Natural and semisynthetic polymers blended orodispersible films of citalopram

Abstract This study was aimed at developing orodispersible films of citalopram using combination of natural and semisynthetic polymers for patients with swallowing problem. Okra biopolymer and moringa gum were utilized in combination with hydroxypropyl methylcellulose (HPMC) and pullulan. The disintegration time was less than 30 seconds and the drug content uniformity was 97.89–102.05% for all film formulations. Films formulated with HPMC (K15 and K4M) combination (F1) and combination of okra and HPMC K15 (F2) had superior mechanical properties as compared with F3 (okra and pullulan) and F4 (moringa gum and HPMC). Thermal analysis revealed stable formulations over the studied temperature range and the crystalline citalopram was completely or partially transformed into amorphous form as revealed by the differential thermal analysis, X-ray diffraction and scanning electron microscopy images. In conclusion, okra biopolymer could be used in combination with HPMC for the development of orodispersible films. Graphical Abstract


Introduction
Among various routes of drug administration, the oral route is considered as the most apt because of its adaptability, non-invasiveness and the ease it offers during drug administration (Gou et al. 2018). Moreover, oral drug administration is far more attractive than any other route of drug administration because it allows conversion of a drug into various types of dosage forms such as tablets, capsules, syrups, suspensions and many more (Cilurzo et al. 2017;Speer, Steiner, et al. 2018). Among aforementioned dosage forms, tablets and capsules are the most common oral solid dosage forms and both can unequivocally deliver an accurate amount of the required active pharmaceutical ingredient. However, tablets and capsules are often associated with the difficulty in swallowing or fear of choking, which makes them inconvenient for pediatric, geriatric and bed-ridden patients. Approximately 35% of the general population, 30-40% of elderly nursing home patients and 25-50% of patients hospitalized for psychological or physiological impairments have difficulty in swallowing, a condition known as dysphagia, which prevents children and elderly patients from ingesting tablets or capsules (El Meshad and El Hagrasy 2011). To overcome these issues, orodispersible films (ODFs) have recently been introduced as an alternative to conventional oral dosage forms (Woertz and Kleinebudde 2015;Speer, Preis, et al. 2018). ODFs are stamp-sized polymeric thin films (Scarpa et al. 2018), which rapidly disintegrate upon contact with saliva, thus offering fast drug release without the need of swallowing of dosage form, which is mandatory for tablets and capsules.
ODFs are usually consisted of plasticized water-soluble polymers in which the drug is dispersed or dissolved (Musazzi et al. 2018). This mixture is most commonly solvent casted and then dried at a suitable temperature to convert into thin sheets, which are subsequently cut into appropriate sizes according to the dosing requirements (Visser et al. 2017). In recent years, several film forming polymers from natural, semi-synthetic and synthetic origin have been proposed for formulating ODFs containing different pharmaceutical ingredients. Many natural polymers have been tested for their film forming properties including starch, maltodextrin, pullulan, pectins and many more (Kawahara et al. 2003;Cilurzo et al. 2008;Tong et al. 2008;Koch et al. 2010;Galgatte et al. 2013). In case of semisynthetic polymers, low molecular weight polymers, typically in the range of 1000-9000 Da, are the best candidates for ODFs intended for rapid disintegration in the oral cavity (Borges et al. 2015). Amongst commonly used semisynthetic polymers, cellulose derivatives such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC) and carboxymethyl cellulose (CMC) have been reported as film-forming polymers (El Meshad and El Hagrasy 2011;Liew et al. 2012;Woertz and Kleinebudde 2015). Among synthetic polymers, polyvinyl alcohol, polyvinyl pyrrolidone, Eudragit and polyethylene oxide have previously been used for film formulations (Borges et al. 2015).
Recently, natural polymers especially polysaccharides have gained considerable attention in the pharmaceutical industry because of their safety, biocompatibility and biodegradability (Sharma et al. 2008;Prajapati et al. 2013). Natural polymers are mostly obtained from plants and microorganism. Okra biopolymer is an example of natural polymer, which is extracted from okra plant (Abelmoschus esculentus L.). Okra plant is abundantly cultivated in Africa, Asia and North America with total trade estimated to over $5 billion (Ghori et al. 2014). Okra biopolymer contains pectin, which is a heterogeneous polysaccharide. Okra biopolymer is considered as an inexpensive, biocompatible and biodegradable polymer, and has a number of applications in pharmaceutical and food industries (Ghori et al. 2017). For example, okra biopolymer has been used as a matrix in controlled release tablets, in the formation of topical gels and emulsions (Rana et al. 2011). Another example of natural polymer is moringa gum, which is obtained from Moringa oleifera, a plant native to Western & sub Himalayan tracts, Pakistan, India, Asia Minor, Africa and Arabia (Anwar et al. 2007;Rimpy & Ahuja 2017). To date okra biopolymer and moringa gum have not been tested for film forming properties especially for ODFs. Therefore, this study was aimed at developing ODFs of citalopram HBr (a selective serotonin reuptake inhibitor used in managing depression) using blends of natural and a commonly used semisynthetic polymer, namely hydroxypropyl methylcellulose.

