Glucagon-like peptide 1 receptor agonists and strategies to improve

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Glucagon-like peptide 1 receptor agonists and strategies to improve their efficiency Seyed Ebrahim Alavi, Peter J. Cabot, and Peter Michael Moyle Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00308 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 5, 2019

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Molecular Pharmaceutics

Glucagon-like peptide 1 receptor agonists and strategies to improve their efficiency Seyed Ebrahim Alavi, *,† Peter J. Cabot,† and Peter M. Moyle

†

School of Pharmacy, The University of Queensland, Pharmacy Australia Centre of Excellence, Woolloongabba 4102, QLD, Australia

ORCID Seyed Ebrahim Alavi: https://orcid.org/0000-0003-4009-4921

*

E-mail: [email protected]

Full Postal Address: School of Pharmacy, The University of Queensland, Pharmacy Australia Centre of Excellence, 20 Cornwall Street, Woolloongabba 4102, QLD, Australia. Phone: +61403521527

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Peter J. Cabot: https://orcid.org/0000-0003-1778-3753

Peter Michael Moyle: https://orcid.org/0000-0002-9247-681X

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ABSTRACT

Type 2 diabetes mellitus (T2DM) is increasing in global prevalence and is associated with serious health problems (e.g. cardiovascular disease). Various treatment options are available for T2DM, including the incretin hormone glucagon-like peptide-1 (GLP1). GLP-1 is a therapeutic peptide secreted from the intestines following food intake, which stimulates the secretion of insulin from the pancreas. The native GLP-1 has a very short plasma half-life, owning to renal clearance and degradation by the enzyme dipeptidyl peptidase-4. To overcome this issue, various GLP-1 agonists with increased resistance to proteolytic degradation and reduced renal clearance have been developed, with several currently marketed. Strategies, such as controlled release delivery systems, methods to reduce renal clearance (e.g. PEGylation and conjugation to antibodies), and methods to improve proteolytic stability (e.g. stapling, cyclization, and glycosylation) provide means to further improve the ability of GLP-1 analogs. These will be discussed in this literature review.

KEYWORDS

3



Combination therapy, Glucagon-like peptide-1, GLP-1 receptor agonists, Peptide delivery, Type 2 diabetes

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INTRODUCTION

Type 2 diabetes mellitus (T2DM) accounts for 90-95% of people affected with diabetes,1 and its prevalence is increasing throughout the world.2 According to the International Diabetes Federation (IDF), 382.5 million people were affected by T2DM in 2015.3 It is expected that this number will exceed 578 million in 20 years.3 Complex genetic predisposition along with behavorial and environmental risk factors, such as physical inactivity and obesity, are involved in the development of T2DM.4 It is a multifunctional complex disease associated with comorbidities, such as dyslipidemia, obesity and hypertension.5 The risk of cardiovascular disease associated with T2DM compared to healthy individuals is more than doubled, and then life expectancy is decreased by an average of 7 years.6 Physical inactivity is also a risk factor for T2DM, accounting for 7% of cases affected with T2DM.7 Bommer et al. reported that, in 2015, 1.8% of the gross domestic product, equal to US$ 1.13 trillion, was expended on diabetes worldwide. Two thirds of this amount, equal to US$ 857 billion, was related to direct medical costs, and one third of this value was related to indirect costs, such as lost productivity.8 Most drugs used to treat T2DM aim to reduce hyperglycemia;

5



however, the treatment of the associated comorbidities is also important to decrease the risk of complications (e.g. cardiovascular disease).5 Lifestyle changes are an effective means to improve glycemic (literally means causing glucose in the blood) control, blood pressure, body weight and lipid profiles.9 However, these behavorial modifications are intrinsically laborious and most patients eventually require medication.9 Insulin resistance and consequently decreased insulin secretion play crucial roles in T2DM pathogenesis.10 Incretins are hormones secreted from the gastrointestinal tract, which are responsible for increasing the insulin secretion.11 Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are the main incretins.12 Furthermore, the activation of GLP-1 and GIP receptors results in non-glycemic effects, such as body weight loss, the reduction of systolic and diastolic blood pressure, the improvement of endothelial dysfunction, and the reduction of plasma C-reactive protein levels,13 on different tissues, through direct effects on tissues expressing incretin receptors and indirect mechanisms mediated by neuronal and endocrine pathways.14 Incretin hormones are responsible for approximately 70% of glucose6


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stimulated insulin release.15 Although, in most T2DM patients, the release of GLP-1 is decreased by 15%,16 the insulinotropic effects of GLP-1 are preserved. However, at supraphysiological doses, GLP-1 is able to elicit strong insulin secretion.17 In contrast to GLP-1, GIP secretion is maintained at physiological or higher concentrations in T2DM patients.18 However, responsiveness to GIP in T2DM patients is decreased considerably even at supraphysiological doses,19 due to GIP-receptor down regulation or desensitization.20 Therefore, among incretin hormones, GLP-1, as a therapeutic agent, has received considerable attention. This literature review provides an overview of peptide and protein drugs in terms of their production, specially their recombinant production and discusses the current state of GLP-1 receptor agonists (GLP-1RAs) and future directions for their development with an emphasis on approaches to obtain agonists with longer plasma half-lives. These include strategies, such as conjugation to antibody fragments, PEGylation, controlled drug delivery systems, cyclization, modification of the GLP-1 sequence, peptidomimetics, stapling, and glycosylation, which will be discussed.

