Insulin Glargine U100 Vs U300: Formulation, Pharmacological Profile, and Clinical Outcomes

Basal Insulin, also known as background insulin is a type of slow-acting insulin that releases constantly to maintain steady glucose levels in the bloodstream during prolonged hours of fasting. This type of insulin is required for those with type 1 or type 2 diabetes or gestational diabetes to prevent glucose spikes. Insulin therapy is common in those with Type 1 Diabetes Mellitus (T1DM) and Type 2 Diabetes Mellitus (T2DM), although dosage is what varies (Davis et al., 2016). Dosage for insulin therapy vastly depends on lifestyle conditions such as obesity, and blood pressure. Body Mass Index (BMI) also plays a crucial role in determination of the dosage. Obese individuals have considerably higher values of BMI, this is also one of the causes for insulin resistance with time, Hence concentrations of insulins and its acting period on the body plays an important role in regulation and maintenance of Insulin. Neutral Protamine Hagedorn (NPH), one of the first forms of synthetic Basal Insulins manufactured, are intermediate acting insulins (Duque et al., 2021a). That regulate glucose flow between meals, or overnight. Short or intermediate acting Insulins require more dosages, in case of NPH, two a day. Glargine, a long acting insulin, one of the newest developments of basal insulins are quite the solution. Ideally formulated in U100 (100 units of insulin in 1 ml), IGlar U100 has shown promising results in safety and efficacy. U300 (300 units of insulin in 1ml) IGlar U300 having a more extended therapeutic action, surpasses action time studies between the two. Although both concentrations have different pharmacokinetic and Pharmacodynamic properties to be explored (Duque et al., 2021b).

EPIDEMOLOGY OF DIABETES AND INSULIN DEPENDENCY:

Although various techniques and definitions of diagnosis have been employed by researchers there is definite evidence about the existence of Type 2 (or non-insulin-dependent) diabetes all across the world. Although it is found in varying proportions in different nations, different races living in the same country, and even in the same race when they settle in different continents within or outside their native land. It is abundantly clear from studies regarding rural-to-urban relocation and migration that the possibility of Type 2 diabetes multiplies manifold when there is a change to the more ‘Western’ style of living (P. Zimmet, 1982).

Towards the end of the 1980s, nearly 70 diabetes registries from more than 40 countries have estimated the incidence of Type 1 insulin-dependent diabetes in children. The major part of these observations comes from regions where the incidence of diabetes is high: Europe and North America. All these estimates provide a chance to compare the frequency of Type 1 diabetes incidence in various regions around the world and in particular within the Northern Hemisphere (Karvonen et al., 1993).

Non-insulin-dependent diabetes mellitus (NIDDM) today is known to contribute to about 85% of diabetes in developed nations and is rapidly spreading to developing nations and also to marginalized populations in developed nations, such as Mexican and African American populations and also to Australia’s Aboriginal and Torres Strait Islander populations. In populations of European origin, it is generally diagnosed in people older than 50 years, whereas in high-risk populations such as Pacific Islanders, Native Americans, and migrant Asian Indians and Chinese in young people, it is diagnosed much earlier. There is enormous variation in the rate of prevalence in different populations; extremely high rates are also observed in populations which change their mode of living to modernized living (e.g. American Pima Indians, Micronesians, Pacific Islanders, Australian Aborigines, migrant Asian Indians, and Mexican- Americans). In the next decade, macro- and microvascular complications of NIDDM are to be expected to pose severe problems (P. Z. Zimmet et al., 1997).

