Protective and Pain-Killer Effects of AMC3, a Novel N-Formyl Peptide Receptors (FPRs) Modulator, in Experimental Models of Rheumatoid Arthritis

Round 1

Reviewer 1 Report

The manuscript "Protective and pain-killer effects of AMC3, a novel N-formyl peptide receptors (FPRs) modulator, in experimental models of rheumatoid arthritis" provides interesting insights into the potential therapeutic use of AMC3 in rheumatoid arthritis. The study is well-designed and the results are presented clearly. However, there are several areas of concern that require further clarification before the study's findings can be considered conclusive.

Firstly, while the study reports the protective and pain-relieving effects of AMC3 in experimental models of rheumatoid arthritis, it is unclear how these effects are mediated. Specifically, the authors should provide a more detailed discussion on the mechanisms by which AMC3 exerts its protective and pain-relieving effects. This would provide a more comprehensive understanding of the potential therapeutic benefits of AMC3 and help inform future research in this area.

Secondly, the study has several limitations that need to be addressed. For example, the sample size in the animal experiments was relatively small, and the study only investigated the effects of AMC3 in male rats. This raises questions about the generalizability of the study's findings to other populations and genders. Moreover, while the in vitro experiments are promising, they do not necessarily translate to in vivo effects, and the relevance of the in vitro findings to human rheumatoid arthritis is not clear.

Finally, the manuscript would benefit from a more detailed discussion of the potential safety concerns associated with the use of AMC3. While the authors state that AMC3 has a good safety profile, they do not provide any evidence to support this claim. Furthermore, given that AMC3 modulates the activity of FPRs, which are involved in various physiological processes, there is a need to investigate potential off-target effects of AMC3 on other systems.

In summary, the manuscript provides an interesting perspective on the potential therapeutic use of AMC3 in rheumatoid arthritis. However, further research is required to clarify the mechanisms by which AMC3 exerts its effects and to address the limitations of the current study. Additionally, the manuscript would benefit from a more comprehensive discussion of potential safety concerns associated with the use of AMC3.

Author Response

Response To Reviewer 1 Comments

Dear authors,

The manuscript "Protective and pain-killer effects of AMC3, a novel N-formyl peptide receptors (FPRs) modulator, in experimental models of rheumatoid arthritis" provides interesting insights into the potential therapeutic use of AMC3 in rheumatoid arthritis. The study is well-designed, and the results are presented clearly. However, there are several areas of concern that require further clarification before the study's findings can be considered conclusive.

Firstly, while the study reports the protective and pain-relieving effects of AMC3 in experimental models of rheumatoid arthritis, it is unclear how these effects are mediated. Specifically, the authors should provide a more detailed discussion on the mechanisms by which AMC3 exerts its protective and pain-relieving effects. This would provide a more comprehensive understanding of the potential therapeutic benefits of AMC3 and help inform future research in this area.

Response: We thank a lot the reviewer for the suggestion. We have added some comments in the discussion section.

Here is the reported text (lines 482-489): “Especially its inhibitory effects are emerged on iNOS overproduction, enzyme primarily linked with the chronic and progressive inflammation [54] and on COX-2, a remarkable enzyme involved in prostaglandin biosynthesis, in particular of Prostaglandin E2 (PGE2), which is responsible of matrix metalloproteinase unbalance [55] and increase the expression of the angiogenic factor VEGF-A [58], that contribute to inflammatory hyperalgesia [59,60], effects potentially promoted by the suppression of NF-κB activity [56], as well as of the influence exerted by the activation of these receptors on MAPK pathways, including ERK, p38 MAPK and JNK [57].”

Secondly, the study has several limitations that need to be addressed. For example, the sample size in the animal experiments was relatively small, and the study only investigated the effects of AMC3 in male rats. This raises questions about the generalizability of the study's findings to other populations and genders. Moreover, while the in vitro experiments are promising, they do not necessarily translate to in vivo effects, and the relevance of the in vitro findings to human rheumatoid arthritis is not clear.

Response: We thank the reviewer for this good point, and although we know the importance of considering the value of including female rats in research, using male rats offers some advantages in terms of obtaining more reliable and less variable data. By excluding the hormonal fluctuations associated with the estrous cycle, we can achieve greater standardization of experimental groups and reduce the complexity of our study design. Furthermore, employing male rats allows us to minimize the number of animals required for the study, aligning with the principles of the 3Rs (reduction, refinement, and replacement). This approach not only reduces the overall impact on animal welfare but also increases the feasibility of long-term studies, as the absence of estrous cycles facilitates consistent assessments and monitoring. Moreover, we obtained formal approval to conduct the described experiments from the Italian Ministry of Health, predicting the right numerosity of each experimental group by using the statistical program G Power 3.1.9.2. We calculated that it was necessary to study 6 rats in each group to detect this difference as statistically significant with 80% power, with an effect size of 1.6 at the 0.05 level. The absence of studies on females is undoubtedly a limitation of this work and a key point to be explored extensively in future studies, as also added in the discussion.

