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Background: The compound 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes - UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity., Methods: UGT-mediated transformations with rat liver (RLM), human liver (HLM), and human intestine (HIM) microsomes and with 10 recombinant human isoenzymes were investigated. Studies on the ability of C-1748 to inhibit UGT were performed with HLM, HT29 colorectal cancer cell homogenate and the selected recombinant UGT isoenzymes. The reactions were monitored using HPLC-UV/Vis method and the C-1748 metabolite structure was determined with ESI-TOF-MS/MS analysis., Results: Pseudo-molecular ion (m/z 474.1554) and the experiment with β-glucuronidase indicated that O-glucuronide of C-1748 was formed in the presence of microsomal fractions. This reaction was selectively catalyzed by UGT2B7 and 2B17. High inhibitory effect of C-1748 was shown towards isoenzyme UGT1A9 (IC 50 =39.7μM) and significant but low inhibitory potential was expressed in HT29 cell homogenate (IC 50 =84.5μM). The mixed-type inhibition mechanism (K i =17.0μM;K i '=81.0μM), induced by C-1748 was observed for recombinant UGT1A9 glucuronidation, whereas HT29 cell homogenate resulted in noncompetitive inhibition (K i =94.6μM)., Conclusions: The observed UGT-mediated metabolism of C-1748 and its ability to inhibit UGT activity should be considered as the potency for drug resistance and drug-drug interactions in the prospective multidrug therapy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug resistance is one of the major causes of pancreatic cancer treatment failure. Thus, it is still imperative to develop new active compounds and novel approach to improve drug efficacy. Here we present 9-amino-1-nitroacridine antitumor agent, C-1748, developed in our laboratory, as a candidate for pancreatic cancer treatment. We examined (i) the cellular response of pancreatic cancer cell lines: Panc-1, MiaPaCa-2, BxPC-3 and AsPC-1, differing in expression levels of commonly mutated genes for this cancer type, to C-1748 treatment and (ii) the role of P450 3A4 isoenzyme and cytochrome P450 reductase (CPR) in the modulation of this response. C-1748 exhibited the highest cytotoxic activity against MiaPaCa-2, while AsPC-1 cells were the most resistant (IC 50 : 0.015, 0.075µM, respectively). A considerable amount of apoptosis was detected in Panc-1 and MiaPaCa-2 cells but only limited apoptosis was observed in AsPC-1 and BxPC-3 cells as indicated by morphological changes and biochemical markers. Furthermore, only AsPC-1 cells underwent senescence. Since AsPC-1 cells were the most resistant to C-1748 as evidenced by the lowest P450 3A4 and CPR protein levels, this cell line was subjected to transient transfection either with P450 3A4 or CPR gene. The overexpression of P450 3A4 or CPR changed the pro-apoptotic activity of C-1748 and sensitized AsPC-1 cells to this drug compared to wild-type cells. However, metabolism was changed significantly only for CPR overexpressing cells. In conclusion, the antitumor effectiveness of C-1748 would be improved by multi-drug therapy with chemotherapeutics, that are able to induce P450 3A4 and/or CPR gene expression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Induction of proteins involved in drug metabolism and in drug delivery has a significant impact on drug-drug interactions and on the final therapeutic effects. Two antitumor acridine derivatives selected for present studies, C-1748 (9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine) and C-1305 (5-dimethylaminopropylamino-8-hydroxy-triazoloacridinone), expressed high and low susceptibility to metabolic transformations with liver microsomes, respectively. In the current study, we examined the influence of these compounds on cytochrome P450 3A4 (CYP3A4) and 2C9 (CYP2C9) enzymatic activity and gene expression in HepG2 tumor cells. Luminescence and HPLC examination, real-time RT-PCR and western blot analyses along with transfection of pregnane X receptor (PXR) siRNA and CYP3A4 reporter gene assays were applied. We found that both compounds strongly induced CYP3A4 and CYP2C9 activity and expression as well as expression of UGT1A1 and MDR1 in a concentration- and time-dependent manner. C-1748-mediated CYP3A4 and CYP2C9 mRNA induction equal to rifampicin occurred at extremely low concentrations (0.001 and 0.01μM), whereas 10μM C-1305 induced three-times higher CYP3A4 and CYP2C9 mRNA levels than rifampicin did. CYP3A4 and CYP2C9 expressions were shown to be PXR-dependent; however, neither compound influenced PXR expression. Thus, the observed drug-mediated induction of isoenzymes occurs on a PXR-mediated regulatory level. Furthermore, C-1748 and C-1305 were demonstrated to be selective PXR agonists. These effects are hypoxia-inhibited only in the case of C-1748, which is sensitive to P450 metabolism. In summary, PXR was found to be a new target of the studied compounds. Thus, possible combinations of these compounds with other therapeutics might lead to the PXR-dependent enzyme-mediated drug-drug interactions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The narrow "therapeutic window" of anti-tumour therapy may be the result of drug metabolism leading to the activation or detoxification of antitumour agents. The aim of this work is to examine (i) whether the diminished toxicity of a potent antitumour drug, C-1748, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine, compared with its 4-demethyl analogue, C-857, results from the differences between the metabolic pathways for the two compounds and (ii) the impact of reducing and/or hypoxic conditions on studied metabolism. We investigated the metabolites of C-1748 and C-857 formed in rat and human liver microsomes, with human P450 reductase (POR) and in HepG2 cells under normoxia and hypoxia. The elimination rate of C-1748 from POR knockout mice (HRN) was also evaluated. Three products, 1-amino-9-hydroxyethylaminoacridine, 1-aminoacridinone and a compound with an additional 6-membered ring, were identified for C-1748 and C-857 in all studied metabolic systems. The new metabolite was found in HepG2 cells. We showed that metabolic rate and the reactivity of metabolites of C-1748 were considerably lower than those of C-857, in all investigated metabolic models. Compared with metabolism under normoxia, cellular metabolism under hypoxia led to higher levels of 1-aminoacridine and aza-acridine derivatives of both compounds and of the 6-membered ring metabolite of C-1748. In conclusion, the crucial role of hypoxic conditions and the direct involvement of POR in the metabolism of both compounds were demonstrated. Compared with C-857, the low reactivity of C-1748 and the stability of its metabolites are postulated to contribute significantly to the diminished toxicity of this compound observed in animals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC(90) and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4x EC(90) concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent beta-galactosidase. On the contrary, no beta-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug., (2009 Elsevier Inc. All rights reserved.)

