Optimization of extrusion spheronization process and gamma scintigraphy of coated rifaximin pelletsAbstract
This study aims to optimize the process of extrusion spheronization for the development of rifaximin pellets and target them to the colon. Microcrystalline cellulose (MCC) is used to spheronizing aid in formulating pellets. MCC concentration, water content, spheronization speed, and time were optimized against roundness and % usable yield using Box-Behnken design. Coating levels of both the polymers were optimized using central composite design for drug release at 2, 4, 7, and 12 h at pH levels of 1.2, 6.8, 7.4, and 6.8 respectively. A gamma scintigraphy study helps in identifying transit pattern of coated pellets in GIT. Optimal conditions for rifaximin pellets with the desired pellet characteristics were identified as 34.19% MCC, 8.6% water, 1004 rpm, and 5.12 minutes. The observed values for drug release at 2, 4, 7, and 12 h at the aforesaid pH conditions were 0.17±0.00%, 3.08±0.13%, 39.01±1.17%, and 99.37±2.73%, respectively. This shows strong concordance between the observed and predicted values. The average durations for colon arrival, small intestine transit, and stomach emptying were 6.26±0.142 h, 5.72±0.408 h, and 0.70±0.265 h, respectively. This shows that the higher number of pellets reaches the colon in the desired time period. The study concluded with the development of optimized pellets and gamma scintigraphy confirms that the coating improves colonic rifaximin delivery.
Keywords:
Pellets; Colon targeting; Rifaximin; Extrusion-spheronization; Optimization; Gamma scintigraphy.
INTRODUCTION
The efficacy of targeted drug delivery systems in administering therapeutic agents to specific regions of the gastrointestinal tract has rendered them significantly relevant in recent years. Due in great part to its potential to treat localized diseases including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), colorectal cancer, and infections, as well as for systemic delivery of protein and peptide drugs broken down in the upper gastrointestinal tract, colon-specific drug delivery has attracted a lot of interest recently. Among the several methods of colon-specific delivery, the use of multiparticulate systems such as pellets is progressively preferred because of their capacity to offer uniform drug distribution, lower dosage dumping, and improved bioavailability. (Lee et al., 2020; Philip, Philip, 2010; Philip, Dabas, Pathak, 2009). Rifaximin, an antibiotic with a broad-spectrum antibacterial profile and poor water solubility, has demonstrated significant therapeutic efficacy in the management of inflammatory bowel disease (IBD). Some conventional tablets of rifaximin are in the market but they incorporate higher doses and these disintegrate in the upper GIT. Though rifaximin reaches to colon but the rate is indefinite, this may vary its therapeutic efficacy. A robust colon-targeted drug delivery system is essential due to the common issues associated with standard rifaximin formulations, such as premature drug release in the upper gastrointestinal tract and inadequate drug concentrations at the colonic site (Guslandi, 2011; Li et al., 2020; Neha, Kaur, 2024; Saadi, McCallum, 2013; Teruel et al., 2023).
Present study involves the applicability and effectiveness of colon targeted pellets for IBD. The unique advantages of pellets, including uniform size, improved drug loading, reduced variability in gastric emptying, and potential for site-specific administration via controlled drug release, render them a versatile drug delivery system. The methodology for preparation of pellets in this work involves extrusionspheronization which is advantageous in its ability to generate spherical, freely flowing pellets characterized by excellent mechanical strength and consistent drug release profiles. (Palugan et al., 2015; Politis, Rekkas, 2011; Di Pretoro et al., 2010; Soh et al., 2013).
A dual-layer polymer coating method employed to coat the pellets with an optimized composition to achieve precise colon targeting. Eudragit NE40D (ENE), constituting the inner layer, was designed to impart controlled-release characteristics, ensuring a consistent release of the medication over an extended duration. Eudragit FS30D (EFS), serving as the outer layer, functioned as a pH-sensitive polymer to facilitate drug release in the colonic milieu (pH > 7) while inhibiting premature drug release in the stomach and small intestine. The dual-coating method can enhance rifaximin's therapeutic efficacy by effectively integrating time-dependent and site-specific release mechanisms (Ahmed, Ayres, 2011; Bose, Elyagoby, Wong, 2014).
