Construction of sublingual trilaminated Eszopiclone fast dissolving film for the treatment of Insomnia: Formulation, characterization and In vivo clinical comparative pharmacokinetic study in healthy human subjects.

BACKGROUND: Disturbed sleep can cause to m health problems such as cognitive impairment, depressed mood, and negative effects on cardiovascular, endocrine, and immune function. This study formulates and optimizes Eszopiclone trilaminate fast dissolving film.
METHODS: Prepared Eszopiclone trilaminate fast dissolving film (Eszopiclone TFDF) was characterized by disintegration time, drug release, tensile strength (TS), percentage elongation (EB%), folding endurance, taste masking test, and in vitro dissolution test. The selected formulas were F2 (0.5% xanthan gum, 10% propylene glycol), F4 (3% sodium alginate, 10% propylene glycol) and F6 (1.5% pullulan, 10% propylene glycol) were subjected to in vivo study compared to conventional Lunesta® tablet.
RESULTS: The results indicated that disintegration time was in the range of 940 m. Drug release was found to be in the field of 78.51%-99.99%, while TS values and EB% differed from 11.12 to 25.74 (MPa) and 25.38%-36.43%, respectively. The folding endurance went between 200 and 300 times. All formulas exhibited acceptable uniformity content, surface pH, film thickness, and a good taste feeling.
CONCLUSION: F4 had the highest Cmax (39.741 ± 6.785-μg/l) and lower Tmax (1.063 hr) among other formulas and conventional tablets. Therefore, FDFs' technology could increase the therapeutic effect of Eszopiclone.

1. Introduction

Insomnia can cause many health problems, such as cognitive impairment, depressed mood, and adverse effects on cardiovascular, endocrine, and immune function [1]. In the US, Eszopiclone is the first hypnotic agent that does not restrict its length of use. It can be used for 6–12 months without evidence of problems (e.g., tolerance/dependence) [2]. The FDA approved Eszopiclone (Lunesta®) on December 15th, 2004.

Eszopiclone (C17H17ClN6O3) is a non-benzodiazepine hypnotic agent of the cyclopyrrone class (hypnotic sedative class of drugs) with a molecular weight of 388.81. It is a white to light-yellow crystalline solid, slightly soluble in water, slightly soluble in ethanol, and soluble in phosphate buffer (pH 3.2) [3, 4]. Eszopiclone is rapidly absorbed following oral administration. Its peak plasma concentrations are achieved about an hour after oral administration. Eszopiclone is weakly bound to plasma protein (52%–59%). The most dramatic side effect of Eszopiclone is its unpleasant taste in the mouth, so it has been formulated as film-coated tablets to be used for oral administration in 1-mg, 2-mg, or 3-mg tablets [4].

Fast dissolving films (FDF) have shown the ability to mask the drug’s bitter taste, enhance the onset of drug action, decrease the dose frequency, convenient dosing, and enhance the drug efficacy [5, 6]. Dose accuracy in comparison to syrup is also advantage of fast dissolving film [7, 8]. The disadvantage of FDF is that high doses cannot be incorporated into the strip. The amount should be between 1–40-mg. There remain many technical limitations with film strip use; the thickness while casting the film. Glass petri- plates cannot be used for casting. The other technical problem with these dosage forms is achieving dose uniformity. Packaging of films needs special equipment, and it is not easy to pack [6, 9, 10].

An ideal fast dissolving delivery system should have the following qualities: high stability, transportability, ease of handling and administration, no special packaging material or processing requirements, no water necessary for application[6, 11].

The formulation of Eszopiclone as a fast-dissolving film may lead to masking the bitter drug taste, very rapid absorption with a fast onset of action, and can also enhance its bioavailability.

This study formulates and optimizes fast dissolving film containing Eszopiclone in addition to studying the influences of formulation parameters on film attributes (in vitro disintegration time, drug content, percent elongation, tensile strength (TS), folding endurance, taste masking test, in vitro dissolution test, and in vivo bioavailability).

2. Material and methods

2.1. Chemicals and excipients

Eszopiclone was bought from the Western company, Egypt. Pullulan, Hydroxypropyl methylcellulose (HPMC) were gifts from EVA Pharma, Egypt. Potassium dihydrogen orthophosphate was bought from Winlab (Leicestershire, United Kingdom). Sodium phosphate dibasic was supplied from Sigma-Aldrich (Missouri, USA). Glycerol, propylene glycol, gelatin, sodium alginate, xanthan gum, clove oil, peppermint oil, and sucralose were bought from Al-Gomhoria Company for medicines and medicals, Cairo, Egypt. All other reagents and solvents used were of analytical grade.

2.2. Methodology

2.2.1. Differential scanning calorimetry (DSC)

DSC test was used to identify the melting temperature and material purity or interaction using a differential scanning calorimeter (Shimadzu DSC TA-50 ESI, Tokyo, Japan). The incompatibilities were detected by appearance, shift, or disappearance of the corresponding peaks.

2.2.2. Construction of Eszopiclone triple fast dissolving films

Taste masking techniques and triple layer films are realistic to overcome the very bitter taste of Eszopiclone as follows.

2.2.3. Preparation of medicated fast dissolving films containing Eszopiclone

FDF were prepared using the solvent casting technique [12]. Polymers (Gelatin, xanthan gum, sodium alginate, and pullulan) and propylene glycol were used as a plasticizer with different concentrations (5%–10%) chosen from the preliminary study to form FDF containing the calculated amount of drug (Eszopiclone). The composition of different formulas is provided in Table 1.

Table 1

Composition of medicated fast dissolving film containing eszopiclone.

Formula	Xanthan gum %w/w	Sodium alginate %w/w	Pullulan %w/w	Gelatin %w/w	Propylene glycol %w/w
F1	0.5%	-	-	-	5%
F2	0.5%	-	-	-	10%
F3	-	3%	-	-	5%
F4	-	3%	-	-	10%
F5	-	-	1.5%	-	5%
F6	-	-	1.5%	-	10%
F7	-	-	-	2.5%	5%
F8	-	-	-	2.5%	10%

a. All formulas containing 63.585-mg Eszopiclone (1-mg in 1-cm2 film).

b. The final volume was completed to 20-gm in all formulas with isopropyl alcohol: water (1:4) w/w.

c. All medicated film surrounded by a sweetening film and flavoring film explained below.

2.2.4. Preparation of plain film for sweetening effect and plain film for flavoring effect

Specified weight of film-forming polymer HPMC (1.5%) was first dissolved in 30-mL casting solvent (distilled water) while stirring on a magnetic stirrer (L32; Bibby, Staffordshire, UK). the polymeric solution was divided into two equal portions. Sweetener (Sucralose) was dissolved in the first portion of the polymeric solution, and the calculated amount of plasticizer propylene glycol (10%) was added to form plain film for sweetening effect. Clove oil, peppermint oil (flavoring agents) was added to the second portion of the polymeric solution to give pain relief and the mouth refreshment feeling. The final volume of each was adjusted to 20-mL with a solution containing (distilled water: isopropyl alcohol (1:1)). And the beaker was covered with aluminum foil to prevent solvent evaporation. The casting solution was subjected to gentle stirring for two hours using a magnetic stirrer (L32; Bibby, Staffordshire, UK).

