From the journal RSC Chemical Biology Peer review history

Berlin, 3 June 2024

Dear Dr Zaykov:

Manuscript ID: CB-ART-04-2024-000078
TITLE: Toward once-monthly insulin therapy via synergy in two pharmacokinetic protractors: Fc-conjugation and fatty acid acylation.

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

************

Reviewer 1

The authors have performed a very detailed and carefully executed study examining dual FC conjugation and lipidation of insulin molecules. The data are comprehensive, the results are well presented and the discussion is fair and balanced, highlighting the limitations and/or potential barriers of this approach moving forward. This study will make and important contribution to the field of peptide-proteins therapeutics as a whole so its significance and impact is not just limited to insulin per se. The authors are to be congratulated on this well designed and executed study.

Reviewer 2

The study by Zaykov et al. presents an innovative approach for generating ultra-long-acting insulin. By doubly protracting insulin with both Fc and fatty acids, they enabled a 20-day glucose-lowering effect in a mouse model for diabetes and observed similar effects in non-diabetic rats and dogs.
The main problem of insulin therapy is iatrogenic hypoglycemia. The data shown also indicated that this risk is present with the modified insulin. It is commendable that the authors have taken this into consideration, discussing the risk of persistent insulin activity in hypoglycemia and proposing possible solutions. The manuscript is well-written, with convincing data and good logic. This study also has great translational potential. Should the strategy prove effective in humans, it could significantly impact diabetes care and treatment.
I have a few comments as follows:
Major:
1. The glycemic data presented are sampled with long intervals (days, except on the day of injection). While this shows a stable reduction in glycemia, it lacks information on the glycemic fluctuations throughout the day of the tested animals. As such, it is difficult to ascertain whether the presence of basal insulin would increase the risk of hypoglycemia. This is particularly relevant given that blood glucose levels are quite low for several days after the injection of Fc/FA-Ins1 (50 mg/dL). It would be useful to show the fluctuation profile for at least a 24-hour period.
2. Insulin therapy can involve both basal insulin and prandial insulin applied shortly before meals to titrate blood glucose. While Fc/FA-Ins1 is effective in setting a low basal glycemia, would the use of fast-acting insulin significantly increase the risk of severe hypoglycemia? Could the authors please discuss this?

Minor:
1. The red trace in Fig. 1A is currently shown as FA-Ins1, while the text/legend indicates it is Fc-Ins1.
2. In Fig. 1A, there is a transient rebound after the injection of the insulin analogues lasting for 2-3 days, with a similar effect observed in Fig. 2A for a few other analogues. Could the authors explain the possible cause for this?
3. Not all supplementary items were referenced in the main text.
4. The labelling of Fig. 3 is inconsistent with the legend.
5. Some supplementary figures have very low resolution
6. in Table 3, the outliers significantly affected data with Ins9, leaving a single data point for both Fc/FA-9 and Fc-ins9. It isn't easy to be conclusive based on this.

Referee: 1

Comments to the Author
The authors have performed a very detailed and carefully executed study examining dual FC conjugation and lipidation of insulin molecules. The data are comprehensive, the results are well presented and the discussion is fair and balanced, highlighting the limitations and/or potential barriers of this approach moving forward. This study will make and important contribution to the field of peptide-proteins therapeutics as a whole so its significance and impact is not just limited to insulin per se. The authors are to be congratulated on this well designed and executed study.

We appreciate the comments and commendations. We share the perspective that this work can lead to design of advanced therapeutics.

Referee: 2

Comments to the Author
The study by Zaykov et al. presents an innovative approach for generating ultra-long-acting insulin. By doubly protracting insulin with both Fc and fatty acids, they enabled a 20-day glucose-lowering effect in a mouse model for diabetes and observed similar effects in non-diabetic rats and dogs.
The main problem of insulin therapy is iatrogenic hypoglycemia. The data shown also indicated that this risk is present with the modified insulin. It is commendable that the authors have taken this into consideration, discussing the risk of persistent insulin activity in hypoglycemia and proposing possible solutions. The manuscript is well-written, with convincing data and good logic. This study also has great translational potential. Should the strategy prove effective in humans, it could significantly impact diabetes care and treatment.

We appreciate the reviewer’s comments and thoughtful review. We are providing our response to individual points below.

