Synergistic combination dry powders for inhaled antimicrobial therapy: Formulation, characterization and in vitro evaluation

European Journal of Pharmaceutics and Biopharmaceutics 83 (2013) 275–284

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Research paper

Synergistic combination dry powders for inhaled antimicrobial therapy: Formulation, characterization and in vitro evaluation Sie Huey Lee a, Jeanette Teo b, Desmond Heng a,⇑, Wai Kiong Ng a, Hak-Kim Chan c, Reginald B.H. Tan a,d,⇑ a

Institute of Chemical and Engineering Sciences, ASTAR (Agency for Science, Technology and Research), Singapore, Singapore Department of Laboratory Medicine, National University Hospital, Singapore, Singapore c Advanced Drug Delivery Group, Faculty of Pharmacy, The University of Sydney, Sydney, Australia d Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore, Singapore b

a r t i c l e

i n f o

Article history: Received 21 June 2012 Accepted in revised form 5 September 2012 Available online 23 September 2012 Keywords: Dry powder inhaler Antimicrobial Combinatorial therapy Synergy Spray-drying

a b s t r a c t In combination antimicrobial therapy, the desired outcome is to broaden the antimicrobial spectrum and to achieve a possible synergistic effect. However, adverse antagonistic species may also emerge from such combinations, leading to treatment failure with serious consequences. It is therefore imperative to screen the drug candidates for compatibility and possible antagonistic interactions. The aim of this work was to develop a novel synergistic dry powder inhaler (DPI) formulation for antimicrobial combination therapy via the pulmonary route. Binary (ciprofloxacin hydrochloride and gatifloxacin hydrochloride, SD-CIP/ GAT) and ternary (ciprofloxacin hydrochloride, gatifloxacin hydrochloride, and lysozyme, SD-CIP/GAT/ LYS) combinations were prepared via spray-drying on a BUCHIÒ Nano Spray Dryer B-90. The powder morphologies were spherical with a slightly corrugated surface and all within the respirable size range. The powders yielded fine particle fractions (of the loaded dose) of over 40% when dispersed using an AerolizerÒ at 60 L/min. Time-kill studies carried out against the respiratory tract infection-causing bacteria Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia, and Acinetobacter baumannii at 1  the minimum inhibitory concentration (MIC) over 24 h revealed no antagonistic behavior for both the binary and ternary combinations. While the interactions were generally found to be indifferent, a favorable synergistic effect was detected in the dual combination (SD-CIP/GAT) when it was tested against P. aeruginosa bacteria. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Infectious diseases caused by bacterial pathogens are a major threat to global human health and are also one of the leading causes of human morbidity and mortality [1]. According to the World Health Organization (WHO), infectious diseases accounted for 32% of deaths worldwide, 68% of deaths in Africa and 37% of deaths in Southeast Asia, in all, killing nearly 14 million people in the developing countries each year [2]. The developed world is not spared either. The number of annual deaths due to infectious diseases was estimated at approximately 170,000 in the United States as of the year 2000 [1,3]. Without doubt, infectious diseases have afflicted the world population with a huge socio-economic cost.

⇑ Corresponding authors. Institute of Chemical and Engineering Sciences, A⇑STAR (Agency for Science, Technology and Research), 1, Pesek Road, Jurong Island, Singapore 627833, Singapore. Tel.: +65 67963861; fax: +65 63166183 (D. Heng), tel.: +65 67963855; fax: +65 63166183 (R.B.H. Tan). E-mail addresses: [email protected], [email protected] (D. Heng), [email protected] (R.B.H. Tan). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2012.09.002

Since the introduction of penicillin into clinical practice in the 1940s, antibiotics have always been used as the first line of defense against many infections. However, the widespread use, misuse, and abuse of antibiotics have contributed to the development of multiply antibiotic-resistant bacteria (sometimes referred to as ‘‘super bugs’’), leading to the emergence of new and the reemergence of old infectious diseases. In the past two decades, 16 new infectious diseases have been identified and five others have been identified as re-emerging according to the US National Institutes of Health (NIH; Bethesda, MD, USA) [4,5]. Antimicrobial combinatorial therapy offers a powerful means of combating the spread of antibiotic-resistant bacterial pathogens [6]. Quite often, bacteria develop resistance via a variety of different mechanisms, and the probability of a bacterium developing resistance to all the antibiotics employed in the combinatorial therapy is much lower than in the case for a single antibiotic in monotherapy [7]. A combination of antibiotics may provide much broader spectrum coverage than any single antibiotic alone. For example, in tuberculosis treatment, combinatorial therapy has been practiced for over 50 years as it has demonstrated a reduced risk of bacterial resistance during therapy [8].

