Peer review of: Molecular engineering of safe and efficacious oral basal insulin (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In the communication from Kjeldsen’s group, the development of ultra-long acting insulin analogs 
for the use of oral insulin therapy for diabetic patients has been reported. This is achieved by the 
combination of the pertinent mutations on the amino acid sequence of human insulin with the 
modification at the B29 lysine residue with an albumin-binding fatty acid. These modifications 
indeed increase the life-time of the bioavailability in the plasma significantly The achievements 
might have a strong impact in the communities. However, in consideration that one of the oral 
insulin analogs, i.e., OI338, was already reported and tested for the pharmacokinetic analysis for 
men in refs 1 and 2, where conceptually the same strategy was employed for the development of 
oral insulin drugs, scientific advances of the submitted paper would be rather limited. Although 
several new experimental data, including new ultra-long acting insulin analogs, such as OI216 and 
OI320, the crystal structures, and their detailed pharmacokinetic and pharmacodynamic behaviors 
for dogs, have been presented, the paper is not recommended for publication in Nature 
Communications due to the lack of enough novelty and innovative data. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In this study, the authors revealed that the combination of changes in three different properties of 
human insulin considerably increased the feasibility of developing oral insulin. The changes include 
enhancing the proteolytic stability, increasing the albumin binding affinity and decreasing insulin-
receptor binding affinity. In addition, they found that the new engineered molecules may provide 
direct protection against hypoglycemia, a condition that is usually resulted from insulin overdose 
and can cause severe health consequences. This research is original and important. The research 
design is well developed, and the experimental procedures and results are clearly described. The 
data from this research study support the feasibility of developing oral insulin for diabetes 
treatment. This work is expected to quickly catch the attention of scientists in the field of insulin. 
Overall, this manuscript is within the scope of Nature Communication and I recommend publication 
as it is. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Nat Com 235956; Hubálek et al 
The paper from Hubálek and colleagues et al is an interesting description of an approach to design 
of insulin analogues for oral administration. The topic of high interest, although whether or not 
these efforts lead to a marketable product is a subject for future clinical work. The manuscript is 
dense, but goes to some trouble to explain the rationale of the steps described, and does this with 
useful clarify. 
My comments are generally editorial: 
1. The authors vary in their usage of types of abbreviation for amino acids, in a way that is not 
good communication and can be confusing. At a simple level most clinical readers will not be 
familiar with the single letter format, and that is not intuitive. In Figure 2a it is confusing, as both 
notations are used, and 'H' is used for hydrogen and for histidine and for human! The reviewer 
suggests revising to the 3-letter form for amino acids throughout. 
2. Figure 2 is not intuitively laid out in any case. It took the rather slow reviewer some 3 minutes 
to work out that the three 'R =' usages without anything following referred to the aliphatic side 
chain displaced further to the right and somewhat lower. Using a brace (curly bracket) with the 
chemical formula immediately adjacent to it would probably largely resolve the problem. 
3. In any case what is the significance of the red coloured symbols in this chemical structure? This 

is not explained in the legend. 
4. Again while 'Ca atoms' will be known to the authors, the reader unfamiliar with the analogues 
and size estimates will wonder if calcium is really meant. Suggest spell it out on first usage. Oddly 
zinc is always spelt out in the supplementary material. 
5. Sodium caprate will not be familiar to many. It is briefly introduced and referenced however. It 
is not a US FDA GRAS substance I believe. Perhaps it is already used in some other orally 
administered substances (apart from being present in some foods). Some statement on its 
regulatory status in Europe and the US would be welcome. 
6. In the Abstract the authors comment on 'efficient' glucose lowering in animals in clamps. While 
it is obvious to the authors, some readers may not realize that IV administration is used in these 
studies. It is suggested to say that. 
7. It is anyway not clear how the IV dosing relative the human insulin was arrived at. To this 
reviewer efficiency would imply that, at steady state with albumin binding saturated, the dose 
giving 100 % equivalent in figure 2 would have the same molar content as the human insulin 
standard. Was that the case? Otherwise efficacy is established not efficiency. Figure 4 on the 
horizontal axis is indeed molar (and shows a halving of potency, which is indeed a minor 
difference), but the authors could be more explicit in the relevant parts of the text as to doses 
used. 
8. In Figure 3 the oral doses are described as 'dose normalized'. This might mean just about 
anything, but perhaps means doses were adjusted for the oral bioavailability? But perhaps not as 
that is surely what is being determined by the oral (when compared to the IV) studies. The 
reviewer is lost and would beg clarification. 
9. At line 183 the authors are comparing variation on days 1 and day 14. Probably then they are 
giving within day variation, reducing due to accumulation in the albumin space. But they then 
immediately go on to talk about 'within patient variability' which could be the same thing (within 
day) or what is usually understood by clinicians and insulin users, namely between days. The 
figure they quote for insulin glargine seems to be within-patient, between-day, CV for fasting 
plasma glucose! The pharmaceutical industry has willingly muddied the water in this area for 
insulin, and clarity would welcome. 
10. In Table 1 relative potency seems to be 50-150 x that of human insulin. Perhaps these are 
percentages, however, but units are not given. 
11. Line 263 et seq: This seems intuitively correct, but again the general reader will have trouble 
in understanding what is meant by central and peripheral compartments. The confusion partly 
arises for clinicians because central and peripheral are also used for fat distribution (and much 
more commonly so), so that 'central' equates to the liver and abdomen. Clarification of meaning is 
suggested. 
12. Incidentally the reviewer also notes that accessibility of albumin bound insulin is different in 
the liver and the periphery, and wonders (as others will) whether this would be more extreme for 
these analogues. A similar issue arises with the blood-brain-barrier, possibly explaining some 
issues relating to insulin detemir. Perhaps the issues could be noted, even if no data are available 
and speculation as to hepatocyte and hypothalamic access is all that can be given. 
13. Figure 5d: Again it is obvious with a little thought which lines are PK and which PD, but the 
positioning of the keys is not intuitive. They could either be moved to be more closely associated 
with their respective axes, or even better labelled as PK and PD. 
14. in various places in the text rats and dogs are described as the animal model, but not how 
they were prepared even in summary form. The information might be in the supplementary 
information, but the animals might at least be described as 'diabetic' or not, and if the former the 
mode of achieving that and subsequent management when first mentioned. 
15. The hypoglycaemia data make sense, but the comparison with human insulin is pretty much 
meaningless (one intended as a basal insulin, the other a meal-time insulin), as in part the authors 
note. The only relevant comparison really would be with an infused square wave of human insulin 
over several hours, which would not be dissimilar to the oral insulin findings perhaps, and thus 
similar to the findings from Halberg et al. I am therefore not sure the human insulin data 
presented here helps at all. Incidentally the issue here is not simply relative affects on Ra and Rd, 
and also ability to switch on Ra in the face of excess insulin after induction of hypoglycaemia due 

to prior relative overdosing, or changes in insulin sensitivity. This problem was seen with the Lilly 
hepato-specific insulin and has not been studied here (I am not suggesting that these insulins in 
any way are similar to PEG-lispro). A little more caution in discussion and further understanding 
are required. 
16. References: The way journal titles are given varies. Some article titles are first letter 
capitalized. 
17. But reference 32 is a mess, with two spelling howlers, as well as style variation.