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Effect of formulation ingredients on the physical characteristics of salmeterol xinafoate microparticles tailored by spray freeze drying
Advanced Powder Technology 24 (2013) 36–42
Contents lists available at SciVerse ScienceDirect
Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
Original Research Paper
Effect of formulation ingredients on the physical characteristics of salmeterol xinafoate microparticles tailored by spray freeze drying Mohammad Reza Rahmati, Alireza Vatanara ⇑, Ahmad Reza Parsian, Kambiz Gilani, Khosrow Malek Khosravi, Majid Darabi, Abdolhossein Rouholamini Najafabadi Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 4 October 2011 Received in revised form 30 December 2011 Accepted 11 January 2012 Available online 26 January 2012 Keywords: Salmeterol Spray freeze drying Cyclodextrin Dissolution
a b s t r a c t Series of microparticles containing salmeterol xinafoate (SX) as active pharmaceutical ingredient (API) and lactose, mannitol or trehalose as a bulking agents were prepared using spray freeze drying (SFD) technique and the effects of sugar type and presence of hydroxy propyl beta cyclodextrin (HPbCD) on the physical properties of powders were evaluated. Precipitation of salmeterol in the presence of lactose and mannitol resulted in the formation of irregular shapes of microparticles with broad size distributions. However application of trehalose resulted in the formation of porous particles with spherical morphology. Addition of cyclodextrin in the formulations was generally helpful for formation of porous and spherical particles with narrow size distribution with a mean size of 10–30 lm. Dissolution of SX from processed particles was substantially higher (90% drug release in 30 min) than that of unprocessed drug and physical mixture of drug and cyclodextrin (22% drug release in 30 min). This study showed that, processing of SX by SFD technique could be a constructive approach to the production of various forms of drug and drastic changes in the physical characteristics of microparticles could be achieved by changing the composition of bulking agent and cyclodextrin. Ó 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
Introduction The efficacy of pharmaceuticals can be significantly influenced by their physicochemical properties such as particle size distribution, morphology, powder flow, compression characteristics, physical stability and bioavailability. Fine pharmaceutical powders are often produced by currently available micronization methods that allow particle engineering. Processes such as air jet milling, spray drying, freeze drying and supercritical fluids based techniques enable scientists to design solid dosage forms tailored to possess optimal physicochemical attributes [1,2]. SFD is a rather new method for particle engineering that is a combination of conventional spray-drying with freeze-drying. A typical SFD technique involves the atomization of the solution containing API and excipients via a nozzle into a chamber filled with a cryogenic liquid such as nitrogen, oxygen or argon. The spraying process can be performed beneath (spray-freezing into liquid) or above the surface of the cryogenic liquid, depending on the position of nozzle. The droplets are quickly frozen because of critical low temperature. Once the spraying process is completed, the fro-
⇑ Corresponding author. Tel.: +98 21 66959057. E-mail address: [email protected] (A. Vatanara).
zen suspension in cryogenic liquid is transferred into lyophilizer to obtain the dried particulate powders [3,4]. SFD technique has several advantages compared to freeze drying and spray drying, including: (1) process with no heat is applicable to thermolabile APIs; (2) producing spherical and porous particles with controllable size; (3) minimizing the crystallization and phase separation of drug [5]. These advantages provide a resourceful process to improve the physicochemical characteristics of powders and producing tailored particles. In this study, fine particles containing SX have been produced by SFD process and the effects of different carbohydrates on the physical characteristics of microparticles have been evaluated. SX was chosen as a model drug since it is a long-acting potent b-adrenoceptor agonist used via inhalation to improve lung function, reduce symptoms and provide a better quality of life for patients with asthma. Solubility of SX in water is limited (sparingly soluble) and it made it an appropriate candidate for this study [6]. In such a way, lactose, mannitol and trehalose were employed as bulking agents and HPbCD was applied as a solubilizing agent for dissolution enhancement of drug. The sugars named above can fulfill many of the requirements to reduce damages to delicate molecules during SFD processing and also appear to be suitable as carriers for dry powder aerosols [3,7]. Cyclodextrins have been used extensively as pharmaceutical
0921-8831/$ - see front matter Ó 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved. doi:10.1016/j.apt.2012.01.007
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M.R. Rahmati et al. / Advanced Powder Technology 24 (2013) 36–42
excipients to increase the solubility of poorly water soluble drugs by the formation of an inclusion complex between the host cyclodextrin molecule and the guest drug molecule [8,9]. Materials and methods Materials Salmetreol xinafoate BP was gifted by Jaber Ebne Hayyan Ltd., Iran. Lactose, mannitol, trehalose and HPbCD was purchased from Sigma–Aldrich, USA. Ethanol of analytical grade was purchased from Merck, Germany. Liquid nitrogen was purchased from Sabalan, Iran. Preparation of spray freeze dried powders
Fig. 1. Schematic diagram of spray freezing stage: (A) formulation solution, (B) high pressure pump, (C) liquid nitrogen, and (D) polymeric nozzle.
