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Pharmacology of GLP-1-based therapies Jens Juul Holst British Journal of Diabetes & Vascular Disease 2008 8: S10 DOI: 10.1177/1474651408100523 The online version of this article can be found at: http://dvd.sagepub.com/content/8/2_suppl/S10

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Pharmacology of GLP-1-based therapies JENS JUUL HOLST

Abstract

G

lucagon-like peptide-1 GLP-1 is a naturally occurring 30-amino acid peptide synthesised in intestinal endocrine L cells. GLP-1 mediates glucose homeostasis through stimulation of glucose-dependent insulin secretion, biosynthesis of insulin and inhibition of glucagon secretion. These effects have potential clinical value in type 2 diabetes. However, because native GLP-1 is rapidly degraded to its inactive form by dipeptidyl peptidase-4 (DPP-4), it has a short half-life in vivo. Strategies to overcome this therapeutic limitation include developing GLP-1 mimetics and analogues with longer half-lives and to inhibit DPP-4. Exenatide (exendin-4) is a 39-amino acid peptide originally derived from the venom of the Gila monster lizard, and shares a 53% sequence identity with human GLP-1. Exenatide has a longer circulating half-life, reflecting relative resistance to DPP-4 degradation, and is administered twice daily. Liraglutide is a once-daily human GLP-1 analogue with high (97%) sequence identity. The specific structural modifications that characterise liraglutide result in increased self-association (allowing slow absorption from the subcutaneous depot), promote albumin binding and reduce susceptibility to DPP-4, giving liraglutide a halflife of 13 hours after once-daily administration. Preliminary studies of exenatide and liraglutide show clinically relevant reductions in glycosylated haemoglobin A1c (HbA1c) compared with placebo, without hypoglycaemia and with weight loss of up to 3 kg. DPP-4 inhibitors, such as vildagliptin (not available in the USA) and sitagliptin can help stabilise postprandial GLP-1 levels and thus produce desirable effects on insulin and glucagon production. The potential for weight reduction with DPP-4 inhibitors appears limited, perhaps reflecting the limited increase in GLP-1 levels achieved with these agents. Br J Diabetes Vasc Dis, 2008; 8 (Suppl 2) : S10–S18

Jens Juul Holst

Introduction GLP-1 is a product of the pro-glucagon gene, which encodes a peptide precursor (pro-glucagon) containing 160 amino acids.1 This gene is expressed in the α cells of the pancreatic islets, the L cells of the intestinal mucosa and in the brain.2–4 The proglucagon peptide contains sequences for GRPP (residues 1–30), glucagon (residues 33–61), GLP-1 (residues 78–107/8) and GLP-2, (residues 126–158).1,5 As shown in figure 1, cleavage of

Figure 1.

Pro-glucagon and its processing in the pancreas vs. the gut and brain1,5–9 1

31 32

62 63

1

30 33

61

77

124 125

Pro-glucagon 70 71

Key words: dipeptidyl peptidase-4, glucagon-like peptide-1, glucagon, incretin, insulin release

Pancreas

Glucagon

GRPP

Gut & brain

Department of Biomedical Sciences, The Panum Institute, University of Copenhagen, Denmark Correspondence to: Dr Jens Juul Holst Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200, Copenhagen, Denmark Tel: +45 (0)353 27518; Fax: +45 (0)353 27537 E-mail: holst@mfi.ku.dk

159 160 158

IP -1 64

1

109 110

72

Major pro-glucagon fragment

69 69

Glicentin

107 NH2

78

GLP-1

126

IP-2 IP-2

158

GL P- 2 GLP-2

111 123 1

30 33

GRPP

69

Oxyntomodulin

Key: GLP = glucagon-like peptide; GRPP = glicentin-related pancreatic peptide; IP = intervening peptide Adapted with permission from Holst.6 Copyright © 2007 American Physiological Society.

