The development of weak DNA-intercalating bioreductive compounds is a new strategy to ensure DNA affinity high enough to produce toxicity yet low enough to permit efficient extravascular diffusion and penetration to hypoxic tumor tissue, as has been exemplified by the lead compound 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257). Indeed, because of its weak DNA-binding, NLCQ-1 demonstrates significant hypoxic selectivity in several rodent and human tumor cell lines that can be increased up to 388-fold with 4.5 h exposure. In vitro reduction studies suggest that cytochrome P450 and b5 reductases play a significant role in NLCQ-1 bioreductive activation. NLCQ-1 synergistically enhances the effect of radiation against hypoxic cells in vitro and murine tumors in vivo and optimizes the effect of radioimmunotherapy in human xenografts. Importantly, NLCQ-1 substantially enhances, in a schedule-dependent manner, the antitumor effect of alkylating agents, as well as 5-fluorouracil and paclitaxel against murine tumors and human xenografts, without a concomitant enhancement in bone marrow or hypoxia-dependent retinal toxicity. In addition, NLCQ-1 exhibits good stability in human plasma and favorable pharmacokinetics in mice. The synthesis of NLCQ-1 has been successfully scaled-up and its excellent recovery from biological fluids has been established. Because of these results and the fact that NLCQ-1 compares favorably with the frontrunner, bioreductive compound tirapazamine, NLCQ-1 is about to enter a Phase I clinical trial.

One of the recognized microenvironmental features of solid tumors is the existence of hypoxic regions, which are resistant to both ionizing radiation and chemotherapy and, thus, can negatively affect cure rates (1, 2, 3, 4). In addition, an increasing number of reports suggest that hypoxia-induced proteome and genome changes in tumors can lead to a more aggressive phenotype and malignant progression (5, 6, 7). However, because tumor hypoxia constitutes a major difference between tumor and normal tissues, it also presents opportunities for exploitation. This can occur by using compounds known as bioreductive drugs or hypoxia-selective cytotoxins, which are inactive prodrugs that are favorably activated by reductive enzymes in a hypoxic environment (8, 9). Once activated, bioreductive prodrugs release toxic metabolites that can cause cell damage and death by various mechanisms (9). The main categories of bioreductive drugs include nitroaromatics, quinones, aromatic or aliphatic-N-oxides, and transition metals (8, 9, 10).

Hypoxia-selective cytotoxins not only supplement other modalities, i.e., radiation or chemotherapy (which primarily attack aerobic, proliferating cells), but often interact in a synergistic way with them. Thus, a therapeutic benefit has been achieved in preclinical studies with a number of bioreductive drugs (10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Recent successful clinical trials of the mitomycin analogue porfiromycin and the hypoxia-selective cytotoxin TPZ1 in combination with radiation or cisplatin have shown that a therapeutic gain can be also achieved in the clinic, in terms of overall response rates and survival (20, 21, 22, 23, 24).

By targeting bioreductive agents to DNA through intercalation, it is possible to increase their potency as selective cytotoxins and radiosensitizers of hypoxic cells in vitro(12, 25, 26, 27). However, chromophores capable of tight DNA binding also demonstrate slow dissociation kinetics, which lead to restricted extravascular diffusion to hypoxic regions of tumors and, thus, ineffectiveness in vivo(27, 28). In addition, such chromophores tend to be potent cytotoxins by mechanisms independent of a bioreductive activation (i.e., by hindering the movement of polymerases or interfering with the action of topoisomerases I or II along DNA), which decreases hypoxic selectivity (29, 30, 31, 32). Furthermore, aerobic radioprotection has also been seen with strong DNA-intercalators, both in vitro and in vivo(25, 26, 27).

The development of weak DNA-intercalating bioreductive compounds was, therefore, undertaken as the next logical step to ensure DNA affinity high enough to produce toxicity yet low enough to permit efficient extravascular diffusion and penetration to hypoxic tumor tissue, as has been exemplified by the lead compound NLCQ-1. For the purpose of our studies, we define as weak DNA-intercalators compounds having a range of concentrations (C50) for 50% EB displacement (from an EB-DNA complex) that lies between 40 and 100 μm (Fig. 1).

Using 4-fluoro-7-chloroquinoline 2, instead of 4,7-dichloroquinoline, as the reagent for the coupling reaction with amine 1 (Fig. 2), we significantly improved the yield of NLCQ-1 (33). The synthesis of the compound was scaled-up successfully by the NCI. Therefore, NLCQ-1 can be formulated properly for future clinical development.

NLCQ-1 binds weakly to DNA through intercalation (33) with a C50 of 44 μm (Fig. 1) and possesses structural similarities with the antimalarial drug chloroquine, which binds to the minor groove of DNA (34). A recent study2 showed that NLCQ-1 diffuses very well through oxic HT29 multilayers with a diffusion coefficient of 6.06 × 10−7 cm2 /s, which makes it a 1.5-fold faster diffuser than TPZ (diffusion coefficient 3.97 × 10−7 cm2 /s). The aqueous solubility of NLCQ-1 is 6 mm whereas its partition coefficient in octanol/water (PCo/w) is 0.22. Stock solutions of NLCQ-1 in water are stable for at least a month at 5°C (33). For in vivo experiments, NLCQ-1 was prepared as a solution in sterile saline at 1.5 mg/ml (32) or in a 5% dextrose solution at 2 mg/ml (35).

