Materials

Febuxostat was gifted by Zydus Healthcare Ltd., Ahmedabad. Crospovidone was obtained from ACS chemicals, Ahmedabad. Ethylcellulose was obtained from Ultrapure lab chem, India. Hydroxy propyl methyl cellulose (HPMC) K15M was purchased from Otto Chemie Pvt. Ltd., Mumbai. Soluplus was purchased from BASF Pvt. Ltd., Mumbai. Aloe vera powder was purchased from Neelkanth Finechem, Jodhpur. Guar gum was obtained from H.B gum industries Pvt. Ltd., Kalol.

Solubility enhancement of febuxostatPreparation of solid dispersion

Solid dispersion was prepared by physical mixing. The powders were passed through a 44# sieve and weighed quantity of drug and soluplus was taken in a glass mortar pestle in different ratios (1:1, 1:2, and 1:3). The powder mixtures were triturated for half an hour [10]. The samples were stored in a screw-capped glass vial until use.

Phase solubility study

According to Higuchi and Connors, the phase solubility study of febuxostat was performed [11]. A surplus of febuxostat/soluplus dispersion was added to acetate buffer with a pH of 4.5 and phosphate buffer with a pH of 6.8. The solution was shaken for 24 h at 100 rpm in an orbital shaker until equilibrium was reached. After filtering the sample, absorbance was measured at λmax (314 nm) against a suitable blank solution using an ultraviolet–visible (UV–VIS) spectrophotometer. The following equation determined the thermodynamic parameter of Gibbs free energy (ΔG°).

$$\Delta G^\circ = - 2.303RT\log \left( }}}}}}} \right)$$

where ΔG° = Gibbs free energy of transfer; R = Gas constant (8.314 J/K-mole); T = Temperature in kelvin; Sc/So = molar solubility ratio of febuxostat/soluplus dispersion [12].

Evaluation of solid dispersionMicromeritic properties of solid dispersion

The flow properties of solid dispersion are important in the pharmaceutical industry, especially in the blending of powders, compression of tablets, and the filling of capsules. Several parameters were used to measure the flow properties of the prepared solid dispersion, such as the bulk density, the tapped density, the angle of repose, Carr’s index, and Hausner’s ratio.

1.

Bulk density

It is the mass-to-bulk volume ratio. Bulk density may influence dissolution and other properties; it is dependent on particulate size, shape, and adhesion tendency. By placing a known mass in a graduated 10 ml measuring cylinder, the bulk density was determined. The cylinder was lowered three times at a rate of two seconds from a height of one inch. The following equation calculated the bulk density,

where ρb = Bulk density; M = Mass of powder; Vb = Bulk volume of powder.

2.

Tapped density

The measurement of tapped density is employed in order to ascertain the packing arrangement and flow characteristics. The tapped density refers to the measurement of the volume of a powder sample obtained by the process of tapping, which involves utilizing a measuring cylinder to contain the predetermined amount of powder. The following equation calculated the tapped density,

where ρt = Tapped density; M = Mass of powder; Vt = Tapped volume of powder.

3.

Carr’s index

The Carr's index is calculated as the percentage of powder compressibility, which is derived from the comparison between bulk density and tapped density. The following equation was used,

$$}^}\;\;}\left( \% \right) = [(\rho_}} - \rho_}} )/\rho_}} ] \, \times 00$$

where ρt = Taped density; ρb = Bulk density.

4.

Hausner’s ratio

The Hausner's ratio serves as a quantitative measure for evaluating the flowability of powders. The ratio being referred to is the relationship between tapped density and bulk density.

$$}^}\;\;}\left( \% \right) = \rho_}} /\rho_}}$$

where ρt = Tapped density; ρb = Bulk density.

5.

Angle of repose

The angle of repose is defined as the possibility of the maximum angle between the surface of the pile and the horizontal plane. The fixed funnel method was used to measure the angle of repose. It has been used for the characterization of interparticle friction between the particles.

$$}\left( \theta \right) = \tan^ \left( \right)$$

where h = Height of the pile; r = Radius of a pile [13].

