BI-LAMINATED ORAL DISINTEGRATING FILM FOR SYMPTOMATIC TREATMENT OF VIRAL NASOPHARYNGITIS: FORMULATION, CHARACTERIZATION, TASTE MASKING, AND STABILITY STUDIES

Objective: Nasopharyngitis (NP) is one of the most common upper respiratory tract infections (URTIs) brought on by viral infections and requires symptomatic treatment. In this study, a new approach to delivering drugs was developed, which is a bi-laminated oral disintegrating film (ODF) containing dextromethorphan hydrobromide (DEX), phenylephrine hydrochloride (PE), and methylcobalamin (MeCbl) to support the symptomatic treatment of NP. Yet, the extreme bitterness of DEX and PE required a taste-masking technique before ODF formulation to enhance patient compliance. Methods: Various complexing agents were tried at different ratios to mask the bitter taste of the drug(s). DEX-PE ODFs were formulated using the solvent casting procedure. A 3 1 .2 1 full factorial design was performed to characterize DEX-PE ODFs, where the effects of polymer grade and plasticizer type were evaluated on the disintegration time (DT) and the percentage of drugs released after 10 min (Q 10 ). Results: Complexation with maltodextrin (MD) and ion exchange resin (Kyron T-314) could successfully mask the bitter taste at a ratio of 1:2:2 of drugs, MD, and Kyron T-314, respectively. The ODF optimized formula (F4) recorded the least DT (5±0.5 s) and highest Q 10 (96.7±2.1%) and (97.4±1.9%) for DEX and PE, respectively. F4 was then combined with the second film layer containing MeCbl to develop the bi-laminated ODF (B1), which was later evaluated and subjected to stability studies. Conclusion: In conclusion, a taste-masked, bi-laminated ODF could be successfully developed for the symptomatic treatment of NP.


INTRODUCTION
Oral disintegrating film (ODF) is one of the most innovative oral drug delivery technologies that enhances patient acceptability.It dissolves rapidly in the mouth without chewing or drinking water.This method of administration is suitable, especially for the geriatric and pediatric populations that have difficulty swallowing traditional oral dosage forms [1,2].Nasopharyngitis (NP), generally known as the common cold, is among the most common upper respiratory tract infections (URTIs).NP's symptoms include weakness, nasal congestion, headache, cough, sore throat, and other symptoms.NP is caused by viral infections of the upper respiratory tract, and its treatment is primarily symptomatic [3,4].Dextromethorphan hydrobromide (DEX) is a non-narcotic antitussive drug that acts centrally on the cough center and alters the brain signals that cause the cough reflex.In 1958, the FDA approved it as an over-the-counter cough suppressant [5,6].According to the Biopharmaceutics Classification System (BCS), DEX is classified as a class II drug [7].Phenylephrine hydrochloride (PE) treats nasal congestion and cough in URTIs, sinusitis, and rhinitis [8].In the BCS classification, phenylephrine hydrochloride is a class I molecule [9].Both drugs have an extremely bitter taste because of the amine functional group [6].Methylcobalamin (MeCbl) is a water-soluble vitamin belonging to the B complex.It has crucial roles in the cardiovascular and circulatory systems, as well as in the control of the immune system and antiviral activities.Because of its role in neuromuscular diseases and its ability to act as an analgesic, vitamin B12 can be considered an adjuvant therapy for mild to severe respiratory viral infections because of its significant role in viral infections [10].MeCbl, as a form of vitamin B12, is classified as BCS class three [11].
One of the biggest challenges in developing drug formulations is masking the bitter-tasting active pharmaceutical ingredients to achieve an acceptable level of palatability.Taste masking can be done using a variety of techniques, including the addition of sweeteners, flavoring agents, ion exchange resins, and complexing agents such as β-cyclodextrins (CDs) and maltodextrins (MDs) [12,13].
This work aimed to formulate a taste-masked formula of DEX-PE to be incorporated into the first film layer of a bi-laminated ODF.Formulation and characterization of several DEX-PE ODFs were done by assessing the formulation factors impacting the ODF characteristics via a 3 1 .2 1 full factorial design using Design-Expert® software version 12.The optimized formula of DEX-PE ODF was combined with a second film layer containing MeCbl, which was evaluated to develop a bi-laminated ODF.The bi-laminated ODF was subjected to shelf and accelerated stability studies to ensure the stability of the final formula.

