Propranolol HCl was received as a gift from Munawar Pharmaceuticals Pvt. Limited Lahore (Pakistan). Carbopol^® 934P, CMC high viscosity grade 500-2500 mPa and EC-100 were purchased from Glow Scientific Traders, Lahore.

The study was completed in three stages, preliminary, main study and confirmation study. The preliminary study was undertaken to select the method of preparation and an estimation of polymers for the delivery of the drug. In the main study, the selected factors were changed within some range of mucoadhesive ingredients in each formulation employing computer-aided approach, artificial neural network on different formula to find the optimized factors for quality attributes for propranolol buccal mucoadhesive tablet formulation. The dose of propranolol HCl was kept fixed throughout the study. The weight of the tablet was kept at 500 mg in each formulation except for preliminary study. All formulations in the study were punched by single punch automatic machine by applying a force of 2 tons for 20 seconds.

The formulation F1 was prepared using the wet granulation method while all others were punched by the direct compression method. The composition of propranolol HCl was 40 mg/tablet. The weight for each tablet designed in preliminary study was 400 mg as depicted in Table 1.

Table 1
Composition and amount of ingredients used in preliminary study.

Based on the preliminary study, composition of ingredients and method of tableting for the main study was decided. The composition of formulation, F3 (Table 1) was taken as formulation which could be manipulated further for the main study. The reason for the selection of F3 was its meetings of maximum specifications for a buccal dosage form. The formulations in preliminary study released the drug much earlier than the desired time due to which HPMC was added in the recipe. Since HPMC retards the release of the drug and has good mucoadhesion [26Karavas, E.; Georgarakis, E.; Bikiaris, D. Application of PVP/HPMC miscible blends with enhanced mucoadhesive properties for adjusting drug release in predictable pulsatile chronotherapeutics. Eur. J. Pharm. Biopharm., 2006, 64(1), 115-126.
[http://dx.doi.org/10.1016/j.ejpb.2005.12.013] [PMID: 16675210] ]. It has been extensively used in the literature [26]. For that reason, it was added in the formulations in the main study (F14-F24). In the main study, the amounts of HPMC, CP, CMC, Na-alginate and EC had been changed to study its effect on formulation. A further aim of study was to find the optimized levels of the above factors to produce an optimized formulation. For this purpose, 11 different formulations (F14- F24) were prepared in the main study (Table 2).

Table 2
Amount of ingredients used in the main study (F14-F24).

The formulations in the main study were evaluated with all the tests listed in the preliminary study. Additionally, in-vitro release, Swellability index and mucoadhesive strength were also performed on the formulations.

2.6.1. Mucoadhesive Strength

To quantify mucoadhesive strength of buccal tablet, a modified instrument was developed as shown in Fig. (1). One arm of the balance was replaced with thread with the help of which two glass slides were tide. The tablet was sandwiched between the slides as shown in Fig. (1). One glass slide was fixed while the other moveable glass slide the attached with arm of the balance to access the degree of mucoadhesion. Between glass slides, both surfaces of tablet were wetted with phosphate buffer solution (pH 6.8).

Fig. (1) Tablet mucoadhesive strength testing apparatus.

On the left pan, drops of water were used in terms of weight to measure the force of detachment. When apparatus was in static equilibrium, water was added [27Mohammadi-Samani, S.; Bahri-Najafi, R.; Yousefi, G. Formulation and in vitro evaluation of prednisolone buccoadhesive tablets. Farmaco, 2005, 60(4), 339-344.
[http://dx.doi.org/10.1016/j.farmac.2005.01.009] [PMID: 15848210] ] until the slide detaches. The force was then accessed for mucoadhesion [28Gupta, A.; Gaud, R.S.; Ganga, S. Development, evaluation and optimization of extended release buccal tablets prepared using progressive hydration technology. Int. J. Drug Deliv., 2011, 2(1)]. Weight in grams was converted to Newton using Equation 2.

