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Tumour hypoxia promotes poor patient outcomes, with particularly strong evidence for head and neck squamous cell carcinoma (HNSCC). To effectively target hypoxia, therapies require selection biomarkers and preclinical models that can accurately model tumour hypoxia. We established 20 patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of HNSCC that we characterised for their fidelity to represent clinical HNSCC in gene expression, hypoxia status and proliferation and that were evaluated for their sensitivity to hypoxia-activated prodrugs (HAPs). PDX models showed greater fidelity in gene expression to clinical HNSCC than cell lines, as did CDX models relative to their paired cell lines. PDX models were significantly more hypoxic than CDX models, as assessed by hypoxia gene signatures and pimonidazole immunohistochemistry, and showed similar hypoxia gene expression to clinical HNSCC tumours. Hypoxia or proliferation status alone could not determine HAP sensitivity across our 20 HNSCC and two non-HNSCC tumour models by either tumour growth inhibition or killing of hypoxia cells in an ex vivo clonogenic assay. In summary, our tumour models provide clinically relevant HNSCC models that are suitable for evaluating hypoxia-targeting therapies; however, additional biomarkers to hypoxia are required to accurately predict drug sensitivity., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue., Methods: Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively., Results: NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug., Conclusions: The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from Escherichia coli has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, E. coli NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise 'off-switch' in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sharrock, McManaway, Rich, Mumm, Hermans, Tercel, Pruijn and Ackerley.)

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug with proven efficacy against hypoxic cells in preclinical tumour models. TH-302 is designed to release the DNA crosslinking agent bromo-isophosphoramide mustard (Br-IPM) when reduced in hypoxic tissue. Br-IPM is considered to diffuse locally from hypoxic regions, eliciting additional tumour cell killing, but the latter 'bystander effect' has not been demonstrated directly. Previous studies with multicellular co-cultures that included cells expressing the E. coli nitroreductase NfsA as a model TH-302 reductase have provided clear evidence of a bystander effect (which we confirm in the present study). However, NfsA is an oxygen-insensitive two-electron reductase that is not expected to generate the nitro radical intermediate that has been demonstrated to fragment to release Br-IPM. Here, we use mass spectrometry methods to characterise TH-302 metabolites generated by one-electron reduction (steady-state radiolysis by ionising radiation and cellular metabolism under hypoxia, including HCT116 cells that overexpress P450 oxidoreductase, POR) or by NfsA expressed in HCT116 cells under oxic conditions, and investigate the stability and cytotoxicity of these products. Br-IPM is shown to have very low cytotoxic potency when added to extracellular culture medium and to be rapidly converted to other hydrophilic products including dichloro-isophosphoramide mustard (IPM). Only traces of Br-IPM or IPM were detected in the extracellular medium when generated by cellular metabolism of TH-302. We identify, in NfsA-expressing cells, the hydroxylamine metabolite of TH-302, and downstream products resulting from rearrangement or hydration of the imidazole ring, and demonstrate that formation of these candidate bystander effect mediators is suppressed by hypoxia. This characterisation of the cellular pharmacology of TH-302 implies that bystander effects from hypoxic activation of TH-302 are unlikely to contribute to its anticancer activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Intra-tumor heterogeneity represents a major barrier to anti-cancer therapies. One strategy to minimize this limitation relies on bystander effects via diffusion of cytotoxins from targeted cells. Hypoxia-activated prodrugs (HAPs) have the potential to exploit hypoxia in this way, but robust methods for measuring bystander effects are lacking. The objective of this study is to develop experimental models (monolayer, multilayer, and multicellular spheroid co-cultures) comprising 'activator' cells with high expression of prodrug-activating reductases and reductase-deficient 'target' cells, and to couple these with agent-based models (ABMs) that describe diffusion and reaction of prodrugs and their active metabolites, and killing probability for each cell. HCT116 cells were engineered as activators by overexpressing P450 oxidoreductase (POR) and as targets by knockout of POR, with fluorescent protein and antibiotic resistance markers to enable their quantitation in co-cultures. We investigated two HAPs with very different pharmacology: SN30000 is metabolized to DNA-breaking free radicals under hypoxia, while the dinitrobenzamide PR104A generates DNA-crosslinking nitrogen mustard metabolites. In anoxic spheroid co-cultures, increasing the proportion of activator cells decreased killing of both activators and targets by SN30000. An ABM parameterized by measuring SN30000 cytotoxicity in monolayers and diffusion-reaction in multilayers accurately predicted SN30000 activity in spheroids, demonstrating the lack of bystander effects and that rapid metabolic consumption of SN30000 inhibited prodrug penetration. In contrast, killing of targets by PR104A in anoxic spheroids was markedly increased by activators, demonstrating that a bystander effect more than compensates any penetration limitation. However, the ABM based on the well-studied hydroxylamine and amine metabolites of PR104A did not fit the cell survival data, indicating a need to reassess its cellular pharmacology. Characterization of extracellular metabolites of PR104A in anoxic cultures identified more stable, lipophilic, activated dichloro mustards with greater tissue diffusion distances. Including these metabolites explicitly in the ABM provided a good description of activator and target cell killing by PR104A in spheroids. This study represents the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the power of combining mathematical modeling of pharmacokinetics/pharmacodynamics with multicellular culture models to dissect bystander effects of targeted drug carriers.

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.

3-(3-Morpholinopropyl)-7,8-dihydro-6 H -indeno[5,6- e ][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1- N -oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

A new series of nitro analogues of the duocarmycins was prepared and evaluated for hypoxia-selective anticancer activity. The compounds incorporate 13 different amine-containing side chains designed to bind in the minor groove of DNA while spanning a wide range of base strength from pK a 9.64 to 5.24. The most favorable in vitro properties were associated with strongly basic side chains, but the greatest in vivo antitumor activity was found for compounds containing a weakly basic morpholine. This applies to single-agent activity and for activity in combination with irradiation or chemotherapy (gemcitabine or docetaxel). In combination with a single dose of γ irradiation 50 at 42 μmol/kg eliminated detectable clonogens in some SiHa cervical carcinoma xenografts, and in combination with gemcitabine using a well-tolerated multidose schedule, the same compound caused regression of all treated A2780 ovarian tumor xenografts. In the latter experiment, three of seven animals receiving the combination treatment were completely tumor free at day 100.

