A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growth-inhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.     

Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.     

Poisoning of topoisomerase I by an antitumor indolocarbazole drug: stabilization of topoisomerase I-DNA covalent complexes and specific inhibition of the protein kinase activity

The authors describe an eye with a central retinal vein occlusion that developed chorioretinal anastomoses following transvitreal venipuncture, a vitreoretinal surgical technique.     

Enhanced binding to DNA and topoisomerase I inhibition by an analog of the antitumor antibiotic rebeccamycin containing an amino sugar residue

Many antitumor agents contain a carbohydrate side chain appended to a DNA-intercalating chromophore. This is the case with anthracyclines such as daunomycin and also with indolocarbazoles including the antibiotic rebeccamycin and its tumor active analog, NB506. In each case, the glycoside residue plays a significant role in the interaction of the drug with the DNA double helix. In this study we show that the DNA-binding affinity and sequence selectivity of a rebeccamycin derivative can be enhanced by replacing the glucose residue with a 2'-aminoglucose moiety. The drug-DNA interactions were studied by thermal denaturation, fluorescence, and footprinting experiments. The thermodynamic parameters indicate that the newly introduced amino group on the glycoside residue significantly enhanced binding to DNA by increasing the contribution of the polyelectrolyte effect to the binding free energy, but does not appear to participate in any specific molecular contacts. The energetic contribution of the amino group of the rebeccamycin analog was found to be weaker than that of the sugar amino group of daunomycin, possibly because the indolocarbazole derivative is only partially charged at neutral pH. Topoisomerase I-mediated DNA cleavage studies reveal that the OH-->NH2 substitution does not affect the capacity of the drug to stabilize enzyme-DNA covalent complexes. Cytotoxicity studies with P388 leukemia cells sensitive or resistant to camptothecin suggest that topoisomerase I represents a privileged intracellular target for the studied compounds. The role of the sugar amino group is discussed. The study provides useful guidelines for the development of a new generation of indolocarbazole-based antitumor agents.     

Topotecan in colorectal cancer: a phase II study of the EORTC early clinical trials group

PURPOSE: This phase II study with the topoisomerase I inhibitor topotecan was performed to determine its clinical activity and toxicity in patients with metastatic or locally unresectable colorectal cancer. PATIENTS AND METHODS: Topotecan 1.5 mg/m2 was administered intravenously by 30-minute infusion for 5 days. Fifty-nine patients entered the study, 2 were considered ineligible and 57 were evaluable for response and toxicity. RESULTS: Partial response was obtained in 4 of 57 evaluable patients (7%). The median duration of the response was 11 months (range 9.3 to 12.2). This topotecan regimen was very well tolerated. A total of 290 courses were given, with a median of 4 courses per patient (range, 1 to 18). The major toxic effects were leuko- and neutropenia (91%), grade 3-4 in 48% and 79% of courses, respectively, but with only 2 infectious complications. Other side effects were grade 1 alopecia (77%) in 46%, nausea (35%), vomiting (10%), and maculo-papular rash (6%). CONCLUSIONS: Topotecan administered as a daily-times-five regimen has only minor activity as a single-agent therapy in colorectal cancer.     

The (8-thiotheophyllinate) (triphenylphosphine) gold (I), (tTAuP): a new gold complex as an anticancer agent

In continuous Friend leukemia cell exposure to tTAuP, the IC50 was 0.2 microM whereas in cells exposed 15 or 60 min to tTAuP followed by 72 h in drug-free medium the IC50 was 2.2 and 1.3 microM respectively. A combination of tTAuP and cisplatin (CDDP) is shown to be more active than either agent alone. Intracellular accumulation studies analysed by SXRF have shown that to achieve a cytotoxic effect, large concentrations of gold are necessary to accumulate in both the nuclear and cytoplasmic fraction. However, at cytostatic doses (1 microM), tTAuP has no effect on the cell cycle but does affect DNA synthesis. At a higher dose, greater than that necessary to induce cytotoxicity, it produces DNA damage, as observed by alkaline elution method. When cells were exposed to toxic doses of tTAuP (10 microM) in the presence of albumin, cytotoxicity was significantly reduced. Similar results were obtained when cells were co-treated with L-cysteine, dithiothreitol or reduced gluthatione. Reduced cytotoxic effect can be related to the interaction with free thiol groups. According to these data it is concluded that tTAuP is highly effective in vitro; whether it is active in vivo remains to be determined.     

