Synthesis of [¹¹C]BIIB104, an α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic-Acid-Positive Allosteric Modulator, and Evaluation of the Bio-Distribution in Non-Human Primate Brains Using Positron Emission Tomography

Round 1

Reviewer 1 Report

I thought this was a well organized and interesting manuscript.  Thank you for sharing your work.  I had a couple questions or requests for added information to be added to select sections.

1) In Section 2.1.4 can you clarify the timing of the image being taken compared to when the injection took place?

2) In the results/discussion section there is a paragraph about the optimization of the radiosynthesis process but this was not discussed in the experimental section.  Please add to experimental section, or cite as appropriate.

3) There is a conclusion that there was not significant different in the blocked vs. unblocked uptake, but in Figure 2 it looks like there is some blocking in NHP1 at early times, but not the similar observation in NHP2, what is the proposed difference in behavior?

4) In Figure 1 can you add the time post-injection that these images represent?

5) In the conclusion section it says the measurements in cynomolgus monkeys- should this be Rhesus monkeys as stated in Section 2.1.4?  Either way, please verify which is correct.

6) It was unclear to me if the C-11 labeling technique was new/novel for the BIIB104, it would appear that way since the first conclusion is that the radiolabeling technique was successful, but can you clarify that in the introduction or discussion section (whichever makes sense depending on the answer)?

Consistent use of subscripts needs to be checked.  Also general grammar in section 2.1.3.  Please check for use of radiation detector (correct) vs. radioactive or rad detector (incorrect).

In Scheme 1 it looks like the degree symbol is a superscripted O, please check and update if needed.

Author Response

I thought this was a well organized and interesting manuscript.  Thank you for sharing your work.  I had a couple questions or requests for added information to be added to select sections.

1) In Section 2.1.4 can you clarify the timing of the image being taken compared to when the injection took place?

Reply: PET measurement started immediately after injection of PET radioligand as described in P6 L24.

2) In the results/discussion section there is a paragraph about the optimization of the radiosynthesis process but this was not discussed in the experimental section.  Please add to experimental section, or cite as appropriate.

Reply: A new table (Table 1) is added with the detail information about the optimisation of the radiosynthesis.

3) There is a conclusion that there was not significant different in the blocked vs. unblocked uptake, but in Figure 2 it looks like there is some blocking in NHP1 at early times, but not the similar observation in NHP2, what is the proposed difference in behavior?

Reply: In NHP1, high brain uptake was observed in PET2, which was pretreatment condition. It often occurred by higher plasma concentration due to peripheral blocking. To avoid this confounding factor, VT values are used for accurate evaluation of brain uptake. Our present results showed no significant difference of VT between baseline and pretreatment conditions, so we concluded no blocking effect.

4) In Figure 1 can you add the time post-injection that these images represent?

Reply: “Images were averaged of total 93minutes just after PET radioligand injection”. It is added in the Figure 1 Legend.

5) In the conclusion section it says the measurements in cynomolgus monkeys- should this be Rhesus monkeys as stated in Section 2.1.4?  Either way, please verify which is correct.

Reply: Corrected to Rhesus monkeys.

6) It was unclear to me if the C-11 labeling technique was new/novel for the BIIB104, it would appear that way since the first conclusion is that the radiolabeling technique was successful, but can you clarify that in the introduction or discussion section (whichever makes sense depending on the answer)?

Reply: C-11 labelling was not new. It was performed following a previously published method (reference no 33 and 34) which is mentioned in the manuscript.

Reviewer 2 Report

The work titled "Synthesis of [11C]BIIB104, an AMPA Positive Allosteric Modulator and Evaluation of the Bio-Distribution in NHP Brain Using PET " is a valuable research work and after some modification and improvement could be acceptable for publication in the molecules Journal the findings seem valuable and suitable contribution to be published in the Molecules Journal after justifying some points.

·       The similarity rate relatively high and should be reduced in some paragraphs

·       Can you add the used method to confirm the synthesized compound [11C]BIIB104 to the abstract

·       The sections Results and discussion should be separated from the method section.

·       The methods section is generally clear, but additional details on the radiolabeling process and the rationale for selecting specific brain regions for PET scans would enhance clarity, and improve the quality of the paper.

·       To reduce the similarity rate you can use some recent wroks regarding the importance of AMPA receptors like “Biomolecules 2023, 13, 1694” and “https://doi.org/10.1007/s12035-022-02913-4” that could improve the importance of the AMPA subunits and the introduction section too.

·        The results highlight successful radiolabeling and brain uptake of [11C]BIIB104, with a notable mild regional heterogeneity in the thalamus. However, the discussion should delve deeper into the implications of these findings, addressing potential limitations and providing context for the observed lack of conclusive evidence for changes in regional VT values post-BIIB104 dosing.

·       It is recommended to write in vivo word should be in italic style through the manuscript

·       Almost all method section should be written again and rephrase them to reduce the similarity rate

·       It is not clear who did you confirmed the chemical structure of your final compound, for example by using HRMS or NMR with very high resolution or what ? and provide us with a the related spectrum

·       You have to uniform the Celsius symbol in the scheme like the text

·       The resolution of figures 2, 3 and 4 should be improved, we could not read the codes inside the figures

·       In the conclusion section, I recommend the authors provide a more nuanced discussion regarding the implications of the observed lack of significant changes in brain uptake after pretreatment with cold BIIB104. Instead of leaning solely towards the conclusion that [11C]BIIB104 may not exhibit strong specific binding to AMPA receptors, consider acknowledging the possibility that the dose of BIIB104 used for pretreatment might have been insufficient for a detectable reduction in specific binding.

