Hypoxia-selective antitumor agents. 16. Nitroarylmethyl quaternary salts as bioreductive prodrugs of the alkylating agent mechlorethamine.

Nitrobenzyl quaternary salts of nitrogen mustards have been previously reported as hypoxia-selective cytotoxins. In this paper we describe the synthesis and evaluation of a series of heterocyclic analogues, including pyrrole, imidazole, thiophene, and pyrazole examples, chosen to cover a range of one-electron reduction potentials (from -277 to -511 mV) and substitution patterns. All quaternary salt compounds were less toxic in vitro than mechlorethamine, and all were more toxic under hypoxic than aerobic conditions, although the differentials were highly variable within the series. The most promising analogue, imidazole 2, demonstrated DNA cross-linking selectively in hypoxic RIF-1 cells, and was active in vivo in combination with radiation or cisplatin. However, 2 also produced unpredictable toxicity in vivo, suggestive of nonspecific nitrogen mustard release, and this has restricted further development of these compounds as hypoxia-selective cytotoxins.

Pharmacokinetics and metabolism of the nitrogen mustard bioreductive drug 5.

PURPOSE: To characterise the pharmacokinetics and metabolism in mice of 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (SN 23862), the lead compound of a new class of bioreductive drugs in which a nitrogen mustard is activated by nitroreduction. Comparison is made with the corresponding aziridine derivative CB 1954. METHODS: Male C3H/HeN mice, bearing s.c. KHT tumours, received 3H-labelled SN 23862 or CB 1954 i.v. at 200 micromol/kg. Plasma, urine and tumour samples were assayed for total radioactivity, and for parent compounds by HPLC. Metabolites were identified by 1H-NMR and mass spectrometry. Cytotoxicity of compounds against Chinese hamster AA8 cells was determined by growth inhibition assay. RESULTS: The plasma pharmacokinetics of SN 23862 and CB 1954 were similar, with half-lives of 1.1 and 1.2 h, respectively. SN 23862 provided tumour/plasma ratios and absolute tumour AUC values almost two times higher than CB 1954. Despite this, SN 23862 was more extensively metabolised than CB 1954, the major route being sequential oxidative dechloroethylation of the nitrogen mustard moiety to the relatively non-toxic half mustard and 5-amine. The inferred chloroacetaldehyde co-product was 260 times more potent than SN 23862. A tetrahydroquinoxaline metabolite resulting from reduction of the 4-nitro group followed by intramolecular alkylation was weakly cytotoxic, while the more cytotoxic 2-amino derivative of SN 23862 was detected in trace amounts. CB 1954 was metabolised by analogous pathways, but the 4- and 2-amine nitroreduction products were the major metabolites while oxidative dealkylation was minor. CONCLUSION: The lesser propensity for SN 23862 to undergo nitroreduction in the host, relative to CB 1954, argues that dinitrobenzamide mustards may be preferable to the corresponding aziridines as bioreductive prodrugs for cancer treatment. However, the toxicological significance of oxidative metabolism of the bis(2-chloroethyl)amine moiety needs to be addressed.

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.

