An inhibitor of RORγ for chronic pulmonary obstructive disease treatment

The role of RORγ as a transcription factor for Th17 cell differentiation and thereby regulation of IL-17 levels is well known. Increased RORγ expression along with IL-17A levels was observed in animal models, immune cells and BAL fluid of COPD patients. Increased IL-17A levels in severe COPD patients are positively correlated with decreased lung functions and increased severity symptoms and emphysema, supporting an urgency to develop novel therapies modulating IL-17 or RORγ for COPD treatment. We identified a potent RORγ inhibitor, PCCR-1 using hit to lead identification followed by extensive lead optimization by structure–activity relationship. PCCR-1 resulted in RORγ inhibition with a high degree of specificity in a biochemical assay, with > 300-fold selectivity over other isoforms of ROR. Our data suggest promising potency for IL-17A inhibition in human and canine PBMCs and mouse splenocytes with no significant impact on Th1 and Th2 cytokines. In vivo, PCCR-1 exhibited significant efficacy in the acute CS model with dose-dependent inhibition of the PD biomarkers that correlated well with the drug concentration in lung and BAL fluid, demonstrating an acceptable safety profile. This inhibitor effectively inhibited IL-17A release in whole blood and BALf samples from COPD patients. Overall, we identified a selective inhibitor of RORγ to pursue further development of novel scaffolds for COPD treatment.


PCCR-1 inhibits RORγ activity and modulate cellular function.
To identify small molecule RORγ inhibitors, we conducted in vitro screening using a TR-FRET binding assay to determine the inhibition of RORγ with a series of different compounds. With extensive effort and from hit identification to lead optimization, PCCR-1 was identified 29 (Fig. 1A). Based on the homology of the ligand binding domains (LBD) between different ROR isoforms (hRORα, hRORβ and hRORγ), slight variations have been observed in the LBD regions which possibly lead to differences in competitive binding between the isoforms (Fig. 1B). Simultaneous binding of a tracer and antibody results in an increase in fluorescence resonance energy transfer (FRET), which decreases with displacement of the tracer upon inhibitor binding. The IC 50 of PCCR-1 was estimated using a specific LBD of human and mouse RORγ and showed an inhibition potency of 34.45 nM and 778.1 nM respectively (Fig. 1C). PCCR-1 showed specific inhibition of human RORγt in a transactivation assay with a potency of 111 nM (Supplementary Fig. 2D) which is comparable to the potency of cellular inhibition of IL-17 release (Fig. 1C).
Inhibition of RORγ by PCCR-1 was also estimated in a functional assay by measuring the inhibition of Th17 dependent cytokine IL-17A release in human peripheral blood mononuclear cells (PBMCs), mouse splenocytes and canine PBMC stimulated with their respective anti-CD3 and anti-CD28 antibodies. PCCR-1 inhibited IL-17A release in human PBMC, mouse splenocytes and canine PBMC with a potency of 117 nM, 766 nM and 504 nM respectively (Fig. 1D). The mouse splenocytes showed relatively lower potency for IL-17A inhibition when compared across species, including rat IL-17 (80.63 nM, Supplementary Fig. 1A). PCCR-1 also showed relatively lower potency for IL-17F levels using hPBMC (788.2 nM, Supplementary Fig. 1B). These results indicate that, PCCR-1 is a potent inhibitor of RORγ and possesses a cellular inhibitory potency for IL-17A release across species.
PCCR-1 treated PBMCs showed downregulation of RORγt and also significantly inhibited IL-17A mRNA expression across healthy human subjects. PCCR-1 reduced the IL-17 expression from human PBMC of IL-17 significantly across healthy subjects in line with RORγt suppression indicating the down-stream effect on IL-17 marker ( Supplementary Fig. 1C).