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PEPTIDE AND PROTEIN DRUGS Peptide drugs are generally obtained from three sources: (a) natural, (b) recombinant (c) and synthetic. Almost all therapeutic peptides have been identified from natural sources21 as bioactive peptides are subjected to natural selection, and consequently their stability is enhanced in vivo environment.22 It has been confirmed that bioactive peptides are highly selective and potent molecules toward various receptors. Several bioactive peptides have been widely studied and are on the market, including GLP-1, exendin-4 and insulin for the treatment of T2DM,23, 24 GnRH agonists and antagonists for the treatment of prostate cancer25 and endometriosis-associated pelvic pain,26 respectively, icatibant for the treatment of hereditary angioedema,

27

and defensins

which display antimicrobial activity.28 Peptides have advantages of both small-molecule drugs and proteins, such as having fewer side effects and specific activity.29, 30 Also, peptides are non-toxic and biodegradable and can be considered as safe metabolites. They have minimal potential for drug-drug interactions and are less probable to produce an immune response compared to larger proteins.31 Over the past decade, peptides have received

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considerable attention for use in medicine. By 2018, more than 60 peptide medicines have been approved by Food and Drug Administration (FDA) and reached to the market. This number is continually increasing, and approximately 140 candidate peptide drugs are now in clinical trials.32 Peptide drugs are great opportunity to treat certain diseases, where small molecule drugs have been proven to be unsuccessful.29 The advantages of peptide drugs can be described as follows: (a) due to highly specific function of therapeutic peptides, their interference with normal biological processes is decreased, resulting in reducing adverse side effects, (b) since many peptides used for therapeutic purposes are produced in the body, therapeutic peptides are therefore likely to be well-tolerated and less likely to stimulate immune responses, (c) in genetic disorders, where a gene is mutated, therapeutic peptides can serve as a replacement in some cases, (d) and therapeutic peptides have received FDA approval faster than small-molecule drugs.33 However, despite their clear advantages, the clinical use of proteins and peptides has faced considerable drawbacks, owing to low bioavailability and metabolic instability.34 Production of Recombinant Peptides and Proteins 9



Mammalian cells, such as Chinese Hamster Ovary (CHO) cells,35 and microorganisms, such as yeast 36 and Escherichia coli (E. Coli),37 are the main options for producing therapeutic proteins. Also, CHO and yeast have capacity to ensure protein folding, assembly and post-translational modifications.36-38 Establishing a cell line for producing therapeutic proteins is initiated by transfecting a mammalian cell line with a plasmid vector containing a gene encoding for a therapeutic protein. Later, the stable transfected cell line with the high productivity of protein is obtained through an antibiotic selection process.39

WHAT IS GLP-1? GLP-1 is a posttranslational proteolytic product and tissue specific peptide derived from the proglucagon gene. GLP-1 is released from intestinal L-cells in response to nutrient ingestion.40 This peptide hormone causes lower blood glucose level (BGL) and people’s weight by reducing appetite and glucagon secretion, increasing glucosedependent insulin release and delaying gastric emptying.41 Also, GLP-1 improves memory and learning;42 has neuroprotective and anti-inflammatory properties;43 has cardioprotective function during myocardial ischemia, which together reduce 10


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cardiovascular disease risk factors, such as hypertension, dyslipidaemia and obesity;44 improve hepatocyte function;45 and regulate energy balance through food intake regulation (energy inflow) and energy consumption (energy outflow). GLP-1 release is triggered by individual nutrients or mixed meals, containing glucose and other sugars, dietary fiber, fatty acids and essential amino acids. In humans, GLP-1 secretion only occurs after oral glucose ingestion not intravenous glucose administration. The secretion of GLP-1 occurs in a biphasic manner initiated with an early phase (within 10-15 min) and continued by a longer second phase (30-60 min), followed by quickly released into the circulatory system.40 As most L-cells, expressing GLP-1, are located in the distal small intestine (ileum) and insulin is secreted before the nutrients reach the ileum, it is unlikely that the early phase of GLP-1 secretion is associated by nutrient contact with L-cells.40, 46 Various studies have reported that the three components of autonomic nervous system including the neurotransmitters acetylcholine, gastrinreleasing peptide (GRP) and GIP contribute to the quick GLP-1 release after nutrients ingestion.40

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GLP-1 gene in humans is 40 kbp, and comprises at least 7 exons located on chromosome 6, band p21.1. There are several types of secreted GLP-1 in vivo, including inactive GLP-1, such as GLP-1(1-37) and GLP-1(1-36)-NH2, and biologically active GLP-1, such as GLP-1(7-37) and GLP-1(7-36)-NH2.40 The major form of circulating GLP-1 in humans is GLP-1(7-36)-NH2.47 The blood half-life of bioactive GLP-1(7-36)-NH2 and GLP-1(7-37) are less than 2 minutes, due to inactivation by the protease enzyme dipeptidyl peptidase-4 (DPPIV) and rapid renal clearance.40,

48, 49

DPPIV cleaves N-terminal dipeptides where the penultimate amino acid residue in the peptide is a proline or alanine.50 Obesity is an appetite disorder 51 and is a risk factor of T2DM.52 In the brain, GLP-1 mimetics are able to protect synapses from the harmful effects of β-amyloid on synaptic plasticity of hippocampus.53 This effect is most probably triggered by the activation of GLP-1 receptors (GLP-1Rs) on neurons, perhaps at the site of presynaptic.53 In rodents, GLP-1 also increases β-cell mass by stimulating neogenesis and proliferation in addition to reducing β cell apoptosis.54

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All GLP-1 functions are mediated through binding to GLP-1 receptors.55 These are class B 7-transmembrane-spanning, heterotrimeric G-protein–coupled receptors.40 Many tissues are reported to express GLP-1Rs at high levels, including the lungs and pancreas, with lower levels in the stomach, intestine, kidney, heart, brain

