International Journal of Pharmaceutics 478 (2015) 288–296

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Design of salmon calcitonin particles for nasal delivery using spray-drying and novel supercritical fluid-assisted spray-drying processes Wonkyung Cho a,b,1, Min-Soo Kim c,1, Min-Sook Jung b , Junsung Park a,b , Kwang-Ho Cha a,b , Jeong-Soo Kim b , Hee Jun Park b , Amjad Alhalaweh d, Sitaram P. Velaga d, ** , Sung-Joo Hwang a, * a

College of Pharmacy, Yonsei University, 162-1 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea College of Pharmacy, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, Republic of Korea c College of Pharmacy, Pusan National University, Busandaehak-ro 63 Beon-gil, Geumjeong-gu, Busan 609-735, Republic of Korea d Department of Health Science, Luleå University of Technology, Luleå S-971 87, Sweden b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 2 July 2014 Received in revised form 3 November 2014 Accepted 22 November 2014 Available online 25 November 2014

The overall aim of this study was to prepare a nasal powder formulation of salmon calcitonin (sCT) using an absorption enhancer to improve its bioavailability. In this work, powder formulations for nasal delivery of sCT were studied using various absorption enhancers and stabilizers. Powders were prepared by two different methods: conventional spray-drying (SD) and novel supercritical fluid-assisted spraydrying (SASD) to investigate the role of CO2 in the particle formation process. The prepared sCT powder formulations were characterized by several analyses; powder X-ray diffractometry (PXRD), scanning electron microscopy (SEM), and the Fourier transform infrared (FT-IR) spectroscopy method. The particle size distribution was also evaluated. In vivo absorption tests were carried out in Sprague-Dawley rat using the prepared powder formulations, and the results were compared to those of raw sCT. Quantitative analysis by high-performance liquid chromatography (HPLC) indicated that sCT was chemically stable after both the SD and SASD processes. Results of PXRD, SEM, and FT-IR did not indicate a strong interaction or defragmentation of sCT. The in vivo absorption test showed that SD- and SASD-processed sCT powders increased the bioavailability of the drug when compared to the nasal administration of raw sCT. In addition, SASD-processed sCT exhibited higher nasal absorption when compared with SDprocessed sCT in all formulations due to a reduction of particle size. The results from this study illustrate that the preparation of nasal powders using the SASD process could be a promising approach to improve nasal absorption of sCT. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Salmon calcitonin Supercritical fluid Nanoparticle Nasal Formulation

1. Introduction Biopharmaceuticals such as peptides and proteins are highly potent and selective drugs with a wide range of therapeutic applications and have been rapidly developing in the last two decades (Frokjaer and Otzen, 2005; Walsh, 2005). Due to the peculiar properties of peptide and protein drugs that include large molecular size and susceptibility to enzymatic degradation, most

* Corresponding author. Tel.: +82 32 749 4518. ** Corresponding author. Tel.: +46 920 493924. E-mail addresses: [email protected] (S.P. Velaga), [email protected] (S.-J. Hwang). 1 Co-first author: Wonkyung Cho, Min-Soo Kim. http://dx.doi.org/10.1016/j.ijpharm.2014.11.051 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

of these biopharmaceuticals are mainly administered via injections for systemic effect. Injections are not always the desired dosage form due to patient compliance, safety and disease management; therefore, safe and effective non-invasive administration routes are required as an alternative to injection. Among the non-invasive routes, nasal administration offers promising potential as a viable alternative for the delivery of some peptide drugs. The nasal epithelium has a relatively high permeability, and only two cell layers separate the nasal lumen from the dense blood vessel network in the lamina propria. Some of the major advantages offered by nasal delivery include rapid absorption, high bioavailability, fast onset of therapeutic action, avoidance of hepatic first pass metabolism, reduced risk of overdose, improved patient compliance and ease of administration (Dondeti et al., 1996). However, most peptides and proteins are not

