RESUMO
A series of novel artemisinin-piperazine-phosphoramide mustard (PPM) hybrids were designed and synthesized by incorporating phosphoramide mustard (PM) into dihydroartemisinin (DHA) via an efficient, catalyst-free two-step sequential substitution. Artemisinin-PPM hybrids showed better cytotoxic potency against HepG2 cells than both the parent DHA and the reference, vincristine (VCR). Structure-activity relationship (SAR) studies showed that the cytotoxicity was significantly enhanced by the introduction of a thiazole moiety. Hybrid 7 h, the most potent compound with the highest selectivity index IC50 (HEK-293T)/IC50 (HepG2)=16, displayed 7.4-fold stronger potency than VCR against HepG2 cells. In addition, hybrid 7 h was substantially more cytotoxic on all human cancer cells tested than on the corresponding non-cancerous cells. Flow cytometric analysis showed that 7 h significantly blocked the cell cycle in the G0/G1 phase and induced apoptosis in a concentration-dependent manner.
Assuntos
Antineoplásicos , Artemisininas , Antineoplásicos/farmacologia , Apoptose , Artemisininas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mostardas de Fosforamida/farmacologia , Piperazina/farmacologia , Relação Estrutura-AtividadeRESUMO
Despite considerable progress has been achieved in hypoxia-associated anti-tumor therapy, the efficacy of utilizing hypoxia-activated prodrugs alone is not satisfied owing to the inadequate hypoxia within the tumor regions. In this work, a mitochondrial targeted nanoplatform integrating photodynamic therapy, photothermal therapy and hypoxia-activated chemotherapy has been developed to synergistically treat cancer and maximize the therapeutic window. Polydopamine coated hollow copper sulfide nanoparticles were used as the photothermal nanoagents and thermosensitive drug carriers for loading the hypoxia-activated prodrug, TH302, in our study. Chlorin e6 (Ce6) and triphenyl phosphonium (TPP) were conjugated onto the surface of the nanoplatform. Under the action of TPP, the obtained nanoplatform preferentially accumulated in mitochondria to restore the drug activity and avoid drug resistance. Using 660 nm laser to excite Ce6 can generate ROS and simultaneously exacerbate the cellular hypoxia. While under the irradiation of 808 nm laser, the nanoplatform produced local heat which can increase the release of TH302 in tumor cells, ablate cancer cells as well as intensify the tumor hypoxia levels. The aggravated tumor hypoxia then significantly boosted the anti-tumor efficiency of TH302. Both in vitro and in vivo studies demonstrated the greatly improved anti-cancer activity compared to conventional hypoxia-associated chemotherapy. This work highlights the potential of using a combination of hypoxia-activated prodrugs plus phototherapy for synergistic cancer treatment.
Assuntos
Hipóxia Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/metabolismo , Nanopartículas/química , Fotoquimioterapia/métodos , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Nitroimidazóis/química , Nitroimidazóis/farmacocinética , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/química , Mostardas de Fosforamida/farmacocinética , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Distribuição TecidualRESUMO
BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive tumor with extremely high mortality that results from its lack of effective therapeutic targets. As an adhesion molecule related to tumorigenesis and tumor metastasis, cluster of differentiation-44 (also known as CD44) is overexpressed in TNBC. Moreover, CD44 can be effectively targeted by a specific hyaluronic acid analog, namely, chitosan oligosaccharide (CO). In this study, a CO-coated liposome was designed, with Photochlor (HPPH) as the 660 nm light mediated photosensitizer and evofosfamide (also known as TH302) as the hypoxia-activated prodrug. The obtained liposomes can help diagnose TNBC by fluorescence imaging and produce antitumor therapy by synergetic photodynamic therapy (PDT) and chemotherapy. RESULTS: Compared with the nontargeted liposomes, the targeted liposomes exhibited good biocompatibility and targeting capability in vitro; in vivo, the targeted liposomes exhibited much better fluorescence imaging capability. Additionally, liposomes loaded with HPPH and TH302 showed significantly better antitumor effects than the other monotherapy groups both in vitro and in vivo. CONCLUSION: The impressive synergistic antitumor effects, together with the superior fluorescence imaging capability, good biocompatibility and minor side effects confers the liposomes with potential for future translational research in the diagnosis and CD44-overexpressing cancer therapy, especially TNBC.