A classical instrument helpful for the optimization of pharmaceutical processes by means of variable interaction is statistical experimental design technique (Soltani et al., 2024; Paterakis et al., 2002). In this research work the process of extrusion spheronization was optimize to analyse the impact of significant independent variables, including the percentage of MCC, water content, spheronization speed, and spheronization duration on roundness and usable yield of pellets, which are essential for ensuring formulation quality and process efficiency. Further, the coating levels of both polymers were also optimized for the release profile under varied gastrointestinal circumstances.
An essential aspect of this research involved evaluating the in vivo performance of the coated pellets by gamma scintigraphy, a non-invasive imaging technique commonly employed to observe the transit pattern of formulation throughout the gastrointestinal tract. The incorporation of a radiolabel into the formulation enabled gamma scintigraphy to provide critical insights into the pellets' transit behaviour across various gastrointestinal regions and their retention in the colon (Katsuma et al., 2004; Nadaf et al., 2004).
One related study has also been done by the authors, but the current study optimizes a greater number of process parameters for development of rifaximin pellets. In addition to this, the coating polymer ratios were also optimized for the release of drugs in different time intervals. The gamma scintigraphy study in the current work also helps in tracking the pattern of pellets in the GIT.
This manuscript focused on the optimization of extrusion-spheronization technology for the development of quality pellets and coating these pellets to target the release of rifaximin in the colon. The manuscript also includes the study of transit pattern of these pellets in the GIT to ensure the targeting effectiveness by gamma scintigraphy. This all may be the basis for developing analogous delivery systems for other drugs requiring site-specific delivery.
MATERIAL AND METHODS
Material
Rifaximin (RFX) was procured from Ankur Drugs and Pharma Ltd., Baddi, Himachal Pradesh, India. Polysorbate 80 was purchased from CDH (New Delhi, India). MCC was supplied by ICPA Health Products Ltd. (Ankaleshwar, India). Evonik Röhm GmbH (Darmstadt, Germany) provided gift samples of Eudragits NE40D and FS30D. All of the chemical reagents utilized to make the coating dispersion and pellets were of analytical grade, as were the other excipients which were of conventional pharmaceutical grade.
Formulation and optimization of rifaximin pellets
Rifaximin pellets were formulated via the extrusion-spheronization technique. The advantages of extrusion-spheronization compared to other techniques include the capacity to incorporate elevated levels of drug without generating excessively large particles; the ease of combining two or more active agents in any ratio within the same unit; the ability to modify the physical characteristics of active ingredients and excipients; and the production of particles with high bulk density, low hygroscopicity, high sphericity, dustfree properties, narrow particle size distribution, and a smoother surface.
The Spheronizer-250 (Anish Pharma) utilised a plate size of 4.2 mm, whereas the Extruder-20 (Anish Pharma) employed a sieve size of 1 mm. Subsequent to thorough amalgamation, the drug and MCC were subjected to sieving through a 400μm mesh. Water was incorporated to achieve a moist aggregate with the requisite plasticity. The extrusion parameters comprised feeder speed, extruder speed, and extruder screen, set at 79 rpm, 30 rpm, and 1.2 mm, respectively. The resultant extrudate was promptly spheronized. Pellets containing the drug were dried for 24 h at 40°C in an oven (Flament et al., 2004; Tho, Sande, Kleinebudde, 2002; Lutchman, Dangor, Perumal, 2005; Trivedi et al., 2007).
Experimental Design
The optimization study was conducted using a Box-Behnken statistical design (BBD) consisting of 27 runs, 4 factors, and 3 levels (Wang et al., 2014). BBD is frequently regarded as superior to other designs in optimization due to its requirement for fewer experimental runs while still delivering precise estimations of a quadratic response surface. This renders it more time and cost-efficient, particularly when managing numerous factors where extreme combinations may be impractical or undesirable. Table I lists the variables that are independent and dependent.
The polynomial equation generated by this experimental design (using State Ease, Design Expert 10) is as follows:
Where b0 is the intercept, Y is the dependent variable and b1 to b33 are the regression coefficients and the independent variables are X1, X2 and X3 selected from preliminary experiments. The models generated contain quadratic terms and can explain nonlinear responses. This Box-Behnken design also resolves two-factor interaction effects from the primary effects of individual variables. ANOVA was computed from the software which gives the magnitude effects of variables over the dependent variables.
