Efficient Synthesis for Altering Side Chain Length on Cannabinoid Molecules and Their Effects in Chemotherapy and Chemotherapeutic Induced Neuropathic Pain

Round 1

Reviewer 1 Report

Comments: 

The article: Efficient Synthesis for Altering Side Chain Length on Cannabinoid Molecules and Their Effects in Chemotherapy is an interesting manuscript, since cannabinoids have emerged as potential therapeutic agents against several diseases.

However, and in spite of the fact that the topic and the work are interesting, the following points must be addressed prior to the publication. 

1-    Introduction. The introduction must be reformulated including some points.

A-    The introduction will be improved when including the reason why the cannabinoids are interesting for the treatments of pain and cancer. For that, authors could include the cannabinoids in clinical use for pain, and the preclinical studies of the cannabinoids against cancer. Also, the authors could include some information regarding the mechanism of action of the cannabinoids in each case. Another important aspect is the use of the in vitro colon cancer cells in this work research. It will useful to include update statistics data about colon cancer. 

B-    Including the chemical structure of the cannabinoids in clinical/preclinical stage. 

C-    I suggest adding a phrase indicating the importance of developing chemical entities for pain and cancer as well, focusing on the patients care.

2-    Result and discussion.

A-    The authors could create the supporting information document with the NMR and MS spectra of the 14-17 compounds. This information will be useful to stablish the chemical characterization of the compounds. For the complete chemical characterization and for the purity of the compounds it is important to include elemental analysis or high-resolution mass spectrometry HRMS of them. The authors did not include any of them. For this reason, I suggest carrying out the HRMS of the compounds and including this information in the manuscript instead of the MS results. The differences between calculated and experimental MS data are a little bit high.

B-    The authors could include some information about the solubility of the compounds. The amount of DMSO Is high. If the compounds are soluble enough the author could think in using less amount of DMSO in the future. 

C-    The animal experiment was well conducted. However, have the authors detected any signals of the toxicity of the synthetic compounds? Including some information regarding the in vivo/in vitro toxicity of the compounds will be important to the potential development of the new compounds 

Finally, change IC50 for IC₅₀ in the Line 336, and add comma instead of  dot in line 68. 

Author Response

1-    Introduction. The introduction must be reformulated including some points

A-    The introduction will be improved when including the reason why the cannabinoids are interesting for the treatments of pain and cancer. For that, authors could include the cannabinoids in clinical use for pain, and the preclinical studies of the cannabinoids against cancer. Also, the authors could include some information regarding the mechanism of action of the cannabinoids in each case. Another important aspect is the use of the in vitro colon cancer cells in this work research. It will useful to include update statistics data about colon cancer. An additional paragraph has been added to the introduction to discuss the role of cannabinoids on cancer growth and reducing pain.  Moreover, information on colon cancer statistics and the selection of these cells for evaluation have been incorporated.   

B-    Including the chemical structure of the cannabinoids in clinical/preclinical stage. We will, of course, defer to the editor on this issue. However, the three approved drugs are CBD, delta-9-THC and nabilone.  These common compounds represent the vast majority of preparations in ongoing clinical trials.  While there are several synthetic compounds under clinical development, they are not the subject of the present report and would seem better suited for a review article.

C-    I suggest adding a phrase indicating the importance of developing chemical entities for pain and cancer as well, focusing on the patients care. We have added a sentence to the introduction discussing this matter.

2-    Result and discussion.

A-    The authors could create the supporting information document with the NMR and MS spectra of the 14-17 compounds. This information will be useful to stablish the chemical characterization of the compounds. For the complete chemical characterization and for the purity of the compounds it is important to include elemental analysis or high-resolution mass spectrometry HRMS of them. The authors did not include any of them. For this reason, I suggest carrying out the HRMS of the compounds and including this information in the manuscript instead of the MS results. The differences between calculated and experimental MS data are a little bit high.  We thank the reviewer for this comment.  Additional analyses have been conducted and the spectral data have been added. 

B-    The authors could include some information about the solubility of the compounds. The amount of DMSO Is high. If the compounds are soluble enough the author could think in using less amount of DMSO in the future. We apologize for this confusion.  Information on the solubility of CBGV and CBG has been added to the results based upon prior literature.  We find below 0.5% DMSO the compounds do not stay in solution, and that colorectal cancer cells tolerate 1% DMSO very well.  We therefore prepare stock solutions in DMSO and then dilute the compounds to reduce the final DMSO concentration to 0.5% in cell culture medium.

C-    The animal experiment was well conducted. However, have the authors detected any signals of the toxicity of the synthetic compounds? Including some information regarding the in vivo/in vitro toxicity of the compounds will be important to the potential development of the new compounds.  We have not yet carried out Maximum Tolerated Dose (MTD) toxicity experiments with these new compounds, but we do know that CBG is well tolerated, as are other cannabinoids such as CBD and THC.  We plan to perform toxicity experiments as we continue to characterize the preclinical efficacy of these compounds. 

Finally, change IC50 for IC₅₀ in the Line 336, and add comma instead of  dot in line 68.  We have corrected these errors, and thank the reviewer for catching them.

