Noninvasive Strategies for Systemic Delivery of Therapeutic Proteins — Prospects and Challenges

It is well established that proteins have great physiological importance, thus possessing great potential for therapeutic use. There is increased interest in protein/peptide pharmaceuticals delivery due to recent improvements in analytical methods, advancements in molecular biology and genetic engineering, and a better understanding of regulatory roles of proteins and peptides. There are however major challenges that need to be overcome for systemic delivery of these biological molecules. The major hurdles that contribute to low biological activity are low stability, immunogenicity, and toxicity. A combination of strategies can be used to overcome these challenges and improve the bioavailability of protein drugs. Alternative delivery routes (e.g., nasal and pulmonary) and the development of new methods for overcoming delivery challenges (e.g., nanomedicine, and PEGylation), along with the development of innovative formulation strategies (e.g., spray-freeze drying, supercritical fluid methods, fluidized-bed spray coating, lyophilization, jet milling and spray drying), have resulted in improved pharmacokinetics of protein drugs and in some cases increased patient compliance

Cardiomyocyte Specific Ablation of p53 Is Not Sufficient to Block Doxorubicin Induced Cardiac Fibrosis and Associated Cytoskeletal Changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity

Combinatorial polymeric conjugated micelles with dual cytotoxic and antiangiogenic effects for the treatment of ovarian cancer

Emerging treatment paradigms like targeting the tumor microenvironment and/or dosing as part of a metronomic regimen are anticipated to produce better outcomes in ovarian cancer, but current drug delivery systems are lacking. We have designed and evaluated paclitaxel (PTX) and rapamycin (RAP) micellar systems that can be tailored for various dosing regimens and target tumor microenvironment. Individual and mixed PTX/RAP (MIX-M) micelles are prepared by conjugating drugs to a poly­(ethylene glycol)-<i>block</i>-poly­(β-benzyl l-aspartate) using a pH-sensitive linker. The micelles release the drug(s) at pH 5.5 indicating preferential release in the acidic endosomal/lysosomal environment. Micelles exhibit antiproliferative effects in ovarian cell cancer lines (SKOV-3 (human caucasian ovarian adenocarcinoma) and ES2 (human ovarian clear cell carcinoma)) and an endothelial cell line (HUVEC; human umbilical vein endothelial cells) with the MIX-M being synergistic. The micelles also inhibited endothelial migration and tube formation. In healthy mice, micelles at 60 mg/kg/drug demonstrated no acute toxicity over 21 days. ES2 xenograft model efficacy studies at 20 mg/kg/drug dosed every 4 days and evaluated at 21 days indicate that the individual micelles exhibit antiangiogenic effects, while the MIX-M exhibited both antiangiogenic and apoptotic induction that results in significant tumor volume reduction. On the basis of our results, MIX-M micelles can be utilized to achieve synergistic apoptotic and antiangiogenic effects when treated at frequent low doses

Climate-Induced Changes in Spring Snowmelt Impact Ecosystem Metabolism and Carbon Fluxes in an Alpine Stream Network

Although stream ecosystems are recognized as an important component of the global carbon cycle, the impacts of climate-induced hydrological extremes on carbon fluxes in stream networks remain unclear. Using continuous measurements of ecosystem metabolism, we report on the effects of changes in snowmelt hydrology during the anomalously warm winter 2013/2014 on gross primary production (GPP), ecosystem respiration (ER), and net ecosystem production (NEP) in an Alpine stream network. We estimated ecosystem metabolism across 12 study reaches of the 254 km2 subalpine Ybbs River Network (YRN), Austria, for 18 months. During spring snowmelt, GPP peaked in 10 of our 12 study reaches, which appeared to be driven by PAR and catchment area. In contrast, the winter precipitation shift from snow to rain following the low-snow winter in 2013/2014 increased spring ER in upper elevation catchments, causing spring NEP to shift from autotrophy to heterotrophy. Our findings suggest that the YRN transitioned from a transient sink to a source of carbon dioxide (CO2) in spring as snowmelt hydrology differed following the high-snow versus low-snow winter. This shift toward increased heterotrophy during spring snowmelt following a warm winter has potential consequences for annual ecosystem metabolism, as spring GPP contributed on average 33% to annual GPP fluxes compared to spring ER, which averaged 21% of annual ER fluxes. We propose that Alpine headwaters will emit more within-stream respiratory CO2 to the atmosphere while providing less autochthonous organic energy to downstream ecosystems as the climate gets warmer

Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines

Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described – OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2) and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby Canine Kidney (MDCK) or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate – previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3

Organic Anion Transporter 1 Is Inhibited by Multiple Mechanisms and Shows a Transport Mode Independent of Exchange

ABSTRACT The mechanism by which drugs inhibit organic anion transporter 1 (OAT1) was examined. OAT1 was stably expressed in Chinese hamster ovary (CHO) cells, and para-aminohippurate (PAH) and 6-carboxyfluorescein were the substrates. Most compounds (10 of 14) inhibited competitively, increasing the Michaelis constant (K m ) without affecting the maximal transport rate (J max ). Others were mixed-type (lowering J max and increasing K m ) or noncompetitive (lowering J max only) inhibitors. The interaction of a noncompetitive inhibitor (telmisartan) with OAT1 was examined further. Binding of telmisartan to OAT1 was observed, but translocation was not. Telmisartan did not alter the plasma membrane expression of OAT1, indicating that it lowers J max by reducing the turnover number. PAH transport after telmisartan treatment and its washout recovered faster in the presence of 10% fetal bovine serum in the washout buffer, indicating that binding of telmisartan to OAT1 and its inhibitory effect are reversible. Together, these data suggest that telmisartan binds reversibly to a site distinct from substrate and stabilizes the transporter in a conformation unfavorable for translocation. In the absence of an exchangeable extracellular substrate, PAH efflux from CHO-OAT1 cells was relatively rapid. Telmisartan slowed PAH efflux, suggesting that some transporter-mediated efflux occurs independent of exchange. Although drug-drug interaction predictions at OAT1 assume competitive inhibition, these data show that OAT1 can be inhibited by other mechanisms, which could influence the accuracy of drug-drug interaction predictions at the transporter. Telmisartan was useful for examining how a noncompetitive inhibitor can alter OAT1 transport activity and for uncovering a transport mode independent of exchange

Characterizing the role of atrial natriuretic peptide signaling in the development of embryonic ventricular conduction system

International audiencePatients born with congenital heart defects frequently encounter arrhythmias due to defects in the ventricular conduction system (VCS) development. Although recent studies identified transcriptional networks essential for the heart development, there is scant information on the mechanisms regulating VCS development. Based on the association of atrial natriuretic peptide (ANP) expression with VCS forming regions, it was reasoned that ANP could play a critical role in differentiation of cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) toward a VCS cell lineage. The present study showed that treatment of embryonic ventricular cells with ANP or cell permeable 8-Br-cGMP can induce gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40). Inhibition of protein kinase G (PKG) via Rp-8-pCPT-cGMPS further confirmed the role of ANP/NPRA/cGMP/PKG pathway in the regulation of HCN4 and Cx40 gene expression. Additional experiments indicated that ANP may regulate VCS marker gene expression by modulating levels of miRNAs that are known to control the stability of transcripts encoding HCN4 and Cx40. Genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation. These results provide mechanistic insights into the role of ANP/NPRA signaling in VCS formation

Quantification of cardiac fibrosis in tissue sections after 3 or 7 days of Dox treatment.

<p>Percent fibrosis values in PSFG-stained heart sections were calculated using Image processing Tool Kit software and image analysis was performed on a minimum of ten slides per animal for each treatment group (n = 3–5 mice per group). Dox (D) treatment significantly increased cardiac fibrosis in wild type (WT) and p53 CKO hearts compared to saline (S) treated hearts after 3 day (A) or 7 day (B) treatment periods. 3 day Dox time point: *<i>P</i><0.05 compared to WT(S), **<i>P</i><0.05 compared to WT(D), ANOVA, Tukey's multiple comparisons test; 7 day Dox time point: #<i>P</i><0.05 compared to WT(S), ANOVA, Tukey's multiple comparison test.</p