Rida Astutik_172210101097

RESUME TEKNOLOGI SEDIAAN SOLIDA “Preformulation studies and optimization of sodium alginate based floating drug delivery system for eradication of Helicobacter pylori”

Guna Melengkapi Tugas Teknologi Sediaan Solida

Dosen Pengampu: “ Eka Deddy Irawan, S.Si.,M.Sc., Apt.”

Disusun oleh:

Sri Eka Agustin

(172210101020)

Rida Astutik

(172210101097)

Talidah Alqibtiyah Roja

(172210101141)

FAKULTAS FARMASI UNIVERSITAS JEMBER 2019

Bentuk Sediaan Tablet apung efferfescent Bahan-bahan yang digunakan dalam preformulasi : Metronidazole digunakan sebagai zat aktif. Natrium alginat sebagai agen pembengkakan. Hydxypropyl cellulose B1 (L-HPC B1) sebagai superdisintegrant. Sodium bikarbonat digunakan sebagai agen effervescent. Bedak, magnesium stearat, dan silikon dioksida koloid hidrofilik sebagai eksipien untuk kompresi tablet. HPLC grade potassium dihydrogen phosphate dan metanol digunakan untuk uji HPLC. Barium sulfat sebagai bahan kontras sinar-X .

Ukuran Parikel 1.

Pengujian isotermal

Memasukkan metronidazole - campuran eksipien dalam botol kaca 15 ml dan vortex selama 2 menit. Kemudian botol disegel dengan gelas karet dan disimpan pada suhu 500C selama 3 minggu. Sampel diperiksa secara berkala untuk menentukan perubahan warna sampel yang tidak normal. Kondisi analisis yaitu fase gerak campuran 300 ml metanol dan 700 ml 0,01 M kalium dihidrogen fosfat, dengan laju aliran 1 ml / menit, deteksi 315 nm, injeksi 10l µl dan semua sampel sebelumnya disaring dengan 0,2 µm. 2. Studi pelepasan metronidazol 2.1 Studi disolusi in vitro Peningkatan jumlah natrium alginat menurunkan deviasi standar pelepasan, akibatnya membuat komposisi lebih dapat direproduksi. 2.2. Kinetika pelepasan obat tablet memiliki disolusi lambat dijelaskan oleh model Higuchi, yang mengacu pada difusi obat dari matriks polimer. Sampel-sampel ini mengandung 10% atau 15% natrium alginat dalam formulasi. 2.3. Optimasi pengapungan yang signifikan dengan bobot 12,75 ± 1,87mN (1300,14 ± 170,69mg).

Kompresibilitas Untuk memperbaiki dari kompresibilitas tablet dilakukan penambahan mucoadhesi secara ex-vivo. Tablet MF_OPT menghasilkan kekuatan detasemen yang jauh lebih tinggi dibandingkan dengan MF_OPT_L-HPC dan MF_OPT_EXC. Tablet tanpa L-HPC B1 memiliki kekuatan detasemen yang jauh lebih tinggi daripada tanpa kedua eksipien

Studi kompatibilitas eksipien obat Studi DTA menunjukkan puncak endotermik tajam metronidazole murni pada 159,9 o

C puncak titik lebur. Pada proporsi komposisi MF OPT, eksipien tidak menunjukkan

perubahan yang signifikan dari titik leleh metronidazol dibandingkan dengan nilai titik lebur (159-163 oC ) menurut literatur. Tidak ada interaksi antara metronidazole dan eksipien. Pengamatan visual dari pengujian stres isotermal tidak menunjukkan perubahan signifikan dalam warna atau bentuk. Setelah 3 minggu dilakukan uji HPLC yang menunjukkan stabilitas yang tepat. Dalam kromatogram, waktu retensi metronidazol sekitar 7 menit dan tidak ada puncak tambahan yang dapat ditemukan mengacu pada produk dekomposisi.

Studi reologi Organisme dijepit dalam cawan petri yang dilapisi Sylgard dan lendirnya diangkat dengan cermat dan hati-hati di bawah mikroskop operasi. Lendir lambung dimasukkan dalam 0,1 M HCl mengandung 0,9% NaCl, dan didispersikan dengan homogenizer 9500 rpm selama 2 menit. Kemudian lendir disentrifugasi dengan 5.500 rpm selama 1 jam. Pelet didialisis dengan Membran sel 4 oC selama 24 jam. Selanjutnya lendir disentrifugasi 15.000 rpm selama 1 jam. Pelet disimpan pada suhu 15 oC. Larutan 3% lendir dan formulasi yang dioptimalkan (MF_OPT) yang diseimbangkan menjadi 3% L-HPC dan natrium alginate. Perilaku viskositas (kurva aliran) sampel diimplementasikan dalam rotasi viscometer. Interval laju geser 0-25 detik dengan pengambilan sampel 6 detik selama 2 menit. MF_OPT diseimbangkan menjadi 3% L-HPC dan natrium alginat dan mucus adalah viskositas larutan lendir 33%. Semua percobaan dilakukan dalam rangkap tiga.

Sifat Fisika dan Kimia Sifat fisikokimia ditunjukkan dalam Tabel 2. Ketebalan tablet 483 berada antara 4,80 ± 0,07 dan 8,80 ± 0,05. Variasi dan keseragaman berat disimpulkam baik di antara batch. Hasil tablet menunjukkan kekerasan yang cukup.

Kesimpulan Hasil eksperimen menunjukkan rentang konsentrasi yang luas dari natrium alginat, hidroksipropil selulosa tersubstitusi rendah dan natrium bikarbonat. Mucoadhesi ex vivo menunjukkan hasil yang signifikan dari komposisi yang dioptimalkan, dapat bekerja dengan mekanisme mengambang untuk mencapai gastroretensi yang lebih baik, kesamaan yang signifikan antara disolusi spektrofotometri dan terdeteksi secara mikrobiologis.

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Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research Paper

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Preformulation studies and optimization of sodium alginate based floating drug delivery system for eradication of Helicobacter pylori

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Péter Diós a,⇑, Sándor Nagy a, Szilárd Pál a, Tivadar Pernecker a, Béla Kocsis b, Ferenc Budán c,d, Ildikó Horváth c, Krisztián Szigeti c, Kata Bölcskei e, Domokos Máthé c, Attila Dévay a a