Chemotherapy in prostate cancer (CaP) even as an adjunct has not been a success. In this communication, we report the pre-clinical efficacy of a nitroacridine derivative, C-1748 (9[2'-hydroxyethylamino]-4-methyl-1-nitroacridine) in CaP cell culture and human xenograft animal models. C-1748, a DNA intercalating agent has been derived from its precursor C-857 that was a potent anti-cancer drug, but failed clinical development due to "high" systemic toxicities. Chemical modifications such as the introduction of a "methyl" group imparted novel properties, the most interesting of which is the difference in the IC(50) values between LnCaP (22.5 nM), a CaP cell line and HL-60, a leukemia cell line (>100 nM). Using gammaH2AX as an intervention marker of DNA double strand breaks, we concluded that C-1748 is more efficacious in CaP cells than in HL-60 cells. In hormone dependent cells, the androgen receptor (AR) was identified as an additional target of C-1748. In xenograft studies, administration of C-1748 intra-peritoneally inhibited tumor growth by 80-90% with minimal toxicity. These studies identify C-1748 as a novel acridine drug that has a high therapeutic index and low cytotoxicity on myelocytic cells with potential for clinical development.

Brain-derived neurotrophic factor (BDNF) modulates glutamate receptors of the NMDA type in many areas of the brain. We assessed whether BDNF exerts an effect on NMDA receptor properties in retinal ganglion cells during early postnatal development. Electrophysiological responses to the glutamate agonist NMDA (500 microM-2 mM) in retinal slices of wildtype and BDNF deficient mice (bdnf-/-) were recorded using the whole-cell patch-clamp technique. Retinal ganglion cells of bdnf-/- mice displayed significantly smaller NMDA currents than those of age-matched wildtype mice. Remarkably, NMDA receptor activity was restored by incubating retinal slices of bdnf-/- mice in BDNF (50 ng/ml) for 1-3 h. We suggest that BDNF plays a role in the activation of functional NMDA receptors in early ganglion cell development.

The cytotoxicity of two nitroheterocyclic compounds (NHCD), Nitracrine, 1-nitro-9(3-3-dimethylaminopropylamino) acridine and Quinifuryl, 2-(5'-nitro-2'-furanyl) ethenyl-4-[N-[4-(N,N-diethylamino)-1'-methylbutyl] carbamoyl] quinoline, towards two lines of leukaemic cells and a line of non-transformed cells, was measured in comparison, on the dark and under illumination with visible light (350-450 nm). Both drugs showed highly elevated cytotoxicity when illuminated with LC(50) values 7-35 times lower after 1 h illumination compared to 1 h incubation of cells incubation with drug on the dark. Cytotoxicity of Nitracrine toward all cell lines studied exceeded that of Quinifuryl, both on the dark and under illumination, so that approximately 10 times lower concentration of former drug was needed to reach the same toxicity as the latter. General toxic effect was calculated as a direct cell kill and a cell proliferation arrest. The effect >80% for both drugs was achieved after 1 h cell illumination with as low drug concentrations as 0.2 microM for Quinifuryl and 0.02 microM for Nitracrine.

