Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration

Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration

Accepted Manuscript Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration Camilla Sander, Katrine Dragsbæ...

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Accepted Manuscript Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration Camilla Sander, Katrine Dragsbæk Madsen, Birgitte Hyrup, Hanne Mørck Nielsen, Jukka Rantanen, Jette Jacobsen PII: DOI: Reference:

S0939-6411(13)00216-6 http://dx.doi.org/10.1016/j.ejpb.2013.05.017 EJPB 11433

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Accepted Date:

8 March 2013 29 May 2013

Please cite this article as: C. Sander, K.D. Madsen, B. Hyrup, H.M. Nielsen, J. Rantanen, J. Jacobsen, Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration, European Journal of Pharmaceutics and Biopharmaceutics (2013), doi: http://dx.doi.org/10.1016/j.ejpb.2013.05.017

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Characterization of spray dried bioadhesive metformin microparticles for oromucosal administration Camilla Sandera, Katrine Dragsbæk Madsena, Birgitte Hyrupb, Hanne Mørck Nielsena, Jukka Rantanena, Jette Jacobsena*

a

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen,

Universitetsparken 2, 2100 Copenhagen, Denmark b

Fertin Pharma A/S, Dandyvej 19, 7100 Vejle, Denmark

*Corresponding author. Tel.: +45 3533 6299; Fax: +45 3533 6030; E-mail address: [email protected]

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Abstract Delivery of drugs into or via the oral cavity offers some distinct advantages due to the easy access to the oral mucosa, fast onset of action, and avoidance of hepatic and intestinal degradation mechanisms. To overcome the effective removal mechanisms existing in this area, bioadhesive drug delivery systems are considered a promising approach as they facilitate a close contact between the drug and the oral mucosa. In this study, bioadhesive chitosan-based microparticles of metformin hydrochloride were prepared by spray drying aqueous dispersions with different chitosan:metformin ratios and chitosan grades with increasing molecular weights. A recently developed ex vivo flow retention model with porcine buccal mucosa was used to evaluate the bioadhesive properties of spray dried microparticles. An important outcome of this study was that microparticles with the desired metformin content could be prepared and analyzed using the ex vivo retention model. We observed an increase in metformin retention on porcine mucosa with increasing chitosan:metformin ratios, while no effect of increasing the chitosan molecular weight was found. Rheological characterization of feeds for spray drying was performed and used for designing the microparticles. This way, novel microparticles with similar particle size distribution, high encapsulation efficiencies, and low moisture content were obtained independent of the chitosan:metformin ratio and the chitosan molecular weight. In conclusion, chitosan:metformin microparticles with significant bioadhesive properties on porcine buccal mucosa were developed.

Key words Bioadhesion, oromucosal drug delivery, spray drying, metformin hydrochloride, chitosan, ex vivo, retention model.

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1. Introduction Oral delivery of drugs and subsequent intestinal absorption presents several significant drawbacks, including delayed onset of action, degradation in the acidic environment of the stomach, enzymatic degradation in the bulk and during transport across the intestine, and hepatic as well as intestinal first pass metabolism. These drawbacks may be avoided by direct absorption of the drug through the oromucosal epithelium as the drug passes directly to the systemic circulation [1,2]. The oral mucosa is easily accessible and buccal as well as sublingual routes of administration are well accepted by the patients. In addition, disorders in the oromucosal epithelium may be treated locally and more effectively without the need of pre-absorption via the intestine [3]. However, oromucosal administration is limited by rapid clearance of the drug from the oromucosal surfaces by movement of the tongue and jaws, flow of saliva, and chewing followed by swallowing [4,5]. This may result in local drug concentrations below the therapeutic level as well as side effects resulting from drug being swallowed. By prolonged retention of the drug on the oromucosal surface, improved mucosal as well as transmucosal drug delivery may be achieved. Incorporating the drug into a bioadhesive formulation is therefore desirable to achieve prolonged mucosal contact and higher drug concentration on the mucosal surface [6,7]. Metformin hydrochloride (metformin) is an oral hypoglycemic drug commonly prescribed to treat diabetes type II. In this study, bioadhesive microparticles were prepared comprising metformin, as an example of a small hydrophilic drug, which may be easily dissolved in saliva and swallowed. Importantly, processing of metformin in the lab is safer compared to handling of for example nicotine, which is another example of a water soluble drug that is commonly used for oromucosal delivery. However, handling of nicotine requires strong safety precautions to ensure a minimal occupational exposure. Microparticles for oromucosal administration were prepared in the current study by spray drying aqueous dispersions of metformin and the bioadhesive polymer, chitosan. Chitosan is a linear Page 3 of 25

