Research Article
Gastric-specific Drug Delivery With Ph-sensitive in-situ Gels: a Novel Approach to Targeting Oseltamivir Phosphate
https://doi.org/10.21203/rs.3.rs-2490064/v1
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Flu
Oseltamivir phosphate
Central composite design
Oral in-situ gel
Flu is a viral infection that affects the upper and lower respiratory tract. It is a very contagious disease and the Influenza is a dangerous pathogen among all the other pathogens. Between 1918 and 1919, it led to the death of millions of people [1, 2]. There were 1,132 H1N1-swine flu cases confirmed till February 2020 and 18 deaths worldwide. Influenza viruses come in four types: A, B, C, and D. It is primarily the human influenza A and B viruses that cause the annual occurrence of illnesses during the winter months in the United States, commonly known as flu season [3, 4]. Influenza A viruses are the only known variety to cause pandemics or global outbreaks of flu, they are the only ones that can spread widely and rapidly. According to the WHO, there have been 3–5 million cases of severe influenza sickness and between 2,90,000 and 6,50,000 respiratory fatalities as a result of the virus. Adults get infected at a rate of 5–10% each year, whereas children are infected at a rate of about 20–30% per year. Serious sickness is more likely in adults over the age of 65 and children under the age of 2yrs [5, 6]. Adsorption, endocytosis, fusion, packaging budding, and release are all part of the influenza life cycle. Viruses connect to host cells via interacting with glycoconjugate receptors that contain sialic acid residues, which are then ingested by the cell via endocytosis. The virus's genetic material is released into the cell after it is uncoated, and the RNA polymerase replicates it. The host cell produces fresh RNA and viral proteins, which are assembled at the cell surface to form a new virus. Viral neuraminidase is required to separate the virus from the host cell once it has budded [7, 8]. Neuraminidase inhibitors are used in clinical practice to target this phase. These inhibitors prevent the enzyme from working. As a result, the spread of illness is limited. Both A and B are affected by them. These are not curing and do not eliminate the virus; instead, they obstruct the virus's ability to multiply [9]. They lessen the duration of flu episodes by a couple of days, minimize the risk of complications, and may reduce the likelihood of the virus being spread. Neuraminidase inhibitors (OP, zanamivir, peramivir) and matrix-2 (M2) protein (amantadine & rimantadine) inhibitors are FDA-approved influenza antiviral medicines [10]. M2 protein inhibitors are not advised for antiviral treatment or chemoprophylaxis of presently circulating influenza A viruses due to significant levels of resistance. The World Health Organization recommends neuraminidase inhibitors as the first-line treatment for those who need antiviral medication for influenza [11, 12]. The most important point to remember is that antiviral resistance to OP is currently low in comparison to other antiviral drugs. The recommended dosage is 75 mg twice a day. Only in serious cases, the dose is increased (150 mg twice a day). The treatment will last for 5 days. The infection is still progressing despite treatment (up to 10 days). Tamiflu is a brand name for OP, which is taken orally as a capsule or dry powder suspension [13]. The inability to swallow the capsule is a common clinical problem, especially in geriatric and juvenile patients. Because OP has a highly unpleasant taste, it causes poor patient compliance. Another key problem is the frequent administration of the drug and the absence of dosage modification [14]. When administered orally, the absorption of OP in the gastrointestinal tract is limited, primarily due to its poor bioavailability resulting from the inadequate release of the drug and a short duration of time spent at the site of absorption. [15]. Decreased patient compliance becomes a major outcome because of the above-said issues. In the pharmaceutical industry, traditional oral administration is commonly utilized to treat disorders. Traditional delivery, on the other hand, has several disadvantages, the most significant of which is the lack of site-specificity. Some medications can only be absorbed in specific locations. They must be released at a precise location [16]. The pharmaceutical industry is currently concentrating on medications that must be site-specific. Gastroretentive delivery is a type of site-specific delivery that involves delivering medications to the stomach or intestine. Oral drug delivery is being revolutionized by in-situ developing drug delivery devices [17]. The ability of in-situ gel-forming technologies to produce prolonged and regulated drug administration has been extensively researched. Recent studies have focused on developing in-situ gel formulations for various administration routes, including oral, nasal, ocular, and vaginal. [18]. In-situ gel formulations utilize polymers that transition from a liquid to a gel-like state due to changes in their physical and chemical properties. These polymers can be triggered to gel by changes in temperature or the presence of certain ions. Formulations often contain polymers such as polycaprolactone, Gellan gum, xyloglucan, alginic acid, chitosan, and various other natural or synthetic gel-forming compounds. [19]. The incorporation of bicarbonates and carbonates into in-situ gel formulations can enhance the floating properties of the gel by creating effervescence and therefore making the gel more lightweight and buoyant. Additionally, using viscosity enhancers can improve the sustained release and consistency of the gel by increasing its viscosity. [20]. OP have a relatively short biological half-life, typically lasting between 1–3 hours, which can make it difficult to achieve effective treatment when administered in traditional dosage forms. To overcome this, high-frequency dosing (35–75 mg per day) is often required. Additionally, the stomach is the primary site of absorption for OP, which further emphasizes the need for frequent dosing to maintain optimal levels in the body. [21, 22]. Therefore, creating an in-situ gel for OP that is hydrodynamically balanced would be beneficial as it would enhance the drug's effectiveness and reduce the need for high-frequency dosing.