Preparation of the taste-masked formulae
Several trials were prepared to select the most taste-masked formula, where each trial was prepared inside a 200-ml glass beaker containing 100 ml of distilled water, 0.5 g of DEX, 0.25 g of PE, 0.2 g of sucralose as a sweetener, and 0.1 g of lemon flavor as a flavoring agent.Then, different combinations of ionexchange resin (Kyron T-314), CD, and MD as taste-masking agents were added at two ratios of (1:1) or (2:1) to the drug(s), as shown in table 1.Each preparation was stirred for 30 min using an Ika Eurostar 20 digital overhead stirrer (Staufen, Germany) [14].Participants were fully aware of the study's nature and goal before it began, and they gave written consent. 1 ml of each prepared formula, equivalent to 5 mg of DEX and 2.5 mg of PE, was administered on the tongue of the six volunteers and then spit out after 5 seconds, followed by a mouth rinse with water before administering the next one.The bitterness degree was recorded to select the most taste-masked formula.The optimized taste-masked formula was subjected to further studies [6,15].

Evaluating the effect of different ODF formulation variables using a full factorial design
Polymer grade (X1) with three levels (HPMC E5, HPMC E15, and HPMC E50) and plasticizer type (X2) with two levels (PG+glycerin and PEG400+glycerin) were investigated as two independent variables using a 3 1 .2 1 full-factorial design via Design-Expert® software version 12 Stat-Ease, Inc., Minneapolis, MN, USA).The dependent variables were disintegration time (DT) (Y1) and percent released after 10 min (Q10) (Y2), as shown in table 2. The compositions of the six formulae developed are shown in table 3.

Formulation of DEX-PE ODFs
DEX-PE ODFs were formulated via the solvent casting procedure using three different polymer grades (HPMC E5, HPMC E15, or HPMC E50) and two different plasticizer combinations (PEG400+glycerin or PG+glycerin), where the polymer and the plasticizers were weighed and dissolved in distilled water to form a polymeric solution.Then, DEX and PE were dissolved in distilled water and stirred with Kyron T314, MD, lemon flavor, and sucralose for 30 min to be transferred to the previous polymeric solution.The preparation was left for 24 h to remove all air bubbles and transferred for coating using an ODF coating machine (Optimags, Germany).The drying process was performed at 60 °C at a speed of 3 m/h.The film was peeled off and cut to the desired size (2 x 3 cm 2 ), then stored in an aluminum foil pouch until evaluation [16].

Visual examination
The homogeneity and color of all the prepared DEX-PE ODFs were assessed visually [17].

Film thickness
As the thickness of the film directly influences the uniformity of the drug(s) content, it is crucial to maintain uniformity in the film's thickness.Using a digital thickness gauge meter (Shanghai, China), the thickness of each film was measured in different areas.The average thickness and the standard deviation (SD) were then determined [18].

Surface pH
The ODF was placed on a petri dish and moistened with 0.5 ml of simulated saliva fluid (SSF) of pH 6.8 for 30 s.A pH meter was used to record the pH.The results were presented as the mean±SD [19].

Drug content
Drug content analysis was performed to confirm the drug loading in the film using high-performance liquid chromatography (HPLC) coupled with an ultraviolet-visible (UV-VIS) detector [20].One film (2 × 3 cm 2 ) was dissolved in 200 ml of SSF, followed by a dilution of 20 ml into 100 ml of SSF to get a final concentration of 5 µg/ml and 2.5 µg/ml of DEX and PE, respectively.The sample is filtered through an Agilent PTFE 0.45 µm syringe filter to be analyzed by HPLC.Chromatographic separation was carried out at a 200 nm wavelength on an Inertsil C18 (4.6 mm x 15 cm, 5 µm) column at a temperature of 40 °C, using a mobile phase composed of 2% acetonitrile and 98% buffer that is prepared by dissolving 3g of sodium perchlorate monohydrate in 1000 ml of distilled water and adjusted to pH 3 by orthophosphoric acid.A membrane filter with a 0.45 μ pore size was used to filter the mobile phase.The analysis was performed at a 1 ml/min flow rate and an injection volume of 100 μl.All measurements were done in triplicate, and the values were represented as the mean±SD.

