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Abstract
Pirfenidone (PFD) is a pharmacological compound with therapeutic efficacy in idiopathic pulmonary fibrosis. It has been chiefly characterized as an antifibrotic agent, although it was initially developed as an antiinflammatory compound because of its ability to diminish the accumulation of inflammatory cells and cytokines. Despite recent studies that have elucidated key mechanisms, the precise molecular activities of PFD remain incompletely understood. PFD modulates fibrogenic growth factors, thereby attenuating fibroblast proliferation, myofibroblast differentiation, collagen and fibronectin synthesis, and deposition of extracellular matrix. This effect is mediated by suppression of TGF-β1 (transforming growth factor-β1) and other growth factors. Here, we appraise the impact of PFD on TGF-β1 production and its downstream pathways. Accumulating evidence indicates that PFD also downregulates inflammatory pathways and therefore has considerable potential as a viable and innovative antiinflammatory compound. We examine the effects of PFD on inflammatory cells and the production of pro- and antiinflammatory cytokines in the lung. In this context, recent evidence that PFD can target inflammasome pathways and ensuing lung inflammation is highlighted. Finally, the antioxidant properties of PFD, such as its ability to inhibit redox reactions and regulate oxidative stress–related genes and enzymes, are detailed. In summary, this narrative review examines molecular mechanisms underpinning PFD and its recognized benefits in lung fibrosis. We highlight preclinical data that demonstrate the potential of PFD as a nonsteroidal antiinflammatory agent and outline areas for future research.
Pirfenidone (PFD) is a unique pharmacological compound and is one of two conditionally recommended therapies for treatment of idiopathic pulmonary fibrosis (IPF) (1–3). PFD has been evaluated in three high-profile, multinational, randomized, placebo-controlled, phase III clinical trials: CAPACITY (Clinical Studies Assessing Pirfenidone in Idiopathic Pulmonary Fibrosis: Research of Efficacy and Safety Outcomes) 004 and 006, and ASCEND (Assessment of Pirfenidone to Confirm Efficacy and Safety in Idiopathic Pulmonary Fibrosis) (4, 5). The pooled data from these clinical trials and subsequent post hoc analyses have demonstrated that PFD improves lung function decline, prolongs progression-free survival, and lowers death rates over 12 months (4–10). Furthermore, real-world studies of PFD treatment in IPF have supported observations that PFD reduces lung function decline, as reported in the phase III clinical trials (11–14).
Surprisingly, although PFD is deemed to be effective in IPF and the compound is now in widespread clinical use, its precise mechanisms of action in the lung are not fully understood. PFD has been characterized as an antifibrotic agent because it modulates fibrogenic growth factors, thereby attenuating fibroblast proliferation, myofibroblast differentiation, collagen synthesis, fibronectin production, and deposition of extracellular matrix (ECM) (15–21). However, PFD was initially developed as an antiinflammatory agent because of its ability to diminish the production of cytokines and accumulation of inflammatory cells (22–27). In addition, PFD regulates and reduces oxidative stress markers in the lung (28–31). This broad spectrum of activities shown by PFD as a pharmacological compound merits consideration, especially given that the combination of fibrosis and inflammation is a key feature of tissue pathology in many chronic lung diseases.
This narrative review details the molecular mechanisms underpinning PFD’s actions and highlights preclinical data that demonstrate the potential of PFD not only as an antifibrotic but also as a viable and innovative antiinflammatory agent. There is tantalizing evidence that in addition to its therapeutic efficacy and putative antifibrotic benefits in IPF, PFD also has benefits for inflammation and oxidative stress–associated pathologies. An improved understanding of the molecular actions and preclinical information pertaining to PFD may pave the way for a broader strategy to use PFD to treat other chronic inflammatory lung diseases.