OP is an acidic drug and is unionized in the upper part of the stomach to act as an absorption window. The upper part of the stomach has more absorptive cells than secretary cells, through columnar epithelial cells drugs entered into the systemic circulation. Utilizing a raft-forming system for OP can increase the duration and length of the gastric stay of the dosage in proximity to the absorption window, this ultimately causes a rise in the bioavailability of the drug (shown in Fig. 1).
This study involves the use of a gelation technique to prepare an in-situ gel of the drug of choice (OP) Sodium alginate is used as the main gelling agent where HPMC K 100 M plays the role of viscosity enhancer. The goal is to optimize the formulation to achieve maximum viscosity and appropriate drug release at 12 hours while minimizing release at 1 hour, using a computer simulation technique was employed that used “response surface methodology” (RSM) and “Central composite design” (CCD) approach. The resulting in-situ gel will be used to create a ‘floating drug delivery’ system.
In our study, Oseltamivir phosphate was obtained as a gift sample from Mylan Laboratories in Hyderabad. Other materials used in the study were also purchased from various suppliers, such as sodium alginate from Otto Kemi, HPMC K 100M and sodium bicarbonate from Loba Chemie, trisodium citrate was received as a gift sample, from Fortz India, calcium carbonate from Avantor Performance Materials India in Maharashtra, and calcium chloride from Kemephasol in Mumbai.
The compatibility of a drug with different excipients was studied using infrared spectroscopy and the KBr disc method. Spectra were obtained for the pure form of the drug and for mixtures of the excipients along with that of the drug, and these spectra were analyzed to identify changes occurring in characteristic peaks of the constituents and the drug. The study was conducted in the infrared range of 4000 − 400 cm− 1.
Method of preparation of in-situ gel
An in-situ gel was obtained by solubilizing a combination of polymers, namely sodium salt of alginic acid (sodium alginate), and HPMC K 100M, in deionized water at 60ºC with continuous stirring. A homogenous mixture formation was achieved by stirring in a hotplate magnetic stirrer at an elevated temperature of 90̊ C. Once the solution had cooled to below 40ºC, various agents were added to enhance the properties of the gel. Calcium Carbonate was added as a gas-forming agent, Calcium Chloride as a cross-linking agent, Sodium Bicarbonate as a buoyancy enhancer, and the drug OP was added to the mixture. Finally, Sodium Citrate was added to maintain the fluidity of the formulation, and the end volume was prepared using deionized water up to 100 mL. [23].
The preparation of an in-situ gel is a critical step in the development of a controlled-release drug delivery system. The use of a combination of polymers, in this case, sodium alginate and HPMC K 100M, provides the gel with the desired properties of viscosity and gelation. The heating and cooling process ensures that a homogeneous solution is formed, which is essential for the consistency of the final product. The addition of various agents such as Calcium Carbonate, Calcium Chloride, Sodium Bicarbonate, and Sodium Citrate, not only enhances the properties of the gel but also assists in maintaining the stability and controlled (sustained or extended) release of the active drug, OP. In conclusion, the preparation of an in-situ gel, as described in this study, provides a promising approach for the controlled release of drugs.
“Design-Expert software (Version 13.0.1.0 Stat-Ease, Inc., Minneapolis, MN)” was employed in the optimization of the in-situ gel, the study of 13 runs, 2 factors, and 3 levels of central composite designs was developed. The model can also be used in the investigation of quadratic response surfaces and to build second-order polynomial models. The main characteristics of this cubic design are the factorial points, the center point of the cubic, and the axial point that restrict the region of limit. The main impacts, interaction effects, and quadratic effects of the formulation components can all be investigated and optimized using a region of interest. 13 experimental trial runs made up a design matrix that was created.
The software created the following nonlinear quadratic design model equation.:
Y = A0 + A1 X1 + A2 X2 + A3 X1 X2 + A4 X12 + A5 X22
Where,
A0 represents the intercept which is the arithmetic mean of all 13 runs
A1, A2, A3, A4, A5 shows the regression coefficient of the observed experimental values of response variable Y
X1 & X2 are independent variables with coded levels
X1 X2 – linear interaction terms
X12, X2 2 – quadratic terms
Concentrations of sodium alginate (X1) and HPMC K 100 (X2) represented the independent variables chosen in this design matrix, + 1 and − 1 codes were assigned to reflect high and low values, respectively. Viscosity (Y1), drug release at 1-hour time (Y2), and 12 hours of drug release (Y3) were named as the dependent variables, as listed in Table 1. The responses attained from all 13 formulations were then fitted onto our design.
Translation-coded values in actual units |
|||
---|---|---|---|
Independent variable |
Levels used, actual (coded) |
||
Low (-1) |
Medium (0) |
High (+ 1) |
|
X1 = concentration of sodium alginate |
1.5 |
2 |
2.5 |
X2 = concentration of HPMC k 100 |
0.4 |
0.5 |
0.6 |
Dependent variable |
|||
Y1 = Viscosity (Cps) |
Maximize |
||
Y2 = Drug release at 1 hour. (%) |
Minimize |
||
Y3 = Drug release at 12 hours. (%) |
Appropriate |
Evaluation of Physical Properties and pH of Floating In-Situ Gels
The evaluation of the physical properties of floating in-situ gels was carried out, including the measurement of the pH of the gel using a digital pH meter. The pH was determined at normal room temperature with the addition of a required amount of the gel to a 50ml beaker [24].
