Amalgamation of solid dispersion and melt adsorption techniques for augmentation of oral bioavailability of novel anticoagulant rivaroxaban

The objective of the present study was to evaluate the potential of solid dispersion adsorbate (SDA) to improve the solubility and bioavailability of rivaroxaban (RXN). SDA of RXN was developed by fusion method using PEG 4000 as carrier and Neusilin as adsorbent. A 32 full factorial design was utilized to formulate various SDAs. The selected independent variables were the amount of carrier (X1) and amount of adsorbent (X2). The responses measured were the time required for 85% drug release (Y1) and saturated solubility (Y2). MTT assay was employed for cytotoxicity studies on Caco-2 cells. In vivo pharmacokinetics and pharmacodynamic evaluations were carried out to assess the prepared SDA. Pre-compression evaluation of SDA suggests the prepared batches (B1–B9) possess adequate flow properties and could be used for compression of tablets. Differential scanning calorimetry and X-ray diffraction data signified the conversion of the crystalline form of drug to amorphous form, a key parameter accountable for improvement in drug dissolution. Optimization data suggests that the amount of carrier and amount of adsorbent significantly (P < 0.05) influence both dependent variables. Post-compression data signifies that the compressibility behavior of prepared tablets was within the official standard limits. A significant increase (P < 0.0001) in the in vitro dissolution characteristics of RXN was noticed in optimized SDA (> 85% in 10 min) as compared to the pure drug, marketed product, and directly compressible tablet. Cytotoxicity studies confirmed the nontoxicity of prepared RXN SDA tablets. RXN SDA tablets exhibited 2.79- and 1.85-fold higher AUC in comparison to RXN suspension and Xarelto tablets respectively indicating improved oral bioavailability. Higher bleeding time and percentage of platelet aggregation noticed with RXN SDA tablets in comparison to RXN suspension further substantiate the efficacy of the prepared formulation. In summary, the results showed the potential of RXN SDA tablets to enhance the bioavailability of RXN and hence can be an alternate approach of solid dosage form for its development for commercial application.


Introduction
For decades, vitamin K antagonists (VKAs) are the only therapeutic category of orally administered anticoagulants. However, VKAs have been associated with demerits including numerous drug-drug and food-drug interactions, a large intra-and inter-individual variation of their dose-response relationship, slow onset and offset of action, and an unpredictable pharmacodynamic response that mandates routine coagulation monitoring and dose adjustment. In recent years, direct oral anticoagulants that are specific in targeting a single coagulation factor (such as Factor Xa or thrombin) have been developed to surpass the limits of VKAs. Rivaroxaban (RXN) is a direct, specific Factor Xa inhibitor that has been approved for clinical use in 2008 to prevent venous thromboembolism in adults undergoing elective hip or knee replacement surgery. It has also been employed for the prevention of stroke, systemic embolism, and atherothrombotic events along with prevention and treatment of deep vein thrombosis and pulmonary embolism. RXN is associated with merits of rapid onset of action, limited drug-drug interactions, well-established pharmacokinetics, and pharmacodynamics, does not require routine coagulation monitoring, making it a potential anticoagulant in simplifying and improving the management of thromboembolic disorders [1,2]. However, RXN is lipophilic, non-ionizable, and, practically insoluble in water with pH-independent solubility of 5-7 mg/L at 25 °C [3].
Solid dispersion consists of a minimum of two different components, usually a hydrophilic inert carrier/matrix and a lipophilic drug. The carrier may be amorphous or crystalline. Typically, it is a dispersion of an active ingredient in a matrix or in an inert carrier formulated by melting, using solvent, or melting solvent method. In this system, the drug experiences particle size reduction, and as a result, dissolution is increased due to an increase in surface area. Additionally, no energy is needed to break the crystal lattice of the drug in the amorphous state throughout the dissolution step [11]. Due to the presence of surrounding hydrophilic carriers, drug wettability, as well as solubility, will be increased further [12]. Attempts were also made to formulate solid dispersions of RXN to enhance the drug dissolution and solubility and thereby enhance the absorption [13][14][15]. However, there are some limitations to this technique as well, which include difficulty in grinding, inadequate flow, poor compressibility, and also less reproducibility of physicochemical properties and instability of the drug and vehicle as well as difficulty in scale-up [5,16]. The concept of solid dispersion adsorbate (SDA) was introduced to control these problems. In this method, solid dispersion is adsorbed on a carrier with a very large surface area to obtain a free-flowing powder and thereby help in an increase in dissolution as well as bioavailability [17][18][19]. There are a variety of commercially available porous carriers like Florite, Neusilin US2, Aerosil, Aerogel, etc. having unique characteristics like pore size, particle size, specific surface area, etc., and have been widely investigated in tablet formulations [20][21][22]. Neusilin US2 is amorphous aluminum magnesium silicate with a high level of specific surface area and good adsorption capacity. On the other hand, polyethylene glycol (PEG) has been widely used in various oral formulations including tablets, microcapsules, soft gelatin capsules, suspensions, and emulsions [23]. Indeed, PEG 4000 (hydrophilic polymer) is one of the most widely studied polymeric carriers in preparing solid dispersion [24][25][26]. Various research groups have emphasized the influence of carrier and adsorbent on the solubility and dissolution of SDAs [17][18][19]. A review of the literature suggests that no work has been done to date on formulating RXN SDA for solubility enhancement. The objective of this study was to formulate SDAs of BCS class II drug-RXN by the amalgamation of solid dispersion and melt adsorption techniques and assess its potential to improve solubility and dissolution. Amounts of PEG 4000 and Neusilin US2 were the critical material attributes that could affect quality attributes such as dissolution and solubility. A 2-factor, 3-level experimental design was employed for statistical optimization, wherein the impact of independent variables [amount of PEG 4000 (X 1 ) and amount of Neusilin US2 (X 2 ) on the dependent variables [time required for 85% drug release (Y 1 ) and saturated solubility (Y 2 ) was assessed. The prepared SDA batches (B1-B9) were assessed for both pre and post-compression properties. Optimized RXN SDA was evaluated for cytotoxicity, pharmacokinetics, and pharmacodynamics properties.

