Swellable and porous bilayer tablet for gastroretentive drug delivery: Preparation and in vitro-in vivo evaluation

Journal Pre-proofs Swellable and porous bilayer tablet for gastroretentive drug delivery: Preparation and in vitro-in vivo evaluation Kyu-Mok Hwang, Thi-Tram Nguyen, Su Hyun Seok, Hyun-Il Jo, Cheol-Hee Cho, Kyu-Min Hwang, Ju-Young Kim, Chun-Woong Park, Yun-Seok Rhee, Eun-Seok Park PII: DOI: Reference:

S0378-5173(19)30828-2 https://doi.org/10.1016/j.ijpharm.2019.118783 IJP 118783

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

15 August 2019 23 September 2019 10 October 2019

Please cite this article as: K-M. Hwang, T-T. Nguyen, S. Hyun Seok, H-I. Jo, C-H. Cho, K-M. Hwang, J-Y. Kim, C-W. Park, Y-S. Rhee, E-S. Park, Swellable and porous bilayer tablet for gastroretentive drug delivery: Preparation and in vitro-in vivo evaluation, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/ j.ijpharm.2019.118783

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Manuscript for International Journal of Pharmaceutics

Swellable and porous bilayer tablet for gastroretentive drug delivery: Preparation and in vitro-in vivo evaluation

Kyu-Mok Hwanga, Thi-Tram Nguyena, Su Hyun Seoka, Hyun-Il Joa, Cheol-Hee Choa, KyuMin Hwanga, Ju-Young Kimb, Chun-Woong Parkc, Yun-Seok Rheed, and Eun-Seok Parka*

a

School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea

b

College of Pharmacy, Woosuk University, Wanju-gun 55338, Republic of Korea

c

College of Pharmacy, Chungbuk National University, Cheongju 19421, Republic of Korea

d

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang

National University, Jinju 52828, Republic of Korea

*Correspondence to: Eun-Seok Park, Ph.D., School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do, Republic of Korea Tel: +82-31-290-7715; Fax: +82-31-290-7729 E-mail address: [email protected]

Abstract The purpose of this study was to develop a novel gastroretentive drug delivery system with immediate buoyancy and high wet strength. The proposed bilayer tablet was composed of a drug layer and a highly porous and swellable gastroretentive (GR) layer. The highly porous GR layer was prepared by sublimating the volatile materials after compaction with swellable polymers. This pore-forming process decreased the density of the GR layer and enabled the tablet to float immediately on the dissolution media. The GR layer formulation was optimized by comparing the swelling, erosion, and mechanical properties of candidate swellable polymers. The release rates were conveniently controlled by changing the polymer content in the drug layer, while the swelling and floating properties were provided by the GR layer. The application of percolation theory revealed that the polymer content above the estimated threshold was required for a reliable drug release profile. In vivo study in fed beagle dogs confirmed the enhanced gastric retention time of the tablets compared to that of conventional single layer tablets. Taken together, our data suggest that the proposed system can be a promising platform technology with superior GR properties and a convenient formulation process.

Keywords Gastric delivery, sublimation, floating, swelling, percolation, release kinetics

1. Introduction

The ultimate goal for any drug delivery system is to achieve high efficacy with minimal side effects. However, some drugs have limited absorption sites located at the upper gastrointestinal (GI) tract such as the duodenum. As orally administered drugs move due to GI motility, conventional sustained release dosage forms have very limited exposure time to the optimal absorption sites. Also, some diseases require targeted drug delivery to the upper GI tract (e.g., gastric ulcer, Helicobacter pylori infection, and gastric cancer). Therefore, several mechanisms have been proposed for the extension of gastric retention time, but only floating, expanding, and bioadhesive systems are reported to be commercialized (Lopes et al., 2016). However, the ability of the bioadhesive system to extend gastric retention time is doubtful due to the high turnover rates of the gastric mucosa (Bardonnet et al., 2006), and only one product is claimed to be commercialized using bioadhesion mechanism (Xifaxan®, Lupin Pharmaceuticals Inc., Maharashtra, India). Even for that product, only IR dosage forms (Xifaxan® 200 mg and 550 mg tablets, Salix Pharmaceuticals, Morrisville, NC, USA) are approved by the FDA. Thus, to the best of our knowledge, only swelling and floating are well-established mechanisms for GR drug delivery. Swelling GR tablets are similar to large non-disintegrating dosage forms and do not require a significant change in the formulation. It is commonly perceived that dosage forms larger than the gastric pyloric diameter, 12 mm, will not be emptied from the fed stomach (Timmermans and Moës, 1993). However, the gastric pylorus is a sphincter muscle, which 1 Abbreviations: GR, gastroretentive; GI, gastrointestinal; MMC, migrating motor complex; RNT, ranitidine hydrochloride; SR, sustained release; ANOVA, analysis of variance

allows even very large capsules (3.5 cm) to be emptied from the stomach at phase 3 of migrating motor complex (MMC) cycle (Berner and Cowles, 2006). Previous reports also signify that most of the proposed GR systems cannot overcome the MMC phase 3 occurring in empty stomach (Waterman, 2007). However, even in fed subjects, securing reliable GR time is not easy. The gastric emptying time of dosage forms in fed volunteers showed a bimodal distribution regardless of their size, although larger tablets had a lower proportion of the rapid emptying group (Timmermans and Moës, 1994). This provides compelling evidence that large tablets cannot eliminate the chance of erratic gastric emptying during the fed state. Likewise, although floating mechanism can efficiently prolong gastric retention time due to the physical separation of the drug from gastric pylorus (Adibkia et al., 2011), the main drawback with this system is that this mechanism loses its efficacy for supine patients (Cowles et al., 2004). Therefore, a combination of both mechanisms may be used to better ensure gastric retention. Most of the previous studies on floating dosage forms incorporate gas-generating agents, which create gas bubbles after immersion into the gastric fluid. However, this inherently requires a lag time for floating (approx. 30 min) and is under the risk of premature gastric emptying. To overcome the difficulties with lag time in floating, dosage forms containing low-density materials (e.g., polypropylene foam, calcium silicate, and lipids) were developed (Ahmed Abdelbary et al., 2015; Asnaashari et al., 2011; Streubel et al., 2003). However, polypropylene foam is not approved by the FDA, and lipids usually require an excessive amount to make the dosage forms float. Hence, highly porous dosage forms that can float immediately without low-density materials were developed using the sublimation technology

2

(Oh et al., 2013). However, the system had limitations with regards to swelling as a high amount of hypromellose (HPMC) will retard the drug release for less soluble drugs. To rectify the abovementioned problems, a bilayer tablet with the porous GR layer and the drug layer was devised. The GR layer is composed of a swellable polymer with a highly porous structure formed by the removal of volatile materials during the sublimation process. The drug layer may contain formulations typical for hydrophilic sustained release matrix. Using this system, the drug layer immerses in water due to the difference in density and releases the drug into the gastric chyme. On the other hand, the porous GR layer provides immediate buoyancy due to its low density and high wet strength by incorporating a large amount of swellable polymer. By separating the roles of increased gastric retention and the desired drug release profile, this procedure is expected to provide an easy formulation process for various types of drugs. In this study, highly porous bilayer tablets containing ranitidine hydrochloride (RNT) as a model drug was developed for the GR drug delivery. The GR layer was optimized for its swelling and floating properties. The release kinetics of the drug layer was investigated using the percolation theory. The polymer percolation threshold can be defined as the concentration of the polymer above which its infinite cluster is formed (Caraballo, 2009). The effect of polymer percentage on the release mechanism was significantly different below and above the percolation threshold (Gonçalves-Araújo et al., 2008). However, to the best of our knowledge, the percolation theory has not been investigated for bilayer tablets. Hence, the aims of the present study were 1) to show if the developed GR layer had high swelling and floating properties, 2) to show if the release rate from the drug layer could be easily changed

3

and if percolation theory could be applied for bilayer tablets to produce a reliable drug release, and 3) to confirm its extended gastric retention time in vivo.