A Brookfield viscometer was used to measure the created in-situ gel's viscosity, consisting of spindle LV-1 set at a rotation speed of 1.5, 3, and 6 rpm at standard room temperature. Three concordant readings were taken to ensure accuracy [25].
To ensure that the medication delivery system floats in the stomach, it is important to measure its density and compare it to the density of stomach fluid (1.004 g/cm3). This can be done by creating a known volume of gel (5 ml) in a Petri plate containing 0.1N HCl, and then measuring its weight using a calibrated balance to calculate its density. This measurement was conducted three times for each formulation to ensure accuracy [26].
To measure the swelling index of a gel, 100mg of the gel was accurately weighed (W1) and kept in a petri dish that contained 50 ml of 0.1N HCl. The gel was left for 24 hours and then weighed again (W2). The formula below was used to determine the swelling index [27].
Where W1 represented the Initial weight of the sample (gel), W2 is the weight of the sample (gel) after 24 hours.
For evaluation of the gelling potential of in-situ gel-forming drug delivery systems, colored solutions were created and their gelling ability was determined visually. The gelling ability of the formulations was determined using the procedure described as follows, 5 ml of 0.1N HCl, pH 1.2 (gelation solution) were added to a 15 ml borosilicate test tube. and keeping it at 37oC. The stiffness of the appearing gel and duration of the stiffness of the gel was used to assess the solution's gelling capacity. The color was added to the gel to make it more visible. Three kinds of in-vitro gelling ability were identified based on the time it took to gel and the amount of time the produced gel remained intact: (+) gels dispersed rapidly after a few minutes, (++) gelation occurred immediately and remained for a few hours, (+++) immediate gelation occurred and lasted more than 24 hours intact [28].
The capacity possessed by the formed gelled mass to retain gastric fluid against peristalsis is measured by its tensile strength or gel strength. To measure the gel strength, a 5 ml solution was added to a cylinder, followed by 25 ml of gastric fluid 0.1 N HCl (pH 1.2) to cause gelation. The HCl drained away after the formation of the gel leaving only the formed gel mass, and a device was placed on top of it. A lightweight pan (4 g) was attached to the device's free end, to which weights were added. The weight needed to pass the device through the formed gel mass was used to determine the gel strength [29].
In-vitro Buoyancy Study
0.5 liters (500ml) of artificial gastric liquid having a pH of 1.2 was prepared and the in-vitro buoyancy investigation was conducted in USP dissolving equipment II. 37°C was maintained as the mean temperature. 10 ml of the produced sample was taken using a disposable syringe and placed on a petri dish, which was later placed carefully in the dissolution flask containing the artificial gastric fluid media. The recorded parameters were the time required for the gel to reach the surface (floating lag time) and the time it stayed there (total floating time) [30].
Accurately measured formulation equivalent to 5 ml of OP in-situ gel was taken and poured into a volumetric flask that held 100 ml of 0.1 N HCl. It was stirred well for 30 minutes and then subjected to sonication for 1 min followed by filtration. After producing dilutions using 0.1N HCL, 221.4 nm (λmax of OP) was fixed as the wavelength and the absorbance was checked. The obtained values were transferred to the equation of the calibration curve to obtain concentration [31].
Study for In-vitro Release of Drug
A drug release study of the formulation was performed using a USP (type-II) paddle apparatus at 37 ± 0.5ºC and 50 rpm with 900 ml of pH 1.2 HCl as the dissolution medium (n = 3). The formulation sample amount used for the study was 10 ml. 5 ml of the sample was withdrawn in regular intervals as per prior planning, followed by filtration, dilution, and analysis by the spectrophotometric method. Equivalent amounts of fresh dissolution media were replaced after each sample withdrawal. A simultaneous equation method was employed in calculating the assay of the drug dissolved in the sample in those particular intervals. [32, 33].
to comprehend the medication release process from the in-situ gel, the dissolution data of the Numerous model-dependent kinetics, including Zero-order, First-order, the Higuchi model, and the Korsemeyer-Peppas equation, were used to examine the optimized formulation. The mechanism of drug release was identified by comparison of the r2 values obtained from each of these models [34].
After receiving approval from the Institutional Ethics Committee, a study strategy was created (ID no: PCP/IAEC/004/2020), and the study was conducted according to the plan. A 2.5 kg healthy albino rabbit was used in the X-ray imaging investigation to assess the gel's retention in the stomach. The rabbit was kept in the study condition for a period of 3 days prior to the commencement of the experiment. The study gel was prepared by incorporating an X-ray contrasting agent. Throughout the trial, the rabbit was not permitted to eat but was given access to water ad libitum. An X-ray machine was used to perform gastric X-ray imaging at predefined intervals of 1 and 8 hours [35, 36].
Stability Studies
The final optimized formulation was stored under monitored conditions having 40 ± 2ºC & 75 ± 5% relative humidity (RH) for 3 months to evaluate its stability as per ICH guideline Q1C. At regular intervals, samples were taken and evaluated for gelation properties, viscosity, gel strength, pH, in-vitro drug release, drug content, floating lag time, total floating time, and floating duration studies to check if there were any changes in the formulation during the stability study period [37, 38].
