International Journal of Pharmaceutics 491 (2015) 367–374
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Inhalable spray-dried formulation of D-LAK antimicrobial peptides targeting tuberculosis Philip Chi Lip Kwoka , Adam Grabarekb , Michael Y.T. Chowa , Yun Lana , Johnny C.W. Lia , Luca Casettaric , A. James Masond , Jenny K.W. Lama,* a
Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, 21 Sassoon Road, Hong Kong School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N A1X, United Kingdom Department of Biomolecular Sciences, University of Urbino, Piazza Rinascimento, 6, Urbino 61029, Italy d Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 March 2015 Received in revised form 29 May 2015 Accepted 1 July 2015 Available online 4 July 2015
Tuberculosis (TB) is a global disease that is becoming more difficult to treat due to the emergence of multidrug resistant (MDR) Mycobacterium tuberculosis. Inhalable antimicrobial peptides (AMPs) are potentially useful alternative anti-TB agents because they can overcome resistance against classical antibiotics, reduce systemic adverse effects, and achieve local targeting. The aims of the current study were to produce inhalable dry powders containing D-enantiomeric AMPs (D-LAK120-HP13 and D-LAK120-A) and evaluate their solid state properties, aerosol performance, and structural conformation. These two peptides were spray dried with mannitol as a bulking agent at three mass ratios (peptide: mannitol 1:99, 1:49, and 1:24) from aqueous solutions. The resultant particles were spherical, with those containing D-LAK120-HP13 being more corrugated than those with D-LAK120-A. The median volumetric diameter of the particles was approximately 3 mm. The residual water content of all powders were 80% purity were purchased from China Peptides (Shanghai, China) and used as provided. Mannitol (Pearlitol 160C) was purchased from Roquette (Lestrem, France). All other reagents and solvents were purchased from Sigma–Aldrich (Poole, UK) and were of analytical grade or better. 2.2. Spray drying of D-LAK peptides Three formulations each of D-LAK120-HP13 and D-LAK120-A were prepared at different peptide to mannitol mass ratios as listed in Table 1. Both peptides and mannitol were dissolved in distilled water to give a final total mass concentration of 1% (w/v) in solution. A Büchi B-290 laboratory spray dryer (Büchi Labortechnik AG, Flawil, Switzerland) was used to prepare the spray-dried powders of each formulation under the following conditions: inlet temperature of 75 C, outlet temperature of 40–42 C, aspiration at 38 m3/min (100%), nitrogen atomization flow rate at 742 norm litre/h, and liquid feed rate at 3.6 ml/min. The spray drying conditions used were slightly modified from those for producing powders containing DNA/siRNA and pH-responsive peptides conducted earlier by the authors (Liang et al., 2015, 2014). The spray-dried powders were collected by a high efficiency cyclone and stored at room temperature with silica gel until further analysis. 2.3. Spray drying yield and recovery of D-AMPs The spray drying yield was calculated as the mass of powder obtained after spray drying divided by the total initial amount of solid ingredients expressed as a percentage. The peptide recovery was determined as the percentage ratio of the amount of peptide measured in the spray dried powder with respect to the theoretical amount of peptide. The amount of peptides in the spray-dried powders was determined using reversed-phase high performance liquid chromatography (HPLC) (HPLC 1260 Agilent Technologies, Santa Clara, USA). A 100 ml aliquot of sample solution was injected by an auto-sampler and passed through a C18 column (140 mm 4.6 mm, 5 mm; VydacTM GraceTM, IL, USA) at ambient
Table 1 Spray dried powders of D-LAK120-HP13 and D-LAK120-A with mannitol, their spray drying yield, peptide recovery, and water content. Data for peptide recovery presented as mean standard deviation (n = 3). Formulation
Peptide
Peptide: mannitol (mass ratio)
Spray drying yield (% w/w)
Peptide recovery (% w/w)
Water content (% w/w)
F1 F2 F3 F4 F5 F6
D-LAK120-HP13 D-LAK120-HP13 D-LAK120-HP13 D-LAK120-A D-LAK120-A D-LAK120-A
1:99 1:49 1:24 1:99 1:49 1:24
46.4 70.0 67.7 66.6 74.1 71.9
94.8 5.6 102.5 2.1 100.2 3.4 98.9 3.8 102.1 3.5 117.6 2.8
2.60 1.58 2.09 1.32 1.33 1.19
P.C.L. Kwok et al. / International Journal of Pharmaceutics 491 (2015) 367–374