2. Materials and methods

2.1. Materials

RNT (SMS Pharmaceuticals Limited, Medak, India), L-menthol (Acros Organics, Geel, Belgium), DL-camphor (Junsei Chemical Co., Ltd., Tokyo, Japan), microcrystalline cellulose (MCC) (Avicel® PH 102, FMC Biopolymer, Philadelphia, PA, USA), lactose (FlowLac® 90 and FlowLac® 100, Meggle, Wasserburg, Germany), fumed silica (Aerosil® 200, Evonik, Darmstadt, Germany), magnesium stearate (Mg St, Daejung Chemical, Korea), HPMC (Methocel™ K100 DC2, K4M DC2, K100M DC2, Dow Chemical Co., Midland, MI, USA), polyethylene oxide (PEO) (Polyox™ WSR-303, Dow Chemical Co., Midland, MI, USA), hydroxyethylcellulose (HEC) (Natrosol™ 250 HHX-Pharm, Ashland, Covington, KY, USA), carbomer

(Carbopol®

974

NF,

Lubrizol,

Wickliffe,

OH,

USA),

sodium

carboxymethycellulose (sodium CMC) (Aqualon™ 7HF PH, Ashland, Covington, KY, USA), xanthan gum (Arthur Branwell & Co. Ltd., Essex, UK), locust bean gum (Sigma-Aldrich, St. Louis, MO, USA), carrageenan (Gelcarin GP 379NF, FMC Biopolymer, Philadelphia, PA, USA), hydroxypropycellulose (HPC) (Klucel™ HF Pharm, Ashland, Covington, KY, USA), pregelatinized starch (Swelstar™ MX-1, Asahi Kasei Chemicals, Tokyo, Japan), and the commercially available GR tablet (Glucophage™ XR 1000 mg, Bristol-Myers Squibb, 4

Princeton, NJ, USA) were used in this study. All other ingredients, reagents used in this study were of analytical grade, and all solvents were of HPLC grade.

2.2. Screening of hydrophilic polymers for the GR layer

2.2.1. Gravimetric swelling and erosion To find the optimal swellable polymer for the GR layer, the commercially available hydrophilic polymers (PEO, HEC, carbomer, sodium CMC, xanthan gum, HPMC, locust bean gum, carrageenan, HPC, pregelatinized starch) were first evaluated for their gravimetric swelling and erosion properties. Among the same type of polymers, high molecular weight grades were chosen as they showed high swelling with low erosion. The polymers were compressed into 450 mg of tablets containing only the polymer using single punch hydraulic press (NP-RD10, Natoli Engineering Company, Inc., Saint Charles, MO, USA) with 10 mm round flat punch at a compression force of 1 ton. The prepared tablets were simpler in shape than the final dosage form (oval and convex) for rapid screening and for reducing the effect of tablet geometry (e.g., tablet height to cup depth ratio) caused by different compressibility of each polymer. Another reason was to obtain tensile strength values and to identify whether the polymer tablet has enough mechanical strength for the downstream manufacturing process after compaction (e.g., transport, storage, and coating), which is 1 MPa (Qiu et al., 2016). The swelling and erosion studies were carried out in triplicate (i.e. 3 tablets for each time point) using USP dissolution apparatus 2 (Vision Elite 8, Hanson Research, Chatsworth, CA, USA). The compressed tablets were put into 900 mL of 0.1 M hydrochloric acid. The 5

temperature of the media was set at 37 ± 0.5 oC and the paddle rotation speed was 50 rpm. Wet tablets were taken out from the dissolution vessels at predetermined time points (1, 2, 4, 6, and 12 h), carefully blotted with tissue paper to remove excess water, and weighed. The measured wet masses of the tablets were used to find changes in mass due to water uptake using Eq. (1) (Mehta et al., 2016):

Gravimetric swelling index

-

100

(1)

where mw and mi are the wet and initial mass of the tablets, respectively. Matrix erosion studies were also carried out for the samples used in the swelling studies. The wet tablets were dried in a convection oven at 60oC until the changes in weight were no longer detected. The change in mass due to matrix erosion was calculated using Eq. (2) (Roy and Rohera, 2002):

Erosion ( )

-

100

(2)

where mi and md are the initial and dried mass of the tablets after the predetermined swelling time, respectively.

2.2.2. Radial swelling The radial swelling properties of pure polymer tablets (same with the one used in section 2.2.1.) and highly porous polymer tablets were analyzed. Highly porous tablets were prepared by mixing 20% w/w of milled camphor, 80% w/w of pure polymer (450 mg in total) and 6

further mixing additional 0.5% w/w of fumed silica and 0.5% w/w of Mg St, and compacting using 10 mm round flat punch and 1-ton compression force followed by a pore-forming sublimation process in a vacuum oven (VT-6130M, Heraeus Instruments, Monroeville, PA, USA) at 60oC for 12 h. The tablets were tested using the dissolution tester under the same conditions described in section 2.2.1, but with different time points for tablet withdrawal (2, 6, and 12 h). The radial swelling index was obtained using a texture analyzer (TA.XT plus, Stable Micro Systems, Godalming, UK) to find the swollen radius of the polymer with and without the compression force. The compression force was applied to mimic compression condition in the human stomach. However, the applied force was slightly higher than the reported maximum antral force (approx. 0.65 N) (Marciani et al., 2001) in order to more clearly observe its effect on radial swelling. The wet tablets were placed vertically (compression of the lateral surface of the tablet) on a plate and a flat, round metal probe with 10 cm diameter pressed the swollen tablet at a speed of 0.1 mm/s. The radial swelling index (with 0 or 1 N compression force) was calculated using Eq. (3):

(3)

where ds and di are swollen diameter with or without compression force, and initial diameter, respectively. The study was carried out in triplicate. In addition, the highly porous oval shaped tablets (17.5 mm × 9 mm) containing 400 mg or 800 mg of PEO was also prepared using the same formulation and compression force as mentioned above (20% w/w of milled camphor, 80% w/w of PEO, with additional 0.5% w/w of fumed silica and 0.5% w/w of Mg St, followed by the sublimation process to remove camphor). The prepared oval tablets were 7

tested for their widths after swelling, and were compared with that of an oval-shaped commercial tablet known for its gastroretentive properties (Glucophage™ XR 1000 mg) (Timmins et al., 2005).

