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Biochemistry Aug 1999Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA...
Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).
Topics: Alkylating Agents; Animals; Antiviral Agents; Cell Line; Cell-Free System; Chlorocebus aethiops; DNA Adducts; DNA Damage; DNA Replication; DNA, Viral; Dose-Response Relationship, Drug; Duocarmycins; Electrophoresis, Gel, Two-Dimensional; HeLa Cells; Hot Temperature; Humans; Indoles; Simian virus 40; Trans-Activators; Urea; Virus Replication
PubMed: 10471303
DOI: 10.1021/bi990598r -
Cancer Chemotherapy and Pharmacology 1994Bizelesin (NSC-615291), a potent, bifunctional analog of the cyclopropylpyrroloindole antitumor antibiotics CC-1065 and adozelesin, has been selected by the National...
Bizelesin (NSC-615291), a potent, bifunctional analog of the cyclopropylpyrroloindole antitumor antibiotics CC-1065 and adozelesin, has been selected by the National Cancer Institute for evaluation as a potential chemotherapeutic agent. All three compounds bind to and alkylate DNA at the N-3 position of adenine in a sequence-selective manner. Bizelesin is unique among the analogs with bifunctional alkylating capability due to two chloromethyl moieties that are converted to the cyclopropyl alkylating species that interact with DNA. A reverse-phase high-performance liquid chromatography (HPLC) assay and an L1210 cell bioassay were developed for bizelesin and subsequently applied to stability and murine pharmacokinetics studies. Following 48 h of incubation with L1210 cells the 50% growth-inhibitory concentrations (IC50) of bizelesin, adozelesin, and CC-1065 were 2.3, 3.4, and 88.1 pM, respectively. Bizelesin was stable in organic solvents but was less stable in aqueous solutions, with the half-life values obtained in buffers at pH 4, 7, and 10 being 9.6, 2.1, and < 1 h, respectively. By HPLC analysis, bizelesin degradation was associated with the appearance of two peaks, the mono- and dicyclopropyl derivatives formed by base-catalyzed intramolecular alkylation of the chloromethyl groups. Bizelesin and the dicyclopropyl derivative were equipotent in the L1210 cell bioassay. Following i.v. administration of bizelesin (15 micrograms/kg) to male CD2F1 mice, the plasma elimination of cytotoxic activity determined with the bioassay was described by a two compartment open model; the alpha-phase (t1/2 alpha) and beta-phase (t1/2 beta) half-lives, steady-state volume of distribution (VSS), and total body clearance (ClTB) were 3.5 min, 7.3 h, 7,641 ml/kg, and 16.3 ml min-1 kg-1, respectively. The systemic drug exposure following i.p. administration was at least 10 times lower than that resulting from i.v. infusion. Following i.v. or i.p. administration the recovery of material in urine was < 0.1% of the delivered dose.
Topics: Alkylating Agents; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzofurans; Chromatography, High Pressure Liquid; Cyclohexanecarboxylic Acids; Cyclohexenes; Duocarmycins; Indoles; Leucomycins; Leukemia L1210; Male; Mice; Mice, Inbred Strains; Urea
PubMed: 8033298
DOI: 10.1007/BF00686039 -
Annals of Oncology : Official Journal... May 2003The aim of this study was to assess the feasibility of administering bizelesin, a cyclopropylpyrroloindole with extraordinarily high potency as a bifunctional... (Clinical Trial)
Clinical Trial
BACKGROUND
The aim of this study was to assess the feasibility of administering bizelesin, a cyclopropylpyrroloindole with extraordinarily high potency as a bifunctional DNA-damaging agent and selectivity for specific AT-rich DNA sequences, as a single i.v. bolus injection every 4 weeks in patients with advanced solid malignancies. The study also sought to determine the maximum tolerated dose (MTD) of bizelesin, characterize its pharmacokinetic behavior, and seek preliminary evidence of anticancer activity.
PATIENTS AND METHODS
Patients with advanced solid malignancies were treated with escalating doses of bizelesin as an i.v. bolus injection every 4 weeks. The selection of the specific starting dose, 0.1 micro g/m(2), which was equivalent to one-tenth the toxic dose low in dogs, factored in large interspecies differences in myelotoxicity as gauged using an ex vivo hematopoietic colony-forming assay. Due to concerns about the high potency of bizelesin and the large interspecies differences in toxicity, a conservative dose-escalation scheme was used for dose-level assignment to determine the MTD levels for both minimally pretreated (MP) and heavily pretreated (HP) patients. A variety of analytical assays were assessed to reliably measure bizelesin concentrations in plasma.