Institute of Pharmaceutical Technology and Biopharmacy, University of Pécs, Rókus Str. 2, H-7624 Pécs, Hungary Department of Microbiology, University of Pécs, Szigeti Str. 12, H-7624 Pécs, Hungary CROmed Translational Research Centers, Baross Str. 91-95, H-1047 Budapest, Hungary d Department of Public Health Medicine, University of Pécs, Szigeti Str. 12, H-7624 Pécs, Hungary e Department of Pharmacology and Pharmacotherapy, University of Pécs, Szigeti Str. 12, H-7624 Pécs, Hungary b c

a r t i c l e

i n f o

Article history: Received 28 February 2015 Revised 21 May 2015 Accepted in revised form 21 July 2015 Available online xxxx Keywords: Floating force Sodium alginate Low substituted hydroxypropyl cellulose Ex vivo mucoadhesion Gastroretention X-ray CT imaging

a b s t r a c t The aim of this study was to design a local, floating, mucoadhesive drug delivery system containing metronidazole for Helicobacter pylori eradication. Face-centered central composite design (with three factors, in three levels) was used for evaluation and optimization of in vitro floating and dissolution studies. Sodium alginate (X1), low substituted hydroxypropyl cellulose (L-HPC B1, X2) and sodium bicarbonate (X3) concentrations were the independent variables in the development of effervescent floating tablets. All tablets showed acceptable physicochemical properties. Statistical analysis revealed that tablets with 5.00% sodium alginate, 38.63% L-HPC B1 and 8.45% sodium bicarbonate content showed promising in vitro floating and dissolution properties for further examinations. Optimized floating tablets expressed remarkable floating force. Their in vitro dissolution studies were compared with two commercially available non-floating metronidazole products and then microbiologically detected dissolution, ex vivo detachment force, rheological mucoadhesion studies and compatibility studies were carried out. Remarkable similarity (f1, f2) between in vitro spectrophotometrically and microbiologically detected dissolutions was found. Studies revealed significant ex vivo mucoadhesion of optimized tablets, which was considerably increased by L-HPC. In vivo X-ray CT studies of optimized tablets showed 8 h gastroretention in rats represented by an animation prepared by special CT technique. Ó 2015 Elsevier B.V. All rights reserved.

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1. Introduction

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The aim of designing gastroretentive drug delivery systems (GDDS) was to increase their bioavailability, to reach better predictability of drug liberation, to decrease adverse effects and – when required – to achieve site-specific drug delivery. In comparison with conventional dosage forms, gastroretentive drug delivery systems have the potential to achieve prolonged gastric residence due to several possible mechanisms and technologies [1]. Floating drug delivery systems (FDDS) belong to GDDS and have the property of flotation in gastric media due to the decrease in the density of the dosage form. Two different technological approaches may be distinguished among FDDSs: (1) non-effervescent systems based on the gel-forming of polymers and (2) effervescent systems utilizing gas generating agents (e.g. carbonates, bicarbonates). In

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⇑ Corresponding author. Tel.: +36 70 5456868; fax: +36 72 503 617. E-mail address: [email protected] (P. Diós).

effervescent floating systems, the formed carbon dioxide gas is entrapped in a swollen matrix. In floating drug delivery systems, characterization of physical floating mechanisms and kinetics may pose a challenge. Floating lag time and total floating time studies are generally performed; however, vertically expressed floating force of dosage forms may also have significant role, as introduced by Timmermans and Moes [2,3]. In this study, sodium alginate was used as a swelling polymer. Sodium alginate generally applied not only in pharmaceutics, but also in food industry [4] is a non-toxic, biodegradable copolymer of L-guluronic and D-mannuronic acid blocks. In water, sodium alginate simultaneously hydrates and swells but in acidic media insoluble rubbery alginic acid is produced after protonation. Our previous studies have shown that a 17.82% or higher concentration of sodium alginate in floating tablets results in a maximum of 35% API release within 4 h [5]. Thus, designing a suitably timed drug release from direct compressed tablets based on sodium alginate may be challenging. Additionally, ex vivo studies [6,7] have

http://dx.doi.org/10.1016/j.ejpb.2015.07.020 0939-6411/Ó 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: P. Diós et al., Preformulation studies and optimization of sodium alginate based floating drug delivery system for eradication of Helicobacter pylori, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.07.020

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reported sodium alginate to be mucoadhesive, which could cause a better gastric retention. In order to control drug release from matrix tablets, not only frame creating polymers but also disintegrants may be applied accelerating the release. There are many types of disintegrants with various mechanisms including swelling, particle repulsion and gas generation. Low substituted hydroxypropyl cellulose (L-HPC) is a disintegrant having a high swelling force via rapid and intense water uptake [8]. The typical concentration of L-HPC for tablet disintegration is 2.5–5.0% [9]. Several articles have aimed at designing floating tablets which can prolong the drug delivery time to 8 or more hours [10–12]. Numerous articles have suggested that a gastric residence of 5 or more hours may only be achieved with sufficient food or beverage consumption [13–16]; however, it results in varying pH and unpredictable gastric residence time of dosage forms. Moreover, dissolution and in vitro floating studies have generally been performed in acidic pH (0.1 M hydrochloric acid) [17], which is considered the media of fasting. During fasting, interdigestive series of electrical events may cycle the gastric content to the duodenum in every 2–3 h [18,19]. Thus, floating drug delivery systems may either be developed for fasting conditions or they may be examined in media at various pHs. Aiming at a gastroretentive local approach for Helicobacter pylori eradication, the present study has applied metronidazole, as it is a nitroimidazole type potent anti-anaerobic, amebicidal, and antiprotozoal active substance [20,21]. Experimental design (DOE) was used for optimization of floating drug delivery tablet compositions based on evaluating the analysis of variance (ANOVA) parameters of in vitro preliminary dissolution and floating studies. The aim was to design a suitable composition having appropriate floating parameters (short floating lag time and high floating force), a maximum of 6 h long total dissolution and significant mucoadhesion contributing to eradication of H. pylori. The X-ray CT technique was used to in vivo visualize the spatial position of the optimized tablets in the gastrointestinal tract.

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2. Materials and methods

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2.1. Materials

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Metronidazole (Molar Chemicals, Hungary) (PubChem CID:4173) was used as active substance. High viscosity grade sodium alginate (Hungaropharma, Hungary) was applied as swelling agent. Viscosity grade was determined to be 213.80 ± 0.83 mPas measured at 100 s1 shear rate. Viscosity determination was carried out with 1% concentrated solution of sodium alginate using rotational viscometer (Anton Paar RheolabQC, Austria) at 30 °C. Low substituted hydroxypropyl cellulose B1 (L-HPC B1) was used as superdisintegrant obtained as a gift from Egis Pharmaceuticals PLC, Hungary. Sodium bicarbonate was used as effervescent agent. Talc, magnesium stearate and hydrophilic colloidal silicon dioxide (Hungaropharma, Hungary) were applied as excipients for tablet compression. HPLC grade potassium dihydrogen phosphate (Szkarabeusz Laboratory, Chemical and Commercial Limited Co., Hungary) and methanol (OptigradeÒ, LGC Standards GmbH, Germany) were used for HPLC assay. Barium sulfate as X-ray contrast material was purchased (Szkarabeusz Laboratory, Chemical and Commercial Limited Co., Hungary).

