FDA-Approved Antitussive Dextromethorphan Enhances Antibrotic Effectiveness of Pirfenidone In Bleomycin-Induced Mice and Patients With Idiopathic Pulmonary Fibrosis

Background: We aimed to investigate whether dextromethorphan (DM), an antitussive, can improve the antifibrotic efficacy of pirfenidone in treating idiopathic pulmonary fibrosis, a fatal interstitial lung disease characterized by progressive and irreversible respiratory failure. Methods: A bleomycin-induced mice pulmonary fibrosis study and an open label randomized clinical trial were performed to evaluate the effectiveness of pirfenidone combined with DM. Results: In the animal study, pirfenidone combined with DM protected mice against bleomycin-induced pulmonary fibrosis with better capabilities than pirfenidone alone or DM alone indicated by lung histologic analysis and hydroxyproline levels. In the clinical study, pirfenidone plus DM markedly mitigated pulmonary functions (FEV1 and FVC) decline and ameliorated chest HRCT imaging scores (ground glass opacities and reticulation) of patients with IPF than pirfenidone alone at one year after administration. There were no significant differences in adverse reactions between pirfenidone-DM group and pirfenidone group. Conclusions: DM significantly potentiates antifibrotic effectiveness of pirfenidone in a mouse IPF model and patients with IPF and does not increase side effects of pirfenidone. fibrosis focal area ratio (B) hydroxyproline measurement the lungs Data Statistical analyses were (C). are the mean ± SD. Statistical analyses performed by an ANOVA one-way test with Tukey’s test for comparisons between groups. *, P< 0.05.


Background
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial In recent years, the inflammation-repression effects of DM have been gradually recognized and discovered. DM reduces serum levels of proinflammatory mediator tumor necrosis factor alpha (TNF-α), IL-6, IL-1 chemokine monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) in mice or patients [11][12][13]. Low dose DM inhibits the expression and activity of microglial NOX2 [14]. The anti-inflammatory and antioxidant properties of DM imply that it could be applied to promote anti-fibrotic capacity of pirfenidone for IPF.
Here, we found that pirfenidone combined with DM showed the best potency in reducing fibrotic pathology compared with pirfenidone alone or DM alone in (1.5mg/kg) bleomycin (Nippon Kayaku Co., Tokyo, Japan) was intratracheally administered to mice except the control group at day 0. Mice in different groups were pretreated with vehicle, pirfenidone alone, DM alone, or pirfenidone plus DM for one day, and continuously administered to day 20. Pirfenidone (Beijing Kangdini Pharmaceutical Co., Ltd., China) was intragastrically administered once a day (100 mg/kg body weight). DM (Selleck biotechnology co., ltd, USA) was subcutaneously injected three times a day (10 ng/kg body weight). The control group and the bleomycin group were given the same volume of saline every day. The mice were sacrificed on D21 and lungs were taken out to prepare for histological staining and hydroxyproline assay.

Histologic analysis
Mice left lungs were dehydrated, paraffin embedded, and cut into 5-μm sections, then stained with H&E and Masson Trichrome staining. The degree of inflammation and fibrosis of lung tissue was observed under an optical microscope. Pulmonary fibrosis area was quantified [15]. Slides were sampled with a random-start systematic sampling scheme using a 6-mm-grid randomly superimposed on the slide to indicate areas to evaluate. Digitized images of the slide were obtained using a ×2 objective on an Olympus BH-2 microscope (Olympus America, Melville, New York, USA) with a Sony DXC970MD camera (Sony America, New York, New York, USA) and an IxTV capture card (IxMicro, San Jose, California, USA) on an Apple Macintosh G3 computer (Apple Computer Inc., Cupertino, California, USA). Images were then opened in Photoshop Elements (Adobe Systems Inc., San Jose, California, USA). The overall area of the lung was obtained by manual outlining. The area of the lung with fibrosis was then outlined and the area obtained. The pixels of total versus fibrotic tissue were then summed over each lung and a percentage was obtained.