Scintigraphic imaging of the hypoxia marker (99m)technetium-labeled 2,2'-(1,4-diaminobutane)bis(2-methyl-3-butanone) dioxime (99mTc-labeled HL-91; prognox): noninvasive detection of tumor response to the antivascular agent 5,6-dimethylxanthenone-4-acetic acid.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) and combretastatin A4 phosphate (CA-4-P) markedly inhibit tumor blood flow in mice and are both currently in clinical trial. One of the challenges in clinical evaluation of antivascular agents is the monitoring of tumor blood flow inhibition in individual patients. This study investigates, using mouse models, whether a new marker for tissue hypoxia, (99m)technetium-labeled 2,2'-(1,4-diaminobutane)bis(2-methyl-3-butanone) dioxime (99mTc-labeled HL-91; Prognox)] has potential for the scintigraphic monitoring of tumor response to antivascular agents. Determination of radioactivity in dissected tissues 3 h after DMXAA (80 micromol/kg) or CA-4-P (227 micromol/kg) was injected indicated that both drugs inhibited blood flow (86RbCl uptake; 84 and 87%, respectively) and increased 99mTc-labeled HL-91 levels (350 and 300%, respectively) selectively in murine RIF-1 tumors. Planar imaging of 99mTc-labeled HL-91 3 h after DMXAA injection showed a dose-dependent increase in tumor levels above a threshold of 50 micromol/kg; this same threshold was observed for the inhibition of tumor blood flow (determined using Hoechst 33342). DMXAA also inhibited blood flow--and increased 99mTc-labeled HL-91 uptake--in MDAH-MCa-4 mouse mammary carcinomas and in NZMN10 human melanoma xenografts. Whether 99mTc-labeled HL-91 might also be useful as a biomarker for tumor cell killing was investigated by clonogenic assay of surviving cells 15 h after imaging 99mTc-labeled HL-91 in RIF-1 tumors. Log cell kill in individual tumors showed a statistically significant linear correlation (P < 0.001) with 99mTc-labeled HL-91 uptake after 60 micromol/kg (r2 = 0.79) and 70 micromol/kg (r2 = 0.44) but not at 80 micromol/kg DMXAA. The lack of correlation at high doses presumably reflects the insensitivity of the tumor-averaged 99mTc-labeled HL-91 signal to small regions in which tumor blood flow is preserved (which will limit log cell kill). The results indicate the potential of 99mTc-labeled HL-91 for the noninvasive imaging of tumor blood flow inhibition by antivascular drugs in humans.

Cisplatin anti-tumour potentiation by tirapazamine results from a hypoxia-dependent cellular sensitization to cisplatin.

Tirapazamine (TPZ) is a new anticancer drug that is activated specifically at the low oxygen level typically found in solid tumours. It exhibits preferential cytotoxicity towards hypoxic cells and has been shown in preclinical studies with transplanted tumours and in phase II and III clinical trials to potentiate the anti-tumour efficacy of cisplatin without increasing its systemic toxicity. At present, the mechanism for this potentiation is unknown. Here we show that there is a schedule-dependent enhancement of cisplatin cytotoxicity by TPZ for cells in vitro that is similar to that seen with transplanted murine tumours. This cisplatin potentiation depends on the TPZ exposure being at oxygen concentrations below 1%, which are typical of many cells in tumours but not in normal tissues. Also, the interaction between TPZ and cisplatin does not occur in cells mutant in ERCC4, a protein essential for repair of DNA interstrand cross-links. Incubation of the cells with TPZ under hypoxia prior to cisplatin treatment increases cisplatin-induced DNA interstrand cross-links with kinetics suggesting that TPZ inhibits or delays repair of the DNA cross-links. In conclusion, we show that the tumour-specific potentiation of cisplatin cytotoxicity is likely the result of an interaction between TPZ and cisplatin at the cellular level that requires the low oxygen levels typical of those in solid tumours. The mechanism of the interaction appears to be through a potentiation of cisplatin-induced DNA interstrand cross-links, possibly as a result of a diminished or delayed repair of these lesions

Enhancement of tumor radiation response by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid.