Assuntos
Quitosana/farmacologia , Lipossomos/química , Nitroimidazóis/farmacologia , Oligossacarídeos/farmacologia , Mostardas de Fosforamida/farmacologia , Fotoquimioterapia/métodos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Quitosana/química , Feminino , Humanos , Receptores de Hialuronatos , Ácido Hialurônico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanomedicina , Nitroimidazóis/química , Oligossacarídeos/química , Imagem Óptica , Mostardas de Fosforamida/química , Fármacos Fotossensibilizantes/química , Pró-Fármacos/química , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Rationale: Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial of non-resectable soft tissue sarcomas, TH-302 did not improve survival in combination with doxorubicin (Dox), possibly due to a lack of patient stratification based on hypoxic status. Therefore, we used magnetic resonance imaging (MRI) to identify hypoxic habitats and non-invasively follow therapies response in sarcoma mouse models. Methods: We developed deep-learning (DL) models to identify hypoxia, using multiparametric MRI and co-registered histology, and monitored response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (radiation-induced fibrosarcoma, RIF-1). Results: A DL convolutional neural network showed strong correlations (>0.76) between the true hypoxia fraction in histology and the predicted hypoxia fraction in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (p<0.05), but not in the RIF-1 model, which had a lower volume of hypoxic habitats. Control studies showed that RIF-1 resistance was due to hypoxia and not other causes. Notably, PDX tumors developed resistance to TH-302 under prolonged treatment that was not due to a reduction in hypoxic volumes. Conclusion: Artificial intelligence analysis of pre-therapy MR images can predict hypoxia and subsequent response to HAPs. This approach can be used to monitor therapy response and adapt schedules to forestall the emergence of resistance.
Assuntos
Hipóxia/tratamento farmacológico , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/farmacologia , Sarcoma/tratamento farmacológico , Animais , Inteligência Artificial , Linhagem Celular Tumoral , Aprendizado Profundo , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Ecossistema , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Neoplasias de Tecidos Moles/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Myeloma cells produce excessive levels of dickkopf-1 (DKK1), which mediates the inhibition of Wnt signaling in osteoblasts, leading to multiple myeloma (MM) bone disease. Nevertheless, the precise mechanisms underlying DKK1 overexpression in myeloma remain incompletely understood. Herein, we provide evidence that hypoxia promotes DKK1 expression in myeloma cells. Under hypoxic conditions, p38 kinase phosphorylated cAMP-responsive element-binding protein (CREB) and drove its nuclear import to activate DKK1 transcription. In addition, high levels of DKK1 were associated with the presence of focal bone lesions in patients with t(4;14) MM, overexpressing the histone methyltransferase MMSET, which was identified as a downstream target gene of hypoxia-inducible factor (HIF)-1α. Furthermore, we found that CREB could recruit MMSET, leading to the stabilization of HIF-1α protein and the increased dimethylation of histone H3 at lysine 36 on the DKK1 promoter. Knockdown of CREB in myeloma cells alleviated the suppression of osteoblastogenesis by myeloma-secreted DKK1 in vitro. Combined treatment with a CREB inhibitor and the hypoxia-activated prodrug TH-302 (evofosfamide) significantly reduced MM-induced bone destruction in vivo. Taken together, our findings reveal that hypoxia and a cytogenetic abnormality regulate DKK1 expression in myeloma cells, and provide an additional rationale for the development of therapeutic strategies that interrupt DKK1 to cure MM.