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Oral IRAK4 inhibitor BAY-1834845 prevents acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a lethal clinical entity that has become an emergency event with the outbreak of COVID-19. However, to date, there are no well-proven pharmacotherapies except dexamethasone. This study is aimed to evaluate IRAK4 inhibitors as a potential treatment for ARDS-cytokine release syndrome (CRS). We applied two IRAK4 inhibitors, BAY-1834845 and PF-06650833 to an inhaled lipopolysaccharide (LPS)-induced ARDS mouse model with control of high dose dexamethasone (10 mg/kg). Unexpectedly, although both compounds had excellent IC50 on IRAK4 kinase activity, only BAY-1834845 but not PF-06650833 or high dose dexamethasone could significantly prevent lung injury according to a blinded pathology scoring. Further, only BAY-1834845 and BAY-1834845 combined with dexamethasone could effectively improve the injury score of pre-existed ARDS. Compared with PF-06650833 and high dose dexamethasone, BAY-1834845 remarkably decreased inflammatory cells infiltrating lung tissue and neutrophil count in BALF. BAY-1834845, DEX, and the combination of the two agents could decrease BALF total T cells, monocyte, and macrophages. In further cell type enrichment analysis based on lung tissue RNA-seq, both BAY-1834845 and dexamethasone decreased signatures of inflammatory cells and effector lymphocytes. Interestingly, unlike the dexamethasone group, BAY-1834845 largely preserved the signatures of naïve lymphocytes and stromal cells such as endothelial cells, chondrocytes, and smooth muscle cells. Differential gene enrichment suggested that BAY-1834845 downregulated genes more efficiently than dexamethasone, especially TNF, IL-17, interferon, and Toll-like receptor signaling.


Introduction
Acute respiratory distress syndrome (ARDS) is a critical respiratory illness associated with infection, autoimmunity, and injuries. The overall mortality rate is as high as 30-40 % [1]. The main pathological features are extensive damage to the barriers of lung epithelial and endothelial cells, diffuse damage to lung capillaries, enhanced permeability, and the neutrophil influx into the lung tissue, resulting in multiple injuries to organ function leading to respiratory failure and high mortality [2][3][4].
Previously, treatment for ARDS focused on lung-protective ventilation that reduced mortality [12]. No specific pharmacotherapies have been identified partially because multiple drug treatments, including inhalation or drip infusion of synthetic surfactants [13], intravenous endotoxin antibodies, ketoconazole, ibuprofen [14] and inhalation nitric oxide (iNO) [15], have proven to be ineffective. Since the COVID-19 pandemic, the increase in ARDS cases has spawned a series of investigations of anti-inflammatory drugs. Among these explorations, only dexamethasone showed benefits on mortality in severe COVID-19 pneumonia [16]. Other medicines, such as the JAK inhibitors tofacitinib and baricitinib, are beneficial to recovery, yet are basically for patients with the moderate disease who receive noninvasive ventilation [17,18], and the IL-6R blocker tocilizumab has failed to improve primary outcomes in COVID-19 patients [19]. Therefore, anti-inflammation is the only pharmaceutical strategy for ARDS that has been clinically proven thus far, although new candidate agents are still greatly needed.
Recently, the theoretical druggability of IRAK4 has been put into practice. BAY-1834845 and PF-06650833 are two IRAK4 inhibitors [34,35] that have published results from phase I clinical trials. This study aimed to investigate the potential efficacy of IRAK4 inhibitors in ARDS.

IC 50 and IRAK4 kinase inhibition efficiency of BAY-1834845 and PF-06650833
The IC 50 (semimaximum inhibitory concentration) values of BAY-1834845 and PF-06650833 were assessed by mobility shift assay (MSA). After the signal was measured, the inhibition rate of the compound at each concentration was calculated. Then nonlinear curve fitting was performed with the logarithmic concentration-inhibition rate to obtain the IC 50 value of the compound.

ARDS mouse model
Modeling. Female Balb/c mice (aged 6-7 weeks) were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd. (China) and were housed in an SPF animal room for seven days to adapt to the environment. The mice were housed under a controlled temperature and humidity with a standard day-night cycle and free access to food and water. For modeling, 50 µl PBS or LPS (700 µg/ml, Sigma) was injected into the trachea of each mouse with a special atomized aerosol needle. All animal experiments were approved by the Animal Care Committee of Shanghai Jiao Tong University.
Treatments. For prevention, all drugs were given 30 min before LPS modeling. In regard to treatment, all mice received twice oral therapeutic agents 4 h and 12 h after LPS modeling.

