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.

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.

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.

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.

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.

Kinetics of the reaction of bleomycin-Fe(II)-O2 complex with DNA.

The kinetics of the reaction of BLM-Fe(II)-O2 with DNA in the absence or presence of 2-mercaptoethanol (2-ME) were studied. The total number of bases released by BLM-Fe(II)-O2 in the presence of 2-ME increased about 6.5 times more than that in the absence of 2-ME in the reaction of 6 hours at 37 degrees C. The molar ratios of the released bases ware little affected by the reaction time, temperature or 2-ME. Among the four bases, thymine was preferentially released. On the basis of a reaction scheme of BLM-Fe(II)-O2 with DNA, the equations were derived by the steady-state method. In the absence of 2-ME, the release of bases from DNA was dependent on the concentration of BLM-Fe(II)-O2, but independent of the concentration of DNA. In the presence of 2-ME, a biphasic reaction was observed; the first one is due to BLM-Fe(II) which originally existed and the second one is due to BLM-Fe(II) produced by the reduction of BLM-Fe(III) with 2-ME. In the second reaction, the rate of the release of bases from DNA was proportional to the concentration of BLM-Fe(II) and 2-ME, but inversely proportional to the concentration of DNA. The rate-determining step in the reaction of BLM-Fe(II)-O2 with DNA in the presence of 2-ME was found to be the reduction of BLM-Fe(III) to BLM-Fe(II). By these kinetic studies, the reaction of BLM-Fe(II)-O2 with DNA in the presence of 2-ME was elucidated to proceed in a catalytic fashion. Furthermore, the maximum number of bases released by BLM from DNA was one base per twelve to thirteen bases.

Lipid peroxidation by bleomycin-iron complexes in vitro.

Lipid peroxidation catalyzed by bleomycin (BLM)-metal complexes was studied in vitro using arachidonic acid as the substrate. Iron complexes of BLM caused extensive lipid peroxidation, but other metal complexes did not. The lipid peroxidation caused by the iron complexes was inhibited by antioxidants such as dl-alpha-tocopherol, ascorbic acid etc., but not by other scavengers of hydroxyl and superoxide radicals, and of singlet oxygen. Cyanide ion suppressed the lipid peroxidation caused by BLM-Fe(II), but did not suppress the peroxidation activity of BLM-Fe(III). The peroxidation activity of BLM-Fe(II) was lost instantly by pre-incubation of the complex at 37 degrees C before mixing with arachidonic acid, but that of BLM-Fe(III) was not. These results indicate that the active form for the lipid peroxidation derived from BLM-Fe(II) differs from that of BLM-Fe(III).

Immunological involvement in pulmonary fibrosis induced by peplomycin.

Pulmonary fibrosis in mice induced by peplomycin (PEP) was suppressed by administration of anti-inflammatory agents such as prednisolone and D-penicillamine during or after the administration of PEP. Pulmonary fibrosis was also suppressed by administration of cyclophosphamide, an immunosuppressive antitumor agent before, during or after the administration of PEP. The pulmonary fibrosis in athymic nude mice induced by PEP was less than that in normal mice. The low response in the nude mice was enhanced by transfer of thymocytes to the same level as that in the normal mice. This suggests that the immune system, especially thymus-dependent immunity, is involved in the pulmonary fibrosis induced by PEP.

Effect of oxygen concentration on pulmonary fibrosis caused by peplomycin in mice.

The pulmonary fibrosis caused by peplomycin (PEP) was studied in terms of oxygen toxicity using ICR mice. When 16 micrograms of PEP was administered intratracheally in mice after exposure to the air containing 75% O2 for 10 days, the pulmonary fibrosis was completely suppressed, while when mice were exposed to 75% O2 after the administration of PEP, the fibrosis was much severe than that of mice raised in atmospheric air. In 50% O2, similar oxygen effect was also observed, but it was weaker than that in 75% O2. In 90% O2, the oxygen toxicity was observed in mice without administration of PEP. When mice were exposed to 75% O2, the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, which are relevant to the detoxication of active oxygen species, were not increased in the lung, but the levels of reducing agents such as glutathione and ascorbic acid, and high molecular substances having 1O2-scavenging activity were enhanced. The results suggest that these materials have some roles to decrease the pulmonary fibrosis caused by PEP.

Biological studies on the degradation products of 3-[(S)-1'-phenylethylamino]propylaminobleomycin: a novel analog (pepleomycin).

