Review History


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Summary

  • The initial submission of this article was received on February 29th, 2016 and was peer-reviewed by 3 reviewers and the Academic Editor.
  • The Academic Editor made their initial decision on March 17th, 2016.
  • The first revision was submitted on April 11th, 2016 and was reviewed by 3 reviewers and the Academic Editor.
  • The article was Accepted by the Academic Editor on May 3rd, 2016.

Version 0.2 (accepted)

· · Academic Editor

Accept

Thank you for your contribution to PeerJ. We are look forward to future contributions from you.

·

Basic reporting

The authors did a good job based on the comments for the revision of this manuscript.

Experimental design

The authors did a good job based on the comments for the revision of this manuscript.

Validity of the findings

The authors did a good job based on the comments for the revision of this manuscript.

Comments for the author

The authors did a good job based on the comments for the revision of this manuscript.

Reviewer 2 ·

Basic reporting

The authors have address most of reviewers' comments.

Experimental design

The authors have address most of reviewers' comments.

Validity of the findings

The authors have address most of reviewers' comments.

Comments for the author

The authors have address most of reviewers' comments.

Reviewer 3 ·

Basic reporting

This manuscript is well written and the result clear demonstrate their hypothesis

Experimental design

The experimental design is straightforward, and the data is clear.

Validity of the findings

The finding could also provide the evidence for the therapy development using CBD in the future.

Comments for the author

This study is clearly demonstrate the idea that CBD is not the substrate of ABC transporters, P-gp and Bcrp. The models they used support their hypothesis and the results could provide the in vivo evidence for future therapy development using CBD.

Version 0.1 (original submission)

· · Academic Editor

Major Revisions

This work used P-gp knockout Abcb1a/b (-/-), Bcrp knockout Abcg2 (-/-), combined P-gp/Bcrp knockout Abcb1a/b(-/-)Abcg2(-/-) and WT mice to examine brain transport of cannabidiol (CBD) and used risperidone and 9-hydroxy risperidone as “positive controls” by measuring their brain and plasma concentrations. In general, the work is well-designed and performed, and the use of knockout animals and LC-MS/MS analysis of drugs are plus. All three reviewers recognize the positive parts of the study but also raised a number of questions which need to be addressed. My additional comments are as follows:

1. Check statistics and use statistic symbols correctly:

Figure 2: “*p<0.05, **p<0.01, ***p<0.001 for comparisons between WT and knockout mice”. However, the way the data presented can be easily confused as differences between 1 h and 3 h. It should be the differences between WT and knockout mice at 1 h time points, and at 3 h time point. In this case the statistic symbol should be put on the individual column, rather covering two columns. To make the figures simple and to the point, *p<0.05 would be sufficient.

Figure 2: “##p<0.01 for comparison between P-gp knockout and P-gp/Bcrp combined knockout”. However, (1) from the Figure B with the label, such a difference is not evident, from Figure C, there are differences but without labels. (2) What are the purpose and significance for such a comparison?
.
Figure 3: “*p<0.05, **p<0.01, ***p<0.001 for comparisons between WT and knockout mice”. Again, the symbol should be labeled on individual columns, as compares to WT mice, not covering two columns easily confused as comparisons between 1 h and 3 h.
2. Since the drug quant illation is the main stone of this study. In reference method (Johnston et al., 2014), there is no representative chromatography presented. The representative pictures of cannabidiol (CBD), risperidone and 9-hydroxy risperidone are desired, as least as supplementary files.

3. Reference citation and list should follow PeerJ format (e.g, journal names, two and three author rules) throughout the manuscript.

·

Basic reporting

The authors present interesting data about distinct properties of CBD compared with other common antipsychotics and anticonvulsants. The authors report that ABC transporters P-gp and Bcrp do not influence the brain uptake of CBD, a novel antipsychotic and anticonvulsant drug which provide evidence for the favourable pharmacokinetic properties of CBD in the treatment of CNS disorders and help build the case for the development of CBD as a therapeutic agent. The manuscript is well written, has important messages, and should be of great interest to the readers. However, there are still some issues need to be revised. In general, I have some comments and suggestions to the authors listed below.

Experimental design

Introduction:
1. The introduction provides a good, generalized background of the topic that quickly gives the reader a clear purpose of this study. However, to make the introduction
more substantial, the author may wish to provide several references to substantiate the possible underlying mechanisms of anticonvulsant and antipsychotic effects.
2. Please describe more about ABC transporter protein family.
3. Add more description about properties of P-gp and Bcrp (eg. molecular structure).
4. “There is evidence that genetic variation in P-gp influences treatment response to
antipsychotic and antiepileptic drugs…” Are these genetic variations related to specific diseases? Please specify.
5. Are Bcrp will be upregulated similar to P-gp during epilepsy and schizophrenia?
6. “If CBD were to accumulate at greater levels in the brain of knockout animals, then this provides evidence that it is an ABC transporter substrate.” It is not accurate. It should be compared with wild-type animals under the same experimental conditions. There is possibility that the transport of CBD has nothing to do with ABC transporter.
7. It is necessary to add the summary, purpose and influence of your current research at the end of Introduction part.

