RESUMEN
The use of patient-derived xenografts (PDXs) in cancer research is increasing due to their ability to closely mimic the features of patient tumors. The ability to quickly and robustly measure protein expression levels in these tissues is a key methodology required in a broad range of experimental designs. Western blotting (WB) is a cost effective and simple tool that is highly specific and sensitive for detecting and quantifying individual proteins, posttranslational modifications and aberrant signaling pathways. Here, we described a method to assess protein expression in PDX tissues using WB to detect proteins involved in cell growth signaling pathways.
Asunto(s)
Western Blotting , Humanos , Animales , Western Blotting/métodos , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Xenoinjertos , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sex steroid hormones have been implicated as disease modifiers in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Androgens, signalling via the androgen receptor (AR), predominate in males, and have widespread actions in the periphery and the central nervous system (CNS). AR translocates to the cell nucleus when activated upon binding androgens, whereby it regulates transcription of target genes via the classical genomic signalling pathway. We previously reported that AR protein is decreased in the lumbar spinal cord tissue of symptomatic male SOD1G93A mice. Here, we further explored the changes in AR within motor neurons (MN) of the CNS, assessing their nuclear AR content and propensity to degenerate by endstage disease in male SOD1G93A mice. We observed that almost all motor neuron populations had undergone significant loss in nuclear AR in SOD1G93A mice. Interestingly, loss of nuclear AR was evident in lumbar spinal MNs as early as the pre-symptomatic age of 60 days. Several MN populations with high AR content were identified which did not degenerate in SOD1G93A mice. These included the brainstem ambiguus and vagus nuclei, and the sexually dimorphic spinal MNs: cremaster, dorsolateral nucleus (DLN) and spinal nucleus of bulbocavernosus (SNB). In conclusion, we demonstrate that AR loss directly associates with MN vulnerability and disease progression in the SOD1G93A mouse model of ALS.
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Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Andrógenos/metabolismo , Animales , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína Fosfatasa 2/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Fosforilación , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Especificidad por SustratoRESUMEN
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder caused by loss of motor neurons. ALS incidence is skewed towards males with typically earlier age of onset and limb site of onset. The androgen receptor (AR) is the major mediator of androgen effects in the body and is present extensively throughout the central nervous system, including motor neurons. Mutations in the AR gene lead to selective lower motor neuron degeneration in male spinal bulbar muscular atrophy (SBMA) patients, emphasising the importance of AR in maintaining motor neuron health and survival. To evaluate a potential role of AR in onset and progression of ALS, we generated SOD1G93A mice with either neural AR deletion or global human AR overexpression. Using a Cre-LoxP conditional gene knockout strategy, we report that neural deletion of AR has minimal impact on the disease course in SOD1G93A male mice. This outcome was potentially confounded by the metabolically disrupted Nestin-Cre phenotype, which likely conferred the profound lifespan extension observed in the SOD1G93A double transgenic male mice. In addition, overexpression of human AR produced no benefit to disease onset and progression in SOD1G93A mice. In conclusion, the disease course of SOD1G93A mice is independent of AR expression levels, implicating other mechanisms involved in mediating the sex differences in ALS. Our findings using Nestin-Cre mice, which show an inherent metabolic phenotype, led us to hypothesise that targeting hypermetabolism associated with ALS may be a more potent modulator of disease, than AR in this mouse model.
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Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/patología , Receptores Androgénicos/metabolismo , Médula Espinal/patología , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Fenotipo , Factores Sexuales , Médula Espinal/metabolismo , Superóxido Dismutasa-1/metabolismoRESUMEN
The Cre-loxP strategy for tissue selective gene deletion has become a widely employed tool in neuroscience research. The validity of these models is largely underpinned by the temporal and spatial selectivity of recombinase expression under the promoter of the Cre driver line. Ectopic Cre-recombinase expression gives rise to off-target effects which can confound results and is especially detrimental if this occurs in germline cells. The Nestin-Cre transgenic mouse is broadly used for selective gene deletion in neurons of the central and peripheral nervous systems. Here we have crossed this mouse with a floxed androgen receptor (AR) transgenic to generate double transgenic neuronal ARKO mice (ARflox ::NesCre) to study germline deletion in male and female transgenic breeders. In male ARflox ::NesCre breeders, a null AR allele was passed on to 86% of progeny regardless of the inheritance of the NesCre transgene. In female ARflox/wt ::NesCre breeders, a null AR allele was passed on to 100% of progeny where ARflox was expected to be transmitted. This surprisingly high incidence of germline recombination in the Nestin-Cre driver line warrants caution in devising suitable breeding strategies, consideration of accurate genotyping approaches and highlights the need for thorough characterization of tissue-specific gene deletion in this model.