Results and discussion
Orodispersible films loaded with citalopram HBr were successfully formulated by employing solvent casting method using combinations of natural and semisynthetic polymers. Initially, various trials were conducted to find optimum combination and concentration of polymers in order to formulate films of required properties, such as transparency, flexibility or brittleness and easy to peel off from petri dish. Specific polymeric proportions which fulfilled the aforementioned criteria were considered for further evaluation. It was noted that okra biopolymer could only be used in combination with HPMC K15 (a high viscosity grade of HPMC) or pullulan for producing suitable ODFs. Similarly, HPMC K15 and moringa gum combination resulted in ODF, and no other combination was possible within the current experimental setup. Furthermore, all the formulated films were smooth apart from the one produced with HPMC and Moringa gum combination (F4), which was slightly rough in texture. However, no other discrepancy was observed in the optimized formulations. It should be noted that quantities of excipients such as plasticizer, superdisintegrant, salivary stimulant and sweetener were kept constant in all the formulations, and the quantities of film forming polymers were optimized. The prepared film formulations were then extensively characterized and the results are reported here.

Physicochemical studies of ODFs
The prepared ODFs were studied for thickness, weight variation, film's surface pH, time to disintegrate and drug content uniformity. The mean thickness of films were found to be 0.18 ± 0.03 mm, 0.18 ± 0.02 mm, 0.19 ± 0.02 mm, and 0.21 ± 0.03 mm for F1, F2, F3 and F4, respectively. No significant difference was observed in thickness of all four formulated films. The average weight of each film formulation was observed to be 65.01 ± 0.20 mg, 61.31 ± 0.15 mg, 68.10 ± 0.43 mg, 73.09 ± 0.73 mg for F1, F2, F3 and F4, respectively. Surface pH of all film formulations was in the range of 6.46 (± 0.12) -6.95 (± 0.45), which is close to the neutral pH and deemed safe for oromucosal drug delivery.
Disintegration time of orodispersible films is an important factor that defines in vivo performance of orodispersible products. Since in vivo methods involve human volunteers which requires strict ethical considerations, such possibilities become difficult to follow in early drug product research. Furthermore, large variations in disintegration time has also been observed with healthy human volunteers in the case of terbutaline sulfate orodispersible films, which further adds uncertainty in the results due to a number of factors such as gender, saliva composition and pressing effects of the tongue (Sayed et al. 2013). Therefore, in vitro tests stand most reliable in such situations, however, analytical methods used to measure disintegration time differs in the published reports (Speer, Steiner, et al. 2018). Alongside, no clear regulatory requirements for ODFs are available in European Pharmacopoeia, therefore, disintegration time threshold for orodispersible tablets (ODT) is often followed, which is less than 180 seconds (Preis et al. 2014). However, the disintegration time threshold is less than 30 seconds for ODT according to US Food and Drug Administration (FDA 2008). In our study, the disintegration time for F1, F2, F3 and F4 was 19.0 ± 1.0 sec, 25.0 ± 2.0 sec, 11.0 ± 1.0 sec and 18.0 ± 1.0 sec, respectively. The disintegration time for all formulations was within the threshold limit set by the European Pharmacopoeia and US Food and Drug Administration, thus deemed suitable for developing ODFs with polymer combinations as described in this study.
Content uniformity was also estimated for all ODFs. Each film formulation was intended to deliver a 10 mg dose of citalopram. The percentage drug content was found to be 98.90 ± 1.06% for F1, 97.87 ± 3.06% for F2, 102.05 ± 5.16% for F3 and 99.95 ± 0.86% for F4. All the ODFs showed excellent uniformity of citalopram content.