RESULTS
Sixty-two patients were treated with 185 courses of bizelesin at eight dose levels ranging from 0.1 to 1.5 micro g/m(2). Myelosuppression, principally neutropenia that was always brief, was the most common toxicity observed. Thrombocytopenia and anemia were uncommon and severe non-hematological effects were not observed. Severe neutropenia alone and/or associated with fever was consistently experienced by HP and MP patients at doses exceeding 0.71 and 1.26 micro g/m(2), respectively. These doses also resulted in functionally non-cumulative myelosuppression as repetitive treatment was well-tolerated. A 40% reduction in measurable disease lasting 24 months was noted in a patient with advanced ovarian carcinoma. Various analytical methods were evaluated but none demonstrated the requisite sensitivity to reliably quantify the minute plasma concentrations of bizelesin and metabolites resulting from administering microgram quantities of drug.
CONCLUSIONS
The highly potent and unique cytotoxic agent, bizelesin can be feasibly administered to patients with advanced solid malignancies. The recommended doses for phase II studies of bizelesin as a bolus i.v. injection every 4 weeks are 0.71 and 1.26 micro g/m(2) in HP and MP patients, respectively. The characteristics of the myelosuppression, the paucity of severe toxicities with repetitive treatment, the preliminary antitumor activity noted, and, above all, its unique mechanism of action as a selective DNA-damaging agent and high potency, warrant disease-directed evaluations of bizelesin in solid and hematopoietic malignancies and consideration of its use as a cytotoxic in targeted conjugated therapeutics.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; DNA; Dose-Response Relationship, Drug; Drug Administration Schedule; Duocarmycins; Female; Humans; Indoles; Intercalating Agents; Male; Middle Aged; Neoplasms; Urea
PubMed: 12702533
DOI: 10.1093/annonc/mdg215 -
Biochimica Et Biophysica Acta Jul 1997Bizelesin, an AT-specific DNA-alkylating antitumor drug, is a potent inhibitor of genomic DNA replication in BSC-1 cells. Fifty percent inhibition of DNA synthesis was...
Bizelesin, an AT-specific DNA-alkylating antitumor drug, is a potent inhibitor of genomic DNA replication in BSC-1 cells. Fifty percent inhibition of DNA synthesis was observed at 10 nM bizelesin compared to 160 nM needed for 50% inhibition of RNA synthesis while no inhibition of protein synthesis was observed up to 200 nM. Sedimentation analysis of nascent genomic DNA showed that bizelesin inhibited new replicon initiation and had significantly less effect on replicon maturation. Bizelesin also suppressed the intracellular synthesis of simian virus 40 (SV40) DNA in virus-infected BSC-1 cells. The analysis of nascent SV40 intermediates synthesized after bizelesin treatment confirmed an initiation-specific inhibition. The inhibitory effects on cellular DNA replication occurred at bizelesin levels resulting in infrequent adducts (one adduct per several replicons). Only one bizelesin adduct per several SV40 molecules was needed for a potent inhibition of intracellular SV40 replication. In contrast, only partial inhibition of SV40 replication in vitro was observed with bizelesin-treated naked SV40 DNA as a template. Overall, the results indicate that infrequent bizelesin lesions impede the cellular replication apparatus at the level of the initiation of new replicons.
Topics: Animals; Antineoplastic Agents, Alkylating; Cell Line; Cross-Linking Reagents; DNA Replication; DNA, Viral; Duocarmycins; Haplorhini; Indoles; Replicon; Simian virus 40; Urea
PubMed: 9256064
DOI: 10.1016/s0167-4781(97)00046-8 -
Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA.Biochimica Et Biophysica Acta Feb 1999Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal... (Comparative Study)
Comparative Study
Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin.
