in vivo evaluation

International Journal of Pharmaceutics 463 (2014) 68–80

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

Enhanced bioavailability of buspirone hydrochloride via cup and core buccal tablets: Formulation and in vitro/in vivo evaluation Mohamed A.A. Kassem, Aliaa N. ElMeshad, Ahmed R. Fares ∗ Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 18 November 2013 Received in revised form 30 December 2013 Accepted 2 January 2014 Available online 8 January 2014 Keywords: Buspirone HCl Mucoadhesive dosage forms Buccal tablets Cup and core tablets Pharmacokinetic study LC/MS/MS

a b s t r a c t This work aims to prepare sustained release buccal mucoadhesive tablets of buspirone hydrochloride (BH) to improve its systemic bioavailability. The tablets were prepared according to 5 × 3 factorial design where polymer type was set at five levels (carbopol, hydroxypropyl methylcellulose, sodium alginate, sodium carboxymethyl cellulose and guar gum), and polymer to drug ratio at three levels (1:1, 2:1 and 3:1). Mucoadhesion force, ex vivo mucoadhesion time, percent BH released after 8 h (Q8h) and time for release of 50% BH (T50% ) were chosen as dependent variables. Additional BH cup and core buccal tablets were prepared to optimize BH release profile and make it uni-directional along with the tablets mucoadhesion. Tablets were evaluated in terms of content uniformity, weight variation, thickness, diameter, hardness, friability, swelling index, surface pH, mucoadhesion strength and time and in vitro release. Cup and core formula (CA10) was able to adhere to the buccal mucosa for 8 h, showed the highest Q8h (97.91%) and exhibited a zero order drug release profile. Pharmacokinetic study of formula CA10 in human volunteers revealed a 5.6 fold increase in BH bioavailability compared to the oral commercial Buspar® tablets. Conducting level A in vitro/in vivo correlation showed good correlation (r2 = 0.9805) between fractions dissolved in vitro and fractions absorbed in vivo. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The buccal mucosa, lining of the oral cavity, is an attractive site for drug administration as it is highly vascular, easily accessible and suitable for retentive dosage forms administration. The buccal transmucosal drug delivery claims advantage over peroral administration as it bypasses the first-pass effect and avoids the presystemic drug elimination within the gastrointestinal tract (Patel et al., 2012; Salamat-Miller et al., 2005; Sudhakar et al., 2006). Buccal mucoadhesive dosage forms include: tablets (Boyapally et al., 2010; Cilurzo et al., 2010), films (Rossi et al., 2003), patches (Nafee et al., 2003; Reddy et al., 2013; Shidhaye et al., 2008), gels (Perioli et al., 2008) and sponges (Portero et al., 2007). Dosage forms designed for buccal drug delivery should possess good bioadhesive properties, high drug loading capacity and cause no irritation. In addition, it should have controlled drug release properties preferably in a unidirectional way toward the mucosa and to be an erodible system so that the dosage form removal at the end of the desired dosing interval is not required (Salamat-Miller et al., 2005). Buccal tablets are the most common dosage forms for buccal drug

∗ Corresponding author at: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, 11562 Cairo, Egypt. Tel.: +20 12 88285866. E-mail address: [email protected] (A.R. Fares). 0378-5173/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2014.01.003

delivery. Buccal tablets are usually prepared by direct compression and intended to dissolve or erode slowly. Buccal tablets may be prepared in different forms including, monolithic, bilayered and cup and core buccal tablets to achieve unidirectional drug release (Salamat-Miller et al., 2005). Many mucoadhesive polymers are employed in the preparation of the buccal mucoadhesive systems: anionic such as carbopol (CP), sodium alginate (SALG) and sodium carboxymethyl cellulose (SCMC); cationic such as chitosan; and non-ionic polymers such as hydroxypropyl methylcellulose (HPMC) and guar gum (GG) (Andrews et al., 2009). Buspirone HCl (BH) is an anxiolytic drug acting by modulating the serotonergic system (Shumilov and Touitou, 2010). It is used in the treatment of generalized anxiety disorder and for anxiety symptoms in depression (Nash and Nutt, 2005). BH undergoes extensive first-pass metabolism leading to very low oral bioavailability (4%) (Gannu et al., 2009; Moffat et al., 2011). The short and variable elimination half-life of BH (mean of 2.4 h) (Moffat et al., 2011; Sakr and Andheria, 2001), its low bioavailability and low molecular weight (422) (Moffat et al., 2011) recommend it a good candidate for sustained release buccal dosage forms. In vitro/in vivo correlation (IVIVC) is the relation between the in vitro dissolution data and the in vivo input rate. A good IVIVC can be used as a surrogate for further bioequivalence studies and to predict in vivo results based on in vitro data. Four levels of IVIVC (level A, B, C and multiple level C) have been described in the FDA guidance.

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Of these levels, level A IVIVC is considered the most informative, representing a point to point relationship between the in vitro dissolution rate and the in vivo absorption rate (Shah et al., 2009; Uppoor, 2001). In the present study, BH mucoadhesive buccal tablets of both matrix and cup and core designs were developed using: CP 934P, HPMC K4M, SALG, SCMC and GG. The effect of these polymers on BH release profile and on the mucoadhesion properties of the tablets was studied. The compatibility between BH and the tablet excipients was studied using differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR). The buccal tablets were evaluated in term of content uniformity, weight variation, thickness, diameter, hardness, friability, surface pH, swelling index, mucoadhesion strength, ex vivo mucoadhesion time and in vitro drug release. The selected formula was further evaluated for its in vivo performance in four healthy human volunteers compared to commercially available BH tablet (Buspar® , 15 mg BH, GlaxoSmith Kline Co., Cairo, Egypt). Level A IVIVC was conducted between the in vitro dissolution data and the in vivo absorption data of the selected formula. 2. Materials and methods 2.1. Materials Buspirone hydrochloride (BH) was kindly supplied by GlaxoSmith Kline Co. (Cairo, Egypt). Carbopol 934P was obtained from Goodrich Chemical Co. (OH, USA). Hydroxypropyl methyl cellulose K4M was purchased from Colorcon (Midland, USA). Sodium alginate (viscosity = 14,000 cps) was purchased from MP chemicals (France). Ketorolac (internal standard), sodium carboxymethyl cellulose, guar gum, ethyl cellulose 100 cps (Ethocel), formic acid and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). Ethyl acetate and n-hexane (HPLC grade) were procured from Tedia Co. (USA) and Leda, Scharlau chemie (European Union) respectively. Spray-dried lactose was purchased from Meggle Co. (Wasserburg, Germany). Human plasma was obtained from VACSERA (Cairo, Egypt). All other chemicals were of analytical grade and used as received. 2.2. Determination of equilibrium solubility of BH in simulated saliva fluid (SSF) The equilibrium solubility of BH in SSF (pH 6.8) was determined according to Tenjarla et al. method (Tenjarla et al., 1998). An excess quantity of BH was added to 3 ml SSF in screw capped glass vials. The vials were placed in a shaking water bath (Model 1083, GLF Corp., Burgwedel, Germany) maintained at 50 rpm at 37 ◦ C for 24 h. Then, the solutions were filtered through a 0.45 Millipore filter and amount of the drug dissolved was analyzed spectrophotometrically at max 240 nm (Shimadzu UV-1601 PC, Kyoto, Japan) and compared to a preconstructed calibration curve (r2 = 0.9998, n = 3). Each experiment was carried out in triplicate and the mean value was deduced. 2.3. Compatibility of BH with tablet excipients Physical mixtures of BH with various excipients namely; CP, HPMC, SALG, SCMC, GG, lactose anhydrous and Ethocel were prepared by mixing in weight ratio of 1:1. The prepared mixtures were evaluated for possible interactions via differential scanning calorimetry and Fourier-transform infrared spectroscopy. 2.3.1. Differential scanning calorimetry (DSC) DSC analysis was performed using Shimadzu differential scanning calorimeter (DSC-60, Shimadzu, Kyoto, Japan). Samples