Evaluation of drug loaded pellets
Particle size analysis was conducted using U.S. standard sieves. The size range of 560-800µm fraction was selected as the usable product. Pellets' sphericity was assessed as roundness score using an image analyser that included a computer system connected to an optical microscope (Olympus CX31, Tokyo, Japan) and a video camera (Olympus SP-350, Tokyo, Japan). Version 3.0 of Magnus Pro software was used to examine the digital photos (Saripella et al., 2015; Sinha et al., 2007).
Coating with pH and time dependent polymers
The RFX pellets were coated in varying amounts with EFS (pH-dependent polymer for the outer coating) and ENE (time-dependent polymer for the inner coating) using a fluidised bed coater (Umang Pharmatech Pvt. Ltd.). For ten minutes, the requisite volume of water, heated to a temperature range of 70 to 80°C, was employed to homogenise the surfactant (polysorbate-80) and antitacking agent (glyceryl monostearate-GMS). The resultant emulsion was combined with the residual water, mixed, and permitted to cool to ambient temperature. The resulting emulsion was incrementally incorporated into the Eudragit dispersion with gentle spinning. The resultant slurry was passed through a 0.5 mm sieve. ENE dispersion was formulated with 5% GMS and 40% polysorbate 80, whereas EFS was composed of 5% GMS, and 40% polysorbate 80. Pellets were coated to achieve 10%, 15%, and 20% weight growth with each coating polymer. Theses coating levels were selected from the preliminary studies by the authors.
Both of these coating polymers possess low minimum film forming temperature which enables smooth surface after coating without or with minimal use of plasticizers. This may also help in reduction of pellet weight and in that place more drug can be incorporated.
The coating process parameters for the fluidized bed coater like inlet temperature, product temperature, blower speed, pump speed, atomization pressure and nozzle diameter was set as 35°C, 25°C, 35 Hz, 3 rpm, 1.2 bar and 1mm respectively (Varshosaz et al., 2011; Mazumder et al., 2021).
Optimization of rifaximin pellets using experimental design
The influence of factors was analyzed to enhance the release of RFX from RFX-pellets utilizing central composite design (CCD, Design-Expert® 10.0.0 version). The Inner Coating Level % of ENE (X1) and the Outer Coating Level % of EFS (X2) were designated as independent factors, while RFX released at pH 1.2 for 2 h (Y1), at pH 6.8 for 4 h (Y2), at pH 7.4 for 7 h (Y3), and at pH 6.8 after 12 h (Y4) were picked as dependent variables. The different runs were conducted, Table II, and the formulation for each run was prepared and analyzed for the release pattern. The regression analysis of all applicable models for each answer was conducted, and the best-fitting model was selected. ANOVA was calculated using software that provides the effect sizes of factors on the dependent variables (Akhgari et al., 2005). The 3D response surface and 2D contour plot were generated for each response. The association between predicted and actual values was established.
Characterization and evaluation of coated pellets
Scanning electron microscopy
The surface morphology of optimized RFX- Pellets was investigated with the help of scanning electron microscope (SEM, Jeol-Japan JSM-840A). Scanning microscope was used to take the pictures with 400X magnification.
Drug release studies
The in vitro release of RFX from the coated pellets was conducted using a USP dissolution device type II (Wang et al., 2014). Different dissolving media, including simulated fluid at pH 1.2, phosphate buffer at pH 6.8 (upper small intestine), phosphate buffer at pH 7.4 (distal intestine), and phosphate buffer at pH 6.8 (colon area), were created. The medium (900 ml) was positioned in a separate dissolving basket and kept at 37°C. The requisite amount of RFX-pellets (equal to 120 mg of RFX) was enclosed in muslin material and secured with the paddle of the dissolution equipment. The procedure was executed at 50 revolutions per minute. After one hour, 5 ml of aliquot was extracted from the dissolving basket, and an equivalent volume of fresh medium was simultaneously introduced to the respective dissolution basket to maintain sink conditions. The aliquots were examined using a UV spectrophotometer (UV 1900, Shimadzu) at 274 nm.
