Anthracyclines and their C-13 alcohol metabolites: growth inhibition and DNA damage following incubation with human tumor cells in culture

Summary Anthracyclines are important antitumor agents used in the treatment of solid tumors, lymphomas, and acute lymphoblastic as well as myelocytic leukemias. The clinical utility of agents such as doxorubicin and daunorubicin and their well-characterized cardiotoxicity have prompted many efforts to develop analogs that retain the desired spectrum of activity but are less cardiotoxic. One such analog is idarubicin (4-demethoxydaunorubicin), which is currently under study in the treatment of adult and pediatric leukemias. The major circulating metabolite of idarubicin is the alcohol product of ketoreductase biotransformation, idarubicinol. Following the administration of idarubicin to adult or pediatric patients, systemic exposure to idarubicinol is greater than that to idarubicin. Moreover, we have also documented the presence of idarubicinol in the cerebrospinal fluid of pediatric patients who have received idarubicin. Idarubicinol has been reported to have greater cytotoxic activity than other anthracycline alcohol metabolites, which are regarded as much less active products of metabolism. We therefore evaluated the growth-inhibitory and DNA-damaging activities of idarubicin, daunorubicin, doxorubicin, epirubicin, and their alcohol metabolites against three relevant (CCRF-CEM lymphoblastic leukemia, K562 myelogenous leukemia, and U87-MG glioblastoma) human tumor cell lines. We found that whereas idarubicin was 2–5 times more potent than the other three anthracycline analogs against these tumor cell lines, idarubicinol was 16–122 times more active than the other alcohol metabolites against the same three cell lines. In addition, idarubicinol and the parent drug idarubicin were equipotent, unlike the other anthracycline alcohol metabolites, which were much less cytotoxic than the corresponding parent drugs. We also assessed the ability of the four parent drugs and their alcohol metabolites to induce DNA single-strand breaks. Idarubicin was more potent than the other three anthracycline analogs and idarubicinol was much more effective than the other alcohol metabolites in inducing DNA damage. These studies in human leukemia and human glioblastoma cell lines support the hypothesis that idarubicinol plays an important role in the antitumor activity of idarubicin and that the activities of idarubicin and idarubicinol are related to their ability to damage DNA.This research was partially supported by funds from Adria Laboratories and by NCI grant CA 15083, DHHS     

DNA damage and cytotoxicity induced by metabolites of anthracycline antibiotics, doxorubicin and idarubicin

This study assessed the ability of major metabolites of two types of anthracycline antibiotics, doxorubicin and idarubicin (4-demethoxydaunorubicin) to damage DNA in mouse fibrosarcoma 935.1 cells. Since DNA lesions by anthracyclines may be mediated by topoisomerase II, we also characterized the ability of the drugs to inhibit this enzyme. The C-13 alcohol and aglycone metabolites of doxorubicin and idarubicin were compared to the parent drugs in terms of induction of DNA single strand breaks measured by filter elution. In whole cells, the maximal DNA strand breakage induced by the C-13 alcohol metabolites was similar to that of their respective parent drugs. In isolated nuclei, however, the alcohol metabolites were two times more potent than the parent drugs. The aglycone metabolites produced very little damage in either whole cells or nuclei. The doxorubicin compounds differed markedly from idarubicin drugs in the way their ability to induce DNA breaks was related to cytotoxic activity. Doxorubicin and doxorubicinol cytotoxic effects (50% cell growth inhibition at 0.2 and 4 microM, respectively) coincided (in terms of drug concentrations) with the induction of significant breakage of cellular DNA. In contrast, the concentrations of idarubicin and idarubicinol needed to produce 50% growth inhibition (0.005 and 0.006 microM, respectively) were about 20 times lower than drug levels that induced significant DNA damage. All six compounds inhibited the catalytic activity of isolated topoisomerase II. While the alcohol metabolites produced this inhibition at concentrations similar to those of their parent drugs (5-10 microM), the aglycones were again much less active.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anthracycline antibiotics with high liposome entrapment: structural features and biological activity

