Smart Approach for Preparing Nanosized Topiramate Loaded Bio-Flexy Films Using Film Former from Piper Nigrum and Screening its In-Vitro Performance

Isolation of Biomaterial from Piper nigrum

50 gm Black Pepper was stirred with 85 ml Methanol + 10 ml Glacial Acetic Acid + 5 ml Conc. Sulphuric Acid to the residue added 10 ml of Sodium Hydroxide solution. Magnetic Stirring was done for 30 mins, Residue was filtered. Added 50 ml of Methanol. Added Calcium Hydroxide (Slaked Lime) until pH 9 was obtained. Mixed, Filtered after 30 mins, Filtrate was concentrated. Added to 25 ml cold water for 5-10 mins, centrifuged. To the supernatant, 50 ml acetone was added and kept for refrigeration. To the precipitate obtained by centrifugation, added Water: Chloroform (1:2) Kept for 2 hrs of Magnetic Stirring, air dried to obtain biopolymer. Isolated biopolymer was powdered, passed through Sieve No. 120, packed and stored. Optimized six times, reported % yield.

Physicochemical Characterization

Physicochemical properties of isolated bio-material were characterized. Physical Parameters: Color, Odor, Texture and Color Changing Point. Color, Odor, Texture of Bio-polymer was examined physically. Color Changing Point was determined by Capillary method by Melting Point Apparatus. Firstly the bio-polymer was kept in a Capillary tube and it was fitted in a Melting Point Apparatus. Temperature at which bio-polymer melts and changes its color was determined by means of a Thermometer and reported. Chemical Parameters: Tests for Carbohydrates, Proteins, Starch and Reducing Sugar. a) Test for carbohydrates Molisch reagent test 2 mL of biopolymer solution (0.1gm dissolved in 2 mL of distilled water) was taken in a test tube. 2 drops of Molisch reagent (Solution of -napthol in 95% ethanol) was added. Solution was then poured slowly into a test tube containing 2mL of concentrated sulfuric acid. Two layers were formed. Purple color appeared at interface of the two layers due to formation of 5-hydroxy methyl furfural. b) Test for proteins Biuret test determines the presence of peptide bonds in protein content in the isolated biomaterial. 2 mL of $$ \mathrm {C l} ^ {-} $$ biomaterial solution (0.1 gm. dissolved in 2 mL of distilled water) was taken in a test tube. 1 mL of sodium hydroxide solution (1%) followed by 1% copper (II) sulphate solution was added drop wise. Test tube was then shaken vigorously. Allowed the mixture to stand for 5 minutes and observed the color change. Biuret test is based on the ability of Cu (II) ions to form a violet- colored chelate complex with the peptide bond (CONH groups) in alkaline conditions. The chelate complex absorb light at 540 nm so it appeared violet. Color change indicated presence of proteins. c) Test for Starch 2 mL of biomaterial solution (0.1 gm. dissolved in 2 mL of distilled water) was taken in a test tube. 1-2 drops of iodine solution was added. Then observed the color change. Appearance of an intense blue black color confirmed the presence of starch in isolated biomaterial (transfer of charge between starch and iodide ion changes the spacing between the energy orbitals, so starch-iodide complex absorb light at higher wavelength) d) Test for Reducing Sugar 2 mL of biopolymer solution (0.1 gm. dissolved in 2 mL of distilled water) was taken in a test tube. Added 1 mL each of Fehling’s A (7 g CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute sulfuric acid) and Fehling's B (35 gm. potassium tartrate, 12gm. of sodium hydroxide in 100 mL of distilled water). The test tube was placed in a water- bath at 60°C. Appearance of brick red precipitate of insoluble copper oxide indicated the presence of reducing sugar in isolated biomaterial.

Drug-Excipient Interaction Study

Drug-excipient interaction study was performed by taking three different ratios of drug and bio-material 1:1, 1:3 and 3:1 [5]. The Ultraviolet (U.V.) absorbance of the three ratios was taken and compared with the absorbance of pure drug.