Reviewer 2 Report

This study elucidates the synthesis and role of cannabinoids derivatives in chemotherapeutic use. 

My comments are as follows:

1. Abstract--Methodology-- These compounds were then tested (delete: to reduce) in an animal model of chemotherapeutic-induced neuropathic pain and cellular model of colorectal cancer.

The outcome of the experiment shall not be included in the Methodology. Please check throughout the manuscript.

2. Expand the results in the abstract - effective dose and name of compound(s)

3.State the source of cannabis plant obtained for this study with voucher no and herbarium (collector)

4. State the ethics approval reference no for animal study Methodology

5.  Do include region and state (location) for American Type Culture Collection (ATCC)

6. Justify the selected dose of  5 mg/kg cisplatin and 10 mg/kg compounds for animal study; and 10uM compound for in vitro study. Did the authors run a pilot study to determine single-dose toxicity testing?

7. For the cellular study, there is no indication of the effects of compounds as potential adjuvants to cancer therapy. It was a cytotoxicity based assays and their related findings. Were the cells treated with cisplatin?

8. Further, cellular study based on MTT assay alone is insufficient to demonstrate the anticancer/cytotoxicity effects of cannabinoids. Various parameters including Western blot and apoptosis analyses, and migration assay shall be included.

8. _{Similarly, only Von Frey test for the assessment of mechanical hyperalgesia in animal model of neuropathic pain is presented. Please also include hot-plate test for the evaluation of thermal hypoalgesia. The main title shall include "chemotherapy-induced neuropathic pain"}

Author Response

1. Abstract--Methodology-- These compounds were then tested (delete: to reduce)in an animal model of chemotherapeutic-induced neuropathic pain and cellular model of colorectal cancer. This change has been made.

The outcome of the experiment shall not be included in the Methodology. Please check throughout the manuscript.  Thank you for this comment, we have restricted the inclusion of results in the methods section (with exception of the chemistry that required description of the reaction products). 

2. Expand the results in the abstract - effective dose and name of compound(s) These changes have been made.

3.State the source of cannabis plant obtained for this study with voucher no and herbarium (collector).  We apologize for any confusion, and have added text clarifying that no plant material was used in our study.  Prior work by others have identified the side chain variants of THC and CBD from plant material (this work has been cited).  Given that CBG is often only found in trace amounts in the plant, the likelihood of isolating the side-chain variants from plant material is very small.  This was the reason for the developing the novel synthetic process highlighted in this paper and this has been clarified. 

4. State the ethics approval reference no for animal study Methodology. Thank you for noting this omission - the protocol number has been added.
5. Do include region and state (location) for American Type Culture Collection (ATCC). These have been added.
6. Justify the selected dose of 5 mg/kg cisplatin and 10 mg/kg compounds for animal study; and 10uM compound for in vitro study. Did the authors run a pilot study to determine single-dose toxicity testing? Thank you for this suggestion.  Previous studies in our laboratory identified 10 mg/kg CBG as the ideal dose for reducing CIPN in mice and we have cited this report.  The use of 5 mg/kg cisplatin is also widely used by numerous laboratories (including our own), and relevant references have been incorporated.
7. For the cellular study, there is no indication of the effects of compounds as potential adjuvants to cancer therapy. It was a cytotoxicity based assays and their related findings. Were the cells treated with cisplatin? This is an outstanding suggestion and these experiments would be of great interest and will be pursued in future studies. However, the focus of this paper was largely on the novel synthetic mechanism and preliminary studies establishing the biological importance of further characterization of the structural variants. 
8. Further, cellular study based on MTT assay alone is insufficient to demonstrate the anticancer/cytotoxicity effects of cannabinoids. Various parameters including Western blot and apoptosis analyses, and migration assay shall be included. We have confirmed the MTT data with trypan blue staining and, as mentioned above, a further examination of these novel molecules is planned for future studies. Our belief is that detailed exploration of molecular mechanisms (western blots, cell signaling, apoptosis/autophagy/necrosis, and cell migration assays) are beyond the scope of the present report (that already has six figures/tables).
9. Similarly, only Von Frey test for the assessment of mechanical hyperalgesia in animal model of neuropathic pain is presented. Please also include hot-plate test for the evaluation of thermal hypoalgesia. The main title shall include "chemotherapy-induced neuropathic pain". The reviewer recommends an important future path of study.  As mentioned above, we plan to further examine these molecules in future studies. They were not explored here as we simply sought to demonstrate that there were reasons to further explore the novel compounds.  We also note that the editorial office gave us only two weeks to return revisions which would have precluded any additional biology.  The title has been modified as suggested.

Round 2

Reviewer 1 Report

The authors have improved the manuscript with suggestions and recommendations. The last recommendation is regarding the HRMS of compound 15. Maybe It was a writing mistake.  There is a big difference between calculated and observed values (303.2326 -303.1941).    

Author Response

We thank the reviewer for noticing that the actual HRMS data is off more than anticipated from the calculated, we double checked the value reported and it is what was received from the analysis.    

Reviewer 2 Report

-

Author Response

We thank the reviewer for his/her time, no additional comment is necessary.