2.2.3. Tensile strength The tensile strengths of round flat pure tablets and highly porous tablets (10 mm diameter, same with the tablets used in section 2.2.2.) were analyzed with indirect diametrical hardness test using Eq. (4):

(4)

where H is the hardness of the tablet, d is the diameter of tablet and t is the thickness of the tablet (Fell and Newton, 1970). The hardness values of tablets were measured with a hardness tester (6D, DR. Schleuniger Pharmatron, Thun, Switzerland).

2.3. Screening of the volatile materials for the sublimating agent

The candidate volatile materials; menthol and camphor were evaluated for the sublimation rate after compression with a swellable hydrophilic polymer, PEO (Polyox™ WSR-303), at various ratios. PEO was chosen due to its widespread use in the pharmaceutical industry (Timmins et al., 2014). As the volatile materials were provided as aggregates of crystals, they were sized using a conical mill (Comil U3, Quadro, Waterloo, Canada) using grater screen (hole diameter of 1.016 mm) and a square impeller at 4,500 rpm. The polymers with various 8

ratios of sublimating agents (0–40% w/w) were mixed, followed by the addition of additional 0.5% w/w fumed silica and 0.5% w/w Mg St. The resultant mixtures were compressed into 600 mg of 16 mm × 8 mm oval convex tablets using single punch hydraulic press (NP-RD10, Natoli Engineering Company, Inc., Saint Charles, MO, USA) at a compression force of 1 ton. The tablets were similar in shape to the final dosage form to better predict the time required for complete sublimation during manufacturing of the final dosage form (oval bilayer tablets). The initial weights of the tablets were measured, and the resultant tablets were vacuum dried at 60oC in a vacuum oven. The tablets were taken out from the vacuum oven at predetermined time points (3, 6, 9, and 12 h) and the percentage of sublimated tablets was calculated using Eq. (5):

(5)

where mt, mi, and mv are the mass of tablet after vacuum drying, the initial weight of the tablet, and initial mass of the volatile material in the tablet, respectively. The hardness values of tablets after complete sublimation were also analyzed using a hardness tester.

2.4. Preparation and in vitro evaluation of tablets containing RNT

Prior to studying the release mechanism from the developed bilayer tablets, the release kinetics of conventional flat round single layer tablets containing the model drug was evaluated. The single layer tablets were prepared in round flat shape to understand the effect of changing from conventional round-shaped sustained release tablets to the oval convex 9

bilayer GR tablets in terms of dissolution kinetics and ultimately provide guidance for applying other candidate APIs to the developed system.

2.4.1. Preparation of single layer round flat tablets The effects of polymer molecular weight, and tablet porosity were investigated using the single layer tablets. The formulations of tablets are shown in Table 1. The powder mixtures were compressed into round flat tablets using a single punch hydraulic press (NP-RD10, Natoli Engineering Company, Inc., Saint Charles, MO, USA) with compression force of 1 ton except for the highly porous single layer tablets (BP1–BP7) where the compression force was adjusted to match the target porosity (two-fold increase compared to B1–B7).

2.4.2. Preparation of highly porous bilayer oval convex tablets Porous bilayer tablets were prepared by direct compression of the drug layer, and the GR layer containing camphor using the formulation shown in Table 2, followed by the sublimation process (Oh et al., 2013). The components for the drug layer and the GR layer were weighed and mixed separately using a powder blender (Turbula® T2F, WAB, Muttenz, Switzerland) at a speed of 72 rpm for 5 min. The lubricant was separately added to the resultant powder mixtures and further mixed at a speed of 72 rpm for 1 min. The powder mixtures were compressed into an oval convex-shaped tablet using a single punch hydraulic press (NP-RD10, Natoli Engineering Company, Inc., Saint Charles, MO, USA). The GR layer was compressed first at a tamping force of 0.1 ton, followed by the compaction of both layers with 1-ton force. The compressed bilayer tablets were put into a vacuum oven and sublimated at 60oC under vacuum for 12 h. The weights of tablets before and after vacuum drying were measured to confirm the complete sublimation of camphor. 10

2.4.3. X-ray micro-computed tomography The internal structures of representative tablets (G5) were analyzed with micro-computed tomography (Inveon™, Siemens medical solutions, Knoxville, TN, USA) (Noguchi et al., 2013). The samples were rotated over 360o, and the operations conditions were voltage of 60 kV, current of 400 µA, exposure time of 400 ms and CCD readout of 2048 × 2048. The reconstructed images of cross-sections and 3D images were obtained with the Cobra software package (Exxim Computing Corp., San Francisco, CA, USA).

2.4.4. In vitro dissolution and buoyancy The release profiles of prepared tablets were conducted in triplicate using the USP dissolution apparatus 2. The dissolution media was 900 mL of 0.1 M acetate buffer (pH 4.0) to mimic the gastric pH after administration of ranitidine (Mojaverian et al., 1990), kept at 37 ± 0.5oC, and the paddle speed was 50 rpm. Helical sinkers were only used for highly porous round flat tablets (BP1–BP7) to focus on evaluating the effect of porosity on drug release and to eliminate the effect of floating (location within the dissolution vessel) on release profiles, which was observed previously for cilostazol (Hwang et al., 2017). As increasing the initial porosity of the tablets increased the tablet volume and made the swelling of tablets to be disturbed by sinkers, the amount of MCC was reduced by 45 mg from the formulation (BP1– BP7). Samples (3 mL) were collected at predetermined time points (0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 4.00, 6.00, 8.00, 10.00, and 12.00 h), and filtered through a 0.2 µm nylon syringe filter (Whatman™, GE Healthcare UK Ltd., Buckinghamshire, UK). The volume removed from each solution was replaced with fresh dissolution medium. The filtrates were analyzed with an HPLC system (1100 series, Agilent, Santa Clara, CA, USA). Zorbax SB-C18 11

analytical column (250 mm x 4.6 mm, 5 μm, Agilent, Santa Clara, CA, USA) was used for analysis and the temperature of the column oven was maintained at 30oC. The detection wavelength was set at 322 nm and the mobile phase consisted of methanol and 0.1 M ammonium acetate solution (85:15) with the final pH value of approx. 7.55. The injection volume was 10 μL and the flow rate was 1.0 mL/min. For the porous bilayer tablets (G1–G7), their buoyancy profiles during the dissolution tests were evaluated.

2.4.5. Application of drug release kinetic models The release kinetics of RNT tablets were analyzed by fitting the dissolution profiles to Korsmeyer-Peppas (Korsmeyer et al., 1983) models. Data points from time zero until 60% of the release were used for the fitting. Contrary to commonly used log-transformation methods, the dissolution data were directly fitted to non-linear equations for more accurate results (Vueba et al., 2004).