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2.2. Methods

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2.2.1. Experimental design, statistical analysis Face centered central composite design (a = 1) was created to view the effects of ingredient alterations on experimental parameters. The composition matrix consisted of three numeric factors:

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sodium alginate (X1), L-HPC B1 (X2) and sodium bicarbonate (X3). In the setup, each factor was examined with three levels (1, 0, +1). The experimental layout is shown in Table 1. Each tablet contained 250.0 mg metronidazole and fixed concentration of tableting excipients contributing tablet compressing ability. Investigated dependent variables were the following: floating lag time, maximal floating force, maximal floating force calculated to 100 mg tablet mass, time needed for maximal floating force, and drug dissolution. Data obtained from all floating tablet formulations were analyzed with Design Expert 7.0.0 software and used to generate the design and the response surface plots. Polynomial models were generated for all dependent variables including linear, and quadratic terms with interactions. The best fitting model was chosen based on the comparison of statistical parameters included: coefficient of variation (CV), coefficient of determination (R2), and adjusted and predicted coefficient of determination (adjusted and predicted R2) provided by Design Expert software. Additionally, influence of factors on response regression coefficients was also evaluated by analysis of variance (ANOVA). F-test and p-values were calculated and evaluated. The following mathematical equation form was evaluated to determine numerically the effects of independent variables on particular response variables:

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Y ¼ b0 þ b1 X 1 þ b2 X 2 þ b3 X 3 þ b12 X 1 X 2 þ b13 X 1 X 3 þ b23 X 2 X 3 þ b11 X 21 þ b22 X 22 þ b33 X 23

ð1Þ

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where Y is the response variable, b0 is the intercept, and bi is the estimated coefficient of factors. X1, X2, and X3 are the main effects representing how responses change, when an individual factor changes. Interaction terms show the effect of simultaneous change

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of factors on responses. X 2i is the quadratic effect for evaluation of non-linear correlations. Subsequently, after determination of optimization criteria, numerical optimization technique was used to generate new formulations with desired responses.

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2.2.2. Preparation of effervescent floating tablets The ingredients of compositions were precisely weighed on analytical scale (Kern, ABJ 220-4M, Germany) based on the design (Table 1). Stirring was performed for 3 min after addition of an ingredient to the blend with the use of pestle and mortar; ultimately all blends were mixed for 10 min in order to reach homogeneity. Flow properties of powder mixtures were qualified to be suitable for direct compression. Tablets were compressed by eccentric single-punch tablet press (Erweka, EP-1, Germany).

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Table 1 Experimental layout of effervescent floating tablets. Samples

Sodium alginate, X1 (%)

L-HPC B1, X2 (%)

NaHCO3, X3 (%)

Total tablet weight (mg)

MF01 MF02 MF03 MF04 MF05 MF06 MF07 MF08 MF09 MF10 MF11 MF12 MF13 MF14 MF15

5.00 15.00 5.00 15.00 5.00 15.00 5.00 15.00 5.00 15.00 10.00 10.00 10.00 10.00 10.00

30.00 30.00 45.00 45.00 30.00 30.00 45.00 45.00 37.50 37.50 30.00 45.00 37.50 37.50 37.50

8.00 8.00 8.00 8.00 13.00 13.00 13.00 13.00 10.50 10.50 10.50 10.50 8.00 13.00 10.50

463.8 569.5 642.7 865.1 511.2 642.7 737.5 1046.0 569.5 737.5 538.8 796.2 603.9 686.8 642.7

Please cite this article in press as: P. Diós et al., Preformulation studies and optimization of sodium alginate based floating drug delivery system for eradication of Helicobacter pylori, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.07.020

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Based on composition weights 8.0, 10.0, 12.0 mm round concave punches were used for compression. 2.2.3. Physicochemical characteristics of floating tablets 2.2.3.1. Tablet weight variation. Tablets (n = 10) were randomly weighed on analytical scale (Kern, ABJ 220-4M, Germany). Mean and standard deviation of weights were evaluated. 2.2.3.2. Tablet thickness. Thickness of randomly selected tablets (n = 10) was measured with vernier caliper. Mean and standard deviation were evaluated. 2.2.3.3. Tablet hardness. Tablet hardness test was performed according to Ph. Eur. 2.9.8. resistance to crushing of tablets (n = 10) using Erweka hardness tester (Erweka TBH 310, Germany). Mean and standard deviation were evaluated. 2.2.3.4. Metronidazole content uniformity. Tablets (n = 10) were selected randomly weighted and crushed individually. Powders of tablets were dispersed in 0.1 M HCl, then filtered through PTFE membrane filter. Metronidazole content of each sample was determined with spectrophotometric method, at the absorption maximum of metronidazole (kmax = 277 nm). Mean and standard deviation of drug content were evaluated.

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filtered through PTFE membrane topping of sampler. All measurements were done in triplicate. Metronidazole content was measured with spectrophotometric method (Jasco V-670, Japan) at the absorption maximum of metronidazole (kmax = 277 nm). Linear calibration curve (R2 = 0.9998) was previously created between 1.39 and 27.9 mg/l concentrations. All samples were measured within this concentration interval. Based on the optimization of compositions according to the floating and release study parameters, the release of the optimized composition was compared with two commercially available metronidazole tablets (KlionÒ 250 mg and SupplinÒ 250 mg).

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2.2.5.2. Kinetics of drug release. Model dependent evaluations of dissolutions were carried out with four mathematical models [25]: zero order model, first order model, Higuchi model and Weibull kinetic model. The following equations were used for data evaluation: Zero order model:

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Mt ¼ kt M1

ð3Þ

First order model:



Mt ln 1  M1

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2.2.4. In vitro evaluation of floating properties 2.2.4.1. Determination of floating lag time. Floating lag time (tlag) is the period of time after immersion of tablets until their buoyancy. Before testing, tablets were placed into desiccator for 24 h. Parameter tlag was studied at 37 ± 0.5 °C in 450 ml 0.1 M HCl in triplicate. Floating lag times were determined visually using a camcorder (Sony, DCR-SX85E). 2.2.4.2. Floating force study. Floating force study was performed based on the theoretical background described by Timmermans and Moes [3,22,23]. The detailed method is described in our previous work [5], during which tablets were immersed into 450 ml 0.1 M hydrochloric acid then a special filtering plate with 2 mm aperture size detected the vertical floating force expressed by the tablets. Filtering plate was connected to a tensiometer (KSV Instruments Ltd., Helsinki, Finland) measuring the force acting upward in the function of time. Floating forces were calculated based on the description by Cromer [24].

F ¼ F bouy  F grav ¼ qf gV  qs gV ¼ ðqf  qs ÞgV ¼ ðdf  M=VÞgV 228

ð2Þ

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F is the total magnitude of force corresponding to the vectorial sum of buoyancy (Fbouy) and gravity forces (Fgrav) acting on the object, qf the fluid density, qs the object density, g the acceleration of gravity, M the object mass and V the object volume. Maximal floating force (Fmax), maximal floating force calculated for 100 mg tablet mass (Fmax/100mg) and the time needed for maximal floating force (tFmax) were evaluated. All experiments were performed in triplicate.