(2)

2.6.2. Swellability Index

The water uptake by the mucoadhesive tablet was checked by calculating swellability index. Tablet was dipped in a Petri dish containing phosphate buffer (pH 6.8). The weight of the tablet was measured initially (W1) and then at every hour interval up to 6 h. The extent of increase in weight was related to water absorbed (W2) for each time. The percentage swellability was calculated by using Equation 3 [28Gupta, A.; Gaud, R.S.; Ganga, S. Development, evaluation and optimization of extended release buccal tablets prepared using progressive hydration technology. Int. J. Drug Deliv., 2011, 2(1)].

(3)

2.6.3. In-vitro Drug Release

The in vitro release study was conducted in USP 28 Type 2 dissolution apparatus (ERWEKA DT-700) with a rotation of paddles at 50 rpm. 500 ml of phosphate buffer (pH 6.8) as dissolution medium was maintained at 37±0.5^oC throughout the experiment. Samples were taken at 0.25, 0.5, 1, 2, 3, 4, 5 and 6 hour and measured against a calibration curve for the quantitative determination of propranolol HCl [29Edavalath, S; Rao, B Design, development and optimization of buccal bioadhesive tablets of diclofenac sodium for the treatment of odontalgia. Ars Pharm., 2011, 2011, 52(2), 05-13.].

A significant mucoadhesion time is required for the attachment of polymers to buccal mucosa so that drug can be released from matrix over time locally. For buccal tablet, the neutral pH is required to avoid irritation.

Results for hardness, thickness, diameter, surface, pH, mucoadhesive time and friability of the tablets prepared in the preliminary study are summarized in Table 4. Though F1 was prepared using wet as well as direct compression method, the results for F1 prepared by the direct compression method have been enlisted (Table 4). All the formulations from F1 to F13 demonstrated 4 to 7.5 kg/cm² hardness, thickness 3 to 3.2 mm, diameter 12 to 12.3mm whereas friability was 0.5 to 0.7%. The mucoadhesive time ranges from 2 to 4 h. The average weight for all formulations in preliminary studies was ranged between 391.6 to 407.4 mg. The deviation of weight of the tablets was within the compendia limits according to USP 32. Surface pH in between 5.8 to 7.0 was considered to be in the physiological range of pH. The surface pH of all formulations was from 5 to7. Only formulations F1, F5, F6 were more acidic and outside the physiological pH range which might be due to the greater concentrations of CMC and CP used as shown in Table 4.

Table 4
Physicochemical response of the formulations in the preliminary study.

The required pH for propranolol HCl is 6.5 ± 0.05 [32Patel, V.M.; Prajapati, B.G.; Patel, M.M. Formulation, evaluation, and comparison of bilayered and multilayered mucoadhesive buccal devices of propranolol hydrochloride. AAPS Pharm. Sci. Tech., 2007, 8(1), 22.
[http://dx.doi.org/10.1208/pt0801022] [PMID: 17408221] ]. In the main study, HPMC was added so as to increase the mucoadhesion properties. Sucrose acted as a diluents, binder and sweetener, thus was kept in further formulation design in the main study