The casting solution (20-mL) was transferred into a previously cleaned and dried petri-plate (Glass petri- dishes; Desiccators, glads funnels (diameter was 9-cm)). Then, the solution was cast on petri-dish and left to evaporate at room temperature for the next 24 h. After drying, the film was removed from the petri-plate and cut into the desired size (1-cm x 1-cm). Then, wrapped in an aluminum foil (to maintain the integrity and elasticity of the films) and were finally stored in a dry place at ambient room temperature.

2.2.5. Formation of trilaminated FDFS (sandwich appearance)

The film containing the flavoring agent was placed on the surface of the film containing the drug after spraying on the drug loaded FDF with small drops of casting solvent (isopropyl alcohol (1): water (4)) w/w on the surface of the film, the film containing the sweetening agent is placed on the other side of the film containing the drug, and the films stick together using a weight and left overnight. The films were subjected to evaluation within one week of preparation.

Visual examination: The smoothness, color, clarity, and transparency of oral films were examined visually. A subjective scoring system was applied to evaluate the prepared films. The score (+++) indicated complete peeling, flexibility, and transparency from the substrate. Films that could not be peeled from the substrate, even though they were transparent, took the score (++), fissured or very brittle films took (+).

Film thickness: Formulations were evaluated using a micrometer screw gage (Mitutoyo, Kawasaki, Japan) with an accuracy of 1-μm. Each sample film was measured using a micrometer at three places, and the calculated mean values [13].

The pH of the surface: The pH was noted by placing the film in a petri-dish, slightly wet with the help of distilled water, then bringing the film’s surface in contact with pH meter electrode (Mettler Toledo—Greifensee, Switzerland) for one minute [14].

Drug content: Eszopiclone-loaded FDF was placed in a 100-ml volumetric flask, then dissolved in 50-ml phosphate buffer (pH 6.8) using a magnetic stirrer. The volume was completed using phosphate buffer (pH 6.8). The resulting solution was filtered to remove undissolved residue. One milliliter of the solution was further diluted to 10-ml with phosphate buffer solution (pH 6.8), and the absorbance was measured at 304-nm using a UV spectrophotometer (Biochrom Libra S22, Biochrom Ltd, Cambridge England) [15]. Drug content uniformity was conducted in triplicate [16]. A mean of three readings and standard deviation was recorded. Three films of each formula were used in this test [17].

Tensile Strength Measurement (TS) is the maximum stress applied to a point at which the film specimen breaks and can be calculated by the following equation: Tensile Strength = Force at break (N)/ Cross-sectional area (mm2) [17].

Tensile testing was conducted using a texture analyzer AG/MC1 (Acquati, Italy) [18], equipped with a 5 N load cell. The film was cut into 30 × 20-mm strips. Tensile tests were conducted following the ASTM International Test Method for Thin Plastic Sheeting (D 882–02) [19]. Each test strip was placed in tensile grips on the texture analyzer. The initial grip separation was 20-mm, and crosshead speed was 1-inch/min. The maximum fracture force, i.e., the force attained just before the film strips ruptured, was recorded [20]. The measurements were repeated in triplicate using three film samples for each formulation type.

Percentage elongation: When stress is applied, a strip sample stretches, and this is referred to as a strain. Strain is the deformation of the strip divided by the original dimension of the sample. Generally, elongation of the strip increases as the plasticizer content increases [21].

Folding endurance (FE): is the most useful index of bending the flexibility of FDFs. It is measured by repeatedly folding the film at the same place until it breaks. The number of times the film could be folded at the same place until it broke or folded up to 300 times, which is considered satisfactory to reveal good film properties defined as FE [22, 23]. Three films of each formula were used in this test [24, 25].

2.2.8. In vitro disintegration time (DT) of fast dissolving films (FDF)

DT was obtained by placing the film (1-cm2) of each formula in a petri-plate in a beaker containing about 25-ml phosphate buffer (pH 6.8), and the time taken by each film to completely disintegrate was noted and considered as DT [16, 26]. Three films of each formula were tested.

2.2.9. In vitro dissolution test of fast dissolving films (FDF)

In vitro dissolution tests of Eszopiclone-loaded oral films were performed using USP dissolution apparatus (Type II) (apparatus Pharma Test Apparatebau PT-DT70—Hainburg, Germany). The test was conducted at 37°C ± 0.5°C with stirring speed of 100-rpm ± 2 rpm in 100-ml phosphate buffer (pH 6.8). Samples were withdrawn at predetermined time intervals (1, 2, 3, 5, and 7 min) and replaced with the same volume of fresh buffer, in which sink conditions were maintained during dissolution. The absorbance was determined at λmax 304-nm using UV–visible spectrophotometer (Biochrom Libra S22, Biochrom Ltd Cambridge England) against a blank made of non-medicated films at the same conditions. The amount of drugs dissolved from each film was calculated. Three films of each formula were tested [27, 28].

2.2.10. Taste masking test (human panel testing) of fast dissolving films (FDF)

Sensory evaluation of the taste of formulations was performed by six healthy volunteers [29] (three female and three male) aged (27–40) years are asked to take the bitter drug, and then the optimum taste-masked formulations selected to in vivo evaluation. Volunteers are then asked to comparatively rate the formulation on different organoleptic properties [30, 31]. A numerical scale was used with the following values: 0 –very pleasant, 1-pleasant, 2 –slightly pleasant, and 3-unpleasant.

Experimental Design and Sample Collection: An in vivo study was conducted to compare the pharmacokinetic parameters of Eszopiclone from the optimized films (test products) and the commercially available, Lunesta® tablet containing 1-mg (reference product), after single oral administration. The protocol and informed consent form were approved by REC-FOPCU (PI 2313). Eight male healthy human volunteers were involved in the study. The study was fully explained to the volunteers before starting the study, and each volunteer signed a written informed consent form as per declaration of Helsinki (Brazil 2013), the study did not include minors, each subject signed the informed consent form by himself.

Two treatments, four periods, randomized, and cross-over design were performed within a 1-week washout period as indicated in Fig 3A. The volunteers were randomly allocated to one of the two groups of equal size. They were only allowed to take water after one hour, and a standard meal was given after four hours from the administration of treatments.

Fig 3

a. Design of Pharmacokinetics study. b. LC-MS_MS chromatograms of Eszopiclone. c. Average plasma drug level of Eszopiclone.

The study was supervised by a physician who was also responsible for the volunteers’ safety and sample collection. Samples of venous blood (5-mL) were collected in heparinized blood tubes through the indwelling cannula immediately before oral dosing and at the predetermined time intervals of 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 24 h after dosing. The plasma samples were obtained by centrifuging the samples at 3500 rpm for ten minutes, and this was frozen at −20°C in labeled tubes until further analysis.

Sample preparation: About 50-μL Aripirazole (from a stock solution of concentration 20-ng/mL) was added to each sample (450-μL plasma) as an internal standard. Eszopiclone and Aripirazole were obtained using ethyl acetate (5-mL), and vortexed for 20 s, then centrifuged for one minute at 5000 rpm (cooling centrifuge, TGL-20 MB). The supernatant was transferred to other vials filtered through a 0.22–l-m Millipore filter, then evaporated to dryness using a vacuum concentrator (Eppendorf Vacufuge plus, Germany).