PFD is an orally administered, synthetic small-molecule derivative of pyridone [5-methyl-1-phenyl-2(1H)-pyridone]. Because of its size and hydrophobic nature, PFD is able to diffuse freely across cell membranes without using a receptor (32). The compound is rapidly absorbed from the gastrointestinal tract (32, 33) and reaches its maximum concentration in blood 1–2 hours after an oral dose (32). It is rapidly metabolized in the liver (up to 80%), primarily by the hepatic cytochrome p450 (CYP)1A2 enzyme and to a lesser extent by other CYP isoforms (33, 34), yielding the inactive metabolites 5-carboxypirfenidone and 5-hydroxypirfenidone (34, 35). It is excreted in the urine ∼6 hours after ingestion (32).
Common adverse effects of PFD include gastrointestinal symptoms, skin rashes, and photosensitivity (4, 5, 12, 33, 36–39). PFD can cause serious liver function abnormalities in <5% of patients, and regular monitoring is recommended (4, 10). Adverse events are dose related and can be improved or resolved upon dose modification (40), dose titration (41), or usually after withdrawal of PFD treatment (10). Comprehensive analyses of safety outcomes in large, well-defined populations (∼1,299 patients with IPF) who received PFD treatment demonstrated that PFD is tolerated in a total dose of up to 2,403 mg/day for a period of up to 9.9 years (4, 5, 42).
Toxicity is a problem that may limit the use of PFD in lung and other diseases. However, adverse effects are generally mild to moderate in severity and dose related, and can be improved or resolved by reducing or titrating doses (40, 41). Potential strategies to modulate the metabolism of PFD to achieve adequate bioavailability in plasma despite the use of lower doses and the development of inhaled formulations require future investigation.
PFD is now in clinical use to treat patients with IPF, but it has taken several years for its benefits to be recognized. Double-blind phase II clinical trials in Japan demonstrated that PFD could significantly reduce the rate of decline in forced vital capacity (FVC) in patients with IPF (36, 37), and a subsequent phase III study demonstrated significant increases in progression-free survival (37). These clinical trials paved the way in 2008 for approval of PFD in Japan for treatment of IPF.
The clinical trials CAPACITY 004 and 006, conducted in North America, Australia, and Europe, have further confirmed the benefits of PFD treatment (5). In CAPACITY study 004, patients showed a reduced decline in FVC during the 72-week treatment period. Surprisingly, CAPACITY study 006 failed to show a similar benefit at 72 weeks, and the U.S. Food and Drug Administration requested additional clinical trials. Patients from both studies have reported common adverse effects, including nausea, dyspepsia, vomiting, anorexia, photosensitivity, and skin rashes. Overall, pooled data from these clinical trials demonstrate that PFD has the ability to improve FVC and 6-minute walk test distance, and prolong progression‐free survival (5).
The ASCEND trial randomized patients with IPF who had FVC 50–90% predicted and DlCO ≥30% predicted. The study reported significant attenuation of FVC declines as well as increases in 6-minute-walk test distance coupled with enhanced progression-free survival during the 52-week follow-up period (4). Based on the pooled results from these trials, PFD was approved for use in IPF by the U.S. Food and Drug Administration in 2014.
Recently, pooled post hoc analyses of the CAPACITY and ASCEND trials confirmed the benefits of PFD treatment to retard disease progression in IPF over 12 months (4–9). PFD was shown to lower the risk of all-cause mortality, FVC decline, and hospitalization, and to increase exercise capacity at 12 months. Similarly, further analyses of patients with advanced baseline lung function impairment who were enrolled in the CAPACITY and ASCEND trials showed that PFD treatment benefits patients with more advanced disease, without an increased risk of discontinuation due to side-effects (43). Finally, akin to the clinical trial data, the benefits of PFD to blunt FVC declines in IPF have been noted in real-world studies of PFD (11–14).
Despite the evidential success of PFD treatment in IPF, there has been limited evaluation of its potential efficacy in targeting other chronic inflammatory lung diseases. The mechanisms and molecular actions of PFD are only partly defined, and apposite investigations are needed to provide a rationale for clinical strategies that aim to repurpose PFD for the treatment of other respiratory diseases.