Pathological assessment of mouse lung tissue
The lungs of the mice were harvested, fixed with formalin solution, embedded in paraffin, and cut into 5-μm-thick slides. Three slices were randomly picked up for each sample with a minimal interval of 20 µm. Haematoxylin and eosin (HE) staining was applied to the slices. A pathologist blindly scored the lung tissue according to the Smith lung injury score [36]. Briefly, the severity of five parameters-inflammation, edema, hemorrhage, atelectasis, and formation of hyaline membrane-was scored on a 0-to 4-point scale: 0, no damage; 1, damaged visual field percentage ≤ 25 %; 2, 25 % <damaged visual field percentage ≤ 50 %; 3, 50 % < damaged visual field percentage ≤ 75 %; and 4, diffuse injury. A total score for a field at 400x magnification (NIKON Eclipse Ci) was calculated by adding the scores of all five parameters. For the selection of the microscopic fields, each slice was divided into quadrants, then 3 non-overlapping fields were randomly pick-up in each quadrant for observation and scoring, and the average of the scores of twelve observation fields was taken as the score for one slice. Each sample contains three discontinuous slices at minimal 20 µm intervals and the average of these three slides is the final score for each sample.

Broncho-alveolar lavage fluid (BALF) analysis
BALF was harvested from each mouse at 4 h and 24 h after LPS modeling. In brief, 0.5 ml PBS was used to wash the lung twice through a tracheal cannula. As much BALF was collected as possible and then centrifuged at 4 • C. Total cell counts and differential cell counts were quantified using an automatic blood cell analyzer (Mindray, BC-5000). According to the manufacturer's instructions, the secretion of IL-1ß, IL-6, and TNF-α in BALF was measured using enzyme-linked immunosorbent assay (ELISA) kits (Beyotime Biotechnology). The levels of lactate dehydrogenase (LDH) activity in the BALF were measured by an LDH Assay Kit (Abcam, ab102526) following the manufacturer's instructions.

Flow cytometry analysis of BALF
Cell suspensions were obtained from BALF and were washed and resuspended in PBS-1 %BSA before FASC analysis. All flow cytometry data was acquired using FACS Canto II (BD) and data was analyzed using

RNA sequencing (RNA-seq) of lung tissue
Lung tissue RNA (RNA integrity number ≥ 7) was extracted with the mirVana miRNA Isolation Kit (Ambion). RNA libraries were prepared with the TruSeq Stranded mRNA kit and sequenced by HiSeqTM 2500 (Illumina). RNA-seq fastq files were processed using bowtie2 [37] and eXpress [38]. Differentially expressed genes (DEGs) were identified using DESeq (2012) [39]. A P value < 0.05 and fold change > 2 or fold change < 0.5 were set as the threshold for significantly differential expression. Hierarchical cluster analysis of DEGs was performed to explore transcript expression patterns. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis [40] and xCell analysis [41] were used to analyze the patterns of differential gene expression of different groups.

Cytokine expression using a Luminex panel
Peripheral blood mononuclear cells from healthy humans (hPBMC, TPCS, PB010C) were incubated with different reagents (PBS/BAY-1834845/PF-06650833) for 20 h at a final concentration of 500nmol/L, then stimulated with LPS (0.1 ug/ml). After 5 h, the supernatant was collected and the release of 48 cytokines was detected using Luminex (X-200) according to the instructions of bioplex Pro Human Cytokine Screening Panel Kit (BiO-RAD, 12007283). Heatmap analysis was used to visualize the changes in 48 cytokines of different reagents.

Statistical analysis
The data conforming to a normal distribution are described as the mean and standard deviation (M±SD), and the or not working to a normal distribution are expressed as the median and interquartile range (IQR). Comparisons between two groups for data conforming to a normal distribution were performed with an unpaired t-test and ANOVA. The Mann-Whitney U test was used for nonparametric testing when comparing data between groups of semi-quantitative data or not conforming to a normal distribution. All data were analyzed using GraphPad Prism 8.0 (La Jolla, USA) and IBM SPSS Statistics 23.0 (IBM, USA), and p < 0.05 was considered indicative of a significant difference.