polysaccharide commonly obtained by deacetylation of chitin. Chitosan is biocompatible, biodegradable, and commonly used in food, cosmetics and agriculture, hence several possibilities for pharmaceutical use have been exploited [8,9]. The cationic character of chitosan results in a high charge density at pH below approximately 6.5, which affects both swelling behavior and solubility of the polymer [10]. Due to the net positive charge chitosan adheres to negatively charged components by ionic interactions, e.g. with sialic acid and sulfonic acid residues of mucins present in saliva and on epithelial surface-bound mucins [9]. Bioadhesive chitosan formulations for oromucosal delivery comprise bioadhesive films [11,12], microspheres [4,13], wafers [14], hydrogels [15], thermosensitive gels [16] and buccal tablets [17]. Spray drying is a continuous process capable of transforming different fluids, solutions, emulsions, and suspensions, into a dry powder [18]. The ability of spray dryers to produce powders with a specific particle size and low moisture content is desirable in the development of multiparticulate drug delivery systems, as is the ability to eliminate organic solvent residuals in the final product. Co-processing of drug and a bioadhesive carrier by spray drying has been used in several cases to improve the bioadhesive properties of the drug [19]. For example Coucke et al. prepared bioadhesive microparticles with metoprolol tartrate [19] while Bowey et al. prepared insulin loaded microparticles [20]. Spray drying of chitosan has been utilized for preparation of bioadhesive formulations for nasal [21,22] and buccal [13] delivery. However, there is a need for further development and evaluation of microparticles with improved bioadhesive characteristics for oromucosal delivery, as the challenge of retaining water soluble drugs persists. The aim of the current study was to use a spray drying approach to prepare drug-loaded chitosan microparticles for oromucosal drug delivery composed of varying chitosan:metformin ratio and varying chitosan molecular weight. These microparticles were characterized with respect to particle size,

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morphology, water content, drug content, and bioadhesive properties using an ex vivo flow retention model simulating oromucosal conditions.

2. Materials and methods 2.1 Materials Metformin hydrochloride was obtained from J. Inc. (Gujarat, India) and Fagron (Rotterdam, The Nederlands). ChitoPharm S, M, and L were a kind gift from Cognis (Monheim, Germany). Deionized water was obtained from a Milli-Q® integral water purification system from Millipore (Billerica, MA). Acetic acid (glacial), ammonium acetate, calcium chloride dihydrate, formic acid (98-100%), hydrochloride acid (fuming 37%), sodium chloride, sodium hydroxid, and sodium hydrogen carbonate were obtaind form Merck (Darmstadt, Germany). Acetonitrile (HPLC grade), mucin - from porcine stomach, type II (PGM), potassium chloride, sodium phosphate dibasic, and sodium phosphate monobasic were from Sigma-Aldrich (St. Louis, Missouri). Methanol (HPLC grade), and sand, sulphuric acid washed (d(10)= 116 and d(90)=312 µm) were from VWR – Bie og Berntsen (Herlev, Denmark). Phosphoric acid (85%) was from J.T. Baker (Deventer, The Netherlands).

2.2 Porcine buccal mucosa Cheeks from freshly slaughtered domestic pigs were collected from the local abattoir (Slagteriskolen, Roskilde, Denmark) and transported on ice. Permission was granted from the Danish Veterinary and Food Administration (approval number: DK-10-3-oth-023). Skin, lips, and excessive connective tissue were removed using scalpel and scissor and only buccal mucosa remained. The mucosa was kept moisturized with isotonic phosphate buffer pH 6.8 during the procedure. Pieces of mucosae (approximately 5 cm2) were stored at -20 °C until use (maximum three months).

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2.3 Preparation of feeds for spray drying Microparticles of metformin and chitosan of increasing molecular weight were prepared by spray drying. Feeds, with compositions as shown in Table 1, were prepared according to the manufacturer’s instructions on how to dissolve chitosan, except in some cases additional HCl was required to obtain a visually clear dispersion. A single batch was prepared in each case, sufficient to prepare material for characterization and bioadhesive evaluation studies. First, metformin was dissolved in deionized water using magnetic stirring followed by addition of chitosan. Chitosan grades, with increasing molecular weight ranges 50-1000 kDa, 100-2000 kDa, and 500-5000 kDa, denoted ChitoPharm S (CPS), ChitoPharm M (CPM), and ChitoPharm L (CPL), respectively, were employed. Polymers were used as received and without further evaluation. The polymer was allowed to hydrate for 10 min (but did not dissolve) before 2.4 M HCl was added in one shot to a final concentration of 0.06 or 0.12 M. The resulting dispersion was stored at room temperature overnight to degas the feed before measuring rheological properties and subsequent spray drying. The Batch ID is used in the current study to name both the feeds and the dried microparticles, which will be clearly indicated from the context.

