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Petter Daleng
Development of new ultra-fast-acting insulin analogues for treatment of type-1 diabetes
KJ2900 Bachelor
Supervisor: Eirik Johansson Solum
April 2021
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Table of contents:
1. Abstract (p. 3)
2. Introduction (p. 3-4) 3. Theory (p. 4-13)
3.1 Diabetes (p. 4-5)
3.2 Insulin (p. 6-14)
4. Discussion (p. 14-15)
5. Conclusion (p. 16)
6. References (p. 17-18)
3 1. Abstract
Despite the successful fabrication of synthetic human insulin many years ago, the subcutaneous injection of human insulin proved inadequate in effectively moderating the blood sugar level of type- 1 diabetics, owing mostly to the long onset of action. The development of rapid-acting insulins by altering the primary structure of the insulin monomer decreased the onset of action by a large margin compared to human insulin, but they are still quite slow-acting. Ultra-fast acting insulins have recently been developed by adding excipients to the rapid-acting insulin formulations that improve the onset of action by local vasodilation, increased monomerization or other. This led to improved PPG values by about -1.55 mmol/L in Lyumjev® and -0.90 mmol/L in FIASP® when injected 1-hour post-meal and comparing them with their respective counterparts, insulin lispro and insulin aspart, while retaining non-inferior HbA1C values. This proved that current ultra-fast acting insulins decrease postprandial hyperglycemia in type-1 diabetics, but their onset of action is still nowhere near instantaneous, meaning that hyperglycemia still occurs in users that reluctantly inject their insulin right before their meal, ignoring the timing required to avoid postprandial hyperglycemia. The development of a glucose- responsive insulin would solve this issue, as it could be tuned to not allow the blood sugar level to rise to the level of hyperglycemia. This would make timing the injection, as well as calculating the exact dosage, unnecessary if a large amount of insulin is injected preprandial. While such an insulin analogue has not been invented yet, there is promise in the structural flexibility of some proof-of-concept structures.
2. Introduction
According to the Diabetes Atlas for 2019 the number of people with diabetes was estimated to be 463 million, with a proposed 4.2 million diabetes-related deaths (1).
The body of a type-1 diabetic is unable to control its blood sugar level (BSL), which is normally automatically managed by the endogenous secretion of precise amounts of insulin from the liver. As of today, there are no means of preventing or curing diabetes mellitus, and type-1 diabetics use insulin treatment to inject synthetic insulin by syringe or pump to manually control the BSL. However, despite that insulin treatment has made it possible to live with type-1 diabetes, many type-1 diabetics still experience frequent hyper- and hypoglycemic events, which in the long-term can lead to a multitude of complications (2).
Most of the dangers and diseases associated with diabetes are results of poorly controlled BSL leading to frequent hyperglycemia. One reason that so many people struggle with maintaining proper BSL is because it is a process that requires a lot of careful calculation of dosage and time of injection in response to digestion of food, exercise, as well as other more unpredictable factors, such as subtle hormonal changes (3). When using rapid-acting insulin such as Humalog®, the insulin takes effect up to 20 minutes after injection, which means that a type-1 diabetic must inject inulin well in advance of the incoming spike in BSL. This is especially troublesome when injecting for meals: The rate of increase in BSL can be very sudden or slow, depending on the ingested food. As a result, episodes of too high BSL (hyperglycemia) and too low BSL (hypoglycemia) happen frequently after meals, which is called postprandial hyper- and hypoglycemia, respectively. Worse, however, is that postprandial hyperglycemia is usually caused by the user not bothering to inject insulin before the meal is consumed. This is quite troublesome, as a high frequency of postprandial hyperglycemic events is considered the major reason for long-term diabetes-related diseases. By lowering the onset of action of the injected insulin, maintaining proper BSL would become much more manageable, leading to fewer hyperglycemic events and therefore less occurrence of diabetes-related diseases. Therefore, it
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is hypothesized that the development of ultra-fast-acting insulins (UFAIs) will decrease the occurrence of hyperglycemic events (2).