A sensitive, specific HPLC method has been developed to measure NLCQ-1 in biological fluids with a lower quantitation limit of 10.4 ng/ml in plasma (35). Ethyl ether was the most efficient organic extraction solvent (50% recovery), with negligible interference from co-extracted plasma constituents. However, a solid-phase extraction (Varian Bond Elut column) is preferable because of the excellent recovery achieved for NLCQ-1 (>90%). Reverse phase HPLC on a C18 Genesis ODS column was used for quantitation of NLCQ-1 with the structurally similar 4-(N-ethyl-piperidinyl-6-amino)-7-chloroquinoline as an internal standard and a 50:50 methanol:10 mm potassium phosphate, 7.5 mm heptanesulfonic acid, 1% triethylamine (pH 3.5) buffer as mobile phase (35).

NLCQ-1, which is a hydrochloride salt, is an off-white crystalline solid and is stable in organic solvents, buffered solutions ranging from pH 2 to pH 10, and human plasma for 48 h at 37°C. NLCQ-1 is stable for 24 h incubation in human whole blood and, to some extent, in fresh dog plasma, but degradation is observed in rodent plasma when incubation is longer than 10 h. The mechanism of degradation during the 10–24 h incubation interval is not known. NLCQ-1 is taken up by erythrocytes (∼30%) after incubation in whole mouse blood and binds to human plasma proteins (>99%). Binding to α1-acid glycoprotein is greater than binding to serum albumin and is concentration-dependent (35).

NLCQ-1 demonstrates up to 30-fold hypoxic selectivity in several rodent or human tumor cell lines. In addition, hypoxic selectivity increases up to 386-fold with 4.5-h exposure time, because of a concomitant increase and decrease in the hypoxic and aerobic potency of NLCQ-1, respectively, over time (33). The hypoxic cytotoxicity of NLCQ-1 (expressed as the product of exposure time and concentration for 50% survival) ranges between 11.5 and 136 μm·h for 5 human and rodent tumor cell lines (A549, OVCAR-3, EMT6, SCCVII, and V79) at 30 μm input concentration. Because uptake is similar in all of these cell lines, the differences in hypoxic potency presumably reflect differences in the enzymatic profile and rate of reductive metabolism among the cell lines.

It has been well established that nitroaromatics are reduced in cells by a number of flavoprotein enzymes, which effect stepwise addition of up to six electrons. Activation is usually restricted to hypoxic cells because oxygen, which has a higher one electron reduction potential than most nitroaromatics (−155 mV versus −330 to −450 mV), can back-oxidize the one-electron anionic radical first formed. The most important (toxic) metabolites in the reductive pathway of nitroaromatic compounds are the nitroradical anion (1e addition), the nitroso (2e), and the hydroxylamine (4e) products (36, 37). The species that lead to the covalent reaction products under hypoxic conditions almost certainly derive from the nitroso and hydroxylamine or their ring-cleavage products, such as glyoxal (37, 38). The cell-free enzymatic metabolism of NLCQ-1 has been investigated under hypoxic or aerobic conditions in the presence of purified reductive enzymes or isolated rat-liver microsomes by monitoring the parent compound with HPLC-UV analysis (39). Enzymatic reduction of NLCQ-1 with isolated rat-liver microsomes and NADPH/NADH shows that, under hypoxic conditions, ∼60% of the parent compound is reduced within 1 h incubation (37°C). Under identical conditions, but in the presence of 2′-AMP (a P450 reductase inhibitor), 6-propyl-2-thiouracil or p-hydroxymercuribenzoate (cytochrome b5 reductase inhibitors), NLCQ-1 reduction is inhibited. Reduction kinetics of NLCQ-1 with recombinant human DTD and NADPH or NADH under hypoxic or aerobic conditions shows that ≤5% of the compound is reduced within 2 h. Finally, reduction kinetics with human P450 reductase-expressing microsomes show ∼75% or ∼50% reduction of NLCQ-1 under hypoxic or aerobic conditions, respectively, after 2 h incubation. These results suggest that DTD is not involved at least in the initial steps of the bioreductive activation of NLCQ-1 (although it could be involved with metabolites of NLCQ-1), whereas P450 and cytochrome b5 reductases play a significant role in NLCQ-1 bioreductive activation (39).

However, these two enzymes may not be the only ones capable of reducing NLCQ-1. A recent toxicology study in rats (Southern Research Institute), with NLCQ-1 given i.v., shows that NLCQ-1 is more toxic to male rather than female rats. This selective NLCQ-1 toxicity indicates that other cytochrome P450s, such as from the CYP2C subfamily, which demonstrate sex-specific expression in rats, may also be involved in the activation of this compound. In particular, CYP2C11 and CYP2C13, which are expressed in the liver of males, may be involved in the activation of NLCQ-1 (in contrast to CYP2C12, which is found in the liver of females rats; ref. 40).

An investigational, in vitro disease-oriented, primary antitumor screen has been performed for NLCQ-1 at the NCI using a panel of 60 cell lines, including leukemia, NSCLC, colon, CNS, melanoma, ovarian, renal, prostate, and breast cancer cells. Varying growth inhibition [measured with the sulforhodamine B (SRB) protein assay] was observed for all cell lines at relatively high NLCQ-1 concentrations and after 48 h aerobic exposure. However, total growth inhibition was observed in only six cell-lines (NCI-H226 NSCLC, HT29 colon cancer, SF-539 CNS cancer, MALME-3M melanoma, SN12C renal cancer, and T-47D breast cancer). Because NLCQ-1 is activated to toxic metabolites primarily under hypoxic conditions, toxicity under aerobic conditions could theoretically correlate with the expression of reductive enzymes in these cells. However, no such correlation exists between tumor cell-sensitivity to NLCQ-1 and DTD, P450 reductase, or cytochrome b5 reductase activity in this panel of tumors. Again, this suggests that other enzymes, possibly from the CYP superfamily, may be involved in the bioactivation of NLCQ-1, or perhaps expression of enzymes that protect aerobic cells from oxidative stress together with DNA repair enzymes may interfere in such correlation (39).