I have a few comments as follows:
Major:
1. The glycemic data presented are sampled with long intervals (days, except on the day of injection). While this shows a stable reduction in glycemia, it lacks information on the glycemic fluctuations throughout the day of the tested animals. As such, it is difficult to ascertain whether the presence of basal insulin would increase the risk of hypoglycemia. This is particularly relevant given that blood glucose levels are quite low for several days after the injection of Fc/FA-Ins1 (50 mg/dL). It would be useful to show the fluctuation profile for at least a 24-hour period.

Daily fluctuations of blood glucose (BG) indeed present a major challenge to insulin therapy, and something that is best addressed by adjustment in prandial insulin dosing rather than basal insulin. The primary aim of this work is to provide an extended-action insulin analog with a low a peak-to-trough ratio as possible. This might be best characterized as an ultra-basal insulin, relative to conventional basal daily insulin therapy. It is not designed to be glucose-sensitive and as such its use will need to be customized for each patient and their ever-changing daily requirements guided by glucose determination, preferably by a continuous monitor.
If we are to zoom in on the data and look at the individual animals, the variability becomes more apparent, although BG levels manage to remain in the 30-60mg/dL range. It is ill-advised to aim for such low blood glucose as witnessed in this profiling of individual diabetic mice, but we know the pharmacology to be dose-responsive, and a lesser dose as with conventional insulins results in less lowering of glucose. However, the keys points in this exploratory report are that the profile is flat and that despite the lower glucose levels these animals tolerate it quite well. While low glucose is to be avoided, it is also erratic glucose control and fluctuations within the hypoglycemic range that are dangerous. We believe that the results we report constitute a means to a “milder” and unique in-vivo activity in these doubly protracted analogs. This provides an important and novel tool for customizing therapy, particularly for the most brittle patients (C-peptide negative) where this ultra-basal approach could be microdosed with more frequent administration, or used to constitute a partial basal replacement that could be supplemented with conventional daily basal insulin for protection against hypoglycemia.



2. Insulin therapy can involve both basal insulin and prandial insulin applied shortly before meals to titrate blood glucose. While Fc/FA-Ins1 is effective in setting a low basal glycemia, would the use of fast-acting insulin significantly increase the risk of severe hypoglycemia? Could the authors please discuss this?

The treatment involving fast-acting analogs comes with an inherent risk of overdose and hypoglycemia. There is certainly a value in creating safer short acting analogs, but until then, it will fall on the patient (or the pump) to decide appropriate dosing. On the flipside, the danger of hypoglycemia arising from fast-acting insulins is shorter-lived and can most often be counteracted with a sugar bolus, or a glucagon injection in more serious cases. It will be much more challenging if this to occur with long-acting analogs. While coadministration of two is possible, it will be safer from a clinical perspective to administer them separately to allow finer titration of the fast-acting analogs and ensuring appropriate dosing of the long-acting component.

Minor:
1. The red trace in Fig. 1A is currently shown as FA-Ins1, while the text/legend indicates it is Fc-Ins1.
Corrected

2. In Fig. 1A, there is a transient rebound after the injection of the insulin analogues lasting for 2-3 days, with a similar effect observed in Fig. 2A for a few other analogues. Could the authors explain the possible cause for this?
We suspect the effect may be caused by delayed biodistribution of the Fc-protracted analogs after the subcutaneous injection. A fraction of the insulin goes directly into the bloodstream providing initial reduction in the blood glucose. The rest of the Fc-Insulin is being captured by lymphatic system (as described in Rosenstock et. al. in Endocrine Reviews 2024), which delays full onset of action for several days. Tmax for insulin efsitora is around day 3-5, which is consistent with this observation.

3. Not all supplementary items were referenced in the main text.
Corrected

4. The labelling of Fig. 3 is inconsistent with the legend.
Corrected

5. Some supplementary figures have very low resolution 6. in Table 3, the outliers significantly affected data with Ins9, leaving a single data point for both Fc/FA-9 and Fc-ins9. It isn't easy to be conclusive based on this.
The result indeed needs to be taken with a grain of salt and the outcome is suggestive rather than conclusive for the PK experiment with Ins9 analogs. The majority of animals in those groups developed ADA’s and a set of data from a single animal does not have the statistical power. The data that is available seems to align well with the results obtained in rodents and supports the notion that Fc and lipid combination improves PK performance in dogs.

Berlin, 17 June 2024

Dear Dr Zaykov:

Manuscript ID: CB-ART-04-2024-000078.R1
TITLE: Toward once-monthly insulin therapy via synergy in two pharmacokinetic protractors: Fc-conjugation and fatty acid acylation.

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 1

The authors have revised the manuscript as suggested.