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Nestina/genética , Receptores Androgénicos/genética , Recombinación Genética , Animales , Femenino , Ingeniería Genética/métodos , Células Germinativas/metabolismo , Integrasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , TransgenesRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting motor neurons which shows sexual dimorphism in its incidence, age of onset, and progression rate. All steroid hormones, including androgens, estrogens, and progestogens, have been implicated in modulating ALS. Increasing evidence suggests that steroid hormones provide neuroprotective and neurotrophic support to motor neurons, either directly or via surrounding glial cell interactions, by activating their respective nuclear hormone receptors and initiating transcriptional regulatory responses. The SOD1G93A transgenic mouse also shows sex-specific differences in age of onset and progression, and remains the most widely used model in ALS research. To provide a more comprehensive understanding of the influences of steroid hormone signaling in ALS, we systemically characterized sex hormone receptor expression at transcript and protein levels, cellular localization, and the impact of disease course in lumbar spinal cords of male and female SOD1G93A mice. We found that spinal motor neurons highly express nuclear androgen receptor (AR), estrogen receptor (ER)α, ERß, and progesterone receptor with variations in glial cell expression. AR showed the most robust sex-specific difference in expression and was downregulated in male SOD1G93A mouse spinal cord, in association with depletion in 5α-reductase type 2 isoform, which primarily metabolizes testosterone to 5α-dihydrotestosterone. ERα was highly enriched in reactive astrocytes of SOD1G93A mice and ERß was strongly upregulated. The 5α-reductase type 1 isoform was upregulated with disease progression and may influence local spinal cord hormone levels. In conclusion, steroid hormone receptor expression is dynamic and cell-type specific in SOD1G93A mice which may provide targets to modulate progression in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/metabolismo , Neuroglía/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Hormonas Esteroides Gonadales/análisis , Hormonas Esteroides Gonadales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Neuroglía/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Médula Espinal/química , Médula Espinal/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa-1/genéticaRESUMEN
BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease typically more common in males, implicating androgens in progression of both patients and mouse models. Androgen effects are mediated by androgen receptor which is highly expressed in spinal motor neurons and skeletal muscles. To clarify the role of androgen receptors in ALS, we therefore examined the effect of androgen receptor antagonism in the SOD1G93A mouse model. EXPERIMENTAL APPROACH: The androgen receptor antagonist, flutamide, was administered to presymptomatic SOD1G93A mice as a slow-release subcutaneous implant (5 mg·day-1 ). Testosterone, flutamide, and metabolite levels were measured in blood and spinal cord tissue by LC-MS-MS. Effects on disease onset and progression were assessed using motor function tests, survival, muscle, and neuropathological analyses. KEY RESULTS: Flutamide was metabolised to 2-hydroxyflutamide achieving steady-state plasma levels across the study duration and reached the spinal cord at pharmacologically active concentrations. Flutamide treatment accelerated disease onset and locomotor dysfunction in male SOD1G93A mice, but not female mice, without affecting survival. Analysis of hindlimb muscles revealed exacerbation of myofibre atrophy in male SOD1G93A mice treated with flutamide, although motor neuron pathology was not affected. CONCLUSION AND IMPLICATIONS: The androgen receptor antagonist accelerated disease onset in male SOD1G93A mice, leading to exacerbated muscle pathology, consistent with a role of androgens in modulating disease severity, sexual dimorphism, and peripheral pathology in ALS. These results also demonstrate a key contribution of skeletal muscle pathology to disease onset, but not outcome, in this mouse model of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Antagonistas de Receptores Androgénicos/farmacología , Flutamida/farmacología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Madre Embrionarias , Femenino , Humanos , Masculino , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Neuroglía/efectos de los fármacos , Próstata/efectos de los fármacos , Próstata/patología , Receptores Androgénicos/metabolismo , Vesículas Seminales/efectos de los fármacos , Vesículas Seminales/patología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Testosterona/sangreRESUMEN
PEGylation typically improves the systemic exposure and tumor biodistribution of polymeric drug delivery systems, but may also restrict enzyme access to peptide-based drug linkers. The impact of dendrimer generation (G4 vs G5) and PEG length (570 vs 1100 Da) on the pharmacokinetics, tumor biodistribution, drug release kinetics, and anticancer activity of a series of PEGylated polylysine dendrimers conjugated with doxorubicin via a cathepsin-B cleavable valine-citrulline linker was therefore investigated in rodents. Although the smallest G4 PEG570 dendrimer showed the most efficient cathepsin-mediated doxorubicin release, systemic exposure and tumor uptake were limited. The largest G5 PEG1100 dendrimer showed good tumor uptake and retention but restricted drug liberation and therefore limited anticancer activity. Superior anticancer activity was achieved using an intermediate sized dendrimer that showed better drug release kinetics, systemic exposure, tumor uptake, and retention. The data suggest that balancing PEG molecular weight and dendrimer size is critical when designing chemotherapeutic dendrimers.
