A novel approach to sustain delivery of leflunomide from floating gastro-retentive in situ gel for treatment of juvenile idiopathic arthritis: development, in-vitro and in-vivo evaluation

Abstract

This study aimed to develop and evaluate a floating oral in situ gel with leflunomide (LEF) as a liquid gastro-retentive drug delivery system to increase the residence time of the drug in the stomach and sustain drug delivery. Floating in situ gel system of leflunomide was prepared using sodium alginate as a gelling polymer, calcium carbonate as floating agent and different polymers to extended release. The physicochemical properties of in situ gels of leflunomide were in the acceptable range. An analysis of the release kinetics showed that the drug release followed a zero order, except for the formula containing hydroxypropylmethylcellulose (HPMC), which followed Higuchi’s model. Formula (F2) (0.4% HPMC K100M) was considered the best formulation as it had a minimum FLT (36 sec), optimum viscosity (230.5 cps), optimum drug release (97.34%) after 24 h, and the highest t₉₀ value (205.17 days). Therefore, formula (F2) was chosen for further ex-vivo and bioavailability studies. The pharmacokinetic parameters were compared to those of commercially tablets (Apetoid^®), and F2 exhibited a relative bioavailability of 197.73%. The prepared floating in situ gel system is a novel approach which indicate a remarkable increase in the residence time of LEF in the stomach and sustain its delivery.

Keywords:
Leflunomide; Gastro-retentive drug delivery system; Floating in situ gel.

INTRODUCTION

About 50,000 children in the United States alone suffer from juvenile idiopathic arthritis (JIA), a condition that causes joint inflammation and stiffness for longer than six weeks in children 16 years of age or under (Ferri, 2021). The joints become heated, swollen, red, and painful due to inflammation. Some types of JIA can cause serious complications, such as growth problems, joint damage, and eye inflammation. (Momah, 2019; Ringold et al., 2019).

leflunomide (LEF) may be utilized as an alternate treatment for JIA in patients with methotrexate intolerance or toxicity.

LEF, an isoxazole derivative, significantly inhibits joint deterioration and improves physical function while reducing the signs and symptoms of active rheumatoid arthritis in adults and adolescent onset arthritis in children. LEF is practically insoluble in water and is absorbed from the gastrointestinal tract at a high rate following oral administration but undergoing extensive first-pass metabolism. To solve these problems, maintain therapeutic plasma levels, and increase bioavailability, a gastro-retentive dosage form may be beneficial, allowing once-a-day administration of the drug with consequent improvement in patient compliance (Lodhi et al., 2012).

After oral administration, LEF, a prodrug, is rapidly metabolized to its major active form (teriflunomide) in the gut wall, plasma, and liver. The oral bioavailability of LEF is 60%, and the peak plasma levels of the active metabolite are reached at 6-12 h. The parent compound is rarely detected in the plasma. The active metabolite is responsible for all LEF in vivo activity (Halima et al., 2011).

Gastroretentive drug delivery systems (GRDDS) were developed as a novel approach for delivering many oral controlled-release drugs. These systems can be retained in the stomach long enough to completely release the active drug from a formulation into the gastric fluid (Bairagi, Gondkar, Saudagar, 2018). Their application comes with several advantages, including improved absorption of drugs and reduced fluctuation in drug blood levels. These benefits lead to increased therapeutic efficacy, minimized adverse effects, and the potential to enable stomach-specific activity (Khanam et al., 2017). The GRDDS can ensure controlled delivery of drugs for an extended period at the desired rate and absorption site. Suitable candidates for GRDDS include drugs with poor absorption in the distal gastrointestinal tract, a narrow absorption window in the proximal small intestine, and stability and/or solubility issues at alkaline intestinal pH (Vrettos, Roberts, Zhu, 2021).

An in situ forming polymeric delivery system has many advantages, such as ease of administration and reduced frequency of administration, improving patient compliance and comfort. The formation of in situ gel occurs because of one or more different stimuli such as pH change, temperature variation, and solvent exchange. GRDDS can increase the bioavailability of drugs compared to the conventional liquid dosage form (Bashir et al., 2019). Oral floating in situ gel dosage forms are increasingly being formulated using hydrophilic polymers, such as cellulose derivatives, alginate sodium, gellan gum, or Carbopol® (Swati et al., 2024).

The aim of this work is to use several polymers to produce a floating in situ gel with LEF that is unique to the stomach. By prolonging the drug’s residence length and extending its release over time, gastroretentive drug delivery of LEF in the form of oral in situ gel will decrease the frequency of dose while boosting bioavailability.

MATERIAL AND METHODS

LEF (Al Hekma Pharma, Egypt) was used with sodium alginate (Al Kahira Co., Egypt) as the gelling polymer. Calcium carbonate (El-Nasr Pharmaceutical Co., Egypt) was used as a gas-generating agent. Hydroxypropyl methylcellulose (HPMC K100M, Dow Chemical Co., USA), methylcellulose (Al Kahira Co., Egypt), carboxymethyl cellulose (EIPICO Pharma, Egypt), and Carbopol (CP934, Delta Pharma, Egypt) were selected as release-retarding polymers. Research-grade teriflunomide (99.9% purity) was purchased from Aventis Pharma Global Pharmaceutical Development (Germany). All other chemicals (El-Nasr Pharmaceutical Co., Egypt) used in this study were analytical grade. This article does not contain any studies with human subjects performed by any of the authors; all institutional and national guidelines for the care and use of laboratory animals were followed. Measurement of pharmacokinetic parameters of the treatments and ex-vivo studies conformed to guide lines of Institutional Animal Ethical of Faculty of Pharmacy, Al-Azhar University (under ethics approval protocol number: 266).