Our initial toxicity studies in non-tumor bearing mice (BALB/c) yielded an LD50 of 35 mg/kg (95 μmol/kg) for NLCQ-1 given by i.p. injection as a single dose, whereas its maximum tolerable dose is 30 mg/kg (81.5 μmol/kg; Ref. 41). In later toxicity studies, however, we showed that NLCQ-1 can be administered as multiple i.p. injections, exceeding the single LD50 dose, without causing any significant systemic toxicity in terms of weight loss or other signs of toxicity (42). Furthermore, in combination with chemotherapeutic agents, NLCQ-1 given i.p. at 10 mg/kg per day for 4 or 5 consecutive days did not cause any additional toxicity in mice bearing human xenografts (43, 44). Moreover, no additional bone marrow toxicity is observed in murine tumor models in combination with various chemotherapeutic agents known to cause myelosuppression (41, 43, 44, 45, 46). However, antitumor activity in combination with radio- or chemotherapy is seen even with a single NLCQ-1 i.p. dose of 10 mg/kg (27 μmol/kg; Refs. 41, 43, 44, 45, 46, 47).

Hypoxia-dependent retinal toxicity has also been addressed in BALB/c mice treated i.p. with NLCQ-1 alone or in combination with CPM (48). No retinal toxicity was observed with 10 or 22 mg/kg of NLCQ-1 alone, or 10 mg/kg NLCQ-1 followed by 100 mg/kg CPM 1 h later, whereas a statistically significant retinal toxicity was observed with 52 mg/kg of TPZ alone, which is equitoxic to 22 mg/kg NLCQ-1 (P < 0.001). Similar toxicity has been reported before for TPZ in female C57B16 mice (49). Investigational new drug-directed toxicological studies with NLCQ-1 are now ongoing, where NLCQ-1 is administered i.v. in rats.

Single-dose pharmacokinetics of NLCQ-1 have been studied in CD2F1 mice after i.v., i.p., or p.o. administration in a 5% dextrose solution (2 mg/ml; Ref. 35). NLCQ-1 is rapidly distributed and eliminated after i.v. administration. The plasma elimination after i.v. administration is described by a two-compartment open model. A peak plasma concentration of 1481 ng/ml is achieved immediately after i.v. injection of 2.5 mg/kg NLCQ-1, and the concentration falls below 10 ng/ml 90 min post injection. The plasma clearance (Cl) and volume of distribution (Vss) after i.v. administration are 69.9 ml/min/kg and 2.04 l/kg, respectively, whereas the NLCQ-1 half-life (t1/2β) is 41.3 min (Table 1).

NLCQ-1 is also rapidly absorbed and eliminated after i.p. administration. A peak plasma concentration of 8900 ng/ml is achieved 5 min after an i.p. dose of 10 mg/kg. After i.p. administration, NLCQ-1 is not detected in plasma after more than 2 h (Table 1).

Finally, NLCQ-1 is rapidly absorbed after oral administration (p.o.) at 10 mg/kg, but eliminated more slowly. In comparison with the exposure after an i.v. dose, i.p. bioavailability is high (85%) and p.o. bioavailability is modest (28%). The apparent elimination half-life is dependent on administration route and it is shortest after i.p. administration. The 24-h urinary recovery of NLCQ-1 is 6.4% of the administered dose. NLCQ-1 plasma concentrations after i.p. administration of an active dose (10 mg/kg) remain above a concentration with demonstrated in vivo activity (3 μm) for ∼30 min. The mouse data suggest that oral administration may achieve plasma concentration and systemic exposure similar to those observed after i.v. administration (35).

With Radiation.

NLCQ-1 is a very potent and efficient radiosensitizer of hypoxic V79 cells in vitro, providing sensitization enhancement ratio values of 2.27–2.56 at 20–80 μm concentration (measured at 10% survival level). The C1.6 of NLCQ-1 is 7.2 ± 0.2 μm, and the in vitro therapeutic index of NLCQ-1 (defined as CT50(Air)/C1.6) varies by exposure time from 57 (1 h exposure) to 145 (4.5 h exposure). The corresponding C1.6 value for TPZ was 16.9 μm and its in vitro therapeutic index was 49 (3 h exposure; Ref. 42).

For in vivo studies with NLCQ-1, both murine tumors and human xenografts have been used, whereas the in vivo-in vitro assay and/or the tumor regrowth assays have been used as endpoints. In general, NLCQ-1 has a weak in vivo antitumor activity on its own. However, in combination with a single radiation dose, a synergistic interaction is observed (>1 log killing) when 10 mg/kg (27 μmol/kg) NLCQ-1 is given between 45 and 60 min before irradiation. An in vivo SER value of 1.58 is obtained with 10 mg/kg NLCQ-1, similar to that obtained with an equitoxic dose of TPZ, in SCCVII tumors, by using a fractionated radiation regimen (42). Recent combination studies with NLCQ-1 (15 mg/kg) and clinically relevant single or fractionated doses of radiation, against SC U251 CNS human tumor xenografts, have also shown a better effectiveness than radiation alone.3 Similar studies are still ongoing.

With Radioimmunotherapy.

In a series of studies using electrophysiology, immunohistochemistry and radiotracers, it has been demonstrated that radioimmunotherapy induces a prolonged state of hypoxia in most tumors, without affecting the Po2 levels in normal tissues, and that the maximum effect is observed ∼14 days posttreatment and independently of initial tumor size (50). When NLCQ-1 was administered i.p. 14 days post a single dose of 131I-MN-14 anti-CEA IgG, in nude mice bearing various human colonic tumor xenografts, tumor size was significantly declined compared with treatment with RAIT alone. A similar decline in tumor size was observed with RAIT and TPZ but at 4.8 fold higher molar dose than NLCQ-1 (50).