a) Wet method

Drug-excipient in the ratio of 1:1, 1:3 and 3:1 were taken in 3 different petridishes. 1 mL of distilled water was added to wet the mixtures. Mixtures were then subjected to drying in oven for 30 mins at 50°C, followed by dilution with 2 mL of methanol. Ultraviolet Spectroscopy study was performed. The shift in λmax was reported in comparison to that of pure Topiramate. b) Dry method Drug-excipient in the ratio of 1:1, 1:3 and 3:1 were taken in their physical forms (dry) in 3 different petridishes. Mixtures were kept at room temperature for 2 hrs, followed by dilution with 2 mL of methanol. Ultraviolet spectroscopy study was then performed. The shift in λmax was reported in comparison to that of pure Topiramate. c) Colorimetry Method: Drug: Polymer 1:1 were mixed with Potassium Permanganate on glass plate. Observed color change, scrapped, Diluted suitably with distilled water, analyzed by UV. Similarly repeated with Drug: Distilled Water and Drug: Potassium Permanganate. Importance of determination of drug-biopolymer interaction by Dry and Wet methods The two methods revealed that no interaction of drug- biopolymer occurred either in dry form on in presence of solvent. Since biopolymer is isolated from natural source and used in formulation, it has to be ascertained whether the biopolymer is inert in both dry (during storage) as well as wet (if used in oral drug delivery) conditions. Thus to confirm inertness and non-reactiveness of biopolymer with drug, these two methods had been performed. The drug was found to be intact with biopolymer

Spectral studies of isolated biopolymer

SEM Analysis: Morphological examination of the surface and internal structure of the biomaterial was performed by using a scanning electron microscope (SEM). A small amount of biomaterial was fixed on aluminum studs and it was coated with gold using a sputter coater under vacuum (pressure: 1 mm Hg). The biomaterial was then analyzed by SEM [5, 6]. FTIR Spectra: The physical form of isolated bio-material was solid. KBr disc method was employed for IR spectroscopy, and in this technique about 1mg of the solid sample was mixed with about 100 mg of pre dried and desiccated solid KBr. The mixture was finely ground in a mortar, preferably under an IR lamp to exclude any water vapors. The finely ground mixture was pressed under pressure of about 10 tons using a hydraulic pump to form a small pellet about 1-2mm in diameter. The resulting KBr disc was removed from the KBr die and is positioned in a special holder into the path of the IR radiation and its spectrum was recorded within the range of 4000-200cm-1. Differential Scanning Calorimetry: (DSC) is a thermo- analytical technique in which the difference in the amount of the heat required to increase the temperature of a sample and reference is measured as a function of temperature. It was performed for determination of Glass Transition temperature (GTT or Tg). For DSC the Perkin Elmer Instrument, Model- JADE DSC will be used, with the Heat flow of 50 - 250°C at the rate of 10°C/minute and Nitrogen flow rate of 20ml/minute will be used. NMR Spectral Analysis: Nuclear magnetic resonance (NMR) is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation. This energy is at a specific resonance frequency which depends on the strength of the magnetic field and the magnetic properties of the isotope of the atoms. Solvent used is DMSO (Dimethyl Sulfoxide). The spectrometer is connected to the process with a 5 mm diameter flow cell. The reaction mixture is pumped with high flow rates to the instrument, a valve switch to stop the flow is activated so the instrument can perform a quick measurement. Afterwards, the valve switches back and the flow cell in the instrument is rinsed again with the reaction mixture. After a measurement is finished, the spectrum is sent to the automation computer where it can be processed and analyzed. Preparation of standard curve of Topiramate: 10 mg of Topiramate was dissolved in 30mL of Phosphate Buffer (pH 7.4) in a 100 mL volumetric flask and diluted up to the mark with Buffer (pH 7.4) (100 µg / mL). Dilutions of Concentrations (0.5,1, 2, 3, 4 and 5µg/mL) were prepared in 10 mL volumetric flasks. Volume was made up to 10 mL with Phosphate Buffer (λmax = 244 nm). Absorbance was measured against solvent blankc [7, 8].

Nanosizing of Topiramate

Nanosizing of Topiramate by Standard Solvent Evaporation Method: 100 mg Topiramate was mixed with 10 mg dextrose, 5 mg fructose and 10 mL methanol in mortar pestle and triturated. The mixture was transferred into beaker, sonicated for 5 cycles (3mins/cycle in ultrasonic bath sonicator). Mixture was diluted with 50 mL distilled water and again sonicated for 5 cycles. Absorbance, % Transmittance, % Blockage (100% Transmittance) was noted after every 5 cycles up to 15 cycles. After 15th cycle, residue was collected, dried, packed and stored. Nanosizing of Topiramate by Sonication method: 100 mg Topiramate was mixed with 10 mg dextrose, 5 mg fructose and 10 mL distilled water in mortar pestle and triturated. The mixture was transferred into beaker, sonicated for 5 cycles (3mins/cycle in ultrasonic bath sonicator). Mixture was diluted with 50 mL distilled water and again sonicated for 5 cycles. Absorbance, % Transmittance, % Blockage (100- %Transmittance) was noted after every 5 cycles up to 15 cycles. After 15th cycle, residue was collected, dried, packed and stored.