Drug and polymer compatibility studies

Fourier transform infrared spectroscopy (FTIR)

Using the potassium bromide disk method, FTIR spectra of drugs and drug-polymer physical combinations have been recorded using an FTIR spectrometer (Shimadzu, Model-8400 S, Japan). From 400 to 4000 cm-1, all spectra were collected using an empty pellet container as a reference (Padmapreetha, Arulkumaran, 2016).

Differential scanning calorimetry (DSC)

DSC (Shimadzu, Model DSC-50, Japan) was used to examine the drug’s thermal characteristics as well as the physical mixtures of the drug and polymer. Weighed samples were sealed in metal pans with flat bottoms and tight-fitting lids. In the presence of nitrogen at a flow rate of 25 mL/min, a scanning speed of 10°C/min from 25°C to 400°C was employed. A thermal analyzer fitted with computer software was used to obtain a thermogram across the temperature range. Pure indium was used as a reference for calibration of the device. Observing any variations in the drug’s melting point allowed for the identification of the materials’ compatibility (Nihal et al., 2017).

Preparation of floating in situ gel

The ideal concentrations of calcium carbonate and sodium alginate were 1.5% and 1% w/v, respectively, based on our earlier preliminary study (Esmaeil, Ramadan, El-Bakry, 2020). LEF floating in situ gel formulations were made with the ingredients listed in Table I. Using a heating magnetic stirrer (Thermolyne, Dubuque, IA, USA), distilled water was heated to 60°C and the measured amount of sodium alginate needed to form a 1.5% (w/v) solution was dissolved in about 75% of the deionized water.

TABLE I
Composition and characterization of prepared floating in situ gels containing LEF

Formula	polymer	Polymer conc. (% w\v)	pH	Invitro gelling capacity	Drug content (%)	Floating properties		Density (g/cm3)	Swelling index %	Gel strength (sec)	Viscosity(cps)
						*FLT Sec	*FD h
F1	HPMC	0.2	8.82±0.1	+++	98.3±3.05	23±14	>24	0.625±0.35	35.4±3.2	15.5±2.26	210.4±21
F2		0.4	9.02±0.2	+++	102±0.85	36±6	>24	0.670±0.23	41.5±4.5	27.60± 2.04	230.5± 34
F3		0.6	9.32±0.05	+++	100.3±1.9	47±4	>24	0.762±0.43	46. 9±2.7	35.76± 0.73	332.5±12
F4	MC	0.2	8.65±0.3	+++	101.5±1.36	27±6	>24	0.606±0.22	37. 8±1.6	32.80 ±1.56	216.7±37
F5		0.4	9.05±0.21	+++	99.5±2.17	33±14	>24	0.710±0.41	40.2±2.5	40.50 ±1.76	243.2±27
F6		0.6	9.15±0.13	+++	98.3±3.15	42±9	>24	0.790±0.26	45. 7±1.7	53.67±1.43	357±38
F7	CMC	0.2	9.3±0.34	+++	102±0.32	24±17	>24	0.723±0.13	47.2±4.1	38.50±0.83	233.4±20
F8		0.4	9.25±0.2	+++	101.2±0.17	28±11	>24	0.705±0.16	51. 3±2.4	43.67±3.01	286.3±26
F9		0.6	9.12±0.06	+++	98.8±0.81	37±8	>24	0.785±0.26	56. 9±6.2	52.66±2.02	303.5±29
F10	CP	0.05	8.61±0.12	+++	99.5±0.75	60±24	>24	0.810±0.02	70.2±2.8	45.24± 2.7	450.2±35
F11		0.075	8.41±0.2	+++	102.3±1.2	62±12	>24	0.950±0.03	75. 3±3.5	59.75±1.06	513.4±42
F12		0.1	8.59±0.26	+++	101.1±0.05	65±7	>24	1.03±0.02	80. 9±2.7	71 ±1.55	600±48

• ^*
  All formulations contain 400mg LEF, 1.5%w/v sodium alginate, 1% w/v calcium carbonate and 0.25%w/v sodium citrate.
• ^*
  FLT: floating lag time, FD: floating duration

The medication LEF (400 mg), the proper amounts of polymer (HPMC K100M, MC, CMC, or CP934), and a gas-generating agent (calcium carbonate, 1% w/v) were dissolved/dispersed uniformly into the sodium alginate solution with constant stirring after the mixture had cooled to below 40°C. Following full addition, the mixture was stirred continuously until a homogenous dispersion was achieved, at which point it was allowed to cool to room temperature. The final preparation was created by adding deionized water to 100% of the volume and thoroughly mixing it. It was then kept in amber bottles until needed (Panwar et al., 2012).

Characterization of floating LEF in situ gel formulations

pH, drug content, density, in vitro gelation, floating, viscosity, and gel strength were evaluated for each prepared LEF in situ gel formulation. The in vitro release data of LEF from various in situ gel formulations was also subjected to kinetic analysis.

Measurement of pH

A pH meter (410A, ORION) was used for measuring the pH of the prepared formulations. The readings were recorded in triplicate for each formulation, and

the averages of the values were considered (Parthiban, Senthilkumar, Vikneswari, 2013; Kim et al., 2022).

