RESUMO
The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor-positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive, triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative, as well as estrogen receptor-positive, breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells, suggesting its mechanism of action differs from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds, as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.
Assuntos
Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Carbocianinas , Citotoxinas , Indóis , Piperazinas , Piridinas , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Células CHO , Carbocianinas/química , Carbocianinas/farmacologia , Cricetulus , Citotoxinas/química , Citotoxinas/farmacologia , Feminino , Células HEK293 , Humanos , Indóis/química , Indóis/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Piridinas/química , Piridinas/farmacologiaRESUMO
This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.
Assuntos
alfa-Globulinas/química , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Haplorrinos , Humanos , Imunoconjugados/química , Camundongos , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.
Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Indóis/farmacologia , Anticorpos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Duocarmicinas , Humanos , Indóis/química , Estrutura Molecular , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antibióticos Antineoplásicos/química , Ciclopropanos/química , Ciclopropanos/toxicidade , Indóis/química , Indóis/farmacologia , Indóis/toxicidade , Animais , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/toxicidade , Cricetinae , Cricetulus , Ciclopropanos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Duocarmicinas , Humanos , Concentração de Íons de Hidrogênio , Indóis/síntese química , Cinética , Camundongos , Prótons , Pirróis/farmacologiaRESUMO
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.
Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Camundongos , Biomarcadores Tumorais/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relevância Clínica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Nitroimidazóis/farmacologia , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Hipóxia TumoralRESUMO
A series of cobalt(III) complexes of the potent DNA minor groove alkylator (1-(chloromethyl)-5-hydroxy-1H-pyrrolo[3,2-f]quinolin-3(2H)-yl)(5,6,7-trimethoxy-1H-indol-2-yl)methanone (3; seco-CPyI-TMI), with cyclam or cyclen auxiliary ligands (L3 and L5) containing a cross-bridging ethylene (CH2CH2) group or the N,N'-dimethyl derivatives of these (L4 and L6), was prepared. Two 8-quinolinato (2) model complexes of these, [Co(L3)(2)](ClO4)2 and [Co(L6)(2)](ClO4)2, and the aquated derivative [Co(L6)(H2O)2](OTf)3 were characterized by X-ray crystallography. Electrochemistry of the 8-quinolinato model complexes showed that the Co(III)/(II) reduction potential was lowered relative to the unsubstituted cyclen ligand. Evaluation of the cytotoxicity of the racemic seco-CPyI cobalt complexes in vitro showed considerable attenuation of their cytotoxicity relative to the free alkylator and marked hypoxic selectivity, especially [Co(L3)(3)](2+) (9), which was 81-212-fold more potent under hypoxia than 20% oxygen in a panel of 10 human tumor cell lines. However, 9 did not elicit significant killing of hypoxic cells in HT29 tumor xenografts, suggesting possible pharmacological limitations in vivo.
Assuntos
Antineoplásicos/química , Cobalto/química , Complexos de Coordenação/química , Compostos Heterocíclicos/química , Pró-Fármacos/química , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Hipóxia Celular , Linhagem Celular Tumoral , Cobalto/farmacologia , Cobalto/uso terapêutico , Complexos de Coordenação/farmacologia , Complexos de Coordenação/uso terapêutico , Cristalografia por Raios X , Ciclamos , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Ligantes , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêuticoRESUMO
Quaternary salt analogues based on the DNA minor groove binder and adenine N3 alkylating agent 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (aminoCBI) show remarkable effects on the body weight of mice (a long-term failure to gain weight relative to matched controls with no loss of appetite or perceptible deterioration in health) following administration of a single (non-toxic) dose between about 0.5-5 µmol/kg. The nature of the quaternizing group was not important, but a related hydroxyCBI analogue was much less effective. Compounds where the chloro group was replaced by a hydrogen or hydroxy group (thus abrogating DNA alkylating capability) showed no weight control activity. It is speculated, based on other studies, that the marked long-term weight control effect is due to inhibition of bile flow into the intestine and reduced absorption of triglycerides, together with accelerated cell death in spleen and white adipose tissues due to drug accumulation there. This class of compound may serve as interesting tools for further study of these phenomena.
