RESUMO
Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.
Assuntos
Proteínas de Membrana , Neoplasias , Animais , Citocinas , Humanos , Imunoterapia/métodos , Interferons , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile.
Assuntos
Imunidade Inata/imunologia , Imunoterapia/métodos , Interferons/metabolismo , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , CamundongosRESUMO
Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.
Assuntos
Antígeno B7-H1 , Imunoterapia , Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Antígeno B7-H1/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente TumoralRESUMO
Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Membrana/metabolismo , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , HumanosRESUMO
BACKGROUND: Constitutive activation of ERK1/2 occurs in various cancers, and its reactivation is a well-described resistance mechanism to MAPK inhibitors. ERK inhibitors may overcome the limitations of MAPK inhibitor blockade. The dual mechanism inhibitor SCH772984 has shown promising preclinical activity across various BRAFV600/RAS-mutant cancer cell lines and human cancer xenografts. METHODS: We have developed an orally bioavailable ERK inhibitor, MK-8353; conducted preclinical studies to demonstrate activity, pharmacodynamic endpoints, dosing, and schedule; completed a study in healthy volunteers (P07652); and subsequently performed a phase I clinical trial in patients with advanced solid tumors (MK-8353-001). In the P07652 study, MK-8353 was administered as a single dose in 10- to 400-mg dose cohorts, whereas in the MK-8353-001 study, MK-8353 was administered in 100- to 800-mg dose cohorts orally twice daily. Safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity were analyzed. RESULTS: MK-8353 exhibited comparable potency with SCH772984 across various preclinical cancer models. Forty-eight patients were enrolled in the P07652 study, and twenty-six patients were enrolled in the MK-8353-001 study. Adverse events included diarrhea (44%), fatigue (40%), nausea (32%), and rash (28%). Dose-limiting toxicity was observed in the 400-mg and 800-mg dose cohorts. Sufficient exposure to MK-8353 was noted that correlated with biological activity in preclinical data. Three of fifteen patients evaluable for treatment response in the MK-8353-001 study had partial response, all with BRAFV600-mutant melanomas. CONCLUSION: MK-8353 was well tolerated up to 400 mg twice daily and exhibited antitumor activity in patients with BRAFV600-mutant melanoma. However, antitumor activity was not particularly correlated with pharmacodynamic parameters. TRIAL REGISTRATION: ClinicalTrials.gov NCT01358331. FUNDING: Merck Sharp & Dohme Corp., a subsidiary of Merck & Co. Inc., and NIH (P01 CA168585 and R35 CA197633).
Assuntos
Indazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirrolidinas/farmacologia , Triazóis/farmacologia , Administração Oral , Adulto , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral , Diarreia/induzido quimicamente , Diarreia/epidemiologia , Cães , Relação Dose-Resposta a Droga , Toxidermias/epidemiologia , Toxidermias/etiologia , Avaliação Pré-Clínica de Medicamentos , Fadiga/induzido quimicamente , Fadiga/epidemiologia , Feminino , Humanos , Indazóis/uso terapêutico , Masculino , Dose Máxima Tolerável , Camundongos , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Náusea/induzido quimicamente , Náusea/epidemiologia , Estadiamento de Neoplasias , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Pirrolidinas/uso terapêutico , Ratos , Triazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Compostos de Piridínio/farmacologia , Proteína bcl-X/metabolismo , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Óxidos N-Cíclicos , Modelos Animais de Doenças , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Dosagem de Genes , Humanos , Indolizinas , Masculino , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias/genética , Fenantrenos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genéticaRESUMO
