In Vivo Evaluation of the Acute Systemic Toxicity of (1S,2E,4R,6R,7E,11E)-Cembratriene-4,6-diol (4R) in Sprague Dawley Rats
, Oné R. Pagán 2, Yancy Ferrer-Acosta 1, Vesna A. Eterović 3 and Peter A. Ferchmin 3
Round 1
Reviewer 1 Report
The authors report a toxicological study on the tobacco cemranoid 4R. They adopted a single-dose toxicity approach. Gross necropsy, blood biochemistry, and histopathology data were also described in detail. My suggestion would be as follows:
Line 18: Omit “the maximum tolerated dose (MTD)” as the study didn’t come up with a toxic dose at which MTD can be determines.
What is the rationale behind selecting 6, 24, 98 mg/kg doses of 4R?
Line 29: Remove “cyclic” as the cembrane is already a macrocycle skeleton
Line 32: Nicotiana and Pinus should be italic.
Line 33. Nicotiana should be italic.
Line 77: “Polyethylene Glycol” should be polyethylene glycol.
Line 82: What is the significance of mentioning that “the Dose preparation was performed under yellow light”? Does the white light harm the compound? Please clarify, otherwise omit yellow light.
Line 101: please report the temperature in “°C” as it is more common worldwide than °F.
Line 102: ad libitum should be italic.
Line 152. Why didn’t the authors collect blood samples before dosing the 4R? then the comparison would be stronger.
Line 281: Omit “for its application as an antidote against AChE-I exposure in male and female SD rats” as the manuscript is only dealing with a toxicological parameter and not a pharmacological study.
Author Response
Response to Reviewer 1 Comments
Point1: Line 18: Omit "the maximum tolerated dose (MTD)" as the study didn't come up with a toxic dose at which MTD can be determines.
Response 1: We thank the reviewer for pointing this out. We have revised the manuscript and omitted the maximum tolerated dose (MTD). The sentences in the abstract and Conclusion section have been removed.
Point2: What is the rationale behind selecting 6, 24, 98 mg/kg doses of 4R?
Response 2: We agree with the reviewer that further elaborating on this point is needed. The 6 mg/kg concentration was used in animal models of Organophosphates exposure, Ischemic stroke, and Parkinson's disease and showed a neuroprotective effect. This study aimed to test the margin of safety of 4R with additional doses of 24 and 98 mg/kg.
The following sentence has been added to Methods 2.3. Single-Dose Toxicity Study Design:
4R was shown to have neuroprotective properties at 6 mg/kg in various models of neurodegenerative diseases 1-3. This study aims to test the margin of safety of 4R with much higher doses. (Line 113)
Point3: Line 29: Remove "cyclic" as the cembrane is already a macrocycle skeleton
Response 3: We have removed "cyclic."
Point4: Line 32: Nicotiana and Pinus should be italic.
Response 4: Please provide your response for Point 1. (in red)
Point5: Line 33. Nicotiana should be italic.
Response 5: Nicotiana tobacum and Pinus genera are corrected in Italic.
Point6: Line 77: "Polyethylene Glycol" should be polyethylene glycol.
Response 6: We agree and have updated
Point7: Line 82: What is the significance of mentioning that "the Dose preparation was performed under yellow light"? Does the white light harm the compound? Please clarify, otherwise omit yellow light.
Response 7: "Yellow light" was removed.
Point8: Line 101: please report the temperature in "°C" as it is more common worldwide than °F.
Response 8: We have fixed the error.
Point9: Line 102: ad libitum should be italic.
Response 9: We have fixed the error.
Point10: Line 152. Why didn't the authors collect blood samples before dosing the 4R? then the comparison would be stronger.
Response 10: We appreciate the reviewer's insightful suggestion and agree that it would have been useful to collect the blood samples before injecting 4R. However, the vehicle control group had the purpose of being the comparison for the effects of the compound.
Point11: Line 281: Omit "for its application as an antidote against AChE-I exposure in male and female SD rats" as the manuscript is only dealing with a toxicological parameter and not a pharmacological study.
Response 11: We agree and have omitted the sentence.
Reference:
- Ferchmin, P.A., Andino, M., Reyes Salaman, R., Alves, J., Velez-Roman, J., Cuadrado, B., Carrasco, M., Torres-Rivera, W., Segarra, A., Martins, A.H., et al. (2014). 4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration. Neurotoxicology 44, 80-90.
