Nanomedicine. Ian Teasdale

Transcription

1 3 Nanomedicine Ian Teasdale Administering a drug and relying on a small percentage to reach the target site could be considered somewhat technically regressive, but it is still how most pharmaceutical agents are administered and with most low molecular weight (M w ) drugs having a nonspecific biodistribution, the vast majority of a given drug is excreted or metabolised before ever reaching its intended target, never mind achieving the desired effect. Nanomedicines attempt to overcome this by reducing systematic elimination and enhancing circulation times, thus extending the loaded drug s therapeutic window, maximising drug activity and reducing side effects. Furthermore, nanomedicines can be designed to induce a controlled release profile of the drug, a triggered release at the active site and/or facilitate a targeted delivery to the required site. This can be achieved, for example, for cancer, vaccinations and inflammatory diseases, and indeed nanomedicines have become widespread in recent years, with a wide range of therapeutic improvements attainable for drug formulations based on nano -sized materials, i.e., materials with dimensions from 1 to 1,000 nm, although most usefully around nm (Figure 3.1) [1]. Since polymers inherently fall into this size range, and polymers have been investigated and used in pharmaceutical technologies for many years, nanomedicine in this sense has been around long before terms such as nanomedicine and nanoparticle became fashionable [2]. The term nanoparticle has been, somewhat confusingly, broadly used in the scientific and medical literature for all polymer formulations, thus in this book we refer to nanoparticles/nanospheres exclusively for conventional drug-delivery devices that noncovalently entrap and control drug release, with microparticles and microspheres referring to their dimensionally larger derivatives. 63

2 Polyphosphazenes for Medical Applications Figure 3.1 Some examples of nanomedicines. Reproduced with permission from T. Lammers, W.E. Hennink and G. Storm, British Journal of Cancer, 2008, 99, 3, , Nature Publishing Group [3] Due to their inherent size in the nanometre region, all polymer-drug conjugates belong to nanomedicines. Drugs can be conjugated to polymers via noncovalent interactions, such as polyplexes and micelles. Indeed, the most widely clinically used nanomedicines are liposomes (e.g., Doxil ), lipid-bilayer-based colloids (also chemically modified) in which drugs can be loaded into the hydrophobic core, prolonging anticancer drug circulation times [4]. The term polymer therapeutics [5], on the other hand, as defined by Duncan [6], refers to multicomponent nanomedicines whereby the drug or protein is covalently bound to the carriers. This is an important differentiation, as chemical conjugation can bring many advantages in terms of structural control and triggered release, but also since polymer therapeutics are classified as new chemical entities and thus fall under different regulatory jurisdiction. Recent advances in synthetic polymer preparation and formulation have enabled a rapid advancement of nanomedicine, with over 40 formulations being approved for routine human use in the last 20 years and many more currently in clinical development [6]. Examples include polymer-drug and protein conjugates [5], block copolymer polymeric micelles [7] and polyplexes developed for 64

3 Nanomedicine gene delivery [8]. The ever-increasing data shows a complex picture, with pharmacokinetics and pharmacodynamics of nanomedicines being governed by a number of factors, determined by size but also architecture [9] and chemical composition of the nanocarrier [10]. Furthermore, the post delivery fate of nanomedicines, which must also be excretable, should also be considered [10]. The structural control, multifunctionality (discussed in Chapter 1), as well as hydrolytic degradability (Chapter 2), indicate that poly(organo)phosphazenes have a large amount to offer in this field and the following chapter details some of the investigations carried out to date in this field. 3.1 Polyphosphazenes in Immunology Vaccine Adjuvants and Delivery Systems A vaccine adjuvant is defined as a component that potentiates immune responses to an antigen and/or modulates it towards the desired immune responses [11] and indeed the use of immunological adjuvants (immunoadjuvants, immunostimulants, immunopotentiators or immunomodulators) to improve the immune response to vaccine antigens has been carried out routinely for decades. Essentially vaccines which suffer from low immunogenicity, a common issue for the safer subunit vaccines, can be enhanced by the use of adjuvants and delivery systems, and furthermore a potential reduction in the number of immunisations required can be achieved. The oldest known and most widely used and investigated adjuvants are based on aluminium hydroxide and aluminium or calcium phosphate gels [1]. Although the precise mechanism of action appears still to be a matter of discussion, it would seem that the antigens are more readily internalised by antigenpresenting cells (APC) when administered in a bound, multivalent particulate form [12]. Formulations based on aluminium salts have been widely used for many years, are well proven and clinically safe and indeed aluminium-based adjuvants are used in standard child vaccination programmes in many countries, and is even more widespread in veterinary medicine [13]. However, aluminium-based adjuvants are not without their disadvantages, the biggest drawback being that, due to their inherent mechanism of activation, for example, 65