2.4. Rheological characterization of feeds Steady state flow viscosity of the polymer dispersions prepared for spray drying was determined using an AR-G2 Rheometer (TA Instruments, New Castle, Delaware) equipped with a cone-and-plate geometry using a 60 mm steel cone with a cone angle of 1°. Truncation of this geometry was 28 µm. Approximately 1 mL sample was applied on the plate using a pipette before lowering the cone while slightly spinning the cone to distribute the sample evenly on the plate. The sample was allowed to equilibrate for 10 minutes at 25 °C before measuring the steady state flow in the range 1-1000 s-1.

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2.5 Spray drying Feeds were spray dried using a Büchi Mini Spray Dryer B-290 (Büchi Labortechnik AG,Flawil, Switzerland). Inlet air was 200 °C corresponding to an outlet air temperature of 75-94 °C, depending on the solids composition of the feed. Compressed air was used as spray gas (flow 0.5 m3/h) and the 0.7 mm nozzle was cleaned regularly using vacuum. An aspirator rate of 32 m3/hour was used together with a pump rate of 3 mL/min.

2.6 Particle size Particle size distribution of spray dried particles was determined by laser diffraction using a Malvern Mastersizer equipped with a dry dispersion unit, Scirocco 2000 (Malvern Instruments, Worcestershire, United Kingdom). Spray dried particles were lightly ground using a mortar and pestle before measurement. Sand particles were used as pre-dispersant to facilitate powder flow. Contribution from the sand particles was subsequently subtracted in the analysis. Median particle sizes dividing the particle volumes after 10, 50 or 90% are reported as d(10), d(50) and d(90), respectively. Measurements were performed in triplicate.

2.7 Particle morphology Morphology of the spray dried microparticles was determined using a scanning electron microscope (SEM), JMS-5200 (JEOL, Tokyo, Japan). The accelerating voltage was set to 10 or 15 kV. Particles were coated with gold prior to evaluation using an Auto Sputter Coater, E5200 (Bio-Rad Laboratories, Hercules, California). Contrast and brightness of micrographs were adjusted using PicasaTM (Picasa Version 3.9.0, Google) to improve picture clarity.

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2.8 Moisture content Water content of microparticles was determined using thermogravimetric analysis (TGA). Approximately 5 mg powder was placed in a small pan at 25 °C for 2 min before heating from 25 to 120 °C at 5 °C/min and finally allowed to equilibrate at 120 °C for another 20 min. Microparticles were stored in closed containers at room conditions and TGA analysis performed shortly after spray drying. Water content is stated as loss on drying.

2.9 Metformin content of spray dried particles Spray dried powder corresponding to 10 mg metformin was accurately weighed and dissolved overnight at room temperature in 0.1 M HCl. Samples were diluted with deionized water to approximately 200 µg/mL metformin and centrifuged at 13,000 rpm for 15 min before determination of metformin content using HPLC-UV as described below. Determinations were performed in triplicate. Encapsulation efficiency (EE) [%] was calculated using equation 1.

(Eq. 1) Where Q is metformin content [mg] of microparticles, m is the mass [mg] of microparticles, cm is metformin concentration [mg/mL] in feed, and c is the total concentration of solid (metformin and chitosan) in feed [mg/mL]. Encapsulation efficiency describes the actual content of metformin in microparticles in relation to the theoretical content and should preferably be close to 100%.

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2.10 Retention studies The bioadhesive properties of microparticles were tested in a recently described [23] flow retention model using porcine buccal mucosa. In short, buccal mucosa was mounted with pins on a rubber pad, at an angle of 30°, and placed in a hydration chamber with approximately 90 % relative humidity (range: 53-92%) and a temperature of approximately 33 °C (range: 32-35). The mucosa was continuously flushed with artificial saliva irrigation medium simulating human whole saliva. The irrigation medium used for retention studies was prepared by a modification of the medium used by Gåserød et al. [24]. The irrigation medium was prepared as a solution of 0.21 g/L of NaHCO3, 0.43 g/L NaCl, 0.75 g/L KCl, 0.22 g/L CaCl2.2H2O, 0.91 g/L NaH2PO4.H2O, and 25 g/L porcine gastric mucin in deionized water and adjusted to pH 6.8 with 10 M NaOH. To humidify the mucosa, an equilibration time of 10 min was allowed prior to administration of the particles. Spray dried particles were lightly ground using a mortar and pestle. An amount of particles corresponding to 50 mg metformin was applied to the mucosa within an area of 1.5 x 3.0 cm using a spatula, except with 3:1 CPS particles an amount corresponding to 25 mg metformin was applied, due to the limited application area and proportionate large volume of particles. The particles were allowed 2 min of equilibration prior to start of irrigation. A flow rate of 4 mL/min was then provided using a peristaltic pump. During the experiment the irrigation medium was magnetically stirred and the temperature was kept constant at 37 °C. The eluate was sampled at time 1, 2, 5, 10, 15 and 20 minutes, diluted with deionized water to an appropriate concentration of metformin, centrifuged (13,000 rpm, 15 min), and the content of metformin in the supernatant was determined using HPLC-UV. Retention studies were performed in triplicates using a new piece of buccal mucosa each time. Differences in metformin retention from different formulations were assessed using Students t-test and considered significant at p<0.05.

