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Preclinical study of therapeutic properties of an innovative drug Doxorubicin-NPh (doxorubicin in the form of ultrafine suspension of phospholipid liposomes) in comparison with free doxorubicin (Doxorubicin-Teva) and protected doxorubicin (Caelyx) was performed on transplanted murine tumor models. All these drugs were efficient in Ca755 breast carcinoma model (tumor growth inhibition ≈100%, increase in lifespan 90.6-114.3%). In P388 lymphocytic leukemia and LLC lung carcinoma, advantages of the protected doxorubicin by the benefit/risk ratio (width of therapeutic interval) were demonstrated: Caelyx>Doxorubicin-NPh>Doxorubicin-Teva. Doxorubicin-NPh and Caelyx exhibited similar therapeutic activity in the LLC model, especially when administered 3 times with 3-day intervals; for Doxorubicin-Teva, the optimal interval between the injections was 7 days.

Conjugates of carminomycin (Cm) with alpha-fetoprotein (AFP) and epidermal growth factor (EGF) were prepared and their cytotoxic activities were studied in vitro. Both conjugates showed cytotoxic activity which exceeded that of free Cm in tumor cell cultures of MCF-7, SKOV3, QOS, P388 and B16 cells. The antitumor effects of the conjugates were studied in vivo in mice with subcutaneous tumors of B16 and P388 cells. The Cm-AFP and Cm-EGF conjugates inhibited tumor growth and noticeably increased the mean life span in experimental animals. Our results suggest that the therapeutic activity of Cm can be significantly enhanced by conjugation to AFP or EGF., (Copyright 2000 S. Karger AG, Basel.)

We have examined the effect of theanine, a specific amino acid in green tea, on idarubicin (IDA)-induced antitumor activity and toxicity. In combination with theanine, IDA (0.25 mg/kg per day x4 days, a dose that does not show antitumor activity) had significant antitumor activity in P388-bearing mice. The IDA concentration in the tumors in the theanine plus IDA group increased to twice the level in the IDA alone group. Furthermore, the decrease in tumor weight caused by IDA at 1.0 mg/kg per day x4 days (at this dose IDA exhibits antitumor activity) was significantly amplified by theanine. The numbers of leukocyte and bone marrow cells decreased significantly on IDA injection. Theanine significantly reversed these changes. These results suggest that theanine selectively moderates the IDA-induced toxicities. Until recently, the antitumor activity and related toxicities of this chemotherapeutic agent in leukemia could not be distinguished. Theanine increases the IDA-induced antitumor activity and ameliorates the toxicities.

It has been reported that aclarubicin inhibits etoposide (VP-16) induced cytotoxicity in human lung cancer cell lines (1, 2). However, it still remains unclear how aclarubicin (ACR) inhibits etoposide-induced cytotoxicity. We report here that the combination of ACR and VP-16 showed antagonistic cytotoxic effect in P388 murine leukemic cells. DNA unwinding assay showed that 1000 ng/ml ACR significantly reduced VP-16 induced early DNA double strand(ds) breaks compared to that of VP-16 alone at a concentration of 10 microM. However, ACR did not inhibit VP-16 induced early DNA double strand breaks at a concentration of 100 ng/ml, a clinically achievable concentration. Furthermore, DNA repair occurred within two hours after removing VP-16 even if ACR was co-cultured at concentrations of 100 and 1000 ng/ml. DNA agarose gel electrophoresis and detection of sub-G1 fraction by flowcytometer showed that 100 ng/ml of ACR inhibited VP-16 induced DNA ladder formation and formation of sub-G1 fraction. Radioactive precursor incorporation studies showed that VP-16 inhibited DNA synthesis rather than RNA synthesis. On the other hand, ACR selectively inhibited RNA synthesis at a concentration of 100 ng/ml. The VP-16 induced increment of [3H]-L-leucine uptake was canceled by addition of 100 ng/ml of ACR. These data suggest that ACR inhibited VP-16 induced apoptosis by the inhibition of RNA synthesis along with protein synthesis, but not early DNA double strand breaks and DNA repair at a concentration of 100 ng/ml in P388 murine leukemic cells.

As a part of a study to exploit anthracycline glycosides effective against resistant tumor cells, the 3'-O-methyl (3), 4'-O-methyl (4), 3'-deoxy (6), 3'-deoxy-3'-fluoro (7), and 3'-deoxy-3'-iodo (8) derivatives of 7-O-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl)daunomycinone have been prepared by coupling suitably protected glycosyl bromides with daunomycinone. The doxorubicin-type analog (5) of 4 was also prepared. Among the compounds prepared, 5 showed the highest antitumor activity. Relationships between chemical structures of the synthetic products and antitumor activities, together with the degree of resistance were discussed.