PCCR-1 inhibited IL-17 release in both mouse and human peritoneal neutrophils with a potency of 245.3 nM and 3661 nM respectively (Supplementary Fig. 2A,C). PCCR-1 also showed inhibition in IL-17 at a lower potency in peritoneal macrophages, with an IC 50  PCCR-1 binds strongly to RORγ. To understand the inverse agonist behavior at the molecular level, interaction with the nuclear receptor RORγ was analyzed in binding assays by estimating its dissociation constant (K off ) and half-life (t 1/2 ). The binding of a ligand stabilizes the conformation of the LBD while binding of a cofactor modifies receptor activity. Binding reversibility was evaluated by adding an excess of competing ligand in the FRET assay and evaluating the displacement of the tracer with inhibitor. Binding of the PCCR-1 with the LBD caused reduction in FRET on antibody addition forming a GST (Glutathione S-transferases) tagged RORγ-antibody complex. The dissociation assay was performed using the jump dilution method with PCCR-1 at a concentration showing maximum binding potency or IC 90 . The dissociation was tested on addition of an excess of a competitive ligand, the tracer. PCCR-1 was observed to be a strong binder showing 20-25% dissociation from the target protein with adequate target residence time for human at t 1/2 290 min and mouse at t 1/2 281.4 min RORγ proteins ( Fig. 2A,B). The absolute dissociation was observed to be 50% in both species till 3 h of measurement.

PCCR-1 selectively inhibits RORγ and no other nuclear receptors or pharmacological off-target receptors.
Based on the homology of the ligand binding domains, 3 different isoforms of ROR (hRORα, hRORβ and hRORγ) are reported 30 . Small variations in the LBD regions possibly lead to differences in competitive binding between the nuclear receptors of the ROR isoforms (Fig. 1b). Similar to the assessment of RORγ binding assay, TR-FRET binding assays were also used to assess the affinity of PCCR-1 for other ROR isoforms. PCCR-1 showed IC 50 of > 10 µM (> 313.7 fold) for the rest of the non RORγ isoforms as well as for other major nuclear receptors (Table 1). This was reconfirmed in cellular functional assays for the selected isoforms and nuclear receptors using Gal4 DBD with luciferase reporter system from Indigo (PA, USA). PCCR-1 was found to have no effect in the transactivation assays done to show RORα inhibition (Indigo, Table 1). In addition, both Schematic diagram of domain structure of RORs with a N-terminal ligand-independent activation function 1 (AF-1) domain, followed by a DNA binding domain (DBD), a hinge domain, and a ligand-binding domain with an activation function 2 (AF-2) domain. Sequence alignment of the ligand binding domain of human RORα, RORβ, and RORγ performed using 56 methodology. Identical and partially conserved residues are labeled with an asterisk and colon respectively. (C) Biochemical activity. Effect on cofactor recruitment to the human (n = 15) and mouse (n = 6) RORγ LBD measured by TR-FRET assay. (D) Cellular activity. Dose dependent inhibition of IL-17 production from human PBMC (n = 24), mouse splenocytes (n = 13) and canine PBMC (n = 6). 'n' is the representative of number of experimental replicates. (The human, mouse and canine basal values were in the range of 58.41 pg/ml, 9.24 pg/ml and 1.5 pg/ml, on induction the IL-17 values were in the range of 1600 pg/ml, 746 pg/ml and 1503 pg/ml respectively). However, PCCR-1 did not show significant inhibition of Th1 (IL-2 and IFN-γ) and Th2 (IL-4, IL-13 and IL-10) dependent cytokines even at 10 µM (Fig. 3A). Similarly, PCCR-1 potency was evaluated under Th17 differen-   (Fig. 3B). Also, PCCR-1 showed IL-17A inhibition in human whole blood under TCR stimulatory conditions with an IC 50 of 3052 nM (Fig. 3C). These results indicate that PCCR-1 is a potent and selective RORγ inverse agonist that specifically inhibits Th17 dependent cytokines under TCR and Th17 dependent stimulatory conditions over Th1 and Th2 cytokines.

