Optimization of Selexipag Cocrystal Encapsulated Formulation to Improve Drug Release

Poor aqueous solubility remains one of the major challenges in pharmaceutical formulation and drug development. A large proportion of newly discovered drug molecules fall under Biopharmaceutical Classification System (BCS) Class II and IV, which exhibit low solubility and variable bioavailability. Insufficient solubility leads to slow dissolution in gastrointestinal fluids, resulting in reduced absorption and therapeutic efficacy. To achieve desired pharmacological action, a drug must first dissolve in the biological medium before it can be absorbed into systemic circulation. Therefore, improving the solubility and dissolution rate of poorly water-soluble drugs is crucial for enhancing bioavailability and therapeutic performance.

Various strategies have been explored to overcome solubility-related challenges, including micronization, salt formation, solid dispersion, and use of surfactants. However, these methods often have limitations depending on the physicochemical nature of the drug. In recent years, pharmaceutical cocrystallization has emerged as an effective and versatile approach for modifying the physicochemical properties of active pharmaceutical ingredients (APIs) without altering their pharmacological activity. Cocrystals are formed by the interaction of the drug with a pharmaceutically acceptable co-former through non-covalent hydrogen bonding. This approach enhances solubility, dissolution rate, and stability, making it a promising technique for improving the oral bioavailability of poorly soluble drugs such as Selexipag.

PRE-FORMULATION STUDIES:¹

1) Organoleptic properties:

The colour, odour, and taste of the drug were characterized and recorded and the results of this are shown in table no 4.

2) Solubility studies:

It determined by dissolving drug substance in water, phosphate buffer pH6.8, methanol, ethanol, DMSO, the solubility study was conducted taking excess amount of the drug in 10ml of solution. Then the samples were kept in the water bath shaker and agitated for 24hrs at 37±0.5?C. the samples were filtered and suitably diluted. The samples were analysed spectrophotometrically at λ max. the concentration of drug was determined using respective standard graph and shown in table no 4.

3) Melting Point:

Melting point of Selexipag was determined by open capillary method. Fine powder of Selexipag was filled in a glass capillary tube (sealed at one end). Capillary tube was tied to thermometer and was placed in the Thieles tube was further placed on flame. The temperature at which the powder starts melting was noted and reported in table no 4.

SPECTROSCOPIC STUDIES; ²

303675789833071400. of λ max:

Most of the drugs absorbs light in UV wavelength region (200-400) nm hence λmax was determined by using UV spectrophotometer. beers range for Selexipag was found to be (10 μg/ml -30 μg/ml). The solution containing 10μg/ml concentration prepared and scanned over the range of 200-400nm phosphate buffer pH 6.8 as blank using double beam UV spectrometer. The maximum wavelength obtained in the graph at Fig No.8 was consider as λmax of the pure drug

PREPARATION OF STANDARD STOCK SOLUTION AND STANDARD CALIBRATION CURVE OF SELEXIPAG

A standard stock solution of Selexipag (1000 μg/mL) was prepared in phosphate buffer (pH 6.8). An intermediate stock (100 μg/mL) was obtained by dilution, followed by serial dilutions (0–30 μg/mL). Absorbance was measured at λmax, and a calibration curve was constructed for analysis.

Screening of Co-formers³

Table.No.01 The data of co-formers considered for the study

Sr. No.	Co-former	Structure	Molecular formula	Molecular weight (g/mol)	Melting point (ºC)	HB Donors	HB Acceptors
1	Nicotinamide		C?H?N?O	122.12g/mol.	128–131°C	2	2
2	Benzoic acid		C?H?O?	122.12g/mol.	122–123°C	1	1

Co-formers were selected based on Hansen solubility parameter and ΔpKa.

Hansen solubility parameter

It divides cohesive energy into dispersion, polar, and hydrogen-bonding components to predict miscibility and compatibility of pharmaceutical materials. Theoretical HSP values of Selexipag and its co-formers were calculated using group contribution methods to evaluate their miscibility and potential for co-crystal formation.