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Table 1 The composition of BH buccal matrix tablets. Formula (100 mg)

Lactose (mg)

CP15 CP30 CP45 HP15 HP30 HP45 A15 A30 A45 CM15 CM30 CM45 G15 G30 G45

70 55 40 70 55 40 70 55 40 70 55 40 70 55 40

Mucoadhesive polymers (mg) CP

HPMC

SALG

SCMC

GG

15 30 45 – – – – – – – – – – – –

– – – 15 30 45 – – – – – – – – –

– – – – – – 15 30 45 – – – – – –

– – – – – – – – – 15 30 45 – – –

– – – – – – – – – – – – 15 30 45

All formulations contain 15 mg BH. CP, carbopol 934P; HPMC, hydroxypropyl methylcellulose K4M; SALG, sodium alginate; SCMC, sodium carboxymethyl cellulose; GG, guar gum.

(3–4 mg) were placed in aluminum pan and heated in the range 10–400 ◦ C at a rate of 10 ◦ C/min, with indium in the reference pan, in an atmosphere of nitrogen. The DSC studies were performed for the drug, the aforementioned excipients and for the drug-excipients powder mixtures. 2.3.2. Fourier-transform infrared spectroscopy (FTIR) FTIR spectra between 4000 and 500 cm−1 of the drug, the aforementioned excipients and for drug-excipients powder mixtures were determined using FTIR spectrophotometer (Model 22, Bruker, UK) according to the potassium bromide disk technique. 2.4. Preparation of BH buccal matrix tablets adopting direct compression technique using various mucoadhesive polymers The composition of BH buccal matrix tablets was listed in Table 1. Accurately weighed quantities of BH, lactose anhydrous and different mucoadhesive polymers; i.e. CP, HPMC, SALG, SCMC and GG, were thoroughly mixed for 30 min by means of a pestle in a glass mortar. The resultant powder mixtures were directly compressed into tablets using a single-punch tablet machine (Royal Artist, Bombay, India) equipped with 7 mm round and flat punches. The force of compression was adjusted so that hardness of all the prepared tablets ranged from 4 to 8 kg. BH buccal tablets were prepared according to 5 × 3 factorial experimental design to investigate the influence of formulation variables on the release profile of the drug and mucoadhesion properties of the formulations. In this design, polymer type (X1 ) and polymer to drug ratio (X2 ) were selected as independent variables, whereas mucoadhesion force (Y1 ), ex vivo mucoadhesion time (Y2 ), percentage of buspirone released after 8 h – Q8h – (Y3 ) and time required for the release of 50% of buspirone – T50% – (Y4 ) were chosen as dependent variables. The levels of the chosen independent variables were illustrated in Table 2. The objective was to prepare buspirone buccal tablets having good mucoadhesion force, sustain the mucoadhesion to the mucosa for 8 h (the release period) and have maximum release extent after 8 h with a suitable release rate. Table 2 Levels of independent variables. Factors (independent variables)

Levels of variables

X1 : polymer type X2 : polymer to drug ratio

CP–HPMC–SALG–SCMC–GG 1:1–2:1–3:1

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2.5. Preparation of BH cup and core buccal tablets In an attempt to optimize the release profile of BH along with the mucoadhesion of the tablets and to achieve uni-directional drug release toward the buccal mucosa, additional cup and core buccal tablets were prepared in which the cup serving as the mucoadhesive element and backing to offer a uni-directional release and the core containing the drug. 2.5.1. Preparation of the mucoadhesive cups The mucoadhesive cups were prepared using CP, HPMC and Ethocel in different ratios. For the purpose of comparison and to select the best mucoadhesive cup, the core tablet was prepared using 100 mg plain lactose anhydrous. All the mucoadhesive cups contained an additional backing layer of Ethocel which was added during the compression stage. Table 3 showed the composition of the mucoadhesive cups. 2.5.2. Preparation of the core tablets Previously prepared A15 and CM15 matrix tablets were used as core tablets. Furthermore two additional core tablets were prepared using the same polymers (SALG and SCMC) in different composition. Table 3 showed the composition of the newly prepared core tablets. The core tablets were directly compressed using a single-punch tablet machine equipped with 7 mm round and flat punches. 2.5.3. Preparation of the cup and core tablets For the preparation of the cup and core tablet, the Ethocel backing layer of the cup was filled into a 10 mm round die of the tablet machine then the 7 mm core tablet was placed above it. The powder mixture of the selected mucoadhesive cup was filled into the 10 mm die around the core tablet and then compressed to the final tablet using 10 mm round and flat punches. The force of compression was adjusted so that hardness of all the prepared tablets ranged from 4 to 8 kg. Different formulations of BH cup and core buccal tablets were listed in Table 3. 2.6. In vitro evaluation of BH buccal tablets 2.6.1. Physical characterization of BH tablets The prepared tablets were evaluated for content uniformity, weight variation, thickness, diameter, hardness, and friability. For the determination of content uniformity, one tablet was crushed and the drug was extracted with 250 ml of SSF (pH 6.8). The solution was then passed through 0.45 Millipore filter and analyzed spectrophotometrically at 240 nm after sufficient dilution with SSF (pH 6.8). The test was done in triplicate. The weight variation test was carried out according to the British Pharmacopoeia (Commission, 2012), where the weight of twenty tablets was determined using an electronic balance (Sartorius GmbH, Gottingen, Germany) and the weight variation was calculated. The thickness and diameter of ten tablets were determined using a micrometer. The hardness of ten tablets was determined by using digital hardness tester (Copley, UK). The friability test was carried out according to British Pharmacopoeia (Commission, 2012), where ten tablets were accurately weighed and placed in the drum of a tablet friabilator (Model DFI1, Veego, Bombay, India), which rotated at 25 rpm for a period of 4 min. The tablets were then removed from the drum, dedusted, and accurately weighed. The percentage weight loss was calculated. 2.6.2. Surface pH study The tablet was allowed to swell by keeping it in contact with 2 ml of SSF (pH 6.8) for 2 h at room temperature. The pH was measured by bringing the electrode of the pH meter in contact with the surface of the tablet and allowing it to equilibrate for 1 min. The surface pH

Fig. 1. Modified balance method for the measurement of in vitro mucoadhesion strength.

