Spectroscopic Measurement of Methylene Blue Distribution in Organs and Tissues of Hamadryas Baboons during Oral Administration

Round 1

Reviewer 1 Report

Kozlikina and co-worker have somewhat improved their submission entitled "Spectroscopic measurement of methylene blue distribution in organs and tissues of Hamadryas baboons during oral administration". While some issues have been addressed, other were not and finally new ones were introduced (see below). As the authors were not able to do a proper revision, the next revision must be checked again.

Figure 1: It is unclear why there is an arrow from the Lewis drawing of the LMB (with the abbreviation of "LMB" below the drawing) to "LMB".

Figure 1: The reagent above the arrow from the PS-MB cycle to LMB is wrong. Instead of H⁺ it should be H^-.

Ref. 2: the ending page is missing.

Ref. 4: Still incorrect: "Others" must be not be used to abbreviate the author list, instead "et al." should be used, if appropriate.

Ref. 12: The article number (194) instead of the page numbers should be mentioned.

Ref. 20: The "1," should be deleted in the reference.

In ref. 21 the "pp." should still be deleted.

Ref. 22: The article number instead of the page numbers should be mentioned.

Ref. 23 and 25 are identical!

Ref. 26: the article number should be mentioned.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The paper entitled "Spectroscopic measurement of methylene blue distribution in organs and tissues of Hamadryas baboons during oral administration" by E.I. Kozlikina, D.V. Pominova, A.V. Ryabova, K.T. Efendiev, A.S. Skobeltsin, N.S. Rudenko, O.G. Kulik, E. I. Muhametzyanova, D.D. Karal-ogly, G.A. Zhemerikin, D.V. Bulgin, A.A. Shiryaev, I.V. Reshetov and V.B. Loschenov reports spectral data on the interstitial distribution of the administered drug in endothelial tissues in primates.

Paper is interesting, nevertheless I’m not sure that “Photonics” is good journal for such kind of studies. Therefore, I recommend to reject and propose more specific journal. In MDPI it could be “Medicina”, “COVID”, “Pharmaceuticals”, “Pharmaceutics” and many others.

Nevertheless, I have few issues about manuscript:

• in introduction in my opinion could be described side-effects of methylene blue

• Figure 3 is unnecessary, it should be removed

• Is in literature available knowledge about MB metabolism? It should be mentioned this issue as a possible factor for low signal in some compartments

• An interesting additional studies could be (based on primary knowledge about the MB activity against SARS-Cov2) treat the surrogate of SARS-Cov2 virus in used animal model. It could exclude or confirm relationship between MB distribution and its activity in in vivo

• On the other hand I’m not sure targeted disease COVID-19 should be highline, the concentration of MB in lung (primary organ affected by virus) is relatively low

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The paper presents original results on Methylen Blue (MB) distribution in primates (babbons) organs in order to study the potential use of MB (oral administration) as a protection from autoimmune response typical of SARS-CoV-2 and as way for macrophage polarization (PDT effect). In the current world situation, such results are of particular interest especially since they are obtained on a primate animal model and are therefore complementary to results obtained on rodents.

I think this paper should be published in order to share such results with a big part of the international scientific community that currently focuses its efforts against SARS-CoV-2. Moreover, as a study studying interactions of light with biological tissues (to determine the localization of MB in tissues by fluorescence spectroscopy and imaging), the topic suits well the Photonics Journal and especially the « Tissue optics » special issue.

Please pay attention to the following minor comments that hopefully will help improve the text quality.

Authors mention the Methylen Blue maximum absorption spectra is set at 665 nm (line 56) : please justify why authors use a 632.8 nm-excitation instead of 665 nm (probably because the available device proposes only one 632.8 nm-excitation ray). At least provide the reader with the MB excitation spectra or with the excitation efficacy ratio at 665 versus 632.8 nm.

Please check the syntax throughout the entire paper in order to get rid of useless « of » like in the following sentence.

Line 143 and 144: […] fluorescence spectra in the 350-1000 nm wavelength range with a 3 nm-resolution.

