Polymerization of the triphosphates of AraC, 2',2'-difluorodeoxycytidine (dFdC) and OSI-7836 (T-araC) by human DNA polymerase alpha and DNA primase

OSI-7836 (4'-thio-araC, T-araC) is a nucleoside analogue that shows efficacy against solid tumor xenograft models. We examined how the triphosphates of OSI-7836 (T-araCTP), cytarabine (araCTP), and gemcitabine (dFdCTP) affected the initiation of new DNA strands by the pol alpha primase complex. Whereas dFdCTP very weakly inhibited primase, both T-araCTP and araCTP potently inhibited this enzyme. Primase polymerized T-araCTP and araCTP more readily than its natural substrate, CTP, and incorporation resulted in strong chain termination. dFdCTP, araCTP, and T-araCTP inhibited pol alpha competitively with respect to dCTP. When exogenously added primentemplates were used, pol alpha incorporated all three analogues into DNA, and incorporation caused either weak chain termination (dFdCTP), strong termination (araCTP), or extremely strong termination (T-araC). Furthermore, pol alpha polymerized T-araCTP only nine-fold less well than dCTP, whereas it polymerized araCTP and dFdCTP 24- and 83-fold less well, respectively. The presence of these three analogues in the template strand resulted in significant pausing by pol alpha, although the site and severity of pausing varied between the analogues. During the elongation of primase-synthesized primers, a reaction that is thought to mimic the normal sequence of events during the initiation of new DNA strands, pol alpha polymerized all three compounds. However, incorporation of araCTP and dFdCTP resulted in minimal chain termination, while incorporation of T-araCTP still caused extremely strong termination. The implications of these results with respect to how these compounds affect cells are discussed.     

Polymerization of 2'-fluoro- and 2'-O-methyl-dNTPs by human DNA polymerase alpha, polymerase gamma, and primase

Studies were undertaken to assess the ability of human polymerase alpha (pol alpha) and polymerase gamma (pol gamma) to incorporate 2'-fluoro- and 2'-O-methyldeoxynucleotides into DNA. In vitro DNA synthesis systems were used to detect incorporation and determine K(m) and V(max) for 2'-FdATP, 2'-FdUTP, 2'-FdCTP, 2'-FdGTP, 2'-O-MedATP, 2'-O-MedCTP, 2'-O-MedGTP, 2'-O-MedUTP, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol alpha incorporated all 2'-FdNTPs except 2'-FdATP, but not 2'-O-MedNTPs. Pol gamma incorporated all 2'-FdNTPs, but not 2'-O-MedNTPs. In general, 2'-fluorine substitution decreased V(max)/K(m) 2'-FdUTP. Because kinetics of insertion of pol alpha can be affected by the nature of the primer, we examined the ability of pol alpha to polymerize 2'-fluoro- and 2'-O-MedATP and dGTP when elongating a primer synthesized by DNA primase. Under these conditions, both 2'-FdATP and 2'-FdGTP were polymerized, but 2'-O-MedATP and 2'-O-MedGTP were not. Primase alone could not readily polymerize these analogs into RNA primers. Previous studies showed that 2'-deoxy-2'-fluorocytosine (2'-FdC) is incorporated by several non-human DNA polymerases. The current studies showed that human polymerases can polymerize numerous 2'-FdNTPs but cannot polymerize 2'-O-MedNTPs.     

Gallium(III) and iron(III) complexes of alpha-N-heterocyclic thiosemicarbazones: synthesis, characterization, cytotoxicity, and interaction with ribonucleotide reductase

A series of gallium(III) and iron(III) complexes with five different 4N-substituted alpha-N-heterocyclic thiosemicarbazones, viz., 2-acetylpyridine N,N-dimethylthiosemicarbazone (1), 2-acetylpyridine N-pyrrolidinylthiosemicarbazone (2), acetylpyrazine N,N-dimethylthiosemicarbazone (3), acetylpyrazine N-pyrrolidinylthiosemicarbazone (4), and acetylpyrazine N-piperidinylthiosemicarbazone (5), with the general formula [GaLCl2] (HL = 1 and 2) and [ML2][Y] (M = Ga, HL = 1-5, Y = PF6; M = Fe, HL = 1-5, Y = FeCl4 and PF6) were synthesized and characterized by elemental analysis, a number of spectroscopic methods (NMR, IR, UV-vis), mass spectrometry, and X-ray crystallography. The in vitro antitumor potency was studied in two human cancer cell lines (41M and SK-BR-3). The central metal ions exert pronounced effects in a divergent manner: gallium(III) enhances, whereas iron(III) weakens the cytotoxicity of the ligands. The capacity of ligand 1 and its Ga(III) and Fe(III) complexes to destroy the tyrosyl radical of the presumed target ribonucleotide reductase is reported.     

