RESUMEN
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.
Asunto(s)
Biomarcadores de Tumor , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Ratones , Biomarcadores de Tumor/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relevancia Clínica , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Nitroimidazoles/farmacología , Profármacos/farmacología , Profármacos/uso terapéutico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Hipoxia TumoralRESUMEN
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.
Asunto(s)
Efecto Espectador/efectos de los fármacos , Hipoxia de la Célula , Indoles/farmacología , Modelos Biológicos , Cromatografía Liquida , Técnicas de Cocultivo , Proteínas de Escherichia coli/genética , Células HCT116 , Humanos , Indoles/farmacocinética , NADPH-Ferrihemoproteína Reductasa/genética , Nitrorreductasas/genética , Profármacos , Esferoides Celulares/citología , Espectrometría de Masas en TándemRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Efecto Espectador , Nitroimidazoles/farmacología , Mostazas de Fosforamida/farmacología , Proteínas de Escherichia coli , Células HCT116 , Humanos , Estructura Molecular , NADPH-Ferrihemoproteína Reductasa , Nitroimidazoles/química , Nitrorreductasas , Mostazas de Fosforamida/química , Profármacos/farmacologíaRESUMEN
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.
Asunto(s)
Nitroimidazoles/química , Nitroimidazoles/farmacología , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Descubrimiento de Drogas , Femenino , Células HCT116 , Humanos , Ratones , Nitroimidazoles/farmacocinética , Nitroimidazoles/toxicidad , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Fármacos Sensibilizantes a Radiaciones/toxicidad , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
3-(3-Morpholinopropyl)-7,8-dihydro-6H-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 â¼8°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.
RESUMEN
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 pKa 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.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Indoles/química , Indoles/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Ovario/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales , Duocarmicinas , Femenino , Humanos , Indoles/farmacología , Ratones , Ratones Desnudos , Nitrocompuestos/química , Nitrocompuestos/farmacología , Nitrocompuestos/uso terapéutico , Neoplasias Ováricas/patología , Neoplasias Ováricas/radioterapia , Ovario/patología , Ovario/efectos de la radiación , Pirrolidinonas/química , Pirrolidinonas/farmacología , Pirrolidinonas/uso terapéuticoRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/química , Ciclopropanos/química , Ciclopropanos/toxicidad , Indoles/química , Indoles/farmacología , Indoles/toxicidad , Animales , Antibióticos Antineoplásicos/síntesis química , Antibióticos Antineoplásicos/toxicidad , Cricetinae , Cricetulus , Ciclopropanos/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Duocarmicinas , Humanos , Concentración de Iones de Hidrógeno , Indoles/síntesis química , Cinética , Ratones , Protones , Pirroles/farmacologíaRESUMEN
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 Tweenâ 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.
Asunto(s)
Antibióticos Antineoplásicos/síntesis química , Química Clic/métodos , ADN/química , Colorantes Fluorescentes/química , Indoles/síntesis química , Alquilación , Alquinos/química , Animales , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Perros , Ensayos de Selección de Medicamentos Antitumorales , Duocarmicinas , Humanos , Indoles/química , Indoles/farmacología , Pirroles/síntesis química , Pirroles/farmacologíaRESUMEN
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.
Asunto(s)
Hipoxia de la Célula/efectos de los fármacos , Nitroimidazoles/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Sulfonamidas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Células HT29 , Humanos , Estructura Molecular , Nitroimidazoles/síntesis química , Nitroimidazoles/química , Fármacos Sensibilizantes a Radiaciones/síntesis química , Fármacos Sensibilizantes a Radiaciones/química , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
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.
Asunto(s)
Flavoproteínas/biosíntesis , Oxidorreductasas/metabolismo , Profármacos/química , Profármacos/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/farmacología , Células HCT116 , Células HEK293 , Células Hep G2 , Humanos , Indoles/administración & dosificación , Indoles/química , Indoles/metabolismo , Oxidación-Reducción , Oxidorreductasas/biosíntesis , Profármacos/administración & dosificación , Triazinas/química , Triazinas/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , NADPH-Ferrihemoproteína Reductasa/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neoplasias/tratamiento farmacológico , Profármacos/farmacología , Antineoplásicos/farmacocinética , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , NADPH-Ferrihemoproteína Reductasa/genética , Proteínas de Neoplasias/genética , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Profármacos/farmacocinéticaRESUMEN
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.
Asunto(s)
Antineoplásicos Alquilantes/síntesis química , Indoles/síntesis química , Neoplasias/metabolismo , Nitrocompuestos/síntesis química , Profármacos/síntesis química , Ácidos Sulfónicos/química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Hipoxia de la Célula , Línea Celular Tumoral , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indoles/química , Indoles/farmacología , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/radioterapia , Nitrocompuestos/química , Nitrocompuestos/farmacología , Profármacos/química , Profármacos/farmacología , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Bromuros/química , Bromuros/farmacología , Profármacos/química , Profármacos/farmacología , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Hipoxia de la Célula/fisiología , Femenino , Humanos , Indoles/química , Indoles/farmacología , Masculino , Ratones , Ratones Desnudos , Fosfatos/química , Fosfatos/farmacología , Pirroles/química , Pirroles/farmacología , Relación Estructura-Actividad , Neoplasias del Cuello Uterino/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Antineoplásicos Alquilantes/síntesis química , Indoles/química , Nitrocompuestos/síntesis química , Profármacos/síntesis química , Alcanosulfonatos/síntesis química , Alcanosulfonatos/química , Alcanosulfonatos/farmacología , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacología , Hipoxia de la Célula/efectos de los fármacos , ADN/química , ADN/metabolismo , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Duocarmicinas , Femenino , Humanos , Indoles/farmacología , Nitrocompuestos/química , Nitrocompuestos/farmacología , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Pirrolidinonas/química , Pirrolidinonas/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
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.
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Antineoplásicos Alquilantes , Indoles/química , Indoles/farmacología , Profármacos/síntesis química , Profármacos/farmacología , Animales , Antineoplásicos Alquilantes/síntesis química , Antineoplásicos Alquilantes/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Duocarmicinas , Humanos , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Pirrolidinonas/química , Pirrolidinonas/farmacología , Radiación Ionizante , Estereoisomerismo , Trasplante HeterólogoAsunto(s)
Aminas/química , Antineoplásicos/química , Indoles/química , Profármacos/química , Aminas/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Duocarmicinas , Humanos , Hipoxia , Indoles/farmacología , Ratones , Estructura Molecular , Pirrolidinonas/química , Pirrolidinonas/farmacologíaRESUMEN
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.