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well absorbed from the nasal cavity when administered as a simple solution. Limiting factors of nasal drug absorption are (i) the enzymatic barrier of the nasal mucosa, (ii) the physical barrier of the epithelium, (iii) the rapid mucociliary clearance limiting the time available for absorption, and (iv) the mucus layer itself (Schmidt et al., 1998). Thus, intranasal formulation development requires special attention to bioavailability, which can be improved by absorption enhancers such as bile salts, fusidate derivatives, fatty acids, surfactants or cyclodextrin (Schipper et al., 1993b, 1995b; Shao and Mitra, 1992). Another key factor for nasal delivery is the dosage form, i.e., liquid or powder. The solid form of the active pharmaceutical ingredient (API) is preferable since it provides good stability, comes in a small packaging size and is easy to handle in comparison to liquid forms (Marttin et al., 1997). In fact, some previous studies showed that the powder forms have better chemical stability and enhanced nasal absorption (Ishikawa et al., 2002; Matsuyama et al., 2006; Schipper et al., 1993a). Protein and peptide drug powders can be prepared by several methods. Spray-drying (SD) is a useful and widely applied method to prepare dry protein and peptide particles, where microparticles and nanoparticles can be formed directly from a drug solution with greater control over particle size, morphology and powder density (Johnson, 1997; Ståhl et al., 2002; Vehring, 2008). In addition, the application of supercritical fluids (SCFs) to particle design has emerged as a promising technique for producing powders for inhalation (Moshashaée et al., 2003a,b,b; Okamoto and Danjo, 2008; Reverchon et al., 2007b). SCFs can be defined as a substance existing as a single fluid phase above its critical temperature and pressure. The particle preparation process of SCFs can take advantage of some specific properties of gases at supercritical conditions, like the modulation of the solubilization power, large diffusivities, solventless or organic solvent-reduced operation and the consequent possibility of controlling powder sizes and distribution. In particular, supercritical carbon dioxide (SC-CO2) is widely used to prepare powder formulations of biopharmaceuticals because of its mild process conditions and non-flammable, non-toxic and inexpensive properties. The techniques proposed are the rapid expansion of the supercritical solution (RESS), the aerosol solvent extraction system (ASES) and supercritical anti-solvent precipitation (SAS). However, in many previous studies, the solubility of many peptides and proteins in SC-CO2 is relatively low, and thus SC-CO2 was used as an anti-solvent for the precipitation of proteins and peptides (Chattopadhyay and Gupta, 2002; Jung and Perrut, 2001; Moshashaée et al., 2003a,b; Winters et al., 1997). For successful SAS precipitation, however, the complete miscibility of the liquid in the SC-CO2 and the insolubility of the solute in it are essential. For this reason, SAS often uses organic solvents such as dimethylsulfoxide, which might cause perturbation of the secondary structure and is not applicable to the water solution of proteins and peptides due to the very low solubility of water in CO2 (Thiering et al., 2000). Recently, supercritical carbon dioxide-assisted atomization techniques were proposed: carbon dioxide-assisted nebulization with a bubble dryer (CAN-BD) and supercritical assisted atomization (SAA) (Reverchon et al., 2007a; Sievers et al., 2003). Based on these theories, a novel supercritical fluid-assisted spray-drying (SASD) process was developed by Hwang’s group as a valid alternative technique to the conventional SD process and SAS process for preparation of nanoparticles (Hwang et al., 2009). In SASD process, SC-CO2 acts both as a co-solvent being partially miscible with solvent to be treated, as well as a pneumatic agent to atomize the solution into fine particles. Therefore, it allows the micronization of the compound from an aqueous solution as well as a water-organic solvent mixture.

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Salmon calcitonin (sCT) is a polypeptide hormone comprised of 32 amino acids with a molecular weight of 3431.88 Da, with a single disulfide bond (Cys1—Cys7) at the NH2-terminus. It is currently formulated as a sterile solution for intramuscular or subcutaneous injection and as a nasal spray in the management of several bone-related diseases including Paget’s disease, hypercalcemia and osteoporosis (e.g., Miacalcic1). It is reported that the bioavailability of market nasal salmon calcitonin is about 3%. Using an absorption enhancer has shown promising results in improving systematic bioavailability of sCT by in vivo study on animals and humans via nasal administration (Gordon et al., 1985; Matsuyama et al., 2006; Schipper et al., 1995a). There are a few studies of the dry powder formulation of sCT using the SD process. These studies showed that the physiochemical stability of sCT powder was not affected during the SD process (Chan et al., 2004; Yang et al., 2007a). However, particle design and formulation of sCT with proper additives using the SD and SCFs processes for nasal delivery with the aim of increasing permeability, bioavailability and maximizing chemical and physical stability have not been systematically investigated. Thus, the objectives of this study were: (i) preparation of sCT nasal powder formulation using the SD and SASD processes with proper additives to improve penetration and bioavailability and (ii) evaluation of physicochemical characteristics and in vivo bioavailability of sCT powders from the SD and SASD processes. 2. Materials and method 2.1. Materials sCT was kindly supplied by Polypeptide Laboratories A/S (Hillerod, Denmark). Inulin (Frutafit1 TEX) was gifted from Sensus (Roosendaal, Netherlands). Trehalose, sodium taurocholate hydrate and heptakis ((2,6-di-o-methyl)-b-cyclodextrin) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Chitosan glutamate [Protasan UP G 213; Mw: 200–600 kDa, DD (degree of deacetylation): 75–90%] was purchased from Nova Matrix/FMC Biopolymer (Sandvika, Norway). Carbon dioxide (CO2, purity 99.9%) was purchased from Hanmi Gas Co., Ltd. (Korea). All organic solvents were of HPLC grade. All other chemicals in this study were analytical reagent grade. 3. Powder preparation 3.1. Powder preparation using the SD process Aqueous solutions (total concentration: 10 mg/ml) of sCT, absorption enhancer, and stabilizer in different weight ratios were prepared as described in Table 1. Inulin and trehalose were used as stabilizers; chitosan, sodium taurocholate, and betacyclodextrin were used as absorption enhancers. The SD process was carried out using a laboratory-scale spray dryer (Mini Spray Table 1 Formulations of sCT powders. Quantity (mg) per formulation sCT Stablizer