temperature and the peptide was detected by UV at wavelength 220 nm. The mobile phase consisted of acetonitrile and ultra-pure water with 0.1% trifluoroacetic acid. A linear gradient from 20 to 80% acetonitrile was applied over 20 min at 1 ml/min. 2.4. Particle size distribution and morphology study The particle size distribution of the spray-dried powders was examined by laser diffractometer (LS 13 320 Beckman Coulter, Pasadena, USA). Approximately 3 mg of each powder was dispersed in 10 ml of chloroform and de-aggregated by ultrasonication for three minutes. The particles suspended in chloroform were checked by optical microscopy (Olympus BX50; Tokyo, Japan) before and after ultra-sonication to confirm no change in their primary particle size and morphology. A few droplets of the dispersed sample were added into a Micro Liquid Module to achieve an obscuration of 8–12 %. The particle size distribution was expressed in terms of D10, D50 and D90 (volumetric diameter under which 10%, 50% and 90% of the sample resided, respectively). The span, which is the width of the size distribution, was calculated using the formula (D90–D10)/D50. Each powder formulation was analyzed in triplicate. The morphology of the spray-dried powders was visualized by scanning electron microscopy (SEM) at 2.0 kV (Hitachi S-4800, Hitachi High-technologies Corp., Tokyo, Japan). A small amount of powder was placed on adhesive carbon discs and sputter-coated with a 15 nm palladium/gold (60/40) coating prior to imaging. The metallic coating is a standard procedure for preparing non-electrically conducting samples to facilitate the discharge of high-energy electrons during imaging. 2.5. X-ray powder diffraction (XRPD) The crystalline structure of the spray dried powders was studied using a X-ray powder diffractometer. About 100 mg of powder was compacted onto a sample holder plate which was subsequently placed into the X-ray diffractometer (X’Pert PRO; PANalytical B.V., Almelo, The Netherlands). The powder compact was measured with Cu Ka radiation at 30 mA and 40 kV, using an angular increment of 0.04 at 2 s per step covering a 2u range from 5 to 50 . 2.6. Differential scanning calorimetry (DSC) The thermal response profiles of each powder sample were assessed by differential scanning calorimetry (DSC) (Model Q1000; TA Instruments, New Castle, DE, USA). Between 1–3 mg of sample powder was loaded into an aluminium crucible and crimped with a non-perforated lid, being heated from 20 C to 250 C at 10 C/min under 50 ml/min nitrogen purge.
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2.7. Thermogravimetric analysis (TGA) The water content of the powders was measured by thermogravimetric analysis (TGA) (Model Q5000; TA Instruments, New Castle, DE, USA). The loss in mass from a sample (1–3 mg) was monitored while heated from 20 C to 180 C at 10 C/min under 25 ml/min nitrogen purge. The loss in mass indicated the amount of residual water evaporated from the sample. 2.8. In vitro aerosol performance In vitro aerosol performance of the spray-dried powders was tested by the pharmacopeial method using a Next Generation Impactor (NGI) (Copley Scientific Limited, Nottingham, UK). Size 3 hydroxypropyl methylcellulose capsules were used for powder loading. Each capsule contained 5 mg of powder which was aerosolized by Osmohaler1 (RS-01; Plastiape, Osnago, Italy). A standard dispersion procedure was conducted for 2.7 s at an airflow rate of about 90 l/min, to obtain 4 l passing air with a pressure drop in the inhaler maintained at 4 kPa. The impactor stages were covered with a thin coat of silicon spray (Dry Film Silicone Lubricant; LPS, Tucker, GA, USA) to minimize particle bounce. After dispersion, powders deposited on the inhaler and NGI parts were assayed using HPLC by dissolving in 4 ml of distilled water. 2.9. Chromatographic assay The HPLC system (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, USA) consisted of a refractive index detector (G1362A, Agilent Technologies, Santa Clara, USA) was used for mannitol assay. Samples (20 ml) were injected and passed through a C18 column (Agilent Hi-Plex Ca, 7.7 300 mm, 8 mm) maintained at 55 C with ultrapure water as the mobile phase running at 1 ml/ min. The loaded dose was the amount of powder weighed into the capsule before the NGI run. The recovered dose was the total amount of powder assayed on all the inhaler and NGI parts in one run. The emitted dose was the total amount of powder assayed from the adaptor downstream to the micro-orifice collector (MOC) of the NGI. This was the amount of powder that was emitted from the inhaler. The emitted fraction (EF) was defined as the percentage fraction of the emitted dose with respect to the loaded dose. The fine particle dose was the amount of powder with aerodynamic diameter
