Absorption of ipratropium and l-carnitine into the pulmonary circulation of the ex-vivo rat lung is driven by passive processes rather than active uptake by OCT/OCTN transporters.

The organic cation transporters OCT and OCTN have been reported to play a significant role in the cellular uptake of substrates within in vitro lung cells. However, no studies to date have investigated the effect of these transporters upon transepithelial absorption of substrates into the pulmonary circulation. We investigated the contribution of OCT and OCTN transporters to total pulmonary absorption of l-carnitine and the anti-muscarinic drug, ipratropium, across an intact isolated perfused rat lung (IPRL). The results obtained from the IPRL were contrasted with active transport in vitro using three human pulmonary cell lines and primary rat alveolar epithelial cells. Ex-vivo studies showed that OCT/OCTN transporters do not play a role in the overall pulmonary absorption of l-carnitine or ipratropium, as evidenced by the effect of chemical inhibition of these transporters upon pulmonary absorption. In contrast, in vitro studies showed that OCT/OCTN transporters play a significant role in cellular accumulation of substrates with preferential uptake of ipratropium by OCTs, and of l-carnitine uptake by OCTNs. The results show that in vitro uptake studies cannot be predictive of airway to blood absorption in vivo. Nevertheless, localised submucosal pulmonary concentrations of inhaled drugs and their pulmonary pharmacodynamic profiles may be influenced by OCT/OCTN transport activity.

Caveolin-1 in renal cell carcinoma promotes tumour cell invasion, and in co-operation with pERK predicts metastases in patients with clinically confined disease.

BACKGROUND: Up to 40% of patients initially diagnosed with clinically-confined renal cell carcinoma (RCC) and who undergo curative surgery will nevertheless relapse with metastatic disease (mRCC) associated with poor long term survival. The discovery of novel prognostic/predictive biomarkers and drug targets is needed and in this context the aim of the current study was to investigate a putative caveolin-1/ERK signalling axis in clinically confined RCC, and to examine in a panel of RCC cell lines the effects of caveolin-1 (Cav-1) on pathological processes (invasion and growth) and select signalling pathways. METHODS: Using immunohistochemistry we assessed the expression of both Cav-1 and phosphorylated-ERK (pERK) in 176 patients with clinically confined RCC, their correlation with histological parameters and their impact upon disease-free survival. Using a panel of RCC cell lines we explored the functional effects of Cav-1 knockdown upon cell growth, cell invasion and VEGF-A secretion, as well Cav-1 regulation by cognate cell signalling pathways. RESULTS: We found a significant correlation (P = 0.03) between Cav-1 and pERK in a cohort of patients with clinically confined disease which represented a prognostic biomarker combination (HR = 4.2) that effectively stratified patients into low, intermediate and high risk groups with respect to relapse, even if the patients' tumours displayed low grade and/or low stage disease. In RCC cell lines Cav-1 knockdown unequivocally reduced cell invasive capacity while also displaying both pro-and anti-proliferative effects; targeted knockdown of Cav-1 also partially suppressed VEGF-A secretion in VHL-negative RCC cells. The actions of Cav-1 in the RCC cell lines appeared independent of both ERK and AKT/mTOR signalling pathways. CONCLUSION: The combined expression of Cav-1 and pERK serves as an independent biomarker signature with potential merit in RCC surveillance strategies able to predict those patients with clinically confined disease who will eventually relapse. In a panel of in-vitro RCC cells Cav-1 promotes cell invasion with variable effects on cell growth and VEGF-A secretion. Cav-1 has potential as a therapeutic target for the prevention and treatment of mRCC.

Selectivity in the impact of P-glycoprotein upon pulmonary absorption of airway-dosed substrates: a study in ex vivo lung models using chemical inhibition and genetic knockout.

P-glycoprotein (P-gp) mediated efflux is recognised to alter the absorption and disposition of a diverse range of substrates. Despite evidence showing the presence of P-gp within the lung, relatively little is known about the transporter's effect upon the absorption and distribution of drugs delivered via the pulmonary route. Here, we present data from an intact isolated rat lung model, alongside two isolated mouse lung models using either chemical or genetic inhibition of P-gp. Data from all three models show inhibition of P-gp increases the extent of absorption of a subset of P-gp substrates (e.g. rhodamine 123 and loperamide) whose physico-chemical properties are distinct from those whose pulmonary absorption remained unaffected (e.g. digoxin and saquinavir). This is the first study showing direct evidence of P-gp mediated efflux within an intact lung, a finding that should warrant consideration as part of respiratory drug discovery and development as well as in the understanding of pulmonary pharmacokinetic (PK)-pharmacodynamic (PD) relationships.