Absorption enhancer

Inulin Trehalose Chitosan Sodium taurocholate F1 F2 F3 F4 F5 F6

50 50 50 50 50 50

400 400 400 – – –

– – – 400 400 400

50 – – 50 – –

– 50 – – 50 –

Beta-cyclodextrin – – 50 – – 50

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Fig. 1. Schematic representation of the SASD process.

Dryer B-290; Büchi, Switzerland) at fixed processing conditions (feed rate: 5 ml/min, atomizing air: 357 l/h, aspirator rate: 80%, inlet temperature: 140
C, outlet temperature: 70
C). 3.2. Powder preparation using SASD process Fig. 1 shows the apparatus used for the SASD process. It is composed of three main feed lines for SC-CO2, the drug solution and hot air and four main chambers for preheating, mixing, evaporation and cyclone action, respectively. Liquid CO2 in the storage tank was pumped to preheating vessel by homemade high pressure plunger pump covered with a cooling jacket. The heated CO2 was introduced to the mixing chamber where it was mixed with the drug solution. The liquid solution was heated and delivered to the mixing chamber by an HPLC pump (P1024B; Chrom Tech Inc., USA). The mixing chamber was loaded with glass beads (diameter 1.5 mm, to provide a large contacting surface between the CO2 and drug solution in order to sufficiently increase residential time to dissolve the SC-CO2 in the drug solution. Once the pressure and temperature had equilibrated using solute-free solvent and SC-CO2, the mixture of the SC-CO2 and drug solution was co-introduced into the evaporator chamber with compressed hot air through the two-flow coaxial spray nozzle (order made, Hanwoul Engineering Inc., Korea), in order to produce a spray of liquid droplets. The diameter of inner nozzle was 0.1 mm. The evaporator chamber was a Pyrex glass vessel operating at near atmospheric pressure. It also received a flow of heated air in order to evaporate the solvent from the droplet and to precipitate the solute in the form of small particles. The formed particles were moved to the cyclone and collected at a cylindrical filter which was wrapped with 200 mesh stainless steel screen, whereas the gaseous stream passed through the filter and was aspirated by an aspiration pump (aspiration rate; 800 l/min; WooSung Vacuum, Korea).

The same formulation of sCT, absorption enhancer, and stabilizer which was prepared in the SD process was dissolved in an ethanol:water (8:2) mixture (total weight 10 mg/ml). The SASD-processed particles were obtained by injecting the solutions into the SASD apparatus at fixed processing conditions (solution feed rate: 5 ml/min, CO2 feed rate: 30 g/min, mixing chamber pressure: 85 bar, mixing chamber temperature: 40
C, blower: 0.8 m3/min, inlet temperature: 140
C, outlet temperature: 50
C, compressed hot air pressure: 10 kPa). 4. Chemical and physical characterizations of the particles 4.1. High performance liquid chromatography (HPLC) analysis of sCT chemical stability About 1.0 mg of powder was weighed into a 1.5 ml Eppendorf tube and mixed with 1 ml of acetate buffer (pH 4.4, 50 mM). The HPLC system consisted of two pumps (LC-10ATvp; Shimadzu Ltd., Japan), a controller (SCL-10Avp; Shimadzu Ltd., Japan), a UV detector (SPD-10Avp; Shimadzu Ltd., Japan), and a column oven (CTO-ASvp; Shimadzu Ltd., Japan). An ODS C18 column (XTerraRP18, 5 mm, 4.6  250 mm; Waters, USA) was used at 25
C. A linear gradient method with following composition and settings was used; mobile phase A was decreased 70–45% in 25 min and then increased back to 70% in 5 min at a flow rate of 1.5 ml/min. The mobile phase A was 0.1% trifluoroacetic acid (TFA) in distilled water, and mobile phase B was 0.1% TFA in acetonitrile. Twenty microliters of sample were injected for each analysis, and the signal was monitored at 220 nm (n = 3). 4.2. Scanning electron microscopy (SEM) The morphology of the particles was examined by scanning electron microscopy (SEM, JSM-6300; Jeol Ltd., Japan). Samples

