Orally Administered N-Oleoyl Alanine Blocks Acute Opioid Withdrawal Induced-Conditioned Place Preference and Attenuates Somatic Withdrawal following Chronic Opioid Exposure in Rats

Round 1

Reviewer 1 Report

The present manuscript deals Orally administered N-Oleoyl alanine blocks acute opiate withdrawal induced-conditioned place preference and attenuates somatic withdrawal following chronic opiate exposure in rats.

The work was introduced well.

objective of the present study was explained well.

The methodology was explained well with enough details.

The methodology was found to appropriate for the present study.

The obtained results were presented well and discussed well.

The conclusion part found to be good and appropriate with the findings.

In Figure 2, the level of significance indicated was not clear and it should be corrected or presented like Fig. 1.

Author Response

Comment 1: The work was introduced well. Objective of the present study was explained well. The methodology was explained well with enough details. The methodology was found to appropriate for the present study. The obtained results were presented well and discussed well. The conclusion part found to be good and appropriate with the findings.
Response 1: We kindly thank the reviewer for such positive remarks on our manuscript.

Comment 2: In Figure 2, the level of significance indicated was not clear and it should be corrected or presented like Fig. 1.
Response 2:  We thank the reviewer for pointing out the lack of clarity.  We have modified the figure accordingly and have modified its description in the text and the legend.

Reviewer 2 Report

To increase the clinical translational appeal the authors have tested the hypothesis if oral route of administration of endogenous N-acyl amino acid 9 N-Oleoyl alanine (OlAla) compared to intraperitoneal injection (i.p.) would offer any benefits in rats. Based on previous studies, OlAla administration via i.p. route has been shown to be effective to reduce both the affective and somatic responses produced by opiate withdrawal in preclinical models. In this study the authors have utilized the same dose range as i.p. route (previous publications) and show that pretreatment at lower dose (5mg/kg) OlAla was effective in attenuating the somatic response such as heroin withdrawal induced-abdominal contractions and diarrhea. However, pretreatment with higher dose (20mg/kg) via oral route the effect was abolished. The authors also establish that orally administered OlAla (20mg/kg) prevented the establishment of an acute naloxone-precipitated MWD-induced conditioned place aversion in male rats.

Minor Comments:

Overall, the study adds promise on the translatability of administration routes for OlAla-based treatment. However, although discussed by the authors the study lacks few critical studies that would make this manuscript more impactful such as a comparison study of the pharmacokinetic profiles of both routes of administration and considering female rats in the study. 

Author Response

Comment 1: Overall, the study adds promise on the translatability of administration routes for OlAla-based treatment. However, although discussed by the authors the study lacks few critical studies that would make this manuscript more impactful such as a comparison study of the pharmacokinetic profiles of both routes of administration and considering female rats in the study. 
Response 1: We agree with the reviewer that these additional studies would strengthen the impact of our findings, however these questions are beyond the scope of our project. We thank the reviewer for pointing out that these limitations were mentioned in our discussion, and we have added several sentences to the limitations section to strengthen this part of the manuscript (see lines 301-313). 

Reviewer 3 Report

This manuscript described the effects of oral administration of N-oleoyl alanine (OlAla) in male rats on opiate withdrawal responses.  Since intraperitoneal OlAla previously showed useful effects on the symptoms followed by the opiate withdrawal, this study is probably meaningful.  To reduce the opiate withdrawal is one of the important problems in medical treatment.  Certainly, the authors’ results in Figures are slightly complicated, so that they are difficult to link focused discussion. However, they are possible to be a trigger for new opiate withdrawal remedies.  Therefore, this reviewer recommends this manuscript as an acceptable article in ''Psychoactives'', after the authors respond to the points, as described below. 

1.     Why did the authors choose heroin among opiates in this study. In the manuscript, the reasons were not described in detail.  They should add several comments about it. 

2.     In Figure 1, this reviewer is difficult to exactly understand what the results of 20mg/kg OlAla showed even after reading the discussion section.  Did the data result from certain bioactivities of OlAla in vivo? 

3.     This reviewer wonders how diarrhea would be induced by single administration of OlAla at various concentrations to male rats.  In general, fatty foods are known to sometimes induce diarrhea at least in human.  In addition, OlAla is a lipid derivative.  Considering them, this reviewer guesses that OlAla itself has a potential to enhance bowel motility and to cause diarrhea in proportion to the intake.  Reduction of diarrhea and abdominal contraction in ‘H-OlAla-5mg-N’ is thought to be truly significant. 

4.     In Figure 2, ‘Diahrrea’ of the vertical axis label should be changed to ‘Diarrhea’. 

5.     Many readers are perhaps difficult to match between the diagrams of Figures and the sentences of ‘Discussion’ and ‘Conclusion’ sections.  The authors should revise it.   

6.     In ‘Conclusion’ section, the authors should explicitly express the novelty of this study again. 

7.     This reviewer could not get clear images about Experiments 1 and 2.  The authors need to additionally insert easy-to-understand information such as illustrations, pictures, or sentences for readers. 