In vitro inhibition of IRAK4 activity by BAY-1834845 and PF-06650833
BAY-1834845 contains an isoindazole as its core structure, while PF-06650833 is quite different and has isoquinoline as its core (Fig. 1A, B). However, although both compounds are very potent against IRAK4, PF-06650833 is several folds better [23,34,42]. We measured the kinase activity of IRAK4 in vitro in the presence of the two compounds and found that the IC 50 values were 3.55 nM and 0.52 nM (Fig. 1C, D), consistent with previously published data. In an in vitro treatment of LPS stimulated human PBMC, both compounds decreased inflammatory cytokines secretion effectively, such as IL-1, IFN-γ, TNF-α, and IL-17 (Fig. 1E).

BAY-1834846 ameliorated lung injury in the LPS-induced ARDS model
To investigate whether IRAK4 inhibitors could work in ARDS, we induced acute lung injury with a single dose of intratracheal LPS. First, the mice were briefly pre-treated with the IRAK4 inhibitors BAY-1834845 or PF-06650833 or with dexamethasone (DEX) as a positive control and received another dose for consolidation 6 h later ( Fig. 2A). Twenty-four hours after LPS inhalation, alveolar cells were destroyed, the cytoplasm leaked out, interstitial edema and hemorrhage were evident, and a hyaline membrane was formed compared with the cells of the normal control group. All these pathological changes were attenuated in the BAY-1834845 group, as well as in the DEX group. However, in the PF-06650833 group, although the alveolar structure and interstitial effusion were somewhat improved, neutrophil infiltration was not reduced (Fig. 2B). Histological scores integrating inflammation, edema, hemorrhage, atelectasis, and hyaline membrane, reflecting overall lung injury, unexpectedly showed statistically significant relief only in the BAY-184845 group but not in the DEX group or PF-06650833 group (Fig. 2C), see more original images in Supplementary data (Supplementary Figs. [1][2][3][4][5]. For each pathological dimension above, BAY-1834845 showed a robust and significant decrease in inflammation infiltration compared with DEX and PF-06650833 (Fig. 2D), while other dimensions were not statistically meaningful (Supplementary Fig 6). To assess the general state of mice, we additionally monitored the weight of each mouse. Although in merely one day, the mice receiving LPS lose near 10 % of body weight. DEX might help prevent inflammation but did not help counteract weight loss, while BAY-1834845 seemed interesting to neutralize the weight reduction (Fig. 2E).
Further, we treated pre-existed ARDS with BAY-1834845, DEX, and the combination of the two agents 4 h of post-modeling (Fig. 2F). All three treatments could reduce the interstitial inflammation, as well as hemorrhage and hyline membrane (Fig. 2G). However, when we calculated the pathological score, again, only BAY-1834845 and the combination therapy significantly decreased the overall pathological score (Fig. 2H) and interstitial inflammation score (Fig. 2I). Other dimensions such as hemorrhage and hyaline membrane are not statistically improved in any treatment groups (Supplementary Fig 7).

BAY-1834845 reduced inflammatory cells in BALF
Based on the histological findings, we investigated the antiinflammatory effects of BAY-1834845 through BALF cell counting (Fig. 3A). The numbers of cells with acute inflammation, including neutrophils and eosinophils, as well as the total cell number in BALF, began to increase rapidly 4 h after LPS modeling and continued to increase at 24 h. Lymphocyte counts increased in the later stage. Both BAY-1834845 and DEX effectively reduced the numbers of all three types of inflammatory cells in BALF 24 h later. However, in the more acute phase at 4 h, only BAY-1834845 showed significant reductions in neutrophil, lymphocyte, and total cell counts. Unexpectedly, a direct comparison between BAY-1834845 and DEX showed that BAY-1834845 reduced the neutrophil count more effectively, which was consistent with the findings of lung histology above (Fig. 3B-E). In addition, DEX and BAY-1834845 showed some extent of reduction of BALF total protein, although no statistical significance was found possibly because of the large dispersion of the data (Supplementary Fig 8).
In the treatment model (Fig. 3F), BAY-1834845, DEX, and the combination of the two agents all reduced BALF LDH activity significantly, while BAY-1834845 showed the most significant effect (Fig. 3G). In further flow cytometry analysis, all these three treatments could decrease total T cells, CD11b+CD14 + monocyte. Especially for total T cells, the combination therapy is superior to monotherapy. For macrophages, both F4/80 + and CD11c+ marked populations could be decreased by all the three treatments. Siglec-F+ resident alveolar eosinophils were not significantly affected by the treatments (Fig. 3H and I).