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Under many clinical circumstances, antimicrobial combinatorial therapy is also advocated for achieving antibiotic synergism (i.e., combined activity is more than the sum of individual activities), to further enhance the bactericidal activity and the rate of killing in vivo relative to those attainable with individual agents [7]. One of the best established examples of such synergism is the use of penicillin with an aminoglycoside in bacterial endocarditis due to Enterococcus faecalis, Staphylococcus aureus, and the viridans group streptococci [9]. Although synergism is often desired, unexpected antagonistic outcomes (i.e., combined activity is less than the sum of individual activities) may be derived from such combinations. There are many examples of antibiotic antagonism, among them, penicillin and aureomycin serves as one of the most classic examples of antibiotic antagonism whereby the survival rate of children treated with penicillin dramatically decreased from 79% to 21% when both penicillin and aureomycin are used together in pneumococcal meningitis treatment [10]. Therefore, scientists and clinicians have to be mindful of the pitfalls of antagonistic combinations. Inappropriate choice of antimicrobial combinations would not only deprive the patient of the therapeutic dose but might also endanger their lives. In treating respiratory infections, delivery of the antibiotics via the pulmonary route is clearly more advantageous over the more traditional routes as the lung is directly targeted [11]. Since the drugs are delivered directly to the target organ, high drug concentrations can be achieved within the lung compared to systemic administration of the agent thereby avoiding effects of systemic toxicity [12]. Additionally, the high local concentration of the inhaled antibiotics could prevent biofilm formation, hence impeding the emergence of drug resistant bacteria [12]. In the delivery of antibiotics to the respiratory tract, nebulization of aqueous solutions obtained from marketed intravenous preparations has been the most common means of administration since the 1940s [11,13,14]. However, nebulizers are often regarded as hospital or home-setting devices [11], delivering fine particle fractions (FPF) of only around 10% or less to the patient [15,16]. Hence, a longer time is required to achieve the therapeutic dose which inadvertently affects patient compliance. Furthermore, when co-nebulizing two or more antibiotics together, there is also the risk of undesired precipitation in solution [17]. Therefore, all these limitations have reflected the need to develop alternative drug delivery devices like the dry powder inhaler (DPI) to improve patient compliance and ease of administration [11]. The DPI, being portable and quick to use, has successfully enhanced patient compliance and improved formulation stability as the powdered drug is used instead of the drug solution or suspension. Currently, there is a drought in antibiotic DPI formulations both in the market and in the clinical trials. The only commercial antibiotic DPI formulation known to date is the NovartisÒ TIP (Tobramycin Inhaled Powder), just recently launched in the UK in September 2011 [11]. ColobreatheÒ is a DPI formulation of colistin, which has recently completed phase III trials, and is expected to be fully launched soon [11]. Cipro InhaleÒ (ciprofloxacin inhaled powder) is currently undergoing Phase II development [11]. There is no known combination of antibiotic DPI available commercially. In the academic scene, DPI antibiotic formulations on tobramycin sulfate, gentamicin, azithromycin, and colistin sulfate have been reported [18–21], with some emerging work into combinatorial therapy to enhance the therapeutic dose. Although Adi et al. [22] had prepared an inhalable co-spraydried antibiotic (ciprofloxacin hydrochloride and doxycycline hydrochloride) formulation with acceptable FPFs, the formulation was found to be non-synergistic via the qualitative disk diffusion test. The result was in line with an earlier report which mentioned the ciprofloxacin–doxycycline pair as being suppressive (or antagonistic at low concentrations) [6].

About a year after the initial effort by Adi et al. [22], Tsifansky et al. [23] reported the co-encapsulation of ciprofloxacin and ceftazidime in a microparticle system containing dipalmitoylphosphatidylcholine (DPPC), albumin, and lactose and demonstrated the additive anti-pseudomonal activity against a tested strain of Pseudomonas aeruginosa. Synergism was not achieved. To date, to the best of the authors’ knowledge, no synergistic DPI antimicrobial combination has ever been reported in the literature. In this work, two inhalable combinatorial DPI antimicrobial formulations, namely, ciprofloxacin hydrochloride/gatifloxacin hydrochloride binary combination (SD-CIF/GAT) and ciprofloxacin hydrochloride/gatifloxacin hydrochloride/lysozyme ternary combination (SD-CIF/GAT/LYS) were developed and quantitatively tested for synergy against the respiratory tract infection-causing bacteria P. aeruginosa, S. aureus, Klebsiella pneumonia, and Acinetobacter baumannii. Ciprofloxacin hydrochloride, gatifloxacin hydrochloride, and lysozyme are currently administered either intravenously or orally and have all yet to be developed and tested for the commercial inhalation market. 2. Materials and methods 2.1. Materials Ciprofloxacin hydrochloride (CIP) and gatifloxacin hydrochloride (GAT) were supplied from Junda Pharmaceutical Co. Ltd. (Changzhou, China). Lyophilized hen-egg white lysozyme (LYS), disodium hydrogen phosphate, phosphoric acid, and trifluoroacetic (TFA) were purchased from Sigma Chemical Co. (Louis, MO, USA). Ultrapure water was used in the experiments. HPLC grade acetonitrile was supplied by Merck (Darmstadt, Germany). The model bacteria used in the study were obtained from the American Type Culture Collection (ATCC) and included P. aeruginosa, S. aureus, K. pneumoniae, and A. baumannii, obtained from the National University Hospital (Singapore). Mueller–Hinton broth (Oxoid, Bashingstoke, UK) was used as the culture media for the antimicrobial activity test. 2.2. Preparation of spray-dried particles Powders of ciprofloxacin hydrochloride (SD-CIP), gatifloxacin hydrochloride (SD-GAT), lysozyme (SD-LYS), binary combination powders of ciprofloxacin hydrochloride/gatifloxacin hydrochloride (SD-CIP/GAT) and ternary combination powders of ciprofloxacin hydrochloride/gatifloxacin hydrochloride/lysozyme (SD-CIP/GAT/ LYS) were obtained by spray-drying aqueous solutions of the antimicrobial agents on a B-90 Nano Spray Dryer (Büchi Labortechnik AG, Flawil, Switzerland) with operating parameters as detailed in Table 1. All solutions were filtered through a 0.45 lm syringe filter (Millipore, Bedford, MA, USA) prior to spray-drying to minimize blockage due to any undissolved particles at the spray mesh. The composition of spray-dried powders prepared in this study is summarized in Table 2. The spray-dried powders were stored in a desiccator at room temperature for further characterization.