56

and

hepatocytes.57 Studies have shown that C cells in the thyroid glands express GLP1Rs.58 In the brain, GLP-1 is expressed in astrocytes and microglia, and GLP-1Rs are expressed on glial cells when they are activated by inflammatory responses.59 Further, neurons in the brain, especially pyramidal neurons of the hippocampus and neocortex, as well as Purkinje cells of the cerebellum, express GLP-1Rs.43 The N-terminal extracellular region of GLP-1Rs is crucial for GLP-1 binding, while distinct domains in the third intracellular loop are essential for receptor to pairing with specific G-proteins, including Gαs, Gαq, Gαi, and Gαo.60, 61 GLP-1Rs are complexed with lipid rafts, and interact with caveolin-1 to adjust their subcellular localization, trafficking and signaling.40 GLP-1RAs are associated with various biological functions in the pancreas, including stimulation of glucose-dependent insulin release.62 GLP-1 binding to GLP-1Rs on 13



pancreatic β-cells gives rise to the activation of adenylate cyclase activity and the generation of cyclic Adenosine Monophosphate (cAMP).40 cAMP subsequently activates protein kinase A, Epac 1 and 2, leading to motivating insulin secretion in a glucose dependent manner 63 by the following mechanisms: (a) the direct blockage of Adenosine triphosphate (ATP)-sensitive K+ channels (K ATP channels), which in turn results in β-cell membrane depolarization; (b) increases in intracellular Ca2+ levels, due to the GLP-1–dependent influx of extracellular Ca2+ through voltage-dependent Ca2+ channels, the activation of non-selective cation channels and the mobilization of intracellular Ca2+ reservoirs; (c) the augmentation of mitochondrial ATP synthesis, leading to membrane depolarization; (d) the closure of voltage-dependent K+ (Kv) channels, resulting in decreased Kv currents, hampering β-cell repolarization; and (e) direct effects on the exocytosis of β-cell insulin storage granules through increasing ATP and intracellular Ca2+ (Figure 1).40

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Figure 1. The signaling mechanism of GLP-1 associates with GLP-1R and Adenylate Cyclase (AC) activation. AC increases the intracellular concentration of cAMP, which consequently activates exchange protein activated by cAMP (EPAC) and increases intracellular Ca2+. Ca2+, in turn, activates the calcineurin/nuclear factor of activated T (NFAT) cells, leading to insulin secretion.

GLP-1 Production Methods

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There are several technologies for protein and peptide synthesis, including recombinant DNA technology (e.g. in E. coli, yeast, mammalian cells, etc.), cell-free expression systems, extraction from natural sources, transgenic animals and plants, and chemical synthesis.64 The methods used for GLP-1 synthesis are summarized in Table 1.

Table 1. Examples of methods for GLP-1 production

Methods Recombinant

Referenc

Description

es

GLP-1(7-36)-NH2 (mini-intein system)

65

GLP-1(7–36)-NH2 (HEK293E cells)

66

Chemical synthesis

GLP-1(7-36)-NH2 (microwave-assisted)

67

Chemical synthesis

GLP-1(7-36, A8G)-NH2, (microwave assisted)

68

Transgenic plants

GLP-1x10 (tobacco plants)

69

Transgenic plants

GLP-1 (transgenic rice plants)

70

expression Recombinant expression

16


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However, as previously mentioned, native GLP-1 is not suitable for treating T2DM, due to its short half-life71 through its inactivation by the enzyme DPPIV.40 Various modifications can be incorporated to prevent GLP-1 cleavage by DPPIV, which is an important enzyme for inactivating GLP-1 in the body. This will be discussed further below.

DIPEPTIDYL PEPTIDASE-4 DPPIV, also known as cluster of differentiation (CD)26, is a ubiquitous enzyme found in various tissues and cell types, including the kidney, lung, adrenal gland, liver, intestine, spleen, testis, pancreas, central nervous system and on the surface of lymphocytes, macrophages and endothelial cells.40 DPPIV is a serine protease that specifically cleaves N-terminal dipeptides from peptide or protein substrates, where the penultimate amino acid residue is an alanine or proline. Since the active native GLP-1 peptides feature a penultimate alanine residue, these species are substrates for DPPIV, leading to their rapid (half-life ˂ 2 min) conversion to GLP-1(9-37) or GLP1(9-36)-NH2, which are inactive (Figure 2).40 Nevertheless, there are reports indicating

17



that GLP-1(9-36)-NH2 cleaved by DPPIV could be a weak partial agonist or antagonist of GLP-1R.72

Figure 2. GLP-1(7-36)-NH2 sequence. The N-terminal dipeptide “histidine alanine” is the proteolytic cleavage site for DPPIV.

Various studies have shown that the inhibition of DPPIV increases the half-life of native and biologically active GLP-1.40 In addition, DPPIV binds to adenosine deaminase and collagen, thereby contributing to T-cell costimulation by some antiCD26 monoclonal antibodies, which is a binding site for adenosine deaminase on Tcells, and proliferation by anti-CD3,40 resulting in further promoting β-cell proliferation and preventing the progression of diabetes.73

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GLP-1 hormone is released from the intestinal L-cells after the ingestion of food.74 Approximately 75% of produced GLP-1 by the intestine is degraded by DPPIV; the rest reaches the portal circulation, and 40-50% of this amount is metabolized in the liver. Therefore, only 10-15% of primary GLP-1 secreted from the gut enters the systemic circulation. For this reason, DPPIV inhibitors have been developed as potential therapeutic agents that specifically inhibit DPPIV activity, resulting in the extension of GLP-1 activity. DPPIV inhibitors enhance insulin secretion and reduce glucagon release. These effects lead to maintaining the BGL in a normal range in patients with T2DM.75,

76

Gliptins, as DPPIV inhibitors, are a group of antidiabetic compounds with increasing clinical use worldwide. They are the competitive reversible inhibitors of DPPIV substrate and act in the extracellular environment. Gliptins include alogliptin and linagliptin as non-peptidomimetic; and anagliptin, saxagliptin, sitagliptin, teneligliptin, vildagliptin,77 and gemigliptin as peptidomimetic agents, used orally with the same mechanism of action.78 As a drug class, they have appropriate clinical profile, owing to proper tolerability, the low risk of hypoglycemia, neutral effect on body weight and 19



once-daily

dosing.78

They

are

usually

prescribed

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with

other

antidiabetic,

antihypertensive and antihyperlipidemic compounds and regarded as a second-line medication for T2DM.77 Due to their effects on the α and β cell function, they improve the profile of glucagon and insulin secretion after meal.78 Generally, this class of drugs has no considerable interaction with other antidiabetic agents.;79,