Determination of drug content uniformity

Precisely measured, 5 mL of in situ gel (equivalent to 20 mg of LEF) was transferred to a 1000 mL volumetric flask, to which 900 mL of 0.1N hydrochloric acid (HCl) was added. The mixture was then stirred for 3 hours using a magnetic stirrer (Thermolyne, Dubuque, IA, USA), and then sonicated for 15 minutes using a (UP400S, Hielscher Ultrasonics, Germany). Once the contents had been completely dispersed, 1 mL of this solution was diluted to 10 mL with 0.1N HCl and filtered. Using a UV spectrophotometer (UV-1601, Shimadzu, Japan), the contents of LEF were measured spectrophotometrically at λmax 260 nm. The drug content measurements were made three times, and the average values were computed (Bobade, Shrikant, 2016).

Determination of density

The water displacement method was used to determine the density of the formulation. The mass and volume of the gels for each formulation were calculated in order to determine the densities of the formulations. The polymeric solution converted into a stiff gel when it contacted an acidic buffer. The gels were placed in a measuring cylinder and their volume was measured once their mass was recorded with a weighing balance (Basu et al., 2021). The density was determined by calculating the gel’s weight and volume. For every formulation, this procedure was used.

In vitro gelation study

1 mL of the formulation was pipetted into a 15-mL test tube after 5 mL of simulated stomach fluid (0.1N HCl, pH 1.2) was added and kept at 37°C. Gently releasing the formulation, the pipette was positioned such that it faced the fluid’s surface in the test tube. The composition rapidly transformed into a gel-like structure upon contact with the gelation medium. It was determined that in vitro gelling capability was based on the gel’s rigidity and how long it stayed that way (Antony, Nair, 2020). Based on both the quick gelation time and the prolonged duration that the gel stayed formed, three groups were identified for in vitro gelling capacity:

(+) = gels within 5 min, dispersed within 4 h,

(++) = gels within 60 s and retains gel structure for up to 8 h,

(+++) = gels immediately and retains gel structure for up to 12 h.

In vitro floating study

In a beaker filled with 100 mL of 0.1N HCl (pH 1.2) at 37°C and moderate agitation, 10 mL of the formulation was added to perform the in vitro floating research. Estimates were made for variables such as the floating lag time (FLT) and floating time, which measure how long the produced gel remained continually floating over the dissolving medium (Patil et al., 2023).

Swelling index (Water uptake by the gel)

The formulation was added to a 40 ml solution of 0.1 N hydrochloric acid with a pH of 1.2. The formulation solution was transformed into a gel using a thermostatically controlled water bath. The resulting gel was then separated from the buffer solution using Whatman filter paper, followed by blotting to remove any excess buffer. The initial weight of the gel was determined, and 10 ml of distilled water was subsequently added to the gel. Water was decanted after 12 hours and the weight of the gel was recorded. This process allowed for the calculation of the change in weight over time (Shastri et al., 2016; Kim et al., 2022; Akshay, Shabaraya, Deekshitha, 2021).

The difference in the weight was calculated and reported as follows:

Water uptake % = final weight - initial weight/ initial weight × 100

Measurement of viscosity

The viscosities of the different formulations were analyzed using Brookfield digital viscometer DV-II+Pro (Brookfield Engineering Laboratories, Middleboro, MA, USA). Twenty milliliters of the formulated solution was taken in a beaker. The T-bar spindle was dropped upright in the center of the beaker containing samples, taking care that the spindle did not touch the bottom of the jar. Viscosities were determined at 50 rpm. The temperature was maintained during the process. The average of three readings was considered for each measurement (Dipal, Kanu, Mukesh, 2016; Shivsharan, Hosmani, Thorat, 2022).

Measurement of gel strength

A 100-mL graduated cylinder was filled with a 50-g sample of the gel produced in 0.1N HCl (pH 1.2). On the middle of the gel’s surface, a 35-g weight was added, and it was let to pass through the gel. For every formulation, the weight’s time to travel 5 cm through the gel was recorded. Every new formulation was subjected to the the same procedure in triplicate, and the mean time was ascertained (Worrawee et al., 2017; Jahnabi, Ananta, 2020).

In vitro release studies

Drug release tests were carried out at 37°C and 50 rpm in a USP type II dissolution test apparatus (DA6D, Bombay, India). This speed maintained the mild agitation conditions thought to exist in vivo while being slow enough not to rupture gelled compositions. 900 mL of 0.1N HCl buffer solution (pH 1.2) used as the dissolving media. Using pipette, 5 mL of in situ gel solution (equivalent to 20 mg of LEF) was added into the dissolution medium without disturbing the medium. Aliquots of 5 mL were withdrawn at predetermined time intervals. The samples were replaced immediately with an equal volume of fresh buffer. Using a spectrophotometer (UV-1601, Shimadzu, Japan) and a blank of 0.1N HCl solution, the samples were filtered and subjected to spectrophotometric analysis at λmax 260 nm. The findings were displayed as the percentage of cumulative LEF released against each matching time, and the measurements were carried out in triplicate (Khadka, Anil Kumar, Junu, 2017; Naveen et al., 2022).

Kinetics analysis of drug release data

Using PCP Disso Version 2.08 software, the dissolution profiles of all investigated formulations were fitted to zero order, first order and Higuchi diffusion to determine the kinetic modeling of drug release. The best fitted model was the one which gave the highest R² value and least slope value. (Darekar et al., 2016; Shabaraya, T Ashwini, Vineetha, 2023).

