The Pharm/Biotech industry’s main focus is on development of therapeutics to address unmet medical needs but sometimes, clinical trials lead to observations that lead to a change in research path for a drug. A good example is sildenafil that was originally studied for use in hypertension and angina pectoris. Observed side effects led to a switch in indication and eventual approval and marketing as Viagra.
One of the hot topics in the media at the moment is the potential of Ozempic, Rybelsus (semaglutide) and Mounjaro (tirzepatide) as weight loss therapeutics. These drugs are analogs to glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), respectively. GLP-1 and GIP are hormones involved in blood sugar control and their analogs have been approved as therapies based on that application for patients with Type-2 diabetes. Clinical studies have also shown that semaglutide and tirzepatide can help with significant weight loss for overweight and obese adults.
A higher dose version of semaglutide (Wegovy) has been approved to help with weight loss and it’s anticipated that similar approval will follow for tirzepatide. Not surprisingly, social media, particularly TikTok, has a lot of discussion about these drugs and there is concern about them being seen as a miracle silver bullet to drive weight loss. Discussion of these drugs is also difficult to avoid it in the print media too with numerous articles appearing in broadsheets like The New York Times, Washington Post & The Guardian over the past six months. Industry insiders speculate that the market for anti-obesity drugs could be between $50 and $100 billion a year.
The GLP-1 analogs are designed to enhance the secretion of insulin to decrease blood sugar levels. Their structures have been modified to enhance their pharmacokinetic properties relative to natural GLP-1 which has a short half life in the body. The most advanced candidates in this compound class tend to combine a peptide molecule with an extended lipid attachment. Whilst this combination of two very different chemical regions within a single moiety enhances the medical utility, it can make the bioanalysis extremely challenging.
To some extent, LC-MS/MS methods for free peptides can follow a fairly standard approach. However, the combination of two distinct chemistries in one moiety such as in the GLP-1 analogues drastically impacts the extraction, chromatography and mass spectrometry performance. Even though they’re chemically “similar”, no single approach for measurement of the intact peptides readily translates from one GLP-1 analog to another.
Adsorption is also a problem to factor into any assay development for the GLP-1 analogs. We have implemented controls in our methods to counter adsorption that we see consistently for all peptides but it can be particularly problematic for peptides with lipid additions. The underlying cause is strong binding to other macromolecules in matrix and surfaces in sample containers, the LC system and autosampler. This often manifests as carryover where we will see signal from one sample to the next. The persistence of lipid substituted peptides throughout the system can require aggressive washing approaches and the use of multiple gradient cycles per injection to clean the system.
We have also briefly looked at using a bottom-up approach for quantitation of the GLP-1 analogs. Using digestion with chymotrypsin to isolate useful peptides removes the problems associated with the lipid chain. Monitoring smaller peptides means that the assay can be more predictable and translates from one GLP-1 analog to another. Some of the analytical issues resulting from adsorption become easier to address with this approach. Having the ability to look at these molecules intact or following digestion adds to KCAS’ flexibility in developing and validating suitable assays for these types of molecules.
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