Here is the reported text (lines 551-557): “Although it is important to recognize that no gender studies have been conducted on the efficacy of AMC3, making further future investigations in females necessary, which will further enrich our knowledge on the subject, to date, it has been possible to highlight the protective antioxidant and anti-inflammatory effects of AMC3 in chondrocytes, as well as its pain-relieving and disease-modifying properties in male RA rats. Protective effects on both joints and the nervous system have been shown. The FPRs agonist AMC3 emerges as a candidate for long-lasting, disease-modifying treatment of RA.”

Furthermore, the choice to study the effects of AMC3 in vitro on rat chondrocytes is justified by the similarity of rat chondrocytes to human chondrocytes in terms of their response to inflammatory stimuli and drug treatments, and the vulnerability of these cells in rheumatoid arthritis (Madry et al., 2010; Khorasani et al., 2017). This allows us to obtain preliminary results and valuable insights into the efficacy of AMC3 in modulating inflammatory responses and chondrocyte degeneration, as well as providing initial insights into the cellular and molecular mechanisms involved in rheumatoid arthritis, although it's important to consider the limitations of animal models and validate findings in human studies and clinical trials.

Finally, the manuscript would benefit from a more detailed discussion of the potential safety concerns associated with the use of AMC3. While the authors state that AMC3 has a good safety profile, they do not provide any evidence to support this claim. Furthermore, given that AMC3 modulates the activity of FPRs, which are involved in various physiological processes, there is a need to investigate the potential off-target effects of AMC3 on other systems.

Response: We thank the reviewer for the fair comment, and although we are aware of the need to study the pharmacological profile of new molecules and to further investigate potential off-target effects, the absence of side effects during in vivo experiments on chronically treated animals for 14 days allowed the pre-clinical studies to continue. We softened the results section and discussion in relation to the safety profile of AMC3, which was investigated only macroscopically, with the aim of conducting further in-depth studies in the future.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments to Author:

Although the investigators put the word ‘Rheumatoid arthritis’ in the manuscript titile, it is not clear this the study is of ‘Rheumatoid arthritis’. It is understandable to use IL-1β to provoke arthritis in rats. However, IL-1β is not a cytokine specific to RA. IL-1β is upregulated other arthritic diseases, such as familial mediterranean fever.

In addition, VEGF is upregulated the other rheumatic diseases, such as polymyalgia rheumatica and RS3PE syndrome.

Considering the above concerns, the title and entire discussion might need re-consideration.

Author Response

Dear authors,

Although the investigators put the word ‘Rheumatoid arthritis’ in the manuscript title, it is not clear this the study is of ‘Rheumatoid arthritis’. It is understandable to use IL-1β to provoke arthritis in rats. However, IL-1β is not a cytokine specific to RA. IL-1β is upregulated other arthritic diseases, such as familial mediterranean fever.

Response: I greatly appreciate the valuable feedback provided by the reviewer however there are numerous studies in the literature confirming the in vitro use of IL-1β to induce rheumatoid arthritis-like features in chondrocytes (Saito et al., 2003; Aigner et al., 2004; Fernandes et al., 2002; Lawyer et al., 2011). These valuable studies mimic the inflammatory environment observed in rheumatoid arthritis and are instrumental in studying the effects of IL-1β on chondrocyte behavior and subsequent disease processes. IL-1β-induced changes in chondrocytes mimic key pathological features of rheumatoid arthritis, including heightened inflammation, cartilage degradation, and altered matrix metabolism.

 In addition, VEGF is upregulated the other rheumatic diseases, such as polymyalgia rheumatica and RS3PE syndrome.

Response: Thanking the reviewer for the suggestion, we have reworded the discussion, clarifying a nonspecific indication of the role of VEGF exclusively in rheumatoid arthritis.

Here is the reported text (lines 505-510): “In recent years, accumulating evidence has suggested the pathological involvement of VEGF-A and its signaling pathways in disease progression and associated pain in various rheumatic diseases, including RA,”

Considering the above concerns, the title and entire discussion might need re-consideration.

Response: We thank the reviewer for this suggestion but as clarified above, the experimental models used (CFA injection in vivo and IL-1β stimulation on rat primary chondrocytes in vitro) are able to reproduce, albeit with due limitations, the classical features of rheumatoid arthritis, consequently we believe that the title is consistent with what has been studied. Discussion was extensively modified.

Round 2

Reviewer 2 Report

(There are no comments)