The aim of the main study was to find the levels of CP, EC, HPMC, SA and CMC for the production of an optimized buccal tablet of propranolol HCl. HPMC as a release retardant and mucoadhesive agent was additionally added in the study. Because the mucoadhesive time for tablet in the preliminary study was comparatively lower than the desired criteria due to which HPMC was predicted to increase mucoadhesion. It has been extensively used in buccal drug delivery and has proven satisfactory results [8Shojaei, A.H. Buccal mucosa as a route for systemic drug delivery: a review. J. Pharm. Pharm. Sci., 1998, 1(1), 15-30.
[PMID: 10942969] ]. The results from the main study are summarized in Table 5. As shown, the average weight and friability of all the formulations were within the USP limit of not more than 0.8%. The average weight of all formulations in the main study was also within in ±5% USP allowed deviation. The pH should be such that it should not disturb the local physiological functions because the buccal tablet has to reside a longer period of time. It should not convert the lining of mucosa into any pathological form being more acidic or so. As in Table 5, the pH of all formulations was within the physiological limit (5.8-7.0) except three [33M. S. Pendekal Ars Pharm P. K. Tegginamat. Formulation and evaluation of a bioadhesive patch for buccal delivery of tizanidine. Acta Pharm. Sin. B, 2012, 2, 318-324.
[http://dx.doi.org/10.1016/j.apsb.2011.12.012] ], F16, F18 and F19 which was 5.5 for all. The hardness of formulations was designed to be around 6-8 Kg/cm² [34Choi, H-G.; Kim, C-K. Development of omeprazole buccal adhesive tablets with stability enhancement in human saliva. J. Control. Release, 2000, 68(3), 397-404.
[http://dx.doi.org/10.1016/S0168-3659(00)00276-5] [PMID: 10974393] ]. A greater value of hardness would alter the release and patterns of swelling of the tablets. The diameter and thickness of the tablets showed a deviation than 5%.

Table 5
Physiochemical properties of buccal tablets in main study.

Time required by the tablet to detach from the oral mucosa is the mucoadhesive time [21Patel, V.M.; Prajapati, B.G.; Patel, H.V.; Patel, K.M. Mucoadhesive bilayer tablets of propranolol hydrochloride. AAPS Pharm. Sci. Tech., 2007, 8(3), E77.
[http://dx.doi.org/10.1208/pt0803077] [PMID: 17915827] ]. The mucoadhesive time of the tablet should be such that it should neither detach instantly from the mucosa due to slight pressure or may not as much strong in mucoadhesion that it may damage the mucosal layer due to sticking action. Highest mucoadhesive time was obtained with the formulations F19 (4.8 h) which was containing 10% of EC only. Higher time was also observed with formulation (F23) containing 5% each of CMC and EC; and 20% percent of HPMC.

In general, swelling behaviour for formulations F17, F18 and F19 was faster compared with the others. Formu-lations containing HPMC i.e. F22-F24 showed least expansion of the gel. Formulations containing low concentrations of CP exhibited greater fragmentation of the gel from the peripheries compared with formulations containing higher concentrations of CP [24Hussain, A.; Syed, M.A.; Abbas, N.; Hanif, S.; Arshad, M.S.; Bukhari, N.I.; Hussain, K.; Akhlaq, M.; Ahmad, Z. Development of an ANN optimized mucoadhesive buccal tablet containing flurbiprofen and lidocaine for dental pain. Acta Pharm., 2016, 66(2), 245-256.
[http://dx.doi.org/10.1515/acph-2016-0020] [PMID: 27279067] ].

The data obtained from the main Study was used as input to optimize ingredient’s output. For this purpose, NeuralPower^® version 3.1, artificial neural network (ANN) software was used for prediction of the optimized levels of above polymers [35Takayama, K.; Morva, A.; Fujikawa, M.; Hattori, Y.; Obata, Y.; Nagai, T. Formula optimization of theophylline controlled-release tablet based on artificial neural networks. J. Control. Release, 2000, 68(2), 175-186.
[http://dx.doi.org/10.1016/S0168-3659(00)00248-0] [PMID: 10925126] ]. In this study, 11 tablet formulations, F14-F24 were prepared and buccal tablet formulation F25 was the confirmation formulation. The tests accomplished in addition to that performed in preliminary study included dissolution test, mucoadhesive strength. Results of different tests are summarized in Table 5. For in-vitro release, standard calibration curve was drawn for propranolol HCl in phosphate buffer (pH 6.8) at concentrations from 0.5 to 40 µg/ml and a regression value of 0.9998 was obtained at 289 nm.

Relative importance generated by software of the factors affecting the overall properties of the buccal tablet formulations as shown in Fig. (2). The two factors, viz, HPMC and EC exhibited more importance for the overall properties of the buccal tablet. The relative importance of CMC, lactose was equal to that of SA and CP showed little importance. Thus to achieve an optimized formulation, by manipulating the HPMC and EC, an optimized buccal table was achieved.

