Development and Characterization of Nanoemulsion-Based Formulation for Oral Delivery of Itopride

Itopride (INN; marketed as Ganaton) is a derivative of prokinetic benzamide. It functions as an acetylcholine esterase inhibitor as well as an antagonist of the dopamine D2 receptor [1]. It has an anti-emetic effect, increases stomach tension and sensitivity, and speeds up gastric emptying [2]. It is prescribed to treat gastrointestinal disorders such as nausea, vomiting, and functional dyspepsia. It affects the part of the brain responsible for controlling vomiting [3-4]. On the other hand, prolonged oral dosing of this drug results in severe adverse effects related to the gastrointestinal tract (GI). Since nausea, vomiting, and postprandial fullness have oral side effects, a different drug delivery method is needed for their long-term therapy [5]. To reduce oral GI side effects, the oral route employing the nanoemulsion technology has been studied. Food moves more easily through the entire gastrointestinal system and the stomach empty more quickly when Itopride, a drug that raises acetylcholine levels and stimulates gastrointestinal peristalsis [6]. Transparent or translucent nano-sized dispersions of water in oil (w/o) or oil in water (o/w) that are thermodynamically stable are known as nano emulsions. They are stabilized by an interfacial coating of surfactant and cosurfactant molecules with droplet sizes ranging from 10 to 100 nm [7].

These systems' thermodynamic stability gives them numerous advantages over unstable dispersions such as gels, emulsions, colloids, and suspensions. Several microemulsion or nano emulsion methods have been studied to improve Itopride bioavailability, transdermal penetration, anti-inflammatory properties, and solubility [8]. There is no literature information on the specific excipient selection used in the development of the nano emulsion formulation. As a result, every excipient used in the current investigation was carefully chosen and optimized for the creation of a nano emulsion formulation intended for Itopride oral delivery [9-10]. One of the most important steps in developing a nano emulsion formulation for transdermal, oral, ocular, or parenteral drug administration is to properly screen various excipients and determine their concentration within the acceptable range. Thus, the current study set out to optimize and choose various excipients that are suitable to pharmaceuticals in order to develop an Itopride nano emulsion formulation [11-12].

Figure 1:  Itopride

MATERIALS AND METHODS

Itopride was purchased from Neuland Laboratories (Hyderabad). Capmul Pg8nf was purchased from Abitec Corporation. Labrafil M1944CS and diethylene glycol monoethyl ether (Transcutol-HP) were purchased from Gattefosse Gas. Ethanol was purchased from Rankem chemicals. Propylene glycol (PG) was purchased from SRL Pvt. Ltd. PEG 200, Oleic acid and PEG 400 was purchased from Fisher Scientific India Pvt. Ltd. Polyoxyethylene sorbitan monolaurate (Tween-20) and polyoxyethylene sorbitan monooleate (Tween-80) were purchased from Croda. All other chemicals and reagents used were of analytical reagent (AR) grade.

PREFORMULATION STUDIES

Pre formulation testing involves examining the physicochemical properties of a drug substance, both on its own and in combination with excipients. This initial step was crucial in the development of dosage forms, as it provides essential information for formulating a stable, bioavailable product that meets key requirements. The identification of the obtained drug sample was carried out through various analytical methods, including IR spectroscopy, UV spectroscopy, and melting point analysis.

1. Organoleptic Characteristics:

Take a small amount of the drug sample, place it on white paper, and visually inspect it to observe its color, odor, and texture.

2. Melting point

The USP method was used to determine the melting point. A small amount of Itopride was placed in a sealed capillary tube, which was then inserted into a melting point apparatus. The temperature was gradually increased, and observations were recorded for the temperature at which Itopride began to melt, as well as when the sample had completely melted. This technique is also known as the capillary method [13].

3. UV spectrum of Itopride

A UV-visible spectrophotometer was commonly used to gather structural information about drugs, specifically targeting the chromophoric regions of molecules in solution. When exposed to light in the visible or ultraviolet spectrum, these chromophores absorb light at specific wavelengths depending on the type of electronic transition involved. The UV spectrum was typically displayed as a graph of absorbance versus wavelength.