PCCR-1 potently inhibits IL-17A in CS mouse model and COPD patient's samples. The effect of
PCCR-1 was evaluated ex vivo using BALf cells from an acute cigarette smoke induced animal model. Animals were exposed to cigarette smoke for 7 days and BALf cells were collected and treated with different doses of PCCR-1 under various stimulatory conditions. IL-17A levels and PCCR-1 inhibitory potentials were compared in basal and LPS or anti-CD3 + anti-CD28 antibody induced conditions and found to be comparable (Fig. 4A), suggesting that RORγ inhibition potentially suppressed inflammation across various inflammatory stimuli. Stimulation by LPS may represent the inflammatory state during a COPD exacerbation, supporting a potential benefit of PCCR-1 in this disease state. A selective up-regulation along with a prominent role of the IL-17A isoform as compared to IL-17F is well documented in the bronchial submucosa and infiltrating inflammatory cells of the small airways of COPD patients 21 (Zhang et al., 2013). In this study, BALf cells, PBMCs and whole blood were evaluated from COPD patients. PCCR-1 showed a concentration dependent inhibition of TCR stimulated IL-17 release from COPD lung BALf cells, PBMCs and whole blood, with potencies of 522.5 nM, 336.1 nM and 5053 nM respectively (Fig. 4B). These results demonstrate a good translation of PCCR-1 activity in COPD patients and also in a mouse CS model. PCCR-1 shows significant efficacy in acute cigarette smoke model. Cigarette smoke exposure for 7 days caused significant increase in the total cell count in BAL fluid, including leukocytes, neutrophils and macrophages as compared to an air exposed control group. PCCR-1 was dosed once a day for 7 days through the intranasal route and showed a dose dependent inhibition of total leukocytes between 1 µg and 50 µg/animal, with inhibition of total leukocytes at 50 µg, similar to that observed with 100 µg BID of Roflumilast (used as a positive control). PCCR-1 at 10, 30, 50 and 100 µg per mouse also significantly obtunded cigarette smoke mediated increase in neutrophil and macrophage counts in BAL fluid (Fig. 4C).

PCCR-1 shows high lung concentrations in mice after intranasal administration.
A single intranasal dose resulted in high lung exposures of PCCR-1 in mice, as suggested by a mean lung to plasma Cmax and AUClast ratios that were found to be > 1000 at both the doses (20 and 50 µg), respectively ( Table 2). Peak lung concentrations at both the doses were observed at 2 to 4 h post dose. Quantifiable levels in lungs were observed up to 24 h post dose. The mean apparent elimination half-life and AUC∞ were not estimated as the data point describing elimination phase were inadequate to get acceptable accuracy in these parameters.
Further, after the 7-day intranasal treatment with PCCR-1 in the acute CS-model in mice, the results observed from the PD study were in line with the PK data, wherein very high concentrations (> 1000-fold) of PCCR-1 were observed in lung as compared to the plasma across all the tested doses (1 to 100 µg). A very good correlation of the lung and BAL fluid concentrations of the compound versus inhibition of the PD biomarkers (total leukocyte count, neutrophils and macrophages) were observed with good dose-dependent inhibition profiles (Supplementary Table 1, Supplementary Fig. 3). Overall, the data indicated a strong PK-PD correlation with the compound. PCCR-1 demonstrates an acceptable toxicology profile. Rats were exposed to PCCR-1 aerosols at 3.4, 10.8 and 32.1 mg/kg/day (delivered dose) for 60 min daily for 14 days using a snout only exposure technique via a modular stainless steel flow past inhalation chamber at indicated doses as mentioned in Table 3. No treatment-related findings noted, including myeloid:erythroid (M:E) ratios in any dose levels except minimal changes in the reticulocytes and adrenal glands.
The TK assessment was done by measuring the concentrations of PCCR-1 in blood. The C max and AUC on both Day 1 and Day 14 appeared to be comparable between male and female rats at the low dose level (Table 3). However, at the mid and high dose levels, the C max and AUC in females were approximately 1.3-1.9 fold and 1.5-2.5 fold higher than males, respectively. The blood concentration profiles indicated that in general, the C max and AUC on both day-1 and 14 increased with an increase in dose level. With once-daily inhalation administration for 14 days, PCCR-1 AUC in blood accumulated about 1.5-2.6-fold relative to that of day-1, except for female rats at 10.8 mg/kg/day dose level where there was no considerable accumulation observed.
A slightly increased reticulocyte count was noted in the mid and high dose group females (1.27 and 1.44 fold compared with controls) and was statistically significant in the high dose group compared with controls (Table 3). A marginal increase in reticulocyte count was also noted in the high dose males compared with the control group (1.09-fold). Statistical significance noted in other haematological parameters was considered incidental. Although statistically not significant, slightly higher group mean adrenal weights (absolute and relative to body weight/brain weight) were observed in the mid and high-dose females when compared with the control group (Table 4). No treatment-related histopathological changes were noticed in any organs, including the respiratory system, except the adrenal gland that showed bilateral diffuse hypertrophy of the zona fasciculata in the mid and high-dose females. This correlated with high adrenal gland weights. Based on these results, the maximum tolerated dose of PCCR-1 was considered to be 32.1 mg/kg/day.