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC(99) concentration delayed progression through the S phase and transiently arrested cells in G(2)/M as found by flow cytometry. Higher drug concentration (2 x IC(99)) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC(99) concentration and irreversible arrest in early S phase at the higher dose (2 x IC(99)). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G(2) arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G(2) arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.

Some N-oxide derivatives of DNA intercalators are bioreductive prodrugs that are selectively toxic under hypoxic conditions. The hypoxic selectivity is considered to result from an increase in DNA binding affinity when the N-oxide moiety is reduced. This study investigated whether differences in DNA binding affinity between N-oxides and their corresponding amines, measured by equilibrium dialysis, can account for the hypoxic cytotoxicity ratios (HCR) of tertiary amine N-oxide (-tO) and aromatic N-oxide (-aO) derivatives of the 1-nitroacridine nitracrine (NC) and its non-nitro analogue 9-[3-(N,N-dimethylamino)propylamino]acridine (DAPA). Cytotoxicity was measured in aerobic and hypoxic suspensions of Chinese hamster ovary (CHO) AA8 cells by clonogenic assay. HCR were much greater for NC-tO (820-fold) than for NC (5-fold) or NC-aO (4-fold), whereas DAPA and its N-oxides lacked hypoxic selectivity (1-fold). DNA binding measurements demonstrated that binding affinity is lowered more by aromatic than tertiary amine (side-chain) N-oxides, an observation that does not correlate with HCR. Compounds were accumulated in cells to high concentrations (C(i)/C(e) approximately 10-200), with the exception of the tertiary amine N-oxides, for which the ratio of intracellular to extracellular drug was less than unity. For NC-tO this probably resulted from low pK(a) values for both the acridine chromophore and the side-chain, whereas DAPA-tO may be too hydrophilic for efficient membrane permeation. Bioreductive drug metabolism, assessed by HPLC, was faster for the NC than the DAPA N-oxides. The high HCR of NC-tO relative to NC-aO is ascribed to the rapid and selective reduction of its N-oxide moiety, followed by activation of the NC intermediate by O(2)-sensitive reduction of its 1-nitro group to the corresponding 1-amine. The metabolism studies suggest that unmasking of DNA binding affinity by reductive removal of the N-oxide moiety, although not the only determinant, is important and needs to occur before nitroreduction for optimal effect.

The tertiary amine N-oxide (nitracrine-N-oxide, 1b) of the 1-nitroacridine nitracrine is a bis-bioreductive agent showing very high hypoxic selectivity (approximately 1000-fold) against tumour cells in culture, but only modest activity against the hypoxic subfraction of tumours in vivo. Because the hypoxic selectivity of 1b was considered to depend significantly on the rate of enzyme-mediated reduction of the N-oxide group, this paper reports the preparation and evaluation of a series of analogues in which the environment of this group was modified. Three analogues contained more weakly basic N-oxides, while two others had varying degrees of steric bulk around the N-oxide. In all but one case (an aromatic N-oxide), the N-oxides were much less cytotoxic (10- to 300-fold) than the corresponding tertiary amines towards AA8 Chinese hamster cells under aerobic conditions. Both the N-oxides and the corresponding amines were more cytotoxic to an ERCC-1 mutant defective in nucleotide excision repair, indicating that DNA alkylation was the cytotoxic event. However, there was no apparent correlation of these parameters with structure. All of the aliphatic N-oxides, with the exception of the aromatic N-oxide example, showed substantial (70- to 800-fold) hypoxic selectivity against AA8 cells in a clonogenic assay. While the weakly basic derivatives were the least selective, there was no apparent relationship between hypoxic selectivity and the steric environment of the N-oxide. Selectivity for hypoxic cells in culture is shown to depend on the hypoxic selectivity of the corresponding tertiary amine (reflecting O2-inhibitable reduction of the 1-nitro group) and the differential in aerobic toxicity between amine and N-oxide (a measure of the potential toxicity increase achievable by reducing the N-oxide). Four analogues whose structures fairly represented the range of steric and electronic modifications of the N-oxide site were evaluated against the hypoxic subfraction of cells in KHT tumours in vivo, but were inactive. These results suggest that either such modifications do not exert significant effects on N-oxide reduction, or that the rate of such reduction is not a factor limiting the in vivo activity of the parent analogue 1b.

The anticancer drug, nitracrine, a 1-nitro-9-aminoalkyl derivative of acridine exhibits potent cytotoxic effects which are due to its metabolic activation, followed by covalent binding to macromolecules--DNA being the target for the drug. The renaturable fraction of DNA from L-1210 cells pretreated with nitracrine is assayed by means of ethidium bromide fluorescence assay and chromatography on hydroxyapatite column. The effect of the drug was compared with furocoumarins of different DNA crosslinking potencies. The existence of crosslinks in DNA upon incubation of cells with nitracrine (1-4 microM) have been confirmed with two different methods under the conditions where 8-methoxypsoralen, a classic crosslinking agent induced the renaturation. The DNA preparation isolated from the drug pretreated cells exhibited decreased transcriptional template activity with E. coli DNA-dependent RNA polymerase.