Firstly, the diseases associated with diabetes were discussed, as well as how insulin treatment is used to combat diabetes. From there, the insulin monomer primary and tertiary structure, dimerization equilibrium, and hexamer formation, followed by purpose, secretion mechanism, and binding to the insulin receptor was presented.
Moving on, the definition and examples of rapid-acting insulin were presented. Lastly, different excipients added to formulations to achieve ultra-fast-acting insulin were reviewed, as well as a newly developed type of self-regulating insulin.
3. Theory 3.1 Diabetes
Diabetes mellitus is a disease characterized by high blood sugar level (BSL), where the body lacks the ability to regulate this. In the body of a non-diabetic, insulin is secreted in response to an internal increase in glucose concentration (2).
There are several versions of diabetes, of which type-1 and type-2 diabetes are the most common:
Type-1 diabetes mellitus is also called “insulin-dependent diabetes mellitus”. In type-1 diabetics, autoimmune antibodies destroy the β-cells responsible for producing insulin. Why this happens is unknown, and there are no current means of preventing it. The body of type-2 diabetics is insulin- resistant, which means that the high BSL of type-2 diabetics leads to a slower rate of absorption of insulin. Like type-1 diabetes, there is no cure (4).
Type-1 diabetes accounts for around 5 – 10 % of all cases of diabetes, where the rest is type-2. Insulin treatment is primarily used by type-1 diabetics, as they are forced to take insulin regularly to maintain proper BSL, while type-2 diabetics can often avoid their usage entirely. Therefore, this report will only concern itself with type-1 diabetes, despite this being the rarer version of diabetes (4).
Insulin treatment is required for type-1 diabetics to be able to live comfortably: It requires balanced and timed injections of insulin to keep the BSL between 4.0 - 7.0 mmol/L, though 5.0 - 10.0 mmol/L is considered healthy for two-hour post-meal BSL in type-1 diabetics (5). There is some time before the insulin takes full effect, meaning that timing the insulin injection in accordance with upcoming spikes or declines in the BSL is of major importance. Exercise typically lowers the BSL, while hormonal changes such as stress may either rise or lower the BSL. As such, achieving complete control over the BSL is next to an impossible task (3).
Diabetes can lead to both micro- and macrovascular diseases in the long term if the BSL is regularly too high. High BSL is called hyperglycemia. These diseases include damage to the eyes (retinopathy), kidney (nephropathy), nerve system (neuropathy), heart and network of blood vessels and veins, as well as most of the muscular system. Hyperglycemia can be caused by not injecting insulin regularly or miscalculation of insulin dosage or time of injection. Hyperglycemia occurs most often in type-1 diabetics following a meal, as many diabetics miscalculate the amount required and when to set the dosage, or they simply do not bother timing the injection. This is called postprandial (after meal) hyperglycemia. Recurring postprandial hyperglycemia is the major culprit behind diabetes-related
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diseases. Reducing the occurrence of hyperglycemic events in type-1 diabetics would drastically lower the onset and severity of these diseases (6).
Diabetes is also associated with hypoglycemia, where the BSL is too low; under 4.0 mmol/L. Symptoms include fatigue, anxiety and numbness in lips, tongue, or cheek, and can develop further into confusion, loss of coordination and loss of consciousness. If the BSL falls even lower, it can in worst case lead to death, as the metabolism of the body will cease to function. Usually, a diabetic will notice when they are in a state of hypoglycemia, though this ability may subside over the years. It is usually a result of overdosage of insulin following meals, meaning postprandial hypoglycemia is the most common form of hypoglycemia. However, hypoglycemia can also occur after exercise or stress as the body no longer facilitates increased gluconeogenesis following a decrease in BSL (7).
The following figure shows factors that influence BSL, which must be kept in mind when trying to maintain a healthy BSL:
Figure 1: The figure shows the different states of the BSL, as well as what factor increases or decreases it. To maintain healthy BSL between 4.0 – 7.0 mmol/L, all the above factors must be taken into consideration, where factors like hormones are not nearly as easily controlled as food-intake and exercise.
