Translational pharmacology of an inhaled small molecule αvβ6 integrin inhibitor for idiopathic pulmonary fibrosis

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and diseaserelated end points. Here we report, GSK3008348 binds to αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy

The urotensin system is up-regulated in the pre-hypertensive spontaneously hypertensive rat

Urotensin II (UII) concentrations are raised both in humans with hypertension and in spontaneously hypertensive rats (SHR). Since the urotensin system acts to regulate glomerular filtration in the kidney it may play a greater role in the pre-hypertensive SHR in which renal dysfunction is known to precede the onset of severe hypertension. This study aimed to determine the renal actions and expression of the urotensin system in the young SHR. Intravenous rat UII (6 pmol. min(-1). 100 g body weight(-1)) had no significant effect on GFR; however urotensin-related peptide (URP) reduced GFR (P<0.05) in 4-5 week old SHR. Administration of the UT antagonist SB-706375 evoked marked increases in GFR (baseline 0.38 ± 0.07 vs antagonist 0.76 ± 0.05 ml. min(-1). 100 g body weight(-1), P<0.05), urine flow and sodium excretion (baseline 2.5 ± 0.4 vs antagonist 9.1 ± 2.1 µmol. min(-1). 100 g body weight(-1), P<0.05) in the SHR. Normotensive Wistar-Kyoto rats showed little response to UT antagonism. Quantitative RT-PCR showed that neither UII nor UT mRNA expression differed between the kidneys of young SHR and WKY rats; however expression of URP was 4-fold higher in the SHR kidney. Renal transcriptional up-regulation indicates that URP is the major UT ligand in young SHR and WKY rats. Enhanced tonic UT activation may contribute to known renal dysfunction in pre-hypertensive SHR

Systemic and renal haemodynamics during rUII and URP infusion.

<p>Effect of exogenous rUII (6 pmol. min^-1. 100 g bwt^-1, solid bars, n = 7 per strain), URP (6 pmol. min^-1. 100 g bwt^-1, hatched bars, n = 5 per strain) or vehicle (0.154 mol/L NaCl, open bars, n = 6 per strain) infusion on mean arterial pressure (A-B), effective renal blood flow (C-D) and glomerular filtration rate (E-F) in anaesthetised 4-5 week-old WKY rats (left column) and pre-hypertensive SHRs (right column). Data shown are mean ± SEM for the baseline control period and the final 15 mins of the treatment period when effects were maximal. * P < 0.05, ** P < 0.01 compared with vehicle-treated rats, Dunnett’s test.</p

Renal water and sodium handling during rUII and URP infusion.

<p>Effect of exogenous rUII (6 pmol. min^-1. 100 g bwt^-1, solid bars, n = 7 per strain), URP (6 pmol. min^-1. 100 g bwt^-1, hatched bars, n = 5 per strain) or vehicle (0.154 mol/L NaCl, open bars, n = 6 per strain) infusion on urine flow rate (A-B), sodium excretion rate (C-D) and fractional sodium excretion (E-F) in anaesthetised 4-5 week-old WKY rats (left column) and pre-hypertensive SHRs (right column). Data shown are mean ± SEM for the baseline control period and the final 15 mins of the treatment period when effects were maximal. * P < 0.05 compared with vehicle-treated rats, Dunnett’s test.</p

Immunolocalisation of urotensin system proteins in the pre-hypertensive SHR and WKY rat kidney.

<p>UII (A-F) and UT (G-L) proteins in the kidneys of 4-5-week-old WKY rats and SHRs. All images are representative and captured at x40 magnification (scale bar 50 µm). Images in the right hand column (C, F, I and L) represent typical negative control sections where primary antibody was omitted. UII-immunoreactivity in both the WKY rat (A) and the SHR (D) cortex was diffuse in distal (arrowheads) and proximal (black arrows) tubules, with no UII-immunoreactivity in glomeruli (red arrow, A). UII-immunoreactivity in both the WKY rat (B) and the SHR (E) medulla was located principally in the collecting ducts (arrowheads), with some immunoreactivity in the loops of Henle (arrows). In both the WKY rat (G) and the SHR (J) cortex UT-immunoreactivity was more intense in the distal (arrowheads) compared to proximal (black arrows) tubules, with UT-immunoreactivity absent in the SHR proximal tubules (black arrow, J); there was also some immunoreactivity in the glomeruli (red arrows). UT-immunoreactivity in both the WJY rat (H) and the SHR (K) medulla was localised to collecting ducts (arrowheads), with little immunoreactivity in the loops of Henle (arrows). </p

Renal haemodynamic and tubular actions of urotensin II in the rat

Urotensin II (UTS) is a potent vasoactive peptide that was originally identified in teleost fish. Mammalian orthologues of UTS and its receptor (UTSR) have been described in several species, including humans and rats. We have shown previously that bolus injections of UTS caused a decrease in urine flow and sodium excretion rates in parallel with marked reductions in renal blood flow (RBF) and glomerular filtration rate (GFR). The aim of this study was to determine the effect of UTS infusion at a dose that has minimal impact upon renal haemodynamics in order to identify a potential direct tubular action of UTS. Infusion of rat UTS (rUTS) at 0.6 pmol/min per 100 g body weight in male Sprague-Dawley rats, which had no effect on RBF and caused a 30% reduction in GFR, resulted in a significant increase in the fractional excretion of sodium (vehicle 2.3+/-0.6 versus rUTS 0.6 pmol 4.5+/-0.6%, P<0.05) and potassium. At the higher dose of 6 pmol/min per 100 g body weight, haemodynamic effects dominated the response. rUTS induced a marked reduction in RBF and GFR (vehicle 1.03+/-0.06 versus rUTS 6 pmol 0.31+/-0.05 ml/min per 100 g body weight, P<0.05) resulting in an anti-diuresis and anti-natriuresis, but no change in fractional excretion of sodium or potassium. Uts2d and Uts2r mRNA expression were greater in the renal medulla compared with the cortex. Together, these data support an inhibitory action of Uts2d on renal tubule sodium and potassium reabsorption in the rat, in addition to its previously described renal haemodynamic effects