**9. Insulins**

In 1921, insulin was introduced as a therapeutic drug, which improved the quality and life expectancy of diabetics. The first available commercial insulin preparations corrected acute diabetic decompensation but were inefficient for chronic use because their duration was too short. Thus, diabetics were required to take four to five injections daily to achieve good metabolic control. Such short-acting insulin was the only commercially available type in 1935. Prolonging the action of insulin to over 24 hours could achieve the aim of decreasing the amount of daily injections, and it was achieved by incorporating certain substances, such as oily solutions, heavy metals (zinc) and protein (protamine). In 1950, by changing the concen‐ tration of protamine and decreasing the amount of zinc, the intermediate insulin called isophane or NPH ("Neutral Protamine Hagedorn" in honor of the scientist) became available. There were still more changes in the formula that affected the time of action, and in 1954, the family of insulins slow, semi-slow, or ultra-slow containing zinc instead of protamine was produced [156, 157].

The use of insulin is essential in the treatment of type 1 diabetes mellitus. In T2DM, it is reserved for patients with severe hyperglycemia with ketonemia or ketonuria, newly diagnosed diabetics or those who do not respond to treatment with diet, exercise, oral hypoglycemic agents and the anti-hyperglycemic action of insulin sensitizers [158].

A milestone in diabetes therapy occurred with the Diabetes Control and Complication Trial (DCCT), which showed that blood glucose levels close to normal drastically reduced or even prevented the complications of diabetes when the carrier of the disease was subjected to intensive insulin treatment and follow-up with a team of diabetes educators. According to the DCCT, to achieve this control, one proposal is to replace conventional insulin treatments (one or two daily applications of insulin) with an intensive treatment of up to four applications per day [159].

Currently, attempts to achieve good metabolic control in patients with diabetes include treatment with exogenous insulin, which is an effective therapy option in cases of partial and/ or total deficiency of insulin secretion by the pancreas. It is estimated that 20-25% of all patients with diabetes are treated with insulin, and 5-10% of these patients are type 1 (who need this hormone to survive) and 15% are type 2 (who show severe insulin deficiency) [157].

Commercial insulin is a protein hormone with two linked chains of amino acids that cannot be administered orally because it is degraded by digestive and intestinal enzymes. Most commercial insulin is manufactured from bovine and porcine pancreases, which are similar to the human pancreas. Bovine and human insulin differ in three amino acids, whereas porcine insulin differs in one amino acid (amino acid thirty). Chemically synthesized insulin is also produced by recombinant DNA techniques that use bacterial cells or other tissues that are free from impurities and have a minor antigenic action [160, 161].

The pharmacokinetics of insulin varies according to its type and kind, injection technique, presence of insulin antibodies, site of injection and the individual [162].

Commercial preparations of insulin are classified according to duration as either short, intermediate or long acting, and the species of origin is also a classifier, with insulin derived from human, porcine, bovine and or porcine bovine mixtures. Because of differences in the amino acid sequences, the bovine and porcine insulins have different physicochemical properties to human insulin. Human insulin has become widely available following the advent and development of recombinant DNA techniques [163]. These techniques have led to different formulations of insulin that differ according to recombinant DNA production techniques, amino acid sequences, concentrations, solubility and time of onset and duration of biological action. However, insulin produced through recombinant DNA technology are more soluble in aqueous solutions. Currently, the commercially available forms are supplied at neutral pH, resulting in improved stability, which is essential for storage over several days at room temperature [164].

The long-acting analogues such as glargine and detemir appeared on the market in 2000 and 2004, respectively [165], and they show a relatively stable profile of action over time [166]. In January 2013, the European Commission authorized the introduction of a new generation of ultra-long insulin analogues. Degludec insulin is an ultra-long acting basal insulin analogue [167].