Assuntos
Indóis/química , Sais/química , Redução de Peso/efeitos dos fármacos , Animais , Ciclopropanos/química , Indóis/síntese química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antineoplásicos Alquilantes/síntese química , Indóis/química , Nitrocompostos/síntese química , Pró-Fármacos/síntese química , Alcanossulfonatos/síntese química , Alcanossulfonatos/química , Alcanossulfonatos/farmacologia , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacologia , Hipóxia Celular/efeitos dos fármacos , DNA/química , DNA/metabolismo , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Duocarmicinas , Feminino , Humanos , Indóis/farmacologia , Nitrocompostos/química , Nitrocompostos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.
Assuntos
Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Brometos/química , Brometos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Hipóxia Celular/fisiologia , Feminino , Humanos , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Nus , Fosfatos/química , Fosfatos/farmacologia , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Neoplasias do Colo do Útero/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of cobalt complexes of the potent DNA minor groove alkylator 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (seco-6-azaCBI-TMI) were prepared from a series of N-substituted cyclen ligands. The final N-substituted complexes carried formal overall charges ranging from +2 to -2 and showed limited improvements in solubility. They showed similar stabilities to that of the complex with the unsubstituted cyclen ligand, and large but variable attenuation of the cytotoxicity of the free alkylator (2-30-fold), compared to 150-fold for the unsubstituted ligand. However, they had oxic/hypoxic ratios (2-22-fold) comparable to that of the unsubstituted cyclen complex (5).
Assuntos
Antineoplásicos Alquilantes/síntese química , Pró-Fármacos/síntese química , Alquilação , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Compostos Azo/química , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cobalto/química , Ciclamos , DNA/química , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Compostos Heterocíclicos/química , Humanos , Hipóxia/metabolismo , Indóis/química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Solubilidade , Oligoelementos/químicaRESUMO
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.
RESUMO
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.
Assuntos
Efeito Espectador/efeitos dos fármacos , Hipóxia Celular , Indóis/farmacologia , Modelos Biológicos , Cromatografia Líquida , Técnicas de Cocultura , Proteínas de Escherichia coli/genética , Células HCT116 , Humanos , Indóis/farmacocinética , NADPH-Ferri-Hemoproteína Redutase/genética , Nitrorredutases/genética , Pró-Fármacos , Esferoides Celulares/citologia , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Hipóxia Celular , Ciclopropanos/química , Ciclopropanos/metabolismo , Indóis/química , Indóis/metabolismo , Neoplasias/tratamento farmacológico , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Animais , Antineoplásicos Alquilantes/farmacocinética , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , DNA/metabolismo , Humanos , Indóis/farmacocinética , Indóis/farmacologia , Camundongos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , SolubilidadeRESUMO
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.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Indóis/química , Indóis/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Ovário/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada , Ensaios de Seleção de Medicamentos Antitumorais , Duocarmicinas , Feminino , Humanos , Indóis/farmacologia , Camundongos , Camundongos Nus , Nitrocompostos/química , Nitrocompostos/farmacologia , Nitrocompostos/uso terapêutico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/radioterapia , Ovário/patologia , Ovário/efeitos da radiação , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Pirrolidinonas/uso terapêuticoRESUMO
Metabolic reduction can be used to activate prodrugs in hypoxic regions of tumours, but reduction by ionising radiation is also theoretically attractive. Previously, we showed that a cobalt(III) complex containing 8-hydroxyquinoline (8-HQ) and cyclen ligands releases 8-HQ efficiently on irradiation in hypoxic solutions [Ahn G-O, Ware DC, Denny WA, Wilson WR. Optimization of the auxiliary ligand shell of cobalt(III)(8-hydroxyquinoline) complexes as model hypoxia-selective radiation-activated prodrugs. Radiat Res 2004;162:315-25]. Here we investigate an analogous Co(III) complex containing the potent DNA minor groove alkylator azachloromethylbenzindoline (azaCBI, 1) to determine whether it releases 1 on radiolytic and/or enzymatic reduction under hypoxia. Monitoring by HPLC, the azaCBI ligand in the Co(III)(cyclen)(azaCBI) complex (2) slowly hydrolysed in aqueous solution, in contrast to the free ligand 1 which readily converted to its reactive cyclopropyl form. Irradiation of 2 (30-50 microM) in hypoxic solutions released 1 with yields of 0.57 micromol/J in formate buffer and 0.13 micromol/J in human plasma. Using bioassay methods, cytotoxic activation by irradiation of 2 at 1 microM in hypoxic plasma was readily detectable at clinically relevant doses (> or = 1 Gy), with a estimated yield of 1 of 0.075 micromol/J. Release of 1 from 2 was also observed in hypoxic HT29 cultures without radiation, with subsequent conversion of 1 to its O-glucuronide. Surprisingly, overexpression of human cytochrome P450 reductase in A549 cells did not increase the rate of metabolic reduction of 2, suggesting that other reductases and/or non-enzymatic reductants are responsible. Thus the cobalt(III) complex 2 is a promising prodrug capable of being activated to release a very potent cytotoxin when reduced by either ionising radiation or cells under hypoxic conditions.