A double-beam UV-visible spectrophotometer (Shimadzu, UV-1800, Japan) was used to determine the λmax of the drug. Accurately weigh 10mg of Itopride was dissolved in 100mL of methanol to make a stock solution of (100µg/ml). A 30 µg/ml solution of Itopride in methanol was scanned over the 200-400 nm range, with λmax observed at 258 nm.

3.1 Calibration curve of Itopride by UV-visible spectrophotometer

The standard curve of Itopride was created in methanol by dissolving 10 mg of Itopride in 100 ml of methanol to prepare a stock solution. This stock was further diluted to achieve concentrations ranging from 10, 15, 20, 25 and 30µg/ml. The absorbance of each solution was measured at 258 nm, using methanol as the blank, on a UV-visible spectrophotometer. A standard curve was then plotted by graphing concentration against absorbance. From this calibration curve, the intercept, slope, equation of the line, and correlation coefficient were determined [14].

4. Solubility studies of Itopride in solvent

For the quantitative study of solubility, 50 mg of the drug was placed in clean culture tubes containing 2ml of different solvents, including methanol, ethanol, DCM, water, phosphate buffer saline at pH 6.8. The tubes were sealed tightly and shaken in a water bath shaker for 24 hours at room temperature. After this period, each sample was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was collected. The supernatant was then filtered, and the filtrates were appropriately diluted and analyzed spectrophotometrically [15].

5. Solubility studies of Itopride in oils, surfactant and Co-surfactant

To determine the solubility of Itopride in various oils, surfactants, and co-surfactants, 50 mg of Itopride was added to 2 ml of selected oils (Oleic acid, Labrafil M1944CS, Capmul PG-8NF, Captex 355, Linalool), surfactants (Tween 80, Tween 20, Kolliphor RH40, Span 80) and co-surfactants (Transcutol HP, PEG 200, PEG 400, and Propylene Glycol) in separate 5 ml stopper vials. The contents were mixed thoroughly using a vortex mixer. The vials were then placed in a water bath shaker at 45 ± 1.0°C for 24 hours to reach equilibrium. After equilibration, samples were removed, centrifuged at 15000 rpm for 15 minutes, and the supernatant was filtered through a 0.45 µm membrane. The concentration of Itopride in each oil, surfactant, and co-surfactant was measured using UV spectroscopy at a wavelength of 258 nm [16].

6. Partition Coefficient of Drug

The partition coefficient (oil/water) was an indicator of a drug's lipophilicity or hydrophilicity and provides insight into its potential to cross cell membranes. The partition coefficient was commonly measured using n-octanol as the oil phase and water as the aqueous phase.

P _o/w = C _n-octanol/C _water

The partition coefficient of Itopride was determined using the shake-flask method. An excess amount of the drug was added to 10 mL of a mixture of two solvents, n-octanol and water (1:1), and allowed to equilibrate for 24 hours. Afterward, the two layers were separated and centrifuged at 15,000 rpm for 15 minutes. Following appropriate dilution, the absorbance of each layer was measured at the λmax of 258 nm using a UV spectrophotometer [17].

7. Shake flask method

The partition coefficient of Itopride was determined using the shake-flask method. An excess amount of the drug was added to 10 mL of a mixture of two solvents, n-octanol and water (1:1), and allowed to equilibrate for 24 hours. Afterward, the two layers were separated and centrifuged at 15,000 rpm for 15 minutes. Following appropriate dilution, the absorbance of each layer was measured at the λmax of 258 nm using a UV spectrophotometer [17].

8. FTIR of Itopride and Excipients

The FT-IR (Fourier Transform Infrared) spectrum, obtained using a Spectrum Two model, provides information on the functional groups present in a compound or drug. FT-IR spectroscopy was utilized for structural analysis. The FT-IR spectra of Itopride and its mixture with excipients were recorded to assess any potential interactions between the drug and excipients. A small amount of Itopride (3-5 mg) was placed on the FT-IR diamond plate, and the infrared spectrum was recorded over the range of 4000 to 400 cm?¹ [18].

