2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT.

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

Comparison of aromatic and tertiary amine N-oxides of acridine DNA intercalators as bioreductive drugs. Cytotoxicity, DNA binding, cellular uptake, and metabolism.

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.

Hypoxia-selective antitumor agents. 15. Modification of rate of nitroreduction and extent of lysosomal uptake by polysubstitution of 4-(alkylamino)-5-nitroquinoline bioreductive drugs.

Studies have shown that 4-(alkylamino)-5-nitroquinolines possess high selectivity (20-60-fold) for hypoxic tumor cells in vitro, but are not active as hypoxia-selective cytotoxins (HSCs) in vivo. The compounds show inadequate rates of extravascular diffusion, likely due both to sequestration of the bisbasic compounds into lysosomes and rapid nitroreduction. A further series of analogues, designed to counteract these limitations, has been synthesized and evaluated. Analogues bearing one to three electron-donating substituents on the quinoline have one-electron reduction potentials up to 100 mV lower than that of the unsubstituted compound (5), but do not have improved biological activity. The relationship between hypoxic selectivity and rates of metabolic reduction suggests at least two mechanisms of cytotoxicity for this series of 5-nitroquinolines. Compounds with high rates of reduction are toxic via oxygen-sensitive net bioreduction, while compounds which are poor substrates for nitroreduction are toxic through an oxygen-insensitive non-bioreductive mechanism. As rates of metabolic reduction are lowered, the non-bioreductive mechanism of toxicity becomes dominant and hypoxic selectivity is lost. A small series of analogues bearing hydrophilic but neutral side chains were also prepared. Compounds with a dihydroxypropyl side chain retained cytotoxic potency and hypoxic cell selectivity in cell culture assays, and had lowered uptake into lysosomes, but none of three analogues evaluated against KHT tumors in mice showed activity as an HSC in vivo.

Synthesis and evaluation of 4-substituted analogues of 5-[N,N-bis (2-chloroethyl)amino]-2-nitrobenzamide as bioreductively activated prodrugs using an Escherichia coli nitroreductase.

2,4-Dinitrobenzamide mustards, exemplified by the parent compound SN 23862 (2) are activated under aerobic conditions by an Escherichia coli nitroreductase enzyme (NR2) via selective reduction of the 2-nitro group, and are thus of interest as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT). A series of related compounds 12a-12d, where the 4-nitro group of 2 was replaced by other substituents of varying electronic properties, were prepared and evaluated as potential ADEPT prodrugs. One-electron reduction potentials of the compounds correlated well with the substituent sigma m values, with the exception of the unsubstituted (4-H) analogue 13, which had a much lower value than expected on electronic grounds, due to a coplanar conformation of the mustard. The cytotoxicities of the compounds towards aerobic UV4 cells correlated positively with the electron-donating ability of the 4-substituent (measured by sigma p values), indicating that the cytotoxicities of the compounds in the absence of the NR2 enzyme are due substantially to the parent (unreduced) compounds. A positive, although less strong, correlation was seen between the electronic properties of the 4-substituent and their cytotoxicities in the presence of the NR2 enzyme, suggesting that, in this closely related series, the degree of activation by the enzyme is significantly dependent on the reduction potential of the 2-nitro group. While the 4-SO2Me derivative 12d was the next most preferred substrate after the parent 2, it was considerably less so (degree of activation as measured by IC50 ratio of 26 compared with 145), despite the similar electronic properties of the two 4-substituents.

Tertiary amine N-oxides as bioreductive drugs: DACA N-oxide, nitracrine N-oxide and AQ4N.

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.

Hypoxia-selective antitumor agents. 13. Effects of acridine substitution on the hypoxia-selective cytotoxicity and metabolic reduction of the bis-bioreductive agent nitracrine N-oxide.

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.

Acquisition of prototype-based information about social groups in adulthood.

Two experiments examined adult age differences in the ability to acquire prototype-based information about a fictitious social group. Young and older adults were presented with 60 descriptions of people who varied in their similarity to a prototypical group member along 12 (Experiment 1) or 10 (Experiment 2) feature dimensions. The prototype represented either an arbitrary combination of features or a coherent set of features based on evaluative content. Younger adults generally performed better than the older adults in learning the arbitrary prototype, whereas age differences were absent or in favor of the older adults when the prototype consisted of evaluatively consistent features. The authors argue that the results can be explained by the age-related variations in the reliance on automatic processing mechanisms and the congruence of these processes with the demands of the task across prototype conditions.