Group contribution method:

The solubility parameter was estimated using Van Krevelen’s, Fedor’s, and Hoy’s group contribution methods. These theoretical approaches aid in selecting co-formers compatible with the drug. Partial solubility parameters were calculated by combining molecular force and molar volume contributions from all structural fragments.

• Fedor’s substituent constant =

CED= ? ΣΔUΣΔV                ….(1)

• Hoy’s substituent constant =

?MA ÷ ??V               ….(2)

• Krevelen =

?F_d² + ?F_p² + ?U_h²     ….(3)

Based on ΔpK_a Values:

The ΔpK_a value (ΔpK_a = pK_a drug - pK_a co-former) is widely accepted as the key to predict the formation of a salt or co-crystal. It is generally considered that if ΔpK_a > 3, the resulting compound will be a salt. A co-crystal may result, if ΔpK_a is < 0. For ΔpK_a between 0 to 3, the outcome can be either a salt or a co-crystal or a complex with partial proton transfer. The pK_a values were collected from the literature, ΔpK_a values were calculated, and summarized. 

METHOD FOR PREPARATION OF CO-CRYSTAL OF SELEXIPAG 

Table No.2. Preparation of Selexipag Co-crystals

SYNTHESIS VIA SOLVENT EVAPORATION METHOD

Cocrystals of Selexipag with Nicotinamide and Benzoic acid were prepared by the solvent evaporation method in 1:1, 1:1.5, and 1:2 molar ratios. The drug was dissolved in methanol and the co-former in water, then mixed with stirring and ultrasonication. The co-former solution was added dropwise to the drug solution under constant stirring until solvent evaporation. The resulting cocrystals were dried overnight in a desiccator.

CHARACTERIZATION OF SELEXIPAG CO-CRYSTALS

Melting point: The melting point of the Selexipag co-crystals were determined using open capillary tubes.

Drug content: Accurately weighed 10 mg of co-crystals were dissolved in methanol and the volume made up to 10 mL. From this, 1 mL was diluted to 10 mL with phosphate buffer (pH 6.8), and absorbance was measured. The percentage drug content was then calculated and recorded.

Aqueous solubility studies: The solubility of pure Selexipag and its co-crystals was evaluated in distilled water. Excess samples were shaken at 25 °C and 75 rpm for 24 hours to reach equilibrium. The equilibrated solutions were filtered, diluted with phosphate buffer (pH 6.8), and analyzed at 298 nm.

In vitro dissolution studies: Dissolution studies were performed in phosphate buffer (pH 6.8) using a USP Type II (paddle) apparatus for 60 minutes. Capsules containing pure Selexipag or co-crystals equivalent to 25 mg of drug were tested in 900 mL of medium at 37 ± 0.5 °C and 50 rpm. Samples were withdrawn at 5, 10, 20, 30, 45, 50, and 60 minutes. Each sample was filtered through a 0.45 μm membrane and suitably diluted. The absorbance was measured at 298 nm using phosphate buffer (pH 6.8) as blank.

FTIR: FTIR spectrum of Selexipag co-crystals were obtained and compared with the pure drug to determine the possibility of co-crystal formation.

Differential Scanning Calorimetry: DSC studies were performed for co-crystals in the manner explained earlier. The DSC thermogram of the co-crystals were compared with that of the pure drug.

X-Ray diffraction studies: PXRD (Powder X-ray Diffraction) was used to study the crystal structure and identify crystalline phases of Selexipag co-crystals

  SEM: Surface morphology of cocrystals was examines by Scanning Electron Microscopy (SEM)

FORMULATION DESIGN FOR DRUG LOADED CAPSULES.  

Table No.3. Formulations design for capsule containing Selexipag co-crystals.

MICRO MERITIC STUDY:

1. Angle of repose: ⁶

The angle of repose (θ) was determined by the fixed funnel method to assess the flowability of Selexipag cocrystals. The funnel height was adjusted so that its tip just touched the pile surface. Accurately weighed cocrystals were allowed to freely pass through the funnel to form a heap.