PURPOSE: 5,6-dimethylxanthenone-4-acetic acid (DMXAA) selectively damages tumor vasculature and is currently in clinical trial as an antitumor agent. Its ability to induce synthesis of tumor necrosis factor (TNF), and its apparent selectivity for poorly-perfused regions in tumors, suggests it possible use in combination with radiotherapy. This investigation examines activity of DMXAA as a radiation modifier using two murine tumors. METHODS AND MATERIALS: Tumor growth delay was evaluated using i.m. RIF-1 and MDAH-MCa-4 tumors irradiated in unanaesthetised, restrained mice (cobalt-60) using single dose or multiple fractions (8 x 2.5 Gy over 4 days) with DMXAA administered i.p. at various times in relation to irradiation. RESULTS: Administration of DMXAA (80 micromol/kg, i.p.) immediately after radiation resulted in a large increase in tumor growth delay, giving a radiation dose modifying factor of 2.3 for RIF-1 and 3.9 for MDAH-MCa-4. The combination was less active when radiation was given 1-4 h after DMXAA, but was highly active 12-48 h after DMXAA. At the latter times, clamping the tumor blood supply caused a large increase in radioresistance. These studies suggest that cells surviving DMXAA are hypoxic for only a short period. DMXAA increased overall growth delay when administered daily during fractionated irradiation, giving an approximately additive response. CONCLUSIONS: The marked synergy between DMXAA and single dose ionising radiation may reflect the complementarity of these agents at the microregional level, with DMXAA preferentially killing hypoxic cells in poorly perfused regions. Despite additional hypoxia shortly after DMXAA treatment, surviving cells appear to reoxygenate quickly which makes it feasible to use DMXAA before and during fractionated radiotherapy. The combination of fractionated radiation and DMXAA appears to be less effective than for single dose radiation (possibly because of the smaller contribution of hypoxia under these conditions), but may be therapeutically useful.

Extravascular diffusion of tirapazamine: effect of metabolic consumption assessed using the multicellular layer model.

PURPOSE: Hypoxia-selective cytotoxic agents, like tirapazamine (TPZ), must diffuse considerable distances in tumors to reach their target cell population. This study uses a new three-dimensional tissue culture model, in which cells are grown as multicellular layers (MCL), to investigate whether metabolic consumption of TPZ is sufficiently rapid to compromise its extravascular diffusion in tumors. METHODS AND MATERIALS: V79-171b and MGH-U1 cells were grown as MCL to thicknesses of approximately 120 and 360 microm respectively. The extent of hypoxia in MCL, as assessed by EF5 binding, was modulated by altering gas-phase O2 content, and flux of TPZ through MCL was investigated by high-performance liquid chromatography (HPLC). Data were fitted to a diffusion-reaction mathematical model to determine the diffusion coefficient of TPZ in the MCL (DM) and the rate of its metabolic consumption under anoxia. These parameters were used to simulate TPZ transport in tumors. RESULTS: The flux of TPZ through well-oxygenated MCL (equilibrated with 95% O2) was well fitted as Fickian diffusion without reaction, with a D(M) of 7.4 x 10(-7) cm2s(-1) (12-fold lower than in culture medium) for V79 and 1.3 x 10(-6) cm2s(-1) for MGH-U1 MCL. Flux of TPZ was suppressed under anoxia, and fitting the data required inclusion of a reaction term with a rate constant for metabolic consumption of TPZ of 0.52 min(-1) for V79 and 0.31 min(-1) for MGH-U1 MCL. These transport parameters would translate into a 43% or 30% decrease respectively in TPZ exposure, as a result of drug metabolism, in the center of a slab of anoxic tissue 100 microm in thickness. CONCLUSIONS: MCL cultures provide an in vitro model for investigating the interaction between metabolic consumption and diffusion of bioreductive drugs. If rates of diffusion and metabolism similar to those measured in V79 and MGH-U1 MCL apply in tumors, then cells in large confluent regions of hypoxia would be partially protected by failure of TPZ penetration. Simulation of extravascular transport of TPZ-like bioreductive drugs demonstrates that the optimum metabolic rate constant is determined by two competing requirements: it should be high enough to ensure potent cytotoxicity under hypoxia, yet low enough that penetration is not severely compromised.

Tirapazamine-induced cytotoxicity and DNA damage in transplanted tumors: relationship to tumor hypoxia.