Particle size analysis of microparticles
Series of solutions containing SX and lactose, mannitol or trehalose were prepared using hydroethanolic solvent (3:1) according to Table 1. SX dissolves into ethanol and the carrier dissolves in water. The concentration of salmeterol was 0.05% in all of the solutions. In the case of solutions containing HPbCD, aqueous solutions of drug and cyclodextrin were prepared and mixed for 24 h. The amount of HPbCD had to be 1% (20 times more than salmeterol concentration). To produce spray freeze dried powder, the feed solution was loaded into the solution cell and then sprayed 10 cm above the surface of 300 ml cryogenic liquid (e.g. liquid nitrogen) through a polyetheretherketone (PEEK) capillary nozzle at the pressure of 400 PSI which was provided by a high pressure pump (Jasco, Japan) with a flow rate of 10 ml/min. Fig. 1 provides a schematic diagram of the spraying set up used in this study. The resulting suspension (frozen droplets of the solution in liquid nitrogen) was transferred into the freeze dryer (Christ, The Netherlands). Vacuum was applied as soon as all nitrogen was evaporated. During the first 24 h, the pressure was set at 0.005 mbar and the shelf temperature at 70 °C. During the second 6 h, the shelf temperature was gradually raised to 20 °C. After removing the samples from the freeze drier, they were stored over silica gel in a desiccator at room temperature. Scanning electron microscopy A Philips Model XL30 scanning electron microscope (Philips, The Netherlands) was used to obtain the SEM photographs. The sample powders were glued onto aluminum stages using double adhesive carbon conducting tape. Particles of representative samples were coated with gold–palladium at room temperature before the examination. The accelerator voltage for scanning was 25.0 kV.
The size distribution of dried powders was measured by laser light scattering using a Malvern Mastersizer X (Malvern Instruments, UK). For particle size analysis, a mass of 10 mg of powder was suspended in 10 ml of pure ethanol and the suspension was then sonicated for 2 min using a water bath sonicator (Starsonic, Italy). Differential scanning calorimetry Thermal behavior of SFD processed particles were studied quantitatively and qualitatively by differential scanning calorimetry using a PL-DSC apparatus (Polymer Laboratories, UK). The samples (5–10 mg) were accurately weighed into standard aluminum pans and sealed. Thermograms were then recorded during heating and cooling runs at a scan rate of 10 °C per minute between 25 and 300 °C. In vitro dissolution testing Dissolution profiles of samples from Run4, Run8 and Run12 formulations were evaluated in comparison with unprocessed SX and physical mixture of drug and HPbCD. In each case, amounts of samples equivalent to 5 mg of drug was prepared for test and the amount of HPbCD was held 20 times more than SX concentration. Drug dissolution was carried out by placing sample in 50 ml freshly prepared deionized water previously heated to 37 °C in a horizontal shaker (Dorsa, Iran) at 100 rpm [10]. Samples of 2 ml were drawn at time interval of 5, 10, 20 and 30 min. Change in volume of solution due to sample withdrawal was considered during concentration determinations. Samples were filtered using 200 nm inline syringe filter (PTFE, 17 mm, Alltech) to remove any suspended particles.
Table 1 Composition of spray freeze dried formulations and the resulted particle sizes. Run no.
Bulking agent
Amount of bulking agent (%)
1 2 3 4 5 6 7 8 9 10 11 12
Lactose Lactose Lactose Lactose Mannitol Mannitol Mannitol Mannitol Trehalose Trehalose Trehalose Trehalose
5 5 10 10 5 5 10 10 5 5 10 10
ND: not determined because of rapid dissolution of powder in ethanol.
Cyclodextrin + + + + + +
d50% (lm) 23.8 ND 22.1 ND 11.1 10.9 17.8 18.9 34.9 23.4 30.8 19.8
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Results
Fig. 2. SEM micrographs of SFD processed particles containing SX and lactose: (A) lactose 5% without cyclodextrin and (B) lactose 10% with cyclodextrin.
Drug concentrations were measured using HPLC analysis (Waters, USA) at 252 nm based on a reported method [11]. The measurements were done in duplicate and averages are reported here.