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2.11 Extraction of metformin from buccal mucosa After completion of a retention study the buccal mucosa was frozen at -20 °C. The frozen mucosa was cut into pieces (approximately 3 mm) using a sharp knife. These pieces of mucosa were homogenized in 15 mL phosphoric acid (85% H3PO4) using a T25 Basic Ultra Turrax homogenizer (Janke & Kunkel, Ika Werke GmbH & Co, Germany) equipped with a S25N-25F dispersing tool. After storage for 48 hours at room temperature, the mucosa was dissolved. The sample was manually flicked 10 times and approximately 1.5 mL of the homogenate was centrifuged at 13,000rpm for 15 minutes. From the middle part of the supernatant 0.5 mL aliquots were withdrawn, diluted with deionized water to approximately 100 µg/ml metformin, filtered through a 0.2 µm filter, and the content of metformin was analyzed using HPLC-UV.

2.12 Recovery Metformin was quantified in the eluate (Meluate)[mg] and extracts of buccal mucosa (Mmucosa) [mg] and compared to the initial amount of metformin applied to the mucosa (Mapplied) [mg]. Recovery [%] was calculated using equation 2. (Eq. 2)

2.13 HPLC-UV Quantification of metformin by HPLC-UV: isocratic elution at room temperature, mobile phase consisting of 45% acetonitrile in 0.15 M ammonium acetate buffer adjusted to pH 6, flow rate 1.0 mL/min, and detection wavelength 234 nm. The column was a strong cationic exchange column Luna SCX 250x4.6 mm 5 µm including security guard cartridge SCX 4x3.0 mm (Phenomenex, Allerød, Denmark). Integration of peak areas was used for quantification of sample concentrations compared with a standard curve. A linear relationship was established in the concentration range 4-512 µg/mL (R2 Page 10 of 25

>0.999). Variability (described by relative standard deviations) at low (4 µg/mL) and high (128 µg/mL) concentrations was below 1% (n=6).

3. Results and discussion Bioadhesive microparticles of metformin were prepared by spray drying aqueous dispersions of drug and chitosan, which is available from this manufacturer in three different grades with increasing molecular weight denoted S, M, and L corresponding to molecular weight ranges 50-1000 kDa, 100-2000 kDa, and 500-5000 kDa, respectively. Assuming that higher molecular weight correlates with an increased polymer chain length, it was hypothesized that the bioadhesive properties of metformin microparticles would be improved by using chitosan grades with increasing polymer chain length, due to a higher degree of interpenetration of mucosal residues and due to higher viscosities of the swelled particles. The exact concentration of chitosan necessary for bioadhesion was unknown and it was decided to pursue three different levels of chitosan:metformin ratios with a single polymer grade.

3.1 Feeds for spray drying Composition of the feeds and drying conditions, including dimensions of the spray dryer, nozzle design, feed rate, air pressure, and drying temperature, strongly affect the droplet size of nebulized solutions and thereby affect the pharmaceutically relevant characteristics of spray dried particles, such as particle size and particle size distribution, moisture content, and morphology [22,25]. In the current study the inlet air conditions were kept constant and microparticles with similar properties with respect to particle size, moisture content, and encapsulation efficiency were targeted by preparing feeds with similar viscosities. Changing the chitosan concentration or increasing the chitosan molecular weight greatly affects the viscosity of the feed, which may influence nebulization of the feed and possibly the feed rate,

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as the spray drier was equipped with a peristaltic pump with limited capacity. For this reason, feeds with a constant level of CPS and increasing metformin concentrations were prepared in order to maintain a similar viscosity of all feeds. As shown in Figure 1 feeds with similar viscosities (i.e. viscosities in the same range) were obtained when metformin was added to the CPS dispersion. The applied polymer concentrations of CPS, CPM, and CPL (Table 1) resulted in similar viscosities of CPS and CPM dispersions while the feed containing CPL possessed a higher viscosity at low shear rates and similar viscosity at higher shear rates (Figure 2). The lower shear rates resembles the conditions experienced by the feed during pumping, while shear rates of 103 s-1 and higher mimics shear conditions of spraying. A cut-off limit of 103 s-1 was used in the current study as turbulence was observed at higher shear rates. Feeds containing only metformin and no polymer showed Newtonian behavior with no shear thinning and markedly lower viscosity (10-3 Pa s) than the dispersions with polymers, similar to that of pure water (data not shown).