Discussion
Th17 cytokines may play a significant role in COPD pathogenesis 31 . The presence of Th17 cells along with the release of various cytokines are predictive of disease severity and airflow limitation 27 . In addition to Th17 cells many other cells have also been shown to release these cytokines including innate lymphocytes, neutrophils and macrophages 18,32,33 . Irrespective of the cell type, the release of Th17 dependent cytokines are regulated primarily by the transcriptional modulator RORγ. Also it has been well documented that the Th17 dependent cytokines are differentially expressed across cell types and are up-regulated based on the disease type and condition 34 .
Although there are some reports correlating the pharmacological regulation of Th17 dependent cytokines and their implication in COPD, this is the first report of an RORγ inhibitor as a potential therapeutic agent for COPD as depicted in the Fig. 5 [35][36][37][38][39] . ROR has a typical nuclear receptor domain structure consisting of four major functional domains: An N-terminal (A/B) domain followed by a highly conserved DNA-binding domain (DBD), a hinge domain, and a C-terminal ligand-binding domain (LBD). The LBDs of nuclear receptors are multifunctional and play a role in ligand binding, nuclear localization, receptor dimerization, and provide an interface for the interaction with co-activators and co-repressors 30 . Therefore, any modulations in the LBD region with a small molecule have the potential to affect the functions of nuclear receptors and influence the downstream signaling in their respective pathways. Modulation of the LBD region of RORγ by an inhibitor is demonstrated in this article and being homologous across species, this inhibition is expected to show and translate across species. PCCR-1 was profiled against a panel of receptors and other isoforms of nuclear receptors and was found to selectively inhibit RORγ, showing minimum off target effects. PCCR-1 showed specific inhibition of the human RORγt in a transactivation assay at a potency that was comparable to the cellular inhibition of IL-17 release. The expression pattern of RORγt also showed a decreasing trend on treatment with PCCR-1 along with significant reduction in IL-17 expression in the human PBMC.
Stimulated Th17 cells release various cytokines, including IL-17A, IL-17F and IL-22 which have been implicated in various inflammatory diseases including COPD 27 . IL-17A is a pro-inflammatory cytokine that modulates www.nature.com/scientificreports/ airway inflammation through recruitment of inflammatory cells, including neutrophils and lymphocytes, which in turn release various chemo-attractants such as CXCLs and inflammatory mediators like TNF-α and IL-6, augmenting the inflammatory state 40 . The use of Simvastatin also showed marked suppression of IL-17A and IL-22 secretion which may contribute to decrease in IL-6 and CXCL8 production in the airways of COPD patients (Fig. 5) 26 . Statins have been well known to cause beneficial effects in COPD regarding lung function decline rates and severity of exacerbations and hospitalization. Long-term use of statins reduced inflammatory factors including CRP and IL-6, increased lung function indices including FEV1% predicted and FEV1/FVC%, and reduced the risk of AECOPD 40 . Literature reports have shown a positive correlation between CRP and IL-17A, providing some evidence of this cytokine having a role in neutrophilic inflammation, a hallmark of COPD 26 . The expression of IL-17A in the bronchial submucosa was also shown to be increased in smoking associated COPD and correlated with disease severity 41 . However, neither a change in IL-17F expression 42 , nor a significant difference was observed in its levels in COPD patients or control groups. Thus IL-17A modulation may have therapeutic benefit in COPD. We evaluated PCCR-1 a selective RORγ inhibitor that demonstrated potent inhibition of IL-17A with lower potency for IL-17F as potential therapeutic agent in COPD.
The compound specifically modulates the Th17 pathway including both IL-17 and IL-22, having no effect on Th1 and Th2 signaling as seen in the cells stimulated under TCR stimulation.