Lonafarnib is a potent, selective farnesyltransferase inhibitor (FTI) undergoing clinical studies for the treatment of solid tumors and hematological malignancies. Preclinically, a number of FTIs, including lonafarnib, interact with taxanes to inhibit cancer cell growth in an additive/synergistic manner. These observations provided rationale for investigating the effects of combining lonafarnib and docetaxel on preclinical prostate cancer models. To date, docetaxel is the only chemotherapeutic agent in clinical use for hormone-refractory prostate cancer. In vitro experiments with 22Rv1, LNCaP, DU-145, PC3 and PC3-M prostate cancer cell lines showed significantly enhanced inhibition of cell proliferation and apoptosis when lonafarnib was added to docetaxel. In human tumor xenograft models, continuous coadministration of lonafarnib with docetaxel caused marked tumor regressions (24-47%) in tumors from all of the cell types as well as parental CWR22 xenografts. Intermittent dosing of lonafarnib (5 days on then 5 days off) coadministered with docetaxel produced similar regressions in hormone-refractory 22Rv1 tumors. 22Rv1 tumors progressing on docetaxel treatment also responded to treatment with intermittent lonafarnib (5 days on then 5 days off). Moreover, animals did not exhibit any signs of toxicity during coadministration of lonafarnib and docetaxel. In conclusion, coadministration of continuous and intermittent lonafarnib enhanced the antitumor activity of docetaxel in a panel of prostate cancer models. An intermittent dosing schedule of lonafarnib coadministered with docetaxel may allow enhanced efficacy to that of continuous dosing by improving the tolerability of higher doses of lonafarnib.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Piperidinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Piridinas/uso terapêutico , Taxoides/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Hormônio-Dependentes/sangue , Neoplasias Hormônio-Dependentes/patologia , Piperidinas/sangue , Piperidinas/farmacocinética , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Piridinas/sangue , Piridinas/farmacocinéticaRESUMO
Ultra-performance hydrophilic interaction liquid chromatography (UPHILIC) interfaced with the electrospray ionization (ESI) source of a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of everolimus in mouse plasma samples. UPHILIC was performed on a sub-2 microm bare silica particle packing with the column pressure under traditional high-performance liquid chromatography (HPLC) to allow fast separation of pharmaceutical compounds within a chromatographic analysis time of 1 min. This UPHILIC technology is comparable with reversed-phase ultra-performance liquid chromatography (RPUPLC) in terms of chromatographic efficiency but demands neither expensive ultra-high-pressure instrumentation nor new laboratory protocols. With the ESI source, multiple reaction monitoring (MRM) of the ammoniated adduct ions of the analyte was used for tandem mass spectrometric detection. The retention mechanism profiles of the test compounds under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of the test compounds in positive ion mode were investigated. A UPHILIC/MS/MS approach following a protein precipitation procedure was applied for the quantitative determination of everolimus at the low ng/mL region in support of a pharmacodynamic study. The analytical results obtained by the UPHILIC/MS/MS approach were fond to be in good agreement with those obtained by the RPUPLC/MS/MS method in terms of assay sample throughput, sensitivity and accuracy.
Assuntos
Cromatografia Líquida/métodos , Sirolimo/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Animais , Everolimo , Camundongos , Sensibilidade e Especificidade , Sirolimo/sangueRESUMO
OBJECTIVES: To determine the effects of combining lonafarnib with paclitaxel on the growth of human ovarian cancer cells and tumor xenografts as well as to monitor a pharmacodynamic marker of farnesyltransferase inhibition (HDJ-2) in peripheral blood mononuclear cells (PBMCs) isolated from tumor-bearing animals after treatment with this combination. METHODS: Proliferation of A2780, PA-1, IGROV-1, and TOV-112D cells was assessed after treatment with lonafarnib and paclitaxel. Cell cycle progression was determined by flow cytometry, and apoptosis was evaluated by assaying for caspase-3 and cleaved PARP. The effects of lonafarnib and paclitaxel on the tumor growth of each model were determined in immunocompromised mice. Proteins extracted from cells, tumors, and PBMCs were assayed for HDJ-2 mobility shifts by Western blotting as well as for farnesyl protein transferase (FTase) enzyme activity by biochemical analyses. RESULTS: In A2780, PA-1, IGROV-1, and TOV-112D cells lonafarnib potentiated the growth inhibitory effects of paclitaxel. In each of the models lonafarnib enhanced paclitaxel-induced mitotic arrest and apoptosis. The combination of lonafarnib plus paclitaxel resulted in marked tumor regressions in A2780, TOV-112D, PA-1, and IGROV-1 tumor xenografts. Western blotting demonstrated that in PBMCs isolated from the animals, paclitaxel treatment suppressed lonafarnib-induced HDJ-2 mobility shifts. Paclitaxel did not affect lonafarnib inhibition of FTase enzyme activity levels in these PBMCs. CONCLUSIONS: Lonafarnib enhances the antiproliferative effects of paclitaxel on ovarian