Safe and Efficient Sigma1 Ligand: A Potential Drug Candidate for Multiple Sclerosis

Round 1

Reviewer 1 Report (Previous Reviewer 1)

The authors answered to all my criticims.

Author Response

We thank the reviewer for its comment.

Reviewer 2 Report (Previous Reviewer 2)

To answer the concerns raised, the Authors (ijms-1938735) have performed additional experiments, additional data have been included in the manuscript and several important issues are now discussed in the Discussion section. However, there are some minor issues left:

1) Table 8 - "Time after injection" should be placed on top of the "min" row and not as a separate column.

2) Figure 5 - the image (A and C) quality could be improved.

3) The Discussion section ends with a reference. Please consider to end Discussion section with some concluding remarks or reorganize the last paragraph of the Discussion that it does not end with a reference.

Re: comment to the response "i.p administration of small molecule results in faster and more complete absorption compared to oral". It highly depends on each individual compound and findings presented by other groups could not be always extrapolated to every small molecule compounds. This should be evaluated for each compound separately.

Overall, after some minor corrections the manuscript could be considered for the publication.

Author Response

Reviewer#2.

To answer the concerns raised, the Authors (ijms-1938735) have performed additional experiments, additional data have been included in the manuscript and several important issues are now discussed in the Discussion section. However, there are some minor issues left:

1) Table 8 - "Time after injection" should be placed on top of the "min" row and not as a separate column.

The table was corrected

2) Figure 5 - the image (A and C) quality could be improved.

The figure was pasted in the document in another format to improve the quality

3) The Discussion section ends with a reference. Please consider to end Discussion section with some concluding remarks or reorganize the last paragraph of the Discussion that it does not end with a reference.

We agree with the reviewer. The reference was cited before, as it does not add anything here, it was deleted in the last sentence of the discussion

Re: comment to the response "i.p administration of small molecule results in faster and more complete absorption compared to oral". It highly depends on each individual compound and findings presented by other groups could not be always extrapolated to every small molecule compounds. This should be evaluated for each compound separately.

We agree with the reviewer, but in general it was the case as the ip administration avoids the crossing of intestinal barrier. The following paper is very interesting to compare administration route : Al Shoyaib A. et al. Pharm. Res. 2020, 37, 12.

Overall, after some minor corrections the manuscript could be considered for the publication.

We would like to thank the Reviewer for careful reading of our manuscript.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The work presented by Bénédicte Oxombre et al. aims at characterizing a novel Sigma-1 receptor agonist, 7i, and to test its protective role in a mouse model of Multiple Sclerosis. The authors used a state of the art set of experiments to characterize in vitro and in vivo this compounds in order to go to clinical trial in MS. They elegantly demonstrated that 7i is a relevant novel Sigma-1 receptor for treating MS. Overall, the experiment are well conducted and the results are of importance.

However, I have a question.

Sigma-1 receptor is well known for its pleiotropic effects. The protein is able to boost IP3R signaling in the mitochondria-associated ER membranes. The protein can also regulate autophagy. Therefore, the precise signaling pathway activated by 7i should be evaluated in order to decipher its mode of action.

Reviewer 2 Report

1. Overview and general recommendation:

The Authors (ijms-1816595) present very dedicated preclinical assessment of novel sigma-1 receptor (S1R)-related compound 7i by demonstrating a quite extensive in vitro analysis and in vivo effects against experimental autoimmune encephalomyelitis, a model resembling clinical features of Multiple Sclerosis (MS). The data presented in the manuscript could be published after major revision, because there are some concerns in the manuscript to which I would draw attention:

2. Major comments:

The biggest concern raised also by the authors is the possible cardiac side-effects of the compound 7i due to inhibition of hERG. Please explain why the effects in vivo on QT interval (Table 8) was evaluated only for up to 9 min after i.p. injection? Although the bioavailability of the 7i was not demonstrated nor discussed, the bioavailability data after p.o. administration states that t_max = 30 min (Table 7). Could it be that the compound 7i after 9 min has not reached its max concentration to cardiac side effects appear? How about the possible cardiac side-effects of metabolite 7i-Me? Please explain and discuss.

Was cytotoxicity (Table 4) tested or considered also in some cardiac cell culture?

Please explain why the in vivo assessment of the possible S1R-related activity in the experimental MS model was done after i.p. and not p.o. injection of the 7i? As seen in the Figure 4, the compound 7i was ineffective after i.p injection. This raised my concerns about the in vivo effects of S1R antagonist BD-1047 on the in vivo activity of 7i.

Please exlplain and discuss why the compound 7i as S1R-related compound is effective against dizocilpine-induced learning deficits and not in the experimental MS model after i.p. administration.

Bi-phasic dose-dependent effect of S1R ligands is a specific concern of the possible therapeutic window of the S1R-related compounds and how this relates to the in vivo activity of 7i. This must be discussed in the Discussion section in regards to possible clinical application.

Figure 2 – Title of the axis is missing.

Figure 2 – For the internal validity of the test a combination between agonist PRE-084 and antagonist NE-100 should be presented. Please explain the reason for the evaluation of the activity of combination of PRE-084 and 7i on BiP dissociation.

Please explain how the t_1/2el of the 7i after p.o. administration (Table 7) was determined and how it is in line with the Figure S2B?

3. Minor comments:

Please reconsider the abbreviation used for the metabolite 7i-Me of the parent compound 7i. People with chemical background might be confused when looking at the chemical structures and abbreviations presented in the Table 1.

Abstract – please rewrite the sentence “They demonstrate a high level of success in limiting new relapses and accumulation of focal lesions using magnetic resonance imaging” in the Abstract (Lines 21-22).

Abstract – mitochondrial disfunction is considered to play a significant role in the development of MS (line 24). However, no specific S1R-dependent mitochondria function-related assays were performed in the present study. Therefore, the abstract should be rewritten.

Figure 3 – since title of the axis are used only for the graphs located left, the values for the axis should also be identical (Figure A vs Figure C).

Please reconsider the use of the term “dose-dependent decrease” after i.p. in line 372-373, since no significant effect was observed.

Table 3 – please transfer Table S1 from the supplementary to the Table 3 in the main body of the text.