We evaluated the entrapment of 21 different water-insoluble aglycones or anthracycline antibiotics in multilamellar liposomes composed of dimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerol at a 7:3 molar ratio. The drug:lipid weight ratio was 1:15 to 1:50. The different analogues tested were modified at position 4 in the aglycone portion (4-demethoxy) and/or positions 2' (halo), 3' (hydroxy, acetoxy), or 4' (epi, acetoxy) in the sugar portion. The entrapment efficiency was assessed by measuring the amount of free drug remaining in the supernatant after centrifugation of the liposomes and by direct examination of the pellets by fluorescent microscopy. Optimal entrapment (greater than 98%) was observed with only four compounds: 4-demethoxyadriamycinone; 2'-iododaunorubicin; 4-demethoxydaunorubicin; and 2'-iodo-3'-hydroxy-4'-epi-4-demethoxydoxorubicin (Compound 22). All other compounds showed significant drug precipitation outside the multilamellar vesicles when observed by fluorescent microscopy. Compound 22, entrapped in liposomes, was evaluated in vivo against i.p. L-1210 leukemia by the i.p. route, and liver metastases of M5076 reticulosarcoma by the i.v. route. In both models, liposome-entrapped Compound 22 was more active than doxorubicin at the optimal dose [median survival (given in percentage) of treated to control animals was for L-1210, greater than 600 versus 212; for M5076, 200 versus 133]. 4-Demethoxy and 2'-iodo are structural modifications that markedly enhance the affinity of anthracycline antibiotics for lipid bilayers without compromising biological activity. These findings will serve as a guideline to obtain liposome-anthracycline preparations, with optimal formulation characteristics, enhanced tumor-targeting properties, and non-cross-resistance with doxorubicin.     

Antitumor activity and pharmacokinetics in mice of 8-carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (CCRG 81045; M & B 39831), a novel drug with potential as an alternative to dacarbazine

A number of 3-alkyl analogues of the experimental antitumor drug mitozolomide [8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H )-one] have been screened against murine tumors in vivo. Only the compounds with a 3-methyl- or 3-bromoethyl group possessed significant antitumor activity against the TLX5 lymphoma. The 3-methyl analogue, 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (CCRG 81045), was investigated further and found to possess good activity, when administered i.p., against the L1210 and P388 leukemias, the M5076 reticulum cell sarcoma, B16 melanoma, and ADJ/PC6A plasmacytoma. The drug was also active when administered p.o. to mice bearing the L1210 leukemia. A daily for 5 days schedule of 100 mg/kg CCRG 81045 produced increases of survival time of treated animals compared to controls of 176 and greater than 235% against the P388 and L1210 leukemias, respectively. In the female C57BL x DBA/2 F1 mouse the 10% lethal dose was 125 mg/kg daily for 5 days. CCRG 81045 was found to undergo mild alkaline hydrolysis and ring fission to form the linear triazene 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, which is the putative metabolite formed upon metabolic activation of the antitumor drug dacarbazine [5-(3,3-dimethyltriazen-1-yl)imidazole-4-carboxamide]. The half-life of CCRG 81045 at 37 degrees C in 0.2 M phosphate buffer (pH 7.4) was 1.24 h, whereas that of 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide at 25 degrees C was reported to be 8 min (F. H. Shealy and C. A. Krauth, J. Med. Chem., 9:34-37, 1966). The half-life of CCRG 81045 in human plasma in vitro at 37 degrees C was 0.42 h. Pharmacokinetic experiments conducted in BALB/c mice produced plasma profiles of CCRG 81045, administered i.p. or p.o., which showed a rapid absorption phase, elimination half-lives of 1.13 h (i.p.) and 1.29 h (p.o.), and a bioavailability of 0.98.     

Antitumor activity and pharmacokinetics of TAS-106, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine.

We examined the effects of dosage schedule on antitumor activity in vitro and in vivo to determine the optimal administration schedule for a new nucleoside antimetabolite 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106). The cytotoxicity of TAS-106 in vitro against human tumors was evaluated at three drug exposure periods. TAS-106 exhibited fairly potent cytotoxicity even with 4 h exposure, and nearly equivalent and sufficiently potent cytotoxicity with 24 and 72 h exposures. These results suggest that long-term exposure to TAS-106 will not be required to achieve maximal cytotoxicity. The antitumor activity of TAS-106 in vivo was compared in nude rat models bearing human tumors on three administration schedules, once weekly, 3 times weekly, and 5 times weekly for 2 or 4 consecutive weeks. TAS-106 showed strong antitumor activity without serious toxicity on all three schedules, but the antitumor activity showed no obvious schedule-dependency in these models. When tumor-bearing nude rats were given a single i.v. dose of [(3)H]TAS-106, tumor tissue radioactivity tended to remain high for longer periods of time as compared to the radioactivity in various normal tissues. Furthermore, when the metabolism of TAS-106 in the tumor was examined, it was found that TAS-106 nucleotides (including the active metabolite, the triphosphate of TAS-106) were retained at high concentrations for prolonged periods. These pharmacodynamic features of TAS-106 may explain the strong antitumor activity without serious toxicity, observed on intermittent administration schedules, in nude rat models with human tumors. We therefore consider TAS-106 to be a promising compound which merits further investigation in patients with solid tumors.     

