RESUMO
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.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Nestina/genética , Receptores Androgênicos/genética , Recombinação Genética , Animais , Feminino , Engenharia Genética/métodos , Células Germinativas/metabolismo , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , TransgenesRESUMO
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.
Assuntos
Catepsinas/química , Dendrímeros/química , Doxorrubicina/química , Polilisina/química , Células A549 , Animais , Catepsina B/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Masculino , Polietilenoglicóis/química , RatosRESUMO
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.
Assuntos
Antivirais/síntese química , Interferon-alfa/síntese química , Sistema Linfático/metabolismo , Polietilenoglicóis/síntese química , Animais , Antivirais/administração & dosagem , Antivirais/química , Antivirais/farmacocinética , Injeções Subcutâneas , Interferon-alfa/administração & dosagem , Interferon-alfa/química , Interferon-alfa/farmacocinética , Masculino , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Distribuição TecidualRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Dendrímeros/química , Portadores de Fármacos/química , Linfonodos/metabolismo , Metotrexato/administração & dosagem , Polietilenoglicóis/química , Animais , Antimetabólitos Antineoplásicos/farmacocinética , Sistemas de Liberação de Medicamentos , Metotrexato/farmacocinética , RatosRESUMO
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.
Assuntos
Dendrímeros/farmacocinética , Portadores de Fármacos/farmacocinética , Sistemas de Liberação de Medicamentos , Pulmão/metabolismo , Linfonodos/metabolismo , Polietilenoglicóis/farmacocinética , Administração por Inalação , Administração Intravenosa , Aerossóis/administração & dosagem , Aerossóis/química , Aerossóis/farmacocinética , Animais , Dendrímeros/administração & dosagem , Dendrímeros/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Masculino , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , OvinosRESUMO
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.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Trastuzumab/administração & dosagem , Trastuzumab/metabolismo , Administração Intravenosa , Animais , Antineoplásicos/química , Biofarmácia , Permeabilidade Capilar , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Injeções Subcutâneas , Linfa/metabolismo , Sistema Linfático/metabolismo , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , Trastuzumab/químicaRESUMO
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.
Assuntos
Dendrímeros/química , Dendrímeros/farmacocinética , Linfonodos/metabolismo , Metotrexato/química , Metotrexato/farmacocinética , Polietilenoglicóis/química , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Masculino , Microscopia Confocal , Neoplasias/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Acrilatos/farmacocinética , Portadores de Fármacos/farmacocinética , Polietilenoglicóis/farmacocinética , Acrilatos/administração & dosagem , Acrilatos/química , Administração Intravenosa , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Humanos , Hidrodinâmica , Masculino , Camundongos , Camundongos Nus , Peso Molecular , Neoplasias/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
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.
Assuntos
Western Blotting , Humanos , Animais , Western Blotting/métodos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Xenoenxertos , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer is a leading cause of death within developed nations, and part of this morbidity is due to difficulties associated with its treatment. Currently, anticancer therapy relies heavily upon the administration of small molecule cytotoxic drugs that attack both cancerous and noncancerous cells due to limited selectivity of the drugs and widespread distribution of the cytotoxic molecules throughout the body. The antitumor efficacy and systemic toxicity of existing chemotherapeutic drugs can, however, be improved by employing formulation and particle engineering approaches. Thus, drug delivery systems can be developed that more specifically target tumor tissue using both passive (such as the enhanced permeation and retention effect) and active (through the use of cancer targeting ligands) modalities. Dendrimers are one such system that can be developed with high structural monodispersity, long plasma circulation times and precise control over surface structure and biodistribution properties. Chemotherapeutic drugs can be associated with dendrimers via covalent conjugation to the surface, or via encapsulation of drugs within the structure. Each of these approaches has demonstrated therapeutic benefit relative to the administration of free drug. Thus far, however, there has not been a systematic review toward which drug association approach will provide the best outcomes in terms of antitumor efficacy and systemic toxicity. Hence, the current literature is reviewed here and recommendations are proposed as to the suggested approach to develop dendrimers as tumor targeted drug-delivery vectors.
Assuntos
Dendrímeros/química , Sistemas de Liberação de Medicamentos/métodos , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Dendrímeros/administração & dosagem , Dendrímeros/efeitos adversos , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/efeitos adversos , Portadores de Fármacos/química , Humanos , Neoplasias/tratamento farmacológicoRESUMO
PEGylated polylysine dendrimers show promise as novel drug delivery systems with the potential to direct site specific deposition patterns and to reduce toxicity at nontarget sites. Here the activity and toxicity profiles of a generation 5 polylysine dendrimer with 50% surface conjugation of PEG1100 and 50% surface conjugation of doxorubicin (via an acid labile 4-hydrazinosulfonyl benzoic acid linker) have been compared in a Walker 256 rat tumor model and a human MDA-MB231 xenograft in mice. A direct comparison was also made to a PEGylated liposomal formulation of doxorubicin and a doxorubicin solution. In both rat and mouse breast cancer models, the dendrimer formulation gave equivalent antitumor efficacy when compared to the liposomal or solution doxorubicin formulations and administration of all three doxorubicin formulations resulted in a significant reduction (>75%) in tumor growth in both models at doses ranging from 2 to 10 mg/kg doxorubicin equivalents. The dendrimer formulation, however, was better tolerated by both rats and mice, and approximately 2-fold higher doses were required to induce similar levels of toxicity (as assessed by organ weight, peripheral white cell counts, body weight and survival curves) when compared to administration of the doxorubicin solution or PEGylated liposomal doxorubicin. In rats the appearance of palmar plantar erythematosis (PPE), or hand foot syndrome, was also less evident after administration of dendrimer doxorubicin when compared to the liposome. Finally, even after administration to mice at 2-fold higher doses, dendrimer-doxorubicin resulted in a reduced incidence of cardiotoxicity when compared with a simple solution formulation of doxorubicin. The data suggest that dendrimer-based doxorubicin formulations may provide advantage over solution and liposomal formulations of doxorubicin via a reduction in systemic toxicity.
