Image adapted from [1].
Ozempic, Weight Loss, and GLP-1 Therapy
Ozempic is a once-weekly injection first approved by the Food and Drug Administration in 2017 to treat type 2 diabetes. This drug has gained significant attention as celebrities like Oprah Winfrey, Kelly Clarkson, and Charles Barkley, along with TikTok influencers, have described taking it to lose weight quickly.
Ozempic is the commercial name given to the synthetic peptide drug Semaglutide, which mimics the action of the naturally occurring glucagon-like peptide in the body known as GLP-1. Ozempic and other GLP-1 analog therapeutics work by stimulating the body to secrete insulin (which lowers blood sugar). They also inhibit glucagon secretion, a hormone that raises blood sugar levels. They are described as GLP-1 receptor agonists. The term receptor agonist indicates that these drugs attach to receptors inside cells or on their surface and cause the same action as the natural substance that usually attaches to the receptor.
Studies have shown that GLP-1 analogs diminish appetite and food intake [2]. In 2021, the FDA approved Wegovy, a higher-dose formulation of Semaglutide, for chronic weight management.
GLP-1 Origin
The preproglucagon gene encodes the amino acid sequences of GLP-1 and related peptides such as glucagon and GLP-2 [3]. This gene is expressed (protein preproglucagon synthesized) in cells present in the pancreas (alpha (α)-cells), gut (L-cells), and brain (certain special neurons). The preproglucogon protein is broken down (cleaved) by enzymes in our body to the protein called proglucogon. Next, depending on the tissue, proglucagon is cleaved into various peptides such as glucagon, GLP-1, GLP-2, oxyntomodulin, etc.
GLP-1 receptors are located on the insulin-producing Beta (β) cells of the pancreas, in the brain, and throughout central nervous system transmission paths. Studies show GLP-1 is distributed throughout the brain [3]. GLP-1 receptors have been discovered in areas of the brain that regulate mood and hunger and are vital for maintaining the body's nutritional and metabolic balance. GLP-1 in the nervous system participates in signals for suppressing food and water intake and reducing appetite. GLP-1 reduces the pleasure from eating by interacting with parts of the brain's dopamine system that are associated with reward and motivation.
The Incretin Effect
A process that stimulates or affects insulin production is known as insulinotropic activity. The incretin effect is one such insulinotropic activity. In response to consuming glucose orally, the incretin effect triggers insulin production. The two primary incretin hormones activated after ingesting glucose or other nutrients are glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) [4]. Glucose taken orally and absorbed via the gut elicits a larger insulin response than intravenously administered glucose.
Pancreatic beta (β)-cells secrete insulin. These cells produce more than half of their meal response insulin through activation from incretin receptors [4]. Incretin peptides have been shown to stimulate insulin production, inhibit glucagon secretion, and reduce appetite and food intake [2].
In Individuals with type 2 diabetes (T2D) the insulinotropic action of GIP is diminished, whereas that of GLP-1 is preserved [5]. Medications such as Liraglutide and Semaglutide, which target the incretin effect for T2D therapy, focus on the GLP-1 pathway. Other treatments, like the peptide drug Tirzepatide, function as dual agonists for GLP-1 and GIP receptors.
Naturally occurring GLP-1 is quickly degraded by an enzyme in our body called dipeptidyl peptidase IV (DPP-IV; also known as CD-26). 50% of GLP-1 degrades within 1.5 – 2.1 minutes after intravenous injection. Therefore, a constant infusion of GLP-1 would be required to provide any therapeutic value, which is impractical [4,6]. Two pharmacological strategies devised to overcome the rapid degradation and clearance of GLP-1 are the development of synthetic long-acting GLP-1 analogs and DPP-IV inhibitors.
Our further discussion focuses on Liraglutide and Semaglutide – two drugs developed using synthetic GLP-1 analog strategies.
GLP-1 Composition
Naturally occurring GLP-1 is a 30 or 31-amino acid sequence closely related to the hormone glucagon. The initial GLP-1 is referred to as GLP-1 (1-37) and is expressed in the pancreas, intestines, and various locations within the brain and central nervous system. GLP-1 (1–37) is susceptible to a chemical reaction called amidation and the breaking of peptide bonds between amino acids (known as proteolytic cleavage). Thus, two biologically active variants of this peptide are more prevalent, named GLP-1(7-37) and GLP-1 (7-36) amide. Both have the same potency; however, most of this active peptide circulating is GLP-1(7-36) amide [4].
Figure 1: Amino acid composition of native human GLP-1 and synthetic GLP-1 analog Liraglutide.
Adapted from Ref. [6].
GLP-1 Analogs
Liraglutide is a GLP-1 analog given by subcutaneous injection once a day. It has demonstrated lasting improvement in glucose levels, weight reduction, and β-cell function (insulin production) in patients with type 2 diabetes.