© SAGE Publications 2008 Los Angeles, London, New Delhi and Singapore

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Abbreviations and acronyms dipeptidyl peptidase-4 glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide glucagon-like peptide-1 glucagon-like peptide-2 glicentin-related pancreatic peptide glycosylated haemoglobin A1c intervening peptide long-acting release left ventricular phosphatidylinositol 3-kinase recombinant glucagon-like peptide-1

GLP-1 GLP-2 GRPP HbA1c IP LAR LV PI3K rGLP-1

Table 1.

Characteristics of the two major incretins: GLP-1 and GIP10–16

GLP-1

GIP

• Produced by L cells

• Produced by K cells in the proximal gut

mainly located in the distaldistal gut (ileum

• Stimulates glucagon secretion

Other effects • Minimal effects on gastric emptying;

glucose output by

no significant effects on satiety or

inhibiting glucagon

body weight

200

Insulin

0 400

GLP-1

200

0 300

GIP

09

13

19

22 09

Hours Key: GIP = glucose-dependent insulinotropic polypeptide; GLP-1 = glucagon-like peptide-1 Adapted with permission from Ørskov et al.24

Physiological effects

secretion in a glucosedependent manner

emptying; reduction of

400

0

dependent insulin release

• Inhibition of gastric

Meal

release

also from proximal gut

• Suppresses hepatic

Meal

• Stimulates glucose-dependent insulin

and colon) but secreted

• Stimulates glucose-

Diurnal mean plasma concentrations (pmol/L) of insulin, GLP-1 and GIP in healthy subjects24

Meal

pmol/L

DPP-4 GIP

Figure 2.

• Potentially enhances β-cell proliferation and survival in islet cell lines

food intake and body weight • Enhances β-cell proliferation and survival in animal models and isolated human islets Key: GIP = glucose-dependent insulinotropic polypeptide; GLP-1 = glucagon-like peptide-1

pro-glucagon in the pancreas differs from that in the gut and brain.1,5,6–9 In the pancreas, cleavage of pro-glucagon results in GRPP, glucagon, an IP-1 and a major pro-glucagon fragment (residues 72–158), whereas in the gut and brain the main products are a glucagon-containing peptide called glicentin (residues 1–69), GLP-1, GLP-2 and a second IP (IP-1, residues 111–123).1,7 In the intestine, part of the glicentin moiety may be further divided into GRPP and a glucagon-like immunoreactant (GLI) called oxyntomodulin.9

Following the ingestion of food, the gut releases so-called incretin hormones, the most important of which are GLP-1 and GIP (table 1).10–16 GLP-1 is produced by L cells located mainly in the ileum and colon, and to a lesser extent by L cells in the duodenum and jejunum, whereas GIP is produced by K cells in the proximal gut.10 Both GLP-1 and GIP powerfully enhance the insulin response to the nutrients that are absorbed from the gut.10 In addition to stimulating insulin release when glucose is elevated, GLP-1 also inhibits glucagon secretion.11,12 These actions are highly glucose dependent.12 In healthy volunteers, administration of GLP-1 has been shown to result in a profound, dose-dependent inhibition of gastric emptying.17 During in vitro and in vivo rodent studies and in isolated human islets, GLP-1 has been shown to promote the expansion of β-cell mass through proliferative and anti-apoptotic pathways.10,13,18–20 Although GIP also stimulates glucose-dependent secretion of insulin, GIP does not appear to markedly affect gastric emptying.10,11,21 When given at supraphysiological doses to subjects with type 2 diabetes, the insulinotropic activity of GIP was less than that observed in normal subjects.22 GIP does not appear to affect satiety or body weight.10 Like GLP-1, GIP may potentially enhance β-cell proliferation and survival in islet cell lines.10,11,14,15

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Effects of incretins on insulin release Stimulation of insulin is greater after oral ingestion of glucose than following intravenous infusion. This phenomenon is called the incretin effect, and 50–70% of the response of insulin to glucose may be caused primarily by GLP-1 and GIP.23

Figure 3.