4 Polyphosphazenes for Medical Applications primarily promoting only Th2, not Th1 immune responses (note: for an explanation of the Th1 and Th2 immune response see [14]), they have little effect on certain critical responses, which are instrumental in protection against many pathogens [12]. Important vaccines used to provide immunity against, for example, human immunodeficiency virus (HIV), influenza and typhoid fever fall into this category [13]. Alternatives include antigen-carrying emulsions, so-called Freund s adjuvants, as well as their various derivations and combinations with an assortment of oils and emulsifiers [15]. Liposomes and virosomes (semisynthetic spherical vesicles reconstituted from virus-derived proteins) have also been shown to be efficient antigen carriers and adjuvant systems [16]. There is significant interest in alternative adjuvants, including polymeric adjuvants and nanoparticles, which can potentially broaden the applicability of immunoadjuvants and offer improved systems with longer lasting and higher immune responses [17, 18]. The structural control and reproducibility offered by synthetic polymers make them a valuable tool, in particular as it is known that properties such as chemical structure size, hydrophobicity and charge, are defining factors in their biodistribution and in the effectiveness of any immunological adjuvants prepared. The advancement in synthetic methods and the ability to prepare polymers with precisely controlled structures, coupled with parallel progress in the biological understanding of immune response mechanisms, opens the door to the rational design of immunological adjuvants tailored to provide optimised results. Furthermore, it is envisaged that polymer/particle surface functionalisation can be used to prepare advanced systems for targeted delivery and triggered and/or controlled release [17]. A number of polymers are currently under investigation including polyelectrolytes [19], dextrans, poly(lactic-co-glycolic acid) (PLGA) and polyanhydrides [18] in various forms, be it as free macromolecules for covalent or noncovalent conjugation to antigens [20], or packaged as nanoparticles, matrices or micelles [17]. Poly(organo)phosphazenes with free carboxylic acid side groups belong to one of the most well-investigated synthetic polymer adjuvants and with its multifunctional, flexible and degradable backbone, can be regarded as one of the most promising of the synthetic polymers tested to date [18]. 66

5 Nanomedicine Polyphosphazene Electrolytes as Immunological Adjuvants The polyacid/polyanionic polyelectrolyte family represent the most thoroughly investigated water-soluble polyphosphazenes used in nanomedicines with most of the work, pioneered by Andrianov and co-workers [21 25], focusing on its application as a polymeric adjuvant. The lead compound, poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) (Figure 3.2) has undergone thorough testing into its production [21, 26], hydrolytic degradation [27, 28], toxicology [29], as well as its combination with a diverse range of antigens [30] in vitro, in vivo, both in mice [31] and large animals [32, 33], and human clinical trials [34]. Figure 3.2 Chemical structures of some polyacid poly(organo) phospahzenes. PCEP: Poly[di(sodium carboxylatoethylphenoxy) phosphazene] The key to the use of PCPP in immunology is the ability of its sodium salt to form noncovalent interactions with antigenic proteins (Figure 3.3), binding multiple protein molecules to form water-soluble complexes [35]. For example, work on model systems with bovine serum albumin 67

6 Polyphosphazenes for Medical Applications (BSA) showed that PCPP binds BSA molecules per polymer, depending on the conditions, with a higher polymer ratio leading to improved binding. Water-soluble conjugates serve to transport, protect and stabilise the loaded antigens, whilst being able to present them to immune competent cells. Although the basic noncovalent interaction of cationic carboxylic acids groups could in theory be achieved with any macromolecular polyacid [19], PCPP has been shown to have superior adjuvant activity to similarly charged aliphatic polymers such as polymethylacrylic acid and polyacrylic acid [30]. The high density of binding sites, two per repeat unit, might be one possible explanation for this experimental observation. Also, the P-N backbone is known to be highly flexible, due to its unique bonding, and there is only minimal hindrance to bond rotation [36]. This flexibility of the backbone leads to a high conformational adaptability, which is thought to play an important role in its observed superior protein binding and presentation [35], and thus excellent activity as an immunoadjuvant. Figure 3.3 PCPP binding to BSA via noncovalent interactions. Reproduced with permission from A.K. Andrianov, A. Marin and B.E. Roberts, Biomacromolecules, 2005, 6, 3, , American Chemical Society [35] 68