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Histona-Lisina N-Metiltransferase/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mieloma Múltiplo/tratamento farmacológico , Proteínas Repressoras/genética , Cromatina/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Nitroimidazóis/farmacologia , Osteoblastos/efeitos dos fármacos , Osteólise/genética , Mostardas de Fosforamida/farmacologia , Hipóxia Tumoral/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Aims: In hypoxic tumor microenvironments, the strongly reducing redox environment reduces evofosfamide (TH-302) to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anticancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO2in vivo. Here, we use quantitative pO2 imaging by electron paramagnetic resonance (EPR) to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models: MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors that do not respond. Results: EPR imaging showed that oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the ex vivo results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in Ktrans observed in dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). Innovations: The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and antiproliferatives post evofosfamide for improved outcomes. Conclusion: This study suggests that reoxygenation after evofosfamide treatment is due to decreased oxygen demand rather than improved perfusion. Following the change in pO2 after treatment may therefore yield a way of monitoring treatment response. Antioxid. Redox Signal. 35, 904-915.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/terapia , Hipóxia Celular/efeitos dos fármacos , Nitroimidazóis/farmacologia , Neoplasias Pancreáticas/terapia , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/química , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Nitroimidazóis/química , Oxirredução , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Mostardas de Fosforamida/química , Pró-Fármacos/químicaRESUMO
PURPOSE: Hypoxia is widely known as one of the mechanisms of chemoresistance and as an environmental condition which triggers invasion and metastasis of cancer. Evofosfamide is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard conjugated with 2-nitroimidazole. Biliary tract cancer (BTC) is known to contain large hypoxic area. This study evaluated the efficacy and safety of evofosfamide as a second-line treatment of advanced BTC. MATERIALS AND METHODS: Patients received evofosfamide at a dose of 340 mg/m2 on days 1, 8, and 15 of every 28-day cycle. Primary end-point was progression-free survival (PFS) rate at 4-months (4m-PFSR). Secondary end-points included overall survival (OS), PFS, disease control rate (DCR), metabolic response by 18F-fluorodeoxyglucose positron emission tomography (PET), hypoxic parameters evaluated by 18F-fluoromisonidazole (FMISO) PET and toxicity. RESULTS: Twenty patients were treated with evofosfamide, with 16 response-evaluable patients. There was no objective response; stable disease was observed in nine patients, with a DCR of 56.25%. 4m-PFSR was 40.6%. Median PFS was 3.60 months (95% confidence interval [CI], 1.68 to 5.52). Median OS was 6.37 months (95% CI, 3.94 to 8.79). Reduction of tumor metabolic activity was observed in eight of 15 patients (53.3%). High baseline hypoxic parameters were associated with poor PFS. Change of hypoxic parameters between pretreatment and post-treatment reflected hypoxic-activated drug response. There was no treatment-related death. CONCLUSION: Evofosfamide as second-line treatment of advanced BTC showed acceptable safety and comparable efficacy to other agents. Changes in volumetric parameters measured with FMISO PET, showing the degree of tumor hypoxia, reflected the response to evofosfamide based on the mode of action.
Assuntos
Neoplasias do Sistema Biliar/tratamento farmacológico , Hipóxia Celular/efeitos dos fármacos , Misonidazol/análogos & derivados , Nitroimidazóis/uso terapêutico , Mostardas de Fosforamida/uso terapêutico , Tomografia por Emissão de Pósitrons/métodos , Neoplasias do Sistema Biliar/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Misonidazol/farmacologia , Misonidazol/uso terapêutico , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Estudos ProspectivosRESUMO
BACKGROUND: Oxazaphosphorines (cyclophosphamide (CPA), ifosfamide (IFO)) are major alkylating agents of polychemotherapy protocols but limiting their toxicity and increasing their efficacy could be of major interest. Oxazaphosphorines are prodrugs that require an activation by cytochrome P450 (CYP). CPA is mainly metabolized (>80%) to phosphoramide mustard while only 10%-50% of IFO is transformed in the alkylating entity, isophosphoramide mustard and 50%-90% of IFO release chloroacetaldehyde, a nephrotoxic and neurotoxic metabolite. Geranyloxy-IFO (G-IFO) was reported as a preactivated IFO to circumvent the toxic pathway giving directly the isophosphoramide mustard without CYP metabolization. The