The following kinetic models were used to evaluate the in vitro drug release: zero order (Eq. 1), first order (Eq.2), Higuchi (Eq. 3), and Korsmeyer-Peppas (Eq. 4).

(1) $$Q_t=Q_0+k_{0}t$$

(2) $$\log Q_t=\log Q_0-k_{1}t/2.303$$

(3) $$Qt=K_H{t}^{1/2}$$

(4) $$M_t/M_{\mathrm{\infty }}=k{t}^n$$

where: Qt is the amount of drug dissolved over time t, Q₀ is the initial amount of drug in solution (equal to zero), k₀ is the zero order release constant (expressed as concentration/time), the constant k₁ is first order (expressed in time units, h^-1), k t^1/2 is the dissolution constant of the Higuchi equation (expressed in time units, h^1/2), Mt is the amount of drug released at time t, M∞ is the total amount of drug released at infinite time, k is the release rate constant (expressed as h^-n) and n is the release exponent which indicates the type of release mechanism.

The release exponent, “n,” values obtained from the Korsmeyer-Peppas power law equation was used to establish the mechanism of drug release from the gels. Indicating the release mechanism is the value of “n”. A release of zero order, or case I transport, occurs when “n” is equal to one; a Super Case transport occurs when “n” is greater than one. When “n” equals 0.5, Fickian diffusion provides the most compelling explanation for the release; however, when 0.5 < “n” < 1, anomalous or non-Fickian diffusion accounts for the release (Guerra-Ponce et al.,2016)

Stability studies

According to ICH recommendations, a stability study was carried out for one month at three different temperatures: room temperature of 25°C ± 2°C/60% ± 5% relative humidity, refrigerator temperature of 5°C ± 2°C, and high temperature of 40°C ± 2°C/75% ± 5% relative humidity. To identify the in situ gel formulations that showed the best properties, an appropriate amount of each formula was prepared, tightly sealed in an amber bottle with an aluminum cap as a closure, and stored at various temperatures (in a refrigerator, room, and thermostatically controlled hot air oven) for one month (Sindhoor, Sneh, Amala, 2018; Patel et al., 2022).

The drug content (measured spectrophotometrically at λmax 260 nm using UV spectrophotometry and 0.1N HCl solution as blank) of these formulations, as well as their pH, FLT (s), floating duration (h), and viscosity, were examined both before and after storage. Every experiment was carried out three times, and the mean ± standard deviation was used to describe the outcomes.

Ex vivo gelation study

The best formula chosen from previous studies (F2) was subjected to an ex vivo gelation study to check in situ gel formation in the stomach of rats. Randomly, we divided nine male albino rats weighing 200-220 g into three groups (three animals per group). Group 1 served as a control, and groups 2 and 3 were treated. All animals were fasted for 24 h prior to the experiment but were allowed free access to water. Using oral feeding tube and syringe, in situ gel formula was feeded to the rats, amount of drug administered orally to test groups was equivalent to 2 mg/kg. Control animals in group 1 were administered normal saline orally and euthanized. The stomach was removed and incised along the greater curvature. Rats in groups 2 and 3 were orally administered the selected in situ gel formulation. After 1 h, group 2 was euthanized, and after 6 h, group 3 was euthanized. Stomachs from these rats were removed and incised along the greater curvature to observe if gel had formed (Ahmed, Kapoor, Adinarayana, 2017).

Pharmacokinetic study

Pharmacokinetic evaluation of floating in situ gel

Twelve male albino rats, weighing between 200 and 220 grams, were split into two groups at random and given the following care:

Treatment (I): using commercial tablet containing 20 mg LEF (Apetoid^®) in suspension form.

Treatment (II): 400 mg LEF, 1.5% sodium alginate, 1% calcium carbonate, and 0.4% HPMC are combined in formula (F2) floating in situ gel. The treatments’ pharmacokinetic parameter measurements were conducted in accordance with Al-Azhar University’s Institutional Animal Ethical Committee rules, using ethical approval protocol number 266.

Rats were given a 24-hour fast before receiving each treatment. Using oral feeding tube and syringe, a single oral dose of a commercial tablet containing LEF (LEF equivalent to 2 mg/kg in suspension form) was given to the rats in the treatment (I) group. Using oral feeding tube and syringe, rats in the treatment (II) group were given a single oral dose of LEF floating in situ gel (F2) (LEF corresponding to 2 mg/kg). Rats’ retro-orbital veins were used to draw blood samples (100 µL) by using capillary tubes into heparinized tubes at 0 (predose), 1, 2, 4, 6, 8, 12, and 24 hours after treatment (Sharma et al., 2014). Samples of blood were immediately centrifuged for 20 minutes at 3000 rpm. Using a micropipette, the plasma was separated into screw-capped tubes and stored at -20°C until analysis. To find the active metabolite of LEF (teriflunomide) in the rat plasma, a conventional HPLC technique was employed as a quick, accurate, and selective approach (Chan, Charles, Tett, 2004). A validated assay method that employed chromatographic conditions with a simple protein precipitation step and HPLC analysis with ultraviolet (UV) detection was used to conduct the analysis. The isocratic mobile phase, consisting of 35% acetonitrile-acetate buffer, was produced by mixing 350 mL of acetonitrile with 650 mL of 0.05 M sodium acetate buffer. The mixture was then degassed and filtered through 0.45-mm pores while under negative pressure. The pH of the mixture was then adjusted using glacial acetic acid. 1.5 mL/min was the column flow rate, and 305 nm was the detector’s setting. Pharmacokinetics were computed with Microsoft Excel 2010 and chromatography was carried out at room temperature. The chromatography was carried out with an HPLC system that included an SPD-10A UV-Vis detector and an LC-10AD pump (Shimadzu, Kyoto, Japan) with a SIL-10AXL autoinjector that were both purchased from the same company. Via a CBM-10A controller (Shimadzu, Kyoto, Japan), the system was managed using Class-LC10 software. A Waters Nova-Pak C18 Sentry guard column (4 _m, 3.9 mm i.d. × 20 mm) and an in-line high-pressure column prefilter SSI (2 _m, 1.5 mm) (Alltech, Deerfield, IL, USA) in front of the guard column protected the Nova-Pak C18 column (4 _m spherical particles, pore size 60 Å, 3.9 mm i.d. × 150 mm) used for the chromatographic separation.

