Materials used in this study were obtained from different sources. Cefixime was a gift sample from Baroque Pharmaceuticals Pvt. Ltd, Gujarat. Soluplus® was a gift sample from BASF, Mumbai. Pearlitol DC 400 was a gift sample from Signet Chemical Corporation Pvt. Ltd, Mumbai. All other chemicals were of analytical grade.

QTPP defines the overall objectives of safety and efficacy of a drug development program. The US FDA document guides QTPP, which describes the components of QTPP for new drug applications. QTPP for granules formulation is listed in Table 1. International Conference on Harmonization (ICH) Q8R2 summarizes them as QTPP. 17 The Ishikawa diagram (Figure 1) was carried out to identify the CMAs and CPPs for Cefixime granules affecting the CQAs of the drug product. Ishikawa fishbone diagram was constructed to establish the potential cause-effect relationship among the product and process variables.18 Based on prior scientific knowledge, physical attributes and diffusion of the drug were considered CQAs of Cefixime granules. These parameters are likely to affect granules' therapeutic efficacy are summarized in Table 2.

Figure 1

a. The Box Behnken design is a technique that allows the identification of factors involved in a process and assesses their relative importance. In addition, any interaction between factors chosen can be identified. This design is suitable for exploring quadratic response surfaces and constructing second-order polynomial models, thus helping to optimize the process using a small number of experimental runs. For the three-level three-factor BB experimental design, a total of 17 experimental runs are required. 22 A selected Box Behnken design describes the proportion in which the independent variables Soluplus®, Croscarmellose sodium and Sodium Bicarbonate were used to formulate Cefixime granules. The % drug release and dispersion time (sec) were selected as dependent variables. The factor X1 (Soluplus®) at a concentration of 10%, 20%, 30%. Three levels of factor X2(Croscarmellose sodium) at a concentration of 5%, 10%, 15%. (% concerning the total weight of granules), three levels of factor X3 (Sodium Bicarbonate) at a concentration of 0.5%, 1%, 1.5% were taken as the rationale for the design of Cefixime granules. Total seventeen Cefixime granules formulations were prepared, and the composition is shown in Table 3. BB experimental design allows the designer to utilize three levels of each factor (with each factor placed at one of each equally spaced value to ensure orthogonality and near rotatability) to adequately quantify second-order response models in 17 runs, inclusive of 5-replicated centre points of a cubical design region. 22

The composition of granules is summarized in Table 3. Thirty gm Cefixime granules containing 100 mg of the drug were prepared by wet granulation and sieving method. PVP K30 (10 % w/v) was used as a binder. A wet coherent mass of Pearlitol DC 400, Soluplus® (Intra granularly – Half), Sodium Bicarbonate, Croscarmellose sodium (Intra granularly – Half) was prepared and passed through a 10# sieve to obtain granules. The wet granules were dried at 50o C for 15 minutes. The dried granules were collected and then screened through a 20# sieve, Soluplus® (Extra granularly -Half) and Croscarmellose sodium (Extra granularly -Half) were added. Granules were lubricated with Aerosil 200 (1%), talc (2%) and magnesium stearate (1%). 23, 24

An excess amount of drug was added to 10 ml vials containing 2 ml of water, and then the vials were sealed. Vials were placed in an orbital shaker at room temperature for the next 24 hours. The Samples were filtered, diluted and then analyzed in a spectrophotometer at 288 nm. The test was performed in triplicate.

Flow or micromeritic properties are essential for the powder or granules for the formulation's ease of production and handling. Therefore, different micromeritic properties were measured thrice for the validation of results.

The angle of Repose: The angle of repose was determined by the fixed funnel method and calculated according to Equation 1. The diameter (d) of the pile and maximum pile height (h) were recorded.

1AngleofRepose=tan-1(2h/d)

Bulk Density and Tapped Density: 10 g of the sample was placed into a 100 ml measuring cylinder, and volume was noted. The ratio of mass (weight) to volume is known as the untapped bulk density of the material. The cylinder was subjected to a fixed number of taps (100). The mass (weight) ratio to volume is known as the tapped density of the material.

Carr's Index: Based on the bulk density and the tapped density, the percentage compressibility of the powder was determined by Equation 2.

2 Carr'sindex = tapped density (ρt)-bulk density (ρb) tapped density (ρt)×100

Hausner's Ratio: Hausner's ratio is an indirect index of ease of measuring the powder flow using tapped density and bulk density as per Equation 3.

3 Hausner's Ratio = tapped δ bulk δ

10 g of granules were subjected to the Roche Friabilator and operated at 25 rpm. After 4 minutes, the granules were sieved on a 200 mesh. The number of granules passed through 200 mesh was calculated as percentage granular friability. The study was performed to check the strength of the granules. The test was performed three times with the given specifications.

The dispersion time was measured in water and repeated thrice. One dose of granules (200 mg) equivalent to 100 mg of Cefixime was added to a petri dish containing water and stirred gently; the time to take granules dispersed in water and make a smooth dispersion was noted as dispersion time of granules.