for each tablet was determined in triplicate and the mean ± SD was calculated (Darwish and Elmeshad, 2009). 2.6.3. Swelling study The swelling index (SI) for each tablet was determined in triplicate and the mean ± SD was calculated. Buccal tablets were weighed individually (W1 ), placed separately on 2% agar gel plates and incubated at 37 ± 1 ◦ C. At regular 2 h time intervals until 8 h, the tablet was removed from the petri dish, and excess surface water was removed carefully with filter paper. The swollen tablet was then reweighed (W2 ) and the swelling index was calculated using the following equation (Patel et al., 2007): Swelling Index =

(W2 − W1 ) W1

(1)

2.6.4. In vitro mucoadhesion strength measurement The in vitro bioadhesive force of the prepared matrix tablets and the mucoadhesive cups was measured using a modified two-armed physical balance (Morsi et al., 2013) as shown in Fig. 1. Freshly excised bovine buccal mucosa (obtained from a local slaughterhouse and stored in normal saline at 4 ◦ C upon collection) was used as a model tissue after removing all fats and debris. The bovine buccal mucosa (B) was fixed on the glass stage (C) using cyanoacrylate adhesive. The prepared tablet (D) was attached to the balance pan and then the glass stage (C) was raised slowly until the tablet surface came in contact with the buccal mucosa. A preload of 50 g (E) was applied over the balance pan above the tablet for 5 min then removed. The weights (F) were raised until the tablet was detached from the buccal mucosa. The minimum weight, in grams, that detached the tablet from the membrane surface was taken as a measure of the bioadhesive strength. The force of adhesion was deduced using the following equation (Darwish and Elmeshad, 2009; Morsi et al., 2013): Force of adhesion (N) =

Bioadhesive strength × 9.81 1000

(2)

2.6.5. Ex vivo mucoadhesion time A freshly cut bovine buccal mucosa was fixed on the internal side of a beaker with cyanoacrylate adhesive. A side of each tablet was wetted with 50 ␮l of SSF and was attached to the buccal tissue by applying a light force with a fingertip for 20 s. The beaker was filled with 800 ml of SSF and kept at 37 ± 1 ◦ C; after 2 min a stirring rate of 150 rpm was applied to simulate the buccal cavity. Mucoadhesive time was monitored until complete detachment or erosion of the tablet occurred. The test was done in triplicate and the mean mucoadhesion time ± SD was calculated (Perioli et al., 2007).

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Table 3 The composition of the mucoadhesive cups, BH core tablets and BH cup and core buccal tablets. Mucoadhesive cups C1 Excipients (mg) – CP 25 HPMC SALG SCMC Ethocel 75 Lactose

Core tablets (100 mg)

C2

C3

C4

C5

C6

C7

C8

C9

12.5 12.5

25 –

– 33.33

16.66 16.66

33.33 –

– 50

25 25

50 –

75

75

66.66

66.66

66.66

Cup and core formulae (250 mg) CA10 CA15 CCM10 CCM15

50

50

A10

A15

10

15

CM15

10

15

75

70

50 75

√ √ √ √

CM10

70

√ √ √ √

All cups contain 50 mg Ethocel as a backing layer. All core tablets contain 15 mg BH. CP, carbopol 934P; HPMC, hydroxypropyl methylcellulose K4M; SALG, sodium alginate; SCMC, sodium carboxymethyl cellulose; Ethocel, ethylcellulose 100.

2.6.6. In vitro drug release studies In vitro drug release studies of the prepared BH buccal tablets as well as the immediate release commercially available Buspar® tablets (Glaxo-Smith Kline Co., Cairo, Egypt) was performed using a modified standard basket apparatus (USP Dissolution Tester, Varian, model VK7000, USA) (Perioli et al., 2007, 2009). One side of the tablet was attached to the bottom flat end of the stirring rod instead of the basket fixture using cyanoacrylate adhesive. This was done to simulate the in vivo conditions. The vessel was filled with 250 ml SSF at 37 ± 1 ◦ C and stirred at 100 rpm. Aliquots each of 3 ml were withdrawn from the release medium at different time intervals and replaced by equal volume of fresh SSF kept at the same temperature. The concentration of the released drug was measured spectrophotometrically at max 240 nm. The experiments were done in triplicate and the average ± SD was calculated. 2.6.7. Kinetic analysis of the release data The release data were kinetically analyzed using Excel 2007 (Microsoft software) to determine the mechanism and the order of drug release from different formulations. Zero order, first order, Higuchi and Korsmeyer–Peppas models were used for the analysis of the release kinetics. 2.6.8. Statistical data analysis Analysis of the factorial design was performed using Social Package for Statistical Study software (SPSS 17® , SPSS Inc., Chicago, USA). 2.7. In vivo pharmacokinetic study in healthy human volunteers 2.7.1. Study design and subjects This study was carried out in order to compare the pharmacokinetics of BH from the selected buccal formula to the commercially available immediate release tablet formulation Buspar® (GlaxoSmith Kline, Egypt) following administration of single doses of 15 mg each, using a non-blind, two-treatment, two-period, randomized, crossover design. Four healthy male volunteers participated in this comparative study after giving informed written consent and were randomly assigned to one of two groups of equal size. Their age ranged from 25 to 35 years, mean body weight was 70.4 ± 7.2 kg and mean height was 172.5 ± 4.5 cm. The biochemical examination of the volunteers revealed normal kidney and liver functions. The study was approved by the Cairo university research ethics committee (serial number of the protocol: PI 577) and the protocol complied with the declarations of Helsinki (Helsinki, 2000) and Tokyo for humans.

All subjects were instructed to abstain from taking medicines and smoking for 1week before the beginning till the end of the study. All subjects fasted for at least 10 h before the study day (FDA, 2002). At 8:00 am the assigned treatment was given. The study was performed on two phases: Phase I, half the number of volunteers received the selected formulation (treatment A) and the remainder received the commercial tablet (Buspar® , treatment B) which is considered as a standard. Treatment A was applied to the buccal mucosa of the cheek without moistening before application by applying light force for 30 s using finger tip and was removed after 8 h. Treatment B was ingested with 200 ml of water. Food and drink (other than water, which was allowed after 2 h) were not allowed until 8 h after dosing and then a standard breakfast, lunch and dinner were given to all volunteers according to a time schedule. A washout period of one week separated the two phases. On the second phase, the reverse of randomization took place. 2.7.2. Sample collection Each group was supervised by a physician who was also responsible for their safety and collection of samples during the trial. Adverse events were spontaneously reported or observed either by the volunteers or the physician and were recorded and evaluated. Venous blood samples (5 ml) were collected into heparinized tubes at the following set points: 0 (pre-dose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12 and 24 h after administration of each treatment. Plasma was obtained by centrifugation at 3500 rpm for 10 min at 4 ◦ C (Centurion Scientific Ltd., West Sussex, UK). The plasma was transferred directly into 5 ml plastic tubes and stored frozen at −20 ◦ C pending drug analysis. 2.7.3. Plasma calibration and quality control standard solutions BH and ketorolac stock solutions with concentrations of 100 ␮g/ml were prepared in the mobile phase and methanol respectively. BH stock solution was serially diluted in the mobile phase to working solutions in concentrations of 0.25–30 ng/ml. Ketorolac stock solution was diluted in methanol to a working solution of 1 ␮g/ml. Plasma calibration standards were prepared fresh daily by spiking BH working standard solutions with blank human plasma to the following concentrations: 0.25, 0.5, 1, 2, 5, 10, 15 and 30 ng/ml. Quality control standards were prepared by spiking BH working solutions with blank human plasma to concentrations of 0.25, 10 and 30 ng/ml. 2.7.4. Sample preparation All frozen human plasma samples were thawed at ambient temperature. A liquid–liquid extraction procedure was used. Human