Materials
RXN was obtained from Symed Labs limited, Hyderabad, India. Neusilin US2 was obtained from Gangwal Chemicals Pvt. Ltd., Mumbai, India. Microcrystalline cellulose (Avicel PH 102) was purchased from Signet, Mumbai, India. Polyethylene glycol (PEG) 4000, crospovidone, magnesium stearate, and talc were acquired from S.D. Fine Chem. Products, Mumbai, India. The cell lines of human colorectal adenocarcinoma (Caco-2) were procured from the National Center for Cell Science, Pune, India. Culturing of cells was performed in Dulbecco's Modified Eagle's medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, 100 µg/ mL streptomycin, and 1% non-essential amino acids in a humidified environment of 5% CO 2 using a carbon dioxide incubator (Thermo Scientific, Waltham, MA, USA) at 37 °C. Xarelto® tablets 10 mg (BAYER, Germany) were purchased from a medical store. All other chemicals were of analytical grade.

Preparation of RXN SDA
SDA of RXN was prepared by a fusion method reported in the literature [17] with minor modifications. In this method, RXN was sifted through sieve number 100. The carrier (PEG 4000) was melted in a porcelain dish and heated till its melting point (57-63 °C) on a hot plate. Then, RXN (0.435 g) was dispersed into melted carrier mass with constant stirring to form solid dispersion. Further, the adsorbent (Neusilin US2) was added and stirred until the blend is converted into a free-flowing powder. The SDA was then passed through sieve number 40.

Experimental design (QBD-DoE approach)
A 2-factor, 3-level full factorial experimental design was chosen to optimize various formulation parameters statistically and the effects of various formulation components on solubility and in vitro release of formulations were evaluated. The selected dependent and independent variables are presented in Table 1 along with their low (− 1), medium (0), and high (+ 1) levels. The amounts of carrier and adsorbent were selected from the results of preliminary experiments and previous literature [27,28]. An experimental design comprising 9 design batches was developed with different amounts of the carrier (X 1 ) and amount of adsorbent (X 2 ) coded and non-coded values of design batches are shown in Table 2.