Construction of a standard calibration curve for DEX and PE
Serial concentrations of 2, 3, 4, 5, 6, 7, 8, and 9 µg/ml for DEX and 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 µg/ml for PE were prepared using SSF.The peak area responses of the prepared solutions were measured by HPLC at the above-mentioned chromatographic conditions.The measured peak area responses were plotted against the corresponding concentrations.The linearity correlation coefficient (r 2 ) and % RSD for peak area response for lower and higher concentrations were calculated.

Folding endurance
It was repeatedly folding the film 180 degrees in the same spot more than 100 times until it broke.The value of folding endurance was determined [21].

In vitro disintegration time (DT)
The pharmacopeial disintegration apparatus was used to carry out the disintegration test.Separately, six films were put into the tubes of the USP apparatus, which were then allowed to move up and down in the disintegration medium (phosphate buffer, pH 6.75 at 37±1 °C) at a rate of 30 times per minute, and the time taken for the films to start disintegration was recorded.The average and SD were determined [22].

In vivo evaluation of ODF disintegration time
Six healthy volunteers participated in the evaluation of the in vivo disintegration time of ODFs.The Research Ethics Committee (REC-FOPCU) at the Faculty of Pharmacy at Cairo University, Egypt, gave the study protocol its permission (protocol No. PI (3189)).Participants were fully aware of the study's nature and goal before it began, and they gave written consent.Participants were instructed to put the film on their tongues, gently slide it on the hard palate of their mouths, and let it dissolve.The lack of the tongue's sensory detection of the films indicated their full disintegration, which was also confirmed by the researcher's monitoring of their absence.The participants were then instructed to wash their mouths with a glass of water.The disintegration time was recorded for each formula [23].

In vitro drug release
The test was done via the USP dissolution system, Agilent (California, USA), USP Apparatus 5 (paddle over disc) at 37±0.5 °C at a speed of 50 rpm using 900 ml of SSF (pH 6.8).Samples were withdrawn at 5, 10, 15, 20, and 30-minute intervals.The percentage of drugs released was measured by HPLC [24].

Optimization of DEX-PE ODFs
An efficient approach for selecting the optimized formula was to use Design Expert® software version 12.The optimum formula was chosen as the ODF with the fastest DT and highest Q10.

Drug-excipient compatibility testing
Testing for drug-excipient compatibility can be performed using various techniques to determine any incompatibility between drug and excipient combinations.A trustworthy result is produced using HPLC technology coupled with a UV-Vis detector in assessing drug excipient compatibility [24].Drug-excipient blends (1:1) underwent isothermal stress testing (IST), which promotes any incompatibilities and speeds up drug interactions with the excipients by being heated to a high temperature (>50 °C) for about 3-4 w [25].

Simultaneous determination of DEX, PE, and MeCbl by HPLC
Chromatographic separation of DEX, PE, and MeCbl in SSF was carried out by HPLC equipped with a UV-Vis detector at the same chromatographic conditions mentioned earlier.A standard calibration curve of MeCbl was constructed where serial concentrations of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 µg/ml of MeCbl were prepared using SSF.The measured peak area responses were plotted against the corresponding concentrations.The r 2 value and % RSD for peak area responses for lower and higher concentrations were calculated.

