Assessing Protein Expression in Patient-Derived Xenografts Using Western Blotting.

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.

Mapping Motor Neuron Vulnerability in the Neuraxis of Male SOD1 Mice Reveals Widespread Loss of Androgen Receptor Occurring Early in Spinal Motor Neurons.

Sex steroid hormones have been implicated as disease modifiers in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Androgens, signalling 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 the classical genomic signalling pathway.

The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

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.

Dissociation of disease onset, progression and sex differences from androgen receptor levels in a mouse model of amyotrophic lateral sclerosis.

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.

Exploring germline recombination in Nestin-Cre transgenic mice using floxed androgen receptor.

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.

Dysregulation of Steroid Hormone Receptors in Motor Neurons and Glia Associates with Disease Progression in ALS Mice.

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.

Androgen receptor antagonism accelerates disease onset in the SOD1 mouse model of amyotrophic lateral sclerosis.

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 SOD1 mouse model.

Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker.

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.

Doxorubicin Conjugation and Drug Linker Chemistry Alter the Intravenous and Pulmonary Pharmacokinetics of a PEGylated Generation 4 Polylysine Dendrimer in Rats.

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.

Distribution of therapeutic proteins into thoracic lymph after intravenous administration is protein size-dependent and primarily occurs within the liver and mesentery.

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.

Lymphatic transport and lymph node targeting of methotrexate-conjugated PEGylated dendrimers are enhanced by reducing the length of the drug linker or masking interactions with the injection site.

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.

An Evaluation of Optimal PEGylation Strategies for Maximizing the Lymphatic Exposure and Antiviral Activity of Interferon after Subcutaneous Administration.

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.

Effect of increased surface hydrophobicity via drug conjugation on the clearance of inhaled PEGylated polylysine dendrimers.

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.

Characteristics of Older Adults Admitted to Hospital versus Those Discharged Home, in Emergency Department Patients Referred to Internal Medicine.

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.

The Pharmacokinetics and Biodistribution of a 64 kDa PolyPEG Star Polymer After Subcutaneous and Pulmonary Administration to Rats.

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.

A Comparison of the Pharmacokinetics and Pulmonary Lymphatic Exposure of a Generation 4 PEGylated Dendrimer Following Intravenous and Aerosol Administration to Rats and Sheep.

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.

Molecular weight (hydrodynamic volume) dictates the systemic pharmacokinetics and tumour disposition of PolyPEG star polymers.

Unlabelled: 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.

PEGylation does not significantly change the initial intravenous or subcutaneous pharmacokinetics or lymphatic exposure of trastuzumab in rats but increases plasma clearance after subcutaneous administration.

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.

Optimal PEGylation can improve the exposure of interferon in the lungs following pulmonary administration.

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.

Methotrexate-conjugated PEGylated dendrimers show differential patterns of deposition and activity in tumor-burdened lymph nodes after intravenous and subcutaneous administration in rats.

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.

Pulmonary administration of a doxorubicin-conjugated dendrimer enhances drug exposure to lung metastases and improves cancer therapy.

Direct administration of chemotherapeutic drugs to the lungs significantly enhances drug exposure to lung resident cancers and may improve chemotherapy when compared to intravenous administration. Direct inhalation of uncomplexed or unencapsulated cytotoxic drugs, however, leads to bolus release and unacceptable lung toxicity. Here, we explored the utility of a 56kDa PEGylated polylysine dendrimer, conjugated to doxorubicin, to promote the controlled and prolonged exposure of lung-resident cancers to cytotoxic drug.

PEGylation of interferon α2 improves lymphatic exposure after subcutaneous and intravenous administration and improves antitumour efficacy against lymphatic breast cancer metastases.

The efficacy of protein-based therapeutics with indications in the treatment of lymphatic diseases is expected to be improved by enhancing lymphatic disposition. This study was therefore aimed at examining whether PEGylation can usefully be applied to improve the lymphatic uptake of interferon α2 and whether this ultimately translates into improved therapeutic efficacy against lymph-resident cancer. The lymphatic pharmacokinetics of interferon α2b (IFN, 19kDa) and PEGylated interferon α2b (IFN-PEG12, 31kDa) or α2a (IFN-PEG40, 60kDa) was examined in thoracic lymph duct cannulated rats.

Association of chemotherapeutic drugs with dendrimer nanocarriers: an assessment of the merits of covalent conjugation compared to noncovalent encapsulation.

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.

Doxorubicin-conjugated PEGylated dendrimers show similar tumoricidal activity but lower systemic toxicity when compared to PEGylated liposome and solution formulations in mouse and rat tumor models.

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.

Differences in colloidal structure of PEGylated nanomaterials dictate the likelihood of accelerated blood clearance.

PEGylated liposomes are known to exhibit accelerated clearance from systemic circulation on repeat administration (the so-called "accelerated blood clearance" or ABC effect); however, little is known about this effect for other PEGylated colloidal drug delivery systems. Furthermore, our understanding of the mechanisms by which the ABC effect is induced is limited. This article further addresses these issues by examining the impact of colloid types [polyethylene glycol (PEG)-liposomes, PEG-micelles] of varying sizes on the appearance of the ABC effect when readministered 7 days after a "priming" dose.