Review
Preparing and evaluating delivery systems for proteins

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Abstract

From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions.

Introduction

Proteins and other biomacromolecules are gaining increased interest as drug molecules. Therefore, formulation development of suitable drug delivery systems is a major issue. The success of a protein as a therapeutic agent depends on the development of a formulation in which the native protein structure and activity during preparation and delivery as well as during shipping and long-term storage is maintained (Allison et al., 1996, Wang, 1999).

The main obstacles for oral delivery of proteins and other biomacromolecules are passing the harsh environment in the gastrointestinal tract, which is designed to degrade proteins, and securing the subsequent effective absorption of the amino acids into the systemic circulation. Therefore, most marketed products are designed for parenteral delivery. However, this entails a need for education of either medical staff or the patients themselves to assure proper administration. Moreover, parenteral delivery is associated with reduced patient compliance, especially for chronic treatment. Many research efforts are therefore aimed at improving compliance, either by employing alternative routes of administration or by reducing the frequency of injections. This review focuses on the latest developments in injectable particulate drug delivery systems for proteins as a method to reduce the frequency of injections. We focus on the use of water-in-oil emulsions as either the final formulation or as an example of the interfaces created in other delivery systems.

The overall purpose of applying an injectable particulate delivery system for proteins is to increase the patient compliance by reducing the number of injections, to decrease the side effects, e.g. by reducing the high peak concentrations of bolus injections, or to achieve long-term local delivery. The more complex drug delivery systems are thus a potential alternative to the conventional formulations of proteins, in which the protein is usually either lyophilized, in suspension, or in an aqueous solution.

The optimal release pattern may vary between proteins and between indications, and adaptable formulations are therefore required. Some proteins require sustained release, while others require controlled, immediate or pulsed release. Release can be obtained with different particulate drug delivery systems, e.g. microspheres, hydrogels, liposomes, and emulsions (Trenktrog and Muller, 1995, Cole and Whateley, 1997, Sadhale and Shah, 1999a, Sadhale and Shah, 1999b, Rosa et al., 2000, Caliceti et al., 2000, Bjerregaard et al., 2001, Kwon et al., 2001, Cleland et al., 2001, Sinha and Trehan, 2003).

One of the drug delivery systems that has been accepted for delivery of peptides and proteins is polymeric microspheres based on poly(lactic-co-glycolic acid) (PLGA) (Sinha and Trehan, 2003). Marketed examples include Nutropin Depot®, a sustained release system for human growth hormone (Cleland et al., 1997, Kwon et al., 2001) and Lupron Depot®, a sustained release system for leuprolide acetate (Okada and Toguchi, 1995, Okada, 1997). In 2004, however, Nutropin Depot was withdrawn from the market, reportedly due to “uncertainties and limitations in product supply required to meet future patient needs as well as the significant resources necessary for manufacturing” (Genentech patient information letter, June 2004). Nevertheless, extensive studies are ongoing using biodegradable microspheres for sustained protein delivery, e.g. prolonged effect of rhVEGF in promoting local angiogenesis has been reported when rhVEGF was encapsulated in PLGA microspheres and administered as implants (Cleland et al., 2001).

Alternatively, the use of microspheres is aimed at vaccine delivery (Johansen et al., 2000). As with the depot formulations mentioned above, PLGA microspheres have been evaluated for administration of vaccines against, e.g. hepatitis B (Shi et al., 2002, Jaganathan et al., 2004), diphtheria (Singh et al., 1998), and pollen (Igartua et al., 2001). Further, multivalent vaccines have successfully been administered in poly(lactic acid)-PLGA microspheres (Boehm et al., 2002). PLGA may also be replaced by the less expensive chitosan polymer as tetanus toxoid encapsulated in chitosan microspheres elicited an equal immune response to tetanus toxoid formulated in PLGA microspheres (Jaganathan et al., 2005). In vaccine development, protein-coated wax-based nanoparticles have also been shown to increase immune responses significantly (Cui and Mumper, 2002, Cui et al., 2004). However, sustained delivery of protein from nanoparticles upon parenteral administration is still awaiting success.

In general, one of the drawbacks associated with the formulation of microspheres and nanoparticles is the initial burst and the incomplete release of the encapsulated protein, which may influence its potential as a drug delivery system (Kim and Park, 1999, Kwon et al., 2001). Even though this to some extend can be controlled by optimising particle size and the manufacturing techniques applied (Fu et al., 2003); it is still a challenge to formulate microsphere and nanoparticulate drug delivery systems for parenteral protein administration.

In maintaining protein integrity and prolonged the effect of a protein, choosing a liposomal formulation is another possible strategy. It is possible to adjust the physical and chemical characteristics of the liposomes by adjusting the lipid composition and thereby to optimise the biological effect. Liposome suspensions are reasonably sturdy and can, e.g. be lyophilized and reconstituted, provided that lyoprotectants are included (van Winden and Crommelin, 1999, Stevens and Lee, 2003, Glavas-Dodov et al., 2005). Encapsulation in or association to liposomes is a mature technology that has proved successful in general pharmaceutical applications (Torchilin, 2005). Numerous studies on liposomal formulations of proteins have been published, and vaccines containing liposomes have been authorized (Langston et al., 2003, Katayama et al., 2003). However, to our knowledge, non-vaccine liposomal formulations of proteins or peptides have not yet reached the market in either Europe or Northern America.