Percentage yield and solubility
Percentage yields of the prepared SDA batches (B1-B9) were calculated using the following equation; (1) % Practical yield = (weight of prepared SDA) Theoretical weight of SDA * 100

Saturation solubility
The solubility of the RXN in prepared SDA batches (B1-B9) was estimated by adding an excess amount of drug-containing SDAs in 10 mL of distilled water in glass vials [29]. The mixture was mixed in a thermomixer C (MixMate, Eppendorf, Germany). The shaking of the mixture continued for 24 h at 25 °C. The solution was then sonicated for 20 min and the supernatant was taken and passed through a syringe filter (0.45 µm). The filtrate was diluted and the absorbance was recorded on a UV spectrophotometer (UV Shimadzu 1800 Scientific Instrument, Japan) at λ max of 248 nm. A similar procedure was appointed to determine the solubility of pure drug-RXN and the value was employed as control.

Pre-compression evaluation
The angle of repose, bulk density, tapped density, Carr's index, and Hausner's ratio of prepared RXN-SDA blends were measured using standard protocols. The precompression parameters were also determined for pure drug-RXN and the values were employed as control [30,31]. Table 3 shows the composition of tablets prepared from RXN SDA. SDA equivalent to 10 mg of RXN was taken followed by the addition of Avicel PH 102 and crospovidone and mixing for 5 min. Then magnesium stearate and talc were sifted through sieve number 100, added to the above blend, and mixed for 2 min. The lubricated blend was compressed into tablets using a 10-mm punch in a tablet compression machine (Rimek mini press-I, Ahmedabad, India). A compression force of 10 kN was applied.

Formulation of directly compressible tablets
The directly compressible tablets were prepared by simple mixing of RXN (10 mg; sifted through sieve number 100), PEG 4000 (142 mg), Neusilin US2 (64 mg), crospovidone (15 mg), and Avicel PH 102 (64 mg) for 5 min. Then, magnesium stearate (2.5 mg) and talc (2.5 mg) were sifted through sieve number 100, added to the above blend, and mixed for 2 min. The lubricated blend was compressed into tablets using a 10-mm punch in tablet compression machine (Rimek mini press-I, Ahmedabad, India). A compression force of 10 kN was applied.

Post-compression evaluation of RXN SDA tablets
The compressed tablets were characterized for general appearance, thickness, weight variation, hardness, friability, disintegration time, and assay according to the standard protocols [32].

Drug content
Ten tablets were weighed and finely powdered using a mortar. An amount of the powdered mass equivalent to 10 mg of RXN was accurately weighed and transferred to a 100 mL volumetric flask. Acetonitrile: water (1:1, 50 mL) was added, and the mixture was sonicated for 15 min to ensure complete extraction of RXN followed by volume make up with acetonitrile: water (1:1). The solution was filtered through a 0.45-µm membrane filter and analyzed spectrophotometrically at 249 nm [33].

In vitro dissolution
The dissolution profile of immediate-release tablets prepared from the SDA batches (B1-B9) was done on USP type II apparatus (Electrolab TDT-08L, Mumbai, India) at 75 rpm.
The dissolution medium used was acetate buffer (pH 4.5; 900 mL) containing 0.4% sodium dodecyl sulfate with a temperature setting at 37 ± 0.5 °C [34]. Samples (5 mL) were withdrawn for analysis at different time points (5,10,15,20,25,30,40,50, and 60 min). The withdrawn volume of the sample from the dissolution vessel was replenished every time with an equal volume of the dissolution media to maintain sink conditions. The sample was analyzed at 248 nm using UV spectroscopy after filtering through a Millipore filter (0.45 µm; Spectrum Medical Inc., San Diego, CA). Using the calibration curve equation, the cumulative percentage of RXN release at various time intervals was calculated. Similarly, in vitro drug release of optimized SDA tablet was performed and the profiles were compared with RXN suspension (Z-average: 989.8 nm; PDI: 1.0), marketed product (Xarelto® 10-mg tablets), and directly compressible tablets.

Generation of polynomial equations and response surface plots
Through the use of the Design-Expert software, polynomial equations and 3D graphs were generated to assess the correlation between independent and dependent variables. Analysis of variance (ANOVA) was employed to assess the significance of the independent variables on the responses [35].

Constrained and graphical optimization of RXN-SDAs
To select the most appropriate formulation, a numerical optimization technique based on the desirability function was employed [36]. The constraints laid down at the start of the experiment on the responses of RXN-SDAs included (i) t 85 (Y 1 ): lowest and (ii) saturated solubility (Y 2 ): highest. The solutions provided by the Design-Expert software were sorted in the descending order of their desirability values and the formulation with the highest desirability factor (values close to 1) was considered for optimization. Graphical optimization was also performed by applying constraints to generate design space [37].