With Chemotherapy.

Various chemotherapeutic agents with different mechanisms of action have been tested in combination with NLCQ-1, in vivo, against murine tumors or human xenografts. A schedule-dependent synergistic interaction is observed in most of the cases. Synergism is optimal when NLCQ-1 is administered before an alkylating agent, such as cisplatin (38), CPM (41, 44), or melphalan (41), and before or after the antimitotic drug taxol (43, 46, 47) and the thymidylate synthase inhibitor 5FU (45, 47).

Dose-response potentiation studies in EMT6 and SCCVII tumor-bearing mice at the optimal administration time-interval, using the clonogenic assay, showed that a single dose of 10 mg/kg NLCQ-1 could modify the efficacy of the chemotherapeutic agent by a factor of 1.7 to 5.7, depending on the drug and tumor model. In the same studies, the dose-modification factor for bone marrow toxicity was always ∼1. Comparison with TPZ in the same tumor models showed that NLCQ-1 is superior to TPZ as a chemosensitizer, in terms of achievable therapeutic indices (44, 45, 46). Significant tumor growth delays are observed in FSaIIC tumors when a single i.p. dose of NLCQ-1 is administered at an optimal interval with melphalan, cisplatin, or CPM (41). For instance, an 18.1 ± 3.2 d delay is observed when NLCQ-1 is combined with 10 mg/kg of melphalan versus a 5.8 ± 3.6 d delay with melphalan alone (41).

Significant tumor growth delays are also achieved when multiple NLCQ-1 doses are combined with multiple small doses of a chemotherapeutic agent against murine tumors or human xenografts (43, 44, 45). Thus, when NLCQ-1 (10 mg/kg) was given i.p. 90 min after an ineffective taxol dose (8 mg/kg) twice a day, 4 h apart, on days 0 and 9, tumor regrowth delay was increased by 10 days compared with taxol alone. This corresponds to 1.53 log cell kill. In the same study, TPZ was responsible for about 4.5 days of extra delay compared with taxol alone, which corresponds to 0.91 log cell kill. In another example, NLCQ-1 potentiated the antitumor activity of marginally active or inactive doses of CPM, against PC-3 prostate human xenografts (44). Thus, a log cell kill of −0.03 (inactive) was calculated for 36 mg/kg per day for four days of CPM given i.p. alone, whereas a log cell kill of 0.87 was calculated for the combination treatment with NLCQ-1 (10 mg/kg per day for four days given i.p. 1.5 h before CPM; Table 2).

As has been mentioned above, NLCQ-1 interacts with chemotherapeutic agents in a synergistic way in a schedule-dependent manner. Synergy with alkylating agents, such as melphalan or cisplatin, requires hypoxic pre-exposure of cells to NLCQ-1 in vitro(51) and administration of NLCQ-1 ∼1 h before melphalan or cisplatin in mice (41, 47). This, together with the fact that NLCQ-1 targets DNA through weak intercalation, suggests that NLCQ-1 may cause DNA lesions on reductive metabolism under hypoxic conditions. To indirectly identify such lesions, rodent cell lines defective in specific DNA repair genes (EM9 and UV41) and their repair-proficient parental AA8, were exposed to NLCQ-1 alone and in combination with melphalan or cisplatin under hypoxic/aerobic conditions and appropriate routes and assessed for clonogenicity. EM9 cells, which lack the functional XRCC1 gene involved in base excision repair and, thus, are unable to efficiently repair DNA ssbs, were 3.7-fold more sensitive to NLCQ-1 than the parental AA8 cells. Similarly, UV41 cells, which are defective in the essential gene for repair of DNA interstrand cross-links (ERCC4/XPF), and, thus, hypersensitive to DNA cross-linking agents, were 4.1-fold more sensitive than AA8 cells to NLCQ-1. Equitoxic concentrations of NLCQ-1 and TPZ gave the same number of ssbs (identified by using the alkaline comet assay) in AA8 or EM9 cells exposed to each compound for 1 h under hypoxic conditions. In potentiation studies with melphalan or cisplatin, synergy was observed in AA8 cells, but not in EM9 or UV41 cells, with either NLCQ-1 or TPZ. These results suggest the involvement of NLCQ-1 in the formation of DNA ssbs and perhaps interstrand cross-links, under hypoxic conditions. The synergistic interaction of NLCQ-1 with melphalan or cisplatin is not yet completely understood; however, it could probably be attributed to an enhancement in the melphalan/cisplatin-induced DNA interstrand cross-links, possibly as a result of an inhibited repair mechanism of these lesions (52).

Mechanisms of taxol and 5FU potentiation by NLCQ-1 have been investigated by use of V79 cells (53). A schedule-dependent synergistic interaction, similar to that seen in transplanted EMT6 murine tumors (47), existed between NLCQ-1 and taxol/5FU in V79 cells. Thus, the optimum potentiation was observed when NLCQ-1 was administered under hypoxic conditions, 2 or 3 h after the antimitotic taxol or the thymidylate synthase inhibitor 5FU. In this case, we found that apoptotic mechanisms induced by taxol or 5FU alone were enhanced and induced earlier in the combination treatment with NLCQ-1 (Fig. 3). Thus, nucleosome formation was enhanced by NLCQ-1 by a factor of 2.5 and 1.3, 24 h and 36 h posttreatment, respectively, compared with taxol alone (Fig. 3,A). Nucleosome formation induced by 5FU alone 12, 24, and 36 h posttreatment was enhanced by NLCQ-1 1.7-, 1.5-, and 1.6-fold, respectively, whereas enhancement was also observed immediately post combination treatment (Fig. 3 B). NLCQ-1 alone did not increase the nucleosome formation compared with the control at all examined time-intervals. Apoptosis was also confirmed by detection of caspase-3 activity, particularly 30 h post combination treatment with either taxol or 5FU plus NLCQ-1. Non-repairable DNA damage and persistent inhibition of DNA, RNA, and protein synthesis were also some of the mechanisms involved in the potentiation of taxol or 5FU by NLCQ-1, only under hypoxic exposure conditions to NLCQ-1 (53).