9. Drug-excipients Compatibility Study by FTIR

The compatibility of the drug with excipients was evaluated using FT-IR spectroscopy. This technique was employed to identify any physical or chemical interactions between the drug and the excipients. The drug and various excipients were mixed in a 1:1 ratio, and the samples were scanned by FT-IR in the range of 400-4000 cm?¹. The spectra of the pure drug and the drug-excipient mixture were compared to detect any potential incompatibilities or physical changes.

OPTIMIZATION OF NANO-EMULSION FORMULATION

To optimize the excipients previously screened for solubility, their ability to emulsify and form a nano-emulsion was assessed through a screening process. The influence of each component on the formulation was then evaluated.

1. Selection of oils

The criterion for choosing or screening oils was the solubility of poorly soluble drug (Itopride) in them. As a result, the amount of drugs in each of the chosen oils (Capmul Pg8nf, Captex 355, Oleic acid, Linalool and Labrafil M1944CS) 50 mg for each was added to 2mL stoppered vials to test the solubility of the drug in different oils. After that, the vials were placed in a hot plate magnetic stirrer and held at 40 ± 1.0°C for 2 hours to reach equilibrium. After that the equilibrated samples were vortex for 2 minutes at 300 rpm and then centrifuged for 15minutes.  Using the appropriate analytical techniques, 100mg of the sample withdraw from culture tube and diluted to 10 ml in a volumetric flask and filled the volumetric flask with distilled water to measure the drug content. The concentration of Itopride was found in each oil at wavelengths of 258 nm by using a previously described UV spectrophotometer analyzed [19].

2. Selection of surfactants

The oil with the highest potential for solubilizing Itopride was chosen. The ability of several surfactants (Span 80, Kolliphor RH40, Tween 80 and Tween 20) to emulsify was tested. In order to investigation the components, 200 mg of surfactant and 200 mg of oily phase were combined, and the mixture was heated to 40–50°C.  After that withdraw 100 mg of each mixture were taken out and diluted to 10 ml in a volumetric flask and filled the volumetric flask with distilled water. Then the emulsions were let to stand for a 2hr. To measure the percentage transmittance UV spectrophotometer was used to analyze the result at λmax=630 nm. Additionally, they were visually examined for turbidity or phase separation [20].

3. Selection of cosurfactants

The major factor in the cosurfactant selection process was the nanoemulsion area in the pseudo-ternary phase diagrams. Choosing a cosurfactant was also based on the mass ratio (Smix ratio) of the surfactant to the cosurfactant for the maximum nanoemulsion area which described in the phase diagrams. Transcutol-HP, Propylene Glycol (PG), PEG 200 and PEG 400 cosurfactants were examined in this study. Cosurfactants that produced the largest nanoemulsion region were chosen for formulation development and for further evaluation [21].

4. Selection of mass ratio of surfactant to cosurfactant (Smix ratio) and pseudo ternary phase diagram study

Labrafil M1944CS was chosen as the oil phase for Itopride and water was used as the aqueous phase. For the purpose of construction of pseudo-ternary phase diagrams, various mass ratios of surfactant to cosurfactant, such as 1:1, 2:1and 1:2, were chosen, taking into consideration the rising concentrations of surfactant relative to cosurfactant and cosurfactant relative to surfactant. For drug, the mass ratio of surfactant to cosurfactant that produced the highest nanoemulsion area in the phase diagram was chosen in order to create the optimal nanoemulsion formulation.

To create the best possible nanoemulsion formulation, the mass ratio of surfactant to cosurfactant that produced the maximum nanoemulsion region in the phase diagram was chosen for drug.