In 1994, Margolin and Lefkowitz reported that PFD prevented and resolved lung fibrosis in a canine lung infection model (44). Earlier studies by Iyer and colleagues (45) and Schelegle and colleagues (46) showed that PFD reduced fibrosis and improved lung function in a hamster model of bleomycin-induced lung injury. Subsequently, the therapeutic efficacy of PFD was noted in several preclinical models of fibrotic disorders in the lung (15–21), renal system (47), liver (47), heart (47), and eye (48). These in vivo and in vitro studies demonstrate that PFD acts by suppressing the induction of fibrogenic mediators and growth factors through a variety of mechanisms (Figure 1).
Figure 1. Pirfenidone (PFD) attenuates profibrotic pathways. Alveolar epithelial cell (AEC) damage due to environmental or other factors induces AECs and other cells (such as endothelial cells) to augment TGF-β (transforming growth factor-β) production. Four key processes (denoted by green circles) are activated: proliferation of fibroblasts, transdifferentiation of fibroblasts into myofibroblasts, collagen synthesis and fibronectin production, and excess extracellular matrix (ECM) production. Targets of PFD include TGF-β itself and TGF-β–induced downstream mediators and products such as SMAD3; α-SMA; tenascin-c; fibronectin; collagen type I, II and III; and the collagen-specific chaperone HSP47 (heat shock protein 47) (gray boxes). PFD also regulates additional growth factors (yellow boxes) such as PDGF (platelet-derived growth factor) and bFGF (basic fibroblast growth factor), thereby modulating collagen production. PFD also inhibits expression of MMP-9 (matrix metalloproteinase 9), TIMP1 (tissue inhibitor of metalloproteinases 1), and MMP-2 (yellow boxes) diectly or by reducing the synthesis of TGF-β and downstream mediators. PFD may also reduce TGF-β activation by MMPs. Red s denote key pathways inhibited by PFD. SMA = smooth muscle actin.
[More] [Minimize]TGF-β1 (transforming growth factor-β1) has a central role in the pathogenesis of fibrosis, but it is also involved in the pathology of other chronic inflammatory lung conditions, such as chronic obstructive pulmonary disease (COPD) and asthma (49). In the lung, TGF-β1 is produced by a variety of cells during epithelial cell damage due to environmental and other factors. These include alveolar epithelial cells (AECs), endothelial cells, fibroblasts, myofibroblasts, and inflammatory cells such as macrophages and neutrophils (50). PFD can act on TGF-β1 in a variety of ways:
| 1. | PFD impacts TGF-β1 and downstream pathways by reducing TGF-β1 protein production (16, 23, 51, 52) and mRNA expression (28). It also suppresses TGF-β–mediated fibroblast proliferation and fibroblast differentiation into myofibroblasts by attenuating TGF-β1/SMAD3-induced signaling (15–17). PFD prevents murine radiation-induced lung fibrosis by reducing expression of TGF-β1 and phosphorylation of SMAD3 (17). Similarly, a novel derivative of PFD (PBD-617) inhibits fibrosis by suppressing expression of TGF-β1 and phosphorylated SMAD3 in a rat bleomycin-induced PF model (16). PFD inhibits human lung fibroblast proliferation and TGF-β1–induced SMAD3 expression in human lung fibroblasts (15, 16). Surprisingly, phosphorylated SMAD3 was unchanged in lung tissue of patients with IPF treated with PFD, a discrepancy that may be due to small patient numbers and the timing of tissue collection (53). | ||||
| 2. | Prior studies have confirmed that TGF-β1–induced mediators such as tenascin-c, fibronectin, and collagen type I, II, and III are the main ECM proteins involved in the fibrotic process (21). Gene and protein expression of these mediators is inhibited by PFD in lung fibroblasts from patients with IPF upon stimulation with TGF-β1 (16, 18) and in bleomycin-treated rats with augmented TGF-β1 expression (16). | ||||
| 3. | PFD reduces TGF-β1–induced expression of α-SMA, a factor that is important for fibroblast-to-myofibroblast transition (16, 18). TGF-β1–induced α-SMA gene expression was suppressed by PFD in bleomycin-induced rat PF (16), in paraquat-induced lung fibrosis in mice (28), and in human lung fibroblasts (16, 18). | ||||