LC-MS/MS Assay of Eszopiclone: Fig 3B represents a chromatogram of a confirmed LC-MS/MS method for analyzing plasma Eszopiclone concentrations, which was employed using LC-MS/MS system (Shimadzu®, Japan) coupled with a triple quadrupole detector (API-4500, AB Sciex, Foster, CA, USA). The mobile phase comprises Methanol: 0.2% Formic acid in water. The chromatographic separation was performed on the Sunfire C18 column (4.6 × 50-mm i.d., 5-μm diameter; Agilent, CA, USA). The injection volume was 10-μL, and the flow was isocratic at a rate of 0.2-mL/min. The method has been validated in terms of selectivity, linearity, precision, accuracy, carryover, extraction recovery, and stability.

Statistical analysis of the Pharmacokinetic results: A non-compartmental pharmacokinetic model was applied to analyze the pharmacokinetic parameters of Eszopiclone after oral administration of two treatments using the PK solver program [32]. These parameters included peak plasma Eszopiclone concentration (Cmax, ng/mL) and time to reach (Tmax, h.), elimination rate constant (Kel, h−1), terminal half-life (t1/2, h.), area under the plasma concentration-time curve from time zero to the last observation time point (AUC(0–24), ng h/mL) and infinity (AUC(0–∞), ng h/mL). IBM SPSS Statistics 20 (Armonk, NY, USA) were employed to analyze all statistical differences in data using a one-way ANOVA test for the established pharmacokinetic parameters, and p-value < 0.05 was statistically significant. Nonparametric Kruskal–Wallis test was conducted to compare the Tmax data obtained from two treatments.

3. Results and discussion

3.1. Differential scanning calorimetry (DSC)

The DSC thermogram of pure Eszopiclone has one main prominent sharp endothermic melting peak at about 206.85°C (onset to 193.24°C and endset to 218.75). The thermogram of xanthan gum was characterized by two thermal events: the first endothermic centered at about 104.01°C, attributed to water loss associated with the hydrophilic groups of the polymer, and the second exothermic centered at about 290.28°C, corresponding to the thermal degradation of xanthan. Three thermal events characterized sodium alginate: two are endothermic at about (93.41°C and 248.91°C), and the third has exothermically centered at about 290.6°C. Gelatin was characterized by two endothermic thermal events: 81.52°C and 216.41°C. Meanwhile, broad peaks were observed at 67.14°C, and 3118.19°C from the DSC thermogram pullulan. The sharp endothermic peak of Eszopiclone was unchanged in the thermogram, confirming the absence of interaction between the drug and excipients used in the formulation. As indicated in Fig 1.

Fig 1

DSC thermograms of drug and excipients.

3.2. Physicochemical In vitro evaluation of fast dissolving films (FDF)

3.2.1. Visual examination

Only films with a score (+++) were conducted for the in vivo evaluation. The formulas that shows the best appearance were F2, F4, and F6, which were prepared from xanthan gum, sodium alginate, and pullulan as polymers, and 10% w/w propylene glycol as a plasticizer that gave the best film flexibility. According to the results in Table 2.

Table 2

In vitro evaluation of medicated fast dissolving films (FDF).

Formula N°	Visual examination	Thickness (mm)	Surface pH range	Drug content	Tensile strength MPa	Elongation% EB%	Folding endurance	Disintegration time (sec)
F1	++	0.23 ± 0.01	6.7	108 ± 0.03	16.18 ± 0.02	27.38 ± 0.01	281 ± 5	11 ± 0.02
F2	+++	0.24 ± 0.01	6.7	91 ± 0.01	20.72 ± 0.02	36.43 ± 0.02	296 ±2	13 ± 0.02
F3	++	0.24 ± 0.02	6.5	99 ± 0.04	19.87 ± 0.02	26.88 ± 0.03	269 ± 2	12 ± 0.01
F4	+++	0.25 ± 0.01	6.8	97 ± 0.03	25.74 ± 0.02	35.23 ± 0.03	291 ±1	14 ± 0.03
F5	+	0.26 ± 0.04	6.6	109 ± 0.03	11.12 ± 0.01	25.38 ± 0.05	200 ±2	9 ± 0.02
F6	+++	0.28 ± 0.02	6.6	103 ± 0.03	15.67 ± 0.01	30.41 ± 0.04	253 ±1	10 ± 0.01
F7	++	0.27 ± 0.01	6.8	99 ± 0.01	12.99 ± 0.01	28.11 ± 0.04	290 ±3	35 ± 0.01
F8	++	0.23 ± 0.05	6.5	100 ± 0.02	14.41 ± 0.02	32.89 ± 0.01	300 ±4	40 ± 0.05

3.2.2. Film thickness

The thickness of the prepared films is illustrated in Table 2. The results demonstrate that all Eszopiclone FDF formulas showed a thickness from 0.23–0.28 mm. This uniform thickness assures the uniform distribution of the drug and polymer inside the film, causing the uniformity in weight and drug content (uniform dosing). The variation in thickness is less than ± 5%.

3.2.3. The pH of the surface

The surface pH of the prepared films ranged from 6.5–6.8 within the physiological range, as indicated in Table 2. Therefore, the prepared films are nonirritant to the lung [33].

3.2.4. Drug content

According to the results indicated in Table 2. All Eszopiclone FDFs formulas showed a drug content range from 97%–109% as F5 > F1 > F6 > F8 > F3 = F4 > F7 > F2. Each 1cm2 film contained ~ 1-mg Eszopiclone as expected.

3.3. Mechanical Invitro evaluation of fast dissolving films (FDF)

3.3.1. Tensile strength (TS), percentage elongation (EB%)

The ideal fast dissolving film has higher tensile strength and percentage elongation. [34]. Table 2 indicates the TS values and EB%, which differed from 11.12 to 25.74 (MPa) and 25.38%–36.43%, respectively. By increasing the plasticizer concentration from (5% to 10%), TS and EB% increase. The addition of pullulan decreases the compatibility of film components, and therefore, film flexibility reduced decreasing in EB% and FE [35]. Pullulan loses its water content easily during the drying process, so it causes less bonding, more phase separation, and larger particle sizes reducing EB%. An increase in particle size causes a loss of mechanical qualities [27]. But xanthan gum, gelatin, and sodium alginate tend to be gelatinous and retain water. According to the literature, TS should be maximized, EB% should be about 20% [36].

3.3.2. Folding endurance

A higher FE value indicates the more mechanical strength of a film. A direct relation exists between mechanical strength and FE of films. The FE varied between 200 and 300 times, as indicated in Table 2, which is considered the sign of good flexibility and elasticity. Mechanical strength is governed by a plasticizer concentration, which directly affects FE value [24]. Also, the FE was increased as the concentration of the polymer increased.