Topics: Animals; Base Sequence; Cell Line; Chemical Fractionation; Chlorocebus aethiops; Cross-Linking Reagents; DNA Adducts; DNA, Viral; Duocarmycins; Electrophoresis, Agar Gel; Genetic Techniques; Indoles; Simian virus 40; Urea
PubMed: 10023060
DOI: 10.1016/s0167-4781(99)00002-0 -
Cancer Chemotherapy and Pharmacology 1996Bizelesin is a potent synthetic derivative of the anticancer agent CC-1065 that preferentially alkylates and binds the minor grove of DNA. Preclinical animal studies... (Comparative Study)
Comparative Study
Bizelesin is a potent synthetic derivative of the anticancer agent CC-1065 that preferentially alkylates and binds the minor grove of DNA. Preclinical animal studies have found bizelesin to be more toxic to beagle dogs than to rodents and that myelosuppression was the dose-limiting toxicity. This toxicity was dose- and time-dependent in all species. Due to the significant difference in the in vivo myelotoxicity between species, it was important to determine which one most closely resembles humans on a pharmacodynamic basis. Therefore, hematopoietic clonal assays were utilized to evaluate the effects of bizelesin on granulocyte-macrophage (CFU-gm) colony formation. Marrow cells were exposed in vitro to bizelesin (0.001-1000 nM) for 1 or 8 h and then assayed for colony formation. There was a 3-log difference in drug concentration at which 100% colony inhibition occurred (1 or 8 h) for murine CFU-gm versus human or canine CFU-gm. The IC70 value after an 8-h bizelesin exposure for human CFU-gm (0.006 +/- 0.002 nM) was 2220-times lower than for murine CFU-gm (13.32 +/- 8.31 nM). At any given concentration, an 8 h drug exposure resulted in greater colony inhibition than a 1 h exposure for all species (P < 0.05). Increasing exposure time from 1 to 8 h increased toxicity to human and canine CFU-gm much more than to murine CFU-gm. The clinically formulated drug solution was a more potent inhibitor of human colony formation than drug dissolved in DMSO. The IC70 value after a 1-h exposure was 1.7 times lower for human CFU-gm with formulated bizelesin (0.106 +/- 0.105 nM) than bulk drug in DMSO (0.184 +/- 0.044 nM). The results of these in vitro clonal assays were qualitatively consistent with those seen in whole animal studies, suggesting that bizelesin will be a potent myelosuppressive agent in the clinic. Since the dose-limiting toxicity in preclinical models is myelosuppression and the in vitro sensitivity of human and canine CFU-gm is similar, the canine maximum tolerated dose (MTD) is better than the murine MTD to determine a safe starting dose for phase I clinical trials.
Topics: Animals; Antineoplastic Agents, Alkylating; Bone Marrow; Dogs; Duocarmycins; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Indoles; Male; Mice; Mice, Inbred BALB C; Species Specificity; Stem Cells; Urea
PubMed: 8995512
DOI: 10.1007/s002800050550 -
Clinical Cancer Research : An Official... Jul 1996Bizelesin (U-77779, NSC 615291), a synthetic analogue of the cytotoxic antibiotic CC-1065, is a bifunctional alkylating agent that produces DNA interstrand cross-links....
Bizelesin (U-77779, NSC 615291), a synthetic analogue of the cytotoxic antibiotic CC-1065, is a bifunctional alkylating agent that produces DNA interstrand cross-links. Bizelesin was evaluated for antitumor activity against a broad spectrum of syngeneic murine tumors and human tumor xenografts in mice. Systemic drug administration produced >6.7 log10 cell kill against i.p. implanted P388 and L1210 leukemias and 80% tumor-free survivors against s.c. implanted L1210. Against i.p. implanted B16 melanoma, i.p. drug administration produced a 158%; increase in life span with 25% tumor-free survivors, whereas i.v. drug administration produced only a 67% increase in life span with no tumor-free survivors. More than 1.0 log10 cell kill was observed at low microgram/kg doses in several human tumor models representing diverse histiotypes (CAKI-1 renal, LX-1 lung, HT-29 colon, LOX IMVI and UACC-62 melanomas, and MX-1 mammary). Less than 1.0 log10 cell kill was exhibited in other tumor models (Lewis lung, colon 38, pancreatic 02, MCF7 mammary, and SK-MEL-3 melanoma). Bizelesin was optimally active when administered i.v. Although antitumor activity was independent of the schedule of administration, greater total doses were tolerated on the more prolonged schedules in any given experiment. Therapeutic doses of bizelesin did not produce delayed deaths, which had previously been observed for the parent compound CC-1065. However, recovery of lost weight was not attained until 16-30 days posttherapy. Bizelesin was as active against murine leukemia sublines resistant to cisplatin, melphalan, and 1,3-bis-(2-chloroethyl)-1-nitrosourea as against the parental line but was totally inactive against a doxorubicin-resistant subline. The complete cross-resistance of the doxorubicin-resistant subline to bizelesin suggests that bizelesin may be a substrate for the efflux pump that causes multidrug resistance. Due to its breadth of antitumor activity, potency, unique mechanism of action, and lack of cross-resistance with other alkylating agents, bizelesin was selected for development in clinical trials by the National Cancer Institute and the Upjohn Company. Toxicological studies and pharmaceutical development have been completed, and clinical trials are planned to start in the summer of 1996.