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plasma samples (0.5 ml) were placed in 7 ml glass tubes, and 50 ␮l of ketorolac solution (1 ␮g/ml) as internal standard (IS) was added to each and vortexed for 30 s. Four ml of ethyl acetate: n-hexane mixture (85:15 v/v) was then added, and samples were then vortexed for 1 min. The tubes were then centrifuged for 10 min at 4000 rpm. The upper organic phases were then transferred to clean glass tubes, and evaporated to dryness using centrifugal vacuum concentrator (Eppendorf 5301, Germany) at 60 ◦ C. Dry residues were then reconstituted with 100 ␮l of mobile phase and vortex mixed for 1 min, and 20 ␮l was injected using the autosampler. 2.7.5. LC/MS/MS assay of BH Plasma concentrations of BH were analyzed adopting a sensitive, selective and accurate liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS). Prior to the study, the method was validated following international guidelines (Chew et al., 2006; FDA, 2002). A Waters series UPLC system (Waters, Milford, MA, USA) equipped with degasser, solvent delivery unit along with autosampler was used to inject 20 ␮l aliquots of the processed samples. The chromatographic separation was carried out on a 50 mm × 2.1 mm ID, 5 ␮m C18 column obtained from Waters. The mobile phase was composed of a mixture of acetonitrile and 0.1% formic acid in water (60:40, v/v). The flow rate was set at 0.6 ml/min. Quantitation was achieved by MS/MS detection in positive ion mode for both BH and IS, using a Waters ACQUITY triple quadrupole LC/MS/MS mass spectrometer, equipped with a Turbo ionsprayTM interface at 450 ◦ C. The ion spray voltage was set at 5500 V. The compound parameters, namely, collision energy (CE) and cone voltage (CV) were 35 V and 40 V for BH and 20 V and 25 V for ketorolac (IS), respectively. Detection of the ions was performed in the multiple reaction monitoring (MRM) mode, monitoring the transition of the m/z 386.5 precursor ion to the m/z 122.1 for BH and m/z 256.3 precursor ion to the m/z 105.1 for IS. The analytical data were processed by EmpowerTM 2 CDS software. 2.7.6. Plasma analysis and quantification The plasma samples withdrawn from the four healthy volunteers after receiving the selected formulation (treatment A) and Buspar® (treatment B) were assayed as described above. The unknown sample concentration was calculated from the following formula: Q =

R±B × dilution factor A

(3)

where, Q is BH concentration, R is the peak area ratio (drug/IS), A is the slope of the standard curve and B is the Y-intercept. 2.7.7. Pharmacokinetic and statistical analysis Plasma concentration–time data of BH was analyzed for each subject by non-compartmental pharmacokinetic models using computer program, KineticaTM 2000 (ver3, InnaPhase Corporation, USA). The maximum drug concentration (Cmax , ng/ml) and the time to reach Cmax (tmax , h) were obtained from the individual plasma concentration–time curves. The area under the curve from zero to 24 h (AUC(0–24) , ng h/ml) and to infinity (AUC(0–∞) , ng h/ml), were calculated using the linear trapezoidal rule. Results were expressed as mean values of 4 volunteers ± SD. The pharmacokinetic parameters, Cmax , AUC(0–24) , and AUC(0−∞) of treatments A and B were compared using two way analysis of variance test (ANOVA) via the software SPSS 17.0 (SPSS Inc., Chicago, USA), in order to investigate the statistical significance among groups. For the untransformed data, the nonparametric Signed Rank Test (Mann–Whitney’s test) was used to compare the mean residence time (MRT) and the medians of tmax for treatments

A and B using the same software. A p value ≤0.05 was considered statistically significant. 2.8. In vitro/in vivo correlation (IVIVC) Level A IVIVC was conducted between the in vitro dissolution data and the in vivo absorption data of the selected formula. The mean plasma concentration–time data of BH was converted into fraction of BH absorbed (FRA) using the Wagner–Nelson equation (Shah et al., 2009; Sreenivasa Rao et al., 2001): FRA =

Ct + k · AUC0→t k · AUC0→∞

(4)

where Ct is the plasma concentration at time t, k is the elimination rate constant, AUC0→t is the area under the curve from 0 to time t and AUC0→∞ is the area under the curve from 0 to infinity. The fraction of BH absorbed (FRA) in vivo was plotted versus the fraction of BH dissolved (FRD) in vitro at the same time and the linear regression coefficient (r2 ) was calculated. 3. Results and discussion 3.1. Solubility of BH in SSF The mean equilibrium solubility of BH in SSF (pH 6.8) was found to be 3.73 ± 0.13 mg/ml. Such high solubility proved that the volume of dissolution medium used in the in vitro study ensured sink condition for the doses of BH used to load the formulations. 3.2. Compatibility of BH with used excipients 3.2.1. DSC Pure BH exhibited an endothermic peak of 203.23 ◦ C corresponding to its melting point (Al-Zoubi et al., 2008). The DSC peak of BH was preserved in its physical mixtures with each of the aforementioned excipients indicating that there was no interaction between the drug and the used excipients. The reduction in BH peak intensity in some thermograms was probably attributed to dilution factor of the mixing process (Botha and Löttter, 1990; Siepmann and Peppas, 2001). 3.2.2. FTIR The FTIR spectrum of pure BH showed characteristic peaks at 3032.1, 2886.22 (aromatic C H stretching), 1724.36, 1678 ( C O stretching), 1589.34, 1486.19 (aromatic C C stretching), and 1273.02 cm−1 ( C N stretching) (Al-Zoubi et al., 2008; Joshi et al., 2010). There were no considerable changes in the IR peaks of BH, when mixed with excipients, indicating the absence of chemical interaction with the used excipients. 3.3. In vitro evaluation of BH buccal tablets 3.3.1. Physical characterization of BH tablets All tablets fulfilled the pharmacopoeial specifications for content uniformity, weight variation and friability. The average drug content ranged from 14.06 ± 0.11 to 16.13 ± 0.76 mg. The average weight ranged from 98.93 ± 1.55 to 101.95 ± 0.62 mg for the matrix tablets and from 232.09 ± 4.60 to 238.94 ± 4.43 mg for the cup and core formulations. The average friability ranged from 0.18 ± 0.08 to 0.67 ± 0.12%. The average thickness ranged from 1.91 ± 0.03 to 2.1 ± 0.05 mm for the matrix tablets and from 2.95 ± 0.04 to 2.98 ± 0.03 mm for the cup and core formulations. The average diameters of the matrix and the cup and core tablets were 7 and 10 mm respectively. All formulations had acceptable hardness values which provided good handling properties without breakage or excessive friability problems.

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Table 4 Mucoadhesion force and ex vivo mucoadhesion time of BH buccal matrix tablets and different mucoadhesive cups prepared. Mucoadhesion force ± SD (N)

Fig. 2. Swelling index of (a) BH buccal matrix tablets and (b) BH cup and core buccal tablets.