An anticancer drug adriamycin (ADR) was incorporated into polymeric micelles forming from poly(ethylene glycol)-poly(aspartic acid) block copolymer by chemical conjugation and physical entrapment. Structural stability of the polymeric micelles was found to be dependent on both the contents of chemically conjugated and physically entrapped ADR. The polymeric micelle with high contents of the chemically conjugated ADR and the physically entrapped ADR expressed very high in vivo antitumor activity against murine C 26 tumor, while the polymeric micelle with only the chemically conjugated ADR showed negligible in vivo activity. This indicates that the physically entrapped ADR played a major role in antitumor activity in vivo. For the polymeric micelle with the high ADR contents, it was found that a dimer of adriamycin molecules formed and that this dimer was physically entrapped in the inner core of the micelle as well as intact ADR.

Female sterility resulting from oocyte destruction is an unfortunate, and in many cases inevitable, consequence of chemotherapy. We show that unfertilized mouse oocytes exposed to therapeutic levels of the antitumor drug, doxorubicin (DXR), undergo apoptosis; however, fertilized oocytes do not initiate apoptosis, but enter cell-cycle arrest, when treated with DXR. Apoptosis induced by DXR in oocytes is blocked by sphingosine-1-phosphate, an inhibitor of ceramide-promoted cell death. Oocytes from Bax-deficient, but not p53-null, female mice display complete resistance to DXR-induced apoptosis in vivo and in vitro. Pretreatment of oocytes with a specific peptide inhibitor of caspases also abrogates the apoptotic response to DXR. These findings indicate that oocyte destruction caused by chemotherapy can be prevented by manipulation of apoptosis-associated signaling pathways.

Tacrolimus hydrate, a potent immunosuppressant produced by Streptomyces tsukubaensis, was examined for its effect on epirubicin activity in multidrug-resistant P388 leukemia (P388/R) cells overexpressing P-glycoprotein and the parent (P388/S) cells. In the absence of modulator, the 50% inhibitory concentration for epirubicin after 48-h incubation, determined using a microculture tetrazolium assay, was 0.8 microgram/ml in P388/R cells and 0.009 microgram/ml in P388/S cells. P388/R cells demonstrated a 90-fold reduction in sensitivity to epirubicin. Tacrolimus hydrate (1 and 10 microM) markedly enhanced epirubicin cytotoxicity by 4.2- and 26.7-fold for P388/R cells. A significant increase in LDH release from cells by tacrolimus hydrate was also observed in P388/R cells treated with epirubicin. Tacrolimus hydrate had a marked effect on epirubicin-induced G2/M blockade in the resistant cells. Both tacrolimus hydrate and cyclosporin A dramatically increased the accumulation of epirubicin by the resistant cells, while these compounds had no effect on epirubicin accumulation in the parent cells. Thus, tacrolimus hydrate is able to down-modulate P-glycoprotein-associated resistance through inhibition of P-glycoprotein function, suggesting that the drug may be a candidate for killing drug-resistant tumor cells.

Antitumor activities of liposomes containing adriamycin (L-ADM) and their distribution process into tumour cells were analysed. The lipid composition of the liposomes was dimyristoylphosphatidylglycerol (DMPG)/egg phosphatidylcholine/cholesterol/adriamycin (ADM) in a molar ratio of 11.4:2:12:1.3. Liver-metastasizing murine tumour models, M5076 and L5178Y-ML, were used. In vivo antitumour effect against these tumour models was assessed from increase in life span (ILS). The survival prolongation effect of L-ADM in mice with liver failure caused by M5076 was significantly higher than that of F-ADM. In contrast, significant enhancement of the effects by encapsulation in liposomes was not observed in L5178Y-ML-bearing mice. In vitro cytostatic activities of L-ADM against M5076 cells as well as against other tumour cell lines were lower than those of F-ADM. The in vitro kinetic study on the distribution of L-ADM to the tumour cells revealed that ADM in L-ADM was taken up into the tumour cells mainly after it was released from the liposomes rather than taken up as the liposomal form. Among the cell lines tested, M5076 cells had the highest phagocytic activity and therefore the highest uptake activity of ADM during incubation with L-ADM. These findings suggest that the augmented antitumour activity of L-ADM in M5076-bearing mice was the result of phagocytosis of L-ADM by M5076 cells as well as the reduction of toxicity, prolonged retention of ADM in systemic circulation, and liver accumulation of ADM after administration of L-ADM.

