Formulation and Evaluation of Colon-Targeted Ozanimod Tablets for Inflammatory Bowel Disease Using a Factorial Design Approach

Figure 1A. Schematic representation of colon-targeted delivery showing protection in the stomach and small intestine followed by drug release in the colon.

Ozanimod is a selective sphingosine-1-phosphate receptor modulator that reduces lymphocyte migration from lymphoid tissue and thereby decreases inflammatory cell trafficking. Because the therapeutic target in ulcerative colitis is the inflamed colonic mucosa, a colon-targeted Ozanimod formulation could potentially improve local action and reduce systemic adverse effects. The present manuscript converts the thesis investigation into a research article focused on formulation design, in vitro evaluation, optimization and stability assessment of colon-targeted Ozanimod tablets.

2. MATERIALS AND METHODS

2.1 MATERIALS

Ozanimod was used as the active pharmaceutical ingredient. Eudragit S100, Eudragit L100 and ethyl cellulose were selected as pH-dependent and release-controlling polymers. Microcrystalline cellulose PH 102, PVP K30, talc and magnesium stearate were used as diluent, binder, glidant and lubricant, respectively. Methanol, ethanol, distilled water and buffer media were used for analytical and dissolution studies.

Figure 2. Chemical structure of Ozanimod used as the active pharmaceutical ingredient.

2.2 Preformulation and analytical studies

Ozanimod was evaluated for color, odor, appearance and texture. Melting point was determined using a capillary method in a digital melting point apparatus and compared with the reported value. Solubility was examined in selected aqueous and organic media after shaking for 24 h, filtration and UV-visible analysis. The UV spectrum of Ozanimod was scanned between 200 and 400 nm, and the analytical wavelength was fixed at 270 nm. Calibration standards of 2, 4, 6, 8 and 10 µg/mL were prepared, and absorbance was measured at 270 nm to construct a calibration curve.

2.3 Drug-excipient compatibility studies

Fourier-transform infrared spectroscopy was performed on pure drug and physical mixtures using the potassium bromide pellet technique over the range of 4000-400 cm⁻¹. Spectra were compared for shifting, disappearance or emergence of major functional-group peaks. Differential scanning calorimetry was performed for pure Ozanimod and drug-excipient mixtures in the range of 30-300 °C under nitrogen, using an empty aluminum pan as reference. Onset, peak, endset and enthalpy values were compared to assess thermal compatibility.

2.4 Experimental design and formulation development

A three-level factorial design was used to study the effects of Eudragit S100, Eudragit L100 and ethyl cellulose concentration on lag time and cumulative drug release at 12 h. Nine formulations were prepared by wet granulation. Ozanimod, microcrystalline cellulose and polymers were passed through sieve #60 and mixed uniformly. PVP K30 binder solution was prepared in isopropyl alcohol and added gradually to form a damp mass. The wet mass was passed through sieve #16, dried at 40 °C, passed through sieve #20 and lubricated with talc and magnesium stearate. Granules were compressed into tablets using a rotary tablet press.

Table 1. Composition of optimized colon-targeted Ozanimod tablet (F7, per tablet).

2.5 Evaluation of tablets

Precompression parameters included angle of repose, bulk density, tapped density, Carr’s index and Hausner ratio. Compressed tablets were evaluated for physical appearance, weight variation, thickness, hardness, friability and drug content uniformity. Drug content was measured after powdering tablets, extracting drug in phosphate buffer pH 7.4, filtration, suitable dilution and UV-visible analysis at the predetermined wavelength.

In vitro dissolution was performed using USP Type II paddle apparatus at 37 ± 0.5 °C and 50 rpm. To simulate gastrointestinal transit, tablets were exposed to 0.1 N HCl for 2 h, phosphate buffer pH 6.8 for the next 3 h and phosphate buffer pH 7.4 until 12 h. Samples were withdrawn at predetermined intervals, filtered, diluted and analyzed spectrophotometrically. Cumulative percentage drug release was calculated and plotted against time.  The optimized formulation was subjected to stability study for three months under controlled storage conditions, and changes in appearance, tablet properties, drug content, release and lag time were recorded.

3. RESULTS AND DISCUSSION

3.1 Preformulation and analytical findings

Ozanimod appeared as a white to off-white, odorless, crystalline and fine free-flowing powder. The observed melting point range was 132-135 °C, close to the reported value of 135 °C, supporting drug identity and acceptable purity. UV analysis showed maximum absorbance at 270 nm. The calibration curve was linear over the concentration range of 2-10 µg/mL, with the regression equation y = 0.0511x + 0.0344 and R² = 0.9955.  This confirmed suitability of the UV method for quantitative analysis of Ozanimod during drug content and dissolution testing.

Figure 3. UV absorption spectrum of Ozanimod showing lambda max at 270 nm.

Figure 4. Calibration curve of Ozanimod at 270 nm.

3.2 Compatibility studies

The FTIR spectrum of Ozanimod showed characteristic bands corresponding to N-H stretching, aliphatic C-H stretching, nitrile C≡N stretching, aromatic C=C stretching, C-N stretching and aromatic out-of-plane C-H bending. The major peaks were retained in the drug-excipient mixture without meaningful disappearance of characteristic signals, indicating absence of major chemical interaction. DSC analysis showed a melting transition for pure Ozanimod with onset, peak and endset temperatures of 130.2, 139.6 and 149.3 °C, respectively. The physical mixture showed onset, peak and endset values of 123.2, 136.5 and 143.1 °C, respectively, with a slight decrease in enthalpy from 118 to 115 mJ. The retention of the characteristic melting behavior suggested no significant incompatibility between Ozanimod and selected excipients.