3.3.2. Surface pH study The surface pH of all tablets ranged from 5.66 ± 0.08 to 6.48 ± 0.36. These results revealed that all formulations provided an acceptable pH in the range of salivary pH (5.5–7.0) and that they would not produce any local irritation to the mucosal surface upon application. 3.3.3. Swelling study Appropriate swelling behavior of a buccal adhesive tablet is essential for uniform and prolonged release of the drug and effective mucoadhesion (Patel et al., 2007). The swelling index was dependent on the polymer type and its ratio in each formulation. As shown in Fig. 2a, matrix tablets containing SALG and SCMC showed maximum swelling indices. The high amount of water uptake by the two polymers and their fast swelling properties might have resulted in the higher rate and extent of swelling of formulations A15, A30, A45, CM15, CM30 and CM45 (Patel et al., 2007). Among all formulations, CM45 showed the highest swelling index (4.9 ± 0.05) by the end of the 8 h. As for cup and core tablets, Fig. 2b, although SALG and SCMC, found in the core tablet, had high rate and extent of swelling (Patel et al., 2007), the decrease in the swelling compared to the matrix tablets might be attributed to the presence of the mucoadhesive cup which contained low swelling polymers (CP and HPMC) and water insoluble polymer, Ethocel. 3.3.4. In vitro mucoadhesion strength measurement Table 4 showed the mucoadhesion force of different buccal matrix formulations. Formula CP45 showed the strongest mucoadhesion force (0.78 ± 0.07 N) whereas formula G15 showed the weakest mucoadhesion force (0.05 ± 0.01 N). A full 5 × 3 factorial design was applied to evaluate the effect of polymer type (X1 ) and polymer to drug ratio (X2 ) on the mucoadhesion force (Y1 ), Fig. 3a. Analysis of factorial design demonstrated

Ex vivo mucoadhesion time ± SD (h)

BH buccal matrix tablets CP15 0.17 ± 0.03 CP30 0.41 ± 0.05 CP45 0.78 ± 0.07 HP15 0.14 ± 0.01 HP30 0.17 ± 0.02 HP45 0.19 ± 0.03 A15 0.12 ± 0.01 A30 0.14 ± 0.01 0.16 ± 0.03 A45 0.12 ± 0.01 CM15 0.14 ± 0.01 CM30 CM45 0.16 ± 0.02 0.05 ± 0.01 G15 0.08 ± 0.01 G30 0.11 ± 0.01 G45

5.25 ± 0.35 >8.00 >8.00 4.50 ± 0.71 >8.00 >8.00 3.00 ± 0.71 5.25 ± 0.35 7.25 ± 0.35 4.13 ± 0.18 5.50 ± 0.71 6.00 ± 0.71 0.50 ± 0.21 1.00 ± 0.39 1.50 ± 0.46

Mucoadhesive cups 0.11 C1 0.20 C2 0.15 C3 0.13 C4 0.36 C5 C6 0.31 0.61 C7 C8 1.20 0.85 C9

1.25 ± 0.35 2.75 ± 0.35 2.00 ± 0.71 1.75 ± 0.35 4.25 ± 1.06 4.75 ± 1.06 >8.00 >8.00 >8.00

± ± ± ± ± ± ± ± ±

0.01 0.06 0.04 0.02 0.04 0.04 0.03 0.10 0.09

that the polymer type had a significant effect on the mucoadhesion force (p < 0.05). The mucoadhesion force of different polymers could be ranked as follow: CP > HPMC > SCMC > SALG > GG. This ranking was almost comparable to that obtained by Nafee et al. (2004) and Wong et al. (1999). The significantly higher mucoadhesion strength of CP might be attributed to the formation of secondary mucoadhesion bonds with mucin (hydrogen bonding due to the great number of the carboxylic groups) and the interpenetration of CP chains into mucus which increased at higher concentrations of CP, while the other polymers only performed superficial mucoadhesion (Boyapally et al., 2010; Park and Munday, 2002; Patel et al., 2007). The weaker mucoadhesion force of HPMC might be due to the absence of a proton-donating carboxyl group which reduce its ability for the formation of hydrogen bonds. On the other hand SALG was fully ionized at pH 6.8 thus reducing the chances of hydrogen bonding. It was believed that polymer hydration was the main mechanism that enabled SALG to adhere to the mucosa (Nafee et al., 2004). Regarding SCMC, the high swelling characterizing SCMC might be responsible for its reduced adhesion, as swelling induced over-extension of hydrogen bonds and other forces (Nafee et al., 2004). This result was in agreement with the result of Bottenberg et al. (1991) who observed that SCMC formulations had the highest swelling rates and low adhesion force compared to other polymers. On the contrary, Varshosaz and Dehghan (2002) observed that SCMC had higher mucoadhesion force than CP and HPMC. This difference in results obtained might be due to the difference in methods used to assess the mucoadhesion force or using mucin substrate instead of fresh bovine buccal mucosa. Furthermore, GG was found to have weak adhesion. This result was in agreement with the results obtained by Park and Munday (2004) who attributed this to the lack of the physical integrity of the formed gel layer. Results also revealed that the polymer to drug ratio showed a significant effect on the mucoadhesion force (p < 0.05). It was obvious that increasing the polymer to drug ratio from 1:1 to 2:1 to 3:1

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Fig. 3. Effect of polymer type and polymer to drug ratio on (a) the mucoadhesion force and (b) the ex vivo mucoadhesion time.

significantly increased the mucoadhesion force. This could be due to the increase in the content of the bioadhesive polymer. None of the prepared matrices fulfilled the desired mucoadhesion in combination with the desired release pattern. Therefore, in an attempt to optimize the release profile of BH along with the mucoadhesion of the tablets and depending on the data obtained from our factorial design, CP and HPMC were used in the preparation of the mucoadhesive cups. Table 4 showed the mucoadhesion force of different mucoadhesive cups prepared. It was obvious that C7, C8 and C9 showed the strongest mucoadhesion forces due to the increase in the ratio of the mucoadhesive polymers CP and HPMC relative to Ethocel. From these three cups, C8, conatining CP and HPMC in ratio (1:1) showed strongest mucoadhesion force. Our result was in agreement with that of Desai and Kumar (2004) who reported that formulations prepared using mucoadhesive cups conatining CP and HPMC in ratio (1:1) exihibited stronger mucoadhesion force than those containing only CP or HPMC alone. 3.3.5. Ex vivo mucoadhesion time By comparing the ex vivo mucoadhesion time of BH matrix tablets, results in Table 4 showed that CP and HPMC matrix tablets

showed the longest mucoadhesion time where formulations CP30, CP45, HP30 and HP45 had adhesion time of 8 h. SALG and SCMC tablets had moderate adhesion time, while GG tablets showed very short mucoadhesion time ranging from 0.5 ± 0.21 to 1.5 ± 0.46 h. A full 5 × 3 factorial design was applied to evaluate the effect of polymer type (X1 ) and polymer to drug ratio (X2 ) on the ex vivo mucoadhesion time (Y2 ), Fig. 3b. Analysis of factorial design demonstrated that the polymer type had significant effect on the mucoadhesion time (p < 0.05). CP and HPMC had significantly longer mucoadhesion time compared to SALG, SCMC or GG. This result was well correlated to the results of the mucoadhesion force and the swelling tests. According to Nafee et al. (2003) as the particles of the tablet swelled, the matrix experienced intra-matrix swelling force promoting leaching of the drug leaving behind a highly porous matrix. Water influx weakened the network integrity of the polymer. The structural resistance of the swollen matrices was thus greatly influenced and erosion of the loose gel layer was more pronounced. Applying this to our case, CP and HPMC had low swelling indices preserving the integrity of the gel matrix in addition to higher mucoadhesion force which led to longer mucoadhesion duration. SCMC and SALG are both highly water