Mechanical properties of films
Primary aim of preparation of ODFs is their sufficient mechanical strength and flexibility that gives them enough stability during handling for packaging and storage. Flexible films resist breakage whilst brittle films are prone to damage at any stage after formulation development. In order to confirm if the ODFs are flexible, the films were manually folded at an approximately 180 angle multiple times at the same place until they break or visible cracks appeared. It was noted that all ODF formulations sustained folding in the range of 220-240 times (Table 1), hence showed sufficient flexibility to withstand industrial packaging and patient's handling.
Mechanical strength of ODFs was estimated using universal testing machine which provides insight into tensile strength, percentage elongation when stress is applied and the Young's modulus, and the values are presented in Table S1, Supplemental material. It should be noted that there is no defined criteria of mechanical properties for ODFs in the European Pharmacopoeia. Therefore, previously reported critical parameters were considered as the quality attributes . According to Visser and coworkers, ODFs with tensile strength greater than 2 N/mm 2 , elongation at break (% E) greater than 10% and the Young's modulus less than 550 N/mm 2 are considered optimum . In this study, F2 and F3 formulations resulted in films with higher tensile strength as compared with F1 and F4. This means that okra based formulations had higher resistance to break as compared with HPMC based films (HPMC K15 and K4M) and the films made with moringa gum and HPMC K15 combination. The percent elongation (% E) for F1 and F2 was found to be 44% and 13.5%, therefore considered more elastic as compared with F3 and F4 which only displayed 2.30% and 8.28% elongation, respectively (Table S1). Young's modulus for all film formulations was less than 550 N/mm 2 , thus confirming that films were sufficiently elastic to withstand the stress.
Based on the mechanical properties of the prepared ODFs, F1 and F2 were considered more suitable. It is noteworthy that okra biopolymer and high viscosity HPMC K15 combination (F2) was found to be superior than HPMC based ODFs (F1) or other polymeric combinations (F3 and F4), thus endorsing suitability of okra biopolymers in developing ODFs.

Solid state characterization
Infrared spectroscopy often leads to determination of interactions between drug and excipients, thus proves to be an important tool in characterizing formulations. In this study, various film forming polymers were used to formulate ODFs loaded with citalopram. Pure drug and all excipients were analyzed for possible interactions using FTIR. It was observed that the characteristic citalopram peaks were present in the film formulations without shifting, although appeared fairly low in intensity, therefore no interaction between polymers and the drug was observed ( Figure S1).
In order to investigate thermal behaviour and the stability of citalopram loaded ODFs, simultaneous thermogravimetric and differential thermal analysis (TG/DTA) was conducted. Thermogravimetric analysis indicates stability of formulations with respect to temperature. Pure citalopram was stable from 30 to 275 C, after which a significant drug loss was observed ( Figure S2-A), thus indicating decomposition of the drug. In comparison to pure drug, film formulations show some initial weight loss when temperature was raised from 30 to 100 C, which is possibly due to the evaporation of the moisture from the polymer matrix. A significant weight loss of ODFs was observed when temperature reached to 250 C, thus confirming the stability of ODFs upto 250 C ( Figure S2-A). Differential thermal analysis of pure citalopram resulted in a sharp melting endotherm appeared at 194 C, indicating crystalline nature of the drug. This sharp endothermic peak was absent in the film formulations as shown in Figure  S2-B. This indicated that the drug was either entrapped with the polymer matrix of the ODFs or drug had undergone transformation from crystalline to amorphous form.
X-ray diffraction patterns could be used to investigate physical state of the drug within a formulation. XRD revealed crystalline nature of citalopram due to presence of several sharp diffraction peaks appearing at 2h angle of 16 , 18 , 20 , 24 , 25 , 27.5 , 28.5 , 31.5 and 32.5 ( Figure S3). XRD patterns of F1 and F3 displayed holographic nature suggesting possible transformation of crystalline drug into amorphous form. However, some peaks were visible in F2 and F4 formulations indicating partial transformation of crystalline drug into amorphous form.
SEM images indicated coarse and rough surface of all film formulations as shown in Figure S4. Most importantly SEM images of F2 and F4 revealed some crystalline drug present in agglomerate form on the film surface, which complements the results of XRD.

Materials
Citalopram HBr, citric acid and fructose were provided as a gift sample by Wilshire Pharmaceuticals (Pvt.) Ltd Lahore, Pakistan. Colorcon UK generously provided HPMC K15 and HPMC K4M. Pullulan and moringa gum was procured from Shaanxi Yuwangtang Biotechnology Development Co., Ltd China. Ludiflash was received as a gift sample from BASF Germany. PEG 400 was sourced from Simz Phama, Lahore Pakistan as a gift sample. Okra biopolymer was extracted from the plant using a previously described method and was used without further purification (Alba et al. 2013). All other reagents and chemicals were of research grade. Distilled water was used throughout the experiments.