Tirapazamine (TPZ) is a hypoxia-selective bioreductive drug currently in Phases II and III clinical trials with both radiotherapy and chemotherapy. The response of tumors to TPZ is expected to depend both on the levels of reductive enzymes that activate the drug to a DNA-damaging and toxic species and on tumor oxygenation. Both of these parameters are likely to vary between individual tumors. In this study, we examined whether the enhancement of radiation damage to tumors by TPZ can be predicted from TPZ-induced DNA damage measured using the comet assay. DNA damage provides a functional end point that is directly related to cell killing and should be dependent on both reductive enzyme activity and hypoxia. We demonstrate that TPZ potentiates tumor cell kill by fractionated radiation in three murine tumors (SCCVII, RIF-1, and EMT6) and two human tumor xenografts (A549 and HT29), with no potentiation observed in a third xenograft (HT1080). Overall, there was no correlation of radiation potentiation and TPZ-induced DNA damage in the tumors, except that the nonresponsive tumor xenograft had significantly lower levels of DNA damage than the other five tumor types. However, there was a large tumor-to-tumor variability in DNA damage within each tumor type. This variability appeared not to result from differences in activity of the reductive enzymes but largely from differences in oxygenation between individual tumors, measured using fluorescent detection of the hypoxia marker EF5. The results, therefore, suggest that the sensitivity of individual tumors to TPZ, although not necessarily the response to TPZ plus radiation, might be assessed from measurements of DNA damage using the comet assay.

Adaptation of human tumor cells to tirapazamine under aerobic conditions: implications of increased antioxidant enzyme activity to mechanism of aerobic cytotoxicity.

Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, SR 4233, WIN 59075) is a bioreductive antitumor agent with a high selective toxicity for hypoxic cells. The selective hypoxic toxicity of TPZ results from the rapid reoxidation of the one-electron reduction product, the TPZ radical, in the presence of molecular oxygen with the concomitant production of superoxide radical. Under hypoxia the TPZ radical kills cells by causing DNA double-strand breaks and chromosome aberrations. However, the mechanism of aerobic cytotoxicity is still a matter of debate. In this study, we investigated the mechanism of aerobic cytotoxicity by adapting human lung adenocarcinoma A549 cells to aerobic TPZ exposure and characterizing the changes associated with drug resistance. The adapted cells were resistant to aerobic TPZ exposures (with dose-modifying factors of up to 9.2), although hypoxic sensitivity was largely unchanged. Relative to the parental A549 cell line, adaptation to continuous aerobic TPZ exposure resulted in increased levels of manganese superoxide dismutase (up to 9.4-fold), moderate increases in glutathione reductase (up to 2.1-fold), and loss of both quinone oxidoreductase (DT-diaphorase) activity and NADPH cytochrome P450 reductase activity. There was essentially no change in the activity of the cytoplasmic form of superoxide dismutase (CuZnSOD), catalase, or glutathione peroxidase. The increased activity of antioxidant enzymes in the resistant cell lines (in particular MnSOD) strongly suggests that reactive oxygen species are, in large part, responsible for the toxicity of TPZ under aerobic conditions, and is consistent with aerobic and hypoxic drug cytotoxicity resulting from different mechanisms.

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.

Nitro reduction as an electronic switch for bioreductive drug activation.

It is well known that the reduction of aromatic nitro groups can give rise to toxic species, and that net nitro reduction by one-electron reductases can usually be inhibited by oxygen. There has been much interest in utilizing this biotransformation to activate drugs in hypoxic regions of tumors, but no clinically useful compound has yet resulted. Nitroreductive activation of prodrugs by oxygen-insensitive (and oxygen-sensitive) reductases is also of current interest because of new methods for introducing specific nitroreductases into tumors (e.g., as antibody-enzyme conjugates or by gene therapy). In most of the compounds investigated previously, cytotoxicity appears to be due to reactive nitroso or hydroxylamine reduction products arising from the nitro group itself. It is argued that there is greater scope for designing potent and selective nitro compounds by using the nitro group as an electronic switch to activate a latent reactive moiety elsewhere in the molecule. Examples of this approach include the nitro(hetero)aromatic mustards (e.g., SN 23816, NSC 646394) in which the nitro group controls the reactivity of a nitrogen mustard to which it is directly conjugated, and the nitro(hetero)aromatic methylquaternary (NMQ) mustards (e.g., SN 25341, NSC 658926) in which reduction of the nitro group triggers fragmentation of the molecule to release a reactive aliphatic nitrogen mustard. Many of these compounds show very high selectivity for hypoxic cells in culture. Some are also active against hypoxic cells in tumors, and provide large tumor growth delays when combined with tumor blood flow inhibitors such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). These prodrug designs also have potential for releasing effectors other than nitrogen mustards, which opens up many possibilities for use of nitro compounds as tumor-selective prodrugs.

Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro.

Tirapazamine (SR 4233), a bioreductive drug selectively toxic towards hypoxic cells, is presently in phase II clinical trials. Since it would not be expected that all tumours would respond equally to the drug, we are exploring ways of predicting the response of individual tumours. In this study we have tested whether the comet assay, which measures DNA damage in individual cells, can provide a simple, surrogate end point for cell killing by tirapazamine. We examined the relationship between the cytotoxicity of tirapazamine under hypoxic conditions and tirapazamine-induced DNA strand breaks in murine (SCCVII, EMT6, RIF-1) and human (HT1080, A549, HT29) tumour cell lines. These results were compared with the relationship between tirapazamine cytotoxicity and another measure of the ability of cells to metabolise tirapazamine; high-performance liquid chromatography (HPLC) analysis of tirapazamine loss or formation of the two electron reduction product SR 4317. The correlation between the hypoxic cytotoxic potency of tirapazamine and DNA damage was highly significant (r = 0.905, P = 0.013). A similar correlation was observed for hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050), while the correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171). The hypoxic cytotoxicity of tirapazamine in vitro can therefore be predicted by measuring tirapazamine-induced DNA damage using the comet assay. This approach holds promise for predicting the response of individual tumours to tirapazamine in the clinic.

Efficient redox cycling of nitroquinoline bioreductive drugs due to aerobic nitroreduction in Chinese hamster cells.

Nitroquinoline bioreductive drugs with 4-alkylamino substituents undergo one-electron reduction in mammalian cells, resulting in futile redox cycling due to oxidation of the nitro radical anion in aerobic cultures, and eventual reduction to the corresponding amines in the absence of oxygen. Rates of drug-induced oxygen consumption (R) due to redox cycling in cyanide-treated AA8 cell cultures were determined for 17 nitroquinolines. There was a linear dependence of log R on the one-electron reduction potential at pH 7 (E(7)1 with a slope of 7.1 V-1, excluding compounds with substituents ortho to the nitro group. The latter had anomalously low rates of oxygen consumption relative to E(7)1, suggesting that interaction with the active site of nitroreductases is impeded sterically for such compounds. Absolute values of R (and the observed E(7)1 dependence) were well predicted by a simple kinetic model that used rates of net nitroreduction to the amines under anoxia as a measure of the rates of one-electron reduction in aerobic cells. This indicates that redox cycling of 4-alkylaminonitroquinolines occurs at high efficiency in aerobic cells, suggesting that there are no quantitatively significant fates of nitro radical anions in cells other than their reaction with oxygen (or their spontaneous disproportionation under hypoxia).

Metabolic and radiolytic reduction of 4-alkylamino-5-nitroquinoline bioreductive drugs. Relationship to hypoxia-selective cytotoxicity.

