Overcoming Radioresistance: Small Molecule Radiosensitisers and Hypoxia-activated Prodrugs.

The role of hypoxia in radiation resistance is well established and many approaches to overcome hypoxia in tumours have been explored, with variable success. Two small molecule strategies for targeting hypoxia have dominated preclinical and clinical efforts. One approach has been the use of electron-affinic nitroheterocycles as oxygen-mimetic sensitisers. These agents are best exemplified by the 5-nitroimidazole nimorazole, which has limited use in conjunction with radiotherapy in head and neck squamous cell carcinoma. The second approach seeks to leverage tumour hypoxia as a tumour-specific address for hypoxia-activated prodrugs. These prodrugs are selectively activated by reductases under hypoxia to release cytotoxins, which in some instances may diffuse to kill surrounding oxic tumour tissue. A number of these hypoxia-activated prodrugs have been examined in clinical trial and the merits and shortcomings of recent examples are discussed. There has been an evolution from delivering DNA-interactive cytotoxins to molecularly targeted agents. Efforts to implement these strategies clinically continue today, but success has been elusive. Several issues have been identified that compromised these clinical campaigns. A failure to consider the extravascular transport and the micropharmacokinetic properties of the prodrugs has reduced efficacy. One key element for these 'targeted' approaches is the need to co-develop biomarkers to identify appropriate patients. Hypoxia-activated prodrugs require biomarkers for hypoxia, but also for appropriate activating reductases in tumours, as well as markers of intrinsic sensitivity to the released drug. The field is still evolving and changes in radiation delivery and the impact of immune-oncology will provide fertile ground for future innovation.

ZD-0473 AstraZeneca.

ZD-0473 (formerly JM-473 and AMD-473) is a sterically hindered platinum (II) complex designed and synthesized by Johnson Matthey Technology and the Cancer Research Campaign (CRC) and under development as a potential treatment for cisplatin-resistant cancer. AstraZeneca is developing both an i.v. and an oral formulation of ZD-0473. In December 1999 the i.v. formulation entered phase II clinical trials for the potential treatment of solid tumors; the oral formulation is undergoing preclinical development [351298,349551]. AstraZeneca expects to file for registration of the drug in the fourth quarter of 2002 [377656]. Phase I clinical trials for the potential treatment of a range of solid tumors, including colorectal cancer were carried out jointly by AnorMED (a subsidiary of Johnson Matthey) and the CRC at the Royal Marsden Hospital, UK [301848,315489,337657]. AstraZeneca has licensed ZD-0473 from AnorMED and is responsible for the worldwide development of the drug beyond phase I trials [337540]. Phase I trials to determine the maximum tolerated dose in patients, the nature of the dose-limiting toxicity and the pharmacokinetics of the drug commenced in the US in November 1999 [347964]. In June 2000, Deutsche Bank predicted sales of US $15 million in 2003 [374500]. In March 1999, Lehman Brothers predicted a 15% probability that the drug would reach worldwide markets, and be launched onto the market in 2003 [336599].

Cellular uptake of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA).

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA), a DNA intercalating dual topoisomerase I/II poison, has high experimental antitumour activity, is able to overcome several forms of multidrug resistance, and is undergoing clinical trial. We prepared 3H-labelled DACA and investigated its uptake using cultured Lewis lung carcinoma cells (LLTC), P388 leukaemia cells and P/DACT cells that were multidrug resistant. The kinetics of uptake and efflux were very rapid and equilibrium was obtained within seconds of drug addition. Fluorescence microscopy of LLTC cells treated with DACA showed punctate fluorescence in the cytoplasm, consistent with uptake into vesicles. To investigate the role of lipophilicity in drug uptake, a fluorimetric assay was developed to measure uptake of a more hydrophilic derivative, 9-amino-5-sulphonylmethyl-DACA (as-DACA). The calculated n-octanol-water partition coefficient for as-DACA was 20-fold lower than that for DACA. On the other hand, as determined by ethidium displacement from DNA, as-DACA bound DNA 16-fold more strongly than did DACA. Uptake and efflux of DACA and as-DACA were very rapid and the uptake ratios in LLTC cells were 550 for DACA and 54 for as-DACA. At equitoxic concentrations (corresponding to the IC50 values), LLTC cell association was estimated to be approximately 1.6 x 10(8) molecules per cell for DACA and 3.0 x 10(6) molecules per cell for as-DACA. It is argued that DACA binds predominantly to lipophilic sites such as proteins and cellular membranes, while as-DACA associates predominantly with DNA. The high affinity of DACA for membranes may contribute to the rapidity of its uptake and efflux, as well as to its ability to overcome multidrug resistance.

A 2-nitroimidazole carbamate prodrug of 5-amimo-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbony l]-1,2-dihydro-3H--benz[E]indole (amino-seco-CBI-TMI) for use with ADEPT and GDEPT.