One-way analysis of variance or ANOVA along with student t-tests were employed to identify any significant differences. If the p-value was 0.05 or less, the results were deemed statistically significant. Utilizing the software GraphPad Prism v.6.0, all data analysis was carried out (GraphPad Software, La Jolla, CA).
Comparison of in-vitro release studies of the optimized batch with marketed formulation
The release of the drug from the marketed formulation of OP (conventional capsule) was studied in-vitro using a USP (type II) dissolution test apparatus of paddle type with a rotation speed of 50. The dissolution medium used was simulated gastric fluid pH 1.2 and the temperature was maintained at 37 ± 0.1ºC. At 5, 10, 15, 20, and 45 min, 5ml of the sample was withdrawn and replaced with 5ml of pH 1.2 buffer. The withdrawn sample was filtered and 1ml of the filtrate was diluted to 10ml with 0.1N HCl. The dissolution data of the optimized formulation was compared with the marketed formulation. The concentration of the drug in the sample was measured by measuring the absorbance at 221.4 nm using a UV spectrophotometer [39, 40].
The compatibility of the drug and polymer was studied using FTIR spectroscopy. The pure drug's IR spectrum (Fig. 2) showed absorption peaks at 3352.42− 1 (O = C-NH), 1716.61− 1 (C = O), 1244.61− 1 (C-O), 1658.87− 1 (C = C), and 1027.08− 1 (C6H5NH2), which are defined as the characteristic peaks of OP. For sodium alginate, IR absorption band peaks were visible at 2925.77− 1 (-OH stretch), 3409.18− 1 (C-H stretch), 1602.08− 1 (C = O stretch), and 1022.80− 1 (C-O stretch) (Fig. 3). The peaks that appeared in the OP in-situ gel were observed at 1613− 1, 872.71− 1, 1613.06− 1, 3448.56− 1, 1153.53− 1, 2964.36− 1, 3448.56− 1, 1594.28− 1, and 616.48− 1 respectively indicating C = C, C-O, C = O, O = C-N-H, C6H5NH2, -OH stretching, C-H stretching, -C = O stretching and, C-O stretching has not altered (Fig. 4). OP signature band was observed to be unaffected by the excipients in the drug-polymer physical mixture IR spectrum. There was no shifting or appearance of peak observed in the compatibility study of the OP in-situ gel. This shows that the formulation's constituents and the drug of choice are compatible.
The statistical analysis of the design variables was carried out using Design-Expert software version 13.0.1.0 from Stat-Ease, Inc. based in Minneapolis, MN. The Concentration of sodium alginate and the concentration of HPMC k 100 is the selected independent variable that places a major role in the conversion of sol to gel and stabilization of gel over a longer period in the upper part of the stomach as an absorption window. These variables which mainly affect the outcome of the product viscosity and the performance of the product drug release at 1 hour and 12 hours are checked and they are the response of the design. The link between the independent factors and the dependent variable of the product was statistically examined using the polynomial equation. Statistical measures produced by the Design-Expert program, such as the multiple correlation coefficient, adjusted multiple correlation coefficient and predicted sum of squares, were used to identify the product's primary effect and interaction effect. The software was used to develop the data based on the independent variables and the experiment was conducted. The responses were then entered into the software (Table 2). The ANOVA function in the software was used to validate the polynomial equation and optimize both the independent and dependent variables of the product. This enables the determination of the optimized batch of the formulation.
Run |
Factor 1 A: sodium alginate mg |
Factor 2 B: HPMC k 100 mg |
Response 1 Viscosity cps |
Response 2 %CDR (1hour) |
Response 3 %CDR (12hours) |
---|---|---|---|---|---|
1 |
2 |
0.5 |
1150 |
34.23 |
89.36 |
2 |
1.29289 |
0.5 |
1050 |
43.55 |
98.21 |
3 |
2 |
0.5 |
1100 |
38 |
93.98 |
4 |
2 |
0.5 |
1100 |
37.8 |
94.56 |
5 |
1.5 |
0.6 |
1250 |
33.56 |
84.98 |
6 |
2 |
0.641421 |
1400 |
31.45 |
82.55 |
7 |
2 |
0.5 |
1100 |
35.67 |
94.32 |
8 |
2.70711 |
0.5 |
1550 |
30.12 |
80.43 |
9 |
1.5 |
0.4 |
1050 |
44.12 |
99.43 |
10 |
2.5 |
0.6 |
1550 |
30.21 |
81.77 |
11 |
2 |
0.5 |
1100 |
36.72 |
95.44 |
12 |
2.5 |
0.4 |
1200 |
33.21 |
86.67 |
13 |
2 |
0.358579 |
1080 |
41.83 |
97.08 |
The response observed for 13 formulations that were prepared was fitted into the design of the experiment by the software ‘Design-expert’.