Phase diagrams for pseudo-ternary systems were created in two stages. In the first stages, the drug oil phase was combined with the appropriate surfactant; various cosurfactants, including (PEG 200, Propylene Glycol, PEG 400 and Transcutol-HP) were used to maximize the choice of cosurfactant. [11–14] Propylene Glycol was chosen as a cosurfactant based on the nanoemulsion region. In the second stage, in order to optimize the mass ratio of surfactant to cosurfactant, specific surfactant (Tween-80) and cosurfactant (Propylene Glycol) [Smix] were mixed in various mass ratios (1:1, 2:1 and 1:2). Each phase diagram was created by mixing oil and Smix ratio in various glass vials with varying mass ratios ranging from 1:9 to 9:1. Using the aqueous phase titration, pseudo ternary phase diagrams of oil, Smix, and aqueous phase were created. Every mass ratio of oil and Smix was subjected to slow titration with aqueous phase and visual observations were conducted for translucent and readily flowable o/w nanoemulsions. A pseudo-ternary phase diagram for the drug indicated the physical condition of the nanoemulsion [22-23].

5. Preparation of drug loaded nanoemulsions

A mass ratio of 1:1 for Smix was chosen for the preparation of drug-loaded nanoemulsions for Itopride due to the presence of a maximal nanoemulsion region at these mass ratios. Several formulations were chosen from the nanoemulsion region at fixed concentrations of Smix based on these phase diagrams; 50mg of drug was dissolved in the corresponding oil phase with constant heating, the aqueous phase was gradually added. Several tests for thermodynamic stability were run on these formulations [24].

Table 1: Solubility of Itopride in various oils

6. Thermodynamic stability tests

Different thermodynamic stability studies were performed on each of the chosen nanoemulsion compositions. We performed three thermodynamic tests, as detailed in our articles: centrifugation, heating and cooling cycles, and freeze-thaw cycles. Formulations that exhibit no phase inversion, stable transmittance and no phase separation, or were chosen for more research [25-26].

7. Determination of Emulsification

Emulsification was performed by adding 0.3 ml of the nanoemulsion formulations into a beaker containing 200 ml of water at 37°C. The mixture was stirred, and the time required for complete emulsification was observed visually [27].

8. pH

One ml of the nanoemulsion was mixed with 100 ml of distilled water, and the pH was measured using a pH meter after vortexing the mixture for 1 minute. The pH was determined in triplicate, and the average values were calculated.

9. Percentage Drug content and Drug Loading

One ml of Itopride nanoemulsion (equivalent to 50 mg of Itopride) was dissolved in 10 mL of methanol and sonicated for 1 minute to obtain a clear solution. The resulting solution was then centrifuged at 15,000 rpm for 15 minutes. Using water as the blank, the drug content and drug loading were determined by analyzing the Itopride nanoemulsion spectrophotometrically at λmax = 258 nm [28].

% Drug content=Final amount of drug Initial amount of drug ×100

Formula……………

% Drug loading=Final amount of drug Amount of nanoemulsion×100

% TRANSMITTANCE

The percentage transmittance of the prepared nanoemulsion formulations was measured spectrophotometrically using a Shimadzu UV-Vis spectrophotometer. One ml of the formulation was diluted 100 times with distilled water and analyzed at 630 nm.

1. Transmission Electron Microscopy (TEM) Analysis

The morphology and structure of the nanoemulsion were examined using transmission electron microscopy (TEM) operating at 200 kV, with point-to-point resolution capabilities. The nanoemulsion formulation was diluted with deionized water and gently mixed. A drop of the diluted sample was placed on copper grids, stained with a 1% phosphotungstic acid solution for 30 seconds, and then observed under the electron microscope to analyze the form and size of the nanoemulsion [29].

2. Particle size and Zeta Potential

The particle size (PS) and zeta potential of the nanoemulsion were assessed using photon correlation spectroscopy and electrophoretic mobility techniques, respectively, with a Zeta-sizer Nano instrument. The prepared emulsion was diluted 100-fold, introduced into the sample cell, and placed in the sample holder unit for the determination of both particle size and zeta potential [30].