Interaction of d-tubocurarine analogs with mutant 5-HT(3) receptors

Apomorphine has been introduced in the treatment of late-stage Parkinson's Disease (PD). The disadvantage of a short half-life of apomorphine is now overcome by the use of a continuous subcutaneous (s.c.) self-delivering system. We examined whether continuous s.c. infusion of apomorphine rescues nigro-striatal dopaminergic neurons from toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Apomorphine was continuously infused in mice by means of a s.c. minipump that delivered the drug at a rate of 0.5 or 3.15 mg/kg/day. MPTP induced a >80% reduction in striatal dopamine (DA) after one day. DA levels were still substantially reduced one month following MPTP injection, in spite of a partial recovery. Similarly, striatal immunoreactivity for tyrosine hydroxylase and dopamine transporter was markedly reduced at this time interval. Continuous s.c. infusion of apomorphine starting 40 h following MPTP injection rescued striatal dopaminergic terminals, as assessed by measurements of DA and its metabolites, as well as TH and DAT immunostaining after one month. The neurorescuing effect was more remarkable at a delivery rate of 3.15 mg/kg/day of apomorphine. In contrast, no rescue was observed when apomorphine was administered as a single daily s.c. bolus of 1 or 5mg/kg starting 40 h following MPTP. We conclude that apomorphine is able to rescue nigro-striatal dopaminergic neurons when continuously delivered at doses that are comparable to those delivered by minipumps in PD patients. These results suggest that continuous s.c. infusion of apomorphine not only relieves the symptoms, but also reduce the ongoing degeneration of nigro-striatal dopaminergic neurons in PD patients.     

Interactions of S-(2-haloethyl)-mercapturic acid analogs with plasmid DNA

A series of related S-(2-haloethyl)-L-cysteine analogs were synthesized and their interaction with DNA was studied with plasmid pBR322. Both S-(2-chloroethyl)-L-cysteine (CEC) and S-(2-bromoethyl)-L-cysteine (BrEC) rapidly induced relaxation of the supercoiled plasmid as determined by agarose gel electrophoresis and electron microscopy, whereas S-(2-fluoroethyl)-L-cysteine did not interact with DNA. The relaxation was most probably due to strand scission at alkylated labile sites in the DNA. When 35S-labeled CEC or BrEC was used as the substrate, covalent binding of 35S to DNA was obtained; CEF displayed a somewhat higher binding than BrEC. No binding of 35S was obtained with (2-hydroxyethyl)-L-[35S]cysteine, [35S]cysteine, or [35S]cystine, substrates which did not induce relaxation of the DNA. Esterification of the carboxyl group resulted in a somewhat lower rate of DNA strand scission, whereas N-acetylation prevented the cysteine analogs from inducing DNA strand breaks. S-(2-Chloroethyl)-glutathione (GSH) did not interact with DNA as determined by lack of effect on the superhelicity of DNA, a finding which is in agreement with the hypothesis that the primary amine groups of CEC or BrEC may participate in the formation of reactive intermediates which can interact with DNA. S-(2-Hydroxyethyl)-GSH and S-(2-hydroxyethyl)-L-cysteine were unable to induce DNA strand breaks. Neutral denaturation of supercoiled pBR322 treated with the analogs revealed that compounds which were able to induce DNA strand breaks also interfered with denaturation of double-stranded circular DNA. No such interference was observed when double-stranded linear DNA (obtained by BamH1 restriction digestion) was treated with the analogs prior to denaturation. These data indicate that a marked difference exists between S-(2-chloroethyl)-L-cysteine and S-(2-chloroethyl)-glutathione in their reaction with supercoiled plasmid DNA. Either a major difference exists in the reactivity of the corresponding episulfonium ions of these conjugates or a separate mechanism of alkylation based on a free alpha-amino of the cysteine conjugate is participating in DNA strand breakage and possible crosslinking. In vivo toxic effects of these S-(2-chloroethyl) conjugates are predicted to be distinctly different.     

