Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF

Rationale Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. Methods We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. Results We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. Conclusions Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. Trial registration number NCT01725139, pre-clinical.

lung tissue biopsies were cut into 1-mm 3

cubes and cultured in Dulbecco's modified
Eagle's medium supplemented with 10% (v/v) newborn calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 2.5 μg/ml amphotericin B (all from Thermo Fisher Scientific). A near confluent monolayer of fibroblasts was obtained after 3 to 4 weeks and passaged. Experiments were conducted on cells between passages 3 and 13. Fibroblast cell line purify was confirmed by immunohistochemical characterization using antibodies to cytokeratin, von Willebrand factor, and desmin to rule out contamination by epithelial, mesothelial, endothelial, or smooth muscle cells.
All lines generated were negative for these markers and > 95% of cells stained positively for the mesenchymal marker, vimentin. Between 20 and 30% of cells were also positive for myofibroblast marker, α-smooth muscle actin. LFs were used between passage 3 and 13, maintained in DMEM at 37°C, 10% CO2, 100% humidity supplemented with L-glutamine, penicillin, streptomycin, fungizone, and 10% FBS (all from Thermo Fisher Scientific). For most experimental series LFs were seeded to become confluent within 24 hours prior to 24 hour serum starvation. The exception to this was for proliferation experiments where LFs were seeded at 7500 cells per well (96 well format), to allow a window for proliferation. Blotting Detection Reagents (GE Healthcare). Following stripping, membranes were blocked and re-probed with antibodies specific for total SMAD2 (#3103, Cell Signalling Technology) or total Akt (#4060, Cell Signalling Technology).
pAkt signalling in BALF cell pellet. BALF cell pellets were prepared by centrifugation (300g, 5 minutes) and differentially counted. Following re-suspension cells were incubated in RPMI containing 0.1% DMSO (vehicle control) or increasing concentrations of GSK2126458 (30 mins, 37 o C, 5% CO2, 100% humidity). Following incubation suspensions were centrifuged (300g, 5 minutes) and cell pellets lysed in ice cold PhosphoSafe® buffer (Merck Millipore). Cell lysates were assayed for total Akt and pAkt S473 using the Meso Scale Discovery (MSD) system. In separate experiments supernatants were harvested for assessment of P1NP by proprietary competitive ELISA as previously described 3 .

Building a dose prediction model for GSK2126458 in IPF.
All in vitro data were first analyzed as a naïve pool using a non-linear mixed effects model 4 as a simulation of the inter-donor variability for fibroblast and BALF cell PD.
Using this approach, the cell assay data were described by the sigmoidal relationship described in Eqn. 1, Figure 5 (for output parameter estimates see Table   1). Next, predicted plasma concentration profiles for GSK2126458 in IPF patients were generated based on population PK data obtained from on-going oncology studies with GSK2126458 in patients with advanced solid tumours (http://www.gskclinicalstudyregister.com/study/112826#rs). The modelled PD and predicted PK data were combined in a stochastic simulation to predict target engagement in IPF patients at twice daily doses of 0.25, 1, 2 and 2.5 mg.

Determination of collagen deposition by molecular crowding assay.
Collagen biosynthesis in 96 well format was measured by a high-content imaging based molecular crowding assay modified from a previously described method 5 . Briefly, confluent LFs were cultured in DMEM containing 0.4% FCS and ascorbic acid

Figure S1. Akt phosphorylation with fibrotic foci in the IPF lung.
Immunoreactivity for Akt phosphorylated at serine 473 (pAkt S473 , A) and threonine 308 (pAkt T308 , C) is shown in serial sections of discrete fibrotic foci, localizing to spindle shaped cells immuno-reactive for SMA (white asterisk, E), as well as capillary endothelium. Immuno-staining for both phosphorylation sites was also observed in epithelial cells overlying the fibrotic focus (pAktS473 A and pAktT308 C, black asterisk E). Signal for pAktS473 and pAktT308 was present in bronchial epithelium (B, D respectively), and macrophages delineated by CD68 (Arrow, G; pAktS473, A; pAktT308,C), with poor signal pAkt signal observed in SMA positive airway smooth muscle (F, pAktS473, B; pAktT308,D); scale bar, 150μm. The fibrotic focus is also shown at high power, scale bar 50μm (inset) Negative immuno-staining with non-immune IgG at equivalent dilutions to pAkt S473 and pAkt T308 specific antibodies also shown (H and I). Data from n=5 C-LF lines are shown and data expressed fold relative to baseline without TGF (0.1%DMSO) (Veh) and shown as mean +/-SEM for n=4 replicate wells per condition; ** P<0.0001 for differences in raw values from no vehicle controls (2 way ANOVA, Tukey's multiple comparisons test).
Compound 1 GSK2126458 pIC50 n (total) Comments pIC50 n (total) Comments PI3K-alpha 10.4 3 2 values of > 9.9 9.5 10 2 values of > 10.0 PI3K-beta 9.5 3 1 value of > 9.9 9.1 10 PI3K-delta 10.6 3 2 values of > 9.6 9.3 10 5 values of > 9.58 PI3K-gamma 9.9 2 1 value of > 9.4 9.3 10 FRAP1 9.5 1 9.6 1 Table S1. pIC50 for PI3K/ mTOR inhibitors in recombinant assays (GSK). pIC50 in recombinant assays (GlaxoSmithKline, Upper Merion, PA). Data points below the concentration limit of the assay are excluded from the summary mean in the PI3K isoform data. An overview of the data is provided in the table. The pIC50 quoted is the mean for the non-modified values; the n is the total of instances the compound has been assayed and the comment field illustrates the instances when the compound tested beyond the limit of the assay.