In conclusion, NLCQ-1 synergistically enhances the antitumor effect of several chemotherapeutic agents or radiation against murine tumors or human xenografts in vivo, in a schedule-dependent fashion without concurrent potentiation of bone marrow, retinal, or systemic toxicity. In addition, NLCQ-1 optimizes the effect of RAIT. NLCQ-1 compares favorably with the frontrunner TPZ, as an adjuvant to both radio- and chemotherapy, at least in our tumor models, perhaps because it is a 1.5-fold faster diffuser than TPZ. NLCQ-1 synthesis has been successfully scaled-up, and a methodology has been developed for its optimal isolation and quantitation from biological fluids. NLCQ-1 demonstrates good stability in human plasma and favorable pharmacokinetics in mice with 85% or 28% bioavailability after i.p. or p.o. administration, respectively, at the moderate, but effective, dose of 10 mg/kg. In view of the above presented encouraging results, we believe that NLCQ-1 can successfully enter clinical trials.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Dr. Maria V. Papadopoulou, Evanston Northwestern Healthcare, The Department of Radiation Medicine, 2650 Ridge Avenue, Evanston, IL 60201. Phone: (847) 570-2262; Fax: (847) 570-1878; E-mail: m-papadopoulou@northwestern.edu

1

The abbreviations used are: TPZ, tirapazamine, (3-amino-1,2,4-benzotriazine-1,4-dioxide, SR-4233); NLCQ-1, 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NSC 709257); C50, concentration of a DNA-intercalator for 50% ethidium bromide displacement from an ethidium bromide-DNA complex; EB, ethidium bromide; HPLC, high-performance liquid chromatography; DTD, DT-diaphorase; CPM, cyclophosphamide; SER, sensitization enhancement ratio; C1.6, concentration for an sensitization enhancement ratio value of 1.6; CT50(Air), concentration for 50% reduction in survival under aerobic conditions; RAIT, radioimmunotherapy; 5FU, 5-fluorouracil; ssbs, single-strand breaks.

2

F. Pruijn and W. R. Wilson, unpublished results and personal communication.

3

M. G. Hollingshead and M. V. Papadopoulou, unpublished results.

Fig. 1.

DNA-binding study of weak DNA-intercalating compounds using the EB-displacement assay. The fluorescence attributable to the EB-DNA complex was recorded and plotted versus compound concentration. The C50 values were determined from the graph and constitute a direct measure of DNA binding. Bars represent SD of quadruplicate measurements.

Fig. 1.

DNA-binding study of weak DNA-intercalating compounds using the EB-displacement assay. The fluorescence attributable to the EB-DNA complex was recorded and plotted versus compound concentration. The C50 values were determined from the graph and constitute a direct measure of DNA binding. Bars represent SD of quadruplicate measurements.

Close modal
Fig. 2.

Schematic synthesis of NLCQ-1.

Fig. 2.

Schematic synthesis of NLCQ-1.

Close modal
Fig. 3.

Nucleosome formation in V79 cells treated with either taxol (A) or 5FU (B) ± NLCQ-1. Cells were exposed to taxol or 5FU for 12 h under aerobic conditions (37°C) while in monolayers and to NLCQ-1 under hypoxia (1 h, 37°C) while in suspension and free of chemotherapeutic drug. NLCQ-1 was added under hypoxia 2 and 3 h later in the taxol- and 5FU-pretreated cells, respectively. Nucleosomes were measured at various time-intervals post NLCQ-1 treatment by using the Cell Death Detection ELISAPLUS kit (Roche Applied Science).

Fig. 3.

Nucleosome formation in V79 cells treated with either taxol (A) or 5FU (B) ± NLCQ-1. Cells were exposed to taxol or 5FU for 12 h under aerobic conditions (37°C) while in monolayers and to NLCQ-1 under hypoxia (1 h, 37°C) while in suspension and free of chemotherapeutic drug. NLCQ-1 was added under hypoxia 2 and 3 h later in the taxol- and 5FU-pretreated cells, respectively. Nucleosomes were measured at various time-intervals post NLCQ-1 treatment by using the Cell Death Detection ELISAPLUS kit (Roche Applied Science).

Close modal
Table 1

Summary of NLCQ-1 pharmacokinetic parameters in CD2F1 micea

ParameterRoute
i.v.i.p.p.o.
Dose (mg/kg) 2.5 10 10 
AUC (ng/ml× min) 35,778 121,774 40,469 
t1/2 (min) 41.3 18.5 83.9 
Bioavailability (%)  85 28 
Cl (ml/min/kg) 69.9   
Vss (l/kg) 2.04   
ParameterRoute
i.v.i.p.p.o.
Dose (mg/kg) 2.5 10 10 
AUC (ng/ml× min) 35,778 121,774 40,469 
t1/2 (min) 41.3 18.5 83.9 
Bioavailability (%)  85 28 
Cl (ml/min/kg) 69.9   
Vss (l/kg) 2.04   
a

Reprinted with permission from Cancer Chemo. Pharmacol. (35).