Based on statistical models of the sequential model sum of squares, lack of fit, and model summary, the answer was analyzed. The sum of squares, mean squares, F-value, P-value, residual; the sum of square, predicted R2, adjusted R2, adeq precision, and “predicted residual error sum of square (PRESS)” values suggest that the Y1 response fit onto the quadratic model. The model is shown to be significant by the Model F-value of 45.69 for the response Y1. An F-value this large might happen to owe to noise only 0.01% of the time. Model terms are considered significant when the P-value is less than 0.0500. In this instance, A, B, A², and B2 are terms that are significant to the model. From this ANOVA we found that the concentration of sodium alginate and the concentration of HPMC k 100 is the independent variable that affects the Y1 response. Y1 had a lack of fit of 6.38, which implies the Lack of Fit is not significant relative to the pure error. Multiple regression term was also analyzed, the Predicted R² of 0.8171 is in reasonable agreement with the Adjusted R² of 0.9490; i.e. the difference is lower than 0.2. which indicates that the model has predicted the response well. Adeq Precision measures the signal-to-noise ratio. A ratio of at least 4 is preferred. The ratio of response Y1 was 19.554 indicating an adequate signal. This model can be used to navigate the design space.
By coded factors, the quadratic equation for the response Y1 was given in the equation
The quantitative effect of all the two independent variables X1 and X2 are the main effects influencing the response Y1 as indicated by this equation. X1, X2, X12, and X22 are denotations for the interaction terms that show a non-linear relationship between the response Y1 and the variable if it was changed simultaneously. The positive sign of X1 and X2 shows a synergistic effect over the response Y1
The factor which influences the response were shown in the perturbation graph (Fig. 5a). The sensitivity of the factor can be determined by a steep slope and curvature of the graph and insensitivity was relative to the flat line. A perturbation plot can determine which of two or more factors has the greatest influence on the response. Both factors A and B for response Y1 exhibit curvature. It suggests that the two factors most responsible for determining the product's viscosity were the concentrations of sodium alginate and HPMC k 100.
A Strong positive correlation suggests that the concentrations of sodium alginate (X1) and HPMC k 100 have a clear correlation with viscosity (X2). In which increase both the independent variable enhances the viscosity of the in-situ gel. Sodium alginate is one of the pH-sensitive polymers which have the mechanism, that an alkaline pH stays in solution form when it enters into the stomach pH and, the presence of Ca2+ ions in the calcium carbonate and calcium citrate which will form cross-linking between the homo and hetero polymeric blocks in the sodium alginate and leads to the formation of alginate gels. An optimum concentration of gelling agent (sodium alginate) was needed to form an in-situ gel. When sodium alginate concentration grew from the lowest to the greatest, the viscosity response increased. The independent variable HPMC K100M was one of the pH-sensitive viscosity enhancers through which the viscosity of the formulation was increased. It is evident from the perturbation graph that increases in the concentration of HPMC K100M increase the viscosity of the formulation. Both HPMC K100M, a co-gelling agent, and this variable sodium alginate, a gelling agent have a synergistic action towards the response which is very important in the case of in-situ gel. Through this optimum viscosity, the drug release from the in-situ gel can be controlled in the predetermined time interval and it will prolong action in the stomach.
The link between the independent variable and the response is depicted by response surface plots, it also shows the possible interaction between the factors. The interaction between the X1 and X2 factors was analyzed over the response of viscosity (Fig. 6a). As it was evident from the response plot, both the concentration of sodium alginate and the concentration of HPMC K 100M show interaction over the response Y1 viscosity. From the plot, it was determined that increasing the concentration of sodium alginate increases the viscosity of the formulation to a particular extent. And also increasing the concentration of HPMC K100M shows a better-increasing viscosity of the formulation. The response surface plot also shows the interaction of both factors towards the response, increasing the concentration of both factors will lead to a drastic increase in the viscosity of the formulation. This viscosity plays a crucial role in the in-situ gel as it controls the drug release over a prolonged period of action in the predetermined time interval.
The sum of squares, mean squares, F-value, P-value, residual; the sum of square, predicted R2, adjusted R2, adeq precision, and PRESS values suggest the Quadratic model for Y2 response. Given that the Model had an F-value for Response Y2 of 42.50, the Model is deemed Significant. An F-value this large might happen to owe to noise only 0.01% of the time. Model terms are considered significant when the P-value is less than 0.0500. A, B, and AB are important model terms in this scenario. This ANOVA found that the independent variables that influence the Y2 response are the concentrations of sodium alginate and HPMC k 100. The F-value for Y1's lack of fit was 0.65, which suggests that the lack of fit is not significant in comparison to the pure error. Multiple regression term was also analyzed, Specifically, the difference is less than 0.2 between the Predicted R2 of 0.8333 and the Adjusted R2 of 0.9121. which indicates that the model has predicted the response well. The signal-to-noise ratio is measured by Adeq precision. A ratio larger than 4 is preferred. The response Y2 ratio was 19.809, which indicates a strong signal. To move around the design space, utilization of this model is possible.
By coded factors, the quadratic equation for the response Y2 was given in the equation
The independent variables, X1 and X2, are seen as having a quantifiable impact on the response, Y2, according to this equation. X1, and X2, are the interaction terms that show the non-linear relationship between the response Y2 and the variable if it was changed simultaneously. The negative sign of X1 and X2 shows an antagonistic effect against the Y2 response.