Pepleomycin (PEP), 3-[(S)-1'-phenylethylamino]propylaminobleomycin has potent activity and is less pulmonary toxic than bleomycin (BLM). Biological activity and toxicity of the following degradation products of PEP have been studied in detail: the product of carbamoyl migration (ISO), the product of decarbamylation (DC), the product of ring closure of the side chain on the pyrimidine moiety (RC), the depyruvamide product (DP) and the product of an enzymatic inactivation (DA). These degradation products showed much lower activity than PEP in vitro: antimicrobial and anti-HeLa activities, inhibition of DNA synthesis in AH66 cells and the DNA strand cleavage. Acute toxicity and pulmonary toxicity were tested in mice. Results indicated much lower acute toxicity corresponding to the decreased in vitro activity when compared to PEP. DP and RC did not cause lung fibrosis in mice, while ISO and DC showed 1/2.6 and 1/5.7 degree of pulmonary toxicity, respectively, in comparison with PEP.

[Studies on antitumor activities and pulmonary toxicity of pepleomycin sulfate (NK631) (author's transl)].

The antimicrobial and antitumor activities, and the pulmonary toxicity of pepleomycin (NK631) were studied in comparison with bleomycin (BLM). NK631 showed a broad antimicrobial spectrum against gram positive and gram negative bacteria equally to BLM, and its activity was about twice higher than BLM. NK631 showed higher activity on cultured HeLa S3 cells and higher antitumor effect on the transplanted tumors of Ehrlich solid carcinoma in mice, AH66 and AH66F ascites hepatoma in rats, and lower antitumor effect on Ehrlich ascites carcinoma in mice than BLM. Similarly to BLM, NK631 did not show satisfactory activity on L1210 leukemia in mice. NK631 showed marked effect on chemically induced squamous cell carcinoma, spontaneous lymph sarcoma of a dog, human and dog gastric cancer heterotransplanted in nude mice equally to BLM. Furthermore NK631 exhibited remarkably higher antitumor activity on lymph node metastasis of AH66 ascites hepatoma of rats and chemically induced gastric carcinoma of rats than BLM. Pulmonary toxicity of NK631 was low as 1/3 in incidence and 1/4 in grade of the BLM in old mice system. This trend was confirmed by chemical analysis of hydroxyproline in lung.

[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.

[Combination effects of etoposide with other antitumor drugs in vitro and in vivo].

Combination effects of etoposide (ET) with each of 10 antitumor drugs were examined with P 388 leukemia cells in vitro and in vivo. Median effect analysis was applied for the evaluation of in vitro effect by the growth inhibition, and the in vivo effect by comparison of the increase of life span (ILS) in a combined group with the sum of ILS's in 2 single agent groups. Among 10 drugs combined with ET, cyclophosphamide (melphalan was used for in vitro study), cisplatin and 6-mercaptopurine exhibited a strong synergism both in vitro and in vivo. The combination of ET with mitomycin C, vincristine, vindesine or cytarabine produced additive or slightly synergistic effect in the both systems. However, methotrexate + ET combination showed an antagonistic effect. Although the combination of ET with doxorubicin or fluorouracil showed a slight synergism in vitro, it was antagonistic in vivo. Thus, the in vitro and in vivo combined effects were consistent in 8 of 10 drugs. The employed methods in the present studies could distinguish high efficacy of ET + cyclophosphamide and ET + cisplatin, which are clinically approved as effective combinations against lung cancer. The methods seem to be useful to assess the drug efficacy in experimental combination.

[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.

[Experimental studies on combination chemotherapy of cisplatin with peplomycin against human squamous cell carcinomas in nude mice].

Combination chemotherapy of cisplatin (CDDP) with bleomycin (BLM) or peplomycin (PEP) has exhibited clinical efficacy against squamous cell carcinomas. The combination of CDDP with PEP was studied using five human squamous cell carcinomas in nude mice. When CDDP was administered before PEP treatment, the combination effect was more pronounced than when CDDP was administered after PEP treatment. Especially, when CDDP was administered 5 or 3 days before PEP treatment, marked delay in growth was shown in 4 out of the 5 human carcinomas. From these results, it is thought that human heterotransplanted tumors in nude mice may provide important information in studies of combination chemotherapy.

[Changes of anticancer activity and pulmonary toxicity of bleomycins in differences of administration schedules and routes in mice].