Adducts generated in vitro by the reaction of 1-nitroacridines with poly(dN)s in the presence of dithiothreitol were used to identify a kind of nucleic base involved in the formation of individual adducts. The patterns of chromatographic spots corresponding to modified nucleotides obtained by 32P-post-labelling assay for synthetic homopolymers of four deoxyribonucleotides were compared with the fingerprints detected in the case of calf thymus DNA reacted with 1-nitroacridines under conditions in which the formation of identical DNA adducts as in cellular models was demonstrated in earlier investigations. Both compounds studied (Ledakrin and C-857) turned out to bind covalently only with purine nucleotides. Ledakrin formed with dG four and C-857 five different adducts. All of them were also detected in ctDNA. The incubation with poly(dA) resulted in four Ledakrin-dA species, two of which were found in ctDNA, and in two C-857-dA adducts that were not, however, observed in DNA containing samples. Modification of purines accounted for all adducts observed in ctDNA. For both compounds studied, the level of total binding to poly(dA) was about one order of magnitude lower than to poly(dG) for which it was comparable with the extent of ctDNA modification. This indicates that dG represents a preferential site of covalent binding of 1-nitroacridines to DNA.

Using agarose gel electrophoresis we confirmed that Ledakrin is capable of incurring covalent crosslinking in pBR322 plasmid DNA and also in poly(dGdC) in the presence of a simple activating system containing DTT. The identification of adducts resulting from DNA crosslinking was carried out by 32P-post-labelling assay. We assumed that such adduct(s) should be brought about more readily with double-stranded than with single-stranded polynucleotides or nucleotides. Since our earlier experiments had shown that guanine is a major site of covalent binding of 1-nitroacridines, we compared DNA adduct formation by Ledakrin for ctDNA, dG-containing synthetic homopolymers and 3'-pdG. 32P-Post-labelling assay revealed two adduct spots that were enhanced in samples containing double-stranded substrates in which interstrand crosslinking between guanines was possible, namely ctDNA and poly(dGdC).

Tertiary amine N-oxides of DNA intercalators with alkylamino sidechains are a new class of bioreductive drugs. N-oxidation masks the cationic charge of the amines, forming prodrugs with low DNA binding affinity and low toxicity which can be activated selectively by metabolic reduction under hypoxic conditions. This study compares three intercalator N-oxides (NC-NO, DACA-NO and AQ4N), which, respectively, give nitracrine (NC), DACA and AQ4 on reduction. In aerobic cell culture all three N-oxide were much less toxic than the corresponding amines, and showed large increases in cytotoxicity under hypoxia. The topoisomerase poisons DACA and AQ4 (and their N-oxides) were less active against non-cycling than cycling cells. However, only AQ4N was active against the mouse mammary tumour MDAH-MCa-4. This dialkylaminoanthraquinone-di-N-oxide has activity at least as great as the reference bioreductive drug RB 6145 against this tumour, both with and without radiation and when combined with the tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetic acid (DMXAA). It is suggested that the high in vivo activity of AQ4N relative to the other topoisomerase-targeted N-oxide, DACA-NO, may be in part due to release in hypoxic cells of an intracalator with sufficiently high DNA binding affinity that it is retained long enough to kill non-cycling cells when they eventually re-enter the cell cycle.

A series of nuclear-substituted derivatives of nitracrine N-oxide (2; a bis-bioreductive hypoxia-selective cytotoxin) were prepared and evaluated, seeking analogues of lower nitroacridine reduction potential. Disubstitution with Me or OMe groups at the 4- and 5-positions did not provide analogues with one-electron reduction potentials significantly lower than those of the corresponding monosubstituted derivatives (E(1) ca. -350 mV for both the 4-OMe and 4,5-diOMe compounds). This appears not to be due to a concomitant raising of the acridine pKa but to a lack of direct electronic effect of substituents in the ring not bearing the nitro group. Conversely, placing two OMe groups in the nitro-bearing ring does result in a substantial further lowering of reduction potential (the 2,4-diOMe analogue has an E(1) of -401 mV). The mono- and disubstituted N-oxides have substantially lower cytotoxicities than the parent nitracrine N-oxide 2 but generally retain very high hypoxic selectivity. The OMe-substituted N-oxides all showed greater metabolic stability than 2 in hypoxic AA8 cell cultures, and the 4-OMe compound 6 had improved activity in EMT6 multicellular spheroids suggesting that this metabolic stabilization may allow more efficient diffusion in tumor tissue. The parent compound 2 was selectively toxic to hypoxic cells in KHT tumors in vivo and clearly superior to nitracrine itself (although only at doses which would eventually be lethal to the host). The analogues of lower E(1), including 6, were not superior to 2 in vivo, indicating that metabolic stabilization of the nitro group is not alone sufficient to improve therapeutic utility.