Using Microsoft Excel 2010, the pharmacokinetic parameters (Cmax, Tmax, Kel, t1/2, AUC0-24, AUC0-∞, and MRT) and relative bioavailability were calculated using the following equations:

MRT (mean residence time) = AUMC_0-24/AUC_0-24,

AUC_0-∞ (area under the curve from 0 to ∞) = AUC_0-t + C_t/K_el,

Vd = volume of distribution = [(dose in mg) × K_ab)] / [antilog a of el × (K_ab - K_el)],

TCR (total clearance rate) = K_el × Vd,

% relative bioavailability = [AUC (0-end) for test/ dose]/ [AUC (0-end) for standard/dose] × 100.

Statistical data analysis of pharmacokinetic parameters

All pharmacokinetic parameter values are shown as the mean ± standard deviation, with a sample size of (n) = 6 unless otherwise specified. A statistical analysis program called Minitab 17 was utilized. Using two-tailed, unpaired Student t-tests, a statistical comparison of the outcomes was performed. P < 0.05 was the threshold for statistical significance.

RESULTS AND DISCUSSION

Drug and polymer compatibility studies

Using DSC and Fourier transform infrared spectroscopy (FTIR), preformulation investigations were carried out to investigate the interactions between the medication and polymer. Based on these findings, interactions between the medication and the polymer were not happening.

FTIR

The LEF alone FTIR spectrum (Figure 1a) shows two distinct features: a sharp peak at 3356 cm-1, which is due to a N-H stretching band of a secondary amine group, and another sharp peak at 3066 cm-1, which is related to a C-H stretching vibration of a benzene ring. A sharp peak at 1693 cm-1 is observed at low frequencies and is ascribed to an HC=N-O group within an isoxazole ring. Furthermore, amide’s C=O is related to a sharp peak at 1604 cm-1, while C=C is attributed to a peak at 1539 cm-1. These findings corresponded with those of Nihal et al. (2017) and Padmapreetha and Arulkumaran., (2016).

FIGURE 1
(a) FTIR spectra of LEF and LEF- Polymer physical mixture, (b) DSC thermograms of LEF and LEF- Polymer physical mixture.

When the drug and several polymers were physically mixed, the distinctive peaks of the drug were seen at the same location, indicating that LEF and the polymers were compatible. Figure 1a displays the corresponding FTIR spectra.

DSC

The DSC thermogram of LEF alone exhibits a distinct endothermic peak at 166.06°C, which is the drug’s melting point and indicates that it is crystalline (Figure 1b). This outcome is consistent with earlier research on the thermal analysis of LEF, as reported by Nihal et al. (2017) and Krishnan et al. (2018). In Figure 1b, comparative DSC thermograms of LEF combined with various polymers are displayed. There is no evidence of any chemical or physical interaction between the drug and the polymers utilized, as seen by the endothermic peak of the drug being well-retained at the same location.

Characterization of floating LEF in situ gel formulations

The characterization tests of gastroretentive in situ gels were significantly influenced by both the type and concentration of the release-retarding polymers used (HPMC, MC, CMC, Carbopol). These results may be as these polymers vary in molecular weight, solubility, and how they interact with ions or pH, which uniquely affects the gel’s performance depending on both their type and concentration These factors directly affect the physicochemical properties of the gel, such as viscosity, gelation capacity, buoyancy, drug release rate, and water uptake, thereby altering the results of characterization tests. (Hala, Yehia, 2019).

The pH of prepared in situ gels

All in situ gel compositions had their pH values evaluated (Table I). The formulations exhibited pH values within the permissible range of 8.41 ± 0.2 to 9.32 ± 0.05. This pH range is considered orally acceptable, indicating that the administration of the formulations will not cause any irritation to the oral cavity. Furthermore, at room temperature, the solutions showed a free-flowing consistency and no signs of gelation. This characteristic suggests that the formulations remain liquid until they come into contact with the gastric fluid in the stomach. The pH range of 4 to 10 is optimal for the stability of aqueous sodium alginate solutions. The alginate solution precipitates alginic acid below pH 3, resulting in a formulation with gel and liquid phases (Temesgen, Belete, Gebre-Mariam, 2016).

Drug content uniformity

All in situ gel formulations had their actual drug content determined (Table I). With standard deviations ranging from 98.3 ± 3.05 to 102.3 ± 1.2, all produced formulations displayed drug content levels that were considered acceptable. These outcomes met the pharmacopeial standards for content consistency.