Asunto(s)
Catepsinas/química , Dendrímeros/química , Doxorrubicina/química , Polilisina/química , Células A549 , Animales , Catepsina B/química , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Humanos , Masculino , Polietilenglicoles/química , RatasRESUMEN
PEGylated polylysine dendrimers have demonstrated potential as inhalable drug delivery systems that can improve the treatment of lung cancers. Their treatment potential may be enhanced by developing constructs that display prolonged lung retention, together with good systemic absorption, the capacity to passively target lung tumors from the blood and highly selective, yet rapid liberation in the tumor microenvironment. This study sought to characterize how the nature of cathepsin B-cleavable peptide linkers, used to conjugate doxorubicin (Dox) to a PEGylated (PEG570) G4 polylysine dendrimer, affects drug liberation kinetics and intravenous and pulmonary pharmacokinetics in rats. The construct bearing a self-emolative diglycolic acid-V-Citrulline linker exhibited faster Dox release kinetics compared to constructs bearing self-emolative diglycolic acid-glycine-leucine-phenylalanine-glycine (GLFG), or non-self-emolative glutaric acid-GLFG linkers. The V-Citrulline construct exhibited slower plasma clearance, but faster absorption from the lungs than a GLFG construct, although mucociliary clearance and urinary elimination were unchanged. Dox-conjugation enhanced localization in the bronchoalveolar lavage fluid compared to lung tissue, suggesting that projection of Dox from the dendrimer surface reduced tissue uptake. These data show that the linker chemistry employed to conjugate drugs to PEGylated carriers can affect drug release profiles and systemic and lung disposition.
Asunto(s)
Dendrímeros/química , Doxorrubicina/química , Pulmón/metabolismo , Polietilenglicoles/química , Polilisina/química , Administración por Inhalación , Administración Intravenosa , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dendrímeros/administración & dosificación , Dendrímeros/farmacocinética , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Pulmón/efectos de los fármacos , Masculino , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacocinética , Polilisina/administración & dosificación , Polilisina/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
Therapeutic proteins can facilitate the targeting and treatment of lymphatic diseases (such as cancer metastases, infections and inflammatory diseases) since they are cleared via the lymphatics following interstitial (SC or IM) administration. However, therapeutic proteins are often administered intravenously (IV). Recently therapeutic proteins have been found to access the thoracic lymph in surprisingly high quantities after IV administration. The aim of this study was to determine, for the first time, the major sites of thoracic lymph access of therapeutic proteins, and the protein properties that enhance lymph access, after IV administration. In order to achieve this, novel methods were developed or optimized to collect hepatic, mesenteric or thoracic lymph from male SD rats. Four different sized PEGylated or non-PEGylated therapeutic proteins (native interferon α2b (IFN, 19kDa), PEGylated interferon α2b (IFN-PEG12, 31kDa), PEGylated interferon α2a (IFN-PEG40, 60kDa) or trastuzumab (150kDa)) were then administered via short IV infusion, and plasma and lymph concentrations of the proteins determined via ELISA. The recovery of the therapeutic proteins in the thoracic lymph duct, which collects lymph from most of the body, was significantly greater for trastuzumab, IFN-PEG40 and IFN-PEG12 (all >3% dose over 8h) when compared to native IFN (0.9% dose). Conversely, the thoracic lymph/plasma (L/P) concentration ratio and thus efficiency of extravasation and transport through the interstitium to lymph was highest for the smaller proteins IFN and IFN-PEG12 (at 90-100% vs 15-30% for trastuzumab and IFN-PEG40). The lower total recovery of IFN and IFN-PEG12 in thoracic lymph reflected more rapid systemic clearance and thus lower systemic exposure. For all therapeutic proteins, the majority (>80%) of lymph access occurred via the hepatic and mesenteric lymphatics. This lymphatic distribution pattern was supported by quantitative imaging of the lymph node distribution of IV administered Cy5 labelled trastuzumab. Optimizing the properties of IV administered therapeutic proteins represents a viable approach to better target and treat pathological states involving the lymphatics, particularly in the liver and mesentery. This includes cancer metastases, infections and inflammatory diseases. Successful development of the novel technique to collect hepatic lymph will also enable future work to evaluate tissue-specific lymph transport in health and disease.