Anticancer activity and pulmonary toxicity of bleomycin and peplomycin in mice were found to be dependent on administration schedules and routes. That is, at the same total dose of 50 mg/kg, the anticancer activity against Lewis lung carcinoma was most strongly exhibited by continuous infusion of peplomycin for 7 days among all schedules tested and decreased in the order of the continuous infusion greater than 4 times injections per day for 10 days greater than daily injection for 10 days greater than daily injection for 2 days. In contrast, the pulmonary toxicity occurred lowest in the continuous infusion and strongest in daily injection for 10 days among the schedules. With respect to administration routes, the anticancer activity and the pulmonary toxicity of them were strongly exhibited in the order of i.v. much greater than i.m. greater than s.c. greater than i.p., except that no route dependency was observed in the continuous infusion. The pulmonary toxicity of peplomycin was lower than that of bleomycin in every administration schedules and routes. From these results, the continuous infusion was concluded to be the best schedule to increase the anticancer activity and to reduce the pulmonary toxicity.

[Experimentally induced bleomycin pulmonary toxicity--comparison of the systemic (intraperitoneal) and local (intratracheal) administration].

Effect of two methods of the systemic (intraperitoneal) administration and the local (intratracheal) instillation in small animal species for the evaluation of BLM-induced pulmonary fibrosis were compared. The grade of BLM-induced pulmonary fibrosis was different among age, species and strains of animals. This was more distinct in the intraperitoneal administration than in the intratracheal instillation. That is, BLM pulmonary fibrosis was severer in 15 and 30 weeks old ICR mice than in 4 weeks old young mice and also severer in the 15 weeks old ICR mice than in 15 weeks old Wistar rats and Hartley guinea pigs. In strains of mice, ICR and C3H mice were suffered from severer BLM pulmonary fibrosis than C57BL/6, BALB/c and DBA/2 mice. Most of BLM analogs produced pulmonary fibrosis to the same extent between the both methods, but three analogs (A5, B2 and BAPP) produced severe pulmonary fibrosis by intratracheal instillation compared to intraperitoneal administration. Furthermore, the pulmonary fibrosis in the intraperitoneal administration group often occurred in widespread subpleural region and that of the intratracheal group instillation group occurred in localized bronchoalveolar region. These results indicated the difference of pathogenesis of the pulmonary fibrosis between the both methods.

[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.

[Therapeutic effect of an antineoplastic agent (peplomycin) adsorbed on activated charcoal (PEP-AC)].

Therapeutic effects of PEP-AC and PEP-saline on pulmonary growth of intratracheally implanted tumor and metastasis into the hilar lymph nodes were studied in mice. Pharmacokinetic studies of PEP-AC and PEP-saline were made by autoradiography (ARG) using 3H-PEP and microbial assay method using B. subtilis. The ARG using 3H-PEP-AC and 3H-PEP-saline demonstrated qualitatively slower elimination of PEP-AC from mouse lung than that of PEP-saline. The half-life time (t1/2) of PEP-AC was estimated to be about 3 days by bioassay method, while about 60 min. was given for PEP-saline. Intratracheal administration of PEP-saline produced no therapeutic effect to pulmonary growth of B16 melanoma, while that of PEP-AC gave a good response depending on doses. Furthermore, PEP-AC inhibited metastasis of B16 melanoma into the hilar lymph nodes. Better therapeutic effects were produced by PEP-AC when decreased inoculum sizes of B16 melanoma or P388 leukemia cells were transplanted.

[Experimental combination chemotherapy of YNK01, a novel analog of cytarabine, with other antitumor agents].

Combined activity of 4-amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone 5'-(sodium octadecyl phosphate) monohydrate, YNK01, with other antitumor agents was studied with mouse P388 and L1210 leukemia in vivo. The two-drug combinations with doxorubicin, daunorubicin, mitomycin C, cisplatin or vincristine showed marked synergistic effects against P388 leukemia. However, YNK01 plus methotrexate showed additive or competitive effect. Among these combinations, the combination with doxorubicin showed greatest activity and the number of cured mice were observed in a wide dose range. In a three-drug combination with daunorubicin and 6-mercaptopurine against L1210 leukemia, the combined effect was more synergistic than two-drug combinations with each of the three drugs. From these results, YNK 01 is expected to be a useful agent in combination chemotherapy.