RESUMEN
PURPOSE: The aim of this study was to evaluate the oral exposure predictions obtained early in drug discovery with a generic GastroPlus Advanced Compartmental And Transit (ACAT) model based on the in vivo intravenous blood concentration-time profile, in silico properties (lipophilicity, pKa) and in vitro high-throughput absorption-distribution-metabolism-excretion (ADME) data (as determined by PAMPA, solubility, liver microsomal stability assays). METHODS: The model was applied to a total of 623 discovery molecules and their oral exposure was predicted in rats and/or dogs. The predictions of Cmax, AUClast and Tmax were compared against the observations. RESULTS: The generic model proved to make predictions of oral Cmax, AUClast and Tmax within 3-fold of the observations for rats in respectively 65%, 68% and 57% of the 537 cases. For dogs, it was respectively 77%, 79% and 85% of the 124 cases. Statistically, the model was most successful at predicting oral exposure of Biopharmaceutical Classification System (BCS) class 1 compounds compared to classes 2 and 3, and was worst at predicting class 4 compounds oral exposure. CONCLUSION: The generic GastroPlus ACAT model provided reasonable predictions especially for BCS class 1 compounds. For compounds of other classes, the model may be refined by obtaining more information on solubility and permeability in secondary assays. This increases confidence that such a model can be used in discovery projects to understand the parameters limiting absorption and extrapolate predictions across species. Also, when predictions disagree with the observations, the model can be updated to test hypotheses and understand oral absorption.
Asunto(s)
Descubrimiento de Drogas/métodos , Preparaciones Farmacéuticas/metabolismo , Animales , Simulación por Computador , Perros , Humanos , Absorción Intestinal/fisiología , Masculino , Modelos Biológicos , Permeabilidad , Ratas , Ratas Sprague-Dawley , SolubilidadRESUMEN
BACKGROUND: Division of the rectum following total mesorectal excision (TME) using intracorporeal stapling devices is technically difficult due to their width and limited roticulation. More than one cartridge is often required and resultant wedging of the stump may be associated with an appreciable leak rate. METHODS: Three-dimensional reconstruction was performed of CT and MRI images from the lower abdomen of six patients undergoing laparoscopic TME using the Amira software environment. The stapling device was virtually reconstructed by in-house developed software, superimposed over the point of division of the rectum and the site of skin entry identified. RESULTS: The 45 degrees angulation of available roticulating stapling devices precludes perpendicular division of the rectum following laparoscopic TME. The optimal angulation for transverse rectal stapling varied between 62 degrees and 68 degrees . CONCLUSION: A roticulating stapler with minimum angulation of 65 degrees would achieve transverse division of the rectum following laparoscopic TME.
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Laparoscopía , Neoplasias del Recto/cirugía , Recto/cirugía , Grapado Quirúrgico/métodos , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Huesos Pélvicos/diagnóstico por imagen , Huesos Pélvicos/patología , Neoplasias del Recto/diagnóstico por imagen , Neoplasias del Recto/patología , Tomografía Computarizada por Rayos X , Interfaz Usuario-ComputadorRESUMEN
The p38 mitogen-activated protein kinase (p38) is a key signaling pathway involved in regulation of inflammatory cytokines. Unexpectedly, several clinical studies using p38 inhibitors found no convincing clinical efficacy in the treatment of chronic inflammation. It was the objective of this study to characterize the population pharmacokinetics (PK) of BCT197 in healthy volunteers and to examine the relationship between BCT197 exposure and pharmacodynamics (PD) measured as inhibition of ex vivo lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα), a downstream marker of p38 activity. PK was characterized using a two-compartment model with mixed-order absorption and limited-capacity tissue binding. The PK-PD relationship revealed that suppression of TNFα was partly offset over time, despite continuous drug exposure. This may indicate a mechanism by which the inflammatory response acquires the ability to bypass p38. Simulations of posology dependence in drug effect suggest that an intermittent regimen may offer clinical benefit over continuous dosing and limit the impact of tolerance development.
RESUMEN
Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells. Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5%. T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells. A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining. When selection for transformation competence is applied, the picture is completely different. Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome. Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors.
Asunto(s)
Agrobacterium tumefaciens/genética , Arabidopsis/genética , ADN Bacteriano/genética , Raíces de Plantas/genética , Recombinación Genética , Proteínas Virales , Arabidopsis/citología , Arabidopsis/microbiología , ADN Bacteriano/fisiología , Eliminación de Gen , Técnicas de Transferencia de Gen , Genes Reporteros , Glucuronidasa/genética , Glucuronidasa/metabolismo , Integrasas/genética , Integrasas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/microbiología , Reacción en Cadena de la Polimerasa , Transformación GenéticaRESUMEN
In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.