Evaluation of the in vitro antiproliferative properties of four novel anthracyclines YM1, 3, 4 and 6 in human leukemia cell lines.

The antitumor activity of novel doxorubicin analogues YM1, YM3, YM4 and YM6 was evaluated against drug sensitive U937 monocytic leukemia and CCRF-CEM lymphoid leukemia cell lines, as well as drug resistant CEM/VLB100 lymphoid multidrug resistant leukemia cell line by a [3H]thymidine incorporation assay. Different antileukemic activities of these new anthracyclines were observed in our studies. These novel anthracyclines produced a dose- and time-dependent inhibition in all the leukemic cell lines tested, while YM1 and YM3 were more effective than YM4 and YM6 against all the leukemic cell lines. The antitumor activity of all these novel analogues was lower than that of doxorubicin or epidoxorubicin in drug sensitive leukemic cells. The relative resistance values (IC50 of resistant cell line/IC50 of sensitive parental cell line) of YM1, 3, 4 and 6 were 27, 7, 5 and 14 respectively. These were lower than the resistance values for ADM and EDR which were 45 and 40 respectively. YM3 had a similar antileukemic activity against the CEM/VLB100 drug resistant leukemic cell line to ADM or EDR with a lower relative resistance value and a slightly increased IC50 value. Our results suggest that YM3 may be used in high dose for the clinical treatment of leukemias with possible less cardiotoxicity as well as less drug resistance.     

4-Demethoxydaunorubicin administered orally in advanced breast cancer. A phase II study

Twenty-five patients with advanced breast cancer were treated with 4-demethoxydaunorubicin (4-DMDR), a new antitumor analogue of daunorubicin, at the dose of 15-20 mg/m2/day x 3 days by oral route every 3-4 weeks. All patients were previously treated with chemotherapy and/or hormone therapy but none with anthracyclines. Of 23 evaluable patients, 1 complete and 5 partial remissions (26%) were observed for a median duration of 4+ months. Leukopenia and nausea occurred in 61% of the patients, vomiting in 30%, diarrhea in 17% and alopecia in 43%. There were 2 cases with minimal and transient EKG variations. 4-DMDR, administered orally in advanced breast cancer, was found to be generally well tolerated. Nevertheless, randomized trials with adriamycin or epirubicin are necessary to compare and to define the therapeutic activity and the toxicity of 4-DMDR.     

Synthesis and antitumor activity of a platinum (II)-doxorubicin complex

Summary A mixed platinum (II) complex with tert-butylamine and doxorubicin (cooordinated via the aminogroup) has been synthesized and tested for antitumor activity. The results indicate that the complex was active against doxorubibin-resistant P388 and cisplatin-resistant L1210 leukemias, while maintaining antitumor activity against sensitive parent lines.     

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.     

Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: evidence for a specific receptor site and a relation to antitumor activity

Twenty-two compounds related to camptothecin, a known inhibitor of eukaryotic topoisomerase I, were studied. The following effects on the actions of topoisomerase I were observed and were well correlated among most of the compounds studied: (a) inhibition of the first-order rate of relaxation of supercoiled DNA; (b) conversion of supercoiled DNA to nicked circles; and (c) single-strand cleavage of linear DNA at specific sites. The locations of the stimulated cleavage sites were the same for all of the active derivatives. Stereochemistry and the positions of substituents were found to be crucial for the presence or absence of effects on topoisomerase I, indicating that the compounds interact with an asymmetrical receptor site on the enzyme or enzyme-DNA complex. From the structure-activity relations, the regions of interaction between the camptothecin ring system and the receptor site were inferred. Striking correlations were observed between activity against topoisomerase I and reported activity against murine leukemias, indicating that an action on topoisomerase I is responsible for the antitumor activity of the camptothecins.     

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.     

Carcinostatic effect of aliphatic aldehydes and aldehyde dehydrogenase activity in Ehrlich carcinoma, Sarcoma 180, and Yoshida AH 130 hepatoma.