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Fig. 4. In vitro release profile of BH buccal matrix tablets prepared using (a) CP; (b) HPMC; (c) SALG; (d) SCMC and (e) GG with different polymer to drug ratios in SSF (pH 6.8) at 37 ◦ C in comparison to Buspar® tablet.

solubility polymers which tend to fragment in water (Perioli et al., 2004). They also had the highest swelling indices among the prepared formulations. This favored the outward diffusion of the drug molecules and weakened the integrity of the polymer matrices, leading to high erosion of the loose gel layer and lower mucoadhesion time. In spite of the low swelling index of GG, it had very weak mucoadhesion force leading to very short mucoadhesion duration. Results also revealed that the polymer to drug ratio showed a significant effect on the mucoadhesion time (p < 0.05). Increasing the polymer to drug ratio from 1:1 to 2:1 to 3:1 led to a corresponding increase in the mucoadhesion time. This

could be due to the increase in the content of the bioadhesive polymer. Results of the ex vivo mucoadhesion time of different cups, Table 4, showed that only C7, C8 and C9 had the required mucoadhesion time (8 h) and this was due to the higher content of the mucoadhesive polymers (CP and HPMC) compared to the rest of the prepared cups. According to the results obtained from both mucoadhesion strength and ex vivo mucoadhesion time tests, cup C8 attained the required mucoadhesion time (8 h) and had higher mucoadhesion force (1.2 ± 0.10 N) than C7 and C9 and so was used in the preparation of the cup and core tablets.

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3.3.6. In vitro drug release studies Results of the in vitro release of BH from the matrix tablets and the market product (Buspar® ) showed that the latter released almost 100% of its buspirone content after 1 h, Fig. 4. A full 5 × 3 factorial design was applied to evaluate the effect of polymer type (X1 ) and polymer to drug ratio (X2 ) on the percentage of BH released after 8 h – Q8h – (Y3 ) and T50% (Y4 ). The results were shown in Fig. 5a and b. Results revealed that the polymer to drug ratio showed a significant effect on the Q8h and the T50% (p < 0.05). Increasing the polymer to drug ratio from 1:1 to 2:1 to 3:1 significantly decreased the Q8h and increased the T50% and significantly retarded the drug release rate (p < 0.05). This was due to the increase in the polymer content which resulted in an increase in the gel viscosity and thickness of the gel layer providing a longer diffusional path. This might cause a decrease in the effective diffusion coefficient of the drug and therefore a reduction in the drug release rate (Khan and Zhu, 1999; Liew et al., 2006; Velasco et al., 1999). Formula HP15 showed a burst drug release of 24.31 ± 0.08% after 30 min. For swellable systems, like those made of HPMC, the gel barrier would be established only after the free dissolution of some drug particles which allow the swelled HPMC particles to come in close contact to permit their adhesion. Furthermore, slow swelling rates of high viscosity HPMC grades, allowed greater time for the free dissolution of the drug before the gel barrier was established leading to a burst release (Campos-Aldrete and Villafuerte-Robles, 1997). Analysis of factorial design also demonstrated that the polymer type had a significant effect on the Q8h and T50% (p < 0.05). HPMC showed the significantly lowest Q8h and longest T50% and exhibited the highest retardation of the drug release rate compared to other polymers. The Q8h could be arranged in a descending order as follows: GG > SALG > SCMC > CP > HPMC, while the T50% could be arranged in an ascending order as follows: GG < SALG < SCMC < CP < HPMC. Although many authors reported that CP retarded the drug release rate more effectively than HPMC (Ceschel et al., 2001; Desai and Kumar, 2004; Ikinci et al., 2004; Li et al., 2003; Streubel et al., 2002), other authors reported the opposite. Jadhav et al. (2004) reported that higher proportions of CP in their formulations showed faster drug release rate, while on the other hand increasing the HPMC content helped in sustaining the release and led to more retardation in the release rate. Jadhav et al. (2004) stated that the viscosities of CP: HPMC solutions of different ratios were in the order of 2:1 < 1:1 < 1:2. So, the formulations containing CP had lower gel viscosity and faster release. The higher viscosity of HPMC gel layer might be due to the hydration of polymer chains, primarily through H-bonding of the oxygen atoms in the numerous ether linkages, causing them to extend and form open random coils. A hydrated random coil was further H-bonded to additional water molecules, entrapping water molecules within, and might be entangled with other random coils. All these factors contributed to larger effective size and increased frictional resistance to drug diffusion (Sankalia et al., 2008). SCMC managed to retard the release rate of BH. The slow release rate might be related to the formation of a less soluble complex between the cationic drug and the anionic SCMC (Palmer et al., 2011; Takka et al., 2001). SCMC matrices released the BH faster than HPMC matrices due to the higher solubility of SCMC at pH 6.8 which led to quick gel erosion rate and a high erosion degree to the whole matrix system (Conti et al., 2007). The higher aqueous solubility of SCMC and the higher erosion rate might be due to the presence of ionized carboxylic acid groups within the polymer structure which possibly led to an increase in the rate and extent of water uptake due to ion-pair repulsion. This led to a stretch in the gel network and break of the bonds responsible for the gel structure (Palmer et al., 2011).

