Advances in Delivery of Biotechnology Based Pharmaceuticals

The use of advanced drug delivery systems for the delivery of biomacromolecules is typically either as particles or as complex liquid formulations. However, relatively few products have reached and are still on the market (83). Part of the developments is centered on exploiting new routes of administration and improving the bioavailability across the biological barriers. The quest for novel approaches to avoid barriers of adsorption and to improve delivery of biomacromolecules is an ever-evolving area. New materials are sought, exploited in search for improvement of the bioavailability or the possibility to deliver the proteins to new targets both locally and systemically.

Pulmonary Delivery

In the development of formulations for pulmonary delivery, control of the particle size is essential, since only particles below 5 |im are able to pass on to the deeper airways (8,84). The pulmonary route of delivery was first employed in 1925 with aqueous nebulization of insulin; however, the marketed product remains scarce (3). Shoyele et al. (85) list potential biomacromolecules that could be delivered through the lungs, but few have reached the market yet. An example is the treatment of cystic fibrosis (local) using Pulmozyme (Roche). It is an inhalation fluid containing dornase alfa in sterile water, pH ~7.0 (other excipients are calcium chloride and sodium chloride) (86). It was marketed in 1996 (3). Another product, Exubera (Pfizer), was withdrawn from the market in October, 2007, not due to safety or efficacy reasons, but because only few patients were treated with it (87). Exubera is a homogeneous insulin powder formulation containing sodium citrate (dihydrate), mannitol, glycine, and sodium hydroxide as excipients. The particle size is distributed so that a fraction of the total particle mass is emitted as fine particles capable of reaching the deep lung. Up to 45% of the 1 mg blister contents, and up to 25% of the 3 mg blister contents, may be retained in the blister (88), this will invariably cause variability in the delivered doses. Other companies were at this point in time still developing inhalable insulin formulations. Examples are Novo Nordisk (AERx), Alkemes (AIR), and MandKind Corporation (Technosphere®insulin), and some argue that the devices are potentially better while others say that their formulation is more rational (3,89). So in that respect, the potential for a viable inhalable protein formulation for systemic administration is still there.

Nasal Delivery

An alternative to the pulmonary route of administration is the nasal route, which is less demanding when it comes to formulation. With regard to, for example, particle size and simpler device development (5,90), examples are Minirin® (Ferring), desmopression, and Suprecur® (Sanofi-Aventis), buserelin, which are proteins formulated as nasal drops or nasal spray, where bioavailabilities of approximately 3% to 10% can be obtained. The formulations are just protein dissolved in purified water containing preservatives: chlorbutanol and benzalkonium chloride (91,92). However, more advanced delivery systems are also used, for example, chitosan formulations where bioavailabilities of 14% to 15% compared to subcutaneous administration can be obtained (90). A recent review by Ulum (2007) gives more details on nanoparticulate systems used for nasal delivery (93) or consult Costantino et al. (2007) on the physiochemical and therapeutic aspects (5).

Oral Delivery

Oral delivery of biomacromolecules, especially peptides and proteins, poses a considerable challenge but also has great potential (94). A recent review on the formulation possibilities can be found in Mahato et al. (2003) (6). Examples of products on the market with desmopresin in oral or sublingual formulations are Minirin® (Ferring) melt tablets and tablets, Nocutil (Gebro Pharma), both with bioavailabilities of approximately 0.25% and 0.1% (95,96). However, the low bioavailabilities do tend to reduce the potential of biomacromolecules being delivered by this route, but intense research is taking place to increase the bioavailability. Alternative formulations are encapsulation into particles, for example, containing chitosan or thiomers or increasing the interaction with the intestinal mucus (10,11,97,98). The sublingual route is also used in Grazax® (ALK-Abelló), a melt tablet containing allergens of grass pollen, however, a systemic effect here is not expected (99).

Cutaneous Delivery

Delivery across the dermis (transdermal) or into the dermis (dermal) poses a considerable challenge for biomacromolecules. Formulation concepts are based on chemical enhancers, iontophoresis, or iontophoresis in combination with electroporation (100,101) or the formation of radio frequency-formed microchannels (102). A review on this topic as well as need-free injections can be found in Schuertz et al. (2005) (103).

Particle Encapsulation

Microparticles By encapsulation of protein in polymeric microspheres or solid micro-particles, a sustained release profile can be obtained as well as a once-daily or once-weekly administration. For example, Zoladex (AstraZeneca), where goserelin is embedded in an absorbable lactid/glycolid copolymer, which releases goserelin continually over 28 days (104). Other examples of products based on polymeric microspheres are Lupron depot (TAP Pharmaceutical Products Inc), Pamorelin® (Ipsen Scandinavia), Sandostatin LAR depot (Novartis), and Decapeptyl (Debiopharm) (104-108). Some are formulated as implants releasing the drug compound over 28 days to one month (Zoladex), while others are suspensions, where a sustained-release is obtained. Nutropin depot, a depot formulation of growth hormone (rhGH) for once-weekly administration, is a formulation of micronized particles of rhGH embedded in biocompatible, biodegradable polylactide-coglycolide (PLG) microspheres (109), which was marketed in 1999 (by Genentech) and withdrawn from the market in 2004.