CLINCIAL NEED FOR LONG ACTING BASAL INSULIN THERAPY:

Despite the advancements made in insulin therapy over the past five decades, there still exist certain issues which act as barriers to its timely and effective utilization. These include, but are not limited to, the fear of experiencing hypoglycemic episodes, issues of increased body weight, the procedure of blood glucose sampling, and the pain of injections. The development of new basal insulins, which have a longer-acting preparation, have shown efficacy in controlling blood glucose levels while reducing the risk of hypoglycemic events. Insulin was historically used for diabetic control in the 1920s, with early commercial preparations having numerous impurities and varying amounts of potency. Then in the 1930s came the development of protamine zinc insulin; this slowed action and had a longer duration of action, which reduced the number of doses per day needed, though it still had instability. Then in 1946 came the development of neutral protamine Hagedorn (NPH) insulin, which could be combined with regular insulin and became the most widely used basal insulin through most of the 20th century. The 1980s brought about a significant shift with the development of human insulin, making it possible to accurately control the treatment. The development of analog insulins that are long acting, such as glargine and detemir, further improved the stability of insulin levels throughout the 24 hours, making it possible for it to be taken on a daily basis (Eliaschewitz et al., 2016).

FORMULATION OF GLARGINE:

Both U100 and U300 are glargine Insulins (Long-acting insulins). The molecular structure of glargine includes amino acid modifications: substitution of asparagine with glycine at position A21, and addition of two arginine residues at position B31 and B32 (González-Beltrán et al., 2021). U100 contains 100 units of glargine per milliliter and U300 has 300 units of glargine per milliliter – Three times more concentrated. The use of higher concentrated insulins give the same effect with a lower volume usage in a singular dose. Both formulations are delivered subcutaneously but via different delivery pens (e.g., Lantus® SoloStar for U100 and Toujeo® SoloStar for U300) (Davis et al., 2016). U300 formulations are designed to release insulin more slowly from the subcutaneous depot due to smaller injection volume and more compact precipitate formation. Upon injecting, both U100 and U300 have depot formations. U300 has a denser and compact depot, causing slower dissolution and release. The difference in depot formations is a key factor in their pharmacodynamics and pharmacokinetics. U100 and U300 are not bioequivalent and not directly interchangeable on a unit-to-unit basis. U300 generally requires a 10–18% higher dose to achieve similar glycemic control due to its lower bioavailability (Davis et al., 2016; D’Souza et al., 2020).

Table 1: Physiochemical and Device Differences Between Gla-100 and Gla-300

RECOMBINANT MANUFACTURING OF INSULIN GLARGINE:

The role of insulin is the regulation of carbohydrate and lipid metabolism by controlling the blood glucose level, which occurs mainly due to increased glucose entry into muscle and fat tissues. Human insulin has two polypeptide chains called the A chain and the B chain that are linked by four disulphide bonds. The molecular weight is about 5,808 Daltons, and it has 51 amino acids. Insulin was originally derived from animals, the pancreas of the cattle and pigs, for therapeutic use. Now, insulin is produced biosynthetically using recombinant DNA technology, allowing for the creation of insulin analogs such as glargine. All the analogs have been produced using microbial organisms such as Escherichia coli and yeast. Insulin lispro, glulisine, and glargine insulin preparations use E. coli, while insulin aspart and insulin detemir preparations use the yeast Saccharomyces cerevisiae. Modifying the insulin by substituting certain residues such as alanine to threonine in glargine and asparagine to glycine, makes the insulin have longer effects and takes longer to be metabolized due to the slowed dissolution of the precipitated insulin after injection. Insulin glargine has two additional arginine residues and the above substitution, which shifts the isoelectric point from 5.4 to 6.7. Insulin glargine is very soluble in slightly acidic solutions but is less soluble in slightly acidic to neutral solutions at which the body normally operates. The insulin hexamer binds to and is stabilized by zinc ions, which, together with the delayed dissolution due to the formation of the insulin precipitate, creates a prolonged absorption pattern after insulin injection into the body (Hwang et al., 2016).

