Cytotoxicity and Thermal Characterization Assessment of Excipients for the Development of Innovative Lyophilized Formulations for Oncological Applications
Round 1
Reviewer 1 Report
In this article, the authors described the characterisation and cytotoxicity of a panel of excipients suitable for freeze-drying. I understand the thermal characterisation that has been done; however, I’m struggling to understand the cytotoxicity assays, why the authors tested the reconstituted freeze-dried excipients at those concentrations, but there is no evidence of those excipients before freeze-drying? I think this should be addressed before accepting this article for publication at Processes. Please also find some comments:
· Line 100: physicochemical instead of physical-chemical.
· Line 101: the phrase that starts with ‘excipients can stabilise…’ sounds like a translation from the authors mother tongue rather than a phrase in English. Please rephrase to make it more fluent.
· Page 3: the two first paragraphs define stabilisers and bulking agents. It’d be useful if the authors provided some examples of each type of excipients.
· Section 2.1: the authors reported a wide number of excipients (four disaccharides, three aminoacids, a polymer, a polyol and a cyclodextrin) at three different concentrations (100 mg/ml, 10 mg/ml and 1 mg/ml). It is quite a lot of information to read, so it’d be useful if the authors could summarise all of this in a table.
· Section 2.2: not sure if the first paragraph is needed.
· Line 179: ‘lyophilization’ instead of ‘lyophlization’.
· Section 3.2: do the authors have any photographs of the cake appearance? It has been described but it is difficult to understand if it’s not visualised.
· Line 282: how did the authors know that the residual moisture content was below 2 %?
Author Response
In this article, the authors described the characterisation and cytotoxicity of a panel of excipients suitable for freeze-drying. I understand the thermal characterisation that has been done; however, I’m struggling to understand the cytotoxicity assays, why the authors tested the reconstituted freeze-dried excipients at those concentrations, but there is no evidence of those excipients before freeze-drying? I think this should be addressed before accepting this article for publication at Processes.
We thank the reviewer for the consideration of our work and we would like to clarify him/her why we have not tested the toxicity of the excipients before freeze-drying from the scientific literature it is recognized that excipient’s compatibility is related to the chemical-physical stability of the drug and of the excipient-drugs complex in solid dosage forms. Excipients may affect API stability in the dosage form through phenomena such as the ion-exchange, transformation of polymorphs, and the formation of eutectic or solid solutions. Most excipients contain water and physical state of water in an excipient rules its performance in the drug–excipient system. Presence of water in the solid-state systems has a significant impact on the stability, not only in causing the hydrolysis of drugs but also its participation as a reaction medium, and in increasing the plasticity and molecular mobility of the whole system [https://doi.org/10.1016/B978-0-444-53242-8.00006-0][ Heljo, V.P., Nordberg, A., Tenho, M. et al. The Effect of Water Plasticization on the Molecular Mobility and Crystallization Tendency of Amorphous Disaccharides. Pharm Res 29, 2684–2697 (2012). https://doi.org/10.1007/s11095-011-0658-4]. For this reason, we decided to quantify the excipients cytotoxicity evaluating it after having been subjected to all the possible chemical-physical modifications due to the various phases of the lyophilization process.
Please also find some comments:
- Line 100: physicochemical instead of physical-chemical.
We thank the reviewer; we fixed it in the main text.
- Line 101: the phrase that starts with ‘excipients can stabilise…’ sounds like a translation from the authors mother tongue rather than a phrase in English. Please rephrase to make it more fluent.
We rephrase it in the text. We thank the reviewer for the help in improving our manuscript.
- Page 3: the two first paragraphs define stabilisers and bulking agents. It’d be useful if the authors provided some examples of each type of excipients.
We added some examples of the two types of excipients in the main text.
- Section 2.1: the authors reported a wide number of excipients (four disaccharides, three aminoacids, a polymer, a polyol and a cyclodextrin) at three different concentrations (100 mg/ml, 10 mg/ml and 1 mg/ml). It is quite a lot of information to read, so it’d be useful if the authors could summarise all of this in a table.