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were coated with gold and palladium using a vacuum evaporator and examined using a SEM at 15 kV accelerating voltage. 4.3. Particle size analysis The particle sizes of unprocessed sCT and SD-processed sCT particles were determined with a HELOS laser diffraction analyzer (Sympatec, Gmbh, Germany) equipped with a RODOS vibrating trough disperser. An air pressure of 0.1 MPa and vacuum of 5.3 kPa were used to produce a uniform powder dispersion for each sample. The size and size distribution of the SASD-processed sCT samples were measured by dynamic light scattering (DLS) (ELS-8000; Otsuka Electronics, Japan) with a He–Ne laser beam at a wavelength of 658 nm at room temperature and a scattering angle of 90
. To obtain complete dispersion of primary particles, the SASD-processed sCT particles were dispersed in mineral oil and sonicated for 2 h (frequency: 40 kHz, Branson 8210; Branson Ultrasonics Co., Danbury, CT, USA). 4.4. Powder X-ray diffractometry (PXRD) PXRD patterns were recorded on a Rigaku Powder X-ray diffraction system (Model D/MAX-2200; Ultima/PC, Japan) with Ni-filtered Cu-Ka radiation. The samples were run over the most informative range from 3
to 40
of 2u. The step scan mode was performed with a step size of 0.02
at a rate of 1
/min.

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solution (corresponding 0.1 mg of sCT) was administered into the nasal cavity using a syringe with a flexible needle. For in vivo study, three rats were used for each group. Serial blood samples (approximately 500 ml) were taken via a jugular vein catheter at pre-dose and 5, 10, 20, 30, 45, 60, 90, 120 min post-dose. The blood samples were held on ice (4
C) until centrifuged at 10,000 rpm, 4
C for 10 min. Separated serum was transferred to individual Eppendorf tubes and stored at 80
C until analyzed. 5.2. Assay of serum concentration sCT serum concentration was measured using an enzyme immunoassay kit (Peninsula Laboratories, CA, USA), an enzymatically amplified “two-step” sandwich-ELISA involving the biotinstreptavidin bridging detection system. In this assay, standards and samples were incubated with biotinylated anti-sCT antiserum in microtitration wells coated with another anti-sCT antibody. After incubation and washing, the wells were incubated with streptavidin labeled with the enzyme horseradish peroxidase. After a second incubation and a washing step, the wells were incubated with tetramethylbenzidine substrate. A stock solution was then added and the degree of enzymatic turnover of substrate was determined by reading the absorbance measurement at 650 (blue color) or 450 (yellow color). The absorbance measured is directly proportional to the concentration of sCT present. The working concentration range of sCT for the ELISA were 0.02–25 ng/ml. 5.3. Pharmacokinetic data analysis

4.5. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra were measured using a Nicolet 380 FT-IR spectrometer (Thermo Fisher, USA) using an attenuated total reflectance method. The scanning range was 650–4000 cm1 and the resolution was 4 cm1. The number of reference scans was 128. 5. In vivo animal study 5.1. Rat study The rat in vivo experiment was performed according to Hirai’s method (Hirai and Mima, 1981) with minor modification. Male Sprague-Dawley rats weighing between 300–350 g were obtained from Samtaco Bio Korea Inc. (Korea). The rats had free access to tap water and a pelleted diet. The rats were housed in a cage and maintained at 12 h light/dark at room temperature (25
C) and a relative humidity of 55  10%. General and environmental conditions were strictly monitored. All animal experiments were performed according to the “Guideline for the Care and Use of Laboratory Animals.” of Chungnam National University. The rats were deprived of food for 20 h before the experiment, anesthetized by intraperitoneal injection of urethane (720 mg/kg) and chloralose (65 mg/kg) and secured on their backs on a board during the experiments. The rats were then tracheotomized, and the outlets of the nasal cavity were sealed by a blind-ended cannula placed in the esophagus to prevent drainage of the drug into the esophagus or trachea and thus avoid overestimation of drug absorption. One milligram of the inulin-formulated nasal powder (corresponding to 0.1 mg of sCT) was administrated into the nasal cavity as previously reported by Todo et al. (2001) with minor modification. In brief, the dry powder was put into the body of a flexible needle and dispersed in the rat nasal cavity by releasing the compressed air in a syringe via opening a three-way stopcock connecting the needle with the syringe. The amount of dry powder administered was calculated by subtracting the needle weight after administration from that measured before administration. For liquid commercial product (Miacalcic1 nasal spray, Novartis), 270 ml of