Duocarmycins are highly cytotoxic natural products that have potential for development into anticancer agents. Herein we describe proposed but previously unidentified NH analogues of the DNA-alkylating subunit and characterise these by solvolysis studies, NMR and computational modelling. These compounds are shown to be the exclusive intermediates in the solvolysis of their seco precursors and to possess very similar structural features to the widely studied O-based analogues, apart from an unusually high basicity. The measured pKa of 10.5 implies that the NH compounds are fully protonated under physiological conditions. Remarkably, their extremely high reactivity (calculated hydrolysis rate 10(8) times higher for protonated NH compared to the neutral O analogue) is still compatible with potent cytotoxicity, provided the active species is formed in the presence of cells. These surprising findings are of relevance to the design of duocarmycin-based tumour-selective therapies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein we report the synthesis of three DNA-alkylating amino analogues of the duocarmycins that carry an alkyne functional group suitable for copper-catalysed click chemistry. The alkyne-containing substituents are connected via a side chain position which projects away from the minor groove, and have only a small effect on DNA alkylation and cytotoxicity. The efficiency of click reactions with fluorophore azides was studied using alkylated ctDNA by analysing the adenine adducts produced after thermal depurination. Click reactions "on DNA" were sensitive to steric effects (tether length to the alkyne) and, surprisingly, to the nature of the fluorophore azide. With the best combination of click partners and reagents, adducts could be detected in the nuclei of treated cells by microscopy or flow cytometry, provided that an appropriate detergent (Triton X-100 and not Tween 20) was used for permeabilisation. The method is sensitive enough to detect adducts at physiologically relevant concentrations, and could have application in the development of nitro analogues of the duocarmycins as hypoxia-activated anticancer prodrugs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nitro-chloromethylbenzindoline prodrug SN29428 has been rationally designed to target tumour hypoxia. SN29428 is metabolised to a DNA minor groove alkylator via oxygen-sensitive reductive activation initiated by unknown one-electron reductases. The present study sought to identify reductases capable of activating SN29428 in tumours. Expression of candidate reductases in cell lines was modulated using forced expression and, for P450 (cytochrome) oxidoreductase (POR), by zinc finger nuclease-mediated gene knockout. Affymetrix microarray mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23 cancer cell lines. Reductive activation and cytotoxicity of prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under hypoxia was strongly attenuated by the pan-flavoprotein inhibitor diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also FAD-dependent oxidoreductase domain containing 2 (FOXRED2), in cancer cell lines. This association persisted after removing the effect of POR enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of hypoxia-targeted prodrugs PR-104A, tirapazamine and SN30000, and increased cytotoxicity of the clinical-stage prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in xenobiotic metabolism. These results will inform future development of biomarkers predictive of SN29428 sensitivity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A novel class of nitroimidazole alkylsulfonamides have been prepared and evaluated as hypoxia-selective cytotoxins and radiosensitisers. The sulfonamide side chain markedly influences the physicochemical properties of the analogues: lowering aqueous solubility and raising the electron affinity of the nitroimidazole group. The addition of hydroxyl or basic amine groups increased aqueous solubility, with charged amine groups contributing to increased electron affinity. The analogues covered the range of electron affinity for effective radiosensitisation with one-electron reduction potentials ranging from -503 to -342mV. Cytotoxicity under normoxia or anoxia against a panel of human tumour cell lines was determined using a proliferation assay. 2-Nitroimidazole sulfonamides displayed significant hypoxia-selective cytotoxicity (6 to 64-fold), while 4- and 5-nitroimidazole analogues did not display hypoxia-selective cytotoxicity. All analogues sensitised anoxic HCT-116 human colorectal cells to radiation at non-toxic concentrations. 2-Nitroimidazole analogues provided modest sensitisation due to the relatively low concentrations used while several 5-nitroimidazole analogues provided equivalent sensitisation to misonidazole and etanidazole at similar molar concentrations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.

The synthesis of 19 (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate prodrugs containing sulfonate leaving groups and 7-substituted electron-withdrawing groups is reported. These were designed to undergo hypoxia-selective metabolism to form potent DNA minor groove-alkylating agents. Analogues 17 and 24, containing the benzyl sulfonate leaving group and a neutral DNA minor groove-binding side chain, displayed hypoxic cytotoxicity ratios (HCRs) of >1000 in HT29 human cancer cells in vitro in an antiproliferative assay. Four analogues maintained large HCRs across a panel of eight human cancer cell lines. In a clonogenic assay, 19 showed an HCR of 4090 in HT29 cells. Ten soluble phosphate preprodrugs were also prepared and evaluated in vivo, alone and in combination with radiation in SiHa human tumor xenografts at a nontoxic dose. Compounds 34 and 39 displayed hypoxic log(10) cell kills (LCKs) of 1.78 and 2.71, respectively, equivalent or superior activity to previously reported chloride or bromide analogues, thus showing outstanding promise as hypoxia-activated prodrugs.

Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sulfonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic cytotoxicity ratios (HCRs) of 52-286 in HT29 cells and 41-43 in SiHa cells. These values compare well with a related previously reported chloride analog. The corresponding more water soluble phosphate pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated activity providing statistically significant hypoxic log(10) cell kills of 0.87-2.80 at non-toxic doses, matching or proving superior to those of their chloride analogs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Racemic 2-{[1-(chloromethyl)-5-nitro-3-{5-[2-(dimethylamino)ethoxy]indol-2-carbonyl}-1,2-dihydro-3H-benzo[e]indol-7-yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia-selective prodrug active against radiation-resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X-ray crystallography. The intermediate was used to prepare the prodrug's enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia-selective cytotoxins, but the "natural" S enantiomer is at least 20-fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia-selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa-tumour bearing animals, the (-)-R enantiomer of the prodrug was inactive, while the (+)-S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)-S-prodrug and radiation eliminated detectable colony-forming cells in four out of five treated tumour-bearing animals., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of 3-substituted (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate (nitroCBI) prodrugs containing sulfonate leaving groups undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. They were evaluated (along with chloride leaving group analogs for comparison) for their cytotoxicity against cultures of SKOV3 and HT29 human tumor cell lines under both aerobic and hypoxic conditions. Sulfonates with neutral side chains (e.g., 5,6,7-trimethoxyindole; TMI) show consistently higher hypoxic cytotoxicity ratios (HCRs) (34-246) than the corresponding chloro analogs (2.8-3.1) in SKOV3 cells, but these trends do not hold for compounds with cationic or polar neutral side chains., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Tirapazamine (TPZ) has attractive features for targeting hypoxic cells in tumors but has limited clinical activity, in part because of poor extravascular penetration. Here, we identify improved TPZ analogues by using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue penetration explicitly during lead optimization., Experimental Design: The SR-PKPD model was used to guide the progression of 281 TPZ analogues through a hierarchical screen. For compounds exceeding hypoxic selectivity thresholds in single-cell cultures, SR-PKPD model parameters (kinetics of bioreductive metabolism, clonogenic cell killing potency, diffusion coefficients in multicellular layers, and plasma pharmacokinetics at well tolerated doses in mice) were measured to prioritize testing in xenograft models in combination with radiation., Results: SR-PKPD-guided lead optimization identified SN29751 and SN30000 as the most promising hypoxic cytotoxins from two different structural subseries. Both were reduced to the corresponding 1-oxide selectively under hypoxia by HT29 cells, with an oxygen dependence quantitatively similar to that of TPZ. SN30000, in particular, showed higher hypoxic potency and selectivity than TPZ in tumor cell cultures and faster diffusion through HT29 and SiHa multicellular layers. Both compounds also provided superior plasma PK in mice and rats at equivalent toxicity. In agreement with SR-PKPD predictions, both were more active than TPZ with single dose or fractionated radiation against multiple human tumor xenografts., Conclusions: SN30000 and SN29751 are improved TPZ analogues with potential for targeting tumor hypoxia in humans. Novel SR-PKPD modeling approaches can be used for lead optimization during anticancer drug development., (©2010 AACR.)

Nitro seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-ones (nitroCBIs) are a new class of prodrugs for antitumor therapy that undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. Although hindered by poor aqueous solubility, several examples have shown activity against hypoxic tumor cells in vivo. Here we investigate structural properties that influence hypoxic selectivity in vitro, and show that for high hypoxic selectivity nitroCBIs should combine an electron-withdrawing group of H-bond donor capacity on the A-ring, with a basic substituent on the minor groove-binding side chain. Substitution on the A-ring is compatible with the introduction of functionality that can improve water solubility., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Nitrochloromethylbenzindolines (nitroCBIs) are a new class of hypoxia-activated prodrugs for antitumor therapy. The recently reported prototypes undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents and are selectively toxic to some but not all hypoxic tumor cell lines. Here we report a series of 31 analogues that bear an extra electron-withdrawing substituent that serves to raise the one-electron reduction potential of the nitroCBI. We identify a subset of compounds, those with a basic side chain and sulfonamide or carboxamide substituent, that have consistently high hypoxic selectivity. The best of these, with a 7-sulfonamide substituent, displays hypoxic cytotoxicity ratios of 275 and 330 in Skov3 and HT29 human tumor cell lines, respectively. This compound (28) is efficiently and selectively metabolized to the corresponding aminoCBI, is selectively cytotoxic under hypoxia in all 11 cell lines examined, and demonstrates activity against hypoxic tumor cells in a human tumor xenograft in vivo.

Hypoxia represents an important therapeutic target in tumors because of the resistance of hypoxic cells to radiotherapy and chemotherapy and because it is more severe in many tumors than in normal tissues. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency. Of three alkylating subunits investigated, the chloromethylbenzindoline (CBI) structure provided the most favorable prodrug properties: aerobic cytotoxic potency of the amines was approximately 90- to 3,000-fold higher than the corresponding nitro compounds, and the nitro compounds showed air/anoxia potency differentials of up to 300-fold. Selective alkylation of adenine N3 in calf thymus DNA by an amino-CBI was shown by characterization of the thermal depurination product; the same adduct was shown in hypoxic RIF-1 cells exposed to the corresponding nitro-CBI prodrug under hypoxic (but not oxic) conditions. The amino metabolite generated from a nitro-CBI by cells expressing Escherichia coli nfsB nitroreductase in multicellular layer cultures was shown to elicit bystander killing of surrounding cells. Nitro-CBI prodrugs were >500-fold less toxic to mice than amino-CBIs by i.p. administration and provided selective killing of hypoxic cells in RIF-1 tumors (although only at maximally tolerated doses). Nitro-CBIs are novel lead hypoxia-activated prodrugs that represent the first examples of hypoxia-selective generation of potent DNA minor groove alkylating agents.

A series of novel tricyclic triazine-di- N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen-, and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUC req) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.