3. In vitro dissolution study

Dissolution studies were conducted to evaluate the dissolution profile of Itopride from the nanoemulsion using USP type 2 methodologies (paddle apparatus, 100 rpm at 37°C). Two milliliters of the nanoemulsion formulation (equivalent to 50 mg of Itopride) and pure drug were introduced into the dissolution medium (pH 1.2 HCl, 900 mL; pH 6.8 phosphate-buffered saline, 900 mL). Samples (2 mL) were collected at specified time points (5, 10, 15, 20, 30, 60, and 120 minutes) and replaced with fresh medium to maintain sink conditions. The drug release from the nanoemulsion was compared to the pure drug suspension. The samples were analyzed for drug content using a UV-Vis spectrophotometer at 258 nm [31-32].

RESULTS AND DISCUSSION

1. Physical Appearance

Table 2: Visual Appearance of nano-emulsion

Discussion: Visual appearance was visualized by naked eye in order to determine successful formulation of nanoemulsion. We have to observe the sudden turbidity of our transparent and clear nanoemulsion. Absence of precipitation and phase separation indicates the stability of formulation

2. Emulsification of nano-emulsion

Table 3: Emulsification of nano-emulsion

Figure 2 : Emulsification of nano-emulsion

Discussion: From the Table 6.2, the rate of emulsification was too fast (within a few seconds) to be accurately measured. The emulsification of all formulation was found to be in a range 2.213±0.021 to 2.310±0.010second.

3.  pH determination

Table 4: pH study of nano-emulsion

Figure 3: pH of nano-emulsion

Discussion: From the Table 6.3 & Figure 6.3, it was found that pH of all formulation was found to be in a range 6.900±0.009 to 6.943±0.040.

4. Drug content

Table 5: Drug content of formulations

Figure 4: Drug content of formulations

Discussion: The drug content was found to be 97.333±0.247 to 99.473±0.377% respectively. Drug content of C10 formulation was found to be 99.473±0.377% which was the higher among all.

5. Drug Loading

Table 6: Drug loading of formulations

Figure 5: Drug loading of formulations

Discussion: From the Figure 6.5 and table 6.5, the drug loading was found to be 4.830±0.012 to 4.974±0.019% respectively. Drug loading of C10 formulation was found to be 4.974±0.018% which was the higher among all.

6. Percentage Transmittance

Table 7: Percentage Transmittance of formulations

Figure 6: Percentage Transmittance of formulations

Discussion: The Percentage Transmittance was found to be 97.647±0.230 to 99.393±0.214% respectively. The percentage Transmittance of all formulations was found to be satisfactory.

7. Viscosity

Table 8: Viscosity of formulations

Figure 7: Viscosity of formulations

Discussion: From the figure 6.7; table 6.7; it was found the viscosity of formulations range from 4.173±0.136 to 4.467±0.068cps.

Among all the formulations, C10 was identified as the most suitable for further assessment, including TEM analysis, FTIR formulation, and in-vitro dissolution study and particle size determination. This selection was based on its promising characteristics, making it ideal for further evaluation.

8. TEM

Figure 8: TEM of Nanoemulsion

Discussion: From the figure 5.34 it was concluded that the prepared nanoemulsion of the optimized formulation was found to be spherical to oval in shape.

9. Particle Size

Figure: 9: Particle size of Formulation (C1)

Discussion: From the Figure 6.9, demonstrated particle size of formulations was ranges from 202.550nm with PDI 0.149.

ZETA POTENTIAL

Figure 10: Zeta potential graph of C10 formulation

Discussion: Figure 7 demonstrated zeta potential of C10 formulation was -15.9 mV represents stability of formulation [33].

1.  FTIR Study

Figure No. 11: FTIR Spectra of Formulation C10

Table 9: Interpretation of FTIR spectrum of Formulation C10

Discussion: FTIR spectrum of final formulation demonstrated that the characteristic peak of drug was either disappeared or slightly shifted with reduced intensity thus indicated the encapsulation of drug in nanoemulsion formulation.