Table 2

Time for the PC-3 human xenografts to grow to 16-fold their original weight, and other parameters regarding the effect and toxicity of the NLCQ-1 ± CPM treatmenta

Treatment groupMedian time (days)Tumor growth delay (days)Log killOptimal % T/Cb (day)Max % Relative Mean Net Wt Loss (day)
Control (vehicle)c 20.5    2.0 (12) 
CPM      
 80 mg/kg (QD× 4)d 37.3 16.8 1.33 9 (21) 13.6 (17) 
 54 mg/kg (QD× 4) 26.9 6.4 0.25 43 (21) no wt loss 
 36 mg/kg (QD× 4) 24.2 3.7 −0.03 62 (21) 6.1 (12) 
NLCQ-1: 10 mg/kg (QD× 4) 23.4 2.9 −0.11 73 (21) no wt loss 
NLCQ-1+ 80 mg/kg CPM 38.9 18.4 1.49 9 (28) 10.2 (12) 
NLCQ-1+ 54 mg/kg CPM 27.6 7.1 0.32 27 (21) 1.2 (31) 
NLCQ-1+ 36 mg/kg CPM 32.9 12.4 0.87 28 (21) 8.3 (31) 
Treatment groupMedian time (days)Tumor growth delay (days)Log killOptimal % T/Cb (day)Max % Relative Mean Net Wt Loss (day)
Control (vehicle)c 20.5    2.0 (12) 
CPM      
 80 mg/kg (QD× 4)d 37.3 16.8 1.33 9 (21) 13.6 (17) 
 54 mg/kg (QD× 4) 26.9 6.4 0.25 43 (21) no wt loss 
 36 mg/kg (QD× 4) 24.2 3.7 −0.03 62 (21) 6.1 (12) 
NLCQ-1: 10 mg/kg (QD× 4) 23.4 2.9 −0.11 73 (21) no wt loss 
NLCQ-1+ 80 mg/kg CPM 38.9 18.4 1.49 9 (28) 10.2 (12) 
NLCQ-1+ 54 mg/kg CPM 27.6 7.1 0.32 27 (21) 1.2 (31) 
NLCQ-1+ 36 mg/kg CPM 32.9 12.4 0.87 28 (21) 8.3 (31) 
a

Data reprinted with permission (44).

b

T and C are median tumor weights of treated and control groups, respectively, on each observation day.

c

Saline.

d

Treatment was initiated on day 10 post xenograft inoculation. Both drugs were given i.p. in saline. In the combination treatment, NLCQ-1 was given 1.5 h before CPM. QD × 4, per day for 4 days.