Liew et al. (2006) stated that SALG matrices could sustain drug release for at least 8 h, even for a water-soluble drug in the presence of a water-soluble excipient as lactose. The faster drug release rate and lower T50% values of SALG matrix tablets compared to CP, HPMC and SCMC tablets could be contributed to the adversely affected integrity of the SALG matrices during the dissolution study. Varying patterns of deformation, manifested by the presence of surface cracks, grooves and lamination were observed. The extent of deformation was greater at higher alginate concentrations. As alginate content increased, the extent of matrix swelling increased due to greater liquid imbibition. The latter caused pressure to be built-up within the matrix which could be released by matrix deformation. These effects might have compromised the gel barrier around the matrix and exposed greater surface area to the dissolution medium (Liew et al., 2006). Fig. 4e showed that different concentrations of GG failed to sustain the drug release. All GG formulations showed very high burst release (100%) after only 2 h. This might be attributed to the lack of the physical integrity and rapid erosion of the gel layer and/or the slow swelling of GG leading to poor hydrogel formation (Streubel et al., 2002). Streubel et al. (2002) reported that using GG formulating tablets led to rapid verapamil HCl release where more than 89% of the drug was released in the first 2 h. Also, Chandran et al. (2011) reported that GG proportions up to 20% could not retard the drug release from the tablets where all formulations showed high burst drug release and released almost 100% drug after 4 h. However, none of the prepared matrices fulfilled the desired release pattern in combination with the desired mucoadhesion time (8 h). From our factorial design it was evident that, although formulations prepared by CP and HPMC had significantly higher mucoadhesion time compared to other polymers, both polymers attained the lowest Q8h, the longest T50% and the most retardation in the drug release rate significantly (p < 0.05). On the other hand, formulations prepared from SALG and SCMC had significantly higher Q8h, reasonable T50% and retardation to release rate, but had significantly lower mucoadhesion time compared to CP and HPMC and failed to sustain the mucoadhesion for 8 h. Therefore, in an attempt to optimize the release profile of BH along with the mucoadhesion of the tablets and to achieve uni-directional drug release toward the buccal mucosa, additional cup and core buccal tablets were prepared. Depending on the data obtained from our factorial design, CP and HPMC were used in the preparation of the mucoadhesive cups while previously prepared A15 & CM15 matrix tablets were used as core tablets in addition to the newly prepared A10 and CM10 tablets. The results of the in vitro release of BH from the different cup and core buccal tablets in comparison to that of immediate release market product (Buspar® ) were shown in Fig. 6. Regarding formulations CA15 and CCM15, it was observed that the Q8h decreased and the T50% increased compared to the matrix core tablets A15 and CM15. This retardation in the release rate might be attributed to the presence of the mucoadhesive cup which contained strong release retarding polymers as CP and HPMC in addition to the water insoluble polymer Ethocel. The presence of this mucoadhesive cup led to the release of the drug from only one surface, so decreased the surface area available for the diffusion of the drug and thus led to more retardation of drug release. In attempt to increase the Q8h of the prepared cup and core formulations to reach complete drug release, new core tablets (A10 and CM10) were prepared. The amount of the polymer in the core tablets was decreased from 15 to 10%. No attempt was made to decrease the amount of the mucoadhesive polymers in the mucoadhesive cup as this cup had the optimum mucoadhesive characters as selected from the previous mucoadhesion force and time studies. Decreasing the release retarding polymer in the core tablets led to higher Q8h in formulations CA10 (97.91 ± 1.65%) and CCM10

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Fig. 5. Effect of polymer type and polymer to drug ratio on (a) the Q8h and (b) the T50% .

(91.73 ± 1.01%) compared to formulations CA15 (83.31 ± 1.27%) and CCM15 (87.7 ± 1.14%). The Q8h of the cup and core formulations were statistically analyzed using one-way ANOVA to test the significance of difference at p < 0.05. Subsequent Tukey Honestly Significant Difference (HSD) test was also performed. There was a significant difference between Q8h for the prepared formulations (p < 0.05). Subsequent Tukey HSD test revealed that Q8h attained by formula CA10 was significantly higher than CCM10, CA15 and CCM15 (p < 0.05). It also revealed that Q8h attained by either CA15 or CCM15 was nonsignificantly different (p > 0.05).

Fig. 6. In vitro release profile of BH cup and core buccal tablets in SSF (pH 6.8) at 37 ◦ C in comparison to Buspar® tablet.

3.3.7. Kinetic analysis of the release data The release kinetic data of BH from different formulations and the corresponding T50% of the drug were presented in Table 5. In order to determine the release model, the in vitro release data were analyzed according to zero order, first order and diffusion controlled mechanism according to simplified Higuchi model (Higuchi, 1963). The preference of a certain mechanism was based on the coefficient of determination (r2 ) deduced for the

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Table 5 Kinetic parameters of the release data from all BH prepared formulations. Formula

Zero

First

Diffusion

Mechanism

r2 CP15 CP30 CP45 HP15 HP30 HP45 A15 A30 A45 CM15 CM30 CM45 G15 G30 G45 CA10 CA15 CCM10 CCM15

0.981 0.999 0.999 0.903 0.999 0.996 0.999 1.000 0.990 0.963 0.983 0.991 0.943 0.939 0.999 0.996 0.998 0.992 0.996

0.855 0.901 0.905 0.806 0.970 0.910 0.895 0.918 0.932 0.817 0.885 0.859 0.923 0.917 0.998 0.881 0.864 0.946 0.920

0.999 0.985 0.979 0.974 0.994 0.992 0.983 0.973 0.945 0.990 0.971 0.981 0.973 0.973 0.989 0.985 0.987 0.950 0.961

Diffusion Zero Zero Diffusion Zero Zero Zero Zero Zero Diffusion Zero Zero Diffusion Diffusion Zero Zero Zero Zero Zero

Korsemeyer–Peppas model

T50% (h)

r2

n

Mechanism

0.994 0.998 0.998 0.999 0.999 0.998 0.999 0.999 0.999 0.999 0.984 0.996 n/a n/a n/a 0.998 0.999 0.978 0.994

0.706 0.813 0.857 0.468 0.598 0.663 0.921 0.838 0.888 1.008 1.168 1.231 n/a n/a n/a 0.985 1.030 1.146 1.199

Anomalous Anomalous Anomalous Anomalous Anomalous Anomalous Super case II Anomalous Case II Super case II Super case II Super case II n/a n/a n/a Super case II Super case II Super case II Super case II

2.54 7.54 7.78 4.91 8.08 9.66 2.18 3.19 3.35 1.04 4.41 4.91 0.71 0.78 1.28 4.05 4.79 4.15 4.32

The underlined and bold values are to specify the chosen mechanism to describe the release data based on the highest r2 underlined.

parameters studied, where the highest coefficient of determination was preferred for the selection of the order of release. However, in practice, polymeric matrices release the drug via a combination of mechanisms (Peppas and Sahlin, 1989). In these cases, the Korsemeyer–Peppas model was adopted to analyze the release kinetics where the release data were fitted to the following general equation (Korsmeyer et al., 1983): Mt = Kt n M∞

(5)

where Mt /M∞ represents the drug dissolved fraction at time t, K is a kinetic constant and n is the diffusional exponent. The diffusional exponent (n) depends on the release mechanism and the shape of the drug delivery device (Ritger and Peppas, 1987). For the case of cylindrical tablets, in particular, n ≤ 0.45 corresponds to a Fickian (case I) diffusion, 0.45 < n < 0.89 to an anomalous (nonFickian) transport (where release is controlled by a combination of diffusion and polymer relaxation), n = 0.89 to a zero order (case II) transport (where the drug release rate is independent of time and involves polymer relaxation), and n > 0.89 to a super case II transport (Harland et al., 1988). However, this equation is valid only for the early stages (≤60%) of drug release (Ritger and Peppas, 1987). It was shown in Table 5 that the release of BH from most matrix tablets formulations and all the cup and core formulations followed zero order kinetics, except the release data of CP15, HP15, CM15, G15 and G30 followed diffusion controlled mechanism according to Higuchi model. According to Korsemeyer–Peppas model, formulations CP15, CP30, CP45, HP15, HP30, HP45 and A30 have n values between 0.45 and 0.89 indicating anomalous (non Fickian) transport. While n for formulations A15, CM15, CM30, CM45 and all the cup and core formulations was above 0.89 indicating super case II transport. Formula A45 showed n value approximately equal to 0.89 indicating case II transport. In case of G15, G30 and G45, data was not sufficient for fitting to this model, so further calculations could not be carried out.