2. Determination of Bulk density :⁷ 

Bulk density is the ratio of the weight of the powder and the volume it occupies; it is expressed in gms/ml. Bulk density is imparted in determining the size of the container needed for handling and processing. A weighed quantity of the cocrystals (W) was carefully taken into a graduated measuring cylinder and the volume occupied by it (V₀) was measured. The bulk density was calculated using the formula 

Bulk density=   weight of the cocrystal(w)Initial volume occupied by the cocrystals        

3. Tapped density:

Tapped density is the ratio of the powder weight to the volume after compaction, expressed in g/ml. It was determined by tapping a graduated cylinder containing a known weight of cocrystals until the volume became constant. The procedure was performed using a bulk density apparatus set for 100 tapings. The initial (V?) and final (Vf) volumes were recorded to calculate tapped density.

Tapped density= Weight of the cocrystal(w)Final volume occupied by the cocrystal

4. Determination of Hausner’s ratio:

It is another parameter for measuring flow ability of the microsphers.it is calculated using the formula.  

Hausner’s ratio = Tapped density Bulk density

5. Determination of compressibility index:

It is indirect measurement of bulk density, size, and shape, surface moisture content and cohesiveness of materials since all that can influence the consolidation index. It is also called as compressibility index. It is denoted by Ci and is calculated using the formula below. 

Ci = Tapped density-Bulk densityTapped density×100

CHARACTERIZATION OF CAPSULE CONTAINING CO-CRYSTALS OF SELEXIPAG.

Percentage weight variation

The test was carried out by weighing 6 capsules individually using analytical balance, then calculating the average weight, and comparing the individual capsule weights to the average. The percentage of weight variation is calculated by using the following formula.

% weight variation = Individual weight Average weight X 100

Disintegration test.⁹  

The disintegration apparatus, described in IP was used for the study. It contains two basket rack assembly. Each basket rack assembly consists of six glass tubes that are the 3 inches long, open at the top and held against the 10-mesh screen at the bottom. Each capsule was placed in each tube, and containing distilled water as medium. 37±2? C temperature was maintained throughout the study. Time required for the complete disintegration of capsule was noted.

In-vitro drug release study: ⁸

In-vitro dissolution studies were performed using a USP Type-I apparatus with 900 ml of 6.8 pH phosphate buffer at 37 ± 0.5 °C and 50 rpm for 1 hour. Samples (5 ml) were withdrawn at 5–60 min intervals and replaced with fresh medium. Drug concentration was analyzed using a UV spectrophotometer at 298 nm. Comparative dissolution studies were conducted for pure Selexipag and formulation F6 under identical conditions.

RESULTS AND DISCUSSIONS

PREFORMULATION STUDIES OF SELEXIPAG

Table.No.4. Preformulation studies of Selexipag

Determination of λ max

Fig No.1: λ max of Selexipag in phosphate buffer pH 6.8.

This was performed by using UV spectrophotometer by using pH 6.8 phosphate buffer as medium. The spectrum of Selexipag (10μg/ml) in pH 6.8 showed the peak at 298nm. The absorption maximum (λ max) of 298 was selected for the study.

Calibration curve of Selexipag:

Table No.5: Spectrometric data for standard cure of Selexipag in Ph 6.8 Phosphate buffer

Fig No.2. Standard plot of Selexipag in phosphate buffer 6.8

The calibration curve of Selexipag in pH 6.8 phosphate buffer exhibited excellent linearity within the concentration range of 0–30 µg/mL. The absorbance values increased proportionally with concentration, following the equation Absorbance = 0.02499 × Concentration + 0.00130, with a correlation coefficient (R²) of 0.9999, indicating high precision and accuracy of the method. The lowest absorbance recorded was 0.0013 at 0 µg/mL, while the highest was 0.7509 at 30 µg/mL. This strong linear relationship confirms the suitability of this method for the quantitative estimation of Selexipag.

Authentication of Selexipag

Melting point: Melting point of Selexipag was found to be 138 °C which agreed to the value 134-139 °C in the literature.

FTIR spectrum:  IR spectrum of Selexipag is shown in Figure 3. The spectra showed peaks at 3360.60cm^-1 corresponding to N-H stretch, 1240.23cm^-1 corresponding to C-N stretching, 1724 cm^-1 and 1558 cm^-1 corresponding to carbonyl group and C stretching respectively.