1. An anticancer drug, nitracrine 1-nitro-9(3'3'-dimethylaminopropylamino)acridine (Ledakrin, C-283) exhibits potent cytostatic effects which can be ascribed to interactions of the drug with DNA. 2. The reduction of the nitro group of nitracrine is one of the activation steps leading to the drug covalent binding to DNA and proteins both in subcellular systems and in the cell. 3. DNA-drug non-covalent interactions and covalent complexes are examined in several model systems and compared with the properties of a number of derivatives with programmed structural changes. 4. DNA-protein crosslinks and interstrand crosslinks are detected in the cells following exposition to the drug. 5. The drug exhibits selective toxicity and radiosensitization effects to hypoxic mammalian cells.

Nitracrine (1-NC) and its nitro-positional analogues are electron-affinic DNA intercalating compounds that act as hypoxic cell radiosensitizers in cell culture, but are too rapidly metabolized to provide radiosensitization in vivo. We have explored the electron-trapping efficiencies of nitroacridines to see if such compounds can scavenge radicals formed in irradiated DNA and if the efficiency of such trapping is related to their radiosensitization properties. We have shown that a correlation does indeed exist as 1-NC, the most potent radiosensitizer, scavenges approximately 45% of the migrating electrons (formed by attachment of eaq- to the DNA) compared with approximately 4% for 4-NC, the least efficient radiosensitizer. The quenching of the delayed fluorescence from acridine orange bound to DNA was also used to probe intracellular uptake and association with DNA. The results are in accord with earlier measurements of average uptake and a suggestion that intercalators which form DNA complexes with short residence times will be preferable to highly bound agents as radiosensitizers. Since 1-NC possesses the smallest association constant with DNA of the four compounds, it is suggested that its high radiosensitization potency may well be related to it being able to interact with more radical sites on the DNA than the other analogues, in addition to its capture of migrating electrons in DNA.

Drugs with two reducible centers, both of which must be metabolized by oxygen-inhibitable processes for full activation ("bis-bioreductive agents"), offer potential for the development of hypoxia-selective cytotoxins with improved oxygen sensitivity. The sidechain N-oxide (1-NCO) of the (mono)bioreductive agent nitracrine (1-NC) has been synthesized and evaluated as a potential example of such an approach. The association constant for reversible DNA binding of 1-NCO was 15-fold lower than that of 1-NC, as measured by equilibrium dialysis in a low ionic strength buffer, indicating that the N-oxide has the potential to act as a less toxic pro-drug of 1-NC. Cell uptake and aerobic cytotoxicity of 1-NCO were much lower than for 1-NC whereas its hypoxic selectivity as a cytotoxin was greatly increased. In stirred suspension cultures of AA8 cells, pure (less than 0.02% 1-NC) 1-NCO was 1000-1500 times more potent under hypoxia than in 20% O2. For 1-NC the corresponding ratio was 10 +/- 1. 1-NCO had greater hypoxic selectivity in this system than misonidazole (ratio 11), RSU 1069 (ratio 25), 8Me-5NQ (ratio 60), or SR 4233 (ratio 80). Studies of 1-NCO metabolism indicate rapid, O2-inhibited reduction to 1-NC. The data are consistent with a two-step bioactivation mechanism, with reduction of the N-oxide generating a DNA intercalator of increased binding affinity, followed by reduction of the nitro group of this DNA-targeted cytotoxin to form reactive cytotoxic metabolites.

The influence of a polish anticancer drug on the cell cycle using Allium test was studied. Methods of aceto-orcein squash slides, curve of labelled mitoses after 3H-thymidine incubation and cytophotometrics after Feulgen's reaction were employed. Ledakrin acts strongly antimitotically, but it does not block the cell cycle completely. The cytostatic activity of ledakrin results from its action on the interphase. The phases G1 and S are prolonged while M is unchanged after 6h incubation with ledakrin. During postincubation in water without ledakrin it was noted, at the beginning, that the mitotic activity decreases and it is brought about the lengthening of S and G2 phases. The duration of the cell cycle phases returns to the control level during further postincubation. The results of analysis of chromatin aberrations and the micronucleus test point to a mutagenic effect of ledakrin.

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.

The mutagenic potential of 9-(3'-dimethylaminopropylamino)-acridine and of its 1-nitro and 2-nitro derivatives was investigated by using histidine-requiring mutants of Salmonella typhimurium. The 9-(3'-dimethylaminopropylamino)-acridine exhibited a weak mutagenic activity only on one of the S. typhimurium tester strains, TA1537. The 1-nitro derivative induced mutations with high frequency in strains TA1537, TA1538 and TA98, whereas the 2-nitro derivative was substantially more mutagenic than the parent compound but it was much less mutagenic than the 1-nitro derivative. Pre-mutational damages made by the 1-nitro derivative were repaired by the uvrB gene-repair system, whereas those caused by the 2-nitro derivative could not be repaired by this system. Both the 1-nitro and 2-nitro derivatives induced some mutations in the base-pair substitution strain TA100 carrying a plasmid. The frequency of the his+ mutation induced both by 1-nitro and by the 2-nitro derivatives in strain TA101 lacking a nitro reductase was lower. These results emphasize the involvement of the nitro group in the interaction of acridine derivatives with the bacterial genome.