Apart from various T-helper cells, neutrophils and macrophages are also believed to play an important role in COPD. An increase in neutrophils is observed in the respiratory secretions of COPD patients, notably during exacerbations, while macrophages are present in small airways and parenchyma and are related to disease severity 32,43 . Recent reports have showed the role of RORγt in the recruitment of macrophages during hydrocarbon oil-induced chronic inflammation 44 . Therefore, modulation of macrophage activity via inhibition of facilitator cytokines by RORγ in activated T-helper cells shows potential therapeutic promise. PCCR-1 also showed inhibition of IL-17 release in other inflammatory cells including neutrophil and macrophages of mouse and neutrophils from human whole blood. The IL-17 inhibition in neutrophils was observed to be much lower www.nature.com/scientificreports/ as compared to the Th17 cells. However, the concentrations of PCCR-1 required for IL-17 inhibition that may impact neutrophil and macrophage inflammation, was significant as seen from the PK-PD correlation. A seven day in vivo acute cigarette smoke exposure model showed a dose dependent inhibition of neutrophil, lymphocyte and macrophage infiltration in BAL fluid by PCCR-1. This indicated the potential benefit of a specific inhibitor of RORγ in modulating cellular infiltration in the lungs of COPD patients in a dose proportional manner. The in vivo effect was further correlated with ex vivo studies on BAL cells of cigarette smoke exposed animals where IL-17 inhibition was observed. The ex vivo model demonstrated favorable potencies across stimulating conditions for IL-17 inhibition by PCCR-1, comparable to its human IL-17 data from the BAL cells of COPD patients. To understand the translation of in vivo animal efficacy to that of human subjects, we used COPD patient samples of banked BALf cells, PBMCs and whole blood. Our data showed equivalent potencies in both human and rodents with comparable inhibition of IL-17 in both BAL cells and whole blood. Inhaled LPS causes an acute increase in airway neutrophil numbers in healthy smokers 45 closely resembling the acute increase in airway neutrophils that occurs during COPD exacerbations. IL-17 expression has been shown to be increased in peripheral blood cells during COPD exacerbations 46 . We demonstrated significant inhibition of IL-17 under conditions of LPS exposure in an in ex vivo cigarette smoke model, where comparable inhibition was observed at similar potency as seen in the BALf from COPD patients.
The mechanism behind the long duration of action for any localized drug relates not only to its PK profile, but also to a slow rate of dissociation (residence time) from its target protein. As in the case of human M3 muscarinic receptor agonist 47 which shows a long duration of action (of approximately 24 h): an important feature of a drug intended to treat chronic diseases, is a prolonged efficacy 48 affording a simple, once-daily dosage regimen resulting in improved patient compliance 49 . There are many drugs currently being evaluated in clinical trials for their ability to function similarly to LAMAs (Long-acting muscarinic antagonists) with a potential for once-daily administration in COPD patients due to the established convenience of the route and once daily dosing regimen. Thus a RORγ inhibitor administered via the inhalation route, showing a strong association with its target, affording a long duration of action would fit this ideal profile of a good candidate drug for COPD. The additional advantage of a long acting RORγ inhibitor drug would be its ease of combining with currently established COPD drugs administered in once or twice daily regimen. The kinetic binding data of PCCR-1 clearly indicates strong binding to its target protein and a long dissociation from its target protein at t 1/2 of 280-290 min (~ 50% absolute value). Literature reports indicate the long acting muscarinic antagonists showed strong binding with dissociation half-lives varying from tiotropium with t 1⁄2 of 27 h to glycopyrrolate with t 1⁄2 of 6.1 h 50 . PCCR-1 demonstrated a t1/2 of > 5 h with a very slow K off , showing only 50% dissociation after 290 min (~ 5 h) supporting a potential for twice daily dosing regimen akin to glycopyrrolate. Based on the efficacy of the molecule in the in vivo model via the intra nasal route and the known toxicities associated with the target inhibition described in the literature, the localized application of this compound was the way forward to understand its overall profile.