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however, their

higher cost compared to insulin, metformin and pioglitazone is considered as an economic drawback.78 As previously mentioned, the native GLP-1 is not practical for therapeutic applications, owing to its very short half-life due to degradation by DPPIV. Therefore, DPPIV resistant GLP-1 analogs have been developed for therapeutic applications. In this regard, GLP-1RAs have been developed and are commercially available for the treatment of T2DM. These include Exenatide, Albiglutide, Liraglutide, Semaglutide, Dulaglutide and Lixisenatide.81 They are administrated subcutaneously,82 stimulate insulin secretion, and suppress glucagon release in a glucose-dependent manner, resulting in lower postprandial glucose levels.83 Due to their glucose-dependent mechanism of action, incidence of hypoglycemia is relatively low.84 In addition, they 20


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reduce gastric emptying and increase satiety, which together promote weight loss and decrease fasting BGLs.85 However, treatment with these agonists is associated with various side effects, such as nausea, diarrhea, headaches, dizziness and vomiting.86 Moreover, using treatment options in T2DM, such as combination therapy (e.g. Liraglutide in combination with metformin),87, 88 antibody fragment conjugation to GLP1 (e.g. Dulaglutide),89, 90 controlled release delivery systems,91 and PEGylation,92 are recommended to treat T2DM more effectively.

GLP-1 RECEPTOR AGONISTS In addition to DPPIV, the low molecular weight (MW) of GLP-1(7-36)-NH2 (3297.7 Da) causes its rapid removal from the blood circulation through renal clearance93 as generally < 20 KDa globular MW is associated with rapid renal clearance.94 One approach to overcome this issue is to increase its MW through conjugation to various compounds, such as human serum albumin. For this purpose, various GLP-1RAs have been developed. Exenatide was the first FDA approved GLP-1RA and is a synthetic version of exendin-4, which is found in the saliva of the glia monster lizard95 and is administered 21



by subcutaneous injection twice daily (Table 2).96 The peptide consists of 39 amino acids, in which the N-terminal 30 amino acids are 53% homologous to human GLP-1, with the C-terminal 9 amino acids, demonstrating no homology with the human GLP-1 sequence (Figure 3).97 Liraglutide was the second GLP-1RA approved by the FDA.98 It has a longer plasma half-life compared to the native GLP-1 (Table 2).99, 100 The structural modifications in Liraglutide include lysine substitution with arginine at position 34 and acylation of the lysine26 ε-amino group with the γ-carboxyl group of N-palmitoyl-L-glutamic acid101 (Figure 3), resulting in slower subcutaneous absorption, reversible albumin binding and resistance to degradation by DPPIV.102 Liraglutide was the first marketed GLP-1 analog and used for obesity treatment. It is injected in the periphery and targets hypothalamic GLP-1Rs placed on arcuate nucleus (ARC) neurons. The ARC neurons are likely involved in Liraglutide-induced body weight loss as the blocking of GLP-1Rs in the ARC neurons decreases Liraglutide-induced weight loss.103

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Figure 3. Structures of GLP-1RAs

Albiglutide is another GLP-1RA expressed in the yeast Saccharomyces cerevisiae104 and composed of a GLP-1 dimer fused to the N-terminus of recombinant human serum

23



albumin. A single amino acid substitution at position 8 (alanine to glycine) in the GLP1 sequence improves its stability to DPPIV,83 and fusion to recombinant human albumin extends the half-life of Albiglutide to approximately 5 days by preventing rapid renal clearance (Figure 3),105 rendering it suitable for weekly dosing (Table 2).83 A clinical study showed that Albiglutide lowers Hemoglobin A1c (HbA1c) concentration more than Exenatide and considerably reduces nausea and vomiting.106 Additionally, Albiglutide plays a significant role in improving cardiac energetics and functions by increasing glucose and lactate oxidation. Therefore, its capability to decrease cardiovascular morbidity compared to insulin therapy makes the drug distinctive.107 Albenatide, as a candidate drug in phase 2 clinical trial, is another GLP-1RA with 40 amino acids, in which an exendin-4 is attached to a maleimide modified human serum albumin through a chemical linker at position 46 (Figure 3). Comparing to Albiglutide, Albenatide is more potent, due to higher therapeutic effect for T2DM treatment at the same dose of Albiglutide (Table 2).108 Semaglutide is structurally based on Liraglutide with two further modifications.108 These include replacing alanine8 with Aib and acylation of lysine26 with C18 fatty acid 24


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side

chain

modified

by

N6-[N-(17-carboxy-1-oxoheptadecyl-l-γ-glutamyl[2-(2-

aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl] residue (Figure 3).108 The effects of these modifications include: (a) better binding to albumin through the fatty acid residues, leading to reduced renal clearance, and (b) reduced degradation by DPPIV, due to the substitution of alanine8 to Aib.109 Semaglutide is once weekly dosed GLP-1 analog (Table 2) and is now in the market.110 Dulaglutide is another GLP-1RA and consists of two identical disulphide-linked chains. Each chain contains an N-terminal GLP-1(7-37) analog sequence with the following substitutions: alanine8 to glycine, glycine22 to glutamic acid, and arginine36 to glycine.90 It is a recombinant fusion protein,111 where the GLP-1 analog sequence is covalently attached to a modified heavy chain of human immunoglobulin G4 (IgG4) via a small peptide linker (Figure 3).112 These modifications result in prolonged half-life, allowing for once weekly dosing (Table 2). Lixisenatide is a 44 amino acid exendin-4 analog incorporating six lysine residues at its C-terminus (Figure 3).113 Its affinity for human GLP-1R (hGLP-1R) is nearly 4-fold higher than endogenous GLP-1(7-37) (Table 2).114 25



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Table 2. GLP-1 agonists available for the treatment of T2DM

GLP-1 agonists

HbA1c

Plasm a half-

Dose

reductio

life

n

daily

0.9%

kg

0.8-

1.1-1.7

1.5%

kg

6-8 d

SC 50 mg/week

Albenatide

8d

SC 2 mg/week

Semaglutid e Dulaglutide

es

2.8–3.1

Albiglutide

h

reduction

0.7-

2.4 h

11-13

Referenc

SC 5 or 10 mg twice

Exenatide

Liraglutide

Weight

1.4%

SC 1.2 mg/daily

1.1-1.7 kg

1.1-

2.0–3.0

1.8%

kg

98, 115

116, 117

108

118, 119

6-7 d

SC 0.1–1.6 mg/week

1.7%

4.8 kg

120-122

4d

SC 1.5 mg/week

1.5%

2.6 kg

120, 123, 124

0.8–

1.8–3.0

0.9%

kg

Lixisenatide 2-4 h

SC 20 mg/daily

125, 126

Abbreviation: SC: subcutaneous administration; HbA1c, hemoglobin A1c.