Density

Floating systems should have a density of less than 1.004 g/cm3, which is less than that of stomach contents. Each formulation’s density satisfied this condition, guaranteeing its buoyancy. In accordance with Monica and Swapnil (2015) and Sindhoor, Sneh and Amala (2018), formulation densities varied from 0.606 ± 0.22 to 1.03 ± 0.02 g/cm3. The results are shown in Table I. The results showed that the densities of the formulations were lower than those of the gastric contents, confirming their ability to float in the stomach. This characteristic facilitates the desired prolonged gastric retention of the formulations, allowing for sustained drug release and improved therapeutic efficacy.

In vitro gelling capacity

The gelation investigation was carried out at pH 1.2 in 0.1N HCl. An ordinal scale with a range of + to +++ was used to evaluate the formulations’ gelation characteristics (Table I). Every formulation that came into contact with the gelation medium experienced a sol-gel transition, and the integrity of the resulting gel was maintained. Findings are displayed in Figure 2.

FIGURE 2
In vitro gelling capacity of in situ gel formulations F1 to F12.

When insoluble calcium carbonate comes into contact with an acidic media, calcium ions and carbon dioxide are released, causing gelation. Instantaneous gelation is produced by the interaction between the calcium ions and the anionic polymer (sodium alginate) in the formulation, creating a gel barrier that limits the release of the medication (Subhashis et al., 2011).

In vitro floating study

In 0.1N HCl (pH 1.2), the floating qualities of the produced formulations were estimated. The duration of floating (the amount of time the formulation floated continuously on the surface of the dissolving medium) and the time needed for the formulation to rise to the medium surface (FLT) were calculated (Table I). In less than a minute, every in-situ gel formulation floated on the medium, and they continued to do so for up to 24 hours. The FLT minimum was displayed by formula (F1), while the FLT maximum was displayed by formula (F12) (65 s). An increase in the concentration of the polymer led to a rise in both the FLT and the floating time of the preparations (Singh, Kim, 2000).

Swelling index (Water uptake by the gel)

The drug’s release from the polymer matrix is significantly influenced by the water content of the drug delivery system. Drug release is mostly caused by water penetrating into the matrix and the drug being released concurrently by dissolution or diffusion. Table I displays the percentage of water taken by each formulation. The in-situ gel formulations exhibited a swelling index that varied between 35.4% ± 3.2% and 80.9% ± 2.7%. The results of formula (F1) indicated the lowest water uptake (35.4% ± 3.2%), whereas formula (F12) displayed the maximum water uptake (80.9%±2.7%).

The higher water uptake observed in F12 can be attributed to the maximum swelling ability of the polymer in this formulation. Gel’s ability to absorb water was directly correlated with the concentration of polymers, and the amount of water absorbed affected the gel’s ability to release drugs (Jyotsana, Bhushan, Kamal, 2015; Akshay, Shabaraya, Deekshitha, 2021).

Viscosity

When administering formulations orally, the viscosity of the gel-forming processes important. The formulations should not be too viscous to pour from the container (Sivannarayana et al., 2013). Table I shows the range of viscosity values for LEF formulations: 210.4 ± 21 (F1) to 600 ± 48 cp (F12). The order of viscosity obtained indicated an increase in viscosity as the concentration of the release-retarding polymer increases. This can be attributed to the increased cross-linking of the polymer, resulting in a higher viscosity. These findings highlight the importance of polymer concentration in determining the viscosity of the formulations. By adjusting the polymer concentration, it is possible to control the viscosity and ensure that it falls within the optimum range for easy administration by the patient. Formulations containing Carbopol 934 had viscosities that were much higher than those of the other cellulose-based formulations. This phenomenon could be explained by the fact that cellulose derivatives are more hygroscopic than carbopol, as well as the thickening and intrinsic swelling properties of carbopol 934 (Wael, Widad, Mohammed, 2018). With the exception of formulas (F11) and (F12), which had viscosity values of 513.4 and 600 cp, respectively, and were difficult to pour, all formulations’ viscosity results showed that they were pourable straight from the container and would be easy to swallow.

Gel strength

Gel strength is an important parameter that reflects the ability of a gelled mass to withstand peristaltic movement in vivo. It indicates the strength and integrity of the gel.

The gel strength of LEF in situ gels is displayed in Table I. Good gel strength was found in all formulations; the range was 15.5 ± 2.26 S for (F1) to 71 ± 1.55 S for (F12). This higher gel strength can be attributed to the higher concentration of different polymers in these formulations (Jahnabi, Ananta, 2020).

In vitro drug release studies

For a duration of 24 hours, the in vitro release analysis of LEF from every in situ gel formulation was carried out in 0.1N HCl (pH 1.2). The results are displayed in Figure 3. In general, the percentage of LEF release decreased as the polymer concentration increased. Both the density of the polymer matrix and the lengthening of the drug molecules’ diffusional path may be responsible for this impact (Monica, Swapnil, 2015).

FIGURE 3
In-vitro release profile of LEF oral floating in situ gel formulations containing different release retarding polymer.

It was found that formula (F1) (HPMC 0.2%) had the highest drug release (100%) and formula (F12) (0.1% Carbopol) had the lowest drug release (81.15%).There was a high release phase (burst effect) at the beginning of the drug’s release, which was followed by a moderate release phase. The medication that was at the gel’s surface and may have released its contents instantly upon coming into touch with 0.1N HCl can be responsible for the burst release. Furthermore, lag time is necessary for the cross-linking of sodium alginate’s glucuronate residues and the release of Ca²⁺ ions from calcium carbonate, both of which are critical processes in the formation of barrier gel (Anyanwu, Adogo, Ajide, 2017).