similarity in structure of CPA and IFO and the similarity in metabolic balance of CPA and G-IFO have led us to explore immunomodulatory effect of these components in mice and to investigate the combination of these oxazaphosphorines with immune checkpoint blockers (ICB). METHODS: The investigation of the immunomodulatory properties of IFO and G-IFO compared with CPA has been conducted through immune cell phenotyping by flow cytometry and analysis of the cytokine profile of T cells after ex-vivo restimulation. T cell-mediated antitumor efficacy was confirmed in CD4+ and CD8+ T cell-depleted mice. A combination of oxazaphosphorines with an anti-programmed cell death 1 (PD-1) antibody has been studied in MCA205 tumor-bearing mice. RESULTS: Studies on a MCA205 mouse model have demonstrated a dose-dependent effect of IFO and G-IFO on T cell immunity. These components in particular favored Th1 polarization when used at low dose (150 and eq. 100 mg/kg, respectively). Antitumor activity at low dose was abolished in mice depleted in CD4+ and CD8+ T cells. G-IFO at low dose (eq. 100 mg/kg) in combination with anti-PD-1 antidody showed high synergistic antitumor efficacy compared with IFO. CONCLUSION: Oxazaphosphorines are characterized by a dual mechanism of antitumor action; low-dose schedules should be preferred in combination with ICB, and dose escalation was found to have better utility in polychemotherapy protocols where a conventional direct cytotoxic anticancer effect is needed. G-IFO, the novel oxazaphosphorine drug, has shown a better metabolic index compared with IFO as its metabolization gives mainly the alkylating mustard as CPA (and not IFO) and a best potential in combination with ICB.
Assuntos
Antineoplásicos/uso terapêutico , Ciclofosfamida/uso terapêutico , Inibidores de Checkpoint Imunológico/uso terapêutico , Mostardas de Fosforamida/uso terapêutico , Animais , Antineoplásicos/farmacologia , Ciclofosfamida/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Mostardas de Fosforamida/farmacologiaRESUMO
The tumor microenvironment in chondrosarcoma (CHS), a chemo- and radio-resistant cancer provides unique hallmarks for developing a chondrosarcoma targeted drug-delivery system. Tumor targeting could be achieved using a quaternary ammonium function (QA) as a ligand for aggrecan, the main high negative charged proteoglycan of the extracellular matrix of CHS, and a 2-nitroimidazole as trigger that enables hypoxia-responsive drug release. In a previous work, ICF05016 was identified as efficient proteoglycan-targeting hypoxia-activated prodrug in a human extraskeletal myxoid chondrosarcoma model in mice and a first study of the structure-activity relationship of the QA function and the alkyl linker length was conducted. Here, we report the second part of the study, namely the modification of the nitro-aromatic trigger and the position of the proteoglycan-targeting ligand at the aromatic ring as well as the nature of the alkylating mustard. Synthetic approaches have been established to functionalize the 2-nitroimidazole ring at the N-1 and C-4 positions with a terminal tertiary alkyl amine, and to perform the phosphorylation step namely through the use of an amine borane complex, leading to phosphoramide and isophosphoramide mustards and also to a phosphoramide mustard bearing four 2-chloroethyl chains. In a preliminary study using a reductive chemical activation, QA-conjugates, except the 4-nitrobenzyl one, were showed to undergo efficient cleavage with release of the corresponding mustard. However N,N,N-trimethylpropylaminium tethered to the N-1 or C-4 positions of the imidazole seemed to hamper the enzymatic reduction of the prodrugs and all tested compounds featured moderate selectivity toward hypoxic cells, likely not sufficient for application as hypoxia-activated prodrugs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Condrossarcoma/tratamento farmacológico , Desenho de Fármacos , Neoplasias de Tecido Conjuntivo e de Tecidos Moles/tratamento farmacológico , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Condrossarcoma/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Neoplasias de Tecido Conjuntivo e de Tecidos Moles/patologia , Mostardas de Fosforamida/síntese química , Mostardas de Fosforamida/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: More than half of patients with soft tissue sarcoma (STS) are aged ≥65â¯years (older), however contemporary data