Table 1 Spray drying parameters. Parameters Spray mesh size (lm) Feed concentration (w/v %) Nitrogen flow rate (L/min) Relative spray rate (mL/h) Inlet temperature (°C) Outlet temperature (°C) Yield (%)

5.5 0.75 120 4 120 40–45 70–80

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Drug content (w/w %)

% of Ideal (relative to feed)

Feed

SD-CIP SD-GAT SD-LYS SD-CIP/GAT SD-CIP/GAT/LYS

Actual

CIP

GAT

LYS

CIP

GAT

LYS

100 – – 28.6 11.1

– 100 – 71.4 27.8

– – 100 – 61.1

99.8 ± 0.4 0 – 27.8 ± 0.6 11.2 ± 0.3

– 99.5 ± 0.4 – 72.0 ± 0.7 27.3 ± 0.5

– – 85.1 ± 0.9 – 51.9 ± 0.8

2.3. Surface morphology The morphology of the powder particles was examined by a field emission scanning electron microscopy (FESEM, JEOL JSM6700). Prior to imaging, the samples were dispersed onto carbon sticky tabs and coated with gold for 80 s using a sputter coater (Cressington 208HR, Watford, UK).

CIP

GAT

LYS

99.8 ± 0.4 99.5 ± 0.4 85.1 ± 0.9 97.2 ± 1.9 100.0 ± 2.7

100.8 ± 1.0 98.2 ± 1.9

84.9 ± 1.3

0.34 lm. Particles with diameter size less than 0.34 lm were captured on a Micro-Orifice Collector (MOC) beyond stage 7. In this study, fine particle fraction (FPF) represents the mass fraction of drug particles smaller than 5 lm in the aerosol cloud relative to the total mass recovered and was obtained by interpolation to the cumulative percent undersize at 5 lm. FPF(emitted) was obtained when the fine particle dose was expressed relative to the emitted dose.

2.4. Particle size analysis 2.7. Drug content quantification The particle size distribution of the spray-dried powders was determined by laser diffraction using Malvern Mastersizer 2000 (Malvern Instruments, UK) using the Scirocco dry dispersion unit. The powders were dispersed in triplicates at 3 bars of pressure using refractive index (RI) of 1.520 for SD-CIP, SD-GAT, SD-CIP/ GAT, and 1.445 for SD-LYS and SD-CIP/GAT/LYS. 2.5. Powder crystallinity Powder crystallinity of the samples was assessed by powder Xray diffraction (pXRD) at room temperature using an X-ray diffractometer (D8 Advance; Bruker AXS GmbH, Karlsruhe, Germany). Samples were scanned from 2° to 50° (2h) at with an angular increment of 0.04° and at 1 s per step using Cu Ka radiation generated at 35 kV and 40 mA. 2.6. In vitro aerosol performance The aerosol performance was assessed using a Next Generation Impactor (NGI, Copley Scientific, Nottingham, UK) coupled with a United State Pharmacopoeia (USP) stainless steel throat. The method followed the procedure specified for DPIs in the British Pharmacopoeia [24]. Prior to testing, all the eight impactor stages were sprayed with MOLYKOTEÒ 316 silicone grease release spray (Dow Corning Corp., Midland, MI) to minimize particle bounce. Approximately 20 ± 2 mg of powder from each formulation was filled into a hydroxypropyl methylcellulose (HPMC) capsule (size 3, CapsugelÒ, NJ, USA), loaded into an AerolizerÒ inhaler (Novartis Pharmaceuticals, Basel, Switzerland), pierced and then actuated for 4 s at 60 L/min. The flow through the NGI was measured using a calibrated flow meter (TSI Model 4040C, TSI Instrument Ltd., Buckinghanshire, UK) and controlled by a high capacity vacuum pump (Model HCP5, Copley Scientific, Nottingham, UK) and a critical flow controller (TPK 2000, Copley Scientific, Nottingham, UK). After actuation, the device, capsule, throat, and each part of the NGI were washed separately and thoroughly using DI water. The solutions were then assayed by high performance liquid chromatography (HPLC) after appropriate sample dilutions were made. Each dispersion test was performed in triplicates to obtain mean values. Temperature and relative humidity (RH) throughout the testing was maintained at 25 °C and 40%, respectively. At a flow rate of 60 L/min, the aerodynamic cut-off diameters of stages 1, 2, 3, 4, 5, 6, and 7 are 8.06, 4.46, 2.82, 1.66, 0.94, 0.55, and

Drug contents in the spray-dried powders and in the aliquots collected from the NGI were analyzed via HPLC (1100 series, Agilent Technologies, CA, USA). For assays of ciprofloxacin hydrochloride and gatifloxacin hydrochloride, a 100 lL aliquot sample was injected into the HPLC system equipped with a Zorbax Extend C18 column (4.6 mm  150 mm, 3.5 lm) (Agilent Technologies, CA, USA) as the stationary phase (column temperature 25 °C), and a mixture of 0.025 M disodium hydrogen phosphate buffer (adjusted to pH 3.0 with phosphoric acid) and acetonitrile (80:20, v/v) as the mobile phase. A flow rate of 0.5 mL/min and an UV absorbance wavelength of 293 nm were employed for the simultaneous detection of ciprofloxacin hydrochloride and gatifloxacin hydrochloride at retention times of 4.2 and 5.7 min, respectively. Assay of lysozyme was conducted via a modified HPLC method originally described by Resmini et al. [25]. Briefly, a reversed-phase polymeric column (PLRP-S 4.6 mm  250 mm, 5 lm) (Varian Inc., Sint-Katelijne-Waver, Belgium) was used, and the mobile phase consisted of solvent A (a mixture of water/0.1% TFA (v/v): acetonitrile/0.1% TFA (v/v) = 100: 38.4 (w/w)) and solvent B (acetonitrile/0.1% TFA (v/v)) delivered at 1 mL/min under a linear gradient: 0–20 min, 100% A; 20–21 min, 100% A to 50% A; 21–23 min, 100% A. Lysozyme retention time was at 18.4 min under a UV wavelength of 280 nm. 2.8. Determination of antimicrobial minimum inhibitory concentration (MIC) The minimum inhibitory concentration (MIC) of ciprofloxacin hydrochloride, gatifloxacin hydrochloride, and lysozyme was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [26]. 2.9. Antimicrobial activity of DPI formulations In vitro antimicrobial activity of the spray-dried powders was determined quantitatively via time-kill studies. Overnight cultures of the test organism were diluted in Mueller–Hinton broth (MHB) to give a starting bacterial density of approximately 5  105 CFU/ mL. Spray-dried powders were added to the cultures such that the final concentrations were at 1  MIC. The cultures were then incubated at 37 °C with shaking. Bacterial cell counts were estimated at time 0 and 24 h. Synergy was defined as a P 2 log 10