Figure 5. FTIR spectra showing retention of the major functional group peaks in the pure drug and drug excipient mixture.

Figure 6. DSC thermograms of pure Ozanimod and the drug-excipient mixture used for compatibility interpretation.

Table 3. Selected FTIR and DSC observations used for compatibility interpretation.

3.3 Precompression and post-compression evaluation

All powder blends displayed satisfactory flow and compressibility. The angle of repose ranged from 27.8° to 31.2°, bulk density from 0.44 to 0.48 g/cm³, tapped density from 0.53 to 0.55 g/cm³, Carr’s index from 12.72% to 16.98% and Hausner ratio from 1.14 to 1.20. The optimized batch F7 showed the best flow characteristics with angle of repose of 27.8°, Carr’s index of 12.72% and Hausner ratio of 1.14. These properties are advantageous for uniform die filling, consistent tablet weight and reproducible drug distribution. Post-compression data indicated that all formulations were acceptable with respect to weight variation, thickness, hardness, friability and drug content. The optimized F7 formulation showed hardness of 3.3 kg/cm², friability of 0.42%, average weight of 200 mg and drug content of 99.6%. The low friability and higher hardness suggest adequate mechanical strength, while the high drug content indicates uniform distribution of Ozanimod in the tablet matrix. The complete post-compression parameter results for individual batches are presented in Table 5, showing acceptable batch-to-batch uniformity and confirming the superiority of optimized batch F7.

Table 4. Summary of tablet evaluation results.

Table 5. Post-compression parameter results of factorial batches F1-F9.

F7 was selected as the optimized formulation because it showed the highest hardness (3.3 kg/cm2), lowest friability (0.42%), ideal tablet weight (200 mg) and maximum drug content (99.6%).

Design of Experiment

The DOE contour and 3D surface plots show the combined effect of Eudragit S100 and Eudragit L100 on the lag time and drug release of Ozanimod colon-targeted tablets. The contour plot for lag time indicates that increasing the concentration of Eudragit S100 increased the lag time because it forms a stronger pH-dependent barrier and protects the drug from premature release in the upper gastrointestinal tract. The 3D plot also confirms this increasing trend, with the maximum lag time observed near formulation F7. The drug release contour and 3D surface plot show that the highest drug release at 12 hours was also obtained near F7, which contained an optimized polymer ratio and showed about 3.2 h lag time and 94% drug release. This indicates that F7 provided the best balance between delayed release and complete drug release in colonic conditions. In contrast, formulations with higher Eudragit L100 and ethyl cellulose levels, such as F8 and F9, showed reduced final release because excessive polymer concentration restricted drug diffusion. Therefore, F7 was selected as the optimized batch for colon-targeted Ozanimod delivery.

Fig 7: The DOE contour and 3D surface plots

3.4 Effect of polymers on lag time and drug release

The polymer system strongly influenced lag time and the extent of Ozanimod release. Lag time varied from 1.7 to 3.2 h across the nine formulations, while cumulative drug release at 12 h ranged from 70% to 94%.  Formulations containing lower Eudragit S100 concentration showed shorter lag phases and lower release performance. Increasing Eudragit S100 generally improved delayed-release behavior because this polymer dissolves at a higher pH threshold and provides stronger protection against premature release in acidic and early intestinal conditions.

F7, containing 60 mg Eudragit S100, 20 mg Eudragit L100 and 10 mg ethyl cellulose, produced the longest lag time of 3.2 h and the highest 12 h drug release of 94%. This combination appears to provide an optimal balance: Eudragit S100 delays drug release until the target pH region is reached, whereas the moderate amount of Eudragit L100 and lower ethyl cellulose content avoid excessive diffusion resistance. In contrast, F8 and F9 contained higher levels of Eudragit L100 and ethyl cellulose and showed lower release values of 77% and 75%, respectively, indicating that excessive polymer loading restricted drug diffusion.

Figure 8. In vitro cumulative release profiles of F1-F9 in sequential dissolution media.

Figure 9. cumulative drug release profile

Figure 10. Comparison of lag time and 12 h drug release among factorial batches.

3.5 Factorial design interpretation

The factorial design confirmed that polymer concentration was a critical formulation variable. The lag-time model was statistically significant, and Eudragit S100 was the most influential factor, showing a strong positive effect on lag time. The drug release model also indicated that Eudragit S100 had the dominant contribution to release behavior. The response pattern suggested that adequate Eudragit S100 is required for colon-targeted delay, but excessive combined polymer concentration may decrease the final amount of drug released.  Therefore, optimization required balancing pH-dependent protection with sufficient permeability after the lag phase.

3.6 Stability of optimized formulation

The optimized formulation F7 remained physically stable over three months. Appearance remained unchanged, thickness decreased only slightly from 2.5 to 2.4 mm, hardness decreased from 3.3 to 3.1 kg/cm² and friability increased marginally from 0.42% to 0.45%, still below the usual 1% limit. Drug content decreased from 99.6% to 98.5%, cumulative drug release changed from 94% to 93%, and lag time remained nearly unchanged at 3.2- 3.1 h. These findings indicate that the optimized formulation retained its physical, chemical and functional properties during the study period.

Table 6. Stability study of optimized F7 formulation.