Fabrication of orodispersible films
The ODFs containing citalopram HBr were prepared using a combination of different polymers, namely HPMC, okra gum, pullulan gum and moringa gum by solvent casting method, as shown in Table 1. Briefly, exact quantity of each polymer combination was dissolved in 10 mL distilled water and this solution was designated as polymeric solution. To this polymeric solution, an accurately weighed amount of citalopram was added with continuous stirring on a magnetic stirrer. Citric acid as saliva stimulant and fructose as sweetener were separately dissolved in 2 mL of distilled water. This solution was mixed with drug containing polymeric solution under continuous stirring. PEG 400 as plasticizer and ludiflash as superdisintegrant were added to the final mixture with continuous stirring and the final volume of each formulation was adjusted to 15 mL. This mixture was continuously stirred until homogeneity reached. Finally, the mixture was casted on a petri dish having a diameter of 9 cm. The mixture was dried by placing petri dish in a hot air oven set at 45 C for 24 hrs. The films were carefully removed from petri dishes and were cut into the size of 2 Â 2 cm 2 to deliver a 10 mg dose of citalopram. Films were packed in aluminum foil and stored in a desiccator until further use.

Physicochemical studies of ODFs
Various physicochemical properties of ODFs were investigated including thickness, weight variation, film surface pH, disintegration time and drug content. Thickness at three different locations of each film was noted using a digital micrometer (Mitutoyo, Japan). Weight of each film was recorded gravimetrically for measuring inter-film weight variations. Film surface pH was measured by placing probe pH meter (BANTE instruments, China) on the surface of films that were already wetted with a drop of water. To measure disintegration time of films, each ODF was placed in a petri dish containing 10 mL distilled water and time to completely disintegrate was recoded using a stop watch. Finally, drug content analysis of each ODF was measured by dissolving respective film in 10 mL of phosphate buffer saline (pH 7.2). This solution was filtered and absorbance was recorded at 240 nm using a UV/Vis spectrophotometer (UV1800 Schimadzu Corporation, Kyoto, Japan). Concentration of drug in the film was then calculated using a calibration curve. The calibration curve was linear in the concentration range of 1.5-50 mg/mL with a R 2 value of 0.999, a slope value of 0.039 and an intercept value of 0.003 ( Figure S5). For statistical significance in the values, each experiment was repeated three times and the mean along with the standard deviation was reported.

Folding endurance
Folding endurance measures the ability of a film to withstand breaking upon folding. For this purpose, films were folded at the same place until cracks appeared or the film broke. Total number of folds were noted and the test was repeated three times (Sultana et al. 2013).

Tensile strength, percent elongation and young's modulus
Mechanical properties in term of tensile strength, percent elongation and Young's modulus were estimated using a universal testing machine (UTM 100-500KN, Testometric Inc. UK) equipped with 5 N load cell at room temperature as per the specifications mentioned in ASTM D638-02b (Pereda et al. 2011). The UTM apparatus had two clamps; the upper fixed clamp while the lower clamp was free to move. A 2 Â 2 cm 2 sized film was held between two clamps having a distance of 10 mm. An increasing force was applied to pull the clamps at a rate of 1 mm/min and the point at which the film broke was observed along with the required force to break the film (Liew et al. 2012). Tensile strength, percentage elongation (% E) and Young's modulus were calculated from the data obtained using following equations (Cilurzo et al. 2011;Liew et al. 2012); Young ' s modulus ¼ slope of stressÀstrain curve f ilm thickness Â cross À head speed (3)

Solid state characterization
To check possible drug and excipients interactions, Fourier transform infrared spectroscopy (FTIR) was conducted. FTIR spectra of drug and excipients were recorded in the range of 1800-650 cm À1 with a scan resolution of 2 cm À1 using FTIR spectrophotometer (Carry 630 FTIR Agilent Technologies, USA). Thermal stability was studied by using simultaneous thermogravimetric and differential thermal analysis (TG/DTA). Thermal analysis of films were carried out in the temperature range of 40-300 C at a heating rate of 10 C/min under dry nitrogen purge flowing at a rate of 50 mL/hr. X-ray diffraction analysis of film formulations and the pure drug was carried out using X-ray diffractometer JDX-3532 (JEOL, Japan) that is furnished with cupper radiation source with 30kv voltage and 30 mA current. Samples were placed on a holder and diffraction patterns were measured in the 2h range of 10 -80 . Images of ODFs were obtained by scanning electron microscope (SEM) (JEOL JSM5910, Japan).

Conclusions
Okra biopolymer has demonstrated potential as a polymer of choice for making orodispersible films in combination with HPMC. This study demonstrated for the first time that a flexible and mechanically strong ODF with a fast disintegration time could be formulated using okra and HPMC. This particular ODF can be used as a potential alternative dosage form for the delivery of an antidepressant drug, namely citalopram.