The Korsmeyer-Peppas model was applied using Eq. (6):

(6) ∞

where Mt/M∞ is the fraction of drug released at time t, k is a kinetic constant, and n is the release exponent. For cylindrically-shaped tablets, the release mechanism is purely diffusionbased when n is 0.43, a combination of diffusion and relaxation (anomalous) when n is between 0.43 and 0.86, and purely relaxation-based (Case II) when n is 0.86 (Korsmeyer et al., 1983). 12

2.4.6. Calculation of physical parameters of the tablets The physical parameters of the tablets that were relevant to the release profiles were calculated. The surface area and volume were calculated based on the actual measurements of tablet diameter and height using a Vernier caliper for the round, flat tablets. For oval convex bilayer tablets, the surface area or volume was calculated using the dimensions of the punch provided by the manufacturer (Natoli Engineering Company, Inc., Saint Charles, MO, USA) assuming a flat interface between the layers and absence of tablet expansion after compression. The thickness of each layer was measured with a Vernier caliper as the interface between each layer could be identified by the difference in color (white GR layer vs. pale yellow drug layer). To calculate the porosity and volumetric percentage of the sustained release agent in the formulation, the true density values of each component were measured using a pycnometer (Accupyc 1330, Micromeritics, Norcross, GA, USA). Helium was used as the measuring gas and the final pressure was 0.738 psig (Wade et al., 2015). The porosity of the tablet was calculated using Eq. (7):

(7)

where Va is the apparent volume and Vs is the skeletal volume of the tablet. The skeletal volume of the tablet was calculated based on the weight fractions and skeletal volumes of each excipient (Cosijns et al., 2007; Hwang et al., 2017). Additionally, the volume fraction of each excipient was also calculated based on its skeletal volume and apparent tablet volume.

13

2.4.7. Determination of the excipient percolation threshold The percolation threshold for the sustained release agent (HPMC) was determined mathematically using various kinetic parameters (Fuertes et al., 2006; Miranda et al., 2006). The derived kinetic parameters (Korsmeyer-Peppas kinetic constant, release exponent) were plotted against the volumetric fraction of HPMC. The range of volumetric percentage below and above the occurrence of significant change, or the inflection point, was regarded as the estimated percolation threshold (Mason et al., 2015).

2.5. In vivo evaluation of porous bilayer tablets containing RNT

2.5.1. Preparation of single and bilayer tablets containing radiopaque threads for in vivo studies The tablets containing radiopaque threads were prepared for the bilayer GR tablet formulation G5 and single layer tablet formulation B5 (Lalloo et al., 2012). For the single layer sustained release (SR) tablets, two 7 mm-long radiopaque threads were placed in the middle of the powder mixture and compressed. For the bilayer GR tablets, two short radiopaque threads (7 mm) were inserted horizontally inside the GR layer, whereas the two long radiopaque threads (15 mm) were inserted vertically into the interface between the drug layer and the GR layer. The dissolution profiles of tablets were compared by calculating the similarity factor (Shah et al., 1998) using Eq. (8):

(8)

14

where n is the number of sampling points, Rt is the dissolution value of the reference at time t, and Tt is the dissolution value of the test batch at time t. Two dissolution profiles having a similarity factor between 50 and 100 were regarded as identical.

2.5.2. Animals Healthy male beagle dogs (age, 12–24 months; body weight, approx.10 kg) were used for in vivo evaluation. The beagle dogs were housed in an animal facility in separate stainless-steel cages at constant room temperature (21 ± 2oC) and humidity (35–65% RH) with an artificial 12/12 h light-dark cycle. Dog diet (300 g, 1050 kcal, Teklad Global 25% Protein Dog Diet, Envigo, Madison, WI, USA) were fed daily with free access to purified water. All animals were cared for in accordance with Institutional Animal Care and Use Committee guidelines.

2.5.3. In vivo gastric retention X-ray imaging was carried out to locate the tablets in vivo, and assess gastric retention as well as the erosion of the porous bilayer gastroretentive tablets (GR) and single layer sustained release tablets (SR). Thirteen beagle dogs were used for the present study, and the dogs were randomly divided into three groups: GR (n = 6), SR (n = 6), and barium sulfate suspension (n=1) group, and the study was conducted in parallel. The animals were fasted overnight prior to drug administration. One dog was used for the preliminary study to locate the organs (e.g., stomach, intestine) by administering 100 mL of 50% w/w barium sulfate suspension. The dogs in the GR and SR group received either GR or SR tablets with radiopaque threads 30 min after being fed with 180 mL of liquidized enteral diet (270 kcal in total, 81 kcal from fat, Carewell 1.5 Plus, Korea Enteral Foods, Co., Ltd., Seoul, Korea). The liquid food and the tablets were all administered by oral gavage followed by 25 mL water 15

rinse. The dogs had free access to water from 1 h post-dosing. A consecutive dog diet (200 g, 700 kcal, Teklad Global 25 % Protein Dog Diet, Envigo, Madison, WI, USA) was provided at 4–5 h post-dosing. Lateral and ventrodorsal x-ray images of the dogs administered with SR or GR tablets were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 12 h. The locations of the tablets within the GI tract were determined from the X-ray images by a licensed veterinarian.

2.6. Statistical analysis A statistical software (SPSS release 21.0, IBM, Chicago, IL, USA) was used to compare means and fit the drug release profile to non-linear equations. Statistical analyses to compare means were performed using Student’s t-test or analysis of variance (ANOVA), followed by post-hoc Tukey’s test. A p-value < 0.05 indicated statistical significance.

3. Results and discussion

3.1. Screening of hydrophilic polymers for the GR layer

The candidate hydrophilic polymers for the GR layer were evaluated for their ability to withstand strong contractile forces of the stomach. It has been reported that the stomach exerts a higher shear and contractile force than the small intestine at phases 2 and 3 of MMC cycle, and at the postprandial state (Sarna et al., 1989; Van Den Abeele et al., 2017). To maximize the swelling property of the developed system, the GR layer was designed to contain only the hydrophilic polymer. Hence, the swelling properties of the candidate materials were compared after being compressed into tablets. Only the hydrophilic polymers 16

that are approved for oral intake were used to ensure the safety of the developed system and rapid product approval. The polymer tablets were tested based on swelling and the erosion properties under in vitro dissolution condition (paddle method). Although this condition did not directly represent the in vivo gastric contraction force impinged on hydrophilic polymers, this enabled the candidate polymer tablets to be compared and ranked.

3.1.1. Gravimetric swelling and erosion To enable rapid screening of polymer tablets, the candidate polymer tablets were first tested for water uptake (gravimetric swelling index) and disentanglement (erosion index). As shown in Fig. 1A, the rank order for the gravimetric swelling indices of the polymer matrices was generally constant throughout the experimental time scale except for carrageenan and PEO. Carrageenan showed very rapid initial swelling followed by a decrease in matrix weight after 4 h, whereas the PEO swelled persistently until 12 h. This is attributed to rapid polymer disentanglement of carrageenan as shown in Fig. 1B. Therefore, the highly eroding polymer tablets with substandard swelling properties (carrageenan, locust bean gum, and sodium CMC) were considered unsuitable for use in the GR layer, hence, they were excluded from further studies. In the case of carbomer and sodium CMC tablets, they disintegrated into several cylindrical fractions during swelling and erosion tests. This may be due to the acidity of the dissolution media and pH-sensitive swelling properties of the two types of the polymer where the binding forces between the polymer particles are reduced. Carbomer is reported to show reduced swelling in acidic media due to its cross-linked acrylic acid structure (Parojĉić et al., 2004), and a similar trend is reported for sodium CMC in which carboxylate ion interacts with hydrogen ion to form a less expanded structure (Yang and Zhu, 2007). 17