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2.2.5. Metronidazole release study 2.2.5.1. In vitro dissolution study. Release study of all floating tablets was carried out according to Ph. Eur. 2.9.3 dissolution test with paddle apparatus at 50 rpm stirring speed (Erweka DT-700, Germany) in 900 ml simulated gastric medium without pepsin (0.1 M HCl) at 37 ± 0.5 °C. All tests were performed for 6 h. During the experiments, 2.5 ml samples were taken at 10, 15, 20, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min. Each sample was

Mt 1=2 ¼ kt M1

ð4Þ

250 251 252 253 254 255

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268 270

ð5Þ

272 273

274

ð6Þ

where k is dissolution rate constant, Mt/M1 is the fraction of dissolved API in time t, t0 is lag time of dissolution, s is mean dissolution time (time when 63.2% of the substance is dissolved) and b is shape parameter of the dissolution curve. During evaluation linear transformation was carried out on each dissolution profile and the equation of the fitted linear line was determined according to the following formula:

Q t ¼ mt þ n

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Weibull model [26]:

 tt b  0 Mt ¼ M 1 1  e s

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Higuchi model: 208

247

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 ¼ kt

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ð7Þ

276 277 278 279 280 281 282 283

284 286

where Qt is the drug dissolved in time t, m is the slope of the line and n is y-intercept.

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2.2.5.3. Optimization. Optimization was performed based on the evaluation of floating behavior and release experimental parameters in order to ascertain the desired floating tablet. Optimization criteria were determined in order to minimize amount of excipients, since less excipient amount is applied, the more active substance can be loaded into tablets. Floating parameters were adjusted to achieve the best floating including minimization of tlag and maximization of Fmax and Fmax/100mg. The release until the first 30 min was minimized, and after 30 min it was maximized.

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2.2.5.4. Microbiologically detected dissolution studies. Antibacterial activities of metronidazole dissolution samples were determined in order to visualize the correlation between dissolution detected with spectrophotometric and microbiological disk diffusion method [27]. A calibration curve was created with standard dilution of pure metronidazole from 0 to 4.0 lg/5 ll. The test bacterium strain Bacteroides fragilis (ATCC 25285) was spread on the surface of Brucella blood agar plates supplemented with haemin, vitamin K1 (Becton Dickinson GmbH, Germany) and 5% defibrinated sheep blood. 30 ll of 105 test bacteria/ml suspension was

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used for inoculation of blood agar plates. After that Whatman 3MM filter paper disks (diameter 6 mm) (Cole-Parmer Instruments Co., USA) were placed onto inoculated plates. These disks were impregnated with 5–5 ll of calibration standards and dissolution samples at different times (5, 10, 15, 20, 30, 45, 90, 120, 180, 240, 300 and 360 min). Inoculated plates were put into an anaerobic jar containing a GENbox anaer (bioMérieux, France) opened just before we closed the jar. The cultures were incubated at 37 °C for 48 h. After incubation time, jars were opened, and determined the diameter of inhibitory zones around the filter paper disk with vernier caliper. Experiments were carried out in triplicate. 2.2.5.5. Determination of difference and similarity factors. Spectrophotometric and microbiologically detected release profiles of dissolution of optimized composition were compared by determination of difference (f1) and similarity factors (f2) [28,29]. Difference factor highlights the percent error of the difference between the test and reference curve over all sample time points. Difference factor was calculated by the following formula:

Pn f1 ¼

j¼1 jRj  T j j Pn 100; j¼1 Rj

ð8Þ

where n is the number of sampling points, and Rj and Tj are the percent of dissolved metronidazole of test and reference tablets at each time point (j). The similarity factor is the logarithmic transformation of the sum of squared error of differences between reference (Rj) and test (Tj) preparations over the time period, which was calculated with the following equation:

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8" 9 #0:5  X < = 1 n 2 f 2 ¼ 50 log 1þ wj jRj  T j j 100 : ; n j¼1

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where wj is an optional weight factor.

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2.2.6. Drug–excipients interaction study 2.2.6.1. Differential thermal analysis (DTA). Differential thermal analyzer (Shimadzu DTA-50, Japan) was used for thermal analysis of metronidazole and metronidazole–excipient blends. Metronidazole and excipients were analyzed separately as well as blends with drug–excipient ratios according to the optimized composition (MF_OPT). 10.0 mg of individual samples and blends were weighed directly into platinum sample holder and scanned in the temperature range of 25–400 °C under air atmosphere. Dried alumina powder (Al2O3) was used as reference material. Temperature rate was 5 °C/min. Peak shifting and melting point were evaluated on thermograms in order to detect interaction between metronidazole and excipients.

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ð9Þ

2.2.6.2. Isothermal stress testing. Isothermal stress testing was carried out with metronidazole and metronidazole–excipient blends based on optimized composition weighed separately, directly into 15 ml glass vials and mixed on vortex mixer for 2 min [30,31]. 10% purified water was measured into each vial and stirred on vortex mixer for 2 min. Then vials were sealed with rubber cups and stored at 50 °C (Binder BF115, Germany) for 3 weeks. Samples were examined periodically in triplicate to determine abnormal color change of samples. As reference, samples without added water were stored in refrigerator. The determination of metronidazole has been performed by using the method according to metronidazole monograph of Ph. Eur. 5th Edition. Applied parameters were the following: mobile phase: mixture of 300 ml methanol and 700 ml 0.01 M potassium dihydrogen phosphate, flow rate: 1 ml/min, detection: 315 nm, injection: 10 ll. All samples were previously filtered with 0.2 lm

GHP membrane (AcrodiscÒ, Pall, USA). The liquid chromatographic system consisted of HPLC device (Class-LC10A, Shimadzu, Japan) and C18 column (LiChrospher 100 RP-18, Teknokroma, Spain) with 25 cm size and 4.6 mm diameter.

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2.2.7. Ex vivo mucoadhesion studies Wistar rats of either sex (250–350 g) were bred in a temperature-controlled room having a 12 h light/dark cycle, provided with standard rodent chow and water ad libitum. For harvesting the gastric mucosa, rats were deeply anaesthetized with sodium thiopental (100 mg/kg i.p.) and killed by cervical dislocation and exsanguination. The abdomen was opened, the stomach was excised and cut open along the lesser curvature. Stomachs were kept in Krebs solution until their further use. Gastric content was carefully emptied and the mucosa was rinsed with 0.1 M HCl solution containing 0.9% NaCl [32].

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2.2.7.1. Detachment force study. Detachment force studies were performed according to the theoretical base of modified surface tensiometer method [33–36]. The inner side of stomach tissues was outspread on 10% agar–agar gel immobilized with pins. Tablets were fixed with ethyl 2-cyanoacrylate on the bottom of a special specimen hanged on a tensiometer arm (KSV Instruments Ltd., Finland). Before measurements, mucosae were wetted with 20.0 ll 0.1 M HCl containing 0.9% NaCl in order to achieve better mucoadhesive performance as published [37,38]. Tablets were left on mucosae surface for 3 min to allow wetting and creation of mucoadhesive bonds [39]. Maximal detachment forces from mucosae were recorded and calculated to mN with the following equation:

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F detach ¼ F total  F tablet

ð10Þ

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386 387 388 389 390 391 392 393 394 395 396 397

398 400

where Fdetach is the detachment force, Ftotal is the measured total weight and Ftablet is the weight of tablet. Structure of measuring method is depicted in Fig 1. Optimized composition (MF_OPT), optimized composition without L-HPC B1 content (MF_OPT_L-HPC) and the optimized composition without sodium alginate and L-HPC B1 (MF_OPT _2EXC) were tested. All measurements were performed in fourfold.