Granules equivalent to 100 mg of drug was taken and dissolved separately in100 ml of phosphate buffer pH 7.2. The filtered solution was diluted to record the absorbance value (288 nm) in a UV-visible spectrophotometer. The actual drug content was calculated using Equation 4. The test was performed three times with the given specifications.

4% Drug content = Practical Drug content  Theoretical Drug content ×100

The particle size distribution measurement was done on the optimized batch using the nest of standard sieves 20, 40, 60, 100, and 120 mesh size. The sieves were agitated mechanically for 10 minutes on a Rotary sieve shaker. The weight of granules retained on each smaller sieve was noted, and the size distribution of optimized batch (granules dispersed in water); the formulation is checked using Malvern zeta sizer instrument.

The dissolution study of granules was conducted using dissolution Type II apparatus (Electrolab TDT 08L, Mumbai, India). The dissolution test was performed using 900 ml phosphate buffer (pH 7.2) at 37 ± 0.5ºC. The paddles were rotated at 100 rpm. Five ml of aliquots were taken at intervals of 5 minutes to determine the immediate drug release. The absorbance of the solutions was taken at 288 nm. Cumulative percentage drug release was calculated using an equation obtained from the standard curve. 32, 33 The study was performed thrice with the given specifications.

Comparison of dissolution profile of granules formulation of the optimized batch with marketed formulation (ZIPRAX – 100 mg Dry Suspension) of Cefixime was carried out. In addition, an in-vitro drug release study of the marketed formulation was carried out using USP apparatus 2, using a paddle at a speed of 100 rpm using 900ml of phosphate buffer (pH 7.2) at 37±0.5ºC.In-vitro drug release study of Cefixime granules was performed as described in the evaluation parameter.

Similarity factor (f2) is approved by FDA to compare the release profiles of the drug from the test with the standard reference formulation and is represented by Equation 5.

5f2=50*log1+1+1nΣ(Rt-Tt)22*100

It has a value ranging between 50 and 100. The f2 values less than 50 indicate the dissimilarity between the drug and test release profiles and reference formulation. If f2 is more excellent than 50 shows the similarity between drug release profile from test and reference formulation.

The stability studies of the optimized granules batch were carried out at 40°C±2°C and 75%±5% RH for the accelerated condition in Alu Alu blister pack according to ICH guideline. The HPMC capsule of Cefixime granules was wrapped in aluminium foil. It was kept in the humidity chamber with well-controlled temperature and humidity conditions. The samples were removed after 10, 20 and 30 days and evaluated for their drug content, in vitro release characteristics and dispersion time.

A phase solubility study was used to check the solubility of drugs in different solvents to identify the drug's pH-dependent solubility (Figure 2). Cefixime is slightly soluble in water and highly soluble in pH 7.2 phosphate buffer. From this study, it is concluded that the Cefixime has a pH-dependent solubility.

Figure 2

The free-flowing powders with good properties can be homogeneously filled in the HPMC capsules. Therefore, flow properties are essential in the formulation and industrial production of the oral solid dosage form. Results of the micromeritic properties are presented in Table 4. For all batches, the angle of repose was found in the range of 25.10±0.08 to 28.80±0.08, Carr's index was found in a range of 10.57±0.01 to 14.45±0.01, and Hausner's ratio was found to be in the range of 1.118±0.01 to 1.169±0.01. All results indicate good flow properties.

In general, friability or strength indicates the ability of granules to withstand the shear forces during handling and various pharmaceutical procedures. All the batches of Cefixime granules were found to have high mechanical strength, as indicated by their friability values (<1% w/w). The friability was less than 1 % (0.3-0.8%) for all the batches.

All the batches of Cefixime granules dispersion time was found to be in the range between 91.0±0.8 to 197.0±0.8 seconds. Croscarmellose Sodium exhibited excellent swelling properties, leading to quick water penetration in granules. 36

Drug content study: Testing for drug content helps ensure that a therapeutic product's strength remains within specified acceptance limits. The drug content in all 17 batches of formulations was uniform and in the range of 97.84±0.29 to 100.14±0.20 % (Table 5) within the permissible limits of USP. 37

The size distribution of the granules was determined using a mechanical sieve shaker fitted with a Standard Sieve number 20, 40, 60 and 100 having size 850µm, 425µm, 250µm and 150µm, respectively. The diameter of the mesh size mainly influenced the size of the granules. 20# remained constant for the granules preparation process, which affects the size, so granules yielded maximum in the range between 425-850 µm. All Batches having granules yielded 1-1.4 % in the range between 1000-850 μm. In all seventeen Batches, 9.1- 18.1% granules range between 250-425μm. 1- 3.8% granules range between 150-250 μm, and 77-88.4% granules range between 425-850 μm.