Interaction of D, L-erythro- and D,L-threo-gamma-fluoromethotrexate with human leukemia cell dihydrofolate reductase

Gamma-fluoromethotrexate (FMTX) is a poorly glutamylated mimic of the anti-cancer drug methotrexate (MTX) which is useful in studies of the roles of MTX poly-gamma-glutamates. A second chiral center occurs at C-4 of the 4-fluoroglutamate used to synthesize FMTX and, as a consequence, FMTX occurs as both D,L-erythro and D,L-threo diastereomers. The interaction of both diastereomers with intracellular dihydrofolate reductase has been examined in the human leukemia cell line CCRF-CEM, using a centrifugal column technique. Measurements of the rate at which radiolabel was displaced from [3H]MTX-saturated dihydrofolate reductase following suspension of the cells in unlabeled drug indicated that MTX and the erythro isomer of FMTX gave essentially the same rate of displacement; the rate of displacement by the threo isomer of FMTX was slower, but the interpretation of these data was ambiguous since the rate of transport of threo-FMTX may have been limiting. In reciprocal experiments in which dihydrofolate reductase was saturated with [3H]erythro-FMTX, the erythro isomer and MTX again behaved equivalently in terms of displacement. When dihydrofolate reductase was saturated with [3H]threo-FMTX, the radiolabel was clearly displaced at a much faster rate than either other radiolabel regardless of whether the displacing agent was MTX or the isomer. These results indicate a distinct stereospecificity for interaction of inhibitor with dihydrofolate reductase in which the threo isomer has a faster off-rate. Of the two FMTX diastereomers, the erythro isomer thus most closely mimics the properties of MTX.     

Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity

Previous studies from our laboratories have shown that (a) Triapine() is a potent inhibitor of ribonucleotide reductase activity and (b) hydroxyurea-resistant L1210 leukemia cells are fully sensitive to Triapine. In an analogous manner, Triapine was similarly active against the wild-type and a hydroxyurea-resistant subline of the human KB nasopharyngeal carcinoma. Triapine was active in vivo against the L1210 leukemia over a broad range of dosages and was curative for some mice. This agent also caused pronounced inhibition of the growth of the murine M109 lung carcinoma and human A2780 ovarian carcinoma xenografts in mice. Optimum anticancer activity required twice daily dosing due to the duration of inhibition of DNA synthesis which lasted about 10 hr in L1210 cells treated with Triapine in vivo. DNA synthesis in normal mouse tissues (i.e. duodenum and bone marrow) uniformly recovered faster than that in L1210 leukemia cells, demonstrating a pharmacological basis for the therapeutic index of this agent. Triapine was more potent than hydroxyurea in inhibiting DNA synthesis in L1210 cells in vivo, and the effects of Triapine were more pronounced. In addition, the duration of the inhibition of DNA synthesis in leukemia cells from mice treated with Triapine was considerably longer than in those from animals treated with hydroxyurea. Combination of Triapine with various classes of agents that damage DNA (e.g. etoposide, cisplatin, doxorubicin, and 1-acetyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazine) resulted in synergistic inhibition of the L1210 leukemia, producing long-term survivors of tumor-bearing mice treated with several dosage levels of the combinations, whereas no enhancement of survival was found when Triapine was combined with gemcitabine or cytosine arabinoside. The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and further suggest that prevention by Triapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.     

Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.     

2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase

It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.     

5'-Phosphoramidates and 5'-diphosphates of 2'-O-allyl-beta-D-arabinofuranosyluracil, -cytosine, and -adenine: inhibition of ribonucleotide reductase.

Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2'-O-allyl-1-beta-D-arabinofuranosyl-uracil and -cytosine and 2'-O-allyl-9-beta-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2'-O-Allyl-araUDP proved to be inhibitory to RNR at an IC(50) of 100 microM, whereas 2'-O-allyl-araCDP was only marginally active (IC(50) 1 mM) and 2'-O-allyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2'-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2'-O-allyl-araU and -araC monophosphates, namely 2'-O-allyl-5'-(phenylethoxy-L-alanyl phosphate)-araU and -araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.     