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decrease in colony count after 24 h by the combination compared to the most active single agent; indifference as a < 2 log 10 increase or decrease in colony count at 24 h by the combination compared with that by the most active single agent and antagonism as a P 2 log 10 increase in colony count after 24 h by the combination compared with that by the most active single agent [7]. 3. Results 3.1. Determination of MICs of raw ciprofloxacin hydrochloride, gatifloxacin hydrochloride, and lysozyme The MICs of raw ciprofloxacin hydrochloride (Raw-CIP), gatifloxacin hydrochloride (Raw-GAT), and lysozyme (Raw-LYS) were determined via the microdilution method on P. aeruginosa, S. aureus, K. pneumoniae, and A. baumannii. These bacteria are among the more common pathogens known to cause respiratory tract infections. The corresponding MIC values are summarized in Table 3. All the bacteria species and strains used in the studies were susceptible to ciprofloxacin hydrochloride and gatifloxacin hydrochloride, showing a MIC of either 0.25 lg/mL or 0.5 lg/mL. Lysozyme was found to have little antimicrobial effect on bacteria at concentrations up to 10 mg/mL. The result was as expected, as lysozyme is known to exhibit only mild antibacterial properties [27]. The antibiotics’ MIC values were important as they provided preliminary guidance on each antibiotic’s required therapeutic dose in the binary (SD-CIP/GAT) or ternary (SD-CIP/GAT/LYS) formulations (i.e., combination ratios). With the exception of P. aeruginosa (i.e., MICCIP/MICGAT = 1:2), all the other bacteria had MICCIP/MICGAT of 1:1 (i.e., Table 3). In this work, a ratio of 1:2 was applied to better accommodate testing across all bacteria species. Therefore, the binary formulation composed of CIP/GAT in the ratio of 1:2, while the ternary composition contained CIP/GAT/LYS in the ratio of 1:2:6. The lysozyme ratio in the ternary formulation was chosen to reflect a (CIP + GAT)/LYS ratio of 1:2. To further enhance the therapeutic efficiency of the antibiotic powders (i.e., to exceed the minimum 1:2 (CIP/GAT) ratio required for P. aeruginosa), the ratios were adjusted to 1:2.5 (CIP/ GAT) and 1:2.5:5.5 (CIP/GAT/LYS) for the binary and ternary formulations, respectively. 3.2. Preparation of the spray-dried powder Spray-dried powders of the single species (SD-CIP, SD-GAT, and SD-LYS), binary combination (SD-CIP/GAT), and ternary combination (SD-CIP/GAT/LYS) were obtained at yields of more than 70% (Table 1). High product yield is one of the unique and desirable features of the Nano Spray Dryer B-90 [28] with published reports

Table 3 Minimum inhibitory concentration (MIC) values of ciprofloxacin hydrochloric, gatifloxacin hydrochloric, and lysozyme against various pathogenic respiratory bacteria. Bacteria species and strain

Pseudomonas aeruginosa ATCC 90207 Staphylococcus aureus ATCC 4330 Klebsiella pneumoniae ATCC 12885 Acinetobacter baumannii ATCC 19606 a

MIC (lg/mL) b

Ratio of MICs

a

c

CIP

GAT

LYS

0.25 0.25 0.25 0.50

0.50 0.25 0.25 0.50

NDd ND ND ND

CIP/GAT 1:2 1:1 1:1 1:1

CIP – ciprofloxacin hydrochloride. GAT – gatifloxacin hydrochloride. LYS – lysozyme. d ND – not determined due to the absence of inhibition even at a high concentration of 10 mg/mL. b