The antitumor activity of 2,3-dihydroxybutyraldehyde on Ehrlich carcinoma, Sarcoma 180, and Yoshida AH 130 hepatoma, as well as the aldehyde dehydrogenase activity in these tumors, was studied. 2,3-Dihydroxybutyraldehyde at nontoxic doses (500 mg/kg body weight i.p. daily for 7 days) slowed down the growth of solid and ascites tumors in mice. The treatment completely prevented the development of Yoshida ascites hepatoma in several rats. 2,3-Dihydroxybutyraldehyde, although it did not influence the growth of Ehrlich carcinoma transplanted in the brain of mice, significantly decreased in the lungs of these animals the number of viable tumour cells that derived from the primary tumor. All the tested tumors, which were sensitive to the action of 2,3-dihydroxybutyraldehyde, were virtually devoid of aldehyde dehydrogenase activity. These results suggest a possible relationship between the lack of this enzyme activity and the antitumor activity of aliphatic aldehydes.     

Phase I and II clinical and pharmacological study of 4-demethoxydaunorubicin (idarubicin) in adult patients with acute leukemia

Fifty-two adults treated previously with either acute leukemia (43 patients) or blastic-phase chronic myelogenous leukemia (nine patients) received 4-demethoxydaunorubicin (20 to 45 mg/sq m) i.v. over 2 to 3 days. Three of the ten patients with acute lymphocytic leukemia achieved a complete remission (CR) lasting 5 to 7 weeks. Five of the 28 patients with acute nonlymphocytic leukemia achieved a CR lasting 5 to 80 weeks. All remissions were induced with one course of treatment with a median time to CR of 28 days (range, 22 to 40 days). None of the patients with blastic chronic myelogenous leukemia or secondary leukemia achieved a CR. The drug was well tolerated; mucositis (36%), nausea and vomiting (35%), and hepatic dysfunction (26%) were the most common side effects. Pharmacokinetic observations on five patients demonstrated multiphasic clearance of 4-demethoxydaunorubicin and extensive formation and prolonged retention of 4-demethoxy-13-hydroxydaunorubicin; that metabolite accumulated in plasma on repeated daily dosing. 4-Demethoxydaunorubicin has sufficient antileukemic activity in both acute lymphocytic leukemia and acute nonlymphocytic leukemia to warrant a prospective comparison, in combination regimens, against the conventional anthracyclines, daunorubicin and/or doxorubicin.     

Antitumoral effect in mice of a new triepoxyde derivative: 1, 3, 5-triglycidyl-S-triazinetrione (NSC 296934)

The antitumor properties of the α and β stereoisomers of 1, 3, 5-triglycidyl-s-triazinetrione (TGT) were investigated on various transplantable mouse tumor systems. Although the two stereoisomers displayed a high therapeutic activity against P388 and L1210 leukemias when administered i.p., α-TGT was superior to the β form in prolonging the lifespan of treated animals and in inducing long-term survival: 70% of long-term survivors over 90 days of treated animals as compared to controls. α-TGT also demonstrated antitumor effect against advanced L1210 leukemia (ILS: 119% at 50 mg/kg × 9) and was still very active when administered orally against i.v. or ascitic L1210 leukemia while its activity when administered i.p. against i.c. L1210 was moderate and yet significant. The i.p. treatment with α TGT significantly inhibited the primary tumor growth and lung metastases of Lewis lung carcinoma. Finally, the high in vivo activity of α-TGT on normal P388 cells and on a subline of this leukemia markedly resistant to cyclophosphamide (Cy) is a further element warranting studies with this agent.     

Antitumor activity of mitoxantrone against murine experimental tumors: Comparative analysis against various antitumor antibiotics

