Strategies to prolong the plasma residence time of peptide drugs

Lisa Pollaro; Christian Heinis

doi:10.1039/C0MD00111B

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DOI: 10.1039/C0MD00111B (Review Article) Med. Chem. Commun., 2010, 1, 319-324

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Strategies to prolong the plasma residence time of peptidedrugs

Lisa Pollaro and Christian Heinis *
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. E-mail: christian.heinis@epfl.ch

Received 15th July 2010 , Accepted 13th September 2010

First published on 21st October 2010

Abstract

Peptides are an attractive class of molecules for the development of therapeutics because they combine unique properties such as high binding affinity, excellent target specificity, low toxicity and a relatively small mass. However, the short in vivo half-life of peptides which is typically only a few minutes had hampered the development of a larger number of peptide leads into drugs. The main reasons for the fast elimination of peptides from the circulation are enzymatic degradation and/or fast renal clearance. To prolong the half-life of peptides, their proteolytic stability can be improved by chemical modification strategies and the rate of clearance can be reduced by conjugating the peptides to molecules that prevent their elimination through the kidney. In this article we review the latter class of strategies that aims at prolonging the in vivo plasma residence time of peptides. Techniques including peptidedrug linkage to large polymers, fusion to long-lived proteins such as albumin or the Fc fragment of immunoglobulin and conjugation to small molecule albumin-binding tags are discussed and the peptide-conjugate half-lives achieved are compared.

1. Introduction

Peptides are an attractive class of molecules that combine favourable properties of small molecule drugs and protein therapeutics.¹ As small molecule drugs, peptides are accessible to chemical synthesis which allows the facile modulation of properties such as stability, solubility and others. Also as small molecules, peptides have shown to penetrate tissue, although generally to a lesser extent due to the slightly larger mass. As protein therapeutics, peptides typically bind to their targets with high affinity and target specificity and they are capable of disrupting protein–protein interactions. And although peptides are generally considered to be cell-impermeable, strategies are now available that facilitate their cellular uptake.^2,3 However, despite their favourable properties, still relatively few peptides are approved as new drugs every year.^1,2,4

One of the main limitation of peptides as drug candidates has been the short half-life in the circulation which is caused mainly by proteolytic degradation and/or fast renal clearance.⁵ To prevent enzymatic peptide degradation, a whole range of strategies based on the chemical modification of the peptides have proved to be effective and generally applicable. These strategies include backbone modification, side chain substitution, use of D-amino acids, cyclisation, termini modification and others.^5,6 To prevent the fast clearance of peptides by the renal route, strategies were developed that hinder the peptides from being filtered through the glomeruli of the kidney and they are subject of this review article. In a first section, the clearance mechanisms of peptides are briefly reviewed and in the further sections half-life extension strategies based on polymer fusion, linkage to long-lived proteins and albumin-binding tags are discussed. While many of the reviewed strategies were applied to both, peptide and proteindrugs,^5,7,8 this article focuses on examples of peptidedrugs but exceptionally also refers to examples of proteins with extended half-lives.

2. Clearance mechanisms of peptidedrugs

Typically peptides are cleared from the bloodstream within minutes after intravenous administration.⁹ The major site of peptide elimination is the kidney, where glomerular ultrafiltrate is pressed out of the plasma. The glomeruli have a pore size of around 8 nm and molecules with a mass below 5 kDa such as peptides are filtered out completely. Larger polypeptides are less rapidly eliminated and proteins with a mass above a threshold which is between 50 and 70 kDa (corresponding to a diameter of approximately 90 Å in the case of globular proteins) are efficiently retained in the circulation. The renal clearance of some peptides and peptidedrugs is reduced due to binding to accessible membraneproteins or serum proteins. For example the cyclic peptidedrug ocreotide (brand name: Sandostatin) has a particularly long half-life of more than an hour due to binding to lipoproteins.¹⁰ Other, generally less relevant routes of peptide clearance, are endocytosis and degradation by the proteasome as well as clearance by the liver.⁹

The lifetime of molecules in the circulation is expressed mostly with the plasma half-life, the time taken to half the concentration of molecules through elimination. When a molecule is administered by a rapid intravenous injection into the vascular system, a disappearance from blood almost always occurs in a biphasic fashion. In a first phase, the drug concentration rapidly declines because of distribution to peripheral tissue. In a second phase, the drug concentration declines less rapidly through elimination. The term ‘plasma half-life’ generally refers to the second phase and it is therefore also termed ‘elimination half-life’ or ‘terminal half-life’.¹¹ In the following sections we compare mostly the ‘terminal half-life’ of peptides and peptide-conjugates. Since these values vary significantly in different species, the terminal half-lives determined in different animals should not be directly compared. Pharmacokinetics in humans may be predicted based on data from animal experiments by allometric scaling.¹²

3. Linking peptides to large polymers to prevent renal filtration

Based on the observation that proteins larger than 50 to 70 kDa are not rapidly filtered out by the kidney, peptides and small proteins have been conjugated to long hydrophilic synthetic or natural polymers, recombinant polymer mimetics or carbohydrates that increase their hydrodynamic volume.^7,8 The polymer that had most widely been employed to increase the size of proteins is polyethyleneglycol (PEG), a molecule built of repeating ethylene oxide (CH₂–CH₂–O) units (Fig. 1; 1).^13,14 One or multiple PEG chains ranging from approximately 5 to 40 kDa are linked to random or specific amino acid positions of peptides or proteins.¹⁵ Since one PEG monomer unit binds multiple water molecules, the hydrodynamic radius of a PEG chain is much larger than the one calculated from its molecular weight. The half-life extension of proteins and peptides through PEGylation varies but is typically between 10 and 100-fold. In addition to reducing the renal clearance, PEG was also found to stabilize peptides from proteolytic degradation, to increase their solubility and to decrease the immunogenicity in some cases. The first PEGylated protein, adenosine deaminase, entered the market in 1990¹⁶ and the efficacy and safety of several other PEGylated proteins have since then been confirmed.^13,17,18 Some of the best selling examples of PEGylated proteindrugs are pegfilgrastim (trade name: Neulasta), a human granulocyte colony-stimulating factor (G-CSF) linked via its N-terminus to a 20 kDa PEG unit with a 12-fold extended terminal half-life (42 h)¹⁹ or peginterferon α-2a (trade name: Pegasys), an interferon linked to a 40kDa branched PEG unit with a 20-fold prolonged half-life (60 h).²⁰ Potential problems that were reported to occur with PEGylation strategies are the inhibition of protein function and the renal toxicity if applied over longer time periods.²¹ In contrast to proteins, none of the PEGylated peptides had yet reached the market but several are currently in development. For example peginesatide (brand name: Hematide), a conjugate of two erythropoietin mimetic peptides that are coupled to a PEG chain. The peptide portion of peginesatide is a completely novel sequence of 20 amino acids isolated by phage display from a random peptide library.²² The peginesatide has a half-life between 21.5 and 59.7 h in rat and monkey when applied intravenously²³ and the drug is currently tested in a phase 3 trial. Another example of a PEGylated peptide is HM-3, a synthetic 17 amino acid integrin-binding peptide that showed anti-angiogenesis activity in rats. Attachment of a 10 kDa PEG unit to the N-terminus extended its terminal half-life in rats by a factor 6 (from 28 to 162 min).²⁴


	Fig. 1 Natural and synthetic polymers. (1) Structure of a dimeric PEG unit. A 40 kDa polymer of this format with l = 420–510 was linked to the N-terminus of interferon α-2a.²⁰ (2) Structure of polysialic acid. N-acetylneuraminic acid units are linked via α-(2 → 8) glycosidic linkages. (3) Structure of a homo-amino-acidpolymer with repeating Gly₄Ser units. Polypeptides of this type with n = 40 and 80 were linked to an antibody Fab fragment.²⁸

Alternatively to the conjugation with synthetic PEG polymers, proteins and peptides were linked to natural polymers such as polysialic acid (PSA) or hydroxyethylstarch (HES). Polysialic acidpolymers are negatively charged and highly hydrated carbohydrate chains (Fig. 1; 2) that have been linked to various proteins and peptides.²⁵Conjugation of 22 or 39 kDa PSA units to insulin extended its glucose-lowering activity in mice suggesting a longer drug half-life (the half-lives of the conjugates were not determined).²⁶Hydroxyethylstarch (HES) is a branched polymer composed of hydroxyethylated D-glucose units that has extensively been used as plasma volume expander. Conjugation of the safe polymer to proteins has shown to prolong their half-lives and suggests that the same effect should be found for peptides.²⁷

Recently, recombinant PEG mimetics based on long unstructured peptides that are expressed as a genetic fusion with the protein or peptidedrug were developed.^28–30 Skerra, A. and co-workers had developed a glycine-rich homo-aminoacidpolymer (HAP) composed of multiple pentapeptide (Gly₄Ser) repeats (Fig. 1; 3). Linkage of such a 200 amino acid sequence to an antibody Fab fragment prolonged its terminal half-life from around 2 h to 6 h.²⁸ Stemmer, P. C. and co-workers developed a longer unstructured polypeptide comprising 864 amino acids, called XTEN. A fusion polypeptide of XTEN and exendin-4, a 39-amino-acidpeptidehomologue of the glucagon-like peptide-1 (GLP-1) found in saliva of the Gila monster, had a terminal half-life of 12, 32 and 60 h in mouse, rat and monkey, respectively.²⁹ This is 71, 65 and 125-fold longer than the half-life of extendin-4 alone (0.17, 0.49 and 0.48 h in mouse, rat and monkey, respectively) which is already used as a drug for the treatment of type 2 diabetes (exenatide, brand name: Byetta). Fusion of a shorter, 288 amino acid XTEN polymer to the peptide glucagon increased its half-life in monkey more than 53-fold to 9 h. The recombinant PEG mimetics have the advantage of not requiring a chemical conjugation step and of yielding a homogenous product. Also, as PSA and HES polymers, the polypeptides are biodegradable and no toxicity was so far been reported. Yet another unstructured polypeptide developed by Skerra, A. and co-workers is based on peptide sequences rich in proline, alanine and serine (PAS) and has shown to extend the half-life of fused polypeptides.³¹ None of the recombinant PEG mimetics had yet been tested in clinic and information about immunogenicity in humans is hence not available.

4. Covalent linkage to plasma proteins with long serum half-lives

The plasma proteins albumin and immunoglobulin display inherently long circulation half-lives of 19 (albumin) and up to 21 days (immunoglobulin; depending on the isotype) in humans. The large size of albumin (67 kDa) and immunoglobulin (e.g. 150 kDa for IgG) prevents fast renal clearance. Additionally, the elimination through pinocytosis by the vascular epithelium is reduced because the plasma proteins bind to neonatal Fcreceptor (FcRn), a MHC class I-related receptor, which recycles them back into circulation where they dissociate at the higher pH.^32,33 Towards the extension of their half-life, peptides have been coupled to albumin and immunoglobulin fragments.

Peptide drugs were linked to human serum albumin either through chemical conjugation or the recombinant expression of peptide-albumin fusion proteins (Table 1). When chemically linked, the peptides are preferentially tethered to the free thiol group of cysteine-34 on the surface of albumin. For example, exendin-4 was conjugated with a chemical linker via its carboxyterminal end to cysteine-34 of albumin. The exendin-4-albumin conjugate (CJC-1134-PC) has a half-life of around 8 days in humans and is currently tested in a phase 1/2 clinical study.³⁴ Other peptidedrugs that have also been chemically linked to albumin with the same strategy are an analogue of GLP-1, an HIV entry inhibitor (C34 peptide) and insulin.³⁵ Examples for a peptide-albumin conjugate that were generated by genetic fusion are albiglutide (formerly known as albugon) and albuBNP. Albiglutide is a 29 amino acid dipeptidyl-protease-IV-resistant GLP-1 analogue that is fused as a dimer to human albumin.^36,37 The fusion peptide has a half-life of 6–8 days in humans when applied subcutaneously and is currently tested in a phase 3 clinical trial.³⁸ AlbuBNP is a 32 amino acidB-type natriuretic peptide (BNP) fused to albumin. Linkage to albumin extended the half-life of the peptide from 3 min to 12–19 h (around 300-fold) in mice.³⁹

Table 1 Peptides covalently linked to plasma proteins. The indicated terminal half-lives were measured after intravenous injection into human (a) or mouse (b). Half-life values marked with an asterisk indicate results obtained after subcutaneous injection

Drug/molecule	Peptide	Peptide conjugate	MW	Stage, company	t_1/2 conjugate	Ref.
CJC-1134-PC	Exendin-4 (peptide hormone from a lizard), 39 aa	Albumin (conjugation to Cys34)	71 kDa	Phase 1/2, Conjuchem	8 days^a	34
Albiglutide (albugon)	Glucagon-like peptide 1 (GLP-1 analogue, 30 aa	Albumin (genetic fusion)	70 kDa	Phase 3, GSK	6–8 days^a,*	36,38
AlbuBNP	B-type natriuretic peptide, 32 aa	Albumin (genetic fusion)	70 kDa	Preclinical, Human Genome Sciences	12–19 h^b	39
Romiplostim (AMG 531, brand name: Nplate)	Thrombopoietin analogue, 41 aa	Fc (genetic fusion)	59 kDa	Launched 2008, Amgen	3.5 days^a, *	41–43
AMG 386 (2xCon4[C])	Angiopoietin-neutralizing peptide, 20 aa	Fc (genetic fusion)	63 kDa	Phase 2, Amgen	3.4–4.1 days^a	44,45
CNTO 528	Erythropoietin mimetic, 20 aa	Fc (genetic fusion)	59 kDa	Phase 1, Centocor	5.9 days^a	46

Immunoglobulins of the subclasses IgG1, IgG2 and IgG4 have the longest half-lives of 2–3 weeks and are therefore most suited as carriers of peptides to extend their circulation time. Most peptides were linked to only a portion of IgGs, the 50 kDa Fc fragment which is a homodimer of the IgG heavy chain domains CH2 and CH3.⁴⁰ A first peptide drug-Fc conjugate, romiplostim (earlier known as AMG 531) has recently been approved for the treatment of chronic immune thrombocytopenia purpura (brand name: NPlate).^41,42 In romiplostim two consecutive copies of a peptide mimetic of thrombopoietin are fused via a glycine linker N-terminally to each monomer of the Fc fragment. The average half-life of romiplostim is 3.5 days when applied subcutaneously.⁴³ A range of other peptide-Fc domain fusions have been developed⁴⁰ and are currently tested in clinical trials including Fc fusion with an angiopoietin-mimicking peptide (AMG 386),^44,45 a B-lymphocyteinhibitor (AMG 623),⁴⁰ an erythropoietin mimetic peptide of 20 amino acids that was isolated from phagepeptide libraries (CNTO 528, CNTO 530)⁴⁶ and a GLP-1-analogue (CNTO 3649)⁴⁰ (Table 1).

5. Conjugation to albumin-binding molecules

As an alternative to the covalent linkage with albumin, peptides have been conjugated to albumin-binding molecules to indirectly tether them to the long-lived serum protein (Table 2). Upon intravenous injection, the peptide conjugates bind to the highly abundant plasma albumin (45 mg mL⁻¹, 600 μM) and are less rapidly filtered out of the blood stream. As albumin-binding molecules, small organic compounds, peptides and whole proteins have been used wherein the molecules with smaller molecular masses have several advantages. Firstly, the site-specific chemical linkage of tags to peptides and the subsequent purification of the conjugates are much facilitated with small molecules compared to large polymers or proteins. Secondly, the conjugation of small tags to peptides does not significantly increase their molecular size which can be advantageous to achieve good tissue penetration (e.g. diffusion into a tumour). If tissue diffusion should be achieved, a fine balance between the free and albumin-bound peptide conjugate must be maintained. Thirdly, the conjugation of peptides to small molecules yields a better activity to mass ratio and generally a more stable drug which together with the small size enables a wider choice of application routes.

Table 2 Albumin-binding molecules. The indicated terminal half-lives were measured after intravenous injection into human (a), rabbit (b), rat (c), and mouse (d). Half-life values marked with an asterisk indicate results obtained after subcutaneous injection. While the fatty acid acylated peptidedrugsinsulin detemir and liraglutide had already been launched in 2004 and 2009, respectively (Novo Nordisk), peptides conjugated to other albumin-binding molecules are all at a pre-clinical development stage

Peptide	Tag	Affinity of tag for albumin (K_d)	t_1/2peptide	t_1/2 conjugate	Factor	Reference
Insulin analogue, 50 aa (conjugate: insulin detemir, NN304, brand name: Levemir)	Myristoyl (C14 fatty acid)	4 μM^a (myristoyl linked to insulin)	1.5 h^a,*	5–7 h^a,*	4^a,*	47,58
Glucagon-like peptide 1 (GLP-1) analogue, 31 aa (conjugate: liraglutide, NN2211, brand name: Victoza)	Palmitoyl (C16 fatty acid)	No data available	1 h^a,*	11–15 h^a,*	15^a,*	48
E76 (fVIIa-inhibiting peptide), 18 aa	Naphthalene acylsulfonamide	No data available	7.6 min^b	24 min^b	3^b	49
Fab D3H44 (TF-inhibiting antibody, 50 kDa)	Peptide SA21, 18 aa, cyclic	467 nM^a	0.88 h^b	32.4 h^b	37^b	53
		320 nM^b	0.4 h^d	10.4 h^d	26^d
		266 nM^c
E76 (fVIIa-inhibiting peptide), 18 aa	Diphenylcyclo-hexanol phosphate ester	No data available	4.2 min^b	3.7 h^b	50^b	50
scFv F8 (anti-EDA domain of fibronectin antibody fragment, homodimer, 51 kDa)	6-(4-(4-iodophenyl)butanamido)hexanoate, ‘Albu’-tag	3.2 μM^a	20–30 min^d	16.7 h^d	50^e	51,52
	6-(4-(4-iodophenyl)butanamido)hexanoate, ‘Albu’-tag	3.6 μM^d				51,52

Early trials to extend the half-life of peptides with small albumin-binding tags were performed with insulin. Saturated fatty acids that bind with weak affinities (10⁻⁴-10⁻⁵ M) to serum albumin were linked to the C-terminal lysine side chain (Lys B29) of an insulin analogue⁴⁷ (Fig. 2; 4). A first molecule of this type, the insulin detemir (NN304) is a conjugate of an insulin analogue and a myristic acid (a fatty acid with 14 carbon atoms) and has been approved for clinical use in 2005 (brand name: Levemir). In patients, insulin detemir is usually applied subcutaneously as an oligomer (insulin forms a hexamer upon addition of zinc) to be slowly released and dissociated into biologically active monomers. The albumin-binding fatty acid tail further extends the half-life of the drug to 5–7 h. A second peptide that was acylated with a fatty acid is a GLP-1 analogue. The 31 amino acidpeptide was linked to palmitic acid (16 carbon atoms) and the resulting conjugate, liraglutide (NN2211), has recently (2009) reached the market (brand name: Victoza). The terminal half-life of subcutaneously applied liraglutide is 11–15 h which is around 15 times longer than the half-life of the peptide alone.⁴⁸ Due to the poor solubility of the resulting conjugates, the strategy of peptideacylation with fatty acids is not generally applicable to all peptidedrugs.⁴⁹


	Fig. 2 Chemical structures of albumin-binding small molecules. (4) Myristic acid,⁴⁷ (5) naphthalene acylsulfonamide,⁴⁹ (6) diphenylcyclohexanol phosphate ester,⁵⁰ and (7) 6-(4-(4-iodophenyl) butanamido) hexanoate (‘Albu’-tag).^51,52

Searching for small organic molecules that could extend the half-life of peptides without impairing their solubility, researchers at Genentech linked a small set of compounds with structural similarities to known albumin-binding drugs to a cyclic coagulation factor VIIa-inhibiting peptide. A naphthalene sulfoneacylamide moiety (Fig. 2; 5) extended the terminal half-life of the peptide from 7.6 to 30 min in rabbits.⁴⁹ Along this line, a small albumin-binding moiety (a diphenylcyclohexanol phosphate ester; Fig. 2; 6) of a contrast agent used in magnetic resonance imaging, was linked to the same peptide and increased its half-life from 4.2 to 222 min in rabbits.⁵⁰ Screening of larger libraries comprising more than 600 small organic molecules by Neri, D. and co-workers yielded several albumin-binding compounds including the 6-(4-(4-iodophenyl)butanamido hexanoate (‘Albu’-tag; Fig. 2; 7) with a K_d for albumin in the low micromolar range.⁵¹Conjugation of the tag to fluorescein and a scFvantibody fragment extended their half-lives in mice from 4.6 and 20–30 min to 495 and 1000 min, respectively.^51,52 Although not tested yet with peptides, the ‘Albu’-tag is likely to extend equally well the plasma half-life of peptides. No information about the toxicity of the newer small molecule albumin-binding tags is yet known.

A number of albumin-binding molecules based on peptides and proteins have also been developed. For example the cyclic 18 amino acidpeptide SA21 isolated by phage selection⁵³ binds to rat, rabbit and human albumin with dissociation constants of 266, 320 and 467 nM, respectively. Conjugation of SA21 to a Fab antibody fragment extended its terminal half-life from 53 min to 32.4 h in rabbits and from 24 min to 10.4 h in mice.⁵³ Fab fragments fused to truncated peptide variants of SA21 had lower affinities for albumin and the weaker binding correlated with shorter half-lives in mouse, rat and rabbit.⁵⁴ Examples for larger albumin-binding tags are domain antibodies (e.g. dAbh8: K_d mouse, rat and human albumin = 34–600 nM),⁵⁵ Fab antibody fragments (e.g. bispecific F(ab′)₂: K_d rat albumin = 26 nM)⁵⁶ and albumin binding domains (e.g. ABD035: K_d = 50–500 fM).⁵⁷ Due to their high albumin-binding affinities, all of these albumin binding proteins showed half-lives longer than 24 h in the various animals.

6. Conclusion

Several strategies are now available to extend the elimination half-life of peptides from minutes to several hours or even days. While some of these strategies have shown to be effective in preclinical tests, others have already been applied for the development of clinically approved therapeutics. Despite these successes, intensive research activities are ongoing to develop new or improved half-life prolongation strategies. For example, in the last few years multiple approaches based on biodegradable recombinant polymer mimetics were developed^28–30 and new companies such as Amunix and XL-protein that exploit these new technologies were founded. Or much progress has recently been made towards the development of small-molecular-weight albumin-binding molecules. As discussed above, due to their small size, these tags offer several advantages over the large polymers and proteins. Different laboratories had developed ligands to albumin that have increasing affinities for the serum protein and show longer retention in the circulation.^49–52 It can be expected that the engineering of half-life prolongation strategies will advance in the future with a similar pace and that new strategies and molecule tags will be developed to improve the therapeutic efficiency of existing and new peptidedrugs.

Acknowledgements

The financial contribution from the Swiss National Science Foundation (SNSF Professorship PP00P3_123524/1 to C.H.) is gratefully acknowledged.

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