The ribonucleotide reductase subunit M2B subcellular localization and functional importance for DNA replication in physiological growth of KB cells

Ribonucleoside diphosphate reductase (EC 1.17.4.1) (RR) is a potential target for antineoplastic agents due to its crucial role in DNA replication and repair. The expression and activity of RR subunits are highly regulated to maintain an optimal dNTP pool, which is required to maintain genetic fidelity. The human RR small subunit M2B (p53R2) is thought to contribute to DNA repair in response to DNA damage. However, it is not clear whether M2B is involved in providing dNTPs for DNA replication under physiological growth conditions. Serum starvation synchronized studies showed that a rapid increase of M2B was associated with cyclin E, which is responsible for regulation of G(1)/S-phase transition. A living cell sorting study that used KB cells in normal growth, further confirmed that M2B increased to maximum levels at the G(1)/S-phase transition, and decreased with DNA synthesis. Confocal studies revealed that M2B redistributed from the cytoplasm to the nucleus earlier than hRRM2 in response to DNA replication. Nuclear accumulation of M2B is associated with dynamic changes in dNTP at early periods of serum addition. By using M2B-shRNA expression vectors, inhibition of M2B may result in growth retardation in KB cells. We conclude that M2B may translocate from the cytoplasm into the nucleus and allow dNTPs to initiate DNA synthesis in KB cells under physiological conditions. Thus, our findings suggested that M2B might play an important role for initiating DNA replication of KB cells in normal growth.     

A novel N-hydroxy-N′-aminoguanidine derivative inhibits ribonucleotide reductase activity: Effects in human HL-60 promyelocytic leukemia cells and synergism with arabinofuranosylcytosine (Ara-C)

Ribonucleotide reductase (RR; EC 1.17.4.1) is responsible for the de novo conversion of ribonucleoside diphosphates into deoxyribonucleoside diphosphates, which are essential for DNA replication. RR is upregulated in tumor cells and therefore considered to be an excellent target for cancer chemotherapy.ABNM-13 (N-hydroxy-2-(anthracene-2-yl-methylene)-hydrazinecarboximidamide), a novel N-hydroxy-N′-aminoguanidine has been designed to inhibit RR activity using 3D molecular space modeling techniques. In this study, we evaluated its effect on human HL-60 promyelocytic leukemia cells. ABNM-13 proved to be a potent inhibitor of RR which was displayed by significant alterations of deoxyribonucleoside triphosphate (dNTP) pool balance and a highly significant decrease of incorporation of radiolabeled cytidine into DNA of HL-60 cells. Diminished RR activity caused replication stress which was consistent with activation of Chk1 and Chk2, resulting in downregulation/degradation of Cdc25A. In contrast, Cdc25B was upregulated, leading to dephosphorylation and activation of Cdk1. The combined disregulation of Cdc25A and Cdc25B was the most likely cause for ABNM-13 induced S-phase arrest. Finally, we combined ABNM-13 with the first-line antileukemic agent arabinofuranosylcytosine (Ara-C) and found that ABNM-13 synergistically potentiated the antineoplastic effects of Ara-C.Due to these promising results, ABNM-13 deserves further preclinical and in vivo testing.     

Enhanced concentration-dependent cytotoxic effect of the dinuclear copper(II) complex of L-carnitine [Cu2(L-carnitine)2Cl2(H2O)2]Cl2, compared to L-carnitine or copper chloride dihydrate, in human leukemic cell lines

We studied the antitumor properties of the dinuclear copper(II) complex of l-carnitine [Cu 2( l-carnitine) 2Cl 2(H 2O) 2]Cl 2, as well as those of l-carnitine and copper chloride dihydrate, in human leukemic cells. The complex was synthesized and characterized using EPR, (1)H NMR, (13)C NMR, IR, and UV-vis analyses. Its cytotoxic effect on the human leukemia cell lines HL-60 and K562 was studied by assessing the metabolic activity of cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT method), the structural integrity of cell membrane using Trypan blue assay, and the proliferation capacity of cells studying growth curves. Both leukemia cell lines showed a concentration-specific increased cytotoxicity of the complex, compared to l-carnitine or copper chloride dihydrate, with distinct underlying mechanisms, which were decreased proliferation efficiency for HL-60 cells and increased necrotic phenomena for K562 cells. Our results are indicative of a concentration-specific enhanced antileukemic effect of the complex, implying its value as a tool in the implementation of leukemia.     

9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp) as a substrate toward replicative DNA polymerases alpha, delta, epsilon, and epsilon*.

The diphosphoryl derivative of the acyclic nucleotide phosphonate analog 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), found previously to weakly inhibit DNA pol delta/proliferating cell nuclear antigen, was studied as a substrate for pol alpha, delta, epsilon, and epsilon*. A comparison of the Vmax and Km for this derivative (PMEApp) and dATP demonstrated that the relative efficiency of the incorporation of this analog into the DNA chain is decreasing in the following order: pol delta approximately equal to pol epsilon approximately equal to pol epsilon* > pol alpha. Under the reaction conditions, this incorporation amounted to 4.4 to 0.7% of dAMP molecules. Similar Km values for PMEApp and dATP in pol epsilon and pol epsilon* catalyzed reactions revealed that proteolysis of the enzyme probably does not affect the dNTP binding site. The DNA polymerases tested were inhibited by the reaction product (PMEA terminated DNA chain) with similar Ki/Km ratios (pol alpha 0.2; pol delta, 0.1; pol epsilon 0.05; and pol epsilon*, 0.06). The associated 3'-5'-exonuclease activity of pol delta, epsilon, and epsilon* was able to excise PMEA from the 3'-OH end of DNA with a rate one order of magnitude lower than that of the dAMP residue.     