Bi-laminated ODF formulation comprising DEX, PE, and MeCbl
The DEX-PE optimized ODF (F4) and the MeCbl-ODF layer formed the composition of the bi-laminated ODF (B1), as shown in table 3. The solvent casting method, as explained earlier, was used to develop each layer using the ODF Coating Machine (Optimags, Germany).The first layer (DEX-PE) was cast and dried at a temperature of 60 °C and a speed of 3 m/h, followed by releasing the second layer (MeCbl) over the first one at a temperature of 40 °C and a speed of 3 m/h to form the bi-laminated ODF (B1), which was subsequently cut and subjected to further evaluation [26].When DEX, PE, and MeCbl were combined into one film as an initial formula (U1), the temperature required for drying the whole film was 65 °C.This could be explained by the higher thickness of the initial formula (U1) than that of the MeCbl layer.Since MeCbl is known to undergo thermal degradation [27], It was recommended to develop a bi-laminated ODF that contained DEX and PE in one layer and MeCbl in the second, with drying temperatures of 60 °C and 40 °C, respectively.Both trials were subjected to further stability studies.

Bi-laminated ODF (B1) characterization
The bi-laminated ODF (B1) was tested using the same characterization tests and procedures previously done on the DEX-PE ODFs.

Stability studies of the bi-laminated ODF (B1)
Since any pharmaceutical formulation's performance is significantly influenced by its stability, the optimized bi-laminated ODF formula (B1) and the initial formula (U1) were packed in sealed triplelaminated aluminum pouches and kept for three months in Thermolab Walk-In Stability Chambers (Thermolab House, Maharashtra, India) at shelf stability conditions (25 °C, 60% relative humidity (RH)) and accelerated stability conditions (40 °C, 75% RH) by the International Council on Harmonization guidelines.After three months, they were examined for their surface pH, folding endurance, film thickness (mm), drug content (%), DT, and Q10 [28,29].

Statistical analysis
The experimental findings of DEX-PE ODFs' characterization were analyzed using GraphPad Prism 5.01 statistical software (GraphPad Software, Inc., La Jolla, CA, USA).A one-way analysis of variance (ANOVA) was performed, followed by Tuckey's test, to assess the significance among the formulated (DEX-PE) ODFs regarding the dependent variables (DT and Q10).Additionally, the in vitro and in vivo DT results were tested for each formula to determine the in vitro/in vivo correlation.The significance or non-significance of stability studies' results on B1 ODF and U1 ODF were also analyzed.The results are considered statistically significant when (*p<0.05).

In vivo taste evaluation of DEX and PE
Table 4 shows the findings of the taste evaluation.The bitterness degree was recorded for each formula.The drug(s) alone in T1 had an extremely bitter taste.In contrast, no bitterness was perceived in T11, which indicates successful taste masking of both DEX and PE using a combination of MD and ion-exchange resin (Kyron T-314) at a ratio of 1:2:2 of drugs: MD: Kyron T-314, respectively.

Standard calibration curves of DEX, PE, and MeCbl
The calibration curve plots demonstrate linearity over a range of 40 to 180% of working level concentration, as shown in fig. 1, with r 2 values of 0.9991, 0.9998, and 0.9998 for DEX, PE, and MeCbl, respectively, and % RSD of 1.41%, 0.52%, and 0.63% for DEX, PE, and MeCbl, respectively.

Characterization of DEX-PE ODFs
Table 5 shows the results of the formulated DEX-PE ODFs.The films were white, thin, and smooth, as shown in fig. 2. The DEX-PE ODFs had thickness values ranging from 0.115 mm±0.01 mm for F1 to 0.125 mm±0.01 mm for F6 and a surface pH ranging from 6.78±0.05for F5 to 6.89±0.05for F3, which was close to the salivary pH (6.8).All the films exhibited values for folding endurance up to 300 folds, demonstrating the films' good flexibility [21].The drug content of the prepared DEX-PE ODFs was found within the pharmacopeia specifications in all the formulae, ranging from 98.5±0.8% to 100.6±0.5% and from 98.7±0.3% to 99.8±0.3% for DEX and PE, respectively, and this shows that the drug(s) were distributed uniformly throughout the film [21].Fig. 3 and 4 reveal the effect of formulation variables on the results of DT and Q10 of DEX-PE ODFs.The grade of the polymer (X1) and the type of plasticizer (X2) had a statistically significant effect on DT and Q10 of DEX-PE ODFs (**p<0.01),where ODF (F4), formulated by HPMC E5 and PEG400, recorded the shortest DT (5±0.5 s) and the greatest Q10 (96.7±2.1% for DEX and 97.4±1.9% for PE).On the other hand, ODF (F3) based on HPMC E50 and PG recorded the longest DT (42±1.2s) and the least Q10 (61±2.5% for DEX and 67.4±2.9% for PE).This could be explained by the fact that as the molecular weight and viscosity of the polymer increase, the disintegration time increases, and thus the release rate (Q10) decreases.The polymer grades can be arranged according to their molecular weight and viscosity, from lowest to highest: HPMC E5<HPMC E15<HPMC E50 [30,31].The surface of the HPMC E5-based films was porous and developed a network-like structure that allowed rapid disintegration and dissolution by Jadhav et al. [32].It was observed that films with PEG400 had faster disintegration and a higher dissolution rate than those with PG.PEG400 had an essential function in the process of dissolution as it not only acted as a plasticizer but also a dissolutionenhancing agent, which could contribute to the strong polarity of PEGs because of the terminal hydroxyl and ether groups in their structure [32,33].Additionally, PEG400 has a good dissolution ability owing to its low molecular weight.It causes rapid drug(s) release as it augments the dissolution, which is in harmony with Chopade et al. [34].

