Crystallographic Modification of Rosuvastatin Calcium: Formulation, Characterization and Pharmacokinetic Evaluation for Enhanced Dissolution, Stability and Bioavailability

Abstract

Rosuvastatin calcium is a promising lipid-lowering agent and the drug of choice in hyperlipidemia. Conventional solid oral delivery of rosuvastatin is limited by its poor solubility and ultimately poor bioavailability. An attempt was made to fabricate the cocrystals of RSC for enhancing solubility and bioavailability. Cocrystals were prepared by a microwave synthesiser-assisted solvent evaporation technique with multiple cocrystal formers. Rosuvastatin-Ascorbic acid (RSC-AA) cocrystals showed the highest solubility (~5-fold increased) amongst all twenty drug-coformer combination (DCC). RSC-AA cocrystals (1:1 ratio) were further characterized by various analytical techniques like FTIR, DSC and XRD to confirm the formation of cocrystals. RSC-AA cocrystals also showed improved flow properties and compressibility in comparison with pure drug, and it was demonstrated using the SeDeM diagram. RSC-AA cocrystals were further formulated into an immediate-release tablet by implementing experimental optimization. Comparative dissolution study of the cocrystal and pure drug tablet revealed improved dissolution after cocrystallization. RSC-AA cocrystal tablet showed the % drug release of 95.61 ± 3.94 while RSC pure drug showed the drug release of 67.83 ± 3.29. In vivo pharmacokinetic analysis showed significant improvement in systemic availability and cumulative absorption of the drug. The peak plasma concentration (Cmax) for RSC pure drug was 13.924 ± 0.477 μg/mL, while RSC-AA cocrystals showed a peak plasma concentration of 22.464 ± 0.484 μg/mL. Area Under Curve (AUC) of RSC-AA cocrystal was also significantly greater compared to the pure drug. In the stability study analysis, the shelf life was calculated from a graphical method and was found to be around 34.58 months for RSC-AA cocrystal tablets and 19.87 months for RSC pure drug tablets, which indicates improved stability with cocrystallization. Overall, the cocrystallization resulted in significant improvement in dissolution and solubility of RSC.

1. Introduction

Rosuvastatin calcium (RSC) is a drug used for the reduction of elevated plasma concentration (hypercholesterolemia) by inhibiting an enzyme called hydroxymethylglutaryl-COA reductase (HMG-COA reductase). Other therapies that include rosuvastatin as an active agent are atherosclerosis, hypertriglyceridemia, and hyperlipidaemia, mainly the diseases characterised by increased concentration of lipid and triglyceride levels in blood. RSC is a promising lipid-lowering agent in comparison with other statins like Atorvastatin, Simvastatin and Lovastatin. It is more potent compared to other statins. It was clinically proven that Rosuvastatin was more efficient in reducing the Low-Density Lipoprotein (LDL) and Triglycerides as compared to other statins and increasing the level of High-Density Lipoprotein (HDL). Despite several therapeutic advantages, Rosuvastatin is limited by poor solubility and varying bioavailability. This resulted in a dose-dependent response and an unnecessary increment in dose due to poor solubility. This dose increment increases the unnecessary hepatic and nephrotic load [1].

Synthesis of multicomponent systems has been a field of focus in recent years. These formulated systems have shown improvement in the dissolution and solubility, which have a superior chance to be chosen to reduce the dosage quantity of a drug in formulation, thereby reducing the adverse outcomes. There have been multiple research studies and inventions of new systems to enhance the solubility of poorly soluble drugs. These studies include the encapsulation of a drug in a lipoidal layer to form multiple lipid vesicular carriers [2], forming a molecular dispersion system of a drug with water-soluble polymers [3], and the use of cocrystallization [4].

Cocrystallization is a process that can be used to overcome the properties of the drug that can be problematic to its efficacy. It is a subpart of solid-state chemistry/supramolecular chemistry. It is based on the principle that two molecules containing specific functional groups are capable of forming a new molecular framework called cocrystals [5].