on the efficacy/safety of anthracycline chemotherapy in older patients with STS are lacking. METHODS: SARC021 randomized patients to receive first-line doxorubicin or doxorubicin plus evofosfamide. The main aim of this study was to compare the outcome and safety of first-line anthracycline-based therapy in older patients compared with those <65â¯years. IRB approval was obtained at all participating sites and this research meets requirements for protection of human subjects. RESULTS: Of 640 patients, 209 (33%) were older, with a median age 70 (range 65-89) years. The median overall survival (OS) was 16.7â¯months (95%CI: 13.2-20.0) in older patients compared to 20.1â¯months (95%CI: 16.9-23.2) in those aged <65â¯years (nâ¯=â¯431), HR 1.21 (95%CI: 0.99-1.48), pâ¯=â¯.057. The median progression-free survival (PFS) in older patients was 6.3â¯months (95%CI: 5.8-7.2) compared to 6.0 (95%CI: 5.1-6.4) in those <65â¯years, HR 0.86 (95%CI: 0.70-1.05), pâ¯=â¯.14. Older patients had significantly more hematological (141 [67%] versus 208 [48%], pâ¯<â¯.0001), non-hematological (131 [63%] versus 215 [50%], pâ¯=â¯.0097) andâ¯≥â¯Grade 3 adverse events (178 [85%] versus 299 [69%], pâ¯=â¯.0002), compared to younger patients. More older patients (30, 14%) stopped treatment due to adverse events compared to younger patients (22, 5%), pâ¯=â¯.0001. CONCLUSIONS: The efficacy of first-line anthracycline-based chemotherapy did not differ significantly between older and younger advanced sarcoma patients. Significantly more older patients stopped chemotherapy due to adverse events. These results provide a benchmark for daily clinical practice and future trials in older patients.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , HumanosRESUMO
Ataxia-telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm+/- mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm+/- mice spent ~25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm+/- mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm+/- mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm+/- than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm+/- mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γH2AX, but fewer γH2AX-positive foci were identified in Atm+/- ovaries. Similarly, cleaved caspase-3 was lower in the Atm+/- PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA/fisiologia , Ovário/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Camundongos , Camundongos Knockout , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Mostardas de Fosforamida/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Esophageal cancer incidence is increasing and is rarely curable. Hypoxic tumor areas cause resistance to conventional therapies, making them susceptible for treatment with hypoxia-activated prodrugs (HAPs). We investigated in vivo whether the HAP evofosfamide (TH-302) could increase the therapeutic ratio by sensitizing esophageal carcinomas to radiotherapy without increasing normal tissue toxicity. MATERIALS AND METHODS: To assess therapeutic efficacy, growth of xenografted esophageal squamous cell (OE21) or adeno (OE19) carcinomas was monitored after treatment with TH-302 (50â¯mg/kg, QD5) and irradiation (sham or 10â¯Gy). Short- and long-term toxicity was assessed in a gut mucosa and lung fibrosis irradiation model, sensitive to acute and late radiation injury respectively. Mice were injected with TH-302 (50â¯mg/kg, QD5) and the abdominal area (sham, 8 or 10â¯Gy) or the upper part of the right lung (sham, 20â¯Gy) was irradiated. Damage to normal tissues was assessed 84 hours later by histology and blood plasma citrulline levels (gut) and for up to 1â¯year by non-invasive micro CT imaging (lung). RESULTS: The combination treatment of TH-302 with radiotherapy resulted in significant tumor growth delay in OE19 (Pâ¯=â¯0.02) and OE21 (Pâ¯=â¯0.03) carcinomas, compared to radiotherapy only. Irradiation resulted in a dose-dependent decrease of crypt survival (Pâ¯<â¯0.001), mucosal surface area (Pâ¯<â¯0.01) and citrulline levels (Pâ¯<â¯0.001) in both tumor and non-tumor bearing animals. On the long-term, irradiation increased CT density in the lung, indicating fibrosis, over time. TH-302 did not influence the radiation-induced short-term and long-term toxicity, confirmed by histological evaluation. CONCLUSION: The combination of TH-302 and radiotherapy might be a promising approach to improve the therapeutic index for esophageal cancer patients.
