RESUMO
Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1-4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.
Assuntos
Triptofano-tRNA Ligase , Triptofano , Códon/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama , Neoplasias/imunologia , Fenilalanina , Linfócitos T , Triptofano/metabolismo , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismo , Triptofano-tRNA Ligase/genética , Triptofano-tRNA Ligase/metabolismoRESUMO
The efficacy of anthracycline-based chemotherapeutics, which include doxorubicin and its structural relatives daunorubicin and idarubicin, remains almost unmatched in oncology, despite a side effect profile including cumulative dose-dependent cardiotoxicity, therapy-related malignancies and infertility. Detoxifying anthracyclines while preserving their anti-neoplastic effects is arguably a major unmet need in modern oncology, as cardiovascular complications that limit anti-cancer treatment are a leading cause of morbidity and mortality among the 17 million cancer survivors in the U.S. In this study, we examined different clinically relevant anthracycline drugs for a series of features including mode of action (chromatin and DNA damage), bio-distribution, anti-tumor efficacy and cardiotoxicity in pre-clinical models and patients. The different anthracycline drugs have surprisingly individual efficacy and toxicity profiles. In particular, aclarubicin stands out in pre-clinical models and clinical studies, as it potently kills cancer cells, lacks cardiotoxicity, and can be safely administered even after the maximum cumulative dose of either doxorubicin or idarubicin has been reached. Retrospective analysis of aclarubicin used as second-line treatment for relapsed/refractory AML patients showed survival effects similar to its use in first line, leading to a notable 23% increase in 5-year overall survival compared to other intensive chemotherapies. Considering individual anthracyclines as distinct entities unveils new treatment options, such as the identification of aclarubicin, which significantly improves the survival outcomes of AML patients while mitigating the treatment-limiting side-effects. Building upon these findings, an international multicenter Phase III prospective study is prepared, to integrate aclarubicin into the treatment of relapsed/refractory AML patients.
Assuntos
Aclarubicina , Antraciclinas , Leucemia Mieloide Aguda , Animais , Feminino , Humanos , Masculino , Aclarubicina/farmacologia , Aclarubicina/uso terapêutico , Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/efeitos adversos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Resultado do TratamentoRESUMO
L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.
Assuntos
Asparaginase/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose , Sistemas CRISPR-Cas , Proliferação de Células , Transportador 1 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 1 de Aminoácido Excitatório/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.
Assuntos
Bilirrubina , Transportadores de Ânions Orgânicos , Masculino , Camundongos , Humanos , Animais , Rosuvastatina Cálcica , Fluvastatina , Pravastatina , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Camundongos Transgênicos , Peptídeos/metabolismo , Ânions/metabolismo , Camundongos KnockoutRESUMO
The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.
Assuntos
Antineoplásicos/efeitos adversos , Cromatina/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Animais , Linhagem Celular , Doxorrubicina/análogos & derivados , Doxorrubicina/síntese química , Doxorrubicina/metabolismo , Doxorrubicina/uso terapêutico , Cardiopatias/induzido quimicamente , Histonas , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , CamundongosRESUMO
Tumour growth and metabolic adaptation may restrict the availability of certain amino acids for protein synthesis. It has recently been shown that certain types of cancer cells depend on glycine, glutamine, leucine and serine metabolism to proliferate and survive. In addition, successful therapies using L-asparaginase-induced asparagine deprivation have been developed for acute lymphoblastic leukaemia. However, a tailored detection system for measuring restrictive amino acids in each tumour is currently not available. Here we harness ribosome profiling for sensing restrictive amino acids, and develop diricore, a procedure for differential ribosome measurements of codon reading. We first demonstrate the functionality and constraints of diricore using metabolic inhibitors and nutrient deprivation assays. Notably, treatment with L-asparaginase elicited both specific diricore signals at asparagine codons and high levels of asparagine synthetase (ASNS). We then applied diricore to kidney cancer and discover signals indicating restrictive proline. As for asparagine, this observation was linked to high levels of PYCR1, a key enzyme in proline production, suggesting a compensatory mechanism allowing tumour expansion. Indeed, PYCR1 is induced by shortage of proline precursors, and its suppression attenuated kidney cancer