3.2. Physicochemical characterization of microparticles The characteristics of spray dried chitosan:metformin particles are shown in Table 2. In most cases a high yield was obtained. For 1:3 CPS microparticles only 50% yield was obtained, which was due to the extra deposition in the equipment. This is a well-known problem when using a small-scale spray dryer as the expansion chamber is very small, which leads to comparable short drying times before the drying droplet/particle touches the chamber wall [18]. Higher yields may be expected in larger spray dryers due to larger expansion chambers with concurrent more efficient drying. Spray dried particles, which did not end up in the collection chamber, were deposited in the expansion chamber or in the cyclone. Keegan et al. [13] noticed a viscosity decreasing effect of adding sodium fluoride to chitosan feed dispersions and a concurrent increase in yield, which they ascribed to more effective atomization and drying of the dispersion. Page 12 of 25

The encapsulation efficiencies of metformin microparticles ranged from 82% to 96% (Table 2). In the current study a tendency of an increased encapsulation efficiency was observed with decreasing chitosan:metformin ratio; the encapsulation efficiency increased from 82% to 89% when chitosan:metformin ration was decreased from 3:1 to 1:3. This is similar to the effect of polymer:drug ratio found by Desai and Park [21]. They also observed an increased encapsulation efficiency by increasing chitosan molecular weight, which they explained by differences in feed viscosities. In the current study no effect or trend with respect to encapsulation efficiencies were obtained from particles with CPS, CPM and CPL (87%, 88%, and 84%, respectively). In all cases high, encapsulation efficiencies were obtained, indicating that metformin and chitosan were deposited to the same extend during the spray drying process and the expected composition of microparticles was achieved despite differences in yield, chitosan:metformin ratio, and chitosan molecular weight. The encapsulation efficiencies and yields presented in Table 2 were considered satisfactory for an exploratory study of this type.

3.3 Particle size distribution and morphology of spray dried microparticles Particles were micron sized with moisture contents below 10% and the spray dried powders appeared cohesive, due to the small particle size. Spray dried microparticles containing only metformin were of uniform size with a rounded and slightly cubic shape, as shown in the SEM micrograph presented in Figure 3A. Individual particles were agglomerated. After spray drying with chitosan (3:1 CPS) SEM micrographs indicated a broader size distribution (Figure 3B), but this was not confirmed by measurements of particle size distribution using laser diffraction (Table 2). Particles containing chitosan were spherical or rounded with dimpled surface characteristics. Examples of such surfaces are shown in Figure 3B. Dimpled surface structures were more prevalent in particles containing CPS, when the

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amount of metformin was decreasing. Particles containing CPL (Figure 3C) were more spherical with less dimpled surface structures compared to CPS particles. Also, CPL particles were slightly larger than particles containing CPS or CPM, which was confirmed by laser diffraction. An increase in particle size was obtained by He et al. [22] by increasing the feed rate, decreasing the compressed air flow, or increasing the nozzle diameter. As these parameters were kept constant in the current study the increase in particle size of CPL microparticles compared with CPS and CPM microparticles can be ascribed to the difference in feed viscosity, which affects nebulization of the feed occurring by the force of compressed air in the spray dryer. Nebulization of feeds with higher viscosity is less efficient and may result in the formation of larger droplets and concurrently larger particles are formed after drying [21]. Dimpled surface characteristics of spray dried chitosan microparticles have previously been observed by Keegan et al. [13], Cervera et al. [26] and Bowey et al. [20]. As explained by Vehring [27], who used the concept of Peclet numbers to explain the formation of dimpled or wrinkled particles, this phenomenon is commonly observed with proteins and polymers. The surface of the drying droplet is enriched with polymer, which initiates different solidification mechanisms once a critical concentration is reached leading to the formation of a shell. Dependent on how quickly the shell dries this may cause the formation of a dimpled surface [27]. Formation of dimples may increase drug diffusion rates and release due to an increased surface area, compared with spherical particles with smooth surfaces. One limitation of the scanning electron microscope is the inability to distinguish between materials, such as chitosan and metformin, in SEM pictures, Figure 3. As such, fine particles in Figure 3B may have different compositions than large particles in the same picture. However, investigating potential differences in the composition of spray dried particles with different particles sizes was not further pursued in the current study.