401

2.2.7.2. Rheological study. Rheological ex vivo mucoadhesion measurements were performed based on literature [40,41]. Stomachs were pinned out in a Sylgard-coated Petri dish and the mucus were meticulously and carefully removed under an operating microscope. Gastric mucus were put into 0.1 M HCl containing 0.9% NaCl, and dispersed with high speed homogenizer (Ultra-TurraxÒ T 25, IKAÒ, Germany) with 9500 rpm for 2 min to achieve homogeneous sample. Then mucus were centrifuged with 5500 rpm for 1 h. Pellets were dialyzed with Membra-CelÒ dialysis tubing (Serva MWCO 3500, Germany) on 4 °C for 24 h. Furthermore mucus were centrifuged (Labogene 1524, Denmark) again with

408

KSV tensiometer measuring arm

wetted surface fixing pins 10% agar gel

tablet sample outspread inner surface of rat stomach

Fig. 1. Structure of tablet detachment force measuring apparatus.

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15,000 rpm for 1 h. Pellets were stored at 15 °C until their further use [42,43]. Solutions of 3% mucus and optimized formulation (MF_OPT) equilibrated to 3% L-HPC and sodium alginate were dispersed separately in 20 ml 0.1 M HCl, then mixture of 3% mucus and MF_OPT equilibrated to 3% L-HPC and sodium alginate were also prepared. Viscosity behaviors (flow curves) of samples were implemented in a rotational viscometer (Anton Paar Rheolab QC, Austria) with standard measuring system (CC27) at 37 °C. Data were recorded in a 0–25 s1 shear rate interval with 6 s sampling for 2 min. Increases in viscosity due to mucoadhesion (gm) were calculated with the following formula:

431 433

gm ¼ gtotal  gtabl  gmucus

419 420 421 422 423 424 425 426 427 428 429

434 435 436 437

ð11Þ

where gtotal is the viscosity of mucus/tablet mixture, gtabl is the viscosity of MF_OPT equilibrated to 3% L-HPC and sodium alginate and gmucus is the viscosity of 3% mucus solution. All experiments were performed in triplicate.

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2.2.8. In vivo X-ray CT evaluation of floating tablets in rat Optimized tablets were studied in male Wistar rats (n = 5) weighing about 300 ± 15 g. For visualization and detection of in vivo behavior of tablets, barium sulfate (BaSO4) was used as X-ray contrast material. 10% of the optimized blend was replaced with BaSO4. The homogenized blend was pressed with 3 mm round concave punches by eccentric single-punch tablet press (TSV-1, OMTKI, Hungary). Thickness of tablets was adjusted to 2.0 mm. The experiment was carried out with the permission from the local institutional animal ethics committee and in compliance with the relevant European Union and Hungarian regulations (EC Directive 86/609/EEC). Images were acquired with a NanoSPECT/CTPLUS (Mediso Ltd., Hungary). To prevent movement, animals were anesthetized with isoflurane (2%) and fixed to a MultiCell™ Imaging Chamber (Mediso Ltd., Hungary) as well as positioned in the center of field of view (FOV). Before the experiment, rats were kept at room temperature in 12 h light and dark cycle. The animals were not fasted, and food and water was supplied ad libitum. Imaging was performed at the following sampling times: 5 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h and 48 h. Image at 48 h after administration was examined and that floating tablets did not cause gastrointestinal obstruction. Experimental parameters of CT were as follows: scan range: 59.8 mm; exposure: 500 ms, 65 kV, projection/rotation: 360; number of rotations: 2; number of frames: 720; pitch: 1; corrections: offset, pixel, quadratic gain; acquisition time: 6 min 1 s; reconstruction: butterworth filter, voxel size: 0.22 ⁄ 0.22 ⁄ 0.22 mm. Reconstructed, reoriented and co-registered images were further analyzed with Fusion (Mediso Ltd., Hungary) and VivoQuant (inviCRO LLC, USA) dedicated image analysis software products by placing appropriate Volume of Interests (VOI) on the tablets. The VOI were delineated manually on each CT scan. Linear attenuation data were reconstructed into Hounsfield units (HU). Then a second, more detailed, lookup table (LUT) (indicated with different colors) was used by image processing to visualize spectacularly the differences of attenuation values of voxels ordered to the tablets VOIs [44]. With the use of a novel CT scaling method, tablet could be imaged besides tissues with another attenuation scale at 8 h after administration.

476

3. Results and discussion

477

3.1. Physicochemical characteristics of floating tablets

478

Physicochemical characteristic examinations were investigated evaluating the manufacturing process and justifying the preparation of further examinations. Characteristics of all prepared

438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474

479 480

floating tablets were determined showing acceptable physicochemical properties represented in Table 2. Thickness of tablets was between 4.80 ± 0.07 and 8.80 ± 0.05. Weight variation and content uniformity were found to be good among batches. Tablet hardness results showed sufficient strength to withstand physical abrasion [45].

481

3.2. In vitro evaluation of floating properties

487

The mechanism of flotation of effervescent floating drug delivery system was previously described in the literature [46,47]. Buoyancy is achieved by bulk density (<1 g/cm3), which is resulted from the hydration of swelling material and carbon dioxide gas generation from carbonates or bicarbonates in acidic media. Swelling polymers may enclose the internal relatively dry core of the tablets causing the decrease of average density of tablets and additionally contributing the carbon dioxide entrapped by swelling polymer. Floating lag time studies evaluated the time, which is needed to achieve flotation. Floating force measurements assessed the force affected by the tablets vertically upward, which were performed for 6 h, during which floating force values were recorded in function of time. These studies may characterize the kinetics of buoyancy. In order to analyze the floating behavior the following parameters were evaluated: floating lag time (tlag), maximal floating force (Fmax), maximal floating force calculated for 100 mg tablet mass (Fmax/100mg) and the time needed for maximal floating force (tFmax). Data of experiments are shown in Table 3. The best fitting model on floating lag time examination was the quadratic (p < 0.01), in which sodium alginate and L-HPC B1 were significant factors (p < 0.01). ANOVA indicated a possible interaction between L-HPC B1 and sodium bicarbonate (p < 0.01), which may be explained by the hydration mechanism of tablets. L-HPC B1 absorbs water/hydrochloric acid [5] rapidly into tablet, which probably leads to more intense carbon dioxide generation from sodium bicarbonate. The final equation of floating lag time analysis is as follows:

488

482 483 484 485 486

489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515

516

tlag ¼ 21:13 þ 66:56X 1  51:48X 2 þ 18:96X 3 þ 3:78X 1 X 2  33:30X 1 X 3  60:30X 2 X 3 þ 77:09X 21 þ 10:25X 22 þ 35:42X 23 ð12Þ

518

Shortest lag times were noticed at tablets having 5.0% sodium alginate and 37.5% or more L-HPC B1. The shortest lag time was observed at MF07, which contains the least sodium alginate and the maximum of L-HPC B1 and sodium bicarbonate amounts.