1
Hockel M., Schlenger K., Mitze M., Schaffer U., Vaupel P. Hypoxia and radiation response in human tumors.
Seminars in Radiat. Oncol.
,
6
:
3
-9,  
1996
.
2
Siemann D. W. Modification of chemotherapy by nitroimidazoles.
Int. J. Radiat. Oncol. Biol. Phys.
,
10
:
1585
-1594,  
1984
.
3
Grau C., Overgaard J. Effect of cancer chemotherapy on the hypoxic fraction of a solid tumor measured using a local tumor control assay.
Radiother. Oncol.
,
13
:
301
-309,  
1988
.
4
Brown J. M. Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies.
Mol. Med. Today
,
6
:
157
-162,  
2000
.
5
Cuvier C., Jang A., Hill R. P. Exposure to hypoxia, glucose starvation and acidosis: effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion.
Clin. Exp. Metastasis
,
15
:
19
-25,  
1997
.
6
Graham C. H., Forsdike J., Fitzgerald C. J., MacDonald-Goodfellow S. Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression.
Int. J. Cancer
,
80
:
617
-623,  
1999
.
7
Hockel M., Schlenger K., Aral B., Mitze M., Schaffer U., Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of uterine cervix.
Cancer Res.
,
56
:
4509
-4515,  
1996
.
8
Stratford I. J., Workman P. Bioreductive drugs into the next millennium.
Anticancer Drug Des.
,
13
:
519
-528,  
1998
.
9
Denny W. A. Prodrug strategies in cancer therapy Eur.
J. Med. Chem.
,
36
:
577
-595,  
2001
.
10
Rockwell S., Sartorelli A. C., Tomasz M., Kennedy K. A. Cellular pharmacology of quinone bioreductive alkylating agents.
Cancer Metastasis Rev.
,
12
:
165
-176,  
1993
.
11
Brown M. J., Lemmon M. J. Potentiation by the hypoxic cytotoxin SR4233 of cell killing produced by fractionated irradiation of mouse tumors.
Cancer Res.
,
50
:
7745
-7749,  
1990
.
12
Roberts P. B., Denny W. A., Wakelin L. P. G., Anderson R. F., Wilson W. R. Radiosensitization of mammalian cells in vitro by nitroacridines.
Radiat. Res.
,
123
:
153
-164,  
1990
.
13
Langmuir V. K., Rooker J. A., Osen M., Mendonca H. L., Laderoute K. R. Synergistic interaction between tirapazamine and cyclophosphamide in human breast cancer xenografts.
Cancer Res.
,
54
:
2845
-2847,  
1994
.
14
Siemann D. W. Chemosensitization of CCNU in KHT murine tumor cells in vivo and in vitro by the agent RB 6145 and its isomer PD 144872.
Radiother. Oncol.
,
34
:
47
-53,  
1995
.
15
Papadopoulou M. V., Ji M., Bloomer W. D. THNLA-1: a DNA-targeted bioreductive agent as chemosensitizer in vitro and in vivo..
In Vivo
,
10
:
49
-58,  
1996
.
16
Papadopoulou M. V., Ji M., Rao M. K., Bloomer W. D. 9-[3-(2-Nitro-1-imidazolyl)-propylamino]cyclopenteno[b]quinoline hydrochloride (NLCPQ-1). A novel DNA-affinic bioreductive agent as cytotoxin and radiosensitizer.
Oncol. Res.
,
8
:
425
-434,  
1996
.
17
Papadopoulou M. V., Ji M., Rao M. K., Bloomer W. D. 9-[3-(2-Nitro-1-imidazolyl)-propylamino]cyclopenteno[b]quinoline hydrochloride (NLCPQ-1): A novel DNA-affinic bioreductive agent as chemosensitizer.
I. Oncol. Res.
,
9
:
249
-257,  
1997
.
18
Dorie M. J., Brown J. M. Modification of the antitumor activity of chemotherapeutic drugs by the hypoxic cytotoxic agent tirapazamine.
Cancer Chemother. Pharmacol.
,
39
:
361
-366,  
1997
.
19
Gallagher R., Hughes C. M., Murray M. M., Friery O. P., Patterson L. H., Hirst D. G., McKeown S. R. The chemopotentiation of cisplatin by the novel bioreductive drug AQ4N.
Br. J. Cancer
,
85
:
625
-629,  
2001
.
20
Haffty B. G., Son Y. H., Wilson L. D., Papac R., Fischer D., Rockwell S., Sartorelli A. C., Ross D., Sasaki C. T., Fischer J. J. Bioreductive alkylating agent porfiromycin in combination with radiation therapy for the management of squamous cell carcinoma of the head and neck.
Radiat. Oncol. Investig.
,
5
:
235
-245,  
1997
.
21
Lee D-J., Trotti A., Spencer S., Rostock R., Fisher C., von Roemeling R., Harvey E., Groves E. Concurrent tirapazamine and radiotherapy for advanced head and neck carcinomas: a Phase II study.
Int. J. Radiat. Oncol. Biol. Phys.
,
42
:
811
-815,  
1998
.
22
Miller V. A., Ng K. K., Grant S. C., Kindler H., Pizzo B., Heelan R. T., von Roemeling R., Kris M. G. Phase II study of the combination of the novel bioreductive agent, tirapazamine, with cisplatin in patients with advanced non-small-cell lung cancer.
Ann. Oncol.
,
8
:
1269
-1271,  
1997
.
23
Treat J., Johnson E., Langer C., Belani C., Haynes B., Greenberg R., Rodriquez R., Drobins P., Miller W., Jr., Meehan L., McKeon A., Devin J., von Roemeling R., Viallet J. Tirapazamine with cisplatin in patients with advanced non-small-cell lung cancer: a phase II study.
J. Clin. Oncol.
,
16
:
3524
-3527,  
1998
.
24
von Pawel J., von Roemeling R., Gatzemeier U., Boyer M., Elisson L. O., Clark P., Talbot D., Rey A., Butler T. W., Hirsh V., Olver I., Bergman B., Ayoub J., Richardson G., Dunlop D., Arcenas A., Vescio R., Viallet J., Treat J. Tirapazamine plus cisplatin versus cisplatin in advanced non-small-cell lung cancer: A report of the international CATAPULT I study group.
J. Clin. Oncol.
,
18
:
1351
-1359,  
2000
.
25
Panicucci R., Heal R., Laderonte K., Cowan D., McClelland R. A., Rauth A. M. NLP-1: A DNA intercalating hypoxic cell radiosensitizer and cytotoxin.
Int. J. Radiat. Oncol. Biol. Phys.
,
16
:
1039
-1043,  
1989
.
26
Papadopoulou M. V., Epperly M. W., Shields D. S., Bloomer W. D. Radiosensitization and hypoxic cell toxicity of NLA-1 and NLA-2, two new bioreductive compounds.
Jpn. J. Cancer Res.
,
83
:
410
-414,  
1992
.
27
Denny W. A., Roberts P. B., Anderson R. F., Brown J. M., Phil D., Wilson W. R. NLA-1: A 2-nitroimidazole radiosensitizer targeted to DNA by intercalation.
Int. J. Radiat. Oncol. Biol. Phys.
,
22
:
553
-556,  
1992
.