cell proliferation when proline was limiting. High PYCR1 is frequently observed in invasive breast carcinoma. In an in vivo model system of this tumour, we also uncover signals indicating restrictive proline. We further show that CRISPR-mediated knockout of PYCR1 impedes tumorigenic growth in this system. Thus, diricore has the potential to reveal unknown amino acid deficiencies, vulnerabilities that can be used to target key metabolic pathways for cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Códon/genética , Neoplasias Renais/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Animais , Asparaginase/metabolismo , Asparagina/genética , Asparagina/metabolismo , Aspartato-Amônia Ligase/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Técnicas de Inativação de Genes , Humanos , Neoplasias Renais/patologia , Camundongos , Prolina/biossíntese , Prolina/deficiência , Biossíntese de Proteínas/genética , Pirrolina Carboxilato Redutases/deficiência , Pirrolina Carboxilato Redutases/genética , Pirrolina Carboxilato Redutases/metabolismo , delta-1-Pirrolina-5-Carboxilato RedutaseRESUMO
Mitogen/extracellular signal-regulated kinase (MEK) inhibitors have been tested in clinical trials for treatment of intracranial neoplasms, including glioblastoma (GBM), but efficacy of these drugs has not yet been demonstrated. The blood-brain barrier (BBB) is a major impediment to adequate delivery of drugs into the brain and may thereby also limit the successful implementation of MEK inhibitors against intracranial malignancies. The BBB is equipped with a range of ATP-dependent efflux transport proteins, of which P-gp (ABCB1) and BCRP (ABCG2) are the two most dominant for drug efflux from the brain. We investigated their impact on the pharmacokinetics and target engagement of a panel of clinically applied MEK inhibitors, in order to select the most promising candidate for brain cancers in the context of clinical pharmacokinetics and inhibitor characteristics. To this end, we used in vitro drug transport assays and conducted pharmacokinetic and pharmacodynamic studies in wildtype and ABC-transporter knockout mice. PD0325901 displayed more promising characteristics than trametinib (GSK1120212), binimetinib (MEK162), selumetinib (AZD6244), and pimasertib (AS703026): PD0325901 was the weakest substrate of P-gp and BCRP in vitro, its brain penetration was only marginally higher in Abcb1a/b;Abcg2-/- mice, and efficient target inhibition in the brain could be achieved at clinically relevant plasma levels. Notably, target inhibition could also be demonstrated for selumetinib, but only at plasma levels far above levels in patients receiving the maximum tolerated dose. In summary, our study recommends further development of PD0325901 for the treatment of intracranial neoplasms.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/fisiologia , Encéfalo/efeitos dos fármacos , MAP Quinase Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Camundongos , Camundongos Knockout , Distribuição TecidualRESUMO
BACKGROUND: Chromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice. METHODS: To investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1-/-;TP53-/- mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination. RESULTS: The enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment. CONCLUSIONS: Our study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Docetaxel/farmacologia , Neoplasias/tratamento farmacológico , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Docetaxel/administração & dosagem , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Camundongos , Mitose/efeitos dos fármacos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Paclitaxel/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Introduction Wee1 is an important kinase involved in the G2 cell cycle checkpoint and frequently upregulated in intracranial neoplasms such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG). Two small molecules are available that target Wee1, AZD1775 and PD0166285, and clinical trials with AZD1775 have already been started. Since GBM and DIPG are highly invasive brain tumors, they are at least to some extent protected by the blood-brain barrier (BBB) and its ATP-binding cassette (ABC) efflux transporters. Methods We have here conducted a comprehensive set of in vitro and in vivo experiments to determine to what extent two dominant efflux transporters in the BBB, P-gp (ABCB1) and BCRP (ABCG2), exhibit affinity towards AZD1775 and PD0166285 and restrict their brain penetration. Results Using these studies, we demonstrate that AZD1775 is efficiently transported by both P-gp and BCRP, whereas PD0166285 is only a substrate of P-gp. Nonetheless, the brain penetration of both compounds was severely restricted in vivo, as indicated by a 5-fold (PD0166285) and 25-fold (AZD1775) lower brain-plasma ratio in wild type mice compared to Abcb1a/b;Abcg2-/- mice. Conclusion The brain penetration of these Wee1 inhibitors is severely limited by ABC transporters, which may compromise their clinical efficacy against intracranial neoplasms such as DIPG and GBM.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Camundongos , Permeabilidade , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , PirimidinonasRESUMO
Poly (ADP-ribose) polymerase (PARP) inhibitors are a relatively new class of anticancer agents that have attracted attention for treatment of glioblastoma because of their ability to potentiate temozolomide chemotherapy. Previous studies have demonstrated that sufficient brain penetration is a prerequisite for efficacy of PARP inhibitors in glioma mouse models. Unfortunately, however, most of the PARP inhibitors developed to date have a limited brain penetration due to the presence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) at the blood-brain barrier. AZD2461 is a novel PARP inhibitor that is unaffected by P-gp mediated resistance in breast cancer models and thus appears to have promising characteristics for brain penetration. We here use a comprehensive set of in vitro and in vivo models to study the brain penetration and oral bioavailability of AZD2461. We report that AZD2461 has a good membrane permeability. However, it is a substrate of P-gp and BCRP, and P-gp in particular limits its brain penetration in vivo. We show that AZD2461 has a low oral bioavailability, although it is not affected by P-gp and BCRP. Together, these findings are not in favor of further development of AZD2461 for treatment of glioblastoma.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Ftalazinas/farmacocinética , Piperidinas/farmacocinética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Permeabilidade , Ftalazinas/administração & dosagem , Piperidinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagemRESUMO
Enhancer of Zeste Homolog 2 (EZH2) has emerged as a promising therapeutic target for treatment of a broad spectrum of tumors including gliomas. We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcb1 and/or Abcg2 knockout mice. All EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by very low membrane permeability. Both P-gp and Bcrp1 restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcb1;Abcg2 knockout mice. These results are likely due to poor membrane permeability and question the clinical usefulness of GSK126. Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Inibidores Enzimáticos/farmacocinética , Complexo Repressor Polycomb 2/antagonistas & inibidores , Administração Oral , Animais , Benzamidas/administração & dosagem , Benzamidas/farmacocinética , Disponibilidade Biológica , Compostos de Bifenilo , Linhagem Celular , Cães , Avaliação Pré-Clínica de Medicamentos , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/administração & dosagem , Humanos , Indazóis/administração & dosagem , Indazóis/farmacocinética , Indóis/administração & dosagem , Indóis/farmacocinética , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Morfolinas , Piridonas/administração & dosagem , Piridonas/farmacocinéticaRESUMO
INTRODUCTION: Palbociclib is a cyclin dependent kinase (CDK) 4/6 inhibitor with nanomolar potency and was recently approved for treatment of breast cancer. The drug may also be useful in glioblastoma (GBM) and diffuse intrinsic pontine gliomas (DIPG), which often have an activated CDK4/6-retinoblastoma signaling pathway. However, GBM and DIPG spread widely into the surrounding brain, which calls for a CDK4/6 inhibitor with sufficient blood-brain barrier penetration. METHODS: We first performed in vitro transwell assays and demonstrate that palbociclib is a substrate of both P-gp and BCRP. Next, we conducted pharmacokinetic studies using wildtype, Abcg2(-/-), Abcb1a/b(-/-) and Abcg2; Abcb1a/b(-/-) mice. RESULTS: The plasma levels were about 3000 and 500 nM and similar in all genotypes at 1 and 4 h after i.v. administration of 10 mg/kg. At 4 h the brain-to-plasma ratios were 0.3 in WT and Abcg2(-/-) mice versus 5.5 and 15 in Abcb1a/b(-/-) and Abcg2; Abcb1a/b(-/-) mice, respectively. The oral bioavailability of palbociclib was high (63 %) in WT mice and increased only modestly and non-significantly in Abcg2; Abcb1a/b(-/-) mice. The plasma level after oral dosing of 150 mg/kg was already much higher than observed in patients (200-400 nM) and exceeded 2500 nM for up to 24 h. This latter dose is commonly used in preclinical studies, which calls into question their predictive value as they were conducted at dose levels causing a clinically non-relevant systemic drug exposure. CONCLUSION: Thus, the brain penetration of palbociclib is restricted by P-gp and BCRP, which may restrict the efficacy against GBM and DIPG. Moreover, preclinical studies with this agent should be conducted at a more clinically relevant dose level.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Piperazinas/farmacologia , Piridinas/farmacologia , Administração Oral , Animais , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Genótipo , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Piperazinas/farmacocinética , Piridinas/farmacocinética , Distribuição TecidualRESUMO