Different particulate structures containing SX was produced by SFD technique using some sugar based excipients. In brief, lactose, mannitol and trehalose showed different behaviors with and without HPbCD during SFD conditions. Depending on the formulation composition, different particle size distributions were observed where d50% of the samples analyzed by laser diffraction varied from 11.1 to 34.9 lm (Table 1). All samples exhibited mono-modal patterns and the span parameter indicated a broad particle size distribution for these samples, which is attributable to the presence of fractured particles. Applying lactose as bulking agent in formulations of Run1 and Run3 resulted in the formation of highly porous particles with irregular morphologies. However, addition of HPbCD as a solubilizing agent in formulations of Run2 and Run4 produced completely spherical porous structures (Fig. 2). Thermal behaviors of SFD processed structures were evaluated using DSC analysis with reference to unprocessed SX, HPbCD and commercial sugars (Fig. 3). The DSC scan of commercial lactose showed the presence of a-lactose monohydrate with two endothermic peaks at approximately 147 and 216 °C due to the loss of crystal water and melting process, respectively (Fig. 4). These two main endothermic peaks were not detected in thermograms of SFD processed particles containing SX, lactose and HPbCD and it was deduced that during the process, the structure of a-lactose has been changed into amorphous configuration [12–14]. In the case of formulations containing mannitol (Run5–Run8), crystalline particles with asymmetrical morphologies were harvested (Fig. 5). DSC analysis of these samples (Fig. 6) showed a sharp endotherm at 167 °C which is similar to the thermograms of commercial crystalline mannitol [15,16]. On the other hand particles produced from 10% mannitol solutions (Run7 and Run8) were completely needle shaped, compared to 5% mannitol solutions (Run5 and Run6) that did not follow any defined shape. Formulations containing trehalose (Run9–Run12) had a better chance of forming highly porous particles than any other employed bulking agent (Fig. 7). However, formulations with lower concentration (Run9 and Run10) produced enhanced particles in terms of
Fig. 3. DSC thermograms of unprocessed SX (Sal) and cyclodextrin (CYC).
M.R. Rahmati et al. / Advanced Powder Technology 24 (2013) 36–42
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Fig. 4. DSC thermograms of commercial lactose (lac) and SFD processed particles containing SX, lactose and cyclodextrin (Run4).
melting at 192 °C in DSC scan of SFD processed particles (Fig. 8) confirmed these findings. The dissolution rate of SX in the form of unprocessed powder, physical mixture with HPbCD and samples from Run4, Run8 and Run12 were determined in distilled water. The dissolution rate of pure SX was extremely low, with only about 22% of drug released during 30 min of the dissolution run (Fig. 9). The dissolution rate of SX from physical mixture of drug with HPbCD was not markedly increased than that of pure SX. Fig. 9 showed increase (more than 90%) in dissolution rate of SFD processed SX in the presence of HPbCD. Discussion
Fig. 5. SEM micrographs of SFD processed particles containing SX and mannitol: (A) mannitol 5% without cyclodextrin and (B) mannitol 5% with cyclodextrin.
morphology and the presence of cyclodextrin improved the morphology of particles. Trehalose is reported to be in amorphous form in freeze dried dosage forms [17]. Nonappearance of the endothermic peak of
Physical characteristics of SFD processed particles including SX were extensively affected by the type and proportion of excipients. Formulations of Run1 and Run2 containing lactose as bulking agent resulted in the formation of highly porous particles with irregular morphologies. This could be related to the physical structure and crystalline nature of this sugar. Lactose as one of the most commonly employed excipients in pharmaceutical processing can be obtained in either two basic isomeric forms namely, a- and b-lactose or as an amorphous form. a-Lactose exists both as monohydrate and as anhydrous forms, the former being the thermodynamically stable and is harder and more elastic than both b-lactose and anhydrous a-lactose [18,19]. SEM micrographs clearly demonstrated that formulations with lower concentrations of lactose, produced particles with improved morphologies. It could be proposed that in higher concentrations of sugar the higher viscosity of sprayed solutions results in lower tendency of droplets toward formation of spherical particles. In the same way, it could be proposed that the solutions with higher concentrations of lactose have lower freezing points which can provide more time to form non-spherical and irregular structures. The SEM images also revealed that the presence of HPbCD in the formulations provided higher possibility to produce porous and spherical structures, which could be the result of lowered adhesion in the solutions and formation of smaller droplets during spraying phase. Correspondingly, there were no alcohol in the formulations containing HPbCD and this provided higher freezing point for droplets and therefore, less crystallinity and rigidity in the particles.
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M.R. Rahmati et al. / Advanced Powder Technology 24 (2013) 36–42
Fig. 6. DSC thermograms of commercial mannitol (Man) and SFD processed particles containing SX, mannitol and cyclodextrin (Run8).
Fig. 7. SEM micrographs of SFD processed particles containing SX and trehalose: (A) trehalose 10% without cyclodextrin and (B) trehalose 10% with cyclodextrin.