7 Nanomedicine Structure Activity Relationships The most common method for the preparation of PCPP is the macromolecular substitution of the polydichlorophosphazene precursor [37] (see Section 1.1). This requires the use of protecting group chemistry, as it is well known that free acid groups lead to skeletal breakdown reactions. Propylparaben, the n-propyl ester of p-hydroxybenzoic acid has been most widely used for this, with subsequent deprotection of the propyl ester by hydrolysis under basic conditions. The requirement for complete macromolecular reactions has been addressed and structurally homogeneous, fully deprotected PCPP can be attained with the correct structure on relatively large (2 kg) scales [21, 26, 38, 39]. As mentioned above, the high density of carboxylic acid groups is essential for antigen binding and presentation. This has been shown with a series of partially deprotected PCPP, which had a defined percentage of propyl ester groups remaining [39]. A direct correlation was observed between the percentage of COOH groups on each macromolecule and its activity as an immunoadjuvant with an influenza antigen for a series of in vivo tests in mice [39]. Not deprotecting all acid groups led to lesser activity, clear proof that the COOH functional groups on the polyphosphazene backbone are an essential component, presumably binding via electrostatic interactions to cationic domains in the protein, in an analogous fashion to aluminium-based adjuvants [13, 40]. Hydrophobic interactions have also been shown to be important for some proteins and hence the hydrophobicity of the aromatic groups could also have a contribution to binding. High-throughput synthesis has been used to prepare a library of 40 polyacids with slightly varying structures to that of PCPP [41]. Of these, at least two (PCEP, and its isomer, named here PCEP-2, see Figure 3.2) have already been identified as having significantly enhanced immunoadjuvant activity in comparison to PCPP [23]. To the best of the author s knowledge, the immunoadjuvant activity of all the other polyacids, prepared in this library, has not yet 69

8 Polyphosphazenes for Medical Applications been reported. The biological data is discussed in more detail in Section 3.1.5, but it is apparent from the available data that the chemical structure of the polyphosphazene is decisive and thus that molecular-level interactions between adjuvant and protein are highly influential in determining the binding of the complex and importantly, the subsequent immunoadjuvant activity of the polymer. Three factors which may contribute to these matters are the flexibility, hydrophobicity and pka of the acid group. In PCEP and PCEP-2, the acid groups are further from the backbone and thus possibly more accessible than in PCPP, and since the flexibility of the polyphosphazene backbone has already been reported to be an important feature, it is also possible that this enhanced flexibility of the COOH moiety contributes to the binding of the polymer to the antigenic protein. Although pure speculation based on the current available data, the ability of the COOH group to bind and/ or penetrate to certain sites may also be important, in a similar fashion to the known enhanced effect of brush-type antimicrobial polymers [42]. This could, for example, explain the higher activity of PCEP compared with PCEP-2 (Figure 3.2), although this hypothesis is yet to be tested. A comparison with aliphatic polyphosphazene polyacids would shed light on this situation. The difference in pka of the aromatic and aliphatic COOH groups (aliphatic PCEP appears to have higher activity than its aromatic analogue PCEP-2), as well as the role of the hydrophobicity of the aromatic groups have also not been tested in this context and may also have an effect, with a combination of these effects, flexibility, hydrophobicity and pka likely to be key. As these effects also alter the solubility (drastically) and the hydrolytic stability of the polymers, a compromise must also be sought. The requirement for a combination of payload binding and presentation ability must also be considered. The ability to produce such libraries from the same polyphosphazene backbone facilitates the direct comparison and, although more work is required, it is an extremely interesting approach toward the targeted design and preparation of conjugates with optimised solubility, binding and activation. The ability to produce polymers 70