#### **9.1. Short or ultra-rapid acting insulin**

**4.** with a sulfonylurea when sulfonylureas alone with diet and exercise do not provide

**5.** with insulin (alone, with metformin or a sulfonylurea or both) when existing therapy

A number of other SGLT2 inhibitors are under investigation, including empagliflozin, canagliflozin and ipragliflozin. In addition, a non-selective SGLT inhibitor (LX4211) is under development. It is likely that these and other agents that share similar pharmacodynamic

In 1921, insulin was introduced as a therapeutic drug, which improved the quality and life expectancy of diabetics. The first available commercial insulin preparations corrected acute diabetic decompensation but were inefficient for chronic use because their duration was too short. Thus, diabetics were required to take four to five injections daily to achieve good metabolic control. Such short-acting insulin was the only commercially available type in 1935. Prolonging the action of insulin to over 24 hours could achieve the aim of decreasing the amount of daily injections, and it was achieved by incorporating certain substances, such as oily solutions, heavy metals (zinc) and protein (protamine). In 1950, by changing the concen‐ tration of protamine and decreasing the amount of zinc, the intermediate insulin called isophane or NPH ("Neutral Protamine Hagedorn" in honor of the scientist) became available. There were still more changes in the formula that affected the time of action, and in 1954, the family of insulins slow, semi-slow, or ultra-slow containing zinc instead of protamine was

The use of insulin is essential in the treatment of type 1 diabetes mellitus. In T2DM, it is reserved for patients with severe hyperglycemia with ketonemia or ketonuria, newly diagnosed diabetics or those who do not respond to treatment with diet, exercise, oral hypoglycemic

A milestone in diabetes therapy occurred with the Diabetes Control and Complication Trial (DCCT), which showed that blood glucose levels close to normal drastically reduced or even prevented the complications of diabetes when the carrier of the disease was subjected to intensive insulin treatment and follow-up with a team of diabetes educators. According to the DCCT, to achieve this control, one proposal is to replace conventional insulin treatments (one or two daily applications of insulin) with an intensive treatment of up to four applications per

Currently, attempts to achieve good metabolic control in patients with diabetes include treatment with exogenous insulin, which is an effective therapy option in cases of partial and/ or total deficiency of insulin secretion by the pancreas. It is estimated that 20-25% of all patients

agents and the anti-hyperglycemic action of insulin sensitizers [158].

along with diet and exercise do not provide adequate glycemic control;

properties may become available in the coming years [146].

adequate glycemic control; and

**9. Insulins**

164 Treatment of Type 2 Diabetes

produced [156, 157].

day [159].

This group of insulins includes regular, lispro, aspart and glulisine analogues.

Regular insulin is usually administered subcutaneously and often in combination with intermediate-acting or long-lasting insulin. Special buffers are used so that a pump is not required to prevent crystallization because of its slow infusion. Monomers of this insulin present as hexamers that reduce the absorption rate. Normally, regular insulin is recom‐ mended for the treatment of diabetic ketoacidosis, and it is associated with human intermediate-acting insulin or basal analogs taken before meals [168]. This insulin should be administered 30-45 minutes before meals to reduce peak postprandial glycemia, and its action lasts between 2 and 4 hours, which contributes to postprandial hyperglycemia and hypoglycemia in the period between meals because regular insulin will peak after food has been metabolized [169].

Insulin lispro is a human insulin analog developed through genetic engineering by reversing the amino acids proline and lysine at positions 28 and 29 of the β chain, which results in the insulin sequence Lys (B28) Pro (B29). This insulin in its pharmaceutical preparation with phenol and zinc form stable hexamers [168], has a reduced tendency to self-aggregate at the site of subcutaneous injection, is absorbed more rapidly than regu‐ lar human insulin, and mimics the physiological profile of insulin in response to a meal. In addition, its onset of action is between 5 and 15 minutes and duration of action is 1-2 hours [170]. The use of these analogues requires an additional dose in the afternoon to compensate for the hyperglycemia that results from an afternoon snack. There is evi‐ dence that insulin lispro reduces postprandial hyperglycemic peaks and the risk of hypoglycemia compared to regular insulin, especially at night [168, 171].