519

Table 2 Physicochemical properties of prepared floating tablets. Samples

Weight variation (%)

Hardness (N)

Thickness (mm)

Content uniformity (%)

MF_01 MF_02 MF_03 MF_04 MF_05 MF_06 MF_07 MF_08 MF_09 MF_10 MF_11 MF_12 MF_13 MF_14 MF_15 MF_OPT

102.03 ± 1.20 99.04 ± 2.81 100.95 ± 1.05 100.44 ± 1.05 100.46 ± 1.82 99.71 ± 1.60 100.65 ± 1.43 98.81 ± 0.52 99.94 ± 1.76 100.89 ± 0.95 100.51 ± 1.15 100.89 ± 1.53 100.69 ± 1.37 101.40 ± 1.20 101.69 ± 1.40 100.39 ± 0.88

47.4 ± 3.0 46.9 ± 3.8 47.2 ± 3.8 46.6 ± 4.3 44.5 ± 4.9 44.3 ± 6.0 44.5 ± 3.7 45.8 ± 3.6 47.7 ± 4.6 49.1 ± 2.3 49.2 ± 5.0 45.8 ± 4.9 44.4 ± 3.4 44.2 ± 2.9 43.8 ± 4.6 44.7 ± 2.3

5.31 ± 0.07 4.86 ± 0.07 5.58 ± 0.59 7.27 ± 0.86 5.65 ± 0.47 5.29 ± 0.05 6.23 ± 0.03 8.80 ± 0.05 4.84 ± 0.03 6.05 ± 0.05 4.58 ± 0.05 6.82 ± 0.06 5.12 ± 0.02 5.74 ± 0.04 5.50 ± 0.04 4.80 ± 0.07

100.30 ± 1.73 100.21 ± 1.66 99.27 ± 1.47 99.66 ± 2.41 99.22 ± 2.51 100.79 ± 1.15 100.99 ± 2.34 99.98 ± 1.93 100.77 ± 2.00 99.79 ± 0.80 99.58 ± 2.03 100.97 ± 1.40 100.66 ± 2.71 100.41 ± 2.09 99.14 ± 1.34 100.43 ± 1.16

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Table 3 Data of floating lag time and floating force studies. Samples

tlag (s)

Fmax (mN)

tFmax (s)

Fmax/100mg (mN)

MF01 MF02 MF03 MF04 MF05 MF06 MF07 MF08 MF09 MF10 MF11 MF12 MF13 MF14 MF15 MF_OPT

29.00 ± 1.00 217.67 ± 43.39 16.67 ± 1.15 220.00 ± 44.93 266.00 ± 50.91 321.00 ± 216.37 12.00 ± 2.37 82.57 ± 26.39 17.33 ± 3.39 165.33 ± 15.01 30.67 ± 0.58 18.33 ± 1.53 54.00 ± 2.00 45.33 ± 3.21 48.67 ± 4.62 13.25 ± 0.50

5.18 ± 0.49 2.2 ± 0.05 12.29 ± 0.03 6.88 ± 0.60 6.17 ± 1.02 5.89 ± 0.29 26.64 ± 1.18 14.13 ± 0.94 16.77 ± 1.86 3.96 ± 0.41 2.68 ± 0.56 8.05 ± 1.47 3.62 ± 0.25 8.49 ± 0.60 7.04 ± 0.04 12.75 ± 1.87

1122.05 ± 400.30 6213.50 ± 338.35 5934.15 ± 1148.90 5179.90 ± 781.42 537.50 ± 334.44 23114.53 ± 1650.89 877.27 ± 21.18 13327.90 ± 2995.73 1352.37 ± 69.55 7058.25 ± 1738.28 2463.40 ± 303.91 7421.10 ± 405.17 4321.07 ± 831.62 21546.00 ± 567.94 20915.05 ± 2881.39 847.68 ± 373.90

1.12 0.39 1.91 0.79 1.21 0.92 3.61 1.35 2.95 0.54 0.50 1.01 0.60 1.24 1.09 2.29

536

Increased sodium alginate content led to higher tlag, which may be explained with increased coherency of the matrix resulting in longer time for hydration. The highest forces could be observed in the case of MF07 expressing 26.64 ± 1.18 mN, which equals 2716.52 ± 120.32 mg resultant weight. This floating value is prominent among floating tablets compared to previous publications [5,48–52]. On the Fmax and Fmax/100mg data, linear model fitted the best (p < 0.01). In the ANOVA evaluation of Fmax, all factors were significant (p < 0.03) referring to having influence on maximal floating force. Final equation of Fmax in terms of coded factors showed the fact that increasing sodium alginate content decreases, increasing L-HPC B1 and sodium bicarbonate contents increase the maximal vertical floating force affected by the tablets.

537 539

F max ¼ 8:68  3:38X 1 þ 4:57X 2 þ 3:14X 3

523 524 525 526 527 528 529 530 531 532 533 534 535

548

549 551

F max=100mg ¼ 1:28  0:68X 1 þ 0:46X 2 þ 0:35X 3

541 542 543 544 545 546 547

ð14Þ

557

The time needed for maximal floating force (tFmax) data did not show significance on linear, quadratic terms and interactions. Linear term had only tendency with p = 0.089, in which only sodium alginate was significant (p < 0.05). The shortest tFmax values could be observed at tablets having lowest sodium alginate content.

558

3.3. Metronidazole release study

559

3.3.1. In vitro dissolution study Dissolution studies of all effervescent floating tablets were performed for 6 h. Metronidazole releases of all floating tablets are depicted in Fig. 2. Quadratic term was fitted on all dissolution data according to the sampling time (p < 0.01). Sodium alginate quantity in tablets was significant for all sampling times (p < 0.0001), and L-HPC B1 was only significant after 30 min (p < 0.05). Sodium bicarbonate has shown only tendency (p < 0.10) to have influence on dissolution in the first two sampling times (5 and 10 min); this

552 553 554 555 556

560 561 562 563 564 565 566 567 568

569

3.3.2. Kinetics of drug release Evaluation revealed that tablets having slow dissolution (MF2, MF4, MF6, MF8, MF10, MF11, MF12, MF13, MF14, MF15) can be described by Higuchi model, which refers to drug diffusion from polymer matrices. These samples contained 10% or 15% sodium alginate in formulations. In the case of low sodium alginate concentration (5%), none of the applied model fitted significantly. Result of kinetics analysis is shown in Table 4.

594

3.4. Optimization

602

Based on the optimization criteria, composition (MF_OPT) with 5.0% sodium alginate (X1), 38.63% L-HPC B1 (X2), and 8.45% sodium bicarbonate (X3) was determined. Floating parameters of MF_OPT were also ascertained and are shown in Table 3 as well as physical characteristics in Table 2. MF_OPT expressed significant floating force with 12.75 ± 1.87 mN (1300.14 ± 170.69 mg resultant weight). Dissolutions of MF_OPT and two commercially available non-floating metronidazole tablets were performed, representing the results in Fig. 3. Dissolution results represent the fact that MF_OPT had biphasic release with the advantage of buoyancy compared to the two reference metronidazole tablets showing rapid dissolution. The best fitted release model of MF_OPT was first order kinetics, but due to the biphasic dissolution coefficient of determination was only R2 = 0.820.