28
Wilson W. R., Denny W. A., Stewart G. M., Fenn A., Probert J. C. Reductive metabolism and hypoxia-selective toxicity of nitracrine.
Int. J. Radiat. Oncol. Biol. Phys.
,
12
:
1235
-1238,  
1986
.
29
Denny W. A., Roos I. A. G., Wakelin L. P. G. Interactions between antitumor activity, DNA breakage, and DNA binding kinetics for 9-aminoacridinecarboxamide antitumor agents.
Anticancer drug design
,
1
:
141
-147,  
1986
.
30
Zwelling L. DNA topoisomerase II as a target of antineoplastic drug therapy.
Cancer Metast. Rev.
,
4
:
263
-276,  
1985
.
31
Rosenzweig H. S., Papadopoulou M. V., Bloomer W. D. A study of the interaction of NLA-compounds with topoisomerases I and II.
Proc. Am. Assoc. Cancer Res. Annu. Meet.
,
35
:
362
1994
.
32
Papadopoulou M. V., Rosenzweig H. S., Doddi M., Bloomer W. D. 9-[3-(2-Nitro-1-imidazolyl)-propylamino]-1, 2, 3, 4-tetrahydroacridine hydrochloride. A novel DNA-affinic hypoxic cell cytotoxin and radiosensitizer. Comparison with NLA-1.
Oncol. Res.
,
6
:
439
-448,  
1994
.
33
Papadopoulou M. V., Ji M., Rao M. K., Bloomer W. D. 4-[3-(2-Nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive compound as a hypoxia-selective cytotoxin.
Oncol. Res.
,
12
:
185
-192,  
2000
.
34
O’Brien R. L., Hahn F. E. Chloroquine structural requirements for binding to deoxyribonucleic acid and antimalarial activity. Antimicrob.
Agents Chemother.
,
:
315
-320,  
1965
.
35
Reid J. M., Squillace D. P., Ames M. M. Single dose pharmacokinetics of the DNA-binding bioreductive agent NLCQ-1 (NSC 709257) in CD2F1 Mice.
Cancer Chem. Pharmacol.
,
51
:
483
-487,  
2003
.
36
McClelland R. A., Panicucci R., Rauth A. M. Products of the reduction of 2-nitroimidazoles.
J. Am. Chem. Soc.
,
109
:
4308
-4313,  
1987
.
37
Noss M. B., Panicucci R., McClelland R. A., Rauth A. M. Preparation, toxicity and mutagenicity of 1-methyl-2-nitrosoimidazole: a toxic 2-nitroimidazole reduction product.
Biochem. Pharmacol.
,
37
:
2585
-2593,  
1988
.
38
Workman P. Keynote address: bioreductive mechanisms.
Int. J. Radiat. Oncol. Biol. Phys.
,
22
:
631
-637,  
1992
.
39
Papadopoulou M. V., Ji M., Rao M. K., Bloomer W. D. Reductive activation of the nitroimidazole-based hypoxia-selective cytotoxin NLCQ-1 (NSC 709257).
Oncol. Res.
,
14
:
21
-29,  
2003
.
40
Tomigahara Y., Onogi M., Saito K., Isobe N., Kaneko H., Nakatsuka I. Metabolism of cyanox in rat. II. Sex-related differences in oxidative dearylation and disulphuration.
Xenobiotica
,
30
:
395
-406,  
2000
.
41
Papadopoulou M. V., Ji M., Bloomer W. D. NLCQ-1, a novel hypoxic cytotoxin: potentiation of melphalan, cisDDP and cyclophosphamide in vivo..
Int. J. Radiat. Oncol. Biol. Phys.
,
42
:
775
-779,  
1998
.
42
Papadopoulou M. V., Ji M., Rao M. K., Bloomer W. D. 4-[3-(2-Nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1). A novel bioreductive agent as radiosensitizer in vitro and in vivo. Comparison with tirapazamine.
Oncol. Res.
,
12
:
325
-333,  
2000
.
43
Papadopoulou M. V., Ji M., Ji X., Bloomer W. D., Hollingshead M. G. Therapeutic advantage from combining Paclitaxel with the hypoxia-selective cytotoxin NLCQ-1 in murine tumor- or human xenograft-bearing mice.
Cancer Chem. Pharmacol.
,
50
:
501
-508,  
2002
.
44
Papadopoulou M. V., Ji M., Bloomer W. D., Hollingshead M. G. Enhancement of the antitumor effect of cyclophosphamide with the hypoxia-selective cytotoxin NLCQ-1 against murine tumors and human xenografts.
J. Exp. Ther. Oncol.
,
2
:
298
-305,  
2002
.
45
Papadopoulou M. V., Ji M., Ji X., Bloomer W. D. Therapeutic advantage from combining 5-fluorouracil with the hypoxia-selective cytotoxin NLCQ-1 in vivo. Comparison with tirapazamine.
Cancer Chem. Pharmacol.
,
50
:
291
-298,  
2002
.
46
Papadopoulou M. V., Ji M., Ji X., Bloomer W. D. Synergistic enhancement of the antitumor effect of taxol by the bioreductive compound NLCQ-1, in vivo. Comparison with tirapazamine.
Oncol. Res.
,
13
:
47
-54,  
2002
.
47
Papadopoulou M. V., Ji M., Bloomer W. D. Schedule-dependent potentiation of chemotherapeutic drugs by the bioreductive compounds NLCQ-1 and tirapazamine against EMT6 tumors in mice.
Cancer Chemother. Pharmacol.
,
48
:
160
-168,  
2001
.
48
Papadopoulou M. V., Ji X., Bloomer W. D. Hypoxia-dependent retinal toxicity of NLCQ-1 and tirapazamine in BALB/c mice.
Proc. Am. Assoc. Cancer Res. Annu. Meet.
,
43
:
1092
2002
.
49
Lee A. E., Wilson W. R. Hypoxia-dependent retinal toxicity of bioreductive anticancer prodrugs in mice.
Toxicol. Appl. Pharmacol.
,
163
:
50
-59,  
2000
.
50
Blumenthal R. D., Taylor A., Osorio L., Ochakovskaya R., Raleigh J., Papadopoulou M., Bloomer W. D., Goldenberg D. M. Optimizing the use of combined radioimmunotherapy and hypoxic cytotoxin therapy as a function of tumor hypoxia.
Int. J. Cancer
,
94
:
564
-571,  
2001
.
51
Papadopoulou, M. V., Ji, X., Xue, C., and Bloomer, W. D. In vitro schedule-dependent potentiation of taxol, 5FU and cisDDP by the hypoxic cytotoxin NLCQ-1. Comparison with tirapazamine. In: The 11th International Conference on Chemical Modifiers of Cancer Treatment. Tumor Physiology and Cancer Treatment, October 5–7, 2000, Banff, Alberta, Canada.
52
Papadopoulou, M. V., Xue, C., and Bloomer, W. D. The involvement of DNA repair genes in the hypoxia-dependent NLCQ-1 (NSC 709257) toxicity and its synergistic interaction with cisplatin or melphalan. In: The 12th International Congress of Radiation Research, August 17–22, 2003, Brisbane, Australia.
53
Papadopoulou M. V., Ji X., Bloomer W. D. Mechanisms involved in the potentiation of paclitaxel or 5-FU by the hypoxic cytotoxin NLCQ-1.
Clin. Cancer Res.
,
7(Suppl.)
:
3679
2001
.