The area under the curve to the last measurable concentration (AUC0 ! 120) was calculated using BA Calc 2007 software (presented by the National Institute of Food and Drug Safety Evaluation of Korea for pharmacokinetic study). The maximal plasma concentration of drug (Cmax) and time to reach maximum plasma concentration (Tmax) were directly obtained from the plasma data. The elimination rate constant (K) of plasma sCT was calculated from the linear regression of semi-logarithmic plot. The half-life (t1/2) was calculated according to following equation: t1=2 ¼

0:693 K

A one-way analysis of variance (ANOVA) test followed by Tukey’s HSD test were performed to demonstrate statistical difference, using SPSS for Windows standard version 12.0 (SPSS, Chicago, IL, USA). Differences between two related parameters were considered statistically significant at p < 0.05. 6. Results and discussion 6.1. Chemical stability of sCT after SD and SASD process The RP-HPLC was a useful tool to evaluate the stability of sCT in various previous studies (Stevenson and Tan, 2000; Yang et al., 1998, 2007a). The chemical integrity of sCTs during the SD and SASD processes was studied by RP-HPLC. Fig. 2 shows that sCT could be recovered completely in the formulation in acetate buffer (pH 4.4). These results indicate that sCT was stable during the SD and SASD processes. When a sCT solution is processed in compressed CO2 to form a precipitate, the sCT is exposed to high pressure, about 100 bar. In general, pressure at this range does not cause irreversible unfolding of protein or peptide (Okamoto and Danjo, 2008; Randolph et al., 1988). In a previous study, it was reported that increases in the proportion of chitosan negatively affected the recovery of sCT powders (Yang et al., 2007b). However, in this study, we confirmed

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microparticles (Fig. 3(d) and (e)). The SASD process resulted in particles that were very spherical, with non-coalescing nanoparticles. The inulin formulation (Fig. 3(g)) showed a smoother surface than the trehalose formulation. The particle size and particle size distribution of SD-processed and SASD-processed particles using chitosan as the absorption enhancer were measured by HELOS and DLS, respectively. The results are summarized in Table 2 and Fig. 4. We failed to measure particle size and distribution of the unprocessed sCT because of its poor dispersibility in air. However, we did observe irregular tile-shaped microparticles with particle sizes larger than 10 mm in the SEM image (Fig. 3(a)). The SASD-processed particles exhibited a narrow particle distribution with a mean particle size of 737.2  35.9 nm and 806.7  55.0 nm for SASD-F1 and SASD-F4 powder, respectively. On the other hand, SD-processed sCT particles showed a broader size distribution with a mean particle size of 2993.3  153.3 nm and 2276.7  115.6 nm, respectively. Fig. 2. Recovery of sCT from SD and SASD processes (n = 3).

6.3. Particle formation mechanism of the SASD process that a 10% proportion of chitosan in the formulation had no effect on the recovery of the sCT in the SD- or SASD-processed powders. 6.2. Particle morphology and size analysis The SEM images of raw sCT, trehalose, inulin, and SD- and SASDprocessed sCT particles are presented in Fig. 3. Absorption enhancer did not affect the morphology of the particles within a process; however, particle morphology was different between the processes. Therefore, only representative particle figures with chitosan are presented hereafter. While unprocessed sCT, trehalose and inulin particles had irregular shapes, drastic changes in the morphology and shape of the drug were observed for all processed drugs. SD-processed particles resulted in irregular-shaped

In our preliminary study, the process of SASD was thought to be characterized by formation of ‘primary droplets’ produced by atomization through a nozzle, from which originated ‘secondary droplets,’ due to the rapid expansion of CO2 from the within the primary droplets (decompressive atomization). Then, these secondary droplets are rapidly dried by hot flow of compressed air through the outer capillary of the two-flow spray nozzle to the evaporator chamber in the SASD process. The formation of primary and secondary droplets in the atomization processes was reported by Lavernia and Wu (1996). Particles from primary droplets sprayed through a nozzle are formed in the conventional SD process. In the SASD process, particles from the secondary droplets are formed by expansion of CO2. Therefore, CO2 acts as a droplet

Fig. 3. SEM images of (a) raw sCT, (b) raw inulin, (c) raw trehalose (d) SD-F1, (e) SD-F4, (f) SASD-F1, and (g) SASD-F4.