Tirapazamine (TPZ) and related 1,2,4-benzotriazine 1,4 dioxides (BTOs) are selectively toxic under hypoxia, but their ability to kill hypoxic cells in tumors is generally limited by their poor extravascular transport. Here we show that removing hydrogen bond donors by replacing the 3-NH2 group of TPZ with simple alkyl groups increased their tissue diffusion coefficients as measured in multicellular layer cultures. This advantage was largely retained using solubilizing 3-alkylaminoalkyl substituents provided these were sufficiently lipophilic at pH 7.4. The high reduction potentials of such compounds resulted in rates of metabolism too high for optimal penetration into hypoxic tissue, but electron-donating 6- and 7-substituents moderated metabolism. Pharmacokinetic/pharmacodynamic model-guided screening was used to select BTOs with optimal extravascular transport and hypoxic cytotoxicity properties for evaluation against HT29 human tumor xenografts in combination with radiation. This identified four novel 3-alkyl BTOs providing greater clonogenic killing of hypoxic cells than TPZ at equivalent host toxicity, with the 6-morpholinopropyloxy-BTO 22 being 3-fold more active.

Pharmacokinetic/pharmacodynamic (PK/PD) modeling has shown the antitumor activity of tirapazamine (TPZ), a bioreductive hypoxia-selective cytotoxin, to be limited by poor penetration through hypoxic tumor tissue. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ to improve activity against hypoxic cells by increasing extravascular transport. The 6 substituents modified lipophilicity and rates of hypoxic metabolism. 3-Alkylamino substituents increased aqueous solubility and also influenced lipophilicity and hypoxic metabolism. PK/PD model-guided screening was used to select six BTOs for evaluation against hypoxic cells in HT29 human tumor xenografts. All six BTOs were active in vivo, and two provided greater hypoxic cell killing than TPZ because of improved transport and/or plasma PK. This PK/PD model considers two causes of therapeutic failure (limited tumor penetration and poor plasma pharmacokinetics) often not addressed early in drug development and provides a general strategy for selecting candidates for in vivo evaluation during lead optimization.

Purpose: To compare oxygen dependence and tissue transport properties of a new hypoxia-activated prodrug, PR-104A, with tirapazamine, and to evaluate the implications for antitumor activity when combined with radiotherapy., Methods and Materials: Oxygen dependence of cytotoxicity was measured by clonogenic assay in SiHa cell suspensions. Tissue transport parameters were determined using SiHa multicellular layers. Spatially resolved pharmacokinetic (PK) and pharmacodynamic (PD) models were developed to predict cell killing in SiHa tumors and tested by clonogenic assay 18 h after treatment with the corresponding phosphate ester, PR-104., Results: The K-value (oxygen concentration to halve cytotoxic potency) of PR-104A was 0.126 +/- 0.021 microM (10-fold lower than tirapazamine at 1.30 +/- 0.28 microM). The diffusion coefficient of PR-104A in multicellular layers (4.42 +/- 0.15 x 10(-7) cm2 s(-1)) was lower than that of tirapazamine (1.30 +/- 0.05 x 10(-6) cm2 s(-1)) but PK modeling predicted better penetration to hypoxic cells in tumors because of its slower metabolism. The tirapazamine PK/PD model successfully predicted the measured activity in combination with single-dose radiation against SiHa tumors, and the PR-104A model underpredicted the activity, which was greater for PR-104 than for tirapazamine (at equivalent host toxicity) both with radiation and as a single agent., Conclusion: PR-104/PR-104A has different PK/PD properties from tirapazamine and superior activity with single-dose radiotherapy against SiHa xenografts. We have inferred that PR-104A is better able to kill cells at intermediate partial pressure of oxygen in tumors than implied by the PK/PD model, most likely because of a bystander effect resulting from diffusion of its activated metabolites from severely hypoxic zones.

A series of 2,4-dinitrobenzamide mustards were prepared from 5-chloro-2,4-dinitrobenzoic acid or the corresponding 5-dimesylate mustard as potential prodrugs for gene-directed enzyme prodrug therapy (GDEPT) with the E. coli nfsB nitroreductase (NTR). The compounds, including 32 new examples, were evaluated in four pairs of NTR+ve/-ve cell lines for selective cytotoxicity (IC50 and IC50 ratios), in multicellular layer (MCL) cultures for bystander effects, and for in vivo activity against tumors grown from stably NTR transfected EMT6 and WiDr cells in nude mice. Multivariate regression analysis of the IC50 results was undertaken using a partial least-squares projection to latent structures model. In NTR-ve lines, cytotoxicity correlated positively with logP, negatively with hydrogen bond acceptors (HA) and donors (HD) in the amide side chain, and positively with the reactivity of the less-reactive leaving group of the mustard function, likely reflecting toxicity due to DNA monoadducts. Potency and selectivity for NTR+ve lines was increased by logP and HD, decreased by HA, and was positively correlated with the leaving group efficiency of the more-reactive group, likely reflecting DNA crosslinking. NTR selectivity was greatest for asymmetric chloro/mesylate and bromo/mesylate mustards. Bystander effects in the MCL assay also correlated positively with logP and negatively with leaving group reactivity, presumably reflecting the transcellular diffusion/reaction properties of the activated metabolites. A total of 18 of 22 mustards showed equal or greater bystander efficiencies in MCLs than the aziridinylbenzamide CB 1954, which is currently in clinical trial for NTR-GDEPT. The dibromo and bromomesylate mustards were surprisingly well tolerated in mice. High MTD/IC50 (NTR+ve) ratios translated into curative activity of several compounds against NTR+ve tumors. A bromomesylate mustard showed superior activity against WiDr tumors grown from 1:9 mixtures of NTR+ve and NTR-ve cells, indicating a strong bystander effect in vivo.