Figure 3 is useless as it shows « empty » black and white boxes. Please replace the scheme by a picture of the setup. In the legend of Figure 3, can the expression « laser spectrum analyzer » be replaced by « spectrometer »?

Lines 146-148, in the description of the optical probe, please add the optical fiber core diameter. In general, please add more information about technical features and devices references (optical fibers, spectrometer, laser, etc.).

Line 150, please specify if the filter used is a high-pass filter and the reference of it : is it the same as the one menioned in line 206 LONG Pass-650 ? Which manufacturer was it bought from ? As said above, please add references.

Is the setup described in section 3. « Metrological characteristics of the equipment » the same as the one described in section 2.2 (LESA-01-Biospec)? If so, please describe the setup only once and specify it is used for both experiments. If not, please stress the differenecs between the two setups.

Please describe at least one in the text acronyms used as :

• Line 178 : T-PMT
• Line 198 : MLT-LCT-Intralipids

Line 233 : replace « of accumulation increase » by « after injection ».

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Kozlikina and co-worker have further improved their submission entitled "Spectroscopic measurement of methylene blue distribution in organs and tissues of Hamadryas baboons during oral administration".

The following issues must be fixed and then publication can proceed.

"Also, Peter C. and et al." should be "Also, Peter and et al."

"Alamdari D.H. and et al." should be "Alamdari and et al."

"Ticino, F.E. and et al." should be "Ticino and et al."

"by the group of Peter C. and et al." should be "by the group of Peter and et al."

Ref. 2: the ending page is 520 not 517.

Ref. 27: instead of "Alamdari, D.; et al." it should be "Alamdari, D.H.; et al."

Reviewer 2 Report

Authors addressed all issues from review. Paper can be published.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

(Also attached as a separate file. )

Review of article:

Spectroscopic measurement of methylene blue distribution in organs and tissues of Hamadryas baboons during oral administration

E.I. Kozlikina 1,2,  , D.V. Pominova 1, K.T. Efendiev 1,2, A.V. Ryabova 1, A.S. Skobeltsin 1,2, N.S. Rudenko 3, D.V. Bulgin 3, E. I. Muhametzyanova 3, D.D. Karal-ogly 3, A.A. Shiryaev 4, G.A. Zhemerikin 4, I.V. Reshetov 4 and V.B. Loschenov 1,2

The presented study aimed to measure the tissue distribution of methylene-blue drug in primates. Authors say that by revealing distribution, accumulation levels may help the investigations about methylene blue as a possible COVID-19 treatment. Methylene-blue would be a repurposed drug in this line because there are a few publications about its in vitro antiviral effects. Although the applied post-mortem fiber-optic spectroscopy can produce quantitative data and laser scanning microscopy can reveal accumulations in specified cells, in this experiment the number of data (1animal/treatment, 2 time points and 1 dosage) does not enough to find strong results. It is only a preliminary setup for further more interesting studies (if they want to investigate anti-COVID effect in monkeys later). In my opinion not really worth for publication at this stage although I appreciate the scientific values of this work.  

Detailed critics:

Methylene-blue has a very wide biological effect profile, it can bind to DNA, proteins, based on its redox character it can alter redox state of biological molecules (most importantly methemoglobin) and produce ROS that can be applied in tumour therapy. The article refer to the few paper in which an in vitro antiviral and even anti-COVID effect was revealed, but anti-COVID effect of methylene blue is not a generally accepted hypothesis. Methylene-blue is a clinically applied drug, but its current application is not an antiviral agent (except that it can be used as an antiviral sterilizing agent of transfused blood in a photosensitive form). Being a clinically applied drug its pharmacokinetics is well known https://monographs.iarc.who.int/wp-content/uploads/2018/06/mono108-06.pdf.