Asunto(s)
Agrobacterium tumefaciens/genética , Arabidopsis/genética , Nicotiana/genética , Plantas Tóxicas , Transformación Genética , ADN Bacteriano/genética , Raíces de Plantas/microbiología , Protoplastos/metabolismoRESUMEN
The aim of this study was to determine the intestinal absorption characteristics of the antiviral agent UC-781 and to optimize the experimental conditions of the in vitro system for low solubility compounds. The absorption potential of UC-781 was studied with the Caco-2 system and with the rat intestinal perfusion technique. The low solubility of UC-781 required the use of solubility/dissolution rate enhancing agents (e.g. VitE-TPGS, Gelucire 44/14). The creation of sink conditions in the receiver compartment of the Caco-2 system was a prerequisite to reliably study the transport of this poorly soluble compound. After inclusion of VitE-TPGS in the acceptor solution, UC-781 could be characterized as a class II drug of the Biopharmaceutical Classification System (low solubility, high permeation across membranes). A significant concentration-dependent decrease in transport of UC-781 was observed upon increasing the concentration of VitE-TPGS in the apical compartment. This observation contrasts to the absorption enhancing properties of VitE-TPGS, and can probably be attributed to a decrease in the concentration of free UC-781 when using higher concentrations of the solubility/dissolution rate enhancing agents. The use of Gelucire 44/14 as a solubilizing agent resulted in a batch-dependent degradation of UC-781. The inclusion of the solubility/dissolution rate-enhancing agent VitE-TPGS did not result in absorption enhancement in the intestinal perfusion technique.
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Anilidas/farmacocinética , Furanos/farmacocinética , Absorción Intestinal , Inhibidores de la Transcriptasa Inversa/farmacocinética , Anilidas/administración & dosificación , Anilidas/química , Animales , Transporte Biológico , Células CACO-2 , Cromatografía Líquida de Alta Presión , Sistemas de Liberación de Medicamentos , Furanos/administración & dosificación , Furanos/química , Humanos , Masculino , Microdiálisis , Perfusión , Ratas , Ratas Wistar , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/química , Solubilidad , Espectrofotometría Ultravioleta , TioamidasRESUMEN
The acquisition of data for cardiac imaging using a C-arm computed tomography system requires several seconds and multiple heartbeats. Hence, incorporation of motion correction in the reconstruction step may improve the resulting image quality. Cardiac motion can be estimated by deformable three-dimensional (3D)/3D registration performed on initial 3D images of different heart phases. This motion information can be used for a motion-compensated reconstruction allowing the use of all acquired data for image reconstruction. However, the result of the registration procedure and hence the estimated deformations are influenced by the quality of the initial 3D images. In this paper, the sensitivity of the 3D/3D registration step to the image quality of the initial images is studied. Different reconstruction algorithms are evaluated for a recently proposed cardiac C-arm CT acquisition protocol. The initial 3D images are all based on retrospective electrocardiogram (ECG)-gated data. ECG-gating of data from a single C-arm rotation provides only a few projections per heart phase for image reconstruction. This view sparsity leads to prominent streak artefacts and a poor signal to noise ratio. Five different initial image reconstructions are evaluated: (1) cone beam filtered-backprojection (FDK), (2) cone beam filtered-backprojection and an additional bilateral filter (FFDK), (3) removal of the shadow of dense objects (catheter, pacing electrode, etc) before reconstruction with a cone beam filtered-backprojection (cathFDK), (4) removal of the shadow of dense objects before reconstruction with a cone beam filtered-backprojection and a bilateral filter (cathFFDK). The last method (5) is an iterative few-view reconstruction (FV), the prior image constrained compressed sensing combined with the improved total variation algorithm. All reconstructions are investigated with respect to the final motion-compensated reconstruction quality. The algorithms were tested on a mathematical phantom data set with and without a catheter and on two porcine models using qualitative and quantitative measures. The quantitative results of the phantom experiments show that if no dense object is present within the scan field of view, the quality of the FDK initial images is sufficient for motion estimation via 3D/3D registration. When a catheter or pacing electrode is present, the shadow of these objects needs to be removed before the initial image reconstruction. An additional bilateral filter shows no major improvement with respect to the final motion-compensated reconstruction quality. The results with respect to image quality of the cathFDK, cathFFDK and FV images are comparable. In conclusion, in terms of computational complexity, the algorithm of choice is the cathFDK algorithm.
