Insights into the Pharmacokinetics and In Vitro Cell-Based Studies of the Imidazoline I₂ Receptor Ligand B06

Round 1

Reviewer 1 Report

The manuscript describes the imidazoline I2 receptor new ligand B06. I felt this manuscript was very important to neurodegenerative disorder therapy. 
However, there are still some concerns to be addressed in the present form.

Major comments:
In Fig.7 using MTT assay, evaluation of neuroprotective effects was  insufficient. Apoptosis data (for example, cleaved-Caspase-3 or cleaved-PARP) should add.

Author Response

We agree with you that additional data is crucial to confirm our results on neuroprotection. As a result of your advice, we have performed an apoptosis study using two different methods: immunofluorescence analysis to measure active caspase 3 expression, and western blot analysis to quantify active caspase 3 levels.

New figure 7 has now included in the revised manuscript and new information has been added as following:

• Page 10, line 353 “For a more in-depth analysis, the level of active caspase 3 was used to determine apoptosis (Figure 7c-d). Qualitatively, our results indicated an increase in the number of SH-SY5Y cells expressing active caspase 3 within 16 h after treatment with 6-OHDA and that this effect was reversed by the treatment with B06. A western blot analysis was used to confirm and quantify these results (Figure 7d).”
• Page 11, line 393,in figure 7 caption “Apoptotic levels were determined by active caspase 3 (green) immunodetection (c) and western blot quantification (d). Representative images of at least three independent experiments are shown. Scale bar, 10 µm. Nuclei were counterstained with DAPI (blue). ***p<0.001 versus 6-OHDA-treated cells. ###p<0.001 versus control (basal) cells.”
• Page 28, line 679, material and methods section: “. To evaluate the extend of apoptotic cell death, SH-SY5Y cultures were grown on glass cover-slips in 24-well cell culture plates for immunocytochemical analysis or in P60 plates for immunoblotting. After treatment, cover-slips were washed, permeabilized and treated as previously described [41]. A rabbit anti-active caspase-3 (1∶200, MAB835 R&D Systems) primary antibody was used followed by incubation with a 488-Alexa-labeled secondary antibody (Invitrogen, San Diego, CA). Images were acquired using a Radiance 2100 confocal microscope (Bio-Rad, Hercules, CA). Immunoblot analysis was performed as previously described [47]. Blots were probed with anti-human active caspase-3 (1∶1000, MAB835 R&D Systems). Secondary peroxidase-conjugated donkey anti-rabbit antibody was purchased from Amersham Biosciences (GE Healthcare, Buckinghamshire, England). Representative images of at least three independent experiments corresponding to three different samples are shown. The images of blotting were quantified using ImageJ software (Wayne Rasband, NIH, Bethesda, MD). Values in the text are the mean of at least three different experiments.”

We are grateful to this reviewer for her/his comment, which helped to complete our study.

Reviewer 2 Report

The article by Bagan et al is an extensive multidisciplinary study, undoubtedly worthy of publication. The problems solved and the combination of methods on the one hand are impressive, but on the other hand the impression remains that the format of the technical data for the publication of the article is clearly excessive. The Materials and Methods section is poorly written, containing a fair amount of inconsistent sentences, unnecessary repetitions, a huge amount of technical details, not to mention errors and misprints. It should be considerably reworked into a readable text format rather than a technical protocol, remove repetitions, and perhaps partially move it to the Supplement. 
For example, the phrase "NADPH solution: A stock solution of 2.66 mM NADPH was prepared by dissolving 577
appropriate amount of NADPH in 100 mM potassium phosphate buffer." does not require repetition, the NADPH solution at the beginning should be removed.  
line 569 - NADPH, 1.3 Mm instead of 1.3 mM
line 571 - Cl2Mg and NaDPH instead of NADPH and MgCl2
line 643 - at 0.2, 0.5, 1, 2, 5, 10 y 20 µM.
The list of abbreviations should be reviewed, technical and perfectly classic abbreviations can be left directly where they are used or full versions can be used.