Fourier transform infrared spectroscopy (FTIR)

Spectroscopy was conducted using FTIR spectrophotometer (Spectrum GX-FT-IR, PerkinElmer, USA) for the untreated febuxostat and solid dispersion. The spectrum was recorded in the range of 4000–400 cm−1. The procedure consisted of dispersing a sample in KBr followed by gentle mixing. The spectrum was scanned at a resolution of 0.15 cm−1 and scan speed was 20 scan/s.

Differential scanning calorimetry (DSC)

DSC (DSC-PYRIS-1, Phillips, Netherlands) was used to study the thermal behavior of the untreated febuxostat and solid dispersion. The experiments were performed in a dry nitrogen atmosphere. The samples (2–4 mg) were heated in hermetically sealed flat-bottomed aluminum pans under nitrogen flow (20 mL/min) at a scanning rate of 10 °C/min from 25 to 200 °C. Empty aluminum pans were used as the reference standard. DSC spectra were recorded in an aluminum pan at a scanning rate of 20 °C/min in an atmosphere of nitrogen gas (50 ml/min) [14].

In vitro dissolution rate studies on solid dispersions

Solid dispersions equivalent to 40 mg of febuxostat were placed in 900 ml of acetate buffer pH 4.5 (to simulate stomach condition) and phosphate buffer pH 6.8 (recommended media as per U.S.FDA) in USP apparatus II for in vitro dissolution rate studies. The paddle was used to maintain 75 revolutions per minute. Throughout the investigation, the temperature of the dissolution medium was maintained at 37 ± 0.5 °C. At regular intervals, approximately 5-ml samples were collected. To maintain the sink's condition, an equal volume of new dissolution medium was added. The withdrawn aliquots were filtered through 0.45 Whatman filter paper, diluted appropriately, and measured for febuxostat at λmax (314 nm) using an UV–VIS spectrophotometer. The dissolution experiments were repeated three times [15, 16].

Development of pulsincap for febuxostat

Pulsincap was manufactured through four stages. (1) Coating of capsule body, (2) Preparation of febuxostat tablets (SR and IR), (3) Optimization of erodible plug to achieve required latency time, and (4) Filling/assembly of tab-SR, erodible plug, and tab-IR into a coated capsule.

Coating of capsule body and optimization

Selecting hard gelatin capsules of the appropriate size, lids and bodies were then separated. As a plasticizer, dibutyl phthalate was combined with ethyl cellulose in methanol to create the coating solution. Only capsule bodies were permitted to be dipped in the coating solution. To meet the requirements of the chronotherapeutic drug delivery system, the number of coatings was optimized to maintain capsule integrity for at least 12/14 h. The integrity of the coated capsule was tested by soaking it in 900 ml of acetate buffer (pH 4.5) for 2 h, followed by phosphate buffer (pH 7.4) for 12 h [17].

Preparation of febuxostat tablets (SR and IR)

The direct compression method was used to prepare SR and IR tablet of febuxostat using various polymers like HPMC K15M, HPMC K100M, crosspovidone, and sodium starch glycolate. All the excipients were passed through 60 mesh sieve separately. The ingredients were weighed and mixed uniformly. The process of tablet compression involved the utilization of flat-faced punches within a rotary tablet punching machine. The compression force was modified in order to sustain the hardness within the range of 3–6 kg/cm2. Polymers were selected based on evaluation parameters. Composition of tablets is depicted in Table 1.

Table 1 Composition of tablets (SR and IR) containing febuxostatEvaluation of prepared SR and IR tablets

All the tablets were evaluated for pre-compression parameters. In vitro dissolution was performed using USP apparatus II. The media for SR tablets were chosen as phosphate buffer pH 6.8 and 7.4, whereas acetate buffer pH 4.5 was used for IR tablets.

1.

Tablet dimensions

Thickness and diameter were measured using a calibrated Vernier caliper. Three tablets of each formulation were picked randomly, and thickness was measured individually [18].

2.

Hardness

Tablet hardness refers to the magnitude of force required to fracture a tablet during diametrical compression, as measured by the Monsanto hardness tester. A total of six tablets from each batch were subjected to testing, wherein the average hardness of the tablets was determined. It is measured in kg/cm2.