Second-phase insulin responses to hyperglycemic clamp during intravenous GIP and GLP-1. All subjects were obese (mean body mass index 29 kg/m2): subjects with type 2 diabetes mellitus (n=8), control subjects (n=6)27

Obese diabetic patients Glucose

The incretin effect in normal subjects

The incretin effect in patients with diabetes mellitus Patients with type 2 diabetes mellitus have defects in incretin function, which involve defects in both incretin secretion and action. Thus there is a significant impairment in secretion of GLP-1 after mixed meals (but not always to oral glucose loads).26 Sensitivity of the β cells to GLP-1 is also decreased, but, importantly, supraphysiological amounts of GLP-1 can nevertheless normalise glucose-induced insulin secretion.27 At the same time, secretion of GIP is slightly impaired, but the effect of GIP is grossly impaired or even abolished.22,23,28 A study by Højberg et al. strongly suggests that incretin hormones have lost their potency in type 2 diabetes. When the glucose level was clamped at 15 mM in subjects with type 2 diabetes, physiological infusions of GLP-1 and GIP had little or no effect on insulin secretion.29 By contrast, physiological concentrations of these hormones in healthy subjects stimulated insulin secretion that was within the normal range.29 Another study compared the effect of GLP-1 and GIP on the ‘early’ and ‘late phase’ insulin response to glucose in eight diabetic subjects (mean BMI 31.6 kg/m2) and six matched healthy subjects.27 A hyperglycaemic clamp (15 mmol/L) experiment was performed in the absence or presence of incretin hormones. GIP

Low GIP

5000

Low GIP

High GIP

GLP-1

4000

Insulin (pmol/l)

Measurement of mean plasma concentrations of insulin, GLP-1 and GIP in healthy subjects over a 15-h day in which they consumed three meals showed that both hormones increased significantly and in parallel with insulin rapidly after each meal (figure 2).24 These findings support the premise that GLP-1 and GIP act as incretins to increase postprandial secretion of insulin.24 Increases in GLP-1, GIP and insulin in response to meals were seen in another study in healthy subjects.25 When physiological amounts (i.e., resulting in plasma concentrations similar to those observed after mixed meal ingestion) of either GIP (1.5 pmol/kg/min) or GLP-1 (0.33 pmol/kg/min) were each infused for 30 min under conditions in which glucose was clamped at each subject’s fasting level (mean 5.1, range 4.8–5.3 mmol/L [86.5–95.5 mg/dL]) and then increased to 6 mmol/L (108 mg/dL) and 7 mmol/L (126 mg/dL), significant increases in insulin were seen. At 7 mmol/L, GLP-1 had a greater effect on insulin concentrations than GIP.25 By comparison, glucose plus saline infusions induced only minor increases in the concentrations of insulin. The responses to GLP-1 and GIP could easily explain the entire insulin response after a meal. In the clamp studies, infusion of glucose alone caused a significant decrease in glucagon concentrations, which was augmented by infusion of GLP-1 but not GIP.25 Researchers concluded that both GLP-1 and GIP contribute approximately equally to the incretin effect of a meal in normal subjects.23

Obese control subjects Glucose

3000 2000 1000 0 −20

80

180

Time (min) Key: GIP = glucose-dependent insulinotropic polypeptide; GLP-1 = glucagon-like peptide-1 Adapted with permission from Vilsbøll et al.27 Copyright © SpringerVerlag 2002.