Dipyridamole, a nucleoside membrane transport inhibitor, enhanced the cytotoxicity of epirubicin for mouse leukemia P388 cells by a factor of 1.8-fold and that for 30-fold doxorubicin-resistant sublines of P388 cells (P388/DOX) by a factor of 6.5-fold. This interaction was shown to be truly synergistic by DNA histogram and median effect analysis. The dipyridamole enhancement of the cytotoxicity of epirubioin was a dose-dependent effect; it was greatest when cells were exposed to dipyridamole before treatment with epirubicin. In cell cycle experiments, 1-5 microM dipyridamole increased the accumulation of G2 + M phase produced by the treatment with 0.5-1 microM epirubicin. Dipyridamole, however, did not appear to alter the patterns of DNA histogram in sensitive cells. These results suggest that the increase of the accumulation of G2 + M phase in resistant cells is an important factor for the interaction between epirubicin and dipyridamole.

We have previously reported that dipyridamole increases the cytotoxicity of epirubicin and alters the cell cycle in doxorubicin-resistant (P388/DOX) cells, increasing the accumulation of G2/M phase by blocking the cell cycle. In cultured cells, dipyridamole increased dose-dependently the intracellular accumulation of epirubicin in the resistant cells. Simultaneous exposure of the resistant cells to epirubicin and 100 microM dipyridamole resulted in a 4.2-fold increase in proportion to the control level of epirubicin after 60 min. Dipyridamole inhibited the enhanced efflux of epirubicin in doxorubicin-resistant cells. However, dipyridamole had no effect on both the influx and efflux of epirubicin in doxorubicin-sensitive cells. In mice, lethal and bone marrow toxicity induced by epirubicin were potentiated by administration of high-dose of dipyridamole. In addition, in vivo results also demonstrated that dipyridamole in combination with epirubicin produced a significant reversal of the in vivo antitumor activity of epirubicin in mice bearing P388/DOX cells. These data imply the enhancement effects of dipyridamole on the efficacy and toxicity of epirubicin.

Doxorubicin and 14-hydroxycarminomycin 14-O-hemiadipates and 14-O-hemipimelates, synthesized from 14-bromo derivatives of daunorubicin and carminomycin and monosodium adipate and pimelate, were converted to the corresponding N-trifluoroacetylated compounds. 13-(4-Methylpiperazine-1-yl)imino derivatives of the anthracycline antibiotics were also obtained. The cytostatic activity of the compounds synthesized was studied using a panel of human and animal tumor cell lines sensitive or resistant to doxorubicin. N-Trifluoroacetylation of the antibiotics resulted in a decrease in the cytostatic activity. The activity of the water-soluble 13-(4-methylpiperazine-l-yl)imino derivatives is close to that of the corresponding parent antibiotics.

Selenium is known to inhibit growth rate of neoplastic cells. We have investigated the role of selenium (Se) in resensitization of the adriamycin (ADR) resistant murine P388/ADR cells to the action of ADR. The experiments were performed in the ADR sensitive parental P388 murine leukemia (P388/S) and its subline P388/ADR, resistant to ADR, developed in our laboratory. The effect of Se was observed to be dose dependent i.e. Se at a concentration of 5 x 10(-8)M resulted in potentiation of DNA biosynthesis whereas 5 x 10(-6)M and 5 x 10(-5)M Se resulted in inhibition of DNA-biosynthesis in P388/S cells. Along with ADR there was a further increase in inhibition of DNA biosynthesis. In P388/ADR cells, Se at 5 x 10(-6)M and 5 x 10(-8)M concentration resulted in inhibition of DNA biosynthesis, which increased further when combined with ADR indicating resensitization of these cells to the action of ADR. The inhibition was observed to be partially irreversible. These results were further confirmed in the in vivo and in vitro bioassays where the Se and Se+ ADR treatments resulted in increased lifespan of tumor bearing mice.