9 Nanomedicine with a wide variety of slightly adjusted structures is especially relevant for bioconjugates, since binding is likely to be different for diverse adjuvants and the wide spectrum of adjuvant structures means a one-hat-fits-all strategy for immunoadjuvants is unlikely to be successful. The fact that these polyanionic polyphosphazenes can be employed as molecular adjuvants, further adds to the control available, in comparison to, for example, alum or other particulate formulations (including polymer-based nanoparticles). The significantly simplified formulation of a molecular system, i.e., simple conjugation of the macromolecule with the biomolecule, brings with it many advantages in terms of the control of structure and loading, thus producing conjugates with reproducible behaviour. The M w of PCPP has also been shown to be of critical importance (Figure 3.4), with increased M w corresponding directly to improved immune response at the lower M w values tested ( kda) for an influenza vaccine [43]. A plateau was observed at M w above 800 kda. The average M w can be controlled to a 10% variation via the ringopening polymerisation used, and indeed reproducible procedures have been developed [26], nevertheless, despite many improvements in the methodology, this polymerisation method inherently produces polymers with broad polydispersities, with M w /weight average M w values of being reported for the samples tested in this work. The use of living polymerisation methods may help to improve this, although current methods do not allow the preparation of such high M w (see Chapter 2). It is also worth noting that the PCPP to antigen ratio has also been tested and shown to be a critical factor in the effective immune response observed for such conjugates [35]. Antibody titres produced from a BSA:PCPP formulation were shown to have an optimal ratio, above which further increasing the amount of BSA saw no improvement in the in vivo performance of the complex. 71

10 Polyphosphazenes for Medical Applications 3,000 2,500 2,000 HAI titre 1,500 1, ,000 1,500 2,000 M W kda Figure 3.4 Representative plot of M w of PCPP versus influenza haemagglutination-inhibition (HAI) antibody response. Reproduced with permission from L.G. Payne, S.A. Jenkins, A.L. Woods, E.M. Grund, W.E. Geribo, J.R. Loebelenz, A.K. Andrianov and B.E. Roberts, Vaccine, 1998, 16, 1, , Elsevier [43] Safety Considerations Many proposed immunoadjuvants fail to advance to clinical studies and/ or market due to safety concerns and thus it is of significant importance that this has been tested in depth for PCPP and to some extent PCEP. A primary concern for all immunoadjuvants is reactogenicity, particularly at the site of injection [43], an effect not observed for PCPP in mice, with the water-soluble polymer being observed to progress rapidly from the site of action, a feature which is clearly a distinct advantage in reducing the injection site reactogenicity [43]. Many studies in mice have shown the safety of PCPP, in various constellations [23]. However, it is well known that safety in mice does not always translate to larger animals 72

11 Nanomedicine and furthermore, humans. The safety of PCPP has, however, also been proven during studies in sheep [44], pigs, primates [33] and indeed human trials [45]. Furthermore, although limited data is available, the presented clinical data is also promising with human phase I clinical trials in combination with influenza antigens [34], as well as HIV-1 vaccines using PCPP, both showing no adverse effects [45]. The more recently developed variant PCEP has also been shown to be safe in recent studies with a recombinant OmlA protein in pigs [32]. Toxicology investigations on PCPP have also been conducted and submitted to the US Food and Drugs Administration as part of studies not yet released to the public [29]. Furthermore, since reproducible synthesis is also of paramount importance, it should also be noted that this has been addressed for PCPP with the development of good manufacturing procedures [26]. Since the synthetic procedures are essentially the same for other polyphosphazene polyacids, this technology should be transferable to other variants. As discussed in Section 2.1.2, the hydrolytically degradable polyphosphazene backbone is in contrast to many synthetic polymers, and indeed to most other polyelectrolytes, and thus opens the door to safe long-term intravenous use, without the risk of long-term accumulation associated with biopersistent polymers. However, when using degradable materials, the safety profile of not only the macromolecules themselves, but also of the degradation products and metabolites must be considered. In the case of PCPP, hydroxybenzoic acid is the major degradation product and is a metabolite of parabens, which are widely used as preservatives in food, cosmetic and pharmaceutical products, and generally regarded as safe [46] Immunological Activity A wide variety of bacterial and viral antigens have been investigated in combination with polyphosphazenes (mostly PCPP) in an assortment of cell and animal models [30]. The most detailed of these has been with influenza vaccines, with significant adjuvanticity being observed in tests for the X:31, H1N1, H3N2 and B/Panama/45/90 vaccines 73