The main purpose of Nanosizing Topiramate by two different methods was to compare novel sonication method with published standard solvent evaporation method (Figure 1).

%T (Without Nanosizing) % T (Novel method) %T (Std. method)

120.00%

100.00%

80.00% % Transmittance

60.00%

40.00%

20.00%

0.00%

200nm 300nm 400nm 500nm 600nm 800nm

λmax Figure 1: Comparative Graph between %Transmittance and λ max of pure Topiramate (without nanosizing) with nanosized Topiramate (by novel sonication and standard solvent evaporation methods).

Formulation of Bio-flexy Films (Solvent Casting Method)

Nanosized Topiramate (anticonvulsant) and Piper nigrum biopolymer (in ratios 1:1, 1:3, 1:5, 1:6, 1:10) (retardant, mucoadhesive agent, film former) were taken in mortar. To this mixture, dextrose (10 mg/mL), fructose (5 mg/mL) (flexicizers) were added and triturated. Added glycerine (10 µl) (plasticizer), pectin (3%) (Film initiator). Distilled water (20 mL) was incorporated, uniformly triturated for 2 mins. Mixtures were subjected to magnetic stirring for 15 mins, followed by sonication for up to 5 cycles (each cycle 3 minutes). Nano-dispersions were poured into petridishes and kept for drying. Prepared films were removed from petridishes by using 1% borax solution (hardening agent) Checked the filmability of prepared films. Same procedure was followed for formulations of Standard Polymer Films (Tables 1 & 2).

Evaluation of Formulated Bio-Flexy Films

Thickness: The thickness of randomly selected bio-flexy films from every batch was determined using standard digital micrometer. The average thickness was determined and reported with appropriate standard deviation [8]. Folding Endurance: Folding endurance of bio-flexy films was determined by repeatedly folding one of the film at the same place till it broke or folded up to 300 times manually, which was considered satisfactory to reveal good properties. The number of times of film could be folded at the same place without breaking gave the value of the folding endurance. This test was done on randomly selected three flexy films from each batch. Surface pH Study: The surface pH of bio-flexy films was determined in order to investigate the possibility of any side effects in vivo. As an acidic or alkaline pH may causes irritation to the soft palatal mucosa, it was determined to keep the surface pH as close to neutral as possible. The bio-flexy films were allowed to swell by keeping it in contact with 1 ml of distilled water for 1 hour at room temperature. The pH was measured by bringing the electrode in contact with the surface of film and allowing it to equilibrate for 1 minute. The experiments were performed in triplicate and avg. values were reported. Surface pH of films was determined because prepared bio-flexy films will be directly placed in soft palatal region, thus it is essential for formulation (rather than polymeric solution) to be neutral to mucosal surface and compatible with soft palatal pH. Weight Uniformity Study: determined by taking weight of ten bio-flexy films of sizes 1 cm2 diameter from every batch and weight individually on electronic balance. The avg. weight was calculated.

Drug Content Uniformity: determined by dissolving the bio-flexy films (1 cm2 in diameter) in 100 ml of phosphate buffer (pH 7.4) for 24 hrs. Under occasional shaking. The 5 ml solution was taken and diluted with phosphate buffer pH 7.4 up to 20 ml, and the resulting solution was filtered through a 0.45 mm Whattman filter paper. The drug content was then determined after proper dilution using a UV spectrophotometer at λmax 750 nm. Swelling Percentage Study: calculated by function of weight and area increase due to swelling. Bio-flexy films of size (1 x 1 cm2) were weighed, kept in a petridish and 10 ml distilled water was added. After one hour, the films were weighed. The difference in the weights gives the weight increase due to absorption of water and swelling of patch. The study was conducted for 24 hours.