The synthesis of a 2-nitroimidazol-5-ylmethyl carbamate prodrug 10 of the potent minor groove alkylating agent amino-seco-CBI-TMI 3 is described. Chemical, radiolytic, and enzymic reductions of a model 2-nitroimidazol-5-yl carbamate 8 show release of the amine effector upon reduction. Prodrug 10 gives a ten fold increase in cytotoxicity against human ovarian carcinoma SKOV3 cells in the presence of E. coli B nitroreductase (NTR) and a 21-fold increase in cytotoxicity against a SKOV3 cell line (SC3.2) transfected with the gene for NTR. The cytotoxicity of 10 increased 15- to 40-fold under hypoxia. Prodrug 10 has significant potential as a prodrug for use in ADEPT and GDEPT applications, and as a hypoxia-selective cytotoxin.

Nitrobenzyl carbamate prodrugs of enediynes for nitroreductase gene-directed enzyme prodrug therapy (GDEPT).

The synthesis and evaluation of the 4-nitrobenzylcarbamate enediyne 6 and related compounds as prodrugs activated by a nitroreductase enzyme (NTR) from E. coli B is described. Expression of NTR in three different cell lines gives increases in cytotoxicity of 21- to 135-fold for 6 (IC50 values 13-24 nM in the NTR-expressing lines), indicating its potential as a prodrug for NTR-mediated Gene-Directed Enzyme Prodrug Therapy. The cytotoxicity of 6 and related enediynes is shown to be oxygen-dependent, especially in nucleotide excision repair-proficient cells, which might limit activity in hypoxic regions of tumours.

Oxygen dependence of the cytotoxicity of the enediyne anti-tumour antibiotic esperamicin A1.

The enediyne anti-tumour antibiotics are extremely potent cytotoxins, apparently because of their conversion to diradical species which induce DNA double strand breaks with high efficiency. The potency of enediynes suggests their possible utility as effector units for prodrugs which can be activated selectively in tumours, such as bioreductive drugs (BD) or radiation-activated cytotoxins (RAC). However, the similarity of the radical-induced DNA breakage reactions of the enediynes to those caused by ionising radiation suggested that resistance of hypoxic cells might be a potential problem. Experiments with AA8 cells in culture demonstrated that the enediyne antibiotics neocarzinostatin and esperamicin A, (ESP) are much less toxic under hypoxic than aerobic conditions. Sensitivity to ESP (concentration for 90% cell kill 10 pM) decreased 15-fold under hypoxia, and was partially restored by simultaneous exposure to misonidazole. ESP induced chromosome breakage (micronucleus formation) with an efficiency similar to gamma radiation at equivalent cell kill, suggesting a clastogenic mechanism of cytotoxicity. In contrast, little micronucleus formation was evident after exposure to ESP under hypoxia, even at concentrations giving equivalent cell killing. These findings suggest that resistance of hypoxic cells may limit the utility of enediynes as cytotoxic effectors for BD or RAC prodrug development, and that further investigation of enediynes as anti-tumour agents should include strategies capable of eliminating hypoxic cells.

Nitroimidazole-based "extruded mustards' designed as reductively activated hypoxia-selective cytotoxins.

A new class of nitroimidazole alkanoic acid amides, designed to extrude para-aminophenyl mustard (3a) by intramolecular cyclization following reduction of the nitro group, have been prepared and evaluated for their ability to function as bioreductively activated prodrugs. The mechanism of activation following (bio) reduction was studied using the model compounds (8a-e, 18 and 23a,b) and the related mustard analogues (11a-c and 26a,b). However, the reduced forms of these compounds were found to be relatively stable and not susceptible to intramolecular cyclization. This is in contrast to the corresponding 2-nitrophenylalkyl amides, where the hydroxylamino or amino reduction products (e.g. 27) undergo intramolecular cyclization via a tetrahedral intermediate, resulting in cleavage of the amide and release of an activated aromatic mustard. One of the 2-nitroimidazole mustards (11b) had 20-fold greater toxicity towards aerobic AA8 cells than RB 6145 (4), and a 51-fold greater toxicity towards UV4 cells (which are defective in DNA cross-link repair and thus hypersensitive to cross-linking agents). The cytotoxicity of 11b against AA8 cells was enhanced 3.3-fold under hypoxic conditions, but the compound was inactive against the hypoxic subfraction of cells in KHT tumours in vivo.

Hypoxia-selective antitumor agents. 10. bis(nitroimidazoles) and related Bis(nitroheterocycles): development of derivatives with higher rates of metabolic activation under hypoxia and improved aqueous solubility.