Hydroxyproline assay
Collagen contents were evaluated with conventional hydroxyproline method in mice right lungs which had been cleared of blood [16]. Samples containing known amounts of purified collagen confirmed the capability of the assay to fully hydrolyze and recover hydroxyproline from collagen. Glucocorticoids or immunosuppressants were not administrated within 1 month. All patients took pirfenidone capsules (Beijing Kangdini Pharmaceutical Co., Ltd., 100mg/capsule, H20133376). An initial dose of 200 mg of pirfenidone was administered three times daily with meals (600 mg/day). Thereafter, the dose was gradually increased by 200 mg every 2 weeks to a maximum of 600 mg per dose (1800 mg/day). Dextromethorphan (DM) hydrobromide tablets (Shijiazhuang Yiling Pharmaceutical Co., Ltd., 15 mg/tablet, H20066348) were added with 7.5-15 mg once a day in pirfenidone-DM group. A total of 16 consecutive eligible patients were enrolled with 8 in pirfenidone group and 8 in pirfenidone-DM group.

Laboratory tests and pulmonary function testing
Blood routine test, C-reactive protein (CRP), liver and kidney function, and pulmonary function testing (PFT) of patients were collected before treatment, 6 months, and 1 year. Pulmonary function tests (PFT) were performed by standard techniques using CHESTAC-33 (Chest MI Co., Tokyo, Japan) and Fudac-77 (Fukuda Denshi, Tokyo). Pulmonary function test indexes included forced vital capacity (FVC), forced vital capacity percent (FVC%) predicted, forced expiratory volume in one second (FEV1), forced expiratory volume in one second percent (FEV1%) predicted and diffusing capacity of the lung for carbon monoxide percent (DLCO%) predicted.

CT images
A high resolution CT scan (Lightspeed-64, GE, America) was used and CT images with 0.5 mm (or 1 mm) slice thickness were obtained (140 kVp and 200 mA). Each recorded extent of normal lung and each lesion (ground glass opacities (GGO), consolidation, reticular abnormality, honeycombing and emphysema) by 5% steps in three zones in each lung based on the method described by Best [18]. The upper zone, middle zone, and lower zone was defined as at or above the aortic arch, between the aortic arch and pulmonary veins, and at or below the pulmonary veins, respectively.
The mean extent of three zones was calculated. HRCT scans were scored by two expert thoracic radiologists using the above method that assesses the degree of ground glass attenuation (HRCT alveolar score) and fibrotic change (HRCT interstitial score and Honeycomb score) [19]. Radiologists were blinded to the patient's diagnosis and grouping. Agreement between the radiologists was very good.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, California, USA). Multiple comparisons were performed using ANOVA tests with post hoc analysis. To compare demographic data and baseline clinical characteristics between pirfenidone group and pirfenidone-DM group, a Chi square test and Fisher's exact test for categorical variables and a Mann-Whitney U test for continuous variables were used as appropriate. The level of interobserver agreement between the two radiologists was evaluated by kappa statistic measure. P values < 0.05 were considered to indicate statistical significance.

3.1.Pirfenidone combined with DM protects mice against bleomycin-induced pulmonary fibrosis
At 21 days after challenge, intratracheal injection of bleomycin resulted in the destruction of normal lung structures, markedly thickened alveolar septum, and mass filling of fibrous tissue indicated by H&E staining of mouse lung sections (Fig. 1A).
The deposition of collagen fibers was largely increased in bleomycin-induced lung injury as shown by Masson's trichrome staining (Fig. 1A). Decreased alveolar damage and collagen fiber deposition were displayed in mice lung sections by administration of pirfenidone, DM, or pirfenidone combined with DM (Fig. 1A). The  1C). Together, the fibrosis focal area ratio quantification and hydroxyproline assay clearly indicated the potency of pirfenidone plus DM in alleviating bleomycin-elicited pulmonary fibrotic pathology.