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Interferones/administración & dosificación , Linfa/metabolismo , Polietilenglicoles/administración & dosificación , Trastuzumab/administración & dosificación , Administración Intravenosa , Animales , Interferones/química , Interferones/farmacocinética , Hígado , Masculino , Mesenterio , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Ratas Sprague-Dawley , Tórax , Trastuzumab/farmacocinéticaRESUMEN
Drug conjugation to dendrimer-based delivery systems has been shown to enhance delivery to the lymphatic system after subcutaneous administration. Dendrimer interaction with components of the interstitium at the injection site, however, may prevent drainage from the injection site. The current study sought to vary the length of a linker employed to conjugate methotrexate (MTX) to a PEGylated dendrimer, in an attempt to reduce MTX interaction with interstitial binding sites and enhance lymphatic drainage. Dendrimers with shorter linkers resulted in higher lymphatic drainage, presumably via shielding of interaction sites by the PEG mantle, but were not retained in lymph nodes. Improved drainage of dendrimers with longer linkers was achieved through coadministration with dextran to mask interactions at the injection site while maintaining retention within the node. Enhanced drug exposure to the lymph node has the potential to enhance the treatment of lymph-node resident cancer metastases.
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Antimetabolitos Antineoplásicos/administración & dosificación , Dendrímeros/química , Portadores de Fármacos/química , Ganglios Linfáticos/metabolismo , Metotrexato/administración & dosificación , Polietilenglicoles/química , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Sistemas de Liberación de Medicamentos , Metotrexato/farmacocinética , RatasRESUMEN
Interferon α2 is an antiviral/antiproliferative protein that is currently used to treat hepatitis C infections and several forms of cancer. Two PEGylated variants of interferon α2 (containing 12 and 40 kDa PEGs) are currently marketed and display longer plasma circulation times than that of unmodified interferon. With increasing realization that the lymphatic system plays an important role in the extrahepatic replication of the hepatitis C virus and in the metastatic dissemination of cancers, this study sought to evaluate PEGylation strategies to optimally enhance the antiviral activity and plasma and lymphatic exposure of interferon after subcutaneous administration in rats. The results showed that conjugation with a linear 20 kDa PEG provided the most ideal balance between activity and plasma and lymph exposure. A linear 5 kDa PEG variant also exhibited excellent plasma and lymph exposure to interferon activity when compared to those of unmodified interferon and the clinically available linear 12 kDa PEGylated construct.
