in preclinical stages, were examined for their action in reversing tumorous phenotypes of H- or K-ras 3T3 cells (NIH3T3 cells transformed by human H- or K-ras oncogene) into normal phenotypes, such as flattened cell morphology, anchorage dependent cell growth, etc. (referred to as anti-ras activity). The study elucidated relationships between the chemical structure of anthracyclines and the anti-ras activity. The human tumor cell line T24, which has a mutated H-ras gene, responded to the anthracyclines, as did K- or H-ras 3T3 cells, in respect to the phenotypic alterations. Pirarubicin was more than 4 times as active as aclarubicin in inhibiting the growth of solid tumors of K-ras 3T3 cells in nude mice, possibly reflecting a difference in anti-ras activity between the two antibiotics.

[Antitumor activity of a new antiestrogenic drug, toremifene (NK622) against human breast cancer xenografts in nude mice].

NK622, a novel tamoxifen(TAM) analog with nonsteroidal structure is an antiestrogenic drug with less toxicity compared with that of TAM. We studied the in vivo antitumor activity against human breast cancer xenografts in nude mice. NK 622 significantly inhibited the growth of estrogen-dependent Br-10 breast cancer but not inhibited the growth of estrogen-independent MC-2-JCK and MC-5-JCK when orally administered once daily for 14 days at the maximum tolerated dose (200mg/kg/day). The dose of NK622 in animal studies was calculated by measuring plasma level in patients receiving 40 mg/body/day oral treatment and clinically equivalent dose (CED) was determined. At the calculated CED, NK622 significantly inhibited the growth of Br-10. These results indicate that NK622 is a promising drug comparable to TAM because of the growth inhibition of estrogen-dependent breast cancers.

Woodfruticosin (woodfordin C), a new inhibitor of DNA topoisomerase II. Experimental antitumor activity.

Woodfruticosin (woodfordin C) (WFC), a new inhibitor of DNA topoisomerase II (topo-II), was isolated from methanol extract of Woodfordia fruticosa Kurz (Lythraceae) and studied for in vitro and in vivo antitumor activities in comparison with Adriamycin (ADR) and etoposide (ETP), well known inhibitors of topo-II. The inhibitory activity against DNA topo-II shown by WFC was much stronger than that shown by ETP or ADR. WFC inhibited strongly intracellular DNA synthesis but not RNA and protein synthesis. On the other hand, WFC had a weaker growth inhibitory activity against various human tumor cells than ETP or ADR, but it showed remarkable activity against PC-1 cells and moderate activity against MKN45 and KB cells. Furthermore, WFC had in vivo growth inhibitory activity against s.c. inoculated colon38. These results indicate that the mechanism by which WFC exhibits antitumor activity may be through inhibition of topo-II.

Characterization of an etoposide-resistant human K562 cell line, K/eto.

An etoposide-resistant K562 cell line (K/eto) was obtained by stepwise exposure, in culture, to increasing concentrations of etoposide, without the use of mutagens. This cell line was resistant to etoposide, and slightly resistant to adriamycin, but sensitive to anti-cancer drugs such as camptothecin, vincristine, actinomycin D and so on. P-Glycoprotein, the mdr1 gene product, was not detected in this cell line, as assessed by immunocytochemistry, immunoprecipitation and flow cytometry. Overexpression of mdr1 mRNA was also not found. Interestingly, expression of 85 kD protein recognized by MRK 20 monoclonal antibody was noted. The level of DNA topoisomerase II protein, detected by antibody staining, decreased concomitantly with a general decrease in DNA topoisomerase II unknotting activity, while DNA topoisomerase I activity was not affected. Cellular accumulation of [3H]etoposide was reduced by 75% in the resistant line compared with parental K562. Karyotype analysis showed that the number of chromosomes in K/eto was 55 and neither a homogeneous staining region nor double-minute chromosomes were detected. These results indicate that this resistance is not due to an altered interaction between the drug and cellular transport machinery, i.e. MDR1, associated with the "classic" multiple drug resistance phenotype, but rather is due to the existence of other mechanism(s) of resistance, decreased transport of the drug and decreased target enzyme, DNA topoisomerase II.

Hematological studies on naturally occurring substances. VI. Effects of an animal crude drug "chan su" (bufonis venenum) on blood coagulation, platelet aggregation, fibrinolysis system and cytotoxicity.

During the screening test of the animal crude drug "Chan su" (Chinese name, toad-cake), the venom of Bufo bufo gargorizans CANTOR (Bufonidae), on blood coagulation, platelet aggregation, fibrinolysis system and cytotoxicity, the ethyl acetate extract showed promotive action on platelet aggregation and remarkable cytotoxic activity on HeLa-S3 cells. Nine kinds of bufadienolides were isolated from the ethyl acetate extract by bioactivity-guided fractionation and were identified by chemical and spectral analysis.

Toxicity and antitumor activity against solid tumors of micelle-forming polymeric anticancer drug and its extremely long circulation in blood.

Toxicity and in vivo antitumor activity against five solid tumors (C 26, C 38, M 5076, MKN-45, MX-1) of Adriamycin (ADR)-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-P[Asp(ADR)]) were evaluated, and its pharmacokinetic behavior in blood and biodistribution by i.v. injection were obtained. PEG-P[Asp(ADR)] was revealed to express higher antitumor activity than ADR against all the examined tumors except MKN-45. Especially against C 26, PEG-P[Asp(ADR)] expressed critical suppression of tumor growth and considerably prolonged life span of the treated mice. PEG-P[Asp(ADR)] was observed in blood at much higher concentrations with a longer half-life than ADR after the i.v. injection. PEG-P[Asp(ADR)] was known to form a micellar structure with a diameter of approximately 50 nm and a narrow distribution in phosphate-buffered saline. Therefore, the stabilized circulation of ADR residue in blood by binding to the block copolymer was considered to result from the micellar structure which possesses the hydrated outer shell composed of the poly(ethylene glycol) chains.