Validation of experimental design
A 3 2 factorial design, employed for the formulating RXN-SDAs, was validated by preparing a checkpoint batch. Based on the values of independent variables obtained from the desirability function and overlay plot, an optimized batch of RXN-SDAs was formulated. Comparisons of the experimental and predicted values of the responses were performed to confirm the validity of the design [37].

Compressibility and flowability
The angle of repose, bulk density, tapped density, Carr's index, and Hausner's ratio of optimized RXN-SDA blend was determined by the procedure described above [30,31].

FTIR spectroscopy
The IR peaks of optimized SDA, RXN, and Neusilin US2 were characterized by FTIR spectroscopy (Bruker Optics GmbH, Ettlingen, Germany). RXN and potassium bromide were mixed and made into pellets by compressing the powder mixture on a potassium bromide press at 20 psi for 10 min [38]. The spectrum was taken from the range of 4000 to 400 cm −1 (Wavenumber).

X-ray powder diffraction
Diffractograms of RXN, Neusilin US2, and SDA were recorded using a diffractometer (Rigaku Corporation-Miniflex, Surat, India). The samples were finely ground in a mortar with the help of a pestle and samples were irradiated with X-ray at a scanning angle in the range of 0 to 40° of 2θ.

Differential scanning calorimetry
DSC thermograms of RXN, Neusilin US2, and SDA were characterized by DSC (DSC-60, Shimadzu Corporation, Kyoto, Japan). The thermal performance of the samples was studied by keeping (3-5 mg) in an aluminum pan and sealing hermetically, while a blank pan was used as standard reference [39]. Thermal behavior was determined at a scanning speed of 10 °C/min at a temperature between 25 and 300 °C under a nitrogen atmosphere (10 mL/min).

Surface morphology
Morphological examination of prepared SDA was carried out by scanning electron microscopy (Hitachi S-3400, India) operated at a specific voltage of 15 kV. Samples were mounted on metal stubs and were made conductive by coating with platinum in neutral condition while maintaining the pressure [40]. Then, the images were captured using a microscope.

Evaluation of optimized SDA (post-compression)
The weight variation, thickness, hardness, friability, drug content, disintegration time, and in vitro dissolution of the optimized RXN SDA tablets were performed as per the procedures mentioned above.

Cytotoxicity test
The cytotoxicity of prepared RXN SDA tablets and suspension containing RXN against the Caco-2 cell line was determined by MTT assay. In brief, cells were seeded at a density of 7.5 × 10 4 cells/mL (200 µL/well) in 96-well culture plates and incubated with 20-100 µM of RXN SDA tablets or RXN suspension. After 24 h of incubation, the MTT reagent (25 µL; 5 mg/mL in PBS, 25 µL/well) was poured into the individual well and kept at 37 °C for 4 h, to metabolize MTT. The formed formazan precipitate was dissolved by adding DMSO (100 μL) to every well. The optical density of the samples was determined at 570 nm with the help of a plate reader (Bio-Rad, iMark, Hercules, CA, USA) and the background was deducted at 630 nm. The estimation of percent cell viability was made based on the formula described in the literature [41].

Pharmacokinetic study
A total of 18 male Sprague-Dawley rats were included in the study. The animals were randomly divided into three groups. Group I, group II, and group III were administered RXN suspension, RXN SDA tablets, and Xarelto tablets respectively (equivalent to 10 mg/kg of RXN) [42]. RXN SDA tablets and Xarelto tablets were powdered and dispersed in 0.5% w/v carboxymethyl cellulose [43]. (Pharsight, Sunnyvale, CA, USA) was employed to perform non-compartmental pharmacokinetic analysis. From the plot of drug plasma level vs. time, pharmacokinetic parameters, namely maximum plasma concentration of RXN (C max ) and the time required to achieve the maximum plasma concentration of RXN (T max ) were calculated. The area under the curve (AUC) was determined using the trapezoidal law from 0 to 24 h (AUC 0−24) , and extrapolating the time to infinity was used to calculate the AUC of the profile from 0 to infinite time (AUC 0−∞ ).