A new dosage formulation (ACR-CH), composed of aclarubicin (ACR) bound to fine activated carbon particles, was developed for the treatment of lymph node metastases in patients with breast cancer. In a mice experimental model, ACR-CH had superior therapeutic effects on lymph node metastases compared to the same dose of ACR aqueous solution. In clinical trials, patients with breast cancer received a local injection of 10 mg/person of ACR in the form of ACR-CH or ACR aqueous solution just before mastectomy. In the regional lymph nodes removed by the operation, the ACR concentration of 40.7 micrograms/g in patients given ACR-CH was higher than the 25.1 micrograms/g in patients given ACR aqueous solution, whereas in blood plasma the concentration was higher in patients given ACR aqueous solution than in those given ACR-CH.

Four new metabolites (1-4) were isolated from mycelium of Aspergillus terreus IFO 6123 producing asterriquinone (ARQ). The structures of 1 and 2 were shown to be 3,6-bis[1-(1,1-dimethyl-2-propenyl)-1H-indol-3-yl]-furo[3,2-b]furan- 2,5-dione (asterridinone) and 2,5-bis[1-(1,1-dimethyl-2-propenyl)-1H-indol-3- yl]-3-acetoxy-6-hydroxy-2,5-cyclohexadiene-1,4-dione (ARQ monoacetate), respectively, by the chemical and spectral data. Compounds 3 and 4 were identified with known asterriquinone isomers, 2-[1-(1,1-dimethyl-2-propenyl)-1H-indol-3-yl]-5-[2-(3-methyl-2-butenyl)- 1H-indol-3-yl]-3,6-dihydroxy-2,5-cyclohexadiene-1,4-dione (isoARQ) and 2,5-bis[2-(3-methyl-2-butenyl)-1H-indol-3-yl]-3,6-dihydroxy- 2,5-cyclohexadiene-1,4-dione (neoARQ), respectively.

New antitumor substance, designated BE-18591, was isolated from the culture broth of a streptomycete, strain BA18591. The active principle was extracted from mycelium by methanol and purified by silica gel chromatography. BE-18591 inhibited the growth of MKN-45 human stomach cancer cell line as well as P388 cell line. In in vivo experiments, BE-18591 inhibited the growth of Ehrlich ascites tumor.BE-18591 showed antimicrobial activity against Gram-positive and some Gram-negative bacteria.

Spicamycin, a nucleoside antibiotic containing fatty acids with a variety of chain lengths (C12-C18), showed potent antitumor activity against human gastric cancer SC-9 and human breast cancer MX-1 in a xenograft model. We have made several semi-synthetic spicamycin analogues (SPMs) which differed in the chain length of the fatty acid moiety, and examined their structure-antitumor activity relationship. The cytotoxic activities of SPMs depended on the chain length of the fatty acid moiety, with dodecanoyl, tetradecanoyl, hexadecanoyl and icosanoyl analogues (SPM VIII, SPM X, SPM XII and SPM XVI) exhibiting the most potent cytotoxic activity against P388 murine leukemia cells. SPM VIII showed the most activity against SC-9 in the human tumor xenograft model with the highest therapeutic index among SPMs. The antitumor activity of SPM VIII was superior to that of mitomycin C.

The effect of the topoisomerase II inhibitor doxorubicin and its non-cross-resistant analogue annamycin on DNA degradation and programmed cell death was examined in murine leukemia P388 cells. P388 parental cells exposed to various concentrations of doxorubicin and annamycin for 24 h displayed dose-dependent DNA cleavage: at 1 microM, both doxorubicin and annamycin were effective in inducing DNA breakdown, but at 10 microM, the effect was markedly decreased or totally absent. In multidrug-resistant P388/Dox cells, doxorubicin did not cause DNA cleavage, while 10 microM annamycin had a significant effect. By agarose gel analysis, drug-induced DNA fragmentation showed the characteristic pattern of internucleosomal ladder. Morphologically, P388 cells treated with 1 microM doxorubicin or annamycin for 24 h showed a reduction in cell volume and condensation of nuclear structures. Similar changes were observed in P388/Dox cells exposed to 10 microM annamycin for 24 h but not in cells exposed to 10 microM doxorubicin. Time course studies demonstrated that DNA fragmentation was detected 12 h after incubation with 1 microM doxorubicin or annamycin, while loss of membrane integrity appeared at 24 h, thus indicating that DNA degradation was a preceding event. DNA fragmentation caused by doxorubicin and annamycin was inhibited by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, and the endonuclease inhibitor aurintricarboxylic acid. Drug-induced cell death was partially prevented by cycloheximide and aurintricarboxylic acid, thus suggesting that the apoptotic process caused by these drugs requires gene expression, synthesis of new proteins, and activation of endogenous nucleases. In contrast, DNA cleavage was not affected by incubating cells with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, thus indicating that intracellular calcium depletion does not affect anthracycline-induced apoptosis. The results obtained demonstrate that the cell killing effect of anthracyclines is mediated, at least in part, by the induction of apoptosis.

