RESUMEN
Protein and peptide drugs hold great promise as therapeutic agents. But there are shortcomings: Many recombinant proteins are quickly degraded by proteolytic enzymes or are rapidly cleared by kidney filtration resulting in a short circulating half-life. Additionally they are prone to be recognized by the immune system resulting in the generation of neutralizing and non-neutralizing antibodies. PEGylation, a process by which polyethylene glycol chains are attached to protein and peptide drugs, can overcome these and other shortcomings. By increasing the molecular mass of proteins and peptides and shielding them from proteolytic enzymes, PEGylation primarily improves pharmacokinetics and helps to prevent adverse drug reactions.
Asunto(s)
Polietilenglicoles/química , Proteínas Recombinantes/administración & dosificación , Animales , Química Farmacéutica , Diseño de Fármacos , Enzimas/química , Humanos , Proteínas Recombinantes/farmacocinética , Compuestos de Sulfhidrilo/químicaRESUMEN
On 11 November 2010, the German Bundestag approved the law on the Reorganization of the Pharmaceutical Market (AMNOG). This act went into force on 01 January 2011. The aim was to "confine the rapidly rising pharmaceutical expenditure of public health insurance." A better way should be defined for a "fair competition" and a "stronger focus on the well-being of patients" through a new balance between innovation and affordability of medicines. Obviously, advocates and opponents of this new law argue differently. Those in favor of the AMNOG argue that a fair evaluation of the incremental clinical benefits of newly approved drugs should build the basis for fair price negotiations, that prices should be paid, which more realistically reflect the value of the innovation, and that the system will receive urgently needed relief from the immense burden of the rather small group of patent-protected drugs. Those opposing the new law see the danger of delayed launches or even withdrawals of new medicines that can benefit patients, erosion of Germany's strength in pharmaceutical innovation, and the loss of high-qualified and high-paying jobs in research and development.
Asunto(s)
Aprobación de Drogas/economía , Aprobación de Drogas/legislación & jurisprudencia , Costos de los Medicamentos/legislación & jurisprudencia , Industria Farmacéutica/economía , Industria Farmacéutica/legislación & jurisprudencia , Necesidades y Demandas de Servicios de Salud/economía , Necesidades y Demandas de Servicios de Salud/legislación & jurisprudencia , Alemania , HumanosAsunto(s)
Fitoterapia/tendencias , Extractos Vegetales/efectos adversos , Extractos Vegetales/uso terapéutico , Plantas Medicinales , Servicios de Información sobre Medicamentos , Alemania , Humanos , Naturopatía , Educación del Paciente como Asunto , Fitoterapia/efectos adversos , Extractos Vegetales/provisión & distribución , Plantas Medicinales/efectos adversosRESUMEN
A physico-chemical method has been developed as an alternative to the current bioassay in normocythaemic mice for estimating the biological activity of erythropoietin batches. Capillary zone electrophoresis was used for quantification of the isoforms and their substructures were further elucidated by N-glycan mapping techniques. The analytical study was carried out on a total of 40 batches of epoetin beta which were selected to cover an adequate range of precisely established potency values. The relationship between the biological and chemical parameters was evaluated statistically in order to identify suitable covariates for the prediction of the biological activity. Out of several alternatives, a prediction model which is based on the percentages of isoforms per batch and the degree of sialidation was selected and tested. This model is comparable in terms of accuracy to the established in vivo bioassay, but is far superior in terms of precision. Further advantages of the method are improved animal welfare and savings in time and effort. The question whether the prediction model already meets the requirements for replacing the bioassay according to the ICH guideline Q6B is discussed.