All the coated runs were calculated for drug release mechanism. The Koresmeyer-Peppas model is a mathematical expression utilized to elucidate the mechanism of drug release from these formulations. The Koresmeyer-Peppas equation is stated as follows: Mt/Mα = Ktn
Where, Mt /Mα is the fractional amount of drug released at time t, K is a kinetic rate constant, and n is the diffusional exponent that characterizes the mechanism of drug release.
Gamma Scintigraphy study
The study was performed as per CCSEA and IAEC guidelines. The animal study protocol was duly approved and the ethical approval number is 838/PO/ Re/S/04/CPCSEA/2023/01. The New Zealand White rabbits (2.6-3.5 kg) were selected and kept at 23 ± 2°C, and 50 ± 10% humidity. The rabbits were free to take the food and water.
The medication was initially radiolabeled with 99mTc-pertechnetate, and pellets were then manufactured. The pellets were subsequently coated according to the optimized coating parameters (Saripella et al., 2015). Three rabbits were utilised to examine the transit pattern of coated RFX-pellets in vivo. Prior to the in vivo imaging research (gamma scintigraphy), the rabbits were subjected to a 12-h fasting period. The radiolabeled pellets (equal to 120 mg of RFX) were orally delivered to rabbits using water. Static photos (60 seconds per image) were acquired using a gamma camera for a duration of 10 h. During the interval between gamma scans, the rabbits were liberated and allowed to engage in their customary activities. They were forbidden from ingesting any food or liquids until the formulation had completely emptied from their stomachs (Flament et al., 2004).
The distribution of radiolabeled pellets was ascertained by quantifying the radioactive counts in the stomach and colon regions of the scintigraphy image. Subsequently, the average of the counts for radioactive decay was calculated and expressed as a dose percentage. The half-life (T50%) for intestinal transit, defined as the interval between T50% values for colon arrival and colon emptying, as well as for colon arrival and stomach emptying, was employed to delineate the transit profile for coated pellets (Kwakye et al., 2004; Sinha et al., 2003). The half-life (T50%) for intestinal transit, colon arrival, and stomach emptying was utilised to delineate the transit profile for coated RFX pellets.
Stability studies
The stability study of coated rifaximin pellets was initiated at accelerated condition of 40ºC at 75% RH condition for a period of 90 days. The formulation was placed in white opaque 60CC thick-walled HDPE bottles with child resistant closure (CRC) and charged at above specified conditions in stability chamber for period of three month. After each 15 days, rifaximin was analyzed for physical appearance, drug content and dissolution profile.
RESULTS AND DISCUSSION
ANOVA reports that the mathematical model is statistically significant (p=0.0005) and describes the data well (R2=0.9881).
Statistical study revealed that four factors-water level, spheronizer speed, spheronization time, and MCC content-as well as additional two-factor interactions, were statistically significant. The result of responses against independent variables in Box Behnken design is given in Table III as 27 runs.
The model's relevance is demonstrated by its Model F value of 4.42. The probability that a F value of this magnitude could result from random variation is about 0.44%. The importance of model terms is denoted by "Prob > F" values under 0.0149. A, AB, and A2 are significant model terms in this context. The lack of fit was not statistically significant (p=0.5122), indicating that the model sufficiently represents this response for the factorial experiments (Table IV).
It has been identified that increase in MCC concentration also facilitates the percentage usable yield of the pellets. Response surface graph (Figure 1) showed that initially water facilitates the pellets formation but at higher concentrations the yield reduced even upon increase in MCC concentration. This may be due to texture of the damp mass which will be unable to spheronized. At lesser concentration of water and MCC together unable to produce sufficient yield because of production of weak extrudates. As the concentration of MCC increases the yield of pellets also increases. This may be because it is able to absorb and retain a large quantity of water due to its large surface area and high internal porosity (Xia et al., 2018), thus facilitating extrusion, improving wetted mass plasticity and enhancing spheronization. According to the ‘crystallite-gel model’, MCC particles are broken down into smaller units and even partly into single crystals of colloidal size during granulation and extrusion in the presence of water. The resulting crystallites and porous particles form a coherent gel-like network (with a high fraction of an insoluble solid phase) and immobilize the granulation liquid. Over a particular range of water, which relates to an acceptable gel strength, extrusion and spheronization becomes possible (Grohn et al., 2020; Muley, Nandgude, Poddar, 2016; Kleinebudde, Knop, 2007; Tomer et al., 2001).