Peptide sequences mediating tropism to intact blood-brain barrier: an in vivo biodistribution study using phage display.

Peptide motifs that demonstrate tropism for the blood brain barrier (BBB) are of real translational value in developing innovative delivery strategies for biological brain targeted therapies. In vivo peptide-phage display affords peptide selection against the full complement of biological markers within the correct cellular macro- and micro-environments. Here a stringent in vivo biopanning protocol was employed in the rat aimed at identifying cyclic 7-mer peptide motifs that mediate tropism to brain microvasculature. Five rounds of biopanning identified 349 unique peptide motifs in the brain tissue gray matter compartment (microvasculature and parenchyma). While in general no consensus was evident linking peptide physico-chemical properties and brain tropism, peptides bearing c-SxTSSTx-c or c-xxxSSTx-c motifs were found to be present in high abundance. Based on amino acid frequency distribution of the 349 unique peptides sequences a theoretical 'idealized' peptide pattern, c-PP(S/P)SSST-c, could be derived. For the most abundant experimental peptide sequence found in brain tissue, c-SYTSSTM-c, an in vivo pharmacokinetic and whole body tissue biodistribution study was performed. Based upon tissue exposure data (i.e. tissue AUC((0-infinity))) the sequence c-SYTSSTM-c efficiently retargeted phage virions to the brain providing an approximate 5-fold greater (P<0.05) accumulation in brain over control phage; in all other organs no significant (P>0.05) difference in tissue tropism between c-SYTSSTM-c and control phages were evident. This peptide and more generally the peptide motifs, -SxTSSTx- or -xxxSSTx-, warrant further investigation as agents mediating sequence-dependent tropism to brain microvasculature potentially able to deliver biologic cargo to the CNS.

Spatial expression and functionality of drug transporters in the intact lung: objectives for further research.

This commentary provides a background appraising evidence in the intact lung on the spatial expression of drug transporters and, where available, evidence in the intact lung of the impact, or otherwise, that such transporters can have upon pulmonary drug absorption and disposition. Ultimately drug discovery and development scientists will wish to identify in a 'pulmonary' context the effect of disease upon transporter function, the potential for drug transporters to contribute to drug-drug interactions and to inter-individual variation in drug handling and response. The rate and extent of lung epithelial permeation of drugs involve an interplay between the dose and the deposition site of drug within the lung and physiological variables operational at the epithelial-luminal interface. Amongst the latter variables is the potential impact of active transporter processes which may well display regio-selective characteristics along the epithelial tract. In pulmonary tissues the spatial pattern of drug transporter expression is generally poorly defined and the functional significance of transporters within the intact lung is explored in only a limited manner. Active transporters in the lung epithelium may affect airway residence times of drug, modulate access of drug to intracellular targets and to submucosal lung tissue, and potentially influence airway to systemic drug absorption profiles. Transporters in the lung tissue may also have the capacity to mediate uptake of drug from the systemic circulation resulting in drug accumulation in the lung. Transporters have physiological roles and new drug candidates while not necessarily serving as transport substrates may modulate transporter activity and hence physiology. The commentary highlights a series of recommendations for further work in pulmonary drug transporter research.

Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide.

With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (p<0.05) to the cumulative transport of less than 0.004% for control phage-clone groups. To characterise phage-independent activity of LTP-1 peptide, the LTP-1 peptide was conjugated to a 53kDa anionic PAMAM dendrimer. Compared to respective peptide-dendrimer control conjugates, the LTP-1-PAMAM conjugate displayed a two-fold (bioavailability up to 31%) greater extent of absorption in the IPRL. The LTP-1 peptide-mediated enhancement of transport, when LTP-1 was either attached to the phage clone or conjugated to dendrimer, was sequence-dependent and could be competitively inhibited by co-instillation of excess synthetic free LTP-1 peptide. The specific nature of the target receptor or mechanism involved in LTP-1 lung transport remains unclear although the enhanced transport is enabled through a mechanism that is non-disruptive with respect to the pulmonary transport of hydrophilic permeability probes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ.