In my opinion the presented spectroscopic method is probably suitable for quantitative evaluation of methylene blue, but by using only 2 time points and 1 drug concentration it is not suitable to assess precise tissue distribution. I also have to mention that it is a post-mortem measurement. The results are not particularly interesting, because in most tissues methylene-blue levels showed very low levels and since it was orally administered it is not surprising that levels are higher along the gut. And along the gut the higher values showed unexpected deviations. Among the examined territories the gall bladder showed a high staining, but the picture showing this has a higher magnification (scale bar is 20 uM whereas 200 uM in others), so the comparison is uncertain to the reader.

Regarding laser scanning microscopy - The staining seems to be quite homogenous on sections and the applied low resolution does not show a clear picture for the evaluation of the accumulation of methylene blue. The authors tried to identify brighter spots as macrophages by CD86 staining. But CD86 is expressed on activated B and T cells, monocytes/macrophages, dendritic cells, and astrocytes as well. But the most important problem is that the background is nearly as strong as the labelled specific staining. It is also problematic that tissue section are from different regions of the organs at different time points. A minor problem is that the label on the top of picture is Alexa-488 and not anti-CD86 staining which would be more appropriate.

Although the starting point in this experiment is to help COVID-19 drug research it is very far from this aim and probably other methods are more suitable to assess in vivo antiviral effects. It is true that in some clinical cases clinicians used methylene-blue as a therapy, but not as a general anti-COVID agent, but to treat a rare symptom, methemoglobinemia, observed in some COVID patients, and even these case studies does not support a conspicous antiviral effect. Before demostration of in vivo antiviral effect I see no motive for measuring tissue distribution. But I appreciate the value of this study as developing technical background for certain studies, may be an intravenous injection would have been more informative if at all methylene-blue can accumulate really in some specific region or cell type.

Comments for author File: Comments.pdf

Reviewer 2 Report

Kozlikina and co-worker present in their submission to Pharmaceutics "Spectroscopic measurement of methylene blue distribution in organs and tissues of Hamadryas baboons during oral administration". The following issues must be addressed. A short re-evaluation will tell, whether the submission can eventually be accepted.

Bad English: "It is also shown that MB is not carcinogenic in vast bio-application" should be "It has been shown that MB is not carcinogenic [1,2]" Further, references 1 and 2 do NOT present any data for that conclusion.

Bad English: "MB oral administration safe dose for a human is a dose of medication in concentration < 2 mg/kg"

"the capability of MB to be both acceptor and donor of hydrogen" MB is not a hydrogen donor, but its reduced product LMB is one.

"By transferring two hydrogen atoms, for example, from nicotinamide adenine dinucleotide (NADH) or dihydroflavine adenine dinucleotide (FADH2) or an appropriate substrate, MB passes into LMB" Not two hydrogen atoms are required to transform MB into LMB, but rather one hydride anion. Therefore, also all 3 drawn reactions in Figure 1 involving LMB (e. g. LMB + ½ O2 -> MB + H2O) are incorrect.

The referee fails to understand, why baboons had to be sacrificed for such an exploratory study. E. g. the ratios in Figure 12 could have been much more easily and more precisely determined with the help of mice experiments.

The author lists in ref. 3, 4, 16 and 22 are incomplete. "Others" must be not be used to abbreviate the author list, instead "et al." should be used, if appropriate.

In ref. 21 no "pp." is necessary.

Reviewer 3 Report

The manuscript deals with the significance of spectral data on the interstitial distribution of the administered drug in endothelial tissues in primates. The work is interesting.

Some comments to improve the document:

1. In the abstract, the objective of the study is not clearly mentioned. Include some lines for this.
2. Fig 3. And line 276 word in vivo must be written in italics.
3. Include the bioethics committee approval number, where the use of primates as an animal model is verified
4. Line 111-112: Could you expand on the selection of primates as an animal model. Likewise, on the number of experimental animals
5. Line 165. Reference the standard method
6. No image of primate control found in spectral study and Immunofluorescence analysis
7. Why figure 12 is not presented in the results section? The first paragraphs of the discussion present results.
8. The discussion is poor. It does not present comparisons with other similar studies. Possibly other drugs or relevant aspects of accumulation in tissues.
9. The paper does not show limitations of the study
10. The conclusion is very long. It is not accurate. Show aspects that should be in the discussion.