Investigating the interactions of amino acid components on a mannitol-based spray-dried powder formulation for pulmonary delivery: A design of experiment approach

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Abstract

Combining an amino acid and a sugar is a known strategy in the formulation of spray or freeze dried biomolecule powder formulations. The effect of the amino acid leucine in enhancing performance of spray-dried powders has been previously demonstrated, but interaction effects of several constituents which may provide multiple benefits, are less well-understood. A 3 factor 2 level (23) factorial design was used to study the effects of leucine, glycine and alanine in a mannitol-based dry powder formulation on particle size, aerosolisation, emitted dose and cohesion. Other qualitative tests including scanning electronic microscopy and X-ray powder diffraction were also conducted on the design of experiment (DoE) trials. The results show that the use of glycine and/or alanine, though structurally related to leucine, did not achieve similar aerosol performance enhancing effects, rather the particle formation was hindered. However, when used in appropriate concentrations with leucine, the combination of amino acids produced an enhanced performance regardless of the presence of glycine and/or alanine, yielding significantly modified particle properties. The results from the DoE analyses also revealed the lack of linearity of effects for certain responses with a significant curvature in the model which would otherwise not be discovered using a trial-and-error approach.

Introduction

Dry powder inhalation has been an attractive delivery method for pulmonary drug administration due to its many advantages: these include ease of administration, convenient portability, relatively simple formulation, low cost and inherent solid state stability, especially for proteins and peptides (Carpenter et al., 1997, Prime et al., 1997, Rave et al., 2004). Dry powder formulations must be readily dispersible and aerosolisable upon inhalation for efficient delivery. However, inter-particulate cohesive forces are particularly dominant in the finely micronised powders (i.e. 1–5 μm) required for pulmonary delivery (Forsyth et al., 2001). These forces influence flowability and de-agglomeration of micronised dry powders and therefore the aerosolisation properties and delivery efficiency of dry powder formulations (Ashurst et al., 2000, Prime et al., 1997).

Spray-drying has gained increased interest for engineering suitable small particles due to its simplicity, adaptability, cost-effectiveness and scalability (Fourie et al., 2008). Spray-drying is a process in which the compound(s) of interest are first prepared in a liquid form, which is then atomised into a drying chamber in which the droplets are dried with heated air. Particle formation is achieved by precipitation of the dissolved compounds as the solvent evaporates from the solution droplets in the drying chamber. The ability to incorporate various ingredients in a single step manufacturing process is a powerful strength of spray-drying. Particles can be engineered to contain various ingredients by adjusting the content of the feed solution. Excipients can therefore be incorporated which may, in principle, manipulate the properties of the dry powder formulation. For example, the amino acid glycine has been added to the sugar mannitol to modify the particle precipitation process, with the benefit of increasing its resulting solid state particle glass transition temperature (Tg) (Sadrzadeh et al., 2010, Schule et al., 2008).

Many excipients have also been considered to improve the aerosolisation properties and performance of inhalable dry powder formulations (Li et al., 2003, Li et al., 2005a, Maa and Prestrelski, 2000, Rabbani and Seville, 2005, Staniforth et al., 2002). Leucine has been demonstrated to improve aerosolisation and performance of dry powder inhaler formulations and more specifically the inclusion of leucine as an additive in a precursor solution for spray-drying has been shown to improve the aerosolisation of the resulting powders (Kamlag et al., 2004, Li et al., 2003, Li et al., 2005b, Lucas et al., 1999, Morton and Kamlag, 2005, Seville et al., 2007). However, this dispersibility enhancing property appears to be specific to leucine and is not necessarily the case with other amino acids (Chew et al., 2005, Minne et al., 2008, Seville et al., 2007). While effects produced from the use of a single amino acid on the performance of a dry powder formulation is more extensively studied, the specific interaction effects resulting from the combined use of different amino acids is not well understood. Such interactions may allow or prevent multiple powder functionalities being achieved.