The impact of OATP drug uptake transporters in drug-drug interactions (DDIs) is increasingly recognized. OATP1B1 and OATP1B3 are human hepatic uptake transporters that can mediate liver uptake of a wide variety of drugs. Recently, we generated transgenic mice with liver-specific expression of human OATP1B1 or OATP1B3 in a mouse Oatp1a/1b knockout background. Here, we investigated the applicability of these mice in OATP-mediated drug-drug interaction studies using the prototypic OATP inhibitor rifampicin and a good OATP substrate, the anticancer drug methotrexate (MTX). We next assessed the possibility of OATP-mediated interactions between telmisartan and MTX, a clinically relevant drug combination. Using HEK293 cells overexpressing OATP1B1 or OATP1B3, we estimated IC50 values for both rifampicin (0.9 or 0.3 µM) and telmisartan (6.7 or 7.9 µM) in inhibiting OATP-mediated MTX uptake in vitro. Using wild-type, Oatp1a/1b-/-, and OATP1B1- or OATP1B3-humanized transgenic mice, we found that rifampicin inhibits hepatic uptake of MTX mediated by the mouse Oatp1a/1b and human OATP1B1 and OATP1B3 transporters at clinically relevant concentrations. This highlights the applicability of these mouse models for DDI studies and may be exploited in the clinic to reduce the dose and thus methotrexate-mediated toxicity. On the other hand, telmisartan inhibited only human OATP1B1-mediated hepatic uptake of MTX at concentrations higher than those used in the clinic; therefore risks for OATP-mediated clinical DDIs for this drug combination are likely to be low. Overall, we show here that OATP1B1- and OATP1B3-humanized mice can be used as in vivo tools to assess and possibly predict clinically relevant DDIs.
Assuntos
Interações Medicamentosas/fisiologia , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Animais , Antineoplásicos/metabolismo , Benzimidazóis/metabolismo , Benzoatos/metabolismo , Transporte Biológico/fisiologia , Células HEK293 , Humanos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , TelmisartanRESUMO
The Polycomb group and oncogene Bmi1 is required for the proliferation of various differentiated cells and for the self-renewal of stem cells and leukemic cancer stem cells. Repression of the Ink4a/Arf locus is a well described mechanism through which Bmi1 can exert its proliferative effects. However, we now demonstrate in an orthotopic transplantation model for glioma, a type of cancer harboring cancer stem cells, that Bmi1 is also required for tumor development in an Ink4a/Arf-independent manner. Tumors derived from Bmi1;Ink4a/Arf doubly deficient astrocytes or neural stem cells have a later time of onset and different histological grading. Moreover, in the absence of Ink4a/Arf, Bmi1-deficient cells and tumors display changes in differentiation capacity.
Assuntos
Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Glioblastoma/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células 3T3 , Animais , Astrócitos/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , Mutação , Estadiamento de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neurônios/patologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Células-Tronco/patologia , Fatores de Tempo , Transdução GenéticaRESUMO
Organic anion-transporting polypeptides (OATPs) are important drug uptake transporters, mediating distribution of substrates to several pharmacokinetically relevant organs. Doxorubicin is a widely used anti-cancer drug extensively studied for its interactions with various drug transporters, but not OATPs. Here, we investigated the role of OATP1A/1B proteins in the distribution of doxorubicin. In vitro, we observed â¼ 2-fold increased doxorubicin uptake in HEK293 cells overexpressing human OATP1A2, but not OATP1B1 or OATP1B3. In mice, absence of Oatp1a/1b transporters led to up to 2-fold higher doxorubicin plasma concentrations and 1.3-fold higher plasma AUC. Conversely, liver AUC and liver-to-plasma ratios of Oatp1a/1b(-/-) mice were 1.4-fold and up to 4.1-fold lower than in wild-type mice, respectively. Decreased doxorubicin levels in the small intestinal content reflected those in the liver, indicating a reduced biliary excretion of doxorubicin in Oatp1a/1b(-/-) mice. These results demonstrate important control of doxorubicin plasma clearance and hepatic uptake by mouse Oatp1a/1b transporters. This is unexpected, as the fairly hydrophobic weak base doxorubicin is an atypical OATP1A/1B substrate. Interestingly, transgenic liver-specific expression of human OATP1A2, OATP1B1 or OATP1B3 could partially rescue the increased doxorubicin plasma levels of Oatp1a/1b(-/-) mice. Hepatic uptake and bile-derived intestinal excretion of doxorubicin were completely reverted to wild-type levels by OATP1A2, and partially by OATP1B1 and OATP1B3. Thus, doxorubicin is transported by hepatocyte-expressed OATP1A2, -1B1 and -1B3 in vivo, illustrating an unexpectedly wide substrate specificity. These findings have possible implications for the uptake, disposition, therapy response and toxicity of doxorubicin, also in human tumors and tissues expressing these transporters.