Summary 1,4-Dihydroxy-5,8-bis{{{{2-[(2-hydroxyethyl)amino]ethyl}amino}}-9,10-anthracenedione dihydrochloride (mitoxantrone) was tested for antitumor activity against experimental tumors in mice and the results were compared with those of seven antitumor antibiotics: adriamycin (ADM), daunomycin (DM), aclarubicin, mitomycin C (MMC), bleomycin, neocarzinostatin, and chromomycin A₃. The drugs were given IP or IV, in general on days 1,5, and 9 following tumor inoculation. Mitoxantrone given IP at the optimal dose (1.6 mg/kg/day; as a free base) produced a statistically significant number of 60-day survivors (curative effect) in mice with IP implanted L1210 leukemia. The curative effect was not observed with any of the other antibiotics. In the case of IV implanted L1210 leukemia, there was an increase in lifespan (ILS) by more than 100% in the mice following IV treatment with mitoxantrone or DM. In IP implanted P388 leukemia, the curative effect was elicited by IP treatment with mitoxantrone or MMC. In IP implanted B16 melanoma, both the curative effect and a more than 100% ILS in mice that did die were produced by IP treatment with mitoxantrone or ADM. In SC implanted Lewis lung carcinoma, mitoxantrone and ADM administered IV also showed effective antitumor activities and produced a 60% and a 45% ILS, respectively. In conclusion, mitoxantrone and ADM had a wider spectrum of antitumor activity against mouse tumors, including two leukemias and two solid tumors, than did the other drugs; however, mitoxantrone elicited higher antitumor effects than ADM on mouse leukemias, especially on L1210 leukemias. Moreover, mitoxantrone possessed much higher therapeutic indices than ADM against IP implanted P388 (optimal dose/ILS₄₀; >128 versus 15.2) and L1210 (optimal dose/ILS₂₅; 72.7 versus 4.8) leukemias. In addition, mitoxantrone showed moderate activity against DM-resistant L1210 leukemia.     

Antitumor activity and novel DNA-self-strand-breaking mechanism of CNDAC (1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine) and its N4-palmitoyl derivative (CS-682).

We have studied the antitumor activity and the novel DNA-self-strand-breaking mechanism of CNDAC (1-(2-Ccyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine) and its N4-palmitoyl derivative (CS-682). In vitro, CS-682 showed strong cytotoxicity against human tumor cells comparable with that of CNDAC; both compounds displayed a similar broad spectrum. In vivo, however, orally administered CS-682 showed a more potent activity against human tumor xenografts than CNDAC, 5'-deoxy-5-fluorouridine, 5-fluorouracil and 2',2'-difluorodeoxycytidine. Moreover, CS-682 was effective against various human organ tumor xenografts at a wide dose range and with low toxicity, and was effective against P388 leukemic cells resistant to mitomycin-C, vincristine, 5-fluorouracil or cisplatin in syngeneic mice. CNDAC, an active metabolite of CS-682, had a prolonged plasma half-life after repeated oral administrations of CS-682 but not after oral administrations of CNDAC itself. This difference may partially explain the higher antitumor activity of CS-682 relative to CNDAC. In both CNDAC- and CS-682-treated carcinoma cells, CNDAC 5'-triphosphate (CNDACTP) was generated and incorporated into a DNA strand. High performance liquid chromatography (HPLC) and mass spectrometric analysis of the nucleosides prepared by digestion of the DNA from the CNDAC-treated cells detected ddCNC (2'-Ccyano-2',3 '-didehydro-2',3 '-dideoxycytidine), which was shown to be generated only when the self-strand-breakage of CNDACTP-incorporated DNA occurred. The cytotoxicity of CNDAC was completely abrogated by the addition of 2'-deoxycytidine and was low against cells with decreased deoxycytidine kinase. Our results suggest that CNDAC is converted to CNDACMP by deoxycytidine kinase and that the resulting CNDACTP incorporated into a DNA strand as CNDACMP may induce DNA-self-strand-breakage. This novel DNA-self-strand-breaking mechanism may contribute to the potent antitumor activity of CS-682.     

Induction of apoptosis by HAC-Y6, a novel microtubule inhibitor, through activation of the death receptor 4 signaling pathway in human hepatocellular carcinoma cells

The present data showed that a novel synthesized compound, 6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido [2,3-b]indole (HAC-Y6), exhibited potent antitumor activity against human hepatocellular carcinoma (HCC) cells in vitro. Western blot and immunofluorescence experiments showed that HAC-Y6 depolymerized microtubules similarly to the effects of colchicine. HAC-Y6-treatment in Hep3B cells resulted in the accumulation of the G2/M phase and induced apoptosis. In addition, HAC-Y6-treatment influenced the expression of cell cycle and apoptosis related proteins in Hep3B cells. HAC-Y6 exposure increased caspases-3, -8, -9 and Bax protein levels, while reducing levels of Bcl-2 family proteins. Moreover, Bid, a substrate of caspase-8, was also activated by HAC-Y6. Treatment of cells caused the up-regulation of the death receptor 4 (DR4) and phosphorylation of p38. Taken together, we show that HAC-Y6 exhibited its antitumor activity by disrupting microtubule assembly, causing cell cycle arrest and apoptosis through both extrinsic and intrinsic pathways in Hep3B cells. Therefore, the novel compound HAC-Y6 is a promising microtubule inhibitor that has great potential for treatment of HCC.     