cancer cells in vitro and ovarian tumor xenografts in vivo. Measuring FTase enzyme activity levels rather than HDJ-2 shifts in PBMCs may be a more accurate biomarker to predict levels of farnesyltransferase inhibition in patients who are also receiving paclitaxel chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Piperidinas/farmacologia , Piridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/sangue , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Farnesiltranstransferase/sangue , Farnesiltranstransferase/metabolismo , Feminino , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Leucócitos Mononucleares/enzimologia , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Piperidinas/administração & dosagem , Piridinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lonafarnib is an orally bioavailable farnesyltransferase inhibitor. Originally developed to block the membrane localization of Ras, subsequent work suggested that farnesyltransferase inhibitors mediate their antitumor activities by altering the biological activities of additional farnesylated proteins. Breast tumor models that express wild-type Ras have been shown to be sensitive to farnesyltransferase inhibitors. We have determined the effects of combining lonafarnib with the antiestrogen 4-hydroxy tamoxifen on hormone-dependent breast cancer cell lines in vitro. The effects of combining lonafarnib with tamoxifen or the aromatase inhibitor anastrozole on the growth of two different MCF-7 breast tumor xenograft models were also evaluated. In four of five human breast cancer cell lines, lonafarnib enhanced the antiproliferative effects of 4-hydroxy tamoxifen. The combination prevented MCF-7 cells from transitioning through the G1 to S phase of the cell cycle and augmented apoptosis. This was associated with reduced expression of E2F-1 and a reduction in hyperphosphorylated retinoblastoma protein. Lonafarnib plus 4-hydroxy tamoxifen also inhibited the mammalian target of rapamycin signal transduction pathway. In nude mice bearing parental MCF-7 or aromatase-transfected MCF-7Ca breast tumor xenografts, lonafarnib enhanced the antitumor activity of both tamoxifen and anastrozole. These studies indicate that lonafarnib enhances the efficacy of endocrine agents clinically used for treating hormone-dependent breast cancer.
Assuntos
Antineoplásicos Hormonais/farmacologia , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Nitrilas/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Tamoxifeno/farmacologia , Triazóis/farmacologia , Anastrozol , Animais , Apoptose/efeitos dos fármacos , Inibidores da Aromatase/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Fase G1/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ovariectomia , Fase S/efeitos dos fármacosRESUMO
Lonafarnib (SCH66336) is a farnesyl transferase inhibitor (FTI) that inhibits the post-translational lipid modification of H-Ras and other farnesylated proteins. K- and N-Ras are also substrates of farnesyl transferase; however, upon treatment with FTIs, they are alternatively prenylated by geranylgeranyl transferase-1. Despite the failure to abrogate prenylation of K- and N-Ras, growth of many tumors in preclinical models is inhibited by FTIs. This suggests that the anti-proliferative action of FTIs is dependent on blocking the farnesylation of other proteins. Rheb (Ras homologue enriched in brain) is a farnesylated small GTPase that positively regulates mTOR (mammalian target of rapamycin) signaling. We found that Rheb and Rheb2 mRNA were elevated in various tumor cell lines relative to normal cells. Peptides derived from the carboxyl termini of human Rheb and Rheb2 are in vitro substrates for farnesyl transferase but not geranylgeranyl transferase-1. Rheb prenylation in cell culture was completely inhibited by SCH66336, indicating a lack of alternative prenylation. SCH66336 treatment also inhibited the phosphorylation of S6 ribosomal protein, a downstream target of Rheb and mTOR signaling. SCH66336 did not inhibit S6 phosphorylation in cells expressing Rheb-CSVL, a mutant construct of Rheb designed to be geranylgeranylated. Importantly, expression of Rheb-CSVL also abrogated SCH66336 enhancement of tamoxifen- and docetaxel-induced apoptosis in MCF-7 breast cancer cells and ES-2 ovarian cancer cells, respectively. Further, inhibition of Rheb signaling by rapamycin treatment, small interfering RNA, or dominant negative Rheb enhanced tamoxifen- and docetaxel-induced apoptosis, similar to FTI treatment. These studies demonstrated that Rheb is modified by farnesylation, is not a substrate for alternative prenylation, and plays a role in SCH66336 enhancement of the anti-tumor response to other chemotherapeutics.