Topics: Animals; Antineoplastic Agents, Alkylating; Body Weight; Drug Administration Schedule; Drug Resistance, Neoplasm; Duocarmycins; Humans; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasms, Experimental; Transplantation, Heterologous; Urea
PubMed: 9816280
DOI: No ID Found -
Biochimica Et Biophysica Acta Jul 2002Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug... (Review)
Review
Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.
Topics: Antineoplastic Agents; Base Sequence; Benzofurans; Binding Sites; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA Damage; DNA, Neoplasm; Distamycins; Drug Design; Duocarmycins; Genome, Human; Humans; Indoles; Nitrogen Mustard Compounds; Tandem Repeat Sequences; Tumor Cells, Cultured; Urea
PubMed: 12084472
DOI: 10.1016/s0925-4439(02)00093-5 -
Biochemistry Oct 1995Bizelesin is a bifunctional covalent minor groove binding agent which forms adducts with 3'-adenines on opposite DNA strands. DNA lesions induced by bizelesin in genomic...
Bizelesin is a bifunctional covalent minor groove binding agent which forms adducts with 3'-adenines on opposite DNA strands. DNA lesions induced by bizelesin in genomic DNA of BSC-1 cells, as well as intracellular and purified simian virus 40 (SV40) DNA, were examined. Alkaline sucrose sedimentation analysis indicated a nonrandom distribution of heat-labile damage in BSC-1 cell genomic DNA with frequencies of 1-60 lesions/10(6) base pairs (bp) for bizelesin concentrations from 10 to 400 nM, respectively. Extrapolation of these data suggested that, at 0.15 nM bizelesin, approximately 10(2) adducts per cell may be sufficient to inhibit cell growth by 90% (D10). While the frequency of bizelesin adducts in intracellular SV40 DNA was comparable to that in genomic DNA, higher levels of lesion formation are observed with purified SV40 DNA. Chromatin structure has little effect on localization of bizelesin adducts since treatment of either infected cells or purified SV40 DNA reveals a similar pattern of drug-induced damage. Bizelesin adduction sites (mapped on the SV40 genome as thermally-induced strand breaks at 50-100 bp resolution) are found in regions centered at 4200, 3900, 4700, and approximately 5200. The location of these regions of intense bizelesin bonding coincides with the sites of potential cross-links predicted using the 5'-T-(A/T)4-A-3' sequence. The analysis of bizelesin adducts at the sequence level in the 3943-4451 SV40 DNA fragment indicated that 40% of total damage was in potential cross-linking sites and an additional 35% in the 5'-A-(A/T)4-A-3' monoalkylating sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Antineoplastic Agents, Alkylating; Base Sequence; Cell Survival; Cells, Cultured; Chlorocebus aethiops; Cross-Linking Reagents; DNA Adducts; DNA Damage; DNA Primers; DNA, Viral; Duocarmycins; In Vitro Techniques; Indoles; Molecular Sequence Data; Mutagens; Nucleic Acid Conformation; Simian virus 40; Urea
PubMed: 7548063
DOI: 10.1021/bi00040a015 -
Molecular Cancer Therapeutics Jul 2003As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions,...
The DNA minor groove-alkylating cyclopropylpyrroloindole drugs adozelesin and bizelesin induce different DNA damage response pathways in human colon carcinoma HCT116 cells.
As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions, whereas bizelesin induces both single-strand lesions and double-strand DNA cross-links. At equivalent cytotoxic concentrations, these agents caused different biological responses. Low adozelesin concentrations (e.g., 0.5 nM) induced a transient S-phase block and cell cycle arrest in G(2)-M, as well as increased induction of p53 and p21, whereas a high drug concentration (e.g., 2.5 nM) caused apoptosis but no p21 induction. In contrast, both low and high bizelesin concentrations enhanced p53 and p21 induction and triggered G(2)-M cell cycle arrest and eventual senescence without significant apoptotic cell death. However, in cells lacking p21, bizelesin, as well as adozelesin, triggered apoptosis, indicating that p21 was crucial to sustained bizelesin-induced G(2)-M arrest. Thus, despite similar abilities to alkylate DNA, the chemotherapeutic agents adozelesin and bizelesin caused a decrease in HCT116 tumor cell proliferation by different pathways (i.e., adozelesin induced apoptosis, and bizelesin induced senescence).
Topics: Antineoplastic Agents, Alkylating; Apoptosis; Benzofurans; Cell Cycle; Cellular Senescence; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Duocarmycins; Humans; Indoles; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Urea
PubMed: 12883038
DOI: No ID Found