Estimation of RXN in plasma
Chromatographic analysis of rivaroxaban was carried out on an HPLC-DAD system consisting of Shimadzu LC-2010-CHT equipped with a binary pump (LC20AT), injection (Rheodyne, 20μL loop), and photodiode array detector (SPD-M20A). The data was captured using LC solution software Ver. 1.25 (Shimadzu, Japan). The wavelength of the detector was set at 249 nm. The chromatographic separation was achieved using a reverse-phase octadecyl silane C 18 (250 mm × 4.6 mm; 5 μm) (GraceSmart) column. The mobile phase consisting of a 90:10 v/v mixture of (A) water containing 0.1% of formic acid and (B) acetonitrile was isocratically pumped at 1 mL/min. The injection volume was 20 μL [44]. All determinations were performed at 25 °C.

Pharmacodynamic studies
Pharmacodynamic studies were performed in Maliba Pharmacy College, Uka Tarsadia University, as per the animal ethics committee approved protocol MPC/IAEC/21/2017 and CPCSEA guidelines.

Tail bleeding time assay
Animals were divided into three groups comprising 6 rats per group, to estimate tail bleeding duration. Group I received 0.5% w/v carboxymethyl cellulose (control); group II received RXN suspension and group III received RXN SDA tablets dispersed in 0.5% carboxymethyl cellulose. Groups II and III received doses equivalent to 10 mg/kg of RXN. All treatments were given orally once. Four hours post-administration; animals were anesthetized by administering pentobarbital sodium (50 mg/kg intraperitoneal) [45]. The method of Wang et al. (2004) with slight modifications was adopted to determine the rat tail bleeding time [46]. A 5-mm rat tail tip was amputated with a scalpel to determine the bleeding time, and blood was blotted onto filter paper every 30 s before the staining of the paper with blood ceased. The bleeding time was described as the time between the amputation of the tail and the cessation of bleeding (min).

Platelet aggregation
This study was performed in rats for all three groups after the tail bleeding time assay. Blood was spontaneously withdrawn from the abdominal aorta and collected in the vial containing anticoagulant, acid-citrate-dextrose (9:1, v/v containing citric acid 130 mM, trisodium citrate 170 mM, dextrose 4%). The platelets were prepared according to the process mentioned previously [47]. Briefly, platelet-rich plasma was collected by centrifuging blood at 3600 rpm for 15 min. In vitro platelet aggregation was evaluated according to the method of Born [48]. Platelet aggregation was performed using an aggregometer at 37 ºC and 1000 rpm. The 240-µL washed platelets were stimulated with an aggregating agent, i.e., collagen (5 mg/ mL). Platelet aggregation was registered for a 10-min period after platelet stimulation.

Data analysis
The statistical interpretation of experimental data was carried out with GraphPad Prism (version 6, GraphPad, San Diego, CA, USA). The difference in values at P < 0.05 is considered statistically significant.

Results and discussion
Flow characteristics of SDA RXN-SDA blends (B1-B9) were evaluated for angle of repose, Carr's index, and Hausner's ratio, and the results are presented in Table 4. The angle of repose, Carr's index, and Hausner's ratio were found in the range of 17.25-27.78°; 9.25-22.78%, and 1.09-1.22 respectively indicating fair flow properties, which is a prerequisite for commercial manufacturing. Neusilin US2 possesses a large surface area and porous nature and has been widely employed for flow improvement. The adsorption of RXN solid dispersions onto Neusilin US2 contributed to improved flowability and compressibility of solid dispersions, which otherwise are known to be sticky.

Percentage yield
The percentage yields of RXN-SDA batches (B1-B9) were found in the range of 95-98% (Table 5). The high values for percentage yield were indicative of the minimum loss, homogeneity, and accuracy of the process, and hence the suitability for scale-up.

Saturated solubility
The aqueous solubility of pure RXN was found to be 0.012 ± 0.009 g/mL whereas the values for batches B1-B9 were found in the range of 0.023-0.041 g/mL. The highest RXN solubility noticed was 0.041 mg/mL, which is 3.4 times higher as compared to the solubility of pure RXN. This improvement in solubility of RXN could be due to PEG 4000 (hydrophilic carrier) that improves the wettability of the drug [49] as well as Neusilin US2 (adsorbent), which increases the surface area of the formulation [50,51].