COMBINATION THERAPY T2DM is recognized as a complex, chronic and progressive disease, in which patients often need to use multiple antidiabetic drugs to control their BGLs. If glycemic 26


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control is not achieved within 3 months, antiglycemic pharmacotherapy is intensifies.127 Treatment intensification, however, is delayed or does not occur in many T2DM patients. The early initiation of combination therapy can delay the progression of T2DM and improve patient outcomes.127 Metformin is the first-line therapy for T2DM. Where this agent is not tolerated, or fails to yield glycemic control, other drugs, such as sulfonylureas,128 meglitinides,129 thiazolidinediones,130 dipeptidyl peptidase-4 inhibitors (gliptins),131-134 GLP-1RAs,135 sodium-glucose co-transporter-2 (SGLT2) inhibitors,136, 137

and α-Glucosidase inhibitors,138, 139 may be added or substituted.

GLP-1 SEQUENCE MODIFICATIONS AND IMPLICATIONS The amino acid sequence of GLP-1 peptides is critical for their functioning.46, 47 The sequence analysis of GLP-1 has shown that the first seven amino acid residues at the N-terminus of the peptide (GLP-1(7-13)) is responsible for interaction with GLP-1R. This region is followed by a helical region (14-20), a linker region (21-23) and a second helical region (24-35).140 Alanine scanning of GLP-1 has shown that residues 7, 10, 12, 13, 15, 28 and 29 are critical for binding to and the activation of GLP-1R, in which

27



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mutation in these residues reduces the GLP-1R affinity by 132-425-fold and potency by 3900-fold.141 It is shown that histidine7 is an essential residue for both receptor binding and activation as determined by alanine scanning.142,

143

In fact, this residue is highly

conserved in the glucagon family. The deletion of histidine7 leads to a considerable loss of affinity (dissociation constant (Kd) = 33 nM compared to 0.3 nM for GLP-1(736)-NH2). Furthermore, the insulinotropic activity of GLP-1 missing histidine7 is 82% lower than that of the native GLP-1.144 Its substitution with alanine also decreases its binding efficiency by more than 100-fold.145 Acetylation of the N-terminus reduces the binding affinity by 5-fold without affecting the GLP-1 potency as well.145 In addition, histidine7 stereochemistry is important for GLP-1 function. For example, D-histidine substitution decreases GLP-1R binding affinity by 25-fold and potency by 34%.145 The second critical residue in GLP-1 peptide is alanine8, due to DPPIV cleavage site at its C-terminus. The replacement of alanine8 with glycine (A8G) results in a DPPIV resistant GLP-1 analog with a significantly longer N-terminal half-life. While, fasting hyperglycemia and glucose intolerance in diabetic mice is normalized by a single 28


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injection of 0.1 nmol of the A8G GLP-1 analog for several hours, 1 nmol of the native GLP-1 only reduces BGLs for a few minutes.144 Glycine10 is another important residue for GLP-1R binding and activation. For instance, the substitution of this residue with hexafluoroleucine results in a dramatic reduction in binding affinity and potency (60and 67-fold, respectively).144 The other important residue is glycine22, which can be substituted with various amino acids (e.g. lysine and glutamic acid). For example, the substitution of this residue with Aib retains binding affinity and potency comparable to the native GLP-1.146 Another important residue is lysine26. Green et al.147 fused a fatty acid chain at the ε-amino group of lysine26, which subsequently increased albuminbinding affinity to overcome renal clearance to extend its half-life and stability to DPPIV. Phenylalanine28 is also a critical residue for both receptor binding and activation,144 and Isoleucine29 is a critical residue for receptor binding.144 In contrast, Aspartic acid15 and Serine17 were showed to be crucial for the insulinotropic activities of GLP1.140 Moreover, glycine35 is an important residue, and its substitution with Aib along with the insertion of Aib at position 8 leads to a more potent analog called Taspoglutide,148 which shows high resistance to DPPIV. 29



However, the C-terminus of GLP-1 appears to be less important for its activity.143 The sequential deletion of amino acids 36 and 35 from the C-terminus produces compounds with decreased GLP-1R affinity and potency, and further deletions lead to the lack of biological activity.143 In addition, exendine-4 has a 9-amino acid sequence at its C-terminus compared to active GLP-1 peptides, resulting in enhanced helicity, metabolic stability and in vivo potency.149 Therefore, efforts have been made to include exendine-4 C-terminal extension into GLP-1 peptides to increase their metabolic stability. For example, the addition of this sequence into C-terminus of GLP-1(7-36)NH2 results in improving in vivo half-life (19.5 ± 3.3 vs. 4.8 ± 0.8 min) and reducing its metabolic and renal clearance in swine.150 Also, the C-terminal extension of GLP-1 does not change its receptor affinity and potency, e.g. GLP-1(7-37) which has similar activity to the native GLP-1, or improve GLP-1R affinity and potency, e.g. Albiglutide with a C-terminal albumin modification which is highly potent compared to the native GLP-1.143 However, peptide drugs are administered subcutaneously, leading to some complications experienced by patients. In this regard, peptide drug delivery systems are recommended.151, 152 30