They have been a long-standing concept in the field to enhance solubility and stability [6,7]. The structural arrangement of cocrystals consists of reversible bonding between an API having low water solubility, belonging to class II and IV of BCS, and a cocrystal former. Coformer is the material that bonds with the API, giving rise to a new system, i.e., cocrystals. The force that binds these components together is mainly hydrogen bonds between functional groups, often including groups such as carboxylic acid, amides, amines, and alcohol groups. Van der Waals attraction between adjacent molecules, interaction of lipophilic moieties and pi-pi stacking are also responsible for cocrystal formation [8,9]. According to the US-FDA, cocrystals are materials that are made up of two or more different compounds in a definite molar ratio, one of which is an API and the other is a conformer [10]. The type of coformer used exhibits a proportion of the cocrystal activity that either increases or has no effect on drug solubility and stability performance. That is why it is critical to assess whether the coformer is compatible with the parent drug compound or not. They are generally chosen from the GRAS list of the US-FDA, or some other approaches can also be employed, such as the ability of a coformer to make hydrogen bonds with the drug or screening for supramolecular synthons (both hetero and homosynthons). Several drugs have shown better physicochemical properties when compounded as cocrystals, among them are tenoxicam [11], praziquantel [12], and telmisartan [13].

To prepare highly soluble cocrystals of it, several techniques can be employed, like dry grinding (solid-state grinding), solvent-assisted grinding (liquid-assisted grinding), the slurry method, microwave-mediated cocrystallization, cocrystals from ultrasonication, using the antisolvent addition method, and the hot melt extrusion technique. All of the preparation methods use the parent API and selected coformers in a stoichiometric ratio.

The method that is most preferred by researchers due to its feasibility and ease is the solvent evaporation technique. In this method, the starting materials, like the parent drug and selected coformers, are combined into a solvent, which is then evaporated using different means of evaporation. As the solvent is reduced in a controlled manner, the dissolved solutes make the solution supersaturated, and further decrease in solvent quantity results in the synthesis of cocrystals. As the solvent is reduced, the nucleation is forced, which leads to the formation of intermolecular hydrogen bonds in the drug and coformers. Instruments that help in the evaporation of solvent can also be used during cocrystallization. One of them is the microwave synthesiser, which irradiates microwaves into the solvent sample. Microwaves are the electromagnetic waves that produce energy in a molecule by aligning and anti-aligning the molecules in the direction of propagation (dielectric heating) [14,15]. The passing of this microwave through the solvent fractures the molecules, leading to the formation of new hydrogen and other intermolecular bonds forming a drug-coformer complex, i.e., cocrystals. It also helps in the controlled evaporation of the solvent, resulting in cocrystals with enhanced physical properties like increased solubility and dissolution.

The goal of the presented research was to improve the solubility and ultimately bioavailability of RSC by crystallographic modification and, furthermore, formulation of a tablet dosage form with improved dissolution and stability.

2. Materials and Methods

2.1. Chemicals

Rosuvastatin calcium was procured from Swapnroop Drugs and Pharmaceuticals, Chhatrapati Sambhajinagar, M.S., India. All the solid carboxylic acids, saccharin and tablet excipients were procured from Sourav Scientific, Pune, M.S., India. Ethanol was procured from the Sugar factory, Kopargaon, M.S., India.

2.2. Preparation of Cocrystals

Rosuvastatin calcium (RSC) cocrystals were prepared by the solvent evaporation technique using a microwave synthesiser. Drug-coformer combination (DCC) was prepared in equimolar (1:1) and non-equimolar (1:2) stochiometric ratios using 10 different cocrystal formers. While selecting coformers, various parameters like hydrogen bond donor and acceptor groups, pKa and absorption maxima were taken into consideration [16,17]. Both the drug and selected cocrystal formers were accurately weighed and co-dissolved in ethanol to obtain a clear solution. The drug and coformers were dissolved in 1 mg/mL volume of ethanol. The resulting solution was further evaporated by heating in a microwave synthesiser at 170 watts, which provides uniform and controlled heating to accelerate solvent removal and promote cocrystal formation [18,19].