Fig. (2) Relative importance of HPMC, EC, CMC, lactose, Na-alginate and CP934 for different physicochemical properties of propranolol HCl generated by the optimization software, NeuralPower 3.1.

The response surface plot of hardness test between HPMC and EC showed that changing amount of HPMC showed inverse relation with hardness of the tablet in the current study. The desired hardness can be achieved if lower amounts of HPMC is used along with higher concentrations of EC used in the study as depicted in Fig. (3a).

The response surface plot showing the combined effect of HPMC and EC indicated the relative importance for physicochemical parameters. The surface pH of all buccal tablet formulations shown to be within the required pH, i.e., 5-7±0.2 [36S Pendekal M. K Tegginamat P. Formulation and evaluation of a bioadhesive patch for buccal delivery of tizanidine. Acta Pharm. Sin. B, 2012, 2(3), 318-324.
[http://dx.doi.org/10.1016/j.apsb.2011.12.012] ]. The response surface plot for surface pH showed that at higher level of HPMC and lower level of EC the pH was higher. The red region in Figs. (3b,c) depicted that pH and mucoadhesive time were mainly dependant on the concentration of HPMC in the formulations. It meant that the increase in the concentration of HPMC was associated with increasing values of pH and mucoadhesion time. The blue colour corresponds to the area where least values of such tests were observed in the curve. The in vitro release was dependent upon the concentration of both polymers i.e. HPMC and EC where the release of drug was dependent upon the lowest concentration of the polymers. At higher concentration, little or least effect was evident at times 1 h 2 h and 3 h according to the surface plots as shown in Figs. (3d,e,f). The release of drug at 4 h was mainly dependant on the concentration of EC where the increase in the concentration of polymer in the formulations was dependent upon the release of the drug as depicted in the red region in Fig. (3g). Conversely, the release of propranolol HCl at 5 h was dependent on the concentrations of both HPMC and EC. Where high impact of EC was found at 5 h and maximum release was relied on EC as shown in red region in Fig. (3h).

The composition provided by the ANN was confirmed by formulating the predicted amounts of the ingredients. The predicted composition is provided in Table 6. The software also generated optimized values of the different physicochemical tests as provided in Table 7.

Fig. (3) Combined effect of HPMC and EC on (a)- hardness, (b)- pH, (c)- mucoadhesive time, (d)- in vitro release at 1 h, (e)- in vitro release at 2 h, (f)- in vitro release at 3 h, (g)- in vitro release at 4 h, (h)- in vitro release at 5 h of propranolol HCl buccal tablet.

Table 6
ANN-predicted composition of optimized mucoadhesive buccal tablet, F25.

Table 7
ANN-predicted physicochemical properties of buccal tablet.

All the tests listed in method section were applied on the confirmation formulation. Additionally, swellability index and release kinetics were employed on the formulation. Results are summarized in Table 8. The formulation F25 showed hardness 8 kg/cm^2, diameter 11.1 mm, thickness 3mm, surface pH 6.5, mucoadhesion time 3.9 h and release was 22% at 15 min, 26% at 30 min, 34% at1 h, 40% at 2 h, 45% at 3 h, 50% at 4 h, 62% at 5h, 76% at 6h, 85% at 7h and 97% at 8 h. However, a slight difference was noted in hardness, surface pH, mucoadhesive time and release profile compared with the predicted values generated by artificial intelligence.

Table 8
Physicochemical characterization of confirmation formulation (F25).