3.4. In vitro disintegration time (DT) test of fast dissolving films (FDF)

In vitro disintegration time (DT) was determined in a phosphate buffer (pH 6.8) as shown in Table 2. The results clearly indicated that the formulated films had a fast disintegrating nature at saliva pH. The polymers used to prepare films had good film-forming properties with good wetting properties [37]. F4 (3% sodium alginate and 10% propylene glycol) show the fast DT while F8 (2.5% gelatin and 10% propylene glycol) show the delayed DT 40 sec therefore, it was excluded from in vivo study. Also, Plasticizer act by inserting themselves between the polymer strands and breaking the polymer-polymer interactions, and increasing the molecular mobility of the polymer strands [38], which plays a role in increasing the disintegration. It was concluded that polymer affects the disintegration time.

3.5. In vitro dissolution test of fast dissolving films (FDF)

The release study was conducted using a phosphate buffer (pH 6.8) to simulate the condition of the buccal cavity. Dissolution samples were taken at 1, 2, 3, and 7 min. The drug release was in the range of 78.51%–99.99%. All polymers used are hydrophilic polymers that absorb the water and form the gel responsible for the retardation of the drug from the film system. Hydrophilic polymers create the pore and channels on the film surface that released the drug from the film [39]. The results of the dissolution test are illustrated in Fig 2.

Fig 2

In vitro dissolution in phosphate buffer pH 6.8.

F7 (2.5% gelatin and 5% propylene glycol) have the lowest percentage drug release within the first seven minutes (78.51%). The percentage of drug released within the first seven minutes from F2, F6, and F2 and formula containing 10% propylene glycol shows higher drug release than other formula (97.7%, 98.52% 99.99%, respectively).

3.6. Taste masking test (human panel testing) of fast dissolving films (FDF)

The drug was very bitter; all three trilaminated formulations are pleasant. F6 > F4 > F2 > Tablet. These results due to the polysaccharide properties of pullulan [40]. The drug does not move from its layer unless it has been wetted with water. So, special packaging is required for products stability and safety [41]. The saliva in the oral cavity wets the film as it consists of hydrophilic polymers and dissolves it rapidly, drug at this point migrate from its layer to sweetening layer so the bitter taste of the drug was masked completely by this technique.

3.7. In vivo pharmacokinetic studies of fast dissolving films (FDF) in human volunteers

The optimized formulations (F2, F4, and F6) were used for in vivo evaluation compared to a marketed Eszopiclone 1-mg tablet (Lunesta ®). The plasma concentration of Eszopiclone versus time for all samples was plotted in Fig 3C.

A one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) as a post hoc test was applied; using SPSS program version 17 software. The differences were considered significant if P<0.05. The column effluent was detected spectrophotometrically at 304 nm. Retention time for aripiprazole was 1.929 min which is well differentiated from the peak of the eszopiclone drug which has retention time 1.865 min as shown in Fig 3B which also revealed that there were no peaks due to formula compounds that might interfere with the assay. Calibration curve were constructed for eszopiclone with an equation that best describe the curve is (y = 0.0137 x– 0.0102) as y is represented by peak area and x for the concentration in (ng/ml) (r2 = 0.9774) as shown in Fig 3D.The limit of detection and the limit of quantitation were 0.05 and 0.5 (ng/ml) respectively.

Key pharmacokinetic parameters, peak plasma concentration (Cmax), time to reach the maximum peak (Tmax), and the extent of absorption (AUC) for the three Eszopiclone FDF and lunesta® were calculated in Table 3. A significant difference (p-value < 0.05) was found between the reference and F4 which contain sodium alginate as polymer in all parameters tested as seen in the sheet attached with our response. Tmax is decreased in F4 formulae which contain sodium alginate as polymer and it was 1.063 h for F4 while 2.25 h only for commercial tablet. Relative bioavailability is increased 1.6 fold for F4, 1.2 fold for F2 and 1.1 fold for F6.

Table 3

In vivo pharmacokinetic parameters & data represent the mean value ± standard deviation (SD).

Treatment	Cmax (ng/mL)	Tmax (hr)	AUC0_t (hr*ng/mL)	AUMC0_t (hr*hr*ng/mL)	AUMC0_INF (hr*hr*ng/mL)
REFRENCE	22.132 ± 2.107	2.25 ± 1.134	138.071 ± 22.571	900.696 ± 207.04	1389.48 ± 469.678
F2	24.862 ± 2.805	1.188 ± 0.259	168.669 ± 20.204	1104 ± 124.554	1507.125 ± 188.939
F4	39.741 ± 6.785	1.063 ± 0.291	225.69 ± 19.68	1402.216 ± 139.466	1948.783 ± 204.245
F6	24.89 ± 7.838	1.344 ± 0.399	160.345 ± 58.375	1123.805 ± 408.136	1641.089 ± 620.144

Eszopiclone FDF containing sodium alginate as polymer indicated the highest Cmax (39.741 ± 6.785-μg/l) and the lowest Tmax (1.063 h) than Eszopiclone FDF containing xanthan, pullulan as polymers, which showed Cmax (24.862 ± 2.805-μg/l and 24.89 ± 7.838-μg/l) respectively and Tmax (1.188 h and 1.344) and the Eszopiclone ® (with Cmax = 22.132 ± 2.107-μg/l and Tmax = 2.25 h), showing a significant difference in their absorption rate. This could be attributed to the faster disintegration rate of FDFs among the conventional sublingual tablets. The calculated AUC values for Eszopiclone FDFs containing sodium alginate, xanthan, pullulan as polymers and Eszopiclone ® were 241.788 ± 19.053-μgh/l, and 180.572 ± 17.31-μg h/l, 175.225 ± 61.746-μg h/l, and 151.92 ± 28.02, respectively, as indicated in Table 3.

The increased AUC showed a better absorption extent of sodium alginate containing FDF than xanthan FDF, pullulan FDF, and conventional tablets. The rate and extent of drug absorption were enhanced by the FDF technique, considering the elimination of the influences of the gastrointestinal tract and the first-pass effect through the buccal trans-mucosal absorption [42, 43].

4. Conclusion

In this study, Eszopiclone was incorporated for the first time in trilaminated FDF using various polymers: pullulan sodium alginate, xanthan gum, and gelatin. Based on the obtained results, trilaminated FDF masks Eszopiclone’s bitter taste with acceptable uniformity content, film thickness, percentage dissolution, disintegration time, FE, EB%, and TS properties. Formulations with F2 (0.05% xanthan gum, 10% propylene glycol), F4 (3% sodium alginate, 10% propylene glycol), and F6 (2.5% pullulan, 10% propylene glycol) were represented as optimized formulations for in vivo study. Pharmacokinetic studies in humans demonstrated that F4 containing sodium alginate as polymer had the highest AUC and Cmax as well as a lower Tmax among other formulations and conventional tablets. Therefore, FDFs’ technology could improve the therapeutic effect of Eszopiclone.

28 Jan 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

José das Neves

Academic Editor

PLOS ONE

Journal Requirements:

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf  and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please amend your Methods section to state the name of your Institutional Review Board and to state any inclusion/exclusion criteria for participants.

3. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://pubmed.ncbi.nlm.nih.gov/24015378/

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Few comments are as follows:

1) pH word in abstract should be corrected.

2) Heading for 2.2.2, what is tril?