c

listing it as between 70% and 90% [28,29]. The antibiotics and lysozyme content in the spray-dried powder formulations were analyzed via HPLC to scrutinize for homogeneity and potential feed loss (Table 2). Ciprofloxacin hydrochloride and gatifloxacin hydrochloride content in all the spray-dried formulations were found to be close to 100% in comparison with the feed. Lysozyme content in either SD-LYS or SD-CIP/GAT/LYS was found to be approximately 15% less than the feed. This could be attributed to a slight loss in lysozyme activity after spray-drying. 3.3. Evaluation of physiochemical properties of the powders The surface morphology of the raw materials and spray-dried powders is shown in Fig. 1. Generally, the Raw-CIP, Raw-GAT, and Raw-LYS particles appeared as irregularly-shaped chips and chunks. In contrast, the spray-dried powders consisted of particles that have more uniform shapes and sizes. SD-CIP, SD-CIP/GAT, and SD-CIP/GAT/LYS (Fig. 1b, g and h) had a spherical geometry with a slightly corrugated surface, whereas SD-LYS (Fig. 1f) was spherical with a smoother surface. Interestingly, SD-GAT was not as spherical as anticipated (Fig. 1d), which might be due to the inherent material properties. The particle size distribution of the formulations is summarized in Table 4. The particle size analysis revealed that all the spray-dried powders were within the respirable size range, with a D50 from 1.75 ± 0.03 to 1.96 ± 0.09 lm. Fig. 2 shows the XRD patterns of the raw materials, physical mixture of the raw materials (with same composition ratio as in spray-dried DPI formulations), and the spray-dried samples. The XRD patterns of Raw-CIP and Raw-GAT displayed intense and sharp peaks, suggesting crystallinity. On the other hand, XRD patterns showed detectable amorphous structure for Raw-LYS, SD-LYS, SD-GAT, SD-CIP/GAT, and SD-CIP/GAT/LYS. Interestingly, reduced crystallinity and not full amorphous structure was observed for SD-CIP. This is in contrast to the work by Adi et al. [22], which reported amorphicity for spray-dried ciprofloxacin hydrochloride obtained on the traditional spray dryer system. Potentially, the Nano Spray Dryer B-90, facilitating a mild and uniform drying at low throughput [28,29], could be useful in preserving the crystallinity of certain compounds. The absence of peaks characterizing CIP in the binary and ternary compounds could be due to the lower ratios of CIP in the formulations and the sensitivity limits of the method as it approaches the lower concentrations (i.e., detection limits 2–10%) [30–33]. The diffraction patterns of the physical mixtures were the summation of the individual diffraction patterns of the raw materials. 3.4. In vitro aerosol performance The in vitro drug deposition and aerosolization performance of the formulation were tested and analyzed. Fig. 3 illustrates the NGI deposition profile of the spray-dried samples following aerosolization from the AerolizerÒ DPI. In general, all the spray-dried DPI formulations had fairly good delivery efficiency with a FPF of approximately 40%, with the exception of SD-LYS (Table 5). SDLYS (i.e., smooth spherical particles) exhibited the highest capsule and inhaler retention (i.e., 11.3 ± 3.6% and 30.8 ± 4.5%, respectively) (Fig. 3c), and the lowest FPF (i.e., 33.3 ± 1.0%), suggesting powder cohesiveness. This was in line with the observations of Kwok et al. [34] on a similar system. A possible strategy to minimize powder adhesion (and hence capsule and inhaler retention) would be to increase the particles’ surface roughness [35,36]. For the binary and ternary combinatorial DPI formulations, there was concomitant deposition observed across all stages (including the throat, inhaler, and capsule), thus suggesting uniformity in the powder and/or aerosol (Fig. 3d and e) [30].

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279

Fig. 1. FESEM images of raw materials and spray-dried powders. (a) Raw-CIP, (b) SD-CIP, (c) Raw-GAT, (d) SD-GAT, (e) Raw-LYS, (f) SD-LYS, (g) SD-CIP/GAT, and (h) SD-CIP/ GAT/LYS.

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Table 4 Particle size (mean ± standard deviation, n = 3) of spray-dried powder.

D10 D50 D90 Span

SD-CIP (lm)

SD-GAT (lm)

SD-LYS (lm)

SD-CIP/GAT (lm)

SD-CIP/GAT/LYS(lm)

0.91 ± 0.01 1.95 ± 0.02 3.92 ± 0.16 1.54 ± 0.06

0.78 ± 0.04 1.75 ± 0.03 4.51 ± 0.38 2.13 ± 0.23

0.90 ± 0.04 1.91 ± 0.15 3.71 ± 0.41 1.53 ± 0.08

0.89 ± 0.06 1.83 ± 0.08 3.53 ± 0.08 1.44 ± 0.06

0.91 ± 0.01 1.96 ± 0.09 4.21 ± 0.44 1.68 ± 0.16

Relative Intensity

D10 – volume diameter under which 10% of the sample resides. D50 – volume median diameter. D90 – volume diameter under which 90% of the sample resides. Span = (D90 D10)/D50.

Raw-CIP Raw-GAT Raw-LYS Physical mix-CIP/GAT Physical mix-CIP/GAT/LYS SD-CIP SD-GAT SD-LYS SD-CIP/GAT SD-CIP/GAT/LYS

5

10

15

20

25

30

35

40

45

50

2θ Fig. 2. XRD patterns of Raw-CIP, Raw-GAT, Raw-LYS, a 1:2.5 (w/w) physical mixture of Raw-CIP/GAT, a 1:2.5.4.8 (w/w) physical mixture of Raw-CIP/GAT/LYS, and spraydried powders SD-CIP, SD-GAT, SD-LYS, SD-CIP/GAT, and SD-CIP/GAT/LYS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.5. Antibacterial activity of DPI formulations The antibacterial activities of the DPI formulations are summarized in Table 6. SD-CIP/GAT and SD-CIP/GAT/LYS reduced the bacterial counts of P. aeruginosa, S. aureus, K. pneumoniae, and A. baumannii strain to 4.1, 4.6, 2.0, and 1.5 log10 CFU/mL, respectively, for the former, and 8.4, 5.6, 0.9, and 1.5, respectively, for the latter within a 24 h test period. From Table 6, it was noted that most of the log10 changes obtained (compared to the most active single agent) were typically less than 2 log10 CFU/mL, indicative of an indifference interaction, with the exception of SD-CIP/GAT against P. aeruginosa. Synergy was observed in SD-CIP/GAT against P. aeruginosa with a change in log10 CFU/mL of 2.8 after 24 h. 4. Discussion Combinations of antibiotics are commonly used in clinical medicine for overcoming bacterial resistance and also for enhancing antibacterial efficacy. Generally, antibiotic combinations can be classified as synergistic, indifferent or antagonistic, depending on whether the combined effect of the combination is larger than, equal to, or smaller than the effect predicted from the sum of each agent (or antibiotic) alone (Fig. 4). The present study showed a synergy interaction against P. aeruginosa and indifference interaction against S. aureus, K. pneumoniae, and A. baumannii between ciprofloxacin hydrochloride and gatifloxacin hydrochloride in their cospray-dried form (SD-CIP/GAT). Combination of ciprofloxacin