Reducing the onset of action of insulin would decrease the occurrence of both hyper- and hypoglycemia. Not only would it be much easier to time the injection, but it would also enable type-1 diabetics to make more informed decisions when setting the dosage: By injection shortly before their meal, they would be less likely to miscalculate by viewing a more relevant current BSL. Furthermore, hyperglycemia would decrease for all those type-1 diabetics that set the injection right before their meal. The only potential downside would be that hypoglycemia would happen more rapid (2).
To measure the amount of hyperglycemia a type-1 diabetic experiences, two parameters are widely used: the HbA1C and the PPG. The HbA1C test gives the percentage of hemoglobin in your blood that has bonded to glucose. This percentage value gives an indication of your average BSL during the last few months. For a normal type-1 diabetic it should be less than 6.5 %, corresponding to 140 mg/dl or 7.0 mmol/L, to be considered healthy (8). The degree of postprandial glycemia (PPG) is usually measured one- or two-hours post-meal, where the number and severity of PPG excursions describes the efficiency of the user´s insulin treatment. Both HbA1C and PPG values are today used in clinical tests to calculate how effective a new insulin formulation is at reducing hyperglycemia in type-1 diabetics. (9). The PPG contributes to the HbA1C value, and both lead to increased chances of cardiovascular diseases if severe (10).
6 3.2 Insulin
Figure 2: The figure shows an unfolded insulin monomer, with the A chain marked in blue and B chain marked in green. In addition, the two sulfide bonds linking chain A and B are showed, as well the internal disulfide bond of chain A. The figure is inspired by a similar figure featured in Insulin on PDB-101: https://pdb101.rcsb.org/global-health/diabetes- mellitus/drugs/insulin/insulin (11).
Insulin Structure: The insulin hormone consists of two peptide chains, designated A and B, composed of 21 and 30 residues each, respectively. They are connected by two disulfide bonds at A7-B7 and A20- B19. The A-chain also has an additional internal disulfide bond at A6-A11 for increased stability. The A chain is composed of two α-helixes in a U-shape. The B chain is composed of a slightly longer α-helix that runs perpendicular to the two α-helixes in the A chain (12).
The monomerization and dimerization of the insulin hormone is driven by the solvent entropy increase due to the burying of hydrophobic sidechains: Burying of hydrophobic amino acid sidechains in proteins increases the overall entropy of the solvent as water-molecules in the surrounding solvation layer can leave to bulk solution, creating more disorder (13).
In the insulin monomer, the hydrophobic side chains Ile A2, Val A3, Leu A16, Tyr A19, Leu B6, Leu B11, and Leu B15 are buried towards the core. The edge of the hydrophobic core, however, slightly exposes Val A3, Ile A13, Val B12, Val B18, Phe B24, and Tyr B26. This is important for dimerization. The insulin monomer can exist in one of two states: If Phe B25 has a hydrogen bond with Tyr A19, the monomer is in the T-state. If this is not the case, it is in the R-state.Insulin exists as a monomer at low concentrations, around 10-6 M. At higher concentrations at neutral pH, dimerization occurs (12).
When dimerization occurs, residues B24 – B28 form an anti-parallel β-sheet, where the hydrophobic side chains of these residues are shielded from the solvent, thus increasing entropy. The dimer molecules can both be in the T- and R-states, or they can be in different states (12).
In the presence of Zn2+ ions, three insulin dimers conform to a hexamer-structure with a threefold axis of rotation, inside of which there are two Zn2+ ions on opposite ends of the “dimer ring”. Each Zn2+ ion is coordinated with three His B10 residues and three water molecules, which means that they are both octahedrally coordinated (12). Figure 3 shows the insulin dimer and hexamer structures:
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Figure 3: To the left is the insulin dimer, composed of one T-state monomer and one R-state monomer. To the right is a top- down view of the insulin hexamer, composed of three dimers coordinated around a Zn2+ ion. The Zn2+ ion is also coordinated to three water molecules. Only the upwards Zn2+ ion is shown here. The figure is inspired by a figure featured in Insulin on PDB-101: https://pdb101.rcsb.org/global-health/diabetes-mellitus/drugs/insulin/insulin (11).