3) Please explain rational for selections of polymer concentration. It is based on Literature search or placebo trials.

4) 2.2.4 : In which layer and when drug should be added is not mentioned.

5) 2.2.5, sandwich approach is good but is drug migration occurs from middle to end layer?

6) P from petri plates should be small at many places.

7) Is for testing in volunteers any ethical approval taken?

8) Why ariprirazole is selected as IS?

9) Line 276, page 14. Gives characteristics of buccal film. Is film described in this manuscript buccal or dissolving?

10) Line 317, page 16, says results are due to polysaccharide properties of pullulan. But maximum good taste is shown by F4 having sodium alginate as polymer. Explain.

11) Figure 1 showed DSC thermogram of excipients and 1 physical mixture. It is physical mixture with which excipient? For better understanding of interaction, authors should show physical mixtures of drug with all other polymers.

12) LC-MS/MS chromatogram of drug and IS should be shown.

13) Statistical analysis is explained but not seen in discussion , in figures please include.

Reviewer #2: This is an interesting paper about formulation of eszopiclone oral film. The paper contains manufacturing of films, in vitro evaluations and test in healthy volunteers. The paper is designed suitably. The main concern is that films disintegrated over 10 minutes, much longer than fast dissolving oral films. These cannot be called fast dissolving oral films, as this type of films disintegrate less than one minute. Therefore, with this long disintegration time, how can they be beneficial to patients? The comments are as the following:

1. There are paragraphs in the Introduction that do not cite references such as the last paragraph in page 3. Also, there is an incomplete sentence on page 3 line 60, and line 68, “better” should be replaced with “bitter”.

2. Could the authors please specify the calibration curve characteristics of eszopiclone for UV measurements on page 8.

3. Were ethics obtained to test the taste of films in volunteers and what were exclusion and inclusion criteria?

4. It is claimed on page 11, Line 225 that the LC-MS data were validated, where is the data or reference? What were the limit of detection and limit of quantification?

5. Please use past tense for line 257.

6. Line 284, please cite a reference.

7. How did the volunteers hold a film for 10 minutes in the mouth, did they chew them and then swallow?

8. Did the authors obtain GMP grades of active ingredient and excipients to prepare the oral films for volunteers? Who supplied these?

9. A statistical method is described in the Method section but the results are not presented.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Comments for PLOS_Jan 22.docx

Click here for additional data file.

20 Feb 2022

Peer Reviewer: 1

Comments to the Author:

1- pH word in abstract should be corrected.

Response: The mistake has been corrected throughout the manuscript and highlighted with yellow color

2- Heading for 2.2.2, what is tril?

Response: it is a written mistake and the correct word is (triple). The correct word has been written in the revised manuscript.

3- Please explain rational for selections of polymer concentration. It is based on Literature search or placebo trials.

Response:

We tried many different concentrations of many polymers in the preliminary study. From which the best results either type of polymer or polymer concentration were selected for construction of eszopiclone triple fast dissolving film. Different researchers in the literature used our polymers in different concentration [1-6].in our work we used this concentrations and additional concentrations in the preliminary study.

Therefore the rational is based on both literature and placebo trials.

The tables of the preliminary study:

formula polymer type polymer conc Plasticizer type Plasticizer conc solvent type

F1 gelatin 40% Propylene Glycol 10% Methanol:Methylene Chloride (1:1)

F2 gelatin 40% Glycerin 20% Isopropyl alcohol:H2O (1:4)

F3 casein 1.50% Glycerin 10% Methanol:Methylene Chloride (1:1)

F4 casein 1.50% Glycerin 10% Isopropyl alcohol:H2O (1:4)

F5 HPMC 1% Propylene Glycol 10% Methanol:Methylene Chloride (1:1)

F6 HPMC 1% Glycerin 20% Methanol:Methylene Chloride (1:1)

F7 gelatin 40% Glycerin 10% Isopropyl alcohol:H2O (1:4)

F8 HPMC 1% Propylene Glycol 20% Isopropyl alcohol:H2O (1:4)

F9 gelatin 40% Propylene Glycol 30% Isopropyl alcohol:H2O (1:4)

F10 gelatin 40% Propylene Glycol 20% Methanol:Methylene Chloride (1:1)

F11 casein 1.50% Glycerin 30% Methanol:Methylene Chloride (1:1)

F12 HPMC 1% Propylene Glycol 30% Methanol:Methylene Chloride (1:1)

F13 gelatin 40% Glycerin 30% Methanol:Methylene Chloride (1:1)

F14 HPMC 1% Glycerin 10% Isopropyl alcohol:H2O (1:4)

F15 casein 1.50% Propylene Glycol 30% Isopropyl alcohol:H2O (1:4)

F16 casein 1.50% Propylene Glycol 10% Isopropyl alcohol:H2O (1:4)

F17 HPMC 1% Glycerin 30% Isopropyl alcohol:H2O (1:4)

F18 casein 1.50% Propylene Glycol 20% Methanol:Methylene Chloride (1:1)

F19 casein 1.50% Propylene Glycol 10% Water

F20 HPMC 2% Propylene Glycol 15% Methanol:Methylene Chloride (1:1)

formula polymer type polymer conc Plasticizer type Plasticizer conc solvent type

F21 casein 1.50% Glycerin 10% Methanol:Methylene Chloride (1:1)

F22 PVA 10% Glycerin 5% Water

F23 casein 1.50% Propylene Glycol 5% Methanol:Methylene Chloride (1:1)

F24 PVA 10% Glycerin 5% Methanol:Methylene Chloride (1:1)

F25 casein 1.50% Glycerin 5% Isopropyl alcohol:H2O (1:4)

F26 PVA 10% Glycerin 15% Methanol:Methylene Chloride (1:1)

F27 casein 1.50% Propylene Glycol 10% Isopropyl alcohol:H2O (1:4)

F28 HPMC 2% Glycerin 10% Water

F29 HPMC 2% Glycerin 15% Isopropyl alcohol:H2O (1:4)

F30 HPMC 2% Propylene Glycol 5% Water

F31 PVA 10% Propylene Glycol 5% Isopropyl alcohol:H2O (1:4)

F32 PVA 10% Glycerin 15% Isopropyl alcohol:H2O (1:4)

F33 casein 1.50% Glycerin 15% Water

F34 HPMC 2% Glycerin 5% Methanol:Methylene Chloride (1:1)

F35 HPMC 2% Propylene Glycol 15% Water

F36 casein 1.50% Propylene Glycol 15% Methanol:Methylene Chloride (1:1)

F37 HPMC 2% Propylene Glycol 10% Isopropyl alcohol:H2O (1:4)

F38 HPMC 2% Propylene Glycol 10% Methanol:Methylene Chloride (1:1)

F39 PVA 10% Propylene Glycol 10% Water

F40 PVA 10% Glycerin 10% Isopropyl alcohol:H2O (1:4)

formula polymer type polymer conc Plasticizer type Plasticizer conc solvent type

F41 HPMC 2% Propylene Glycol 10% ,Isopropyl alcohol:H2O (1:4)

F42 HPMC 2% Glycerin 5% Isopropyl alcohol:H2O (1:4)