BAY-1834845 decreased immune cell signatures while preserved stromal cell signatures through digital sorting based on lung tissue RNA-seq
To further identify the detailed cell types that BAY-1834845 could regulate, we used cell type signatures based on lung tissue RNA-seq, harvested 4 h after LPS modeling. Principal component analysis (PCA) plots showed significant and clear differences in the interaction between the four groups (Fig. 4A). The degree of individual dispersion within the components was small, indicating that the results were stable and reliable. We used xCells for digital sorting based on a total of 27,818 genes that showed significant differences in 26 cell types across the four groups (Fig. 4B). Both BAY-1834845 and DEX decreased the signatures of most innate immune cells, including neutrophils, M1 and M2 macrophages, eosinophils, and plasmacytoid dendritic cells (pDCs), and classic dendritic cells (cDCs). At the same time, BAY-1834845 was more effective than DEX in decreasing the signatures of neutrophils, eosinophils, and cDCs (Fig. 4C).
Regarding adaptive immune cells, both BAY-1834845 and DEX could downregulate the enrichment score of effector or memory cells (Fig. 4B,  C). Interestingly, compared with DEX which decreased both naïve and effector cell signatures nonselectively, BAY-1834845 seemed to target memory lymphocytes specifically and preserve naïve B and naïve CD4 T cell signatures close to those of the normal control (Fig. 4C). In addition, BAY-1834845 showed similar preservation of lung stromal cells, including signatures of smooth muscle cells, chondrocytes, and endothelial cells (Fig. 4D).

BAY-1834845 regulated inflammation and innate immunity genes more efficiently than dexamethasone
We picked up a total of 111 DEGs upon BAY-1834845 treatment (Pvalue < 0.05 and fold change ≥ 2, Fig. 5A, Supplementary Fig 9). GO, and KEGG enrichment identified these genes mainly on immune and inflammation responses such as IL-17, TNF, TLR, and NF-κB signaling ( Supplementary Fig 10A, B). DEX had similar DEG enrichment results with BAY-1834845 ( Supplementary Fig 10C, D). As a validation, the secretion of representative cytokines in BALF, including TNF-α, IL-6, and IL-1β, was decreased by both BAY-1834845 and DEX (Fig. 5B). We noticed 48 genes not as effectively suppressed by DEX as BAY-1834845 (Fig. 5A, red boxes). To our surprise, these 48 genes were highly concentrated in IL-17 signaling, COVID-19, rheumatoid arthritis, and Toll-like receptor signaling (Fig. 5C, D).
In addition, to identify possible off-target effects of BAY-1834845, we compared the DEGs of BAY-1834845 versus LPS in our study with data GSE9037, transcriptome data of monocytes from IRAK4 kinasedead mice. We found that 97 out of 111 DEGs (87.3 %) overlapped with GSE9037, as only minor DEGs may be associated with off-target change (Fig. 5E). The left 14 genes not overlapping with GSE9037 seem to have no direct connection with IRAK4 according to known signaling, and their functions are scattered, such as spermatogenesis (speer4c and ccdc155) [43], body-weight maintenance (gpr82) [44], and dietary lipid absorption (noct and oacyl) [45,46] (Fig. 5F). Therefore, they cannot be attributed to a definite off-target effect unless further evidence is present. More importantly, the change of these fourteen genes upon BAY-1834845 is close to normal controls, suggesting beneficial effects rather than unexpected side effects.