| 4. | TGF-β1 has been shown to increase mRNA expression of HSP47 (heat shock protein 47), a collagen-specific chaperone, which is involved in the processing, assembly, and secretion of procollagen during fibrotic processes (54, 55). PFD has directly inhibited HSP47 mRNA expression in TGF-β1–induced human lung fibroblasts (20) and in a mouse model of lung fibrosis (19) | ||||
| 5. | A similar inhibitory effect of PFD on HSP47-mediated protein and gene expression was demonstrated, which resulted in a reduction of collagen synthesis in A549 alveolar epithelial type II cells after TGF-β1 stimulation (54). In addition, epithelial cells can undergo epithelial–mesenchymal transition (EMT), an important mechanism of myofibroblast production during lung fibrosis (56) that is induced by TGF-β1 in rat alveolar epithelial cells (57) and A549 cell cultures (58). PFD can oppose TGF-β–induced loss of E-cadherin, the chief mediator protein in EMT in A549 cells (54) and PF in the rat silicosis model (59), indicating that PFD also inhibits EMT. | ||||
Taken together, these data indicate that PFD downregulates TGF-β1 as well as a number of downstream TGF-β1–associated signaling mechanisms. These actions have a potential impact on fibrosis, as well as on other TGF-β1–associated inflammatory and immune pathways.
| 1. | PFD regulates additional growth factors, such as PDGF (platelet-derived growth factor) (23, 24, 60) and bFGF (basic fibroblast growth factor) (24, 51). PDGF is a fibroblast mitogen that is upregulated in experimental models of fibrosis (24, 60). PFD inhibited synthesis of both PDGF-A and -B isoforms by lung macrophages in a bleomycin hamster model of lung fibrosis (24), as well as in a rat bleomycin-induced IPF model (60). In addition, bFGF is a mitogen of fibroblasts and myofibroblasts, and PFD was shown to suppress the expression of bFGF in bleomycin-induced preclinical models of IPF (24, 51). To date, the precise actions of PFD on PDGF- and bFGF-driven lung fibrosis are not understood, but it has been shown that PFD inhibits myofibroblast differentiation by regulating PDGF receptor activation (61) or by modulating collagen type I accumulation and α(I) procollagen expression (62). | ||||
| 2. | MMPs (matrix metalloproteinases) are a major group of proteases that regulate the degradation of fibrogenic growth factors and ECM proteins (63). Development of fibrosis is associated with an imbalance between MMPs and TIMP (tissue inhibitor of metalloproteinases), which leads to production of excessive components of the ECM (63). Reduced MMP9/TIMP1 ratios are associated with changes in collagen deposition in the lungs of bleomycin-treated mice (64, 65). In a mouse model of PF induced by chronic exposure to LPS, collagen deposition was associated with increases in MMP2 and MMP9 activities in BAL fluid (66). Pretreatment with PFD reduced MMP9 secretion and fibrosis in this model (67). Similarly, PFD has shown therapeutic efficacy by inhibiting expression of TIMP1 (68, 69) and MMP2 (69) in bleomycin and paraquat models of lung fibrosis. Finally, studies have demonstrated that MMP9 and MMP2 contribute to TGF-β1 activation, in addition to impacting several TGF-β1–driven responses, including activation of the Smad3 gene and production of fibronectin (70, 71). In support of this notion, PFD was shown to modulate expression of MMP2 and MMP9 while reducing the synthesis and secretion of TGF-β1 in IPF, as well as in cardiac and renal fibrosis models (68, 69, 72, 73). | ||||
Inflammatory responses are integral host reactions to infections, toxins, allergens, and pollutants. This process is protective and also plays a key role in initiating the healing process (74). However, inflammation persists in many chronic lung diseases, including asthma, IPF, and COPD (74, 75). Although it is not widely appreciated, PFD has been shown to downregulate inflammatory pathways, and the compound may have considerable potential as a nonsteroidal antiinflammatory agent (Figure 2).