Both factors A and B show a negative slope for response Y2 (Fig. 5b). It suggests that the main variables influencing the product's drug release were the concentrations of sodium alginate and HPMC k 100. The increase in the concentration of factors shows a decrease in the drug release of the gel. This can be understood by increasing the concentration of both the gelling and co-gelling agent there will be an increase in the viscosity of the formulation. The higher the viscosity of the formulation the drug has to come out of this extent to exert its action. This drug release pattern at a predetermined time was necessary for the prolonged action in the upper part of the stomach. Response surface plots express the relationship between the independent variable and the response, it also shows the possible interaction between the factors. The interaction between the X1 and X2 factors was analyzed over the response of drug release at 1 hour (Fig. 6b). As it was evident from the response plot that both the concentration of sodium alginate and the concentration of HPMC K 100M show interaction over the response Y2. From the plot, it was determined that increasing the concentration of sodium alginate showed a negative regression coefficient by increasing the viscosity of the formulation in which the drug release of the gel was decreased. And also increasing the concentration of HPMC K 100M shows also show a negative regression coefficient by better increasing the viscosity of the formulation which also decreases the drug release. The interaction of both factors towards the response was increasing the concentration of both the factors will lead to a decrease in the drug release of the formulation at 1 hour. This viscosity plays a crucial role in the gel as the drug has to be released from the higher viscosity of the formulation to the stomach to provide its action. The concentration of both factors should be optimized to attain the specific viscosity of the formulation when it reaches the stomach, this viscosity will have the drug released over some time in a controlled manner.
The sum of squares, mean squares, F-value, P-value, residual; sum of square, predicted R2, adjusted R2, adeq precision, and PRESS values suggest the Quadratic model for Y3 response. The model is likely significant because the Model F-value for the response Y3 was 18.75. An F-value this large might happen as a result of noise only 0.06% of the time. Model terms are considered significant when the P-value is less than 0.0500. A, B, A2, and B2 are significant model terms in this situation. This ANOVA found that the independent factor that influences the Y3 response is the concentration of sodium alginate and the concentration of HPMC k 100. According to the Lack of Fit F-value of 0.84 for Y3, the Lack of Fit is not significant relative to the pure error. Multiple regression term was also analyzed, the Predicted R² of 0.7429 is in reasonable agreement with the Adjusted R² of 0.8809; i.e. the difference is less than 0.2. which indicates that the model has predicted the response well. Adeq Precision measures the signal-to-noise ratio. A ratio greater than 4 is desirable. The ratio of response Y3 was 12.915 indicating an adequate signal. This model can be used to navigate the design space.
By coded factors, the quadratic equation for the response Y3 was given in the equation
Y3 = 93.53–5.14 X1 − 4.99 X2 + 2.39 X1 X2 – 2.44 X12 – 2.20 X22
The quantitative impact of each of the two independent variables, X1 and X2, is shown by this equation. Those are the primary factors affecting the reaction Y3. X1, X12, and X22 are the interaction terms that demonstrate a non-linear link between the response Y3 and the variable if it was changed simultaneously. The negative sign of X1 and X2 shows an antagonistic effect on response Y3.
Both factors A and B for response Y3 exhibit curvature (Fig. 5c). It suggests that the main variables influencing the product's drug release were the concentrations of sodium alginate and HPMC k 100. A strong negative coefficient of both factors is inversely proportional to the response. This can be understood by increasing the concentration of both the gelling and co-gelling agent there will be an increase in the viscosity of the formulation and the drug release at 12 hours was decreased. This drug release pattern at a predetermined time was necessary for the prolonged action in the upper part of the stomach.
The interaction between the X1 and X2 factors was analyzed over the response of drug release at 12 hours (Fig. 6c). Both the concentration of sodium alginate and that of HPMC k 100 exhibit interaction over the response Y3 as can be seen from the response plot. From the plot, by raising the viscosity of the formulation, it was found that increasing the sodium alginate concentration lowered the drug release from the gel by showing a negative regression coefficient. And also increasing the concentration of HPMC K100M shows also show a negative regression coefficient by better increasing the viscosity of the formulation which also decreases the drug release. The interaction of both factors towards the response was increasing the concentration of both factors will lead to a decrease in the drug release of the formulation at 12 hours. This viscosity plays a crucial role in the gel as the drug has to be released from the higher viscosity of the formulation to the stomach to provide its action. The concentration of both factors should be optimized to attain the specific viscosity of the formulation when it reaches the stomach, this viscosity will have the drug released over some time in a controlled manner.
Optimization and validation:
Figure 8: Optimized result by numerical optimization obtained for the OP in-situ gel
To identify the optimal formulation, The Design-Expert software was used to analyze the desirability criterion. (Fig. 7) which was based on specific criteria such as maximum viscosity and minimal drug release at 1 hour and 12 hours. To validate the optimization procedure, a fresh batch of the in-situ gel was made using the expected values of the variables. The predicted optimized formula was composed of 2.5 mg of sodium alginate and 0.6 mg of HPMC k 100, which met the optimization requirements (Fig. 8). The optimized formulation had a viscosity of 1573.71 Cps, a drug release of 30.3934% at 1 hour, and 81.1508% of drug release at 12 hours.
To prevent throat discomfort, oral medications must have their pH measured. A digital pH meter was used to measure the in-situ gel's pH, calibrated with pH 4 and pH 7 to make sure the solution's pH was alkaline. The pH values were recorded immediately after the preparation. The formulation was in a free-flowing form and had no gelation when left to stand at room temperature (Fig. 9). The optimized batch had a milky white appearance and the pH was 8.3 ± 0.04 which is suitable for oral administration. Therefore, no adjustments to the pH were necessary. The most stable pH range for sodium alginate aqueous solutions is 4–10, as alginic acid precipitates from the alginate solution below pH 3, making the gel and liquid phases of the formulation unappealing.