FTIR
The FTIR spectra of pure RXN, PEG 4000, Neusilin US2, and SDA are presented in Fig. 1. From the figure, it is observed that the infrared spectrum of pure drug and SDA have a significant difference in the absorption peak intensity, probably due to the low concentration of drug compared to the excipients used. A slight shift towards the lower wavelength or broadening was noticed in a few peaks. These subtle changes could be attributed to the intermolecular hydrogen bonding and/or van der Waals interactions between RXN and PEG 6000. Hydrogen bonds are essential in solid dispersions because they reduce molecular mobility, depress the driving force of crystallization, and thus enhance the stability and solubility of the solid dispersion compared to pure RXN. A similar finding has been reported by another research group [52].

XRD
The diffraction spectra of RXN, PEG 4000, Neusilin US2, and RXN SDA are shown in Fig. 2. The graph of pure RXN showed the high crystalline nature of the drug with main diffraction peaks. The X-ray diffractogram of SDA did not show any characteristic peaks which are observed in pure drug diffraction patterns, which indicates the transformation of crystalline to an amorphous form of the drug. The possible reason for this change could be due to the annihilation of RXN crystal lattice, because of homogenous dispersion of drugs into molten carriers. Therefore, the reduction in crystallinity of the drug might be causing improvement in the dissolution of RXN [53].

DSC
The DSC thermogram of a pure drug, Neusilin US2, PEG 4000, and SDA are presented in Fig. 3. RXN showed a distinct strong endothermic peak at 230 °C (which is the drug melting point), which indicates its crystalline nature. However, the DSC of SDA did not show any peak of pure RXN; this might be due to the complete dissolution of RXN in the melted polymer which indicated that the drug can be in an amorphous state [54].

Surface morphology
SEM images were captured to observe the surface morphology of the SDA (Fig. 4). The SEM image of the RXN powder exhibited its crystalline nature and irregular shape. The

Post-compression characterization of SDA tablets
Physical examination of tablets from each batch showed that all the tablets were circular, white to off white, flat, and without any physical defects. The thickness of SDA tablets ranged from 3.10 ± 0.02 to 3.48 ± 0.02 mm (  (Table 5), which assures the ability of the tablets to withstand wear and tear during handling and transportation. Friability was in the range of 0.32 ± 0.02 to 0.48 ± 0.05% (Table 5), which ensures acceptable resistance by tablets to withstand mechanical shocks. Disintegration time ranged between 25.33 ± 0.57 to 44.23 ± 1.52 s ( Table 5). The percentage drug contents of all the batches of SDA were found in the range of 96.15-99.60%, indicating the uniformity of the drug in the developed formulation. All the post-compression parameters were satisfactory suggesting that the developed formulation can be scaled up with success.  Table 6. A positive number indicates an effect that favors the optimization, on the other hand, a minus number suggests that the relationship between factor and response is negative. It is evident that both the independent variables, viz., the amount of carrier (X 1 ) and the amount of adsorbent (X 2 ) have positive effects on the responses, viz., t 85 (time required for 85% drug release) and saturated solubility (g/mL).