Patient characteristics
Based on the findings in the mice experiments, we further studied whether DM could potentiate the treatment effectiveness of pirfenidone in patients with IPF. We enrolled 20 patients but 4 patients (2 from pirfenidone group and 2 from pirfenidone-DM group) died of infections. Therefore, 16 patients were statistically analyzed with PFTs and HRCT scores. There were no significant differences in gender, age, age at diagnosis, clinical-radiological diagnosis, smoking years, prior treatment received steroids, and IPF treatment at baseline (Table 1). Three patients (50.00%) in the pirfenidone group and two patients (37.50%) in the pirfenidone-DM group showed the usual interstitial pneumonia (UIP) pattern on HRCT, but there was no statistical difference between the two groups (Table 1). 3.3. Pirfenidone combined with DM mitigates pulmonary function decline for IPF patients Pulmonary function tests (PFTs) were performed before medication, 6 months and 1 year. Before antifibrotic treatment and 6 months later, there were no significant differences in the performances of PFTs between pirfenidone group and pirfenidone-DM group (Table 2). Some PFT indexes of the two groups showed an overall downward trend, and DLCO%pred was not different between the two groups 1 year later (Fig. 2E). However, pulmonary function tests including FVC, FVC%pred, FEV1, and FEV1%pred in pirfenidone-DM group were statistically better at 1 year after treatment than those in pirfenidone group (P<0.05) ( Fig. 2A-D). Therefore, pirfenidone combined with DM significantly mitigates pulmonary function decline compared with pirfenidone alone in patients with IPF.

Pirfenidone combined with DM ameliorated pulmonary imaging scores of patients with IPF
The inter-observer agreement between two radiologists with regard to changes in alveolar score, reticulation, and honeycombing was good (Cohen's kappa = 0.74 for alveolar score, k = 0.78 for reticulation, k = 0.72 for honeycombing). There was no significant difference in HRCT alveolar score, interstitial score and honeycomb score between the two groups of patients before treatment (Table 3). After 1 year of antifibrotic treatment, HRCT displayed that the alveolar score and interstitial score of patients in pirfenidone-DM group were markedly improved comparing to pirfenidone group (P=0.035, P=0.037, respectively) (Table3, Fig. 3 A, B), although honeycomb scores remained similarly between the two groups (P=0.999) (Table3, Fig. 3C). Thus, pirfenidone combined with DM significantly ameliorated pulmonary imaging scores of patients with IPF. A patient's chest HRCT showed evidently alleviated ground glass opacities and reticulation upon 1 year administration of pirfenidone plus DM (Fig. 4).
3.5. DM did not increase adverse reactions of pirfenidone 2 patients (1 from pirfenidone group and 1 from pirfenidone-DM group) had gastrointestinal reactions, and 1 patient from pirfenidone group felt slight dizziness.
There was no between-group difference in the risk of adverse reactions (t-test, P>0.5).
The liver and kidney function and blood routine test of patients showed no statistical differences between two groups and no significant changes were observed before and after medication (P>0.5).