Background: Because of the inefficient vasculature of solid tumors, anticancer drugs must penetrate relatively long distances through the extravascular compartment. The requirement for such diffusion may limit their activity, especially that of hypoxia-targeted drugs. We tested whether a three-dimensional pharmacokinetic/pharmacodynamic (PK/PD) model based on a representative mapped tumor microvascular network could predict the therapeutic activity of anticancer drugs in mouse xenograft tumors., Methods: Diffusion coefficients of the hypoxia-activated anticancer drug tirapazamine (TPZ) and of 15 TPZ analogs were estimated by measuring their transport through HT29 colon cancer multicellular layers (MCLs). Anoxic cytotoxic potency (by clonogenic assay) and metabolism of the TPZ analogs were measured in HT29 cell suspensions, and their plasma pharmacokinetics was measured in CD-1 nude mice. This information was used to create a spatially resolved PK/PD model for the tumor microvascular network. Model predictions were compared with actual hypoxic cell kill as measured by clonogenic assays on HT29 xenograft tumors 18 hours after treatment with each TPZ analog., Results: Modeling TPZ transport in the tumor microvascular network showed substantial drug depletion in the most hypoxic regions, with predicted maximum cell kill of only 3 logs, compared with more than 10 logs if there were no transport impediment. A large range of tissue diffusion coefficients (0.027 x 10(-6)-1.87 x 10(-6) cm2/s) was observed for the TPZ analogs. There was a strong correlation between model-predicted and measured hypoxic cell kill (R2 = 0.89) but a poor correlation when the model did not include extravascular transport (R2 = 0.32)., Conclusions: Extravascular transport in tumors, and its consequences for tumor cell killing, can be predicted by measuring drug penetration through MCLs in vitro and modeling pharmacokinetics at each position in three-dimensional microvascular networks.

The extravascular diffusion of antitumor agents is a key determinant of their therapeutic activity, but the relationships between physicochemical properties of drugs and their extravascular transport are poorly understood. It is well-known that drug lipophilicity plays an important role in transport across biological membranes, but the net effect of lipophilicity on transport through multiple layers of tumor cells is less clear. This study examines the influence of lipophilicity (measured as the octanol-water partition coefficient P) on the extravascular transport properties of the hypoxic cytotoxin tirapazamine (TPZ, 1) and a series of 13 neutral analogues, using multicellular layers (MCLs) of HT29 human colon carcinoma cells as an in vitro model for the extravascular compartment of tumors. Flux of drugs across MCLs was determined using diffusion chambers, with the concentration-time profile on both sides of the MCL measured by HPLC. Diffusion coefficients in the MCLs (D(MCL)) were inversely proportional to M(r)(0.5) (M(r), relative molecular weight), although this was a minor contributor to differences between compounds over the narrow M(r) range investigated. Differences in lipophilicity had a larger effect, with a sigmoidal dependence of D(MCL) on log P. Correcting for M(r) differences, lipophilic compounds (log P > 1.5) had ca. 15-fold higher D(MCL) than hydrophilic compounds (log P < -1). Using a pharmacokinetic/pharmacodynamic (PK/PD) model in which diffusion in the extravascular compartment of tumors is considered explicitly, we demonstrated that hypoxic cell kill is very sensitive to changes in extravascular diffusion coefficient of TPZ analogues within this range. This study shows that simple monosubstitution of TPZ can alter log P enough to markedly improve extravascular transport and activity against target cells, especially if rates of metabolic activation are also optimized.

The hypoxic cytotoxin tirapazamine (TPZ) is currently in phase III clinical trial and appears to have clinical activity. One hypothesis as to why TPZ has been used more successfully in the clinic than most other bioreductive drugs is that its unusual O(2) dependence allows killing of radioresistant cells at "intermediate" O(2) concentrations. We have determined the O(2) dependence of the metabolism of TPZ to its reduction product SR 4317, and its cytotoxicity, in stirred suspensions of HT29 colon carcinoma cells while monitoring O(2) in solution with an Oxylite trade mark probe. The O(2) dependence of the cytotoxicity of TPZ is entirely accounted for by its inhibition of the metabolism of TPZ, with a K(O(2)) value (O(2) concentration for 50% inhibition) of 1.21 +/- 0.09 (SEM) microM. We used this experimental O(2) dependence to extend a recent (Hicks et al., Cancer Res. 63, 5970-5977, 2003) pharmacokinetic/pharmacodynamic model for the cytotoxicity of TPZ in anoxic HT29 multicellular layers to model cell killing in tumors. The model indicates that the O(2) dependence of killing by TPZ complements that of radiation well during fractionated radiotherapy. It predicts that lowering K(O(2)) would decrease killing in radioresistant cells at intermediate O(2) concentrations, while higher K(O(2)) values would exacerbate metabolic consumption of TPZ and thus further impede its penetration into hypoxic regions. Raising K(O(2)) would also increase metabolic activation at physiological O(2) concentrations, thereby compromising hypoxic selectivity. We conclude that the K(O(2)) value of TPZ is indeed close to the optimum for a bioreductive drug of this class (i.e. one that kills only cells in which it is reduced).

Tirapazamine (TPZ), a bioreductive drug with selective toxicity for hypoxic cells in tumors, is currently in Phase III clinical trials. It has been suggested to have a dual mechanism of action, both generating DNA radicals and oxidizing these radicals to form DNA breaks; whether the second (radical oxidation) step is rate-limiting in cells is not known. In this study we exploit the DNA radical oxidizing ability of the 1-N-oxide metabolite of TPZ, SR 4317, to address this question. SR 4317 at high, but nontoxic, concentrations potentiated the hypoxic (but not aerobic) cytotoxicity of TPZ in all four of the human tumor cell lines tested (HT29, SiHa, FaDu, and A549), thus providing a 2-3-fold increase in the hypoxic cytotoxicity ratio. In potentiating TPZ, SR 4317 was 20-fold more potent than the hypoxic cell radiosensitizers misonidazole and metronidazole but was less potent than misonidazole as a radiosensitizer, suggesting that the initial DNA radicals from TPZ and radiation are different. SR 4317 had favorable pharmacokinetic properties in CD-1 nude mice; coadministration with TPZ provided a large increase in the SR 4317 plasma concentrations relative to that for endogenous SR 4317 from TPZ. It also showed excellent extravascular transport properties in oxic and anoxic HT29 multicellular layers (diffusion coefficient 3 x 10(-6) cm(2)s(-1), with no metabolic consumption). Coadministration of SR 4317 (1 mmol/kg) with TPZ at a subtherapeutic dose (0.133 mmol/kg) significantly enhanced hypoxic cell killing in HT29 tumor xenografts without causing oxic cell killing, and the combination at its maximum tolerated dose was less toxic to hypoxic cells in the retina than was TPZ alone at its maximum tolerated dose. This study demonstrates that benzotriazine mono-N-oxides have potential use for improving the therapeutic utility of TPZ as a hypoxic cytotoxin in cancer treatment.