3.

Weight variation

This test was conducted by randomly selecting and weighing 10 tablets from each batch, and calculating the average weight.

$$\begin \% } & = \left( } \right. \\ & \quad \quad \left. }} \right) \\ & \quad \times 100 \\ \end$$

4.

Friability

Each set of 6.25 g tablets was placed in the revolving drum (25 rpm) of a Roche friability apparatus, which subjected the tablets to rolling and repetitive shock caused by free-fall within the apparatus. The tablets were removed after four minutes, dedusted, and reweighed. The weight was then recorded, and the friability was computed as a percentage of weight loss. Each experiment was repeated three times.

$$\begin \% } & = \left( } \right. \\ & \quad \quad \left. }} \right) \\ & \quad \times 100 \\ \end$$

5.

Disintegration time of IR tablets

The disintegration apparatus was used to analyze the disintegration of IR tablets. As a medium, 900 ml of acetate buffer pH 4.5 to replicate gastric conditions and phosphate buffer pH 6.8 (U.S.FDA-approved media) were kept at 37 ± 0.5 °C. A total of six tablets, denoted as  = 6, selected at random, were introduced into individual glass tubes and subsequently subjected to operation. The disintegration time should be duly recorded to represent the average duration necessary for tablets to completely disintegrate. It is imperative that no residue be left behind on the sieve.

6.

Drug content of prepared SR and IR tablets

A total of six tablets were subjected to crushing and subsequent weighing in this study. A quantity of 40 mg of powder was dissolved in methanol and subsequently subjected to filtration using a Whatman filter paper. The resulting filtrate was then subjected to analysis for drug content at the wavelength of maximum absorption (λmax) of 314 nm, utilizing a UV–VIS spectrophotometer.

7.

Comparison of prepared formulation with marketed formulation

Comparison of dissolution profile of febuxostat formulation of the optimized batch with marketed formulation (40 mg febuxostat tablet) (FEXANTO 40 mg ER and FEBUVEL 40 mg IR) was carried out. In vitro drug release study of the marketed formulation was carried out using USP apparatus II, at a speed of 75 rpm using 900 ml of dissolution media phosphate buffer pH 6.8 and 7.4 for SR and acetate buffer pH 4.5 for IR at 37 ± 0.5 ℃.

Selection of polymers for erodible plug

A study was conducted to prepare an erodible plug for sealing the capsule body. This was achieved by compressing different polymers, namely HPMC K15M, guar gum, and aloe vera using various punches and dies on a rotary tablet press. The thickness and hardness values of the erodible plug were intentionally varied during the experimentation process. The erodible plug was subsequently inserted into the interior of a gelatin capsule that had been coated. Polymers for erodible plug were selected based on hardness, friability, swelling index, % erosion, and lag time [18].

Evaluation of polymers for erodible plug

Pre-compression parameters and post-compression parameters such as hardness and friability were performed.

1.

Swelling index of erodible plug

Erodible plugs were weighed individually (W1) and kept at pH 4.5 for 2 h, 6.8 for 3 h followed by pH 7.4 for the remaining time in a petri dish. The erodible plugs were removed from the petri dish after each hour, and excess surface water was removed carefully using tissue paper. The swollen erodible plug was then reweighed (W2), and the swelling index was calculated using the following formula,

$$\% } = \frac - W_ }} }}* 100$$

2.

% Erosion of erodible plug

Initial weight of plug was noted down (W1). Swollen plugs were dried at 60 °C for 24 h in an oven, kept in a desiccator for 48 h and reweighed (W3). % matrix erosion was calculated using the following formula [19]

$$\% } = \frac - W_ }} }}*100$$

3.

Determination of lag time

The determination of lag time was conducted by employing an SR tablet and an erodible plug, followed by the assessment of in vitro drug release using a coated capsule body. The capsule body was hermetically sealed using a cap that is soluble in water. The release of the drug was monitored in an acetate buffer with a pH of 4.5 for a duration of 2 h. This was followed by a phosphate buffer with a pH of 6.8 for a duration of 3 h. Finally, the release was observed in a phosphate buffer with a pH of 7.4 for the remaining hours. The USP apparatus II was employed for this investigation. Aliquot samples were withdrawn at each hour for 12 h, replaced with fresh media and analyzed spectrophotometrically [20].

