Qualification of Human Liver Microsomes for Antibacterial Activity Screening of Drug Metabolites

Round 1

Reviewer 1 Report

Overall, the manuscript is interesting, and the data support the main conclusion.

Author Response

Dear Reviewer 1,

We highly appreciate your review of the manuscript and that you found the manuscript suitable for publication in its current form. Thank you for your time.

Sincerely,

Navid

Reviewer 2 Report

The authors have written a well-structured and thought article. The background of the introduction is sufficiently described. The experimental part and result and discussion are clearly described.

I do not have any significant suggestions. I only have a few minor comments:

-  Staphylococcus aureus (e.g. page 1, line 19) should be written in italics,

-  please unify the symbol for degrees celcius.

Author Response

Thank you for your positive comments on the manuscript. Please see below response for your review.

-  Staphylococcus aureus (e.g. page 1, line 19) should be written in italics,

This has been fixed - thank you.

-  please unify the symbol for degrees celcius.

The symbol has been unified throughout the mansucript. 

Reviewer 3 Report

Please see  attached file for comments.

Comments for author File: Comments.pdf

Author Response

Thank you for your positive review of the manuscript. Please see below the response to your comments:

1. Qualification of human liver microsomes for antibacterial activity 2 screening of drug metabolites – this has been added. Thank you.
2. 50 uL human liver microsomes (10 mg/mL, commercial) – additional details has been added.
3. Thank you for this point. Using a different cell line may produce additional metabolites that was not formed by microsomes. As the aim of the downstream experiment was to test the microsomal metabolites and their antibacterial activity, using Caco-2 cell line is out of the scope of this manuscript but will be an excellent experiment for future similar studies.
4. Thank you for pointing out about the matrix effect. We used LC-MS as a qualitative tool to demonstrate the utility of our in vitro metabolism system where we can see gradual decline of parent drugs and corresponding gradual formation of the major metabolites. As this is not done in a quantitative way, it will now be out of scope of this manuscript. Should the LC-MS assay be used quantitatively for assessing in vitro metabolism, matrix effect would have been evaluated. We hope the reviewer would agree with us in this and will support our efforts.
5. Percent and rate of parent depletion again were done in a qualitative manner to show that the in vitro metabolism is working. The major focus of this manuscript was to assess the quality of microsome and sterilize them so that the microsomal metabolite mix can be screened for antibacterial activity. Hence no other statistical analysis on metabolic performance was not performed and is out of scope at this moment. We hope the reviewer would see it affirmatively.
6. Gram staining: fixed. Thank you.
7. M/z for the control CYP substrates (not the control antibiotics) are listed on table 1, a column has been now added to table 1 for retention times. These control CYP substrates were used to confirm the metabolic performance of our in vitro metabolism system. A new figure (Figure 3_ for these control drugs and their proposed biotransformation with their m/z has been incorporated as well . Thank you for this suggestion.
8. Part of this work was – this has been fixed. Thank you!