- Martins, A.H., Hu, J., Xu, Z., Mu, C., Alvarez, P., Ford, B.D., El Sayed, K., Eterovic, V.A., Ferchmin, P.A., and Hao, J. (2015). Neuroprotective activity of (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (4R) in vitro and in vivo in rodent models of brain ischemia. Neuroscience 291, 250-259.
- Hu, J., Ferchmin, P.A., Hemmerle, A.M., Seroogy, K.B., Eterovic, V.A., and Hao, J. (2017). 4R-Cembranoid Improves Outcomes after 6-Hydroxydopamine Challenge in Both In vitro and In vivo Models of Parkinson's Disease. Frontiers in neuroscience 11, 272.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The authors performed a single-dose toxicity study of (1S,2E,4R,6R,7E,11E)-cembratriene-4,6-diol (4R) in males and females of Sprague Dawley rats as a potential medicine against neurotoxic organophosphates. They found that subcutaneous injections of 4R at doses of 6, 24, or 98 mg/kg did not evoke significant changes in body weight, appearances and behaviour of the animals. Furthermore, the gross necropsy, as well as histopathological and biochemical analysis did not reveal any adverse effects related to a single 4R administration. On the other hand, several blood chemistry parameters and hematology were statistically different in subjects treated with 4R compared to vehicle controls, but these changes were rather inconsistent and considered clinically irrelevant. It was concluded that the MTD for 4R was greater than 98 mg/kg in rats. Generally this study has been well designed and conducted. The methods are sound and the results have been clearly described and presented in tables.
Specific comments:
- The main limitation of this study is that only one animal species has been used.
- A rationale for choosing the specific range of doses of 4R is missing. More basic information from other studies on the tobacco cembranoid toxicities, e.g. their LD50, should be given
- Table 1. Body weight and mortality of Sprague Dawley rats after the single dose 4R injection. Is the value 64.8±32.1 correct?
- Page 10: “for clinal application” (should be: clinical)
Author Response
Response to Reviewer 2 Comments
Point1: The main limitation of this study is that only one animal species has been used.
Response1: We agree with the reviewer that this is a limitation. We hope we will be able to test the compound on non-human primates in the near future.
Point2: A rationale for choosing the specific range of doses of 4R is missing. More basic information from other studies on the tobacco cembranoid toxicities, e.g. their LD50, should be given
Response 2: We agree with the reviewer that further elaborating on this point is needed. The 6 mg/kg concentration was used in animal models of Organophosphate exposure, Ischemic stroke, and Parkinson's disease and showed a neuroprotective efficacy. This study aimed to test the margin of safety of 4R with additional doses of 24 and 98 mg/kg.
The following sentence has been added to Methods 2.3. Single-Dose Toxicity Study Design:
4R was shown to have neuroprotective properties at 6 mg/kg in various models of neurodegenerative diseases 1-3. This study aimed to test the margin of safety of 4R in much higher doses. (Line 113)
To the best of our knowledge, studies on tobacco cembranoid toxicity have not been performed. Cembranoids seem to be highly tolerable in high doses.
Point3: Table 1. Body weight and mortality of Sprague Dawley rats after the single dose 4R injection. Is the value 64.8±32.1 correct?
Point3: We have fixed the error.
Point4: Page 10: "for clinal application" (should be: clinical)
Point4: We have fixed the error. Line 341
References:
- Ferchmin, P.A., Andino, M., Reyes Salaman, R., Alves, J., Velez-Roman, J., Cuadrado, B., Carrasco, M., Torres-Rivera, W., Segarra, A., Martins, A.H., et al. (2014). 4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration. Neurotoxicology 44, 80-90.
- Martins, A.H., Hu, J., Xu, Z., Mu, C., Alvarez, P., Ford, B.D., El Sayed, K., Eterovic, V.A., Ferchmin, P.A., and Hao, J. (2015). Neuroprotective activity of (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (4R) in vitro and in vivo in rodent models of brain ischemia. Neuroscience 291, 250-259.
- Hu, J., Ferchmin, P.A., Hemmerle, A.M., Seroogy, K.B., Eterovic, V.A., and Hao, J. (2017). 4R-Cembranoid Improves Outcomes after 6-Hydroxydopamine Challenge in Both In vitro and In vivo Models of Parkinson's Disease. Frontiers in neuroscience 11, 272.
Author Response File: Author Response.pdf