In Figure 3, the T3R3 insulin hexamer structure is shown. This is one of several structures, such as T6
and R6, but the precise function of each of these structures remains unknown. As a result, this aspect of the insulin hexamer structure will not be discussed any further in this text (12).
Insulin production, secretion, and function: Insulin is produced in the pancreas, in the hormone- producing part known as Islets of Langerhans, in response to elevated BSL in these islets. It is stored in vesicles as Zn2+-coordinated hexamers in preparation for secretion. Secretion of insulin is a result of elevated plasma glucose concentration leading to an increase in its ATP-yielding pathway, which depolarizes the cell membrane by closing ATP-gated K+ channels. This, in turn, opens the voltage gated Ca2+ channels, which ultimately trigger insulin secretion by exocytosis (12). This is shown in Figure 4:
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Figure 4: The secretion of insulin is triggered by an overabundance of glucose entering the cell. The figure is taken from p.
924 in Lehninger: Principles of biochemistry (13).
Following secretion, insulin binds in its monomer state to the insulin receptor (IR), which triggers a series of metabolic pathways, the mechanisms of which are complex and poorly understood. This influences synthesis of proteins and DNA, as well as cell growth, but it mainly decreases plasma glucose by increasing the number of glucose-transporters on cell membranes: These transporters trap plasma glucose and moves them into cells mainly for use as either short- or long-term energy. Insulin also suppresses hepatic gluconeogenesis (12).
When there is no intake of glucose in the form of food, a small amount of insulin is being continuously secreted to maintain a proper BSL. This amount is called the basal insulin (14). When food is ingested, a larger amount of insulin is secreted simultaneously to offset the impending spike in BSL. This amount is called a bolus (2).
Insulin treatment seeks to mimic the endogenous secretion of human insulin by subcutaneously injecting both long-lasting and fast-acting insulin in a balance: The long-lasting insulin replaces the basal insulin, while the fast-acting insulin takes care of the bolus insulin during mealtimes (14). For the fast-acting insulin to mimic endogenous secretion of bolus insulin, it is important that it has a similar response curve, meaning three parameters should be equal: The onset of action, the onset of the insulin peak and the overall duration of the insulin (2). Figure 5 is a visualization of these three parameters in a typical insulin response curve:
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Figure 5: The graph shows the plasma insulin as a function of time, starting from injection. The colored lines note the time of onset of action [red], onset of peak [green] and overall duration [blue]. Notice the tailing of the insulin after the main peak.
The figure is inspired by a graph featured in Insulin Chart: What You Need to Know About Insulin Types and Timing:
https://www.healthline.com/health/type-2-diabetes/insulin-chart (15).
Any other uncharacteristic increases in BSL are also handled by the fast-acting insulin. There are other insulins of varying onsets of action that can be combined to more accurately control the BSL, but using only two types of insulin is more common. This is not the case when using insulin pumps, however:
These can be programmed to release insulin continuously, meaning that fast-acting insulin can be used both as basal and bolus insulin (16).
Insulin is usually injected into the subcutaneous (SC) tissue by either syringes or pumps. The site of injection influences the rate of absorption of insulin into the bloodstream: It is common to inject fast- acting insulin in the stomach, and long-lasting insulin in the thighs. In the syringes/pumps used for these injections, the insulin is usually stored as Zn2+-coordinated hexamers, to provide better shelf-life for the product (3).
Subcutaneous injection of insulin: Upon being injected into the SC tissue, the insulin must traverse the extracellular matrix of the SC tissue, followed by the interstitium, before being absorbed into the bloodstream by the lymph capillaries. The time spent traversing this area is restricted by the structural macromolecules, which increases the onset of action. Furthermore, to absorb into the lymph capillaries, the insulin must be in the monomer conformation, as anything larger will absorb much slower. This is a problem for most insulin formulations, as the insulin is typically stored as a Zn2+- coordinated hexamer. The insulin formulation tends slightly towards monomerization in the SC tissue for two reasons: Firstly, there are few Zn2+ ions to form hexamers with. Secondly, the insulin formulation will spread into a greater area in the interstitium, favoring separation into smaller, more numerous particles. The latter factor is slightly hindered, however, by the macromolecules present in the interstitium.