It was demonstrated that Ledakrin, an antitumour drug, causes mobilization of free fatty acids and glycerol from the epididymal adipose tissue of rat in vitro. The disproportion observed in the release of glycerol and free fatty acids following Ledakrin administration suggested a biphasic effect of this drug, with initial stimulation and later inhibition of the processes of lipolysis and lipogenesis. Blockade of adrenergic membrane receptors (with propranolol or regitine) abolished the lipolytic effect of Ledakrin.

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.

Ledakrin (Nitracrine) and its three analogues (C-846, C-857, C-1006) selected in primary in vitro and in vivo screening systems have been tested for antitumor activity in Lewis lung carcinoma, B16 melanoma of 16/C mammary adenocarcinoma bearing mice. Their effect on mouse bone marrow stem cells has also been evaluated by means of CFU-S technique. None of the tested compounds, including Ledakrin, revealed anti-tumor activity. Their bone marrow toxicity was minimal and comparable with that of Bleomycin.

By using a new mild procedure for detecting DNA crosslinks it has been shown that adriamycin and daunomycin are able to form interstrand DNA crosslinks in HeLa cells. This effect seems to be preceded by transformation of the parent antibiotics in the cell to active forms. In addition, interstrand DNA crosslinks formed by adriamycin and daunomycin were found to be temperature- and alkali-labile.

The mechanism of cytotoxicity of a series of 4-substituted derivatives of 9-[[3-(dimethylamino)propyl]amino]-1-nitroacridine (nitracrine) has been studied, using a panel of DNA repair-defective mutants of the Chinese hamster ovary cell line AA8. Cell lines UV-4 and UV-5 were hypersensitive to nitracrine, with sensitivities approximately 10-fold greater than that of AA8, while EM-9 showed a hypersensitivity factor (HF) of about 2-fold. This pattern suggests the major cytotoxic lesions induced by nitracrine are bulky DNA monoadducts, rather than DNA interstrand cross-links as previously suggested. The desnitro analogue of nitracrine, which retains the intercalative potential of the latter but cannot be metabolically activated by nitro reduction, showed no hypersensitivity, indicating the specificity with which this panel of cell lines can discriminate different types of DNA damage. Several of the highly cytotoxic 4-substituted nitracrine derivatives showed HFs similar to that of the parent, but the less potent 4-dialkylamino and 4-COOMe derivatives showed much lower HFs for UV-4, suggesting that different mechanisms of cytotoxicity contribute. All compounds showed similar HFs under both aerobic and hypoxic conditions, indicating that hypoxia-selective toxicity in this series is due to a quantitative rather than qualitative change in the presence of oxygen. Rates of metabolic consumption of the compounds were measured under both aerobic and hypoxic conditions by bioassay against the sensitive UV-4 cell line. The results agreed well with previous inferences on metabolic stability derived from cell-killing kinetics and showed that electron-donating 4-substituents can be used to increase metabolic stability in vitro. Such stabilization may enhance the therapeutic utility of the nitroacridines in cancer therapy since rapid metabolism of nitracrine appears to prevent its activity against hypoxic cells in solid tumors.

Ledakrin effect on the bioelectrical parameters of sodium ion transport (potential difference -- PD and short-circuit current -- SSC) and on Na22 transport across frog (R. esculenta) skin was studied in vitro. Ledakrin induced a biphasic action, after a phase of increase of PD, SCC and sodium flux the transport of sodium ions decreased gradually. The effect of this nitro-derivative of acridine was compared with that of Amiloride. When both these agents were given simultaneously only one phase developed, that is inhibition of sodium ion transport. In both situations there was a significant stimulation of outflux. The obtained results suggest that the external apical cell membrane of epithelial cells is the site of action of Ledakrin.

The effect of Ledakrin (an acridine derivative with antineoplastic action), free or liposome-bound, on the bioelectric activity of frog skin was studied by the method of Ussing and it was shown that this activity (being a function of sodium transport) depended on the chemical composition of the liposomes as well as on the calcium content of the experimental medium. Two conclusions have been drawn: 1) the first phase of the response triggered by Ledakrin is due to its action on the cell membrane, 2) the second phase depends on an intracellular mechanism due, probably, to Ledakrin effect on the cytoskeleton.