Author Response

Reviewer 3:

Comment 1: Overall, the study adds promise on the translatability of administration routes for OlAla-based treatment. However, although discussed by the authors the study lacks few critical studies that would make this manuscript more impactful such as a comparison study of the pharmacokinetic profiles of both routes of administration and considering female rats in the study. 
Response 1: We agree with the reviewer that these additional studies would strengthen the impact of our findings, however these questions are beyond the scope of our project. We thank the reviewer for pointing out that these limitations were mentioned in our discussion, and we have added several sentences to the limitations section to strengthen this part of the manuscript (see lines 301-313). 

Comment 2: Why did the authors choose heroin among opiates in this study. In the manuscript, the reasons were not described in detail.  They should add several comments about it. 
Response 2: The reviewer raises an excellent point not previously described in our manuscript. Our previous work has detailed that OlAla (and OlGly) reduce withdrawal responses following morphine or heroin exposure. Our published reports, and the current experiments, used heroin for chronic opioid exposure via minipump given the solubility limits for the appropriate doses of morphine via minipumps, and to increase the translational appeal of our findings to commonly misused recreational opioids (i.e. heroin). We have now added a sentence in the introduction of the manuscript to explain our selection (see line 85-88).

Comment 3: In Figure 1, this reviewer is difficult to exactly understand what the results of 20mg/kg OlAla showed even after reading the discussion section.  Did the data result from certain bioactivities of OlAla in vivo? 
Response 3: We thank the reviewer for letting us know that our CPA result descriptions were unclear. We have now made several adjustments to the manuscript to account for this (see lines 194-195, 198-201, 239-242). In brief, the rats that received oral VEH exhibited an acute naloxone-precipitated morphine withdrawal induced conditioned place avoidance, as demonstrated by less time spent on the MWD-paired floor. This was expected given our acute naloxone precipitated MWD regimen consistently produces this behavior. Rats that were pretreated with 5 mg/kg oral OlAla group also exhibited this avoidance of the MWD-paired floor, but not rats administered 20 mg/kg oral OlAla. We have already published several experiments demonstrating that OlAla does not affect MWD-induced conditioned place avoidance on its own (Ayoub et al. 2020; Ayoub et al. 2021). Therefore, we concluded that a higher 20 mg/kg dose of oral OlAla was necessary to prevent this withdrawal behavior. This was a higher dose than was necessary to prevent this withdrawal behavior when OlAla was delivered by intraperitoneal injection, and possible reasons for this are mentioned in the discussion of the manuscript.

Comment 4: This reviewer wonders how diarrhea would be induced by single administration of OlAla at various concentrations to male rats.  In general, fatty foods are known to sometimes induce diarrhea at least in human.  In addition, OlAla is a lipid derivative.  Considering them, this reviewer guesses that OlAla itself has a potential to enhance bowel motility and to cause diarrhea in proportion to the intake.  Reduction of diarrhea and abdominal contraction in ‘H-OlAla-5mg-N’ is thought to be truly significant. 
Response 4: We believe there is a misunderstanding here that we have tried to correct in the manuscript (see method, results and figure caption for Figure 2).  Diarrhea was produced by naloxone-precipitated withdrawal from heroin (Group HVN) when compared with the saline control group (Group SVN).  It was not produced by OlAla.  Group H-5 mg/kg OlAla-N showed suppressed diarrhea compared with Group HVN, suggesting that it reduced this heroin withdrawal behavior.  We have tried to clarify our method, results and discussion.

Our earlier work with intraperitoneally administered OlAla has demonstrated that OlAla does not produce any effects on our recorded behaviors of interest when administered alone (Ayoub et al. 2020; Ayoub et al. 2021), and has never produced diarrhea. OlAla also has no effect on body temperature, nociception and activity levels in rats (Rock et al. 2021).

Comment 5: In Figure 2, ‘Diahrrea’ of the vertical axis label should be changed to ‘Diarrhea’. 
Response 5: Thank you for catching this error. The axis has been changed.

Comment 6:  Many readers are perhaps difficult to match between the diagrams of Figures and the sentences of ‘Discussion’ and ‘Conclusion’ sections.  The authors should revise it.   
Response 6: To enhance the clarity of our observed results and interpretations we have altered the figure legends of both graphs and text within the discussion and conclusions section.

Comment 7: In ‘Conclusion’ section, the authors should explicitly express the novelty of this study again. 
Response 7: We have now inserted a sentence into the conclusion section that clearly states the novelty of our manuscript. We thank the reviewer for the suggestion. (see lines 315-316)

Comment 8: This reviewer could not get clear images about Experiments 1 and 2.  The authors need to additionally insert easy-to-understand information such as illustrations, pictures, or sentences for readers. 
Response 8: We thank the reviewer for giving us the opportunity to enhance the clarity of our work. To address this comment we have altered the figure legends of both graphs and have clarified text within the discussion and conclusions section. We have also inserted a figure to better describe the place conditioning procedure used in our experiments (see lines 159-166). If further clarification is needed, we kindly ask the reviewer to describe what remains unclear.