hydrochloride, gatifloxacin hydrochloride, and lysozyme in their co-spray-dried form (SD-CIP/GAT/LYS) resulted in an indifference interaction against P. aeruginosa, S. aureus, K. pneumoniae, and A. baumannii. This work has demonstrated that co-spray-drying using the Nano Spray Dryer B-90 is a useful formulation platform for facilitating simultaneous delivery of multi-active pharmaceutical ingredients to the same site of action for potential synergistic outcomes. In addition, the approach also showed the simplicity of preparing particles in the inhalable size range with good content uniformity. An important drawback of current aerosol antibiotic treatments is their inability to penetrate into the deep lung. This sets up an antibiotic concentration gradient (i.e., high levels in the proximal airway but extremely low levels in the distal airway), which might lead to antimicrobial resistance [37]. There is considerable evidence to suggest that antibiotic concentrations below the MIC, at sub-inhibitory levels, select and enrich for resistant bacteria [38]. Therefore, it is imperative for formulators to develop robust antibiotic therapy with adequate penetration potential. The FPF in this work represents a single cut-off (mass percent of particles with an aerodynamic diameter < 5 lm) and is represented by the total amount of particles depositing from stage 3 down to the microorifice collector in the in vitro deposition graphs (Fig. 3). On an in vitro level, the aerodynamic particle size distribution and the respirable fraction could be readily obtained from the impactors (Fig. 3). However, to be able to accurately predict regional deposition, in-depth in vivo lung deposition studies in humans would have to be carried out [39]. An FPF value that is significantly higher than 30% is an index of good aerosol performance [40,41]. In this work, SD-CIP/GAT, SDCIP, and SD-GAT already had FPFs of 40% at 60 L/min, which was considered a low air flow rate achievable with minimal inhalation effort. This flow rate was used to simulate the reduced inspiratory effort achieved by patients with compromised lung functions (e.g., cystic fibrosis and pneumonia). The reasonably robust FPFs obtained in this work indicated that the powders of SD-CIP/GAT, SD-CIP, and SD-GAT were not particularly cohesive. Respiratory infections can affect any part of the respiratory system consisting of the upper and lower divisions. Bacterial lower respiratory infections are more common and have the potential to cause serious infections such as bronchitis and pneumonia. The young are particularly susceptible to bacterial infections during or soon after upper respiratory infections. P. aeruginosa, S. aureus, K. pneumoniae, and A. baumannii are the common infective pathogens found in both hospitals and community settings and were reported as the most frequent respiratory isolates from adults with hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) in Asian countries [42]. These bacterial droplets can be generated from sneezing and coughing in public. In this work, SD-CIP/GAT has been shown to interact synergistically against P. aeruginosa bacteria isolate. This is in good agreement with the results of a previous in vitro study that reported synergistic interaction between ciprofloxacin and gatifloxacin for

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50 40 30 20

40 30 20 10

0

0

60

70

(b)

60

50 40 30 20

30 20

0

0

Deposition (%)

CIP GAT

40

10

60

(d)

50

10

70

(c)

50

10

70

Deposition (%)

60

Deposition (%)

Deposition (%)

60

70

(a) Deposition (%)

70

(e)

CIP GAT LYS

50 40 30 20 10 0

Fig. 3. In vitro deposition of spray-dried powders at 60 L/min. Data presented as mean ± standard deviation (n = 3). (a) SD-CIP, (b) SD-GAT, (c) SD-LYS, (d) SD-CIP/GAT, and (e) SD-CIP/GAT/LYS. S1–S7 denote impactor stages 1–7, followed by their corresponding aerodynamic cutoff diameter in parentheses. MOC is the NGI micro-orifice collector.

Table 5 Deposition parameters (mean ± standard deviation, n = 3) of different formulations measured by NGI at 60 L/mL. SD-CIP

FPFa (%) FPF(emitted)b (%) a b

42.3 ± 1.5 56.8 ± 2.1

SD-GAT

39.6 ± 2.8 53.7 ± 3.5

SD-LYS

33.3 ± 1.0 57.7 ± 2.6

SD-CIP/GAT

SD-CIP/GAT/LYS

CIP

GAT

CIP

GAT

LYS

41.3 ± 2.6 53.6 ± 6.7

40.9 ± 2.7 52.8 ± 6.8

42.56 ± 1.1 57.8 ± 4.4

42.2 ± 1.7 57.2 ± 5.1

42.0 ± 2.8 56.5 ± 5.3

FPF – fine particle fraction. FPF(emitted) – emitted fine particle fraction.

P. aeruginosa [43]. Gatifloxacin, a new synthetic broad-spectrum 8methoxyfluoroquinolone that has similar substituent configuration to reserpine (a known efflux pump inhibitor) is believed to interfere with the efflux pumping of ciprofloxacin, hence potentiating synergism in the combination [43]. P. aeruginosa is not only considered to be one of the most common pathogens in chronic obstructive pulmonary disease (COPD) patients, but also the leading cause of morbidity and mortality

for patients with cystic fibrosis (CF). Furthermore, more than 80% of CF patients will battle a lung infection caused by P. aeruginosa at some point of their lives, made not much easier with the increased prevalence of antibiotic resistance [44]. Therefore, the newly-developed synergistic SD-CIP/GAT combinatorial DPI formulation may be a promising alternative in the fight against P. aeruginosa and its resistant forms for the various respiratory diseases.