The mechanism of the biological activity of the 1-nitro and 2-nitro aminoacridine derivatives containing the dimethylaminopropyl side chain was studied. RNA synthesis in the isolated rat liver nuclei was only slightly influenced by both compounds. They do not differ in their ability to form an intercalative complex with DNA. Only the 1-nitro derivative exhibited strong inhibitory effect on RNA biosynthesis and caused distinct ultrastructural changes (nucleolar segregation, chromatine margination etc.) in a living cell. The 1-nitro derivative binds covalently to DNA in vivo resulting in crosslink formation. It is concluded that the biological activity of 1-nitro acridine derivatives depends more on their crosslinking activity than on their ability to intercalate into DNA.

Hypoxic cells in solid tumours are resistant to ionizing radiation and may be refractory to treatment by many chemotherapeutic agents. For these reasons the identification of drugs with selective toxicity towards hypoxic cells is an important objective in cancer chemotherapy. Nitroimidazoles such as misonidazole demonstrate such hypoxia-selective toxicity but have very low dose potency. The 1-nitroacridine derivative 1-nitro-9-(dimethylaminopropylamino)acridine (nitracrine) binds reversibly to DNA but also forms covalent adducts with DNA in vivo. We have found nitracrine to be selectively toxic to the Chinese hamster ovary cell line AA8 under hypoxic conditions in culture, with a potency approximately 100,000 times higher than that of misonidazole. The effect of oxygen is not a simple dose-modifying one in this system, probably in part because of rapid metabolic inactivation of nitracrine under hypoxic conditions. Viscometric studies with the mini col E1 plasmid PML-21 confirmed that nitracrine binds to DNA by intercalation, and provided an unwinding angle of 16 degrees (relative to 26 degrees for ethidium). It is proposed that the cytotoxicity of nitracrine under hypoxia is due to reductive metabolism to form an alkylating species, but that intercalation of the chromophore may enhance reactivity towards DNA and hence contribute to the marked enhancement of potency with respect to simple nitroheteroaromatic drugs.

Among the optical isomers of N, N'-dimethyl-N, N'-bis[1-(3', 4', 5-trimethoxybenzoyloxy)-butyl-2]-ethylenediamine dihydrochloride only the isomer with the (2S, 2'S) configuration (Craviten) in a concentration of 0.2 microgram/ml inhibits the ADP-induced aggregation of human platelets. The (2R, 2'R) isomer and quinidine exert a similar effect but only in a concentration ten times higher. Procaine amide (2 micrograms/ml) has an opposite effect, stimulating human platelet aggregation.

The mutagenic and recombinogenic potential of 9-[(3-dimethylaminopropyl)amino]acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied using 3 different strains of Saccharomyces cerevisiae. The parent compound slightly enhanced the frequency of total aberrant colonies in diploid strains D5 and D7, but showed no evidence of recombinogenic effects. Each of the nitroacridines enhanced the frequency of total aberrant colonies in strains D5 and D7, but only the 1- and 4-nitro compounds significantly enhanced mitotic crossing-over (measured as twin spotted colonies in strains D5 and D7) or gene conversion in D7. The 3- and 4-nitro derivatives were effective mitochondrial mutagens, substantially increasing the frequency of 'petite' mutants in strains D5 and 5178B.

The authors observed changes in activity of leucine amino-peptidase (E.C. 3.4.1.1--LAP), gamma-glutamyl transpeptidase (F.C 2.3.2.2--GGTP) and Co++-activated acylase in blood serum of patients with ovarian carcinoma, with or without ascites, treated with Ledakrin (1-nitro-9-[3-dimethylaminopropylamino] acridine). It was stated that the activities of LAP and GGTP increased during the treatment and during tumor progression. The level of Co++-activated acylase increased only slightly during the treatment and therefore, the determination of this enzyme appeared to be of no use in monitoring the therapy of ovarian carcinoma.

These studies were aimed at characterizing the capability of an antitumor DNA-damaging drug, Ledakrin, and its analogs to inhibit DNA replication in HeLa S3 cells. The studied agents are extremely potent inhibitors of [3H]thymidine incorporation in whole cells. These compounds produced also a potent dose- and time-dependent inhibition of DNA synthesis in subcellular systems derived from drug-treated cells, as found by [3H]dGTP incorporation in cellular lysates and nuclei. Experiments in which nuclei from control and drug-treated cells were supplemented with cytoplasmic fractions from either control or drug-treated cells, or with exogenous DNA, demonstrate that Ledakrin and other 1-nitro-9-aminoacridines inhibit DNA replication in HeLa S3 cells by interfering with the DNA template, while not affecting DNA polymerase(s) or other enzymes and replication factors. The negligible effect of Ledakrin added to lysates or nuclei from untreated cells suggests that metabolic activation is a prerequisite for replication inhibition by Ledakrin. Analysis of the size of newly synthesized DNA, by alkaline sucrose gradient sedimentation, indicates that Ledakrin does not inhibit the initiation of replication but does interfere with chain growth. Impairment of DNA replication by 1-nitro-9-aminoacridines seems to originate from DNA damage and to result in the inhibition of cellular growth.