In aspart insulin, one proline amino acid is replaced by aspartic acid, which is negatively charged, at position 28 of the β chain, producing electrical repulsion between the insulin molecules and reducing their self-association tendency; in vials or cartridges, it occurs as hexamers, but in subcutaneous tissue, there is rapid dissociation to dimers and monomers, which ensures its rapid absorption and onset of action between 5 and 15 minutes and duration of action of 1 to 2 hours[168].

Insulin glulisine is another ultra-rapid insulin analogue obtained by the exchange of aspara‐ gine for lysine at position 3 of the β chain and lysine for glutamic acid at position 29 of the same chain. Thus far, there have been few studies with glulisine insulin, which appears to be similar to lispro and aspart in efficacy and hypoglycemic events. Because of its faster absorp‐ tion, its administration should be performed only 5-10 minutes before meals to provide greater flexibility for the patient and thereby improve their quality of life. Its shorter half-life reduces the need to eat food 2-3 hours after its administration, which is necessary with regular insulin, whose longer half-life causes postprandial hypoglycemia [172].

A recent direct and indirect meta-analysis published by Sanches et al. (2013) for glycated hemoglobin reduction outcomes compared rapid action insulin (aspart, glulisine and lispro) with human insulin (regular), and the direct meta-analysis only showed a statistically signif‐ icant difference for aspart, favoring the insulin analog. However, the results of indirect metaanalyses of HbA1c reduction outcomes showed that the result of rapid-acting insulin are consistent and the difference between them is not clinically significant. The ranking suggests that the probability of selecting the short-acting insulin brings the following provision: the first choice should be regular followed by glulisine, lispro and finally aspart. No significant differences were found in the comparison of tolerability outcomes in the rapid-acting insulin (aspart and lispro) and human insulin (regular) [173].

Recent studies have attempted to alter the pharmacokinetics of fast acting insulin analogues that are associated with recombinant human hyaluronidase, and they revealed that absorption was accelerated two-fold during the first half hour of exposure, which resulted in an onset of action between 13 and 25 minutes faster and a shorter duration of effect (40 to 49 minutes). The ultra-rapid action arising from this association may be beneficial in furthering the control of postprandial blood glucose, and administering insulin lispro and hyaluronidase immedi‐ ately before meals in patients with type 1 or 2 diabetes may be beneficial. Studies are being conducted for the commercial production of HUMALOG ® (Eli Lilly Nederland B.V) as part of an intensive basal-bolus insulin treatment for these patients [174].

#### **9.2. Intermediate insulins**

hypoglycemia in the period between meals because regular insulin will peak after food has

Insulin lispro is a human insulin analog developed through genetic engineering by reversing the amino acids proline and lysine at positions 28 and 29 of the β chain, which results in the insulin sequence Lys (B28) Pro (B29). This insulin in its pharmaceutical preparation with phenol and zinc form stable hexamers [168], has a reduced tendency to self-aggregate at the site of subcutaneous injection, is absorbed more rapidly than regu‐ lar human insulin, and mimics the physiological profile of insulin in response to a meal. In addition, its onset of action is between 5 and 15 minutes and duration of action is 1-2 hours [170]. The use of these analogues requires an additional dose in the afternoon to compensate for the hyperglycemia that results from an afternoon snack. There is evi‐ dence that insulin lispro reduces postprandial hyperglycemic peaks and the risk of

In aspart insulin, one proline amino acid is replaced by aspartic acid, which is negatively charged, at position 28 of the β chain, producing electrical repulsion between the insulin molecules and reducing their self-association tendency; in vials or cartridges, it occurs as hexamers, but in subcutaneous tissue, there is rapid dissociation to dimers and monomers, which ensures its rapid absorption and onset of action between 5 and 15 minutes and duration

Insulin glulisine is another ultra-rapid insulin analogue obtained by the exchange of aspara‐ gine for lysine at position 3 of the β chain and lysine for glutamic acid at position 29 of the same chain. Thus far, there have been few studies with glulisine insulin, which appears to be similar to lispro and aspart in efficacy and hypoglycemic events. Because of its faster absorp‐ tion, its administration should be performed only 5-10 minutes before meals to provide greater flexibility for the patient and thereby improve their quality of life. Its shorter half-life reduces the need to eat food 2-3 hours after its administration, which is necessary with regular insulin,