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Antivirales/síntesis química , Interferón-alfa/síntesis química , Sistema Linfático/metabolismo , Polietilenglicoles/síntesis química , Animales , Antivirales/administración & dosificación , Antivirales/química , Antivirales/farmacocinética , Inyecciones Subcutáneas , Interferón-alfa/administración & dosificación , Interferón-alfa/química , Interferón-alfa/farmacocinética , Masculino , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Distribución TisularRESUMEN
PEGylated polylysine dendrimers are attractive and well tolerated inhalable drug delivery platforms that have the potential to control the release, absorption kinetics and lung retention time of conjugated drugs. The clinical application of these systems though, would likely require partial substitution of surface PEG groups with drug molecules that are anticipated to alter their lung clearance kinetics and clearance pathways. In the current study, we therefore evaluated the impact of increased surface hydrophobicity via substitution of 50% surface PEG groups with a model hydrophobic drug (α-carboxyl OtButylated methotrexate) on the lung clearance of a Generation 5 PEGylated polylysine dendrimer in rats. PEG substitution with OtBu-methotrexate accelerated lung clearance of the dendrimer by increasing polylysine scaffold catabolism, improving systemic absorption of the intact dendrimer and low molecular weight products of scaffold catabolism, and enhancing mucociliary clearance. These results suggest that the conjugation of hydrophobic drug on the surface of a PEGylated dendrimer is likely to accelerate lung clearance when compared to a fully PEGylated dendrimer.
Asunto(s)
Dendrímeros/química , Metotrexato/química , Polietilenglicoles/química , Polilisina/química , Animales , Sistemas de Liberación de Medicamentos/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Pulmón/metabolismo , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Frail older adults present to the Emergency Department (ED) with complex medical, functional, and social needs. When these needs can be addressed promptly, discharge is possible, and when they cannot, hospital admission is required. We evaluated the care needs of frail older adults in the ED who were consulted to internal medicine and seen by a geriatrician to determine, under current practices, which factors were associated with hospitalization and which allowed discharge. METHODS: We preformed a chart-based, exploratory study. Data were abstracted from consultation records and ED charts. All cases had a standard Comprehensive Geriatric Assessment (CGA which records a Clinical Frailty Scale (CFA) and allows calculation of a Frailty Index (FI). RESULTS: Of 100 consecutive patients, 2 died in the ED, 75 were admitted, and 23 were discharged, including one urgent placement. Compared with discharged patients (0.39 ± SD 0.16), those admitted had a higher mean FI-CGA (0.48 ± 0.13; p < .01). Greater mobility dependence (2% in discharged vs. 32% in admitted; p < .05) was notable. CONCLUSIONS: Discharge decisions require assessment of medical, functional, and social problems. Ill, frail patients often can be discharged home when social and nursing support can be provided. The degree of frailty, impaired mobility, and likely delirium must be taken into account when planning for their care.
RESUMEN
PolyPEG star polymers have potential utility as cost-effective polymeric drug delivery vehicles, and as such, it is important to develop an understanding of their biopharmaceutical behavior. Moreover, although a number of studies have evaluated the utility of PolyPEG stars in vitro, investigation of these novel materials in vivo has been limited. Herein, we evaluated the pharmacokinetics of a 64 kDa tritiated PEG-based star polymer after subcutaneous and pulmonary administration in rats. After subcutaneous administration, the star polymer showed near complete bioavailability (â¼80%) and a similar organ biodistribution profile to the polymer after intravenous administration. After intratracheal instillation to the lungs, the star polymer showed limited bioavailability (â¼3%), and most of the administered radiolabel was recovered in lung tissue and feces after 6 d. The data reported here suggest that star polymers display similar pharmaceutical behavior to PEGylated dendrimers after subcutaneous and inhaled delivery and may therefore be used as similar, but more cost-effective drug delivery vehicles.
Asunto(s)
Polietilenglicoles/farmacocinética , Administración por Inhalación , Animales , Disponibilidad Biológica , Biofarmacia , Líquido del Lavado Bronquioalveolar , Sistemas de Liberación de Medicamentos , Heces/química , Inyecciones Subcutáneas , Pulmón/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
PURPOSE: Cancer metastasis to pulmonary lymph nodes dictates the need to deliver chemotherapeutic and diagnostic agents to the lung and associated lymph nodes. Drug conjugation to dendrimer-based delivery systems has the potential to reduce toxicity, enhance lung retention and promote lymphatic distribution in rats. The current study therefore evaluated the pharmacokinetics and lung lymphatic exposure of a PEGylated dendrimer following inhaled administration. METHODS: Plasma pharmacokinetics and disposition of a 22 kDa PEGylated dendrimer were compared after aerosol administration to rats and sheep. Lung-derived lymph could not be sampled in rats and so lymphatic transport of the dendrimer from the lung was assessed in sheep. RESULTS: Higher plasma concentrations were achieved when dendrimer was administered to the lungs of rats as a liquid instillation when compared to an aerosol. Plasma pharmacokinetics were similar between sheep and rats, although some differences in disposition patterns were evident. Unexpectedly, less than 0.5% of the aerosol dose was recovered in pulmonary lymph. CONCLUSIONS: The data suggest that rats provide a relevant model for assessing the pharmacokinetics of inhaled macromolecules prior to evaluation in larger animals, but that the pulmonary lymphatics are unlikely to play a major role in the absorption of nanocarriers from the lungs.