The influence of the cytostatic drug Ledakrin on immunologic induction of PF and RF cells was studied. In low doses, the drug had a negligibly small effect on the numbers of PFC and RFC, but in high doses nearly entirely prevented their appearance. Cells producing IgM were more susceptible to this action than IgG-producing cells. Absence of a linear relation between the dose of the drug and its action was noteworthy. Drug-induced immunosuppression occured in "all-or-none" fashion. The possibility of oncostatic effect without interfering with the immunologic system is discussed.

Nitracrine [1-nitro-9-(dimethylaminopropylamino)-acridine] is a clinical antitumour agent with hypoxia-selective cytotoxicity and radiosensitizing activity. Developments in tumour therapy using either nitracrine itself or some of its analogues under development are likely to be targeted at anaerobic regions of tumours. We have investigated the effects of anaerobiosis on mutagenic activity of nitracrine and a small series of analogues in three derivatives of Salmonella typhimurium frameshift strain hisC3076. Nitracrine-induced mutagenicity was apparently enhanced by a period of anaerobic incubation followed by an aerobic period, with maximal mutagenic effectiveness (as revertants/nmol) being seen at 24 h anaerobiosis. However, the maximal number of nitracrine-induced revertants was not increased by anaerobic incubation, and the relationship between mutagenicity and toxicity remained similar. Comparisons of the effects of anaerobiosis on mutagenicity of bacterial strains with different DNA repair capacities suggested that anaerobiosis was not simply depressing 'SOS' repair. Comparisons of nitracrine with four of its analogues showed that this effect was not a universal characteristic of either acridine derivatives or of nitracrine analogues. Rather, the dose displacements obtained paralleled hypoxic cell selective cytotoxicity in mammalian cells. If this result extends to other compounds, experiments of this nature using the bacterial strains could provide a novel and useful screening system to identify hypoxia-selective cytotoxic anticancer drugs of potential clinical value.

The 1-nitroacridine nitracrine [NC,1-nitro-9-(dimethylaminopropyl-amino)acridine] is a potent hypoxia-selective cytotoxic agent in culture, but lacks activity against hypoxic tumor cells in vivo at therapeutically accessible doses. To clarify reasons for this failure in vivo the metabolism of NC was investigated in stirred suspension cultures of Chinese hamster ovary cells, in EMT-6 spheroids, and in mice. One major low molecular weight metabolite (identical to that generated by NaBH4/Pd/C reduction) was observed in hypoxic (less than 10 ppm O2) single cell suspensions, while [G-3H-acridinyl]NC formed trichloroacetic acid- and acetonitrile-insoluble macromolecular adducts (MA) at a rate seven-fold higher than in aerobic (20% O2) cultures. Formation of these adducts correlated with cytotoxicity under air or nitrogen, and hence may provide a dosimeter for NC-induced damage. Autoradiographic investigation of the distribution of MA in spheroids equilibrated with 5% O2 showed that the label was restricted to the outer cell layers rather than being localized in the hypoxic central region. Thus metabolic activation is probably too rapid, even in well-oxygenated cells, to allow adequate distribution to hypoxic microenvironments in tumors. In mice, levels of MA were higher in liver, kidney, spleen and lung than in Lewis lung tumors, indicating that oxygen concentration does not exert a dominant influence on relative rates of metabolic activation in vivo. The development of nitroacridines with useful hypoxic selectivity in vivo will require identification of analogs for which reductive metabolism is more completely inhibited at oxygen concentrations found in normal tissues.

Encouraging results of preclinical pharmacologic analysis prompted the following study on the chronic action of N10-oxide 1-nitro-9-/3-dimethylaminopropylamino/-acridine (C-666) and 1-nitro-9- [/2-dimethylamino/ -1-methylethylamino] -acridine (C-829). Toxic influence of both compounds on liver, kidneys, gonads and intestine epithelium was observed. These histological changes were not, however, followed by any disorders in functions of liver and gonads. Only administration of compound C-666 was followed by the decrease of filtration of renals glomuses. A slight involutional influence of acridine derivatives on lymphopoetic reproductive center of thymus and lymph nodes was observed. At the same time there were no disorders in the value of circular leukocyte system. It was stated that the investigated compounds had no effect on the system of the red cells of the blood; only blood clotting time was prolonged. Compounds C-666 and C-829 did not inhibit the growth of tested animals.

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied. The des-nitro compound resembled 9 aminoacridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA- strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1- and polA3- strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA- strain, and showed some increased toxicity in polA1- and polA3- strains. The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB- strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB- or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This 'high-dose' mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracrine is a cross-link, but that this is not the major mutagenic lesion.