The traversal time of the SC tissue and interstitium, and the following rate of absorption into the lymph capillaries, are the major contributors to the onset of action of insulin formulations. This time can be lowered by increasing the insulin spread in the SC tissue, decreasing the resistance of the interstitium or increasing insulin monomerization (2).
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Insulin formulation improvement: Insulin monomerization can be favored by editing the primary structure of the insulin monomer. When doing so, it is important to preserve the biological activity of the structure, so that it still binds correctly to the IR and retains the same effectiveness (12). There are three well-known insulin types that have undergone primary structure amino acid substitution. They are known as rapid-acting insulin analogues (RAIAs) (2): In insulin lispro, the proline at position B28 and the lysine at position B29 have switched places. This means that there is no longer any hydrogen- bonding between B23 and B28, which usually contributes to anti-parallel dimerization, followed by hexamer formation (17). In insulin aspart, the proline at B28 has been replaced with aspartic acid (18).
In insulin glulisine asparagine at B3 and lysine at B29 have been replaced with lysine and glutamic acid, respectively. This lowers the isoelectric point, which increases the solubility at a physiologic pH. This makes insulin glulisine more prone to dimerization in the absence of ligands. There are no Zn2+ ions present in this formulation, only Polysorbate (“Tween”) 20 added as a stabilizer and trometamol added as a buffer (19). Despite having an earlier onset of action, the duration of action is still too long for the RAIAs, requiring injection 20 minutes before mealtime. As a result, there is still a huge need for even faster-acting insulins. It is worth briefly mentioning that the onset of action, onset of peak and overall duration for the different RAIAs vary from source to source, making it difficult to give exact estimates on their properties.
Today, most insulin formulations for use in vials or syringes contain one RAIA, in addition to excipients that either increase the shelf-life of the product or promote a faster onset of action. In this text, we define ultra-fast acting insulins (UFAIs) as RAIAs that have a significantly faster onset of action by additions of such excipients (2).
Niacinamide is added in faster insulin aspart (FIASP®) to promote monomerization in the SC tissue. It is a hydrotrope, meaning it can increase the solubility of the hydrophobic part of the insulin monomer surface, which shifts the equilibrium towards monomerization. Furthermore, it increases absorption of the insulin monomer by local vasodilation (expansion of lymph capillaries) (20). A measured combination of zinc and niacinamide in the faster-aspart insulin formulation is needed to allow the optimal amount of hexamer stability for shelf-life and efficiency. L-arginine is also added for further stabilization (21).
Similarly, treprostinil provides local vasodilation in Lyumjev® (previously named ultra-rapid insulin lispro (URLi) (22)), accompanied by sodium citrate, which enhances vascular permeability (10).
Alternate methods of administration include jet-injection and sprinkler needles: Jet-injection allows for a needle-less injection of insulin and facilitates greater spreading in the SC tissue, leading to faster insulin absorption. However, it is not widely used due to issues of cost and ease of use (23). Similarly, sprinkler needles are shaped to spread injected insulin in all directions through multiple holes on its sides, but are not typically used (2,24).
Other administration methods include inhalation in the nose and lungs (pulmonary) through use of an inhaler, though there is little ongoing research on these, despite adequate results in terms of pharmacokinetics (2).
The following table lists the different rapid-acting insulin formulations on the market today for use in vials, displaying their respective RAIAs in parentheses. Their onset of action, onset of peak and overall duration, are shown in hourly intervals.
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Table 1: Rapid-acting insulin formulations (with RAIAs) for use in vials, with their onset of action, onset of peak and duration in hours. The table can be found in 2020 List of Insulin Products: http://www.diabetesincontrol.com/wp- content/uploads/2020/07/Insulin_Chart_07112020_DiabetesinControl_1050.pdf (25).