2.3. Solubility Analysis of DCC

RSC and DCC were subjected to saturated solubility analysis. For saturated solubility analysis, an excess amount of RSC and various DCC were dissolved in 10 mL of water. The saturated solutions were kept shaking on a rotary shaker for 24 h. All the solutions were filtered using a Whatman filter and subjected to UV spectrophotometric (Shimadzu 1900i, Kyoto, Japan) analysis at 241 nm to determine soluble concentration [20].

2.4. Characterization of Cocrystals

2.4.1. Fourier Transform Infrared (FTIR)

FTIR spectroscopy (Agilent, Santa Clara, CA, USA) was employed to identify the functional group present in RSC, Ascorbic acid (AA). Samples of RSC, AA and cocrystals were placed on diamond ATR and scanned over the range of 4000–650 cm⁻¹ with a resolution of 8 cm⁻¹ and 32 scans per spectrum. The obtained spectra were analysed to determine the vibrational modes of chemical bonds present in the RSC-Ascorbic acid (RSC-AA) cocrystals [21,22].

2.4.2. Differential Scanning Calorimetry (DSC)

RSC, Ascorbic acid (AA), and RSC-Ascorbic acid (RSC-AA) cocrystals were subjected to DSC analysis (Mettler Toledo, Greifensee, Zurich, Switzerland). For thermogram analysis, a 2 mg sample was used, and the thermogram was recorded between 35 and 300 °C under atmospheric nitrogen at a heating rate of 10 °C per minute [23,24].

2.4.3. X-Ray Diffraction (XRD)

Pure drug samples of RSC and Ascorbic acid, as well as RSC-Ascorbic acid (RSC-AA) cocrystals, were acquired for XRD (Bruker D2 Phaser, 2nd generation, Karlsruhe, Germany) testing using a voltage of 40 kV, current of 55 mA, and 2θ over a range of 5 to 50° at a scan rate of 0.05°/s. Each step took 180 s [25,26].

2.5. Flow Property Analysis and SeDeM Model

To evaluate the impact of co-crystallization on flow properties, various parameters such as bulk density, tapped density, angle of repose, Hausner’s ratio, and Carr’s index were compared for both RSC and its cocrystals with ascorbic acid. This comparison aimed to determine the extent of improvement in flow properties after the cocrystal formation. The SeDeM model was applied for the calculation of good compressibility of cocrystals in comparison to the drugs. SeDeM is the Sediment Delivery model. It is the model used in preformulation to demonstrate the suitability of a powder blend for direct compression. The SeDeM model calculation is based on various parameters like dimensions, compressibility, flowability and lubricity (stability and dosage). Radii values are calculated from the conversion factor, and on the basis of the radii value, the radar plot known as the SeDeM diagram is created. In the SeDeM model, two important parameters are calculated to understand the compressibility of tablet blend, namely, Parameter Profile Index (IPP) and Good Compressibility Index (IGC), using the following formulas [27].

$$\mathrm{P}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{p}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{l}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x} \left(\mathrm{I}\mathrm{P}\mathrm{P}\right)=Average r of all parameters$$

(1)

$$\mathrm{G}\mathrm{o}\mathrm{o}\mathrm{d} \mathrm{c}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x} \left(\mathrm{I}\mathrm{G}\mathrm{C}\right)=IPP X f$$

(2)

where f is the reliability factor calculated from polygon area and circle area in the radar plot, and for 12 parameters, the standard value is 0.952.

2.6. Powder Dissolution Study

A powder dissolution study was performed using the USP type-II (rotating paddle) apparatus (Labindia, India) at 50 rpm and 900 mL phosphate buffer pH 6.8 as a dissolution media at 37 ± 0.5 °C [28]. Sampling was carried out for 1 h and 6 samples were collected at the interval of 10 min each. Sink condition was maintained by adding fresh media after sample collectionPure drug powder (20 mg RSC) and RSC-AA cocrystals (equivalent to 20 mg RSC) were subjected to dissolution, and comparative drug release analysis was carried out using a UV spectrophotometric method at 241 nm (Shimadzu 1900i, Japan) [29].