content, weight variation, friability and hardness. Thus, CA10 was selected for the comparative in vivo study, with the commercially available immediate release tablets Buspar® using four healthy human volunteers. LC/MS/MS method used for the determination of the plasma concentrations of BH was validated following international guidelines and proven to be sensitive, selective and accurate. Under the described conditions, the retention time of BH and IS were 0.74 and 2.56 min, respectively. A standard plasma calibration curve was constructed by plotting the peak-area ratio of BH to IS against BH concentrations. The standard calibration curve was found to be linear across the concentration range of 0.25–30 ng/ml with a correlation coefficient (r2 ) 0.9998. The lower limit of quantification was 0.25 ng/ml. The inter-day and intra-day precision is expressed as % relative standard deviation (%RSD), while the inter-day and intra-day accuracy is expressed as recovery %. The inter-day % RSD and recovery % ranged from 0.9 to 4.9% and 103.7 to 106%, respectively. The intra-day % RSD and recovery % ranged from 1.3 to 7.8% and 96.4 to 107.6%, respectively. Fig. 7 illustrated the average plasma-concentration time profiles of BH following the administration of the selected tablet CA10 and Buspar® tablets. The CA10 tablets showed higher Cmax ,

3.4. In vivo pharmacokinetic study in healthy human volunteers Results obtained from the in vitro evaluation revealed that the cup and core formula CA10, sustained the mucoadhesion time to the buccal mucosa for 8 h, had the significantly highest Q8h (97.91 ± 1.65%) and optimum T50% (4.05 h) (p < 0.05) in addition to a zero order release profile. Besides, CA10 exhibited acceptable drug

Fig. 7. The mean plasma concentration time curve after the administration of BH cup and core buccal tablets CA10 and Buspar® tablets to four human volunteers.

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Table 6 Mean pharmacokinetic parameters of BH following the administration of BH cup and core buccal tablets CA10 and Buspar® tablets to four human volunteers. PK parameter

BH cup and core buccal tablet

Buspar®

Statistical test

Cmax (ng/ml) tmax (h)a MRT (h)a AUC(0–24) (ng h/ml) AUC(0–∞) (ng h/ml)

11.61 ± 0.82 3.5 23.38 75.71 ± 1.15 111.1 ± 11.05

4.61 ± 0.81 1 9.69 17.57 ± 3.49 19.65 ± 3.12

p = 0.013 p = 0.011 p = 0.043 p = 0.001 p = 0.004

a

Median.

tmax and AUC values when compared to the commercially available immediate release formula. The estimates of the mean pharmacokinetic parameters obtained by non-compartmental fitting of the concentration–time data of BH after buccal administration of the cup and core tablets and oral administration of Buspar® tablets were given in Table 6. The mean Cmax values for BH cup and core tablets was 11.61 ± 0.82 ng/ml and it was reached at a tmax of 3.5 h, whereas the mean Cmax following the administration of Buspar® tablets was 4.61 ± 0.81 ng/ml and it was reached at a tmax of 1 h. Statistically significant differences (p < 0.05) between the two treatments for Cmax and the median tmax were obtained. In addition a statistically significant (p < 0.05) prolongation in the MRT was obtained by formula CA10. The significantly longer tmax and MRT values attained by the buccal formula CA10 compared to Buspar® tablets indicate a significant difference in the rate of drug absorption and the time the drug remains in the body. This could be attributed to the sustained nature of the cup and core tablet CA10 as compared to the immediate release Buspar® tablets. Regarding the extent of drug absorption as indicated by the AUC(0–∞) values, significantly higher values were obtained from the cup and core buccal tablets, where the mean AUC(0–∞) was found to be 111.1 ng h/ml compared to 19.65 ng h/ml in the case of the market formula Buspar® tablets. The relative bioavailability was found to be 565.44% based on the mean AUC(0–∞) . The significant increase in the extent of BH absorption and the higher Cmax values of the cup and core buccal formula CA10 might be attributed to the avoidance of first-pass hepatic metabolism and enhanced BH bioavailability after buccal administration. In a previous study, Gannu et al. (2010) reported a 2.65 fold improvement in the bioavailability of BH after transdermal administration compared to oral solution in rabbits. Based on the abovementioned results, it could be concluded that the enhanced bioavailability of BH obtained by the sustained release cup and core buccal tablet CA10, with a 5.6 fold increase in bioavailability than that obtained after oral administration of immediate release Buspar® tablets and higher Cmax , could be due to the avoidance of BH first-pass hepatic metabolism by the buccal route. These results suggest that a promising BH sustained release cup and core buccal tablet was successfully developed and can provide an effective management of anxiety but dose adjustment might be necessary due to the enhanced BH bioavailability. Yet, because of the small number of volunteers, the results can be considered preliminary, and the efficacy of prepared BH cup and core tablets should be further studied with a larger number of volunteers to prove its clinical efficacy. 3.5. In vitro/in vivo correlation (IVIVC) Level A IVIVC between FRA and FRD of the selected formula CA10 was investigated using linear regression analysis to their plot as shown in Fig. 8. Good level A IVIVC was observed between FRA and FRD with regression coefficient of 0.9805. The close

Fig. 8. Fraction of BH dissolved (FRD) in vitro versus fraction of BH absorbed (FRA) in vivo for BH cup and core buccal tablet CA10.

correlation between the in vitro dissolution and in vivo absorption data indicates that the mentioned dissolution method can be used to predict the in vivo data of the sustained release cup and core buccal tablet CA10. 4. Conclusion BH mucoadhesive buccal tablets prepared and showed acceptable physical properties. The analysis of the factorial design revealed that none of the prepared BH buccal matrix tablets fulfilled the desired release pattern in combination with the desired mucoadhesion time (8 h). Further optimization through preparation of BH cup and core buccal tablets was performed. Mucoadhesive cup C8 had the required optimum mucoadhesion time (8 h) and highest mucoadhesion force (1.2 N) so it was used in the preparation of the cup and core tablets. The in vitro release studies showed that the cup and core buccal tablet CA10 released 97.91% of the drug at the end of the 8 h. The in vivo evaluation, in four healthy human volunteers, of formula CA10 revealed a 5.6 fold increase in bioavailability than that obtained after oral administration of the commercially available Buspar® tablets. So it could be concluded that a promising BH sustained release cup and core buccal tablet was successfully developed and can provide an effective management of anxiety after dose adjustment. A good level A IVIVC (r2 = 0.9805) was observed for formula CA10 which can be used to waive bioequivalence studies. However, further studies on the BH sustained release cup and core buccal tablet using larger number of volunteers are needed to assure its clinical efficacy and safety. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm. 2014.01.003. References Al-Zoubi, N., Alkhatib, H.S., Bustanji, Y., Aiedeh, K., Malamataris, S., 2008. Sustainedrelease of buspirone HCl by co spray-drying with aqueous polymeric dispersions. Eur. J. Pharm. Biopharm. 69, 735–742. Andrews, G.P., Laverty, T.P., Jones, D.S., 2009. Mucoadhesive polymeric platforms for controlled drug delivery. Eur. J. Pharm. Biopharm. 71, 505–518. Botha, S., Löttter, A., 1990. Compatibility study between naproxen and tablet excipients using differential scanning calorimetry. Drug Dev. Ind. Pharm. 16, 673–683. Bottenberg, P., Cleymaet, R., de Muynck, C., Remon, J.P., Coomans, D., Michotte, Y., Slop, D., 1991. Development and testing of bioadhesive, fluoride-containing slow-release tablets for oral use. J. Pharm. Pharmacol. 43, 457–464.

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