Figure 2. Antiinflammatory activities of PFD. Airway AECs generate inflammatory responses after injury caused by environmental or other factors. PFD has broad antiinflammatory activities and can curtail damaging inflammatory responses by modulating the activities of inflammatory cells such as dendritic cells (DCs), CD4+ and CD8+ T cells, macrophages, neutrophils, and eosinophils (blue boxes). It can also oppose the activities of cytokines secreted by cells, either directly (yellow boxes) or by disrupting downstream signaling pathways (gray boxes). Uniquely, PFD can attenuate oxidative stress pathways (white boxes) and inflammasome activation (yellow boxes). ASC = apoptosis-related speck-like protein containing a caspase recruitment domain; COPD = chronic obstructive pulmonary disease; DAMPs = danger-associated molecular patterns; IPF = idiopathic pulmonary fibrosis; JAK-STAT = Janus kinase/signal transducers and activators of transcription; MCP-1 = monocyte chemoattractant protein 1; NOX = NADPH oxidase; Th1 = T-helper cell type 1; Th2 = T-helper cell type 2.
[More] [Minimize]| 1. | Dendritic cells (DCs) reside in both lymphoid and nonlymphoid tissues, such as airway epithelium and alveolar septa, and express a variety of PRRs (pattern recognition receptors), including Toll-like receptors and Nod-like receptors (NLRs) (76). Upon activation, DCs produce cytokines that mediate inflammatory responses and stimulate the proliferation and differentiation of naive CD4+ and CD8+ T cells (76). Conversely, several cytokines (e.g., TNF-α and IL-1) can trigger DC activation, which has been implicated in human chronic inflammatory lung diseases such as asthma and COPD (77, 78). PFD’s immune-modulating activities exert suppressive effects upon DC activation by reducing CD103+ cells, which control naive CD8+ T-cell activation (22). In addition, PFD can reduce the expression of a number of proinflammatory cytokines produced by DCs, including colony-stimulating factor 3, IL-10, MCP-1 (monocyte chemoattractant protein 1), CCL12, TNF receptor I, and TNF-α (22). Furthermore, in vitro DC cultures with LPS and allogeneic stimulation have demonstrated that PFD impairs the capacity of DCs to stimulate T-cell activation (22). | ||||
| 2. | Macrophages reside in the airways, alveoli, and lung interstitial spaces, and their function is upregulated by DCs (79). Macrophages phagocytose pathogens and are also a key source of cytokines, chemokines, and other inflammatory mediators that propagate or suppress immune responses (79). Activated macrophages produce a range of proinflammatory and antiinflammatory cytokines that initiate immune responses and promote tissue repair (80). In this context, PFD attenuates PF by suppressing macrophage-driven cytokines such as IL-1, TNF-α, TGF-β1, PDGF (24, 29), HSP47 (19), and MCP-1 (81), as well as macrophage numbers (82, 83). Similarly, PFD was shown to significantly blunt TGF-β1 production induced by IL-4/IL-13 in rat alveolar macrophages stimulated in vitro (27). Taken together, the current evidence indicates that PFD’s ability to suppress macrophage inflammatory activities is likely to be an important advantage of this compound. | ||||
| 3. | Neutrophils and their products are considered to be highly active in disease initiation and progression in chronic inflammatory lung diseases such as COPD, asthma, and cystic fibrosis (84). Although antiinflammatory drugs (such as corticosteroids) (85) and biologics (TNF inhibitors such as infliximab (86) and IL-6 receptor-α inhibitors such as tocilizumab) (87) do not specifically target neutrophil function, many of these compounds may exert inhibitory effects on neutrophils. To date, no pharmacological agents that impact neutrophil numbers or function are in clinical use for lung diseases, and PFD may have potential in this context. PFD inhibited neutrophil numbers in a bleomycin-induced mouse model of lung fibrosis after 3 weeks of treatment (82). Similarly, treatment with PFD suppressed neutrophils in a bleomycin-induced hamster model of lung inflammation (83) and in a nonallergic rat model of LPS-induced pulmonary inflammation (26). Unexpectedly, PFD failed to suppress neutrophil influx in a hamster model of acute injury and fibrosis (88), indicating that the effectiveness of PFD may depend on the particular tissue microenvironment and experimental disease models used. | ||||