3.1.2. Radial swelling The polymer tablets with low erosion properties were compared for their robustness under compression force using a texture analyzer. This was to further investigate the performance of candidate polymer tablets because the swollen gel layer has a significantly lower mechanical strength than the dry core in previous studies (Ali et al., 2017; Jamzad et al., 2005; Mason et al., 2016). The physiologically relevant compression force exerted by the texture analyzer was set at 1 N as the previous report of gastric contraction force was lower than 1 N (Marciani et al., 2001). The tablets were analyzed for their radial swelling instead of axial swelling due to several reasons. Considering the dimensions of the developed bilayer dosage form, the radial swelling of the GR layer was regarded more important as it occurs in two directions whereas the axial swelling of the GR layer occurs in only one direction (i.e. the swelling of GR layer cannot occur rapidly at the interface between the two layers as the exposure to the dissolution media is inhibited by the drug layer). Furthermore, the radial swelling of the bilayer tablet was determined by the highly swellable GR layer and less affected by the swelling of the drug layer, enabling the independent control of the drug release profile and GR properties. The radial swelling index without any compression force showed a similar trend with the gravimetric swelling index (Fig. 1C). However, all tablets showed decreased radial swelling when compressed with texture analyzer (Fig. 1D) and the rank order for radial swelling after applying 1 N compression force did not corroborate with the results without compression (Figs. 1A and 1C). For xanthan gum, although gravimetric swelling and erosion were similar to HPMC, its resistance to compression was weaker than that of HPMC. On the contrary, as opposed to the other types of polymers, the diameter of pregelatinized starch continuously 18

increased for 12 h. The discrepancy between the gravimetric swelling index and the radial swelling index under compression force could be due to the higher mechanical strength of the dry core than the gel layer (Jamzad et al., 2005). This signifies the importance of evaluating wet strength in addition to commonly used volumetric or gravimetric swelling rates to take into account how the matrix will behave in vivo. Hence, xanthan gum and HPC, the polymers with weak wet strength, were considered unsuitable for the GR layer and were excluded from further studies.

3.1.3. Swelling and erosion of high porosity polymer tablets The polymer tablets were evaluated for their swelling, erosion, and resistance to compression after being made into porous tablets to resemble the inner structure of the actual GR layer. As expected, the pore formation by the sublimation method led to tablets with lower resistance to compression (Fig. 2A). In addition, highly porous tablets showed a decreased extent of gravimetric swelling and increased erosion (data not shown). This could be due to the

detrimental effect of high porosity to the integrity of the swollen gel layer.

Unexpectedly, the highly porous tablet containing pregelatinized starch disintegrated during the test, which could be due to the combined effect of low swelling rate and less binding ability between the polymer particles. Among the three types of polymer matrices, PEO generally showed the highest swelling index, although the difference was non-significant at 12 h. In addition, the tensile strengths of the polymer tablets were measured before and after sublimation to assess their suitability for scale-up. PEO showed the highest tensile strength, and was less affected by sublimation (2.8 ± 0.1 MPa before sublimation and 2.1 ± 0.0 MPa after sublimation), meeting the tensile strength requirements for commercial scale 19

manufacturing (1 MPa) (Qiu et al., 2016). In contrast, the tensile strengths of HEC and HPMC decreased significantly after sublimation (HEC: 1.0 ± 0.0 MPa, HPMC: 1.9 ± 0.1 MPa before sublimation and HEC: 0.4 ± 0.0 MPa, HPMC: 0.6 ± 0.0 MPa after sublimation). The high tensile strength of PEO could be due to its highly plastically deforming nature (Klevan et al., 2010; Picker-Freyer, 2006). The significantly reduced tensile strength of the for HEC and HPMC tablets (approx. 50% reduction) may have lowered the ability of the dry cores to resist compression force during the swelling test. Overall, PEO was regarded as the most feasible polymer for GR layer, and used as the optimal GR layer for bilayer tablets. The swelling property of the optimal GR layer was compared with that of the commercial product (Glucophage™ XR 1000 mg). Only the widths of tablets were evaluated as the lengths were much longer for all tablets and tablets should be long enough in two dimensions to prevent gastric emptying (Timmermans and Moës, 1994). As shown in Fig. 2B, the GR layers were initially smaller than the commercial product before swelling to aid in easier administration, but soon became similar or superior to the commercial product after 2 h. Considering that drug layer was not attached to the GR layer during evaluation and that a physiologically relevant compression force (1 N) was applied when measuring the widths, it is logical to say that even the GR layer with lower PEO amount (400 mg) can swell with enough physical integrity to provide extended gastric residence time while maintaining acceptable initial tablet size for easy swallowing.

3.2. Screening of the sublimating agents for the GR layer

20

Candidate volatile materials were included in the GR layer of the system and sublimated after compaction by vacuum drying to increase the porosity and enable the tablet to float on water. The tablets were tested for their sublimation rate and mechanical hardness. Although camphor and menthol were both completely removed from the tablets after 12 h of vacuum drying, the sublimation rates for the two volatile materials were compared as the time required for the sublimation of volatile materials were directly related to the productivity of the manufacturing process. In addition, the hardness of the GR layer after sublimation was also important as low hardness could lead to tablet breakage during transport, storage, and coating. Therefore, oval convex tablets containing various ratios of volatile materials were prepared with the PEO as the hydrophilic polymer and their effect on sublimation rate and tablet mechanical strength was investigated. As shown in Fig. 3A, the sublimation rate was decreased for the increased initial amount of menthol in the formulation. This could be due to a disproportional increase in the total amount of menthol to be sublimated and the surface area available for sublimation. On the contrary, the sublimation of tablets containing camphor was completed in 3 h regardless of the initial camphor content. This difference could be caused by the symmetrical molecular structure of camphor, leading to low dipole moment strength and intermolecular force (Jones, 1960; Pavia et al., 2015). As expected, the increasing amount of sublimating agent decreased the hardness of the tablets (Fig. 3B); it has long been known that increase in tablet porosity correlates with a decrease in tablet hardness (Grymonpré et al., 2016). In addition, there were clear differences between the hardness values of tablets prepared using camphor and those prepared using menthol which could be caused by the difference in compressibility (Leuenberger, 1982). This was partially proved by the fact that the tablets prepared with camphor were always 21

thinner than those prepared with menthol (data not shown). Hence, camphor was chosen as the sublimating agent for optimal GR layer in subsequent studies.

3.3. In vitro evaluation of the tablets containing RNT

The developed system was first analyzed for its internal structure to observe the homogeneous distribution of pores within the GR layer of the tablet, followed by in vitro buoyancy and dissolution testing. The effects of various factors including tablet shape, multilayer structure, HPMC grade, and HPMC ratio on the dominant release mechanisms were evaluated. Additionally, percolation theory was applied to the tablets with various formulations and dimensions to mechanistically understand the factors that may affect drug release from bilayer tablets.