Materials and methods:
Generally, the experimental apparatus is quite standard, and is appropriate for the study.
1. In Animal part, please add light condition in order to maintain day/light cycle.
2. Please add references substantiate a. s.c. injection for all of the drugs (why not i.v.?); b. blood collection method; c. separation of plasma from the blood.
3. What is the post-hoc for two-way ANOVA?

Validity of the findings

Results:
Basically, the description of Results section is too simple, more detailed information should be added into Results section. It is not good that Results section look likes figure legends.
1. Please add the chemical structures of THC, CBD, risperidone and 9-hydroxy risperidone.
2. In Figure 1 legend, is it means + SEM or means ± SEM? The same as Figures 2 and 3.
3. In Figure 2, panel A, it does not look like that Bcrp has significant difference from the wild-type from the bar graph.
4. In Figure 3, both panels B and C do not have brain/plasma ratios from 2h time point. Please be consistent with panel A.

Discussion:
1. “CBD has a growing list of distinctive properties to THC”, please list more.
2. Please list current method in order to avoid resistance from ABC transporters or other related transporters if applicable.
3. There are no limitations of the study discussed. Think about what are the major limitations that should be discussed in the manuscript.
4. Need to write future directions of current research.

Comments for the author

My recommendation:
Due to the suggestions listed above, I would suggest that this manuscript need to be revised with minor revision before getting accepted.

Reviewer 2 ·

Basic reporting

The paper applied LC-MS/MS analysis to measure CBD concentrations in blood and plasma in Pgp and BCRP knockout mice. The author concluded that CBD is not a substrate of Pgp and BCRP. CBD, as a drug for schizophrenia and epilepsy, is already being tested in clinical trials. The drug resistance has not been reported as a serious issue for CBD although it could be a problem for other epileptic drug candidates. Therefore, the significance of this study is less important. Also, the paper used only one assay- LC-MS to complete the study, which was not mechanistic, but rather descriptive, so it carries less weight.

Experimental design

There are a few questions regarding the figures.

1) In Figure 1 and 3, three time points were reported, but in Figure 2, only 2 time pointes were reported. Why?
2) In Figure 2, both compounds tested have been already established as ABC transporter substrates, so the findings were not novel. Since authors used them as positive controls, they should not be reported in a separate figure, but should be compared to CBD side by side.
3) In Figure, 3, the ratio of brain and plasma could be useful in some way, but all the values were already reported in previous figures. So, this figure is not new date and authors should do either way.

Validity of the findings

In general, I do not recommend publishing as Research Article since this paper presented negative results and data were not organized well. However, the journal may consider Short Communication if needed.

Comments for the author

The paper applied LC-MS/MS analysis to measure CBD concentrations in blood and plasma in Pgp and BCRP knockout mice. The author concluded that CBD is not a substrate of Pgp and BCRP. CBD, as a drug for schizophrenia and epilepsy, is already being tested in clinical trials. The drug resistance has not been reported as a serious issue for CBD although it could be a problem for other epileptic drug candidates. Therefore, the significance of this study is less important. Also, the paper used only one assay- LC-MS to complete the study, which was not mechanistic, but rather descriptive, so it carries less weight. There are a few questions regarding the figures.

1) In Figure 1 and 3, three time points were reported, but in Figure 2, only 2 time pointes were reported. Why?
2) In Figure 2, both compounds tested have been already established as ABC transporter substrates, so the findings were not novel. Since authors used them as positive controls, they should not be reported in a separate figure, but should be compared to CBD side by side.
3) In Figure, 3, the ratio of brain and plasma could be useful in some way, but all the values were already reported in previous figures. So, this figure is not new date and authors should do either way.

Reviewer 3 ·

Basic reporting

This is a clear and well written manuscript. The background info and literature citation are appropriate.

Experimental design

The experimental design is straightforward and the data is clear cut. Meanwhile the results from double knockout mice strengthen the finding.

Validity of the findings

This study well demonstrate the CBD is not the substrate for the P-gp and Bcrp transporters in vivo. The finding will help the usage of CBD in therapy in the future.

Comments for the author

Brzozowska and colleagues used P-gp, Bcrp and double knockout mice to demonstrate that CBD is not the substrate of these ABC transporters. This paper is well-written, the idea is clear and the discussion covers most of the concerns of the study. I have only minor concerns for consideration.
1. In the introduction line 106: It is therefore important to establish whether CBD is an ABC transporter substrate. Here the author proved that CBD is not the substrate for P-gp and Bcrp, but not sure how about other ABC transporter, CBD may have effects at different time point.
2. In the discussion section, the authors and other groups have found that the CBD could inhibit P-gp and Bcrp (line 268-270), although CBD is not the substrate of these transporters, but it could change the expression of these transporters. So, the authors may consider the long-term study for CBD treatment, and combine with resperidone, the result may similar to P-gp KO. Therefore, is CBD safe for long-term use in patients?
3. Spell out the THC in abbreviation and introduction sections.

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