"Development of Petrelintide: a Potent, Stable, Long-Acting Human Amylin Analogue"
Today, we share a research paper published in the Journal of Medicinal Chemistryby Henrik Fischer Munch's team. This study systematically details the rational design and optimization process of Petrelintide, a novel human amylin analogue. The research successfully overcame multiple drug development challenges associated with native amylin and its analogues, including physical instability (prone to fibrillation), chemical instability (susceptible to degradation and dimerization), and formulation incompatibility (requiring acidic pH for preparation). The final candidate molecule exhibits not only potent and long-acting pharmacological properties but, crucially, remains stable under neutral pH conditions and has been successfully co-formulated with the GLP-1 receptor agonist semaglutide, representing a breakthrough for next-generation combination therapies in weight management.
01 Research Background
The continuously rising global prevalence of obesity poses a significant public health challenge. Although medications like glucagon-like peptide-1 (GLP-1) receptor agonists have shown remarkable efficacy in weight management, limitations remain, including gastrointestinal side effects, high patient discontinuation rates, and weight regain. Therefore, developing novel therapeutic agents acting on different energy balance pathways is particularly urgent. Amylin, a 37-amino acid peptide hormone co-secreted with insulin postprandially by pancreatic β-cells, is an attractive pharmacological target due to its role as a physiological satiety signal. Amylin acts primarily in the central nervous system (particularly in the area postrema and nucleus of the solitary tract) by activating amylin receptors (AMYR1-3), which are complexes of the calcitonin receptor (CTR) and receptor activity-modifying protein (RAMP1-3) subtypes, effectively suppressing appetite, delaying gastric emptying, and inhibiting glucagon secretion.
However, developing amylin as a therapeutic agent faces significant challenges:
Physical Instability: Native human amylin has a strong propensity to aggregate, readily forming insoluble amyloid fibrils.
Chemical Instability: The molecule contains multiple asparagine (Asn) residues susceptible to chemical degradation (e.g., deamidation). Furthermore, the disulfide bond between Cys2 and Cys7 near the N-terminus is unstable near neutral pH, prone to ring opening leading to covalent dimer formation – a key degradation pathway thoroughly investigated and addressed in this study.
Poor Pharmacokinetics: Native amylin has a short half-life, and the first approved synthetic analogue, pramlintide, requires multiple daily injections.
Formulation Limitations: To maintain solubility and slow degradation, analogues like pramlintide must be formulated under acidic conditions (pH ~4), which prevents their co-development with most peptide drugs requiring neutral pH, such as GLP-1 analogues.
Therefore, developing a long-acting, stable amylin analogue that simultaneously addresses these issues and is suitable for combination therapy holds high clinical value.
02 Strategy and Innovative Highlights
Synergistic Use of N-Methylation and Sequence Optimization to Suppress Fibrillation
Unlike the strategy used in the marketed drug pramlintide, which introduces three proline residues (mimicking rat amylin) to prevent β-sheet aggregation, this study innovatively introduced N-methylation modifications within the amyloidogenic core region of human amylin (at glycine 24 and isoleucine 26). This strategy effectively disrupts intermolecular hydrogen bonding networks to inhibit fibrillation while maximizing retention of the human sequence. Research showed that N-methylation alone (e.g., Peptide 1) or combined with an H18R mutation (Peptide 2) was insufficient to fully suppress fibrillation; synergistic action with other sequence optimizations was required to resolve the issue completely.
Systematic Eradication of Chemical Instability: From Deamidation to Dimerization
The research team first improved basic chemical stability by deleting or replacing deamidation-prone Asn residues. A key finding was that deleting Asn at positions 21 and 22 (e.g., Peptide 11) significantly enhanced chemical stability while perfectly maintaining nanomolar-level in vitro potency (Figure 1).
Figure 1. Effect of single or double amino acid deletions in human amylin analogues on in vitro functional potency of AMY3R.
Systematic evaluation indicated an overall improvement in chemical stability as the number of potential deamidation sites in the sequence decreased (Figure 2). Additionally, introducing charged amino acids (e.g., glutamate (E) at position 14 and arginine (R) at position 18, potentially forming a salt bridge) helped stabilize the α-helical structure and improve physical stability.
Figure 2. Chemical stability, fibrillation, and AMY3R potency of selected human amylin analogues, demonstrating an overall improvement of chemical stability with reduction of potential deamidation sites.
More critically, this study thoroughly investigated and resolved the covert degradation pathway mediated by disulfide bond-driven covalent dimerization. The researchers abandoned the traditional disulfide bond and innovatively introduced a lactam bridge as a replacement. Through systematic structure-activity relationship studies, they precisely defined the lactam ring size and amide bond connectivity that simultaneously maintained high receptor activity and excellent chemical stability (Figure 3). Results showed that analogues with the amide bond closer to residue 2 (rather than 7) and a bridge size of 6-8 atoms (e.g., Peptide 18) fully retained the high receptor activity (EC50 < 0.5 nM) of their parent peptides.