Competitive solvation and complexation of Cu(I), Cu(II), Pb(II), Zn(II), and Ag(I) in aqueous ethanol,acetonitrile, and dimethylsulfoxide solutions containing chloride ion with applications to hydrometallurgy

The changes in reduction potential and single ion activity of Cu(I), Cu(II), Pb(II), Zn(II), and Ag(I) have been measured in a range of aqueous ethanol (EtOH), acetonitrile (AN), and dimethylsulfoxide (DMSO) compositions containing excess chloride ion. The results are compared with changes in such solutions in the absence of chloride ion and with the changes in strong brines and rationalized in terms of the various competitive ion-solvent and ion-chloride interactions. Organic solvents are shown to generally enhance chloride ion activity and promote complex ion formation. But AN is a stronger ligand for Cu(I) and DMSO is a stronger ligand for Cu(II) and Zn(II) than is Cl⁻ or the other solvents. The decrease in metal ion activity in mixed aqueous solvents containing Cl⁻ is greater than that in concentrated aqueous chloride salt solutions, according to the strength of the chloro- or solvo-complex. These fundamental changes lead to applications in the extraction of metal ion complexes and promote the dissolution of AgCl, PbCl₂, and CuCl in aqueous DMSO containing Cl⁻. Formerly Doctoral Student, Murdoch University     

A phase I clinical and pharmacokinetic study of the new topoisomerase inhibitor GI147211 given as a 72-h continuous infusion

GI147211 is a novel, totally synthetic camptothecin with promising preclinical and early clinical activity. This study was designed to determine the maximum tolerated dose of Gl147211 as a 72-h infusion and to describe its pharmacokinetics and pharmacodynamics on this schedule. In a single-arm, rising-dose study in patients with advanced cancer, eight cohorts of three or more patients received 72-h infusions of Gl147211 at doses ranging from 0.25 to 2.5 mg m(-2) day(-1). Forty-four patients received a total of 124 cycles. All patients had refractory tumours and 40 had received prior chemotherapy and/or radiotherapy. Whole-blood Gl147211 lactone, total blood and total concentrations were measured during and over the 12 h following the infusion. Myelosuppression was observed at all dose levels. Neutropenia was dose limiting at 2.0 mg m(-2) day(-1) in minimally pretreated patients, while both neutropenia and thrombocytopenia were limiting at 1.5 mg m(-2) day(-1) in those more heavily pretreated. Phlebitis occurred with infusions through peripheral veins early in this study, necessitating the use of central venous access. Other toxicities included mild nausea and vomiting, fatigue, headache, central venous catheter infections and alopecia. Three partial and two minor responses lasting 8-34+ weeks were noted in patients with ovarian, colon and breast carcinomas and hepatoma. Mean steady-state concentrations of Gl147211 increased with dose over a range of 0.25-1.24 ng ml(-1). The mean terminal elimination half-life was 7.5 h, and the clearance averaged 1074 ml min(-1) m(-2) over the doses studied. The mean fractional excretion of unchanged drug in urine was 0.114. Gl147211 lactone exposure correlated with haematological toxicity. The recommended phase II doses for this regimen are 1.75 mg m(-2) day(-1) and 1.2 mg m(-2) day(-1) for minimally pretreated and heavily pretreated patients respectively. At these doses, steady-state Gl147211 concentrations within the range of those effective in vitro were achieved. Extensive phase II evaluation of this compound and further phase I trials evaluating more prolonged infusions are ongoing.     