The drug release profiles of Cefixime granules from the prepared formulations are shown in Figure 3. The drug release study of granules containing Soluplus®, Croscarmellose sodium and Sodium bicarbonate indicated that the concentration of all three excipients influenced the release of the drug. The concentration of Soluplus® was an essential factor and affected the release behaviour of the drug from granules. Soluplus® (SP) is a graft copolymer consisting of polyethylene glycol (PEG), polyvinyl caprolactam, and polyvinyl acetate (13% PEG 6000/57% vinyl caprolactam/30% vinyl acetate); it has a PEG 6000 backbone with one or two side chains consisting of vinyl acetate randomly copolymerized with vinyl caprolactam. Due to the amphiphilic character of the polymer, it forms micelles in aqueous media. It can be used to solubilize poorly water-soluble drugs and increase the drug release of the granules. 14 30 % Soluplus® containing batches(F2, F4, F6, F8) showed more significant release than 10 % Soluplus®. Batch F2, F4, F6 and F8 showed more excellent release 98.15±0.28, 102.33±0.17, 99.99±0.34 and 102.43±0.07 in 30 minutes.

Figure 3

The causal factor and response variables were related using a polynomial equation with statistical analysis through Design-Expert® software. A positive coefficient sign indicates a synergistic effect, while a negative term indicates an antagonistic effect upon the response. The 2D and 3D response surface plots are shown in Figure 4 and Figure 5.

The Design-Expert® software suggested a Quadratic model with a correlation coefficient (R²) value is 0.9974, with (p<0.001). A high R²value (0.99) indicates a good fit between the independent and first dependent variables (% CDR). The evolved mathematical model is

Y1 (% CDR in 20 minutes) = 90.53 + 16.94 X1+ 4.59 X2+ 5.76 X3 – 2.00 X1X2 – 1.63 X1X3 – 0.9850 X2X3 – 10.87 X₁² – 3.17 X₂² + 0.1132 X₃².

Significant effect on % CDR in 20 minutes of the concentration of Soluplus®. The change in % CDR of the granules can also be obtained by changing the croscarmellose sodium and sodium bicarbonate.The 2D and 3D response surface plot derives that as the concentration of Soluplus® (X1) increase, the %CDR increase (Figure 4).

The design expert software suggested a quadratic model with an R2 value equal to 0.9867, with (p < 0.001). The evolved mathematical model is

Y2 (Dispersion time)= 118.46 - 6.34 X1 – 28.96 X2 – 21.03 X3+ 0.7500 X1 X2 –12.58 X1 X3 – 5.83 X2 X3 + 4.94 X₁²+ 1.69 X₂²+ 29.67 X₃².

The 2D and 3D response surface plot represented that the concentration of croscarmellose sodium significantly affected the granules' dispersion time (Figure 5). The overlay plot is shown in Figure 6, which is generated by superimposing the contour plots of all the regions. The common region obtained is the overlay plot by which we can get some predicted standard batches called Checkpoint analysis. The range selected for the overlay plot was ± 10 as per USFDA criteria.

Figure 6

The relative error should not be more than 5%. The optimized batch was selected based on the % CDR in 20 minutes (NLT 90%) and Dispersion Time (< 120 seconds).

After generating the polynomial equations relating the dependent and independent variables, the Cefixime granules were optimized for the Y1 and Y2. The optimum formulation was prepared according to the above values of the factors and subjected to the % drug release and dispersion time to verify the evolved models. As shown in Table 6, it demonstrated that the observed value of a checkpoint batch was relatively closer to the predicted value. The prediction error was less than 5% which validated the model applied.

All batches were evaluated through micromeritics and other evaluation parameters such as drug content, granules' strength, dispersion time and % CDR (in 20 minutes); the results are within the permissible range as shown in Table 7.

Figure 7

Figure 7 show the size distribution study of optimized batch (before dispersion). Maximum yield of granules 79.1 % fall in the range of 425-850 µm, whereas 0.9 % granules were in the range of 1000-850 µm.

Figure 8

The granules were dispersed in water. The results revealed that the average diameter of the granules was found to be 868.2 (d. nm), which shows that the granules were converted from micrometre size to nanometer size after being dispersed in water. The results of prepared nanodispersion in water are shown in Figure 8.

The dissolution profile of the optimized batch and the marketed product showed 99.56±0.20% and100.12±0.20% drug release in 30 minutes, respectively. The results revealed that the Cefixime granules have a similar dissolution profile like the marketed product.

Developing pediatric formulations can be quite scientifically challenging due to unique requirements and limitations. Cefixime granules in capsule formulation will prove alternative to traditional dosage formulations. These formulations in which the capsules can be opened to release small granules of drug that can be dispersed in water or swallowed whole for adults. Granules dispersed in water help improve the compliance of children and their caretakers. In addition, these granules capsule serve as alternative formulations for infants, children, and patients with dysphagia.

The Cefixime granules formulation was systematically developed using the standard wet granulation method. The concentration of Soluplus, Croscarmellose sodium and Sodium bicarbonate had a significant effect on the formulation of Cefixime granules. The findings of the current study can be helpful to draft the control strategy for the product's life cycle management. In addition, the Cefixime will give better absorption and lead to better bioavailability.

Briefly, a patient-friendly dosage form of Cefixime was developed with sound knowledge of the impacting variables to supply quality products. In addition, a control strategy was being proposed for the industry.

QTPP (Quality target product profile), CQA (Critical Quality Attribute), CMA (Critical Material Attribute), CPP (Critical Process Parameter), QbD (Quality by Design), RH (Relative humidity), CDR (Cumulative drug release).