Best wishes

Author Response

The work titled "Synthesis of [11C]BIIB104, an AMPA Positive Allosteric Modulator and Evaluation of the Bio-Distribution in NHP Brain Using PET " is a valuable research work and after some modification and improvement could be acceptable for publication in the molecules Journal the findings seem valuable and suitable contribution to be published in the Molecules Journal after justifying some points.

• Can you add the used method to confirm the synthesized compound [11C]BIIB104to the abstract.

      Reply: The identity of [¹¹C]BIIB104 was confirmed by co-injecting it with an authentic reference standard using an HPLC. This is added in the abstract section.

• The sections Results and discussion should be separated from the method section.

      Reply: Results and discussion section is separated from the method section.

• The methods section is generally clear, but additional details on the radiolabeling process and the rationale for selecting specific brain regions for PET scans would enhance clarity, and improve the quality of the paper.

Reply: A new table (Table 1) is added with the detailed information about the optimisation of the radiosynthesis.

We do not have a-priori assumption for brain regionality of this PET radioligand, so relatively broad regions were selected according to our laboratory standard procedures.

• To reduce the similarity rate you can use some recent works regarding the importance of AMPA receptors like “Biomolecules 2023, 13, 1694” and “https://doi.org/10.1007/s12035-022-02913-4” that could improve the importance of the AMPA subunits and the introduction section too.

      Reply: Updated accordingly and new reference 13 is added.

• The results highlight successful radiolabeling and brain uptake of [11C]BIIB104, with a notable mild regional heterogeneity in the thalamus. However, the discussion should delve deeper into the implications of these findings, addressing potential limitations and providing context for the observed lack of conclusive evidence for changes in regional VT values post-BIIB104 dosing.

      Reply: The following explanation is added to the result and discussion section. ``Generally, the brain regionality suggested that PET radioligand binds to target specifically, but that of the present study was relatively small. Additionally, VT did not change after pretreatment. These results suggested that [<sup>11</sup>C]BIIB104 may not have sufficient specific binding to AMPA receptors``.

• It is recommended to write in vivoword should be in italic style through the manuscript

      Reply: Corrected accordingly.

• Almost all method section should be written again and rephrase them to reduce the similarity rate

      Reply: Updated as recommended.

• It is not clear who did you confirmed the chemical structure of your final compound, for example by using HRMS or NMR with very high resolution or what? and provide us with the related spectrum

      Reply: The structural conformation was not done. The identity of the final product [¹¹C]BIIB104 was confirmed by co-injecting it with an authentic reference standard using an HPLC.

• You have to uniform the Celsius symbol in the scheme like the text

      Reply: Updated the Celsius as recommended.

• The resolution of figures 2, 3 and 4 should be improved, we could not read the codes inside the figures

      Reply: Figures 2, 3 and 4 are updated with higher resolution.

• In the conclusion section, I recommend the authors provide a more nuanced discussion regarding the implications of the observed lack of significant changes in brain uptake after pretreatment with cold BIIB104. Instead of leaning solely towards the conclusion that [11C]BIIB104 may not exhibit strong specific binding to AMPA receptors, consider acknowledging the possibility that the dose of BIIB104 used for pretreatment might have been insufficient for a detectable reduction in specific binding.

Reply: We agree with the reviewer. But as we performed PET measurement after pretreatment with a single dose of cold BIIB104 (0.0032 mg/kg), we could not conclude the lack of significant changes in brain uptake is due to too low dose of BIIB104. That’s why we acknowledged both the possibilities that [¹¹C]BIIB104 either may not exhibit strong specific binding to AMPA receptors or too low dose of BIIB104 as pretreatment used for detectable reduction of specific binding.

Round 2

Reviewer 2 Report

The authors improved the work with title "Synthesis of [11C]BIIB104, an AMPA Positive Allosteric Modulator and Evaluation of the Bio-Distribution in NHP Brain Using PET " but still some points need improving

·       Firstly, I should receive a clean version of the manuscript, without track changes, it is better to use the highlighted text

·       The “co-injecting” method to confirm the chemical structure or to identifying the [11C]BIIB104 should be discussed well in the manuscript

·       The HPLC spectrum should be provided in the supplementary file or in the main text too with the ref. standard

·       It was recommended to cite the recent work with title “Thiazole Derivatives as Modulators of GluA2 AMPA Receptors: Potent Allosteric Effects and Neuroprotective Potential” “Biomolecules 2023, 13, 1694” and “https://doi.org/10.1007/s12035-022-02913-4” which is considered important recent works on AMPA receptors which could improve the importance of the AMPA subunits and the introduction section too.

·       It is important to add the error bars to the figure 5, and state the statistical methods if used or not applicable in your work??

Best wishes

Author Response

• Firstly, I should receive a clean version of the manuscript, without track changes, it is better to use the highlighted text

Reply: It’s recommended by the journal to upload both clean and including track changed. We followed the Journal´s recommendation.

• The “co-injecting” method to confirm the chemical structure or to identifying the [11C]BIIB104 should be discussed well in the manuscript

Reply: The “co-injecting” method to confirm the identity of the PET radioligand is most common and well known. We have added the explanation as the supplementary information.

• The HPLC spectrum should be provided in the supplementary file or in the main text too with the ref. standard

Reply: The HPLC chromatograms are provided in the supplementary file.

• It was recommended to cite the recent work with title “Thiazole Derivatives as Modulators of GluA2 AMPA Receptors: Potent Allosteric Effects and Neuroprotective Potential” “Biomolecules 2023, 13, 1694” and “https://doi.org/10.1007/s12035-022-02913-4” which is considered important recent works on AMPA receptors which could improve the importance of the AMPA subunits and the introduction section too.

Reply: It is cited. Please see reference no 14.

• It is important to add the error bars to the figure 5, and state the statistical methods if used or not applicable in your work??

Reply: Not applied here.

Author Response File: Author Response.pdf