Composite particle formulations of colistin and meropenem with improved in-vitro bacterial killing and aerosolization for inhalation

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Abstract

Antibiotic combination therapy is promising for the treatment of lower respiratory tract infections caused by multi-drug resistant Gram-negative pathogens. Inhaled antibiotic therapy offers the advantage of direct delivery of the drugs to the site of infection, as compared to the parenteral administrations. In this study, we developed composite particle formulations of colistin and meropenem. The formulations were characterized for particle size, morphology, specific surface area, surface chemical composition, in-vitro aerosolization performance and in-vitro antibacterial activity. The combinations demonstrated enhanced antibacterial activity against clinical isolates of Acinetobacter baumannii N16870 and Pseudomonas aeruginosa 19147, when compared with antibiotic monotherapy. Spray-dried meropenem alone showed a poor aerosolization performance as indicated by a low fine particle fraction (FPF) of 32.5 ± 3.3%. Co-spraying with colistin improved the aerosolization of meropenem with up to a two-fold increase in the FPF. Such improvements in aerosolization can be attributed to the enrichment of colistin on the surface of composite particles as indicated by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), and the increases in particle porosity. Intermolecular interactions between colistin and meropenem were observed for the combination formulations as measured by FT-IR. In conclusion, our results show that co-spray drying with colistin improves the antibacterial activity and aerosol performance of meropenem and produces a formulation with synergistic bacterial killing.

Introduction

Lower respiratory tract infections (or lung infections) cause high mortality and morbidity (Liang et al., 2011, Mizgerd, 2006, WHO, 2014). Antibiotics administered via systemic routes are often not effective for lung infections, as for many antibiotics such as the polymyxins (polymyxin B and colistin) only a small fraction of the drug is available at the sites of infection (i.e. in the lungs) (Velkov et al., 2015). Simply increasing parenteral dose often causes severe systemic adverse effects (Traini and Young, 2009). For instance, high-dose parenteral colistin can lead to neurotoxicity and nephrotoxicity (Garonzik et al., 2011).

Colistin is often used for treatment of respiratory infections caused by multidrug-resistant (MDR) Gram-negative bacteria (Levin et al., 1999, Velkov et al., 2015). Recently, there is a marked increase in the incidence of colistin-resistant infections (Cai et al., 2012, Marchaim et al., 2011, Paterson and Harris, 2016). Due to the dry development pipeline of novel antibiotics, combination therapy can be a practical and swift approach for treating the infections caused by colistin-resistant pathogens (Cai et al., 2012). However, synergistic antibacterial effects of systemically administered combination antibiotics can be compromised due to the different pharmacokinetic profiles (Weers, 2015), which may not allow both drugs to attain effective drug concentrations at the same time at the infection sites.

Antimicrobial therapy via the inhalation route has attracted increasing attentions for the treatment of lower respiratory infections (Velkov et al., 2015, Zhou et al., 2015). Inhalation therapy substantially improves drug concentration on the airway surfaces with much reduced systemic exposure, hence maximizes the treatment efficacy and reduces the systemic toxicities (Cipolla and Chan, 2013, Montgomery et al., 2014, Yapa et al., 2013). Additionally, inhalation therapy may be able to deliver combinational antibiotics to the same targeted infection sites simultaneously allowing greater opportunity to achieve intended synergistic effects. In addition, dry powder inhalers (DPIs) may enable the delivery of high-doses of antibiotics directly to the respiratory tracts (Zhou et al., 2015).

Typically, the inhaled drug particles produced by traditional jet-milling approach are highly cohesive and have poor flowability and poor aerosolization performance (Lin et al., 2015). Addition of excipients such as fine lactose particles may improve the aerosolization of cohesive powders to some extent (de Boer et al., 2012, Grasmeijer et al., 2014, Smyth and Hickey, 2005). However, for high-dose drugs like antibiotics, addition of excipients may increase the inhalation powder mass that needs an excessive number of inhalations to complete the dose and a bulky inhaler to accommodate the large dose (Zhou et al., 2015).

Our earlier studies have indicated that the spray dried colistin particles without any excipient had high aerosol performance with fine particle fraction (FPF) >80% with an Aerolizer® device (Zhou et al., 2013). It was proposed that such high aerosol performance of the spray dried colistin powders is attributed to its surfactant-like properties (Mestres et al., 1998, Wallace et al., 2010), which allows self-assembly of non-polar tail at the air-liquid interface during spray drying resulting in the formation of low surface energy particles (Jong et al., 2016). Previous studies have shown that surface-active components could also potentially self-assemble on the surface, when co-sprayed with a secondary component altering its surface physico-chemical and aerosolization properties (Chew et al., 2005a, Chew et al., 2005b, Momin et al., 2018a, Momin et al., 2018b, Rabbani and Seville, 2005, Sou et al., 2013, Zhou et al., 2016). The aim of this study was to investigate the effect of colistin on the aerosol performance of the co-spray dried formulations in synergistic combination with meropenem (Lenhard et al., 2016).

In this study, colistin was co-spray dried with meropenem to develop combinational DPI formulations. The resultant DPI formulations were characterized regarding particle size, morphology, surface chemical composition and specific surface area. The in-vitro aerosol performance and in-vitro antibacterial activity were characterized.

Section snippets

Materials

Colistin sulfate and meropenem trihydrate were purchased from ßetaPharma® (Shanghai) Co., Ltd (Wujiang City, JiangSu Province, China). Acetonitrile (HPLC grade) and sodium sulfate were purchased from Fisher Scientific (Fair Lawn, New Jersey, USA). Tryptone soy broth and Mueller-Hinton Broth were supplied by Oxoid Ltd, Basingstoke, UK and glycerol by Astral Scientific Pty Ltd, Taren Point, Australia.

Time-kill assays

Clinical isolates of A. baumannii N16870 and Pseudomonas aeruginosa 19147 were stored in tryptone

Time kill assay

Fig. 1 shows the time-kill profiles against clinical isolates of A. baumannii N16870 and P. aeruginosa 19147. For A. baumannii N16870, meropenem showed no antibacterial effect at 16 mg/L in 24 h, and marginal antibacterial activity at 48 mg/L in 2 h, albeit regrowth was observed in 4 h (Fig. 1A). Colistin showed bacterial killing at both 16 and 48 mg/L; but at 16 mg/L regrowth was evident in 4 h. The combination of Colistin:Meropenem_1:1 (16:16 mg/L) demonstrated superior antibacterial activity

Discussion

Combination antibiotic therapies are increasingly used to combat MDR Gram-negative lung infections (Mouton, 1999). This has spurred the search for effective colistin combinations that exhibit greater bacterial killing, improved safety and superior pharmacokinetics (Biswas et al., 2012, Zhou et al., 2014, Zhou et al., 2016). Typically, the antibiotics with different mechanisms of action can act synergistically to greatly enhance antibacterial killing. Colistin is a lipopeptide antibiotic that

Conclusions

In the present study, we developed and characterized co-spray dried colistin and meropenem DPI formulations which showed superior antibacterial activity. Incorporation of colistin improved the aerosolization performance of meropenem as evidenced by an almost two-fold increase in FPF, attributable to the enrichment of colistin on the particle surface and the increased porosity. FT-IR spectra demonstrated intermolecular interactions between colistin and meropenem. Such synergistic antimicrobial

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. Jian Li and Tony Velkov are supported by a research grant from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01AI111965). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of

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