Applying mannitol as bulking agent resulted in the production of crystalline particles with asymmetrical morphologies. Mannitol
is usually supplied as a commercial product in the crystalline form, and several polymorphic forms have been reported in the literature. The numerous polymorphic forms of mannitol that have been described in the literature present a confusing picture as to the number of polymorphs that actually exist. The different polymorphic forms reported 2–7 were examined by Burger et al. who concluded that three polymorphs exist. These were named modification 1 (b), modification 2 (a), and modification 3 (d) in line with Walter-Levy’s classification [20]. Despite rapid freezing of solutions during spraying phase of SFD, mannitol showed tendency to form crystalline structure and referring to DSC thermograms, it was entirely similar to the structures which has been reported for lyophilized products containing this sugar [15,16]. Mannitol in higher concentration produced needle shaped particles which were different from particles resulted from lower concentration of sugar. The reason could be explained according to the concentration of the solutions, where, solutions with 10% mannitol had lower freezing point and therefore they had more time to form elongated structures and the rate of this process was accelerated because of the higher concentration. Evaluation of the dissolution rate of pure SX showed that it dissolved slowly and this might be attributed to poor wettability and particle agglomeration during the run that caused the powder to float on the surface of dissolution medium. Porous particles are expected to show faster dissolution considering the fact that they could dissolve more easily and release the drug more rapidly compared to other particles. Furthermore, presence of cyclodextrin in the composition of produced particles is expected to enhance drug solubility due to its capability of forming inclusion complexes with drug molecule into a hydrophobic cavity area. This molecular encapsulation will increase the solubility, chemical stability, and absorption of the drug [21,22]. Generally, complexation between cyclodextrins and drug molecules need longer times than the period of this dissolution rate study. The presence of HPbCD in SFD processed particles enhanced dissolution rate of SX, where, it could be attributed to the simultaneously effects of porous structure of processed particles and presence of cyclodextrin as a solubilizing enhancer.
M.R. Rahmati et al. / Advanced Powder Technology 24 (2013) 36–42
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Fig. 8. DSC thermograms of commercial trehalose (Tre) and SFD processed particles containing SX and trehalose (Run12).
Fig. 9. Dissolution profile of unprocessed SX, physical mixture of SX and cyclodextrin and SFD processed SX.
Conclusion
Acknowledgments
Particle engineering by SFD technique provides an attractive and efficient procedure to design variety of particulate forms. There are several operational and formulation based parameters in this method which should be studied in order to make it practicable in the field of pharmaceutical processing. In the formation of SX microparticles by this method the type of sugar-based bulking agent and the presence of cyclodextrin showed extensive effects on physical characteristics of precipitated particles. Changing the physical characteristics of SX particles could be a preliminary study to find more efficient particles for drug delivery and particularly for respiratory rout. In this way, evaluation of other operative parameters such as spraying conditions and nozzle properties could be proposed for production of finer particles.
This study was funded and supported by Tehran University of Medical Sciences (TUMS); Grant No. 6365-33-04-86.
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[5] T. Niwa, H. Shimabara, M. Kondo, K. Danjo, Design of porous microparticles with single-micron size by novel spray freeze-drying technique using four-fluid nozzle, International Journal of Pharmaceutics 382 (2009) 88–97. [6] A. Jouyban Gharamaleki, P. York, M. Hanna, B.J. Clark, Solubility prediction of salmeterol xinafoate in water–dioxane mixtures, International Journal of Pharmaceutics 216 (2001) 33–41. [7] G. Pilcer, K. Amighi, Formulation strategy and use of excipients in pulmonary drug delivery, International Journal of Pharmaceutics 392 (2010) 1–19. [8] B. Yang, J. Lin, Y. Chen, Y. Liu, Artemether/hydroxypropyl-b-cyclodextrin host– guest system: characterization, phase-solubility and inclusion mode, Bioorganic and Medicinal Chemistry 17 (2009) 6311–6317. [9] T.H. Hoang Thi, N. Azaroual, M.P. Flament, Characterization and in vitro evaluation of the formoterol/cyclodextrin complex for pulmonary administration by nebulization, European Journal of Pharmaceutics and Biopharmaceutics 72 (2009) 214–218. [10] H. Friedrich, B. Fussnegger, K. Kolter, R. Bodmeier, Dissolution rate improvement of poorly water-soluble drugs obtained by adsorbing solutions of drugs in hydrophilic solvents onto high surface area carriers, European Journal of Pharmaceutics and Biopharmaceutics 62 (2006) 171–177. [11] A. Handoko, L. Ian, S. Peter, Influence of the polydispersity of the added fine lactose on the dispersion of salmeterol xinafoate from mixtures for inhalation, European Journal of Pharmaceutical Sciences 36 (2009) 265–274. [12] M. Mazzobrea, G. Sotob, J. Aguilerab, M. Bueraa, Crystallization kinetics of lactose in systems co-lyofilized with trehalose. Analysis by differential scanning calorimetry, Food Research International 34 (2001) 903–911. [13] A. Gombas, P. Szabo Revesz, M. Kata, G. Regdon Jr., I. Eros, Quantitative determination of crystallinity of alpha-lactose monohydrate by DSC, Journal of Thermal Analysis and Calorimetry 68 (2002) 503–510.