Encapsulation of proteins in liposomes can yield a longer half-life by protecting the protein from degradation and by releasing the protein slowly during degradation of the liposomes. External association of proteins to the surface of liposomes may likewise yield a prolonged biological effect. Association of interleukin-2 to liposomes gave a prolonged effect (Kanaoka et al., 2003), as was observed for interferon-gamma (van Slooten et al., 2001). The secondary structural integrity is of concern since it may change on association to the liposomal surface (Janshoff et al., 1999, Halter et al., 2005), but studies on human interferon-gamma have shown that the secondary and tertiary structure of this particular protein is retained on association to and dissociation from liposomes (van Slooten et al., 2000). The potential drawbacks of i.v. liposomal formulations include that liposomes are subject to opsonization in the human body, and they are thus not suited to a treatment scheme demanding frequent administration.

Hydrophilic polymers can be transformed into a microparticulate system using a simple water-in-oil emulsification procedure (Poncelet et al., 1992). More advanced technology uses a water-in-water emulsion technology, as has been shown with modified dextran (Stenekes et al., 1998, Stenekes et al., 1999) and starch (Ekman and Lindahl, 1989, Gustaffson et al., 2000). The particles may be stabilised by a cross-linking procedure or by coating the particles with a hydrophobic polymer. Release from these particles is controlled by diffusion through the pores that are formed by degradation or swelling of the matrix.

So far, there has been little research into the physicochemical stability of proteins entrapped in these hydrogel microparticles (Cadee et al., 2002, Reslow et al., 2002). The all-aqueous preparation procedures were developed with the objective to eliminate protein degradation by organic solvents or by the water–organic solvent interface. However, theoretically, the water–water interface in the preparation procedure may still result in protein denaturation, since the water structure of the two water phases differs. Any protein that adsorbs at the interface may unfold resulting in further degradation, such as aggregation. In addition, other steps in the preparation procedure, such as the cross-linking or hardening procedure, may result in protein degradation (Cadee et al., 2002).

Water-in-oil emulsions can also provide controlled release of a drug, e.g. following intramuscular administration (Davis et al., 1985, Bjerregaard et al., 2001). The systems are flexible formulations, and the release of incorporated hydrophilic molecules of various sizes and characteristics can be modified by adjusting parameters such as volume fraction of the disperse phase, droplet size and osmotic gradient (Bjerregaard et al., 1999, Jorgensen et al., 2004c). In vivo prolonged release of aprotinin, a 6.5 kDa protein, from a water-in-oil emulsion has been demonstrated in mice (Bjerregaard et al., 2001). Also, the incorporation of ovalbumin into an emulsion has been shown to be more efficient in enhancing the immunogenic response when compared to an aqueous solution or a plain emulsion without protein (Masuda et al., 2003).

The joint characteristics during the preparation of these delivery systems are the presence of either two phases, an interface and/or application of various types of mechanical stress to prepare the drug delivery systems. This is illustrated in Fig. 1, where the preparation procedure is shown for microspheres (top) and a water-in-oil emulsion (bottom).

When preparing these particulate systems, e.g. emulsions, the protein is exposed to mechanical stress from the homogenisation as well as to the different excipients in the emulsion. In addition, the protein is exposed to the mixing of the oil and aqueous phase, and last but not least, to the creation of the oil–water interface. Proteins are amphiphilic molecules, and they have been used for their surfactant properties in, e.g. food science to stabilise oil-in-water emulsions (Burnett et al., 2002). This also makes them susceptible to structural changes when exposed to the interfaces created during the preparation of the particulate system (Dickinson and Matsumura, 1994, Wilde et al., 2004).

The potential detrimental effect of this exposure to interfaces on the structural stability of proteins in emulsions is of great importance for pharmaceutical proteins. Altered protein species may not only reduce the activity, but also increase the risk for side effects, such as immunogenicity (Thurow and Geisen, 1984, Frokjaer and Otzen, 2005) or changes in the pharmacological activity of the released protein (Kondo and Urabe, 1995). Steps to prevent structural changes are therefore needed. However, a rational stabilisation requires methods to study the potential changes, preferably in situ (Sadana, 1993, Zoungrana et al., 1997). Therefore, one of the most challenging tasks in the development of protein pharmaceuticals is dealing with the physical and chemical stability of proteins when they are incorporated into drug delivery systems. It should always be verified that the native structure is retained in the formulation or that the structural changes are fully reversible (Wang, 1999, Cauchy et al., 2002, Frokjaer and Otzen, 2005).

Section snippets

Stability of the delivery system and incorporated protein with emulsions as an example

Essentially, in developing delivery systems, both delivery system and the incorporated molecules should be stable and the delivery system should be able to release (the major part of) the incorporated protein unaltered. Therefore, the stability of the delivery system (e.g. the emulsion), the physical stability of the incorporated protein, and the possibility of achieving release of protein from the emulsion is discussed below.

Methods to obtain and/or increase protein stability in water-in-oil emulsions

Despite the promising features in terms of protein structure retention and release, further formulation optimisation of water-in-oil emulsion is still required. Future studies could include the effects of additional excipients that could either increase the stability of the protein or decrease the tendency for the protein to adsorb to the interface.

The physical stability and the structural changes of the model proteins in the water-in-oil emulsion vary, and the effects cannot be predicted from

Conclusion

Overall, particulate systems are promising drug delivery systems for the parenteral delivery of proteins. Nevertheless, further optimisation of the formulation to protect the proteins even further against the stress effects from the preparation and for instance exposure to the interface is required. As well as studies of, e.g. the activity of the released protein, both in vitro and in vivo, would have to be performed.

Additionally, further development of techniques for exploring the structural

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