Response surface and contour plots of %usable yield as a function of (a) %MCC and water, (b) %MCC and spheronization time, and (c) spheronizer speed and time.
At reduced spheronizing durations and speed, elevated concentrations of MCC resulted in increased yields of usable pellets. Extended spheronizing time and elevated spheronizing speed, along with moderate quantities of MCC and water, resulted in increased yields within the desirable particle size range (Mahdi, El-Shhibia, 2017; Singh, Pai, Devi, 2012).
Greater spheronizing speed and time generally favours the formation of pellets. It was observed that a longer spheronization time at low spheronizer speed allows moist extrudate particles to aggregate with fine particles to reduce the loss of fine particles from the spheronizer. A longer spheronization time allows the material in the spheronizer to dry to a greater extent, and dry material can fragment into particles that are smaller than the usable yield size range. The size distribution was more dispersed when pellets were produced at low spheronization speed, but at high rates the resulting pellets were larger and had a narrow size distribution. On the other hand, the spheronization time did not have major contribution on size distribution. Thus, the spheronization rate rather than spheronization time was more determining for pellet growth.
The model's significance is evidenced by its Model F value of 5.47. The likelihood of obtaining a F value of this size due to random fluctuation is around 0.54%. The significance of model terms is indicated by "Prob > F" values below 0.0001. A, AB, and A2 are essential model terms in this context. The lack of fit was not statistically significant (p=0.4713), suggesting that the model adequately depicts this response for the factorial tests (Table IV).
In Figure 2 longer spheronization periods produced more spherical pellets at elevated MCC concentrations. An increase in the spheronization time allows more time for rounding up these plasticized polymer particles. The data revealed that spheronizer speed has a significant influence on the quality of the pellets. Very low speed produced no significant shape changes in the extrudate and very high speed resulted in a size reduction of the particles.
Response surface and contour plots of roundness as a function of (a) MCC % and spheronization speed, (b) MCC % and spheronization time (c) Spheronizer speed and spheronizer time.
Results showed that an extension of the spheronization duration enables additional time for the rounding of these plasticised polymer particles. A reduction in spheronizer speed during prolonged spheronization in this study enhances roundness by creating an environment less conducive to the energy required for more fragmentation of the extrudate particles and reducing the likelihood of further drying the material in the spheronizer. At reduced spheronizer speed, prolonging the spheronization duration can facilitate sufficient time to achieve material rounding polymer particles increased opportunity to achieve a with an appropriate spheronizer speed. Extended rounded morphology (Bolcskei et al., 2011; Umprayn, spheronization duration allows these plasticised Chitropas, Amarekajorn, 1999).
The optimized variables for the development of pellets were identified from the overlay plot in graphical optimization. This suggested the optimal conditions for producing rifaximin pellets with the requisite features were determined to be 34.19% MCC, 8.6% water, a spheronizing speed of 1004 rpm, and a spheronization time of 5.12 minutes for 88.56% usable yield and sphericity value of 1.02. The observed values of %usable yield and sphericity were found to be 89.01% and 1.00 respectively. The predicted errors for both responses %usable yield and sphericity were ±0.50% and ±1.96% respectively.
Coating of RXN pellets:
The drug-loaded pellets were further coated with Eudragit NE40D (inner coating) and EFS (outer coating) to facilitate targeted drug delivery to the colon. ENE was employed to coat the interior of the drug-loaded pellets. ENE is a time-dependent polymer that swells in hydrophilic media systems, forming a gel layer structure, making it a potential component of controlled release formulations. The physicochemical properties of drugs, including solubility and particle size; the physical properties of polymers, such as drug/polymer ratio, polymer viscosity, and particle size; and fabrication factors, including stirring speed, formulation excipients, and processing methods, all significantly influence drug release from ENE matrices (Dvorackova et al., 2011; Zhang, Chen, Gao, 2006; ElMalah, Nazzal, 2008).