[14] D. Chiou, T.A.G. Langrish, R. Braham, The effect of temperature on the crystallinity of lactose powders produced by spray drying, Journal of Food Engineering 86 (2008) 288–293. [15] L. Bond, S. Allen, M. Davies, C. Roberts, A. Shivji, S. Tendler, P. Williams, J. Zhang, Differential scanning calorimetry and scanning thermal microscopy analysis of pharmaceutical materials, International Journal of Pharmaceutics 243 (2002) 71–82. [16] W. Hulse, R. Forbes, M. Bonner, M. Getrost, The characterization and comparison of spray-dried mannitol samples, Drug Development and Industrial Pharmacy 35 (2009) 712–718. [17] N. Jovanovic, A. Bouchard, G. Hoflandb, G. Witkamp, D. Crommelin, W. Jiskoot, Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying, European Journal of Pharmaceutical Sciences 27 (2006) 336–345. [18] H. Larhrib, X.M. Zeng, G.P. Martin, C. Marriott, J. Pritchard, The use of different grades of lactose as a carrier for aerosolised salbutamol sulphate, International Journal of Pharmaceutics 191 (1999) 1–14. [19] K. Haque, Y. Roos, Crystallization and X-ray diffraction of spray-dried and freeze-dried amorphous lactose, Carbohydrate Research 340 (2005) 293–301. [20] A. Hawe, W. Frieb, Impact of freezing procedure and annealing on the physicochemical properties and the formation of mannitol hydrate in mannitol–sucrose–NaCl formulations, European Journal of Pharmaceutics and Biopharmaceutics 64 (2006) 316–325. [21] J. Wang, Y. Cao, B. Sun, C. Wang, Characterisation of inclusion complex of trans-ferulic acid and hydroxypropyl-b-cyclodextrin, Food Chemistry 124 (2011) 1069–1075. [22] Z. Wang, Y. Deng, S. Sun, X. Zhang, Preparation of hydrophobic drugs cyclodextrin complex by lyophilization monophase solution, Drug Development and Industrial Pharmacy 32 (2006) 73–83.
Contents lists available at SciVerse ScienceDirect
Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
Original Research Paper
Effect of formulation ingredients on the physical characteristics of salmeterol xinafoate microparticles tailored by spray freeze drying Mohammad Reza Rahmati, Alireza Vatanara ⇑, Ahmad Reza Parsian, Kambiz Gilani, Khosrow Malek Khosravi, Majid Darabi, Abdolhossein Rouholamini Najafabadi Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 4 October 2011 Received in revised form 30 December 2011 Accepted 11 January 2012 Available online 26 January 2012 Keywords: Salmeterol Spray freeze drying Cyclodextrin Dissolution
a b s t r a c t Series of microparticles containing salmeterol xinafoate (SX) as active pharmaceutical ingredient (API) and lactose, mannitol or trehalose as a bulking agents were prepared using spray freeze drying (SFD) technique and the effects of sugar type and presence of hydroxy propyl beta cyclodextrin (HPbCD) on the physical properties of powders were evaluated. Precipitation of salmeterol in the presence of lactose and mannitol resulted in the formation of irregular shapes of microparticles with broad size distributions. However application of trehalose resulted in the formation of porous particles with spherical morphology. Addition of cyclodextrin in the formulations was generally helpful for formation of porous and spherical particles with narrow size distribution with a mean size of 10–30 lm. Dissolution of SX from processed particles was substantially higher (90% drug release in 30 min) than that of unprocessed drug and physical mixture of drug and cyclodextrin (22% drug release in 30 min). This study showed that, processing of SX by SFD technique could be a constructive approach to the production of various forms of drug and drastic changes in the physical characteristics of microparticles could be achieved by changing the composition of bulking agent and cyclodextrin. Ó 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
Introduction The efficacy of pharmaceuticals can be significantly influenced by their physicochemical properties such as particle size distribution, morphology, powder flow, compression characteristics, physical stability and bioavailability. Fine pharmaceutical powders are often produced by currently available micronization methods that allow particle engineering. Processes such as air jet milling, spray drying, freeze drying and supercritical fluids based techniques enable scientists to design solid dosage forms tailored to possess optimal physicochemical attributes [1,2]. SFD is a rather new method for particle engineering that is a combination of conventional spray-drying with freeze-drying. A typical SFD technique involves the atomization of the solution containing API and excipients via a nozzle into a chamber filled with a cryogenic liquid such as nitrogen, oxygen or argon. The spraying process can be performed beneath (spray-freezing into liquid) or above the surface of the cryogenic liquid, depending on the position of nozzle. The droplets are quickly frozen because of critical low temperature. Once the spraying process is completed, the fro-
⇑ Corresponding author. Tel.: +98 21 66959057. E-mail address: [email protected] (A. Vatanara).