Assuntos
Adenocarcinoma/radioterapia , Neoplasias Esofágicas/radioterapia , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Radiossensibilizantes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Nitroimidazóis/efeitos adversos , Mostardas de Fosforamida/efeitos adversosRESUMO
Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7-7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd × 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes-MKI67, POR, and SLFN11-did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Hipóxia Tumoral/efeitos dos fármacos , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Proteínas Nucleares/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.
Assuntos
Transporte de Elétrons/efeitos dos fármacos , Mitocôndrias/genética , Neoplasias/genética , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Análise de Sequência de RNA/métodos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HCT116 , Humanos , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Pró-Fármacos , RNA Interferente Pequeno/farmacologiaRESUMO
Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Tumores Neuroendócrinos/genética , Neoplasias da Próstata/genética , Proteína Smad3/genética , Fatores de Transcrição/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Conjuntos de Dados como Assunto , Progressão da Doença , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Tumores Neuroendócrinos/patologia , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Próstata/patologia , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor hypoxia contributes to resistance to anticancer therapies. Hypoxia-activated prodrugs (HAPs) selectively target hypoxic cells and their activity can extend to well-oxygenated areas of tumors via diffusion of active metabolites. This type of bystander effect has been suggested to be responsible for the single agent activity of the clinical-stage HAP evofosfamide (TH-302) but direct evidence is lacking. To dissect the contribution of bystander effects to TH-302 activity, we implemented a Green's function pharmacokinetic (PK) model to simulate the spatial distribution of O2, TH-302 and its cytotoxic metabolites, bromo-isophosphoramide mustard (Br-IPM) and its dichloro derivative isophosphoramide mustard (IPM), in two digitized tumor microvascular networks. The model was parameterized from literature and experimentally, including measurement of diffusion coefficients of TH-302 and its metabolites in multicellular layer cultures. The latter studies demonstrate that Br-IPM and IPM cannot diffuse significantly from the cells in which they are generated, although evidence was obtained for diffusion of the hydroxylamine metabolite of TH-302. The spatially resolved PK model was linked to a pharmacodynamic (PD) model that describes cell killing probability at each point in the tumor microregion as a function of Br-IPM and IPM exposure. The resulting PK/PD model accurately predicted previously reported monotherapy activity of TH-302 in H460 tumors, without invoking a bystander effect, demonstrating that the notable single agent activity of TH-302 in tumors can be accounted for by significant bioreductive activation of TH-302 even in oxic regions, driven by the high plasma concentrations achievable with this well-tolerated prodrug.
Assuntos
Efeito Espectador , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Pró-Fármacos , Algoritmos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Transporte Biológico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Hidroxilamina/administração & dosagem , Hidroxilamina/farmacocinética , Hidroxilamina/farmacologia , Hipóxia/metabolismo , Modelos Biológicos , Nitroimidazóis/administração & dosagem , Nitroimidazóis/farmacocinética , Mostardas de Fosforamida/administração & dosagem , Mostardas de Fosforamida/farmacocinética , Espectrometria de Massas em TandemRESUMO
Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.
Assuntos
Adenocarcinoma/terapia , Imunoterapia , Neoplasias Experimentais/terapia , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Neoplasias da Próstata/terapia , Linfócitos T/imunologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Hipóxia Celular/genética , Hipóxia Celular/imunologia , Linhagem Celular Tumoral , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Linfócitos T/patologiaRESUMO
Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line-derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias de Cabeça e Pescoço/terapia , Nitroimidazóis/uso terapêutico , Mostardas de Fosforamida/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Adulto , Idoso , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Concentração Inibidora 50 , Pessoa de Meia-Idade , Nitroimidazóis/farmacologia , Papillomaviridae/isolamento & purificação , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/administração & dosagem , Intervalo Livre de Progressão , Critérios de Avaliação de Resposta em Tumores Sólidos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Efeito Espectador , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/farmacologia , Proteínas de Escherichia coli , Células HCT116 , Humanos , Estrutura Molecular , NADPH-Ferri-Hemoproteína Redutase , Nitroimidazóis/química , Nitrorredutases , Mostardas de Fosforamida/química , Pró-Fármacos/farmacologiaRESUMO
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.