The 4-alkylamino-5-nitroquinolines (5NQs) are a new series of bioreductive drugs that exhibit varying degrees of selective toxicity (up to 60-fold) under hypoxic conditions in cell culture. This study tested the hypothesis that differences in hypoxia-selective cytotoxicity in this series reflect differences in the efficiency with which oxygen inhibits metabolic reduction. The products of reduction of six 5NQs were characterized and rates of reduction compared in aerobic and hypoxic AA8 cells. The major stable products of both radiolytic and metabolic reduction under anoxic conditions were the corresponding amines, which were not responsible for the toxicity of the parent nitro compounds. Metabolism of each compound was inhibited completely in aerobic cells, indicating that differences in hypoxia-selective toxicity in this series are not due to variations in efficiency as substrates for oxygen-insensitive nitro reduction. Rates of hypoxic metabolism correlated broadly with hypoxia-selective cytotoxicity; the 5NQ derivatives with high rates of hypoxic metabolism had good hypoxia-selective cytotoxicity, whereas the compounds with low rates of reduction (the 3,6-dimethyl and 8-methylamino compounds; 3,6diMe-5NQ and 8NHMe-5NQ) were non-selective. Low rates of drug-induced oxygen consumption by 3,6-diMe-5NQ and 8NHMe-5NQ in respiration-inhibited cells confirmed that these compounds are poor substrates for enzymatic nitro reduction. While there was an overall correlation between one-electron reduction potential at pH 7 (E1(7)) and rate of metabolic reduction, the relatively high E1(7) of 3,6diMe-5NQ (-367 mV) indicates that rates of reduction, and hypoxic selectivity of cytotoxicity, cannot be predicted from reduction potential alone. 3,6diMe-5NQ and 8NHMe-5NQ are cytotoxic through a non-bioreductive mechanism, the variable contribution of which may underlie the differences in hypoxia-selective cytotoxicity within this series of bioreductive drugs.

Oxygen dependence of the cytotoxicity and metabolic activation of 4-alkylamino-5-nitroquinoline bioreductive drugs.

The cytotoxic potency of 4-alkylamino-5-nitroquinoline drugs in AA8 cell cultures is enhanced up to 60-fold under hypoxia, with wide variations in selectivity for hypoxic cells observed for different members of this series. This study uses three representative 5-nitroquinolines to examine whether these differences in hypoxia-selective cytotoxicity are cell line specific, and to explore quantitatively the oxygen dependence of the cytotoxicity and metabolism of these compounds. The parent compound 5NQ, its 5NQ, its 8-methyl analogue (8Me5NQ) and the 8-methylamino analogue (8NHMe-5NQ) each showed similar hypoxic selectivity (ratio of concentration x time for 90% kill for zero versus 20% oxygen of 13-18-, 30-69- and 1.2-1.4-fold respectively in the three cell lines tested (AA8 Chinese hamster ovary, EMT6/Ak mouse mammary tumour and FME human melanoma). The cytotoxicity and metabolism (covalent binding) of radiolabelled 8Me-5NQ was investigated in AA8 cultures over a range of oxygen tensions (0-95%). The oxygen tension in solution required for 50% inhibition of log cell kill or adduct formation observed under anoxia (C50) was 0.01 and 0.02% oxygen respectively, suggesting that bioreductive alkylation is the mechanism of 8Me-5NQ toxicity. The K-value (oxygen concentration for cytotoxic potency equal to the mean of the potencies at zero and infinite oxygen) was similar (0.02% oxygen). Calculations based on measured rate constants for formation of the nitroradical anion of 8Me-5NQ and rates of radical loss through disproportionation or reaction with oxygen, predict a K-value for 8Me-5NQ of 0.025% oxygen, in good agreement with the experimentally determined value. Modelling of cell killing expected by the combination of 8Me-5NQ plus radiation suggested that tumour cells at intermediate oxygen tensions (0.01-1%) will be partially resistant to this treatment, and would limit the use of these 5-nitroquinolines in combination with radiation, unless sufficient drug could be delivered to cause extensive killing in the anoxic compartment.

Does DNA targeting affect the cytotoxicity and cell uptake of basic nitroquinoline bioreductive drugs?