2.7. Formulation and Optimization of a Tablet

An immediate-release tablet containing pure RSC and RSC-OA cocrystal was formulated to determine comparative enhancement in dissolution following cocrystallization. The Cocrystal tablet was optimized using 2² factorial designs, and data analysis was done with the help of statistical software Design Expert (Trial version 13.0) by considering binder (sodium starch glycolate, X1) and lubricant (magnesium stearate, X2) as independent parameters while drug release was a dependent response.

Table 1. Coded and actual values for experimental batches.

Formulation Code	Variables Level (Coded Values)		Variables Level (Actual Values)
	X1	X2	X1 (mg)	X2 (mg)
F1	−1	−1	4	1.5
F2	−1	+1	4	3
F3	+1	−1	6	1.5
F4	+1	+1	6	3

and

Table 2. Formula for experimental tablet batches.

Sr. No	Ingredients (mg)	Batch Code
		F1	F2	F3	F4
1	Rosuvastatin calcium/cocrystal	23.52	23.52	23.52	23.52
2	Lactose monohydrate	85	85	85	85
3	MCC PH102	63.98	62.58	61.98	60.58
4	Starch	20	20	20	20
5	Sodium starch glycolate	4	4	6	6
6	Talc	2	2	2	2
7	Magnesium stearate	1.5	3	1.5	3
	Total (mg)	200	200	200	200

illustrate coded and actual values for independent variables and the formula for various tablet batches, respectively [21,30].

2.8. Characterization of Tablet Blend

Preformulation characteristics like bulk density, tapped density, angle of repose, Hausner’s ratio and Carr’s index of tablet blend were studied. Bulk and tapped density analysis was performed with 20 gm tablet blend using bulk density apparatus (Labtronics, Panchkula, Haryana, India). Furthermore, the post-compression parameters like thickness, hardness, friability and weight variation were determined for the tablets. Friability analysis was performed with number tablets equivalent to initial weight of 6 gm, using friability apparatus (Labtronics, India) and friability was calculated from following formula [31].

$$\mathrm{\%} \mathrm{F}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=Initial weight-Final weight ÷Final weight\times 100$$

2.9. Dissolution Study of a Tablet

The dissolution behaviour of the optimized RSC-AA cocrystal tablet and pure RSC tablet was evaluated in a USP type II rotating paddle apparatus (Labindia, India) at 50 rpm using 900 mL phosphate buffer (pH 6.8) at 37 ± 0.5 °C by maintaining standard conditions. Sink condition was maintained by adding fresh sample after each sampling interval of 10 min. Sampling was carried out for 60 min. The pure RSC tablet was prepared using the same formulation and process parameters as those used for the optimized batch cocrystal tablet. Samples were collected at pre-determined intervals with maintenance of sink condition and further analyzed spectrophotometrically, and the dissolution profile was compared to assess the enhancement in drug release achieved through crystallization [32,33].

2.10. Pharmacokinetic Study

The animal study protocol was approved by the Institutional Animal Ethical Committee (IAEC) of Invitox R&D Institute with approval no. IRDI/IAEC/T-01/2024-25 dated 2 February 2025. Pharmacokinetic evaluation was performed using a liquid-liquid extraction technique to isolate rosuvastatin and its calcium cocrystal from rat plasma. Wistar rats (n = 4, 180–250 g, male) were fasted for approximately 12 h with free access to water, followed by oral administration of either the standard drug or cocrystal formulation at an equivalent dose of 2.06 mg/kg. Blood samples (100 μL) were collected in triplicate from each animal at predetermined intervals (0.5, 1, 3, 6, 12, 24, 36, and 48 h) via the retro-orbital plexus into polypropylene tubes containing Na₂EDTA as an anticoagulant. Samples were vortexed for 10 min, centrifuged at 4000 rpm, and stored at −20 °C until analysis. The supernatant was evaporated at 40 °C to dryness, reconstituted with (500 μL) acetonitrile, vortexed briefly, and transferred into vials for injection. Drug concentrations in plasma were quantified, and pharmacokinetic parameters, including total area under the Curve (AUC), were calculated versus time by using the linear trapezoidal rule (Pk Solver) [34,35].