603

3.5. Drug–excipients compatibility study

617

DTA studies showed a sharp endothermic peak of pure metronidazole at 159.9 °C, which is considered to be the peak of the melting point. At the proportion of the MF_OPT composition, excipients did not show significant alteration of melting point of metronidazole compared to the melting point value (159–163 °C) according to the literature [53]. DTA thermograms of metronidazole and blends are shown in Fig. 4. This result inferred the fact that there are no interactions between metronidazole and excipients. Visual observations of isothermal stress testing did not show significant changes in color or in appearance. After 3 weeks under stress, metronidazole contents were determined with HPLC method showing appropriate stability. In chromatograms,

618

570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

595 596 597 598 599 600 601

ð13Þ

At Fmax/100mg, only sodium alginate and L-HPC B1 were significant factors (p < 0.01). P-value of sodium bicarbonate shows only tendency with p = 0.079. L-HPC B1 amount increase caused increase of Fmax/100mg, contrary to higher sodium alginate concentration, which decreased it. The tendency of sodium bicarbonate behaved the same as at Fmax evaluation according to final equation. Analysis of Fmax/100mg and Fmax resulted similarly; however, Fmax/100mg forces were standardized with tablet weight individually to 100 mg.

540

phenomenon may be due to the disintegrative effect of sodium bicarbonate reacting with acidic medium on the tablet surface. ANOVA indicated interaction (p < 0.05) between sodium alginate and L-HPC B1 on the dissolution, which negatively influenced the drug release. The fastest dissolution was observed at MF07 dissolution containing 45% L-HPC B1, 13% sodium bicarbonate and 5% sodium alginate, resulting in total dissolution of metronidazole at 60 min. Metronidazole could not be released more than 22.7% at floating tablets containing more than 5% sodium alginate content (10.0%, 15.0%). In the case of 5% sodium alginate content at least 78.39 ± 6.39% metronidazole dissolution was observed. In contrast to the standard deviation of release data at all sampling times were also evaluated with ANOVA resulting in sodium alginate as a significant influence (p < 0.05). Increased sodium alginate amount decreased the standard deviation of release, consequently made the compositions more reproducible. Two commercially available non-floating metronidazole tablets (KlionÒ 250 mg and SupplinÒ 250 mg) were also studied for comparison of two approved and the optimized compositions. KlionÒ 250 mg released 96.98 ± 2.99% metronidazole in the first 30 min, while SupplinÒ 250 mg released 100.90 ± 2.99% after 20 min. Comparative dissolution study of optimized composition (MF_OPT), KlionÒ 250 mg and SupplinÒ 250 mg is represented in Fig. 3.

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MF01

100.0

MF03

80.0

MF05

60.0

MF07 40.0 MF09 20.0 MF_OPT 0.0 0

50

100

150

200

250

300

350

time (min)

metronidazole release (%)

MF02 25.0

MF04 MF06

20.0

MF08 MF10

15.0

MF11 MF12

10.0

MF13 5.0

MF14 MF15

0.0 0

50

100

150

200

250

300

350

time (min)

metronidazole release (%)

Fig. 2. Metronidazole dissolution of all floating tablets.

100.00 80.00 60.00 MF_OPT

40.00

Supplin® 250 mg Klion® 250 mg

20.00 0.00 0.00

50.00

100.00 150.00 200.00 250.00 300.00 350.00

time (min) Fig. 3. Comparison of two commercially approved non-floating metronidazole tablets with the optimized floating tablet (MF_OPT – 5.0% sodium alginate, 38.63% L-HPC B1, 8.45% sodium bicarbonate).

633

retention times of metronidazole were about 7 min and no additional peaks could be found referring to decomposition products. Data of recovered metronidazole from control and stressed samples are shown in Table 5.

634

3.6. Microbiologically detected dissolution studies

635

Microbiological inhibition activity of metronidazole released from MF_OPT floating tablets was evaluated to present the pharmacological effect in the function of time. Detection limit of method was determined on blood agar plates to be 0.5 lg/5 ll/disk and maximally 4 lg/5 ll/disk metronidazole. Calibration results provided the further calculation of microbiologically detected

630 631 632

636 637 638 639 640

dissolution. Results of spectrophotometric and microbiological detection of dissolution of MF_OPT were compared and are shown in Fig. 5. Difference (f1) and similarity factor (f2) were calculated being used for qualitative, model independent comparison [25,54,55] of dissolution profiles endorsed by Food and Drug Administration. Difference factor (f1) 5.23 and similarity factor (f2) 66.61 were found to be statistically significant, which revealed similar dissolution results of spectrophotometric and microbiological detection methods.

Table 4 Model dependent evaluation of dissolution data. Formulations

Zero order model R2

First order model R2

Higuchi model R2

Weibull model R2

MF1 MF2 MF3 MF4 MF5 MF6 MF7 MF8 MF9 MF10 MF11 MF12 MF13 MF14 MF15 MF_OPT KlionÒ SupplinÒ

0.509 0.964 0.518 0.857 0.373 0.808 0.658 0.900 0.481 0.911 0.934 0.954 0.958 0.897 0.960 0.524 0.497 0.833

0.678 0.969 0.896 0.883 0.500 0.812 0.817 0.909 0.771 0.919 0.942 0.963 0.963 0.907 0.961 0.820 0.618 0.897

0.693 0.980 0.693 0.944 0.546 0.888 0.789 0.972 0.657 0.940 0.946 0.977 0.962 0.959 0.946 0.690 0.671 0.899

0.728 0.939 0.860 0.758 0.664 0.917 0.891 0.919 0.785 0.906 0.942 0.900 0.909 0.858 0.887 0.816 0.866 0.953

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Metronidazole Metronidazole + sodium alginate Metronidazol + L- HPC B1

µV

Metronidazole + NaHCO3 Metronidazole + talc

50

100

150

200

250

300

350

400

Temperature (˚C)

Metronidazole + magnesium stearate Metronidazole + silica dioxide

Fig. 4. DTA thermograms of metronidazole and metronidazole–excipient blends.

Table 5 Result of isothermal stress testing of metronidazole after 3 weeks stressed storage. Recovered quantity Samples

Drug: excipient ratio

Control samples (%)

Stressed samples (%)

Metronidazole Metronidazole + sodium alginate Metronidazole + L-HPC B1 Metronidazole + NaHCO3 Metronidazole + talc Metronidazole + magnesium stearate Metronidazole + silica dioxide

– 1:0.1116 1:0.8619 1:0.1885 1:0.0446 1:0.0223 1:0.0022

97.25 ± 1.83 95.61 ± 2.42 98.74 ± 4.07 97.34 ± 2.68 99.74 ± 3.92 96.23 ± 1.61 99.20 ± 3.49

95.26 ± 4.95 96.04 ± 2.38 99.32 ± 0.58 95.65 ± 0.82 98.14 ± 1.67 97.06 ± 1.80 96.52 ± 1.82

600

90 500

80 70 60 spectrophotometric method

50

microbiologically detected dissolution

40 30 20 10

Detachment force (mN)

metronidazole release (%)

100

400 300 200 100

0 0

50

100

150

200

250

300

350

400

time (min) Fig. 5. Comparison of spectrophotometric and microbiologically detected dissolutions of MF_OPT tablets (5.0% sodium alginate, 38.63% L-HPC B1, 8.45% sodium bicarbonate).