3.3.1. Observing internal structures of bilayer tablets with X-ray micro-computed tomography The comparison of Figs. 4A and 4B shows the effect of the sublimating agent on the internal structure of the GR layer. It can be seen that only the GR layer was affected by the pore formation (sublimation) process. Additionally, the pores are distributed homogeneously throughout the matrix, suggesting that buoyancy will be maintained even after a slight surface erosion of the GR layer in vitro or in vivo. The difference in the porosity of each layer suggests that the porous GR layer will float above the drug layer, inducing the drug layer to be completely immersed into the dissolution medium or the gastric fluid.

3.3.2. Dissolution testing for single and bilayer tablets 22

The release kinetics of RNT was investigated by varying the ratio of sustained release agent, HPMC, observing its release profiles, and fitting them to Korsmeyer-Peppas model. To understand the release kinetics of the model drug, RNT, the release kinetics of the single layer tablets were also studied and compared with that of porous bilayer tablets. Independent of the drug layer formulation, the prepared bilayer tablets floated immediately, i.e., the tablets sank less than 5 cm when they were first dropped into the vessel and floated above the dissolution media within 3 s. Further, the tablets remained buoyant for longer than 12 h during the dissolution test. This implies that the buoyancy can be ensured regardless of the drug layer formulation. As illustrated in Fig. 5A, the release of the drug was highly affected by the amount of the sustained release agent, HPMC 4,000 cps. This indicates that the drug release profiles could be fine-tuned to meet the clinical requirement of the product (e.g., b.i.d. or q.d. dosing). However, the formulations with low HPMC amounts (5% and 10% w/w HPMC) were similar to immediate-release dosage forms, and the release rate was highly variable. For the single layer tablets (Fig. 5B), the release rate was less influenced by the amount of HPMC and they generally showed slower dissolution rates compared to the porous bilayer tablets containing identical formulations of the drug layer. Furthermore, the release profiles for tablets containing HPMC ratios equal to or higher than 30% w/w were almost identical to each other. The differences in dissolution profiles between single and bilayer tablets could be attributed to either the increase in tablet porosity (Aguilar-de-Leyva et al., 2012; Hiremath and Saha, 2008; Nokhodchi et al., 1996) or the tablet surface area to volume ratio (Reynolds et al., 2002). It could be seen that porosity was almost doubled whereas the surface area to volume ratio was decreased (B1–B7 and G1–G7 from Tables 1 and 2). The increase in porosity could be due to the convex shape of the bilayer tablet (Diarra et al., 2015). Considering that the surface area to volume ratio was decreased for the bilayer tablets, it 23

appears that the increased release rate was due to the higher porosity of the drug layer in the bilayer tablets.

3.3.3. Release kinetic modeling of single and bilayer tablets The drug release profiles of round, flat and porous bilayer tablets were evaluated by fitting their drug release profiles to the Korsmeyer Peppas model, and plotting the kinetic constants against the HPMC content. These plots were also used to mathematically determine the percolation threshold (Mason et al., 2015). The exact value may change due to the variability of experimental conditions in the homogeneous mixing of HPMC, and the selection of different HPMC ratios for regression. This is why the threshold value is indicated as a range of HPMC % v/v, instead of a specific value (Gonçalves-Araújo et al., 2008). The release kinetic parameter for the standard round flat single layer tablet (Fig. 6A) shows that the kinetic constant decreased gradually by increasing the HPMC content until a lower limit is reached (approx. 0.3). This trend could also be interpreted as the existence of the percolation threshold, or the point where slope changes, between 14.23 and 28.21% v/v HPMC. The release exponent was generally higher than 0.43, but lower than 0.86, indicating anomalous release (combined effect of diffusion and relaxation) except for the tablets with a very low amount of HPMC (5 % w/w), which implied Fickian diffusion. As shown in Fig. 6B, the tablets with high porosity (BP1–BP7) showed highly variable kinetic constant at the HPMC contents below the estimated percolation threshold (13.30– 25.06% v/v). The trend was also similar for the tablets with low molecular weight polymer (Fig. 6C, HPMC 100 cps), which also showed similar percolation threshold range (13.38– 26.31% v/v) and increased variation below the threshold. This corroborates with previous studies indicating the existence of a percolation threshold value near 30% w/w (Ghimire et al., 24

2010; Tajarobi et al., 2009). According to percolation theory, the rapid release with high variability is due to the formation of non-continuous and discrete HPMC clusters. On the contrary, the release parameters for formulations above the threshold became similar to each other, indicating the formation of a continuous cluster to retard the drug release. Interestingly, the kinetic constants and the release exponents of the formulations above the percolation threshold (BP5–BP7 and A5–A7) were similar to those of the conventional round flat tablets with HPMC 4,000 cps (B5–B7). This suggests that it is important to have the polymer content above the percolation threshold to ensure reliable drug release from tablets regardless of the polymer molecular weight or tablet porosity. The release kinetic parameters for the porous bilayer tablets (Fig. 6D) also followed a similar trend to those of highly porous and low HPMC content formulations. In fact, the release rate of G1 was similar to that of BP1, and was too high to be fitted to any of the kinetic models. Although the formulation was equivalent to those of single layer tablets containing HPMC 4,000 cps, the highly variable and rapid dissolution rate below the percolation threshold could be attributed to the increased porosity of convex tablets as previously discussed. In addition, it showed significantly low (below 0.43) release exponent values for formulations with low HPMC contents, which is rarely found in conventional SR tablets. The lower release exponent value indicates a rapid initial release of the drug followed by a sudden decrease in release rate. This could be due to the unique shape and geometry of the tablet as the Korsmeyer-Peppas equation was not originally used for tablets with the GR layer attached (Ritger and Peppas, 1987). Nevertheless, the result indicated the threshold value is located between 11.48 and 21.69% v/v. This is similar to the threshold value obtained for round, flat, single layer tablets of the same formulation (B1–B7). This indicates that percolation theory is applicable to the porous bilayer tablets, and it emphasizes that HPMC 25

amount above a critical point (threshold) is necessary to obtain a reliable dissolution profile (Ghimire et al., 2010).

3.4. In vivo evaluation of the tablets containing RNT

In vivo performance of floating porous bilayer GR tablets were compared to that of nonfloating tablets to confirm prolonged gastric retention time. Beagle dogs were used in this study as they are the most widely used non-rodent species used to assess the in vivo performance of GR dosage forms (Klausner et al., 2003) with similar gastric anatomy and postprandial myoelectric activity to humans (Gill et al., 1985). As the higher restrictive nature of the pylorus is reported for dogs compared to humans (i.e., short pyloric diameter), the GR time in dogs for non-disintegrating tablets may not be directly transferrable to humans (Lalloo et al., 2012). However, the focus of this study was to analyze the ability of the developed system to maintain its integrity and stay in the fed stomach. Especially, the ability for the developed GR system to withstand canine gastric contractile force (Martinez and Papich, 2009) can provide compelling evidence that the dosage form will stay intact and float in human stomach (markedly weaker contraction than that observed in dogs). Therefore, the dogs were tested at fed state, and the consecutive meal was provided at 4 h to test the tablets in the stomach. Formulation G5 (GR tablet) and B5 (SR tablet) were chosen as they had the same drug layer formulation with similar dissolution profiles (f2 = 72.92). After inserting the radiopaque threads, their buoyancy and dissolution profiles were not affected and remained similar to each other (f2 = 63.51 between GR and SR tablets).