% S = (Xt – Xo / Xo) x 100

where, Xt = weight or area of the swollen bio-flexy film after time t Xo = original bio-flexy film weight or area at zero time Percentage moisture uptake (PMU) was carried out to check the physical stability of the bio-flexy films at high humid conditions. Three 1cm diameter bio-flexy films were cut out and weighed accurately then the films were placed in desiccator containing saturated solution of aluminium chloride, keeping the humidity inside the desiccator at 79.5 %. After 3 days the bio-flexy films were removed, weighed and percentage moisture absorption was calculated. Average percentage moisture absorption of three films was found. Percentage moisture uptake = [(Final weight – Initial weight)/ Initial weight] X 100

In-Vitro Mucoadhesivity study of Isolated Biopolymer: The mucoadhesive property of Piper nigrum polymer was assessed by Modified Shear Stress Method at different concentrations 1%, 2%, 4%, 6%, 8% and 10% of biopolymer. The weight required to slide a glass plate by using various concentrations of the biomaterial solutions from 0-30 mins was determined and compared with Sodium Carboxyl Methyl Cellulose (Sodium CMC) (1%). Mucoadhesion study of formulated bio-flexy films by rotating cylinder method: In Rotating Cylinder method, the mucoadhesive property of bio-flexy films was evaluated by Capra aegagrus (goat) intestinal mucosa. Bio-flexy films of area 1cm2 of each formulation were cut down using sharp blade. Tied the goat intestinal mucosa over the rotating basket of i-disso apparatus. The Dissolution medium was composed of 900 ml buffer pH 7.4, maintained at 37°C, subjected for rotation at 50 rpm. It was applied over the inner surface of goat intestinal mucosa until it got dislodged. The detachment and dislodgement of film from mucosal substrate was noted at regular intervals. Mucoretention study of formulated bio-flexy films: Bio-flexy films of area 1cm2 of each formulation were cut down using sharp blade. Tied the Capra aegagrus (goat) intestinal mucosa over slanting condenser over which buffer was allowed to flow from a burette. It was applied over the inner surface of goat intestinal mucosa until it got dislodged. The detachment and dislodgement of film from mucosal substrate was noted at regular intervals. In-vitro drug release (by modified M .S. apparatus) [9, 10]: A thermostatically controlled compartment with vials containing buffer pH 7.4 was prepared. Egg membranes tied to donor compartment (contained formulations) inserted into receiver compartment. Temperature was maintained at 37ºC using orbital shaker incubator. Samples were withdrawn at regular intervals ranging from 10 mins to 30hrs. Complete Replacement of buffer each time. Ultra Violet Spectral analysis was performed. Stability studies: Stability studies were conducted as per ICH Guidelines. Stability testing of pharmaceutical product is done to ensure the efficacy, safety and quality of active drug substance and dosage forms and shelf life or expiration period. The stability studies of the formulations were conducted at 40ᵒC ± 2°C and ± 45± 5% RH, 25± 2°C and 60 ± 5% RH and 2 ± 5°C temperature and RH values respectively. After every 15 days, the aggregation, nature, color change, and in-vitro drug release of formulations was determined [11, 12].

Isolation of Biomaterial

The biopolymer was isolated from pulp of Piper nigrum by simplified economic process. The optimization of biopolymer isolation process was repeated six times for and the % yield was calculated. During optimization the results obtained were reproducible with insignificant variation and can be adopted for scaling up in bulk manner. The % yield for biomaterial from Piper nigrum was found to be 2.2%±0.02.

Physicochemical Properties of Isolated Biomaterial

It was tested positive i) for proteins and carbohydrates, amino acids were not present.

(i) Physical Properties: The biomaterial obtained from pulp of Piper nigrum was obtained in powdered texture that was dark brown in color, pungent odour, soluble in methanol. Its colour changing point was found to be 190ºC±1. (ii) Chemical Properties (a) Test for Carbohydrates: Isolated biomaterial showed presence of Carbohydrates. (b) Test for Protein: Isolated biomaterial showed presence of Protein. (c) Test for Starch: Isolated biomaterial showed absence of Starch. (d) Test for Reducing Sugar: Isolated biomaterial showed presence of Reducing Sugar.