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Inhalable spray-dried formulation of D-LAK antimicrobial peptides targeting tuberculosis Philip Chi Lip Kwoka , Adam Grabarekb , Michael Y.T. Chowa , Yun Lana , Johnny C.W. Lia , Luca Casettaric , A. James Masond , Jenny K.W. Lama,* a
Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, 21 Sassoon Road, Hong Kong School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N A1X, United Kingdom Department of Biomolecular Sciences, University of Urbino, Piazza Rinascimento, 6, Urbino 61029, Italy d Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 March 2015 Received in revised form 29 May 2015 Accepted 1 July 2015 Available online 4 July 2015
Tuberculosis (TB) is a global disease that is becoming more difficult to treat due to the emergence of multidrug resistant (MDR) Mycobacterium tuberculosis. Inhalable antimicrobial peptides (AMPs) are potentially useful alternative anti-TB agents because they can overcome resistance against classical antibiotics, reduce systemic adverse effects, and achieve local targeting. The aims of the current study were to produce inhalable dry powders containing D-enantiomeric AMPs (D-LAK120-HP13 and D-LAK120-A) and evaluate their solid state properties, aerosol performance, and structural conformation. These two peptides were spray dried with mannitol as a bulking agent at three mass ratios (peptide: mannitol 1:99, 1:49, and 1:24) from aqueous solutions. The resultant particles were spherical, with those containing D-LAK120-HP13 being more corrugated than those with D-LAK120-A. The median volumetric diameter of the particles was approximately 3 mm. The residual water content of all powders were 80% purity were purchased from China Peptides (Shanghai, China) and used as provided. Mannitol (Pearlitol 160C) was purchased from Roquette (Lestrem, France). All other reagents and solvents were purchased from Sigma–Aldrich (Poole, UK) and were of analytical grade or better. 2.2. Spray drying of D-LAK peptides Three formulations each of D-LAK120-HP13 and D-LAK120-A were prepared at different peptide to mannitol mass ratios as listed in Table 1. Both peptides and mannitol were dissolved in distilled water to give a final total mass concentration of 1% (w/v) in solution. A Büchi B-290 laboratory spray dryer (Büchi Labortechnik AG, Flawil, Switzerland) was used to prepare the spray-dried powders of each formulation under the following conditions: inlet temperature of 75 C, outlet temperature of 40–42 C, aspiration at 38 m3/min (100%), nitrogen atomization flow rate at 742 norm litre/h, and liquid feed rate at 3.6 ml/min. The spray drying conditions used were slightly modified from those for producing powders containing DNA/siRNA and pH-responsive peptides conducted earlier by the authors (Liang et al., 2015, 2014). The spray-dried powders were collected by a high efficiency cyclone and stored at room temperature with silica gel until further analysis. 2.3. Spray drying yield and recovery of D-AMPs The spray drying yield was calculated as the mass of powder obtained after spray drying divided by the total initial amount of solid ingredients expressed as a percentage. The peptide recovery was determined as the percentage ratio of the amount of peptide measured in the spray dried powder with respect to the theoretical amount of peptide. The amount of peptides in the spray-dried powders was determined using reversed-phase high performance liquid chromatography (HPLC) (HPLC 1260 Agilent Technologies, Santa Clara, USA). A 100 ml aliquot of sample solution was injected by an auto-sampler and passed through a C18 column (140 mm 4.6 mm, 5 mm; VydacTM GraceTM, IL, USA) at ambient
Table 1 Spray dried powders of D-LAK120-HP13 and D-LAK120-A with mannitol, their spray drying yield, peptide recovery, and water content. Data for peptide recovery presented as mean standard deviation (n = 3). Formulation
Peptide
Peptide: mannitol (mass ratio)
Spray drying yield (% w/w)
Peptide recovery (% w/w)
Water content (% w/w)
F1 F2 F3 F4 F5 F6
D-LAK120-HP13 D-LAK120-HP13 D-LAK120-HP13 D-LAK120-A D-LAK120-A D-LAK120-A
1:99 1:49 1:24 1:99 1:49 1:24
46.4 70.0 67.7 66.6 74.1 71.9
94.8 5.6 102.5 2.1 100.2 3.4 98.9 3.8 102.1 3.5 117.6 2.8
2.60 1.58 2.09 1.32 1.33 1.19
P.C.L. Kwok et al. / International Journal of Pharmaceutics 491 (2015) 367–374
