Objetivos Específicos

The physiological metabolism of fatty acids as well as their ability to bind albumin with a high affinity inspired the use of post-translational acylation as a safe and natural platform for prolonging the half-life and the mode of action of peptides and small proteins. So far, this strategy was successfully exploited by the Danish pharma company Novo Nordisk that launched three acylated peptide drugs on the market for the treatment of diabetes and obesity.99

Kurtzhals et al. pioneered the development of the first clinically available acylated insulin analogue in 1995.227 _{Lys29 in the B chain of insulin was conjugated to different}

saturated fatty acids (10-16 carbons), and Thr30 was removed. The insulin analogue carrying the C14 myristic acid [LysB29_{-tetradecanoyl des(B30) insulin] bound albumin with}

Kd values of 4.2 and 10 PM at 25°C and 37°C, respectively, which was two to three orders

of magnitude lower than free fatty acids (Fig. 27a). Subcutaneous injection of this C14 conjugate in rabbits resulted in a prolonged effect on blood-glucose levels.227 _Similar

results were confirmed in pig experiments and showed an important correlation between binding affinity for albumin and in vivo half-life.218 _{In addition, its binding affinity was un-}

affected by the co-presence of potential competitors, such as free fatty acids and several well-known albumin-binding drugs.229 _{This myristoylated insulin analogue was approved}

in 2004 as insulin detemir (Levemir) for the treatment of type 1 diabetes. The drug has a half-life of five to seven hours upon s.c. injection in human, which is 50 to 100-fold longer than insulin alone in blood (four to six minutes, i.v.; 2.3 hours, s.c.)110,111_{, allowing either}

once or twice-daily dosing.230 _{The half-life prolongation is not exclusively related to its}

ability to bind albumin in blood circulation, but is also associated with the route of admin- istration and the formulation. In presence of phenol and Zn2+_{, insulin detemir self-assem-}

bles into hexameric–dihexameric units, protracting its blood absorption from the subcu- taneous deposit site.231

Ten years later, Jonassen et al. developed a longer-acting acylated insulin for once-daily injection.228 _{The new insulin analogue, approved as insulin degludec}

(Tresiba), conjugated a longer 16 carbons dicarboxylic fatty acid to the LysB29 through a J-Glu linker, thus introducing two net negative charges (Fig. 27c) that led to improved affinity and solubility. Insulin deglutec has a 2.4-fold stronger affinity for albumin than insulin detemir at 25°C, and a much larger oligomerization upon s.c. injection thanks to the terminal carboxylic acid.228 _{These changes resulted in a half-life of 25 hours in hu-}

best pharmacokinetic and pharmacodynamic profiles among the currently approved in- sulin formulations.232

As broadly reported in the previous section, the improvement of the GLP-1 half-life is of great interest and several extension platforms have been successfully applied. Ac- ylation also led to the approval of liraglutide (Victoza) in 2009, a once-daily GLP-1 ana- logue used in the treatment of type 2 diabetes and later obesity.123 _{Knudsen et al. per-}

formed a structure-activity relationship (SAR) study of 36 acylated analogues of GLP-1, varying i) peptide sequence, ii) site of modification, iii) fatty acid, and iv) linker. Several derivatives were equipotent or better than GLP-1, and showed a more than ten-fold pro- longed half-life in pigs upon s.c. injection.123 _{Among them, the future liraglutideretained}

the potency of GLP-1 and resulted in promising pre-clinical studies in animals. The final structure is composed of GLP-1 conjugated to Lys26 with a 16-carbon palmitic acid via a J-Glu linker, and with the Lys34 replaced by Arg (Fig. 27b).233 _{A direct measurement}

of its affinity has not been performed, but dialysis studies confirmed a strong albumin binding.234 _{The half-life in human has been calculated as eight hours and 11-15 hours}

upon i.v. and s.c. injection, respectively, a remarkable improvement compared to the native GLP-1 (1.5 minutes, i.v.; 1-1.5 hours, s.c.).123,235 _{As with insulin analogues, the}

long half-life of liraglutide is also related to self-association with a slow release from the site of injection. In addition to this, a reduced susceptibility to enzyme degradation was noted, probably due to an albumin protective effect upon binding.235