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Table 2 The particle size and SPAN of unprocessed SCT, SASD processed sCT and SD processed sCT. Formulation SASD-F1 SASD-F2 SASD-F3 SASD-F4 SASD-F5 SASD-F6 SD-F1 SD-F2 SD-F3 SD-F4 SD-F5 SD-F6

Mean particle size (nm) a

466.1  48.2 681.5  97.5a 590.96  38.4a 504.3  62.9a 428.25  1.8a 511.9  66.3a 2993.3  153.3b 3340  56.5b 4280  339.4b 2276.7  115.6b 2690  169.7b 3960  282.8b

SPANc 1.14 1.37 1.04 1.37 1.02 1.42 1.94 1.95 1.77 1.92 2.05 1.82

a Mean particle size calculated by cumulative method using dynamic light scattering measurement (n = 3). b Mean particle size calculatied by cumulative method using HELOS laser diffraction analyzer (n = 3). c SPAN = D90  D10/D50.

exploder for atomization of the primary droplet and the atomization propellant through the inner capillary of the nozzle. The mean particle size of SASD-processed particles was decreased by the atomization of CO2. In addition, spherical nanoparticles with narrow particle size distribution were formed due to the rapid expansion of CO2 by enhanced mass transfer using compressed air through the outer capillary of the two-flow spray nozzle, in the SASD process. The difference between SD-processed particles and SASD particles can depend on the different strength of cohesive forces (surface tension and viscosity) operating on the formed droplets. An even more important factor is that the dissolution of gas in liquid strongly reduces the viscosity and the surface tension of the formed solution (Reverchon et al., 2007b). The greater the quantity of gas dissolved, the more the cohesive force is reduced (Brunner, 1994). Moreover, the solubility of CO2 in ethanol is much larger than that in water; thus, the viscosity and surface tension of the solution were reduced. As a result, smaller particles with a narrow particle distribution can be obtained by the SASD process in comparison to the SD process.

Fig. 5. Powder X-ray diffraction patterns of (a) raw sCT, (b) raw inulin, (c) raw trehalose, (d) SD-F1, (e) SD-F4, (f) SASD-F1, and (g) SASD-F4.

samples. All of the samples showed an amorphous nature regardless of the process type or formulation. Therefore, representative PXRD patterns are shown in Fig. 5. Raw sCT and inulin did not show any peak confirming an amorphous nature, while raw trehalose was in a crystalline form based on the presence of numerous peaks. However, after the SD and SASD processes, no PXRD patterns of processed particles showed any characteristic diffraction peaks, confirming that trehalose was no longer present in its crystalline form. These results indicate amorphous sCT was not changed during the SD or SASD process.

6.4. Powder X-ray diffractometry (PXRD) PXRD study was performed on the raw materials such as sCT, inulin, and trehalose and all of the SD- and SASD-processed

Fig. 4. Cumulative particle size distribution curves for representative SD- and SASD-processed sCT formulations.

Fig. 6. FT-IR spectra of (a) raw sCT, (b) raw inulin, (c) raw trehalose, (d) SD-F1, (e) SD-F4, (f) SASD-F1, and (g) SASD-F4.

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6.5. FT-IR analysis FT-IR spectra of all processed sCT particles were observed to confirm the presence of the amino and carboxyl groups on the surface of SASD-processed particles and SD-processed particles, respectively. Fig. 6 shows FT-IR spectra of SD- and SASD-processed sCT, respectively. sCT showed characteristic peaks at 1650 cm1 (for the amide I region, which is primarily due to C¼O stretching vibration) and 3300 cm1 (for the O—H stretching vibration) wave numbers. As can be observed from Fig. 6, peaks of sCT were preserved after the SASD and SD processes, indicating no chemical degradation. In addition, no aggregate peak of protein was observed in any of the FT-IR spectra, which would be expected around 1625–1630 cm1 6.6. In vivo absorption study The serum concentration–time profiles of sCT after nasal administration of the inulin-formulation of sCT powder samples, prepared by SD and SASD processes, unprocessed raw sCT and the marketed product, Miacalcic1 nasal solution, to the rats are shown in Fig. 7. The pharmacokinetic parameters calculated from the time profiles are presented in Table 3. The minimum effective concentration of sCT that inhibits pit formation by osteoclasts in vitro is known to be lower than 1 pM (3.4 pg/ml) (Nakamuta et al.,