The role of RORα as a novel contributor of structure and function of adrenal cortex is well known and its inactivation in both sexes of mice have shown to cause structural disorganization of the adrenal cortex with increased adrenal cortex size in female mice and increased cell proliferation in males 51 . Even though PCCR-1 showed a clean profile for RORα, administration of PCCR-1 showed slight increase in reticulocyte count in the mid and high dose group females without any changes in other hematological parameters. In females, a minimal increase in the adrenal gland weight and diffused bilateral hypertrophy was observed with mid and high dose groups. This may be due to increased stimulation by adrenocorticotropic hormone (ACTH) from the pituitary gland by stress or due to adrenocortical insufficiency as a result of this effect 52,53 . The cause of the hypertrophy of the adrenal cortex in the current study was not apparent 54,55 and both stress and adrenocortical insufficiency were ruled out due to the lack of thymic atrophy, and hematological findings (such as reduced numbers of blood lymphocytes and/or increased numbers of blood neutrophils). The adrenal cortical hypertrophy occurred only in female rats and was not seen in any of the treated male rats due to higher exposures seen in females. Based on these results, the maximum tolerated dose of PCCR-1 was considered to be 32.1 mg/kg/day in the rat. As the compound was primarily being explored for the inhalation route, the systemic levels were quite low to expect any of these AEs. With more lead optimization efforts, an advanced compound-PCCR-2 was identified and advanced to clinical trials. PCCR-2, has shown a favorable safety profile in all Phase 1 enabling 4-week repeat dose administration studies in both rats and dogs, and has been successfully progressed to Phase 1 human Clinical Trial evaluation in USA as a potent RORγ inhibitor for COPD via-inhalation route (Clinical trial IND 144,906). Table 2. Concentrations of PCCR-1 observed in mice exposed to cigarette smoke at 1 µg, 10 µg, 30 µg, 50 µg and 100 µg on day-8 after intranasal dose of PCCR-1.    Corn cob (BioCobb, AT&T) was used as the bedding material. Commercial pellet diet (Altromin, Germany) and community tap water passed through a reverse osmosis system (Millipore) were given. Water was provided ad libitum throughout the study period. Food was also provided ad libitum throughout the study period.

Methods. RORγt-LBD and other isoforms co-activator ligand binding assay. The human and mouse RORγt
cDNA clones were obtained from OriGene (MD USA). The RORγt LBDs of human (accession no. NM_00100152; RORC-LBD region 229-497aa) and mouse (accession no. NM_011281.2; RORγt-LBD region 264-516aa) were sub-cloned between Sal I and Not I, in pGEX-4T1-modified plasmid (pGEX-4T1-modified plasmid was a generous gift from Prof. Orly Reiner, from Weizmann Institute of Science, Rehovot, Israel). The correct sequence was verified by dideoxynucleotide sequencing (outsourced to Saf Labs, place). pGEX-4T1 plasmid containing human RORC -LBD was transformed and expressed in Escherichia coli BL21-DE3 cell stock. Protein expression was checked by western blotting. 10 nM human/mouse RORC-LBD prepared in assay buffer (25 mM HEPES; pH 7.4 with100 mM NaCl, 5 mM DTT, 0.01% BSA, and 10% Glycerol) was incubated for 60 min at 22 °C and detection mixture of 300 nM Fluorescein-D22 coactivator peptide (Invitrogen) and 10 nM Lantha screen Tbanti GST antibody (Invitrogen. MS USA) prepared in assay buffer were added into a 384-well white plate. The plate was then incubated for 60 min at 22 °C on shaker and kept overnight at 4 °C. The next day, plate was read on Infinite F500 reader (Magellan Tecan Switzerland). TR-FRET signal was defined as the ratio 520/495. The percent activity of each dilution was determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC 50 values were determined by fitting percent inhibition data in GraphPad Prism (version-5.01) software. www.nature.com/scientificreports/ For selectivity, the ROR isoforms RORα, FXR, LXRα, LXRβ, RXR, RARα were incubated with compound for 60 min at 22 °C followed by detection mix containing respective agonist (All trans retinoic acid for RARα, T0901317 for LXRα, LXRβ, 9-cis Retinoic acid for RXR, GW-4064 for FXR) and coactivator peptide (FD22 for RORα, PGC1α for RXR, RARα, TRAP220 for LXRα, LXRβ, SRC2-2 for FXR) and Tb-anti GST antibody prepared in assay buffer were added into a 384-well white plate. Reading taken after overnight incubation and the data analysis done as per the protocol mentioned in RORγt-LBD and co-activator ligand binding assay.
For whole blood assay the heparinized whole blood was diluted (1:1) with saline and added in 96-well plate pre-coated with human anti-CD3 mAb (10 µg/mL). Compound treatment was done for 30 min followed by costimulation with anti-CD28 mAb (2 µg/mL). After 48 h of incubation supernatant was used for IL-17 estimation by ELISA.