Optimization of DEX-PE ODFs
The F4 formula was chosen by Design Expert® software version 12 to be the optimum formula with the shortest DT (5±0.5 s) and the greatest Q10 (96.7±2.1%) and (97.4±1.9%) for DEX and PE, respectively.Characterization and evaluation of the Bi-Laminated ODF (B1)

Drug-excipient compatibility testing by HPLC
Table 5 shows the results of the characterization tests performed on the bi-laminated ODF (B1).The DEX-PE layer (F4) was a smooth, thin, and white layer, whereas the MeCbl layer was red since the drug itself has a red color, as shown in fig. 2. B1 ODF has a folding endurance of up to 300 folds, which ensures the bilaminated film's flexibility.The drug content (%) of the drugs was accepted within the pharmacopeia specifications.The in vitro release (Q10) of DEX, PE, and MeCbl in B1 was 96.2%, 96.7%, and 97.1%, respectively.

Stability studies of the bi-laminated ODF (B1)
The optimized formula (B1) was packed in triple-laminated aluminum pouches.The results over the specified time under accelerated stability conditions are shown in table 6, where visual examination, surface pH, film thickness, folding endurance, DT, and Q10 were not significantly impacted.The drug content (%) decreased from 99.8% to 98.6% for DEX and from 99.5% to 98.8% for PE (p-value>0.05).Drug content (%) of MeCbl decreased from 101.9% to 97.1% and from 98.8% to 92.3% in B1 and U1, respectively, indicating a statistically significant difference (*p<0.05) between B1 and U1 for MeCbl.This demonstrates that the optimized formula B1 was stable over the three months and confirms its preference over the initial formula U1 regarding MeCbl stability.

CONCLUSION
A taste-masked, bi-laminated ODF could be successfully developed for the symptomatic treatment of NP.The taste-masking technique before ODF formulation was achieved by the complexation of DEX and PE with MD, along with the use of ion exchange resin (Kyron T-314) at a ratio of 1:2:2 of drugs: MD: Kyron T-314, respectively.A 3 1 .2 1 full factorial design was performed to formulate and characterize DEX-PE ODFs.The optimized ODF (F4), based on HPMC E5, PEG400, and glycerin, recorded the least DT and highest Q10.The optimized formula (F4) was then combined with the second film layer containing MeCbl to develop the bi-laminated ODF (B1), which was later evaluated and subjected to shelf and accelerated stability studies to ensure the stability of the final formula.

Fig. 6
Fig. 6 presents HPLC chromatograms of the bi-laminated ODF excipients, the drug(s) in combination with the excipients initially, and the drug(s) combined with the excipients after being stored under IST for four weeks.No changes were detected in the peak area responses

Fig. 5 :Fig. 6 :
Fig. 5: Bar chart of in vitro and in vivo DT of DEX-PE ODFs.There was no significant difference between the in vitro and in vivo DT in all the formulae (p>0.05)