2.11. Stability Study and Shelf-Life Analysis

Formulations of optimized batch (F1) were subjected to accelerated stability study at 40 ± 2 °C and 75 ± 5% RH in a desiccator for three months and evaluated for various parameters like hardness, disintegration time % drug release and drug content. Furthermore, shelf life was calculated using SigmaPlot 15.0 software [36].

3. Results and Discussion

3.1. Solubility Analysis of Cocrystals

The saturated solubility analysis revealed notable differences between pure rosuvastatin calcium (RSC) and its DCC with various coformers. Pure RSC exhibited a solubility of 0.1932 ± 0.0174 mg/mL, whereas cocrystallization generally led to improved aqueous solubility, with the extent of enhancement dependent on the type of coformer and stoichiometric ratio. The greatest enhancement was observed for RSC–Ascorbic acid (1:1) (0.8841 ± 0.0323 mg/mL), followed by RSC–Oxalic acid (1:2) and RSC–Tartaric acid (1:2). Citric acid, benzoic acid, and oxalic acid (1:1) offered moderate improvement, while acetyl salicylic acid, maleic acid, saccharin, and sulpho benzoic acid showed minimal effect (Figure 1) [37,38].

The hydrophilicity and high intrinsic solubility of AA result in improved wettability and dissolution of RSC. The lactone and hydroxyl group present in AA can form hydrogen bonding with carboxyl, sulphonamide and hydroxyl groups present in RSC, resulting in the formation of a more water-soluble crystal lattice.

3.2. FTIR Analysis

The FTIR spectrum of rosuvastatin calcium exhibited characteristic bands at 3511, 3388 and 3309 cm⁻¹ corresponding to O-H stretching vibrations, while bands at 2949–2851 cm⁻¹ indicate aliphatic C-H stretching. The peaks at 1585 and 1554 cm⁻¹ were assigned to asymmetric stretching of the carboxylate (COO-) group, whereas symmetric COO-stretching was observed near 1392 cm⁻¹, consistent with its salt form. Strong absorptions between 1312–1148 cm⁻¹ correspond to S = O stretching of the sulfone group, and bands near 1228–1143 cm⁻¹ indicated C-F stretching from the fluorophenyl moiety. Ascorbic acid displayed a broad O-H stretching band in the range 3651–3205 cm⁻¹, and C-O-C stretching vibration between 1246–1020 cm⁻¹.

The cocrystal spectrum retained the major characteristic peaks of both pure RSC and ascorbic acid with slight shifts and changes in intensity in the O-H (3420–3232 cm⁻¹) and carbonyl/carboxylate region (around 1656–1554 cm⁻¹), suggesting the weak intermolecular interaction, like hydrogen bonding between the drug and co-former, responsible for enhancing solubility. The absence of significant new peaks or major shifts indicates no strong covalent bond formation [39,40].

3.3. DSC Analysis

The DSC thermogram of pure rosuvastatin calcium exhibited a sharp endothermic peak at 230.86 °C (onset: 211.70 °C), corresponding to its melting point. Ascorbic acid displayed a characteristic endothermic peak at 194.89 °C (onset: 191.25 °C). In contrast, the cocrystal demonstrated a significantly reduced melting point, with a peak at 174.87 °C (onset: 165.59 °C), which is lower than both the drug and the conformer. This marked depression in melting point suggests the formation of a new solid phase with weaker intramolecular forces, thereby enhancing molecular mobility and potentially improving solubility. The distinct shift in thermal events confirms successful cocrystal formation between rosuvastatin calcium and ascorbic acid [41].