Microspheres are prepared by encapsulating the protein in a polymeric matrix either by solvent evaporation, coaservation, lyophilization, or spray drying. During the process, exposure to agitation, interfaces, and other types of denaturing stress should be minimized. Polymers introduced to prepare microspheres for controlled delivery of peptide and proteins can be either synthetic or purified from natural resources. The choice of material will depend on the desired release profile as well as the administration route, since many of the polymers are biodegradable. Some examples of polymers used are alginate, poly(lactic-co-glycolic acid) (PLGA), chitin, chitosan, or sodium hyaluronate. (9,110-113). The release mechanism from polymeric microspheres is diffusion, polymer degradation, or a combination of the two (111,114). However, one of the main drawbacks typically associated with the formulation of proteins in microspheres is the initial burst and the incomplete release of all the encapsulated protein (115,116). The amount of protein released from microspheres reaches approximately 28.2% to 54.7% for different formulations within the first day but with no subsequent release the following days (116). Thus, an incomplete release is exhibited. The incomplete release is likely to arise from noncovalent aggregates formed either within the microspheres or from the lyophilization process (116,117).

Nanoparticles Nanoparticles can be formed from surfactants, polymers, or polyamino acids. The concept is mainly that viable particles are formed, they can maintain their shape and use during preparation, they are stable, and the particles formed are in the nanometer range. An example of nanoparticles is the Medusa® concept by Flamel Technologies.

Nanoparticles of 10 to 50 nm are formed by poly L-glutamate grafted with a hydrophobic a-tocopherol (Medusa II) or of synthetic copolymer of natural leucine and glutamate amino acids (Leu hydrophobic and Glu hydrophilic) (Medusa I) (118,119). The amphiphilic character of the polyamino acid polymers drives the self-assembling of the nanoparticles in water; the poly-Leu chains are packed inside the structure, whereas those of Glu amino acids are exposed to water. The nanoparticles, which are 20 to 50 nm in diameter, are composed of 95% water and 5% Leu-Glu polymer. They are robust over a wide range of pH levels and can be stored as either stable liquid or stable dry forms (118,119). The Medusa I is used together with insulin in Basulin, and this formulation is the first controlled-release insulin formulation that uses recombinant human insulin rather than an insulin analog as in, for example, Lantus (Lilly) or Levemir (Novo Nordisk) (119). Other examples include the case of bone engineering (InductOs from Wyeth), where several different types of nanoparticulate systems have been tested (120). Solid lipid nanoparticles (SLN) are used for both parenteral and nonparenteral delivery, where mucosal membranes, blood-brain, and topical barriers may be surpassed (31,121). The encapsulations efficiencies vary immensely with the method of preparation and the excipients and protein used, but incorporation efficiencies from 1% to almost 100% can be obtained (31). The release from SLN can be a burst or an incomplete release, depending on the way the protein is incorporated (31).

One of the few potential successful sustained-release lipid formulations of biomacromolecules is the Depofoam™ concept by SkyePharma. It is different from the conventional liposomes by the increased surface area available, which makes the aqueous volume larger (122). It has been shown to successfully encapsulate (60-85%) and sustain the release of insulin, luteinizing hormone-releasing factor and others (123).

Chemical enhancers Another approach is a combination of particulate formation and enhancement of the delivery by use of enhancers that increase the ability of the biomacromolecules to cross membranes. Some examples are Eligen® and the SMART concept. The Eligen® technology employs low molecular weight compounds, for example, Ar-[8-(2-hydroxybenzoyl)amino]caprylat (SNAC) that interact weakly or noncovalently with the protein increasing its lipophilicity. Consequently, this increases the ability of the proteins to cross membranes and into the blood stream (12,13). The SMART concept of pH sensitive and membrane-destabilizing polymers enhances the uptake of biomacromolecules into the cytoplasm. These polymers undergo conformational changes as the pH changes in the endosome. The polymers are of the following types; pyridyl disulfide acrylate (PDSA) or poly(ethylarylic acid) (PEAA)(14,15).