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3.4 Ex vivo bioadhesion studies The bioadhesive properties of the spray dried microparticles were evaluated using a recently described ex vivo flow retention model [23]. This model describes the duration and extend of bioadhesion from multiparticulate formulations to porcine buccal mucosa utilizing a thorough evaluation of the rheological and physicochemical properties of the irrigation medium in comparison with human whole saliva. Spray drying of metformin with chitosan improved the bioadhesive properties of metformin on porcine buccal mucosa compared to spray dried metformin without chitosan (Figure 4). Retention of metformin was highest during the first 5 minutes period and then leveled off approaching zero after 20 minutes. Compared with the amount of metformin retained from particles without chitosan (SD MF), the amount of metformin retained from particles containing CPS (3:1 CPS and 1:1 CPS) was statistically significantly higher at time points from 1-20 minutes, while particles containing the lowest amount of CPS (1:3 CPS) only showed significant differences at time points up to 5 minutes (Figure 4). As can be seen from the figure, increasing chitosan:metformin ratio resulted in improved retention of metformin, which may be explained by a higher amount of polymer, which retains metformin in the polymer matrix, with a concurrent better bioadhesion of the formulation to the mucosa. The bioadhesive properties of solid dosage forms have previously been reported to be improved by increasing polymer concentration [28]. Metformin retention on porcine buccal mucosa was improved using equal CPS and metformin amounts. Changing the molecular weight by increasing polymer chain length with equal chitosan and metformin ratios (1:1 CPM and 1:1 CPL) did not further improve metformin retention (Figure 5). A tendency of 1:1 CPS particles being slightly less retentive than 1:1 CPM and 1:1 CPL particles was observed, but this was not statistically significant. It is well accepted that a large molecular weight is essential for diffusion, entanglement and hence mucoadhesion, although each polymeric system is unique preventing a general definition of an optimal chain length [28,29]. Chitosan molecular weight did not affect metformin retention on porcine buccal mucosa in the current study. Although increasing polymer chain length has Page 15 of 25

previously been related to improved bioadhesive properties [28], a threshold limit may exist above which chitosan retention is not further improved. Excessively long chain length may lose their ability to diffuse and interpenetrate the mucosal surface, but no indication of this was observed in the current study. In the best case, approximately 50% of the metformin dose was retained after 10 minutes at the porcine mucosa, which was reduced to approximately 40% after 20 minutes (3:1 CPS microparticles in Figure 4).The clinical significance of this strongly depends on the drug of interest. This may be sufficient for achieving higher transmucosal drug delivery for fast acting drugs, but may be insufficient for prolonged treatment of mucosal conditions. Also, individual factors, such as mouth feel, perception, and alteration of taste, which were not included in the current study, has to be taken into account. High recovery of metformin in retention studies was established, with the main amount of metformin found in the eluate collected after irrigation of the mucosa. Only a small amount of metformin was extracted from the porcine mucosa after completion of the retention studies. The net amount of metformin in eluate and mucosal extracts in some cases exceeded 100% recovery, which is ascribed to experimental variations.

3.5 Buccal drug delivery of metformin Metformin is used in the current study mainly as an example of a small hydrophilic drug, which might be easily dissolved in saliva and swallowed. However, buccal absorption of metformin has some appealing features as the oral administration of metformin is challenged by its high solubility in water and narrow oral absorption window. Metformin is primarily absorbed in duodenum [30] and only to a small extend from the stomach, jejunum and ileum [31], and approximately one third of the dose is excreted unchanged in the feces [31]. Common side effects include nausea and gastrointestinal disturbances.

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Extending the absorption site to buccal administration could therefore potentially improve the bioavailability and limit the gastrointestinal-related side effects due to less drug being swallowed from bioadhesive formulations. No previous attempts of bioadhesive metformin formulations for oromucosal delivery have been reported and future studies will have to elucidate the possibilities of metformin buccal absorption. Previously, bioadhesive formulations of metformin have been prepared to prolong metformin retention in the stomach [32,33] using hydrophilic polymers with combined controlled release and mucoadhesive properties. A bioadhesive formulation using prosopis gum, which is a naturally occurring source of polysaccharides, was shown to improve the glucose lowering effect of metformin [34].

4. Conclusion Metformin microparticles with effective drug encapsulation and bioadhesive properties were prepared by spray drying. Rheological characterization of feeds for spray drying was performed and used for designing the microparticles. This way, novel microparticles with similar particle size distribution, high encapsulation efficiencies, and low moisture content were obtained independent of the chitosan:metformin ratio and the chitosan molecular weight. The bioadhesive properties of spray dried metformin microparticles on porcine buccal mucosa were significantly improved after spray drying with chitosan as determined by the ex vivo flow retention model.

Acknowledgements The authors would like to thank the Drug Research Academy, University of Copenhagen, for the material grant to support purchase of analytical equipment for this study.

