Viewpoint Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Chemical Strategies for Half-Life Extension of Biopharmaceuticals: Lipidation and Its Alternatives Esben M. Bech,†,‡ Søren L. Pedersen,‡ and Knud J. Jensen*,† †
Department of Chemistry, University of Copenhagen, Frederiksberg DK-1871, Denmark Gubra Aps, Hørsholm DK-2970, Denmark
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‡
ABSTRACT: Strategies for half-life extension are often required in the design of new biopharmaceuticals. This Viewpoint focuses on chemical moieties that convey protraction by albumin binding or by self-assembly to form larger structures, with GLP-1 and insulin as examples.
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BACKGROUND The direct use of native polypeptides as biopharmaceuticals is often limited by their very short systemic half-lives resulting from a rapid metabolism, enzymatic degradation, and, for smaller proteins and peptides, effective renal clearance. The most common approach to improve PK properties of peptides and proteins has been PEGylation where polyethylene glycol (PEG) chains are attached covalently.1 However, there are concerns regarding the long-term safety of PEGylation, as it might lead to hypersensitivity, antibody formation, and bioaccumulation.1 Another approach to improve the pharmacokinetic (PK) properties of peptides relies on chemical moieties that convey protraction by binding to albumin and promote self-assembly to form larger structures. When bound to human serum albumin (HSA), molecules are sterically shielded from proteolytic degradation and protected from rapid renal filtration due to the relatively large size of HSA of approximately 66 kDa. Following albumin binding, molecules are gradually released into circulation. Thus, biopharmaceuticals modified to bind to albumin have an extended mode of action, which in principle can approach the 19-days half-life of albumin.2 Albumin affinity may also prolong the PK of subcutaneously injected compounds, as albumin binding in the subcutaneous interstitial fluid, theoretically, can protract the absorption into circulation.3 Noncovalent binding of peptides to albumin has been achieved by coupling of different moieties, including simple lipids, lipid dicarboxylic acids, lipids with additional moieties, small-molecule albumin binders, and streptococcal protein G’s albumin-binding domain (ABD).1,2 Lipidation is well-established in approved peptide pharmaceuticals from Novo Nordisk (Figure 1). Alternative strategies to improve PK properties rely on covalent conjugation or fusion to long-lived macromolecules with low immunogenicity, in particular fragment crystallizable (Fc) region, and HSA.1,2 Additionally, PK properties of peptides have been improved through native O- and N© XXXX American Chemical Society
glycosylation, anchoring to large carbohydrate polymers, control of nanoscale self-assembly, and chemical attachment of unstructured polypeptides.1,2 Here we focus on chemical moieties that convey protraction by albumin binding or by self-assembly to form larger structures, with GLP-1 and insulin as examples. It is increasingly popular within biopharmaceutical drug development, especially for peptide drugs but also, gradually, for larger proteins. For other PK enhancing strategies, see the review by van Witteloostuijn et al.1
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LIPIDATION HSA contains nine different fatty acid binding sites, which may bind free fatty acids as well as fatty acids conjugated to larger molecules.1,2 In 1996, pioneering studies on fatty acid-modified insulins revealed how in vivo half-life correlated with HSA affinity, such that strong albumin affinity resulted in prolonged action. The first lipidated biopharmaceutical to obtain regulatory approval was insulin detemir in 2004. Insulin detemir, a basal insulin for the treatment of diabetes, consists of desB30 human insulin conjugated to myristic acid (C14) through the Nε-amine of LysB29. Insulin detemir has a protracted absorption after s.c. administration and a terminal half-life of 4−7 h,1 which is a significant extension compared to native insulin and makes it suitable for once-daily use. The mechanism underlying its extended half-life involves both albumin binding and protraction of absorption by oligomerization.1 The myristic acid of insulin detemir is believed to stabilize a hexamer− dihexamer equilibrium as well as hexamer−albumin complexes in the subcutis, both of which could protract insulin absorption into the bloodstream.1 Upon dissociation and absorption to the bloodstream, the insulin detemir monomers can bind to
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DOI: 10.1021/acsmedchemlett.8b00226 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
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Figure 1. Structures of lipid chains in approved insulin variants and GLP-1 analogs, as well as in the hGH analog somapacitan.
