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.

An ex vivo investigation into the transurothelial permeability and bladder wall distribution of the nonsteroidal anti-inflammatory ketorolac.

Transurothelial drug delivery continues to be an attractive treatment option for a range of urological conditions; however, dosing regimens remain largely empirical. Recently, intravesical delivery of the nonsteroidal anti-inflammatory ketorolac has been shown to significantly reduce ureteral stent-related pain. While this latest development provides an opportunity for advancing the management of stent-related pain, clinical translation will undoubtedly require an understanding of the rate and extent of delivery of ketorolac into the bladder wall. Using an ex vivo porcine model, we evaluate the urothelial permeability and bladder wall distribution of ketorolac. The subsequent application of a pharmacokinetic (PK) model enables prediction of concentrations achieved in vivo. Ketorolac was applied to the urothelium and a transurothelial permeability coefficient (Kp) calculated. Relative drug distribution into the bladder wall after 90 min was determined. Ketorolac was able to permeate the urothelium (Kp = 2.63 × 10(-6) cm s(-1)), and after 90 min average concentrations of 400, 141 and 21 µg g(-1) were achieved in the urothelium, lamina propria and detrusor respectively. An average concentration of 87 µg g(-1) was achieved across the whole bladder wall. PK simulations (STELLA) were then carried out, using ex vivo values for Kp and muscle/saline partition coefficient (providing an estimation of vascular clearance), to predict 90 min in vivo ketorolac tissue concentrations. When dilution of the drug solution with urine and vascular clearance were taken into account, a reduced ketorolac concentration of 37 µg g(-1) across the whole bladder wall was predicted. These studies reveal crucial information about the urothelium's permeability to agents such as ketorolac and the concentrations achievable in the bladder wall. It would appear that levels of ketorolac delivered to the bladder wall intravesically would be sufficient to provide an anti-inflammatory effect. The combination of such ex vivo data and PK modeling provides an insight into the likelihood of achieving clinically relevant concentrations of drug following intravesical administration.

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.