Drug Excipient Interaction Studies

Drug-polymer interaction study of biomaterial isolated was done by UV techniques. The drug interaction study was performed by using wet and dry method.

a) Wet method λmax was observed at 752 nm, no significant difference than that of pure drug. b) Dry method λmax was observed at 752 nm, no significant difference than that of pure drug. Thus, no drug-excipient interaction occurred. c) Colorimetry 0.05 gm. Topiramate showed color change with potassium permanganate from pink to brown, indicating the reaction of potassium permanganate because of saturation of double bonds. Drug (0.05 g) +bio-polymer (0.05 g) also showed a drug. As no interaction was found, it can be concluded that the biomaterial may be useful in the formulation of Bio-Flexy films. Drug: Polymer 1:1 were mixed with Potassium Permanganate on glass plate. Observed color change, scrapped, Diluted suitably with distilled water, analyzed by UV. Similarly repeated with Drug: Distilled Water and Drug: Potassium Permanganate. Drug showed color change from pink to brown with Potassium Permanganate while polymer showed no color change. No significant difference in shift of λmax than that of pure Topiramate was observed (Table 3).

similar color change with potassium permanganate. This indicated that the drug was not entrapped. With the use of U.V. method λmax of the drug–excipient mixture was found to be close to that of the pure drug. Thus, the drug–excipient interaction study revealed no interaction between the drug and the biomaterial and the biomaterial was found to be compatible with the

100µm. SEM showed flakes type fine structure with smooth texture (Figure 2).

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inbuilt mucoadhesive property with functional groups RCOOH, RCH2OH, RCONH2, S=O, C=C-COOH, RNH2 (Figure 3).

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Polymer’s Melting Temperature Tm is 130°C. Thus bio- polymeric nature was confirmed (Figure 4).

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NMR Spectral Analysis: The presence of proton and their environment was confirmed by 1HNMR. The 1HNMR Spectra confirms the presence of carbohydrates residue within the biopolymer extracted as the shift of carbohydrate ring protons are 3-6 ppm and the spectra when compared reflects the peak at 3.4191 ppm (Figure 5).

Preparation of Calibration Curve of Drug: Calibration curve of Topiramate was prepared in Buffer (pH 7.4) showed linearity. $R^2$ value was found to be 0.9945 (Figure 6).

Nanosizing of Topiramate

The main purpose of Nanosizing Topiramate by two different methods was to compare novel method with published Standard Solvent Evaporation method which showed nanosized drug showed increase in % Transmittance with increasing Wavelength in UV while non-nanosized drug showed almost constant % Transmittance (Figure 1).

Weight uniformity of formulations: Weight of nanosized Topiramate loaded bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5) was found to be in range of 0.020±0.05 to 0.031±0.04. Drug content uniformity: Drug Content Uniformity of nanosized Topiramate loaded bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5) was found to be in range of 90.4%±0.65 to 98.6%±0.60. This indicates that drug was uniformly dispersed in bio-flexy films. Swelling percentage study: Swelling Percentage Study of bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5) ranged from 70%±0.5 to 73%±0.3. Swelling increased on increasing biopolymer content. It indicates that drug released through bio-flexy films by swelling followed by erosion. Percentage Moisture Uptake (PTU) of bio-flexy films: Percentage Moisture Uptake (PTU) of bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5) was found to be in range of 2.2%±0.13 to 2.6%±0.12 Mucoadhesivity study of isolated biopolymer: Shear Stress study of Piper nigrum biopolymer revealed that 10% concentration showed promising mucoadhesivity comparable to standard Sodium Carboxyl Methyl (Sodium CMC) polymer (Table 4).

Evaluation Parameters of formulated Films

Thickness of formulations: Thickness of nanosized Topiramate loaded bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5) was in range 0.027±0.006 mm to 0.037±0.002 mm. As concentration of biopolymer was increased in formulations, thickness also increased although not much significant difference was found. Folding endurance of formulations: The folding endurance was obtained in the range of 102-167 for nanosized Topiramate loaded Bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5). As concentration of biopolymer was increased in formulations, folding endurance also increased as their flexibility got enhanced due to biopolymer. Surface pH of formulations: Surface pH of nanosized Topiramate loaded bio-flexy films containing Piper nigrum biopolymer (FNO1-FNO5) was found to be 7.01±0.05. Prepared formulations suitable for soft palatal delivery platform as they are in the range of physiological pH.

Mucoadhesion Study of Formulated nanosized Topiramate loaded Bio-flexy Films by Rotating Cylinder method (Table 5).

Dynamic Method

In-Vitro Drug Release of Formulations FNO1 to FNO5 by Modified M.S. Apparatus) and Figure 8 (comparative graph of In-Vitro Drug Release of Formulations FSO1 to FSO5 by Modified M.S. Apparatus) (Tables 7-10).

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Stability studies: The formulations showed no physical changes, related to the color, odor, taste etc. The drug content and in-vitro release was found to be the same, no significant change was observed. So it was concluded that the formulation bio-flexy films of Topiramate was found to be stable.