at 4 and 16 pmol/kg/min or GLP-1 (7–36) at 1 pmol/kg/min were infused continuously to provide prolonged stimulation of the β cell.27 Plasma insulin and glucagon levels were measured before and for up to 240 min after the increase of plasma glucose in diabetic subjects and for up to 120 min in healthy subjects.27 As shown in figure 3, the late-phase (20–120 min) insulin response to glucose was lower in subjects with type 2 diabetes than in normal subjects.27 In subjects with type 2 diabetes, intravenous administration of GLP-1 at 1 pmol/kg/min led to a significant increase in the late glucose-independent insulin response compared with GIP administration at 4 pmol/kg/min (area under the curve 20–120; 97.2 vs. 22.2 [100 min·nmol/L], p<0.01). This GLP-1 effect was similar to the one observed in healthy subjects after glucose infusion.27 However, GIP infused at 4 pmol/kg/min or even 16 pmol/kg/min was not able to increase the glucosedependent insulin response in subjects with type 2 diabetes. Glucose infusion without incretins decreased glucagon levels from baseline (fasting) at 120 min (10 vs. 2.7 pmol/L) in healthy subjects, but induced only a small decrease in subjects with type 2 diabetes (8.4 vs. 5.8 pmol/L). However, GLP-1 stimulation led to a marked decrease in glucagon from baseline (9.5 vs. 2.9 pmol/L) in subjects with type 2 diabetes. Administration of GIP (4 pmol/kg/min) had no effect on the glucagon response in either healthy subjects or those with type 2 diabetes.27 Thus, in obese subjects with type 2 diabetes, amplification of the late-phase insulin response by GIP is defective, whereas the late-phase insulin response to GLP-1 is preserved.27 The strong inhibition of glucagon secretion by GLP-1 in subjects with type 2 diabetes shows that, although potency is reduced, pharmacological doses

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Table 2.

Therapeutic potential of GLP-1 in type 2 diabetes mellitus

Type 2 diabetic phenotype Impaired β-cell function

Actions of GLP-1 • Increases insulin secretion and biosynthesis • Improves β-cell function (glucose sensitivity, proinsulin: insulin ratio) • Upregulates other genes essential for β-cell function (e.g., GLUT-2, glucokinase) ↓

Reduced β-cell mass

β-cell proliferation/differentiation

Clearly, there are many reasons to believe that GLP-1 has therapeutic potential in type 2 diabetes mellitus. As shown in table 2, if β-cell function is impaired, GLP-1 can increase insulin secretion and biosynthesis, improve β-cell function (i.e. glucose sensitivity and the proinsulin: insulin ratio) and upregulate other genes essential for β-cell function (e.g. GLUT-2, glucokinase).31 If β-cell mass is reduced, animal studies have shown that GLP-1 can increase β-cell proliferation or differentiation. In vitro studies in human islet cells have also shown that GLP-1 can inhibit β-cell apoptosis.32 A very important parameter in type 2 diabetes – glucagon hypersecretion – is decreased by GLP-1. GLP-1 can also improve overeating and obesity by decreasing gastric emptying and appetite, and increasing satiety.6 As will be described subsequently, GLP-1 can also have beneficial cardiovascular effects.6

(animal studies) ↓ β-cell apoptosis (in vitro studies) Glucagon hypersecretion

↓ Glucagon secretion

Overeating, obesity

↓ Gastric emptying,

satiety,

↓ appetite, ↓ food intake, weight loss Macrovascular complications

Beneficial cardiovascular effects

Key: GLP-1 = glucagon-like peptide-1; GLUT-2 = glucose transporter isoform-2

of this hormone are able to restore the normal response to glucose of both α and β cells of the pancreatic islets.27

Therapeutic potential of GLP-1 in type 2 diabetes mellitus The preliminary physiological clinical investigations of incretins raise an intriguing question: if the impaired incretin response contributes significantly to the defective insulin secretion in type 2 diabetes mellitus, will restoration of incretin action by pharmacological amounts of GLP-1 improve metabolism? Results of some preliminary studies are beginning to suggest that it can. In a pilot study in which patients with type 2 diabetes were assigned alternately to continuous subcutaneous infusions of GLP-1 (n=10) or saline (n=9) for 6 weeks, the only significant change in the saline group was an increase in fructosamine concentrations (p=0.0004). By contrast, in the GLP-1 group, fasting and mean plasma glucose were reduced by 4.3 and 5.5 mmol/L, respectively (p<0.0001); HbA1c decreased by 1.3% (p=0.003); and fructosamine decreased to normal levels (mean 282 µmol/L, range 246–319; p=0.0002).30 GLP-1 significantly inhibited gastric emptying (p=0.014), and body weight decreased steadily throughout the 6 weeks, with a mean decrease of 1.9 kg at week 6, presumably because of decreased appetite.30 Insulin sensitivity in the GLP-1 group increased by 77.3% (p=0.002) and β-cell function improved significantly, despite the fact that the dose and mode of administration were suboptimal.30 GLP-1 administration was not associated with any important side effects.30