Four bispyrazole compounds have been prepared as potentially more stable analogues of the DNA minor groove binding polypyrrole compounds netropsin and distamycin, which are susceptible to oxidative breakdown. These compounds bind less strongly to DNA, and show much lower specificity for binding to AT-rich DNA sequences in comparison with distamycin. N.m.r. studies show that two of these compounds cause a downfield shift of the DNA imino proton resonances on interaction with the oligonucleotide d(ATATATATAT)2, suggesting that these isomers can adopt low-energy conformations similar to that shown by distamycin in its DNA minor groove binding site. The benzoic acid mustard analogue of one of the minor groove binding bispyrazoles was prepared, and showed in vitro cytotoxicity comparable with that of the previously-reported distamycin mustard, but only a low level of activity in vivo.

Two new derivatives of glycosyl mitomycin C, 7-N-[4-O-(beta-D-glucopyranosyl and alpha-sialosyl)phenyl]-9a- methoxymitosanes, were synthesized, and their structures were elucidated by analysis of the nuclear magnetic resonance spectra. Field desorption mass spectrometry was successfully used for the confirmation of these structures. The cytotoxic, antibacterial, and antitumor activities of 7-N-(4-glycosylphenyl)-9a- methoxymitosanes were also examined.

The experimental antitumor activity of a new mitomycin derivative, 7-cysteaminomitosane (RR-150), was evaluated in mice. When administered i.p. to mice bearing i.p.-implanted tumors, RR-150 was superior to mitomycin C (MMC) in increasing the life span of animals bearing P388 leukemia, B16 melanoma, and a line of L1210 leukemia partially resistant to MMC. RR-150 appeared comparable to MMC in increasing life span of mice bearing Madison 109 lung carcinoma, Colon 26 carcinoma, or parental (nonresistant) L1210 leukemia. Mice immunosuppressed with 550 rads whole-body irradiation prior to i.p. implantation of B16 still benefited (e.g., 40% cure rate) following optimal RR-150 therapy when compared to nonirradiated, B16-implanted mice given RR-150 (e.g., 70% cure rate). RR-150 had inconsistent activity in the treatment of s.c.-implanted tumors. In toxicity evaluations, RR-150 was comparable to MMC in suppression of total while blood cell counts but appeared to be less neutropenic. RR-150 also caused less cumulative leukopenia than did MMC in a weekly chronic dose experiment. Based on serum chemistries, RR-150 did not have significant nephrotoxicity, but there was evidence of possible liver toxicity at doses near the 50% lethal dose. Because of the balance of favorable antitumor and toxicity properties of RR-150, work is under way to prepare a more bioavailable form for advanced evaluation.

We have previously utilized N-(p-azidobenzoyl)daunorubicin (NABD), a photoactive analogue of daunorubicin (DNR), to identify unique anthracycline-binding polypeptides in rodent tissues and in tumor cells. Using cultured P388 tumor cells, we have now compared the cellular pharmacology and antitumor activity of NABD with that of DNR. Although rapidly accumulated by cells, the intracellular concentration of NABD was less than 20% that of DNR at steady-state levels. The cellular uptake of both drugs by P388 cells was dependent on extracellular drug concentration in the medium and on temperature. The rapid efflux of NABD and DNR from P388 cells in drug-free medium was reduced at lowered temperature (0 degrees C). Cytofluorescence microscopy demonstrated that NABD was predominantly localized in the cytoplasm, in contrast to the nuclear localization of DNR. NABD produced dose-dependent inhibition of [3H]thymidine (IC50 = 10.0 microM) and [3H]uridine (IC50 = 1.60 microM) incorporation in P388 cells to a lesser degree than DNR ([3H]thymidine, IC50 = 0.15 microM and [3H]uridine, IC50 = 0.70 microM). Continuous exposure to NABD inhibited P388 cell proliferation with an IC50 of 0.27 microM, compared with an IC50 of 0.017 microM for DNR. NABD is a pharmacologically active, photoactive analogue of DNR, which possesses properties different from those of the parent drug but similar to those of other anthracycline analogues. Photoaffinity labeling studies with NABD may identify important cytoplasmic constituents which interact with this type of anthracycline and perhaps with the anthracycline antibiotics in general.