F43 PVA 10% Propylene Glycol 10% Water

F44 PVA 10% Glycerin 5% Water

F45 HPMC 2% Propylene Glycol 5% Isopropyl alcohol:H2O (1:4)

F46 HPMC 2% Glycerin 5% Water

F47 PVA 10% Glycerin 5% Isopropyl alcohol:H2O (1:4)

F48 HPMC 2% Glycerin 10% Isopropyl alcohol:H2O (1:4)

F49 HPMC 2% Propylene Glycol 5% Water

F50 HPMC 2% Glycerin 10% Water

F51 PVA 10% Propylene Glycol 5% Water

F52 PVA 10% Propylene Glycol 5% Isopropyl alcohol:H2O (1:4)

F53 HPMC 2% Propylene Glycol 10% Water

F54 PVA 10% Propylene Glycol 10% Isopropyl alcohol:H2O (1:4)

F55 PVA 10% Glycerin 10% Water

F56 PVA 10% Glycerin 10% Isopropyl alcohol:H2O (1:4)

F57 HPMC:Na alginate (1:1) propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F58 PVA 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F59 Pollulan 2% Glycerin 5% Isopropyl alcohol:H2O (1:4)

F60 Pollulan:Maltodextrin (1:1) Glycerin 5% Isopropyl alcohol:H2O (1:4)

F61 HPMC 2% propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F62 HPMC:Pollulan (1:1) Glycerin 5% Isopropyl alcohol:H2O (1:4)

F63 HPMC:Pollulan (1:1) Glycerin 15% Isopropyl alcohol:H2O (1:4)

F64 PVA 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F65 PVA 2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F66 HPMC:Na alginate (1:1) Glycerin 15% Isopropyl alcohol:H2O (1:4)

F67 PVA 2% Glycerin 10% Isopropyl alcohol:H2O (1:4)

F68 PVA propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F69 HPMC:Pollulan (1:1) propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F70 HPMC:PVA (1:1) Glycerin 5% Isopropyl alcohol:H2O (1:4)

F71 HPMC:Na alginate (1:1) propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F72 HPMC:PVA (1:1) propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F73 HPMC:PVA (1:1) propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F74 HPMC 2% Glycerin 5% Isopropyl alcohol:H2O (1:4)

F75 HPMC:PVA (1:1) propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F76 PVA 2% Glycerin 5% Isopropyl alcohol:H2O (1:4)

F77 HPMC 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F78 HPMC:PVA (1:1) propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F79 HPMC 2% Glycerin 15% Isopropyl alcohol:H2O (1:4)

F80 pullulan 2% propylene glycol 5% Isopropyl alcohol:H2O (1:4)

formula polymer type polymer conc Plasticizer type Plasticizer

conc solvent type

F81 Pollulan:Maltodextrin (1:1) propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F82 HPMC:Pollulan (1:1) propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F83 HPMC:PVA (1:1) Glycerin 15% Isopropyl alcohol:H2O (1:4)

F84 Pollulan:Maltodextrin (1:1) Glycerin 15% Isopropyl alcohol:H2O (1:4)

F85 pullulan 2% Glycerin 15% Isopropyl alcohol:H2O (1:4)

F86 HPMC:PVA (1:1) Glycerin 10% Isopropyl alcohol:H2O (1:4)

F87 Pollulan:Maltodextrin (1:1) propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F88 pullulan 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F89 PVA 2% Glycerin 15% Isopropyl alcohol:H2O (1:4)

F90 HPMC:Na alginate (1:1) Glycerin 5% Isopropyl alcohol:H2O (1:4)

F91 HPMC 2% propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F92 HPMC 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F93 HPMC 2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F94 HPMC , Sodium alginate ,Pullulan 1%,1%,1% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F95 HPMC , Sodium alginate ,Pullulan 1%,1%,1% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F96 HPMC , Sodium alginate ,Pullulan 1%,1%,1% propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F97 Sodium alginate 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F98 Sodium alginate 2% propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F99 Sodium alginate 2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F100 Pullulan 2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

formula polymer type polymer conc Plasticizer type Plasticizer

conc solvent type

F101 Pullulan 2% propylene glycol 15% Isopropyl alcohol:H2O (1:4)

F102 Pullulan 2% propylene glycol 5% Isopropyl alcohol:H2O (1:4)

F103 Pullulan , HPMC 1.5%, 0.5 % propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F104 Pullulan , HPMC 1%, 1% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F105 Pullulan , HPMC 1%, 2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F106 Pullulan , Maltodextrin 2%,1% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F107 Pullulan , HPMC 1.5%, 1 % propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F108 Pullulan , Maltodextrin 1.5%,1% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F109

F110 Pullulan , Maltodextrin 2.5%,2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F111 Pullulan 5% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F112 Pullulan , HPMC 2.5%,2.5% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F113 HPMC, Xanthan gum 2.5%, 1% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F114 Pullulan, Xanthan gum 2% ,2% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F115 Pullulan 1.50% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F116 Xanthan gum 0.5% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

F117 Sodium alginate 3% propylene glycol 10% Isopropyl alcohol:H2O (1:4)

4- 2.2.4 : In which layer and when drug should be added is not mentioned.

Response: first we prepare the three films separately:

1) The first film consists of flavoring agent was prepared using casting method in which flavoring agent (clove oil and peppermint oil), HPMC as film forming polymer, propylene glycol as plasticizer, casting solvent (distilled water:isopropyl alcohol (1:1)) were used.

2) The second film consists of the drug was prepared using casting method in which drug (eszopiclone) ,( xanthan gum,sodium alginate or pullulan ) as film forming polymer, propylene glycol as plasticizer, casting solvent (distilled water:isopropyl alcohol (1:1)) were used.

3) The third film consists of sweetening agent was prepared using casting method in which sweetening agent (sucralose), HPMC as film forming polymer, propylene glycol as plasticizer, casting solvent (distilled water:isopropyl alcohol (1:1)) were used.

4) After drying of the three films we stick the first film (flavoring film) and the third film (sweetening film) in the both sides of the second film (drug containing film) using small drops of casting solvent. As mentioned in 2.2.5 and in the figure below.

5- 2.2.5, sandwich approach is good but is drug migration occurs from middle to end layer?

Response:

The saliva in the oral cavity wets the film as it consists of

hydrophilic polymers and dissolves it rapidly. Thus the drug is

released into the saliva and is absorbed via the highly

vascularized oro mucosal tissues.

The drug does not move from its layer unless it has been wetted with water. So it is Require special packaging for products stability and safety[7].The saliva in the oral cavity wets the film as it consists of hydrophilic polymers and dissolves it rapidly. Thus the drug is released into the saliva and is absorbed via the highly vascularized oro mucosal tissues[7]. The discussion was enriched by your valuable comments in section 3.6 page 16 in the revised manuscript.

6- P from petri plates should be small at many places.

Response: All words have been revised, corrected and highlighted.

7- Is for testing in volunteers any ethical approval taken?

The affirmed protocol set by the ethics committee of the Faculty of Pharmacy, Cairo University, Egypt with serial number: PI (2313) and the date of this approval (26 / 11 /2018).