Discussion
The recommendations for pharmacotherapy in IPF patients was in clinical practice guidelines 2015, and pirfenidone were recommended conditionally for treatment as antifibrotic therapy [8] for it can only moderately slowed down the decline of lung function in patients with IPF. Since then, little clinical progress has been made.
However, in the current study, DM significantly potentiates the antifibrotic efficacy of pirfenidone not only in mice induced by bleomycin but also in patients with IPF.
Someone propose that the core pathway that mediates fibrosis may be a better target to develop drugs for anti-fibrosis in multiple organ systems drug [21] and address that age-related redox imbalances has been recognized as one of these core approaches [22]. DM, as a broad and safe antitussive drug, has been found to have obvious anti-inflammatory and antioxidative effects in recent years. In order to explore the anti-fibrotic effect of the combination of pirfenidone and DM on IPF, we conducted this study and found that the combination of the two can significantly improve pulmonary fibrosis. In animal studies, compared with other groups, pirfenidone combined with DM can improve bleomycin-induced pulmonary fibrosis indicated by decreased proliferation of alveolar epithelium, production of fibroblasts, and levels of hydroxyproline. In clinical studies, we have observed that pirfenidone combined with DM can improve the PFT of patients, delay the reduction of FEV1 and FVC, and mitgate the ground glass opacities and reticulation of HRCT.
During the development of IPF, oxidative stress refers to the imbalance between the production and removal of oxygen free radicals in the body or cells, which leads to an increase in reactive oxygen species (ROS). ROS regulates cell growth, proliferation and death by stimulating related genes, and promote the release of various inflammatory mediators and cytokines, such as transforming growth factor-β(TGF-β), etc. Then ROS promotes pulmonary fibrosis. TGF-β increases the production of mitochondrial ROS in different cells, which in turn will promote the apoptosis[23], epithelial-mesenchymal transition(EMT)[5], fibrosis gene expression and fibroblast differentiation [24]. Although the exact role of inflammation in fibrosis is still controversial, there is clear evidence that chronic inflammation also exists in IPF [25].
There are activated inflammatory cells in the lungs of IPF patients, such as alveolar macrophages and neutrophils [26]. These phagocytes may be involved in ROS-mediated epithelial cell damage. In addition, the lung tissues of IPF patients showed accumulation of activated non-proliferative T cells and B cells, with follicular dendritic cell aggregation [27,28]. These inflammatory cells produce a large number of cytokines, such as TGF-β, TNF-α, IFN-γ, IL-4, IL-10, IL-12, IL-1, MCP and so on.
They form a complex cytokine network, regulating the apoptosis of alveolar epithelial cells and the proliferation of mesenchymal fibroblasts, and ultimately facilitating the formation of lung fibrosis [4,29].
In patients with IPF, after individual treatment and various immunotherapies, the epithelial cells and blood vessels in the lung are damaged, resulting in cell destruction, uncontrolled repair, and the development of progressive pulmonary fibrosis.
Combining various hypotheses, when exposed to pathogenic factors, once fibrosis has formed, anti-fibrosis treatment may be initiated. Previously, progressive fibrosis was mostly classified as ordinary pulmonary fibrosis. Once fibrosis occurs, immunosuppression is no longer effective, and anti-fibrosis therapy is needed to dampen the decline in lung function during pulmonary fibrosis. Pirfenidone is the world's first therapeutic drug to obtain indications for idiopathic pulmonary fibrosis.
Human lung tissue is exposed to higher concentrations of oxygen than other organs, therefore being more susceptible to damage from oxidative stress. When there is too much ROS production or insufficient antioxidant capacity in the body, excessive ROS exists in tissues or cells, which can induce oxidative stress and inevitably lead to lung tissue damage and remodeling [30]. NOX2 is universally expressed and stable in the lung, and its expression will increase under inflammatory conditions [31]. The evidence of NOX2 in IPF mainly comes from animal models, which show that mice with NOX2 genetic defects can reduce AEC damage induced by bleomycin or carbon nanotubes [32]. In addition to the role of NOX2 in inflammatory cells, studies have shown that the NOX2/ROS/NLRP3 inflammasome signaling pathway can promote the occurrence of IPF [33]. DM is derived from non-opioid morphinans. As a widely used and safe over-the-counter cough suppressant, it has a history of more than 50 years [34]. Structurally, it belongs to morphine drugs. In view of its right-handed structure, DM has a weak affinity with opioid receptors, and it is difficult to form addiction. DM can specifically bind to the phosphorylated form of the subunit gp91 in NOX2, thereby inhibiting the catalytic activity of NOX2, thereby inhibiting the production of ROS. In addition to repressing the production of superoxide, DM also reduces the production of NO, thereby reducing pulmonary fibrosis. Previous studies have confirmed that DM has the effect of antagonizing the inflammation in the nervous system and preventing neurodegeneration. Moreover, DM significantly lower the production of superoxide in neutrophils.
As a functional examination, PFTs mainly reflect the damage to lung ventilation and ventilation function caused by disease. Although FEV1 and FVC are affected by many factors such as alveolar elasticity and airway resistance during expiration, pirfenidone combined with DM in this study is better than pirfenidone treatment in delaying the decline of FEV1 and FVC in IPF patients. Pulmonary diffusion function depends on the gas diffusion area in the lung, the thickness of alveolar capillary membrane and the blood flow in the lung. The deterioration of lung diffusion during IPF is aggravated by the excessive proliferation of fibroblasts and the continuous deposition of extracellular matrix in alveoli and lung interstitium accompanied by pulmonary vascular remodeling and occlusion. In terms of diffusion function, DLCO%pred after treatment was no statistical difference between the two groups before and after treatment, although pirfenidone plus DM appeared to slightly increase DLCO%pred than pirfenidone alone. it has a significant therapeutic effect on pulmonary function testing, but We have also observed that it has no significant improvement in improving the HRCT of IPF patients' honeycomb. The DLCO% of IPF patients decreased, and the pathological changes on HRCT mainly showed reticulation and honeycombing, which is consistent with the good correlation between reticulation and honeycombing and DLCO%.
Adverse reactions of pirfenidone are common and mainly gastrointestinal manifestations, skin diseases and dizziness [37]. The combination of pirfenidone and DM was well-tolerated by patients in this study and DM did not increase side effects of pirfenidone. The add-on therapy containing DM has exerts potent effects in many clinical settings. NEW YORK, Dec. 08, 2020, Very recently, Axsome Therapeutics  Prior treatment received steroids-n (%) 1 (13) 2 (25) .522