albumin through their fatty acids.3 More than 95% of circulating insulin detemir is albumin bound.1 Current lipidated biopharmaceuticals have hydrophilic spacers, typically γGlu or OEG (8-amino-3,6-dioxaoctanoic acid), in between the lipid and peptide moieties to increase parameters such as albumin affinity, potency, water-solubility, and oligomerization.3−5 One example is liraglutide, a once-daily glucagon-like peptide 1 (GLP-1) analog marketed for treatment of diabetes and obesity. The liraglutide sequence is identical to that of native GLP-1 except for a Lys34Arg substitution, which enables selective palmitoylation through the Nε of Lys26 via a γGlu spacer.4 Liraglutide binds albumin in sera (∼99%), and absorption from the subcutis to the bloodstream is protracted.4 Because of albumin binding and slow absorption, liraglutide has a significantly extended half-life (11−15 h, s.c.) compared to native GLP-1 (1−1.5 h, s.c.).1,4 From the dietary fatty acids used in insulin detemir and liraglutide, the preferred fatty acid for lipidation has advanced to the nondietary dicarboxylic fatty acids used in insulin degludec, a once-daily basal insulin, and semaglutide, a onceweekly GLP-1 analog (Figure 1). Insulin degludec is lipidated at LysB29 with a γGlu-spaced palmitic diacid.3 Upon injection into the subcutis, insulin degludec forms large chain-like multihexamer (MW > 5 MDa) with an architecture resembling “pearls on a string”.3 The palmitic diacid assembles the individual hexamers, which are anchored to each other through the interaction between Zn2+ (an excipient in basal insulin formulations that stabilizes hexamer formation) and the terminal carboxylic acid of palmitic diacid.3 Subsequent diffusion of Zn2+ releases the bioactive monomer, which is absorbed into the bloodstream and bound by albumin, with higher affinity than insulin detemir.3 As a result of both absorption protraction and albumin affinity, insulin degludec has a half-life of 25 h in humans (s.c.), near-optimal for oncedaily administration.3 The peptide backbone of semaglutide is similar to liraglutide’s, except for a substitution of Ala8 to 2-aminoisobutyric acid (Aib), which reduces degradation by dipeptidyl peptidase IV (DPP-4).4 Semaglutide is lipidated at Lys26 with an octadecanoic diacid through a spacer consisting of γGlu and two OEG units (Figure 1), which elicits an albumin affinity 5.6-
fold larger than liraglutide’s.4 The high albumin affinity as well as the DPP-4 resistance gives semaglutide a half-life of approximately 1 week in humans (s.c.).1 Impressively, this prolonged half-life is obtained without decreasing the GLP-1 receptor potency compared to the native ligand.4 Recently, lipidation has also been shown as a viable strategy for half-life extension of larger proteins as demonstrated by somapacitan, a once-weekly human growth hormone in phase 3 clinical trials.6 The lipidation of somapacitan includes a significantly longer spacer region and a noncarboxylic fatty acid with a tetrazole headgroup (Figure 1). Somapacitan was found to be more than 99% bound to plasma proteins in human plasma of which albumin was the primary carrier.6 Lipidation with dietary fatty acids such as myristic acid and palmitic acid is generally perceived as a safe approach to half-life extension, and so far, clinical evidence indicates that the same is true for the structurally similar fatty diacids. Generation of antibodies against lipidated biopharmaceuticals have been reported, but the levels were low and without clinical relevance.1 However, the long-term safety profiles associated with modern lipid motifs containing fatty diacids, OEG spacers, noncarboxylic head-groups, and more is yet unknown. In this context it is important to recall that lipidation is a common post-translational modification of peptides and proteins. Native lipidation of polypeptides by, e.g., Spalmitoylation is known to induce anchoring to the lipid bilayer of cells. Membrane anchoring depends on the nature and number of the lipid chains, with polyunsaturated lipids providing a more dynamic membrane binding. Like the native lipidations, non-naturally lipidated peptides might also transiently bind to membranes. In fact, pepducins, a class of small lipopeptides that target GPCR’s intracellularly, relies on palmitylation for membrane tethering and flipping across the cellular membrane.7 Additionally, lipidated peptides and proteins might interact with lipid binding proteins other than albumin. Thus, conjugation of fatty acids could potentially prolong the in vivo action of peptides through several mechanisms. B
DOI: 10.1021/acsmedchemlett.8b00226 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
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PHARMACOLOGY AND DISTRIBUTION