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.

In common with other bioreductive drugs, metabolic reduction is required for activation of the benzotriazine-di-N-oxide tirapazamine (TPZ) in hypoxic regions of tumors. This same metabolism also consumes the drug as it diffuses, impeding its penetration into hypoxic tissue. In this study, we develop a pharmacokinetic (PK)/pharmacodynamic (PD) model for TPZ that explicitly includes its diffusion characteristics as measured in multicellular layer (MCL) cultures of HT29 colon carcinoma cells. The kinetics of TPZ metabolism to its mono-N-oxide derivative SR 4317, determined by high-performance liquid chromatography using anoxic HT29 single cell suspensions, demonstrated both a first order and saturable (K(m) = 3.6 micro M) component. Cell killing, assessed by clonogenic assay under the same conditions, demonstrated an approximately quadratic concentration dependence and linear time dependence. TPZ transport through MCLs, determined under hyperoxic conditions (95% O(2)) to suppress reductive metabolism, provided a concentration-independent diffusion coefficient of 0.40 x 10(-6) cm(2)s(-1). Under anoxia, this transport was strongly suppressed and was well predicted by the single cell metabolism parameters (scaled to the cell density in MCLs). These PK (transport) and PD (cytotoxicity) parameters were used to calculate cell killing as a function of distance in anoxic HT29 MCLs after the addition of TPZ to both sides of the MCL. The predicted average cell kill was in good agreement with measured values, which showed much less killing than for single cell suspensions under the same conditions. The success of this PK/PD model in predicting response in MCL shows that inefficient transport, rather than changes in intrinsic sensitivity, is responsible for TPZ resistance in these three-dimensional cell cultures and suggests that optimization of transport properties is a high priority in developing second-generation TPZ analogues.

To improve the potency of the hypoxic cytotoxin tirapazamine (TPZ), we have constructed an analog, SN26955, with the TPZ moiety attached to an acridine chromophore to target the drug to DNA. The underlying reason for this is our previous finding that the hypoxic cytotoxicity of TPZ is a result of its ability to produce DNA double-strand breaks, whereas many of the toxicities of the drug in clinical use are likely the result of its metabolism in the cytoplasm and effects on mitochondria. We found that the DNA-targeted TPZ analog was more potent than TPZ in killing hypoxic cells by 1-2 orders of magnitude, yet it retained the hypoxic selectivity for cell killing of TPZ. We show that SN26955 is only active in producing DNA damage when it is enzymatically reduced while bound to, or in close association with, the DNA. We also show that it has a different cofactor dependence than TPZ for reduction leading to DNA double-strand breaks, suggesting the involvement of a different reductase for production of the lethal lesion than for TPZ. These results show the promise of DNA-targeting of TPZ to produce a DNA compound with greater clinical efficacy than TPZ itself.

The dinitrobenzamide aziridine CB 1954 (1) and its nitrogen mustard analogue SN 23862 (6) are prodrugs that are activated by enzymatic nitroreduction in tumors. Bioactivation of 1 is considered to be due to reduction of its 4-nitro group to the hydroxylamine and subsequent formation of the N-acetoxy derivative; this acts as a reactive center, in concert with the aziridine moiety, to provide a bifunctional DNA cross-linking agent (Knox model). It is currently unclear whether bioactivation of 6 occurs by the same mechanism or results from the electronic effects of nitroreduction on reactivity of the nitrogen mustard moiety. To discriminate between these mechanisms, we have synthesized the hydroxylamine and amine derivatives of 1 and 6, plus related compounds, and determined their alkylating reactivities in aqueous solution, using LC/MS to identify reaction pathways. The relationships between substituent electronic effects, reactivity, and cytotoxicity were determined using the UV4 cell line, which is defective in nucleotide excision repair (thus avoiding differences in repair kinetics). Alkylating reactivity correlated with the electron-donating character of the ortho or para substituent in the case of the mustards, with a less marked electronic effect for the aziridines. Importantly, there was a highly significant linear relationship between cytotoxic potency and alkylating reactivity in both the aziridine and the mustard series, with the notable exception of 4, the 4-hydroxylamine of 1, which was 300-fold more toxic than predicted by this relationship. This demonstrates that the high potency of 4 does not result from activation of the aziridine ring, supporting the Knox model. The single-step bioactivation of 6, to amino or hydroxylamine metabolites with similar potency to 4, is a potential advantage in the use of dinitrobenzamide mustards as prodrugs for activation by nitroreductases.

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxic cytotoxin currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. As part of a program to develop TPZ analogues with improved solubility/potency and therapeutic indices, we synthesized 34 1,2,4-benzotriazin-3-amine 1,4-dioxides (BTO) to examine structure-activity relationships (SAR) for ring substitution. The electronic, hydrophobic, and steric parameters of substituents at the 5-, 6-, 7-, and 8-positions were systematically varied, and the aqueous solubility and one-electron reduction potentials [E(1)] of the analogues were determined. For each compound, we determined cell killing of mouse SCCVII tumor cells in vitro under aerobic and hypoxic conditions by clonogenic survival and determined their relative hypoxic toxicity (RHT; relative to TPZ) and hypoxic cytotoxicity ratio (HCR). A subset of compounds was independently evaluated using a 96-well SRB proliferation assay, the data from which correlated well with that derived by the clonogenic endpoint. Most substituents, except 5- and 8-dimethylamino and 8-diethylamino, gave analogues less soluble than TPZ. E(1) values ranged from -240 mV through -670 mV (with TPZ having a value of -456 mV) and correlated well with the electronic parameter sigma for substituents at the 5-, 6-, 7-, and 8-positions. Aerobic cytotoxic potency showed a strong positive correlation with E(1) (i.e., electron-withdrawing substituents increased aerobic toxicity). Hypoxic cytotoxicity also generally increased with increasing E(1), with a maximum (RHT up to 3.9-fold) seen in halo- and trifluoromethyl-substituted BTO derivatives having E(1) between ca. -370 to -400 mV. Analogues with high HCRs (>50) all had E(1)s in the range -450 to -510 mV (weakly electron-donating substituents) with the exception of the 8-CF(3) analogue, which had an HCR of 112 against SCCVII despite a high E(1) of -372 mV). The results suggest that ring-A substituents in BTO analogues can be used to predictably vary one-electron reduction potentials and also provide a much better definition than previously of the optimum range of these reduction potentials for a desirable biological activity profile (high HCR, RHT, and solubility).