3-Bromoacetylamino benzoylurea (3-BAABU), a new antimicrotubule cancericidal agent applied in cytogenetic analysis in hematology

3-Bromoacetylamino benzoylurea (3-BAABU) is a newly synthesized antimicrotubule cancericidal compound. In the present study, we investigated the possibility of using 3-BAABU as a mitotic blocking agent for hematologic karyotyping. Treatment with 3-BAABU caused scattering of metaphase chromosomes throughout the cytoplasm both in phytohemagglutinine (PHA)-stimulated human lymphocytes and in human leukemic cells. Kinetic showed a rapid uptake of 3-BAABU by treated cells and irreversibility of its effect. Using 3-BAABU in routine procedure, a karyotype of lymphocytes from a normal male was 46, XY, with normal structure and CEM leukemic line was 85, XX, in a representative spread with abnormalities similar to reports using other blocking agents. Using 3-BAABU in spectral karyotyping, details of translocations in CEM leukemic cells were readily detected in several chromosomes, such as 7 [t(7;11)], 8 [t(8;9)], 9 [t(8;9) & t(9;19)], 11 [t(7;11)], 16 [t(16;18;20)] and 20 [t(1;20)]. 3-BAABU displayed two important characters in cytogenetics, 1) it caused dispersion of chromosomes, avoiding chance of overlap; 2) compared to the conventional mitotic blocking agent, vinblastine sulfate, 3-BAABU exhibited much gentle effect on chromosomes, thus providing more flexibility in time to perform karyotyping.     

Simulation as a design tool for phase I/II clinical trials: an example from bone marrow transplantation

We discuss the design and analysis of a proposed phase I/II clinical trial in bone marrow transplantation whereby dose modifications that decrease the risk of one complication increase the risk of another. Trials of this type are carried out to determine whether a dose can be found that balances the risks of each complication. Three different scenarios describing potential relationships between each risk and the treatment dose are postulated. The scenarios encompass both favorable situations in which several acceptable doses exist and unfavorable situations in which no acceptable dose exists. The operating characteristics of three sequentially developed trial designs were examined by simulation under each dose-response scenario. The first design was derived from seemingly reasonable rules, but simulations showed that performance fell far short of what was desired, thus motivating modifications. Subsequent designs showed improved performance characteristics compared to the original design. Without close examination of the operating characteristics, the original design would have been implemented, leading to high risk of an erroneous conclusion.     

Anti-arrhythmic properties of acebutolol (Bay c 7705), a new cardioselective beta-adrenergic blocking agent: a preliminary report

A study was carried out in 20 ambulatory patients with various heart diseases and different forms of cardiac arrhythmias to evaluate the anti-arrhythmic effect of acebutolol when given orally(400 mg/day). Long-term(4 hour) ECGs were monitored before and after 3 days of treatment. The results showed that after acebutolol treatment 60% of all the arrhythmias were prevented or abolised, 16% were improved, and only 24% persisted unchanged.     

A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent

NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.     

Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones. 2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1-100 microM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na+ currents (TTXR). From a holding potential (Vh) of -90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC50 value of approximately 103 microM. 3. The inhibitory effect of 4030W92 on TTX(R) was both voltage- and use-dependent. Currents evoked from a Vh of -60 mV were inhibited by 4030W92 with a mean IC50 value of 22 microM, which was 5 fold less than the value obtained at -90 mV. Repeated activation of TTX(R) by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 microM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX(R) and markedly slowed the recovery from channel inactivation. 4. Fast TTX-sensitive Na+ currents (TTXs) were also inhibited by 4030W92 in a voltage-dependent manner. The IC50 values obtained from Vhs of -90 mV and -70 mV were 37 microM and 5 microM, respectively. 4030W92 (30 microM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTXs. 5. High threshold voltage-gated Ca2+ currents were only weakly inhibited by 4030W92. The reduction in peak Ca2+ current amplitude produced by 100 microM 4030W92 was 20+/-6% (n=6). Low threshold T-type Ca2+ currents were inhibited by 17+/-8% and 43+/-3% by concentrations of 4030W92 of 30 microM and 100 microM, respectively (n=6). 6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10-30 microM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7+/-1.5 to 4.2+/-1.0 and 2.6+/-1.1 in the presence of 10 microM and 30 microM 4030W92, respectively (n=5). 7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na+ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na+ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.     