Name Onset of action [h] Onset of peak [h] Duration [h]
Adipira®
(glulisine) 0.2 – 0.5 1.6 – 2.8 3 – 4
Admelog®
(lispro) 0.25 – 0.5 0.5 – 2 3 – 5
FIASP®
(aspart) 0.2 – 0.3 1.5 – 2 5 – 7
Humalog®
(lispro) 0.25 – 0.5 0.5 – 2 3 – 5
Novolog®
(aspart) 0.2 - 0.3 1 – 3 3 – 5
Lyumjev®
(lispro - aabc) 0.2 – 0.3 2 - 3 2.5 – 7.5
FIASP® has a 4.9 min faster onset of action than its counterpart insulin aspart (26). Lyumjev® has an onset of action of 20 min, which is 11 min faster than its counterpart insulin lispro (in Humalog®) (2).
The following table lists the different rapid-acting insulin formulations on the market today for use in insulin pens, displaying their respective RAIAs in parentheses. Their onset of action, onset of peak and overall duration, are shown in hourly intervals.
Table 2: Rapid-acting insulin formulations (with RAIAs) or use in insulin pens, with their onset of action, onset of peak and overall duration hours. The table can be found in 2020 List of Insulin Products: http://www.diabetesincontrol.com/wp- content/uploads/2020/07/Insulin_Chart_07112020_DiabetesinControl_1050.pdf (25).
Name (RAIA)
Onset of action [h] Onset of peak [h] Overall duration [h]
Adipira Solostar®
(glulisine) 0.2 – 0.5 1.6 – 2.8 3 – 4
Admelog Solostar®
(lispro) 0.25 – 0.5 0.5 – 2 3 – 5
FIASP FlexTouch®
(aspart) 0.2 – 0.3 1.5 – 2 5 – 7
Humalog U-100 KwikPen®
(lispro)
0.25 – 0.5 0.5 – 2 3 – 5
Humalog U-100 Junior KwikPen®
(lispro)
0.25 – 0.5 0.5 – 2 3 – 5
Humalog U-200 KwikPen®
(lispro)
0.25 – 0.5 0.5 – 2 3 – 5
NovoPen Echo®
(aspart) 0.2 - 0.3 1 – 3 3 – 5
Novolog FlexPen®
(aspart) 0.2 - 0.3 1 – 3 3 – 5
12 Lyumjev U-100
KwikPen®
(lispro - aabc)
0.2 – 0.3 2 - 3 4.5 – 7.5
Lyumjev U-200 KwikPen®
(lispro - aabc)
0.2 – 0.3 2 - 3 4.5 – 7.5
HbA1C, PPG and hypoglycemia for UFAIs vs. RAIAs: In a recent study, Lyumjev® (named URLi at this time) was compared against Humalog® (insulin lispro) for differences in HbA1C and PPG values:
Lyumjev® was administered both right before eating the meal (mealtime), as well as 20 min after starting (post-meal). Regarding HbA1C, Lyumjev® showed non-inferiority with a difference of -0.08 %, with a 95 % confidence interval (IC) of [-0.16 %, -0.00 %], for mealtime and +0.13 % [+0.04, +0.22] for post-meal. Lyumjev® showed superiority in PPG values where the difference in PPG values after 1-hour post-meal amounted to -1.55 mmol/L [-1.96, -1.14], in favor of Lyumjev®. The difference was even greater after 2-hours post-meal, which amounted to -1.73 mmol/L [-2.28, -1.18]. It is worth noting that the rate and severity of hypoglycemia between the two was similar, however, the occurrence of hypoglycemia >4 hours after meals was 37 % lower and 30 % lower for mealtime and post-mealtime Lyumjev® compared with Humalog® (27).