Purpose: To determine whether there is a therapeutic interaction between the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and nine chemotherapy drugs against an early-passage mouse mammary tumour (MDAH-MCa-4), and to investigate the mechanism of any such interaction., Methods and Results: Female C3H/HeN mice bearing intramuscular MDAH-MCa-4 tumours were injected intraperitoneally with DMXAA (80 micro mol/kg) or chemotherapy drug (at a range up to the maximum tolerated dose) alone, or coadministered. A small reduction in the dose of the chemotherapy drug was required in most cases, but the increase in antitumour effect was much greater than the increase in host toxicity (body weight loss). The therapeutic gain increased in the order 5-fluorouracil (no gain)<(etoposide, carboplatin, cyclophosphamide, doxorubicin, cisplatin)<(docetaxel, vincristine)

There is considerable evidence that DNA intercalating drugs fail to penetrate tumor tissue efficiently. This study used the multicellular layer (MCL) experimental model, in conjunction with computational modeling, to test the hypothesis that a DNA intercalator in phase II clinical trial, N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA), has favorable extravascular transport properties. Single cell uptake and metabolism of DACA and the related but more basic aminoacridine 9-[3-(dimethylamino)propylamino]acridine (DAPA), and penetration through V79 and EMT6 MCL, were investigated by high-performance liquid chromatography. DACA was accumulated by cells to a lesser extent than DAPA and was metabolized to the previously unreported acridan by V79 but not EMT6 cells. Despite this metabolism, flux of DACA through MCL was much faster than that of DAPA. Modeling MCL transport as diffusion with reaction (metabolism and reversible binding) showed that the faster flux of DACA was due to a 3-fold higher free drug diffusion coefficient and 10-fold lower binding site density. The MCL transport parameters were used to develop a spatially resolved pharmacokinetic model for the extravascular compartment in tumors, which provided a reasonable prediction of measured average tumor concentrations from plasma pharmacokinetics in mice. Area under the curve was essentially independent of distance from blood vessels, although the combined pharmacokinetic/pharmacodynamic model predicted a modest decrease in cytotoxicity (from 1.8 to 1.1 logs of cell kill) across a 125-microm region. In conclusion, this study demonstrates that it is possible to design DNA intercalators that diffuse efficiently in tumor tissue, and that there is little impediment to DACA transport over distances required for its antitumor action.

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.

Cobalt(III) complexes [Co(R2dtc)2(L)]+ containing two dithiocarbamate ligands (R = Me, Et, pyrrolidine) and a bidentate nitrogen mustard ligand (L) have been prepared as potential hypoxia-selective cytotoxins. The complexes were synthesized by treatment of the binuclear precursor [Co2(R2dtc)5]+ with the diamine mustards N,N'-bis(2-chloroethyl)ethylenediamine (BCE) and N,N-bis(2-chloroethyl)ethylenediamine (DCE), or their non-alkylating analogues [N,N-diethylethylenediamine (DEE) and N,N'-diethylethylenediamine (BEE)]. Cyclic voltammetry of the complexes in MeCN reveals quasi-reversible behaviour for the Co(III)/Co(II) couple, with E1/2 increasing in the order DCE < DEE approximately BCE < BEE. In MeCN/H2O electrochemical reduction is irreversible, indicating rapid substitution of H2O into the coordination sphere of the Co(II) intermediate. This fast ligand loss was confirmed by pulse radiolysis of [Co(Me2dtc)2(DEE)]+, while steady-state radiolysis showed that the initial intermediate disproportionates to [CoII(H2O)6]2+ + 2[CoII(Me2dtc)3]. The latter species reduces additional parent complex to give an overall stoichiometry of 3 mol parent complex/mol reductant. [Co(Me2dtc)2(DCE)]+ decays rapidly by an analogous mechanism in hypoxic culture medium. This reaction is not inhibited by O2, indicating that reoxidation of the Co(II) intermediate by O2 is not rapid enough to compete with ligand dissociation. The resulting free R2dtc-ligands, rather than the released mustards, are primarily responsible for growth inhibition by [Co(R2dtc)2(L)]+ complexes, although DCE release does contribute to clonogenic cell killing. Clonogenic cell killing is not appreciably enhanced under hypoxic conditions for any of the dithiocarbamato complexes. This finding, coupled with their instability in culture medium, suggests that [Co(R2dtc)2(L)]+ complexes are probably not suited for further development as bioreductive anticancer drugs.