Antitumor activity of IHL-305, a novel pegylated liposome containing irinotecan, in human xenograft models

The antitumor effect of IHL-305, a novel pegylated liposome containing irinotecan, was investigated in human xenograft models. After subcutaneous transplantation of several human cancer cell lines (colorectal, non-small cell lung, small cell lung, prostate, ovarian and gastric cancer cells) to nude mice, IHL-305 or CPT-11 was administered intravenously 3 times at 4-day intervals. In all xenograft models tested, IHL-305 showed superior antitumor activity to that of CPT?11 and a comparable tumor-growth-inhibitory effect at one-eighth or less of the dose of CPT-11, even against HT-29 colorectal and NCI-H460 non-small cell lung cancer cell lines, which show intrinsic resistance to CPT-11. A single injection or 2 injections of IHL-305 on several dosing schedules also resulted in a significant antitumor effect compared to that of vehicle control in a dose-dependent manner and showed comparable antitumor activity at about one-fifth the dose of the maximum tolerated dose of CPT-11. The analysis of the concentrations of irinotecan and SN-38, an active metabolite of CPT-11, in plasma and tumors revealed that irinotecan was maintained at high concentrations, and the prolonged presence of SN-38 in plasma and tumors in IHL-305 treated mice compared with CPT-11-treated mice. Therefore, the stronger tumor inhibitory effect of IHL-305, as compared to CPT-11, was associated with the difference in the concentration of irinotecan in plasma or tumors after each agent was administered and with the maintainance of a higher concentration of SN-38. These results indicate that IHL-305 demonstrated superior antitumor activity against a wide range of tumors at lower doses than CPT-11.     

Fluorescence assay of tissue distribution of 4-demethoxydaunorubicin and 4-demethoxydoxorubicin in mice bearing solid tumors

Summary The tissue distribution of 4-demethoxydaunorubicin and 4-demethoxydoxorubicin was studied in comparison with that of their parent compounds, daunorubicin and doxorubicin, in mice bearing transplanted tumors. The doses administered were equal or equitoxic to those of their parent compounds. The levels of total fluorescence due to initial drugs and metabolites were determined on tissue extracts by fluorometry. After administration of equal doses of daunorubicin and 4-demethoxydaunorubicin, the calculated Cxt values of 4-demethoxydaunorubicin equivalents were higher than those for daunorubicin in all the organs tested except the heart. In animals treated with equitoxic doses, lower 4-demethoxydaunorubicin levels were found in all the organs tested. In mice treated with equitoxic doses of doxorubicin and 4-demethoxydoxorubicin, 4-demethoxydoxorubicin reached higher drug concentrations than doxorubicin in spleen and liver, whereas in all the other organs tested lower drug levels were found. The rate of drug disappearance from organs was slower in animals treated with 4-demethoxyderivatives than in those treated with their parent drugs.     

Antitumor Activity and Pharmacokinetics of TAS-106, l-(3-C-Ethynyl-β-D-ribo-pentofuranosyl)cytosine

We examined the effects of dosage schedule on antitumor activity in vitro and in vivo to determine the optimal administration schedule for a new nucleoside antimetabolite l-(3- C -ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106). The cytotoxicity of TAS-106 in vitro against human tumors was evaluated at three drug exposure periods. TAS-106 exhibited fairly potent cytotoxicity even with 4 h exposure, and nearly equivalent and sufficiently potent cytotoxicity with 24 and 72 h exposures. These results suggest that long-term exposure to TAS-106 will not be required to achieve maximal cytotoxicity. The antitumor activity of TAS-106 in vivo was compared in nude rat models bearing human tumors on three administration schedules, once weekly, 3 tunes weekly, and 5 tunes weekly for 2 or 4 consecutive weeks. TAS-106 showed strong antitumor activity without serious toxicity on all three schedules, but the antitumor activity showed no obvious schedule-dependency in these models. When tumor-bearing nude rats were given a single i.v. dose of [³H]TAS-106, tumor tissue radioactivity tended to remain high for longer periods of time as compared to the radioactivity in various normal tissues. Furthermore, when the metabolism of TAS-106 in the tumor was examined, it was found that TAS-106 nucleotides (including the active metabolite, the triphosphate of TAS-106) were retained at high concentrations for prolonged periods. These pharmacodynamic features of TAS-106 may explain the strong antitumor activity without serious toxicity, observed on intermittent administration schedules, in nude rat models with human tumors. We therefore consider TAS-106 to be a promising compound which merits further investigation in patients with solid tumors.