Protection against β-cell apoptosis A very interesting effect of GLP-1 appears to be that it protects β cells against cell death induced by elevated glucose or nonesterified fatty acids. One of the most interesting studies demonstrating this was conducted by Buteau and colleagues.32 Human islet cells were exposed to 5 or 25 mmol/L glucose in the presence or absence of palmitate. The combination of a high glucose concentration with palmitate increased cell death to 50%. Treatment with GLP-1 decreased cell death to 20%, demonstrating protection against β-cell glucolipotoxicity.32

Effects on gastric emptying and energy intake Others have investigated the effects of GLP-1 on gastric emptying and energy intake.17,33 Nauck and colleagues studied the effects of physiological and pharmacological doses of GLP-1 on gastric emptying in normal volunteers given a liquid meal intragastrically (50 g sucrose, 8% amino acids, 440 ml, 327 kcal).17 Intravenous infusion of GLP-1 (7–36 amide) at doses of 0.4, 0.8 and 1.2 pmol/kg/min beginning 30 min prior to the meal and continuing for 240 min after the meal inhibited gastric emptying in a dose-dependent manner.17 The increase in plasma glucose following infusion of the test meal began to decline with initiation of the intravenous infusion of GLP-1, and eventually meal-related increases in glycaemia were abolished.17 A meta-analysis of data from seven previously published studies and two then-unpublished studies examined the effects of GLP-1 on the rate of gastric emptying and energy intake in both lean and overweight healthy subjects and people with type 2 diabetes.33 As in the previous study, three studies in overweight subjects showed that infusion of GLP-1 decreased the rate of gastric emptying.33 Pooled observations from all nine studies (147 observations) showed that, compared with those given a control infusion, subjects receiving the GLP-1 infusion demonstrated a mean reduction in ad libitum energy intake of 11.7%.33 Although lean subjects had a higher absolute reduction in energy intake than overweight subjects, the relative reductions in the two groups did not differ significantly (13.2 and 9.3%, respectively). The only independent predictor of the reduction in energy intake was the rate of GLP1 infusion.33 Importantly, the infusion of GLP-1 did not have

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Table 3.

Cardiovascular effects of GLP-1

Table 4.

Possible categories of incretin mimetics and incretin enhancers

1. There are GLP-1 receptors in the heart34,35 •

Metabolically stable activators of the GLP-1 receptor (exendin and

3. Anti-hypertensive effects of GLP-1 in salt-sensitive Dahl rats

Slow-release formulations of exendin or GLP-1 analogues

4. GLP-1 improves cardiac function in dogs with chronic heart failure

Covalent or non-covalent association of GLP-1 with large proteins

2. Mice with a GLP-1 receptor knockout have impaired left

exendin derivatives)

36

ventricular contractility and diastolic functions

37

(albumins) to lengthen half-lives

(re: treatment)38 5. GLP-1 protects rat myocardium against subsequent ischaemia/ 39

reperfusion injury (re: prevention)

6. Administration of GLP-1 to patients with acute myocardial

Inhibitors of DPP-4 (small orally active molecules)

Small molecule activators of GLP-1 receptor

Key: DPP-4 = dipeptidyl peptidase-4; GLP-1 = glucagon-like peptide-1

infarction and LVEF of 29% after angioplasty improved LVEF to 39%40 7. GLP-1 improves endothelial dysfunction in type 2 diabetes mellitus 41

patients with coronary heart disease

Key: GLP-1 = glucagon-like peptide-1; LVEF = left ventricular ejection fraction

any adverse effect on well-being. The investigators suggest that the increased satiety seen with GLP-1 may be due, but only partly, to the reduced rate of gastric emptying.33