The human neutrophils were isolated by Ficoll-Hypaque by centrifugation of blood from healthy subjects at 700 g for 30 min, followed by erythrocyte lysis with distilled water for 30 s. Cells were washed in PBS and post 1 h treatment with PCCR-1, cells were stimulated with 50 ng/mL PMA and 2 µg/mL Ionomycin. After 18 h the supernatant was collected for IL-17 estimation by ELISA.
Splenocytes were prepared from 6-8 week old BALB/c mice and cells were seeded in 96-well plates coated with mouse anti-CD3 mAb (10 µg/mL). Cells were treated with test compounds for 30 min followed by stimulation with mouse anti-CD28 mAb (3 µg/mL) for 72 h at 37 °C in 5% CO incubator. The supernatant was used for estimating mouse IL-17 by ELISA.
The peritoneal neutrophils or macrophages were isolated from C57BL/6 male mice or BALB/c female mice (6-8 wks; 18-22 g) injected with 3% or 4% thioglycollate broth. After 4 h or 4 days, RPMI medium was injected into the peritoneal cavity of mouse and peritoneal lavage was collected. The lavage was pooled and spun at 800 rpm for 10 min. The pellet was re-suspended in 1X Gey's solution for 5 min followed by PBS wash. In case of neutrophils, the isolated cells were seeded in 48 well plate for 1.5 h. After 1.5 h, the supernatant containing peritoneal neutrophils were collected and centrifuged at 2000 rpm for 10 min and the pellet were re-suspended in 2% RPMI. In case of isolated peritoneal macrophages, the cells were seeded in 48 well plate for 3-4 h followed by PBS wash. Both the neutrophils and macrophages were treated with PCCR-1 in 2% RPMI for 1 h followed by stimulation with 10 µg/ml or 5 µg/ml of LPS for 18 h or 24 h. The supernatant collected for IL-17 estimation by ELISA.
Canine PBMCs were procured from Lonza (Cologue, Germany). Frozen canine PBMCs was quickly thawed in a 37 °C water bath and cells were seeded in 96-well plates coated with canine anti-CD3mAb (30 µg/mL). Cells were further treated with the compound similar to human PBMC with co-stimulation of canine anti-CD28 mAb (10 µg/mL). After 72 h of incubation the supernatant was collected for estimating canine IL-17 by ELISA.
For RORg transactivation assay the HEK293 cells were seeded in 100 mm dish and transfected with 4. www.nature.com/scientificreports/ Whole blood and BAL samples from COPD patients were obtained from Multispecialty hospital, Mumbai post approval from ethics committee and informed consent of patients. 30-40 mL Broncho alveolar lavage (BALf) was collected per patient and stored in ice. Blood was collected in heparin vaccutainer as per approved protocol. Both the samples were processed within 2 h of collection. BALF was passed through 100-µm and 40-µm cell strainer to remove debris and mucus, the samples were centrifuged and the cells were re-suspended in RPMI 1640 complete medium. PBMCs from blood were isolated using Histopaque-1077 by density gradient centrifugation. Isolated BAL cells and PBMCs were seeded in 96 well coated with 10 µg/ml anti-CD3 and 2 µg/ml anti-CD2. Cells were further treated with different concentration of test compound for 60 min followed by stimulation with 20 µg/ml anti-CD28 and incubated for 48-72 h. The supernatant was used for estimating IL-17 by ELISA. .In addition to that, all animal experimentation complies with the guidelines of Animal Research: Reporting In Vivo Experiments (ARRIVE). Male C57BL/6 mice were exposed to either room air or cigarette smoke (CS) from 10 cigarettes (Kentucky Research Cigarettes 3R4F) for 50 min, twice daily for 7 days in a whole body box exposure system (SIU24, ProMech Lab Holding AB, Sweden). PCCR-1 and Roflumilast were sonicated in phosphate buffered saline (PBS) containing 0.005% tween 80 for 20 min. The animals were administered vehicle or test compounds intranasally (i.n.) 40 µl (µl) per animal under isoflurane anesthesia. Cigarette smoke exposed mice were dosed intranasal either with vehicle or PCCR-1 (1, 10, 30, 50 and 100 µg per animal. n = 7 per group) once daily or Roflumilast 100 µg twice daily 1 h before the cigarette smoke exposure. Control animals were given vehicle one hour before exposure to fresh air or cigarette smoke. PCCR-1 and Roflumilast were sonicated in phosphate buffered saline (PBS) containing 0.005% tween 80 for 20 min. For localized delivery to lung, animals were administered vehicle or test compounds intranasal (i.n.) 40 µl (µl) per animal under isoflurane anesthesia. Animals were sacrificed 20 h after the last smoke exposure and broncho alveolar lavage fluid (BALf) was collected after euthanizing with a urethane, trachea was exposed and BALf was collected 4 times using 0.3 ml PBS. All aspirates of BAL were pooled and total number of cells were determined using a hemocytometer. BALf was centrifuged in cold and cell pellet was used for preparation of smears. BALf smear slides were stained with Leishmans stain and differential cell count of 500 cells based on standard morphology was performed manually. All data are presented as mean ± S.E.M. of animals.