temperature and the peptide was detected by UV at wavelength 220 nm. The mobile phase consisted of acetonitrile and ultra-pure water with 0.1% trifluoroacetic acid. A linear gradient from 20 to 80% acetonitrile was applied over 20 min at 1 ml/min. 2.4. Particle size distribution and morphology study The particle size distribution of the spray-dried powders was examined by laser diffractometer (LS 13 320 Beckman Coulter, Pasadena, USA). Approximately 3 mg of each powder was dispersed in 10 ml of chloroform and de-aggregated by ultrasonication for three minutes. The particles suspended in chloroform were checked by optical microscopy (Olympus BX50; Tokyo, Japan) before and after ultra-sonication to confirm no change in their primary particle size and morphology. A few droplets of the dispersed sample were added into a Micro Liquid Module to achieve an obscuration of 8–12 %. The particle size distribution was expressed in terms of D10, D50 and D90 (volumetric diameter under which 10%, 50% and 90% of the sample resided, respectively). The span, which is the width of the size distribution, was calculated using the formula (D90–D10)/D50. Each powder formulation was analyzed in triplicate. The morphology of the spray-dried powders was visualized by scanning electron microscopy (SEM) at 2.0 kV (Hitachi S-4800, Hitachi High-technologies Corp., Tokyo, Japan). A small amount of powder was placed on adhesive carbon discs and sputter-coated with a 15 nm palladium/gold (60/40) coating prior to imaging. The metallic coating is a standard procedure for preparing non-electrically conducting samples to facilitate the discharge of high-energy electrons during imaging. 2.5. X-ray powder diffraction (XRPD) The crystalline structure of the spray dried powders was studied using a X-ray powder diffractometer. About 100 mg of powder was compacted onto a sample holder plate which was subsequently placed into the X-ray diffractometer (X’Pert PRO; PANalytical B.V., Almelo, The Netherlands). The powder compact was measured with Cu Ka radiation at 30 mA and 40 kV, using an angular increment of 0.04 at 2 s per step covering a 2u range from 5 to 50 . 2.6. Differential scanning calorimetry (DSC) The thermal response profiles of each powder sample were assessed by differential scanning calorimetry (DSC) (Model Q1000; TA Instruments, New Castle, DE, USA). Between 1–3 mg of sample powder was loaded into an aluminium crucible and crimped with a non-perforated lid, being heated from 20 C to 250 C at 10 C/min under 50 ml/min nitrogen purge.
369
2.7. Thermogravimetric analysis (TGA) The water content of the powders was measured by thermogravimetric analysis (TGA) (Model Q5000; TA Instruments, New Castle, DE, USA). The loss in mass from a sample (1–3 mg) was monitored while heated from 20 C to 180 C at 10 C/min under 25 ml/min nitrogen purge. The loss in mass indicated the amount of residual water evaporated from the sample. 2.8. In vitro aerosol performance In vitro aerosol performance of the spray-dried powders was tested by the pharmacopeial method using a Next Generation Impactor (NGI) (Copley Scientific Limited, Nottingham, UK). Size 3 hydroxypropyl methylcellulose capsules were used for powder loading. Each capsule contained 5 mg of powder which was aerosolized by Osmohaler1 (RS-01; Plastiape, Osnago, Italy). A standard dispersion procedure was conducted for 2.7 s at an airflow rate of about 90 l/min, to obtain 4 l passing air with a pressure drop in the inhaler maintained at 4 kPa. The impactor stages were covered with a thin coat of silicon spray (Dry Film Silicone Lubricant; LPS, Tucker, GA, USA) to minimize particle bounce. After dispersion, powders deposited on the inhaler and NGI parts were assayed using HPLC by dissolving in 4 ml of distilled water. 2.9. Chromatographic assay The HPLC system (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, USA) consisted of a refractive index detector (G1362A, Agilent Technologies, Santa Clara, USA) was used for mannitol assay. Samples (20 ml) were injected and passed through a C18 column (Agilent Hi-Plex Ca, 7.7 300 mm, 8 mm) maintained at 55 C with ultrapure water as the mobile phase running at 1 ml/ min. The loaded dose was the amount of powder weighed into the capsule before the NGI run. The recovered dose was the total amount of powder assayed on all the inhaler and NGI parts in one run. The emitted dose was the total amount of powder assayed from the adaptor downstream to the micro-orifice collector (MOC) of the NGI. This was the amount of powder that was emitted from the inhaler. The emitted fraction (EF) was defined as the percentage fraction of the emitted dose with respect to the loaded dose. The fine particle dose was the amount of powder with aerodynamic diameter