8- Why ariprirazole is selected as IS?

Response: ariprirazole was selected as IS to enhance the calculation using the peak area ratio instead of peak area only to cancel the error due to injection or the error sampling on HPLC and the method was attached in the mail.

Ariprirazole according to its chemical structure has close polarity to our drug (eszopiclone). So it is peak appeared at a retention time 1.929 which is well differentiated from the peak of the drug which has retention time 1.865 as seen in the figure 3b.

Figure 3b: sample of Chromatogram of Eszopiclone (drug) and Aripiprazolr (IS)

Figure of aripiprazole chemical structure

Figure of eszopiclone chemical structure

9- Line 276, page 14. Gives characteristics of buccal film. Is film described in this manuscript buccal or dissolving?

Response:

Formula was prepared as fast dissolving film to achieve higher plasma concentration at lower tmax and also to decrease the residence time in mouth to enhance patient compliance for this drug regarding its bitter taste so buccal film was written as representation of the mouth area. So to correct this misunderstanding, the word buccal has been replaced with fast dissolving film in line 276 page 14.

10- Line 317, page 16, says results are due to polysaccharide properties of pullulan. But maximum good taste is shown by F4 having sodium alginate as polymer. Explain.

Response:

The preliminary study consists of 117 formula from which we choose the best eight formula to continue the advanced tests. In line 317 page 16 the point 3.6 taste masking test the pullulan was given better taste than sodium alginate. Because of this result, we searched for a reason and found that the pullulan has polysaccharide properties so this arrangement was overlapped by mistake and the correct arrangement is F6 > F4> F2 > tablet. This mistake was corrected in page 16, line 316 and highlighted.

11- Figure 1 showed DSC thermogram of excipients and 1 physical mixture. It is physical mixture with which excipient? For better understanding of interaction, authors should show physical mixtures of drug with all other polymers.

Response:

Figure 1: DSC

The physical mixture containing (eszopiclone: pullulan: sodium alginate: xanthan gum: gelatin) in a ratio (1:1:1:1:1) w/w.

12- LC-MS/MS chromatogram of drug and IS should be shown.

Response:

The chromatogram was attached in the main manuscript line 329 page 17.

Fig 3b. LC-MS_MS chromatograms of Eszopiclone.

13- Statistical analysis is explained but not seen in discussion, in figures please include.

Response:

All statistical results are attached with the revised manuscript.

A one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) as a post hoc test was applied; using SPSS program version 17 software. The differences were considered significant if P<0.05. The column effluent was detected spectrophotometrically at 304 nm. Retention time for aripiprazole was 1.929 min which is well differentiated from the peak of the eszopiclone drug which has retention time 1.865 min as shown in figure (3b) which also revealed that there were no peaks due to formula compounds that might interfere with the assay. Calibration curve were constructed for eszopiclone with an equation that best describe the curve is (y = 0.0137 x – 0.0102 ) as y is represented by peak area and x for the concentration in (ng/ml) (r2 = 0.9774 ) as shown in figure (3d).The limit of detection and the limit of quantitation were 0.05 and 0.5 (ng/ml) respectively.

Discussion: When we compare the reference tablet with the three fast dissolving films containing sodium alginate or xanthan gum or pullulan as polymer to study its effect on Cmax , AUC 0-24 and AUC 0-INF. A significant difference (p-value < 0.05) was found between the reference and F4 which contain sodium alginate as polymer in all parameters tested as seen in the sheet attached with our response. Tmax is decreased in F4 formulae which contain sodium alginate as polymer and it was 1.063 h for F4 while 2.25 h only for commercial tablet . Relative bioavailability is increased 1.6 fold for F4, 1.2 fold for F2 and 1.1 fold for F6.

Peer Reviewer: 2

• The main concern is that films disintegrated over 10 minutes, much longer than fast dissolving oral films.

Response:

It is typing error and the correct time is (sec). The correct time has been written in the revised manuscript.

The disintegration time of all formula were found to be between 10 to 40 sec so it is called fast dissolving film. And seen in Table 2 (In vitro evaluation of medicated fast dissolving films (FDF)), page 14 in the main manuscript.

1. There are paragraphs in the Introduction that do not cite references such as the last paragraph in page 3. Also, there is an incomplete sentence on page 3 line 60, and line 68, “better” should be replaced with “bitter”.

Response:

a. We revised the manuscript and the references were added to uncited paragraph and highlighted with yellow color. Additional references were added to other paragraphs which lack of references such as line 154,156,159.

b. The incomplete sentence was revised and completed.

c. “Better” word has been replaced with “bitter” in line 69.

2. Could the authors please specify the calibration curve characteristics of eszopiclone for UV measurements on page 8.

Calibration table

Abs at λmax 304 Concentration µg/ml

0 0

0.197 6

0.281 9

0.359 12

0.447 15

0.521 18

0.607 21

0.696 24

0.764 27

0.859 30

0.934 33

Figure 4 : calibration curve of eszopiclone

This figure was added to line 146 page 8 and highlighted.

3. Were ethics obtained to test the taste of films in volunteers and what were exclusion and inclusion criteria?

Response:

The affirmed protocol set by the ethics committee of the Faculty of Pharmacy, Cairo University, Egypt with serial number: PI (2313) and the date of this approval (26 / 11 /2018).

Inclusion criteria:

• Human Subjects from 18 to 55 years of age (inclusive of both).

• Subjects within the BMI range from 18 to 35 kg/m2.

• Subjects with normal range of vital signs (Blood Pressure, Pulse Rate, Respiratory Rate and Body Temperature).

• Subjects with normal Medical and Surgical history without illness within the last 4 weeks prior to start of the study.

• Subjects with normal functioning of Cardiovascular, Respiratory, Gastrointestinal, Nervous System, Musculoskeletal, Vascular, GenitoUrinary, Endocrine/Metabolic systems.

• Subjects with normal Lymph nodes, head, neck, eyes, ears, nose, throat and skin.

• Subjects with normal laboratory investigations

• Subjects able to communicate effectively.

• Subjects willing to provide informed consent and adhere to the protocol requirements.

• Urine analysis for narcotic drug (for all subjects) / Pregnancy test (for females) will be conducted prior to each phase.

Exclusion Criteria:

• Contraindications or Hypersensitivity to Eszopiclone or related group of drugs.

• History or presence of any medical condition or disease according to the opinion of the physician.

• History or presence of significant alcoholism or drug abuse in the past one year.

• History or presence of significant smoking (more than 09 cigarettes/day or consumption of tobacco products and refusal to restrain from smoking or consumption of tobacco products for 48.00 hours before dosing until checkout).

• History or presence of significant renal or hepatobiliary problems.

• History or presence of significant asthma, urticaria or other allergic reactions.

• History or presence of significant gastric and/or duodenal ulcers.

• Difficulty in donating blood.

• Difficulty in swallowing film coated tablet or capsules.

• Systolic blood pressure less than 90 mm Hg or more than 140 mm Hg.

• Diastolic blood pressure less than 60 mm Hg or more than 100 mm Hg.

• Pulse rate less than 50/minute or more than 100/minute.