Discussion
ARDS is an emergency and lethal clinical entity [47], especially against the background of the COVID-19 outbreak. It is characterized by excessive inflammation, the leading cause of death [48,49]. Unfortunately, the pharmaceutical treatment of ARDS was not well investigated until the pandemic, and only dexamethasone was identified as effective for both moderate and severe ARDS cases.
IRAK4 is downstream of all TLRs, except TLR3 [22]. To pharmaceutically target IRAK4, small-molecule IRAK4 inhibitors were designed almost a decade ago. These compounds can inhibit the inflammatory signal transduction induced by TLRs (including TLR4, TLR7, TLR8, and TLR9) in vitro and in vivo [50][51][52] and reduce gouty inflammation in uric acid peritonitis [51] and ischaemic inflammation in most nephrectomized rats [53]. Supported by this evidence, many pharmaceutical companies are vigorously developing potent and safe IRAK4 inhibitors for clinical diseases [23], which are represented by a series of preclinical models, including collagen-induced arthritis [42,51], gout [51], lupus [54,55], inflammatory dermatitis [51], and activated B cell-like (ABC) subtypes of diffuse large B cell lymphoma (DLBCL) [51]. Two major small-molecule IRAK4 inhibitors, BAY-1834845 and PF-06650833, have finished phase I clinical trials. In addition, PF-06650833 has shown efficacy in a phase II clinical trial in patients with rheumatoid arthritis (RA) who responded insufficiently to methotrexate (NCT02996500). Other ongoing clinical trials of these two compounds are for the treatment of RA (NCT04413617), psoriasis (NCT03493269), hidradenitis suppurativa (NCT04092452), and COVID-19 pneumonia (NCT04933799). The initiation of this trial supports our hypothesis that IRAK4 inhibitor may treat lung inflammation, although this trial NCT04933799 is focused on moderate-to-severe COVID-19 pneumonia (but not including the most critical patients with anticipated survival <72 h), a population not largely overlapping with the ARDS population.
Our study is the first to compare the two compounds head-to-head in one animal model. To our surprise, the two compounds, BAY-1834845 and PF-06650833, seemed not equally effective on ARDS, as assessed by histological examination. BAY-1834845 exhibited a much more substantial reduction in lung inflammation, especially neutrophil infiltration, than PF-06650833. Given the equally excellent IC 50 of the two compounds, we infer that pharmacokinetic characteristics might contribute to the different in vivo effects. The single-dose pharmacokinetics of PF-06650833 published by Pfizer showed quite a high clearance rate in rats of 56 ml/min/kg, leading to a T 1/2 of merely 0.6 h. For larger animals, the T 1/2 of dogs and monkeys was 1.1 h and 1.7 h, respectively. The oral bioavailability in rats is moderate (34-50 %), and the oral bioavailability in dogs and monkeys is low (41 % and 6.9 %) [42]. With these parameters, the therapeutic efficiency of PF-06650833 has driven concerns in a preclinical model: in a collagen-induced arthritis model, the arthritis score of the PF-06650833 group was inferior to that of the group treated with tofacitinib, a JAK inhibitor that has been approved for RA treatment for years [56]. As result, Pfizer has developed a modified release tablet to improve the pharmacokinetics of the compound in a recent RA trial (NCT02996500). On the other hand, the favorable pharmacokinetic profiling of BAY-1834845 in rats was identified by our group to have a lower clearance rate of 6.68 ml/min/kg, a longer T 1/2 of 3.09 h, and one hundred percent bioavailability, which is entirely consistent with its excellent efficacy with more than 80 % inhibition of cytokine release even at 16 h following induction by LPS in mice.
To our surprise, the in vivo therapeutic effect of BAY-1834845 is not only statically different from PF-06650833 but also from dexamethasone, the only medicine currently proven to be effective for the survival of ARDS patients [57]. We are fully aware of the limitation of the single-dose design of our experiment, which cannot demonstrate a definite superior effect of BAY-1834845. However, the dexamethasone (10 mg/kg once) we applied is a short-term pulse dose and is higher than typical amounts of ARDS [58]. According to the classic dose translation [59], 10 mg/kg equals approximately 50 mg of dexamethasone once in humans, while the common dose for ARDS in clinical trials is 6-20 mg daily [16,57]. For BAY-1834845 and PF-06650833, we applied the same dose as reported in patent documents by Bayer and Pfizer, respectively, at which the two compounds have been verified to be fully effective. Besides, the results of fewer BALF inflammatory cell counts and specific inhibition of IL-17 signaling, COVID-19, rheumatoid arthritis, and Toll-like receptor signaling upon BAY-1834845 treatment are supportive of our histological findings. It would be interesting and informative to analyze more detailed immune cell phenotypes by flow cytometry in the future, particularly on lung macrophage and dendritic cells, with an up-to-date identification panel. [60].
In terms of safety, infection is a major concern. Although IRAK4 is widely involved in innate immunity and inflammation, susceptibility to bacterial, fungal, viral mycobacterial, or parasitic infections in adults was not increased. Children genetically lacking IRAK-4 are susceptible to certain purulent infections, including G+ purulent bacteria, such as Streptococcus pneumoniae and Staphylococcus aureus [29]; as these patients enter puberty, susceptibility to infection becomes increasingly rare, and there have been no reports of serious viral or parasitic infections [61][62][63][64] or infection-related deaths after the age of 8 [65]. In our study, BAY-1834845 preserved naïve lymphocyte signatures and stromal cell signatures of lung tissue. Such preservation may help this compound avoid some side effects such as impaired pathogen defense and delayed tissue repair, which are common in patients receiving steroids.

Conclusions
IRAK4 kinase inhibiting is a feasible approach to treating ARDS cascade and needs further investigation.