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[15] R. Pignatello, A.H.S. Stancampiano, C.A. Ventura, and G. Puglisi, Dexamethasone sodium phosphate-loaded Chitosan based delivery systems for buccal application, J Drug Target, 15 (2007) 603-610. [16] S. Rossi, M. Marciello, M.C. Bonferoni, F. Ferrari, G. Sandri, C. Dacarro, P. Grisoli, and C. Caramella, Thermally sensitive gels based on chitosan derivatives for the treatment of oral mucositis, Eur J Pharm Biopharm, 74 (2010) 248-254. [17] N. Langoth, H. Kahlbacher, G. Schoffmann, I. Schmerold, M. Schuh, S. Franz, P. Kurka, and A. Bernkop-Schnurch, Thiolated chitosans: Design and in vivo evaluation of a mucoadhesive buccal peptide drug delivery system, Pharm Res, 23 (2006) 573-579. [18] K. Masters, Spray drying handbook, fifth ed., Longman Scientific & Technical, Harlow Essex, 1991. [19] D. Coucke, C. Vervaet, P. Foreman, P. Adriaensens, R. Carleer, and J.P. Remon, Effect on the nasal bioavailability of co-processing drug and bioadhesive carrier via spray-drying, Int J Pharm, 379 (2009) 67-71. [20] K. Bowey, B.E. Swift, L.E. Flynn, and R.J. Neufeld, Characterization of biologically active insulinloaded alginate microparticles prepared by spray drying, Drug Dev Ind Pharm, 39 (2013) 457465. [21] K.G.H. Desai and H.J. Park, Preparation and characterization of drug-loaded chitosantripolyphosphate microspheres by spray drying, Drug Dev Res, 64 (2005) 114-128. [22] P. He, S.S. Davis, and L. Illum, Chitosan microspheres prepared by spray drying, Int J Pharm, 187 (1999) 53-65. [23] K.D. Madsen, C. Sander, S. Baldursdottir, AM.L. Pedersen, and J. Jacobsen, Development of an ex vivo retention model simulating bioadhesion in the oral cavity using human saliva and physiologically relevant irrigation media, Int J Pharm, 448 (2013) 373-81. [24] O. Gåserød, I.G. Jolliffe, F.C. Hampson, P.W. Dettmar, and G. Skjak-Braek, The enhancement of the bioadhesive properties of calcium alginate gel beads by coating with chitosan, Int J Pharm, 175 (1998) 237-246. [25] M.I. Re, Formulating drug delivery systems by spray drying, Drying Technology, 24 (2006) 433446. [26] M.F. Cervera, J. Heinamaki, N. de la Paz, O. Lopez, S.L. Maunu, T. Virtanen, T. Hatanpaa, O. Antikainen, A. Nogueira, J. Fundora, and J. Yliruusi, Effects of spray drying on physicochemical properties of chitosan acid salts, AAPS PharmSciTech., 12 (2011) 637-649. [27] R. Vehring, Pharmaceutical particle engineering via spray drying, Pharm Res, 25 (2008) 9991022. [28] G.P. Andrews, T.P. Laverty, and D.S. Jones, Mucoadhesive polymeric platforms for controlled drug delivery, Eur J Pharm Biopharm, 71 (2009) 505-518. Page 19 of 25

[29] N. Salamat-Miller, M. Chittchang, and T.P. Johnston, The use of mucoadhesive polymers in buccal drug delivery, Adv Drug Deliv Rev, 57 (2005) 1666-1691. [30] G.T. Tucker, C. Casey, P.J. Phillips, H. Connor, J.D. Ward, and H.F. Woods, Metformin Kinetics in Healthy-Subjects and in Patients with Diabetes-Mellitus, Br J Clin Pharmacol, 12 (1981) 235-246. [31] N. Vidon, S. Chaussade, M. Noel, C. Franchisseur, B. Huchet, and J.J. Bernier, Metformin in the digestive tract, Diabetes Res Clin Pract, 4 (1988) 223-229. [32] J. Piao, J.E. Lee, K.Y. Weon, D.W. Kim, J.S. Lee, J.D. Park, Y. Nishiyama, I. Fukui, and J.S. Kim, Development of novel mucoadhesive pellets of metformin hydrochloride, Arch Pharm Res, 32 (2009) 391-397. [33] P.P. Ige and S.G. Gattani, Design and in vitro and in vivo characterization of mucoadhesive matrix pellets of metformin hydrochloride for oral controlled release: a technical note, Arch Pharm Res, 35 (2012) 487-498. [34] M.U. Adikwu, Y. Yoshikawa, and K. Takada, Bioadhesive delivery of metformin using prosopis gum with antidiabetic potential, Biol Pharm Bull, 26 (2003) 662-666.

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Figure captions Fig. 1. Shear rate dependent steady state viscosity (25°C) of aqueous chitosan:metformin feeds containing 15 mg/mL ChitoPharm S with increasing concentrations of metformin intended for spray drying. The legends refer to the composition of feeds outlined in Table 1.

