Effects of Pirfenidone and Nintedanib on Markers of Systemic Oxidative Stress and Inflammation in Patients with Idiopathic Pulmonary Fibrosis: A Preliminary Report

Round 1

Reviewer 1 Report

The study by Fois et al. aimed to evaluate the effects of two drugs (i.e. pirfenidone and nintedanib) for the treatment of idiopathic pulmonary fibrosis on systemic redox balance. To address this objective the authors measured a number of putative biomarkers of oxidative damage and antioxidant defense (besides some not conventional markers of inflammation) at baseline and after 24-week treatment in 18 patients affected by idiopathic pulmonary fibrosis (10 treated with 25 pirfenidone and 8 with nintedanib) and in healthy controls.

In my opinion the manuscript has some merits: it is well-written, clear and easy to follow also for readers that are not familiar with the topic. However, the work also suffers from severe methodological shortcomings that limits its overall scientific quality .

• The main issues of the study are due to the markers used for measuring the systemic redox homeostasis. None of the (several) molecules measured by the authors can be considered as reliable and analytically accurate markers for oxidative stress.

The choice of the authors can, at least in part, be justified by the absence of universally accepted “golden-standard” biomarker for the in vivo assessment of oxidative stress. However, according to some articles from major expert in redox biology field [1–6], there are very good alternatives such as F2-isoprostanes (as assessed by GS/MS), MDA (but only determined by HPLC plus florescence) and 8-oxo-2'-deoxyguanosine (8-oxo-dG) [2,4,7,8].

Regarding some markers considered in the study, there are some considerations to make (and always report in the manuscript as evident intrinsic methodological limitations):  1)  TBARS is widely considered as a highly unspecific biomarker of lipid peroxidation  (“TBA can react with several compounds, including sugars, amino acids, bilirubin, and albumin, producing interferences in the measurement”[9]) 2) Homocysteine is not a direct marker of oxidative damage and its concentration is influenced by a wider range of other endogen/exogen factors 3) GSH and thiols are not biomarkers of antioxidant power, especially when they are measured in plasma/serum, because of their great chemical instability. The ratio GSH/GSSG is a good way to measure the degree of oxidative stress in a cell, but not in the more complex and variegate environment of blood.

• The authors should not describe the biological significance and the function of the markers in the “Methods” section. This description can be made in the “Discussion” or, briefly, in the “Introduction”.
• The (very) small size of the sample limits the validity and reliability of the statistical analysis
• The role of NRF2 is crucial in the response to oxidative insult. However, there are just a few research papers dealing with the measurement of this transcription factors in an extracellular fluid. The authors should justify and support with published evidence the use of this molecule as peripheral biomarkers of antioxidant defensive system.

1. Kalyanaraman, B.; Darley-Usmar, V.; Davies, K.J.A.; Dennery, P.A.; Forman, H.J.; Grisham, M.B.; Mann, G.E.; Moore, K.; Roberts, L.J.; Ischiropoulos, H. Measuring reactive oxygen and nitrogen species with fluorescent probes: Challenges and limitations. Free Radic. Biol. Med. 2012, 52, 1–6, doi:10.1016/j.freeradbiomed.2011.09.030.
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3. Kadiiska, M.B.; Peddada, S.; Herbert, R.A.; Basu, S.; Hensley, K.; Jones, D.P.; Hatch, G.E.; Mason, R.P. Biomarkers of oxidative stress study VI. Endogenous plasma antioxidants fail as useful biomarkers of endotoxin-induced oxidative stress. Free Radic. Biol. Med. 2015, 81, 100–6, doi:10.1016/j.freeradbiomed.2015.01.006.
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7. Klawitter, J.; Haschke, M.; Shokati, T.; Klawitter, J.; Christians, U. Quantification of 15-F2t-isoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared. Rapid Commun. Mass Spectrom. 2011, 25, 463–468, doi:10.1002/rcm.4871.
8. Milne, G.L.; Gao, B.; Terry, E.S.; Zackert, W.E.; Sanchez, S.C. Measurement of F2- isoprostanes and isofurans using gas chromatography-mass spectrometry. Free Radic. Biol. Med. 2013, 59, 36–44, doi:10.1016/j.freeradbiomed.2012.09.030.
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Author Response

The study by Fois et al. aimed to evaluate the effects of two drugs (i.e. pirfenidone and nintedanib) for the treatment of idiopathic pulmonary fibrosis on systemic redox balance. To address this objective the authors measured a number of putative biomarkers of oxidative damage and antioxidant defense (besides some not conventional markers of inflammation) at baseline and after 24-week treatment in 18 patients affected by idiopathic pulmonary fibrosis (10 treated with 25 pirfenidone and 8 with nintedanib) and in healthy controls.