zen suspension in cryogenic liquid is transferred into lyophilizer to obtain the dried particulate powders [3,4]. SFD technique has several advantages compared to freeze drying and spray drying, including: (1) process with no heat is applicable to thermolabile APIs; (2) producing spherical and porous particles with controllable size; (3) minimizing the crystallization and phase separation of drug [5]. These advantages provide a resourceful process to improve the physicochemical characteristics of powders and producing tailored particles. In this study, fine particles containing SX have been produced by SFD process and the effects of different carbohydrates on the physical characteristics of microparticles have been evaluated. SX was chosen as a model drug since it is a long-acting potent b-adrenoceptor agonist used via inhalation to improve lung function, reduce symptoms and provide a better quality of life for patients with asthma. Solubility of SX in water is limited (sparingly soluble) and it made it an appropriate candidate for this study [6]. In such a way, lactose, mannitol and trehalose were employed as bulking agents and HPbCD was applied as a solubilizing agent for dissolution enhancement of drug. The sugars named above can fulfill many of the requirements to reduce damages to delicate molecules during SFD processing and also appear to be suitable as carriers for dry powder aerosols [3,7]. Cyclodextrins have been used extensively as pharmaceutical
0921-8831/$ - see front matter Ó 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved. doi:10.1016/j.apt.2012.01.007
37
M.R. Rahmati et al. / Advanced Powder Technology 24 (2013) 36–42
excipients to increase the solubility of poorly water soluble drugs by the formation of an inclusion complex between the host cyclodextrin molecule and the guest drug molecule [8,9]. Materials and methods Materials Salmetreol xinafoate BP was gifted by Jaber Ebne Hayyan Ltd., Iran. Lactose, mannitol, trehalose and HPbCD was purchased from Sigma–Aldrich, USA. Ethanol of analytical grade was purchased from Merck, Germany. Liquid nitrogen was purchased from Sabalan, Iran. Preparation of spray freeze dried powders
Fig. 1. Schematic diagram of spray freezing stage: (A) formulation solution, (B) high pressure pump, (C) liquid nitrogen, and (D) polymeric nozzle.
Particle size analysis of microparticles
Series of solutions containing SX and lactose, mannitol or trehalose were prepared using hydroethanolic solvent (3:1) according to Table 1. SX dissolves into ethanol and the carrier dissolves in water. The concentration of salmeterol was 0.05% in all of the solutions. In the case of solutions containing HPbCD, aqueous solutions of drug and cyclodextrin were prepared and mixed for 24 h. The amount of HPbCD had to be 1% (20 times more than salmeterol concentration). To produce spray freeze dried powder, the feed solution was loaded into the solution cell and then sprayed 10 cm above the surface of 300 ml cryogenic liquid (e.g. liquid nitrogen) through a polyetheretherketone (PEEK) capillary nozzle at the pressure of 400 PSI which was provided by a high pressure pump (Jasco, Japan) with a flow rate of 10 ml/min. Fig. 1 provides a schematic diagram of the spraying set up used in this study. The resulting suspension (frozen droplets of the solution in liquid nitrogen) was transferred into the freeze dryer (Christ, The Netherlands). Vacuum was applied as soon as all nitrogen was evaporated. During the first 24 h, the pressure was set at 0.005 mbar and the shelf temperature at 70 °C. During the second 6 h, the shelf temperature was gradually raised to 20 °C. After removing the samples from the freeze drier, they were stored over silica gel in a desiccator at room temperature. Scanning electron microscopy A Philips Model XL30 scanning electron microscope (Philips, The Netherlands) was used to obtain the SEM photographs. The sample powders were glued onto aluminum stages using double adhesive carbon conducting tape. Particles of representative samples were coated with gold–palladium at room temperature before the examination. The accelerator voltage for scanning was 25.0 kV.