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Table 6 Time-kill test results of spray-dried powder against different pathogenic respiratory bacteriaa. Bacterial strain

Remaining countb (24-h) (log10 CFU/mL)

Pseudomonas aeruginosa ATCC 90207 SD-CIP 9.9 SD-GAT 6.9 SD-LYS 12.3 SD-CIP/GAT 4.1 SD-CIP/GAT/LYS 8.4 Staphylococcus aureus ATCC 4330 SD-CIP 8.7 SD-GAT 5.5 SD-LYS 12.7 SD-CIP/GAT 4.6 SD-CIP/GAT/LYS 5.6 Klebsiella pneumoniae ATCC 12885 SD-CIP 1.8 SD-GAT 1.0 SD-LYS 11.3 SD-CIP/GAT 2.0 SD-CIP/GAT/LYS 0.9 Acinetobacter baumanii ATCC 19606 SD-CIP 8.1 SD-GAT 1.7 SD-LYS 11.6 SD-CIP/GAT 1.5 SD-CIP/GAT/LYS 1.5

Time-kill assayc (log10 change)

Interaction

– – –

– – – Synergy Indifference

2.8 1.4 – – – 1.0 0.0

– – – Indifference Indifference

– – – 0.9 0.2

– – – Indifference Indifference

– – –

– – – Indifference Indifference

0.27 0.24

CIP – ciprofloxacin hydrochloride; GAT – gatifloxacin hydrochloride; LYS – lysozyme; SD – spray-dried. a Performed in triplicate. b After 24-h incubation with spray-dried powder (binary or ternary combination) at 1  MIC. c Values represent the log10 change in CFU/mL in the time-kill assay after 24-h exposure to the spray-dried powder (binary or ternary combination) compared to the most active drug.

Fig. 4. Schematic representation of synergy, indifference, and antagonism.

Against S. aureus, K. pneumonia and A. baumannii, SD-CIP/GAT elucidated an indifferent response. When the combination is indifferent or synergistic, the combined drugs could be used to achieve the same or improved efficacy. Respiratory infections in asthma, chronic obstruction pulmonary disease (COPD), and cystic fibrosis (CF) are usually due to severe inflammatory conditions of the airways in which mucus hypersecretion is a pathophysiological feature. During infection or inflammation, the airway mucosa typically responds by increasing the volume of mucus that is secreted due to the phenomenon of secretory hyperresponsiveness [37]. As mucus hypersecretion and airway inflammation are closely linked and related to respiratory infections, the work was therefore extended to include a ternary compound with dual anti-inflammatory and mucolytic properties to the synergistic SD-CIP/GAT formulation (i.e., ternary formulations). Lysozyme, also commonly called muramidase, is a mucolytic enzyme that exhibits anti-inflammatory and natural antibiotic properties. Furthermore, it is abundant in a number of bodily secretions, such as tears, saliva, human milk and mucus, and has

long been shown to play an important role in pulmonary host defenses [45]. The antimicrobial activity of lysozyme is primarily due to its bacteriolytic actions, capable of hydrolyzing the b-1,4-glycosidic bond in the peptidoglycan cell wall of gram-positive bacteria, hence leading to cell wall rupture and bacteria kill [46]. The anti-inflammatory action of lysozyme is closely linked to the neutralization of acidic substances released during the inflammatory process [47]. The mucolytic properties of lysozyme come from its ability to catalyse the depolymerization of highly polymerized mucopolysaccharides (the chief ingredient in mucus), leading to the breakdown of complicated mucoids into much simpler forms [48]. P. aeruginosa in CF patients has been known to be a difficult bacteria to eradicate due to the overproduction of an exopolysaccharide known as alginate that leads to the formation of mucoid biofilms [44]. Therefore, a suitably-developed antibiotic–mucolytic combinatorial formulation would indeed be timely for the CFafflicted population. The SD-CIP/GAT/LYS ternary formulation in the present study had FPF values similar to the SD-CIP/GAT binary formulation (Table 5). SD-LYS was found to have a lower FPF value (33.3 ± 1.0%) in comparison with that of SD-CIP (42.3 ± 1.5%) and SD-GAT (39.6 ± 2.8%). When the ternary species were combined via cospray-drying, each component in the co-spray-dried powder (SDCIP/GAT/LYS) was found to have an FPF value much closer to that for SD-CIP and SD-GAT than SD-LYS (i.e., 42.6 ± 1.1% for CIP, 42.2 ± 1.7% for GAT, and 42.0 ± 2.8% for LYS). This observation suggested that ciprofloxacin hydrochloride and gatifloxacin hydrochloride may have a dominating effect on the aerosolization efficiency even though the lysozyme composition was higher when compared to the other two components. Furthermore, all three components in SD-CIP/GAT/LYS had similar deposition patterns (Fig. 3), implying that the co-spray-dried formulation was homogeneous, with minimal molecular segregation during the spray-drying process. SD-CIP/GAT/LYS was observed to be indifferent to all four respiratory pathogen isolates when tested via the time-kill assay. It was surprising that synergy was not detected when SD-CIP/GAT/LYS was tested against the P. aeruginosa isolate, as SD-CIP/GAT was previously shown to exhibit synergy against the same isolate. Although not fully understood at this stage and beyond the scope of the present work, possible explanations could be due to the insensitivity of lysozyme to gram-negative bacteria at ambient pressure [49,50] or the presence of a lysozyme-induced change to the Pseudomonas cell wall which serves to provide resistance to lysozyme mediated killing [51]. Inadvertently, the cell wall changes may also result in reduced susceptibility to antibiotics and hence the apparent loss of antibacterial activity by the fluoroquinolones. Although indifference was detected in the ternary DPI formulation (SD-CIP/GAT/LYS) when tested against all four respiratory bacteria, this work nonetheless describes a useful first proof-ofconcept for co-delivering antibiotics with a mucolytic and antiinflammatory agent (i.e., lysozyme) for the treatment of pulmonary infections.

