Design, development, in vitro evaluation and pharmcokinetic studies of bioadhesive buccal patches for pioglitazone and glimepiride

The buccal ilm know-how has arisen as an advanced substitute to the other conventional types of drug delivery systems. The buccal mucosa is a suitable route of administration. In this study, a bioadhesive buccal therapeutic system using Glimepiride and Pioglitazone to prolong the residence time at its point of administration. The FTIR studies showed no chemical incompatibility between drugs and excipients. The DSC of the product showed two separate peaks for Pioglitazone and Glimepiride, indicating no potential incompatibility. It was found that the breadth of all buccal ilms ranges from 0.28 to 0.30 mm. In all formulations, drug content was determined to be in the range of 1.97 to 2.02 mg for glimepiride and 14.95 to 15.01 mg for pioglitazone. Total percentage of cumulative of drug released from all formulations are from 64.12% to 87.0% for glimepiride and 70.0% to 92.2% for pioglitazone. The predicted values by the model agree with actual values. The drugs were released for 12 h, and it was found to be Cmax for pioglitazonewas 467.5 ng/mL and for glimepiride was 65 ng/mL. Tmax was determined for pioglitazone was three h and for glimepiride was 2.8 h. The drugs are released for 12 h AUC of pioglitazone is 4031.25 h ng/mL, and AUC of glimepiride is 505 h ng/mL. Buccal patch for glimepiride and pioglitazone was prepared successfully.


INTRODUCTION
The advantages of the buccal ilm are low therapy cost, the cosiness of administration and self-medication (Gupta et al., 2009). Paediatric and elderly patients circumvent taking the solid oral preparations such as tablets and capsules due to choking hazard, dif iculty in swallowing and do not follow the doctor's direction (Gupta et al., 2012).
The use of buccal membranes as sites for drug administration has been a topic of interest for long time onwards. The absorption of the drug from the buccal membrane, evading irst-pass hepatic metabolism and gastrointestinal drug degradation.
As buccal ilms are attached to the buccal mucosa, they can be formulated to exhibit local and systemic action. The buccal ilm may be advanta-geous over a buccal tablet for lexibility and comfort. The bioavailability will be improved because of circumventing the irst-pass metabolism. These dosage forms do not require an expert to administer, economical and has better patient compliance (Kurosaki and Kimura, 2000;Lee et al., 2000).
A buccal ilm is a dosage form that uses water dissolving polymer that dissolves the drug in mouth and absorbs into blood (Sudhakar et al., 2006). The main advantage of the buccal ilm is the large surface area of the buccal membrane that increases absorption of the drug fast when compared to tablets (Joseph et al., 1987). Bioadhesive buccal ilms had been formulated for local action to cure fungal infections in the oral cavity (Adhikari et al., 2010).

Mechanism of action (MOA)
Glimepiride (GLM) belongs to Sulphonylureas (SU) which increases insulin secretion from beta-cells of the pancreas and recovers both irst and second phases of insulin secretion.

MOA
Pioglitazone (PLG) is a thiazolidinedione group that acts as a peroxisome proliferator activating receptor (PPAR)-γ agonist that increases whole-body insulin sensitivity. These drugs have bene icial effects on atherogenic diabetic dyslipidemia and recover several atherosclerotic risk markers and lipoproteins. These two drugs act synergistically to treat type 2 diabetes. To develop a formulation based on mucoadhesive patch system (Derosa, 2007).

Objectives
In the present investigation, an attempt is made to fabricate bioadhesive buccal therapeutic system of Glimepiride and Pioglitazone to sustain the residing time at the site of application to enhance the bioavailability.

FT-IR spectra
Separately the FTIR of pioglitazone, glimepiride and buccal ilm with potassium bromide was done. This mixture was punched to form a pellet. FTIR spectrophotometer (FTIR 8400 S, Shimadzu) is used for this study.

Differential scanning calorimetry (DSC)
Differential scanning calorimetry was carried out at the rate of 10 • C/min in the Atmosphere of Nitrogen from 30 to 300 • C. Mettler DSC1 is used for this study.

Design
After doing the preliminary studies, Central composite design (CCD) was selected. The design was given (Table 1).