ENE coated RFX-pellets were coated with varying thicknesses using the EFS. It offers both mechanical resistance and resistance to gastric juice effects. EFS possesses a subtle, unique odour and is a milky white liquid characterised by low viscosity. A water-dispersed anionic copolymer composed of methyl acrylate, methyl methacrylate, and methacrylic acid is utilised. It dissolves by the formation of salt at pH levels over 7.0, but remains insoluble in acidic environments. Its degradation at elevated pH levels facilitates the precise delivery of colon-targeted drugs, complementing its gastrointestinal properties. The glass transition temperature, acid value, and molar mass are around 48 °C, 70 mg KOH/g polymer, and 280,000 g/mol, respectively. Because of the low minimum film forming temperature (MFT), no or only minimal quantities of plasticizer are necessary to achieve a smooth film formation (Moustafine et al., 2012; Potesta, 2001).
For ENE and EFS, all batches were evaluated for optimal coating efficiency, which varied from 97.61±2.06% to 98.30±2.31%. The pH and residence time at different gastrointestinal tract regions are the determining factors for targeted colonic drug release. EFS's distinctive pH sensitivity safeguards the pellets in the stomach milieu, but ENE's pH-independent permeability of the water-insoluble film progressively rises, rendering it an appropriate choice for the formulation of oral sustained release dosage forms. A mix of pH and time-dependent methods was utilised for efficient rifaximin dispersion in the colon. The variable pH medium method was employed for dissolving research, aligning with the fluctuating pH of the gastrointestinal tract. The polymers utilised for colon targeting must decompose at the neutral to slightly alkaline pH of the terminal ileum while withstanding the lower pH levels of the stomach and the proximal small intestine (McCoubrey et al., 2023). Consequently, it was essential to evaluate the lag time prior to medicine release in the small intestine at different pH levels.
Central composite design was employed to optimise the coating levels of ENE and EFS. This design generates 13 runs for the formulations of rifaximin-coated pellets. The drug release data for these RXN pellets indicates that a 10% coating level of EFS was inadequate to achieve the specified lag time for drug release. At a 10% concentration of both polymers, drug release was observed at 18.93±0.74%, 47.58±0.08%, 76.39±0.39%, and 99.48±0.68% at 2, 4, 7, and 12 h, respectively. As the inner coating level rises to 20%, the drug release percentages are 10.71±0.31%, 34.85±0.84%, 61.96±0.59%, and 99.38±0.09% at 2, 4, 7, and 12 h, respectively. The data indicates that this degree of outer coating is inadequate to prevent the release of the drug in the upper gastrointestinal tract, which is the objective of this investigation. An elevation in the coating level of EFS results in an extended lag period and safeguards drug release in the upper gastrointestinal tract. In formulation run 3, 4.39±0.36% and 11.28±0.08% of the medication were released at 2 and 4 h, respectively. In run 4, utilizing a 20% coating level of both polymers, the drug release was measured at 0±0.00% after 2 h and 0.19±0.02% after 4 h. At these concentrations, less than 20% of the medication is released after 12 h. Coating levels over 20% were not employed, as a formulation with a diminished coating level provides advantages such as decreased processing time, reduced cost, and a more compact, lighter final dosage form. The Eudragit FS 30D was dissolved, and carboxylic groups were converted into carboxylate groups as the pH above 6.5 (Umprayn, Chitropas, Amarekajorn, 1999). Nonetheless, due to the similarity in pH levels between the colon and small intestine, it may be infeasible to attain or predict drug release at the colonic location utilizing solely pH-dependent polymers. The mean pH of the small intestine, namely in the proximal, middle, and distal regions, is 6.6, 7.4, and 7.5, respectively (Fallingborg, 1999). Consequently, coatings that degrade at pH 7 would facilitate medication release in the ileum rather than the colon. Furthermore, undesirable side effects and systemic absorption may arise from medication release in the ileum. To resolve this issue, a combination of timeand pH-sensitive polymers may be employed.
The marketed formulation (Rifagut 400mg, by Sun Pharmaceutical Industries Ltd) was found to release 88.71±2.06% drug release in first 2h. Conventional formulations encounter numerous challenges on their way to the colon, such as physiological barriers and disease severity. Treatment could be more effective if drugs could be specifically targeted to act directly on the colon because site-targeted drug release allows to obtain a high drug concentration in injured tissues.
All the coated formulations were assessed for drug release mechanism. The computed n values for various formulations ranged from 0.996 to 2.183. Values of n > 0.89 have been recorded, indicating super Case-II transport in the drug release process, which involves both diffusion and the relaxation of the polymer chain.