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PEPTIDE DELIVERY Generally peptides are administered by subcoutaneus injection, due to their poor stability and short plasma half-life. However, patients receiving peptide drugs as injection often experience inconvenience and pain.151 Peptide drug delivery technologies aim to improve patient adherence and convenience, and enhance safety and efficacy through approaches, including sustained-release injected formulations or by allowing the alternative routes of administrations.152 Of the available drug administration routes, the oral route is the most popular, due to its convenience;152 however, some challenges still remain.33 The challenges which restrict the oral delivery of peptides and proteins include: (a) their high MW and the presence of hydrophilic charged groups, resulting in poor cell permeability;153 (b) the loss of protein tertiary structure in exposure to stomach acid as digestion, resulting in the loss of biological activity;153 (c) their instability towards chemical and proteolytic digestion;33 (d) renal clearance of peptides;33 (e) the adverse effects resulted from immune responses elicited towards proteins or peptides;33 and (f) the high costs of therapeutic peptides and proteins synthesis and/or expression.154, 155

31



Various strategies have been assessed to overcome these difficulties, including delivery with absorption enhancers and/or enzyme inhibitors, conjugation to polyethylene glycol (PEG), glycolipids or fatty acids, chemical modifications, etc., often in combination.152 The oral delivery of GLP-1 and its analogs can be a promising target for the treatment of T2DM.156 Biotinylated GLP-1 is another strategy to enhance membrane permeability. For this purpose, the peptide surface is modified by adding a site-specific bioconjugation, such as fatty acids and vitamins, leading to increasing contact surface between the peptide and the apical membrane of enterocytes, facilitating cell absorption and crossing the intestinal epithelium.157-159 Also, some studies reported that coupling PEG to GLP-1 increased its enzyme resistance and lowered its clearance rate.160 It has been demonstrated that the nasal route allows the systemic administration of peptides and proteins. The nasal mucosa is well perfused and provides a large surface area for absorption (150–180 cm2). Moreover, drug administration through the nasal mucosa can bypass the blood-brain barrier and delivers drugs to the central nervous system (CNS).161, 162 32


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Lipophilic molecules are generally absorbed quickly through the nasal mucosa with bioavailabilities that can reach more than 75%. However, this is not the case for peptide and protein drugs which generally show poor absorption and bioavailabilities (less than 1%). Therefore, the addition of absorption enhancers to facilitate the absorption of peptide/protein drugs across the nasal membrane is recommended. The examples of nasal absorption enhancers include ChiSys® (based on chitosan), Critical Sorb®(based on Solutol HS15) and Intravail® (based on alkylsaccharides).162 In recent years, various studies have reported safety and efficacy data for nasally administered GLP-1.163,

164

The results of one study on humans showed that nasal

GLP-1 could be a potential treatment for T2DM. The compound helped insulin secretion and the inhibition of glucagon release.163 The results of the study showed that the plasma glucose level was lower in GLP-1 treated group compared to the placebo group. In another study, exendin-4 was administered intranasally, and the results showed that this administration route was 4-fold more effective in peptide delivery to the olfactory bulbs than intravenous administration, and the peptide efficiency was enhanced by combination with cyclodextrin.164 33



Another means to enhance the efficacy and plasma half-life of peptide drugs is the use of controlled drug delivery systems, such as biodegradable poly lactic-co-glycolic acid (PLGA) in the form of triblock copolymer PLGA-PEG-PLGA.165

CONTROLLED RELEASE DRUG DELIVERY SYSTEMS With increasing knowledge in the field of nanomedicine, the development of nanosized delivery systems have revolutionized the pharmaceutical field by improving the therapeutic effectiveness of most drugs and their bioavailability. In this regard, nanoparticles are promising drug carriers and have received considerable attention for oral protein delivery.166 Nanoparticles can be prepared from biomaterials and tailored based on the administration route. Also, they can be conjugated with other molecules, including targeting ligands, to improve their delivery to target organs.167 Poly lactic-co-glycolic acid (PLGA) and hydroxypropylmethylcellulose acetylsuccinate (HPMC-AS) are two polymers received FDA approved for parenteral and the components of food products, respectively. They are widely used as drug delivery systems, due to their lack of toxicity after acute and chronic administration, biocompatibility, favorable degradation 34


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characteristics and physical strength.156,

168-170

One such successful experiment is

exenatide long-acting release (LAR; Bydureon). In exenatide, exendin-4 peptide noncovalently entrapped into poly(D,L-lactide-co-glycolide) to form 60 µm microspheres. Exendin-4 is slowly released by the diffusion and hydrolysis of the microspheres, and the microspheres prolong the half-life of exendin-4 to 5-6 days in humans, making once-weekly subcutaneous injection.171 Choi et al.165 used a thermosensitive hydrogel constructed from a biodegradable triblock copolymer of poly [(DL-lactide-co-glycolide)-b-ethylene glycol-b-(DL-lactidecoglycolide)] for the controlled release of GLP-1 in a rat model of T2DM. The formulation was administered subcutaneously and the blood concentration of GLP-1, insulin and glucose were monitored every day after the administration. The results showed that GLP-1 was constantly released from the formulation without initial burst. Also, one injection of formulation controlled the BGL for 2 weeks. Also, chitosan is a polysaccharide used in nanoparticulate formulations and has the ability to affect the permeability of the nasal mucosa through interactions between positively charged amino groups in chitosan and anionic groups in the mucosa.172 This makes chitosan 35



a promising compound for enhancing the transport of hydrophilic molecules across the nasal mucosa.162 Illum et al.173 evaluated the capacity for a chitosan glutamate solution for the nasal delivery of insulin in a sheep model. They observed a 43% decrease in plasma glucose levels within 90 min using chitosan glutamate at the concentration of 0.5%. In addition to these polymers, there are other strategies to increase the plasma half-life of peptide drugs, including the cyclization of linear peptides,174 pegylation, stapling,175 and peptide and protein glycosylation,176 the insertion of non-natural amino acids (mainly D amino acids), terminal region modification (acetylation or amidation), and the use of non-peptide backbones (peptidomimetics such as peptoids).174, 175

GLP-1 MODIFICATIONS Cyclization Cyclization provides a mean to stabilize peptides against proteolysis through hiding the mobile ends of peptides in the space and preventing exopeptidases from cleaving; therefore, protease degradation becomes more difficult. This is important because proteases are abundant throughout the human body, particularly trypsin-like proteases, which can reduce the effectiveness of peptide-based therapeutics.177 36