• Use of any prescribed medication during last two weeks or OTC medicines or medicinal products during the last one week preceding the first dosing.

• Major illness during 3 months before screening.

• Subjects who have been on an abnormal diet during the four weeks before screening.

• Participation in a drug research study within past 3 months.

• Donation of blood in the past 3 months before screening.

• Refusal to abstain from water for at least one hour prior to study drug administration and for at least one hour post dose.

• Refusal to abstain from food for at least ten hours before dosing and for at least 3 - 4 hours’ post dose.

• Refusal to abstain from alcohol or methylxanthine-containing beverages or foods (coffee, tea, carbonated drinks, chocolate) from 2 days prior to dosing till last sample collection.

• HIV positive.

• Anti-HCV antibodies positive.

• Found positive in the Urine drugs of abuse done at the time of check-in at the beginning of each period.

• Evidence of an uncooperative attitude.

4. It is claimed on page 11, Line 225 that the LC-MS data were validated, where is the data or reference? What were the limit of detection and limit of quantification?

Response:

LC-MS were validated as seen in the calibration curve:

conc (ng/ml) peak area of IS peak area of drug PAR

413949 0 0

0.5 418411 29767 0.07114297

5 456679 36747 0.08046571

10 408909 66537 0.16271836

15 437259 108534 0.24821444

25 497676 169853 0.34129233

50 489784 263185 0.53734912

75 401758 405673 1.00974467

100 441067 653450 1.48152095

The limit of detection is 0.05 ng/ml and the limit of quantification is 0.5 ng/ml.

5. Please use past tense for line 257.

Response: past tense was used and corrected in the revised manuscript.

6. Line 284, please cite a reference.

Response: the reference was added to line 284.

7. How did the volunteers hold a film for 10 minutes in the mouth, did they chew them and then swallow?

Response: our formula was prepared as fast dissolving film to achieve higher plasma concentration in lower tmax and also to decrease residence time in the mouth to enhance patient compliance for this very bitter drug. The disintegration time of all formula were found to be between 10 to 40 seconds. A written mistake of the time was correct to sec. The correct time has been written in the revised manuscript in Table 2 (In vitro evaluation of medicated fast dissolving films (FDF)), page 14.

8. Did the authors obtain GMP grades of active ingredient and excipients to prepare the oral films for volunteers? Who supplied these?

Response:

In the material section point 2.1 line 76 page 4. Chemicals and Excipients we described where we obtained each material and each chemical used was supplied with material safety data sheet (MSDS).

9. A statistical method is described in the Method section but the results are not presented.

Response:

All statistical results are attached with the revised manuscript.

A one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) as a post hoc test was applied; using SPSS program version 17 software. The differences were considered significant if P<0.05. The column effluent was detected spectrophotometrically at 304 nm. Retention time for aripiprazole was 1.929 min which is well differentiated from the peak of the eszopiclone drug which has retention time 1.865 min as shown in figure (3b) which also revealed that there were no peaks due to formula compounds that might interfere with the assay. Calibration curve were constructed for eszopiclone with an equation that best describe the curve is (y = 0.0137 x – 0.0102 ) as y is represented by peak area and x for the concentration in (ng/ml) (r2 = 0.9774 ) as shown in figure (3d).The limit of detection and the limit of quantitation were 0.05 and 0.5 (ng/ml) respectively.

Discussion: When we compare the reference tablet with the three fast dissolving films containing sodium alginate or xanthan gum or pullulan as polymer to study its effect on Cmax , AUC 0-24 and AUC 0-INF. A significant difference (p-value < 0.05) was found between the reference and F4 which contain sodium alginate as polymer in all parameters tested as seen in the sheet attached with our response. Tmax is decreased in F4 formulae which contain sodium alginate as polymer and it was 1.063 h for F4 while 2.25 h only for commercial tablet . Relative bioavailability is increased 1.6 fold for F4, 1.2 fold for F2 and 1.1 fold for F6.

1. Ponrasu, T., et al., Fast Dissolving Electrospun Nanofibers Fabricated from Jelly Fig Polysaccharide/Pullulan for Drug Delivery Applications. Polymers, 2021. 13(2): p. 241.

2. Abu-Huwaij, R., et al., Formulation and in vitro evaluation of xanthan gum or carbopol 934-based mucoadhesive patches, loaded with nicotine. AAPS PharmSciTech, 2011. 12(1): p. 21-27.

3. Phaechamud, T. and G.C. Ritthidej, Formulation variables influencing drug release from layered matrix system comprising chitosan and xanthan gum. AAPS PharmSciTech, 2008. 9(3): p. 870-877.

4. Senturk Parreidt, T., K. Müller, and M. Schmid, Alginate-Based Edible Films and Coatings for Food Packaging Applications. Foods (Basel, Switzerland), 2018. 7(10): p. 170.

5. Duckworth, P.F., et al., Alginate films augmented with chlorhexidine hexametaphosphate particles provide sustained antimicrobial properties for application in wound care. Journal of materials science. Materials in medicine, 2020. 31(3): p. 33-33.

6. Zulkiflee, I. and M.B. Fauzi, Gelatin-Polyvinyl Alcohol Film for Tissue Engineering: A Concise Review. Biomedicines, 2021. 9(8): p. 979.

7. Karthikeyan, D., S. Sri, and C.S. Kumar, Development of Fast Dissolving Oral Film Containing of Rizatriptan Benzoate as an Anti Migraine Medication. Indo American Journal of Pharmaceutical Research, 2013. 3(3): p. 2642-2654.

Submitted filename: Peer Reviewer 2 comments.docx

Click here for additional data file.

14 Mar 2022

Construction of Sublingual Trilaminated Eszopiclone Fast Dissolving Film for Treatment of Insomnia: Formulation, Characterization and In-vivo Clinical Comparative Pharmacokinetic Study in Healthy Human Subjects.

PONE-D-21-37028R1

Dear Dr. Teaima,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

José das Neves

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please address the comment of Reviewer #1 regarding language during proofing of the article.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments for ‘Construction of Sublingual Trilaminated Eszopiclone Fast Dissolving Film for

Treatment of Insomnia: Formulation, Characterization and In-vivo Clinical Comparative

Pharmacokinetic Study in Healthy Human Subjects.’

Ref. No. PONE-D-21-37028R

I thank the editor for giving me an opportunity to review this manuscript. All major corrections are done but still language is required to improve.

Eg pg 16 of manuscript, line 318 -So it is Require special packaging for products stability and safety’ this can be written as Special packaging is required for stability and safety of product.

Please note, paper can be accepted after minor /grammatical correction, no need to review again.

Reviewer #2: Dear authours many thanks for addrssing the points and making alterations in the manuscript. It is an interesting study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dr Touraj Ehtezazi, Reader in Pharmaceutics

17 Mar 2022

PONE-D-21-37028R1

Construction of Sublingual Trilaminated Eszopiclone Fast Dissolving Film for the Treatment of Insomnia: Formulation, Characterization and In vivo Clinical Comparative Pharmacokinetic Study in Healthy Human Subjects

Dear Dr. Teaima:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. José das Neves

Academic Editor

PLOS ONE