Fig. 7. Serum concentration–time profiles of sCT after intranasal administration: (a) SD processed sCT (b) SASD processed sCT. * Indicates p < 0.05, compared with unprocessed sCT.

1997). In pharmacokinetic parameters of raw material (as powder), the area under the concentration–time curve (AUC0 ! 120), Cmax and Tmax were 2026.44  503.31 ng min/ml, 76.97  18.64 ng/ml and 13.33  5.77 min, respectively. These values for unprocessed sCT were in agreement with the result of a previous study (Matsuyama et al., 2006). After the administration of Miacalcic1 nasal solution, the area under the concentration–time curve (AUC0 ! 120), Cmax and Tmax were 4431.68  994.40 ng min/ml, 108.32  26.33 ng/ml and 5.77  8.33 min, respectively. The analysis of variance by Tukey’s HSD test showed that there are no significant differences (p > 0.05) between the unprocessed sCT and the commercial product in terms of AUC0 ! 120 and Cmax. Fig. 7(a) shows the serum concentration–time profile of sCT in a rat after nasal administration of SD processed sCT at a dose equivalent to 0.1 mg of sCT. All of the SD processed samples exhibited increased AUC0 ! 120 and Cmax in comparison with those of unprocessed sCT and the commercial product, although the difference was not statistically significant except in the SD-F1 samples. However, compared to the SASD-processed samples (Fig. 7(b)), they showed lower AUC0 ! 120 and Cmax values. AUC0 ! 120 and Cmax of all SASD processed samples were significantly higher than those of unprocessed sCT and the commercial product (p < 0.05). In addition, there were no significant differences between SASD-F1, SASD-F2, and SASD-F3 in terms of either AUC0 ! 120 or Cmax. The increased absorption of SASD-processed sCT powders was probably attributed to a reduction of particle size since smaller particles generally show higher uptake by nasal epithelia than larger ones (Amidi et al., 2006; Brooking et al., 2001; Desai et al., 1996; Donovan and Huang, 1998). In all formulations, the AUC0 ! 120 and Cmax of SASD processed samples were much higher than those of the SD processed samples. This higher absorption of SASD processed samples may be due to the difference of particle size, as discussed in Section 3.2. In SD- and SASD-processed powders, absorption enhancers possibly contributed to the increase in the bioavailability of formulated sCT in comparison to unprocessed sCT and commercial product. Among the three formulations, chitosan (SASD-F1, SD-F1) showed the highest AUC0 ! 120 and Cmax in both the SD- and SASD-processed samples. A similar result was reported in many previous studies using chitosan as the nasal absorption enhancer (Amidi et al., 2006; Aspden et al., 1996; Hinchcliffe et al., 2005; Sinswat and Tengamnuay, 2003; Tengamnuay et al., 2000). Chitosan is a cationic polysaccharide which improves the uptake of hydrophilic drugs such as polypeptides across nasal epithelia. The enhancing mechanisms were reported to be a decrease mucociliary clearance due to combination of mucoadhesion (Henriksen et al., 1996; Witschi and Mrsny, 1999) and an effect on the gating properties of the tight junction (Artursson et al., 1994). Improved nasal absorption by bile salt and b-cyclodextrin has also been widely reported. Their absorption-enhancing mechanism is different from that of chitosan. Bile salts promote transmembrane movement of endogenous and exogenous lipid and the transcellular and/or paracellular movement of several small and polar large molecules (Gordon et al., 1985). Cyclodextrins are thought to enhance peptide absorption by interaction with calcium ions and the nasal mucosal membrane lipids of the nasal mucosa, which may reduce the barrier function of the nasal membrane. Another mechanism is inhibition of enzymatic degradation of sCT in the nasal mucosa (Schipper et al., 1995c). However, bile salt and b-cyclodextrin have been reported to damage the membrane (Schipper et al., 1995b). Indeed, Tengamnuary et al. reported that chitosan showed a higher bioavailability and a decreased membrane-irritating effect than b-cyclodextrin in rats after nasal administration in solution (Sinswat and Tengamnuay, 2003; Tengamnuay et al., 2000). Based on the results of our