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Generally, the potential cleavage sites of a protein are more sensitive to protease attack if they are placed in the flexible regions of the backbone.178 Cyclization results in conformational constraint, decreases in peptide flexibility and improvements of the stability and membrane permeability of peptides. Peptide cyclisation can be performed using enzyme mediated ligation as a method carried out under mild reaction condition with high specificity and catalytic efficiency. In this method, linear peptides containing non-natural amino acids, D-amino acid and non-peptidic linkage are cyclized using various enzymes, such as sortase, peptiligase, subtiligase, butelase, thioesterase, trypsin and protease OaAEP1b.179 Peptides can be cyclised through different means, including head/tail-to-side-chain, head-to-tail and side-chain-to-side-chain (Figure 4). Generally, cyclization is performed via lactonization, lactamization or disulfide bridges.180 Research has been performed to evaluate the effect of cyclisation on the different peptide properties.181-185

37



Figure 4. Four different ways of cyclising peptides

In one study, it was found that a cyclic enkephalin analog is greatly resistant to in

vitro enzymatic degradation by serum peptidases/proteases. The results showed no significant degradation of the cyclic analog after 240 min, while native enkephalin was rapidly metabolized (half-life of 15 min).184 Another example is a cyclic herpes simplex virus glycoprotein peptide epitope that is 100% stable in 50% human serum after 96 h, while the linear peptide was completely degraded after 24-h incubation in phosphate buffer containing 10% human serum.182

38


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In addition, cyclosporine A (CSA) demonstrates the benefits of cyclization for oral peptide/protein therapy. CSA is a cyclic non-ribosomal 11-amino acid peptide isolated from fungus, containing a single D-amino acid.34 Most natural peptides and proteins are constituted of L-amino acids, while some natural non-ribosomal proteins have Damino acids in their structure. The site of D-amino acid insertion is resistant to proteolytic cleavage, resulting in greater in vivo stability of such analogs.186 CSA is resistant to proteolytic degradation and demonstrates absorption via oral route187 with 14-36% oral bioavailability.188 Also, it was demonstrated that the oral absorption of somatostatin and encephalin could be improved by cyclization.181, 183 In most cases, peptide cyclization considerably improves antimicrobial activity against Gram-positive and -negative strains of bacteria and serum stability of peptides compared to linear counterparts.185 The results of one study showed that cyclization by backbone peptide linkage improved the effectiveness of short antimicrobial peptides, while disulfide bridging between two added terminal disulfides appeared to be even more effective. This might be due to amidated C-termini of the disulfide bridged peptides.185 In another study, cyclization was applied for an indolicidin analog 39



and a bovine neutrophil-derived antimicrobial peptide which yielded disulfide-bridged peptides; and the results showed improved resistance to degradation by trypsin compared to their linear counterparts.177 However, the common modes of cyclization, including the formation of a lactam bridge through carboxyl and amino functional groups or disulfide bridges through thiol groups, resulting in side-chain-to-side-chain bridge formation, have two main issues: (a) cyclization can result in a loss of biological activity, owing to the contribution of the side chain groups involved in cyclization that are critical for bioactivity; and (b) the number of cyclization possibilities is limited.189 The lack of appropriate amino acids to perform cyclization may result in the need to substitute amino acids bearing amine (lysine, Ornithine), carboxyl (glutamic acid, aspartic acid) or thiol (cysteine) groups to enable cyclization, provided that the substitutions do not affect the peptide activity.190, 191 In this regard, Gilon et al.192 used a novel and easy strategy of backbone cyclization. According to this technique, cyclization is carried out through binding atoms to each other in the peptidic backbone rather than atoms in side chains or terminal groups, in which an NH and/or Cα are 40


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replaced with ω-functional alkylidene chains which can next be linked together to produce the appropriate cyclic peptide.193 Although cyclization can be applied for some peptides, its use is limited for larger therapeutic peptides and proteins. PEGylation is another strategy for some peptides that are not able to be cyclized,34 due to the lack of proper amino acid with carboxyl and amino functional group or thiol group.190, 191 PEGylation PEG is a polyether compound widely used in medicine and the most used polymer in polymer-based drug delivery, including protein drugs.194 PEGylation of peptides involves the site-specific or random covalent attachment of PEG to a peptide.195 PEGylation is one of the most successful strategies to enhance peptide delivery.196 This technique has the ability to improve the pharmakokinetic parameters of peptide drugs by for example reducing their clearance by the kidneys (e.g. by increasing the MW and the size of the peptide); reducing their clearance by the reticulo endothelium system (RES) through increasing biocompatibility; and reducing their proteolytic degradation (by masking the protein surface). In addition, the attachment of PEG to proteins can substantially decrease immune responses towards heterologous proteins, 41



which in turn reduces the risk of adverse effects, such as anaphylactic reactions. PEGylation improves protein application through enhancing water solubility and protecting the protein from enzymatic degradation and as a result improves the pharmacokinetics and pharmacodynamics of these compounds.195 Moreover, it keeps proteins partly intact from organic solvents, leading to protection against denaturation.197 For example, PEGylated bacteriorhodopsin, which is a membrane protein, increases stability against the high concentrations of ethanol (30%), while the native protein is denatured in 30% ethanol.197 PEG as an FDA approved compound is non-toxic polymer for human use.198 To achieve simple and controlled conjugation of PEG to peptides and proteins, PEG can be functionalized with thiol binding functional groups, such as pyridyl disulphides, maleimide and vinyl sulfonates or amine binding terminal functional groups, such as N-hydroxysuccinimide and aliphatic aldehydes.199 The terminal α-amine or lysine εamines are the main targets for protein PEGylation. These amines present in high frequency (approximately 10% of amino acids in protein) with only a few residues involved in the protein’s active site.200 42