Preparation
The drugs pioglitazone and glimepiride were dissolved in ethanol (10 mL) (API solution) and the other ingredients were dissolved in a separate container using distilled water (10 mL) under continuous stirring for 4 hours. After stirring, mix the API solution and polymer solution. Propylene glycol (plasticiser) was added into an API solution with constant stirring. The viscid solution was allowed overnight to get a clear, bubble-free solution. The solution was poured into a glass Petri dish and left to dry, and a lexible dry patch was produced. The patch was removed carefully, checked for air bubbles. The patch was cut into pieces of the measured area. Aluminium foil was used to pack the patches. The packed patches were kept in desiccators to preserve (Balamurugan et al., 2001;Li et al., 1997). Tables 2 and 3 shows the composition and design.

Film thickness
The thickness of each ilm was determined using "Dial Caliper". Detection limit 0.05 mm

Folding endurance
Folding of the ilm was done repeatedly until it breaks. The number of times the ilm could be folded without breaking is the value of folding endurance.

Swelling index
Buccal ilms were initially weighed (W 1 ) accurately and immersed in 50 mL of water. Films were taken out after two excess water is drained with ilter paper which was present on their surface and weighed (W 2 ). The swelling index was calculated using the following formula.

Diffusion Studies
Diffusion studies were conducted in Franz diffusion cells with a volume of 125 mL in the acceptor compartment. These are carried out at 37 • C, and 480 rpm for eighth. Samples were withdrawn in ixed timings. First 2 h in pH 1.2 and then pH 6.8 is used. A cellulose membrane with molecular weight cut-off  Ethanol 95% (ml) 10.000 10 Puri ied Water (ml) 10.000 Each formulation contains 2 mg of GLIM and 15 mg of P at 12 k Da. was used. The released drug was estimated by HPLC and is given below.

HPLC method
The method was developed using Shimadzu LCLC which consisted of a column Agilent BDS Hypersil C18 (250 x 4.6mm) with 5 µm particle size, isocratic pump mode.

Chromatographic conditions
The assay was detected at 225 nm, and the injection volume 10 µL.
A mixture of Buffer: Methanol: Acetonitrile 37:42:21 was used as mobile phase and a low rate of 1.5 mL/min. A buffer of 0.05M Disodium hydrogen Phosphate was adjusted to pH 3.0. All solutions were degassed by ultra-sonication. The mobile phase was iltered through a 0.45 µ nylon ilter before using (Mallu et al., 2011).

In-vitro bioadhesive strength studies
Bioadhesive quality of the patches was analysed by the marginally adjusted method utilising the porcine gastric mucosa as the model ilm. The instrument is comprehensively made out of two physical arm balance in which the correct container had been sup-   planted by a detailing holding glass plate and offset a water gathering skillet hanging to one sidearm. The container got a siphon tube from a 10 L bottle that was kept back at a high spot so that water head in the jug, consistently stays over the water gathering dish. The siphon tube bears a stream controlling gadget. Nylon string was utilised to suspend both the glass plate and the dish. An adhesive tissue mounting stage was joined to the focal point of a glass container. Glass measuring utensil was loaded up with phosphate buffer (pH 6.8) to re-enact in-vivo salivation conditions. A stirrer furnished with temperature control was utilised to keep up the temperature of phosphate buffer (pH 6.8) in a glass dish at 37 ± 0.5 • C. The uncovered ilm surface was dampened with phosphate buffer (pH 6.8) and left for quite a while for hydration and expanding. At that point glass plate (with the ilm) was kept on the mucosal tissue made sure about on the tissue mounting stage so that ilms stayed in contact with the mucosa. The entire set up was saved undisturbed for a few moments (preload time) to set up the grip between the ilm and mucosal tissue. The glass plate (weight 50 g) itself went about as a preload. After the preload time, water gathering dish was suspended to one sidearm and water was included it, by the siphon tube, at a steady pace of 200 drops for every moment until the separation of the ilm from mucosal surface occurred-the water gathering dish to hold it at the hour of separation. Weight of water gathered in the container at the hour of separation was estimated. The examination was acted (n =3). Bioadhesive quality of buccal movies was estimated on the "Adjusted Physical Equalisation". Rodent peritoneal ilm as a model mucosal layer (Sharma et al., 2019).