5. Conclusion A synergistic DPI formulation against P. aeruginosa was successfully prepared by co-spray-drying the binary system containing ciprofloxacin hydrochloride and gatifloxacin hydrochloride (weight ratio of 1:2.5). The powder consisted of respirable-sized particles (D50 of 1.83 ± 0.08 lm) capable of achieving a FPF of approximately 40%. In addition, a concomitant and uniform in vitro deposition profile could be achieved across all impaction stages when dispersed at 60 L/min. Co-spray-dried powder of

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ciprofloxacin hydrochloride and gatifloxacin hydrochloride may therefore offer an alternative anti-pseudomonal therapy for the control of airway infections associated with cystic fibrosis and pneumonia. The potential use of lysozyme as an antimicrobial agent has also been explored in the co-spray-dried powders of ciprofloxacin hydrochloride, gatifloxacin hydrochloride, and lysozyme. Although synergy was not detected in the ternary formulation, the inclusion of lysozyme in the formulation is potentially beneficial for its dual mucolytic and anti-inflammatory properties. Acknowledgements This work was supported by the Science and Engineering Research Council of ASTAR (Agency for Science, Technology and Research), Singapore (Grant No. ICES/09-122A02) and partially by the Australian Research Council (Grant No. DP120102778). We are grateful to Miss Agnes Nicole Phua, Mr. Ng Jun Wei, Mr. Wan Xian Chao, and Miss Cicilia Amex for their assistance in the experiments. References [1] R.W. Pinner, S.M. Teutsch, L. Simonsen, L.A. Klug, J.M. Graber, M.J. Clark, R.L. Berkelman, Trends in infectious diseases mortality in the United States, Journal of the American Medical Association 275 (1996) 189–193. [2] WHO, Removing Obstacles to Healthy Development, World Health Organization, Geneva, Switzerland, , 1999 (Retrieved 30.04.12). [3] D.F. Gordon, The Global Infectious Disease Threat and its Implications for the United States, National Intelligence Council, Washington, DC, USA, , 2000 (Retrieved 30.04.12). [4] NIH, Emerging and Re-emerging Infectious Diseases, National Institutes of Health, Bethesda, MD, USA, , 2008 (Retrieved 30.04.12). [5] A.S. Fauci, N.A. Touchette, G.K. Folkers, Emerging infectious diseases: a 10-year perspective from the National Institute of Allergy and Infectious Diseases, Emerging Infectious Diseases 11 (2005) 519–525. [6] R. Chait, A. Craney, R. Kishony, Antibiotic interactions that select against resistance, Nature 446 (2007) 668–671. [7] V. Lorian, Antibiotics in Laboratory Medicine, Williams and Wilkins, Baltimore, MD, 2005. [8] G. Marshall, J.W.S. Blacklock, C. Cameron, N.B. Capon, R. Cruickshank, J.H. Gaddum, F.R.G. Heaf, A. Bradford Hill, L.E. Houghton, J. Clifford Hoyle, H. Raistrick, J.G. Scadding, W.H. Tytler, G.S. Wilson, P. D’Arcy Hart, Streptomycin treatment of pulmonary tuberculosis: a medical research council investigation, British Medical Journal 2 (1948) 769–782. [9] B. Fantin, C. Carbon, In vivo antibiotic synergism: contribution of animal models, Antimicrobial Agents and Chemotherapy 36 (1992) 907–912. [10] M.H. Lepper, H.F. Dowling, Treatment of pneumococcic meningitis with penicillin compared with penicillin aureomycin, Archives of Internal Medicine 88 (1951) 489–494. [11] D. Traini, P.M. Young, Delivery of antibiotics to the respiratory tract: an update, Expert Opinion on Drug Delivery 6 (2009) 897–905. [12] T. Abu-Salah, R. Dhand, Inhaled antibiotic therapy for ventilator-associated tracheobronchitis and ventilator-associated pneumonia: an update, Advances in Therapy 28 (2011) 728–747. [13] R. Beasley, C. Burgess, S. Holt, Call for worldwide withdrawal of benzalkonium chloride from nebulizer solutions, Journal of Allergy and Clinical Immunology 107 (2001) 222–223. [14] M.J. Asmus, J. Sherman, L. Hendeles, Bronchoconstrictor additives in bronchodilator solutions, Journal of Allergy and Clinical Immunology 104 (1999) S53–S60. [15] G. Loughlin, H. Eigen, Respiratory Disease in Children: Diagnosis and Management, Williams and Wilkins Baltimore, MD, 1994. [16] A.L. Coates, P.D. Allen, C.F. MacNeish, S.L. Ho, L.C. Lands, Effect of size and disease on estimated deposition of drugs administered using jet nebulization in children with cystic fibrosis, Chest 119 (2001) 1123–1130. [17] R.L. Elmore, M.E. Contois, J. Kelly, A. Noe, A. Poirier, Stability and compatibility of admixtures of intravenous ciprofloxacin and selected drugs, Clinical Therapeutics 18 (1996) 246–255. [18] D.E. Geller, M.W. Konstan, J. Smith, S.B. Noonberg, C. Conrad, Novel tobramycin inhalation powder in cystic fibrosis subjects: pharmacokinetics and safety, Pediatric Pulmonology 42 (2007) 307–313. [19] N.R.C. Labiris, A.M. Holbrook, H. Chrystyn, S.M. Macleod, M.T. Newhouse, Dry powder versus intravenous and nebulized gentamicin in cystic fibrosis and bronchiectasis – a pilot study, American Journal of Respiratory and Critical Care Medicine 160 (1999) 1711–1716.

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