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Cysteine residues are also a good site for PEG conjugation. However, the frequency of cysteine in proteins is generally low, or they exist in the active site of proteins or are involved in disulphide bands. However, the mutations of specific amino acids to cysteine or their insertion provides a mean to site-specifically incorporate PEG chains into proteins, where no other free cysteine residue is present. Sugar portions of glycoproteins are other choices for PEGylation. They can have some modification sites for PEGylation, including reducing aldehyde end groups, hydroxyl groups, primary amines, carboxylates or phosphates. Moreover, vicinal-hydroxyl groups can be oxidized by periodate, producing two reactive aldehyde groups.200-202 PEG used for conjugation can be categorized into three groups according to their configuration; (a) linear PEGs prepared by chemical modification of PEG terminal hydroxyl moieties, (b) Y-shaped PEGs which reduce resistance to proteolysis and immunogenicity, due to its umbrella-like shape, and (c) branched (comb-shaped) PEGs prepared by the ring opening polymerization of ethylene oxide (Figure 5); and they all have wide range of MW.199 Immunogenicity is the ability of a substance to induce an immune response.203

43



Figure 5. Three different types of PEG-peptide conjugates based on the macromolecular architecture of the conjugating polymers. A) Linear PEG; B) Y-shaped PEG; and C) Branched PEG.

In earlier attempts, PEGs with MW below 5kDa were used; however, in recent attempts longer PEGs (MW˃20 kDa) have been used, due to greater activity for longer PEG conjugate, including higher specific activity and the longer circulating half-life of peptides for longer PEG conjugates. Also, the PEGylation of proteins results in the reduction of immune responses associated with protein drugs.30 In one study, Nojima 44


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et al.198 prepared mono-PEGylated bovine lactoferrin (20kDa-PEG-bLf) with branched 20 kDa (2 × 10 kDa) PEG. The results of in vitro studies, such as iron binding, Interleukin 6 (IL-6) cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid showed that the PEGylated peptide was bound iron and exhibited 69.6 +/2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. Moreover, the proteolytic half-life of the pegylated peptide increased 2-fold compared to unmodified lactoferrin (17 min vs. 35 min, respectively). In addition, a ~10-fold increase in absorption was observed from the intestinal tract compared to unmodified lactoferrin. Furthermore, the intravenous injection of the peptide (1 mg/kg) demonstrated that PEGylated peptide compared to unmodified counterpart prolonged serum half-life of the peptide by approximately 5.4-fold. Also, the area under the curve was increased to approximately 9.2-fold compared to that of unmodified peptide. In another study, bLf was conjugated to a 40-kDa branched PEG molecule (40 kDa-PEG-bLf) and in vitro activities and pharmacokinetic properties were evaluated. The conjugate was fully active in iron binding and indicated 97.1 +/- 5.5% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. Also, the in vitro proteolytic half-life of peptide was increased 45



Page 46 of 94

at least 6-fold compared to that of unmodified lactoferrin. In addition, the plasma halflife of peptide was increased 8.7-fold for conjugated protein compared to that of unmodified lactoferrin in rats.198 Internal PEGylation can be achieved through the mutation of specific amino acid residue to a cysteine residue204 or pacetylphenylalanine.205

The

cysteine

residue

or

p-acetylphenylalanine

are

subsequently used for conjugation to PEG.206 PEGylation at the site of pacetylphenylalanine prolongs the plasma half-life of FGF21 to more than 30 h in rodents.205 It has been demonstrated that PEGylation at certain positions, including Nterminal residue and p-acetylphenylalanine, preserves FGF21 activity both in vivo and

in vitro.205, 207 GLP-1 PEGylation was also evaluated in some studies.91, 208, 209 GLP-1 has three possible amine PEGylation sites, including the N-terminus (histidine7) and two lysine residues (lysine26 and 34). In one study, two series of mono-PEGylated GLP-1, including PEG2kDa-Nter-GLP-1, where GLP-1 is N-terminally modified and PEG2kDa-Lys-GLP-1 which is a mixture containing pegylated lysine26 or 34 were constructed using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. The results of the study showed that the in vitro insulinotropic effect of 46


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PEG2kDa-Lys-GLP-1 was approximately equal to the native GLP-1 in isolated rat pancreatic islet and was considerably more potent than the PEG2kDa-Nter-GLP-1. Moreover, the resistance of PEG2kDa-Lysine-GLP-1 against DPP-IV was increased by 50-fold compared to that of the native GLP-1. Also, when PEG2kDa-Lysine-GLP-1 was administered intravenously and subcutaneously into rats, PEGylation increased the mean plasma residence time by 16-fold for intravenous (54.21 ± 21.10 min vs. 3.34 ± 1.45 min for pegylated and intact GLP-1, respectively) and 3.2-fold for subcutaneously administration (43.81 ± 5.90 min vs. 14.35 ± 3.66 min).208 In another study, Chae et al.91 constructed PEGylated GLP-1 at which lysine34 residue of the native GLP-1 was PEGylated with PEGs of 2, 5 and 10 kDa. The results showed that lysine34-PEG10kDa-GLP-1 extended the plasma half-life from 8.5 min to 105 min compared to that of native GLP-1 in ICR mice.91 Lee et al.209 prepared two potent enzyme-resistant forms of GLP-1, including N-PEG/GLP-1, where GLP-1 was Nterminally modified and histidine was PEGylated by mPEG2KDa-aldehyde, and Lysine-PEG/GLP-1, in which lysine34 was PEGylated by mPEG2KDa-succinimidyl propionate. They examined stability of the conjugates in plasma and tissue extracts. 47



Furthermore, in vitro insulinotropic effects of the conjugates were evaluated using isolated rat pancreatic islets, and in vivo potency was measured in db/db mice to control the glycemic conditions. The results of this study showed that half-lives of Lysine-PEG/GLP-1 were 40-, 10- and 28-fold compared to that of the native GLP-1 for plasma, liver, and kidney homogenates, respectively. The half-lives of the native GLP1 in rat plasma, liver, and kidney homogenates in vitro were 114 ± 28, 6 ± 0.5, and 1 ± 0.2 min, respectively, while these values for Lysine-PEG/GLP-1 were found to be 4471 ± 1822, 62 ± 23, and 28 ± 1 min, respectively (p

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