Modification of Peptides and Proteins

Another approach to optimize the release and biological activity of protein drugs is to modify the drug compound itself (18). Protein conjugation is an expanding field in the search for ways to improve the efficacy of protein drugs (124). Conjugation can be defined as the covalent binding of one or more molecules to a drug molecule (125,126). Different conjugation strategies can be applied in the field of proteins. These include glycosylation (127,128) and attachment of fatty acids, i.e. acylation (129,130). Additionally, a large area of protein conjugation involves the attachment of polymers, for example, polyethylene glycol (PEG) (125,126). This invariably also leaves many choices and experiments as to which modifications to use and also subsequent formulation studies just with a modified protein. On the market are amino acidnsubstituted analogues, PEGylated and acylated proteins, for example, Proleukin (Novartis), Humalog (Lilly), NovoRapid (Novo Nordisk), Lantus® (Sanofi-Aventis), and Apidra® (Sanofi-Aventis), PEGylated: Neulasta (Amgen),

Pegasys (Roche), Somavert (Pfizer) and Peglntron (SP Europe), and acylated: Levemir (Novo Nordisk), and in Phase 3 trials Liraglutide (Novo Nordisk)(131).

Modification of the amino acid sequence The stability, biological activity, and distribution of proteins can be changed by alteration in the amino acid sequence. The main purpose is to increase or alter the pharmacological effect, but exchange of the chemical liable amino acids can also be employed. By altering the sequence, exchanging, or adding amino acids the pharmacological profile can be changed. Insulin has the alteration of proline at B28 to Asp, insulin aspart, NovoRapid (Novo Nordisk) and it has increased the onset of action to approximately 10 to 20 minutes after injection. This is due to the reduced tendency for insulin aspart to form hexamer, which increases the absorption rate upon injection (132-134). Similar profiles are obtained with insulin lispro, Humalog (Lilly), where Lis and Pro are exchanged at B28 and B29 and with insulin glulisin, Apidra® (Sanofi-Aventis), where Asp at position B3 is replaced by Lys, and Lys at position B29 is replaced by Glu (135-137).

A prolonged action can also be obtained, for example, insulin glargin, Lantus® (Sanofi-Aventis). It differs from native human insulin by three amino acids; Gly in position A21, and 2 Arg at the C-termial in the B-chain at position B31 and B32 (138,139). These alterations shift the pi of the protein from 5.4 to 7.0, which make the protein more soluble at acidic pH and insoluble at neutral pH. This leads to the formation of micro precipitates upon injection. The insulin is subsequently released slowly from the precipitate and absorbed from the injection site (138,139). The substitution at position A21 also alters the association properties, which makes the hexamer more stable. The release profile of insulin glargin is more flat, avoiding the peak concentrations seen for other insulin types (140). A prolonged effect and faster acting is also obtained in Proleukin (Novartis), where aldesleukin is produced as: nonglycosylated, containing no N-terminal alanine, and a replacement of Cysl25 by Serl25 (141).

Modification by PEGylation Protein PEGylation is defined as the covalent conjugation of PEG to proteins (142). Conjugation of PEG, PEGylation, is now widely used due to its advantageous effects on therapeutic efficacy, low toxicity, relatively simple chemistry, and commercial availability. The main objective of protein PEGylation is to maintain the inherent therapeutic activity after modification while also optimizing the pharmacokinetic profile of the drug (125,126). In first generation PEGylation, typically small (2-5 kDa) PEG was randomly attached to the protein chain resulting in different PEGylated species with various PEGylation sites. The random attachment could potentially diminish biological activity (125). Second generation PEGylation includes improved PEGylated proteins using site-specific PEGylation with a better potential for preserving biological activity. Larger and sometimes branched PEGs are applied (20-40 kDa), and usually the proteins are monoPEGylated. The first PEGylated protein product to reach the market was adenosine deamidase (Adagen® by Enzon Inc.) in 1990. Adagen® contains multiple PEGylated isomers (143). The marketed products differ with respect to PEGylation site, size of PEG attached, and the number of PEGs attached.

Modification by acylation Acylation is the conjugation of an acylchain to the proteins (129,130). The rationale for acylating peptides is alteration in the circulation time and increased stability against degradation. One of the protein products on market is an insulin product, Levemir (Novo Nordisk). Determir has an increased circulation time due to the attachment to serum albumin; approximately 98% is bound (129,144,145). In Determir, a C-14 acylchain is attached to insulin at B29. Another product in Phase 3 trials is

Liraglutide, where a C-16 acylchain is attached to glucagons-like peptide (GLP-1) at position 26. For Liraglutide, the attachment of the acylchain decreases the enzymatic degradation, and thereby elimination in the kidneys. This results in prolonged half-life, also because of binding to serum albumin (130).

Modification by glycosylation Glycosylation is one of the naturally occurring modifications to the proteins (127,128). It describes the pattern of sugar moieties attached to the protein. It varies with the cell type used during the production. In other words, it is only human cells that produce the identical human proteins with regard to glycosylation pattern. It can be used in formulation to increase stability (146), but it is mostly the cause of differences between the produced drug compound and the naturally occurring protein. In the formulation of EPO, the alteration in the glycosylation patterns is thought to cause increased immunogenicity (69).

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