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Table 3 Pharmacokinetic parameters of sCT in rat after nasal administration (Mean  S.D, n = 3). Formulation

Cmax(ng/ml)

Tmax (min)

AUC0 ! 120 (ng min/ml)

Elimination rate constant (K)

Half-life (t1/2,min)

SASD-F1 SASD-F2 SASD-F3 SD-F1 SD-F2 SD-F3 Unprocessed sCT Commercial product

558.39  83.02a 523.12  107.44a 458.8  189.06a 425.34  42.68a 306.45  43.02 342.97  67.01a 76.97  18.64 108.32  26.33

16.66  5.77 23.33  5.77 13.33  5.77 16.67  5.77 20.00  10.00 10.00  0.00 13.33  5.77 5.77  8.33

21218.78  7320.53a 18079.86  3116.93a 13212.32  2541.46a 12192.65  2173.70a 8441.01  973.33 10431.76  2581.37 2026.44  503.31 4431.68  994.40

0.026  0.008 0.035  0.021 0.035  0.013 0.025  0.005 0.038  0.006 0.023  0.001 0.035  0.011 0.022  0.006

28.39  9.06 23.59  10.27 22.02  8.60 28.71  4.85 18.75  3.23 30.63  1.52 21.01  7.02 33.25  7.42

a

Indicates p < 0.05 between unprocessed sCT and SASD or SD processed sCT.

study, the absorption-enhancing effect of chitosan could be maximized through size reduction by the SASD process. 7. Conclusions The overall aim of this study was to prepare a nasal powder formulation of sCT with enhanced bioavailability using proper excipients such as absorption enhancers. Fine spherical sCT nasal powders were successfully prepared using the SD and SASD processes. The prepared powders were characterized by HPLC, SEM, particle sizing, PXRD and FT-IR. These results showed that sCT powders were chemically stable, and the amorphous form of sCT was not changed during the SD or SASD process. In the in vivo pharmacokinetic study in rats, processed sCT samples showed much higher nasal absorption than unprocessed sCT. Among three absorption enhancers, chitosan showed the highest bioavailability enhancing effect by its properties of mucoadhesion. In particular, SASD-processed sCT exhibited higher nasal absorption when compared with SD processed sCT in all formulations due to a reduction of particle size. Therefore, the results from this study suggest that the preparation of fine nasal powder with a proper absorption enhancer using the SASD process could be a promising approach to improving nasal absorption of biopharmaceuticals such as sCT. Acknowledgements This work was supported by the National Research Foundation (NRF) of Korea grant (No. 951-0504-027-2, Korea–Sweden Research Co-operation Program), funded by Ministry of Education, Science and Technology (MEST) of the Korean government. Authors also thanks to STINT Sweden for Sweden–Korea collaboration grant. References Amidi, M., Romeijn, S.G., Borchard, G., Junginger, H.E., Hennink, W.E., Jiskoot, W., 2006. Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system. J. Control. Release 111, 107–116. Artursson, P., Lindmark, T., Davis, S.S., Illum, L., 1994. EFfect of chitosan on the permeability of monolayers of intestinal epithelial-cells (Caco-2). Pharm. Res. 11, 1358–1361. Aspden, T.J., Illum, L., Skaugrud, O., 1996. Chitosan as a nasal delivery system: evaluation of insulin absorption enhancement and effect on nasal membrane integrity using rat models. Eur. J. Pharm. Sci. 4, 23–31. Brooking, J., Davis, S.S., Illum, L., 2001. Transport of nanoparticles across the rat nasal mucosa. J. Drug Target. 9, 267–279. Brunner, G., 1994. Gas Extraction. Springer-Verlag, New York. Chan, H.K., Clark, A.R., Feeley, J.C., Kuo, M.C., Lehrman, S.R., Pikal-Cleland, K., Miller, D.P., Vehring, R., Lechuga-Ballesteros, D., 2004. Physical stability of salmon calcitonin spray-dried powders for inhalation. J. Pharm. Sci. 93, 792–804. Chattopadhyay, P., Gupta, R.B., 2002. Protein nanoparticles formation by supercritical antisolvent with enhanced mass transfer. AIChE J. 48, 235–244. Desai, M.P., Labhasetwar, V., Amidon, G.L., Levy, R.J., 1996. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharm. Res. 13, 1838–1845.

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