| 4. | T lymphocytes, with two major subsets (CD4+ and CD8+), provide cell-mediated immunity. CD4+ T cells drive cellular immunity, whereas CD8+ T cells are mainly cytotoxic or regulatory (89). CD4+ T cells are further subdivided into Th1 (T-helper cell type 1) and Th2 cells, which have different cytokine profiles (90). A delicate balance between Th1 and Th2 is required for immunological stability, and a deregulated response has been implicated in a variety of chronic inflammatory conditions, including asthma, COPD, and chronic bronchitis (91, 92). PFD inhibits proliferation of both CD4+ and CD8+ cells, and suppresses key proinflammatory cytokines linked to Th1 cells (IFN-γ, IL-1β, and TNF-α) and Th2 cells (IL-4) (25). However, PFD does not impact T-regulatory cells in vivo and in vitro in response to T-cell receptor activation (25). PFD inhibited CD4+ cells in vitro and in vivo and significantly diminished Th2 cytokines in BAL in a model of chronic allergen challenge with ovalbumin (23, 25). In a bleomycin-induced mouse model of lung fibrosis, PFD prevented reductions in IFN-γ, potentially restoring the Th1/Th2 balance (24). | ||||
| 5. | Eosinophils are associated with parasitic infections and allergic diseases such as asthma, and are often localized to chronic tissue inflammation (93, 94). The cells are potentially cytotoxic and have the ability to release damaging mediators (94). PFD was shown to prevent eosinophil accumulation in allergic airway inflammation and diminish airway hyperresponsiveness after chronic allergen challenge in mice (23). Similar effects have been noted in antigen-induced allergic models of mice and guinea pigs (26), and in a hamster model of acute lung injury and fibrosis (88). Surprisingly, it has been reported that PFD administration may lead to an elevation of lung eosinophil counts (95). This may have contributed to the eosinophilic pleurisy observed in patients treated for lung fibrosis in one study (96). Discontinuation of PFD in that study reversed the eosinophilia, but it is not clear whether other treatments the patients received may have played a role. | ||||
In summary, there is considerable preclinical evidence that PFD modulates inflammatory cell numbers as well as proinflammatory mediators. For now, it is not clear whether this is a direct effect or PFD acts chiefly on inflammatory cells by modulating cytokines and other mediators.
Several key questions relating to the actions of PFD on the regulation of inflammatory mediators remain unanswered. However, there is evidence that PFD can impact the production of TNF-α, IL-1β, IL-6, IL-8, IL-10, and IFN-γ (all of which are key cytokines that are capable of regulating inflammatory cell activity), and adhesion molecules, and expression of matrix remodeling factors (26, 51, 60, 67, 97–99). PFD has been reported to modulate cytokine expression in a number of ways:
| 1. | PFD was shown to significantly reduce the levels of bioactive and cell-associated TNF-α in fibrosis models (60, 67, 69) and after stimulation with LPS (97). PFD has also been shown to inhibit TNF-α activity through protection against acute allograft injury (52, 98) and acute lung injury (26, 99). In addition, PFD can downregulate intercellular adhesion molecule-1 through suppression of TGF-β1 and TNF-α, resulting in cell–cell interaction between lymphocytes and fibroblasts, and thereby preventing activation in other tissues (100, 101). The ability of PFD to suppress the activities of TNF-α in matrix remodeling has also been reported (67, 69). | ||||