Assuntos
Alquil e Aril Transferases , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neoplasias/tratamento farmacológico , Neuropeptídeos/metabolismo , Piperidinas/metabolismo , Proteínas Quinases/metabolismo , Piridinas/metabolismo , Tamoxifeno/uso terapêutico , Taxoides/uso terapêutico , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Antineoplásicos/uso terapêutico , Caspases/metabolismo , Linhagem Celular Tumoral , Farnesiltranstransferase , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Neuropeptídeos/genética , Fosforilação , Proteínas Quinases/genética , Prenilação de Proteína , RNA Mensageiro/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TORRESUMO
Ovariectomized mice bearing tumor xenografts grown from aromatase-transfected estrogen receptor (ER)-positive human breast cancer cells (MCF-7Ca) were injected s.c. with 10 microg/d letrozole for up to 56 weeks. Western blot analysis of the tumors revealed that ERs (ERalpha) were increased at 4 weeks but decreased at weeks 28 and 56. Expression of erbB-2 and p-Shc increased throughout treatment, whereas growth factor receptor binding protein 2 (Grb2) increased only in tumors proliferating on letrozole (weeks 28 and 56). In cells isolated from tumors after 56 weeks and maintained as a cell line (LTLT-Ca) in 1 micromol/L letrozole, ERalpha was also decreased whereas erbB-2, adapter proteins (p-Shc and Grb2), and the signaling proteins in the mitogen-activated protein kinase (MAPK) cascade were increased compared with MCF-7Ca cells. Growth was inhibited in LTLT-Ca cells but not in MCF-7Ca cells treated with MAPK kinase 1/2 inhibitors U0126, and PD98059 (IC(50) approximately 25 micromol/L). PD98059 (5 micromol/L) also reduced MAPK activity and increased ERalpha to the levels in MCF-7Ca cells. Epidermal growth factor receptor kinase inhibitor, gefitinib (ZD1839) inhibited growth of LTLT-Ca cells (IC(50) approximately 10 micromol/L) and restored their sensitivity to tamoxifen and anastrozole. In xenografts, combined treatment with ER down-regulator fulvestrant and letrozole, prevented increases in erbB-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results indicate that blocking both ER- and growth factor-mediated transcription resulted in the most effective inhibition of growth of ER-positive breast cancer cells.
Assuntos
Inibidores da Aromatase/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Triazóis/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores da Aromatase/administração & dosagem , Linhagem Celular Tumoral , Esquema de Medicação , Ativação Enzimática/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Letrozol , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/administração & dosagem , Tamoxifeno/administração & dosagem , Triazóis/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To optimize treatment strategies for postmenopausal breast cancer patients, we investigated the efficacy of the steroidal aromatase inhibitor exemestane alone or in combination with the antiestrogen tamoxifen in a xenograft model of postmenopausal breast cancer. We also determined the effects of these agents in sequential second-line therapy and the effect of the nonsteroidal aromatase inhibitor letrozole on tumors that progressed on the above treatments. EXPERIMENTAL: Aromatase-transfected human estrogen receptor-positive breast cancer cells (MCF-7Ca) were grown as tumors in ovariectomized athymic mice. Animals received subcutaneous injection with vehicle, tamoxifen, exemestane, tamoxifen plus exemestane, and letrozole. Tumor volumes were measured weekly. RESULTS: All treatments were effective initially in suppressing tumor growth as first-line therapy compared with vehicle treatment. Exemestane suppressed tumor growth to a greater extent than tamoxifen. However, the combination of tamoxifen plus exemestane was more effective than either drug alone. After tumor volumes doubled on initial treatment, the mice were crossed over to receive exemestane or tamoxifen. Tumor growth slowed briefly in mice treated with tamoxifen and crossed over to exemestane, but tumor growth continued unabated in those changed from exemestane to tamoxifen. However, letrozole was effective in both groups as third-line therapy for a limited period. Letrozole as initial single agent was the best overall treatment in terms of the degree of tumor suppression and the length of effectiveness of treatment. CONCLUSION: Exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.