Assuntos
Dendrímeros/química , Doxorrubicina/análogos & derivados , Lipossomos/química , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/uso terapêutico , Animais , Linhagem Celular Tumoral , Dendrímeros/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos/efeitos adversos , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Ratos , Ratos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The pharmacokinetics, biodistribution, and antitumor efficacy of three doxorubicin formulations (doxorubicin in saline, conjugated to a polylysine dendrimer, and encapsulated within a stealth liposome) were investigated in Walker 256 tumor-bearing rats. Liposomal and dendrimer-based delivery systems resulted in more prolonged plasma exposure of total doxorubicin when compared to administration of doxorubicin in saline, although concentrations of free doxorubicin remained low in both cases. Biodistribution profiles revealed enhanced accumulation of dendrimer- and liposome-associated doxorubicin in tumors when compared to doxorubicin alone, although all three doxorubicin formulations reduced tumor growth to a similar extent. Markers of systemic toxicity (spleen weight, white blood cell counts, body weight, and cardiotoxicity) were more pronounced in rats that received doxorubicin and liposomal doxorubicin when compared to dendrimer-doxorubicin. The data provide preliminary evidence that dendrimer-doxorubicin displays similar antitumor efficacy to PEGylated liposomal doxorubicin, but with lower systemic toxicity (resulting from reduced drug exposure to nontarget organs). FROM THE CLINICAL EDITOR: In this manuscript, three different doxorubicin preparations are compared and preliminary evidence suggests that dendrimer-doxorubicin displays similar antitumor efficacy to PEGylated liposomal doxorubicin, but with lower systemic toxicity.
Assuntos
Antibióticos Antineoplásicos/sangue , Dendrímeros/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/sangue , Polietilenoglicóis/administração & dosagem , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/uso terapêutico , Carcinoma 256 de Walker/tratamento farmacológico , Dendrímeros/farmacocinética , Dendrímeros/uso terapêutico , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Contagem de Leucócitos , Polietilenoglicóis/uso terapêutico , Ratos , Cloreto de Sódio , Distribuição TecidualRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Androgênios/metabolismo , Animais , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
A generation 5 PEGylated (PEG 1100) polylysine dendrimer, conjugated via a stable amide linker to OtBu protected methotrexate (MTX), was previously shown to have a circulatory half-life of 2 days and to target solid tumors in both rats and mice. Here, we show that deprotection of MTX and substitution of the stable linker with a matrix metalloproteinase (MMP) 2 and 9 cleavable linker (PVGLIG) dramatically increased plasma clearance and promoted deposition in the liver and spleen (50-80% of the dose recovered in the liver 3 days post dose). Similar rapid clearance was also seen using a scrambled peptide suggesting that clearance was not dependent on the cleavable nature of the linker. Surprisingly, dendrimers where OtBu capped MTX was linked to the dendrimer surface via the hexapeptide linker showed equivalent in vitro cytotoxicity against HT1080 cells when compared to the uncapped dendrimer and also retained the long circulating characteristics of the stable constructs. The OtBu capped MTX conjugated dendrimer was subsequently shown to significantly reduce tumor growth in HT1080 tumor bearing mice compared to control. In contrast the equivalent dendrimer comprising uncapped MTX conjugated to the dendrimer via the same hexapeptide linker did not reduce tumor growth, presumably reflecting very rapid clearance of the construct. The results are consistent with the suggestion that protection of the α-carboxyl group of methotrexate may be used to improve the circulatory half-life and reduce the liver accumulation of similar MTX-conjugated dendrimers, while still retaining antitumor activity in vivo.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Dendrímeros/farmacologia , Fibrossarcoma/tratamento farmacológico , Metotrexato/farmacologia , Polietilenoglicóis/farmacologia , Polilisina/farmacologia , Animais , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/farmacocinética , Proliferação de Células/efeitos dos fármacos , Dendrímeros/farmacocinética , Sistemas de Liberação de Medicamentos , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Rim/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica , Metotrexato/sangue , Metotrexato/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Polietilenoglicóis/farmacocinética , Polilisina/farmacocinética , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/patologia , Receptores Androgênicos/metabolismo , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Fenótipo , Fatores Sexuais , Medula Espinal/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/metabolismo , Neuroglia/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/análise , Hormônios Esteroides Gonadais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/patologia , Neuroglia/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Medula Espinal/química , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1/genéticaRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Antagonistas de Receptores de Andrógenos/farmacologia , Flutamida/farmacologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Células-Tronco Embrionárias , Feminino , Humanos , Masculino , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Neuroglia/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/patologia , Receptores Androgênicos/metabolismo , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Testosterona/sangueRESUMO
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.
Assuntos
Dendrímeros/química , Doxorrubicina/química , Pulmão/metabolismo , Polietilenoglicóis/química , Polilisina/química , Administração por Inalação , Administração Intravenosa , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dendrímeros/administração & dosagem , Dendrímeros/farmacocinética , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Pulmão/efeitos dos fármacos , Masculino , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacocinética , Polilisina/administração & dosagem , Polilisina/farmacocinética , Ratos , Ratos Sprague-DawleyRESUMO
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.