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In Novo’s Own Words: Degradation of Amylin Analogs Such as Cagrilintide (and How to Test For It)
“A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort.”
So begins Novo Nordisk’s excellent design study of cagrilintide.[1] In the very first sentence, the creators of cagrilintide lay out their challenge: formulating an amylin analog that doesn’t form amyloid fibrils.Human amylin is a hormone that's released after eating along with insulin within the pancreas and helps regulate food intake and body weight. Researchers have long been interested in the potential of human amylin to address obesity.
“Human amylin is also involved in the cytotoxic amyloid formation [2] seen in … patients with type 2 diabetes. It is therefore pivotal that drug development of amylin mimetics addresses the propensity of human amylin to form fibrils.”
But the problem is that amylin is highly unstable. It degrades rapidly and begins to aggregate, which is where degraded amylin molecules combine with other amylin molecules and develop into fibrils.Fibrils are like a bunch of perfectly straight strands of yarn (peptides) that have become tangled up together, forming a messy ball of yarn. These fibrils then form the basis of amyloid deposits that have been implicated in a variety of degenerative diseases, including Type 2 Diabetes and Alzheimer’s Disease.
This is the challenge that Novo’s researchers were facing when they set out to design an analog (a synthtic version) of human amylin.
“The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life… This suggests an option for improvement by reducing the frequency of the injection. ”
Pramlintide was the first analog of human amylin to be FDA approved in 2005 for diabetes treatment. Researchers noticed that rat amylin was more stable than human amylin in that it was less prone to aggregation which led to amyloid fibril formation. Rat amylin differs from human amylin by six amino acids of which three are prolines that researchers discovered were responsible for the suppression of fibril formation. So they created an analog of human amylin with those three proline substitutions and they called it "pramlintide."Novo researchers are bringing up pramlintide because it’s a more stable analog, but it has a very short half life that makes it impractical compared to the once-weekly dosing of other weight loss peptides. This presents their second challenge which eventually led to the development of cagrilintide.
“Published data on pramlintide [17, 18] suggested a need for improvement of chemical stability, primarily targeting deamidation of asparagine residues to enable formulation at neutral pH.”
Even though pramlintide was formulated more like rat amylin to be more stable and prevent fibrillation, the researchers are acknowledging here that pramlintide also has problems with instability. In particular, is is prone to a particular kind of degradation called “deamidation”—but only when formulated at a neutral pH of ~7.0."Deamidation of asparagine residues" refers to a chemical reaction where the asparagine (Asn) side chain loses its amide group (-NH2), converting it into aspartic acid (Asp). This leads to a whole slew of consequences.
Deamidation changes the charge of the molecule, which affects how the peptide interacts with other molecules. It makes the peptide bend or kink, changing its shape. Both the changes in charge and shape affect the peptide’s function, affecting its ability to perform its intended action in the body. Deamidation can make the peptide immunogenic (able to trigger an allergic reaction).But most importantly, deamidation weakens the peptide, making it more susceptible to further degradation and aggregation. This is what enables fibrillation.
The researchers attempted to solve this problem with cagrilintide. Did they succeed?
"We found that formulation [of cagrilintide] at pH 7 caused chemical instability including deamidation of asparagine residues, as reported for pramlintide [17, 18], and this could not be solved by formulations or minor pH adjustments.”
The cagrilintide researchers at Novo Nordisk are acknowledging here that they failed in their attempt to make cagrilintide stable at a neutral pH, and they were unable to solve that problem using special formulations.“In order not to deviate too much from [human]-amylin, formulation at low pH seemed necessary. We initiated a third branch of the program focused on the chemical stability at neutral pH to secure the option of co-formulation with other peptides. This part of the program led to NN1558 and will be reported elsewhere."
Their failure to prevent deamidation of cagrilintide at a neutral pH led to the development of a new program aimed at solving that problem. They labeled this new compound, “NN1558.”What were the results?
“Results: NN1558 monotherapy increased body weight after an initial period (~3 days) of weight loss. This increase in body weight was accompanied by increased food intake.”
This is what Novo researchers published in the research journal “Obesity” about NN1558.[3] They attempted to make a cagrilintide that was stable at pH 4.0, and it actually resulted in an **increase** of body weight. In other words, they failed.Novo researchers also recently published a report about *another* amylin analog only a few months ago called NN1213.[4] This new report contained some interesting new insights about amylin that Novo hadn’t shared in their cagrilintide report. But first, we’ll cover the goal of this new analog and whether they succeeded with it.
“The first series of peptides were synthesized based on the human amylin scaffold, aiming at formulation at neutral pH.”
Once again, the goal was to create a “cagrilintide” that was stable at a neutral pH of ~7.0.Did they succeed?
“It was found that the chemical stability at neutral pH required for a drug candidate was not sufficient… This was partly related to deamidation and isomerization at asparagine residues but also the disulfide bridge.”
The new analogs had the same problems as cagrilintide at pH 7, but also suffered from instability of the disulfide bridge. The disulfide bridge is critical for the biological functionality of amylin and also protects it from aggregation. This was likely an issue for cagrilintide as well, though it wasn’t reported—the researchers in that report said that instability of cagrilintide “included” deamidation, implying there were other kinds of degradation.“Degradation of the asparagine residues and general stability were significantly improved at pH 4.”
Just like cagrilintide, these new amylin analogs were found to only be stable at a pH of 4.“Initially, it seemed likely that a neutral formulation was possible… However, as development of a neutral formulation not only caused deamidation but also involved disulfide bridge instability, it was decided to revert to using an acidic formulation.”
Again like cagrilintide, and then NN1558, the researchers finally gave up on making this new amylin analog stable at a neutral pH.Now, onto the interesting insights they hadn’t shared in their cagrilintide report. Within the context of discussing the potential of amylin to form fibrils, here’s what they said:
“The amino acid sequence of human amylin enables a process of misfolding whereby monomeric amylin initially forms soluble beta-sheet-rich oligomers that may be cytotoxic.”
For the first time, Novo researchers are discussing not only fibrils, but also oligomers. To understand oligomers, imagine you have a box of LEGO bricks. Each individual brick is a **monomer** (mono meaning "one"). Now, if you connect a few of these bricks together, you create a small chain called an **oligomer** (oligo meaning "a few"). When two bricks connect, that's called a **dimer**, and three is a **trimer**.These oligomers, or short chains of amylin, then form bonds with each other in a side-by-side arrangement, forming sheet-like structures called beta sheets.
They note here that oligomers “may be cytotoxic,” meaning toxic to cells. The research on amylin oligomers is actually much more conclusive than “maybe:” oligomers disrupt cell membranes, induce stress in the endoplasmic reticulum, cause mitochondrial dysfunction, increase oxidative stress, trigger inflammation, and damage DNA.[5]
, 1mg/wk