A significant mucoadhesion is required or the attachment of tablet on to the mucosal membrane. Mucoadhesive strength of optimized formulation F25 was 0. 0338N which was comparable [21Patel, V.M.; Prajapati, B.G.; Patel, H.V.; Patel, K.M. Mucoadhesive bilayer tablets of propranolol hydrochloride. AAPS Pharm. Sci. Tech., 2007, 8(3), E77.
[http://dx.doi.org/10.1208/pt0803077] [PMID: 17915827] ]. Swellability index is the direct measurement of sorption of water into the mucoadhesive matrix. The ability of water intake by the polymers used in the formulations makes them swell and creates a passage for drugs to release either by erosion of the matrix or by movement of drug out of the intact matrix [37Boyapally, H.; Nukala, R.K.; Bhujbal, P.; Douroumis, D. Controlled release from directly compressible theophylline buccal tablets. Colloids Surf. B. Biointerfaces, 2010, 77(2), 227-233.
[http://dx.doi.org/10.1016/j.colsurfb.2010.01.031] [PMID: 20188529] ]. Swelling index continuously increased from 42% to more than 100% during time 1 h to 6 h reaching a plateau at 6 h. Swellable weight equal to the dry weight was achieved at 5 h. The maximum swelling was observed at 6 h as observed in Fig. (4).

Fig. (4) Swellability index of Formulation, F25 with predicted trendline.

The desirability of buccal tablet was to release the drug in a continuous manner upto 8 hours. This design was to avoid fluctuation of concentration of drug at the serum. Propranolol HCl is a potent drug and slighter change in the amounts of drug in plasma is associated with adverse effects [38Lardinois, C.K.; Neuman, S.L. The effects of antihypertensive agents on serum lipids and lipoproteins. Arch. Intern. Med., 1988, 148(6), 1280-1288.
[http://dx.doi.org/10.1001/archinte.1988.00380060044012] [PMID: 2897834] ]. So, a continuous release was preferred. The data was used for optimization of the ingredients. The release data for formulation F25 was taken up to 8 h based on the predicted release of at least 65.70% at 6 h, as given in Table 7. The in vitro release data is presented in Table 9. It revealed that the initial release was neither slow nor fast and at time 5 h, half of the drug was released from tablet matrix. The release of the drug was almost complete and at 8 h, more than 97% of propranolol HCl was released.

Table 9
In vitro release data propranolol HCl from the confirmation formulation, F25.

Kinetic behaviour of the confirmation formulation, F25 was studied using software “DD Solver. The coefficient of relationship (R²) values of the models are given in Table 10. Based on the highest R² value, the Korsmeyer-Peppas model (Fig. 5) fitted the release data well and has been indicated in Fig. (4). The n value of the formulation F25 was found to be 0.534 (R² = 0.9235) which suggests the non-Fickian drug release [39Pygall, S.R.; Kujawinski, S.; Timmins, P.; Melia, C.D. Mechanisms of drug release in citrate buffered HPMC matrices. Int. J. Pharm., 2009, 370(1-2), 110-120.
[http://dx.doi.org/10.1016/j.ijpharm.2008.11.022] [PMID: 19100822] ]. Such a system shows that the rate of drug diffusion and the polymer erosion are same [40Dash, S.; Murthy, P.N.; Nath, L.; Chowdhury, P. Kinetic modeling on drug release from controlled drug delivery systems. Acta Pol. Pharm., 2010, 67(3), 217-223.
[PMID: 20524422] ]. It suggests that drug is released by an “initial burst” and drug resides in the surrounding gel layer of the polymer. After that erosion of polymer will take place following the diffusion of drug into the dissolution medium [39Pygall, S.R.; Kujawinski, S.; Timmins, P.; Melia, C.D. Mechanisms of drug release in citrate buffered HPMC matrices. Int. J. Pharm., 2009, 370(1-2), 110-120.
[http://dx.doi.org/10.1016/j.ijpharm.2008.11.022] [PMID: 19100822] ]. The amount of the drug released depends upon the penetration of dissolution medium through the surrounding gel of CP into the HPMC matrix.

Table 10
The value of R² in different kinetic models to predict the release kinetics of optimized mucoadhesive formulation, F25.

Fig. (5) Fitting of release of propranolol HCl in optimized formulation, F25 according to Korsmeyer-Peppas Model.