Effects of the antibiotic component on in-vitro bacterial killing, physico-chemical properties, aerosolization and dissolution of a ternary-combinational inhalation powder formulation of antibiotics for pan-drug resistant Gram-negative lung infections

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Abstract

Combinational antibiotic formulations have emerged as an important strategy to combat antibiotic resistance. The main objective of this study was to examine effects of individual components on the antimicrobial activity, physico-chemical properties, aerosolization and dissolution of powder aerosol formulations when three synergistic drugs were co-spray dried. A ternary dry powder formulation consisting of meropenem (75.5 %w/w), colistin (15.1 %w/w) and rifampicin (9.4 %w/w) at the selected ratio was produced by spray drying. The ternary formulation was characterized for in-vitro antibacterial activity, physico-chemical properties, surface composition, aerosol performance and dissolution. All of the formulations demonstrated excellent aerosolization behavior achieving a fine particle fraction of >70%, which was substantially higher than those for the Meropenem-SD and Colistin-Meropenem formulations. The results indicated that rifampicin controlled the surface morphology of the ternary and binary combination formulations resulting in the formation of highly corrugated particles. Advanced characterization of surface composition by XPS supported the hypothesis that rifampicin was enriched on the surface of the combination powder formulations. All spray-dried formulations were amorphous and absorbed substantial amount of water at the elevated humidity. Storage at the elevated humidity caused a substantial decline in aerosolization performance for the Meropenem-SD and Colistin-Meropenem, which was attributed to increased inter-particulate capillary forces or particle fusion. In contrast, the ternary combination and binary Meropenem-Rifampicin formulations showed no change in aerosol performance at the elevated storage humidity conditions; attributable to the enriched hydrophobicity of rifampicin on the particle surface that acted as a barrier against moisture condensation and particle fusion. Interestingly, in the ternary formulation rifampicin enrichment on the surface did not interfere with the dissolution of other two components (i.e. meropenem and colistin). Our study provides an insight on the impact of each component on the performance of co-spray dried combinational formulations.

Introduction

Respiratory infections due to multi-drug resistant (MDR) Gram-negative pathogens are associated with high mortality and morbidity rates, which represent an unmet healthcare problem worldwide (Mizgerd, 2006, WHO, 2014). Traditionally, antibiotics are administered via oral or parenteral routes; but for some systemically administered antibiotics only a small amount/proportion of drugs can reach the site of infections in the deep lungs which can compromise treatment efficacy (Cheah et al., 2015, Cipolla et al., 2016, Ritrovato and Deeter, 1991, Yapa et al., 2014). Simply increasing the dose of oral and parenteral administrations is often unfeasible because of dose limiting systemic toxicity, as is the case with the polymyxins (i.e. polymyxin B and colistin) (Bergen et al., 2012, Hartzell et al., 2009).

Antimicrobial resistance has become a serious global health challenge (Boucher et al., 2009, Payne et al., 2007). In the absence of antibiotics with novel mechanisms of action (Boucher et al., 2009, Payne et al., 2007), synergistic combinations of antibiotics with different mechanisms are the most economic and effective strategy to ward off an impending post-antibiotic era (Mouton, 1999). For such combinations to be optimally effective in-vivo, both drugs should localize and accumulate at the infections site concomitantly (Mukker et al., 2015). However, drugs often exhibit variable pharmacokinetic profiles, for example the rate and extent of drug absorption in the lungs following systemic administration is highly dependent on biological factors and drug properties (Levison and Levison, 2009). This raises concerns regarding the effectiveness of systemic therapies of combinational antibiotics against lower respiratory tract infections.

Inhalation therapies have advantages for the treatment of respiratory tract diseases (Frijlink and De Boer, 2004). Drugs can be delivered directly to the lungs via inhalation in order to achieve high drug concentrations at the infection site and reduced systemic exposure, as compared with the oral and parenteral delivery systems (Hickey et al., 2016). The achievement of higher drug concentrations in the airways and lowering the systemic exposure often translates into enhanced antimicrobial activity, minimized systemic toxicity and reduced emergence of resistance (Cipolla and Chan, 2013, Lu et al., 2012). Dry powder inhalers (DPIs) are portable, possess better chemical stability and higher delivery efficiencies in comparison to the traditional nebulization (Zhou et al., 2015a). Nevertheless, inhalation of large amount of drug/excipient powders may cause local adverse events including bronchospasm and coughing, which may compromise patient compliance (Claus et al., 2014, Zhou et al., 2015a). Such adverse reactions are more prevalent with the high-dose medications such as inhaled antibiotics, likely attributable to large amount of powders deposited in the upper respiratory tracts and the inhalation maneuver of the patients (Claus et al., 2014, Velkov et al., 2015, Weers, 2015). Thus, it is important to minimize the deposition of inhaled powder in the upper respiratory tract and maximize aerosol performance to reduce the mass of the drug powders to be inhaled for improved patient compliance and adherence (Zhou et al., 2014b).