A recent direct and indirect meta-analysis published by Sanches et al. (2013) for glycated hemoglobin reduction outcomes compared rapid action insulin (aspart, glulisine and lispro) with human insulin (regular), and the direct meta-analysis only showed a statistically signif‐ icant difference for aspart, favoring the insulin analog. However, the results of indirect metaanalyses of HbA1c reduction outcomes showed that the result of rapid-acting insulin are consistent and the difference between them is not clinically significant. The ranking suggests that the probability of selecting the short-acting insulin brings the following provision: the first choice should be regular followed by glulisine, lispro and finally aspart. No significant differences were found in the comparison of tolerability outcomes in the rapid-acting insulin

Recent studies have attempted to alter the pharmacokinetics of fast acting insulin analogues that are associated with recombinant human hyaluronidase, and they revealed that absorption was accelerated two-fold during the first half hour of exposure, which resulted in an onset of action between 13 and 25 minutes faster and a shorter duration of effect (40 to 49 minutes).

hypoglycemia compared to regular insulin, especially at night [168, 171].

whose longer half-life causes postprandial hypoglycemia [172].

(aspart and lispro) and human insulin (regular) [173].

been metabolized [169].

166 Treatment of Type 2 Diabetes

of action of 1 to 2 hours[168].

NPH insulin was released in 1946, and it is an insulin suspension in a zinc complex and protamine phosphate buffer. Its dosage is usually once a day before breakfast or twice a day. It has an absorption peak approximately 4-6 hours after subcutaneous administration, which is followed by a steady decline in plasma insulin concentrations [175].

The major disadvantages of NPH are the wide daily variations in the timing and duration of peaks among and between individuals, which, when compared to the timing and duration of long-acting analogs, may result in non-optimal metabolic control and an increased risk for nocturnal hypoglycemia. [172].

With respect to better glycemic control and safety when comparing the use of NPH with longterm insulin analogues, a meta-analysis published in 2013 showed that in type 2 diabetic patients, glycemic control does not seem to differ among different classes, although there is evidence for a possible reduced risk of nocturnal hypoglycemia.

Other information found in the study was that only detemir (and not glargine) may be associated with less of a weight gain than is associated with NPH [176]. In an indirect metaanalysis of long-term insulin, the results of reducing HbA1c are consistent with data from direct comparison meta-analyses and allowed a ranking of probability choice of insulins to improve the reduction of HbA1c as follows: NPH, glargine and detemir [173, 177].

#### **9.3. Basal insulins**

Glargine and detemir insulin analogs represent groups referred to as long-term or basal [168]. Detemir is produced by means of recombinant DNA technology with expression in *Saccharomyces cerevisiae* followed by chemical modification [178]. A fatty acid (myristic acid) is attached to the lysine at position 29, and it binds to circulating albumin, forming a complex that dissociates slowly, thereby prolonging its action time. Detemir is soluble at neutral pH but cannot be mixed with the rapid analogs. Detemir has shown potential benefits in body weight control, with weight loss or decreased weight gain in adults and in children and adolescents [179].

Glargine is synthesized from changes in the amino acid chain of human insulin through a substitution of asparagine by glycine at position A21 and the addition of two arginines at position B30. These modifications result in a unique pattern of release from the injection site, meaning that this analog precipitates in the subcutaneous tissue, allowing a gradual absorption into the bloodstream [180].

Basal insulin has been developed to promote basal levels of insulin over 24 hours and can be administered once a day or at bedtime. When comparing conventional long-acting insulin with glargine insulin, the insulin analog is observed to have a constant concentration profile without prominent peaks [181]. In addition, the onset is between 1 and 2 hours, plateau of biological action is between 4 and 6 hours and termination of effect is between 20 and 24 hours. Because of its slightly acidic pH, glargine cannot be mixed with other insulins in the same syringe; therefore, children may sometimes complain of a burning sensation at the application site [168]. The timing of administration of glargine appears to have no impact on its efficacy for glycemic control, but the administration should occur at approximately the same time each day so that its effectiveness as an insulin is maintained without peak action. If a dose is missed, 50% of the daily insulin will be missing that day [172].