Several studies show that the antitumour activity of melphalan (MEL) and other alkylating agents can be enhanced by the selective inhibition of tumour blood flow, although the mechanism(s) underlying these interactions are unclear. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer agent currently in phase I clinical trial, inhibits blood flow in murine tumours. DMXAA increased the activity of MEL against the MDAH-MCa-4 mouse mammary tumour maximally when MEL was given about 2 h after DMXAA, without compromising the maximal dose of the alkylating agent that could be given. The plasma pharmacokinetics of MEL were unchanged by DMXAA pretreatment, but the area under the concentration-time curve (AUC) for the tumour increased by 33% as a result of decreasing clearance (consistent with falling tumour blood flow). However, inhibition of tumour blood flow also leads to microenvironmental changes (e.g. acidosis and hypoxia) that might influence sensitivity to MEL. The sensitivity of KHT cells (freshly isolated from tumours) to MEL in vitro was increased by lowering of either pH or oxygen concentration (pO2), with an overall dose-modifying factor of 15 being recorded for aerobic cells at pH 7.4 versus hypoxic cells at pH 6.5. The cellular uptake of MEL by KHT cells was increased by 74% under hypoxia. Thus, DMXAA appears to augment the antitumour activity of MEL through two different mechanisms, increased exposure (via decreased tumour clearance of MEL) and increased sensitivity resulting from changes to the tumour microenvironment, both of which result from inhibition of tumour blood flow.

1. Hypoxia arises in solid tumour because of inefficient blood supply. While hypoxic cells are resistant to radiotherapy and probably to many chemotherapeutic drugs they can, in principle, be turned to advantage through the development of hypoxia-activated cytotoxic drugs (bioreductive drugs). 2. Three general approaches to exploiting tumour hypoxia are discussed. The first relies on fluctuating blood flow in tumours and the consequent cycling of cells through the hypoxic compartment. The second incorporates a prodrug approach in which drug activation gives rise to cytotoxic metabolites which diffuse out of hypoxic zones. The third utilizes selective inhibitors of tumour blood flow to induce additional hypoxia and thus enhance bioreductive drug activation. 3. The latter two approaches are illustrated by recent studies with the dinitrobenzamide nitrogen mustard class of bioreductive drugs and their combination with the tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetic acid.

In order to establish the mechanism of perturbation of hormonally regulated calcium homeostasis in hepatocytes caused by menadione, the effects of menadione on hepatic alpha 1-adrenergic receptors and on alpha 1-adrenergic receptor-mediated increase in cytosolic free calcium concentration were determined. Menadione had no detectable effect on the alpha 1-adrenergic receptor but significantly inhibited (-)-epinephrine-dependent increases in intracellular free calcium concentration in Quin2 acetoxymethyl ester-loaded hepatocytes. The hormonally induced increase in intracellular free calcium concentration is caused by formation of inositol 1,4,5-trisphosphate (IP3) which binds to a specific receptor and causes a release of intracellular ATP-dependently sequestrated calcium. The IP3-stimulated release of calcium from intracellular pools in hepatocytes was inhibited to a great extent after treatment with menadione. This inhibition could also be observed after treatment of hepatocytes with p-benzoquinone and N-ethylmaleimide and could not be reversed by the thiol-reducing reagent dithiothreitol which indicated covalent binding to an essential free sulfhydryl group. The inhibition of IP3-dependent release of intracellular calcium was accompanied by a large increase in the number of detectable IP3 receptors without any change in the dissociation constant as determined in permeabilized hepatocytes. The increase in IP3 receptors caused by menadione could be reversed by dithiothreitol which suggests the involvement of free sulfhydryl groups. It is concluded that the IP3 receptor plays an important role in the mechanism of menadione-induced perturbation of hormonally regulated calcium homeostasis in rat hepatocytes.

We investigated the effects of the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) and menadione--a sulfhydryl-arylating agent, which can undergo redox cycling--on the [3H]inositol-1,4,5-trisphosphate ([3H]IP3) binding properties of rat plasma membranes. Rat liver plasma membranes were incubated for 15 min at 37 degrees with 0.1 mM, 0.2 mM, 0.5 mM NEM or 0.3 mM menadione and subsequently diluted for use in [3H]IP3 binding studies. An incubation as such (15 min at 37 degrees) already caused the dissociation constant (Kd) of [3H]IP3 binding to increase from 1.9 +/- 0.2 nM to 3.4 +/- 0.2 nM, with only a small change in the maximal number of IP3 binding sites (Bmax-values of 401 +/- 32 and 349 +/- 13 fmol/mg protein, respectively). Incubation with NEM (0.1, 0.2 and 0.5 mM) resulted in a dose dependent decrease in the Bmax with 41, 87 and 99%, respectively, without a significant change in the Kd compared to the time matched controls. Menadione (0.3 mM) decreased the Bmax with 54% without affecting the Kd. In contrast to our findings at 37 degrees, incubation of the plasma membranes with NEM (0.5 mM) at 0 degrees for 30 min did not affect [3H]IP3 binding. In order to account for this discrepancy, the reaction rate of NEM with glutathione was examined at both 0 degrees and 37 degrees by recording the changes in the UV-spectrum of NEM (lambda max = 302 nm) after addition of 1 mM NEM to 1 mM glutathione. A similar reaction rate was observed at both temperatures. These data suggest that alkylation of a sulfhydryl-moiety in the IP3-receptor molecule causes inactivation of the receptor function. Since at 0 degrees NEM is still able to react with sulfhydryl groups, but not able to inactivate the IP3-receptor, it can be suggested that the sulfhydryl-moiety of the IP3-receptor is masked and cannot be reached by a sulfhydryl-alkylating agent at 0 degrees.

Liver plasma membranes of non-treated rats or rats treated with carbon tetrachloride were prepared. Liver plasma membranes obtained from non-treated rats were preincubated with cumene hydroperoxide, N-ethylmaleimide or hydrogen peroxide in the absence or presence of Fe2+. The effects of in-vivo or in-vitro treatment on hepatic alpha 1-adrenergic receptors were assessed in a [3H]prazosin binding assay. All treatments except preincubation with hydrogen peroxide alone caused a decrease in the number of alpha 1-adrenergic receptors without a change in the affinity of [3H]prazosin. The decrease in receptor number was accompanied by an increase in the level of lipid peroxidation in the plasma membranes. The results indicate that the hepatic alpha 1-adrenergic receptor is vulnerable to free radical stress.