Optimization of erodible plug

32 full factorial design was chosen to statistically optimize the polymer concentration of erodible plug. The employed methodology facilitated the identification of key factors and optimal combinations that effectively fulfill the necessary characteristics of erodible plugs. Table 2 presents the recorded values of the formulation parameters, in both their actual and coded forms, as part of the comprehensive 32 full factorial design. The independent variables chosen for this study were the concentration of HPMC K15M (X1) and the concentration of a 1:1 mixture of aloe vera and guar gum (X2). These variables were investigated at three different levels, namely − 1, 0, and + 1. To generate the experimental runs, a 32 full factorial design was employed using design expert 11 software. This resulted in a total of nine experimental runs. The erodible plugs were fabricated through the process of direct compression, in accordance with the experimental runs outlined by the software. Subsequently, these plugs were assessed for their percentage swelling index (Y1), hardness (Y2), and lag time (Y3) [17].

Table 2 Actual and coded values of formulation parameters for 32 full factorial design

The present study employed the Design Expert software to conduct multiple regression analysis (MLR) and analysis of variance (ANOVA). The objective was to investigate the relationship between two independent variables, namely X1 and X2, and three dependent variables, namely Y1, Y2, and Y3, within a factorial design. The selection of the optimal mathematical model involves considering the outcomes of MLR, specifically the correlation coefficient and coefficient values, as well as ANOVA, which includes the Fisher's ratio and associated P values. These metrics are utilized in the decision-making process to determine the most suitable model.

The selection of the most appropriate mathematical model was made by evaluating various statistical parameters, such as the coefficient of variation (CV), the multiple correlation coefficient (r2), the adjusted multiple correlation coefficient (adjusted r2), and the predicted residual sum of square (PRESS). This evaluation was conducted using the design expert® software. The PRESS metric is utilized to assess the goodness of fit of a model to the given data. It is desirable for the chosen model to have a relatively small PRESS value compared to the other models being evaluated.

Linear model:

$$Y \, = \, \beta_ + \beta_ X_ + \beta_ X_ + \beta_ X_ X_$$

Quadratic model:

$$Y = \beta_ + \beta_ X_ + \beta_ X_ + \beta_ X_ X_ + \beta_ X_^ + \beta_ X_^ + \beta_ X_ X_^ + \beta_ X_ X^$$

where β0 is the intercept representing the arithmetic average of all quantitative outcomes of factorial runs; β1 and β2 are the coefficients computed from the observed experimental values of Y, and X1 and X2 are the coded levels of the independent variable(s). The terms X1X2 and X12 represent the interaction and quadratic terms, respectively. The statistical validity of the polynomials was established based on ANOVA; subsequently, the feasibility and grid searches were performed to locate the composition of the optimum formulation.

Validation of experimental design

The validation of the experimental design was conducted by determining the relative error through the utilization of the following equation formula,

$$}\left( \% \right) = \frac} - }}}}}$$

A relative error of less than 10% is expected to confirm the preciseness of the design.

Fabrication of optimized formulation

A formulation that has been optimized was prepared by utilizing concentrations of erodible plug weight that have also been optimized. The final formulation was fabricated by filling the SR tablet and optimized erodible plug in coated capsule body. IR tablet was placed on the erodible plug and should conform to the coated capsule body. Subsequently, the capsule body, which had been previously coated, underwent the process of being hermetically sealed with an uncoated cap. The formulation that had been optimized was subjected to in vitro dissolution studies, as previously described.

Stability studies of optimized formulation

Stability studies were performed for optimized formulation. A sufficient quantity of capsules was carefully placed into glass bottles and sealed with a rubber cork. These bottles were subsequently placed inside stability chambers that were meticulously regulated at a temperature of 40 ± 2 °C, along with a relative humidity of 75 ± 5%. At different time intervals, samples were collected and analyzed for in vitro drug release.