In my opinion the manuscript has some merits: it is well-written, clear and easy to follow also for readers that are not familiar with the topic. However, the work also suffers from severe methodological shortcomings that limits its overall scientific quality.

• The main issues of the study are due to the markers used for measuring the systemic redox homeostasis. None of the (several) molecules measured by the authors can be considered as reliable and analytically accurate markers for oxidative stress. The choice of the authors can, at least in part, be justified by the absence of universally accepted “golden-standard” biomarker for the in vivo assessment of oxidative stress. However, according to some articles from major expert in redox biology field [1–6], there are very good alternatives such as F2-isoprostanes (as assessed by GS/MS), MDA (but only determined by HPLC plus florescence) and 8-oxo-2'-deoxyguanosine (8-oxo-dG) [2,4,7,8].Regarding some markers considered in the study, there are some considerations to make (and always report in the manuscript as evident intrinsic methodological limitations):  1)  TBARS is widely considered as a highly unspecific biomarker of lipid peroxidation  (“TBA can react with several compounds, including sugars, amino acids, bilirubin, and albumin, producing interferences in the measurement”[9]) 2) Homocysteine is not a direct marker of oxidative damage and its concentration is influenced by a wider range of other endogen/exogen factors 3) GSH and thiols are not biomarkers of antioxidant power, especially when they are measured in plasma/serum, because of their great chemical instability. The ratio GSH/GSSG is a good way to measure the degree of oxidative stress in a cell, but not in the more complex and variegate environment of blood.

We agree with the Reviewer that more reliable and analytically accurate biomarkers could be used to evaluate OS. However this primarily depends on access to highly complex instruments such as GS/MS or HPLC-Flu, which we do not have. That said, as also highlighted by the Reviewer, there is no universally accepted “golden-standard” biomarker for the in vivo assessment of OS. We acknowledge the limitations of the TBARS assay, widely discussed in our previous reports [1-4]. However, we have also demonstrated the utility of this assay in evaluating OS in several diseases [5-12].

We also agree that homocysteine is not a direct marker of oxidative damage. In fact,  we state “…Hcy can cyclize to give homocysteine thiolactone, a five-membered heterocycle. Subsequent cleavage of this molecule during protein N-homocysteinylation reaction may generate reactive species [13]. In addition, elevated Hcy concentrations inhibit mitochondrial respiration resulting in ROS accumulation [14]” (page 9, lines 270-273 ). Thus, Hcy plasma concentrations may be associated with OS [15-17].

Although we agree in principle with the comment on GSH, its measurement in blood has often been reported in the literature as an approach to evaluate OS. For example, 22 studies investigating GSH as an OS biomarker have been published in relation to a single condition such as chronic obstructive pulmonary disease [18-39].

• The authors should not describe the biological significance and the function of the markers in the “Methods” section. This description can be made in the “Discussion” or, briefly, in the “Introduction”.

We thank the Reviewer for the suggestion. This section has been moved to the discussion.

• The (very) small size of the sample limits the validity and reliability of the statistical analysis

We agree with the Reviewer. This is reflected in the title, “preliminary report”, and study limitations (page 11, lines 397-399).

• The role of NRF2 is crucial in the response to oxidative insult. However, there are just a few research papers dealing with the measurement of this transcription factors in an extracellular fluid. The authors should justify and support with published evidence the use of this molecule as peripheral biomarkers of antioxidant defensive system.

We thank the Reviewer for the suggestion. Accordingly, several sentences and references have been added in the discussion to provide additional evidence regarding the use of this molecule as a peripheral biomarker of the antioxidant defensive system (page 10 lines 330-334).

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Reviewer 2 Report

The authors demonstrated the antioxidant effects of pirfenidone and nintedanib, which has therapeutic effects for pulmonary fibrosis, in patients with that disease. The findings are quite important in clinical data in actual patients. However, there are several points which should be clarified for publication in this journal.