A series of analogues of the previously described compound N-[2-(2-methyl-5-nitroimidazol-1H-yl)ethyl]-4-(2-nitroimidazol- 1H-yl)butanamide (4), a novel hypoxic cell cytotoxin and radiosensitizer, have been prepared and evaluated for hypoxia-selective cytotoxicity and hypoxic cell radiosensitization in vitro. The new derivatives were designed to overcome the low aqueous solubility of 4 and its slow kinetics of killing under hypoxia. The nitroheterocycle unit had a significant effect on solubility, with 3-nitrotriazoles being about 6-fold more soluble than the corresponding 2-nitroimidazoles. Analogues with a range of neutral linker chains (polyhydroxy, alkanesulfonamide, and bisamide) showed only slightly improved solubility and were unable to be fully evaluated. However, a series of analogues with cationic amine linkers had adequate aqueous solubility (up to 280 mM). The amine analogues could not be prepared by direct reduction of precursor amides such as 4 and were most conveniently synthesized by aza-Wittig condensation of the appropriate azide and aldehyde components. The amine-linked compounds were more cytotoxic than 4, with the symmetrical bis(2-nitroimidazole) derivatives (13 and 14) up to 9-fold more potent. They showed hypoxic selectivities comparable to that of 4 (ca. 200-fold) but had much more rapid kinetics of killing under hypoxia, resulting in high hypoxic selectivity at early times in culture. The nature of the mechanism of cytotoxicity of these compounds remains unclear but appears not to be DNA cross-linking, with the compounds showing a lack of hypersensitivity toward repair-deficient UV4 cells. The enhanced solubility and hypoxia-selective cytotoxicity (at early times) of 13 compared with 4 represent significant potential advantages.

N-[2-(2-methyl-5-nitroimidazolyl)ethyl]-4-(2-nitroimidazolyl)butanamide (NSC 639862), a bisnitroimidazole with enhanced selectivity as a bioreductive drug.

Compounds containing two redox centers, both of which must be reduced for full expression of cytotoxicity by oxygen-inhibitable pathways (bis-bioreductive drugs), have potential as cytotoxins with high selectivity for hypoxic tumor cells. The bisnitroimidazole N-[2-(2-methyl-5-nitroimidazolyl)ethyl]-4-(2-nitroimidazolyl)butanamide (NNB, NSC 639862), in which a 2-nitroimidazole and 5-nitroimidazole moiety are joined via a carboxamide linker, is highly selective for hypoxic AA8 Chinese hamster cells (200-fold by 8 h) relative to mononitroimidazoles (5-25-fold). A bis-bioreductive mechanism is consistent with the marked increase in hypoxic potency and selectivity of NNB with time and the apparent requirement that the two nitro groups be present in the same molecule. NNB differed from mononitroimidazoles in inducing fewer DNA single strand breaks at equivalent toxicity, suggesting that a duplex DNA lesion (locally doubly damaged site) may be responsible for cell killing. Alkaline elution studies and the lack of hypersensitivity of the repair-defective UV4 cell line indicate that the cytotoxic lesion is not a DNA interstrand cross-link. NNB shows greater hypoxic selectivity than the alkylating 2-nitroimidazole RB 6145 against AA8 cells and is active in combination with radiation when administered in multiple doses against the MDAH-MCa-4 mouse mammary carcinoma.

Hypoxia-selective antitumor agents. 8. Bis(nitroimidazolyl)alkanecarboxamides: a new class of hypoxia-selective cytotoxins and hypoxic cell radiosensitisers.

A series of novel bis(nitroimidazolyl)alkanecarboxamides has been prepared and evaluated for hypoxia-selective cytotoxicity and hypoxic cell radiosensitisation in vitro and in vivo. The compounds were prepared by direct coupling of preformed side chain acid and amine components, using diethyl phosphorocyanidate at room temperature. Although designed to be bis-bioreductive prodrugs of DNA cross-linking agents, none of the compounds showed evidence of DNA cross-linking activity, being equally potent against cell lines deficient and proficient in repair of cross-links. However, one of these compounds, N-[2-(2-methyl-5-nitro-1H-imidazolyl)ethyl]-4-(2-nitro-1H- imidazolyl)butanamide (10; SN 24699), showed high hypoxic selectivity as a cytotoxin (rising to 200-fold after exposure to the drug for several hours) in the repair-proficient Chinese hamster cell line AA8. This selectivity was greater than observed for the alkylating 2-nitroimidazole (4; RB 6145) (40-fold) or simple mononitroimidazoles (5-25-fold). Investigation of structure-activity relationships for hypoxic selectivity of bis(nitroimidazoles) was restricted by their low aqueous solubility, but a certain minimum separation of the two nitroimidazole units (by more than five atoms) appears desirable. All the compounds radiosensitized hypoxic cells in vitro but were little more potent as radiosensitizers than the corresponding monomeric nitroimidazoles. Compound 10 caused additional cell killing in the KHT tumor when multiple drug doses were administered in combination with a single dose of radiation. It is not yet clear whether this activity reflects hypoxic cell radiosensitization or cytotoxicity toward hypoxic cells, but this new class of bis-bioreductive agent clearly warrants further investigation.