Acute Toxicity Evaluation of Non-Innocent Oxidovanadium(V) Schiff Base Complex

Round 1

Reviewer 1 Report

Despite the changes made, I maintain that this report lacks originality/novelty and should not be published in Inorganics.

Author Response

Reviewer 1

We would like to thank the reviewer for the suggestions and appreciate the opportunity to improve our manuscript. Furthermore, we have done our best to improve the English writing and the manuscript has been now carefully revised. The changes made in the manuscript are described below as well as being highlighted in the manuscript for the convenience of the reviewer.

Considering that toxicity is a critical property of compounds and the fact this is one of the first reports on the toxicity of vanadium compounds and the very first oxidovanadium(V) Schiff base coordination compound, this topic indisputably is innovative. From that point of view, we believe this study is scientifically important and is not necessary that the compounds are new. What is however important is the fact that the compounds have anticancer properties superior to cisplatin.

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper can be accepted in present form.

Author Response

Reviewer 2

We would like to thank the reviewer for recognizing the value of our manuscript. Furthermore, we have done our best to improve the English writing and the manuscript has been now carefully revised. The changes made in the manuscript are described below as well as being highlighted in the manuscript for the convenience of the reviewer.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

1. The authors need to restructure and shorten the introduction section which includes repetitive statements of why it is important to evaluate the acute toxicity of a compound in vivo
2. The authors claim improvement of the synthetic procedure in the scaling-up, in previous reports by the group degassed solvent, nitrogen atmosphere and at least 24 h reaction time has been mentioned for very closely related products [77] and the reported yield is of the same order of magnitude. Hence, the degree of inorganic novelty is very limited. I would suggest that the manuscript would fit better in an MDPI toxicology journal.
3. There are still plenty of syntactic and grammatical errors which make the manuscript difficult to read. Moreover typos and spaces need to be corrected. 
4. Although there is a clear observation based on previous work [19, 56] about the hydrolysis of the complex and the Schiff base ultimately leading to the formation of the aldehyde and the amine still there is no experimental evidence on the toxicity of either (HSHED) or the aldehyde or the amine. Please explain and provide some data even by mentioning relevant refs of the non toxic nature of the components.
5. Line 258: "However, our results showed an elevated level of creatinine, which is suggestive of some hepatic toxicity issues with the catechol group. Indeed, both [VO(HSHED)dtb] and 3,5-di(t-butyl)catechol was rated as the same degree which LD50 values between 300 and 2000mg/kg (category 4)." Please discuss this observation a bit more connecting it to ref[77]. It is not really clear what category 4 is on what scale/ranking. 

Author Response

Reviewer 3

We would like to thank the reviewer for the suggestions and appreciate the opportunity to improve our manuscript. We have responded to the reviewer by addressing the points highlighted and all suggestions were addressed by the authors. Furthermore, we have done our best to improve the English writing and the manuscript has been now carefully revised. The changes made in the manuscript are described below as well as being highlighted in the manuscript for the convenience of the reviewers.

1.The authors need to restructure and shorten the introduction section which includes repetitive statements of why it is important to evaluate the acute toxicity of a compound in vivo.

The introduction has been restructured and shortened.

2.The authors claim improvement of the synthetic procedure in the scaling-up, in previous reports by the group degassed solvent, nitrogen atmosphere and at least 24 h reaction time has been mentioned for very closely related products [77] and the reported yield is of the same order of magnitude. Hence, the degree of inorganic novelty is very limited. I would suggest that the manuscript would fit better in an MDPI toxicology journal.

The target compound has been previously reported, however, the scientific interest in this vanadium Schiff base complex has been increased because of its biological effects as an antitumoral agent. Since there is no study with a focus on the toxicity of the vanadium-Schiff base catecholate complexes in vivo and in vitro, the paper is of interest in the context of the development of vanadium-based medicaments.  Furthermore, this manuscript is submitted to a special issue entitled “New Trends on Vanadium Chemistry, Biochemistry, and Medicinal Chemistry” which title suggests that articles with an interdisciplinary content are to be presented.

3.There are still plenty of syntactic and grammatical errors which make the manuscript difficult to read. Moreover, typos and spaces need to be corrected. 

The language and English writing have been carefully revised.

4.Although there is a clear observation based on previous work [19, 56] about the hydrolysis of the complex and the Schiff base ultimately leading to the formation of the aldehyde and the amine still there is no experimental evidence on the toxicity of either (HSHED) or the aldehyde or the amine. Please explain and provide some data even by mentioning relevant refs of the non-toxic nature of the components.

We have now addressed this question in two ways. We recorded the stability of the compound in the presence of Tween as used during the oral gavage treatment, and this data is now included as a Figure 2 in the manuscript.  We saw surprisingly that the Tween 80 resulted in an immediate stabilization of the VO(HSHED)dtb suggesting that the compound would stay intact for some time after administration. We did not measure if the ligand hydrolysis products would be toxic. Previous works have been reported that simple aldehyde and amine-based compounds are safe and used for designing new drugs. We have now added references to this fact as you requested. In contrast, catechols are known to be more toxic.  Considering the expense associated with these types of studies, we chose to test the toxicity of the most toxic part of the ligand.  In addition, we have now modified our discussion including this point so that issue is not ignored.