(1) Table 2 indicated that, after 24-week treatment, two agents significantly decreased oxidative stress in pulmonary fibrosis patients. Nevertheless, it seems that lung functions deteriorated. It is quite strange. Are there any advantage in antioxidant effects in treatment of pulmonary fibrosis? The relation of antioxidant effects and treatment outcome should be investigated and shown.

(2) Tables 3 and 4 indicated that antioxidant effects of two agents were not identical. Antioxidant effects and treatment outcome should be compared between two agents.

(3) d-ROMS, which is referred to as a crude marker of LOOH, may well-reflect total oxidative stress status in human more than other specific markers (Medicine (Baltimore). 2018 Nov;97(47):e12845. doi: 10.1097/MD.0000000000012845). For understanding overall oxidative stress and inflammation status in pulmonary fibrosis patients, this analysis should be performed and presented.

Author Response

The authors demonstrated the antioxidant effects of pirfenidone and nintedanib, which has therapeutic effects for pulmonary fibrosis, in patients with that disease. The findings are quite important in clinical data in actual patients. However, there are several points which should be clarified for publication in this journal.

(1) Table 2 indicated that, after 24-week treatment, two agents significantly decreased oxidative stress in pulmonary fibrosis patients. Nevertheless, it seems that lung functions deteriorated. It is quite strange. Are there any advantage in antioxidant effects in treatment of pulmonary fibrosis? The relation of antioxidant effects and treatment outcome should be investigated and shown.

We wish to thank the Reviewer for highlighting this point. Although counterintuitive, lung function deterioration during treatment is common. As described in the introduction “In 2014, the Food and Drug Administration approved nintedanib and pirfenidone for IPF treatment because of their ability to retard disease progression”. Thus, these drugs do not improve lung function but, rather, retard disease progression. The FVC deterioration observed in our study, 96mL with pirfenidone and 92mL with nintedanib, is similar to that previously reported in clinical trials [1-2]. The relationship between antioxidant effect and treatment outcomes should be properly investigated in larger placebo controlled studies. For these reason, our final sentence states “All reported data, including non-significant differences, may assist with the adequate design (i.e. sample size and/or biomarker selection) of larger interventional trials to confirm these findings and to establish whether such effects are associated with clinical improvement in this group”.

(2) Tables 3 and 4 indicated that antioxidant effects of two agents were not identical. Antioxidant effects and treatment outcome should be compared between two agents.

Please see response for question 1.

(3) d-ROMS, which is referred to as a crude marker of LOOH, may well-reflect total oxidative stress status in human more than other specific markers (Medicine (Baltimore). 2018 Nov;97(47):e12845. doi: 10.1097/MD.0000000000012845). For understanding overall oxidative stress and inflammation status in pulmonary fibrosis patients, this analysis should be performed and presented.

We thank the Reviewer for this suggestion. However, the use of d-ROMS to directly detect reactive oxygen species (ROS) is marred by several issues, particularly the ROS instability in biological samples [3]. Therefore, their quantification is inevitably affected by the time interval between sample collection and the assay, with longer delays associated with greater loss of ROS. Another concern is the possible interference from ceruloplasmin (a copper-containing enzyme responsible for oxidation of serum iron and aromatic amines and phenols) activity, which creates false positive signals [3-5]. Furthermore, iron, albumin, and thiol levels can also alter the test’s outcome [3]. For these and other reasons [6-8], we used other assays that are able to detect stable products of ROS.

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Round 2

Reviewer 1 Report

The authors addressed all my comments. All the responses are clear and, I appreciated the attempt to explain the missing points and the shortcomings of the study. Nevertheless, in my opinion, the fact that they did not measure any of the most reliable markers of oxidative stress, remains an intrinsic and unsolved issue of the work.  How can we know whether the two drugs really work against oxidative stress, if we do not evaluate this condition properly? This methodological problem is relevant especially considering the high impact and important ranking of the journal in the redox-biology field

Author Response

While we respect the Reviewer’s comments, we remain of the opinion that the oxidative stress biomarkers measured in our study have been widely investigated in previous studies. Such studies, described in our initial rebuttal, have described not only their pathophysiological role but also their clinical significance. In particular, as also previously highlighted, over the last decade our group has published a considerable number of studies on GSH and MDA as robust biomarkers of oxidative stress.

Reviewer 2 Report

I have no further criticism for this revised manuscript.

Author Response

Thank you