Assuntos
Antineoplásicos/farmacocinética , Doxorrubicina/farmacocinética , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Animais , Antineoplásicos/administração & dosagem , Transporte Biológico , Western Blotting , Cromatografia Líquida de Alta Pressão , Doxorrubicina/administração & dosagem , Feminino , Células HEK293 , Humanos , Fígado/efeitos dos fármacos , Transportador 1 de Ânion Orgânico Específico do Fígado , Camundongos , Camundongos Transgênicos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto , Distribuição TecidualRESUMO
BMS-275,183 is a novel oral C-4 methyl carbonate analogue of paclitaxel. Recently, a drug-drug interaction between BMS-275,183 and benzimidazole proton pump inhibitors (PPIs) was suggested in clinical trials resulting in elevated drug exposure and toxicity. We explored whether the interaction takes place at the level of P-glycoprotein (Pgp, MDR1, ABCB1), Breast Cancer Resistance Protein (BCRP, ABCG2) and MRP2 (ABCC2) using in vitro and in vivo models. In vitro cell survival, drug accumulation, efflux and transport studies with BMS-275,183 were performed employing MDCKII (wild-type, MDR1, BCRP, MRP2) and LLCPK (wild-type and MDR1) cells. In vivo the pharmacokinetics and tissue distribution of BMS-275,183 after p.o. and i.v. administration were explored in Mdr1a/1b(-/-) and wild-type mice, in presence or absence of the PPI pantoprazole. Results In vitro, BMS-275,183 was found to be a good substrate for MDR1, a moderate substrate for MRP2 and not a substrate for BCRP. In vivo, oral bioavailability, plasma AUC0-6h and brain concentrations were significantly 1.5-, 4-, and 2-fold increased, respectively, in Mdr1a/1b(-/-) compared with wild-type mice (p < 0.001). However, oral co-administration of pantoprazole (40 mg/kg) did not alter the pharmacokinetics of BMS-275,183 in wild-type mice. Conclusions BMS-275,183 is efficiently transported by Pgp and to a lesser extent by MRP2 in vitro. Genetic deletion of Pgp significantly altered the pharmacokinetics and brain distribution of p.o. and i.v. administered BMS-275,183 in Mdr1a/1b-/- compared to wild-type mice. Oral co-administration of BMS-275,183 with pantoprazole did not affect the pharmacokinetics of BMS-275,183 in wild-type mice, suggesting no interaction with PPI at the dose employed.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacocinética , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , 2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacologia , Encéfalo/metabolismo , Hidrocarbonetos Aromáticos com Pontes/sangue , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dibenzocicloeptenos/farmacologia , Cães , Interações Medicamentosas , Feminino , Humanos , Rim/metabolismo , Pulmão/metabolismo , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Miocárdio/metabolismo , Proteínas de Neoplasias/genética , Paclitaxel/farmacologia , Pantoprazol , Quinolinas/farmacologia , Baço/metabolismo , Suínos , Distribuição TecidualRESUMO
BRAFV600 -mutated melanoma brain metastases (MBMs) are responsive to BRAF inhibitors, but responses are generally less durable than those of extracranial metastases. We tested the hypothesis that the drug efflux transporters P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) expressed at the blood-brain barrier (BBB) offer MBMs protection from therapy. We intracranially implanted A375 melanoma cells in wild-type (WT) and Abcb1a/b;Abcg2-/- mice, characterized the tumor BBB, analyzed drug levels in plasma and brain lesions after oral vemurafenib administration, and determined the efficacy against brain metastases and subcutaneous lesions. Although contrast-enhanced MRI demonstrated that the integrity of the BBB is disrupted in A375 MBMs, vemurafenib achieved greater antitumor efficacy against MBMs in Abcb1a/b;Abcg2-/- mice compared with WT mice. Concordantly, P-gp and BCRP are expressed in MBM-associated brain endothelium both in patients and in A375 xenografts and expression of these transporters limited vemurafenib penetration into A375 MBMs. Although initially responsive, A375 MBMs rapidly developed therapy resistance, even in Abcb1a/b;Abcg2-/- mice, and this was unrelated to pharmacokinetic or target inhibition issues. Taken together, we demonstrate that both intrinsic and acquired resistance can play a role in MBMs.