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Artefactos , Tomografía Computarizada de Haz Cónico/métodos , Corazón/diagnóstico por imagen , Corazón/fisiología , Imagenología Tridimensional/métodos , Movimiento , Animales , Técnicas de Imagen Sincronizada Cardíacas , Fantasmas de Imagen , PorcinosRESUMEN
There is increasing use of three-dimensional rotational angiography (3DRA) during cardiac ablation procedures. As compared with 2D angiography, a large series of images are acquired, creating the potential for high radiation doses. The aim of the present study was to quantify patient-specific effective doses. In this study, we developed a computer model to accurately calculate organ doses and the effective dose incurred during 3DRA image acquisition. The computer model simulates the exposure geometry and uses the actual exposure parameters, including the variation in tube voltage and current that is realized through the automatic exposure control (AEC). We performed 3DRA dose calculations in 42 patients referred for ablation on the Siemens Axiom Artis DynaCT system (Erlangen, Germany). Organ doses and effective dose were calculated separately for all projections in the course of the C-arm rotation. The influence of patient body mass index (BMI), dose-area product (DAP), collimation and dose per frame (DPF) rate setting on the calculated doses was also analysed. The effective dose was found to be 5.5 +/- 1.4 mSv according to ICRP 60 and 6.6 +/- 1.8 mSv according to ICRP 103. Effective dose showed an inversely proportional relationship to BMI, while DAP was nearly BMI independent. No simple conversion coefficient between DAP and effective dose could be derived. DPF reduction did not result in a proportional effective dose decrease. These paradoxical findings were explained by the settings of the AEC and the limitations of the x-ray tube. Collimation reduced the effective dose by more than 20%. Three-dimensional rotational angiography is associated with a definite but acceptable radiation dose that can be calculated for all patients separately. Their BMI is a predictor of the effective dose. The dose reduction achieved with collimation suggests that its use is imperative during the 3DRA procedure.
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Algoritmos , Ablación por Catéter/métodos , Angiografía Coronaria/métodos , Imagenología Tridimensional/métodos , Dosis de Radiación , Planificación de la Radioterapia Asistida por Computador/métodos , Arritmias Cardíacas/diagnóstico por imagen , Arritmias Cardíacas/cirugía , Automatización , Índice de Masa Corporal , Simulación por Computador , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Rotación , Tomografía Computarizada por Rayos X/métodosRESUMEN
The transgene locus KH15, which is highly susceptible to silencing in Arabidopsis thaliana, contains two inversely repeated beta-glucuronidase (gus) genes separated by a palindromic sequence and has a low GUS activity, was found to be heavily methylated in the gus coding sequence and in the center of the inverted repeat. The locus KHsb67, which is less prone to silencing, was found to be less densely methylated in the non-repetitive region that separates the inversely repeated gus genes. After the removal of one of the gus genes by Cre-mediated recombination, methylation in both loci decreased or was totally lost. Despite the presence of a 732-bp palindromic sequence in the deletion line derived from KH15, this sequence was not methylated. Whereas the KH15 locus triggers methylation of homologous gus genes when placed in trans to them, the deletion derivative did not, suggesting that the capacity for cross-talk was severely affected by disruption of the palindromic arrangement. This result suggests that the transcribed palindromic sequences are required to maintain the methylation of both symmetrically and non-symmetrically arranged cytosines.
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Arabidopsis/genética , Glucuronidasa/genética , Plantas Modificadas Genéticamente/genética , Metilación de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Silenciador del Gen , Glucuronidasa/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
Locus KH15, containing two invertedly repeated beta-glucuronidase (gus) genes separated by a palindromic sequence showed low gus expression in both hemizygous and homozygous seedlings, in all plant tissues tested and throughout development assayed up to 70 days after sowing. Removal of one of the gus genes by Cre-mediated recombination resulted in a 10- to 100-fold increase of gus expression, confirming that inverted repeats and transgene silencing are strictly correlated. Crosses between the locus KH15 and highly expressing gus transformants further revealed that invertedly repeated gus genes could induce in trans silencing of gus copies at allelic and nonallelic positions. Double-stranded RNA produced by this KH15 may be responsible for this in trans inactivation.