Effect of amount of PEG 4000 (X 1 ) and amount of Neusilin US2 (X 2 ) on time required for 85% drug release (Y 1 )
An in vitro dissolution profile for B1-B9 batches is shown in Fig. 5. The time required for 85% release of a drug from a formulation (t 85 ) is a crucial parameter in evaluating its quality. The effects of the amount of PEG 4000 (X 1 ) and amount of Neusilin US2 (X 2 ) on t 85 for RXN-SDA tablets were studied. The t 85 values for batches B1-B9 were recorded in Table 6 and were found in the range of 8.55-16.10 min.
The lower values of t 85 for batches B1-B9 in comparison to RXN suspension could be attributed to the molecular dispersion and solubilization of RXN in hydrophilic carrier PEG 4000. Additionally, improved wettability of RXN due to PEG 4000, prevention of drug aggregation and decreased particle size of RXN in SDAs also contribute to lowering of t 85 (enhanced dissolution rate). Furthermore, reduction in the crystallinity of RXN when dispersed in PEG 4000 in SDAs, is considered a crucial factor in the enhancement of the dissolution rate.  Neusilin US2, a porous adsorbent, employed in formulating RXN-SDAs possesses an excellent adsorption capacity (3.2 mL/g) and a high specific surface area (300 m 2 /g). The adsorption of RXN solid dispersions onto Neusilin US2 in RXN-SDAs would provide an increased effective surface area, which would lead to rapid desorption of the drug upon exposure to dissolution medium [56].
The linear model is selected for this variable. The polynomial equation for Y 1 is as shown below: (2) Y 1(t85) = +10.62 − 1.53X 1 − 3.01X 2 0 The 2D and 3D plots for Y 1 are shown in Fig. 6. From Eq. (2) and Fig. 6, it is clear that X 1 was found to have a negative impact on Y 1 . An increase in the amount of PEG 4000 led to a decrease in the time required for 85% drug release. This could be explained as follows: Hydrophilic carrier-PEG 4000 is known to improve the wettability of the drugs [44]. RXN would be present in different proportions of the crystalline and amorphous forms based on the amount of PEG 4000 employed in the preparation of RXN solid dispersion. As the amount of PEG 4000 increases, the amorphous form of RXN is expected to increase, which can cause augmentation in the dissolution of RXN and reduction in t 85 . A similar observation was recorded by our research group while formulating Ticagrelor solid dispersion adsorbates [57]; Kapsi and Ayers [58] while formulating Itraconazole-PEG solid dispersions; Chutimaworapan et al. [59] while formulating Nifedipine solid dispersions in presence of water-soluble carriers like PEG, hydroxyl propyl β-cyclodextrin (HP-β-CD), and poloxamer 407.
As seen from the above equation, the amount of Neusilin US2 (X 2 ) was also found to have a negative impact on t 85 . Neusilin US2 provides an enhanced effective surface area [46] leading to the rapid diffusion of RXN into the dissolution medium. The enhanced dissolution rate is suggestive of a reduced t 85 .

Effect of amount of PEG 4000 (X 1 ) and amount of Neusilin US2 (X 2 ) on saturated solubility (Y 2 )
Solubility is one of the crucial parameters to achieve the desired concentration of drug in the plasma for anticipated therapeutic response. RXN solubility was found to be 0.012 ± 0.009 g/mL whereas the solubilities for B1-B9 were found to be in the range of 0.023-0.041 g/mL ( Table 6). The results indicated that Y 2 is affected by the amount of PEG 4000 and the amount of Neusilin US2. 2D and 3D plots, representing the influence of both independent variables on Y 2 are shown in Fig. 7. The response (Y 2 ) obtained for two independent variables was subjected to multiple regression to get a quadratic polynomial equation: From Eq. (3) and Fig. 7, it is evident that Neusilin US2 has a positive impact on the RXN solubility when SDAs were formulated. An increase in the amounts of Neusilin leads to a linear increment in the solubility. This could be attributed to the increased surface area provided by the novel adsorbent. At constant levels of Neusilin US2, solubility was increased marginally when PEG 4000 was increased from − 1 to 0 levels. This could be due to interaction between PEG 4000 and RXN mainly by electrostatic forces and occasionally by other types of forces like hydrogen bonds [60]. A further increase from 0 to + 1 levels, led to a slight decrease in the solubility.

Optimization and validation overlay and checkpoint analysis
The numerical and graphical optimization approaches were employed to obtain RXN-SDAs with desirability functions close to unity. The overlay plot is shown in Fig. 8, which helps to obtain acceptable fields for the independent variables. The yellow area in the overlay plot exhibits the required design space within which the anticipated results for the responses can be obtained. An optimized formulation with X 1 and X 2 values of 5.7 g and 2.52 g respectively was chosen. The anticipated Y 1 and Y 2 values were 9.27 min. and 0.0395 g/mL respectively. To check the validity of the optimization process, an optimized batch was formulated with the chosen levels of X 1 and X 2 . The composition of the optimized RXN SDA tablets is shown in Table 7. The predicted and experimental values of the responses (Y 1 and Y 2 ) are tabulated in Table 8. The differences between expected and observed values were negligible. The RSM model's validity was verified by the strong agreement between the predicted and experimental values.

Pre-compression parameters
The optimized RXN-SDAs were evaluated for flowability and compressibility. The values for the angle of repose, Carr's index, and Hausner's ratio were found to be 17.25 ± 0.29°, 9.20 ± 0.65%, and 1.17 ± 0.08 respectively. The values are suggestive of excellent flowability and compressibility, which would suffice the requirements of scale-up and commercial manufacturing. The saturated solubility of RXN-SDAs was found to be 0.041 ± 0.0007 mg/ mL, which is 3.42 folds higher than the pure RXN.