Encapsulation of the topoisomerase I inhibitor GL147211C in pegylated (STEALTH) liposomes: pharmacokinetics and antitumor activity in HT29 colon tumor xenografts

The topoisomerase I inhibitor GL147211C [7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-campto thecin trifluoroacetate], a camptothecin analogue, has significant activity in tumor cell cytotoxicity assays in vitro and antitumor activity in both animal tumor models and human patients. Its toxicity is significant, however, effectively limiting the amount of drug that can be administered and its clinical utility. To determine whether the therapeutic index of GL147211C could be improved, the drug was encapsulated in long-circulating, pegylated (STEALTH) liposomes (SPI-355). The pharmacokinetics and antitumor activity of SPI-355 were compared to those of nonliposomal GL147211C. The plasma pharmacokinetics of SPI-355 in rats were typical of those of other pegylated liposomal formulations, with significantly increased blood circulation time; the dose-corrected area under the curve and Cmax of SPI-355 (10 mg/kg) were 1250- and 35-fold higher, respectively, than those of nonliposomal GL14711C (8.72 mg/kg). The comparative antitumor activity of SPI-355 and nonliposomal GL1472211C was evaluated in nude mice implanted with HT29 colon carcinoma xenografts. SPI-355 was 20-fold more effective than GL147211C in inhibiting tumor growth (1 mg/kg SPI-355 and 20 mg/kg GL147211C) and produced durable complete remissions of tumors at well-tolerated dose levels that were >5-fold lower than the maximally tolerated dose of GL147211C, which induced no durable complete responses. Signs of toxicity were similar between the two drugs, but liposome encapsulation increased the toxicity of drug approximately 4-fold, with increased weight loss and several deaths with SPI-355 (5 mg/kg SPI-355 versus 20 mg/kg GL147211C). Despite the increased toxicity seen with SPI-355, the therapeutic index of the liposomal formulation was increased approximately 5-fold over that of nonliposomal GL147211C, suggesting that such a pegylated liposomal formulation could demonstrate increased therapeutic index in human patients.     

Synthesis,characterization and biological studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes involving a potentially tetradentate Schiff base ligand

Co(II), Ni(II), Cu(II) and Zn(II) complexes of the Schiff base derived from vanillinidene-4-aminoantipyrine and o-phenylenediamine were synthesized and characterized by elemental analysis, ¹H-NMR, ¹³C-NMR, molar conductance, infrared, electronic, magnetic measurements, cyclic voltammetry, powder XRD and SEM. The IR results demonstrate that the co-ordination sites are the four azomethine nitrogen atoms. The electronic spectral and magnetic measurement data indicate that the complexes exhibit octahedral geometry. Powder XRD displays the crystalline nature of Co(II) and Zn(II) complexes. The SEM images reveal that all the complexes have almost similar morphologies with broken-stone like structure. The in vitro biological screening effects of the synthesized compounds were tested against the bacterial species, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus; fungal species, Aspergillus niger, Aspergillus flavus and Candida albicans by the disc diffusion method and the results show that the metal complexes are more biological active than the ligand.     

Treatment of malignant melanoma (clinical stages I and II) (author's transl)

The uncorrected cumulative five-year survival rate ("actuarial method") among 195 patients with infiltrative malignant melanoma of the skin was 58% (160 cases) in clinical stage I, 26% (35 cases) in clinical stage II. In addition to clinical staging, microstaging - i.e. the histologically determined depth of invasion of the primary tumour - is of great prognostic significance. In microstages 2 and 3 with the largest vertical tumour diameter below 0.76 mm, five-year survival rate was 100% while in microstage 3 with greater vertical tumour diameter it was 66% in microstage 4 55% and microstage 5 31%. Results of treatment can be reliably interpreted only if they are divided according to microstage. Propylactic dissection of the regional lymph-nodes (dissection in clinical stage I) need not be undertaken in microstages 2 and 3 with vertical tumours diameter below 0.76 mm. Whether prophylactic dissection was done in one or two sessions has apprarently no significant influence on survival rate. A single X-radiation dose to the primary tumour of 4 000 -6000 R immediately before excision of the tumour did not significantly increase the results. The results were particularly bad when the primary tumour was removed after inadequate manipulation.