5.Line 258: "However, our results showed an elevated level of creatinine, which is suggestive of some hepatic toxicity issues with the catechol group. Indeed, both [VO(HSHED)dtb] and 3,5-di(t-butyl)catechol was rated as the same degree which LD50 values between 300 and 2000mg/kg (category 4)." Please discuss this observation a bit more connecting it to ref[77]. It is not really clear what category 4 is on what scale/ranking. 

We have modified the discussion as suggested by the reviewer in response to this comment. We also corrected a typo. In addition, it clear that the values showed a slight increase of creatinine levels in animals treated with 3,5-di(t-butyl) catechol, which are still within reference values. We have also added a discussion about the categories of toxicity on page 5, as described bellow “The classification of the test substance is based on the determination of the mortality dose(s), when there are no effects observed this concentration will be the highest dose tested by gavage in a single dose. Thus, the compounds could be rated as categories 1 to 5, from the most to less toxic, according to the LD₅₀ (median lethal dose) values in mg/kg b.w from each category [60]. [VO(HSHED)dtb] and 3,5-di(t-butyl)catechol was found to be category 4, with median lethal dose LD₅₀ estimated to be between 300 mg/kg and 2.000 mg/kg b.w, and orthovanadate is more toxic than both (category 3) with LD₅₀ estimated to be between 50mg/kg and 300 mg/kg b.w.”.

 

 

Author Response File: Author Response.pdf

Reviewer 4 Report

see attached

Comments for author File: Comments.pdf

Author Response

Reviewer 4

We would like to thank the reviewer for the suggestions and appreciate the opportunity to improve our manuscript. We have responded to the reviewer by addressing the points highlighted and all suggestions were addressed by the authors. Furthermore, we have done our best to improve the English writing and the manuscript has been now carefully revised. The changes made in the manuscript are described below as well as being highlighted in the manuscript for the convenience of the reviewers.

Line 15: “non-exhibiting of toxic properties” ??

We have modified this sentence.

Lines 39/40 and 41-43: The issues addressed here lack clarity; reformulation afforded

Those paragraphs have been rewritten.

Lines 48/49: “anti-cancer labelled” ?; – “metil” ? Throughout the manuscript: Replace oxovanadium for oxidovanadium (following IUPAC regulations for nomenclature). Line 50: ethylenediamine (instead of ethanediamine)

Those typo writing were fixed in the manuscript. Also, we have replaced for oxidovanadium.

Line 51: “pheol-im” ? Line 126: ADME ??

L-Pheol-im referred to the L-phenylalaninol. ADME is the four-letter acronym for Absorption, Distribution, Metabolism and Excretion. We have added those abbreviations in the “List of abbreviations”.

 

Lines 109/110: What kind of cells are the authors referring to?

The cells used in these studies include breast, brain, bone and prostate cancer cells.

 

Lines 124-126 lack clarity. Line 58: “vanadium vanadium compounds” ? Line 68: “according with the animal model”: this formulation affords clarification. Line 78: “… are different than most …” ?? Lines 91-95: The statement/information provided here lacks clarity. Lines 129-133: lack clarity Line 138: Define (quantify) “high dose”. Lines 197-198: The statement “no experimental significant differences … support the interpretation that …” lacks clarity

We have now rewritten and included relevant and clearer discussion to support our interpretations.

Line 222: For dipicolinato complexes see also J. Gätjens et al., Eur. J. Inorg. Chem. 2006, 3575-3585

Thank you for this suggestion. We have included this reference in the manuscript.

Line 262: “The vanadate salt has been demonstrated insulin-enhancement”: Which vanadate salt? Also, those vanadium coordination compounds for which an antidiabetic effect has been noted, did not primarily enhance insulin production, but rather acted as phosphatase inhibitor (and thus mimicking the role of insulin).

The materials in the solid state was sodium orthovanadate, but this is converted to H₂VO₄^- when added to an aqueous environment at neutral pH. We have improved this sentence as suggested by the reviewer.

Line 325: Tween ?

We used Tween 80, a surfactant that facilitate the dissolution of the complex into water.  We added this information in the manuscript as well as added the data showing the stability of the compound under those conditions.

Line 377: “simple vanadium salt” is supposed to indicate the anion H2VO4- ?

We have now referred to the anion H₂VO₄^- as suggested. 

Line 388: The phrasing “… attributed to the redox properties obtained when combining …” is insufficiently clear. As far as I can see, the redox behavior of the complex has not been quantified. Further, it should have been possible to check whether the complex is sufficiently stable in aqueous solution.

We have changed the discussion including the data we just measured on the hydrolysis of the complex in aqueous solution and in Tween 80. We did not investigate the redox chemistry of the system. We have modified the paragraph and the abstract in response to this comment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have not addressed all queries made by the referees. The report should not be published.

Author Response

Reviewer 1
We have addressed all the queries made by the referees. Furthermore, we have done changes in the introduction and the results section to make it clearer as suggested by the reviewers. Also, we have added relevant references to improve the background and the quality of the paper. The English language has been now carefully revised by native speakers. We would like to thank the reviewer for the opportunity to improve our paper. The changes made in the manuscript are described below as well as being highlighted in the manuscript for the convenience of reviewers and the editor.  

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have adequately addressed all points of concern. Thank you

Author Response

We would like to thank the reviewer for recognizing the value of our manuscript.

Author Response File: Author Response.docx

Reviewer 4 Report

I recommend publication of the revised manuscipt in the present form

Author Response

We would like to thank the reviewer for recognizing the value of our manuscript.

Author Response File: Author Response.docx