Asunto(s)
Dendrímeros/farmacocinética , Portadores de Fármacos/farmacocinética , Sistemas de Liberación de Medicamentos , Pulmón/metabolismo , Ganglios Linfáticos/metabolismo , Polietilenglicoles/farmacocinética , Administración por Inhalación , Administración Intravenosa , Aerosoles/administración & dosificación , Aerosoles/química , Aerosoles/farmacocinética , Animales , Dendrímeros/administración & dosificación , Dendrímeros/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Femenino , Masculino , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Ratas , Ratas Sprague-Dawley , OvinosRESUMEN
Herein we report for the first time the biological fate of poly[(oligoethylene glycol) acrylate] (POEGA) star polymers synthesised via a versatile arm-first reversible addition-fragmentation chain transfer (RAFT) polymerisation approach. The biopharmaceutical behaviour of three different molecular weight (49, 64 and 94kDa) POEGA stars was evaluated in rats and nude mice bearing human MDA MB-231 tumours after intravenous administration. The 94kDa star polymer exhibited a longer plasma exposure time than the 49kDa or 64kDa star polymer; an observation attributable to differences in the rates of both polymer biodegradation and urinary excretion. Tumour biodistribution also correlated with molecular weight and was greatest for the longest circulating 94kDa star. Different patterns of liver and spleen biodistribution were observed between mice and rats for the different sized polymers. The polymers were also well-tolerated in vivo and in vitro at therapeutic concentrations. FROM THE CLINICAL EDITOR: Advances in nanotechnology has enabled scientists to produce nanoparticle as drug carriers in cancer therapeutics. In this article, the authors studied the biological fate of poly[(oligoethylene glycol) acrylate] (POEGA) star polymers of different size, after intravenous injections. This would allow the subsequent comparison to other drug delivery systems for better drug delivery.
Asunto(s)
Acrilatos/farmacocinética , Portadores de Fármacos/farmacocinética , Polietilenglicoles/farmacocinética , Acrilatos/administración & dosificación , Acrilatos/química , Administración Intravenosa , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Femenino , Humanos , Hidrodinámica , Masculino , Ratones , Ratones Desnudos , Peso Molecular , Neoplasias/metabolismo , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The lymphatic system plays a major role in the metastatic dissemination of cancer and has an integral role in immunity. PEGylation enhances drainage and lymphatic uptake following subcutaneous (sc) administration of proteins and protein-like polymers, but the impact of PEGylation of very large proteins (such as antibodies) on subcutaneous and lymphatic pharmacokinetics is unknown. This study therefore aimed to evaluate the impact of PEGylation on the sc absorption and lymphatic disposition of the anti-HER2 antibody trastuzumab in rats. PEG-trastuzumab was generated via the conjugation of a single 40 kDa PEG-NHS ester to trastuzumab. PEG-trastuzumab showed a 5-fold reduction in HER2 binding affinity, however the in vitro growth inhibitory effects were preserved as a result of changes in cellular trafficking when compared to native trastuzumab. The lymphatic pharmacokinetics of PEG-trastuzumab was evaluated in thoracic lymph duct cannulated rats after iv and sc administration and compared to the pharmacokinetics of native trastuzumab. The iv pharmacokinetics and lymphatic exposure of PEG-trastuzumab was similar when compared to trastuzumab. After sc administration, initial plasma pharmacokinetics and lymphatic exposure were also similar between PEG-trastuzumab and trastuzumab, but the absolute bioavailability of PEG-trastuzumab was 100% when compared to 86.1% bioavailability for trastuzumab. In contrast to trastuzumab, PEG-trastuzumab showed accelerated plasma clearance beginning approximately 7 days after sc, but not iv, administration, presumably as a result of the generation of anti-PEG IgM. This work suggests that PEGylation does not significantly alter the lymphatic disposition of very large proteins, and further suggests that it is unlikely to benefit therapy with monoclonal antibodies.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Trastuzumab/administración & dosificación , Trastuzumab/metabolismo , Administración Intravenosa , Animales , Antineoplásicos/química , Biofarmacia , Permeabilidad Capilar , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Inyecciones Subcutáneas , Linfa/metabolismo , Sistema Linfático/metabolismo , Masculino , Tasa de Depuración Metabólica , Modelos Biológicos , Polietilenglicoles/efectos adversos , Polietilenglicoles/química , Ratas , Ratas Sprague-Dawley , Trastuzumab/químicaRESUMEN