In a direct meta-analysis comparing the insulin analogues (glargine and detemir) to NPH insulin in reducing glycated hemoglobin, the results were statistically significant and favored the twice-daily administration of detemir alone. The safety and tolerability results also showed minor differences between the insulin analogues and NPH insulin [173].

The insulin degludec belongs to a new class of insulin analogues and has a unique absorption mechanism that allows for an ultra-slow and stable pharmacokinetic profile. Its structure differs from human insulin at the β chain termination with removal of threo‐ nine at position B30 and a 16-carbon fatty acid attached to lysine at B29 by glutamic acid. This change allows for the formation of a deposit of soluble multi-hexamers, which accumulate in the subcutaneous tissue and have a slow release because of the dissocia‐ tion of zinc ions; therefore, degludec insulin monomers are circulated in a slow and sustained fashion. In clinical trials, it was observed that the pharmacokinetic variations are four times smaller than in the other long-acting insulin analogues. Studies show that this insulin is related to a lower risk of nocturnal hypoglycemia, and because of these character‐ istics, administration can occur at intervals of up to 40 hours [182, 183].

New insulins, such as U300 and LY2605541 insulins, are still under investigation. Insulin glargine U300 is a new formulation containing glargine in a 300 U/mL concentration (the usual concentration is 100 U/mL). This change alters the pharmacokinetic and pharmacodynamic properties of glargine. For subcutaneous injections, a compact deposit of U300 is administered with a smaller deposit surface, and this produces a more gradual and prolonged release than conventional glargine; therefore, its pharmacokinetic profile is more regular and shows plasma concentrations even beyond 24 hours [184].

Another new insulin, LY2605541, is a long-acting insulin that is a modification of lispro insulin with a 20 kDa polyethylene glycol and half of lysine B28 through covalent urethane, which increases the hydrodynamic size of the insulin complex. This surface provides a greater delay in absorption and reduces the clearance, resulting in a prolongation of its action. This modified insulin has a low affinity for binding to the growth factor receptors linked to insulin, which reduces its mitogenic potential compared to human insulin. Its average life is 24-45 hours, and the duration of action may exceed 36 hours. Animal studies suggest selective action on hepatic metabolism [184].

#### **9.4. Inhalable insulin**

Basal insulin has been developed to promote basal levels of insulin over 24 hours and can be administered once a day or at bedtime. When comparing conventional long-acting insulin with glargine insulin, the insulin analog is observed to have a constant concentration profile without prominent peaks [181]. In addition, the onset is between 1 and 2 hours, plateau of biological action is between 4 and 6 hours and termination of effect is between 20 and 24 hours. Because of its slightly acidic pH, glargine cannot be mixed with other insulins in the same syringe; therefore, children may sometimes complain of a burning sensation at the application site [168]. The timing of administration of glargine appears to have no impact on its efficacy for glycemic control, but the administration should occur at approximately the same time each day so that its effectiveness as an insulin is maintained without peak

action. If a dose is missed, 50% of the daily insulin will be missing that day [172].

minor differences between the insulin analogues and NPH insulin [173].

istics, administration can occur at intervals of up to 40 hours [182, 183].

concentrations even beyond 24 hours [184].

metabolism [184].

168 Treatment of Type 2 Diabetes

In a direct meta-analysis comparing the insulin analogues (glargine and detemir) to NPH insulin in reducing glycated hemoglobin, the results were statistically significant and favored the twice-daily administration of detemir alone. The safety and tolerability results also showed