Cell selective glucocorticoid induction of caveolin-1 and caveolae in differentiating pulmonary alveolar epithelial cell cultures.

Increased caveolin-1 expression is a marker of the differentiation of lung alveolar epithelial type II cells into a type I phenotype. Here, we show in both a primary differentiating rat alveolar culture, and a human alveolar cell line (A549) that caveolae formation and caveolin-1 expression are dependent upon dexamethasone Dex, and is inhibited by the glucocorticoid receptor (GR) antagonist, mifepristone. Study of a panel of 20 different cell types showed the effect of (Dex) upon caveolin-1 expression to be highly cell selective for lung alveolar epithelial cells. The actions of glucocorticoid upon caveolin-1 appear indirect acting via intermediary genes as evidenced by cycloheximide (CHX) abolition of Dex-induced increases in caveolin-1 mRNA and by recombinant transfection studies using the caveolin-1 promoter cloned upstream of a reporter gene. Treatment with actinomycin D (ACD) revealed that the effects of Dex are also, at least in part, mediated by stabilisation of caveolin-1 mRNA. Collectively, these results indicate that glucocorticoids modulate the expression of caveolin-1 and caveolae biogenesis within alveolar epithelial cells via both transcriptional and translational modifications. The cell-selective effects of glucocorticoid upon caveolin may represent a previously unrecognised mechanism by which glucocorticoids affect lung development.

Phage display identification of functional binding peptides against 4-acetamidophenol (Paracetamol): an exemplified approach to target low molecular weight organic molecules.

Peptide-phage display has been widely used to explore protein-protein interactions, however, despite the potential range of applications the use of this technology to identify peptides that bind low molecular weight organic molecules has not been explored. In this current study, we identified a phage clone (PARA-061) displaying the cyclic 7-mer peptide sequence N' AC-NPNNLSH-CGGGS C' that binds the low molecular weight organic molecule 4-acetamidophenol (4-AAP; paracetamol). To avoid occupancy of key functional groups on the target 4-AAP molecule our panning strategy was directed against insoluble complexes of 4-AAP rather than against the target linked to a stationary support or bearing an affinity tag. To augment the panning procedure we deleted phage that also bound the 4-AAP isomers, 2-AAP and 3-AAP. The identified PARA-061 peptide-phage clone displayed functional binding properties against 4-AAP in solution, able in a peptide sequence-dependant manner to prevent the in vitro hepatotoxicity of 4-AAP and reduce ( approximately 20%) the permeability of 4-AAP across a semi-permeable membrane. Molecular dynamic simulations generated a stable binding conformation between the PARA-061 peptide sequence and 4-AAP. In conclusion, we show that a phage display library can be used to identify peptide sequence-specific clones able to modulate the functional binding of a low molecular weight organic molecule. Such peptides may be expected to find utility in the next generation of hybrid polymer-based biosensing devices.

Endocytosis at the blood-brain barrier: from basic understanding to drug delivery strategies.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful xenobiotics and endogenous molecules. Anatomically, it comprises the brain microvasculature whose functionality is nevertheless influenced by associated astrocyte, pericyte and neuronal cells. The highly restrictive paracellular pathway within brain microvasculature restricts significant CNS penetration to only those drugs whose physicochemical properties afford ready penetration into hydrophobic cell membranes or are capable of exploiting endogenous active transport processes such as solute carriers or endocytosis pathways. Endocytosis at the BBB is an essential pathway by which the brain obtains its nutrients and affords communication with the periphery. The development of strategies to exploit these endocytic pathways for the purposes of drug delivery to the CNS is still an immature field although some impressive results have been documented with the targeting of particular receptors. This current article initially provides an overview of general endocytosis processes and pathways showing evidence of their functional existence within the BBB. Subsequent sections provide, in an entity-specific manner, comprehensive reviews on BBB transport investigations of endocytosis involving: transferrin and the targeting of the transferrin receptor; hormones; cytokines; cell penetrating peptides; microorganisms and toxins, and nanoparticles aimed at more effectively delivering drugs to the CNS.