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Arabidopsis/genética , Silenciador del Gen , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Plantas Modificadas Genéticamente , Secuencias Repetitivas de Ácidos Nucleicos , TransgenesRESUMEN
To analyse experimentally the correlation between transgene silencing and the presence of an inverted repeat in transgenic Arabidopsis thaliana plants, expression of the beta-glucuronidase (gus) gene was studied when present as a convergently transcribed inverted repeat or as a single copy in otherwise isogenic lines. In transformants containing two invertedly repeated gus genes separated by a 732 bp palindromic sequence, gus expression was low, as exemplified by the expression levels in the parental line KH15. The parental KH15 locus could induce efficiently in trans silencing of gus copies at allelic and non-allelic positions. In transformants containing two invertedly repeated gus genes separated by a 826 bp non-repetitive spacer region, gus expression was high or intermediate, especially in hemizygous state and at late developmental stages, as demonstrated in detail for line KHsb67. Removal of one of the gus copies by Cre recombinase resulted in all cases in constitutively high gus expression in hemizygous as well as in homozygous state. The derived deletion lines could no longer induce in trans silencing of homologous gus copies. The results show that convergent transcription of transgenes in an inverted repeat is an important parameter to trigger their silencing and that co-transformation of two T-DNAs with identical transgenes can be used to obtain inverted repeats and targeted co-suppression of the homologous endogenes. Moreover, the data suggest that the spacer region in between the inverted genes plays a role in the efficiency of initiating and maintaining silencing.
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Eliminación de Gen , Silenciador del Gen , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transgenes/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrolloRESUMEN
This study aimed at determining whether transgene expression variability is observed in single-copy T-DNA plants and whether it can be correlated with the T-DNA integration position. Among a population of 135 Arabidopsis thaliana transformants, selected on the basis of antibiotic resistance marker expression, 21 single-copy T-DNA transformants were identified and characterized. In 19 of these 21 lines, 35S-beta-glucuronidase transgene expression, measured in two subsequent generations, was similar. This observation means that the intra-transformant variability was as high as the inter-transformant variability. Integration into an intergenic or genic region, into an exon or intron, in sense or antisense orientation, did not result in differential transgene expression. Remarkably, single-copy transformants were not always the highest expressers, implying that low transgene expression is not always induced by multicopy transformants. In only 2 of the 21 single-copy plants was the transgene expression more than 20-fold lower. However, characteristics of the insertion position in one of these lines did not differ significantly when compared to high-expressing lines. In the remaining line, methylation of the transgene was clearly demonstrated. In conclusion, screening for single-copy T-DNA transformants greatly enriches for stable and high transgene expression, because the integration position is not a major determinant of transgene expression variability in Arabidopsis.
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Arabidopsis/genética , ADN Bacteriano/química , Genoma de Planta , Glucuronidasa/metabolismo , Plantas Modificadas Genéticamente , ADN Complementario/metabolismo , ADN de Plantas , Fluorometría , Genes de Plantas , Modelos Genéticos , Oligonucleótidos Antisentido/farmacología , Estructura Terciaria de Proteína , TransgenesRESUMEN
Nicotiana protoplasts and Arabidopsis leaf discs or roots were co-cultivated with two Agrobacterium strains each carrying a different T-DNA. Co-transformed plants were selected and the integration of the different T-DNAs was analysed at the genetic and genomic level. Genetic analysis showed that the T-DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used. Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co-transferred T-DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage. Overall, our data support the hypothesis that ligation of separate T-DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T-DNAs. We propose a scheme which could explain the formation of T-DNA repeats and the preferential involvement of right borders in T-DNA linkages.
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ADN Bacteriano/genética , Plantas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Rhizobium/genética , Transformación Genética , Arabidopsis/genética , Arabidopsis/microbiología , Ligamiento Genético , Meiosis , Modelos Genéticos , Plantas/microbiología , Plantas Tóxicas , Selección Genética , Especificidad de la Especie , Nicotiana/genética , Nicotiana/microbiologíaRESUMEN
After Agrobacterium-mediated plant transformation, multiple T-DNAs frequently integrate at the same position in the plant genome, resulting in the formation of inverted and direct repeats. Because these inverted repeats cannot be amplified and analyzed by PCR, Arabidopsis root cells were co-transformed with two different T-DNAs with distinct sequences adjacent to the T-DNA borders. Nine direct or inverted T-DNA border junctions were analyzed at the sequence level. Precise end-to-end fusions were found between two right border ends, whereas imprecise fusions and filler DNA were present in T-DNA linkages containing a left border end. The results suggest that end-to-end ligation of double-stranded T-DNAs occurs especially between right T-DNA ends and that illegitimate recombination on the basis of microhomology, deletions, repair activities and insertions of filler DNA is involved in the formation of left border T-DNA junctions. Therefore, a similar illegitimate recombination mechanism is proposed that is involved in the formation of complex T-DNA inserts as well as in the integration of the T-DNA in the plant genome.
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Agrobacterium tumefaciens/genética , Arabidopsis/genética , ADN Bacteriano/genética , Genoma de Planta , Transformación Genética , Secuencia de Bases , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADNRESUMEN
Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.
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Proteínas de Plantas/biosíntesis , Plantas/genética , Reactores Biológicos , Silenciador del Gen , Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Transgenes/genéticaRESUMEN
The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed.