Albumin and its application in drug delivery.

Review

Albumin and its application in drug delivery

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Ohio State University Libraries on 12/19/14 For personal use only.

Darrell Sleep 1.

Introduction

Novozymes Biopharma UK Ltd., Nottingham, UK

2.

Albumin in drug delivery

3.

Conclusions

4.

Expert opinion

Introduction: Rapid clearance of drugs from the body results in short therapeutic half-life and is an integral property of many protein and peptide-based drugs. To maintain the desired therapeutic effect patients are required to administer higher doses more frequently, which is inconvenient and risks undesirable side effects. Drug delivery technologies aim to minimise the number of administrations and dose-related toxicity while maximising therapeutic efficacy. Areas covered: This review describes albumin’s inherent biochemical and biophysical properties, which make it an attractive drug delivery platform and the developmental status of drugs that are associated, conjugated or genetically fused with albumin. Albumin interacts with a number of cell surface receptors including gp18, gp30, gp60, FcRn, cubilin and megalin. The importance of albumin’s interaction with the FcRn receptor, the basis for albumin’s long circulatory half-life, is described, as are engineered albumins with improved pharmacokinetics. Albumin naturally accumulates at tumours and sites of inflammation, a characteristic which can be augmented by the addition of targeting ligands. The development of albumin drug conjugates which reply upon this property is described. Expert opinion: Albumin’s inherent biochemical and biophysical properties make it an ideal drug delivery platform. Recent advances in our understanding of albumin physiology and the improvement in albumin-based therapies strongly suggest that albumin-based therapies have a significant advantage over alternative technologies in terms of half-life, stability, versatility, safety and ease of manufacture. Given the importance of the albumin:FcRn interaction, the interpretation of the pharmacokinetic and pharmacodynamic profiles of albumin-based therapeutics with disturbed albumin:FcRn interaction may have to be reassessed. The FcRn receptor has additional functionality, especially in relation to immunology, antigen presentation and delivery of proteins across mucosal membranes, consequently albumin-based fusions and conjugates may have a future role in oral and pulmonary-based vaccines and drug delivery. Keywords: albumin, cancer, conjugation, drug binding, drug delivery, drug development, FcRn, fusion, half-life, inflammation, oncology, pharmacokinetics Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

The constant demand for superior therapies for poorly served medical conditions provides the incentive for the development and approval of new and improved drugs. It is clear that the pharmacodynamics of many small molecules, peptide and protein drugs are sub-optimal. Innovative drug formulations, drug delivery technologies and medical devices strive to improve the pharmacodynamic profile through the use of advanced formulations; improved routes of administration; optimised pharmacokinetics, including drug release, absorption, distribution and 10.1517/17425247.2015.993313 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. . .

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The inherent biochemical and biophysical properties of albumin make it an ideal drug delivery platform. While maintaining therapeutic efficacy, drug load and toxicity can be reduced by means of association, conjugation or genetic fusion to albumin. A number of albumin-enabled drugs are approved for human use or are in clinical development for the treatment of diabetes, cancer, inflammation and haemophilia. The albumin:FcRn interaction is critical for albumin’s long circulatory half-life. The elucidation of the albumin: FcRn interaction has enabled the creation of engineered albumin with significantly improved circulatory half-lives. Conjugation to multiple sites on the surface of albumin may disturb the albumin:FcRn interaction and consequently the pharmacokinetic and pharmacodynamic profiles of drugs previously conjugated in this manner may have to be reassessed. Albumin is known to accumulate at inflamed and malignant tissues due to a combination of effects including EPR, catabolism and the presence of albumin receptors. This can be enhanced by incorporating additional targeting ligands; furthermore, the addition of chemotherapeutic agents had led to the development of albumin--drug conjugates.

This box summarises key points contained in the article.

elimination; reduced toxicity and targeted drug delivery to the required anatomical location while avoiding accumulation or action of the drug in healthy tissue. Protein and peptide-based drugs have attracted a significant investment in recent years due to their potential to deliver greater efficacy with increased specificity and decreased side effects. However, the use of such drugs is often limited due to their inherent short therapeutic half-life, sometimes as short as a few minutes. Rapid renal clearance of drugs from the body may, therefore, require frequent administration of higher drug doses. This leads to an inconvenient medication regimen, higher costs and greater risks of undesirable side effects. Development of many drugs with promising therapeutic value may be limited by this factor. Improved drug delivery technologies aim to minimise drug load, while maximising therapeutic efficacy and patient convenience and thereby improve patient compliance. A range of technologies has been used in the recent past to overcome some of these challenges using both chemical and biological drug carriers including PEGylation, fusion to the crystallisable fragment (Fc) portion of antibodies and to non-natural designed, disordered proteins. However, while a number of drugs chemically conjugated to PEG have been approved, more recent safety concerns regarding the use of this polymer have made this a less attractive option. The more biological solutions such as Fc fusion have not raised similar safety signals but their benefit in delivering dosing regimens longer than once-weekly has yet to be proven and the use of disordered proteins has yet to 2

be fully validated by the approval of any candidate using this approach. The association, fusion or conjugation of protein and peptide-based drugs as well as small molecules to albumin has enabled the development of approved therapies with superior therapeutic efficacy for a range of clinical indications. 2.

Albumin in drug delivery

Human albumin is a single non-glycosylated polypeptide chain of 585 amino acids with a molecular weight of 66,439 Da. Synthesised predominantly in the liver, albumin performs a number of important functions including the maintenance of oncotic pressure, transport and distribution of a variety of endogenous and exogenous ligands and contributing to the maintenance of plasma pH. The average 70 kg person has a total albumin pool of ~ 360 g, the ~ 120 g intravascular albumin pool being in constant exchange with the extravascular pool [1-4]. Human albumin is the most abundant plasma protein with an average plasma concentration of 42 g/l (632 µM) and a circulatory half-life of 19 days. Albumin’s long circulatory half-life is the basis for a number of half-life extension strategies [5]. Albumin is a component of numerous secretions including milk, saliva, sweat and tears [1-5]. As will be described later in this review, albumin accumulates at inflamed and malignant tissues, a property which can be enhanced by the addition of specific targeting ligands. Given albumin’s abundance, stability, long circulatory half-life and inherent binding capacity, albumin is an important determining factor in the pharmacokinetics of many drugs and is an excellent drug delivery platform (Figure 1). 2.1

Albumin -- Nature’s own drug delivery vehicle Structure and stability

2.1.1

Based upon an analysis of the human albumin primary structure, the molecule is formed from three homologous domains, DI -- III which themselves comprise two separate helical subdomains (named A and B) [1,6,7]. A heart-shaped protein with 67% a-helix and no b-sheet, albumin has 17 disulphide bonds and one free thiol from an unpaired cysteine (Cys34) in DI [1,7]. As a consequence, human albumin is very stable to changes in pH, exposure to heat and denaturing solvents. In response to changes in pH, albumin undergoes a number of structural changes. The only physiologically relevant change occurs above pH 8, when albumin undergoes an isomerisation known as the N to B transition with significant changes in ligand binding associated with alterations to the structure of DII and to a lesser extent DI, with little or no change to DIII [1,7,8]. Due to albumin’s high level of charged amino acids, 83 basic (lysine and arginine) and 98 acidic (glutamic acid and aspartic acid) albumin is very soluble in aqueous solutions around neutral pH. Commercially, albumin solutions of up to 35% (w/v) can be prepared from pooled human plasma and are stable for up to 3 years if stored below 25 C in an appropriate formulation [1]. Formulation can further enhance the resistance of albumin to heat by addition of

Expert Opin. Drug Deliv. (2014) 12(6)


C-terminus Cys34 DI

DIII N-terminus

Physical properties Single polypeptide chain Very soluble Flexible Stable to adverse pH and temperature conditions Biochemical properties Very abundant Highly disulphide bridged Single free thiol Long circulatory half-life


Biopharmaceutical properties Long history of safe use Low intrinsic activity Recombinant versions available Fusions and conjugates approved, safe and well tolerated Low immunogenicity Inherent tumour accumulation

DII

Figure 1. Biochemical and biophysical properties of albumin for drug delivery. Albumin’s inherent physical, biochemical and biopharmaceutical properties collectively ensure albumin is an excellent drug delivery platform. The 17 disulphide bonds are indicated in red while the free thiol at Cys34 within DI is shown in cyan. Figure used with permission from Novozymes and prepared by K. Bunting and D. Sleep.

sodium octanoate and/or N-acetyltryptophan resulting in a thermal protectant conformational change in DIII. This effect is exploited by manufacturers of human serum albumin (HSA) from blood plasma who are required to ‘pasteurise’ their products at 60 C for 10 h to control the potential transmission of infectious agents. Ligand binding and allosteric interaction between binding sites

2.1.2

Due to its abundance and inherent properties, albumin possesses an extraordinary ligand-binding capacity and makes albumin the most important carrier/transporter protein in the human body for both endogenous and exogenous ligands, enabling some drugs to be available in quantities beyond their natural plasma solubility, decreasing their toxicity, lowering clearance rates and increasing circulatory half-life [7,9]. Albumin bound ligands include up to 35 different proteins and peptides [7,10] and a wide range of small molecules and transition metal ions [7,11-15]. Bulky heterocyclic anions such as warfarin bind to a site located in the core of DIIA, known as the major drug-binding site I or Sudlow’s site I, while aromatic carboxylates such as ibuprofen and diazepam bind to a site in DIIIA, known as the major drug-binding site II, or Sudlow’s site II [12-14]. Remarkably, 70 -- 80% of circulating serum albumin possesses a free sulphydryl group, Cys34. The highly reduced nature of this sulphydryl group is ascribed to its positioning within a 9.5 A˚ deep pocket within DIA and the modulating function of surrounding amino acids both lowering the pKa of the thiol and contributing to its high redox potential [16]. Cys34 acts as the binding site for Au(I), Hg(II) and complexed Pt(II) in the form of cisplatin [15] and represents a natural conjugation site for nitric oxide (NO),

with over 80% of blood NO being carried in a S-nitrosothiol bound form where it represents an endogenous pool of NO to be liberated in response to physiological demand (Table 1) [13,17]. Not unsurprisingly, this site has been exploited extensively for the conjugation of small molecules as well as protein and peptide-based drugs as will be described later. Distributed throughout its tertiary structure, albumin can bind up to 7 -- 9 molar equivalents of long chain (14 -- 18 carbons) fatty acids Table 1, [18]. Seven fatty acid-binding sites have been identified by crystallographic studies [14,19,20]. Albumin is the major plasma fatty acid delivery vehicle to tissues based upon metabolic need. Under normal physiological conditions albumin binds 0.1 -- 2 moles of unesterified fatty acids, however, it can bind more under certain disease states [7,21]. The albumin bound fatty acid profile largely mirrors the free plasma fatty acid profile, with 98% of plasma unesterified fatty acids bound to albumin [22]. Fatty acid binding induces significant conformation change in the albumin structure, impacting the binding of other drugs when their binding sites are co-located with the fatty acid-binding sites [11,20,21]. Ligand association and disassociation is concentration driven but given albumin’s structure, multiplicity of ligand-binding sites and inherent flexibility, there are significant implications for co-administration of multiple albuminbinding drugs, whereby ligand binding at one site can displace a ligand at the same site or modify the affinity for a second ligand at a distal site [7,11,12]. Albumin receptors, half-life and the importance of the albumin:FcRn interaction

2.1.3

Albumin interacts with a number of cell surface receptors including gp18, gp30, gp60, FcRn, cubilin and megalin.


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Table 1. Distribution of ligand-binding sites with the albumin tertiary structure. Binding site

Location

Ligands

Notes

Ref.

N-terminal site

DIA

Co(II), Ni(II) and Cu(II)

[15]

Cys34 FA1

DIA DIB

FA2 MBS (also known as site A or cadmium site A) FA7

Between DIA and DIIA DI/DII inter-domain contact region

Au(I), Hg(II), Pt(II) and NO Fatty acids, haem-Fe(III), bilirubin, hemin, synthetic Fe (II) porphoryrins and Al(III) phthalocyanines (tumour localising photosensitisers) and prostaglandins Fatty acids Cu(II), Ni(II), Cd(II) and Zn(II)

Consists of the 3 N-terminal amino acids, Asp-Ala-His Cisplatin-binding site Low-affinity FA-binding site

High-affinity FA-binding site Surrounded by FA1, FA2 and FA7

[7,18,19,21] [15]

Major drug-binding site I, or Sudlow’s site I, Low-affinity FA site

[6,7,12,13,18,21]

FA6 Met298 FA3-FA4

Between DIIA and DIIB Between DIIA and DIIB DIIIA

Low-affinity FA-binding site

[7,18,21] [15] [6,7,9,12,13,18,19,21]

FA5 FA8-FA9

DIIIB Between DIA--DIB--DIIA and DIIB--DIIIA--DIIIB

Secondary MBS (also known as site B or cadmium site B)

Currently not defined

DIIA

Fatty acids, thyroxine, bulky heterocyclic anions such as warfarin, CMPF, phenylbutazone, tolbutamide, iodipamide and indomethacin Fatty acids Cisplatin (Pt(II))-binding site Fatty acids, aromatic carboxylates ibuprofen, diazepam, diflunisal, diclofenac, iopanoic acid and thyroxine Fatty acids and thyroxine Fatty acids

Major drug-binding site II, or Sudlow’s site II. FA3 lowaffinity FA binding, while FA4 high-affinity FA binding High-affinity FA binding Supplementary sites only. FA8 short-chain FA and FA9 induced during FA saturation

Cd(II), Co(II), Mn(II) and Zn(II)

[13,15,17] [6,7,9,13,18,19,21]

[7,9,13,18,19,21] [7]

[15]

Albumin is the most important carrier/transporter protein in the human body for both endogenous and exogenous ligands due to its extraordinary ligand-binding capacity. Bulky heterocyclic anions bind to the major drug-binding site I or Sudlow’s site I located in the core of DIIA, while aromatic carboxylates bind to the major drug-binding site II, or Sudlow’s site II located in DIIIA. Albumin can bind up 9 molar equivalents of long chain fatty acids at nine sites, FA1-FA9. The albumin-bound fatty acid profile largely mirrors the free plasma fatty acid profile, with 98% of plasma unesterified fatty acids bound to albumin. NMR studies have indicated that FA2, 4 and 5 represent high-affinity long chain fatty acid-binding sites, while FA1, 3, 6 and 7 represent low-affinity long chain fatty acidbinding sites. CMPF: 3-Carboxy-4-methyl-5-propyl-2-furanpropionic acid; MBS: Multimetal binding site.

These receptors play a central role in the transport of albumin between compartments as well as its degradation, salvage and recycling [5,23]. Gp18 and gp30 are widely distributed among a range of tissues and have higher affinity for conformationally modified albumins but not native albumin from which it has been concluded that gp18 and gp30 resemble scavenger proteins mediating the endocytosis and degradation of damaged albumins. Gp60 binds native albumin but not chemically modified albumin, has a narrower distribution being restricted to vascular continuous endothelium and alveolar epithelium and plays a pivotal role in the transcytosis of albumin and the bulk flow of plasma proteins across the intact 4

endothelium from the apical to the basal membrane. Albumin binding induces the formation of invaginations which subsequently pinch off, trapping plasma components including free unbound albumin in vesicles (caveolae), which traffic across the endothelial cell layer before fusing with the basal membrane. As a testament to their importance in endothelial transport, caveolae occupy 50 -- 70% of the surface plasma membrane and 10 -- 15% of the total cell volume. Despite extensive biochemical characterisation, the gene for gp60 has yet to be identified [24,25]. The reabsorption and metabolism of proteins from the glomerular filtrates by receptor-mediated endocytosis is important function of renal proximal tubular




Albumin is released from the cell

Neutral pH Albumin

Acidic pH

Misc proteins

Non-receptor bond proteins are degraded

FcRn receptor

Albumin binds FcRn sorting to FcRn Artist’s impression showing indicative albumin recycling process

Figure 2. FcRn-mediated albumin recycling. Albumin and other plasma components are continuously sampled by the pinocytosis. Upon acidification of the early endosome the affinity of albumin for the FcRn receptor significantly increases and albumin binds to FcRn in a stoichiometric ratio of 1:1. FcRn-bound albumin is recycled to the plasma membrane whereupon albumin is released from FcRn when the pH returns to 7.4. Plasma components which did not bind to FcRn, which could include damaged or conformationally modified albumin as well as albumin to which additional components have been randomly conjugated to surface amino acid residues such as radiolabel tracers, sugars or targeting ligands, are sorted away from the recycling route and degraded in the lysosome. Figure prepared by Novozymes and used with permission from Novozymes.

epithelial cells. Megalin forms a complex with cubilin and another protein, amnionless. This complex is responsible for the receptor-mediated reabsorption of albumin, after which the albumin is degraded in the lysosome [26,27]. The extraordinarily long circulatory half-life of human albumin is partially due to its size of ~ 67 kDa being above the kidney filtration threshold but also to a receptor-mediated salvage mechanism, which rescues albumin and IgGs from degradation, facilitated by the neonatal Fc receptor (FcRn) [28-34]. FcRn is widely distributed in many mammalian tissues and cell types including renal, brain and vascular endothelia; gut, upper airway and alveolar epithelia and antigen-presenting cells. Expression can differ between animal species, for example, while FcRn is expressed by human intestinal epithelial cells in both the neonate and adult, rodent FcRn expression is highest in neonatal intestinal epithelial cells but levels decline rapidly after weaning [29,32,33]. There is growing interest in the role of FcRn in the transport and distribution of both albumin and IgG, antigen retrieval and presentation [35]. A heterodimeric type I glycoprotein, FcRn consists of a glycosylated heavy chain (HC) composed of three a-domains (a1, a2 and a3), a single transmembrane domain, a 44 amino acid cytoplasmic tail and has structural homology to the transmembrane extracellular domains of the HC of MHC class I molecules. The HC is non-covalently associated with the common soluble light chain consisting of 12 kDa b2-microglobulin (b2m) [30-32,36-39].

The importance of FcRn in albumin homeostasis and transcytosis has been well documented [3,30,31,33]. The extracellular environment is continuously sampled by pinocytosis and although FcRn is present at the cell surface, under normal physiological conditions albumin has low affinity for FcRn. The FcRn extends the circulatory half-life of albumin by binding albumin with high affinity at low pH within the acidic endosome and diverting it from lysosomal degradation. Albumin is returned to the extracellular compartment where at physiological pH it is released from FcRn, thereby prolonging the circulatory half-life of albumin in a strictly pH-dependent manner (Figure 2) [28]. The magnitude of FcRn rescue in humans is significant as demonstrated by an example of familial hypercatabolic hypoproteinemia, attributed to a mutant b2m gene [32,33,40], where serum levels of albumin are reduced to 19 -- 21 g/l (normal range 34 -- 54 g/l) [41]. Animal models are widely used to determine the pharmacokinetic profile of albumin-enabled drugs. Therefore, it is important to understand the cross-species binding of human albumin-based drugs to animal FcRn to predict the possible impact of drug conjugation or fusion to the affinity of the albumin component for FcRn and the resulting impact on the pharmacokinetic profile. Mouse albumin binds to mouse FcRn with 10-fold higher affinity than human albumin, and surprisingly has a 5-fold higher affinity to human FcRn than human albumin so reducing the expected benefit of


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performing pharmacokinetic studies of human albumin-based drugs in mice in which the endogenous mouse FcRn has been replaced with human FcRn [42]. Molecular modelling, X-ray crystallography and sitedirected mutagenesis studies have elucidated the basis of the pH-dependent binding of albumin to FcRn [43-46]. Albumin binds to the FcRn HC with a 1:1 stoichiometry. DIII is important for functional pH-dependent interaction with FcRn; DII does not contribute to the albumin:FcRn interaction, while DI has a lesser role compared with DIII [28,34,43,44,46]. Albumin binding to FcRn at a crevice between DI and DIII results in movement of both DI and DIII relative to the rest of the molecule, with DIII’s movement being the most significant [46]. Analysis of albumin:FcRn co-crystals suggests that interaction of DIII with FcRn is predominantly hydrophobic in nature and centres around W53 and W59 within the FcRn HC [44-46]. Affinity of albumin for FcRn at acidic pH can be modulated by the introduction of selective amino acid substitutions within the albumin primary sequence. Further, albumin variants with improved affinity for FcRn under acidic conditions but retaining low affinity for FcRn at physiological pH have improved pharmacokinetics in rodent and nonhuman primate preclinical animal models [44,47]. Albumin: a safe and low immunogenic protein Human albumin is highly polymorphic; currently 79 naturally occurring mutations within the human albumin coding region have been characterised [48,49]. Despite this heterogeneity within the general population, albumin purified from pooled plasma has a long, safe and successful history of use in medical interventions including plasma volume expansion, detoxification, imaging/diagnostics, coating medical devices and drug delivery [1]. Immune responses to protein therapeutics can be influenced by a large number of factors but occur either because the body detects the molecule as foreign, or immune tolerance to an autologous protein has been breached. One result is the production of antidrug antibodies (ADAs) by B-cells. ADAs have been observed at varying levels of incidence and with varying consequences in the vast majority of biotherapeutic products. Recombinant albumins are one of the few exceptions to date [50], even when an immunologically high-risk dose regimen was used. It is all the more surprising that human albumin auto-antibodies can be detected in healthy individuals, which are elevated in various disease states including cirrhosis, diabetes, familial dysautonomia, hepatitis and inflammatory bowel disease. Some human albumin auto-antibodies may actually be specific for bovine serum albumin, which shares 75% homology to human albumin and may arise due to the ingestion of cow’s milk and so the identification of some human albumin auto-antibodies may actually be assay artefacts to assay reagents or may nonspecifically recognise HSA. Nevertheless, it would appear that detection of antibodies to albumin is a common phenomenon, and does not lead to immune complex disease, significant reduction in albumin levels or any other directly 2.1.4

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related clinical condition, even when at highly elevated levels [51-57]. Recombinant albumins While circulating endogenous plasma albumin is a viable target by which drug developers have sought to manipulate the solubility, distribution, pharmacokinetics and pharmacodynamics of exogenously administered drugs, the use of plasma-derived albumin to modify the same ex vivo has met with increasing safety concerns. To meet these needs, a number of systems have been assessed for the manufacture of recombinant albumin for therapeutic applications [7], however, only yeast and rice-based systems are currently used for the manufacture cGMP grade material [58]. Yeast-derived recombinant human albumin is structurally identical to serum-derived albumin [59], with the advantages of animalfree production, consistency, exceptional purity (host cell protein levels < 1 ppm) and very high levels of free thiol (> 95% [w/w]). Clinical studies with yeast-derived recombinant albumin have shown comparable safety, tolerability and pharmacokinetic/pharmacodynamic profiles, with no evidence of any immunological response with cumulative i.v. doses up to 80 g and i.m. doses up to 300 mg per volunteer [50,60]. The ability to express recombinant human albumins has also enabled a detailed examination of albumin ligand-binding properties through site-directed mutagenesis, an examination of the biochemical and biophysical properties of isolated albumin domains and the introduction of new functionality, for example, the ability to reversibly bind oxygen [6-8,13,16,61,62]. The availability of a reactive, natural single free thiol in circulating and recombinant albumins has enabled the development of prodrugs and therapeutics peptides designed specifically to conjugate to albumin in vivo and in vitro, respectively, as described further later in this review. The high number of surface lysine residues which contribute to albumin’s high aqueous solubility have also attracted attention as conjugation sites for small molecules, peptides and sugars. An important consideration is how a conjugated moiety may impact the biochemical/biophysical properties of albumin. For example, a natural variant of albumin which becomes glycosylated in DIII has reduced circulatory halflife [63,64]. Moreover, forced glycation, oxidation of albumin at basic amino acids (lysine and arginine) and variants with increased hydrophobicity and net charge all increase elimination of albumin primarily by hepatic clearance [65-67]. Chemically modified albumins are also known to have over 1000-fold higher affinity for two widely distributed albumin scavenger proteins termed gp18 and gp30 found in liver, heart, lung, muscle, kidney, fat, brain, adrenal and pancreas tissue, which internalise and degrade conformationally modified but not native albumins [68,69]. Consequently, drugs conjugated to albumin in such a way that results in a reduced affinity of albumin for FcRn and/or increase affinity for gp18/gp30 might be expected to have reduced half-life and increased accumulation in certain tissues such as the liver. 2.1.5



2.2

Albumin-based half-life extension

Albumin has an exceptionally long 19-day circulatory half-life due to a combination of reduced renal filtration and FcRnmediated recycling. Consequently, various strategies have sought to extend the circulatory half-life of small molecules, peptides and proteins by engaging them covalently or transiently with albumin [5,70,71]. Non-covalent association Albumin’s endogenous ligand-binding capacity has been exploited in a number of ways to increase the circulatory half-life of therapeutic molecules, which do not have natural affinity for albumin. A small organic molecule based upon 4-(p-iodophenyl) butanoic acid derivatives was identified due to its ability to specifically bind to albumin [72]. Attachment of the ‘albutag’ to either small molecules or recombinant proteins has been shown to be able to increase their half-lives. Albutag was able to extend the half-life of a single chain antibody (scFv) by a factor of 40-fold in a tumour-bearing mouse model [72]. Similarly, a series of phosphate esters of albuminbinding small molecules to a FVIIa anticoagulant peptide were shown to significantly extend the half-life in a rabbit pharmacokinetic study; a diphenylcyclohexanol phosphate ester was particularly effective, increasing the half-life over 50-fold [73]. The covalent attachment of fatty acids to peptides, proteins and small molecules has proven to be a very successful strategy, both clinically and commercially, to extend the plasma half-life by association with albumin’s fatty acid-binding sites. Insulin detemir (NN304, Levemir) is an insulin analogue myristoylated (C14) at the B29 lysine. Upon subcutaneous (s.c.) administration, insulin detemir exists in the presence of zinc predominantly as reversible aggregates of hexamers that delays absorption of monomer [74], which is enhanced by the presence of the fatty acid component; however, once released from the site of injection insulin detemir rapidly associates with circulating albumin (insulin detemir is 98% albumin bound) through DIIIA prolonging the half-life from 4 -- 6 min to 5 -- 7 h in humans [5,74,75]. The slow dissociation of the acylated insulin analogue from albumin further contributes to blood glucose lowering ability of the insulin analogue [70,71,74]. Insulin degludec (NN1250, Tresiba) is an ultra-long acting insulin with a stable pharmacokinetic/ pharmacodynamic profiles achieved through the attachment of palmitic diacid (C16) via a g-L-glutamyl spacer to B29 lysine of desB30 insulin molecule. Upon s.c. administration, insulin degludec exists as long chains of zinc stabilised hexamers. The chain gradually disassembles releasing the monomers, which rapidly associate with circulating albumin. The longer fatty acid side chain prolongs the plasma half-life of insulin degludec to just over 24 h in humans [76]. Following the same principle, liraglutide (NN2211, Victoza) is a glucagon-like peptide 1 (GLP-1) (7 -- 37) analogue in which Lys37 is substituted by arginine and Lys26 is palmitoylated


2.2.1

(C16) via g-L-glutamyl spacer. The resultant modifications increase self-association into heptamers, reduce susceptibility to protease degradation by dipeptidyl peptidase-4 (DPP-4) and allow reversible association with albumin with a concomitant increase in plasma half-life from ~ 2 min to 11 -- 15 h in humans. At a clinically relevant concentration (10 nM), greater than 98.9% of liraglutide is protein bound [5,70,71,77-79]. Semaglutide (NN9535) is a new acylated, long-acting GLP-1 analogue with a half-life of 160 h in humans allowing once-weekly dosing. The improved pharmacokinetics is achieved by substituting Ala8 with a-aminobutyric acid, while Lys26 is modified with stearic diacid (C18) via a g-Lglutamyl and a bis-aminodiethoxyacetyl containing spacer. As with liraglutide, Lys37 was substituted with arginine to simplify manufacture [80]. Small molecules can also be modified by acylation, for example, studies with cisplatin-bearing axial succinate and a series of unbranched aliphatic (carbon chain length C2-C16) carbamate species have shown C16 derivatised cisplatin to have the highest affinity for human albumin, binding in a 1:1 stoichiometry. Moreover, the C16 derivatised cisplatin was taken up more efficiently by cancer cells and therefore had the greatest cytotoxicity [81]. Proteins and peptides that naturally, or by protein engineering, can specifically bind albumin have also been used to extend the circulatory half-life of drugs. Albumin-binding domains (ABD) have a relatively small size (below the renal filtration threshold) and possess specific and non-covalent binding to albumin, which delays their renal elimination through association with circulating albumin. ABD include bacterial proteins, peptides and antibody fragments [82-92]. A number of the ABD bind to albumins from different animal species, which is an advantage during preclinical development; however, while those ABD with the highest affinity for albumin have the longest life-life, the theoretical upper halflife limit will always be the half-life of the endogenous albumin [87,88]. The plasma half-life achieved by use of an ABD depends on the type of the ABD used, its affinity for albumin, the impact of ABD binding to albumin on FcRn recycling and the clearance mechanism of the drug attached to the ABD [5]. Conjugation As described previously, albumin possesses a single natural free thiol Cys34 within DI, which acts as a natural binding site for metal ions and a conjugation site for metabolites. Not surprisingly, a number of groups have sought to extend the circulatory half-life of proteinaceous drugs through conjugation to albumin via Cys34 including granulocyte colony stimulating factor (G-CSF) [93], Kringle domain [94], DARPin domain [95], the antiretroviral gp41 targeting peptide C34 (PC-1505) [96], insulin [97], the opioid agonist dynorphin A (CJC-1008) [98], YY peptide [99] and GLP-1/exendin-4 (CJC-1131 and CJC-1134-PC) [100-103] through chemical conjugation to this site. The availability of cGMP recombinant human albumins with high free thiol content has significantly advanced this 2.2.2


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approach [50]. In situations where manufacturing processes for the therapeutic molecule are well established, conjugation to albumin is a viable and attractive life-cycle management approach for drug developers. Moreover, dependent upon the nature of therapeutic molecule, de novo chemical synthesis of the drug with linker and thiol-compatible conjugation chemistry, with the option to incorporate non-natural amino acids to improve protease resistance, can be accomplished in a single step, as exemplified by the approaches taken by ConjuChem LLC to develop GLP-based diabetic therapies. CJC-1131 consists of a synthetic peptide similar to GLP-1 (7 -- 36), in which Ala8 is substituted with D-alanine to provide resistance to DPP-4 and the addition of a C-terminal lysine (Lys37) with the selective attachment of (2-(2-(2-maleimidopropionamido(ethoxy)ethoxy)acetamide)) to the epsilon amino group of Lys37, with CJC-1131 being administered parenterally, conjugating in vivo with circulating endogenous albumin [100,101]. In clinical studies, CJC-1131 was well tolerated with no evidence of neutralising antibodies or increases in specific IgG/IgE antibodies. CJC-1131 demonstrated significant improvement in circulatory half-life from a 2 -- 3 min in humans expected for free GLP-1 to ~ 9 -- 15 days for CJC-1131 [5,100]. In a second approach, a synthetic exendin4 peptide, with the addition of the same maleimide derivatised C-terminal lysine used in CJC-1131, was conjugated in vitro to recombinant albumin [50] creating CJC-1134-PC, the preformed albumin-exendin-4 conjugate being administered parenterally [103]. CJC-1134-PC is active in vitro and in vivo with no loss of activity and demonstrated significant improvement in circulatory half-life from a 2 -- 3 h in humans for the free exendin-4 peptide to over a week for the preformed conjugate, representing almost a 200-fold improvement in circulatory half-life, allowing once-weekly dosing [5]. Albumin fusion As an alternative to chemically coupling albumin to the proteinaceous drug in vitro, the application of genetic engineering allows the DNA of albumin and the therapeutic molecule to be cloned as one continuous open reading frame, coupling the two through a peptide bond formed in vivo during protein translation within the manufacturing host of choice, typically yeast or mammalian cells, as an albumin fusion [5,104]. The safety and general utility of albumin fusions can be seen by considering the number of albumin fusions approved, under review by the Regulatory Authorities or in clinical development (Table 2). The advantage for the drug developer is a greatly simplified commercial manufacturing process with the albumin fusion being secreted fully folded into the fermentation broth from which it can be purified utilising albumin as a purification tag, facilitating the development of platform manufacturing processes [104]. Typically, the therapeutic molecule is fused either to the N- or C-terminus of albumin, however, they can be fused simultaneously to both termini creating bivalent or bispecific albumin fusions. Over the last 20 years, a large 2.2.3

8

variety of peptides and proteins with diverse activities as well as biochemical/biophysical properties have been genetically fused to albumin to produce biologically active molecules. These have included peptides (GLP-1/exendin-4, BNP and ANF), growth factors (erythropoietin and G-CSF), coagulation factors (FVIIa, FIX and von Willebrand factor), anticoagulants (hirudin, infestin and barbourin) cytokines (IL-2, IL-1ra, interferon-a-2b and interferon-b), hormones (growth hormone and insulin), enzymes (human butyrylcholinesterase), redox modulators (thioredoxin) and a variety of antibody fragments and alternative antibody scaffolds [5,104]. As discussed previously, albumin is a very stable molecule, unlike a number of the therapeutic proteins and peptides which have been fused to albumin. Where the phenomenon has been studied, conformational and colloidal stability of the therapeutic protein/peptide can increase following fusion to albumin, however, the improvement in colloidal stability is the primary cause of improved stability. The impact of the effect is affected by the nature of the therapeutic protein/ peptide and in general drug formulations selected to stabilize the therapeutic protein/peptide maybe preferred to enhance the overall stability of the albumin fusion [105-107]. The potency of the therapeutic molecule may be impacted by fusion to albumin, however, this can be enhanced by the use of linkers between the albumin and the therapeutic protein (Table 2). As with the development of all therapeutic proteins, the potential appearance of antibodies to the product must be considered as emergence of such antibodies can impact the drug efficacy, drug clearance or cross-react with related proteins. In this regard, fusion to albumin was superior to PEGylation, with just over 20% of patients receiving a PEGylated interferon-a raising antibodies to the product compared with less than 1% receiving the clinical dose of albinterferon a-2b (ClinicalTrials.gov identifier: NCT00115908) [108]. Of the 2098 patients from 7 combined Phase III albiglutide studies, 5.5% tested positive for antialbiglutide antibodies, none was neutralising or impacted the change in haemoglobin A1c [109,110]. Analysis of a clinical study of balugrastim revealed that 6.9% of subjects tested positive for anti-albumin antibodies pre-dosing, while treatment-emergent anti-albumin antibodies were detected in 1.8% of subjects. The responses were weak and transient, being undetectable at the end of the study. In relation to anti-G-CSF-emergent antibodies, the occurrence rate was 0.5% in the balugrastim subject group and 0.9% in the PEGylated G-CSF subject group; none of the antibody responses was neutralising [111]. While the clinical data set is limited, there is nothing to indicate that fusion to albumin potentiates the immune response to the therapeutic protein or to albumin itself. In fact, the evidence suggests that the inclusion of albumin might have the opposite effect. It has been suggested that albumin may contain regulatory T-cell epitope sequences (Tregitopes) that tolerise immune responses to protein therapeutics [112,113].



Type II diabetes

Chemotherapyinduced neutropenia Chronic hepatitis C

Haemophilia B

Cocaine-addiction

Gastric and breast cancer

Haemophilia A Haemophilia B

GLP-1

G-CSF

IFN-a-2b

Factor IX

Butyrylcholinesterase

Anti-HER2 scFv and anti-HER3 scFv

Factor VIIa

CCN: rVIIa-FP, CSL689

CCN: MM-111

CCN: AlbuBChE, TV-1380

INN: Albutrepenonacog alfa CCN: rIX-FP, CSL654

INN: Albinterferon alpha-2b

INN: Balugrastim

INN: Albiglutide

International nonproprietary name or company code name

CSL Behring

Merrimack

Teva

CSL Behring

HGS

Teva

GSK

Company

MAA submitted (Europe) BLA and MAA withdrawn

1 -- 2 (18 -- 40 h)

Phase II

Phase II

Phase I

Unknown 4 (86 -- 90 h)

0.3 (6.1 -- 9.7 h)

4 (92 h)

Phase III

Approved (USA and Europe)

6 -- 7 (138 -- 163 h)

6 (140 -- 159 h)

Status

Half-life in humans (days)

GLP-1 (7 -- 36) dimer fused to N-terminus of albumin. Marketed as Tanzeum (USA) and Eperzan (Europe) G-CSF fused to C-terminus of albumin IFN-a-2b fused to C-terminus of albumin. Development terminated FIX fused to N-terminus of albumin via a linker incorporating a FVIIa/TF cleavage site. ClinicalTrials.gov identifiers: NCT01496274, NCT02053792 and NCT01662531 ClinicalTrials.gov identifier: NCT01887366 onceweekly administration ClinicalTrials.gov identifier: NCT01304784 onceweekly administration in combination with other small molecules and mAbs ClinicalTrials.gov identifier: NCT01542619 FVIIa fused to N-terminus of albumin via a 31 amino acid serine/ glycine linker

Notes

Fusion to human albumin has been successfully accomplished with a range of proteins and peptides [5,104] and a number of these molecules have either been approved for human use or are currently in clinical development. The improvement to circulatory half-life in humans varies with the therapeutic molecule fused to albumin from 5-fold to over 5000-fold [5].

Indication

Therapeutic protein

Table 2. Clinical progress of albumin fusions.



9

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2.2.4

Modulating the albumin:FcRn interaction

While the circulatory half-lives of albumin fusions in humans (1 -- 6 days) far exceed those of their unfused counterparts, they are usually less than that of native albumin (~ 19 days) due to the clearance of the active therapeutic protein component of the albumin fusion through proteolysis or receptormediated clearance. Consequently, dosing frequency is usually limited to no more than once weekly [5,104]. Research currently being performed by several groups is directed at closing this performance gap by characterising the albumin/FcRn interaction, identifying key amino acid residues at the albumin/FcRn interface and the generation of albumin variants with higher affinity for FcRn and extended circulatory halflives [43,44,46,47]. Consistent with the important role of DIII in the functional pH-dependent interaction with FcRn [34], the double amino acid substitution Glu505Gly and Val547Ala resulted in a 53% increase in the circulatory halflife in a non-human primate pharmacokinetic study [44], while the Lys573Pro substitution resulted in a 60% increase in circulatory half-life [47]. By combining various substitutions, it has been possible to increase circulatory half-life up to 2.5-fold in a non-human primate model (Figure 3) opening the prospect for once-monthly therapeutic dosing [114]. The role of albumins in targeting selective tissues and disease states 2.3.1 Accumulation of albumin at disease sites 2.3

Albumin and proteins, in general, are emerging as an extremely versatile and effective delivery tool in cancer therapy [23,70,71,115-117]. Albumin accumulates at inflamed and malignant tissues, commonly believed to involve an effect known as enhanced permeability and retention (EPR), which occurs due to the extensive neovascularisation in these tissues and the combined effects of a leaky capillary network and an impaired lymphatic drainage. Albumin and other plasma proteins are also catabolised by tumours, providing nutrition and energy to the fast growing tumour [118]. A number of albumin-binding proteins may also contribute to the accumulation of albumin at tumour sites, including FcRn, gp60 and SPARC [23,24,32,119]. Adult expression of SPARC, a 43 kDa glycoprotein, is largely restricted to tissues undergoing repair or remodelling such as wound healing, arthritis and cancer (including head and neck, melanoma, glioma, bladder, colon, prostate, lung, breast and lung). SPARC modulates cellular interaction with the extracellular matrix, binds cytokines/chemokines and is reported to have high affinity for albumin and may in part be responsible for the accumulation of albumin in the vicinity of tumours and inflamed tissues [24]. More recently, a new way to interpret the role of SPARC has been proposed in which SPARC acts to reduce the inflammatory response in healthy tissue surrounding the tumour caused by the presence of the tumour, thereby suppressing tumour growth. SPARC may also have a protective function by restricting migrating cancer 10

cell attachment at new metastatic sites. In aggressive tumours, the endogenous expression of SPARC is reduced, while in surrounding tissue it is increased, consistent with high-level SPARC expression being a poor prognostic indicator for cancer progression [24,119,120]. Compared with normal tissue, solid tumours present a slightly more acidic tumour environment. The pH of normal tissue ranges from pH 7.3 to 7.4, while the malignant tissue range is larger from pH 6.2 to 6.9, with considerable variation within different regions of the same tumour [121]. This may have implications for FcRn-mediated interaction of albumin in solid tumours as it is known that a range of cell lines express FcRn and albumin affinity for FcRn increases at the more acidic pHs [32]. Albumin facilitated imaging and drug accumulation

2.3.2

Given albumin’s natural ability to accumulate at such disease sites, it is not surprising that albumin is integral to a number of tumour imaging and diagnostic techniques, for example, macroaggregated technetium99m-labelled albumin particles are available in a number of sizes and are used in the imaging of various organs such as lungs, heart, liver and kidneys. Evaluation of an aminofluorescein-labelled albumin in Phase I/II clinical studies of fluorescence-guided malignant brain tumour resection demonstrated that the pharmacokinetics of aminofluorescein-labelled albumin was characterised by a low clearance rate, a small volume of distribution and a long circulatory half-life of 12.8 days. Furthermore, these studies demonstrated that albumin was a suitable carrier system for selective targeting into malignant glioma [122,123]. As previously discussed, Cys34 represents a natural conjugation site for NO. While NO effects a number of physiological processes including platelet aggregation and vascular smooth muscle relaxation, at high concentrations NO causes DNA damage and cell death. By combining the inherent tumour accumulation properties of albumin or albumin dimers with mono- or poly-S-nitrosylation, nitrosylated albumin has been shown to be cytotoxic in vitro and reduced tumour growth in vivo [124,125]. The antifolate drug methotrexate was conjugated to albumin via surface lysine residues in an attempt to increase the circulatory half-life of this small molecule while allowing for the passive tumour accumulation. Unfortunately, high drug-to-albumin loading resulted in denaturation of the albumin--methotrexate conjugate, accumulation in the liver and clearance via the mononuclear phagocyte system. Consequently, preclinical and clinical studies were performed on albumin--methotrexate conjugates with close to 1:1 albumin:drug loading as these conjugates enjoyed the same favourable tumour targeting properties, low liver uptake rates and a long circulatory half-life [126]. However, the drug substance would still be expected to have had a degree of heterogeneity as albumin surface lysines were the conjugation target for the methotrexate. An alternative approach would have been to conjugate to albumin via the



WT 10,0000

- 1.0-fold

V0098 - 1.6-fold V0354 - 2.1-fold V0311 - 2.4-fold

Mean serum conc. (ng/ml) ± SEM


10000

T1/2 320 h

1000

T1/2 286 h

100

T1/2 211 h 10

T1/2 132 h 1 0

10

20

30

40

50

Time (day)

Figure 3. Half-life extension through albumin engineering. Through selective amino acid substitution the affinity of human albumin for human FcRn can be increased over 50-fold at pH 5.5 while minimising the affinity for human FcRn at pH 7.4. In a cynomolgus monkey, pharmacokinetic study increased affinity towards FcRn correlated to an increase in the terminal plasma half-life by up to 2.5-fold. Study conditions: dose 1 mg/kg; bolus administration, i.v. Figure prepared by Novozymes and used with permission from Novozymes.

unpaired free thiol at Cys34; however, given the safety concerns related to the use of plasma-derived human albumin, a 6-maleimidocaproic acid (EMC) methotrexate derivative prodrug, EMC-D-Ala-Phe-Lys-Lys(g-MTX) (AW054 [Medac GmbH, Hamburg, Germany]), was developed which rapidly and selectively conjugated to form a 1:1 conjugate with the free-thiol on circulating endogenous plasma albumin. AW054 incorporated a plasmin/cathepsin B cleavage site to liberate Lys(g-MTX) at the target site. In a human ovarian carcinoma xenograph model, AW054 showed a significant reduction in tumour volume, whereas methotrexate had no effect [127], while in a collagen-induced arthritis (CIA) model AW054 showed suppression of the disease and was superior to methotrexate alone given at higher doses [128]. Replacing the protease cleavage site with a pH-sensitive hydrazone group and incorporating a DNA intercalating agent doxorubicin created the albumin-binding prodrug aldoxorubicin (formally DOXO-EMCH, or INNO-206 [CytRx Corp., Los Angeles, California, USA]). The acid sensitivity of the linker allows doxorubicin to be released intracellularly following uptake into the endosome/lysosomal pathway or extracellularly in the slightly more acidic tumour environment. In vivo conjugation with circulating albumin increased the circulatory half-life of aldoxorubicin to 20 h [129], compared with only 5 min for doxorubicin, reduced toxicity and accumulation

in non-target tissues while increasing the maximum tolerated dose. Aldoxorubicin is currently undergoing clinical assessment for the treatment of relapsed metastatic, locally advanced or unresectable soft tissue sarcoma (ClinicalTrials. gov identifier: NCT02049905), glioblastoma (ClinicalTrials. gov identifier: NCT02014844) and Kaposi’s sarcoma (ClinicalTrials.gov identifier: NCT02029430) [119], while a Phase IIb small cell lung cancer study (ClinicalTrials.gov identifier: NCT02200757) was initiated in Q3 2014 as were Phase Ib studies in combination therapies for the treatment of various solid tumours (ClinicalTrials.gov identifiers: NCT02235688 and NCT02235701). Paclitaxel is a chemotherapeutic agent used in the treatment of ovarian, breast, head and neck cancer, Kaposi’s sarcoma and non-small cell lung cancer. Its hydrophobic nature requires that as Taxol, paclitaxel is formulated in a 1:1 blend of Cremophor EL (polyethoxylated castor oil) and dehydrated ethanol, being diluted 5- to 20-fold in normal saline or dextrose solution (5%) before administration. Paclitaxel binds to albumin with high affinity but is readily released to tissues. Unfortunately, the use of Cremophor EL has toxic side effects including hypersensitivity reactions, nephrotoxicity and neurotoxicity [130]. Paclitaxel’s hydrophobicity has been exploited in an alternative, albumin-based formulation. ABI-007 (Abraxane) is an albumin bound


11


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form of paclitaxel composed of water-soluble ~ 130 nm albumin paclitaxel nanoparticles. The formulation avoids the use of Cremophor EL, the need for premedication with antihistamines and corticosteroids and undesirable biological effects associated with the use of Cremophor EL [131]. Following administration, the nanoparticles dissociate and the paclitaxel circulates associated with albumin resulting in a similar paclitaxel half-life for ABI-007 compared with the Cremophor EL formulation but with higher clearance and volume of distribution [24]. ABI-007 is approved for the treatment of breast, pancreatic and non-small cell lung cancer and is in clinical development for the treatment of multiple myeloma (ClinicalTrials.gov identifier: NCT02075021), malignant melanoma (ClinicalTrials.gov identifier:NCT00864253), metastatic colorectal cancer (ClinicalTrials.gov identifier: NCT02103062), bladder (ClinicalTrials.gov identifier: NCT00583349) and ovarian (ClinicalTrials.gov identifier: NCT00407563) cancers [23,24,119,132-137]. The in vitro and in vivo performances of paclitaxel differ in crosslinked and non-crosslinked albumin nanoparticles [138]. In an in vitro simulation, transfer of paclitaxel to plasma albumin was observed from both crosslinked and non-crosslinked albumin nanoparticles, however, drug release was greater from the non-crosslinked albumin nanoparticles. Pharmacokinetic analysis revealed that initially plasma paclitaxel concentrations were higher from the crosslinked nanoparticles but these rapidly declined and were lower than the non-crosslinked albumin nanoparticles. However, due to the slower clearance of paclitaxel from the crosslinked nanoparticles, the terminal half-life of paclitaxel from crosslinked nanoparticles was greater than that from non-crosslinked nanoparticles. Tissue distribution studies revealed that paclitaxel from crosslinked nanoparticles rapidly accumulates in tissues with active mononuclear phagocyte systems (e.g., liver, spleen and lung), while paclitaxel from non-crosslinked nanoparticles rapidly accumulated in local tissues such as heart, kidney and intestine and was then rapidly cleared, consistent with the disintegration of the noncrosslinked nanoparticles. Both nanoparticle formulations showed antineoplastic activity, however, non-crosslinked nanoparticles showed greater efficacy but with slightly greater toxicity [138]. The accumulation of drugs at selected disease sites through conjugation to albumin is not limited to metal ions or small molecules. A conjugate of HSA and the 66 kDa tumour necrosis factor-related apoptosis-induced ligand (TRAIL) not only displayed a circulatory half-life nearly 27-fold longer than TRAIL in a rat pharmacokinetic study, but retained biological activity in vitro and enhanced therapeutic activity in a CIA mouse model. Furthermore, HSA--TRAIL conjugate accumulated in the inflamed paws of the CIA mice and was retained at this site for 3 days, while the accumulation of TRAIL in the inflamed paws was negligible and disappeared within 1 day [139]. Likewise, a genetic fusion of IL-1ra to albumin not only extended the circulatory half-life by almost 30-fold in healthy mice, but also showed selective 12

accumulation and retention of the albumin-IL-1ra fusion in the inflamed paws of CIA mice and with lower accumulation in other organs compared with IL-1ra alone [140]. Active drug targeting Drug tumour targeting facilitated by conjugation or binding to albumin may be further enhanced by incorporating an additional targeting ligand to a feature of the tumour itself, be that the cancer cells themselves or the neovasculature which develops around the tumour to supply nutrients and oxygen. The blood--brain barrier is a specialised layer of capillary endothelial cells with tight junctions, which separates blood from the underlying brain cells. The blood--brain barrier prevents entry into the brain of most drugs with almost 100% of large-molecule drugs and greater than 98% of smallmolecule drugs being unable to cross the blood--brain barrier. Albumin with an isoelectric point of ~ 5.2 has a net negative charge at physiological pH. The blood--brain barrier is also anionic (negatively charged) at physiological pH creating an environment for selective transport of cationic substances from the blood to the brain. Albumin can be cationised by treatment with ethylenediamine and it has been shown that cationic albumin not only binds to brain capillary endothelial cells, but also increases the uptake of proteins and nanoparticles conjugated with cationic albumin both in vitro and in vivo [141]. Removal of the terminal sialic acid residues from glycoproteins exposes galactose residues resulting in their rapid clearance from the circulation through interaction with the high-affinity hepatocyte asialoglycoprotein receptor (ASGPR) followed by internalisation and degradation in lysosomes. Attachment of galactose to albumin can very easily be achieved by conjugating lactose, a disaccharide composed of galactose and glucose, to lysine residues on the surface of albumin to create lactosaminated albumin. Attachment of 20 -- 30 galactose residues is sufficient to ensure that lactosaminated albumin is selectively internalised by hepatocytes. Not surprisingly, lactosaminated albumin has been exploited as a liver small molecule delivery vehicle, however, an important consideration is that cellular uptake via the ASGP-R is saturable and imposes important selection criteria on the drugs conjugated to lactosaminated albumin. Attachment of the antiviral drug adenine arabinoside monophosphate (ara-AMP) to surface lysines and histidines of lactosaminated albumin via a phosphoamide bond ensured that cleavage and release of adenine arabinoside (ara-A) occurred following internalisation. Lactosaminated albumin--ara-AMP conjugates have been assessed in the treatment of chronic hepatitis B. Treatment inhibited viral replication to a similar degree as the free drug but without the side effects. However, it was not able to eradicate the virus which reappeared following cessation of treatment [142]. Attachment of doxorubicin via a pH-sensitive hydrazone bond to the free thiols in a partially reduced lactosaminated albumin created a liver targeting doxorubicin with a doxorubicin:albumin molar ratio of 5:1 for the treatment of 2.3.3



Properties Stable Inert Abundant Long circulatory half-life Non-immunogenic Safe Albiglutide Balugrastim rIX-FP and rVIIa-FP CJC-1134-PC MM-111 Targeted

Abraxane Aldoxorubicin AW054


albumin drug conjugates Proteins and peptides Therapeutic GLP-1, G-CSF, FIX, FVIIa, scFv Targeting αvβ3 ligands scFv

Small molecules Paclitaxel Doxorubicin Methotrexate Auristatin Radionucleotides Gemcitabine Sunitinib

Figure 4. Albumin as a versatile drug delivery platform. Albumin’s inherent biochemical and biophysical properties make it an ideal platform molecule from which to develop a range of drug delivery technologies, including therapeutic peptides with long circulatory half-lives, albumin prodrug conjugates and targeted albumin drug conjugates. Figure used with permission from Novozymes and prepared by D. Sleep.

hepatocellular carcinoma. In rats with chemically induced hepatocellular carcinoma, i.v. administration of lactosaminated albumin--doxorubicin conjugate significantly inhibited tumour growth while selectively accumulating in the liver tumours and the surrounding liver tissue but not in heart, intestine or kidney tissue. This was in contrast to doxorubicin which did not accumulate in the liver but did show greater accumulation in heart, intestine and kidney tissue, from which it was concluded that lactosaminated albumin--doxorubicin conjugate increased both the anticancer efficacy and safety of doxorubicin in rats with hepatocellular carcinoma [142]. The integrin avb3 is an adhesion receptor that is highly expressed by the endothelial cells of the tumour neovasculature and in some cases the tumour cells but is poorly expressed on established vasculature and normal tissues. Integrin avb3 binding to extracellular matrix proteins including fibronectin, vitronectin, fibrinogen and osteopontin is mediated through binding to an Arg-Gly-Asp (RGD) peptide [143]. To investigate the potential of such a targeting approach, albumin was modified by conjugation of 4 mole per mole of the cytotoxic agent monomethyl auristatin F (MMAF) through a valine-citrulline containing linker and with 7 moles per mole of a RGD containing cyclic peptide. In vitro the RGD-targeted MMAF--albumin conjugate efficiently killed human umbilical vein endothelial cells with EC50 values of 20 -- 80 nM conjugated drug equivalent, while RGD-targeted MMAF--albumin conjugates killed avb3 expressing C26 carcinoma cells with EC50 values of 9 -- 150 nM. Pronounced tumour

accumulation of RGD-auristatin--albumin conjugates was observed following administration of the conjugates to C26 murine colon carcinoma-bearing mice and while the conjugates could not be detected in spleen or kidney, some offtarget accumulation in the liver was observed which declined over time in contrast to the tumour accumulation which was retained indicting the avb3 targeting can have antitumor activity in vivo through the direct killing of tumour cells and the vasculature supporting the tumour growth [144,145]. Antibodies and antibody fragments have been used extensively to provide selective tissue targeting. The accumulation of a radiolabelled albumin fusion to a scFv with high affinity targeting to the carcinoembryonic antigen (CEA) was studied in a mouse LS174T human colorectal carcinoma xenograph model. Accumulation and retention of the albumin fusion protein at the tumour was observed, plateauing at 23 or 37% of the initial dose depending on the radiolabelling procedure, significantly higher than the scFv alone which plateaued at 5% of the initial dose. Accumulation was ascribed to a combination of albumin’s accumulation at tumour sites due to EPR and the CEA-targeted accumulation. The CEA tumour targeting albumin fusion protein also showed lower normal tissue accumulation than a scFv-CH3 to the same antigen [146]. A bivalent scFv--albumin fusion ‘HSAbody’ targeting CEA specifically localised to LS174T human colon carcinoma xenographs reaching almost 25% of the initial dose while being rapidly cleared from normal tissues with a tumour:blood ratio of 7:1. The introduction of N-linked glycosylation site at a position between the scFv and albumin with subsequently clearance through mannose receptors increased the tumour:blood ratio of 22:1 but decreased the tumour uptake to around 4% of the initial dose [147]. Taking a similar approach a DARPin, an alternative antibody scaffold, targeting epithelial cell adhesion molecules (EpCAM) was conjugated to MMAF and mouse albumin in such a way as to maintain affinity for mouse FcRn. As expected, the circulatory half-life of EpCAM-MMAF was increased 100-fold by conjugation to albumin. The in vitro cytotoxicity of the EpCAM-targeted drug to cell lines expressing EpCAM was over 100-fold higher than a non-targeting DARPin--MMAF control, while the additional conjugation of mouse albumin improved the in vitro cytotoxicity a further 10-fold [95]. In another study, the use of antibody-targeted albumin nanoparticles was investigated as a means to overcome the toxic side effects associated with the use of a multikinase inhibitor, sunitinib by promoting the accumulation of the multikinase inhibitor in tumour cells and preventing the exposure of healthy cells to the inhibitor. A camelid antibody (Nanobody) with specificity for the EGF-receptor (EGFR) was conjugated to 100 nm glutaraldehyde crosslinked albumin nanoparticles loaded with a multikinase inhibitor sunitinib analogue (named 17864) coupled with cysteine and methionine residues of albumin in such a way that it can be released in a reductive environment. In vitro the


13


D. Sleep

Nanobody-targeted albumin nanoparticles showed a 40-fold higher binding to EGFR-positive 14C squamous head and neck cancer cells relative to PEGylated albumin nanoparticles. Internalisation of the albumin nanoparticles via clathrinmediated endocytosis and successful release of the kinase inhibitor suppressed cell proliferation, whereas the PEGylated albumin nanoparticles had no antiproliferative effects on 14C cells [148]. Similarly, RGD-conjugated albumin nanoparticles loaded with the chemotherapeutic agent gemcitabine, a nucleoside analogue, were shown to be internalised by the pancreatic cancer cell line BxPC-3 in a RGD-dependent manner consistent with the expression of the avb3 integrin on the cell surface. An in vivo investigation of the antitumour efficacy of gemcitabine-loaded albumin nanoparticle in a nude mouse BxPC-3 pancreatic cancer xenograph model revealed that the nanoparticles were superior to the equivalent dose of gemcitabine and that the addition of the RGD tumour targeting ligand improved the efficacy still further such that while the gemcitabine treatment suppressed the growth of the tumour the gemcitabine-loaded RGD-conjugated albumin nanoparticles significantly reduced the size of the tumours emphasising yet again that the additive effects of EPR and tumour targeting in the delivery of toxic payloads to selected anatomical sites can be demonstrated in the appropriate animal model systems [149]. 3.

Conclusions

As a safe, inert, abundant, non-immunogenic and stable protein with an extraordinarily long circulatory half-life, albumin is an ideal platform molecule from which to develop a range of drug delivery technologies (Figure 4). It is less than 10 years since the discovery that albumin interacts with FcRn and that this interaction is responsible for a significant proportion of albumin’s long circulatory half-life. Through association, conjugation and genetic fusion to albumin, the circulatory half-life of a diverse range of therapeutic molecules has been significantly improved and a number of products are already approved for human use or are in late-stage clinical development in the fields of metabolic diseases, neutropenia, haemostasis, substance abuse and oncology. Despite these undoubted successes, the circulatory half-life of the albuminenabled drugs is not as long as the 3-week circulatory halflife of albumin. In the past 2 years, the elucidation of the albumin:FcRn interaction has progressed rapidly. The ability to engineer albumin variants combined with albumin’s inherent stability which tolerates such changes has greatly accelerated this process and has led to the development of engineered albumins with superior pharmacokinetics. The availability of simple and scalable platform albumin-based manufacturing processes will further encourage the adoption of engineered albumins. The observation that albumin accumulates at certain sites of disease is well established and consequently the exploitation of albumin as an effective drug delivery tool in areas such as 14

cancer therapy is accelerating. The reason for this is likely to be multifaceted combining the EPR effect, the catabolism of albumin by tumours, the expression of albumin-binding proteins at the tumour site and the local conditions created by the tumour itself. Furthermore, albumin’s natural ability to accumulate at solid tumours can be supplemented by additional targeting ligands in the form of peptides, sugars and antibody fragments providing additional selectivity. Circulating albumin possesses an endogenous conjugation site, which is used in the body to carry and distribute small molecules. Therefore, this site offers a natural solution as to where on albumin to conjugate toxic payloads to create a homogeneous product without disturbing albumin’s structure. However, care must be exercised when developing albumin--drug conjugates, with or without additional targeting ligands, not to disturb the albumin structure as this can lead to unwanted recognition by albumin scavenging receptors such as gp18/30, clearance by the mononuclear phagocyte system and loss of affinity for FcRn resulting in increased clearance and unwanted accumulation in non-target tissues such as the liver. Given albumin’s unique set of inherent properties, it is surprising that albumin has not been exploited more extensively in the field of drug delivery. Our understanding and control over albumin’s interaction with normal and disease state cellular processes is growing and albumin is emerging as an extremely versatile and effective platform in drug delivery. 4.