Fig. 2. Shear rate dependent steady state viscosity (25 °C) of aqueous chitosan:metformin feeds containing chitosan of increasing molecular weight and a constant chitosan:metformin ratio of 1:1. The legends refer to the composition of feeds outlined in Table 1.

Fig. 3. Representative SEM micrographs of A) Spray dried metformin (SD MF), B) spray dried metformin microparticles with ChitoPharm S (3:1 CPS), and C) spray dried metformin microparticles with ChitoPharm L (1:1 CPL). Scale bar corresponds to 5 µm.

Fig. 4. Metformin retention on porcine buccal mucosa using an ex vivo flow retention model, when metformin was applied as microparticles with different chitosan:metformin ratios. By increasing chitosan:metformin ratio a higher retention of metformin was achieved. Data are presented as mean + SD (n=3) and adjacent points are connected with straight lines. CPS = ChitoPharm, SD MF = spray dried metformin hydrochloride, 1:3, 1:1, and 3:1 denotes chitosan:metformin ratio (w/w). Data points at similar sampling time, which are significantly different from those of SD MF, are marked with *(p < 0.05) or **(p < 0.01).

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Fig. 5. Metformin retention on porcine buccal mucosa using an ex vivo flow retention model, when metformin was applied as microparticles with different chitosan grades and same chitosan:metformin ratio. Increasing chitosan molecular weight did not change the retention of metformin. Data are presented as mean + SD (n=3) and adjacent points are connected with straight lines. The differences between data points at each point in time are not statistically significant. CPS = ChitoPharm S, CPM = ChitoPharm M, CPL = ChitoPharm L, 1:1 denotes chitosan:metformin ratio (w/w).

Page 22 of 25

Table 1 Composition of dispersions for spray drying and theoretical composition of spray dried particles. Batch ID

SD CPS

3:1 CPS

1:1 CPS

1:3 CPS

SD MF

1:1 CPM

1:1 CPL

Chitosan

15

15

15

15

0

10

7.5

Metformin

0

5

15

45

120

10

7.5

FEED COMPOSITION (mg/mL)

Total solids content

15

20

30

60

40

20

15

Total volume (mL)

340*

250*

170*

100*

50*

250**

333**

0

25

50

75

100

50

50

100

75

50

25

0

50

50

S

S

S

S

-

M

L

THEORETICAL SOLID COMPOSITION (% (w/w)) Metformin Chitosan Chitosan type

*final concentration of hydrochloric acid was 0.12 M **final concentration of hydrochloric acid was 0.06 M

Page 23 of 25

Table 2 Characteristics of spray dried chitosan:metformin microparticles. CPS = ChitoPharm S, CPM = ChitoPharm M, CPL = ChitoPharm L, SD MF = spray dried metformin hydrochloride, 1:3, 1:1, and 3:1 denotes chitosan:metformin ratio (w/w). Batch ID

a

Particle size a [µm]

Yield

Moisture content

Encapsulation efficiency a

[% (w/w)]

[% (w/w)]

d(10)

d(50)

d(90)

SD CPS

84%

8.1

2±0

5±0

18 ± 0

n.a.

3:1 CPS

90%

4.8

2±0

5±0

17 ± 0

82 ± 1

1:1 CPS

82%

3.6

2±0

5±0

19 ± 0

87 ± 1

1:3 CPS

50%

3.4

3±0

7±0

18 ± 0

89 ± 1

SD MF

82%

0

3±0

7±0

18 ± 0

96 ± 2

1:1 CPM

88%

2.4

2±0

6±0

22 ± 1

88 ± 1

1:1 CPL

82%

0

2±0

12 ± 0

33 ± 1

84 ± 2

[%]

Data are mean values ± standard deviations (n = 3)

Page 24 of 25

Table 3 Recovery of metformin in irrigation medium eluate (Meluate) and extracts from porcine mucosa (Mmucosa) after completion of retention studies. Mean ±SD (n=3).

Batch ID Mapplied (mg) Concentration dependency study SD MF 50 ± 0 3:1 CPS 21 ± 0 1:1 CPS 43 ± 0 1:3 CPS 44 ± 0 Molecular weight dependency study 1:1 CPS 47 ±0 1:1 CPM 45 ±0 1:1 CPL 42 ±0

Meluate (mg)

Mmucosa (mg)

Recovery (%)

51 ± 5 14 ± 1 37 ± 3 36 ± 8

< LOQ 4 ±1 3 ±3 2 ±1

104 ± 9 87 ± 7 93 ± 9 86 ± 17

47 ±3 42 ±1 39 ±4

4 ±1 4 ±3 6 ±2

109 ±5 102 ±4 107 ±5

Page 25 of 25

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 3C

Figure 4

Figure 5