In vivo studies
Healthy rabbits weighing 1.5 to 2.0 kg each were used for the pharmacokinetic studies Institutional Animal Ethics Committee's approval (ref no. VIPS/1454/12-13) was obtained before the commencement of the study. Newzeland Rabbits (6) were fasted 12 h before the study. A blank blood sample was taken from the marginal ear vein of each rabbit (control). Animals were anaesthetised by giving ketamine (40 mg/kg) and xylazine (5 mg/kg) through the intramuscular route. Then the buccal ilm was wetted with a small quantity of water (30 mL) and attached to the buccal mucosa of the rabbits. Blood samples were collected at the intervals of time (0, 1, 2, 3, 4, 6, 8, 10, and 12 h).

Analysis of plasma samples
The HPLC system is Shimadzu. The column kept at ambient temperature. The mobile consisted of a mixture of methanol and ammonium acetate buffer (pH-3.5) ixed in the ratio of 55: 45 at a low rate of 0.5 mL/min.

Extraction of drugs from rabbit plasma
The extraction of drugs from was done by using ether as a solvent.
The HPLC system consisted of injector valve with a 10 µL loop, the column used was Aqua RP-C18 column (250 x 4.6 mm internal diameter, 5 µm particle diameter), (Phenomenex, USA) and a precolumn (guard column with C18 precolumn inserts) (Waters, USA), a Knauer Model K-2500 UV variable wavelength detector, the chromatograms were saved, the eluent was iltered through a 0.45 µm membrane ilter using vacuum iltration unit (Phenomenex, USA). 20 µL aliquots were injected (n = 3) and eluted with the mobile consisted of a mix-ture of methanol and ammonium acetate buffer (pH-3.5) ixed in the ratio of 55: 45 at a low rate of 0.5 mL/min the eluent was monitored at 252 nm (Lakshmi et al., 2009).

Inference
No chemical incompatibility between drugs and excipients.

Differential scanning calorimetry (DSC)
Inference DSC of the product has two separate melting peaks for Pioglitazone and Glimepiride -No potential incompatibility ( Figure 2).

Film thickness
It was found that the thickness of all buccal ilms ranges from 0.28 to 0.29 mm in all formulations. The physical parameters of buccal ilms were found and were given (Table 4).

Content
The assay for drug content was to determined, and those are in the range 1.97 to 2.02 mg for GLIM and14.95 to 15.01 mg for PLG. The example of the chromatogram is given in Figure 3.

Bioadhesive Strength
Effect of variables on bioadhesive strength, the constant and regression coef icient for bioadhesive strength is given by R3 = 8.51 + 0.7153*Xantham gum + 0.508*Carrageenan. The linear model F-value of 13.00 and probability value 0.0017, which is less than 0.05 means the model is signi icant. 3D graph and predicted vs actual are given for bioadhesive strength (Figures 6 and 7).

Effect of variables on release
The linear model is found signi icant for drug release with model F-value 13.35 and p-value 0.0015 for glimepiride.
2. The percentage of cumulative drug release of the optimised formula is given in Table 5. A good relationship is observed between experimental and predicted values as below (Table 6).

ANOVA, Pure Error, Absence of Fit
Results of ANOVA (Table 7) demonstrate model is signi icant for all dependent variables. All the independent variables (factors) found to be significant for all R1, R2, R3 and R4 response variables. The absence of it F-value of 5.97 implies (folding endurance) there is a 5.27% coincidental that an absence of Fit F-value this large could occur due to noise. The absence of Fit F-value of 1.24 implies (for bioadhesive strength) not substantial relative to the pure error. Non-signi icant lack of it is good -we want the model to it.

In vivo evaluation
The drugs were released for 12 h and it was found to be C max for pioglitazone is 467.5 ng/mL and for glimepiride is 65 ng/mL. t max for pioglitazone was found to be 3 h and for glimepiride was 2.8 h (Figure 12). AUC of pioglitazone is 4031.25 h ng/mL and AUC of glimepiride is 505 h ng/mL.

CONCLUSIONS
Effect of Xanthan Gum is more pronounced than that of Carrageenan. The experimental independent variables found to be very close to predicted values of the optimised formulation, which proves the possibility of the optimisation method in the successful development of buccal ilms containing Glimepiride and Pioglitazone.