Optimization by Analysis of Response surface method
CCD was employed to optimise RFX-loaded pellets with various polymer compositions. A total of 10 formulations were created with varying compositions of the independent variable, and the dependent variables (release of RFX at different time intervals) were analyzed. The outcomes of all formulations are presented in Table V. The regression analysis for each model (linear, second-order, and quadratic) was conducted to ascertain the optimal fit model. The quadratic model was determined to be the optimal fit for each answer (Y1, Y2, Y3, and Y4) due to its superior R2 value (0.9881) compared to other models (0.7181 and 0.6936). The ANOVA of the fitted model for each answer was analyzed, elucidating the impact of the independent variable on the response (Table VI). The contour and 3D response plots were generated for each response, illustrating the impact of independent variables on the response both collectively and individually, Figure 3.
response surface and countour plot showing the effect of conceration of polymer on release profile of RFX from the pellets (a) at pH 1.2 in first 2 h, (b) at pH 6.8 in 4 h, (c) at pH 7.2 in 7 h, and (d) pH 6.8 in 12 h were considered as target points.
The quadratic second-order polynomial equations were constructed from the software based on the release of RFX in different time intervals.
The % drug release of RFX from the pellets was assessed for 2h at pH 1.2 (Y1), up to 4h at pH 6.8 (Y2), up to end of 7h at pH 7.4 (Y3) and up to 12h at pH 6.8 (Y4), respectively. X1 is the ratio of ENE and EFS and X2 is coating level percentage.
For Y1 RFX release was <1% at pH 1.2 in first 2 h, for Y2 it was < 3% at pH 6.8 in 4 h, for Y3 30-40% at pH 7.2 in 7 h, and for Y4, RFX release ≥90% at pH 6.8 in 12 h were considered as target points.
Figure 3 indicates that as the coating level of X2 increases, the release of rifaximin diminishes due to EFS being a pH-dependent polymer, which exhibits pH-dependent solubility. The internal coating of ENE aids in time-dependent drug release, as this polymer expands upon the ingress of dissolving fluid into the pellets, thereby promoting the gradual release of the drug. Another problem with the coating level of both X1 and X2 is the pellet density, which may hinder the filling of the pellets (equal to a 120mg dose) into the capsule. ANOVA (analysis of variance) was employed to perform the statistical analyses of the models, as shown in Table VI. A significance level of 5% was utilised for the statistical analysis to ascertain the model's significance.
The Model F-value of each response indicates the model's significance. An F-value of this magnitude might occur due to noise in only 0.02% of instances. Values of "Prob > F" ≤ 0.0500 indicate the relevance of the model terms. Table VI presents the pertinent facts. Graphical optimization through overlay plot of the data suggests that at the 12.32% of X1 and 19.64% of X2, the values for Y1, Y2, Y3, and Y4 were 0.06±0.00%, 1.39±0.08%, 37.86±1.03%, and 99.07±2.81%, respectively. The observed values for Y1, Y2, Y3, and Y4 were 0.17±0.00%, 3.08±0.13%, 39.01±1.17%, and
99.37±2.73%, respectively. It was found that there was strong concordance between the observed and predicted values.
Scanning electron microscopy (SEM)
Figure 4 displayed the SEM image of formulation with optimized coating level. The cross section of the coated pellets reflects the proper coating of drug and polymer mixture. SEM images indicated that the coating of the pellets effectively concealed the inherent defects, such as fractures and pores.
SEM image of coated pellets (a), Cross sections of coated pellets showing the layers of coating dispersions (b-i, ii).
Results of Gamma Scintigraphy study
The percentage of RFX released from the coated pellets, both labelled and unlabelled, is presented in Figure 5 under simulated gastrointestinal conditions. The drug release from unlabelled pellets was recorded as 0% at 2 h, 0.99±0.03% at 4 h, 33.16±0.41% at 7 h, and 91.01±1.73% at 12 h. The RFX release from radiolabeled pellets was determined to be 0% at 2 h, 1.03±0.02% at 4 h, 34.01±0.63% at 7 h, and 90.96±1.84% at 12 h. The medication release patterns from both formulations were observed to be similar. The RFX release pattern was unaffected by radiolabeling. Notwithstanding the radiolabeling, the release percentages were consistent across the specified time intervals. Notably, radiolabeling exerted no substantial effect on drug release at the examined pH circumstances.