The utility of inhaled protein therapeutics to treat lung-resident diseases is limited by protein degradation in the lungs and rapid clearance. This study therefore aimed to evaluate the impact of PEGylation on the lung and systemic exposure of interferon (IFN) α2 after intratracheal administration to rats. An inverse correlation was observed between PEG chain length and systemic exposure, where bioavailability was 5.5% for the 31 kDa PEGylated construct and <0.4% for the 60 kDa PEGylated construct when compared with 15% for native IFN (19 kDa). Retention of PEGylated IFNα within the lungs increased 2.5-fold to threefold when compared with native IFN. When comparing the lung and systemic exposure of PEGylated and native IFN in terms of protein biological activity, the 31 kDa PEGylated construct increased exposure by 50% and 100%, respectively, when compared with native IFN, but the 60 kDa PEG construct offered no benefit. Preliminary work also indicated that the conjugation of IFNγ with 10 kDa PEG significantly increases the retention of the protein within the lung. Optimal PEGylation may therefore be used as a means to improve the exposure of lung-resident diseases to therapeutic cytokines and potentially reduce systemic exposure and side effects as well as dosing frequency.
Asunto(s)
Antineoplásicos/administración & dosificación , Portadores de Fármacos , Interferón-alfa/administración & dosificación , Interferón gamma/administración & dosificación , Pulmón/metabolismo , Polietilenglicoles/administración & dosificación , Administración por Inhalación , Animales , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Humanos , Interferón alfa-2 , Interferón-alfa/sangre , Interferón-alfa/química , Interferón-alfa/farmacocinética , Interferón gamma/sangre , Interferón gamma/química , Interferón gamma/farmacocinética , Modelos Lineales , Pulmón/efectos de los fármacos , Pulmón/patología , Neoplasias Pulmonares/patología , Masculino , Modelos Biológicos , Peso Molecular , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/sangre , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Tecnología Farmacéutica/métodosRESUMEN
The current study sought to explore whether the subcutaneous administration of lymph targeted dendrimers, conjugated with a model chemotherapeutic (methotrexate, MTX), was able to enhance anticancer activity against lymph node metastases. The lymphatic pharmacokinetics and antitumor activity of PEGylated polylysine dendrimers conjugated to MTX [D-MTX(OH)] via a tumor-labile hexapeptide linker was examined in rats and compared to a similar system where MTX was α-carboxyl O-tert-butylated [D-MTX(OtBu)]. The latter has previously been shown to exhibit longer plasma circulation times. D-MTX(OtBu) was well absorbed from the subcutaneous injection site via the lymph, and 3 to 4%/g of the dose was retained by sentinel lymph nodes. In contrast, D-MTX(OH) showed limited absorption from the subcutaneous injection site, but absorption was almost exclusively via the lymph. The retention of D-MTX(OH) by sentinel lymph nodes was also significantly elevated (approximately 30% dose/g). MTX alone was not absorbed into the lymph. All dendrimers displayed lower lymph node targeting after intravenous administration. Despite significant differences in the lymph node retention of D-MTX(OH) and D-MTX(OtBu) after subcutaneous and intravenous administration, the growth of lymph node metastases was similarly inhibited. In contrast, the administration of MTX alone did not significantly reduce lymph node tumor growth. Subcutaneous administration of drug-conjugated dendrimers therefore provides an opportunity to improve drug deposition in downstream tumor-burdened lymph nodes. In this case, however, increased lymph node biodistribution did not correlate well with antitumor activity, possibly suggesting constrained drug release at the site of action.