26

As detailed in the representative X-ray images of SR and GR tablets on the left-hand side of Fig. 7, pairs of radiopaque threads were incorporated in the formulation not only to aid the location of the tablets in the organs but also to identify matrix erosion and layer separation for bilayer tablets. Radiographic imaging after inserting a pair of radiopaque threads into the dosage form is a convenient technique which provides the location and intactness of the dosage form while not significantly changing its formulation and density. This technique was applied for unfolding polymeric films as well as single layer tablets (Klausner et al., 2003; Klausner et al., 2002; Lalloo et al., 2012). Typical images of intact tablets and eroded tablets are shown on the right-hand side of Fig. 7. The separation of the pair of threads in the SR tablets after 12 h indicates the erosion of the tablet. Similarly for GR tablets, separation of long threads with short threads remaining close to each other indicates complete erosion of only the drug layer, but not the GR layer. As evident from Table 3, the gastric retention time of non-floating SR tablets was highly variable (3 to 12 h). For three dogs, the tablets eroded in the fed state before being emptied from the stomach, indicating the tablets were not strong enough to withstand the high shear force impinged by the stomach. For two dogs, the radiopaque tablets were not found. This could be due to the erosion of the tablet before or after gastric emptying, or the tablet could be excreted from the body, which is less probable. One dog showed gastric emptying of the intact tablet. These unreliable results in gastric retention time could be attributed to the low wet strength of the drug layer, compromising the gastric retention properties of the dosage form. On the contrary, increased and reliable gastric retention time compared to SR tablets for GR tablets can be seen from Table 4. The gastric retention time for GR tablets was longer than 8 h for all the dogs. Gastric emptying between 8 and 12 h occurred only for the dogs that 27

refused to eat solid food provided at 4–5 h or excreted liquid stool (diarrhea). For the three dogs that had consecutive meals, GR tablets were not emptied from the stomach until 12 h. For two of the dogs that had the tablets in the stomach at 12 h (Dogs no. 3 and 5), only the long threads placed between GR layer and the drug layer disappeared. This indicates that the GR layer could maintain its integrity while the drug layer was completely eroded, which confirms the robustness of the GR layer against gastric contraction and ultimately its ability to increase the retention time in human by swelling and floating.

4. Conclusion

In this study, a novel bilayer GR drug delivery system containing a porous swellable polymer layer and a drug layer was successfully developed. Various release profiles could be achieved by altering the ratio of HPMC, a sustained release agent, in the drug layer without affecting the floating and swelling properties of the bilayer tablets. The effect of changing tablet porosity and HPMC grades was more significant for tablets with HPMC ratios below the estimated percolation threshold, accompanied with highly variable release profiles, again implying the significance of the percolation theory in obtaining a robust formulation. The in vivo study clearly demonstrated higher gastric retention times for the GR tablets than conventional SR tablets in fed dogs, indicating the ability of the GR layer to maintain its shape against strong gastric motility. The formulation study is required for only the drug layer; hence, the developed system could be a promising platform technology for efficient stomachspecific delivery of drugs.

28

Acknowledgments

This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors.

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Timmermans, J., Moës, A.J., 1993. The cutoff size for gastric emptying of dosage forms. J. Pharm. Sci. 82, 854-854. https://doi.org/10.1002/jps.2600820821. Timmermans, J., Moës, A.J., 1994. Factors controlling the buoyancy and gastric retention capabilities of floating matrix capsules: new data for reconsidering the controversy. J. Pharm. Sci. 83, 18-24. https://doi.org/10.1002/jps.2600830106. Timmins, P., Donahue, S., Meeker, J., Marathe, P., 2005. Steady-state pharmacokinetics of a novel extended-release

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formulation.

Clin.

Pharmacokinet.

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721-729.

https://doi.org/10.2165/00003088-200544070-00004. Timmins, P., Pygall, S.R., Melia, C.D., 2014. Hydrophilic Matrix Tablets for Oral Controlled Release. Springer. Van Den Abeele, J., Rubbens, J., Brouwers, J., Augustijns, P., 2017. The dynamic gastric environment and its impact on drug and formulation behaviour. Eur. J. Pharm. Sci. 96, 207-231. https://doi.org/10.1016/j.ejps.2016.08.060. Vueba, M., De Carvalho, L.B., Veiga, F., Sousa, J., Pina, M., 2004. Influence of cellulose ether polymers on ketoprofen release from hydrophilic matrix tablets. Eur. J. Pharm. Biopharm. 58, 51-59. https://doi.org/10.1016/j.ejpb.2004.03.006. Wade, J., Martin, G., Long, D., 2015. The evolution of granule fracture strength as a function of impeller tip speed and granule size for a novel reverse-phase wet granulation process. Int. J. Pharm. 488, 95-101. https://doi.org/10.1016/j.ijpharm.2015.04.033. Waterman, K.C., 2007. A Critical Review of Gastric Retentive Controlled Drug Delivery. Pharm. Dev. Technol. 12, 1-10. 10.1080/10837450601168680. Yang, X.H., Zhu, W.L., 2007. Viscosity properties of sodium carboxymethylcellulose solutions. Cellulose 14, 409-417. https://doi.org/10.1007/s10570-007-9137-9.

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Figure captions

Figure 1. (A) Gravimetric swelling index, (B) erosion index, (C) radial swelling index without compression force, and (D) radial swelling index with 1 N compression force as a function of hydration time for polymer tablets (n=3, mean ± SD). Figure 2. (A) Radial swelling index under 1 N compression force of high porosity polymer tablets and (B) swollen tablet width for the optimal GR layers and the commercial product (Glucophage XR 1000 mg) (n=3, mean ± SD). Figure 3. Effect of sublimating agent type and content on (A) sublimation rate and (B) hardness of highly porous PEO tablets (n=3, mean ± SD). Figure 4. Representative 3D reconstructed X-ray micro-computed tomographic images of bilayer GR tablets (G5) (A) before sublimation and (B) after sublimation (GR layer facing up). Figure 5. Effect of HPMC content on dissolution profiles of (A) GR bilayer tablets (G1–G7) and (B) single layer tablets (B1–B7) (n=3, mean ± SD). Figure 6. Effect of HPMC content on Korsmeyer Peppas kinetic constant and release exponent for (A) round flat tablets with HPMC 4,000 cps (B1–B7), (B) highly porous round flat tablets with HPMC 4,000 cps (BP1–BP7), (C) round flat tablets with HPMC 100 cps (A1–A7), and (D) oval convex GR bilayer tablets with HPMC 4,000 cps (G1–G7) (n=3, mean ± SD). Figure 7. Representative X-ray images of tablets with radiopaque threads and beagle dog no. 5 (ventrodorsal view) after administration of (A) single layer SR and (B) bilayer GR tablets. 35

Table 1. Composition, surface area to volume ratio, and initial porosity for the 10 mm round flat single layer SR tablets

(mg)

