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@KruseEtAl_2021_DevelopmentCagrilintideLongActing

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Reference

Kruse, T., Hansen, J. L., Dahl, K., Schäffer, L., Sensfuss, U., Poulsen, C., Schlein, M., Hansen, A. M. K., Jeppesen, C. B., Dornonville de la Cour, C., Clausen, T. R., Johansson, E., Fulle, S., Skyggebjerg, R. B., & Raun, K. (2021). Development of Cagrilintide, a Long-Acting Amylin Analogue. Journal of Medicinal Chemistry, 64(15), 11183–11194. https://doi.org/10.1021/acs.jmedchem.1c00565

Blue: Important conclusions

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This pointed to 23 as the remaining candidate that constituted the best compromise between in vitro and in vivo potency, species selectivity, solubility in the pH range 4.0−8.0, tendency to fibrillation, duration of action, and a low number of mutations relative to h-amylin.

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When looking beyond the selection of compounds chosen for this paper, the SAR of our lipidated amylins can be summarized as follows. The human amylin sequence is very prone to fibrillate, and this tendency was difficult to reduce sufficiently while keeping the number of mutations low. In trend, introduction of oppositely charged residues to support a helix-stabilizing salt bridge in the central region and proline mutations in the C-terminal region to reduce β-sheet propensity seemed to be beneficial for physical stability. The prolines could be placed in other positions than 25, 28, and 29 but with no major improvement in potency or fibrillation tendency. Near the N-terminus, the disulfide bridge was needed in order to keep potency, but the lysine in position 1 could be deleted.

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Cterminal amide was found essential for activity. In contrast, the middle part of the sequence accepted considerable modifications without loss of potency. The pI of h-amylin is in the basic range but could be lowered by introduction of negative charges in the lipidation moiety. The lipidation was possible in several positions in the middle part of the sequence without compromising activity, but we found no advantage relative to N-terminal lipidation that is easiest in terms of synthesis

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Cagrilintide comprises 14E/17R mutations expected to stabilize the central helix via a salt bridge, 25P/ 28P/29P mutations as in rat amylin to reduce β-sheet propensity and fibril formation, a C-terminal proline to improve potency on CTR in particular, and an N-terminally linked C20 fatty acid to give long in vivo presence.

Yellow: Interesting

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Amylin1 is a 37 amino acid peptide, produced in pancreatic beta cells and co-secreted with insulin

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contains three proline substitutions relative to human amylin. These prolines are also present in rat amylin (Table 1) that is known not to be fibrillating.

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There is a substantial homology between calcitonin and amylin, and they both bind to the same receptors although with different selectivity

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The calcitonins we have studied bind both the calcitonin receptor (CTR) and AMY receptors, although with varying affinity, but s-calcitonin stands out as a particular strong and balanced agonist on both receptors

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area postrema (AP) is a central target in mediating the anorectic effects of amylin analogues

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Although AP has been shown to contain the CTR and all three RAMPs,22 which allows for expression of all three AMY receptors, RAMP3 has been suggested to be especially important for amylin signaling in the hindbrain.23 Knock out studies had also demonstrated an effect of RAMP3 on body weight,24

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The common backbone chosen was similar to pramlintide (see Table 1) rather than h-amylin in order to reduce issues with fibril formation.

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certain positions in the backbone could be mutated to lysine and subsequently acylated on the lysine sidechain with weakly albumin-binding fatty acids similar to the one used in liraglutide with no or modest loss of potency

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These structures predict that the CTR/RAMP complex has a more open structure than the GLP-1 receptor. Furthermore, while the N-terminus of GLP-1 penetrates as an α-helix into the transmembrane (TM) domain (see Figure S-5 in Supporting Information), the N-terminus of the amylin peptide is predicted to form a loop, stabilized by a disulfide bond, that points out of the TM area, thereby tolerating Nterminal lipidation.

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As fibrils of h-amylin are known to be cytotoxic,31 the new design strategy therefore focused at identifying compounds with better solubility in the pH range 4.0−7.4 and with low tendency to form fibrils

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Well-known means to reduce fibril formation propensity are the introduction of prolines and salt bridges. Amylin has been reported to possess an α-helix from position 5−20.33

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Compound 2 introduced a salt bridge (14E and 18R) and had a much longer duration of action and improved in vivo potency relative to 1, but it only had moderately improved solubility

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Apart from introduction of salt-bridges in the central region of the amylin sequence, introduction of solubilizing mutations at other positions were investigated.

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We could not obtain sufficient chemical stability except at low formulation pH

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The duration of action of 23 was indicated from the longlasting reduction of appetite in the food intake screening model (Figure 3) and was further confirmed in a pharmacokinetic study in rats (Figure 4 and Table 7)

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A particular focus was on the combination of semaglutide and cagrilintide as the combination of amylin and GLP-1 therapy has been suggested to work using partly complimentary mechanisms.

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The half-life was found to be 159−195 h.

  • 6-7 días.

Red: Disagree with the paper

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Human amylin is also involved in the cytotoxic amyloid formation2 seen in pancreatic islets of Langerhans in patients with type 2 diabete

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Formulation at pH 4 was preferred for chemical stability based on published pramlintide data, as described above.18 However, when 1 was taken into further preclinical development and high doses were given subcutaneously to rats, there were quite severe injection site reactions with signs of necrosis.

Purple: To learn more

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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.

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