Rheological Property
According to the rheological analysis, the polymer concentration grew significantly increased as the formulations' viscosities significantly increased. This boost in viscosity was related to a rise in sodium alginate content, it results in more double-helical alginate segments aggregating and calcium cation binding sites increasing, forming a three-dimensional network. Additionally, the system's enhanced viscosity was also influenced by the presence of HPMC K100M. It was found that increasing the calcium carbonate concentration also led to an increase in viscosity, probably due to the presence of calcium carbonate as insoluble particles in the dispersion. However, for the formulation to be suitable, it must have an optimal viscosity that allows for easy ingestion as a liquid and instantly gels after consumption. The concentration of calcium carbonate was adjusted to optimize the formulation further. The results showed that all of the formulations had viscosity within the desired range. The viscosity of the optimized formulation was 1573.71 ± 1.73 Cps, indicating that the formulation can be easily administered orally within this range.
When evaluating buoyancy, density is an important evaluation factor of the gastro retentive dosage form. The formulation must have a density that is lower than or equal to that of gastric contents (1.004 gcm− 3), in order for it to float on the contents of the stomach. The formulation's density was discovered to be 0.90 ± 0.7, which is less than the specified value. Therefore, the formulation has the ability to float on the stomach, promoting the gastro-retentive in-situ gel.
The swelling behavior of the in-situ gel is affected by the concentrations of the primary polymers (sodium alginate) as well as the addition of secondary polymers (HPMC K100M). High percentages of hydration are obtained by increasing the sodium alginate concentrations, and sodium-calcium ion exchange leads to the formation of insoluble calcium alginate regions, which then results in easy hydration and rapid swelling of Na alginate. The water uptake statistics are closely related to the molecular weight and concentration of HPMC; as higher molecular weight HPMC grades are used or the concentration of specific HPMC is increased, the swelling index will significantly increase. The system's water content has an impact on how quickly drugs are released from the polymer matrix. The drug may be released by diffusion or dissolution after the water has penetrated the matrix. The in-situ gel exhibited a swelling index of 53.8%.
Gelation is the process of forming a three-dimensional network through the formation of double-helical systems via hydrogen bonding and complexation with cations. Instant gelation in ten seconds was seen with the formulation (+++) and lasted longer than 12 hours unchanged. The term "gel strength" describes the gelled mass's tensile strength and its capacity to sustain peristaltic motions in vivo. The concentration of the gelling agent and the cation source affect the formulation's gel strength. The optimized batch had a high gel strength. This implies that the formulation's stomach residence duration is longer, and its gel strength is higher.
In-vitro floating ability
The floating “lag time” and floating time duration were studied for the prepared gel. In-vitro floating research was conducted using 500 ml of 0.1N HCl (pH 1.2) at a temperature of 37℃. 10 ml of the formulation was added to the dissolution basket without disturbing the medium. When the formulation was inserted into the medium, the CO2 it released was caught in the gel network, creating a buoyant formulation. Furthermore, when sodium alginate and calcium ions interact, a crosslinked 3-D network formation of gel takes place, which expands and captures more CO2. The trapping in the network structure kept the buoyancy and flotation going for a long time. Additionally, Drug release was hindered by the gel network, showing an extended-release pattern as a result. An in-vitro floating latency of fewer than two minutes was observed for the formulation. For the formulation, the total floating time was longer than 12 hours.
The gel's drug content was discovered to be reliable, with minimal loss of the drug during the formulation process and an even distribution of the drug throughout the gel. The drug content of the in-situ gel was shown to be affected by the concentration of Sodium alginate and HPMC K 100M.
Drug release (In-vitro)
The release study of the drug OP in 0.1N HCL was carried out over a period of 12 hours from the optimized formulation batch, and the results are shown in a graph (Fig. 10). The rate and amount of medication released were shown to significantly decrease when the in-situ gel's polymer concentration was raised. This can be traced back to the longer diffusion channel for the drug molecules to follow and increased polymer matrix density. The release rate is also affected by the use of Sodium alginate and HPMC K 100M, which play a key role in the transition from a liquid to a gel state, and also affect the buoyancy of the gel. It was seen that the drug's release from the in-situ gel continued even after the 12-hour timeframe.
Kinetics Of Drug Release
Mathematical models were applied in the investigation of drug release from the in-situ gel, such as the zero-order, Higuchi, and first-order kinetics. The results showed that the Higuchi model provides the best explanation for drug release from the in-situ gel, as evidenced by the highest correlation coefficient of 0.9857. The mechanism of drug release was also studied using the Hixson Crowell and Korsemeyer Peppas models, with the Peppas model showing the highest correlation coefficient of 0.9810. The results indicate that the drug release from the in-situ gel is by diffusion, as indicated by the ‘n’ value of 0.522, which is greater than 0.45, indicating non-fickian transport. Overall, the findings imply that the Higuchi model is followed by the drug release from the OP in-situ gel with a non-fickian diffusion mechanism.
The OP in-situ gel was stored in a sealed vial with a rubber cap for three months, in a humidity chamber set at 40 ± 2°C and 75 ± 5% relative humidity. Samples were taken at 30, 60, and 90 days to evaluate the drug content, drug release, pH, and viscosity results shown in Table 3. The findings revealed that the gel's appearance had not changed significantly, and also in the other parameters through the stability analysis (P < 0.05).