In inhalation industrial practice, raw drug materials are jet milled to obtain small particle sizes with aerodynamic diameters 1–5 μm (Lin et al., 2015). Such fine milled particles are inherently cohesive and difficult to disperse into individual particles upon inhalation, which result in low aerosol efficiency (Buttini et al., 2012). Controlling the powder cohesion via particle engineering methods is a straightforward approach to improve the aerosolization performance of DPI formulations (Buttini et al., 2012, Qu et al., 2015). One of the popular approach of particle engineering is spray drying (Bohr et al., 2014, Son et al., 2013, Vehring, 2008). Our research has been focused on developing antibiotic combinations, which not only exhibit synergistic antibacterial activity but also augment the aerosol and dissolution performance when combined into DPI formulations (Mangal et al., 2018a, Mangal et al., 2019, Shetty et al., 2018a, Wang et al., 2016, Zhou et al., 2014a, Zhou et al., 2015b, Zhou et al., 2016). For instance, a DPI formulation containing amorphous colistin and crystalline rifapentine was developed by spray drying rifapentine suspension in colistin aqueous solution (Zhou et al., 2015b). The formulation has shown synergistic antimicrobial activities against planktonic and biofilm of P. aeruginosa. In addition, crystalline rifapentine particles act as carriers that prevented moisture-induced deterioration in aerosol performance for hygroscopic colistin (Zhou et al., 2015b). In other studies, co-spray drying colistin with hydrophobic azithromycin or rifampicin was shown to form a hydrophobic coating on the surface of co-spray dried composite particles, which improved the stability of colistin against moisture-induced particle fusion/agglomeration at elevated humidity (Zhou et al., 2014a, Zhou et al., 2016).

Ternary antibiotic combinations are becoming an important avenue for eradication of MDR ‘superbugs’ that have developed resistance to almost all clinically available antibiotics (Urban et al., 2010). The literature evidence has shown the potential of ternary combinations of polymyxin B, carbapenem (or its family antibiotics) and rifampicin for rapid killing against pan-drug resistant (PDR) A. baumannii (Yoon et al., 2004), K. pneumoniae (Diep et al., 2017) and P. aeruginosa (Urban et al., 2010). Thus far there has been one attempt to develop DPI formulations of ternary antibiotic combinations (Lee et al., 2016). However, the effects of each component and surface composition on the aerosol performance at elevated humid conditions were not been investigated; this is important as the latter has been demonstrated to critically affect the aerosol stability of DPIs containing hygroscopic compounds such as colistin (Zhou et al., 2014a, Zhou et al., 2016, Zhou et al., 2013).

The aim of this study was to examine the role of colistin and rifampicin on the antimicrobial activity, physico-chemical properties, aerosolization and dissolution of meropenem as co-spray dried formulations. We employed colistin for this study, and not polymyxin B, given the potential toxic effects of polymyxin B on lung epithelial cells noted in our previous report (Ahmed et al., 2017).

Section snippets

Materials

Meroepenem trihydrate, colistin sulphate and rifampicin were purchased from Beta Pharma (Beta Pharma (Shanghai) Co. Ltd, Wujiang City, China). Acetonitrile (HPLC grade) was purchased from Merck (Fair Lawn, New Jersey, USA).

MDR P. aeruginosa 20143 n/m and A. baumannii 03-149.2 (colistin-resistant) were clinical samples from respiratory infections. The bacterial strains were maintained at −80 °C in tryptone soy broth containing glycerol (20% v/v).

Static time-kill experiment

Antimicrobial activities were examined by static

Antimicrobial activity

Time-kill profiles for colistin, meropenem, and rifampicin per se and in binary and ternary formulations against A. baumannii 03-149.2 and P. aeruginosa 20143 n/m are shown in Fig. 1. Colistin monotherapy produced no bacterial killing against both strains, which was similar to the behaviour of the bacteria-only control; this is in line with the polymyxin resistant nature of both strains. Meropenem monotherapy, produced substantial bacterial killing within 4–6 h against A. baumannii 03-149.2 and

Discussion

Inhalation is an promising route for anti-infective drug delivery targeting respiratory tract infections, as it offers direct access of the drugs to the airway surfaces, limits the systemic exposure and hence unwanted toxicity to the off-target sites (Bruinenberg et al., 2009, Ritrovato and Deeter, 1991). Inhaled antibiotics achieve relatively high drug concentrations in the lungs quickly (Gontijo et al., 2014, Yapa et al., 2013), thus reducing the risk of sub-optimal exposure which promotes

Conclusions

This study has examined effects of individual components on the antimicrobial activity, physico-chemical properties, aerosolization and dissolution of triple antibiotics when co-spray dried. The combination of the three antibiotics with varying antimicrobial mechanisms showed synergistic antimicrobial activities against colistin-resistant Gram-negative bacteria that may cause fatal respiratory tract infections. It is noteworthy that formulation process of spray drying did not compromise the

Declaration of interests

None.

Acknowledgement

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01AI132681. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Qi (Tony) Zhou is partically supported by the Ralph W. and Grace M. Showalter Research Trust Award. Jiayang Huang was supported by Purdue College of Pharmacy Dean’s

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