Investigation in this study is focused on the production of insulin glargine through recombinant E. coli BL-21(DE3) in a fed-batch fermentation culture. Cell disruption through high-pressure homogenization was employed to release the glargine precursor in the form of inclusion bodies. Incorporating citraconylation enabled efficient removal of impurities such as Arg (B31)-insulin. This is an improvement over previous methods. After refolding and enzymatic processing of this precursor protein to produce mature glargine insulin, purification and polishing were done with ion exchange chromatography and reverse phase high performance liquid chromatography to attain a high purity of 98.6%. Approximately 0.3 g/L of highly purified recombinant glargine insulin could be obtained in only two purification steps out of 20 L of cultured bacterial solution (Kaki et al., 2022).

Since recombinant therapeutics are produced inside living expression systems, such as bacterial, yeast, and mammalian systems, there could be a final molecular structure of the therapeutic influenced both by the expression host and culture conditions. There could be post-translational modifications, such as glycosylation, due to various enzymatic reactions, which could affect key characteristics of the therapeutic. Such final molecular structure could occur both naturally and as a result of processing, where conditions could include cell line, media, temperature, pH, and other culture conditions. An example could be pH variations causing aggregation, as observed where there could be multiple glycoforms of a particular therapeutic, as is commonly observed in yeast expression systems. The differences could occur both due to natural conditions, where there could be variations from batch to batch, and other conditions where there could be specific processing considerations to improve productivity or cut costs. Since insulin possesses a clear higher-order structure, any slight change in either processing or purification could translate to a different structure, influencing receptor binding, biological activity, mitogenic, stability, among other factors, which could contribute to either aggregation or susceptibility to oxidation (Heinemann et al., 2023).

STRUCTURAL AND PHYSIOSTABILITY CONDITONS:

Insulin Glargine is a long-acting insulin and a first-generation long-acting insulin analog. It requires a maximum of one and a half hours to take effect and works for a whole day. The reason it was designed this way was because of lower solubility at a biological pH, so it precipitates on being given to the body. The biosimilar insulin glargine has to prove similarity to the original insulin regarding its therapeutic and safety profiles, and finally, quality of manufacturing. In this process of proving similarity, comparisons at multiple levels are being performed: at a structural level, comparisons involve spectrometry and chromatography, at a functional level, comparisons involve cell-based bioassays, and finally, at a level involving stability, comparisons involve stress testing.

In this study, the biosimilar scenario of insulin glargine available in the Indian market was assessed comparatively to the original formulation, Lantus® (Sanofi Aventis). Two batches each of the four available biosimilar insulin products, Glaritus (Wockhardt, India), Basaglar (Eli Lilly, India), Basalog (Biocon, India), and Basugine (Lupin, India), were tested using an extensive analytical procedure. Methods such as circular dichroism, fluorescence, and FT-IR were utilized to determine the structure, while RP-HPLC-MS/ESI-TOF-MS is utilized to determine the amino acid composition, molecular weights, PT modification, and disulfide bond patterns of the insulin molecules. Parameters related to quality like concentration of protein, purity level, and product variant were evaluated by taking RP-HPLC and size exclusion chromatographic measurements. Excipients were analyzed by making use of atomic absorption spectroscopy and RP-HPLC. Degradation properties at higher temperatures were used for testing thermal resistance by subsequent measurements of aggregation by SE-HPLC. Biological activity was compared by performing in vitro glucose uptake assays among different proteins.

On the whole, the biosimilars investigated were shown to have equal architecture and biological activity to the reference, but certain low levels of variant forms cast doubts on their long-term stability. Percentages of aggregates after two weeks of storage were significantly correlated both with initial levels of aggregates and shelf life prior to expiration of samples. On the principle of the degree of closeness to the reference, the sequence of closeness turned out to be: Lantus® > Biosimilar 2 > Biosimilar 4 > Biosimilar 1 > Biosimilar 3 (Vishwakarma et al., 2023).