Assuntos
Alquilantes/farmacologia , Compostos Azo/farmacologia , Hipóxia Celular , Cobalto/química , Indóis/farmacologia , Pró-Fármacos/química , Alquilantes/farmacocinética , Compostos Azo/farmacocinética , Biotransformação , Cromatografia Líquida de Alta Pressão , Células HT29 , Humanos , Indóis/farmacocinética , Espectrometria de Massas , Radiação IonizanteAssuntos
Aminas/química , Antineoplásicos/química , Indóis/química , Pró-Fármacos/química , Aminas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Duocarmicinas , Humanos , Hipóxia , Indóis/farmacologia , Camundongos , Estrutura Molecular , Pirrolidinonas/química , Pirrolidinonas/farmacologiaRESUMO
A set of 10 compounds, each combining the seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (seco-CBI) and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores, was designed and prepared. These compounds were anticipated to cross-link between N3 of adenine and N2 of guanine in the minor groove of DNA. The compounds, which differ in the chain length separating the two alkylation subunits, and the configuration of the CBI portion, showed great variation in cellular toxicity (over 4 orders of magnitude in a cell line panel) with the most potent example exhibiting IC50s in the pM range. Cytotoxicity correlated with the ability of the compounds to cross-link naked DNA. Cross-linking was also observed in living cells, at much lower concentrations than for a related symmetrical PBD dimer. A thermal cleavage assay was used to assess sequence selectivity, demonstrating that the CBI portion controlled the alkylation sites, while the PBD substituent increased the overall efficiency of alkylation. Several compounds were tested for in vivo activity using a tumor growth delay assay against WiDr human colon carcinoma xenografts, with one compound (the most cytotoxic and most efficient cross-linker) showing a statistically significant increase in survival time following a single iv dose.
Assuntos
Antineoplásicos Alquilantes/síntese química , Benzodiazepinas/química , Benzodiazepinonas/síntese química , Reagentes de Ligações Cruzadas/síntese química , Ciclopropanos/química , DNA de Neoplasias/química , Indóis/química , Pirróis/química , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Benzodiazepinonas/química , Benzodiazepinonas/farmacologia , Ensaio Cometa , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Nitroarylmethyl quaternary (NMQ) ammonium salts have potential as prodrugs for enzymatic or radiolytic reduction to release amine effectors under hypoxia. Earlier studies demonstrated one-electron release of the cytotoxic amine mechlorethamine (HN2) from 4-nitroimidazolyl and 2-nitropyrrolyl NMQ prodrugs (but not from nitrobenzyl analogs) through intramolecular electron transfer. In this study we determined whether this is a general feature of heterocyclic NMQ prodrugs of HN2 and examined the reductive pathways in detail using pulse and steady-state radiolysis. The kinetics of radical fragmentation varied by more than four orders of magnitude, independently of the one-electron reduction potential, within the series of eight nitroheterocycles examined. In addition to the compounds identified previously, new 2-nitropyrrole and 3-nitrothiophene NMQ prodrugs were found to provide efficient HN2 release (G > 0.5 micromol/J in anoxic formate buffer). However, the nitrothiophene was sensitive to nucleophilic displacement of HN2, making it less promising. Product analysis by HPLC/mass spectrometry identified symmetrical dimers arising from benzyl-type radical intermediates but also demonstrated that these dimers are not reliable markers for the intramolecular fragmentation of the initial nitro radical anion. This study elucidated multiple competing pathways for reductive fragmentation of NMQ prodrugs and identified the preferred electron acceptors for use in the development of analogs that release more potent cytotoxins.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Mecloretamina/farmacologia , Nitrocompostos/farmacologia , Pró-Fármacos/farmacologia , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta à Radiação , Elétrons , Cinética , Modelos Químicos , Nitrocompostos/química , Fatores de TempoRESUMO
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.