2. In vitro dissolution study

Table 10 : In vitro dissolution study of Itopride- loaded nanoemulsion & pure drug

Figure 12: In vitro dissolution study of Itopride- loaded nanoemulsion & Pure Drug

OPTIMIZATIONS OF NANO-EMULSION FORMULATION

All excipients (oils, surfactants, and cosurfactants) were chosen primarily based on their pharmacological acceptability and classification as generally recognized as safe (GRAS). Different criteria applied to oils, surfactants, cosurfactants, and the aqueous phase for choosing excipients. Every excipient chosen for the research was safe to use, non-toxic, and acceptable from a pharmaceutical standpoint.

1. Selection of oils

The ability of drug to dissolve in oils was a crucial factor in the creation of nanoemulsions for all forms of drug delivery systems. Therefore, the solubility of drug in various oils was conducted. Table 1.1 displays the maximum solubility of Itopride in Labrafil M1944CS oil. Therefore, Labrafil M1944CS was chosen as the oil phase for the creation of the nanoemulsion formulation.

2. Selection of surfactants

Nanoemulsion formation was achieved with the selection of surfactants for the research whose transmittance was greater than 90%. In this study, 2 surfactants (Tween-20 and Tween-80) were used. The final choice of surfactants was determined by how well certain oils (Labrafil M1944CS for Itopride) could dissolve in the surfactant. Tween-80 was discovered to have the highest solubilization of Itopride; for this reason, it was chosen as the surfactant for the creation of the nanoemulsion formulation.

(a)

(b)

(c)

(d)

Figure 13: Pseudo-ternary phase diagrams of Itopride, indicating nanoemulsion region of Labrafil M1944CS (oil), Tween80 (surfactant) with different cosurfactants indicated in (a) PEG 200, (b) Transcutol-HP, (c)PEG 400 and (d) propylene glycol [1:1].

3. Selection of cosurfactants

Pseudo-ternary phase diagrams were created for each cosurfactant to aid in the selection process. Using the aqueous phase titration approach as outlined in our previously published works, pseudo-ternary phase diagrams were created. As seen in Figures 8.3, four distinct cosurfactants (PEG 200, Transcutol-HP, PEG 400 and propylene glycol) were used to generate pseudo-ternary phase diagrams for each medication. Propylene glycol was used to determine the maximal nanoemulsion zone for each medication (Figures 8.3). Consequently, Propylene glycol was chosen as a cosurfactant in order to create the best possible nanoemulsion formulation of Itopride. Propylene glycol has also been studied in earlier studies as a superior cosurfactant.

(a)

(b)

(c)

(d)

Figure 14: Pseudo-ternary phase diagrams of Itopride, indicating nanoemulsion region of Labrafil M1944CS (oil), Tween 80 (surfactant) with different mass ratio of surfactant to cosurfactant (Smix) indicated in (a) Smix 1:1, (b) Smix 2:1 and (c) Smix 1:2 (d) Overall.

Discussion: From figure 8.3, it can be concluded that the formation of nanoemulsion depend on the ratio’s in which the oil, surfactant and co-surfactant were added. The pseudo-ternary diagram depicts the region (shade as black) where there was the probability of formation of nanoemulsion from the figure 8.3 and the red crossed region indicates turbidity of the emulsion and the blue six-star region indicates light bluish emulsion and the blue diamond dots has been assigned to the highly transparent emulsion.

4. Selection of mass ratio of surfactant to cosurfactant (Smix ratio) and pseudo- ternary phase diagram study

For drug nanoemulsion formulation, the mass ratio of surfactant to cosurfactant that produced the maximum nanoemulsion zone in the phase diagram was chosen. In Figure 6.2, phase diagrams with a different mass ratio were used in order to develop Itopride nanoemulsion formulation. A pseudo-ternary phase diagram can be used to illustrate how the phase behavior of nanoemulsions and their constituent parts relate to one another [34].

To optimize the mass ratio, cosurfactant, and surfactant of Smix, pseudo-ternary phase diagrams were created independently for each Smix ratio. Propylene glycol was determined to be the best cosurfactant among all those evaluated in terms of the nanoemulsion region, as seen in Figures 8.4

Table 11: Composition of selected nanoemulsion formulations of Itopride from each ratio