Assuntos
Androstadienos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Tamoxifeno/administração & dosagem , Animais , Linhagem Celular Tumoral , Progressão da Doença , Relação Dose-Resposta a Droga , Feminino , Humanos , Letrozol , Neoplasias Mamárias Animais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Nitrilas/administração & dosagem , Pós-Menopausa , Distribuição Aleatória , Tamoxifeno/farmacologia , Fatores de Tempo , Triazóis/administração & dosagemRESUMO
A facile preparation of azolyl steroids, VN/85-1 and VN/87-1 (potent inhibitors of CYP17) has been developed. This process without tedious chromatographic separations improved the overall yields from 55 and 45% to 70 and 65% for VN/85-1 and VN/87-1, respectively. Pharmacokinetic studies of VN/85-1 were conducted in male SCID mice. Following subcutaneous (s.c.) administration of 100mg/kg of VN/85-1, peak plasma level of 16.73 microg/ml occurred after 45 min, and the compound was cleared rapidly with a t(1/2) of 52.34 min. The bioavailability of VN/85-1 after s.c. administration was 83.0%. VN/85-1 was also rapidly metabolized to the corresponding 3-oxo-4-ene analog, 17-(1H-imidazol-1-yl)androsta-4,16-diene-3-one (VN/108-1). In our attempt to optimize the anti-tumor efficacy of these two CYP17 inhibitors, we studied their anti-tumor efficacies in male SCID mice bearing LNCaP tumor xenografts, utilizing various drug doses and drug scheduling. Three times a day dose regimen (3 x dose regimen) of VN/85-1 was more effective than a once daily dose. In contrast, 3 x dose regimen doses of VN/87-1 were less effective than the once daily dose. However, at their effective dosage regimes, VN/85-1 and VN/87-1 were each as effective as castration and more effective than finasteride or casodex, an anti-androgen used for prostate cancer (PC) therapy. For all of the treatments, there was a strong correlation between the tumor volumes and other associated parameters, such as, tumor weights, and serum testosterone (T) and PSA levels. These results indicate that VN/85-1 or VN/87-1 may be useful in the treatment of hormone-dependent prostate cancer.
Assuntos
Androstadienos/farmacocinética , Androstanóis/farmacocinética , Imidazóis/farmacocinética , Neoplasias da Próstata/tratamento farmacológico , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Triazóis/farmacocinética , Androstadienos/farmacologia , Androstanóis/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Finasterida/farmacologia , Humanos , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/enzimologia , Fatores de Tempo , Transplante Heterólogo , Triazóis/farmacologiaRESUMO
BACKGROUND: The antiestrogen tamoxifen has potent activity against estrogen receptor-positive breast cancer, but two nonsteroidal aromatase inhibitors, letrozole and anastrozole, show considerable advantages over tamoxifen with respect to patient survival and tolerability. To determine the optimal way to use letrozole and tamoxifen, we studied their effects on a breast tumor xenograft model, MCF-7Ca, that is responsive to both antiestrogens and aromatase inhibitors. METHODS: Female ovariectomized BALB/c athymic nude mice carrying xenograft tumors were treated daily subcutaneously with one of the following first-line therapies for varying durations: no drug (control), tamoxifen (100 microg/day) alone, letrozole (10 microg/day) alone, both drugs at the same time, or alternating 4-week courses of each drug (beginning with a course of tamoxifen or beginning with a course of letrozole). Tumor volumes and weights were estimated using linear mixed-effects models. The time to tumor doubling was calculated, and tumor weights in the treatment groups were compared, with adjustments for multiple comparisons being made with either Tukey's or Dunnett's procedure. Second-line therapies (with tamoxifen, letrozole, or fulvestrant) were initiated when tumors doubled in size under first-line therapies. All statistical tests were two-sided. RESULTS: The times for doubling of tumor volume were as follows: control, 3-4 weeks; tamoxifen alone, 16 weeks; tamoxifen alternating with letrozole, 17-18 weeks; tamoxifen plus letrozole, 18 weeks; letrozole alternating with tamoxifen, 22 weeks; letrozole alone, 34 weeks. First-line treatment with letrozole was superior to treatment with tamoxifen alone or with the two drugs combined (at week 16, both P<.001). Alternating tamoxifen and letrozole and alternating letrozole and tamoxifen were also not as effective as letrozole alone (at week 16, P =.002 and P<.001, respectively). Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole compared with those remaining on tamoxifen at the end of treatment (week 28, P<.001), whereas tumors progressing on letrozole were unaffected by second-line treatment with the antiestrogens tamoxifen or fulvestrant. CONCLUSIONS: First-line letrozole therapy extends time for tumor progression in this model relative to the other treatment regimens tested. However, further studies are needed to determine the most effective second-line therapy for tumors that progress on letrozole.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores da Aromatase , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Moduladores de Receptor Estrogênico/administração & dosagem , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Nitrilas/administração & dosagem , Tamoxifeno/administração & dosagem , Triazóis/administração & dosagem , Animais , Modelos Animais de Doenças , Progressão da Doença , Esquema de Medicação , Inibidores Enzimáticos/sangue , Moduladores de Receptor Estrogênico/sangue , Feminino , Humanos , Letrozol , Modelos Lineares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nitrilas/sangue , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Tamoxifeno/sangue , Transplante Heterólogo , Triazóis/sangue , Útero/efeitos dos fármacosRESUMO
Aromatase inhibitors have recently been reported to be more effective than the antiestrogen tamoxifen (Tam) in treating breast cancer. Here, we studied the mechanisms and signaling pathways of cell growth, cell cycle progression, and apoptosis induced by three aromatase inhibitors: letrozole (Let), anastrozole, and 4-hydroxyandrostenedione in comparison with estrogen withdrawal (E2W) and antiestrogens Tam and faslodex. Estrogen-dependent human breast cancer cells stably transfected with aromatase (MCF-7Ca) were used. All treatments induced growth suppression and cell cycle arrest at the G(0)-G(1) phase that was associated with up-regulation of p53 and p21 protein and mRNA levels and down-regulation of cyclin D1 and c-myc mRNA. The apoptotic index was increased 4-7 fold, Bcl-2 protein expression decreased, Bax increased, and caspase-9, caspase-6, and caspase-7 were activated but not caspase-3 and caspase-8. Let and E2W caused regression of tumors of MCF-7Ca cells grown in nude mice and increased the number of cells undergoing apoptosis. In contrast, Tam and faslodex did not induce tumor regression and a lower number of apoptotic cells was detected. Cleavage of poly(ADP-ribose) polymerase was detected. Treatment with Let, Tam, or E2W resulted in a dose- and time-dependent increase in active caspase-7 and up-regulation of p53 and p21 protein. Although the mechanisms involved appeared to be similar for antiestrogens and aromatase inhibitors, the most significant effects occurred with Let, which were significantly greater than with E2W and consistent with marked effects of Let on tumor and cell growth.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores da Aromatase , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Animais , Apoptose/genética , Apoptose/fisiologia , Aromatase/genética , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ovariectomia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2RESUMO
We have developed a tumor model for studying the effects of aromatase inhibitors and antiestrogens in vivo. The model simulates postmenopausal breast cancer patients with estrogen-dependent tumors. This model utilizes human estrogen-dependent breast cancer cells transfected with the aromatase gene (MCF-7(CA)), which are inoculated in Matrigel s.c. into ovariectomized nude mice. The estrogen receptor-positive cells produce sufficient estrogen by aromatization to stimulate their proliferation and the formation of tumors in the mice. Using several different strategies, we have compared the aromatase inhibitors (letrozole and anastrozole) with antiestrogens (tamoxifen and fulvestrant). Aromatase inhibitors were more effective than tamoxifen and were more effective alone than when combined with antiestrogens. In addition, letrozole had a longer duration of effect than tamoxifen. The possibility of delaying the development of resistance to antiestrogens and aromatase inhibitors was investigated by alternating letrozole with tamoxifen every 4 weeks. However, although alternating treatment proved more effective than tamoxifen alone (tumors doubled in 16 weeks), tumors doubled in volume at about 18 weeks when treatment began with tamoxifen. When treatment started with letrozole and alternated with tamoxifen, tumors doubled in volume in about 22 weeks. However, tumors of mice treated with letrozole alone did not double in size until 35 weeks. These results demonstrate that this aromatase inhibitor is more effective and has a longer duration of response as a single agent than tamoxifen or in combination with tamoxifen.