We added a new Table 1 in the materials and method section.
- Section 2.2: not sure if the first paragraph is needed.
Since the reviewer had a concern that the first paragraph might be unnecessary, based on the fact that both we and the other reviewer agree to keep it, we decided to leave it in the main text ·
Line 179: ‘lyophilization’ instead of ‘lyophlization’.
Thank, we fixed the typo in the manuscript.
- Section 3.2: do the authors have any photographs of the cake appearance? It has been described but it is difficult to understand if it’s not visualised.
As requested we reported below three representative images of mannitol at 100, 10 and 1 mg/ml
- Line 282: how did the authors know that the residual moisture content was below 2 %?
We measured the residual moisture content with Karl Fisher titration.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Common: in the introduction section it is necessary to justify the selected range of concentrations studied, and in the discussion section to compare these concentrations with the expected plasma concentrations when drugs using these excipients, taking into account in vivo pharmacokinetics.
Below there are some comments.
11. In the discussion section, it is desirable to give the concentrations of some of the most used excipients in known preparations and compare them with those studied in the work , taking into account in vivo pharmacokinetics.
22. In Figures 2-5, black bars are not present everywhere. Even if the viability at 100mg/mL is zero, this should be displayed.
33. It is better to mark the reliability in the figures with brackets, and it is necessary to write in the note what does the red and black asterisks mean.
44. In some cases, the stimulation of cell proliferation was observed, but the authors do not explain or discuss this. It should be discussed.
Comments for author File: 
Comments.pdf
Author Response
Common: in the introduction section it is necessary to justify the selected range of concentrations studied, and in the discussion section to compare these concentrations with the expected plasma concentrations when drugs using these excipients, takinginto account in vivo pharmacokinetics.
We thank the reviewer for the opportunity she/he gave us to improve the manuscript. As requested, in the introduction of the revised manuscript we specified the reasons that prompted us to use the concentrations of excipients with which the tests were conducted. Furthermore, in the discussion section of the revised manuscript we compared the concentrations we tested in vitro and the ones of some excipients already used in commercial products. These results were comparable using the QIVIVE method (quantitative in vitro in vivo extrapolation) which allows the conversion from the in vitro to the in vivo dosage.
Below there are some comments.
- In the discussion section, it is desirable to give the concentrations of some of the most used excipients in known preparations and compare them with those studied in the work, taking into account in vivo
As requested, we updated the discussion section in the revised main text.
- In Figures 2-5, black bars are not present everywhere. Even if the viability at 100mg/mL is zero, this should be displayed.
We thank the reviewer for her/ his comment. We proceeded to highlight in the text thanks to the addition of table 1, that both for methionine and for isoleucine it was not possible to detect cytotoxicity at 100 mg/ ml concentration for their low solubility in water, respectively 57 mg/ml and 34 mg/ml.
- It is better to mark the reliability in the figures with brackets, and it is necessary to write in the note what does the red and black asterisks mean.
In accordance with the reviewer's request we modified the graphs and the related captions.
- In some cases, the stimulation of cell proliferation was observed, but the authors do not explain or discuss this. It should be discussed.
We thank the reviewer for pointing out that we had not referred to the proliferative effect observed for some treatments. This phenomenon has been commented in details in the discussion section of the revised manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors addressed all the comments accordingly
Author Response
We thank the reviewer for his/her appreciation of our work and for his/her profitable advice.
Reviewer 2 Report
The manuscript has been significantly improved.
There are formal remarks - in the new version, many words have been changed to the same ones, for example:
"2.1. Preparation of formulations to be lyophilizedlyophilized".
In addition, in the "References" section, both full and abbreviated names of the journals are listed.
Author Response
We thank the reviewer for the carefulness with which she/he reviewed our manuscript. Furthermore, we apologize for the inconveniences incurred with the tracks and for the error formatting some of the references.