3.4. XRD Analysis

The XRD pattern of pure rosuvastatin calcium displays distinct characteristic peaks at 2θ values of 9.550°, 11.907°, 18.883°, and 22.357°, confirming its crystalline nature. Ascorbic acid exhibited sharp peaks at 2θ values of 20.077°, 25.575°, 28.328°, and 30.280°, corresponding to its crystalline structure. In the cocrystal diffractogram, several peaks of the parent compound either shifted in position or disappeared, while new peaks emerged at different 2θ values, indicating the formation of a new crystalline phase. These modifications in the diffraction pattern confirm successful cocrystal formation, reflecting a change in the molecular arrangement within the crystal lattice [42].

3.5. Flow Property and SeDeM Analysis

The flowability of the RSC-AA cocrystal was notably enhanced compared to pure rosuvastatin calcium. These improvements may be attributed to the altered particle shape, size distribution, and surface properties resulting from cocrystallization, as shown in

Table 3. Flow property analysis of drug and cocrystals.

Properties	Angle of Repose	Bulk Density	Tapped Density	Carr’s Index	Hausner’s Ratio
Rosuvastatin calcium	33.77	0.35	0.48	27.08	1.37
RSC-AA Cocrystal	29.87	0.54	0.63	16.66	1.17

[43].

The SeDeM analysis was carried out on the basis of various dimensions, compressibility, flowability and lubricity parameters. All the values and their conversion into radius values are well illustrated in Table provided in Supplementary File.

From the radii (r) value of 12 different parameters included in SeDeM analysis, a radar plot is created, which is also described as a SeDeM diagram (Figure 2a,b). From the SeDeM analysis and diagram, two important parameters, namely, IPP and IGC, were calculated for both pure RSC and RSC-AA cocrystals. The IPP value for pure RSC was 4.88, and the IGC value was 4.65, while the IPP value for RSC-AA cocrystals was found to be 5.50, and the IGC value was found to be 5.24. The IGC value of more than 5 for RSC-AA cocrystals indicates better compressibility and tabletting efficiency [27,44].

3.6. Powder Dissolution Study

The dissolution profile revealed a marked improvement in drug release from the RSC-AA cocrystal compared to pure rosuvastatin calcium. The pH 6.8 is used to simulate the in vivo absorption site pH of RSC. As RSC is a BCS class-II drug, the pH plays a crucial role in dissolution and ultimately absorption. Improved dissolution of RSC after cocrystallization in Phosphate Buffer pH 6.8 demonstrates the possibility of improved absorption from the GIT. This enhanced release trend persisted throughout the study, with cocrystal achieving 90.03 ± 4.70% release at 50 min and 98.82 ± 3.80% at 60 min, whereas the pure drug reached only 58.94 ± 4.10% at the same time, as shown in Figure 3. The significant improvement in dissolution rate may be attributed to the cocrystal’s lattice, improved wettability, and enhanced solubility, showing faster drug release into the dissolution medium [45].

3.7. Formulation and Optimization of Tablet

A 2² factorial design was employed to optimize the cocrystal tablet of RSC-AA, while the Response Surface Methodology (RSM) was used to identify key interactions among the variables to achieve proper drug release. The results of the experimental batches for % drug release by DoE are illustrated in Figure 4. Furthermore, the pre-compression and post-compression parameters of factorial formulation were tabulated in

Table 4. Evaluation of pre-compression parameters of factorial formulations.