Cardiovascular effects The cardiovascular effects of GLP-1 are summarised in table 3.34–41 GLP-1 receptors have been identified in the heart.34 Mice with genetic deletion of the GLP-1 receptor show impaired left ventricular contractility and diastolic function following the administration of insulin, providing evidence that GLP-1 plays a key role in the control of cardiac structure and function.36 Many of the phenotypic characteristics of salt-sensitive hypertension in humans are exhibited by Dahl salt-sensitive (Dahl S) rats. In rats fed a high-salt diet and infused with rGLP-1 or vehicle for 14 days, rGLP-1 had anti-hypertensive effects, reduced cardiac and renal end organ damage and improved endothelial function.37 The antihypertensive effect of rGLP-1 was due mainly to diuretic and natriuretic actions, preventing retention of sodium and water.37 GLP-1 also has favourable effects on the myocardium. In one study, conscious dogs with advanced dilated cardiomyopathy induced by 28 days of rapid pacing received either a 48-h infusion of rGLP-1 (1.5 pmol/kg/min) or saline (3 mL/day). Dogs given rGLP-1 showed significant (p<0.02) increases in LV dP/dt (98%), stroke volume (102%) and cardiac output (57%), and decreases in LV end-diastolic pressure, heart rate and systemic vascular resistance. Myocardial insulin sensitivity and glucose uptake were increased also. These effects suggest that rGLP-1 may be useful in the treatment of heart failure.38 In another study of rats subjected to 30 min of regional ischaemia induced by coronary artery occlusion and followed by 120 min of reperfusion, administration during stabilisation prior to occlusion of GLP-1 and valine pyrrolidide (VP), an inhibitor of DPP-4 which degrades native GLP-1, significantly (p<0.001) reduced infarction

compared with groups given VP or saline alone.39 In vitro experiments in rat hearts showed that the myocardial protective effect of GLP-1 was abolished by the GLP-1 antagonist exendin (9–39), the cyclic AMP inhibitor Rp-cAMP, and a PI3K inhibitor. These findings suggest that each of these pathways is needed for protection by GLP-1.39 Studies in patients with acute myocardial infarction and in those with type 2 diabetes and stable coronary artery disease have also documented beneficial effects following GLP-1 administration.40,41 In one study, patients with acute myocardial infarction and LV dysfunction (LV ejection fraction < 40%) underwent successful primary angioplasty followed by a 72-h infusion of GLP-1 (1.5 pmol/kg/min) in addition to medical and interventional therapy.40 Echocardiograms obtained after reperfusion and after completion of the GLP-1 infusion showed that, compared with untreated controls, patients receiving GLP-1 had significantly (p<0.01) improved LV ejection fraction (from 29 to 39%), global wall motion score indexes (1.94→1.63) and regional wall motion score indexes (2.53→2.02).40 The benefits of GLP-1 were seen in both diabetic and non-diabetic patients, as well as in patients with anterior and non-anterior myocardial infarction.40 In patients with type 2 diabetes and stable coronary artery disease, a randomised crossover study demonstrated that rGLP-1 acutely improved endothelial dysfunction, but not insulin resistance.41

Exploiting the therapeutic potential of GLP-1 Two problems have to be overcome to develop a pharmacologically active agent: rapid degradation of GLP-1 by DPP-4 and rapid elimination via the kidneys. In human plasma the GLP-1 molecule (7–36 amide) is degraded by DPP-4, which cleaves the two N-terminal amino acids. This leaves GLP-1 (9–36 amide), which is inactive with respect to insulin secretion.42 This process occurs very rapidly, decreasing the half-life of intact GLP-1 in the circulation to 1–2 min.43 The plasma clearance of GLP-1 after intravenous bolus administration of high (15 or 25 nmol) doses to both diabetic and healthy subjects was calculated to be 3–10 L/min, which is two to four times the cardiac plasma output.43 A number of strategies to overcome the problems of rapid degradation and elimination are listed in table 4. VOLUME 8 SUPPL 2 . NOVEMBER/DECEMBER 2008