Figure 3. Effect of amide bond location and of lactam ring size and orientation on functional potency of amylin analogues.
Achieving the Breakthrough: Neutral pH Formulation and Combination Therapy
The stability optimizations described above, combined with adjustments to charged amino acids, successfully shifted the isoelectric point (pI) of the final candidate peptide to the acidic range (e.g., pI 4.0 for Peptide 40). This allows it to remain stably soluble under neutral pH conditions (6.1-7.4), fundamentally solving the formulation bottleneck. This characteristic is a prerequisite for its co-formulation with mainstream GLP-1 drugs like semaglutide.
03 Results and Discussion
3.1 Potency and Stability Optimization
In potency optimization, the research team found the C-terminal structure crucial for receptor activation. Inspired by salmon calcitonin, they replaced the C-terminal tyrosine of human amylin with proline (P) or hydroxyproline (Hyp). This modification significantly enhanced agonist activity at AMY3R and CTR; for instance, Peptide 5 (Pro37) and Peptide 7 (Hyp37) achieved EC50 values of 0.16 nM and 0.21 nM, respectively, outperforming many earlier compounds (Table 1).
Table 1. Overview of Peptides and Potency in the Human Amylin and Calcitonin Assay
Chemical stability studies confirmed that replacing the disulfide bond with a lactam or triazole bridge completely eliminated dimeric degradation products and significantly improved overall chemical stability. For example, after 14 days under accelerated conditions at pH 7.5, no dimers were detected for Peptide 18 (lactam bridge) and Peptide 40, with very low chemical degradation (≤3%).
3.2 Final Selection and Characterization of the Candidate Molecule Petrelintide (Peptide 40)
Based on comprehensive data on in vitro potency, chemical/physical stability, and developability, Peptide 40 was selected as the clinical candidate and named Petrelintide. Its key modifications include: an N-terminal C20 fatty acid chain for extended half-life; N-methylglycine at position 24 and N-methylisoleucine at position 26 to inhibit fibrillation; deletion of Asn at positions 21/22 and replacement of other unstable residues to reduce deamidation; and replacement of the Cys2-Cys7 disulfide bond with a lactam bridge to eradicate dimerization.
Pharmacokinetic studies in Sprague-Dawley (SD) rats showed that a single subcutaneous injection of Petrelintide resulted in a desirable long half-life of 33.8 hours, supporting a once-weekly dosing frequency (Figure 4).
Figure 4. Pharmacokinetic half-life of selected amylin analogues in male SD rats.
Pharmacodynamic studies demonstrated that Petrelintide produced dose-dependent and sustained body weight reduction in both SD rat and diet-induced obese (DIO) rat models. In the DIO model, injections of Petrelintide (15 nmol/kg) every other day for 20 days significantly suppressed food intake and effectively controlled weight gain, confirming its therapeutic potential for chronic weight management (Figures 5, 6).
Figure 5. Percentage of mean body weight loss in male SD rats dosed with selected amylin analogues.
3.3 Formulation and Co-formulation Studies
Formulation studies confirmed that Petrelintide exhibits excellent chemical and physical stability in various physiologically compatible buffer systems within the neutral pH range (6.1-7.4). As proof of concept, Petrelintide was successfully co-formulated with semaglutide in two different neutral pH formulations. Accelerated stability studies indicated good compatibility, as the co-formulation did not significantly affect Petrelintide's chemical degradation rate or aggregation tendency, providing strong experimental support for its future development as a fixed-dose combination product.
04 Conclusion and Future Perspectives
This study is a successful example of rational design in peptide drug development. Through a series of precise and synergistic optimization strategies – including N-methylation to inhibit fibrillation, substitution/deletion of key unstable residues, and replacement of the unstable disulfide bond with a tailored lactam bridge – a natural hormone with poor drug-like properties was successfully transformed into an ideal clinical candidate.
The success of Petrelintide lies in its combination of high receptor activity, excellent physical/chemical stability, long-acting pharmacokinetics, and, crucially, the ability to be formulated at neutral pH and co-formulated. These properties make it not only a promising monotherapy but also an ideal partner for combination therapy with GLP-1 receptor agonists. This work not only presents a highly promising candidate molecule but, more importantly, demonstrates a systematic methodology for addressing common, interconnected challenges in peptide drug development, offering significant guidance for the future design of complex peptide therapeutics. It is reported that Petrelintide has entered clinical development as a potential weight management therapy, and its clinical results are highly anticipated.
Original Article:
Fischer Munch H, Just R, Mosolff Mathiesen J, Eriksson PO, Skodborg Villadsen J, Vestergaard B, Deryabina M, Demmer O, Skarbaliene J, Hamprecht DW, Rist W, Baader-Pagler T, Grube A, Heim-Riether A, Giehm L. Development of Petrelintide: a Potent, Stable, Long-Acting Human Amylin Analogue. J Med Chem. 2025 Nov 11.