These positive results stimulated research into new variants of insulin and GLP-1, and expanded the acylation strategy towards other peptides. Recently, Novo Nordisk developed a new analogue of GLP-1, semaglutide, and the documents for its approval have been recently submitted to the FDA.236 _{Starting from the sequence of liraglutide}

and applying the same criteria of optimization, Lau et al. synthesized 45 new variants. They tested i) longer fatty acids or diacids, as done with insulin deglutec, ii) different linkers between Lys26 and fatty acids, and iii) new amino acid substitutions for increasing the enzymatic stability against DDP-4.237 _{The selected candidate semaglutide was mod-}

ified at Lys26 with a octadecanoic diacid (18 carbons and two carboxylic acids) through a long linker composed of two 8-amino-3,6-dioxaoctanoic acid (O2Oc) units and a J-Glu (Fig. 27d). It also has Ala8 substituted by the unnatural amino acid 2-aminoisobutyric acid. By applying only these few modifications, semaglutide displayed much better prop- erties than liraglutide,similarly to what has been already observed with insulin detemir and insulin deglutec. Despite a lower binding for the receptor, the potency of semaglutide was still equivalent to GLP-1 and liraglutide. Its binding affinity for albumin was six-fold

stronger than liraglutide providing semaglutide with a four-fold longer half-life in pigs upon i.v. injection, and a 48 hour lowering-effect on blood glucose in mice after s.c ad- ministration.237 _{In humans semaglutide showed a half-life of 155-185 hours, ten and 100-}

fold longer than liraglutide and GLP-1, respectively, allowing once weekly s.c. dosing.238

Figure 27. Examples of lipid moieties used for half-life extension of biopharmaceuticals. (a) Myristic acid

(insulin detemir). (b) Palmitic acid linked to a J-Glu linker (liraglutide). (c) Hexadecanoic diacid with a J-Glu linker (insulin degludec). (d) Octadecanoic diacid coupled to a linker consisting of J-Glu and two 8-amino- 3,6-dioxaoctanoic acid units (semaglutide). Reprinted with permission from 99_{, reprinting license nr.} 4047140757234, copyright © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Several other peptides were modified with acylated moieties and showed promis- ing pre-clinical results. A pancreatic polypeptide was modified with the J-glu-C16 fatty acid moiety used in liraglutide. It exhibited retained in vitro stability and affinity for the receptor, and a seven-fold longer half-life in rats upon i.v. injection.239 _{Insulin end exen-}

din-4 were modified with a 16-carbon hexadecanoic acid terminating in a sulfonate anion. The derivatives had better affinity for albumin and much higher solubility than insulin detemir. This translated into five to ten-fold prolonged glucose-lowering effects in mice after both i.v. and s.c. administration.240 _{The same acylated motif used for semaglutide}

was also applied to insulin on Lys22, increasing the number of fatty acid carbons to 22. The resulting conjugate, called insulin-327, showed interesting liver-specific effects in dogs and a prolonged half-life.241 _{Several other lipidated peptides and small proteins}

were differently acylated, such as neuromedin U, a GLP-1/GIP co-agonist, calcitonin, oxyntomodulin, human growth hormone and others, providing derivatives with enhanced pharmacokinetic profiles.99

Finally, an innovative combination of fatty acids and glyco moieties was recently proposed by Jensen and co-workers for extending the half-life of GLP-1.242 _{The improved}

backbone of liraglutide was conserved and Lys26 was modified with one or two units of mucic acid, a galactose derivative, linked to three different fatty acids via a J-Glu linker. All of the new conjugates had activity and solubility in the same range of GLP-1 and liraglutide, and could form oligomers as well. The apparent binding affinities for albumin were approximately 1-2.5 PM for all of the glycolipidated peptides, equivalent to what they measured for liraglutide, and this translated into comparable in vivo pharmacologi- cal effects after s.c. dosing in mice.242