Assuntos
Inibidores da Aromatase , Inibidores Enzimáticos/uso terapêutico , Antagonistas de Estrogênios/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Animais , Aromatase/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Feminino , Humanos , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Hormônio-Dependentes , Células Tumorais Cultivadas/transplanteRESUMO
PURPOSE: The aromatase inhibitors letrozole and anastrozole have been approvedrecently as first-line treatment options for hormone-dependent advanced breast cancer. Although it is established that a proportion of patients who relapse on first-line tamoxifen therapy show additional responses to aromatase inhibitors, it has not been determined whether tumors that acquire resistance to aromatase inhibitors in the first line remain sensitive to second-line therapy with antiestrogens. The aim of this study was to determine whether aromatase-transfected and hormone-dependent MCF-7Ca human breast cancer cells remain sensitive to antiestrogens after: (a) long-term growth in steroid-depleted medium in vitro; and (b) long-term treatment with the aromatase inhibitor letrozole in vivo. METHODS: In the first approach, a variant of the MCF-7Ca human breast cancer cell line was selected that had acquired the ability to grow in estrogen-depleted medium after 6-8 months of culture. Steroid-deprived UMB-1Ca cells were analyzed for aromatase activity levels, hormone receptor levels, and sensitivity to estrogens and antiestrogens in vitro and in vivo. In the second approach, established MCF-7Ca breast tumor xenografts were treated with letrozole 10 microg/day for 12 weeks followed by 100 microg/day for 25 weeks until tumors acquired the ability to proliferate in the presence of the drug. Long-term letrozole-treated tumors were then transplanted into new mice, and the effects of antiestrogens and aromatase inhibitors on tumor growth were determined. RESULTS: Steroid-deprived UMB-1Ca breast cancer cells continued to express aromatase activity at levels comparable with the parental cell line. However, compared with MCF-7Ca cells, UMB-1Ca cells expressed elevated levels of functionally active estrogen receptor. The growth of UMB-1Ca cells in vitro was inhibited by the antiestrogens tamoxifen and faslodex and tumor growth in vivo was inhibited by tamoxifen. In the second approach, the time for MCF-7Ca tumor xenografts to approximately double in volume after being treated sequentially with the increasing doses of letrozole was thirty-seven weeks. Long-term letrozole-treated tumors continued to express functionally active aromatase. When transplanted into new mice, growth of the long-term letrozole-treated tumors was slowed by tamoxifen and inhibited more effectively by faslodex. Tumor growth was refractory to the aromatase inhibitors anastrozole and formestane but, surprisingly, showed sensitivity to letrozole. CONCLUSIONS: Steroid-deprived UMB-1Ca human breast cancer cells selected in vitro and long-term letrozole-treated MCF-7Ca breast tumor xenografts remain sensitive to second-line therapy with antiestrogens and, in particular, to faslodex. This finding is associated with increased expression of functionally active estrogen receptor after steroid-deprivation of MCF-7Ca human breast cancer cells in vitro.