Author Response

Dear reviewer,

Taking into consideration your suggestions we have partially move technical details of the Material and Methods section to the Supporting Information Section, we have corrected the mistakes you indicated and revised all the manuscript.

In detail:

3. The 3.2. point has been modified for a more concise information detail.
4. The paragraphs 3.4. Metabolic stability of B06 in human liver microsomes and 3.5. Metabolic profiling of B06 in human and mouse liver microsomes are now included in the Supporting Information Section Pages S1 and S3.
5. The phrase indicated by the reviewer in line 577 “NADPH solution: A stock solution of 2.66 mM NADPH was prepared by dissolving appropriate amount of NADPH in 100 mM potassium phosphate buffer." does not require repetition, the NADPH solution at the beginning should be removed”, is not in the revised manuscript.
6. The paragraph 3.6. Chemical synthesis included a general experimental condition that is now in the Supporting information to reduce the Material and Methods section in page S19.
7. We thanks to the reviewer for the detection of the following mistakes that are now being corrected: line 569 - NADPH, 1.3 Mm instead of 1.3 mM; line 571 - Cl2Mg and NaDPH instead of NADPH and MgCl2, and line 643 - at 0.2, 0.5, 1, 2, 5, 10 y 20 µM.
8. As the reviewer indicated the text contained errors and misprints that are now corrected and highlighted with the “Track Changes” function in the revised manuscript.
9. The list of abbreviations has been reviewed and some classic abbreviations removed.
10. The title 2.4. was “Metabolic profiling of B06 liver microsome” and now is “Metabolic profiling of B06 in liver microsome”.
11. The titles of the page S2 were “Table S3. Metabolic stability of B06 and verapamil in human liver” and “Figure S2. Time course of metabolic stability of B06 in human liver microsomes” and now is “Table S3. Metabolic stability of B06 and verapamil in human liver microsomes” and “Figure S2. Time course of metabolic stability of B06 in human liver microsomes”, respectively.
12. The title of the page S3 was “Table S7. Metabolic stability of B06 and verapamil in mouse liver” and now is “Table S7. Metabolic stability of B06 and verapamil in mouse liver microsomes”.
13. The title of the page S4 was “Figure S4. Time course of metabolic stability of B06 in mouse liver microsomes” and now is “Figure S4. Time course of metabolic stability of B06 in mouse liver microsomes”.
14. The titles of the page S5 were “Table S9. Metabolite time course in HLM incubations for B06” and “Figure S5. Metabolite time course in HLM incubations from 0 to 60 min for B06” and now are “Table S9. Metabolite time course in HLM incubations for B06” and “Figure S5. Metabolite time course in HLM incubations from 0 to 60 min for B06”, respectively.
15. The titles of the page S6 were “Table S10. Metabolite time course in MLM incubations for B06” and “Figure S6. Metabolite time course in MLM incubations from 0 to 60 min for B06” and now are “Table S10. Metabolite time course in MLM incubations for B06” and “Figure S6. Metabolite time course in MLM incubations from 0 to 60 min for B06”, respectively.
16. The titles of the page S7 were “Table S11. Metabolite time course in vivo incubations for B06” and “Figure S7. Metabolite time course in HLM incubations from 0 to 60 min for B06” and now are “Table S11. Metabolite time course in vivo incubations for B06” and “Figure S7. Metabolite time course in HLM incubations from 0 to 60 min for B06”, respectively.
17. The titles of the page S8 were “Table S12. Specificity of B06 and internal standard in mouse plasma samples”, “Table S13. Auto-sampler carry over test of B06 and internal standard in mouse plasma samples”, “Table S14. Back calculated values (ng/ml) data of cc standard for B06 in mouse plasma samples” and “Table S15. Accuracy and precision of B06 in mouse plasma samples” and now are “Table S12. Specificity of B06 and internal standard in mouse plasma samples”, “Table S13. Auto-sampler carry over test of B06 and internal standard in mouse plasma samples”, “Table S14. Back calculated values (ng/ml) data of cc standard for B06 in mouse plasma samples” and “Table S15. Accuracy and precision of B06 in mouse plasma samples”, respectively.