IPF treatment at baseline
Oxygen therapy at baseline-n (%) 3 (38) 2 (25) .590 Hypertension-n (%) 2 (25) 1 (13) .522 Diabetes mellitus-n (%) 1 (13) 2 (25) .522 GERD-n (%) 2 (25) 1 (13) .522 Emphysema-n (%) 2 (25) 1 (13) .522 Values are expressed as numbers and (%) or median and ranges as appropriate. To compare demographic data and baseline clinical characteristics between two groups, Chi square test for categorical variables.   bleomycin-induced lung injury. Pirfenidone combined with DM alleviated pulmonary pathological changes and reduced the production of collagen. Pirfenidone plus DM significantly decreased fibrosis focal area ratio (B) and hydroxyproline measurement in the lungs (C). Data are the mean ± SD. Statistical analyses were performed by an ANOVA one-way test with Tukey's test for comparisons between groups. *, P< 0.05.  Figure 1 Pirfenidone combined with DM reduce bleomycin-induced pulmonary brosis in mice at D21. (A) As indicated by H&E staining of lung sections, the intratracheal injection of bleomycin led to the destruction of normal pulmonary architecture, the prominently thickening of alveolar septum and the mass lling of brous tissue. As illustrated by Masson's trichrome staining, the deposition of collagen bers was largely increased in bleomycin-induced lung injury. Pirfenidone combined with DM alleviated pulmonary pathological changes and reduced the production of collagen. Pirfenidone plus DM signi cantly decreased brosis focal area ratio (B) and hydroxyproline measurement in the lungs (C). Data are the mean ± SD. Statistical analyses were performed by an ANOVA one-way test with Tukey's test for comparisons between groups. *, P< 0.05. Figure 2 PFT indexes of two groups of patients. Except DLCO%pred, the comparison of FVC, FEV1, FVC%pred, FEV1%pred showed statistical differences between the two groups (PFVC=0.038, PFEV1=0.044, PFVC%pred=0.044, and PFEV1%pred=0.049). Reference baseline for lung function in IPF patients comes from other studies [20]. *p<0.05 and NS (no signi cance) compared to pirfenidone group. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test was performed.

Figure 3
Alveolar score, interstitial score, and honeycomb score of patients treated with pirfenidone (P) or pirfenidone plus dextromethorphan (P+D). Comparison of Alveolar score, Interstitial score and Honeycomb score of HRCT between two groups of patients before and after treatment. Two-way ANOVA followed by Bonferroni post hoc multiple comparison test was performed.

Figure 4
Comparisons of HRCT images of a patient received pirfenidone and DM before and after treatment. Male, 61y, with a history of pulmonary brosis for 4 years and a history of smoking. Initial HRCT (A and D), 6 months later HRCT(B and E), and one year later HRCT (C and F) displayed apparently less ground glass opacities, reticulation and honeycombing after treatment.