[Antitumor activity by long-term administration of low-dose etoposide].

Antitumor activity by long term administration of low dose etoposide (ETP) was investigated in mice. In a spontaneous metastasis system, intraperitoneal consecutive administration of ETP at 2 mg/kg/day inhibited the number of metastatic nodules of Lewis lung carcinoma in the lung and showed a greater activity than 5-fluorouracil (5-FU) at the dose of 5 mg/kg/day. Alternative administration of ETP and 5-FU also exhibited the anti-metastatic activity, but mean survival time of mice was similar in all of these groups. Mean survival time of ETP-treated mice was prolonged when administration interval was shortened. Also in an artificial metastasis system, ETP inhibited metastasis of Lewis lung carcinoma in the lung. ETP showed antitumor activity against Colon adenocarcinoma 38 as 5-FU, when drugs were administered orally for long term, and the activity did not declined during the experimental period. These results suggest that long term administration of low dose ETP is clinically useful for post-operative and maintenance chemotherapy.

Human ovarian cancer cell lines resistant to cisplatin, doxorubicin, and L-phenylalanine mustard are sensitive to delta 7-prostaglandin A1 and delta 12-prostaglandin J2.

The antitumor activity of delta 7-prostaglandin A1 (delta 7-PGA1) or delta 12-prostaglandin J2 (delta 12-PGJ2) on human ovarian cancer cell lines resistant to cisplatin (CDDP), doxorubicin (ADR), and L-phenylalanine mustard (l-PAM) was studied in vitro. A2780AD, A2780 (parent cells of A2780AD), 2008DDP, and 2008 cells (parent cells of 2008DDP) were used. The antitumor activities of the drugs were defined with 50% inhibitory concentration (IC50) estimated from growth inhibition curves, which were obtained by an indirect colorimetric method. Drug-resistance ratios obtained from IC50 values, by comparing A2780AD and A2780 cells, were 62.5 for ADR, 4.6 for CDDP, 4.9 for l-PAM, 1.5 for delta 7-PGA1, and 1.8 for delta 12-PGJ2. Those obtained by comparing 2008DDP and 2008 cells were 1.1 for ADR, 16.0 for CDDP, 2.9 for l-PAM, 2.3 for delta 7-PGA1, and 3.2 for delta 12-PGJ2. Thus some human ovarian cancer cells resistant to ADR, CDDP, and l-PAM remain sensitive to antitumor PGs.

Platelet aggregation inhibitors from the seeds of Swietenia mahagoni: inhibition of in vitro and in vivo platelet-activating factor-induced effects of tetranortriterpenoids related to swietenine and swietenolide.

The ether extract from the seeds of Swietenia mahagoni Jacq. (Meliaceae) was found to inhibit platelet-activating factor (PAF)-induced platelet aggregation. Systematic separation of the extract afforded twenty eight tetranortriterpenoids related to swietenine and swietenolide. Among them, several new compounds, named swietemahonin A, D, E, and G and 3-O-acetylswietenolide and 6-O-acetylswietenolide, showed a strong inhibition against PAF-induced aggregation in vitro and in vivo assays.

Constituents of the leaves of Woodfordia fruticosa Kurz. I. Isolation, structure, and proton and carbon-13 nuclear magnetic resonance signal assignments of woodfruticosin (woodfordin C), an inhibitor of deoxyribonucleic acid topoisomerase II.

Woodfruticosin (woodfordin C), a new cyclic dimeric hydrolyzable tannin having an inhibitory activity toward deoxyribonucleic acid (DNA) topoisomerase II, has been isolated from the leaves of Woodfordia fruticosa Kurz (Lythraceae) along with three known flavonol glycosides and three known flavonol glycoside gallates. The structure of wood fruticosin (woodfordin C) was determined by the use of two-dimensional nuclear magnetic resonance (2-D NMR) spectroscopy including heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC) techniques. Detailed analyses of the proton and carbon-13 NMR (1H- and 13C-NMR) spectra of six known flavonoids were performed.

[Antitumor activity of a novel analog of cytarabine, 4-amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01)].

4-Amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate (YNK01) was an orally active depot form of 1-beta-D-arabinofuranosylcytosine (Ara-C). In the present study, antitumor activity of YNK01 was compared with it of Ara-C in vitro and in vivo. The activity of a main metabolite of YNK01, 5'-carboxypropylphosphate of Ara-C (C-C3PCA), was also studied. Growth inhibitory activity of YNK01 against various cultured tumor cells was 1/32-1/1,100 of that of Ara-C. YNK01 exhibited antitumor activity against L1210 leukemia in mice after i.v., i.p. or p.o. administration. The activity did not depend on the administration routes. Compared with Ara-C, the activity was comparable in both i.v. and i.p. administrations, but greater in p.o. administration. Oral administration of YNK01 showed similar antitumor spectrum to i.p. administration of Ara-C. Oral activity of YNK01 against L1210 leukemia did not depend on the administration schedules but depended on a total administration dose. In contrast, activity of Ara-C greatly depended on the schedules, and the frequent i.p, administration showed greatest activity. Growth inhibitory activity of C-C3PCA against cultured tumor cells was 1/2-1/7 of Ara-C. The metabolite exhibited activity against L1210 leukemia in mice after i.p. administration. These results suggest that YNK01 is a clinically useful drug with p.o. administration for cancers as well as Ara-C.