FIASP® showed a similar trend when compared to insulin aspart in a recent study: Regarding HbA1C, FIASP® showed non-inferiority with a difference of -0.02 % [-0.11, +0.07] for mealtime and -0.10 % [+0.004, +0.19] for post-meal. The difference in PPG after 1-hour post-meal was -0.90 mmol/L [-1.36, -0.45], favoring FIASP®. Like Lyumjev®, it showed less occurrence of hypoglycemia in the 3-to-4-hour range after injection (21).
On glucose-responsive insulin: A recent study published a report on a glucose-responsive insulin (GRI).
The idea being that you inject large amount of “caged” insulin monomers that disassociate and bond to the IR based on the plasma glucose concentration.
In this GRI, the Lys at B29 in the insulin monomer is acylated and covalently bound to hydrazone or thiazolidine, followed by a fatty acid. This is shown in Figure 6:
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Figure 6: The glucose-sensitive insulin (GRI). Hydrazone and thiazolidine as links between the insulin monomer and the fatty acid have been experimented with. Four of these are bonded to albumin by the fatty acid, which is not shown. The figure is inspired by a figure featured in An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins:https://chemistry- europe.onlinelibrary.wiley.com/doi/10.1002/chem.202004878 (28).
The acylation of B29 does not impair its effectiveness as an insulin monomer. Four of these GRI are bonded to the fatty-acid sites of albumin (not shown in the figure), and water hydrolyses the hydrazone or thiazolidine bonds to give either a hydrazide or aminothiol product, respectively, in addition to an aldehyde. Usually, the hydrazide and aminothiol groups are attached to the acylated insulin, thus giving a fatty acid with aldehyde and an acylated insulin with hydrazide or thiazolidine following hydrolysis. This is shown in Figure 7:
Figure 7: Simplified reaction mechanisms for the hydrazone and thiazolidine links, respectively: R1 and R3 are bonded to the acylated insulin monomer and the fatty acid/glucose, respectively. They can also be inverted for a different relationship. R1
and R2 can be varied to give desired glucose sensitivity. The figure is inspired by a figure featured in An Aldehyde Responsive,
Cleavable Linker for Glucose Responsive Insulins: https://chemistry-
europe.onlinelibrary.wiley.com/doi/10.1002/chem.202004878 (28).
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Glucose can then, in its open state, act as an aldehyde and restore the hydrazone or thiazolidine group by reacting with hydrazide or aminothiol. This is shown in Figure 8:
Figure 8: Simplified illustration of the open form of glucose binding to hydrazide and aminothiol, respectively restoring the hydrazone and thiazolidine links. The figure is inspired by a figure featured in An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins:https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202004878 (28).
The presence of glucose makes the hydrolysis preferable, as in this state the insulin molecule is small enough to bind to IR as usual. Thus, the concentration of glucose in the blood determines the amount of insulin that can bind to the IR. Different substituents on the hydrazone/thiazolidine parts, as well as different fatty acids, were tested to find the optimal responsiveness with glucose. The team concluded with that a thiazolidine-based GRI was the most effective one, and it demonstrated clear glucose- sensitivity. Furthermore, the overall structure was quite flexible to changes without obstructing its sensitivity (28).
4. Discussion
Looking at Table 1 and 2, the interval of earliest and latest possible onset of action is smaller for Lyumjev® than for Humalog®, though the table gives the impression that Lyumjev® does not necessarily work faster than Humalog®. This gives a wrong impression according to the clinical trial for Lyumjev® versus Humalog®, where it works 11 min faster than Humalog®. Furthermore, the clinical trial claims that Lyumjev® has an onset of action of 20 minutes, which is much more than the earliest 12 min from Table 1 and 2. This is one of several examples of conflicting information regarding the pharmacodynamic effects of different insulin formulations.
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No information on Novolog® versus its counterpart insulin aspart regarding HbA1C and PPG values could be found, despite it appearing to have onsets of action similar to Lyumjev® and FIASP®. It is likely to show a similar trend, meaning improves PPG values and non-inferior HbA1C values, though this is not confirmed in this report.