Fig.No.3. FTIR spectrum of pure Selexipag

DSC

Thermogram obtained from DSC study is shown in figure 4.

Fig.No.4. DSC of pure Selexipag

Selection of Co-formers

Hansen solubility parameter: The HSPs of the co-formers and drug were calculated using group contribution methods and the values are tabulated in Table 6,7,8

Table.No.6 Calculation of solubility parameter of Selexipag based on Fedor’s substituent constants

Table. No.7 Calculation based Hoy’s Molar attraction constants

Table No.8. Calculation of solubility parameter of Selexipag based on partial solubility parameter or Van Krevelen method

HSP values of co-formers by different methods

Table No.9. Calculated HSP values of co-formers by different methods

Table No.10. Differences in the solubility parameter values of drug & co-former and possibility of co-crystal formation.

Based on ΔpK_a Values: The pK_a values were collected from the literature, ΔpK_a values were calculated, and summarized as shown in Table.No.11

Table No.11. The data of pK_a of co-formers and ΔpK_a values

Comparing all formulation to Hansen solubility parameter

Table No.12 Interpretation of Hansen solubility parameters

Comparing pKa values helps predict cocrystal or salt formation. Selexipag has a weakly acidic group (pKa 4.8). Nicotinamide (pKa 3.3) and Benzoic acid (pKa 4.2) show ΔpKa < 3, indicating a tendency toward cocrystal formation rather than salt. This supports selecting these coformers for screening.

Preparation of Selexipag co-crystal

Co-crystals were prepared by solvent evaporation method. Nicotinamide and Benzoic acid were selected hence; the co-crystals were successfully obtained with these co-formers. The prepared co-crystals were further subjected to evaluation and characterization.

Fig No.5. Selexipag Cocrystal

Characterization of Selexipag co-crystals

1. Melting point

Table No.13. Melting point of Selexipag co-crystals

The melting point of pure Selexipag was 139°C. All cocrystal formulations showed a decrease in melting point, with F3 and F6 exhibiting the greatest reduction, confirming stable co-crystal formation and improved solubility.

Drug content determination:

Table No.14. Drug content determination

All the value represents are mean of 3 readings (n-3)

The drug content results show uniformity among formulations, indicating consistent mixing and encapsulation. F1 and F4 have the highest drug content (89.24 mg), followed by F2 and F5 (78.03 mg), while F3 and F6 have slightly lower content (75.00 mg). All values are within acceptable limits, confirming formulation accuracy and reproducibility.

2. Solubility studies

Table No.15. Solubility profile of Selexipag cocrystals

The aqueous solubility of Selexipag cocrystals increased with higher co-former ratios for both Nicotinamide (F1–F3) and Benzoic acid (F4–F6). F6 (1:2 BNZ) exhibited the highest solubility (0.016 mg/mL), while lower-ratio formulations showed moderate solubility.

3. In vitro dissolution studies:

The in vitro drug release in 6.8 pH phosphate buffer from the prepared cocrystals

Table.No.16. Dissolution data of Selexipag co-crystals

Table.No.17.  Dissolution data of Selexipag Nicotinamide co-crystals

Fig No.6. Dissolution profile of F1, F2 and F3

The dissolution study revealed that F2 (1:1.5 NIC) and F3 (1:2 NIC) showed significantly enhanced drug release compared to F1 (1:1 NIC). F2 achieved 91.36% release at 30 minutes, while F3 showed a slightly higher initial release (93.02% at 30 minutes) but declined slightly afterward. In contrast, F1 exhibited a slower and lower release (42.86% at 60 minutes). This indicates that increasing Nicotinamide concentration improves dissolution, with F2 providing optimal performance.

Dissolution study of Selexipag- Benzoic acid Co-crystals

Table No.18. Dissolution data of Selexipag-Benzoic acid co-crystals

Figure 07: Dissolution profile of F4, F5 and F6

The dissolution profile of Benzoic Acid cocrystals showed that F6 (1:2 BNZ) had the highest drug release, reaching 94.32% at 30 minutes and maintaining 95.24% at 60 minutes. F5 (1:1.5 BNZ) demonstrated slightly lower but comparable release (91.75% at 30 minutes), while F4 (1:1 BNZ) exhibited slower and incomplete release (48.12% at 60 minutes).