In vitro dissolution
In vitro release of pure RXN, RXN-SDA tablets, marketed tablets (Xarelto® 10 mg), and directly compressible tablets were carried out using USP type-II apparatus. The release profile is represented in Fig. 9 Our developed formulation (RXN-SDA tablets) exhibited a higher rate and extent of dissolution at all the time points in comparison to the Xarelto tablets. The higher in vitro dissolution, attributable to a multitude of mechanisms described above, could be the causative reason for augmented in vivo dissolution. Higher in vivo dissolution might, in turn, provide a faster T max and a higher C max , AUC, and bioavailability along with reduced dose, sideeffects, cost of therapy, better patient compliance, and improved management of hypertension.

Cytotoxicity test
To evaluate the cytotoxic effect of RXN SDA tablet and RXN suspension, an MTT assay was performed on Caco-2 cells [62,63]

Pharmacokinetic study
Based on the plasma concentration-time profile (Fig. 10), corresponding pharmacokinetic parameters were determined and tabulated in  (Table 9). The results of this study are in-line with the in vitro release studies. These values clearly indicated augmentation in bioavailability of RXN from SDAs when compared to its suspension and marketed formulation, confirming an increase in solubility and dissolution rate thus enhancing the oral bioabsorption. A similar observation was noticed in earlier studies with other drugs as well [64]. The outcomes of this study may be extrapolated towards the possibilities of dose reduction upon the administration of the test formulation. Literature suggests that food does not affect the AUC or C max when a 10-mg oral dose is administered [2]. However, for higher doses (20 mg), the fed state exhibits improved bioavailability. Hence, the food intake along with the fold in solubility improvement has to be jointly considered for dose escalation studies.

Tail bleeding time assay
To discern the antithrombotic and associated antihemostatic effects of 0.5% CMC (control), RXN suspension,  and RXN SDA tablets; bleeding time was investigated in the rat tail model. The bleeding times recorded with control, RXN suspension, and RXN SDA tablets were found to be 68.00 ± 9.08 s, 126.00 ± 6.52 s, and 189.00 ± 7.70 s respectively. RXN SDA tablets exhibited significantly higher (P < 0.05) prolongation of bleeding time in comparison to control and RXN suspension. The higher efficacy of the developed formulation along with the reversible binding of RXN to Factor Xa would probably provide greater clinical benefits compared to other antiplatelet agents.

Platelet aggregation
The effects of various formulations [0.5% CMC (control), RXN suspension, and RXN SDA tablets] on collagen-induced platelet aggregation were evaluated 4 h post-dosing. The Control group exhibited a value of 55.62 ± 2.95% whereas the values exhibited by RXN suspension and RXN SDA tablets were 24.37 ± 1.86% and 13.78 ± 1.62%, respectively. The highest reduction in percentage platelet aggregation registered by RXN SDA tablets can be ascribed to the improved bioavailability of RXN, which ultimately inhibits Factor Xa leading to potentiation of the antiplatelet action. The data are in agreement with pharmacokinetic studies asserting the clinical benefit of the developed novel formulation of RXN. A similar finding has been reported by another research group [65].

Conclusion
This work demonstrated the amalgamation of solid dispersion and melt adsorption technologies in formulating RXN-SDAs, wherein the ternary system comprised of RXN (insoluble drug), PEG 4000 (hydrophilic carrier), and Neusilin US2 (high surface area adsorbent). A DoE approach was employed in the optimization of RXN-SDAs. RXN-SDA blends exhibited better flow properties and higher solubility as compared to pure drugs. RXN-SDA tablets exhibited a lower t 85 in comparison to the pure drug, marketed tablets, and directly compressible tablets. MTT assay confirmed the absence of cytotoxicity in the developed formulation. RXN-SDA tablets exhibited improved bioavailability and rapid onset of action in comparison to the pure drug and marketed formulation when evaluated in a rodent model. Similarly, bleeding time and platelet aggregation studies suggested superior anticoagulant efficacy of RXN-SDA tablets in comparison to a pure drug. The improved bioabsorption would provide a reduction in dose, dose-related side effects, cost of therapy, and better patient compliance. The studies are proof of the concept that RXN-SDAs prepared by this simple, economic, scalable, and solvent-free process can prove to be a viable alternative in the management of stroke and embolism. However, scale-up, validation, and clinical studies are required for the successful translation of this dosage form from bench to bedside.