The attachment of half-life extending moieties can in some cases affect receptor-mediated uptake of biopharmaceuticals as reported by Beck-Sickinger and co-workers for pancreatic polypeptide (PP).8 Receptor-mediated internalization was increased by palmitoylation but reduced by PEGylation.8 Receptor-mediated internalization can subsequently lead to desensitization (tachyphylaxis).9 Hence, avoiding receptor internalization might be used to achieve superagonists, as suggested for efpeglenatide, an aglycosylated Fc-conjugated exendin-4 analog.9 Receptor internalization should be considered when choosing a half-life extension strategy. Half-life extending moieties may also affect the biodistribution of the polypeptide conjugate. Liraglutide is administered subcutaneously, but can subsequently be found in the brain from where weight-loss induced by long-term GLP-1R agonists is likely mediated.10 GLP-1R agonists with larger molecular sizes, i.e., the once-weekly albiglutide (HSA-conjugated) and dulaglutide (Fc-conjugated) have a reduced effect on weightloss in clinical trials compared to liraglutide.11 This difference might be a result of suboptimal dosing regimen for the onceweekly GLP-1R agonists or, alternatively, differential degrees of brain-localized GLP-1R activation.11 It has been hypothesized that the large molecular sizes of albiglutide and dulaglutide would hinder transport across the blood−brain barrier or through fenestrated capillaries.10 Additionally, liraglutide shows greater GLP-1R mediated internalization than dulaglutide in vitro,9 which might be important for brain entry.10 Accordingly, brain uptake and distribution could be likely explanations for the difference in weight-loss efficacy between liraglutide and the larger GLP-1R agonists. However, one cannot rule out other explanations or alternative routes for the molecules to access the brain.
Figure 2. Structures of some experimental, chemical albumin binders. Cholesterol-like5 and small-molecule albumin binders.12
compared to its monovalent equivalent. This demonstrates how albumin avidity arising upon application of divalent binders could significantly improve half-life extending strategies utilizing drug binding sites I and II of albumin. Nonchemical methods for obtaining albumin binding have also been pursued. These rely on fusion of the biopharmaceutical to polypeptides with albumin affinity, for example, the albumin binding domain (ABD) of streptococcal protein G, antibody fragments, single-domain antibodies, and albumin binding peptides.2 Common for these methods are that they often elicit albumin affinities higher than lipidation and other chemical strategies.
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ALTERNATIVES TO LIPIDATION Chemical attachment of cholesterol can, in some cases, extend the half-life and the potency of biopharmaceuticals through membrane anchoring. Cholesterylation of peptides have been hypothesized to direct the membrane anchoring of the conjugate, especially to lipid rafts, in a quasi-irreversible fashion, which otherwise would require two long-chain lipids.5 It has been hypothesized that tethering of peptides to membranes can increase drug potency toward membrane bound receptors.5 In line with this, an oxyntomodulin analogue, with an optimized cholesterol tag had increased PK and potency compared to the native peptide (Figure 2).5 In a few cases, targeting albumin’s two structurally dissimilar drug binding sites, drug sites I and II, has been reported as an alternative to lipidation. By chemical conjugation of small molecules with affinity for drug site I or II, the in vivo half-lives of biopharmaceuticals can be extended.1,2 We have taken a multivalent approach to targeting drug sites I and II.12 A divalent small molecule albumin binder would have multiple affinities (avidity) for its target, and the avidity of a multivalent ligand can be much higher than the sum of affinities for its inherent components. To optimize albumin binding, we studied the effect of mono- versus divalent small-molecule albumin binders (Figure 2).12 Studies on GLP-1 analogs functionalized with diflunisal, indomethacin, or both showed that albumin affinity was increased for divalent analogs. In lean mice, the divalent GLP-1 analogs showed superior biological efficacy and a promising gain in circulatory half-life and absorption time
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CONCLUSION Lipidation has emerged as the most preeminent alternative to PEGylation for achieving half-life extension of biopharmaceuticals by chemical modification. This is largely due to the approved biopharmaceuticals drugs developed by scientists at Novo Nordisk A/S. Lipidation has since been adopted by numerous academic and industrial researchers. However, lipidation suffers from inherent limitations, including hydrophobicity. A largely unanswered question is to which degree lipidated biopharmaceuticals bind other proteins than albumin, for example, ApoA1 in high-density lipoprotein particles, and to which extend they bind membranes. Alternative ways to achieve albumin binding by chemical means are being developed. This includes ligands that bind other sites on albumin than the fatty acid binding sites. Another important aspect for injectable biopharmaceuticals is the extent to which they form a depot on injection and the rate with which molecules are released from the depot. These micro- and C
DOI: 10.1021/acsmedchemlett.8b00226 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
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nanoscale properties are also very important for the overall pharmacokinetic properties.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Knud J. Jensen: 0000-0003-3525-5452 Notes
Views expressed in this editorial are those of the authors and not necessarily the views of the ACS. The authors declare no competing financial interest.