A1

A2

A3

A4

A5

A6

A7

B1

B2

B3

B4

B5

B6

B7

BP1

BP2

BP3

BP4

BP5

BP6

BP7

RNT

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

150

MCC

95

95

95

95

95

95

95

95

95

95

95

95

95

95

50

50

50

50

50

50

50

Lactosea

225

200

175

150

100

50

0

225

200

175

150

100

50

0

225

200

175

150

100

50

0

HPMC 100 cpsb

25

50

75

100

150

200

250

-

-

-

-

-

-

-

-

-

-

-

-

-

-

HPMC 4,000 cpsc

-

-

-

-

-

-

-

25

50

75

100

150

200

250

25

50

75

100

150

200

250

Fumed silica

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

Mg St

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

Total weight

500

500

500

500

500

500

500

500

500

500

500

500

500

500

455

455

455

455

455

455

455

9.08 13.38 17.82 26.31 33.91 42.51 4.87

% v/v HPMCd

4.62

Surface area to volume ratiod

0.794 0.787 0.781 0.780 0.774 0.761 0.762 0.803 0.797 0.792 0.790 0.787 0.769 0.760 0.782 0.778 0.767 0.759 0.747 0.739 0.729

Initial porosityd

0.146 0.154 0.164 0.159 0.161 0.179 0.166 0.123 0.123 0.131 0.125 0.118 0.150 0.159 0.237 0.239 0.256 0.266 0.280 0.283 0.295

9.66 14.23 18.96 28.21 35.68 43.44 4.63

a

Flowlac® 90 K100 DC2 c Methocel™ K4M DC2 d n=3, mean values are shown b Methocel™

36

9.14 13.30 17.34 25.06 33.42 41.77

Table 2. Composition, surface to volume ratio, and initial porosity for the 17.5 mm × 9 mm oval convex bilayer GR tablets

Drug Layer

GR Layer

(mg)

G1

G2

G3

G4

G5

G6

G7

RNT

150

150

150

150

150

150

150 95

MCC

95

95

95

95

95

95

Lactosea

225

200

175

150

100

50

0

HPMC 4,000 cpsb

25

50

75

100

150

200

250

Fumed silica

2.5

2.5

2.5

2.5

2.5

2.5

2.5

Mg St

2.5

2.5

2.5

2.5

2.5

2.5

2.5

Camphor (milled)c

100

100

100

100

100

100

100

PEO

400

400

400

400

400

400

400

Fumed silica

7.5

7.5

7.5

7.5

7.5

7.5

7.5

Mg St

2.5

2.5

2.5

2.5

2.5

2.5

2.5

910.0

910.0

910.0

910.0

910.0

910.0

910.0

Total weight after sublimation % v/v HPMCd

3.95

7.89

11.48

15.08

21.69

28.33

34.31

Surface area to volume ratiod,e

0.559

0.558

0.552

0.549

0.540

0.536

0.530

Initial porosityd

0.288

0.284

0.299

0.304

0.322

0.325

0.336

a

Flowlac® 90 b Methocel™ K4M DC2 c Removed after compaction by sublimation process d Calculated based on only the drug layer (n=3, mean values are shown) e Surface area available for dissolution (excluding the interfacial surface area) divided by the bulk volume of the drug layer

37

Table 3. Location and physical integrity (e.g. erosion) of non-floating SR tablets in vivo determined with X-ray imaging at predetermined time points

Time (h)

GRTa (h)

Dog no. 0

0.5

1

1.5

1

2

2.5

3

4

5

6

7

12

N/Ab

Stomach (intact)

2c

8

5–6 N/Ab

Stomach (intact)

3

Stomach (intact)

4

Complete erosion

Stomach (intact)

Complete erosion

5

Stomach (intact)

6

Stomach (intact)

Intestined

8–12 6–7

3–4

Complete erosion Complete erosion

5–6 5–6

a

Estimated gastric retention time b Disappearance of tablet possibly due to disintegration or emptying from body c Dog no. 2 did not eat consecutive solid meal at 4 h d Gastric emptying of intact tablet

Table 4. Location and physical integrity (e.g. erosion) of floating GR bilayer tablets in vivo determined with X-ray imaging at predetermined time points

Time (h) GRTa (h)

Dog no. 0

0.5

1

1.5

2

2.5

3

4

5

6

7

8

12

1b

Stomach (intact)

N/Ac

8–12

2d

Stomach (intact)

Intestinee

8–12

3

Stomach (intact)

Intact GR layerf

> 12

4

5 6d

Stomach (intact)

> 12

Stomach (intact)

Intact GR layerf

> 12

Stomach (intact)

Intestinee

8–12

a

Estimated gastric retention time b Dog no. 1 excreted watery feces at ~ 11.5 h c Disappearance of tablet possibly due to emptying from body d Dog no. 2 and 6 did not eat consecutive solid meal at 4 h e Gastric emptying of bilayer tablet (intact GR layer and drug layer) f Separation of long threads indicating erosion of only the drug layer (parallel short threads indicating intact GR layer in the stomach)

38

B

120

120

100

100

Cumulative drug release (%)

Cumulative drug release (%)

A

80

60

G1 (5% HPMC) G2 (10% HPMC) G3 (15% HPMC) G4 (20% HPMC) G5 (30% HPMC) G6 (40% HPMC) G7 (50% HPMC)

40

20

80 B1 (5% HPMC) B2 (10% HPMC) B3 (15% HPMC) B4 (20% HPMC) B5 (30% HPMC) B6 (40% HPMC) B7 (50% HPMC)

60

40

20

0

0 0

2

4

6 Time (h)

8

10

12

0

2

4

6 Time (h)

8

10

12

A

B 500

100

400

80

60 10% camphor 20% camphor 30% camphor 40% camphor 10% menthol 20% menthol 30% menthol 40% menthol

40

20

0

Tablet hardness (N)

Percentage sublimated (% w/w)

120

Camphor Menthol 300

200

100

0 0

2

4

6 Time (h)

8

10

12

0

10

20

30

Amount of sublimating agent (% w/w)

40

B 180

Swollen tablet width (mm, 1 N compression force)

Radial swelling index (%, 1 N compression force)

A 160 PEO HEC HPMC

140 120 100 80 60 40 20 0 0

2

4

6 Time (h)

8

10

12

16 14 12 10 8 6 Optimal GR layer (PEO 400 mg) Optimal GR layer (PEO 800 mg) Commercial product

4 2 0 0

2

4

6 Time (h)

8

10

12

A

B 80

1000

70 PEO HEC Carbomer Sodium CMC Xanthan gum HPMC Locust bean gum Carrageenan HPC Pregelatinized starch

600

400

60

Erosion index (%)

Gravimetric swelling index (%)

800

Carrageenan Locust bean gum Sodium CMC HPMC Xanthan gum Pregelatinized starch PEO HEC HPC Carbomer

50 40 30 20

200

10 0 2

4

Radial swelling index (%, 0 N compression force)

C

6

8

10

0

12

0

180 160 140 120

PEO HEC HPMC Xanthan gum Pregelatinized Starch HPC

100 80 60 40 20 0 0

2

4

6 Time (h)

2

4

D

Time (h)

8

10

12

Radial swelling index (%, 1 N compression force)

0

6

8

10

12

Time (h) 180 160 140 120

Pregelatinized starch HPMC PEO HEC HPC Xanthan gum

100 80 60 40 20 0 0

2

4

6 Time (h)

8

10

12

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

39