Formulation Code	Bulk Density (gm/cm3)	Tapped Density (gm/cm3)	Hausner’s Ratio	Compressibility Index (%)	Angle of Repose (θ)
F1	0.55 ± 0.021	0.61 ± 0.018	1.09 ± 0.010	9.83 ± 0.785	26.03 ± 0.011
F2	0.57 ± 0.032	0.64 ± 0.025	1.12 ± 0.019	10.97 ± 1.525	26.88 ± 0.016
F3	0.58 ± 0.012	0.66 ± 0.029	1.13 ± 0.027	12.07 ± 2.05	27.17 ± 0.027
F4	0.56 ± 0.028	0.63 ± 0.013	1.12 ± 0.033	11.15 ± 2.61	26.95 ± 0.047
Pure RSC tablet	0.43 ± 0.017	0.54 ± 0.0	1.25 ± 0.050	20.37 ± 3.15	28.69 ± 0.053

±SD is calculated n = 3.

and

Table 5. Evaluation of post-compression parameters for tablets obtained from factorial batches.

Parameters	F1	F2	F3	F4	Pure RSC Tablet
Weight Variation (mg)	200 ± 0.63	200 ± 0.74	200 ± 0.91	200 ± 0.88	200 ± 0.82
Hardness (kg/cm2)	4.2 ± 0.28	4.3 ± 0.29	4.2 ± 0.25	4.1 ± 0.19	4.2 ± 0.0.22
Thickness (mm)	3.22 ± 0.11	3.20 ± 0.12	3.23 ± 0.15	3.26 ± 0.17	3.24 ± 0.16
Friability (%)	0.86 ± 0.06	0.74 ± 0.4	0.71 ± 0.04	0.78 ± 0.05	0.91 ± 0.07
Disintegration time (min)	11 ± 1.0	10 ± 1.0	12 ± 1.5	12 ± 1.5	12 ± 1.0
Drug content (%)	99.41 ± 0.67	99.18 ± 0.37	98.99 ± 0.51	99.01 ± 0.17	99.02 ± 0.28

±SD is calculated n = 3.

, indicating the suitability of the blend for direct compression and formulation of tablets following all the criteria of evaluations as per compendia [21,46]. The comparative dissolution clearly demonstrated the improved dissolution RSC after cocrystallization. Pure RSC tablet showed the % drug release of 67.83 ± 3.29 while RSC-AA cocrystal tablet showed % drug release of 95.61 ± 3.94. This improved dissolution is indicating the solubility enhancement of drug resulting from hydrogen bond formation between RSC and AA.

In the optimization study, the probability (p) value of less than 0.05 indicated that all of the model terms were significant, and both the independent variables, sodium starch glycolate concentration (X1) and magnesium stearate concentration (X2), significantly affect the drug release from the tablet. The R² value for the drug release is 0.9979, which is in accordance with the adjusted R² value of 0.9938, which correlates with the better fitting of the model as shown in

Table 6. Model fitting and statistical analysis of ANOVA for drug release analysis.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value
Model	31.58	2	15.79	242.81	0.0453	significant
A-Sodium Starch Glycolate	7.81	1	7.81	120.14	0.0579
B-Magnesium stearate	23.77	1	23.77	365.48	0.0333
Residual	0.0650	1	0.0650
Cor Total	31.64	3

.

3-D response surface graph and contour plot were constructed to visualize the effect of independent variables, i.e., binder (sodium starch glycolate) and lubricant (magnesium stearate), on the response parameters, i.e., drug release. Both plots demonstrated the significant impact of these independent variables on drug release. Minimum concentration of SSG and MS showed the highest drug release (Figure 5a,b) [47].

The perturbation plot displays each factor’s impact on drug release while holding the others constant. Influence is higher on steeper slopes. In this case, magnesium stearate (B) and sodium starch glycolate (A) both adversely impact drug release, although the slope of (B) is marginally stronger. Drug release is further reduced by the combined effect of both factors [48].

From the desirability analysis, F1 was selected as an optimized batch. For desirability analysis, the maximum % drug release was selected as a desired response, and the highest desirability value (0.983), showing batch was selected as an optimized batch [49].

3.8. Pharmacokinetic Study

In vivo pharmacokinetic studies were conducted to demonstrate the improved bioavailability and pharmacokinetics of the drug after transforming into the cocrystals. The comparative improvement in bioavailability is presented in a plasma concentration vs. time graph illustrated in Figure 6. The pharmacokinetic parameters of pure RSC and RSC-AA Co-crystal after oral administration at various time points are provided in

Table 7. Pharmacokinetic parameters of RSC pure drug and RSC-AA cocrystals.