650

3.7. Ex vivo mucoadhesion studies

651

Two most frequently used ex vivo mucoadhesion studies were carried out to represent gastroretentive properties of MF_OPT samples. Detachment force studies were performed with MF_OPT and three other tablets with modified composition as reference having sodium alginate, L-HPC B1 (MF_OPT_L-HPC) or both excipient (MF_OPT_EXC) absences from composition. The result of detachment force study is shown in Fig. 6. MF_OPT tablets have resulted in considerably higher detachment force (505.49 ± 45.62 mN) compared to MF_OPT_L-HPC (314.91 ± 37.88 mN) and MF_OPT_EXC (264.68 ± 15.42 mN). Tablets without L-HPC B1 had significantly higher detachment force than the reference without both

652 653 654 655 656 657 658 659 660 661 662

0 MF_OPT

MF_OPT_L-HPC MF_OPT_2EXC

Fig. 6. Result of the detachment force study of MF_OPT (5.0% sodium alginate, 38.63% L-HPC B1, 8.45% sodium bicarbonate) and reference tablets without sodium alginate and/or L-HPC B1 (MF_OPT – optimized composition, MF_OPT_L-HPC – MF_OPT without L-HPC B1, MF_OPT_2EXC – MF_OPT without sodium alginate and L_HPC).

excipients. This study may highlight the possibility that there may be a physical synergistic effect between the applied swelling polymer and disintegrant affected with rapid water absorption. Result of MF_OPT tablets with the absence of sodium alginate resulted in splitting of tablets due to extensively rapid hydration effect of L-HPC B1. Without sodium alginate, less coherence of the tablet structure was experienced in the presence of acidic medium; therefore, these tablets were not evaluated. Rheological studies were performed in order to confirm the mucoadhesive property of MF_OPT. Rheological ex vivo mucoadhesion measurements evaluate the floating tablet’s mucoadhesion in a different way. Detachment force study evaluates more likely the

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1,000.0 900.0

viscosity (mPas)

800.0 700.0

tablet mucus tablet+mucus mucoadhesion

600.0 500.0 400.0 300.0 200.0 100.0 0.0 0.0

5.0

10.0

15.0

20.0

25.0

30.0

shear rate (1/s) Fig. 7. Result of ex vivo rheological mucoadhesion studies of 3% mucus, MF_OPT equilibrated to 3% L-HPC and sodium alginate (tablet), their mixture (tablet + mucus) and calculated viscosity increase signed as mucoadhesion.

689

possible mucoadhesion in relatively dry status, which rather correlates to measure mucoadhesion based on dehydration theory [56]. In contrast to the rheological method, which indirectly measures mucoadhesion based on macromolecular interpenetration [57], rheological method interprets the viscosity changes, when the tablet components are in dissolved and swollen form. Result of rheological method is depicted in Fig. 7. Low viscosity values were observed at 3% mucus (7.63 ± 1.24 mPas) and at MF_OPT tablet dispersion (27.57 ± 23.22 mPas). Mixture of tablet and mucus showed significant increase in viscosity (846.89 ± 78.25 mPas at 2.63 1/s). At low shear rates which has the greatest interest [40], eightfold increase could be observed, which decreased to twice more viscosity at high shear rates than mucus and tablet dispersion. Flow curve of mixture of tablet and mucus showed plastic flow behavior.

690

3.8. In vivo X-ray CT evaluation of floating tablets in rat

675 676 677 678 679 680 681 682 683 684 685 686 687 688

691 692 693

In vivo gastric retention of MF_OPT tablets having 10% BaSO4 was studied using rat model, which was performed as a correlation to human model in a cost-effective way to gain valuable

(a)

(b)

1h

5 min

1h

4h

6h

2h

3h

preliminary information. HU values of tablets with 10% BaSO4 (HUMF_OPT = 1214.12 ± 120.14) at sampling times resulted in considerably higher HU values in comparison with the liver’s HU as reference (HUliver = 800). Thus, tablets could be significantly (p < 0.0001) distinguished from tissues. Based on the results (Fig. 8), MF_OPT tablets remained in stomach for at least 8 h, which may be enough gastric retention considering the fact that more than 96% API content was released within 6 h based on the in vitro spectrophotometric and microbiologically detected dissolution studies (Fig. 5). After 48 h, tablets could be identified in the intestinal tract. The prolonged intestinal retention may be due to the effect of anesthesia, and this effect is published by Torjman et al. [58]. The position of MF_OPT tablet in gastrointestinal tract at 8 h is shown with yellow color in Fig 9. The homogeneity parameter of the tablet is visualized in a spectacular manner. Besides the expected discerning from background attenuation of tissues, fine attenuation pattern of the voxels of tablet can be visualized quantitatively with the use of CT approach. This method could have prominent role in in situ examination and visualization of tablet structure.

Fig. 9. Snapshot of animation of the position of MF_OFT tablets in rat at 8 h. Two different lookup tables (LUT) were used to visualize voxels: for the VOIs of tablet (indicated with yellow colors) and for VOIs of background (a conventional gray scale) (video data). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2h

3h

48 h

8h

4h

6h

8h

48 h

Fig. 8. X-ray CT images of MF_OPT (5.0% sodium alginate, 38.63% L-HPC B1, 8.45% sodium bicarbonate) tablets loaded with 10% barium sulfate at different time periods in transverse (a) and in sagittal plane (b) (location of tablets indicated with arrows).

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4. Conclusion

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In present study, wide concentration ranges of sodium alginate, low substituted hydroxypropyl cellulose and sodium bicarbonate were tested, evaluated and optimized, in which 15 batches and the optimized floating drug delivery compositions were successfully prepared. All tablets had acceptable physicochemical properties. Optimized tablets had remarkable floating behavior and biphasic release compared to two commercially available metronidazole products. Studies showed no incompatibility among components. Experimental results revealed significant ex vivo mucoadhesion of the optimized composition, which may cooperate with the floating mechanism to achieve better gastroretention. Studies demonstrated significant similarity between spectrophotometric and microbiologically detected dissolution studies showing remarkable effect correlation. In vivo studies represented 8 h retention of tablets in the gastric region. A promising controlled-release floating tablet of metronidazole was developed, which highlights the possibility to increase the local effect of anti-Helicobacter pylori agents via gastroretentive system based on floating and mucoadhesion.

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Acknowledgments

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This research was supported by Bagoly Teréz, Mária Zöldhegyi (Department of Pharmacology and Pharmacotherapy, University of Pécs) granting rat stomachs and assisting in harvesting gastric mucosae. We are also thankful to Aleksandar Secenji from the Department of General and Physical Chemistry (University of Pécs) for DTA analyses. We thank Miklós Kellermayer Jr. (Department of Biophysics and Radiation Biology, Semmelweis University), Zoltán Gyöngyi and István Kiss (University of Pécs, Medical School, Department of Public Health Medicine) the helpful discussions.

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Appendix A. Supplementary material

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