Stability duration |
pH |
Viscosity (Cps) |
Gelling time (sec) |
Floating lag time (sec) |
Floating duration (hours) |
Drug content (%) |
Q12 (%) |
---|---|---|---|---|---|---|---|
Before storage |
8.2 ± 0.3 |
1572 ± 1.69 |
8.33 ± 0.47 |
53 ± 0.43 |
> 12 |
99.75 ± 0.27 |
81.52 ± 0.16 |
After 1 month |
8.2 ± 0.25 |
1575 ± 1.77 |
8.2 ± 0.43 |
52 ± 0.44 |
> 12 |
99.75 ± 0.22 |
81.42 ± 0.35 |
After 3 months |
8.1 ± 0.28 |
1572 ± 1.75 |
8.2 ± 0.41 |
52 ± 0.47 |
> 12 |
99.75 ± 0.25 |
81.28 ± 0.28 |
Gastroretentive X-ray Imaging Study
The in-vivo x-ray studies were conducted after obtaining approval from the institutional animal ethics committee. The studies were performed to determine the gastric retention of the in-situ gel in healthy male albino rabbits at different time intervals (0, 1, and 8 hours) using an x-ray machine. The results of the study showed that the gel remained in the upper part of the stomach, confirming its ability to float in-vivo. The change in position of the gel in the 1st -hour and 8th -hour x-ray images also demonstrated that it does not adhere to the mucous and continues to float. Additionally, the size of the gel was observed to increase, as seen in the comparison of the 1st -hour and 8th -hour x-ray images, which is an indication of the swelling of the gel. The results were represented in a Fig. 11a-c.
Comparison of the Optimized Formulation and the Marketed Formulation's In-vitro Drug Release
The study discovered that the OP in-situ gel was designed to have a good swelling index and to sustain drug release for up to 12 hours, as opposed to the marketed formulation, which did so only for 30 minutes. The results of the comparison are presented in Fig. 12.
Also, dissolution profiles of two materials (OP in-situ gel with the conventional commercial capsule used in this study) were compared using the dissimilarity factor f1 and the similarity factor f2, Dissimilarity factor f1 was calculated from the following Eq. (41, 42):
where n is the number of pull points, Tt is the test assay at time point t, Rt is the reference assay at time point t, and Wt is the optional weight factor.
Similarity factor f2 was calculated for dissolution profile comparison of OP capsule with OP in-situ gel, f2 was 16. This shows that the commercial products examined for this study are not identical or similar (f2 > 50, dissolution profiles are defined as similar)
Dissimilarity factor f1 was calculated for dissolution profile comparison of OP capsule with OP in-situ gel, f1 was 64. This indicates that commercial products tested in this study are different (% error increases as the dissimilarity between two profiles increases). The dissolution rate of the capsule was high compared to OP in-situ gel, indicating the capsule has burst release and causes serious side effects.
The purpose of the current study was to use the central composite model to create a floating in-situ gel for the sustained delivery of OP, a highly potent antiviral medication. The findings showed that the concentration of sodium alginate and HPMC K100M substantially impacted the formulation's viscosity and drug release. The formulation's optimization through desirability and overlay plot met all of the target set's requirements. The drug release kinetics was consistent with the Korsemeyer Peppas model, indicating a non-fickian “diffusion-controlled” formulation release. The in-vivo floating ability of the gel was found to be retained in the human stomach for more than 12 hours. The calculated values for f1 and f2 were found to be considerably different based on the in-vitro results, indicating that the in-situ gel had controlled the rate of drug release for an extended period of time. In conclusion, the development of a floating in-situ gel for the oral sustained delivery of OP is a promising approach for the treatment of flu. The formulation was able to fulfill all the criteria set as a target and it also showed a good swelling index and sustained release of the drug till 12 hours. This novel formulation has demonstrated the potential to enhance the efficacy of the drug while minimizing adverse effects, making it a promising option for improving patient outcomes
OP- Oseltamivir Phosphate, HPMC- Hydroxypropyl methylcellulose, WHO- World health organization, %CDR- Percentage cumulative drug release, CCD- Central composite design, RSM- Response surface methodology, ANOVA- analysis of variance, PRESS- Predicted residual error sum of square, M2-Matrix 2 protein, H1N1- Swine flu, FDA-Food and drug administration.
Acknowledgment
The authors would like to thank the India Council of Medical Research (ICMR)-New Delhi for awarding the Senior Research Fellowship (SRF)-2021. The authors would like to thank the Department of Science and Technology-Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions, New Delhi for their infrastructure support for our department. The figures and graphical abstract in this manuscript were created with Biorender.com and the support of http://biorender.com under a paid subscription.
Author contributions
All of the authors played a significant role in the development and design of the study, the collection and analysis of data, and the creation and revision of the article. They all approved the final version of the article and are responsible for its content.
Funding: This is no funding to report.
Availability of data and materials The data and materials will be made available if asked for.
Ethics approval All animal experimental procedures were performed in accordance with international rules and approved by the Institute Animal Ethics Committee of PCPS, Tiruchirapalli, Tamil Nadu, India (approval number PCP/IAEC/005/2020, 08th May 2020).
Consent to participate This manuscript does not include any clinical study or patient data.
Consent of publication Consent has been asked where appropriate.
Competing interests The authors do not have any competing interests.
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