The size distribution of dried powders was measured by laser light scattering using a Malvern Mastersizer X (Malvern Instruments, UK). For particle size analysis, a mass of 10 mg of powder was suspended in 10 ml of pure ethanol and the suspension was then sonicated for 2 min using a water bath sonicator (Starsonic, Italy). Differential scanning calorimetry Thermal behavior of SFD processed particles were studied quantitatively and qualitatively by differential scanning calorimetry using a PL-DSC apparatus (Polymer Laboratories, UK). The samples (5–10 mg) were accurately weighed into standard aluminum pans and sealed. Thermograms were then recorded during heating and cooling runs at a scan rate of 10 °C per minute between 25 and 300 °C. In vitro dissolution testing Dissolution profiles of samples from Run4, Run8 and Run12 formulations were evaluated in comparison with unprocessed SX and physical mixture of drug and HPbCD. In each case, amounts of samples equivalent to 5 mg of drug was prepared for test and the amount of HPbCD was held 20 times more than SX concentration. Drug dissolution was carried out by placing sample in 50 ml freshly prepared deionized water previously heated to 37 °C in a horizontal shaker (Dorsa, Iran) at 100 rpm [10]. Samples of 2 ml were drawn at time interval of 5, 10, 20 and 30 min. Change in volume of solution due to sample withdrawal was considered during concentration determinations. Samples were filtered using 200 nm inline syringe filter (PTFE, 17 mm, Alltech) to remove any suspended particles.
Table 1 Composition of spray freeze dried formulations and the resulted particle sizes. Run no.
Bulking agent
Amount of bulking agent (%)
1 2 3 4 5 6 7 8 9 10 11 12
Lactose Lactose Lactose Lactose Mannitol Mannitol Mannitol Mannitol Trehalose Trehalose Trehalose Trehalose
5 5 10 10 5 5 10 10 5 5 10 10
ND: not determined because of rapid dissolution of powder in ethanol.
Cyclodextrin + + + + + +
d50% (lm) 23.8 ND 22.1 ND 11.1 10.9 17.8 18.9 34.9 23.4 30.8 19.8
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Results
Fig. 2. SEM micrographs of SFD processed particles containing SX and lactose: (A) lactose 5% without cyclodextrin and (B) lactose 10% with cyclodextrin.
Drug concentrations were measured using HPLC analysis (Waters, USA) at 252 nm based on a reported method [11]. The measurements were done in duplicate and averages are reported here.
Different particulate structures containing SX was produced by SFD technique using some sugar based excipients. In brief, lactose, mannitol and trehalose showed different behaviors with and without HPbCD during SFD conditions. Depending on the formulation composition, different particle size distributions were observed where d50% of the samples analyzed by laser diffraction varied from 11.1 to 34.9 lm (Table 1). All samples exhibited mono-modal patterns and the span parameter indicated a broad particle size distribution for these samples, which is attributable to the presence of fractured particles. Applying lactose as bulking agent in formulations of Run1 and Run3 resulted in the formation of highly porous particles with irregular morphologies. However, addition of HPbCD as a solubilizing agent in formulations of Run2 and Run4 produced completely spherical porous structures (Fig. 2). Thermal behaviors of SFD processed structures were evaluated using DSC analysis with reference to unprocessed SX, HPbCD and commercial sugars (Fig. 3). The DSC scan of commercial lactose showed the presence of a-lactose monohydrate with two endothermic peaks at approximately 147 and 216 °C due to the loss of crystal water and melting process, respectively (Fig. 4). These two main endothermic peaks were not detected in thermograms of SFD processed particles containing SX, lactose and HPbCD and it was deduced that during the process, the structure of a-lactose has been changed into amorphous configuration [12–14]. In the case of formulations containing mannitol (Run5–Run8), crystalline particles with asymmetrical morphologies were harvested (Fig. 5). DSC analysis of these samples (Fig. 6) showed a sharp endotherm at 167 °C which is similar to the thermograms of commercial crystalline mannitol [15,16]. On the other hand particles produced from 10% mannitol solutions (Run7 and Run8) were completely needle shaped, compared to 5% mannitol solutions (Run5 and Run6) that did not follow any defined shape. Formulations containing trehalose (Run9–Run12) had a better chance of forming highly porous particles than any other employed bulking agent (Fig. 7). However, formulations with lower concentration (Run9 and Run10) produced enhanced particles in terms of
Fig. 3. DSC thermograms of unprocessed SX (Sal) and cyclodextrin (CYC).
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Fig. 4. DSC thermograms of commercial lactose (lac) and SFD processed particles containing SX, lactose and cyclodextrin (Run4).
melting at 192 °C in DSC scan of SFD processed particles (Fig. 8) confirmed these findings. The dissolution rate of SX in the form of unprocessed powder, physical mixture with HPbCD and samples from Run4, Run8 and Run12 were determined in distilled water. The dissolution rate of pure SX was extremely low, with only about 22% of drug released during 30 min of the dissolution run (Fig. 9). The dissolution rate of SX from physical mixture of drug with HPbCD was not markedly increased than that of pure SX. Fig. 9 showed increase (more than 90%) in dissolution rate of SFD processed SX in the presence of HPbCD. Discussion
Fig. 5. SEM micrographs of SFD processed particles containing SX and mannitol: (A) mannitol 5% without cyclodextrin and (B) mannitol 5% with cyclodextrin.