Adult age differences in impression change processes.

Three experiments examined the factors influencing impression change in young and older adults. In each study, Ss formed an impression of a fictitious target person and then read additional behavioral information that varied in its consistency with this initial impression. On the basis of previous work, older adults were expected to be less likely than younger adults to integrate new, inconsistent information in the schema-based memory representation, which would result in less impression change. No support for this prediction was found; instead, young and older adults varied in their weighting of different types of information (e.g., negative behaviors), which subsequently affected their impressions and memory for specific behavioral information. These results highlight the importance of considering the impact of age differences in implicit theories about behavior on social cognition.

Nitrobenzyl mustard quaternary salts: a new class of hypoxia-selective cytotoxins capable of releasing diffusible cytotoxins on bioreduction.

PURPOSE: To explore the utility of a new class of compounds, nitrobenzyl mustard quaternary salts, as hypoxia-selective prodrugs of diffusible cytotoxins. METHODS AND MATERIALS: The parent compound N,N-bis(2-chloroethyl)-N-methyl-N-(2-nitrobenzyl)ammonium chloride (SN 25246) was prepared by reaction of 2-nitrobenzyl chloride with N-methyldiethanolamine, and reaction of the resulting quaternary diol with thionyl chloride at room temperature. The rate of release of mechlorethamine from this compound in the presence of cells under aerobic and hypoxic conditions was determined by trapping with diethyldithiocarbamate. Cytotoxicity was assessed by clonogenic assay of stirred suspension cultures of EMT6 cells, and also in intact and dissociated EMT6 spheroids. In vivo activity was evaluated in mice bearing SC KHT tumors. RESULTS: The parent compound is a stable, water-soluble compound with an E(1) of -369 mV. It releases mechlorethamine, and shows selective toxicity towards hypoxic EMT6 cells in culture, increasing with time to several 1000-fold after 4 h. It is much more active against intact than dissociated EMT6 spheroids, and shows low but statistically significant activity against KHT tumors in vivo. CONCLUSION: Nitrobenzyl mustard quaternary salts are a new class of hypoxia-selective cytotoxin. The parent compound shows very high selectivity for hypoxic cells in vitro, and undergoes reduction in hypoxic cells, releasing mechlorethamine which can back-diffuse to kill surrounding oxygenated cells.

Hypoxia-selective antitumor agents. 3. Relationships between structure and cytotoxicity against cultured tumor cells for substituted N,N-bis(2-chloroethyl)anilines.

A series of aniline mustards with a wide range of electron-donating and -withdrawing substituents in the 3- and 4-positions has been synthesized and evaluated for cytotoxicity in cell culture to examine the potential of using nitro group deactivated nitrogen mustards for the design of novel hypoxia-selective anticancer drugs (Denny, W. A.; Wilson, W. R. J. Med. Chem. 1986, 29, 879). Hydrolytic half-lives in tissue culture media, determined by bioassay against a cell line (UV4) defective in the repair of DNA interstrand cross-links showed the expected dependence on the Hammett electronic parameter, sigma, varying from 0.13 h for the 4-amino analogue to greater than 100 h for analogues with strongly electron-withdrawing substituents. Cytotoxic potencies in aerobic UV4 cultures showed a similar dependence on sigma. This dependence predicted that the 4-nitroaniline mustard would be 7200-fold less potent than its potential six-electron reduction product, the 4-amino compound, in growth inhibition assays using a 1-h drug exposure. The measured differential was much lower (225-fold) because of the instability of the latter compound, but a differential of 17,500-fold was observed in the initial rate of killing by using a clonogenic assay. The potential for formation of reactive mustards by reduction to the amine or hydroxylamine was demonstrated by the 4-nitroso compound, which had an aerobic toxicity similar to that of the amine. Although these features confirmed the original rationale, the 3-nitro- and 4-nitroaniline mustards had only minimal hypoxic selectivity against UV cells. Toxicity to hypoxic cells appears to be limited by the low reduction potentials of these compounds and consequent lack of enzymatic nitroreduction. However, this study has demonstrated that nitro groups can be used to latentiate aromatic nitrogen mustards and indicates that examples with higher reduction potentials could provide useful hypoxia-selective therapeutic agents.