Several studies investigating amino acids as spray-drying additives used concentrations based on mass ratios (Chew et al., 2005, Minne et al., 2008, Seville et al., 2007, Shur et al., 2008). However, it is the interactions between the amino acids and other formulation components at the molecular level that eventually determines the behaviour of the final formulation. The extent of molecular interactions may hence relate to the number of molecules, as opposed to mass, which does not provide the same number of molecules of each component for comparability. Compounds with a lower molecular mass obviously contain more molecules than larger compounds in the same mass. Molar concentrations were therefore used in the present study instead of mass ratio in order to investigate and compare the effects achieved with the same number of molecules of glycine, alanine and leucine.

Studying the effect of several formulations with multiple excipients in different compositions by trial-and-error or ‘changing one separate factor at a time’ (COST) approaches are often inefficient as the results from these experiments do not allow the identification of interaction effects between formulation ingredients (Naelapää et al., 2010). A previous study investigating the effect of leucine, glycine and alanine on the performance of dry powder formulations did not provide information on the combination use of these excipients (Minne et al., 2008). Recently, the design of experiment (DoE) approach has been successfully used to optimise spray-drying process conditions by identifying combination of parameters, including drying temperature, airflow rate, pump setting, aspiration setting, feed concentration and solution feed rate, that produced formulations with the best performance (Baldinger et al., 2011, Tajber et al., 2009). The DoE approach was therefore used in the present study to screen in a systematic manner a range of formulations, however, in this case with a focus on various compositions. The study utilised a 23 factorial design to investigate the effect of the three amino acids in various compositions.

In the current study, three amino acids, glycine, alanine and leucine were selected specifically with increasing hydrocarbon chain lengths respectively, in order to control specific properties of a mannitol-based dry powder formulation. It was proposed that the hydrocarbon chain length may influence the mass transport and self-assembly of each amino acid upon droplet drying. The subsequent influence of these amino acids on the physical properties of the spray-dried particles including particle size distribution, dispersibility, aerosolisation properties, inter-particle interaction, surface morphology and crystallinity were therefore investigated. The study aims to screen a range of formulations using a DoE approach to determine the potential utility of these amino acids as performance enhancing excipients for inhalable dry powder formulations. To the best of our knowledge, the effects produced from the combination use of these or similar additives on a spray-dried powder formulation for pulmonary delivery have not previously been investigated.

Section snippets

Materials

d-Mannitol was obtained from VWR International Ltd. (Poole, BH15 1TD, England). l-Leucine (LEU), glycine (GLY) and l-alanine (ALA) were obtained from Sigma–Aldrich Chemicals (Castle Hill, NSW, Australia).

Preparation of spray-dried powders

Aqueous solutions containing mannitol and selected amino acids (LEU, GLY, ALA) in various compositions as shown in Table 1 were dissolved in 200 mL of Milli-Q water. A small amount of methylene blue dye (10 mg) was incorporated in each formulation to allow a simple quantification of powder by

Factorial design methodology and analysis

In the present study, the design of experiment methodology was employed to systematically evaluate the effect of varying the amount of glycine, alanine and leucine, as well as to identify any interaction among these excipients on the particle size distribution, dispersibility, aerosolisation and inter-particle interaction of the mannitol dry powder formulations. The summary of result data obtained of various responses is presented in Table 3. The DoE approach facilitated the identification of

Conclusion

This study indicates that interactions between combinations of excipients used in spray dried formulations can be identified hence leading to a deeper study of such combination effects and greater understanding of the study design space. The results from the present study show that the use of glycine and/or alanine, though being structurally similar to leucine, provide detrimental rather than beneficial effects on particles both during as well as after spray-drying with mannitol. In addition,

Acknowledgements

T.S. would like to acknowledge the support of Australian Postgraduate Award (APA) scholarship. L. M. K. was supported by an NHMRC Australian Biomedical Training Fellowship.

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