Assuntos
Neoplasias Encefálicas , Melanoma , Humanos , Animais , Camundongos , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sulfonamidas/farmacologia , Indóis/farmacologia , Indóis/uso terapêutico , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genéticaRESUMO
BACKGROUND: Pharmacotherapy for brain diseases is severely compromised by the blood-brain barrier (BBB). ABCB1 and ABCG2 are drug transporters that restrict drug entry into the brain and their inhibition can be used as a strategy to boost drug delivery and pharmacotherapy for brain diseases. METHODS: We employed elacridar and tariquidar in mice to explore the conditions for effective inhibition at the BBB. Abcg2;Abcb1a/b knockout (KO), Abcb1a/b KO, Abcg2 KO and wild-type (WT) mice received a 3 h i.p. infusion of a cocktail of 8 typical substrate drugs in combination with elacridar or tariquidar at a range of doses. Abcg2;Abcb1a/b KO mice were used as the reference for complete inhibition, while single KO mice were used to assess the potency to inhibit the remaining transporter. Brain and plasma drug levels were measured by LC-MS/MS. RESULTS: Complete inhibition of ABCB1 at the BBB is achieved when the elacridar plasma level reaches 1200 nM, whereas tariquidar requires at least 4000 nM. Inhibition of ABCG2 is more difficult. Elacridar inhibits ABCG2-mediated efflux of weak but not strong ABCG2 substrates. Strikingly, tariquidar does not enhance the brain uptake of any ABCG2-subtrate drug. Similarly, elacridar, but not tariquidar, was able to inhibit its own brain efflux in ABCG2-proficient mice. The plasma protein binding of elacridar and tariquidar was very high but similar in mouse and human plasma, facilitating the translation of mouse data to humans. CONCLUSIONS: This work shows that elacridar is an effective pharmacokinetic-enhancer for the brain delivery of ABCB1 and weaker ABCG2 substrate drugs when a plasma concentration of 1200 nM is exceeded.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Acridinas , Barreira Hematoencefálica , Encéfalo , Camundongos Knockout , Tetra-Hidroisoquinolinas , Animais , Tetra-Hidroisoquinolinas/farmacologia , Tetra-Hidroisoquinolinas/administração & dosagem , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Camundongos , Acridinas/farmacologia , Acridinas/administração & dosagem , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Quinolinas/farmacologia , Quinolinas/farmacocinética , Quinolinas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Masculino , Camundongos Endogâmicos C57BLRESUMO
ATP-binding cassette (ABC) transporters facilitate the movement of diverse molecules across cellular membranes, including those within the CNS. While most extensively studied in microvascular endothelial cells forming the blood-brain barrier (BBB), other CNS cell types also express these transporters. Importantly, disruptions in the CNS microenvironment during disease can alter transporter expression and function. Through this comprehensive review, we explore the modulation of ABC transporters in various brain pathologies and the context-dependent consequences of these changes. For instance, downregulation of ABCB1 may exacerbate amyloid beta plaque deposition in Alzheimer's disease and facilitate neurotoxic compound entry in Parkinson's disease. Upregulation may worsen neuroinflammation by aiding chemokine-mediated CD8 T cell influx into multiple sclerosis lesions. Overall, ABC transporters at the BBB hinder drug entry, presenting challenges for effective pharmacotherapy. Understanding the context-dependent changes in ABC transporter expression and function is crucial for elucidating the etiology and developing treatments for brain diseases.