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Exenatide: a metabolically stable activator of the GLP-1 receptor Exendin-4 from the saliva of the Gila monster (Heloderma suspectum) is 53% homologous with GLP-1; it is a potent, degradation-resistant agonist on the GLP-1 receptor.44,45 Exendin-4 is not the native GLP-1 of this animal, but a special peptide that is produced in their salivary glands.2 A synthetic form of the 39-peptide amino acid peptide exendin-4 called exenatide has been developed.46 In subjects with type 2 diabetes, exenatide exhibited dose-proportional kinetics following administration of single subcutaneous doses of 0.1, 0.2, 0.3 and 0.4 µg/kg 5 min before a meal.46 Peak plasma concentrations of exenatide were achieved between 2 and 3 h, and drug concentrations of at least 100 pg/ml were present at 5 h post-dose.46 At doses of 0.2 µg/kg and higher, exenatide was still detectable at 15 h post-dose, confirming its prolonged half-life and resistance to DPP-4 degradation.46 The mean terminal half-life of exenatide is 2.4 h, and in most patients, drug concentrations are measurable for approximately 10 h post-dose.47 Therefore, 10 µg exenatide given twice daily (the maximal recommended dose) may not provide complete coverage after midday meals and overnight.48 Results of three 30-week, randomised, triple-blind, placebo-controlled trials in type 2 diabetic patients with inadequate glycaemic control on maximally effective doses of a sulphonylurea, metformin or both, showed that the addition of exenatide given subcutaneously in doses of 5 or 10 µg twice daily before breakfast and dinner significantly reduced HbA1c and was associated with dose-dependent, progressive weight loss.49–51 Exenatide treatment was associated with a dosedependent decrease in the proinsulin-to-insulin ratio toward more physiological proportions, indicating that the drug had a beneficial effect on the β cell.49,50 A modest reduction in fasting plasma glucose was seen at week 30.49–51 The percentages of patients with detectable anti-exenatide antibody titres at 30 weeks ranged from 41 to 49%, but the presence of antibodies had no predictive effect on the extent of a subject’s glycaemic response or the occurrence of adverse events.49–51 Sustained reductions in HbA1c and progressive reductions in body weight were seen in patients who continued adjunctive open-label treatment with exenatide for a total of 1.5 years (82 months).52 A sustained LAR formulation of exenatide incorporating biodegradable polymeric microspheres is under development.48 A randomised, placebo-controlled, phase II study compared exenatide LAR 0.8 mg (n=16) or 2.0 mg (n=15) and placebo injected subcutaneously once weekly for 15 weeks in 45 subjects with type 2 diabetes who failed to achieve optimal control with metformin or diet and exercise.48 At week 15, both the 0.8 and the 2.0 mg doses of exenatide LAR significantly reduced mean HbA1c (−1.4±0.3% and −1.7±0.3%, p<0.0001 for both) and fasting plasma glucose (−2.4±0.9 mmol/L [−43.2±16.2 mg/dL] and −2.2±0.5 mmol/L [−39.6±9.0 mg/dL], p<0.001 for both) from baseline.48 Significant reductions in body weight (−3.8±1.4 kg, p<0.05) were recorded only in subjects receiving the 2.0 mg dose of exenatide LAR.48

Albumin-based GLP-1 analogues and conjugates Another approach to developing a pharmacological agent that resists degradation by DPP-4 is to conjugate GLP-1 with various moieties that will facilitate binding to albumin, thereby slowing elimination kinetics.53 Three compounds using different methods to achieve this are in development: an acetylated derivative of GLP-1 that binds non-covalently to albumin (liraglutide; NovoNordisk, Denmark); a conjugate of exendin-4 and albumin (Conjuchem, Canada); and a fusion protein consisting of a DPP4-resistant GLP-1 analogue covalently bound to human albumin (Albugon; Human Genome Sciences, USA).53 Of these, only liraglutide is currently at a late stage of clinical development. Liraglutide is a fatty-acid derivative of GLP-1 that has improved pharmacokinetics compared with native GLP-1 while maintaining biological activity.54 In a double-blind, randomised, dose-escalation, placebo-controlled study, liraglutide was administered subcutaneously at five consecutive dose levels of 1.25, 5.0, 7.5, 10.0 and 12.5 µg/kg to healthy subjects. Maximum plasma concentrations were reached at 10–14 h post-dose.54 The mean elimination half-life of liraglutide was 12.6±1.1 h.54 Following repeated administration of all but the 5.0 µg/kg dose, there was statistically significant accumulation of liraglutide (Rac=1.4–1.5).54 The increased half-life seen with liraglutide supports once-daily dosing, and is probably due to a combination of factors, including stability against DPP-4 degradation, a high degree of albumin binding and slow absorption from the injection site.55 A recent study also found that administration of a single subcutaneous injection of liraglutide (7.5 µg/kg) to subjects with type 2 diabetes 9 h before a graded glucose infusion restored β-cell responsiveness to physiological hyperglycaemia.56