Pyrrolo(l ,4)benzodiazepine Antitumor Antibiotics: Chemistry,Interaction with DNA,and Biological Implications

The pyrrolo(l,4)benzodiazepine (P(1,4)B) antitumor antibiotics, anthramycin, tomaymycin, sibiromycin and the neothramycins A and B, are potent anticancer agents that form covalent adducts through the exocyclic amino group of guanine in DNA. This review describes the chemistry important for both the DNA reactivity and synthesis of the carbinolamine containing drugs and the strategy for elucidation of the three-dimensional form of the adduct with DNA. The high DNA sequence specificity as well as some of the observed biological consequences of DNA damage caused by these agents in human and yeast cells are rationalized through the proposed structure of the drug-DNA adducts. Parallel toxicological studies have led to a proposal for the underlying mechanism for the cardiotoxicity of certain members of this group of agents. A rationale for designing drugs which should retain their potent antitumor activity without the associated cardiotoxicity is also proposed. Lastly, the application of the P(l,4)B's as probes for monitoring drug binding to DNA and drug-induced conformational changes is described.     

Phase I study of the ribonucleotide reductase inhibitor 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) in combination with high dose cytarabine in patients with advanced myeloid leukemia

Summary Purpose: This Phase I dose escalation study was based on the hypothesis that the addition of 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) to cytarabine would enhance cytarabine cytotoxicity. The primary objective of the study was to establish the maximum tolerated dose of 3-AP when given in combination with a fixed dose of cytarabine. Experimental design: Twenty-five patients with relapsed or refractory myeloid leukemia were enrolled to three dose levels of 3-AP. Cytarabine was administered as a 2 h infusion at a fixed dose of 1,000 mg/m²/day for 5 consecutive days. Escalating doses of 3-AP as a 2 h infusion were administered on days 2 through 5. The 3-AP infusion preceded the start of the cytarabine infusion by 4 h. Results: In general, the toxicities observed with the combination were similar to the expected toxicity profile for cytarabine when utilized as a single agent at this dose and schedule. However, two of three patients developed dose-limiting methemoglobinemia at the highest 3-AP dose studied (100 mg/m²). Transient reversible methemoglobinemia was documented in 11 of 15 patients enrolled at the 75 mg/ m² dose level. Objective evidence of clinical activity was observed in four patients. Conclusions: The combination of 3-AP and cytarabine given on this schedule is feasible in advanced myeloid leukemia. The recommended Phase II dose is 75 mg/m²/day of 3-AP on days 2–5 given prior to cytarabine administered at a dose of 1,000 mg/m²/day over 5 consecutive days. Methemoglobinemia is a common toxicity of this combination and requires close monitoring.     

羧甲基壳聚糖-Co(II)配合物与鲱鱼精DNA的相互作用

目的 研究了羧甲基壳聚糖-Co(II)配合物与鲱鱼精DNA之间的作用方式。方法 采用循环伏安法和光谱法研究了羧甲基壳聚糖-Co(II)配合物与鲱鱼精DNA之间的作用机制。结果 (1) 羧甲基壳聚糖-Co(II)的存在导致Fe(CN)63-/4-探针分子氧化还原峰电流下降,峰值电位正移,显示羧甲基壳聚糖-Co(II)和Fe(CN)63-/4-与DNA之间存在竞争性作用。(2) 随着DNA的不断加入,羧甲基壳聚糖-Co(II)的特征吸收峰强度逐渐降低,峰位红移;同时,中性红的加入导致羧甲基壳聚糖-Co(II)/DNA体系的特征吸收峰发生增色效应、峰位红移并伴有等吸收点出现,表明羧甲基壳聚糖-Co(II)、DNA与中性红三者存在新的共平衡体系,中性红对羧甲基壳聚糖-Co(II)与DNA的作用有竞争抑制作用。结论 羧甲基壳聚糖-Co(II)能够以嵌插方式与鲱鱼精DNA发生相互作用。