Sr No	Pk Parameter	Pure Rosuvastatin Calcium	Rosuvastatin Ascorbic Acid Cocrystal
1	t1/2	22.398 ± 1.08	34.604 ± 5.93
2	Tmax (h)	6	6
3	Cmax (μg/mL)	13.924 ± 0.477	22.464 ± 0.484
4	AUC0–t μg/mL∗h	272.572 ± 4.18	414.605 ± 18.57
5	AUC0–∞ μg/mL∗h	365.137 ± 10.92	600.867 ± 69.57
6	AUC0–t/0–∞	0.7469 ± 0.0211	0.69417 ± 0.05467
7	AUMC0–∞ μg/mL∗h2	12,444.78 ± 999.17	25,733 ± 6834.34
8	MRT0–∞ h	34.051 ± 1.823	42.2964 ± 6.956

±SD is calculated n = 4.

.

The RSC-AA cocrystal showed higher systemic availability and peak concentrations in comparison with pure rosuvastatin calcium (AUC_0–t and AUC_0–∞ increased by 52% and 64%, respectively. The peak plasma concentration (C_max) increased from 13.92 to 22.46 µg/mL. The cocrystal showed a lower elimination rate constant and a longer t_1/2, while T_max and T_lag were unchanged. The overall observations from the pharmacokinetic studies endorsed an enhanced systemic availability of cocrystals compared to the pure drug [50].

3.9. Stability Study and Shelf-Life Analysis

Stability and shelf-life analysis revealed the stability of the formulation for the long term. There was no significant change in important evaluation parameters of the optimized formulation.

Table 8. Stability study analysis.

Formulation Parameter	Observation at 40 ± 2°/75 ± 5% RH
Color	White
Odor	No
Hardness (kg/cm2)	4.1 ± 0.33
Friability (%)	0.79 ± 0.16
Drug content (%)	98.57 ± 0.21
Disintegration time (min)	10 ± 1
Percent drug release	95.84 ± 0.46

illustrates the stability study analysis of the optimized formulation. The shelf life (t_90%) was found to be 34.58 months, which is significantly higher than the shelf life of pure RSC tablet (19.87 months), indicating the long-term stability of cocrystal tablets (Figure 7) [36,51].

4. Conclusions

The solubility limitation of rosuvastatin was resolved by crystallographic modification and fabrication of cocrystals. Microwave-assisted solvent evaporation resulted in controlled heating and formation of high-quality cocrystals. Out of all 20 drug-coformer combination (DCC) from 1:1 and 1:2 ratios, RSC-AA (1:1 ratio) cocrystals exhibited the highest solubility (an increase in solubility by 5-fold). Formation of cocrystals was further confirmed by FTIR, DSC, and XRD analysis. Powder dissolution study showed the comparatively better dissolution of cocrystals than the pure drug. Cocrystallization resulted in improved flow properties and compressibility, and it was additionally illustrated using SeDeM analysis and a diagram. Furthermore, the cocrystals were formulated as a tablet with the implementation of experimental design and optimization. The comparative dissolution of a pure RSC tablet and an RSC-AA cocrystal tablet showed the superior dissolution after crystallization. The in vivo pharmacokinetic analysis performed using the Wistar rat model also demonstrated the improvement in systemic availability of RSC when administered as cocrystals. The pharmacokinetic parameters like Cmax, AUC_0–t, AUC_0–∞ and MRT were significantly higher in RSC-AA cocrystals in comparison with the pure drug. The stability study and shelf-life analysis performed for the RSC pure drug tablet and RSC-AA cocrystal tablet demonstrated the comparatively better stability of the cocrystal tablet. This study concludes that the fabrication of cocrystals of RSC with AA resulted in improved solubility, bioavailability, micromeritics and stability.