Inhibitors of DPP-4 Another strategy to improve the pharmacokinetics of GLP-1 is to develop selective protease inhibitors that prevent degradation by DPP-4.57 Agents undergoing clinical investigation

Table 5.

Incretin-based therapy of type 2 diabetes mellitus: incretin mimetics vs. incretin enhancers

Mimetics •

High plasma concentrations of GLP-1-receptor activator

Strong effects on all GLP-1 receptors; appetite, food intake, body weight, cardiovascular effects, β cells, α cells

Tendency to cause gastrointestinal side effects

Injectables (so far)

Enhancers •

Orally active, once daily

Very few side effects

Modest elevations of incretin hormone concentrations

Weight neutral, and no gastrointestinal side effects

Key: GLP-1 = glucagon-like peptide-1

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the potential to protect β cells in humans, but data are lacking. A final, crucial question concerns efficacy: will these agents prove sufficiently potent to lower HbA1c to a target of 6.5%, the goal of antidiabetic therapy? Future research is needed to answer these questions.

Key messages

GLP-1 enhances insulin secretion and insulin sensitivity and inhibits glucagon secretion, hepatic glucose production, gastric emptying, appetite and food intake, and has beneficial actions on the cardiovascular system. GLP-1 therefore targets several metabolic disturbances in type 2 diabetes ● GLP-1 causes proliferation of β-cells and recruitment of new β-cells from pancreatic duct epithelium in rodents and inhibits β-cell apoptosis rates in rodent and human β-cells. It may therefore exert protective effects on the diabetic β cells and thereby modify disease progression. ●

References 1. 2.

3.

4. 5.

6.

include saxagliptin and alogliptin.57,58 Randomised, controlled trials lasting from 12 weeks to up to 1 year conducted with vildagliptin (not available in the USA) and sitagliptin, both of which are given orally, demonstrated modest efficacy.59 Noninferiority has been established in trials comparing sitagliptin with glipizide and vildagliptin with thiazolidinediones, but not when vildagliptin was compared with metformin.59–63 Less information is available regarding alogliptin and saxagliptin.59

7.

Discussion

11.

Current knowledge regarding incretin-based therapies of type 2 diabetes mellitus is summarised in table 5. Two therapeutic approaches – incretin mimetics and incretin enhancers – are possible. The incretin mimetics exenatide and liraglutide, which are related to the GLP-1 molecule, achieve high plasma concentrations of the GLP-1 receptor activator and have strong effects on all GLP-1 receptors. These agents positively affect appetite, food intake, body weight, the cardiovascular system and the pancreatic β and α cells. They tend to cause gastrointestinal side effects, and at present these agents are administered via subcutaneous injection. Exenatide is administered twice daily, but a long-acting release formulation is undergoing clinical trials. The incretin action enhancers vildagliptin and sitagliptin are orally active and can be administered once daily. They are associated with very few side effects and are devoid of gastrointestinal side effects. These agents cause modest elevations of incretin hormone concentrations and are weight neutral. A number of questions regarding these incretin mimetics and incretin action enhancers remain unanswered at present. Incretin mimetics with relatively low amino acid sequence homology like exendin are antigenic, and it has not been excluded that they may have effects apart from their actions on GLP-1. The mechanism of action of DPP-4 inhibitors remains unclear, and the consequences of prolonged DPP-4 inhibition are not known. These are small molecules and may affect other targets. Both incretin mimetics and the DPP-4 inhibitors have

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