The compositional differences between native GLP-1 and Liraglutide are illustrated in Figure 1. In Liraglutide, a fatty acid chain is added to the native GLP-1 template, and an amino acid substitution is made at position 34 (letters highlighted in blue). These modifications mean that liraglutide shares 97% amino acid identity with native human GLP-1. The fatty acid side chain is added specifically to increase residence time in the body by promoting binding with human serum albumin (HSA) circulating in the bloodstream. Docking with HSA shields the specific cleavage sites DPP-IV targets on the liraglutide peptide chain, slowing down its degradation as well as preventing fast renal clearance - how quickly Liraglutide is filtered out from the plasma by the kidney and excreted in urine. These chemical changes to Liraglutide increased the time required for the blood concentration of Liraglutide to reduce to half its initial value (also known as blood half-life) to approximately 13 hours [6]. Recall naturally occurring GLP-1 has a blood half-life of 1.5-2.1 minutes.
Successful clinical trials with liraglutide and other GLP-1 analogs (e.g., exenatide, based on the venom of the Heloderma lizard) led to increased interest in GLP-1-based therapies. To develop Liraglutide, endogenous human GLP-1 was used as the starting point for the drug discovery program. The next GLP-1 analog discussed here, Semaglutide, used Liraglutide as its road map.
The aim of developing Semaglutide was to extend the therapeutic life (blood half-life) of a GLP-1 analog dose and achieve a once-weekly administration cycle. The strategy employed to maintain adequate levels of the drug in the body for prolonged periods of time was to bind it with the large stable plasma protein HSA (similar to the Liraglutide strategy). An optimal balance between sufficiently long circulation times and high efficacy was found by modifying the Liraglutide peptide, as illustrated in Figure 2. A subtle change in the fatty acid chain and an additional amino acid substitution (highlighted in red) resulted in a final dosage form with the desired attributes [7].
Figure 2: Chemical compositions of liraglutide and semaglutide.
Adapted from Ref. [7].
GLP-1 Analogs Manufacturing
The successful deployment of GLP-1 analogs in the treatment of obesity and type 2 diabetes, two of the most prevalent afflictions facing the developed world, is causing prolonged drug shortages.8 Demand for these products is high and rising.
Native GLP-1, comprising natural amino acids, can be created using recombinant techniques or chemical synthesis. However, GLP-1 analogs that contain non-natural amino acids cannot currently be manufactured efficiently using recombinant expression methods. These polypeptides are usually made using chemical synthesis. The most common method for making peptides is solid-phase peptide synthesis, where protected amino acids are added step by step using a polymer for support. Considerable research and development focus on addressing and overcoming this limitation of recombinant techniques. Further, new solid-state peptide manufacturing strategies and alternative compounds are being developed to sustainably address the high demand for these drugs.
Purification techniques and technology are also critical steps in the production chain to get these compounds from the manufacturing floor to pharmaceutical-grade drugs in the hands of patients. Downstream processing (purification) is a resource-intensive section of pharmaceutical manufacturing, cited as high as 50% of production costs. Advances in the liquid chromatography technology used to purify these blockbuster drugs could signal greater access, reduced drug costs to patients, and higher profit margins for drug developers.
Millennial Scientific has taken up the challenge of developing a liquid chromatography stationary phase media specifically designed to purify GLP-1 analogs. Please see our blog and related application note at: https://www.millennialscientific.com/post/separation-of-semaglutide-and-liraglutide-an-optimized-method-using-nanopak-c-all-carbon-microbeads
Contact us to discuss how we can support your GLP-1 peptide purification challenges. Please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.
Author: Michael Jack Parente
References
1) Yabe, D. & Seino, Y. Liraglutide in adults with type 2 diabetes: global perspective on safety, efficacy and patient preference. Clin Med Insights Endocrinol Diabetes 4, 47-62 (2011).
2) Holst, J. J. From the incretin concept and the discovery of GLP-1 to today's diabetes therapy. Frontiers in Endocrinology 10, 453933 (2019).
3) Maigen Bethea, M., Bozadjieva-Kramer, N., & A Sandoval, D. Preproglucagon Products and Their Respective Roles Regulating Insulin Secretion. Endocrinology. 162, bqab150 (2021).
4) Al-Sabah, S. Molecular pharmacology of the incretin receptors. Medical Principles and Practice 25, 15-21 (2016).
5) Seino, Y. Vol. 96 934-935 (Oxford University Press, 2011).
6) Sjöholm, Å. Liraglutide therapy for type 2 diabetes: overcoming unmet needs. Pharmaceuticals 3, 764-781 (2010).
7) Knudsen, L. B. & Lau, J. The discovery and development of liraglutide and semaglutide. Frontiers in endocrinology 10, 440904 (2019).
8) Whitley, H. P., Trujillo, J. M. & Neumiller, J. J. Special report: potential strategies for addressing GLP-1 and dual GLP-1/GIP receptor agonist shortages. Clinical Diabetes 41, 467-473 (2023).
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