morphology and the presence of cyclodextrin improved the morphology of particles. Trehalose is reported to be in amorphous form in freeze dried dosage forms [17]. Nonappearance of the endothermic peak of
Physical characteristics of SFD processed particles including SX were extensively affected by the type and proportion of excipients. Formulations of Run1 and Run2 containing lactose as bulking agent resulted in the formation of highly porous particles with irregular morphologies. This could be related to the physical structure and crystalline nature of this sugar. Lactose as one of the most commonly employed excipients in pharmaceutical processing can be obtained in either two basic isomeric forms namely, a- and b-lactose or as an amorphous form. a-Lactose exists both as monohydrate and as anhydrous forms, the former being the thermodynamically stable and is harder and more elastic than both b-lactose and anhydrous a-lactose [18,19]. SEM micrographs clearly demonstrated that formulations with lower concentrations of lactose, produced particles with improved morphologies. It could be proposed that in higher concentrations of sugar the higher viscosity of sprayed solutions results in lower tendency of droplets toward formation of spherical particles. In the same way, it could be proposed that the solutions with higher concentrations of lactose have lower freezing points which can provide more time to form non-spherical and irregular structures. The SEM images also revealed that the presence of HPbCD in the formulations provided higher possibility to produce porous and spherical structures, which could be the result of lowered adhesion in the solutions and formation of smaller droplets during spraying phase. Correspondingly, there were no alcohol in the formulations containing HPbCD and this provided higher freezing point for droplets and therefore, less crystallinity and rigidity in the particles.
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Fig. 6. DSC thermograms of commercial mannitol (Man) and SFD processed particles containing SX, mannitol and cyclodextrin (Run8).
Fig. 7. SEM micrographs of SFD processed particles containing SX and trehalose: (A) trehalose 10% without cyclodextrin and (B) trehalose 10% with cyclodextrin.
Applying mannitol as bulking agent resulted in the production of crystalline particles with asymmetrical morphologies. Mannitol
is usually supplied as a commercial product in the crystalline form, and several polymorphic forms have been reported in the literature. The numerous polymorphic forms of mannitol that have been described in the literature present a confusing picture as to the number of polymorphs that actually exist. The different polymorphic forms reported 2–7 were examined by Burger et al. who concluded that three polymorphs exist. These were named modification 1 (b), modification 2 (a), and modification 3 (d) in line with Walter-Levy’s classification [20]. Despite rapid freezing of solutions during spraying phase of SFD, mannitol showed tendency to form crystalline structure and referring to DSC thermograms, it was entirely similar to the structures which has been reported for lyophilized products containing this sugar [15,16]. Mannitol in higher concentration produced needle shaped particles which were different from particles resulted from lower concentration of sugar. The reason could be explained according to the concentration of the solutions, where, solutions with 10% mannitol had lower freezing point and therefore they had more time to form elongated structures and the rate of this process was accelerated because of the higher concentration. Evaluation of the dissolution rate of pure SX showed that it dissolved slowly and this might be attributed to poor wettability and particle agglomeration during the run that caused the powder to float on the surface of dissolution medium. Porous particles are expected to show faster dissolution considering the fact that they could dissolve more easily and release the drug more rapidly compared to other particles. Furthermore, presence of cyclodextrin in the composition of produced particles is expected to enhance drug solubility due to its capability of forming inclusion complexes with drug molecule into a hydrophobic cavity area. This molecular encapsulation will increase the solubility, chemical stability, and absorption of the drug [21,22]. Generally, complexation between cyclodextrins and drug molecules need longer times than the period of this dissolution rate study. The presence of HPbCD in SFD processed particles enhanced dissolution rate of SX, where, it could be attributed to the simultaneously effects of porous structure of processed particles and presence of cyclodextrin as a solubilizing enhancer.
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Fig. 8. DSC thermograms of commercial trehalose (Tre) and SFD processed particles containing SX and trehalose (Run12).
Fig. 9. Dissolution profile of unprocessed SX, physical mixture of SX and cyclodextrin and SFD processed SX.
Conclusion
Acknowledgments
Particle engineering by SFD technique provides an attractive and efficient procedure to design variety of particulate forms. There are several operational and formulation based parameters in this method which should be studied in order to make it practicable in the field of pharmaceutical processing. In the formation of SX microparticles by this method the type of sugar-based bulking agent and the presence of cyclodextrin showed extensive effects on physical characteristics of precipitated particles. Changing the physical characteristics of SX particles could be a preliminary study to find more efficient particles for drug delivery and particularly for respiratory rout. In this way, evaluation of other operative parameters such as spraying conditions and nozzle properties could be proposed for production of finer particles.
This study was funded and supported by Tehran University of Medical Sciences (TUMS); Grant No. 6365-33-04-86.
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