Lyumjev® and FIASP® showed non-inferior HbA1C values and significant decreases in PPG values versus their counterparts, as expected based on their lower onsets of action: The reduction of PPG values 1-hour post-meal for Lyumjev® and FIASP® at -1.55 mmol/L and -0.90 mmol/L, as well as the 2- hour post-meal PPG reduction in Lyumjev® at -1.73 mmol/L, show that especially Lyumjev® improves upon insulin lispro remarkedly by exhibiting a faster onset of action, made possible by the simple addition of two excipients. This clearly shows that the RAIAs can be greatly enhanced by the addition of excipients that promote local vasodilation and insulin monomerization.
The question then becomes whether further addition of excipients can improve upon these formulations so that the onset of action becomes minimal, enabling users to inject at mealtime without experiencing hyperglycemia. For this to work, the insulin must be nearly instantaneous, and current UFAIs are nowhere near that goal. After all, despite the addition of further excipients, the SC tissue must still be traversed before absorption into the lymph capillaries, followed by insulin binding to the IR. If hyperglycemia is to be avoided in type-1 diabetics that inject insulin at mealtime, the development of a successful GRI is perhaps more worthwhile. In the meantime, however, Lyumjev®
and FIASP® have proven to reduce PPG values by exhibiting faster onsets of action.
Regarding hypoglycemia, neither Lyumjev® nor FIASP® showed an increase. In fact, there was less occurrence of hypoglycemia four hours post-dose for Lyumjev® when compared with Humalog® . This is surprising, as hypoglycemia theoretically should occur faster and be harder to offset when using a UFAI. Despite this, I still personally believe statistics sometime in the future will reveal more severe hyperglycemic events in type-1 diabetics using Lyumjev® and FIASP®, as many people probably won´t bother switching to a faster-acting snack.
The development of a GRI that only takes effect when the BSL is elevated beyond 7.0 mmol/L would dramatically change insulin treatment for type-1 diabetics: If a large amount of GRI was injected well before a meal, there would be no danger of hyperglycemia due to mistiming the injection or miscalculating the required dosage. If enough insulin were present in the blood, it would react spontaneously to always keep the BSL at 7.0 mmol/L. This would also mean that the user would not need to inject insulin to correct for hyperglycemia, which is an important factor for many people who despise needles. Furthermore, hyperglycemia due to overdosage would not be possible. As such, it would then only arise from exercise or hormonal changes. A possible problem could arise in users that purposefully allow some degree of hyperglycemia during exercise: By allowing a slightly elevated BSL, it is easier to avoid hypoglycemic events when exercising, which are much more dangerous compared to hyperglycemia. If the GRI does not allow the BSL to elevate beyond 7.0 mmol/L, it is possible that residual plasma GRI during exercise would lead to more occurrence of hypoglycemic events. Perhaps a GRI that only corrects for BSL beyond 10.0 mmol/l, for instance, could be invented for use in exercise.
Regarding the GRI, it seems that much more experimentation with different structures, linkers and substituents are required for the discovery of an effective GRI. However, the proposed GRI seems very accommodating to structural changes without completely losing its glucose-responsive effect.
This gives a lot of hope for the future development of GRIs.
16 5. Conclusion
Lyumjev® and FIASP® are two UFAIs that exhibit faster onsets of action from the addition of excipients to their respective RAIAs. They both demonstrate markedly lower PPG values for 1-hour post-meal injections while retaining non-inferior HbA1C values, and show a decreased risk of hypoglycemia after 4 hours plus. Their faster onsets of action will in practice also make it easier for type-1 diabetics to time their injections. However, despite showing clear superiority over their respective RAIAs, neither can eliminate postprandial hyperglycemia due to their onsets of action being far from instantaneous. If traversal of the SC tissue, followed by absorption into the lymph capillaries, continue to be limiting factors for the onset of action, UFAIs will probably never achieve this goal. As such, the invention of a successful GRI is the true next step for insulin treatment: In addition to being self-regulating, the GRI has already traversed the SC tissue and absorbed into the lymph capillaries when the concentration of plasma glucose starts to increase, enabling instantaneous insulin secretion, truly mimicking the endogenous insulin secretion.
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