Table. No.19. Comparing drug release of F3 and F6

Figure 08: Dissolution profile of F3 and F6

The dissolution data show that both F3 (1:2 NIC) and F6 (1:2 BNZ) exhibit significantly improved drug release compared to the pure drug. F6 demonstrates slightly higher release, reaching 97.10% at 60 minutes, while F3 achieves 93.98%. Rapid release is observed within the first 20 minutes, with over 87% for F3 and 90% for F6. This enhancement indicates that cocrystallization with benzoic acid and nicotinamide improves solubility and dissolution, with F6 showing superior performance.

4. FT- IR Analysis

Fig No 09 a) Pure drug, b) Selexipag-Nicotinamide c) Selexipag- Benzoic acid

Table No.20. Interpretation of IR data

5. Differential Scanning Calorimetry

Fig No.10. DSC curves for SLP-NIC cocrystal

Fig No.11. DSC curves for SLP-BNZ cocrystal

DSC thermograms showed endothermic peaks for Selexipag, Nicotinamide, and Benzoic acid at 139.0, 134.1, and 133.9 °C, respectively. Co-crystals (F3 and F6) exhibited different melting points from the pure drug, indicating co-crystal formation. Absence of co-former peaks confirmed the formation of true co-crystals rather than physical mixtures. The thermal variations provided clear evidence of successful co-crystallization.

6. Powder X-RAY diffractometry

Fig No.12: a)Powder X-RAY Diffractometry of Selexipag, b) SLP-NIC c) SLP-BNZ

Table No .21: XRD spectrum of Selexipag-NIC, BNZ co-crystal

7. SEM

A                                                                                    B

C

Figure 13: a) SEM images of Selexipag b) SLP-NIC c) SLP-BNZ

Micromeritic Study:

Table No.22. Micromeritic properties of Selexipag cocrystals

*All the Values represents are mean of 6 readings (n=6)

1. Angle of Repose:

The value of angle of repose of co-crystals of Selexipag was found to be in the range of 27.12±0.015- 32.11 ± 0.013 which indicates the good flow property. An obtained value of angle of repose was depicted in Table No.22.

2. Bulk density & Tapped density:

The bulk density and tapped density values ranged from 0.38 ± 0.015 to 0.44 ± 0.017g/ml for bulk density and 0.46 ± 0.016to 0.52 ± 0.015g/ml for tapped density respectively So, it shows that all formulations having good flow properties and packability and which were within the limit as per IP specifications.

3. Carr’s index:

The Carr’s index values for Selexipag were found to be in the range 16.0%-17.4% which lies between the normal values indicating good flow property. An obtained value of Carr’s index was depicted in Table No.22. 

4. Hausner’s ratio:                                                                                                                       

The Hausner’s ratio values for Selexipag cocrystals were found to be in the range 1.18 to 1.21. These values are within the range of 1.25 which indicated that the powder mixture exhibited good flow property. An obtained value of Hausner’s ratio was depicted in Table No.22.

CHARACTERIZATION OF CAPSULE CONTAINING CO-CRYSTALS OF SELEXIPAG 

%Weight variation and Disintegration time

Table No.23. Weight variation and Disintegration time

All six formulations demonstrated excellent uniformity, with mean weights ranging from 99.6 to 100.6 mg and %RSD values between 0.8% and 1.3%, which are well within the acceptable. Disintegration times varied according to coformer ratio. Formulations with higher coformer content, such as F3 (1:2 NIC) and F6 (1:2 BNZ), exhibited the fastest disintegration, around 4 minutes, due to improved wettability and porosity. In contrast, F1 (1:1 NIC) and F4 (1:1 BNZ) showed slightly longer disintegration times, approximately 6–7 minutes.

Fig No.14: Capsule formulation loaded with Selexipag cocrystals

In-vitro drug release of capsule containing Selexipag

Table No.24. In-vitro drug release of pure drug, F3 and F6 encapsulated Selexipag

Fig No.15: Dissolution profile of cocrystal loaded capsule