18. The titles of the page S9 were “Figure S8. Blank sample chromatogram of B06 and internal standard in mouse plasma samples” and “Figure S9. Chromatogram of B06 and internal standard in lloq sample in mouse plasma samples (concentration 5 ng/ml)” and now are “Figure S8. Blank sample chromatogram of B06 and internal standard in mouse plasma samples” and “Figure S9. Chromatogram of B06 and internal standard in lloq sample in mouse plasma samples (concentration 5 ng/ml)”, respectively.
19. The title of the page S10 was “Figure S10. Standard curve of B06 in mouse plasma samples” and now is “Figure S10. Standard curve of B06 in mouse plasma samples”.
20. The titles of the page S11 were “Table S16. Specificity of B06 and internal standard in mouse brain samples”, “Table S17. Auto-sampler carry over test of B06 and internal standard in mouse brain samples”, “Table S18. Back calculated values (ng/ml) data of cc standard for B06 in mouse brain samples” and “Table S19. Accuracy and precision of B06 in mouse brain samples” and now are “Table S16. Specificity of B06 and internal standard in mouse brain samples”, “Table S17. Auto-sampler carry over test of B06 and internal standard in mouse brain samples”, “Table S18. Back calculated values (ng/ml) data of cc standard for B06 in mouse brain samples” and “Table S19. Accuracy and precision of B06 in mouse brain samples”, respectively.
21. The titles of page S12 were “Figure S11. Blank sample chromatogram of B06 and internal standard in mouse brain samples” and “Figure S12. Chromatogram of B06 and internal standard in LLOQ sample in mouse brain samples (concentration 2.5 ng/ml)”, and now are “Figure S11. Blank sample chromatogram of B06 and internal standard in mouse brain samples” and “Figure S12. Chromatogram of B06 and internal standard in LLOQ sample in mouse brain samples (concentration 2.5 ng/ml)”,”, respectively.
22. The title of page S13 was “Figure S13. Standard curve of B06 in mouse brain samples” and now is “Figure S13. Standard curve of B06 in mouse brain samples”.
23. The titles of page S14 were “Figure S14. Calibration curve for B06 in plasma samples” and “Table S20. Quantitation for B06 in plasma samples at dosing 10 mg/kg and samples grouping and statistics in plasma samples” and now are “Figure S14. Calibration curve for B06 in plasma sample” and “Table S20. Quantitation for B06 in plasma samples at dosing 10 mg/kg and samples grouping and statistics in plasma samples”, respectively.
24. The titles of page S15 were “Figure S15. B06 concentration in plasma samples vs. extraction time” and “Figure S16. Calibration curve for B06 in brain samples” and now are “Figure S15. B06 concentration in plasma samples vs. extraction time” and “Figure S16. Calibration curve for B06 in brain samples”, respectively.
25. The title of page S16 was “Table S22. Quantitation for B06 in brain samples at dosing 10 mg/kg and samples grouping and statistics in brain samples” and now is “Table S22. Quantitation for B06 in brain samples at dosing 10 mg/kg and samples grouping and statistics in brain samples”.
26. The title of page S17 was “Figure S17. B06 concentration in brain samples vs. extraction time” and now is “Figure S17. B06 concentration in brain samples vs. extraction time”.

 

 

Round 2

Reviewer 1 Report

The manuscript describes the imidazoline I2 receptor new ligand B06. I felt this manuscript was very important to neurodegenerative disorder therapy.

Minor comment:

1) Page 11: In Fig 7d, please change ‘‘Active 3-caspase’’ to ‘‘Active caspase-3’’

Author Response

Dear Reviewer,

Thank you for the correction of the mistake in the Figure 7d, that is now active caspase-3.

"1) Page 11: In Fig 7d, please change ‘‘Active 3-caspase’’ to ‘‘Active caspase-3’’".