When using different polymers, the percentage of drug release varied and was as follows: HPMC K100M > MC > CMC > CP 934. This order might be attributed to the viscosity of Carbopol, which is higher than other polymers and encourages the production of extremely viscous gels when in contact with aqueous fluids, hence delaying the rate of drug release more than other polymers.

In vitro release kinetic parameters of LEF in situ gels

Table II shows the calculated correlation coefficient for the in vitro release of LEF employing different kinetic orders or systems.

TABLE II
In-vitro release kinetic data of LEF floating in situ gel formulations

Formula code	Regression value (R2)			Order of drug release	Parameter’s for peppas equation			Mechanism of drug release
	Zero	First	Higuchi		N	K	R2
F1	0.993	-0.987	0.998	Higuchi	0.3934	0.0687	0.9972	Fickian
F2	0.982	-0.996	0.998	Higuchi	0.4572	0.0449	0.9934	Fickian
Zero	First	Higuchi	N	K	R2
F3	0.989	-0.992	0.993	Higuchi	0.4530	0.0305	0.9794	Fickian
F4	0.988	-0.957	0.962	Zero	0.5151	0.0388	0.9942	Non Fickian
F5	0.994	-0.982	0.978	Zero	0.5289	0.0464	0.9873	Non Fickian
F6	0.999	-0.987	0.987	Zero	0.5803	0.0171	0.9877	Non Fickian
F7	0.993	-0.967	0.974	Zero	0.6586	0.0102	0.9813	Non Fickian
F8	0.982	-0.943	0.946	Zero	0.5371	0.0194	0.9849	Non Fickian
F9	0.992	-0.962	0.963	Zero	0.6680	0.0083	0.9705	Non Fickian
F10	0.999	-0.986	0.991	Zero	0.5786	0.0148	0.9918	Non Fickian
F11	0.998	-0.994	0.987	Zero	0.6434	0.0093	0.9880	Non Fickian
F12	0.998	-0.993	0.988	Zero	0.7290	0.0050	0.9923	Non Fickian

The correlation coefficient (r²) indicates that, with the exception of formulas (F1), (F2), and (F3), which showed Higuchi’s diffusion model, the in vitro release of LEF from various in situ gels followed a zero-order model suggesting the release rate was time independent.

The in vitro release of LEF from various in situ gel formulations was seen to exhibit kinetic treatments with the value of the release exponent n in the Korsmeyer-Peppas model approached a value (greater than 0.5), which corresponds to non-Fickian diffusion transport mechanism. This suggests that the drug release mechanism could involve a combination of diffusion and chain relaxation mechanisms. Consequently, the swelling of the polymer, drug diffusion through the polymer, and gradual erosion of the polymer regulate the release of the drug from the formed gels, with the exception of (F1), (F2), and (F3), which have “n” values (less than 0.5) showing Fickian diffusion transport which indicates a rapid initial release, followed by a decrease in release owing to a chemical potential gradient. These findings are in agreement with previous reports that have shown that poorly soluble drugs are released mainly by attrition mechanisms from hydrophilic and swellable polymeric matrices (Guerra-Ponce et al., 2016)

Stability studies

Drug content

The percentage of drug remaining after a month of storage at 5°C ± 2°C and 40°C ± 2°C/75% ± 5% RH is displayed in Figure 4. For (F1), (F2), (F4), and (F7), the percentages of drug that remained after 30 days at 5°C ± 2°C were 100.22%, 99.06%, 99.97%, and 98.45%, respectively. For (F1), (F2), (F4), and (F7), the percentages of medicines that remained after 30 days at 40°C ± 2°C were 99.75%, 98.75%, 99.65%, and 98.18%, respectively. Based on the correlation coefficient (r), the degradation reaction was zero-order.

FIGURE 4
Percent LEF remained from floating in situ gel formulations at a) 5 ˚C and b) 40 ˚C.

The values of Ea, estimated K₂₀, t₉₀, and t_1/2 at 20°C for each of the examined LEF in situ gel formulations are shown in Table III.

TABLE III
Kinetic data for LEF in situ gels based on accelerated stability testing according to zero order

Formula No.	Ea cal/mol.	Calculated K (day)-1 20	t1/2 (days)	t90 (days)
F1	1320.4640	0.0503	994.4133	198.8827
F2	988.3041	0.0492	1015.8505	205.1701
F4	795.7982	0.0505	989.7542	197.9508
F7	556.6084	0.0606	824.4692	164.8938

Physical evaluation

Table IV displays the physical properties of the stored LEF in situ gels, which showed slight changes in pH, viscosity, and in vitro floating experiments, all within allowed limits. We selected the (F2) formula to perform further ex vivo and in vivo tests because it displayed the highest t90 value (205.17 days).

TABLE IV
Various physical characteristics of LEF floating In situ gel after stability study for one month at 5±2 and 40 ±2˚C with 75% RH

Stability Duration	pH			Viscosity(cp)			Floating lag time Second
	At zero day	After 30days at 5˚C	After 30days at 40˚C	At zero day	After 30 days at 5˚C	After 30 days at 5˚C	At zero day	After 30 days at 5˚C	After 30days at 40˚C
F1	8.82±0.1	8.88±0.33	8.93±0.1	210.4±21	245.3±102	290±53	23±14	28±3	29±4
F2	9.02±0.2	9.15±0.11	8.93±0.5	230.5±34	253.2±78	310.5±79	36±6	42±8	43±7
F4	8.65±0.3	8.42±0.21	8.52±0.2	216.7±37	243.4±38	305.4±92	27±6	37±10	37±3
F7	9.3±0.34	9.18±0.24	9.38±0.4	233.4±20	256±88	275.2±45	24±17	30±5	31±8

• ^*
  The measurements were carried out in triplicates
• ^*
  All values are Mean ± SD

Ex vivo gelation study

To investigate gel production in the stomach, an ex vivo investigation with rats was carried out. Figure 5a represents stomach mucosal layer of control group 1 rats. The findings showed that after one hour, gel developed in the stomachs of the group 2 rats (Figure 5b). For more than six hours, the developed gel stayed on the stomach mucosal layer in rats in group 3 (Figure 5c).