Ex vivo BAL studies. C57BL/6 mice were exposed to as per above mentioned protocol. On 7th day, animals were sacrificed and a tracheal cannula was inserted. Three times via the trachea, 1 mL of plain RPMI medium was instilled and recovered by gentle manual aspiration. The recovered BALf was centrifuged, the cell pellet washed twice and finally re-suspended in 5% FBS-RPMI. Isolated BAL cells were pre-incubated with different concentration of PCCR-1 for 60 min, followed by stimulating with 1 µg/ml soluble mouse anti-CD3 and 3 µg/ ml mouse anti-CD28 mAbs and/or 100 ng/ml of LPS and incubated for 24-48 h at 37 °C. The supernatant was used for estimating IL-17 by ELISA.

Pharmacokinetic (PK) studies in mice.
A single dose intranasal PK study of PCCR-1 was conducted in male C57 mice at 20 and 50 µg/animal dose. The formulation was prepared in saline with 0.1% v/v tween-80 as the wetting agent. Animals were anesthetized briefly using gaseous isoflurane and oxygen mixture and a 40 µL of the formulation was instilled into both the nostrils (20 µL each) once the animal reached a regular breathing pattern and at pre-determined time points (0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h), approximately 0.3 mL of blood samples were collected using retro-orbital puncture in suitable anticoagulant (K3 EDTA). The animals were immediately sacrificed and lung was isolated. Blood samples were centrifuged (3000 rpm, 5 min) and plasma was harvested. Lung samples were homogenized in PBS buffer (pH 7.4) and centrifuged to collect the supernatant. Both plasma and lung samples were extracted using methyl t-butyl ether, mixed and centrifuged. The supernatant samples obtained after centrifugation were dried and reconstituted with acetonitrile-ammonium acetate buffer (90:10). The samples were mixed and analyzed in LCMS/MS for the analyte concentrations. Concentration vs time values were subjected to non-compartmental analysis using Phoenix WinNonlin® (Version 6.4) to estimate the appropriate pharmacokinetics parameters.
Additionally, after 7-day intranasal treatment with PCCR-1 in the CS-model in mice as described above, blood, BAL fluid and lung tissues were collected on day-8 approximately 1 h after the last treatment dose for estimating the drug concentrations for PK-PD correlation.
Toxicology studies. The  www.nature.com/scientificreports/ The toxicology study was done in SD (Sprague Dawley) rats that were exposed to PCCR-1 aerosol for 60 min daily at 3.4, 10.8 and 32.1 mg/kg/day (delivered dose) for 14 days using a snout only exposure technique via a modular stainless steel flow past inhalation chamber. A 60% w/w blend of PCCR-1 in Lactose LH201 was prepared by adding the appropriate amount of PCCR-1 to the appropriate amount of lactose. The estimated delivered doses were derived based on analytical aerosol concentration, and respiratory minute volume calculated based on body weight, exposure duration, and animal body weight (Supplementary section B).
Statistical analysis. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test using Graph Pad Prism (version-5.01, GraphPad Software Inc., CA). Graph Pad Prism software (version 5.01, GraphPad Software Inc., CA) was used for the generation of graphs. Statistical difference between two groups were determined by pared t-test and p value < 0.05 were considered statistical significant.