Comparison of % cumulative release profile of drug from radiolabeled (blue) and unlabeled pellets (orange).
Figure 6 presents a gamma image of the coated pellets marked with 99mTc. Each rabbit achieved a gastric retention period of around 6 h. An adequate count of RFX pellets labelled with 99mTc-DTPA over 10 h exhibited excellent colon arrival and retention periods. The gamma scintigraphy images indicated that the coated pellets remained intact and were uniformly scattered throughout the colon after 10 h of research.
Gamm scintigraphy image of radiolabeled RFX coated pellets after oral administration (A), in stomach (B) intestine (C) colon.
The majority of pellets were observed to remain intact in the stomach and small intestine regions. The Gamma pictures indicated that a sufficient quantity of pellets was present in the small intestine at 150 minutes, and at 450 minutes, the pellets had reached the proximal colon. At the 10th h, the picture indicated that the pellets swiftly dissolve in the distal colon.
The transit time (T50%, indicating the duration for 50% of pellets to traverse a certain region of the gastrointestinal tract) of radiolabeled RFX-loaded coated pellets was examined in Wistar albino rabbits (n=3). Three rabbits were utilised to examine the transit length of radiolabeled pellets. The transit duration of pellets in each gastrointestinal tract region is illustrated in Table VII. The transit durations for the stomach and small intestine were determined to be 0.70±0.265 h and 5.72±0.408 h, respectively. The time for pellets to reach the colon was shown to be 6.26±0.142 h (Table VII). This transit pattern correlates with the overall behaviour of humans (Sinha et al., 2003). The acquired in vitro drug release data indicates that the highest quantity of drug is released in the colon region, with negligible or minimal release at the gastric site. Because of great physiological differences and the natural complexity of the human body relative to animal models, translating gamma scintigraphy data from rabbits to humans presents various challenges. These constraints affect radiopharmaceutical biodistribution, absorption, and general study results by means of variations in metabolism, anatomy, and disease processes. Furthermore, complicating the translation process are elements like age, sex, and personal differences in persons.
Stability Studies
The % drug content in the formulation was 99.07±2.71% and found no significant differences in it at the end of 90 days (99.06±2.37%) when compared to day zero. In addition, there was no change appeared in physical appearance of rifaximin pellets. % drug release from the pellets were estimated at 2,4,7 and 12h was 0.068±0.04%, 2.67±0.37%, 38.63±0.91% and 96.37±2.37% respectively at the end of 90 days. The results showed that the formulation is stable under the tested conditions.
CONCLUSION
Extrusion-spheronization proved to be an effective pelletization technique for achieving the pellets of desired properties. MCC concentration, water concentration, spheronizing speed, and spheronizing duration found to affect the properties like % usable yield percentage and roundness under study. The surface response graphs and optimisation facilitated the layering of rifaximin pellets using a combination of ENE and EFS to achieve the required release profile and assures precise drug targeting to the colon. These formulations were shown to have super Case-II transport in the drug release process, which involves both diffusion and the relaxation of the polymer chain. The optimal coating level of 12.32% of X1 and 19.64% of X2 found to get the desired release pattern of the drug release at 2, 4, 7, and 12 h at pH levels of 1.2, 6.8, 7.4, and 6.8 respectively. Gamma scintigraphy imaging provides a useful perspective on the transit behaviour of pellet formulations, since the colonic arrival time (T50%) varies from 6.11 to 6.39 with an average of 6.26±0.142. The pellets offer a scalable platform for administering other drugs requiring colon-specific release, potentially enhancing therapeutic outcomes in colonic disorders.
ACKNOWLEDGEMENT
The authors are indebted to Dr K N Modi Institute of Pharmaceutical Education and Research for all kind of technical advice and kind support. This work forms a part of Ph.D. thesis of Ashwani Kumar Chaturvedi under AKTU, Lucknow.
DATA AVAILABILITY
The raw/processed data required to reproduce these findings are available on request from the corresponding author. The data are not publicly available at this time as the data also forms part of an ongoing study.
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Associate Editor:
Rilton Freitas