FIGURE 5
Ex-vivo examination study for stomach of rat, a: group 1 serve as control, b: group 2 serve as treated group scarified after 1hr, c: group 3 serve as treated group scarified after 6h from oral administration of LEF in situ gel.

Pharmacokinetic study

To measure the plasma concentration of LEF following oral administration of an in-situ gel formula (F2) and a commercial LEF tablet (Apetoid ®), in vivo studies were carried out. Table V and Figure 6 show the different pharmacokinetic parameters as well as the plasma concentration - time profile of the commercial tablet and (F2) floating in situ gel. Due to the drug’s gradual release from the formulations, the test formula’s rise of Tmax showed delayed drug absorption. AUC0-24 increases are indicative of better bioavailability.

TABLE V
Pharmacokinetic parameters of Teriflunomide estimated after oral administration of treatment I (commercial tablet) and oral administration of treatment II (F2 floating in situ gel) to six rats and Statistical analysis of Pharmacokinetics parameters

Parameter	Commercial tablet	F2 in situ gel	P value
Dose(mg/kg)	2±0.000	2±0.000	--------
Cmax(µg/ml)	3.263±0.314	4.353±0.235	<0.0003
Tmax(hr)	4±0.000	6±0.000	<0.0001
Kel(hr ) -1	0.136±0.047	0.105±0.011	---------
t1/2el (hr)	5.487±1.461	6.975±0.636	-------
AUC0-24 (µg.hr/ml)	27.764±2.432	54.621±2.046	<0.0001
AUC0-∞ (µg.hr/ml)	29.332±3.370	60.771±3.601	<0.0001
MRT(hr)	7.851±1.134	10.120±0.554	-------
RB%	-	196.733	-------

• ^*
  All values are Mean ± SD, P-value (< 0.05) = significant difference

FIGURE 6
Teriflunomide mean plasma concentration-time curve after oral administration of commercial tablet (Treatment I) and oral administration of treatment II (F2 floating in situ gel) to six rats.

Peak plasma concentration (Cmax) for the commercial pill was 3.26 μg/mL at 4 h (Tmax), and the area under the curve (AUC0-24) was 27.764 μg/h/mL.

For the controlled-release floating oral in situ gel, the C_max was 4.353 μg/mL at 6 h (T_max). The AUC_0-24 was 54.62 μg/h/mL, which is significantly higher than that of the commercial tablet. The elimination rate constant for LEF in the in situ gelling systems was found to be 0.105/h. The relative bioavailability of LEF was 196.73%. The improved bioavailability of LEF could be attributed mainly to the increase in residence time of the drug at the absorption site.

Statistical data analysis of pharmacokinetic parameters

Tmax, AUC (0-24), AUC (0-∞), and Cmax value significantly improved when the bioavailability characteristics of the (F2) in situ gel were statistically analyzed in comparison to the commercial tablet (Table V).

CONCLUSION

The goal of the current study was to improve the oral bioavailability and offer sustained drug release for LEF, an oral medication, by creating a gastro-retentive in situ gel. The developed formulations satisfied every requirement needed to prepare a gastro-retentive in situ gel system that instantly gelled and floated in the stomach’s pH range. Therefore, in comparison to traditional formulations for the treatment of JIA, stomach-specific in situ forming gel with LEF can be regarded as a promising effective formulation with better efficacy, longer drug release, reduced side effects, enhanced patient compliance, and cost-effectiveness.

• FUNDING
  This research follows Al- Azhar University and the funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian knowledge bank (EKB). The authors asseverate that no assets, grants, or other support were gained during the preparation of this manuscript.

ACKNOWLEDGEMENTS

The authors would like to give a privilege to all the members of Pharmaceutics and Pharmaceutical Technology Department, Faculty of Pharmacy (Girls), Al-Azhar University, for providing excellent facilities and deep support for carrying out the research work. And editing, and supervision.

HUMAN AND ANIMAL RIGHTS

This article does not contain any studies with human subjects performed by any of the authors; all institutional and national guidelines for the care and use of laboratory animals were followed. Measurement of pharmacokinetic parameters of the treatments and ex-vivo studies conformed to guide lines of Institutional Animal Ethical of Faculty of Pharmacy, Al-Azhar University (under ethics approval protocol number: 266).

DECLARATION OF GENERATIVE AI AND AI ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

The authors declare that they do not use generative AI and AI Assisted technologies in the Writing Process.

Data Availability:

The datasets produced and /or analyzed during the present work are available from the corresponding author on reasonable request.

DATA AVAILABILITY STATEMENT

Use of data not disclosed.

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Edited by

• Associate Editor:
  Marlus Chorilli

Publication Dates

• Publication in this collection
  31 Aug 2026
• Date of issue
  2026

History

• Received
  13 Jan 2025
• Accepted
  09 June 2025