Expert opinion

Albumin is an extremely abundant protein throughout the human body. This is an advantage to drug developers as there are few anatomical sites which are not already exposed to albumin. Consequently, administration of albumin-enabled drugs is unlikely to result in additional accumulation in healthy tissues through association of the albumin component of the drug to albumin receptors or cryptic albumin-binding sites as these are already occupied by endogenous albumin. The transport and distribution of albumin-binding drugs and prodrugs such as ABI-007 and aldoxorubicin take advantage of albumin’s long circulatory half-life and ubiquitous high abundance, ensuring rapid and efficient distribution. The albuminbinding drug or prodrug is subsequently released at sites of inflammatory and oncogenic disease due to the accumulation of albumin at these sites and the particular environmental conditions present at these disease sites. The potential of albuminbinding prodrugs such as aldoxorubicin is that they combine the high water-solubility and high plasma stability from the albumin component with the acid-sensitive hydrazone linker allowing the therapeutic agent, in this case doxorubicin, to be released either extracellularly in the slightly acidic environment of the tumour or intracellularly in acidic endosomal compartments after uptake of the albumin conjugate by the tumour cell. By this mechanism, the drug, once released from albumin, evades FcRn-mediated cellular recycling.




It is well established that through conjugation and genetic fusion to albumin, the circulatory half-life of a diverse range of therapeutic molecules has been significantly improved. However, the recent elucidation of the albumin:FcRn interaction allows a reinterpretation of previous pharmacokinetic and pharmacodynamic data generated with native albumin fused or conjugated to payloads such as radiolabel tracers, diagnostic agents, drugs in the form of small molecules, proteins or peptides, sugars or targeting ligands. This is especially true for payloads randomly conjugated to surface amino acids in an attempt to increase their stoichiometric loading onto albumin. The reasons are twofold: first, such modifications could led to the formation of damaged or conformationally modified albumins so promoting their rapid clearance through gp18/gp30 and second, given the importance of the functional interaction with FcRn to ensure a long circulatory half-life and possible targeting to sites of disease, any disruption of this interaction will have a significant negative impact on the observed pharmacokinetic profile and possibly on the magnitude of the observed biological effect. There is a growing realisation of the importance of selecting the correct preclinical animal models and the interpretation of the subsequent data. Despite significant homology between mammalian albumins, human albumin affinity for animal FcRns varies considerably. Administration of clinically relevant low doses of human albumin-based drugs, combined with high levels of endogenous animal albumin and poor affinity for the endogenous animal FcRn has led to significant underestimation of the impact of human albumin in drug delivery, especially as evident from studies performed in commonly used preclinical animal models. Such preclinical assessment would be greatly aided by the development of transgenic animal models, in which human albumin and human FcRn replace their endogenous animal counterparts. The availability of appropriate human albumin:animal FcRn (including human FcRn) affinity assays such as those based upon surface plasmon residence, competitive ELISA or BioLayer Interferometry will continue to assist the development and understanding of the impact of payload composition, location and presentation on the affinity of albumin for FcRn and the impact on circulatory half-life in the chosen preclinical animal model. Preclinical and clinical data clearly show that albumin can be used to deliver payloads (small molecules and proteins/ peptides) conjugated or genetic fused to albumin to solid tumours or sites of inflammation. It is becoming increasingly clear that the FcRn receptor has additional functionality, especially in relation to immunology, antigen presentation and delivery of IgGs and Fc-fusions across mucosal membranes. In humans, FcRn is functionally expressed in the epithelial mucosa of the intestine, genitourinary tract and the central and upper airways. In recent years, the transepithelial delivery of Fc-fusions of erythropoietin, follicle-stimulating hormone

and interferons has been demonstrated across the pulmonary epithelial mucosa [150], while the transepithelial delivery of Fc-tagged nanoparticle loaded with insulin has been demonstrated across the intestinal epithelial mucosa [151]. Moreover, intranasal immunisation of Fc-fusion to herpes-simplex virus glycoprotein gD induced a strong immune response affording protecting against intravaginal challenge with a virulent form of herpes-simplex virus [152]. Successful pulmonary drug delivery requires the drug to be presented in a particulate form and delivered via a device to ensure delivery to the correct location within the lung, increasing the importance of drug stability in this form and within the device. Oral drug delivery of large protein-based drugs has its own challenges including high inter- and intra-subject variability, low product stability primarily due to proteolysis and poor absorption leading to low bioavailability. Possible solutions to these challenges include the use of encapsulation, the use of protease inhibitors, targeting ligands and permeability enhancers, including fatty acids. In this regard, albumin’s ability to bind fatty acids and interact with FcRn, combined with the availability and ease of manufacture of albumin-based fusions and conjugates as monomeric proteins and nanoparticles, combined with their inherent high solubility and stability, it is surprising that oral and pulmonary-based vaccination and drug delivery has yet to be fully explored. Ultimately, the goal of future research should be aimed at developing therapies with increased efficacy and specificity, decreased side effects and a route of administration and dosing regimen, which maximises patient convenience and thereby patient compliance. Recent advances in our understanding of albumin physiology and the improvement in albumin-based therapies which have developed based upon this understanding strongly suggest that albumin-based therapies have a significant advantage over alternative technologies in terms of half-life, stability, versatility, safety and ease of manufacture.

Acknowledgements The author would like to thank J Waters and P Chamberlain for their assessment of the immunogenicity risk associated with albumin-based products and S Kjærulff, L Evans, D Pearson, D Shelly and K Bunting for their critical review of the manuscript.

Declaration of interest The author is an employee of Novozymes Biopharma UK Ltd and a named inventor on a number of patents assigned to Novozymes Biopharma DK A/S in the subject field of this review. The author has received no payment for the preparation of this manuscript, nor does the author hold stocks in Novozymes A/S.


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Affiliation Darrell Sleep PhD Director, Biopharma R&D, Novozymes Biopharma UK Ltd., Castle Court, 59 Castle Boulevard, Nottingham, NG7 1FD, UK Tel: +44 115 9551286; Fax: +44 115 9551299; E-mail: [email protected]

Albumin nanostructures as advanced drug delivery systems.

Carrageenan and its applications in drug delivery.

Cys34-PEGylated Human Serum Albumin for Drug Binding and Delivery.

Body temperature reduction of graphene oxide through chitosan functionalisation and its application in drug delivery.

Supramolecular hybrid hydrogel based on host-guest interaction and its application in drug delivery.

Albumin corona on nanoparticles - a strategic approach in drug delivery.

Albumin-based drug delivery: harnessing nature to cure disease.

Application of gold nanoparticles in biomedical and drug delivery.

Constructing tunable nanopores and their application in drug delivery.

Application of liposomes in medicine and drug delivery.

Situational awareness and its application in the delivery suite.

Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices.

Application of chitosan-based nanocarriers in tumor-targeted drug delivery.

Application of Various Types of Liposomes in Drug Delivery Systems.

Biocompatibility of chitosan carriers with application in drug delivery.

Formulation and characterization of atropine sulfate in albumin-chitosan microparticles for in vivo ocular drug delivery.

Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization.

Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery.

In vivo biocompatibility, clearance, and biodistribution of albumin vehicles for pulmonary drug delivery.

Bioavailability of capsaicin and its implications for drug delivery.

The Smart Drug Delivery System and Its Clinical Potential.

Particle margination and its implications on intravenous anticancer drug delivery.

calcium phosphate hybrids for drug delivery application.

Albumin acts like transforming growth factor β1 in microbubble-based drug delivery.