Asunto(s)
Alternativas a las Pruebas en Animales , Electroforesis Capilar , Eritropoyetina/análisis , Hematínicos/análisis , Secuencia de Aminoácidos , Alternativas a las Pruebas en Animales/métodos , Alternativas a las Pruebas en Animales/normas , Animales , Resinas de Intercambio Aniónico , Bioensayo , Cromatografía por Intercambio Iónico , Eritropoyetina/química , Eritropoyetina/farmacología , Eritropoyetina/normas , Glicosilación , Hematínicos/química , Hematínicos/farmacología , Hematínicos/normas , Hematopoyesis/efectos de los fármacos , Modelos Lineales , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Análisis de Componente Principal , Conformación Proteica , Isoformas de Proteínas , Control de Calidad , Proteínas Recombinantes , Reproducibilidad de los Resultados , Ácidos Siálicos/análisis , Resonancia por Plasmón de SuperficieRESUMEN
The chromosomal translocation t(4;11)(q21;q23) is a frequent genetic aberration of the mixed lineage leukemia (MLL) gene, predominantly associated with high-risk acute lymphoblastic leukemia (ALL) in pediatric patients. Previous studies demonstrated that mice transplanted with hematopoietic cells expressing the AF4-MLL fusion protein develop proB ALL. The AF4-MLL oncoprotein becomes activated by Taspase1-mediated hydrolysis, which subsequently leads to a heterodimer of the cleavage products AF4-MLL·N and MLL·C. This protein-protein interaction is due to the FYRN and FYRC interaction domains present in both protein fragments. Heterodimerization subsequently induces high-molecular-weight protein complex formation that is protected against SIAH1/2-mediated polyubiquitinylation. Here, we attempted to selectively block this initial heterodimerization step, aiming to prevent the oncogenic activation of the AF4-MLL multiprotein complex. The minimal interaction interface was experimentally defined first in a bacterial two-hybrid system, and then in mammalian cells by using a biosensor assay. Expression of the FYRC domain, or smaller portions thereof, resulted in the inhibition of heterodimer formation, and blocked AF4-MLL multiprotein complex formation with subsequent destruction of the AF4-MLL oncoprotein. Thus, it is in principle possible to specifically target the AF4-MLL protein. This knowledge can now be exploited to design inhibitory decoys in order to destroy the AF4-MLL oncoprotein.
Asunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 4/genética , Proteínas de Unión al ADN/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Translocación Genética , Proteínas de Unión al ADN/genética , Células HeLa , N-Metiltransferasa de Histona-Lisina , Humanos , Immunoblotting , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Multimerización de Proteína , Estructura Terciaria de Proteína , Factores de Elongación TranscripcionalRESUMEN
Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Epigénesis Genética , Leucemia/etiología , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Proteínas Nucleares/fisiología , Proteínas de Fusión Oncogénica/fisiología , Factor B de Elongación Transcripcional Positiva/metabolismo , Cromatografía en Gel , Proteínas de Unión al ADN/aislamiento & purificación , Activación Enzimática , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Metilación , Proteína de la Leucemia Mieloide-Linfoide/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Proteínas de Fusión Oncogénica/aislamiento & purificación , Fosforilación , Factores de Elongación TranscripcionalAsunto(s)
Anticuerpos Monoclonales/farmacología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Hipoglucemiantes/uso terapéutico , Inmunoterapia , Insulina/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Complejo CD3/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Diabetes Mellitus Tipo 1/patología , Humanos , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patologíaRESUMEN
Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.
Asunto(s)
Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Recombinación Genética , Translocación Genética , Enfermedad Aguda , Adulto , Biopsia , Médula Ósea/química , Médula Ósea/patología , Niño , Rotura Cromosómica , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/ultraestructura , Biología Computacional , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Duplicación de Gen , N-Metiltransferasa de Histona-Lisina , Humanos , Reacción en Cadena de la PolimerasaAsunto(s)
Vino , Antioxidantes/química , Antioxidantes/farmacología , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/prevención & control , Humanos , Enfermedades Neurodegenerativas/epidemiología , Enfermedades Neurodegenerativas/prevención & control , Resveratrol , Sirtuina 1 , Sirtuinas/genética , Estilbenos/química , Estilbenos/farmacología , Vino/análisisAsunto(s)
Inversión Cromosómica , Proteínas de Unión al ADN/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Adolescente , Cromosomas Humanos Par 11 , Supervivencia sin Enfermedad , Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Neoplasias Primarias Secundarias/genética , Proteínas de Fusión Oncogénica , TransactivadoresAsunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 19/genética , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Trans-Empalme/genética , Factores de Transcripción/genética , Transcripción Genética , Translocación Genética/genética , Enfermedad Aguda , Adulto , Animales , Niño , Rotura Cromosómica , Cromosomas Humanos Par 11/ultraestructura , Cromosomas Humanos Par 19/ultraestructura , ADN de Neoplasias/genética , Exones/genética , N-Metiltransferasa de Histona-Lisina , Humanos , Intrones/genética , Ratones , Proteína de la Leucemia Mieloide-Linfoide/química , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesisRESUMEN
The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA double-strand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.
Asunto(s)
Proteína de la Leucemia Mieloide-Linfoide/genética , Regiones Promotoras Genéticas , Recombinación Genética , Transcripción Genética , Animales , Cromatina , Etopósido , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones , ARN Polimerasa IIRESUMEN
The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.
Asunto(s)
Apoptosis , Ciclo Celular , Cromosomas Humanos Par 4/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Animales , Células Cultivadas , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Perfilación de la Expresión Génica , Humanos , Ratones , Mutación/genética , Reacción en Cadena de la Polimerasa , Factores de Transcripción/genéticaRESUMEN
Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.