The insulin degludec belongs to a new class of insulin analogues and has a unique absorption mechanism that allows for an ultra-slow and stable pharmacokinetic profile. Its structure differs from human insulin at the β chain termination with removal of threo‐ nine at position B30 and a 16-carbon fatty acid attached to lysine at B29 by glutamic acid. This change allows for the formation of a deposit of soluble multi-hexamers, which accumulate in the subcutaneous tissue and have a slow release because of the dissocia‐ tion of zinc ions; therefore, degludec insulin monomers are circulated in a slow and sustained fashion. In clinical trials, it was observed that the pharmacokinetic variations are four times smaller than in the other long-acting insulin analogues. Studies show that this insulin is related to a lower risk of nocturnal hypoglycemia, and because of these character‐

New insulins, such as U300 and LY2605541 insulins, are still under investigation. Insulin glargine U300 is a new formulation containing glargine in a 300 U/mL concentration (the usual concentration is 100 U/mL). This change alters the pharmacokinetic and pharmacodynamic properties of glargine. For subcutaneous injections, a compact deposit of U300 is administered with a smaller deposit surface, and this produces a more gradual and prolonged release than conventional glargine; therefore, its pharmacokinetic profile is more regular and shows plasma

Another new insulin, LY2605541, is a long-acting insulin that is a modification of lispro insulin with a 20 kDa polyethylene glycol and half of lysine B28 through covalent urethane, which increases the hydrodynamic size of the insulin complex. This surface provides a greater delay in absorption and reduces the clearance, resulting in a prolongation of its action. This modified insulin has a low affinity for binding to the growth factor receptors linked to insulin, which reduces its mitogenic potential compared to human insulin. Its average life is 24-45 hours, and the duration of action may exceed 36 hours. Animal studies suggest selective action on hepatic The benefit of injectable insulin is often limited because of the difficulty of convincing the patient to adhere to proper treatment, which is related to the need for multiple injections to ensure adequate glycemic control [185].

To alleviate this discomfort, the first inhalable insulin (Exubera®, Pfizer/Nektar) was approved in the U.S. in January 2006. Exubera® consists of a dry powder formulation containing 1 to 3 g of human insulin administered via a single inhaler lung [186]. The technology used in this product was the development of an inhaler for polyethylene glycol in a dry powder that releases the equivalent to 3 UI and 8 UI of short-acting insulin subcutaneously [187]. Exubera® has demonstrated efficacy and a low risk of hypoglycemia; however, there was a poor acceptance by the prescriber and patient. In April 2008, clinical trials showed the first case of cancer, and there were six subsequent cases of lung cancer and a case of primary malignant lung tumor in a patient who had a history of smoking. Other important aspects are coughing, decline of lung function, and increase of anti-insulin antibodies [188]. These facts led the manufacturer to withdraw Exubera® from the market.

The insulin AERx was developed by the Aradigm Corporation and Novo Nordisk. This system generates aerosol droplets from liquid insulin, and the devices guide the user to inhale the insulin. Moreover, it offers the ability to download data on the use of insulin, such as the frequency of inhalation, which can allow for the monitoring of treatment, which is important because of the experience with Exubera® [189].

AFREZZA™ (insulin Technosphere®) overcomes some of the barriers that contributed to the withdrawal of Exubera® from the market. Studies have shown that Technosphere® is a unique formulation of ultra-rapid insulin with a relatively short duration that effectively improves glycemic control without contributing to an increase in weight gain or hypoglycemia com‐ pared to other prandial insulins. Additionally, Technosphere® insulin has shown a favorable safety and tolerability profile in clinical studies to date [190]. Technosphere® insulin (TI) combines the post-dried recombinant human insulin (Mannkind Corp.) with the MedTone® Inhaler (Pharmaceutical Discovery Corp.) [191, 192.]. Recently the FDA approved this insulin; however, a continuation of the studies is required in the post-marketing period.

### **10. Conclusion**

Despite the variety of drugs currently available for the treatment of type 2 diabetes, there was no observed decrease in the number of patients who have inadequate glycemic control keeps the last 10 years. This occurs for a variety of reasons, such as non-adherence to treatment, inappropriate prescribing of medication, lack of efficacy of medicine, among other reasons.

The search of glycemic control in patients with T2DM is still a challenge for patients and health professionals. Importantly, the success of drug treatment also depends on the association with non-pharmacological measures such as healthy diet and exercise.