PURPOSE: A series of 4-(N,N-dimethylaminopropylamino)-5-nitroquinoline bioreductive drugs was studied to determine whether DNA binding influences cytotoxic potency, hypoxic selectivity or cellular uptake in cell culture. METHODS AND MATERIALS: Cytotoxicity was assessed by clonogenic assay of stirred suspension cultures of aerobic (20% O2) or hypoxic (< 10 ppm O2) late log-phase AA8 cells. Drug uptake was measured by high performance liquid chromatography of acetonitrile-extracted cell pellets and extracellular medium, or by using radiolabelled drug. Drug binding to calf-thymus DNA was measured by equilibrium dialysis. Intracellular pH was determined using the [14C]-5,5-dimethyl-2,4-oxazolidinedione method and intralysosomal pH using the fluorescein isothiocyanate-labelled dextran method. RESULT: The compounds were weak DNA binders under physiological conditions, with association constants in the range 25-480 M-1. There was no correlation between DNA binding affinity and hypoxic or aerobic cytotoxic potency, or hypoxic selectivity. These compounds were accumulated by cells to high concentrations (25-60 fold higher than extracellular), but cell uptake also showed no relationship to DNA binding affinity. Ammonium chloride selectively raised intralysosomal pH and inhibited the cellular accumulation of these drugs. CONCLUSION: These results indicate that DNA binding is not the major determinant of cytotoxic potency, hypoxic selectivity, or cellular uptake of the 5-nitroquinolines. Instead, the variable contribution of a nonbioreductive mechanism of toxicity appears to underlie the differences in cytotoxic potency and hypoxic selectivity within this series. The high intracellular drug concentrations of these diprotic bases appear to be due primarily to lysosomal uptake rather than DNA binding. Lysosomal uptake might restrict diffusion of basic bioreductive drugs to the target hypoxic regions of solid tumors.

5-Nitro-4-(N,N-dimethylaminopropylamino)quinoline (5-nitraquine), a new DNA-affinic hypoxic cell radiosensitizer and bioreductive agent: comparison with nitracrine.

Targeting of electron-affinic radiosensitizers to DNA via noncovalent binding (e.g., intercalation) may offer the potential for increasing sensitizing efficiency. However, it has been suggested that high-affinity DNA binding may compromise sensitization by restricting the mobility of sensitizers along the DNA, and by decreasing rates of extravascular diffusion in tumors. The weak DNA intercalator nitracrine (1-NC) is a more efficient radiosensitizer than related nitroacridines with higher DNA-binding affinities (Roberts et al., Radiat. Res. 123, 153-164, 1990). The present study investigates whether electron-affinic agents of even lower DNA-binding affinity may be superior to nitroacridines. The quinoline analog of 1-NC, 5-nitraquine (5-NO), was shown to have an intrinsic association constant for calf thymus DNA in 20 mM phosphate buffer which was 12-fold lower than that of 1-NC. 5-Nitraquine was not accumulated as efficiently as 1-NC by AA8 cells, but, despite a similar one-electron reduction potential, was 2- to 3-fold more potent than 1-NC as a hypoxia-selective radiosensitizer in vitro when compared on the basis of average intracellular concentration. Thus the radiosensitizing potency of 5-NQ appears not to be compromised by its low DNA-binding affinity. The cytotoxic mechanisms of 5-NQ and 1-NC appear to be similar (hypoxia-selective formation of DNA monoadducts), but 5-NQ is 1200-fold less potent than 1-NC as a cytotoxin. Despite this advantage, 5-NQ was not active in vivo as a radiosensitizer in SCCVII tumors. This lack of activity appears to be due to its relatively high toxicity in vivo (intraperitoneal LD50 of 105 mumol kg-1 in C3H/HeN mice), high one-electron reduction potential (-286 mV), and rapid metabolism to the corresponding amine in mice. The in vitro therapeutic index (hypoxic radiosensitizing potency/aerobic cytotoxic potency) of this weak DNA binder was lower than that of the non-DNA targeted radiosensitizer misonidazole, suggesting that DNA targeting enhances cytotoxicity more than radiosensitization. Development of useful DNA-targeted radiosensitizers may require the exploitation of DNA binding modes different from those of the nitroacridines and nitroquinolines.