| 2. | PFD was found to reduce the levels of IL-6, IL-10, and IFN-γ in studies of fibrosis, acute lung injury, and allergic disease models (26, 27, 51, 77, 102). These cytokines use JAK-STAT (Janus kinase/signal transducers and activators of transcription) pathways to regulate cellular mechanisms that maintain lung homeostasis (103). Normal fibroblasts cultured on IPF-conditioned matrix have increased levels of phospho-STAT3 and fibrotic mediators such as α-SMA. PFD was shown to prevent IPF-conditioned, matrix-induced fibroblast phenotype alterations, although it is not clear whether phospho-STAT3 is directly inhibited by PFD (104). Treatment of IPF fibroblasts with PFD downregulated the expression of various ECM-associated genes and protein levels of phosphorylated STAT3 (105). Finally, reductions in the activities of STAT6, JAK1, JAK3, and tyrosine kinase 2 (proteins in the JAK-STAT pathway) were observed when a human macrophage cell line (U937 cells) was treated with PFD (106). Clearly, PFD has the potential to inhibit JAK-STAT signaling, but whether PFD primarily suppresses cytokines that are important for initiating this pathway has not been clarified. | ||||
| 3. | PFD inhibits the cytokines IL-8, MCP-1, and IL-12p40 in the lung (51). These mediators activate NF-κB signaling pathways, and limited studies have demonstrated that phosphorylation of IKKβ, IκBα, and NF-κB (proteins that are upregulated in a bleomycin rat fibrosis model) can be downregulated by PFD (107). Similarly, PFD was shown to suppress NF-κB pathways in a rat model of ischemia-reperfusion injury (108). | ||||
In summary, inhibition of inflammatory cytokine production as well as downstream pathways is likely to explain key aspects of the clinical benefit observed after PFD treatment.
Innate immune responses within the lung mucosa depend on the tightly coordinated activation of a series of PRRs, which are widely expressed in both epithelial and immune cells. These PRRs collectively trigger inflammatory responses after recognition of diverse ligands, such as DAMPs (danger-associated molecular patterns). Members of the PRR families form the core of distinct inflammasomes that lead to the recruitment and oligomerization of the key adaptor protein ASC (apoptosis-related speck-like protein containing a caspase recruitment domain [CARD]) to facilitate the activation of caspase-1, which in turn catalyzes the maturation of pro–IL-1β or pro–IL-18 proteins into secreted bioactive cytokines (109, 110).
Inflammasomes drive acute innate immune responses that resolve inflammation and maintain tissue homeostasis, and in recent years, numerous data from clinical studies and mouse disease models have demonstrated that excessive inflammasome activation promotes several chronic autoimmune and inflammatory diseases, including COPD, IPF, cystic fibrosis, and acute respiratory distress syndrome (ARDS) (109). Emerging data show that PFD suppresses inflammasome signaling components such as IL-1β (15, 23, 27, 51, 58, 65, 100) and DAMPs, including reactive oxygen species (ROS) (28, 111). Recent studies also demonstrated that PFD inhibited silica-induced NLRP3 inflammasome activation in a human bronchial epithelial cell line (112), and protein expression of NLRP3, ASC, cleaved caspase-1, and mature IL-1β in LPS-induced acute lung inflammation (111). The latter study also confirmed that PFD ameliorates LPS-induced acute lung injury and fibrosis by blocking NLRP3 inflammasome activation (111).
Although studies to date are limited, recent evidence that PFD can target inflammasome pathways expands the potential scope of treatment with this compound, and supports the concept that PFD may have substantial potential as an antifibrotic and antiinflammatory therapeutic agent.
At a physiological level, respiratory cells produce ROS as byproducts of cellular metabolism, a process that is tightly regulated by cytoplasmic ROS-generating enzymes such as NADPH oxidase (NOX) to prevent oxidative stress (113). Lung diseases such as ARDS, COPD, IPF, and asthma are associated with oxidative stress, as evidenced by altered redox status (production of free radicals and peroxides such as superoxide radical, hydroxyl radical, and hydrogen peroxide [H2O2]) (114). This results in irreversible oxidative modifications in proteins or DNA (lipid peroxidation), mitochondrial dysfunction, and enhanced activity of NOX enzymes and antioxidant enzyme systems (114). Current therapeutic strategies using antioxidant therapies have been relatively ineffective (115) and there is a need for alternative therapies to address this pathology. Again, PFD has potential because it has been shown to inhibit redox-based mechanisms in animal models and in vitro systems, as detailed below.