PHARMACOKINETICS AND PHARMACODYNAMICS:

The action of insulin is mediated through its binding to the insulin receptor (IR), a transmembrane tyrosine kinase receptor composed of two extracellular alpha subunits and two intracellular beta subunits. When insulin binds to the alpha domains, it triggers a conformational shift that activates the tyrosine kinase activity of the beta domains. This leads to auto phosphorylation of the beta subunits and the recruitment of intracellular docking proteins, which in turn initiate downstream signaling cascades. Among these, the phosphatidylinositol 3-kinase (PI3K) pathway is key, ultimately activating protein kinase B (Akt). Akt modulates various cellular responses, including translocation of glucose transporter type 4 (GLUT4) to the cell surface, and interacts with other pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK)(De Meyts, 2000).

Structurally, insulin glargine is modified to delay its absorption and prolong its activity. These modifications include a substitution of glycine for asparagine at position A21 and the addition of two arginine residues at positions B31 and B32 (Davis et al., 2016). These changes increase its solubility in acidic conditions (pH 4.0) and promote the formation of micro precipitates upon injection into subcutaneous tissue, where the pH is neutral. These precipitates gradually dissolve, allowing a steady release of insulin over a period of up to 24 hours. The onset of action typically occurs within 1.5 to 2 hours after administration. Gla-300, being three times more concentrated than Gla-100, forms a smaller subcutaneous depot with reduced surface area, leading to slower, prolonged absorption compared to Gla-100. At steady state (daily dosing 0.4 U/kg in T1DM for 8 days), Gla-300 exhibits a remarkably flatter insulin concentration profile over 24 hours, with delayed early-phase exposure and elevated later-phase exposure, compared to Gla-100. Gla-300 half-life after one week: ~19 hours vs. ~13.5 hours for Gla-100. Duration of action with Gla-300 may extend up to ~32 hours depending on dose. Time to 50% of total area under curve (AUC????): approximately 3 hours later for Gla-300 vs. Gla-100, reflecting delayed absorption onset. In T1DM subjects under clinically individualized dosing, within-day PK variability was 50% lower with Gla-300 than Gla-100. Day-to-day variability also significantly reduced with Gla-300. Under clamp studies at 0.4 U/kg fixed doses, Gla-300 demonstrated a more even and extended glucose-lowering effect than Gla-100, maintaining euglycemia (~≤ 105 mg/dL) for a median of ~30 h versus ~24 h with Gla-100. At steady state with individualized clinical dosing, both insulins achieved equivalent overall 24-h glucose-lowering. However, Gla-300 suppressed endogenous glucose production (EGP) more in late post-dose hours (18–24 h) and less in early hours compared to Gla-100. Gla-300 also produced greater suppression of glucagon, lipolysis, and ketogenesis over 24 h, particularly in the second half, providing additional metabolic stability(Becker et al., 2015; Heise et al., 2017).

CLINICAL EFFICACY:

HbAc1 is the percentage of hemoglobin that is glycated or coated with sugar, this test is often done to determine level of glucose levels in the bloodstream. In the EDITION clinical trials, Gla- 300 consistently demonstrated non-inferior reductions in HbA?c compared to Gla-100 over six months. Both insulins lowered mean HbA?c to a similar extent in diverse populations, including type 2 diabetes on basal-bolus or oral antidiabetic regimens. Real-world data from the DosInGlar study in insulin-naïve type 2 patients confirmed these findings over 18 months, with both cohorts achieving equivalent long-term glycemic control (~8.1% HbA?c). Fasting Plasma Glucose (FPG) is a blood test done to determine blood glucose levels after a person has undergone fasting for at least 8 hours, higher values of FPG level (mg/dl) can be indicative of prediabetes or T2DM. In In EDITION 1 and related phase III trials, fasting glucose levels declined similarly in both treatment arms, with no statistically significant differences in reductions between Gla-300 and Gla-100 at six months. The real-world cohort study also showed comparable declines in mean FPG over 18 months (e.g., −0.8 mmol/L for Gla-300 versus −0.6 mmol/L for Gla-100 at 18 months) (Duque et al., 2021b; Oriot et al., 2018).

Table 3: Clinical outcomes of Insulin Glargine U300 and U100