RESUMEN
Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.
Asunto(s)
Antineoplásicos , Alcaloides de Pirrolicidina , Humanos , Citocromo P-450 CYP3A/metabolismo , Hígado , Hepatocitos , Alcaloides de Pirrolicidina/metabolismo , Daño del ADN , Antineoplásicos/farmacologíaRESUMEN
Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure-toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1-10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.
Asunto(s)
Neoplasias Hepáticas , Alcaloides de Pirrolicidina , Humanos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Alcaloides de Pirrolicidina/metabolismo , Hepatocitos , Pruebas de Mutagenicidad , Neoplasias Hepáticas/metabolismoRESUMEN
Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.
Asunto(s)
Anomalías Inducidas por Medicamentos/prevención & control , Anticonvulsivantes/efectos adversos , Epilepsia/tratamiento farmacológico , Ácido Valproico/efectos adversos , Adolescente , Adulto , Utilización de Medicamentos , Femenino , Humanos , Farmacovigilancia , Embarazo , Medición de Riesgo , Adulto JovenRESUMEN
Duchenne muscular dystrophy (DMD) is a rare, severe, progressive muscle-wasting disease leading to disability and premature death. Patients lack the muscle membrane-stabilizing protein dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach that aims to induce production of partially functional dystrophins. Recently, an AON targeting exon 51 became the first of its class to be approved by the United States regulators [Food and Drug Administration (FDA)] for the treatment of DMD. A unique aspect of the exon-skipping approach for DMD is that, depending on the size and location of the mutation, different exons need to be skipped. This challenge raises a number of questions regarding the development and regulatory approval of those individual compounds. In this study, we present a perspective on those questions, following a European stakeholder meeting involving academics, regulators, and representatives from industry and patient organizations, and in the light of the most recent scientific and regulatory experience.
Asunto(s)
Aprobación de Drogas , Distrofina/genética , Exones/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/uso terapéutico , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Mutación , Participación de los Interesados , Estados Unidos , United States Food and Drug AdministrationRESUMEN
On 31 March 2016, the European Commission issued a decision for a marketing authorisation valid throughout the European Union (EU) for pitolisant (Wakix) for the treatment of narcolepsy with or without cataplexy in adults. Pitolisant is an antagonist/inverse agonist of the human histamine H3 receptor. The dose should be selected using an up-titration scheme depending on individual patient response and tolerance and should not exceed 36 mg/day. The main evidence of efficacy of pitolisant was based on two Phase III clinical trials. The improvement on excessive daytime sleepiness was shown against placebo in the Harmony I study (-3.33 points; 95% confidence interval (CI) [-5.83; -0.83]; p = 0.024) and in Harmony CTP (-3.41 points; 95% CI [-4.95; -1.87]; p < 0.0001). The daily cataplexy rate in Harmony I improved against placebo with a rate ratio (rR) of 0.38 whilst in the Harmony CTP the ratio of improvement on weekly cataplexy rate against placebo was 0.512. The most commonly reported adverse reactions were headache, insomnia and nausea. This article summarizes the scientific review leading to approval of pitolisant in the EU. The assessment report and product information are available on the European Medicines Agency website (http://www.ema.europa.eu).
Asunto(s)
Narcolepsia/tratamiento farmacológico , Piperidinas/uso terapéutico , Receptores Histamínicos H3/efectos de los fármacos , Adolescente , Adulto , Anciano , Animales , Atención/efectos de los fármacos , Cataplejía/tratamiento farmacológico , Ensayos Clínicos Fase III como Asunto , Cognición/efectos de los fármacos , Agonismo Inverso de Drogas , Humanos , Persona de Mediana Edad , Piperidinas/administración & dosificación , Piperidinas/efectos adversos , Piperidinas/metabolismo , Receptores Histamínicos H3/fisiología , Resultado del Tratamiento , Promotores de la Vigilia/farmacología , Adulto JovenRESUMEN
Duchenne muscular dystrophy is a rare, progressive, muscle-wasting disease leading to severe disability and premature death. Treatment is currently symptomatic, but several experimental therapies are in development. Implemented care standards, validated outcome measures correlating with clinical benefit, and comprehensive information about the natural history of the disease are essential for regulatory approval of any treatment. However, for Duchenne muscular dystrophy and other rare diseases, these requirements are not always in place when potential therapies enter the clinical trial phase. A cooperative effort of stakeholders in Duchenne muscular dystrophy-including representatives from patients' groups, academia, industry, and regulatory agencies-is aimed at addressing this shortfall by identifying strategies to overcome challenges, developing the tools needed, and collecting relevant data. An open and constructive dialogue among European stakeholders has positively affected development of treatments for Duchenne muscular dystrophy; this approach could serve as a paradigm for development of treatments for rare diseases in general.
Asunto(s)
Distrofia Muscular de Duchenne/terapia , Producción de Medicamentos sin Interés Comercial , Asociación entre el Sector Público-Privado , Aprobación de Drogas , Terapia Genética , Humanos , Distrofia Muscular de Duchenne/genética , Evaluación de Resultado en la Atención de SaludRESUMEN
Regulatory agencies have a key role in facilitating the development of new drugs for Alzheimer disease, particularly given the challenges associated with early intervention. Here, we highlight the strategies of the European Medicines Agency to help address such challenges.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Unión Europea , Industria Farmacéutica , Humanos , Agencias InternacionalesAsunto(s)
Codón sin Sentido , Aprobación de Drogas , Distrofina/genética , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Oxadiazoles/uso terapéutico , Adolescente , Adulto , Niño , Preescolar , Ensayos Clínicos Fase II como Asunto , Humanos , Masculino , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del Tratamiento , Adulto JovenAsunto(s)
Fármacos del Sistema Nervioso Central/normas , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Aprobación de Drogas , Regulación Gubernamental , Fármacos del Sistema Nervioso Central/economía , Aprobación de Drogas/legislación & jurisprudencia , Aprobación de Drogas/estadística & datos numéricos , Europa (Continente) , Humanos , Mercadotecnía/legislación & jurisprudenciaRESUMEN
Improving and facilitating the process of making new drugs available to patients with multiple sclerosis (MS) requires cooperation among the regulators and other stakeholders. This cooperation will also positively contribute towards developing guidelines of the highest quality in medical, regulatory and scientific aspects. This would be beneficial both in areas that require further guideline development, but also in fields where existing guidance should be adapted to take into account evolution in science. Considering the input from all stakeholders, the European Medicines Agency confirmed its intention to update the relevant guideline and apply a flexible approach towards new drug development strategies in MS. This article is the first official position from the EU regulators, presenting the main changes to be expected in the guidance document.
Asunto(s)
Descubrimiento de Drogas/legislación & jurisprudencia , Descubrimiento de Drogas/normas , Unión Europea , Regulación Gubernamental , Guías como Asunto/normas , Esclerosis Múltiple/tratamiento farmacológico , Proyectos de Investigación/legislación & jurisprudencia , Proyectos de Investigación/normas , Conducta Cooperativa , Evaluación de la Discapacidad , Descubrimiento de Drogas/tendencias , Determinación de Punto Final/normas , Humanos , Comunicación Interdisciplinaria , Relaciones Interinstitucionales , Esclerosis Múltiple/diagnóstico , Selección de Paciente , Valor Predictivo de las Pruebas , Proyectos de Investigación/tendencias , Resultado del TratamientoRESUMEN
The publication of the fifth version of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) in 2013 raises important questions for clinical trials and associated regulatory decisions on new drugs for psychiatric disorders.