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REFERENCES
(1) van Witteloostuijn, S. B.; Pedersen, S. L.; Jensen, K. J. Half-Life Extension of Biopharmaceuticals Using Chemical Methods: Alternatives to PEGylation. ChemMedChem 2016, 11, 1−23. (2) Sleep, D.; Cameron, J.; Evans, L. R. Albumin as a Versatile Platform for Drug Half-Life Extension. Biochim. Biophys. Acta, Gen. Subj. 2013, 1830, 5526−5534. (3) Jonassen, I.; Havelund, S.; Hoeg-Jensen, T.; Steensgaard, D. B.; Wahlund, P. O.; Ribel, U. Design of the Novel Protraction Mechanism of Insulin Degludec, an Ultra-Long-Acting Basal Insulin. Pharm. Res. 2012, 29 (8), 2104−2114. (4) Lau, J.; Bloch, P.; Schäffer, L.; Pettersson, I.; Spetzler, J.; Kofoed, J.; Madsen, K.; Knudsen, L. B.; McGuire, J.; Steensgaard, D. B.; et al. Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide. J. Med. Chem. 2015, 58 (18), 7370−7380. (5) Santoprete, A.; Capitò, E.; Carrington, P. E.; Pocai, A.; Finotto, M.; Langella, A.; Ingallinella, P.; Zytko, K.; Bufali, S.; Cianetti, S.; et al. DPP-IV-Resistant, Long-Acting Oxyntomodulin Derivatives. J. Pept. Sci. 2011, 17 (4), 270−280. (6) Thygesen, P.; Konradsen, G.; Schjødt, C. B.; Nielsen, P. F. Somapacitan (NNC0195−0092) a Novel Long Acting Human GH Derivative Binds Tightly, but Reversibly to Albumin in Plasma. In Endocrine Society’s 98th Annual Meeting and Expo. Boston, MA, 2016; poster SAT-034. (7) Tsuji, M.; Ueda, S.; Hirayama, T.; Okuda, K.; Sakaguchi, Y.; Isono, A.; Nagasawa, H. FRET-Based Imaging of Transbilayer Movement of Pepducin in Living Cells by Novel Intracellular Bioreductively Activatable Fluorescent Probes. Org. Biomol. Chem. 2013, 11 (18), 3030. (8) Mäde, V.; Babilon, S.; Jolly, N.; Wanka, L.; Bellmann-Sickert, K.; Diaz Gimenez, L. E.; Mörl, K.; Cox, H. M.; Gurevich, V. V.; BeckSickinger, A. G. Peptide Modifications Differentially Alter G ProteinCoupled Receptor Internalization and Signaling Bias. Angew. Chem., Int. Ed. 2014, 53 (38), 10067−10071. (9) Choi, I. Y.; Park, S. H.; Trautmann, M.; Moon, M. J.; Kim, J. Y.; Lee, Y. M.; Hompesch, M.; Kwon, S. C. Underlying Superagonistic Mechanisms of Efpeglenatide in Glycaemic Control and Weight Loss Potency. In American Diabetes Association’s (ADA) 76th Scientific Sessions. New Orleans, LA, 2016; poster 1068−P. (10) Secher, A.; Jelsing, J.; Baquero, A. F.; Hecksher-sørensen, J.; Cowley, M. A.; Dalbøge, L. S.; Hansen, G.; Grove, K. L.; Pyke, C.; Raun, K.; et al. The Arcuate Nucleus Mediates GLP-1 Receptor Agonist Liraglutide-Dependent Weight Loss. J. Clin. Invest. 2014, 124 (10), 4473−4488. (11) Madsbad, S. Review of Head-to-Head Comparisons of Glucagon-like Peptide-1 Receptor Agonists. Diabetes, Obes. Metab. 2016, 18, 317−332. (12) Bech, E. M.; Martos-Maldonado, M. C.; Wismann, P.; Sørensen, K. K.; Van Witteloostuijn, S. B.; Thygesen, M. B.; Vrang, N.; Jelsing, J.; Pedersen, S. L.; Jensen, K. J. Peptide Half-Life Extension: Divalent, Small-Molecule Albumin Interactions Direct the Systemic Properties of Glucagon-Like Peptide 1 (GLP-1) Analogues. J. Med. Chem. 2017, 60, 7434−7446.
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DOI: 10.1021/acsmedchemlett.8b00226 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