PFD directly inhibits redox reactions. The agent was shown to suppress oxidative stress induced by toxic hydroxyl radicals, but not superoxide radicals, in bleomycin-induced PF in mice (31), potentially via inhibition of the NOX isoforms Nox4 and Nox1 (30). PFD also significantly suppressed genes belonging to the ROS enzymatic system, such as iNos, Nox1, Nox4, and the antioxidant Gpx1, while reducing lipid peroxidation and restoring antioxidant enzymes (e.g., superoxide dismutase and catalase) in a murine model of paraquat-induced lung injury and fibrosis (28). Similarly, studies in hamster models of fibrosis indicated that PFD ameliorates bleomycin-induced lung fibrosis by suppressing oxidative stress mediators (29, 45). Collectively, these preclinical studies indicate that reductions in ROS formation and oxidative stress might be additional mechanisms by which the antifibrotic effects of PFD are mediated. However, it is noteworthy that in clinical trials of IPF, plasma PFD concentrations were ∼8 mg/L, a level at which the hydroxyl-scavenging effect may not be effective (32).
Finally, a protective effect of PFD mediated by decreases in oxygen radicals was demonstrated in experimental models of ARDS (112). In response to PFD, Sod1 mRNA expression decreased as a result of reduced H2O2 during Streptococcus pneumoniae infection in macrophages. PFD treatment further improved mitochondrial respiration, possibly by detoxifying mitochondrial peroxidase enzymes such as glutathione peroxidase, thereby revealing a capacity to preserve normal mitochondrial function (116).
Preclinical research has unambiguously demonstrated that PFD may be an effectual antiinflammatory treatment. However, these putative benefits have not been exploited in a relevant clinical context. For example, COPD and asthma are diseases characterized by elevated TGF-β1, chronic airway inflammation, and a degree of fibrosis (92, 117), but therapy for these conditions still predominantly relies on glucocorticosteroids (GCS). These compounds are often limited in efficacy and have detrimental side-effects (118). Moreover, individuals with COPD and/or asthma are prone to exacerbations (despite chronic GCS treatments) representing flares of airway inflammation that then require much higher doses of systemic GCS to control symptoms and aid recovery. In this setting, PFD treatment is an appealing option because the compound has antiinflammatory, antioxidant, and antifibrotic properties (47). Repurposing PFD for this indication is worth consideration, and such a strategy might be particularly attractive if “tactical” treatment during times of frequent viral and other infections could mitigate inflammation or attenuate COPD or asthma exacerbations. More preclinical studies against this background could provide proof-of-concept evidence to pursue this approach.
Despite the success of PFD in IPF, the compound has some clinical pharmacokinetic drawbacks that necessitate innovation. It is rapidly absorbed after oral ingestion and metabolized, with only high doses reaching therapeutic plasma concentrations (32). Therefore, novel forms of PFD delivery may help to ensure adequate bioavailability. In a recent study, PFD was used as an inhaled powder to treat PF in a rodent model (119). The investigators reported that inhaled administration provided greater benefit than oral dosing, but this approach has not yet been considered and tested in human trials. Finally, strategies to inactivate or modulate CYP2 system enzymes to retard the metabolism of PFD may have potential benefits.
PFD has therapeutic efficacy in treating IPF because of its ability to suppress TGF-β1 and other growth factors. It also has compelling antiinflammatory capabilities because of its ability to diminish the production of cytokines, reduce accumulation of inflammatory cells, prevent inflammasome activation, and limit oxidative stress responses. The therapeutic use of PFD in IPF and the repurposing of this compound to treat chronic inflammation in lung disorders and other analogous diseases are appealing prospects.
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Originally Published in Press as DOI: 10.1165/rcmb.2019-0328TR on January 22, 2020
Author disclosures are available with the text of this article at www.atsjournals.org.
